Model Of Global Brain Ischemia Research Articles

Effects of Sildenafil on Cognitive Function Recovery and Neuronal Cell Death Protection after Transient Global Cerebral Ischemia in Gerbils.

Cerebral ischemic stroke is a major cause of death worldwide due to brain cell death resulting from ischemia-reperfusion injury. However, effective treatment approaches for patients with ischemic stroke are still lacking in clinical practice. This study investigated the potential neuroprotective effects of sildenafil, a phosphodiesterase-5 inhibitor, in a gerbil model of global brain ischemia. We investigated the effects of sildenafil on the expression of glial fibrillary acidic protein and aquaporin-4, which are markers related to astrocyte activation and water homeostasis, respectively. Immunofluorescence analysis showed that the number of cells co-expressing these markers, which was elevated in the ischemia-induced group, was significantly reduced in the sildenafil-treated groups. This suggests that sildenafil may have a potential mitigating effect on astrocyte activation induced by ischemia. Additionally, we performed various behavioral tests, including the open-field test, novel object recognition, Barnes maze, Y-maze, and passive avoidance tests, to evaluate sildenafil's effect on cognitive function impaired by ischemia. Overall, the results suggest that sildenafil may serve as a neuroprotective agent, potentially alleviating delayed neuronal cell death and improving cognitive function impaired by ischemia.

Chronic Kombucha Beverage Consumption Attenuates Inflammatory Markers and Histopathology of Brain Tissue in Transnet Global Brain Ischemia in Rats.

There is evidence that kombucha beverage (KB), a traditional fermented beverage, has a preventive effect on experimental brain ischemia. According to our previous studies, pre-treatment of KB attenuates brain edema and improves motor skills and oxidative stress in a rat model of global brain ischemia. This study was designed to evaluate the effects of the pre-treatment of KB, as a novel agent, on pro-inflammatory parameters and brain histopathology changes following global brain ischemia. Adult male Wistar rats were randomly divided into the sham, the control, and the groups treated with kombucha (KB1 and KB2 groups). KB at doses 1 and 2 mL/kg was prescribed two-week consecutive days before induction of global brain ischemia. Global brain ischemia was induced by blocking common carotid arteries for 60min and the following reperfusion by 24h. The serum and brain levels of tumor necrosis factor-α(TNF-α), IL-1β, histopathological change, and infarct volume are determined using the ELISA, hematoxylin and eosin (H&E), and 2,3,5-triphenyl tetrazolium chloride (TTC) staining, respectively. This study indicated that pre-treatment of KB significantly reduced infarct volume, the serum, and brain levels of TNF-α and IL-1β. The histopathological finding of the brain tissue confirmed a protective role for pre-treatment KB in the ischemic rats. Thus, the present study showed that the beneficial effects of KB pre-treatment on brain ischemic may be mediated by decreasing pro-inflammatory parameters.

Effect of Methionine Diet on Time-Related Metabolic and Histopathological Changes of Rat Hippocampus in the Model of Global Brain Ischemia.

Hyperhomocysteinemia (hHcy) represents a strong risk factor for atherosclerosis-associated diseases, like stroke, dementia or Alzheimer’s disease. A methionine (Met)-rich diet leads to an elevated level of homocysteine in plasma and might cause pathological alterations across the brain. The hippocampus is being constantly studied for its selective vulnerability linked with neurodegeneration. This study explores metabolic and histo-morphological changes in the rat hippocampus after global ischemia in the hHcy conditions using a combination of proton magnetic resonance spectroscopy and magnetic resonance-volumetry as well as immunohistochemical analysis. After 4 weeks of a Met-enriched diet at a dose of 2 g/kg of animal weight/day, adult male Wistar rats underwent 4-vessel occlusion lasting for 15 min, followed by a reperfusion period varying from 3 to 7 days. Histo-morphological analyses showed that the subsequent ischemia-reperfusion insult (IRI) aggravates the extent of the sole hHcy-induced degeneration of the hippocampal neurons. Decreased volume in the grey matter, extensive changes in the metabolic ratio, deeper alterations in the number and morphology of neurons, astrocytes and their processes were demonstrated in the hippocampus 7 days post-ischemia in the hHcy animals. Our results suggest that the combination of the two risk factors (hHcy and IRI) endorses and exacerbates the rat hippocampal neurodegenerative processes.

Inhibitory modulation of cytochrome c oxidase activity with specific near-infrared light wavelengths attenuates brain ischemia/reperfusion injury

The interaction of light with biological tissue has been successfully utilized for multiple therapeutic purposes. Previous studies have suggested that near infrared light (NIR) enhances the activity of mitochondria by increasing cytochrome c oxidase (COX) activity, which we confirmed for 810 nm NIR. In contrast, scanning the NIR spectrum between 700 nm and 1000 nm revealed two NIR wavelengths (750 nm and 950 nm) that reduced the activity of isolated COX. COX-inhibitory wavelengths reduced mitochondrial respiration, reduced the mitochondrial membrane potential (ΔΨm), attenuated mitochondrial superoxide production, and attenuated neuronal death following oxygen glucose deprivation, whereas NIR that activates COX provided no benefit. We evaluated COX-inhibitory NIR as a potential therapy for cerebral reperfusion injury using a rat model of global brain ischemia. Untreated animals demonstrated an 86% loss of neurons in the CA1 hippocampus post-reperfusion whereas inhibitory NIR groups were robustly protected, with neuronal loss ranging from 11% to 35%. Moreover, neurologic function, assessed by radial arm maze performance, was preserved at control levels in rats treated with a combination of both COX-inhibitory NIR wavelengths. Taken together, our data suggest that COX-inhibitory NIR may be a viable non-pharmacologic and noninvasive therapy for the treatment of cerebral reperfusion injury.

Abstract 20928: Non-Invasive Mitochondrial Modulation: A Novel Approach to Reduce Brain Injury Following Cardiac Arrest

Loss of oxygen delivery to the brain during cardiac arrest can promote significant brain injury. While prompt resuscitation is critical, the reintroduction of oxygen can potentiate injury by promoting reactive oxygen species (ROS) generation. While mitigating ROS damage may serve as a therapeutic avenue for cerebral reperfusion injury, traditional attempts to scavenge ROS have failed. This failure is thought to be due to inherent difficulties with delivery to the brain and sub-cellular targets within the early minutes of reflow. We found that specific infrared light (IRL) wavelengths penetrate the brain and reversibly reduce the activity of cytochrome c oxidase (CcO). Accordingly, we developed a non-pharmacologic therapy using specific IRL wavelengths to non-invasively modulate CcO and circumvent these delivery barriers. Our previous data shows that IRL wavelengths that reduce CcO activity also directly modulate mitochondrial respiratory rate and membrane potential, block mitochondrial ROS production, and limit neuronal death following oxygen-glucose deprivation. We hypothesized that these wavelengths could be used to inhibit ROS generation following resuscitation from cardiac arrest and thus, provide neuroprotection. IRL therapy was first evaluated for neuroprotection in the rat model of global brain ischemia (bilateral carotid occlusion and systemic hypotension - 8 minutes). Rats were randomly enrolled into IRL treatment groups, initiated immediately upon reperfusion. After 14 days, animals subjected to ischemia demonstrated an 86% loss of neurons in the CA1 hippocampus. Strikingly, for the IRL-treated animals, loss of neurons was significantly less, ranging from 17% with the best treatment to 35% with the least efficacious regimen (n=8-12, p<0.05) and preserved neurologic function, assessed by radial arm maze (n = 10, p<0.05). Finally, preliminary data from a swine model of cardiac arrest/resuscitation show a reduction in neurologic deficit score (0-150 scale 0=no deficit, 150=brain dead) with IRL treatment (Cardiac Arrest/Resuscitation = 112, Cardiac Arrest/Resuscitation + IRL treatment = 1: N = 4-6/group). These data demonstrate that IRL therapy may provide a novel strategy for the treatment of post-cardiac arrest brain injury.

Inactivation of NSF ATPase Leads to Cathepsin B Release After Transient Cerebral Ischemia.

Neurons have extraordinary large cell membrane surface area, thus requiring extremely high levels of intracellular membrane-trafficking activities. Consequently, defects in the membrane-trafficking activities preferentially affect neurons. A critical molecule for controlling the membrane-trafficking activities is the N-ethylmaleimide-sensitive factor (NSF) ATPase. This study is to investigate the cascade of events of NSF ATPase inactivation, resulting in a massive buildup of late endosomes (LEs) and fatal release of cathepsin B (CTSB) after transient cerebral ischemia using the 2-vessel occlusion with hypotension (2VO+Hypotension) global brain ischemia model. Rats were subjected to 20min of transient cerebral ischemia followed by 0.5, 4, 24, and 72h of reperfusion. Neuronal histopathology and ultrastructure were examined by the light and electron microscopy, respectively. Western blotting and confocal microscopy were utilized for analyzing the levels, redistribution, and co-localization of Golgi apparatus and endosome or lysosome markers. Transient cerebral ischemia leads to delayed neuronal death that occurs at 48-72h of reperfusion mainly in hippocampal CA1 and neocortical (Cx) layers 3 and 5 pyramidal neurons. During the delayed period, NSF ATPase is irreversibly trapped into inactive protein aggregates selectivelyin post-ischemic neurons destined to die. NSF inactivation leads to a massive buildup of Golgi fragments, transport vesicles (TVs) and late endosomes (LEs), and release of the 33kDa LE type of CTSB, which is followed by delayed neuronal death after transient cerebral ischemia. The results support a novel hypothesis that transient cerebral ischemia leads to NSF inactivation, resulting in acascade of events offatal release of CTSB and delayed neuronal death after transient cerebral ischemia.

MicroRNA-137 protects neurons against ischemia/reperfusion injury through regulation of the Notch signaling pathway

MicroRNAs (miRNAs) have emerged as novel regulators in various pathological processes including ischemic stroke. However, the precise role of miRNAs in ischemic stroke remains largely unknown. In this study, we investigated the role of miR-137 in the regulation of neuronal ischemia/reperfusion injury with oxygen-glucose deprivation and reoxygenation (OGD/R), a model of global brain ischemia. The results showed that miR-137 was significantly downregulated in neurons subjected to OGD/R treatment: OGD/R-induced cell injury was markedly inhibited by miR-137 overexpression and exacerbated by miR-137 suppression. Moreover, Notch1 was predicted as a target gene of miR-137 and verified by dual-luciferase reporter assay, real-time quantitative polymerase chain reaction, and western blot analysis. Through targeting of Notch1, miR-137 regulated the Notch signaling pathway. The blockade of the Notch signaling pathway reversed the effect of miR-137 suppression, whereas overexpression of the Notch intracellular domain abrogated the effect of miR-137 overexpression on OGD/R-induced cell injury. Overall, our study suggests that miR-137 regulated the Notch signaling pathway by targeting Notch1 to protect neurons from OGD/R-induced cell injury, providing a novel insight into understanding the molecular basis of ischemia stroke and a potential therapeutic target.

Proliferating cells in the adult primate cerebellar cortex after ischemia

INTRODUCTION: Brain ischemia is a devastating neurological condition with significant medical and social impact. Here we investigated the effect of experimental ischemia of different duration on the ability of the adult macaque cerebellum to produce new cells of specific phenotypes. MATERIAL AND METHODS: We used a well-established model of global brain ischemia in young adult Japanese monkeys applying bromodeoxyuridine (BrdU) for 5 days х 100 mg/kg daily. Animals were distributed into different experimental and control groups depending on postischemic survival periods: 4, 9, 15, 23, 44 days (D) and BrdU starting day. Immunohistochemical detection of BrdU+ cells, Iba1+ microglia and GFAP+ astroglia was performed on cryosections. Statistical evaluation of newly generated cells with phenotyping for microglia and astrocytes in various cortical layers of the cerebellum were done. RESULTS and CONCLUSIONS: The numbers of BrdU+ cells in some ischemic groups were significantly higher compared to control animals. By cerebrocerebellum, there was an increasing value by D4 group compared to the control, then slightly reducing in D9 and D15 groups and increasing again by D23 and D44 groups. In the spinocerebellum, an increase was detected only in D44 group. The newly generated cells were dispersed in all cerebellar cortical layers with highest concentration in Purkinje cell layer. Our data show that ischemia stimulates cellular proliferation in the cerebellum but this effect declines with time after ischemia. We found evidence for generation of new microglia but not for astroglia. Our data may contribute to a better understanding of regeneration in the cerebellum after brain ischemia.

Mitochondrial dynamics following global cerebral ischemia

Global brain ischemia/reperfusion induces neuronal damage in vulnerable brain regions, leading to mitochondrial dysfunction and subsequent neuronal death. Induction of neuronal death is mediated by release of cytochrome c (cyt c) from the mitochondria though a well-characterized increase in outer mitochondrial membrane permeability. However, for cyt c to be released it is first necessary for cyt c to be liberated from the cristae junctions which are gated by Opa1 oligomers. Opa1 has two known functions: maintenance of the cristae junction and mitochondrial fusion. These roles suggest that Opa1 could play a central role in both controlling cyt c release and mitochondrial fusion/fission processes during ischemia/reperfusion. To investigate this concept, we first utilized in vitro real-time imaging to visualize dynamic changes in mitochondria. Oxygen-glucose deprivation (OGD) of neurons grown in culture induced a dual-phase mitochondrial fragmentation profile: (i) fragmentation during OGD with no apoptosis activation, followed by fusion of mitochondrial networks after reoxygenation and a (ii) subsequent extensive fragmentation and apoptosis activation that preceded cell death. We next evaluated changes in mitochondrial dynamic state during reperfusion in a rat model of global brain ischemia. Evaluation of mitochondrial morphology with confocal and electron microscopy revealed a similar induction of fragmentation following global brain ischemia. Mitochondrial fragmentation aligned temporally with specific apoptotic events, including cyt c release, caspase 3/7 activation, and interestingly, release of the fusion protein Opa1. Moreover, we uncovered evidence of loss of Opa1 complexes during the progression of reperfusion, and electron microscopy micrographs revealed a loss of cristae architecture following global brain ischemia. These data provide novel evidence implicating a temporal connection between Opa1 alterations and dysfunctional mitochondrial dynamics following global brain ischemia.

Curcumin reverses neurochemical, histological and immuno-histochemical alterations in the model of global brain ischemia

Curcumin, a curcuminoid from Curcuma longa, presents antioxidant and anti-inflammatory actions and, among pathological changes of cerebral ischemic injury, inflammation is an important one. The objectives were to study the neuroprotective action of curcumin, in a model of global ischemia. Male Wistar rats (sham-operated, ischemic untreated and ischemic treated with curcumin, 25 or 50 mg/kg, p.o.) were anesthesized and their carotid arteries occluded, for 30 min. The SO group had the same procedure, except for carotid occlusion. In the 1st protocol, animals were treated 1 h before ischemia and 24 h later; and in the 2nd protocol, treatments began 1 h before ischemia, continuing for 7 days. Twenty four hours after the last administration, animals were euthanized and measurements for striatal monoamines were performed, at the 1st and 7th days after ischemia, as well as histological and immunohistochemical assays in hippocampi. We showed in both protocols, depletions of DA and its metabolites (DOPAC and HVA), in the ischemic group, but these effects were reversed by curcumin. Additionally, a decrease seen in 5-HT contents, 1 day after ischemia, was also reversed by curcumin. This reversion was not seen 7 days later. On the other hand, a decrease observed in NE levels, at the 7th day, was totally reversed by curcumin. Furthermore, curcumin treatments increased neuronal viability and attenuated the immunoreactivity for COX-2 and TNF-alpha, in the hippocampus in both protocols. We showed that curcumin exerts neuroprotective actions, in a model of brain ischemia that are probably related to its anti-inflammatory activity.

Downregulation of GABAA Receptor Recycling Mediated by HAP1 Contributes to Neuronal Death in In Vitro Brain Ischemia.

Downregulation of GABAergic synaptic transmission contributes to the increase in overall excitatory activity in the ischemic brain. A reduction of GABAA receptor (GABAAR) surface expression partly accounts for this decrease in inhibitory activity, but the mechanisms involved are not fully elucidated. In this work, we investigated the alterations in GABAAR trafficking in cultured rat hippocampal neurons subjected to oxygen/glucose deprivation (OGD), an in vitro model of global brain ischemia, and their impact in neuronal death. The traffic of GABAAR was evaluated after transfection of hippocampal neurons with myc-tagged GABAAR β3 subunits. OGD decreased the rate of GABAAR β3 subunit recycling and reduced the interaction of the receptors with HAP1, a protein involved in the recycling of the receptors. Furthermore, OGD induced a calpain-mediated cleavage of HAP1. Transfection of hippocampal neurons with HAP1A or HAP1B isoforms reduced the OGD-induced decrease in surface expression of GABAAR β3 subunits, and HAP1A maintained the rate of receptor recycling. Furthermore, transfection of hippocampal neurons with HAP1 significantly decreased OGD-induced cell death. These results show a key role for HAP1 protein in the downmodulation of GABAergic neurotransmission during cerebral ischemia, which contributes to neuronal demise.

Ischemic postconditioning protects against ischemic brain injury by up-regulation of acid-sensing ion channel 2a.

Ischemic postconditioning renders brain tissue tolerant to brain ischemia, thereby alleviating ischemic brain injury. However, the exact mechanism of action is still unclear. In this study, a rat model of global brain ischemia was subjected to ischemic postconditioning treatment using the vessel occlusion method. After 2 hours of ischemia, the bilateral common carotid arteries were blocked immediately for 10 seconds and then perfused for 10 seconds. This procedure was repeated six times. Ischemic postconditioning was found to mitigate hippocampal CA1 neuronal damage in rats with brain ischemia, and up-regulate acid-sensing ion channel 2a expression at the mRNA and protein level. These findings suggest that ischemic postconditioning up-regulates acid-sensing ion channel 2a expression in the rat hippocampus after global brain ischemia, which promotes neuronal tolerance to ischemic brain injury.

Brain ischemia downregulates the neuroprotective GDNF-Ret signaling by a calpain-dependent mechanism in cultured hippocampal neurons.

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected, and the functional consequences, have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51), but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD), a model of global brain ischemia, as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity, the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore, GDNF protected hippocampal neurons when present before, during or after OGD, and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF.

Sulbactam Plays Neuronal Protective Effect Against Brain Ischemia via Upregulating GLT1 in Rats.

The study was undertaken to investigate whether sulbactam protects cerebral neurons against ischemia and whether the protection is mediated by regulating the expression and uptake activity of glial glutamate transporter-1 (GLT1) in a rat global brain ischemia model. The CA1 hippocampus was selected as the observing target. Real time quantitative PCR, Western blot and immunohistochemistry assays were used to detect GLT1 expression. Neuropathological evaluation was performed after thionin staining to determine the extent of the delayed neuronal death (DND) of pyramidal neurons. It was found that cerebral ischemia for 8min induced obvious DND of pyramidal neurons and GLT1 downregulation. Sulbactam pretreatment significantly upregulated GLT1 expression in sham rats and prevented or reversed the GLT1 downregulation normally induced in the ischemic rat brain. Meanwhile, sulbactam pretreatment effectively prevented the DND of pyramidal neurons normally induced by brain ischemia in a dose-dependent manner. Sulbactam posttreatment also protected pyramidal neurons against DND induced by brain ischemia although the magnitude of the protective effect was weaker than that in sulbactam pretreatment. Furthermore, either antisense knockdown of GLT1 expression or inhibition of the GLT1 uptake activity with dihydrokainate, a selective inhibitor of GLT1, significantly blocked the neuronal protective effect of sulbactam. These findings indicate that sulbactam has a neuronal protective effect though upregulating GLT1.

Cytochrome c Is Tyrosine 97 Phosphorylated by Neuroprotective Insulin Treatment

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Epidermal growth factor and growth hormone-releasing peptide-6: Combined therapeutic approach in experimental stroke

Stroke is the second cause of mortality worldwide, with a high incidence of disability in survivors. Promising candidate drugs have failed in stroke trials. Combined therapies are attractive strategies that simultaneously target different points of stroke pathophysiology. The aim of this work is to determine whether the combined effects of epidermal growth factor (EGF) and growth hormone-releasing peptide-6 (GHRP6) can attenuate clinical signs and pathology in an experimental stroke model. Brain global ischemia was generated in Mongolian gerbils by 15 minutes of carotid occlusion. After reperfusion, EGF, GHRP6 or EGF+GHRP6 were intraperitoneally administered. Clinical manifestations were monitored daily. Three days after reperfusion, animals were anesthetized and perfused with an ink solution. The anatomy of the Circle of Willis was characterized. Infarct volume and neuronal density were analyzed. EGF+GHRP6 co-administration reduced clinical manifestations and infarct volume and preserved neuronal density. No correlation was observed between the grade of anastomosis of the Circle of Willis and clinical manifestations in the animals receiving EGF+GHRP6, as opposed to the vehicle-treated gerbils. Co-treatment with EGF and GHRP6 affects both the clinical and pathological outcomes in a global brain ischemia model, suggesting a suitable therapeutic approach for the acute management of stroke.

Global brain ischemia in Mongolian gerbils: assessing the level of anastomosis in the cerebral circle of Willis.

The Mongolian gerbil has been widely used as a global brain ischemia model because of its incomplete cerebral circle of Willis. However, the inter-individual anatomic variability of this vascular structure interferes with the reliability of the model. The aim of this work was to introduce modifications to the protocol of global brain ischemia experiments in Mongolian gerbils in an attempt to increase the reliability and usefulness of this model. Our study focused on the assessment of the level of anastomosis of the cerebral circle of Willis in order to evaluate its contribution to clinicopathological outcomes in this model. Sham-operated, Ischemic, and Ischemic + Hypothermia animals were subjected to a 15-minute occlusion of the common carotid arteries. Transcardiac perfusion with bromophenol blue / gelatin solution was performed 72 hours after ischemia. Brains were processed for anatomopathological analysis. Tissue damage was observed in the hippocampus, caudate-putamen nucleus, neocortex, and thalamic nuclei of animals from the Ischemic group. The circles of Willis of the Sham-operated animals showed bilateral (38 percent), unilateral (48 percent) or no posterior communicating arteries (14 percent). A negative correlation between infarct volume and the level of anastomosis was revealed for the Ischemic, but not for the Ischemic + Hypothermia group. Additionally, Analysis of covariance (ANCOVA) was performed to assess the contribution of the level of anastomosis to the clinicopathological outcomes. It was confirmed that the infarct volume decreased in the Ischemic + Hypothermia group when compared to the Ischemic group. Since the level of anastomosis cannot be predicted, this variable should necessarily be considered when analyzing the results of global brain ischemia in Mongolian gerbils.

The “Refrige-a-RAT-or”: An Accurate, Inexpensive, and Clinically Relevant Small Animal Model of Therapeutic Hypothermia

Physical and molecular mechanisms for the neuroprotective effect of therapeutic hypothermia are not completely understood, and new therapeutic applications incorporating hypothermia remain to be developed and tested. Clinically relevant animal models of therapeutic hypothermia are not well established or consistent. The objective was to develop and test an inexpensive small animal therapeutic hypothermia system that models those in widespread clinical use and verify that such a system confers neuroprotection in a rat model of global brain ischemia. A water-cooled extracorporeal system and attendant anesthesia/sedation protocol were developed and tested. In Stage 1, animals were instrumented for brain, temporalis, and rectal temperature monitoring, and the system was tested for its effect on temperature and hemodynamics. In Stage 2, animals were instrumented for rectal temperature only, subjected to global brain ischemia by two-vessel occlusion and hypotension for 8 minutes, and given either sham therapy (37°C) or hypothermia (32°C) for 4 hours. Viable CA1 neurons were counted at 7 days. The system was well tolerated, provided exquisite control of animal core and brain temperatures, and conferred robust neuroprotection at 7 days. The median and interquartile ranges (IQRs) of viable neurons per 300-μm field were 130 (IQR = 128 to 135) for sham control, 19 (IQR = 15 to 30) for untreated ischemic animals, and 101 (IQR = 94 to 113) for ischemic animals treated with hypothermia (p < 0.05 for comparison between all groups). Like human protocols, this model incorporates sedation and analgesia, results in robust neuroprotection, is well tolerated, and offers exquisite temperature control. The system is noninvasive and inexpensive and offers a model that is similar to methods used in clinical practice. This system will be of interest to investigators using small animal models to examine neuroprotective mechanisms of hypothermia and translational strategies that combine hypothermia with targeted pharmacotherapy.

Controlled low-flow reperfusion after warm brain ischemia reduces reperfusion injury in canine model

Acute occlusion of the carotid artery caused by acute type A aortic dissection (AAD) induces on-going warm brain ischemia. The purpose of this study was to elucidate the hypothesis that low-flow reperfusion could mitigate reperfusion injury after warm ischemic damage to the brain. Experiments were performed using a canine global brain ischemia model, with 15 minutes of ischemia followed by 3 hours reperfusion, which was established by a simple brain reperfusion circuit with a roller pump. The right common carotid artery (RCCA) flow ratio was determined as the mean RCCA flow during reperfusion divided by the mean RCCA flow during pre-ischemia. Animals were divided into two groups according to the RCCA flow ratio; low RCCA flow ratio of 0.3 to 0.6 (Group L, n=5) and control RCCA flow ratio of 1.0 to 1.4 (Group C, n=5). At the 3-hour reperfusion time point, physiological and histopathological assessments were performed in both groups. Electroencephalographic activity recovered in four of five animals (80%) animals in Group L, whereas no recovery (0%) in activity was observed in Group C. Brain water content in Group L animals was significantly less than that in Group C. Apoptosis, number of perivascular edematous regions and NFkappaB expression were apparently suppressed in Group L compared with Group C. There were significant positive correlations of RCCA flow with brain water content, apoptosis and number of perivascular edematous regions. Controlled low-flow reperfusion mitigated reperfusion-induced brain edema and apoptosis, leading to rescue of brain function in the canine model.

RNAi targeting µ-calpain increases neuron survival and preserves hippocampal function after global brain ischemia

The calpain family of cysteine proteases has a well-established causal role in neuronal cell death following acute brain injury. However, the relative contribution of calpain isoforms has not been determined in in vivo models. Identification of the calpain isoform responsible for neuronal injury is particularly important given the differential role of calpain isoforms in normal physiology. This study evaluates the role of m-calpain and µ-calpain in an in vivo model of global brain ischemia. Adeno-associated viral vectors expressing short hairpin RNAs targeting the catalytic subunits of µ- or m-calpain were used to knockdown expression of the targeted isoforms in adult rat hippocampal CA1 pyramidal neurons. Knockdown of µ-calpain, but not m-calpain, prevented calpain activity 72 h after 6-min transient forebrain ischemia, increased long-term survival and protected hippocampal electrophysiological function. These findings represent the first in vivo evidence that reducing expression of an individual calpain isoform can decrease post-ischemic neuronal death and preserve hippocampal function.