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Open Access

Peer-reviewed

Research Article

Hippocampal transcriptome profiling reveals status epilepticus-induced early changes in gene expression mainly implicating in neuroinflammation and immune responses linked to microglial dysfunction

Abstract

Status epilepticus (SE) is a severe type of epileptic seizure and induces molecular and cellular changes in the brain tissues which contribute to neuron injury. Here we used RNA sequencing to determine changes in hippocampal gene expression in pilocarpine-induced SE mice at 3-hour (SE-3h) and 24-hour (SE-24h) time points, a crucial stage of SE-induced brain acute damage. A total of 366 differentially expressed genes (DEGs) were identified from the SE-3h hippocampus and 570 DEGs from the SE-24h hippocampus, and most of them were up-regulated upon SE induction. Bioinformatical analyses showed that, compared to SE-3h up-regulated genes with poor scores in functional and pathway enrichment, the SE-24h up-regulated genes were predominantly enriched in inflammatory and immune response, positive regulation of response to external stimuli and inflammatory response (GO function), and Microglia pathogen phagocytosis pathway and Tyrobp causal network in microglia (WikiPathway). Specifically, a subset of DEGs such as Tyrobp, C1qc, Itgb2, Ncf2, and Nckap1l involved in the two pathways are present in the inflammatory and immune cascades. Therefore, this study delineates early altered transcriptional profiles in the hippocampus after SE, and highlights up-regulation of a subset of genes might be involved in the activation of microglia-mediated inflammatory and immune responses linked to the early pathogenesis of SE-induced brain injury.

Citation: Tang H-L, Min Y, Long Y-S (2026) Hippocampal transcriptome profiling reveals status epilepticus-induced early changes in gene expression mainly implicating in neuroinflammation and immune responses linked to microglial dysfunction. PLoS One 21(3): e0344387. https://doi.org/10.1371/journal.pone.0344387

Editor: Stephen D. Ginsberg, Nathan S Kline Institute, UNITED STATES OF AMERICA

Received: November 14, 2025; Accepted: February 18, 2026; Published: March 20, 2026

Copyright: © 2026 Tang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This project was funded by the Key Medical and Health Project of the Panyu District Science and Technology Program (grant numbers 2022-Z04-107).

Competing interests: The authors have declared that no competing interests exist.

1. Introduction

Status epilepticus (SE) is a life-threatening neurological emergency characterized by markedly prolonged seizures or recurrent seizures within a short period without full recovery between episodes [1,2]. The annual incidence of SE is estimated to be 10–40 cases per 100,000 population [3,4], with an overall mortality rate of nearly 20% [5]. Accumulating evidences have highlighted that SE induces structural brain damage and neuronal dysfunctions, which are believed to contribute to long-term cognitive and behavioral impairments [6]. Clinical neuroimaging studies have demonstrated that those patients suffered from SE developed hippocampal atrophy, temporal lobe sclerosis, and cortical volume loss, and these structural abnormalities were identified to be correlated with memory deficits and executive dysfunction [7–9]. Longitudinal follow-up studies further revealed that SE-control patients also exhibited persistent attention deficits, impaired working memory, and emotional disturbances [10,11]. These phenomena can be partially replicated from some SE animal models induced by electrical stimulation or treatment with chemoconvulsants, who presented similar cognitive and other behavioral impairments [12,13], as well as neuronal injury and hippocampal damage [14,15].

Over the past years, a number of studies have promoted the understanding of the cellular and molecular pathogenesis of SE-induced neuronal injuries. It is known that sustained explosive discharges during SE induce the excessive generation of reactive oxygen species which then lead to oxidation stress-mediated neuronal destruction and even death [15,16]. Multiple downstream pathological processes including neuroinflammation and immune responses are identified to be initiated in response to the SE-induced neuronal injuries [6,17,18]. Furthermore, excessive microglial activation and abnormal synaptic pruning further exacerbate brain damages, disrupt neuronal networks and aggravate SE-related cognitive deficits [13,19,20]. Recent studies have also revealed that the contributions of molecular changes to the SE-induced injured brain tissues. In the early phase (within several hours to 24 hours after SE induction), some pro-inflammatory mediators such as IL-1β, TNF-α, and CCL2 and their downstream pathways were identified to be activated, which are involved in inflammatory responses, apoptosis, and glial activation [21–24]. The early-phase molecular changes not only directly execute acute neuronal injury programs (e.g., pro-inflammatory and pro-apoptotic pathways) but also initiate subsequent glial responses and circuit remodeling processes in the subacute and chronic phases [25–27]. Clinical and animal intervention studies further demonstrate that early blockade of key acute molecular pathways (e.g., administration of the IL-1 receptor antagonist anakinra or anti-HMGB1 antibody after SE) reduces neuronal death by suppressing the inflammatory and immune cascades, which markedly decreases the risk of cognitive deficits and spontaneous seizures [25,28–30]. These findings suggest that early molecular changes upon SE should be associated with acute neuronal injuries, as well as subsequent epileptogenesis and brain dysfunctions. Using mRNA sequencing, several studies have determined temporal changes in mouse hippocampal transcriptome at different time points, which provide some valuable insights into the early molecular and cellular events linked to SE-induced neuronal injuries. For example, Hansen et al. [31] identified that those genes changed at 12 hours post-SE are mainly that regulate transcription and synaptic physiology. Galvis-Montes et al. [32] found that differentially expressed genes (ranging from 6 hours to 72 hours post-SE) in the hippocampal CA1 region are related to inflammation and excitation stress. Recently, Popova et al. [33] investigated differentially expressed mRNAs in the mouse hippocampus at 1 hour, 8 hours, 36 hours and 120 hours after SE, and found that temporal changes in metabolic reactions linked to immediate responses (e.g., mitochondrial dysfunction, oxidative stress and inflammatory response) to injury followed by recovery and regeneration. Therefore, further elucidating the early molecular markers after SE induction is helpful for investigating the pathology of SE-related brain injury.

To monitor early molecular markers in response to SE, this study used a high-throughput RNA sequencing to detect the hippocampal transcriptomes in the pilocarpine-induced SE mice at both 3-hour (SE-3h) and 24-hour (SE-24h) time points which should represent a crucial stage of the SE-induced neural damage. We identified more differentially expressed genes (DEGs) upon SE induction at SE 24h compared to SE 3h, with few overlaps between the two time points. Of them, the DEGs tent to be up-regulated at both time points. Compared to the SE-3h up-regulated genes with lower scores in GO function and biological pathway enrichments, the SE-24h up-regulated genes were highly enriched in inflammatory and immune responses (top enriched GO terms) linked to microglia pathogen phagocytosis and Tyrobp causal network (top enriched pathways). Furthermore, a subset of the SE-24h up-regulated genes were identified as microglia M1 type- and M2 type-related genes, suggesting that microglia should be activated at 24 hours after SE induction. Thus, this study identified that the hippocampal transcriptomic alterations upon SE induction were dominantly enriched in neuroinflammatory and immune responses linked to microglia activation, which may be associated with the early pathogenesis of SE-induced hippocampal injuries and also provide potential clues for future therapeutic strategies.

2. Materials and methods

2.1. Animals

C57BL/6 mice were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China), and were subsequently bred and housed in a temperature-controlled environment (22 ± 2 °C) under a 12-h light/dark cycle with water and food ad libitum. All efforts regarding drug treatment and surgical procedure were made to minimize pain, and the animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University.

2.2. SE induction

Adult male mice (weight: 20–25 g, age: 6–8 weeks) were used to generate the pilocarpine-induced SE model according to our previous descriptions [34]. Briefly, the animals were intraperitoneally injected with 1 mg/kg atropine methyl nitrate, followed 30 minutes later with a subcutaneous injection of pilocarpine hydrochloride (325 mg/kg, MedChem Express, NJ, United States). The seizure activities of the mice treated with pilocarpine were observed via videoing, and the seizure severity was assessed using the modified Racine scale. Only those mice with generalized convulsions (up to stage 5) and lasted for at least 5 min were used for the following studies. The convulsions were terminated by injection of diazepam (15 mg/kg) after 2 hours of SE. The control animals were injected with saline (instead of pilocarpine) followed by injecting the same dose of diazepam to reduce false positives.

2.3. RNA isolation and sequencing

At 3 hours and 24 hours after SE induction, all mice were anesthetized by placing the animals in a plexiglass chamber with 5% isoflurane for 5 min. When fully sedated measured by a lack of active paw reflex, the mice were decapitated by cervical dislocation for RNA extraction. Bilateral hippocampi were collected and subjected to total RNA extraction using TRIzol reagent (Invitrogen, CA, United States) according to the manufacturer’s protocol. The concentration and purity of total RNA were detected by a NanoDrop™ 2000 spectrophotometer, and RNA integrity was assessed with an Agilent 2100 Bioanalyzer. Three total RNA samples per group were used for the following RNA sequencing. For RNA sequencing, qualified RNA was purified using Oligo(dT) magnetic beads. The isolated mRNA was randomly fragmented using fragmentation buffer, and first-strand cDNA was synthesized with random oligonucleotide primers. Double-stranded cDNA was then generated and purified, followed by end repair, 3′-end adenylation (A-tailing), and adapter ligation. The cDNA fragments were size-selected using AMPure XP beads (Beckman Coulter, CA, USA) and amplified by PCR to complete library construction. The libraries were sequenced on the Illumina HiSeq platform (Illumina, San Diego, CA, United States) at Biomarker Technologies Corporation in Beijing.

2.4. Bioinformatical analyses

The sequence data were processed using FastQC software for quality control. After filtering, clean reads were mapped to the reference genome using HISAT2 software. DESeq2 was used to analyze DEGs. The gene with a fold change >2 and a false discovery rate (FDR) ≤ 0.01 was defined as DEG. All identified genes were displayed by a Volcano Plot according to their change folds and P values, and the DEGs were clustered to show as a hierarchical heatmap. Venn analyses were performed to compare our data against other datasets. Enrichments of Gene Ontology (GO) functions and 3 pathways including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, WikiPathways and Reactome Gene Sets for the up-regulated genes were conducted using the Metascape platform (https://metascape.org/gp/index.html).

2.5. Real-time quantitative PCR (RT-qPCR)

For RT-PCR experiments, a TRIzol reagent was used to extract total RNAs from the hippocampus of the SE mice and control mice under the same condition as those animals in the RNA sequencing experiment, and cDNA was subsequently synthesized using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). Approximate 200 ng of cDNAs were used for qPCR analysis with Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan). All primers are listed in S1 Table. Relative mRNA expression was calculated by the 2-ΔΔCT method.

2.6. Statistical analyses

All data were analyzed using SPSS 19.0 software and are presented as mean ± SEM. Two-tailed unpaired Student’s t test was used to analyze statistical differences between two groups. Chi-square test was applied for Venn analysis. A P value < 0.05 was considered as statistically significant.

3. Results

3.1. Characterization of RNA sequencing data identified from the hippocampi of SE-induced mice and control mice

To identify early transcriptional changes in the hippocampus of pilocarpine-induced SE mice, we performed mRNA sequencing on the hippocampal tissues isolated at 3 hours and 24 hours after SE induction, respectively. In the constructed sequencing libraries, the percentages of more than Q30 bases from all 12 samples were as high as over 87% (S2 Table). Comparison of our data against the reference genome data showed that the unique mapped reads were more than 82% (S3 Table), and more than 83% of the identified reads were distributed within exons (S4 Table). At a cut of FDR ≤ 0.01, we identified 375 DEGs (307 up-regulated, 68 down-regulated) and 584 DEGs (498 up-regulated, 86 down-regulated) in the SE-3h hippocampus and SE-24 hippocampus, respectively (Fig 1A, S5-S6 Tables), suggesting that induction of SE tends to induce up-regulation of hippocampal gene expression. At both time points, the up-regulated genes outnumbered the down-regulated ones accompanied by a greater change extent. Among them, 13 up-regulated genes (i.e., Cxcl10, Ccl4, Tgm1, Zbp1, Ccl12, Ccl2, Ccl3, Timp1, Gcg, Mx1, Mnda, Trh, and Ch25h) changed more than 10 folds in the SE-24h hippocampus, whereas only 5 up-regulated genes (i.e., Fgf3, Atf3, Krt75, Inhba, and Ccl3) were identified from the SE-3h hippocampus with the same filtering criteria (Fig 1A). These data suggest more pronounced changes in gene expression in the SE-24h hippocampus compared with those in the SE-3h hippocampus. Hierarchical clustering analyses showed that these DEGs could be separated between the SE-induced group and the control group at both time points (Fig 1B), suggesting a high biological repetitiveness of these transcriptomic data.

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Fig 1. Evaluation of differentially expressed genes (DEGs) in the hippocampus at 3 hrs and 24 hrs after SE.

(A) Volcano plots showing changes of all identified transcripts in the hippocampus at 3 hrs and 24 hrs after SE (SE-3h and SE-24h). Red and green dots representing up-regulation and down-regulation, respectively. The genes with the most significant up-regulation were labeled. (B) Heatmaps showing the hierarchical clustering of DEGs in SE-3h and SE-24h hippocampus, respectively. The color bars on the right of each figure indicated the magnitude of expression changes of the DEGs.

https://doi.org/10.1371/journal.pone.0344387.g001

3.2. Comparisons of our transcriptomic data against other datasets

We firstly compared all the identified DEGs between the SE-3h and SE-24h time points, and found that a total of 58 up-regulated genes were overlapped between the two time points, significantly higher (P = 0.0075) than the overlap (3 genes) of the down-regulated genes (Fig 2A, S7 Table). Given that SE induction tends to up-regulate gene expression, we then compared the up-regulated genes identified in this study against other published datasets. By comparing the up-regulated genes in our data with the 1642 up-regulated genes at the SE-12h time point identified by Hansen et al. [31], we found that 30 DEGs were overlapped between the SE-3h and SE-12h time points, significantly higher (P = 0.0012) than the overlap (22 up-regulated genes) between the SE-24h and SE-12h time points (Fig 2B, S7 Table). By comparing our data against the transcriptomic data at three time points after SE in another study [32], we found that the up-regulated genes at the SE-3h time point overlapped with those at the SE-12h time point were more than (P = 0.00015) overlap between the SE-24h and SE-12h time points(Fig 2C, S7 Table). However, the overlap of the SE-3h up-regulated genes against the SE-36h and SE-72h up-regulated genes were respectively lower than (P < 0.00015) those of the SE-24h up-regulated genes against the SE-36h and SE-72h up-regulated genes (Fig 2C). Our data combined with other published datasets suggest a time-dependent fashion of alterations in hippocampal gene expression after SE induction.

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Fig 2. Venn analyses for comparison of the DEGs identified in our study against other published datasets.

(A) A significantly higher overlap of the up-regulated genes between the SE-3h and SE-24h hippocampus, compared to the down-regulated genes. (B) Comparison of the SE-3h and SE-24h up-regulated genes against the up-regulated genes in SE-12h hippocampus identified by Hansen et al. [31]. (C) Comparisons of the SE-3h and SE-24h up-regulated genes against the up-regulated genes in SE-12h, SE-36h and SE-72h CA1 region identified by Galvis-Montes et al. [32].

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3.3. Comparison of GO function and biological pathway enrichments between the SE-3h and SE-24h up-regulated genes

To understand the roles of the up-regulated genes in the brain damage upon SE-induction, we performed GO function enrichment analyses with the SE-3h and SE-24h up-regulated genes, respectively. The SE-3h up-regulated genes were mainly enriched in protein modification and signaling cassettes, i.e., enzyme-linked receptor protein signaling pathway (GO:0007167, 30 genes), positive regulation of protein phosphorylation (GO:0001934, 31 genes), signaling receptor regulator activity (GO:0030545, 29 genes) and so on. (Fig 3A, S8 Table). Compared to the SE-3h up-regulated genes with lower enriched scores (Fig 3A), the SE-24h up-regulated genes were highly enriched in four enriched terms, i.e., innate immune response (GO:0045087, 104 genes), positive regulation of response to external stimulus (GO:0032103, 80 genes), inflammatory response (GO:0006954, 60 genes), and positive regulation of cell migration (GO:0030335, 61 genes) (Fig 3A, S9 Table). As shown in Fig 3B, a subset of genes was common in at least 3 of the top enriched terms, and 9 of them (i.e., Ccl2, Clec7a, Csf1, Lyn, Pycard, Tac1, Tlr4, Tlr2, and Trem2) were overlapped among the 4 top-enriched terms.

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Fig 3. Enrichment of GO functions of the SE-3h and SE-24h up-regulated genes by Metascape software.

(A) Histograms showing the top 10 enriched terms with discrete color scales to show statistical significance and gene number of each term on the right of each column. (B) A Venn diagram showing the overlaps among DEGs involved in the top four enriched terms, the red box indicates the up-regulated genes overlapped with 4 top enriched terms at SE 24h. Those genes overlapped among at least 3 terms were shown in the box.

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Pathway enrichment analyses using the up-regulated genes showed that, similar to the GO enrichment described above, the SE-3h up-regulated genes were enriched at lower scores no more than 10 (-Log10) compared to the SE-24h up-regulated genes (Fig 4A, S10 Table). The SE-3h up-regulated genes were dominantly enriched in Lung fibrosis (Wikipathway) and MAPK signaling pathway (KEGG pathway) with enriched scores more than 7 (Fig 4A), and 9 pathways were enriched using the SE-24h up-regulated genes with higher enriched scores more than 10, of them two Wikipathways (Microglia pathogen phagocytosis pathway and Tyrobp causal network in microglia) represented the most enriched pathways with the enriched score more than 20 (Fig 4A, S11 Table). By Venn analysis, we found that 5 genes (C1qc, Itgb2, Ncf2, Nckap1l, and Tyrobp) were overlapped between the top two enriched pathways (Fig 4B). RT-qPCR analyses confirmed significant up-regulation of the 5 genes in the SE-24h hippocampus compared to the control mice (Fig 4C). Furthermore, Venn analysis indicated that a subset of genes involved in Microglial pathogen phagocytosis pathway and Tyrobp causal network in microglia were substantially overlapped with inflammation and immune response-related genes, respectively (Fig 4D). Of them 3 genes, i.e., C1qc, Itgb2, and Tyrobp were common in the two enriched pathways and GO function terms (Fig 4D). Taken together, both the function and pathway analyses highlight that the SE-24h up-regulated genes dominantly participate in neuroinflammation and immune responses regarding microglial dysfunctions.

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Fig 4. Enrichment of pathways using the SE-3h and SE-24h up-regulated genes.

(A) Histograms showing the top 10 enriched pathways of the SE-3h and SE-24h hippocampus, respectively. (B) A Venn diagram showing the 5 overlapped genes between the two top enriched pathways from SE-24h hippocampus. (C) RT-qPCR showing that the relative levels of the 5 overlapped genes (i.e., C1qc, Itgb2, Ncf2, Nckap1l, and Tyrobp) in the SE-24h hippocampus were significantly increased compared with the control hippocampus. The relative mRNA levels (target genes/β-actin) in the control mice were normalized as “1”. n = 5, *P < 0.05. (D) Venn diagrams showing the overlapped genes of the up-regulated genes in the Microglia pathogen phagocytosis pathway and Tyrobp causal network in microglia against those genes involved in inflammation & immune responses. Red fonts indicated those genes presenting in the two overlaps.

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3.4. Illustration of the roles of some SE-24h up-regulated genes involved in the two top-enriched pathways

As shown in Fig 5A, most genes in Microglial pathogen phagocytosis pathway were identified as up-regulated genes under a SE-24h condition. Of them, the encoded proteins of 8 genes (including three C1q family genes, Trem2, Tyrobp, Itgb2, Fcgr1, and Fcer1g) represent key upstream components in this pathway, and they are also shown as inflammatory and immune response-related factors. In Tyrobp causal network in microglia pathway, more than one-third of the genes (22 genes) are shown to be up-regulated upon SE 24h, and of them 10 genes are associated with inflammatory and immune responses (Fig 5B). Furthermore, C1qc/Itgb2/Tyrobp represent one of the upstream axes in Tyrobp causal network in microglia pathway and are also important in Microglial pathogen phagocytosis pathway (Fig 5). Thus, descriptions of the roles of these critical genes further suggest a strong relationship between inflammatory/immune responses and microglial dysfunctions at 24 hours after SE induction.

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Fig 5. Diagrams of the two top enriched pathways Microglia pathogen phagocytosis pathway (A) and Tyrobp causal network in microglia (B).

The protein products of those up-regulated genes were shown as color boxes. The common genes of the two pathways were displayed in bold font.

https://doi.org/10.1371/journal.pone.0344387.g005

3.5. Some SE-24 up-regulated genes are either microglial M1-type markers or M2-type markers

Previous studies have shown that SE induce neuronal injuries by microglial activation [23,35]. Here, we further investigated which type of microglia might be activated. By comparing the SE-24h up-regulated genes to those known M1-type markers and M2-type markers [36], we found that 17 genes and 9 genes were common in M1 markers and M2 markers, respectively (Fig 6A). We then performed RT-qPCR analyses to determine the mRNA levels of 6 represented genes (M1-related genes: Ccl3, Ccl4, and Ccl7; M2-related genes: Ccl2, Clec7a, and Ctsc). The relative mRNA levels of all these genes in the SE-24h hippocampus were significantly higher than those in the control hippocampus (Fig 6B). These data suggest that SE induction could activate both M1 type and M2 type of microglia.

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Fig 6. Up-regulation of both microglial M1-type and M2-type genes upon SE induction.

(A) A Venn diagram showing the overlapped genes between the SE-24h up-regulated genes and microglia polarization–related markers [35]. (B) The relative mRNA levels (target genes/β-actin) of the representative genes (Ccl3, Ccl4, Ccl7, Ccl2, Clec7a and Ctsc) in the SE-24h hippocampus were significantly increased compared to the control mice. The relative mRNA levels (target genes/β-actin) in the control mice were normalized as “1”. n = 5, *P < 0.05.

https://doi.org/10.1371/journal.pone.0344387.g006

4. Discussion

Previous evidence has documented that SE can induce brain injury [37], but its molecular linkage during an early phase after SE induction remains elusive. Here, we identified that the SE-3h up-regulated genes are mainly associated with protein modification, receptor signaling cassettes and MAPK signaling pathway, and the SE-24h up-regulated genes are predominantly associated with inflammatory and immune processes linked to microglial dysfunction. Our data revealed molecular changes in the hippocampus at 3h and 24h after SE induction，which should reflect the early pathological changes following SE, i.e., from acute injury to inflammatory amplification in early phase. This study should provide novel candidates for further investigating the underlying molecular mechanisms of SE-induced brain injury.

The present study determined altered transcriptomes in the SE-3h and SE-24h hippocampus, and found that most genes were up-regulated upon induction of SE, which is similar to previous findings that SE tends to induce the increase in gene expression [32,33,38]. During recent years, a few studies have determined the hippocampal transcriptomes at several time points (i.e., 12h, 36h and 72h) under the same SE condition as our study [31,32]. By respectively comparing the up-regulated genes from our data against their datasets (Fig 2), we found more overlapping genes at adjacent time points, suggesting the reliability of this study. Overall, our datasets at SE-3h and SE-24h, combing with previous datasets, reveal early dynamic changes of hippocampal genes upon SE induction, implicating in the early pathological changes after SE insult.

By GO function enrichment analysis, the present study identified the different enrichment terms of the up-regulated genes from the SE-3h and SE-24h hippocampus respectively. Although substantial genes were up-regulated in the hippocampus at 3h after SE, no specific GO terms were significantly enriched at this early stage, suggesting that the initial molecular events following SE are likely dominated by rapid and heterogeneous responses. Similarly, Popova et al. [33] reported that early transcriptomic alterations after SE were characterized by rapid and diverse changes in gene expression, which should support our above finding. Different to the SE-3h hippocampus, the up-regulated genes in the SE-24h hippocampus were highly enriched in several GO terms regarding inflammatory and immune responses. We propose that the inflammatory and immune responses triggered in the early phase after SE (approximately 24 hours) may represent one of the important factors associated with the SE pathologies. This hypothesis is partially supported by several evidences that up-regulation of a subset of genes at early stage after SE are mainly associated with neuroinflammation and hippocampal dysfunctions [32,33,39], and inhibition of inflammatory and immune responses in the SE model with captopril was found to alleviate neuronal damage and improving cognitive function [13]. A previous study showed that enhancement of inflammatory and immune responses was associated with epileptogenesis in patients with epilepsy and animal models of epilepsy [40], and Serrano et al. [41] found that COX-2 upregulation after SE markedly amplified the inflammatory and immune responses in the hippocampus, which are related to the SE-induced neuronal injuries. In addition to amplifications of immune and inflammatory responses, previously transcriptomic studies also identified that SE induced prominent alterations in immediate early genes and synaptic function-associated genes [31–33], suggesting diverse changes and complex pathophysiology of the SE-induced hippocampal injuries at different stages.

Further biological pathway enrichment analysis of the up-regulated genes found that the pathways were not dominantly enriched at SE 3h, compared to the SE-24h up-regulated genes. The SE-3h up-regulated genes were mainly enriched in fibrosis, MAPK and NF-kB signaling pathways, and etc., implicating early hippocampal injuries occur upon SE insult due to some of them being pathological traits in the brain tissues during the SE-induced acute phase period [42,43]. Interestingly, the SE-24h up-regulated genes could be highly enriched in two pathways, i.e., Microglial pathogen phagocytosis pathway and Tyrobp causal network in microglia linked to inflammation and immune responses. Our finding suggests potential roles of microglial activation in the enhancement of inflammatory and immune responses upon SE induction, due that microglia could be rapidly activated during seizures [44–46]. The strong relationship between microglial activation and enhancement of inflammatory and immune responses has been well established to contribute to neuronal injury. For example, overactivation of microglia is involved in neuroinflammation, aberrant phagocytosis, synaptic loss and neural circuit imbalance which are key mechanisms contributing to cognitive impairment [19,47]. Furthermore, suppression of microglial activation has been demonstrated to mitigate cognitive deficits by inhibiting microglia-mediated inflammatory responses and synaptic phagocytosis [13,48,49]. As shown in Fig 4D, several key genes including C1qc, Itgb2, Ncf2, Nckap1l, and Tyrobp in the two pathways are involved in inflammatory and immune responses. Previous studies have identified the critical roles of their protein products in brain functions and diseases. TYROBP (also named as DAP12), as a signaling hub molecule of microglia, was identified to be increased in the hippocampus of epilepsy patients linking with greater response to microglial phagocytosis [50], and overactivation of the TYROBP–TREM2 pathway in cell and mouse models of Alzheimer’s disease can lead to excessive synaptic pruning and aberrant neural circuit remodeling, thereby impairing learning and memory functions [51,52]. Microglial TREM2 deficiency has been identified to impair phagocytic clearance of damaged neurons correlated with increased spontaneous seizures after SE induction [53]. In addition, C1QC acts as an initiating factor of the classical complement pathway to regulate synaptic pruning, and Up-regulation of C1QC could induce aberrant activation of the complement–phagocytic system in various neurodegenerative diseases and epilepsy models has been shown to promote synaptic loss and memory impairment [54,55]. As a member of the leukocyte integrin family, ITGB2 is implicated in various neurological disorders such as Alzheimer’s disease and traumatic brain injury, inhibition of its upregulation could alleviate neurological symptoms [56,57]. Notably, the simultaneous up-regulation of Ncf1, Ncf2, and Cybb genes has been demonstrated to enhance oxidative burst activity linked to Parkinson’s disease [58,59]. Above evidences suggest that dysregulations of these genes are mainly associated with neurodegenerative disorders, as well as epileptic conditions, but their pathogenic roles in the epilepsy are still poorly known. Thus, the present study should provide some new candidates for further investigating the SE pathologies. Taken together, comparisons of the function and pathway enrichments between the SE-3h and SE-24h up-regulated genes suggest that the dynamic molecular changes might be associated with the early pathological process after SE insult.

This study further showed that some M1-type inflammation-related genes (such as Ccl3, Ccl4 and Ccl7) and M2-type repair-related genes (such as Ccl2, Clec7a and Ctsc) were simultaneously up-regulated, suggesting that microglia should exhibit two polarization states, which could maintain a dynamic balance between inflammatory and reparative responses upon SE induction. This finding is evidenced by a previous study showing mixed or transitional phenotypes of microglial activation after SE [60]. As the primary immune cells of the central nervous system, the polarization state of microglia is critical for neuro-inflammation and cognitive dysfunction [61]. In addition, it has been reported that microglia play protective roles by limiting neuronal hyperexcitability [62,63], and microglia depletion was identified to increase seizure susceptibility [64,65]. Therefore, we propose that SE-induced microglial activation identified in the present study should play both detrimental and protective effects at early stage of SE onset.

Finally, a few limitations of this study should be mentioned. Firstly, transcriptomic analyses in this study were performed using a bulk hippocampal tissue, which could not distinguish whether the observed DEGs reflect altered transcription within microglia, neurons or other cell types. Secondly, it is reported that there exist some differences in hippocampal neurodegenerative phenotypes (including deteriorative reactive gliosis in microglia) between male SE mice and female SE mice [66]. Our findings are derived from only male mice, which could not exclude effects of sex on susceptibility to seizure response and neural injury process.

5. Conclusion

In summary, this study provides early transcriptomic profiles of the mouse hippocampus with an up-regulation trend at both 3 hours and 24 hours after SE induction. The SE-3h up-regulated genes are related to protein modification, receptor signaling cassettes and MAPK signaling pathway, and the SE-24h up-regulated genes are mainly involved in microglial activation-associated inflammatory and immune processes. Our findings might reflect time-related changes of the hippocampal injury process in early phase after SE induction. Taken together, this study provides a subset of novel candidates for further exploring the underly mechanisms of seizure-induced brain injury, which might be helpful for future therapeutic strategies.

Supporting information

S1 Table. The primer sequences for qPCR.

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S2 Table. RNA sequencing data analysis.

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S3 Table. Comparison of the RNA sequencing data from this study against the reference genome data.

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S4 Table. Distribution of the reads in different regions of the genome.

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S5 Table. A list of DEGs in the hippocampus of mice at SE 3h.

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S6 Table. A list of DEGs in the hippocampus of mice at SE 24h.

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S7 Table. Comparison of our data with other datasets.

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S8 Table. GO function enrichment of up-regulated genes in the hippocampus of mice at SE 3h.

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S9 Table. GO function enrichment of up-regulated genes in the hippocampus of mice at SE 24h.

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S10 Table. Pathway enrichment of up-regulated genes in the hippocampus of mice at SE 3h.

https://doi.org/10.1371/journal.pone.0344387.s010

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S11 Table. Pathway enrichment of up-regulated genes in the hippocampus of mice at SE 24h.

https://doi.org/10.1371/journal.pone.0344387.s011

(XLSX)

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