In vitro high glucose increases apoptosis, decreases nerve outgrowth, and promotes survival of sympathetic pelvic neurons

Abstract

AbstractBackgroundDiabetes mellitus (DM) is a common cause of erectile dysfunction (ED), yet the molecular basis of DM neurogenic ED remains unknown.AimIn this study we examined the impact of high glucose on survival and growth of primary cultured pelvic neurons in a rat model and assessed whether coculturing with healthy Schwann cells (SCs) can rescue pelvic neuron growth in patients with DM.MethodsMajor pelvic ganglia (MPGs) from adult male Sprague Dawley rats (n = 8) were dissociated and plated on coverslips. Neurons were exposed to high glucose (45 mM) for 24 or 48 hours and compared to time-matched controls (25 mM). Neurons were stained for neuron-specific beta-tubulin, neuronal nitric oxide synthase, vesicular acetylcholine transferase, tyrosine hydroxylase, and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling) assay. Schwann cells were dissociated from MPGs of healthy male Sprague Dawley rats (n = 4) and grown to confluence. Additional Sprague Dawley rats were made diabetic with streptozotocin (50 mg/kg, n = 4), and 5 weeks later MPGs were collected from these rats, dissociated, and cocultured on healthy SCs. Neurons and SCs were stained with beta-tubulin and S100.OutcomesLength, branching, and survival of nitrergic, parasympathetic, and sympathetic neurons was assessed in neurons exposed to normal or high glucose concentrations, and neuron length was measured in neuron-SC coculture.ResultsThe total number of neurons and the length and number of branches were significantly decreased after 24 and 48 hours of high glucose (P < .05). The percentage of nitrergic neurons decreased 10% after 24 hours and 50% after 48 hours of high glucose (P < .05). After 24 hours of high glucose, cholinergic-positive neurons were unchanged; however, these neurons decreased 30% after 48 hours (P < .05). The proportion of sympathetic neurons increased 25% after 48 hours of high glucose (P < .05). At both timepoints, there was a 2-fold increase in the total apoptotic neurons with high glucose (P < .05). Neurite outgrowth recovered to control lengths after coculture of diabetic neurons with healthy SCs (P < .05).Clinical TranslationGlucose can be used as a tool to investigate the direct effects of DM on neuritogenesis. Our data suggest that an effective treatment for DM ED protects and repairs the penile neuronal supply.Strengths and LimitationsExposing MPG neurons to high glucose offers a quick and, inexpensive proxy for DM-related conditions. A limitation of our study is that our model reflects type 1 DM, whereas clinically, most diabetic ED patients have type 2 DM.ConclusionCulturing pelvic neurons in high glucose can be used as a tool to elucidate how to protect proerectile neurons from cell death and may lead to new therapeutic strategies for diabetic men suffering from ED.