Protective Effects Of ?-melanocyte Stimulating Hormone On Glutamate-induced Retinal Excitotoxicity

Background The three major blind-causing eye diseases in the world include glaucoma,diabetic retinopathy and age-related macular degeneration.All of that have their own typical pathological processes and at the same time there is a common pathological condition ? increasion of the retinal excitatory neurotransmitter glutamate,whereas excessive glutamate in this tissue may induce retinal excitotoxicity.It has been reported that ?-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 vitro.Intraocular injection were given to newborn chicks,and study the protective effects of a-MSH on glutamate-induced excitotoxicity in vivo.Methods Part 1:The retinas were isolated from chick embryos at embryonic day 9(E9)and cultured as explants in 10%and 15%FBS,The explants at 3,5 and 7 days in vitro and the retinas at corresponding embryonic day 12.14 and 16(E12,E14,E16)were collected.The morphology of explant cultures was examined by HE staining.In the experiment of glutamate-induced excitotoxicity,the retinal explants at 4 days in vitro were treated with glutamate for24?48 hours,?-MSH was incubated with the explants 30 minutes before and during the glutamate treatment period.Then the activity of LDH in the culture media were detected by CCK-8 at the 2 points,the apoptotic cells were detected by TUNEL staining and quantified at the 2 points.The expression of Nitric oxide synthase(NOS including eNOS?nNOS?iNOS),glial fibrillary acidic protein(GFAP)at 24?48 hours post-treatment in all retinal explants were analyzed by real-time PCR.Part 2:Intraocular injection were given to 1 day newborn chicks in glutamate group??-MSH+ glutamate group?normal group?24h later glutamate were given,and after 48h all objects were tested ERG and their retina were examined by HE staining and TUNEL staining.Results Part 1:1.Compared with retinas cultured in 10%FBS complete medium.the total thickness of retina cultured in 15%FBS complete medium increased at 3DIV(P<0.05).what's more,the thickness of outer nuclear layer(P<0.05),inner nuclear layer(P<0.001),and the whole layer(P<0.001)increased significantly at 5DIV.but there were no difference of layer thickness at 7DIV(P>0.05),developmental state of retinal explants cultured in 15%FBS complete medium was closer to that of chicken embryo.2.24 hours after glutmate treatment,LDH activity in medium of glutamate alone group increased significantly than that of normal control group(P<0.001),whereas there were no statistically significant difference of LDH activity between a-MSH+glutamte group and normal control group(P =0.197),suggesting that a-MSH can prevent increase of LDH activity.48 hours after glutmate treatment,there were no statistically significant difference of LDH activity among groups(P>0.05).3.TUNEL staining revealed that after treatment of the retinal explant cultures with glutamate 24h?48h,the retinal inner nuclear layer and ganglion cell layer have lots of apoptotic cells.even the outer nuclear layer has a few apoptotic cells,DAPI staining revealed the retinal structure disorder after 48h.but order in the a-MSH+ glutamate group.Quantitative results showed that treatment of the retinal explant cultures with a-MSH substantially and significantly reduced number of apoptotic cells induced by glutamate in both 24h and 48h(P<0.001).4.Relative GFAP and eNOS mRNA expression increased significantly after 24h in the retinal glutamate group than normal group(P<0.00),and treatment of the retinal explant cultures with a-MSH had significant suppression of glutamate-induced eNOS?GFAP?up-regulation(P<0.001).At 48h relative GFAP mRNA expression increased significantly in the retinal glutamate group than ?-MSH+ glutamate group(P=0.000),while eNOS?nNOS?iNOS mRNA have no difference in glutamate group and a-MSH+glutamate group(P>0.05).Part 2:1.48h after glutamate treatment ERG data shows:implicit time of photopic ERG in glutamate group and ?-MSH+ glutamate group were reduced than normal group(P<0.01),while there was no difference between glutamate group and P-MSH-glutamate group(P>0.05).Photopic ERG and Photopic 30Hz flicker amplitude were reduced in glutamate group than normal group(P=0·000).and treatment of a-MSH can prevent it significantly(P<0.01).2.There were no retinal hemorrhage,edema,atrophy and detachment in all three groups by HE staining.3.Quantitative results showed that the average number of every chicken retinal section was different in 3groups(P=0.000),treatment of the retinal explant cultures with ?-MSH substantially and significantly reduced number of apoptotic cells induced by glutamate(P<0.001).Conclusions Application of a-MSH dramatically ameliorated glutamate-induced tissue damage,cell apoptosis and function damage.This protective effect may be due to a-MSH-mediated suppression of NO production and astrogliosis caused by abnormal elevation of glutamate.