TISSUE-SPECIFIC STEM CELLS Subventricular Zone Localized Irradiation Affects the Generation of Proliferating Neural Precursor Cells and the Migration of Neuroblasts a a a c d PRAGATHI ACHANTA, VIVIAN CAPILLA-GONZALEZ, DAVID PURGER, JUVENAL REYES, KURT SAILOR, d b e c HONGJUN SONG, JOSE MANUEL GARCIA-VERDUGO, OSCAR GONZALEZ-PEREZ, ERIC FORD, a ALFREDO QUINONES-HINOJOSA aDepartment of Neurosurgery, cDepartment of Radiation Oncology and Molecular Radiation Sciences, and dInstitute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; bLaboratory of Comparative Neurobiology, Instituto Cavanilles, University of Valencia, Valencia, CIBERNED, Spain; eLaboratory of Neuroscience, Facultad de Psicologia, Universidad de Colima, Colima, Mexico Key Words. Radiation therapy • Neural stem cells • Migrating neuroblasts • Subventricular zone ABSTRACT Radiation therapy is a part of the standard treatment for the ability to generate migrating neuroblasts. Neuroblasts brain tumor patients, often resulting in irreversible neuro- from the unirradiated posterior SVZ did not follow their psychological deficits. These deficits may be due to perma- normal migratory path through the irradiated anterior nent damage to the neural stem cell (NSC) niche, damage SVZ. Our results indicate that the migrating neuroblasts to local neural progenitors, or neurotoxicity. Using a com- were not replenished, despite the presence of type B cells puted tomography-guided localized radiation technique, in the SVZ post-irradiation. This study provides novel we studied the effects of radiation on NSC proliferation insights into the effects of localized SVZ radiation on neu- and neuroblast migration in the mouse brain. Localized rogenesis and cell migration that may potentially lead to irradiation of the subventricular zone (SVZ) eliminated the development of new radiotherapy strategies to mini- the proliferating neural precursor cells and migrating neu- mize damage to NSCs and neuroblast migration. STEM roblasts. After irradiation, type B cells in the SVZ lacked CELLS 2012;30:2548–2560 Disclosure of potential conflicts of interest is found at the end of this article. nervous system, such as neurons, oligodendrocytes, or astro- INTRODUCTION cytes [12–15]. Damaging NSCs during radiation therapy can have detrimental effects [4, 7]. Mitigating the neurotoxic Brain irradiation is an integral component of the treatment of effects of radiation is important particularly in the context of patients with brain tumors [1, 2]. Despite its effectiveness in brain tumor patients who survive longer due to new combina- controlling the progression of some cancers, radiation is often tion therapies [1, 2, 6]. Furthermore, NSCs have the potential associated with neuropsychological deficits [3–6]. These defi- to be used to treat brain cancer in two different ways. First, cits may be partly due to radiation-induced damage to neuro- endogenous NSCs in the brain can participate in the repair of genic niches, as shown in animal models [7–11]. In the adult normal tissue and/or inhibit tumor growth [16]. Second, engi- mammalian brain, these niches are localized in the subventric- neered stem cells may be used to deliver cytotoxic agents and ular zone (SVZ) and the subgranular zone of the hippocampal prodrug-activating enzymes to the tumor [17–21]. Both of dentate gyrus [12–14]. The neural stem cells (NSCs) in these these possibilities require that NSCs generate neuroblasts to two regions are of great interest for therapeutic applications migrate toward the tumor site, a process that is not well- in the brain because of their ability to proliferate and produce understood in the context of radiation therapy. new cells. These newly generated cells are able to migrate The SVZ is the largest source of NSCs in the adult brain, and differentiate into any of the main cell types of the central located underneath the layer of ependymal cells lining the 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. Correspondence: Alfredo Quinones-Hinojosa, M.D., Department of Neurosurgery, Johns Hopkins University, 1550 Orleans St, CRB-II, Room 247, Baltimore, Maryland 21201, USA. Telephone: 410-502-2869; Fax: 410-502-7559; e-mail: [email protected] Received April 13, 2012; accepted for publication July 21, 2012; first published online in STEM CELLS EXPRESS September 4, 2012. VC AlphaMed Press 1066-5099/2012/$30.00/0 doi: 10.1002/stem.1214 STEM CELLS 2012;30:2548–2560 www.StemCells.com Achanta, Capilla-Gonzalez, Purger et al. 2549 ventricles [13, 22–24]. NSCs, or type B1 cells in the SVZ, are and were frozen in Tissue-Tek OCT Compound. Ten-microme- astrocyte-like glial fibrillary acidic protein (GFAPþ) cells that ter-thick coronal slices were sectioned using a cryostat. Sections divide and give rise to transit amplifying neural precursor cells were treated with 0.01 M sodium citrate at 95Cfor10minutes (NPCs), or type C cells, which in turn give rise to migrating neu- for antigen retrieval. Sections were incubated in PBS containing roblasts, or type A cells [25]. Neuroblasts migrate long distances 0.1% Triton X-100, and 10% normal goat serum (NGS) for 30 through the rostral migratory stream (RMS) before differentiating minutes, and then incubated with primary antibodies for 16 into olfactory bulb (OB) interneurons [26, 27]. The mouse SVZ- hours at 4C. Primary antibodies used in this study were mouse RMS-OB migration route represents a well-characterized system anti-rH2Ax (Ser139) (1:700), rabbit anti-Ki67 (1:200), mouse of endogenous neuroblast migration that serves as a model to anti-GFAP (1:500), rabbit anti-doublecortin (DCx) (1:500), study migration in humans receiving radiotherapy [27, 28]. mouse anti-nestin (1:100), mouse anti Mash-1 (1:100), cleaved Current radiotherapy treatments, such as intensity-modu- caspase-3 (1:100), and mouse anti-CD31 (1:200). More details lated radiation therapy, are intended to deliver a maximum on the primary antibodies are summarized in supporting infor- dose of radiation to the tumor while reducing radiation expo- mation Table S1. The sections were washed with PBS and incu- sure to the surrounding normal tissue [29, 30]. To more accu- bated with secondary antibodies conjugated with fluorophores rately reproduce human radiotherapy, we used a small animal for 1 hour at room temperature (RT). Anti-mouse, anti-rat, and radiation research platform (SARRP) developed in-house [31]. anti-rabbit Alexa 488 and/or 594 secondary antibodies (1:500; Using this technology, we recently reported that local irradia- Invitrogen, Green Island, NY, USA) were used. Nuclei were tion of hippocampus significantly decreased the number of stained with 4’,6-diamidino-2-phenylindole (DAPI) (1:500) All proliferating cells in the irradiated dentate gyrus compared to the sections were air-dried and coverslipped with .Aquamount the unirradiated side [32]. Furthermore, the number of new- mounting media (Vector Labs, Burlingame, CA, USA). Fluores- born cells differentiating into mature neurons 2 months after cent images were taken with an ORCA II camera (Hamamatsu) irradiation was significantly reduced [32]. In this study, we connected to an Olympus IX81 inverted microscope. examined the effect of SVZ-restricted irradiation on NSC and NPC proliferation and neuroblast migration in the adult mouse SVZ Whole Mount Dissection and Immunostaining SVZ-RMS-OB system. The two main findings from this study Mice (n ¼ 3 per group) were sacrificed by cervical dislocation and are that type B cells lacked the ability to regenerate NPCs their brains were immediately extracted into L-15 Leibovitz and neuroblasts even a month after irradiating SVZ, and that medium (Gibco, Green Island, NY, USA). A whole mount of SVZ neuroblasts failed to migrate through the irradiated regions. tissue (2–4 mm) with the lateral ventricle and striatum was dis- These results will help inform future radiotherapy protocols to sected under microscope, and the hippocampus and septum were minimize damage to neurogenic niches in the human brain. removed as described [24, 33]. The SVZ whole mounts were fixed in 4% PFA/0.1% Triton X-100 overnight at 4C. They were then washed with PBS and blocked with 10% NGS/0.5% Triton for 30 MATERIALS AND METHODS minutes at RT. The tissues were incubated in primary and second- ary antibodies diluted in blocking buffer for 48 hours at 4C. After Subjects staining, the SVZ whole mounts were further dissected from l The subjects were 6–8-week-old male C57BL6/J mice from the underlying striatum to 200–300- m thick tissue, transferred to a glass slide, and coverslipped with