No abstract

Diabetes mellitus-induced hyperglycemia is associated with a number of pathologies such as retinopathy, nephropathy, delayed wound healing, and diabetic peripheral neuropathy (DPN). Approximately 50% of patients with diabetes mellitus will develop DPN, which is characterized by disrupted sensory and/or motor functioning, with treatment limited to pain management. Zebrafish ( Danio rerio) are an emerging animal model used to study a number of metabolic disorders, including diabetes. Diabetic retinopathy, nephropathy, and delayed wound healing have all been demonstrated in zebrafish. Recently, our laboratory has demonstrated that following the ablation of the insulin-producing β-cells of the pancreas (and subsequent hyperglycemia), the peripheral nerves begin to show signs of dysregulation. In this study, we take a different approach, taking advantage of the transdermal absorption abilities of zebrafish larvae to extend the period of hyperglycemia. Following 5 days of 60 mM d-glucose treatment, we observed motor axon defasciculation, disturbances in perineurial glia sheath formation, decreased myelination of motor axons, and sensory neuron mislocalization. This study extends our understanding of the structural changes of the peripheral nerve following induction of hyperglycemia and does so in an animal model capable of potential DPN drug discovery in the future. NEW & NOTEWORTHY Zebrafish are emerging as a robust model system for the study of diabetic complications such as retinopathy, nephropathy, and impaired wound healing. We present a novel model of diabetic peripheral neuropathy in zebrafish in which the integrity of the peripheral nerve is dysregulated following the induction of hyperglycemia. By using this model, future studies can focus on elucidating the underlying molecular mechanisms currently unknown.

Diabetic peripheral neuropathy (DPN) is estimated to affect 50% of diabetic patients. Although DPN is highly prevalent, molecular mechanisms remain unknown and treatment is limited to pain relief and glycemic control. We provide a novel model of acute DPN in zebrafish ( Danio rerio) larvae. Beginning 5 days postfertilization (dpf), zebrafish expressing nitroreductase in their pancreatic β-cells were treated with metronidazole (MTZ) for 48 h and checked for β-cell ablation 7 dpf. In experimental design, this was meant to serve as proof of concept that β-cell ablation and hyperglycemia are possible at this time point, but we were surprised to find changes in both sensory and motor nerve components. Compared with controls, neurod+ sensory neurons were often observed outside the dorsal root ganglia in MTZ-treated fish. Fewer motor nerves were properly ensheathed by nkx2.2a+ perineurial cells, and tight junctions were disrupted along the motor nerve in MTZ-treated fish compared with controls. Not surprisingly, the motor axons of the MTZ-treated group were defasciculated compared with the control group, myelination was attenuated, and there was a subtle difference in Schwann cell number between the MTZ-treated and control group. All structural changes occurred in the absence of behavioral changes in the larvae at this time point, suggesting that peripheral nerves are influenced by acute hyperglycemia before becoming symptomatic. Moving forward, this novel animal model of DPN will allow us to access the molecular mechanisms associated with the acute changes in the hyperglycemic peripheral nervous system, which may help direct therapeutic approaches.

One of the most common comorbidities associated with diabetes mellitus is diabetic peripheral neuropathy (DPN), which is the progressive degeneration of peripheral nerves. The manner in which DPN develops and the underlying mechanisms of peripheral nerve degeneration are currently unknown. Our lab has characterized a glucose‐induced model of DPN in zebrafish (Danio rerio) in which we observe defasciculation/degeneration of motor axons, decreased myelination, and a decrease in ensheathment by perineurial glia. Currently, we are interested in characterizing the molecular pathways involved in the degeneration we observe. To accomplish this, we are testing dexamethasone, a FDA‐characterized small molecule drug, in an effort to decrease the degree of nerve degeneration present after DPN onset. Beginning 5 days post fertilization (dpf) experimental larvae were immersed in 120 mM d‐glucose for 7 seven days to induce DPN. At 10 dpf, larvae were additionally incubated with 0.5% dimethyl sulfoxide (DMSO) and a 1:1000 dilution of dexamethasone. As a control, a group of larvae were exposed to the same conditions without the addition of the drug treatment. Tg(nkx2.2a:meGFP) fish were used to allow visualization of perineurial glia 12 dpf using EVOS fluorescent microscopy. The percentage of motor axons ensheathed by perineurial glia was measured and quantified using FIJI. An increase in motor axons ensheathed by perineurial glia was apparent, furthermore the number of perineurial cells surrounding the motor axons appeared to increase as well. This increase in perineurial glia is meaningful because these cells form the blood‐nerve‐barrier, have phagocytic capabilities, and have also been shown to provide guidance cues for axonal regrowth. Future work will focus on characterizing the pathways affected by dexamethasone to uncover the molecular mechanisms potentially responsible for facilitating nerve regeneration after DPN onset. Support or Funding Information Support was provided by the Henson School of Science and Technology at Salisbury University.