Preparation and Analysis of New 1,3,5-Trisubstituted-2-Pyrazolines Derivative for Their Analgesic Potential

The goal of the study was to develop, synthesise, and characterise a novel 1,3,5-trisubstituted-2-pyrazolines derivative, as well as to assess its analgesic potential. The reaction of chalcone derivatives with 4-hydrazinylbenzene sulfonamide hydrochloride and phenyl hydrazine hydrochloride yielded 1,3,5-tri-substituted-2-pyrazolines derivatives. The IR, 1HNMR, and mass spectrum analyses were used to characterise a total of sixteen substances. Analgesic activity of the proposed substances has been tested. The analgesic effect of the produced compounds was tested using two methods: the hot plate test technique and acetic acid induced writhing in mice. To compare the effectiveness, pentazocine and acetyl acetic acid were utilised as reference drugs. The hot plate test technique and acetic acid induced writhing in mice were used to assess the analgesic effect of the 16 produced chemical series A1-A8, and B1-B8. The evaluation's outcomes were viewed using Pentazocine and acetyl acetic acid as the standard drugs. In a 90-minute hot plate test, compounds A2 (10.30 s), A4 (9.45 s), A7 (11.65 s), and A8 (11.26 s) showed a delay in paw withdrawal latency time. Compounds B2 (9.10 s) and B7 (10.42 s) prolong the paw withdrawal latency time after 90 minutes in series B1-B8, reduce the pain feeling, and inhibit pain induced by heat methods. Compounds A2, A5, A6, A7, and A8 from Series A1-A8 showed 83.00, 76.01, 80.34, 86.99, 88.15 percent inhibition, substantially (p0.05 and p0.001, respectively), and decreased the number of wriths caused by 0.6 percent acetic acid at a dosage of 10 mg/kg. Acetylsalicylic acid (10 mg/kg) appears to be more successful in lowering the number of wriths, with a 99.0% reduction in the number of wriths (p0.001). B1, B3, and B4 have the least amount of active activity. These all finding suggest that these synthesized compounds have the potential as analgesic agent.

TailTimer: A device for automating data collection in the rodent tail immersion assay

The tail immersion assay is a widely used method for measuring acute thermal pain in a way which is quantifiable and reproducible. It is non-invasive and measures response to a stimulus that may be encountered by an animal in its natural environment. However, quantification of tail withdrawal latency relies on manual timing of tail flick using a stopwatch, and precise temperatures of the water at the time of measurement are most often not recorded. These two factors greatly reduce the reproducibility of tail immersion assay data and likely contribute to some of the discrepancies present among relevant literature. We designed a device, TailTimer, which uses a Raspberry Pi single-board computer, a digital temperature sensor, and two electrical wires, to automatically record tail withdrawal latency and water temperature. We programmed TailTimer to continuously display and record water temperature and to only permit the assay to be conducted when the water is within ± 0.25°C of the target temperature. Our software also records the identification of the animals using a radio frequency identification (RFID) system. We further adapted the RFID system to recognize several specific keys as user interface commands, allowing TailTimer to be operated via RFID fobs for increased usability. Data recorded using the TailTimer device showed a negative linear relationship between tail withdrawal latency and water temperature when tested between 47–50°C. We also observed a previously unreported, yet profound, effect of water mixing speed on latency. In one experiment using TailTimer, we observed significantly longer latencies following administration of oral oxycodone versus a distilled water control when measured after 15 mins or 1 h, but not after 4 h. TailTimer also detected significant strain differences in baseline latency. These findings valorize TailTimer in its sensitivity and reliability for measuring thermal pain thresholds.

Evaluation of the paw withdrawal latency for the comparison between tramadol and butorphanol administered locally, in the plantar surface of rat, preliminary study

The aim of the study was to evaluate the analgesic efficacy of tramadol compared to butorphanol administered locally in ventral surface of the hind paw of rats. Prospective, randomized experimental study; twenty-one adult male Wistar rats were selected. Heart rate (beats minute-1), respiratory rate (breaths minute-1), and paw withdrawal latency (onset of radiant heat until paw withdrawal/seconds) were measured prior (T0) and after (T5, T10, T15, T20) intraplantar injection with saline solution 0,9% (group S), butorphanol 1 mg kg-1 (group B), and tramadol 1 mg kg-1 (group T). Shapiro-Wilk normality test and Friedman test were used to analyze the data expressed by median and range. Statistical significance was set at p < 0.05. Statistical analysis of heart rate showed that there were significant differences between groups at different monitoring times. There were no significant differences in respiratory rate after intraplantar injection in any of the treatment groups. The paw withdrawal latency values at T5, T10, and T15 minutes after intraplantar injection in the group B were significantly higher compared to baseline value and to the values of the other groups. The paw withdrawal latency were no significant changes in the measurements of intragroup in S and T. Intraplantar administration of butorphanol provides a good analgesia and significantly increases paw withdrawal latency compared to tramadol. Intraplantar injection of butorphanol could be useful and safe and safe technique to achieve local analgesia for minor surgical procedures in rats.

Increase in the glutamate transporter 1 and time withdrawal latency following wet cupping therapy in chronic constriction injury in rats

Objective: Neuropathic pain (NP) is a chronic debilitating pain and is caused by disease or lesion of somatosensory system. NP respond worse to the pharmacological drugs leading to this pain still a big problem in medical treatment and furthermore make many patients seek alternative treatment. Wet cupping therapy (WCT) has been widely used to relief both of acute and chronic pain, but the mechanism for reducing pain has not yet been clear. Recent studies have shown that NP is associated with alteration of GLT-1/EAAT2, and WCT has beneficial role to reduce the pain in various pain models. This is the pilot study, no other study has applied WCT in chronic constriction injury (CCI) models, the most commonly employed animal model of NP. Therefore, we investigate the association between WCT and the reducing pain by looking at the increase of GLT-1 and time withdrawal latency (TWL) in rats with CCI. Methodology: The study design was randomized, post-test only, controlled trial with a total of 21 male rats (Rattus Norvegicus) with CCI, aged 4 months, weighing 220 to 250 g, randomly divided into three groups, P1 (sham CCI group), P2 (CCI group), and P3 (CCI group plus WCT). WCT had been applied 2 times/week for 3 weeks to all of the groups in paralumbar region, both left and right side. TWL was recorded to assess pain threshold of the rats by hot plate and the expression of GLT-1 on glial cells in spinal cord were counted. Results: This study revealed that mean ± SD values for P1, P2, and P3 were 7.20 ± 1.30, 2.57 ± 1.27, and 18.20 ± 3.50 respectively. There were significant differences in the TWL between groups P1-P2, P1-P3, and P2-P3 (p = 0.003, p = 0.0001, and p = 0.0001 respectively) and GLT-1 increase was significant between groups P2-P3 (p = 0.009). Conclusion: It can be concluded that wet cupping therapy decreases the pain by increasing the time withdrawal latency and GLT-1 in chronic constriction injury models. We suggest that wet cupping therapy as a promising method to reduce pain in peripheral neuropathic pain models. However, further investigation is still needed to confirm its mechanism of action. Key words: GLT-1/EAAT2; Neuropathic pain; Wet cupping therapy; Chronic constriction injury; CCI, TWL Citation: Hidayati HB, Machfoed MH, Kuntoro, Subadi I, Khaerunnisa S, Widjiati. Increase in the glutamate transporter 1 and time withdrawal latency following wet cupping therapy in chronic constriction injury in rats. Anaesth. pain & intensive care 2021;25(1):48-54. DOI: 10.35975/apic.v25i1.1441 Received: 24 January 2019, Reviewed: 4 January 2019, 14 January 2019, Revised: 24 January 2019, Accepted: 20 May 2019

Antiarthritic Potential of Calotropis procera Leaf Fractions in FCA-Induced Arthritic Rats: Involvement of Cellular Inflammatory Mediators and Other Biomarkers

Calotropis procera (commonly known as Swallow wort) is described in the Ayurvedic literature for the treatment of inflammation and arthritic disorders. Therefore, in the present work, the antiarthritic activity of potential fractions of Swallow wort leaf was evaluated and compared with standards (indomethacin and ibuprofen). This study was designed in Wistar rats for the investigation of antiarthritic activity and acute toxicity of Swallow wort. Arthritis was induced in Wistar rats by injecting 0.1 mL of Freund’s complete adjuvant (FCA) on the 1st and 7th days subcutaneously into the subplantar region of the left hind paw. Evaluation of our experimental findings suggested that antiarthritic activity of methanol fraction of Swallow wort (MFCP) was greater than ethyl acetate fraction of Swallow wort (EAFCP), equal to standard ibuprofen, and slightly lower than standard indomethacin. MFCP significantly reduced paw edema on the 17th, 21st, 24th, and 28th days. It also showed significant effect (p < 0.01) on arthritic score, paw withdrawal latency, and body weight. The inhibition of serum lysosomal enzymes and proinflammatory cytokines along with improvement of radiographic features of hind legs was also recorded with MFCP. Finally, it was concluded that MFCP can be a feasible therapeutic candidate for the treatment of inflammatory arthritis.

Interferon-β suppresses inflammatory pain through activating µ-opioid receptor

Interferons (IFNs) are cytokines secreted by infected cells that can interfere with viral replication. Besides activating antiviral defenses, type I IFNs also exhibit diverse biological functions. IFN-β has been shown to have a protective effect against neurotoxic and inflammatory insults on neurons. Therefore, we aimed to investigate the possible role of IFN-β in reducing mechanical allodynia caused by Complete Freund’s Adjuvant (CFA) injection in rats. We assessed the antinociceptive effect of intrathecal IFN-β in naïve rats and the rats with CFA–induced inflammatory pain. After the behavioral test, the spinal cords of the rats were harvested for western blot and immunohistochemical double staining. We found that intrathecal administration of IFN-β in naïve rats can significantly increase the paw withdrawal threshold and paw withdrawal latency. Further, the intrathecal injection of a neutralizing IFN-β antibody can reduce the paw withdrawal threshold and paw withdrawal latency, suggesting that IFN-β is produced in the spinal cord in normal conditions and serves as a tonic inhibitor of pain. In addition, intrathecal injection of IFN-β at dosages from 1000 U to 10000 U demonstrates a significant transient dose-dependent inhibition of CFA-induced inflammatory pain. This analgesic effect is reversed by intrathecal naloxone, suggesting that IFN-β produces an analgesic effect through central opioid receptor-mediated signaling. Increased expression of phospho-µ-opioid receptors after IFN-β injection was observed on western blot, and immunohistochemical staining showed that µ-opioids co-localized with IFN-α/βR in the dorsal horn of the spinal cord. The findings of this study demonstrate that the analgesic effect of IFN-β is through µ-opioid receptors activation in spial cord.

Analgesic Efficacy of Tramadol and Morphine in White’s Tree Frogs (Litoria caerulea)

Published data are sparse regarding the recognition of clinically relevant pain and appropriate analgesia in amphibians. The amphibian analgesia literature has primarily focused on nociceptive pathways in a single species, the northern leopard frog (Rana pipiens). The objective of the current study was to assess the analgesic efficacy and safety of oral tramadol and subcutaneous morphine in a commonly maintained zoo and pet species, White’s tree frog (Litoria caerulea). We hypothesized that tramadol and morphine would provide dose-dependent antinociception, as measured by significant increases in hindlimb withdrawal latency after exposure to a noxious thermal stimulus. Two randomized, placebo-controlled, complete crossover studies were performed, with tramadol (n = 12) administered at 15, 25, and 40 mg/kg PO and morphine (n = 12) administered at 5 and 10 mg/kg SC. Hindlimb withdrawal latency was measured for a maximum of 72 h. No adverse side effects or signs of sedation were observed with any dose or drug evaluated. No significant difference in withdrawal latency was detectedbetween the control and either tramadol or morphine. These negative results were surprising, suggesting that the thermalnociceptive model may not be biologically relevant in amphibian species.

Lidocaine activates autophagy of astrocytes and ameliorates chronic constriction injury-induced neuropathic pain

Lidocaine is a commonly used drug to alleviate neuropathic pain (NP). This work aims to investigate the mechanism of lidocaine in alleviating NP. Chronic constriction injury (CCI) rats were established by surgery to induce NP. We observed the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of rats. Immunofluorescence staining was performed to determine the LC3/glial fibrillary acidic protein (GFAP)-positive cells. Rat astrocytes were treated with lipopolysaccharide (LPS) to induce CCI, and then treated with lidocaine or 3-MA (autophagy inhibitor). CCK-8 was performed to detect cell proliferation. Western blot and enzyme-linked immunosorbent assay were performed to detect the level of protein and inflammatory factor. CCI rats exhibited a decrease of MWT and TWL, which was effectively abolished by lidocaine. Lidocaine enhanced the number of LC3/GFAP-positive cells in CCI rats. Moreover, lidocaine inhibited the expression of GFAP and p62, and enhanced LC3-II/LC3-I expression in the LPS-treated astrocytes. Lidocaine inhibited the level of TNF-α and IL-1β in the LPS-treated astrocytes. The influence conferred by lidocaine was effectively abolished by 3-MA. In conclusion, our work demonstrates that lidocaine activates autophagy of astrocytes and ameliorates CCI-induced NP. Thus, our study provides a further experimental basis for the mechanism of lidocaine to alleviate NP.

Mannitol Enhances the Antinociceptive Effects of Diphenhydramine as an Alternative Local Anesthetic

Mannitol has recently been reported to be effective in enhancing the antinociceptive efficacy of lidocaine. No single study to date, however, has compared diphenhydramine with and without mannitol for nociceptive processing as an alternative local anesthetic. In this study, we examined the antinociceptive efficacy enhancements of diphenhydramine when combined with mannitol. Male Sprague-Dawley rats weighing 230–260 g were used in a hot plate test to evaluate the antinociceptive effects of diphenhydramine. All chemicals were dissolved in isotonic normal saline and administered subcutaneously into the plantar surface of the right hind paw at 10 min before the hot plate test. A subcutaneous injection of 0.5% or 1% diphenhydramine produced significant inhibition of the withdrawal latency time compared with the vehicle treatment. Antinociceptive effects appeared 10 min after the diphenhydramine injections and persisted for over 30 min. The antinociceptive effects of 1% diphenhydramine were not statistically different from those of 1% lidocaine. Although a subcutaneous injection of a 0.5 M mannitol solution alone did not affect the withdrawal latency time, 1% diphenhydramine with 0.5 M mannitol significantly enhanced antinociception. A subcutaneous injection of 1% diphenhydramine with epinephrine (1 : 100,000) solution did not increase the antinociceptive effect of the diphenhydramine. These results suggest that diphenhydramine with mannitol can be used as an alternative local anesthetic.