WeiGang Gu

Cortical Neurogenesis in Adult Rats After Transient Middle Cerebral Artery Occlusion

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 double-immunofluorescent 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, &bgr;-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.

Cortical Neurogenesis in Adult Rats after Reversible Photothrombotic Stroke

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 neuron-specific 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.

Early and delayed induction of immediate early gene expression in a novel focal cerebral ischemia model in the rat

This study aimed at evaluating changes in expression of immediate early genes in a new photothrombotic focal ischemia model that exhibits late spontaneous reperfusion and morphological restoration in the region‐at‐risk within the cerebral cortex. Gene expression was studied with Northern blots, in situ hybridization and immunohistochemistry. At early time points (1–4 h), nerve growth factor‐induced gene A and B, and c‐fos mRNAs, were quickly induced throughout the ipsilateral cortex, with no obvious differences between the region‐at‐risk and remote cortical areas. High concentrations of nerve growth factor‐induced gene A and c‐Fos proteins were present within the region‐at‐risk even when cortical cerebral blood flow was as low as 40% of control values. At 4 h the nerve growth factor‐induced gene A mRNA and protein expression was significantly decreased in the hippocampus vs. naive controls. However, a small decrease was also found in sham‐operated and anaesthetized controls. A late induction, at 5 days, of c‐fos and nerve growth factor‐induced gene B mRNAs was seen bilaterally in the hippocampus and also, in the case of nerve growth factor induced‐gene B, in the contralateral cortex. A complex pattern of changes in immediate early gene expression occurs after reversible focal cortical ischemia. This may be important for tissue recovery as well as neuropsychiatric symptoms after stroke.

A photothrombotic ring stroke model in rats with remarkable morphological tissue recovery in the region at risk

Abstract 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.

Long-term cortical CBF recording by laser-Doppler flowmetry in awake freely moving rats subjected to reversible photothrombotic stroke.

This study aimed at developing a laser-Doppler flowmetry (LDF) device suitable for long-term cortical cerebral blood flow (cCBF) measurement in awake, freely moving rats. The device included a flow probe adapter for permanent fixation to the skull bone and a connector that held the flow probe in the adapter in exactly the same position during repeated cCBF recordings. With this LDF recording system, cCBF values were stable and unaltered in awake, freely moving rats up to 4 days after operation compared with initial recordings during anesthesia. Repeated cCBF measurements in rats after transient removal and reattachment of the flow probe revealed a coefficient of variation of 7.0-17.4%. The LDF recording system was applied to rats subjected to a photothrombotic ring stroke lesion. cCBF in the region-at-risk declined to 59-34-26-33% of baseline values (P < 0.01) at 1-2-24 48 h after irradiation with gradually restored cCBF values of 56-87% at 72-96 h post-irradiation (P < 0.01 vs. 24 h). Transcardial carbon black perfusion examination of the brains confirmed the sustained hypoperfusion in the region at risk up to 48 h post-ischemia followed by a consistently occurring late spontaneous reperfusion. In conclusion, a novel laser-Doppler cortical CBF recording system has been set up that allows stable long-term cortical CBF follow-up in awake, freely moving rats.

Progressive and reproducible focal cortical ischemia with or without late spontaneous reperfusion generated by a ring-shaped, laser-driven photothrombotic lesion in rats

Clinical stroke is mostly of thromboembolic origin, in which the magnitude of brain damage resulting from arterial occlusions depends on the degree and duration of the concomitant ischemia. To facilitate more controllable and reproducible study of stroke-related pathophysiological mechanisms, a photothrombotic ring stroke model was initially developed in adult rats. The ring interior zone comprises an anatomically well confined cortical region-at-risk which is gradually encroached by progressive hypoperfusion, thus mimicking the situation (albeit in inverse fashion) of an ischemic penumbra or stroke-in-evolution. Modification of this model using a thinner ring irradiation beam resulted in late spontaneous reperfusion in the cortical region-at-risk and a remarkable morphological tissue recovery in this ostensibly critically injured region. On the other hand, doubling the thin irradiating beam intensity facilitates a complementary situation in which lack of reperfusion in the region-at-risk after stroke induction leads to tissue pannecrosis. The dual photothrombotic ring stroke model, effectuated either with or without reperfusion and thereby tissue recovery or pannecrosis, may be well suited for the study of events related to postischemic survival or cell death in the penumbra region. To popularize the photothrombotic ring stroke model, we present a detailed protocol of how this model is induced in either version as well as protocols for transcardial carbon black perfusion and laser-Doppler flowmetry experiments.

Neurotransmitter synthesis in poststroke cortical neurogenesis in adult rats.

Neurogenesis occurs in the cerebral cortex of adult rats after focal cerebral ischemia. Whether or not the newborn neurons could synthesize neurotransmitters is unknown. To elucidate such a possibility, a photothrombotic ring stroke model with spontaneous reperfusion was induced in adult male Wistar rats. The DNA duplication marker BrdU was repeatedly injected, and the rats were sacrificed at various times after stroke. To detect BrdU nuclear incorporation and various neurotransmitters, brain sections were processed for single/double immunocytochemistry and single/double/triple immunofluorescence. Stereological cell counting was performed to assess the final cell populations. At 48 h, 5 days, 7 days, 30 days, 60 days and 90 days after stroke, numerous cells were BrdU-immunolabeled in the penumbral cortex. Some of these were doubly immunopositive to the cholinergic neuron-specific marker ChAT or GABAergic neuron-specific marker GAD. As analyzed by 3-D confocal microscopy, the neurotransmitters acetylcholine and GABA were colocalized with BrdU in the same cortical cells. In addition, GABA was colocalized with the neuron-specific marker Neu N in the BrdU triple-immunolabeled cortical cells. This study suggests that the newborn neurons are capable of synthesizing the neurotransmitters acetylcholine and GABA in the penumbral cortex, which is one of the fundamental requisites for these neurons to function in the poststroke recovery.

A photothrombotic ring stroke model in rats with or without late spontaneous reperfusion in the region at risk

This study aimed at developing a dual setup of the photothrombotic ring stroke model with or without late spontaneous reperfusion in the region at risk and to explore the morphological consequences. The exposed crania of adult male Wistar rats were subjected to a ring-shaped laser-irradiation beam (o.d. 5.0 mm, 0.35 mm thick) for 2 min simultaneously with intravenous erythrosin B (17 mg/kg) infusion. Transcardial carbon-black perfusion revealed that a laser intensity of 0.90 W/cm(2) resulted in late, that is, starting at 72 h, spontaneous reperfusion, whereas the lowest laser intensity that produced lack of reperfusion at 7 days post-irradiation was 1.84 W/cm(2). Laser-Doppler flowmetry showed prompt cortical cerebral blood flow (cCBF) reduction both in the ring lesion and region at risk (12% and 25% of control values) after high-intensity irradiation; these reduced flow values were more rapid and pronounced than in the low-intensity irradiation setup as previously shown. The high- compared with low-intensity irradiation setup produced more frequent occurrence of thrombi in the ring-lesion region and a larger ischemic cortical lesion with a more rapid pace of ischemic cellular changes in the ring-lesion region and the region at risk. The region at risk transformed into pannecrosis in the high-intensity, but recovered morphologically in the low-intensity irradiation setup. This dual photothrombotic setup with or without spontaneous reperfusion enables the study of events related to ischemic cell survival or death in an anatomically predefined region at risk.

Real-time cortical cerebral blood flow follow-up in conscious, freely moving rats by laser Doppler flowmetry

This article describes a laser Doppler flowmetry (LDF) system that enables repeated measurements and thereby long-term followup of cortical cerebral blood flow (CBF) in awake and freely moving rats. The system consists of a specially designed flow probe adapter, a flow probe connector, and a LDF flow probe, which may thereby rotate through its own axis. During the experiment, the flow adapter is permanently mounted onto the rats skull bone. A thin layer of skull bone is left intact at the site for cortical CBF measurements. The probe connector and the flow probe may be repeatedly detached and remounted to the adapter, which allows for cortical cerebral blood flow recording from exactly the same anatomical location. The laser Doppler flowmetry system enables stable cortical CBF recordings in the conscious rat while it moves freely in a bowl cage.

Cell division in the cerebral cortex of adult rats after photothrombotic ring stroke.

Neurogenesis has been shown to occur in the cerebral cortex in adult rats after ischemic stroke. The origin of the newborn neurons is largely unknown. This study aimed to explore cell division in the poststroke penumbral cortex. Adult male Wistar rats were subjected to photothrombotic ring stroke. After repeated delivery of the DNA duplication marker BrdU, the animals were sacrificed at various times poststroke. BrdU was detected by immunohistochemistry/immunofluorescence labeling, as was the M-phase marker Phos H3 and the spindle components alpha-tubulin/gamma-tubulin. DNA damage was examined by TUNEL staining. Cell type was ascertained by double immunolabeling with the neuronal markers Map-2ab/beta-tubulin III and NeuN/Hu or the astrocyte marker GFAP. From 16h poststroke, BrdU-immunolabeled cells appeared in the penumbral cortex. From 24h, Phos H3 was colocalized with BrdU in the nuclei. Mitotic spindles immunolabeled by alpha-tubulin/gamma-tubulin appeared inside the cortical cells containing BrdU-immunopositive nuclei. Unexpectedly, the markers of neuronal differentiation, Map-2ab/beta-tubulin III/NeuN/Hu, were expressed in the Phos H3-immunolabeled cells, and NeuN was detected in some cells containing spindles. This study suggests that in response to a sublethal ischemic insult, endogenous cells with neuronal immunolabeling may duplicate their nuclear DNA and commit cell mitosis to generate daughter neurons in the penumbral cortex in adult rats.