Integrating Sensory Neurons with Keratinocytes to Model Painful Diabetic Neuropathy on a Chip

Abstract

Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. PDN is characterized by small-fiber degeneration and neuropathic pain. Uncovering the mechanisms underlying neurodegeneration in PDN remains a major challenge to finding effective and disease-modifying therapy. Keratinocytes are closely juxtaposed to cutaneous nerve terminals potentially enabling communication between keratinocytes and cutaneous afferents. The aim of this study is to explore mechanisms by which keratinocytes communicate with cutaneous afferents and how this communication impacts DRG neuron axonal degeneration underlying neuropathic pain in PDN. We have established a Xona microfluidic coculture device to compartmentalize murine DRG neurons and keratinocytes to model the skin in vitro and investigated the effects of activated K14 keratinocytes on DRG neurite outgrowth and neuron excitability. We found that cocultured DRG neurites grew towards keratinocytes within 7 days. Additionally, we found that once the neurites grew in the microchannels, they are unable to turn back and continue to grow into the adjacent compartment, where they form connections with cultured keratinocytes. Using electrophysiological and calcium images studies on these microfluidic devices we revealed a dynamic interplay between the neuronal activity and keratinocytes. For example, we have used this system to test the effects on DRG neurons neurite outgrowth and excitability upon application different stimuli including class III semaphorins and chemokines (neutrophil chemo-attractant genes such as CXCL10), known to be secreted by keratinocytes. More recently we have used this platform to study interaction between human iPSCs derived DRG neurons and human keratinocytes. In conclusion, this platform can be used to explore mechanisms by which keratinocytes communicate with cutaneous afferents and how this communication impacts DRG neuron axonal degeneration underlying neuropathic pain in PDN. Grant support from 1R01AR77691-01. Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. PDN is characterized by small-fiber degeneration and neuropathic pain. Uncovering the mechanisms underlying neurodegeneration in PDN remains a major challenge to finding effective and disease-modifying therapy. Keratinocytes are closely juxtaposed to cutaneous nerve terminals potentially enabling communication between keratinocytes and cutaneous afferents. The aim of this study is to explore mechanisms by which keratinocytes communicate with cutaneous afferents and how this communication impacts DRG neuron axonal degeneration underlying neuropathic pain in PDN. We have established a Xona microfluidic coculture device to compartmentalize murine DRG neurons and keratinocytes to model the skin in vitro and investigated the effects of activated K14 keratinocytes on DRG neurite outgrowth and neuron excitability. We found that cocultured DRG neurites grew towards keratinocytes within 7 days. Additionally, we found that once the neurites grew in the microchannels, they are unable to turn back and continue to grow into the adjacent compartment, where they form connections with cultured keratinocytes. Using electrophysiological and calcium images studies on these microfluidic devices we revealed a dynamic interplay between the neuronal activity and keratinocytes. For example, we have used this system to test the effects on DRG neurons neurite outgrowth and excitability upon application different stimuli including class III semaphorins and chemokines (neutrophil chemo-attractant genes such as CXCL10), known to be secreted by keratinocytes. More recently we have used this platform to study interaction between human iPSCs derived DRG neurons and human keratinocytes. In conclusion, this platform can be used to explore mechanisms by which keratinocytes communicate with cutaneous afferents and how this communication impacts DRG neuron axonal degeneration underlying neuropathic pain in PDN. Grant support from 1R01AR77691-01.