Increased subventricular zone-derived cortical neurogenesis after ischemic lesion
Research Highlights
►Using transgenic mice and BrdU injections we labeled all post-stroke-generated SVZ neuroblasts. ►Stroke enhances SVZ neurogenesis in ipsi and contralateral hemispheres. ►SVZ neuroblasts migrate as a stream to the injury zone. ►SVZ-derived neurons survive in the peri-infarct zone at least for 35 days. ►Post-stroke-generated SVZ neuroblasts differentiate into mature cortical neurons.
Introduction
Stroke, one of the most common causes of death and disability in adulthood, is triggered by occlusion of a cerebral artery leading to brain infarction. The resulting functional neurological deficits are mainly due to the subsequent massive loss of neurons, suggesting that cell replacement therapy might reduce the long-term consequences in stroke patients.
There is an increasing body of evidence that stroke, which experimentally can be induced in rats and mice by transient or permanent middle cerebral artery occlusion, can trigger neurogenesis in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (Arvidsson et al., 2002, Dempsey et al., 2003, Jin et al., 2001, Komitova et al., 2005, Parent et al., 2002, Zhang et al., 2001), reviewed in (Wiltrout et al., 2007).
Under physiological conditions, neuroblasts generated in the SVZ migrate tangentially along the rostral migratory stream (RMS) to the olfactory bulb where they continue to migrate radially to reach their final destination and differentiate into GABAergic periglomerular and granule neurons (Ghashghaei et al., 2007, Khodosevich et al., 2009, Lledo et al., 2006). Neurogenesis in the SGZ gives rise to glutamatergic granule cells in the dentate gyrus (Abrous et al., 2005, Kempermann et al., 2004). Ischemic brain injury can increase neurogenesis in both neurogenic regions and promote migration of neuroblasts to the ischemic boundary in the cortex and striatum (Arvidsson et al., 2002, Jin et al., 2001, Parent et al., 2002). In the striatum it has been described that these newborn neuroblasts differentiate into GABAergic medium spiny neurons or calretinin-positive GABAergic aspiny inhibitory interneurons (Arvidsson et al., 2002, Hou et al., 2008, Liu et al., 2009, Parent et al., 2002, Yang et al., 2008) and that the latter are integrated into neuronal networks receiving synaptic input and firing action potentials (Hou et al., 2008, Yamashita et al., 2006). In contrast, relatively little is known about the fate of SVZ-derived neuroblasts that are directed to the cortex following cortical stroke.
In the present study we analyzed the contribution of SVZ progenitor cells to cortical neurogenesis in a cortex-specific stroke model caused by middle cerebral artery occlusion (MCAO) using 5HT3A-EGFP transgenic mice, where EGFP is expressed under promoter of serotonin receptor 5HT3A (Inta et al., 2008). In these mice all SVZ-derived doublecortin-positive cells (= neuroblasts) are EGFP-labeled. Upon maturation EGFP expression decreases in the majority of neurons, but persists in those neurons, that normally express the 5HT3A receptor (e.g., periglomerular cells in olfactory bulb or layer 1 cortical interneurons). In addition, EGFP never co-localizes with glial markers. Thus, by combining EGFP detection and BrdU injections we were able to follow the migratory routes of post-stroke-generated SVZ-derived neuroblasts. We found an increase in post-stroke-generated neuroblasts in the SVZ of ischemic mice in both the ipsilateral and contralateral hemisphere. However, while the increase in neuroblast generation in the ipsilateral SVZ could be detected already 14 days after stroke, in the contralateral SVZ it was observed only 35 days after stroke. In the peri-ischemic zone, 1% and 2% of all BrdU-positive cells were EGFP/BrdU double-positive neuroblasts/neurons 14 days and 35 days after stroke, respectively. In the cortex the majority of EGFP/BrdU double-positive neuroblasts survived for at least 35 days after stroke. Many of them expressed mature neuronal marker NeuN and were not labeled by apoptotic marker activated caspase-3. In the frontal cortex, a portion of post-stroke generated SVZ-derived neurons co-expressed calretinin, a marker for a GABAergic interneuron subclass.
Section snippets
Animals
For all our experiments, we used 5HT3A-EGFP transgenic mice (Inta et al., 2008). All procedures with animals were performed according to the Heidelberg University Animal Care Committee.
Materials and reagents
The following antibodies were used in the study: rabbit anti-EGFP (1:10000; Molecular Probes, USA), chicken anti-EGFP (1:1500; Abcam, UK), mouse anti-calretinin (1:1000; Swant, Switzerland), rabbit anti-calretinin (1:1500; Swant, Switzerland), rabbit anti-activated caspase-3 (1:1000; R&D Biosystems, USA), rat
Generation of SVZ neuroblasts is enhanced after ischemic stroke
Although several studies demonstrated an increase in SVZ neurogenesis after ischemic stroke, they did not directly estimate the increase in SVZ-derived neuroblasts in the cortex (Arvidsson et al., 2002, Jiang et al., 2001, Jin et al., 2003, Leker et al., 2007, Parent et al., 2002, Zhang et al., 2006, Zhang et al., 2001, Ziv et al., 2007). We investigated cortical neurogenesis after stroke combining BrdU injections and EGFP detection in SVZ-derived neurons. BrdU injections allow the labeling of
Discussion
In the present study we investigated post-stroke neurogenesis and its contribution to the generation of neurons in the cortex using the MCAO model in 5HT3A-EGFP transgenic mice with EGFP expression in SVZ-derived neurons. These transgenic mice in combination with BrdU injections provided us a novel tool to track SVZ-derived neuroblasts produced during the first days after stroke. We found that following stroke the neuroblast production in the SVZ is enhanced in both the ipsilateral and
Acknowledgments
This work was supported in part by the DFG (SFB488 grant), the BMBF (RUS 09/B38), and European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreements 201024 and 202213 (European Stroke Network).
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