The present study was approved by the Animal Care and Ethics Committee of The Affiliated Suzhou Hospital of Nanjing Medical University and Shandong University (Jinan, China). A total of 36 male Sprague-Dawley rats (weight, 50–60 g; age, 21 days) were obtained from The School of Medicine, Shandong University. All the rats were housed with 3–4 animals per cage with ad libitum access to tap water and food. The temperature was set at 24°C and the relative humidity was 50–70%. They were kept under natural light in 12-h light/dark cycles. All rats were anesthetized with 4% isoflurane and placed in prone position in a 23EX linear accelerator (Varian Medical Systems, Inc.). A total of 1–2% isoflurane with 0.6–0.8 l/min flow rate maintained anesthesia during this process. Each rat was treated with WBI using a 4-MeV electron beam at a single dose of 10 or 0 Gy for the control group. The beam was directed downwards towards the head, whilst the body was shielded using a customized block.

After WBI, each rat received a twice daily intraperitoneal injection of bromo-deoxyuridine (BrdU; 50 mg/kg body weight; Sigma-Aldrich; Merck KGaA) for 4 days prior to irradiation exposure. All rats were anesthetized with 4% isoflurane followed by 1–2% isoflurane with 0.6–0.8 l/min flow rate to maintain anesthesia. Adeno-associated virus (serotype 8, AAV8) encoding GFP was stereotaxically infused into the dorsal hippocampus within 24 h after WBI. All Sprague-Dawley rats were randomly apportioned into the following groups (n=12 per group): i) AAV-control, consisting of control rats infused with AAV encoding TrkA scramble sequence; ii) AAV-irradiation, consisting of irradiated rats infused with AAV encoding TrkA scramble sequence; and iii) AAV-overexpression (ovp)-TrkA, consisting of irradiated rats infused within AAV encoding overexpression TrkA. Morris water maze and open field test was performed at 1 and 3 months, following which all rats were sacrificed with carbon dioxide. The animals were placed into a box and CO₂ was infused into the box at a rate of 10–30% of the volume of the euthanasia box every minute. After 5 min, the animals exhibited no movements, no breathing, no heartbeat, and pupil dilation. Then, the infusion of CO₂ was terminated and the rats were observed for another 2 min to confirm the death of the animal. Thus, a total of 7 min observation was used to confirm animal sacrifice. All the euthanasia procedures on the use of experimental animals adhered to the AVMA Guidelines for the Euthanasia of Animals (17) and the best efforts were made to minimize animal suffering throughout the experiment. The humane endpoints of this study included rats quickly losing 20% of their original body weight and rats that were unable to eat and drink on their own due to weakness. Immunofluorescence staining or western blotting were performed to evaluate the effects of irradiation on neurogenesis and protein expression.

Each rat received an intraperitoneal injection of BrdU (50 mg/kg body weight), twice daily at 8-h intervals, for a total period of 4 days before WBI.

An AAV serotype 8 encoding GFP vector was used in the present study. AAV8 encoding TrkA scramble sequence and overexpressing TrkA were constructed by Jikai Biological Technology Co., Ltd. AAV8 expressing a TrkA scramble sequence served as control. After anesthesia, rats were bilaterally infused with AAV8 into the dorsal hippocampus as previously described (18). Briefly, the injection site was −3.7 mm anteroposterior from the bregma, ±2.2 mm mediolateral from the bregma and 3.5 mm below the surface of the skull. The titer of AAV8 virus was 3.92×10¹² v.g./ml (vector genomes/ml), where a total volume of 2 µl was injected. The rate of infusion was 0.2 µl/min, where the cannula was left in place for 5 min to ensure the complete diffusion of the virus into the brain.

Place navigation tests were conducted on days 1–5, where all rats underwent 4 trials per day. A square platform, ~9 cm in diameter, was submerged 1.5 cm beneath the water. The location of the platform remained constant throughout the trials, but the starting location changed for each trial. Each rat was given 60 sec to locate the platform, where it was then permitted to rest for a further 10 sec before being assisted back into the home cage. If the rat failed to locate the platform in the allotted time, it was guided to the platform and allowed to remain on it for 10 sec.

A spatial probe test took place on day 6, where the platform was removed and the rats were allowed to swim freely for 60 sec. The test was performed within 24 h after the trials for 4 days. The time taken to reach the platform, path length, swimming speed and the number of times the target zone was crossed were recorded.

Using an opaque open field (410×410×505 mm), the middle and inner areas were termed the central region. Each rat was placed in the central region, and was allowed to move freely around the open field for 10 min. An automated video-tracking system (Shanghai Jiliang Software Technology Co., Ltd.) was used to assess voluntary locomotor activity. Total track length, recorded as the total distance traveled and the time spent in the central region, were measured using the automated video-tracking system.

Immediately after the behavior experiment, the experimental animals were sacrificed with carbon dioxide. The hippocampus tissue homogenates were lysed in RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) and subjected to 10% SDS-PAGE (cat. no. ST628; Beyotime Institute of Biotechnology). Protein concentrations were determined using the bicinchoninic acid protein assay kit (cat. no. P0009; Beyotime Institute of Biotechnology). The polyvinylidene difluoride membranes were blocked in 5% milk in TBST for 1 h at room temperature, incubated with primary antibodies overnight at 4°C, and then incubated with secondary antibodies for 1 h at room temperature. The following antibodies were used: Anti-NGF (1:1,000; product code ab6199; Abcam), anti-TrkA (1:2,000; product code ab76291; Abcam), anti-phosphorylated (p)-PI3K (1:1,000; cat. no. 511120; ZenBioscience), anti-p-AKT (1:1,000; cat. no. 310022, ZenBioscience) and anti-p-Erk1/2 (1:1,000; cat. no. 310064; ZenBioscience). Anti-GAPDH (1:5,000; cat. no. ABS16), anti-β-actin (1:10,000; cat. no. AV40173) and anti-tubulin (1:10,000; cat. no. T3526) were purchased from Sigma-Aldrich, Merck KGaA. Goat anti-mouse HRP (1:10,000; code no. 115-035-166) and goat anti-rabbit HRP (1:10,000; code no. 111-035-003) were purchased from Jackson ImmunoResearch, Laboratories Inc. Immunolabeling of membranes was detected by ECL chemiluminescence (P0018S; Beyotime Institute of Biotechnology). The integrated densities of each band were quantified using ImageJ Software (version 2006.02.01; National Institutes of Health).

Immediately after the behavior experiment, the rats were anesthetized and perfused with ice-cold saline followed by ice-cold 4% paraformaldehyde. Brain tissues were then removed and post-fixed overnight in 4% paraformaldehyde at 4°C followed by equilibration in 30% sucrose. Sagittal sections at 30-µm-thick were cut at −20°C by frozen slicer (Leica CM1950; Leica Microsystems, Inc.). The sections were treated with 2 M HCl at 37°C for half an hour and then washed in 1X Tris-buffered saline pH 8.5 before incubation in 5% BSA (cat. no. ST023; Beyotime Institute of Biotechnology) and 5% Triton X-100 (Beyotime Institute of Biotechnology) in PBS for 1 h at room temperature. Tissue sections were incubated with rabbit anti-doublecortin (DCX; 1:100; cat. no. D9818; Sigma-Aldrich; Merck KGaA), mouse anti-neuronal nuclei (NeuN; 1:50; cat. no. MAB377; Thermo Fisher Scientific, Inc.), rabbit anti-glial cell marker glial fibrillary acidic protein (GFAP; 1:100; cat. no. ab207165, Abcam) or rabbit anti-BrdU (1:500; cat. no. B8434; EMD Millipore) primary antibodies at 4°C overnight, followed by a fluorescent secondary antibody conjugated with Alexa Fluor^® 488 goat anti-mouse (1:500; cat. no. A11001; Invitrogen; Thermo Fisher Scientific, Inc.) or Cy3-conjugated goat anti-rabbit (1:500; cat. no. 111-165-144; Jackson ImmunoResearch Laboratories, Inc.) secondary antibodies for 2 h at room temperature. Cell nuclei were stained with 100 ng/ml DAPI (cat. no. C1002; Beyotime Institute of Biotechnology) for 10 min at room temperature.

A confocal laser-scanning microscope (Olympus Corporation) with ×20 objective was used to analyze the tissue staining. Cell counts were evaluated in 5–10 tissue sections per rat using a multi-channel configuration with ImageJ software (version 2006.02.01). Cell counts were limited to regions in the granule cell layer and hilus. The volumes of the granule cell layer and hilus were used to normalize the numbers of positive cells.

SPSS 22.0 software (IBM Corp.) was used to analyze all statistical data, and the results are expressed as the mean ± standard error of the mean (SEM). Data were analyzed using unpaired t-test, one-way analysis of variance (ANOVA), or two-way ANOVA. Student-Newman-Keuls was used as the post hoc test following ANOVA. Mann-Whitney U-test was used to analyze non-normally distributed behavior test data. P<0.05 was considered to indicate a statistically significant difference.

The effects of radiation on spatial learning and memory were investigated using spatial navigation tests 3 months after WBI. In the place navigation test, all rats displayed improved performance during days 1–4. On days 4 and 5, the irradiated rats exhibited significantly increased latency times before locating the submerged platform, compared with those of the non-irradiated rats (P<0.05; Fig. 1A). Furthermore, in the spatial probe test, the irradiated rats spent significantly less time in the target quadrant compared with that of the non-irradiated rats (P<0.01; Fig. 1B and C). These results demonstrated that irradiation was associated with spatial memory dysfunction.

An open field test was subsequently applied to assess the anxiety levels and spontaneous movement. The results revealed no significant differences in spontaneous movement or anxiety-like behavior between the two groups (Fig. 1D and E), which eliminates the influences of exercise and emotion on spatial memory dysfunction.

The results of the BrdU-labeling assay revealed that irradiated rats exhibited 74.2 and 73.9% fewer proliferating cells (P<0.01; Fig. 2A and B) compared with those in non-irradiated rats after 1 and 3 months, respectively. Immature neurons were labeled with BrdU and DCX, a microtubule-associated protein expressed in migrating neuroblasts (19). The results revealed an 85.4 and 73.7% decrease in the number of BrdU- and DCX-positive cells after cranial irradiation (P<0.01; Fig. 2C and D). In addition, double labeling of BrdU and the glial cell marker GFAP also revealed reduced staining (32.9 and 66.7%) in irradiated rats compared with those in the non-irradiated rats at 1 and 3 months (P<0.01; Fig. 2E and F). Mature neurons that had undergone recent division were labeled with BrdU and NeuN (BrdU⁺/NeuN⁺). The irradiated rats exhibited 68.5 and 88.6% fewer BrdU⁺/NeuN⁺ cells compared with those in the non-irradiated rats at 1 and 3 months, respectively (P<0.01; Fig. 2G and H). These results indicated WBI inhibited normal neuronal proliferation, differentiation, maturation and survival in the hippocampus.

Continuity detection was performed via western blotting, which was used to assess changes in the expression of NGF and TrkA in the rat hippocampus after irradiation. No differences in NGF protein expression were detected between irradiated and non-irradiated rats from 1 day to 3 months post-WBI (Fig. 3A and C). However, compared with that in the non-irradiated group, TrkA protein expression was found to be increased on days 1 and 3, in addition to after 1 week (P<0.05), which was then decreased after the 1-month time-point and was significantly reduced after 3 months (P<0.05; Fig. 3B and D).

The effects of TrkA overexpression on irradiation-induced memory deficit were subsequently investigated. AAV8s encoding TrkA scramble sequence and overexpressing TrkA (AAV-ovp-TrkA) were injected into the hippocampus of irradiated rats within 24 h after WBI (Fig. S1). In control rats, AAV8s encoding a TrkA scramble sequence were injected into irradiated rats (AAV-irradiation) and non-irradiated rats (AAV-ctl), respectively. Western blotting demonstrated the successful overexpression of TrkA in the hippocampus (Fig. S2). The influence of AAV8 injection on irradiated rats was also ruled out (Fig. S3). The levels of hippocampal TrkA expression in these three groups of rats were then detected by western blotting (Fig. 4A and B), which verified the effectiveness of AAV-ovp-TrkA infection. Morris water maze test was used to evaluate spatial memory function 3 months after WBI.

In the place navigation test, rats in the AAV-ovp-TrkA group exhibited shorter latency times prior to arrival at the hidden platform compared with those in the AAV-irradiation group on days 4 and 5 (P<0.05; Fig. 4C). In the spatial probe trial, rats in the AAV-ovp-TrkA group also spent significantly more time in the target quadrant compared with those in the AAV-irradiation group (P<0.05; Fig. 4D). There was no statistical difference between the AAV-ctl and AAV-ovp-TrkA groups. The swim tracks revealed that rats in the AAV-irradiation group spent more time searching for the submerged platform compared with those in the other two groups (Fig. 4E).

At 3 months after WBI, intrahippocampal infusion of AAV-ovp-TrkA significantly prevented a reduction in neurogenesis when compared with rats in the AAV-irradiation group (Fig. 5A-D). The rats in AAV-ovp-TrkA group exhibited a 41.6% increase BrdU⁺/NeuN⁺ cells compared with those in the AAV-irradiation group (P<0.05; Fig. 5D). These results indicated TrkA overexpression in the irradiated rat hippocampus restored neurogenesis.

The effect of cranial irradiation on the PI3K/AKT and ERK1/2 pathways was subsequently evaluated. The results revealed that the phosphorylation of PI3K, AKT and ERK1/2 was decreased following WBI (all P<0.05; Fig. 6A and B). However, tissues from the AAV-ovp-TrkA group exhibited increased PI3K, AKT and ERK1/2 phosphorylation compared with those in the AAV-irradiation group (Fig. 6A and B).