| Objective This study was aimed to explore the pathological changes of rat retinal glial cells after acute methanol poisoning. The study would provide experimental evidences for further research about visual impairment caused by acute methanol poisoning.Materials and Methods100healthy adult Sprague-Dawley (SD) rats of either sex with a body weight280g±30g were used. They were randomly divided into four groups:Group A (n=25), B (n=25), and C (n=25) were exposed to a mixture of N2O/O2and given a high-dose of methanol (Group A,2g/kg), a low-dose methanol (Group B, lg/kg) or saline by gavage (Group C), respectively; Group D (n=25) was saline air control group. Each group was further randomly divided in which the rats were killed at5time points at2,12,24and72h and lw after methanol administration (n=5at each time point). Gas chromatography was used for determination of methanol concentration in the blood. The pathological changes of Aquaporin4(AQP4), GS (glutamine synthetase, a retinal Muiler cell marker), GFAP (glial fibrillary acidic protein, an astroglial cell marker), lectin (a microglial cell marker) in the retina after acute methanol poisoning were investigated by immunohistochemical as well as double immunofiuorescence labeling.Results (1) Results of methanol concentration determination:Compared with Group C and D, the methanol concentration of Group A was higher at2h,12h and24h (p<0.05) and the methanol concentration of Group B was higher at2h and12h (p<0.05) peaking at2h. The methanol concentration was not detected in each group at72h and lw.(2) Histopathological changes of rat retina:①AQP4expression was up-regulated at2h,12h and24h in Group A. The average optical density of AQP4positive reaction in retina of Group A was higher than Group B and C (p<0.01, p<0.05, respectively), notably at2h; thereafter, the average optical density of AQP4decreased with time. At72h and lw there were no significant differences in the AQP4optical density between Group A and Group C or D (p>0.05). AQP4expression was up-regulated at2h and12h in Group B. The average optical density of AQP4immunoreactivity in the retina of Group B was higher than Group C or D (p<0.01,p<0.05, respectively). After24h it decreased progressively so that it was not significantly different between them (p>0.05). Histologically, the retina can be divided into inner layer (including the nerve fiber layer, ganglion cell layer, inner plexiform layer and inner nuclear layer) and the outer layer (outer plexiform layer, outer nuclear layer, outer limiting membrane, photoreceptor cell layer). In Group A, at2h after methanol treatment, AQP4immunoexpression in the inner layer of the retina was increased but it was evidently decreased in the outer layer. AQP4immunoreactivity was significantly different in the inner and outer layers between Group A and other groups (p<0.01, p<0.05, respectively). At12h in Group A, AQP4immunoexpression in the inner layer of the retina was markedly increased. AQP4immunoreactivity was significantly different in the inner layer between Group A and othergroups (p<0.01,p<0.05, respectively). In the outer layer of the retina in Group A, AQP4immunoexpression was noticeably increased at12h. AQP4immunoreactivity in the outer layer was significantly different between Group A and Group C (p<0.05), but it was comparable between Group A and Group B. At24h,72h and lw, AQP4immunorexpression, either in the inner or the outer layer, was comparable between Group A, B and C.②GS immunoexpression in Group A was up-regulated at2h peaking at12h. It was significantly different between Group A and Group C (F=6.592, p<0.01). At24h, GS expression gradually diminished but it remained a higher level than Group C (p<0.01). GS immunopositive cell processes appeared discontinuous and furthermore the cellular structures were in disarrayed after acute methanol poisoning. At12h and24h in Group A, GS immunoexpression was higher than that in Group B (p<0.05) and it gradually returned to normal levels (p>0.05) at72h and1w. In Group B, GS immunoexpression of was up-regulated at2h peaking at12h which was higher than Group C and D at the corresponding time point (p<0.05); however, the expression gradually returned to the normal level after24h. In the inner layer of retina, GS immunoexpression in Group A was most intense at12h, and was markedly higher than that in the saline control group (p<0.05). In the outer layer of the retina, GS immunoexpressions between Group B and the saline control group was not significantly different (p>0.05).(3)In saline control group, GFAP immunoexpression was detected in the nerve fiber layer and ganglion cell layer. In Group A and at2h after methanol treatment, GFAP processes which were clearly hypertrophic extended over a much longer distance towards the outer layer of the retina when compared with that in the saline control groups (p<0.05). GFAP immunoexpression was detected as far as in the inner plexiform layer and inner nuclear layer at12and24h. This was especially consipicuous at12h in which the means of OD value were higher than the saline control group and Group B (p<0.01,p<0.05). With time, GFAP immunopositive cells projected their long processes from the inner nuclear layer towards the inner and outer limiting membrane but the expression was gradually attenuated. There was no significant difference GFAP immunoexpression between Group A and saline control group at72h or1w. The alteration in GFAP expression pattern in Group B followed that of Group A but the immunoreactivity at each corresponding time point was lower in Group B. In the retina inner layer of Group A, GFAP immunoexpression was drastically increased at2h but it peaked at12h; thereafter (24h), it was decreased, but the expression was higher than in the saline control group (p<0.05). In the retina inner layer in Group B, GFAP immunopositive expression was higher than Group C (p<0.05) at12h and24h. GFAP expression in Group A and B decreased at72h and lw. GFAP positive cell processes extended in great length towards the outer limiting membrane.④Double immunofluorescence labeling of GS and GFAP, AQP4and GS showed that in the inner retinal layer, GS and GFAP were co-expressed in long slender processes extending from the inner limiting membrane to the inner nuclear layer. Muller cells located in the inner nuclear layer and whose long processes extending from inner to outer limiting membrane co-expressed AQP4in the cell bodies and processes.⑤In Group A and at12h,24h,72h of and Group B at24h,72h, the number of lectin-positive cells gradually increased peaking at72h. A striking feature was that they migrated from the inner to the outer retina and their cell processes became stouter and shorter. They were significantly different from the control group (F=17.664, p<0.01). The difference was statistically significant between Group A and Group B (p<0.05). In Group A in the inner retina at12h,24h,72h, and in Group B at72h, large numbers of lectin-positive cells were observed in the ganglion cell layer, inner plexiform layer and inner nuclear layer being most numerous at72h. When this compared with the saline control group, the difference in cell numbers was significantly different (p<0.01). In Group A in the outer retina, at24h and72h, and in Group B at72h, the number of lectin-positive cells in the outer plexiform layer appeared to be increased compared with the saline control group (p<0.01) but the difference was not significantly different.Conclusions (1) Acute methanol poisoning in a rat model was successfully established by gas mixture (N2O/O2) inspiration and high or low-dose methanol administered via intragastric infusion.(2) Muller cells in the inner layer exhibited enhanced glutamine synthetase (GS) immunoexpression which was at early onset. A major finding after methanol poisoning was the dramatic increase in AQP4and GFAP which may account for the retinal edema. It would appear that such a phenomenon may be dose dependent. In other words, more intense AQP4and GFAP immunoexpressin occurred in rats treated with a higher dose of methanol. After methanol poisoning, microglial cells were activated. Remarkably, the activated microglia appeared to immigrate from the inner to outer layer of retina. The roles of activated microglia however remain uncertain although the possibility of them being protective for the retinal Miiller cells or other local cells is considered. On the contrary, activated microglia may also exacerbate retinal inflammation and hence are neurodestructive; this awaits further investigation. |
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