Regenerative response in ischemic brain restricted by p21cip1/waf1 - PubMed (original) (raw)

Regenerative response in ischemic brain restricted by p21cip1/waf1

Jianhua Qiu et al. J Exp Med. 2004.

Abstract

Neural precursor cells from adults have exceptional proliferative and differentiative capability in vitro yet respond minimally to in vivo brain injury due to constraining mechanisms that are poorly defined. We assessed whether cell cycle inhibitors that restrict stem cell populations in other tissues may participate in limiting neural stem cell reactivity in vivo. The cyclin-dependent kinase inhibitor, p21cip1/waf1 (p21), maintains hematopoietic stem cell quiescence, and we evaluated its role in the regenerative response of neural tissue after ischemic injury using the mice deficient in p21. Although steady-state conditions revealed no increase in primitive cell proliferation in p21-null mice, a significantly larger fraction of quiescent neural precursors was activated in the hippocampus and subventricular zone after brain ischemia. The hippocampal precursors migrated and differentiated into a higher number of neurons after injury. Therefore, p21 is an intrinsic suppressor to neural regeneration after brain injury and may serve as a common molecular regulator restricting proliferation among stem cell pools from distinct tissue types.

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Figures

Figure 1.

Increased cell proliferation in p21−/− brain tissues after MCAO. BrdU was injected into the littermate or age-matched p21−/− or p21+/+ mice on day 7 and 8 or day 14 and 15; and animals were killed 9 d or 16 d after MCAO for 20 min. Immunohistochemistry was performed on free-floating 40-μm coronal sections pretreated by denaturing DNA, and a specific stereological analysis (18) was applied to enumerate the labeled cells per volume as detailed in Materials and Methods. A few BrdU-labeled cells were detected in the noninjured p21−/− and p21+/+ mice. There was an increased BrdU labeling (black cells) in the region corresponding to the SGZ of dentate gyrus in hippocampus (A) or the SVZ in lateral ventricle (B) after MCAO. Mean values from multiple experiments are summarized in the graphs under each histological picture. The noninjured samples are marked as control in the graph. Black and white bars indicate mean values ± SD from p21−/− and p21+/+ genotype, respectively. A significant increase of BrdU-labeled cells in both genotypes and a larger fraction of BrdU-positive cells in p21−/− genotype was observed 9 d after MCAO (P < 0.01, n = 5, assessed by ANOVA with Bonferroni's post hoc analysis). To further confirm the increased cell proliferation, the injured brain tissue sections were incubated with anti-PCNA antibody, and second antibody was labeled with Rhodamine X (red). The PCNA-expressing cells were significantly more abundant in p21−/− SVZ than in p21+/+ SVZ (color pictures in B). The color images were taken with confocal microscopy.

Figure 1.

Figure 2.

Up-regulation of p21 expression in brain tissues after MCAO. RT-PCR for p21 or GAPDH was performed on the noninjured and injured (MCAO) tissues from hippocampus (Hipp) or subventricular zone (SVZ) 4 d after MCAO. An enhanced intensity of p21 expression was noted on the RT-PCR gels, and there was a significant increase of p21 mRNA levels if normalized to the transcript of a housekeeping gene (GAPDH) after MCAO (black bars) in both anatomic sites (two experiments, two pairs of animals in each experiment) as shown in the graph. Numbers above the gels indicate samples from different individual animals. The noninjured samples are marked as control in the graph. Detailed RT-PCR conditions are described in the methods.

Figure 3.

Expression of the neuronal migration marker (DCX) by the proliferating cells in SGZ 1 wk after MCAO. BrdU was injected on day 7 and 8, and animals were killed on day 9 after MCAO. Immunohistochemistry was performed on floating 40-μm coronal sections pretreated by denaturing DNA, and a specific stereological analysis (18) was applied to enumerate the labeled cells per volume as detailed in Materials and Methods. Brain sections were stained for BrdU immunoreactivity (cy2, green) with the neuronal migration indicator, DCX (cy3, red) and examined in SGZ of dentate gyrus with confocal microscopy for BrdU/DCX-coexpressing cells (merged, yellow) (A). A significant increase of BrdU/DCX double-positive cells in p21−/− SGZ especially after MCAO was visualized among the different samples (B). The morphology of the neuroblast cell expressing both BrdU and DCX is shown by a single cell in two-dimension (C) and three-dimension (D). Based on the data from multiple experiments, these differentiating cells positive for DCX/BrdU are significantly more in p21−/− SGZ than in p21+/+ SGZ (P < 0.01, n = 4, assessed by ANOVA with Bonferroni's post hoc analysis) (E). The black and white bars in the graphs indicate mean values ± SD from the p21−/− and p21+/+ genotype, respectively. The noninjured samples are marked as control and Day 9 indicates 9 d after MCAO in the graph (E).

Figure 3.

Figure 4.

Expression of the neuronal maturation markers (NeuN and Calbindin) by the proliferated cells in GCL 5 wk after MCAO. BrdU was injected on day 7 and 8, and animals were killed on day 9 or 35 after MCAO. Brain sections were stained for BrdU immunoreactivity (cy2, green) and the neuronal differentiation marker (cy3, red) NeuN (A) or calbindin (C) and examined with confocal microscopy. Immunohistochemistry was performed on floating 40-μm coronal sections pretreated by denaturing DNA and a specific stereological analysis (18) was applied to enumerate the labeled cells per volume as detailed in Materials and Methods. NeuN was extensively examined on day 9 and 35 after MCAO. 9 d after MCAO, most BrdU-positive cells in SGZ did not express NeuN (A, first column). Strikingly, 35 d after MCAO a significant increase of BrdU/NeuN double-positive cells in both genotypes and a larger fraction of BrdU/NeuN-positive cells in p21−/− brain was observed and relocated from SGZ to GCL after MCAO (P < 0.01, n = 5, assessed by ANOVA with Bonferroni's post hoc analysis) (A and B). The black and white bars in the graphs indicate mean values ± SD from p21+/+ and p21−/− genotype, respectively. The noninjured samples are marked as control and MCAO indicates 35 d after MCAO in the graph. Calbindin was used as an independent differentiation marker showing its coexpression in with BrdU in the cells located in GCL 35 d after injury (C, merged yellow color a representative image from the p21−/− brain).

Figure 5.

No difference of the cell proliferation in SGZ between p53−/− and p53+/+ mice. Identical methods were used as described in the study on BrdU incorporation in p21−/− or p21+/+ mice (Fig. 1), and there was no difference of BrdU incorporation between p53−/− and p53+/+ in SGZ either before or after MCAO (P > 0.05, n = 5, assessed by ANOVA with Bonferroni's post hoc analysis). Mean values from multiple experiments are summarized in the graph under the histological picture. White and black bars in the graphs indicate mean values ± SD from p53−/− and p53+/+ genotype, respectively. The noninjured samples are marked as control in the graph.

Figure 6.

No difference in the levels of FGF-2 growth factor in hippocampus between p21−/− and p21+/+ mice. FGF-2 concentration in hippocampus before and 1 wk after MCAO was measured by EIA as described in Materials and Methods. Black and white bars in the graph indicate mean values ± SD from p21−/− and p21+/+ samples, respectively. Five animals in each group were used in this experiment.

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Regenerative response in ischemic brain restricted by p21cip1/waf1 - PubMed (original) (raw)