Sex- and injury-dependent nociceptive C-fibre temporal relay

Chronic pain is a debilitating condition, affecting significantly more females than males. It poses a significant clinical burden due to the currently limited effective treatments, reflecting limited understating of the mechanism underlying chronic pain states. Chronic pain symptoms include increased sensitivity to noxious heat stimuli (heat hypersensitivity), which is known to involve changes in the function of the pain-sensing C-fibres. It can also cause pain triggered by innocuous stimuli (allodynia), which is thought to involve central plasticity driven by the ongoing C-fibre activity. Given that majority of the studies investigating chronic pain mechanisms have been conducted primarily in males, it is therefore important to study the function of the pain-sensing C-fibres in both sexes. C-fibres display a unique phenomenon termed activity-dependent slowing (ADS) whereby repetitive stimulation results in a progressive slowing of action potential conduction velocity, which manifests as a progressive increase in response latency in both humans and animals. It has been shown to also alter the temporal relay of pain signals to the spinal cord, where pain processing occurs. In addition, C-fibre ADS has been demonstrated to be altered in neuropathic pain states and following tissue inflammation in a sex-dependent manner. Given the accumulating evidence for sex difference in acute and chronic pain sensitivity along with the recent findings of sex differences in the temporal relay of C-fibre mediated pain signals to the spinal cord in inflammatory pain states, this thesis aimed to study C-fibre ADS in both sexes in normal physiology and under pathological conditions. In addition, it aimed to determine the impact of C-fibre ADS on spinal heat pain processing in both sexes. C-fibre ADS was investigated, using compound action potential recordings in dorsal roots ex vivo, in normal physiology and in the incision-induced model of postoperative pain in juvenile rats of both sexes, with findings of incision-induced enhancement of ADS, which was more pronounced in females. This is proposed to contribute to the less pronounced reduction in peak heat hypersensitivity observed following incision in females. Given that the voltage-gated sodium (NaV) channels are implicated in C-fibre ADS, the impact of NaV channels on ADS in normal physiology and in the pathology of postoperative pain was explored in both sexes. Pharmacological manipulation of the tetrodotoxin-sensitive NaV channels suggests sex- and incision-dependent changes in NaV functional expression in different C-fibre subtypes. It has been previously demonstrated that blood plasma levels of the glycolytic metabolite methylglyoxal (MG) differentiate between diabetic neuropathy patients with or without pain symptoms, with one proposed mechanism of the MG-induced pain involving post-translational modifications of the NaV channels, which are also implicated in C-fibre ADS. Chronic but not acute application of MG was found to alter C-fibre ADS in a sex-dependent manner in juvenile rats, with females showing enhancement of ADS and males showing a reduction in ADS. These ADS findings can potentially contribute to the observed MG-induced heat hypersensitivity in males only. ADS has been suggested to provide a ‘memory’ of previous levels of activity, which could in turn influence responses to subsequent high-frequency stimuli, such that ADS induced by prolonged low-level firing, similar to spontaneous C-fibre firing observed in pain states, could impact subsequent responses to high frequency inputs by altering their ADS profile. This thesis has shown that prolonged low-frequency C-fibre stimulation could alter ADS levels in juvenile rats and adult mice in a sex-dependent manner. Given the fact that sex differences in thermal pain sensitivity have been shown to be strain- and species-dependent, the findings in this thesis of a more pronounced ADS in juvenile female Sprague-Dawley (SD) rats and in adult male C57BL/6 mice suggest that these may contribute to the previously observed higher heat thresholds in female SD rats and in male C57BL/6 mice. Patch clamp recording from noxious heat sensitive superficial dorsal horn neurons in adult mouse spinal cord slices, in both sexes, was used to assess the impact of ADS and dynamic memory on noxious heat processing on an individual spinal neuron level. In line with the observed more pronounced C-fibre ADS in the male C57BL/6 mice, there was more ADS in monosynaptic C-fibre inputs to the heat sensitive spinal neurons, which, however, was associated with an overall less pronounced evoked activity in those neurons in males. This finding likely reflects the initial less pronounced evoked activity of the heat sensitive spinal neurons in males, independent of C-fibre ADS. Using the length-dependency of ADS, it was demonstrated that pronounced ADS in monosynaptic C-fibre inputs is associated with less pronounced reduction in the activity of the heat sensitive spinal neurons in both sexes, suggesting C-fibre ADS as a potential mechanism involved in maintaining the activity of the heat sensitive spinal neurons following repetitive simulation. Dynamic memory significantly altered monosynaptic C-fibre input to noxious heat sensitive superficial dorsal horn neurons in both sexes, increasing the number of C-fibre synaptic input failures, which was reflected in the pronounced reduction of the activity of the heat sensitive spinal neurons in both sexes, suggesting that dynamic memory may act as an intrinsic self-inhibitory mechanism to limit the activity of the heat sensitive spinal neurons. This thesis proposes a role for C-fibre ADS in modulating noxious heat sensitivity in a sex-dependent manner in normal physiology and in different pain pathologies. In addition, this work highlights the potential influence of ADS on spinal circuits involved in noxious heat processing

Macrophage-derived insulin-like growth factor-1 is a key neurotrophic and nerve-sensitizing factor in pain associated with endometriosis

Endometriosis is a common incurable inflammatory disorder that is associated with debilitating pelvic pain in women. Macrophages are central to the pathophysiology of endometriosis: they dictate the growth and vascularization of endometriosis lesions and more recently have been shown to promote lesion innervation. The aim of this study was to determine the mechanistic role of macrophages in producing pain associated with endometriosis. Herein, we show that macrophage depletion in a mouse model of endometriosis can reverse abnormal changes in pain behavior. We identified that disease-modified macrophages exhibit increased expression of IGF-1 in an in vitro model of endometriosis-associated macrophages and confirmed expression by lesion-resident macrophages in mice and women. Concentrations of IGF-1 were elevated in peritoneal fluid from women with endometriosis and positively correlate with their pain scores. Mechanistically, we demonstrate that macrophage-derived IGF-1 promotes sprouting neurogenesis and nerve sensitization in vitro. Finally, we show that the Igf-1 receptor inhibitor linsitinib reverses the pain behavior observed in mice with endometriosis. Our data support a role for macrophage-derived IGF-1 as a key neurotrophic and sensitizing factor in endometriosis, and we propose that therapies that modify macrophage phenotype may be attractive therapeutic options for the treatment of women with endometriosis-associated pain.—Forster, R., Sarginson, A., Velichkova, A., Hogg, C., Dorning, A., Horne, A. W., Saunders, P. T. K., Greaves, E. Macrophage-derived insulin-like growth factor-1 is a key neurotrophic and nerve-sensitizing factor in pain associated with endometriosis

The formation of paranodal spirals at the ends of CNS myelin sheaths requires the planar polarity protein Vangl2

During axonal ensheathment, noncompact myelin channels formed at lateral edges of the myelinating process become arranged into tight paranodal spirals that resemble loops when cut in cross section. These adhere to the axon, concentrating voltage-dependent sodium channels at nodes of Ranvier and patterning the surrounding axon into distinct molecular domains. The signals responsible for forming and maintaining the complex structure of paranodal myelin are poorly understood. Here, we test the hypothesis that the planar cell polarity determinant Vangl2 organizes paranodal myelin. We show that Vangl2 is concentrated at paranodes and that, following conditional knockout of Vangl2 in oligodendrocytes, the paranodal spiral loosens, accompanied by disruption to the microtubule cytoskeleton and mislocalization of autotypic adhesion molecules between loops within the spiral. Adhesion of the spiral to the axon is unaffected. This results in disruptions to axonal patterning at nodes of Ranvier, paranodal axon diameter and conduction velocity. When taken together with our previous work showing that loss of the apico-basal polarity protein Scribble has the opposite phenotype—loss of axonal adhesion but no effect on loop–loop autotypic adhesion—our results identify a novel mechanism by which polarity proteins control the shape of nodes of Ranvier and regulate conduction in the CNS

LGI3/2-ADAM23 interactions cluster Kv1 channels in myelinated axons to regulate refractory period

Along myelinated axons, Shaker-type potassium channels (Kv1) accumulate at high density in the juxtaparanodal region, directly adjacent to the paranodal axon-glia junctions that flank the nodes of Ranvier. However, the mechanisms that control the clustering of Kv1 channels, as well as their function at this site, are still poorly understood. Here we demonstrate that axonal ADAM23 is essential for both the accumulation and stability of juxtaparanodal Kv1 complexes. The function of ADAM23 is critically dependent on its interaction with its extracellular ligands LGI2 and LGI3. Furthermore, we demonstrate that juxtaparanodal Kv1 complexes affect the refractory period, thus enabling high-frequency burst firing of action potentials. Our findings not only reveal a previously unknown molecular pathway that regulates Kv1 channel clustering, but they also demonstrate that the juxtaparanodal Kv1 channels that are concealed below the myelin sheath, play a significant role in modifying axonal physiology

LGI3/2-ADAM23 interactions cluster Kv1 channels in myelinated axons to regulate refractory period

Along myelinated axons, Shaker-type potassium channels (Kv1) accumulate at high density in the juxtaparanodal region, directly adjacent to the paranodal axon-glia junctions that flank the nodes of Ranvier. However, the mechanisms that control the clustering of Kv1 channels, as well as their function at this site, are still poorly understood. Here we demonstrate that axonal ADAM23 is essential for both the accumulation and stability of juxtaparanodal Kv1 complexes. The function of ADAM23 is critically dependent on its interaction with its extracellular ligands LGI2 and LGI3. Furthermore, we demonstrate that juxtaparanodal Kv1 complexes affect the refractory period, thus enabling high-frequency burst firing of action potentials. Our findings not only reveal a previously unknown molecular pathway that regulates Kv1 channel clustering, but they also demonstrate that the juxtaparanodal Kv1 channels that are concealed below the myelin sheath, play a significant role in modifying axonal physiology