Development of an in vitro model of "pain"

Pain is a complex multidimensional sensory experience. By definition pain is function of higher brain functions. However, there is now very strong evidence that the majority of clinically relevant pain depends on abnormal primary sensory neurone activity. This is not to say that changes within the brain are not important, but these central changes depend in large measure on abnormal peripheral neurone excitability. The overarching aim of this application is to exploit the knowledge to replace and reduce the use of animal models in pain research. Application of disease modelling using in vitro platforms could potentially be the most effective method of reducing number of animals used in biomedical research. The challenge, particularly in neurological conditions, is to develop relevant models that can recapitulate salient features of a given pathology while preserving, as much as possible, the relationship between cells in a tissue. The use of microfluidic cultures in neuroscience has enabled preserving the connectivity of different neuronal cells while isolating them in their own environment. Our work has shown that microfluidic cultures of pain sensing neurons respond to damage in a similar way, by becoming sensitized, as do in animal models of pain.
The aim of this project is to develop a tissue culture platform and associated methodologies that will allow us to investigate the changes in many aspects of pain signalling in exquisite detail in response to injuries that result in chronic pain in animals and humans. This versatile platform that would be based on a number of advanced enabling technologies including microfluidic systems and optogenetic activation of nerve cells, would allow researchers to simulate aspects of nerve injury, in a culture dish and study the way these nerve cells respond to injury and how they change their communication to other neurons through altered synapses.
Pain signalling in humans and mammals in general, is highly integrated and complex, involving the immune system, specialized pain sensing neurons in the periphery and the spinal cord as well as pain processing in the brain. We can evaluate changes in signalling in neurons after injury in relation to immune cells and neuroimmune regulators in order to better understand the interactions between these components of the pain system. Ultimately this platform has to potential to significantly impact use of lesion based animal models in pain research.
The current advances in growing human neurons in a dish affords a great opportunities to model neurological diseases in human neurons. Our aim is is extend the use of in vitro platform in neurological research by studying injury responses of human neurons obtained from reprogrammed skin cells in a dish which is common to many neurological disorders. Our project will deliver tools that will impact animal use in pain research and therapeutic development.

Pathological changes in axonal function are integral features of many neurological disorders, yet our knowledge of the molecular basis of axonal dysfunction remains limited. This is due difficulties in using animal model to study detailed neurophysiology of nociceptors. Nerve injury results in long lasting pain hypersensitivity in animal models of pain. We show that microfluidic chambers can provide unique insight into the axonal compartment independent of the soma and can be used to assay the physiological properties of the axonal and somal compartments. We illustrate the ease and versatility to assay electrogenesis and conduction of action potentials (APs) in naïve, damaged or sensitized (using chemotherapeutic agents for instance) nociceptive axons using calcium imaging at the soma or patch-clamp electrophysiology for detailed biophysical characterisation. Impact of non-neuronal cells can be readily studied on the co-culture system through additional microfluidic compartments. We further demonstrate adaptability of the system for co-culturing and studying synaptic function between sensory neurons and dorsal horn neurons of the spinal cord. Hence we describe a novel in vitro platform for the study peripheral pain signalling and as surrogate model for nerve injury and sensitization in animals. We further show the iPSC-derived neurons can be used in this platform and inform major insights into the physiological responses of human neurons to injury.
This toolbox can have significant advantages over in vivo models in deciphering intricate molecular relationships underpinning pain hypersensitivity. As such it has immense potential to impact replacement of animal models in pain research.

Application of medically relevant, in vitro models of disease can significantly impact the number of animals used in biomedical research. However, up until now development of in vitro models of pain has been slow and challenging. This is primarily due to limitations of the existing in vitro and ex vivo models to capture the complexities of tissue injury and pain signalling. We have shown that a microfluidic based tissue culture system has the potential to overcome these limitations and offer significantly greater insight into mechanisms of axonal injury and sensitization. We posit that the wider use of in vitro models of axonal injury will lead to a reduction in the number of painful procedures performed in rodents by up to 30%. A brief meta-analysis of studies published during 2009-2013 validates this point: only 779 publications report having used cultures of sensory neurons in study of pain hypersensitivity. In contrast 3590 publications addressed pain mechanisms using injury to peripheral nerves [1]. Using our microfluidic culture system the physiological responses of sensory neurons following axonal injury in culture can be directly addressed hence requiring fewer animals to undergo painful procedures to study these properties. In addition a culture model of axonal injury will enable research projects to demand fewer animals since tissue extracted from one animal can be used to seed multiple culture dishes.
Further reductions in animal use in pain research is achievable through the use of hiPSC-derived neurons as a model system. Our proposal to develop tools to study the physiological responses of hiPSC-derived neurons (hiPSC-N) to axonal injury, will directly impact the number of animals needed for in vitro studies. We estimate 20% of published reports on pain mechanisms involve cultures of injured primary DRG neurons from rat or mouse which can be replaced by hiPSC-Ns [1]. As the protocols for obtaining enriched population of hiPSC-derived sensory neurons are becoming rapidly accessible [2], a medically relevant surrogate model of nerve damage will provide an alternative to animal models of nerve injury. Further impact will be in industry where an in vitro human model of neuronal injury will be of much value and significant reductions in animal use in preclinical drug development programs would be expected.
1-source: Google Scholar 2- PLOS One 2012 7(12):e53010