Markus F. Stevens

Apoptosis induction by different local anaesthetics in a neuroblastoma cell line.

BACKGROUND Local anaesthetics are known to induce apoptosis in clinically relevant concentrations. Hitherto, it is unknown what determines the apoptotic potency of local anaesthetics. Therefore, we compared apoptosis induction by local anaesthetics related to their physicochemical properties in human neuronal cells. METHODS Neuroblastoma cells (SHEP) were incubated with eight local anaesthetics, two of the ester and six of the amide types. At least, five concentrations of each local anaesthetic were evaluated. After incubation for 24 h, rates of cells in early apoptotic stages and overall cell death were evaluated by annexin V and 7-amino-actinomycin D double staining by flow cytometry. The concentrations that led to half-maximal neurotoxic effects (LD50) were calculated and compared for all local anaesthetics. RESULTS All local anaesthetics were neurotoxic in a concentration-dependent manner. All drugs induced similar rates of early apoptotic cell formation at low concentrations, whereas at high concentrations, late apoptotic or necrotic cell death predominated. Comparison of LD50 values of the different local anaesthetics resulted in the following order of apoptotic potency from high to low toxicity: tetracaine>bupivacaine>prilocaine=mepivacaine=ropivacaine>lidocaine>procaine=articaine. The toxicity correlated with octanol/buffer coefficients and also with experimental potency of the local anaesthetic, but was unrelated to the structure (ester or amide type). CONCLUSIONS All commonly used local anaesthetics induce neuronal apoptosis in clinically used concentrations. The neurotoxicity correlates with lipid solubility and thus with the conduction blocking potency of the local anaesthetic, but is independent of the chemical class (ester/amide).

Failed epidural: causes and management

Failed epidural anaesthesia or analgesia is more frequent than generally recognized. We review the factors known to influence the success rate of epidural anaesthesia. Reasons for an inadequate epidural block include incorrect primary placement, secondary migration of a catheter after correct placement, and suboptimal dosing of local anaesthetic drugs. For catheter placement, the loss of resistance using saline has become the most widely used method. Patient positioning, the use of a midline or paramedian approach, and the method used for catheter fixation can all influence the success rate. When using equipotent doses, the difference in clinical effect between bupivacaine and the newer isoforms levobupivacaine and ropivacaine appears minimal. With continuous infusion, dose is the primary determinant of epidural anaesthesia quality, with volume and concentration playing a lesser role. Addition of adjuvants, especially opioids and epinephrine, may substantially increase the success rate of epidural analgesia. Adjuvant opioids may have a spinal or supraspinal action. The use of patient-controlled epidural analgesia with background infusion appears to be the best method for postoperative analgesia.

Ketamine induces apoptosis via the mitochondrial pathway in human lymphocytes and neuronal cells

BACKGROUND Ketamine has been shown to have neurotoxic properties, when administered neuraxially. The mechanism of this local toxicity is still unknown. Therefore, we investigated the mechanism of cytotoxicity in different human cell lines in vitro. METHODS We incubated the following cell types for 24 h with increasing concentrations of S(+)-ketamine and racemic ketamine: (i) human Jurkat T-lymphoma cells overexpressing the antiapoptotic B-cell lymphoma 2 protein, (ii) cells deficient of caspase-9, caspase-8, or Fas-associated protein with death domain and parental cells, and (iii) neuroblastoma cells (SHEP). N-Methyl-d-aspartate (NMDA) receptors and caspase-3 cleavage were identified by immunoblotting. Cell viability and apoptotic cell death were evaluated flowcytometrically by Annexin V and 7-aminoactinomycin D double staining. Mitochondrial metabolic activity and caspase-3 activation were measured. RESULTS Ketamine, in a concentration-dependent manner, induced apoptosis in lymphocytes and neuroblastoma cell lines. Cell lines with alterations of the mitochondrial pathway of apoptosis were protected against ketamine-induced apoptosis, whereas alterations of the death receptor pathway did not reduce apoptosis. S(+)-Ketamine and racemic ketamine induced the same percentage of cell death in Jurkat cells, whereas in neuroblastoma cells, S(+)-ketamine was slightly less toxic. CONCLUSIONS Ketamine at millimolar concentrations induces apoptosis via the mitochondrial pathway, independent of death receptor signalling. At higher concentrations necrosis is the predominant mechanism. Less toxicity of S(+)-ketamine was observed in neuroblastoma cells, but this difference was minor and therefore unlikely to be mediated via the NMDA receptor.

Value of single-injection or continuous sciatic nerve block in addition to a continuous femoral nerve block in patients undergoing total knee arthroplasty: a prospective, randomized, controlled trial

Background and Objectives: Continuous femoral nerve block in patients undergoing total knee arthroplasty (TKA) improves and shortens postoperative rehabilitation. The primary aim of this study was to investigate whether the addition of sciatic nerve block to continuous femoral nerve block will shorten the time-to-discharge readiness. Methods: Ninety patients undergoing TKA were prospectively randomized to 1 of 3 groups: patient-controlled analgesia via femoral nerve catheter alone (F group) or combined with a single-injection (Fs group) or continuous sciatic nerve block (FCS group) until the second postoperative day. Discharge readiness was defined as the ability to walk and climb stairs independently, average pain on a numerical rating scale at rest lower than 4, and no complications. In addition, knee function, pain, supplemental morphine requirement, local anesthetic consumption, and postoperative nausea and vomiting (PONV) were evaluated. Results: Median time-to-discharge readiness was similar: F group, 4 days (range, 2-16 days); Fs group, 4 days (range, 2-7 days); and FCS group, 4 days (range, 2-9 days; P = 0.631). No significant differences were found regarding knee function, local anesthetic consumption, or postoperative nausea and vomiting. During the day of surgery, pain was moderate to severe in the F group, whereas Fs and FCS groups experienced minimal pain (P < 0.01). Patients in the F group required significantly more supplemental morphine on the day of surgery and the first postoperative day. Until the second postoperative day, pain was significantly less in the FCS group (P < 0.01). Conclusions: A single-injection or continuous sciatic nerve block in addition to a femoral nerve block did not influence time-to-discharge readiness. A single-injection sciatic nerve block can reduce severe pain on the day of the surgery, whereas a continuous sciatic nerve block reduces moderate pain during mobilization on the first 2 postoperative days.

Does interscalene catheter placement with stimulating catheters improve postoperative pain or functional outcome after shoulder surgery? A prospective, randomized and double-blinded trial

BACKGROUND: In this prospective, randomized, double-blind trial we investigated the use of stimulating catheters in patients during and after shoulder surgery; functional improvement being the primary outcome measurement. METHODS: After eliciting an adequate muscular twitch at ≤0.5 mA nerve stimulation output, the perineural catheter was advanced either blindly (conventional catheter = CC group, n = 20) or guided by stimulation via the catheter (stimulating catheter = SC group, n = 20). A bolus of 40 mL prilocaine 1% and 10 mL ropivacaine 0.75% was injected, followed by a patient-controlled infusion of ropivacaine 0.2% (8 mL/h infusion rate, bolus 2 mL, lockout time 20 min). RESULTS: Onset of motor block was faster in the SC group, whereas sensory block did not differ between groups. Median pain scores on two postoperative days were equal. Improvement of the objective shoulder function score (Constant Murley Score) 6 wk postoperatively was enhanced to a clinically relevant extent in the SC group compared to the CC group (P < 0.01). CONCLUSIONS: We conclude that the use of a stimulating catheter results in a faster onset of motor block, unaltered postoperative pain, and a significantly improved functional outcome 6 wk after shoulder surgery.

Skin Temperature After Interscalene Brachial Plexus Blockade

Background and Objectives: In neuraxial anesthesia, increase of skin temperature is an early sign of successful block. Yet, during peripheral nerve block of the lower extremity, increase in skin temperature is a highly sensitive, but late sign of a successful block. We hypothesized that after interscalene brachial plexus block, a rise in skin temperature follows impairment of sensation during successful nerve block and occurs only distally, as observed in the lower extremity. Methods: In the present study, we prospectively evaluated the changes in skin temperature after interscalene brachial plexus blockade in 45 patients scheduled for elective shoulder surgery. We assessed pinprick and cold sensation as well as skin temperature at sites of the skin innervated by the median, ulnar, radial, axillary and musculocutaneous nerve. Results: At the skin areas innervated by the axillary and musculocutaneous nerve, skin temperature did not increase after successful block. At the distal sites, innervated by the median, ulnar, and radial nerve, skin temperature increased significantly (1.9-2.1°C within 30 min) after successful block while it did not after failed nerve block or on the contralateral side. In these areas attenuation of skin sensation preceded a measurable rise in skin temperature (≥1°C) in 56.3% of nerve blocks, occurred at the same time in 35.2%, and in 8.5% the temperature rise occurred first. Conclusions: Assessment of skin temperature cannot predict the success of an interscalene brachial plexus block of the axillary and musculocutaneous nerve. Distally, the increase of skin temperature has a high sensitivity and specificity but occurs later than the loss of sensory and motor functions. Therefore, the measurement of skin temperature during interscalene blockade is of limited clinical value.

Skin temperature during regional anesthesia of the lower extremity

Increase in skin temperature (Ts) occurs early during neuraxial blocks. However, the reliability of Ts to predict successful peripheral block is unknown. Therefore, we investigated whether an increase in Ts more than 1°C precedes or follows an impairment of sensation after combined femoral and sciatic nerve block as well as after epidural anesthesia. In this prospective, nonrandomized study we determined Ts changes in 33 patients undergoing knee or foot surgery under femoral and sciatic nerve block and 10 patients undergoing epidural anesthesia. Perception and motor function were assessed every 5 min. An increase in Ts (≥1°C) at the foot occurred later after sciatic nerve block than after epidural anesthesia (10.3 ± 2.8 versus 5.0 min; P < 0.01). Alterations of Ts at skin innervated by the femoral nerve were <1°C. Ts increase preceded sensory block after sciatic nerve block in 6.6% of patients but indicated a successful block (sensitivity, specificity, and accuracy = 100%). We conclude that an increase of Ts is a reliable, but late, sign of successful sciatic nerve block. Therefore it is of limited clinical value. Ts changes after femoral nerve block are negligible and late.

Midazolam activates the intrinsic pathway of apoptosis independent of benzodiazepine and death receptor signaling.

Background and Objectives: Midazolam has neurotoxic properties when administered neuraxially in vivo. Furthermore, midazolam induces neurodegeneration in neonatal animal models in combination with other general anesthetics. Therefore, this study focuses on the mechanism of neurotoxicity by midazolam in neuronal and nonneuronal cells. The study aims to evaluate the apoptotic pathway and to investigate the protective effects of the benzodiazepine antagonist flumazenil and the caspase inhibitor N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)-methylketone. Methods: The apoptosis-inducing effect of preservative-free midazolam on human lymphoma and neuroblastoma cell lines was evaluated using flow cytometric analysis of early apoptotic stages (annexin V/7AAD) and caspase 3 activation. B-cell lymphoma (Bcl2) protein overexpressing and caspase 9-deficient lymphoma cells were used to determine the role of the mitochondrial (intrinsic) pathway. Caspase 8-deficient and Fas-associated protein with death domain (FADD)-deficient cells were used to evaluate the death receptor (extrinsic) pathway. The protective effects of flumazenil and the caspase inhibitor N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)-methylketone were investigated in neuroblastoma cells and primary rat neurons using metabolic activity assays (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and immunofluorescence microscopy. Results: Midazolam induced apoptosis in all investigated cell types in a concentration-dependent manner, indicated by flow cytometry. Bcl2-overexpression and caspase 9 deficiency protected against toxicity, whereas caspase 8 or FADD deficiency had no effect. Pancaspase inhibition had a strong protective effect, whereas flumazenil did not inhibit midazolam-induced apoptosis. Conclusions: Midazolam induces apoptosis via activation of the mitochondrial pathway in a concentration-dependent manner. The mechanism of midazolam toxicity switches from caspase-dependent apoptosis to necrosis with increasing concentrations. The induction of apoptosis and necrosis by midazolam is presumably unrelated to GABAA receptor pathway signaling.

The Influence of Adjuvants Used in Regional Anesthesia on Lidocaine-Induced Neurotoxicity In Vitro

Background: Neurotoxic properties of local anesthetics can rarely lead to irreversible neuronal damage as in cauda equina syndrome. Clinically, local anesthetics are often combined with adjuvants to improve or prolong the anesthetic effect, whereas the impact of such adjuvants on lidocaine-induced apoptosis is unclear. Therefore, we investigated the influence of different adjuvants on the neurotoxicity of lidocaine. Methods: Human neuroblastoma cells and primary rat astrocytes were incubated for 24 hrs with lidocaine at a toxic concentration alone and in combination with morphine, sufentanil, clonidine, epinephrine, neostigmine, ketamine, and midazolam. Subsequently, the rates of cell death and early apoptosis were measured by flow cytometry in neuroblastoma cells, whereas astrocyte viability was analyzed by mitochondrial activity assay. In addition, isobolograms were calculated to describe the additive effects of lidocaine with ketamine or midazolam, respectively. Results: Coadministration of lidocaine with sufentanil, clonidine, epinephrine, and neostigmine did not alter the rates of cell death compared with cells treated with lidocaine alone. Morphine improved the viability of astrocytes only at concentrations beyond those occurring clinically. In contrast, coincubation of lidocaine with ketamine or midazolam led to significantly increased rates of cell death. The combined toxicity of ketamine and lidocaine was additive, whereas the combined toxicity of midazolam and lidocaine was subadditive. Conclusions: Sufentanil, clonidine, epinephrine, and neostigmine do not influence the neurotoxicity of lidocaine in vitro. Morphine may have some cytoprotective effect at concentrations greater than those seen intrathecally in humans. In contrast, ketamine and midazolam increase the neurotoxicity of lidocaine in vitro, presumably by additive induction of mitochondrial apoptosis.

Long-term pain and functional disability after total knee arthroplasty with and without single-injection or continuous sciatic nerve block in addition to continuous femoral nerve block: a prospective, 1-year follow-up of a randomized controlled trial.

Background and Objectives This is a follow-up to determine long-term outcomes after total knee arthroplasty (TKA) in patients enrolled in a previous randomized trial that found reduced postoperative pain after addition of sciatic nerve block to continuous femoral nerve block for TKA. Methods Physical function after TKA was evaluated at 3 and 12 months in patients (n = 89) receiving continuous femoral nerve block alone (group F), combined with a single-injection (group Fs) or continuous sciatic nerve block (group FCS) after TKA, until the second postoperative day. Physical function, stiffness, and pain were measured by using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Oxford Knee Score 12-item knee questionnaires, and visual analog scale at rest and during mobilization before TKA and 3 and 12 months afterward. Post hoc, a median split on poor functioning (WOMAC) was analyzed. Results Western Ontario and McMaster Universities Osteoarthritis Index, Oxford Knee Score 12-item knee, and visual analog scale scores improved significantly in all patients, without any differences among groups. Median (range) WOMAC at 3 months were in group F, 83 (20–97); group Fs, 72 (25–99); and group, FCS 76 (28–100) and at 12 months 87 (35–98), 77 (43–100), and 89 (35–100), respectively. Conclusions No differences were detected in the secondary outcomes we examined. Thus, improved postoperative outcome did not translate into improved functional outcome or long-term pain.