Correlation between amino acid release and neuropathologic outcome in rat brain following middle cerebral artery occlusion

Using in vivo brain microdialysis, we studied amino acid release in the striatum and cortex of eight rats following permanent middle cerebral artery occlusion. We then processed all brains for histopathologic assessment of the volume of ischemic damage 4 hours after occlusion. Ischemic damage was varied by occlusion of the middle cerebral artery at a point either proximal (n = 4) or distal (n = 4) to the lenticulostriate vessels. Proximal occlusion elevated the dialysate contents of all amino acids. The largest increases occurred for the potentially neurotoxic amino acids aspartate and glutamate and for taurine (800-2,800% of basal efflux). We observed smaller increases for the "metabolic" amino acids (280-580% of basal efflux). Distal occlusion did not affect amino acid efflux in the striatum, and release in the cortex was significantly lower than that following proximal occlusion. We compared release data with acute histopathologic outcome. Proximal occlusion resulted in a large volume of ischemic damage in the cortex and striatum (25-48% of hemispheric volume). A smaller volume of ischemic damage was noted following distal occlusion (0-21% of hemispheric volume). The volume of ischemic damage and the amount of amino acid release were significantly correlated (p less than 0.05).     

Serum amino acid levels after permanent middle cerebral artery occlusion in the rat

BACKGROUND AND PURPOSE: High levels of glutamate in plasma and cerebrospinal fluid (CSF) have been demonstrated in patients with acute ischemic stroke. Whereas this glutamate increase in CSF is only evidenced during the first 6 h in stable ischemic stroke, it is sustained for 24 h in progressing stroke. The aim of this investigation was to study the evolution of serum glutamate levels after stroke in a rat model of permanent cerebral artery occlusion. METHODS: Glutamate, glycine, aspartate, taurine and tryptophan were measured by high-performance liquid chromatography from serum samples taken before and at different times after permanent middle cerebral artery occlusion (MCAO) and from sham-operated rats. RESULTS: After MCAO, a 3-fold increase in glutamate and a 2-fold increase in glycine and aspartate were observed in rat serum. The onset of this amino acid increase began 4-6 h after ischemic induction, reached peak values at 8-24 h and returned to preischemic values by 48-72 h. Serum concentrations of taurine and tryptophan were not modified after MCAO. Sham-operated rats did not exhibit changes of basal amino acid concentrations in serum. CONCLUSIONS: The serum excitatory amino acid profile in this experimental model confirms that the early detection of increased concentrations of glutamate and glycine at systemic circulation observed in patients with acute stroke is a consequence of the cerebral ischemic process.     

Changes in regional cerebral catecholamines following middle cerebral artery occlusion in the rat

Ischemic brain injury affects the content and metabolism of brain monomines. Our aim was to know the time course of changes in regional cerebral catecholamines during focal cerebral ischemia, and whether focal cerebral ischemia may affect the metabolism of catecholamines in distant area of the brain. Methods Fifty-five rats were subjected to occlusion of the middle cerebral artery (MCA) on the olfactory tract, under halothane anesthesia. Fourteen animals were sham-operated group. Animals were decapitated at 1/2, 1,2,3,6,12 and 24 hours post-occlusion (PO), respectively. The brains were removed, and the brain structures dissected out include bilateral corpus striatum, cerebral cortex (MCA territory) and cerebellar hemisphere. Catecholamines were extracted by alumina procedure, and determined by high-performance liquid chromatography with electrochemical detection. Results Dopamine (DA) contents, in ipsilateral corpus striatum and cerebral cortex to the ischemia, decreased at 1 hour PO, and reached, at 6 hours PO, to 40% of control value in corpus striatum and 30% in cerebral cortex, respectively. After 6 hours PO, DA remained low. Norepinephrine (NE) content in the ipsilateral corpus striatum gradually reduced and reached to 60% of control value at 24 hours PO. NE in the ipsilateral cerebral cortex decreased to 50% of control at 1 hour PO, and thereafter remained reduced. In the contralateral corpus striatum and cerebral cortex, either DA or NE showed no significant changes, except 1/2 hour PO. NE contents in bilateral cerebral cortex showed a transient increase at 1/2 hour PO. Cerebellar NE content, bilaterally, reduced slowly to 70% of control at 24 hours PO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elevated immunoreactivity for glutamic acid decarboxylase in the rat cerebral cortex following transient middle cerebral artery occlusion

We investigated the expression of glutamic acid decarboxylase (molecular weight 67,000; GAD67) immunohistochemically in the rat cerebral cortex following transient middle cereral artery occlusion (MCAO) capable of producing slowly progressive neuronal damage. An increase in GAD67 immunoreactivity was observed in the cerebral cortex ipsilateral to the ischemic insult, most prominent in lamina IV, 3 to 14 days after MCAO. At this stage, light microscopy showed GAD67-positive puncta to be larger and more strongly immunoreactive in the ipsilateral cortex than those in the contralateral side. The elevated expression of GAD67 in the insulted cortex may reflect part of the adaptive functional changes in GABA transmission with slowly progressive cortical ischemic damage.Supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan     

Outcome following occlusion of the middle cerebral artery

Outcome was studied prospectively in 28 consecutive patients with occlusion of the middle cerebral artery (MCA). They comprise a subgroup of 101 consecutive patients with TIA or stroke less than or equal to 75 years of age, admitted within 72 h after the stroke. Cerebral angiography and CT-scan were performed within 1-2 days of admission. CT-scan was repeated 6 months later. Functional status on admission, 3 and 6 months after the stroke was evaluated using the Rankin disability scale (score 1-2: independent of others care, score 3-5: dependent on others care). The degree of hemiparesis was measured using the Medical Research Council's score. Thirteen had infarcts with a diameter less than or equal to 3 cm (mean 2.5 +/- 0.9 cm); 15 had infarcts greater than 3 cm (mean 6.3 +/- 1.4 cm); 10 had trunk occlusions; 18 had branch occlusions. MCA occlusions with large infarcts and severe hemiparesis on admission carried a poor outcome. Eleven (85%) of 13 patients with the case in only 1 (7%) of the 15 with infarcts greater than 3 cm, the remaining 14 (93%) had either died (40%) or were dependent (53%) (p less than 0.00005). Eleven (85%) of 13 patients with mild hemiparesis on admission were independent, while 13 (87%) of 15 with moderate or severe hemiparesis on admission had either died (40%) or were dependent on others' care (47%) 6 months after the stroke (p less than 0.0004). Type of occlusion (branch trunk) was a poor predictor of outcome.     

Induction of Rheb mRNA following middle cerebral artery occlusion in the rat.

Rheb is a recently identified member of the Ras super-family and is an immediate early gene that is rapidly and transiently induced in the hippocampal granule cells by NMDA-dependent synaptic activity in the long term potentiation paradigm. The close homologies with Ras and its rapid inducibility strongly suggest that Rheb shares many biochemical and signaling properties with Ras. The present study investigated the effect of middle cerebral artery (MCA) occlusion on the expression of Rheb mRNA in the rat brain. In situ hybridization autoradiography showed that Rheb mRNA was induced in the extensive regions of cerebral cortex and medial striatum surrounding the ischemic region and bilateral hippocampal formation following MCA occlusion. The induction of Rheb mRNA in the cingulate cortex persisted prominently at 24 h of MCA occlusion. Although the Rheb mRNA induction in the medial striatum and hippocampal formation decreased after 8h of occlusion, it still remained significant at 24h of occlusion. The data suggest the possibility that Ras signaling pathways can be implicated in the cerebral ischemia-elicited events through NMDA receptor activation.     

Atrophy of the ipsilateral substantia nigra following middle cerebral artery occlusion in the rat

Following occlusion of the left middle cerebral artery in the rat, marked atrophy was observed in the ipsilateral substantia nigra in and after the second week. The mechanism of this neuropathological change in the substantia nigra, which is remote from the site of infarction, may be explained by transsynaptic, neurotransmitter-mediated disinhibition as a result of infarction of the striatum.     

Physostigmine induced reversal of ischemia following acute middle cerebral artery occlusion in the rat

Cerebral cortical ischemia was induced in anesthetized rats by occlusion of the middle cerebral artery (MCA). Cerebral blood flow (CBF) was measured with the H2 clearance technique in the center and periphery of the ischemic territory. A decrease of CBF to about 50% of pre-occlusion values was observed in both areas. Administration of Physostigmine, a cholinesterase inhibitor, at a dose of 0.15 mg/Kg by intravenous route, induced an increase of CBF in the ischemic cortex. This change in CBF reached 120% of pre-occlusion level in the periphery and 80% of pre-occlusion value in the center of the area of distribution of the occluded artery. Although Physostigmine induced an increase in arterial blood pressure, the cerebral hyperemia observed both in normal and ischemic cortex could still be demonstrated after blockade of the pressor effect by bleeding or Phentolamine administration.     

实验性脑缺血再灌流后不同时程热休克蛋白基因表达的变化

为探讨脑缺血再灌流后热休克蛋白(HSP70)基因表达的变化,采用原位杂交和免疫组化方法检测了脑缺血2h再灌流后不同时程应激蛋白—热休克蛋白(hsp70)mRNA和蛋白表达的变化。结果显示再灌流后早期即可见hsp70mRNA的蛋白表达增加,以18～24h阳性染色最强,HSP70阳性细胞主要分布于缺血周围半暗带区,提示HSP70蛋白表达增加可抵御缺血性脑损伤,对神经元具有保护作用。     

Ischemic thresholds of cerebral protein synthesis and energy state following middle cerebral artery occlusion in rat

The ischemic threshold of protein synthesis and energy state was determined 1, 6, and 12 h after middle cerebral artery (MCA) occlusion in rats. Local blood flow and amino acid incorporation were measured by double tracer autoradiography, and local ATP content by substrate-induced bioluminescence. The various images were evaluated at the striatal level in cerebral cortex by scanning with a microdensitometer with 75 microns resolution. Each 75 x 75 microns digitized image pixel was then converted into the appropriate units of either protein synthesis, ATP content, or blood flow. The ischemic threshold was defined as the flow rate at which 50% of pixels exhibited complete metabolic suppression. One hour after MCA occlusion, the threshold of protein synthesis was 55.3 +/- 12.0 ml 100 g-1 min-1 and that of energy failure was 18.5 +/- 9.8 ml 100 g-1 min-1. After 6 and 12 h of MCA occlusion, the threshold of protein synthesis did not change (52.0 +/- 9.6 and 56.0 +/- 6.5 ml 100 g-1 min-1, respectively) but the threshold of energy failure increased significantly at 12 h following MCA occlusion to 31.9 +/- 9.7 ml 100 g-1 min-1 (p less than 0.05 compared to 1 h ATP threshold value; all values are mean +/- SD). In focal cerebral ischemia, therefore, the threshold of energy failure gradually approached that of protein synthesis. Our results suggest that with increasing duration of ischemia, survival of brain tissue is determined by the high threshold of persisting inhibition of protein synthesis and not by the much lower one of acute energy failure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Internal borderzone infarction following acute middle cerebral artery occlusion

In 36 patients suffering acute middle cerebral artery (MCA) occlusion, we studied the angiographic findings within 6 hours of the ictus and the chronic CT results at 3 months. Seven patients suffering distal pial MCA branch occlusion developed a pattern of internal borderzone infarction on follow-up CT. Carotid artery or carotid siphon stenosis or occlusion was absent in all seven. Proximal MCA branch occlusions, prior to the origin of the lenticulostriate arteries, were associated with extensive cortical and deep infarction in the entire MCA territory in 14 patients. There was proximal carotid artery or siphon stenosis or occlusion in 12 of these 14 patients. The remaining 15 patients showed a mixture of proximal and distal MCA occlusions and patchy ischemic damage in the MCA territory. There were no cases of superficial cortical watershed infarction. These data show that internal borderzone infarctions may result from intracranial MCA branch occlusions alone and need not be associated with hemodynamic alterations due to large vessel extracranial disease.     

大鼠大脑中动脉缺血和再灌注后环氧合酶-2mRNA表达的研究

目的研究大鼠大脑中动脉缺血和再灌注模型中环氧合酶－２（ＣＯＸ－２）基因的表达。方法原位杂交方法。结果缺血３０ｍｉｎ再灌注组,血流再通４ｈ后缺血区的大脑皮层有很强的表达并持续２４ｈ。缺血９０ｍｉｎ再灌注组和持续缺血组在缺血区以外的广泛大脑皮层显著表达,在海马的齿状回两侧及纹状体也发现表达。ＣＯＸ－２ｍＲＮＡ的表达可被ＭＫ－８０１抑制。而ＮＢＱＸ和倍他米松对其表达没有影响。结论在缺血区、缺血周边部、缺血远隔区有ＮＭＤＡ受体介导的ＣＯＸ－２表达,阐明上述区域花生四烯酸代谢活性化     

Changes in xanthine and uric acid in rat brain after middle cerebral artery occlusion

Xanthine and uric acid, products of purine metabolism, were measured by reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection in rat forebrain following focal cerebral ischemia. Focal cerebral ischemia was induced in the rat by permanent occlusion of the left middle cerebral artery (MCA). Sprague-Dawley rats were anesthetized with halothane inhalation and left MCA was occluded via trans-retro-orbital approach. Normal and sham-operated rats were used as control animals. The animals were decapitated 2 (MCA = 5, Sham = 5), 4 (MCA = 7, Sham = 6), 8 (MCA = 5, Sham = 5), and 16 (MCA = 6, Sham = 6) hours or 1 (MCA = 5, Sham = 5), 2 (MCA = 6, Sham = 6), 7 (MCA = 7, Sham = 6), 14 (MCA = 6, Sham = 5), and 28 (MCA = 7, Sham = 5) days after the operation. The brains were removed and divided into right and left hemisphere. Each hemisphere was homogenized and centrifuged. The supernates were filtered with membrane filter. An aliquot of the filtrate was used for measurement of xanthine and uric acid in both of the ischemic and contralateral hemisphere by a HPLC system. In the normal group, xanthine and uric acid in the brain was 12.4 +/- 0.4 and 2.2 +/- 0.1 nmol/g tissue (mean +/- SEM), respectively. In the ischemic hemisphere, xanthine increased up to 57.7 +/- 5.2 nmol/g tissue 2 hours after MCA occlusion and reached a maximum value of 59.42 +/- 4.91 nmol/g tissue 4 hours following the induction of ischemia. Xanthine level was still high 8 hours after ischemia and then rapidly decreased to the normal value at day 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of local cerebral blood flow, glucose utilization, and tissue pH following a middle cerebral artery occlusion in the rat

The use of three sets of the double-tracer autoradiographic technique to measure topographical changes of local cerebral blood flow (LCBF), glucose utilization (LCGU), and tissue pH following a 3 h middle cerebral artery (MCA) occlusion in the rat is described. In a sham-operated group of animals there was 10% reduction of LCBF and 7% reduction of LCGU in the most affected areas as compared to the contralateral homologous regions. However, the ratio of LCGU/LCBF in the affected areas remained within normal limits. In the MCA-occluded animals, LCGU showed a bimodal response to decreased LCBF. LCGU decreased with reduced LCBF until LCBF fell to 38% of normal. Below this LCBF level LCGU increased, most likely implying anerobic glycolysis. Decline of tissue pH corresponds to the mismatch of LCBF and LCGU. These results suggest that brain tissue pH change cannot be predicted on the basis of LCBF or LCGU alone.     

Regional flow-metabolism couple following middle cerebral artery occlusion in cats

The regional flow-metabolism couple was studied during the recovery period after 1 h of left middle cerebral artery (MCA) occlusion in cats. Local CBF (LCBF) was assessed at the end of ischemia as well as at the end of 4 h of recirculation by the microsphere technique. Local CMRgl (LCMRgl) was measured at the end of the recirculation period with [14C]2-deoxyglucose. Histology was evaluated by light microscopy from coronal brain blocks adjacent to those used for the determination of LCBF and LCMRgl. When LCBF in the central and peripheral MCA territories during the recovery period was between 40 and 115% of the value in sham occlusion studies, LCMRgl was greater than the control level found in the sham studies, and was accompanied by slight histological damage. This finding suggests that anaerobic glycolysis may persist after transient ischemia in spite of the recovery of LCBF to a level that is normally greater than the threshold for the activation of anaerobic glycolysis (less than 40% of the control). Persistent anaerobic glycolysis in the reperfusion period following an ischemic insult may be a sign of early tissue damage. Some of the regions in the peripheral MCA territory with LCBF between 40 and 110% of the levels during the recovery period in the sham studies showed a mild to moderate depression in LCMRgl so that the flow-metabolism ratio remained normal. These regions did not exhibit histological damage. This possible protective mechanism of the tissue in response to ischemia is discussed from the standpoint of the relationship between flow and metabolism.     

A change of P-selectin immunoreactivity in rat brain after transient and permanent middle cerebral artery occlusion

Abstract

Although the role of an adhesion molecule such as P-selectin may be important in the pathogenesis of stroke, temporal, spacial, and cellular profiles of the expression ofsuch a protein has not been fully studied in the case ofthe middle cerebral artery (MCA) occlusion and reperfusion in rat brain. Change in expression of immunoreactive P-selectin was examined in rat brain after transient MCA occlusion (MCAO) in comparison to that of permanent occlusion with an anti-P-selectin monoclonal antibody. Western blot analyses were performed to ensure the selective detection of immunoreactive P-selectin protein with the monoclonal antibody using brain homogenates before and after MCAO. Temporal, spacial, and cellular changes of P-selectin expressions were evaluated with rat brain sections at 2, 8 h, 1 and 3 days of permanent MCAO, and at 2, 8 h, 1, 3 and 7 days of reperfusion after 1 h of transient MCAO. Western blot showed a single band with a molecular weight of 140 kOa for both cases with permanent occlusion and reperfusion. P-selectin immunoreactivity was not normally present in rat brain sections. However, it was expressed mainly in the post-capillary venules of the cerebral cortex and caudate in the MCA territory with a peak at 2-8 h after permanent occlusion and at 8 h to 1 day after the reperfusion. The expression was diminished by 1 day ofpermanent occlusion and 3 days of reperfusion. The maximum staining in the case of permanent MCAO was stronger than the case with reperfusion. However, spacial distribution of the staining was similar in the cerebral cortex and caudate between the cases with permanent or transient MCAO. These results suggest a different temporal but similar spacial and cellular expression of P-selectin immunoreactivity between permanent occlusion and reperfusion of MCA in rat brain. [Neural Res 1998; 20: 463–469]     

Morphological consequences of early reperfusion following thrombotic or mechanical occlusion of the rat middle cerebral artery

Summary The early morphological consequences of recirculation following middle cerebral artery (MCA) occlusion were studied in two rat models. The proximal MCA was occluded for 1 h by either a surgical clip or platelet thrombus; subsequently, 1 h of recirculation was facilitated. Following clip occlusion and recirculation, mild astrocytic swelling, especially around blood vessels, was detected in reperfused cortical and striatal areas. Neuronal changes included slight chromatin clumping and dilation of the rough endoplasmic reticulum. In contrast, severe structural abnormalities were detected following recanalization of the thrombosed MCA segment. Marked astrocytic swelling of cell bodies and perivascular processes with neuropil vacuolation were commonly seen. A heterogeneous pattern of neuronal alterations, including a high frequency of dense shrunken neurons surrounded by swollen astrocytic processes was documented in cortical and striatal regions. Severe neuronal changes were documented in brain regions exhibiting a wellperfused microcirculation. Vascular endothelial cells contained large numbers of pinocytotic vesicles associated with luminal and abluminal surfaces. Pronounced and rapid morphological changes evolve with reperfusion when thrombotic and ischemic events occur simultaneously. The basis for these rapid parenchymal changes following vascular thrombosis may involve acute alterations in cerebral microvascular permeability which exacerbate ischemic consequences.Supported by USPHS grants NS-05820 and NS-23244, National Parkinson Foundation Grant YR 660068, and by the American Heart Association Grant-in-Aid 87-1012, with funds contributed by the Florida Affiliate. W. Dalton Dietrich is an Established Investigator of the American Heart Association.     

Enhanced protein synthesis in the ipsilateral substantia nigra following middle cerebral artery occlusion in the rat

Following focal cerebral ischemia, neuronal cell death is detected in remote areas of the brain, including the ipsilateral thalamus and substantia nigra (SN), as well as in the ischemic core. We have investigated protein synthesis in the remote areas of rats exposed to focal ischemia using autoradiography. The proximal portion of the left middle cerebral artery (MCA) was permanently occluded, and at various periods (6 h, 2, 4 and 7 days and 2 and 4 weeks following ischemia) animals received a single dose of l-[2,3-³H]valine (6.7 mCi/kg). Brain sections containing the thalamus and SN were processed for autoradiography. In the ipsilateral cerebral cortex and striatum, marked impairment of protein synthesis was observed and was never completely recovered during the experiment. No changes in protein synthesis in the ipsilateral thalamus were detected during the experiment. However, a change in protein synthesis was demonstrated in the ipsilateral SN. At 2 days after MCA occlusion, incorporation of [³H]valine into the whole zona reticulata of the ipsilateral SN was slightly enhanced and the increase became evident at 4 days after ischemia. Increased incorporation of [³H]valine began to be localized in the lateral portion of the zona reticulata after 7 days and continued up to 4 weeks following ischemia. Enhanced protein synthesis during the early stage (2 and 4 days after ischemia) may be due to the activated function of the neurons in the zona reticulata and that during the late stage (7 days and 2 and 4 weeks) after ischemia to astroglial proliferation Received: 22 July 1997 / Revised, accepted: 13 November 1997