| Background The major blind-causing eye diseases in the world include Glaucoma,diabetic retinopathy and other eye diseases. The sustained high intraocular pressure in glaucoma and the retinal microvessel injury in diabetic retinopathy both lead to retina ischemia, which in turn causes elevated glutamate concentration in retina, whereas excessive glutamate in this tissue may induce retinal excitotoxicity. Therefore, glutamate induced retinal excitotoxicity is the common pathology observed in glaucoma,diabetic retinopathy and other eye diseases, the major blinding causing eye diseases. It has been reported that a-melanocyte stimulating hormone rescues the hippocampal neurons from death and inhibits reactive astrogliosis caused by glutamate induced excitotoxicity.Objective Construct a chicken embryonic retinal explants culture system and study protective effects of a-MSH on glutamate-induced excitotoxicity in a chicken embryonic retinal explant culture system firstly in China.Methods The retinas were isolated from chick embryos at embryonic day9(E9) and cultured as explants. The explants at3,5and7days in vitro and the retinas at corresponding embryonic day12,14and16(E12, E14, E16) were collected. The morphology of explant cultures was examined by Hematoxylin and Eosin staining, and the expression of melanocortin receptors (MCRs) analyzed by real-time PCR. In the experiment of glutamate-induced excitotoxicity, the retinal explants at4days in vitro were treated with glutamate for48hours, a-MSH was incubated with the explants30minutes before and during the glutamate treatment period. Then the apoptotic cells were detected by TUNEL staining and quantified. The glutamate alone treated explants and those treated with culture media were included as controls. The expression of glial fibrillary acidic protein (GFAP) at48hours post-treatment in all retinal explants was analyzed by real-time PCR.Results1.Hematoxylin and Eosin staining showed that the retinal explants exhibited similar morphology to those observed in the retinas from chick embryos at the corresponding developmental stages. 2.The real time PCR analyses of chick retinas showed that MC1R mRNA levels at E9,12,14and16were significantly lower than that in post-hatch day1(P＜0.01); whereas the transcript levels of MC5R were. significantly increased from E9to E12and E14(both P＜0.001), then gradually decreased from E14to P1. The expression of these genes showed similar temporal patterns in the retinal explant cultures.3. TUNEL staining revealed that after treatment of the retinal explant cultures with glutamate48h, the retinal inner nuclear layer and ganglion cell layer have lots of apoptotic cells,also outer nuclear layer has few apoptotic cells, DAPI staining revealed the retinal structure disorder,but order in the α-MSH+glutamate group. Quantitative results showed that the average number of every chicken embryonic retinal section in glutamate group is (600.80±24.19)apoptotic cells,(61.80±15.77) apoptotic cells in a-MSH+glutamate group,(72.4±12.93) apoptotic cells in normal group. Treatment of the retinal explant cultures with a-MSH substantially and significantly reduced number of apoptotic cells induced by glutamate (P＜0.001).4. Relative GFAP mRNA expression in the retinal explant cultures of glutamate group is2.23±0.06, in α-MSH+glutamate group is1.14±0.15, in normal group is1.05±0.04, so treatment of the retinal explant cultures with a-MSH had significant suppression of glutamate-induced GFAP up-regulation (P＜0.001).Conclusions Application of a-MSH dramatically ameliorated glutamate-induced cell death in retinal explant cultures, this protective effect may be due to a-MSH-mediated suppression of astrogliosis caused by abnormal elevation of glutamate. This work provides the primary theoretical and experimental basis on which the neuro-protective reagent used in glaucoma and diabetic retinopathy will be further developed. |
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