Subcutaneous Injection Of Formalin Research Articles

Mode of delivery alters sensitivity to thermal and chemical stimuli in adult rats: An experimental study.

The mode of delivery might prompt a long-lasting alteration in physiological and behavioral responsiveness in offspring. This study was intended to evaluate if the mode of delivery could alter sensitivity to thermal and chemical stimuli in female rats. 56 adult female Wistar rats (200-220 gr) that were born by vaginal or cesarean section (C-section) were used (n = 28/each). Inflammatory pain was induced by subcutaneous injection of formalin into the hind paw. The thermal nociceptive threshold was determined by tail-flick and hot plate tests. Besides, the Western blot test was used to evaluate the spinal cord levels of c-Fos and c-Jun proteins. Formalin-induced inflammation was significantly decreased in C-section group as compared to vaginally born rats (p 0.001). The baseline nociceptive threshed and morphine-induced analgesia were significantly increased in C-section groups in comparison to vaginally born rats. In addition, the levels of c-Fos and c-Jun proteins were significantly decreased in the spinal cord of C-section rats as compared to vaginally born animals (p 0.01). Morphine treatment could decrease the expression of c-Fos and c-Jun in the C-section group (p 0.05). Overall, C-section rats showed lower spinal nociceptive processing and neuronal activity later in life, compared to the vaginal born rats.

Ivabradine-Induced Bradycardia is Accompanied by Reduced Stress-Related Anxiety.

Hypertensive individuals with higher heart rates and anxiety have greater cardiovascular morbidity and mortality. Despite the correlation between hypertension, heart rate, and anxiety, scant attention has been paid to the effect of hypertension drug therapy on behavioral outcomes in cardiovascular disease. Ivabradine, an inhibitor of hyperpolarization-activated, cyclic nucleotide-gated funny channels (HCNs), has been used clinically to reduce heart rates and has been shown to improve quality of life in patients with angina and heart failure. We postulated that in addition to lowering heart rate, ivabradine could reduce anxiety in mice exposed to a significant stress paradigm. Mice underwent a stress induction protocol, subsequently they received either vehicle or ivabradine (10 mg/kg) via osmotic minipumps. Blood pressure and heart rates were measured with tail cuff photoplethysmography. Anxiety was assessed quantitatively through the open field test (OFT) and the elevated plus maze (EPM). Cognition was assessed with an object recognition test (ORT). Pain tolerance was measured by the hot plate test or subcutaneous injection of formalin. HCN gene expression was measured with RT-PCR. Ivabradine reduced resting heart rate in the stressed mice by 22%. Stressed mice treated with ivabradine displayed significantly greater exploratory behavior in the OFT, EPM, and ORT. The expression of central HCN channels was significantly reduced following stress. It is suggested from our findings that ivabradine can reduce anxiety following significant psychological stress. Reductions in heart rate may directly improve quality of life by reducing anxiety in patients with hypertension and high heart rates.

Behaviors associated with formalin-induced pain in the Japanese quail (Coturnix coturnix japonica)

The present study aimed to investigate behaviors associated with formalin-induced nociception in the Japanese quail (Coturnix japonica). The tarsometatarsus region of the male quail's foot (shank) was injected with 40µl of formalin (0.3, 0.6, and 0.9%) solutions or normal saline. Two distinct nociceptive behaviors that is injected foot lifting and flinching/shaking behaviors were recorded over 45 minutes. Also, unstimulated preening, eye closing, and chamber wall pecking behaviors were recorded during the observation period. Subcutaneous injection of formalin 0.6 and 0.9% significantly increased the foot lifting activity in the 0-5 minutes (acute phase) and 15-45 minutes (inflammatory phase) after administration. But the injection of formalin 0.3% significantly increased foot lifting activity only in the acute phase. In the other recorded behaviors, we did not find this biphasic time course pattern. All formalin concentrations induced significant foot flinching/shaking activity only in the acute phase. Subcutaneous injection of formalin 0.9% but not 0.6 and 0.3% significantly increased the duration of eye closure behavior between 20-45 minutes after administration. Injection of all formalin concentrations caused a significant decrease in the unstimulated preening activity between the period of 10 to 45 minutes after administration. Injection of all formalin concentrations caused a significant decrease in the observation chamber wall pecking activity between the period 30 to 45 minutes after administration. These results suggest that the quail formalin test can be considered a relevant, sensitive, and specific technique that helps researchers to use it in the determination of the neurobiology and pharmacology of nociception in the avian species.

Analgesic Efficacy of a Combination of Fentanyl and a Japanese Herbal Medicine "Yokukansan" in Rats with Acute Inflammatory Pain.

Background: Fentanyl can induce acute opioid tolerance and postoperative hyperalgesia when administered at a single high dose; thus, this study examined the analgesic efficacy of a combination of fentanyl and Yokukansan (YKS). Methods: Rats were divided into control, formalin-injected (FOR), YKS-treated+FOR (YKS), fentanyl-treated+FOR (FEN), and YKS+FEN+FOR (YKS+FEN) groups. Acute pain was induced via subcutaneous injection of formalin into the paw. The time engaged in pain-related behavior was measured. Results: In the early (0–10 min) and intermediate (10–20 min) phases, pain-related behavior in the YKS+FEN group was significantly inhibited compared with the FOR group. In the late phase (20–60 min), pain-related behavior in the FEN group was the longest and significantly increased compared with the YKS group. We explored the influence on the extracellular signal-regulated kinase (ERK) pathway in the spinal cord, and YKS suppressed the phosphorylated ERK expression, which may be related to the analgesic effect of YKS in the late phase. Conclusions: These findings suggest that YKS could reduce the use of fentanyl and combined use of YKS and fentanyl is considered clinically useful.

The Rostral Agranular Insular Cortex, a New Site of Oxytocin to Induce Antinociception.

The rostral agranular insular cortex (RAIC) is a relevant structure in nociception. Indeed, recruitment of GABAergic activity in RAIC promotes the disinhibition of the locus ceruleus, which in turn inhibits (by noradrenergic action) the peripheral nociceptive input at the spinal cord level. In this regard, at the cortical level, oxytocin can modulate the GABAergic transmission; consequently, an interaction modulating nociception could exist between oxytocin and GABA at RAIC. Here, we tested in male Wistar rats the effect of oxytocin microinjection into RAIC during an inflammatory (by subcutaneous peripheral injection of formalin) nociceptive input. Oxytocin microinjection produces a diminution of (1) flinches induced by formalin and (2) spontaneous firing of spinal wide dynamic range cells. The above antinociceptive effect was abolished by microinjection (at RAIC) of the following: (1) L-368899 (an oxytocin receptor [OTR] antagonist) or by (2) bicuculline (a preferent GABAA receptor blocker), suggesting a GABAergic activation induced by OTR. Since intrathecal injection of an α2A-adrenoceptor antagonist (BRL 44408) partially reversed the oxytocin effect, a descending noradrenergic antinociception is suggested. Further, injection of L-368899 per se induces a pronociceptive behavioral effect, suggesting a tonic endogenous oxytocin release during inflammatory nociceptive input. Accordingly, we found bilateral projections from the paraventricular nucleus of the hypothalamus (PVN) to RAIC. Some of the PVN-projecting cells are oxytocinergic and destinate GABAergic and OTR-expressing cells inside RAIC. Aside from the direct anatomic link between PVN and RAIC, our findings provide evidence about the role of oxytocinergic mechanisms modulating the pain process at the RAIC level.SIGNIFICANCE STATEMENT Oxytocin is a neuropeptide involved in several functions ranging from lactation to social attachment. Over the years, the role of this molecule in pain processing has emerged, showing that, at the spinal level, oxytocin blocks pain transmission. The present work suggests that oxytocin also modulates pain at the cortical insular level by favoring cortical GABAergic transmission and activating descending spinal noradrenergic mechanisms. Indeed, we show that the paraventricular hypothalamicnucleus sends direct oxytocinergic projections to the rostral agranular insular cortex on GABAergic and oxytocin receptor-expressing neurons. Together, our data support the notion that the oxytocinergic system could act as an orchestrator of pain modulation.

Evaluation of Ficus carica Linn Fruit Extracts on Formalin Induced Pain in the Male Rat Paw

Background: One of the most significant symptoms in many acute and chronic diseases is pain. While many researches reveal side effects of many synthetic analgesic drugs, which are commonly used, new surveys are searching for new alternative analgesic drugs (especially by use of medicinal plants) with few side effects. Objectives: This study was designed to evaluate the analogous function of Ficus carica L. (one of the important medicinal plants in Persian medicine) as an anti-nociceptive in allopathic medicine. Methods: Subcutaneous injection of formalin in rat paw was performed to establish the pain. Formalin test was applied to assess the severity of pain every 15 seconds for one hour. In two parts of this study, two different types of Ficus extract were used in intraperitoneal injections; the aqueous boiled Ficus extract (500, 1000, 2000 mg/kg) in comparison with distilled water (DW) and the petroleum ether extract (500, 1000 mg/kg) in comparison with DMSO. Results: The aqueous boiled Ficus extract showed no significant difference between control (DW) group and groups that had received extracts (P > 0.05). While a significant difference between the petroleum ether extract (1000 mg/kg) and the DMSO group in the last five minutes of the study (P < 0.05) was recorded. Anti-inflammatory examination of aqueous and ethanolic extract of Ficus fruit showed no anti-inflammatory effect; however, the petroleum ether extract displayed a dose-dependent (1000 mg/kg) anti-inflammatory effect even in comparison with sodium salicylate. Conclusions: According to the achieved results, it was concluded that there are no anti-nociceptive and anti-inflammatory effects of Ficus carica fruit aqueous extract on the formalin test in the male rat paw in doses mentioned above. However, there is a delayed anti-nociceptive effect dose-dependently and a dose-dependent anti-inflammatory effect of petroleum ether extract.

(256) Peripherally-Restricted Dual-Acting Kappa/Delta Opioid Agonist (CA1001) Prevents Formalin-Induced Hyperalgesia

Peripheral mu-opioid receptors (MOR) are responsible for opioid-induced constipation, but in the uninjured state do not participate significantly in the pain pathway. On the other hand, kappa- (KOR) and delta-opioid (DOR) receptors are constitutively present in the periphery. However, the DOR only becomes active following the induction of the inflammatory response. The relative contribution of DOR agonism to KOR agonism was examined in the formalin model. Subcutaneous plantar injection of formalin causes a bi-phasic nocifensive behavioral response in rodents. The early phase (phase 1) lasts for about 5-10 minutes, following which an interphase occurs without any discernible nociceptive reactions, after which the late phase (phase 2) nociceptive reaction ensues continuing from about 20-60 minutes. Phase 2 of the formalin model is a model of continuously present persistent pain. The late phase (phase 2), in particular, is considered as a pharmacodynamic surrogate of central sensitization. A novel dual-acting, peripherally-restricted kappa-delta opioid agonist (CA1001) was compared to a peripherally-restricted kappa agonist (ICI204448) in the formalin model in the mouse. Following IACUC approval, mice were randomly pretreated with inert vehicle, ICI204448 1mg/kg, ICI204448 10mg/kg, CA1001 1mg/kg, or CA1001 10 mg/kg. Spontaneous nocifensive behaviors were blindly assessed (video recording). Neither agent was effective in reducing the acute (0-5 minutes) response to formalin injection (phase 1). CA1001 1 mg/kg was as effective as ICI204448 10 mg/kg in reducing formalin-induced responses at 20-35 minutes; CA1001 10 mg/kg was significantly more effective than ICI204448 10 mg/kg (phase 2; p

Erratum: Effect of Nigella sativa (Kalozira) oil on pain in Long Evans rats. J Bangladesh Soc Physiol 2018, June;13(1):1-7

The author of this article brought attention to publishers that there were errors on page 1,3,4,5 published in previous issue. These errors have been corrected to enhance credibility to readers and the whole corrected article has been published in this issue of JBSP.&#x0D; The online version of the original article can be found at doi:http:/ /dx.doi.org/10.3329/jbsp,v13i1.37840(http://banglajol.info/ index.php/JBSP/article/view/37840)&#x0D; Background: For thousands of years, Nigella sativa has been used in many countries as a spice as well as a protective and curative remedy for numerous health disorders. Nigella sativa oil (NSO) suppressed pain in animal models.&#x0D; Objective: To assess the effects of Nigella sativa oil on pain after single administration with three increasing doses in rat models.&#x0D; Methods: This experimental study was conducted in the Department of Physiology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, from March 2016 to February 2017. For this, 20 (twenty) Long Evans rats (200±20) of both sexes were divided into control ( 10 ml/kg of 1% solution of Tween 20) and experimental groups (NSO 0.5, with 0.5 ml/kg NSO; NSO 1, with 1 ml/kg NSO; NSO 1.5, with 1.5 ml/kg NSO) with 5 rats in each group. To assess nociceptive pain, central analgesic system and inflammatory pain early phase (1st-5th minutes); inter phase (6th-15th minutes) and late phase (16th-60th minutes) of formalin test respectively were observed. All the agents were given intraperitoneally in a single dose just one hour before the formalin test. In all phases, total frequency of jerking and total duration of flexing and licking of right hind paw were counted after administration of subcutaneous formalin (50 mL, 2.5%) injection. Statistical analysis was done by ANOVA, followed by Bonferroni’s post hoc test.&#x0D; Results: In early phase, NSO significantly reduced the jerking (p ≤0.001, in all doses) and flexing and licking (p ≤0.001, in higher 2 doses). In interphase, jerking was significantly (p ≤0.01) lowered after 1.5 ml/kg of NSO but flexing and licking significantly lowered by all 3 doses (p ≤0.05, p ≤ 0.001, p≤ 0.001). In addition, in late phase jerking and flexing and licking was reduced by higher 2 doses (p ≤0.001) and all 3 doses respectively (p ≤ 0.001, in all doses).&#x0D; Conclusion: From this study, it may be concluded that, NSO possess analgesic effect and higher two doses were more effective than lower doses.&#x0D; J Bangladesh Soc Physiol. 2018, December; 13(2): 84-90

Involvement of central opiate receptors in modulation of centrally administered oxytocin-induced antinociception.

Objective(s):Oxytocin is involved in modulation of many brain-mediated functions. In the present study, we investigated the central effects of oxytocin and its receptor antagonist, atosiban on inflammatory pain. The contribution of opiate receptors was explored using non-selective and selective antagonists. Materials and Methods:The fourth ventricle of the brain of anesthetized rats was implanted with a guide cannula. Inflammatory pain in the orofacial region was induced by subcutaneous (SC) injection of formalin into the vibrissa pad, and time duration of face rubbing behavior was measured for 45 min. Results:A typical biphasic pain was observed after formalin injection. This biphasic pain behavior was attenuated by intra-fourth ventricle administration of oxytocin (12.5, 50, and 200 ng/rat). Central prior administration of 400 ng/rat atosiban (an oxytocin receptor antagonist), naloxone (a non-selective opiate receptor antagonist), naloxonazine (a selective µ-opiate receptor antagonist), and nor-binaltorphimine (a selective κ-opiate receptor antagonist), but not naltrindole (a δ-opiate receptor antagonist), prevented oxytocin-induced (200 ng/rat) antinociception. Except for naltrindole, other antagonists increased pain intensity when used alone. Above-mentioned drugs did not alter locomotor activity. Conclusion:Oxytocin, as a neuropeptide neurotransmitter, may be involved in the supraspinal modulation of inflammatory pain through µ- and κ-, but not δ-opiate receptors.

Effect of Nigella Sativa (Kalozira) Oil on Pain in Long Evans Rats

Background: For thousands of years, Nigella sativa has been used in many countries as a spice as well as a protective and curative remedy for numerous health disorders. Nigella sativa oil (NSO) suppressed pain in animal models.Objective: To assess the effects of Nigella sativa oil on pain after single administration with three increasing doses in rat models.Methods: This experimental study was conducted in the Department of Physiology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, from March 2016 to February 2017. For this, 20 (twenty) Long Evans rats (200±20) of both sexes were divided into control ( 10 ml/kg of 1% solution of Tween 20) and experimental groups (NSO 0.5, with 0.5 ml/ kg NSO; NSO 1, with 1 ml/kg NSO; NSO 1.5, with 1.5 ml/kg NSO) with 5 rats in each group.. To assess nociceptive pain, central analgesic system and inflammatory pain ,early phase (1st-5th minutes); inter phase (6th-15th minutes) and on late phase (16th-60th minutes) of formalin test respectively were observed. All the agents were given intraperitoneally in a single dose just one hour before the formalin test. In all phases, total frequency of jerking and total duration of flexing and licking of right hind paw were counted after administration of subcutaneous formalin (50 µL, 2.5%) injection. Statistical analysis was done by ANOVA, followed by Bonferroni’s post hoc test.Results: In early phase, NSO significantly reduced the jerking (p ≤0.001, in all doses) and flexing and licking (p≤0.001, in higher 2 doses). In interphase, jerking was significantly (p ≤ 0.01) lowered after 1.5 ml/kg of NSO but flexing and licking were significantly lowered by at all 3 doses (p ≤0.05, p ≤0.001, p ≤0.001,). In addition, in late phase jerking and flexing and licking were reduced by higher 2 doses (p ≤0.001, ) and all 3 doses respectively (p ≤0.001, in all doses).Conclusion: From this study, it may be concluded that, NSO possess analgesic effect and higher two doses were more effective than lower doses.J Bangladesh Soc Physiol. 2018, June; 13(1): 1-7

Mouse mast cells and mast cell proteases do not play a significant role in acute tissue injury pain induced by formalin.

Subcutaneous formalin injections are used as a model for tissue injury-induced pain where formalin induces pain and inflammation indirectly by crosslinking proteins and directly through activation of the transient receptor potential A1 receptor on primary afferents. Activation of primary afferents leads to both central and peripheral release of neurotransmitters. Mast cells are found in close proximity to peripheral sensory nerve endings and express receptors for neurotransmitters released by the primary afferents, contributing to the neuro/immune interface. Mast cell proteases are found in large quantities within mast cell granules and are released continuously in small amounts and upon mast cell activation. They have a wide repertoire of proposed substrates, including Substance P and calcitonin gene-related peptide, but knowledge of their in vivo function is limited. We evaluated the role of mouse mast cell proteases (mMCPs) in tissue injury pain responses induced by formalin, using transgenic mice lacking either mMCP4, mMCP6, or carboxypeptidase A3 (CPA3), or mast cells in their entirety. Further, we investigated the role of mast cells in heat hypersensitivity following a nerve growth factor injection. No statistical difference was observed between the respective mast cell protease knockout lines and wild-type controls in the formalin test. Mast cell deficiency did not have an effect on formalin-induced nociceptive responses nor nerve growth factor-induced heat hypersensitivity. Our data thus show that mMCP4, mMCP6, and CPA3 as well as mast cells as a whole, do not play a significant role in the pain responses associated with acute tissue injury and inflammation in the formalin test. Our data also indicate that mast cells are not essential to heat hypersensitivity induced by nerve growth factor.

Role of dorsal hippocampal orexin-1 receptors in modulation of antinociception induced by chemical stimulation of the lateral hypothalamus

The Hippocampus has a role not only in nociception but also in modulation of pain perception. In addition, orexinergic neurons present in the lateral hypothalamus (LH) have a recognized role in pain modulation. The presence of orexinergic projections from the lateral hypothalamus (LH) to the dorsal hippocampal Cornu Ammonis 1 (CA1) region raises the question of whether pain modulatory role of LH is mediated through the CA1. To elucidate the interactions between the LH and neural substrates involved in modulation of formalin-induced nociception, the study aimed to test the pain modulatory role of CA1 orexin receptors in the formalin test. Seventy-one male Wistar rats were unilaterally implanted with two cannulae above the LH and CA1. In the treatment groups, intra-CA1 administration of SB-334867, as an orexin-1 receptor antagonist, was performed 5min before intra-LH microinjection of carbachol, as a cholinergic receptor agonist. In dimethyl sulfoxide (DMSO)-control group, DMSO and saline as well as in carbachol-control group, DMSO and carbachol were microinjected into the CA1 and LH, respectively. In all rats, the procedure was followed by subcutaneous injection of formalin after 5-min time interval. Carbachol reduced both phases of formalin-induced nociception. Intra-CA1 administration of SB-334867 antagonized the LH-induced analgesia during both phases in a dose-dependent manner. It seems that the blockade of orexin-1 receptors has more effects on reduction of antinociception during the late phase compared to the early phase. Pain modulatory role of orexinergic system in the formalin test through a neural pathway from the LH to CA1 provides the evidence that orexins can be useful therapeutic agents for chronic pain treatment.

Metabolomics and genomics: revealing the mechanism of corydalis alkaloid on anti-inflammation in vivo and in vitro

As one of the major active components of Qizhiweitong (QZWT) prescription, corydalis alkaloid (CA) has been reported to have noticeable effect of anti-inflammation in pharmacological studies. However, the anti-inflammatory function and potential mechanism of CA in anti-inflammation remain to be clarified. Hence, the aim of this study was to investigate the anti-inflammation efficacy and potential mechanism of CA in anti-inflammation by metabonomic approach coupled with related gene analyzes in vivo and in vitro. Mice acute inflammation was induced by subcutaneous injection of formalin in hind paws, and evaluated the anti-inflammatory effect of CA via detecting the index of paw edema. The results indicated that CA has an anti-inflammatory effect on mice through alleviating the paw edema significantly. Moreover, metabolic profiling was performed by high performance liquid chromatography quadrupole-time-of-flight mass spectrometer (HPLC-QTOF-MS) combined with multivariate data analysis, such as principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). Thirteen metabolic targets (including lysophosphatidylcholine (LysoPC), choline, arachidonic acid, phosphodimethylethanolamine) and related pathways of the cholinergic anti-inflammatory and arachidonic acid (AA) metabolism were identified, Meanwhile, the relevant target genes, like iNOS, NF-κB, TNF-α, were detected and verified in vitro. The results indicated that CA has a good pharmacological effect of anti-inflammation through regulating multiple disordered targets and metabolic networks, which provides a theoretical basis for further research on CA. Furthermore, there is a great potential for the development of CA to be a new anti-inflammatory drug with high effective activities, little side effects and weak drug resistance.

Effects of intra-hippocampal microinjection of vitamin B12 on the orofacial pain and memory impairments induced by scopolamine and orofacial pain in rats

In the present study, we investigated the effects of microinjection of vitamin B12 into the hippocampus on the orofacial pain and memory impairments induced by scopolamine and orofacial pain. In ketamine-xylazine anesthetized rats, the right and left sides of the dorsal hippocampus (CA1) were implanted with two guide cannulas. Orofacial pain was induced by subcutaneous injection of formalin (1.5%, 50μl) into the right vibrissa pad, and the durations of face rubbing were recorded at 3-min blocks for 45min. Morris water maze (MWM) was used for evaluation of learning and memory. Finally, locomotor activity was assessed using an open-field test. Vitamin B12 attenuated both phases of formalin-induced orofacial pain. Prior administration of naloxone and naloxonazine, but not naltrindole and nor-binaltorphimine, prevented this effect. Vitamin B12 and physostigmine decreased latency time as well as traveled distance in Morris water maze. In addition, these chemicals improved scopolamine-induced memory impairment. The memory impairment induced by orofacial pain was improved by vitamin B12 and physostigmine used alone. Naloxone prevented, whereas physostigmine enhanced the memory improving effect of vitamin B12 in the pain-induced memory impairment. All the above-mentioned chemicals did not alter locomotor activity. The results of the present study showed that at the level of the dorsal hippocampus, vitamin B12 modulated orofacial pain through a mu-opioid receptor mechanism. In addition, vitamin B12 contributed to hippocampal cholinergic system in processing of memory. Moreover, cholinergic and opioid systems may be involved in improving effect of vitamin B12 on pain-induced memory impairment.

Why depression and pain often coexist and mutually reinforce: Role of the lateral habenula

The interrelation of depression and pain is increasingly coming under scrutiny. Although the lateral habenula (LHb) is widely implicated in the pathogenesis of depression and pain, its role in the interaction of depression and pain remains unknown. Thus, the aim of current study was to investigate the role of LHb in rat depression-pain comorbidity. Single extracellular firing recording and immunofluorescence methods were used to compare firing rates and c-Fos expression of the LHb neurons in normal and model rats. Following subcutaneous injection of formalin into the hind paw to simulate natural pain, we assessed pain behavior in rats subjected to the chronic, unpredictable mild stress procedure (CUMS, a model of depression). Pain sensitivity in the model rats was increased over that of controls. These rats showed a significant increase in the firing activity of LHb neurons compared with normal rats. Significantly, about 73% of neurons with high discharge frequency in LHb of model rats were pain-activated neurons (PANs), and the firing rates of PANs were inhibited by intraperitoneal injection of a tricyclic antidepressant, clomipramine. Immunofluorescence showed that the percentage of c-Fos positive cells in LHb was significantly increased in rats receiving CUMS alone, rats receiving pain stimulation alone, and rats receiving both CUMS and pain stimulation, but especially the last. The interaction effect was inhibited by injection of clomipramine. The LHb lesion can improve both depression-like behavior and pain sensitivity in depression model rats with pain. These suggest that hyperactivity of the LHb neurons contributes to depression-pain comorbidity in rats.

Role of orexin-2 receptors in the nucleus accumbens in antinociception induced by carbachol stimulation of the lateral hypothalamus in formalin test.

Orexins, which are mainly produced by orexin-expressing neurons in the lateral hypothalamus (LH), play an important role in pain modulation. Previously, it has been established that the nucleus accumbens (NAc) is involved in the modulation of formalin-induced nociceptive responses, a model of tonic pain. In this study, the role of intra-accumbal orexin-2 receptors (OX2rs) in the mediation of formalin-induced pain was investigated. A volume of 0.5 μl of 10, 20, and 40 nmol/l solutions of TCS OX2 29, an OX2r antagonist, were unilaterally microinjected into the NAc 5 min before an intra-LH carbachol microinjection (0.5 μl of 250 nmol/l solution). After 5 min, animals received a subcutaneous injection of formalin 2.5% (50 μl) into the hind paw. Pain-related behaviors were assessed at 5 min intervals during a 60-min test period. The findings showed that TCS OX2 29 administration dose dependently blocked carbachol-induced antinociception during both phases of formalin-induced pain. The antianalgesic effect of TCS OX2 29 was greater during the late phase compared with the early phase. These observations suggest that the NAc, as a part of a descending pain-modulatory circuitry, partially mediates LH-induced analgesia in the formalin test through recruitment of OX2rs. This makes the orexinergic system a good potential therapeutic target in the control of persistent inflammatory pain.

Anti-Inflammation Effects and Potential Mechanism of Saikosaponins by Regulating Nicotinate and Nicotinamide Metabolism and Arachidonic Acid Metabolism.

Inflammation is an important immune response; however, excessive inflammation causes severe tissue damages and secondary inflammatory injuries. The long-term and ongoing uses of routinely used drugs such as non-steroidal anti-inflammatory drugs (NSAIDS) are associated with serious adverse reactions, and not all patients have a well response to them. Consequently, therapeutic products with more safer and less adverse reaction are constantly being sought. Radix Bupleuri, a well-known traditional Chinese medicine (TCM), has been reported to have anti-inflammatory effects. However, saikosaponins (SS) as the main pharmacodynamic active ingredient, their pharmacological effects and action mechanism in anti-inflammation have not been reported frequently. This study aimed to explore the anti-inflammatory activity of SS and clarify the potential mechanism in acute inflammatory mice induced by subcutaneous injection of formalin in hind paws. Paw edema was detected as an index to evaluate the anti-inflammatory efficacy of SS. Then, a metabolomic method was used to investigate the changed metabolites and potential mechanism of SS. Metabolite profiling was performed by high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). The detection and identification of the changed metabolites were systematically analyzed by multivariate data and pathway analysis. As a result, 12 different potential biomarkers associated with SS in anti-inflammation were identified, including nicotinate, niacinamide, arachidonic acid (AA), and 20-carboxy-leukotriene B4, which are associated with nicotinate and nicotinamide metabolism and arachidonic acid metabolism. The expression levels of biomarkers were effectively modulated towards the normal range by SS. It indicated that SS show their effective anti-inflammatory effects through regulating nicotinate and nicotinamide metabolism and arachidonic acid metabolism.

Differential contribution of orexin receptors within the ventral tegmental area to modulation of persistent inflammatory pain.

Orexinergic neurons in the lateral hypothalamus (LH) play an important role in pain modulation. In addition, ventral tegmental area (VTA) is known as a part of descending pain modulatory circuitry. Little is known about the interaction between the LH and neural substrates involving in modulation of formalin-induced nociception. Accordingly, we aimed to examine the pain modulatory role of VTA orexin receptors in the formalin test. Seventy-eight male Wistar rats were unilaterally implanted with two cannulae above the LH and VTA. Intra-VTA administration of SB-334867 (orexin-1 receptor antagonist) or TCS OX2 29 (orexin-2 receptor antagonist) was performed 5min before intra-LH microinjection of carbachol (a cholinergic receptor agonist). The procedure was followed by subcutaneous injection of formalin after 5-min interval time. Carbachol attenuated formalin-induced biphasic pain responses and SB-334867 or TCS OX2 29 administration dose-dependently antagonized the LH-induced analgesia during both phases. Blockade of orexin-1 and -2 receptors had more profound effects on the reduction of antinociception during the late phase compared to the early phase. Also, contribution of orexin-1 receptors in mediation of LH-induced analgesia was greater than orexin-2 receptors during the late phase. Formalin test, a model of persistent inflammatory pain, mimics the conditions encountered in clinical situations. Pain modulatory role of orexinergic system in the formalin test through a neural pathway from the LH to the VTA provides the evidence that orexins can be useful therapeutic targets for chronic pain treatment. WHAT DOES THIS STUDY ADD?: There is a pathway from the lateral hypothalamus (LH) to the ventral tegmental area (VTA) which modulates biphasic formalin-induced pain. Blockade of VTA orexin receptors dose-dependently reduces LH-induced analgesia during both phases. Anti-analgesic effect of orexin receptor antagonists is more considerable during the late phase. Contribution of orexin-1 receptors to mediation of LH-induced analgesia is more than orexin-2 receptors during the late phase.

Sleep Deprivation and Recovery Sleep Prior to a Noxious Inflammatory Insult Influence Characteristics and Duration of Pain.

Insufficient sleep and chronic pain are public health epidemics. Sleep loss worsens pain and predicts the development of chronic pain. Whether previous, acute sleep loss and recovery sleep determine pain levels and duration remains poorly understood. This study tested whether acute sleep deprivation and recovery sleep prior to formalin injection alter post-injection pain levels and duration. Male Sprague-Dawley rats (n = 48) underwent sleep deprivation or ad libitum sleep for 9 hours. Thereafter, rats received a subcutaneous injection of formalin or saline into a hind paw. In the recovery sleep group, rats were allowed 24 h between sleep deprivation and the injection of formalin. Mechanical and thermal nociception were assessed using the von Frey test and Hargreaves' method. Nociceptive measures were performed at 1, 3, 7, 10, 14, 17 and 21 days post-injection. Formalin caused bilateral mechanical hypersensitivity (allodynia) that persisted for up to 21 days post-injection. Sleep deprivation significantly enhanced bilateral allodynia. There was a synergistic interaction when sleep deprivation preceded a formalin injection. Rats allowed a recovery sleep period prior to formalin injection developed allodynia only in the injected limb, with higher mechanical thresholds (less allodynia) and a shorter recovery period. There were no persistent changes in thermal nociception. The data suggest that acute sleep loss preceding an inflammatory insult enhances pain and can contribute to chronic pain. The results encourage studies in a model of surgical pain to test whether enhancing sleep reduces pain levels and duration.

Intra-accumbal Orexin-1 Receptors are Involved in Antinociception Induced by Stimulation of the Lateral Hypothalamus in the Formalin Test as an Animal Model of Persistent Inflammatory Pain.

Orexin, mainly produced by orexin-expressing neurons in the lateral hypothalamus (LH), plays an important role in pain modulation. Moreover, it is shown that the nucleus accumbens (NAc) is one of the important areas involved in this modulation. Orexin-1 (OX1) and orexin-2 (OX2) receptors are densely distributed in the NAc. The study investigated the involvement of OX1 receptors in the NAc on antinociception induced by intra-LH administration of carbachol in formalin test. Rats were unilaterally implanted by two separate cannulae into the LH and NAc. Different doses of SB334867, as an OX1 receptor antagonist, were microinjected into the NAc (1, 3 and 10 nM/0.5 µL DMSO) prior to intra-LH carbachol injection (250 nM/0.5 µL saline). Formalin test was applied as an animal model of persistent inflammatory pain. The animals received a subcutaneous injection of formalin into the hind paw, 5 min after SB334867 administration. Pain scores were calculated at 5-min blocks for a 60-min test period. Results showed that the administration of SB334867 into the NAc decreased LH chemical stimulation-induced antinociception dose-dependently in early and second phase of formalin test. Our findings showed that OX1 receptors in the NAc may be involved in modulation of inflammatory pain.