| Objective Stroke is one of the most common diseases which is amajor public health problem threaten to human health worldwide andshows an increase in prevalence as population ages. Stroke has alreadyinduced severe economic burden on the family and society in developingcountries. Cerebral ischemia generally leads to persistent and severefunctional deficits. Accumulated evidence has indicated that the limitedcapacity of the adult central nervous system (CNS) to support therearrangement and re-extension of axonal connections is the majordeterminant of failed functional recovery. Axonal regeneration failure ismost likely attributable to the non-permissive adult CNS environment.There are many axonal regeration inhibitors near the site of injury. Therepulsive guidance molecule A (RGMa) is a membrane-bound proteinoriginally identified as an axon guidance molecule in chicken temporalretina. It recently has been identified as a powerful myelin-derived neuriteoutgrowth inhibitor in adult CNS. RGMa is mediated by the RhoA/ROCKpathway. The active ROCK regulates the phosphorylation level of MLC in an indirect manner by phosphorylating myosin light chains phosphatase(MLCP), these lead to up-regulation of phosphorylated MLC, which play akey role in neurite retraction and growth cone collapse. Minocycline is asecond-generation semi-synthetic tetracycline drug, which freely crossesthe blood-brain barrier and possesses a wide-spectrum anti-inflammatory,anti-apoptotic, antioxidative activities and vascular protective properties inmany animal models of cerebral ischemia. Accumulating clinical trialssuggest that minocycline could improve neurological function recovery inpatients with acute cerebral ischemia. However, the underlying cellular andmolecular bases for its neuroprotective effects have not been fullyelucidated. In this study, we investigated whether minocycline maydown-regulate the expression of RGMa protein and improve neurologicalfunction recovery after focal cerebral ischemia reperfusion. Furthermore,we investigated whether minocycline could enhance neurite outgrowth ofPC12cells exposed to oxygen glucose deprivation, which was associatedwith the activation of MCLP/MLC signaling pathway.MethodsPart I1. Seventy-two adult male healthy Sprague-Dawley (SD) rats wererandomly divided into the following four groups: sham-operated (n=18),cerebral ischemia and reperfusion (n=18), saline vehicle (n=18) andminocycline treated groups (n=18). The focal cerebral ischemia/reperfusion model (I/R) was induced by ligation the right middle cerebral artery of ratswith nylon monofilament for2h and3mg/kg minocycline was injectedintravenously immediately after reperfusion twice a day for14days in ratsof minocycline treated group. The permeability of blood-brain barrier wasassessed by Evan’s blue dye extravasation (EB) in the infarct brain24h afterMCAO/R.2. The expression of RGMa protein in ischemic cortex andhippocampus was measured by immunohistochemical staining and Westernblot. Neurofilament protein200(NF-200) immunohistochemical stainingwas used to assess axonal damage2weeks after MCAO/R.3. The staircase test and modified neurological severity score wereperformed to evaluate functional outcome at2,7,14and28days afterMCAO/R.Part II1. PC12cells were subjected to oxygen glucose deprivation andreoxygenation (OGD/R) to mimic cerebral ischemia reperfusion injury invivo. The cultured PC12cells were randomly divided into three groups:normal control group, OGD/R group (PC12cells were exposed to2,4,6,8h of OGD) and various doses of minocycline (0.1,1,10μM) treated group.24hours after reoxygenation, the cell viability of PC12cells was detectedwith CCK-8assay.2. Twenty four hours after OGD/R, The expression of neurite outgrowth maker protein, growth associated protein (GAP-43) wasmeasured by western blot, and the mean length of neurite outgrowth wasalso measured with microtubule associated protein-2immunostainingunder fluorescence microscope.3. PC12cells were randomly divided into normal control group,OGD/R group (6h), minocycline treated group (1μM) and MLCP inhibitorgroup (1nM, Calyculin A). The expression of myosin light chain (MLC),p-MLC and GAP-43protein were measured by Western blot and neuriteoutgrowth was visualized by immunofluorescence staining with MAP-2antibody24h after OGD/R.ResultsPart I1. The Evans blue extravasation in ischemic brain tissue of rats wasobvious24h after I/R（4.40±0.73μg/g. Minocycline at a dose of3mg/kg(caudal vein) significantly reduced the extravasation of EB (2.71±0.68μg/g, P<0.05).2. The expression of RGMa protein in ischemic cortex andhippocampus was significantly increased in the I/R group compared to thesham group assessed by both immunochemical staining and Western blottwo weeks after I/R, axonal regrowth was enhanced in the minocyclinetreated group compared to the I/R group (P<0.05). In addition, minocyclinesignificantly reduced the expression of RGMa protein2weeks after I/R. (P<0.05).3. Minocycline could decrease mNSS and improve forelimb motorfunction as assessed by the staircase test of rats compared to the rats in theI/R group (P<0.05).Part II1. Oxygen glucose deprivation injury induced a time-dependentdecrease in cell viability of PC12cells, and the cell viability was46.1±2.9%at6h after OGD. Minocycline elicited a non-linear concentration-dependentneuroprotective effect on cell viability of PC12cells exposed to OGD for6h.The maximal neuroprotective effect of minocycline was achieved at1μM(77.0±2.5%, P<0.05).2. OGD/R injury induced neurite retraction, minocycline improvedneurite outgrowth and increased the expression of GAP-43protein in PC12cells after OGD/R injury (P<0.05). Calyculin A (the inhibitor of MLCP)could abolish the promotive effects of minocycline. In addition, OGD/Rinduced an increase in the phosphorylation of MLC and minocyclinedecreased the phosphorylation of MLC in PC12cells after6h of OGD/Rinjury (P<0.01).Conclusions Minocycline at a dose of3mg/kg promotes neurologicalfunctional recovery and axonal regeneration in rats after I/R injury, whichmight be mediated by down-regulating RGMa expression. Minocyclinecould activate MLCP/MLC signaling pathway, induce lower levels of p-MLC in PC12cells after OGD/R, which leads to the enhancement ofneurite outgrowth. |
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