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.     

Exo-focal postischemic neuronal death in the rat brain

We describe delayed neuronal damage in ipsilateral areas remote from the ischemic area of rat brain after transient focal ischemia induced by embolization of the right middle cerebral artery (MCA). After 15, 30, 60 and 90 min 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 method, and the mechanism involved was investigated by measuring local cerebral glucose metabolism. Depending on the duration of ischemia, 45Ca accumulation extended to the lateral segment of the caudate putamen and to the cerebral cortex, both supplied by the occluded MCA. Moreover, 3 days after ischemic insult, 45Ca had accumulated in the ipsilateral substantia nigra and ventral posterior nucleus of the thalamus. Histological examination revealed that the neurons in both areas suffered damage and were selectively reduced in number. Cerebral glucose utilization decreased in the thalamus, but increased approximately 30% (P less than 0.01) in the substantia nigra compared with the value in the corresponding contralateral area. Both areas lie outside the ischemic area, but have transsynaptic connections with the ischemic focus. Based on the present study, we suggest that the mechanisms of delayed neuronal death in these two remote areas may not be identical, but that this phenomenon may be caused by a transsynaptic process associated with the ischemic focus.     

Alterations of 45Ca accumulation and [3H]inositol 1,4,5-trisphosphate binding using autoradiography in the exo-focal postischemic brain areas of the rat.

We studied the alterations of calcium accumulation and intracellular signal transduction using autoradiography of the second messenger system in order to clarify the mechanisms of the delayed neuronal changes in the remote areas of rat brain after transient focal ischemia. Chronological changes of 45Ca accumulation and [3H]inositol 1,4,5-trisphosphate (IP3) binding sites were determined after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by different periods of recirculation. After the ischemic insult, 45Ca accumulation extended to the lateral segment of the caudate putamen and to the cerebral cortex, both supplied by the occluded MCA. One day after the ischemia, [3H]IP3 binding sites decreased significantly compared with the control values in these ischemic areas. Moreover, 3 days after the ischemia, 45Ca accumulation was first detected in the ipsilateral thalamus and the substantia nigra, which lay outside the ischemic areas. In the substantia nigra, a significant decrease of [3H]IP3 binding sites and concurrent 45Ca accumulation were observed. In the thalamus, however, there was not alteration until 1 week after the ischemia, and then [3H]IP3 binding sites increased significantly 2 weeks (P less than 0.05) and 4 weeks (P less than 0.01) after the ischemia. Based on the present study, we speculate that different mechanisms associated with signal transduction systems may be responsible for exo-focal postischemic delayed neuronal changes in the thalamus and the substantia nigra. The increase of [3H]IP3 binding sites of the thalamus in the chronic stage may be new evidence of plasticity related to neurotransmission.     

Exo-focal postischemic neuronal damage in the rat brain: alteration of [3H]forskolin binding using in vitro autoradiography.

We studied the alteration of intracellular signal transduction using quantitative autoradiography of the second messenger system in order to clarify the mechanisms of delayed neuronal damage in the remote areas of rat brain after transient focal ischemia. Chronological changes of [3H]forskolin binding sites were measured to demonstrate the striatal-nigral pathway after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by 3 h, 6 h, 1 day, 3 days, 1 week, 2 weeks and 4 weeks of recirculation. [3H]Forskolin binding sites were found to be markedly decreased in the lateral segment of the caudate putamen supplied by the occluded MCA after 90 min of ischemia with no recirculation. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, marked reduction of [3H]forskolin binding sites was observed in the ipsilateral substantial nigra which lay outside the ischemic areas. This postischemic delayed phenomenon observed in the substantia nigra developed concurrently with 45Ca accumulation, which was detected there in our previous study. The delayed reduction of [3H]forskolin binding sites in the substantia nigra observed in the present study indicates that striatonigral terminal degeneration at presynaptic sites is caused by precedent ischemic damage of the ipsilateral caudate putamen and that exo-focal postischemic neuronal death is caused by a transsynaptic process associated with the ischemic foci.     

Autoradiographic analysis of second messenger and neurotransmitter receptor bindings in the strionigral system of the postischemic rat brain.

We studied the postischemic alterations of second messenger and receptor systems focusing on the strionigral pathway in order to clarify the mechanism of the delayed neuronal changes in remote areas of the rat brain after transient focal ischemia. Chronological changes of [3H]forskolin and [3H]SCH 23390 binding sites and 45Ca accumulation were determined by using autoradiographic methods after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by different periods of recirculation. After the ischemic insult, 45Ca accumulation extended to the lateral segment of the caudate putamen (CPu-L) and to the cerebral cortex, both supplied by the occluded MCA. After the ischemia, [3H]forskolin binding sites were found to be markedly decreased in the early stage in the CPu-L, the ischemic focus in this model, but reduction of the dopamine D-1 receptor sites was first detected there 1 day after the ischemia. On the contrary, in the exo-focal remote areas, there was no alteration of either [3H]forskolin or D-1 receptor binding sites on day 1. However, 3 days after the ischemia, marked reduction of both these binding sites was first observed in the ipsilateral substantia nigra, which had not been directly affected by the original ischemic insult. These postischemic delayed phenomena observed in the substantia nigra developed concurrently with abnormal 45Ca accumulation. These results suggest that strionigral terminal degeneration in the substantia nigra is caused by precedent ischemic damage of the ipsilateral caudate putamen and that intracellular signal transduction including both second messenger and receptor systems may be involved prior to the neuronal damage in the exo-focal postischemic brain areas.     

Exo-focal postischemic neuronal damage in the rat brain: alteration of [H]forskolin binding using in vitro autoradiography

We studied the alteration of intracellular signal transduction using quantitative autoradiography of the second messenger system in order to clarify the mechanisms of delayed neuronal damage in the remote areas of rat brain after transient focal ischemia. Chronological changes of [³H]forskolin binding sites were measured to demonstrate the striatal-nigral pathway after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by 3 h, 6 h, 1 day, 3 days, 1 week, 2 weeks and 4 weeks of recirculation. [³H]Forskolin binding sites were found to be markedly decreased in the lateral segment of the caudate putamen supplied by the occluded MCA after 90 min of ischemia with no recirculation. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, marked reduction of [³H]forskolin binding sites was observed in the ipsilateral substantia nigra which lay outside the ischemic areas. This postischemic delayed phenomenon observed in the substantia nigra developed concurrently with ⁴⁵Ca accumulation, which was detected there in our previous study. The delayed reduction of [³H]forskolin binding sites in the substantia nigra observed in the present study indicates that striatonigral terminal degeneration at presynaptic sites is caused by precedent ischemic damage of the ipsilateral caudate putamen and that exo-focal postischemic neuronal death is caused by a transsynaptic process associated with the ischemic foci.     

Mitochondrial response to transient forebrain ischemia and recirculation in the rat

Recovery of brain mitochondrial function was studied following forebrain ischemia induced in rats by common carotid artery occlusion in combination with hypotension caused by bleeding. A reversible insult was induced by 15-min ischemia in fasted animals (hypoglycemic ischemia), and an irreversible one by 30-min ischemia in fed animals (normoglycemic ischemia), the latter procedure causing exaggerated lactic acidosis as well. Mitochondrial function recovered during a 30-min recirculation period after 15-min hypoglycemic ischemia, although a small amount of Ca2+ accumulated during recirculation. Thirty-minute normoglycemic ischemia induced irreversible mitochondrial damage that was not associated with Ca2+ accumulation during recirculation. Ischemia of 15 and 30 min caused a loss of mitochondrial Mg2+ (approximately 25%) that persisted during recirculation but did not influence recovery. Based on our earlier data obtained on isolated brain mitochondria in vitro, it is suggested that the lack of full recovery following 30 min of normoglycemic ischemia was due to the profound lactic acidosis during this insult.     

Neuronal damage and calcium accumulation following repeated brief cerebral ischemia in the gerbil

We investigated the distribution of neuronal damage following brief cerebral transient ischemia and repeated ischemia at 1-h intervals in the gerbil, using light microscopy and 45Ca autoradiography as a marker for detection of ischemic damage. The animals were allowed to survive for 7 days after ischemia induced by bilateral carotid artery occlusion. Following 2-min ischemia, neuronal damage determined by abnormal calcium accumulation was not observed in the forebrain regions. Following 3-min ischemia, however, abnormal calcium accumulation was recognized only in the hippocampal CA1 sector and part of the striatum. Two 2-min ischemic insults caused extensive abnormal calcium accumulation in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus, the substantia nigra and the inferior colliculus. The ischemic insults were more severe than that of a single 3-min ischemia. However, three 1-min ischemic insults caused abnormal calcium accumulation only in the striatum. On the other hand, three 2-min ischemic insults caused severe abnormal calcium accumulation in the brain. The abnormal calcium accumulation was found in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus, the medial geniculate body, the substantia nigra and the inferior colliculus. Gerbils subjected to three 3-min ischemic insults revealed most severe abnormal calcium accumulation. Marked calcium accumulation was seen not only in the above sites, but also spread in the neocortex, the septum and the hippocampal CA3 sector. Morphological study after transient or repeated ischemia indicated that the distribution and frequency of the neuronal damage was found in the sites corresponding to most of the regions of abnormal calcium accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time profile of calcium accumulation in hippocampus,striatum and frontoparietal cortex after transient forebrain ischemia in the gerbil

Summary The topical and temporal relationship between neuronal injury and calcium loading was investigated in gerbils following bilateral carotid artery occlusion for 5 or 10 min and recirculation times from 15 min to 7 days. The association of histochemically visible calcium deposits with neuronal death was assessed by combining two calcium stains, alizarin red and arsenazo III, with conventional histological techniques. Neuronal calcium accumulation was evaluated morphometrically in the striatum, the frontoparietal cortex and the CA1 and CA4 sectors of the hippocampus. After 5-min ischemia and 1–2 days of recirculation numerous calcium-containing neurons appeared in the CA4 sector but only a few were present in the CA1 sector. After 4 days of recirculation calcium accumulation was visible in the whole CA1 sector and the dorso-lateral part of striate nucleus. After 10-min ischemia calcium accumulation started in these regions, as well as in the cortex, already after 1 day. In the CA1 sector calcium accumulation followed a typical time course: on day 2 only the lateral parts were affected, while on day 4 the whole CA1 neuronal band was calcium positive. The regional distribution of histological lesions matched that of calcium loading and, furthermore, the lesions appeared after a corresponding delay in the respective regions. Morphometric evaluations of calcium staining and histological lesions in the CA1 sector revealed a high correlation, indicating that calcium accumulation and neuronal death are closely associated both topically and temporally. This suggests that disturbances of calcium homeostasis such as those measured by this histochemical technique are the consequence of and not the reason for ischemic cell death.     

Alteration of protein kinase C activity in the postischemic rat brain areas using in vitro [3H]phorbol 12,13-dibutyrate autoradiography

Summary Chronological changes of protein kinase C (PKC) activity were measured using in vitro [³H]phorbol 12,13-dibutyrate (PDBu) autoradiography to investigate the postischemic alteration of this second messenger system in the rat brain. Transient ischemia was induced by the occlusion of the middle cerebral artery (MCA) for 90 min and such occlusion followed by various recirculation periods of up to 4 weeks. After 90 min of ischemia followed by 3 hours of recirculation, [³H]PDBu binding sites were found to be significantly decreased in the cerebral cortex and lateral segment of the caudate putamen, both supplied by the occluded MCA; thereafter, the binding sites decreased progressively in those ischemic foci. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, a significant decrease of [³H]PDBu binding sites was first detected in the ipsilateral thalamus and the substantia nigra, which both areas had not been directly affected by the original ischemic insult. This postischemic delayed phenomenon observed in the thalamus and the substantia nigra developed concurrently with⁴⁵Ca accumulation, which was detected there in our previous study. These results suggest that alteration of second messenger (PKC) pathways may be involved not only in the ischemic foci, but also in neuronal degeneration of the exo-focal remote areas in relation to the disruption of intracellular calcium homeostasis which plays a key role in the pathogenesis of postischemic neuronal damage and that marked alteration of intracellular signal transduction may precede the neuronal damage in the exo-focal postischemic brain areas.     

The effect of hyperglycemia on intracellular calcium in stroke.

The effect of hyperglycemia on cytosolic free calcium ([Ca2+]i) during temporary focal cerebral ischemia was investigated in cats using a fluorometric technique. The middle cerebral artery (MCA) was occluded for a period of 1 h, after which the clip was removed. In seven animals, plasma glucose was raised to 500-700 mg/dl by infusion of a 50% glucose solution starting 30 min after MCA occlusion, while eight animals were kept normoglycemic during and following occlusion. MCA occlusion induced a significant, but identical, elevation of the [Ca2+]i signal ratio (400/506 nm) in both the normoglycemic group (from 1.40 to 1.97 +/- 0.34, p less than 0.01) and in the hyperglycemic group (from 1.40 to 2.00 +/- 0.53, p less than 0.01) at the end of the occlusion. Between 10 and 30 min after reopening, the [Ca2+]i signal ratio decreased to control levels in the normoglycemic group (1.40 +/- 0.11 and 1.36 +/- 0.08 at 10 and 30 min after reopening, respectively), but remained elevated in the hyperglycemic group (1.69 +/- 0.18 and 1.65 +/- 0.21 at 10 and 30 min after reopening, respectively). There was a statistically significant difference between the two groups (p less than 0.01). These data suggest that hyperglycemia may be harmful to calcium recovery during the early recirculation period following focal cerebral ischemia.     

Blockade of central histaminergic H2 receptors facilitates catecholaminergic metabolism and aggravates ischemic brain damage in the rat telencephalon

Blockade of central H(2) receptors aggravates ischemic neuronal damage. Since changes in the activity of the monoaminergic system are contributing factors in the development of ischemic neuronal damage, the authors evaluated the effects of ranitidine on the monoaminergic system and ischemic neuronal damage in the middle cerebral artery (MCA) occlusion model of rats. Wistar rats pretreated with saline or ranitidine (3 and 30 nmol, i.c.v.) were subjected to reversible occlusion of MCA for 2 h. The total infarct volume was determined 24 h after reperfusion. The relationship between dopaminergic activity and the histologic outcome was estimated by lesioning the substantia nigra 2 days before MCA occlusion. In a second experiment, the animals were subjected to 15 min of MCA occlusion, and the effects of ranitidine on the histologic outcome was evaluated 7 days after ischemia. In a third experiment, the tissue concentrations of monoamines and their metabolites were determined in the cerebral cortex and striatum 2 h after reperfusion following MCA occlusion for 2 h. The turnover of norepinephrine and dopamine was compared between animals treated with saline and those treated with ranitidine by estimating the alpha-methyl-p-tyrosine-induced depletion of norepinephrine and dopamine, respectively. The turnover of 5-hydroxytryptamine was evaluated by the probenecid-induced accumulation of 5-hydroxyindoleacetic acid. Treatments with ranitidine markedly increased the infarct volume 24 h after reperfusion. Ranitidine also aggravated delayed neuronal death 7 days after ischemia. The aggravation was abolished by the lesion of the substantia nigra before MCA occlusion. The MCA occlusion increased the turnover of cortical norepinephrine and striatal dopamine. The turnover was further facilitated by ranitidine. Although ranitidine suppressed the 5-hydroxytryptamine turnover in the cerebral cortex, the extent of this effect was similar in both the ischemic and non-ischemic sides. These results suggest that facilitation of the catecholaminergic systems is involved in the aggravation of ischemic neuronal damage by H(2) blockade.     

Regional neuroprotective effects of pentobarbital on ischemia-induced brain damage

We investigated the neuroprotective effect of pentobarbital, a GABAA receptor-effector, on ischemic neuronal damage in the gerbils. The animals were allowed to survive for 7 days after 10-min ischemia induced by bilateral occlusion of the common carotid arteries. Morphological changes and abnormal calcium accumulation were evaluated in selectively vulnerable areas after ischemia. Pentobarbital (40 mg/kg, IP), administered 30 min prior to ischemia, significantly reduced neuronal cell loss in the neocortex, the striatum, and the hippocampal CA3 sector. However, pentobarbital failed to prevent the damage to the hippocampal CA1 sector and the thalamus. 45Ca autoradiographic study also revealed that a marked calcium accumulation was found in the selectively vulnerable regions after ischemia, which was consistent with the extent of histological neuronal damage. The abnormal calcium accumulation was reduced in the sites corresponding to most of the regions in which the protective effect of pentobarbital was found. The results suggest that ischemia-induced neuronal damage may be partly caused by an imbalance between excitatory and inhibitory input.     

Hyperglycemia and focal brain ischemia.

The influence of hyperglycemic ischemia on tissue damage and cerebral blood flow was studied in rats subjected to short-lasting transient middle cerebral artery (MCA) occlusion. Rats were made hyperglycemic by intravenous infusion of glucose to a blood glucose level of about 20 mmol/L, and MCA occlusion was performed with the intraluminar filament technique for 15, 30, or 60 minutes, followed by 7 days of recovery. Normoglycemic animals received saline infusion. Perfusion-fixed brains were examined microscopically, and the volumes of selective neuronal necrosis and infarctions were calculated. Cerebral blood flow was measured autoradiographically at the end of 30 minutes of MCA occlusion and after 1 hour of recirculation in normoglycemic and hyperglycemic animals. In two additional groups with 30 minutes of MCA occlusion, CO2 was added to the inhaled gases to create a similar tissue acidosis as in hyperglycemic animals. In one group CBF was measured, and the second group was examined for tissue damage after 7 days. Fifteen and 30 minutes of MCA occlusion in combination with hyperglycemia produced larger infarcts and smaller amounts of selective neuronal necrosis than in rats with normal blood glucose levels, a significant difference in the total volume of ischemic damage being found after 30 minutes of MCA occlusion. After 60 minutes of occlusion, when the volume of infarction was larger, only minor differences between normoglycemic and hyperglycemic animals were found. Hypercapnic animals showed volumes of both selective neuronal necrosis and infarction that were almost identical with those observed in normoglycemic, normocapnic animals. When local CBF was measured in the ischemic core after 30 minutes of occlusion, neither the hyperglycemic nor the hypercapnic animals were found to be significantly different from the normoglycemic group. Brief focal cerebral ischemia combined with hyperglycemia leads to larger and more severe tissue damage. Our results do not support the hypothesis that the aggravated injury is caused by any disturbances in CBF.     

Plastic changes and nitric oxide synthase induction in neurons which innervate the regenerated tail of the lizard Gekko gecko. II. The response of dorsal root ganglion cells to tail amputation and regeneration

NS-7 is a novel blocker of voltage-sensitive Ca(2+) and Na(+) channels, and it significantly reduces infarct size after occlusion of the middle cerebral artery. Persistent activation of cyclic AMP response element binding protein (CREB), which can be induced by increase in intracellular Ca(2+) concentrations or other second messengers, has recently been found to be closely associated with neuronal survival in cerebral ischemia. The present study was therefore undertaken to evaluate the neuroprotective effects of NS-7 by analyzing changes in CREB phosphorylation in a focal cerebral ischemia model. CREB phosphorylation in the brain of rats was investigated immunohistochemically at 3.5-48-h recirculation after 1. 5-h occlusion of the middle cerebral artery. NS-7 (1 mg/kg; NS-7 group) or saline (saline group) was intravenously injected 5 min after the start of recirculation. The NS-7 group showed significantly milder activation of CREB phosphorylation in various cortical regions after 3.5 h of recirculation than the saline group. The inner border zone of ischemia in the NS-7 group subsequently exhibited a moderate, but persistent, increase in number of phosphorylated CREB-positive neurons with no apparent histological damage. By contrast, the saline group displayed a marked, but only transient, increase in number of immunopositive neurons in this border zone after 3.5 h of recirculation, and this was followed by clear suppression of CREB phosphorylation and subsequent loss of normal neurons. These findings suggest that: (1) the marked enhancement of CREB phosphorylation in the acute post-ischemic phase may be triggered largely by an influx of calcium ions as a result of activation of the voltage-sensitive Ca(2+) and Na(+) channels; and that (2) the neuroprotective effects of NS-7 may be accompanied by persistent activation of CREB phosphorylation in the inner border zone of ischemia.     

Effect of vagus nerve stimulation during transient focal cerebral ischemia on chronic outcome in rats

The aim of this study was to investigate the effect of vagus nerve stimulation (VNS) on infarct volume and neurological recovery up to 3 weeks following transient focal cerebral ischemia. Transient ischemia was produced by filament occlusion of the proximal middle cerebral artery (MCA) in rats. The right vagus nerve was stimulated starting 30 min after MCA occlusion and consisted of 30-sec pulse trains (20 Hz) delivered to the animal's right vagus nerve every 5 min for a total period of 60 min (n = 10). All the procedures were duplicated, but no stimulus was delivered, in a control group (n = 10). Neurological evaluations were performed in all animals at 24 hr, 48 hr, 1 week, 2 weeks, and 3 weeks after MCA occlusion; animals were euthanized; and neuronal damage was evaluated in hematoxylin-eosin-stained sections. The ischemic lesion volume was smaller in the VNS-treated animals in comparison with the nonstimulated group (P < 0.02). Although the functional score in both treated and untreated groups improved over the 3-week observation period (P < 0.001), there was still a statistically significant improvement reszulting from VNS treatment compared with control animals (P < 0.05). Cerebral blood flow changes in the MCA territory during ischemia did not differ between the VNS-treated animals (31.9% ± 10.4% of baseline) and control animals (29.9% ± 9.1%; P = 0.6). Stimulation of the vagus nerve for only a brief period early in ischemia provides neuroprotection in transient ischemia, with neuroprotection persisting for at least 3 weeks.     

Penumbral tissues salvaged by reperfusion following middle cerebral artery occlusion in rats.

BACKGROUND AND PURPOSE: The rat is now extensively used for studies on focal cerebral ischemia, and several novel pharmacological principles have been worked out in rat models of middle cerebral artery occlusion. The objective of the present study was to assess how ischemic tissue can be salvaged by reperfusion in a model of transient focal ischemia that gives infarction of both the caudoputamen and the neocortex. METHODS: The middle cerebral artery of anesthetized rats was occluded for 15, 30, 60, 90, 120, or 180 minutes by an intraluminal filament, and recirculation was instituted for 7 days to allow assessment of the density and localization of ischemic brain damage using histopathologic techniques. Local cerebral blood flow was measured in separate animals to verify that removal of the filament was followed by adequate recirculation. RESULTS: Following 15 minutes of middle cerebral artery occlusion seven of eight rats showed selective neuronal necrosis in the caudoputamen, while the neocortex was normal. After 30 minutes of occlusion, seven of eight animals had infarcts localized to the lateral caudoputamen, and four of eight had selective neuronal necrosis in the neocortex. Prolongation of the ischemia to 60 minutes induced cortical infarction in all eight rats. The infarct size increased progressively with increasing occlusion time, up to 120-180 minutes, when the infarcts were as extensive as those observed following 24 hours of permanent middle cerebral artery occlusion. CONCLUSIONS: The results demonstrate a time window for salvage of penumbral tissues by reperfusion that is shorter than that suggested on the basis of previous data in other species. The results probably reflect a lower collateral blood flow in the rat than in other species. This should be taken into account when the effect of pharmacological agents is studied in rats.     

大鼠大脑中动脉缺血/再灌注模型中Caspase-3的表达

目的 研究Caspase-3在缺血性脑损伤中的作用，进一步探讨缺血性脑血管病的分子机制。方法 用Belayev改良的Longa线栓法制备大鼠局灶性大脑中动脉(MCA)缺血／再灌注模型，TTC染色观察梗死灶的形成，分别用原位杂交及免疫组化技术检测鼠脑中Caspase-3 mRNA与活性蛋白的表达。结果 缺血2小时再灌注24小时，TTC染色见明显的梗死灶形成，正常脑组织、假手术组及MCAO缺血对侧脑中有少量的Caspase-3 mRNA表达，但活性蛋白几无表达；再灌注24小时后，缺血侧脑中Caspase-3 mRNA表达明显增强，蛋白质活化增多，再灌注48小时进一步增加。结论 细胞凋亡机制参与了缺血后迟发性神经元死亡，Caspase-3在其中起重要作用。     

Middle cerebral artery occlusion in the mouse by intraluminal suture coated with poly-L-lysine: neurological and histological validation.

The present study was conducted to validate a modified method of temporary focal cerebral ischemia in the mouse; neurobehavioral function and histopathological infarction were quantitated following various periods of middle cerebral artery occlusion (MCAo). Male C57BL/6 mice were anesthetized with 3% halothane in a mixture of 30%O2/70%N2O delivered by face mask and were subjected to 30- to 180-min of temporary middle cerebral artery occlusion (MCAo) by an intraluminal suture coated with poly-l-lysine. Twenty-eight of 40 mice showed an initial high-grade neurological deficit (30-min MCAo, n=7; 60-min, n=8; 120-min, n=8; 180-min, n=5) when examined during MCAo; these were used for subsequent study. One day after MCAo, behavioral function was re-evaluated, and brains were perfusion-fixed and infarct volumes were measured. The initial neurological deficit improved at 24 h in mice with 30- or 60-min of prior MCAo but tended to persist in mice with 120- or 180-min insults. Following each duration of ischemia, mice exhibited ipsilateral infarcts. Small, inconsistent predominantly subcortical infarcts were present after 30-min MCAo, while longer occlusion periods gave rise to consistent foci of subcortical infarction involving striatum, septum, thalamus, and hippocampus, as well as areas of frontoparietal cortical infarction. The major advantages of the improved intraluminal MCAo model reported here, incorporating sutures coated with poly-l-lysine, include: a 100% incidence of infarction of predictable location and size in mice having an initial neurological deficit. Periods of 60- to 180-min MCA occlusion in this model yield sufficiently reproducible sequelae to permit the effects of various therapeutic agents on neurological outcome and size of infarction to be readily studied.     

Expression of the RNA-binding protein TIAR is increased in neurons after ischemic cerebral injury

T-cell restricted intracellular antigen-related protein (TIAR) is an RNA recognition motif-type RNA-binding protein that has been implicated in the apoptotic death of T-lymphocytes and retinal pigment epithelial cells. Western blots prepared with a monoclonal antibody against TIAR showed expression in normal rat hippocampus, and induction by 15 min of global cerebral ischemia. This increased expression was evident at 8 hr after ischemia and maximal at 24 hr, whereas expression at 72 hr was reduced below basal levels. Expression of TIAR protein was also increased in parietal cortex 6 and 24 hr after 90 min of focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, as well as in cultured cortical neurons and astroglia after exposure to hypoxia in vitro. Immunocytochemistry showed that increased expression of TIAR occurred mainly in the CA1 sector of hippocampus 24 hr after global ischemia, and in cortical and striatal neurons 24 hr after 20 or 90 min of focal ischemia. Double-labeling studies showed that TIAR protein expression was co-localized with DNA damage in neuronal cells. The findings suggest that TIAR may be involved in neuronal cell death after cerebral ischemic injury.