Study Of Mechanism Underlying Bone Marrow Mesenchymal Cells Used In AMD Animal Model

BackgroundAge-related macular degeneration?AMD?is the leading cause of irreversible loss of vision in the elderly worldwide,the World Health Organization study shows that AMD accounts for 8.7%of all blindness.The majority of AMD patients are over 50years old.With the increase of the aging population in China,AMD patients are also increasing year by year.According to pathological and clinical manifestations,AMD can be divided into exudative and atrophic types.In recent years,research on treatment of exudative AMD has made great progress.Anti-vascular endothelial growth factor?VEGF?has been proven to be effective in AMD therapy,which may provide a new way to treat AMD.However,there is no effective treatment for atrophic AMD,so the prevention and therapy of atrophic AMD is the focus of reducing AMD blindness.NaIO₃ can selectively damage retinal pigment epithelium?RPE?leading to secondary photoreceptor cell lesions.These features are similar to human atrophic AMD.The NaIO₃ animal model can be used to simulate the lesion characteristics of human atrophic AMD.The NaIO₃ model have many advantages,including the ability to induce degeneration in animals of different species and/or strains.Therefore,we can adjust the earliest onset and progression of retinal lesions according to the needs of our research.Additionally,the toxins are easier to apply,and the retina of some animals has the potential to regenerate under special conditions,which is beneficial to research on atrophic pathogenesis,drug or stem/precursor cell therapy and regenerative medicine.The remarkable curative efficacy of stem cell transplantation in the treatment of retinal degenerative diseases has been widely confirmed in domestic and foreign research.Previous studies have proved that stem cell transplantation has great therapeutic potential in the treatment of AMD,and is gradually shifting from more than ten years of basic research to phase I/II clinical trials.The results of these clinical trials initially indicate that the effect of stem cell therapy is as expected.The mechanism of stem cell therapy include cell replacement and neurotrophy,but little is known about the action on retinal glial cells.There are three main types of glial cells that maintain homeostasis in the retina:microglia,astrocytes,and Müller cells.Müller cells are the main glia of the neural retina and display intimate contact with other neurons and retinal blood vessels as the only cells across the entire layer of the retina.Due to this arrangement,Müller cells play significant roles in supporting neuronal function in the healthy retina.In pathological conditions,Müller cells in fish and amphibians can dedifferentiate into retinal progenitor cells to play a role in nerve regeneration,which can completely restore retinal function,but the stem cell potential of Müller cells is greatly restricted in mammals.Seeking a safe and efficient way to stimulate the proliferation and dedifferentiation of Müller cells to promote retinal regeneration is a key point to achieveendogenousregenerationofmammalianretina.Exploringthe dedifferentiation and regulation mechanism of Müller cells during retinal degeneration and regeneration can provide a reference for exploring the mechanism of retinal regeneration in mammals.Studies have shown that mammalian retina damage stimulates the characteristics of potential endogenous retinal precursor cells in Müller cells,and Müller cells cantransdifferentiate into neuronal cells and photoreceptor cells.Multiple signaling pathways are involved in drug-induced retinal degeneration in Müller cell activation,such as Jak/Stat,mTor,ERK,Wnt-catenin,and Notch,which up-or down-regulate Müller cell proliferation.Participate in the process of retinal regeneration.Transforming growth factor?₂?TGF?₂?as a highly efficient and versatile signal transduction pathway can regulates cell proliferation,differentiation,growth,migration and apoptosis.Inhibition of TGF?-Smad_2/3/3 in NMDA and MNU-induced retinal degeneration can promote the dedifferentiation of Müller cells into retinal progenitor cells,but there is no research on the role of TGF?-Smad_2/3/3 and Müller cell proliferation and dedifferentiation in the NaIO₃ model.Therefore,this study was to investigate the effect of BMSCs on Müller activation in AMD induced by NaIO₃,and the relationship between TGF?-Smad_2/3/3 and Müller cell proliferation and dedifferentiation.ObjectTo observe themorphological and pathological changes in the retina of the AMD model induced by NaIO₃ and changes in Müller activation during retinal degeneration.The protective effect of BMSCs on the retina and its effect on Müller activation.To investigate the relationship between TGF?-Smad_2/3/3 and Müller cell proliferation and dedifferentiation.Method1.NaIO₃ was injected into the tail vein of rats to induce retinal degeneration.The morphology and pathological changes of retinal tissue were observed by HE staining,in situ terminal transferase labeling,immunohistochemistry and Western blot,changes in Müller cell activation during retinal degeneration progression were studied by using markers of Müller cells and retinal progenitor cells.2.BMSCs were isolated,cultured,identified,and passed to P₃ for subretinal transplantation.SD rats were randomly divided into four groups:NaCl group,NAIO₃group,BMSCs group and PBS group.The BMSCs group injected 5×10 ⁴/4 ul of BMSCs cell suspension into the retina of SD rats;the PBS group only injected 4 ul of PBS into the subretinal.The protective effect of BMSCs on the retina was observed by HE staining.The animal behavioral test was used to observe the effect of BMSCs on the recoveryofvisualfunction.Immunofluorescenceandwesternblot immunoblotting were used to observe the activation of Müller cells.3.The relationship between TGF?-Smad_2/3/3 signaling pathway and Müller cell proliferation and dedifferentiation was detected by immunofluorescence double staining,western blot and RT-qPCR.Result1.The damage of RPE cells appeared after 6 hours of NaIO₃ treatment.The cell damage was more obvious after 1d.In the next 3-7d,the damage of RPE cells was relieved,and the RPE cells gradually lost with the progress of the disease.2.After treatment with NaIO₃ for 3-7 days,Müller cells were activated and proliferated and dedifferentiated into retinal progenitor cells.As the disease progressed,the proliferation and dedifferentiation ability of Müller cells gradually weakened and the activity of retinal progenitor cells was lost.3.Retinal detachment was completely reattach on the 7th day after intraocular transplantation of BMSCs.Frozen sections HE staining showed that BMSCs had obvious protective effect on the retina after intraocular transplantation.This protective effect was reflected in the rescue of RPE morphology and the thickness of the outer nuclear layer of the retina.Morris test showed that the visual function of rats was improved after intraocular transplantation of BMSCs by detecting changes in animal behavior.4.BMSCs were observed for 7d,14d and 28d after intraocular transplantation,which significantly promoted Müller cell proliferation and dedifferentiation into retinal precursor cells compared with other groups.5.The TGF?₂-Smad_2/3/3 signaling pathway was activated in the retina after treatment with NaIO₃.The TGF?₂-Smad_2/3/3 signaling pathway was inhibited in Müller cells after intraocular transplantation in BMSCs.The activation of TGF?₂-Smad_2/3signaling pathway in PBS group was always activated.Conclusion1.NaIO₃ selectively damages RPE cells and subsequently causes apoptosis in photoreceptor cells..2.During the process of retinal degeneration induced by NaIO₃,Müller cells were activated and proliferated and dedifferentiated.3.Intraocular transplantation of BMSCs has a protective effect on the retina and can save vision function.4.Intraocular transplantation of BMSCs can activate Müller cells and promote Müller cell proliferation and dedifferentiation.5.The TGF?₂-Smad_2/3/3 signaling pathway was activated during NaIO₃-induced retinal degeneration.BMSCs can promoted Müller cell proliferation and dedifferentiation by inhibiting TGF?₂-Smad_2/3/3 after intraocular transplantation.