Transient focal ischemia in hyperglycemic rats is associated with increased cerebral infarction

To study whether transient ischemia is influenced by hyperglycemia, the middle cerebral artery was occluded for 5, 10 and 15 min in normo- and hyperglycemic rats. Five-minute ischemia induced minor lesions in both groups. After 10-min ischemia a significant greater infarct volume was found in hyperglycemia compared with normoglycemia (29 +/- 9 mm3 vs 4 +/- 4 mm3, P less than 0.001). Fifteen-minute artery occlusion induced even more damage in both hyper- and normoglycemia (63 +/- 20 mm3 vs 13 +/- 12 mm3, P less than 0.006). The lateral part of striatum was infarcted in all hyperglycemic animals exposed to 10 or 15 min of ischemia. In the same area selective neuronal injury occurred in 6 out of 9 normoglycemic animals. The findings show that hyperglycemia increases brain damage during transient ischemia by conversion of selective neuronal injury into cerebral infarction.     

Mannitol infusion immediately after reperfusion suppresses the development of focal cortical infarction after temporary cerebral ischemia in gerbils

Previously we found that, after temporary cerebral ischemia, microvasculogenic secondary focal cerebral cortical ischemia occurred, caused by microvascular obstruction due to compression by swollen astrocytic end‐feet, resulting in focal infarction. Herein, we examined whether mannitol infusion immediately after restoration of blood flow could protect the cerebral cortex against the development of such an infarction. If so, the infusion of mannitol might improve the results of vascular reperfusion therapy. We selected stroke‐positive animals during the first 10 min after left carotid occlusion performed twice with a 5‐h interval, and allocated them into four groups: sham‐operated control, no‐treatment, mannitol‐infusion, and saline‐infusion groups. Light‐ and electron‐microscopic studies were performed on cerebral cortices of coronal sections prepared at the chiasmatic level, where the focal infarction develops abruptly in the area where disseminated selective neuronal necrosis is maturing. Measurements were performed to determine the following: (A) infarct size in HE‐stained specimens from all groups at 72 and 120 h after return of blood flow; (B) number of carbon‐black‐suspension‐perfused microvessels in the control and at 0.5, 3, 5, 8, 12 and 24 h in the no‐treatment and mannitol‐infusion groups; (C) area of astrocytic end‐feet; and (D) number of mitochondria in the astrocytic end‐feet in electron microscopic pictures taken at 5 h. The average decimal fraction area ratio of infarct size in the mannitol group was significantly reduced at 72 and 120 h, associated with an increased decimal fraction number ratio of carbon‐black‐suspension‐perfused microvessels at 3, 5 and 8 h, and a marked reduction in the size of the end‐feet at 5 h. Mannitol infusion performed immediately after restitution of blood flow following temporary cerebral ischemia remarkably reduced the size of the cerebral cortical focal infarction by decreasing the swelling of the end‐feet, thus preventing the microvascular compression and stasis and thereby microvasculogenic secondary focal cerebral ischemia.     

Evolution of brain infarction after transient focal cerebral ischemia in mice.

The evolution of brain infarction after transient focal cerebral ischemia was studied in mice using multiparametric imaging techniques. One-hour focal cerebral ischemia was induced by occluding the middle cerebral artery using the intraluminal filament technique. Cerebral protein synthesis (CPS) and the regional tissue content of adenosine triphosphate (ATP) were measured after recirculation times from 0 hours to 3 days. The observed changes were correlated with the expression of the mRNAs of hsp-70, c-fos, and junB, as well as the distribution of DNA double-strand breaks, visualized by TUNEL. At the end of 1 hour of ischemia, protein synthesis was suppressed in a larger tissue volume than ATP in accordance with the biochemical differentiation between core and penumbra. Hsp70 mRNA was selectively expressed in the cortical penumbra, whereas c-fos and junB mRNAs were increased both in the lateral part of the penumbra and in the ipsilateral cingulate cortex with normal metabolism. During reperfusion after withdrawal of the intraluminal filament, suppression of CPS persisted except in the most peripheral parts of the middle cerebral artery territory, in which it recovered between 6 hours and 3 days. ATP, in contrast, returned to normal levels within 1 hour but secondarily deteriorated from 3 hours on until, between 1 and 3 days, the ATP-depleted area merged with that of suppressed protein synthesis leading to delayed brain infarction. Hsp70 mRNA, but not c-fos and junB, was strongly expressed during reperfusion, peaking at 3 hours after reperfusion. TUNEL-positive cells were detected from 3 hours on, mainly in areas with secondary ATP depletion. These results stress the importance of an early recovery of CPS for the prevention of ischemic injury and suggest that TUNEL is an unspecific response of delayed brain infarction.     

Heterogeneous hyperactivity and distribution of ischemic lesions after focal cerebral ischemia in Mongolian gerbils

Various types of poststroke hyperactivity exist in humans, but studies of each mechanism using animal models are scarce. We aimed to analyze the heterogeneity of postischemic hyperlocomotion and to identify the ischemic lesions responsible for postischemic hyperlocomotion in rodent models of focal ischemia. Mongolian gerbils underwent right common carotid artery occlusion (CCAO) for 10 or 20 min. At 24 h, 2 days, and 7 days postischemia, we performed quantitative and qualitative locomotor analysis and correlated these results with the extent of ischemic lesions. Intermittent explosive hyperlocomotion was induced transiently in a 10‐min CCAO group at 24 h after ischemia and continual unexplosive hyperlocomotion persisted for 7 days in the 20‐min CCAO animals. Selective neuronal death, confined to the hippocampal cornu ammonis 1 (CA1), was observed in the 10‐min CCAO group and widespread cortical and basal ganglia infarction was observed in the 20‐min CCAO group. Amyloid precursor protein was transiently observed in the hippocampus at 24 h postischemia in the 10‐min CCAO animals, while it was widely distributed over the ischemic regions throughout the 7 days postischemia in the 20‐min CCAO animals. Incidence maps and correlation analysis revealed hippocampal neuronal death of the CA1 sector and widespread hemispheric infarction, including the cortex, as the region responsible for the 10‐min and 20‐min CCAO‐induced hyperactivity, respectively. Two distinct types of locomotor hyperactivity were observed that varied with regard to the distribution of the ischemic lesion, that is, hippocampal neuronal death and widespread infarction involving the cortex. These two types of locomotor hyperactivity appear to be models of the different types of poststroke hyperactivity seen in stroke patients.     

Experimental cerebral ischemia in Mongolian gerbils

The behavior of the BBB in cerebral ischemia was studied in symptom-positive Mongolian gerbils subjected to left common carotid artery occlusion using Evans Blue dye as indicator of BBB injury. The BBB damage was demonstrable grossly by the presence of areas of blue discoloration, and microscopically by the presence of a bright red fluorescent tracer, localized mostly in the neurons. The survey of various groups of animals revealed a direct relationship between the incidence and time of appearance of the BBB lesions and the duration of the ischemic occlusion. This relationship can be interpreted as another example of the previously described "maturation" phenomenon. A relatively late occurrence of the BBB injury in cerebral ischemia, at the time when the affected brain tissue shows severe, edematous histopathologic changes indicates that the brain edema, as the main complication of ischemia, could be regarded as being primarily of the cytotoxic type.     

Experimental cerebral ischemia in Mongolian gerbils

Summary Light microscopic observations were carried out on Mongolian gerbils (Meriones unguiculatus) subjected to a partial cerebral ischemia by occlusion of the left common carotid artery at the neck. About 30% of gerbils developed an ischemic injury in the ipsilateral hemisphere and their brains revealed the following histopathologic features: 1. the changes were related to the intensity (duration) of the ischemic insult and to the time elapsed following release of the occlusion. The ischemic lesions appear to progress after re-establishment of the circulation and this presents one facet of a maturation phenomenon which seems to be a general principle applicable to various parameters of ischemic injury. The rate of maturation of the lesions is related to the intensity of the ischemic insult, a lesser intensity resulting in longer development of lesions. 2. The changes were either focal or diffuse in character. The former were assumed to be directly related to a vascular involvement; among the latter the topistic distribution of the hippocampal changes suggested a feature of selective vulnerability. 3. An indirect indication of neuronal recovery was surmised from observations on animals sacrificed after different periods following occlusions of the same duration. Also capable of recovery was a reactive change observed in the H3 neurons of the hippocampus. This change was characterized by central chromatolysis and resembled the primäre Reizung of Nissl.     

Experimental cerebral ischemia in Mongolian gerbils

Summary Behaviour of biogenic amines was studied in the brains of Mongolian gerbils subjected to unilateral occlusion of the common carotid artery. Assays on the hemispheres ipsilateral to occlusion revealed in symptom-positive animals a progressive decrease in norepinephrine and dopamine, and an increase in serotonin throughout the duration of an ischemic insult. In post-ischemic periods following the release of the clip, changes in biogenic amine levels generally conformed to the principles of a previously described maturation phenomenon, with delayed reactions occurring after the shorter ischemic insults.     

Experimental cerebral ischemia in mongolian gerbils

Summary Mongolian gerbils exposed to relatively short (7 or 15 min) unilateral or bilateral occlusions of the common carotid artery develop, 20 h after release of the clipping, characteristic morphologic changes in the H3 sector of the hippocampus. Ultrastructural study of these changes revealed an eccentric shift of the nuclei associated with chromatolytic perikarya which showed a dense accumulation of lysosomes and mitochondria in their central parts. The Golgi apparatus was recognizable only by clusters of vesicles, and this change was associated with a negative thiamine pyrophosphatase reaction.     

Experimental cerebral ischemia in Mongolian gerbils

Summary A cerebral ischemia was produced by unilateral ligation of the common carotid artery in the neck of Mongolian gerbils (Meriones unguiculatus), which are frequently characterized by deficiencies in the circulus of Willis. Concentrations of glucose, lactate, pyruvate and glycogen were measured in the hemisphere on the side of occlusion and in the contralateral control hemisphere of animals sacrificed after 5, 15 and 30 min, as well as after 1, 3, 5 and 9 hrs of carotid clamping. Significant decrease of glucose, and increase in lactate and pyruvate concentration were found in the hemisphere ipsilateral to occlusion; the extent of the changes was proportional to the duration of the ischemia. After an initial fall, an increase in the glycogen content occurred in the later stages of ischemia. Glycogen, glucose, lactate and pyruvate were determined also at 1, 5, 20 hrs and 1 week intervals following release of an occlusion lasting for 1 hr. Return to normal values of glucose and pyruvate was seen at 1 hr after release. The lactate and glycogen levels were significantly raised on the occluded side after 20 hrs release. An increased level of glycogen was observed as long as 1 week after a 1-hr carotid occulusion.     

Delayed AMPA receptor blockade reduces cerebral infarction induced by focal ischemia.

The potent and selective AMPA receptor antagonist NBQX was tested for cytoprotective properties in an adult rat model of transient focal neocortical ischemia. Nineteen spontaneously hypertensive rats sustained 2 h of middle cerebral artery occlusion, followed by 22 h of recirculation. Ninety minutes following the onset of ischemia, at the time of, and 30 min following reperfusion, they received i.p. injections of either saline (n = 10) or 30 mg kg-1 of NBQX (n = 9). Saline-treated rats had a mean volume of neocortical infarction ( +/- s.d.) of 181 +/- 31 mm3, while NBQX-treated rats sustained significantly less damage, 125 +/- 19 mm3 (p less than 0.001). Regional cerebral blood flows during ischemia and reperfusion were not affected by the drug. We suggest that the AMPA receptor may play an important role in ischemic cerebral infarction.     

Convulsive activity in gerbils subjected to cerebral ischemia.

Convulsive activity observed in the mongolian gerbil following carotid artery occlusion is of extracerebral origin. The clinical discharge is demonstrated to be primarily in the spinal cord.     

Immunohistochemical investigation of cerebral ischemia in gerbils

Experimental cerebral ischemia was produced in gerbils by occlusion of the right common carotid artery in the neck. The evolution of the ischemic lesions was followed from five minutes to six hours by using the immunohistochemical techniques for tubulin and creatine kinase BB-isoenzyme. The earliest lesion was found in the subiculum-CA1 and CA2 regions of the hippocampus in five minutes. There was loss of staining in the apical dendrites and perikarya of the pyramidal cells. The earliest lesion in the cerebral cortex, visible in ten minutes, was a laminar loss of staining for tubulin. Evolution of the ischemic lesions in the thalamus and caudoputamen was delayed. However, in two hours widespread ischemic lesions were seen there. Evolution of the ischemic lesions was slightly slower with the reaction for creatine kinase BB-isoenzyme as compared to the reaction for tubulin, but was far more sensitive than hematoxylin-eosin staining. The distribution of ischemic lesions detected by the immunohistochemical method compared to ischemic areas detected by an India ink perfusion study suggested that both the extent of regional ischemia and regional difference in tissue vulnerability were contributing factors for the emergence of early ischemic lesions. The mechanism for prompt disappearance of the immunohistochemical reaction for tubulin is not clear, but the present investigation demonstrates the usefulness of the immunohistochemical technique for detecting early ischemic lesions and provides a possible biochemical mechanism for cellular damage after ischemic insults.     

C-Phycocyanin is neuroprotective against global cerebral ischemia/reperfusion injury in gerbils

Although the huge economic and social impact and the predicted incidence increase, neuroprotection for ischemic stroke remains as a therapeutically empty niche. In the present study, we investigated the rationale of the C-Phycocyanin (C-PC) treatment on global cerebral ischemia/reperfusion (I/R) injury in gerbils. We demonstrated that C-PC given either prophylactically or therapeutically was able to significantly reduce the infarct volume as assessed by triphenyltetrazolium chloride (TTC) staining and the neurological deficit score 24h post-stroke. In addition, C-PC exhibited a protective effect against hippocampus neuronal cell death, and significantly improved the functional outcome (locomotor behavior) and gerbil survival after 7 days of reperfusion. Malondialdehyde (MDA), peroxidation potential (PP) and ferric reducing ability of plasma (FRAP) were assayed in serum and brain homogenates to evaluate the redox status 24h post-stroke. The treatment with C-PC prevented the lipid peroxidation and the increase of FRAP in both tissue compartments. These results suggest that the protective effects of C-PC are most likely due to its antioxidant activity, although its anti-inflammatory and immuno-modulatory properties reported elsewhere could also contribute to neuroprotection. To our knowledge, this is the first report of the neuroprotective effect of C-PC in an experimental model of global cerebral I/R damage, and strongly indicates that C-PC may represent a potential preventive and acute disease modifying pharmacological agent for stroke therapy.     

Temporal thresholds for neocortical infarction in rats subjected to reversible focal cerebral ischemia.

We investigated the temporal threshold for focal cerebral infarction in the spontaneously hypertensive rat. The right middle cerebral artery and common carotid artery were occluded for 0, 1, 2, 3, 4, or 24 hours, and all the animals were sacrificed 24 hours after the onset of ischemia. Cortical infarct volumes and edema volumes were quantified in serial frozen sections of hematoxylin and eosin-stained tissue using image analysis. Upon occlusion, blood flow in the core of the ischemic zone, measured with laser-Doppler flowmetry, fell to a mean +/- standard deviation of 21 +/- 7% of the preocclusion baseline value (n = 26). During the first hour of ischemia, blood flow in the densely ischemic zone rose to 27 +/- 8% of baseline (n = 25). Release of the middle cerebral artery and common carotid artery occlusions rapidly restored cortical blood flow to 213 +/- 83% of baseline (n = 21). Focal ischemia of 1 hour's duration caused little or no infarction, while ischemic intervals of 2 and 3 hours produced successively larger volumes of infarcted cortex. Ischemic intervals of 3-4 hours' duration followed by approximately 20 hours of recirculation yielded infarct volumes that were not significantly different from those after 24 hours of permanent focal ischemia. The results indicate that 3-4 hours of focal cerebral ischemia in this rat model is sufficient to attain maximal infarction and suggest that recirculation or pharmacological interventions after this time will provide little benefit.     

Magnetoencephalography of focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: The purpose of this study was to use magnetoencephalography to record magnetic field changes in the brain during middle cerebral artery occlusion. METHODS: A direct-current electrocorticogram (two channels) and a direct-current magnetoencephalogram (seven channels) were simultaneously recorded from five rats subjected to middle cerebral artery occlusion for 1-2 hours. RESULTS: Direct-current electrocorticographic and direct-current magnetoencephalographic signal deflections were observed after the onset of middle cerebral artery occlusion and occurred repeatedly throughout the ischemic period, with a mean +/- SD time interval of 12 +/- 5 minutes. A one-to-one correspondence of the electrocorticographic and magnetoencephalographic signal deflections was demonstrated. CONCLUSIONS: Direct-current magnetoencephalography can provide a new noninvasive technique for studying depolarization and/or spreading depression in focal cerebral ischemia.     

7—nitro—indazole减小大鼠暂时性局灶性脑梗塞灶范围

目的探讨神经元型一氧化氮合酶（ｎＮＯＳ）在暂时性局灶性脑缺血中的作用。方法用栓线法建立了大脑中动脉阻塞（ＭＣＡＯ）模型的大鼠上，观察特异性ｎＮＯＳ抑制剂７－ｎｉｔｒｏ－ｉｎｄａｚｏｌｅ（７－ＮＩ）对大鼠缺血３ｈ、再灌注３ｈ脑梗塞灶范围的影响。结果７－ＮＩ（２５ｍｇ／ｋｇ）可减小大鼠脑梗塞灶范围，且主要减小大脑皮质梗塞灶，其作用可被Ｌ－精氨酸（３００ｍｇ／ｋｇ）逆转，Ｄ－精氨酸（３００ｍｇ／ｋｇ）则否。结论ｎＮＯＳ产生的ＮＯ在暂时性局灶性脑缺血中起损害作用     

Effects of tetrahydrobiopterin on cerebral infarction after transient focal ischemia in rats

Abstract

Objectives: The present study investigated the effects of tetrahydrobiopterin (BH4) on cerebral infarction after transient focal ischemia in rats.

Methods: Focal ischemia (1·5 hours) was created in male Sprague-Dawley rats (250-280 g) by middle cerebral artery occlusion. Some rats were treated with 20 mg/kg tetrahydrobiopterin by intraperitoneal injection 30 minutes before reperfusion. At 2, 6, and 12 hours of reperfusion, the brains were harvested for the nitric oxide synthase (NOS) activity and nitric oxide (NO) level assays. At 12 hours of reperfusion, the brains were harvested for infarct size measurement.

Results: NOS activity and NO level were all augmented after reperfusion. BH4 treatment significantly further increased NOS activity and NO level. Cerebral infarct size was significantly bigger in BH4 treatment group compared to that in no treatment group.

Conclusions: The data indicate that BH4 enhances cerebral infarction after transient focal ischemia in rats, through NOS and NO pathway.     

L-arginine in focal cerebral ischemia

Nitric oxide and its precursor, L-arginine, have a great importance in cerebrovascular studies. In this study, we elucidate the dose dependent L-arginine effects on cerebral ischemia. The study involved 96 New Zealand albino rabbits, which were randomly allocated into four groups. The middle cerebral artery was occluded after a modified transorbital approach. Before the occlusion of MCA, each group was intravenously administered three doses of L-arginine i.e. 2.5 mg kg-1 for Group 1, 7.5 mg kg-1 for Group 2, and 12.5 mg kg-1 for Group 3. Thus, each group consisting of 24 animals was listed as 2.5 mg kg-1 (Group 1), 7.5 mg kg-1 (Group 2), 12.5 mg kg-1 (Group 3), and control group (receiving no intervention). Cerebral tissue oxygenazation was measured in parietal area by near infrared spectroscopy in all animals prior to and at 5, 30, and 60 min after MCA occlusion. Six hours after MCA occlusion, all the animals were studied for the area of ischemia (n = 40), edema formation (n = 32), and blood nitrite-nitrate levels (n = 24). At the dose of 2.5 mg kg-1 of L-arginine no differences were detected on ischemic tissue volume, brain edema, cerebral tissue oxygenazation, blood nitrite-nitrate levels when compared to the values of control group. However, with the dose of 7.5 mg kg-1, there were significant improvements in the levels of ischemic tissue volume, brain edema, and nitrite-nitrate levels compared to those of the control group and the 2.5 mg kg-1 group. At a dose of 12.5 mg kg-1, there were further improvements in the levels of ischemic tissue volume, brain edema, penumbral zone nitrite-nitrate levels. After 30 min of occlusion, cerebral tissue oxygenazation values increased in a dose dependent fashion. L-arginine's protective effect on cerebrovascular ischemia shows a dose dependent effect on infract size and tissue water content that may prove beneficial in the treatment of ischemia. However, further dose-dependent studies are needed.