# Long-term Impact of Early-Life Stress on Hippocampal Apoptotic Gene Expression in Mice

Aida Nurul Barokah, İhsan Kıvanç Gürsoy, Merve Hilal Dönmez, Juliette Fitremann, Arslan Bayram, Keziban Korkmaz Bayram

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Aida Nurul Barokah, İhsan Kıvanç Gürsoy, Merve Hilal Dönmez, Juliette Fitremann, Arslan Bayram, et al.. Long-term Impact of Early-Life Stress on Hippocampal Apoptotic Gene Expression in Mice. 2025. [hal-05390932](https://hal.science/hal-05390932v1)

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# Long-term Impact of Early-Life Stress on Hippocampal Apoptotic Gene Expression in Mice

**Aida Nurul Barokah**

Ankara Yıldırım Beyazıt University

**İhsan Kıvanç Gürsoy**

Ankara Yıldırım Beyazıt University

**Merve Hilal Dönmez**

Technical University of Berlin

**Juliette Fitremann**

Université de Toulouse, CNRS UMR 5623

**Arslan Bayram**

GENTAN Genetic Diseases Evaluation Centre

**Keziban Korkmaz Bayram**

k.korkmaz.bayram@aybu.edu.tr

Ankara Yıldırım Beyazıt University

## Research Article

**Keywords:** Apoptosis, cell survival, maternal deprivation, 3D hippocampal cell culture, nucleofection.

**Posted Date:** April 1st, 2025

**DOI:** https://doi.org/10.21203/rs.3.rs-6267468/v1

**License:** This work is licensed under a Creative Commons Attribution 4.0 International License.

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**Additional Declarations:** No competing interests reported.
**Abstract**

Early-life stress (ELS) such as maternal separation has been associated with neuronal apoptosis and impaired hippocampal function in rodent models. This study investigated the long-term effects of early-life unpredictable maternal separation (MS) and MS combined with unpredictable maternal stress (MSUS) from postnatal days 1 to 14 on the mRNA expression levels of the proapoptotic genes *Bax*, *Tp53* and *Casp3*, and the prosurvival gene *Bcl2* in the hippocampus of mice (*in vivo*). Additionally, total RNA from the MSUS hippocampus was nucleofected at different concentrations into healthy mouse hippocampal cells, followed by 3D neuronal cell culture using N-heptyl-D-galactonamide (GalC7) hydrogels as scaffolds for hippocampal cell growth and apoptotic gene expression studies (*in vitro*). Quantitative real-time PCR was conducted to assess the expression of target genes, which were subsequently analyzed using the comparative Ct method ($2^{-\Delta\Delta Ct}$). *Bax* mRNA expression was significantly lower in the MS group than in the control group, whereas both the MS and MSUS groups presented significant increases in *Bcl2* mRNA expression. In addition, the expression ratio of *Bcl2/Bax* was significantly greater in the MS and MSUS groups. No significant differences in *Tp53* or *Casp3* mRNA expression levels were detected between the groups. Although the *in vitro* mRNA expression levels were not significantly different, the mRNA expression ratio of *Bcl2/Bax* reached equilibrium as the concentration of total RNA nucleofected increased. Our results suggested that, in response to ELS, hippocampal cells adapt to prioritize survival over apoptosis.

**Introduction**

The early postnatal period is a critical time for neurodevelopment, particularly in the hippocampus. During this period, environmental factors can significantly impact brain development and potentially lead to long-lasting alterations in neural circuitry and behavior. One such environmental factor is early-life stress (ELS), which is associated with an increased risk for various neuropsychiatric disorders later in life [1]–[3]. Studies have shown that the hippocampus, a brain region critical for memory formation [4], [5], is influenced through various pathways under uncontrolled stress conditions [6]. Stress is known to impair memory performance, alter synaptic plasticity and neuronal firing patterns, and induce structural modifications in the hippocampus. These stress-induced alterations in the hippocampus have been associated with stress-related disorders, including posttraumatic stress disorder (PTSD) [6]. The hippocampus plays a crucial role in learning and memory, and early-life challenges that begin as early as pregnancy can have long-term effects by influencing neuronal apoptosis in the hippocampus, which in turn contributes to learning deficiencies in offspring [7]. In animal models, excessive exposure to glucocorticoids due to stress has been associated with hippocampal dysfunction and neuronal loss [8]. Studies in both humans and rodents have provided significant insights into how early-life stress and maternal separation affect hippocampal function, particularly apoptosis and neuronal proliferation. ELS, such as maternal separation, is linked to a reduction in mature neurons in the CA3 region of the hippocampus, affects cognitive functions and maternal behavior in adult female mice [9], and leads to epigenetic upregulation of corticotropin-releasing hormone in the hippocampus, which is associated
with synaptic dysfunction and memory defects [10]. In addition, research suggests that early maternal and social deprivation can expand neural stem cell populations, increase neurogenesis in the hippocampus and amygdala, and reduce fear memory [11]. Research in animal models consistently demonstrates that early-life stressors, such as maternal separation, have detrimental effects on behaviors in adulthood [12], [13]. Studies have demonstrated that early MS can increase apoptosis in the hippocampal region of litters [14]–[16]. Therefore, the effects of postnatal two-week MS, which is a crucial period with some postnatal maturation characteristics [17], on the trends of apoptosis in five-week-old litters have not been reported before.

To examine the potential long-term impacts of ELS on hippocampal gene expression, we focused on four genes involved in regulating apoptosis: *Bax*, *Bcl2*, *Casp3*, and *Tp53*. These genes play crucial roles in determining cell fate during neurodevelopment and in response to stress. *Bax* promotes apoptosis [18]–[20], whereas *Bcl2* inhibits it [21]. *Casp3* is a key executioner caspase in the apoptotic cascade [22], and *Tp53* is a tumor suppressor gene that can induce apoptosis in response to cellular stress [23]. Using quantitative real-time PCR (qPCR), we analyzed the expression levels of these genes in the hippocampi of the mice at PND35, three weeks after the cessation of the stress paradigms. This time point allows us to assess the persistent effects of ELS on gene expression beyond the immediate stress response period. Additionally, we performed nucleofection procedures to explore how different RNA doses from MSUS affect apoptosis-related gene expression in healthy hippocampal cells cultured on N-heptyl-D-galactonamide (GalC7) hydrogels as a 3D cellular model. The GalC7 hydrogel was selected because it is composed of a single, pure molecule, which means that its composition is highly reproducible. The gels formed have low moduli, on the order of a few kPa, making them suitable for neuronal cell culture. Finally, the gel architecture is made up of quite wide and sparse fibers which enable the cells to grow partly embedded within the gel. The degree of cell adhesion is also low, encouraging cells to organize themselves in small clusters along the fibers, thus reproducing a 3D culture quite well [24], [25]. Previous studies have reported the microinjection of RNA to create mouse models for autism [26], [27]. Similarly, our approach involves nucleofecting RNA from the hippocampi of stressed mice into healthy cells to replicate the stress environment *in vitro*. This method allows us to explore how ELS-induced gene expression changes *in vivo* can be mimicked in a controlled, cellular model, providing further insights into the molecular mechanisms underlying stress-related disorders.

# Materials and methods

## Animals

This study was conducted in accordance with the institutional guidelines for the care and use of laboratory animals. Ethical clearance was obtained from Ankara University Laboratory Animals and Research Laboratory (Ethics Committee Approval No: 2023-3-20). The eight-week-old *BALB/c* mice (n = 6 per group) were housed under a 12:12 h light/dark cycle (lights at 7 a.m. to 7 p.m.) with constant room temperature (22 ± 2°C) and humidity (45 ± 5%) in standard plastic mouse cages (22 cm × 38 cm × 15 cm). Food and tap water were available *ad libitum*. All the cages were subjected to weekly cage cleaning.
The breeding procedures included housing one female and one male in the same cage. After the mating period, the males were removed, and the female mice remained alone throughout the gestation period.

## Maternal stress procedures

This study included three groups of litter from related dams: control, unpredictable maternal separation (MS), and unpredictable maternal separation combined with unpredictable maternal stress (MSUS) groups. The maternal stress model was re-established following the same procedures described in our previous study [28]. Briefly, dams and litters were separated for 3 hours each day from postnatal day (PND) 1 to PND 14. Additionally, MSUS dams experienced unpredictable maternal stress in conjunction with the MS procedure. A total of 72 five-week-old litters, with 24 (12 female litters and 12 male litters) from each group, were used in the *in vivo* study. Additionally, five-week-old control male litters (n = 4) were used for the *in vitro* 3D hippocampal cell culture experiments.

## Tissue collection and Hippocampal Dissociation into Single Cells

At PND35, the mice were sacrificed by cervical dislocation. The total brains were carefully removed, and the brain hemispheres were gently lifted toward the forebrain. The right and left hippocampi were carefully removed via fine forceps. The surgical instruments were cleaned with 75% ethanol between each transition to prevent cross-contamination. The hippocampal tissues were frozen in cryotubes in liquid nitrogen and stored at -80°C until RNA isolation. For *in vitro* 3D hippocampal cell culture, the hippocampi of control male mice were transferred to 15 mL Falcon tubes containing 5 mL of dissection solution and kept on ice until they were transported to the cell culture laboratory. The dissection solution was prepared before the tissue collection step by mixing 500 mL of Hank's balanced salt solution (HBSS), 5 mL of penicillin/streptomycin (Gibco, Life Technologies), 5 mL of 1 M MgCl₂, 3.5 mL of 1 M HEPES (pH 7.3) (Lonza, Switzerland) and 5 mL of 200 mM L-glutamine (Gibco, Life Technologies) in a Falcon tube. The samples in the dissection mixture were centrifuged at 80 × g for 5 minutes, and the supernatant was carefully discarded. Subsequently, 1.5 mL of trypsin solution was added to the pellet, and the samples were incubated at 37°C for 10–20 minutes. After trypsinization, the samples were centrifuged at 80 × g for 5 minutes, and the supernatant was discarded. The pellet was washed twice with 5 mL of HBSS. After the second wash, 1.5 mL of prewarmed (37°C) medium was added to the pellet. All the tissue pieces were gently triturated 20–30 times via a fire-polished Pasteur pipette until a homogeneous suspension was achieved. This process was repeated for 1 minute with a fresh fire-polished Pasteur pipette.

## Cell counting and viability assessment

Five milliliters of culture medium was added to the hippocampal cells, and the cells were counted using a TC20 cell counter (Bio-Rad, USA). The average hippocampal cell count was 5.91 × 10⁶ cells/mL. The
counted cells were centrifuged at 80 × g for 10 minutes, and the supernatant was discarded. Cell viability was assessed under a microscope using 0.4% trypan blue solution (Gibco, Life Technologies) at a 1:1 ratio. Once cell viability was confirmed to be suitable, nucleofection was performed.

## Total RNA isolation from hippocampal tissue

The collected hippocampal tissues were transferred into Eppendorf tubes containing 500 mL of NucleoGene Tri Reagent Lysis Reactive (NucleoGene, Türkiye). The tissues were disintegrated via an ultrasonic tissue homogenizer. Total RNA was isolated from hippocampal tissue (n = 72) via the phenol-chloroform extraction method as described in the previous study [29]. The total RNA used for nucleofection was selected on the basis of the criteria of increased *Bcl2* and decreased *Bax* expression, and was obtained from the hippocampus of a male litter in the MSUS group. Male mice were used to avoid the potential confounding effects of hormonal fluctuations associated with the estrous cycle in female mice. The total RNA was further purified via the High Pure RNA Isolation Kit (Roche, Germany) to eliminate any DNA-induced effects. The purity and concentration of the total RNA were measured with a NanoDrop 2000c (Thermo Fisher Scientific, USA).

## Preparation of the Hydrogel

The GalC7 molecule was synthesized as previously described [24], and the GalC7 hydrogel was prepared for culture as described in our previous study [29]. Briefly, for each sample, 9 mg of GalC7 powder was weighed and placed into a glass vial. Subsequently, 2 mL of nuclease-free water was added to the vial and the vial was slightly closed with a cap. The vial was then placed in a programmable oven preheated to 115°C to dissolve the GalC7 powder completely. Once dissolved, the hot solution is quickly transferred to culture plate wells. The culture plate was closed to prevent the gels from drying out, and the temperature is allowed to decrease slowly until gelation occured. The gel prepared the day before was saturated with medium and incubated at 37°C in an incubator containing 5% CO₂.

## Nucleofection and 3D cell culture using the GalC7 hydrogel

Exogenous RNA (total RNA, RNAse-treated total RNA (mock) and 0.5, 1, and 2 ng/µL diluted from total RNA) was nucleofected into control male hippocampal cells using The Amaxa® 4D-Nucleofector® Protocol for Primary Mammalian Neurons (Lonza, Switzerland) Kit at the previously optimized DR-114 program, as indicated in the kit protocol. After nucleofection, the nucleocuvette was incubated for 10 minutes at room temperature. After incubation, the cells were resuspended in prewarmed medium and gently mixed by pipetting up and down 2-3 times. The nucleofected cells were cultured with GalC7 gel prepared in a 48-well plate. A total of 250 µL of DMEM containing 10% penicillin/streptomycin (Gibco, Life Technologies), and 1X B27 supplement (Gibco, Life Technologies) were added. The samples were incubated at 37°C with 5% CO₂ for seven days. Four samples from each group were prepared: a control group without nucleofection; a mock-nucleofected group; 0.5, 1 and 2 ng/µL total RNA-nucleofected
groups. These samples were cultured in GalC7 for a 7-day culture period. After 7 days, the medium was removed, and the samples were washed with PBS. The samples were lysed with 1 mL of NucleoGene Tri Reagent Lysis Reactive. Total RNA was extracted from 3D hippocampal cell culture using a phenol-chloroform method that was previously established [29].

## cDNA synthesis and quantitative real-time PCR

cDNA synthesis from total RNA was performed with the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). Each sample was adjusted to approximately 1 µg of total RNA. The reaction mixture for each sample consisted of 10 µL of 2x RT Mix, 2 µL of HiScript II Enzyme Mix, 1 µL of Oligo-(dT)23 VN (50 µM), 1 µL of random hexamers (50 ng/µL), 4 µL of total RNA, and 2 µL of nuclease-free water, yielding a total reaction volume of 20 µL. cDNA synthesis was carried out via T100 Thermal Cycler PCR (Bio-Rad, USA) with the following profile: 5 minutes at 25°C, 15 minutes at 50°C, and 2 minutes at 85°C. The resulting cDNA samples were diluted 1:5 with nuclease-free water.

Quantitative real-time PCR was performed on a Rotor-Gene Q (Qiagen, Germany) with specific primers (Table 1) and 2x ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). The reaction mixture consisted of 10 µL of 2x ChamQ Universal SYBR qPCR Master Mix, 0.8 µL of 10 µM forward and reverse primer mixture, 6.2 µL of nuclease-free water, and 3 µL of 1:5 diluted cDNA, totaling 20 µL per qPCR

The qPCR profile was as follows: 30 s at 95°C for initial denaturation, 10 s at 95°C for denaturation, and 30 s at 58°C for annealing and extension, followed by melting curve analysis. The cycle count was set to 45. Each sample was repeated at least twice. The results were analyzed via the delta-delta-Ct (2^(-ΔΔCt)) method. The data were normalized to the reference gene *Gapdh*, and the mean values from the control group were used as the calibrator.
<table><caption>Table 1
Sequences of the primers.</caption><thead><tr><th>Primer</th><th>Sequence (5'-3')</th><th>Amplicon Length (bp)</th></tr></thead><tbody><tr><td><i>Gapdh</i></td><td>Forward: CTCTCTGCTCCTCCCTGTTC<br>Reverse: TACGGCCAAATCCGTTCACA</td><td>105</td></tr><tr><td><i>Bax</i></td><td>Forward: TTTGCTACAGGGTTTCATCCA<br>Reverse: ATATTGCTGTCCAGTTCATCTCC</td><td>147</td></tr><tr><td><i>Bcl2</i></td><td>Forward: CTGGGATGCCTTTGTGGAAC<br>Reverse: TCAAACAGAGGTCGCATGCT</td><td>51</td></tr><tr><td><i>Casp3</i></td><td>Forward: CAGCACCTGGTTACTATTCCTG<br>Reverse: TTCCTGTTAACGCGAGTGAG</td><td>130</td></tr><tr><td><i>Tp53</i></td><td>Forward: CAACAGCTCCTGCATGGGGGGC<br>Reverse: AGGACAGGCACAAACACGAACC</td><td>121</td></tr></tbody></table>

## Statistical Analyses

Statistical analyses were conducted via GraphPad Prism 9.1.0 (GraphPad Software, USA). The distribution of the data was assessed through histograms, q-q plots, and Shapiro-Wilk tests. The outliers were identified and removed via the ROUT method in GraphPad Prism (Q = 1%). Independent t tests or Mann-Whitney U tests were used to compare data between two groups. For calculation of the *Bcl2/Bax* ratio, the relative quantification units of *Bcl2* were divided by those of *Bax* for each sample. For comparisons involving three or more groups, the Kruskal-Wallis H test was employed to assess the significance of the difference in accordance with the distribution of the data. Dunn's test was used for post hoc analysis. The relationships between the data were evaluated via Spearman correlation. Statistical significance was considered at a p value of < 0.05.

## Results

The mRNA expression levels of *Bax*, *Bcl2*, *Casp3*, and *Tp53* in hippocampal tissue are presented in Fig. 1a-d. Prior to conducting the main analysis, including the Kruskal-Wallis test, an independent t-test was performed to confirm the absence of significant differences between male and female offspring, allowing us to combine the data. Given that the data did not meet the assumption of a normal distribution, a nonparametric Kruskal-Wallis test was performed. Our study revealed significant differences in the mean ranks of *Bax* mRNA expression among the groups ($H(2, n = 72) = 6.38, P = 0.04$; Fig. 1a). Dunn's multiple comparison test revealed significant downregulation in the MS group compared with the control group ($P = 0.05$). For *Bcl2*, significant differences in mean ranks were observed ($H(2, n = 72) = 16.22, P = 0.0003$; Fig. 1b). Dunn's multiple comparison test revealed significant upregulation in
both the MS and MSUS groups compared with the control group (P = 0.0005 and P = 0.0057, respectively). No significant differences were detected among the groups for either *Casp3* (*H* (2, n = 72) = 3.43, *P* > 0.05; Fig. 1c) or *Tp53* (*H* (2, n = 72) = 5.50, *P* > 0.05; Fig. 1d). Furthermore, our study revealed significant differences in the mean ranks of the *Bcl2/Bax* expression ratios among the groups (*H* (2, n = 72) = 20.16, *P* < 0.0001; Fig. 1e). Dunn's multiple comparison test revealed a significant increase in the *Bcl2/Bax* expression ratio in both the MS and MSUS groups compared with the control group (P = 0.0001 and P = 0.0010, respectively).

<table><caption>Table 2
Spearman correlation coefficients between Bax, Bcl2, Casp3 and Tp53 mRNA expression in the hippocampus of litters within each experimental group.</caption><thead><tr><th></th><th></th><th>1. Bax</th><th>2. Bcl2</th><th>3. Casp3</th><th>4. Tp53</th></tr></thead><tbody><tr><td rowspan="4">Control</td><td>1.</td><td>1</td><td></td><td></td><td></td></tr><tr><td>2.</td><td>-0.40</td><td>1</td><td></td><td></td></tr><tr><td>3.</td><td>0.24</td><td>0.01</td><td>1</td><td></td></tr><tr><td>4.</td><td><strong>0.61**</strong></td><td><strong>-0.43*</strong></td><td>0.21</td><td>1</td></tr><tr><td rowspan="4">MS</td><td>1.</td><td>1</td><td></td><td></td><td></td></tr><tr><td>2.</td><td>-0.18</td><td>1</td><td></td><td></td></tr><tr><td>3.</td><td><strong>0.61**</strong></td><td>-0.10</td><td>1</td><td></td></tr><tr><td>4.</td><td><strong>0.74****</strong></td><td>-0.11</td><td>0.37</td><td>1</td></tr><tr><td rowspan="4">MSUS</td><td>1.</td><td>1</td><td></td><td></td><td></td></tr><tr><td>2.</td><td>0.07</td><td>1</td><td></td><td></td></tr><tr><td>3.</td><td><strong>0.68***</strong></td><td>0.23</td><td>1</td><td></td></tr><tr><td>4.</td><td><strong>0.65***</strong></td><td>0.04</td><td><strong>0.58**</strong></td><td>1</td></tr></tbody><tfoot><tr><td colspan="6">Note. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001</td></tr></tfoot></table>

Spearman's rank correlation was performed to further explore the relationships between gene expression in the hippocampal tissue of the litters. As shown in Table 2, in the control group, a significant positive correlation was observed between *Bax* and *Tp53* expression (r = 0.61, *P* = 0.002), whereas *Bcl2* and *Tp53* expression was significantly negatively correlated (r=-0.43, *P* = 0.034). In the MS group, significant positive correlations were observed between *Bax* and *Tp53* (r = 0.74, *P* < 0.0001) and between *Bax* and *Casp3* (r = 0.61, *P* = 0.0015). In the MSUS group, significant positive correlations were detected between *Bax* and *Tp53* (r = 0.65, *P* = 0.0006), between *Bax* and *Casp3* (r = 0.68, *P* = 0.0003), and between *Casp3* and *Tp53* (r = 0.58, *P* = 0.0027).
Trypan blue staining confirmed that most cells remained viable postnucleofection (Fig. 2a). Additionally, the development of hippocampal cells in GalC7 was monitored over time via an Olympus CKX41 inverted microscope at 20x magnification (Fig. 2b-e). Initially, the cells appeared sparsely distributed with minimal neurite outgrowth. Over time, increased cell clustering was observed, along with more prominent interactions between cells and the surrounding GalC7 fibers. In later stages, the cells appeared more densely distributed and organized within the hydrogel, suggesting progressive maturation within the 3D environment.

The *Bcl2/Bax* expression ratios in 3D hippocampal cell culture samples from different groups (control, mock, 0.5, 1, and 2) are presented in Fig. 3a. No significant differences were observed across groups (H (4, n = 20) = 3.77, P > 0.05). Notably, the *Bcl2/Bax* ratios in the control and mock groups were similar, which is important for ruling out potential effects of nucleofection on gene expression. Although no significant differences were found, the *Bcl2/Bax* ratio was lower in the nucleofected groups than in the control and mock groups. The highest variability in the *Bcl2/Bax* ratio was observed in the 0.5 group, suggesting a heterogeneous response at this concentration, whereas the variability decreased at higher concentrations.

Furthermore, the mRNA expression levels of *Casp3* in 3D hippocampal cell culture samples from different groups (control, mock, 0.5, 1, and 2) are presented in Fig. 3b. No significant differences were observed across groups for *Casp3* (*H* (4, *n* = 20) = 7.91, *P* > 0.05).

Discussion

In this study, we investigated the long-term effects of PND1-14 postnatal unpredictable maternal separation (MS) and unpredictable maternal separation combined with unpredictable maternal stress (MSUS) on apoptosis-related gene expression in the hippocampus of five-week-old mice. While several studies have reported that maternal separation leads to increased apoptotic cell death in the hippocampus of rats and mice, notably at various time intervals [14]–[16], [30], our findings revealed that survival tends to be promoted instead of apoptotic pathways in the hippocampus. These findings are consistent with those of the study by Coccurello R. et al. (2014), who reported an increase in *Bcl2* mRNA expression in the hippocampal region [31]. In our study, the expression of the *Bcl2* mRNA was significantly greater in the MS and MSUS groups than in the control group (*P* = 0.0005 and *P* = 0.0057, respectively). Compared with that in the control group, *Bax* was significantly downregulated in the MS group (*P* = 0.05) but not in the MSUS group. However, MSUS *Bax* expression still consistently decreased, similar to MS. In the control correlation results, *Bax* and *Bcl2* showed a nonsignificant moderate negative correlation (*r* = -0.40), reflecting their classical antagonistic role in regulating apoptosis. However, this correlation weakened in MS (*r* = -0.18) and disappeared in MSUS (*r* = 0.07), suggesting that their regulatory relationship is disrupted under MS and MSUS conditions. The upregulation of *Bcl2* in MS and MSUS indicates the promotion of hippocampal neuronal survival while simultaneously suppressing apoptotic processes. Additionally, the significantly increased *Bcl2/Bax* ratios in the MS (*P* = 0.0001) and MSUS (*P* = 0.0010) groups compared with those in the control group further support a shift toward cell survival
mechanisms. Previous studies have shown that ELS, such as low-level maternal care, increases vulnerability to hippocampal neuron loss via apoptosis in PND90 rats [32]. In humans, early-life adversity has been linked to a smaller hippocampus volume in male depressed in-patients [33]; this alteration may stem from the apoptotic pathway and contribute to severe neuropsychiatric outcomes. Moreover, early MS has been associated with long-term consequences [34]. Our findings suggest that hippocampal cells adapt to promote survival, counteract apoptosis and preserve neuronal integrity. This response may help mitigate the long-term impact of ELS and prevent more severe consequences later in life. Neuronal survival can be initiated through increased intracellular Ca²⁺, which activates prosurvival signal transduction pathways (the phosphoinositide 3-kinase (PI3K)/Akt pathway, the protein kinase C (PKC)/extracellular signal-regulated kinase (ERK) pathway [14], and the Ca²⁺/calmodulin pathway), which further inhibits the transcription of cell death-related genes and activates key transcription factors such as CREB and NF-κB. This leads to an increase in *Bcl2* expression [35], [36] and an increase in the ratio of antiapoptotic to proapoptotic factors within the cell [37], possibly due to an increase in hippocampal IL-1β and TNF-α via those pathways, as reported in some short- and long-term maternal separation studies [38]. On the basis of our unpublished data, which revealed a decrease in the expression of Ca²⁺-permeable *Gria2* in MS and MSUS, it is reasonable to hypothesize that Ca²⁺ influx may involve the upregulation of *Bcl2*, further supporting neuronal survival.

There were no significant differences in *Casp3* expression among the groups. There was variability in expression, but there was no strong trend toward upregulation or downregulation. This result is consistent with the notion that the transcription of the prosurvival protein *Bcl2* and the repression of the proapoptotic protein *Bax* likely prevent the activation of the subsequent caspase cascade, including the *Casp3*-dependent apoptotic cascade. Despite the lack of significant differences, *Casp3* maintained strong positive correlations with *Bax* under MS (r = 0.61) and MSUS (r = 0.68) conditions. *Tp53* expression did not significantly change; however, its significant strong correlation with *Bax* expression in MS (r = 0.74) and MSUS (r = 0.65) suggests that while survival conditions are maintained, there may not be complete suppression of apoptosis.

In addition to our *in vivo* study, we evaluated how different RNA doses (0.5, 1, and 2 ng/µL) of MSUS affect apoptosis-related gene expression in healthy hippocampal cells cultured on GalC7 hydrogels. Although statistical significance was not reached, several key observations provide insights into the potential of this *in vitro* model. The similarity in gene expression between the control and mock-nucleofected groups indicates that the nucleofection process itself does not induce apoptosis-related changes. Moreover, in the 3D hippocampal cell culture nucleofected with 0.5 ng/µL total RNA, the *Bcl2/Bax* ratio was lower than that in the control and mock-nucleofected groups; however, this decrease was not statistically significant. As the nucleofected total RNA concentration increased, the ratio approached the levels observed in the control and mock groups, with notably reduced variability. Neuronal cells, which are particularly challenging to culture in 3D, are expected to undergo prominent apoptosis. However, the upregulated *Bcl2* transcript observed in the hippocampus of stressed mice may contribute to maintaining healthy hippocampal cells in a survival mode, potentially depending on the concentration of nucleofected RNA. Our 3D neuronal culture findings indicate that the microenvironment
of stressed mice sustains adaptive processes *in vitro*, leading to a more stable survival response at higher RNA nucleofection concentrations. Our *in vitro* models shed light on the importance of concentration-dependent nucleofection for ELS-related apoptosis studies. Despite the limitations of this study, particularly the lack of detailed characterization of the total RNA extracted from the hippocampus of male mice in the MSUS group used for nucleofection, we confirmed that the RNA sample used for nucleofection was extracted from tissue exhibiting an *in vivo* expression profile of increased *Bcl2* and decreased *Bax* levels, whereas the mRNA levels of *Casp3* and *Tp53* did not show comparable expression to those in the control group. Another limitation of this study is that neuronal markers were not assessed in 3D hippocampal culture. However, our previous study demonstrated that the mRNA expression levels of genes expressed by neurons increased when they were cultured with GalC7 [29].

## Conclusion

We concluded that unpredictable early-life MS and MSUS stress significantly affect the mRNA expression of prosurvival genes, leading to an increase in the latter, whereas the expression of proapoptotic genes decreases in the hippocampus of five-week-old *BALB/c* mice. These gene expression profiles are similar in both sexes, suggesting the existence of nonsex-driven mechanisms. The alterations observed may be an adaptive mechanism supporting neuronal survival in adolescents. If early-life maternal deprivation or MS leads to widespread apoptosis in the hippocampus — a region where neurogenesis continues into adulthood — the detrimental effects are likely to manifest as an increased susceptibility to neurodegenerative diseases. However, the brain possesses adaptive mechanisms to mitigate severe neuronal loss. In this study, the activation of survival pathways in hippocampal cells may reflect an adaptive response, protecting against major stress-induced damage.

## Statements and declarations

### Acknowledgments

We are very grateful for the financial support from Ankara Yıldırım Beyazıt University Scientific Research Project Coordination Unit (AYBU BAP). We would also like to express our sincere appreciation for the molecular laboratory facilities provided by Ankara Yıldırım Beyazıt University (AYBU) Central Research Laboratory (AYBU Merlab) for this study.

### Funding

This study received financial support from the Ankara Yıldırım Beyazıt University Scientific Research Project Coordination Unit (AYBU BAP) (No. TYL-2023-2484).

### Author contributions

Funding acquisition was carried out by K.K.B and İ.K.G. The mouse model and wet-lab experiments were performed by K.K.B., A.N.B., İ.K.G and M.H.D. Figures and graphs were prepared by A.N.B. Statistical
analyses were performed by A.N.B. Conceptualization and data interpretation were performed by K.K.B. and A.B. K.K.B., A.N.B, and J.F. prepared the manuscript. J.F. provided the GalC7. All the authors reviewed and approved the final version of the manuscript.

**Competing interests**

The authors declare that they have no competing interests.

**Data availability**

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Figures

<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

Violin plots showing the relative mRNA expression of *Bax* (a), *Bcl2* (b), *Casp3* (c), and *Tp53* (d) and the *Bcl2/Bax* expression ratio (e) in the hippocampus of litters subjected to early-life maternal separation stress. The y-axis represents relative mRNA expression, and the x-axis represents the control (n=24), MS (n=24), and MSUS (n=24) groups. Each plot includes individual data points, the first quartile (Q1), the
third quartile (Q3), and the median. The descriptive statistics (mean ± SD, median, Q1, and Q3) for each gene are as follows: *Bax*: control (1.12 ± 0.46, 1.20, 0.82, and 1.55), MS (0.74 ± 0.77, 0.45, 0.14, and 1.22), and MSUS (0.86 ± 0.79, 0.64, 0.20, and 1.63); *Bcl-2*: control (1.13 ± 0.49, 1.11, 0.64, and 1.50), MS (6.58 ± 6.86, 3.26, 1.50, and 10.85), and MSUS (4.94 ± 4.64, 2.92, 0.96, and 8.75); *Casp3*: control (1.12 ± 0.52, 1.06, 0.67, and 1.52), MS (0.95 ± 1.07, 0.55, 0.04, and 2.09), and MSUS (1.49 ± 1.40, 1.06, 0.40, and 2.49); *Tp53*: control (1.03 ± 0.23, 1.06, 0.89, and 1.20), MS (1.27 ± 0.70, 1.16, 0.79, and 1.81), and MSUS (1.45 ± 0.70, 1.44, 0.88, and 1.75); and *Bcl2/Bax*: control (0.01 ± 0.41, 0.02, -0.07, and 0.19), MS (1.11 ± 1.10, 0.89, 0.16, and 1.98), and MSUS (1.03 ± 1.39, 0.56, 0.30, and 1.56)

<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>

Viability and development of hippocampal cells in 3D GalC7 hydrogels. (a) Trypan blue staining revealed mostly viable cells postnucleofection. (b-e) Hippocampal cells within the GalC7 hydrogels on Day 1 (b), Day 3 (c), Day 5 (d), and Day 7 (e), as captured via an Olympus CKX41 inverted microscope at 20x magnification
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>

Violin plot showing the *Bcl2/Bax* expression ratio in 3D cultured hippocampal cells (a) and the relative mRNA expression of *Casp3* (b) in 3D cultured hippocampal cells. The y-axis represents relative mRNA expression, and the x-axis represents the control (n=4), mock (n=4), 0.5 (n=4), 1 (n=4), and 2 (n=4) groups. Each plot includes individual data points, the first quartile (Q1), the third quartile (Q3), and the median. The descriptive statistics (mean ± SD, median, Q1, and Q3) for each group are as follows:

*Bcl2/Bax*: control (1.78 ± 1.21, 2.09, 0.51, and 2.74), mock (2.16 ± 0.81, 2.34, 1.31, and 2.82), 0.5 (1.15 ± 2.18, 0.08, 0.04, and 3.34), 1 (1.57 ± 1.69, 1.10, 0.25, and 3.34), and 2 (1.09 ± 0.54, 1.21, 0.52, and 1.54);

*Casp3*: control (1.14 ± 0.74, 0.83, 0.69, and 1.89), mock (0.69 ± 0.49, 0.70, 0.23, and 1.14), 0.5 (0.17 ± 0.13, 0.16, 0.05, and 0.30), 1 (0.91 ± 0.30, 0.86, 0.66, and 1.20), and 2 (0.81 ± 1.39, 0.16, 0.04, and 2.23)