Transplantation of BDNF-secreting mesenchymal stem cells provides neuroprotection in chronically hypertensive rat eyes

Abstract

Purpose: To evaluate the ability of mesenchymal stem cells (MSCs) engineered to produce and secrete brain-derived neurotrophic factor (BDNF) to protect retinal function and structure after intravitreal transplantation in a rat model of chronic ocular hypertension (COH).

Methods: COH was induced by laser cauterization of trabecular meshwork and episcleral veins in rat eyes. COH eyes received an intravitreal transplant of MSCs engineered to express BDNF and green fluorescent protein (BDNF-MSCs) or just GFP (GFP-MSCs). Computerized pupillometry and electroretinography (ERG) were performed to assess optic nerve and retinal function. Quantification of optic nerve damage was performed by counting retinal ganglion cells (RGCs) and evaluating optic nerve cross-sections.

Results: After transplantation into COH eyes, BDNF-MSCs preserved significantly more retina and optic nerve function than GFP-MSC-treated eyes when pupil light reflex (PLR) and ERG function were evaluated. PLR analysis showed significantly better function (P = 0.03) in BDNF-MSC-treated eyes (operated/control ratio = 63.00% ± 11.39%) than GFP-MSC-treated eyes (operated/control ratio = 31.81% ± 9.63%) at 42 days after surgery. The BDNF-MSC-transplanted eyes also displayed a greater level of RGC preservation than eyes that received the GFP-MSCs only (RGC cell counts: BDNF-MSC-treated COH eyes, 112.2 ± 19.39 cells/section; GFP-MSC-treated COH eyes, 52.21 ± 11.54 cells/section; P = 0.01).

Conclusions: The authors have demonstrated that lentiviral-transduced BDNF-producing MSCs can survive in eyes with chronic hypertension and can provide retina and optic nerve functional and structural protection. Transplantation of BDNF-producing stem cells may be a viable treatment strategy for glaucoma.

Figure 1.

MSCs engineered with lentiviral vectors express GFP (green) and BDNF (red). MSCs transduced with lentiviral BDNF vectors (A–C) showed increased expression of BDNF (A, arrows). Merged image (C) demonstrates perinuclear localization of BDNF immunoreactivity in BDNF-MSCs (arrows). Control MSCs transduced with lentiviral GFP vectors (D–F) show diffuse BDNF immunoreactivity throughout the cell (D, arrows). Robust GFP expression is apparent using conventional fluorescence microscopy (E). Merged image (F) demonstrates the BDNF localization in GFP-MSCs.

Figure 2.

MSCs secrete bioactive BDNF. ELISA analysis revealed BDNF-MSCs secrete 41.40 ± 1.32 ng BDNF/10⁶ cells/day, which was significantly higher than control GFP-MSCs secreting 0.97 ± 0.52 ng BDNF/10⁶ cells/day (A). Media conditioned by BDNF-MSCs or GFP-MSCs were collected and applied to E17 rat dorsal root ganglia explant cultures to assay and quantitate BDNF bioactivity (B). DRGs were treated with MSC growth media (C), media conditioned by GFP-MSCs (D) or BDNF-MSCs (E), or MSC growth media supplemented with 50 ng rhBDNF (F). Montage images of DRGs were collected and subsequently scored by naive observers (B). No significant difference was observed in the modest outgrowth of neurites induced by MSC media only (C) or GFP-MSC–conditioned media (B, D; P > 0.05). BDNF-MSC conditioned media induced robust outgrowth of neurites from DRGs (E) and did not differ significantly from DRG outgrowth induced by rhBDNF-supplemented media (B, F; P > 0.05). Each group of DRGs exposed to BDNF, however, had significantly more neurite outgrowth than each of the control groups (P < 0.05). Scale bar, 400 μm (C–F). GFP-MSC CM, GFP-MSC conditioned media; BDNF-MSC CM, BDNF-MSC conditioned media; rhBDNF, recombinant human brain-derived neurotrophic factor. *P < 0.05.

Figure 3.

Laser cauterization of trabecular meshwork and episcleral veins resulted in the development of COH. (A) Laser cauterization resulted in the significant IOP increase in operated eyes at 10 and 25 days after surgery (*P < 0.05). BDNF-MSCs, hypertensive eyes that received transplants of BDNF-producing MSCs; BDNF-Fellow, opposite (control eye) that did not receive laser surgery or an MSC transplant; GFP-MSCs, hypertensive eyes that received transplants of GFP-expressing MSCs not modified to produce BDNF; GFP-Fellow, opposite (control eye) that did not undergo laser surgery or MSC transplantation. (B) Statistical analysis revealed no difference in the ΔIOP (IOP operated eye − IOP control eye) between BDNF-MSC and GFP-MSC groups at different time points during the experiment (mean ± SEM). (C) Analysis of the IOP integral revealed no significant difference in the ΔIOP integral between BDNF-MSC– and GFP-MSC–treated groups.

Figure 4.

Transplantation of BDNF-MSCs preserved retinal function in COH eyes. Retinal electrical activity was significantly higher in BDNF-MSC–treated eyes than in GFP-MSC–treated eyes at 20 (A) and 40 (B) days after transplantation for the rod b-wave, cone b-wave, maximum combined b-wave (Max b-wave), and flicker responses. Oscillatory potentials (OPs) were not significantly different between BDNF-MSC– and GFP-MSC–treated animals. *P < 0.05.

Figure 5.

Computerized pupillometry analysis of the pupil light response at 42 days after transplantation showed significantly better pupil constriction in BDNF-MSC–treated eyes than in GFP-MSC–treated eyes (*P < 0.05). The pupil light reflex is presented as a ratio of constriction amplitudes measured from the control (nonoperated) eye after illumination of the operated and control eyes.

Figure 6.

MSCs survive after transplantation into hypertensive eyes. Lentiviral-transduced MSCs were detected in the retina using an anti-GFP antibody at the end of the experiment (A, D). MSCs (BDNF-MSCs, A–C; GFP-MSCs, D–F) were predominantly detected in the retinal ganglion cell layer (B, C) and the inner retinal layers at (E, F). Autofluorescence was detected in the outer retina associated with photoreceptor outer segments (A–C). DIC, differential interference contrast.

Figure 7.

BDNF-MSC–transplanted cells served to protect Brn3a-IR RGCs in COH eyes. BDNF-MSC–treated COH eyes had significantly fewer Brn3a-IR cells per section than untreated fellow eyes (A–D; P < 0.05). GFP-MSC–treated COH eyes also had significantly fewer Brn3a-IR cells per section than untreated fellow eyes (E, F; P < 0.01). However, quantitative analysis of BDNF-MSC– and GFP-MSC–treated eyes revealed significantly more Brn3a-IR cells per section in BDNF-MSC–treated eyes than in GFP-MSC–treated COH eyes (G; *P < 0.05).

Figure 8.

Effect of BDNF-MSC transplants on optic nerve integrity in COH eyes. Optic nerves were assigned to 1 of 5 grades of damage, with 1 representing healthy optic nerves and 5 representing severely damaged optic nerves. No significant difference in optic nerve grades was observed between the optic nerves of COH eyes treated with BDNF-MSCs (A) and the fellow optic nerves (B). A significant difference was observed between optic nerves of COH eyes treated with GFP-MSCs (C) and control optic nerves of fellow, untreated eyes (D, P < 0.001). However, analysis of optic nerve scores between BDNF-MSC– and GFP-MSC–treated eyes did not reveal a statistically significant difference (E).