Pioneering Translational Models of Pain
Uncovering the Path for Future Target-Based Treatments
Chronic Pain – A need for new approaches
Chronic pain is a devastating disease that affects approximately one in five individuals in Canada. Despite the personal, social, and economic challenges associated with poorly managed chronic pain, there are a lack of safe and effective treatments. To identify potential new therapeutic targets, the specific sequence of events that leads to chronic pain need to be determined. At a cellular level, chronic pain arises when neurons in the pain signalling pathway lose their ability to properly regulate their excitability (or output) by balancing excitatory versus inhibitory inputs. Researchers have identified many molecular candidates that are proposed to be involved in these pathological pain processes. However, the functional roles of individual molecular players in physiological (good) pain processing as well as how molecular functions are disrupted to cause runaway excitability and pathological (bad) pain are not well understood.
To compound the challenge of developing new approaches for treating chronic pain, most mechanistic research is based on rodent models of pain. These powerful models enable precise biochemical, genetic and pharmacological manipulations to tease out the roles of specific molecular, cellular, and network processes in driving pain hypersensitivity. The challenge arises once potential molecular drug targets are identified based on these rodent models. How do we know whether these targets are involved in the same pathological processes in specific human pain conditions? In the current pain research pipeline, there is a large translational gap between mechanistic pain studies in rodents and the testing of individual drug candidates in clinical trials. Our research aims to bridge this therapeutic divide by investigating the molecular players that underlie physiological and pathological pain using a combination of parallel rodent and human models of spinal pain processing.
Research Program Overview
The spinal cord is an essential component of the pain transmission pathway and is a major site of dysfunction in chronic pain syndromes. Within the dorsal horn of the spinal cord, the superficial layers (laminae I and II) form a neuronal pain processing network that integrates sensory signals from the periphery with modulatory signals from the brain and then relays processed messages up to brain pain centers. A myriad of pain-producing factors and events can shift the balance towards dorsal horn excitation and pain hypersensitivity, yet the underpinning molecular and cellular mechanisms are poorly understood. Our research program centers around the ion channel and receptor proteins that control the excitability of laminae I and II spinal neurons. We combine electrophysiological, biochemical, pharmacological, and behavioural approaches to explore the function and regulation of ion channels and receptors in dorsal horn neurons of both rodent and human spinal cord. Two main research themes in the lab are:
I. Investigating the physiological roles of ion channels and receptors in dorsal horn neurons across development.
The excitation of dorsal horn neurons is mediated by two main classes of proteins – ligand gated ion channels and voltage-gated ion channels. Within the voltage-gated channel superfamily, voltage-gated sodium channels and voltage-gated calcium channels are expressed in dorsal horn neurons and contribute to spinal pain processing. Many of the frontline treatments for pain, including opioids, anticonvulsants, and antidepressants, block voltage-gated calcium and/or sodium channel activity through direct and indirect mechanisms. However, the molecular, functional, and modulatory properties of voltage-gated sodium and calcium channels in dorsal horn neurons remain unresolved.
Synaptic excitation in the CNS, including the spinal cord dorsal horn, is primarily mediated through the activation of ligand-gated ion channels by glutamate. These ionotropic glutamate receptors generate depolarization, which activates voltage-gated ion channels and shapes neuronal excitability. The NMDA receptor (NMDAR) subtype of glutamate receptor has unique properties, enabling it to also function as an integrator of synaptic inputs and intra- as well as inter-cellular signalling. NMDARs are therefore critical in physiological mechanisms of learning and memory as well as pathological mechanisms of hyperexcitability. Biochemical, electrophysiological, and behavioural experiments have linked NMDAR activity to the amplification of pain outputs from the dorsal horn. Despite the central role of NMDARs in spinal cord pain signalling, the specific variants of NMDARs that mediate synaptic currents in dorsal horn neurons are unknown. Moreover, how NMDAR activity is directly modulated by pain producing signals remains to be tested.
Our lab is characterizing the functional properties and regulation of voltage-gated channels and NMDARs in rodent and human superficial dorsal horn neurons. We hypothesize that the differential expression of channel/receptor isoforms across development and between neuronal subpopulations contributes to functional heterogeneity and specialization within the superficial dorsal horn. We are also investigating whether mechanisms that regulate dorsal horn excitability are conserved or diverge between sexes and species.
II. Exploring mechanisms that lead to ion channel/receptor dysregulation and dorsal horn hyperexcitability in rodent and human models of pathological pain.
Pathological pain arises when neurons in the pain signalling pathway become sensitized by repeated exposure to pain-producing chemical and electrical inputs. These sensitized neurons fire action potentials in response to subthreshold inputs or even in the absence of inputs, leading to enhanced pain sensitivity and spontaneous pain, respectively. The molecular mechanisms underlying sensitization of peripheral nociceptors have been well explored, partly due to the ability to isolate and study the molecular, chemical, and electrical properties of nociceptors in situ. In contrast, although many cellular signalling pathways and molecules have been associated with dorsal horn central sensitization, experimental challenges have limited the ability to determine the actual functional outcome of these dorsal horn signalling pathways on ion channel activity.
Our lab is studying molecular pathways that cause an upregulation in the activity of excitatory voltage-gated channels and NMDARs. Importantly, we are investigating these pathological processes in both rodent and human model models of pathological pain. We use a combination of cell culture, ex vivo spinal models, and in vivo rodent models of chronic pain to test our hypotheses at levels ranging from molecular mechanisms to behavioural outcomes. As specific molecular targets are validated in the human model of spinal pathology, we use behavioural assays to test whether modulating these targets specifically reverses rodent pain hypersensitivity in vivo. Thus, our research aims to provide a foundation for the rational development of new target-based therapeutics that reverse human pain pathology.
The approaches described below are examples of the techniques that we combine in the lab to explore spinal cord physiology and synaptic signalling within distinct rodent and human models of pain processing.
Models of Pain
Rodent In Vivo
Rodent models of chronic inflammatory and neuropathic pain, with associated behavioural measurements of pain hypersensitivity. For example, Complete Freund’s Adjuvant (CFA) is injected into the rat hindpaw to induce inflammation and mechanical allodynia (decreased paw withdrawal thresholds) that persists for days.
Rodent Ex Vivo
Treating isolated spinal cord tissue from naïve rats with specific chemical drivers of pain hypersensitivity is used to study determinants of spinal hyperexcitability ex vivo. For example, treatment of viable rat spinal tissue with BDNF elicits the same pathological coupling between loss of inhibition and potentiation of excitatory NMDAR receptors as that observed in rat in vivo models of neuropathic (nerve injury) and inflammatory (CFA injection) pain.
Human Ex Vivo
Through collaboration with Dr. Eve Tsai’s research team at the Ottawa Hospital Research Institute, we are able to collect viable spinal tissue from neurologic determination of death organ donors. By treating adjacent spinal segments with chemical amplifiers of pain (such as BDNF) versus control saline, mechanisms of pathological pain processing are investigated in the human spinal cord. Physiological mechanisms of synaptic signalling and excitability are also studied in this viable human tissue.
Spinal Cord Physiology
Electrophysiological recordings of individual spinal neurons from either ex vivo tissue slices or in vitro culture preparations (see below). Overall membrane excitability as well as the biophysical and pharmacological properties of specific subtypes of ligand-gated and voltage-gated channels and receptors are measured and compared between species, sexes, pain states, and developmental stages.
Stem cells (spinal cord ependymal cells) and neurons are isolated and cultured from viable spinal cord and dorsal root ganglion tissue of both rats and humans. Responses of cells to various endogenous signalling factors and external agents are studied using a combination of electrophysiological, pharmacological and biochemical approaches.
A segment of viable rodent or human spinal cord is acutely sectioned into thin (300 to 500 um) parasagittal or transverse slices. Changes in excitability and molecular signalling are studied within individual and subpopulations of dorsal horn neurons using a combination of electrophysiological, pharmacological and biochemical approaches.
In Vivo Pharmacology
Molecular determinants of spinal hyperexcitability are targeted through IP or intrathecal delivery of specific antagonists or agonists in the above rodent models of pathological pain. The effects of the blockers or activators on pain hypersensitivity and spinal signalling are evaluated using a combination behavioural, electrophysiological, and biochemical assays.
Ex Vivo Pharmacology
Spinal sections from naïve rats as well as ex vivo and in vivo pathological pain models (see above) are treated with antagonists and agonists of specific molecular players involved in spinal pain processing. Experimental outcomes are measured using electrophysiological and biochemical approaches.
Fluorescent labelling of the localization and intensity of individual molecular players involved in pain processing in dorsal horn neurons from spinal cord sections as well as in cultured DRG and spinal cord neurons. Parallel experiments are conducted on cells collected from rodent as well as human tissue.
Western Blot/ Synaptosomes
The quantity of total and phosphorylated proteins in specific regions of the spinal cord (such as the superficial dorsal horn) are assayed using western blot analysis on rodent and human tissue from control and various pain model conditions. Potential changes specific to synaptic proteins are tested using synaptosome versus cell homogenate conditions.
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