Therapeutic Effects Of Mesenchymal Stem Cells Administered At Later Phase Of Recurrent Experimental Autoimmune Uveitis

Objective Uveitis is an inflammatory disease which involves the uvea, retina, retinal vessel and vitreous body. The disease mainly affects middle age and young people with a variety of clinical manifestations and is often recurrent. ICommon therapy includes corticosteroids and immunosuppressants. However, numerous adverse effects associated with long-term usage of corticosteroids and immunosuppressants have pushed the development of new therapies. Previously, we reported that mesenchymal stem cells(MSCs) could prevent a rat model of recurrent experimental autoimmune uveitis(r EAU) when given at the onset of the disease. Although several pilot clinical studies provide a strong impetus for translating MSC therapy to widespread clinical use, the appropriate protocols for MSC administration still lack rigorous standards. Further data are necessary to evaluate and optimize the therapeutic regimens and establish clear guidelines for MSC-based therapies. In this study, we tested the therapeutic effects of delayed treatment of MSCs in uveitis by using animal model of r EAU, to provide additional referential basis for future clinical trials.Methods 1. Culture of MSCs: MSCs were cultured by adherent culture, seperated from Wistar rats’ bone marrow, and amplified in vivo. The phenotype of cultured MSCs was characterized by flow cytometry. 2. The EAU was first induced in Lewis rats by active subcutaneous immunization with an emulsion containing IRBP peptide R16 and Mycobacterium tuberculosis in complete Freund’s adjuvant, was injected subcutaneously into one hind footpad of rats. A single cell suspension was prepared on day 10 after immunization, from lymph nodes and spleens of EAU rats, and irradiated with 30 Gy, to serve as APCs. The T cells then were incubated with APCs for 48 hours with stimulation by R16. For induction of recurrent uveitis, activated T cells were injected intravenously into Lewis rat. 3. Treatment with MSCs :The immunized rats were intravenously treated once daily with 5 × 106 allogeneic MSCs suspended in 1 ml of PBS or with an equalvolume of PBS in control groups for three consecutive days. The treatment started on day 4(onset therapy), day 20(later therapy), or on both day 4 and day 20(double therapy). 4. Clinical assessment of r EAU:Slit-lamp microscopy was used to clinically examine the EAU activity, and the rats were followed up for 40 days after transfer. The incidence and severity of inflammation were scored in a masked manner on a scale of 0 to 4, in accordance with the standard of Caspi. 5. Histological assessment of r EAU: eyes were enucleated on day 40 after transfer, histopathological changes of retina were examined by hematoxylin and eosin staining. The sections were scored on a scale of 0(no disease) to 4(maximum disease). The degree of retinal damage was also assessed by measuring the thickness of both the retina and the outer nuclear layer(ONL). 6. Electroretinogram(ERG) recordings:The retinal function of rats was evaluated by recording dark-adapted ERG at 10, 15, 20, 25, 30, 35, and 40 days after T cell transfer. 7. Optical coherence tomography(OCT) imaging:OCT images were acquired using Heidelberg Spectralis HRA+OCT system on day 40 after transfer. Semi-quantitative scoring of the OCT was evaluated on a scale of 0–4. 8. Immunohistochemistry observation: expression of glial fibrillary acidic protein(GFAP) and rhodopsin(RHO) in the retina was detected by immunohistochemistry on day 40 after transfer.Results 1. MSCs were successfully separated and identified. 2. Rat models of r EAU were successfully induced. 3. Inflammation was observed until 40 days after transfer. Compared to the control group, the incidence and severity of the intraocular inflammation was significantly decreased in both double therapy and onset therapy groups during the entire 40 days’ observation, whereas later therapy significantly alleviated the clinical severity but slightly affected the frequency of relapse. 4. Histological evaluations showed that all the MSC therapeutic regimens significantly ameliorated the histological scores, and improved the thickness of boththe retina and ONL. However, no significant difference was found between the double therapy and onset therapy groups. 5. ERG shows an obvious increase occurred in the 0.01 a/b- and 3.0 a-wave amplitudes of rats treated at later stage in the MSC-treated group compared with those in the control group, an improving tendency can also be found in other components. 6. OCT images in all the three MSC therapy groups demonstrated better outer retinal structure, strikingly reduced highly reflective dots(inflammatory cells) in the vitreous and highly reflective masses(inflammatory cells) in the retina. 7. All MSC therapies, including later therapy, significantly reduced the GFAP expression, early therapy resulted in relatively preserved RHO immunoreactivity in terms of thickness and intensity, whereas later therapy contributed to retain the RHO expression compared with the control group.Conclusion In conclusion, our results showed that although MSC treatment during the early phase of r EAU produced more satisfactory clinical efficacy, later administration during the recurrent phase can also prevent structural and functional damage of the retina. Furthermore, double therapies with longer interval demonstrated additional therapeutic advantage.