Effectiveness of maturity of Rubus occidentalis on hyperalgesia induced by acidic saline injection in rats

Background Rubus occidentalis, also known as black raspberry, contains several bioactive components that vary depending on the maturity of the fruit. The goal of this study was to evaluate the efficacy of immature Rubus occidentalis extract(iROE) on acid-induced hyperalgesia, investigate the mechanism involved, and compare the antihyperalgesic effect of immature and mature ROEs. Methods In adult male Sprague-Dawley rats, chronic muscle pain was induced via two injections of acidic saline into one gastrocnemius muscle. To evaluate the dose response, the rats were injected intraperitoneally with 0.9% saline or iROE (10, 30, 100, or 300 mg/kg) following hyperalgesia development. To evaluate the mechanism underlying iROE-induced analgesia, the rats were injected intraperitoneally with saline, yohimbine 2 mg/kg, dexmedetomidine 50 μg/kg, prazosin 1 mg/kg, atropine 5 mg/kg, mecamylamine 1 mg/kg, or naloxone 5 mg/kg 24 h after hyperalgesia development, followed by iROE 300 mg/kg administration. To compare immature versus mature ROE, the rats were injected with mature ROE 300 mg/kg and immature ROE 300 mg/kg after hyperalgesia development. For all experiments, the mechanical withdrawal threshold(MWT) was evaluated using von Frey filaments before the first acidic saline injection, 24 h after the second injection, and at various time points after drug administration. Data were analysed using multivariate analysis of variance(MANOVA) and the linear mixed-effects model(LMEM). We compared the MWT at each time point using analysis of variance with the Bonferroni correction. Results The iROE 300 mg/kg injection resulted in a significant increase in MWT compared with the control, iROE 30 mg/kg, and iROE 100 mg/kg injections at ipsilateral and contralateral sites. The iROE injection together with yohimbine, mecamylamine, or naloxone significantly decreased the MWT compared with iROE alone, whereas ROE together with dexmedetomidine significantly increased the MWT. According to MANOVA, the effects of immature and mature ROEs were not significantly different; however, the LMEM presented a significant difference between the two groups. Conclusions Immature R. occidentalis showed antihyperalgesic activity against acid-induced chronic muscle pain, which may be mediated by the α2-adrenergic, nicotinic cholinergic, and opioid receptors. The iROE displayed superior tendency regarding analgesic effect compared to mature ROE.

β-elemene relieves neuropathic pain in mice through the regulation on C-X-C motif chemokine receptor 3 and GABAA receptor

β-elemene (Bel) is a sesquiterpene compound has shown potential in the antinociceptive treatment. This study focused on the function of Bel in neuropathic pain relief in mice. A murine model with spared nerve injury (SNI) was established and treated with Bel. The paw withdrawal thresholds in response to mechanical and thermal stimulations were examined using von Frey filaments. The L4-L6 spinal dorsal horn tissue samples were collected for histological examination. Bel treatment reduced the sensitivities of model mice to mechanical and thermal stimulations, and it inhibited activation of microglia and the secretion of inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in tissues. Bel treatment reduced the expression of nociceptor excitatory NMDAR whereas enhanced the expression of nociceptor inhibitory GABAA receptor to relieve the nociception of mice. CXCR3 was a downstream molecule mediated by Bel. Either overexpression of CXCR3 or downregulation of GABAA receptor in the tissues aggravated the neuropathic pain in SNI mice which was initially relieved by Bel. In conclusion, this study suggested that Bel might serve as a drug for nociception management by inhibiting CXCR3 and upregulating GABAA receptor. This study may offer novel insights into the field of neuropathic pain relief.

Metformin Attenuates Bone Cancer Pain by Reducing TRPV1 and ASIC3 Expression

Bone cancer pain (BCP) is a common pathologic pain associated with destruction of bone and pathological reconstruction of nervous system. Current treatment strategies in clinical is inadequate and have unacceptable side effects due to the unclear pathology mechanism. In the present study, we showed that transplantation of Walker 256 cells aggravated mechanical allodynia of BCP rats (**p < 0.01 vs. Sham), and the expression of ASIC3 (Acid-sensitive ion channel 3) and TRPV1 was obviously enhanced in L4-6 dorsal root ganglions (DRGs) of BCP rats (**p < 0.01 vs. Sham). ASIC3 and TRPV1 was mainly expressed in CGRP and IB4 positive neurons of L4-6 DRGs. While, TRPV1 but not ASIC3 was markedly upregulated in L4-6 spinal dorsal horn (SDH) of BCP rats (**p < 0.01 vs. Sham). Importantly, intrathecal injection of CPZ (a TRPV1 inhibitor) or Amiloride (an ASICs antagonist) markedly increased the paw withdraw threshold (PWT) of BCP rats response to Von Frey filaments (**p < 0.01 vs. BCP + NS). What’s more, intraperitoneally injection of Metformin or Vinorelbine markedly elevated the PWT of BCP rats, but reduced the expression of TRPV1 and ASIC3 in L4-6 DRGs and decreased the TRPV1 expression in SDH (*p < 0.05, **p < 0.01 vs. BCP + NS). Collectively, these results suggest an effective analgesic effect of Metformin on mechanical allodynia of BCP rats, which may be mediated by the downregulation of ASIC3 and TRPV1.

Frozen Vein Wrapping for Chronic Nerve Constriction Injury Reduces Sciatic Nerve Allodynia in a Rat Model

BackgroundAutologous vein wrapping (VW) is used in the treatment of recurrent chronic constriction neuropathy and traumatic peripheral nerve injury. However, use of autologous veins is limited by the inability to obtain longer veins of sufficient length for larger sites. Frozen allograft tissue has several advantages, including its availability for large grafts, avoidance of donor-site morbidity, and shorter operation time. Here, we investigated the effect of frozen vein wrapping (FVW) in Wistar rats as a model of sciatic nerve injury. MethodsThe rats were grouped by treatment as (i) untreated after chronic constriction injury surgery (CCI; control group), (ii) treated with vein wrapping using freshly isolated vein (VW), and (iii) treated with vein wrapping using frozen vein (FVW). Mechanical allodynia was assessed with von Frey filaments on postoperative days (PODs) 1, 3, 5, 7, and 14. ResultsThe response of heme oxygenase-1 gene, Hmox-1, expression to VW and FVW was assessed by RT-PCR. Both VW and FVW significantly increased withdrawal threshold levels compared to the untreated control group on POD 1, 3, and 5. Both VW and FVW also showed increased HO-1 expression compared to the CCI group. ConclusionsOur results suggest that FVW may be a suitable therapeutic optionas a source of large grafts.

111 Artificial Light-Triggered Sleep Deprivation May Lead to Allodynia in Rodent Model

Introduction The combination of artificial light and lack of exposure to natural light can delay the circadian clock, dysregulate the circadian cycle, and decrease alertness upon waking. This effect has been especially significant during the COVID-19 pandemic, where overexposure to artificial light at improper hours has contributed to increased rates of clinical insomnia. Artificial light may also contribute to concomitant neurological conditions such as primary headache, but the mechanisms by which light triggers sleep deprivation-induced headache are not well-understood. Methods To measure pain sensitivity, we habituated 13 wild-type male mice to von Frey filaments applied to the periorbital area until there was no response to 0.6g stimulus. We then applied 5 lux of continuous dim light to mice during their usual 12-hour dark cycle. The 12-hour light cycle remained unchanged with 200 lux continuous light. Three groups of mice experienced the dim light stimulus for one, three, or five consecutive days. Ambulation and rest activity were measured using SOF-812 Activity Monitor machines. After the experiment concluded, we waited 24 hours and measured mechanical threshold using von Frey filaments at 1, 3, 5, 8, and every 3 days subsequently until mice no longer responded to 0.6g stimulus. Results Artificial light triggered changes in circadian behavior including increased number of rest periods during 12-hour dark (dim light) cycle and shortened sleep duration during 12-hour light cycle. Following the artificial light stimulus, there was a significant decrease in mechanical threshold (P<0.05), representing allodynia. The one-day group displayed one day of significant allodynia. The three-day group displayed three days of significant allodynia. The five-day group displayed five days of significant allodynia. Conclusion Artificial light may trigger circadian dysregulation, and the duration of artificial light exposure seemed to be directly correlated to the duration of allodynia up to one week after the stimulus was removed. We will repeat these experiments and analyze CNS and PNS tissue samples to understand the underlying physiological and biochemical bases of how artificial light triggers sleep deprivation-induced headache. This knowledge could increase our understanding of the pathophysiology and comorbidity of sleep deprivation and headache. Support (if any) Funding was received from the National Institute of Health (NS104200).

Role of Muscarinic Receptors in Hypoalgesia Induced by Crocin in Neuropathic Pain Rats

Objective. Crocin as an important constituent of saffron has antineuropathic pain properties; however, the exact mechanism of this effect is not known. The aim of this study was whether the hypoalgesic effect of crocin can be exerted through muscarinic receptors. Materials and Methods. In the present project, 36 male Wistar rats (200 ± 20 g) were used. Animals randomly divided into six groups (sham, neuropathy, neuropathy + crocin, neuropathy + atropine 0.5 mg/kg, neuropathy + atropine 1 mg/kg, and neuropathy + atropine 1 mg/kg + crocin). Neuropathy was induced by the chronic constriction injury (CCI) method on the sciatic nerve. Crocin and atropine was administered intraperitoneally during 14 days following the 14th day after surgery. Pain response was detected every three days, two hours after each injection and 3 days following last injection. Mechanical allodynia and thermal hyperalgesia were detected using the Von Frey filaments and plantar test device, respectively. Results. CCI significantly reduced the paw withdrawal response to mechanical and thermal stimulus ( P < 0.01 and P < 0.05 , respectively). Crocin therapy significantly reduced mechanical allodynia and thermal hyperalgesia induced by CCI ( P < 0.05 ). Atropine pretreatment significantly blocked the hypoalgesic effect of crocin ( P < 0.05 in mechanical allodynia and P < 0.01 in thermal hyperalgesia). Fourteen days administration of atropine alone at a dose of 0.5 mg/kg but not 1 mg/kg significantly reduced CCI-induced mechanical allodynia at day 30 after surgery. Conclusion. Crocin significantly decreased CCI-induced neuropathic pain. The hypoalgesic effect of crocin was blocked by atropine pretreatment, which indicates an important role for muscarinic receptors in the effect of crocin.

Assessment of Lingual Tactile Sensitivity in Children and Adults: Methodological Suitability and Challenges

Few methodological approaches have been developed to measure lingual tactile sensitivity, and little information exists about the comparison between children and adults. The aims of the study were to: verify the cognitive and perceptive suitability of Von Frey filaments and a gratings orientation test in children of different ages; compare lingual tactile sensitivity between children and adults; investigate the relationships between lingual tactile sensitivity, preference and consumption of foods with different textures and level of food neophobia. One hundred and forty-seven children aged 6–13 years and their parents participated in the study, in addition to a separate sample of seventy adults. Participants filled in questionnaires, and lingual tactile sensitivity was evaluated through filaments and gratings. Results showed that gratings evaluation was more difficult than filaments assessment but enabled a better separation of participants according to their performance than filaments. R-indices from filaments were not correlated with those of gratings, suggesting that the tools measure different dimensions of lingual tactile sensitivity. No differences were found in lingual tactile sensitivity between children and adults, nor between children of different ages. Food neophobia was negatively associated with preferences of hard foods in children. Although a multifactor analysis concluded that neither texture preferences nor food consumption were strongly correlated with lingual tactile sensitivity, there was a weak but significant positive correlation between lingual tactile sensitivity to the finest Von Frey filament and food neophobia in the youngest age group, indicating that children with higher levels of food neophobia are more sensitive to oral tactile stimuli. Suitable child-friendly adaptations for the assessment of lingual sensitivity in children are discussed.

Electroacupuncture Alleviates Osteoarthritis by Suppressing NLRP3 Inflammasome Activation in Guinea Pigs

Osteoarthritis (OA) is an increasingly prevalent disease affecting synovial joints, which includes joint degeneration, inflammation, and joint pain. The activation of nucleotide-binding and oligomerization domain-like receptor containing protein 3 (NLRP3) could promote synovial inflammation. Previous studies have shown that electroacupuncture (EA) has potential anti-inflammatory effect. However, the effect of EA treatment on OA remains unclear. The aim of this study was to investigate the effect of applied EA on OA and joint pain and its relationship with NLRP3 inflammasome. The Hartley guinea pigs with naturally occurring OA at age 18 months were chosen as the OA model and treated with EA for 4 weeks. Mechanical allodynia was quantified by using von Frey filaments. The expression of NLRP3 inflammasome and the downstream proinflammatory factors in the cartilage tissue were quantified. Our results showed that EA treatment significantly reduces mechanical allodynia, improves the articular cartilage structure, and decreases the fibrillation on the cartilage surface in guinea pigs with spontaneous osteoarthritis. Moreover, we also found that EA treatment attenuates the NLRP3 inflammasome activation and suppresses the protein expression levels of caspase-1 and IL-1β in the cartilage tissue. Our findings suggest that EA treatment attenuates OA and joint pain by suppressing NLRP3 inflammasome activation and support further investigation of the potential therapeutic tactics.

Functional Block of Interleukin-6 Reduces a Bone Pain Marker But Not Bone Loss in Hindlimb-Unloaded Mice

Interleukin-6 (IL-6) is widely accepted to stimulate osteoclasts. Our aim in this study was to examine whether the inhibitory effect of IL-6 on bone loss and skeletal pain associated with osteoporosis in hindlimb-unloaded (HU) mice in comparison with bisphosphonate. Eight-week-old male ddY mice were tail suspended for 2 weeks. Starting immediately after reload, vehicle (HU group), alendronate (HU-ALN group), or anti-IL-6 receptor antibody (HU-IL-6i group) was injected subcutaneously. After a 2-week treatment, pain-related behavior was examined using von Frey filaments. The bilateral distal femoral and proximal tibial metaphyses were analyzed three-dimensionally with micro-computed tomography. Calcitonin gene-related peptide (CGRP) expressions in dorsal root ganglion (DRG) neurons innervating the hindlimbs were examined using immunohistochemistry. HU mice with tail suspension developed bone loss. The HU mice showed mechanical hyperalgesia in the hindlimbs and increased CGRP immunoreactive neurons in the L3-5 DRG. Treatment with IL-6i and ALN prevented HU-induced mechanical hyperalgesia and upregulation of CGRP expressions in DRG neurons. Furthermore, ALN but not IL-6i prevented HU-induced bone loss. In summary, treatment with IL-6i prevented mechanical hyperalgesia in hindlimbs and suppressed CGRP expressions in DRG neurons of osteoporotic models. The novelty of this research suggests that IL-6 is one of the causes of immobility-induced osteoporotic pain regardless improvement of bone loss.

Neurokinin 1 receptor activation in the rat spinal cord maintains latent sensitization, a model of inflammatory and neuropathic chronic pain

Latent sensitization is a model of chronic pain in which a persistent state of pain hypersensitivity is suppressed by opioid receptors, as evidenced by the ability of opioid antagonists to induce a period of mechanical allodynia. Our objective was to determine if substance P and its neurokinin 1 receptor (NK1R) mediate the maintenance of latent sensitization. Latent sensitization was induced by injecting rats in the hindpaw with complete Freund’s adjuvant (CFA), or by spared nerve injury (SNI). When responses to von Frey filaments returned to baseline (day 28), the rats were injected intrathecally with saline or the NK1R antagonist RP67580, followed 15 min later by intrathecal naltrexone. In both pain models, the saline-injected rats developed allodynia for 2 h after naltrexone, but not the RP67580-injected rats. Saline or RP67580 were injected daily for two more days. Five days later (day 35), naltrexone was injected intrathecally. Again, the saline-injected rats, but not the RP67580-injected rats, developed allodynia in response to naltrexone. To determine if there is sustained activation of NK1Rs during latent sensitization, NK1R internalization was measured in lamina I neurons in rats injected in the paw with saline or CFA, and then injected intrathecally with saline or naltrexone on day 28. The rats injected with CFA had a small amount of NK1R internalization that was significantly higher than in the saline-injected rats. Naltrexone increased NK1R internalization in the CFA-injected rats but nor in the saline-injected rats. Therefore, sustained activation of NK1Rs maintains pain hypersensitivity during latent sensitization.