Whole-Mount Adult Ear Skin Imaging Reveals Defective Neuro-Vascular Branching Morphogenesis in Obese and Type 2 Diabetic Mouse Models

Tomoko Yamazaki,Elise Bernhard,Sei-ichiro Motegi,Takahisa Nakamura,Haiming Cao,Ling Yang,Mark C. Udey,Ping Li,Yoh-suke Mukouyama,Wenling Li

doi:10.1038/s41598-017-18581-7

Abstract

Obesity and type 2 diabetes are frequently associated with peripheral neuropathy. Though there are multiple methods for diagnosis and analysis of morphological changes of peripheral nerves and blood vessels, three-dimensional high-resolution imaging is necessary to appreciate the pathogenesis with an anatomically recognizable branching morphogenesis and patterning. Here we established a novel technique for whole-mount imaging of adult mouse ear skin to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels. Whole-mount immunostaining of adult mouse ear skin showed that peripheral sensory and sympathetic nerves align with large-diameter blood vessels. Diet-induced obesity (DIO) mice exhibit defective vascular smooth muscle cells (VSMCs) coverage, while there is no significant change in the amount of peripheral nerves. The leptin receptor-deficient db/db mice, a severe obese and type 2 diabetic mouse model, exhibit defective VSMC coverage and a large increase in the amount of smaller-diameter nerve bundles with myelin sheath and unmyelinated nerve fibers. Interestingly, an increase in the amount of myeloid immune cells was observed in the DIO but not db/db mouse skin. These data suggest that our whole-mount imaging method enables us to investigate the neuro-vascular and neuro-immune phenotypes in the animal models of obesity and diabetes.

Highlights

Though diabetes is the most common recognized cause, human epidemiology and animal studies have demonstrated a link between obesity and neuropathy: diabetic neuropathy in type 2 diabetes appears to associate www.nature.com/scientificreports/
Our high-resolution whole-mount immunohistochemistry of the ear skin was performed with antibodies to platelet endothelial cell adhesion molecule (PECAM-1) as a pan-endothelial cell marker, neuron-specific class III β-tubulin (Tuj1) as a pan-neuronal marker, and α smooth muscle actin as a vascular smooth muscle cells (VSMCs) marker
The inner skin has smaller-diameter nerve bundles (

Summary

Introduction

Though diabetes is the most common recognized cause, human epidemiology and animal studies have demonstrated a link between obesity and neuropathy: diabetic neuropathy in type 2 diabetes appears to associate www.nature.com/scientificreports/. Together with the onset of glucose intolerance, high-fat diet (HFD) induces inflammatory cytokine expression, resulting in the activation of the innate immune system and macrophage infiltration Whether such a peripheral immune cell activation is a primary cause of nerve injury remains to be elucidated[3]. In the peripheral ear skin region, diet-induced obesity (DIO) mice exhibit defective VSMC coverage, while there is no significant change in the amount of nerves. An increase in the amount of myeloid immune cells was observed in the DIO, but not in db/db skin, suggesting that leptin signaling regulates the infiltration of these cells These data suggest that our whole-mount imaging method enables us to investigate the neuro-vascular and neuro-immune phenotypes in mouse models of obesity and diabetes

Methods

Results

Conclusion