Microglia Negatively Regulate the Proliferation and Neuronal Differentiation of Neural Stem/Progenitor Cells Isolated from Poststroke Mouse Brains

Abstract

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.

Neonatal Brains Exhibit Higher Neural Reparative Activities than Adult Brains in a Mouse Model of Ischemic Stroke

“…Postnatal day 13 (P13) and 8-to-12-week-old wild-type mice (CB-17/Icr-+/+Jcl) were purchased from CLEA Japan (Tokyo, Japan). Permanent ischemic stroke was induced in neonatal [ 16 ] and adult mice [ 14 , 15 , 17 ], as previously described. Briefly, under isoflurane anesthesia, left MCAO was induced by electrocoagulation in adult and neonatal mice.…”

“…Ischemic volume was measured in the poststroke neonatal and adult mouse brains, as previously described [ 15 ]. Briefly, 1 day after MCAO, mice were anesthetized with a mixture containing medetomidine, midazolam, and butorphanol [ 14 , 17 ]. After removing the brains, 2 mm thick coronal sections were prepared.…”

“…Poststroke mice were deeply anesthetized and transcardially perfused with 4% PFA, as described previously [ 14 , 17 ]. Whole brains were removed and fixed with 4% PFA.…”

“…The ischemic and peri-ischemic areas were defined as regions within the border of the poststroke area and within a diameter of 100 around the poststroke area, respectively. Areas positive for nestin and DCX and the number of cells positive for Sox2 were measured in the ischemic and peri-ischemic areas of coronal brain sections (3 regions per section from 3 mice for 9 datapoints), as described previously [ 15 , 17 ].…”

“…To investigate the presence and properties of iNSPCs within ischemic areas, ischemic tissues were removed from neonatal (P13) and adult mice on poststroke day 7. Retrieved tissues were then dissociated and cultured under conditions that promote the formation of neurosphere-like cell clusters, as described previously [ 14 , 15 , 17 ]. Briefly, tissues were mechanically dissociated by sequential passage through 18-, 23-, and 27-gauge needles.…”