Sorbonne Université UM80, INSERM UMR 968, CNRS UMR 7210, Institut de la Vision, IHU FOReSIGHT, 17 rue Moreau, 75012, Paris, France.
Service 3, CHNO des Quinze-Vingts, INSERM-DGOS CIC 1423, 28 rue de Charenton, 75012, Paris, France.
J Neuroinflammation. 2022 Mar 2;19(1):63. doi: 10.1186/s12974-022-02418-w.
Glaucoma is a blinding degenerative neuropathy in which the death of retinal ganglion cells (RGCs) causes progressive loss of visual field and eventually vision. Neuroinflammation appears to be a key event in the progression and spread of this disease. Thus, microglial immunomodulation represents a promising therapeutic approach in which mesenchymal stem cells (MSCs) might play a crucial role. Their neuroprotective and regenerative potentials have already raised hope in animal models. Yet no definitive treatment has been developed, and some safety concerns have been reported in human trials. In the present study, we investigated the neuroprotective and immunomodulatory properties as well as the safety of MSCs in an ex vivo neuroretina explant model.
Labeled rat bone marrow MSCs were placed in coculture with rat retinal explants after optic nerve axotomy. We analyzed the neuroprotective effect of MSCs on RGC survival by immunofluorescence using RBPMS, Brn3a, and NeuN markers. Gliosis and retinal microglial activation were measured by using GFAP, CD68, and ITGAM mRNA quantification and GFAP, CD68, and Iba1 immunofluorescence stainings. We also analyzed the mRNA expression of both 'M1' or classically activated state inflammatory cytokines (TNFα, IL1β, and IL6), and 'M2' or alternatively activated state microglial markers (Arginase 1, IL10, CD163, and TNFAIP6).
The number of RGCs was significantly higher in retinal explants cultured with MSCs compared to the control group at Day 7 following the optic nerve axotomy. Retinal explants cultured with MSCs showed a decrease in mRNA markers of gliosis and microglial activations, and immunostainings revealed that GFAP, Iba1, and CD68 were limited to the inner layers of the retina compared to controls in which microglial activation was observed throughout the retina. In addition, MSCs inhibited the M1 phenotype of the microglia. However, edema of the explants was observed in presence of MSCs, with an increase in fibronectin labeling at the surface of the explant corresponding to an epiretinal membrane-like phenotype.
Using an ex vivo neuroretina model, we demonstrated a neuroprotective and immunomodulatory effect of MSCs on RGCs. Unfortunately, the presence of MSCs also led to explant edema and epiretinal membrane formation, as described in human trials. Using the MSC secretome might offer the beneficial effects of MSCs without their potential adverse effects, through paracrine signaling.
青光眼是一种致盲性退行性神经病变，视网膜神经节细胞 (RGC) 的死亡导致视野进行性丧失，最终导致视力丧失。神经炎症似乎是疾病进展和扩散的关键事件。因此，小胶质细胞免疫调节代表了一种有前途的治疗方法，间充质干细胞 (MSC) 可能在此过程中发挥关键作用。它们的神经保护和再生潜力已在动物模型中带来希望。然而，尚未开发出明确的治疗方法，并且在人类试验中报告了一些安全问题。在本研究中，我们使用体外神经视网膜外植体模型研究了 MSC 的神经保护和免疫调节特性以及安全性。
在视神经切断后，将标记的大鼠骨髓 MSC 与大鼠视网膜外植体共培养。我们通过使用 RBPMS、Brn3a 和 NeuN 标志物进行免疫荧光分析，来评估 MSC 对 RGC 存活的神经保护作用。通过 GFAP、CD68 和 ITGAM mRNA 定量和 GFAP、CD68 和 Iba1 免疫荧光染色来测量神经胶质增生和视网膜小胶质细胞激活。我们还分析了两种“M1”或经典激活状态炎症细胞因子 (TNFα、IL1β 和 IL6) 和“M2”或替代激活状态小胶质细胞标志物 (精氨酸酶 1、IL10、CD163 和 TNFAIP6) 的 mRNA 表达。
与视神经切断后第 7 天的对照组相比，培养有 MSC 的视网膜外植体中的 RGC 数量明显更高。与对照组相比，培养有 MSC 的视网膜外植体中神经胶质增生和小胶质细胞激活的 mRNA 标志物减少，免疫染色显示 GFAP、Iba1 和 CD68 仅局限于视网膜内层，而对照组中观察到整个视网膜的小胶质细胞激活。此外，MSC 抑制了小胶质细胞的 M1 表型。然而，在外植体中存在 MSC 时观察到外植体水肿，外植体表面的纤维连接蛋白标记增加，类似于人类试验中描述的视网膜前膜样表型。
使用体外神经视网膜模型，我们证明了 MSC 对 RGC 具有神经保护和免疫调节作用。不幸的是，MSC 的存在也导致了外植体水肿和视网膜前膜形成，正如人类试验中所描述的那样。通过旁分泌信号，使用 MSC 分泌组可能会带来 MSC 的有益作用而没有其潜在的不良影响。
