Neuroprotective Effect Of Mesenchymal Stem Cells Against Light-induced Retinal Injury And Its Mechanism

Conventional concept that mesenchymal stem cells (MSCs) are adult stem cells widelyexisted in mesenchymal tissues and capable of multilineage differentiation has altered toMSCs are heterogenous cell source that involve in various tissue injury. MSCs may promoterepair process during tissue injury through paracrine of cytokines or modulation of tissuemicroinvironment. However, the exact machnism how MSCs work during tissue injury is stillelusive. In this study, MSCs were stimulated by light-induced retinal injury to inducecytokine expression change and the role of cytokine expression change in retinal protectiveeffect was evaluated in vitro. Consequently, MSCs were systemically administered in light-induced retinal injury rat model to investigate their retinal protective effect andtransplantation-induced host reaction.Chapter1Rat model for light-induced retinal injuryObjective: To investigate structural change, and consequently endogenous cytokineexpression change in the retinas of rats after intense light exposure.Methods: Sprague-Dawley rats (aged6weeks to8weeks) were exposed to cyclic white light(5klux,12-hour on-off) with pupil dilation. The rats were sacrificed1d,2d,5d,7d,14d afterlight exposure. Ultrastructure change after early light exposure was studied usingtransmission electronic microscopy. Histological change in the retinas was observed throughhematoxin-eosin (HE) staining. Terminal deoxynucleotidyl transferase-mediated nick endlabeling (TUNEL) was conducted to identify apoptosis in retinal cells after light exposure.Retinal vascular permeability change was evaluated by in perfusion. The expression ofcytokines bFGF, BDNF and CNTF was determined in the retinas after light exposure byimmunofluorescence.Results: Ultrastructural change after early light exposure was induced as the destruction ofouter segments of the retinas, swelled mitochondrial membrane and increased electrondensity in the nuclear. Progressive reduction of the outer nuclear layer (ONL) thickness wasdetected. Light exposure also induced apoptosis in retinal cells. Cells death mainly occurred in the ONL and retinal pigment epithelium (RPE) layer. Permeability of retinal vesselsincreased after light injury, revealed by dot-like ink exudation2days after light exposure andpatchy exudation after14days exposure. Light injury up-regulated the expression of bFGF,BDNF, CNTF and GFAP. In addition, timed and continuous up-regulation of CNTF andGFAP was identified during the exposure.Conclusions: Intense light exposure is able to induce retinal injury, accompanied bycytokines up-regulation and glial reaction.Chapter2In vitro study on MSCs in response to light-induced retinal injury and retinalprotective effect of MSCs after injury stimulationPurpose: To investigate neurotrophin expression change in MSCs during light-inducedretinal injury and the role of neurotrophin expression change in protection against retinaldegeneration.Methods: MSCs were cultured and underwent flowcytometry analysis for surface markersand induction for osteogenic/adipogenic differentiation. Neurotrophins (bFGF, BDNF andCNTF) expression in MSCs was detected by western-blotting after stimulation bysupernatants of homogenized retina (SHR) from normal and light-injured rats for2days and5days. Control MSCs (CM-MSCs), MSCs stimulated by normal SHR (CM-NSHR), andMSCs stimulated by light-injured SHR (CM-ISHR) were examined regarding their ability ofpreventing degeneration of retinal explants in conditioned media by RT-qPCR analysis forbcl-2/bax expression in retinal explant and LDH assay in the media. Basic fibroblast growthfactor in MSCs was knockdown by lentivirus-mediated mRNA interference. TransfectedMSCs were stimulated by SHR, and retinal preservation was reevaluated in the resultantconditioned media.Results: The expression of bFGF, BDNF and CNTF was identified in normal MSCs.Significant up-regulation of bFGF and CNTF was detected in MSCs stimulated by light-injured SHR, demonstrated by superior retinal neurotrophic effects compared to thoseobserved in normal SHR-stimulated and control MSCs. In the light-injured SHR group,significant up-regulation of bcl-2(P＜0.05) and inhibition of bax (P＜0.05) was detected incomparison with other groups. LDH release from retinal explants was also significantlyinhibited in the light-injured SHR group (P＜0.05), suggesting improved membrane integrity.Down-regulation of MSCs bFGF effectively inhibited this protective effect.Conclusions: Retinal injury may enhance neurotrophin expression in MSCs and promote the repair process. Basic fibroblast growth factor may play a critical role in the MSCs response toretinal injury.Chapter3Therapeutic effect of MSCs against light-induced retinal injury aftersystemic administrationObjective: To investigate retinal distribution of MSCs, MSCs-induced host reaction andinhibition of apoptosis in light-induced retinal injury after systemic administration.Methods: Retinal injury was induced by intense light exposure. MSCs were intravenouslyinjected at a dose of5×106cells/ml48hours later. Light-injured rats without treatment andlight-injured rats received only DMEM injection served as controls. TUNEL analysis wasconducted14days after light exposure for comparison of retinal injury among groups. TheONL thickness was also compared among groups14days later. The expression of CNTF andGFAP in the retinas of different groups was determined by Western-blotting at1d,2d,5d,7d,14d respectively. Double-staining was conducted for locating CNTF and GFAP in the retinas.GFP-labeled MSCs was also intravenously injected for retinal distribution analysis.Results: MSCs were able to migrate out of retinal vessels after administration and distributedmainly in the ONL and choroid vessels. Delayed reduction of the ONL thickness wasdetected in MSCs-injected group (P＜0.05). Systemic administration of MSCs alsosignificantly inhibited apoptosis in retinal cells (P＜0.05). MSCs injection induced significantup-regulation of CNTF and GFAP during the exposure (P＜0.05). Immunofluorescencerevealed colocalization of CNTF and GFAP.Conclusions: Systemic administration of MSCs exerts protective effect against light-inducedretinal injury. MSCs transplantation-induced glial reaction plays a potential role in retinalprotection.