The aim of the present study was to characterize the poststroke neural proliferation in this new mouse model of neonatal ischemic strokes by studying the trends as a function of time. The areas of interest were neurogenic niches in the ipsilateral injured and contralateral uninjured SGZ and in the ipsi- and contralateral SVZ. This study has four findings: 1) In line with the age-dependent decrease of neurogenesis detected in rats (Kuhn et al., 1996
; Seki and Arai, 1995
), control CD-1 mice showed age-dependent reduction in BrdU-labeled cells both in the SGZ and in the SVZ, which at P33 was ~50% of the counts at P19. 2) In ligated mice, SGZ cell proliferation was persistently negatively modulated at all three time points investigated, suggesting long-term impairment of the process in the injured hippocampi. In contrast, contralateral uninjured hippocampi showed proliferation rates comparable to control. 3) In ligated mice, SVZ cell proliferation was higher than control at 7 DAI ipsilaterally but returned to control levels by 21 DAI. 4) Percentage cell type distribution profiles in the SGZ did not change drastically between ligated and control mice for nestin- or DCX-positive newborn cells. However, a significantly higher percentatge of GFAP-positive BrdU-labeled cell counts was noted ipsilaterally at 21 DAI. In summary, as noted by others investigators, SVZ cell proliferation was enhanced in response to the neonatal insult (Arvidsson et al., 2002
; Goings et al., 2004
; Plane et al., 2004
; Hayashi et al., 2005
), whereas SGZ neural proliferation was lowered ipsi- but not contralaterally.
Prior studies with this model of unilateral ischemia unexpectedly found that, in addition to ipsilateral SGZ neurogenesis, contralateral neurogenesis in the SGZ was also negatively modulated for cells born 1 week after ischemia that survived maturation and integration for 3 weeks following BrdU labeling. These findings could have resulted from either decreased SGZ neural proliferation or decreased survival of the newborn neurons. From the present study, we can conclude that the previously reported long-term decrease in SGZ neurogenesis results, at least in large part, from a poststroke decrease in SGZ neural proliferation ipsilaterally. We also wanted to know whether this decrease in SGZ neural proliferation was a transient or sustained phenomenon at temporally distant time points as the neonatal brain matured. The study reported here reveals that the comparable proliferation rates seen contralaterally may indicate poor maturation and survival over time. Ipsilaterally, SGZ poststroke neural proliferation remained low at all three time points; this suggests that therapeutic measures introduced after the stroke that are also neurally proliferative could boost ipsilateral SGZ neurogenesis and help prevent loss of new neurons during maturation contralaterally.
With the current study, the SVZ neural proliferation at 7 DAI was clearly higher than control, ipsi- more than contralaterally, indicating that SVZ neural proliferation was enhanced for a specific period after the insult in the model. We can conclude based on these results that the poststroke increased long-term SVZ-derived neurogenesis previously reported results in large part from a poststroke increase in neural proliferation. This transient poststroke enhancement in neural proliferation, together with findings from our chronic survival studies showing migration of SVZ-derived BrdU-labeled cells into the injured cortex and striatum (Kadam et al., 2008
), is encouraging. Future studies aimed at modulating the cell fate commitment or survival of the newborn cells could take advantage of this window, which likely starts soon after the stroke but returns to control levels by P26. In addition, the age-dependent decline of neuroblast proliferation rates seen in controls and mirrored in the contralateral uninjured hemispheres of ligated mice further defines the advantageous window for intervention present in the developing brain with a higher capacity for neuroregeneration compared with a juvenile brain.
BrdU is available for ~30 min (Packard et al., 1973
) after an injection, so only a proportion of dividing cells is labeled by the single-injection protocol used in this study. Also, the duration of the S phase for proliferative cells in the dentate gyrus of the mouse has been estimated to be ~8 hr (Nowakowski et al., 1989
). Therefore, by perfusing the mouse brains at 2 hr following the BrdU injection, we labeled the pool of dividing progenitor cells that were in the DNA synthetic phase of the cell cycle for ~1.5–2 hr prior to fixation. Because this pool of BrdU-labeled cells would not have had adequate time to show mature cell commitment markers, early progenitor cell markers (nestin, GFAP, and DCX) that label the type 1, 2a, 2b, and 3 cells (Kempermann et al., 2004
) were examined. These cell types represent the first four of six total developmental milestones of hippocampal neurogenesis, with type 1 cells (GFAP/nestin+
) representing the stem cell pool (Gage et al., 1995
). Type 2a (GFAP−
), type 2b (nestin+
), and type 3 (nestin−
) cells represent the next three consecutive stages of transiently amplifying putative progenitor cells that also represent increasing neuronal differentiation. The type 3 cells denote the end of the cell cycle, which is followed by the postmitotic state, in which cell maturation and survival dictate cell fate. Type 1 cells have been shown to divide into intermediate GFAP+
progenitors (i.e., type D cells described by Seri et al., 2001
), and investigation of these subtypes in future studies may provide valuable insight into post-stroke susceptibility and modulation of rates of proliferation of these subsets that survive the ischemic insult.
In this study, ~90% of the BrdU-labeled cells in the SGZ of control mice were nestin positive, which likely represented the entire population of GFAP+
progenitors [i.e., radial glial (type 1) and intermediate type D cells] and the putative progenitors with undetermined (type 2a) or determined (type 2b) lineages [Gage et al., 1995
; for details see review by Kronenberg et al., 2003
)]. The 20–30% BrdU-labeled cells in the SGZ that colabeled with GFAP likely represent the type 1 progenitors and their intermediates and therefore were a subset of the nestin-positive pool (Filippov et al., 2003
). This indicates that the remaining 60–70% of the nestin-positive newborn cells were either type 2a or type 2b cells. The DCX-positive newborn cells amounted to 5–10% of the BrdU-labeled cell population. Insofar as both type 2b and type 3 cells show DCX expression, a portion of this DCX-positive pool represented a subset of the nestin-positive pool (type 2b). The remainder represented the pool of type 3 DCX-positive but nestin-negative newborn cells. Approximately 10% of the BrdU-labeled cell population did not colabel with nestin; some of these cells might have been the type 3 DCX-positive-only cells. Insofar as all three cell types are putative progenitors, the larger pool of putative type 2a cells may represent the higher capacity for proliferation reported for this cell type compared with the type 1 (i.e., putative neural stem cell), type 2b, and type 3 cells (Kronenberg et al., 2003
). Type 1 cells have been described (Kronenberg at al., 2003
; Kempermann et al., 2004
) to have low proliferative activity (Seri et al., 2001
) and to have pyramidal somas with a dominant apical process traversing the granule cell layer, with processes in the inner molecular layer. Therefore, by staining with nestin, GFAP, and DCX, the new cell populations labeled <2 hr before fixation in our study showed early cell type proliferation profiles in immature CD-1 mice similar to those found in prior studies (Gage et al., 1995
; Filippov et al., 2003
; Kronenberg et al., 2003
) that have characterized the early stages of SGZ neurogenesis.
The ligated mice used in this study did not show significant differences from controls, for the profile described above, except for significant increases of percentage GFAP-positive newborn cells at P33. This may suggest reversal of more committed progenitors (types 2a and 2b) back to more immature forms (i.e., radial glial cell types or cell type 1) over time to compensate for the consistently lowered neural proliferation in the ipsi-lateral SGZ or symmetric divisions of type 1 cells to replenish the neural stem cell pool. However, the mean percentage nestin-positive newborn cells was about 60–70% at 7 and 14 DAI ipsilaterally; this may also indicate suppression of the type 2a proliferation in the injured hippocampi. Percentage nestin-positive newborn cells returned to match control by 21 DAI; this may indicate recovery of the proliferation capacity back to control levels by 3 weeks. Seaberg and van der Kooy (2002)
have previously used culture methods to show that the stem cells derived from the lateral ventricle and other ventricular subependymal regions directly adjacent to the hippocampus exhibit long-term self-renewal and multi-potentiality and can produce separate neuronal and glial progenitors compared with the limited self-renewal capacity of SGZ-derived cell proliferation. This suggests that neuron-specific progenitors and not multipotential stem cells are the source of newly generated DG neurons throughout adulthood. This may explain the paradoxical response of the SVZ cell proliferation compared with that of the SGZ and the absence of an altered cell type commitment profile following stroke in the SGZ.
An important feature of this neonatal mouse model is the occurrence of acute seizures following the stroke; this feature models the frequent presentation of neonatal stroke with acute seizures. A significant finding of this study was the predictive value of the acute seizure scores for the severity of the stroke injury in the hippocampus (i.e., overall atrophy). The negative but not predictive correlation of acute seizure scores to the lengths of SGZs in the injured brains emphasizes the nonuniform stroke injury within subpopulations of primary neurons in the DG. Severe seizures during early childhood have been associated with adverse effects on postnatal granule cell neurogenesis (McCabe et al., 2001
; Mathern et al., 2002
). Therefore, in addition to the ischemic stroke, the acute seizures may affect early brain development. The negative modulation of the neural maturation after long survival delays in our previous study (Kadam et al., 2008
) may among other possibilities be a reflection of the adverse effect of seizures on cell proliferation, survival, and maturation. Additionally, the reduced neurogenesis may play a pivotal role in epileptogenesis following perinatal brain injury. The data show that long-term proliferation rates were affected by the ligation injury. Future studies to investigate the effect of successful acute seizure suppression following stroke on postnatal neurogenesis are planned. Interventions that help both to reduce the ischemic injury and to block the acute seizures in this model may provide valuable insight into neonatal stroke management.