McMains, Jennifer C.

ABSTRACTBackgroundProstatic radiation therapy (RT) leads to erectile dysfunction by damaging peri-prostatic pro-erectile nerves of the pelvic ganglion. Schwann cells (SC) facilitate neuronal repair after mechanical injury, however, their role in repair of pelvic neurons post-radiation hasn’t been explored.AimTo determine if SCs cocultured with primary pelvic neurons can rescue neuronal survival and growth after ex vivo RT.MethodsMajor pelvic ganglia (MPG) were collected from adult male Sprague-Dawley rats (n = 12) to isolate SCs. SCs received RT (0 or 8 Gy), were plated on coated coverslips and grown to confluence before the addition of neurons. Additional MPGs were irradiated (0 or 8 Gy) and digested to isolate pelvic neurons. Dissociated neurons were plated alone or atop SC-coated coverslips to create 6 experimental groups (n = 3/grp): (i) Control (CON) MPG, (ii) RT MPG, (iii) CON SC + CON MPG, (iv) CONSC + RT MPG, (v) RT SC + CON MPG, and (iv) RT SC + RT MPG. After 72 hours, coverslips were fixed and stained for beta-tubulin (neuron marker), S100 (SC marker), neuronal nitric oxide synthase (nitrergic marker), tyrosine hydroxylase (sympathetic marker), and terminal deoxynucleotidyl transferase dUTP nick-end labeling.OutcomesWe measured neurite length, branching, specific neuron populations and apoptosis.ResultsEx vivo RT decreased MPG neuron length, increased apoptosis and decreased nitrergic neurons in monoculture. Compared to all other groups, CON SC + RT MPG cocultures demonstrated increased neurite outgrowth (P < .001). Neurite branching was decreased in the RT MPG + RT SC coculture, but unchanged in other cocultures. Groups containing RT MPG neurons exhibited increased apoptosis, but coculture with CON SC reduced the degree of RT-induced apoptosis (P < .01). The number of tyrosine hydroxylase positive neurons was unchanged while nitrergic neurons were significantly lower in RT neurons and coculture with CON SCs was unable to prevent nitrergic loss.Clinical TranslationThese findings suggest that SCs may be an important target in prostate cancer patients with radiation-induced pelvic neuropathy to promote MPG neuron survival and neuronal repair after RT.Strengths and LimitationsThis is the first study to characterize the ex vivo ability of SCs to rescue pelvic nerve growth and survival. The study is limited by little supporting mechanistic molecular data and the need to confirm the ability of healthy SCs to promote pelvic neuron survival and repair following prostatic RT in vivo.ConclusionUnirradiated SCs partially mitigated RT-induced MPG apoptosis but did not affect the loss of nitrergic neuron populations suggesting that SCs promote irradiated MPG neuron survival and facilitate intrinsic repair functions.

ABSTRACT Introduction Prostatic radiation therapy (RT) leads to erectile dysfunction (ED) by damaging adjacent pro-erectile nerves of pelvic ganglion. Preclinical cell culture studies have shown that RT induces pelvic neuron death and impairs neurite outgrowth. However, ex vivo RT and culture of whole pelvic ganglia leads to enhanced neurite outgrowth. Schwann cells (SC), which are present in intact ganglia, facilitate neuron repair after mechanical injury. The role of SCs in supporting neuron growth and survival in the context of RT has not been investigated. Objective Determine if SCs cocultured with primary pelvic neurons improve neuronal survival and growth after ex vivo RT. Methods Major pelvic ganglia (MPG) were collected from male Sprague-Dawley rats (n=12) and digested in collagenase/dispase to isolate SCs. SCs were plated on poly-L lysine-coated coverslips and grown in SC-selective medium to confluence for 24 hours. Confluent SCs received RT (0 or 8 Gy) before the addition of neurons. Additional MPGs (n=18) were irradiated (0 or 8 Gy) and digested to isolate pelvic neurons. Dissociated neurons were plated separately or atop SC-coated coverslips to create 6 experimental groups (n=3/grp): 1) CON MPG, 2) RT MPG, 3) CON SC+CON MPG, 4) CONSC+RT MPG, 5) RT SC+CON MPG, and 6) RT SC+RT MPG. After 72 hours, coverslips were fixed and stained for beta-tubulin (neuron marker), S100 protein (myelinating SC marker), neuronal nitric oxide synthase (nNOS; nitrergic marker), tyrosine hydroxylase (TH; sympathetic marker), and TUNEL to assess neurite length, branching, specific neuron populations and apoptosis. Results Consistent with previous findings, ex vivo RT decreased MPG neuron length, increased apoptosis and decreased nitrergic neurons in monoculture. In the current study, CON SC+RT MPG cocultures demonstrated increased neurite outgrowth compared to all other groups (p<0.001). Neurite branching was decreased in the RT MPG+RT SC coculture, but unchanged in other cocultures. Groups containing RT MPG neurons exhibited increased proportions of apoptosis, but coculture with CON SC reduced the degree of RT-induced apoptosis (p<0.01). The proportion of adrenergic TH positive neurons was unchanged while nitrergic neurons were significantly lower in RT neurons and coculture with CON SCs was unable to prevent nitrergic loss. Conclusions RT induces MPG neuronal apoptosis, inhibits post-injury neuritogenesis, and diminishes erectogenic nitrergic neuron populations. The presence of CON SCs in coculture augments MPG neuron repair mechanisms such as neurite outgrowth and branching. CON SCs partially mitigated overall RT MPG apoptosis, but did not affect the loss of nitrergic neuron populations after RT. These findings suggest that SCs promote MPG neuron survival and facilitate intrinsic repair functions acutely after RT. Further in vivo studies are needed to confirm the potential for healthy SCs to promote pelvic neuron survival and repair following prostatic RT. Disclosure No