Background
Spinal cord injury (SCI) can lead to sensory and motor dysfunction, manifesting a wide range of functional deficits depending on the level of injury [
1,
2]. Following the mechanical injury that rapidly kills neurons and glia at the initial injury site, devastating and delayed secondary cellular and molecular events occur. These include enlarged cell death through necrosis and apoptosis, which is exacerbated by the presence of inflammatory cells, such as neutrophils, macrophages, and T cells [
3,
4]. Among these inflammatory cells, macrophages, a type of phagocyte, have a dominant role, which not only damage neurons and glia but also are essential for the reconstruction of injured tissues by phagocytosis [
5]. Chemokines, a type of small chemoattractant peptide, which can provide directional cues for the cell trafficking, are also necessary for the inflammatory responses [
6]. Microarray analysis has been proven reliable and efficient in elucidating the molecular events following SCI [
7‐
9]. For example, microarray analysis together with immunohistochemical studies has validated neuronal regeneration after SCI based upon the up-regulation of neurite growth and regeneration-associated genes after SCI, such as those encoding VGF, SPRR1A, and GAP-43 [
10‐
12]. Chamankhah et al. have identified gene expression profiles at day 1, day 3, week 1, week 2, and week 8 in rats following SCI, and they have found that the adaptive and induced innate immune responses span the acute and subacute phases and also persist throughout the chronic phase of SCI, despite the induced innate responses are more active during the acute phase, while the adaptive immune response processes are more significant during the chronic phase [
13].
In the present study, the GSE45006 microarray data submitted by Chamankhah et al. that were collected from rats at different time points after SCI were downloaded and further analyzed. Here, we identified differentially expressed genes (DEGs) that were common throughout different time points. Then, based on the common DEGs, pathway enrichment analysis was performed and an interaction network was also constructed. These common DEGs may be essential for the pathological responses in rats after SCI, thus can be alternatives for screening new therapeutic targets for spinal cord-injured patients.
Discussion
Chamankhah et al. have performed both time point and time-series analyses of the identified DEGs, which were further analyzed using Gene Ontology (GO) [
13]. In the present study, after we identified genes differentially expressed at each time point, only those expressed successively across different time points were further studied by investigating the pathways these genes were involved in, through which it is found that the common DEGs were significantly enriched in three pathways, namely Fcγ R-mediated phagocytosis pathway, MAPK signaling pathway, and chemokine signaling pathway, and may have a profound implication for the pathological responses in rats at the cellular and molecular level after SCI. The chemokine signaling pathway is transduced by chemokine receptors (G protein-coupled receptors) expressed on the immune cells, which mediate the activation-diverse downstream pathways resulting in cellular polarization and actin reorganization [
6]. The Fcγ R-mediated phagocytosis is mediated by the Fcγ receptors (Fc receptor for immunoglobulin G) [
23]. Despite phagocytosis being universally acknowledged after SCI [
5,
24], Fcγ R-mediated phagocytosis is seldom reported in spinal cord-injured rats. These two enrichment pathways further confirmed the key role of inflammation after SCI, conforming with numerous previous studies [
3,
4,
25,
26] that have demonstrated the involvement of two major types of inflammatory cells within certain time periods after SCI. For the MAPK cascade pathway, it is a highly conserved module that is involved in various cellular functions. Two MAPKs,
ERK1/2 (extracellular signal-related kinases) and p38 MAPK (p38alpha/beta/gamma/delta), mediate inducible nitric oxide synthase (iNOS)-induced spinal neuron degeneration, which is a general way to cause neuronal apoptosis by generating nitric oxide (NO) after acute traumatic SCI [
27].
AKT3 and
RAC2 were enriched in all the three pathways, indicating their important role after SCI. The AKT (also called protein kinase B) family consists of three members, Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ, which share a high degree of structural similarity [
28]. Peviani et al. have proposed that down-regulation or lack of induction of the P13K/AKT prosurvival pathway may be responsible for the defective response of spinal cord motor neurons to injury and their consequent cell death [
29]. The finding that
AKT3 is significantly down-regulated across all the time points overall in our study further validates their proposal.
The Rho guanosine triphosphatases (GTPases) comprising RAC1 and RAC2 are key regulators of cytoskeletal dynamics and affect many cellular processes, including cell polarity, migration, vesicle trafficking, and cytokinesis [
30]. Overall, the expression of
RAC2 was up-regulated after SCI in our study, which is consistent with the finding that Rho activity is raised after SCI [
31]. Activation of Rac GTPase has been proven to be involved in the p75 neurotrophin receptor-dependent apoptosis [
31,
32]. Furthermore, Harrington et al. have reported that this apoptosis pattern is mediated by activation of c-jun N-terminal kinase [
32], which can phosphorylate c-JUN, a major component of the JUN family protein (c-JUN, JUNB, and JUND) members that are necessary for the assembly of the AP-1 transcription factor complex [
33]. c-JUN, involved in the MAPK signaling pathway, is highly induced in response to neuronal injury [
34,
35], which is consistent with the up-regulation of its expression observed in our study.
Additionally,
VAV1,
LYN, and
HCK were involved in both the Fcγ R-mediated phagocytosis and the chemokine signaling pathway.
VAV1 is engaged in the regulation of T cell migration [
36], and the up-regulation of its expression proves the participation of T cells in immune response after SCI. The expression of two Src family members,
HCK and
LYN, with similar roles in a signal pathway, were both up-regulated in our study, which is consistent with the up-regulation of
LYN observed by Yang et al., who also studied the peripheral nerve injury-induced modification of genes in Sprague-Dawley rat dorsal spinal cords using both microarray analysis and immunohistochemistry [
9]. Meanwhile,
RAP1B (Ras-related protein Rap-1B) was also observed to participate in both the MAPK signaling pathway and the chemokine signaling pathway. Schwamborn and Püschel have discovered that localization of the GTPase RAP1B (Ras-related protein Rap-1B) to the tip of a single neurite is a decisive step in determining which neurite becomes the axon [
37]; thus, the up-regulation of RAP1B expression in our study may suggest the occurrence of neuronal regeneration after SCI.
Conclusions
In summary, we identified genes that were differentially expressed at different time points after SCI in rats, as well as three pathways, Fcγ R-mediated phagocytosis pathway, MAPK signaling pathway, and chemokine signaling pathway, through which these DEGs were speculated to respond to SCI. This further proves that neuronal death, inflammation, and neuronal regeneration occur after SCI, especially inflammation. And, DEGs, AKT3, RAC2, VAV1, RAP1B, HCK, and LYN, that are involved in the above three pathways may be essential for the pathological responses to SCI, which may be considered as candidate genes for the targeted therapy of SCI. Our work has deepened our understandings into the molecular mechanisms underlying SCI. However, since our findings here were obtained by bioinformatics methods, they should be taken prudently and further validated by experimental proofs before any clinical use.