Impact of diet and aging on the mouse corneal innervation at the epithelial-stromal interface

Purpose: The cornea is the most highly innervated structure in the body, undergoing changes in sensitivity and distribution throughout life and in response to insult such as the introduction of an obesogenic diet. However, the mechanisms governing corneal nerve reorganization are currently unknown. While imaging corneal nerves using SBF-SEM, we discovered a novel event whereby axons fused with basal epithelial cells such that portions of the nerve bundle do not penetrate into the epithelium. Here we morphologically define neuronal-epithelial cell fusion, and determine its correlation with age and obesogenic diet-induced nerve loss. Methods: Corneas were collected from C57BL/6 mice, prepared for either SBF-SEM or light-microscopy, and evaluated for neuronal-epithelial cell fusion frequency. In aim 1, 9 week-old mice were evaluated for fusion frequency. In aim 2, 9, 16, and 24 week-old mice were assessed for correlation between aging and frequency of fusion. In aim 3, 5 week-old C57BL/6J mice were fed a normal diet, or an obesogenic diet, for 10 weeks before being assessed for a correlation between diet and fusion frequency. Results: In 9 week-old animals 47% of central cornea stromal nerve bundles contain axons that fuse with basal epithelial cells. The average surface-to-volume ratio of a penetrating nerve was 3.32, while the average fusing nerve was 1.39. Fusing axons were swollen, electron-translucent, contained autophagic vesicles, and lacked mitochondria near sites of fusion. Penetrating and fusing nerves passed through similar sized basal lamina discontinuities. Most corneal nerves labeled with DiI (membrane tracer) after trigeminal ganglion application, and fusion sites were identified by DiI transfer to basal epithelial cells. Fusion frequency increased with aging and the introduction of an obesogenic diet. 74% of central nerves showed fusion in 24 week-old animals, 82% showed fusion in 6 week-old mice fed a 10 week obesogenic diet, and 57% contained fusion in 16 week-old normal diet controls. Conclusion: This is, to our knowledge, the first report of neuronal-epithelial cell fusion in the mouse cornea. The fusion event is positively correlated with nerve loss reported in aging and obesogenic-diet fed mice and as such, it represents a potential mechanism for nerve organization and loss in the central cornea

Neuron-Epithelial Cell Fusion Occurs in the Central Murine Cornea

Of the 21 interactions observed in the central cornea, 43% were fusing with basal corneal epithelial cells. Nerves undergoing fusion were twice as large in diameter as standard corneal stromal nerves. Fusing nerves lack mitochondria and functional microtubules. In this study a novel form of cell interaction in the cornea was discovered and phenotypically defined; this form of cell interaction occurs exclusively in the central cornea, involves a nerve axon twice as large as nerves penetrating the basal lamina and contributing to the epithelial nerve plexus, and is devoid of mitochondria and microtubules.Vision Science

MyD88 contribution to ocular surface homeostasis

<div><p>The cornea must maintain homeostasis, enabling rapid response to injury and microbial insult, to protect the eye from insult and infection. Toll-like receptors (TLRs) are critical to this innate immune response through the recognition and response to pathogens. Myeloid differentiation primary response (MyD88) is a key signaling molecule necessary for Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R)-mediated immune defense and has been shown to be necessary for corneal defense during infection. Here, we examined the intrinsic role of TLR signaling in ocular surface tissues by determining baseline levels of inflammatory mediators, the response to mechanical stimuli, and corneal infection in MyD88-deficient mice (MyD88^-/-). In addition, cytokine, chemokine, and matrix metalloproteinase (MMP) expression was determined in ocular surface cells exposed to a panel of TLR agonists. Compared to wild-type (WT) animals, MyD88^-/- mice expressed lower MMP-9 levels in the cornea and conjunctiva. Corneal IL-1α, TNFα, and conjunctival IL-1α, IL-2, IL-6, and IL-9 levels were also significantly reduced. Additionally, CXCL1 and RANTES expression was lower in both MyD88^-/- tissues compared to WT and IL-1R^-/- mice. Interestingly, MyD88^-/- mice had lower corneal sensitivities (1.01±0.31 gm/mm²) than both WT (0.59±0.16 gm/mm²) and IL-1R^-/- (0.52±0.08 gm/mm²). Following <i>Pseudomonas aeruginosa</i> challenge, MyD88^-/- mice had better clinical scores (0.5±0.0) compared to IL-1R^-/- (1.5±0.6) and WT (2.3±0.3) animals, but had significantly more corneal bacterial isolates. However, no signs of infection were detected in inoculated uninjured corneas from either MyD88 or IL-1R-deficient mice. This work furthers our understanding of the importance of TLR signaling in corneal defense and immune homeostasis, showing that a lack of MyD88 may compromise the baseline innate response to insult.</p></div

TLR agonist treatment induces cytokine expression in human corneal cells.

<p>hTCEpi and primary HCEC were treated with TLR agonists [TLR2/1 (PAM), TLR2 (HKLM), TLR4 (LPS), TLR5 (FLAG), TLR6/2 (FLS-1), TLR7 (IMQ), TLR8 (ssRNA), or TLR9 (ODN)] for 24 hours and IL-8 (A, B) and IL-6 (C, D) expression was determined in cell supernatants by Luminex bead assay (hTCEpi) or ELISA (HCEC). Graphs represent mean ±SEM of 3 independent experiments (n = 3). Analysis was performed by ANOVA with Bonferroni’s test for multiple comparisons. p < *0.05, **0.01, ****0.0001.</p

MyD88-deficient mice have lower levels of MMP expression in the cornea and conjunctiva.

<p>(A) MMP-9 expression was determined in untreated corneal lysates by RT-PCR. Graph represent mean ±SEM of 3 independent experiments with each sample pooled from 4 mice. (B) MMP-9 protein expression was quantitated in conjunctival homogenates by Luminex multiplex assay. Samples represent 10 pooled corneas per genotype. Graph represent mean ±SEM of 3 independent experiments (n = 3). Analysis was performed by ANOVA with Bonferroni’s test for multiple comparisons. p < *0.05, **0.01.</p

MyD88^-/- mice infected with PA were protected from inflammatory corneal infiltrates; however, more bacteria were isolated from MyD88^-/- corneas compared to both WT and IL-1R^-/-.

<p>Corneas of WT (C57), IL-1R^-/-, and MyD88^-/- mice were scratched and infected with 1.0 × 10⁶ CFU GFP-PA01. (A-B) After 24 hours, eyes were imaged by slit lamp microscopy and graded based on severity of infection. Images are representative of 3 animals per group. Analysis was performed by ANOVA with Bonferroni’s test for multiple comparisons. p<*0.05, **0.01 (B) For bacterial counts, corneas were harvested 24 hours post-infection, homogenates plated, and incubated for 16 hours at 37°C. The number of colonies was counted and data represent 3 corneas per group.</p

Lack of TLR signaling (MyD88^-/-) results in decreased levels of cytokines in the cornea.

<p>IL-1α (A, C) and TNFα (B, D) gene expression was determined in untreated corneal lysates by RT-PCR (top) and protein expression quantitated in corneal homogenates by Luminex multiplex assay (bottom), in wild-type (WT), IL-1R^-/-, and MyD88^-/- mice. Graphs represent mean ± SEM of 3 independent experiments with each sample pooled from 5 mice. Analysis was performed by ANOVA with Bonferroni’s test for multiple comparisons, with comparison to WT samples. p<*0.05, **0.01, nd = not determined.</p

MyD88^-/- mice have decreased corneal sensitivity.

<p>Corneal sensitivity was measured in untreated mice using a Cochet-Bonnet esthesiometer, with greater pressure (gm/mm²) indicating lower sensitivity. Graph represents mean ± SEM of 9 mice per genotype. Analysis was performed by ANOVA with Bonferroni’s test for multiple comparisons. p<***0.001.</p

MyD88^-/- mice have decreased levels of cytokines in the conjunctiva.

<p>IL-1α (A), IL-6 (B), IL-2 (C), and IL-9 (D) expression were determined in untreated conjunctival homogenates from wild-type (WT), IL-1R^-/-, and MyD88^-/- mice by Luminex multiplex assay. Data represent mean ± SEM of 3 independent experiments with each sample pooled from 5 mice. Analysis was performed by ANOVA with Bonferroni’s test for multiple comparisons, with comparison to WT samples. p<*0.05, **0.01.</p