The effect of natural antioxidants, NAO and apocynin, on oxidative stress in the rat heart following LPS challenge.

Oxidative damage plays a key role in septic shock induced by lipopolysaccharide (LPS) which is known to enhance the formation of reactive oxygen species (ROS). In this study, biochemical parameters indicative of oxidative stress were tested in the rat heart following LPS challenge, with and without pretreatment with the antioxidants NAO (natural antioxidant) and apocynin. NAO is a natural antioxidant isolated and purified from spinach and its main components are flavonoids and coumaric acid derivatives. Treatment with LPS alone significantly (P<0.05) increased the malondialdehyde (MDA) level in heart, both in cytosolic and mitochondrial fractions by 1.5- and 2.4-fold, respectively, and in plasma (2.66 fold). In the heart homogenate, the level of hydroperoxides also increased significantly (P<0.05). In addition, LPS treatment significantly (P<0.05) increased NADPH oxidase activity in the heart microsomal fraction by approximately 10-fold compared to control. Pretreatment for 7 days with either apocynin or NAO prior to the LPS challenge significantly (P<0.05) improved rat survival, decreased MDA levels in both fractions and decreased microsomal NADPH-oxidase activity, compared to LPS alone. Catalase (CAT) activity slightly increased at 24 h post-LPS injection in LPS group and returned to the control level in the apocynin treated group. No meaningful changes were indicated for glutathione peroxidase activity among all the treatment groups. The activities of cytosolic and mitochondrial superoxide dismutase (SOD) enzymes significantly (P<0.05) increased approximately 20% in the LPS-treated group, compared to control. Apocynin significantly (P<0.05) decreased SOD level in the mitochondrial fraction with no effect on the cytosolic fraction; whereas, NAO had no important effect on SOD level in both fractions. The beneficial pretreatment effects of the antioxidants against oxidative stress in the rat heart presented in this study may suggest a potential chemopreventive effect of this compound in sepsis prevention.

Metabolic and ionic responses to global brain ischemia in the newborn dog in vivo: II. Post-natal age aspects.

The main difference between newborn and adult brains is expressed in the relative resistance of the newborn brain to oxygen deprivation. The aim of the present study was to examine the effect of global ischemia in canine puppies of three different ages on the metabolic, ionic and electrical activity of the brain and to study the basic mechanisms underlying the relative resistance of the newborn brain in ischemic episode. The puppies were divided into three age groups. The young group included 0-6-day-old puppies (n = 16), the intermediate group included 7-19-day-old puppies (n = 21), and the 'adult' group included puppies aged 20 days or more (n = 17). Statistical analysis of the results led to the following conclusions: The younger the puppy, the longer is the time until the occurrence of the secondary reflectance increase SRI (13.0 +/- 1.9 min vs. 5.3 +/- 0.5 min). The younger the puppy, the longer the time until onset of potassium leakage from the cells (0.9 +/- 0.1 min vs. 0.35 +/- 0.05 min) and the lower the amount of potassium leakage (9.6 +/- 2.8 mM vs. 21.7 +/- 4.8 mM). The rate of pumping of the potassium ions into the cells during the recovery stage was higher in the oldest group (1.2 +/- 0.2 mM min-1 vs. 0.38 +/- 0.1 mM min-1). It was possible to speculate that in the young puppies there is uncoupling of the oxidative phosphorylation from respiration and as a result, there is a lower, if any, rate of ATP synthesis. It seems that the newborn brain is able to cope with a decrease in available energy for a longer period of time. This is apparently due to differences in membrane characteristics and an improved ability to retain ionic equilibrium across both sides of the membrane.

The effect of hyperbaric hyperoxia on brain function in the newborn dog in vivo.

Age is a natural factor that has been found to significantly affect sensitivity to hyperbaric hyperoxia (HBO). Exposure to HBO may lead to damages in the energy metabolism of the brain cells. The aim of this study was to test the effect of HBO on the metabolic, hemodynamic and electrical activities in the newborn dog. The study was performed using one-day- to 70-day-old puppies. The puppies were placed in a pressure chamber. The pressure of pure O2 in the chamber was raised by 5 atmospheres (ATA, 75 psi = 6 ATA) within 10 min. The first biochemical change to take place during HBO was oxidation of mitochondrial NADH. The age of the puppy was found to affect the time to the initiation of seizures. In the puppies under the age of 24 days, the average time was 35.1 +/- 5.9 min. In the puppies of 24 days old and older, the average time was 5.1 +/- 0.8 min. In the younger puppies, there was a later occurrence of blood vessel contractions and a longer life span compared to the older puppies. The comparison between the puppies of different ages during exposure to HBO showed differences in the metabolic response, hemodynamic changes and electrical activity. These differences can partially explain the higher resistance in the younger puppies to HBO.

Hypovolemia in rats increases mortality rates following endotoxin administration.

The aim of the present study was to examine whether acute or chronic hypovolemia increase the mortality rates of rats following endotoxin injection. Another aim of this study was to examine whether this increase in sensitivity can be explained by increased leakage of endotoxin from the digestive tract to the blood stream. Chronic hypovolemia was caused by water deprivation for 8 days. Acute hypovolemia was caused by injection of sucrose (300mg/100g) or by hemorrhage of a volume of up to 2.1 ml/100g. The hypovolemia was examined by measuring the plasma volume using Evans Blue (EB). Endotoxin at various doses was injected to the hypovolemic rats and the lethality of the various treatments was examined. Acute and chronic hypovolemia caused a significant increase inthe mortality rates of rats injected IP with a dose of 1-1.5 mg/100g endotoxin. Endotoxin administration (6mg/100g) by drinking to hypovolemic rats did not cause death at all. In contradistinction, injection of galactoseamine to rats that underwent similar treatments caused 100% mortality. Endotoxin tolerance reduced the mortality rates following galactoseamine injection to the control level of administration of endotoxin without hypovolemia (p < 0.001). Injection of the antibiotic polymixin B following the galactoseamine injection also decreased mortality rates to 40% (p < 0.05). Examination of plasma endotoxin concentration exhibited a significant increase following administration of endotoxin by drinking to hypovolemic rats (p < 0.001) compared to rats that received the same amount of endotoxin but without hypovolemia. These results indicate that one possible explanation for the increased mortality rate of the hypovolemic rats after endotoxin injection is due to leakage of endotoxin from the digestive tract.

Effect of natural antioxidants and apocynin on LPS-induced endotoxemia in rabbit.

The objective of this study was to compare the prophylactic effects of the natural antioxidant from spinach (NAO) and apocynin, on the hepatic oxidative stress and liver damage induced by lipopolysaccharide (LPS). Male New Zealand rabbits were challenged with LPS with or without 8 days of antioxidant pretreatment. Pretreatment with NAO, but not apocynin, significantly (p < 0.05) decreased the levels of hydroperoxides and malondialdehyde (MDA) in the liver cytosolic fraction and the activity of NADPH oxidase-generated superoxide in the microsomal fraction, compared to LPS alone. The activity of glutathione peroxidase (G-POX) was significantly (p < 0.05) increased in the LPS-treated group, whereas treatment with NAO, but not apocynin, significantly (p < 0.05) decreased G-POX activity. Pretreatment with the same antioxidants had no significant effects on superoxide dismutase (SOD) activity, whereas an increased level of catalase (CAT) was obtained in all LPS-treated groups. TUNEL immunohistochemical staining in the LPS-treated animals indicated that there was no increase in apoptosis outside of necrotic foci. However, apoptotic hepatocytes were observed within areas of focal necrosis in animals exposed to LPS alone or LPS plus apocynin. Hepatocyte cell proliferation was tested by the proliferating-cell nuclear antigen (PCNA) tool, which indicated a proliferative effect in the LPS group, whereas the effect disappeared in the antioxidant-treated groups. The prophylactic effect of NAO on liver pathology and the significant decreases in lipid peroxidation products and NADPH oxidase activity suggest the use of NAO as an efficient strategy for treatment of endotoxemia.

Brain metabolic and ionic responses to systemic hypoxia in the newborn dog in vivo.

Newborns are less sensitive than adults to hypoxic/ischemic injury. However, research into the mechanism of the newborn's relative resistance to reduced brain oxygen levels is relatively scarce, and the time-scale for the disappearance of resistance is not known. The multiprobe assembly (MPA) has enabled us to examine the resistance of puppies at various ages to hypoxia via continuous, simultaneous, on-line measurement of various ionic, metabolic and electrical parameters from the cerebral cortex. The parameters measured included electrocorticogram (ECoG), direct current (DC) steady state potential, extracellular potassium (Ke+) and calcium ion concentrations and intra-mitochondrial Nicotine amide adenine dinucleotide NADH redox levels. These parameters were measured under various degrees of hypoxia (fraction of inspiration oxygen was between 0-10%) in 6-h-old to 24-week-old puppies (n = 44). Sensitivity to hypoxia increased with age, being expressed in the leakage of potassium ions out of the cells (0.3 +/- 0.07 mM in the younger puppies and 3.0 +/- 1.3 mM in the older puppies) following an increase in intra-mitochondrial NADH redox levels. Potassium ion (Ke+) leakage was apparently due to depleted energy stores resulting from an impairment in the balance between oxygen supply and demand. Although the overall effect was similar, the kinetics of these changes were much faster in the older puppies. The time to initial increase of extracellular K+ was 2.5 +/- 0.1 min in the younger puppies and 0.9 +/- 0.1 min in the older puppies. The time to maximum increase of NADH was 3.2 +/- 0.2 min in the younger puppies and 1.4 +/- 0.1 min in the older puppies. Our results indicate that the older puppies utilize the existing oxygen faster than the younger puppies. It is concluded that the increased resistance of newborn puppies to hypoxia is due to intrinsic properties of the brain itself, like the ability of the membrane to maintain ionic homeostasis.

Lipopolysaccharide-induced oxidative stress in the liver: comparison between rat and rabbit.

In the present study the effect of LPS on biochemical systems involved in radical formation and scavenging processes in tissues from rabbit (LPS-sensitive) and rat (LPS-resistant) was investigated. The results obtained show a significant enhancement in the endogenous antioxidative enzyme system in rats as a result of LPS injection. In rats, 24 h after LPS injection, glutathione peroxidase (G-POX) and superoxide dismutase (SOD) activities were increased by 60% and 120%, respectively, compared to the control. However, in rabbits the increase in these activities was relatively mild. Moreover, NADPH-oxidase activity, which produces superoxide radical, was increased about twofold in rabbit, 15 h following LPS injection. In rats, injection of LPS did not result in any significant changes in the activity of this enzyme. In rats, a decrease in malonaldehyde (MDA) levels appeared after injection of LPS, while in contradistinction, the peroxidative levels of lipids in the rabbit's liver were increased about 3-fold. Injection of D-galactosamine (Gal-N) in combination with LPS significantly increased the sensitivity of rats to LPS characterized by a significant increase in NADPH-oxidase activity. This study indicates that one possible mechanism (among others) that may explain the relative sensitivity of rabbits compared to rat, may be related to the increase in the production of reactive oxygen substances (ROS) which is not accompanied by a concomitant increase of the protective antioxidative enzymes. Furthermore, the relative resistance of the rat was found to be related to an increase in the activity of the protective antioxidative systems following administration of LPS.

Real time monitoring of rat liver energy state during ischemia.

Hepatic failure is one of the major problems developed during the posttransplantation period. A possible cause of hepatic failure is the prolonged ischemia induced during the implantation procedure. Hepatic ischemia leads to a reduction in oxygen supply, ATP level decline, liver metabolism impairment, and finally organ failure. The purpose of this study was to estimate the functional state of the liver by monitoring liver blood flow and the mitochondrial NADH redox state simultaneously and continuously during in situ liver ischemia followed by reperfusion. Measurements were performed using the multiprobe developed in our laboratory consisting of fibers for the measurement of relative liver blood flow (laser Doppler flowmetry) and mitochondrial redox state (NADH fluorescence). The experimental procedure included the temporary interruption of blood flow to the liver using three types of ischemia, hepatic artery occlusion, portal vein occlusion, and simultaneous occlusion of hepatic artery and portal vein, followed by a reperfusion period. These preliminary experiments showed a significant decrease in liver blood flow, following the three types of liver ischemia, and a significant increase in NADH levels. The probe used in this study incorporates the advantage of monitoring NADH and liver blood flow simultaneously and continuously from the same area on the surface of the liver. Since each of these two parameters is not calibrated in absolute units, the simultaneous monitoring decreases possible artifacts. Also, it will allow us to determine of the coupling between tissue blood flow and oxidative phosphorylation. It is believed that the measurements of respiratory chain dysfunction might predict organ viability in clinical organ transplantation situations. Using this probe may also help to decrease the variability in liver blood flow monitoring since liver blood flow monitoring is supported simultaneously with the mitochondrial redox state, which supplies the information on liver metabolic and functional state.

Reduction of risk following endotoxin injection in unilaterally nephrectomized rats.

In this study the consequences of unilateral nephrectomy on the survival rate of rats were examined under severe pathological conditions including injection of a lethal dose of endotoxin (exogenic LPS), cecal puncture (endogenic LPS), injection of LPS concomitantly with renal ischemia as well as a simultaneous injection of LPS and glycerol to the leg muscle in order to induce acute renal failure. Sham operated rats did not exhibit higher survival rates than the nephrectomized rats. In most cases there was even a decrease in the percent age rate of deaths in the nephrectomized rats. Improvement in the resistance of the nephrectomized rats to pathophysiological stress occurred when stress was induced immediately following nephrectomy as well as when stress was induced 30 days following nephrectomy. No significant differences were found in blood pressure, heart rate, hematocrit, rate of respiration and body temperature in the nephrectomized rats as compared to rats that did not undergo nephrectomy. A possible explanation for our results is that the solitary kidney excretes more LPS into the urine.

Unilateral renal ischemia reperfusion in the rat: effect of blood volume trapped in the kidney, sucrose infusion, and antioxidant treatments.

This study was carried out in order to examine whether the severity of acute renal failure observed during the four hours following a 45 min period of unilateral occlusion of the renal pedicle could be reduced by various treatments. These include intrarenal flush with saline immediately before the occlusion, by sucrose infusion immediately before reperfusion, or by injection of NAO (natural antioxidant) and vitamin E before the occlusion. After renal pedicle occlusion, creatinine levels increased to 165% of their pre-ischemic values. Urine flow, GFR, renal cortex blood flow and NADH decreased by 99%, 99%, 50% and 36%, respectively. A decrease in the Na and K reabsorption (15% and 32%, respectively) was also observed. Partial protection of renal function against ischemic damage was observed when kidney tissue remained blood-free, by exposing it to saline throughout the period of ischemia. Significant protection was observed after treatment with sucrose, vitamin E and NAO. This study demonstrates that it is possible to attenuate the injury to the ischemic kidney by inducing ischemia in a bloodless kidney, by inducing diuresis in the first phase of reperfusion, or by antioxidant treatment, such as vitamin E or NAO.

Increased sensitivity to bilateral nephrectomy in rat caused by endotoxemia.

In this study the possibility that the kidneys play a role in the removal of endotoxin from the blood was examined. This was carried out by an IP injection of endotoxin (at a dosage of 1 mg/100 gr) to rats that had undergone bilateral nephrectomy, and examining the times of death following various treatments. Furthermore, the rats underwent treatment for endotoxin tolerance or received treatment with the antioxidants superoxide dismutase (SOD) and natural antioxidant (NAO) or were injected with an inhibitor of the enzyme NOS (NGmethyl l-arginine 1-NMA). The endotoxin concentration in the plasma was also measured. The results of this study indicate the rats were more sensitive to bilateral nephrectomy in the presence of endotoxin. This increased sensitivity in the binephrectomized rat decreases following treatment inducing tolerance to endotoxin, or following treatment with an antioxidant or with the inhibitor of NOS. A possible explanation for the increased sensitivity following endotoxin injection is that it apparently results from an increased concentration of endotoxin in the blood due to the impossibility of removing the endotoxin via the kidneys. Therefore, endotoxin tolerance or abolishing the potential injuries that may be caused by endotoxin negate the sensitivity in the binephrectomized rats injected with endotoxin.

Reduction of risk following endotoxin injection in unilaterally nephrectomized rats.

In this study the consequences of unilateral nephrectomy on the survival rate of rats were examined under severe pathological conditions including injection of a lethal dose of endotoxin (exogenic LPS), cecal puncture (endogenic LPS), injection of LPS concomitantly with renal ischemia as well as a simultaneous injection of LPS and glycerol to the leg muscle in order to induce acute renal failure. Sham operated rats did not exhibit higher survival rates than the nephrectomized rats. In most cases there was even a decrease in the percent age rate of deaths in the nephrectomized rats. Improvement in the resistance of the nephrectomized rats to pathophysiological stress occurred when stress was induced immediately following nephrectomy as well as when stress was induced 30 days following nephrectomy. No significant differences were found in blood pressure, heart rate, hematocrit, rate of respiration and body temperature in the nephrectomized rats as compared to rats that did not undergo nephrectomy. A possible explanation for our results is that the solitary kidney excretes more LPS into the urine.

The effects of food and drink on the survival of binephrectomized rats.

Previous studies have shown that the survival time of rats following binephrectomy is between 48 and 120 hours. The aim of the present study was to examine the assumption that the eating and drinking protocol before and after binephrectomy affects the survival time following surgery in male, female and young rats. In this study we used protocols which included various combinations of eating and drinking or their prevention before and after binephrectomy. Survival time was determined by examining the kinetics of death following binephrectomy. Examination of the azotemia and physiological condition of the rat was performed by testing BUN, creatinine, plasma Na and K concentrations as well as the hematocrit. All rats that had eaten and drunk before and after binephrectomy, in various combinations, died within 48 hours. Rats that did not eat or drink before and after binephrectomy died within 72 hours (p < 0.001). Rats that did not eat before and after the binephrectomy but had free access to drink died within 90 hours of the surgery (p < 0.001). From this study it can be concluded that the eating and drinking protocol before and after binephrectomy affects the survival time of male, female and young rats. The effect varies between a survival time of 48 and 90 hours following binephrectomy. A possible explanation for this effect is that the eating and drinking protocol affects the rate of azotemia formation.

Antioxidants attenuate endotoxin-gentamicin induced acute renal failure in rats.

The synergistic mechanism by which endotoxin enhances the nephrotoxic potential of gentamicin is unknown. In this study, we attempted to shed light on this mechanism by injecting rats with endotoxin plus gentamicin. Renal injury was assessed by measuring creatinine, inulin and PAH clearance, NADH levels and electrolyte reabsorption, for 24 hr following this injection. Gentamicin alone (20 mg/100 g) induced no renal injury, while endotoxin without gentamicin (0.075 mg/100 g) induced mild injury. However, endotoxin plus gentamicin resulted in acute renal failure. In an attempt to halt the progressive renal dysfunction, the antioxidants NAO (5 mg/100 g), Vitamin E (0.2 mg/100 g per day) and dimethylthiourea (DMTU-50 mg/100 g) were administered, or early endotoxin tolerance was induced before injecting the rats with endotoxin plus gentamicin. The reduction in renal function was markedly slower in rats administered with antioxidants compared with untreated rats. Similar results were obtained with endotoxin tolerance. These data suggest that NAO, vitamin E, DMTU and endotoxin tolerance are potentially beneficial in arresting progressive renal damage associated with endotoxin plus gentamicin.

Composition and viscosity of interstitial fluid of rabbits.

Open-ended plastic tubes were used as capsules for obtaining interstitial fluid from rabbits. The capsule was a 2.5 cm long plastic tube with an inner diameter of 6 mm. Three small incisions were made in the dorsal mid-line of the anaesthetized rabbit; two capsules were inserted into each incision. A sample of capsular fluid was obtained 6 weeks later by inserting a hypodermic needle through the skin. The volume of fluid obtained from the capsule was sufficient for the analysis of total protein, albumin, albumin:globulin ratio, colloid osmotic pressure and the fluid viscosity. Despite the significantly lower total protein, albumin, globulin and colloid pressure of intracapsular fluid compared with plasma, the intracapsular fluid was found to have a greater viscosity. It is our opinion that the increased viscosity of the intracapsular fluid is due to the presence of a high molecular weight substance other than albumin and globulin, possibly hyaluronan.

Antioxidants attenuate endotoxin-induced acute renal failure in rats.

Acute kidney dysfunction, manifested by a reduction in renal blood flow and in the glomerular filtration rate, is a common finding in septic shock. The pathogenetic mechanisms responsible for the renal dysfunction observed in the endotoxemic murine model are not completely understood. In this study, an attempt was made to halt the progressive renal dysfunction in the rats by administration of the antioxidants dimethylthiourea (DMTU) (50 mg/100 g) and superoxide dismutase (SOD) (0.4 mg/100 g) before endotoxin infusion (0.5 mg/100 g), or by inducing endotoxin tolerance. Renal function, assessed by creatinine, inulin, and p-aminohippuric acid clearance, nicotinamide adenine dinucleotide, and electrolyte reabsorption, was measured 4 hours after the endotoxin infusion. Renal function declined in all rats throughout the study period. However, the reduction in renal function was markedly slower in endotoxemic rats administered DMTU and SOD compared with untreated rats. Similar results were found following induction of endotoxin tolerance. These data suggest that DMTU, SOD, and endotoxin tolerance may be potentially beneficial in halting progressive renal damage associated with endotoxemia.

Glycerol-induced augmentation of sensitivity to endotoxin in rats.

Combined sepsis and rhabdomyolysis result in a mortality rate much higher than that caused by each process alone. An analogous rat model is obtained by simultaneous i.p. administration of a nonlethal dose of lipopolysaccharide (LPS 0.025 mg/100 g) and a nonlethal i.m. injection of glycerol (1 ml/100 g). The aim of this study was to determine the factors contributing to the high mortality rate in this rat model. The factors examined include: Dehydration, plasma volume expansion, 'immunization' to glycerol, induction of LPS tolerance and the effect of free radicals formed in this model. Neither dehydration nor volume expansion affected mortality. 'Immunization' with glycerol was also not effective. In contradistinction, tolerance to LPS achieved by a daily injection with gradual increasing doses of LPS (from 0.05 mg/100 g to 1 mg/100 g) for 6 days reduced the mortality rate by 60% (P < 0.001). Moreover, decreasing free radical activity using the natural antioxidant (NAO) (5 mg/100 g) reduced mortality rates by 50%. A different antioxidant, dimethylthiourea (DMTU) (50 mg/100 g) failed to reduce mortality rates. This study suggests that the synergism between glycerol and LPS is apparently due to an increase in the rats' sensitivity to endotoxin following glycerol injection. However, endotoxin apparently does not enhance sensitivity to glycerol in the rat. The new antioxidant NAO significantly reduced the high mortality rate.

Models of glycerol-induced acute renal failure in rats.

Acute renal failure (ARF) following rhabdomyolysis is not uncommon in man. The popular model for ARF formation following rhabdomyolysis in experimental animals is glycerol injection into the leg muscle following a 24 hour period of water deprivation. A large percentage of patients developing ARF following rhabdomyolysis do not suffer from such long periods of water deprivation. On the contrary, fluid loss in patients developing ARF is relatively fast and is the result of excessive sweating or hemorrhage. Since it is known that the hydration state of the body during rhabdomyolysis considerably affects the development of ARF, it seems that the popular model of glycerol injection following a prolonged period of water deprivation in experimental animals is, to a certain extent, deficient. The aim of the present study was to examine two models of ARF formation in the rat following glycerol injection and acute diminution of the body's water content: 1) by sucrose injection (200 mg/100 g), 2) by hemorrhage (0.7 ml/100 g). A number of differences were found between the various models of ARF formation by glycerol. The differences are mainly expressed in the urine volume three hours after the glycerol injection. In the sucrose and hemorrhage groups a decrease of 29% and 66% (p < 0.001) in urine volume was found at the end of the experiment. In contradistinction, in the group that underwent water deprivation for a period of 24 hours prior to the glycerol injection, an increase of 46% (p < 0.001) in the urine volume was observed at the end of the experiment. Differences were also found in potassium uptake and in the extent of the decrease in renal cortex blood flow as measured by the laser Doppler flowmetry technique. From this study it may be concluded that glycerol injection to the rat leg muscle results in ARF in all three methods of decreasing the body's fluid content. It is possible that the models of sucrose injection or hemorrhage prior to glycerol injection are better suited for reflecting the hydration condition of humans suffering from rhabdomyolysis than 24 hours of water deprivation prior to this injection.

Fiber optic surface fluorometry-reflectometry technique in the renal physiology of rats.

Most current knowledge on events in the mitochondria leading to acute renal failure originates from studies in which indirect methods were used. The disadvantage of these methods is that they cannot measure the turnover rate of various metabolites, and only one result per animal can be obtained. Chance et al. /9/ developed a method using optical techniques for continuously monitoring the fluorescence of intramitochondrial NADH, which has been applied mainly to the brain. This optical method has not yet been examined quantitatively in the kidney and no attempt has been made to adapt this method for routine measurement in kidney tissue. The purpose of this study was to adapt the surface fluorometry method for monitoring renal NADH redox state in situ, and to determine whether the hemodynamic artifacts involved in fluorometric studies of the renal surface in situ could be eliminated by using a correction factor. Another purpose was to understand the relationship between the changes in reflectance and blood volume in the rat kidney. This was achieved by measuring the reflectance after: a) blood exchange by FC-43 emulsion; b) intrarenal saline flush; c) occlusion of the renal vein, renal artery and reopening of the renal vein; d) calculation of the correlation between changes in kidney weight after renal artery occlusion, and the reflectance. Our results suggest that in the rat kidney, as opposed to the brain, a correction factor of 1:1 is not always applicable. This factor may vary between animals, and it is therefore necessary to adjust it electronically for each rat kidney. This observation contradicts the view suggesting a constant correction factor of 1:1 in the kidney. The results reported herein indicate that changes in the reflectance in the ischemic rat kidney are due to changes in blood volume. In conclusion, it seems that optical techniques for monitoring fluorescence are suitable for localized, continuous and non-invasive recording of tissue mitochondrial NADH redox states under various conditions in the rat kidney.

Effects of changes in plasma volume on fatal rhabdomyolysis in the rat induced by glycerol injections.

Rhabdomyolysis can be fatal in both experimental animals and man, but very little is known of the factors causing increased mortality in rhabdomyolysis. The aims of this study were to create an animal model of fatal rhabdomyolysis in rats by a glycerol injection into the leg muscle, and to elucidate some of the factors affecting mortality as a result of rhabdomyolysis formation. In this study, two factors which can result in increased mortality in rats as a result of glycerol injection, were examined. These factors include varying doses of 50% glycerol (0.5-2 ml/100 g) and various stages of dehydration prior to glycerol injection. Dehydration was induced by 1: chronic dehydration, in which the rats underwent water deprivation for a period of 24 to 72 hours prior to injection of glycerol; 2: acute dehydration, by the induction of either diuresis, by injecting sucrose (200-600 mg/100 g) to the femoral vein, or hemorrhage (0.7-2.1 ml/100 g). The results demonstrate that the mortality rate in rats increased in all three models of dehydration as the dose of glycerol injected to the rats increased (above a dose of 0.75 ml/100 g) and as the extent of dehydration increased. Use of a blood substitute before or after glycerol injection in order to compensate for the loss of body fluids did not increase the survival rate of the glycerol-injected rats. In contradistinction, rats treated with non-lethal doses of glycerol exhibited substantial resistance to a second lethal dose of glycerol, injected two weeks following the first injection.