Neurotrophic effects of vascular endothelial growth factor and neurotrophins on cultured major pelvic ganglia

Abstract

Objective: To investigate the feasibility of using a ganglial culture system to screen various growth factors as potential therapeutic agents for pelvic nerve injuries.

Materials and methods: The major pelvic ganglia (MPG) were isolated from male rats and attached to culture dishes with the aid of Matrigel (Becton Dickinson, Mountain View, CA, USA). Alternatively, the dorso-caudal region (DCR) of MPG, from which the cavernous nerves originate, was dissected and then attached to a Matrigel-coated coverslip. The MPG or DCR was cultured in serum-free medium supplemented with phosphate-buffered saline (PBS, control), 50 ng/mL of vascular endothelial growth factor (VEGF), 20 ng/mL of a neurotrophin (BDNF, NT3, or NT4), or combinations of these growth factors. After 2 days of incubation, the ganglial tissues with their outgrowing nerve fibres were stained for the expression of NADPH-diaphorase, tyrosine hydroxylase (TH) and acetylcholinesterase (AChE). The length and staining intensity of nerve fibres were analysed.

Results: The outgrowing fibres were significantly longer in MPG treated with any of the four tested growth factors than in PBS-treated MPG. The combination of VEGF and NT3 induced the best fibre growth. Improvements to the culturing conditions allowed a histological examination of the outgrowing fibres for the expression of nitric oxide synthase (NOS), TH and AChE. VEGF and BDNF were equally capable of inducing NOS- and TH-expressing fibres. BDNF was much weaker than VEGF for inducing AChE-expressing fibres.

Conclusions: This improved culturing system is potentially useful for screening nerve-regenerating factors; VEGF had neurotrophic effects comparable with BDNF, NT3, or NT4.