A novel animal model of neuropathic corneal pain-the ciliary nerve constriction model

Abstract

Introduction: Neuropathic pain arises as a result of peripheral nerve injury or altered pain processing within the central nervous system. When this phenomenon affects the cornea, it is referred to as neuropathic corneal pain (NCP), resulting in pain, hyperalgesia, burning, and photoallodynia, severely affecting patients' quality of life. To date there is no suitable animal model for the study of NCP. Herein, we developed an NCP model by constriction of the long ciliary nerves innervating the eye.

Methods: Mice underwent ciliary nerve constriction (CNC) or sham procedures. Safety was determined by corneal fluorescein staining to assess ocular surface damage, whereas Cochet-Bonnet esthesiometry and confocal microscopy assessed the function and structure of corneal nerves, respectively. Efficacy was assessed by paw wipe responses within 30 seconds of applying hyperosmolar (5M) saline at Days 3, 7, 10, and 14 post-constriction. Additionally, behavior was assessed in an open field test (OFT) at Days 7, 14, and 21.

Results: CNC resulted in significantly increased response to hyperosmolar saline between groups (p < 0.0001), demonstrating hyperalgesia and induction of neuropathic pain. Further, animals that underwent CNC had increased anxiety-like behavior in an open field test compared to controls at the 14- and 21-Day time-points (p < 0.05). In contrast, CNC did not result in increased corneal fluorescein staining or decreased sensation as compared to sham controls (p > 0.05). Additionally, confocal microscopy of corneal whole-mounts revealed that constriction resulted in only a slight reduction in corneal nerve density (p < 0.05), compared to naïve and sham groups.

Discussion: The CNC model induces a pure NCP phenotype and may be a useful model for the study of NCP, recapitulating features of NCP, including hyperalgesia in the absence of ocular surface damage, and anxiety-like behavior.

Keywords: animal model; chronic constriction injury; neuropathic corneal pain; nociception; ocular pain.

Figure 1

Reproducibility and distribution of responses following CNC or sham surgeries. (A) A schematic of the CNC, showing key steps of the procedure. Notice that the ciliary nerves run parallel to the optic nerve. A suture is carefully placed around the ciliary nerves (and optic nerve) with slight tension so as not to sever the ciliary nerves nor damage the optic nerve. (B) Pre- and post-CNC hyperosmolar responses across three different surgeons (n = 10, 20, and 20, respectively). (C) A histogram view of all pre- and post-CNC hyperosmolar responses from A reveals a normal distribution of data in both settings. The mean of baseline (pre) responses +3 standard deviations is indicated. (D) The same data shown as a scatter plot allows for visualization of the cut-off value for a successful surgery. (E) Pre- and post-sham surgery hyperosmolar responses, indicating a normal distribution of the data. (F) The same data from E shown as a scatter plot demonstrates no difference in pre- and post-sham surgery responses.

Figure 2

Assessment of spontaneous and evoked pain responses. (A) Depiction of the measurements taken for calculation of palpebral opening ratios, an indicator of spontaneous pain. (B) Quantification of palpebral opening ratios as a marker of spontaneous pain at baseline and Days 7 and 14 (n = 9/group). (C) Depiction of hyperosmolar saline response, resulting in characteristic paw wipe response to this stimulus. (D) Quantification of the paw wipes as a marker of an evoked pain response to application of hyperosmolar saline. Note the pronounced increase in the CNC animals from Day 3 post-CNC until termination of the study (Sham: n = 11; CNC: n = 19). Statistical analysis: One-Way ANOVA; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Panels A,C were created with BioRender.

Figure 3

Open field test shows anxiety-like behavior following CNC. A 10-min open field test was performed on sham and CNC animals on Days 7, 14, and 21. The following parameters were analyzed: total distance traveled (A), center distance (B), periphery distance (C), immobility (D), center entries (E), and periphery entries (F). Statistical analysis was conducted by a One-Way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. # denotes significance compared to Day 7 group with #p < 0.05 and ## p < 0.01. Normal n = 10; Sham n = 7; CNC n = 5.

Figure 4

Characterization of molecular alterations in trigeminal ganglia following CNC. The fold change of cytokines (A), ion channels (B), and neurotrophic factors (C) were determined by RT-qPCR. Fold change was calculated by the ddCt method and normalized to sham group. Data are presented as means ± SEM. Statistical analysis was conducted using unpaired t-test; *, p < 0.05; **, p < 0.01. n = 5-6/group.

Figure 5

The ocular surface remains undamaged after CNC. (A) Representative slit-lamp microscopy images from sham and CNC animals as well as (B) quantification of the corneal fluorescein staining according to the NEI scale (C). Note that there is minimal staining of the ocular surface, and there is no significant difference between sham and CNC animals (sham: n = 12, CNC: n = 30). Statistical analysis: Groups were compared at respective time points by a Kruskal-Wallis test (p = 0.4978).

Figure 6

Corneal innervation remains intact following CNC or sham surgery. (A) Quantification of corneal mechanosensation as determined by Cochet-Bonnet esthesiometry at Days 3, 7, 10 and 14. (B) Representative confocal images of βIII-tubulin staining from corneal whole-mounts in the central cornea – the top row is of the subbasal plexus, and the bottom row is of the stromal nerves. (C) Quantification of nerve density in the central cornea – white bars indicate the subbasal plexus and the black bars represent the stromal nerves. (D) Representative confocal images of β_III-tubulin staining from corneal whole-mounts in the peripheral cornea – the top row is of the subbasal plexus, and the bottom row is of the stromal nerves. (E) Quantification of nerve density in the central cornea – white bars indicate the subbasal plexus and the black bars represent the stromal nerves. For A, Sham = 12 and CNC = 30 and for B–E n = 5/group. Statistical analysis for A was conducted by Kruskal-Wallis test; statistical analysis for C,E was conducted using One-Way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.