Presentation Authors: Joshua Randolph*, Elena Pak, Johanna Hannan, Greenville, NC
Introduction: Prostatic radiation therapy (RT) causes nerve damage to the major pelvic ganglia (MPG) resulting in erectile dysfunction (ED). To understand the impact of neuronal injury, MPGs have previously been studied in vitro using whole ganglia or dissociated neuron cultures. To date, these two methods have not been directly compared. This study investigates effects of ex vivo radiation on MPG neuron survival and growth in dissociated vs whole culture.
Methods: MPGs from male Sprague-Dawley rats (n=14) were removed and irradiated ex vivo (0 or 800cGy). For dissociated culture, MPGs were digested in collagenase/dispase and neurons cultured on coverslips for 72 hours. Immunofluorescent staining for beta-tubulin (neuron marker), neuronal nitric oxide synthase (nNOS; nitrergic marker), tyrosine hydroxylase (TH; sympathetic marker), and TUNEL was performed to assess neurite length, branching and apoptosis. For whole culture, MPGs were grown in Matrigel and neurite growth was measured at 72 hours. Following whole culture, MPG gene expression of apoptosis markers (Caspase 1, 3), myelination (Sox10, Krox10), glial dedifferentiation (GFAP), nNOS, TH, and TUBB3 (beta-tubulin) was measured.
Results: Irradiated dissociated MPGs demonstrated less neurite outgrowth (p < 0.01) and no change in neurite branching. Apoptosis was markedly increased in irradiated dissociated neurons (p < 0.001). The prevalence of nNOS+ neurons was strikingly decreased in irradiated dissociated culture (p < 0.05), while the proportion of TH+ neurons was unchanged. In contrast, irradiated MPGs grown in Matrigel had slightly increased neurite length (p < 0.08). Gene expressions of Caspase 3, Sox10, Krox10, nNOS, and TUBB3 was increased in irradiated whole culture (p < 0.05). Overall the two different culture methods had opposing neurite growth following irradiation. Dissociated culture provided clearer neurites that allowed for measurement of length, branches and could be stained for specific neuron types, whereas whole MPG culture neurites were harder to identify and could not be clearly stained.
Conclusions: This study is the first to compare dissociated and whole MPG culture methods. The opposing trends in outgrowth with radiation may be due to the other factors present in whole MPG culture such as Schwann cells (SCs) which are known to facilitate repair and may be providing a neurotrophic environment. Future studies will investigate the mechanisms by which SCs increase neuritogenesis following ex vivo radiation so that we may identify novel targets for therapy.
Source of Funding: BSOM Summer Scholars Research Award