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Purpose: The most common protocols in the initial diagnostic of acute ischemic stroke do not assess cardiogenic or aortic causes of embolism. These are usually evaluated later by transthoracic (TTE) or transesophageal (TEE) echocardiography. This study aimed to evaluate the feasibility of a diagnostic tool for thoracic cardiovascular thrombi according to the first experience with a new extended cardio-stroke protocol (Big 5-Jena eCS protocol) in acute stroke patients., Methods: Retrospective analyses of the tomography scans database of the Jena University Hospital were performed. We included a total of 67 patients in the feasibility analyses, based on the evaluation of three outcomes., Results: Primary outcome: the Big 5-Jena eCS protocol was able to detect thoracic cardiovascular thrombi in a total of 20 patients in different locations including the arch of the aorta, the aortic valve, the left atrium, the left atrial appendage, the left ventricle, and the pulmonary arteries. Secondary outcome: implementating the protocol did not result in a significant elevation of the radiation exposure compared to traditional protocols. Tertiary outcome: the new protocol identified seven cases that were considered negative by echocardiography., Conclusion: The implementation of an extended cardio-stroke protocol is feasible, no significantly time-consuming, acquiring assessable imaging, and maintaining radiation exposure acceptable. The Big 5-Jena eCS protocol was also able to detect some thrombi not reported by TTE or TEE; however, due to our data's explorative character, a conclusive comparison with cardiac ultrasound is not possible. A prospective pilot study and clinical trials should be conducted to assess the diagnostic accuracy of this protocol compared to echocardiography and determine the potential impact on diagnostic and treatment decisions., (© 2021. The Author(s).)

Stroke-induced immunodepression is a major risk factor for severe infectious complications in the immediate post-stroke period. We investigated the predictive value of heart rate variability (HRV) to identify patients at risk of post-stroke infection, systemic inflammatory response syndrome, or severe sepsis during the post-acute interval from days 3 to 5 after stroke onset. A prospective, observational monocentric cohort study was conducted in a university hospital stroke unit of patients with ischemic infarction in the territory of the middle cerebral artery without an ongoing infection at admission. Standard HRV indices were processed from Holter ECG. Recording started within the first day after the onset of stroke. Infection (primary endpoint: pneumonia, urinary tract, unknown localization) was assessed between days 3 and 5. The predictive value of HRV adjusted for clinical data was analyzed by logistic regression models and area under the receiver operating characteristic curve (AUC). From 287 eligible patients, data of 89 patients without event before completion of 24-h Holter ECG were appropriate for prediction of infection (34 events). HRV was significantly associated with incident infection even after adjusting for clinical covariates. Very low frequency (VLF) band power adjusted for both, the National Institutes of Health Stroke Scale (NIHSS) at admission and diabetes predicted infection with AUC = 0.80 (cross-validation AUC = 0.74). A model with clinical data (diabetes, NIHSS at admission, involvement of the insular cortex) performed similarly well (AUC = 0.78, cross-validation AUC = 0.71). Very low frequency HRV, an index of integrative autonomic-humoral control, predicts the development of infectious complications in the immediate post-stroke period. However, the additional predictive value of VLF band power over clinical risk factors such as stroke severity and insular involvement was marginal. The continuous HRV monitoring starting immediately after admission might probably increase the predictive performance of VLF band power. That needs to be clarified in further investigations.

Phosphoinositide 3-kinase γ (PI3Kγ) is linked to neuroinflammation and phagocytosis. This study was conducted to elucidate conjectural differences of lipid kinase-dependent and kinase-independent functions of PI3Kγ in the evolvement of brain damage induced by focal cerebral ischemia/reperfusion. Therefore, PI3Kγ wild-type, knockout, and kinase-dead mice were subjected to middle cerebral artery occlusion followed by reperfusion. Tissue damage and cellular composition were assessed by immunohistochemical stainings. In addition, microglial cells derived from respective mouse genotypes were used for analysis of PI3Kγ effects on phagocytic activity, matrix metalloproteinase-9 release, and cAMP content under conditions of oxygen/glucose deprivation and recovery. Brain infarction was more pronounced in PI3Kγ-knockout mice compared to wild-type and kinase-dead mice 48 h after reperfusion. Immunohistochemical analyses revealed a reduced amount of galectin-3/MAC-2-positive microglial cells indicating that activated phagocytosis was reduced in ischemic brains of knockout mice. Cell culture studies disclosed enhanced metalloproteinase-9 secretion in supernatants derived from microglia of PI3Kγ-deficient mice after 2-h oxygen/glucose deprivation and 48-h recovery. Furthermore, PI3Kγ-deficient microglial cells showed a failed phagocytic activation throughout the observed recovery period. Lastly, PI3Kγ-deficient microglia exhibited strongly increased cAMP levels in comparison with wild-type microglia or cells expressing kinase-dead PI3Kγ after oxygen/glucose deprivation and recovery. Our data suggest PI3Kγ kinase activity-independent control of cAMP phosphodiesterase as a crucial mediator of microglial cAMP regulation, MMP-9 expression, and phagocytic activity following focal brain ischemia/recirculation. The suppressive effect of PI3Kγ on cAMP levels appears critical for the restriction of ischemia-induced immune cell functions and in turn tissue damage.

Following cerebral injuries such as stroke, a structural and functional reorganization of the impaired tissue occurs, which is often accompanied by a re-expression of developmental genes. During brain development, embryonic splice variants of the GABA-synthesizing GAD67 gene (collectively termed EGAD) participate in cell proliferation, migration, and neuronal differentiation. We thus hypothesized an involvement of EGAD in post-ischemic plasticity. EGAD transcripts were up-regulated at early reperfusion times in the injured area following transient middle cerebral artery occlusion (with a peak expression of 4.5-fold at 6h in C57BL/6 mice). Cell-specific analysis by a combination of radioactive in situ hybridization and immunolabeling revealed EGAD up-regulation in TUNEL-positive neurons. This unexpected cell death-associated expression of EGAD was confirmed in cell culture models of ischemia (combined oxygen-glucose deprivation) and apoptosis (staurosporine). Staurosporine-mediated cell death led to cleaved Caspase-3 activation, a key regulator of apoptosis following stroke. Blocking of staurosporine-associated EGAD expression via antisense RNA treatment reduced cleaved Caspase-3 activation by ~30%. In addition to the involvement of EGAD in proliferative processes during brain development, we found here that EGAD participates in cell death under pathophysiological conditions in the adult brain. Re-expression of EGAD in neurons following stroke may play a role in aberrant cell cycle activation, consequently being pro-apoptotic. Our observation of a new GABA related pro-apoptotic mechanism and its successful modification might be of significant clinical relevance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The purpose of the present study was to determine whether a unilateral photothrombotic brain lesion induces bilateral ischemic tolerance towards a subsequent severe ischemia performed 5 days later. Severe ischemia was induced by transient (1h; t) or permanent (p) occlusion of the middle cerebral artery (MCAO). Rats were sacrificed 24h later. Preconditioning reduced the size of subsequent infarcts in both hemispheres. This effect was most prominent with tMCAO, and ipsilateral preconditioning was more effective than contralateral preconditioning (% of hemispheric volume, mean ± SD: 31.9 ± 3.7 to 19.0 ± 10.3 with ipsilateral tMCAO; 31.9 ± 3.7 to 22.9 ± 4.9 with contralateral tMCAO; 64.7 ± 4.3% to 47.2 ± 12.5% with ipsilateral pMCAO; 64.7 ± 4.3% to 53.1 ± 8.9% with contralateral pMCAO). Ischemic preconditioning was associated with a successive bilateral up-regulation of superoxide dismutases which may be involved in the development of ischemic tolerance. Our data suggest that a focal ischemic brain lesion induces neuroprotective mechanisms in extensive brain areas and thus cause bilateral ischemic tolerance within a certain time window., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Infection is the most important complication after acute stroke. This is substantially based on a stroke-induced immunosuppression. Heart rate variability (HRV) represents the autonomic nervous system activity in connection with stroke-induced immunomodulation and infections. We demonstrated in a feasibility study that HRV indices obtained in patients without acute post-stroke infections can predict infections in the subsequent sub-acute phase., Methods/design: The study PRED-SEP is a prospective observational study. Adult patients with acute ischemic infarction in the territory of the middle cerebral artery and severe neurological deficit (National Institutes of Health Stroke Scale: NIHSS ≥ 8) are recruited. Primary endpoint is the development of infections, secondary endpoints are SIRS and severe sepsis in the sub-acute phase (day 3-5) after stroke and the functional outcome after 3 months. Infection is defined according to the PANTHERIS study and comprises pneumonia, urinary tract infection and infections without determined focus. SIRS and severe sepsis are defined according to German Sepsis Society guidelines. Functional outcome is measured by lethality and neurological scores (modified Rankin Scale, Barthel Index). Prognostic factors are HRV risk indices calculated from selected intervals of 24 h ECG measurements within 48 hours after symptom onset. It is planned to recruit 240 patients.HRV risk indices (predictors) will be calculated according to standards and procedures previously developed and published by the authors. The predictive effects of HRV indices on infections will be estimated by fitting logistic regression models and estimating odds ratios with 95% confidence intervals. A prespecified modelling procedure will be applied to estimate unadjusted and confounder adjusted odds ratios. Secondary endpoints will be analysed in the same way. The functional outcome scales will be dichotomized. The association between HRV indices and pro- and anti-inflammatory markers will be quantified by calculating the appropriate correlation coefficients according to scale (Person or Spearman)., Discussion: Since a general prophylactic antibiotic treatment after stroke is not recommended, results of this study could have essential implications for an early identification and hence, timely appropriate treating of stroke-induced infections., Trial Registration: Prädiktoren für die Sepsis - Pred Sep, German Clinical Trials Register: DRKS00003392.

Background and Purpose: Tirofiban is a highly selective, fast-acting nonpeptide glycoprotein IIb/IIIa platelet receptor antagonist with a short half-life time. Glycoprotein IIb/IIIa antagonists are effective for the treatment of acute coronary syndromes proven in large clinical trials. Safety and efficacy in patients with ischemic stroke are uncertain. This was addressed in the Safety of Tirofiban in acute Ischemic Stroke (SaTIS) trial., Methods: Two hundred sixty patients with acute ischemic stroke were randomized in a placebo-controlled, prospective, open-label treatment, blinded outcome reading multicenter trial. Subjects with a National Institutes of Health Stroke Scale between 4 and 18 received intravenously either tirofiban or placebo within 3 to 22 hours after symptom onset for 48 hours. The primary end point was the rate of cerebral bleeding as measured in follow-up CT scans 2 to 7 days after inclusion. The secondary end point was clinical efficacy within 1 week (National Institutes of Health Stroke Scale, modified Rankin Scale) and after 5 months (Barthel Index, modified Rankin Scale)., Results: The rate of cerebral hemorrhagic transformation (I/II) and parenchymal hemorrhage (I/II) did not differ between both groups (tirofiban 36 of 120; placebo 33 of 124: OR, 1.18; 95% CI, 0.66 to 2.06). Mortality after 5 months was significantly lower in patients treated with tirofiban (3 of 130 [2.3%] versus 11 of 126 [8.7%]; OR, 4.05; 95% CI, 1.1 to 14.9). No difference in neurological/functional outcome was found after 1 week and after 5 months., Conclusions: We conclude that tirofiban might be safe in acute moderate ischemic stroke even when administered within a large time window after symptom onset and might save lives in the late outcome. Clinical Trial Registration- URL: www.strokecenter.org/trials/. Trial name: SaTIS. Enrollment began before July 1, 2005.

Background and Purpose: We aimed to validate the usefulness of the Essen Stroke Risk Score (ESRS) to predict stroke recurrence in a hospital-based follow-up study., Methods: We followed up 730 consecutive patients admitted to a neurological stroke unit in Berlin, Germany, with ischemic stroke (IS) or transient ischemic attack (TIA). The mean follow-up time was 13.4 months (SD 5.9). We calculated the time of event-free survival by Kaplan-Meier estimates., Results: The cumulative rate of a recurrent stroke was 10.4% (76 of 730). The risk of a recurrent stroke was significantly higher in patients with an ESRS >2 (p = 0.01; log rank). The area under the curve (AUC) was 0.59. The risk of a vascular event (VE), defined as a fatal or nonfatal IS or myocardial infarction, was significantly higher in patients with an ESRS >2 (p = 0.003; log rank). The AUC was 0.58. Stratifying the data at exact 1-year follow-up, 9 of 185 (10.3%) patients with an ESRS ≤2 suffered a recurrent stroke, while 56 of 311 (18%) patients with an ESRS >2 did so (p < 0.02; χ(2)). Thirty-six of 196 (18.4%) patients with an ESRS ≤2 suffered a VE, while 156 of 353 (35.7%) patients with an ESRS >2 did so (p < 0.001; χ(2))., Conclusions: The ESRS identifies groups of patients with TIA or IS who are at significantly increased risk for a recurrent stroke and cardiovascular event. Patients with a high ESRS require short-term follow-up and reevaluation of secondary prevention strategies., (Copyright © 2011 S. Karger AG, Basel.)

The clinical differentiation between stroke and seizure is usually straightforward but postictal neurological deficits can be mistaken for stroke in case no detailed medical history is available. Up to now, the imaging findings of Todd's paresis are not well described. This case report demonstrates that postictal paresis can be accompanied by a reversible global hemispheric hypoperfusion as measured with perfusion MRI indicating transient but profound cerebrovascular dysfunction in postictal paresis. Extensive postictal perfusion changes must be discriminated from emerging stroke to avoid potentially harmful therapy like thrombolysis. Further investigations are warranted to clarify the role of cerebrovascular dysfunction in the pathophysiology of postictal paresis., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: Aging is a major risk factor for a variety of neurobiological diseases leading to variations of transcriptional expression in affected tissues. Reverse transcription of RNA followed by quantitative PCR is a powerful technique for detection and quantification of specific transcripts differentially expressed. An essential prerequisite for accurate interpretation of quantitative PCR data obtained from expression studies is an appropriate normalization process. Therefore we validated the expression of the most frequently used reference genes consisting of Gapdh and Actb as well as Hmbs, Hprt1 and Gusb in an animal model of mice in respect to two major influence factors, aging and ischemia. In the experimental settings we intended to reflect variations in both, the local and systemic immune response., Results: The consistency in gene expression of the tested transcripts was quantified based on standard deviation, correlation analysis and two algorithms available as Visual Basic Applications (VBA) termed GeNorm and Normfinder. Overall, the results of the study proofed the suitability of Actb in combination with Gapdh and with tissue-specific limitations Hmbs in brain and Gusb in white blood cells as the most stable transcripts for accurate normalization. We clearly demonstrated that both, the aging process per se and aging in combination with ischemia are confounding factors with respect to the expression stability of Hprt1., Conclusions: The present study emphasizes the urgent need to validate the expression stability also from bona fide unvaried transcripts under specific conditions of investigation to ensure adequate normalization of qPCR data. Based on the expression stability, the use of Gapdh and Actb as highly abundant transcripts for normalization of qPCR data under conditions of aging and ischemia in a mouse model was evaluated. However, for low abundant genes the use of Hmbs in brain and Gusb in white blood cells is recommended., (Copyright (c) 2008 Elsevier Inc. All rights reserved.)

Rehabilitation therapy is known to drive motor improvement in stroke patients. However, the interplay of functional recovery and compensation in postischemic motor behavior is poorly understood. This study focused on the time course of functional recovery versus motor compensation in skilled forelimb movements after cerebral ischemia in rats. Young adult male rats underwent a focal cerebral ischemia by unilateral photothrombotic lesion of the motor cortex related to the preferred forelimb. In a first set of experiments animals were exposed to small cortical lesions comprising the forelimb motor cortex (n=8) or to larger lesions additionally extending into the hind limb motor area (n=8). In a second set of experiments animals with large lesion were either housed in standard (n=10) or enriched environment (n=14). Skilled reaching was assessed for 25 to 28 days postischemia. This task allows the distinction between recovery and compensation by parallel quantitative (reaching success) and qualitative (movement pattern) analysis. The results reveal that lesion size determines the initial magnitude of motor deficits, but not the degree of chronic impairments in movement pattern in all experimental groups. Compensatory movements represent the major mechanism of functional improvement and were accompanied by a partial functional restitution. Enriched environment facilitates effective compensation in skilled reaching, while it does not promote restitution of function. In particular, rotating movements of the forelimb during reaching were permanently impaired and required functional compensation through intensified use of the upper body. We conclude an activity dependent postischemic restoration of movement success. Enriched environment provides benefit by increased motor activity mainly due to compensation. Furthermore, these findings emphasize the power of comprehensive movement analysis to gain insight into recovery processes after stroke.

Cerebral ischemia activates endogenous neurogenesis in the subventricular zone (SVZ) and the dentate gyrus. Consecutively, SVZ-derived neural precursors migrate towards ischemic lesions. However, functional relevance of activated neurogenesis is limited by poor survival of new-born precursors. We therefore employed the HI-virus-derived fusion protein TAT-Bcl-x(L) to study the effects of acute anti-apoptotic treatment on endogenous neurogenesis and functional outcome after transient cerebral ischemia in mice. TAT-Bcl-x(L) treatment led to significantly reduced acute ischemic cell death (128+/-23 vs. 305+/-65 TUNEL+ cells/mm(2) in controls) and infarct volumes resulting in less motor deficits and improved spatial learning. It significantly increased survival of doublecortin (Dcx)-positive neuronal precursors (389+/-96 vs. 213+/-97 Dcx+ cells in controls) but did not enhance overall post-ischemic cell proliferation or lesion-specific neuronal differentiation 28 days after ischemia. Our data demonstrate that post-stroke TAT-Bcl-x(L)-treatment results in acute neuroprotection, improved functional outcome, and enhanced survival of lesion-specific neuronal precursor cells after cerebral ischemia in mice.

Background: Investigations following stroke first of all require information about the spatio-temporal dimension of the ischemic core as well as of perilesional and remote affected tissue. Here we systematically evaluated regions differently impaired by focal ischemia., Methodology/principal Findings: Wistar rats underwent a transient 30 or 120 min suture-occlusion of the middle cerebral artery (MCAO) followed by various reperfusion times (2 h, 1 d, 7 d, 30 d) or a permanent MCAO (1 d survival). Brains were characterized by TTC, thionine, and immunohistochemistry using MAP2, HSP72, and HSP27. TTC staining reliably identifies the infarct core at 1 d of reperfusion after 30 min MCAO and at all investigated times following 120 min and permanent MCAO. Nissl histology denotes the infarct core from 2 h up to 30 d after transient as well as permanent MCAO. Absent and attenuated MAP2 staining clearly identifies the infarct core and perilesional affected regions at all investigated times, respectively. HSP72 denotes perilesional areas in a limited post-ischemic time (1 d). HSP27 detects perilesional and remote impaired tissue from post-ischemic day 1 on. Furthermore a simultaneous expression of HSP72 and HSP27 in perilesional neurons was revealed., Conclusions/significance: TTC and Nissl staining can be applied to designate the infarct core. MAP2, HSP72, and HSP27 are excellent markers not only to identify perilesional and remote areas but also to discriminate affected neuronal and glial populations. Moreover markers vary in their confinement to different reperfusion times. The extent and consistency of infarcts increase with prolonged occlusion of the MCA. Therefore interindividual infarct dimension should be precisely assessed by the combined use of different markers as described in this study.

Astroglial proliferation and delayed neuronal death are two common pathological processes in the ischemic brain. However, it is not clear if astrogliosis causes delayed neuronal death. In this study, we addressed this potential linkage by examining the relationship between attenuated astrocyte proliferation, induced by cyclin-dependent kinase (CDK) inhibition, and delayed neuronal death in rat ischemic hippocampus. Our results show that following middle cerebral artery occlusion (MCAO), astrocyte hypertrophy and proliferation were closely associated with delayed neuronal death. Importantly, administration of olomoucine, a selective CDK inhibitor, not only suppressed astroglial proliferation and glial scar formation, but also decreased neuronal cell death in the ischemic boundary zone and hippocampal CA1 region at days 1 and 30 after MCAO. These results indicate that reactive astrogliosis and delayed neuronal death, at least in rat hippocampus, are sequential pathological events following MCAO. Therefore, suppressing astroglial cell cycle progression in acute focal cerebral ischemia may be beneficial to neuronal survival. Our study also implies that cell cycle regulation should be considered as a promising future therapeutic intervention in treating those neurological diseases characterized by an excessive astrocyte proliferation.

Purpose: The physiological function of the cellular prion protein (PrPC) is still unclear. A growing body of evidence suggests that PrPC has neuroprotective properties and that its deletion increases susceptibility to focal cerebral ischemia. The purpose of this study was to elucidate the role of PrPC overexpression in ischemic brain injury in vivo., Methods: PrPC overexpressing (TG35) and wild type (WT) mice were subjected to a 90-minute transient focal cerebral ischemia followed by infarct volume analysis 24 hours after lesion. To identify effects of PrPC overexpression on signalling pathways important for the regulation of ischemic cell death, we studied postischemic activation and expression of Akt and Erk1/2 using quantitative Western Blot analysis., Results: TG35 mice displayed significantly smaller infarct volumes and showed reduced early postischemic Erk1/2 phosphorylation, a pathway known to exacerbate neuronal injury following transient cerebral ischemia. In contrast, PrPC overexpression did not change postischemic Akt phosphorylation, which acts anti-apoptotic and is reduced in PrPC knockout animals., Conclusions: These results demonstrate that PrPC overexpression reduces deleterious Erk1/2 activation but does not affect Akt activation after transient cerebral ischemia, suggesting a role for distinct cytosolic signalling pathways in PrPC mediated neuroprotection.

Several types of CNS injury and various diseases are associated with the development of a glial scar. Astrocytes are major components of the glial scar. They are interconnected by gap junctions, with connexin43 (Cx43) being the most prominent channel protein. We applied a model of focal cerebral ischemia to study the spatio-temporal expression of glial fibrillary acidic protein, as well as of Cx43 mRNA and protein in gliotic tissue up to 60 days after injury. Reactive astrocytes enveloping the lesion up-regulated their Cx43 mRNA and protein. A band of reactive astrocytes filling in the lesion exhibited elevated Cx43 and showed a high degree of proliferation. Because of these findings, we hypothesize a role for Cx43 in glial scar formation, specifically in the proliferation of astrocytes.

Initial functional impairments after cerebral ischemia often improve considerably during the early period after the insult. Although pathological changes associated with post-lesion improvements have been widely investigated, it has not been resolved whether behavioral improvement represents true restoration of function (recovery) or development of new strategies (compensation). This study investigated whether early motor improvements after focal cerebral ischemia reflect recovery or compensation. Adult female Wistar rats were trained to retrieve food pellets in a skilled reaching task prior to receiving a unilateral cortical infarction induced by photothrombosis in forelimb motor cortex. Animals were continuously tested in the reaching task up to 3 weeks after lesion. The end point measures revealed that reaching success rates remained at pre-lesion levels, however, qualitative analysis of reaching movements indicated permanent changes in forelimb movement patterns. Similar observations were made in a skilled walking task and a test for forelimb asymmetry. These data indicate that lesion animals adopted alternative movement strategies in order to successfully perform the tasks. The changes in postoperative performance were compared to anatomical data in individual animals. The finding that reaching success was not related to lesion size supports the idea that the degree of adaptive behavior after cortical ischemia depends on plastic properties of the remaining intact tissue.

1alpha,25-(OH)(2)-vitamin-D(3) (1,25-D(3)) and 17beta-estradiol are both known to act neuroprotective in certain experimental in vitro and in vivo settings. We studied the effects of 1,25-D(3) or 17beta-estradiol or their combined application on heat shock protein-27 (HSP-27) distribution after focal cortical ischemia using the photothrombosis model. HSP-27 is a well-established marker of the cerebral oxidative stress response and a potent inhibitor of apoptosis. Lesioned rats were injected i.p. one hour after injury with either 1 microg 1,25-D(3)/kg or 7 microg 17beta-estradiol/kg or a combination of both steroids. Groups of non-lesioned steroid-treated rats and lesioned, solvent-treated rats served as controls. Treatment with both steroids did not affect the size of the lesion. In addition, 17beta-estradiol resulted in significant reduction of HSP-27 induction, whereas the combination of 1,25-D(3)+17beta-estradiol resulted in a highly significant reduction of HSP-27 within the infracted cerebral cortex, indicating that both steroids act synergistically in a protective manner.

Delayed neuronal death (DND) of pyramidal neurons in the CA1 and CA3 regions of the hippocampus has been extensively studied following global brain ischemia, whereas only little is known about DND in this highly vulnerable brain region after focal brain ischemia. In the present study, the distribution and time course of hippocampal neuronal apoptosis were studied following transient middle cerebral artery occlusion (MCAO) in rats 1, 3, 7, 14, and 30 days after the insult. In 60% of the animals, more than 90% of CA1 pyramidal neurons showed strong nick-end labeling (TUNEL) staining at day 3 with fragmentation and marginalization of the nuclei in approximately 40% of these cells. The number of TUNEL-positive cells decreased within the next days, but 30 days after MCAO, some apoptotic neurons were still present. Analysis of the expression of the glial cell line-derived neurotrophic factor (GDNF) and its receptors GFRalpha1, GFRalpha2, and GFRalpha3 using triple immunofluorescence and confocal laser scanning microscopy revealed that in all animals showing marked hippocampal DND, the neuronal staining for GFRalpha1, GFRalpha3, and GDNF decreased prior to the onset of TUNEL staining in CA1. After 7 days, some apoptotic neurons still expressed GFRalpha3, whereas only few showed GFRalpha1 immunoreactivity, indicating that GFRalpha1 may be beneficial for the survival of hippocampal neurons. The data suggest that reduced expression of GDNF and impairment of GFRalpha1/3 may contribute to hippocampal DND after focal brain ischemia.

Background and Purpose: The most important effect of cerebral ischemia is brain infarction. In this magnetic resonance imaging (MRI) study, the authors aimed at assessing postischemic brain atrophy., Methods: Ten patients suffering from their first acute cerebral ischemia in the territory of the middle cerebral artery were studied retrospectively. Three-dimensional MRI volume scans were recorded in the acute and chronic stage after infarction and analyzed voxel by voxel intraindividually with the newly developed voxel-guided morphometry., Results: Shrinkage of brain tissue was detected in all patients, not only in the perilesional cortical structures but also in contralateral homolog cortex areas and subcortically in the striatum and thalamus. This secondary shrinkage was not related to the size of the infarcts or to the clinical outcome of patients., Conclusions: Our study suggests that delayed brain atrophy after acute ischemic stroke involved areas anatomically connected with the ischemic brain lesion but nevertheless was accompanied by a simultaneous improvement of the neurological deficit.

1alpha,25-(OH)(2)-vitamin D(3) (1,25-D(3)) and 17beta-estradiol are both known to act neuroprotectively in certain experimental in vitro and in vivo settings and it has been noted that both steroids lead to an upregulation of certain neurotrophic factors. Here, we studied the effects of 1alpha,25-(OH)(2)-vitamin D(3) or 17beta-estradiol or their combined application on heat shock protein-32 (HSP-32) distribution after focal cortical ischemia using the well established photothrombosis model. Heat shock protein-32 is a well-established marker of the cerebral oxidative stress response and contributes to neuroprotection by metabolising cytotoxic free heme to carbon monoxide, iron and biliverdin. Photothrombotically lesioned rats were injected i.p. 1h after injury with either 1 microg 1alpha,25-(OH)(2)-vitamin D(3)/kg or 7 microg 17beta-estradiol/kg or a combination of both steroids. Groups of non-lesioned steroid-treated rats and lesioned, solvent-treated rats served as controls. In contrast to non-lesioned rats, in lesioned animals a significant increase in heat shock protein-32 expression occurred which was slightly, but non-significantly altered in the groups treated either with 1alpha,25-(OH)(2)-vitamin D(3) or 17beta-estradiol alone when compared to the solvent-treated control group. Only the combined treatment with 1alpha,25-(OH)(2)-vitamin D(3) and 17beta-estradiol resulted in a significant reduction of glial heat shock protein-32 immunoreactivity within the lesion-remote cortical areas supplied by the affected middle cerebral artery (MCA), indicating that both steroids act synergistically in a protective manner.

In the surrounding of focal ischemic brain lesions dysfunctional neuronal zones emerge often resulting in pathological oscillatory activity. Using whole-head magnetoencephalography we recorded brain activity during rest in 23 patients with ischemic cortical lesions to find out whether we can localise and characterise low-frequency oscillatory activity. We measured patients at different times after stroke and partly in a follow-up approach to determine the time course of slow-wave activity. Using the analysis tool Dynamic Imaging of Coherent Sources we computed tomographic maps of oscillatory power in the delta-band (0.5-3 Hz). Fifteen of 23 patients with cortical strokes showed delta-activity, which was localised in an area not more than 2 cm away from the lesion. We found this perilesional low-frequency activity in the acute as well as in the chronic stage of stroke. Follow-up measurements of individual patients revealed persistence of perilesional low-frequency activity for months and even years. No consistent relation between perilesional activity and clinical symptoms was observed. Our results indicate that perilesional delta activity is common after ischemic cortical stroke. However, the functional significance remains to be elucidated.

Focal cerebral lesions in rat brain induced by photothrombosis lead to an impaired inhibitory neurotransmission. A reduced gamma-aminobutyric acid (GABA)-mediated inhibition has been revealed by electrophysiological recordings associated with a diminished immunostaining of GABA handling proteins. Changes were found in ipsi- as well as in contralateral brain areas. Inhibition is mediated by interneurons using GABA as neurotransmitter. These cells use GAD (glutamate decarboxylase) to synthesize GABA. To analyze the vulnerability of GABAergic neurons in rats with a lesioned hindlimb area, cells expressing GAD65/67 mRNA were labeled using in situ hybridization. Positive somata were counted 7 and 30 days after focal ischemia in different cortical (hindlimb cortex, frontal cortex, primary and secondary somatosensory cortex) and hippocampal subsectors (pyramidal cell layer, stratum oriens and stratum radiatum/lacunosum-moleculare). The lesioned hemispheres were compared with the intact brain sides and with control brains. GABAergic interneurons survived the injury for up to 30 days in all investigated brain regions. Therefore it is unlikely that a loss of GABAergic neurons contributes to the reduced inhibition.

Objective: To detect perfusion reductions in patients with acute cerebral infarcts using near-infrared spectroscopy (NIRS) with indocyanine green (ICG) as tracer., Methods: Kinetics of an intravenous bolus of ICG were monitored by NIRS in 13 patients with acute infarction in the territory of the middle cerebral artery (mean (SD) age, 62.2 (13.0) years) and 12 controls (64.2 (9.1) years) at 2.8 (2.8) days after onset. NIRS optodes were placed bitemporally, with an interoptode distance of 4-5 cm. Absolute concentration changes in ICG were calculated. The following were assessed: time to peak, maximum ICG concentration, time interval between 0% and 100% maximum ICG concentration (interval), rise time (time between 10% and 90% ICG maximum), slope (maximum Delta ICG/interval), and blood flow index (BFI = maximum Delta ICG/rise time) of each hemisphere. Intraindividual differences were calculated between the two hemispheres., Results: Patients with ischaemic stroke had increased time to peak (p<0.01), interval (p<0.01), and rise time (p<0.01), while maximum ICG concentration (p<0.03), slope (p<0.01), and BFI (p<0.01) were diminished at the site of infarction compared with the unaffected hemisphere. In stroke patients, intraindividual differences in time to peak (p<0.001), interval (p<0.001), rise time (p = 0.001), maximum ICG concentration (p<0.02), slope (p<0.001), and BFI (p<0.001) were greater than in the controls, with excellent sensitivity and specificity for Delta time to peak (100% and 100%, respectively) and Delta time interval (100% and 91.7%)., Conclusions: Measurement of interhemispheric differences in ICG kinetics by NIRS detects perfusion reductions in patients with acute middle cerebral artery infarction. This non-invasive bedside test is rapid, repeatable, without major side effects, and avoids transportation of critically ill patients.

Background and Purpose: After focal cerebral ischemia, depending on its localization and extent, secondary neuronal damage may occur that is remote from the initial lesion. In this study differences in secondary damage of the ventroposterior thalamic nucleus (VPN) and the reticular thalamic nucleus (RTN) were investigated with the use of different ischemia models., Methods: Transient middle cerebral artery occlusion (MCAO) leads to cortical infarction, including parts of the basal ganglia such as the globus pallidus, and to widespread edema. Photothrombotic ischemia generates pure cortical infarcts sparing the basal ganglia and with only minor edema. Neuronal degeneration was quantified within the ipsilateral RTN and VPN 14 days after ischemia. Glial reactions were studied with the use of immunohistochemistry., Results: MCAO resulted in delayed neuronal cell loss of the ipsilateral VPN and RTN. Glial activation occurred in both nuclei beginning after 24 hours. Photothrombotic ischemia resulted in delayed neuronal cell loss only within the VPN. Even 2 weeks after photothrombotic ischemia, glial activation could only be seen within the VPN., Conclusions: Pure cortical infarcts after photothrombotic ischemia, without major edema and without effects on the globus pallidus of the basal ganglia, only lead to secondary VPN damage that is possibly due to retrograde degeneration. MCAO, which results in infarction of cortex and globus pallidus and which causes widespread edema, leads to secondary damage in the VPN and RTN. Thus, additional RTN damage may be due to loss of protective GABAergic input from the globus pallidus to the RTN or due to the extensive edema. Retrograde degeneration is not possible because the RTN, in contrast to the VPN, has no efferents to the cortex.

Structural and functional reorganization in the vicinity of damaged neocortex and other connected brain areas seems to be responsible for postlesional functional recovery. To better understand the molecular mechanisms underlying this type of plasticity, gene expression patterns were analyzed by using DNA macroarrays comprising 1176 genes. Circumscribed unilateral infarcts consistently affecting the forelimb area of the motor cortex were induced photochemically in adult rats. Ten days after lesioning, cortical gene expression fingerprints were evaluated from an area adjacent to the lesion as well as two contralateral areas of motor and somatosensory cortex. Discrete regions showed distinct expression patterns. Upregulation was observed of different members of transcription factors, immediate early genes, neuronal signaling as well as neuronal growth and structure-associated genes, ipsilaterally (six genes) and/or contralaterally (eight genes in the motor and seven in the somatosensory cortex). In contrast, downregulations were restricted to ipsilateral areas and included genes coding for ion channels, transport proteins, mediators of metabolic pathways, and intracellular transducers (14 genes). A subset of these regulations were further confirmed by real-time polymerase chain reaction (TaqMan assay). At least part of the detected regulations, in particular those of the contralateral hemisphere, are likely to underlie plasticity processes.

Neuronal nitric oxide-I is constitutively expressed in approximately 2% of cortical interneurons and is co-localized with gamma-amino butric acid, somatostatin or neuropeptide Y. These interneurons additionally express high amounts of glutamate receptors which mediate the glutamate-induced hyperexcitation following cerebral injury, under these conditions nitric oxide production increases contributing to a potentiation of oxidative stress. However, perilesional nitric oxide synthase-I containing neurons are known to be resistant to ischemic and excitotoxic injury. In vitro studies show that nitrosonium and nitroxyl ions inactivate N-methyl-D-aspartate receptors, resulting in neuroprotection. The question remains of how these cells are protected against their own high intracellular nitric oxide production after activation. In this study, we investigated immunocytochemically nitric oxide synthase-I containing cortical neurons in rats after unilateral, cortical photothrombosis. In this model of focal ischemia, perilesional, constitutively nitric oxide synthase-I containing neurons survived and co-expressed antioxidative enzymes, such as manganese- and copper-zinc-dependent superoxide dismutases, heme oxygenase-2 and cytosolic glutathione peroxidase. This enhanced antioxidant expression was accompanied by a strong perinuclear presence of the antiapoptotic Bcl-2 protein. No colocalization was detectable with upregulated heme oxygenase-1 in glia and the superoxide and prostaglandin G(2)-producing cyclooxygenase-2 in neurons. These results suggest that nitric oxide synthase-I containing interneurons are protected against intracellular oxidative damage and apoptosis by Bcl-2 and several potent antioxidative enzymes. Since nitric oxide synthase-I positive neurons do not express superoxide-producing enzymes such as cyclooxygenase-1, xanthine oxidase and cyclooxygenase-2 in response to injury, this may additionally contribute to their resistance by reducing their internal peroxynitrite, H(2)O(2)-formation and caspase activation.

Metabotropic glutamate receptors (mGluRs) seem to be involved both in neuronal excitotoxicity evoked by pathological conditions such as ischemia, and in processes associated with neuronal plasticity and regulation of synaptic strength. Using semiquantitative reverse transcription-polymerase chain reaction, we measured the messenger RNA levels of mGluR2-5, 24 h and 7 days after experimentally induced focal cortical infarcts in rat motor cortex. The mRNA level of mGluR3 was strikingly decreased ipsilaterally after 24 h. On day 7, the expression of mGluR2 was down-regulated both ipsi- and contralaterally. Other receptors of this family showed either a slight or no regulation at both time points. The early changes in the expression pattern of mGluRs might be primarily linked to excitotoxicity processes which occur immediately after an ischemic lesion in cortex, whereas the delayed changes might represent one chain link in the cascade of compensatory mechanisms contributing to functional amelioration.

The effect of the neuroprotective drug lubeluzole on cortical receptor binding was investigated in animals with photothrombotic ischemic lesions. Control animals were treated with the inactive stereoisomer of the drug. Lubeluzole was applied intravenously as a single bolus (0.31 mg/kg) followed by a 1-h infusion of 0.31 mg/kg. Lubeluzole selectively increased gamma-amino-butyric acid(A) (GABA(A)) receptor binding but had no significant and/or consistent effects on N-methyl-D-aspartate (NMDA), (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and kainate receptors. Lubeluzole caused a significant up-regulation of GABA(A) receptor binding in the lesioned area as well as in unimpaired cortical areas of both hemispheres. This effect appeared in the hours following the lesion and peaked at 24 h. Our findings suggest that reduced cortical excitability brought about by increased binding capacities of GABA(A) receptors may contribute to the neuroprotective effect of lubeluzole.

Heme oxygenase-1, an inducible heat shock protein, is upregulated by oxidative stress, and its expression is modulated by proinflammatory cytokines such as IL-1 and IL-6. In the present study, we investigated the effects of postlesional, orally applied ebselen, a neuroprotective antioxidant, on serum levels of IL-6 and cerebral heme oxygenase-1 expression following focal ischemia induced by photothrombosis. Ebselen (50 mg/kg body weight) was given 30 min postlesion to male Wistar rats. Animals were divided into four groups: sham-operated vehicle control (n = 9), sham-operated ebselen control (n = 8), lesioned vehicle control (n = 14), and lesioned ebselen-treated (n = 17). Ebselen treatment resulted in a significant lowering of IL-6 plasma levels (26 +/- 5 pg/ml) as compared with that seen in lesioned vehicle controls (48 +/- 9 pg/ml) at 24 h postlesion. In sham-operated rats IL-6 was not detectable. Heme oxygenase-1-positive glial cells were quantitated within topographically determined perilesional brain regions. Within the 0.5-mm-wide rim region directly associated with the lesion core, no differences in the amount of heme oxygenase-1-positive glial cells were found. However, in the more remote ipsilateral perilesional cortex, significantly fewer heme oxygenase-1-positive glial cells were present within the supragranular cortical layers of lesioned ebselen-treated rats compared to lesioned vehicle controls (P < 0.001). In sham-operated rats, no glial heme oxygenase-1 induction occurred. The results indicate that postlesional ebselen treatment lowered plasma IL-6 levels subsequent to a photothrombotic lesion concomitant with a lowering of the heme oxygenase-1 response in glial cells., (Copyright 2000 Academic Press.)

Transient and permanent focal cerebral ischemia results in a series of typical pathophysiologic events. These consequences evolve in time and space and are not limited to the lesion itself, but they can be observed in perilesional (penumbra) and widespread ipsi- and sometimes contralateral remote areas (diaschisis). The extent of these areas is variable depending on factors such as the type of ischemia, the model, and the functional modality investigated. This review describes some typical alterations attributable to focal cerebral ischemia using the following classification scheme to separate different lesioned and perilesional areas: (1) The lesion core is the brain area with irreversible ischemic damage. (2) The penumbra is a brain region that suffers from ischemia, but in which the ischemic damage is potentially, or at least partially, reversible. (3) Remote brain areas are brain areas that are not directly affected by ischemia. With respect to the etiology, several broad categories of remote changes may be differentiated: (3a) remote changes caused by brain edema; (3b) remote changes caused by waves of spreading depression; (3c) remote changes in projection areas; and (3d) remote changes because of reactive plasticity and systemic effects. The various perilesional areas are not necessarily homogeneous; but a broad differentiation of separate topographic perilesional areas according to their functional state and sequelae allows segregation into several signaling cascades, and may help to understand the functional consequences and adaptive processes after focal brain ischemia.

Functional as well as structural reorganization takes place in the surrounding and remote brain areas after focal ischemic lesions. In particular, reactive or regenerative processes have been described to occur in the contralateral hemisphere. We used mRNA differential display to gain more insight into the molecular mechanisms underlying this type of neuronal plasticity. Circumscribed unilateral infarcts consistently affecting the forelimb area of the primary motor cortex were induced photochemically in adult male Wistar rats. The lesion produced significant behavioral asymmetry with subsequent partial recovery within 1 week. Cloning the genes with altered expression profiles identified the 20S proteasome subunit C2 as a gene whose expression level is decreased in contralateral homotopic cortex. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed approximately twofold lower proteasome C2 mRNA levels in the lesion group as compared with the sham-operated group. The proteasome serves as the central enzyme of non-lysosomal protein degradation. It is responsible for intracellular protein turnover and is critically involved in a variety of regulation processes, such as cell cycle, metabolism and differentiation. Our results suggest that proteasome activity may play also a role in contralateral cortical plasticity occurring after focal cerebral ischemia.

Following focal brain lesions, complex adaptive processes take place in remote intact areas. The present study examines changes in NMDA (N-methyl-D-aspartate), AMPA ((+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and kainate receptors following focal photothrombotic ischemic lesions using quantitative receptor autoradiography. Increases in binding density of NMDA receptors were seen in both hemispheres for up to 30 days. In the contralateral hemisphere, this increase of NMDA receptors occurred as early as 4 h after lesion whereas it appeared with a delay for 14 days on the lesioned side. Binding density of [3H]AMPA and [3H]kainate was unchanged. We suggest that the translational process is differentially regulated by spreading depressions. The delayed up-regulation of NMDA receptor binding on the lesioned side may be due to a translation block similar to that previously described for GABA(A) receptor subunits.

Remote changes in brain function following stroke are called diaschisis. These remote effects may contribute to the neurological deficit following brain infarction; in addition they may lead to post-stroke epilepsy and affect functional recovery. In the present study we addressed the question of whether an increase in excitability can be observed contralateral to middle cerebral artery (MCA) infarction. Permanent occlusion of the middle cerebral artery (MCAO) was induced experimentally in rats with an intraluminal silicon-coated filament. Seven days later, brain excitability was tested with extracellulare recording techniques in neocortical coronal brain slices using a paired-pulse stimulus protocol. In rats with MCAO, excitability was increased in the neocortex contralateral to the infarction compared with the control group. These alterations extended through wide parts of the contralateral neocortex. The study demonstrates that MCAO causes transcallosal electrophysiological diaschisis. Together with results obtained previously with photothrombotic cortical lesions, it can be concluded that these remote effects are not due to characteristics of the individual lesion model, but are common consequences of brain lesions.

Focal cortical lesions are associated with a functional downregulation of the GABAergic system in perilesional tissue lasting (at least) several weeks. The molecular mechanisms underlying this phenomenon are still poorly understood. Here we used RT-PCR to investigate whether mRNA-levels of two alpha-subunits of the GABAA-receptor (alpha1- and alpha2-subunits) change following ischemic cortical lesioning. The results show that 7 days after lesion induction mRNA-levels for both the alpha1- and alpha2-subunits are increased threefold in perilesional tissue ipsilateral, but not contralateral to the lesion. Taken together with the results of a previous immunohistochemical study in which a moderate decrease of the alpha1-subunit-protein and no change for the alpha2-subunit [T. Neumann-Haefelin, J.F. Staiger, C. Redecker, K. Zilles, J.M. Fritschy, H. Mohler, O.W. Witte, Immunohistochemical evidence for dysregulation of the GABAergic system ipsilateral to photochemically induced cortical infarcts in rats. Neuroscience (Oxford) 87 (4) (1998) 871-879] was observed, this is interpreted as a partial block of translation in the perilesional tissue surrounding cortical ischemic lesions., (Copyright 1999 Elsevier Science B.V.)

It has been shown that cortical lesions are associated with an increase of excitability in surrounding brain regions, and with a downregulation of GABA(A) receptors. In the present study we investigated whether this increased excitability affects the cortical map of inputs represented in areas surrounding the lesioned brain area. Focal lesions with a diameter of 2-2.5 mm were induced photochemically in the hindlimb area at the border of the primary somatosensory cortex of the rat. One week after lesioning, the cortical representation of the B3 vibrissa was studied using 14C-deoxyglucose (DG) autoradiography. In all animals mechanical stimulation of the B3 vibrissa produced a column-shaped DG-labeling in the somatosensory cortex, corresponding to the B3-barrel with a maximum of the glucose uptake in layer IV. In control animals without cortical lesions (n=6), stimulation increased the glucose uptake rate by 50.8+/-10.5% in layer IV. In lesioned animals (n=6) maximum DG-uptake in layer IV (54.8+/-8.6%) did not differ significantly from that in controls. However, as compared to control animals, lesioned animals showed also increased glucose uptake within the activated column in layers II/II (51.+/-11.1%, lesioned animals; 31.8+/-11.2%, controls; P<0.05, lesioned vs. control) and V (47.5+/-5.8%, lesioned animals, 28.8+/-10.5%, controls; P<0.05, lesioned vs. control). The diameter of the metabolically activated B3-barrel area of layer IV was expanded from 461.8+/-77.6 microm in control animals to 785.5+/-103.6 microm; P<0.01) in lesioned animals. Lesioned animals also showed expansion of the activated area in layers II/III (890.4+/-134.8 microm, lesioned animals; 430.6+/-95.1 microm, controls; P<0.01) and layer V (1117.5+/-163.6 microm, lesioned animals; 648.7+/-114.1 microm, controls; P<0.01). The depth profile of the activation columns showed a maximum in layer IV in control animals, which was expanded towards layers II/III and layer V in lesioned animals. It is concluded that cortical lesions alter the representational area of neighboring afferent inputs through disinhibition or 'unmasking' of pre-existing silent or ineffectual intracortical synapses. The present observations raise the possibility that the brain supports recovery from lesions by decreasing GABAergic inhibition, thereby facilitating a remapping of the cortical representation in neighboring brain areas.

Copper/zinc-superoxide dismutase (Cu/Zn-SOD) belongs to a class of enzymes, identified as essential and highly effective endogenous scavengers of cytotoxic oxygen radicals. These radicals contribute to postlesional neurotoxicity. In order to determine the superoxide-scavenging potential of regions affected by unilateral cortical photothrombosis, we studied the changes in the distribution of Cu/Zn-SOD and the appearance of activated microglia by immunohistochemistry and isolectin B4 binding. Four hours postlesion, Cu/Zn-SOD increased significantly within a homotopic area of the contralateral hemisphere and in ipsilateral thalamic nuclei, whereas isolectin B4-positive microglia were upregulated at days 5 and 7 postlesion within the same regions. The contralateral increase in the amount of the superoxide-scavenging Cu/Zn-SOD indicates that this enzyme is induced by a retrograde reaction carried through callosal connections.

The effect of an ischaemic focal cortical lesion on the excitability of surrounding and remote brain areas was investigated. Infarcts were produced photothrombotically in rat frontal cortex and brain excitability was assessed by a extracellular paired-pulse stimulation in coronal slices 7 days later. The cortical lesions caused a reduction of inhibition. The extent and grade of these electrophysiological effects depended on the depth of the lesion: in animals with a lesion affecting the deeper cortical layers a pronounced transcortical diaschisis was found, whereas animals with a shallow lesion showed only a slight ipsilateral affliction. The study shows that focal lesions in the motor cortex cause widespread disinhibition, probably resulting from deafferentation, and these may have a significant impact on recovery of function.

Global cerebral ischemia leads to long lasting hyperexcitability and a reduced protein synthesis in non-infarcted tissue surrounding the lesion. In this study we investigated whether protein synthesis inhibition by pharmacological means itself changes neocortical excitability. Two hours after the last of three i.p. injections with the protein synthesis inhibitor cycloheximide (12 h interval, 1.5 mg/kg body weight) we observed a widespread reduction of neocortical inhibition. The study indicates that inhibition of protein synthesis may contribute to the altered brain excitability following ischemia.

In this study we examined the time course of apoptotic cell death after photochemically induced focal ischemia of the rat cerebral cortex. For unequivocal differentiation between apoptosis and necrosis two criteria of programmed cell death were used: terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and morphological evidence of fragmentation and marginalization of nuclei. After photothrombosis, many TUNEL-positive cells were found within the infarct region from 12 h to 3 days. By day 6 they were preferentially located in the boundary zone of the infarct, and by day 14 they had disappeared. A high proportion of TUNEL-positive cells displayed fragmentation or marginalization of their nuclei, indicating apoptosis. Neurons, but not T cells and macrophages, were apoptotic. Inflammatory infiltrates were in close contact to apoptotic neurons throughout the infarct areas at day 1 and in the boundary zone between days 2 and 6 after photothrombosis. In summary, our study shows that neuronal apoptosis after cerebral ischemia is a prolonged process to which leukocyte-derived cytokines may contribute. In contrast to autoimmune diseases of the nervous system, termination of the local inflammatory response after cerebral ischemia does not involve apoptosis.

This study describes local immune responses in cerebral ischemia induced by permanent occlusion of the middle cerebral artery (MCAO) in the rat. The temporal and spatial pattern of leukocyte infiltration was characterized immunocytochemically using monoclonal antibodies against CD5, a pan T cell marker, against CD4 and CD8 for subtyping of T lymphocytes, and ED1, a marker for macrophages. CD5+ T cells were present in some animals on the pial surface at day 1 and with increasing numbers mainly at the edges of the infarcts at days 3 and 7. By day 14 their number had significantly decreased. Subtyping of T lymphocytes revealed that CD4+ helper/inducer T cells were rare, while CD8+ lymphocytes were abundant. Moreover, CD8+ lymphocytes outnumbered CD5+ T cells indicating the presence of CD5-/CD8+ natural killer (NK) cells. ED1+ macrophages primarily infiltrated the core of the infarct starting on day 1. Infiltrating leukocytes expressed leukocyte function associated antigen-1 and MHC class I and II antigens. Early after infarction, increased expression of the intercellular adhesion molecule-1 was found on vessels and leukocytes. In conclusion, this study shows that lymphocytes enter the nervous system not only in autoimmune diseases, but also in response to primarily 'non-immune' neuronal damage such as stroke.