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. 2015 Feb 5;56(2):1357-66.
doi: 10.1167/iovs.14-15472.

Intravitreal Delivery of Human NgR-Fc Decoy Protein Regenerates Axons After Optic Nerve Crush and Protects Ganglion Cells in Glaucoma Models

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Free PMC article

Intravitreal Delivery of Human NgR-Fc Decoy Protein Regenerates Axons After Optic Nerve Crush and Protects Ganglion Cells in Glaucoma Models

Xingxing Wang et al. Invest Ophthalmol Vis Sci. .
Free PMC article

Abstract

Purpose: Glaucoma is a major cause of vision loss due to retinal ganglion cell (RGC) degeneration. Therapeutic intervention controls increased IOP, but neuroprotection is unavailable. NogoReceptor1 (NgR1) limits adult central nervous system (CNS) axonal sprouting and regeneration. We examined NgR1 blocking decoy as a potential therapy by defining the pharmacokinetics of intravitreal NgR(310)-Fc, its promotion of RGC axonal regeneration following nerve crush, and its neuroprotective effect in a microbead glaucoma model.

Methods: Human NgR1(310)-Fc was administered intravitreally, and levels were monitored in rat vitreal humor and retina. Axonal regeneration after optic nerve crush was assessed by cholera toxin β anterograde labeling. In a microbead model of glaucoma with increased IOP, the number of surviving and actively transporting RGCs was determined after 4 weeks by retrograde tracing with Fluro-Gold (FG) from the superior colliculus.

Results: After intravitreal bolus administration, the terminal half-life of NgR1(310)-Fc between 1 and 7 days was approximately 24 hours. Injection of 5 μg protein once per week after optic nerve crush injury significantly increased RGCs with regenerating axons. Microbeads delivered to the anterior chamber increased pressure, and caused 15% reduction in FG-labeled RGCs of control rats, with a 40% reduction in large diameter RGCs. Intravitreal treatment with NgR1(310)-Fc did not reduce IOP, but maintained large diameter RGC density at control levels.

Conclusions: Human NgR1(310)-Fc has favorable pharmacokinetics in the vitreal space and rescues large diameter RGC counts from increased IOP. Thus, the NgR1 blocking decoy protein may have efficacy as a disease-modifying therapy for glaucoma.

Keywords: glaucoma; optic neuropathy; regeneration.

Figures

Figure 1
Figure 1
RGC rescue using rat NgR1(310)-Fc after episcleral vein cauterization. (A) Significant RGC loss was observed in all EVC + treatment eyes (IV) compared to non-EVC left eyes. The RGC loss in NgR1(310)-Fc treatment groups (IIIV) is significantly less than control groups (I and II). (B) Large RGC density (per mm2). Significant reductions (P < 0.05) in large RGCs density were observed in EVC eyes versus non-EVC eyes in all groups. The reduction of large RGC in NgR1(310)-Fc treatment groups (IIIV) is significantly (P < 0.05) less than in control groups (I and II). (C) Percentage IRL thickness (% IRL/total retina thickness). Hematoxylin and eosin (H&E) and immunohistology staining demonstrate the significant reduction (P < 0.05) of IRL (RGC and inner plexiform layer [IPL]) thickness in EVC + treatment eyes versus the non-EVC eyes. A significant reduction (P < 0.05) IRL thickness occurs in nontreated EVC eyes (I and II) comparing to NgR1(310)-Fc–treated eyes (IIIV). Data are mean ± SEM.
Figure 2
Figure 2
Pharmacokinetics of intravitreal human NgR1(310)-Fc. Rats received a single dose of 5 μg hNgR1(310)-Fc injected in the vitreal humor in 1 μL of saline. Tissue was collected at the indicated times after injection and hNgR1(310)-Fc protein was measured. Data are mean ± SEM for n = 8 rats per time point. (A) The hNgR1(310)-Fc concentration in the vitreous is plotted as a function of time after injection. (B) Data from (A) for 1 to 7 days after injection are replotted on a log scale and fit to an exponential decay curve. (C) The hNgR1(310)-Fc concentration in the retinal tissue is plotted as a function of time after injection. (D) Data from (C) for 1 to 7 days postinjection are replotted on a log scale and fit to an exponential decay curve.
Figure 3
Figure 3
Intravitreal human NgR1(310)-Fc treatment of ON crush. (A) Experimental timeline of hNgR1(310)-Fc intraocular injection in a rat ON crush model. Rats received bilateral RGCs retrograde labeling with 6% FG at Day 0. At day 5 of FG labeling, the left ON was crushed by using a #5/45 Dumont forceps. Right after the ON crush, 5 μL of either hNgR1(310)-Fc or hIgG-Fc (1 μg/μL) was injected into the anterior chamber of the left eye (n = 10 per group). At 1 week after the first intravitreous treatment, the same dose of either hNgR1(310)-Fc or hIgG-Fc was injected into the anterior chamber of the left eye. At 1 week after the second treatment, both eyes received anterograde ON axonal tracing with Alexa Fluor 555 conjugates of CTB. Rats were killed two to three days after the CTB tracing (15 or 16 days after crush injury). (B) Representative images of FG labeled RGCs from the ON crushed eyes of hNgR1(310)-Fc or hIgG-Fc–treated rats. Scale bar: 100 μm. (C) Quantification of FG-labeled RGCs without injury, or after ON crush from hNgR1(310)-Fc or hIgG-Fc–treated groups. The FG-labeled RGC counts were higher in hNgR1(310)-Fc–treated rats compared to the hIgG-Fc–treated rats, but the difference did not reach statistical significance. Data are mean ± SEM (P = 0.18, Student's 2-tailed t-test). (D) The FG-labeled RGCs after axotomy were measured as in (C), except that only large diameter cells (>120 μm2) were measured. Data are mean ± SEM (**P < 0.01, Student's 2-tailed t-test).
Figure 4
Figure 4
Increased axonal regeneration after intravitreal human NgR1(310)-Fc. (A) Representative images of ON from two hNgR1(310)-Fc– and one hIgG-Fc–treated rats. The CTB-labeled RGC axons are white. The eye is the left and the brain to the right. Proximal to the crush injury labeling is strong and individual fibers are not visualized. These images are projections of confocal Z stacks through the entire ON. Scale bar: 500 μm. (B) The number of regenerating ON fibers is presented as a function of distance central from the crush site. The number of regenerating axon increased in the NgR-Fc–treated group from 100 to 1000 μm distal to the crush site compared to the hIgG-Fc group. Data are mean ± SEM, n = 9 for the NgR-Fc group and n = 8 for the IgG-Fc group. P < 0.05, by repeated measures ANOVA.
Figure 5
Figure 5
Microbead model of glaucoma and human NgR1(310)-Fc administration. (A) Experimental timeline of hNgR1(310)-Fc intraocular injection in a rat microbeads occlusion glaucoma model. Rats received microbeads injection into the anterior chamber. At day 5 of the injection, eyes with IOP ≥ 15 mm Hg were included in the experiment and divided randomly into two groups (n = 12 per group). Then, 5 μL of either hNgR1(310)-Fc or hIgG-Fc (1 μg/μL) were injected into the anterior chamber. The IOPs were measured once a week afterwards. At day 26 of intraocular treatment, RGCs were retrogradely labeled with 6% FG. Rats were killed 7 days after FG tracing. (B) The hNgR1(310)-Fc treatment does not affect the IOP in the rat microbeads induced ocular hypertension model. The IOPs were measured by using the tonometer before the microbeads injection (day 0), at day 5 (right before the intraocular treatment), and every 7 days after treatment. The IOP is plotted as a function of time. There is no significant difference in IOP between the hNgR1(310)-Fc–treated group and the hIgG-Fc–treated group. Data are mean ± SEM. P > 0.05, by repeated measures ANOVA.
Figure 6
Figure 6
The RGC preservation by intravitreal human NgR1(310)-Fc in elevated IOP model. (A) Representative images of FG-labeled RGC in retina from hNgR1(310)-Fc–treated and hIgG-Fc-treated animal after microbead IOP elevation. Scale bar: 100 μm. (B) Quantification of FG-labeled RGCs in the microbead occlusion rats after hNgR1(310)-Fc or hIgG-Fc treatment, or rats without microbeads injection. There is significant increase of FG-labeled RGCs in the NgR-Fc–treated group compared to the hIgG-Fc–treated group. The FG-labeled RGCs counts from the NgR-Fc–treated group was similar to naïve rats (no beads). Data are mean ± SEM. *P < 0.05, by 1-way ANOVA, Dunnett's multiple comparisons tests. (C) The large (> 120 μm2) FG-labeled RGC counts from the hNgR1(310)-Fc–treated groups were significantly higher than the hIgG-Fc–treated rats, but were similar to the naïve rats (no beads). Data are mean ± SEM. *P < 0.05 and **P < 0.001, by 1-way ANOVA, Dunnett's multiple comparisons tests.

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