| ObjectiveA variety of retinal degenerative diseases are characterized by loss of retinal cells, and there still is no effective treatment available in clinical practice. Mesenchymal stem cells (MSCs) continue to show their unique advantages in the research of neuroprotection and repairment for retinal damage. To explore new ways for future clinical application of MSCs for the treatment of retinal degenerative diseases and provide theoretical reference, in this study we observed the morphological changes of the injured retina and retinal cell apoptosis after MSCs treatment with laser-induced retinal injury model. The associated molecular mechanisms were also investigated.Methods1. Cultivation of MSCs:Green fluorescent protein (GFP) labeled MSCs from C57BL/6mice were cultured and passaged in vitro, and the GFP expression were observed by seeding cells on the cover slips.2. Establishing laser-induced retinal injury model:135C57BL/6mice were divided into three groups including normal control group (n=15), injured control group (n=60) and MSCs treatment group (n=60). Laser retinal injuries were induced by laser photocoagulation.3. MSCs treatment:One day after photocoagulation,0.2ml cell suspension, which contained1×106GFP-MSCs, were injected into the mice in treatment group via tail vein, and the mice in injured control group were given equal volume of PBS.4. Histopathological changes:Animals were executed on3,7,14and21days following laser damage. Paraffin sections and hematoxylin-eosin staining were performed to assess the changes of injured retinas. The diameters of laser spots and areas with total loss of cells in outer nuclear layer (ONL) were analysed by image processing software.5. Retinal cell apoptosis:Animals were executed at each time points to perform frozen sections. The apoptosis of retinal cells was examined by TUNEL staining.6. MSCs migration:The migration of GFP-MSCs into the retina was observed by fluorescence microscope. 7. GFAP and MMP-2gene expression:Animals were executed at each time points. The total RNA was extracted from the eyes of each mouse. cDNA was synthesized by reverse transcription, and real-time PCR were performed to detect the mRNA expression of glial fibrillary acidic protein (GFAP) and matrix metalloproteinase-2(MMP-2).Results1. GFP-MSCs were successfully cultivated, and there was a high ratio of GFP expression.2. Laser-induced retinal injury model was well established.3. HE staining shows that the retinal structures were integrated in normal control group. Retinal damages were milder in MSCs treatment group than in injured control group. Though the average diameter of area with total loss of cells in ONL of MSCs treatment group was less than that of the injured control group (t=5.769, P<0.05), the diameters of laser spots show no difference (t=0.964, P>0.05) on day3. Both the average diameter of laser spots (t=5.180,5.417,2.381) and area with total loss of cells in ONL (t=3.530,3.224,3.162) were less in the MSCs treatment group on day7,14and21(P<0.05).4. TUNEL staining shows that the apoptosis were decreased after MSCs transplantation on day3,7,14and21(t=11.142,7.479,6.678,3.953, P<0.05). No apoptosis was observed in normal control group.5. Very few GFP-MSCs were observed in the retina at all time points. They were only seen in the subretinal and choroidal neovascularization occasionally on day7and14.6. Real-time PCR results showed that GFAP mRNA levels in MSCs treatment group were lower than the levels in the injured control group14and21days after laser photocoagulation (t=2.481, t=2.562, P<0.05). MMP-2mRNA expression were reduced on day7,14and21in the MSCs treatment group (t=2.466, t=4.687, t=4.001P<0.05).ConclusionIntravenous injection of MSCs can inhibit apoptosis of retinal cells after laser injury in mice, reduce the inflammatory response, improve retinal tissue structure and limit the spreading of the damage. |
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