Animal Studies

Compressive neuropathy is a recurring and challenging disease for patients, regardless of medical or surgical treatment. Neuropathological severity is associated with the force of mechanical compression. Available animal models do not address mechanical issues with reproducible outcomes. We used a chronic constriction injury model to analyze tension-controlled compressive neuropathy and achieve reproducible functional outcomes.

Anti-inflammatory corticosteroids are a common treatment for different conditions involving chronic pain and inflammation. Clinically used steroids target the glucocorticoid receptor (GR) for its anti-inflammatory effects. We previously reported that GR in sensory neurons may play central roles in some pain models and that GR immunoreactivity signal in dorsal root ganglia (DRG) decreased after local inflammation of the DRG (a model of low back pain). In the current study, we aimed to determine if similar changes in GR signal also exist in a skin inflammation model, the complete Freund's adjuvant (CFA) model (a model of peripheral inflammatory pain), in which the terminals of the sensory neurons rather than the somata are inflamed.

Neuropathic pain causes severe suffering, and most patients are resistant to current therapies. A core element of neuropathic pain is the loss of inhibitory tone in the spinal cord. Previous studies have shown that foetal GABAergic neuron precursors can provide relief from pain. However, the source of these precursor cells and their multipotent status make them unsuitable for therapeutic use. Here, we extend these findings by showing, for the first time, that spinally transplanted, terminally differentiated human induced pluripotent stem cell-derived GABAergic (iGABAergic) neurons provide significant, long-term, and safe relief from neuropathic pain induced by peripheral nerve injury in mice. Furthermore, iGABAergic neuron transplants survive long term in the injured spinal cord and show evidence of synaptic integration. Together, this provides the proof in principle for the first viable GABAergic transplants to treat human neuropathic pain patients.

Spinal cord injury (SCI) produces both locomotor deficits and sensory dysfunction that greatly reduce the overall quality of life. Mechanisms underlying chronic pain include increased neuro-inflammation and changes in spinal processing of sensory signals, with reduced inhibitory GABAergic signaling a likely key player. Our previous research demonstrated that spinal transplantation of GABAergic neural progenitor cells (NPCs) reduced neuropathic pain while intensive locomotor training (ILT) could reduce development of pain and partially reverse already established pain behaviors. Therefore, we evaluate the potential mutually beneficial anti-hypersensitivity effects of NPC transplants cells in combination with early or delayed ILT. NPC transplants were done at 4 weeks post-SCI. ILT, using a progressive ramping treadmill protocol, was initiated either 5 days post-SCI (early: pain prevention group) or at 5 weeks post-SCI (delayed: to reverse established pain) in male Sprague Dawley rats. Results showed that either ILT alone or NPCs alone could partially attenuate SCI neuropathic pain behaviors in both prevention and reversal paradigms. However, the combination of ILT with NPC transplants significantly enhanced neuropathic pain reduction on most of the outcome measures including tests for allodynia, hyperalgesia, and ongoing pain. Immunocytochemical and neurochemical analyses showed decreased pro-inflammatory markers and spinal pathology with individual treatments; these measures were further improved by the combination of either early or delayed ILT and GABAergic cellular transplantation. Lumbar dorsal horn GABAergic neuronal and process density were nearly restored to normal levels by the combination treatment. Together, these interventions may provide a less hostile and more supportive environment for promoting functional restoration in the spinal dorsal horn and attenuation of neuropathic pain following SCI. These findings suggest mutually beneficial effects of ILT and NPC transplants for reducing SCI neuropathic pain.

Unique features of sensory neuron subtypes are manifest by their distinct physiological and pathophysiological functions. Using patch-clamp electrophysiology, Ca imaging, calcitonin gene-related peptide release assay from tissues, protein biochemistry approaches, and behavioral physiology on pain models, this study demonstrates the diversity of sensory neuron pathophysiology is due in part to subtype-dependent sensitization of TRPV1 and TRPA1. Differential sensitization is influenced by distinct expression of inflammatory mediators, such as prostaglandin E (PGE), bradykinin (BK), and nerve growth factor (NGF) as well as multiple kinases, including protein kinase A (PKA) and C (PKC). However, the co-expression and interaction of TRPA1 with TRPV1 proved to be the most critical for differential sensitization of sensory neurons. We identified N- and C-terminal domains on TRPV1 responsible for TRPA1-TRPV1 (A1-V1) complex formation. Ablation of A1-V1 complex with dominant-negative peptides against these domains substantially reduced the sensitization of TRPA1, as well as BK- and CFA-induced hypersensitivity. These data indicate that often occurring TRP channel complexes regulate diversity in neuronal sensitization and may provide a therapeutic target for many neuroinflammatory pain conditions.

Currently, cancer pain is viewed as a process orchestrated by the release of pronociceptive molecules and the invasion of neural structures, referred to as perineural invasion (PNI). Cancer pain resulting from PNI is well-documented, but the mechanisms leading to peripheral sensitization because of tumor growth are not fully known.

Microglia activation following peripheral nerve injury has been shown to contribute to central sensitization of the spinal cord for the development of neuropathic pain. In a recent study, we reported that the amount of nerve damage does not necessarily correlate with chronic pain development. Here we compared the response of spinal microglia, using immunohistochemistry as a surrogate of microglial activation, in mice with two different types of crush injury of the sciatic nerve. We confirmed that incomplete crush of the sciatic nerve (partial crush injury, PCI) resulted in tactile hypersensitivity after the recovery of sensory function (15 days after surgery), whereas the hypersensitivity was not observed after the complete crush (full crush injury, FCI). We observed that immunoreactivity for Iba-1, a microglial marker, was greater in the ipsilateral dorsal horn of lumbar (L4) spinal cord of mice 2 days after FCI compared to PCI, positively correlating with the intensity of crush injury. Ipsilateral Iba-1 reactivity was comparable between injuries at 7 days with a significant increase compared to the contralateral side. By day 15 after injury, ipsilateral Iba-1 immunoreactivity was much reduced compared to day 7 and was not different between the groups. Our results suggest that the magnitude of the early microgliosis is dependent on injury severity, but does not necessarily correlate with the long-term development of chronic pain-like hypersensitivity after peripheral nerve injury.

Neuropathic pain following peripheral nerve injury (PNI) is linked to neuroinflammation in the spinal cord marked by astrocyte activation and upregulation of interleukin 6 (IL6 chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-X-C motif) ligand 1 (CXCL1), with inhibition of each individually being beneficial in pain models.