Evaluation of hippocampal arylalkylamine N-acetyltransferase activity in amyloid-ß neurotoxicity.

Arylalkylamine N-acetyltransferase (AANAT), a rate-limiting enzyme in melatonin synthesis, is present in extra-pineal tissues such as the hippocampus. The hippocampal AANAT activity in amyloid ß (Aß) neurotoxicity has not been exactly defined. Adult male rats received bilateral intra-CA1 Aß administration. The hippocampus tissue sampling was performed 2, 12, and 24 h after Aß injection in the morning and night. The inflammation was monitored using tumor necrosis factor-alpha (TNF-α) immunohistochemistry. The AANAT enzyme activity and melatonin levels were measured using western blotting and high-performance liquid chromatography. The sampling in the morning vs night showed no significant differences in the AANAT activity. The Aß increased the area of TNF-α positive staining 24 h after injection, which indicated the induction of an inflammatory context. It was accompanied by a significant reduction in AANAT activity and hippocampal melatonin. A reverse correlation was also detected between TNF-α and AANAT activity in the 24-h group. The TNF-α positive area was significantly increased in the 24-h group as compared to the 12-h group. Data showed that inflammatory processes began 12 h after the Aß injection and augmented 24 h later. In the second experiment, the impact of Aß injection on hippocampus AANAT activity was examined in the pinealectomized (PIN×) animals. The PIN× per se did not affect the hippocampal AANAT and melatonin levels. However, there was a significant decrease in hippocampal melatonin in the PIN×+Aß group. The findings suggest the accompanying hippocampal inflammatory context and AANAT enzyme activity reduction in early stages after Aß administration. Understanding the underlying mechanism of the decreased AANAT activity may suggest new treatment strategies.

A surgical modification in the technique of rat pinealectomy.

BACKGROUND: Several experimental intents require pineal gland removal. The main challenge of the pinealectomy surgical procedure is the hemorrhage due to the transverse sinus torn. The study aimed to modify the rat pinealectomy surgical procedure to reduce the risk of bleeding and the mortality rate. METHODS: Adult male rats experienced pinealectomy surgery. A mini-drill was used to remove a small skull area in the junction of the lambda and sagittal sutures. The pineal gland was removed using a curved-head hook. Animals experienced intensive post-surgical care. Locomotion, cerebellar motor function, working memory, and anxiety were evaluated 2 weeks after pinealectomy by the open field, rotarod, Y maze, and the elevated plus maze, respectively. RESULTS: Surgical modification reduced the bleeding risk and animal mortality rate. No significant alteration was found in locomotion and working memory. However, the pinealectomy was anxiogenic and decreased entry to the open arm. The cerebellar motor performance did not change in the rotarod test. Hematoxylin-Eosin staining of removed tissue confirmed the histology of the pineal gland. CONCLUSION: Advantages of this technique were removing a small skull area, modifying the hook insertion point to prevent damaging the brain veins, reducing the bleeding risk and the mortality rate. Surgery modification was associated with a decreased final number of animals used. Regardless of the melatonin shortage, pinealectomy affects different organs, which should be considered in the research study design.

Increment of CSF fractalkine-positive microvesicles preceded the spatial memory impairment in amyloid beta neurotoxicity.

BACKGROUND: Fractalkine (CX3CL1) is a key chemokine, affects neuronal cell communication and involves in Alzheimer's disease pathogenesis. Microvesicles (MVs) participate in neuronal cells' cross-talk in physiological and pathological states. Microvesicles released in cerebrospinal fluid (CSF) may provide a valuable footprint of brain changes. Little information is available regarding the release of fractalkine-positive MVs (CX3CL1+ -MVs) in the nervous system. METHODS: We induced cognitive impairment by bilateral injection of amyloid-beta (Aß) into the cerebral ventricles. We analyzed the CSF by flow cytometry in two experiments (trained and untrained) to elucidate the presence of CX3CL1+ -MVs. The hippocampal TNF-α as an inflammatory factor was assessed by immunohistochemistry. RESULTS: The Aß induced spatial memory impairment after two weeks, verified by a decrease in the escape latency in Morris water maze test. It caused an increase in the anxiety-like behaviors demonstrated by a decrease in entries into the open arms of elevated plus maze test. The Aß increased the percent of the positive area for TNF-α staining. Histological evaluation of the hippocampus confirmed the tissue injuries. The CSF levels of CX3CL1+ -MVs, increased 2 and 7 days after Aß injection. The Aß increased the TNF-α staining and provided an inflammatory context to facilitate the MVs release. The rise of CX3CL1+ -MVs was transient and subsided after two weeks. Both trained and untrained experiments showed a similar rise pattern of CX3CL1+ -MVs. CONCLUSION: Increase of fractalkine-positive microvesicles preceded the cognitive impairment, more studies are required to approve the CX3CL1+ -MVs as a potential biomarker in the early diagnosis of Alzheimer's disease.

Inhibition of brain 17ß-estradiol synthesis by letrozole induces cognitive decline in male and female rats.

BACKGROUND: Hippocampal aromatase is responsible for local synthesis of 17ß-estradiol (E2) that has much higher concentrations than serum levels in males and females. Letrozole, an aromatase inhibitor, passes through the brain barriers, distributes to the brain, and affects local E2 synthesis. Here, the effects of intra-cerebroventricular (ICV) letrozole administration in the presence and absence of gonads were examined on the cognitive abilities of male and female rats. METHOD: Animals received intra-ICV injection of letrozole or vehicle for 14 consecutive days. Spatial working memory, novel object recognition memory, and anxiety-related behavior, were evaluated using Y-maze, object recognition test, and elevated plus maze, respectively. The E2 levels in the serum and hippocampal tissue were measured by the ELISA technique. RT-PCR was performed to assess the hippocampal estrogen receptors (ER) expression. Moreover, letrozole effect on neuronal activity of CA1 pyramidal neurons was studied by in vivo single-unit recording. RESULTS: Letrozole (0.2, 0.4, and 0.8 µg) significantly decreased the hippocampal E2 levels compared to the vehicle group. Letrozole caused cognitive impairments in a dose-dependent manner in male and female rats in the presence or absence of gonads. Dose-response analysis revealed that the minimum effective dose of letrozole on the behavioral measures was 0.4 µg. Letrozole also caused an up-regulation of ERα and ERß and a down-regulation of GPR30 gene expression. The firing rate of pyramidal neurons was reduced by letrozole in gonadal-intact animals. CONCLUSION: The detrimental effects of letrozole treatment on cognitive abilities in the presence and absence of gonads indicate that local E2 synthesis in the hippocampus is a crucial factor in normal cognitive performance. The suppressive effect of letrozole on hippocampal neuronal firing might alter synaptic plasticity that is critical for memory formation. These data potentially suggest that memory deficits following letrozole administration should be monitored.

GnRH protective effects against amyloid ß-induced cognitive decline: A potential role of the 17ß-estradiol.

INTRODUCTION: The 17ß-estradiol (E2) enhances hippocampal dendritic spine synapses, facilitates learning processes, and exerts neuroprotection. Brain estrogen decline has been reported in Alzheimer's disease. The role of GnRH in modulating steroid biosynthesis convinced us to examine whether hippocampal GnRH administration could enhance the local E2 levels and overcome the development of cognition decline in amyloid ß (Aß) neurotoxicity. To explore if GnRH acts through regulating E2 synthesis, letrozole, an aromatase inhibitor, has been applied in combination with GnRH. METHODS: Female rats received an intracerebroventricular injection of Aß. The GnRH and, or letrozole were injected into the CA1 for 14 consecutive days. Working memory, novel object recognition memory, and anxiety-like behavior were evaluated. Serum and hippocampal E2 levels were measured. Hippocampal mRNA expression of GnRH (GnRH-R) and E2 (ERα and ERß) receptors was assessed. GnRH effect on the excitability of pyramidal cells was studied by in vivo single-unit recording. RESULTS: GnRH increased hippocampal E2 levels, evoked an increase in the spontaneous firing of pyramidal neurons, and caused mRNA overexpression of hippocampal GnRH receptors. GnRH prevented the adverse effects of Aß on working memory, NOR index, and anxiogenic behavior. Letrozole did not reverse GnRH modulatory effects on hippocampal E2 levels and neuroprotection. CONCLUSION: GnRH prevented the Aß-induced memory deficit, which may be mediated through hippocampal E2 levels enhancement. The electrophysiological analysis revealed the enhanced neuronal excitability in the CA1 region. All these data suggest that GnRH might be a promising candidate that reduces anxiety and improves memory indices in the context of Aß neurotoxicity.

Opioid Use Disorder Induces Oxidative Stress and Inflammation: The Attenuating Effect of Methadone Maintenance Treatment.

Objective: Frequent use of opioids produces reactive oxygen species, upregulates inflammatory factors, and contributes to opiate dependence. In this study, we examined perturbations of plasma oxidative and inflammatory markers in patients with opioid use disorder in two phases. In the first phase, we compared the oxidative status in patients with opioid use disorders and in healthy controls; and in the second phase, we examined oxidative changes before and after methadone maintenance treatment. Method: To explore whether oxidative changes were associated with opioid use disorder, we compared plasma oxidative and inflammatory markers in patients with opioid use disorder and in smoking and non-smoking healthy participants. All participants completed measures of catalase (CAT), glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), matrix metalloproteinase (MMP-9), and TNF-α at baseline. Baseline measures were compared using Kruskal-Wallis test. In the second phase, to explore oxidative changes during transition from opium use to methadone, blood and urine samples of patients with opioid use disorder were re-evaluated on Days 3, 7, and 14 after methadone therapy. Repeated measures analysis was used to determine the relative contribution of intervention to changes in CAT, GSH, MDA, SOD, MMP-9, and TNF-α level over time. Results: We observed lower SOD and catalase activities, and higher TNF-α and MMP-9 level in patients compared to the two comparison groups. Opioids exacerbated the oxidative imbalance and superimposed the underlying oxidative injury in smoker comparison group. Methadone therapy was associated with lower MMP-9 and TNF-α level, and higher SOD and catalase activities two weeks after therapy; showing an improvement in oxidative profile. Conclusion: This was an investigation indicating an oxidative imbalance before methadone therapy and during early days of transition from opium use to methadone. Being aware of redox status is crucial for determining an appropriate antioxidant therapy in opioid use disorder.

Simvastatin enhances the hippocampal klotho in a rat model of streptozotocin-induced cognitive decline.

Brain oxidative status is a crucial factor in the development of sporadic Alzheimer's disease (AD). Klotho, an anti-aging protein, diminishes oxidative stress by the induction of manganese superoxide dismutase (MnSOD). Thus, the substances that increase klotho expression could be considered as a potential treatment for Alzheimer's disease when the oxidative imbalance is present. Statins are suggested to up-regulate klotho expression. We examined the effect of simvastatin (5mg/kg, daily for 3weeks) on hippocampal klotho and MnSOD expression in the cognitive declined animal model induced by intracerebroventricular (ICV)-streptozotocin (STZ) administration. Cognitive assessment was performed by the Morris Water Maze (MWM) test. The results indicated that mean escape latency and distance were prolonged in the ICV-STZ group compared with the control group. The expression of klotho and MnSOD were also down regulated in the hippocampus. Furthermore, improved spatial performance was observed in simvastatin-treated animals. This effect could be related to increase in oxidative stress tolerance as evidenced by klotho and MnSOD up-regulation. Our current study indicates that klotho upregulation may be a neuroprotective mechanism of simvastatin against cognitive decline in AD.

Noisy galvanic vestibular stimulation enhances spatial memory in cognitive impairment-induced by intracerebroventricular-streptozotocin administration.

There are several anatomical connections between vestibular system and brain areas construct spatial memory. Since subliminal noisy galvanic vestibular stimulation (GVS) has been demonstrated to enhance some types of memory, we speculated that application of noisy GVS may improve spatial memory in a rat model of intracerebroventricular streptozotocin (ICV-STZ)-induced cognitive impairment. Moreover, we attempted to determine the effect of repeated exposure to GVS on spatial memory performance. The spatial memory was assessed using Morris water maze test. The groups received 1 (ICV-STZ/GVS-I) or 5 (ICV-STZ/GVS-II) sessions, each lasting 30 min, of low amplitude noisy GVS, or no GVS at all (Control, ICV-saline, ICV-STZ/noGVS). Hippocampal morphological changes investigated with cresyl violet staining and the immediate early gene product c-Fos, as a neuronal activity marker, was measured. Hippocampal c-Fos positive cells increased in both GVS stimulated groups. We observed significantly improved spatial performance only in ICV-STZ/GVS-II group. Histological evaluation showed normal density in ICV-STZ/GVS-II group whereas degeneration observed in ICV-STZ/GVS-I group similar to ICV-STZ/noGVS. The results showed the improvement of memory impairment after repeated exposure to GVS. This effect may be due in part to frequent activation of the vestibular neurons and the hippocampal regions connected to them. Our current study suggests the potential role of GVS as a practical method to combat cognitive decline induced by sporadic Alzheimer disease.

Curcumin as a double-edged sword for stem cells: dose, time and cell type-specific responses to curcumin.

BACKGROUND: The beneficial effects of curcumin which includes its antioxidant, anti-inflammatory and cancer chemo-preventive properties have been identified. Little information is available regarding the optimal dose and treatment periods of curcumin on the proliferation rate of different sources of stem cells. METHODS: In this study, the effect of various concentrations of curcumin on the survival and proliferation of two types of outstanding stem cells which includes bone marrow stem cells (BMSCs) and adult rat neural stem/progenitor cells (NS/PCs) at different time points was investigated. BMSCs were isolated from bilateral femora and tibias of adult Wistar rats. NS/PCs were obtained from subventricular zone of adult Wistar rat brain. The curcumin (0.1, 0.5, 1, 5 and 10 µM/L) was added into a culture medium for 48 or 72 h. Fluorescent density of 5-bromo-2'-deoxyuridine (Brdu)-positive cells was considered as proliferation index. In addition, cell viability was assessed by MTT assay. RESULTS: Treatment of BMSCs with curcumin after 48 h, increased cell survival and proliferation in a dose-dependent manner. However, it had no effect on NSCs proliferation except a toxic effect in the concentration of 10 µM of curcumin. After a 72 h treatment period, BMSCs and NS/PCs survived and proliferated with low doses of curcumin. However, high doses of curcumin administered for 72 h showed toxic effects on both stem cells. CONCLUSIONS: These findings suggest that curcumin survival and proliferative effects depend on its concentration, treatment period and the type of stem cells. Appropriate application of these results may be helpful in the outcome of combination therapy of stem cells and curcumin.

Modulation of ventral tegmental area dopamine receptors inhibit nicotine-induced anxiogenic-like behavior in the central amygdala.

Nicotine, the major addictive substance in tobacco, increases the activity of the central amygdala (CeA). Amygdala is directly implicated in anxiety modulation and sends projections to the vicinity of the midbrain dopamine neurons, including the ventral tegmental area (VTA) which is a key area that controls nicotine dependence processes. In this study, the role of dopamine D(1) and D(2)/(3) receptors of the VTA on anxiogenic-like behavior induced with intra-CeA injection of nicotine has been investigated. Male Wistar rats with cannula aimed to the left CeA and the left VTA were submitted to the elevated plus-maze (EPM). The nicotine injection (1 µg/rat) into the CeA decreased the percentage of open arm time and open arm entries, but not locomotor activity, indicating an anxiogenic-like response. Intra-VTA injection of a dopamine D1 receptor antagonist, SCH23390 (0.25 µg/rat), and a dopamine D2/3 receptor antagonist, sulpiride (0.7 µg/rat), inhibited the anxiogenic-like response caused by intra-CeA injection of nicotine. These results suggest that the relationship between the VTA and the CeA may be involved in nicotine-induced anxiogenic-like behavior via dopamine D(1) and D(2)/(3) receptors. An understanding of these cellular processes will be crucial for the development of new intervention to combat nicotine effect.

Involvement of rat dopaminergic system of nucleus accumbens in nicotine-induced anxiogenic-like behaviors.

The objective of the present study was to investigate the involvement of dopamine D1 and D2 receptors of the nucleus accumbens (NAc) shell in the anxiogenic-like effect of intra-central amygdala (CeA) nicotine administration. Male Wistar rats with cannula implants in the left CeA and the left shell of NAc were submitted to the elevated plus-maze (EPM). Intra-CeA injections of nicotine (1 µg/rat) decreased % open arm time spent (%OAT) but not % open arm entries (%OAE) and locomotor activity, indicating the possibility of an anxiogenic-like response. Intra-NAc injection of D1 dopamine receptor antagonist, SCH23390 (0.5 µg/rat) but not other doses of the antagonist (0.06, 0.125 and 0.25 µg/rat) increased both %OAT and %OAE, showing an anxiolytic-like effect for the higher dose of the drug. Similar administration route of sulpiride (0.25, 0.5, 0.75 and 1 µg/rat), a selective antagonist at dopamine D2 receptor, had no significant effect on OAT%, OAE% and locomotor activity. Moreover, intra-CeA injection of nicotine (1 µg) with intra-NAc injection of sub-threshold doses of antagonists increased %OAT and %OAE without significant effect on locomotor activity. These findings may suggest the involvement of dopamine transmission, through D1 and D2 receptors of NAc shell, in the anxiogenic-like effect of nicotine in the EPM task.

Evaluation of renal oxidative stress in the development of DOCA-salt induced hypertension and its renal damage.

To ascertain the onset of renal oxidative stress and its interrelation with the increase in blood pressure (BP) and kidney injury in rats subjected to Deoxycorticosterone (DOCA)-salt treatment, BP, renal antioxidants, renal damage indices, and histological changes were studied weekly. In the two other groups, 200 mg/kg/day vitamin E or C were co-administered with DOCA-salt for 4 weeks. Blood Pressure was increased at week one. Urinary N-acetyl-B-diglucosaminidase (NAG) and proteinuria increased and renal catalase decreased at 2nd week. Histological changes and decreased glothatione (GSH) and Ferric reducing antioxidant power (FRAP) were demonstrated at three week. Vitamin therapy increased renal antioxidants and decreased BP, NAG, proteinuria, and histological damage. Thus, elevation in BP precedes the onset of renal oxidative stress in DOCA-salt treated rats. Enhanced renal oxidative stress contributes to kidney damage. In this study, treatment with vitamin C or vitamin E preserved renal antioxidant levels, prevented renal damage, and partially inhibited elevation of BP in the DOCA-salt treatment.

Oxidative stress status in renal transplant recipients.

OBJECTIVES: Despite the demonstration of oxidative stress in patients with end-stage renal disease, the oxidative status during and after a renal transplant are not completely understood. Hepatocyte growth factor is reported to act as an endogenous factor against oxidative stress. The aim of this study was to evaluate the pattern of changes in plasma oxidative status and hepatocyte growth factor levels in living-donor renal transplant recipients during the early phase after transplant. MATERIALS AND METHODS: Nineteen patients who underwent a renal transplant were included. All were on cyclosporine-based immunosuppression. Plasma levels of malondialdehyde, ferric reducing activity, hepatocyte growth factor, vitamin E, erythrocyte glutathione, and superoxide dismutase activities were determined before, and on the second, seventh, and 12th days after the transplant. RESULTS: High malondialdehyde concentration and low superoxide dismutase activity were seen before and 48 hours after transplant compared with healthy subjects. Significant reductions in plasma ferric reducing activity, malondialdehyde, and hepatocyte growth factor were seen on the seventh and twelfth days after transplant, compared with the before-transplant data. Direct correlations were found between hepatocyte growth factor levels and ferric reducing activity of plasma as well as hepatocyte growth factor and creatinine or uric acid. CONCLUSIONS: Renal transplant recipients display persistent oxidative stress during the early phase of transplant. The pattern of oxidative changes should be considered for appropriate time, dosage, type, and the duration of antioxidant therapy in these patients.

Flow cytometric evaluation of sperm superoxide anion production in rats with experimental varicocele.

OBJECTIVES: To determine whether intracellular superoxide anion production changes in the sperm of rats with experimental varicocele and to study mitochondrial membrane potential (MMP), sperm viability, antioxidant activity, Bax and Bcl-2 changes, and sperm count and motility in these rats. METHODS: The study groups consisted of 32 male rats divided into the following 4 groups: control, sham, varicocele-1, and varicocele-2. Experimental varicocele was established by partial ligation of the left renal vein in the latter 2 groups. Animals were killed 2 and 6 months after surgery, and dilation of the internal spermatic veins was observed. Then, superoxide anion production, MMP, antioxidant activity, and sperm characteristics were evaluated. Flow cytometry was used to study sperm superoxide anion production, assessed by dihydroethidium and MMP with rhodamine 123. RESULTS: Our results showed intracellular superoxide anion production significantly increased, and MMP, sperm viability, sperm count, and motility decreased in rats with experimental left varicocele. More Bax-positive cells were seen in the varicocele groups. However, there was no significant difference for seminal plasma antioxidant activity among all groups. CONCLUSIONS: These findings suggest that a main source of reactive oxygen species production in varicocele condition is intracellular.

Effects of different periods of renal ischemia on liver as a remote organ.

AIM: To assess the hepatic changes after induction of different periods of renal ischemia. METHODS: Rats were subjected to either sham operation or ischemia (30, 45 and 60 min) followed by 60 min reperfusion. Liver and renal functional indices were measured. Hepatic glutathione (GSH) and ferric reducing antioxidant power levels and the concentration of interleukin (IL)-10 and tumor necrosis factor (TNF-alpha) were evaluated. Portions of liver and kidney tissues were fixed for histological evaluation. RESULTS: Forty-five minutes renal ischemia followed by 60 min reperfusion caused significant changes in liver structure and a significant reduction in renal function. These rats showed a significant decrease in liver GSH, as well as a significant increase in TNF-alpha and IL-10 concentrations. These results demonstrated that renal ischemia caused changes in liver histology, function, oxidative stress and inflammatory status, which led to a reduction in hepatic antioxidant capacity. With 30 min ischemia, the magnitude of these changes was less than those with 45 or 60 min ischemia. CONCLUSION: A minimum of 45 min ischemia is needed to study the effects of renal injury on the liver as a remote organ.

Ischemic and non-ischemic acute kidney injury cause hepatic damage.

Recent studies have documented that remote organs are affected by ischemic injury to the kidney. Here we studied whether the liver also suffers damage during induction of renal ischemia-reperfusion in rats and compared this to bilateral nephrectomy. Hepatic levels of tumor necrosis factor-alpha increased significantly after 6 and 24 h of renal ischemia or nephrectomy. Malondialdehyde, an index of lipid peroxidation, increased while total glutathione was decreased in the liver in both the renal ischemia and nephrectomy groups, suggesting activation of oxidative stress. Expression of liver spermine-spermidine acetyl transferase, an enzyme upregulated in early phases of hepatic injury was significantly increased 6 h after either kidney ischemia or nephrectomy. Apoptosis was increased in hepatocytes 24 h after nephrectomy. We also found histological evidence of hepatocyte injury following both ischemia and bilateral nephrectomy. Infusion of reduced glutathione, before the induction of renal ischemia, significantly improved liver architecture and was associated with a reduction in hepatic malondialdehyde and serum alanine transaminase levels. Our study shows that acute kidney ischemia or renal failure activates oxidative stress and promotes inflammation, apoptosis, and tissue damage in hepatocytes.

Assessment of plasma antioxidant status in hemodialysis patients.

The risk of atherosclerosis and cancer is high in patients on hemodialysis. A breakdown in the natural balance between the activity of the body's antioxidant system and the production of oxidizing agents is suggested to be involved. To investigate the oxidative stress status in Iranian hemodialytic patients, in this study we evaluated plasma vitamin E, malondialdehyde (MDA), reduced glutathione (GSH), and ferric reducing antioxidant power (FRAP) levels in these patients. Twenty-four hemodialytic patients and 24 control subjects (age and sex matched) were included in this study. Each patient was under dialysis, three times per week, four hours in each session. Before and after dialysis, blood was taken for biochemical measurements as well as oxidative stress tests. There was a significant decrease in FRAP and GSH levels after dialysis comparing to before treatment levels. MDA was increased by dialysis and vitamin E levels were less in dialytic patients, both before and after treatment, compared to controls. This study indicates that there is a significant level of oxidative stress in chronic renal patients and this stress is augmented by dialysis. Antioxidant therapy could be considered in these patients.

Beneficial effects of MnTBAP, a broad-spectrum reactive species scavenger, in rat renal ischemia/reperfusion injury.

BACKGROUND: In recent years, several lines of evidence have implicated reactive species as contributors to renal ischemia/reperfusion injury (I/R). This study was designed to investigate the effect of Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a broad-spectrum reactive species scavenger, in the prevention of renal I/R injury. METHODS: Experiments were performed on rats anesthetized with pentobarbital. After tracheotomy, the right femoral artery was cannulated and the mean arterial pressure and heart rate were recorded. A midline laparatomy was performed, and the renal arteries were carefully separated from surrounding tissues. After surgery and a stabilization period (60 min), the animals were randomly assigned to four groups: sham-operated; sham+MnTBAP; I/R; I/R+MnTBAP. In I/R groups, the rats were subjected to bilateral renal artery occlusion for 40 min followed by 6 h reperfusion. Other groups underwent the surgery protocol but did not undergo renal artery occlusion, and were maintained under anesthesia for the duration of the experiment. Rats were administered either MnTBAP (10 mg kg(-1), i.v. bolus, 15 min prior to I/R) or saline. Renal function was assessed by plasma creatinine (Cr), blood urea nitrogen (BUN), and aspartate aminotransferase (AST) measurements. The fractional excretion of Na(+) (FE(Na+)) and urinary N-acetyl-beta-D-glucosaminidase (NAG) activities were also measured. Renal section damage was evaluated by light microscopy, and oxidative stress status was evaluated by measurements of plasma and renal vitamin E levels. RESULTS: We found that MnTBAP significantly reduced the I/R-mediated increases in plasma Cr, BUN, AST, FE(Na+), and NAG and improved the renal tissue histology. CONCLUSION: Our results showed that MnTBAP was effective in preventing the development of I/R-induced renal injury.

Effects of co-supplementation of vitamins E and C on gentamicin-induced nephrotoxicity in rat.

Gentamicin (GM) is an effective antibiotic against severe gram-negative infections. However it can produce nephrotoxicity in human. Reactive oxygen species (ROS) have been proposed as the causative factors of the renal side effects the drug. This study was performed to investigate the protective role of antioxidant vitamins against GM-mediated nephropathy in an in situ model of isolated rat kidney. Male Sprague-Dawley rats were randomly assigned to one of the following groups of seven rats: group 1 (Control) was perfused with Tyrode solution; group 2 (GM), 200 microg ml(-1) GM was added to the perfusate; group 3 (GM + Vit C), as group 2 with vitamin C added to the drinking water for 3 days (200 mg l(-1)) and to the perfusate (100 mg l(-1)); group 4 (GM + Vit E), as group 2 with vitamin E (100 mg (100 g body weight)(-1), i.m.) injected 12 h before the start of the experiment; group 5 (GM + Vit C + Vit E) as group 2 with vitamin E and C co-administered (concentrations and conditions as in groups 3 and 4). To compare the groups, urinary lactate dehydrogenase (LDH), N-acetyle-beta-D-glucosaminidase (NAG) and alkaline phosphatase (ALP) activities, inulin clearance (glomerular filtration rate, GFR) and renal tissue glutathione (GSH) content were measured. GM caused a significant nephrotoxicity demonstrated by an increase in urinary LDH, NAG and ALP activities. Reduction in GSH content and a marked decrease in GFR were observed compared to controls. Vitamin C inhibited the GM-induced increase in urinary enzyme activities but did not show a significant effect on the GSH content or GFR. Vitamin E prevented the GM-induced reduction in GSH level without a significant improvement in GFR. Co-administration of vitamins C and E significantly prevented the GM-induced nephrotoxicity demonstrating by preservation of GFR and GSH levels and prevention of increase in urinary enzyme activities. We conclude that co-administration of moderate doses of vitamins C and E has beneficial effects on renal preservation in GM-induced nephrotoxicity.

Protection of rat renal vitamin E levels by ischemic-preconditioning.

BACKGROUND: During renal transplantation, the kidney remains without blood flow for a period of time. The following reperfusion of this ischemic kidney causes functional and structural injury. Formation of oxygen-derived free radicals (OFR) and subsequent lipid peroxidation (LP) has been implicated as the causative factors of these injuries. Vitamin E is known to be the main endogenous antioxidant that stabilizes cell membranes by interfering with LP. The present study was designed to examine the role of ischemic-preconditioning (repeated brief periods of ischemia, IPC) in prevention of renal injury caused by ischemia-reperfusion (IR) in rats. METHODS: IPC included sequential clamping of the right renal artery for 5 min and release of the clamp for another 5 min for a 3 cycles. IR was induced by 30 min ischemia followed by 10 min reperfusion. Four groups of male rats were used: Control, IPC, IR and IPC-IR. Vitamin E, an endogenous antioxidant and as an index of LP, was measured by HPLC and UV detection in renal venous plasma and tissue. Renal function was assessed by serum creatinine and BUN levels. Renal damage was assessed in sections stained with Haematoxylin and Eosin. RESULTS: In the IR group, there was a significant decrease in vitamin E in plasma and tissue compared to a control group (p,0.05). In the IPC-IR group, vitamin E concentration was significantly higher than in the IR group (p,0.01). The results showed that 30 min ischemia in the IR group significantly (p,0.05) reduced renal function demonstrated by an increase in serum creatinine levels as compared with the control group. These results in the IPC group also showed a significant difference with the IR group but no significant difference in serum BUN and creatinine between IR and IPC-IR group were detected. Histological evaluation showed no structural damage in the IPC group and an improvement in the IPC-IR group compared to IR alone. CONCLUSIONS: In this study, IPC preserved vitamin E levels, but it could not markedly improve renal function in the early phase (1-2 h) of reperfusion. IPC may be a useful method for antioxidant preservation in organ transplantation.