'Ischemic tolerance' phenomenon detected in various brain regions.

We investigated the effects of mild and non-lethal ischemic insult on neuronal death following subsequent lethal ischemic stress in various brain regions, using a gerbil model of bilateral cerebral ischemia. Single 10-min ischemia consistently caused neuronal damage in the hippocampal CA1, CA2, CA3 and CA4, layer III/IV of the cerebral cortex, dorsolateral part of the caudoputamen and ventrolateral part of the thalamus. On the other hand, in double ischemia groups, 2-min ischemic insult 2 days before 10-min ischemia exhibited significant protection in the CA1 and CA3 of the hippocampus, the cerebral cortex, the caudoputamen and the thalamus. Five-min ischemic insult 2 days before 10-min ischemia also showed protective effect in the same areas as those of 2-min ischemia except for the CA1 region of the hippocampus, while 1-min ischemic insult exhibited no protective effect in any brain regions. In the immunoblot analysis, both 2- and 5-min ischemia caused increased synthesis of heat shock protein 72 (HSP 72) in the hippocampus, but 1-min ischemia did not. The present study demonstrated that the 'ischemic tolerance' phenomenon was widely found in the brain and also suggested that ischemic treatment severe enough to cause HSP 72 synthesis might be needed for induction of 'ischemic tolerance'.     

‘Ischemic tolerance’ phenomenon detected in various brain regions

We investigated the effects of mild and non-lethal ischemic insult on neuronal death following subsequent lethal ischemic stress in various brain regions, using a gerbil model of bilateral cerebral ischemia. Single 10-min ischemia consistently caused neuronal damage in the hippocampal CA1, CA2, CA3 and CA4, layer III/IV of the cerebral cortex, dorsolateral part of the caudoputamen and ventrolateral part of the thalamus. On the other hand, in double ischemia groups, 2-min ischemic insult 2 days before 10-min ischemia exhibited significant protection in the CA1 and CA3 of the hippocampus, the cerebral cortex, the caudoputamen and the thalamus. Five-min ischemic insult 2 days before 10-min ischemia also showed protective effect in the same areas as those of 2-min ischemia except for the CA1 region of the hippocampus, while 1-min ischemic insult exhibited no protective effect in any brain regions. In the immunoblot analysis, both 2- and 5-min ischemia caused increased synthesis of heat shock protein 72 (HSP 72) in the hippocampus, but 1-min ischemia did not. The present study demonstrated that the ‘ischemic tolerance’ phenomenon was widely found in the brain and also suggested that ischemic treatment severe enough to cause HSP 72 synthesis might be needed for induction of ‘ischemic tolerance’.     

Regional differences in the rate of energy impairment after threshold level ischemia for induction of cerebral infarction in gerbils

The development of infarction and/or selective neuronal death in the brain after transient cerebral ischemia depends on the severity of the ischemic episode. After transient cerebral ischemia of the threshold level for the induction of infarction, both changes evolve slowly in various postischemic regions. We examined the relationship of disturbances of energy metabolism to infarction and selective neuronal death in various regions of the postischemic brain subjected to two 10-min occlusions of the unilateral common carotid artery. Our results indicated that in various cerebral regions that developed infarction, the tissue ATP content, in parallel with the succinic dehydrogenase activity, fell to their lowest levels at different times over a 4-day period after circulation had been restored (earliest to latest: dorsolateral thalamus > dorsolateral caudate > chiasmal level cortex > hippocampal CA3 sector > hippocampal CA1 sector). In the cortex at the infundibular level, disseminated selective neuronal death developed over a 7-day period following restoration of circulation; it was accompanied by only a slight alteration in energy metabolism. The present results indicate that regional differences existed in the rate of energy impairment and evolving infarction in the postischemic gerbil brain. Energy impairment, in association with mitochondrial enzymatic dysfunction, seems to be indispensable for the delayed manifestation of cerebral infarction but not for disseminated selective neuronal death. Received: 1 December 1999 / Revised, accepted: 6 March 2000     

Reversal of acute apparent diffusion coefficient abnormalities and delayed neuronal death following transient focal cerebral ischemia in rats.

Twenty-two rats were subjected to 8, 15, 30, or 60 minutes of temporary middle cerebral artery occlusion (n = 5 per group) or sham occlusion (n = 2) in the magnetic resonance imaging unit. Diffusion-, perfusion-, and T2-weighted imaging were acquired before and during occlusion, and after reperfusion. A coregistration method was used to correlate the acute changes of the average apparent diffusion coefficient (ADCav) with the histology after 72 hours at the same topographic sites. The initially reduced ADCav values recovered completely in both the lateral caudoputamen and upper frontoparietal cortex in the 8-, 15-, and 30-minute groups, partially in the cortex, and not at all in the caudoputamen in the 60-minute group. The histology showed that the caudoputamen was either normal or had mild neuronal injury in the 8-minute group and invariably had some degree of neuronal death in the 15-, 30-, and 60-minute groups, whereas the cortex was either normal or had varying degrees of neuronal injury in all groups. No histological abnormalities were seen in the sham-operated rats. Our data suggest that acute ADCav reversal does not always predict tissue recovery from ischemic injury and that temporary focal ischemia for even 8-minute duration can cause delayed neuronal death that is more severe in the caudoputamen where the initial ADCav decline was greater than in the cortex.     

Characterization of a recovery global cerebral ischemia model in the mouse.

Transgenic/knockout murine variants allow roles of specific proteins to be studied in cerebral ischemia. Because of the size of mice, however, study of prolonged recovery from global ischemia has been limited. This project characterized an adaptation of the rat two-vessel occlusion model of global ischemia for use in the mouse. C57B1/6J mice (8 weeks old; 21 +/- 1 g) were overnight fasted, anesthetized with halothane, intubated and mechanically ventilated. The right internal jugular vein and femoral artery were cannulated. Pericranial temperature was held at 37.0 degrees C. The carotid arteries were occluded and mean arterial pressure was reduced to 35 mmHg with 0.3 mg intra-arterial trimethaphan and venous exsanguination. Electroencephalographic isoelectricity was confirmed in cohort mice. Ten minutes later ischemia was reversed. Mice were allowed 1, 3 or 5 days survival followed by histologic analysis. Regional cerebral blood flow (CBF) was determined autoradiographically. Outcome effects of intra-ischemic hyperglycemia (approximately 350 mg/dl) or hypothermia (34 degrees C) were also examined. The mortality rate was less than 10% in all recovery groups. Ischemia caused reduction of CBF to < 2% of sham values in cortex, hippocampus, and caudoputamen. CBF was unchanged in thalamus, brainstem and cerebellum. CA1 damage, greater after 3 days vs. 1 day reperfusion, was not further increased at 5 days. Histologic injury was increased by hyperglycemia although seizures did not occur. Hypothermia reduced CA1 damage. This study demonstrates feasibility of using the two-vessel occlusion + hypotension recovery model in the mouse. Recovery intervals of > or = 3 days are required to account for delayed CA1 neuronal necrosis. Histologic outcome can be modulated by known physiologic determinants of ischemic brain damage.     

Isoforms of protein kinase C in postsynaptic densities after cerebral ischemia

Relatively mild ischemic insult can lead to delayed neuronal cell death in vulnerable brain regions. We provide evidence that the protein composition of the postsynaptic densities (PSD) undergoes rapid modification after 15 min postdecapitative as well as 5 min transient global ischemia. We observed a significant increase in cPKC and nPKC protein content in the postischemic PSD. Of the calcium-regulated PKC isoforms, the alpha and beta subtypes increase in PSD over ten times above the control values whereas gamma PKC, an isoform most abundant in the native PSD structure, shows relatively smaller changes under ischemic conditions. For the first time, the PSD membrane translocation of Ca(2+)-independent isoforms delta and epsilon is shown. The yield of the PSD protein preparation from the postischemic cortex was two times higher compared with control. This correlated with an abundant increase in electron density and changes in ultrastructure of PSD isolated from postischemic cortex. Also sections from CA1 gerbils hippocampus after transient ischemia showed persistent enlargement of postsynaptic densities up to 24 h of reperfusion. This was accompanied by elevation of the PSD/cytoskeleton-associated alpha, beta PKC immunoreactivity and other changes in neuronal and glial cell morphology typical of the early postischemic degeneration. Sustained changes in PKC composition and organization of postsynaptic membranes during and after ischemia may cause persistent alteration in synaptic transmission and subsequently contribute to delayed neuronal injury.     

Calpain activity in the rat brain after transient forebrain ischemia.

Activity of the Ca(2+)-dependent protease calpain is increased in neurons after global and focal brain ischemia, and may contribute to postischemic injury cascades. Understanding the time course and location of calpain activity in the post-ischemic brain is essential to establishing causality and optimizing therapeutic interventions. This study examined the temporal and spatial characteristics of brain calpain activity after transient forebrain ischemia (TFI) in rats. Male Long Evans rats underwent 10 min of normothermic TFI induced by bilateral carotid occlusion with hypovolemic hypotension (MABP 30 mm Hg). Brain calpain activity was examined between 1 and 72 h after reperfusion. Western blot analysis of regional brain homogenates demonstrated a bimodal pattern of calpain-mediated alpha-spectrin degradation in the hippocampus, cortex, and striatum with an initial increase at 1 h followed by a more prominent secondary increase at 36 h after reperfusion. Immunohistochemical analysis revealed that calpain activity was primarily localized to dendritic fields of selectively vulnerable neurons at one hour after reperfusion. Between 24 and 48 h after reperfusion neuronal calpain activity progressed from the dorsal to ventral striatum, medial to lateral CA1 hippocampus, and centripetally expanded from watershed foci in the cerebral cortex. This progression was associated with fragmentation of dendritic processes, calpain activation in the neuronal soma and subsequent neuronal degeneration. These observations demonstrate a clear association between calpain activation and subsequent delayed neuronal death and suggest broad therapeutic window for interventions aimed at preventing delayed intracellular Ca(2+) overload and pathologic calpain activation.     

大鼠脑缺血再灌注后星形胶质细胞反应性变化差异与海马CA1区的迟发性神经元死亡

BACKGROUND： Blood supply to the hippocampus is not provided by the middle cerebral artery. However, previous studies have shown that delayed neuronal death in the hippocampus may occur following focal cerebral ischemia induced by middle cerebral artery occlusion.
OBJECTIVE： To observe the relationship between reactive changes in hippocampal astrocytes and delayed neuronal death in the hippocampal CA1 region following middle cerebral artery occlusion.
DESIGN, TIME AND SETTING： The immunohistochemical, randomized, controlled animal study was performed at the Laboratory of Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from July to November 2007.
MATERIALS： Rabbit anti-glial fibrillary acidic protein （GFAP） （Neomarkers, USA）, goat anti-rabbit IgG （Sigma, USA） and ApoAlert apoptosis detection kit （Biosciences Clontech, USA） were used in this study. METHODS： A total of 42 healthy adult male Wistar rats, aged 3–5 months, were randomly divided into a sham operation group （n = 6） and a cerebral ischemia/reperfusion group （n = 36）. In the cerebral ischemia/reperfusion group, cerebral ischemia/reperfusion models were created by middle cerebral artery occlusion. In the sham operation group, the thread was only inserted into the initial region of the internal carotid artery, and middle cerebral artery occlusion was not induced. Rats in the cerebral ischemia/reperfusion group were assigned to a delayed neuronal death （＋） subgroup and a delayed neuronal death （–） subgroup, according to the occurrence of delayed neuronal death in the ischemic side of the hippocampal CA1 region following cerebral ischemia.
MAIN OUTCOME MEASURES： Delayed neuronal death in the hippocampal CA1 region was measured by Nissl staining. GFAP expression and delayed neuronal death changes were measured in the rat hippocampal CA1 region at the ischemic hemisphere by double staining for GFAP and TUNEL.
RESULTS： After 3 days of ischemia/reperfusion, astrocytes with abnormal morphology were detected in the rat hippocampal CA1 region in the delayed neuronal death （＋） subgroup. No significant difference in GFAP expression was found in the rat hippocampal CA1 region at the ischemic hemisphere in the sham operation group, delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup （P 〉 0.05）. After 7 days of ischemia/reperfusion, many GFAP-positive cells, which possessed a large cell body and an increased number of processes, were activated in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression in the hippocampal CA1 region was greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.01）. Moreover, GFAP expression was significantly greater in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.01）. After 30 days of ischemia/reperfusion, GFAP-positive cells were present in scar-like structures in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression was significantly greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.05）. GFAP expression was significantly lower in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.05）. The delayed neuronal death rates were 42% （5/12）, 33% （4/12） and 33% （4/12） at 3, 7 and 30 days, respectively, followingischemia/reperfusion. No significant differences were detected at various time points （χ2 = 0.341, P 〉 0.05）.
CONCLUSION： The activation of astrocytes was poor in the hippocampal CA1 region during the early stages of ischemia, which is an important reason for delayed neuronal death. Glial scar formation aggravated delayed neuronal death during the advanced ischemic stage.     

Neuroprotective effect of postischemic administration of sodium orthovanadate in rats with transient middle cerebral artery occlusion.

Orthovanadate is a competitive inhibitor of protein tyrosine phosphatases. Some of its reported biologic effects are its insulin mimetic property and its activation of phosphoinositide 3-kinase and extracellular-signal regulated kinase (ERK). The authors previously reported its neuroprotective effect on delayed neuronal death of gerbil hippocampal CA1 neurons via Akt and ERK activation after transient forebrain ischemia. In the present study, the neuroprotective effect of postischemic intraperitoneal administration of sodium orthovanadate (2 l/kg of 50-mmol/l sodium orthovanadate in saline) was investigated in rats with transient middle cerebral artery occlusion. Ischemic neuronal injury was evaluated 1 day and 28 days after ischemia. The neuroprotective effect of orthovanadate was significant in the cortex but not the caudate putamen (ischemic core) at both 1 and 28 days after ischemia. In orthovanadate group, the activities of Akt and ERK were maintained after reperfusion; they were decreased in saline group. Blood glucose level decreased but within normal range. Regional cerebral blood flow was lower than that of saline group only at 0 hours after reperfusion. These data suggest that orthovanadate has neuroprotective effects in rats with transient middle cerebral artery occlusion and that these effects are mediated by Akt and ERK activation. Furthermore, low blood glucose levels and gradual recovery of regional cerebral blood flow may contribute to neuroprotection.     

Brain damage in a mouse model of global cerebral ischemia. Effect of NMDA receptor blockade

The importance of particular genes in neuronal death following global cerebral ischemia can readily be studied in genetically modified mice provided a reliable model of ischemia is available. For that purpose, we developed a mouse model of global cerebral ischemia that induces consistent damage to different regions of the brain and with a low mortality rate. Twelve minutes of ischemia was induced in C57BL/6 mice by bilateral common carotid artery occlusion under halothane anesthesia and artificial ventilation. Body and brain temperature were monitored and cortical cerebral blood flow in each hemisphere was measured by laser Doppler flowmeter before, during, and for 5 min after ischemia. Extensive damage was found in the striatum and marked cell damage was observed in the CA1 and CA2 regions of hippocampus and in thalamus. Mild damage was seen in the CA3 region, dentate gyrus and cortex. Hippocampal damage in the CA1 region is delayed and developed over 48 h. Intraischemic hypothermia of 33 degrees C provided a robust neuroprotection. The non-competitive N-methyl-D-aspartate receptor blocker, MK-801, did not provide protection in the hippocampus, cortex, striatum or thalamus when administered 30 min prior to ischemia or 2 h after the end of ischemia, but selectively mitigated damage in the hippocampus, when administered immediately following ischemia. This model of global cerebral ischemia may be useful in pharmacological and genomic studies of ischemic brain damage.     

Temporal evolution of ischemic injury evaluated with diffusion-, perfusion-, and T2-weighted MRI

OBJECTIVE: Ischemic lesions seen on diffusion-weighted imaging (DWI) are reversible if reperfusion is performed within minutes after the onset of ischemia. This study was designed to determine whether acute reversibility of DWI abnormalities is transient following brief temporary focal brain ischemia and to characterize the temporal evolution of in vivo ischemic lesions. METHODS: Eight rats were subjected to 30 minutes of temporary middle cerebral artery occlusion and underwent diffusion-, perfusion-, and T2-weighted MRI during occlusion; immediately after reperfusion; 30, 60, and 90 minutes after reperfusion; and 12, 24, 48, and 72 hours after reperfusion. Average apparent diffusion coefficient (ADCav) values and the cerebral blood flow index (CBFi) ratio were calculated in both the lateral caudoputamen and overlying cortex at each time point. The size of the in vivo ischemic abnormalities was calculated from the ADCav and the T2 maps. Postmortem triphenyltetrazolium chloride (TTC) staining was used to verify ischemic injury. RESULTS: Both the CBFi ratio and ADCav values declined significantly in the two regions during occlusion. The CBFi ratio recovered immediately after reperfusion and remained unchanged over 72 hours. However, ADCav values returned to normal at 60 to 90 minutes and secondarily decreased at 12 hours after reperfusion as compared with those in the contralateral hemisphere. The extent of the in vivo ischemic lesions maximized at 48 hours and was highly correlated with TTC-derived lesion size. CONCLUSIONS: Acute recovery of initial ADCav-defined lesions after reperfusion is transient, and secondary ADCav-defined lesions develop in a slow and delayed fashion.     

Activation of cell death pathway after a brief period of global ischemia in diabetic and non-diabetic animals

Mitochondria play a critical role in the pathogenesis of cerebral ischemia. Acute hyperglycemia has been shown to activate the mitochondria-initiated cell death pathway after an intermediate period of ischemia. The objective of the present study was to determine if diabetic hyperglycemia induced by streptozotocin activates the cell death pathway after a brief period of global ischemia. Five minutes of global ischemia was induced in nondiabetic and diabetic rats. Brain samples were collected after 30 min, 6 h, 1, 3, and 7 days of recirculation as well as from sham-operated controls. Histopathological examination in the hippocampal CA1, CA3, hilus, and dentate gyrus regions, as well as in the cortical and thalamic areas, showed that neuronal death in diabetic animals increased compared to nondiabetic ischemic controls. Neuronal damage maturation occurred after 7 days of recovery in nondiabetic rats, while it was shortened to 3 days of recovery in diabetic animals. Western blot analyses revealed that release of cytochrome c markedly increased after 1 and 3 days of reperfusion in diabetic rats. Caspase-3 activation was evident in the nuclear fraction of the cortex of diabetic rats after 3 days recovery and it was preceded by activation of caspase-9, but not activation of caspase-8. Electron microscopy demonstrated that chromatin condensation and mitochondrial swelling were features of the diabetes-mediated ischemic neuronal damage. However, no apoptotic bodies were observed in any sections examined. These results suggest that a brief period of global ischemia in diabetic animals activates a neuronal cell death pathway involving cytochrome c release, caspase-9 activation, and caspase-3 cleavage, all of which are most likely initiated by early mitochondria damage.     

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.     

Multi-focal delayed neuronal damage after transient regional ischemia--mechanism of vascular dementia

We describe multi-focal delayed neuronal death of rat brain after transient regional ischemia induced by embolization of the right middle cerebral artery (MCA). After sixty minutes of MCA occlusion, recirculation was achieved by removal of the embolus. Chronological changes in the distribution of the neuronal damage were determined by using the 45Ca autoradiographic technique and the histological examination. Sixty minutes after MCA occlusion, 45Ca accumulation extended to the lateral segment of the caudate putamen and the cerebral cortex supplied by the occluded MCA. Moreover, three days after ischemic insult, 45Ca had accumulated in the ipsilateral thalamus and substantia nigra. Histological examination revealed that the neurons in both area suffered damage and were selectively reduced in number. Both areas lie outside the ischemic area, but have transsynaptic connections with the ischemic focus. We suggest that the postischemic delayed neuronal death in exo-focal remote areas may be caused by a transsynaptic process associated with the infarcted areas and that these delayed multi-focal brain damage may exacerbate clinical symptoms in the chronic stage of stroke.     

Mild intraischemic hypothermia suppresses consumption of endogenous antioxidants after temporary focal ischemia in rats

Oxidative damage by free radicals has been proposed as a mechanism of cerebral injury due to ischemia and reperfusion. Hypothermia protects against ischemic necrosis; however, its effect on oxidative stress has not been investigated. In this study, the effects of hypothermia on oxidative stress were studied by determining consumption of endogenous antioxidants after temporary focal ischemia in rats. Thirty-two Sprague-Dawley rats anesthetized with 1.5% isoflurane underwent 3 h of middle cerebral artery occlusion under hypothermic (33°C) or normothermic (37°C) conditions followed by 3 h of normothermic reperfusion. In the first study (n = 8per group), intraischemic hypothermia suppressed the reduction of tissue concentrations of endogenous antioxidants, ascorbate (P≤ 0.05), and glutathione (P≤ 0.05) in ischemic cortex but not in caudoputamen. In a parallel study (n = 8per group), hypothermia reduced tissue damage in ischemic frontoparietal cortex (P ≤ 0.05), but not in caudoputamen. Laser-Doppler estimates of cortical blood flow showed that intraischemic hypothermia significantly attenuated early postischemic hyperperfusion (P ≤ 0.01) and delayed postischemic hypoperfusion (P ≤ 0.01). These results demonstrate that intraischemic mild hypothermia reduces oxidative stress and cell injury after prolonged focal ischemia followed by reperfusion. The reduction of oxidative stress by hypothermia may be related indirectly to attenuation of postischemic blood flow changes.     

Delayed hypoperfusion after incomplete forebrain ischemia in the rat. The role of polymorphonuclear leukocytes

The role of polymorphonuclear leukocytes (PMNLs) in postischemic delayed hypoperfusion in the rat brain was investigated. Cerebral ischemia was accomplished by reversible bilateral occlusion of the common carotid arteries for 15 min combined with bleeding to an MABP of 50 mm Hg. The animals of one group were depleted of their circulating. PMNLs by intraperitoneal injections of an antineutrophil serum (ANS) prior to the experiment. All animals included in this group had fewer than 0.2 x 10(9) circulating PMNLs/L at the start of the experiments. In another group ANS was injected intravenously for 5 min starting 2 min after the ischemic insult. After 4 min of recirculation, the number of circulating PMNLs in this group was below 10% of the normal. Control animals were injected with the same amount of normal sheep serum or were not treated at all. Sixty minutes after termination of ischemia, the local blood flow in previously ischemic cerebral structures was 40-50% of the normal as measured with the [14C]iodoantipyrine technique. In animals treated with ANS prior to the ischemic insult, the postischemic blood flow in the frontal, sensorimotor, and parietal cortex as well as caudoputamen and thalamus was significantly higher than that in non-ANS-treated animals. Treatment with ANS immediately after the ischemic period caused no improvement of the local CBF. It is concluded that PMNLs are involved in the cerebral postischemic flow derangements seen in this model. Their effects seem to be exerted during ischemia or immediately upon reinstitution of blood flow.     

Necrosis, apoptosis and hybrid death in the cortex and thalamus after barrel cortex ischemia in rats

Focal ischemia in the cerebral cortex results in acute and delayed cell death in the ischemic cortex and non-ischemic thalamus. We examined the hypothesis that neurons in ischemic and non-ischemic regions died from different mechanisms; specifically, we tested whether a mixed form of cell death containing both necrotic and apoptotic changes could be identified in individual cells.Focal barrel cortex ischemia in rats was induced by occlusion of small branches of the middle cerebral artery (MCA) corresponding to the barrel cortex, local blood flow was measured by quantitative autoradiography. Cell death was visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H&E) staining, the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and caspase-3 staining 1 to 10 days after the ischemia. Electron microscopy was used for ultrastructural examination. Cell death occurred in the ipsilateral cortex 24 h after ischemia, followed by selective neuronal death in the ventrobasal (VB) thalamus 3 days later. TUNEL positive neurons were found in these two regions, but with striking morphological differences, designated as type I and type II TUNEL positive cells. The type I TUNEL positive cells in the ischemic cortex underwent necrotic changes. The type II TUNEL positive cells in the thalamus and the cortex penumbra region represented a hybrid death, featured by concurrent apoptotic and necrotic alterations in individual cells, including marked caspase-3 activation, nuclear condensation/fragmentation, but with swollen cytoplasm, damaged organelles and deteriorated membranes. Cell death in the thalamus and the cortex penumbra were attenuated by delayed administration of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD-FMK). Our data suggest that TUNEL staining should be evaluated with morphological changes, the hybrid death but not typical apoptosis occurs in the penumbra region and non-ischemic thalamus after cerebral ischemia.     

Response of Hippocampal Neurons and Glial Cells to Alternating Magnetic Field in Gerbils Submitted to Global Cerebral Ischemia

The purpose of this study was to determine whether exposure to an extremely low-frequency magnetic field (ELF-MF, 50 Hz) affects the outcome of postischemic damage in the hippocampus of Mongolian gerbils. After 10-min bilateral carotid occlusion, the gerbils were continuously exposed to ELF-MF (average magnetic induction at the center of the cage was 0.5 mT) for 7 days. The impact of ELF-MF was estimated immediately (the 7th day after reperfusion) and 7 days after cessation of exposure (the 14th day after reperfusion) compared with ischemic gerbils without ELF-MF exposure. Applying stereological methods, histological evaluation of changes in the hippocampus was done for determining its volume, volume densities of degenerating neurons and astrocytes, as well as the number of microglial cells per unit area. ELF-MF per se did not induce any morphological changes, while 10-min global cerebral ischemia led to neuronal death, especially in CA1 region of the hippocampus, as expected. Ischemic gerbils exposed to ELF-MF had significantly a lower degree of cell loss in the examined structure and greater responses of astrocytes and microglial cells than postischemic gerbils without exposure on the seventh day after reperfusion (immediate effect of ELF-MF). Similar response was observed on the 14th day after reperfusion (delayed effect of ELF-MF); however, differences in measured parameters were low and insignificant. Applied ELF-MF has possible neuroprotective function in the hippocampus, as the most sensitive brain structure in the model of global cerebral ischemia, through reduction of neuronal death and activation of astrocytes and microglial cells.     

Effects of intracarotid arterial injection of cyclosporin A and spontaneous hypothermia on brain damage incurred after a long period of global ischemia

A recent study showed that a single intracarotid arterial injection of cyclosporin A (CsA) can dramatically reduce infarct volume in rats subjected to transient focal ischemia. The present experiments were undertaken to investigate whether intracarotid arterial injection of CsA reduces brain damage after global ischemia. Since hypothermia is also an efficacious factor in preventing ischemic brain damage, in the second part of the experiments we tested whether a combination of hypothermia and CsA would provide additional brain protection. Global ischemia of a 30-min duration was induced in the rat. CsA (10 mg/kg) was injected into the carotid artery immediately after reperfusion. Hypothermia was instituted after ischemia by allowing spontaneous head temperature to fall to 30–32°C, while body temperature was upheld at 37°C. The results demonstrated that vehicle-treated animals could not survive beyond 1–2 days after reperfusion, and the histopathological outcome in a separate group of rats perfusion-fixed after 1 day reperfusion showed 80–100% brain damage in the caudoputamen, and in the hippocampal CA1, CA3, CA4 and dentate gyrus subregions. Microinfarction and grade 3 damage were frequently observed in the cingulate and parietal cortex and in the thalamus. CsA moderately prolonged animal survival to 3 days after reperfusion and reduced brain damage to grade 2 in the cortical areas and the thalamus. Hypothermia further increased animal survival to at least 6 days after reperfusion and reduced brain damage to 30% in the caudoputamen, to close to zero in the CA3, CA4, and dentate gyrus, and to grade 1–2 in the cortical areas and the thalamus. The combination of hypothermia and CsA did not give additional protection.     

Effect of calcium antagonists on postischemic protein biosynthesis in gerbil brain.

BACKGROUND AND PURPOSE: Prolonged inhibition of protein synthesis precedes delayed neuronal death in the CA1 sector of the hippocampus after transient cerebral ischemia. Organic calcium antagonists have been recommended for alleviation of ischemic neuronal damage. The present study was undertaken to investigate whether these drugs improve the recovery of protein biosynthesis after interruption of cerebral blood flow. METHODS: Cerebral protein synthesis was measured biochemically and autoradiographically in gerbils submitted to 5 minutes of bilateral occlusion of the common carotid arteries followed by 2 hours or 2 days of recirculation. Flunarizine (25 mg/kg) or nimodipine (1.5 mg/kg) were applied intraperitoneally shortly after ischemia. RESULTS: Treatment with either calcium antagonist did not markedly influence postischemic recovery of protein synthesis in the resistant regions of the brain and did not prevent the persisting inhibition in the vulnerable stratum pyramidale of the CA1 sector of the hippocampus. CONCLUSIONS: The postischemic application of the organic calcium antagonists nimodipine and flunarizine does not promote postischemic recovery of protein synthesis. The beneficial effects of these drugs must, therefore, be based on other mechanisms.