Blood-brain barrier disruption and exacerbation of ischemic brain edema after restoration of blood flow in experimental focal cerebral ischemia

Summary The mechanism of exacerbation of ischemic brain edema after blood flow restoration was studied in 20 cats under ketamine and alpha-chloralose anesthesia. Regional cerebral blood flow was measured by the hydrogen clearance method, and the left middle cerebral artery (MCA) was occluded for 6 h in group A, and for 3 h with subsequent 3 h recirculation in group B. Severity of brain edema was assessed by specific gravity measurement of tissue samples taken from coronal brain sections at the MCA area, while severity of blood-brain barrier (BBB) disruption was determined by measuring the amount of extravasated serum albumin by using [¹²⁵I]albumin and tissue-uptake method in the same samples as those used for gravimetry. Structural and ultrastructural change was correlated with the severity of ischemic brain edema and BBB disruption. The results obtained showed that: (i) ischemic brain edema observed in group A was not associated with BBB opening to serum proteins; (ii) ischemic edema in group B was exacerbated significantly after recirculation in correlation with serum protein extravasation in most of the postischemic area; (iii) in the severely edematous area, serum protein extravasation reached a plateau and morphological examination at this type of area revealed cell membrane disruption especially of astrocytes, with leakage of intracellular substances. Our study indicated that the increase of extracellular osmotic pressure due to leakage of serum proteins via the disrupted BBB and of intracellular substances via the ischemically injured cell membrane into the extracellular space is the mechanism responsible for edema fluid accumulation in exacerbated ischemic brain edema.     

Effects of dextromethorphan on regional cerebral blood flow in focal cerebral ischemia

Dextromethorphan (DM), a noncompetitive NMDA antagonist, has been demonstrated to reduce ischemic neuronal damage and edema, but DM's influence on cerebral blood flow has not been extensively studied. In this investigation, it is shown that DM has significant effects on regional cerebral blood flow (rCBF) patterns in a rabbit model of focal cerebral ischemia. rCBF was measured using radioactive microspheres following a 1 h permanent occlusion of the left internal carotid, anterior cerebral, and middle cerebral arteries in rabbits. Somatosensory evoked potentials (SEPs) were used to assess the degree of ischemia; only animals where SEPs were completely abolished were used for a frequency distribution analysis of rCBF. It was found that there were significantly more regions with lower flows in animals treated with normal saline (NS) (n = 7) compared to animals treated with DM (n = 7) (p less than 0.05, ipsilateral left side; p less than 0.001, contralateral right side). The frequency distribution medians were 27.5 ml 100 g-1 min-1 (left) and 70.0 ml 100 g-1 min-1 (right) in the NS group vs. 34.5 ml 100 g-1 min-1 (left) and 80.5 ml 100 g-1 min-1 (right) in the DM group. The left and right hemispheric regional means were 29.4 +/- 20 and 74.3 +/- 23 ml 100 g-1 min-1, respectively, in the NS group vs. 34.4 +/- 16 and 91.0 +/- 28 ml 100 g-1 min-1, respectively, in the DM group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of propentofylline on cerebral blood flow in a gerbil focal cerebral ischemia

BACKGROUND AND PURPOSE: Neuroprotection and improvement of cerebral blood flow are two basic principles of pharmacological intervention in acute stroke. Propentofylline, an adenosine uptake and phosphodiesterase inhibitor, has been shown to be neuroprotective in various models of cerebral ischemia. However, its effect on cerebral circulation in ischemic conditions is not yet fully elucidated. Present experiments were designed to investigate the effect of propentofylline on regional cerebral blood flow (rCBF) in the gerbil permanent focal cerebral ischemia model. METHODS: Focal cerebral ischemia in gerbils was produced by clipping one common carotid artery and contralateral external carotid artery. rCBF was measured in both parietal cortices concurrently by the hydrogen clearance. RESULTS: Propentofylline at 10 mg/kg administered intraperitoneally 30 min after induction of cerebral ischemia significantly increased rCBF in ischemic regions (increase of 94.6%). Effects were dose dependent. Higher dosage (30 mg/kg) induced reductions of ischemic rCBF, which were associated with significant decreases of mean arterial blood pressure. Lower dosage (5 mg/kg) was without significant effect. CONCLUSIONS: Results suggest that propentofylline at suitable dosage improves rCBF in ischemic brain areas. Taking into account neuroprotective potentials of propentofylline, results offer additional rationale for clinical trials investigating efficacy of propentofylline in treatment of acute stroke.     

Nimodipine pretreatment improves cerebral blood flow and reduces brain edema in conscious rats subjected to focal cerebral ischemia

The effect of nimodipine pretreatment on CBF and brain edema was studied in conscious rats subjected to 2.5 h of focal cortical ischemia. An infusion of nimodipine (2 micrograms/kg/min i.v.) or its vehicle, polyethylene glycol 400, was begun 2 h before the ischemic interval and was continued throughout the survival period. Under brief halothane anesthesia, the animals' right middle cerebral and common carotid arteries were permanently occluded, and 2.5 h later, they underwent a quantitative CBF study ([14C]iodoantipyrine autoradiography followed by Quantimet 970 image analysis). Nimodipine treatment improved blood flow to the middle cerebral artery territory without evidence of a "vascular steal" and reduced the volume of the ischemic core (cortex with CBF of less than 25 ml/100 g/min) and accompanying edema by approximately 50% when compared with controls (p = 0.006 and 0.0004, respectively). Mild hypotension induced by nimodipine did not aggravate the ischemic insult. The ischemic core volumes, however, were 50-75% smaller than the 24-h infarct volumes generated in a similar paradigm that demonstrated 20-30% infarct reduction with continuous nimodipine treatment. These results suggest that nimodipine pretreatment attenuates the severity of early focal cerebral ischemia, but that with persistent ischemia, cortex surrounding the ischemic core undergoes progressive infarction and the early benefit of nimodipine treatment is only partly preserved.     

TMB—8对脑缺血大鼠脑血流量的作用

目的 研究8－（N，N－二乙胺）－n－辛基－3，4，5－三甲氧基苯甲酸酯（TMB－8）对局灶性脑缺血大鼠脑血流量（CBF）的作用。方法 用激光多谱勒血流仪测量大脑中动脉阻断（MCAO）大鼠脑血流量。分别于阻断前30分钟和阻断后20分钟给予TMB－8进行干预。结果 MCAO后，CBF迅速下降，维持恒定。阻断前30分钟给予TMB－8 0．5、1和2mg/kg，可剂量依赖性抑制CBF下降，阻断后20分钟给予TMB－8 1mg/kg，也能明显增加CBF。结论 TMB－8能预防和治疗MCAO局灶性脑缺血大鼠CBF减少，改善缺血区血供。     

Quantified EEG, somatosensory evoked potentials and cerebral blood flow in monitoring experimental brain ischemia

Cerebral ischemic lesions were induced in rabbits through internal carotid artery embolization by means of microspheres. Different models of stroke ranging from single focal cortical lesion to massive bilateral brain edema were reproduced and monitored up to the 24th hour by electroencephalogram (EEG and QEEG), somatosensory evoked potentials (SEPs), cerebral blood flow (CBF), platelet aggregation. The site and extent of the lesions were verified by histological examinations. Results obtained and physiopathological implications are discussed.

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Sommario Lesioni ischemiche cerebrali sono state indotte sperimentalmente nel coniglio mediante embolizzazione con microsfere attraverso l'arteria carotide interna. Modelli diversi, dall'ischemia corticale singola fino all'edema cerebrale bilaterale massivo, sono stati monitorizzati per 24 ore mediante l'elettroencefalogramma (EEG e QEEG), potenziali evocati somatosensoriali (SEPs), flusso ematico cerebrale (CBF), aggregazione piastrinica. Vengono discussi sia i risultati ottenuti sia alcune considerazioni fisiopatologiche.

     

亚低温对局灶脑缺血再灌注后脑血流、血脑屏障、神经细胞损伤作用的研究

目的对比研究不同时期亚低温（ＭＨＴ３２℃±０．２℃）对局灶脑缺血再灌注损伤的作用。方法６４只雄性ＳＤ鼠被随机分成常温（ＮＴ）、缺血期亚低温（ＭＨＴｉ）、再灌注期亚低温（ＭＨＴｒ）、缺血期加再灌注期亚低温（ＭＨＴｉ＋ｒ）四组,并用改良Ｋｏｉｚｕｍｉ＇ｓ局灶脑缺血模型,分别观察了动物缺血３小时再灌注３小时过程中,缺血周边和核心区局部脑血流（ｒＣＢＦ）改变,再灌注３小时后血脑屏障（ＢＢＢ）破坏及再灌注７２小时后缺血梗塞灶体积。结果ＭＨＴｉ＋ｒ及ＭＨＴｉ均有改善缺血周边区再灌注后急性高灌注和继发低灌流及核心区持续低灌流、减轻血脑屏障破坏、减少缺血梗塞灶体积的作用。该作用尤以ＭＨＴｉ＋ｒ为明显。ＭＨＴｒ作用有限。结论ＭＨＴｉ＋ｒ对皮层的保护作用较ＭＨＴｉ好。由此可推知,局灶脑缺血再灌注损伤是个持续的过程,亚低温治疗不但要考虑开始时间,其持续时间对疗效具有同样重要价值     

Mechanisms of brain damage in focal cerebral ischemia

Ischemic stroke is a major disabling disease. There are 500,000 new cases in U.S. every year, and the middle cerebral artery (MCA) is the artery most often occluded. In this paper recent results of experimental MCA occlusion are reviewed, with special emphasis on those factors contributing to irreversible damage. Occlusion of MCA in the rat causes a pronounced decline of flow in the neostriatum to less than 10% of normal. The area of low flow is surrounded by a zone 0.2-0.5 mm wide, across which blood flow increases steeply. Beyond this zone, changes in flow are more gradual, and perfusion is reduced to about 1/3 of normal in the adjacent ipsilateral cortex. The MCA occlusion leads to a sharply demarcated infarct and to scattered neuronal injury in the adjacent cortical tissue. It is suggested that the ischemic core is identical with the tissue infarct, i.e. that it is the initial pattern of blood flow which determines the volume and topography of infarction. Waves of spreading depression are detected in the cortical low perfusion area during the first hours of MCA occlusion, and glucose consumption is increased, presumably due to an increased demand for ionic transport. In hyperglycemic animals, the number of spreading depressions is reduced as is the glucose consumption. The repeated waves of spreading depression in combination with partial energy depletion may induce selective neuronal injury in the peri-infarct zone, a suggestion which finds support in the fact that hyperglycemia ameliorates neuronal injury around the infarction.     

Plasminogen activation in experimental permanent focal cerebral ischemia

BACKGROUND: Previous experimental work using in situ zymography has shown very early increased plasminogen activation in ischemic regions after 3 h of ischemia with and without reperfusion. The objective of the present study was to evaluate the time course and extent of plasminogen activation in long-term permanent focal cerebral ischemia. MATERIAL AND METHODS: The middle cerebral artery in male Fisher rats was irreversibly occluded by electrocoagulation. Duration of ischemia was 48, 72, and 168 h. Occlusion was controlled in vivo by MRI at day 2. Plasminogen activation was detected by in situ zymography of 10 microm cryosections with an overlay containing plasminogen and the plasmin substrate caseine. Areas of plasminogen activation were compared to structural lesions (immunohistochemical loss of microtubule-associated protein 2; MAP 2). RESULTS: Compared to controls, increased plasminogen activation was observed in the basal ganglia and the cortex of the ischemic hemisphere after 48, 72, and 168 h (affected area of basal ganglia: 44.5+/-21.9, 70.1+/-2.3 and 66.6+/-2.8%, respectively; affected area of cortex: 63.4+/-9.8, 67.7+/-0.7 and 64.0+/-3.7%, respectively). The duration of ischemia had no significant influence on the extent of plasminogen activation. Areas of increased plasminogen activation significantly overlapped with and exceeded areas of MAP 2 loss (P<0.005). DISCUSSION: Permanent focal cerebral ischemia leads to increased plasminogen activation in ischemic regions. This plasminogen activation remains elevated at persistent levels over days. It may contribute to extracellular matrix (ECM) disruption, secondary hemorrhage, and brain edema in subacute stages of ischemic stroke.     

High-dose methylprednisolone treatment in experimental focal cerebral ischemia

Previous studies using steroids for experimental focal stroke have demonstrated conflicting results, possibly related to dose used or ischemic models employed. In this study we examined high-dose methylprednisolone treatment following permanent and temporary focal cerebral ischemia in the rat. Focal stroke was induced in spontaneously hypertensive rats by permanent right common carotid and either permanent or 3 h of temporary middle cerebral artery (MCA) occlusion. Methylprednisolone (105 mg/kg) was administered intra-arterially. Infarct volume was measured at 24 h after permanent and temporary MCA occlusion. Cerebral edema was determined by measuring right and left hemispheric volumes and water content 24 h after permanent MCA occlusion in one experiment. Methylprednisolone, whether administered in divided doses over 12 h (n = 15 in each group) or a single bolus (n = 9 per group), had no effect on infarct volume after permanent MCA occlusion. Methylprednisolone treatment also had no influence on cerebral edema (n = 9 per group). In two different experiments, methylprednisolone given in divided doses over 12 h (n = 11, n = 25) after temporary MCA occlusion decreased infarct volume (P < 0.05) by 20% compared with saline controls (n = 10, n = 25). High dose methylprednisolone decreased infarct volume following temporary, but not permanent, focal ischemia. The benefit suggests that high dose methylprednisolone may prove useful clinically if reperfusion can be established with thrombolytic agents. Furthermore, the differential treatment effect in the setting of comparable ischemic insults implies that different modifiable biochemical processes may be present during temporary but not permanent focal ischemia, thus providing indirect evidence for reperfusion injury.     

Superoxide dismutase activity in experimental focal cerebral ischemia

The activity of the free radical scavenger, superoxide dismutase, was studied in focal cerebral ischemia produced in Mongolian gerbils (Meriones unguiculatus) by occluding the right common and left external carotid arteries under halothane anesthesia. After recovery from anesthesia animals were classified according to their neurologic symptoms. Five animals exhibiting neurologic symptoms such as hemiparesis and rolling seizures were reanesthetized 120 min after vascular occlusion and their brains frozen in situ with liquid nitrogen. A series of 20-micron-thick coronal sections was cut in a cryostat; pictorial representations of tissue pH, ATP, and glucose were obtained using fluorescent and bioluminescent techniques. Using a highly sensitive bioluminescent technique, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and Mn-superoxide dismutase (Mn-SOD) activities were then measured in samples from both ischemic and nonischemic regions of the remaining tissue block. Cu,Zn-SOD and Mn-SOD activities were, respectively, 13.9 +/- 0.7 X 10(3) units/g and 5.4 +/- 0.3 X 10(3) units/g in the nonischemic tissue, and 13.2 +/- 0.6 X 10(3) units/g and 5.0 +/- 0.2 X 10(3) units/g within the ischemic tissue. Thus focal cerebral ischemia does not lead to a global decrease in SOD activity, as observed by others after heart and liver ischemia.     

Autoregulation of cerebral blood flow during controlled hypotension in baboons

The effect of graded, progressive hypotension on the autoregulation of cerebral blood flow was studied in anaesthetised baboons. Progressive hypotension was produced over a period of four to five hours, either by graded haemorrhage or by the administration of increasing concentrations of hypotensive drugs. During haemorrhagic hypotension autoregulation was maintained until the mean arterial pressure had decreased to 65% of its baseline value, below which cerebral blood flow was pressure passive. In those animals subjected to drug-induced hypotension, autoregulation persisted to lower levels of mean arterial pressure (35-40% of baseline). It is postulated that under conditions of haemorrhagic hypotension, constriction of the extraparenchymal cerebral vessels in response to sympathetic stimulation decreases the possible range of autoregulation in the anaesthetised baboon.     

流式细胞仪检测大鼠局灶性脑缺血再灌流后细胞凋亡的研究

本研究采用流式细胞仪（ＦＣＭ）检测大鼠局灶性脑缺血再灌流后凋亡细胞ＤＮＡ断裂百分率。结果表明：随再灌流时间的增加，ＤＮＡ断裂百分率逐渐增加，再灌流１ｄ时达高峰，再灌流３～７ｄ逐渐下降。结果提示：细胞凋亡在局灶性脑缺血再灌流损伤中呈动态过程且系神经细胞死亡的重要形式。     

Time course of cerebral blood flow and histological outcome after focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: The relation between time-dependent changes in cerebral blood flow and the appearance of infarction after focal cerebral ischemia is still a matter for debate. The aim of this study was to measure perfusion after simultaneous occlusions of the left middle cerebral artery and ipsilateral common carotid artery in rats and correlate it with the timing and distribution of histological changes. METHODS: We studied histological and cerebral blood flow changes 5 minutes and 4, 24, and 48 hours after the onset of focal ischemia. Blood flow was determined autoradiographically using [14C]iodoantipyrine. A coronal template subdivided into regions of interest was applied to the autoradiographs and the histological data. RESULTS: In some regions of the nonoccluded hemisphere, cerebral blood flow 5 minutes after occlusion fell below 50% of normal. Many ischemic structures showed stable blood flow for 48 hours after occlusion, confirming that in this model reperfusion is minimal. Infarction occurred eventually in all areas in which blood flow at 5 minutes fell below 10% of that in control rats, but infarction appeared earlier in regions in which blood flow at 5 minutes was below 5% of that in control rats. When blood flow at 5 minutes rose above 12% of that in control rats, the occurrence of infarction became unpredictable. CONCLUSIONS: Despite the general dependence of infarction on perfusion levels, blood flow was not a reliable indicator of those regions committed to infarction.     

Increased intracellular calpain detection in experimental focal cerebral ischemia

Calpains are intracellular proteinases whose proteolytic activity is directed mainly against the cytoskeleton and regulatory proteins. We studied the presence of calpain by immunohistochemistry in a rat model of reversible focal cerebral ischemia (3 h) at various times of reperfusion. The numbers of calpain-positive cells on the ischemic side were compared with the non-ischemic side. In controls only 2 +/- 1% cells were positive, whereas the cortex of the ischemic vs the non-ischemic side showed 88 +/- 3% vs 13 +/- 4% calpain-positive cells (p < 0.001), and the basal ganglia 47 +/- 3% vs 13 +/- 4% (p < 0.01) after 3 h ischemia and 24 h reperfusion. This is the first demonstration of elevated intracellular levels of calpains in areas of cerebral ischemia. Longer reperfusion resulted in an increase in calpain positivity.     

Cerebral blood flow and regional potassium distribution during focal ischemia of gerbil brain

In 8 gerbils (Meriones unguiculatus) focal cerebral ischemia was produced by occlusion of the left common carotid artery and the opposite external carotid artery. After two hours blood flow was measured with iodoantipyrine labeled with carbon 14, and evaluated by means of quantitative autoradiography. Thereafter the same brain sections were stained for regional potassium by means of a histochemical technique. Changes in tissue potassium content were assessed by measuring the differences in optical densities in homotopic brain regions of the stained sections. The correlation between blood flow and tissue potassium level revealed that below a flow threshold of about 0.23 ml/gm/min, a definite potassium loss from the tissue was observed. The combination of autoradiographic methods with a technique for measuring the regional distribution of potassium may be useful in providing additional information on the occurrence of disturbed electrolyte homeostasis after the onset of focal ischemia.     

A gelatin in situ-overlay technique localizes brain matrix metalloproteinase activity in experimental focal cerebral ischemia

To determine the activity of matrix metalloproteinases (MMP), especially MMP-2 and MMP-9, which play an important role in ischemic stroke and intracerebral hemorrhage, we adapted a simple and rapid method for localizing gelatinase activity to a gelatin film in situ-overlay technique previously used in cancer research. Ten micrometer cryosections of rat brain from controls and animals subjected to 3 h of ischemia and 48 h of reperfusion (suture model for transient cerebral ischemia) were used. After thawing, a gelatin film with a polyester base was put on the slide, incubated for 24 h at 37 degrees C, stained with Ponceau S, and then discolored in bi-distilled water. Non-staining areas on the film corresponded to lysis zones, caused by activated MMPs. This was proven by MMP incubation at various concentrations on the plain gelatin film and pretreatment with EDTA (an MMP inhibitor), which prevents lysis zones in normal and ischemic brains. As confirmatory tests, SDS-PAGE zymography was used to define MMP activity, and also MMP-2 immunohistochemistry to detect the possibly cellular origin of MMPs. Normal rat brain exhibited a low background activity, which was visible as a light halo-like lysis zone over and around the brain. Areas in normal brain with medium MMP activity were within the white matter (corpus callosum, anterior commissure, and cerebellum). Ischemic brain exhibited high activity lysis zones within the infarcted area (detected by microtubuli associated protein-2 staining). These zones consisted of microscopically small lysis holes with a diameter of about 10-20 microm. Immunohistochemistry showed that especially microvessels expressed MMP antigen. SDS-PAGE zymography differentiated between a high level of activated MMPs in the ischemic area and a low level in the non-ischemic basal ganglia. The gelatin film in situ-overlay technique is able to localize MMP activity in ischemic rat brain tissue on a microscopic level.