Corneal pain and experimental model development

Abstract

The cornea is a valuable tissue for studying peripheral sensory nerve structure and regeneration due to its avascularity, transparency, and dense innervation. Somatosensory innervation of the cornea serves to identify changes in environmental stimuli at the ocular surface, thereby promoting barrier function to protect the eye against injury or infection. Due to regulatory demands to screen ocular safety of potential chemical exposure, a need remains to develop functional human tissue models to predict ocular damage and pain using in vitro-based systems to increase throughput and minimize animal use. In this review, we summarize the anatomical and functional roles of corneal innervation in propagation of sensory input, corneal neuropathies associated with pain, and the status of current in vivo and in vitro models. Emphasis is placed on tissue engineering approaches to study the human corneal pain response in vitro with integration of proper cell types, controlled microenvironment, and high-throughput readouts to predict pain induction. Further developments in this field will aid in defining molecular signatures to distinguish acute and chronic pain triggers based on the immune response and epithelial, stromal, and neuronal interactions that occur at the ocular surface that lead to functional outcomes in the brain depending on severity and persistence of the stimulus.

Keywords: Cornea; Dry eye; Neuropeptides; Nociception; Pain; Tissue engineering.

Fig. 1.

Current approaches for studying corneal toxicity and pain. ^a Albino rabbit eye following application of the standardized Draize test, a common toxicity assay using clinical scoring post-topical irritant application to appropriately label chemicals based on their propensity to cause corneal damage (Wilhelmus, 2001). Image reproduced from (Liu et al., 2015) with permission. ^b Bright field image of an enucleated porcine eye and isolated cornea proper following chemical application. ^c Stratified epithelium cultured on curved cellulose filters at an air-liquid interface to assess ocular toxicity. Image reproduced from (Postnikoff et al., 2014) with permission. ^d Functional magnetic resonance imaging (MRI) image of a human patient exposed to bright light to induce pain sensations. The red box denotes location of pain activation distinct from eye blinking. Sensory homunculus depicts location of corneal pain in the somatosensory cortex. Images reproduced from (Moulton et al., 2012) with permission. Pictorials generated using Servier Medical Art based on a Creative Commons Attribution 3.0 Unported License available at https://creativecommons.org/licenses/by/3.0/.

Fig. 2.

Sensory nerve fibers innervating the cornea. (A) Neuronal extensions present between the stroma and epithelial layer in the mouse cornea (top inset: βIII-tubulin: red). z-stack of the corneal epithelium (bottom inset: βIII-tubulin: red, DAPI: blue) from the sub-basal nerve (large arrow) and extending intraepithelial nerve endings reaching into the epithelium (small arrows) in corneas isolated from adult C57/BL6 mice. (B) En face view of spatially dispersed intraepithelial nerve endings (small arrow) innervating the ocular surface. Modified from (Li et al., 2011) and reproduced with permission. (C) Stereofluorescent image of corneal nerves in the transgenic mouse cornea (YFP-labelled immune cells). Inset highlights limbus region containing a high density of YFP+-immune cells. Modified from (Sarkar et al., 2013) and reproduced with permission.

Fig. 3.

Interplay of the epithelium, stromal, immune cell, and sensory nerves following exposure to mechanical or chemical stimuli that result in a wound healing response. Images generated using Servier Medical Art under a Creative Commons Attribution 3.0 Unported License available at https://creativecommons.org/licenses/by/3.0/.

Fig. 4.

TRPV1 expression in the apical layer of the central and limbal corneal epithelium of the human cornea. (red- TRPV1, blue- DAPI). Reproduced from (Zhang, 2007) with permission. Scale bar = 25 μm.

Fig. 5.

TRP-mediated signaling in sensory nerve fiber following exposure to noxious stimuli. TRPV1, TRPA1, and TRPM8 voltage-gated receptors are co-expressed on C and Aδ-nerve fibers and are responsive to chemical, mechanical, and thermal stimulation. Bradykinin release from resident inflammatory cells is known to increase sensitization of TRP receptors on sensory nerves acting primarily via phospholipase C (PLC) and protein kinase A (PKA) signaling pathways. Pictorials modified from Servier Medical Art under a Creative Commons Attribution 3.0 Unported License available at https://creativecommons.org/licenses/by/3.0/ .

Fig. 6.

Transmission of sensory perception of stimuli detected at the ocular surface to the trigeminal ganglion, brainstem, and ultimately to the somatosensory cortex where pain is registered. Images of organs modified from Servier Medical Art under a Creative Commons Attribution 3.0 Unported License available at https://creativecommons.org/licenses/by/3.0/.

Fig. 7.

(A) Sub-basal nerve plexus in normal subject. (B) Patient with DED showing increased immune cells (arrow) and reduced nerve density. (C) Herpes simplex keratitis patient with increased immune cells (arrows), presence of micro-neuromas (arrow-head) and reduced nerve density. (D) Patient with herpes zoster ophthalmicus showing decreased nerve density and presence of immune cells. (E) Patient with NCP showing the presence of micro-neuromas (arrow-head). (F) Patient with diabetes showing increased nerve tortuosity (arrow-head), decreased nerve density, and presence of immune cells (arrow). All scale bars represent 100 μm (unpublished data).

Fig. 8.

(A) Sub-basal nerve plexus in a normal subject; (B, C, D, E) NCP patient with different forms of micro-neuroma presentations (arrows) and presence of immune cells (dashed arrows); F. Sub-basal nerve plexus in NCP patient showing the presence of beading, axonal nerve swelling and nerve tortuosity (arrow-heads). All scale bars represent 100 μm (unpublished data).

Fig. 9.

Tissue engineered innervated corneal model. Customized silk scaffolds containing appropriate cell types assembled to mimic the native cornea. The neural cell component (DRGs) are seeded in a silk sponge on the periphery with innervation into the stroma promoted using high NGF loading (50ng/mL) on the anterior film. The stromal layers are formed using Arg-Gly-Asp (RGD)-coated porous silk films assembled with interlaying collagen to develop biomimetic silk lamellae. (Abbreviations: hCECs (human corneal epithelial cells), hCSSCs (human corneal stromal stem cells), DRGs (dorsal root ganglion). Re-printed with permission (Wang et al., 2017).

Fig. 10.

Considerations for developing a functional corneal tissue model to study irritancy in vitro. A) NIH 3T3 fibroblasts grown in 3D collagen hydrogels or 2D polystyrene culture dishes. TRITC-conjugated phalloidin (red) and DAPI (blue). Adapted from (Bohm et al., 2017) with permission. B) Morphological differences between human corneal fibroblasts (hCFs) (elongated, expanded cytoplasm) and keratocytes (dendritic-like) depending on cell culture conditions maintained in vitro (Adapted from (Wilson et al., 2012) with permission). Immunofluorescence imaging of BIII-tubulin of DRGs grown on silicon micro-pillar substrates (Repic et al., 2016) and stem cell-derived nociceptors (Wainger et al., 2015) reproduced with permission. C) Dynamic maintenance of ex vivo tissue showing higher tight-junctions maintenance in the dynamic environment comparable to native lamellar corneas (NLC) in auto-tissue-engineered lamellar cornea (ATELC). Adapted from (Wu et al., 2014b) and reproduced with permission. D) Electrophysiological experiments in stem-cell derived nociceptors post-chemical (250 μm menthol, 100 μm mustard oil, 1 μm capsaicin, or 40 mM KCl) application. The number of nociceptors responding to stimuli is shown in a Venn diagram with capsaicin exhibiting the most robust response comparative to the extracellular microelectrode array recording. Adapted from (Wainger et al., 2015) and reproduced with permission. Potential modeling of pain responses will depend on exposure time and epithelial, stromal, and nerve contributions to the biochemical and electrophysiological responses to specific chemicals of interest (unpublished). Ideal validation of in vitro responses should correspond to reported in vivo responses, thereby establishing predictability for unknown chemicals.