Animal Studies

Pancreatitis-associated proteins (PAPs) display multiple functions in visceral diseases. Previous studies showed that the expression level of PAP-I was low in the dorsal root ganglion (DRG) of naïve rats but was expressed after peripheral nerve injury. However, its role in neuropathic pain remains unknown. We found that PAP-I expression was continuously up-regulated in the DRG neurons from rat spared nerve injury (SNI) models, and transported towards the spinal dorsal horn to act as a pro-inflammatory factor. Intrathecal delivery of PAP-I enhanced sensory hyperalgesia, whereas PAP-I deficiency by either gene knockout or antibody application alleviated tactile allodynia at the maintenance phase after SNI. Furthermore, PAP-I functioned by activating the spinal microglia via C-C chemokine receptor type 2 (CCR2) that participated in neuropathic pain. Inhibition of either microglial activation or CCR2 abolished the PAP-I-induced hyperalgesia. Thus, PAP-I mediates the neuron-microglial crosstalk after peripheral nerve injury, and contributes to the maintenance of neuropathic pain.Neuropathic pain is maladaptive pain condition and the maintaining mechanism is largely unclear. Here we reveal that after peripheral nerve injury, PAP-I can be transported to the spinal dorsal horn and is crucial in the progression of neuropathic pain. Importantly, we prove that PAP-I mainly functions through activating the spinal microglia via CCR2-p38 MAPK pathway. Furthermore, we confirm that the pro-inflammatory effect of PAP-I is more prominent after the establishment of neuropathic pain, thus indicating that microglia also participates in the maintenance phase of neuropathic pain.

Voltage-gated T-type Ca (Cav3) channels regulate diverse physiological events including neuronal excitability and have been linked to several pathological conditions such as absence epilepsy, cardiovascular diseases, and neuropathic pain. It is also acknowledged that Ca/Calmodulin-dependent protein kinase II (CaMKII) and protein kinases A and C (PKA and PKC) regulate the activity of T-type channels. Interestingly, peripheral nerve injury induces tactile allodynia and up-regulates Cav3.2 channels and Cdk5 in dorsal root ganglia (DRG) and spinal dorsal horn (SDH). Here, we report that recombinant Cav3.2 channels expressed in HEK-293 cells are regulatory targets of cyclin-dependent kinase 5 (Cdk5). Site-directed mutagenesis showed that the relevant sites for this regulation are residues S561 and S1987. We also found that Cdk5 may regulate Cav3.2 channel functional expression in rats with mechanical allodynia induced by spinal nerve ligation (SNL). Consequently, the Cdk5 inhibitor olomoucine affected the compound action potential (cAP) recorded in the spinal nerves, as well as the paw withdrawal threshold. Likewise, Cdk5 expression was upregulated after SNL in the DRGs. These findings unveil a novel mechanism for how phosphorylation may regulate Cav3.2 channels and suggest that increased channel activity by Cdk5-mediated phosphorylation after SNL contributes nerve injury-induced tactile allodynia.Neuropathic pain is a current public health challenge. It can develop as a result of injury or nerve illness. It is acknowledged that the expression of various ion channels can be altered in neuropathic pain, including T-type Ca channels that are expressed in sensory neurons where they play a role in the regulation of cellular excitability. The present work shows that the exacerbated expression of Cdk5 in a preclinical model of neuropathic pain increases the functional expression of Cav3.2 channels. This finding is relevant for the understanding of the molecular pathophysiology of the disease. Additionally, this work may have a substantial translational impact, since it describes a novel molecular pathway that could represent an interesting therapeutic alternative for neuropathic pain.

Many diseases display unequal prevalence between sexes. The sex-specific immune response to both injury and persistent pain remains underexplored and would inform treatment paradigms. We utilized high-dimensional mass cytometry to perform a comprehensive analysis of phenotypic and functional immune system differences between male and female mice after orthopedic injury. Multivariate modeling of innate and adaptive immune cell responses after injury using an elastic net algorithm, a regularized regression method, revealed sex-specific divergence at 12 h and 7 days after injury with a stronger immune response to injury in females. At 12 h, females upregulated STAT3 signaling in neutrophils but downregulated STAT1 and STAT6 signals in T regulatory cells, suggesting a lack of engagement of immune suppression pathways by females. Furthermore, at 7 days females upregulated MAPK pathways (p38, ERK, NFkB) in CD4T memory cells, setting up a possible heightened immune memory of painful injury. Taken together, our findings provide the first comprehensive and functional analysis of sex-differences in the immune response to painful injury.

Despite growing evidence suggesting that spinal microglia play an important role in the molecular mechanism underlying experimental neuropathic pain (NP) in male rodents, evidence regarding the sex-dependent role of these microglia in NP is insufficient. In this study, we evaluated the effects of microglial regulation on NP using Gi-designer receptors exclusively activated by designer drugs (Gi-DREADD) driven by the microglia-specific promoter. For the Cre-dependent expression of human Gi-coupled M4 muscarinic receptors (hM4Di) in CX3C chemokine receptor 1-expressing (CX3CR1) cells, R26-LSL-hM4Di-DREADD mice were crossed with CX3CR1-Cre mice. Mouse models of NP were generated by partial sciatic nerve ligation (PSL) and treatment with anti-cancer agent paclitaxel (PTX) or oxaliplatin (OXA), and mechanical allodynia was evaluated using the von Frey test. Immunohistochemistry revealed that hM4Di was specifically expressed on Iba1 microglia, but not on astrocytes or neurons in the spinal dorsal horn of CX3CR1-hM4Di mice. PSL-induced mechanical allodynia was significantly attenuated by systemic (intraperitoneal, i.p.) administration of 10 mg/kg of clozapine N-oxide (CNO), a hM4Di-selective ligand, in male CX3CR1-hM4Di mice. The mechanical threshold in naive CX3CR1-hM4Di mice was not altered by i.p. administration of CNO. Consistently, local (intrathecal, i.t.) administration of CNO (20 nmol) significantly relieved PSL-induced mechanical allodynia in male CX3CR1-hM4Di mice. However, neither i.p. nor i.t. administration of CNO affected PSL-induced mechanical allodynia in female CX3CR1-hM4Di mice. Both i.p. and i.t. administration of CNO relieved PTX-induced mechanical allodynia in male CX3CR1-hM4Di mice, and a limited effect of i.p. CNO was observed in female CX3CR1-hM4Di mice. Unlike PTX-induced allodynia, OXA-induced mechanical allodynia was slightly improved, but not significantly relieved, by i.p. administration of CNO in both male and female CX3CR1-hM4Di mice. These results suggest that spinal microglia can be regulated by Gi-DREADD and support the notion that CX3CR1 spinal microglia play sex-dependent roles in nerve injury-induced NP; however, their roles may vary among different models of NP.