An integrated approach for morphofunctional analysis of DRGs in normal and diabetic mice

Introduction: Nociceptive sensory neurons in dorsal root ganglia (DRGs) are first-order neurons conveying pain information to higher centers. Physiological pain has a protective role, which is disrupted in several pathologies leading to abnormal inflammatory or neuropathic pain. Diabetic polyneuropathy (DPN) is a common complication of diabetes and affects up to fifty percent of diabetic patients, some of which display neuropathic pain. Aims: To develop an integrated method for investigating the neurochemical and functional properties of DRG neurons in normal and diabetic mice; to analyze the spatial relationships of DRG neurons in a whole-mount preparations. Methods: CD1 male mice were made diabetic after a single intraperitoneal injection of streptozotocin (150 mg/Kg) at P30, while controls received vehicle only. Glucose levels and nociceptive behaviour were weekly monitored. All animals were sacrificed at P60. DRGs were acutely excised and the connective tissue was dissolved by incubation in 5-10 mg/mL collagenase. The entire DRGs were then used for patch-clamp recordings and, subsequently, for immunofluorescence, as the elimination of the connective capsule facilitated the access of the recording pipette as well as the penetration of primary antibodies. DRGs were stained for two classical phenotypic markers of nociceptors, i.e. the calcitonin gene-related peptide (CGRP) and the isolectin B4 (IB4) by a rabbit antibody and a biotin-conjugate, respectively. Whole DRGs Z Stacks were then collected using confocal microscopy. 3D quantitative analysis was performed using home-developed software, which provided automated counting of the immune-labeled neurons and quantification of their 3D morphological characteristics. Results: Pre-treatment with collagenase was found to improve reagent penetration in our whole-mount preparations, allowing an easy identification of the main populations of nociceptors (CGRP+ peptidergic and IB4+ non-peptidergic). The phenotypic identification of recorded neurons was possible thanks to the injection of a fluorescent tracer in the recorded neurons. Functional analysis confirmed an increase of pain hypersensitivity in diabetic mice which was paralleled by a decreased firing latency in DRG neurons. This difference was prominent in small cells (<25 µm). Consistent with functional analysis, 3D quantitative immunofluorescence revealed significant changes of CGRP and IBP-labeled structures between control and diabetic mice that can be related to DPN. Conclusions: This method is a flexible in vitro approach that preserves the neuroanatomical relationships between individual neurons, allowing also a functional analysis of their electrophysiological properties. The application of such a method in DRGs obtained from diabetic mice will shed new light on the morphofunctional alterations occurring in DPN.