YEXNR-11641; No. of pages: 11; 4C: Experimental Neurology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr

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Yeonghoon Son a,f,1, Miyoung Yang a,d,f,1, Joong-Sun Kim b, Juhwan Kim a,f, Sung-Ho Kim a,f, Jong-Choon Kim a,f, Taekyun Shin c, Hongbing Wang d, Sung-Kee Jo e, Uhee Jung e, Changjong Moon a,f,⁎

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Hippocampal dysfunction during the chronic phase following a single exposure to cranial irradiation

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Department of Veterinary Anatomy, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, South Korea Research Center, Dongnam Institute of Radiological & Medical Sciences (DIRAMS), Busan 619-953, South Korea Department of Veterinary Anatomy, College of Veterinary Medicine, Jeju National University, Jeju 690-756, South Korea d Department of Physiology and Neuroscience Program, Michigan State University, MI 48824, USA e Radiation Research Division for Bio-Technology Institute, Jeongeup Campus of Korea Atomic Energy Research Institute, Jeonbuk 580-185, South Korea f Department of Toxicology, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, South Korea b

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Article history: Received 5 December 2013 Revised 21 January 2014 Accepted 26 January 2014 Available online xxxx

a b s t r a c t

Ionizing radiation can significantly affect brain functioning in adults. The present study assessed depression-like behaviors in adult C57BL/6 mice using the tail suspension test (TST) at 30 and 90 days following a single cranial exposure to γ-rays (0, 1, or 10 Gy) to evaluate hippocampus-related behavioral dysfunction during the chronic phase following cranial irradiation. Additionally, hippocampal neurogenesis, inflammatory cytokines, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF) were analyzed. At 30 and 90 days following irradiation with 10 Gy, mice displayed significant depression-like behaviors. We observed a persistent decrease in the number of cells positive for doublecortin, an immunohistochemical marker for neurogenesis, in the hippocampus from 1 to 90 days after irradiation with 10 Gy. Changes in the mRNA expression of inflammatory cytokines, including interleukin (IL)-1β, tumor necrosis factor-α, IL-6, and interferon-γ, were not correlated with the decrease in hippocampal neurogenesis or the appearance of depression-like behavior during the chronic phase following irradiation. However, at 30 and 90 days after irradiation with 10 Gy, the number of microglia was significantly decreased compared with age-matched sham-irradiated controls. The reduction in the chronic phase was consistent with the significant down-regulation in the mRNA expression of iNOS, COX-2, BDNF, and GDNF in the hippocampus. Therefore, hippocampal dysfunction during the chronic phase following cranial irradiation may be associated with decreases in the neurogenesis- and synaptic plasticity-related signals, concomitant with microglial reduction in the hippocampus. © 2014 Published by Elsevier Inc.

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Introduction

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The hippocampus is located within the ventromedial aspect of the temporal cortex in the brain and plays important roles in memory and emotional regulation (Richardson et al., 2004; Richter-Levin, 2004; Squire, 1992; Zola-Morgan and Squire, 1993). It is established that new neurons are produced in the subgranular zone (SGZ) of the dentate gyrus (DG) within the hippocampus and that neural stem cells are produced throughout life (Altman and Das, 1965). When necessary, stem cells from these specific areas of the brain can migrate and repopulate damaged areas following brain stimulation (Doetsch et al., 1999; Morshead et al., 1994). The rate of neurogenesis in the

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Keywords: Ionizing radiation Hippocampus Depression-like behavior Neurogenesis Microglia Neurotrophic factor

⁎ Corresponding author: Department of Veterinary Anatomy, College of Veterinary Medicine, Chonnam National University, 300 Yongbong-Dong, Buk-Gu, Gwangju 500757, South Korea. Fax: +82 62 530 2941. E-mail address: [email protected] (C. Moon). 1 The first two authors equally contributed to this study.

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hippocampus likely plays a pivotal role in hippocampus-dependent functions, including learning and memory and regulation of emotion (Bruel-Jungerman et al., 2005; Fike et al., 2009; Sahay and Hen, 2007; Shors et al., 2001), and it may be altered by several factors, including chemicals (Seo et al., 2010; Yang et al., 2010), radiation (Kim et al., 2008), and environmental enrichment (van Praag et al., 1999). The brain may be exposed to ionizing irradiation following nuclear accidents or during space travel, atomic weapon testing and use, and medical treatment. Although the adult brain is less vulnerable to irradiation than other organs, it is likely that even relatively low doses of irradiation can induce cognitive impairment (Butler et al., 2006; Kim et al., 2008; Meyers and Brown, 2006). Moreover, in adults, cranial irradiation can induce a variety of side effects, including long-lasting declines in cognitive functioning (Abayomi, 1996; Robison et al., 2005), even if few structural changes occur (Sheline et al., 1980; Snyder et al., 2005). In experimental animals, acute irradiation has resulted in the transient or prolonged loss of proliferating cells in the DG, as well as in learning and memory impairment (Kim et al., 2008;

0014-4886/$ – see front matter © 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.expneurol.2014.01.018

Please cite this article as: Son, Y., et al., Hippocampal dysfunction during the chronic phase following a single exposure to cranial irradiation, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.01.018

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Male C57BL/6 mice (8 weeks of age; Daihan-Biolink Co.; Chungbuk, Korea) were housed in a room maintained at 23 ± 2 °C with a relative humidity of 50 ± 5%, artificial lighting from 08:00 to 20:00 h, and 13–18 air changes every hour. The animals were given ad libitum access to tap water and commercial rodent chow (Jeil Feed Co.; Daejeon, Korea). After acclimatization, mice were randomly divided into three groups (n = 36 mice for sham-irradiated group, n = 60 mice for the

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Irradiation and tissue sampling

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Animals

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The mice were anesthetized with tiletamine/zolazepam (Zoletil 50®; Virak Korea; Seoul, Korea) and immobilized, and single radiation fractions of 1 and 10 Gy apiece were delivered through the whole brain. The brain received whole-brain irradiation using six MV highenergy photon rays (ELEKTA; Stockholm, Sweden) at a dose rate of 3.8 Gy/min. A radiation dosimeter (Semiflex Ionization Chamber 31013, PTW Co., Freiburg, Germany) was used to determine that the radiation doses ranged from 99 to 100% at a point 3 cm below the surface of the simulated mouse head. We aligned the center of the head to the beam line center using the mouse holder. The distance of the skin from radiation source was 1 m. Sham-irradiated mice were anesthetized with tiletamine/zolazepam (Zoletil 50®) and immobilized for the same period of time without irradiation. Mice from each group were subdivided (1 and 2 experiments, Fig. 1): (1) Eight mice per group were used for hippocampal collection to extract mRNA. In the chronic phase, mice were subjected to TST, followed by hippocampal collection for mRNA extraction 4 h later; (2) Four mice per group were sacrificed, and the brains were quickly removed and divided at the midline. The left hemisphere was fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for immunohistochemistry and the hippocampus was removed from the right hemisphere and immediately stored at −70 °C for mRNA extraction.

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Tail suspension test (TST)

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The TST was conducted as reported previously (Steru et al., 1985). Briefly, mice were suspended from a plastic rod mounted 50 cm above a surface by fastening the tail to the rod with adhesive tape. Immobility was measured for 6 min using the SMART video-tracking system (Panlab; Barcelona, Spain).

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Immunohistochemistry

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The immunohistochemical procedures were performed as described previously (Kim et al., 2008; Yang et al., 2011; 2010). Briefly, after 0-, 1-, or 10-Gy irradiation (n = 4 mice/group), 5-μm thick sagittal sections were deparaffinized, hydrated, and allowed to react with polyclonal rabbit anti-DCX (1:400; Cell Signaling Technology; Beverly, MA, USA), anti-GFAP (1:2000; Dako; Glostrup, Denmark), and anti-Iba-1 (1:1000; Wako; Osaka, Japan) for 2 h at room temperature (RT). Then, the sections were reacted with biotinylated goat anti-rabbit IgG (Vector ABC Elite Kit; Vector; Burlingame, CA, USA) for 60 min at RT. Immunoreactivity was performed for 60 min at RT using the avidin–biotin peroxidase complex (Vector ABC Elite Kit) prepared according to the manufacturer's instructions, and the peroxidase reaction was developed using a diaminobenzidine substrate kit (SK-4100; Vector). As a control, the primary antibodies were omitted for a few test sections in each experiment. All sections were counterstained with Harris' hematoxylin prior to being mounted.

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Materials and methods

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1- and 10-Gy-irradiated groups), and further subdivided into 13 groups (n = 12 mice/group) based on the days after irradiation (1, 2, 8, 30, and 90 days). To minimize the number of mice used in this study, the same group was used for the acute phase sham-irradiated control (1, 2, and 8 days). The Institutional Animal Care and Use Committee of Chonnam National University approved the protocols used in this study (CNU IACUC-YB-2012-38), and the animals were cared for in accordance with internationally accepted principles for laboratory animal use and care as found in the National Institutes of Health Guidelines (USA). The number of animals used and the suffering caused was minimized in all experiments.

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Raber et al., 2004). The acute effects of 2 and 4 Gy irradiation on adult neurogenesis, and the associated memory deficits, were fully reversed 2 weeks and 1 month later, respectively (Ben Abdallah et al., 2007; Kim et al., 2008). However, radiation-induced reductions in hippocampal neurogenesis have also been associated with cognitive deficits in young adult rodents 3 months after a single exposure to wholebrain irradiation with 5 or 10 Gy (Raber et al., 2004; Rola et al., 2004). Thus, cranial irradiation with relatively high doses (≥5 Gy) may result in the prolonged reduction of hippocampal neurogenesis and may also be related to delayed cognitive impairments. It has been suggested that this process is associated with neuroinflammation (Hong et al., 1995; Kyrkanides et al., 2002; Monje et al., 2003; Rola et al., 2008). Previous studies have reported that excessive high-dose irradiation (25–35 Gy) induced an up-regulation of pro-inflammatory cytokines such as interleukin (IL)-1β, tumor-necrosis factor (TNF)-α, IL-6, and interferon (IFN)-γ during the hyper-acute phase of 4 or 6 h (Hong et al., 1995; Kyrkanides et al., 2002). In contrast, a loss of microglia was detected in the subacute phase 7 days following a single dose of 8 Gy to immature rat brain (Kalm et al., 2009) and at 1 and 10 weeks following a single 10 Gy dose to adult rats (Schindler et al., 2008). Thus, there is some controversy regarding the effects of inflammation on the reduction in hippocampal neurogenesis and associated hippocampal functions following irradiation, requiring further investigations of specific time-point and radiation-dose analyses. Nevertheless, the precise mechanisms associated with hippocampal dysfunction following cranial irradiation remain unknown. Neurotrophic factors play critical roles in the synaptic activity and plasticity of mature neurons (Murer et al., 2001), as well as in the proliferation, differentiation, and survival of neurons in the central nervous system (McAllister, 2001). Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) genes are abundantly expressed in the mouse hippocampus (McCarthy, 2006) and have important roles in the development and function of dopaminergic neurons and the regulation of a variety of functions (Lapchak et al., 1996; Lewin and Barde, 1996; Shen et al., 1997). Decreased BDNF expression in the hippocampus is related to cognitive deficits (Gooney et al., 2004; Hattiangady et al., 2005; Linnarsson et al., 1997), and previous research suggests that depression and neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease, are linked to a lack of neurotrophic factors, including BDNF and GDNF (Altar, 1999; Shirayama et al., 2002; Siegel and Chauhan, 2000). However, there is a relative lack of knowledge concerning the possible involvement of BDNF and GDNF in hippocampal dysfunction following cranial irradiation. Thus, the present study assessed depression-like behavior using the tail suspension test (TST) and measured levels of doublecortin (DCX), an immature progenitor neuron marker, using immunohistochemistry in adult mice following cranial exposure to γ-rays (1 or 10 Gy). Additionally, changes in the expression levels of inflammatory cytokines (IL-1β, TNF-α, IL-6, and IFN-γ), pro-inflammatory enzymes, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), markers for microglia (Iba-1) and astrocytes (glial fibrillary acidic protein [GFAP]), and neurotrophic factors, including BDNF and GDNF were examined in the mouse hippocampus.

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Y. Son et al. / Experimental Neurology xxx (2014) xxx–xxx

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Please cite this article as: Son, Y., et al., Hippocampal dysfunction during the chronic phase following a single exposure to cranial irradiation, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.01.018

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Fig. 1. Schematic diagram of the experimental procedure. Filled and open arrowheads indicate the times of tissue collection from sham-irradiated (0 Gy) controls and the 1- or 10-Gyirradiated group, respectively.

RNA extraction and cDNA synthesis

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Total RNA was isolated from the hippocampus using an RNeasy® Lipid Tissue Mini Kit (Qiagen; Valencia, CA, USA) according to the manufacturer's instructions. The concentration of RNA samples was ascertained by measuring optical density using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific; Waltham, MA, USA). Firststrand complementary DNA (cDNA) was prepared using random primers (TaKaRa Bio Inc.; Tokyo, Japan) with Superscript™ II reverse transcriptase (Invitrogen; Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA was diluted to 8 ng/μL with RNase-free water and stored at −70 °C.

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Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

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qRT-PCR amplifications were performed using TOPreal™ qPCR 2 × PreMIX SYBR Green (Enzynomics; Daejeon, Korea) on a Stratagene

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Statistical analysis

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The data are reported as mean ± SE. Non-repeated-measures twoway analysis of variance (ANOVA) was used to test for the main effects of time (1, 2, 8, 30, and 90 days after irradiation) and dose (0, 1, and 10 Gy) and the interactions between them. The Newman–Keuls post hoc test was used to test for multiple comparisons when warranted by a significant omnibus F-statistic. The ANOVA results are provided in Table 2, and the results of the test for multiple comparisons are provided in the relevant figures below. In all analyses, a p-value less than 0.05 was deemed to indicate statistical significance.

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Results

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Cranial irradiation-induced depression-like behavior during the chronic phase

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The TST, which is a hippocampus-related behavioral paradigm, is useful for the assessment of depression-like behavior and the activity of antidepressants (Seo et al., 2010; Steru et al., 1985; Yang et al., 2011). The sham-irradiated (0 Gy) controls and mice irradiated with 1 or 10 Gy were examined using the TST at 30 and 90 days following irradiation (Fig. 2). The duration of immobility during the TST was increased in a dose-dependent manner, although there were no significant differences in mice irradiated with 1 Gy. Immobility times were significantly longer in mice irradiated with 10 Gy at 30 days (176.4 ± 10.3 s; n = 8, p b 0.01) and at 90 days (209.6 ± 10.6 s; n = 8, p b 0.001) post-irradiation than in sham-irradiated controls (30 days: 106.8 ± 11.8 s, n = 8; 90 days: 117 ± 21.3 s, n = 8). This indicates that cranial irradiation induces depression-like behavior during the chronic phase.

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The brain from each mouse was sampled at approximately 1.56 mm lateral to the midline, and a standardized counting area that contained 5-μm thick sagittal sections from a 1-in-10 series of sections representing the rostral/mid-hippocampus was used. For each mouse, two non-overlapping sections, one from each of the two regions of the hippocampus approximately 50 μm apart were analyzed. For quantification of immunopositive cells, an observer blinded to the sample identity scored the number of cells. For counting of DCXpositive cells, the number of positive cells in the hippocampus showing specific characteristics of immature progenitor cells immunopositive for DCX was scored using a histomorphometric approach (Kim et al., 2009; Yang et al., 2011; 2010). The numbers of immunopositive cells, as determined from the values obtained from each DG in the two brain sections, within the SGZ of the supra- and infra-pyramidal blades of the DG were quantified. Cells located in the granular cell layer (GCL) adjacent to the hilus of the DG in brain section and exhibiting cytoplasmic staining for DCX were counted as DCX-immunopositive if the nuclei were counterstained with hematoxylin. The number of immunopositive cells in the two sections of each mouse was averaged and expressed as the mean ± standard error (SE) for each group (n = 4). For quantifying GFAP- and Iba-1-positive cells, the numbers of positive cells with a detectable cell body in the dentate GCL, molecular layer, and hilus in stained sections were counted by a blinded observer. The mean number of immunopositive cells in two non-overlapping sections from each mouse was taken as n = 1. The number of immunopositive cells per group was averaged and expressed as the mean ± SE (n = 4 per group) and number of cells per 1 mm2.

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MX3000P (Agilent Technologies; Palo Alto, CA, USA) according to the manufacturer's instructions. The thermal cycling profile consisted of a preincubation step at 94 °C for 10 min, followed by 45 cycles of denaturation (94 °C, 15 s), annealing (55–58 °C, 30 s), and elongation (72 °C, 20 s). Melting curves were subsequently carried out by heating the product at 94 °C for 15 s, cooling it to 50 °C for 30 s, and then slowly heating it to 94 °C in increments of 0.5 °C. A melt-curve analysis was conducted to verify that only one product was amplified. The gene primer sequences for qRT-PCR are presented in Table 1. The readings were normalized using the housekeeping gene glyceraldehyde3-phosphate dehydrogenase (GAPDH) and the reference dye included in the SYBR master mix. The threshold generated by the software was used to calculate specific mRNA expression with the cycle at threshold (Ct) method, and the results were expressed as fold change over the control for each transcript using the 2−ΔΔCT method.

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Cell counts

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Please cite this article as: Son, Y., et al., Hippocampal dysfunction during the chronic phase following a single exposure to cranial irradiation, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.01.018

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Table 1 Primer sequences for real-time qPCR analysis.

t1:1

Gene

GenBank® accession number

Primer sequence

t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1 t1:1

IL-1β

NM_008361.3

TNF-α

NM_013693.2

IL-6

NM_031168.1

IFN-γ

NM_008337.3

iNOS

NM_010927.0

COX-2

NM_011198.3

Iba-1

D86382.1

GFAP

NM_001131020.1

BDNF

NM_001048142.1

GDNF

NM_010275.2

GAPDH

NM_008084.2

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

Amplicon size 5′-CTCGCAGCAGCACATCAACAAG-3′ 5′-CCACGGGAAAGACACAGGTAGC-3′ 5′-CATCTTCTCAAAATTCGAGTGACAA-3′ 5′-TGGGAGTAGACAAGGTACAACCC-3′ 5′-TGGAGTCACAGAAGGAGTGGCTAAG-3′ 5′-TCTGACCACAGTGAGGAATGTCCAC-3′ 5′-TTCTTCAGCAACAGCAAGGC-3′ 5′-ACTCCTTTTCCGCTTCCTGA-3′ 5′-TTGAAATCCCTCCTGATCTTGT-3′ 5′-TCACAGAAGTCTCGAACTCCAA-3′ 5′-GAAGTCTTTGGTCTGGTGCCTG-3′ 5′-GTCTGCTGGTTTGGAATAGTTGC-3′ 5′-CGAGGAGACGTTCAGCTACT-3′ 5′-CCAGTTGGCCTCTTGTGTTC-3′ 5′-AAGGTTGAATCGCTGGAGGA-3′ 5′-ACCACTCCTCTGTCTCTTGC-3′ 5′-TGGCTGACACTTTTGAGCAC-3′ 5′-GTTTGCGGCATCCAGGTAAT-3′ 5′-AGGCCCAGCTACAGAAAACT-3′ 5′-ACCACAGGAGATACACTGGC-3′ 5′-TCCATGACAACTTTGGCATT-3′ 5′-GTTGCTGTTGAAGTCGCAGG-3′

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exposed to 1-Gy irradiation, but persistent in those exposed to 10-Gy 295 irradiation (Fig. 3C). 296 mRNA expression of inflammatory cytokines in the mouse hippocampus 297 after cranial irradiation 298

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To evaluate the effects of cranial irradiation on adult hippocampal neurogenesis, levels of DCX, an immunohistochemical marker of neurogenesis, were examined in the DG of brain sections at 1, 2, 8, 30 and 90 days post-irradiation. Representative photographs show the dose- and time-dependent distribution of DCX-positive cells in the hippocampal DG with irradiation (Fig. 3A). DCX-positive cells in the sham-irradiated mice had well-developed dendrites with tertiary branches that extended into the GCL of the DG in brain sections, but this morphological feature was not observed in 10-Gy-irradiated mice (Fig. 3A). The number of DCX-positive cells in the hippocampus of sham-irradiated (0 Gy) controls decreased gradually from 1 to 90 days post sham-irradiation in a time-dependent manner, indicating an aging-related decrease in hippocampal neurogenesis (Fig. 3B). In mice irradiated with 1 Gy, the number of DCX-positive cells was significantly reduced from 1 to 8 days after irradiation, compared with shamirradiated controls (Fig. 3B). However, there were no differences in the number of immunopositive cells between sham- and 1-Gy-irradiated mice at 30 and 90 days after irradiation. In contrast, in mice irradiated with 10 Gy, the number of DCX-positive cells began to decrease significantly in the hippocampal DGs within 1 day post-irradiation; the reduction continued up to 90 days after irradiation (Fig. 3B). Thus, the reduction in hippocampal neurogenesis tended to be transient in mice

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Table 2 Results of two-way ANOVA testing for effects of time and dose on the radiation-induced change in each dependent variable.

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t2:2 t2:2 t2:2 t2:2 t2:2 t2:2 t2:2 t2:2 t2:2 t2:2 t2:2 t2:2 t2:2 t2:2 t2:2

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To examine changes in the inflammatory response in the mouse hippocampus following cranial irradiation, mRNA expression of the inflammatory cytokines IL-1β, TNF-α, IL-6, and IFN-γ was assessed using qRT-PCR. There were no differences in the expression levels of IL-1β (Fig. 4A), TNF-α (Fig. 4B), IL-6 (Fig. 4C) and IFN-γ (Fig. 4D) among sham-, 1- and 10-Gy-irradiated mice at each time-point. This indicates that marked alterations in inflammatory cytokines did not occur in the hippocampus after cranial irradiation.

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Time spent to immobile DCX-positive cells IL-1β mRNA levels TNF-α mRNA levels IL-6 mRNA levels IFN- γ mRNA levels iNOS mRNA levels COX-2 mRNA levels Iba-1-positive cells Iba-1 mRNA levels GFAP-positive cells GFAP mRNA levels BDNF mRNA levels GDNF mRNA levels

Figure

Dose

Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.

F(2,42) F(2,39) F(2,91) F(2,91) F(2,91) F(2,91) F(2,91) F(2,91) F(2,39) F(2,39) F(2,39) F(2,39) F(2,91) F(2,91)

2 3B 4A 4B 4C 4D 5A 5B 6B 6C 7B 7C 8A 8B

To examine the effects on pro-inflammatory enzymes, iNOS and COX-2 mRNA levels were assessed using qRT-PCR. The expression of iNOS decreased significantly from 1 to 90 days following irradiation with 10 Gy (Fig. 5A). However, there were no significant differences in iNOS expression between 1-Gy-irradiated mice and sham-irradiated controls. A significant down-regulation of COX-2 was observed from 8 to 90 days following irradiation with 10 Gy, although the expression

Time = = = = = = = = = = = = = =

17.163, p b 0.001 235.51, p b 0.001 1.29, p = 0.281 0.32, p = 0.724 1.92, p = 0.152 0.14, p = 0.867 18.71, p b 0.001 14.74, p b 0.001 16.68, p b 0.001 4.78, p = 0.014 1.63, p = 0.208 2.33, p = 0.111 12.27, p b 0.001 3.30, p = 0.041

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mRNA expression of pro-inflammatory enzymes in the mouse hippocampus 307 after cranial irradiation 308

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Cranial irradiation induced a transient or persistent decrease in hippocampal neurogenesis

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F(1,42) F(4,39) F(4,91) F(4,91) F(4,91) F(4,91) F(4,91) F(4,91) F(4,39) F(4,39) F(4,39) F(4,39) F(4,91) F(4,91)

Interaction = = = = = = = = = = = = = =

2.991, p = 0.091 49.51, p b 0.001 1.89, p = 0.118 0.66, p = 0.625 5.20, p = 0.001 0.18, p = 0.949 1.22, p = 0.308 3.94, p = 0.005 21.90, p b 0.001 2.29, p = 0.077 1.03, p = 0.402 11.14, p b 0.001 2.99, p = 0.023 0.88, p = 0.479

F(2,42) F(6,39) F(6,91) F(6,91) F(6,91) F(6,91) F(6,91) F(6,91) F(6,39) F(6,39) F(6,39) F(6,39) F(6,91) F(6,91)

= = = = = = = = = = = = = =

0.379, p = 0.687 12.92, p b 0.001 0.87, p = 0.520 0.42, p = 0.865 1.06, p = 0.390 1.20, p = 0.315 0.68, p = 0.665 3.26, p = 0.006 3.92, p = 0.004 0.70, p = 0.653 0.85, p = 0.542 3.48, p = 0.008 1.35, p = 0.243 1.21, p = 0.307

Please cite this article as: Son, Y., et al., Hippocampal dysfunction during the chronic phase following a single exposure to cranial irradiation, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.01.018

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Cranial irradiation down-regulated GFAP mRNA in the mouse hippocampus during the chronic phase

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Down-regulation of neurotrophic factor mRNA expression in the mouse hippocampus following cranial irradiation

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Alterations in the expression of the neurotrophic factors BDNF and GDNF were assessed by qRT-PCR following cranial irradiation. With

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To evaluate the time- and dose-dependent changes in the number of astrocytes in response to cranial irradiation, the astrocytic marker (GFAP) in the hippocampus was examined using immunohistochemistry and qRT-PCR. Representative photographs show the dose- and timedependent distribution of GFAP-positive cells in the hippocampal DG with irradiation (Fig. 7A). The number of GFAP-positive astrocytes decreased at 8 and 30 days in 10 Gy-irradiated mice compared to sham-irradiated controls, although this difference between the groups was not statistically significant (Fig. 7B). The mRNA expression of GFAP in the hippocampus was sharply elevated 1 day after irradiation, in a dose-dependent manner, but rapidly returned to the basal level 2 days post-irradiation (Fig. 7C). These data demonstrate that 10-Gy irradiation did not change the number of astrocytes, but altered GFAP mRNA expression in the hippocampus.

Discussion

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The present study demonstrated that cranial irradiation significantly induces depression-like behavior in the chronic phase and reduces hippocampal neurogenesis. Although the altered patterns of inflammatory cytokine expression were not consistent with changes in hippocampal neurogenesis or dysfunction following cranial irradiation, the expression levels of iNOS, COX-2, BDNF, and GDNF were significantly down-regulated in the chronic phase. Cranial irradiation is used clinically to treat primary and metastatic brain tumors but because ionizing irradiation can damage normal cells, patients suffer from a variety of side effects, particularly cognitive impairments and depression (Meyers and Brown, 2006; Sehlen et al., 2003). In experimental animals, X-irradiation with 5 or 10 Gy reduced hippocampal neurogenesis and induced cognitive deficits 3 months later (Raber et al., 2004; Rola et al., 2004), suggesting that cranial irradiation may induce hippocampal-dependent memory deficits during the chronic phase. In addition, it is established that animals irradiated with 10 Gy show cognitive impairment without damage to the structure of the brain (Belarbi et al., 2013; Mizumatsu et al., 2003; Raber et al., 2004). Previous studies have shown that cognitive impairments and depression are closely related to hippocampal neurogenesis, and the disruption of this phenomenon by chemical cell cycle inhibitors or Xirradiation with 15 Gy blocks hippocampal-dependent learning (Shors et al., 2001) and antidepressant action (Santarelli et al., 2003), respectively. The TST, in which inescapable stress is induced via suspension by the tail, is used widely for the assessment of behavioral despair in experimental animals (Cryan et al., 2005) and has been used to evaluate depression-like behaviors (Cryan et al., 2005; Pollak et al., 2010). Hippocampal neurogenesis modulates the behavioral function of the DG in terms of memory and regulation of emotion (Sahay and Hen, 2007). While radiation-induced cognitive impairment affecting neurogenesis in the DG of the hippocampus may be involved (Madsen et al., 2003; Mizumatsu et al., 2003; Raber et al., 2004; Rola et al., 2004), the effects of cranial irradiation on emotional regulation have yet to be determined. Thus, the present study utilized the TST to assess depression-like behavior following cranial irradiation and used DCX immunohistochemistry to examine hippocampal neurogenesis. In the present study, cranial irradiation led to increased immobility, a depression-like behavior, in the TST. There was a moderate to severe dose-dependent decrease in the number of DCX-positive cells in the acute phase. While the decreased number of cells immunopositive for DCX after 1 Gy returned to sham-irradiated control levels in the chronic phase, these decreases persisted with 10-Gy irradiation. The appearance of a depression-like behavior is consistent with a significant reduction in the number of DCX-positive cells in the DG of brain sections at 30 and 90 days following γ-irradiation with 10 Gy. In contrast, there was a non-significant increase in the immobility of mice irradiated with 1 Gy. This suggests that the acute transient decrease in neurogenesis in mice irradiated with 1 Gy may affect hippocampal-related behavior in the chronic phase. These findings suggest that the reduction in hippocampal neurogenesis may be associated with the depression-like behavior during the chronic phase following cranial irradiation. Previous reports have suggested that radiation-induced neuroinflammation mediated by activated microglial cytokines is one of the pathways that results in brain damage following irradiation (Chiang

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To assess the time- and dose-dependent changes in the number of microglia following cranial irradiation, the expression of the microglial marker Iba-1 in the mouse hippocampus was examined using immunohistochemistry and qRT-PCR. Representative photographs show the dose- and time-dependent distribution of Iba-1-positive cells in the hippocampal DG with irradiation (Fig. 6A). The number of Iba-1 immunopositive cells in sham-irradiated (0 Gy) controls and 1Gy-irradiated mice increased significantly with aging (Fig. 6B). However, the numbers of Iba-1-positive cells in the DG at 30 and 90 days after 10-Gy irradiation were significantly lower than in age-matched shamirradiated controls (Fig. 6B). The mRNA expression of Iba-1 in the hippocampus was reduced significantly at 8 days following irradiation with 10 Gy (Fig. 6C). This trend was also observed 90 days after irradiation, although it did not reach statistical significance (Fig. 6C). These data demonstrate that 10-Gy irradiation significantly reduced the number of microglia and Iba-1 mRNA expression in the hippocampus.

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Cranial irradiation decreased the number of microglia in the mouse hippocampus during the chronic phase

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was transiently elevated 1 day after 1-Gy-irradiation (Fig. 5B). These data demonstrate a marked decrease in the levels of pro-inflammatory enzymes in the hippocampus following cranial irradiation.

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10-Gy irradiation, expression of BDNF tended to decrease gradually 90 days post-irradiation; a significant decrease was observed between days 8 and 90 (Fig. 8A). Similar to BDNF, GDNF expression decreased significantly 90 days post-irradiation in 10-Gy-irradiated mice (Fig. 8B). However, BDNF and GDNF levels in 1-Gy-irradiated mice did not differ from those in sham-irradiated controls (Figs. 8A and B). These data demonstrate a marked decrease in the mRNA expression of neurotrophic factors in the chronic phase following cranial irradiation.

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Fig. 2. Depression-like behavior during the chronic phase following cranial irradiation. In the tail suspension test (TST), the immobility time was increased at 30 and 90 days following irradiation. The data are reported as means ± SE (n = 8 per group). **p b 0.01, ***p b 0.001 vs. age-matched sham-irradiated (0 Gy) controls.

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Fig. 3. Down-regulation of hippocampal neurogenesis following cranial irradiation. (A) Representative photomicrographs of DCX-positive cells in the DGs of sham-irradiated (0 Gy) controls and 1 or 10 Gy-irradiated mice. Cells were counterstained with hematoxylin. GCL, granular cell layer; DG, dentate gyrus. (B) Bar graphs show the mean numbers of DCX-positive cells in the DG of brain sections. (C) Bar graphs show the percentages of DCX-positive cells in the DG of brain sections compared to controls. The data are reported as means ± SE (n = 4 per group). A scale bar indicates 100 μm. *p b 0.05, **p b 0.01, ***p b 0.001 vs. age-matched sham-irradiated (0 Gy) controls; #p b 0.05, ##p b 0.01, ###p b 0.001 vs. 2 days groups after irradiation with 0 and 1 Gy, respectively; †††p b 0.001 vs. 30 days groups after irradiation with 0 and 1 Gy, respectively.

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et al., 1997; Gaber et al., 2003; Hong et al., 1995; Moravan et al., 2011; Olschowka et al., 1997). Single-fraction X-irradiation (20 Gy) in the whole brain significantly increases the mRNA expression of TNF-α in the mouse brain 2–6 h post-irradiation (Gaber et al., 2003). Other

reports have identified an acute up-regulation of TNF-α, IL-1β, and IL6 in the mouse brain 4–8 h after X-irradiation with 25 Gy (Hong et al., 1995), and a persistent increase in TNF-α, GFAP, and MHC II gene expression levels in the mouse brain following cranial irradiation with

Please cite this article as: Son, Y., et al., Hippocampal dysfunction during the chronic phase following a single exposure to cranial irradiation, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.01.018

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2–6 h but did not persist to 1 day following cranial irradiation with doses ≤ 15 Gy (Hong et al., 1995; Moravan et al., 2011). Furthermore, these inflammatory cytokine expression patterns were not correlated with decreased hippocampal neurogenesis and behavioral dysfunction after irradiation. It is generally accepted that pro-inflammatory enzymes, such as iNOS and COX-2, are related to the inflammatory response subsequent to brain injury (Heneka and Feinstein, 2001; Kyrkanides et al., 2002). However, previous studies have found that the selective inhibition of the iNOS gene results in a down-regulation of neurogenesis following focal cerebral ischemia (Zhu et al., 2003) and reduces synaptic potentiation in somatosensory cortical slices (Buskila and Amitai, 2010). Additionally, the constitutive expression of COX-2 plays an important role in brain functions such as synaptic plasticity and memory consolidation (Rall et al., 2003; Shaw et al., 2003; Yamagata et al., 1993). In the present study, cranial radiation decreased the mRNA levels of hippocampal iNOS and COX-2 during the chronic phase, suggesting that the downregulation of iNOS and COX-2 expression may be related to hippocampus-related behavioral dysfunction. In addition, decreased iNOS mRNA expression was observed from 1 day after irradiation with 10 Gy, which is consistent with the decreased neurogenesis, and

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≥25 Gy (Moravan et al., 2011). The up-regulation of new microglia and astrocytes was also reported during the chronic phase following cranial irradiation, although the total number of each cell phenotype was not increased (Rola et al., 2004; 2008). In contrast, the number of microglia was reduced 7 days post-irradiation after a single dose of 8 Gy to the developing rat brain (Kalm et al., 2009), and at 1 and 10 weeks after a single dose of 10 Gy to the adult rat brain, although the number of activated microglia was increased (Schindler et al., 2008), which might be related to cognitive deficits following irradiation. Moreover, no activated microglia were observed in the rat brains after γ-irradiation of 10 Gy (Wojtowicz, 2006). Therefore, it remains controversial whether neuroinflammation occurs following irradiation and influences hippocampal activity and function. A more comprehensive study investigating neuroinflammation, especially in the hippocampus, in relation to hippocampal changes during the chronic phase after cranial irradiation is necessary. In the present study, altered patterns of proinflammatory cytokine activity, including IL-1β, TNF-α, IL-6, and IFN-γ activities, were not observed after cranial irradiation from 1 day following irradiation, a finding that is consistent with previous studies on the alterations of inflammatory cytokines during the acute phase, which indicated that pro-inflammatory cytokine activity was increased at

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Fig. 4. Changes in the mRNA expression of inflammatory cytokines in the mouse hippocampus. mRNA expression of IL-1β (A), TNF-α (B), IL-6 (C), and IFN-γ (D) was examined following cranial irradiation. The data are reported as means ± SE (n = 8 per group). #p b 0.05 vs. 2 days group after irradiation with 1 Gy; †p b 0.05 vs. 30 days group after irradiation with 1 Gy.

Fig. 5. Changes in the mRNA expression of iNOS and COX-2 in the mouse hippocampus. Cranial irradiation with 10 Gy decreased mRNA expression of iNOS (A) and COX-2 (B) in the mouse hippocampus during the chronic phase following irradiation. The data are reported as means ± SE (n = 8 per group). *p b 0.05, **p b 0.01, ***p b 0.001 vs. age-matched sham-irradiated (0 Gy) controls; #p b 0.05, ##p b 0.01 vs. 2 days group after irradiation with 10 Gy; †p b 0.05 vs. 30 days group after irradiation with 1 Gy.

Please cite this article as: Son, Y., et al., Hippocampal dysfunction during the chronic phase following a single exposure to cranial irradiation, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.01.018

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Fig. 6. Changes in the number of microglia and mRNA expression of Iba-1 in the mouse hippocampus following cranial irradiation. (A) Representative photomicrographs of Iba-1-positive microglia in the DGs of sham-irradiated (0 Gy) controls and 1- or 10-Gy-irradiated mice. Cells were counterstained with hematoxylin. The scale bar indicates 100 μm. (B) Bar graphs show the numbers of Iba-1-positive cells (cells/mm2) in the DG. (C) Bar graphs show the Iba-1 mRNA levels in the hippocampus. The data are reported as means ± SE (n = 4 per group, for B and C). *p b 0.05, ***p b 0.001 vs. age-matched sham-irradiated (0 Gy) controls; ###p b 0.001 vs. 2 days groups after irradiation with 0 and 1 Gy, respectively.

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indicates expression of iNOS to be closely related to altered neurogenesis in the hippocampus. The down-regulated levels of iNOS and COX-2 also support the notion that the inflammatory state was not induced in the chronic phase after cranial irradiation. Thus, the

decreased expression of iNOS and COX-2 during the chronic phase may be correlated with altered hippocampal neurogenesis and the appearance of depression-like behavior in mice following cranial irradiation.

Please cite this article as: Son, Y., et al., Hippocampal dysfunction during the chronic phase following a single exposure to cranial irradiation, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.01.018

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Fig. 7. Changes in the number of astrocytes and mRNA expression of GFAP in the hippocampus following cranial irradiation. (A) Representative photomicrographs of GFAP-positive astrocytes in the DGs of sham-irradiated (0 Gy) controls and 1- or 10-Gy-irradiated mice. Cells were counterstained with hematoxylin. The scale bar indicates 100 μm. (B) Bar graphs show the number of GFAP-positive cells (cells/mm2) in the DG. (C) Bar graphs depicts mRNA expression of GFAP in the hippocampus. The data are reported as means ± SE (n = 4 per group, for B and C). ***p b 0.001 vs. age-matched sham-irradiated (0 Gy) controls.

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In addition to inflammatory and pro-inflammatory enzymes, microglia and astrocytes play important roles in inflammatory responses, and a previous study reported reduced neurogenesis in T-cell deficient mice, which suggested a beneficial role of microglia (Butovsky et al.,

2006). In the present study, the basal number of Iba-1-positive microglia in the DG increased gradually with age, but the number of microglia did not increase during the chronic phase after 10-Gy irradiation. Iba-1 mRNA expression in the hippocampus was lower in the early and

Please cite this article as: Son, Y., et al., Hippocampal dysfunction during the chronic phase following a single exposure to cranial irradiation, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.01.018

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2012M2A2A7049062). The animal experiment in this study was sup- 536 ported by the Animal Medical Institute of Chonnam National University. 537

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intermediate phases after 10-Gy irradiation compared to that of the age-matched control. While GFAP mRNA expression was increased significantly 1 day after irradiation in a dose-dependent manner, although the number of GFAP-positive astrocytes in the hippocampus did not change. Thus, the change in the number of Iba-1- and GFAP-positive cells in the hippocampus after irradiation is inconsistent with the changes in Iba-1 and GFAP mRNA levels, suggesting that the alterations in the number of them are different to the cellular mRNA levels in the hippocampus with irradiation. Additionally, these findings suggest that the patterns of changes in the numbers of microglia and astrocytes in the hippocampus do not reflect the inflammatory response during the chronic phase after cranial irradiation. Conversely, these results suggest that this loss of microglia contributed to the hippocampal dysfunction detected after irradiation. Neurotrophic factors, such as BDNF and GDNF, play important roles in hippocampal neurogenesis, hippocampal-dependent learning and memory, and emotional regulation (Lipsky and Marini, 2007; Pardon, 2010; Shirayama et al., 2002; Taliaz et al., 2010), and increased neurotrophins attenuate neuronal injury (Mattson and Scheff, 1994). Previous studies reported that neurons, as well as astrocytes and microglia, express BDNF and GDNF, and play a pivotal role in neuronal survival and plasticity by releasing neurotrophic factors (Batchelor et al., 1999; Chen et al., 2006; Parpura and Zorec, 2010; Trang et al., 2011), although the primary role of microglia is to protect neurons from a variety of insults (Kempermann and Neumann, 2003). In this study, the hippocampal BDNF and GDNF mRNA levels were not changed during the acute phase, but were significantly decreased during the chronic phase following irradiation with 10 Gy, which was consistent with a delayed decrease in the number of microglia. These findings suggest that the down-regulated levels of BDNF and GDNF may be involved in the incidence of depression-like behavior or altered hippocampal neurogenesis during the chronic phase following cranial irradiation, an effect possibly mediated by the decreased number of microglia. Further studies are needed to verify the specific mechanisms underlying the dysregulation of hippocampal neurogenesis and its associated behavioral functions following irradiation. In conclusion, the present study shows that cranial irradiation induces depression-like behavior in mice during the chronic phase following cranial irradiation, possibly via decreased hippocampal neurogenesis. Levels of inflammatory-related cytokines were not correlated with the decreased neurogenesis or the appearance of depression-like behavior. However, the decreased number of microglia during the chronic phase was consistent with down-regulated mRNA expression of iNOS, COX-2, BDNF, and GDNF in the hippocampus. Consequently, we suggest that hippocampal dysfunction in the chronic phase may be related to the decreased levels of neurogenesis- and synaptic plasticity-related signals, concomitant with microglial reduction in the hippocampus.

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Fig. 8. Changes in the mRNA expression of BDNF and GDNF in the mouse hippocampus. Cranial irradiation with 10 Gy decreased mRNA expression of BDNF (A) and GDNF (B) in the mouse hippocampus during the chronic phase following irradiation. The data are reported as means ± SE (n = 8 per group). *p b 0.05, **p b 0.01 vs. age-matched sham-irradiated (0 Gy) controls.

Please cite this article as: Son, Y., et al., Hippocampal dysfunction during the chronic phase following a single exposure to cranial irradiation, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.01.018

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Please cite this article as: Son, Y., et al., Hippocampal dysfunction during the chronic phase following a single exposure to cranial irradiation, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.01.018

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