Aldose Reductase Regulates Microglia/macrophages Polarization To Influence Locomotion Recovery After Spinal Cord Injury In Mice

The central nervous Spinal Cord Injury is a lead to severe paralyzed disease,and it is difficult in treatment and rehabilitation is hardly, for the individual, thefamily and the society are created a lot of pressure. So far, in clinical, there is noeffective treatment methods for SCI or drug yet. The factors which havedisadvantage roles for axonal regeneration in injured environment areconsidered as one of the main inhibitory causes for recovery after SCI. Theinflammation in the most important pathological processes which determinesdirectly the functional recovery in SCI. The microglia/macrophages is the mostcritical inflammatory population after SCI. They polarize to M1or M2typescontain different characteristics in physiological functions.Microglia/macrophages in M1type secretes pro-inflammatory factors topromote inflammation with disadvantage in axonal regeneration, however, M2type secretes anti-inflammatory factors to inhibit inflammation with woundhealing and axonal regeneration. Aldose reductase （AR） is the key limit enzyme in polyols pathway, which is metabolized glucose to fructose. In the case ofhyperglycemia, about33%of the glucose passes into the polyols metabolicpathway to produce diabetic complications. In addition, AR also play criticalroles in the process of inflammation. The Aldose reductase inhibitors （ARI） candecrease the inflammatory factors secretion of macrophages and alleviateinflammatory responses. In this project, we were planning to detect the ARexpression pattern and elucidate the roles of AR in inflammatory responses incontribution to motor functional recovery after SCI in mice. The study wasconsisted with two parts as below.The first part of the experiment was histopathological researches of AR,which included AR expression pattern and the location of cell types in injuredspinal cord. We also detected AR expression level in relationship with motorfunctional recovery after SCI in mice. We used the AR^+/+mice to generate severespinal cord crushing model, and separated the injured mice in sham group,post-injury at4hour （h）,8h,12h,1day （d）,3d,7d and14d groups,respectively. We collected the tissues and extracts of protein and mRNA form1cm spinal cord which included the0.5cm rostral and caudal spinal cord tissuesform epicenter of injured spinal cord in mice. Immunofluorescence, QPCR andWestern Blot were used to detect the expression pattern of AR after SCI. Wealso compared the motor functional recovery with BBB evaluation and thechange of injured areas at3,7and14day post-injury with GFAPimmunofluorescent staining between AR^+/+and AR-/-mice in SCI.In the second part of the experiment, we detected the roles and mechanismof AR in polarization of microglia/macrophages in SCI. We collected the tissuesand extracts of protin and mRNA as Test1. We compared the specific genesexpressions of M1and M2, such as iNOS and ArgⅠ, between AR^+/+and AR-/- mice in SCI. At same time, we also observed the expression of transcriptionalfactors of NF-κB and CREB （cAMP response element-binding protein, CREB）,which are believed in regulating expression of iNOS and ArgⅠ, respectively. Inaddition, we gave the different polarized stimulation to microglial cell line N9combined with ARI to observe the expression of M1or M2specific genes andrelative transcriptional factors in culture condition.We found AR upregulated gradually after SCI and reached its highestexpression at3day post-injury. The neurons and the microglia/macrophagesupregulated AR expression levels in protein after SCI. These phenotypes wereconfirmed by N9cell line with inflammatory cytokines stimulation in vitro.Compared with AR^+/+mice, more ArgⅠ-positive, but not iNOS-positivemicroglia/macrophages recruited to lesion sites after SCI in AR-/-mice. Furtherexperiments showed that M2specific genes, such as ArgⅠand CD206, werehighly expressed after SCI in AR-/-mice, compared with AR^+/+mice. The M1specific genes, however, such as iNOS and CD86, expressed much higher inAR^+/+than AR-/-mice after SCI. These results suggested that AR mightregulate the polarization of microglia/macrophages after SCI in mice.NF-κB was low expression in AR-/-compared with AR^+/+mice in SCI. TheCREB was high expression in early stage of injury and reached highest level at7day post-injury, then decreased significantly after SCI in AR-/-mice. In AR^+/+mice, however, the CREB expression was significantly increase at14daypost-injury after SCI. ARI treatment can inhibit iNOS and promote ArgⅠ andCREB expression in N9cell line in vitro.So we got the conclusion that AR deficiency could inducemicroglia/macrophages polarized to M2direction with benefit to motorfunctional recovery in SCI. AR regulated microglia/macrophages to M1/M2 polarization through NF-κB and CREB transcriptional factors pathways.