Neurogenesis occurs throughout life in the dentate gyrus of hippocampus and subventricular zone, but this phenomenon has rarely been observed in other brain regions of adult mammals. The aim of the current study was to investigate the cell proliferation process in the ischemically challenged region-at-risk after focal cerebral ischemia in the adult rat brain. A reversible photothrombotic ring stroke model was used, which features sustained hypoperfusion followed by late spontaneous reperfusion and a remarkable morphologic tissue recovery in the anatomically well defined somatosensory cortical region-at-risk. Twelve-week-old male Wistar rats received repeated intraperitoneal injections of the cell proliferation specific marker 5-bromodeoxyuridine (BrdU) after stroke induction. Immunocytochemistry of coronal brain sections revealed that the majority of BrdU-positive cells were of glial, macrophage, and endothelial origin, whereas 3% to 6% of the BrdU-positive cells were double-labeled by BrdU and the neuronspecific marker Map-2 at 7 and 100 days after stroke onset in the region-at-risk. They were distributed randomly in cortical layers II-VI. Three-dimensional confocal analyses of BrdU and the neuronal-specific marker Neu N by double immunofluorescence confirmed their colocalization within the same cells at 72 hours and 30 days after stroke induction. This study suggests that, as a potential pathway for brain repair, new neurons can be generated in the cerebral cortex of adult rats after sublethal focal cerebral ischemia.

Background and Purpose-This study explored the possible occurrence of newly generated nerve cells in the ischemic cortex of adult rats after middle cerebral artery occlusion and reperfusion. Methods-Nine-to 10-week-old male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion by the monofilament method. Rats received repeated intraperitoneal injections of the cell proliferation-specific marker 5-bromodeoxyuridine (BrdU) after stroke induction. Brain sections were processed for immunohistochemistry with an avidin-biotin complex-alkaline phosphatase and/or -peroxidase method. Brain sections processed with doubleimmunofluorescent staining were further scanned by confocal microscopy. Results-Interspersed among the predominantly newly formed glial cells, some cells were double labeled by BrdU and 1 of the neuron-specific markers, Map-2, ␤-tubulin III, and Neu N, at 30 and 60 days after stroke onset. These cells were randomly distributed throughout cortical layers II through VI, occurring with highest density in the ischemic boundary zone. Three-dimensional confocal analyses of BrdU and the neuron-specific marker Neu N confirmed their colocalization within the same cortical cells. Conclusions-This study suggests that new neurons can be generated in the cerebral cortex of adult rats after transient focal cerebral ischemia. Cortical neurogenesis may be a potential pathway for brain repair after stroke.

The photothrombotic ring stroke model with sustained underperfusion followed by late spontaneous reperfusion (Gu et al. 1999) was employed to study its morphological consequences. The exposed crania of adult male Wistar rats were subjected to a ring-shaped laser irradiation beam simultaneously with intravenous erythrosin B infusion. The ischemic volume was calculated from serial sections throughout the ischemic lesions at 4, 10, 24, 48, and 72 h and 7 days and 28 days after irradiation. The ischemic volume, expressed as a percentage of the ipsilateral hemispheric volume, increased steadily from 4 to 10 to 24 h to reach its maximum value at 48 h after irradiation; at 3 days, 7 days, and 28 days, the ischemic volume was reduced to 75%, 24%, and 22% of the value at 48 h. Evaluation of ischemic volumes at different anteroposterior levels revealed that the reduced ischemic volume at 72 h and later was mainly due to morphological restoration in the centrally located, nonirradiated region at risk. An initial enlargement and development of cystic coagulation necrosis was observed in the cortical areas corresponding to the ring lesion itself. In the region at risk, a gradually deteriorating neuropil and nerve cell morphology were observed over time, with maximum severity at 48 h postirradiation. At this time, most laminae II and III neurons in the region at risk exhibited eosinophilia and pyknosis but no incrustations, with small islands of less damaged neurons randomly scattered. At 72 h and up to 28 days after irradiation, these cell characteristics were no longer observed and the region at risk was well populated with neurons that had a chiefly unremarkable cytological appearance. Neuronal counts in the central part of the region at risk were performed; no significant difference in neuronal density was observed between sham-operated controls and at 28 days after irradiation. In conclusion, the consistent, late spontaneous reperfusion coincided with remarkable tissue recovery as assessed morphologically in the region at risk. The data suggest that nerve cell repair may occur even after the detection, by conventional morphological methods, of prolonged critical ischemic neuronal damage in the setting of acute ischemic stroke.

In clinical thromboembolic stroke, spontaneous late recanalization is a common feature, but one which has been very sparsely studied experimentally. This study aimed at enabling the study of spontaneous reperfusion and exploring its consequences by modifying a recently developed photothrombotic-stroke model that focuses on the region-at-risk located within an ischemic ring-locus. The exposed crania of male Wistar rats (280-340 g) were subjected to a ring-shaped (5.0 mm outer diameter and 0.35 mm thick) laser-irradiation beam (514.5 nm; 0.89 W/cm2) for 2 min simultaneously with intravenous erythrosin B (17 mg/kg) infusion for 30 s. Transcardial carbon-black perfusion experiments revealed a ring-shaped cortical perfusion deficit at 4 h post-irradiation, which progressively increased at 10, 24, and 48 h, at which time the whole region-at-risk was pale with single distal branches of the middle cerebral artery being extensively narrowed, but not occluded. At 72 h, spontaneous reperfusion was observed in the region-at risk, which was even more pronounced at 7 and 28 days. Cortical cerebral blood flow (cCBF), measured by laser-Doppler flowmetry, was distinctly reduced at 2 min post-irradiation and further decreased slightly during 4 h of recording to ca. 24% of baseline values at the ring locus and 40% in the region-at-risk. In the region-at-risk, cCBF flow values were 23-30% of the baseline at 2448 h post-irradiation, followed by a relative cCBF increase to 71 and 77% at 72 and 96 h post-irradiation. Brain water content in the ischemic part of the cortex increased steadily from 4 to 48 h post-irradiation; at 72 h, it leveled off and returned to control values at 7 days. In conclusion, by employing a laser beam in the shape of a thin ring, critically sustained cCBF reduction was followed by late, consistent spontaneous reperfusion in the region-at-risk in this novel photochemically induced stroke-in-evolution model.

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