Identification of molecular pathway changes after spinal cord injury by microarray analysis

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.

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.