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.

Intrathecal IGF2 siRNA injection provides long-lasting anti-allodynic effect in a spared nerve injury rat model of neuropathic pain

Previous studies have shown an increase of insulin-like growth factor-2 (IGF2) in animal models of neuropathic pain. We aimed to examine the hypothesis that reducing the expression of IGF2 using intrathecal IGF2 small-interfering RNA (siRNA) would attenuate the development of neuropathic pain in rats after spared nerve injury (SNI). Male Wistar rats were divided into three groups: sham-operated group, in which surgery was performed to cut the muscles without injuring the nerves; SNI group, in which SNI surgery was performed to sever the nerves; and SNI + siRNA IGF2 group, in which SNI surgery was performed, and IGF2-siRNA was administered intrathecally 1 day after SNI. The rats were assessed for mechanical allodynia and cold allodynia 1 day before surgery (baseline), and at 2, 4, 6, 8, and 10 days after siRNA treatment. The rat spinal cord was collected for quantitative polymerase chain reaction and western blot analysis. Compared with the SNI group, rats that received IGF2 siRNA showed a significantly increased SNI-induced paw-withdrawal threshold to metal filament stimulation from Day 4 to Day 10 after SNI surgery. IGF2 siRNA significantly decreased the response duration from the acetone test from Day 2 to Day 10 following SNI surgery. SNI increased IGF2 mRNA expression on Day 2 and increased IGF2 protein expression on Day 8 and Day 10 in the spinal cord of the SNI rats. However, the above-mentioned effects of IGF2 mRNA and protein expression were significantly inhibited in the SNI + IGF2 siRNA group. We demonstrated that intrathecal administration of IGF2 siRNA provided significant inhibition of SNI-induced neuropathic pain via inhibition of IGF2 expression in the spinal cord. The analgesic effect lasted for 10 days. Further exploration of intrathecal IGF2 siRNA administration as a potential therapeutic strategy for neuropathic pain is warranted.

Effect of Ropivacaine-Loaded Magnetic Nanoparticles on Ankle Nerve Block in Rats

Aim. Our study is to determine the influence of ropivacaine-loaded magnetic nanoparticles (MNP/Rop) on ankle nerve block in rats. Materials and Methods. MNP/Rop was prepared and then injected intravenously into rats to evaluate its anesthetic effect on rat limbs. Mechanical pain thresholds paw withdrawal threshold (PWT) and paw withdrawal thermal latency (PWL) were employed for the assessment of ankle nerve block in rats. Results. PWT increased from T1 to T4 in each group ( P < 0.05 ). The intergroup comparison determined no distinct difference in the PWT value among the three series at T1 ( P > 0.05 ); however, PWT values at T2-T4 were higher in nerve block control group (NBCG) and MNP/Rop group than in blank group (BG), and they remained slightly higher in MNP/Rop group than in NBCG. The intragroup comparison revealed that from T1 to T4, PWL in each group showed a rising trend. The PWL at T1 showed no evident difference among the three series ( P > 0.05 ); however, PWL values at T2-T4 were higher in NBCG and MNP/Rop group than in BG, and they remained slightly higher in MNP/Rop group than in NBCG. In MNP/Rop group, both PWT and PWL increased with the increase of Fe3O4 load in MNP/Rop ( P < 0.05 ), while PWT and PWL remained unchanged when the load was 2.189%; moreover, PWT and PWL elevated as Rop concentration increased in MNP/Rop ( P < 0.05 ), while they kept unaltered under 40 μL 1% Rop. Conclusion. Intravenous injection of MNP/Rop into rats and inhalation of MNP into the ankle joint can effectively block ankle nerve conduction in rats.

Propofol Alleviates Neuropathic Pain Induced by Chronic Contractile Injury by Regulating the Spinal glun2b-p38mapkepac1 Pathway

Background. Propofol acts as an intravenous anesthetic cure which is widely used as a therapy for the craniocerebral injury that comprised surgical anesthesia as well as the sedation done in the intensive care units. Propofol is one of the most commonly used and efficient anesthetics where the painful effects are followed by an injection of propofol. In many cases, patients experience pain followed by anxiety, boredom, fear, and even myocardial ischemia. Objective. This study was performed to investigate the underlying mechanism of propofol and its effect on regulating spinal glun2b-p38mapkepac1 pathways in chronic contractile injury. Material and Methods. Contractile injury was performed by ligation around the nerve of the thigh region postanesthesia. Rats were divided into three groups to analyze the changes like mechanical allodynia by the paw withdrawal threshold and histopathological analysis for assessing cellular degradation. L4-L6 from the spinal dorsal horns were isolated and harvested for studying protein expression, by the method of western blotting and immunofluorescence analysis. Results. The pain caused due to mechanical allodynia in the paw region was highest at 1 hour postinduction and lasted for three days postinjury. Pain was significantly less in the group receiving propofol when compared with the isoflurane group for the first two hours of injury. In the propofol group, EPAC1, GluN2B, and p38 MAP K were significantly lower. Conclusion. In the rat model of induced chronic contractile injury, postsurgery there was a suppression of the GluN2B-p38MAPK/EPAC1 signaling pathway in the propofol group. As the p38MAPK/EPAC pathway has a significant role in the postoperative hyperalgesia, thus our experiment suggests that propofol has analgesic effects.

Dexmedetomidine Alleviates Hyperalgesia in Arthritis Rats Through Inhibition of the p38MAPK Signaling Pathway

Background: Dexmedetomidine (DEX) has showed significant analgesic effects in neuropathic pain, but the underlying mechanism has remained elusive. Our present study aimed to explore the effect of DEX on hyperalgesia with the involvement of p38MAPK signaling pathway a rat model of monoarthritis (MA).Methods: MA rat model was induced by injection of Complete Freund's Adjuvant (CFA). Pathological changes of ma rats were observed by HE staining and Safranin-O/Fast Green staining. Ankle circumference, paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) was measured to judge the degree of hyperalgesia in MA rats. Immunohistochemistry and ELISA were applied to observe the degree of inflammation in rats. Western blot analysis was conducted to detect expression of p38MAPK signaling pathway-related factors. The mechanism of p38MAPK signaling pathway in MA rats was observed via treatment of Anisomycin or SB203580 combined with DEX.Results: After 8 h of CFA induction, joint swelling and hyperalgesia occurred in rats. There were obvious pathological changes in the joint cavity, the joint cavity space became narrow and synovial bursa became rough. A large number of inflammatory cell infiltration was observed under microscope. After injection of DEX and SB203580, PWT and PWL was prolonged, the expression of serum inflammatory factors was decreased, and the expression of p38MAPK signaling pathway-related factors was decreased; while all the detected indexes were recovered in MA rats after treated with DEX and Anisomycin.Conclusions: Our study provided evidence that DEX could alleviate hyperalgesia in arthritis rats through inhibition of the p38MAPK signaling pathway.

5-HT1F Receptor Agonist Ameliorates Mechanical Allodynia In Neuropathic Pain Via Induction of Mitochondrial Biogenesis and Suppression of Neuroinflammation

Background: Neuropathic pain is a devastating disease affecting millions of people worldwide. Serotonin (5-hydroxytryptamine, 5-HT) has long been involved in pain modulation. Several lines of evidence indicate that 5-HT1F receptor agonists are potent inducers of mitochondrial biogenesis. In this study, we tested the hypothesis that 5-HT1F receptor agonist ameliorates mechanical allodynia in neuropathic pain via induction of mitochondrial biogenesis and suppression of neuroinflammation. Methods: Male Sprague-Dawley rats were used to establish neuropathic pain via spared nerve injury (SNI). Paw withdrawal threshold was used to evaluate mechanical allodynia. Real time polymerase chain reaction was used to examine the mitochondrial DNA (mtDNA) copy number. Western blot and immunofluorescence were used to examine the expression of target proteins. Results: Our results showed that mitochondrial biogenesis was impaired in the spinal cord of SNI rats. Moreover, activation of PGC-1α, the master regulator of mitochondrial biogenesis, attenuated established mechanical allodynia in neuropathic pain rats. Besides, neuronal 5-HT1F receptor was significantly downregulated in the spinal cord of neuropathic pain rats. Furthermore, selective 5-HT1F receptor agonist Lasmiditan attenuated established mechanical allodynia in neuropathic pain rats. Finally, Lasmiditan treatment restored mitochondrial biogenesis and suppressed neuroinflammation in the spinal cord of SNI rats. Conclusion: These results provide the first evidence that Lasmiditan ameliorates mechanical allodynia in neuropathic pain via induction of mitochondrial biogenesis and suppression of neuroinflammation in the spinal cord. Mitochondrial biogenesis inducers may become encouraging therapeutic option for the management of neuropathic pain.

Pannexin 1-Mediated ATP Signaling in the Trigeminal Spinal Subnucleus Caudalis Is Involved in Tongue Cancer Pain

Pain is one of the most severe concerns in tongue cancer patients. However, the underlying mechanisms of tongue cancer pain are not fully understood. We investigated the molecular mechanisms of tongue cancer-induced mechanical allodynia in the tongue by squamous cell carcinoma (SCC) inoculation in rats. The head-withdrawal threshold of mechanical stimulation (MHWT) to the tongue was reduced following SCC inoculation, which was inhibited by intracisternal administration of 10Panx, an inhibitory peptide for pannexin 1 (PANX1) channels. Immunohistochemical analyses revealed that the expression of PANX1 was upregulated in the trigeminal spinal subnucleus caudalis (Vc) following SCC inoculation. The majority of PANX1 immunofluorescence was merged with ionized calcium-binding adapter molecule 1 (Iba1) fluorescence and a part of it was merged with glial fibrillary acidic protein (GFAP) fluorescence. Spike frequencies of Vc nociceptive neurons to noxious mechanical stimulation were significantly enhanced in SCC-inoculated rats, which was suppressed by intracisternal 10Panx administration. Phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive (IR) neurons increased significantly in the Vc after SCC inoculation, which was inhibited by intracisternal 10Panx administration. SCC inoculation-induced MHWT reduction and increased pERK-IR Vc neuron numbers were inhibited by P2X7 purinoceptor (P2X7R) antagonism. Conversely, these effects were observed in the presence of P2X7R agonist in SCC-inoculated rats with PANX1 inhibition. SCC inoculation-induced MHWT reduction was significantly recovered by intracisternal interleukin-1 receptor antagonist administration. These observations suggest that SCC inoculation causes PANX1 upregulation in Vc microglia and adenosine triphosphate released through PANX1 sensitizes nociceptive neurons in the Vc, resulting in tongue cancer pain.

Peripherally Administered Botulinum Toxin Type A Localizes Bilaterally in Trigeminal Ganglia of Animal Model

Peripheral nerve injury leads to sensory ganglion hyperexcitation, which increases neurotransmitter release and neuropathic pain. Botulinum toxin type A (BoNT/A) regulates pain transmission by reducing neurotransmitter release, thereby attenuating neuropathic pain. Despite multiple studies on the use of BoNT/A for managing neuropathic pain in the orofacial region, its exact mechanism of transport remains unclear. In this study, we investigated the effects of BoNT/A in managing neuropathic pain in two different animal models and its transport mechanism in the trigeminal nerve. Intraperitoneal administration of cisplatin induced bilateral neuropathic pain in the orofacial region, reducing the head withdrawal threshold to mechanical stimulation. Unilateral infraorbital nerve constriction (IONC) also reduced the ipsilateral head withdrawal threshold to mechanical stimulation. Unilateral peripheral administration of BoNT/A to the rat whisker pad attenuated cisplatin-induced pain behavior bilaterally. Furthermore, contralateral peripheral administration of BoNT/A attenuated neuropathy-induced behavior caused by IONC. We also noted the presence of BoNT/A in the blood using the mouse bioassay. In addition, the Alexa Fluor-488-labeled C-terminal half of the heavy chain of BoNT/A (BoNT/A-Hc) was localized in the neurons of the bilateral trigeminal ganglia following its unilateral administration. These findings suggest that axonal and hematogenous transport are involved in the therapeutic effects of peripherally administered BoNT/A in the orofacial region.

Notch1 Signaling Contributes to Mechanical Allodynia Associated with Cyclophosphamide-Induced Cystitis by Promoting Microglia Activation and Neuroinflammation

Aims. Notch1 signaling regulates microglia activation, which promotes neuroinflammation. Neuroinflammation plays an essential role in various kinds of pain sensation, including bladder-related pain in bladder pain syndrome/interstitial cystitis (BPS/IC). However, the impact of Notch1 signaling on mechanical allodynia in cyclophosphamide- (CYP-) induced cystitis is unclear. This study is aimed at determining whether and how Notch1 signaling modulates mechanical allodynia of CYP-induced cystitis. Methods. CYP was peritoneally injected to establish a bladder pain syndrome/interstitial cystitis (BPS/IC) rat model. A γ-secretase inhibitor, DAPT, was intrathecally injected to modulate Notch1 signaling indirectly. Mechanical withdrawal threshold in the lower abdomen was measured with von Frey filaments using the up-down method. The expression of Notch1 signaling, Iba-1, OX-42, TNF-α, and IL-1β in the L6-S1 spinal dorsal horn (SDH) was measured with Western blotting analysis and immunofluorescence staining. Results. Notch1 and Notch intracellular domain (NICD) were both upregulated in the SDH of the cystitis group. Moreover, the expression of Notch1 and NICD was negatively correlated with the mechanical withdrawal threshold of the cystitis rats. Furthermore, treatment with DAPT attenuated mechanical allodynia in CYP-induced cystitis and inhibited microglia activation, leading to decreased production of TNF-α and IL-1β. Conclusion. Notch1 signaling contributes to mechanical allodynia associated with CYP-induced cystitis by promoting microglia activation and neuroinflammation. Our study showed that inhibition of Notch1 signaling might have therapeutic value for treating pain symptoms in BPS/IC.

Low-Dose Cyclophosphamide Induces Nerve Injury and Functional Overactivity in the Urinary Bladder of Rats

Aim: This research aimed to investigate the neurotoxicity of low-dose cyclophosphamide (CYP) on the urinary bladder of rats by in vivo and in vitro studies.Methods: To establish CYP-induced cystitis rat model, rats were treated with three intraperitoneal injections of CYP (25 mg/kg) in a week. During treatment, the up-down method was used to assess the mechanical withdrawal threshold. On day 8, urodynamic test and bladder smooth muscle contractility study, including the contraction of bladder strips to electrical field stimulation (EFS, 2–64 Hz), carbachol (CCh, 10–8–10–5 M) and KCl (120 mM), were performed to evaluate the function of bladder function. Body weight and bladder weight were also recorded. Morphometric analysis using an optical microscope and transmission electron microscope was performed to observe the changes of microstructure and submicrostructure of the bladder. The major pelvic neurons were isolated and treated with acrolein (the main CYP metabolite) to assess apoptosis in vitro. RT-PCR assays were used to quantify the mRNA expression levels of Nlrp6, Asc, Casp11 and Casp1 in bladder tissues and primary neurons.Results: After CYP injections, the body weights decreased, but the bladder weights increased in the model group. The mechanical withdrawal threshold of the cystitis model remained at a low level. The morphometric analysis suggested bladder inflammation and neuroinflammation in the bladder of the cystitis rat model. Urodynamic test revealed that, the amplitude, the pressure baseline, the peak pressure and pressure threshold of model rats significantly increased after CYP treatment. The muscle strips of model rats exhibited significantly higher contractility caused by EFS and CCh than the controls. Apoptotic cells appeared at the highest concentration group (100 μM acrolein) after 6 h of acrolein incubation in apoptosis assay of primary neurons. The mRNA expression levels of Nlrp6 and Casp11 were significantly increased in the cystitis rat model and in the acrolein-treated neurons.Conclusions: Low-dose CYP treatment was confirmed to induce nerve injury, which leading to bladder pain and overactive bladder in female rats, and the up-regulation of Nlrp6 and Casp11 may contribute to these pathological changes.