Persistent effects of the orexin-1 receptor antagonist SB-334867 on naloxone precipitated morphine withdrawal symptoms and nociceptive behaviors in morphine dependent rats.

AIM OF THE STUDY: In this study, we investigated the effect of long-term administration of orexin receptor 1 (OXR1) antagonist on naloxone-precipitated morphine withdrawal symptoms and nociceptive behaviors in morphine-dependent rats. MATERIALS AND METHODS: Wistar rats received subcutaneous (s.c.) injections of morphine (6, 16, 26, 36, 46, 56, and 66 mg/kg, 2 ml/kg) at an interval of 24 h for 7 days. In chronic groups, the OXR1 antagonist, SB-334867 (20 mg/kg, i.p.), or its vehicle, was injected repetitively from postnatal day 1 (PND1)-PND23 and then for the following seven days before each morphine injection. Meanwhile, in acute groups, SB-334867, or its vehicle, was administered before each morphine injection. In groups of rats that were designated for withdrawal experiments, naloxone (2.5 mg/kg, i.p.) was administered after the last injection of morphine. In the formalin-induced pain, the effect of OXR1 inhibition on the antinociceptive effects of morphine was measured by injecting formalin after the final morphine injection. RESULTS: Animals that received long-term SB-334867 administration before morphine injection demonstrated a significant reduction in chewing, defecation, diarrhea, grooming, teeth chattering, wet-dog shake, and writhing. Inhibiting OXR1 for a long time increased formalin-induced nociceptive behaviors in interphase and phase II of the formalin-induced pain. CONCLUSIONS: Our results indicated that the inhibition of OXR1 significantly reduces the development of morphine dependence and behavioral signs elicited by the administration of naloxone in morphine-dependent rats. Furthermore, the prolonged blockade of OXR1 might be involved in formalin-induced nociceptive behaviors.

Formalin-induced pain prolongs sub- to supra-second time estimation in rats.

BACKGROUND: Temporal estimation can be influenced by pain, which is a complex psychological and physiological phenomenon. However, the time range in which perception is most sensitive to pain remains unclear. METHODS: In the present study, we explored the effects of acute inflammatory pain on time perception in the sub- to supra-second (0.6-2.4-s) and supra-second (2-8-s) ranges in rats. Plantar formalin injection was used to induce acute inflammatory pain, and a temporal bisection task was used to measure time perception. Task test sessions were held for five consecutive days (one per day): the day before injection (baseline), immediately after injection, and the three post-injection days. The point of subjective equality (PSE, which reflects the subjective duration) and Weber fraction (which reflects temporal sensitivity) were calculated and analysed. RESULTS: In the 0.6-2.4-s range, the PSE was significantly lower, indicating prolonged subjective duration, in the formalin group relative to the saline group (p = 0.049) immediately after injection. Formalin-induced pain also tended to lengthened time perception in the 0.6-2.4-s range on post-injection days 2 (p = 0.06) and 3 (p = 0.054). In the 2-8-s range, formalin injection did not affect the PSE or Weber fraction. CONCLUSIONS: The enhanced effect of pain on temporal perception in the sub- to supra-second range is observed in this study and this effect is attenuated with the prolongation of estimated time, even in rats.

Analgesic Effect of Intrathecal Melissa officinalis in the Rat Model of Hot-Water and Formalin-Induced Pain.

Melissa officinalis (MO) is one of the oldest herbal medicines commonly used in traditional medicine, which some studies have investigated for its analgesic effect. This study is an attempt to investigate the effects of intrathecal administration of Melissa officinalis on the pain induced by heat and formalin. In this experimental study, 70 male Wistar rats with an average weight of 270-320 g were randomly divided into five groups: control; sham that received 25 µl of saline through the spinal catheter; and three experimental groups that received 5, 10 or 20 mg/kg M. officinalis via the spinal catheter respectively. Five days after catheterization of the spinal cord from the lumbar region under anesthesia, the effects of Intrathecal administration of M. officinalis on heat- and formalin-induced pain were evaluated. Data were analyzed by using one-way ANOVA. Intrathecal injection of M. officinalis blocked heat-induced pain compared to sham group (p = 0.001). Maximum analgesia was observed 30 min after the injection. Furthermore, intrathecal administration of MO alleviated both acute (p = 0.007) and chronic (p = 0.001) phases of formalin-induced pain. Motor block was not observed in any of the above mentioned groups. The results showed that intrathecal administration of MO could significantly improve hot-water and formalin-induced pain in male Wistar rats.

Manganese-enhanced magnetic resonance imaging of the spinal cord in rats with formalin-induced pain.

Manganese-enhanced magnetic resonance imaging (MEMRI) is based on neuronal activity-dependent manganese uptake, and provides information about nervous system function. However, systematic studies of pain processing using MEMRI are rare, and few investigations of pain using MEMRI have been performed in the spinal cord. Herein, we investigated the pain dependence of manganese ions administered in the rat spinal cord. MnCl2 was administered into the spinal cord via an intrathecal catheter before formalin injection into the right hind paw (50 µL of 5% formalin). The duration of flinching behavior was recorded and analyzed to measure formalin-induced pain. After the behavioral test, rats were sacrificed with an overdose of urethane (50 mg/kg), and spine samples were extracted and post-fixed in 4% paraformaldehyde solution. The samples were stored in 30% sucrose until molecular resonance (MR) scanning was performed. In axial Mn2+ enhancement images of the spinal cord, Mn2+ levels were found to be significantly elevated on the ipsilateral side of the spinal cord in formalin-injected rats. To confirm pain-dependent Mn enhancement in the spinal cord, c-Fos expression was analyzed, and was found to be increased in the formalin-injected rats. These results indicate that MEMRI is useful for functional analysis of the spinal cord under pain conditions. The gray matter appears to be the focus of intense paramagnetic signals. MEMRI may provide an effective technique for visualizing activity-dependent patterns in the spinal cord.

Involvement of endoplasmic reticulum stress in formalin-induced pain is attenuated by 4-phenylbutyric acid.

BACKGROUND: Endoplasmic reticulum (ER) stress is involved in many neurological and inflammatory responses. Peripheral inflammatory responses can induce central sensitization and trigger inflammatory pain. However, there is little research on the relationship between ER stress and inflammatory pain. In this study, we examined whether the ER stress response is involved in peripheral inflammatory pain using a formalin-induced rat pain model. METHODS: Rats were divided into the following five groups: control, formalin, formalin + vehicle, formalin + 4-phenylbutyric acid (4-PBA) (40 mg/kg) and formalin + 4-PBA (100 mg/kg). Formalin-induced pain was assessed behaviorally by recording licking activity. The expression levels of immunoglobulin-binding protein (BIP), activating transcription factor-6 (ATF6), phosphorylated inositol-requiring enzyme-1 (p-IRE1), phosphorylated protein kinase RNA-like ER kinase (p-PERK) and c-fos were quantitatively assessed by Western blot, and the distribution of BIP, ATF6 and c-fos in the lumbar enlargement of spinal cord were identified by immunohistochemistry in spinal dorsal horn slices. In addition, the concentrations of nitric oxide (NO) and prostaglandin E2 (PGE2) in the spinal cord were tested by biochemical measurement and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: Intraperitoneal injection of 4-PBA at the dose of 100 mg/kg before formalin injection significantly decreased nociceptive behavior in the second phase compared with control, formalin, formalin + vehicle and formalin + 4-PBA (40 mg/kg) (P<0.05). Western blot showed that formalin injection significantly upregulated the expression of BIP, ATF6, p-PERK and c-fos in the spinal cord. This upregulation was reduced by peritoneal injection of 4-PBA (P<0.05), while expression of p-IRE1 was not altered by formalin treatment. Immunohistochemistry revealed markedly increased staining density for BIP, ATF6 and c-fos in the superficial spinal dorsal horn after formalin injection. This was significantly decreased by administration of 4-PBA (P<0.05). Compared with the formalin + vehicle group, 4-PBA inhibited the release of NO and PGE2 in the spinal cord (P<0.05). CONCLUSION: These results suggest that ER stress is involved in formalin-induced inflammatory pain and that inhibition of ER stress may attenuate central sensitization induced by peripheral inflammatory stimulation.

Antinociceptive properties of shikonin: in vitro and in vivo studies.

Shikonin possess a diverse spectrum of pharmacological properties in multiple therapeutic areas. However, the nociceptive effect of shikonin is not largely known. To investigate the antinociceptive potential of shikonin, panel of GPCRs, ion channels, and enzymes involved in pain pathogenesis were studied. To evaluate the translation of shikonin efficacy in vivo, it was tested in 3 established rat pain models. Our study reveals that shikonin has significant inhibitory effect on pan sodium channel/N1E115 and NaV1.7 channel with half maximal inhibitory concentration (IC50) value of 7.6 µmol/L and 6.4 µmol/L, respectively, in a cell-based assay. Shikonin exerted significant dose dependent antinociceptive activity at doses of 0.08%, 0.05%, and 0.02% w/v in pinch pain model. In mechanical hyperalgesia model, dose of 10 and 3 mg/kg (intraperitoneal) produced dose-dependent analgesia and showed 67% and 35% reversal of hyperalgesia respectively at 0.5 h. Following oral administration, it showed 39% reversal at 30 mg/kg dose. When tested in first phase of formalin induced pain, shikonin at 10 mg/kg dose inhibited paw flinching by ∼71%. In all studied preclinical models, analgesic effect was similar or better than standard analgesic drugs. The present study unveils the mechanistic role of shikonin on pain modulation, predominantly via sodium channel modulation, suggesting that shikonin could be developed as a potential pain blocker.

Effects of microinjection of histamine into the anterior cingulate cortex on pain-related behaviors induced by formalin in rats.

BACKGROUND: The present study was aimed to investigate the effects of microinjection of histamine and its H1, H2 and H3 receptor antagonists, mepyramine, ranitidine and thioperamide, respectively, into the anterior cingulate cortex (ACC) on pain-related behaviors induced by formalin in rats. METHODS: Two stainless steel guide canulas were bilaterally implanted into the ACC of anaesthetized rats. For induction of pain, intraplantar (ipl) injection of a 2.5% formalin solution was performed. The duration of paw licking/biting and the number of paw flinching were recorded in 5 min blocks for 60 min. Locomotor activity was assessed using an open-field test. RESULTS: Formalin produced a marked biphasic pattern of pain. Histamine reduced the second phases of paw licking/biting and flinching. Mepyramine (2 µg/side) prevented the suppressive effect of histamine (1 µg/side) on second phase of pain, but at a dose of 8 µg/side it did not inhibit the suppressive effects of 4 µg/side of histamine. Ranitidine at doses of 2 and 8 µg/side prevented histamine (1 and 4 µg/side)-induced antinociception. Thioperamide not only suppressed the second phases of pain, but also increased the suppressive effect of histamine. Naloxone prevented suppressive effects of histamine and thioperamide on pain. Mepyramine (8 µg/side) suppressed locomotor activity. CONCLUSION: The results of the present study showed pain suppressing effects for histamine. Histamine H2 and H3, and to a lesser extent, H1 receptors might be involved in histamine-induced antinociception. Opioid receptors might be involved in suppressive effects of histamine and thioperamide.

Evaluation of analgesic, anti-inflammatory and CNS depressant activities of methanolic extract of Lawsonia inermis barks in mice.

OBJECTIVES: The study was carried out to assess the analgesic, anti-inflammatory, and CNS depressant activity of the methanolic extract of the Lawsonia inermis barks (MELIB). MATERIALS AND METHODS: Anti-inflammatory effects of MEBLI were studied using carrageenan-induced inflammatory method at the dose of 300 and 500 mg/kg b.wt., p.o. Analgesic activity was measured using acetic acid-induced writhing model and formalin-induced licking and biting in mice. The CNS depressant activity was evaluated by observing the reduction of locomotor and exploratory activities in the open field and hole cross tests at a dose of 300 and 500 mg/kg body weight. RESULTS: Statistical analysis showed that dose of 500 mg/kg exhibited higher analgesic activity against acetic acid-induced pain in mice than the standard drug diclofenac sodium. Furthermore, doses of 300 and 500 mg/kg caused higher percent of protection (91.16% and 95.03%, respectively) of licking and biting of formalin-induced mice than diclophenac sodium (70.72%). The Lawsonia inemis methanolic extract (300 and 500 mg/kg) also exhibited sustained inhibition (54.97% and 65.56%) of paw edema at the 4(th) hour compared with standard indomethacin (74.17%). Besides, the plant extract also had significant (p<0.05) dose-dependent CNS depressant activity. CONCLUSION: this study recommends that the methanolic extract of Lawsonia inermis barks has significant analgesic, anti-inflammatory, and CNS depressant properties.

Effects of administration of histamine and its H1, H2, and H3 receptor antagonists into the primary somatosensory cortex on inflammatory pain in rats.

OBJECTIVE(S): The present study investigated the effects of microinjection of histamine and histamine H1, H2, and H3 receptor antagonists, chlorpheniramine, ranitidine and thioperamide, respectively into the primary somatosensory cortex (PSC) on inflammatory pain. MATERIAL AND METHODS: Two stainless steel guide canulas were bilaterally implanted into the PSC of anaesthetized rats. Inflammatory pain was induced by subcutaneous (SC) injection of formalin (50 µl, 2.5%) in the ventral surface of right hind paw. Time durations of licking/biting of the injected paw were recorded as a pain measure. RESULTS: Formalin produced a biphasic pattern of licking/biting of the injected paw. Histamine at doses of 0.5, 1, and 2 µg decreased the intensity of pain. Chlorpheniramine and ranitidine at the same doses of 1 and 4 µg had no effects, whereas thioperamide at a dose of 4 µg suppressed both phases of formalin-induced pain. Pretreatments with chlorpheniramine and ranitidine at the same dose of 4 µg prevented histamine (2 µg)-induced antinociception. Antinociceptive effects were observed when thioperamide at doses of 1 and 4 µg was used with 0.25 and 1 µg of histamine, respectively. The antinociceptive effects induced by histamine (2 µg) and thioperamide (4 µg) were prevented by prior treatment with naloxone (4 µg). CONCLUSION: These results indicate that at PSC levels, histamine through post-synaptic H1, H2, and pre-synaptic H3 receptors might be involved in pain modulation. The endogenous opioid system may be involved in histamine- and thioperamide-induced antinociception.

The effect of sciatic nerve injection of dexmedetomidine on the nociceptive behavior and spinal c-fos expression in formalin mice / 重庆医学

Objective To investigate the effect of sciatic nerve injection of dexmedetomidine on the nociceptive behavior and spi-nal c-fos expression in formalin mice .Methods Adult male Kunming mice ,20-25 g ,were divided into 4 groups randomly :mice in group A and D were given an injection of NS (10 μL) and 0 .1 μg dexmedetomidine in 10 μL adjacent to the right sciatic nerve , sepretely .Mice in group B received intacutaneous injection of 10μL formalin(5% ) in the right hindpaws .Mice in group C were giv-en an injection of 0 .1 μg dexmedetomidine in 10 μL adjacent to the right sciatic nerve 15 min before formalin injection in the right hindpaws .Paw licking/biting time was counted every 5 min for an hour after hindpaw injections ,calculated pain score .1 hour after behavior tests ,the L4-5 of spinal cord were harvested to immunofluorescence for c-fos expression in the dorsal corn of spinal cord . Results Mice in group A、C and D showed no obvious foot licking/biting behaviors and a low level of c-fos expression in the dorsal corn of spinal cord .Compared with group A ,mice in group B displayed obvious two phases foot licking/biting behaviors(P<0 .05 , both in Phase Ⅰ and Phase Ⅱ) and a significant increasing expression of c-fos(P<0 .05) .Compared with group B ,dexmedetomi-dine injected to the adjacent of right sciatic nerve decreased the pain score and spinal c-fos expression in goup C(P<0 .05) .Conclu-sion Injection of dexmedetomidine to the right sciatic nerve alleviated intraplantar injected formalin-induced acute inflammatory pain ,and reduced the expression of spinal c-fos protein .

Sertraline inhibits formalin-induced nociception and cardiovascular responses

The objective of the present study was to determine the antihyperalgesic effect of sertraline, measured indirectly by the changes of sciatic afferent nerve activity, and its effects on cardiorespiratory parameters, using the model of formalin-induced inflammatory nociception in anesthetized rats. Serum serotonin (5-HT) levels were measured in order to test their correlation with the analgesic effect. Male Wistar rats (250-300 g) were divided into 4 groups (N = 8/per group): sertraline-treated group (Sert + Saline (Sal) and Sert + Formalin (Form); 3 mg·kg-1·day-1, ip, for 7 days) and saline-treated group (Sal + Sal and Sal + Form). The rats were injected with 5 percent (50 µL) formalin or saline into the right hind paw. Sciatic nerve activity was recorded using a silver electrode connected to a NeuroLog apparatus, and cardiopulmonary parameters (mean arterial pressure, heart rate and respiratory frequency), assessed after arterial cannulation and tracheotomy, were monitored using a Data Acquisition System. Blood samples were collected from the animals and serum 5-HT levels were determined by ELISA. Formalin injection induced the following changes: sciatic afferent nerve activity (+50.8 ± 14.7 percent), mean arterial pressure (+1.4 ± 3 mmHg), heart rate (+13 ± 6.8 bpm), respiratory frequency (+4.6 ± 5 cpm) and serum 5-HT increased to 1162 ± 124.6 ng/mL. Treatment with sertraline significantly reduced all these parameters (respectively: +19.8 ± 6.9 percent, -3.3 ± 2 mmHg, -13.1 ± 10.8 bpm, -9.8 ± 5.7 cpm) and serum 5-HT level dropped to 634 ± 69 ng/mL (P < 0.05). These results suggest that sertraline plays an analgesic role in formalin-induced nociception probably through a serotonergic mechanism.

Role of central muscarinic cholinergic receptors in the formalin-induced pain in rats.

OBJECTIVES: In the present study, central effects of physostigmine and atropine have investigated in the formalin-induced pain in rats. MATERIALS AND METHODS: In conscious rats implanted with an intracerebroventricular (i.c.v.) cannula, the effects of i.c.v. injection of physostigmine and atropine were investigated on the formalin test in the rat. Formalin test was induced by subcutaneous (s.c.) injection of formalin (50 mul, 1%) in ventral surface of left hind paw, and durations of licking and biting of the injected paw were measured in 5-min blocks for 1 h. RESULTS: FORMALIN PRODUCED A BIPHASIC RESPONSE (FIRST PHASE: 0-5 and second phase: 15-40 min) in durations of licking and biting of the injected paw. Physostigmine at doses of 2.5, 5 and 10 ug significantly (P < 0.05) attenuated both first and second phases of pain response. Atropine (5 and 10 ug), used alone, produced no significant effect on pain, but pretreatment with atropine (10 ug) significantly (P < 0.05) blocked antinociception induced by physostigmine (5 ug). CONCLUSION: These results indicate that i.c.v. physostigmine can affect both neurogenic and inflammatory phases of formalin-induced pain through a mechanism in which the muscarinic cholinergic receptors are involved.