Occlusion Model Of Cerebral Ischemia Research Articles

Reduction of Astrocyte and Microglial Activation in Insulin/Saline‐treated Diabetic Ketoacidosis by the KCa3.1 Blocker TRAM‐34

Previous studies have provided evidence that diabetic ketoacidosis (DKA) and/or its treatment with insulin and saline (I/S) causes brain injury in children. However, mechanisms underlying DKA‐related brain injury have been poorly understood. In recent studies using immunohistochemistry (IHC) in a streptozotocin (STZ)‐induced DKA juvenile rat model, we examined expression of glial fibrillary acidic protein (GFAP) and CD‐11b protein (OX42) in hippocampus, cortex and striatum at 4, 24 and 72 hr after I/S treatment. We found increased GFAP in hippocampus as well as increased OX42 in both hippocampus and cortex, suggesting that reactive gliosis and microglial activation occur during DKA‐related brain injury. Other studies have shown that TRAM‐34, a KCa3.1 channel blocker, reduces microglial activation in the rat middle cerebral artery occlusion model of cerebral ischemia. The present study was conducted to determine whether TRAM‐34 could also reduce DKA‐related inflammatory changes in the brain. We used IHC to investigate the effects of TRAM‐34 on GFAP and OX‐42 expression in brain sections from juvenile rats with STZ‐induced DKA before, during and after treatment with insulin and saline. As in previous studies, OX42 staining intensity was increased in the hippocampus and cortex during DKA and increased further 4 hours after I/S treatment in a manner sustained through 24 and 72 hours after recovery from DKA. GFAP staining intensity was also increased over the same time course in hippocampus but not cortex. However, in rats administered TRAM‐34 (IP, 40 mg/kg) at the start of I/S treatment, staining intensities of both OX42 and GFAP were significantly reduced at all time points after DKA. OX42 staining intensity was also decreased during DKA prior to I/S treatment in TRAM‐34 treated rats. We conclude that DKA causes inflammatory changes in the brain including reactive gliosis and activation of microglia. Treatment with TRAM‐34 not only decreases microglial activation but also reduces reactive gliosis, suggesting an overall decrease in the brain's inflammatory response to DKA.Support or Funding InformationSupported by ADA

Neuroprotective Effects of Bone Marrow Mesenchymal Stem Cells on Bilateral Common Carotid Arteries Occlusion Model of Cerebral Ischemia in Rat.

Cell therapy is the most advanced treatment of the cerebral ischemia, nowadays. Herein, we discuss the neuroprotective effects of bone marrow mesenchymal stem cells (BMSCs) on rat hippocampal cells following intravenous injection of these cells in an ischemia-reperfusion model. Adult male Wistar rats were divided into 5 groups: control, sham (surgery without blockage of common carotid arteries), ischemia (common carotid arteries were blocked for 30 min prior to reperfusion), vehicle (7 days after ischemia PBS was injected via the tail vein), and treatment (injections of BMSC into the tail veins 7 days after ischemia). We performed neuromuscular and vestibulomotor function tests to assess behavioral function and, finally, brains were subjected to hematoxylin and eosin (H&E), anti-Brdu immunohistochemistry, and TUNEL staining. The ischemia group had severe apoptosis. The group treated with BMSCs had a lower mortality rate and also had significant improvement in functional recovery (P < 0.001). Ischemia-reperfusion for 30 min causes damage and extensive neuronal death in the hippocampus, especially in CA1 and CA3 regions, leading to several functional and neurological deficits. In conclusion, intravenous injection of BMSCs can significantly decrease the number of apoptotic neurons and significantly improve functional recovery, which may be a beneficial treatment method for ischemic injuries.

Gel-based Protease Proteomics for Identifying the Novel Calpain Substrates in Dopaminergic Neuronal Cell

Calpains are a family of calcium-dependent cysteine proteases that are ubiquitously expressed in mammals and play critical roles in neuronal death by catalyzing substrate proteolysis. Here, we developed two-dimensional gel electrophoresis-based protease proteomics to identify putative calpain substrates. To accomplish this, cellular lysates from neuronal cells were first separated by pI, and the immobilized sample on a gel strip was incubated with a recombinant calpain and separated by molecular weight. Among 25 altered protein spots that were differentially expressed by at least 2-fold, we confirmed that arsenical pump-driving ATPase, optineurin, and peripherin were cleaved by calpain using in vitro and in vivo cleavage assays. Furthermore, we found that all of these substrates were cleaved in MN9D cells treated with either ionomycin or 1-methyl-4-phenylpyridinium, both of which cause a calcium-mediated calpain activation. Their cleavage was blocked by calcium chelator or calpain inhibitors. In addition, calpain-mediated cleavage of these substrates and its inhibition by calpeptin were confirmed in a middle cerebral artery occlusion model of cerebral ischemia, as well as a stereotaxic brain injection model of Parkinson disease. Transient overexpression of each protein was shown to attenuate 1-methyl-4-phenylpyridinium-induced cell death, indicating that these substrates may confer protection of varying magnitudes against dopaminergic injury. Taken together, the data indicate that our protease proteomic method has the potential to be applicable for identifying proteolytic substrates affected by diverse proteases. Moreover, the results described here will help us decipher the molecular mechanisms underlying the progression of neurodegenerative disorders where protease activation is critically involved.

Seizure activity in the rat hippocampus, perirhinal and prefrontal cortex associated with transient global cerebral ischemia

Epileptiform EEG activity associated with ischemia can contribute to early damage of hippocampal neurons, and seizure activity may also lead to dysfunction in extrahippocampal regions. In this study, seizure activity associated with the four-vessel occlusion model of cerebral ischemia was monitored using chronically implanted electrodes in the CA1/subicular region, the perirhinal cortex, and the prefrontal cortex of the rat. Background EEG amplitude was reduced in all recording sites during occlusion, but spiking and bursting activity was also observed. Seizure activity occurred in most animals during the first several hours of reperfusion, but was not observed on subsequent days. Epileptiform spikes and bursts were often synchronous between two or three recording sites, and spikes in the CA1 region also often occurred just prior to spikes in other sites. These results demonstrate that the four-vessel occlusion model can lead to patterns of seizure activity in the hippocampus, prefrontal and perirhinal cortices.

Protective effect of exogenous administration of alpha-tocopherol in middle cerebral artery occlusion model of cerebral ischemia in rats.

Oxidative stress due to increased free radical generation and depletion of endogenous antioxidants have been implicated in ischemia-reperfusion-induced neuronal injury. The present study was carried out to study the effect of acute administration of alpha-tocopherol in the middle cerebral artery (MCA) occlusion model of stroke in rats. Rats were anesthetized using chloral hydrate (400 mg/kg i.p.) and subjected to 2 h of transient MCA occlusion. alpha-Tocopherol was administered at the dose of 125 and 250 mg/kg orally 1 h prior to the occlusion of MCA. Motor performance test (grip test, foot fault test, rotarod performance test, spontaneous locomotor activity), markers of oxidative stress and 2,3,5-triphenyl tetrazolium chloride staining were carried out 24 h after MCA occlusion. A vehicle-treated group was run parallel. It was observed that alpha-tocopherol at the dose of 125 mg/kg neither improved neurologic deficit, nor decreased the raised level of oxidative stress markers in comparison with the MCA-occluded rats. However, higher dose of alpha-tocopherol (250 mg/kg p.o.) afforded significant protection as evident by increase in motor performance tests and a decrease in the volume of infarction. The raised levels of malondialdehyde after MCA occlusion were also significantly attenuated. The results demonstrate that exogenous administration of alpha-tocopherol is able to reduce the neuronal damage caused during ischemia-reperfusion, which can be attributed to its antioxidant activity.

Differential effects of phospholipase inhibitors on free fatty acid efflux in rat cerebral cortex during ischemia-reperfusion injury

Free fatty acid (FFA) elevation in the brain has been shown to correlate with the severity of damage in ischemic injury. The etiology of this increase in FFA remains unclear and has been hypothesized to result from phospholipase activation. This study examines the effects of specific phospholipase inhibitors on FFA efflux during ischemia-reperfusion injury. A four-vessel occlusion model of cerebral ischemia was utilized to assess the effects of PLA 2 and PLC inhibitors on FFA efflux from rat cerebral cortex. In addition, FFA efflux from non-ischemic cortices exposed to PLA 2 and PLC was measured. Concentrations of arachidonic, docosahexaenoic, linoleic, myristic, oleic, and palmitic acids in cortical superfusates were determined using high performance liquid chromatography (HPLC). Exposure to the non-selective PLA 2 inhibitor 4-bromophenylacyl bromide (BPB) significantly inhibited FFA efflux during ischemia-reperfusion injury ( P<0.01 arachidonic, oleic and palmitic; P<0.05 all others); exposure to the PLC inhibitor U73122 had no observed effect. The effects of the Ca 2+-dependent PLA 2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF 3) mirrored the effects of BPB and led to reductions in all FFA levels ( P<0.01 arachidonic, oleic and palmitic; P<0.05 all others). Exposure to the secretory PLA 2 inhibitor 3-(3-acetamide-1-benzyl-2-ethyl-indolyl-5-oxy) propane sulfonic acid (LY311727) and to the Ca 2+-independent PLA 2 inhibitor bromoenol lactone (BEL) had only minimal effects on FFA efflux. Application of both PLA 2 and PLC to non-ischemic cortices resulted in significant increases in efflux of all FFA ( P<0.05). The study suggests that FFA efflux during ischemia-reperfusion injury is coupled to activation of Ca 2+-dependent PLA 2 and provides further evidence of the potential neuroprotective benefit of Ca 2+-dependent PLA 2 inhibitors in ischemia.

Design and pharmacological activity of phosphinic acid based NAALADase inhibitors.

A novel series of phosphinic acid based inhibitors of the neuropeptidase NAALADase are described in this work. This series of compounds is the most potent series of inhibitors of the enzyme described to date. In addition, we have shown that these compounds are protective in animal models of neurodegeneration. Compound 34 significantly prevented neurodegeneration in a middle cerebral artery occlusion model of cerebral ischemia. In addition, in the chronic constrictive model of neuropathic pain, compound 34 significantly attenuated the hypersensitivity observed with saline-treated animals. These data suggest that NAALADase inhibition may provide a new approach for the treatment of both neurodegenerative disorders and peripheral neuropathies.

Changes in mRNA for ryanodine receptors after transient cerebral ischemia and tolerance induction

AbstractDisturbance of Ca2+ homeostasis is assumed to precede ischemia‐induced neuronal death. Using the 4‐vessel occlusion model of cerebral ischemia and induction of ischemic tolerance in the rat, we studied the intracellular Ca2+ activated Ca2+ channel, the ryanodine receptor (RyR), by means of in situ hybridisation and immunohistochemistry. We found the level of RyR2 mRNA increased in CA1 and CA3 of the ischemic vulnerable hippocampus following 3 minutes of tolerance inducing ischemia, whereas 9 minutes of ischemia lead to an increased mRNA level in CA1 in contrast to a decrease in CA3. In ischemic tolerant animals (3 + 8.5 minutes of ischemia) the mRNA level was decreased in CA3. In all experimental groups, RyR1 mRNA remained unaltered, whereas RyR3 mRNA decreased in DG. Immunohistochemistry revealed no alterations of the RyR protein level in all the ischemic groups compared to sham‐operated animals. Taken together, these changes do not clarify if the RyR is involved in tolerance induction or in the degenerative process leading to ischemic delayed neuronal death.

Baclofen does not protect against cerebral ischemia in rats.

Presynaptic release of glutamate into the extracellular compartment and activation of receptor-operated calcium channels may contribute to ischemic neuronal damage. We evaluated the effect of baclofen, a selective inhibitor of presynaptic glutamate release, on mortality, working memory, and light microscopic hippocampal and cortical damage in the four-vessel occlusion model of cerebral ischemia using 64 male Wistar rats. Baclofen (10 mg/kg i.p.) given 1 hour before and 30-60 minutes after 20 minutes of global ischemia did not lessen mortality, prevent ischemic cellular damage, or significantly improve working memory compared with no treatment. We conclude that preischemic and postischemic administration of baclofen does not protect neurons from ischemic injury.

P-089 - Alteration of baroreceptor reflex in the rat four-vessel occlusion model of cerebral ischemia

P-089 - Alteration of baroreceptor reflex in the rat four-vessel occlusion model of cerebral ischemia

Moderate hypothermia reduces postischemic edema development and leukotriene production.

Using the bilateral carotid artery occlusion model of cerebral ischemia in the gerbil, we studied the effect of moderate hypothermia (30 to 31 degrees C) on the postischemic production of prostanoids (cyclooxygenase pathway) and leukotrienes (lipoxygenase pathway) and accompanying changes in cerebral edema formation. Hypothermia capable of slowing central evoked potential conduction time was studied over the course of 40 minutes of cerebral ischemia and for up to 2 hours of reperfusion. The successful induction of cerebral ischemia was confirmed by somatosensory evoked potential amplitude changes. Measurements of 6-ketoprostaglandin F1 alpha (PGF1 alpha) and leukotriene B4 (LTB4) (radioimmunoassay) and cerebral edema (specific gravity) were made at early (10 minutes) and late (2 hours) reperfusion times. Although both white and gray matter showed no early significant difference in edema accumulation between normothermic and hypothermic gerbils at 10 minutes of reperfusion, hypothermic animals demonstrated significantly less white matter edema (specific gravity, 1.0397 +/- 0.0010 vs. 1.0341 +/- 0.0012, P less than 0.01) and gray matter edema (specific gravity, 1.0408 +/- 0.0009 vs. 1.0365 +/- 0.0008, P less than 0.01) by 2 hours of reperfusion. Production of PGF1 alpha was not significantly different between normothermic and hypothermic animals during the reperfusion period; however, hypothermic gerbils demonstrated significantly lower production of LTB4 at 10 minutes reperfusion time compared to normothermic animals (1.49 +/- 0.79 vs. 5.28 +/- 1.49 pg/mg of protein, P less than 0.05). This difference between the two groups in LTB4 levels was no longer detectable at 2 hours of reperfusion time.(ABSTRACT TRUNCATED AT 250 WORDS)