Animal Studies

HIV-associated neuropathic pain (HNP) is a common complication for AIDS patients. The pathological mechanism governing HNP has not been elucidated, and HNP has no effective analgesic treatment. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophic factor family related to the plasticity of the central nervous system. BDNF dysregulation is involved in many neurological diseases, including neuropathic pain. However, to the best of our knowledge, the role and mechanism of BDNF in HNP have not been elucidated. In this study, we explored this condition in an HNP mouse model induced by intrathecal injection of gp120. We found that Wnt3a and β-catenin expression levels increased in the spinal cord of HNP mice, consequently regulating the expression of BDNF and affecting hypersensitivity. In addition, the blockade of Wing-Int/β-catenin signaling, BDNF/TrkB or the BDNF/p75NTR pathway alleviated mechanical allodynia. BDNF immunoreactivity was colocalized with spinal microglial cells, which were activated in HNP mice. Inhibition of spinal microglial cell activation by minocycline relieved mechanical allodynia in HNP mice. This study helped to elucidate the role of the Wing-Int/β-catenin/BDNF signaling axis in HNP and may establish a foundation for further research investigating the Wing-Int/β-catenin/BDNF signaling axis as a target for HNP treatment.

Low-threshold mechanosensitive C fibers (C-LTMRs) that express the vesicular glutamate transporter VGLUT3 are thought to signal affective touch, and may also play a role in mechanical allodynia. However, the nature of the central termination of C-LTMRs in the dorsal horn remains largely unexplored. Here, we used light and electron microscopy in combination with VGLUT3 immunolabeling as a marker of C-LTMR terminations to investigate this issue. VGLUT3 C-LTMRs formed central terminals of Type II glomeruli in the inner part of lamina II of the dorsal horn, often establishing multiple asymmetric synapses with postsynaptic dendrites but also participating in synaptic configurations with presynaptic axons and dendrites. Unexpectedly, essentially all VGLUT3 C-LTMR terminals showed substantial VGLUT1 expression in the rat, whereas such terminals in mice lacked VGLUT1. Most VGLUT3 C-LTMR terminals exhibited weak-to-moderate VGLUT2 expression. Further, C-LTMR terminals formed numerous synapses with excitatory protein kinase Cγ (PKCγ) interneurons and inhibitory parvalbumin neurons, whereas synapses with calretinin neurons were scarce. C-LTMR terminals rarely if ever established synapses with neurokinin 1 receptor (NK1R)-possessing dendrites traversing lamina II. Thus, VGLUT3 C-LTMR terminals appear to largely correspond to neurofilament-lacking central terminals of Type II glomeruli in inner lamina II and can thus be identified at the ultrastructural level by morphological criteria. The participation of C-LTMR terminals in Type II glomeruli involving diverse populations of interneuron indicates highly complex modes of integration of C-LTMR mediated signaling in the dorsal horn. Furthermore, differences in VGLUT1 expression indicate distinct species differences in synaptic physiology of C-LTMR terminals.

Chronic pain is a pathological manifestation of neuronal plasticity supported by altered gene transcription in spinal cord neurons that results in long-lasting hypersensitivity. Recently, the concept that epigenetic regulators might be important in pathological pain has emerged, but a clear understanding of the molecular players involved in the process is still lacking. In this study we linked Dnmt3a2, a synaptic activity-regulated de novo DNA methyltransferase, to chronic inflammatory pain. We observed that Dnmt3a2 levels are increased in the spinal cord of adult mice following plantar injection of Complete Freund's Adjuvant (CFA), an in vivo model of chronic inflammatory pain. In vivo knockdown of Dnmt3a2 expression in dorsal horn neurons blunted the induction of genes triggered by CFA injection. Among the genes whose transcription was found to be influenced by Dnmt3a2 expression in the spinal cord is Ptgs2, encoding for Cox-2, a prime mediator of pain processing. Lowering the levels of Dnmt3a2 prevented the establishment of long-lasting inflammatory hypersensitivity. These results identify Dnmt3a2 as an important epigenetic regulator needed for the establishment of central sensitization. Targeting expression or function of Dnmt3a2 may be suitable for the treatment of chronic pain.

The cyclic nucleotide signaling, including cAMP-PKA and cGMP-PKG pathways, has been well known to play critical roles in regulating cellular growth, metabolism and many other intracellular processes. In recent years, more and more studies have uncovered the roles of cAMP and cGMP in the nervous system. The cAMP and cGMP signaling mediates chronic pain induced by different forms of injury and stress. Here we summarize the roles of cAMP-PKA and cGMP-PKG signaling pathways in the pathogenesis of chronic pain after nerve injury. In addition, acute dissociation and chronic compression of the dorsal root ganglion (DRG) neurons, respectively, leads to neural hyperexcitability possibly through PAR2 activation-dependent activation of cAMP-PKA pathway. Clinically, radiotherapy can effectively alleviate bone cancer pain at least partly through inhibiting the cancer cell-induced activation of cAMP-PKA pathway. Roles of cyclic nucleotide signaling in neuropathic and inflammatory pain are also seen in many other animal models and are involved in many pro-nociceptive mechanisms including the activation of hyperpolarization-activated cyclic nucleotide (HCN)-modulated ion channels and the exchange proteins directly activated by cAMP (EPAC). Further understanding the roles of cAMP and cGMP signaling in the pathogenesis of chronic pain is theoretically significant and clinically valuable for treatment of chronic pain.

Neuropathic pain is a type of chronic pain induced by either central or peripheral nerve injury. MicroRNAs (miRs) have been recently linked to many diseases, including neuropathic pain. However, the role of miR-7a in neuropathic pain still remains elusive. Thus, we aim to investigate the effects of miR-7a on neuropathic pain based on the spinal nerve ligation (SNL) rat model. After establishment of SNL rat models, rats were infected with adeno associated virus (AAV)-neurofilament light polypeptide (NEFL), AAV-miR-7a or treated with metformin. The paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were assessed afterward, and the expression of miR-7a and NEFL as well as their interaction was determined. Subsequently, miR-7a was overexpressed or silenced in dorsal root ganglion (DRG) cells to investigate the role of miR-7a in neuropathic pain. Furthermore, the regulatory effect of NEFL on neuropathic pain was detected using plasmid overexpressing NEFL. SNL rat model exhibited upregulation of NEFL but downregulation of miR-7a. Additionally, NEFL accumulation or miR-7a inhibition decreased PWT and PWL. Then, NEFL accumulation or miR-7a inhibition was observed to increase the phosphorylation level of STAT3. miR-7a was found to directly target NEFL and downregulate NEFL. In addition, inhibiting the STAT3 signaling pathway was also revealed to increase PWT and PWL. Collectively, our study demonstrated that miR-7a ameliorated neuropathic pain via blocking the STAT3 signaling pathway by repressing NEFL. These findings, if taken further, can be of important clinical significance in treating patients with neuropathic pain.

Background Uremic neuropathy commonly affects patients with chronic kidney disease (CKD), with painful sensations in the feet, followed by numbness and weakness in the legs and hands. The symptoms usually resolve following kidney transplantation, but the mechanisms of uremic neuropathy and associated pain symptoms remain unknown. As blood urea levels are elevated in patients with CKD, we examined the morphological and functional effects of clinically observed levels of urea on sensory neurons. Methods Rat DRG neurons were treated with 10 or 50 mMol/L urea for 48 hours, fixed and immunostained for PGP9.5 and βIII tubulin immunofluorescence, ,. Neurons were also immunostained for TRPV1, TRPM8 and Gap43 expression, and the capsaicin sensitivity of urea or vehicle treated neurons was determined. Results Urea treated neurons had degenerating neurites with diminished PGP9.5 immunofluorescence, and swollen, retracted growth cones. βIII tubulinappeared clumped after urea treatment. Neurite lengths were significantly reduced to 60 ± 2.6 % (10 mMol/L, **P<0.01), and to 56.2 ± 3.3 %, (50 mMol/L, **P<0.01), urea treatment for 48 hours, compared with control neurons. Fewer neurons survived urea treatment, with 70.08 ± 13.3% remaining after10 mMol/L (*P<0.05), and 61.49 ± 7.4 % after 50 mMol/L urea treatment (**P<0.01), compared with controls. The proportion of neurons expressing TRPV1 was reduced after urea treatment, but not TRPM8 expressing neurons. In functional studies, treatment with urea resulted in dose-dependent neuronal sensitization. Capsaicin responses were significantly increased to 115.29 ± 3.4 % (10 mMol/L, **P<0.01) and 125.3 ± 4.2% (50 mMol/L, **P<0.01), compared with controls. Sensitization due to urea was eliminated in the presence of the TRPV1 inhibitor SB705498, the MEK inhibitor PD98059, the PI3 kinase inhibitor LY294002, and the TRPM8 inhibitor AMTB. Conclusion Neurite degeneration and sensitization are consistent with uremic neuropathy, , and provide a disease-relevant model to test new treatments.