Animal Studies

Fibromyalgia (FM), a disease of unknown etiology characterized by chronic generalized pain, is partly recapitulated in an animal model induced by repeated acid saline injections into the gastrocnemius muscle. Here, we attempted to investigate the sex difference in pain hypersensitivity (mechanical allodynia and hypersensitivity to electrical stimulation) in the repeated acid saline-induced FM-like generalized pain (AcGP) model. The first unilateral acid injection into gastrocnemius muscle at day 0/D0 and second injection at D5 (post day 0, P0) induced transient and long-lasting mechanical allodynia, respectively, on both sides of male and female mice. The pretreatment with gonadectomy did not affect the first injection-induced allodynia in both sexes, but gradually reversed the second injection-induced allodynia in male but not female mice. Moreover, the AcGP in male mice was abolished by intracerebroventricular minocycline treatments during D4-P4 or P5-P11, but not by early treatments during D0-D5 in male but not female mice, suggesting that brain microglia are required for AcGP in late-onset and sex-dependent manners. We also found that the intravenous treatments of splenocytes derived from male but not female mice treated with AcGP caused allodynia in naive mice. In addition, the purified CD4 T cells derived from splenocytes of acid-treated male mice retained the ability to cause allodynia in naive mice. These findings suggest that FM-like AcGP has multiple sexual dimorphic mechanisms.

Ischemic neuropathy is common in subjects with critical limb ischemia, frequently causing chronic neuropathic pain. However, neuropathic pain caused by ischemia is hard to control despite the restoration of an adequate blood flow. Here, we used a rat model of ischemic-reperfusion nerve injury (IRI) to investigate possible effects of hepatocyte growth factor (HGF) against ischemic neuropathy. Hemagglutinating virus of Japan (HVJ) liposomes containing plasmids encoded with HGF was delivered into the peripheral nervous system by retrograde axonal transport following its repeated injections into the tibialis anterior muscle in the right hindlimb. First HGF gene transfer was done immediately after IRI, and repeated at 1, 2 and 3 weeks later. Rats with IRI exhibited pronounced mechanical allodynia and thermal hyperalgesia, decreased blood flow and skin temperature, and lowered thresholds of plantar stimuli in the hind paw. These were all significantly improved by HGF gene transfer, as also were sciatic nerve conduction velocity and muscle action potential amplitudes. Histologically, HGF gene transfer resulted in a significant increase of endoneurial microvessels in sciatic and tibial nerves and promoted nerve regeneration which were confirmed by morphometric analysis. Neovascularization was observed in the contralateral side of peripheral nerves as well. In addition, IRI elevated mRNA levels of P2X3 and P2Y1 receptors, and transient receptor potential vanilloid receptor subtype 1 (TRPV1) in sciatic nerves, dorsal root ganglia and spinal cord, and these elevated levels were inhibited by HGF gene transfer. In conclusion, HGF gene transfer is a potent candidate for treatment of acute ischemic neuropathy caused by reperfusion injury, because of robust angiogenesis and enhanced nerve regeneration.

Neuropathic pain (NP) is a complex, chronic pain condition caused by injury or dysfunction affecting the somatosensory nervous system. This study aimed to identify crucial mRNAs and microRNAs (miRNAs) in the prelimbic cortex (PL) of NP rats. mRNA and miRNA microarrays were applied in the present study. The miRNA-mRNA regulatory network was constructed by using ingenuity pathway analysis (IPA). A total of 35 differentially expressed (DE) RNAs (24 miRNAs and 10 mRNAs) were identified in the spared nerve injury (SNI) group compared with the control group. The DE miRNA-mRNA network showed that IL-6 and tumor necrosis factor (TNF) were core components. Mir-30c-5p and mir-16-5p were the most connected miRNAs in the network. Interestingly, four mRNAs (Rnase 4, Egr2, Rexo4, and Klf2) with significantly increased expression were abundantly expressed in microglia, which was verified by the real-time quantitative polymerase chain reaction (qPCR). Furthermore, the expression of Rnase4 and Egr2 decreased in M1-polarized macrophages and increased in M2-polarized macrophages. In conclusion, we screened dozens of DE mRNAs and miRNAs in the PL of SNI rats. The core of the DE mRNA and miRNA network pointed to molecules associated with inflammation. Four mRNAs (Rnase4, Egr2, Rexo4, and Klf2) might be the potential markers of M2 polarization.

Infants spending extended time in the neonatal intensive care unit are at greater risk of developing a variety of mental health problems later in life, possibly due to exposure to painful/stressful events. We used a rodent model of inflammatory neonatal pain to explore effects on fear conditioning, somatosensory function and maternal behavior. Hindpaw injections of 2% λ-carrageenan on postnatal days 1 and 4 produced an attenuation in conditioned freezing during the postweaning period, similar to our previous work with acute pain, but did not cause lasting impacts on contextual freezing nor somatosensory function. Additionally, we assessed maternal behavior to observe dam-pup interactions during the neonatal period. Results showed dams of litters which experienced pain spent similar amounts of time with pups as undisturbed controls. However, the specific behaviors differed per condition. Dams of pain litters exhibited less time licking/grooming, but more time nursing than controls. These results suggest changes in maternal care following pain could be a contributing factor underlying the long-term effects of neonatal trauma. Furthermore, our laboratory has previously shown acute, but not inflammatory pain, disrupted conditioned freezing; the current experiment observed the long-term effects of neonatal inflammatory pain on conditioned fear using a weak conditioning protocol.

Inflammatory pain is the most common type of pain treated clinically. However, the currently available treatments for inflammatory pain have limited effects and can cause severe side effects. The aim of this study is to describe the effect of miRNA-485-5p on osteoarthritis-related inflammatory pain.

The chemokine receptor, CXCR4, and the transforming growth factor-beta receptor, ALK5, both contribute to various processes associated with the sensation of pain. However, the relationship between CXCR4 and ALK5 and the possible mechanisms promoted by ALK5 in the development of pain have not been evaluated.

The aims of this study were to investigate if Toll-like receptor 4 (TLR4) is expressed in the medullary dorsal horn (MDH) and if medullary application of a TLR4 antagonist (lipopolysaccharides from , LPS-RS) can attenuate changes in nociceptive sensorimotor responses or TLR4 expression that might be evoked by mustard oil (MO) application to the right maxillary first molar tooth pulp. Of 41 adult male Sprague-Dawley rats used in the study, 23 received intrathecal application of the TLR4 antagonist LPS-RS (25 μg/10 μl; LPS-RS group) or isotonic saline (10 μl; vehicle control group) 10 min before pulpal application of MO (95%; 0.2 μl). Bilateral electromyographic (EMG) activities of the anterior digastric and masseter muscles were recorded continuously before and until 15 min after the MO application to the pulp. In 6 of these 23 rats and an additional 18 rats, the caudal medulla containing the ipsilateral and contralateral MDH was removed after euthanasia for subsequent Western Blot analysis of TLR4 expression in LPS-RS ( = 8) and vehicle ( = 8) groups and a naïve group ( = 8). The % change from baseline in the MO-evoked EMG activities within the anterior digastric muscles were significantly smaller in the LPS-RS group than the control group (two-way ANOVA, Bonferroni, < 0.0001). Western Blot analysis revealed similar levels of TLR4 expression in the caudal medulla of the naïve, vehicle and LPS-RS groups. These novel findings suggest that TLR4 signaling in the caudal medulla may mediate MO-induced acute dental inflammatory pain in rats.

Resident microglia of the central nervous system are being increasingly recognized as key players in diseases such as neuropathic pain. Biochemical and behavioral studies in neuropathic pain rodent models have documented compelling evidence of the critical role of ATP mediated-P2X4R-brain-derived neurotrophic factor (BDNF) signaling pathway in the initiation and maintenance of pain hypersensitivity, a feature driving neuropathic pain-related behavior. The goal of this study was to develop and characterize an in vitro cell line model of activated microglia that can be subsequently utilized for screening neuropathic pain therapeutics. In the present study, we characterized the SIM-A9 microglia cell line for key molecules in the P2X4R-BDNF signaling axis using a combination of biochemical techniques and developed an ATP-activated SIM-A9 microglia model. We present three novel findings: first, SIM-A9 cells expressed P2X4R and BDNF proteins, second, ATP, but not LPS, was cytocompatible with SIM-A9 cells and third, exposure of cells to optimized ATP concentrations for defined periods increased intracellular expression of Iba1 and BDNF proteins. Increased Iba1 levels confirmed microglia activation and increased BDNF expression confirmed ATP-mediated stimulation of the P2X4R signaling pathway. We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.