In vitro sepsis up-regulates Nociceptin/Orphanin FQ receptor expression and function on human T- but not B-cells.

BACKGROUND AND PURPOSE: In animal models of sepsis, increased activation of the Nociceptin/Orphanin FQ (N/OFQ) receptor NOP is associated with mortality and NOP antagonists improved survival. We have explored the role of the N/OFQ-NOP system in freshly isolated volunteer human B- and T-cells incubated with lipopolysaccharide (LPS) and peptidoglycan G (PepG) as a model of in vitro sepsis. EXPERIMENTAL APPROACH: B- and T-cell NOP expression was measured using the NOP fluorescent probe N/OFQATTO594 , N/OFQ content was measured using immunofluorescence, N/OFQ release was tracked using a CHOhNOPGαiq5 biosensor assay and NOP function was measured using transwell migration and cytokine/chemokine release using a 25-plex assay format. Cells were challenged with LPS/PepG. KEY RESULTS: CD19-positive B-cells bound N/OFQATTO594 ; they also contain N/OFQ. Stimulation with CXCL13/IL-4 increased N/OFQ release. N/OFQ trended to reduced migration to CXCL13/IL-4. Surface NOP expression was unaffected by LPS/PepG, but this treatment increased GM-CSF release in an N/OFQ sensitive manner. CD3-positive T-cells did not bind N/OFQATTO594 ; they did contain N/OFQ. Stimulation with CXCL12/IL-6 increased N/OFQ release. When incubated with LPS/PepG, NOP surface expression was induced leading to N/OFQATTO594 binding. In LPS/PepG-treated cells, N/OFQ reduced migration to CXCL12/IL-6. LPS/PepG increased GM-CSF release in an N/OFQ sensitive manner. CONCLUSIONS AND IMPLICATIONS: We suggest both a constitutive and sepsis-inducible N/OFQ-NOP receptor autocrine regulation of B- and T-cell function, respectively. These NOP receptors variably inhibit migration and reduce GM-CSF release. These data provide mechanistic insights to the detrimental role for increased N/OFQ signalling in sepsis and suggest a potential role for NOP antagonists as treatments.

Effect of a continuous perineural levobupivacaine infusion on pain after major lower limb amputation: a randomised double-blind placebo-controlled trial.

OBJECTIVES: Randomised controlled trial of the effect of a perineural infusion of levobupivacaine on moderate/severe phantom limb pain 6 months after major lower limb amputation. SETTING: Single-centre, UK university hospital. PARTICIPANTS: Ninety patients undergoing above-knee and below-knee amputation for chronic limb threatening ischaemia under general anaesthesia. Exclusion criteria were patients having surgery under neuraxial anaesthesia; inability to operate a patient-controlled analgesia device or complete a Visual Analogue Scale; amputation for trauma or malignancy; or contraindication to levobupivacaine. INTERVENTIONS: Either levobupivacaine 0.125% or saline 0.9% (10 mL bolus, infusion of 8 mL/hour for 96 hours) via a sciatic or posterior tibial nerve sheath catheter placed under direct vision during surgery. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome measure was the presence of phantom limb pain, residual limb pain and phantom limb sensations up to 6 months after amputation. Secondary outcome measures included early postoperative pain and morphine requirements after surgery. RESULTS: Data from 81 participants were analysed; 6-month follow-up data were available for 62 patients. Pain and morphine requirements varied widely before and after amputation in both groups. The incidences of moderate/severe phantom limb pain, residual limb pain and phantom limb sensations were low from 6 weeks with no significant differences between groups in phantom limb pain at rest (OR 0.56, 95% CI 0.14 to 2.14, p=0.394) or movement (OR 0.58, 95% CI 0.15 to 2.21, p=0.425) at 6 months. Early postoperative pain scores were low in both groups with no between-group differences in residual limb pain or phantom limb sensations (rest or movement) at any time point. High postoperative morphine consumption was associated with worsening phantom limb pain both at rest (-17.51, 95% CI -24.29 to -10.74; p<0.001) and on movement (-18.54, 95% CI -25.58 to -11.49; p<0.001). The incidence of adverse effects related to the study was low in both groups: postoperative nausea, vomiting and sedation scores were similar, and there were no features of local anaesthetic toxicity. CONCLUSIONS: Long-term phantom limb pain, residual limb pain and phantom limb sensations were not reduced significantly by perineural infusion of levobupivacaine, although the study was underpowered to show significant differences in the primary outcome. The incidence of phantom limb pain was lower than previously reported, possibly attributable to frequent assessment and early intervention to identify and treat postoperative pain when it occurred. There were large variations in postoperative pain scores, high requirements for analgesics before and after surgery and some problems maintaining recruitment and long -term follow-up. Knowledge of these potential problems should inform future research in this group of patients. Further work should investigate the association between perioperative morphine requirements and late phantom limb pain. TRIAL REGISTRATION NUMBERS: EudraCT 2007-000619-27; ISRCTN68691928.

Pathophysiological and clinical considerations in the perioperative care of patients with a previous ischaemic stroke: a multidisciplinary narrative review.

With an ageing population and increasing incidence of cerebrovascular disease, an increasing number of patients presenting for routine and emergency surgery have a prior history of stroke. This presents a challenge for pre-, intra-, and postoperative management as the neurological risk is considerably higher. Evidence is lacking around anaesthetic practice for patients with vascular neurological vulnerability. Through understanding the pathophysiological changes that occur after stroke, insight into the susceptibilities of the cerebral vasculature to intrinsic and extrinsic factors can be developed. Increasing understanding of post-stroke systemic and cerebral haemodynamics has provided improved outcomes from stroke and more robust secondary prevention, although this knowledge has yet to be applied to our delivery of anaesthesia in those with prior stroke. This review describes the key pathophysiological and clinical considerations that inform clinicians providing perioperative care for patients with a prior diagnosis of stroke.

Post-carotid Endarterectomy Hypertension. Part 2: Association with Peri-operative Clinical, Anaesthetic, and Transcranial Doppler Derived Parameters.

OBJECTIVE/BACKGROUND: The first paper in this series observed that pre-operative baroreceptor dysfunction and poorly controlled hypertension were independently predictive for identifying patients who went on to require treatment for post-endarterectomy hypertension (PEH). The second paper examines the influence of intra-operative patient, transcranial Doppler (TCD) ultrasound, and anaesthetic variables on the incidence of PEH. METHODS: In total, 106 patients underwent carotid endarterectomy (CEA) under general anaesthesia. Systolic blood pressure (SBP) changes, anaesthetic and vasoactive agents, analgesia, and post-operative pain scores, as well as TCD derived changes in middle cerebral artery (MCA) velocity during surgery were recorded. Patients who met pre-existing unit criteria for treating PEH after CEA (SBP > 170 mmHg without symptoms or SBP > 160 mmHg with headache/seizure/neurological deficit) were treated according to an established and validated protocol. RESULTS: In total, 40/106 patients (38%) required treatment for PEH following CEA (26 in theatre recovery [25%], 27 back on the vascular surgery ward [25%]), whereas seven (7%) had SBP surges > 200 mmHg on the ward. Patients requiring treatment for PEH had significantly higher pre-induction SBP (174 ± 21 mmHg vs. 153 ± 21 mmHg; p < .001), the greatest decreases in SBP after induction of anaesthesia (median decrease 100 ± 32 mmHg vs. 83 ± 24 mmHg; p = .01) and were significantly more likely to experience moderate/severe pain scores post-operatively (p = .003). Logistic regression analysis of the pre- and intra-operative data revealed that higher pre-induction mean SBP and lower pre-operative (impaired) BRS were the only independent predictors of PEH. CONCLUSION: This analysis of intra-operative variables has demonstrated that patients with poorly controlled and/or labile hypertension at induction of general anaesthesia were those at greatest risk of requiring treatment for PEH in the post-operative period after CEA. No other variables, including use of vasopressors, treatment of hypotension, anaesthetic agents, or changes in MCA velocity after clamp release and restoration of flow were able to predict who might go on to require treatment for PEH. Identification of at-risk individuals and aggressive blood pressure control in the post-operative period remains the mainstay of treatment.

Exploring LPS-induced sepsis in rats and mice as a model to study potential protective effects of the nociceptin/orphanin FQ system.

The nociceptin receptor (NOP) and its ligand nociceptin/orphanin FQ (N/OFQ) have been shown to exert a modulatory effect on immune cells during sepsis. We evaluated the suitability of an experimental lipopolysaccharide (LPS)-induced sepsis model for studying changes in the nociceptin system. C57BL/6 mice BALB/c mice and Wistar rats were inoculated with different doses of LPS with or without a nociceptin receptor antagonist (UFP-101 or SB-612111). In C57BL/6 mice LPS 0.85 mg/kg injection produced no septic response, whereas 1.2mg/kg produced a profound response within 5h. In BALB/c mice, LPS 4 mg/kg produced no response, whereas 7 mg/kg resulted in a profound response within 24h. In Wistar rats LPS 15 mg/kg caused no septic response in 6/10 animals, whereas 25mg/kg resulted in marked lethargy before 24h. Splenic interleukin-1ß mRNA in BALB/c mice, and serum TNF-α concentrations in Wistar rats increased after LPS injection in a dose-dependent manner, but were undetectable in control animals, indicating that LPS had stimulated an inflammatory reaction. IL-1ß and TNF-α concentrations in LPS-treated animals were unaffected by administration of a NOP antagonist. Similarly NOP antagonists had no effect on survival or expression of mRNA for NOP or ppN/OFQ (the N/OFQ precursor) in a variety of tissues. In these animal models, the dose-response curve for LPS was too steep to allow use in survival studies and no changes in the N/OFQ system occurred within 24h. We conclude that LPS-inoculation in rodents is an unsuitable model for studying possible changes in the NOP-N/OFQ system in sepsis.

The Nociceptin/Orphanin FQ system is modulated in patients admitted to ICU with sepsis and after cardiopulmonary bypass.

BACKGROUND AND OBJECTIVES: Nociceptin/Orphanin FQ (N/OFQ) is a non-classical endogenous opioid peptide that modulates immune function in vitro. Its importance in inflammation and human sepsis is unknown. The objectives of this study were to determine the relationship between N/OFQ, transcripts for its precursor (pre-pro-N/OFQ [ppNOC]) and receptor (NOP), inflammatory markers and clinical outcomes in patients undergoing cardiopulmonary bypass and with sepsis. METHODS: A prospective observational cohort study of 82 patients admitted to Intensive Care (ICU) with sepsis and 40 patients undergoing cardiac surgery under cardiopulmonary bypass (as a model of systemic inflammation). Sixty three healthy volunteers, matched by age and sex to the patients with sepsis were also studied. Clinical and laboratory details were recorded. Polymorph ppNOC and NOP receptor mRNA were determined using quantitative PCR. Plasma N/OFQ was determined using ELISA and cytokines (TNF- α, IL-8, IL-10) measured using radioimmunoassay. Data from patients undergoing cardiac surgery were recorded before, 3 and 24 hours after cardiopulmonary bypass. ICU patients with sepsis were assessed on Days 1 and 2 of ICU admission, and after clinical recovery. MAIN RESULTS: Plasma N/OFQ concentrations increased (p<0.0001) on Days 1 and 2 of ICU admission with sepsis compared to matched recovery samples. Polymorph ppNOC (p= 0.019) and NOP mRNA (p<0.0001) decreased compared to healthy volunteers. TNF-α, IL-8 and IL-10 concentrations increased on Day 1 compared to matched recovery samples and volunteers (p<0.0001). Similar changes (increased plasma N/OFQ, [p=0.0058], decreased ppNOC [p<0.0001], increased IL-8 and IL-10 concentrations [both p<0.0001]) occurred after cardiac surgery but these were comparatively lower and of shorter duration. CONCLUSIONS: The N/OFQ system is modulated in ICU patients with sepsis with similar but reduced changes after cardiac surgery under cardiopulmonary bypass. Further studies are required to clarify the role of the N/OFQ system in inflammation and sepsis, and the mechanisms involved.

Cell and tissue responses of a range of Urotensin II analogs at cloned and native urotensin II receptors. Evidence for coupling promiscuity.

Urotensin II (U-II) is the peptide ligand for the G-protein-coupled U-II receptor (UT). U-II has been dubbed "the most potent vasoconstrictor identified to date". However, in vivo studies with this system are hampered by the paucity of available ligands. Here, we characterise Chinese hamster ovary (CHO) cells expressing the human UT receptor in the following assays; (1) [(125)I]U-II binding, (2) GTPgamma[(35)S] binding, (3) cAMP formation, and (4) intracellular Ca(2+). We assess activity of 9 U-II analogues using these paradigms and examine their ability to contract isolated rat aorta. CHO(hUT) cells bound [(125)I]U-II with a B (max) and K (d) of 1,110+/-70 fmol/mg protein and 742 pM, respectively. hU-II stimulated GTPgamma[(35)S] binding (pEC(50) 8.38), optimal at low (0.1 muM) GDP concentrations. The hU-II GTPgamma[(35)S] response was partially PTx sensitive and there was a potent (pEC(50) 9.23) low efficacy ( approximately 20% inhibition) coupling to adenylyl cyclase. In CHO(hUT) cells hU-II stimulates calcium release from intracellular stores (pEC(50) 8.80) and calcium influx in a PTx-insensitive manner. In our structure-activity relationship study most ligands acted as full agonists. However, urantide behaved as a partial agonist (pEC(50) 7.67/pK(B) 7.55) in GTPgamma[(35)S] binding, a full agonist (pEC(50) 8.11) for increases in intracellular Ca(2+) and a competitive antagonist in the rat aorta bioassay (pK(B) 8.59). Collectively, these data show promiscuity at high expression and indicate the need for careful multi-assay evaluation of novel U-II analogues. Further modification of urantide, in order to eliminate residual agonist activity and to identify novel ligands for in vivo cardiovascular studies are clearly warranted.

A comparison of cerebrospinal fluid and plasma urotensin II concentrations in normotensive and hypertensive patients undergoing urological surgery during spinal anesthesia: a pilot study.

UNLABELLED: Urotensin II is a novel endogenous vasoconstrictor. There are no data describing cerebrospinal fluid (CSF) concentrations in humans. Therefore, in this study, we aimed to quantify and compare plasma and CSF urotensin II concentrations in patients with essential hypertension and matched controls. Twenty male patients (10 receiving >6 mo of treatment for essential hypertension and 10 normotensive controls scheduled to undergo urological surgery under spinal anesthesia) were recruited into this single-blinded cohort study. Plasma and CSF urotensin II concentrations were measured by radioimmunoassay, along with mean arterial blood pressure (MAP), before admission, on the day of admission, and immediately before anesthesia. CSF and plasma urotensin II concentrations were low. Median (range) values in CSF for all 20 patients were significantly lower than plasma by approximately 15% (19.0 pg/mL [10.6-24.9 pg/mL] compared with 22.3 pg/mL [17.7-28.4 pg/mL]; P = 0.004). There were no significant differences between normotensive and hypertensive patients in either CSF or plasma concentrations. However, there was a significant positive correlation between average MAP and CSF urotensin II concentrations (r(2) = 0.44; P = 0.036) in the hypertensive group. IMPLICATIONS: Urotensin II is the most potent known endogenous human vasoconstrictor. In this pilot study, we report for the first time that cerebrospinal fluid levels are smaller than plasma levels and that there may be some association with increased blood pressure.