Effects of alamandine on monocrotaline-induced pulmonary hypertension in rats.

Objectives: Pulmonary arterial hypertension (PAH) is a severe and often fatal disease that is associated with oxidative stress and inflammation. Alamandine, a component of the renin-angiotensin system, known for its antioxidative, anti-inflammatory, and antifibrotic effects, has been investigated in this study to determine if it has protective effects against PAH induced by monocrotaline (MCT) and if these effects are associated with oxidative stress, inflammatory factors, and inducible nitric oxide synthase (iNOS). Materials and Methods: Rats were administered MCT (40 mg/kg) on day 0 and then received alamandine (50 mg/kg/day) via mini-osmotic pumps for 21 days starting one day later. Hemodynamic parameters, electrocardiograms, superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), inflammatory cytokines (TNF-α, IL-1ß, and NF-κB), iNOS, and MrgD receptor expression in lung tissue were evaluated at the end of the 21-day period. The MrgD receptor was quantified through immunofluorescent staining, and the histopathology of lung tissues was evaluated using hematoxylin and eosin staining. Results: The results showed that alamandine treatment significantly improved hemodynamic parameters, oxidative stress markers, inflammatory factors, and electrocardiographic data. Furthermore, treatment with alamandine decreased the levels of iNOS. Additionally, alamandine treatment decreased the expression levels of MrgD receptors in the lung tissue of MCT-induced PAH. Conclusion: In summary, this study indicates that alamandine has protective effects against monocrotaline-induced PAH, and these effects may be attributed to the inhibition of oxidative stress, inflammatory parameters, and iNOS.

Dexamethasone release from hyaluronic acid microparticle and proanthocyanidin-gelatin hydrogel in sciatic tissue regeneration.

Biodegradable microparticles are useful vehicles for the controlled release of bioactive molecules in drug delivery, tissue engineering and biopharmaceutical applications. We developed dexamethasone (Dex) encapsulation into tyramine-substituted hyaluronic acid microparticles (Dex-HA-Tyr Mp) mediated by horseradish peroxidase (HRP) crosslinking using a microfluidic device and infollowing crosslinked gelatin (Gela) with proanthocyanidin (PA) as a semi-confined bed hydrogel for the repair of sciatic tissue injury. It was found that the simultaneous use of Dex-HA-Tyr Mp and cross-linked Gela-PA hydrogel improved the physical properties of the hydrogel, including mechanical strength and degradability. The designed composite also provided a sustained release system for Dex delivery to the surrounding sites, demonstrating the applicability of the fabricated hydrogel composite for sciatic nerve tissue engineering and regeneration. The encapsulated cells were viable and showed adequate growth ability and morphogenesis during prolonged incubation in Gela-PA/HA-Tyr Mp hydrogel compared to control conditions. Interestingly, histological analysis revealed a significant increase in the number of axons in the injured sciatic nerve following treatment with Dex-HA-Tyr Mp and injectable Gela-PA hydrogel compared to other control groups. In conclusion, the results demonstrated that fabricated Dex-loaded MPs and injectable hydrogel from biomimetic components are suitable systems for sustained delivery of Dex with adequate biocompatibility and the approach may have potential therapeutic applications in peripheral nerve regeneration.

Enhancement of bactericidal effects of bacteriophage and gentamicin combination regimen against Staphylococcus aureus and Pseudomonas aeruginosa strains in a mice diabetic wound model.

Diabetic patients are more susceptible to developing wound infections resulting in poor and delayed wound healing. Bacteriophages, the viruses that target-specific bacteria, can be used as an alternative to antibiotics to eliminate drug-resistant bacterial infections. Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) are among the most frequently identified pathogens in diabetic foot ulcers (DFUs). The aim of this study was assessment of bacteriophage and gentamicin combination effects on bacterial isolates from DFU infections. Specific bacteriophages were collected from sewage and animal feces samples and the phages were enriched using S. aureus and P. aeruginosa cultures. The lytic potential of phage isolates was assessed by the clarity of plaques. We isolated and characterized four lytic phages: Stp2, Psp1, Stp1, and Psp2. The phage cocktail was optimized and investigated in vitro. We also assessed the effects of topical bacteriophage cocktail gel on animal models of DFU. Results revealed that the phage cocktail significantly reduced the mortality rate in diabetic infected mice. We determined that treatment with bacteriophage cocktail effectively decreased bacterial colony counts and improved wound healing in S. aureus and P. aeruginosa infections, especially when administrated concomitantly with gentamicin. The application of complementary therapy using a phage cocktail and gentamicin, could offer an attractive approach for the treatment of wound diabetic bacterial infections.

Alamandine injection in the periaqueductal gray and rostral ventromedial medulla attenuates allodynia induced by sciatic nerve ligation in rats.

Alamandine, a peptide known to interact with Mas-related G protein-coupled receptor subtype D (MrgD), has been implicated in moderating inflammatory signals. MrgD receptors are abundantly found in pain transmission pathways, but the role of alamandine/MrgD in pain modulation has not been thoroughly explored. This study aimed to investigate the effects of alamandine (10, 40, and 100 pmol) in a rat model of allodynia induced by sciatic nerve ligation, with a specific focus on examining the involvement of MrgD receptors, NMDAR1, and serotonin transporter (SERT) in the ventrolateral periaqueductal gray (vlPAG) and rostral ventromedial medulla (RVM). Microinjection of alamandine into the vlPAG at a dose of 100 pmol and into the RVM at doses of 40 and 100 pmol resulted in a significant increase in paw withdrawal threshold (PWT). Additionally, co-administration of D-Pro7-Ang-(1-7) at 50 pmol, an MrgD receptor antagonist, effectively blocked the analgesic effects of alamandine. Immunofluorescence analysis confirmed the presence of MrgD receptors in both the vlPAG and RVM regions. Importantly, an upregulation of MrgD receptor expression was observed following allodynia induction, suggesting a potential compensatory mechanism in response to pain. Our findings support the co-localization of MrgD receptors with NMDAR1 in vlPAG neurons, suggesting their ability to initiate analgesic pathways similar to those activated by NMDA receptors in the vlPAG. Furthermore, our results underscore a significant co-localization of MrgD receptors with the SERT in the RVM, underscoring their potential impact on serotonergic neurons involved in promoting analgesic effects.

Alamandine: Potential Protective Effects in SARS-CoV-2 Patients.

Coronavirus disease 2019 (COVID-19) can occur due to contracting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has no confined treatment and, consequently, has high hospitalization and mortality rates. Moreover, people who contract COVID-19 present systemic inflammatory spillover. It is now known that COVID-19 pathogenesis is linked to the renin-angiotensin system (RAS). COVID-19 invades host cells via the angiotensin-converting enzyme 2 (ACE2) receptor-as such, an individual's susceptibility to COVID-19 increases alongside the upregulation of this receptor. COVID-19 has also been associated with interstitial pulmonary fibrosis, which leads to acute respiratory distress, cardiomyopathy, and shock. These outcomes are thought to result from imbalances in angiotensin (Ang) II and Ang-(1-7)/alamandine activity. ACE2, Ang-(1-7), and alamandine have potent anti-inflammatory properties, and some SARS-CoV-2 patients exhibit high levels of ACE2 and Ang-(1-7). This phenomenon could indicate a failing physiological response to prevent or reduce the severity of inflammation-mediated pulmonary injuries. Alamandine, which is another protective component of the RAS, has several health benefits owing to its antithrombogenic, anti-inflammatory, and antifibrotic characteristics. Alamandine alleviates pulmonary fibrosis via the Mas-related G protein-coupled receptor D (MrgD). Thus, a better understanding of this pathway could uncover novel pharmacological strategies for altering proinflammatory environments within the body. Following such strategies could inhibit fibrosis after SARS-CoV-2 infection and, consequently, prevent COVID-19.

Possible Risk of Thrombotic Events following Oxford-AstraZeneca COVID-19 Vaccination in Women Receiving Estrogen.

People who receive the ChAdOx1 nCoV-19 vaccine, particularly perimenopausal women who are on birth control or postmenopausal women who take estrogen supplements, may experience thrombosis and thrombocytopenia. Estrogen and the ChAdOx1 nCoV-19 vaccine both have the potential to cause thrombus in different ways. Some postmenopausal women who are also taking estrogens may develop thrombosis and thrombocytopenia after receiving the ChAdOx1 nCoV-19 vaccine. Therefore, women are encouraged to stop taking drugs containing estrogen before receiving this vaccine. Furthermore, consuming fish oil can help reduce the risk of developing blood clots among women who are in the luteal phase and, thus, have high estrogen levels. In addition, ChAdOx1 nCoV-19's side effects in young women could be mitigated by administering it during the follicular phase.

Protective effect of alamandine on doxorubicin­induced nephrotoxicity in rats.

BACKGROUND: This study aimed to evaluate the protective effects of alamandine, a new member of the angiotensin family, against doxorubicin (DOX)-induced nephrotoxicity in rats. METHODS: Rats were intraperitoneally injected with DOX (3.750 mg/kg/week) to reach a total cumulative dose of 15 mg/kg by day 35. Alamandine (50 µg/kg/day) was administered to the rats via mini-osmotic pumps for 42 days. At the end of the experiment, rats were placed in the metabolic cages for 24 h so that their water intake and urine output could be measured. After scarification, the rats' serum and kidney tissues were collected, and biochemical, histopathological, and immunohistochemical studies were carried out. RESULTS: DOX administration yielded increases in pro-inflammatory cytokines, including interleukin (IL)-1ß and IL-6, pro-fibrotic proteins transforming growth factor-ß (TGF-ß), pro-inflammatory transcription factor nuclear kappa B (NF-κB), kidney malondialdehyde (MDA), creatinine clearance, blood urea nitrogen (BUN), and water intake. On the other hand, the DOX-treated group exhibited decreased renal superoxide dismutase (SOD), renal glutathione peroxidase (GPx) activity, and urinary output. Alamandine co-therapy decreased these effects, as confirmed by histopathology and immunohistochemical analysis. CONCLUSIONS: The results suggest that alamandine can prevent nephrotoxicity induced by DOX in rats.

Alamandine significantly reduces doxorubicin-induced cardiotoxicity in rats.

Doxorubicin (DOX) is an anthracycline antibiotic. Despite its unwanted side effects, it has been successfully used in tumor therapy. Given that oxidative stress and inflammatory factors are essential to cardiotoxicity caused by DOX, we assumed that alamandine, which enhances endogenous antioxidants and has anti-inflammatory effects, may prevent DOX-induced cardiotoxicity. Rats received DOX (3.75 mg/kg) i.p on days 14, 21, 28, and 35 (total cumulative dose = 15 mg/kg) and alamandine (50 µg/kg/day) via mini-osmotic pumps for 42 days. At the end of the 42-day period, we evaluated hemodynamic parameters, electrocardiogram, cardiac troponin I (cTnI), superoxidase dismutase (SOD), total antioxidant capacity (TAC), malondialdehyde (MDA), inflammatory cytokines (tumor necrosis factor-α (TNF-α), IL-1ß, NF-κB), apoptosis markers (caspase 3), and histopathology of haemotoxylin- and eosin-stained cardiac muscle fibers were evaluated. DOX significantly increased QT, corrected QT (QTc), and RR intervals. Alamandine co-therapy prevented ECG changes. Alamandine administration restored DOX-induced disruptions in the cardiac muscle architecture and vascular congestion. Alamandine co-therapy also alleviated other effects of DOX, including cardiac contractility, decreased systolic and diastolic blood pressure, and increased left ventricular end-diastolic pressure. Moreover, alamandine co-therapy substantially decreased the elevation of oxidative stress markers, inflammatory cytokines, and caspase 3 in DOX-treated rats. The results suggest that alamandine reduced DOX-induced cardiotoxicity via antioxidant, anti-inflammatory, and anti-apoptotic activities.

Angiotensin (1-7) and Apelin co-therapy: New strategy for heart failure treatment of rats.

OBJECTIVE: Isoproterenol (ISO)-induced heart failure is a standardized model for the study of beneficial effects of various drugs. Both apelin and angiotensin 1-7 have a cardiac protective effect. We assumed that co-therapy with apelin and angiotensin 1-7 (Ang (1-7)) may have synergistic cardioprotective effects against isoproterenol-induced heart failure. Methods The animals were randomly assigned to one of eight groups of seven animals in each group as follows: (1) control I (saline; IP injection) (1) control II (saline; via mini-osmotic pump) (3) ISO (5 mg/ kg; IP), (4) Apelin (20µg/ kg; IP), (5) Ang (1-7) (30 µg/kg/day; via mini-osmotic pump), (6) Apelin+ISO, (7) Ang (1-7)+ISO, (8) Apelin+Ang (1-7)+ISO. Rat myocardial injury was induced by intraperitoneal injection of 5mg/kg of ISO for ten days. Apelin and Ang (1-7) were administered 30 minutes before ISO injection. RESULTS: A decrease in systolic blood pressure (SBP; p<0.001), diastolic blood pressure (DBP; p<0.05), left ventricular systolic pressure (LVSP; p<0.001), left ventricular contractility (dP / dt max; p<0.001), relaxation (dP / dt min; p<0.001) and an increase in left ventricular end-diastolic pressure (LVEDP; p<0.001) were observed in ISO-treated rats. Plasma LDH and myocardial and plasma MDA were higher in the ISO heart than in controls (P<0.001). Histopathological examination of cardiac tissue showed myocardial fibrosis and leukocyte infiltration in ISO-treated rats as compared to control. Co- therapy with apelin and Ang (1-7) was more effective than either agent used alone in restoring these parameters to that of control rats. CONCLUSION: The results of this study showed that the combination of apelin and ang (1-7) had a more cardioprotective effect than either used alone against ISO-induced heart failure, and co-therapy may be a useful treatment option for myocardial injuries and heart failure.

Effect of Prolonged Infusion of Alamandine on Cardiovascular Parameters and Cardiac ACE2 Expression in a Rat Model of Renovascular Hypertension.

Alamandine is a new member of the angiotensin family. Here, we studied the mRNA and protein expression of cardiac angiotensin-converting enzyme 2 (ACE2) in the chronic phase of a rat model of 2-kidney, 1-clip hypertension (2K1C), and the effects of 2-week alamandine infusion on blood pressure, cardiac indices, and ACE2 mRNA and protein expression in the hearts. The rats were subjected to to sham-operation or placement of plexiglass clips around the left renal artery. Alamandine, at a dose of 600 µg/kg/d, was administered for 2 weeks via an osmotic mini-pump. At 18 weeks, after induction of hypertension, blood pressure and cardiac indices of contractility were measured using a Powerlab Physiograph system. The ACE2 mRNA and protein levels were determined using real time-PCR and Western blotting, respectively. In the hypertensive rats, alamandine caused a significant decrease in systolic blood pressure (p < 0.001), diastolic blood pressure (p < 0.001), left ventricular end-diastolic pressure (p < 0.001) and, left ventricular systolic pressure (p < 0.001) and increase in the maximum rate of pressure change in the left ventricle (dP/dt(max)) (p < 0.05). Also, the ACE2 mRNA expression in the heart increased in the hypertensive rats compared to the normotensive rats (p < 0.05), and alamandine restored this to normal values, although these changes were only seen at the mRNA and not the protein level. Histological analysis of cardiac tissue confirmed that alamandine decreased cardiac fibrosis and hypertrophy in 2K1C hypertensive rats. Our results indicate that alamandine, which acts as a depressor arm of the renin-angiotensin system, could be developed for treating hypertension.

Differences in Cardiovascular Responses to Alamandine in Two-Kidney, One Clip Hypertensive and Normotensive Rats.

BACKGROUND: Alamandine is a newly discovered component of the renin-angiotensin system, which regulates blood pressure. In this study, the effect of alamandine on cardiovascular parameters in two-kidney, one clip (2K1C) hypertensive rats and normotensive rats, and the possible roles of the angiotensin II type 1 receptor (AT1R) and the PD123319-sensitive receptors in mediating this effect was investigated.Methods and Results:The cardiovascular parameters were monitored for 10 min before the infusion of the drugs or saline, and for 30 min afterward. In the 2K1C hypertensive rats, alamandine caused brief increases in mean arterial pressure (MAP), left-ventricular systolic pressure (LVSP) and maximum rate of pressure change in the left ventricle (dP/dt(max)). This was followed by decreases in these parameters, which extended throughout the remainder of the infusion period. Losartan, an AT1R blocker, abolished alamandine's initial pressor effect and PD123319, which can block AT2R and Mas-related G protein-coupled receptor D (MrgD) receptors, partially decreased the late depressor effect. Left ventricular end-diastolic pressure (LVEDP) decreased during alamandine infusion; this effect was reduced by PD123319. In the normotensive rats, alamandine increased MAP, LVSP, dP/dt (max), and it decreased LVEDP during the infusion period. These effects of alamandine were reduced by losartan. CONCLUSIONS: The results of this investigation suggest that, under normal conditions, alamandine acts via AT1R, but in pathological conditions such as hypertension, its effect on PD123319-sensitive receptors masks its effect on AT1R.

Prevalence and risk factors of alcohol and substance abuse among motorcycle drivers in Fars province, Iran.

PURPOSE: The aim of this present study is to investigate the prevalence of alcohol and substance abuse (ASA) and its relationship with other risky driving behaviors among motorcycle drivers. METHODS: This is a cross sectional study which is performed at Shiraz city of Iran. Data from motorcycle drivers were collected using a standard questionnaire in eight major streets at different times of the day. The data includes consumption of alcohol and other substances two hours before driving and some of the risky behaviors during driving. RESULTS: A total of 414 drivers with a mean ± SD age of (27.0 ± 9.3) years participated in the study. Alcohol or substance consumptions two hours before driving was significantly associated with risky driving behaviors such as using mobile phone during driving, poor maneuvering, and driving over the speed limit (both p < 0.001). It was also associated with carelessness about safety such as driving with technical defects (p < 0.001) and not wearing a crash helmet (p=0.008). CONCLUSION: Screening for alcohol and substance consumption among motorcycle drivers is an efficient way to identify drivers that are at a greater risk for road traffic accidents.

The prevalence and effective factors of crash helmet usage among motorcyclists in Iran.

BACKGROUND: Crash helmet plays an important role in protecting the deriver's head during crashes and reduces the rate of severe injuries and fatalities. Although it has been proved that wearing the crash helmet can save the deriver's life by around 42%; previous studies showed that the rate of wearing crash helmet has not been acceptable in Iran. Due to the huge number of motorcyclists on the roads in Iran, the use of crash helmet is an important area of research. The aim of this study was to assess the factors that could possibly relate to or affect the use of crash helmet by the motorcyclists. METHODS: This is an observational study on 414 motorcyclists in Shiraz, Southern Iran. All participants completed a questioner containing demographic features, crash helmet use, motorcycle license, and the reasons for using motorcycles. RESULTS: All the participants were males and aged from 16 to 64 years with mean age 27±9.28. The results of logistic regression model revealed that only the drivers who had motorcycle license (OR=2.73, C.I: 1.40-7.24), employed the motorcycle for reasons other than pleasure (OR=3.18, C.I: 1.42-7.37) and been driving for 10 or more years (OR=1.92 95% C.I: 1.12-3.30) had greater rate of wearing crash helmet. Interestingly, educational levels, age, and other demographical variables had no relationship with crash helmet usage. CONCLUSIONS: It is believed that in order to increase the rate of crash helmet use, it is necessary to enact obligatory requirement for driving license by motorcyclists and increase the legal age for motorcycle driving.

Cardiovascular responses to apelin in two-kidney-one-clip hypertensive rats and its receptor expression in ischemic and non-ischemic kidneys.

BACKGROUND: Apelin and its receptor APJ have been shown to have beneficial effects on cardiovascular function. Apelin was shown to elicit hypotensive effects and also a positive inotropic effect on failing hearts. In this study, we investigated the effect of apelin on blood pressure and cardiac contractility in a two-kidney-one-clip (2K1C) hypertension model. We also assessed the changes in the level of apelin and some other hemodynamically effective hormones in serum and apelin receptor gene expression in nonischemic and ischemic kidneys. METHODS: 2K1C was produced by placing a Plexiglas clip around the left renal artery. Four weeks later, blood pressure (BP) and cardiac indices of contractility were measured by power lab system. The sample venous blood was drawn from the jugular vein for biochemical variable measurements. The mRNA and protein level of APJ were determined in the kidneys by RT-PCR and Western blot methods respectively. RESULTS: The findings showed that, 2K1C increased BP from 116/75 in sham group to 200/140 mmHg in test group. Furthermore, intravenous administration of apelin-13 to hypertensive rats significantly decreased systolic (SBP) and diastolic (DBP) blood pressures in dose of 20 µg/kg with maximal responses within 2 min of injection. This reduction was long lasting and prominent in dose of 40 µg/kg. Apelin at dose of 20 µg/kg increased +LVdp/dt max and -LVdp/dt max. However at dose of 40 µg/kg SBP, DBP, +LVdp/dt max and -LVdp/dt max strongly decreased. All of the observed effects were completely blocked by apelin antagonist F13A. 2K1C did not change serum apelin, aldosterone and arginine-vasopressin levels but significantly increased angiotensin II level. 2K1C hypertension decreased apelin receptor mRNA and protein expression in contra lateral (nonischemic) kidney, but these were not affected in clipped kidney. CONCLUSION: Apelin induces hypotensive and positive inotropic effects in medium doses. However, in higher doses it elicits hypotensive and negative inotropic effects in 2K1C rats. Down regulation of apelin receptor in nonischemic kidney of hypertensive rats may play a role in pathophysiology of renovascular hypertension. Apelin together with renin-angiotensin antagonism may play a useful role in treatment of this type of hypertension.

Delayed post ischemic treatment with Rosiglitazone attenuates infarct volume, neurological deficits and neutrophilia after embolic stroke in rat.

Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor-gamma (PPAR-gamma), has been shown to provide neuroprotective and anti-inflammatory effects in the acute phase of cerebral ischemia, and traumatic or surgical brain injuries. However, the effect of delayed post ischemia administration of this compound is still unclear. This study was designed to evaluate the neuroprotective effects of RGZ when first administered at 24 h after the embolic model of stroke. Embolic focal cerebral ischemia was induced in rats by placing a preformed clot into the middle cerebral artery (MCA). RGZ (5 mg/kg, intraperitoneally) was injected at 24 and 48 h after MCA embolization. Neurological deficits were evaluated at 24, 48 and 72 h after stroke, and blood and brain tissues were then collected for differential blood cell counts and assessments of infarct volume and DNA fragmentation, respectively. Compared to the control group, the administration of RGZ, starting 24 h after cerebral ischemia, reduced infarct volume by 56% (P<0.05) and decreased neurological deficits at 72 h after cerebral ischemia (P<0.05). Also, delayed administration of RGZ prevented neutrophilia in blood (P<0.005) and significantly decreased DNA fragmentation (P<0.05), 72 h after MCA occlusion. Therefore, our data demonstrate that treatment with RGZ, starting 24 h after stroke, can reduce ischemic injury, improve neurological outcome, and prevent neutrophilia. These findings may support the idea that RGZ has an extended therapeutic window for the treatment of ischemic stroke, as it targets delayed pathways.

A potent and selective inhibitor of endocannabinoid uptake, UCM707, potentiates antinociception induced by cholestasis.

Cholestasis is associated with changes including analgesia. The endocannabinoid system can reduce pain sensitivity. Considering the interaction between the endogenous opioid and endocannabinoid systems in nociception processing, we studied the effect of UCM707 as a potent and selective inhibitor of endocannabinoid uptake on modulation of nociception in a model of elevated endogenous opioid tone, cholestasis. Cholestasis was induced in male Sprague-Dawley rats by ligation of the main bile duct using two ligatures and transecting the duct at the midpoint between them. Seven days later, tail-flick latencies were measured 10 min after injection of UCM707 (0.1, 1 and 10 mg/kg, i.p.) alone or with co-administration of a CB(1) receptor antagonist, AM251 (1 mg/kg, i.p.), with UCM707 (10 mg/kg, i.p.) in experimental groups. A significant increase (P < 0.01) in tail-flick latency was observed in cholestatic rats compared with rats belonging to unoperated and sham groups. Administration of UCM707 (1 and 10 mg/kg) to cholestatic animals significantly increased tail-flick latency compared with the vehicle-treated cholestatic group (P < 0.05 and P < 0.001, respectively). UCM707 injection in unoperated and sham groups did not alter baseline tail-flick latency compared with vehicle-treated groups. The effect of UCM707 in the cholestatic group was blocked by co-administration of AM251 (1 mg/kg, i.p.) with UCM707. These data showed that the endocannabinoid system is involved in nociception processing during cholestasis and that the effects of UCM707 on the pain threshold in cholestatic rats may be a result of CB(1) receptor activation by the increased extracellular levels of endocannabinoids.

GABAA receptors in the central nucleus of amygdala (CeA) affect on pain modulation.

The central nucleus of the amygdala (CeA), the nociceptive amygdala, serves as the major output nucleus of the amygdala and participates in receiving and processing pain information. Considering the abundance of GABA(A) receptors in the CeA and also the attributed bidirectional roles for GABA in controlling nociception, we examined the effects of bilateral intra-CeA microinjection of a different dose of the GABA(A) receptor agonist, muscimol, and the GABA(A) receptor antagonist, bicuculline, on pain modulation using a tail-flick test. Adult rats were exposed to intra-CeA microinjection of a selective GABA(A) receptor antagonist, bicuculline, (50,100,200,400 ng/side) or a selective GABA(A) receptor agonist, muscimol, (62.5, 125,250,500 ng/side) and subjected to the tail-flick test. Tail-flick latencies were measured every 5 min after drug microinjection for 60 min. Microinjection of bicuculline and muscimol into the CeA increased and decreased tail-flick latency, respectively in a dose-dependent fashion. The hyperalgesic effect of muscimol (500 ng) microinjected into the CeA was attenuated (P<0.001) by a prior microinjection of bicuculline (50 ng) at the same site. The results of the present study showed that locally released GABA in the CeA is involved in pain modulation and suggests the existence of a GABA(A) mediated inhibitory system in the CeA on pain control.

CB1 receptor activation in the basolateral amygdala produces antinociception in animal models of acute and tonic nociception.

1. Recent studies have suggested that the basolateral nucleus of the amygdala (BLA) participates in the processing of pain information, especially noxious somatic information. Cannabinoid receptors or CB1 mRNA are expressed more in the BLA than in other nuclei of the amygdala. Thus, the aim of the present study was to examine whether CB1 receptors in the BLA may be involved in modulating acute and/or tonic nociceptive processing. 2. Adult rats were exposed to intra-BLA microinjection of the cannabinoid receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo [1,2,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate [WIN 55,212-2 (1, 2.5, 5 or 10 microg/side)] and subjected to the tail flick and formalin tests. 3. The rats demonstrated a dose-dependent increase in latency to withdraw from a thermal noxious stimulus in the tail flick test and a decrease in formalin-induced pain behaviours. The antinociceptive effects of the CB1 receptor agonist WIN 55,212-2 (10 microg/side) in both tests were attenuated in the presence of the selective CB1 receptor antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3- carboxamide (AM251; 0.55 ng/side). Administration of the CB1 receptor antagonist AM251 (0.55, 5.5, or 55.5 ng/side) alone did not alter the nociceptive thresholds in either test. Bilateral microinjection of the selective CB2 receptor antagonist N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528; 1 microg/side) had no effect on the antinociception produced by WIN 55,212-2, suggesting that the antinociceptive actions of WIN 55,212-2 are mediated by CB1 receptors. 4. The findings suggest the existence of a CB1-mediated inhibitory system in the BLA that, when activated, can diminish responsivity to acute and tonic noxious stimuli, but that normally has no tonic effect on the response threshold of these stimuli.

Modulation of cholestasis-induced antinociception in rats by two NMDA receptor antagonists: MK-801 and magnesium sulfate.

Acute cholestasis is associated with increased activity of the endogenous opioid system that results to changes including analgesia. N-methyl-d-aspartate (NMDA) receptors are involved in the nociceptive pathway and play a major role in the development of morphine induced analgesia. The magnesium acts as a non-competitive NMDA receptor antagonist by blocking the NMDA receptor channel. Considering the reported antinociceptive effect of magnesium sulfate as a NMDA receptor antagonist and the existence of close functional links between NMDA receptor antagonists and magnesium with the opioid system, we studied the effect of acute and chronic administration of MK-801 as a NMDA antagonist and magnesium sulfate on modulation of nociception in an experimental model of elevated endogenous opioid tone, acute cholestasis, using the tail-flick paradigm. Cholestasis was induced by ligation of the main bile duct using two ligatures and then transsection of the duct at the midpoint between them. A significant increase (P<0.001) in nociception threshold was observed in bile duct ligated rats compared to unoperated and sham-operated animals. In acute treatment, MK-801 (0.1 mg/kg, b.i.d), but not magnesium (150 mg/kg magnesium sulfate, i.e. 30 mg/kg of Mg(+2), i.p., b.i.d.) increased antinociception in cholestatic rats compared to saline treated cholestatics (P<0.05). In chronic treatment, administration of MK-801 or magnesium sulfate for 7 consecutive days, increased tail-flick latency (P<0.05, P<0.01) in cholestatic animals compared to saline treated cholestatics. These data showed that NMDA receptor pathway is involved in modulation of cholestasis-induced antinociception in rats and that repeated dosages of magnesium sulfate similar to MK-801 is able to modulate nociception in cholestasis.