Animal Studies

Spinal cord injury (SCI) usually causes a devastating lifelong disability for patients. After a traumatic lesion, disruption of the blood-spinal cord barrier induces the infiltration of macrophages into the lesion site and the activation of resident glial cells, which release cytokines and chemokines. These events result in a persistent inflammation, which has both detrimental and beneficial effects, but eventually limits functional recovery and contributes to the appearance of neuropathic pain. Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that regulate the expression of inflammatory genes by interacting with acetylated lysine residues. While BET inhibitors are a promising therapeutic strategy for cancer, little is known about their implication after SCI. Thus, the current study was aimed to investigate the anti-inflammatory role of BET inhibitors in this pathologic condition.

The cation channel TRPA1 transduces a myriad of noxious chemical stimuli into nociceptor electrical excitation and neuropeptide release, leading to pain and neurogenic inflammation. Despite emergent evidence that TRPA1 is regulated by the membrane environment, it remains unknown whether this channel localizes in membrane microdomains or whether it interacts with cholesterol. Using total internal reflection fluorescence microscopy and density gradient centrifugation we found that mouse TRPA1 localizes preferably into cholesterol-rich domains and functional experiments revealed that cholesterol depletion decreases channel sensitivity to chemical agonists. Moreover, we identified two structural motifs in transmembrane segments 2 and 4 involved in mTRPA1-cholesterol interactions that are necessary for normal agonist sensitivity and plasma membrane localization. We discuss the impact of such interactions on TRPA1 gating mechanisms, regulation by the lipid environment, and role of this channel in sensory membrane microdomains, all of which helps to understand the puzzling pharmacology and pathophysiology of this channel.

Immunohistochemical characterization of primary afferent fibers (intact or after nerve damage) is traditionally performed in thin sections from dorsal root ganglia (DRGs) or in teased fibers, as light scattering in whole-mounts compromises visualization. These procedures are time-consuming, require specific equipment and advanced experimental skills. Lipid-clearing techniques are increasing in popularity, but they have never been used for the peripheral nervous system. We established a modified, inexpensive clearing method based on lipid-removal protocols to make transparent peripheral nerve tissue (inCLARITY). We compared retrograde-labeling and free-floating immunostaining with cryo-sections. Confocal microscopy on whole-mount transparent DRGs showed neurons marked with retrograde tracers applied to experimental neuromas (Retrobeads, Fluoro-ruby, Fluoro-emerald, DiI, and Fluoro-gold). After immunostaining with calcitonin gene-related peptide (peptidergic) or isolectin IB4 (non-peptidergic), nociceptors were visualized. Immunostaining in transparent whole-mount nerves allows simultaneous evaluation of the axotomized branches containing the neuroma and neighboring intact branches as they can be mounted preserving their anatomical disposition and fiber integrity. The goal of our study was to optimize CLARITY for its application in peripheral nerve tissues. The protocol is compatible with the use of retrograde tracers and improves immunostaining outcomes when compared to classical cryo-sectioning, as lack of lipids maximizes antibody penetration within the tissue.

Hyperexcitability of the anterior cingulate cortex (ACC) is thought to drive aversion associated with chronic neuropathic pain. Here, we studied the contribution of input from the mediodorsal thalamus (MD) to ACC, using sciatic nerve injury and chemotherapy-induced mouse models of neuropathic pain. Activating MD inputs elicited pain-related aversion in both models. Unexpectedly, excitatory responses of layer V ACC neurons to MD inputs were significantly weaker in pain models compared to controls. This caused the ratio between excitation and feedforward inhibition elicited by MD input to shift toward inhibition, specifically for subcortically projecting (SC) layer V neurons. Furthermore, direct inhibition of SC neurons reproduced the pain-related aversion elicited by activating MD inputs. Finally, both the ability to elicit pain-related aversion and the decrease in excitation were specific to MD inputs; activating basolateral amygdala inputs produced opposite effects. Thus, chronic pain-related aversion may reflect activity changes in specific pathways, rather than generalized ACC hyperactivity.

Neuropathic pain, resulting from somatosensory nervous system dysfunction, remains a serious public health problem worldwide. microRNAs are involved in the physiological processes of neuropathic pain. However, the biological roles of miR-98 in neuropathic pain development have not been investigated. Therefore, in our current study, we focused on the effects of miR-98 in neuropathic pain. It was shown that miR-98 was significantly downregulated in chronic sciatic nerve injury (CCI) rat models. In addition, high mobility group A2 (HMGA2) was obviously upregulated in CCI rats. Overexpression of miR-98 inhibited neuropathic pain progression, including mechanical and thermal hyperalgesia. By a bioinformatics analysis, HMGA2 was predicted as a direct target of miR-98. The negative correlation between miR-98 and HMGA2 was validated in our present study. Furthermore, overexpression of miR-98 dramatically repressed HMGA2 protein and messenger RNA (mRNA) expression. Neuroinflammation participates in neural-immune interactions, which can contribute to the neuropathic pain development. Meanwhile, we found that inflammatory cytokine (interleukin [IL]-6, IL-1β, and COX-2) protein expression in rats infected with LV-miR-98 was greatly suppressed. Taking these results together, we concluded that miR-98 might depress neuropathic pain development through modulating HMGA2.

The present study aimed to investigate cerebral metabolic changes in a neuropathic pain model following deafferentation. A total of 24 Sprague-Dawley rats were included for modeling of right brachial plexus avulsion (BPA) through the posterior approach. As nerve injury would cause central sensitization and facilitate pain sensitivity in other parts of the body, thermal withdrawal latency (TWL) of the intact forepaw was assessed to investigate the level of pain perception following BPA-induced neuropathic pain. [Fluorine-18]-fluoro-2-deoxy-D-glucose (F-FDG) positron emission tomography (PET) was applied to the brain before and after brachial plexus avulsion to explore metabolic changes in neuropathic pain following deafferentation. The TWL of the left (intact) forepaw was significantly lower after BPA than that of baseline (p<0.001). Using TWL as a covariate, standardized uptake values (SUVs) of F-FDG significantly increased in the ipsilateral dorsolateral thalamus and contralateral anterodorsal hippocampus after BPA. Conversely, SUVs in multiple brain regions decreased, including the contralateral somatosensory cortex, ipsilateral cingulate cortex, and ipsilateral temporal association cortex. The Pearson correlation analysis showed that the SUVs of the contralateral anterodorsal hippocampus and ipsilateral dorsolateral thalamus were negatively related to the TWL of the intact forepaw, whereas the SUVs in the contralateral somatosensory cortex and ipsilateral cingulate cortex were positively related to it (p<0.05). These findings indicate that upregulation of metabolism in the anterodorsal hippocampus and dorsolateral thalamus and downregulation metabolism in the contralateral somatosensory cortex and ipsilateral cingulate cortex could be a unique pattern of metabolic changes for neuropathic pain following brachial plexus avulsion.