Leptomeningeal-derived doublecortin-expressing cells in poststroke brain

Abstract

Increasing evidence indicates that neural stem/progenitor cells (NSPCs) reside in many regions of the central nervous system (CNS), including the subventricular zone (SVZ) of the lateral ventricle, subgranular zone of the hippocampal dentate gyrus, cortex, striatum, and spinal cord. Using a murine model of cortical infarction, we recently demonstrated that the leptomeninges (pia mater), which cover the entire cortex, also exhibit NSPC activity in response to ischemia. Pial-ischemia-induced NSPCs expressed NSPC markers such as nestin, formed neurosphere-like cell clusters with self-renewal activity, and differentiated into neurons, astrocytes, and oligodendrocytes, although they were not identical to previously reported NSPCs, such as SVZ astrocytes, ependymal cells, oligodendrocyte precursor cells, and reactive astrocytes. In this study, we showed that leptomeningeal cells in the poststroke brain express the immature neuronal marker doublecortin as well as nestin. We also showed that these cells can migrate into the poststroke cortex. Thus, the leptomeninges may participate in CNS repair in response to brain injury.

FIG. 1.

Immunohistochemistry (A) showed that nestin- and vimentin-positive cells were present in the poststroke pia mater and cortex (nestin: B, D, E, green; vimentin: C–E, red; DAPI: B–E, blue). Three days after plating in the cell-differentiation-inducing medium, some of the nestin-positive cells isolated from the poststroke pia matter coexpressed DCX (nestin: F, H, green; DCX: G, H, red; DAPI: F–H, blue). DCX-positive cells were also confirmed by RT-PCR (I) and western blot (J) analyses. Scale bar: 100 μm (B) and 50 μm (E and F). Results shown are representative of 5 replicates of the experimental protocol. DAPI, 4′,6-diamino-2-phenylindole; DCX, doublecortin; L1, cortical layer 1; P, pia mater; RT-PCR, reverse transcription–polymerase chain reaction.

FIG. 2.

DCX-positive cells were observed near nestin-positive cells in the poststroke pia mater and cortex (DCX: A, red; nestin: A, green; DAPI: A, blue), although they were not observed in the nonischemic ipsilateral pia mater and cortex (DCX: B, red; nestin: B, green; DAPI: B, blue). Some pial cells, which were labeled with a GFP-expressing vector (C, GFP: D, E, green; laminin: E, red; DAPI: D, E, blue), migrated into the poststroke cortex after ischemia where they expressed nestin (arrows) (GFP: F, green; nestin: F, red; DAPI: F, blue) and DCX (arrows) (GFP: G, green; DCX: G, red; DAPI: G, blue). However, the number of DCX-positive cells in the poststroke pia mater and cortex decreased gradually at later time points (H). To investigate the potential contribution of leptomeningeal cells to cortical repair, brain slices were incubated in a medium that accelerated the proliferation of pial iNSPCs. Although DCX immunohistochemistry using the DAB reaction showed that DCX-positive cells were not observed in the nonischemic ipsilateral pia mater and cortex (I), it was confirmed that DCX-positive cells could survive and proliferate in the poststroke pia mater and cortex (J) as well as in the expanding nestin-positive cells (nestin: K, M, green; DCX: L, M, red; DAPI: K–M, blue). Scale bar: 100 μm (D, I, and K) and 50 μm (A, E, and F). Results shown are representative of 5 replicates of the experimental protocol. DAB, diaminobenzidine; GFP, green fluorescent protein; iNSPCs, ischemia-induced neural stem/progenitor cells.