Traumatic brain injury (TBI) is a lethal and disabling injury and is a major challenge to public health and social development worldwide [1]. The global incidence of TBI has been continuously increasing over the past several decades [1, 2]. In 2016, there were more than 27 million new cases of TBI, and the total number of prevalent cases exceeded 55 million [2]. TBI is caused by the application of an external mechanical force to the head, followed by a series of primary and secondary pathological mechanisms, such as cell death in the brain parenchyma, disruption of the neurovascular unit, axonal injury, neuroinflammation, and neurodegeneration [3]. Due to the complex pathological mechanisms of TBI, developing effective neuroprotective therapeutics for afflicted patients has been challenging [3]. In recent years, increasing emphasis has been placed on the progressive and chronic consequences of TBI. Many preclinical and clinical studies have suggested that TBI is a significant risk factor for neuropsychiatric and cognitive disorders such as posttraumatic stress disorder (PTSD), chronic traumatic encephalopathy (CTE), and Alzheimer’s disease (AD) [4, 5, 6, 7]. The hippocampus is the main locus responsible for cognitive processing involving learning and memory [6, 8]. It has been shown that hippocampus dysfunction is associated with cognitive deficits following a TBI [6, 8, 9]. Existing treatments for TBI primarily focus on immediate neurosurgical procedures and long-term behavioral rehabilitation, but few treatments are sufficient to improve TBI-induced cognitive deficits [10, 11]. TBI differs from other diseases in that it involves different periods after the injury. However, subacute phase studies of TBI functional recovery have received substantially less attention than acute phase studies [12, 13]. Since effective therapeutic strategies for the subacute phase of TBI are limited, it is essential to explore optimal therapeutic targets at this stage [12, 13].

Understanding the molecular mechanisms underlying pathological processes is a critical step toward developing effective treatments for TBI. Based on the systematic bioinformatics analysis, microarray and high throughput sequencing techniques have emerged as essential tools for exploring the pathogenesis of complex human diseases [14, 15]. Many bioinformatics studies have been conducted in the field of oncology and some non-tumor diseases, but relatively few have focused explicitly on traumatic nerve injuries such as TBI [16]. The Gene Expression Omnibus (GEO) is a publicly available database consisting of an invaluable resource of mass gene expression data that can be systematically analyzed for biomarker or therapeutic target discovery [17]. In this study, we used two datasets from the GEO database to screen the differentially expressed genes (DEGs) of the hippocampus in the subacute phase of TBI. Gene functional enrichment analyses, protein-protein interaction (PPI) network construction, and hub gene identification were also performed. Furthermore, we observed the main histopathological features of the injured hippocampus and selected several DEGs for verification in vivo. The schematic workflow of our study is presented in Fig. 1. This study may provide new insight into potential biomarkers and therapeutic targets for cognitive deficits induced by TBI.

Fig. 1.

Schematic illustration of bioinformatics analyses and experimental validation in the study.

TBI is one of the most common traumatic injuries which can cause long-lasting cerebral damage and cognitive deficits [25]. Elucidating the potential molecular mechanisms and therapeutic targets for improving cognitive function helps to improve the clinical prognosis and alleviate the burden on patients suffering from TBI [26]. Transcriptomic profiling is a powerful tool that can identify the genes differentially expressed in specific physiological and pathological states and discover new diagnostic or therapeutic targets [27]. In this study, we conducted a systematic bioinformatics analysis of the two GEO datasets (GSE111452 and GSE173975) to reveal the potential molecular mechanisms responsible for hippocampal damage in rats in the subacute phase of TBI.

Initially, we performed GSEA on the basis of all transcriptome data of the two GEO datasets, separately. The GSEA results revealed notable enrichment in the MAPK signaling pathway, PI3K/Akt signaling pathway, focal adhesion, and cellular senescence. The MAPK family in mammals consists of c-Jun N-terminal kinase, p38 MAPK, and extracellular signal-regulated kinase [28]. Previous findings suggested that inhibiting the activation of the MAPK signaling pathway might produce neuroprotective effects in TBI [29, 30]. As a critical regulator of organismal growth and multiple cellular processes, the PI3K/Akt signaling pathway plays an important role under various pathophysiological conditions [31]. Preclinical studies of TBI revealed that the PI3K/Akt signaling pathway exerted positive effects on neural repair by regulating neuronal apoptosis and neurogenesis [32, 33]. Focal adhesions are protein complexes that mediate cell adhesion by linking the extracellular matrix to the cytoskeleton [34]. A recent in vitro study demonstrated that inhibiting focal adhesion kinase phosphorylation could mitigate astrocyte activation induced by mechano-stimulation [35]. Cellular senescence refers to a cell state triggered by various stress factors and some specific physiological conditions [36]. It is characterized by proliferative arrest, secretory phenotype, macromolecular damage, and alternation in metabolism [36, 37]. Several studies suggested that DNA damage-induced cellular senescence was a possible driver of TBI-associated sequelae such as cognitive impairment [38, 39, 40]. Among the common DEGs, several genes might play a role in the response to DNA damage. Du et al. [41] found that the activation of complement C3 contributed to retinal DNA damage and C3 deficiency alleviated alkylation-induced retinal degeneration in mice. Raso et al. [42] reported that Isg15 upregulation promoted DNA replication fork progression, leading to extensive DNA damage and chromosomal aberrations in a human osteosarcoma cell line. Xie et al. [43] found that downregulating the expression of CX3C chemokine receptor 1 (Cx3cr1) accelerated double-strand DNA damage in irradiated ovarian cancer cells. These experiments were primarily in the field of oncology, and the studies that investigated how these genes regulated DNA damage response to impact the progression of TBI are still limited.

To further explore potential biological functions and signaling pathways involved in hippocampal damage, we performed GO and KEGG analyses based on the common DEGs of the two datasets. We found that the DEGs were mainly involved in an array of critical immune and inflammatory processes, including antigen processing and presentation, leukocyte and lymphocyte mediated immunity, adaptive immune response, phagosome, lysosome, and complement and coagulation cascades. Following the initial insult triggered by TBI, peripheral leukocytes and lymphocytes were successively recruited to the injury site [44]. Antigen processing and presentation by antigen-presenting cells promote the transition between innate and adaptive immune responses [45]. Moderate immune responses promote the removal of cellular debris, repair, and regeneration in the central nervous system (CNS) post-TBI, but excessive immune responses result in reactions causing destructive neuroinflammation and maladaptive secondary injuries [3, 44]. In view of the enrichment of phagosome- and lysosome-related genes after TBI, it is reasonable to speculate that active phagocytosis plays a critical role in neural injury and repair [46]. Microglia act as important phagocytes in the brain and can rapidly remove cellular and myelin debris after brain injury [47]. Moderate microglial phagocytosis is beneficial for remodeling the brain microenvironment and alleviating secondary injury [47, 48], whereas excessive phagocytosis by microglia of neurons, synapses, or myelin may impede tissue and functional recovery [49, 50]. Current evidence indicates extensive crosstalk between the complement and coagulation systems, which have fundamental clinical implications for inflammation, immunity, and tissue damage [51]. A previous proteomics study suggested that complement and coagulation cascades were activated during the acute and subacute phases of TBI, which was consistent with our findings [52].

In addition to the functional enrichment and pathway analyses described above, we proceeded to construct the PPI network of the DEGs and then identified the top 15 hub genes which were upregulated in both datasets. Among the hub genes, we found several microglia signature genes such as Csf1r, Aif1, and Trem2. Csf1r is a crucial regulator of myeloid lineage cells and is also necessary for maintaining the cell viability of microglia [53]. Several experiments adopted the method of Csf1r inhibition to attenuate microglia-mediated neuroinflammation in rodent models of TBI [54, 55]. However, given that microglia plays a key role in maintaining homeostasis and normal function of the brain [56], inhibiting Csf1r may increase the risk of failure in clinical translation. In contrast, the infiltrating peripheral macrophages in the brain after TBI may be promising targets for developing potential treatment strategies [57]. Aif1 is a calcium-binding protein that regulates immune and inflammatory responses [58, 59]. Increased expression of Aif1 is an indication of microglia and macrophage activation in response to brain trauma [60, 61]. A previous experimental study identified Trem2 and Tyrobp as significant hub genes in TBI mice expressing human APOE [62]. Trem2 is an innate immune receptor mainly expressed on various tissue macrophages, such as microglia in the brain [63]. It transmits intracellular signals through binding with the adaptor protein Tyrobp, which in turn regulates many crucial biological processes, including phagocytosis, chemotaxis, inflammatory responses, and lipid metabolism [56, 63, 64]. It was reported that Trem2 deficiency alleviated the acute peripheral macrophage infiltration and attenuated chronic hippocampal atrophy and cognitive deficits in a murine TBI model [65]. Given the vital role of Trem2 in the progression of neurodegeneration, it was worthwhile to explore it as a potential therapeutic target in managing TBI-mediated neurodegenerative pathologies [56, 57, 65].

Ctss is a cysteine protease expressed by diverse immune cells that is responsible for cleaving certain extracellular matrix proteins and cell adhesion molecules to promote immune cell motility [66, 67]. In the acute phase after TBI, the upregulation of Ctss expression was mainly observed in microglia; inhibiting Ctss could reduce the level of inflammatory factors, alleviate brain edema, and improve neurobehavioral function [68]. The Cd74 molecule is the cognate receptor of macrophage migration inhibitory factor that is recognized to be a pleiotropic inflammatory cytokine [69, 70]. Tobin et al. [45] found that TBI-induced neurodegeneration depended on antigen processing and presentation that required Cd74. Cd74 deficiency could decrease peripheral lymphocyte activation and neurodegeneration following TBI, suggesting that targeting the switch between innate and adaptive immunity might be a potential therapeutic strategy [45]. C1qa, C1qb, and C1qc are three distinct subunits of complement component C1q, which is a crucial protein in the complement cascade within the innate immune system [71, 72]. One recent clinical study showed that elevated serum C1q levels were significantly correlated with traumatic severity and could serve as an independent prognostic factor for long-term outcomes after TBI [73]. After TBI, microglia and neurons were the primary source of C1q [74, 75]. The increased expression of C1q might lead to neuron loss and chronic neuroinflammation and correlate with sleep spindle loss and epileptic spikes. Blocking C1q was beneficial to counteract these effects, indicating that C1q might be a potential target for treating the devastating long-term outcomes of TBI [75].

We also discovered two upregulated Fc receptor genes. It was reported that Fcer1g might be a potential microglial biomarker related to aging and neurodegeneration [72, 76]. Among the Fc receptors, Fcgr2b could serve as one marker of the pro-inflammatory phenotype of microglia after TBI [77, 78, 79]. There was some evidence that Laptm5 had pro-inflammatory properties in macrophages [80]. In a chronic constriction injury in rats, Laptm5 was identified as a significant upregulated neuroinflammation-related gene that influenced neuropathic pain behavior [81]. However, due to the lack of relevant literature, it is unknown whether Laptm5 plays a role in the pathological processes of TBI.

TF co-factors play an essential role in diverse biological processes by interacting with TFs to suppress or activate gene transcription [21, 82]. Among the DEGs shared in common between the GSE111452 and GSE173975 datasets, we found two inflammation-related TF co-factors (Aebp1 and Pycard). Aebp1 has been shown to promote inflammatory processes in macrophages by activating the nuclear factor kappa B [83, 84]. Shijo et al. [85] found that Aebp1 was highly expressed in hippocampal neurons and glial cells in AD patients, implying its potential role in the progression of AD pathology. The Pycard gene encodes a critical adaptor protein in activating inflammasomes [86]. Recent research has shown that inflammasome proteins such as Pycard are elevated in the blood of TBI patients and are closely associated with injury severity and pathological outcomes [87]. Experimental inhibition of inflammasomes exerted anti-inflammatory and neuroprotective effects in rodent models of TBI, implying that developing inflammasome-targeting therapeutics might be a potential direction for future research [88, 89, 90].

CTE refers to a progressive Tau-dependent neurodegenerative disease that remains relatively understudied [91, 92]. In several animal models of TBI, the immunocytochemical signature for CTE has been presented, which is in agreement with clinical studies [4, 7]. Among the common DEGs identified in this study, several genes (e.g., Trem2, Tyrobp, and Cx3cr1) might be involved in the progression of tauopathy based on the current literature. Jiang et al. [93] found that Trem2 attenuated tau kinase activity by inhibiting neuroinflammation and Trem2 deficiency exacerbated tau pathology and neurodegeneration in P301S tau transgenic mice. Another study reported that Trem2 deletion enhanced tau dispersion through microglia exosomes [94]. In a tauopathy mouse model, silencing of Tyrobp reduced C1q levels and improved learning behavior and electrophysiological properties despite increasing tau phosphorylation [95]. Cx3cr1 was deemed as a potential target for treating tauopathy, since its deficiency impaired the phagocytosis and internalization of extracellular tau by microglia [96, 97].

Recent evidence suggested that endoplasmic reticulum (ER) stress resulted in extensive behavioral changes related to a CTE-like phenotype after TBI [91, 98]. Inhibiting ER stress ameliorated cognitive deficits by reducing tau hyperphosphorylation in rodent models of CTE [91, 98]. Several genes within the common DEGs might be involved in ER stress response. It was reported that Glycoprotein nonmetastatic melanoma protein B (Gpnmb) increased the expression of chaperone protein BiP to increase the protein-folding capacity of the ER and attenuate cell death caused by ER stress [99]. In a cerebral ischemia mouse model, the neuronal damage of Gpnmb-transgenic mice was significantly alleviated compared with wild-type mice [99]. Kam et al. [100] found that Fcgr2b mediated amyloid-$\beta$ neurotoxicity by activating ER stress and caspase-12. It has been reported that the translocator protein (Tspo) is a potential biomarker in multiple inflammatory and neurodegenerative diseases [101, 102]. Loss of Tspo in hepatocytes caused free cholesterol accumulation, which subsequently induced ER stress [103]. More importantly, recent clinical studies suggested that PET imaging with tracers binding to Tspo could be applied to detect the neuroinflammatory changes in subjects with CTE [102, 104]. Interestingly, a multi-tracer PET Study showed that tau deposition and increased Tspo expression were co-localized in the cortex and hippocampus in a tau transgenic mouse model; however, the complex interaction between tauopathy and Tspo expression remains to be further studied [105]. Neuroimaging modalities could be conducted longitudinally at various time points on the same subject, showing an advantage in observing dynamic change during the progression of CTE [92]. In future studies, molecular imaging through PET technology such as Tspo-PET may be used as a novel tool to study CTE on preclinical models and help to elucidate the complex pathophysiological mechanisms leading from TBI to symptomatic CTE [102, 104]. In addition, Gouna and colleagues [106] demonstrated that Trem2 deficiency in microglia/macrophages resulted in ER stress due to impaired lipid droplet formation. The potential critical role of these genes in both tauopathy progression and ER stress implied that they might be of considerable value in the field of CTE modeling, although the specific molecular mechanisms remain to be determined in future studies. Tau transgenic rodent models combined with targeted gene deletion might be instrumental for the study of CTE modeling and help to reveal the potential pathophysiological processes of CTE.

In summary, our research was a comprehensive bioinformatics study analyzing the transcriptome data of rat hippocampus in the subacute phase of TBI. We used multiple tools for enrichment analysis to reveal the underlying biological mechanisms involved in TBI-related hippocampal impairment. The enrichment analysis showed that TBI might result in a significant dysregulation of multiple inflammation and immune processes. We further identified the hub genes, TF co-factors, and IRGs, which likely play a critical role in the progression of hippocampal impairment.

In the validation experiment, we found some apparent histopathologic changes in the hippocampus during the subacute phase of TBI, including neuronal necrosis and reductions in the number of intact neurons. A previous study supports the pathological changes described in our model [107]. Furthermore, we used immunohistochemical staining to observe the number of microglia. The staining results suggested that the level of microglia activation was markedly elevated in the hippocampus of rats in the TBI group when compared with the sham group. Through immunofluorescence staining analysis, Luo et al. [52] also found that the number of microglia was significantly increased in the hippocampus during the subacute phase of TBI, which was consistent with our results. Similarly, the immune-related DEGs identified by bioinformatic analyses were significantly enriched in BP terms associated with the activation of microglia and macrophages. Microglia is the major type of glial cells involved in neuroinflammation in the mammalian CNS [108]. The peripheral immune cells (especially macrophages) are also essential responders to tissue injuries in the CNS [3, 109]. These findings suggest that these inflammatory cells might be valuable targets in the therapy of hippocampal impairment in the subacute phase after TBI. Moreover, we also detected the mRNA expression levels of the 15 hub genes identified by bioinformatics analyses. Overall, the RT-qPCR results were consistent with the transcriptome data, indicating that the transcriptome data were reliable. Overall, traumatic homogeneity between the datasets and our validation experiment was relatively high. Both the datasets and our experiment used adult male SD rats as the experimental objects, and the location and extent of the brain injury were similar. In addition, the tissue type and disease stage were consistent.

However, there were several limitations of this study. First, it specifically focused on the hippocampus rather than other brain regions. The principal reason was that hippocampal impairment acts as a key player in the pathogenesis of TBI-induced cognitive decline [6, 8, 9], which was one of the most dominant clinical manifestations in the subacute phase [110, 111, 112]. Developing effective treatments to promote cognitive rehabilitation in TBI patients during the subacute phase is of high clinical importance [110]. The bioinformatics analyses and experimental validation performed in this study mainly focused on the subacute phase (14 days), which was important for assessing cognitive function in the rodent model of TBI [13, 113]. Notably, there was some evidence that several potential biomarkers were significantly upregulated in the acute phase. For instance, Saber et al. [65] found that both the protein and mRNA expressions of Trem2 were significantly increased in the ipsilateral cortex in mice on the third day after experimental TBI. The study by Luo and colleagues [52] revealed that the multiple proteins (e.g., C1qa, Pycard, Aif1, Ptpn6, and Fcer1g) in the ipsilateral hippocampus were markedly upregulated in a rat model of TBI on the third day. In addition, Wang et al. [54] reported that the gene expression levels of C1qa, Aif1, and Csf1r were prominently increased in the perilesional area on the fifth day post injury in TBI mice. Therefore, these biomarker genes might become important therapeutic targets during the early phase of TBI. Further study on these crucial genes might provide useful insight for treating TBI-induced cognitive deficits in fundamental, translational, and clinical research.

Second, in this study, only male rats were used in the experiments. Previous observations have suggested that there may be a link between sex and clinical outcomes after TBI [114, 115]. Some studies demonstrated that females had a lower risk of mortality and cognitive dysfunction than males, while others failed to find such advantages [114, 115]. Given the predominant role of microglia in posttraumatic neuroinflammation, it is worthwhile to determine whether sex differences exist in microglial activation after TBI [114]. Several studies have revealed that male rodents may exhibit more significant microglial activation than female rodents [114, 116, 117]. However, there has also been conflicting evidence about the influence of sex on microglial activation in rodent models of TBI [79, 114]. Regrettably, there is currently an apparent male bias in the field of TBI research, both in preclinical rodent studies and clinical trials [116, 118]. Thus, future studies are needed before drawing definite conclusions about the role of sex in TBI. Finally, traditional microarray and mRNA sequencing techniques mainly investigate the global gene expression patterns of the same tissues. Single-cell mRNA sequencing techniques may help to reveal the exact role of specific cell types in the complex pathophysiological mechanisms underlying TBI.

In conclusion, this study identified several critical genes and key signaling pathways involved in hippocampal impairment induced by TBI. Several genes involved in neuroinflammation (e.g., Trem2, C1q, and Pycard) might represent potential targets for treating TBI-induced cognitive deficits.

TBI, Traumatic brain injury; PTSD, posttraumatic stress disorder; CTE, chronic traumatic encephalopathy; AD, Alzheimer’s disease; GEO, Gene Expression Omnibus; DEGs, differentially expressed genes; PPI, protein-protein interaction; FPI, fluid percussion injury; SD, Sprague-Dawley; GSEA, gene set enrichment analysis; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, standardized enrichment score; BP, biological process; CC, cellular components; MF, molecular function; STRING, Search Tool for the Retrieval of Interacting Genes; TF, Transcription factor; IRGs, immune-related genes; SPF, specific pathogen-free; CCI, controlled cortical impact; HE, hematoxylin and eosin; BSA, bovine serum albumin; DAB, Diaminobenzidine; RT-qPCR, quantitative Real-Time PCR; SEM, standard error of the mean; MAPK, mitogen-activated protein kinase; PI3K/Akt, phosphatidylinositol 3-kinases/protein kinase B; Ctss, Cathepsin S; Aif1, allograft inflammatory factor 1; Csf1r, colony-stimulating factor 1 receptor; Trem2, triggering receptor expressed on myeloid cells 2; Tyrobp, TYRO protein tyrosine kinase-binding protein; Arhgdib, Rho GDP dissociation inhibitor-$\beta$; Laptm5, lysosomal-associated protein transmembrane 5; Fcer1g, Fc epsilon receptor Ig; Fcgr2b, Fc gamma receptor IIb; Ptpn6, protein tyrosine phosphatase non-receptor type 6; Aebp1, adipocyte enhancer-binding protein 1; Pycard, PYD and CARD domain containing; Ifngr1, interferon $\gamma$ receptor 1; Unc93b1, Unc-93 homolog B1; Lgmn, legumain; Grn, granulin precursor; Anxa2, annexin A2; Isg15, interferon-stimulated gene 15; Cx3cr1, CX3C chemokine receptor 1; CNS, central nervous system; ER, endoplasmic reticulum; Gpnmb, Glycoprotein nonmetastatic melanoma protein B; Tspo, translocator protein.

The transcriptomic datasets are available in the GEO database https://www.ncbi.nlm.nih.gov/geo/.

HZ and YZ contributed to the study design. SZ, SW, and ZP participated in data collation and statistical analysis. HZ, SZ, SW, and ZP finished the experimental validation. HZ and LC wrote the manuscript. YZ and SW revised the manuscript. All authors read and approved the final manuscript.

The animal experiment was approved by the Laboratory Animal Ethics Committee of Jinan University (ethical number: 14075).

The authors appreciate the GEO database and contributors for providing their meaningful datasets.

This study was supported by the National Natural Science Foundation of China (nos. 81873362, 82174483, and 82205249), the Natural Science Foundation of Guangdong Province (nos. 2021A1515011219 and 2021A1515110146), China Postdoctoral Science Foundation (2022M710912), and the Administration of Traditional Chinese Medicine of Guangdong Province (no. 20231086).

The authors declare no conflict of interest.