Animal Studies

A well-recognized relationship exists between ageing and increased susceptibility to chronic pain conditions, underpinning the view that pain signaling pathways differ in aged individuals. Yet despite the higher prevalence of altered pain states among the elderly, the majority of preclinical work studying mechanisms of aberrant sensory processing are conducted in juvenile or young adult animals. This mismatch is especially true for electrophysiological studies where patch clamp recordings from aged tissue are generally viewed as particularly challenging. In this study we have undertaken an electrophysiological characterization of spinal dorsal horn neurons in young adult (3-4 months) and aged (28-32 months) mice. We show that patch clamp data can be routinely acquired in spinal cord slices prepared from aged animals and that the excitability properties of aged dorsal horn neurons differ from recordings in tissue prepared from young animals. Specifically, aged dorsal horn neurons more readily exhibit repetitive action potential discharge, indicative of a more excitable phenotype. This observation was accompanied by a decrease in the amplitude and charge of spontaneous excitatory synaptic input to dorsal horn neurons and an increase in the contribution of GABAergic signalling to spontaneous inhibitory synaptic input in aged recordings. While the functional significance of these altered circuit properties remains to be determined, future work should seek to assess if such features may render the aged dorsal horn more susceptible to aberrant injury or disease induced signaling and contribute to increased pain in the elderly.

The formation of neuromas involves expansion of the cellular components of peripheral nerves. The onset of these disorganized tumors involves activation of sensory nerves and neuroinflammation. Particularly problematic in neuroma is arborization of axons leading to extreme, neuropathic pain. The most common sites for neuroma are the ends of transected nerves following injury; however, this rodent model does not reliably result in neuroma formation. In this study, we established a rodent model of neuroma in which the sciatic nerve was loosely ligated with two chromic gut sutures [1]. This model formed neuromas reliably (~95%), presumably through activation of the neural inflammatory cascade. Resulting neuromas had a disorganized structure and a significant number of replicating cells. Quantification of changes in perineurial and Schwann cells showed a significant increase in these populations. Immunohistochemical analysis showed the presence of β-tubulin 3 (TUJ1) in the rapidly expanding nerve and a decrease in neurofilament heavy chain compared to the normal nerve, suggesting the axons forming a disorganized structure. Measurement of the permeability of the blood-nerve barrier (BNB) shows that it opened almost immediately and remained open as long as 10 days. Studies using an antagonist of the 3-adrenergic receptor (ADRβ3) (L-748,337) or cromolyn showed a significant reduction in tumor size and cell expansion as determined by flow cytometry, with an improvement in the animal's gait detected using a Catwalk system. Previous studies in our laboratory have shown that heterotopic ossification (HO) is also a result of the activation of neuroinflammation. Since HO and neuroma often occur together in amputees, they were induced in the same limbs of the study animals. More heterotopic bone was formed in animals with neuromas as compared to those without. These data collectively suggest that perturbation of early neuroinflammation with compounds such as L-748,337 and cromolyn may reduce formation of neuromas.

Multiple sclerosis (MS) is a neurodegenerative autoimmune disease with many known structural and functional changes in the central nervous system (CNS). A well-recognized, but poorly understood, complication of MS is chronic pain. Little is known regarding the influence of sex on the development and maintenance of MS-related pain. This is important to consider, as MS is a predominantly female disease. Using the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, we demonstrate sex differences in measures of spinal cord inflammation and plasticity that accompany tactile hypersensitivity. While we observed substantial inflammatory activity in both sexes, only male EAE mice exhibit robust staining of axonal injury markers and increased dendritic arborisation in morphology of deep dorsal horn neurons. We propose that tactile hypersensitivity in female EAE mice may be more immune-driven, while pain in male mice with EAE may rely more heavily on neurodegenerative and plasticity-related mechanisms. Morphological and inflammatory differences in the spinal cord associated with pain early in EAE progression supports the idea of differentially regulated pain pathways between the sexes. Results from this study may indicate future sex-specific targets that are worth investigating for their functional role in pain circuitry.

The anterior cingulate cortex (ACC) modulates emotional responses to pain. Whereas, the caudal ACC (cACC) promotes expression of pain affect, the rostral ACC (rACC) contributes to its suppression. Both subdivisions receive glutamatergic innervation, and the present study evaluated the contribution of NMDA receptors within these subdivisions to rats' expression of pain affect. Vocalizations that follow a brief noxious tail shock (vocalization afterdischarges, VAD) are a validated rodent model of pain affect. The threshold current for eliciting VAD was increased in a dose-dependent manner by injecting NMDA into the rACC, but performance (latency, amplitude and duration) at threshold was not altered. Alternately, the threshold current for eliciting VAD was not altered following injection of NMDA into the cACC, but its amplitude and duration at threshold were increased in a dose-dependent manner. These effects were limited to Cg1 of the rACC and cACC, and blocked by pre-treatment of the ACC with the NMDA receptor antagonist D-2-amino-5-phosphonovalerate (AP-5). These findings demonstrate that NMDA receptor agonism within the cACC and rACC either increase or decrease emotional responses to noxious stimulation, respectively. Perspective: NMDA receptor activation of the rostral and caudal anterior cingulate cortex respectively inhibited or enhanced rats' emotional response to pain. These findings mirror those obtained from human neuroimaging studies; thereby, supporting the use of this model system in evaluating the contribution of ACC to pain affect.

Bone cancer pain (BCP) is common in patients with advanced cancers as tumors metastasize to bone. Pathogenesis of BCP is complex and poorly understood which leads to inefficiency of clinical treatment. During pathological pain status, astrocytes are activated and release various inflammatory cytokines, which result in the development of peripheral and central sensitization. As energy factory, mitochondria undergo frequent fusion and fission and play essential role for astrocyte function. 2-bromopalmitate (2-BP) is an inhibitor of protein palmitoylation, and its function on BCP was unclear. In this paper, we aimed to investigate the potential curative effects and mechanisms of 2-BP on bone cancer pain. BCP rat model was established through intratibial inoculation of rat mammary gland carcinoma cells (MRMT-1) into the left tibia of Sprague-Dawley female rats. As a result, BCP rats exhibited bone destruction and sensitive nociceptive behavior. And increased leukocyte infiltration, activation of astrocytes, and imbalance of mitochondrial fission and fusion dynamics were observed in spinal cord of BCP rats. Intrathecal 2-BP administration significantly attenuated pain behavior of BCP rats. Meanwhile, 2-BP administration reduced spinal inflammation, reversed spinal mitochondrial fission and fusion dynamic imbalance, and further inhibited spinal mitochondrial apoptosis in BCP rats. In C6 cell level, 2-BP treatment decreased Drp1 expression and increased OPA1 expression in a dose-dependent manner, and inhibited CCCP-induced mitochondrial membrane potential change. These data illustrated that 2-BP attenuated bone cancer pain by reversing mitochondrial fusion and fission dynamic imbalance in spinal astrocytes.

Aim of this study was to detect the expression of miR-15a in rats following chronic constriction injury (CCI) and to investigate the regulatory functions of miR-15a during neuropathic pain (NP) development.