Serap Sirvanci

L-Carnitine ameliorates methotrexate-induced oxidative organ injury and inhibits leukocyte death

Methotrexate (MTX), a folic acid antagonist widely used for the treatment of a variety of tumors and inflammatory diseases, affects normal tissues that have a high rate of proliferation, including the hematopoietic cells of the bone marrow and the gastrointestinal mucosal cells. To elucidate the role of free radicals and leukocytes in MTX-induced oxidative organ damage and the putative protective effect of L-carnitine (L-Car), Wistar albino rats were administered a single dose of MTX (20 mg/kg) followed by either saline or L-Car (500 mg/kg) for 5 days. After decapitation of the rats, trunk blood was obtained, and the ileum, liver, and kidney were removed for histological examination and for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity, and collagen content. Our results showed that MTX administration increased the MDA and MPO activities and collagen content and decreased GSH levels in all tissues, while these alterations were reversed in L-Car-treated group. The elevated serum TNF-α level observed following MTX treatment was depressed with L-Car. The oxidative burst of neutrophils stimulated by Annexin V was reduced in the saline-treated MTX group, while L-Car abolished this inhibition. Similarly, flow cytometric measurements revealed that leukocyte apoptosis was increased in MTX-treated animals, while L-Car reversed these effects. Severe degeneration of the intestinal mucosa, liver parenchyma, and glomerular and tubular epithelium observed in the saline-treated MTX group was improved by L-Car treatment. These results suggest that L-Car, possibly via its free radical scavenging and antioxidant properties, ameliorates MTX-induced oxidative organ injury and inhibits leukocyte apoptosis. Thus, supplementation with L-Carnitine as an adjuvant therapy may be promising in alleviating the systemic side-effects of chemotherapeutics.

The Effects of Riluzole on Neurological, Brain Biochemical, and Histological Changes in Early and Late Term of Sepsis in Rats

OBJECTIVE One of the underlying mechanisms of sepsis is thought to be the oxidative damage due to the generation of free radicals. Glutamate, the major excitatory amino acid in the brain, is known to play an important role in blood brain barrier (BBB) permeability, brain edema, and oxidative damage in pathological conditions. Riluzole, a glutamate release inhibitor, has been shown to have neuroprotective effects in several animal models. The aim of our study was to investigate the putative protective effect of riluzole against sepsis-induced brain injury. METHODS Sepsis was induced by cecal ligation and puncture in Wistar albino rats. Sham operated (control) and sepsis groups received either saline or riluzole (6 mg/kg, s.c.) 30 min after the surgical procedure, and every 12 h as continuing treatment. The effect of riluzole on the survival rate, weight loss, fever, leukocyte count, brain edema, BBB permeability, oxidative damage, and histological observations were evaluated for early (6 h) and late (48 h) phase of sepsis. RESULTS Riluzole, when administered 6 mg/kg s.c., diminishes the sepsis-induced augmentation in weight loss, body temperature, brain edema, increase in BBB permeability, oxidative damage, and brain injury that is observed histologically. Besides increasing the survival rate in sepsis, it has also improved neurological examination scores and the prognosis of the disease. CONCLUSION According to the results of this study, riluzole appears to have a protective effect for sepsis-induced encephalopathy.

Effect of Various Nerve Decompression Procedures on the Functions of Distal Limbs in Streptozotocin-induced Diabetic Rats: Further Optimism in Diabetic Neuropathy

It is known that diabetic neuropathy is the result of endoneurial edema caused by various biochemical reactions triggered by hyperglycemia. This sequence of events can cause cessation of circulation at the perineurial level, or the tough layer, which is not resilient enough to spread intraneural pressure. Internal and external limiting structures create a double crush phenomenon to the nerve structure. Decompression of the nerve trunk at separate levels is one of the adjuncts to the overall treatment plan for diabetic neuropathy. In this study, the right sciatic nerves of 30 rats with streptozotocin-induced diabetes were used; three groups were created. In the control group, the sciatic nerves were explored and dissected only. In group II, tarsal tunnel release was performed and accompanied by epineurotomy of the sciatic nerve and its peroneal and tibial extensions. In group III, in addition to the procedures performed in group II, perineural sheaths, exposed through the epineurotomy sites at both the peroneal and tibial nerves, were incised for decompression of the fascicles. Improvement in diabetic neuropathy was evaluated by using footprint parameters. The last print length values, estimated according to the 38-month measurements, were 26.1 ± 0.12 mm in the control group, 23.2 ± 0.07 mm in group II, and 22.2 ± 0.1 mm in group III. The toe spread and intermediate toe spread values of the groups were parallel to improvements in print lengths throughout the study. The best improvement was observed in the perineurotomy group. Finally, an electron microscopic study revealed variable degenerative changes in all groups, but they were milder in groups II and III. This experimental study reveals that adding internal decompression to external release doubled the effect in reducing derangement in the sciatic nerves of the rats and, in the authors’ opinion, offers cause for further optimism in the treatment of diabetic neuropathy.

Intra-Amygdaloid Injection of Kainic Acid in Rats with Genetic Absence Epilepsy: The Relationship of Typical Absence Epilepsy and Temporal Lobe Epilepsy

We showed previously that genetic absence epilepsy rats from Strasbourg (GAERS) resist secondary generalization of focal limbic seizures after electrical kindling. We now investigate the effect of intra-amygdaloid injection of kainic acid, as another model of temporal lobe epilepsy, focusing on epileptogenesis, spike-and-wave discharges (SWDs), and the transition from basal to SWD states in GAERS. The EEG was recorded from the hippocampus and cortex of adult GAERS and Wistar rats before kainic acid injections into the basolateral amygdala and for 3 months thereafter. EEG and video recordings monitored SWDs and convulsive seizures. We analyzed spectral changes of the EEG during kainic acid-induced status epilepticus, SWDs, for 10 s before (silent period) and for 2 s before (transition period) SWDs. After the injection of kainic acid, all animals experienced convulsive seizures for at least 3 h. The first convulsive seizure was significantly delayed in GAERS compared with Wistar rats. SWDs and increases in power of the delta, alpha, and beta frequency ranges during the transition period disappeared after the kainic acid injection for 1–3 d and gradually reappeared. Power increases in the delta and alpha ranges were significantly correlated with the number of SWDs, in the beta and alpha ranges with their mean duration. Neo-Timms staining at the end of experiments demonstrated that mossy fiber sprouting in GAERS is less pronounced than in Wistar rats. Our findings show that mechanisms underlying absence epilepsy and temporal lobe epilepsy interact with each other, although a site of this interaction remains to be defined.

Protective effect of MESNA (2-mercaptoethane sulfonate) against hepatic ischemia/reperfusion injury in rats.

PurposeReoxygenation of ischemic tissue generates various reactive oxygen metabolites (ROMs), which have a deleterious effect on various cellular functions. We evaluated the possible protective effect of 2-mercaptoethane sulfonate (MESNA) on hepatic ischemia/reperfusion (I/R) injury.MethodsWistar albino rats were subjected to 45-min hepatic ischemia, followed by 60-min reperfusion. 2-Mercaptoethane sulfonate, 150 mg/kg, or saline was given intraperitoneally (i.p.) twice, 15 min before ischemia and immediately before reperfusioin. We measured serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels to assess liver function. Liver tissue samples were taken to measure the levels of malondialdehyde (MDA), an end-product of lipid peroxidation; glutathione (GSH), a key antioxidant; and myeloperoxidase (MPO) activity, as an indirect index of neutrophil infiltration. We also measured hepatic collagen content, as a fibrosis marker.ResultsPlasma ALT and AST levels were higher in the I/R group than in the control group, but this increase was significantly decreased by MESNA treatment. Hepatic GSH levels, which were significantly depressed by I/R, increased back to the control levels in the MESNA-treated I/R group. Increases in tissue MDA levels and MPO activity caused by I/R injury decreased back to the control levels after MESNA treatment. Similarly, the increased hepatic collagen content in the I/R group decreased to the level of the control group after MESNA treatment.ConclusionThe fact that MESNA alleviated I/R-induced injury of the liver and improved hepatic structure and function suggests that its antioxidant and oxidant scavenging properties may be of therapeutic value in protecting the liver against oxidative injury caused by I/R.

Protective role of resveratrol against cisplatin induced ototoxicity in guinea pigs

OBJECTIVE The aim of this study was to evaluate the effectiveness of systemic administration of resveratrol against cisplatin-induced ototoxicity in guinea pigs. MATERIALS AND METHODS Healthy guinea pigs (n=24) were randomly divided into four groups. Group 1 (n=6) received resveratrol+cisplatin, group 2 (n=6) received 4% ethanol+cisplatin, group 3 (n=6) received cisplatin, and group 4 (n=6) received saline. Cisplatin was administered at a dose of 10mg/kg/day on days 14 and 15 of the study. Resveratrol (10mg/kg/day), 4% ethanol, and saline were administered throughout the study. Baseline auditory brainstem responses (ABR) (4 kHz, 8 kHz, and click stimulus) were determined for all groups. ABR was repeated 72 h after the last dose of cisplatin in order to record the threshold shifts. The ABR threshold shifts for the click stimulus, 4-kHz- and 8-kHz-frequency stimuli were compared after drug administration. After follow-up ABRs the animals sacrificed under deep sedation and their cochleae were removed. Left cochleae were immediately harvested for measurement of level of reactive oxygen species (ROS). Right cochleae were prepared for histological changes which were observed by scanning electron microscopy (SEM). RESULTS For the all stimulus, there was a significant threshold difference among the groups (p<0.01). Group 3 had a significantly higher threshold shift at all stimuli when compared with groups 1 and 4. There was no significant threshold shifts in all stimuli between groups 2 and 3. The resveratrol-treated group 1 showed preservation of threshold in ABR (p ≤ 0.05). SEM showed that inner and outer hair cells were preserved in the group 1. Level of reactive oxygen species (ROS) were significantly higher in groups 2 and 3 compared with groups 1 and 4 (p ≤ 0.05). CONCLUSION These results indicated that systemic administration of resveratrol afforded statistically significant protection to the cochlea of guinea pigs from cisplatin toxicity. Experimental dose of resveratrol injections may have a protective effect against cisplatin ototoxicity in guinea pigs.

Aqueous garlic extract alleviates ischaemia‐reperfusion‐induced oxidative hepatic injury in rats

This study was designed to examine the effects of aqueous garlic extract (AGE) on hepatic ischaemia‐reperfusion (I/R) injury in rats. For this purpose, Wistar albino rats were subjected to 45 min of hepatic ischaemia, followed by a 60‐min reperfusion period. AGE (1 mL kg−1, i.p., corresponding to 500 mg kg−1) or saline was administered twice, 15 min before ischaemia and immediately before the reperfusion period. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined to assess liver functions. Liver tissues were taken for the determination of malondialdehyde (MDA) levels, an end product of lipid peroxidation; glutathione (GSH) levels, a key antioxidant; and myeloperoxidase (MPO) activity, as an indirect index of neutrophil infiltration. Hepatic collagen content, as a fibrosis marker, was also determined. Plasma ALT and AST activities were elevated in the I/R group as compared with the control group, while these increases were significantly decreased by AGE treatment. Hepatic GSH levels, significantly depressed by I/R, were elevated back to control levels in the AGE‐treated I/R group. Increases in tissue MDA levels and MPO activity due to I/R injury were reduced back to control levels by AGE treatment. Similarly, increased hepatic collagen content in the I/R group was reduced to the control level with AGE treatment. Since AGE administration alleviated the I/R‐induced injury of the liver and improved the hepatic structure and function, it seems likely that AGE, with its antioxidant and oxidant‐scavenging properties, may be of potential therapeutic value in protecting the liver against oxidative injury due to ischaemia‐reperfusion.

Effects of statins on experimental colitis in normocholesterolemic rats.

Objective. The results of previous studies suggest that statins have a direct anti-inflammatory effect that is not directly related to their cholesterol-lowering activity. The aim of this study was to investigate the effect of simvastatin (SIM) and fluvastatin (FLU) on trinitrobenzene sulfonic acid (TNBS)-induced colonic inflammation in rats. Material and methods. The drugs were given for 3 days (0.1 and 1 mg/kg day−1; intraperitoneally) after induction of colitis. The lesions in the distal colon were scored at the macroscopic and microscopic level. Tissue malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and collagen content were assessed and formation of reactive oxygen species and peroxynitrite was monitored by chemiluminescence (CL) assay. Trunk blood was collected for the measurement of serum tumor necrosis factor (TNF)-α level. Results.Treatment with SIM reduced the lesion score of the colitis group at macroscopic level (p<0.05), but there was no effect of treatment with FLU. The increase in colonic MDA level of the colitis group was reduced by both drugs at all doses (p<0.05–0.001). The decrease in GSH and the an increase in MPO activity in the colitis group were reversed by SIM at all doses (p<0.01), but FLU had no effect. An increase in colonic lucigenin CL value in the colitis group was reduced by SIM and FLU at all doses (p<0.001) and an increase in peroxynitrite ratio in the colitis group showed a significant reduction in SIM-treated groups; FLU reduced this effect at a dose of 1 mg/kg (p<0.01). An increase in tissue collagen content and serum TNF-α level in the colitis group was reversed by both drugs at all doses (p<0.001). Conclusions. SIM and FLU seemed to be beneficial in a TNBS-induced rat colitis model through the prevention of lipid peroxidation, superoxide generation, cytokine production and neutrophil accumulation.

Synthesis and characterization of poly(ethylene glycol)-poly(D,L-lactide-co-glycolide) poly(ethylene glycol) tri-block co-polymers modified with collagen : a model surface suitable for cell interaction

This study focused on the synthesis and characterization of poly(ethylene glycol)poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) tri-block co-polymer (PEG-PDLLG-PEG), and its modification with type-I collagen. To this aim, a PEG-PDLLG-PEG tri-block co-polymer was synthesized in two steps by reacting poly(ethylene glycol)bis(carboxymethyl)ether with thionyl chloride to obtain an acyl-halide-terminated poly(ethylene glycol) and subsequently coupling this compound to hydroxyl-terminated poly(D,L-lactide-co-glycolide) (PDLLG). The new carboxyl end-groups of PEG-PDLLG-PEG were subsequently reacted with N-hydroxysuccinimide (NHS) in the presence of the hetero-bifunctional cross-linking agent dicyclohexylcarbodiimide (DCC) in order to activate the co-polymer for coupling with collagen. PEG-PDLLG-PEG and its activated form PEG-PDLLG-NHS were characterized by Fourier transform infrared (FT-IR) and 1H-NMR spectroscopy. Molecular weights of the polymeric products were determined by SEC. Type-I collagen in phosphate buffer was reacted with PEG-PDLLG-NHS. The resultant product, PEG-PDLLG-Col, was characterized by FT-IR. This biopolymer was used for preparation of a suitable surface for cell growth experiment. To measure the degree of cell proliferation, the films prepared with PDLLG, PEG-PDLLG-NHS and PEG-PDLLG-Col were seeded with L929 mouse fibroblasts. Cell growth was followed by SEM photography and quantitated by the neutral red uptake assay. It was shown that the attachment of collagen significantly increased the number of cells on the co-polymers.

Glutamate and GABA immunocytochemical electron microscopy in the hippocampal dentate gyrus of normal and genetic absence epilepsy rats

It is generally accepted that absence epilepsy results from the impairment of GABAergic and glutamatergic neurotransmission. In particular, besides excessive GABA mediation within the thalamo-cortico-thalamic circuit in absence epilepsy, neuronal networks of the hippocampus have recently received attention. In the present study, we examined the density of glutamate and GABA neurotransmitter immunolabeling in the dentate gyrus of the hippocampus of genetic absence epilepsy rats from Strasbourg (GAERS) compared to the control group. GABA and glutamate were found to exist in synaptic vesicles of the mossy fiber terminals of the control and GAERS groups. The density of glutamate immunolabeling within the mossy fiber terminals in the hilar region of GAERS hippocampus was found to be significantly decreased compared to the control group. There was no difference in the density of immunolabeling within GABA nerve terminals between GAERS and control group. The findings of this study suggest that mechanisms underlying absence seizures in GAERS may also manifest themselves in other brain regions such as the hippocampus. The presence of GABA within synaptic vesicles of mossy fiber terminals, as revealed by high resolution ultrastructural immunocytochemistry, has provided additional evidence to the possible modulatory role of GABA on synaptic transmission between the mossy fiber and the target cell.