| Retinal degeneration (RD) is a set of inherited eye diseases that results in blindness and ispathologically characterized by retinal photoreceptor cell degeneration and apoptosis. It presents clinically and genetically as a heterogeneous condition [1]. The prevalence of RD is approximately 1:3000 to 1:4000.Rdl mice were used as an animal model for RD. In this model, there is a mutation in the rod photoreceptor-specific cyclic guanidine monophosphate-phosphodiesterase 6?(PDE6?), which causes the accumulation of cyclic guanidine monophosphate, thereby resulting in the degeneration of the rod photoreceptor[2].Microglia, as the only innate immune cells in the retina, acts as active sensors of the microenvironment trough their rapid transformation[3]. Increasing experimental evidence has provided a comprehensive overview of the microglia physiology and pathology in RD[4]. Before RD occurs, microglia cells are ramified and rest in the inner layers of retina.As degeneration continues, the endogenous environment induces the proliferation and migration of microglia, enhanced phagocytosis, and secretion of cytokines, chemokines, and neurotoxins. Then, the activated microglia cells contribute significantly to retinal tissue damage and photoreceptor apoptotic events, which promote retinal degeneration [5, 6].Although not thoroughly understood, a number of therapeutic strategies have been reported for the treatment of RD. The recent progress of basic scientific research regarding stem cell therapy may soon benefit RD patients [7]. Different stem cell types have been used to perform transplantation therapy, including mesenchymal stem cells (MSCs)[8],neural stem cells (NSCs)[9] and embryonic stem cells (ESCs)[10]. In our lab, we have already transfect C17.2 NSCs with green fluorescence protein (GFP) to mark them. So, it is quite easy to track the transplanted cellsin the retina[11].Following transplantation, either systemically or focally administered stem cells exert their beneficial effects, not only through cell replacement effect and the release of trophic factors but also immunomodulation effect in the central nervous system [12]. However, it remains unclear whether stem cell administration is also beneficial in RD therapy.The immunosuppressive properties of stem cells have been demonstrated in a wide range of adaptive and innate immune cells, including T/B lymphocytes and microglia cells in the CNS [12, 13]. The transplantation of stem cells into the subretinal space might facilitate the ability of stem cells to "cross-talk" with resident retinal microglia. During "cross-talk," stem cells use different pathways[14], including cell-to-cell contact (juxtacrine), the production of soluble factors gradients (paracrine), the secretion of factors into the blood (endocrine),and the release of extracellular membrane vesicles (EVs). But though which way may the stem cells modulate the immune microenvironment in RD still remains unknown.Based on the above research status, we hypothesize that in the retinal degeneration,microglia cells were activated by the endogenous environment and then contribute to retinal tissue damage and photoreceptor apoptotic events, which promote retinal degeneration.When neural stem cells were transplanted into the subretinal space when degeneration started, the activation of microglia cells might be inhibited and the degeneration might be delayed.This study consists of three parts:Part One: Study on the character of photoreceptor apoptosis and microglia activation of retinal degeneration in rd1 mice.By TUNEL assay and immunofluorescence staining, we used a rapid retinal degeneration mouse model, rd1 mice, to confirm that the number of TUNEL-positive cells in the ONL began to increase on P8 and peaked on P14 before declining on P16. Similarly,the number of Ibal-positive microglia in the ONL increased on P10 and peaked on P14. The gradual decrease in the INL and increase in the ONL in the number of Ibal-positive microglia cells suggested microglia migration through degeneration process. Additionally,by Western-blot assay we found Fractalkine, expressed by photoreceptors [15], rised to the peak on P16d. Meanwhile, CX3CR1 expressed by microglia, as the sole receptor of Fractalkine, also increased and peaked on P16d. This phenomenon might indicate the possible migration mechanism of microglia.Part Two: Study on the survival of photoreceptor cells and immune modulation effect on retinal microglia activation after NSCs transplantation.To study the immune modulation effect of NSCs in RD, we transplanted GFP-C17.2-NSCs into the subretinal space of rd1 mice. The retinal frozen sections were made. By TUNEL assay, we found that after transplantation of NSCs, the apoptosis of photoreceptors was suppressed and the thickness of the ONL remains higher than that of the control group. Moreover, through immunofluorescence staining method we discovered that the morphology of the Ibal-positive was more ramified following the transplantation of NSCs compared to the massive amoeboid morphology that developed in the PBS group 7 daysafter operation, accompanied by the reduction of the microglia numbers in the retina.Therefore, we demonstrated that photoreceptors could be partially protected from degeneration by inhibiting the activation of microglia following NSC transplantation.Part Three: Study on the immune modulation effects on microglia activation by NSCs in vitro.In order to study the possible cross talk mechanisms between NSCs and microglia cells,we constructed a Transwell system allowing cytokine exchanges for the two cells but preventing cell-to-cell contact[16]. To mimic microglia activation in the degenerative microenvironment,we applied 1?g/ml of Lipopolysaccharide(LPS) to stimulate BV2 cells for 4 hours before co-culturing started, as previously described [17].Throughimmunofluorescence staining of Ki67 and the TUNEL assay, we confirmed that the proliferation of activated BV2 cells was inhibited while apoptosis was promoted by co-culturing with NSCs. Moreover, FACS analysis showed that the proliferation index (PI)of BV2 cells was suppressed by co-culturing with NSCs.Not only did the cell cycle and morphology change, but also inflammation factor appearedtransformation following microglia activation, which was consistent with the research of Yoshida [18]. Though RT-qPCR and ELISA assay, we confirmed that LPS stimuli quickly increased the expression level of TNF-? and IL-1?, while co-culturing with NSCs blocked their increase.Several studies have demonstrated that tissue inhibitor of metalloproteinase 1 (TIMP1)and its target protein metalloproteinase 9 (MMP9) might participate in the "cross-talk"between NSCs and microglia [19, 20]. We confirmed this by using RT-qPCR and ELISA test and demonstrated that increasing TIMP1 in the co-culturing system could significantly decrease MMP9 expression in microglia. This indicates that TIMP1/MMP9 may be a possible "cross-talk" between NSCs and microglia cells.In summary, we draw the following conclusions:1. The number of TUNEL-positive cells in the ONL began to increase on P8 and peaked on P14 before declining on P16. Similarly,the activation of microglia cells occurred with the same trend.2. The damaged photoreceptors secreted Fractalkine to attract microglia cells migrated to ONL of the retina through its sole receptor CX3CR1, which was expressed by microglia cells only.3. We demonstrated that photoreceptors could be partially protected from degeneration by inhibiting the activation of microglia following NSC transplantation.4. The proliferation ability was suppressed and apoptosis of microglia cells was promoted by co-culturing with NSCs. Additionally, the secretion of inflammatory factor by microglia cells, such as TNF-? and IL-1?,was also attenuated.5. TIMP1, the only inhibitor of MMP9 expressed by NSCs, could significantly decrease MMP9 expression in microglia. This indicates that TIMP1/MMP9 may be a possible "cross-talk" mechanism for immunomodulation of NSCs. |
PDF Full Text Request