Incidence of Postoperative Opioid Overdose and New Diagnosis of Opioid Use Disorder Among US Veterans.

BACKGROUND AND OBJECTIVES: Perioperative exposure to opioids is associated with adverse outcomes. We aim to determine the associations between surgery and subsequent opioid overdose, an acute event, and a new diagnosis of opioid use disorder (OUD), a chronic relapsing disease, in parallel. METHODS: This retrospective cohort study of US veterans used surgery as exposure and the two outcomes were (1) occurrence of overdose and (2) new diagnosis of OUD in the first postoperative year. Surgical group was matched to the reference controls based on the propensity score of having surgery, and matched logistic regression was used to calculate the odds ratio (OR). RESULTS: A total of 261 208 surgical patients were matched to 479 531 controls. Overdose occurred in 1893 (0.7%) of the surgical patients and in 518 (0.1%) of the matched controls in the first postoperative year (OR, 6.71; 95% confidence interval [CI], 5.80-7.75; P < .001). Among patients with no history of OUD, surgery was also associated with a new diagnosis of OUD in the first postoperative year (OR, 1.13; 95% CI, 1.02-1.24; P = .015). DISCUSSION AND CONCLUSIONS: The postoperative period is strongly associated with opioid overdose, but only weakly associated with new diagnosis of OUD. This is likely due to the difficulty of diagnosing OUD in the postoperative period. SCIENTIFIC SIGNIFICANCE: This is the first study that has examined opioid overdose and new-onset OUD in the postoperative period in parallel. Our analysis suggests different risk factors for each, as well as different strengths of association with surgery. More sensitive diagnostic criteria for postoperative OUD are needed to promptly diagnose and treat this condition. (Am J Addict 2020;00:00-00).

Does Thrombolysis Have a Place in the Cardiopulmonary Resuscitation of Patients With Acute Pulmonary Embolism? A Case of Successful Thrombolysis During Pulmonary Embolism Induced Cardiopulmonary Arrest.

OBJECTIVE: Pulmonary embolism often causes cardiac arrest. When this occurs, thrombolytic therapy is not routinely administered. There are multiple reasons for this, including difficulty with rapidly adequately diagnosing the embolus, the lack of good data supporting the use of thrombolytics during resuscitation, the belief that thrombolytic therapy is ineffective once a patient has already arrested, the difficulty of obtaining thrombolytics at the bedside rapidly enough to administer during a code, and the increased risks of bleeding, particularly with ongoing chest compressions. In this case report, we present a patient who was successfully treated with thrombolytic therapy during pulmonary embolism-induced cardiopulmonary arrest and discuss the role of thrombolytics in cardiopulmonary resuscitation. DESIGN: Case report. SETTING: Surgical ICU in a comprehensive cancer center. PATIENT: A 56-year-old man who developed hypotension, dyspnea, hypoxia, and pulseless electrical activity 10 days after resection of a benign colon lesion with a right hemicolectomy and primary end-to-end anastomosis. INTERVENTIONS: After a rapid bedside echocardiogram suggesting pulmonary embolus, thrombolytic therapy was administered during cardiopulmonary resuscitative efforts. MEASUREMENTS AND MAIN RESULTS: The patient had a return of spontaneous circulation and showed improvement in repeat echocardiographic imaging. He had a prolonged course in the ICU and hospital, but eventually made an essentially complete clinical recovery. CONCLUSION: As bedside echocardiographic technology becomes more rapidly and readily available, the rapid diagnosis of pulmonary embolism and use of thrombolytics during cardiopulmonary resuscitation may need to be more routinely considered a potential therapeutic adjunctive measure.

The incidence of opioid misuse among the surgical patients with persistent opioid use.

OBJECTIVE: To calculate the monthly incidence of opioid-related adverse events in the opioid-naïve patients who contin-ue to use opioids in the post-operative period. BACKGROUND DATA: An estimate of 1-6 percent of surgery patients con-tinues to persistently use the opioids longer than 90 post-operative days. However, the association of the opioid ad-verse events with the persistent post-operative use is not clearly known. METHODS: This is a historical (retrospective) cohort study of the opioid-naïve US Veterans between the ages of 18 and 85. The surgical group (who had major surgery between January 1, 2008 and December 31, 2015) was compared to the control references. The outcome of opioid misuse was either opioid overdose or the development of opioid use disorder. Each case was followed for 13 months. The monthly incidence of opioid misuse was calculated by dividing the number of new opioid misuse divided by the number of patients who are still filling the prescription for opioid medications in that specific month. RESULTS: A total of 150,088 surgical patients and 1,145,553 controls met the inclusion criteria. The risk of opioid misuse was between 0.3 and 1.8 percent for first 11 post-operative months among both groups. The incidence of opioid misuse increases to 4.6 percent (95 percent CI: 3.2-6.4) at 12 months and to 8.1 percent (95 percent CI: 5.6-11.5) at 13th post-operative month. The incidence also increases to 2.3 percent (95 percent CI: 1.6-3.4) at 12th and 3.9 percent (95 percent CI: 2.5-6.0) at 13th follow-up month among the control references. CONCLUSIONS: After initiation of opioid pre-scription, the opioid misuse occurs, and the incidence increases dramatically after the 11th month regardless of the reason for opioid therapy initiation (surgery vs chronic conditions). The risks/-benefits of opioid use must be evaluated more frequently with the longer opioid use.

The incidence of persistent postoperative opioid use among U.S. veterans: A national study to identify risk factors.

OBJECTIVE: To calculate the incidence and identify the predictors of persistent postoperative opioid use at different postoperative days. BACKGROUND DATA: A subset of surgical patients continues to use long-term opioids. The importance of the risk factors at different postoperative days is not known. DESIGN: A historical cohort. SETTING: Postoperative period. PATIENTS: Opioid-naive U.S. veterans. INTERVENTIONS: The surgical group had any one of 19 common invasive procedures. The control group is a 10% random sample. Each control was randomly assigned a surgery date. MEASUREMENTS: The outcomes were the presence of persistent opioid use as determined by continued filling of prescriptions for opioids on postoperative days 90, 180, 270, and 365. MAIN RESULTS: A total of 183,430 distinct surgical cases and 1,318,894 controls were identified. 1.0% of the surgical patients were using opioids at 90 days, 0.6% at 180 days, 0.4% at 270 days, and 0.1% at 365 days after the surgery. Surgery was strongly associated with postoperative persistent opioid use at day 90 (OR 3.67, 95% CI, 3.43-3.94, p < 0.001), at day 180 (OR 2.85, 2.67-3.12, p < 0.001), at day 270 (OR 2.63, 2.38-2.91, p < 0.001) and at day 365 (OR 2.11, 1.77-2.51, p < 0.001) compared to non-surgical controls. In risk factor analysis, being male and single were associated with persistent opioid use at earlier time points (90 and 180 days), while hepatitis C and preoperative benzodiazepine use were associated with persistent opioid use at later time points (270 and 365 days). CONCLUSIONS: Many surgeries or invasive procedures are associated with an increased risk of persistent postoperative opioid use. The postoperative period is dynamic and the risk factors change with time.

Cerebrovascular responses in mice deficient in the potassium channel, TREK-1.

We tested the hypothesis that TREK-1, a two-pore domain K channel, is involved with dilations in arteries. Because there are no selective activators or inhibitors of TREK-1, we generated a mouse line deficient in TREK-1. Endothelium-mediated dilations were not different in arteries from wild-type (WT) and TREK-1 knockout (KO) mice. This includes dilations of the middle cerebral artery to ATP, dilations of the basilar artery to ACh, and relaxations of the aorta to carbachol, a cholinergic agonist. The nitric oxide (NO) and endothelium-dependent hyperpolarizing factor components of ATP dilations were identical in the middle cerebral arteries of WT and TREK-1 KO mice. Furthermore, the NO and cyclooxygenase-dependent components were identical in the basilar arteries of the different genotypes. Dilations of the basilar artery to alpha-linolenic acid, an activator of TREK-1, were not affected by the absence of TREK-1. Whole cell currents recorded using patch-clamp techniques were similar in cerebrovascular smooth muscle cells (CVSMCs) from WT and TREK-1 KO mice. alpha-linolenic acid or arachidonic acid increased whole cell currents in CVSMCs from both WT and TREK-1 KO mice. The selective blockers of large-conductance Ca-activated K channels, penitrem A and iberiotoxin, blocked the increased currents elicited by either alpha-linolenic or arachidonic acid. In summary, dilations were similar in arteries from WT and TREK-1 KO mice. There was no sign of TREK-1-like currents in CVSMCs from WT mice, and there were no major differences in currents between the genotypes. We conclude that regulation of arterial diameter is not altered in mice lacking TREK-1.

Postoperative opioid misuse in patients with opioid use disorders maintained on opioid agonist treatment.

BACKGROUND: Patients recovering from opioid use disorders (OUD) may be prone to relapse and opioid misuse in the postoperative period due to re-exposure to prescription opioids for pain control. This retrospective study analyzed the incidence of confirmed opioid misuse in the postoperative period in patients with OUDs enrolled in an opioid agonist treatment (OAT) program. METHODS: The study population was US veterans with a diagnosis of OUD who enrolled in the OAT program at VA Maryland Health Care System (Baltimore, Maryland, USA) between 1/1/2000 and 12/31/2016. The patients were excluded if they were enrolled in OAT for less than a year, or if they had surgery within the first 180 days after OAT admission. The surgical group consisted of veterans who had surgery or an invasive procedure during their enrollment in the OAT program. The control (reference) group consisted of enrolled veterans who did not have any invasive procedure. The primary outcome was the first opioid misuse within 365 days after surgery date in the surgical group or a randomly assigned sham surgery date in controls. Opioid misuse was defined as either inappropriate use of opioids detected via urinalysis or admission with a diagnosis of an opioid overdose. RESULTS: From a total of 1352 patients enrolled in the OAT program, 413 were excluded because they were enrolled for less than a year, and 26 were excluded because they had surgery within the first 180 days after admission to the OAT program. Of the 923 eligible patients, 87 had surgery while enrolled and 836 did not. Using propensity scores, all 87 of the surgical cases were matched to 249 of the control cases. In the matched groups, surgery was positively associated with postoperative opioid misuse (odds ratio (OR) of 1.91, 95% CI 1.05-3.48, p = 0.034) in logistic regression. CONCLUSION: Among patients with a history of opioid use disorders, the postoperative period was associated with an increased risk of opioid misuse. Moreover, opioid misuse among patients in an opioid agonist treatment program may well be considered a surgical hazard.

Impact of Multidisciplinary Spine Conferences on Surgical Planning and Perioperative Care in Elective Lumbar Spine Surgeries.

STUDY DESIGN: Pre- and post-implementation analysis. PURPOSE: We examined the impact of implementing multidisciplinary spine conferences-"spine board" reviews-on the general utilization of elective lumbar spine surgeries in a tertiary medical institute. OVERVIEW OF LITERATURE: A multidisciplinary approach to spine care reportedly improves the appropriate utilization of surgical spine procedures. METHODS: A multidisciplinary spine board was established to review candidates selected for elective lumbar spine surgery. The board comprised representatives from orthopedic spine surgery, neurosurgery, psychology, physical therapy, radiology, pharmacy, primary care, pain management, anesthesiology, and veteran advocacy. Two similar 6-month periods were selected to study the impact of this implementation: pre-implementing (June 1, 2015 to November 30, 2015) and post-implementation (June 1, 2016 to November 30, 2016) periods. RESULTS: Between March 1, 2016 and December 30, 2016, the spine board discussed 11 patients. All patients underwent clinical examinations and radiological assessments findings that warranted elective lumbar surgery. The board recommended non-surgical interventions before proceeding with the planned surgeries in all cases. In the pre-implementation period, a total of 101 elective lumbar spine surgeries were performed. In the post-implementation period, a total of 51 elective lumbar spine surgeries were performed (p <0.05). The surgical plan for elective lumbar spine surgery in the post-implementation period was not directly influenced by the review of spine board because none of the cases were discussed in the conferences; however, the care occurred at a hospital where the spine board was implemented. There was no significant change in the number of cervical spine surgeries performed (66 preimplementation vs. 56 post-implementation). The average surgery duration was 52 minutes shorter in the post-implementation period compared with that in the pre-implementation period (p <0.05). CONCLUSIONS: Implementation of a multidisciplinary spine board was concurrent with an overall decrease in the utilization of lumbar spine surgeries for elective cases of low back pain in a tertiary medical center.

Use of an optimized transient occlusion of the middle cerebral artery protocol for the mouse stroke model.

Intraluminal occlusion of the middle cerebral artery in rodents is widely used for investigating cerebral ischemia and reperfusion injury. Two types of filaments used for occlusion were tested in terms of surgical success, incidence of subarachnoid hemorrhage, and mortality: a standard 6-0 monofilament coated with methyl methacrylate glue (rigid probe) and an 8-0 monofilament coated with silicone (flexible probe). In 98 wild-type (WT) mice, the flexible probe produced significantly (P < .05) more successful strokes (73.5%) than the rigid probe (46.6%). The incidences of subarachnoid hemorrhage (3.7%) and mortality (5.6%) with the flexible probe were significantly lower than those with the rigid probe (26.6% and 11.1%, respectively). Rigid and flexible probes were also compared in heme oxygenase 1 knockout (n = 17) and WT littermates (n = 17), because knockout mice have been suggested to have more fragile blood vessels. All mice receiving the flexible probe had successful strokes, with no cases of subarachnoid hemorrhage or mortality; however, with the rigid probe, the success rate was only 80% in the WT mice and 60% in the knockout mice. The rates of subarachnoid hemorrhage and mortality were also significantly higher with the rigid probe in both genotypes, but the infarct volumes produced by each type of probe did not differ significantly between the 2 groups. We conclude that the flexible silicone-coated 8-0 probe is superior to the more rigid glue-coated probe, because it produces infarct volumes of equal size with a higher success rate and lower risk of subarachnoid hemorrhage and mortality.

alpha2-Adrenoceptor subsensitivity in mesenteric vascular bed of cholestatic rats: the role of nitric oxide and endogenous opioids.

Cholestasis is associated with vascular changes and in previous studies decreased response of visceral vessels of cholestatic animals to phenylephrine and acetylcholine has been shown. In the present study, the response of mesenteric vascular bed of cholestatic rats to clonidine (an alpha2-adrenoceptor agonist) was investigated and we also examined the role of endogenous opioids and nitric oxide (NO). Seven-day ligation of bile duct was used as the model to study cholestasis. Six groups of rats, each of which divided into two subgroups (bile duct-ligated and sham-operated), were examined. Three groups of animals were chronically treated with either normal saline, naltrexone (an opioid receptor antagonist, 20 mg/kg/day, s.c.) or aminoguanidine (a selective inducible nitric oxide synthase inhibitor, 150 mg/kg/day, s.c.) for 7 days. After 7 days the response of the mesenteric vascular bed to subsequent doses of clonidine was studied. In other two groups, 7 days after the operation, the response of the mesenteric vascular bed to clonidine in the presence of either yuhimbine, an alpha2-adrenoceptor antagonist, or N(omega)-nitro-L-arginine methyl ester (L-NAME), a non-selective nitric oxide synthase inhibitor, was studied. In the last group, vasodilation response to sodium nitroprusside (an endothelium-independent vasorelaxant) was evaluated. Clonidine caused vasodilation in a dose-dependent manner by acting on endothelial alpha2-adrenoceptors since its effect was antagonized by yohimbine, and this vasodilation was through the L-arginine pathway since there was no response in the presence of L-NAME in the perfusate. Compared to sham-operated rats, there was a significant right shift in the clonidine concentration curves of cholestatic animals. Maximum response in cholestatic rats was significantly lower comparing to the sham group (P<0.01) and the dose of clonidine that causes 50% of maximum response (ED50) was significantly higher in cholestatic rats (P<0.05). Vasodilation response to sodium nitroprusside was the same in cholestatic and sham-operated rats. Seven-day treatment with aminoguanidine recovered the effect of cholestasis. Seven-day treatment with naltrexone caused an increase in maximum response (P<0.01) and a decrease in ED50 (P<0.05) in cholestatic rats, while this treatment in sham-operated rats caused a decrease in the maximum response (P<0.01) and an increase in ED50 (P<0.05). This study showed that cholestasis is associated with decreased responsiveness of mesenteric vascular bed to clonidine and the cholestasis-associated NO overproduction and increased level of endogenous opioids may contribute to this process.

Stroke outcomes in mice lacking the genes for neuronal heme oxygenase-2 and nitric oxide synthase.

Heme oxygenase-2 (HO-2) has been suggested to be a cytoprotective enzyme in a variety of in vivo experimental models. HO-2, the constitutive isozyme, is enriched in neurons and, under normal conditions, accounts for nearly all of brain HO activity. HO-2 deletion (HO-2-/-) leads to increased neurotoxicity in cultured brain cells and increased damage following transient cerebral ischemia in mice. Moreover, pharmacologic inhibition of HO activity significantly augments focal ischemic damage in wildtype (WT) mice, but does not further exacerbate it in HO-2-/- mice. The HO system shares some similarities with nitric oxide synthase (NOS), notably their syntheses of carbon monoxide (CO) and nitric oxide (NO), respectively, which are diffusible gases with numerous biological actions, including neurotransmission and vasodilation. While deletion of HO-2 results in greater stroke damage, the pharmacologic inhibition of neuronal nitric oxide synthase (nNOS), or its gene deletion, confers neuroprotection in animal models of transient cerebral ischemia. To investigate the interactions, the outcome of focal cerebral ischemia-reperfusion in double knockout (HO-2-/- X nNOS-/-) mice lacking both genes was compared to control WT mice. Wildtype and double knockout male mice underwent intraluminal middle cerebral occlusion for 2 hours, followed by reperfusion for 22 hours. Outcomes in neurologic deficits and infarct size were determined. No difference was observed between WT and double knockout mice in the volume of infarction, neurologic signs, decrease in relative cerebral blood flow during ischemia, or core body temperature. The results suggest that the deleterious action of nNOS would counteract the role of HO-2 in neuroprotection.

Prostaglandins of J series control heme oxygenase expression: potential significance in modulating neuroinflammation.

Cyclopentenone prostaglandins (cyPGs) are a subfamily of prostaglandins that are characterized by the cyclopentenone ring in their structure. They exert their effect after active transportation into the cell, probably by interacting with cellular target proteins or DNA sequences. The cyPGs have anti-inflammatory activities, especially important during the resolution of inflammation, anticancer, and cytoprotective properties. Here, we show that the cyPGs, especially the 15-deoxy-Delta(12,14) PGJ(2), can specifically induce heme oxygenase 1 in mouse primary neuronal cells. Heme oxygenase is the enzyme responsible for the degradation of heme into biliverdin, ferrous iron, and carbon monoxide. This enzyme conveys protection to oxidative cellular injury by degrading the pro-inflammatory heme; producing biliverdin and bilirubin, potent antioxidants; producing carbon monoxide, a neurotransmitter that also has anti-inflammatory and vasodilatory properties; and assisting in keeping iron cellular homeostasis. CyPGs appear to possess a promising future in designing therapeutics for many neurologic diseases, such as Alzheimer's disease, vascular-related dementia, multiple sclerosis, ischemic conditions, and many others in which inflammation is a part of the pathophysiology.

The effect of cyclosporin A on morphine tolerance and dependence: involvement of L-arginine/nitric oxide pathway.

Cyclosporin A is known to decrease nitric oxide (NO) production in nervous tissues. The effects of systemic cyclosporine A on the induction and expression of morphine tolerance and dependence, acute morphine-induced antinociception, and the probable involvement of the L-arginine/nitric oxide pathway in these effects were assessed in mice. Cyclosporin A (20 mg/kg), N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) and a combination of the two at lower and per se non-effective doses (5 and 3 mg/kg, respectively) showed a similar pattern of action, inhibiting the induction of tolerance to morphine-induced antinociception and increasing the antinociception threshold in the expression phase of morphine tolerance. These agents also inhibited the expression of morphine dependence as assessed by naloxone-precipitated withdrawal signs, while having no effect on the induction of morphine dependence. L-Arginine, at a per se non-effective dose (60 mg/kg), inhibited the effects of Cyclosporin A. Moreover, acute administration of Cyclosporin A (20 mg/kg) or L-NAME (10 mg/kg) enhanced the antinociception induced by acute administration of morphine (5 mg/kg), while chronic pretreatment with Cyclosporin A (20 mg/kg) or L-NAME (10 mg/kg) for 2 days (twice daily) did not affect morphine-induced antinociception. The inducible nitric oxide synthase inhibitor, aminoguanidine (100 mg/kg), did not alter morphine antinociception, tolerance or dependence. In conclusion, decreasing NO production through constitutive nitric oxide synthase may be a mechanism through which cyclosporin A differentially modulates morphine tolerance, dependence and antinociception.

Inhibition by immunophilin ligands of morphine-induced tolerance and dependence in guinea pig ileum.

Immunophilin ligands, cyclosporine A and FK506 (tacrolimus), besides their immunosuppressive action, have several effects on different neural functions, such as modulation of the release of many neurotransmitters, the reduction of nitric oxide (NO) production by the inhibition of dephosphorylation of neuronal nitric oxide synthase (nNOS) and the alteration of the expression of certain genes. Many of these actions apparently occur through the inhibition of calcineurin, a calcium-calmodulin-dependent phosphatase. On the other hand, several studies have shown that NO has a critical role in opioid-induced tolerance and dependence in both in vivo and in vitro models. In the present study, the effect of cyclosporine A and FK506 on the development of tolerance to and dependence on morphine in the guinea pig ileum was assessed. Morphine inhibited electrically stimulated twitch of ileum in a concentration-dependent manner (pD(2)=7.45+/-0.07). Tolerance to this effect was induced by incubation of ileum with 2 x IC(50) or 4 x IC(50) of morphine for 2 h that induced a degree of tolerance of 6.81 and 18.10, respectively. The co-incubation of ileum with morphine along with either cyclosporine A or FK506 reduced the degree of tolerance significantly (P<0.05) and restored the sensitivity of ileum to the morphine inhibitory effect. Dependence was induced by incubation with 4 x IC(50) of morphine for 2 h and was assessed based on naloxone-induced contractions (10(-5) M). Cyclosporine A (10(-9) M) and FK506 (10(-9) M) can attenuate the development of dependence to morphine as shown by the significant decrease in naloxone-induced contractions (P<0.05). These results suggest that immunophilin ligands at very low concentrations (nanomolar) can reduce the induction of acute tolerance to and dependence on morphine in the myenteric plexus of guinea pig ileum.

The nonadrenergic noncholinergic relaxation of anococcygeus muscles of bile duct-ligated rats.

Previous studies have shown the naloxone-induced withdrawal syndrome and the development of tolerance in the tissues of cholestatic animals. Increased neuronal nitric oxide synthase (nNOS) expression is reported to exist in morphine-tolerant animals. This, together with evidence for nitric oxide (NO) overproduction in cholestasis, suggested the possibility of an alteration of nonadrenergic noncholinergic (NANC) relaxation of anococcygeus muscles of cholestatic rats. To study this, we used three main groups of animals: unoperated, sham-operated and bile duct-ligated. Electrical field stimulation, in the presence of atropine and guanethidine, caused NANC relaxation in the anococcygeus muscle which was enhanced in bile duct-ligated animals. N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS blocker, caused a dose-dependent inhibition of the NANC relaxation. The IC(50)'s of L-NAME in 7-day (7.30+/-0.87 microM), 14-day (6.98+/-0.70 microM) and 21-day (8.25+/-1.40 microM) bile duct-ligated groups were significantly different from those of unoperated (1.69+/-0.30 microM) and sham-operated groups (1.90+/-0.27 microM). L-NAME (100 microM) completely inhibited the NANC relaxation response, suggesting that NANC relaxation in the rat anococcygeus muscle is mediated mainly via NO. The contraction response of the intact muscle to phenylephrine, an alpha(1)-adrenoceptor agonist, and the relaxation response of the phenylephrine-contracted muscle to sodium nitroprusside, an NO donor, were not different in unoperated, sham-operated and 7-day bile duct-ligated groups. These results showed that the smooth muscle component of NANC relaxation is not altered in anococcygeus muscles of bile duct-ligated rats. It can thus be concluded that the NANC relaxation in the anococcygeus of cholestatic rats is more resistant to a NOS blocker, providing evidence for increased nitrergic neurotransmission in the anococcygeus muscles of cholestatic rats.

The role of nitric oxide in anticonvulsant and proconvulsant effects of morphine in mice.

Acute subcutaneous administration of lower doses of morphine (0.5, 1 and 3 mg/kg) increase the threshold of seizures induced by pentylenetetrazole (PTZ) in mice, whereas higher doses of morphine (15, 30 and 60 mg/kg) have proconvulsant effects. The effect of systemic administration of nitric oxide synthase (NOS) inhibitors N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-nitro-L-arginine (L-NNA) and nitric oxide synthase (NOS) L-arginine on biphasic effect of morphine was investigated. Acute administration of both L-NAME (1, 3 and 10 mg/kg) and L-NNA (1 and 10 mg/kg) as well as chronic pretreatment with L-NAME (1 and 10 mg/kg, 4 days) dose-dependently inhibited both the anticonvulsant and proconvulsant effects of morphine (1 and 30 mg/kg, respectively). The inhibition was complete for anticonvulsant effect while partial for proconvulsant effect. L-arginine at doses that did not affect seizure threshold per se (acute, 30 and 60 mg/kg; chronic, 60 mg/kg) potentiated both anticonvulsant and proconvulsant properties of less potent doses of morphine (0.5 and 15 mg/kg, respectively). The L-arginine induced potentiation of both phases of morphine effect was blocked by L-NAME (0.5-30 mg/kg). Moreover, low and per se non-effective doses of naloxone (0.1 mg/kg) and L-NAME (0.3, 0.5 or 1 mg/kg) showed additive effects in inhibiting both phases of morphine effects. These results support the involvement of L-arginine/nitric oxide pathway in the modulation of seizure threshold by morphine.

Alpha-2-adrenoceptor hyporesponsiveness in isolated tissues of cholestatic animals: involvement of opioid and nitric oxide systems.

In the present study, the status of alpha(2)-adrenoceptors during cholestasis was investigated by the inhibitory effect of clonidine on the electrically stimulated contractions of mice vas deferens (MVD) and guinea pig ileum (GPI). Clonidine inhibited the contractions in both tissues in a dose-dependent manner. Compared to unoperated animals, there was a significant right-shift in the clonidine concentration-curves of both tissues obtained from 5-day bile-duct ligated (BDL) animals (p < 0.01), implying the hyporesponsiveness of alpha(2)-adrenoceptors during cholestasis. Chronic treatment with naltrexone (3 mg/kg/day) reversed the right-shift induced by cholestasis in both tissues. Administration of N(omega)-nitro-L-arginine methyl ester (20 mg/kg/day) also partially reversed cholestasis-induced effect on IC(50) of clonidine. These two treatments had no effect on IC(50) of tissues from controls. Chronic yohimbine treatment (5 mg/kg/day) recovered the effect of cholestasis on MVD, but sensitized the ileum of unoperated and BDL guinea pigs to clonidine to a similar extent, providing evidence for the role of the augmented adrenergic state of cholestasis in the hyporesponsiveness of norepinephrine-releasing neurons of MVD. We concluded that cholestasis is associated with the decreased responsiveness of alpha(2)-adrenoceptors and the cholestasis-associated augmented opioidergic tone and increased NO production contribute to this process.

Do endogenous opioids contribute to the bradycardia of rats with obstructive cholestasis?

Endogenous opioids have nitric oxide (NO)-dependent cardiovascular actions. In the light of biological evidence of accumulation of endogenous opioids in cholestasis and also existence of NO-dependent bradycardia in cholestatic subjects, this study was carried out to evaluate the role of endogenous opioids in the generation of bradycardia in a rat model of cholestasis. Male Sprague-Dawley rats were used to induce cholestasis by surgical ligation of the bile duct, with sham-operated animals serving as a control. The animals were divided into six groups which received naltrexone [20 mg/kg/day, subcutaneously (s.c.)], N(G)-L-nitro-arginine methyl ester (L-NAME, 3 mg/kg/day, s.c.), aminoguanidine (200 mg/kg/day, s.c.), L-arginine (200 mg/kg/day, s.c.), naltrexone + L-NAME (20 and 3 mg/kg/day, s.c) or saline. One week after the operation, a lead II electrocardiogram (ECG) was recorded and the spontaneously beating atria of the animals were then isolated and the chronotropic responses to epinephrine evaluated. The plasma L-nitro-tyrosine level and alanine amino transferase and alkaline phosphatase activities were also measured. The heart rate of cholestatic animals was significantly lower than that of control rats in vivo and this bradycardia was corrected with daily adminstration of naltrexone or L-NAME. The basal spontaneous beating rate of atria in cholestatic animals was not significantly different from that of sham-operated animals in vitro. Cholestasis induced a significant decrease in the chronotropic effect of epinephrine. This effect was corrected by daily injection of naltrexone or L-NAME, or concurrent administration of naltrexone + L-NAME, and was not corrected by aminoguanidine. L-arginine had an equivalent effect to L-NAME and increased the chronotropic effect of epinephrine in cholestatic rats but not in control animals. Bile duct ligation increased the plasma activity of liver enzymes as well as the level of L-nitro-tyrosine. L-arginine and naltrexone treatment significantly decreased the elevation of liver enzymes in bile duct-ligated rats. Pretreatment of cholestatic animals with naltrexone or L-NAME decreased the plasma L-nitro-tyrosine level. The results suggest that either prevention of NO overproduction or protection against liver damage is responsible for recovery of bradycardia after naltrexone administration.

Comparison of UV and tandem mass spectrometric detection for the high-performance liquid chromatographic determination of diclofenac in microdialysis samples.

High-performance liquid chromatography (HPLC) was used to analyze microdialysis samples obtained in vivo from human subcutaneous adipose tissue after topical application of the nonsteroidal anti-inflammatory drug diclofenac. For the reliable determination of diclofenac two different detection principles were applied in two different laboratories. One HPLC method utilized UV-detection at 280 nm, the other one used selected reaction monitoring mass spectrometry (MS). The HPLC-UV and -MS methods offered low limits of quantification of 10 and 1 ng/ml and an accuracy between 94.0-126.7 and 89.3-110.9%, respectively. However, a comparison showed that the HPLC-UV method failed to determine diclofenac in biological matrices, as both false negative and positive values were found. HPLC-MS is clearly superior to HPLC-UV due to a much more selective detection, increased sensitivity and shorter run times.

Traumatic brain injury in mice lacking the K channel, TREK-1.

The purpose of this study was to determine whether the potassium channel, TREK-1, was neuroprotective after traumatic brain injury (TBI). Since there are no selective blockers, we used TREK-1 knockout (KO) mice for our study. Wild-type (WT) and TREK-1 KO mice were anesthetized and subjected to controlled-cortical impact injury (deformation of the brain by 1.5 mm by a 3-mm diameter rod traveling at a 3 m/s). Laser Doppler perfusion (LDP) decreased by ∼80% in the injured cortex and remained at that level in both WT and TREK-1 KO mice (n=10 and 11, respectively). Laser Doppler perfusion decreased by 50% to 60% in cortical areas directly adjacent to the site of injury. There were no statistical differences in LDP between genotype. The contusion volume, determined 15 days after the TBI using hematoxylin and eosin-stained coronal brain sections, was 4.1±0.8 (n=10) and 5.1±0.5 (n=11) mm(3) for WT and TREK-1 KO, respectively (not significant, P=0.34). Cell counts of viable neurons in the CA1 and CA3 regions of the hippocampus were similar between WT and TREK-1 KO mice (P=0.51 and 0.84 for CA1 and CA3, respectively). We conclude that TREK-1 expression does not provide brain protection after TBI.

Characterization of TWIK-2, a two-pore domain K+ channel, cloned from the rat middle cerebral artery.

TWIK-2, a member of the Two-Pore Domain K channel family, is expressed in a number of mammalian tissues including the vascular system. The function of TWIK-2 is not known. The purpose of this study was to clone the TWIK-2 channel from the rat middle cerebral artery, express it in CHO cells, and characterize the channel's electrical properties. In light of the fact that there are no specific TWIK-2 inhibitors or activators, a better characterization of the channel should enhance our understanding of its role in the vascular system. TWIK-2 was cloned from the rat middle cerebral artery and expressed with an N-terminal green fluorescence protein (GFP) in CHO cells. We report that rTWIK-2-GFP currents were relatively linear at physiological K(+) concentrations but become slightly inwardly rectifying in symmetrical K(+). rTWIK-2-GFP was insensitive to 10 mM TEA, 3 mM 4-aminopyridine, and 10 microM glibenclamide. However, rTWIK-2-GFP was inhibited by Ba(2+) with 50% of the current being blocked at 80 microM. rTWIK-2-GFP activity was enhanced 60% by 100 microM arachidonic acid. The electrophysiological characteristics of TWIK-2 indicate that it could serve an important role in ion homeostasis and regulation of the membrane potential in arteries and arterioles.