4-bromopropofol decreases action potential generation in spinal neurons by inducing a glycine receptor-mediated tonic conductance.

BACKGROUND AND PURPOSE: Impaired function of spinal strychnine-sensitive glycine receptors gives rise to chronic pain states and movement disorders. Therefore, increased activity of glycine receptors should help to treat such disorders. Although compounds targeting glycine receptors with a high selectivity are lacking, halogenated analogues of propofol have recently been considered as potential candidates. Therefore we asked whether 4-bromopropofol attenuated the excitability of spinal neurons by promoting glycine receptor-dependent inhibition. EXPERIMENTAL APPROACH: The actions of sub-anaesthetic concentrations of propofol and 4-bromopropofol were investigated in spinal tissue cultures prepared from mice. Drug-induced alterations in action potential firing were monitored by extracellular multi-unit recordings. The effects on GABAA and glycine receptor-mediated inhibition were quantified by whole-cell voltage-clamp recordings. KEY RESULTS: Low concentrations of 4-bromopropofol (50 nM) reduced action potential activity of ventral horn neurons by about 30%, compared with sham-treated slices. This effect was completely abolished by strychnine (1 µM). In voltage-clamped neurons, 4-bromopropofol activated glycine receptors, generating a tonic current of 65 ± 10 pA, while GABAA - and glycine receptor-mediated synaptic transmission remained unaffected. CONCLUSIONS AND IMPLICATIONS: The highest glycine levels in the CNS are found in the ventral horn of the spinal cord, a region mediating pain-induced motor reflexes and participating in the control of muscle tone. 4-Bromopropofol may serve as a starting point for the development of non-sedative, non-addictive, muscle relaxants and analgesics to be used to treat low back pain.

Plasma endotoxin in horses presented to an equine referral hospital: correlation to selected clinical parameters and outcomes.

REASONS FOR PERFORMING STUDY: Endotoxaemia is frequently presumed on the basis of clinical signs in horses with colic. OBJECTIVE: Measurements of plasma endotoxin (LPS) are rarely made in clinical cases and there is little information on the correlations between this variable, clinical variables and outcomes. OBJECTIVES: To measure LPS levels in plasma of horses presented to the Philip Leverhulme Equine Hospital on admission and daily for up to 4 days and to relate LPS levels to selected clinical parameters, such as heart rate and packed cell volume, and outcomes. METHODS: Blood samples were collected and stored at -20°C prior to assay of the plasma using a validated kinetic chromogenic Limulus amoebocyte lysate (LAL) assay. Clinical parameters and outcome variables were collected from hospital records. Associations were determined by Chi-squared test and logistic regression analysis. RESULTS: Daily blood samples were collected from 234 horses. LPS was detected in 26.5% of the study population and in 29% of those horses presented for colic. Horses providing samples with detectable LPS were more likely to die whilst in the hospital than those that did not (P = 0.045). Horses presenting with colic were more likely to have detectable LPS in their plasma than noncolic cases (P = 0.037), although LPS was detected in the plasma of 8 out of 42 noncolic horses. A horse that did not meet the study definition of clinical endotoxaemia was 10 times less likely to provide a positive LPS sample (OR 0.10, 95% CI: 0.05-0.22). CONCLUSIONS: The proportion of horses providing samples with detectable LPS was similar to other studies. POTENTIAL RELEVANCE: LPS was detected in the minority of horses presented with colic. Increased levels of LPS positively correlated with packed cell volume and with risk of mortality in colic cases.

High-affinity blockade of voltage-operated skeletal muscle and neuronal sodium channels by halogenated propofol analogues.

BACKGROUND AND PURPOSE: Voltage-operated sodium channels constitute major target sites for local anaesthetic-like action. The clinical use of local anaesthetics is still limited by severe side effects, in particular, arrhythmias and convulsions. These side effects render the search for new local anaesthetics a matter of high interest. EXPERIMENTAL APPROACH: We have investigated the effects of three halogenated structural analogues of propofol on voltage-operated human skeletal muscle sodium channels (Na(V)1.4) and the effect of one compound (4-chloropropofol) on neuronal sodium channels (Na(V)1.2) heterologously expressed in human embryonic kidney cell line 293. KEY RESULTS: 4-Iodo-, 4-bromo- and 4-chloropropofol reversibly suppressed depolarization-induced whole-cell sodium inward currents with high potency. The IC(50) for block of resting channels at -150 mV was 2.3, 3.9 and 11.3 microM in Na(V)1.4, respectively, and 29.2 microM for 4-chloropropofol in Na(V)1.2. Membrane depolarization inducing inactivation strongly increased the blocking potency of all compounds. Estimated affinities for the fast-inactivated channel state were 81 nM, 312 nM and 227 nM for 4-iodopropofol, 4-bromopropofol and 4-chloropropofol in Na(V)1.4, and 450 nM for 4-chloropropofol in Na(V)1.2. Recovery from fast inactivation was prolonged in the presence of drug leading to an accumulation of block during repetitive stimulation at high frequencies (100 Hz). CONCLUSIONS AND IMPLICATIONS: Halogenated propofol analogues constitute a novel class of sodium channel-blocking drugs possessing almost 100-fold higher potency compared with the local anaesthetic and anti-arrhythmic drug lidocaine. Preferential drug binding to inactivated channel states suggests that halogenated propofol analogues might be especially effective in suppressing ectopic discharges in a variety of pathological conditions.

Pre-operative and anaesthesia-related risk factors for mortality in equine colic cases.

Mortality rates for horses that have undergone emergency abdominal surgery are higher than for other procedures. Here, multivariable modelling of data from 774 surgical colic cases is used to identify pre-operative and anaesthesia-related variables associated with intra- and post-operative mortality. Intra-operative mortality was significantly (P<0.05), and positively associated with heart rate and packed cell volume (PCV) at admission, and negatively associated with the severity of pain. Post-operative mortality increased with increasing age and PCV at admission. Draught horses, Thoroughbreds and Thoroughbred-cross horses carried a significantly worse prognosis. We detected a small but significant variability in the risk of intra-operative death amongst referring veterinary surgeons. Different anaesthetic induction agents, inhalation maintenance agents and the use, or not, of intermittent positive pressure ventilation had no significant effect on risk of death. We conclude that cardiovascular compromise, level of pain, age, and breed are all associated with the risk of mortality in equine surgical colic cases.

High-affinity blockade of voltage-operated skeletal muscle sodium channels by 2,6-dimethyl-4-chlorophenol.

BACKGROUND AND OBJECTIVE: The aromatic alcohol most closely resembling the aromatic tail of lidocaine is 2,6-dimethylphenol. This agent is as potent as lidocaine in blocking voltage-operated sodium channels. The aim of this study was to show the effect of halogenation in the para-position on the potency of this compound to block voltage-operated sodium channels. METHODS: Insertion of the halogen chloride into the para-position of the molecule 2,6-dimethylphenol yielded 2,6-dimethyl-4-chlorophenol. Block of sodium currents by this compound was studied using heterologously expressed voltage-operated rat neuronal (rat IIa) sodium channels. RESULTS: 2,6-dimethyl-4-chlorophenol reversibly suppressed depolarization-induced whole-cell sodium inward currents. The ECR50 for block of resting channels at a hyperpolarized holding potential (-150 mV) was 127 micromol, the Hill coefficient nH 1.7. Membrane depolarization inducing either fast or slow-inactivation strongly increased the blocking potency. This is an important feature of a local-anaesthetic-like action. The estimated half-maximum effect concentration for the fast-inactivated channel state ECI50 was 28 micromol, the Hill coefficient nH 3.8. When 20-30% of channels were slow-inactivated using long (2.5 s) prepulses, followed by a 10 ms repolarization period to allow recovery from fast inactivation, the IC50 at -100 mV holding potential was reduced to 53 micromol. CONCLUSION: These results, which show that 2,6-dimethyl-4-chlorophenol blocks voltage-operated sodium channels in a lidocaine-like manner while having a several fold higher potency than the non-halogenated parent compound, highlight a potentially meaningful principle of increasing the sodium channel blocking potency of phenol derivatives.

Terminal complement complex in septic shock with capillary leakage: marker of complement activation?

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the value of terminal complement complex (C5b-9) plasma levels as a marker for complement activation in septic shock with concomitant capillary leak syndrome. METHODS: In a prospective animal study 10 fasted, anaesthetized, mechanically ventilated and multi-catheterized pigs (20.6 +/- 1.3 kg) were investigated over a period of 8 h. Sepsis was induced by faecal peritonitis (1 g kg(-1) body weight faeces, n = 5) and compared to controls (n = 5). The animals received 6% hydroxyethyl starch 200/0.5 to maintain a central venous pressure of 12 mmHg. To quantify capillary leak syndrome, albumin escape rate was measured using 99mTc-labelled human serum albumin. Plasma levels of terminal complement complex were measured in a double antibody immunoassay (neoepitope-specific MoAb aE 11 as catching antibody). Immunohistological studies of renal specimens were performed to detect terminal complement complex deposition. RESULTS: Albumen escape rate increased in septic animals (+ 52%) compared to controls (+ 3%, P < 0.05). Plasma levels of terminal complement complex decreased during the study period in both groups. In septic animals this finding was accompanied by a significant deposition of terminal complement complex in renal specimens (P < 0.05). CONCLUSION: We found an activation of the complement system proven by marked deposition of terminal complement complex in renal specimen, while its plasma levels decreased during the study period in septic and control animals. These results suggest that in septic shock with capillary leak syndrome plasma level of terminal complement complex may not be a reliable marker of complement activation.

Comparison of cardiac output measurements by arterial trans-cardiopulmonary and pulmonary arterial thermodilution with direct Fick in septic shock.

BACKGROUND AND OBJECTIVE: The aim of this study was to compare cardiac output (CO) measurements by arterial trans-cardiopulmonary thermodilution (ATD) and pulmonary arterial thermodilution (PATD) with CO estimated on the basis of the Fick calculation via a metabolic monitor in septic shock. METHODS: In a prospective animal study 20 anaesthetized, ventilated pigs (20.9 +/- 1.9 kg) were investigated. Septic shock was induced with faecal peritonitis. A pulmonary artery catheter was used for conventional measurement of CO. Simultaneously ATD was measured with a thermistor tipped catheter inserted into right carotid artery. Whole body oxygen consumption was measured by indirect calorimetry. Eighty data pairs of simultaneous CO measurements were analysed. RESULTS: CO measured with Fick and that measured with PATD were significantly correlated (r = 0.94, r = 0.87, P < 0.001). Mean CO measured by PATD was 94.3 +/- 40.1 mL min(-1) kg(-1). Bias was 10.1 mL min(-1) kg(-1) (95% confidence interval (CI): 6.0-14.2 mL min(-1) kg(-1)) with limits of agreement of -26.8 to 47.0 mL min(-1) kg(-1). Correlation between Fick derived CO estimation and ATD CO was similar (r = 0.91, r2 = 0.83, P < 0.001). Mean CO measured by trans-cardiopulmonary thermodilution was 104.3 +/- 43.2 mL min(-1) kg(-1). Bias was 0.75 mL min(-1) kg(-1) (95% CI: -3.8 to 5.3 mL min(-1) kg(-1)) with limits of agreement of -39.7 to 41.2 mL min(-1) kg(-1). CONCLUSIONS: Even during haemodynamic instability in septic shock the correlation of arterial trans-cardiopulmonary thermodilution and PATD derived CO with direct Fick was good. As arterial trans-cardiopulmonary thermodilution is less invasive than PATD, the former may offer practical advantages.

Ringer's solution but not hydroxyethyl starch or modified fluid gelatin enhances platelet microvesicle formation in a porcine model of septic shock.

BACKGROUND: Sepsis is associated with volume deficit and clotting system activation. Platelet activation in sepsis results in an increased formation of microvesicles, which in turn, have been associated with increased mortality. We hypothesized an effect of different volume replacement solutions on platelet-derived microvesicle formation in septic shock. METHODS: Anaesthetized, mechanically ventilated and multi-catheterized pigs received 1 g kg(-1) body weight faeces into the abdominal cavity to induce sepsis and were observed over 8 h. Five animals in each group received volume replacement therapy with modified fluid gelatin 4% or 8% (MFG4%, MFG8%), 6% hydroxyethylstarch (HES) 200/0.5 or Ringer's solution (RS) to maintain a central venous pressure of 12 mm Hg. Flow cytometry was used for determination of microvesicles before induction of sepsis (baseline) and after 8 h. Platelets and microvesicles were identified with an anti-platelet monoclonal Ab and a secondary antibody. Microvesicles were determined as the smallest 1-3% positive cells in forward scatter. Intergroup comparisons were performed using Wilks-Lambda and Ryan-Einot-Gabriel-Welsh F-test. Differences within groups were compared using a two-tailed Student's t-test. RESULTS: Baseline values were considered as 100%. While microvesicle formation was reduced in HES (73 (sd 19)%), MFG4% (63 (41)%) and MFG8% groups (53 (17)%), an increase in the RS-group (210 (121)%) was observed. Eight hours after induction of sepsis, formation of microvesicles was significantly higher in the RS group compared to all colloid-treated groups. CONCLUSION: In this porcine septic shock model the formation of platelet-derived microvesicles was significantly increased by volume replacement with Ringer's solution in comparison to colloid solutions.

Assessing fluid responsiveness by stroke volume variation in mechanically ventilated patients with severe sepsis.

BACKGROUND AND OBJECTIVE: Our hypothesis was that stroke volume variation during mechanical ventilation of the lungs would allow accurate prediction and monitoring of changes in cardiac index in response to fluid loading in patients with severe sepsis. METHODS: This was a prospective clinical study in a university hospital. Ten mechanically ventilated patients with severe sepsis or septic shock were given fluid loading with 500 mL 10% hydroxyethylstarch 200/0.5 over 30 min. Before and after fluid loading pulmonary arterial occlusion pressure and central venous pressure were measured. Intrathoracic blood volume index, stroke volume variation and cardiac index were measured by the transpulmonary thermodilution technique. After verifying normal distribution of the data (skewness < 1.0) the paired t-test was used for statistical analysis. RESULTS: After fluid loading stroke volume variation decreased significantly, whereas central venous pressure, pulmonary arterial occlusion pressure, intrathoracic blood volume index and cardiac index increased significantly. Changes of cardiac index in response to fluid loading were correlated to baseline values of stroke volume variation (r = 0.64, P = 0.02) and intrathoracic blood volume index (r = -0.73, P = 0.009). Changes in cardiac index were significantly correlated to percentage changes in stroke volume variation (r = -0.65, P < 0.001) and changes in intrathoracic blood volume index (r = 0.52, P = 0.002), whereas changes in cardiac index revealed no significant correlation to changes in central venous pressure (r = 0.28, P = 0.07) and changes in pulmonary arterial occlusion pressure (r = 0.29, P = 0.06). CONCLUSIONS: Measuring stroke volume variation may be a useful way of guiding fluid therapy in ventilated patients with severe sepsis because it allows estimation of preload and prediction of cardiac index changes in response to fluid loading.

High-affinity block of voltage-operated rat IIA neuronal sodium channels by 2,6 di-tert-butylphenol, a propofol analogue.

BACKGROUND AND OBJECTIVE: Propofol is a phenol derivative (2,6 di-isopropylphenol) with a unique effect profile including activating effects on GABA(A) and blocking effects on voltage-operated sodium channels. If the substituents in the 2- and the 6-positions are replaced by tert-butyl groups, the resulting phenol derivative, 2,6 di-tert-butylphenol, despite being a close structural propofol analogue, completely lacks GABA(A) receptor effects. The aim of this in vitro study was to investigate the effects of 2,6 di-tert-butylphenol on voltage-operated neuronal sodium channels in order to determine whether and, if so, how these structural changes alter the sodium channel-blocking effect seen with propofol. METHODS: Whole-cell sodium inward currents through heterologously expressed rat type IIA sodium channels were recorded in the absence and presence of definite concentrations of 2,6 di-tert-butylphenol and propofol. RESULTS: When applied at concentrations > or = 30 micromol, 2,6 di-tert-butylphenol completely and irreversibly blocked sodium inward currents. The blockade equilibrium time was about 2 min. A partial washout was possible only if the application was stopped before the equilibrium of the blockade was achieved. CONCLUSIONS: 2,6 Di-tert-butylphenol exerts a high-affinity block of neuronal sodium channels. Apparently, the slight structural differences of 2,6 di-tert-butylphenol in comparison with propofol--which account for the lack of GABA(A) receptor effects--enhance its voltage-operated sodium channel-blocking effects. As 2,6 di-tert-butylphenol is much more potent than most sodium channel blockers in clinical use, it might be of interest in the development of local anaesthetics.

Voltage-dependent block of neuronal and skeletal muscle sodium channels by thymol and menthol.

BACKGROUND AND OBJECTIVE: Thymol is a naturally occurring phenol derivative used in anaesthetic practice as a stabilizer and preservative of halothane, usually at a concentration of 0.01%. Although analgesic effects have long been described for thymol and its structural homologue menthol, a molecular basis for these effects is still lacking. We studied the blocking effects of thymol and menthol on voltage-activated sodium currents in vitro as possible molecular target sites. METHODS: Whole cell sodium inward currents via heterologously (HEK293 cells) expressed rat neuronal (rat type IIA) and human skeletal muscle (hSkM1) sodium channels were recorded in the absence and presence of definite concentrations of either thymol or menthol. RESULTS: When depolarizing pulses to 0 mV were started from a holding potential of -70 mV, half-maximum blocking concentrations (IC50) for the skeletal muscle and the neuronal sodium channel were 104 and 149 mumol for thymol and 376 and 571 mumol for menthol. The blocking potency of both compounds increased at depolarized holding potentials with the fraction of inactivated channels. The estimated dissociation constant Kd for thymol and menthol from the inactivated state was 22 and 106 mumol for the neuronal and 23 and 97 mumol for the skeletal muscle sodium channel, respectively. CONCLUSIONS: The results suggest that antinociceptive and local anaesthetic effects of thymol and menthol might be mediated via blockade of voltage-operated sodium channels with the phenol derivative thymol being as potent as the local anaesthetic lidocaine.

Hydroxyethyl starch and modified fluid gelatin maintain plasma volume in a porcine model of septic shock with capillary leakage.

OBJECTIVE: To compare the effects of different volume replacement therapies on maintenance of plasma volume in septic shock and capillary leakage syndrome. DESIGN AND SETTING: Prospective randomized, controlled animal laboratory study in a university animal laboratory. MEASUREMENTS AND RESULTS: Twenty-five fasted, anaesthetized, mechanically ventilated and multi-catheterized pigs (20.8+/-1.8 kg) received 1 g/kg body weight faeces into abdominal cavity to induce sepsis and were observed over 8 h. Five animals each received volume replacement therapy with modified fluid gelatin 4% or 8% (MFG4%, MFG8%), 6% HES 200/0.5, or Ringer's solution and were compared to controls receiving 6% HES 200/0.5. Infusion rate was titrated to maintain a central venous pressure of 12 mmHg. Plasma volume was determined using (51)Cr-labelled erythrocytes and standard formulae. Albumin escape rate was calculated using technetium (99m)Tc-labelled albumin. Colloid osmotic pressure, systemic haemodynamics and oxygenation were obtained before and 4 and 8 h after induction of sepsis. Plasma volume was reduced in the Ringer's solution group (-46%) but was maintained in HES (+/-0%), MFG4% (+4%), MFG8% (+23%) groups. Albumin escape rate increased in HES (+52%), MFG4% (+47%), MFG8% (+54%) and the Ringer's solution group (+41%) compared to controls. CONCLUSION: In this porcine septic shock model with concomitant capillary leakage syndrome, confirmed by an increased albumin escape rate, the artificial colloids HES, MFG4%, and MFG8% maintained plasma volume and colloid osmotic pressure. These results suggest the intravascular persistency of artificial colloids in the presence of albumin leakage. An editorial regarding this article can be found in the same issue (http://dx.doi.org/10.1007/s00134-002-1283-9)

Effects of C1 inhibitor and r-SP-C surfactant on oxygenation and histology in rats with lavage-induced acute lung injury.

OBJECTIVE: To assess the effects of C1 inhibitor (INH) administration and r-SP-C surfactant application on oxygenation and lung histology in an acute respiratory distress syndrome model. DESIGN AND SETTING: Randomized, controlled experimental study in an animal research laboratory. MATERIAL: 36 adult male Sprague-Dawley rats. INTERVENTIONS: Animals were subjected to repetitive lung lavage. Four experimental groups and two control groups were studied: groups 1 and 2 served as controls. Animals of groups 3-6 received 200 U/kg body weight C1-INH (group 3), 25 mg/kg r-SP-C surfactant (group 4) or both (group 5) at 60 min postlavage (pl). Animals of group 6 were treated with 200 U/kg C1-INH1 at 10 min pl. Animals of group 1 were killed 60 min (min) pl, animals of groups 2-6 were killed at 210 min pl. Thereafter the lungs were excised for histological examination. MEASUREMENTS AND RESULTS: Hyaline membrane formation, intra-alveolar neutrophil (PMN) accumulation and intra-alveolar/perivascular haemorrhage were graded semiquantitatively (0-4). Blood gases were determined 120, 150, 180 and 210 min pl. At 210 min pl pO(2) in group 4 (456+/-74 mmHg) and group 5 (387+/-155 mmHg) was significantly higher than in controls (72+/-29 mmHg) or after C1-INH monotherapy (group 3: 120+/-103, group 6: 63+/-12 mmHg). PMN infiltration after C1-INH monotherapy was significantly less severe than in controls. The combination of r-SP-C surfactant and C1-INH led to significantly lower PMN infiltration than surfactant monotherapy. CONCLUSION: In this lavage-induced acute respiratory distress syndrome model the administration of C1-INH might be followed by a higher clinical efficacy of exogenously supplied recombinant SP-C surfactant.

Structural requirements of phenol derivatives for direct activation of chloride currents via GABA(A) receptors.

Propofol directly activates gamma-aminobutyric acid (GABA(A)) receptors in the absence of the natural agonist. This mechanism is supposed to contribute to its sedative-hypnotic actions. We studied the effects of seven structurally related phenol derivatives on chloride inward currents via rat alpha1beta2gamma2 GABA(A) receptors, heterologously expressed in HEK 293 cells in order to find structural determinants for this direct agonistic action. Only compounds with the phenolic hydroxyl attached directly to the benzene ring and with aliphatic substituents in ortho position to the phenolic hydroxyl activated chloride currents in the absence of GABA. Concentrations required for half-maximum effect were 980 microM for 2-methylphenol, 230 microM for 2,6-dimethylphenol, 200 microM for thymol, and 23 microM for propofol. Drug-induced chloride currents showed no desensitisation during the 2-s application. These results show that the position of the aliphatic substituents with respect to the phenolic hydroxyl group is the crucial structural feature for direct GABA(A) activation by phenol derivatives.

Postoperative reading speed does not indicate implicit memory in elderly cardiac patients after propofol and remifentanyl anaesthesia.

BACKGROUND: A recent study in young patients undergoing propofol-alfentanil-nitrous oxide anaesthesia demonstrated implicit memory for stories presented during operation using a postoperative reading speed task. In this study we investigated whether patients who tolerate only small amounts of anaesthetics are prone to develop implicit and explicit memories about intraoperative events. METHODS: Thirty patients with poor physical status (ASA III-IV) undergoing cardioverter defibrillator implantation were included in the study. Patients were premedicated with intravenous midazolam and anaesthesia was maintained using propofol and remifentanil infusions. During surgery one of two audio-tapes containing two short stories was played to the patients. Reading speed for the stories played during surgery and two similar stories from the other tape was tested 4 h later. Explicit memory was tested at 4 h and 24 h after audiotape presentation using a structured interview and a forced-choice recognition test pertaining to the story content. Thirty additional awake subjects served as controls. RESULTS: Although half of the patients seemed to be awake one or more times during the operation, no explicit memories of intraoperative events were reported. The forced-choice recognition of the stories was at chance level. No effect on reading speed was found in either the patients or the control subjects. CONCLUSIONS: The possible reasons for reduced explicit and implicit memory performance in elderly patients are age and poor physical status of the patients and the modality change between study and test phases. A non-anaesthetised control group of the same age and physical status should therefore be included in all studies of implicit memory.