Abidol Reduces Anti-aquaporin-4 Antibody-positive Serum Induced Neurotoxicity In Vivo And In Vitro

Objective: Neuromyelitis optica(NMO) is an inflammatory demyelinating disease that produces visual and neurological function impairment in the central nervous system. Aquaporin 4(AQP4) is a water channel protein expressed in astrocyte foot, anti-AQP4 antibody(AQP4-Ab) could cause the pathology by bingding to AQP4, which induces antibody-dependent cytotoxicity(ADCC) and complement-dependent cytotoxicity(CDC) and the downstream inflammatory, blood-brain barrier(BBB) disruption, results in demyelination, astrocytes and neuronal injury. While, Current NMO therapies mainly induce immunosuppressants, immunemosuators and plasma exchange, which have limited efficacy to prevent recurrence of NMO. Furthermore, all of these are not involved in the pathology. A recent research investigates that abidol, a small molecule inhibitor, effectively alleviated CDC and ADCC in AQP4-transfection cells and reduced the size of NMO-like lesion in mouse. However, the protection of abidol in rat cortical cells primary culture or intracerebral injection model of NMO in rats have not yet reported. Here, we developed an animal model of NMO following intracerebral injection of AQP4-Ab positive serum, and to investigate the protective effect of abidol on AQP4 antibody-induced neurotoxicity in vivo and in vitro.Methods: The cerebral cortical cells of newborn SD rat were conventional culture at 37℃ 5%CO2 in DMEM/F12 supplemented with 10% fetal bovine serum. After 4 days, cells were used for experments. We randomly divided the cells into four groups and respectively added the normal control serum, serum with AQP4 antibody positive, AQP4 antibody positive serum after abidol preconditioning with different concentrations(12.5μM, 25μM, 50μM) and Dimethyl sulfoxide(DMSO 1%). After 6h cultivation, cells were stained with Hoechst33258 and propidium iodide(PI) to research the most effective concentration of abidol. Then we aboserved the morphological and the total area changes of astrocytes and neurons through immunofluorescence staining. In addition, the rats were also randomly divided into four groups, respectively suffered intracerebral injection of AQP4 antibody positive serum or control serum, AQP4 antibody positive serum after intraperitoneal injection of abidol and DMSO. After 3 days, we detected the expression levels of AQP4 and glial fibrillary acidic protein(GFAP) and the lesion size.Results:1 The effective concentration screening of abidolWe found large neurosis in AQP4 antibody positive serum group, nor in the nomal serum group and DMSO group, the neurosis apparently decreased in abidol precondioning group. There was significantly statistical different in total(χ2=261.7,P<0.05). Cells survival rate in 12.5μM and 25μM group were 64.24% and 78.92%, it was significantly increased compared with AQP4-Ab positive serum group(48.62%)(P<0.01). However, there was no significantly different between 25μM group and AQP4-Ab positive serum group(P=0.014).2 Effects of abidol and AQP4 antibody positive sera on changes of astrocytes and neuronsIn vitro, the total area of positive cells(astrocytes and neurons) in AQP4-Ab seropositive group were 405.17±79.41 and 295.26±43.31(μm2) respectively. It was significantly decreased compared with normal control group(3226.85±334.56 and 1990.03±63.27)(P<0.01); while abidol preconditioning in AQP4-Ab positive serum could apparently increase the area of positive cells(1086.17±148.31 and 955.75±132.05)(P<0.01); in addition, there was no statistical significantly difference between normal control group and DMSO group(2903.61±162.50 and 1864.10±67.39)(P=0.102).3 Intracerebral injection of AQP4-Ab positive serum caused NMO-like pathologyWe observed large inflammatory infitration in injection-side hemisphere, less in non-injected side through HE staining.We also found the loss of AQP4 and GFAP expression within lesion, the strong GFAP immunoreactivities around the lesion but not AQP4.4 Effects of abidol preconditioning on lesion size in vivoIn vivo, The loss area size of AQP4 and GFAP express in AQP4-Ab positive group were 5.5864±0.72756mm2 and 18.0692±1.21775mm2 respectively,significantly increased as compared with the normal control serum(4.6205±0.3178 and 3.8589±0.28073)(P<0.05); howerver, the lesion size apparently decreaded in abidol preconditioning group(9.7107±0.41437 and 8.3793±0.59119), there was significantly different compared with AQP4 antibody positive group(P<0.05).Conclusions:1 Intracerebral injection of AQP4-Ab positive serum caused NMO-like pathology, including leukocyte infiltration, loss of AQP4 and GFAP expression.2 Abidol alleviated the neurotoxicity induced by AQP4-Ab positive serum in vivo and in vitro.3 Abidol would be effective on the treatment of NMO.