Male and female Wistar rats, nearly three months old and weighing approximately 200–250 grams, were housed in plastic cages. A total of 11 female and 11 male rats were utilized for the experimental procedures. Each female rat was involved in a single pregnancy only, and none were subjected to a second pregnancy during the study. All animals were kept in standard laboratory conditions at a temperature of 22 ± 2 degrees Celsius, with around 50% humidity, and a 12-h light/dark cycle. Access to food and water was provided ad libitum. Mating cages were set up for male and female rats. The appearance of vaginal plugs was considered as the zero-day of pregnancy (Goyeneche et al., 2003). Pregnant rats received a single intraperitoneal injection of 500 mg/kg VPA (Sajaddaru Co., Iran) dissolved in 3.3 mL/kg saline on embryonic day 12.5, except for the control group which did not receive any injections (Favre et al., 2013). Pups were weaned on postnatal day 21 and they were examined for morphological malformation (Saft et al., 2014). Subsequent experiments were conducted on the male offspring. The pups were categorized into four groups:

(1) The intact control group consisted of wildtype rats (no interventions were administered), (2) Sham, VPA-exposed rats, received prenatal VPA but did not undergo any oxygen treatment, (3) HBOT-2 group comprised VPA-exposed rats that received hyperbaric oxygen therapy at 2 ATA and (4) the HBOT-2.5 comprised VPA-exposed rats that received hyperbaric oxygen therapy at 2.5 ATA.

All Experiments were carried out in accordance with the international ethical codes and complied with local institutional regulations at Ferdowsi University of Mashhad (Iran National Committee for Ethics in Biomedical Research). The project obtained ethical approval from the local FUM Ethical Review Committee (IR.UM.REC.1399.117). The study’s reporting follows the ARRIVE guidelines.

Four days after separation from their mother, the pups underwent the three-chamber social interaction test, Y-maze, elevated plus maze, and Morris Water Maze (MWM) tests. These evaluations assessed social behaviors, repetitive behaviors, anxiety, and spatial memory, respectively.

All behavioral assessments were conducted on 25-day-old rats, between 9 am and 4 pm. One hour before testing, all animal-containing cages were transferred to the laboratory to allow the animals to habituate to the testing room (Hånell and Marklund, 2014).

This test assesses two key aspects of social behavior: “Sociability,” which is defined as the propensity to spend time with another rat rather than with a nonliving object, and “Social Novelty” which is the preference for spending time with an unfamiliar rat over a familiar one (Moy et al., 2004). The apparatus used was a box (100 × 40 × 40 cm) divided by separating walls, allowing free access to each chamber. The side chambers contained two identical wire cages. The test comprised four 10-min stages separated by 5-min intervals. To prevent residual odors from influencing the test, all three chambers and cages were cleaned with 75% ethanol both initially and before each stage. In the first stage (habituation), animals were allowed to freely explore the empty chambers and cages. In the second stage called pre-test, two identical nonliving objects were placed in the cages. The third stage was the Social Stimulus Stage: One wire cage contained an unfamiliar rat (social stimulus), while the opposite cage contained an unfamiliar inanimate object (non-social stimulus). In the last stage (Social Novelty Stage), the non-social object was replaced with a novel unfamiliar rat as the novel social stimulus and the social stimulus were at the same place. During these stages, interactions such as following, touching, grooming, and sniffing any body part were recorded using a video camera. The duration of interaction with each stimulus was manually measured. Animals used as stimuli were randomly chosen, matching the test rats in age, sex, and strain (Rein et al., 2020).

This test aimed to measure anxiety-like behavior in rats. Rats were placed for 5 min in an apparatus consisting of two open arms and two closed arms (each arm 50 × 10 cm and the height 50 cm). Animals were positioned in the middle of the maze and time spent in each arm was recorded with a video camera connected to a computer using ANY-maze software. The maze was cleaned with 75% ethanol and allowed to dry after each test to eliminate the odors of previously tested rats. The total distance traveled and time spent in each arm was recorded (Markram et al., 2008; Hånell and Marklund, 2014).

The Y-maze can be used to measure short-term memory, exploratory, and stereotyped patterns of behavior (Onaolapo, 2012; Wöhr et al., 2015). Hence, spontaneous alternation was determined using a Y-maze. The maze, consisting of three equal arms (40 × 5 × 30 cm) separated by 120-degree angles, resembles the shape of a Y. Rats were placed individually in the start arm and allowed to explore freely for 5 min (Onaolapo, 2012). The arms were labeled A, B, and C. A video camera connected to a computer running ANY-maze software tracked and recorded the sequence of arm entries and the total distance traveled. An alternation was defined as entry into all three arms consecutively. The percentage of alternation was computed using the formula: [(number of alternations) / (total arm entries −2)] × 100. After each trial, the apparatus was cleaned with 75% ethanol and allowed to dry to remove any residual odors from previous rat trials (Onaolapo, 2012; Hånell and Marklund, 2014; Jung et al., 2020).

The MWM assesses spatial learning and memory capabilities (Vorhees and Williams, 2006). The apparatus consisted of a circular black pool, 150 cm in diameter and 50 cm deep, filled halfway with water maintained at approximately 25 ± 1°C. A platform, matching the pool’s color and measuring 9 cm in diameter and 25 cm in height, was submerged 1 cm below the water’s surface, making it nearly invisible during training sessions. On the first day, the platform was positioned 1 cm above the water’s surface and marked with a red flag for visibility by the animals. The pool was enclosed with curtains to minimize distractions for the animals. Inside the pool, above the water’s surface, four high-contrast spatial cues were placed. The pool was divided into four quadrants by two axes, with directional markers (North, South, East, and West) at the ends of each line. Rats underwent 5 days of spatial training, with each session comprising five 60-s trials and 60-s inter-trial intervals. At the start of each trial, the rat began facing the pool’s wall at one of four positions. If the rat located the platform within 60 s, it remained on the platform for 5 s. However, if the rat did not find the platform, it was gently placed on the platform for 20 s. The escape latency and total distance traveled were automatically recorded using the ANY-maze video tracking software (Vorhees and Williams, 2006; Markram et al., 2008; Bromley-Brits et al., 2011).

HBOT was administered to 35-day-old rats using a hyperbaric chamber (Irsa Sakht Asia Co., IR). For this purpose, rats were placed in the hyperbaric chamber. After sealing the chamber, the pressure inside the chamber was gradually increased over a 5-min period until it reached the designated pressure levels. One group was pressurized to 2 ATA, while the second group underwent pressurization to 2.5 ATA. Once the chamber pressure attained the desired levels, the rats remained exposed to this pressure condition for 50 min. Throughout this time, a 100% oxygen environment was maintained. The oxygen flow rate was 6 liters per minute. Following the 50-min exposure, the chamber pressure was gradually reduced over a 5-min period to return to atmospheric conditions, totaling the rats’ exposure duration within the chamber to 60 min. After each HBOT session, the rats were returned to their cages. These sessions were conducted once daily for 14 consecutive days (Simsek et al., 2012; Körpınar and Uzun, 2019).

All administrations were conducted between 9 AM and 11 AM to minimize potential effects.

Post-HBOT behavioral tests:

All behavioral tests were repeated after the completion of the HBOT sessions.

For tissue sampling, CO2 was used for euthanizing the animals. After opening the skulls, the whole brain was extracted, and the frontal lobe was immediately excised. The excised frontal lobe samples were placed in microtubes containing RNA later solution (Sinaclon Co., Iran) and stored at −80°C until molecular examinations commenced.

RNA extraction and cDNA synthesis were performed using RNX Plus (Sinnaclon Co, Iran) for total cellular RNA extraction from frontal lobe samples, following the manufacturer’s instructions. Primer sequences for the target gene and GAPDH gene (used as a reference gene) were designed and determined (shown in Table 1). The RNA was reverse transcribed into single-stranded cDNA using a cDNA synthesis kit (Pars tous Co, Iran) in accordance with the manufacturer’s protocol.

The real-time PCR procedure was conducted utilizing a Corbett real-time PCR apparatus. The cDNA samples were amplified employing SYBR-Green in a 20 μL reaction mixture comprising 10 μL of 2X TaqMan Universal PCR Master Mix, 1 μL of 20X TaqMan Gene Expression Assay, and 9 μL of cDNA template. The amplification process involved an initial denaturation step at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min. Subsequently, the real-time PCR data were analyzed using the comparative Ct method to ascertain the relative expression levels of the target genes, which were then normalized to the expression of the reference gene (Schmittgen and Livak, 2008). To confirm the specificity of the PCR products, a melting curve analysis was performed. Additionally, non-template controls were included in each run to identify any potential contamination or background signal.

The Behavioral tests and Real-time PCR results underwent analysis in R software (R Core Team, 2022). Initially, the data for each test were evaluated for normal distribution using the Shapiro–Wilk test. Subsequently, for the three-chamber social interaction test, exploratory behavior, and spontaneous alternation in the Y-maze test, the Welch t-test was used to compare the control group to each treatment group. For the elevated plus maze data, the non-parametric Mann–Whitney test was employed. Then, the paired t-test was applied to assess the pre-and post-treatment data for comparison. The MWM data underwent analysis using a repeated measure analysis of variance (ANOVA), adjusted with the Bonferroni correction, followed by Tukey’s post hoc test. Gene expression data were analyzed using one-way ANOVA, and Tukey’s post hoc test was conducted to determine the specific differences among groups. The repeated measure ANOVA was performed using the nlme package (Pinheiro and Bates, 2000), while post hoc tests were conducted using functions from the multcomp package (Hothorn et al., 2008). A significance level of p-value ≤0.05 was considered. The statistics reported in the text and figures represent the mean ± SEM.

Out of the 36 rats exposed to VPA in utero, 28 exhibited tail malformations, indicating that 80% of the VPA-exposed rats showed abnormalities in their tails. Apart from tail malformations, no other body malformations were observed (Figure 1).

The observed tail malformations were classified into two groups: (1) Rats that exhibited a single bend along their tail, and (2) Rats that displayed multiple bends along their tail, often having two bends, with one case showing four bends (Figure 1).

In the novelty preference test, the percentage of s interaction’animal with the unfamiliar social stimulus has been measured by calculating the interaction time with the unfamiliar stimulus compared to the total interaction time (time interacted with unfamiliar social stimulus/(time interacted with unfamiliar social stimulus + time interacted with familiar social stimulus) × 100).

Before treatment, VPA-exposed rats exhibited impaired preference in interacting with the unfamiliar social stimulus compared to the control group [HBOT-2 and control (t = 2.5196, df = 11.854, p-value = 0.02714) and HBOT-2.5 and control (t = 1.3114, df = 16.765, p-value = 0.2074)] (Figure 2C).

Following HBOT administration, interaction with the unfamiliar social stimulus increased in both groups, with statistical significance observed only in HBOT-2 [HBOT-2 before and after (t = −4.4773, df = 9, p-value = 0.001538)], while not in HBOT-2.5 [HBOT-2.5 before and after (t = −2.0733, df = 10, p-value = 0.06492)] (Figure 2C).

Comparison of post-treatment data with the control group did not reveal a significant difference [HBOT-2 and control (t = −0.58468, df = 10.445, p-value = 0.5712), HBOT-2.5 and control (t = −0.058536, df = 10.545, p-value = 0.9544)] (Figure 2C).

In this test, the percentage of time spent in the open arms relative to the total test time was calculated to measure anxiety (time spent in the open arms/total test time × 100). Similarly, using the same equation, the percentage of distance traveled in the open arms was also computed.

Compared to control rats, VPA-exposed rats exhibited increased time spent in the open arms. This difference was statistically significant in HBOT-2.5 (t = −2.3226, df = 14.61, p-value = 0.03509), while not significant in HBOT-2 (t = −1.5596, df = 14.427, p-value = 0.1405) (Figure 3A).

Post HBOT administration, both groups displayed a decrease in the percentage of time spent in the open arms. This reduction was statistically significant in HBOT-2.5 (t = 3.7104, df = 11, p-value = 0.003439), but not in HBOT-2 (t = 1.7032, df = 10, p-value = 0.1194) (Figure 3A).

Comparing control rats with HBOT-2 rats after treatment did not reveal a significant difference (t = 0.47785, df = 17.177, p-value = 0.6388). However, a significant difference was observed between control rats and HBOT-2.5 rats after HBOT sessions (t = 2.7111, df = 9.7132, p-value = 0.02243) (Figure 3A).

While VPA-exposed rats showed increased distance traveled in the open arms compared to the control group, this difference was not statistically significant [control and HBOT-2 (W = 42.5, p-value = 0.6213), control and HBOT-2.5 (W = 37, p-value = 0.2469)]. Post HBOT sessions, there was a decrease in the percentage of distance traveled in the open arms, notably significant in the HBOT-2.5 group [HBOT-2.5 before and after (V = 75, p-value = 0.002441), HBOT-2 before and after (V = 47, p-value = 0.2402)]. Furthermore, a significant difference was observed between HBOT-2.5 after receiving HBOT and the control group [HBOT-2.5 and control (W = 91.5, p-value = 0.006846), HBOT-2 and control (W = 59.5, p-value = 0.4703)] (Figure 3B).

The maze was used to assess any impairments in learning and memory. Results from the repeated measure ANOVA indicated significant differences between groups (p-value <0.0001). As shown in Figure 5, the learning process was observed in the sham rats, yet no statistical differences were found between the control and the sham animals throughout all five days of training (p-value = 0.491).

Post-treatment analysis revealed a noteworthy decrease in the time taken by HBOT-2 rats to find the platform after receiving hyperbaric oxygen therapy, compared to the sham and control groups that did not receive HBOT (Sham and HBOT-2: p-value <2e-16, Sham and HBOT-2.5: p-value <2e-16, Control and HBOT-2: p-value = 1.75e-11, Control and HBOT-2.5: p-value = 1.24e-11) (Figure 5A).

However, no statistical difference was found between HBOT-2 and HBOT-2.5 (p-value = 1.000) (Figure 5A).

Gene expression analysis was conducted in 58-day-old rats. The gene fold data revealed significant differences between groups (p-value = 1.82e-12). Tukey’s post hoc analysis illustrated that the expression of the GRIN2B gene was markedly higher in control rats compared to the sham rats (p-value ~0) (Figure 6).

It has been demonstrated in Figure 6 that the GRIN2B gene expression has significantly increased in rats after undergoing HBOT [sham and HBOT-2 (p-value = 0.0062312), sham and HBOT-2.5 (p-value = 0.0088335)].

Despite this observed increase in gene expression post-treatment, the GRIN2B expression in both HBOT groups remained notably lower than in the control group [control and HBOT-2 (p-value ~0), control and HBOT-2.5 (p-value ~0)] (Figure 6).

Furthermore, following the administration of hyperbaric oxygen, there was no significant difference in gene expression between the HBOT-2 and HBOT-2.5 groups (p-value = 0.9970551) (Figure 6).

Our initial finding revealed that rats exposed to prenatal VPA exhibited tail malformations in 80% of the rats consistent with previous reports (Foley et al., 2012; Kim et al., 2013). In contrast, two studies, one utilizing 500 mg/kg of VPA and the other 600 mg/kg, reported tail malformations in 10 and 34% of rats, respectively (Favre et al., 2013; Saft et al., 2014). These studies suggest that exposure to valproic acid during critical stages of pregnancy, such as neural tube closure, may trigger teratogenic effects in offspring, however, the exact mechanism requires further investigation. Despite these varying observations, understanding the higher incidence of this anomaly in our rats compared to previous studies requires additional research into genetic and embryonic factors.