Human neural stem cell transplantation provides long-term restoration of neuronal plasticity in the irradiated hippocampus

Abstract

For the majority of CNS malignancies, radiotherapy provides the best option for forestalling tumor growth, but is frequently associated with debilitating and progressive cognitive dysfunction. Despite the recognition of this serious side effect, satisfactory long-term solutions are not currently available and have prompted our efforts to explore the potential therapeutic efficacy of cranial stem cell transplants. We have demonstrated that intrahippocampal transplantation of human neural stem cells (hNSCs) can provide long-lasting cognitive benefits using an athymic rat model subjected to cranial irradiation. To explore the possible mechanisms underlying the capability of engrafted cells to ameliorate radiation-induced cognitive dysfunction we analyzed the expression patterns of the behaviorally induced activity-regulated cytoskeleton-associated protein (Arc) in the hippocampus at 1 and 8 months postgrafting. While immunohistochemical analyses revealed a small fraction (4.5%) of surviving hNSCs in the irradiated brain that did not express neuronal or astroglial makers, hNSC transplantation impacted the irradiated microenvironment of the host brain by promoting the expression of Arc at both time points. Arc is known to play key roles in the neuronal mechanisms underlying long-term synaptic plasticity and memory and provides a reliable marker for detecting neurons that are actively engaged in spatial and contextual information processing associated with memory consolidation. Cranial irradiation significantly reduced the number of pyramidal (CA1) and granule neurons (DG) expressing behaviorally induced Arc at 1 and 8 months postirradiation. Transplantation of hNSCs restored the expression of plasticity-related Arc in the host brain to control levels. These findings suggest that hNSC transplantation promotes the long-term recovery of host hippocampal neurons and indicates that one mechanism promoting the preservation of cognition after irradiation involves trophic support from engrafted cells.

Figure 1

The yield of hNSC transplant-derived cells at 1, 4, and 8 months postsurgery. Transplant-derived cells were tracked by BrdU immunofluorescence staining and quantified using fluorescence stereology and an optical fractionator by counting BrdU-positive nuclei. Data are represented at mean ± SEM of four individual observations.

Figure 2

Representative fluorescent micrographs of OX6⁺ microglia (MHC-II, red and nucleus, DAPI, blue) in sham surgery control (Cont-Sham), irradiated (IRR-Sham), and irradiated with hNSC transplantation (IRR + hNSC) at 1 month (A, C, E) and 8 months (B, D, F) postsurgery. Quantification of OX6 in the hippocampus (DG, DH, CA1, and CA3) indicated significant upregulation (*p < 0.008) in the transplanted group (IRR + hNSC) at 1 month postsurgery (D). The trend was reversed (higher OX6 in IRR sham) at 8 months postsurgery group (H), albeit not significant (p < 0.06) at this time point. Data are represented as mean ± SEM of four individual observations. Scale bar: 50 μm (A–F).

Figure 3

Representative confocal fluorescent micrographs of location of transplanted cells and its close proximity with activated microglia. Intrahippocampal location of transplanted hNSC (green, BrdU⁺ and blue, DAPI⁺ nucleus) is shown at 5× and 40× magnification (A, B). Transplanted hNSCs and host OX6⁺ microglia (red) were found in the close vicinity at 8 months postsurgery (arrows, C and D). The activated microglia extended process to interact with transplanted hNSCs (arrows, E, F). Scale bar: 100 μm (A–B), 50 μm (C–D), and 5 μm (E–F).

Figure 4

Representative fluorescent micrographs of Arc protein expression (red) 30 min after memory test in the DG neurons (blue) of the hippocampus. Memory test induced a significant increase in Arc⁺ neurons compared to caged control animals for both 1 and 8 months postgrafting in all treatment groups except the irradiated animals (*p < 0.0001 vs. caged). Scale bar: 200 μm.

Figure 5

Representative fluorescent micrographs of Arc protein expression (red) 30 min after memory test in the CA1 pyramidal neurons (blue) of the hippocampus. Thirty minutes after behavioral exploration in the memory test the percentage of Arc⁺ pyramidal neurons was significantly higher than the cage control in all the treatment except irradiated animals (*p < 0.0001, vs. caged) both 1 and 8 months postirradiation and was significantly reduced compared to sham control animals (+p < 0.001, vs. Cont-Sham). Scale bar: 50 μm.