Peripheral neuropathy is a devastating complication of diabetes conferring vast morbidity and mortality. Despite prolonged efforts to elucidate the mechanisms underlying diabetic related neuropathic phenomena and develop effective therapies, current treatment is for the most part glycemic control and symptomatic care. This is partially due to the intricate pathophysiology of diabetic neuropathy and the scarcity of valid experimental models. The aim of the study was to establish novel systems enabling monitoring and dissection of significant processes in the development of diabetic neuropathy. In a non-invasive in vivo model, two-photon microscopy is applied to evaluate mechanoreceptors (Meissner corpuscles) within an intact footpad of transgenic mice expressing a fluorescent neuronal tracer. By applying this advanced technology, which couples potent tissue penetration with superb resolution, we documented qualitative and quantitative diabetes-specific alterations in these sensory structures. Detection of such changes previously required laborious invasive histopathological techniques. In parallel, we present an ex vivo system that mimics the native microenvironment of the nerve ending via a unique co-culture of primary sensory neurons and thin skin slices. In conjunction with innovative high-throughput digital axonal measurements and computerized quantification tools, this method enables an unbiased exploration of neuronal autonomous and non-autonomous malfunctions. Using this setup we demonstrate that while the diabetic nerve retains a near-normal growth and regeneration capacities, the diabetic skin exhibits a decreased ability to support axonal outgrowth. Thus, an early target organ failure rather than intrinsic neuronal failure may initiate the neuropathy. Overall, the illustrated experimental platforms may greatly facilitate the holistic investigation of diabetic neuropathy.
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We are grateful to Dr. Guy Shakhar (Depratment of Immunology, the Weizmann Institute) and Mr. Yosef Addadi (Department of Biological Regulation, the Weizmann Institute) for their help with the two-photon microscopy and anesthesia, Dr. Rony Paz (Department of Neurobiology, the Weizmann Institute) for the statistical analysis and Dr. J Sanse, (Department of Cellular and Molecular Biology, Harvard University) for the YFP-16 mouse line. The research was funded by the Nella and Leon Benoziyo Center for Neurological Diseases of the Weizmann Institute.
Conflict of interest
The authors declare no conflict of interest.
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Supplementary figure 1 Whole embryonic dorsal root ganglia (DGR) explants were isolated and grown either alone (a) or in the presence of NGF (b) or healthy skin (d). NGF neutralizing antibodies abolished the NGF-induced axonal growth, yet not the skin-induced axonal growth (c and e respectively) (PDF 6123 kb)
Supplementary figure 2 Digital assessment of Meissner corpuscles: a. Serial footpad acquisitions are stacked to create a 2D image of the 3D projection. b. An automatic digital threshold is activated to enhance bright areas. c. Particle analysis tool is applied to sum the total square pixels of above-threshold intense areas (TIFF 1521 kb)
Supplementary video clips Pad clips a and b depict a 360º view of a 3D reconstruction of two-photon images of footpads (3rd pad of right hind limb) of control (clip a) or diabetic (clip b) mice sacrificed immediately before acquisition. Pad clip c depicts a reconstruction of a two-photon acquisition performed on a live anesthetized mouse (MPG 602 kb)
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Amit, S., Yaron, A. Novel systems for in vivo monitoring and microenvironmental investigations of diabetic neuropathy in a murine model. J Neural Transm 119, 1317–1325 (2012). https://doi.org/10.1007/s00702-012-0808-9
- Diabetic neuropathy
- In vivo imaging
- High-throughput analysis