Brain lactate uptake increases at the site of impact after traumatic brain injury

Neuronal death in the hippocampal CA1 subregion has been shown to occur in a delayed manner after transient global ischemia. The 2-vessel occlusion model is one of the most frequently used global ischemia paradigms in rodents. Although researchers often fail to induce bilateral delayed CA1 neuronal death, the importance of hypotension severity has not been fully discussed. We induced 10 min of global ischemia with 2-vessel occlusion and various severities of hypotension in rats, and the subsequent neuronal damage and neurogenesis in the hippocampal CA1 pyramidal cell layer were immunohistochemically studied. Neuronal apoptosis after global ischemia was also characterized by terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL). The mean arterial blood pressure of 31-35 mmHg was the most appropriate range of hypotension in this model because of low mortality and consistent bilateral CA1 injury. Most of the neurons in the CA1 pyramidal cell layer lost neuron specific nuclear protein and became TUNEL-positive 3 days after ischemia. There was no evidence of apoptosis or neurogenesis at 7-28 days. There were ischemia-tolerant neurons in the CA1 pyramidal cell layer that survived delayed neurodegeneration, however, further studies are necessary to characterize the property of these neurons.     

Prevention of postischemic hyperthermia prevents ischemic injury of CA1 neurons in gerbils

Halothane-anesthetized Mongolian gerbils were submitted to 5-min bilateral carotid artery occlusion. After ischemia, halothane anesthesia was continued for various periods of up to 85 min, and the degree of CA1 neuronal injury was estimated 7 days later by counting the number of surviving pyramidal cells. During ischemia and postischemic halothane anesthesia, rectal and cranial temperature was kept at control level (37.7 and 37.0 degrees C, respectively) using a feedback-controlled heating system. When anesthesia was discontinued after ischemia, transient hyperthermia occurred. In animals with 0- and 15-min postischemic halothane anesthesia, both cranial and rectal temperature rose by approximately 1.5 degrees C, and the number of surviving CA1 neurons amounted to less than 25% of control. After 45- or 85-min postischemic anesthesia, hyperthermia was significantly reduced and the number of surviving neurons increased to 65 and 89%, respectively. The protective effect of postischemic anesthesia was lost when anesthetized animals were submitted to the same hyperthermic profile as nonanesthetized ones, using a feedback-controlled heating system (16% surviving neurons in hyperthermia vs. 89% in normothermia, respectively). These observations demonstrate that postischemic anesthesia with 1% halothane protects against delayed neuronal death by preventing postischemic hyperthermia and not by its anesthetic effects.     

Postischemic changes in the immunophilin FKBP12 in the rat brain

An immunosuppressant tacrolimus (FK506) protects against neuronal damage following cerebral ischemia. On the other hand, the major physiological role of the immunophilin FK506-binding protein-12 (FKBP12) is a modulation of intracellular calcium flux. Since an increase in intracellular calcium concentration is a major mediator of ischemic neuronal death, we investigated the changes in FKBP12 following cerebral ischemia in the rat. We induced focal cerebral ischemia by intraluminal occlusion of the middle cerebral artery for 1 h, and global cerebral ischemia for 10 min by bilateral carotid artery occlusion combined with hypotension. The animals were killed at 4 h to 7 days after reperfusion. Immunohistochemistry was performed on paraffin sections using a monoclonal antibody raised against recombinant FKBP12. Immunoreactivity to FKBP12 in control brains was most pronounced in the CA1 subfield of the hippocampus and the striatum, the localization being primarily neuronal. Following focal ischemia, FKBP12 immunoreactivity decreased rapidly in the ischemic core by 4 h, but increased in surviving neurons in penumbra areas (4 h-7 days). Within an area of infarction, invading leukocytes and macrophages exhibited immunoreactivity to FKBP12 (3-7 days). Following global ischemia, FKBP12 immunoreactivity in CA1 neurons decreased after 1 day, and then it was lost between 2 and 7 days, although many CA1 neurons showed a transient increase in FKBP12 at 2 days. No FKBP12 immunoreactivity was observed in reactive glial cells. Thus, FKBP12 declined in dying neurons, whereas FKBP12 was upregulated in less severely injured neurons. The findings suggest that (1) FKBP12 plays an important role in the process of neuronal survival and death following cerebral ischemia, and (2) FKBP12 is involved in inflammatory reactions that occur within an area of infarction.     

Delayed neuronal death and damage of GDNF family receptors in CA1 following focal cerebral ischemia

Delayed neuronal death (DND) of pyramidal neurons in the CA1 and CA3 regions of the hippocampus has been extensively studied following global brain ischemia, whereas only little is known about DND in this highly vulnerable brain region after focal brain ischemia. In the present study, the distribution and time course of hippocampal neuronal apoptosis were studied following transient middle cerebral artery occlusion (MCAO) in rats 1, 3, 7, 14, and 30 days after the insult. In 60% of the animals, more than 90% of CA1 pyramidal neurons showed strong nick-end labeling (TUNEL) staining at day 3 with fragmentation and marginalization of the nuclei in approximately 40% of these cells. The number of TUNEL-positive cells decreased within the next days, but 30 days after MCAO, some apoptotic neurons were still present. Analysis of the expression of the glial cell line-derived neurotrophic factor (GDNF) and its receptors GFRalpha1, GFRalpha2, and GFRalpha3 using triple immunofluorescence and confocal laser scanning microscopy revealed that in all animals showing marked hippocampal DND, the neuronal staining for GFRalpha1, GFRalpha3, and GDNF decreased prior to the onset of TUNEL staining in CA1. After 7 days, some apoptotic neurons still expressed GFRalpha3, whereas only few showed GFRalpha1 immunoreactivity, indicating that GFRalpha1 may be beneficial for the survival of hippocampal neurons. The data suggest that reduced expression of GDNF and impairment of GFRalpha1/3 may contribute to hippocampal DND after focal brain ischemia.     

Therapeutic window of halothane anesthesia for reversal of delayed neuronal injury in gerbils: relationship to postischemic motor hyperactivity

The effect of postischemic halothane anesthesia on locomotor activity and delayed neuronal injury in the hippocampal CA1 sector was examined in gerbils subjected to 5-min forebrain ischemia. Locomotor activity was assessed for 48 h after ischemia using an animal activity monitor, and CA1 injury was evaluated by counting the number of surviving neurons following 7 days of recirculation. Sham-treated animals exhibited a slight decrease of motor activity for about 1 day after surgery. Gerbils subjected to ischemia without postischemic halothane anesthesia developed significant motor hyperactivity (18 times higher than control activity) between 1.7 h and 6.7 h of recirculation. Surviving CA1 neurons in this group amounted to only 17% of those in the control animals. Postischemic halothane anesthesia during the initial 1.7 h of recirculation abolished subsequent motor hyperactivity and protected 84% of all CA1 neurons. Postischemic halothane anesthesia during 1.7 h-3.3 h of recirculation and 3.3-5 h of recirculation did not abolish motor hyperactivity except during the period of anesthesia, and did not protect hippocampal CA1 neurons (only 24% and 10% neuronal survival, respectively). These results demonstrate that the therapeutic window of halothane anesthesia for protection of hippocampal injury precedes the phase of locomotor hyperactivity, and that the appearance of the latter predicts delayed neuronal death.     

钾通道阻断剂TEA对大鼠全脑缺血后海马神经元损伤的保护作用

在大鼠四血管夹闭前脑缺血模型上,观察了侧脑室给予钾通道阻断剂四乙基铵（TEA）和4-氨基吡啶（4-AP）对脑缺血后海马CA1区锥体神经元迟发性死亡的保护作用。结果发现：再灌流30min后给予TEA组CA1区存活的锥体细胞数明显高于生理盐水对照组,而再灌流30min后给予4-AP组和缺血前30min给予TEA组的存活细胞数则与生理盐水对照组无明显差别。表明再灌流后给予TEA对脑缺血诱导的海马CA1区锥体神经元死亡具有明显的保护作用,提示钾通道可能在缺血后海马CA1区锥体细胞的迟发性死亡中发挥重要的作用。     

Calpain inhibitor entrapped in liposome rescues ischemic neuronal damage

Transient forebrain ischemia induces activation of calpain and proteolysis of a neuronal cytoskeleton, fodrin, in gerbil hippocampus. This phenomenon precedes delayed neuronal death in hippocampal CA1 neurons. We examined effects of a calpain inhibitor on delayed neuronal death after transient forebrain ischemia. In gerbils, a selective calpain inhibitor entrapped in liposome was given transvenously and 30 min later, 5-min forebrain ischemia was produced by occlusion of both common carotid arteries. On day 7, CA1 neuronal damage was examined in the hippocampal slices stained with cresyl violet. Calpain-induced proteolysis of fodrin was also examined by immunohistochemistry and immunoblot. Additionally, to assure entrapment of the inhibitor by CA1 neurons, the inhibitor-liposome complex was labeled with FITC and given to gerbils. Fluorescence in the hippocampal slices was examined by confocal laser scanning microscope. Selective CA1 neuronal damage induced by forebrain ischemia was prevented by administration of the inhibitor in a dose-dependent manner. Calpain-induced proteolysis of fodrin was also extinguished by the calpain inhibitor in a dose-dependent manner. Bright fluorescence of the FITC-labeled inhibitor was observed in the CA1 neurons. The data show an important role of calpain in the development of the ischemic delayed neuronal death. Calpain seems to produce neuronal damage by degrading neuronal cytoskeleton. Our data also show a palliative effect of the calpain inhibitor on the neurotoxic damage, which offers a new and potent treatment of transient forebrain cerebral ischemia.     

Temporospatial expression of HSP72 and c-JUN, and DNA fragmentation in goat hippocampus after global cerebral ischemia

The role of gene induction (expression of HSP72 and c-JUN proteins) and delayed ischemic cell death (in situ labeling of DNA fragmentation) have been investigated in the goat hippocampus after transient global cerebral ischemia. The animals were subjected to 20-min ischemia (bilateral occlusion of the external carotid arteries plus bilateral jugular vein compression) and allowed to reperfuse for 2 h, and then 1, 3, and 7 days. Histological signs of cell loss were not found in the hippocampus at 2 h, 1 day, or 3 days of reperfusion. However, such an ischemic insult produced extensive, selective, and delayed degeneration in the hippocampus, as 68% of the neurons in CA1 had died at 7 days, but cell loss was not detected in CA3 and dentate gyrus fields. Concomitantly, a high percentage of TUNEL-positive CA1 neurons (60+/-9%, mean +/- SEM) was seen at 7 days, but not at the earlier time points. Mild induction of HSP72 was detected in the goat hippocampus after ischemia. The maximum percentage of HSP72-positive neurons (10-15%) was shown at 3 days of reperfusion and was concentrated mainly in the CA3 field, subiculum, and hilus, rather than in the CA1 field, whereas HSP72 expression was hardly detected at 7 days. At this later time point, scattered induction of nuclear c-JUN was found in a few neurons. The results show that: 1) postischemic delayed neuronal death selectively affects the CA1 field in the goat hippocampus, a phenomenon which seems to take longer to develop than in previously reported rodent models; and 2) postischemic expression of c-JUN does not appear to be related to cell death or survival, while the inability of most CA1 neurons to express HSP72 could contribute to neuronal death.     

Global cerebral ischemia due to cardiocirculatory arrest in mice causes neuronal degeneration and early induction of transcription factor genes in the hippocampus

To analyze the role of specific genes and proteins in neuronal signaling cascades following global cerebral ischemia, it would be useful to have a reproducible model of global cerebral ischemia in mice that potentially allows the investigation of mice with specific genomic mutations. We first report on the development of a model of reversible cardiocirculatory arrest in mice and the consequences of such an insult to neuronal degeneration and expression of immediate early genes (IEG) in the hippocampus. Cardiocirculatory arrest of 5 min duration was induced via ventricular fibrillation in mechanically ventilated NMRI mice. After successful cardiopulmonary resuscitation (CPR), animals were allowed to reperfuse spontaneously for 3 h (n=7) and 7 days (n=7). TUNEL staining revealed a selective degeneration of a subset of neurons in the hippocampal CA1 sector at 7 days. About 30% of all TUNEL-positive nuclei showed condensed chromatin and apoptotic bodies. Immunohistochemical studies of IEG expression performed at 3 h exhibited a marked induction of c-Fos, c-Jun, and Krox-24 protein in all sectors of the hippocampus, peaking in vulnerable CA1 pyramidal neurons and in dentate gyrus. In contrast, sham-operated animals (n=3) did not reveal neuronal degeneration or increased IEG expression in the hippocampus when compared with untreated control animals (n=3). In conclusion, we present a new model of global cerebral ischemia and reperfusion in mice with the use of complete cardiocirculatory arrest and subsequent CPR. Following 5 min of ischemia, a subset of CA1 pyramidal neurons was TUNEL-positive at 7 days. The expression of IEG was observed in all sectors of the hippocampus, including selectively vulnerable CA1 pyramidal neurons. This appears to be a good model which should be useful in evaluating the role of various genes in transgenic and knockout mice following global ischemia.     

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia.

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.     

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.     

BCL-2 transduction, using a herpes simplex virus amplicon, protects hippocampal neurons from transient global ischemia

Transient global ischemia results in selective neuronal damage of hippocampal CA1 neurons. Five minutes of bilateral common carotid artery occlusion, in the Mongolian gerbil, effectively restricts forebrain blood flow, resulting in a delayed neuronal death of CA1 pyramidal cells. While there is a delay of approximately 72 h in the appearance of cell death, markers related to the mechanism of ischemic death become apparent well before neurons die. Ischemia-induced increases in the cell-death-promoting protein, bax, may disrupt the bcl-2/bax ratio necessary for normal neuronal functioning and thus promote transient ischemic death. In order to locally maintain this critical bcl-2/bax ratio and thus protect CA1 neurons from delayed neuronal death, a herpes simplex viral (HSV) vector was used to selectively introduce human bcl-2, under the control of the herpes IE 4/5 promoter, into the CA1 region of the gerbil hippocampus. Twenty-four hours prior to ischemia surgery, 1 microl of HSVbcl-2 was infused unilaterally into the CA1 region at a rate of 2 nl/min. Seventy-two hours after ischemia the animals were sacrificed and processed using Nissl, silver degeneration, and immunohistochemical (anti-human bcl-2) staining. Immunohistochemistry demonstrated both glial and neuronal bcl-2 expression around the HSVbcl-2 infusion site. The evaluation following silver degeneration staining indicated a further degeneration of CA1 neurons in the immediate area of the viral vector infusion. This damage seems to be the result of cellular debris associated with the processing of the viral amplicons. Silver degeneration staining is not present in the areas that demonstrate bcl-2 staining. These neurons appear to have been rescued from ischemic damage. This result was confirmed using the Nissl staining. Therefore, by altering the local ratio of bcl-2/bax using the HSVbcl-2 vector one may protect CA1 pyramidal cell from the delayed neuronal death of transient global ischemia.     

Histochemical study of Ca2+-ATPase activity in ischemic CA1 pyramidal neurons in the gerbil hippocampus

Although cytosolic Ca²⁺ accumulation plays a pivotal role in delayed neuronal death, there have been no investigations on the role of the cellular Ca²⁺ export system in this novel phenomenon. To clarify the function of the Ca²⁺-pump in delayed neuronal death, the plasma membrane Ca²⁺-ATPase activity of CA1 pyramidal neurons was investigated ultracytochemically in normal and ischemic gerbil hippocampus. To correlate enzyme activity with delayed neuronal death, histochemical detection was performed at various recirculation times after 5 min of ischemia produced by occlusion of the bilateral carotid arteries. At 10 min after ischemia, CA1 pyramidal neurons showed weak Ca²⁺-ATPase activity. Although enzyme activity had almost fully recovered 2 h after ischemia, it was reduced again 6 h after ischemia. Thereafter, Ca²⁺-ATPase activity on the plasma membrance of CA1 pyramidal neurons decreased progressively, losing its localization on day 3. On day 4 following ischemia, reaction products were diffusely scattered throughout the whole cell body. Our results indicate that, after once having recovered from ischemic damage, severe disturbance of the membrane Ca²⁺ export system proceeds from the early stage of delayed neuronal death and disturbs the re-export of accumulated cytosolic Ca²⁺, which might contribute to delayed neuronal death. Occult disruption of Ca²⁺ homeostasis seems to occur from an extremely early stage of delayed neuronal death in CA1 pyramidal cells.     

亚低温对大鼠短暂全脑缺血后神经元凋亡的影响

目的 探讨亚低温对大鼠脑缺血后神经元凋亡的影响，揭示亚低温的部分神经保护机制。方法 采用“双侧颈总动脉阻断＋全身低血压”方法来建立大鼠短暂性全脑缺血模型。用神经元尼氏体亚甲兰特殊染色法观察大鼠脑缺血后海马CA1区神经元损害情况；原位细胞凋亡检测法（TUNEL染色）及电镜观察脑缺血后CA1区神经元凋亡情况。结果 与假手术组、低温缺血组相比，常温缺血组海马CA1区神经元缺失明显（P＜0．01）。常温及低温缺血组海马CA1区均存在神经元凋亡，但低温缺血组海马CA1区凋亡神经元数明显少于缺血组（P＜0．01）。结论 经“双侧颈总动脉阻断＋全身低血压”方法建立的大鼠短暂全脑缺血模型证实了亚低温的脑保护作用。全脑缺血后的迟发性神经元死亡很可能经由凋亡途径，而亚低温可通过抑制缺血性神经元凋亡而发挥一定的神经保护作用。     

Nuclear localization of the hypoxia-regulated pro-apoptotic protein BNIP3 after global brain ischemia in the rat hippocampus

The 19 kD interacting protein 3, Nip3/BNIP3, is a pro-apoptotic member of the Bcl-2 family induced during hypoxia via the hypoxia-inducible factor (HIF) 1. BNIP3 has been linked to both apoptotic and necrotic cell death involving mitochondrial permeability transition. Since apoptotic and necrotic mechanisms may occur in brain ischemia, immunohistochemical changes of BNIP3 were studied at 1, 2, 3 and 7 days after transient global brain ischemia (12.5 min) in ventilated normothermic rats. In control brains, BNIP3-like immunoreactivity was moderately strong in neuronal processes or cytoplasm and absent in the nucleus. In the ischemia-vulnerable CA1 neurons, BNIP3-positive granules were seen in the nucleus at 1 and 2 days, and these neurons were damaged at 3 and 7 days. The resistant CA3 neurons showed nuclear BNIP3 labeling by 1 day and then returned to the normal state. BNIP3-positive granules did not overlap with the nucleolus. Constitutively expressed BNIP3 may participate in apoptotic and necrotic processes after brain ischemia. Nuclear location of BNIP3 after brain ischemia indicates a novel role for the regulation of cell survival in neurons or a general disturbance of the nuclear envelope.     

The effect of blood pressure (37 vs 45 mmHg) and carotid occlusion duration (8 vs 10 min) on CA1-4 neuronal damage when using isoflurane in a global cerebral ischemia rat model

This study presents our findings on the extent of neuronal damage in the hippocampal CA1-4 subfields following global (forebrain) cerebral ischemia in rats when using different blood pressure levels (37 vs 45 mmHg) and bilateral carotid occlusion durations (8 vs 10 min) under isoflurane anesthesia. We observed that global ischemia induced at a blood pressure of 37 mmHg resulted in high-grade CA1 neuron injury (>90%) at either duration of carotid occlusion. In contrast, global ischemia induced at a blood pressure of 45 mmHg resulted in either high-grade CA1 neuronal loss or a neuronal loss of ≈50% or less. We also noted that a post-reperfusion EEG recovery time (return of burst suppression spikes) of >12 min was associated with an 85% rate of high-grade CA1 neuronal injury. Neuronal loss in the other hippocampal subfields did not differ significantly between any of the 4 different model parameters tested. In these subfields ≈55% neuronal loss occurred in the CA2 subfield, and ≈30% in the CA3 and CA4 subfields. These findings highlight the need to assess different model parameters in order to achieve consistent high-grade CA1 neuronal damage, which, among other experimental outcomes, will improve the ability to uncover therapeutic effects using the least possible animals when assessing a neuroprotective treatment.     

Involvement of 5-hydroxytryptamine4 receptor in the exacerbation of neuronal loss by psychological stress in the hippocampus of SHRSP with a transient ischemia

A transient forebrain ischemia produced a delayed neuronal death of the hippocampus pyramidal cells in stroke-prone spontaneously hypertensive rats (SHRSP). Long term exposure of rats to stress has been reported to induce deleterious effects on the brain including morphological neuronal degeneration in the hippocampus. The present study was designed to examine the effects of psychological and physical stress on the ischemia-related neuronal death and the effects of 5-hydroxytryptamine(4) (5-HT(4)) receptor antagonist. SHRSP were exposed to the psychological or physical stress for 60 min in the communication box once or repeatedly for 3 days and occluded. SB204070, a 5-HT(4) receptor antagonist was injected before the occlusion. Seven days after the occlusion, the number of the neurons damaged morphologically was examined. A transient bilateral carotid occlusion produced a neuronal death of the CA1 subfield of the hippocampus in a time-dependent manner between 3 and 10 min. A 4 min occlusion induced very little morphological damage and a 5 min one produced a significant neuronal death. Exposure of rats to the psychological stress during 60 min for 3 days before the ischemic insults damaged the pyramidal cells by 4 min ischemia much more than without stress. Physical stress daily for 3 times also increased the damaged neurons. Pretreatment of SB204070 0.1 mg/kg after the stress exposure for 3 days significantly decreased the neuronal damage exacerbated by the stress exposure; however, it did not alter the damage induced by 4 or 10 min occlusion without stress. These results suggest that the repeated exposure of animals to the stress dramatically exacerbates the neuronal death by a transient ischemia and the 5-HT(4) receptor may be involved in the stress-induced exacerbating mechanism of the neuronal damage.     

A new method for producing temporary complete cerebral ischemia in rats

A new model of temporary complete cerebral ischemia was developed and tested in 64 rats. With use of microsurgical techniques, both pterygopalatine and external carotid arteries were occluded and the basilar artery was coagulated to reduce potential collateral CBF during ischemia. After this preliminary five-vessel occlusion, temporary global ischemia was induced by occluding the common carotid arteries (CCAs) with microclips. To validate the method, CBF was measured autoradiographically in 24 anatomical regions at death after 5 min of ischemia or after 15 min of ischemia followed by 5 min of reperfusion. Mean arterial blood pressure and arterial blood gases remained stable under controlled endotracheal ventilation and anesthesia (halothane, 70% N2O, and 30% O2) throughout the CBF experiments, except for a 10-15% increase in mean arterial blood pressure for 1-5 min after bilateral CCA occlusion. After the initial five-vessel occlusion, the EEG did not change, and local CBF levels were comparable to those in anesthetized non-surgical controls. When the CCAs were occluded, the EEG flattened rapidly; after 5 min of ischemia, autoradiography showed no detectable blood flow in the forebrain and cerebellum. The local CBF levels measured after 15 min of temporary global ischemia and 5 min of reperfusion demonstrated relatively homogeneous postischemic hyperperfusion; only two of eight rats had several 1- to 3-mm areas of no-reflow. Survival studies showed increasing motor impairment after 10, 15, 30, and 60 min of temporary CCA occlusion. Ischemic neuronal damage was observed histologically in the hippocampus and basal ganglia 24 h after 10 min of temporary ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apoptosis and necrosis occur in separate neuronal populations in hippocampus and cerebellum after ischemia and are associated with differential alterations in metabotropic glutamate receptor signaling pathways.

It was evaluated whether postischemic neurodegeneration is apoptosis and occurs with alterations in phosphoinositide-linked metabotropic glutamate receptors (mGluRs) and their associated signaling pathways. A dog model of transient global incomplete cerebral ischemia was used. The CA1 pyramidal cells and cerebellar Purkinje cells underwent progressive delayed degeneration. By in situ end-labeling of DNA, death of CA1 and Purkinje cells was greater at 7 days than 1 day after ischemia, whereas death of granule neurons in dentate gyrus and cerebellar cortex was greater at 1 than at 7 days. Ultrastructurally, degenerating CA1 pyramidal neurons and cerebellar Purkinje cells were necrotic; in contrast, degenerating granule neurons were apoptotic. In agarose gels of regional DNA extracts, random DNA fragmentation coexisted with internucleosomal fragmentation. By immunoblotting of regional homogenates, mGluR1alpha, mGluR5, phospholipase Cbeta (PLCbeta), and Galphaq/11 protein levels in hippocampus at 1 and 7 days after ischemia were similar to control levels, but in cerebellar cortex, mGluR1alpha and mGluR5 were decreased but PLCbeta was increased. By immunocytochemistry, mGluR and PLCbeta immunoreactivity dissipated in CA1 and cerebellar Purkinje cell/ molecular layers, whereas immunoreactivities for these proteins were enhanced in granule neurons. It was concluded that neuronal death after global ischemia exists as two distinct, temporally overlapping forms in hippocampus and cerebellum: necrosis of pyramidal neurons and Purkinje cells and apoptosis of granule neurons. Neuronal necrosis is associated with a loss of phosphoinositide-linked mGluR transduction proteins, whereas neuronal apoptosis occurs with increased mGluR signaling.     

In vivo administration of corticotropin-releasing hormone at remote intervals following ischemia enhances CA1 neuronal survival and recovery of spatial memory impairments: a role for opioid receptors

The contribution of corticotropin-releasing hormone (CRH) in the modulation of ischemia-induced cell death in vivo remains unclear. We characterized the impact of pre-ischemic administration of CRH (0, 0.1, 1, 5 microg, i.c.v., 15 min prior to vessel occlusion) on neuronal damage following global ischemia in rats. The injection of 5 microg CRH led to a 37% increase in CA1 neuronal survival compared to vehicle-treated ischemic animals, while pre-treatment with alpha-helical CRH (9-41) abolished this neuronal protection. A second objective aimed to determine whether CRH protection is maintained over weeks when the peptide is administered at remote time intervals following ischemia. Compared to vehicle-treated ischemic animals, administration of CRH 8h following global ischemia led to a 61% increase in CA1 neuronal survival observed 30 days post-ischemia. Neuronal protection translated into significant improvement of ischemia-induced spatial memory deficits in the radial maze. Finally, our findings demonstrated that selective blockade of kappa- and delta-opioid receptors (using nor-binaltorphimine and naltrindole, respectively) prior to CRH administration significantly reduced CA1 neuronal protection. These findings represent the first demonstration of enhanced neuronal survival following in vivo CRH administration in a global model of ischemia in rats. They also support the idea that CRH-induced neuroprotection involves opioid receptors activation.