309 Radiation-induced Schwann Cell Dedifferentiation Leads to Opposing Growth in Whole Ganglia vs Dissociated Cultured Pelvic Neurons

Abstract

Prostatic radiation therapy (RT) is a highly effective treatment for localized low-grade prostate cancer. Unfortunately, in 67-85% of patients, RT causes erectile dysfunction (ED). The direct molecular mechanisms underlying RT-induced ED are not understood, and no effective therapies have been identified. The major pelvic ganglia (MPG) in rats is anatomically equivalent to the hypogastric plexus in humans and can be used to assess radiation-induced nerve injury. Both whole and dissociated MPG cultures have examined neurite growth and apoptosis in models of ED; however, the impact of radiation on MPG neuron growth and survival has not been directly compared in these culture conditions. Determine the effects of ex vivo radiation on MPG neuron survival, growth, and gene expression in whole vs dissociated culture. MPGs from male Sprague-Dawley rats (n=14) were removed and irradiated ex vivo (0 or 800 cGy). For dissociated culture, MPGs were digested in collagenase/dispase. Neurons were collected by trituration and cultured on coverslips for 72 hours. Immunofluorescent staining for beta-tubulin III (Tubb3; neuron marker), neuronal nitric oxide synthase (nNOS; nitrergic marker), tyrosine hydroxylase (TH; sympathetic marker), and TUNEL assessed neurite length, branching, autonomic neuron density, and apoptosis. For whole culture, MPGs were grown in Matrigel and neurite growth was measured at 72 hours. Gene expression of apoptotic markers (Caspase 1, 3), myelination (Sox2, Sox10, Krox20), glial dedifferentiation (GFAP), Tubb3, nNOS, and TH was measured in irradiated cultures of dissociated MPG neurons, isolated Schwann cells (SCs), and whole cultured MPGs by qPCR.