Intraocular Delivery of a Collagen Mimetic Peptide Repairs Retinal Ganglion Cell Axons in Chronic and Acute Injury Models

Abstract

Vision loss through the degeneration of retinal ganglion cell (RGC) axons occurs in both chronic and acute conditions that target the optic nerve. These include glaucoma, in which sensitivity to intraocular pressure (IOP) causes early RGC axonal dysfunction, and optic nerve trauma, which causes rapid axon degeneration from the site of injury. In each case, degeneration is irreversible, necessitating new therapeutics that protect, repair, and regenerate RGC axons. Recently, we demonstrated the reparative capacity of using collagen mimetic peptides (CMPs) to heal fragmented collagen in the neuronal extracellular milieu. This was an important step in the development of neuronal-based therapies since neurodegeneration involves matrix metalloproteinase (MMP)-mediated remodeling of the collagen-rich environment in which neurons and their axons exist. We found that intraocular delivery of a CMP comprising single-strand fractions of triple helix human type I collagen prevented early RGC axon dysfunction in an inducible glaucoma model. Additionally, CMPs also promoted neurite outgrowth from dorsal root ganglia, challenged in vitro by partial digestion of collagen. Here, we compared the ability of a CMP sequence to protect RGC axons in both inducible glaucoma and optic nerve crush. A three-week +40% elevation in IOP caused a 67% degradation in anterograde transport to the superior colliculus, the primary retinal projection target in rodents. We found that a single intravitreal injection of CMP during the period of IOP elevation significantly reduced this degradation. The same CMP delivered shortly after optic nerve crush promoted significant axonal recovery during the two-week period following injury. Together, these findings support a novel protective and reparative role for the use of CMPs in both chronic and acute conditions affecting the survival of RGC axons in the optic projection to the brain.

Keywords: collagen mimetic peptides; collagen reparative; extracellular matrix; glaucoma; neuroprotection; optic nerve crush; optic neuropathy.

Figure 1

Collagen mimetic peptide does not influence IOP. (A) Mean daily intraocular pressure (IOP) in mice treated with vehicle (○, n = 6) vs. CMP 13A (Δ, n = 7) prior to (day 0) and following (days ≥ 1) a single unilateral injection of either polystyrene microbeads or an equivalent volume of saline in the contralateral eye; intravitreal injection of CMP or vehicle occurred on day 10. (B) Microbead injection significantly elevated IOP for both cohorts: vehicle (20.88 ± 0.67 vs. 15.27 ± 0.59 mmHg; *, p < 0.001); CMP 13A (20.58 ± 0.68 vs. 15.41 ± 10.57 mm Hg; *, p < 0.001). IOP did not differ between cohorts for either the saline- or microbead-injected eye (p ≥ 0.33). Data = mean ± SEM.

Figure 2

Collagen mimetic peptide protects anterograde axon transport. (A). Single sections through superior colliculus (top) showing regions of intact CTB transport (red) for mice receiving either vehicle (n = 6) or CMP 13A (n = 7) via intravitreal injection. In vehicle mice, the colliculus serving the optic projection from the saline-injected eye had fully intact transport, while microbead-induced IOP elevation in the fellow eye created regions of degraded transport (dashed lines). Retinotopic maps (bottom) reconstructed from serial sections through colliculus show levels of intact CTB signal ranging from 0% (blue) to 50% (green) to 100% (red). Medial (M) and rostral (R) orientations are indicated, as is the representation of the optic disc (white circles). Scale = 500 μm. (B). For vehicle-treated mice, intact transport declined by 67% with microbead-induced elevations in IOP compared to colliculus from the saline eye (*, p < 0.0001), while microbead eyes from the CMP 13A cohort demonstrated a two-fold improvement in transport compared to vehicle microbead (&, p = 0.002) and did not differ significantly from intact transport in the vehicle saline eye (p = 0.06). Interestingly, transport from the saline eye in the CMP 13A cohort exceeded that from the saline eye in the vehicle cohort (#, p = 0.01). Data = mean ± SEM.

Figure 3

Collagen mimetic peptide promotes axon repair following nerve crush. (A) Stitched montage of confocal micrographs of longitudinal section through optic nerve two weeks following crush from eye receiving an intravitreal injection of vehicle (DMSO) three days after the injury. Axons containing cholera toxin B (CTB, false color white) extend to the site of crush (dashed line) but generally not beyond distally towards the brain (arrow). Astrocyte glia labeled for glial fibrillary acidic protein (GFAP, red) are shown for comparison. (B) In contrast, CTB-containing axons in section of nerve from eye receiving CMP 13A extend beyond the crush site and are apparent even at more distal locations along the nerve. Repaired axons are largely coincident with localized patches of CMP 13A (green), visualized through its attached chromophore (Tide Fluor™ 2, see Methods). Scale = 200 μm (A) or 100 μm (B).

Figure 4

Collagen mimetic peptide increases axon recovery following nerve crush. (A) Number of CTB+ axon segments at specific distances distal to the injury site two weeks following optic nerve crush in individual samples (symbols), normalized to nerve width as described (see Methods). At each location, the median number of scored axons (dashed lines) in nerves from CMP 13A eyes (left) exceeded that in nerves from DMSO eyes (right); this trend is summarized in the inset. (B) The number of CTB+ axon segments above the median (top 50%) from (A) averaged at distances from crush site as indicated. Nerves from eyes receiving CMP 13A demonstrate a significantly greater number of axons at each location (*, p ≤ 0.02). (C) CTB-containing RGC axon segments in nerves from eyes receiving intravitreal CMP 13A extend further compared to axon segments from vehicle-treated eyes, both by mean length and the longest 25 segments in each group (*, p < 0.001).