The major finding of this study is that EE increased the number of progenitor cells in the ipsilesional granule cell layer and hilus of the dentate gyrus following a FP injury. This finding is in general agreement with several previous studies that have demonstrated that EE can increase neural progenitors in the hippocampus [6
]. However, this finding was also novel and somewhat unexpected in comparison to previous studies because 1) in intact animals, EE produced bilateral increases in neural progenitors in the granule cell layer, and no increases in the hilus [6
], and 2) following an ischemic injury, two weeks of EE did not have an additive effect on the number of progenitor cells in the dentate gyrus [5
]. There are several possible explanations for the differences between our findings and those of previous studies.
One possibility is that the increases in BrdU labeling that we observed in the ipsilesional dentate reflect cell death rather than proliferation. We believe this is unlikely for the following reasons. First, we observed no Fluoro Jade staining in the dentate gyrus in any of our animals, suggesting that neuronal degeneration is no longer present in this region of the brain 3 weeks post-injury. This observation is in agreement with a previous time course study, which reported an absence of neuronal degeneration (based on both Fluoro Jade and TUNEL labeling) in the hippocampus between 2 and 4 weeks post-FP injury [65
]. Second, although we did observe chronic neuronal degeneration in the cortex following FP injury, we have not found any cells that are double-labeled with Fluoro Jade and BrdU, suggesting that at least in the penumbra of the cortical lesion, neural progenitors and degenerating cells are two distinct populations (unpublished data). Third, although following a hypoxic-ischemic injury dying cells appear to re-enter the cell cycle and become positively labeled with BrdU and Ki67, this phenomenon was not observed following TBI [44
]. Thus, it appears that reactivation of cell cycling in apoptotic neurons may or may not occur, and depends upon unknown factors that may be associated with specific types of CNS injury. Fourth, if apoptosis and re-entry into the cell cycle pathway was responsible for the unilateral increases in BrdU labeling then one might also expect to see unilateral increases in anti-Ki67 labeling, but that was not the case. Ki67-labeled cells in the dentate were symmetrical between the right and left hemispheres.
Another possible explanation for the lack of a bilateral effect of EE on cell proliferation in the dentate is that we provided animals with just 1 hour of daily EE, whereas most previous studies provided EE for 24 h/d [79
]. However, 1 h/d of EE was sufficient to attenuate deficits in spatial learning in the MWM following FP (), and 3 h/d of EE produced increases in neurogenesis and improvements in memory and learning in intact mice [18
] and rats [7
]. Even shorter durations, as little as 40 min/d, was sufficient to produce alterations in mRNA and brain weight in developing animals [13
]. Thus, it appears that alterations to the brain can be produced with relatively short periods of EE, but whether this confers optimal benefits remains to be determined.
In addition to the daily amount of EE, there are also several other factors that could explain why we did not observe bilateral alterations in the number of progenitor cells. For example, EE has been reported to improve behavior following FP injury in male rats, but not in female rats [77
]. Besides gender [54
], there are numerous other factors, including species, [6
], age [81
], the onset and duration of the EE [20
], and the type of injury [5
] that may also explain the differential effects of EE among various studies. Furthermore, differences in the number, timing, and dose of BrdU injections [8
] may also contribute to different study outcomes. Interestingly, in sham injured animals EE did not improve behavior [31
], nor did it increase the number of BrdU+ cells in the dentate (unpublished pilot data). Therefore, the lack of an effect of EE on cell proliferation in the contralesional dentate following FP injury, as well as the lack of an effect following sham injury suggests that numerous factors can interact with EE and modulate its effects on behavior and neurogenesis.
A second important finding of this study is that progenitor cell fate varied by region and by group. In the left granule cell layer following SH injury, approximately 85% of the progenitors had a neuronal phenotype, compared to 62% following FP, and 68% following FP+EE. Our data for FP injury is in close agreement to that reported following an ischemic injury, where approximately 60% of the progenitor cells in the granule cell layer were neurons [70
]. The reason for the proportional decrease in neurogenesis in the left granule cell layer following FP is unknown. Although it may be caused by inhibition of neurogenesis, this seems unlikely because the absolute number of new neurons is not decreased after FP, and is actually increased after FP+EE. Rather than inhibition of neurogenesis, it may be the increase in astrogenesis that explains the proportional decrease of newly born neurons following FP injury.
Progenitor cell fate following FP injury was not further modulated by EE. The lack of an effect of EE on cell fate has also been reported following seizures, where the percent of BrdU/NeuN cells in the dentate granule cell layer ranged from 70 – 80% in animals that did and did not receive enrichment [17
]. In contrast to the lack of an effect of EE on progenitor fate following FP injury, age did have an effect, with greater neurogenesis in younger animals [74
In the left hilar region, EE increased progenitor cells following FP, but neurogenesis was rare. Most of the progenitors in the FP and FP+EE groups were astrocytes or single-labeled BrdU cells. The left hilus was of particular interest because it has a significant loss of neurons following FP injury [23
], and is adjacent to the subgranular zone. Since recent studies suggest that stem cell proliferation, fate and migration are modulated by the extracellular environment [25
], we hypothesized that the neuropathology in the hilus following FP injury, in combination with unknown molecular events associated with EE, would provide a permissive environment for neurogenesis. However, it appears that FP+EE does not enhance neurogenesis in this region, but it does provide a permissive environment for progenitor cells, as single-labeled BrdU cells in the hilus were significantly increased in FP+EE compared to FP. This is important because if these progenitors are undifferentiated, then they could provide a substrate for additional manipulations aimed at driving them to a neuronal fate.
Although we did not see evidence of neuronal replacement following brain injury with our intervention (EE), a recent study suggests that other interventions may be effective. Following an ischemic injury, administration of endothelial growth factor (EGF) and fibroblast growth factor (FGF-2) significantly increased neurogenesis in the CA1 pyramidal cell layer [53
]. Interestingly, FGF-2 is increased in the hippocampus by exercise [21
]. Since EE includes motor, social, and sensory components, it is possible that the increases in progenitors in the hippocampus following FP+EE may be linked to increases in FGF-2 protein levels, as well. Whether neuronal replacement in CA1 is required for behavioral recovery, though, is unclear. Even without growth factor administration, neurogenesis increased in the dentate gyrus and was correlated with recovery of electrophysiological and behavioral function, despite the loss of cells in the CA1 region [78
]. Similarly, the attenuation of cognitive deficits associated with EE following FP injury may be attributable to the increases in newly born neurons in the granule cell layer, rather than neuronal replacement in the hilus.
FP injury increased increase astrogenesis in the left hilus, but EE did not have an additive effect. This data is in agreement with a previous study on intact animals, which demonstrated that wheel-running, but not EE, increased new astrocytes [73
]. In comparison to adult rats, juveniles had less astrogenesis in the hilus following FP injury [74
], and we observed a similar trend in the FP+EE group compared to FP. Several previous studies have reported an increase in glial fibrillary acidic protein (GFAP), a marker for astrocytes, in the hippocampus following experimental TBI [3
]. Although one might predict that astrogenesis would account for the increase in GFAP, actually very few GFAP+ cells were found to be co-localized with BrdU [3
]. Furthermore, the number of astrocytes in the dentate gyrus was not significantly increased following FP injury [23
]. Thus, it appears that number of new astrocytes in the hilus following FP injury is small relative to the total number of astrocytes in the entire dentate gyrus or hippocampus. In addition, it is possible that the number of new astrocytes may be even smaller than our estimates, as a recent report suggests that S100β may also label oligodendrocytes [26
Astrogenesis, but not neurogenesis, varied by rostral-caudal levels within the dentate. Astrocytes were more common in the left granule cell layer and hilus in sections collected 4.9 mm posterior to bregma than in more rostral sections following FP and FP+EE. The craniotomy, where the fluid percussive wave is transmitted, is centered approximately at bregma − 4.5 mm. The rostral-caudal variability observed in this study highlights the need for sampling BrdU+ cells at multiple levels of the brain when determining cell phenotypes following brain injury. Although neural stem cell fate may be relatively homogenous in naïve and sham injured animals, it appears that proximity to the injury may be a major factor following injury. This data also suggests that neurogenesis and astrogenesis are differentially regulated following brain injury, as the proximity to the impact site only affected the BrdU/S100β+ cells.
We also evaluated the effect of EE on cell cycle activation at the time of euthanasia. Unlike the BrdU data, which demonstrated increased survival of cells that were proliferating at the time of injury, the ki67 labeling did not differ between groups or sides of the brain. These findings are in agreement with previous studies where EE was demonstrated to increase cell survival, but not ongoing proliferation in control animals [6
]. Interestingly, running increases both cell proliferation and survival of progenitors in the dentate gyrus [16
], which underscores the importance of clarifying how specific components of these behavioral interventions can differentially modulate progenitor cells.
In conclusion, this study suggest that 1 hour of daily EE following a FP injury increases the survival of endogenous neural progenitors in the ipsilesional dentate gyrus, but does not alter their on going rate of proliferation or their fate. Whereas a majority of the progenitors in the granule cell layer of the dentate differentiate into neurons, neurogenesis in the hilus is rare. Thus, the beneficial effects of EE on behavioral recovery following FP injury do not appear to be attributable to neuronal replacement in the hilus, but may be related to increased neurogenesis in the granule cell layer. This study lays the groundwork for further investigations on the regulation of endogenous neural stem cells following brain injury and their role in recovery from FP injury.