Prenatal diesel exhaust exposure alters hippocampal synaptic plasticity in offspring

Effects of prenatal exposure to DEPs on the body weight and organ weight of male offspring

The male offspring mean body weight, but not brain weight, was found to be affected by prenatal exposure to DEPs from birth to adulthood (Figure 1). During the first 6 weeks, the body weight of the control group’s offspring remained higher than the other groups. Starting from week 7, the low-dose group’s offspring exhibited faster growth compared to the control group. Nevertheless, the average body weight of the medium and high-dose group’s offspring remained lower than that of the control group on postnatal day 56 (PND56). However, these differences were not statistically significant due to individual variations within the groups (Figure 1A, 1C). Interestingly, compared to the control group, the mean body weight and brain weight of male offspring in the high-dose group were significantly lower on postnatal day 7 (PND7), both brain weight and the brain-to-body weight ratio were significantly lower in the low-dose groups (Figure 1B). This indicates that prenatal exposure to DEPs may impair the growth and development of the offspring.

Figure 1. Body weight and organ weight of male offspring after prenatal DEPs exposure. (A) The body weight of male offspring. (B) The body weight, brain weight and relative brain of 7 days male offspring. (C) The body weight, brain weight, relative brain, hippocampal weight and relative hippocampal of 56 days male offspring. The values shown are the mean ± SD. Compared to control; * p < 0.05, ** p < 0.01. N0,7day (Con: Control group; LD: low dose group; MD: medium dose group) =22, N0, 7 day (HD:High dose group)=12, N14-56day (each group)=12.

Histological effects of prenatal DEPs exposure on PND7 male offspring

We assessed the histology of the hippocampus in PND7 male offspring using H&E staining to investigate the impact of prenatal exposure to DEPs on the hippocampus of early postnatal male mice. In the control group, hippocampal neurons were organized into dense neuronal layers, exhibiting a normal structure and no signs of damage to the hippocampus (Figure 2A). However, in the DEPs treatment groups, the arrangement of hippocampal neuron cells appeared disordered, accompanied by cell swelling, slight cytoplasm staining, cytoplasmic vacuolization, intense nuclear staining, and nuclear pyknosis (Figure 2B–2D). These findings suggest that exposure to DEPs throughout pregnancy significantly influences the development and morphology of the hippocampus in male offspring mice.

Figure 2. Histopathological changes in the hippocampus tissues of male offspring mice following pregnancy exposure to DEPs through hematoxylin and eosin (H&E) staining. 100 or 200 × magnification. (A) Control group. The morphology of hippocampus tissues was normal. (B–D) DEPs (0.235, 0.47 and 0.94 μg/mouse) treatment groups. Scale bar: 100 μm. N=4.

The impact of exposure to DEPs throughout pregnancy on cognitive function of mature male offspring

The spatial learning and memory abilities were employed to assess by the Morris water maze test, which reflected the cognitive function of mature male offspring (PND49-PND56) following exposure to DEPs throughout pregnancy (Figure 3D). The results indicated that the escape latency of the trained mice decreased as the training days progressed, suggesting that all groups of mice possessed a learning ability (Figure 3A). Nevertheless, the differences between each group were dependent on their respective treatments. Specifically, apart from the high-dose (HD) group, there were no significant differences in the escape latency among the other three groups on the first day of spatial training. However, from the second to the fifth day of training, the DEPs treatment groups exhibited longer escape latencies compared to the control group. After the completion of the spatial navigation test, the platform was removed, and the mice were allowed to choose one of the original entry points into the water. The number of mice crossing the target quadrant within a span of 60 seconds was recorded. The results indicated that the average frequency of crossing the platform of the DEPs treatment group was significantly lower than that of the control group (P<0.01) (Figure 3C). Furthermore, the male offspring exposed to DEPs spent less time in the target quadrant compared to the control group (P<0.01) (Figure 3B). Collectively, these findings suggest that exposure to DEPs throughout pregnancy impairs the cognitive abilities of male offspring.

Figure 3. Performance of the different groups of mice in the Morris water maze test. (A) The time required to locate the hidden platform in the water maze during the learning stage. (B) The time elapsed in the correct quarter of the water maze during the probe trial. (C) Number of platform crossings in the four quadrants of the Morris maze during the probe trial. (D) Representative spatial and nonspatial probe trial tracings of four subjects. In the spatial version, the placement of the quadrant remained constant over trials. Con: Control group; LD: low dose group; MD: medium dose group; HD: high dose group. The values shown are the mean ± SD (n_{each group}=12), Compared to control; ** p < 0.01.

The influence of exposure to DEPs throughout pregnancy on the hippocampal synaptic proteins expression, PKA/CREB and CPEB3 activation in PND7 offspring

In this study, the Western blot analysis was conducted to examine the expression levels of NeuN, NMDA receptors (N1, N2A, and N2B), PSD95, and SYP in the hippocampus of PND7 male offspring (Figure 4A, 4B). However, due to the limited number of male offspring in the HD group, the Western blot data for the PND7 mice in the high-dose group was not available. This was done to ensure an adequate sample size for later adult behavioral and molecular experiments. The results showed that in the medium-dose (MD) group, the levels of SYP and NeuN expression were significantly lower compared with the control group (P<0.01). Additionally, the levels of N1, N2A, and PSD95 were decreased, while the NR2B expression was dramatically increased in both the low-dose (LD) and MD groups (P<0.01). To further investigate the changes in protein expression and cellular localization, immunohistochemical techniques were employed to compare the levels of these proteins between the control and MD groups (Figure 4C, 4D). The results confirmed that PSD95, SYP, NeuN, N1, and N2A subunits exhibited reduced expression in the MD group mice. The change in the cell membrane of the N2B subunit was not obvious, but an increase in the expression of the cell membrane space was observed. These findings were consistent with the Western blot results. Overall, these results suggest that exposure to DEPs throughout pregnancy leads to alterations in the expression of hippocampal synaptic transmission-related proteins and structural proteins in early male offspring (PND7).

Figure 4. Protein expression of NMDA/PKA/CREB and CPEB3 signaling pathway-associated genes in hippocampus samples of 7 days male offspring mice. (A) Western blot analysis of p-CREB, CREB, PKA, N2A, N2B and NR1 protein in hippocampus. (B) Western blot analysis of CPEB3, NEUN, PSD-95 and SYN protein in hippocampus. (C) Immunohistochemical analysis of NR1, N2B and N2A protein in hippocampus. (a–c) Control group (CON). (d–f) DEPs treatment groups (DEPs, the MD group). (D) Immunohistochemical analysis of SYN, PSD-95 and NEUN protein in hippocampus. (a–c) Control group (CON). (d–f) DEPs treatment groups (DEPs, the MD group). The values shown are the mean ± SD (NAB=6, NCD=4), Compared to control; ** p < 0.01.

In order to investigate whether the NMDA/PKA/CREB and CPEB3 signaling pathways are involved in the neurotoxic effects induced by DEPs, the levels of PKA, p-CREB, CREB, and CPEB3 proteins in the hippocampus were measured using Western blotting (Figure 4A, 4B). The results confirmed that the level of PKA was significantly increased in the MD group (P<0.01), but not in the LD group. Additionally, the levels of p-CREB were up-regulated, while the levels of CPEB3 were significantly down-regulated in both the LD and MD groups (P<0.01). These findings suggest that the NMDA/PKA/CREB signaling pathway may be activated, while the CPEB3 signaling pathway could be suppressed in early postnatal male offspring exposed to DEPs throughout pregnancy.

The impact of DEP exposure throughout pregnancy on the expression of hippocampal synaptic proteins, as well as the activation of PKA/CREB and CPEB3 in mature offspring

The same proteins that were detected in PND7 were also found in the mature offspring of mice (Figure 5A, 5B). In the DEPs treatment groups, except for NR2B, the levels of hippocampal proteins such as SYP, NeuN, N1, N2A, and PSD95 were reduced compared to the control group (P<0.01). The results of immunohistochemical analysis in the mature offspring were similar to those in PND7 (Figure 5C, 5D). The expression of N1 and N2A subunits in the DEPs treatment groups was lower than in the control group, and there were no significant changes in the cell membrane of the N2B subunit, but the expression of the cell membrane space increased. Additionally, there was a reduction in PSD95, SYP, and NeuN protein expression in the DEPs treatment groups. These findings indicate that the hippocampal synaptic transmission function and structural related proteins changed in mature male offspring with prenatal exposure to DEPs. Furthermore, the levels of PKA and p-CREB were significantly increased in the DEPs treatment groups (P<0.01), while the levels of CPEB3 were significantly down-regulated in MD and HD groups (P<0.01) (Figure 5A, 5B). This suggests that the NMDA/PKA/CREB pathway could still be activated, but the CPEB3 signaling pathway might be suppressed in mature male offspring exposed to DEPs throughout pregnancy.

Figure 5. Protein expression of NMDA/PKA/CREB and CPEB3 signaling pathway-associated genes in hippocampus samples of 56 days male offspring mice. (A) Western blot analysis of p-CREB, CREB, PKA, N2A, N2B and NR1 protein in hippocampus. (B) Western blot analysis of CPEB3, NEUN, PSD-95 and SYN protein in hippocampus. (C) Immunohistochemical analysis of NR1, N2B and N2A protein in hippocampus. (a–c) Control group (CON). (d–f) DEPs treatment groups (DEPs, the MD group). (D) Immunohistochemical analysis of SYN, PSD-95 and NEUN protein in hippocampus. (a–c) Control group (CON). (d–f) DEPs treatment groups (DEPs, the MD group). The values shown are the mean ± SD of (N_AB=9, N_CD=3). Compared to control; * p < 0.05, ** p < 0.01.

Animals and treatments

8-week-old C57BL/6 mice weighing 21±0.5 g were housed at the SPF Experimental Animal Center of Nantong University for 1 week for acclimation before being mated in a 2:1 cage ratio with 32 female mice and 16 male mice. The vaginal smear on the second day of cage-closure was marked as pregnancy 0 day. On the first day of pregnancy, the mother mice were randomly assigned to a control group, low-dose exposure group, middle-dose exposure group, and high-dose exposure group. To ensure experiment reproducibility and particle composition stability, commercial DEPs (the National Institute of Standards and Technology, SRM 2975; Gaithersburg, MD, USA) were used. Sterile PBS was used to prepare suspensions of different concentrations, with each suspension packed separately in 1 mL volumes. Prior to exposure, the suspensions were sonicated for 15 minutes and then heated to 37° C.

To minimize potential errors in exposure dose among offspring within the same group or even the same experimental animal, this study utilized tail vein injection as the method for DEPs exposure. This approach ensures a more accurate and consistent delivery of DEPs to the offspring brain tissue. Other exposure methods such as pharyngeal nasal drip and tracheal drip may result in variations in conversion efficiency and DEPs absorption, leading to significant errors in the actual exposure levels in the offspring brain. By employing tail vein injection, the study aims to accurately investigate the cross-generational toxicity of DEPs.

To facilitate tail vein injection, the mouse’s tail was immersed in 45° C water for 1 minute to dilate the blood vessels. A sterile 27-gauge needle syringe was prepared for the injection. Consistent with previous research reports, intravenous injection was employed to expose the mice to diesel exhaust particulate matters [44]. The injection procedure begins at the tip of the tail and progresses towards the base of the tail, with the injection site changing each time to target different blood vessels. The injection volume administered per mouse is 0.1 mL.

According to the 2012 China Environmental Air Quality Standard (GB3095-2012), the maximum allowed concentration of secondary PM2.5 for a 24-hour period is 75 μg/m³. To determine the exposure level of mice via the respiratory tract at this limit, we consider the breathing rate of the mice (24 ml/min) and the conversion factor for equivalent dose to the human body surface area (9.1). The calculation for the exposure level of mice through the respiratory tract is as follows: 75*24*10-6*24*60*9.1=2.35 μg. Recent research data shows that the efficiency of particulate matter in air pollution entering the brain through blood circulation may be 8 times higher than that through the nose [45]. Based on this, we used a dose equivalent to 10% (0.235 μg/mouse/time) of the maximum concentration allowed for respiratory exposure as the lowest dose (low dose group, LD) for tail vein injection. The medium dose group (MD) received 0.47 μg/mouse/time, and the high dose group (HD) received 0.94 μg/mouse/time. The tail vein injections started on the first day and were administered every other day, for a total of 10 injections. The purpose of this study is to investigate any potential neurotoxic effects of DEP exposure during pregnancy.

After birth, the newborn offspring were weighed and counted. Male offspring were specifically collected, with a breastfeeding ratio of 4 male mice per mother mouse. Subsequently, the male offspring were weighed once a week. At postnatal day 7 (PND 7), ten male mice from each group were sacrificed. Hippocampal tissues were collected from each group for tissue sections (n=4) and protein expression analysis (n=6). The remaining male offspring mice underwent a water maze experiment at PND 49. After the experiment, all mice were euthanized, and their body weight, brain weight, and hippocampus weight were measured.

Morris water maze experiment

The Morris water maze experiment was conducted in a circular pool with a diameter of 150 cm and a depth of 22 cm. The pool was divided into four quadrants: North (N), East (E), South (S), and West (W). The water in the pool was warm and opaque, maintained at a temperature of 24 ± 2° C. A black platform with a diameter of 14 cm and a height of 21 cm was submerged in the pool. The purpose of the submerged platform was to provide a target for the mice to locate during the experiment. On the first day of the experiment, the animals were placed in the pool for a duration of 90 seconds. This initial session allowed the mice to become familiar with the maze environment, following protocols that have been previously reported [46]. The mice underwent training for five consecutive days, with each day consisting of four trials. During each trial, the mice were dropped into one of the four quadrants of the pool and allowed to swim freely until they located the submerged goal platform. Once the mice found the platform, they were required to stay on it for at least 3 seconds. If a mouse was unable to find the platform within 1 minute, it was gently guided to the platform and allowed to stay on it for 15 seconds. On the seventh day of the experiment, the platform was removed, and the mice were dropped into the pool for a duration of 60 seconds. Throughout the experiment, the swimming route, time spent in the water, and number of times the mice appeared on the platform were recorded. The SLY-WMS Morris water maze experiment system was used to conduct the test and gather the necessary data.

Histological preparation

For morphometric analysis, fresh hippocampal tissues were harvested from the animals, fixed in 4% paraformaldehyde, and subsequently embedded in paraffin. Paraffin-embedded sections of the hippocampus, 5 μm thick, were stained with Hematoxylin-eosin (HE) for microscopic examination. All sections were examined using light microscopy.

Immunohistochemistry analysis

The 5 μm thick paraffin-embedded hippocampal slices were mounted on slides coated with 3-aminopropyl-triethoxysilane (APES) for immunohistochemistry analysis as previous literature report [47]. After deparaffinization and drying, the paraffin sections were rinsed with NaCl solution, dehydrated, and then incubated in a solution of 3% H₂O₂-methanol-PBS (0.01 M, pH 7.2) for 10 minutes. Antigen retrieval was performed using trisodium citrate buffer (0.01 mol/L, pH 6.0), followed by three washes with PBS. Subsequently, the sections were treated with 10% bovine serum albumin (BSA) to block the activity of endogenous peroxidases for 1 hour. For immunostaining, the sections were incubated overnight at 4° C with primary antibodies, including neuron-specific nuclear protein (NeuN, Proteintech, 26975-1-AP), NR1 (LMAI Bio, LM-23343R), N2B (LMAI Bio, LM-0222R), N2A (Merck, AB1555P), SYP (CUSABIO, CSB-PA004200), and PSD-95 (LMAI Bio, LM-20649R). To recover the antigens, the sections were boiled in 0.01 mol/L trisodium citrate buffer (pH 6.0) and then washed three times with PBS (every 3 minutes). The sections were then stained using 3, 3’-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO, USA). Negative controls were obtained by omitting the primary antibodies. The sections were examined and photographed using NIS-Elements BR 2.30 (40×), and subsequently counterstained with hematoxylin.

Western blot analysis

The WB analysis is consistent with the literature [48]. The mice hippocampus was subjected to centrifugation at 12,000 g and 4° C for approximately 10 min to obtain total proteins. The concentration of the total protein was measured using the BCA Protein Assay kit (Beyotime, P0009, China). The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Millipore Corporation, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk at room temperature for 1 h, followed by incubation with specific primary antibodies. Specifically, β-actin (ACTB) was detected using (Sigma, A5316), neuron-specific nuclear protein (NeuN) with (Proteintech, 26975-1-AP), and the primary antibodies used for NR1, N2B, N2A, PKA, SYP were the same as those used in immunohistochemistry. Additionally, PSD-95 was detected with (GeneTex, GTX112846), p-CREB with (PerkinElmer, TRF0200-M), and CPEB3 with (PL0306681, PLLABS) for western blot. Following incubation with primary antibodies, the membrane was washed with TBST and treated with a peroxidase-conjugated secondary antibody (HRP-labeled goat anti-IgG, Zhongshan Biology Company, Beijing, China) diluted at 1:1000 in blocking solution for 1 hours at room temperature. Densitometry analysis of the target protein bands was performed by ImageJ software.

Statistical analysis

The data were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA) and presented as mean ± SD of independent experiments. The Wilcoxon Rank-Sum test was used for analysis of non-normally distributed data, while Student’s t-test or analysis of variance (ANOVA) was used for data with a normal distribution. A p-value of less than 0.05 was considered statistically significant.