Contribution of constitutively proliferating precursor cell subtypes to dentate neurogenesis after cortical infarcts

Abstract

Background: It is well known that focal ischemia increases neurogenesis in the adult dentate gyrus of the hippocampal formation but the cellular mechanisms underlying this proliferative response are only poorly understood. We here investigated whether precursor cells which constitutively proliferate before the ischemic infarct contribute to post-ischemic neurogenesis. To this purpose, transgenic mice expressing green fluorescent protein (GFP) under the control of the nestin promoter received repetitive injections of the proliferation marker bromodeoxyuridine (BrdU) prior to induction of cortical infarcts. We then immunocytochemically analyzed the fate of these BrdU-positive precursor cell subtypes from day 4 to day 28 after the lesion.

Results: Quantification of BrdU-expressing precursor cell populations revealed no alteration in number of radial glia-like type 1 cells but a sequential increase of later precursor cell subtypes in lesioned animals (type 2a cells at day 7, type 3 cells/immature neurons at day 14). These alterations result in an enhanced survival of mature neurons 4 weeks postinfarct.

Conclusions: Focal cortical infarcts recruit dentate precursor cells generated already before the infarct and significantly contribute to an enhanced neurogenesis. Our findings thereby increase our understanding of the complex cellular mechanisms of postlesional neurogenesis.

Figure 1

Infarct model, experimental design and quantification of the number of bromodeoxyuridine (BrdU)-positive cells in the subgranular zone. (A) Location and morphology of a photothrombotic infarct in the sensorimotor cortex on the brain surface (dotted line) and on a coronal BrdU-stained section at day 4 postsurgery. Scale bars represent 5 mm. (B) Schematic illustration of the experimental design. Prior to surgery all animals received intraperitoneal injections of the proliferation marker BrdU twice daily for 4 consecutive days and survived till day 4, 7, 14 or 28. (C) Immunohistochemically stained sections with antibodies against BrdU through the dentate gyrus of the hippocampus (two magnifications). (D) Diagram of the total number of BrdU-positive cells in the subgranular zone of the dentate gyrus at day 4, 7, 14 and 28. BrdU-positive cells significantly increase at day 14 and 28 postsurgery. Error bars represent S.E.M.. Significant differences (P < 0.05) are indicated by an asterisk.

Figure 2

Overview and distribution of distinct precursor cells in the dentate gyrus of the transgenic nestin-GFP mouse. (A) labelled for bromodeoxyuridine (BrdU; red), green fluorescent protein (GFP; green) and glial fibrillary protein (GFAP; blue). Scale bar represent 100 μm. (B) Schematic illustration of distinct precursor cell subtypes leading to new neurons in the dentate gyrus. Type 1 cells exhibit characteristic morphology with a triangle-shaped soma, long and strong apical processes reaching into the granular cell layer (GCL). These radial glia-like cells express precursor cell markers like nestin and additionally the astrocytic marker glial fibrillary protein (GFAP). Arising from type 1 cells transient amplifying progenitor cells (type 2) express nestin, have plump, short processes orientated parallel to the subgranular zone (SGZ). They do not express GFAP. Type 2 cells exist in two subtypes, one negative (type 2a) and one positive for doublecortin (DCX) (type 2b). While type 2 cells express the marker nestin, type 3 cells are only positive for doublecortin and comprise a transition from a proliferative stage to postmitotic immature neurons. After exit from the cell cycle the terminal post mitotic differentiation of granular cells start the NeuN marker expression for mature neurons. (C) Confocal images of double- and triple-labelled immunofluorescent sections showing the distinct cell types (marked with dotted rectangles). Scale bars represent 10 μm. SGZ, subgranular zone; GCL, granular cell layer.

Figure 3

Quantification of BrdU-positive precursor cell subtypes and newborn mature neurons in the dentate gyrus at day 4, 7, 14 and 28 after surgery. Note the increase of BrdU-labelled type 2a cells at day 7, type 3/immature neurons at day 14 and mature neurons at day 28 after focal infarcts. Error bars represent S.E.M.. Significant differences (P < 0.05) between the experimental groups are indicated by an asterisk.

Figure 4

Expression of different proliferation markers in type 2a cells at day 7 and type 3 cells/immature neurons at day 14 after surgery. (A) Schematic illustration of the expression of the proliferation markers BrdU (red) and Ki67 (blue) during the cell cycle. (B) Quantification of BrdU- and Ki67-positive type 2a cells at day 7 and type 3 cells/immature neurons at day 14. Error bars represent S.E.M.. Significant differences (P < 0.05) are indicated by an asterisk. (C, D) Confocal images of Ki67-expressing type 2a (C) and type 3 cells (D). Scale bars represent 10 μm. GFP: green fluorescent protein, DCX: doublecortin