Neuroanatomy and Neurochemistry of Mouse Cornea

Abstract

PurposeTo investigate the entire nerve architecture and content of the two main sensory neuropeptides in mouse cornea to determine if it is a good model with similarities to human corneal innervation.MethodsMice aged 1 to 24 weeks were used. The corneas were stained with neuronal-class βIII-tubulin, calcitonin gene–related peptide (CGRP), and substance P (SP) antibodies; whole-mount images were acquired to build an entire view of corneal innervation. To test the origin of CGRP and SP, trigeminal ganglia (TG) were processed for immunofluorescence. Relative corneal nerve fiber densities or neuron numbers were assessed by computer-assisted analysis.ResultsBetween 1 and 3 weeks after birth, mouse cornea was mainly composed of a stromal nerve network. At 4 weeks, a whorl-like structure (or vortex) appeared that gradually became more defined. By 8 weeks, anatomy of corneal nerves had reached maturity. Epithelial bundles converged into the central area to form the vortex. The number and pattern of whorl-like structures were different. Subbasal nerve density and nerve terminals were greater in the center than the periphery. Nerve fibers and terminals that were CGRP-positive were more abundant than SP-positive nerves and terminals. In trigeminal ganglia, the number of CGRP-positive neurons significantly outnumbered those positive for SP.ConclusionsThis is the first study to show a complete map of the entire corneal nerves and CGRP and SP sensory neuropeptide distribution in the mouse cornea. This finding shows mouse corneal innervation has many similarities to human cornea and makes the mouse an appropriate model to study pathologies involving corneal nerves.

Recovery of Corneal Sensitivity and Increase in Nerve Density and Wound Healing in Diabetic Mice After PEDF Plus DHA Treatment

“…After washing with PBS (three times for 10 min each), the corneas were incubated with the corresponding secondary antibodies Alexa Fluor 488 goat anti-rabbit Ig G (H+L) or Alexa Fluor 594 goat anti-rat Ig G (H+L) for 24 h at 4°C and then washed thoroughly with PBS. Images were taken as described previously (13,17). Briefly, four radial cuts were performed on each cornea, and the tissue was mounted flat on a slide with the endothelium side up.…”

“…The nerve area and the total area of the image were obtained by using the histogram tool. The percentage of total nerve area was quantified for each image as described previously (13,17). …”

“…More recently, we have disclosed the anatomy of corneal innervation in the mouse, which shares many common features with human cornea, making the mouse an appropriate model to study pathologies involving corneal nerves (13). In the current study, we used a diabetic mouse model to investigate the effect of PEDF+DHA on sensitivity, tear secretion, wound healing, and nerve regeneration in corneas with or without injury.…”

Intraepithelial dendritic cells and sensory nerves are structurally associated and functional interdependent in the cornea

“…Corneas were washed in PBS, incubated in 20 mM pre-warmed EDTA for 30 minutes at 37 °C to weaken epithelium-stroma adhesion and cell-cell junction to allow antibodies to penetrate into the subbasal space, and incubated with 0.025% hyaluronidase 37 °C for 1 day. For blocking, corneas were incubated in PBS containing 0.2% Triton-X, 1% bovine serum albumin, 10% serum of the host species used to generate the first antibody, or Fc blocking antibody (BD Biosciences) for 20 minutes at room temperature50. After blocking, the corneas were incubated overnight at 4 °C with 100 μl of mouse anti-CD11c (BD Biosciences), anti-β-tublin III (Covance) antibodies.…”

Subconjunctival Bevacizumab Injection Impairs Corneal Innervations and Epithelial Wound Healing in Mice

“…In the subbasal layer, the corneal nerve fibers form a distinctive pinwheel pattern plexus and project toward the apical surface of the corneal epithelium. 16,17 The intact corneal innervations play an important role in the regulation of blink reflex, epithelial homeostasis, and tear production and secretion. 10 However, the attenuation of corneal innervations, caused by herpetic viral infection, trigeminal nerve damage, or diabetic mellitus, can cause impaired corneal sensation, chronic inflammation, delayed corneal epithelial wound healing, and even persistent defects.…”