Morphofunctional alterations in nociceptors of diabetic mice

Diabetic polyneuropathy is among the most common long-term complications of diabetes mellitus and affects up to fifty percent of diabetic patients, some of which (15-25%) display chronic neuropathic pain. Nociceptive pathways are activated by a specific subset of peripheral sensory receptors, the nociceptors, that provide information about tissue damage from the soma and/or viscera. The cell bodies of nociceptors are located in the dorsal root ganglia (DRGs) that relay nociceptive stimuli to the central nervous system. Since previous studies showed an involvement (among others) of the glial-derived neurotrophic factor (GDNF) in neuropathic pain, here we investigate the intervention of GDNF in the alterations of DRGs that are consequent to the diabetic neuropathy by combining electrophysiological and morphological approaches. To mimic type 1 diabetes, four week-old male mice are injected intraperitoneally with streptozotocin (STZ, 150 mg/kg) that selectively kills insulin-producing pancreatic β-cells. Diabetes-related alterations in DRGs are analyzed by immunofluorescence on fixed tissues. The main populations of nociceptors are identified by an IB4 biotin-conjugate and an anti-CGRP antibody that respectively label non-peptidergic and peptidergic nociceptors. An anti-p-ERK antibody is used to report the activation of the MAPK/ERK pathway that plays a critical role in central sensitization and transduction of nociceptive signals. Electrophysiological recordings are carried out on intact DRGs, after treatment with collagenase (5 mg/ml) to remove the connective capsule of the ganglia. Neurons are recorded in whole cell configuration and different protocols are applied to analyze the firing pattern (current clamp) or the threshold for the activation of sodium currents (voltage clamp). Recorded neurons are intracellularly labelled by Lucifer yellow or Alexa 568 for post-recording identification. Preliminary results indicate that neuronal activation measured by p-ERK increases in nociceptors of diabetic mice in comparison to normal controls. This is paralleled by an increased firing activity in recorded neurons. Administration of GDNF during electrophysiological recordings delays the firing in a subset of neurons. The phenotype (peptidergic vs non-peptidergic) of recorded neurons is presently being addressed by combining patch clamp and immunohistochemical analysis. If confirmed, our data indicate a protective role of GDNF against the increased activity of nociceptors in diabetes.