Abstract
Purpose: :
Our previous data demonstrated that vascular endothelial growth factor (VEGF) can regulate the growth of corneal nerves. We sought to identify the VEGF receptor sub-types (VEGFR1, VEGFR2, or neuropilin-1(NPR1)) which mediate the potent effect of VEGF on isolated trigeminal neurons in vitro.
Methods: :
Thy1-YFP mice were sacrificed and trigeminal ganglia (TG) isolated following craniotomy. Isolated neurons were isolated from TG by limited enzymatic treatment with papain and collagenase/dispase followed by Percoll gradient centrifugation. Neurons were seeded on poly-D-lysine coated culture plates and incubated in Neurobasal A media containing 1% B27 supplement. Two hours after plating, neurons were treated for 1 hour with either VEGFR2 kinase inhibitors (SU1498 and Ki8751), specific neutralizing antibodies for VEGFR1, VEGFR2 or NPR1 or random IgG. Subsequently, 50ng/ml VEGF was added to each pre-treated well to evaluate for VEGF-dependent neuronal growth. Neurons were imaged by fluorescence microscopy at multiple time points. Images were analyzed using quantitative neuronal tracing software (Neurolucida).
Results: :
Primary cultured trigeminal neurons extended dendrites 24 hr after plating. By 72 hours, 50 ng/ml VEGF increased dendrite numbers, branches, length and tortuosity by 3-fold. SU1498 (5uM) or Ki 8751 (5nM) completely suppressed dendrite elongation in the presence of VEGF. Neutralizing antibodies for VEGFR1, VEGFR2, and NPR1 inhibited dendrite growth in a dose-dependent manner with nearly complete inhibition achieved by 10ug/ml anti-VEGFR1, 2.5 ng/ml anti-VEGFR2, and 2ug/ml anti-NPR1. Random IgG did not affect dendrite growth.
Conclusions: :
VEGF promotion of trigeminal neuron growth is mediated through multiple receptors, including VEGFR receptors type 1, 2 and NPR 1. VEGF represents a potential drug target to promote restoration of corneal innervation following corneal injury. Further study is needed to identify opportunities to optimize VEGF-mediated neurogenesis while minimizing its possible angiogenic effects.
Keywords: cornea: basic science • innervation: sensation • vascular endothelial growth factor