Cytotoxic Effect Of Anti-aquaporin-4Antibody To Cultured Rat Cortical Cells

Objective: Neuromyelitis optica (NMO) is an inflammatorydemyelinating disease of the central nervous system(CNS), which primarilyaffects the optic nerves and spinal cord. Although the identification of AQP4antibody in the sera of patients with NMO has improved the diagnostic criteriaof NMO and expanded the NMO disease spectrum, it still remains unclearwhether AQP4antibody plays a directly role in the pathogenesis of NMO. Theintracerebral co-injection of AQP4antibody from NMO patients with humancomplement reproduces the key histological features of NMO in experimentalanimals, including inflammatory cell infiltration, demyelination, loss of AQP4and glial fibrillary acidic protein(GFAP) expression, and perivasculardeposition of activated complement. In vitro study, AQP4antibody bound tothe extracellular domain of AQP4of astrocytes, activated classicalcomplement pathway, and caused antibody-dependent complement andcell-mediated astrocyte damage. However, the effect of AQP4antibody to themixed cultured rat cortical cells has not been reported. In the current study weobserved the changes of morphology and quantitative of astrocytes, neuronsand microglias, the levels of interleukin-6(IL-6) in primary cultured ratcortical cells incubated with AQP4antibody positive or negative sera ofpatients with NMO at different time points, and to investigated the cytotoxiceffect and pathogenic mechanism of AQP4antibody on cultured rat corticalcells.Methods: The routine methods were used to extract cortical cell from16-19days Wistar embryonic rats. The cortical cell were planted into culturedishes of35mm and maintained in DMEM contained20%fetal bovine serum.The medium was changed after24h and72h. The cortical cell cultures at3days were used for experiments. The cells were treated with the sera of healthy control, of AQP4antibody positive and negative patients with NMOfor2h,4h and6h respectivly. The cellular immunofluorescence staining ofMAP-2, GFAP and OX42were carried out at different time point and thechanges of morphology and quantitative of MAP-2, GFAP, OX-42positivecells were observed by fluorescence microscope. The numbers of cells wereassesed using Image Pro-Plus software. The quantitative of cells werecalculated as the percentage of cells compared to the total population.Statistics analyses were performed by SPSS13.0software. Data are presentedas mean±SEM. Statistical comparisons were made using ANOVA test.Significance is indicated with P≤0.05.Results:1. The effect of AQP4antibody on of astrocytes and neurons in primarycultured rat cortical cellsNone changes of the morphology and quantitative of astrocytes andneurons were observed at the different time points in control group. Theastrocytes exhibited cellular swelling and hypertrophy, accompanied byneuronal processes fragmentation in the AQP4antibody positive and negativegroups at2h. The percentage of astrocytes and neurons in the AQP4antibodypositive group at2h were33.32±12.25%and46.67±9.48%respectively. Nostatistical significant differences compared with the control group(P>0.05).The percentage of astrocytes and neurons at4h (24.73±5.27%and35.49±8.43%respectively) and6h (19.04±5.80%and29.39±7.62%respectively) were significantly decreased as compared with the controlgroup(P<0.01). There were also significant differences of the percentage ofcells among the different time point(P＜0.01). The exposure of cultured ratcortical cells to the sera of AQP4antibody negative patients inducedsignificant decrease of neurons at4h and6h(40.89±5.67%and37.40±3.95%, P<0.01compared with the control group). The percentage ofastrocytes were29.67±3.40%and25.62±6.85%at4h and6hrespectively(P>0.05at4h and P<0.01at6h compared with the control group).There were significant differences of the percentage of astrocytes among the different time point(P<0.01).2. The effect of AQP4antibody to microglias in primary cultured rat corticalcellsThere were obviously morphological changes of microglias includingcellular swelling and hypertrophy, the processes reduced, shorten andfragmentation when exposed to the sera of AQP4antibody positive andnegative patients at2h. The percentage of microglias in AQP4positive andnegative group at2h were18.55±9.78%and18.39±5.03%respectively, andno signifIcantly different from the control group(15.76±6.66%, P>0.05).However, the percentage of microglias significantly increased at4h and6h inAQP4positive group(27.35±13.17%and40.37±13.28%, P<0.01comparedwith the control group) and AQP4negative group(24.70±7.27%and32.60±10.77%, P<0.01compared with the control group) and no significantdifferences of the percentage of microglias between AQP4antibody positiveand negative group among the different time point(P>0.05).3. The effect of AQP4antibody to the levels of IL-6in primary cultured ratcortical cellsThe levels of IL-6in the rat cortical cells culture exposed to AQP4antibody positive sera at2h,4h and6h were22.00±0.24,28.56±0.15and30.78±0.08respectively. There were significant decrease compared to thecontrol group(P<0.01), although the levels of IL-6gradually increased withtime prolonged. However, The IL-6levels in AQP4negative group werelower at2h(29.53±0.37), and higher at4h(41.03±0.62) and6h(34.01±0.10)than that of the control group(P<0.01).Conclusion: In summary, the present study showed that the primarycultured rat cortical cells exposed to AQP4antibody positive sera of patientswith NMO induces the loss of astrocytes and neurons, microglial activation,which is similar to histological features of NMO lesions. Our study stronglysupports the primary role of AQP4antibody in the pathogenesis of NMO andalso suggest that IL-6is involved in NMO pathogenesis.