Neuroprotective effects of intravitreal mesenchymal stem cell transplantation in experimental glaucoma

Abstract

Purpose. Retrograde neurotrophic factor transport blockade has been implicated in the pathophysiology of glaucoma. Stem cell transplantation appears to ameliorate some neurodegenerative conditions in the brain and spinal cord, in part by neurotrophic factor secretion. The present study was conducted to determine whether local or systemic bone marrow-derived mesenchymal stem cell (MSC) transplantation can confer neuroprotection in a rat model of laser-induced ocular hypertensive glaucoma. Methods. MSCs were isolated from the bone marrow of adult wild-type and transgenic rats that ubiquitously express green fluorescent protein. MSCs were transplanted intravitreally 1 week before, or intravenously on the day of, ocular hypertension induction by laser photocoagulation of the trabecular meshwork. Ocular MSC localization and integration were determined by immunohistochemistry. Optic nerve damage was quantified by counting axons within optic nerve cross-sections 4 weeks after laser treatment. Results. After intravitreal transplantation, MSCs survived for at least 5 weeks. Cells were found mainly in the vitreous cavity, though a small proportion of discrete cells migrated into the host retina. Intravitreal MSC transplantation resulted in a statistically significant increase in overall RGC axon survival and a significant decrease in the rate of RGC axon loss normalized to cumulative intraocular pressure exposure. After intravenous transplantation, MSCs did not migrate to the injured eye. Intravenous transplantation had no effect on optic nerve damage. Conclusions. Local, but not systemic, transplantation of MSCs was neuroprotective in a rat glaucoma model. Autologous intravitreal transplantation of MSCs should be investigated further as a potential neuroprotective therapy for glaucoma.

Figure 1.

MSC characterization. Cultured MSCs exhibited typical fibroblast morphology under phase contrast (A). MSCs differentiated into osteocytes, as indicated by positive staining for calcium deposits with alizarin red S (B) and adipocytes, as indicated by positive staining for lipid vacuoles with oil red O (C). Immunocytochemistry revealed that undifferentiated MSCs expressed fibronectin (D), vimentin (E), laminin (F), and collagen IV (H). Some MSCs expressed nestin (G). MSCs did not express the monocyte marker CD11b (I). WT (GFP⁻) Lewis MSCs are shown. Scale bars, 100 μm (A–D). DAPI, blue (D–I).

Figure 2.

IOP analysis. Laser photocoagulation of the trabecular meshwork to induce OHT took place on days 1 and 8, and each treatment was followed by an elevation of IOP (A, C, E). Cumulative IOP exposure in untreated control eyes and eyes that received laser-induced OHT was calculated as the integral of IOP over the 4-week experimental period (B, D, F). Error bars represent SEM. *P ≤ 0.05 by unpaired t-test comparing OHT groups at the indicated time point; ***P < 0.001 by unpaired t-test. The number of animals in each group can be found in Table 1.

Figure 3.

MSC localization after intravitreal transplantation. Immunohistochemistry revealed that GFP⁺ mesenchymal stem cells (MSCs, green) survived in the posterior eyecup up to 5 weeks after transplantation. (A, B) Most MSCs were found as a bolus within the vitreous, often adherent to the posterior lens capsule (strongly immunoreactive for laminin; red; A, low magnification; B, high magnification). (C–H) In rare cases, discrete MSCs migrated to the host retina and were almost exclusively localized in the nerve fiber layer or ganglion cell layer. (D, F) Higher magnification of the areas within the yellow squares in (C) and (E), respectively. (G, H) Single orthogonal sections from the maximal projection confocal z-stack depicted in (F). L, lens; V, vitreous; R, retina; RGC, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Yellow arrows: blood vessels. Scale bars: 500 μm (A); 50 μm (B–F).

Figure 4.

Absence of GFP⁺/ED1⁻ MSCs in the eye after intravenous transplantation. Confocal analysis revealed that ED1 expression (macrophage/monocyte marker) colocalized with GFP⁺ cells within the posterior eyecups of animals that received intravenous GFP⁺ MSC transplants, indicating erroneous GFP labeling of infiltrating macrophages or microglial cells rather than engrafted MSCs (A–C). (A, arrows) GFP⁺/ED1⁺ macrophages/microglia with activated morphology. (A, arrowheads) GFP ⁻/ED1⁺ microglia with a resting, ramified morphology. (D, E) Orthogonal sections from the maximal projection confocal z-stack depicted in (A) to (C). Scale bar, 50 μm.

Figure 5.

RGC axon survival after intravitreal MSC transplantation. (A, B) Results of experiments using Lewis MSCs and recipients (dead MSCs, n = 10; live MSCs, n = 10). (C, D) Results of experiments using SD MSCs and recipients (dead MSCs, n = 9; live MSCs, n = 10). (A, C) RGC axon survival was calculated as the percentage of surviving RGC axons in ocular hypertensive optic nerves compared with contralateral control tissue. (B, D) RGC axon loss normalized to cumulative IOP exposure was calculated as the percentage of RGC axon loss divided by the integral IOP exposure experienced by the ocular hypertensive eye over 4 weeks. (E) Representative micrographs of healthy control optic nerves, (F) ocular hypertensive optic nerves from animals that received dead MSC transplants, and (G) ocular hypertensive optic nerves from animals that received live MSC transplants. Error bars represent SEM. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05 (unpaired t-tests). Scale bar, 20 μm.

Figure 6.

RGC axon survival after intravenous MSC transplantation. PBS (n = 8) or SD MSCs (dead MSCs, n = 8; live MSCs, n = 9) were injected intravenously into SD recipients. (A) RGC axon survival was calculated as the percentage of surviving RGC axons in ocular hypertensive optic nerves compared with contralateral control tissue. (B) RGC axon loss normalized to cumulative IOP exposure was calculated as the percentage of RGC axon loss divided by the integral IOP exposure experienced by the ocular hypertensive eye over 4 weeks. Error bars represent SEM. No significant differences between groups were found after ANOVA.