Background
Inbred strains of mice with identical or nearly identical genotypes have been developed and used extensively in experimental research. They provide a valuable means to study genetic influence on various biological determinants. Susceptibility to, or the severity of, experimental models of neurological disease and injury is often strain-dependent [
1‐
3]. In such systems, inflammatory mediators and immunological activation are recognized as key factors. Gene linkage analysis of hybrids from two strains of mice and rats have implicated many immunologically relevant genes that may regulate in clinical susceptibility or severity [
2,
4‐
15].
SJL/J mice are a strain commonly used in animal models of neurological disease. Variations in immune responsiveness have also been well defined between C57BL/6J and BALB/cJ mice in non-CNS tissues. It is not fully known whether the immediate response to injury in CNS tissue differs among these strains. A simple ex vivo system was devised to address this. The transcriptional response of inflammatory-related genes was measured in cerebral cortical tissue that was incubated in explant medium for less than five hours from resection. The inflammation-related transcriptional targets selected for analysis were based on their previously documented involvement in models of neurological disease and injury [
16‐
18]. This included pro-inflammatory cytokines, chemokines, CD74, and antisecretory factor. CD74 is differentially regulated among inbred strains following CNS injury [
4,
19]. Antisecretory factor is an understudied molecule with anti-inflammatory activity that has been implicated in severity of experimental autoimmune encephalomyelitis [
20], a model system known to exhibit well established strain-dependent variability [
21,
22]. In this study, the levels of mRNAs were compared in freshly isolated cerebral cortex and cortical explants among three mouse strains. A classic injury response of pro-inflammatory mediators was observed in cortical explants, yet differences based on genetic background were also observed.
Discussion
The data reported in this study establish that differences in the immediate gene response to damage of central nervous system (CNS) tissue occur among mouse strains. Message for several genes involved in CNS injury and neurological diseases with autoimmunity or chronic innate immune activation were strain-dependently altered. Short-term explants of cortical sections provided a reliable system for defining immunologically relevant transcriptional changes in CNS tissue and the model may serve as a cost-effective method to test novel immunomodulating pharmaceuticals.
One millimeter-thick explants of adult cerebral cortex were used in this study. Acute CNS explants of this thickness have been previously demonstrated to induce pro-inflammatory mRNA expression consistent to the temporal profile observed following injury
in vivo [
24,
25]. The production of immunologically relevant mRNAs is likely caused by a combination of tissue damage at the periphery of the explant due to tissue sectioning and axotomy of projection neurons throughout the explant, and some undefined amount of ischemia in the center of the explant. Pro-inflammatory mRNA increase occurs in cortex within hours following lesioning [
26] or ischemia [
27,
28]
in vivo. Since there were no established foci of inflammation in the tissue prior to sectioning in our explant model, the influence of the miniscule number of leukocytes in the vasculature would contribute only marginally and is limited to the residual cells in the vasculature at the time of sectioning. Therefore, the extent of the measured pro-inflammatory response is predominantly by resident CNS cells.
Pro-inflammatory cytokines and chemokines expression are activated in cells of the monocyte/macrophage lineage in the innate response to injury or infections. Microglia are considered the primary cell type within the CNS parenchyma that carry out this function. Pattern recognition receptors recognize specific molecules released from damaged host cells or foreign microbes leading to the activation of transcription factors that induce transcription of certain inflammatory genes. Sections of cerebral cortex incubated ex vivo in explant medium were demonstrated to increase mRNA for pro-inflammatory cytokines and chemokines within five hours. The constitutive amounts in uninjured cortical tissue were mostly very low and transcripts that could be confidently quantified did not differ significantly among mouse strains. However the amount measured in cortical explants from these strains was different for several of the transcripts suggesting strain-related alterations in their induction. The chemokines evaluated were CCL2 (MCP-1), CCL3 (MIP-1α), and CCL4 (MIP-1β). These transcripts were found in increasing abundance in BALB/cJ, SJL/J, and C57BL/6J, respectively. Interestingly, the abundance of IL-1α and COX-2 mRNA revealed a similar pattern to the chemokines suggesting these inflammatory mediators could have a common regulatory mechanism that is distinct among these mouse strains. Messages for IL-1β and TNF-α were similar in SJL/J and C57BL/6 explants, but higher than BALB/cJ. Taken together, BALB/cJ appears to have a dampened immunological response to tissue damage in cerebral cortex.
Expression of genes associated with autoimmunity was also examined in this study. Experimental autoimmune encephalomyelitis is a widely studied autoimmune model, and involves infiltration of CD4+ lymphocytes and immunological activation of microglia within the CNS [
21,
22,
29]. Although MHC class II haplotype and its binding to specific myelin autoantigen play a pivotal role in this model, non-MHC class II effectors are also implicated [
30]. BALB/cJ is EAE resistant while SJL/J and C57BL/6J are susceptible [
2,
3,
31]. Cytokines IL-12 and IL-23 are implicated in the pathogenesis of autoimmune diseases including EAE [
17]. The p35 subunit of IL-12 and p19 subunit of IL-23 form respective cytokines with a common p40 subunit. Blocking the IL-12/23 p40 subunit with inhibiting antibodies is effective in non-CNS autoimmune diseases such as psoriasis [
32]. In cortical explants, the p40 mRNA was upregulated. Its levels were considerably lower in the resistant BALB/cJ explants. This suggests that inherent difference within the CNS tissues may contribute to strain susceptibility to autoimmunity.
Antisecretory factor (ASF) has been shown to affect the severity of EAE. Blocking its activity with an inhibiting antibody increases clinical severity implying it has an anti-inflammatory property [
20]. Our results showed that differences in ASF mRNA expression occurred in normal cortical tissue and in cultured cortical explants. C57BL/6J mice had high constitutive ASF mRNA. Its levels decreased after injury in cortical explants in each strain, but to a lesser degree in BALB/cJ. This supports the hypothesis that higher amounts of ASF due to genetic background may contribute to EAE resistance.
Antigen presentation by MHC class II is critical for many autoimmune diseases. CD74 (invariant chain, Ii) acts as an MHC class II chaperone [
33,
34]. CD74 mRNA was higher in SJL/J cortex relative to BALB/cJ and C57BL/6. A similar trend among these strains was reported in facial nucleus two weeks following facial nerve axotomy [
19]. Our study revealed that higher levels were found in uninjured cortex and that these differences were increased further in explants due to down-regulation in C57BL/6J and BALB/cJ tissues. CD74 also mediates transcription by NF-κB [
35,
36] and regulates dendritic cell migration [
37]. This suggests its altered expression among strains could influence a wide range of effects.
Now, identifying the inherent genetic polymorphisms that control the variations in transcriptional response to an injury stimulus among strains is important for understanding genetically variable responses to a spectrum of neurological disorders. Approaches such as quantitative trait locus analysis and/or haplotype-based computational genetic mapping can be utilized with the cortical explant model. Haplotype-based computational genetic mapping has recently pinpointed genetic variation of Nalp1 as a contributor to interstrain differences in the inflammatory response to injured skin [
38]. It is important to recognize that the involvement of multiple genes may be required and that such analyses will likely require data from additional strains and transcripts.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DJG carried out the experiments and evaluated the data. DJG, BTH, and WFH participated in the design and assisted with the preparation of the manuscript. All authors have read and approved the final version of the manuscript.