The main findings of the present study were: 1) EC-SOD KO mice had a lower baseline hippocampal neurogenesis compared to WT mice; 2) proliferating precursor cells and immature neurons of the dentate SGZ in EC-SOD KO mice showed acute (6–48 hr) sensitivity to irradiation and the level was comparable to that of WT mice; 3) survival of newly generated cells in the dentate SGZ after 5 Gy irradiation was substantially higher in EC-SOD KO mice compared to WT controls; and 4) contrary to what was seen in WT mice, irradiation did not reduce the survival of newly generated neurons and glia in EC-SOD KO mice. These findings showed that after irradiation, an environment lacking EC-SOD was much more permissive in the context of hippocampal neurogenesis. The mechanism(s) behind this observation is not yet known, but this effect might constitute a basis for future interventions aimed at rescue or at least amelioration of the risks for cognitive dysfunction in individuals subjected to CNS irradiation.
The effects of irradiation on hippocampal structure and function have been extensively studied (reviewed in [5
]) but only recently has it been suggested that radiation-induced injury of the neurogenic cell populations within dentate gyrus [10
] may play a role in cognitive sequelae of cranial irradiation. Studies from our lab [10
] and others [12
] provide a quantitative description of the impact that ionizing radiation exerts on hippocampal neurogenesis. Those studies show that the stem/precursor cell populations within the neurogenic areas are very vulnerable to injury and that changes are associated with radiation-related impairments of hippocampal-dependent cognitive tasks [12
]. Furthermore, radiation-induced changes in neurogenesis have been shown to be associated with microenvironmental factors including neuroinflammation [56
], vascular changes [10
] and oxidative stress [28
In this study we were interested in determining if and how the absence of EC-SOD would impact neurogenesis. EC-SOD immunoreactivity is found throughout the brain, and is particularly prominent in the hippocampus [62
]. It is of particular interest that alterations in EC-SOD expression in mice is associated with impaired learning [34
], and that over-expression of EC-SOD protects synaptic plasticity and learning and memory against oxidative damage [63
]. Here we found that the absence of EC-SOD was not associated with compensatory changes in other SODs or other anti-oxidant enzymes (), although given the intracellular localization of these molecules it might be unlikely to expect them to have compensatory functions in terms of affecting extracellular superoxide free radicals. It could be possible that circulating antioxidants, including ascorbic acid, tocopherol, uric acid, bilirubin, proteins and other compounds, could be increased as a compensatory mechanism, but we did not address this in the current study. While total antioxidant capacity in the serum can be assessed quantitatively [64
], without knowing the amount of circulating EC-SOD and its contribution to total antioxidant capacity in the serum, how these compounds may or could affect regional effects in the SGZ may be difficult to interpret. However, a consideration of such ideas is worthy of further study. Regardless, our data show that at least qualitatively, 2-month-old EC-SOD KO mice showed indications of a persistent oxidative stress (). As a result, and based on data relating EC-SOD with cognitive function [34
], we expected neurogenesis in the KO mice to be lower than that of WT mice. This was, in fact, what we observed (). Whether these changes are responsible for the cognitive impairment observed in EC-SOD KO mice is not clear. However, given the extensive data available associating changes in neurogenesis with cognitive performance [11
], it certainly seems possible, and at the very least may play a contributory role. The baseline reduction in new neuron production in KO mice was different from what was seen in glia () and inflammatory cells (), where there were apparent increases in cell number in KO mice relative to WT. While the significance of these latter findings may be affected by the variability in the data, increased numbers of astrocytes and activated microglia would be consistent with an elevated level of oxidative stress in the KO animals.
After irradiation with a relatively low dose of x-rays, the early responses, including apoptosis, altered cell proliferation, and changes in numbers of immature neurons, were similar between WT and KO mice. This suggested that the absence of EC-SOD and the increased oxidative stress status of KO animals did not influence mechanisms responsible for acute cell death after irradiation. Furthermore, there were no major differences between WT and KO mice in cell proliferation and numbers of immature neurons at 1 month after irradiation (). It was of interest, therefore, to see substantial and significant differences in the survival of newly generated cells after irradiation as a function of genotype, with KO mice showing almost 4 times the number of surviving newly generated cells than WT (). This surprising result also translated into a very significant and nearly 4-fold difference in the number of surviving newly generated neurons in irradiated KO as compared to irradiated WT mice. In fact, while radiation caused an 85% reduction in newly generated neurons in WT mice, the same dose resulted in virtually no difference in KO mice. To the best of our knowledge, this is the first report to show that an EC-SOD deficient environment provides a ‘protective’ effect in hippocampal neurogenesis after irradiation. In a general sense, this resembles the preconditioning seen in stroke [69
], heart attack [70
] or acute lung injury [71
] where oxidative mechanisms plays a crucial role in the pathogenesis of the disease. While the precise mechanism(s) responsible for this effect has not yet been clarified, our study rules out simple compensatory changes in expression and activities of other antioxidant enzymes (). Our findings clearly suggest that EC-SOD deficient mice have developed a resistance to radiation-induced inhibition of neurogenesis that may involve some type of adaptation within the microenvironment, without compensatory changes in other major intracellular antioxidants. These ideas are being tested further using inducible in vitro and in vivo models of EC-SOD expression. Furthermore, it is possible that a specific set of trophic factors and signaling molecules that favor differentiation and long-term survival of newly-generated neurons in the SGZ is up-regulated or activated in the irradiated EC-SOD KO brains to counter the chronic inflammatory environment created from irradiation. These trophic factors and signaling molecules may include brain derived trophic factor (BDNF), vascular endothelial growth factor (VEGF), and nitric oxide (NO), all of which have been shown to favor differentiation and survival of neurons [72
]. At the same time, the phenotype of the activated microglia [76
] in the irradiated EC-SOD null environment may also play a role in enhanced neurogenesis. Whatever the mechanism(s) involved, our data clearly show that the production of new neurons is more sensitive to irradiation in WT animals than in the EC-SOD KO mice.
Due to the relatively lower numbers of newly generated astrocytes and oligodendrocytes, there is more variability in the data for these cells, but clearly there appears to be a trend toward increased numbers in KO vs. WT mice after 5 Gy of x-rays (). This suggests that perhaps the same mechanism responsible for the relative protective effect of neurons in EC-SOD KO mice is operative in glial cells as well. Given recent data showing that neural stem cells in the SGZ express GFAP [78
], it is possible that the increased numbers of GFAP+ cells seen in KO mice represent, in part, increased stem cell or regenerative responses. On the other hand, the observed changes in BrdU+/GFAP+ cells may very well be a sign of the well-described astrogliosis seen in the oxidative stress conditions [79
] as well as after irradiation [80
Neurogenesis depends upon a complex microenvironment that involves signaling between multiple cell types [81
], and changes in the oxidative status along with irradiation could affect any or all of these cells or their interactions. While the precise nature of such effects has not yet been clarified, previous studies have suggested chronic inflammatory changes as one of the important factors [10
]. We found significant increases in the total numbers of activated microglia in both WT and KO mice after irradiation as compared to non-irradiated mice (). Furthermore, there appeared to be increases in numbers of newly generated activated microglia (BrdU+/CD68+) in both genotypes although the magnitude of the differences was not statistically significant. That increasing numbers of activated microglia after irradiation had no apparent association with neurogenesis in EC-SOD KO mice is somewhat surprising, given recent studies showing that increased neuroinflammation was linked to an inhibition
of hippocampal neurogenesis [13
]. It is particularly interesting, therefore, that a recent study has suggested that microglial phenotype critically influences their ability of these cells to support or impair cell renewal processes in the adult brain [77
]. It may be that in a microenvironment characterized by persistent oxidative stress (i.e., EC-SOD KO), the subsequent activation of microglia may, in fact have a beneficial effect, at least in terms of neurogenesis. This would suggest that microglia may respond differently to a given stimulus (irradiation) depending upon the presence of another and preceding stimulus (oxidative stress); such ideas have recently been reviewed [83
]. While intriguing, such a hypothesis is speculative at this time, but further studies seem warranted.
While the mechanistic relationship between EC-SOD and forebrain neurogenesis is not yet known, the data shown here clearly suggest that the lack of EC-SOD provides some sort of survival advantage to neurogenic cell populations after irradiation. This could have major implications with respect to protecting or ameliorating the adverse effects of radiation on specific brain functions if the underlying changes in the molecular network can be identified.