Role Of Müller Cell-specific Reactive Gliosis In Acute Ocular Hypertension-induced Retinal Damage Of Rats

Glaucoma is a common cause of blindness, which pathological basis is the lossof retinal ganglion cells (RGCs) and nerve fiber. Many factors are involved in theoccurrence of glaucoma. High intraoeular pressure not only can lead to retinalischemia and hypoxia, but also can make RGCs degeneration and cause apoptosis.The apoptosis of RGCs is critical for glaucoma’s pathological damage; thereforestudy of drugs that can protect RGCs from damage has important implications for thetherapy of glaucoma. Müller cells are the principal glial cell in retina, not onlysupport and nourish the neuron cells, but also help in maintaining the balance ofextracellular irons and participate in glutamate cycle, synaptic transmission and so on.Besides, Müller cells are involved in RGCs’ metabolism, homeostasis, thetransportation and intake of the neurotransmitter, so they play important role inRGCs’ growth, injury, repair and regeneration. New evidences indicate that Müllercells participate in many pathological and physiological processes, and Müllercell-specific reactive gliosis is found in virtually every disease of the retina. Theupregulation of glial fibrillary acidic protein (GFAP) is the main characteristic changeof Müller cell-specific reactive gliosis. In order to understand the molecularmechanism underlying acute ocular hypertension (AOH)-induced retinal injuries andprovide an effective therapeutic method to acute angle-closure glaucomatousretinopathy in clinic, the AOH rat model was estabolished to explore the Müllercell-specific reactive gliosis in AOH rat retina and the effect of glial toxinα-aminoadipic acid (AAA) onAOH-induced retinal damage.Experiments were performed at room tempreture (20-22℃) on male SD rats (8-10W,200-250g). All animal procedures were in strict accordance with theAssociation for Research in Vision and Ophthalmology (ARVO) Statement for theuse of animals in Ophthalmic and Vision Research. In addition, the Animal Care andUse Committee of Capital Medical University approved this study. The glial toxin,AAA, which can selectively inhibite Müller cell-specific reactive gliosis, wasdelivered by intravitreal injection. Animals were randomly divided into Control,AOH (14.63kPa,1h) and AOH+AAA treated groups (1,3and5d subgroupsaccording the reperfusion time), and AAA treatment or AOH+PBS control group. TheGFAP immunofluorescent staining was performed to determine Müller cell-specificreactive gliosis; terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) was used to detect cell apoptosis; Thy-1staining was applied tomark retinal ganglion cells (RGCs); and Western blot was used to detect the proteinexpression levels of tumor necrosis factor-α (TNF-α). Statistical analysis wasconducted by One-Way Analysis of Variance followed by all pairwise multiplecomparison procedures using Bonferroni test. Significance was regarded as p<0.05.The results were reported as follows:1. Effects of AOH on rat retinaThe Hochest staining results demonstrated that AOH (14.63kPa,1h) couldattenuate the thicknesses of inner plexiform layer (IPL) and inner nuclear layer (INL)after1-5d reperfusion, and cause cell disorganization and cell loss in ganglion celllayer (GCL, p<0.05, n=4per group). The results of GFAP staining showed that therewere no or less expressions of GFAP in Müller cells of normal Control rat retina; theMüller cells started to express GFAP at1d reperfusion following AOH, while thesignificant increase of GFAP expressions in Müller cells could be observed even inthe whole retinal layers at3-5d reperfusion after AOH treatment (p<0.05, n=4pergroup). These results confirmed that AOH could induce the retinal damage and the Müller cell-specific reactive gliosis, suggesting the successful estabolishment of AOHrat model of acute angle-closure glucoma.2. Effects of AAA on Müller cell-specific reactive gliosis in retina of AOH ratsCompared with AOH groups at different time points of reperfusion, theintravitreal injection of selective inhibitor of Müller cell-specific reactive gliosis,AAA (5μl,50μg/μl) could significantly inhibit GFAP expressions in retinal Müllercells (not astrocytes) from AOH+AAA groups. In addition, the GFAP expressions inretina from AOH+PBS groups were similar with that of AOH groups, which ruled outthe influence of solvent for AAA (p<0.05, n=4per group). These results suggestedthat AAA could selectively inhibit the onset of Müller cell-specific reactive gliosis inretina of AOH rats.3. Effects of AAA on retinal injuries of AOH ratsCompared with Ctrl and AAA groups, AOH caused a significant increase of cellapoptosis (TUNEL positive cell number), but this AOH-induced cell apoptosis couldbe blocked by the AAA treatment in GCL layers of retina from AOH+AAA group(p<0.05, n=4per group). Compared with Ctrl group, AOH could induce significantRGCs (Thy-1positive) cell loss, while AAA attenuated this AOH-induced RGCs celldamage in AOH+AAA group (p<0.05, n=4per group). By using Hochest staining,AAA could relief AOH-induced cell disorganization and layer’s getting thin in retinaof AOH+AAA rats when compared with that of AOH group. These results suggestedthat using the selective inhibitor of Müller cell-specific reactive gliosis, AAA couldprotect rat retina against AOH-induced damage.4. Effects of the inhitibiton of Müller cell-specific reactive gliosis on TNF-αprotein levels in retina of AOH ratsThe results of Western blot showed that the protein expression levels of TNF-αcould be up regulated in retina from AOH group or down regulated in retina from AOH+AAA group when compared with that of Ctrl and AOH groups (p<0.05, n=4per group), respectively. Similarly, the TNF-α expressions in retina from AOH+PBSgroups were similar with that of AOH groups, which ruled out the influence ofsolvent for AAA (p<0.05, n=4per group). These results suggested that AAA inhibitsAOH-induced Müller cell-specific reactive gliosis through down-regulating TNF-αprotein expression levels, and then attenuating its toxicity on RGCs and promotingRGCs survival.In conclusion, our results indicated that Müller cell-specific reactive gliosis wasinvolved in AOH-induced retinal damage, and its inhibition by AAA could decreaseTNF-α protein levels and effectively protect retina against RGCs loss and apoptosisin retina of AOH rats. This achievement may enrich our understanding of themolecular mechanism underlying AOH-induced retinal injuries and provide aneffective therapeutic method to acute angle-closure glaucomatous retinopathy inclinic.