Yehuda Zurovsky

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.

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 rabbits 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.

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.

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.

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 bodys 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 bodys 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.

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.

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

Abstract 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 02 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. [Neurol Res 2000; 22: 404-408]

Multiorgan Monitoring of Hemodynamic and Mitochondrial Responses to Anoxia and Cardiac Arrest in the Rat

All tissues in the body are dependent upon the continuous supply of energy to perform their various functions. Glucose and oxygen are brought to the tissue through the bloodstream to fulfill this demand. The basic need of glucose as an energy substrate and oxygen for respiration is common to all tissues. However, the different organs in the body have various levels of demand for these substances. For this reason, the body distributes its resources differently to different organs.

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.

Optical monitoring of tissue viability using reflected spectroscopy in vivo

Only few techniques that could provide real-time continuous multiparametric physiological data have been developed. Therefore, experimental and clinical monitoring devices for organ and tissue viability evaluation are still lacking. In this study, we present the new concept of tissue vitality defined as a product of a few parameters monitored in real- time by a combined measurement of tissue blood flow and volume as well as the oxidation reduction state of the mitochondria. The hypothesis behind the new approach is that in order to evaluate in real-time tissue vitality, it is necessary to monitor both microcirculatory blood flow and volume as well as the intracellular O2 balance as reflected in the mitochondrial redox state.