Collagen mimetic peptide repair of the corneal nerve bed in a mouse model of dry eye disease

Lauren K Wareham, Joseph M Holden, Olivia L Bossardet, Robert O Baratta, Brian J Del Buono, Eric Schlumpf, David J Calkins, Lauren K Wareham, Joseph M Holden, Olivia L Bossardet, Robert O Baratta, Brian J Del Buono, Eric Schlumpf, David J Calkins

Abstract

The intraepithelial sub-basal nerve plexus of the cornea is characterized by a central swirl of nerve processes that terminate between the apical cells of the epithelium. This plexus is a critical component of maintaining homeostatic function of the ocular surface. The cornea contains a high concentration of collagen, which is susceptible to damage in conditions such as neuropathic pain, neurotrophic keratitis, and dry eye disease. Here we tested whether topical application of a collagen mimetic peptide (CMP) is efficacious in repairing the corneal sub-basal nerve plexus in a mouse model of ocular surface desiccation. We induced corneal tear film reduction, epithelial damage, and nerve bed degradation through a combination of environmental and pharmaceutical (atropine) desiccation. Mice were subjected to desiccating air flow and bilateral topical application of 1% atropine solution (4× daily) for 2 weeks. During the latter half of this exposure, mice received topical vehicle [phosphate buffered saline (PBS)] or CMP [200 μm (Pro-Pro-Gly)7, 10 μl] once daily, 2 h prior to the first atropine treatment for that day. After euthanasia, cornea were labeled with antibodies against βIII tubulin to visualize and quantify changes to the nerve bed. For mice receiving vehicle only, the two-week desiccation regimen reduced neuronal coverage of the central sub-basal plexus and epithelial terminals compared to naïve, with some corneas demonstrating complete degeneration of nerve beds. Accordingly, both sub-basal and epithelial βIII tubulin-labeled processes demonstrated increased fragmentation, indicative of nerve disassembly. Treatment with CMP significantly reduced nerve fragmentation, expanded both sub-basal and epithelial neuronal coverage compared to vehicle controls, and improved corneal epithelium integrity, tear film production, and corneal sensitivity. Together, these results indicate that topical CMP significantly counters neurodegeneration characteristic of corneal surface desiccation. Repairing underlying collagen in conditions that damage the ocular surface could represent a novel therapeutic avenue in treating a broad spectrum of diseases or injury.

Keywords: collagen mimetic peptides (CMPs); collagen reparative; dry eye; extracellular matrix; neuropathy; ocular collagen.

Conflict of interest statement

RB, BD, and ES were employed by Stuart Therapeutics, Inc., and DC served as a consultant for Stuart Therapeutics, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2023 Wareham, Holden, Bossardet, Baratta, Del Buono, Schlumpf and Calkins.

Figures

FIGURE 1
FIGURE 1
Schematic of dry eye desiccation model and treatment regimen. Baseline tear film production (wick test) and corneal sensitivity measurements were taken in all mice before commencement of the study (baseline = day 0). Animals were then treated bilaterally with topical 1% atropine 4× daily with desiccation for a total of 7 days. At day 5 and day 7, tear film and corneal sensitivity measurements were taken. At day 7, in addition to atropine + desiccation, mice were then treated topically with application of vehicle or CMP 03A (200 μm), 1× per day bilaterally for a further 7 days. Tear film production and corneal sensitivity measurements were carried out at day 10 and upon completion of the study at day 14. In vivo corneal fluorescein analysis was completed at baseline (day 0) and on day 14, after which mice from all cohorts were euthanized and corneal tissue processed for analysis.
FIGURE 2
FIGURE 2
CMP 03A improves tear film production and corneal sensitivity. (A) The two-week desiccation regimen significantly reduced tear production in mice after 5 and 7 days (#, p < 0.001) compared to baseline (day 0). Tear film production did not improve in vehicle-treated mice (day 7 vs. day 14, p = 0.07), but did with CMP 03A treatment (day 7 vs. day 14; *, p < 0.001). (B) Corneal sensitivity was significantly reduced after 7 days of the desiccation regimen (#, p < 0.001). After 3 days of vehicle treatment, sensitivity was further reduced (day 10 vehicle vs. CMP 03A; p = 0.057), but not with CMP 03A (p = 0.29). On day 14 after 7 days, CMP 03A treatment improved sensitivity (*, p = 0.01), while vehicle did not (p = 0.29). N = 11–29 (vehicle), 11 (CMP 03A), and 13 (naïve).
FIGURE 3
FIGURE 3
Central corneal nerve bed coverage in naïve, vehicle-, and CMP 03A-treated mice. Representative confocal images of βIII-tubulin-stained nerve fibers in the whole cornea with higher magnification images of the central sub-basal plexus and epithelial terminals. The dashed boxes indicate central swirl location. (A) Naïve animals have an intact central nerve fiber swirl and robust sub-basal plexus and nerve terminal coverage. (B) Desiccation with vehicle treatment only led to reduced sub-basal plexus and epithelial terminal coverage. (C) CMP 03A-treated mice did not exhibit a reduced nerve coverage at the sub-basal or terminal epithelial plexus.
FIGURE 4
FIGURE 4
CMP reduces nerve fragmentation. Representative pseudo-colored nerve fragmentation images from (A) naïve, (B) desiccation + vehicle-, and (C) desiccation + CMP 03A-treated mice. Images show fragmentation in the (i) sub-basal plexus and (ii) epithelial terminals. Dashed boxes indicate location of enlarged inset images (iii). Desiccation + vehicle increased nerve fragmentation as shown by the increase in frequency of distinctly colored fragments in the sub-basal plexus (B,i). Although reduced in number, remaining epithelial terminals were more fragmented than in naïve and CMP 03A-treated mice (B,ii; inset). (C) CMP 03A-treated mice exhibited less nerve fragmentation in the sub-basal plexus (C,i) and in epithelial terminals (C,ii). Scale bars as indicated.
FIGURE 5
FIGURE 5
CMP 03A prevents desiccation-induced corneal nerve degeneration. (A) Desiccation regimen significantly reduced sub-basal nerve coverage in vehicle-treated mice compared to naïve (#, p < 0.001). CMP 03A improved coverage compared to vehicle (p = 0.001). (B) Vehicle significantly reduced epithelial coverage compared to naïve (#, p = 0.001). CMP 03A increased epithelial coverage compared to vehicle (p = 0.001) to a level that did not significantly differ from naïve (p = 0.13). (C) The desiccation regimen caused significantly higher sub-basal fragmentation in vehicle-treated mice compared to naïve (#, p < 0.001); CMP 03A reduced fragmentation (*, p < 0.001) similar to naïve levels (p = 0.06). (D) In the vehicle group, epithelial terminal fragmentation increased compared to naïve (#, p < 0.001); CMP 03A reduced this trend compared to vehicle (*, p = 0.02). Results obtained from imaging as described in Figures 3, 4 and replicated as follows: n = 10 (naïve), 22 and 10 (vehicle sub-basal and epithelial, respectively), and 15 and 9 (CMP 03A sub-basal and epithelial, respectively).
FIGURE 6
FIGURE 6
Corneal nerve bed is intact after 1 week of desiccation. (A) Representative confocal images of βIII-tubulin-stained nerve fibers in the (i) central sub-basal plexus and (ii) epithelial terminals after 1 week of desiccation. (B) Representative pseudo-colored nerve fragmentation images from the (i) central sub-basal plexus and (ii) epithelial terminals. (C) After 1 week, the desiccation regimen did not cause significant fragmentation in the sub-basal plexus compared to naïve (p = 0.60). (D) Similarly, there was no significant fragmentation of epithelial terminals (p = 0.30). Compared to 1 week, nerve fragmentation in both the sub-basal (C) and epithelial plexus (D) at 2 weeks with vehicle treatment was significantly increased (#, p ≤ 0. 01). Scale bars as indicated. Experiment replicated as in Figure 5 for naïve and vehicle (2 weeks); n = 5 (vehicle 1 week).
FIGURE 7
FIGURE 7
CMP 03A prevents desiccation-induced disruption of the corneal epithelium. Representative images of the corneal epithelial surface in (A) naïve, (B) vehicle- and (C) CMP-treated mice visualized in vivo by fluorescein staining. CMP 03A prevented corneal epithelial pitting observed in the vehicle group. Representative DAPI-stained confocal images of the epithelial layer in (D) naïve, (E) vehicle- and (F) CMP 03A-treated mice. Vehicle treatment caused epithelial disruption that is absent in the CMP 03A-treated cohort and comparable to naïve animals. Scale bars as indicated.
FIGURE 8
FIGURE 8
CMP 03A preserves the integrity of the corneal epithelial cell layer. Examples of fluorescence intensity and first-derivative line plots, respectively, in naïve (A,D), vehicle (B,E), and CMP-treated (C,F) mice. Desiccation reduces the amplitude of the derivative compared to naïve; CMP reverses this trend. (G) The transition delta is significantly reduced in the vehicle group compared to naive (#, p = 0.01), which is reversed by CMP 03A (*, p = 0.035). (H) The area under the curve (AUC) is significantly reduced by vehicle compared to naïve (#, p = 0.002); CMP 03A improves this trend compared to vehicle (*, p < 0.001), elevating to naïve levels. Similarly, the area under the curve per transition (I) is significantly reduced in vehicle compared to naïve (#, p = 0.008). CMP 03A improves this compared to vehicle (p = 0.001), once again to naïve levels. Results obtained from imaging as described in Figure 7 and replicated as follows: n = 3 (naïve), 6 (vehicle), and 8 (CMP 03A).

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