Author contributions: P.A.: conception and design of experiments, data collection, assembly, analysis and interpretation, manuscript writing, final approval of manuscript, and contributed to the manuscript editing; D.P.: data collection, assembly, analysis, and contributed to the manuscript editing; J.R.: data collection and contributed to the manuscript editing; V.C.G. and J.M.G.V.: data collection, assembly, analysis and interpretation from electron microscopy, and contributed to the manuscript editing; K.S. and H.S.: provided the GFP expressing retrovirus, assisted with stereotactic injections, and contributed to the manuscript editing; O.G.P.: experimental design, data interpretation, and contributed to the manuscript editing; E.F.: conception and design of experiments, SARPP technology, data interpretation, and contributed to the manuscript editing; A.Q.H.: conception and design of experiments, final approval of manuscript, financial support, and contributed to the manuscript editing.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS September 4, 2012.
Radiation therapy is a part of the standard treatment for brain tumor patients, often resulting in irreversible neuropsychological deficits. These deficits may be due to permanent damage to the neural stem cell (NSC) niche, damage to local neural progenitors, or neurotoxicity. Using a computed tomography-guided localized radiation technique, we studied the effects of radiation on NSC proliferation and neuroblast migration in the mouse brain. Localized irradiation of the subventricular zone (SVZ) eliminated the proliferating neural precursor cells and migrating neuroblasts. After irradiation, type B cells in the SVZ lacked the ability to generate migrating neuroblasts. Neuroblasts from the unirradiated posterior SVZ did not follow their normal migratory path through the irradiated anterior SVZ. Our results indicate that the migrating neuroblasts were not replenished, despite the presence of type B cells in the SVZ post-irradiation. This study provides novel insights into the effects of localized SVZ radiation on neurogenesis and cell migration that may potentially lead to the development of new radiotherapy strategies to minimize damage to NSCs and neuroblast migration. STEM CELLS2012;30:2548–2560
Brain irradiation is an integral component of the treatment of patients with brain tumors [1, 2]. Despite its effectiveness in controlling the progression of some cancers, radiation is often associated with neuropsychological deficits [3–6]. These deficits may be partly due to radiation-induced damage to neurogenic niches, as shown in animal models [7–11]. In the adult mammalian brain, these niches are localized in the subventricular zone (SVZ) and the subgranular zone of the hippocampal dentate gyrus [12–14]. The neural stem cells (NSCs) in these two regions are of great interest for therapeutic applications in the brain because of their ability to proliferate and produce new cells. These newly generated cells are able to migrate and differentiate into any of the main cell types of the central nervous system, such as neurons, oligodendrocytes, or astrocytes [12–15]. Damaging NSCs during radiation therapy can have detrimental effects [4, 7]. Mitigating the neurotoxic effects of radiation is important particularly in the context of brain tumor patients who survive longer due to new combination therapies [1, 2, 6]. Furthermore, NSCs have the potential to be used to treat brain cancer in two different ways. First, endogenous NSCs in the brain can participate in the repair of normal tissue and/or inhibit tumor growth . Second, engineered stem cells may be used to deliver cytotoxic agents and prodrug-activating enzymes to the tumor [17–21]. Both of these possibilities require that NSCs generate neuroblasts to migrate toward the tumor site, a process that is not well-understood in the context of radiation therapy.
The SVZ is the largest source of NSCs in the adult brain, located underneath the layer of ependymal cells lining the ventricles [13, 22–24]. NSCs, or type B1 cells in the SVZ, are astrocyte-like glial fibrillary acidic protein (GFAP+) cells that divide and give rise to transit amplifying neural precursor cells (NPCs), or type C cells, which in turn give rise to migrating neuroblasts, or type A cells . Neuroblasts migrate long distances through the rostral migratory stream (RMS) before differentiating into olfactory bulb (OB) interneurons [26, 27]. The mouse SVZ-RMS-OB migration route represents a well-characterized system of endogenous neuroblast migration that serves as a model to study migration in humans receiving radiotherapy [27, 28].
Current radiotherapy treatments, such as intensity-modulated radiation therapy, are intended to deliver a maximum dose of radiation to the tumor while reducing radiation exposure to the surrounding normal tissue [29, 30]. To more accurately reproduce human radiotherapy, we used a small animal radiation research platform (SARRP) developed in-house . Using this technology, we recently reported that local irradiation of hippocampus significantly decreased the number of proliferating cells in the irradiated dentate gyrus compared to the unirradiated side . Furthermore, the number of newborn cells differentiating into mature neurons 2 months after irradiation was significantly reduced . In this study, we examined the effect of SVZ-restricted irradiation on NSC and NPC proliferation and neuroblast migration in the adult mouse SVZ-RMS-OB system. The two main findings from this study are that type B cells lacked the ability to regenerate NPCs and neuroblasts even a month after irradiating SVZ, and that neuroblasts failed to migrate through the irradiated regions. These results will help inform future radiotherapy protocols to minimize damage to neurogenic niches in the human brain.
MATERIALS AND METHODS
The subjects were 6–8-week-old male C57BL6/J mice from the National Cancer Institute maintained on a 12:12 hour light/dark cycle. Food and water were available ad libitum. All experiments described were performed with the approval of the Johns Hopkins Animal Care and Use Committee under standard protocols.
Mice were anesthetized with the injection of 100 mg/kg ketamine + 10 mg/kg xylazine intraperitoneally. To directly visualize the ventricles and target the SVZ, 70 μl of iodine contrast was injected intrathecally and computed tomography (CT) images were obtained as described previously . A single dose of 10 Gy was delivered using CT-based tissue visualization. Previous studies have demonstrated that the overall geometric targeting accuracy of this technique is 0.2 mm . A radiation beam of 3 × 3 mm2 was used to target right SVZ while left brain structures served as controls. For migration studies, a radiation beam of 1-mm diameter was used to target the anterior dorsal region of the SVZ. Additionally, a radiation beam of 5 × 9 mm2 was used to target right OB, RMS, and/or anterior SVZ (aSVZ). For sham irradiation, control animals were anesthetized, brought into the treatment room, and handled and positioned identically to irradiated animals without radiation delivery.
Mice (n = 5 per group) were deeply anesthetized and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS). Brains were removed from the skull and postfixed in 4% PFA overnight at 4°C. The brains were then equilibrated in 30% sucrose and were frozen in Tissue-Tek OCT Compound. Ten-micrometer-thick coronal slices were sectioned using a cryostat. Sections were treated with 0.01 M sodium citrate at 95°C for 10 minutes for antigen retrieval. Sections were incubated in PBS containing 0.1% Triton X-100, and 10% normal goat serum (NGS) for 30 minutes, and then incubated with primary antibodies for 16 hours at 4°C. Primary antibodies used in this study were mouse anti-rH2Ax (Ser139) (1:700), rabbit anti-Ki67 (1:200), mouse anti-GFAP (1:500), rabbit anti-doublecortin (DCx) (1:500), mouse anti-nestin (1:100), mouse anti Mash-1 (1:100), cleaved caspase-3 (1:100), and mouse anti-CD31 (1:200). More details on the primary antibodies are summarized in supporting information Table S1. The sections were washed with PBS and incubated with secondary antibodies conjugated with fluorophores for 1 hour at room temperature (RT). Anti-mouse, anti-rat, and anti-rabbit Alexa 488 and/or 594 secondary antibodies (1:500; Invitrogen, Green Island, NY, USA) were used. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (1:500) All the sections were air-dried and coverslipped with .Aquamount mounting media (Vector Labs, Burlingame, CA, USA). Fluorescent images were taken with an ORCA II camera (Hamamatsu) connected to an Olympus IX81 inverted microscope.
SVZ Whole Mount Dissection and Immunostaining
Mice (n = 3 per group) were sacrificed by cervical dislocation and their brains were immediately extracted into L-15 Leibovitz medium (Gibco, Green Island, NY, USA). A whole mount of SVZ tissue (2–4 mm) with the lateral ventricle and striatum was dissected under microscope, and the hippocampus and septum were removed as described [24, 33]. The SVZ whole mounts were fixed in 4% PFA/0.1% Triton X-100 overnight at 4°C. They were then washed with PBS and blocked with 10% NGS/0.5% Triton for 30 minutes at RT. The tissues were incubated in primary and secondary antibodies diluted in blocking buffer for 48 hours at 4°C. After staining, the SVZ whole mounts were further dissected from underlying striatum to 200–300-μm thick tissue, transferred to a glass slide, and coverslipped with Aquamount mounting media for imaging. A Nikon C1si True Spectral Imaging Confocal Laser Scanning Microscope was used to image the SVZ whole mounts.
Semithin Sections, Electron Microscopy, and Ultrastructural Analysis
Mice (n = 5 per group) were anesthetized and perfused with 0.9% saline, followed by 2% PFA and 2.5% glutaraldehyde. Heads were removed and postfixed in the same fixative overnight. After postfixation, brains were dissected and washed in 0.1 M phosphate buffer (PB) (pH 7.4), cut into 200 μm coronal sections with a VT 1000M vibratome (Leica, Wetzlar, Germany), and treated with 2% osmium tetraoxide in 0.1 M PB for 2 hours. Sections were then rinsed, dehydrated through increasing ethanol solutions, and stained with 2% uranyl acetate in 70% ethanol. Following dehydration, slices were embedded in araldite (Durcupan, Fluka BioChemika, Ronkokoma, NY). To study the cellular organization of the SVZ and RMS, we cut serial 1.5 μm coronal semithin sections with a diamond knife and stained them with 1% Toluidine blue. Sections were visualized under E200 light microscope (NIKON, Tokyo, Japan). To identify cell types, 60–70 nm ultrathin sections were cut with a diamond knife, stained with lead citrate, and examined under a Spirit transmission electron microscope (FEI Tecnai, Hillsboro, OR). The different cell types in the SVZ and RMS were identified based on their ultrastructural characteristics as described previously .
SVZ Neurosphere Cultures
Adult mouse brains were removed into sterile cold Neurocult basal medium (Stem Cell Technologies, Vancouver, BC, Canada). The SVZ was dissected out from each hemisphere as described in Ferron et al. . The left and right SVZ were collected separately and pooled from four different mice. The tissue was triturated gently until all the pieces were dissociated and cell suspension was homogenous. The cells were washed in the complete mouse Neurocult proliferation medium with Neurocult proliferation supplement, recombinant growth factors (10 μg/ml epidermal growth factor and 10 μg/ml fibroblast growth factor), and 2 μg/ml heparin (Stem Cell Technologies, Vancouver, BC, Canada). Viable cells were counted and plated at 2 × 104 cells per square centimeter. The cultures were incubated at 37°C in a 5% CO2 humidified incubator adding fresh medium every 4 days. Neurospheres of 100–200 μm size were passaged and replated.
Retrovirus Preparation and Stereotactic Injections
Engineered self-inactivating murine oncoretrovirus expressing green fluorescent protein (GFP), with the Woodchuck hepatitis virus post-transcriptional regulatory element under the Ubiquitin-C promoter, was produced as previously described . A day after irradiation, mice (n = 5 per group) were anesthetized with ketamine (100 mg/kg)/xylazine (10 mg/kg) anesthesia and injected with buprenorphine (0.02 mg/kg) for postoperative analgesia. The head was secured to a stereotaxic frame (David Kopf, Tujunga, CA, USA). The skull surface was exposed and retrovirus was injected (1 microliter per site) into the SVZ using a glass needle at the following two different coordinates bilaterally relative to bregma: anterior = 1.0 mm, lateral = ±1.0 mm, ventral = 2.2 mm; anterior = 0. 0 mm, lateral = ±1.8 mm, ventral = 1.6 mm. The scalp was sutured and the mice were returned to their standard housing. Mice were sacrificed a week after retrovirus injections, the brains were fixed, sectioned sagittally, and immunostained against Ki67. Sections were visualized under a microscope for GFP-expressing migrating neuroblasts and Ki67+ cells. The experimental paradigm is shown in Figure 7A.
Cell Quantification and Statistical Analysis
In the coronal sections of the mouse brain, the quantification of Ki67+ cells in the SVZ, RMS, and OB was performed on every 20th section evenly spaced along the entire rostral-caudal length of the lateral ventricle of the SVZ using a fluorescent microscope. A total of 7, 9, and 10 sections were counted for SVZ, RMS, and OB, respectively, for each animal. The cell counts for all mice in a given group were averaged per section and the SEM was calculated. To quantify the number of cells within the SVZ under electron microscope, we considered the area comprised of the first 20 μm adjacent to the ventricle lumen (0–1 mm anterior to bregma). The counts were performed on three different levels per animal, and the average was shown in cells per mm. Blood vessel analysis was performed using semithin sections. We considered the area comprising the first 100 μm adjacent to the ventricle lumen (0–1 mm anterior to bregma). The number and size of blood vessels were measured with Image Tool software (Evans Technology, Roswell, Georgia). Changes in RMS size were determined by measuring the area occupied by RMS in semithin sections using a light microscope, at three different levels per animal. The analysis was performed with Image Tool software. An unpaired t test was performed using SigmaPlot 11.0 software (Systat Software Inc, San Jose, CA) to determine the difference between the left and right SVZ for all our data quantified. The differences were considered significant at a p-value less than .05.
Localized Radiation Eliminates Actively Proliferating Cells in the SVZ
A series of pilot experiments was performed to validate the localized nature of the radiation beams from the SARRP. A 10-Gy, 3 × 3 mm2 field of localized radiation was delivered to the right ventricle while the nonirradiated left ventricle served as a control (Fig. 1A). The accuracy of localized radiation delivery in the targeted right SVZ region was validated by immunohistochemical staining of γH2Ax, a marker of DNA double-strand breaks, 1 hour post-irradiation. The irradiated right ventricle of the mouse brain showed restricted expression of γH2Ax (Fig. 1B). Cell proliferation in the SVZ was assessed by immunostaining against Ki67 in response to the localized radiation. A strong lateral asymmetry in cell proliferation was observed on every coronal section 1-day post-irradiation (Fig. 1C) and on SVZ whole mounts 1-month post-irradiation (Fig. 1D). There was a significant decrease in the number of Ki67+ cells in the right SVZ at 1 day (left SVZ: 85.05 ± 12.84 cells per section vs. right SVZ: 14.27 ± 2.35 cells per section) (Fig. 1E) and 1 month (left SVZ: 56 ± 5.26 cells per section vs. right SVZ: 7 ± 1.67 cells per section) (Fig. 1F) after irradiation (p < .001). Remarkably, the number of proliferating cells in the RMS and OB was not affected by localized radiation of the SVZ at either 1 day or 1 month after irradiation (Fig. 1E, 1F). These results indicate that the vast majority of proliferating cells in the mouse SVZ were permanently eliminated in a highly localized fashion using SARRP technology.
Localized Radiation of SVZ Decreases NPCs and Neuroblasts
We then explored the effect of localized radiation on different cell types in the SVZ, such as NSCs/type B1 cells, NPCs/type C cells, and migrating neuroblasts/type A cells, using cell specific markers and electron microscopy 1-month post-irradiation. Nestin and GFAP immunostaining was used to label type B cells, Mash-1 for type C cells, and DCx for migrating neuroblasts . While GFAP and nestin expression appeared to remain stable (Fig. 2A, 2C), there was a strong qualitative reduction of Mash-1+ cells (Fig. 2B) and DCx+ cells (Fig. 2C). The GFAP+ type B1 cells in the coronal sections were distinguished by their contact with the ventricular surface (arrowheads in Fig. 2A) and long distal processes (arrows in Fig. 2A) from the parenchymal astrocytes or type B2 cells that form glial tubes and are also positive for GFAP . GFAP and DCx costaining of lateral ventricle whole mounts further confirmed the presence of GFAP+ cells and the absence of DCx+ cells in the irradiated SVZ (Fig. 2D). These findings from SVZ immunostaining were further confirmed with electron microscopy, and quantitative analysis of different cell types was performed using electron microscopy (Fig. 3).
Light and electron microscope examination of the SVZ showed a clear disruption in both the antero-posterior and dorso-ventral axes in irradiated hemisphere 1 month after irradiation. While the cell organization in nonirradiated SVZ appeared normal, with a monolayer of ependymal cells and typical chains of migratory neuroblasts (dark cells) surrounded by type B cells (light cells), SVZ in irradiated hemisphere was reduced to ependymal cells with dispersed astrocytes and neuronal bodies close to the ventricle (Fig. 3A, 3C). There were no migratory chains after irradiation (Fig. 3A, 3C), but dispersed type A cells were occasionally observed. Consequently, cell density was drastically decreased in irradiated SVZ (left SVZ: 236.1 ± 26.2 cells per mm vs. right SVZ: 97.9 ± 6 cells per mm; p < .005) (Fig. 3B). This loss of cell density was attributed to a decrease in the number of type B cells (left SVZ: 76.8 ± 7.2 cells per mm vs. right SVZ: 38.2 ± 6.6 cells per mm; p = .004), type C cells (left SVZ: 25.5 ± 5.9 cells per mm vs. right SVZ: 0.5 ± 0.3 cells per mm; p = .01), and type A cells (left SVZ: 77.0 ± 17.0 cells per mm vs. right SVZ: 3.5 ± 0.6 cells per mm; p = .01), while ependymal cells remained unaltered (left SVZ: 46.9 ± 2.9 cells per mm vs. right SVZ: 46.8 ± 2.3 cells per mm) (Fig. 3D and supporting information Table S2). Most importantly, type B1 cells, identified by their apical process extending into the lateral ventricle and primary cilia, were observed in the right SVZ 1 month after irradiation (Fig. 3E, 3E′, and Table 2). Extensive portions of basal lamina were found frequently between astrocytes and/or ependymal cells in the irradiated SVZ, compared to nonirradiated hemisphere (Fig. 3F). As a result of the decreased cell density, neuropil structures were shifted medially toward the ependymal layer in the right SVZ following irradiation. Mylenic and amylenic axons, including synaptic contacts, were observed adjacent to the ependymal layer, establishing direct contact with ependymal and/or astrocytic cells in some cases (Fig. 3G). Together, these results indicate that the migrating neuroblasts were not replenished even 1 month after irradiation, despite the presence of type B cells in the SVZ post-irradiation (Figs. 2, 3E).
Localized Radiation Affects the Generation of NPCs and Migrating Neuroblasts from NSCs
The presence of type B1 cells after irradiation raised the question if their proliferation was intact. Double labeling of the SVZ against nestin and Ki67 1 month after radiation showed proliferation in few of the residual type B cells (Fig. 4A). These proliferating type B cells were further found to be in proximity to blood vessels (Fig. 4B). CD31 immunostaining for endothelial cells in the SVZ appeared to be no different between the control and irradiated SVZ (supporting information Fig. S1A). These results were also confirmed by electron microscopy, where the basal lamina and endothelial cells were no different between the left (unirradiated) SVZ and the right (irradiated) SVZ (supporting information Fig. S1B). There was no difference in the number of blood vessels (left SVZ: 66 ± 4.93 vs. right SVZ: 51 ± 7.57; p = .172) and in the size of the blood vessels (left SVZ: 50.93 ± 11.6 vs. right SVZ: 45.09 ± 7.29; p = .692) as quantified in the semithin section (supporting information Fig. S1C, S1D). Interestingly, when the dissected SVZ tissue was dissociated and cultured in vitro, the cells from the left SVZ gave rise to neurospheres, whereas the cells from the irradiated right SVZ failed to form neurospheres (Fig. 4C). Taken together, these results indicate that the residual type B cells are able to proliferate after irradiation but lack the ability to generate NPCs and migrating neuroblasts.
Localized Radiation of SVZ Affected the Size of the RMS
Although there was no significant difference in the number of proliferating cells in the RMS (Fig. 1F), there were fewer DCx+ migrating neuroblasts in the right RMS 1 month after localized right SVZ radiation as compared to the left RMS (Fig. 5B). In addition, there was a significant reduction in cross-sectional size of the RMS (left RMS: 9,892 ± 737 mm2 vs. right RMS: 7,202 ± 474 mm2) (Fig. 5C). Ultrastructural analysis with electron microscopy revealed no morphological alterations in cell populations of the RMS despite the reduction in size (Fig. 5D). Furthermore, we noticed few cells in the irradiated RMS in their mitotic phase, which based on their ultrastructural features, were identified as neuroblasts (Fig. 5D, inset).
Localized Radiation Disrupts the Migration of Neuroblasts
The anterior dorsal SVZ is one of the regions with the largest NSC population . In this experiment, we irradiated the anterior dorsal region of the SVZ with a single 10-Gy lateral beam of 1-mm diameter (supporting information Fig. S2A, S2B). Whole mount staining of SVZ against GFAP and DCx showed no neuroblasts in the irradiated region after 1 week (supporting information Fig. S2C). Additionally, no neuroblasts from the posterior dorsal SVZ were found to migrate into the irradiated region indicating that radiation may have affected the migration of neuroblasts in the SVZ (supporting information Fig. S2C′, S2C″). We then examined the effect of irradiation on anterior SVZ (aSVZ) while sparing the posterior SVZ. A 5 × 9 mm2 beam of 10 Gy was used to deliver radiation to the aSVZ, RMS, and OB (Fig. 6A). Immunostaining of SVZ whole mount against γH2Ax, 1-hour post-irradiation, showed a precise radiation field confined only to the aSVZ, sparing the posterior SVZ (Fig. 6B). DCx+ neuroblasts were seen 1 hour post-irradiation, but were absent after 1 day and 1 month post-irradiation (Fig. 6C). The neuroblasts from the posterior SVZ did not migrate through the irradiated aSVZ, consistent with our results in supporting information Figure S2. Furthermore, there were no apoptotic cells observed 1 week after localized irradiation of the anterior SVZ, as assessed by immunohistochemistry against activated Caspase-3 (data not shown).
To evaluate whether the migratory properties of neuroblasts were affected by irradiation, we injected GFP-expressing retrovirus into the SVZ to label NSCs and track the migratory pattern of newly generated neuroblasts, 1 day after radiation treatment. Three separate groups of mice received either no radiation or a single 10-Gy dose to their RMS/OB (sparing the entire SVZ) or to their aSVZ/RMS/OB. One day after irradiation, mice were injected with GFP-expressing retrovirus in the SVZ to label dividing NSCs and track the migration of neuroblasts (Fig. 7A). As predicted, sham-irradiated mice had GFP+ cells in the SVZ, RMS, and OB 1 week after injection (Fig. 7B, insets 1–4). Mice that received radiation to the RMS/OB showed GFP+ cells in their SVZ but not in RMS and OB (Fig. 7C, inset 5). Mice that received radiation to the aSVZ/RMS/OB showed no GFP+ cells (Fig. 7D). Quantification of Ki67+ cells further revealed that the RMS/OB irradiation did not affect the SVZ proliferation (supporting information Fig. S3). These results indicate that irradiation of the RMS alone (sparing SVZ) can inhibit the migration of neuroblasts. Our findings suggest that irradiation did not disrupt the intrinsic mechanism of neuroblast migration, but the local microenvironment permissive of cell migration.
The effect of radiation on NSC/NPC proliferation and neuroblast migration cannot be disregarded, given that radiation therapy is a first-line treatment for many brain tumors, including glioblastoma . Numerous studies have examined the decrease in proliferating cells in SVZ and subgranular zone after radiation [8, 9, 11, 36–38]. The antiproliferative effects last for several months after a single radiation treatment and are dose-dependent [9, 11, 37–39]. Most studies to date have been restricted to irradiating relatively large areas of the brain [7, 10, 39]. Very few studies used localized brain irradiation using custom-designed radiation shields or the Gamma Knife unit (Elekta Inc., Sweden) to irradiate subregions of the brain [9, 40–42]. We used SARRP technology, capable of delivering millimeter-scale beams to the mouse brain under CT guidance. This novel technology accurately mimics clinical radiation treatment, on the murine scale . The localized radiation treatment used in our study is validated by immunohistochemistry against γH2Ax, a marker for DNA strand breaks . Localized irradiation of right SVZ affected the proliferation in that region but not in the RMS and OB, further confirming the precise nature of the SARRP technology. There was a significant decrease in the proliferative population of cells, including NPCs and neuroblasts [24, 44], in the SVZ 1 day after localized radiation. The same effect was noticed even 1 month after localized radiation of SVZ (Fig. 1D, 1E). The decrease in the proliferative population after irradiation appeared to be due to the loss of NPCs and neuroblasts as shown by immunohistochemistry (Fig. 2B, 2C). To determine whether this was not due to changes in antibody binding efficiency, we used electron microscopy. The ultrastructural analyses from electron microscopy confirmed the elimination of NPCs and neuroblasts and the presence of NSCs in the SVZ after localized radiation. This could be explained by the quiescent nature of NSCs (cell cycle time ∼ 15 days) [44, 45]. Proliferating cells such as NPCs (cell cycle time ∼ 13 hours) and migrating neuroblasts are highly susceptible to ionizing radiation [9–11, 44, 45]. Our results on the depletion of NPCs and neuroblasts after radiation treatment are similar to the effects of antimitotic drugs such as cytosine-β-D-arabinofuranoside (AraC)  or the alkylating reagent N-ethyl-N-nitrosourea (ENU) on SVZ [46, 47]. The NPCs and neuroblasts regenerate from NSCs and replenish the entire SVZ in approximately 4 days after AraC treatment [44, 45]. In case of ENU-exposure, residual NSCs allowed a partial recovery after treatment [46, 47]. On the contrary, we did not observe the regeneration of NPCs and neuroblasts from NSCs even 1 month after localized radiation of SVZ in our study (Figs. 2, 3). This suggests that irradiation might have caused permanent damage to the NSCs and/or the microenvironment that supports the proliferation and maintenance of the NSCs [10, 48–52].
NSCs are known to associate closely with the vascular niche in the SVZ [50–52]. At higher doses of irradiation, the endothelial cells that support NSCs undergo apoptosis, resulting in vascular damage and decreased neurogenesis [53–55]. We did not notice any morphological changes in endothelial cells after localized irradiation, as shown by CD31 immunohistochemistry and electron microscopy. There was no difference in the number or size of blood vessels in the irradiated SVZ as compared to the unirradiated SVZ (supporting information Fig. S1). Furthermore, the radiation dose used in our study is less than that which has been shown to cause such vascular damage [49, 56]. Therefore, the inability of NSCs to give rise to NPCs cannot be attributed to vascular niche damage. However, we cannot rule out the possibility that, even though the gross morphology of endothelial cells is intact, the molecular signals that trigger NSCs to proliferate and give rise to NPCs could be affected by irradiation [57–59]. Basal lamina in the neurogenic niche is known to play a role in the proliferation of NSCs . There were no differences in the ultrastructural features of basal lamina between the irradiated and nonirradiated hemispheres. However, we found it more often and extensive in the irradiated right SVZ compared to left SVZ (Fig. 3F). The extensive basal lamina observed in our animals could be a response due to the cell loss post-irradiation. Cranial irradiation is known to disrupt the microenvironment and to activate reactive microglia, affecting neurogenesis [10, 48, 49, 60]. When NSCs isolated from irradiated brain were transplanted into normal brain, NSCs regained the ability to proliferate and gave rise to neuronal and glial cell lineages . In our study, we isolated cells from the irradiated and unirradiated SVZ and cultured them in NSC growth medium. While cells from irradiated SVZ remained as single cells, the cells from unirradiated SVZ divided and gave rise to neurospheres, nonadherent clusters of cells generated from NSCs in vitro  (Fig. 4C). Although the specific phenomena are not completely understood, our interpretation of these data is that immediately following irradiation, the rapidly dividing NPCs and migrating neuroblasts are eliminated and the NSC compartment becomes unable to fully repopulate. To better understand the effects of localized radiation on SVZ neurogenesis, detailed analysis of gene expression and/or molecular signaling pathways is warranted .
The ability of NPCs and neuroblasts to migrate to the site of damage and to participate in repair is well-documented [63–65]. Radiation may compromise this ability, since radiation kills NPCs and neuroblasts in the SVZ and also inflicts long-term damage to the NSCs in this region, as shown in this and previous studies [9–11, 37–39, 41, 60, 66]. Furthermore, we show that there are fewer neuroblasts in the right RMS 1 month after irradiating the right SVZ (Fig. 5). The few neuroblasts observed in the right RMS could have been generated locally from the multipotent, self-renewing NSCs present in the RMS that were not affected by SVZ irradiation (Fig. 1F) [67–69]. The depletion of migrating neuroblasts in the right SVZ after radiation also resulted in the reduction in the size of the right RMS, suggesting that the NSCs within the RMS does not compensate for the loss of migrating neuroblasts from the SVZ and also the number of neuroblasts generated in the SVZ can affect the size of the RMS [70, 71].
With recent advancements in clinical radiotherapy planning and delivery, neurogenic niches can be spared from radiation without compromising dose coverage of the tumor [29, 30]. We have recently shown that it is possible to spare the NSC niche during radiation therapy in a retrospective clinical radiation treatment planning study . NSCs may be used either to inhibit tumors by causing apoptosis or to repair tissue damage that results from radiation treatment [16–19, 65, 72]. Both uses rely on the migration of the neuroblasts to locations where they are needed, but very little is known about the effect of radiation on the migration of neuroblasts.
The anterior SVZ is one of the hotspots in the adult mouse brain with the highest number of NSC clusters . When the anterior SVZ was irradiated, we noticed that neuroblasts from the posterior SVZ did not migrate through the irradiated area (Fig. 6). Even when a part of the anterior dorsal SVZ received a radiation beam as small as 1 mm in diameter, neuroblasts did not migrate through the irradiated region of the SVZ (supporting information Fig. S2). This is important in the context of emerging studies on the transplantation of modified stem cells into the mouse brain to migrate and undergo repair at the site of damage [17, 19, 65, 70]. To further confirm these findings, we labeled NSCs locally in the SVZ with GFP-expressing retrovirus 1 day after irradiation of RMS/OB or SVZ/RMS/OB. The inclusion of RMS/OB irradiation allowed us to study the effect of radiation on the migrating neuroblasts when the SVZ is spared from irradiation. The GFP-expressing cells from the SVZ failed to migrate through the RMS in the RMS/OB-irradiated mice, indicating that neuroblasts did not follow their normal migratory path through the irradiated SVZ-RMS-OB route (Fig. 7). This migratory alteration is probably not due to an intrinsic disruption in the migratory machinery of new-formed neuroblasts, but rather due to a disturbance of parenchymal migratory signaling in situ [59, 73–78]. To our knowledge, this is the first report showing a strong inhibitory effect on the migration of neuroblasts after localized radiation. The mechanisms involved in this effect are unknown. At this point, we cannot predict if this inhibitory effect on neuroblast migration after irradiation will be seen outside the SVZ-RMS-OB migration route. Also, we cannot disregard the possibility that the effect of RMS/OB irradiation would have functional impact on the SVZ, despite the proliferation in the SVZ was not compromised in these mice compared to sham mice. Although our data on the effects of localized radiation on SVZ NSCs, NPCs, and neuroblast migration are striking, much more work is needed before the mechanisms are fully understood and the clinical implications of the use of NSCs for damage repair in brain tumor patients can be fully appreciated.
In summary, we show that (a) SVZ-restricted irradiation affects the proliferation of type C and A cells in a highly localized manner; (b) the residual type B cells lack the ability to regenerate NPCs, which in turn give rise to migrating neuroblasts; (c) SVZ-restricted irradiation reduces the size of the RMS; and (d) neuroblasts failed to migrate through the irradiated regions of the SVZ-RMS-OB route. Future studies are indicated to investigate the effects of localized radiation not only on the molecular signals involved in the proliferation of NSCs/NPCs but also on those involved in neuroblast migration. This will allow for a thorough understanding of the effects of localized irradiation on the neurogenic regions of the brain both at the cellular and molecular levels to better improve radiotherapy protocols in the clinical setting.
This research was supported by the Maryland Stem Cell Research Fund (A.Q.H.), The Robert Wood Johnson Foundation (A.Q.H.), The Howard Hughes Medical Institute (A.Q.H.), and The National Institutes of Health (A.Q.H.). Dr. Oscar Gonzalez-Perez was supported in part by the Consejo Nacional de Ciencia y Tecnologia's grant (CONACyT, CB-2008-101476)) and Dr. Jose Manuel Garcia-Verdugo by the Prometeo grant (GVPROMETEO-2009/011). We thank Dr. Zaman Mirzadeh for technical help with whole mount dissections. We thank Dr. Michael Bonaguidi for fruitful discussions on the experimental design and data interpretation. We thank Dr. Hugo Guerrero-Cazares for helpful technical hints and critical reading of manuscript. E.F. is currently affiliated with the Department of Radiation Oncology, University of Washington Medical Center, Seattle, Washington, DC.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.