Immunocytochemical Localisation of the Neuropeptide WE-14 in the Developing Mouse Cornea

Abstract

Abstract: : Purpose: The cornea possesses an elaborate neuronal architecture emanating from the corneoscleral limbal junction, terminating in the corneal epithelium and it has been shown to possess a complex array of neurochemical agents. A previous study had observed WE-14, a 14-residue neuropeptide derived from the cell-specific post-translational processing of the neuroendocrine secretory granule protein chromogranin A (CgA) in corneal tissue. The aim of this study was to map the distribution of WE-14 in the normal developing mouse cornea. Methods: Enucleated neonatal (N2, 6, 8, 10, 15, 18) and adult mouse eyes (n=5/age) were dissected and intact corneas immersion-fixed in 4% PFA (w/v) in 0.1M phosphate buffered saline, pH 7.4 (24 hr, 4₀C). The corneas were washed in PBS containing 0.1% (v/v) Triton X-100 (24 hr, 4₀C) and immunostained with rabbit anti-WE-14 sera (24 hr, 4₀C), wash and primary antisera binding visualised employing SWAR-FITC with propidium iodide counter staining. The specimens were viewed with confocal laser scanning microscope (CLSM). Latterly the immunostained tissue specimens were cryoprotected and transverse 60µm tissue sections were re-examined with CLSM. Results: Analysis of the intact adult cornea revealed a dense network of nerve fibres exhibiting intense WE-14 immunostaining. CLSM revealed a minor population of outer sub-epithelial varicose nerve fibres with a dense fibre network resident in the upper mid stromal region. A limited number of fibres were evident deep within the stroma. A radiate pattern of WE-14 immunopositive fibre tracks progressed toward the central cornea within the upper mid stromal region with an intervening fine fibre network. Analysis of transverse sections (60 um) revealed that mid-stromal varicose fibres tracks extended toward Bowman’s layer, which was penetrated by fibres entering the epithelial layer some of which extended toward the surface epithelium. Weak WE-14 immunostaining was first detected in neonatal corneal stromal tissue at N10. The intensity and distribution of WE-14 immunostaining increased with age and at N18 mirrored that observed in adult cornea. Conclusion: Developmental analysis has revealed that WE-14 is present in a simple nerve net at N10 and the distribution and intensity of WE-14 immunostaining increases with age. In the adult cornea WE-14 was observed in the dense peripheral nerve system and in the network innervating the central cornea terminating in individual nerve fibres in the epithelium. The pathophysiological impact of WE-14 on the developing and mature cornea requires investigation.

Keywords: 370 cornea: basic science • 488 neuropeptides • 434 immunohistochemistry