Global Brain Ischemia In Rats Research Articles

Transient global ischemia in rat brain promotes different NMDA receptor regulation depending on the brain structure studied

The mRNA expression of the major subunits of N-methyl- d-aspartate receptors (NR1, NR2A and NR2B) following ischemia–reperfusion was studied in structures with different vulnerabilities to ischemic insult in the rat brain. The study was performed using quantitative real-time PCR on samples from 3-month-old male Sprague–Dawley rats after global transient forebrain ischemia followed by 48 h of reperfusion. Expression of NMDA receptor subunits mRNAs decreased significantly in all structures studied in the injured animals as compared to the sham-operated ones. The hippocampal subfields (CA1, CA3 and dentate gyrus) as well as the caudate-putamen, both reported to be highly ischemic-vulnerable structures, showed outstandingly lower mRNA levels of NMDA receptor subunits than the cerebral cortex, which is considered a more ischemic-resistant structure. The ratios of the mRNA levels of the different subunits were analyzed as a measure of the NMDA receptor expression pattern for each structure studied. Hippocampal areas showed changes in NMDA receptor expression after the insult, with significant decreases in the NR2A with respect to the NR1 and NR2B subunits. Thus, the NR1:NR2A:NR2B (1:1:2) ratios observed in the sham-operated animals became (2:1:4) in insulted animals. This modified expression pattern was similar in CA1, CA3 and the dentate gyrus, in spite of the different vulnerabilities reported for these hippocampal areas. In contrast, no significant differences in the expression pattern were observed in the caudate-putamen or cerebral cortex on comparing the sham-operated animals with the ischemia-reperfused rats. Our results support the notion that the regulation of NMDA receptor gene expression is dependent on the brain structure rather than on the higher or lower vulnerability of the area studied.

Protection effect of hypothermia used in ischemia reperfusion model in rats.

20世纪80年代中期发现了中度脑低温在心跳骤停期间和非失血性心跳骤停后复苏对于生物活性保存的保护作用,开创了一个新的时代研究治疗性低温.在1987年Busto等[1]发现了浅低温对于缺血损伤的神经细胞保护作用.低温的脑保护作用在体外培养的神经细胞、全脑以及局部脑缺血的实验中已经充分证明了其有效性,作为一种脑保护措施已经在外科手术中广泛应用,目前在心肺复苏治疗中也常常应用低温来保护神经细胞损伤。

Age-dependent MRI-detected lesions at early stages of transient global ischemia in rat brain

Although ischemic stroke has higher incidence and severity in aged than in young humans, the age factor is generally neglected in ischemia animal models. This study was aimed at comparing age-dependent effects at early stages of transient global cerebral ischemia (TGCI) in rats. TGCI was induced in two groups of rats (3-6 and 20-24 months old, respectively) by exposure to 15% oxygen and 15 min occlusion of the two common carotid arteries. Brains were analysed in vivo by MRI-apparent diffusion coefficient (ADC) and T2 maps--at 1-3 h post-TGCI and in vitro by histochemical examination of triphenyltetrazolium chloride (TTC)-stained slices. At 1-3 h post-TGCI, a higher incidence of lesions was found in aged than in young rats especially in the hippocampus and cortex (occipital plus parietal) but not in the thalamus. The lesioned regions showed lower ADC values in aged than in younger rats. The most substantial ADC decreases were associated with enhanced spin-spin relaxation and lower TTC staining. The different responses of the two age groups support the use of aged animals for investigations on different ischemia models. Our model of brain ischemia appears appropriate for further studies including drug effects.

Nuclear factor-κB decoy attenuates neuronal damage after global brain ischemia: A future strategy for brain protection during circulatory arrest

Objectives: Recent studies have reported that cis element decoy oligodeoxynucleotides against nuclear factor-κB block the activation of genes that mediate ischemic injury. To improve brain protection during circulatory arrest in cardiac surgery, we evaluated the efficacy of nuclear factor-κB decoy oligodeoxynucleotides in preventing neuronal damage after global brain ischemia. Methods: Hemagglutinating virus of Japan–liposome complex with fluorescein isothiocyanate–labeled nuclear factor-κB decoy oligodeoxynucleotides was injected through the carotid artery during 20 minutes of global brain ischemia in rats to evaluate the efficacy of transfecting the decoy oligodeoxynucleotides. The messenger RNA levels of several factors related to ischemia-reperfusion injury in the hippocampus were estimated by a real-time polymerase chain reaction method 1 hour after reperfusion. Neuronal damage was evaluated by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling staining and by using immunohistochemical study of microtubule-associated protein 2 in the hippocampus CA-1 region 7 days after ischemia. Results: Introduction of the nuclear factor-κB decoy oligodeoxynucleotides into rat brain neurons through the carotid artery during global brain ischemia was markedly successful. The polymerase chain reaction study showed that the transfected nuclear factor-κB decoy oligodeoxynucleotides effectively inhibited the expression of tumor necrosis factor α interleukin 1β and intracellular adhesion molecule 1 messenger RNA 1 hour after global brain ischemia. Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling staining and microtubule-associated protein 2 immunohistochemistry showed that the transfected nuclear factor-κB decoy oligodeoxynucleotides significantly attenuated the neuronal damage 7 days after global brain ischemia. Conclusions: Therapeutic transfection of nuclear factor-κB decoy oligodeoxynucleotides during brain ischemia may be useful for attenuating neuronal damage, suggesting a strategy for cerebral protection against global ischemia.

Expression of Nuclear Orphan Receptors Ngfi-B/Tr3, Nurr1 And Nor-1 after Global Brain Ischemia in Rat.

INTRODUCTION. Neuron-derived orphan receptor (NOR-1) was cloned from cultured embryonal neurons undergoing apoptotic cell death. NOR-1 is related to the nuclear orphan receptors, NGFI-B and NURR1, which interact with the retinoic acid receptor, RXR. NGFI-B and NURR1 are immediate early genes in the ischemic brain, suggesting a role in repair or cell death. Very recently, NGFI-B/TR3 was found to exert a pro-apoptotic effect through mitochondrial targeting and cytochrome release leading to apoptosis (1). Such dual role of NGFI-B (and related orphans) in nuclear and mitochondrial function could be important for neuronal damage in brain ischemia (stroke). We evaluated novel gene expression (mRNA levels) of nuclear orphan receptors after short brain ischemia. This model allows to correlate changes in gene and protein expression with pathological outcome, i.e. selective vulnerability occurs in CA1 neurons of the hippocampus while the adjacent CA3 cells and the dentate granule cells survive.

Induction of vascular endothelial growth factor and hypoxia-inducible factor-1α by global ischemia in rat brain

Vascular endothelial growth factor is an angiogenic and neurotrophic peptide whose expression is transcriptionally induced in hypoxic tissues through the action of hypoxia-inducible factor-1α. To determine if this signaling pathway is activated in the ischemic brain, and might therefore participate in adaptive processes such as angiogenesis and neuroprotection, we examined the expression of vascular endothelial growth factor and hypoxia-inducible factor-1α in cerebral cortex and hippocampus following transient global cerebral ischemia in the rat. Northern analysis showed ischemia-inducible expression of multiple vascular endothelial growth factor messenger ribonucleic acid splice variants between 4 and 24 h. Western analysis and immunocytochemistry demonstrated the concerted induction of vascular endothelial growth factor and hypoxia-inducible factor-1α in the same, apparently neuronal, cells in vulnerable regions of cortex and hippocampus after 15 min of ischemia, which persisted for as long as 4 to 72 h of reperfusion. These findings demonstrate that hypoxia-sensitive vascular endothelial growth factor signaling can be induced in neurons in global cerebral ischemia in vivo, and are consistent with the hypothesis that ischemic insults trigger hypoxia-sensing and adaptive downstream molecular responses in central neurons.

Selective glial vulnerability following transient global ischemia in rat brain.

Global cerebral ischemia selectively damages neurons, but its contribution to glial cell death is uncertain. Accordingly, adult male rats were sacrificed by perfusion fixation at 1, 2, 3, 5, and 14 days following 10 minutes of global ischemia. This insult produces CA1 hippocampal neuronal death at post-ischemic (PI) day 3, but minor or no damage to neurons in other regions. In situ end labeling (ISEL) and immunohistochemistry identified fragmented DNA of dead or dying glia and distinguished glial subtypes. Rare ISEL-positive oligodendroglia, astrocytes, and microglia were present in control brain. Apoptotic bodies and ISEL-positive glia significantly increased at PI day 1 in cortex and thalamus (p < 0.05), but were similar to controls in other regions and at other PI intervals. Most were oligodendroglia, although ISEL-positive microglia and astrocytes were also observed. These results show that oligodendroglia die rapidly after brief global ischemia and are more sensitive than neurons in certain brain regions. Their selective vulnerability to ischemia may be responsible for the delayed white matter damage following anoxia or CO poisoning or that associated with white matter arteriopathies. Glial apoptosis could contribute to the DNA ladders of apoptotic oligonucleosomes that have been found in post-ischemic brain.

Transient expression of GAP-43 within the hippocampus after global brain ischemia in rat.

Neuroanatomical methods have been used to study selective vulnerability after global brain ischemia. A consistent pattern of ischemic neuronal damage is found in the rodent hippocampus with loss of CA1 neurons and of some cells in the hilus of the dentate gyrus. Very little is known about plastic changes that would be expected in ischemia-resistant areas such as CA3 neurons and granule cells. Neuronal plasticity after lesions may be indicated by changes in labeling with antibodies to the growth-associated protein 43 (GAP-43). Expression of GAP-43 as a marker for neuronal plasticity was studied here in the hippocampus after global brain ischemia. Halothane-anesthetized rats were subjected to 20 min of transient forebrain ischemia using four-vessel occlusion. In situ hybridization was used to study GAP-43 mRNA at 1, 3, 6, and 12 h and at 1, 3, and 7 days after ischemia. Immunostaining was carried out with two different antibodies to GAP-43 in brains which were perfusion-fixed after 1, 2, 4, and 7/8 days. In the control hippocampus, GAP-43 mRNA was localized to CA1-CA3 and the hilus. Moderate increases in cellular signals were seen in hilar cells and granule cells early after ischemia, and some changes occurred in CA3 at late stages. Hybridization was lost in CA1 due to cell death. With immunostaining, GAP-43 was not seen in the cytoplasm of neurons, whereas dense labeling occurred in a differentiated pattern in the axonal and dendritic layers. At 1 day after ischemia, neurons in the hilus of the dentate gyrus and in the stratum pyramidale and lucidum of CA3 showed strong cytoplasmic labeling for GAP-43. Few cells were labeled in these regions at 2 days, and none at later stages. Pyramidal cells in CA1 and CA3 areas and granule cells were never labeled. These studies demonstrate a transient expression of GAP-43 mRNA and protein in a subset of vulnerable neurons after transient brain ischemia. The cytoplasmic localization in hilar neurons could be due to increased synthesis of GAP-43 or to changes in axoplasmic transport. It is suggested that axonal damage occurs in hilar cells which stimulates GAP-43 expression. The increased production of trophic factors after ischemia in granule cells could also cause plastic changes in hilar cells. Since hilar neurons are in a strategic position to control the excitability of the dentate area, increased expression of GAP-43 may indicate an important pathophysiological process. In seizure experiments, strong expression of GAP-43 mRNA in granule cells was associated with abnormal mossy fiber sprouting and development of chronic epilepsy. The relevance of the minor GAP-43 mRNA upregulation after ischemia must be considered. The changes in CA3 neurons at several days after ischemia might represent a plastic response to a loss of CA1 neurons.

Cerebral resuscitation from cardiac arrest

Both the period of total circulatory arrest to the brain and postischemic-anoxic encephalopathy (cerebral postresuscitation syndrome or disease), after normothermic cardiac arrests of between 5 and 20 mins (no-flow), contribute to complex physiologic and chemical derangements. The best documented derangements include the delayed protracted inhomogeneous cerebral hypoperfusion (despite controlled normotension), excitotoxicity as an explanation for selectively vulnerable brain regions and neurons, and free radical-triggered chemical cascades to lipid peroxidation of membranes. Protracted hypoxemia without cardiac arrest (e.g., very high altitude) can cause angiogenesis; the trigger of it, which lyses basement membranes, might be a factor in post-cardiac arrest encephalopathy. Questions to be explored include: What are the changes and effects on outcome of neurotransmitters (other than glutamate), of catecholamines, of vascular changes (microinfarcts seen after asphyxia), osmotic gradients, free-radical reactions, DNA cleavage, and transient extracerebral organ malfunction? For future mechanism-oriented studies of the brain after cardiac arrest and innovative cardiopulmonary-cerebral resuscitation, increasingly reproducible outcome models of temporary global brain ischemia in rats and dogs are now available. Disagreements exist between experienced investigative groups on the most informative method for quantitative evaluation of morphologic brain damage. There is agreement on the desirability of using not only functional deficit and chemical changes, but also morphologic damage as end points.

Cerebral resuscitation from cardiac arrest

Both the period of total circulatory arrest to the brain and postischemic-anoxic encephalopathy (cerebral postresuscitation syndrome or disease), after normothermic cardiac arrests of between 5 and 20 mins (no-flow), contribute to complex physiologic and chemical derangements. The best documented derangements include the delayed protracted inhomogeneous cerebral hypoperfusion (despite controlled normotension), excitotoxicity as an explanation for selectively vulnerable brain regions and neurons, and free radical-triggered chemical cascades to lipid peroxidation of membranes. Protracted hypoxemia without cardiac arrest (e.g., very high altitude) can cause angiogenesis; the trigger of it, which lyses basement membranes, might be a factor in post-cardiac arrest encephalopathy. Questions to be explored include: What are the changes and effects on outcome of neurotransmitters (other than glutamate), of catecholamines, of vascular changes (microinfarcts seen after asphyxia), osmotic gradients, free-radical reactions, DNA cleavage, and transient extracerebral organ malfunction? For future mechanism-oriented studies of the brain after cardiac arrest and innovative cardiopulmonary-cerebral resuscitation, increasingly reproducible outcome models of temporary global brain ischemia in rats and dogs are now available. Disagreements exist between experienced investigative groups on the most informative method for quantitative evaluation of morphologic brain damage. There is agreement on the desirability of using not only functional deficit and chemical changes, but also morphologic damage as end points. (Crit Care Med 1996; 24(Suppl):S57-S68)

HSP70 heat shock protein induction following global ischemia in the rat.

Stress proteins, including the 70 kD heat shock protein (HSP70), are induced in injured cells. The present study was designed to characterize the cells injured by global ischemia in rat brain. Adult rats were subjected to forebrain ischemia using bilateral carotid occlusion and systemic hypotension. HSP70 protein immunostaining of brain sections was performed using the C92 monoclonal antibody one day later. HSP70 immunoreactive cells were found in many brain regions including cortex. HSP70 positive neurons in cortex were found in certain laminae, especially layers 2 and 3. Acid fuchsin positive neurons, cells presumed to be dead, were located only in the layers of cortex where HSP70 immunoreactive neurons were found and were infrequent compared to the large number of HSP70 positive neurons. HSP70 immunoreactive glial cells were detected at the margins of ischemic areas, and were mostly OX42 immunoreactive microglia plus some GFAP immunoreactive astrocytes. In some animals HSP70 stained bipolar cells were detected in the striatum and in white matter which may be type 2 astrocytes. These findings confirm that global ischemia injures microglia and astrocytes, and that cells in a given ischemic region sustain varying degrees of injury--from the HSP70 stained neurons that likely survive the ischemia to acid fuchsin stained cells that die.

NMDA and non-NMDA receptor gene expression following global brain ischemia in rats: effect of NMDA and non-NMDA receptor antagonists.

Transient forebrain or global ischemia in rats induces selective and delayed damage of hippocampal CA1 neurons. In a previous study, we have shown that expression of GluR2, the kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit that governs Ca2+ permeability, is preferentially reduced in CA1 at a time point preceding neuronal degeneration. Postischemic administration of the selective AMPA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), protects CA1 neurons against delayed death. In this study we examined the effects of NBQX (at a neuroprotective dose) and of MK-801 (a selective NMDA receptor antagonist, not protective in this model) on kainate/AMPA receptor gene expression changes after global ischemia. We also examined the effects of transient forebrain ischemia on expression of the NMDA receptor subunit NMDAR1. In ischemic rats treated with saline, GluR2 and GluR3 mRNAs were markedly reduced in CA1 but were unchanged in CA3 or dentate gyrus. GluR1 and NMDAR1 mRNAs were not significantly changed in any region examined. Administration of NBQX or MK-801 did not alter the ischemia-induced changes in kainate/AMPA receptor gene expression. These findings suggest that NBQX affords neuroprotection by a direct blockade of kainate/AMPA receptors, rather than by a modification of GluR2 expression changes.

Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain

Laddered DNA indicative of apoptosis was observed in the CA1 layer of hippocampus and in dorsolateral striatum following a global cerebral ischemic insult produced by transient two vessel occlusion in rats. The extent of this DNA damage was proportional to the duration of the ischemic episode. Breaks in DNA were demonstrated in situ in sections from post-ischemic brain in neurons of the hippocampal CA1 which undergo selective neuronal death but not in other cell types. It is concluded that there is an apoptotic component to selective neuronal death following global ischemia in rat brain.

Selective brain cooling during and after prolonged global ischemia reduces cortical damage in rats.

Studies of the cerebroprotective effects of selective brain cooling have failed to show amelioration of ischemic injury in the cerebral cortex. This study was designed to test the hypothesis that mild-to-moderate selective brain cooling initiated after the onset of global brain ischemia in rats protects the cerebral cortex and improves neurological outcome. Global forebrain ischemia for 30 minutes in 27 fasted adult male Wistar rats was achieved by bilateral carotid occlusion and hypotension. In group 1, brain temperature, measured in the temporalis muscle, was maintained at 37-38 degrees C throughout the experiment. In group 2, brain temperature fell spontaneously during ischemia to 34.7 +/- 0.1 degrees C and rose spontaneously to 36-37 degrees C after 10 minutes of recirculation. In group 3, brain temperature was lowered with ice packs placed around the head after 15 minutes of ischemia to 24.1 +/- 0.9 degrees C by the end of ischemia, maintained at 30.0 +/- 1.0 degrees C for the first hour of recirculation, then allowed to rise to 36-37 degrees C. Seven-day survival was 0% (0 of 6) in group 1, 73% (8 of 11) in group 2, and 100% (6 of 6) in group 3. Severity of neuronal damage was less in group 2 than in group 1 in the cortex (p < 0.05) and hippocampal CA1 (p < 0.05) and CA3 regions (p < 0.05). Group 3 had less neuronal damage than group 2 in both cortex (p < 0.02) and striatum (p < 0.02). Furthermore, postischemic weight loss was less and neurobehavioral scores were significantly higher in group 3. This study shows that selective brain cooling increases survival from prolonged global ischemia and reduces neuronal injury in the cerebral cortex as well as the striatum and hippocampus.

Regional differences in rat brain lipids during global ischemia.

Membrane lipid degradation plays an important role in the pathogenesis of ischemic brain damage, but there is little information on changes in cerebrosides, sulfatides, and sphingomyelin. We studied regional changes in the quantities of these lipids during complete global brain ischemia in rats. Nitrous oxide-anesthetized rats were subjected to ischemia by a high-pressure neck cuff and arterial hypotension for 0 (control), 3, 10, or 30 minutes (n = 5 at each time). Brain temperature was allowed to fall spontaneously during ischemia, and the brain was frozen in situ with liquid N2 without recirculation. The frontal cortex, hippocampus, and basal ganglia were dissected at -15 degrees C. The lipids were separated by column and high-performance thin-layer chromatography and quantified by charring and densitometry. Total lipid content was higher (p less than 0.01) in the hippocampus (72.6 +/- 2.8 mg/g wet wt, mean +/- SD) than in the frontal cortex and basal ganglia (57.7 +/- 2.1 and 62.6 +/- 1.5 mg/g wet wt, respectively). Ischemic changes occurred only in the frontal cortex, where total lipid content fell (p less than 0.01) by 11% after 30 minutes of ischemia because sulfatide and cerebroside contents fell by 44% and 38%, respectively. Despite a marked accumulation of free fatty acids during complete global brain ischemia in rats, the only detectable changes in brain lipids were in the amounts of cerebrosides and sulfatides in the frontal cortex.

Lipid peroxidation in vivo induced by reversible global ischemia in rat brain.

It has been hypothesized that ischemia, followed by reperfusion, facilitates peroxidative free-radical chain processes in brain. To resolve this question, rats were subjected to reversible global ischemia. From coronal sections of brains frozen in situ, small (ca. 2 mg) amounts of tissue were sampled from neocortex, hippocampus, and thalamus of both cerebral hemispheres of four groups of rats exposed to 30 min cerebral ischemia followed by 0, 30, 60, and 240 min of reperfusion, and from a control group subjected to the same operative procedures, except for the induction of ischemia. Heptane-solubilized total lipid extracts from these samples were analyzed spectroscopically in the 190-330 nm range for content of isolated (nonconjugated) double bonds and of conjugated diene structures; the latter are formed from isolated double bonds during peroxidation of unsaturated fatty acids. Spectra derived from tissue regions of rats subjected to ischemia, or ischemia followed by reperfusion, were compared to averaged, region-specific control spectra and were normalized to the original content of isolated double bonds in the peroxidized samples. The resultant difference spectra were analyzed in terms of ratios of conjugated diene concentration to the concentration of isolated double bonds originally at risk in the specific tissue zones considered. The peak representing conjugated diene formation was centered at 238 +/- 1 nm and was usually well resolved when the molar ratio [conjugated diene]/[isolated double bonds], expressed as a percentage [( CD]/[IDB]), was greater than 0.25%.(ABSTRACT TRUNCATED AT 250 WORDS)