Rat Retinal INL Degeneration And Regeneration Induced By Ouabain And Regulation By Gelatinases

Retina is an organ forming the vision. Horizontal cells (HCs) are, together with amacrine cells, retinal interneurons that lie within the inner nuclear layer (INL).They play a role in color vision formation. Vertebrate retinal HCs have recently been shown to exhibit a variety of unique biological properties, as compared with other nerve cells, that challenge many long-standing assumptions in the fields of neural development and cancer biology. These features include their unusual migratory behavior, their unique morphological plasticity, and their propensity to divide at a relatively late stage during development. At adulthood, fully differentiated mouse HCs have the ability to re-enter cell cycle and to give rise to metastatic retinoblastoma. In addition, the latest study showed intrinsic light response of retinal HCs of teleosts. There results suggest their significance. However, few papers have reported on HCs damage and regeneration since less attention is paid to HCs. In our study, we establish the animal model of INL degeneration, in SD rats and observe stem cell potential of Muller cell,including horizontal cells regeneration, which may broaden our understanding of HCs.; explore the roles of gelatinases.The methods are as following:1 Establishment of INL, especially HCs degeneration animal model.①INL degeneration was induced by intravitreal injection of the different doses of ouabain into Sprague-Dawley rats.②The degree of INL damage and cell death pathway were investigated by HE staining, TUNEL and transmission electron microscopy.③The types of ouabain-injured retinal cells were identified by immunoflurorescent staining of specific antigens of retinal cells and synapses and western-blot semi-quantitive analysis of calbindin D-28k protein.2 INL degeneration induced stem cell potential of Muller cell.①Muller cell activation was detected by immunostaining, single and double, of GS, GFAP and Nestin. The change of GS and GFAP protein was assessed by western-blot.②the mitosis in INL was observed by transmission electron microscopy.③Double staining of GS/brdu and GS/PCNA was applied to analyze the number of proliferating Mullercells in ouabain-induced retinal degeneration.④Double staining of brdu/prox1 and brdu/calbindin D-28k were used to detect new HCs and ACs regeneration in adult rats. The effect of SHH-N on retinal regeneration was assessed by the same indicator.3 Endogenous MMPs, especially MMP9 functions in neuronal death and Muller cell proliferation.①The expression and activity of MMP9 in ouabain-treated retina. Gelatin zymography and in situ zymography were used to detect MMP2/9 activity; western-blot assays and immunohistochemistry were used to analyse MMP9 expression.②The effect of MMP9 inhibitor on endogenous MMP9 activity was assessed by gelatin zymography and in situ zymography.③The influence of MMP9 inhibitor on ouabain-induced retinal damage. The indicators were INL thickness, the number of calbindin D-28k and syntaxin immnuopositive cells, respectively, and the change of calbindin D-28k protein by western-blot.④The effect of MMP9 inhibitor on INL apoptosis was determined by the number of apoptosis-like nuclear by DAPI staining and the change of caspase 3 protein by western-blot.⑤The effect of MMP9 inhibitor on Muller cell proliferation. Double immunostaining of GS/GFAP, GS/brdu and GS/PCNA were used to detect Muller cell activation and proliferation. GFAP protein was analyzed by western-blot.Results and conclusions:1 Rat HCs and ACs were sensitive to ouabain.At 3 dpi (day post injection), ouabain induced a rapid and obvious cell loss in the INL at all doses≥2.75 nmol, with little effect on GCL and ONL. The effect was dose- and time-dependent; at 22nmol dose and 2dpi, the damage was the severest. TUNEL staining showed there were many apoptotic nuclear in INL; at 12hpi, the number of TUNEL positive cells reached a peak. Immunostaining showed calbindin D-28k and syntaxin positive cells were lost, with horizontal cells the most susceptible.2 HCs and ACs were regenerated from adult mammalian retina.After ouabain treatment, actived Muller cells dedifferentiated into stem cells via downregulation of GS expression (mature Mullercells marker) and upregulation of Nestin expression (neuronal stem cell marker), and proliferated through brdu incorporation and upregulation of PCNA, with 2dpi the most pronounced. At 15dpi,21dpi and 35dpi(single ouabain injection), a portion of brdu positive cells expressed prox1 and calbindin D-28k, respectively (marker for all HCs and a fraction of amacrine cell), and these double immunopositive cells were located in horizontal cell and amacrine cell layer. These suggest HCs and amacrine cells regeneration from adult mammalian retina. SHH-N did not promote the two types of cell regeneration.3 MMP9 protected retinal interneurons from ouabain-induced non-apoptotic cell death and enhanced Muller cell proliferation.Ouabain induced acute MMP9 expression and resulted in an increase in MMP9 activity. At the highest dose (22nmol) and 2dpi, MMP9 active was the strongest. Muller cells and neurons in INL and cells in GCL upregulated MMP9 protein and activity. While, there was MMP2 activity in retinas at 7-21 days after ouabain treatment. At 2dpi, MMP9 inhibitor blocked most of endogenous MMP9 activity. The effect of MMP9 inhibitor on ouabain-induced retinal damage was to accelerate the change through the decrease in INL thickness and the number of calbindin D-28k and syntaxin immunopositive cells in INL. However, it resulted in the decrease in the number of apoptotic-like nuclear in INL and caspase 3 protein. Therefore, endogenous MMP9 may protect retinal interneurons from ouabain-induced non-apoptotic cell death. With respect to Muller cell reaction, the inhibitor inhibited Muller cell activation through downregulation of GFAP in NF layer and proliferation via the decrease of brdu incorporation and PCNA expression in Muller cell.