CCR5 tyrosine sulfation heterogeneity generates cell surface receptor subpopulations with different ligand binding properties.

BACKGROUND: Chemokine receptor tyrosine sulfation plays a key role in the binding of chemokines. It has been suggested that receptor sulfation is heterogeneous, but no experimental evidence has been provided so far. The potent anti-HIV chemokine analog 5P12-RANTES has been proposed to owe its inhibitory activity to a capacity to bind a larger pool of cell surface CCR5 receptors than native chemokines such as CCL5, but the molecular details underlying this phenomenon have not been elucidated. METHODS: We investigated the CCR5 sulfation heterogeneity and the sensitivity of CCR5 ligands to receptor sulfation by performing ELISA assays on synthetic N-terminal sulfopeptides and by performing binding assays on CCR5-expressing cells under conditions that modulate CCR5 sulfation levels. RESULTS: Two commonly used anti-CCR5 monoclonal antibodies with epitopes in the sulfated N-terminal domain of CCR5 show contrasting binding profiles on CCR5 sulfopeptides, incomplete competition with each other for cell surface CCR5, and opposing sensitivities to cellular treatments that affect CCR5 sulfation levels. 5P12-RANTES is less sensitive than native CCL5 to conditions that affect cellular CCR5 sulfation. CONCLUSIONS: CCR5 sulfation is heterogeneous and this affects the binding properties of both native chemokines and antibodies. Enhanced capacity to bind to CCR5 is a component of the inhibitory mechanism of 5P12-RANTES. GENERAL SIGNIFICANCE: We provide the first experimental evidence for sulfation heterogeneity of chemokine receptors and its impact on ligand binding, a phenomenon that is important both for the understanding of chemokine cell biology and for the development of drugs that target chemokine receptors.

Lidocaine inhibits cytoskeletal remodelling and human breast cancer cell migration.

BACKGROUND: The metastatic potential of breast cancer cells has been strongly associated with overexpression of the chemokine CXCL12 and the activity of its receptor CXCR4. Lidocaine, a local anaesthetic that can be used during breast cancer excision, inhibits the growth, invasion, and migration of cancer cells. We therefore investigated, in a breast cancer cell line, whether lidocaine can modulate CXCL12-induced responses. METHODS: Intracellular calcium, cytoskeleton remodelling, and cell migration were assessed in vitro in MDA-MB-231 cells, a human breast cancer epithelial cell line, after exposure to lidocaine (10 µM or 100 µM). RESULTS: Lidocaine (10 or 100 µM) significantly inhibited CXCR4 signalling, resulting in reduced calcium release (Fluo 340 nm/380 nm, 0.76 mean difference, p<0.0001), impaired cytoskeleton remodelling (F-Actin fluorescence mean intensity, 21 mean difference, P=0.002), and decreased motility of cancer cells, both in the scratch wound assay (wound area at 21 h, -19%, P<0.0001), and in chemotaxis experiments (fluorescence mean intensity, 0.16, P=0.0047). The effect of lidocaine was not associated with modulation of the CD44 adhesion molecule. CONCLUSIONS: At clinical concentrations, lidocaine significantly inhibits CXCR4 signalling. The results presented shed new insights on the molecular mechanisms governing the inhibitory effect of lidocaine on cell migration.

Prevention of arterial hypotension after spinal anaesthesia using vena cava ultrasound to guide fluid management.

BACKGROUND: Significant hypotension is frequent after spinal anaesthesia and fluid administration as therapy is usually empirical. Inferior vena cava (IVC) ultrasound (US) is effective to assess fluid responsiveness in critical care patients. The aim of this study was to evaluate the IVCUS-guided volume optimization to prevent post-spinal hypotension. METHODS: In this prospective, randomized, cohort study, 160 patients scheduled for surgery under spinal anaesthesia were randomized into a study group (IVCUS-group), consisting of an IVCUS analysis before spinal anaesthesia with IVCUS-guided volume management and a control group (group C) with no IVCUS assessment. The primary outcome was a relative risk reduction in the incidence of hypotension between the groups; secondary outcomes were the need for vasoactive drugs and the amounts of fluids required after spinal anaesthesia. We also tested the hypothesis of a correlation between IVC collapsibility index and hypotension after spinal anaesthesia. RESULTS: The relative risk reduction of hypotension between the groups was 35% (IVCUS-group 27.5%, Group C 42.5%, P=0.044, CI=95%). The need for vasoactive drugs in the IVCUS-group was significantly lower compared to the C-group (P=0.015), while the total amount of fluids was significantly superior higher in the IVCUS group (P<0.0001) compared to Group C. IVC collapsibility index was correlated with the amount of fluid administered (r2=0.32), but could not be used to predict postspinal anaesthesia hypotension. CONCLUSIONS: IVCUS is an effective method to prevent postspinal anaesthesia hypotension by IVCUS-guided fluid administration before spinal anaesthesia. CLINICAL TRIAL REGISTRATION: www.clinicaltrials.gov - NCT02271477.

Parecoxib, propacetamol, and their combination for analgesia after total hip arthroplasty: a randomized non-inferiority trial.

BACKGROUND: This study assessed non-inferiority of parecoxib vs. combination parecoxib+propacetamol and compared the opioid-sparing effects of parecoxib, propacetamol, and parecoxib+propacetamol vs. placebo after total hip arthroplasty. METHODS: In this randomized, placebo-controlled, parallel-group, non-inferiority study, patients received one of four IV treatments after surgery: parecoxib 40 mg bid (n = 72); propacetamol 2 g qid (n = 71); parecoxib 40 mg bid plus propacetamol 2 g qid (n = 72); or placebo (n = 38) with supplemental IV patient-controlled analgesia (morphine). Patients and investigators were blinded to treatment. Pain intensity at rest and with movement was assessed regularly, together with functional recovery (modified Brief Pain Inventory-Short Form) and opioid-related side effects (Opioid-Related Symptom Distress Scale) questionnaires up to 48 h. RESULTS: After 24 h, cumulative morphine consumption was reduced by 59.8% (P < 0.001), 38.9% (P < 0.001), and 26.8% (P = 0.005) in the parecoxib+propacetamol, parecoxib, and propacetamol groups, respectively, compared with placebo. Parecoxib did not meet criteria for non-inferiority to parecoxib+propacetamol. Parecoxib+propacetamol and parecoxib significantly reduced least-squares mean pain intensity scores at rest and with movement compared with propacetamol (P < 0.05). One day after surgery, parecoxib+propacetamol significantly reduced opioid-related symptom distress and decreased pain interference with function compared with propacetamol or placebo. CONCLUSION: Parecoxib and parecoxib+propacetamol provided significant opioid-sparing efficacy compared with placebo; non-inferiority of parecoxib to parecoxib+propacetamol was not demonstrated. Opioid-sparing efficacy was accompanied by significant reductions in pain intensity on movement, improved functional outcome, and less opioid-related symptom distress. Study medications were well tolerated.

Clinically relevant concentrations of lidocaine and ropivacaine inhibit TNFα-induced invasion of lung adenocarcinoma cells in vitro by blocking the activation of Akt and focal adhesion kinase.

BACKGROUND: Matrix-metalloproteinases (MMP) and cancer cell invasion are crucial for solid tumour metastasis. Important signalling events triggered by inflammatory cytokines, such as tumour necrosis factor α (TNFα), include Src-kinase-dependent activation of Akt and focal adhesion kinase (FAK) and phosphorylation of caveolin-1. Based on previous studies where we demonstrated amide-type local anaesthetics block TNFα-induced Src activation in malignant cells, we hypothesized that local anaesthetics might also inhibit the activation and/or phosphorylation of Akt, FAK and caveolin-1, thus attenuating MMP release and invasion of malignant cells. METHODS: NCI-H838 lung adenocarcinoma cells were incubated with ropivacaine or lidocaine (1 nM-100 µM) in absence/presence of TNFα (20 ng ml(-1)) for 20 min or 4 h, respectively. Activation/phosphorylation of Akt, FAK and caveolin-1 were evaluated by Western blot, and MMP-9 secretion was determined by enzyme-linked immunosorbent assay. Tumour cell migration (electrical wound-healing assay) and invasion were also assessed. RESULTS: Ropivacaine (1 nM-100 µM) and lidocaine (1-100 µM) significantly reduced TNFα-induced activation/phosphorylation of Akt, FAK and caveolin-1 in NCI-H838 cells. MMP-9 secretion triggered by TNFα was significantly attenuated by both lidocaine and ropivacaine (half-maximal inhibitory concentration [IC50]=3.29×10(-6) M for lidocaine; IC50=1.52×10(-10) M for ropivacaine). The TNFα-induced increase in invasion was completely blocked by both lidocaine (10 µM) and ropivacaine (1 µM). CONCLUSIONS: At clinically relevant concentrations both ropivacaine and lidocaine blocked tumour cell invasion and MMP-9 secretion by attenuating Src-dependent inflammatory signalling events. Although determined entirely in vitro, these findings provide significant insight into the potential mechanism by which local anaesthetics might diminish metastasis.

The effect of continuous popliteal sciatic nerve block on unplanned postoperative visits and readmissions after foot surgery--a randomised, controlled study comparing day-care and inpatient management.

Regional anaesthesia has been shown to have several advantages over general anaesthesia in reducing the need for, and hence cost of, unscheduled outpatient visits or readmission to hospital. However, the benefit has not been evaluated in a direct comparison between day-care patients and inpatients. We randomly allocated 120 patients undergoing unilateral foot surgery to either inpatient (two-day postoperative stay) or day-care management under continuous regional anaesthesia, and compared the impact on unscheduled postoperative outpatient visits, readmissions to hospital and the associated costs. The operations were performed under popliteal sciatic nerve block. A perineural catheter was inserted before surgery and removed from all patients on the third postoperative day. We found no significant difference in the incidence of outpatient visits (3.3% day-care vs 5.0% inpatient, p = 0.640), readmissions (6.7% day-care vs 3.3% inpatient, p = 0.395) or complications between the two groups. Costs were also significantly lower in the day-care group (net difference €8011 (£6684; $10 986) per patient, p < 0.001). We conclude that continuous regional anaesthesia allows foot surgery to be performed as a day-care procedure more cheaply than in inpatients, without an increase in clinical complications.

Cerebral oxygenation in patients undergoing shoulder surgery in beach chair position: comparing general to regional anesthesia and the impact on neurobehavioral outcome.

BACKGROUND: Ischemic brain damage has been reported in healthy patients after beach chair position for surgery due to cerebral hypoperfusion. Near-infrared spectroscopy has been described as a non-invasive, continuous method to monitor cerebral oxygen saturation. However, its impact on neurobehavioral outcome comparing different anesthesia regimens has been poorly described. METHODS: In this prospective, assessor-blinded study, 90 patients undergoing shoulder surgery in beach chair position following general (G-group, n=45) or regional anesthesia (R-group; n=45) were enrolled to assess the prevalence of cerebral desaturation events comparing anesthesia regimens and their impact on neurobehavioral and neurological outcome. Anesthesiologists were blinded to regional cerebral oxygen saturation values. Baseline data assessed the day before surgery included neurological and neurobehavioral tests, which were repeated the day after surgery. The baseline data for regional cerebral oxygen saturation/bispectral index and invasive blood pressure both at heart and auditory meatus levels were taken prior to anesthesia, 5 min after induction of anesthesia, 5 min after beach chair positioning, after skin incision and thereafter all 20 min until discharge. RESULTS: Patients in the R-group showed significantly less cerebral desaturation events (p<0.001), drops in regional cerebral oxygen saturation values (p<0.001), significantly better neurobehavioral test results the day after surgery (p<0.001) and showed a greater hemodynamic stability in the beach chair position compared to patients in the G-group. CONCLUSIONS: The incidence of regional cerebral oxygen desaturations seems to influence the neurobehavioral outcome. Regional anesthesia offers more stable cardiovascular conditions for shoulder surgery in beach chair position influencing neurobehavioral test results at 24h.

Regional anaesthesia and cancer metastases: the implication of local anaesthetics.

Clinical and basic science studies have demonstrated the anti-inflammatory properties of local anaesthetics. Recent studies have begun to unravel molecular pathways linking inflammation and cancer. Regional anaesthesia is associated in some retrospective clinical studies with reduced risk of metastasis and increased long-term survival. The potential beneficial effects of regional anaesthesia have been attributed mainly to the inhibition of the neuroendocrine stress response to surgery and to the reduction in the requirements of volatile anaesthetics and opioids. Because cancer is linked to inflammation and local anaesthetics have anti-inflammatory effects, these agents may participate in reducing the risk of metastasis, but their mechanism of action is unknown. We demonstrated in vitro that amide local anaesthetics attenuate tumour cell migration as well as signalling pathways enhancing tumour growth and metastasis. This has provided the first evidence of a molecular mechanism by which regional anaesthesia might inhibit or reduce cancer metastases.

Deltoid, triceps, or both responses improve the success rate of the interscalene catheter surgical block compared with the biceps response.

BACKGROUND: The influence of the muscular response elicited by neurostimulation on the success rate of interscalene block using a catheter (ISC) is unknown. In this investigation, we compared the success rate of ISC placement as indicated by biceps or deltoid, triceps, or both twitches. METHODS: Three hundred (ASA I-II) patients presenting for elective arthroscopic rotator cuff repair were prospectively randomized to assessment by biceps (Group B) or deltoid, triceps, or both twitches (Group DT). All ISCs were placed with the aid of neurostimulation. The tip of the stimulating needle was placed after disappearance of either biceps or deltoid, triceps, or both twitches at 0.3 mA. The catheter was advanced 2-3 cm past the tip of the needle and the block was performed using 40 ml ropivacaine 0.5%. Successful block was defined as sensory block of the supraclavicular nerve and sensory and motor block involving the axillary, radial, median, and musculocutaneous nerves within 30 min. RESULTS: Success rate was 98.6% in Group DT compared with 92.5% in Group B (95% confidence interval 0.01-0.11; P<0.02). Supplemental analgesics during handling of the posterior part of the shoulder capsule were needed in two patients in Group DT and seven patients in Group B. Three patients in Group B had an incomplete radial nerve distribution anaesthesia necessitating general anaesthesia. One patient in Group B had an incomplete posterior block extension of the supraclavicular nerve. No acute or late complications were observed. CONCLUSIONS: Eliciting deltoid, triceps, or both twitches was associated with a higher success rate compared with eliciting biceps twitches during continuous interscalene block.

Histological Consequences of Needle-Nerve Contact following Nerve Stimulation in a Pig Model.

Background. Nerve stimulation can facilitate correct needle placement in peripheral regional anesthesia. The aim of this study was to determine whether the high threshold current is associated with reduced nerve injury due to fewer needle-nerve contacts compared with low current. Methods. In anaesthetized pigs, thirty-two nerves of the brachial plexus underwent needle placement at low (0.2 mA) or high current (1.0 mA). The occurrence of needle-nerve contact was recorded. After 48 hours, the nerves were analyzed for occurrence of histological changes. Nerve injury was scored ranging from 0 (no injury) to 4 (severe injury). Results. The frequency of needle-nerve contact was 94% at low compared to 6% at high current. The score was significantly higher at low (median [interquartile range] 2.0 [1.0-2.0]) compared to high current (0.0 [0.0-1.0] P = .001). Conclusions. Inflammatory responses were directly related to needle-nerve contacts. Hence, posttraumatic inflammation may be diminished using higher current for nerve localization.

Additional femoral catheter in combination with popliteal catheter for analgesia after major ankle surgery.

BACKGROUND: The contribution of the saphenous nerve in pain after major ankle surgery is unknown. The aim of this study was to evaluate its contribution in this context. METHODS: Fifty patients were included in this prospective, randomized, controlled study. In all patients [Group P (popliteal) and Group F (popliteal+femoral)], a popliteal catheter was placed before operation and ropivacaine 0.5% (30 ml) administered via this catheter; major ankle surgery was then performed under spinal anaesthesia. In Group PF patients, an additional femoral catheter was sited before operation and ropivacaine 0.5% (10 ml) administered. Six hours after spinal anaesthesia (defined as T(0)), a continuous infusion of ropivacaine 0.3% (14 ml h(-1)) was started through the popliteal catheter until T(24). Then, the concentration was reduced to 0.2% until T(48). Patients in Group PF received continuous ropivacaine 0.2% (5 ml h(-1)) through the femoral catheter from T(0) to T(48). I.V. morphine patient-controlled analgesia was used as a rescue analgesia. Pain at rest, pain with movement, adverse effects, and i.v. morphine consumption were assessed. Pain at rest and on movement was evaluated 6 months after operation. RESULTS: Pain at rest was comparable in the two groups. In Group PF, patients had significantly reduced pain during movement in the postoperative period (P=0.01) and 6 months after operation (P=0.03). Morphine consumption was significantly reduced in Group PF at T(0)-T(24) and T(24)-T(48) (P=0.01). Adverse effects were comparable in both groups. CONCLUSIONS: The addition of continuous femoral catheter infusion of ropivacaine to a continuous popliteal catheter infusion improved postoperative analgesia during movement after major ankle surgery. This effect was still present 6 months after surgery.

In vitro exposure of human fibroblasts to local anaesthetics impairs cell growth.

Lidocaine, bupivacaine or ropivacaine are used routinely to manage perioperative pain. Sparse data exist evaluating the effects of local anaesthetics (LA) on fibroblasts, which are involved actively in wound healing. Therefore, we investigated the effects of the three LA to assess the survival, viability and proliferation rate of fibroblasts. Human fibroblasts were exposed to 0·3 mg/ml and 0·6 mg/ml of each LA for 2 days, followed by incubation with normal medium for another 1, 4 or 7 days (group 1). Alternatively, cells were incubated permanently with LA for 3, 6 or 9 days (group 2). Live cell count was assessed using trypan blue staining. Viability was measured by the tetrazolium bromide assay. Proliferation tests were performed with the help of the colorimetric bromodeoxyuridine assay. Production of reactive oxygen species (ROS) was determined, measuring the oxidation of non-fluorescent-2,7'-dichlorofluorescin. Treatment of cells with the three LA showed a concentration-dependent decrease of live cells, mitochondrial activity and proliferation rate. Group arrangement played a significant role for cell count and proliferation, while exposure time influenced viability. Among the analysed LA, bupivacaine showed the most severe cytotoxic effects. Increased production of ROS correlated with decreased viability of fibroblasts in lidocaine- and bupivacaine-exposed cells, but not upon stimulation with ropivacaine. This study shows a concentration-dependent cytotoxic effect of lidocaine, bupivacaine and ropivacaine on fibroblasts in vitro, with more pronounced effects after continuous incubation. A possible mechanism of cell impairment could be triggered by production of ROS upon stimulation with lidocaine and bupivacaine.