Animal Studies

Visceral pain originating from chronic inflammation of the pancreas is often intractable and difficult to manage clinically. However, the pathogenesis of the central nervous system underlying visceral pain is still poorly understood. The aim of the present study was to investigate the role of the midbrain ventrolateral periaqueductal gray (vlPAG) in a rat model of chronic visceral pain induced by pancreatitis. In the present study, we used a well-established rat model of chronic pancreatitis induced by tail vein injection of dibutyltin dichloride (DBTC). To assess the DBTC-induced visceral pain, we examined the abdominal withdrawal by von Frey filament test. We further studied the synaptic transmission in the vlPAG by whole-cell patch-clamp electrophysiological recordings. Rats receiving DBTC injection exhibited a significantly increased withdrawal frequency to mechanical stimulation of the abdomen compared to rats injected with vehicle. Interestingly, compared to rats injected with vehicle, we found that neurons dissected from DBTC-treated rats exhibited a significantly decreased synaptic strength, which was revealed by a diminishedα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/ N-methyl-D-aspartic acid (AMPA/NMDA) ratio in the vlPAG. Moreover, our results further demonstrated that neurons obtained from DBTC-treated rats displayed a higher paired-pulse ratio, as well as less frequent and smaller amplitudes of miniature excitatory postsynaptic currents in the vlPAG compared to rats injected with vehicle. Furthermore, intra-vlPAG microinjection of AMPA alleviated DBTC-induced abdominal hypersensitivity. Taken together, our findings suggest that diminished glutamatergic synaptic strength via both presynaptic and postsynaptic mechanisms in the midbrain vlPAG is associated with DBTC-induced abdominal hypersensitivity. In addition, activation of AMPA receptors in the vlPAG alleviates DBTC-induced abdominal hypersensitivity.

Group III/IV muscle afferents transduce nociceptive signals and modulate exercise pressor reflexes (EPRs). However, the mechanisms governing afferent responsiveness to dually modulate these processes are not well characterized. We and others have shown that ischemic injury can induce both nociception-related behaviors and exacerbated EPRs in the same mice. This correlated with primary muscle afferent sensitization and increased expression of glial cell line-derived neurotrophic factor (GDNF) in injured muscle and increased expression of GDNF family receptor α1 (GFRα1) in dorsal root ganglia (DRG). Here, we report that increased GDNF/GFRα1 signaling to sensory neurons from ischemia/reperfusion-affected muscle directly modulated nociceptive-like behaviors and increased exercise-mediated reflexes and group III/IV muscle afferent sensitization. This appeared to have taken effect through increased cyclic adenosine monophosphate (cAMP) response element binding (CREB)/CREB binding protein-mediated expression of the purinergic receptor P2X5 in the DRGs. Muscle GDNF signaling to neurons may, therefore, play an important dual role in nociception and sympathetic reflexes and could provide a therapeutic target for treating complications from ischemic injuries.

Chronic pain has been shown to depend on nociceptive sensitization in the spinal cord, and while multiple mechanisms involved in the initiation of plastic changes have been established, the molecular targets which maintain spinal nociceptive sensitization are still largely unknown. Building upon the established neurobiology underlying the maintenance of LTP in the hippocampus, this present study investigated the contributions of spinal atypical PKC isoforms PKCι/λ and PKM and their downstream targets (p62/GluA1 and NSF/GluA2 interactions, respectively) to the maintenance of spinal nociceptive sensitization in male and female rats. Pharmacological inhibition of atypical PKCs by ZIP reversed established allodynia produced by repeated intramuscular (i.m.) acidic saline injections in male animals only, replicating previously demonstrated sex differences. Inhibition of both PKCι/λ and downstream substrates p62/GluA1 resulted in male-specific reversals of i.m. acidic saline-induced allodynia, while female animals continued to display allodynia. Inhibition of NSF/GluA2, the downstream target to PKM, reversed allodynia induced by i.m. acidic saline in both sexes. Neither PKCι/λ, p62/GluA1 or NSF/GluA2 inhibition had any effect on formalin response for either sex.This study provides novel behavioural evidence for the male-specific role of PKCι/λ and downstream target p62/GluA1, highlighting the potential influence of ongoing afferent input. The sexually divergent pathways underlying persistent pain are shown here to converge at the interaction between NSF and the GluR2 subunit of the AMPA receptor. Though this interaction is thought to be downstream of PKM in males, these findings and previous work suggest that females may rely on a factor independent of atypical PKCs for the maintenance of spinal nociceptive sensitization.

Descending nociceptive modulation from the supraspinal structures has an important role in cancer-induced bone pain (CIBP). Midbrain ventrolateral periaqueductal gray (vlPAG) is a critical component of descending nociceptive circuits; nevertheless, its precise cellular and molecular mechanisms involved in descending facilitation remain elusive. Our previous study has shown that activation of p38 MAPK in vlPAG microglia is essential for the neuropathic pain sensitization. However, the existence of potential connection between astrocytes and JNK pathway in CIBP has not yet been elucidated. The following study examines the involvement of astrocyte activation and up-regulation of p-JNK in vlPAG, using a CIBP rat model. Briefly, CIBP was mimicked by an intramedullary injection of Walker 256 mammary gland carcinoma cells into the animal tibia. A significant increase in expression levels of astrocytes in the vlPAG of CIBP rats was observed. Furthermore, stereotaxic microinjection of the astrocytic cytotoxin L-α-aminoadipic acid decreased the mechanical allodynia, as well as established and reversed the astrocyte activation in CIBP rats. A significant increase in expression levels of p-JNK in astrocytes in vlPAG of CIBP rats was also observed. Moreover, the intrathecal administration of JNK inhibitors SP600125 reduced the expression of GFAP, while microinjection of the SP600125 decreased the mechanical allodynia of CIBP rats. These results suggested that cancer-induced bone pain is associated with astrocyte activation in the vlPAG that probably participates in driving descending pain facilitation through the JNK MAPK signaling pathway. To sum up, these findings reveal a novel site of astrocytes modulation of CIBP.

Research into potentially novel biomarkers for chronic pain development is lacking. microRNAs (miRNAs) are attractive candidates as biomarkers due to their conservation across species, stability in liquid biopsies, and variation that corresponds to a pathologic state. miRNAs can be sorted into extracellular vesicles (EVs) within the cell and released from the site of injury. EVs transfer cargo molecules between cells thus affecting key intercellular signaling pathways. The focus of this study was to determine the plasma derived EV miRNA content in a chronic neuropathic pain rat model. This was accomplished by performing either spinal nerve ligation (SNL; n=6) or sham (n=6) surgery on anesthetized male Sprague-Dawley rats. Mechanosensitivity was assessed and plasma derived EV RNA was isolated at baseline (BL), day 3, and 15 post-nerve injury. EV extracted small RNA was sequenced followed by differentially expressed (DE) miRNAs and gene target enrichment/signaling pathway analysis performed using R packages and TargetScan/Ingenuity pathway analysis (IPA), respectively. Seven of the DE miRNAs were validated by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR). The data indicated that SNL rats displayed a time-dependent threshold reduction in response to evoked stimuli from day 3 to day 15 post-nerve injury. The data also revealed that 22 and 74 miRNAs at day 3 and 15, respectively, and 33 miRNAs at both day 3 and 15 were uniquely DE between the SNL and sham groups. The key findings from this proposal include 1) the majority of the DE EV miRNAs, which normally function to suppress inflammation, were downregulated, and 2) several of the plasma derived DE EV miRNAs reflect previously observed changes in the injured L5 nerve. The plasma derived DE EV miRNAs regulate processes important in the development and maintenance of neuropathic pain states and potentially serve as key regulators, biomarkers, and targets in the progression and treatment of chronic neuropathic pain.

Over 100 million people are challenged by the effects of chronic pain in the United States alone. This burden also impacts the U.S. economy; 600 billion dollars annually is spent on medical care, medications, and lost productivity in the workplace.1 Current opioid treatments cause adverse effects including nausea, constipation, tolerance, and addiction liability.2 Nociceptive sensitization is thought to perpetuate chronic pain, but too little is known about its mechanisms. Components of the pathways that sensitize the nociceptors after injury are likely to be valuable targets for novel medications for the relief or prevention of chronic pain. Utilizing the Drosophila melanogaster cell targeting and RNA interference toolkit, we are investigating the Bone Morphogenetic Protein (BMP) pathway and its role in ultraviolet light (UV) injury-induced nociceptive sensitization. BMPs are well known as secreted developmental morphogens that control development, but other functions are known.3 We have previously identified BMP signaling components used in nociceptors to modulate injury-induced allodynia, including Decapentaplegic (Dpp, orthologous to mammalian BMP 2/4), and its downstream signaling components.4 The morphogen Hedgehog has also been shown to be necessary for allodynia following injury.5 Here, we show that two membrane-embedded regulators of the Dpp and Hedgehog pathways, Dally and Dally-like, are necessary for injury-induced thermal allodynia, as the formation of sensitization was reduced when either component was suppressed. These BMP components are highly conserved and, because dysregulation of nociceptor sensitization underlies chronic pain, the homologs of Dally and Dally-like may represent novel therapeutic targets in humans challenged by chronic pain. Furthermore, because of their extracellular location, Dally and Dally-like represent attractive therapeutic drug targets because such drugs would not need to cross the plasma membrane.

The expression of potassium ion channel subunit 1.2 (Kv1.2) in the dorsal root ganglion (DRG) influences the excitability of neurons, which contributes to the induction and development of neuropathic pain (NPP); however, the molecular mechanisms underlying the downregulation of Kv1.2 in NPP remain unknown. Histone deacetylase (HDAC) inhibitors are reported to attenuate the development of pain hypersensitivity in rats with NPP. Whether HDAC inhibitors contribute to regulation of Kv1.2 expression, and which specific HDAC subunit is involved in NPP, remain unexplored. In this study we established a chronic constrictive injury (CCI) model and used western blot, quantitative real-time PCR, immunostaining, intrathecal injection, and siRNA methods to explore which HDAC subunit is involved in regulating Kv1.2 expression to mediate NPP. Our results demonstrated that nerve injury led to upregulation of HDAC1 expression in the DRG, and of HDAC2 in the DRG and spinal cord. Double-labeling immunofluorescence histochemistry showed that Kv1.2 principally co-localized with HDAC2, but not HDAC1, in NF200-positive large neurons of the DRG. Intrathecal injection with the HDAC inhibitor, suberoylanilide hydroxamic acid, attenuated mechanical and thermal hypersensitivity and reversed the decreased expression of Kv1.2 in rats with CCI. Furthermore, treatment with HDAC2, but not HDAC1, siRNA also relieved mechanical and thermal hypersensitivity and upregulated the Kv1.2 expression in this model. In vitro transfection of PC12 cells with HDAC2 and HDAC1 siRNA confirmed that only HDAC2 siRNA could regulate the expression of Kv1.2. These findings suggest that HDAC2, but not HDAC1, is involved in NPP through regulation of Kv1.2 expression.