Paraspecific neutralization capacity of polyvalent snake antivenom against Montivipera wagneri venom.

PURPOSE: This study aims to measure the paraspecific neutralization capacity of nationally produced HSGM polyvalent snake antivenom (HSGM-PSAV), produced using Macrovipera lebetina obtusa, Montivipera xanthina, and Vipera ammodytes montandoni venom, against the lethal effect of the venom of Montivipera wagneri, which is endemic to the Eastern Black Sea and Eastern Anatolia regions of Turkey. METHODS: The neutralization capacity of HSGM-PSAV against the lethal effect of M. wagneri venom was studied using the potency determination testing method specified in the Turkish and European Pharmacopoeia. Lethal dose 50 (LD50) values of the venoms used in immunization, M. wagneri venom in mice, and effective dose 50 (ED50) values of HSGM-PSAV against four types of venoms were calculated using two-fold dilutions. RESULTS: HSGM-PSAV neutralized the lethal effect of M. wagneri venom in mice. The ED50 of the HSGM-PSAV against M. wagneri venom was calculated as 304.42 LD50/mL. CONCLUSION: As a result of this in-vivo study, it was determined that HSGM-PSAV neutralized M.wagneri venom above the antivenom neutralization capacity threshold values (≥50 LD50/mL) specified in the Turkish and European Pharmacopoeia. This result is important preclinical data regarding the usability of HSGM-PSAV in the treatment of poisoning due to M. wagneri bites.

Composition characterization of various viperidae snake venoms using MS-based proteomics N-glycoproteomics and N-glycomics.

Viperidae snake species is widely abundant and responsible for most envenomation cases in Turkey. The structural and compositional profiles of snake venom have been investigated to study the venom component variation across different species and to profile the venom biological activity variation against prey. In this context, we used proteomics, glycoproteomics and glycomics strategies to characterize the protein, glycoproteins and glycan structural and compositional profiles of various snake venoms in the Viperidae family. Moreover, we compared these profiles using the downstream bioinformatics and machine learning classification modules. The overall mass spectrometry profiles identified 144 different proteins, 36 glycoproteins and 78 distinct N-glycan structures varying in composition across the five venoms. A high amount of the characterized proteins belongs to the glycosylated protein family Trypsin-like serine protease (Tryp_SPc), Disintegrin (DISIN), and ADAM Cysteine-Rich (ACR). Most identified N-glycans have a complex chain carrying galactosylated N-glycans abundantly. The glycan composition data obtained from glycoproteomics aligns consistently with the findings from glycomics. The clustering and principal component analyses (PCA) illustrated the composition-based similarities and differences between each snake venom species' proteome, glycoproteome and glycan profiles. Specifically, the N-glycan profiles of M. xanthina (Mx) and V. a. ammodytes (Vaa) venoms were identical and difficult to differentiate; in contrast, their proteome profiles were distinct. Interestingly, the variety of the proteins across the species highlighted the impact of glycosylation on the diversity of the glycosylated protein families. This proposed high throughput approach provides accurate and comprehensive profiles of the composition and function of various Viperidae snake venoms.

Comparative effects of methylprednisolone and tetracosactide (ACTH1-24) on ischemia/reperfusion injury of the rabbit spinal cord.

INTRODUCTION: Tetracosactide is an engineered peptide that applies the same biological impacts as the endogenous adrenocorticotropic hormone. Previous studies indicated that tetracosactide has anti-inflammatory, antioxidant and neurotrophic activity. In this study, we hypothesized that tetracosactide may have protective effects in spinal cord ischemia-reperfusion injury. MATERIAL AND METHODS: Rabbits were randomized into the accompanying four groups of eight animals each: group 1 (control), group 2 (ischemia), group 3 (methylprednisolone) and group 4 (tetracosactide). In the control group, just a laparotomy was performed. In the various groups, the spinal cord ischemia model was made by the impediment of the aorta only caudal to the renal vein. Neurological assessment was conducted with the Tarlov scoring system. Levels of myeloperoxidase, malondialdehyde and catalase were analyzed, similar to the activities of xanthine oxidase and caspase-3. Histopathological and ultrastructural assessments were additionally performed. RESULTS: After ischemia-reperfusion injury, increments were found in the tissue myeloperoxidase levels (p < 0.001), malondialdehyde levels (p < 0.001), xanthine oxidase action (p < 0.001) and caspase-3 movement (p < 0.001). Conversely, both serum and tissue catalase levels were diminished (p < 0.001 for both). After the administration of tetracosactide, declines were seen in the tissue myeloperoxidase levels (p < 0.001), malondialdehyde levels (p = 0.003), xanthine oxidase action (p < 0.001) and caspase-3 movement (p < 0.001). Conversely, both the serum and tissue catalase levels were expanded (p < 0.001). Besides, tetracosactide treatment indicated enhanced results related to the histopathological scores (p < 0.001), the ultra-structural score (p = 0.008) and the Tarlov scores (p < 0.001). CONCLUSIONS: The findings showed for the first time that tetracosactide shows significant neuroprotective activity against ischemia-reperfusion injury of the spinal cord.

Comparative effects of vitamin D and methylprednisolone against ischemia/reperfusion injury of rabbit spinal cords.

INTRODUCTION: Recent studies have demonstrated the neuroprotective and immunomodulatory effects of 1,25-dihydroxyvitamin D3 (calcitriol), but no previous study has examined these effects on spinal cord ischemia/reperfusion (I/R) injury. Therefore, the present study aimed to evaluate whether calcitriol protects the spinal cord from I/R injury. METHODS: Rabbits were randomized into four groups of eight animals: group 1 (laparotomy control), group 2 (ischemia control), group 3 (30mg/kg intraperitoneal methylprednisolone at surgery), and group 4 (0.5µg/kg, intraperitoneal calcitriol for 7 days before I/R injury). The rabbits in the laparotomy control group underwent laparotomy only, whereas all rabbits in the other groups were subject to spinal cord ischemia by aortic occlusion for 20min, just caudal to the renal artery. Malondialdehyde and catalase levels, myeloperoxidase and xanthine oxidase activities, and caspase-3 concentrations were analyzed. Finally, histopathological, ultrastructural, and neurological evaluations were performed. RESULTS: After I/R injury, increases in malondialdehyde levels, myeloperoxidase and xanthine oxidase activities, and caspase-3 concentrations were found (p<0.001 for all); by contrast, catalase levels decreased (p<0.001). Calcitriol pretreatment was associated with lower malondialdehyde levels (p<0.001), reduced myeloperoxidase (serum, p=0.018; tissue, p<0.001) and xanthine oxidase (p<0.001) activities, and caspase-3 concentrations (p<0.001), but increased catalase levels (p<0.001). Furthermore, calcitriol pretreatment was associated with better histopathological, ultrastructural, and neurological scores. CONCLUSION: Calcitriol pretreatment provided significant neuroprotective benefits following spinal cord I/R injury.

Antioxidant and antiapoptotic effects of darbepoetin-α against traumatic brain injury in rats.

INTRODUCTION: In this study, we tried to determine whether darbepoetin-α would protect the brain from oxidative stress and apoptosis in a rat traumatic brain injury model. MATERIAL AND METHODS: The animals were randomized into four groups; group 1 (sham), group 2 (trauma), group 3 (darbepoetin α), group 4 (methylprednisolone). In the sham group only the skin incision was performed. In all the other groups, a moderate traumatic brain injury modelwas applied. RESULTS: Following trauma both glutathione peroxidase, superoxide dismutase levels decreased (p < 0.001 for both); darbepoetin-α increased the activity of both antioxidant enzymes (p = 0.001 and p < 0.001 respectively). Trauma caused significant elevation in the nitric oxide synthetase and xanthine oxidase levels (p < 0.001 for both). Administration of darbepoetin-α significantly decreased the levels of nitric oxide synthetase and xanthine oxidase (p < 0.001 for both). Also, trauma caused significant elevation in the nitric oxide levels (p < 0.001); darbepoetin-α administration caused statistically significant reduction in the nitric oxide levels (p < 0.001). On the other hand, malondialdehyde levels were increased following trauma (p < 0.001), and darbepoetin α significantly reduced the malondialdehyde levels (p < 0.001). Due to the elevated apoptotic activity following the injury, caspase-3 activity increased significantly. Darbepoetin-α treatment significantly inhibited apoptosis by lowering the caspase-3 activity (p < 0.001). In the darbepoetin group, histopathological score was lower than the trauma group (p = 0.016). CONCLUSIONS: In this study, darbepoetin-α was shown to be at least as effective as methylprednisolone in protecting brain from oxidative stress, lipid peroxidation and apoptosis.

Vasoconstrictive effects of levobupivacaine on the basilar artery in the rabbit.

INTRODUCTION: Spinal anesthesia is a widely used technique of the modern practice of anesthesia. Spinal cord ischemia is a rare but catastrophic complication of spinal anesthesia which may be caused by a direct vasoconstrictive effect of the local anesthetic. Although the vasoconstrictive effects of levobupivacaine have been widely studied, the vasoconstrictive effects of this drug on the intradural arteries have never been studied. The aim of this study is to evaluate whether levobupivacaine has vasoconstrictive effects on the basilar artery in rabbits. MATERIAL AND METHODS: Thirty male New Zealand white rabbits were divided randomly into three groups of ten rabbits each: group 1 (control); group 2 (0.125% levobupivacaine); group 3 (0.25% levobupivacaine). The cisterna magna was punctured as described below, then 1 ml of saline or 0.125% or 0.25% levobupivacaine was injected into the cisterna magna in 10 min by an infusion pump in groups 1, 2 and 3 respectively. All animals were euthanized by perfusion-fixation 30 min after the procedure. The luminal area and the size of the cross-sectional area for each basilar artery were measured. RESULTS: Both 0.125% and 0.25% levobupivacaine infusion caused significant vasoconstriction. Vasoconstriction was more significant for the 0.125% concentration. CONCLUSIONS: The results of this study indicated that both 0.125% and 0.25% concentrations of levobupivacaine caused significant vasoconstriction of the basilar artery when administered into the subarachnoid space. This may constitute proof that subarachnoid administration of levobupivacaine may diminish the spinal cord blood flow, causing ischemia.

The comparative effects of recombinant human erythropoietin and darbepoetin-alpha on cerebral vasospasm following experimental subarachnoid hemorrhage in the rabbit.

BACKGROUND: Darbepoetin alpha is a hypersialylated analogue of erythropoietin effective for activating erythropoietin-receptors. This study investigated the vasodilator and neuroprotective effects of darbepoetin alpha on an experimental subarachnoid hemorrhage model and compared it with erythropoietin. METHODS: Forty adult male New Zealand white rabbits were randomly divided into four groups of ten rabbits each: group 1 (control), group 2 (subarachnoid hemorrhage), group 3 (erythropoietin), and group 4 (darbepoetin alpha). Recombinant human erythropoietin was administered at a dose of 1,000 U/kg intraperitoneally after the induction of subarachnoid hemorrhage and continued every 8 h up to 72 h. Darbepoetin alpha was administered at a single intraperitoneal dose of 30 µg/kg. Animals were killed 72 h after subarachnoid hemorrhage. Basilar artery cross-sectional areas, arterial wall thicknesses, hippocampal degeneration scores and biochemical analyses were measured in all groups. RESULTS: Both erythropoietin and darbepoetin alpha treatments were found to attenuate cerebral vasospasm and provide neuroprotection after subarachnoid hemorrhage in rabbits. Darbepoetin alpha revealed better morphometric and histopathological results than erythropoietin among experimental subarachnoid hemorrhage-induced vasospasm. CONCLUSIONS: Our findings, for the first time, showed that darbepoetin alpha can prevent vasospasm and provides neuroprotection following experimental subarachnoid hemorrhage. Moreover, darbepoetin alpha showed better results when compared with erythropoietin.

The protective effect of low-dose methotrexate on ischemia-reperfusion injury of the rabbit spinal cord.

Methotrexate was developed as a cytostatic agent, but at low doses, it has shown potent anti-inflammatory activity. Previous studies have demonstrated that the anti-inflammatory effects of methotrexate are primarily mediated by the release of adenosine. In this study, we hypothesized that low-dose methotrexate has protective effects in spinal cord ischemia-reperfusion injury. Rabbits were randomized into the following four groups of eight animals each: group 1 (control), group 2 (ischemia), group 3 (methylprednisolone) and group 4 (methotrexate). In the control group only a laparotomy was performed. In all the other groups, the spinal cord ischemia model was created by the occlusion of the aorta just caudal to the renal artery. Neurological evaluation was performed with the Tarlov scoring system. Levels of myeloperoxidase, malondialdehyde and catalase were analyzed, as were the activities of xanthine oxidase and caspase-3. Histopathological and ultrastructural evaluations were also performed. After ischemia-reperfusion injury, increases were found in the serum and tissue myeloperoxidase levels, tissue malondialdehyde levels, xanthine oxidase activity and caspase-3 activity. In contrast, both serum and tissue catalase levels were decreased. After the administration of a low-dose of methotrexate, decreases were observed in the serum and tissue myeloperoxidase levels, tissue malondialdehyde levels, xanthine oxidase activity and caspase-3 activity. In contrast, both the serum and tissue catalase levels were increased. Furthermore, low-dose methotrexate treatment showed improved results concerning the histopathological scores, the ultrastructural score and the Tarlov scores. Our results revealed that low-dose methotrexate exhibits meaningful neuroprotective activity following ischemia-reperfusion injury of the spinal cord.

The protective effect of 2-mercaptoethane sulfonate (MESNA) against traumatic brain injury in rats.

BACKGROUND: The agent, 2-mercaptoethane sulfonate (MESNA), is a synthetic small molecule, widely used as a systemic protective agent against chemotherapy toxicity, but is primarily used to reduce hemorrhagic cystitis induced by cyclophosphamide. Because MESNA has potential antioxidant and cytoprotective effects, so we hypothesized that MESNA may protect the brain against traumatic injury. METHOD: Thirty-two rats were randomized into four groups of eight animals each; Group 1 (sham), Group 2 (trauma), Group 3 (150 mg/kg MESNA), Group 4 (30 mg/kg methylprednisolone). Only skin incision was performed in the sham group. In all the other groups, the traumatic brain injury model was created by an object weighing 450 g falling freely from a height of 70 cm through a copper tube on to the metal disc over the skull. The drugs were administered immediately after the injury. The animals were killed 24 h later. Brain tissues were extracted for analysis, where levels of tissue malondialdehyde, caspase-3, glutathione peroxidase, superoxide dismutase, nitric oxide, nitric oxide synthetase and xanthine oxidase were analyzed. Also, histopathological evaluation of the tissues was performed. RESULTS: After head trauma, tissue malondialdehyde levels increased; these levels were significantly decreased by MESNA administration. Caspase-3 levels were increased after trauma, but no effect of MESNA was determined in caspase-3 activity. Following trauma, both glutathione peroxidase and superoxide dismutase levels were decreased; MESNA increased the activity of both these antioxidant enzymes. Also, after trauma, nitric oxide, nitric oxide synthetase and xanthine oxidase levels were increased; administration of MESNA significantly decreased the levels of nitric oxide, nitric oxide synthetase and xanthine oxidase, promising an antioxidant activity. Histopathological analysis showed that MESNA protected the brain tissues well from injury. CONCLUSIONS: Although further studies considering different dose regimens and time intervals are required, MESNA was shown to be at least as effective as methylprednisolone in the traumatic brain injury model.

Effects of darbepoetin-α in spinal cord ischemia-reperfusion injury in the rabbit.

BACKGROUND: Darbepoetin-alpha (DA) is a novel erythropoiesis-stimulating agent developed for treating anemia. In animal models, recombinant human erythropoietin has been reported to be beneficial for neuroprotection. In this study, we determined whether DA would protect the spinal cord against ischemia-reperfusion injury in a rabbit model. METHODS: Forty rabbits were randomized into five groups of eight animals each: group 1 (sham), group 2 (ischemia), group 3 (vehicle), group 4 (30 mg/kg methylprednisolone), group 5 (30 µg/kg DA). Only laparotomy was performed in the sham group. In all the other groups, the spinal cord ischemia model was created by a 20-min occlusion of the aorta just caudal to renal artery with an aneurysm clip. The drugs were administered immediately after the clamp was removed. The animals were killed 24 h later. Spinal cord segments between L2 and L5 were harvested for analysis. Neurological evaluation was performed with the Tarlov scoring system just before the animals were killed. Level of tissue malondialdehyde was analyzed as a marker of lipid peroxidation and tissue caspase-3 activity as a marker of apoptosis. Also, histopathological evaluation of the tissues was performed. RESULTS: Both malondialdehyde and caspase-3 levels were significantly decreased by DA administration. Histopathological evaluation of the tissues also demonstrated decrease in neuronal degeneration and infiltration parameters after DA administration. In the DA group, neurological outcome scores were statistically significantly better compared with the ischemia and the vehicle groups. CONCLUSIONS: Although further studies considering different dose regimens and time intervals are required, DA was shown to be at least as effective as methylprednisolone in spinal cord ischemia/reperfusion model.