Cerebral Ischemia In Rat Brain Research Articles

Repeated Oral Administration of Human Serum Albumin Protects from the Cerebral Ischemia in Rat Brain Following MCAO.

Albumin is known to have neuroprotective effects. The protein has a long half-life circulation, and its effects can therefore persist for a long time to aid in the recovery of brain ischemia. In the present study, we investigated the neuroprotective effects of human serum albumin (HSA) on brain hemodynamics. Albumin is administrated using repeated oral gavage to the rodents. Sprague-Dawley rats underwent middle cerebral artery occlusion procedures and served as a stroke model. Afterwards, 25% human serum albumin (1.25 g/kg) or saline (5 ml/kg) was orally administrated for 2 weeks in alternating days. After 2 weeks, the rodents were assessed for levels of brain ischemia. Our testing battery consists of behavioral tests and in vivo optical imaging sessions. Modified neurological severity scores (mNSS) were obtained to assess the levels of ischemia and the effects of HSA oral administration. We found that the experimental group demonstrated larger hemodynamic responses following sensory stimulation than controls that were administered with saline. HSA administration resulted in more significant changes in cerebral blood volume following direct cortical electric stimulation. In addition, the mNSS of the treatment group was lower than the control group. In particular, brain tissue staining revealed that the infarct size was also much smaller with HSA administration. This study provides support for the efficacy of HSA, and that long-term oral administration of HSA may induce neuroprotective effects against brain ischemia.

Comparison between single and combined post-treatment with S-Methyl-N,N-diethylthiolcarbamate sulfoxide and taurine following transient focal cerebral ischemia in rat brain

We have recently reported on the efficacy of an N-methyl-d-aspartate (NMDA) receptor partial antagonist, S-Methyl-N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO), in improving outcome following stroke, including reduced infarct size and calcium influx, suppressing the endoplasmic reticulum (ER) stress-induced apoptosis as well as improving behavioral outcome. DETC-MeSO was shown to suppress the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway, one of the major ER stress pathways. Several studies including ours have provided evidence that taurine also has neuroprotective effects through reducing apoptosis and inhibiting activating transcription factor 6 (ATF6) and inositol requiring enzyme 1 (IRE-1) pathways. We hypothesized that a combined treatment with DETC-MeSO and taurine would ameliorate ischemia-induced brain injury by inhibiting all three ER stress pathways. Twenty four hours following reperfusion of a 2-h ischemic stroke, rats received either 0.56-mg/kg DETC-MeSO or 40-mg/kg of taurine, either alone or in combination, subcutaneously for 4days. Our study showed that combined DETC-MeSO and taurine, but not DETC-MeSO alone at the dose used, greatly reduced the infarct size, improved performance on the neuro-score test and attenuated proteolysis of αII-spectrin. Meanwhile, the level of the pro-apoptotic protein, Bax, declined and the anti-apoptotic protein, B-cell lymphoma 2 (BCL-2), expression was markedly increased. Combination therapy decreased both caspase-12 and caspase-3 activation by preventing the release of Cytochrome-c from mitochondria, indicating attenuation of apoptosis in ischemic infarct. Glucose-regulated protein (GRP)78 as a marker of the unfolded protein response decreased and levels of the key ER stress protein markers p-PERK-ATF4, p-eIF2α and cleaved-ATF-6 were found to significantly decline. NeuN expression levels indicated that more neurons were protected in the presence of DETC-MeSO and taurine. We also showed that combined treatment can prevent gliosis and increase p-AKT a pro-survival marker in the penumbra. Therefore, we conclude that combined treatment with both DETC-MeSO and taurine synergistically inhibits all three ER stress pathways and apoptosis and therefore can be a novel and effective treatment after ischemic stroke.

Expression of chemokine-like factor 1 after focal cerebral ischemia in the rat

Cerebral ischemic injury is associated with the induction of a series of pro-inflammatory mediators such as cytokines and chemokines. The chemokine-like factor 1(CKLF1), as a novel human cytokine, displays chemotactic activities in a wide spectrum of leukocytes. The present study was conducted to determine if CKLF1 was produced in the brain of rats subjected to transient middle cerebral artery occlusion (TMCAO). Therefore, RT-PCR, Western blot and immunohistochemistry were utilized to characterize the expression of CKLF1 at different times after TMCAO. The result showed that almost no expression of CKLF1 was found in the sham-operated or contralateral cerebral cortex and hippocampus. CKLF1 expression significantly increased in the ischemic cerebral cortex and hippocampus, elevating at 12 h and peaking at 2 days after reperfusion. CKLF1 positive staining was mainly present in the cerebral cortex, hippocampus, thalamus, and hypothalamus. These results demonstrate that the expression of CKLF1 increases after focal cerebral ischemia in rat brain. Thus, CKLF1 may be a potential therapeutic target for cerebral ischemia.

Regulation of endothelial nitric oxide synthase by agmatine after transient global cerebral ischemia in rat brain

Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) plays a protective role in cerebral ischemia by maintaining vascular permeability, whereas NO derived from neuronal and inducible NOS is neurotoxic and can participate in neuronal damage occurring in ischemia. Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane if brain vessels to promote cell death and tissue injury. We previously reported that agmatine, synthesized from L-arginine by arginine decarboxylase (ADC) which is expressed in endothelial cells, has shown a direct increased eNOS expression and decreased MMPs expression in bEnd3 cells. But, there are few reports about the regulation of eNOS by agmatine in ischemic animal model. In the present study, we examined the expression of eNOS and MMPs by agmatine treatment after transient global ischemia in vivo. Global ischemia was induced with four vessel occlusion (4-VO) and agmatine (100 mg/kg) was administered intraperitoneally at the onset of reperfusion. The animals were euthanized at 6 and 24 hours after global ischemia and prepared for other analysis. Global ischemia led severe neuronal damage in the rat hippocampus and cerebral cortex, but agmatine treatment protected neurons from ischemic injury. Moreover, the level and expression of eNOS was increased by agmatine treatment, whereas inducible NOS (iNOS) and MMP-9 protein expressions were decreased in the brain. These results suggest that agmatine protects microvessels in the brain by activation eNOS as well as reduces extracellular matrix degradation during the early phase of ischemic insult.

Effect of dietary sesame oil as antioxidant on brain hippocampus of rat in focal cerebral ischemia

Oxidative stress may be regarded as an imbalance between free radical production and opposing antioxidant defenses. Free radical oxidative stress is implicated in rat cerebral ischemia and naturaceutical antioxidants are dietary supplements that have been reported to have neuroprotective activity. Many studies have reported dietary sesame oil (SO) as an effective antioxidant. In the present study the neuroprotective effect of dietary SO was evaluated against middle cerebral artery occlusion (MCAO)-induced cerebral ischemia injury in rats. Rats were fed on diet (20% SO) for 15 days. The middle cerebral artery of adult male Wistar rat was occluded for 2 h and reperfused for 22 h. The antioxidant properties of brain were measured as levels of reduced glutathione (GSH), glutathione- S-transferase (GST), glutathione peroxide (GPx), glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD) and thiobarbituric acid reactive substance (TBARS). A decrease in the activity of all the enzymatic and non-enzymatic antioxidants was observed along with an increase in lipid peroxidation (LPO) in MCAO group. The neurobehavioral activity of rats was also observed by using videopath analyzer. Dietary SO improved the antioxidant status in MCAO + SO group when compared with MCAO group. The results of neurobehavioral activity also support our biochemical data. The results obtained suggest protective effect of SO against cerebral ischemia in rat brain through their antioxidant properties.

Redistribution of glucose-6-phosphate dehydrogenase in response to cerebral ischemia in rat brain

Glucose-6-phosphate dehydrogenase (G6PD), a cytoplasmic enzyme, plays a protective role during oxidative stress in eucaryotic cells, since they provide coenzymes and substrates to the primary antioxidant enzymes. The redistribution of G6PD in the hippocampus was studied post-ischemia (PI). There was a characteristic localisation of G6PD in pyramidal cell layers of the rat hippocampus. In hippocampus CA1 cells were stained weakly whereas CA3 cells showed strong histochemical staining. Ischemia induced up-regulation of G6PD in the hippocampus was in a specific manner. First, the activity increased in the whole hippocampus (at 4 hours PI) which persisted 6 hrs PI in CA1 area. However G6PD activity decreased in the CA3 area & dentate gyrus. At 10 & 24 hrs PI, activity decreased in CA1 area but normalised in CA3 area & dentate gyrus compared to controls. This suggests that the sensitive CA1 neurons are transiently capable of generating an anti-oxidative arsenal to cope with the oxidative stress in the first few hours PI. We can conclude that the brain contains inducible endogenous mechanisms that are capable of enhancing the ability of neurons to withstand lethal ischemic challenge.

Effect of intra-ischemic hypothermia on the expression of c-Fos and c-Jun, and DNA binding activity of AP-1 after focal cerebral ischemia in rat brain

It is unknown whether immediate early gene (IEG) induction and subsequent late gene regulation after ischemia is beneficial or deleterious. The aim of this study was to examine the effect of hypothermia on expression of c-Fos and c-Jun, and AP-1 DNA binding activity, after transient focal cerebral ischemia in rat brain, and clarify the role of IEGs and AP-1 after insults. Male Wistar rats underwent right middle cerebral artery occlusion for 1 h with the intraluminal suture method. During ischemia, animals were assigned to either normothermic (NT) or hypothermic (HT) groups. In the NT group, brain temperature was observed to spontaneously increase to 40 °C during ischemia. In the HT group, brain temperature decreased to 30 °C. Infarct volume in cortex was decreased in the HT group, compared with that in the NT group ( P<0.001). Increased c-Fos immunoreactivity in the cortex was observed at 3 h after reperfusion in the HT, but not the NT group, while c-Jun expression was not affected by HT treatment. There was also a significant increase in AP-1 DNA binding activity at 3 h in the HT group when compared to the NT group ( P<0.01). In conclusion, hypothermia decreased cerebral infarction in association with early increases in c-Fos expression and AP-1 DNA binding activity in peri-infarct cortex. It remains to be established whether such responses are a cause or consequence of cell survival, but these results clearly establish that altered transcription is a key feature of tissue spared following hypothermic focal ischemia.

Persistent phosphorylation of cyclic amp responsive element-binding protein and activating transcription factor-2 transcription factors following transient cerebral ischemia in rat brain

The transcription factors cyclic AMP responsive element-binding protein (CREB) and activating transcription factor-2 were studied in rat brains subjected to 15 min ischemia followed by varied periods of reperfusion using western blot and immunocytochemical analyses. The total amounts of both CREB and activating transcription factor-2 were not altered in the hippocampus after ischemia. In contrast, levels of the phosphorylated forms of both transcription factors decreased during ischemia but rebounded following reperfusion. The phospho-forms of CREB and activating transcription factor-2 showed regional and temporal differences in their expression. Phospho-CREB was increased relative to control levels at 30 min, and continued to increase for at least three days postischemia, mainly in dentate granule cells. The level of phospho-activating transcription factor-2 appeared to be higher in CA1 pyramidal cells than in dentate granule cells after ischemia. The present findings suggest that the signaling pathways for phosphorylation of CREB may be neuroprotective for dentate cells, which are relatively resistant to ischemic insults. The increased phospho-activating transcription factor-2 may reflect increased stresses in these neurons. The more modest activation of CREB pathways in CAI neurons may not be enough to overcome the increased stresses in these neurons, contributing to delayed neuronal death.

P-selectin antibody reduces hemorrhage and infarct volume resulting from MCA occlusion in the rat.

We investigated the effect of an anti-P-selectin antibody (RMP-1) on ischemic cell damage and hemorrhage after transient middle cerebral artery occlusion (MCAo) in the rat. Animals were divided into four groups: (1) antibody (Ab) 1 group (n = 14) RMP-1 (2 mg/kg) was administered to rats 1 h prior to induction of 2 h of MCA occlusion; (2) control-vehicle group Ab2 (n = 12) rats were subjected to the same experimental protocol, except that an isotype-matched control antibody was administered; (3) Abl group (n = 10) rats were subjected to 2 h of MCA occlusion and RMP-1 (2 mg/kg) was administered upon reperfusion; (4) control-vehicle group Ab2 (n = 10) rats were subjected to the same experimental protocol, except that an isotype-matched control antibody was administered. Animals were sacrificed 48 h after onset of the MCAo for histological evaluation of infarction and hemorrhage, and to quantify number of neutrophils. The lesion volume was significantly smaller only in pretreated rats (RMP-1 group, 18.7+/-3.1%) compared to the vehicle-treated (31.6+/-2.6%) group (P<0.01). Total area of hemorrhage (5.94 x 10(3)+/-2.86 x 10(3) microm2) in the pre MCAo RMP-1 treated group animals was significantly reduced (P<0.02) compared to the vehicle group (6.1 x 10(4)+/-3.42 x 10(4) microm2), respectively. Our data demonstrate that administration of the anti-P-selectin antibody before transient focal cerebral ischemia in rat brain reduces ischemic cell damage and petechial hemorrhage.

Effects of long-term recovery from transient cerebral ischemia in rat brain: tissue levels of acetylcholine, monoamines, and their metabolites.

Concentrations of acetylcholine and the monoaminergic neurotransmitters dopamine, serotonin and their respective metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxy-phenylacetic acid (HVA), 5-hydroxyindolacetic acid (5-HIAA) and choline were simultaneously determined in the corpus striatum of rats after 15 min. complete cerebral ischemia (CCI) and in different intervals (1, 24, 48, 72, 96 hours) of postischemic cerebral reperfusion. Results were compared to respective sham-operated control animals. After 15 min. CCI acetylcholine concentration decreased to 15%, and dopamine concentration to 56% of the control values. The metabolite levels of DOPAC decreased to 40% and HVA to 64% of the control values. Acetylcholine, dopamine, serotonin and choline concentrations were not changed significantly after reperfusion. The metabolites HVA and 5-HIAA showed their maximum increases after 1 and 24 hours of reperfusion, additionally HVA was decreased both, after 72 and 96 hours of reperfusion. The data indicate that surprisingly little permanent damage could be caused by a 15 min. ischemia in the striatum. Tissue levels of the neurotransmitters appeared differentially altered but similarly regulated during ischemia and subsequent recirculation. Acetylcholine and dopamine levels decreased profoundly during ischemia. However, acetylcholine levels could be compensated rapidly during reperfusion, whereas the dopaminergic system showed a long-lasting change in its turnover rate. Although serotonin levels were unaffected by CCI, there was an increase of its presumed turnover rate during reperfusion.

Investigation of cerebral ischemia using magnetization transfer contrast (MTC) MR imaging

The effects of cerebral ischemia in rat brain were monitored as a function of time using proton MR imaging. Spinspin relaxation time ( T 2), proton density, and magnetization transfer contrast (MTC) were measured by MR imaging at various time intervals during a 1-week period following the induction of ischemic damage. Ischemic injury was characterized by a maximization of both T 2 value and MTC appearance at 24 hr postischemic injury. These changes were accompanied by a gradual increase in MR observable water density over the first few days of ischemia. A reduction in the magnetization exchange rate between “free” and “bound” water protons as measured by MTC imaging is at least partially responsible for the elevation in T 2 values observed during ischemia, and may accompany breakdown of cellular structure.