Decrease of IL-1β and TNF in the Spinal Cord Mediates Analgesia Produced by Ankle Joint Mobilization in Complete Freund Adjuvant-Induced Inflammation Mice Model

ObjectiveThis study aims to investigate the effects of ankle joint mobilization (AJM) on mechanical hyperalgesia and peripheral and central inflammatory biomarkers after intraplantar (i.pl.) Complete Freund’s Adjuvant (CFA)-induced inflammation.MethodsMale Swiss mice were randomly assigned to 3 groups (n = 7): Saline/Sham, CFA/Sham, and CFA/AJM. Five AJM sessions were carried out at 6, 24, 48, 72, and 96 h after CFA injection. von Frey test was used to assess mechanical hyperalgesia. Tissues from paw skin, paw muscle and spinal cord were collected to measure pro-inflammatory (TNF, IL-1β) and anti-inflammatory cytokines (IL-4, IL-10, and TGF-β1) by ELISA. The macrophage phenotype at the inflammation site was evaluated by Western blotting assay using the Nitric Oxide Synthase 2 (NOS 2) and Arginase-1 immunocontent to identify M1 and M2 macrophages, respectively.ResultsOur results confirm a consistent analgesic effect of AJM following the second treatment session. AJM did not change cytokines levels at the inflammatory site, although it promoted a reduction in M2 macrophages. Also, there was a reduction in the levels of pro-inflammatory cytokines IL-1β and TNF in the spinal cord.ConclusionTaken together, the results confirm the anti-hyperalgesic effect of AJM and suggest a central neuroimmunomodulatory effect in a model of persistent inflammation targeting the pro-inflammatory cytokines IL-1β and TNF.

Effects of buprenorphine on model development in an adjuvant-induced monoarthritis rat model

Complete Freund’s adjuvant (CFA)-induced arthritis in rats is a common animal model for studying chronic inflammatory pain. However, modelling of the disease is associated with unnecessary pain and impaired animal wellbeing, particularly in the immediate post-induction phase. Few attempts have been made to counteract these adverse effects with analgesics. The present study investigated the effect of buprenorphine on animal welfare, pain-related behaviour and model-specific parameters during the disease progression in a rat model of CFA-induced monoarthritis. The aim was to reduce or eliminate unnecessary pain in this model, in order to improve animal welfare and to avoid suffering, without compromising the quality of the model. Twenty-four male Sprague Dawley rats were injected with 20 μl of CFA into the left tibio-tarsal joint to induce monoarthritis. Rats were treated with either buprenorphine or carprofen for 15 days during the disease development, and were compared to a saline-treated CFA-injected group or a negative control group. Measurements of welfare, pain-related behaviour and clinical model-specific parameters were collected. The study was terminated after 3 weeks, ending with a histopathologic analysis. Regardless of treatment, CFA-injected rats displayed mechanical hyperalgesia and developed severe histopathological changes associated with arthritis. However, no severe effects on general welfare were found at any time. Buprenorphine treatment reduced facial pain expression scores, improved mobility, stance and lameness scores and it did not supress the CFA-induced ankle swelling, contrary to carprofen. Although buprenorphine failed to demonstrate a robust analgesic effect on the mechanical hyperalgesia in this study, it did not interfere with the development of the intended pathology.

Dexmedetomidine Alleviates Microglia-Induced Spinal Inflammation and Hyperalgesia in Neonatal Rats by Systemic Lipopolysaccharide Exposure

Noxious stimulus and painful experience in early life can induce cognitive deficits and abnormal pain sensitivity. As a major component of the outer membrane of gram-negative bacteria, lipopolysaccharide (LPS) injection mimics clinical symptoms of bacterial infections. Spinal microglial activation and the production of pro-inflammatory cytokines have been implicated in the pathogenesis of LPS-induced hyperalgesia in neonatal rats. Dexmedetomidine (DEX) possesses potent anti-neuroinflammatory and neuroprotective properties through the inhibition of microglial activation and microglial polarization toward pro-inflammatory (M1) phenotype and has been widely used in pediatric clinical practice. However, little is known about the effects of DEX on LPS-induced spinal inflammation and hyperalgesia in neonates. Here, we investigated whether systemic LPS exposure has persistent effects on spinal inflammation and hyperalgesia in neonatal rats and explored the protective role of DEX in adverse effects caused by LPS injection. Systemic LPS injections induced acute mechanical hyperalgesia, increased levels of pro-inflammatory cytokines in serum, and short-term increased expressions of pro-inflammatory cytokines and M1 microglial markers in the spinal cord of neonatal rats. Pretreatment with DEX significantly decreased inflammation and alleviated mechanical hyperalgesia induced by LPS. The inhibition of M1 microglial polarization and microglial pro-inflammatory cytokines expression in the spinal cord may implicate its neuroprotective effect, which highlights a new therapeutic target in the treatment of infection-induced hyperalgesia in neonates and preterm infants.

The Peripheral Role of CCL2 in the Anti-Nociceptive Effect of Sigma-1 Receptor Antagonist BD1047 on Inflammatory Hyperalgesia in Rats

Our recent study demonstrated that the CC-chemokine ligand 2 (CCL2) present in primary afferent fibers (PAFs) plays an important role in the microglia-dependent neuronal activation associated with zymosan-induced inflammatory pain. The present study was aimed to evaluate whether BD1047 (a prototypical sigma-1 receptor (Sig-1R) antagonist) is capable of modifying elevated levels of inflammation-evoked CCL2 as a peripheral antinociceptive mechanism. In DRG primary culture, zymosan dose-dependently increased CCL2 release from isolectin B4 (IB4)-positive DRG neurons, a process that was inhibited by co-culture with BD1047. Single treatment of BD1047 before intraplantar injection of zymosan in rats significantly reduced thermal hyperalgesia and mechanical hyperalgesia, as well as CCL2 expression in DRG neurons and microglia activation in the spinal dorsal horn. In the Complete Freund’s adjuvant (CFA)-induced inflammation model, repeated administration of BD1047 dramatically attenuated thermal hyperalgesia and mechanical hyperalgesia, and significantly diminished CCL2 immunoreactivity and microglia activation. Notably, CFA-induced inflammation significantly increased Sig-1R immunoreactivity in DRG neurons, which was co-localized with CCL2 and IB4, respectively. Taken together, our results suggest that BD1047′s anti-nociceptive property was substantially mediated by the inhibition of CCL2 release in unmyelinated PAFs and that this may, in turn, have attenuated the spinal microglia activation that is associated with inflammatory pain.

Modulation of the N13 component of the somatosensory evoked potentials in an experimental model of central sensitization in humans

The N13 component of somatosensory evoked potential (N13 SEP) represents the segmental response of dorsal horn neurons. In this neurophysiological study, we aimed to verify whether N13 SEP might reflect excitability changes of dorsal horn neurons during central sensitization. In 22 healthy participants, we investigated how central sensitization induced by application of topical capsaicin to the ulnar nerve territory of the hand dorsum modulated N13 SEP elicited by ulnar nerve stimulation. Using a double-blind placebo-controlled crossover design, we also tested whether pregabalin, an analgesic drug with proven efficacy on the dorsal horn, influenced capsaicin-induced N13 SEP modulation. Topical application of capsaicin produced an area of secondary mechanical hyperalgesia, a sign of central sensitization, and increased the N13 SEP amplitude but not the peripheral N9 nor the cortical N20-P25 amplitude. This increase in N13 SEP amplitude paralleled the mechanical hyperalgesia and persisted for 120 min. Pregabalin prevented the N13 SEP modulation associated with capsaicin-induced central sensitization, whereas capsaicin application still increased N13 SEP amplitude in the placebo treatment session. Our neurophysiological study showed that capsaicin application specifically modulates N13 SEP and that this modulation is prevented by pregabalin, thus suggesting that N13 SEP may reflect changes in dorsal horn excitability and represent a useful biomarker of central sensitization in human studies.

Cryo-electron microscopy revealed TACAN is extensively and specifically associated with membrane lipids

TACAN is not a mechanosensitive ion channel but significantly linked to the mechanical hyperalgesia. In this study, we show that the human TACAN is a homodimer with each monomer consisting of a body, a spring and a blade domains. The body domain contains six transmembrane helices that forms an independent channel. The spring domain adapts a loop-helix-loop configuration with the helix running within and parallel to the membrane. The blade domain is composed of two cytoplasmic helices. In addition, we found that all the helices of the body and the spring domains are specifically associated with membrane lipids. Particularly, a lipid core, residing within a cavity formed by the two body and spring domains, contacts with the helices from the body and spring domains and extends to reach two symmetrically arranged lipid clusters. These results extremely imply that the membrane lipids coordinate with the membrane-embedded protein to sense and transduce the mechanic signal.

Tmem45b is essential for inflammation and tissue injury-induced persistent mechanical hyperalgesia

Persistent mechanical hyperalgesia, associated with peripheral inflammation and tissue injury, impairs patient’s quality of life and daily activity. However, its molecular mechanism and treatment are yet to be deciphered. Herein, we report that Tmeme45b is expressed in a subset of unmyelinated primary sensory neurons and plays an essential role in inflammation and tissue injury-induced mechanical hyperalgesia. Our findings provide new insights into the mechanisms and the treatment of mechanical hyperalgesia.

Voluntary Forelimbs Exercise Reduces Immobilization-Induced Mechanical Hyperalgesia in the Rat Hind Paw

Voluntary exercise is sufficient to protect against neuropathic pain. However, it is unclear whether voluntary exercise reduces immobilization-induced hyperalgesia. We examined the effect of voluntary forelimb exercise on immobilized-induced hyperalgesia in hind paws of rats. Wistar rats were randomly divided into the (1) both hind limbs immobilized group (IM group), (2) immobilization and exercise with nonimmobilized fore limbs group (EX group), and (3) control group. In the IM and EX groups, the bilateral ankle joints of each rat were immobilized in full plantar flexion with a plaster cast for eight weeks. In the EX group, voluntary exercise using nonimmobilized forelimbs in the running wheel was administered during the immobilization period, while hind limbs were kept immobilized (60 min/day, 5 days/week). Mechanical hyperalgesia in the hind paw was measured using a digital von Frey device every week. To investigate the abnormality of primary sensory neurons and central sensitization, the number of calcitonin gene-related peptide-positive cells in the dorsal root ganglion and the expression level of calcitonin gene-related peptide in the spinal dorsal horn were analyzed by immunohistochemical staining. Immobilization-induced mechanical hyperalgesia was inhibited in the EX group compared to the IM group at three weeks after immobilization. In the EX group, the number of calcitonin gene-related peptide-positive cells in the dorsal root ganglion and the expression level of calcitonin gene-related peptide were significantly decreased compared to those in the IM group. Our results therefore suggest that voluntary forelimb exercise during hind limb immobilization partially reduces immobilization-induced hyperalgesia by suppressing that the plastic changes of the primary sensory nerves that excessively transmit pain and increased responsiveness of nociceptive neurons in the spinal dorsal horn.

17β-Estradiol Exacerbated Experimental Occlusal Interference-Induced Chronic Masseter Hyperalgesia by Increasing the Neuronal Excitability and TRPV1 Function of Trigeminal Ganglion in Ovariectomized Rats

Pain symptoms in temporomandibular disorders (TMD) predominantly affect reproductive women, suggesting that estrogen regulates pain perception. However, how estrogen contributes to chronic TMD pain remains largely unclear. In the present study, we performed behavioral tests, electrophysiology, Western blot and immunofluorescence to investigate the role and underlying mechanisms of estrogen in dental experimental occlusal interference (EOI)-induced chronic masseter mechanical hyperalgesia in rats. We found that long-term 17β-estradiol (E2) replacement exacerbated EOI-induced masseter hyperalgesia in a dose-dependent manner in ovariectomized (OVX) rats. Whole-cell patch-clamp recordings demonstrated that E2 (100 nM) treatment enhanced the excitability of isolated trigeminal ganglion (TG) neurons in OVX and OVX EOI rats, and EOI increased the functional expression of transient receptor potential vanilloid-1 (TRPV1). In addition, E2 replacement upregulated the protein expression of TRPV1 in EOI-treated OVX rats. Importantly, intraganglionic administration of the TRPV1 antagonist AMG-9810 strongly attenuated the facilitatory effect of E2 on EOI-induced masseter mechanical sensitivity. These results demonstrate that E2 exacerbated EOI-induced chronic masseter mechanical hyperalgesia by increasing TG neuronal excitability and TRPV1 function. Our study helps to elucidate the E2 actions in chronic myogenic TMD pain and may provide new therapeutic targets for relieving estrogen-sensitive pain.