An accumulation of evidence shows that endogenous neural stem/progenitor cells (NSPCs) are activated following brain injury such as that suffered during ischemic stroke. To understand the expression patterns of these cells, researchers have developed mice that express an NSPC marker, Nestin, which is detectable by specific reporters such as green fluorescent protein (GFP), i.e., Nestin-GFP mice. However, the genetic background of most transgenic mice, including Nestin-GFP mice, comes from the C57BL/6 strain. Because mice from this background strain have many cerebral arterial branches and collateral vessels, they are accompanied by several major problems including variable ischemic areas and high mortality when subjected to ischemic stroke by occluding the middle cerebral artery (MCA). In contrast, CB-17 wild-type mice are free from these problems. Therefore, with the aim of overcoming the aforementioned defects, we first crossed Nestin-GFP mice (C57BL/6 background) with CB-17 wild-type mice and then developed Nestin-GFP mice (CB-17 background) by further backcrossing the generated hybrid mice with CB-17 wild-type mice. Subsequently, we investigated the phenotypes of the established Nestin-GFP mice (CB-17 background) following MCA occlusion; these mice had fewer blood vessels around the MCA compared with the number of blood vessels in Nestin-GFP mice (C57BL/6 background). In addition, TTC staining showed that infarcted volume was variable in Nestin-GFP mice (C57BL/6 background) but highly reproducible in Nestin-GFP mice (CB-17 background). In a further investigation of mice survival rates up to 28 days after MCA occlusion, all Nestin-GFP mice (CB-17 background) survived the period, whereas Nestin-GFP mice (C57BL/6 background) frequently died within 1 week and exhibited a higher mortality rate. Immunohistochemistry analysis of Nestin-GFP mice (CB-17 background) showed that GFP⁺ cells were mainly obverted in not only conventional neurogenic areas, including the subventricular zone (SVZ), but also ischemic areas. In vitro, cells isolated from the ischemic areas and the SVZ formed GFP⁺ neurosphere-like cell clusters that gave rise to various neural lineages including neurons, astrocytes, and oligodendrocytes. However, microarray analysis of these cells and genetic mapping experiments by Nestin-CreERT2 Line4 mice crossed with yellow fluorescent protein (YFP) reporter mice (Nestin promoter-driven YFP-expressing mice) indicated that cells with NSPC activities in the ischemic areas and the SVZ had different characteristics and origins. These results show that the expression patterns and fate of GFP⁺ cells with NSPC activities can be precisely investigated over a long period in Nestin-GFP mice (CB-17 background), which is not necessarily possible with Nestin-GFP mice (C57BL/6 background). Thus, Nestin-GFP mice (CB-17 background) could become a useful tool with which to investigate the mechanism of neurogenesis via the aforementioned cells under pathological conditions such as following ischemic stroke.

Rehabilitative exercise following a brain stroke has beneficial effects on the morphological plasticity of neurons. Particularly, voluntary running exercise after focal cerebral ischemia promotes functional recovery and ameliorates ischemia-induced dendritic spine loss in the peri-infarct motor cortex layer 5. Moreover, neuronal morphology is affected by changes in the perineuronal environment. Glial cells, whose phenotypes may be altered by exercise, are known to play a pivotal role in the formation of this perineuronal environment. Herein, we investigated the effects of voluntary running exercise on glial cells after middle cerebral artery occlusion. Voluntary running exercise increased the population of glial fibrillary acidic protein-positive astrocytes born between post-operative days (POD) 0 and 3 on POD15 in the peri-infarct cortex. After exercise, transcriptomic analysis of post-ischemic astrocytes revealed 10 upregulated and 70 downregulated genes. Furthermore, gene ontology analysis showed that the 70 downregulated genes were significantly associated with neuronal morphology. In addition, exercise reduced the number of astrocytes expressing lipocalin 2, a regulator of dendritic spine density, on POD15. Our results suggest that exercise modifies the composition of astrocytic population and their phenotype.

Increasing evidence shows that the administration of mesenchymal stem cells (MSCs) is a promising option for various brain diseases, including ischemic stroke. Studies have demonstrated that MSC transplantation after ischemic stroke provides beneficial effects, such as neural regeneration, partially by activating endogenous neural stem/progenitor cells (NSPCs) in conventional neurogenic zones, such as the subventricular and subgranular zones. However, whether MSC transplantation regulates the fate of injury-induced NSPCs (iNSPCs) regionally activated at injured regions after ischemic stroke remains unclear. Therefore, mice were subjected to ischemic stroke, and mCherry-labeled human MSCs (h-MSCs) were transplanted around the injured sites of nestin–GFP transgenic mice. Immunohistochemistry of brain sections revealed that many GFP⁺ cells were observed around the grafted sites rather than in the regions in the subventricular zone, suggesting that transplanted mCherry⁺ h-MSCs stimulated GFP⁺ locally activated endogenous iNSPCs. In support of these findings, coculture studies have shown that h-MSCs promoted the proliferation and neural differentiation of iNSPCs extracted from ischemic areas. Furthermore, pathway analysis and gene ontology analysis using microarray data showed that the expression patterns of various genes related to self-renewal, neural differentiation, and synapse formation were changed in iNSPCs cocultured with h-MSCs. We also transplanted h-MSCs (5.0 × 10⁴ cells/µL) transcranially into post-stroke mouse brains 6 weeks after middle cerebral artery occlusion. Compared with phosphate-buffered saline-injected controls, h-MSC transplantation displayed significantly improved neurological functions. These results suggest that h-MSC transplantation improves neurological function after ischemic stroke in part by regulating the fate of iNSPCs.

We previously demonstrated that neural stem/progenitor cells (NSPCs) were induced within and around the ischemic areas in a mouse model of ischemic stroke. These injury/ischemia-induced NSPCs (iNSPCs) differentiated to electrophysiologically functional neurons in vitro, indicating the presence of a self-repair system following injury. However, during the healing process after stroke, ischemic areas were gradually occupied by inflammatory cells, mainly microglial cells/macrophages (MGs/MΦs), and neurogenesis rarely occurred within and around the ischemic areas. Therefore, to achieve neural regeneration by utilizing endogenous iNSPCs, regulation of MGs/MΦs after an ischemic stroke might be necessary. To test this hypothesis, we used iNSPCs isolated from the ischemic areas after a stroke in our mouse model to investigate the role of MGs/MΦs in iNSPC regulation. In coculture experiments, we show that the presence of MGs/MΦs significantly reduces not only the proliferation but also the differentiation of iNSPCs toward neuronal cells, thereby preventing neurogenesis. These effects, however, are mitigated by MG/MΦ depletion using clodronate encapsulated in liposomes. Additionally, gene ontology analysis reveals that proliferation and neuronal differentiation are negatively regulated in iNSPCs cocultured with MGs/MΦs. These results indicate that MGs/MΦs negatively impact neurogenesis via iNSPCs, suggesting that the regulation of MGs/MΦs is essential to achieve iNSPC-based neural regeneration following an ischemic stroke.

Brain injuries, such as ischemic stroke, cause cell death. Although phagocytosis of cellular debris is mainly performed by microglia/macrophages (MGs/MΦs), excessive accumulation beyond their phagocytic capacities results in waste product buildup, delaying brain cell regeneration. Therefore, it is essential to increase the potential for waste product removal from damaged brains. Lipocalin-type prostaglandin D synthase (L-PGDS) is the primary synthase for prostaglandin D2 (PGD2) and has been reported as a scavenger of waste products. However, the mechanism by which the L-PGDS–PGD2 axis exerts such an effect remains unelucidated. In this study, using a mouse model of ischemic stroke, we found that L-PGDS and its downstream signaling pathway components, including PGD2 and PGD2 receptor DP1 (but not DP2), were significantly upregulated in ischemic areas. Immunohistochemistry revealed the predominant expression of L-PGDS in the leptomeninges of ischemic areas and high expression levels of DP1 in CD36⁺ MGs/MΦs that were specifically present within ischemic areas. Furthermore, PGD2 treatment promoted the conversion of MGs/MΦs into CD36⁺ scavenger types and increased phagocytic activities of CD36⁺ MGs/MΦs. Because CD36⁺ MGs/MΦs specifically appeared within ischemic areas after stroke, our findings suggest that the L-PGDS–PGD2–DP1 axis plays an important role in brain tissue repair by regulating phagocytic activities of CD36⁺ MGs/MΦs.