Neuroprotection And Mechanism Of Recombinant Human Interleukin-2 In Retina Of Chronic Elevated Intraocular Pressure Model Rat

Objective: To observe the neuroprotection of recombinant human interleukin-2(rhIL-2) by intravitreous injection on the retinal ganglion cells(RGCs) following chronic elevated IOP model in Wistar rats .Methed: Chronic elevated IOP model of Wistar rats were established by cauterizing the limbal episcleral veins to block the reflux of aqueous humor by 532nm viridis-lite diode laser in the eyes.(1) After chronic elevated IOP model of right eyes established, 12 adult male Wistar rats were randomly divided into 1^st day,3^rd days, 1^st week,2^nd weeks,4^th weeks groups, IOP was measured by Tonopen .(2)64 adult male Wistar rats were randomly divided into normal control,modle control, PBS treatment and rhIL-2 treatment groups. Intravitreous injection of 2μl PBS or 2μl rhIL-2 for eyes were administered 1 week after chronic elevated IOP model established. Rats were sacrificed after intravitreous injection 3 days,7 days, 14 days,21 days. To identify RGC,the 3% fluorogold was used to the epithalamus 7 days before sacrifice. The retina were examined through a fluorescence microscope,retinal ganglion cells were quantitatively analyzed by computer. Ocular tissue was immunostained to investigate ED-1 expressions . (3)48 adult male Wistar rats were randomly divided into normal control,modle control, rhIL-2 treatment and LY29400,AG490 mixed liquor treatment groups,that was to say,2μl PBS,2μl rhIL-2 or 2μl LY29400,AG490 mixed liquor were injection for eyes. Western blotting analysis Akt1 and STAT3 protein in the retina after intravitreous injection 3h,12h,24h,96h.The other 12 rats LY29400,AG490 mixed liquor treatment rats were sacrificed after intravitreous injection 3 days ,7 days, 14 days ,21 days,to identify and quantitaty RGC,the same was used to the epithalamus 7 days before sacrifice.Results : (1) The IOP was prominently elevated by cauterizing 3/4 circle of limbal episleral blood vessel by 532nm diode laser. The difference of the amount of rat's HE staining RGCs between its right eye and left eye was significant(P<0.01).(2)Numerous ED-1 positive macrophages were seen in the vitreous and along the inner retinal surface in rhIL-2 treatment group .(3) The numbers of RGCs in rhIL-2 group was 182.23±7.16/4 visual fields, 186.74±8.78/4 visual fields,188.53±12.99/4 visual fields,190.54±10.39/4 visual fields on the 3^rd,7^th,15^th and 21^st day, the number of RGCs were much higher than that in control groups and PBS group(P<0.01).The numbers of RGCs in rhIL-2 and LY29400, AG490 mixed liquor group was as much lower than that in rhIL-2 group (P<0.01).(4) Western blotting analysis showed very little Akt1 and STAT3 protein in the retina after 3h intravitreous injections of rhIL-2 in rats,started to increase rapidly at 12 reperfusion,96h decreased slightly. LY294006 and AG490 prevented the Akt1 and STAT3 protein in the retina.Conclution:(1) By means of 532nm diode laser cauterization of occlusion the rat's 3/4 peripheral limbal episcleral vein can establish a stable chronic elevated IOP model of Wistar rats.(2)rhIL-2 has neuroprotective effection after intravitreous injection. It can decrease or avoid RGCs layer damage in chronic glaucoma model of rats.(3) Intravitreal injection of rhIL-2 activates macrophages. Macrophages derived factors might enhance the RGCs survival.(4)After intravitreal injection of rhIL-2 PI3K/Akt,Jak/Stat3 signal transductions may play an important role following a chronic elevated IOP model,and LY294006, AG490 can block these effect.