Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells, and it promotes angiogenesis in vivo. Here we report that VEGF(165) has neurotrophic actions on cultured adult mouse superior cervical ganglia (SCG) and dorsal root ganglia (DRG), measured as axonal outgrowth. Maximal effect was observed at 10-50 ng/ml for SCG and 100 ng/ml for DRG. VEGF-induced axonal outgrowth was inhibited by the mitogen-activated protein kinase kinase inhibitor PD 98059 but not by the protein kinase inhibitor K252a. VEGF also increased survival of both neurons and satellite cells and the number of proliferating Schwann cells. Immunocytochemistry and immunoblotting revealed that VEGF was expressed in virtually all nerve cells in the SCG but only in a population of small-diameter (<35 micrometers) neurons representing approximately 30% of the neurons in DRG. Immunostaining showed that the VEGF receptor fetal liver kinase receptor (flk-1) was found on nerve cell bodies in DRG and to a lesser extent on neurons in SCG. Growth cones of regenerating axons from both types of ganglia exhibited flk-1 immunoreactivity, as did Schwann cells. We conclude that VEGF has both neurotrophic and mitogenic activity on cells in the peripheral nervous system.

Vascular endothelial growth factor (VEGF) is an angiogenic factor that stimulates axonal outgrowth. Here we used in situ hybridization and immunocytochemistry to study the VEGF receptor flk-1 in cultured superior cervical ganglia (SCG) and dorsal root ganglia (DRG) from adult mice, and also the effects of VEGF on regeneration in vitro. Neurons in both ganglia contained the flk-1 receptor and showed an increased mRNA expression and immunoreactivity for flk-1 after 48 h in culture. In SCG, but not in DRG, double immunostaining for flk-1 and VEGF revealed coexpression in many neurons, implying that VEGF may exert both autocrine and paracrine actions. One proportion of the flk-1-positive neurons in DRG stained positive for the large neuron marker RT97 and another proportion expressed calcitonin gene-related peptide (CGRP). Small IB4-positive neurons were devoid of flk-1 immunoreactivity. Most flk-1-positive neurons in the DRG, but not in the SCG, were also immunoreactive to neuropilin-1. VEGF was found to stimulate axonal outgrowth from DRG, both by an action on the growing axons and the nerve cell bodies. The latter effect could be mediated by retrograde axonal transport as revealed by the use of a two compartment system to assay axonal outgrowth. We also found that the VEGF-induced axonal outgrowth was blocked by the flk-1 inhibitor SU5416. The results strongly suggest that VEGF acts as a neurotrophic factor and plays an important role during the regeneration of peripheral nerves.

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The possibility that collateral sprouting could occur from intact axons in an undamaged sciatic nerve was studied in the rat by suturing either a 7-day predegenerated or a fresh nerve segment in an end-to-side fashion to the sciatic nerve proper. Following a 14- or 35-day recovery period, the pinch reflex test was performed on the transplanted segment to demonstrate the presence of sensory axons. The majority of cases, using a predegenerated nerve segment but not a fresh segment, responded positively. Neurofilament staining and histological examination confirmed the presence of axons in the attached nerve segment. In another series of experiments, the proximal peroneal fascicle was ligated and cut. Following a 7-day predegeneration period the distal stump was sutured end-to-side to the ipsilateral tibial fascicle. After 90 days, stimulation of the tibial nerve proximal to the attached site induced substantial contraction in both the native gastrocnemius muscle and the foreign tibialis anterior muscle. These findings suggest that collateral sprouting may occur from intact axons, perhaps induced by factors emanating from the attached nerve segment, and subsequently make functional peripheral connections.