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Biomed Grid | Antihypertensive Peptides in Dairy Products
Introduction
Milk and dairy products are rich in protein content, and main proteins in milk casein and whey proteins are the principle bioactive peptide precursors [1]. Although there are numerous studies on bioactive peptides, the first discoveries of bioactive peptides from foods are dairy products. These bioactive peptides have different functions such as antihypertensive, opioid, immune-modulatory, antimicrobial, and antioxidant [2, 3, 4, 5]. Hypertension is a condition in which the blood vessels have persistently raised pressure [6]. It affects approximately 30% of the adult population worldwide [7]. It is a prominent risk factor for cardiovascular diseases such as coronary heart disease, peripheral artery disease, and stroke. Even though it is a controllable disease high prevalence, and serious consequences make hypertension an important comprehensive health threat [7]. ACE (peptidyldipeptide hydrolase, EC 3.4.15.1) is an exopeptidase which splits various peptides from C-terminal and forms dipeptides. ACE performs a critical role in regulating the blood pressure by the renin-angiotensin and bradykinin pathways. Angiotensin I is a decapeptide and inactive in its intact form. ACE catalyzes hydrolyzation of angiotensin I to the potent vasoconstrictor octapeptide angiotensin II[8]. Bradykinin, a vasodilator, is involved in the blood pressure system. ACE also controls the blood pressure by degrading bradykinin. Thus, inhibition of ACE results in an antihypertensive effect. Consequently, synthetic ACE inhibitors such as captopril and enalapril, are used in the treatment of hypertension and other related heart diseases [9]. However, they can cause adverse effects such as hypotension, cough, altered taste, rash and angioedema [10]. Bioactive peptides are natural and healthier alternatives to synthetic ACE inhibitors without side effects [11]. Although there are many different protein sources of ACE inhibitor peptides, milk proteins are accepted as the primary sources [12]. Chemical structures of peptides are important for binding to the catalytic sites of ACE [13, 14]. The presence of aromatic or branched hydrophobic structures in the tripeptide structure at the carbon end of the peptide is necessary for binding [13, 15]. Several functional dairy products are present in the market comprising antihypertensive peptides. Calpis sour milk in Japan (Calpis Co Ltd, Tokyo, Japan) cultured via Saccharomyces cerevisiae and Lactobacillus helveticus poses two potent ACE inhibitory peptides, Val-Pro-Pro and Ile-Pro-Pro [15]. These two peptides are also present in calcium-enriched fermented milk drink in Finland [16]. In short- and long-term human studies have shown that IPP and VPP peptides decrease blood pressure [4]. BioZate contains β-lactoglobulin fragments as functional bioactive peptides [16]. This paper will review studies on antihypertensive especially, ACE inhibitor peptides and their production in dairy products.
Factors Affecting Occurrence of Bioactive Peptides in Dairy Products
The bioactive peptide profile of a dairy product is tightly dependent on processes used such as thermal processes, homogenization, pressure applications, coagulation of milk, fermentation, and ripening [17]. Thermal processes are essential in the production of almost all dairy products. Reactions that occurred during thermal processes can affect the structure of proteins and the bioactive peptide content of the product [17, 18]. Thermal processes affect activities of natural enzymes found in milk, thus affect the peptide profile of the last product. Caseins are hydrolyzed through the action of enzymes from different sources such as casein residue coagulants, natural milk enzymes, starter culture enzymes, enzymes of seconder cultures and non-starter lactic acid bacteria [5].
ACE Inhibitor Peptides Naturally Found in Milk and Dairy Products
In general, dairy products, in particular, fermented dairy products, are the most popular foods for the intake of bioactive peptides with their sensory properties and high levels of consumption favored by consumers [1]. Some of these studies summarized in (Table 1). Among the dairy products, ripened cheeses contain numerous peptides, affecting the properties of the final product such as taste, odor, and texture due to the variety and complexity of the production methods. ACE inhibitor peptides in Spanish cheeses (Cabrales, Idiazábal, Roncal, Manchego, Mahón and goat’s milk) are identified [14]. In this study, researchers confirmed ACE inhibition effect of 8 synthetic peptides (VRGP, PFP, QP, DKIHP, PKHP, FP, PP, and DKIHPF). Since proteolysis and peptide formation continue during cheese ripening, the ACE inhibitor effect may alter during the cheese maturation period. Further proteolysis during ripening may cause hydrolyzation of bioactive peptides and inactivation of them. Gomez-Ruiz et al. [19] determined the ACE-inhibitor peptides in Manchego cheese. The antihypertensive activity reached the maximum level after eight months of maturation and decreased again after twelve months of maturation. Likewise, Gouda ripened for 8 months decreased more strongly the blood pressure of spontaneously hypertensive rats than 24-month-old Gouda, although they have a similar ACE inhibitor activity in vitro [3]. In view of composition rich in proteins, cheese whey can be considered as a valuable source of bioactive proteins [20]. Alongside studies on bioactivities of cheese varieties some researchers identified ACE inhibitor peptides (FVAPFPE, NLHLPLPLLQ, FVAPFPEVFG, NLHLPLPLQ originated from αs1-casein, β-casein, αs1-casein, β-casein, respectively) in a liquid waste deriving from Ricotta cheese production [21]. Probiotic fermented milk beverage from milk of different species also have antihypertensive activity [22, 23]. Caseins are the best precursors for the production of angiotensin I am converting enzyme (ACE) [de Gobba et al. 2014].
Table 1: Antihypertensive peptides found in dairy products.
Production of ACE Inhibitor Peptides from Milk Proteins
Basically, there are two approaches to generate ACE-inhibitor peptides from milk proteins. One approach is to utilize the proteolytic enzymes of lactic acid bacteria in fermented dairy products. The other approach is to hydrolyze milk proteins in vitro by one protease or a combination of various proteases or peptidases.
Production of ACE Inhibitor Peptides with Enzymes
Most of the researches about the production of bioactive peptides with enzymes have utilized digestive enzymes, and commercial dry cheese whey, purified whey proteins or microfiltration permeates as a substrate [27]. Besides, other digestive enzymes from different sources and various milk protein preparations have been studied to generate antihypertensive peptides (Table 2). Different bioactive peptides are produced from caseins of milk from different species, which implicates the sequence and conformation of the caseins affect the bioactive peptide yield [28]. Minervini et al. [29] used a proteinase from Lactobacillus helveticus PR4 to obtain ACE inhibitor and antimicrobial peptides from casein of milk from six different species (bovine, sheep, goat, pig, buffalo, and human). Abdel-Hamid et al. [29] identified new peptide sequences (FPGPIPK, IPPK, QPPQ) showing ACE inhibitor activity generated from buffalos’ skim milk hydrolyzed with papain.
Table 2: Using proteases to generate ACE inhibitor peptides from milk proteins.
ACE inhibitor and antioxidant capacity of 6 synthetic peptides (WY, WYS, WYSL, WYSLA, WYSLAM, WYSLAMA) deriving from β-lactoglobulin were evaluated [30]. Dipeptide WY β-lactoglobulin fragment f (19-20) showed potent ACE inhibitor activity. ACE inhibitor activity depends on the amino acid sequence in the C-terminus of the peptide, and the amino acid Ser at the C –terminus showed a potential decreasing effect on ACE inhibitor activity. Sheep cheese whey hydrolyzed using proteinase from Bacillus sp. P7 to generate ACE inhibitor peptides [20]. ACE inhibitor activity was dependent on hydrolysis time. In a recent work, trypsin from bovine pancreas employed to hydrolyze whey from the production of panela cheese to generate bioactive peptides [27]. The researchers found a significant correlation between antioxidant and ACE inhibitor activity.
Production of ACE Inhibitor Peptides through Fermentation
In the dairy industry mainly highly proteolytic starter cultures are preferred. Bioactive peptides can be generated by the starter culture or non-starter bacteria added as an adjunct culture (Table 3).
Table 3: Obtaining ACE inhibitor peptides by using adjunct culture and fermentation.
Ahtesh et al. [1] produced a new fermented functional dairy product with combination of L. helveticus and Flavourzyme® using a bioreactor. They have achieved to obtain an acceptable product with high ACE inhibitor activity. L. helveticus is a highly proteolytic bacterium, thus, there are many studies on both fermentation with this bacterium and hydrolysis with proteinases of this bacterium [22, 28].
Similarly, researchers utilized L. helveticus LH-B02 strain in order to improve the ACE inhibitor activity in Prato cheese [5]. They observed that levels of ACE inhibitor peptides β-casein (f193-206) and β-casein (f194-209) increased while relative intensity of αS1- casein (f1-9) reduced. Gonzalez Gonzalez et al. [25] isolated highly proteolytic lactic acid bacteria from Chiapas cheese and evaluated tendency of releasing bioactive peptides of selected strains. They employed four selected strains for fermentation of milk and observed that most proteolytic strain has lowest ACE inhibitor activity, presumably according to further breakdown peptides to inactive amino acids. Solieri et al. [31], fermented bovine milk with non-starter lactic acid bacteria (Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus strains) to evaluate their potential to produce fermented milk with enhanced ACE inhibitor activity [32, 34, 35]. They concluded that the strains used in the study especially L.casei PRA205 can produce high amounts of VPP and IPP peptides.
Conclusion
In recent years, the tendency to consume functional health-promoting foods has increased the interest in bioactive peptides. There are numerous studies on bioactive peptides in foods in the literature. Dairy products, which are an indispensable part of a healthy and balanced diet, are considered as ideal sources for bioactive peptides and natural alternatives to therapeutic drugs due to their high protein content and technological processes in production. However, the mechanism of action of bioactive peptides is not fully described. Molecular studies employing new technologic enhancements and peptidomics approach are necessary to understand the mechanisms of antihypertensive peptides as well as to design functional products.
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Biomed Grid | Assessment of Brain Metabolic Score (BMS) In Vivo Based on Mitochondrial Activity in Neuropathology
Introduction
The discovery of oxygen occurred toward the end of the 18th Century (1771-1775) by three scientists including Carl Wilhelm Scheele, Joseph Priestley and Antoine de Lavoisier. It took more than 100 years to discover the intracellular organelle, named mitochondrion by Carl Benda in 1898 [1], that utilized 90-95% of the oxygen taken up and consumed by the body of patients as summarized by Waltemath in 1970 [2].
The aim of the current review is to describe the historical R&D process of using light in order to study brain biochemical and physiological activities. I will deal with the function and regulation of oxygen in supplying energy to this unique central organ of the body. The relationship between the activities of the brain and using optical technologies are presented in (Figure 1). Most of the information on mitochondrial function has been collected using in vitro studies. Small portion of publications dealt with monitoring in vivo of brain mitochondrial function in real-time. Prof. Britton Chance was the leader in the field of using the light, seen in (Figure 1) in studying mitochondrial function (Figure 1) especially the brain under in vivo conditions. The study of mitochondrial function in vivo was expanded later by our group that developed the multiparametric monitoring system used as seen in (Figure 1) [3, 4, 5]
Historical Overview
The functional capacity of any tissue, and especially the brain, is related to its ability to perform its work. The assessment of this ability could be done by checking tissue oxygen balance, i.e. the ratio of oxygen supply to demand. As seen in (Figure 2) a similar description was made by Barcroft 105 years ago [6].
He showed the relationship between tissue activity, oxygen consumption as well as increase in blood supply serving as a compensation mechanism. This observation that was published in 1914 was and is supported by many studies published since then. Presentation of the balance between tissue oxygen supply and demand in a typical organ is shown in (Figure 2). Oxygen supply is dependent upon the microcirculatory blood flow (TBF), blood volume (TBV) and the hemoglobin saturation level (HbO2) in the small blood vessels, namely, the microcirculation. The saturation of the hemoglobin in the microcirculation is affected by 2 factors, namely, oxygen consumption by the mitochondria and the microcirculatory blood flow. The demand for oxygen is affected by the specific activities taking place in each organ as seen in the right side of the figure. The mitochondrial NADH (the reduced form) level is a parameter directly related to the oxygen balance.
Figure 1: The story of brain bio-photonics. A - Citation regarding the creation of light. B - The use of the UV part of the spectrum as a tool for the monitoring of mitochondrial function (C). D - Schematics of the elements that represent part of the brain tissue seen in part E.
Figure 2: The “hypothesis” formulated by Barcroft in 1914 regarding the connection between organ activity, oxygen consumption and blood flow (6). B - Presentation of tissue oxygen balance related to the energy supply and demand. Oxygen supply could be evaluated by measurement of tissue blood flow (TBF), blood volume (TBV) and hemoglobin saturation (HbO2). Oxygen demand varies between different tissues and include Ionic Homeostasis, Signal Conduction, Glandular Secretion, Muscle Contraction, and G-I tract and kidney function. Mitochondrial NADH serve as an indicator for tissue oxygen balance (16).
(Figure 3) shows the gradient of oxygen levels between air inspired to the lungs, heart, large arteries and small arterioles to the brain intracellular compartment and finally the mitochondria. The various points of patients’ clinical monitoring are shown. As seen the largest gradient of oxygen occur between the oxygen level in the large arteries and the microcirculation. The delivery of oxygen is done in the microcirculation, therefore the level of oxygen in large arteries is very high (about 100 mmHg). The last usual parameter, in the oxygen gradient, that is monitored clinically is the pulse oximeter that measures the saturation of hemoglobin in the systemic arteries. At this point the HbO2 is highly saturated as indicated in point 1 at part A of the figure. The saturation of the HbO2 at the microcirculation is depending on the organ that is evaluated. In the brain, heart or kidney (very active organs), the saturation will be in the range of 50%-60% and in the resting muscle it will be around 80%. Monitoring of the microcirculation and especially mitochondrial function in vivo is not a standard approach in daily clinical activities.
Figure 3: The gradient of O2 from air to the mitochondria in nervous system. Monitoring of patients include various parameters along the oxygen gradient (96). In the insert A, the dissociation curve of O2 and hemoglobin is presented.
The historical milestones in the development of mitochondrial NADH monitoring after its discovery in 1906 by Harden and Young are listed in (Table 1). Most of the milestones were achieved by Prof. Chance. The collaboration with the physiologist, Prof. Jobsis, led to many studies where various organs in vitro or in vivo were monitored. Most of the studies published in this field were expanded by the team working with Prof. Chance in Philadelphia and then moved to other universities around the world.
Table 1: The main Milestones in NADH Measurements.
(Figure 4) shows the pictures of the 5 scientists who affected significantly the development of the theoretical and experimental technology for the monitoring of mitochondrial NADH function in vitro and in vivo.
Figure 4: The scientists contributed significantly to our knowledge on mitochondrial function and NADH monitoring under in vitro and in vivo conditions.
Figure 5: A - Mitochondrial metabolic state, defined in vitro, by Chance and Williams, and opened up a new era in measurements of respiratory chain enzyme’s redox state in vitro as well as in vivo (43). B - The fluorescence emission spectrum showing the difference between an anaerobic an d an aerobic suspension of liver mitochondria) .The excitation wavelenght was 353nm (97). C - The emission spectra of rat cerebral cortex under aerobic conditions (lower trace) and under anoxic conditions (upper trace) (98).
It is now more than 60 years since the pioneering work of Chance & Williams on mitochondrial metabolic state in vitro shown in (Figure 5), was published [7, 8, 9, 10]. Harden & Young described the pyridine nucleotides almost 110 years ago [11, 12] followed by the description of its full structure by Warburg and collaborators 30 years later [13]. All those studies initiated the first detailed experiments, by Chance et al. [14], in which NADH (Nicotine amide adenine dinucleotide) fluorescence, was used as a marker of mitochondrial function of the brain and kidney in vivo in the anesthetized animals.
Principles of Monitoring NADH Fluorescence
Monitoring of NADH by the difference in the absorption spectrum of its reduced form, led to a limitation of that technique to the study of mitochondria in vitro, and in very thin tissue samples (e.g. muscle) or in cell suspension. More specific and better method is fluorescence spectrophotometry in the near-ultraviolet range (UVA).
The discovery of the optical properties of reduced Nicotineamide Adenine Dinucleotide – NADH (earlier names: DPNH - diphosphopyridine nucleotide, or PN - pyridine nucleotide), led to an intensive research activity since the early 1950’s. The reduced form of this molecule, NADH, shown in Figure 6A1 & 6A2 [15], absorbs light at 320-380 nm (Figure 6) and emits fluorescent light at 420-480 nm range (Figure 6) [16]. The oxidized form NAD+ does not absorb light in the UV range, therefore it was possible to measure the redox state of the mitochondria by monitoring the UV absorbance or Blue fluorescence of NADH. (Figure 6) present NADH fluorescence spectra monitored from the brain of anesthetized rat exposed to anoxia as described in detail [17].
Figure 6: A. The structures of NAD+. The nicotinamide group (broken ring) is the “functional” part of both molecules (A1) i.e. the portion of the molecules where oxidation and reduction take place (15,99). A2. The transition between oxidized and reduced NADH. A3. Excitation and emission spectra of NADH (16). B. Emission spectra of the brain under excitation of 366 nm light (A1, B2, B1, B2, and C1) or laser 324 nm light (C2). C1 and C2 were measured from a dead brain (23).
Figure 7: A. Apparatus for measurement of “fluorescence spectra”. The wavelength drum was driven by a synchronous motor. A sectioned disc d, mounted on another synchronous motor, modulated the incident light. The a.c. component of the current, caused by the light falling on the iP 21 multiplier, was amplified, rectified and fed into the recorder. B. Fluorescence spectra of NADH. The open circles are for excitation by wavelength 313 mμ, the black ones for excitation by 366 mμ. The spectrum for 313 mμ has been multiplied by a certain factor to make its maximum of equal height as the maximum of the spectrum excited by 366 mμ. The spectra appear to be identical; the maximum is at about 462 mμ (22).
The first model of fluorescence recorder was described by Theorell & Nygaard [18, 19] and Theorell, Nygaard and Bonnichsen [20]. Boyer & Theorell in 1956 showed that the fluorescence of DPNH was shifted and the intensity was increased upon combination of DPNH and liver alcohol dehydrogenase-ADH [21]. The 1st study using fluorescence spectrophotometry of NADH in intact Baker’s yeast cells and Algae cells, was published in 1957 [22] as seen in (Figure 7) .
The features of NADH fluorometers consist of the 4 components:
a. A light source (including appropriate filters).
b. An optical path to the monitored object and back to the detection unit.
c. Signal detection and processing units.
d. Signal recording and data storage units.
In our 1984 review, we specified the light-guide-based fluorometry used in our studies [23]. Ince et al. [24], included many other technical aspects of the methodology in their review. Duysens & Amez [22] schematized the first fluorescence spectrophotometer used for intact cells. They utilized the “classical” light source – the mercury arc – providing a very strong band at 366 nm, even though not at the maximal NADH absorption peak of NADH (340 nm). Using a monochromator, they were able to obtain the NADH fluorescence spectrum in baker’s yeast cells and photosynthesizing cells. They concluded that “the fluorescence excited by 366 nm can be used for measuring reduced pyridine nucleotide in vivo”.
Chance & Legallais [25] described a differential fluorometer that opened a new era in monitoring NADH fluorescence in vivo. They used a microscope, serving as the fluorometer basis, with two light sources: tungsten and mercury lamps with appropriate filters. Chance & Jobsis [26] and Chance [27] showed that mechanical muscle activity ends up with NADH oxidation measured in excised muscle. This study was the bridge from the subcellular (mitochondria) and cellular (intact cell) monitoring approaches toward actual in vivo applications.
Figure 8: 1 - Microspectrofluorometer developed and used in the 1960’s. In addition to the interference filter, a Wratten type 2C filter is also placed in the back aperture of the objective. The wave-length-range of the interference filter is 400-700 mμ, and the specification on its spectra interval is 30-40 mμ. Other features of the high pressure mercury arc excite the fluorescence of the specimen at 366 mμ by means of an ‘Eppendorf’ primary filter. Fluorescence excitation and emission pass through the Leitz Ultrapak objective and a ocular (98). 2 – Experimental setup for microfluorometry of brain and kidney cortex in the rat measured simultaneous. Two microfluorometers were focused on the exposed surfaces of the 2 organs. The oxidation-reduction level of the intracellular pyridine nucleotide was altered by changes in ventilation and the corresponding fluorescence changes was recorded by the two microfluorometers. For the kidney fluorometer, the water-cooled lamp housing attached to the Leitz “Ultrapak” illumination system is shown. 3 –Recordings of the kinetics of increases in fluorescence observed in oxygennitrogen transition for kidney (A) and brain (B) cortex of anesthetized rats. The increases in fluorescence are recorded as a downward deflection. The times when gas in the tracheal cannula was changed from oxygen to nitrogen and the times when breathing stopped and started again are indicated.
The in vivo NADH monitoring system was introduced during in the late 1950’s and early 1960’s. The effects of scattered light and tissue absorption due to blood were not evaluated or measured when NADH fluorescence was measured. The first results of in vivo NADH fluorescence measurements appeared in 1962 [14]. These “classical” papers described two microfluorometers that were modifications of previous designs [25, 28]. This micro fluorometer (Figure 8) type employed Leitz “Ultrapack” illumination, which had been used for many years until the development of UV transmitting optical fibers. To avoid movement artifacts, rats were deeply anesthetized, and their heads were fixated in a special holder on the operation table. The same instrumentation was used in other in vivo studies, including those of Chance’s group and other investigators cited in a previous review [17].
The effect of blood on NADH fluorescence was discussed early by Chance et al. [14]. In order to monitor NADH in vivo, it was necessary to avoid large blood vessels in the monitored area which interfere with the emission and excitation light. The monitoring of a second parameter in tissue fluorometry in vivo was reported in 1963 [29]. It was shown that “changes due to the deoxygenation of oxyhemaglobin do not interfere with measurement of the time course of fluorescence changes in the tissue studies”. The addition of a second monitoring signal, namely, tissue reflectance at the excitation wavelength was reported in 1968 [30]. It was based on a previous model described by Jobsis et al. in 1966 [31]. In another two papers [32, 33] , the measurement of 366 nm reflectance was used for the correction of the NADH fluorescence signal from the brain. The reflectance signal was subtracted from the fluorescence signal. The same type of fluorometer was used in by Gyulai et al. [34].
In studies of the cerebral cortex the skull was removed carefully in order to minimize bleeding; about 25 square millimeters of the cortex was exposed. The dura was left intact. The head was held similarly to that used in stereotaxic studies of the brain and was kept sufficiently motionless for continued observation of a small area between major blood vessels. In a few studies, measurements were made simultaneously on the cortex of the brain and on the kidney by means of two independent microfluorometers. In such cases the kidney was held in a clamp on the back of the animal. The apparatus is seen in Figure 8-1 & 8-2.
Areas of the brain and kidney were selected which showed a bright and uniform distribution of fluorescent material. Areas containing large blood vessels were avoided. On the brain cortex a field was selected in which the number of visible capillaries was minimal. Since continuous viewing is possible during photoelectric measurement of the fluorescence, these positions were monitored to make sure that mechanical artifacts were avoided. Figure 8A (lower left side) show the response of the kidney. The increase in fluorescence observed at the time respiration ceases is 11 percent of the total increase during anoxia. As soon as a plateau is reached, the lungs are ventilated with oxygen three times. Three seconds after ventilation is ended an abrupt decrease in fluorescence is observed and breathing starts as the decrease reaches its peak. Irregularities in the extent of oxidation in the fluorescence signal are observed for the next 0.5 minute, but the fluorescence remains above the initial value for 2½ minutes.
A Figure 8B illustrates the response of the brain cortex. No fluorescence changes are observed for 30 seconds after the inspired gas is changed from oxygen to nitrogen. A small but consistently observed diminution of fluorescence occurs, and 10 seconds thereafter hyperventilation was observed. After a plateau is established, ventilation with oxygen is commenced, and 3 seconds thereafter an abrupt decrease in fluorescence reaches its plateau, breathing commences.
These results indicate that similar fluorescence changes under anoxia in vivo, were observed in excised slices, and in isolated mitochondria.
Fiber Optic based Fluorometer/Reflectometer
A flexible means was needed to connect the tested brain to the fluorometer in order to monitor of Brain NADH fluorescence in intact anesthetized or unanesthetized animals. This happened in 1972, when UV transmitting quartz fibers became available (Schott Jena Glass, Germany). The light-guide-based fluorometer for in vivo monitoring of the brain [35, 36] subjected to anoxia or cortical spreading depression was developed and used.
Laboratory device
The development of light-guide-based fluorometry-reflectometry is shown in (Figure 9). The original fluorometer reflectometer was based on the time-sharing principle (Figure 9). Four filters were placed in front of a 2 arms light guide. Filters 1 and 3 enabled the measurement of NADH fluorescence, while filters 2 and 4 were used to measure tissue 366nmreflectance. The reflectance trace was used to correct the NADH signal for possible hemodynamic artifacts, and to indicate relative changes in the blood volume of the monitored tissue.
In this system, one photomultiplier tube was used to detect the two signals namely fluorescence and reflectance. Figure 9B presents the first in vivo brain monitoring time sharing setups, connected to the brain [36]. In order to simplify the system, the time-sharing approach (AC mode) was replaced by splitting the light emitted from the tissue into two unequal fractions for the measurement of fluorescence and reflectance signals. This was achieved by using 2 photomultipliers. This device, named the DC fluorometer, contained two arms light guide probe. In the two configurations, the reflectance signal was used for the correction of the fluorescence signal (see details [17]). This model was used in studying the brain [46, 47, [48, 49, 50], the kidney [41] and the heart [42].
The responses of the rat brain to anoxia using two size diameters of the fiber optic bundle are shown in (Figure 9) . When the brain was exposed to concentrated KCl solution, repeated cycles of NADH oxidation (decreased signal) as seen in (Figure 9). The MitoViewer Another type of fluorometer/reflectometer device (MitoViewer) was developed in 2007 by - Prizmatix Ltd as seen in (Figure 10) & B.
Another type of fluorometer/reflectometer device (MitoViewer) was developed in 2007 by - Prizmatix Ltd as seen in (Figure 10) & B.
The MitoViewer
Figure 9: The time sharing fluorometer reflectometer and B - The brain of a small animal connected to this time sharing device. Ex - Excitation, Em - Emission optical fibers for the monitoring of NADH redox state. H.V. - high voltage, PM-photomultiplier. C - The effect of the monitored tissue volume (the diameter of the fiber optic probe) was tested under anoxia as shown in C upper 3 traces (2 mm diameter) and C lower 3 traces (1 mm diameter). (A+C - (23), B - (47), D - The responses of NADH fluorescence and reflectance to cortical spreading depression initiated by exposing the cortex to 0.3M KCl (39).
Figure 10: A. The MitoViewer fluorometer uses 365 nm UV LED for excitation of the NADH fluorescence (Fluor signal). This excitation wavelength is also used for the correction of hemodynamic artifacts by measuring the reflection light intensity (the Refl signal). The light is transmitted from and to the device by means of a flexible fiber optic bundle. The software displays the Reflectance, Fluorescence and the NADH corrected fluorescence signal B. The view of the MitoViewer.
System overview
The MitoViewer device comprises the following subunits:
Light Source Unit: Provides UV light at 365nm for NADH excitation and tissue reflectance measurements. Also included is a reference photodiode that enables correction of signals in cases of intensity changes of the LED during measurement.
Fiber Optic bundle: Transmits the UV light from the Light Source to the measured tissue and transmits the collected light (the reflection (Refl) and the fluorescence (Fluor) from the tissue, to the Detection Unit.
Detection Unit: Provides detection to transform the Refl and Fluor light into electrical signals which are transmitted to the electronics circuit for amplification.
Detection Unit: Provides detection to transform the Refl and Fluor light into electrical signals which are transmitted to the electronics circuit for amplification.
USB-6009 module: Provides A/D conversion for the Refl and Fluor signals and D/A conversion for control signals sent from a PC to control the function of the MitoViewer.
PC: Personal Computer controlling of the MitoViewer operation using the MitoViewer software.
Power Adaptor: Provides the DC voltage for operation of the MitoViewer.
The fiber optic probe of the MitoViewer is connected to the surface of the brain via an appropriate holder cemented to the skull with acrylic cement. Rats (200-250 grams) anesthetized and operated as discussed in our various papers cited in the attached list.
Typical responses of the brain to oxygen depletion by exposing the rat to 100% nitrogen are presented in (Figure 11) . The Fluor signal (blue) is elevated due to inhibition of the respiratory chain activity. The Refl signal (green) is decreasing, as expected under this anoxic condition, due to the elevation in blood volume in the monitored tissue. The corrected NADH (black) shows a symmetrical increase and decrease signals during the anoxic cycle. The effects of the increase in energy utilization, were induced by exposure of the brain to Cortical Spreading Depression (by high level of potassium.) as seen in (Figure 11) . Since the oxygen supply is not limited, the Fluor (blue) and the NADH (red) decreased due to the oxidation of NADH. Under this stimulation the ATP turnover was dramatically increased, and the extra oxygen supply was provided by an increase in microcirculatory blood flow (not measured in this animal). The effects of hypoxia (6% oxygen) and hyperoxia (100% oxygen) are presented in(Figure 11).
Figure 11: Typical responses to metabolic perturbations measured in the rat brain using the MitoViewer. A - Responses to Anoxia; B - Effects of cortical spreading depression; C –The effect of hypoxia (6% oxygen) and hyperoxia (100% oxygen) on the measured signals.
Animal Preparation for NADH monitoring
An operation table and probe holding device was used to perform brain as well as other body organs preparation for the measurement period. The table for the operation procedure is shown in (Figure 12). The head is connected to a special head holder for the period of the brain operation (20-30 minutes) and then could be released, for the monitoring period, as shown in (Figure 12) . If needed, the other monitored organs i.e. muscle, kidney or liver, must be held by a micromanipulator during the measurement period. The cerebral cortex was the main organ monitored by other investigators as well as in our group.
Figure 12: A –The surgical system used to prepare and measured up to 4 organs simultaneously including the brain. The same system enables to perform a craniotomy while the animal is connected to a special head holder. B+C Stages in the preparation of the rat brain for NADH monitoring. B – The location of screws needed for the fixation of the light guide holder to the skull by dental cement. C - The view of the head at the end of the operation. D - The fiber optic probe is inserted to its holder and the animal is ready for monitoring (16).
The entire protocol of the preparation of the rat is given here. Adult male Wistar rats (180–250g) were anesthetized by intraperitoneal (IP) injection of Equithesin solution (each ml contains: Pentobarbital 9.72 mg, Chloral Hydrate 42.51 mg, Propylene Glycol 44.34%, Magnesium Sulfate 21.25 mg, Alcohol 11.5% water) 0.3 ml/100g body weight. A midline incision is made in the skin in order to expose the skull. Three holes were drilled in the skull and appropriate screws were inserted to the skull as shown in (Figure 12) . The craniotomy (3-5 mm in diameter) was drilled in the right or left parietal bone for the fixation of a light guide holder. The light guide holder and the 3 screws were then fixated to the skull using acrylic cement (Figure 12) . Ten minutes later the head was released from the holder and the fiber optic probe was inserted to the appropriate depth and fixed by a set screw (Figure 12) .
From Single Parameter to Multiparameter Monitoring Approach
(Figure 13) illustrates the various experimental and clinical perturbations used in our laboratory during the last 45 years. (Table 2) lists the studies published by Mayevsky and his collaborators on brain NADH fluorescence. The papers are classified according to the type of perturbation used. In all those papers published in the period of 1973 and 1978 we monitored the mitochondrial NADH as a single parameter monitoring system. For the physiological interpretation of the changes in NADH measured in vivo, it was necessary to move from a single parameter monitoring system to the multiparametric monitoring approach) MPA).
Table 2: Classification of the papers on brain NADH monitoring published by Mayevsky et al (1973-2017).
As described in (Figure 25) the redox state of NADH represent also the balance between oxygen demand and supply. Therefore, the multiparametric monitoring system results could provide better understanding of the pathophysiological processes developed. The term “Brain physiological mapping” based on the various parameters that could be monitored in vivo using a minimally invasive techniques presented in (Figure 14)(Figure 15) [5].
Figure 13: Presentation of the various perturbations used in monitoring brain NADH fluorescence in experimental animals and patients.
Figure 14: The technology developed for real time evaluation of energy metabolism at the tissue level. Part A shows the coupling between the macro-circulation measured by Pulse oximetry and the microcirculation. B-D The main monitoring technique of cellular and intracellular compartments (16).
Figure 15: Schematic presentation of the “basic building stones” of a typical cerebral cortex tissue. During an ischemic or other 3 events shown, the sequence of the 8 early responses developed are presented in numbered circles.
The aims are to monitor, a small volume of the cerebral cortex containing all the tissue elements that are part of a typical functioning brain tissue. The interest is in the microenvironment of the brain tissue containing neurons, glial cells, synapses and the microcirculatory elements (small arterioles and capillaries). During the process we pursued the goal of being minimally invasive to the cortical tissue itself. It was obvious that the various probes could not monitor the same volume of tissue due to the size of each probe used. Therefore, we minimized the diameter of the various probes placed in the multiparametric assembly (MPA) that had a 5-6 mm contact area with the cerebral cortex. In many perturbations used, (global ischemia, anoxia, hypoxia or hemorrhage), most of the areas in the cortex will respond in the same way. The development of the MPA after the establishment of the fiber-optic-based NADH monitoring system in 1972 when the first UV transmitting fibers appeared [36]. The connection of the brain to the fluorometer via optical fibers enabled us to monitor, for the first time, the monitoring unanesthetized brain. The initial data using this technology appeared in 2 papers [35,36]. All details of the technological aspects and animal preparation appear in the original relevant publications; our approach was to develop a new upgraded version of the monitoring system and present initial preliminary results. In the next step a large well-designed study on few groups of animals were done and the data was analyzed for its statistical significance.
Responses of Brain NADH Fluorescence to various Experimental Conditions
In the current section, the effects of various experimental treatments in animal models on brain NADH will be describe in detail. It is important to note that most of the published data on NADH monitoring, have been accumulated from the cerebral cortex.
Changes of Oxygen supply in vivo
Introduction: Chance and Williams [9, 43], found that the complete depletion of O2 from the mitochondria inhibits oxidative phosphorylation terminates ATP production. This situation affects the normal function of the tissue, and cell death can ensue. We defined anoxia as a complete deprivation of O2 caused by breathing 100% N2. Under Hypoxia the deprivation of O2 from the breathing mixture is partial and ranges between 21% (normal air) and 0% (anoxia). Under Ischemia the decrease in O2 supply is due to a decrease in blood flow to the monitored organ. The level of ischemia can vary from a full absence of flow (complete ischemia) to different levels of blood flow (partial ischemia). Although oxygen deficiency is the main event in each of the three experimental conditions (anoxia, hypoxia and ischemia), other physiological factors may differ as well. For example, brain microcirculatory blood flow is decreased under ischemia, but increases under brain hypoxia. Thus, changes in the tissue due to other blood flow related factors are not identical.
Hypoxia and Anoxia: The responses to hypoxia and anoxia are very similar; therefore, they will be discussed together. According to Chance & Williams [8, 9], a shift toward State 5 in the metabolic state of the mitochondria, involves an increase in NADH proportional to a decrease in O2 supply. Figure 16A shows the response of the brain NADH to anoxia in vivo [44, 45]. At those days the fluorescence was measured and displayed without correction for hemodynamic artifacts which was developed later in time. A clear increase in NADH fluorescence was recorded under the deprivation of oxygen. Similar responses of the kidney to anoxia are presented in the lower part of A.
In 1972, we used UV transmitting optical fibers and applied the quartz fibers to the in vivo monitoring of NADH fluorescence in the brain. It was assumed that the response of NADH fluorescence to hypoxia or anoxia, induced in vivo, should be very similar to the response of isolated mitochondria that were investigated until those days.
(Figure 16) presents an interesting two responses of the brain to anoxia. NADH and the electrical activity of the brain were measured [37]. In these experiments the rats were slightly anesthetized by Equithesin. The nitrogen was applied via a nasal mask. In part B1, the duration of the anoxia was 70 sec and in part B2, 100 sec. (Figure 16)1 shows the effect of N2 on the NADH fluorescence, reflectance, EEG and blood pressure. The top trace shows the reflectance which in all animals decreases during the N2 cycle. This decrease of reflectance was in two phases. The first decrease was small (in comparison to the second one) and occurred while the animal was breathing spontaneously. A second decrease occurred after the animal stopped breathing (SB). The recovery of the reflectance to the baseline occurred about 10 min after the rat started breathing again. The second trace from the top, the fluorescence, shows a large increase in NADH fluorescence during the first phase of the N2 cycle. In order to correct for hemodynamic artifacts induced by various treatments, we used the correction technique suggested by Jobsis et al. [32, 33] and Harbig et al. [46]. The reflectance signal at 366 nm was subtracted from the fluorescence signal at 450 nm at a 1:1 ratio. The difference between the fluorescence and reflectance signals is shown in the third trace, the ‘corrected’ fluorescence. After the cessation of respiration, a large decrease in reflectance occurs; an apparent decrease in fluorescence (oxidation) is observed which is almost undetectable in the corrected trace. The small decrease shown in the corrected trace is due to imperfection of the correction factor in this special animal. After the N2 administration had been discontinued (SN), artificial respiration (AR) was applied to induce spontaneous breathing. After the animal started breathing, a fast decrease of NADH is observed in the uncorrected fluorescence as well as in the corrected fluorescence signal. The recovery of the NADH level to the baseline was very fast in comparison to the recovery of the reflectance. The EEG of both hemispheres reaches low amplitude when the NADH level reaches 80-90% of the maximum increase during the N2 cycle. The response of the two hemispheres was identical. The recovery of the EEG follows the NADH recovery to the normoxic level. (Figure 16) shows the response of the same animal to a longer N2 cycle. The animal was exposed to N2 for 100 sec. The main differences between the two cycles are that after the recovery of the NADH to the normoxic level a further decrease in NADH occurred (third trace) and at this time the EEG was depressed and recovered to normal only later. This oxidation of NADH following the N2 cycle was observed in many animals after exposure to a long N2 cycle. The pattern of changes in reflectance, fluorescence and the corrected traces were similar to those observed in the cortical spreading depression (SD) elicited by KCl as shown also in (Figure 9)(Figure 11).
Figure 16: A - Simultaneous recordings of fluorescence changes in rat brain and kidney in the same cycle of anoxia. Fluorescence increases are indicated in a downward direction (45). B1+B2 - The effects of anoxia on brain NADH fluorescence, 366 nm reflectance, EEG, and blood pressure. SB, the animal stopped breathing; SN, stop nitrogen; AR, short artificial respiration (37).
(Figure 17) shows the responses of a dog/puppy brain to graded hypoxia (A-C) and to brain anoxia (D) [17]. As seen, the changes in the corrected fluorescence signals (CF), which represent the NADH redox state, were inversely correlated to the decrease in FiO2 levels (from 6% to 0% O2). In four records, the intensity of the decrease in the reflectance trace was also correlated with the level of hypoxia.
Figure 17: A - Simultaneous recordings of fluorescence changes in rat brain and kidney in the same cycle of anoxia. Fluorescence increases are indicated in a downward direction (45). B1+B2 - The effects of anoxia on brain NADH fluorescence, 366 nm reflectance, EEG, and blood pressure. SB, the animal stopped breathing; SN, stop nitrogen; AR, short artificial respiration (37).
The correlation between the NADH fluorescence and the FiO2 levels are presented in (Figure 17) . It was found that the FiO2 had a significant effect on the NADH redox state (F = 113.6, df = 6, p < 0.0001). However, age did not significantly affect the NADH response (F = 0.25, df = 4, p < 0.91). The NADH response of the puppies to various oxygen concentrations could be divided into four levels of hypoxia which are statistically different from each other:
In order to understand better the response of the mitochondrial NADH to anoxia/hypoxia it was necessary to monitor more physiological parameters from the same brain simultaneously. The results obtained when the multiparametric monitoring system was used are presented in (Figure 18) . The effects of complete deprivation of oxygen (anoxia) on the brain were detected when the animal was exposed to hypoxia as shown in Figure 18B [23]. The rat was exposed to 10%, 5% or to 100% N2. The decrease in oxygen supply resulted in a gradual decrease in brain pO2, as well as in an increase in NADH. The ECoG showed a clear response only to 100% N2. This response corresponded to a slight increase in extracellular K+.
Figure 18: A – The Multiprobe Assembly (MPA) used in hyperbaric chamber. The relative location of various probes above brain are in the lower part of A. Ref, reference; Ag-AgCl electrode; f, refill tube for Ref or DC electrode; c, Lucite cannula; s, Plexiglas sleeve; L, light guide; h, cable holder; Ek, K+ electrode; DCk, DCL, Ag-AgCl electrode; PO2, O2 electrode; ECoG, electrocorticography electrode (91). B - The effects of hypoxia (10% O2, 5% O2) and anoxia (100% N2) on the metabolic, ionic and electrical activities of the rat brain. In this animal the oxygenation of the brain responses to breathing air as compared to 95% O2 are shown (23).
Partial and complete Ischemia: Under partial or complete ischemia, blood flow to the monitored organ is decreased and, as a result, O2 delivery is limited or even abolished. The use of ischemia in animal models provides information relevant to clinical situations such as brain stroke. The primary factor starting the pathological state is the decrease in O2 supply, making the tissue energy balance negative.,
In the early 1960’s, Chance [45] tested the effect of irreversible ischemia on brain NADH using the decapitation model. As shown in (Figure 19), about 8 secs were required between the start of NAD reduction and the attainment of half-maximal reduction. In an attempt to observe the time for NAD reduction in ischemia, we have employed a decapitation technique with the mouse; the optical system is arranged so that the slight mechanical artifact occurring on decapitation would not disturb the fluorescence excitation.
Using the fiber optic based fluorometer, we measured, in 1976, the effect of decapitation on NADH and ECoG in the awake rat and typical response is shown in (Figure 19) [47].
Figure 19: A. Fluorescence changes of the mouse brain cortex in ischemia caused by decapitation. (45). B – Metabolic, reflected-light and electrical responses to decapitation (measured bilaterally). The upper four traces were measured from the right hemisphere and the lower four from the left hemisphere. R = reflectance; F = fluorescence; CF = corrected fluorescence (F –R); ECoG = electrocorticogram.
Later, we tested the effects of age on NADH redox state in the awake and anesthetized rat exposed to decapitation [48]. The NADH was monitored from the two hemispheres of the rat brain. Here we are presenting the four upper traces that were measured from the right hemisphere. The differences in the responses between the two hemispheres were insignificant in most cases. The 366 nm reflected light (R) shows a very small initial response to the decapitation (Figure 19) . However, a very large secondary reflectance increase was recorded
1-2 min after ECoG = 0 when NADH reached its maximal level. The uncorrected (F) and corrected (CF) 450 nm fluorescence signals were like those described previously. In order to analyze the effects of age on the responses to decapitation, various parameters were measured and calculated from the analog signals, as shown in Figure 19B.
The definitions for the various parameters are as follows:
T0 = Time (sec) when electrical activity was very low and close to 0.
T1 = Interval between decapitation and the point when corrected fluorescence started increasing.
T2 = Time when the maximum level of NADH was reached after decapitation.
T3 = Time when NADH reached a level which is half of its maximum increase (CFmax/2).
T4 = Time when a large increase of the reflectance was measured (SRI = secondary reflectance increase).
CFmax = Maximum percentage increase of NADH above baseline after decapitation.
CFo = Percentage increase of NADH above baseline when ECoG = 0.
= Percentage of NADH increase when ECoG = 0 in proportion to the maximum increase measured in the same rat.
CFN2 = Percentage increase of NADH above baseline in nitrogen environment (anoxia).
The same type of data analysis could be used in other models of ischemia such as blood vessel reversible occlusion. It was clear that under severe ischemia a hemodynamic response was measured after reaching the maximal level of NADH. We named it “Secondary Reflectance Increase” (SRI), which appears at time T4 shown in Figure 19B.
In the late 1980’s a new technique enables the monitoring of microcirculatory blood flow (laser Doppler flowmetry) was incorporated into our MPA monitoring system as seen in Figure 20A [49].
Figure 20: A - The experimental setup used. LG - Light guide for the monitoring the NADH redox state and CBF (LDF), ECoG - Electro Corticography electrodes, K, Ca, - Minielectrodes for Potassium and Calcium monitoring, To - Thermistor probe, DC - DC steady potential electrodes, DA - Dental acrylic cement, Ref - Reference electrode, KCI - cannula for KCI application. ), Ex, Em - Excitation and Emission fibers of the fluorometer, LDin, LDout - Fibers connected to the Laser Doppler flowmeter, C - Plexiglass probe holder, h - connectors holder, s - Plexiglass sleeve, f - feeling tube of reference electrode. B - The effects of unilateral (Roccl) and bilateral (Loccl) carotid occlusion on the metabolic, hemodynamic, ionic and electrical activities in the gerbil brain. R 366 nm reflectance F -450 nm fluorescence CF -NADH corrected fluorescence, LDF LDvol LDvel - Laser Doppler flow, volume and velocity. K+e(1), K+e(2), Ca2+e - Extracellular potassium and calcium electrodes. DCK+l DCK+2, DCCa2+ - DC steady potential measured concentric to the three electrodes, ECoG – Electrocorticogram (49).
(Figure 20) shows typical responses to unilateral (ROCCl) and bilateral (LOCCl) in CBF (LDF) and an increase in NADH levels (CF). During the period of ischemia, accumulation of K+ in the extracellular space was recorded (K1+, K2+) but the DC steady potential and the Ca2+ levels remained unchanged during the occlusion period. The ECoG reached the isoelectric level very soon after the second occlusion. During the reopening of the carotid arteries a fast reperfusion was recorded together with the oxidation of NADH. A spontaneous wave of SD was developed during the recovery phase, characterized by a large increase in K+e and a decrease in Ca2+e together with a negative shift in the DC steady potential. During the recovery from the SD wave a large increase in CBF (300%) was recorded accompanied by an oxidation wave of the NADH.
Hyperoxia (increase in FiO2): In order to expose an organ in vivo to elevated oxygenation
hyperoxia, it is possible to use one of the two options:
a. Normobaric hyperoxia the animal breathes elevated FiO2, between 21% O2 to 100% O2 at atmospheric pressure.
b. Hyperbaric hyperoxia (HBO) A hyperbaric chamber, in which oxygen pressure is elevated while the animal is located in the chamber, was used.
Providing animals or man with elevated oxygen will lead to the development of “oxygen toxicity.” The development of this toxic event is inversely proportional to the level of oxygenation, namely the higher the pO2, the shorter the time. Providing more O2 may be beneficial in conditions such as carbon monoxide toxicity, body oxygenation pathology. Therefore, it became necessary to understand the relationship between the level of oxygenation and the function of the mitochondria in vivo. Chance and collaborators [50, 51, [52, 53, 54] developed the setup that enabled the exposure of various types of mitochondria or the entire small animal to the hyperbaric chamber. They found that the NADH of the brain, liver and kidney became more oxidized under hyperbaric oxygenation, and this effect was correlated with a decrease in NADH measured by biochemical analysis of fixed tissue. Figure 21A shows the system that enabled the monitoring of NADH under in vitro or in vivo exposure to hyperbaric oxygenation. This technique was used also when various organs of the rat were exposed in vivo to HBO as seen in (Figure 21) [52].
Figure 21: A - Apparatus for the fluorometric measurement of changes in NADH redox state in the organs of an anesthetized rat. The fluorometer components are mounted on the top of the window of the hyperbaric chamber. The compensating photomultiplier is show in the left, center is the excitation lamp, and right is the measuring photomultiplier (52). B - Responses of rat liver to repetitive compression and decompression with oxygen. The sensitivity for measuring fluorescence changes is also indicated (52).
Rat Liver in vivo. As found previously, the fluorescence of reduced NADH observed at the surface of the rat liver decreased at high oxygen pressures. In further support of No distinctive plateau in the relationship between fluorescence decrease and oxygen pressure was observed up to oxygen pressures of 10 atm. When pressurization was rapid, the tank artifact mentioned earlier became apparent and was followed by the slower biochemical response of the liver. More satisfactory results were obtained with pressurization through a needle valve. With this method of pressurization, the oxidation of the nucleotides appeared to “keep pace” with the increase in pressure, so that little further effect was observed after final pressure was reached.
(Figure 21) demonstrates that the cycle of oxidation of the pyridine nucleotides is reversible and can be repeated. This pressurization technique was used in later experiments where the effects of uncoupling agents or amobarbital on the hyperbaric response were studied. Depending upon the susceptibility of the animal to irreversible oxygen poisoning and the final pressure of the exposure, two to four such reversible cycles can be obtained. Analyses of results for 14 animals in which such compression cycles were obtained gave decreases of 13±1% of the initial fluorescence level at pressures of 120-150 psi. The response to anoxia in rat livers, also shown in (Figure 21) , was transient, and the maximum fluorescence increase was approximately 60% of the anoxic response in this preparation.
Using the light-guide-based fluorometry, we exposed an awake brain to hyperbaric oxygenation as seen in (Figure 22) .
Results recorded from the unanesthetized rat brain exposed to 75 psi (6ATA) of oxygen are shown in (Figure 22) [55]. The reflectance at 366 nm increases during the pressure elevation period, and a few minutes later a large decrease of reflectance was recorded. The third trace from the top-the corrected fluorescence – represents the difference between the fluorescence emission at 450 nm and the reflectance at 366 nm. This correction technique is now used by several investigators [36, 39, [46, 56, 57]. During the compression, an oxidation of NADH of 10% of the normoxic fluorescence level is observed, which is maintained for 15 min. A series of oxidation-reduction cycles of NADH were recorded. About ten minutes before the animal stops breathing, the NADH increased by 50-60% at the end.
The fourth and fifth traces of Figure 22B present the EEG measured from the two hemispheres. The two hemispheres of the brain respond to HBO in the same way. A few minutes after compression, the EEG changes from the typical ‘awake’ pattern to the activated pattern, and then the seizures activity appear. The number of bursts of convulsive activity differs between animals. The EEG becomes flat just before the animal stops breathing, and the increase of NADH was recorded.
A quantitative analysis of the signals was made as seen in Figure 22C including the number of convulsions and oxidation cycles. The three parameters shown in Part B left side [55] are probably connected to each other and in most conditions occurred in the same order Between 30 and 60 psi the slopes of the changes of all three parameters were very sharp, whereas between 60 and 150 they were moderate. Thus, the 60-psi pressure is a breaking point of the line. The other three parameters shown in part C right side, are affected differently by the pressure. The maximum effect was observed at 60 psi, and the curves had a bell shape. The differences between the 60-psi point and the 30- or 150-psi level are statistically significant (P<0.005), as calculated by the Student’s t test.
Figure 22: A – The Time-sharing fluorometer/reflectometer is attached to the hyperbaric chamber for the measurement of NADH from the cortex of the awake rat exposed to HBO (40). B - Effects of pressure level during hyperbaric oxygenation on hemodynamic, metabolic, and electrical activity of the brain. C - Effects of pressure level on electrical activity and its concomitant phenomena of convulsions and spreading depression (55).
Responses of NADH Fluorescence to Increase in Energy Consumption
Chance and Williams showed that the activation of the mitochondria by increased ADP is coupled with oxidation of NADH (decreased NADH levels) and is known as the State 4 – State 3 transition in isolated mitochondria [15, 31]. As shown in (Figure 2) , the demand for energy (ATP) by various tissues is dependent on the specific tasks of each organ or tissue. Nevertheless, the stimulation of mitochondrial function is common in all tissues in the body. The effects of tissue activation on NADH fluorescence under normoxic conditions as well as during limitation of O2 supply (hypoxia, ischemia) is presented.
Effects of Cortical Spreading Depression (CSD): Brain Cortical Spreading Depression [58] was
Figure 23: A - The first publication described the development of cortical spreading depression. Gradual spread of depression from A to F. Electrodes arranged as shown in the inset. A. Before stimulation L. 7 min after K. Unless otherwise mentioned, the stimuli were induction shocks at “tetanizing” frequency, applied for 3 to 5 sec. through electrodes S (58). B - Repetitive responses of the cerebral cortex to spreading depression evoked by application of 0.4 M KCI on the dura. The third trace is on an expanded amplitude scale. The arrow direction shows an increase in the optical signals. (39).
discovered by Aristides Leao in 1944 (Figure 23), After cortical electrical stimulation he recorded a wave of EEG depression propagated from the stimulation point to the rest of the hemisphere. In parallel to the EEG depression, the steady electrical potential shows a negative shift [59]. I The wave of depolarization passing through the tissue increases energy consumption [58]. In another study Leao described the effects of CSD wave on the diameter of the blood vessels on the brain surface [59]. The involvement of the microcirculation in the response to CSD was described by Van Harreveld et al few years later [60, 61]. In other studies, the The PO2 in the brain had indicated that demand for energy and oxygen consumption increased during the CSD wave [62, 63, 64]. Bures and Krivanek [65] used a special approach to check ion shift in the rat cortex and showed that extracellular potassium is elevated [66]. In the early 1970s, Vyskocil et al. used a potassium-selective microelectrode and showed clearly that extracellular K+ was elevated significantly and lead to increase in oxygen consumption [67].
When dealing with CSD and hypoxia or ischemia it is important to define the situations clearly. The discovery of the CSD event happened when rabbits’ brain was exposed to electrical stimulation in order to elicit “experimental epilepsy”. Oxygen supply to the brain was normal. Under ischemia, the EEG is also depressed as soon as the oxygen supply to the brain is limited but the mechanism behind the EEG depression is different from that operated in CSD (Figure 31).
The effects of CSD on brain mitochondrial NADH was first described in 1973, by Rosenthal and Somjen, where a clear oxidation wave was recorded [68]. In the same year [36] our team showed the same NADH oxidation response to CSD in the rat brain (Figure 09). A year later, we demonstrated the coupling between the elevated extracellular K+ induced by CSD and the NADH oxidation needed to recover the normal ionic homeostasis by the mitochondria [38]. Also, we studied in detail the nature of the NADH response to CSD as seen in (Figure 23) [39]. In order to initiate a wave of spreading depression (SD), KCl solution was passed through one of the pairs of tubes located above to the Dura mater. At threshold (~0.1 M KCl) only one wave of SD developed, while at maximum (~0.4 M KCl) many cycles were recorded. (Figure 23) represents the latter condition, in which six SD waves developed by 0.4 M KCl were terminated by washing the brain surface with 0.9% NaCl. The oxidation of NADH is corresponding to states 4-3-4 transitions and are related to the changes in energy demand during the SD cycle.
In order to show that the CSD wave recovery is dependent on the availability of oxygen another experiment performed and the results appear in (Figure 24). If energy production is in the normal range, the pumping of potassium into the cells starts immediately and its level recover back to the normal value- 3 mM. When energy production is inhibited, the pumping of K+ is inhibited, as shown in (Figure 24) [69]. In this animal, waves of CSD developed spontaneously after a few hypoxic episodes and the left side of the figure shows the regular response to CSD. The efflux of K+ reached a level of 14-15 mM and was then pumped back into the cells and the NADH (CF) shows a clear oxidation cycle as shown also in Figure 23B. In the second CSD cycle shown in (Figure 24) , hypoxia of 4% oxygen was induced at the beginning of the recovery period and the recovery process was inhibited until 100% O2 was provided again to the rat.
Figure 24: A - The experimental setup used (see details in (Figure 18)). B - The effects of hypoxia (4% O2) on the metabolic, ionic and electrical responses to cortical spreading depression developed spontaneously in the rat brain. Abbreviations are as in Figure 18 (69).
Figure 25: A - Presentation of the experimental setup used to study brain cortical spreading depression (see details in (Figure 20)). B - Metabolic, hemodynamic, ionic and electrical responses to CSD. The MPA used in this study contained two different bundles of fibers for monitoring the relative CBF and NADH redox state. R, CF - reflectance and corrected fluorescence; LDF LDv - Laser Doppler flow and volume monitored from another optic fiber located in the MPA; ECoG - electrocorticogram; K+e , ECa2+, Cae2+ - corrected potassium, uncorrected and corrected calcium ion concentrations, respectively; DCK+, DCca2+ - DC steady potential around the K+ and Ca2+ electrodes (49).
(Figure 25) shows a laser Doppler flowmeter added in order to record microcirculatory blood flow [70]. Part B shows two sets of responses to CSD recorded after 0.1M KCl application 2 mm anterior to the MPA. A clear correlation between the oxidation of NADH (CF) and large increase in relative CBF can be seen during the six cycles recorded. The changes in relative CBF are calculated in relation to the control values before the waves were initiated, The cycles of CSD were not so clear on the volume trace and we suspect it was a reading problem, since in other studies a clear connection between flow and volume was found using the same laser Doppler flowmeter.
Effects of Epileptic activity: Jobsis et al. in 1971 [32] described the influence of epileptiform activity on brain mitochondrial NADH redox state. Using anesthetized cats exposed to an epileptogenic drug (Metrazol or Strychnine), a marked expected oxidation of NADH was recorded. The changes in the electrical activity, increased the demand for energy in order to restore ionic homeostasis during the epileptic activity. The connection between the concentration of K+ and the oxidation of mitochondrial NADH was shown in cat hippocampus [71]. The effect of seizures on mitochondrial NADH was investigated later by other groups, [72, 73, [74, 75, 76, 77, 78, [79, 80, 81]. Vern et al. showed that epileptic activity, induced in hypotensive cats, caused an increase in NADH instead of a decrease [80].
In 1975, we described the mitochondria NADH responses to epileptic activity measured in non-anesthetized rats [37]. The Metrazol (100 mg/ml) was applied epidurally, to the surface of the rat brain, using a special cannula [37]. The typical response to Metrazol occurs 3-5 min after the administration of the drug and the results are shown in Figure 26-part A. The Metrazol was applied to the right hemisphere and the left one served as a control. An increase in electrical activity was recorded together with an oxidation of NADH which was in the range of 5-10% of the normoxic level. After a period of a very intense activity, the EEG became isoelectric while the NADH showed a very large oxidation cycle A different response was recorded in other animals as. In these cases, the small oxidation under Metrazol effect was recorded but the recovery to the normoxic level showed no large oxidation cycle (data not shown).(Figure 26) presents the progression of seizure activity in a special strain of gerbils [[84], developing epileptic effects as a result of monotonic noise. As seen in the (Figure 26) the exposure of the awake gerbil to noise resulted in the development of epileptic activity followed by a CSD wave [17]. The coupling between the two pathological events is clear and manifested by the electrical activity (ECoG) as well as extracellular K+ levels and NADH redox state. During the epileptic stage, extra cellular potassium increased and started to recover to the baseline, but then a larger elevation was recorded. There is a clear correlation between the various parameters during epilepsy and CSD. The decrease in NADH was smaller during the first stage, followed by an oxidation cycle typical for CSD.
Figure 26: A - The effects of topical application of Metrazol on brain NADH fluorescence, reflectance and the EEG of the two hemispheres. Note that the amplitude of the EEG calibration in the right hemisphere is 1 mV (37). B - The development of epileptic activity followed by cortical spreading depression (CSD) wave in the brain of a seizure-prone gerbil. ECoG, electrocorticogram; PO2, partial pressure of O2; EK+, DCK+, extracellular potassium and DC steady potential measured around the K+ electrode; R, F, and CF, reflectance, NADH fluorescence, and corrected NADH fluorescence (17).
NADH responses to activation of the brain under restriction of oxygen supply: We used 2 ways to decrease oxygen supply to the brain while exposing it to an increase in oxygen demand by local brain ischemia or systemic hypoxia.
In order to study the effects on the metabolic response of the brain resulting from the limitation of its blood supply, the carotid artery ligation technique was used. The common carotid arteries were dissected in the neck, and loops of ligature were placed around the vessels. Measurements of NADH redox state were taken by a light guide holder located above the right hemisphere. Initiation of cortical spreading depression (CSD) cycles was initiated by flushing KCl solution through a small push-pull cannula implanted above the right hemisphere so that NADH fluorescence measurements were taken at the same time. KCl was applied topically to the cortex and NADH fluorescence and ECoG measurements were taken.
This, response represents the normal brain and served as a control. Three hours after the operation when the animals were fully awake, the carotid arteries were ligated.
Figure 27: A - The effect of acute bilateral carotids occlusion on the metabolic response to cortical spreading depression (SD). Parts B, C and D were measured 15 min, 90 min and 18h after occlusion, respectively (85). E -The metabolic responses of the normoxic and ischemic (F) brain to a wave of CSD R-366 nm reflectance, CF-NADH change(101).
(Figure 27) shows the response of an awake brain to a bilateral carotid artery ligation and CSD [85]. Before ligation, 0.5 M KCl was applied to the cortex and the normal metabolic response to CSD was recorded (Figure 27) . The bilateral ligation of the carotid arteries caused an increase in NADH, and since the KCl remained on the cortex during this period (about 3 min), the response to SD was immediately apparent. In this animal, the duration of the oxidation cycle became very long after the ligation, as a response to spreading depression. A similar long oxidation cycle was recorded again later (lower Part-B), but within 2h the metabolic response to SCD disappeared (not shown).
In this set of experiments 10 rats were involved. Figure 27 shows a typical response of the brain to acute bilateral carotid arteries ligation. Parts B, C and D show the response of NADH to spreading depression elicited 15 min, 90 min and 18h, respectively, after the ligation. In this animal the changes of the reflectance (upper trace in each part) was minimal during the CSD cycles. In part D, a very long biphasic cycle of the reflectance was recorded. The effect of the ligation on the NADH was transient, namely, that a small increase was recorded during the ligation, but within two minutes it recovered to the base line. The main effect of the ligation is on the response of the brain to CSD which increases oxygen utilization. During the first 60 min after ligation the oxidation cycle can be detected as a response to CSD, but the amplitude of the oxidation (decrease) was diminished and the duration of the cycle became much longer. The qualitative change in the response of the brain to CSD started several hours after the ligation. In part C, the response to CSD was recorded 90 min after the ligation. A biphasic response of NADH to CSD was recorded. At the beginning of the cycle an increase in NADH was recorded, while afterwards the NADH decreased below the base line level. In part D (18h after ligation) the response of the brain was completely opposite to what was measured before the ligation (part A). Instead of an ‘oxidation cycle’ as a response to CSD, we found a ‘reduction cycle’ namely an increase in NADH redox state. (Figure 27) -right side illustrate schematically the difference in response to CSD recorded in normoxic (E) and ischemic brain (F).
The effects of hypoxia on the response to epileptic activity induced by Metrazol is shown in (Figure 28) [37]. Matrazol was applied under hypoxic condition. The animal was exposed to air (A), 10% O2 (B), 7.5% O2 (C), and 5% O2 (D). The line N.L. represents the normoxic level of NADH. In all oxygen concentrations, an increase in activity followed by a flat EEG was recorded. The changes in NADH are the same as in Figure 26A, namely, that two phases in the oxidation were found. By giving the animal lower concentrations of O2 the baseline shifted to a more reduced level depending on the O2 level.
Figure 28: The effects of graded hypoxia on the response of the awake brain to local application to Metrazol. The fluorescence responses occur 3-5 min after the application of Metrazol. The levels of O2 were in A, air; B, 10%; C, 7.5%; and D, 5% (37).
In order to test the validity of the 2 types of NADH responses to CSD induced in the normoxic and ischemic brain we used a different approach. We constructed and tested a mathematical model [86], capable of simulating changes in brain energy metabolism under various pathophysiological conditions. The model incorporates the following parameters: cerebral blood flow, partial oxygen pressure, mitochondrial NADH redox state, and extracellular potassium. Accordingly, all the model variables are only time dependent (‘point-model’ approach). Numerical runs demonstrate the ability of the model to mimic pathological conditions, such as complete and partial ischemia, cortical spreading depression under normoxic or partial ischemic conditions. The pathological state of cortical spreading depression was induced by “potassium injection”. When CSD was induced, the increase in energy requirement leads to the activation of Na+-K+ ATPase in order to restore the normal potassium distribution and causes an increase in blood flow and a decrease in NADH. When the blood flow decreases mildly (mimicking the ischemic period, an infusion of potassium was modeled. Accordingly, the blood flow slightly decreases as a result of the infusion, and then increases to the new resting level.
Monitoring of the Human Brain in Neurosurgical OR and ICU
After accumulation of preliminary results using the 1st clinical multiparametric monitoring system [4], a new commercial device was developed. In the new device-the TiSpec, we monitored only three out of the 8 parameters monitored by the multiparametric monitoring system [4]. We used only the optical based probes in order to simplify the technology and to shorten the “time to market” of the new device. The TiSpec provided parameters of microcirculation (blood flow and volume) and mitochondrial NADH. The TiSpec and the various probes developed for this device are presented in (Figure 29). This device was tested in animal models as well as in neurosurgical patients. In (Figure 29) , one of the various holders of the fiber optic probe are shown. In. In Figure 29B the probe is located on the surface of the brain during operation. More details regarding the Tispec were published [87, 88].
The first attempt to monitor NADH from the human brain by our group was in 1990. We monitored few neurosurgical patients in the Hospital of the University of Pennsylvania after IRB approval. We monitored in those patients only Mitochondrial NADH and microcirculatory blood flow since extra time availability during the surgical procedure is very limited. The responses of the human brain to ischemia are presented in (Figure 29) In this patient a short brain ischemic episode was induced by the occlusion of the common carotid artery during the preparations for brain aneurysm procedure. As seen in (Figure 29) an immediate decrease of CBF was recoded simultaneously with an increase in NADH redox state with only very small changes in the reflectance recorded (R). The recovery of CBF and NADH oxidation were very fast. The time to reach 50% of the change in NADH during the recovery was less than 3 seconds. In contrary to the NADH recovery to the pre-ischemic level, the CBF signal shows a large hyperemic response (large overshoot in CBF).
Figure 29: A - The Tissue spectroscope (TiSpec) device used in monitoring patients in the neurosurgical operation room. B - The application of the standard optical probe with the floating arm during an operation for the removal of a brain tumor [136,137]. C - Responses of the human cortex to a decrease in blood flow induced by common carotid artery occlusion. R, F, CF – Reflectance, Fluorescence and corrected fluorescence. D - Effects of IV infusion of Mannitol on brain hemodynamic, metabolic and ionic activities in head injured patient (4).
In the 1980’s we developed the first multiparametric monitoring system that was adapted to experimental animals exposed to various pathological conditions [89-92]. Later on, we improved the system in order to introduce it to the neurosurgical operating room or intensive care unit (ICU) for patient monitoring. The concept behind the development was that the more parameters that are monitored, the better the diagnosis of the situation will be. The ideal system was to monitor eight parameters from the brain (right side) in addition to the various systemic parameters monitored in every patient hospitalized. The long-term vision was that all parameters monitored by the 2 monitoring systems will be integrated in the same data bank and an expert system will be developed for better diagnosis of the patients. The translation of this concept into a practical tool and monitoring device is presented in (Figure 30) [93-98].
Figure 30: A - Schematic representation of the multiprobe assembly (MPA) used for monitoring the brain of head-injured patients in the ICU. The MPA is connected to the brain via a special holder screwed into the skull. ICP, intracranial pressure probe; CBF, NADH-fiber optic light guide probe to measure local cerebral blood flow, (CBF) and mitochondrial redox state (NADH); K+, DC, extracellular K+ mini surface-electrode surrounded by a DC steady potential monitoring space; ECoG, bipolar electrocortical electrodes (4). B - The first clinical monitoring setup (“Brain Monitor”) used in the neurosurgical intensive care unit. The Multiparametric monitoring system consists of various devices installed in the same cart. The MPA shown in (Figure 30) is connecting the brain of the patient to the Brain monitor (96). C+D - Two responses of the brain to a wave of cortical spreading depression developed spontaneously in a severely head-injured patient. ICP intracranial pressure; R - 366 nm reflectance, NADH - 450 nm corrected fluorescence; CBF, CBV cerebral blood flow and volume measured by laser Doppler flowmetry; Ke, DC - extracellular potassium levels and the DC steady potential measured around the K+ electrode; ECoG electrocorticography.
Based on the well-developed MultiProbe Assembly (MPA) used for animal experiments [3] a new MPA was developed and applied to patients monitored in the neurosurgical operation rooms and ICU. This device allowed us to monitor, in real-time, the hemodynamic, metabolic ionic and electrical activities in the brain of comatose patients. All details regarding the technology and the clinical setup appear in our published paper [4]. Figure 30A shows the longitudinal section of the MPA (measuring 8 parameters) used in the neurosurgical operating room and ICU. The MPA was connected to the multiparametric monitoring system shown in Figure 30B.
The next step was to use the multiparametric monitoring system in the neurosurgical ICU. In this group, 14 patients were monitored (Figure 30). Only one of the 14 monitored patients had developed spontaneous changes in all parameters like the typical responses to SD in animals. These changes were recorded 4.5h after the beginning of monitoring, which was 7 hours after admittance to the hospital. During the measuring period, this patient was bilaterally irresponsive to pain, his pupils were dilated and non-reactive to light. He was mechanically ventilated, and his brain CT scan showed evidence of severe brain edema in the left hemisphere and right parietal hemorrhagic contusion. The measurements were taken from the right frontal lobe. As seen in (Figure 30) , the ECoG became depressed for 10-15 minutes and at the same time a cycle of elevated extracellular K+ and a small negative shift in the DC potential were recorded. These changes are typical to transient depolarization, which is a dominant part of cortical spreading depression. NADH was oxidized while blood flow and volume increased. This patient exhibited repetitive SD cycle every 20-30 minutes. However, the following SD like cycles that were recorded from this patient (after the first ones) showed different hemodynamic and metabolic responses (Figure 30) . While extracellular level of K+ and the pattern of the DC potential were very similar, NADH oxidation cycles were replaced by a biphasic cycle comprised mainly of a phase of increased NADH followed by a small oxidation phase. The compensation of blood flow and volume was also reversed at this time. The monophasic increase in CBF and CBV was replaced by an initial decrease followed by a smaller increase. Significant correlations were seen between CBF, CBV and NADH (CF) and extracellular K+ levels [95, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 131, 132, 133], 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198].
The severe head injured patient who showed high levels of ICP was treated with Mannitol infusion as seen in Figure 29D. The infused bolus led to a clear decrease in ICP associated with a large increase of CBF and a beneficial effect on tissue oxygenation state (oxidation of NADH) [99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109].
Discussion and Future perspectives
(Figure 31) [16] presents, in a schematic way, the relationship between various physiological parameters monitored in vivo under different experimental perturbations. The upper two factors (left side), namely, systemic hypoxia and cerebral ischemia affect the availability of oxygen so that energy metabolism is inhibited (blue arrows). The other two perturbations shown are cortical spreading depression and epileptic activity that induced cortical depolarization (purple arrows). All four perturbations are affecting the same type of processes in the brain such as CBF, NADH and extracellular levels but in the opposite direction as seen in the scheme. Under hypoxia and ischemia, the production of ATP by the mitochondria is decreased and becomes a limiting factor in the system. Due to the inhibition of the ionic pumps, the ionic homeostasis is disrupted, and hypoxic or ischemic cortical depolarization is developed[110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130]. The end points of this process are changes in the electrical activities recorded as a depression of the ECoG and a negative shift in the DC steady potential. In addition, due to the depolarization event the extracellular potassium is elevated. If this situation persists for more than a few minutes, irreversible damage to the cortex may develop. When the brain is activated by CSD or epileptic activity (lower left side) the events will propagate toward the activation of the brain and energy consumption is increased (purple arrows) [131-149]. The coupling between the two pathological events is clear and manifested by the electrical activity (ECoG) as well as extracellular K+ levels and NADH redox state. During the epileptic stage, extracellular potassium increased and started to recover to the baseline, but then a larger elevation was recorded. There is a clear correlation between the various parameters during epilepsy and CSD. The decrease in NADH was smaller during the first stage, followed by an oxidation cycle typical for CSD [150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198].
Figure 31: A schematic representation of the effects of various pathological conditions on the energetic-mitochondrial function, ionic and hemodynamic state of the cerebral cortex (details are in the text) (16).
After the accumulation of huge amounts of experimental results, we decided to develop a preliminary tool named Brain Metabolic Score-BMS as seen in (Figure 32) . In the initial developmental stage, the BMS was calculated by using two parameters, namely the NADH and CBF. In the present study we are presenting the preliminary developed Brain Metabolic Score- BMS that could be used in evaluating the metabolic state of the cerebral cortex in real time. We used the brain as a typical organ for the calculation of the Score, but the same approach could be used for other organs or tissues. The goal of our study is to develop the Tissue Metabolic Score that could be monitored in the intensive care units or in the operating rooms. In those medical environments the need for objective approach for decision making processes is very important as discussed previously [183-199]
Figure 32: A-G - Schematic presentation of the elements involved in the concept “Brain Metabolic Score “(BMS). H+I - The calculated BMS of human brain during the development of two cortical spreading depression cycles in a head injured patient.
In order to develop the BMS we used the multiparametric monitoring system described in detail in (Figure 15) and the development of the score appeared in our publication [200, 201, 202, 203, 204, 205, 206, 207, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235].
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Biomed Grid | Relation of a Pulse Transit Time to the Blood Pressure in Bifurcated Cardiovascular Networks
Introduction
Pulse Arrival Time (PAT) is the generally established empirical marker for continuous non-intrusive blood pressure monitoring, which is defined as a time required for a pulse wave to travel from the heart to a peripheral site. A popular estimate of PAT is the timebased delay between R wave peak of Electrocardiogram (ECG) and a characteristic point of a Photoplethysmogram (PPG). PAT consists of two components: the non-constant Pre-Ejection Period (PEP), which is a duration of the ventricle contraction up to aortic valve opening, and the Pulse Transit Time (PTT), which defines the period for the arterial pulse wave to travel from the aortic valve to the peripheral site.
A simple measurement setup consisting of arm Electrocardiogram (ECG) and Peripheral Site Photoplethysmogram (PPG) allows to assess PAT as the time delay between ECG R peak and one of the optional points in the PPG waveform: peak, foot, maximum values of the slope, or the second derivative of PPG waveform. PEP can be derived noninvasively using for instance thoracic Impedance Plethysmogram (IPG) as described in [1, 2, 3]. Estimation of a systolic and diastolic BP is based on equivalence of the measured and model- based prediction of PTT. In general, prediction methods can be categorized into data-driven, physics-based and hybrid approaches. Data-driven approaches investigate relationship between BP and PTT through the linear or nonlinear regression analysis, employing a simple set of basic functions, or using artificial intelligence (neural network). Physics-based approaches assume that a reliable physical model describing connection of a PTT to BP is available. Hybrid approaches combines the methods to calibrate the personalized bio-physical properties, improving prediction.
As follows from the physical modelling, PTT and PWV are mainly affected by four factors: arterial compliance, cardiac output, peripheral resistance, and a blood pressure. Most data driven approaches select the only single parameter as an independent variable, which is a PTT or the averaged PWV, to predict systolic and diastolic blood pressures. The physics-based approach automatically accounts for the full set of factors affected BP according to the physical model, i.e. cardiac output, stroke volume, vascular compliance, peripheral resistance. The following sections describe PTT based blood pressure estimation according to the classification. Since many of the papers using data driven regression analysis are listed in several reviews, we will not cite relating individual papers, focusing mainly on a physical modeling as a foundation for linking PTT to systolic and diastolic BP.
Data-Driven BP Estimation
Multiple linear and nonlinear regressions have been explored by different authors using combinations of exponential, power, logarithm, polynomial and logistics functions to fit the experimental dataset of PTT (or PWV) vs BP [3, 4, 5]. In [6] the heart rate as a second independent variable in addition to PTT is introduced in a linear regression, which according to the authors improves the accuracy of BP prediction. In the monograph [7] the Young’s modulus is presented as an exponential function of pressure, where E0- is the Yong modulus at zero pressure, and α -empirical coefficient. As a result, the formal substitute of the modified elastic modulus E into the Moens-Korteweg expression for the PWV results in a pulse wave velocity dependent on a blood pressure, is the Moens-Korteweg speed of propagation at zero pressure, α-is a calibrating constant.
The mentioned approach is completely empirical, since it does not fit the paradigm of classical mechanics, which specifies physical nonlinearity by appropriate constitutive equations in terms of stress – strain components. The described expression cannot be derived from the fluid-structure interaction model using any constitutive equations. It could be converted to the linear regression (in a log scale) by application of logarithm to the expression for PWV, which results in
where is the distance of a pulse propagation, a, b-are determined from the best fit procedure. Equation (2) remains nowadays a one of the most widely used technique for noninvasive continuous BP monitoring. The modified logarithm-based regression was successfully applied in [8] to monitor BP as a function of PTT under the effect of hydrostatic pressure due to hand elevations. The effect of including PEP in BP estimation is under investigation in different papers based on empirical regression analysis over different cohorts of human subjects [4, 9, 10]. The simplest approach is an attempt to estimate PEP as a percentage of the RR interval, with the following subtraction from PAT to obtain PTT [11]. There is still a controversial evidence from different authors regarding effect of PEP on BP. The impact of PEP on the overall PAT decreases with distance from the heart, so that for the short PATs, like ones extracted from the ear-worn device, correction with PEP is required.
Neural Network (NN) modeling has recently been in place predicting BP as a function of a set of measured parameters. In [12] a total of 17 parameters were selected as the set of independent variables being chosen as characteristic feature points from ESG and PPG signals. Two different neural networks have been used to predict separately brachial systolic and diastolic blood pressures as functions of ECG and PPG measurements. The maximum error range in the brachial BP prediction is reported in terms of a root mean square error RMSE=±5.2mmHg. In [13] the SVR (Support Vector Machine Regression) algorithm is applied to establish relationship between human physiological data and systolic and diastolic BPs. The different number of main physiological indexes, obtained from ECG and PPG, include PTT, HR, SPO2 and others, are explored in application of NN modeling. The maximum error range of a brachial BP prediction is reported as ±10mmHg.
Few studies managed to compare different noninvasive BP estimations in a wide physiological BP range. None of data driven approaches proved to be ubiquitous, being able to monitor with a reasonable accuracy the only single feature of a BP, either systolic, or diastolic or a mean [3, 4, 5].
Physical Modeling BP Estimation
In this section, we assess physics-based models’ capabilities to predict systolic and diastolic BP as a function of model required independent parameters. Considering an arbitrary pressure-area connection, P = P(A) ,we present system of conservation laws in the following non-conservative quasi-linear form
where fluid density. This system could be transformed to the decoupled system of equations for the characteristic variables (Riemann variables), which read
Relating characteristic directions (eigenvalues) read
and forward and backward running characteristics can be found accordingly
Since the slope of a forward running characteristic line is determined by PWV =
Equation (7) serves to calculate the PTT required for the pulse wave to propagate through the Nv vessels, each of the length along the flow pathway from the left ventricle to the peripheral site.
Nonlinear Vs Linear Models
In this section three type of nonlinear models are reviewed following the papers [14, 15]: the infinitesimally Small Deformation Linear Elasticity Model (SDL), Small Deformation Hyperplastic Model (SDH) and Finite Deformation hyper elastic Model (FDH). The Fung’s exponential descriptor for passive behavior of arteries [16] presents strain energy density function for the pseudo elastic wall deformation in a form
Here are material constants, are the circumferential and axial strain components. In a 1D problem strain energy of the wall can be simplified by setting Equilibrium condition results in a generalized tube law for the hyperelastic wall the Moens-Korteweg speed at
Equations (10) and (6) present the instantaneous PWV for the SDH model in a compliant hyperelastic artery as the following
Model SDL is achieved by setting hyperelastic material coefficient to zero so that the expressions for PWV in model 2 follows from (10) at
Model FDH, which considers finite deformation, is derived based on the same expression for strain energy (8), where and is interpreted as the Green-Lagrange strain components in circumferential and axial directions accordingly. Relating Cauchy stress components.
are governed by equilibrium conditions
Here: are the stretch ratios in circumferential, radial and axial directions accordingly; r, h-are the luminal radius and thickness in a deformed state,
All three models have been tested against Histand and Anliker results on a PWV measurements presented in [17, 18] and reproduced in (Figure 1) by square markers. The experimental curve notably exhibits curvature starting from elevated level of pressure exceeding 140mmHg. Material parameters have been identified for each model independently, based on a best fit procedure. The Finite Deformation Hyper Elasticity (FDH) model and Small Deformation Hyper Elasticity (SDH) model have the highest quality of fitting process, creating practically the same regression line in (Figure 1) within the physiological range of BP. The Small Deformation Model with Linear Elasticity (SDL) was not able to fit the experimental curve at the quality of FDH or SDH models.
Figure 1: The nonlinear model FDH produced the best fit of the PWV vs. transmural pressure function.
Figure 2: Simulation results show that within a physiological longitudinal pre-stress load effects PWV by ~ 3%. Tz denotes the axial physiological Lagrangian stress.
Dash lines indicate theoretical prediction. Square markers illustrate the total set of experimental points. Using the properties extracted from the nonlinear model the lower (solid) line shows the effect on PWV using the partially nonlinear model SDH, combining hyper elasticity with small deformation. To illustrate the effect of a longitudinal force on PWV the variation of PWV due to the variability of a longitudinal pre-stress force is presented in (Figure 2). According to simulation within the realistic physiological range of a longitudinal stress, the relative deviation in PWV does not exceed 3%.
Several PWV estimations presented in literature is based on its correlation with the BP and an arterial wall compliance. The study in [19]examined the impact of a systolic flow correction of a measured PWV on blood pressure prediction accuracy using data from two published in vivo studies. Both studies examined the relationship between PWV and blood pressure under pharmacological manipulation, one in mongrel dogs and the other in healthy adult males. Systolic flow correction of the measured PWV improves the R2 correlation to blood pressure from 0.51 to 0.75 for the mongrel dog study, and 0.05 to 0.70 for the human subjects’ study. The results support the hypothesis that systolic flow correction is an essential element of non-invasive, cuff-less blood pressure estimation based on PWV measures.
Thick Wall Vessels
A novel mathematical model predicting PWV propagation with rigorous account of, blood vessel elasticity, and finite deformation of multi-layer thick wall arterial segments was studied in [20]. It was found that the account for the multilayer model affects distribution of local parameters in the proximity of the external layer (adventitia) and does not affect stiffness related to the internal layer. The latter means that the single thick layer model is enough to predict PWV of an arterial segment. (Figure 3) depicts the dependence of PWV on pressure for the Systole Phase (marked as “SBP”) and a Diastole Phase (marked as “DBP”) for three vessels of different thicknesses of a human aorta. All results have been compared with the simplified thin walled model of a membrane shell interacting with an incompressible fluid.
Figure 3: Simulation results show that within a physiological longitudinal pre-stress load effects PWV by ~ 3%. Tz denotes the axial physiological Lagrangian stress.
To explore inaccuracies induced by use of the less complex thin wall model, error in both PWV and blood pressure were calculated for a blood pressure of SBP/DBP = 150/95mmHg representing the median of stage 1 hypertension. The single layer thick wall model improves PWV accuracy by (4.0-8.4%) corresponding to the relative wall thickness (H/R1) range of 0.07-0.38. One of the goals for the model is PWV based blood pressure prediction, where the thick wall model offers an improvement of (3.3-19.4%).
Calibration Free Approaches
Willemet et al. [21, 22] proposed approach to use cardiovascular simulator for generation of a database of “virtual subjects” with sizes limited only by computational resources. In their study, the databases were generated using one-dimensional model of wave propagation in an artery network comprising of 55 largest human arteries. A linear elastic model was employed to describe deformation of arterial walls. The database is created by running the cardiovascular model repeatedly. The seven model parameters were varied: elastic artery PWV, muscular artery PWV, the diameter of elastic arteries, the diameter of muscular arteries, Heart Rate (HR), SV and peripheral vascular resistance. 3325 healthy virtual subjects presented a diversity of hemodynamic, structural and geometric characteristics. For each virtual subject, all characteristics are known at every point of the systemic arterial tree, i.e. anatomical and structural properties, as well as pressure, flow, pulse wave velocity and area waves at the larger arteries, therefore allowing the computation of the exact value of the diagnostic tool.
Huttunen et al. [23] used cardiovascular modelling of the entire adult circulation to create a database of “virtual subjects”, which is applied with machine learning to construct predictors for health indices. They carry out theoretical assessment of estimating aortic pulse wave velocity, diastolic and systolic blood pressure and stroke volume using pulse transit/arrival timings derived from photoplethysmography signals. The generated database was then used as training data for Gaussian process regressors applied finally to simulation. Simulated results provide theoretical assessment of accuracy for predictions of the health indices. For instance, aortic pulse wave velocity was estimated with a high accuracy (r>0.9. Similar accuracy has been reached for diastolic blood pressure, but predictions of systolic blood pressure proved to be less accurate (r > 0.75).
Conclusion
Developed technologies in general allow to implement a PTT/ PAT-based system to predict continuously cardiovascular health markers such as arterial blood pressure, cardiac output, arterial stiffness. However, none of approaches is able so far to monitor accurately all cardiac markers for the wide range of physiological conditions. The limitations to be addressed in future are the following. First, each model must be investigated for its limitations. We believe that a calibration stage is required to build a reliable simulator within the range of investigated conditions. Also, most of the research addresses healthy population, which is characterized by different behavior of a vascular system rather than group with medical conditions. In the current review we only consider pulse transit and arrival type of time information as the input to the predictor. It would be beneficial to develop approaches that do not need reference measurement for the aortic valve opening/R-peak.
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Biomed Grid | Future of Biotech Equipment/Medical Device Research & Development: A Case for Redemption of the Neglect
Introduction
The global healthcare industry is undergoing a progressive transformation at a rapid pace. Ground breaking innovation across multiple fields including biotechnology, pharmaceutical and medical devices is driving this transformative journey, the main outcome of which is improved patient health and decreased cost burden resulting in enhanced quality of life.
Small molecule drug development and advances in bio therapeutic formulations by various pharmaceutical and biotechnological companies respectively, either alone, or in collaboration with dedicated research groups in academia tend to garner the majority of government and non-governmental funding. However, a critical area of biomedical research that lags behind in comparison with its peers in this category are biomedical devices that are also an integral part of patient therapy. While this disparity is most evident in the developing countries beset with social inequalities and a lack of medical infrastructure [1] , even in developed nations leading in biotech innovation such as the US there has been a steady decline in the investment poured into this field with a fall in available funding from 11% in 2010 to approximately 5% in 2016 [2] . Collectively, this imbalance could perhaps be applied to the term “10/90 Gap” coined by Global Forum for Health Research [3] to reflect that only 10% of health research funds are spent on the problem of developing optimum biotech equipment and devices for 90% of the world’s population.
Traditionally, most of the research and development (R&D) of new and efficient biotech equipment especially smaller medical devices are undertaken by startup companies which invariably depend on funding provided by private equity/venture capitalists. But the risks associated with market stability coupled with slow pace of development including the prolonged process of getting FDA approval oftentimes result in the redirection of finances from these sources towards more lucrative non-medtech sectors wherein the time gap from development to actualization of return is less. The big players, namely the corporate sharks on the other hand are more prone to withhold investing in smaller startups and rather play a wait and watch approach to let the completion of the development and testing process before paying to acquire the business in its entirety.
Despite these setbacks the tides are starting to turn in favor of greater opportunities for those involved in R&D in the biotech equipment/medical device sector at both the industry and academic levels. The potential for crowd sourcing funds to support startups and establishment of grants to sponsor seed money by Research Institutions is a step in the right direction. It is indeed heartening to note that in the 2018 cycle of the LEAP Inventor Challenge Awards administered by the Skandalaris Center for Interdisciplinary Innovation & Entrepreneurship at the Washington University in St Louis, 3 out of the 5 awards conferred were in the area of medical device development. As the transformative journey in this previously eclipsed field gathers renewed momentum the hope for further advancements in the future holds promise.
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Biomed Grid | Will the Modern Technology ever Catch up with the ‘God’s Formula’ of Dietary Fiber?
Opinion
The history of dietary fiber began back in ancient Europe during Hippocratic era, where function of wheat bran was already known as a laxative. The development of powdering system in France during 18th century has led to a debate between white bread and brown bread. However, this debate was based solely on their nutritional values, and depended on the opinions of food manufacturing companies and certain dietitians, thus the significance of dietary fiber was not well explored.
In the USA, Kellogg et al. [1] had found a great interest in the function of wheat bran and advanced the scientific research on it. During the process, Williams et al. [2] have found that although cellulose, hemicellulose and lignin are degraded in digestive tracts, they increase the amount of stool and softens it. Also, McCarrison [3] concluded from the research that eating whole-wheat bread, beans and fruits are beneficial for human health. However, the debate on dietary fiber was still depended on the opinions of food manufacturing companies and certain dietitians.
In terms of the relationship between dietary fiber and chronic lifestyle disease, Walker [4] conducted a research on Bantu tribes in South Africa in 1940s, and suggested that low prevalence of heart disease and atherosclerosis among the tribe was due to their lowfat and high-fiber dietary habit. Cleave [5] claimed that high consumption of refined carbohydrate food, especially sugar and white flour led to obesity, diabetes, colon diseases etc., which are also known as the Saccharin disease. The interest of dietary fiber then finally caught attention of medical doctors and researchers.
In 1953, dietary fiber was originally defined by Hipsley [6], and it included the definition of cellulose, hemicellulose and lignin as well.
In 1960s, Trowel [7] reported that the reasons why certain diseases are common in western Europe and not in Africa (constipa tion, diverticulosis, colorectal cancer, ulcerative colitis etc.) are due to the amount of dietary fiber intake in their diet.
In 1971, Burkitt [8] introduced the dietary fiber hypothesis, where he stated a significant association between dietary fiber and colorectal cancer. As a result, more researches on dietary fiber and diseases associated with large bowel (cancer, diverticulosis, constipation etc.…) were conducted. Also, it led to more research not only on diseases related to large bowel, but also other diseases such as diabetes mellitus, obesity and ischemic heart disease as well. The newly discovered function of dietary fiber other than that on large bowel function, including inhibition of calorie intake and preventive effect of absorption, had a large impact on the researchers.
The dietary fiber hypothesis was supported for many years, however, Fuchs et al. [9] published the research in 1999 reporting the completely opposite results. Their cohort research included administering food-related surveys to 88,757 females (34-59 years old) in 1980, and then followed them up for the next 16 years. During the period, 787 cases of colorectal cancer were found. As an overall result, there was no significant association between the amount of dietary fiber intake and incidence of colorectal cancer. There were several researches conducted in the USA afterwards, which resulted in supporting Fuchs’ findings. There were a few researches that still supported the dietary fiber hypothesis; however, the impact of Fuchs’ research was tremendous, and medical research on dietary fiber became stagnant in 21st century.
Such trend was ceased by the increasing interests in intestinal bacteria research. Notable progression and development of method to analyze intestinal bacteria using DNA analysis have accelerated the research of intestinal bacteria, which was once known as the ‘black box’ of human body. Since most of the substrate for degradation and fermentation is dietary fiber, the interest of intestinal bacteria subsequently increased the interest for dietary fiber (prebiotics).
At the same time, many developed countries started facing ageing society, that involved increased number of elderly adults with dementia. Due to its association with dementia, mastication then gained attention as preventive factor for cognitive function decline [10]. Thus, dietary fiber that promotes mastication has been gaining attention.
Five major functions of dietary fiber that are currently known are as follows:
a. To increase stool volume and to maintain colon functions
b. To prevent obesity by reducing calorie intake
c. To prevent diabetes mellitus by delaying absorption of nutrients
d. To affect the health of whole body through intestinal bacteria
e. To prevent dementia by promoting mastication
As well as dementia, the world is now facing an increasing prevalence of overeating and subsequent obesity, diabetes mellitus, arteriosclerosis and cardiovascular diseases. The same trend was emphasized when the dietary fiber hypothesis was introduced by Burkitt. 50 years on, the amount of attention gained due to the importance of dietary fiber is greater than ever. This is since we have reached the era of food satiation, where controlling the amount of dietary intake became an important medical and societal issues.
Development of refining technology in recent years have been allowing us to consume highly nutritious food, in other words, less dietary fiber-rich food, as dietary fiber is considered non-nutritive. This is causing obesity and then to increased prevalence of related diseases. Thus, it is important to consume non-nutritive (rich in dietary fiber) food. Such concept did not exist when people were suffering from famine and shortage of food in the past. Therefore, dietary fiber is a big hope in the food satiety era.
In addition, we need to pay attention to the way dietary fiber is consumed. We often focus only on the amount of dietary fiber intake; however, the type and the contents of dietary fiber is also extremely important. There are variety of dietary fiber in foods, from soluble and non-soluble, to different types such as cellulose, hemicellulose, lignin, pectin, guar gam etc.…, and they all have different functions. If dietary fiber functions when they are balanced, each dietary fiber needs to be consumed in the appropriate/balanced amount. To be able to achieve that, variety of plant-based food need to be consumed. Some plant-based food contains the dietary fiber in the perfect balance, known to have ‘God’s formula’, although people have rapidly diverted from consuming such food in the past half century. As humans have been having eating habit that is rich in dietary fiber for centuries, we must aim to consume the same amount of dietary fiber as the past, with food rich in the right amount and balance of dietary fiber, or ‘God’s formula’, as that’s how our body have adapted and developed in the first place. Before dietary fiber gained attention, the world of dietetics was only about nutrients. Many scientists emerged in this field, producing Noble prize winners as well. When dietary fiber gained more attention, many researchers warned the excessive intake of nutrients, which created a different dimension in the world of dietetics - with nutrients being a ‘light’ and dietary fiber being a ‘shadow’ in the dietetics field.
Dietary fiber has become a big hope in dietetics field with its wide variety of functions including the effect on intestinal bacteria as well as prevention of constipation, Saccharin disease, colon diseases, obesity, diabetes mellitus and dementia. The existence of dietary fiber has changed from something that only certain dietitian cared about, to something that all the scientists as well as any ordinary people consider or think about in their everyday lives.
The future of dietary fiber should consist of an establishment of methodology that can maximize the amount of dietary fiber intake, despite its commonly unpleasant flavor and texture. Therefore, what need to be done to increase dietary fiber consumption are; for each individual to understand the importance of dietary fiber, and for food manufacturers and scientists to develop a plant-based food material as well as cooking method to significantly improve flavor and texture of fiber-rich food, and to invent a technology to produce them.
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Biomed Grid | Real-Time Three-Dimensional Reconstruction of Intraoperative Liver Surface Based on Structured Light Technique
Mini Review
The incidence of liver tumor is increasing year by year, and liver tumor resection is a routine operation for liver lesions. Liver tumor resection is more difficult than general surgery, mainly reflected in the liver itself is easy to deform, vascular density, complex structure and other aspects [1]. If the three-dimensional information of the liver surface can be obtained in real time during the operation, it will be helpful for doctors to observe the cutting status of the liver surface in real time and avoid the possibility of being cut by mistake to a large extent. Clinical studies have shown that failure to accurately locate the focal point of the liver during surgery, to obtain real-time three-dimensional information on the liver surface and to plan the surgical path are the most important reasons for the failure of such surgery [2].
Traditional liver tumor resection surgery mainly relies on the doctor’s own experience, hand feel, visual observation to determine the location of the tumor, to obtain real-time information of the liver cutting surface. In the field of computer-assisted surgery, there is no good way to provide real-time three-dimensional reconstruction of the liver surface during surgery. It also fails to provide color and high definition on the surface of the doctor’s liver so that the texture of the liver surface can be well recognized.
In order to solve these problems, surgical navigation technology (IGS), which is represented by real-time three-dimensional reconstruction of intraoperative liver surface, has gradually entered scientists’ research field of vision [3]. Real-time three-dimensional reconstruction of intraoperative liver surface based on structured light is one of the hot topics in the field of medicine and engineering. It is a combination of medical, optical, instrument and information technology, aiming at using structural light for real-time threedimensional reconstruction of liver surface during surgery [4]. In this way, the accuracy and timeliness of the three-dimensional reconstruction of the organ surface during the operation can be further improved [5]. Especially in operations with significant soft-tissue manipulation or deformation, such as tumor resection of the liver, where the deformation is larger, the incision is deeper, and the operation is more extensive. At this point, real-time threedimensional liver reconstruction based on structural light can give full play to the advantages of good real-time performance and high accuracy, thus greatly reducing the risk of surgery. Compared with the traditional method of intraoperative liver surface information collection, the real-time intraoperative liver surface information collection method and system based on structural light scanning has the advantages of no contact, no radiation, anti-interference and strong adaptability, which is suitable for various operating environments.
Starting from the 21st century, the 3D reconstruction technology based on structural light has been relatively mature in industrial and architectural industries. In the medical industry, the technology of structural light for 3d reconstruction is not yet mature, and only a few technologies can be used for 3D reconstruction of endoscopic intestinal tract. Currently, there are few researches on the introduction of structural light to achieve real-time reconstruction of intraoperative liver surface. The United States, the United Kingdom and Canada are among the world’s leading researchers in this field, and some research teams have made outstanding achievements in applying structural light technology to three-dimensional reconstruction of liver, lung, spine and other organs or human body parts.
In 2012, the university of nuremberg in Germany proposed a new endoscope 3D scanning system based on monocular structured light, integrating the camera receiving the pattern and the pattern projection unit into the sensor. The sensor head is only 3.6 mm in diameter and 14 mm in length and is mounted on a flexible shaft [6]. Capture 3D video at a rate of 30 frames per second, typically generating about 5,000 3D points per frame. In 2015, imperial college London in the United Kingdom developed an endoscopic structured light system for intestinal examination, called the miniature structured light (SL) system [7]. It is mainly used for rapid recovery of tissue surface shape in minimally invasive surgery (MIS). The average accuracy of three-dimensional reconstruction of the intestinal inner surface has reached less than 1mm, and the accuracy has completely reached the clinical requirement of less than 2mm. At the 34th IEEE EMBS international conference, the university of Strasbourg, France, presented a novel three-dimensional laparoscopic device based on structured light for minimally invasive surgery [8]. In October 2018, structured light was used by the university of Toronto in Canada for high-speed, high-density intraoperative 3D reconstruction, which was used for effective registration of MRI and CT images in the navigation of cranial and spinal surgery [9]. The experimental navigation system of optical topology imaging (OTI) was developed to obtain the 3D surface anatomy of the surgical cavity. The system is much faster than commercial reference systems and does not affect spatial accuracy.
At present, the research on 3D reconstruction technology of human organs has made a breakthrough to a certain extent. The 3D reconstruction effect of similar rigid bodies (such as spine and bone, etc.) has basically met the needs of doctors in the process of surgery. However, for soft tissue (such as liver, heart and lung, etc.), there has not been a breakthrough. For the existing 3D soft tissue reconstruction system, most of them can only stay in the experimental stage, failing to meet the clinical requirements. The three-dimensional reconstruction of the spine based on structured light technology has achieved good results, which can be combined with medical principles and introduced into the real-time threedimensional reconstruction of intraoperative liver.
The need to achieve better three-dimensional reconstruction of liver surface is also closely related to optics, information technology, human-computer interaction and other disciplines. A new, more suitable, more perfect and more humane scheme should be proposed from the perspective of both patients and doctors. Real-time acquisition of intraoperative liver surface information based on structured light is the basis of liver surgery navigation, so it requires high real-time and precision of three-dimensional reconstruction. At present, the liver surface information collection and real-time 3D reconstruction based on structured light still have some problems, such as poor real-time performance, low precision, and light shielding. With the integration of structured light technology into medical surgery and modern computer technology, three-dimensional reconstruction of liver surface based on structured light is possible. It will bring great breakthroughs to the medical robot industry and create great social benefits for human medical surgery [10].
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Biomed Grid | New Understanding of Fanconi Anemia Signaling Network upon Studying FANCD2
Abstract
The Fanconi Anemia (FA) pathway is activated upon replication stress and DNA damage. With the accumulated studies, this pathway has emerged as a fundamental signaling network to defend genome stability. The Fanconi Anemia complementation group D2 protein (FANCD2) sits at the center of the pathway, orchestrating many players to prevent our genome from going awry, leading to diseases including cancer. Here, we highlight recent advances in our understanding of FA signaling, emphasizing on a rarely known form of FANCD2, FANCD2-V2.
Fanconi Anemia
Fanconi anemia is a rare genetic disease, characterized by developmental and physical abnormalities, bone marrow failure and increased cancer susceptibility [1, 2, 3, 4, 5, 6, 7, 8, 9]. The majority of children with FA are likely to develop myelodysplastic syndrome or acute myeloid leukemia (AML), while adults are predisposed to head and neck squamous cell carcinoma, hepatocellular carcinoma, gynecological, and gastrointestinal cancers [1, 2, 3, 5, 6, 8, 10, 11]. FA is caused by germline mutations in any of the 22 FA genes (FANCA/B/C/D1/ D2/E/F/G/I/J/L/M/N/O/P/Q/R/S/T/U/V/W). FA gene products maintain genomic integrity and participate in a common DNA repair pathway- the FA/BRCA pathway [2, 4, 12, 14]. Proteins involved are specialized more in resolving DNA interstrand cross-links (ICL), a lesion that blocks both DNA replication and transcription [2, 5, 14]. Patients suffering from FA display high frequencies of chromosomal abnormalities and are hypersensitive to DNA crosslinking agents (i.e. mitomycin C, cisplatin, diepoxybutane) [1, 3, 5, 6, 8]. If DNA is not properly repaired it can lead to genomic instability, apoptosis, senescence and tumorigenesis [4, 7].
FA Signaling
Proteins implicated in the FA/BRCA pathway coordinate nearly all known DNA repair mechanisms in order to resolve ICLs [1, 2, 3, 4, 5]. Given accumulated studies showing much crosstalk between FA and other important DNA damage repair proteins, FA signaling pathway emerges as a significant signaling network, responding to a variety of genotoxic stresses. This network is activated upon replication stress or DNA damage. FANCM is a DNA translocase and forms a protein complex with FAAP24 (FA-associated protein 24) and others. FANCM-FAAP24 recognizes stalled replication forks due to the ICL [1, 2]. Histone-fold containing kinetochore protein MHF1- MHF2 allow for the stable association of FANCM to chromatin [5]. ATR checkpoint kinase phosphorylates FANCM proceeding its recruitment to the site of damage [3]. Eight FA proteins assemble and form the FA core complex (FANCA, B, C, E, F, G, L, M) in addition to FAAP100, FAAP20, and FAAP24 to act as E3 ubiquitin ligase. Similarly, to FANCM, FANCD2 and FANCI are phosphorylated in an ATR-dependent manner. FANCT acts as an E2 together with the FA complex E3 responsible for monoubiquitinating FANCD2 and FANCI at Lys 561 and Lys 523, respectively [15]. The monoubiquitination of FANCD2-FANCDI (ID2) complex is a key step, representing FA signaling network activation. Therefore, FANCD2 must be tightly regulated to maintain DNA repair. The ID2 complex moves to the DNA lesion and recruits’ nucleases (FANCP/SLX4 and ERCC1/ FANCQ/XPF) to unhook the ICL [2, 16]. The incision is converted to a double strand break (DSB) and translesion synthesis (TLS) allows for continued replication of the leading strand, while the lagging strand is mediated by homologous recombination (HR) [3, 5, 14, 17, 18, 19, 20]. However, it remains largely unclear as to how each individual DNA damage repair mechanism works in concert to fully repair the damage within the expanded FA signaling network, including many FA and non-FA proteins that are yet to be identified.
FA Signaling and Cancer
Therefore, if the FA pathway is impaired due to mutations in involved genes, DNA remains damaged, promoting genotoxic stress, genomic instability, and tumorigenesis [3, 21, 22]. For instance, unresolved ICLs cause DNA breakage and chromosomal rearrangement, leading to cancer development [4]. Mutations or epigenetic silencing of FA genes are found in breast, ovarian and pancreatic cancers, and heterozygous germline mutations in BRCA2/FANCD1, BRIP1/FANCJ, PALB2/FANCN, RAD51C/FANCO, and BRCA1/FANCS are important cancer risk alleles [6, 12, 23, 24, 25]. Recently, researchers performed whole genome sequencing on three patients presented with three up to five primary cancers. Interestingly, the varied genes in each patient are part of the FA pathway [26]. FA gene defects are found in a variety of human cancers. Of the FA genes, FANCA has the highest mutation rate and is associated with AML, pancreatic, cervical, oral and prostate cancers [1, 27]. An elevated FANCA expression determines a worse outcome for patients with chronic lymphocytic leukemia (CLL) and is due to a reduction of p53 genes, p21 and ΔNp73 [28]. Another group found FANCA amplification correlates with reduced progression-free survival in head and neck squamous cell carcinoma after radiotherapy [27]. FANCC has the next highest mutation rate and is similarly associated with pancreatic, cervical and oral cancers in addition to breast cancer [1, 6, 29]. Like any other FA gene, mutations in FANCD2 gene are involved in a variety of malignancies, including testicular and esophageal squamous cell carcinoma [1]. However, its importance is far beyond cancer implications, as FANCD2 sits at the center of the FA signaling network, and its activation/monoubiquitination represents the activation of FA signaling. With our recent studies on FANCD2, the previous unrecognized variant of FANCD2, FANCD2-V2 appears to be a more potent tumor suppressor than the commonly known form of FANCD2, FANCD2-V1. This review will focus on our current knowledge of FANCD2-V2. The related studies will aid in further understanding the roles that FANCD2 and/or FA signaling play in the development of human cancer and other diseases.
Overlooked FANCD2 Variant
Previously, our lab reported the first study on an alternate variant of FANCD2, named FANCD2-V2 (NCBI RefSeq accession#NM_033084.4) [30]. We refer to the long-known form as FANCD2-V1. FANCD2-V2’s cDNA is 60 bp longer than FANCD2-V1, encoding a 1471 amino acid (aa) transcript compared to FANCD2-V1 at 1451 aa. Both variants share 1427 aa at the N-terminus, resulting in 95% aa identity. We checked the expression of both FANCD2 variants in human lung tumor and matched normal samples. Interestingly, V2/V1 (ratio of V2 to V1) expression is higher in normal/non-malignant tissue compared to malignant tissues. We further assessed a panel of normal (CRL-1790, HEK293, WI-38) and tumor (HEK293T, PA-1, U2OS, HCT116, HT-29, RKO, LoVo) cell lines, which demonstrate the same trend.
In examining FANCD2 gene structure, we found a proximal and distal polyadenylation site (PAS). When the proximal PAS is used the FANCD2-V2 transcript loses the last intron; however, this intron is incorporated in the FANCD2-V1 transcript. We learned through RNA polymerase II ChIP that more DNA fragments were pulled down at the distal PAS in malignant cells compared to nonmalignant cells. U2 snRNP RNA Immunoprecipitation (RIP) showed SF3A1 antibody, a component of U2 snRNP, pulled down fragments representing the last FANCD2-V1 intron with greater binding in malignant cells than non-malignant cells. This finding suggests FANCD2-V1 transcripts are more prevalent in malignant cells. Additionally, human bladder and ovarian cancer samples present an increased V2/V1 expression in lower stage cancers. These data demonstrate FANCD2-V2 could be a more potent tumor suppressor as its expression is high in non-malignant cells and tissues.
Studies propose more than 50% of mammalian genes have multiple PAS [30]. How are sites of polyadenylation determined? A possible cause is DNA methylation. We recently reported, treating HEK293 with DNA methyltransferases reduced FANCD2-V2 expression and DNA methylation intensity near the proximal APS by methylated DNA immunoprecipitation (MeDIP) [31]. Our findings were validated with publicly available datasets (TCGA) in lung, ovarian, kidney, endometrial, colon and breast cancer, displaying an elevated methylated distal to methylated proximal APS ratio (Me-D/Me-P) is associated with malignancy. A high level of DNA methylation in the distal APS promotes FANCD2-V1 expression. Conversely, a high level of DNA methylation in the proximal APS results in FANCD2-V2 expression. To this end, we identified how two forms of FANCD2 are differentially expressed and connected with human tumorigenesis. However, it is largely unknown as to which traits in contributing to the functions are common or different between them. Simply, we would also ask; do additional FANCD2 variants exist? How are they expressed and/or function?
Conclusion and Prospective
In this short review, we aimed to update our understanding of FA signaling network with a focus on the center player, FANCD2. We described a recent recognized form of FANCD2, FANCD2-V2. This variant is expressed higher in non-malignant cell lines and tissues comparing to the corresponding malignant ones, due to proximal APS usage. These data suggest the long-known form FANCD2-V1 may be rather oncogenic compared to FANCD2-V2, which is more tumor suppressive. Of note, another group identified an alternatively splice isoform of FANCE, FANCEΔ4, which is expressed in breast cancer patients without BRCA1/2 mutations [32]. FANCEΔ4 blocked cells into G2/M phase after MMC treatment, reduced cell survival, and restricted FANCD2 and FANCI from monoubiquitination. Perhaps additional FA genes rely on alternative splicing for proper expression and gene regulation. More studies are needed to define the function(s) of alternative isoforms as wild-type protein function maybe disrupted or the isoform could gain new functions. These studies will allow for further characterization of FA genes and a deeper understanding of the FA signaling network.
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Biomed Grid | The Right Little Fingerprint of Rev. Dr. Martin Luther King, Jr.
Abstract
In the frequency of NCIC FPC patterns, there is a range in the spectrum. The populous direction is one percent or greater. Anything less than one present is on the other side in the series. We must note the infinite regions of both sides of the confines. When a pattern falls in the million frequency or greater of less than one percent, it is in a category for further scrutiny. In as much as the brain and fingerprints develop simultaneously, we have a comparison between the thought processes of those from each side of the scale between two extreme or opposite points. Individuals on the unpopulous side of the spectrum may display dermal ridge arrangements reflecting a different time in the evolution of fingerprint patterns. As such, their natural thinking may be different from those of the populous direction (Figure 1).
Keywords: Dr. MLK, Jr Tented Arch; Fingerprint, Left Little; NCIC FPC Frequency
Introduction
The right little fingerprint of Rev. Dr. Martin Luther King, Jr. receives the classification of a tented arch pattern. As a pattern, it cannot classify simply as an ulnar loop or radial loop. Dr. King’s right little fingerprint is a combination of both radial and ulnar loop. However, there is only one delta. Therefore, it is appropriate to the category of a technical tented arch in the tented arch series of fingerprint patterns [1] (Figure 2).
In 1993, the FBI conducted a study on the frequency of fingerprint patterns. In that study, 17,951,192 individuals were included from the male population. The tented arch pattern appeared on the right little finger of 114,881 individuals. Therefore, it can be noted that Dr. King’s display of the tented arch pattern on the right little finger is 0.639963073 % of the total [2] which is six hundred thirty-nine million, nine hundred sixty-three thousand, seventy-three billionths (Figure 3).
We are looking at a tented arch pattern from the right little finger of Dr. MLK, Jr., which displays both a radial and ulnar loop formation in juxtaposition; this is another rare phenomenon with Dr. King’s right little fingerprint. As radial and ulnar loops are in juxtaposition in the spectrum of fingerprint patterns, it is an impression in juxtaposition within its own display. I have often wondered if a tented arch pattern like the one that was on the right little finger of Dr. King resides midway between the radial and ulnar loop in the evolution of fingerprint patterns. In view of the fact that radial development precedes ulnar development for the hands, it is logical to assume that radial loops appeared on the human person prior to ulnar loops. The ulnar loop is therefore more recent and maintains the highest frequency of all patterns. As a result, ulnar loops are not likely to dissipate genetically in current time. In comparison, having a low frequency, the radial loop indicates a fading out of evolutionary existence.
In the display of this pattern, the loop formation on the left occupies a near vertical profile and is radial in appearance while the formation on the right is diagonal and ulnar. This is the fourth noted rarity of Dr. King’s fingerprint. The fact that a radial loop formation was on the right little finger is a clear departure from the expected frequency of the dermal ridge arrangements [3] rarity number five (Figure 4). The frequency of the radial loop on the right little finger is 0.532%.
Conclusion
Dr. Martin Luther King, Jr.’s right little fingerprint had a tented arch frequency of 0.639963073 %. As a frequency, it is less than 1%. One percent is the first whole unit in the populous direction. Anything less than one percent is on the other side of the spectrum. A person as such may be able to see things in a different light [4] .
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Biomed Grid | Heavy Metals in Barnacles Balanus Sp.: From Biomonitoring to Coastal Management
Introduction
The first preliminary study on the heavy metal concentrations in the soft tissue and shells of Balanus sp. (Subphylum: Crustacea; Class: Cirripedia) from Malaysia was reported by Yap et al. [1]. As part of regular monitoring of heavy metal contamination and bioavailability’s in the coastal waters of Peninsular Malaysia, the data of this study should serve as biomonitoring data for longterm reference in the future. Barnacles have been used to assess the bioavailability of heavy metals in the coastal waters in many countries including India [2, 3], Mexico, Hong Kong [4, 5], China [6] and Poland [7]. According to [8], barnacles are among crustaceans which appear most able to fulfil the characteristics of ideal bio monitors. The objective of this study is to determine the levels of heavy metals in the soft tissues and shells of Balanus sp. collected from four sites in Peninsular Malaysia.
Materials and Methods
Samples of barnacles Balanus sp. and their habitat surface sediments were collected at the same time in 2008. Sampling locations and site descriptions are shown in (Figure 1) & (Table 1), respectively. The collected samples were placed in polyethene bags and stored in the low temperature cold iceboxes and taking back to the laboratory. In the laboratory, the samples were stored at -10°C. The measurements of length-width-height of the biological specimens were recorded by using a Vernier caliper to an accuracy of 0.01cm. The identification of the barnacles was based on the book authored by [9] and George (1979). For metal analysis, the Balanus samples were thawed at room temperature on a clean tissue paper to defrost. After cleaning, the soft tissues of the barnacles were dissected from the hard tissues. They were dried in 60°C for 72 hours in an oven until constant dry weights. Dried samples were weighed for 0.5g and triplicates were analyzed for each pooled sample. They were digested with 10ml concentrated HNO3 (Analar grade. BDH 69%) in a hot-block digester first at low temperature (40°C) for 1 hour and were completely digested at a high temperature (140°C) for 3 hours [10]. The digested samples were diluted up to 40 ml with DDW and filtered with Whatman filtered paper No. 1 into acid-washed polyethene bottles.
Figure 1: Map showing the sampling sites for barnacles in the west coast of Peninsular Malaysia (1 = Kuala Juru; 2= Sebatu; 3= Sg. Ayam; 4= Kg. Pasir Puteh).
Table 1: Global Positioning System (GPS), date of sampling and description of all sampling sites for Balanus sp.
The collected surface sediments were oven-dried and were sieved using 63μm mesh size. For the surface sediment samples, the geochemical fractions of easily, freely, Exchangeable or Leachable (EFLE), acid-reducible (AR), Oxidizable-Organic (OO) and Resistant (Res) were fractionated by sequential extraction technique as suggested by [11]. The concentrations of Cd, Cu, Fe, Ni and Zn were determined by an air-acetylene flame Atomic Absorption Spectrophotometer (AAS) Perkin-Elmer Model Analyst 800. The data were presented in μg/g dry weight. Multiple-level calibration standards were analyzed to generate calibration curves against which sample concentrations were calculated. Standard solutions for each metal were prepared from 1000mg/L per stock solution of each metal (MERCK Titrisol).
For quality control, all the glassware and equipment used were acid-washed with 10% diluted nitric acid solution for one night long. Procedural blanks were prepared for every digestion made for standardization. Quality control samples made from standard solution for Cd, Cu, Fe, Ni and Zn were analyzed after five to ten samples to check for accuracy of the samples. The recoveries percentages were acceptable at 80-110% for each of the heavy metal analyses. The blank samples were subtracted from the results to avoid the contamination possibility. The analytical procedures for the samples were checked with the Certified Reference Material (CRM) for dogfish liver (DOLT-3, National Research Council Canada) (Table 2).
Table 2: Analytical results for the certified reference material (DOLT-3 Dogfish liver) and the measured values for each metal (μg/g dry weight).
Results and Discussion
(Table 3) shows the allometric data for all populations of Balanus sp. from the four sampling sites in Peninsular Malaysia. The levels of Cd, Cu, Fe, Ni and Zn in the soft tissues and shells of Balanus sp. collected from the four sites are presented in (Table 4). (Table 5) shows the levels of the five heavy metals in the four geochemical fractions of surface sediments collected from the four sampling sites. For Cd levels in the shells: Sg. Ayam> Sebatu> Kuala Juru > Kg. Pasir Puteh. For Cd levels in the soft tissues: Sg. Ayam> Kg. Pasir Puteh> Sebatu> Kuala Juru. This highest bioavailable Cd levels to the Balanus collected from Sg. Ayam is well supported by the highest contamination level of Cd in the surface sediments represented by the geochemical fractions of EFLE and AR, and the highest percentage of non-resistant faction (59.3%) (Table 5).
Table 3: Allometric parameters for all Balanus sp. populations.
Table 4: Heavy metal concentration (mean μg/g dry weight ± SE) in the total Soft Tissues (ST) and shells of barnacles Balanus sp. collected from the intertidal areas of Peninsular Malaysia.
Table 5: Concentrations (μg/g dry weight) of heavy metals in the four geochemical fractions of surface sediments collected from the 4 sampling sites.
For Fe in the shells and soft tissues: Sg. Ayam> Sg. Sebatu> Kg. Pasir Puteh> Kuala Juru. However, these Fe bioavailability results do not agree with the Fe levels in the geochemical fractions in the surface sediments from Sg. Ayam (Table 5) due to Fe is not an anthropogenic metal and complications of other factors affecting the Fe accumulation in the Balanus and physicochemical factors being involved in the sedimentation processes. For Cu, the highest Cu level was found in the Balanus soft tissues collected from Kuala Juru, followed by Kg. Pasir Puteh, Sebatu and Sg. Ayam. The highest Cu level in the shells was also found in the Balanus from Kuala Juru. These Balanus results are well supported by the Cu levels in the surface sediments from Kuala Juru in the geochemical fractions of EFLE, AR and OO, with the highest non-resistant fraction (48.2%) (Table 5). This indicated the high bioavailable of Cu to the Balanus collected from Kuala Juru, which is a Cu contaminated site as evidenced in the surface sediment results.
Inconsistent results are found for Ni levels between soft tissues and shells of Balanus. For Ni the shells: Sg. Ayam> Kg. Pasir Puteh> Sg. Sebatu> Kuala Juru. The reverse pattern was found for the Ni in the soft tissues: Kuala Juru> Sebatu> Kg. Pasir Puteh> Sg. Ayam. Only Balanus soft tissues in the Kuala Juru is supported by the highest Ni levels in the EFLE and AR geochemical fractions ((Table 5)). For Zn levels in the shells: Kg. Pasir Puteh> Sg. Ayam> Kuala Juru> Sebatu. For Zn levels in the soft tissues: Kg. Pasir Puteh> Sebatu > Sg. Ayam> Kuala Juru. The highest Zn levels in both shells and soft tissues are supported by EFLE fraction in the surface sediments (Table 5). Although Balanus sp. is not a direct food source to the human, they are feeding source for birds and ducks which become a potential food chain to the end consumers- human. They are potential bio monitor of heavy metal pollution the surrounding environment [7, 8, 12, 13] because they provide integrated measures of the metals supply available to them in the local environment, accumulating the metal taken up from food [12]. According to [13, 14] Cu is accumulated by barnacles in Cu- and sulphur-rich deposits, probably representing end products of the lysosomal breakdown of Cu-containing metallothionein’s.
The metal concentrations (μg/g dry weight) in the soft tissues of Balanus sp. collected from four sites in Peninsular Malaysia ranged from 2.93-4.17 for Cd, 20.2-92.5 for Cu, 480-1193 for Fe, 6.40-18.0 for Ni, and 224-414 for Zn (Table 4). The present results are comparable to and lower than those in the Balanus Amphitrite for Hong Kong coastal waters reported by [5] (Cd: 0.69-9.45; Cu: 52.4-1810; Fe: 313-1470; Ni: 1.25-98.9; Zn: 2860-23300). Our results are also comparable to and within those in Balanus sp. collected from Penang’s Bridge and Semilang (Peninsular Malaysia) reported by [1, 15, 16, 17, 18, 19] (Cd: 4.72-6.66; Cu: 10.3-20.3; Fe: 633-670; Ni: 21.6-23.2; Zn: 361-434).
Conclusion
This preliminary study on heavy metal levels provides a new baseline against which future local changes can be assessed. This is highly recommended that further studies should be focused on the genetic structures and taxonomy on this potential Balanus sp. that can be established as a good bio monitor in Malaysian coastal waters in the future. Overall, this preliminary baseline data can be used for regular ecological monitoring for the effective management of the coastal area in Malaysia.
Acknowledgement
The authors wish to acknowledge the financial support provided through the Research University Grant Scheme (RUGS), [Vote no: 91229], by University Putra Malaysia and through e-Science Fund [Vote no: 5450338], by the Ministry of Science, Technology and Innovation, Malaysia.
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Biomed Grid | Transient AV Complete Heart Block: A Rare Complication Following Regadenoson Injection
Case Report
A 49-year-old woman with hypertension, diabetes mellitus type II, chronic Kidney disease stage III, hyperlipidemia, asthma, moderate obstructive sleep apnea, neuropathy and recent cystourethroscopy and stent placement. A transthoracic echocardiogram was obtained as a part of pre-operative evaluation for decreased exercise tolerance showed decreased Left ventricle ejection fraction (LVEF). She was referred for cardiology evaluation post-surgery for newly diagnosed cardiomyopathy. She reported atypical chest pain with limited exercise capacity. She was referred for pharmacological nuclear stress test, given multiple coronary artery disease risk factors and decreased LVEF. Pre-test electrocardiogram showed normal sinus rhythm with heart rate of 86 bpm, normal axis without ST/T segment changes and normal QT segment. Resting blood pressure was 156/85 mmHg. Pre-stress physical exam was unremarkable.
Figure 1: Resting ECG in normal sinus rhythm. Heart rate at 86 bpm.
Figure 2: Initial Development of 2:1 AV block and subsequent progression AV complete Heart block and Ventricular escape rhythm following 40 second. of regadenoson injection and lasted for 20 seconds.
Figure 3: Immediate recovery of the conduction as sinus tachycardia following intravenous 75mg aminophylline injection.
Following intravenous injection of regadenoson (0.4 mg) patient developed sinus bradycardia which progressed to AV complete heart block for 20 seconds. Normal AV conduction was recovered as sinus tachycardia following immediate intravenous 75 mg aminophylline injection. Patient did not lose consciousness however felt extremely nauseated and subsequently vomited a few times. Patient was observed in the stress laboratory for 30 minutes, she remained hemodynamically stable and there was no recurrence of AV block. Patient completed Myocardial Perfusion Imaging (MPI) scan which showed moderate size, moderate intensity partially reversible defect extending from the apex to the base of the anterior wall on SPECT images, mildly enlarged LV size and moderately decreased systolic function on gated images. Further evaluation with diagnostic catheterization as outpatient showed non-obstructive coronary artery disease (Figure 1) (Figure 2) (Figure 3)
Discussion
Regadenoson, is easier to use and associated with better safety and tolerability profiles than non-selective agents such as adenosine and dipyridamole. Vast majority of these side effects are short-lived, benign, and spontaneously terminate. On rare occasions, however, more serious cardiovascular and neurological adverse events may develop, namely symptomatic myocardial ischemia, infarction, high-grade AV block, asystole, and seizures [5].
Although its affinity at the A1 receptor has been reported to be 10-15-fold weaker, this remains important as these receptors are found at the sinoatrial and atrioventricular node in addition to atrial and ventricular myocytes [6] which is likely the potential source of bradyarrhythmic complications. Also, there has been a theoretical concern of increased and prolonged side effects in patients with chronic kidney disease; however, prior work has demonstrated safety and tolerability in end stage renal disease patients [7, 8]. Among those totals of 7 reported high degree AV block patients only 2 reportedly had chronic kidney failure. Furthermore, clinical information regarding the 47 cases of complete heart block and 25 cases of sinus arrest reported via the FDA adverse event reporting system (FAERS) is not available [9] hence the real prevalence of regadenoson-induced high-degree AV block remains unknown. It is difficult to predict if a patient with a normal ECG will develop a complete heart block following regadenoson injection. It is important to stress the importance of recognizing potential side effects and treating them immediately [10]. In our case immediate reversal with aminophylline restored sinus rhythm with normal conduction and improvement in symptoms.
We believe the awareness of this potential side effect in nuclear laboratories and readiness of the equipment and drugs in emergency situations is important.
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Biomed Grid | The Mediterranean Diet: Plant Lectins as Essential Components
Opinion
In the U.S. News and World Report annual rankings (published in 2020) we note that the Mediterranean diet (Md) has, for the third year in row, been named as the overall best healthy diet. This diet is well-known for its emphasis on a high content of fruits, nuts, vegetables and whole grains. In some meals in this class of diet one can find more than 20 individual components belonging to these groups of foodstuffs. For many years now the Mediterranean way of life has been highly looked upon for health inspiration. Based on studies of Md it is now well recognised that diets rich in fruits, vegetables, fish, and healthy fats are good for us, particularly our hearts. Numerous studies have clearly shown that eating like they do in countries such as Greece, Italy, and Turkey then there is a reduced risk of suffering from heart disease. Furthermore, according to health professionals at the Mayo Clinic, those that adhere to the Md show a reduced risk of developing cancer, Parkinson’s disease, and Alzheimer’s disease. So, what makes the Md exceptional?
In my opinion one should look for a common factor among the ingredients of the Md. A recent survey of the content of fruits, nuts, vegetables and whole grains has shown that all these commodities contain varying amounts of protein molecules termed lectins. Health authorities in Western society strongly advise members of the population to adopt the “five a day” principle i.e. one should include at least five portions of plant-based material in the diet per day (approx. 400g). In actual fact health benefactors are thus advising us that a diet rich in lectins is beneficial to health! This is in stark contrast to what can be described as anti-lectin propaganda that we read in the popular press and in opinions expressed on numerous web sites. It is indeed correct that there are reports in the scientific literature (animal experiments) that have demonstrated negative effects of diets highly enriched in certain lectins.
The amounts added to these artificial diets fed to animals are, however, in most cases so high that it would be impossible for an individual following a normal diet to attain a lectin intake likely to be detrimental to health. Furthermore, there is no evidence that individuals that keep to a vegetarian diet, i.e. daily are exposed to a diet enriched in lectins, show any signs of poor health. So what are lectins? Plant lectins were discovered by Stillmark in 1888 when he observed that castor bean extracts caused agglutination of red blood cells in vitro. The word lectin (legere; from latin, means to select) was coined by Boyd in 1954 to cover a group of hemagglutinins that were able to discriminate between blood types in the ABO system. This hemagglutination property of lectins has been exploited as a useful method to identify the presence of lectins in, for example, food extracts. This approach was used by Nachbar and Oppenheim in 1980 when they reported that lectins were present in >20 common food commodities including potatoes, carrots, tomatoes, beans and peas.
Lectins are proteins that are in general resistant to low pH in the stomach and to proteolytic enzymes in the small intestine, and for some of these molecules >90% can survive passage through the gut. It is therefore evident that biologically active lectins will be present in the intestine after a meal containing raw plant material. About 80% of our immune system is associated with the alimentary canal, such that dietary lectins will provide an activation of our immunedefense apparatus. One can surmise that a diet lacking active plant lectins would result in a weakened immune system. Plant lectins have physiological effects, such as binding to glycoproteins on the epithelial surface of the small intestine, where they may elicit local and/or systemic reactions, e.g. modulation of the immune system and the micro flora in the gastrointestinal tract.
That different lectins bind to specific regions of the small intestine has been clearly demonstrated in experiments using human biopsies. In 1996 Sharma and colleagues used a panel of 27 lectins to study binding to M-cells, enterocytes, goblet cells, lymphocytes and macrophages. They clearly established that lectins with known differences in binding properties showed individual specificity with regard to which region of the small intestine they bound. For example, only 13/27 were found to bind to the M-cells of the follicle-associated epithelium and of these 5 bound to goblet cells. Although the biological significance of this is currently not known, some lectinologists speculate that lectins which have the property of binding to different cell types associated with the small intestine will initiate or trigger, separate biological responses positive for our health/well-being. The lectin, following binding to the cell, can send information into the cell´s interior via second messengers, often modulating gene expression or the lectin itself may enter the cell through endocytosis.
A large number of animal studies have clearly shown that dietary supplementation with various lectins can reduce growth of a series of different types of tumors. In their review published in 2005 Gonzalez De Mejia and Prisecaru indicate that use of plant lectins may provide novel strategies in the future for the development of new forms of cancer treatment. Beneficial effects have been reported in cases of terminal cancer where lectin-based preparations have been utilized. As mentioned earlier lectins are protein molecules meaning that they are denatured by the high temperatures met during cooking, frying, grilling etc. resulting in loss of biological activity. Thus, the importance of emphasising the need to consume raw plant material.
Dietary lectin studies are complicated by the fact that humans have consumed lectins in their habitual diet for thousands of years, making the study of individual lectins thus virtually impossible, since many may well act in concert. Furthermore, a meal would seldom consist of e.g. a single vegetable. There is indeed information indicating that when different lectin-containing preparations are mixed separately and in various proportions, then the biological responses elicited are quite unlike. Plant lectins represent an unavoidable component of a balanced diet. During the past 10- 20 years more and more studies have been addressed to the conception that lectin molecules may have an important positive impact on our health in general. Current opinion concerns the potential that lectins may have in relation to preventing illness and should therefore be regarded as health promoting components in our diet along with vitamins, minerals and other micronutrients.
In conclusion there can be no doubt that the Md is enriched in lectins and that these protein molecules can promote both positive and beneficial effects on our health. It is therefore not difficult to voice the opinion that a balanced diet containing a rich variety of fruits, vegetables and nuts will provide the necessary diversity of lectins required to stimulate biological processes in the body, forming the basis of a healthy lifestyle. Although there is much talk of the benefits of Md there is no information readily available to the general public on why a plant-based diet is positive for our health. For good health it is not simply a matter of obtaining sufficient amounts of nutrients from our food and vitamins and minerals etc. by an intake of tablets from the local drugstore, since lectins can only be obtained, not in the form of a pill, but by ingestion of raw plant material purchased at the supermarket! In my opinion more public awareness needs to be directed to the content of Md.
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Biomed Grid | Bio Polymerization as One of The Stimulation Cause of The Cancer Cell Multiplication
Introduction
As it is known one of the general properties of the carcinogen materials and other influencers on the cancer evolution is their polymerizing properties. Therefore, we can assume that one of the factors stimulating the process of blocking of intercellular feedback is the bio polymerization process. Carcinogenic materials and other factors contribute to the blocking of intercellular metabolism and exchange of genetic information what complicates the genetic balance of biological processes [1] . As a result, the management of genetic processes by the central nervous system is weakened. After a certain concentration of carcinogenic substances and other factors acting on the cells, an uncontrolled polymerization process occurs, for example, of the membrane system between cells, and an uncontrolled process of cell reproduction begins, that is, cancer growth and the appearance of metastases. So, one of the important mechanisms of the cancer generation must be uncontrolled additional process of bio polymerization in the organism [2] .
Overview
Let’s list of the some carcinogens - substances of various chemical structures that can cause malignant tumors (cancer) and/ or benign neoplasms: Aflatoxins; Benzene; Benz (a) pyrenes; Vinyl chloride; Dioxins; Nitrates, nitrites; Cadmium and its compounds; Peroxides; Formaldehyde and so on. All of these materials are activators of polymerization processes.
Most chemical carcinogens are organic compounds, only a small number of inorganic substances have this ability [3] . According to Miller, all carcinogens are, to one degree or another, electrophiles that easily interact with nucleophilic groups of nitrogenous bases of nucleic acids, in particular DNA, forming strong covalent bonds with them. The negative effects of carcinogens are manifested in the chemical modification of nucleic acids [4] . The consequences of such modifications are manifested in the impossibility of the proper course of DNA transcription and replication, which leads to the formation of the so-called DNA adducts associated with it. For example, in the replication of modified DNA, nucleotides that are associated with a carcinogen may not be read correctly by DNA polymerase, resulting in mutations. The accumulation of a large number of mutations in the genome leads to the transformation of normal cells into tumor cells, which is carcinogenesis.
Chemical carcinogens can be divided into two large groups:
a) Genotoxical
b) Non-genotoxical
Genotoxic carcinogens are chemical compounds in the interaction of which with DNA components can cause damage and mutations of the cell genome [5] . Mutations in turn can lead to the formation of tumor cells. Non-toxic carcinogens are chemicals that can cause damage to the genome only in high concentrations, with a very long and almost continuous exposure [6] . They cause uncontrolled cell proliferation, inhibit apoptosis, disrupt the interaction between cells (cell adhesion). Most non-genotoxic carcinogens are carcinogenesis promoters, such as organochlorine pesticides, hormones, fibrous materials, asbestos, especially dust.
According to the mode of action, genotoxic carcinogens can be divided into:
i. Substances containing alkyl and acetylating substances- N-nitrosyl alkylurea (NAM), epoxides (especially PAHs), ethyleneimine and its derivatives, chloroethylamine, etc.).
ii. indirect - low-activity substances that form covalently bound DNA adducts only after enzymatic activation, which occurs with the formation of highly active electrophilic metabolites that can interact with nucleophilic DNA groups (PAHs and their derivatives).
The most famous physical carcinogens are various types of ionizing radiation (α, β, γ radiation, x-ray x radiation, neutron radiation, proton radiation, cluster radioactivity, ion fluxes, fission fragments), although they are also used to treat cancer. Ultraviolet is completely absorbed by the skin, and therefore can only cause melanoma. Whereas ionizing radiation, freely penetrating into the body, can cause radiogenic tumors of any tissues and organs of the body (quite often hematopoietic, due to high sensitivity). Microwave radiation.
The role of biological factors in carcinogenesis is not as great as that of chemical and physical factors, but in the etiology of some malignant tumors it is very significant [7] . So, up to 25% of cases of primary liver cancer in Asia and Africa are associated with hepatitis B virus infection. About 300,000 cases of cervical cancer per year and a significant proportion of cases of cancer of the penis are associated with sexually transmitted papillomaviruses (primarily HPV-16, HPV-18, HPV-33). Approximately 30-50% of cases of Hodgkin’s lymphoma are associated with damage to the human body by the Epstein-Barr virus.
In the 1990s, convincing data were obtained on the dependence of most varieties of gastric cancer on infection with the bacterium Helicobacter pylori [8] . In any case, all processes of initiation and propagation of cancer cells can be associated with uncontrolled processes of bio polymerization.
Conclusion
According to the author, the role of polymerization processes in the body should be taken into account when studying and treating cancer [9] . From this point of view, one should look for ways to slow down or reverse the polymerization processes in the body. Based on the author’s many years of experience, one of the most effective ways to prevent and treat cancer is the effects of passive radiation on the body in the optical range of 0.6-0.90.6-0.9μm of the corresponding lasers or LED which increase the activity of weak intermolecular bonds.
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Biomed Grid | Regulation of Oxidative Imbalances in Mitochondria and Endoplasmic Reticulum
Introduction
Intracellular oxidative stress has been described as a double-edged sword, at the physiological level these molecules complements cellular functioning, but excessive burden may cause damaged to cellular macromolecules and organelles [1, 2]. Notably, cells dissipate the reactive oxygen species (ROS) induced oxidative stress through arsenal of antioxidant enzymes (such as glutathione peroxidase, catalase, thioredoxin reductase and superoxide dismutase) and by redox systems (such as oxidized glutathione/ reduced glutathione (GSSG/GSH), NAD+/NADH, NADP+/NADPH) to maintain cellular homeostasis [3]. Interestingly, the oxidative imbalance in the cells are mostly generated through organelles in the process of building cellular architecture [4, 5]. For instance, the mitochondrial respiratory chain or electron transport chain (ETC) comprises of complex I to IV are the prime site for production of reactive oxygen species (ROS). The mitochondrial ETC generates superoxide anion that converts to hydrogen peroxide (H2O2) through mitochondrial dismutase and may generates highly reactive hydroxyl radical such as super oxide (O2•−) and hydroxyl radical (OH•) [5, 6]. Similarly, reactive nitrogen species (RNS) is derived from nitrogen species also known as call peroxynitrite (ONOO−) in the term of ions.
Thus, nitric oxide (NO.) which is also derives from mitochondria (NO•), can alter the cellular activity like respiration, oxidative stress and mitochondrial biogenesis by enhancing the production of ROS and RNS [7]. Therefore, the term oxidative stress represents both ROS and RNS. On the other side, organelles such as peroxisome and the endoplasmic reticulum (ER) at the physiological levels generates oxidative stress within in the cells [8]. The exacerbated accumulation of terminally misfolded proteins been removed through activation of unfolded protein response (UPR) and ER-associated degradation (ERAD) pathway to restore homeostasis [9]. In fact, immature protein folding within the oxidized lumen of ER generates oxidative stress within the cells [10]. Importantly, the enzymatic cascade of endoplasmic reticulum oxidoreductin (ERO-1), and NADPH oxidase complexes balances redox levels of ER lumen to regulate oxidative stress in the cells [11]. However, failure to resolve mitochondrial and ER induced oxidative burden leads to cell death [12, 13].
Mitochondrial ETC complex: source of reactive radicles
The role of mitochondrial complex I and III are more significant in the production of ROS [14]. Although the major site for ROS production under oxidizing complex I is still not clear [15]. Previous studies showed that pathological and physiological site site in complex 1 for ROS is flavin mononucleotide group (FMN) not the ubiquinone of complex III [16, 17]. For the production of superoxide radicals (O2•−), Complex III might be more responsible specially from the mitochondria of heart and lungs [18], but the superoxide formation with complex I might be the primary source for mitochondria of brains [19]. The production of ROS is measured in term of H2O2 because of quenching property of mitochondrial superoxide dismutase (SOD), which quickly control the superoxide production and finally dismutated by SOD. Thus, the production of superoxide/ROS has been measured in term of H2O2 only. Although the enzymatic source of super oxide is NADPH oxidase which can be present on endothelial cells, microphases and cell membrane [20]. To confirm the production site in different complexes of respiratory chain several study have been reported, study reveals that rotenone quencher of complex I not enhancing the ROS generation in mitochondria but very significantly increased in sub-mitochondrial particles linked with NADH oxidation [21]. In the pathogenesis of myocardial ischemia, negative effect of rotenone in the production of ROS have been reported [22]. Study further reveals that when complex III is ceased by antimycin A, then the oxidation of both complex I and II favor the generation of ROS [23]. Proper balance between ROS generation and anti-oxidant defenses is required to control the pathogenesis linked with various disease. The balance concentration of anti-oxidant in mitochondria is mentioned by anti-oxidant defense and repair enzyme [18]. Mitochondrial membrane potential (MMP) is another evidence of ROS induced damage of inner membrane of mitochondria. Lower the MMP higher the chance of impairment of inner membrane of mitochondria, which favor the release of apoptotic signals like Bax and cell death via final activation of Caspase3 [24]. Our previous report also concluded that release of Ca++ from endoplasmic reticulum (ER) upregulated the stress marker of ER like AFT6 and CHOP at gene level. CHOP directly correlate the MMP and finally active the chain of caspase via elevated level of Bax/Bcl2 ratio [25]. Effect of xenobiotic on mitochondria directly affects the rate of superoxide production, increasing rate of generation of O2•− by blocking the electron transport which enhance the reduction level of carriers present on inhibition site or if the xenobiotic accepts the electron from respiratory carrier and transfer to oxygen molecule by redox cycling, which also result in elevated level of O2•− [18]. Thus, the clinical pathogenesis, apoptosis and aging is dependent to cellular respiration by regulation of ROS and RNS by mitochondria membrane potential.
ER stress induces oxidative stress: Ca2+ connection
The ER serves as a site for synthesis and proper folding of proteins through cascade of reactions that depends on the redox status within the lumen of ER [26]. In the lumen of ER, protein disulfide isomerase (PDI) enzymes catalyzes the disulfide bond formation in nascent proteins and accounts for balancing redox status of the environment [27]. Notably, the oxidizing environment of ER lumen contains high ratio of oxidized to reduced glutathione (GSSG/ GSH) for the synthesis of disulfide bonds during protein folding [3]. Concomitantly, cellular redox status reflects the balance between oxidized to reduced glutathione (GSSG/ GSH) and protein disulfide bond synthesis [28]. The consequences of ER stress induced oxidative stress has detrimental effect on the stored Ca2+ ions within the lumen of ER [29]. The import and export of Ca2+ to the ER has been regulated by sarcoendoplasmic reticulum Ca2+ transport ATPase (SERCA) members and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) members respectively [29]. An earlier report states that, the reduced GSH induced oxidative stress triggers release of Ca2+ from ER through inositol trisphosphate receptor (IP3R) [30].
In fact, excessive release ofCa2+ to cytosol amplify the stress to mitochondria that leads to production of ROS through multiple mechanisms (Figure 1). Indeed, a fraction of the ER interacts with mitochondria known as mitochondria-associated ER membranes (MAMs) serves as a hotspots for the cross-talk between theses organelles [31]. It has been reported that, 5% to 20% fraction of mitochondria physically located to the ER serves as an exchange site ofCa2+ between ER to mitochondria through multiple channels [32]. In addition, the leakedCa2+ from the ER via inositol triphosphate receptors (IP3R) accumulates and rapidly internalized within t e h mitochondria to generat excessive ROS, leading to alteration in mitochondrial membrane potential and mitochondrial damage [33, 34]. Accumulation ofCa2+ within the mitochondria produces superoxide and creates oxidative stress [35]. In addition, calcium overload to mitochondria triggers mitochondrial dependent apoptotic instigation and lead to cell death [36].In a feedback manner the mitochondrial Na+/Ca2+ exchanger (mNCX) releases some Ca2+ that taken up by the sarco- endoplasmic reticulum Ca2+-ATPase (SERCA) channel resides to ER [37, 38, 39].
Figure 1: Oxidative regulation in Mitochondria and ER. Interface of mitochondria and ER (MAMs) shares an important site for the exchange of biomolecules and perturbation in balance leads to apoptotic cell death.
Moreover, the Ca2+ regulates opening if IP3R in a biphasic manner; at low concentration Ca2+ stimulates IP3R, whereas at high level Ca2+ inhibits ER Ca2+ release [40]. Interestingly, the mitochondrial associated protein such as B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax/Bak) and the Bcl-2- interacting killer (BIK) may enhance Ca2+ transfer from ER to mitochondria leading to apoptosis via release of cytochrome c [12, 41, 42, 43]. Indeed, levels of ROS can also tune Ca2+ release from ER; for instance, ROS at nanomolar concentrations enhances Ca2+ release through calmodulin-sensitized ryanodine receptor (RyR) whereas at micromolar concentrations ROS inhibits the function of calmodulin and restricts RyR activity [40]. Conversely, in a feedback mechanism the ROS originating from mitochondria triggers calcium release from ER to sensitize the calcium release channel at the ER membrane that reinforces ROS production in both organelles [45]. In addition, mitochondrial ROS can cross-influence ER functioning through dictating the cellular disulfide bond formation [46].
ER-stress coping: UPR and Ca2+ signaling:
In order to cope with exacerbated ER stress cells, activate unfolded protein response (UPR) through a signaling network consists of proteins IRE1α, PERK, and ATF6α [47, 48]. The IRE1α, PERK, and ATF6α are ER-spanning transmembrane proteins that through the ER chaperone BiP [47, 49].BiP has been reported in Ca2+ transport across the ER to the mitochondria through an ER membrane sigma-1 receptor (Sig-1R) [50]. Moreover, in response to ER stress IRE1α autophosphorylates and activated to splice X-box binding protein 1 (XBP1) mRNA that subsequently translocated into to the nucleus to control transcriptional machinery related to ER quality control and ERAD pathway [51]. Similar to IRE1α, PERK activation initiates phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) to attenuates further translation machinery through activating Transcription Factor 4 (ATF4) and restore ER stress [52]. In addition, the activation of ATF6α generates the cleaved fragment of ATF6α that translocate to the nucleus and induces transcriptional activation of UPR associated genes involved in IREα and PERK signaling [53- 55]. Importantly, ER Ca2+ imbalance can also activate PERK arm of UPR by interacting with calcineurin that act as a conserved Ca2+ activated phosphatase [56,57]. An earlier report shown that, ER stress subjected to PERK-knockout mouse fibroblasts leads to induction of ROS levels [28]. In contrast, UPR signaling network has been shown in regulation of ER–mitochondrial Ca2+ transfer through mitochondrial associated ubiquitin ligase Parkin [58, 59]. In addition, the pro-apoptotic component of UPR such as CHOP may induce oxidative stress in response to ER stress. Notably, the CHOP target gene ERO1L contributes in disulfide bond formation during protein synthesis. In addition, ERO1α regulates IP3R mediated Ca2+ leakage from the ER through interacting with kinase MKII and leading to the execution of oxidative stress induced apoptosis [60, 61, 62].
Conclusion
The mitochondrial and ER associated membranes serves as regulatory sites for several biochemical dynamics relevant to human disease and remained elusive to further dissect. Failure to resolve cellular stress leads to results in pathological abnormalities and leading to cell death. For instance, mitochondria undergo continuous biochemical arrangement during mitochondrial respiration and generates several reactive oxygen species (ROS) molecules, however improper resolution of ROS molecules leads to oxidative stress within cells. Similarly, ER lumen is the prime site for protein synthesis and folding, but immature protein folding within ER leads to transient ER stress that may results in cell death. In addition, mitochondria and ER serves as a storage site for calcium ions (Ca2+) that play a key role in several pathologies. Notably, the transport channels such as IP3 and mNCX at the interface of ER and mitochondria respectively balance the excessive Ca2+ concentration in a feedback mechanism to minimize oxidative imbalance within cells. Importantly, the interface of mitochondria and endoplasmic reticulum (ER) are critical to cell functions including redox regulation, calcium signaling and lipid biosynthesis. Notably UPR signaling partially regulates the (Ca2+) imbalance to the ER and reduce oxidative burden. Thus, a healthy tuning between mitochondria and ER determines the fate of oxidative stress resolution in the cells.
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Biomed Grid | Intima-Media Thickness Measurement in Children; Techniques and Reference Values
Introduction
Intima-media thickness (IMT) is a subclinical marker of vascular disease in obese children, diabetics and with other conditions predisposing to cardiovascular diseases. It is defined as the arterial wall thickness (frequently the common carotid artery) from the lumen-intima interface to the media-adventitia interface. Before conventional clinical and radiological manifestations (atherosclerotic plaque on Doppler ultrasound), the vascular disease is under diagnosed [1, 2, 3, 4]. There are several techniques for measuring IMT and reference values in children are rare. These references depend on the technique (B mode or radio-frequency ultrasound) and on how IMT is measured (manually, semi-automatic or automatic) [5]. The IMT values across age in the same pediatric population and according to the different techniques do not exist in the literature. It is therefore difficult today to decide whether an IMT value in an obese child is normal or high.
IMT and Obesity
Atherosclerosis begins in childhood, especially in children with risk factors. These risk factors include and are not limited to obesity, dyslipidemia, diabetes, genetic factors, and high fat and sugar diet [1, 3]. Obese children tend to remain obese as adults. The first stage of the disease is endothelial dysfunction followed by impaired vascular vasodilation and increased intimamedia thickness, fat deposition and ultimately the formation of atherosclerotic plaque. IMT measurement is one of the radiological examinations that can assess the early manifestation of the disease before the formation of atherosclerotic plaques. IMT has been shown to be increased in children with risk factors [6].
Radiological Imaging of IMT
Since the variability of this thickness depends on the measurement site, the Mannheim consensus has been established [7]. In addition, the Association of European Pediatric Cardiologists (AEPC) has also issued recommendations to standardize the measurement of IMT in the literature [8]. Even with these guidelines, the measurement can still be carried out according to conventional B mode or radiofrequency ultrasound. The radio-frequency method analyzes raw ultrasound data before post-processing. Theoretically this measurement should be more precise. The measurement can be done manually by placing the markers on the screen or semiautomatically by indicating to the software where to measure the IMT. An example of the latter is the semi-automatic method which gives the average of sequential measurements along the vessel wall on a line drawn by the operator in front of the desired vascular wall segment. This line could be of variable length. The software will calculate an average of the thickness along the indicated line (Figure 1).
Figure 1: Longitudinal ultrasound view of the common carotid artery. A) shows the intima-media thickness. B) shows the green line drawn by the operator indicating to the automated software where the intima-media thickness is desired to be measured. The average thickness between the yellow and the pink lines is given automatically by the software. I.Q. is Quality Index indication the percentage of measurements taken into account to calculate the average thickness. The desired Q.I. is at least 0.50.
Reference Charts
Doyon et al have published a multicenter study giving normal values of IMT and vascular distensibility of the carotid artery in children. However, this is a manual measurement according to B mode ultrasound [9]. Knowing that IMT is in the range of 0.3 to 0.5 mm, manual measurement has a lot of room for error. In addition, in children the techniques according to B mode and radio frequency do not correlate [5]. Therefore, we will not be able to extrapolate from these reference tables to decide on a measurement obtained using the radio-frequency method. Jourdan et al did a similar study using B mode ultrasound [10]. Engelen et al. [11] on the other hand, measured according to radio-frequency method [11]. However, their population begins at the age of 15. In summary, there are no studies in pediatrics that used a semi-automatic non-manual method for measuring IMT according to B mode and radio-frequency methods in children under 15 years of age. In the absence of these reference tables, it is difficult to diagnose early vascular disease in children at risk.
Clinical Perspectives
Studies of healthy subjects in pediatrics have shown that there is no difference in the measurement of IMT between the right and left sides with regard to the common carotid artery. There is possibly a role of puberty hormones on the cardiovascular system and IMT as evidenced by increased IMT and reduced elasticity after puberty [12]. Several pediatric pathologies affect IMT. These include kidney failure [13], kidney and liver transplantation, Kawasaki disease, diabetes, genetic predisposition, chronic systemic inflammation and dyslipidemia. Obesity, lifestyle and lack of exercise are associated with increased IMT [14] and remain the most common causes of cardiovascular diseases. For the time being, IMT in children is measured mainly in research.
Conclusion
Age has a very important role in the arterial aging process. Blood pressure also changes with age. Hence, extrapolation from the adult IMT reference tables would not be accurate in pediatrics. For all these reasons, it is important to correlate with custom technique specific reference charts taking into account the age range of children [15, 16].
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Biomed Grid | The Dream as Foetus
Introduction
Nanshe, the Babylonian Goddess of dream interpretation is at the same time the Goddess of water and fertility; “her symbol a vessel with water and a fish in it, symbolising her gravid womb”. [1] Indeed, like a fish, the foetus swims in the amniotic fluid of the womb, and like a foetus, the dream germinates in an internal world.
There is no better support of such a view than a woman’s dream announcing her pregnancy by means of a baby floating in her swimming pool. Yet a more frequent sign of her pregnancy is a dream of fish. Here, like always, the context is of prime importance, for a fish in a particular scenario may simply stand for the penis, as in a dream of a woman, for instance, where she is grasping a fish. On the other hand, a dream of an aquarium full of fish would be more certain to foretell a pregnancy. Such a dream will not only alert the woman to the fact that she is pregnant but will at the same time also indicate whether or not the occasion would be welcome. The latter will obviously become evident in her reaction to her dream.
I know of a case where a young woman, just recently married, recalled just such a dream. She described the beauty of the fish in glowing terms, relishing their colours and exquisite patterns. The upshot was, of course, equal excitement at the arrival of a healthy boy.
The symbol for Nanshe testifies to the Babylonians’ perfect understanding of the pathway and function of the dream. It clearly makes evident that the land’s priesthood understood that the dream was the germ for corresponding waking situations and events. It explains in one simple picture that the dream engenders an internal world, which like a germ is a rudimentary and compact potential that will develop to its concisely described fullness upon passing into the external, waking world. In other words, just as a foetus is more of an indication what will be, so the dream in its elemental form merely suggests what the fully matured waking counterpart might look like and how its condensed drama might unfold.
It is mostly because of the dream’s ‘foetal form’ and compactness that the uninitiated finds the dream a most bewildering experience. “I’ve had a weird dream” is a common exclamation. The nocturnal dream experience is found to be such because it usually does not align with ‘common sense’ scenarios and sequences. Situations look bizarre and surreal. Surreal is, of course, a most apt term for the dream because it is ‘sub waking reality’. It underlies all of what is happening on the waking surface. This sub-fact, acknowledged in the term ‘surreal’ ought to make us stop and think about the role of the dream in that position. Does it not intimate that the dream might well be the foundation of the waking experience?
Generally, the circumstance that the dream is in a sub position earns itself an inferior status. It is looked down upon and considered something of lesser importance than what is above it. It is like forgetting that the ground we stand on is also the substance and framework of our physical bodies. “Earth produces food”, so we say, but more often than not we call it ‘dirt’, forgetting that we eat it in its transmuted state, so nourishing and building our very body.
This is often the stance we assume when we are confronted with the dream experience. “Just a bit of weird surrealism of no consequence”, we might remark. But when in time we realise that nightmares cease at the moment we have understood them, it will dawn on us that dreams must have a wider influence on the waking process than what the first encounter with a nightmare permits us to see. Indeed, upon reflection we may even get to appreciate the fact that such disturbing dreams not only affect our wellbeing when they occur, but that they also must have a distinct directing function since they ultimately lead to the termination of their traumatic disturbances.
The classic example for this fact is the nightmare Michael Barnsley had. It began in his student days and only stopped twenty years later when a more begnine dream showed him that the task the nightmare had assigned to him was to invent image compression softwareM. Curiously enough, his nightmare had withheld its ultimate aim and kept the circuitry for the software secret till the day of the resolving dream. [2] As it turned out later, it had held its aim back because a certain computer program had to be devised first, without which the resolving dream could not have been understood and practically exploited.
Barnsley’s nightmare and its resolution in a new invention not only sheds light on the function of nightmares, but also demonstrates that the dream state is in charge of the inventive process; that it knows where it is heading even when the dreamer himself has no inkling of it. There is no better and more convincing case that demonstrates the guiding principle of the dream. (For a full exposition of this theme see my essay online, “To what extent does the dream influence the creative process? IJoDR, University of Heidelberg, Germany)
In light of this, we are compelled to see the source of Nanshe’s symbolic fishbowl as an invention of the dream itself. In other words, it was the dream that likened its function to a vessel containing a fish, thus symbolising the more organic situation of a pregnant womb. From this we must conclude that dreaming is a thoroughly feminine matter. Indeed, the sleeping body of the dreamer becomes the womb itself in which the foetal dream world unfolds, and as the sleeper awakes, the birthing process begins, bringing forth a completely new world, perfectly analogous to the birth of a completely new being.
For some, this comparison may seem somewhat contrived, but when we reflect on the waking process a little, we soon see that it is not at all as abiding as it generally impresses us. Matter, as constant as it may seem to our eyes is in permanent flux much like a river, and so, upon waking, we can never step into the same world twice, just as we never can step into the same river twice, or more precisely, into the same mass of water. In other words, as suggested, every awaking is a new birth of the ‘nocturnal foetus’ that begins its daytime unravelling with the end of the last dream.
Nanshe’s fishbowl has its counter part in the Vesica Piscis, ‘vessel of the fish’. Its design is that of a pointed oval resulting from the intersection of two circles. It represents the vulva of various Mother Goddesses including that of Mary, Mother of Christ. It is called the ‘vessel of the fish’ because it is said that the vagina has a ‘fishy smell’, which description became, in fact, the name of the Hindu Goddess Kali in her aspect as the virgin [3].
“The Vesica Piscis was an unequivocally genital sign of the sheila- na-gig figures of old Irish churches. The squatting naked Goddess displayed her vulva as a vesica as did the temple-door images of Kali in India” [4]. So, while Nanshe’s fishbowl represents the gravid womb, the Vesica Piscis stands for the birthing process, the departure of the newly formed being from the inner world in order to grow in the outer realm. And parallel to this, the same vessel stands for the emergence of the dream in order to be transformed to a waking reality.
The worship of Nanshe continued well into the Christian era, overlapping with it for a considerable time. So it is not surprising that Christians adopted the Vesica Piscis for their own icon, especially in view of the fact that the Greek acrostic for ‘Jesus Christ, Son of God the Saviour’ resulted in the word FISH.
As this renewed icon, where a transcendental entity is conceived in a virgin’s womb in order to become a being of the waking world, the vesica is given a further dimension. It reminds us of the fact that biblical dream interpretation maintains that dreams are unalterable instructions from God [5] Just how this plot of a Saviour in fish gestalt is engrained in our psyche, becomes clearly evident in the fact that the Avatar and Saviour of Vishnu is not only a fish, but emerges from the mouth of a fish [6]
In this context it is of interest to discover that the ancient Egyptians said “Abtu, the Abyss, was a ‘fish that swallowed the penis of Osiris’, but that abyss was also ‘The Fish of Isis’, therefore a sexual metaphor” [7] Such erotic mythology of the ancients recalls the fact that for Freud the meaning of the dream was not exhausted until it revealed its sexual sense. For Jung, on the other hand, the critical meaning was in its spiritual associations. The views of both interpreters taken together bring back the full perspective of the ancients. In other words, their understanding of intercourse between man and woman was at the same time also intercourse between heaven and earth. This is not so surprising when we remember that the mystics were not at all averse to speak of the love for God in erotic terms.
With this the cycle and function of the Vesica Piscis is complete. Conception and birth are exposed and with it a further parallel to the dream cycle is made manifest. This is the fact that dreaming and waking constitute a round like breathing in and out. While on the one hand the dream is transmitting new adventures from beyond, some of the waking experiences are fed back into the womb of dreaming. This is parallel to the creative formula of the Mandelbrot set where infinite iteration occurs. Quite generally, the dreaming/ waking round agrees with Einstein’s formula that describes energy converting to matter, or dream energy to waking matter.
If we now return to the Egyptian Abyss or the Fish of Isis, we find that Nanshe and her fishbowl are also associated with an Abyss or Abaton. “Also called a mundus or earth-womb, the Abaton was a real pit, standard equipment in a pagan temple. Those who entered it to ‘incubate’, or to sleep overnight in magical imitation of the incubatory sleep in the womb, were thought to be visited by an ‘incubus’ or spirit who brought prophetic dreams” [8].
Just as biblical authors saw the dream being transported by an angel, so the priests of Nanshe conceived of the dream’s vehicle as a spirit. In both cases the dream is seen to have originated in a transcendental realm, while the earth womb pictures an augmented womb, or an enlarged sleeping body made from earth.
In our non-theistic psychology this pit has become the Unconscious, which Freud saw as the receptacle of our total destiny. As he states in the final chapter of his ‘Psychopathology’, “all examples imply that both the conscious and unconscious life is determined absolutely” [9].
Priests of Nanshe would agree with this without reservation. Curiously enough, Freud never believed that the dream could give us knowledge of the future, insisting that it was, instead, giving us knowledge of the past [10] It makes us wonder why he never sought to reconcile his findings of his research in psychopathology with that of dream analysis of which he said that it was the Royal Road to the Unconscious. Surely, if psychopathology shows that all is determined, the Royal Road to the Unconscious must inevitably lead us to the same conclusion, from which follows that dreams must be messengers of the future.
Indeed, just as the foetus embodies a future human being, so the dream encapsulates what is to occur in waking. While current science would accept this only in as much as the DNA of the foetus determines the future evolution of the physique and mind, but not the life path, our forebears of Nanshe’s days would insist that the dream outlined both the subject’s constitution and its concomitant environment.
A good example for this is Gilgamesh’s dream which promised victory over Humbaba, the monster guardian of the Lebanese forest where he and his companion Enkidu were to cut the cedar for the royal palace: “In my second dream again the mountain fell, it struck me and caught my feet from under me. Then came an intolerable light blazing out, and in it was one whose grace and his beauty were greater than the beauty of this world. He pulled me from under the mountain, he gave me water to drink and my heart was comforted, and he set my feet on the ground.” [11]
The mountain that fell on Gilgamesh’s feet was, of course, a reference to the mountainous monster guardian, while the bright light Saviour was Shamash, the Sun God. As for the rest, this dream story is also its interpretation. Enkidu, Gilgamesh’s companion rightly confirms this when he says: “Your dream is good, your dream is excellent, the mountain which you saw is Humbaba. Now surely, we will seize and kill him, and throw his body down as the mountain fell on the plain”. (Ibid)
This example demonstrates that the dream in the view of our ancestors predicted complete events where there was no room for ‘free will’. But this example demonstrates as well that the interpreters of Nanshe’s days also understood what I have called associative manifestation or ego-transference. The present case in question is the inversion of Humbaba falling on Gilgamesh’s feet in the dream; while in waking Humbaba’s body is thrown down on the plain by Gilgamesh (and his companion).
This example alone is sufficient evidence showing that the Sumerians and their Babylonian heirs understood the function and structure of the dream better than most present day dream researchers do. Of course, there are standardised dream dictionaries of Babylonian extraction inherited by the Roman interpreter Artemidorus, for instance, which indicate that their fixed meanings listed could be misleading. But if we can go by Artemidorus himself, it quickly becomes evident that a good diviner took into account the current, personal circumstances of the dreamer before he ventured a prediction, and thus individualised the standard meaning listed [12] Another thing that needs to be considered is a certain amount of corruption occurring in the course of time.
One aspect current dream researchers often neglect completely is the close connection between dreaming of the future and the Near Death Experience. Most authors of NDE reports maintain that aftereffects of NDE episodes feature an increase in dreams, or to put this more correctly, an increase of dream recalls. Yet a further aftereffect is that the NDE subjects find to their astonishment that their dreams are all of the future. Part of this discovery is that they now have the gift of clairvoyance and precognition [13] Both of these phenomena are, of course, rooted in dreams. Thus, clairvoyance and precognition, which are principally identical occurrences, are simply cryptomnesic recalls of dreams, all of which are inevitably of the future [14]
Earlier on we have seen that Nanshe’s fishbowl was associated with an earth womb in which incubation of dreams was sought. For Nanshe’s priests it served that very purpose, but for novices it was also an initiation ritual of “descent into the ‘pit’, a symbolic death and resurrection” [15] Obviously its purpose was to endow the novices with the gift of interpreting the dreams of other men and women.
We discover here an interesting parallel between the Near Death Experience and that of the death and resurrection ritual. In one case we note that there is an increase of dreams that moreover see the future, while in the other we find that the ‘resurrected’ candidates have now the ability of interpreting the future consequences of dreams.
As well as that, we note that both the NDE and the initiatory ritual clearly end in rebirth, a second emerging from the womb that transforms the ‘foetus’ and gives it a new ability in life. In fact, the connections between the two situations are close enough for us to suspect that the death ritual could well have its origin in a Near Death Experience.
Certainly, being struck down by a severe and life-threatening illness is often part of a spontaneous initiation into Shamanism. For the ‘born Shaman’ such illness is transformative. He or she becomes a new ‘foetus’ as it were, one that will grow into a completely new being with visionary and healing qualities, diminished ego and a totally new world perspective. The parallels between the NDE and being struck down by such life-changing illness are clearly apparent. Indeed, as an author on Shamanism once remarked: “For Shamans, death shows up as a spiritual being, an ever present spirit teacher- guide whom you get to know, and eventually integrate with. It is an on-going relationship” [16]
Of interest in connection with the resurrection from a pit is the lore of Kundalini. The root of this word is Kunda, a sacrificial fireplace in form of a pit [17] Kun is the Earth; the K refers to a hollow, an aperture, which is often symbolised by a yoni or an open vagina. In light of this it would seem that Kunda is the root of the English vulgarism of ‘cunt’. Certainly, the vagina as pit of transformation leads us directly to the womb where the foetus awaits its birth.
In chakra lore, Kundalini is said to be at the base of the spine in the genital area. It is pictured as a serpent, which is, of course, a representation of the life force; of energy that gradually spirals along the spine towards higher chakra centres. Literally, chakra means wheel or circle. In chakra lore it is seen as a centre of whirling energy. The higher Kundalini rises, the closer is the individual to enlightenment.
The top chakra is called Sahasrara or crown chakra, symbolised as a thousand-petalled lotus. It is also associated with death. And indeed, enlightenment is a particular death, the death of the ego. But it is also concomitant with the death of the body. There is no better evidence of this than Pam Reynolds’ artificially induced death from which she emerged after an hour alive and well. As her body reached hypothermic arrest, her etheric or soul left her body through the top of the skull, so affirming the ancient Hindu wisdom that sees Sahasrara as portal to the transcendental world [18]. There are seven chakra centres in all. But since Sahasrara is the portal to the transcendental world and thus well above the six other centres, much as the Sun is among the seven classic planets (Sun, Moon, Mercury, Venus, Mars, Jupiter and Saturn) it is truer to life to say that there are six plus one chakras rather than seven. [19] Indeed, if we consider the diagram of the ‘Seed of Life’, the six plus one in place of seven is graphically enhanced. This diagram is constructed by drawing a circle, which represents the realm of creation. Within it are drawn seven overlapping circles with the same diameter. Six of them are regularly spaced within the seventh, producing a rosette with eighteen lens shaped petals: six smaller ones inside and twelve larger ones outside. (Ibid) Each of these lens shapes can quite rightly be regarded as a Vesica Piscis, a birth-giving yoni.
Incidentally, this six plus one relationship alerts us to the fact that the biblical creation story with six days of activity and one day of rest was not to be understood literally, but instead, in terms of mathematical ratios, the ratios of life. But above all, it explains the special position of Sahasrara in Kundalini lore.
As Kundalini reaches Sahasrara we are face to face with the microcosmic counterpart of the annihilation of the old world making way for a new creation. Death and rebirth in the Abaton is its parallel. And indeed, we need not dig a pit in the earth at all in order to descend in it for the purpose of life changing transformation, for the human body is a ready-made Abaton in which death and rebirth is constantly re-enacted as immersion in sleep endowed with dreaming that comes to fruition in its subsequent waking manifestation.
Certainly, apart from daily little deaths and revivals that enhance our psyche, there are on occasions transformations of such momentous proportions that they stand out like a rite of passage with effects that are in the league of NDEs. One example is the encounter of transcendental light between dreams. In other words, it is possible to ‘wake up’ to the cosmic consciousness that underlies our waking and dream states, which is normally hidden by overlaid dream and waking imagery.
Western dream research is not aware of this, so far as I can assess. On the other hand, Hinduism has always been cognisant of it. It claims that in deep sleep, which for the West is delta sleep, it is possible to experience this cosmic consciousness in form of radiant light, the same that NDE subjects report after they have passed through the realm of darkness.
I can vouch for the veracity of this claim thanks to personal experience. In a dream I was led to a temple in which a tiny yogi in padmasana reached out to me as I approached him. He touched my forehead with his finger. I swooned instantly. All thought and imagery vanished, making way for blissful, pure and amazingly bright light.
It can also happen that the dream itself will provide the Abaton in which an oneiric death and resurrection will take place. In such a dream I once was whisked into the womb of Persephone who was emerging from the ground as the Goddess of spring. In her womb, which was a massive, dark hall I floated in frozen angst just below the ceiling. Soon, a fluorescent disk and a fluorescent swastika appeared in the pitch dark. Not long after that I looked down and discovered a TV set that screened a cartoon-like figure whose eyes were bulging out of their socket like tennis balls. It reminded me of a Zen painting of Daruma. He held a huge sceptre in his hand that was constructed from the axel and wheel of a railway car.
As I gradually woke from this nightmarish dream, I thought I was going to sleep because waking reality seemed so insipid compared with the dream. But, more importantly, I found that this experience had endowed me with a better understanding of my dreams, allowing me to see how the dream story unravelled itself in terms of corresponding waking facts. At the same time, I also recognised that the power of Nanshe is still as present today as it was thousands of years ago. After all, time is an illusion as the mysterium coniunctions, where past, present and the future are all experienced as one single event demonstrated to Carl Jung [20].
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Biomed Grid | BODIPY Dyes for Biomedical Applications: Recent Advances
Introduction
BODIPYs are fluorescent dyes with attention-grabbing properties such as high fluorescence quantum yields (0.6-1.0) and absorption coefficients (60000-80000M-1cm-1), comparatively sharp emission spectra and straightforward functionalization. In addition, they have low sensitivity to each solvent polarity and pH, additionally as wonderful photostability [1, 2, 3, 4]. Because of these wonderful properties, BODIPY dyes are utilized in totally different application areas, particularly wide applied in medical specialty areas like biolabeling, bioimaging, PDT etc. [5, 6, 7]. This mini review will focus on recent studies about BODIPY dyes in biomedical applications.
In recent years, BODIPY derivatives have been generally applied as fluorescent probes for bioimaging [8, 9, [10, 11, 12>4] and as Photosensitizers (PSs) for PDT [13, 14, [15, 16, 17]. Cheng et al. [18] examined the switching mechanism of a Near-Infrared (NIR) aza-BODPIY-based fluorescent dye [18] and its potential usages a Switchable Fluorescent Probe (SFP) in several formats, together with wash-free cell imaging, in vivo tissue imaging, temperature sensing, and tissue Ultrasound- Switchable Fluorescent probe (USF) imaging [19] . These results indicated that this aza-BODIPY based fluorescent dye is highly environment-sensitive dye and showed very weak fluorescence in aqueous solution whereas strong fluorescence in non-polar and high viscosity media. Because of the unique switching property of this dye, it has been successfully applied in wash-free live-cell imaging, fluorescence imaging in live animals, temperature sensing and USF imaging.
Meares et al. [20] synthesized a series of Polyethylene Glycol (PEG) substituted BODIPY-chlorin/bacterochlorin arrays. They examined optical and fluorescence properties of these arrays in organic solvents and aqueous surfactants. They reported two series of arrays: The first series of arrays contains BODIPYs with PEG substituents attached to the boron, whereas in the second series, PEG substituents are attached to the aryl at the meso positions of BODIPY. For both series of arrays, excitation of BODIPY at 500nm results in efficient energy transfer to and bright emission of hydro porphyrin in the deep red (640-660nm) or near-infrared (740- 760nm) spectral windows. These arrays possess large effective Stokes shift (115-260nm), multiple excitation wavelengths, and narrow, tunable deep red/near-IR fluorescence in aqueous surfactants and reported as promising candidates for a variety of biomedical-related applications [20] .
Ogata et al. [21] developed two types of activatable NIR fluorophores derived from bacteriochlorin: chlorin-bacteriochlorin energy transfer dyads and BODIPY-bacteriochlorin energy-transfer dyads. They characterized these fluorophores by multiple narrow excitation bands with comparatively strong emission in NIR. PEG chains were added to enhance the hydrophilicity and decrease aggregation, resulting in a viable in vivo imaging agent that detected cancer with high sensitivity in murine models. They reported that these fluorophores supply potential for multicolored fluorescence imaging and are a promising tool for susceptible medicine [21] .
Belali et al. [22] have improved a straightforward and efficient way to prepare extremely water-soluble neutral BODIPY formulations by cross-linking them in chitosan-based 3D hydrogel networks. 3,5-diformyl-BODIPY is used as a crosslinker to generate a new class of chitosan-based hydrogels with Fluorescence Resonance Energy Transfer (FRET) dynamics and good solubility in water. The dynamic character of the hydrogel was approved with some rheological, macroscopic and microscopic self-healing tests. The fluorescence lifetime was found to increase in aqueous solution of the BODIPY-chitosan hydrogel compared to the 3,5-diformyl-BODIPY monomer. Experimental results such as red-shift and decreased intensity of the emission spectrum of highly dye-concentrated hydrogel in comparison to dilute hydrogels, together with changes in the fluorescence lifetime of the hydrogel at different concentration of dyes, suggest that the BODIPY-chitosan hydrogels fluorescence dynamics obeyed FRET. They reported that their approach of creating fluorescent hydrogels creates opportunities for new optical polymer designs and uses for biomedical applications such as implantable fluorescent hydrogels [22] .
A series of novel fluorescent BODIPY-anionic boron cluster conjugates bearing [B12H12]2−, [3,3′-Co(1,2-C2B9H11)2]−, and [3,3′-Fe(1,2-C2B9H11)2]−anions have been synthesized from meso- (4-hydroxyphenyl) substituted BODIPY by Chaari et al. [23] .The syntheses and complete characterization of these compounds were described along with the photophysical properties. Furthermore, the cytotoxicity of the conjugates has been evaluated and their cellular uptake by HeLa cells have been compared by flow cytometry in order to assess their potential as fluorescent dyes for cell tracking. Bioimages of HeLa cells incubated with compounds have been also analyzed by confocal laser microscopy and photoluminescence properties of them have been investigated. Linking anionic boron clusters to the BODIPY does not alter significantly the luminescent properties of the final fluorophores, showing all of them similar emission fluorescent quantum yields (3-6%). According to the literature, these are the first BODIPY-anionic boron cluster conjugates developed as fluorescent dyes aiming at prospective biomedical applications [23] .
Chen et al. [24] investigated the molecular interactions of two 2,6-diiodo-BODIPY derivatives with Human Serum Albumin (HSA) using the combination of experimental and computational studies. Their results demonstrated that the binding to HSA boosted the photodynamic efficiencies of BODIPYs. The BODIPY/HSA complexes exhibited notably enhanced water solubility and singlet oxygen generation efficiency with respect to the BODIPY alone. Additionally, molecular docking, molecular dynamics simulations, and binding free energy calculations provided the structural and energetic insights into the binding mechanism of BODIPY-based derivatives to HSA. According to these results they reported that the formation of complexes of BODIPYs with HSA is a promising strategy for the design and development of BODIPY-based PSs with improved bioavailability and biocompatibility for cancer therapeutic applications [24] .
Two PEGylated BODIPY compounds which could self-assemble into Nanoparticles (NPs) in aqueous media were synthesized via Passerini reaction by Zhu et al. [25]. Optical properties including FRET were studied in detail. These results showed that the obtained NPs possess good cytocompatibility and could be used for living cell imaging and effective PDT and shed light on one-pot synthesis of PEGylated fluorescent nanoparticles via multicomponent reaction for biomedical application [25] . Su et al. [26] developed a strategy to fabricate a kind of nanocomposite (CBNPs) via the noncovalent interactions between Carbon Dots (CDs) and BODIPY, by which the solubility and PDT effects of BODIPY were improved through the Fluorescence Resonance Energy Transfer (FRET) mechanism. The CBNPs exhibit good water solubility, excellent singlet oxygen quantum yield, and high biocompatibility and PDT efficiency. They declared that these compounds may have potential applications in biomedical fields and cancer treatment [26] .
Conclusion
According to all these recent studies, it was shown that great advances have been made in biomedical applications especially on living cell imaging and PDT, with using BODIPY dyes due to their excellent photophysical and photochemical properties. Soon, its waited for the studies with these dyes in biomedical applications are expected to potentially increase.
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Biomed Grid | Indisputable Proof about the Effects of Eye Rubbing on the Cornea, the Lens and the Globe
Introduction
As we herald the beginning of the new decade, we welcome a lot of innovations in the field of ophthalmology, especially the dawn of digitalization in the healthcare industry. And it is through one of these dynamic technologies, Magnetic Resonance Imaging, which saw prominence during the last two decades, that the myth about eye rubbing not influencing the development of keratoconus has been completely demystified and debunked. Dr. Damien Gatinel, Dr. Giullaume Debellumaniere and Dr. Julian Savatovsky of the Rothschild Foundation Hospital in France showed in You Tube in November 10, 2019 the actual footage of a healthy volunteer rubbing his eye while explicit digital images were being taken and recorded using MRI https://youtu.be/tM4z3MYZeNK.
The images were very impressive and revealed how the cornea, the lens, the globe and surrounding orbital structures were distorted by the seemingly innocuous gesture of rubbing the eyes. Eye rubbing is usually an attempt to get a slight sense of relief for the unpleasant condition afflicting the individual’s eye/eyes at that moment. Keratoconus is a thinning and deforming of the cornea. The importance of eye rubbing in the development of keratoconus has been underestimated, if not completely ignored by the medical community. Now, through the striking images captured in real time, there is indisputable proof that keratoconus is, without a doubt, a disease of friction. The propensity for individuals to develop the disease, I believe, depends on the congenital structure of the cornea the individual is born with, which answers the question on why some persons are prone to develop and progress into keratoconus while others don’t.
There are several types of eye rubbing:
Type 4 : Back of the hand rub
Type 5 : Shoulder rub
Type 6 : Face in the pillow rub
Several causes for chronic eye rubbing have been raised, namely, atopy/allergy, dry eye, contact lens wear, pollution, presence of foreign body, night compression, habit/compulsory and pleasant sensation, which leads to bio-cellular and bio-mechanical consequences.
a) Bio-cellular consequences (extracellular matrix degradation, impaired surface barrier, collagenase and enzymatic activity, cell mechanosensitivity, reduction of proteoglycans and microfibrils, and reduction of collagen maintenance)
b) Bio-mechanical consequences (shearing forces lead to viscoelastic and ground substance displacement, fiber and fibril slippage, transient deformation, corneal weakening reduction of collagen maintenance and central stromal thinning)
Both ultimately and unfortunately lead to corneal macroscopic deformity like keratoconus. In some instances, there is a disparity in the corneal distortion in both eyes, and the more affected eye would be the one the patient sleeps on.
Conclusion
So, this is a timely reminder to our colleagues to admonish their patients on the dangers of eye rubbing, unless they want to rub their eyes blind. It is, therefore, recommended that we educate our patients to be more vigilant in order to preserve their corneal integrity, prevent eye asymmetry and conserve their vision.
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