Chronic pain is the single biggest global disease burden. Chronic pain conditions originate after periods of intense neuronal activity but the cellular and molecular mechanisms underlying nociception, particularly the transition from acute to chronic pain, are not well understood. Nociceptive circuits are complex and involve interplay between several different neuron types. This complexity is difficult to model with current in vitro models and studies of nociception typically rely on in vivo systems. However, there have been issues with animal models accurately predicting clinical efficacy of analgesics.
Why we funded it
This PhD Studentship aims to reduce the requirements of animal models in the study of nociception by developing a compartmentalised microfluidic cell culture system.
An in vivo study of a behavioural test of nociceptive function comparing a single dose of a candidate drug to a control group requires approximately 20 rats. A hypothetical study of 15 different experimental conditions, for example screening 15 different drugs, would therefore typically require approximately 300 rats. The microfluidic systems developed in this proposal have the potential to reduce the number of rats required by 90%, reducing the rats needed for the aforementioned hypothetical study from 300 to 30.
Nociceptive circuits extend from the central nervous system (CNS) to the periphery, connecting a multitude of target tissues to the CNS. Sub-cellular compartments of the nervous cells are separate and exist in differing environments, for example the cell body is in the dorsal root ganglion whereas the terminals are in either the CNS or in peripheral tissues. This compartmentalisation and the polarisation of the nociceptive circuits are not currently reflected in in vitro models. This proposal uses a compartmentalised microfluidic system to allow the culturing of primary neurons isolated from rat embryos whilst retaining the morphological development seen in vivo. Embryos isolated from one rat are able to generate enough neurons to culture in ~30 microfluidic chambers. Once developed, this system will be used to determine the role of microRNA (miRNA) regulation in the transition from acute to chronic nociception. The microfluidic system has the capacity to allow investigation of novel molecular mechanisms in addition to the potential for screening of compounds in pain research.
The mechanisms that control the development of chronic nociception are not yet fully understood. Despite recent advances, the complex interplay achieved by different neuronal types in nociceptive circuits has not been adequately modelled in vitro, with cell culture experiments ignoring the incredible anatomical polarisation of dorsal root ganglia (DRG) neurons.
In this context, there is a clear need to implement physiologically relevant in vitro systems that could allow the investigation of cellular and molecular mechanisms in a context of multi-cellular circuit complexity. Culture platforms with fluidically isolated compartments allow the functional separation of neuronal domains in more physiologically relevant contexts. We have already demonstrated the ability to replicate the pseudo-unipolar nature of DRG neurons in functional experiments by measuring Ca2+ signals in the cell body compartment of microfluidic systems.
We will further develop these novel culture systems by sequentially adding levels of network complexity. Dorsal horn primary neurons and keratinocytes will be seeded into the lateral compartments of three-channel microfluidic devices that have DRG neurons placed in the middle channel. This system will model central and peripheral domains of DRG neuron circuitry, but with greater experimental accessibility.
Our preliminary work identified key miRNAs that are changed in hyperalgesic priming, an in vivo model for studying the transition from acute to chronic pain. Although local protein expression has been suggested in hyperalgesic priming mechanisms, the role of miRNAs has not been investigated. Using the proposed system, miRNA inhibitors will be added into the peripheral and central compartments while neuron excitability and expression of synaptic markers will be evaluated. This will allow us to determine the potential role of miRNAs in the control of protein expression and local regulation of neuronal circuits during nociception.