Effects of the Human Amniotic Membrane on Axonal Outgrowth of Dorsal Root Ganglia Neurons in Culture

Abstract

Purpose: In order to investigate the potential underlying neurotrophic mechanisms of human amniotic membrane (AM) in the treatment of neurotrophic corneal ulcers, we evaluated whether or not there are significant differences in the neuritic growth of neuronal cell cultures on different surfaces of AM. Methods: Neurite outgrowth of dorsal root ganglia neurons was examined under two separate conditions: (1) in serum-free medium consisting of minimal essential medium (MEM), glucose, and L-glutamine, (2) in same medium additionally supplemented with horse serum, chick embryonic extract, and nerve growth factor. Neuritic outgrowth was labeled with antibodies against neurofilaments and tubulin and screened by confocal laser scanning microscopy. Moreover, Western blot analysis was performed with antibodies to neuronal cell adhesion molecule (NCAM), L1, pan-cadherin, semaphorin-3F, as well as various ephrins. Results: The basement membrane and the stromal surface promoted the outgrowth of an extensive neuritic network, whereas the epithelial surface did not. Interestingly, these differences of neuritic growth were evident in cell cultures treated with serum-free medium lacking neurotrophic factors and in standard medium containing neurotrophic factors. Western blot analysis revealed an abundant expression of ephrin A4 in intact AM but not in epithelium-denuded AM, and no significant difference of pan-cadherin and NCAM expression was found in intact AM compared with denuded AM. Additionally, no expression of L1, semaphorin-3F, and the ephrins A1, A2, B1, and B3 was detected. Conclusions: This study shows that AM exhibits location-dependent neuritic growth–promoting effects that might influence the clinical outcome of (amniotic membrane transplantation) in neurotrophic conditions.

KEYWORDS:

• amniotic membrane
• cell culture
• corneal wound healing
• neurotrophic keratopathy