Neurogenic Homeostasis of Corneal Endothelial Cells: Peripheral Innervation Maintains Endothelial Cell Survival Through Vasoactive Intestinal Peptide

Abstract

Purpose: Loss of corneal endothelial cell (CEC) density in various pathologic states and after corneal transplantation can lead to vision loss. Currently, no treatments are available to prevent the loss of CECs. We recently reported an inverse correlation of corneal nerve with CEC density in patients with early Fuchs Endothelial Cell Dystrophy. The purpose of this study was to evaluate the functional role of corneal nerves on CEC in vivo.

Methods: Trigeminal axotomy or sham procedures were performed in 6-8 week-old male BALB/c mice. Central corneal thickness (CCT) was obtained using anterior segment optical coherence tomography (AS-OCT) under anesthesia on 7, 14 and 21 days thereafter. Normal, axotomized and sham control corneas were excised on days at 7, 14 and 21, and immunohistochemistry was performed with anti-βIII tubulin to evaluate corneal nerve density, and with anti-ZO-1 and TO-PRO-3 iodide to evaluate CEC density. Further, vasoactive intestinal peptide (VIP) protein and gene expression levels were determined using enzyme immunoassay kit and by RT-PCR. The above studies were repeated with daily intraperitoneal VIP or sham saline injections.

Results: Axotomy resulted in continuous loss of CEC centrally (2330 ± 100 cells/mm2 vs. 1430 ± 100; p < 0.001) and peripherally (2450 ± 55 vs. 1770 ± 137; p < 0.001) comparing baseline to 21 days respectively. No decrease in CEC was noted in the sham group. Increased CCT was observed from 66.7 ± 2.5 μm (normal) and 67.3± 0.7 (sham-treated) to 99.3 ± 10.7 after axotomy (p < 0.003). Gene and protein levels of VIP in the cornea were significantly decreased at days 7 and 14 after axotomy (p < 0.05) compared to controls. Daily injection of intraperitoneal VIP after axotomy prevented loss of central CEC (2100 ± 68 cells/mm2, p = 0.37), did not prevent loss of peripheral CEC (1970 ± 63 cells/mm2, p < 0.001). Daily inejection of VIP after axotomy prevented increase in CCT after corneal denervation (p = 0.437), as compared to sham saline group (p = 0.02).

Conclusions: Our results identify neurogenic homeostasis as a critical process whereby the peripheral nervous system maintains corneal endothelial cell survival. In the absence of corneal nerves, CEC loss can be prevented by exogenous VIP treatment.

Keywords: 481 cornea: endothelium • 614 neuropeptides • 564 innervation: neural regulation