Effects Of Brimonidine Tartrate On Ischemic Changes Induced By Repeated Intravitreal Injections Of Endothelin-1 In Rabbit Retina

Objective: Glaucoma is a common cause of blindness, which injury pathogenesis of optic nerve is the loss of retinal ganglion cells (RGCs) and nerve fiber. In addition to the mechanical stress of intraocular pressure, optic nerve head ischemia has been suggested as an important cause of glaucomatous optic neuropathy, especially in normal-tension glaucoma (NTG). There has recently been an increase in the recognition of endothelin-1(ET-1) as an important factor in glaucomatous optic neuropathy.The purpose of this study was to investigate the effects of brimonidine tartrate 0.2% (BMD), anα2-receptor agonist, on the depression of visual function and the loss of RGCs induced by repeated intravitreal injections of ET-1in rabbits.Methods:1 Animal and groups: Twenty-eight big healthy New Zealand White rabbits (male/female) were randomly divided into four groups comprising fourteen eyes in each. In Group A1, ET-1 was delivered without any treatment. In Group B1,topical BMD therapy was applied together with ET-1 delivery. Eyes in Groups A2 andB2 were the fellow eyes of rabbits in Group A1 and B1, respectively, and taken as control groups.2 Methods: ET-1(10?6 M, 20μl) was injected into the posterior vitreous of the right eye of rabbits(GroupA1,GroupB1) through the pars plana via a 30-gauge needle Micro-syringe (under local anesthesia induced using 0.5% proparacaine hydrochloride) . The vehicle for ET-1 was injected into the left eye on the same schedule (GroupA2,GroupB2) . The injections were given twice a week for four weeks. Eyes in Groups B1,B2 were given topical 0.2% BMD therapy, twice a day till the time for taking specimens.3 Measurement of Intraocular pressure (IOP): The IOP of rabbits were measured by schiotz tonometer at nine AM of the day before the first injection,7day,14day,28day,42day after the first injection.4 Measurement of F-VEP: Flash visual evoked potentials were recorded at 1day before the first injection,14day,28day,42day after the first injection. Let the implicit time of wave P1 (ms) , which were automatically calculated by the computer, be our observation.5 Histopathology: 28days,42days after the first ET-1 injection, seven eyes in each group were enucleated and placed in FAA stationary liquid. The eyes were made into paraffin sections for hematoxylin and eosin (HE) stain and TUNEL stain.The numbers of TUNEL-positive cells in the RGC layer were counted in five high power field (HP) of three sections.The mean cell counts of these sections were then used to determine the propotion of cells undergoing apoptosis of each eye.6 Transmission electron microscope (TEM) observation: 28 days,42days after the first ET-1 injection, the two optic nerve in each group were cut and made into ultrathin sections for TEM observation.Results:1 IOP: There was no distinguishable differences in this study about the IOP in the four groups before experiment (P>0.05) .The IOP of groupsB1,B2 turned down from the seventh day ,the minimum value was observed at 42nd day(15.93±1.10mmHg, 15.54±1.01mmHg). A two-way interation analysis revealed significant differences between group A1 and group B1, (P<0.01). However, the IOP between group B1 and group B2 and between group A1 and group A2 were not different from each other (P>0.05) .2 The latency time of F-VEP: There was no distingu- ishable differences in this study about the LP1in the four groups(P>0.05).In group A1, the LP1 started to show prolon- gation 14th day and remained prolonged even after the injections were stopped.The maximum value was observed at 28th day(47.86±3.07ms). In group B1, the LP1 remained relatively stable , and those in groupsA2 and B2 did not change significantly throughout the study period. A two-way interation analysis revealed significant differences between group A1 and group A2 and between group A1 and group B1 (P<0.01). However, the of group B1 and group B2 were not different from each other (P>0.05) .3 Histological analysis:3.1 HE staining: The structure of retina in group A2 and group B2 were clear, well arranged. There was no cellular necrosis and degeneration in the 28th,42nd day. There was less RGCs but more cellular degeneration in group A1. We can see more RGCs in group B1 compared with group A1.3.2 TUNEL staining: We can't find apoptotic cells in RGC of group A2 and group B2. There was significant differernces of the four groups(P<0.05). At 28th day, group A1 was different from other three groups; group B1 was different from other three groups; group A2 was not different from group B2. At 42nd day, group A1 was different from other three groups. There were no differences among the other three groups.4 TEM observation: The ultra structure of optic nerve in group A2 and group B2 showed normal structure of axons with tight and regular myelin sheath uranium, we can see microfilament,mitochondria etal organelles. At 28th day, the structure in group A1 showed axons degeneration and arranging disorderly and less organelles; the structure in group B1 showed apporoximately normal structure of axons with regular myelin sheath uranium. At 42nd day, the structure of group A1 was not changed so much.The same to groupB1.Conclusion:1 Repeated intravitreal injections of ET-1 lead to alterations in flash-visually evoked potentials (F-VEPs) and apoptosis of retinal ganglion cells.2 The intravitreal injections of ET-1 (10?6 M, 20μl) for twice a week for four weeks, make no influnce in IOP .3 Topically applied BMD seems to be neuroprotetive in the ET-1-induced optic nerve ischaemia model, besides can reduce IOP.