Time- and pressure-dependent changes in blood-brain barrier permeability after temporary middle cerebral artery occlusion in rats

Summary After 180 min of temporary middle cerebral artery occlusion in rats, the affect of phenylephrine-induced hypertension on blood-brain barrier permeability was assessed. One of the following blood-pressure regimens was maintained during either a 30- or 120-min period of reperfusion: (a) 30/Norm, 30 min of normotensive reperfusion was allowed; (b) 30/HTN, mean arterial blood pressure was increased by 35 mm Hg during 30 min of reperfusion; (c) 120/Norm, 120 min of normotensive reperfusion was allowed; or (d) 120/HTN, mean arterial blood pressure was increased by 35 mm Hg during 120 min of reperfusion. Evans blue (30 mg/kg) was given, and brains were analyzed for Evans blue by spectrophotometry. Evans blue (g/g brain tissue, mean ± SD) was greater (P<0.05) in both hypertensive groups versus their time matched normotensive groups (30/HTN: 80±16 versus 18±6 in the 30/Norm group; 120/HTN: 17±6 versus 8±3 in the 120/Norm group). In addition, Evans blue was greater (P<0.05) in both 30-min groups versus their pressure matched 120-min groups (30/Norm: 18±6 versus 8±3 in the 120/Norm group; 30/HTN: 80±16 versus 17±6 in the 120/HTN group). The data are consistent with previous studies which have demonstrated an opening of the blood-brain barrier at the onset of reperfusion. In addition, the data support a hypothesis that changes in blood-brain barrier permeability are more sensitive to hypertension in the early period of reperfusion.     

The biphasic opening of the blood-brain barrier to proteins following temporary middle cerebral artery occlusion

Summary The behavior of the blood-brain barrier (BBB) was studied in cats following release after 1-h middle cerebral artery (MCA) occlusion. The regional cerebral blood flow (rCBF) was determined by hydrogen clearance method in the caudate nucleus and the cerebral cortex. The BBB was assayed with Evans blue (EB) tracer and by immunohistochemical peroxidaseantiperoxidase (PAP) method. Following release of MCA occlusion, there were two openings of the BBB, separated by a refractory period. The first opening, occurred shortly after recirculation; this was associated with rCBF below 15 ml/100 g/min during the ischemic period and a pronounced reactive hyperemia promptly following release of MCA occlusion. A refractory period of the BBB was indicated by the absence of EB leakage in cats injected with the tracer 30 min before killing at 3 h after recirculation, although the rCBF values in these animals were even lower (6±1 ml/100 g/min) during occlusion, and all of them showed a pronounced hyperemia after recirculation. The occurrence of the previous BBB opening in these animals was confirmed by the PAP staining. The second opening of the BBB was observed at 5 and 72 h after recirculation in cats which were injected with EB 30 min before killing, and which showed rCBF below 15 ml/100 g/min during occlusion, followed by a pronounced reactive hyperemia. No EB extravasations were observed at any time in cats in which the rCBF during occlusion was above 15 ml/100 g/min and which failed to show a marked reactive hyperemia.     

Oedema-related tissue damage after temporary and permanent occlusion of the middle cerebral artery

Oedema-related tissue damage after temporary and permanent occlusion of the middle cerebral arteryEleven adult spontaneously–hypertensive male rats (SHR) were studied 2 2 h or 7 days after a 2 h unilateral occlusion of the right middle cerebral artery (MCA). Another 11 SHR were studied after 24 h or 7 days of permanent MCA ligation. The brain infarcts were significantly larger ( P < 0.05) after permanent occlusion than after a 2h occlusion. More extensive and widespread vasogenic oedema, emanating from the infarcts, was visualized immunohistochemically in the temporarily–ligated animals and the relative number of astrocytes in their contralateral hemispheres was greater, thereby indicating that the vasogenic oedema influences the degree of gliosis. An immunopositivity for albumin but not for fibrinogen extended via the white matter into the ipsilateral thalamic nuclei, where cytolytic nerve cell damage, severely shrunken and karyorrhectic nerve cells as well as gliosis were found one week after permanent and temporary MCA ligation. The histological changes in the thalamus indicated a difference in timing between lateral and medial parts of the lesion as well as between temporarily–and permanently–ligated SHR. These findings together with the close spatial correlation with albumin immunoreactivity indicate that the spread of extravasated plasma constituents or degradation products with the oedema bulk flow from the infarct influences the timing, character and extent of thalamic lesions after cerebral infarction.     

Effect of difluoromethylornithine on reperfusion injury after temporary middle cerebral artery occlusion.

Polyamines have been shown to play an important role in the disturbance of the blood-brain barrier (BBB) in a number of pathological states including ischemia. BBB disturbances may be almost completely prevented by treating animals with the ornithine decarboxylase (ODC) inhibitor, alpha-difluoromethylornithine (DFMO). DFMO has been also shown to prevent N-Methyl-D-aspartate (NMDA) toxicity in tissue cultures. It has been suggested that the pathological disturbances in polyamine metabolism observed following cerebral ischemia, particularly the post-ischemic increase in putrescine, may contribute to the ischemic injury that is most evident in the CA1 subfield of the hippocampus. In this study, effects of DFMO in cerebral ischemia and reperfusion were examined. The results showed that inhibition of the polyamine system by DFMO decreased ischemic injury volume and brain tissue water content in a dose-dependent manner, without change in vital signs, including systemic arterial blood pressure, arterial partial oxygen pressure, regional cerebral blood flow and body temperature.     

Hypervolemic-hemodilution and hypertension during temporary middle cerebral artery occlusion in rats: the effect on blood-brain barrier permeability

The effect of hypervolemic-hemodilution, with and without hypertension, on blood-brain barrier permeability was investigated in rats, after 180 minutes of middle cerebral artery occlusion (MCAo), and 60 minutes of reperfusion. One of the following conditions was maintained during MCAo: 1) Control--hematocrit and blood pressure were not manipulated; 2) Hypervolemic-Hemodilution/Normotension--the hematocrit was decreased to 30%; 3) Hypervolemic-Hemodilution/Hypertension--the hematocrit was decreased to 30% and mean arterial pressure increased by 30 mmHg with phenylphrine. In all groups, Evans Blue was administered, and its concentration determined by spectrophotometric assay. Evans Blue (micrograms (g-1 of brain tissue [mean +/- SD]) was greater in the Hypervolemic-Hemodilution/Hypertension group (71 +/- 20) versus the Control (13 +/- 9) and Hypervolemic-Hemodilution/Normotension (17 +/- 10) groups (p less than 0.05). No other differences were present. These results support the hypothesis that during MCAo, hypervolemic-hemodilution/hypertensive therapy effects an increase in blood-brain barrier permeability in the early period of reperfusion.     

Estradiol reduces activity of the blood-brain barrier Na-K-Cl cotransporter and decreases edema formation in permanent middle cerebral artery occlusion.

Estrogen has been shown to protect against stroke-induced brain damage, yet the mechanism is unknown. During the early hours of stroke, cerebral edema forms as increased transport of Na and Cl from blood into brain occurs across an intact blood-brain barrier (BBB). We showed previously that a luminal BBB Na-K-Cl cotransporter is stimulated by hypoxia and arginine vasopressin (AVP), factors present during cerebral ischemia, and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema in rats subjected to permanent middle cerebral artery occlusion (MCAO). The present study was conducted to determine whether estrogen protects in stroke at least in part by reducing activity of the BBB cotransporter, thereby decreasing edema formation. Ovariectomized rats were subjected to 210 mins of permanent MCAO after 7-day or 30-min pretreatment with 17beta-estradiol and then brain swelling and 2,3,5-triphenyltetrazolium chloride staining were assessed as measures of brain edema and lesion volume, respectively. Diffusion-weighed imaging was used to monitor permanent MCAO-induced decreases in apparent diffusion coefficient (ADC) values, an index of changes in brain water distribution and mobility. Na-K-Cl cotransporter activity of cerebral microvascular endothelial cells (CMECs) was assessed as bumetanide-sensitive K influx and cotransporter abundance by Western blot analysis after estradiol treatment. Estradiol significantly decreased brain swelling and lesion volume and attenuated the decrease in ADC values during permanent MCAO. Estradiol also abolished CMEC cotransporter stimulation by chemical hypoxia or AVP and decreased cotransporter abundance. These findings support the hypothesis that estrogen attenuates stimulation of BBB Na-K-Cl cotransporter activity, reducing edema formation during stroke.     

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.     

Reduction of ischemic brain injury by anti-P-selectin monoclonal antibody after permanent middle cerebral artery occlusion in rat.

The selectin family of adhesion molecules is involved in adhesion of leukocyte to microcirculatory system and the transmigration into brain parenchyma. Although the role of P-selectin may be important in the pathogenesis of brain ischemia, a possible protective effect on ischemic brain injury by blocking P-selectin has not been reported. We have examined the effects of a novel anti-P-selectin antibody on ischemic brain injury after 24 h of permanent middle cerebral artery occlusion (MCAO) in rat. Male Wistar rats were subjected to MCAO by an insertion of a silicone rubber cylinder for 24 h. Anti-rat P-selectin monoclonal antibody, ARP 2-4, was injected intravenously at a dose of 1 mg kg-1 at 5 min before the induction of MCAO. Control animals received the same volume of vehicle solution. Regional cerebral blood flow (rCBF) was measured immediately after and at 8 h of MCAO. At decapitation of rats at 24 h of permanent MCAO, infarct size was compared between the antibody and vehicle treated group. In addition, immunohistochemistry for leukocyte infiltration and HSP72, and histochemistry for TUNEL were also, compared. Pretreatment with ARP 2-4 improved rCBF at 8 h of MCAO (55.4% +/- 11.7% of control, n = 5) as compared to vehicle group (24.2% +/- 11.8%, n = 5, p < 0.02). Although leukocyte infiltration was not normally detected by monoclonal antibodies for CD11a and CD18, it became remarkably evident at 1 day of MCAO. Although HSP72 and TUNEL were not also detected in sham control brains, they were induced in neurons of the MCA area at 1 day of MCAO. Treatment with ARP 2-4 significantly reduced the numbers of leukocyte and neurons with positive HSP72 and TUNEL stainings. These results demonstrated that an administration of a monoclonal antibody against P-selectin improved rCBF, and attenuated infarct size that was associated with reduction of leukocyte infiltration. Furthermore, treatment with the antibody reduced both HSP72 and TUNEL stainings. These data suggest an important role of P-selectin in ischemic brain damage, and a future therapeutic potential to human stroke patients.     

Reduction of ischemic brain injury by anti-P-selectin monoclonal antibody after permanent middle cerebral artery occlusion in rat

Abstract

The selectin family of adhesion molecules is involved in adhesion of leukocyte to microcirculatory system and the transmigration into brain parenchyma. Although the role of P-selectin may be important in the pathogenesis of brain ischemia, a possible protective effect on ischemic brain injury by blocking P-selectin has not been reported. We have examined the effects of a novel anti-P-selectin antibody on ischemic brain injury after 24 h of permanent middle cerebral artery occlusion (MCAO) in rat. Male Wistar rats were subjected to MCAO by an insertion of a silicone rubber cylinder for 24 h. Anti-rat P-selectin monoclonal antibody, ARP 2-4 was injected intravenously at a dose of 1 mg kg^–1 at 5 min before the induction of MCAO. Control animals received the same volume of vehicle solution. Regional cerebral blood flow (rCBF) was measured immediately after and at 8 h of MCAO. At decapitation of rats at 24 h of permanent MCAO, infarct size was compared between the antibody and vehicle treated group. In addition, immunohisto- chemistry for leukocyte infiltration and HSP72, and histochemistry for TUNEL were also compared. Pretreatment with ARP 2-4 improved rCBF at 8 h of MCAO (55.4% ± 11.7% of control, n = 5) as compared to vehicle group (24.2% ± 77.8%, n = 5, p < 0.02). Although leukocyte infiltration was not normally detected by monoclonal antibodies for CD11a and CD18, it became remarkably evident at 7 day of MCAO. Although HSP72 and TUNEL were not also detected in sham control brains, they were induced in neurons of the MCA area at 7 day of MCAO. Treatment with ARP 2-4 significantly reduced the numbers of leukocyte and neurons with positive HSP72 and TUNEL stainings. These results demonstrated that an administration of a monoclonal antibody against P-selectin improved rCBF, and attenuated infarct size that was associated with reduction of leukocyte infiltration. Furthermore, treatment with the antibody reduced both HSP72 and TUNEL stainings. These data suggest an important role of P-selectin in ischemic brain damage, and a future therapeutic potential to human stroke patients. [Neurol Res 1999; 21: 269-276]     

Effects of dexmedetomidine after transient and permanent occlusion of the middle cerebral artery in the rat

Summary. Increased sympathetic tone is a consequence of cerebral ischemia. Although the role of catecholamines in ischemic damage is still unclear, in some experimental ischemia models α₂-adrenergic agonism has proved to be neuroprotective. In the present work we have compared the effects of transient and permanent middle cerebral artery occlusion (MCAO) on the infarct volume, and, also, examined whether a selective α₂-adrenergic receptor agonist, dexmedetomidine (9 μg/kg or 15 μg/kg i.v.), is able to reduce ischemic damage after transient or permanent MCAO in rats. Permanent MCAO led to a significantly larger infarct volume than transient occlusion (p < 0.05). The rats receiving the higher dose of dexmedetomidine were detectected to have smaller (statistically non-significant) infarct volume in the cortex (30.9%) and in the striatum (20.3%) after transient occlusion. Additionally, dexmedetomidine caused significant variations in the physiological parameters. Received December 22, 1999; accepted September 20, 2000     

Detrimental effects of 17beta-oestradiol after permanent middle cerebral artery occlusion.

Oestrogen is a complex hormone whose role as a neuroprotectant in experimental stroke has been published in numerous studies. However, although some clinical studies report a reduction in stroke incidence by oestrogen replacement therapy in postmenopausal women, others have found increased mortality and morbidity after stroke. Diathermy occlusion of the proximal middle cerebral artery (MCAO), one of the most reproducible rodent stroke models, has consequently been employed to investigate physiologic and supraphysiologic doses of 17beta-oestradiol on ischaemic brain damage. Lister Hooded rats were ovariectomised (OVX) and a 21-day release pellet (placebo, 0.025 or 0.25 mg 17beta-oestradiol) implanted in the neck. At 2 weeks after OVX, animals underwent MCAO and were perfusion fixed 24 hours later. Neuronal perikaryal damage was assessed from haematoxylin and eosin-stained sections and in adjacent sections, axonal pathology was assessed with amyloid precursor protein and Neurofilament 200 (NF-200) immunohistochemistry. 17beta-Oestradiol induced a dose-dependent increase in neuronal perikaryal damage, 0.025 and 0.25 mg 17beta-oestradiol increased damage by 20% (P<0.05) and 27.5% (P<0.01) compared with placebo. 17beta-Oestradiol did not influence axonal pathology compared with placebo. Our results suggest that 17beta-oestradiol treatment can exacerbate brain damage in experimental stroke. Thus, further investigation into the role of oestrogen and the mechanisms which mediate its effects in stroke is required.     

Evolution of microcirculatory disturbances after permanent middle cerebral artery occlusion in rats.

Nonischemic brain capillaries show a continuous and heterogeneous plasma perfusion. In the current study, plasma perfusion was investigated in rats during 2 to 168 hours of permanent middle cerebral artery occlusion. Perfused capillaries were detected in brain cryosections by fluorescein isothiocyanate (FITC) dextran after 10 minutes of circulation time. Heterogeneity of capillary perfusion was identified by Evans blue (EB), which circulated for 3 seconds. In this setting, the heterogeneity of intracapillary EB concentrations reflects heterogeneities in capillary flow velocities. The CBF was quantified by simultaneous iodo[14C]antipyrine autoradiography. When moving from normal flow to low-flow areas in the ischemic hemisphere, three states of capillary filling could be distinguished: state 1--fast perfusion, filling by FITC dextran and EB (CBF 0.33 mL x g(-1) x min(-1)); state 2--delayed perfusion, only FITC dextran filling (CBF 0.104 mL x g(-1) x min(-1)); state 3--minimal perfusion, no dye filling (CBF 0.056 mL x g(-1) x min(-1)). In tissue of state 1 at the borderline to ischemic tissue, a higher heterogeneity of intracapillary EB concentration (85.7%) was found than in the contralateral nonischemic hemisphere (76.4%) (P < 0.05), indicating a compromised microcirculation. The adjacent ischemic areas were filled by FITC dextran (state 2) 2 to 4 hours after middle cerebral artery occlusion, indicating a maintained, although slow, perfusion at this time. Later, minimal perfused areas (state 3) progressively replaced the delayed perfused areas (state 2). This study shows, for the first time, the evolution of microvascular disturbances in relation to CBF. In the low-flow areas, an early residual plasma perfusion is later followed by a lack of perfusion or minimal perfusion. In areas of higher, although reduced flow at the border between normal and ischemic tissue, an extreme capillary perfusion heterogeneity indicates permanent microcirculatory abnormalities.     

Neurological deficit and cerebral infarction after temporary middle cerebral artery occlusion in unanesthetized cats

Forty-four unanesthetized cats underwent temporary middle cerebral artery (MCA) occlusion with an implanted, externally controlled balloon cuff occluder. The occlusion was reversed to allow reperfusion of the MCA after 2 min to 24 hr of ischemia. Fourteen cats had temporary occlusions lasting 2 min to 3 hr; their neurological deficits improved or resolved after reperfusion, and brain sections showed only scattered microscopic areas of necrosis. After a 4-hr occlusion, five of nine cats (55%) recovered completely within 24 hr; two had persistent deficit when sacrificed, 10 days later, and each had a circumscribed infarct. All 18 cats undergoing 5-, 6-, 8-, and 24-hr occlusions sustained permanent neurological deficits. Three 3-hr occlusions at 2-day intervals in three cats resulted in permanent deficits and infarcts that were 25% larger than those after single 8-hr occlusions. Ten cats underwent permanent MCA occlusion; three deteriorated neurologically and died, and the survivors showed no improvement. Infarcts after 5-, 6-, and 8-hr occlusions followed by reperfusion were 66% smaller (p less than 0.05) than those after permanent occlusion; reperfusion after 24 hr of occlusion did not reduce infarct size. Hemorrhagic infarction occurred after two permanent occlusions, but after only one 5-hr temporary occlusion. The results obtained with this method of temporary regional ischemia indicate that restoration of flow after 1-8 hr, but not after 24 hr, of MCA occlusion resulted in less severe neurological deficit and smaller infarcts than did permanent occlusion. The infarct size correlated with the duration of MCA occlusion (p less than 0.05) rather than with the degree of deficit during occlusion.     

Bumetanide inhibition of the blood-brain barrier Na-K-Cl cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke.

Increased transport of Na+ across an intact blood-brain barrier (BBB) participates in edema formation during the early hours of cerebral ischemia. In previous studies, the authors showed that the BBB Na-K-Cl cotransporter is stimulated by factors present during ischemia, suggesting that the cotransporter may contribute to the increased brain Na+ uptake in edema. The present study was conducted to determine (1) whether the Na-K-Cl cotransporter is located in the luminal membrane of the BBB, and (2) whether inhibition of the BBB cotransporter reduces brain edema formation. Perfusion-fixed rat brains were examined for cotransporter distribution by immunoelectron microscopy. Cerebral edema was evaluated in rats subjected to permanent middle cerebral artery occlusion (MCAO) by magnetic resonance diffusion-weighted imaging and calculation of apparent diffusion coefficients (ADC). The immunoelectron microscopy studies revealed a predominant (80%) luminal membrane distribution of the cotransporter. Magnetic resonance imaging studies showed ADC ratios (ipsilateral MCAO/contralateral control) ranging from 0.577 to 0.637 in cortex and striatum, indicating substantial edema formation. Intravenous bumetanide (7.6-30.4 mg/kg) given immediately before occlusion attenuated the decrease in ADC ratios for both cortex and striatum (by 40-67%), indicating reduced edema formation. Bumetanide also reduced infarct size, determined by TTC staining. These findings suggest that a luminal BBB Na-K-Cl cotransporter contributes to edema formation during cerebral ischemia.     

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]     

Periinfarct and remote excitability changes after transient middle cerebral artery occlusion.

Transient middle cerebral artery (MCA) occlusion results in substantially smaller cortical infarcts than permanent MCA occlusion if reperfusion is initiated within the first few hours. Only little information is available on the long-term functional outcome of the cortical regions "salvaged" by early reperfusion. To address this issue we examined basic electrophysiologic parameters in vitro using standard extracellular recording techniques at 7 and 28 days after transient MCA occlusion (1- and 2-hour ischemia) in rats. Both neocortical areas ipsi- and contralateral to MCA occlusion were systematically mapped to delineate the extent of periinfarct and remote alterations. In the periinfarct region we found a significant reduction of field potential amplitudes up to 3 mm when measuring from the infarct border at 7 days and up to 7 mm at 28 days. Paired-pulse inhibition, an indicator of GABAergic transmission, was only moderately impaired in this region at 7 days and not significantly different from control at 28 days. Remote effects were observed both ipsi- and contralaterally. Ipsilaterally they were restricted to a region close to the midline (presumably motor cortex) and were most likely attributable to the degeneration of corticostriatal connections. The extent of the contralateral excitability changes was clearly related to the size of the neocortical infarcts with large infarcts resulting in the widespread reduction of field potential amplitudes and an impairment of paired-pulse inhibition. The results show that there is a relatively large periinfarct region with decreased overall excitability after transient MCA occlusion which is likely to have a profound effect on perilesional processes involved in functional recovery. Remote excitability changes may contribute to the functional deficit and are probably related to deafferentation.     

Distinct physiologic properties of microglia and blood-borne cells in rat brain slices after permanent middle cerebral artery occlusion.

The authors investigated the time course of leukocyte infiltration compared with microglial activation in adult rat brain slices after permanent middle cerebral artery occlusion (MCAO). To distinguish peripheral leukocytes from microglia, the blood cells were prelabeled in vivo with Rhodamine 6G (Rhod6G) i.v. before induction of ischemia. At specific times after infarct, invading leukocytes, microglia, and endothelial cells were labeled in situ with isolectin (IL)B4-FITC (ILB4). Six hours after MCAO only a few of the ILB4+ cells were colabeled by Rhod6G. These cells expressed the voltage-gated inwardly and outwardly rectifying K+ currents characteristic of macrophages. The majority of the ILB4+ cells were Rhod6G- and expressed a lack of voltage-gated channels, recently described for ramified microglial cells in brain slices, or exhibited only an inward rectifier current, a unique marker for cultured (but unstimulated) microglia. Forty-eight hours after MCAO, all blood-borne and the majority of Rhod6G- cells expressed outward and inward currents indicating that the intrinsic microglial population exhibited physiologic features of stimulated, cultured microglia. The ILB4+/Rhod6G- intrinsic microglial population was more abundant in the border zone of the infarct and their morphology changed from radial to ameboid. Within this zone, the authors observed rapidly migrating cells and recorded this movement by time-lapse microscopy. The current findings indicate that microglial cells acquire physiologic features of leukocytes at a later time point after MCAO.