Effects of sevoflurane on dopamine, glutamate and aspartate release in an in vitro model of cerebral ischaemia.

Release of excitatory amino acids and dopamine plays a central role in neuronal damage after cerebral ischaemia. In the present study, we used an in vitro model of ischaemia to investigate the effects of sevoflurane on dopamine, glutamate and aspartate efflux from rat corticostriatal slices. Slices were superfused with artificial cerebrospinal fluid at 34 degrees C and episodes of 'ischaemia' were mimicked by removal of oxygen and reduction in glucose concentration from 4 to 2 mmol litre(-1) for < or = 30 min. Dopamine efflux was monitored in situ by voltammetry while glutamate and aspartate concentrations in samples of the superfusate were measured by HPLC with fluorescence detection. Neurotransmitter outflow from slices was measured in the absence or presence of sevoflurane (4%). After induction of ischaemia in control slices, there was a mean (SEM) delay of 166 (7) s (n = 5) before sudden efflux of dopamine which reached a maximum extracellular concentration of 77.0 (15.2) micromol litre(-1). Sevoflurane (4%) reduced the rate of dopamine efflux during ischaemia (6.90 (1.5) and 4.73 (1.76) micromol litre(-1) s(-1) in controls and sevoflurane-treated slices, respectively; P<0.05), without affecting its onset or magnitude. Excitatory amino acid efflux was much slower. lschaemia-induced glutamate efflux had not reached maximum after 30 min of ischaemia. Basal (pre-ischaemic) glutamate and aspartate efflux per slice was 94.8 (24.8) and 69.3 (31.5) nmol litre(-1) superfusate (n = 4) and was not significantly reduced by 4% sevoflurane. lschaemia greatly increased glutamate and aspartate efflux (to a maximum of 919 (244)% and 974 (489)% of control, respectively). However, ischaemia-induced efflux of both glutamate and aspartate was significantly reduced by 4% sevoflurane (P < 0.001 for glutamate, P < 0.01 for aspartate). In summary, sevoflurane may owe part of its reported neuroprotective effect to a reduction of ischaemia-induced efflux of excitatory amino acids and, to a lesser extent, dopamine.

Comparison of ketamine stereoisomers on tissue metabolic activity in an in vitro model of global cerebral ischaemia.

Ketamine (2-o-chlorophenenyl-2-methylaminocyclohexanone hydrochloride) is a dissociative general anaesthetic with neuroprotective properties. Since ketamine is optically active, we compared the neuroprotective efficacy of the (+)- or (-)-enantiomers in global cerebral ischaemia. Rat corticostriatal slices superfused with, or incubated in, artificial CSF at 34 degrees C were subjected to a brief ischaemic insult. Dopamine efflux was measured using fast cyclic voltammetry. Tissue metabolism was determined with 2,3,5-triphenyltetrazolium chloride staining, a marker of mitochondrial enzyme activity. In control slices, ischaemia caused rapid striatal dopamine release (to 122 microM over 18 s) after an initial delay of 149s. Racemic ketamine (100 micromol/l) significantly delayed (by 24%, P<0.05), slowed (by 63%, P<0.01) and reduced (by 27%, P<0.05) ischaemia-induced dopamine release. Ischaemia (10 min) also caused significant decreases in striatal (25%, P<0.01) and cortical (31%, P<0.001) metabolic activity, manifested as a drop in mean TTC staining intensity. Racemic ketamine and its (+)- and (-)-enantiomers (each 100 microM) attenuated the loss of metabolic activity in the striatum. However, in the cortex, only (+)-ketamine (100 microM) was significantly neuroprotective. We conclude that neuroprotection by ketamine in cerebral ischaemia is both region- and isomer-dependent.

Rapid quantification of ischaemic injury and cerebroprotection in brain slices using densitometric assessment of 2,3,5-triphenyltetrazolium chloride staining.

2,3,5-Triphenyltetrazolium chloride (TTC), a marker of mitochondrial enzyme activity, is widely used to assess the effects of cerebral ischaemia in vivo. In the present study, we characterised its utility as a simple rapid macrohistological measure of ischaemic damage in brain slices. Coronal rat corticostriatal slices were incubated in oxygenated artificial cerebrospinal fluid (aCSF) until subjected to 'ischaemia' (deoxygenated, hypoglycaemic aCSF) for up to 12 min. After a further 30 min to 16 h of reincubation in oxygenated aCSF, slices were stained with TTC, fixed with formalin and transferred to cover slips. The slices were scanned in 8-bit greyscale using a standard desktop scanner and the staining analysed by densitometry of the acquired images. Control slices stained a rich pink/red. Ischaemia (10 min) reduced both the area and intensity of staining. Both measures of striatal staining were negatively correlated with the duration of ischaemia (0-12 min). Furthermore, staining in the striatum correlated significantly with cortical TTC staining. The effects of TTC concentration (0.063-0.5% w/v) and post-ischaemic interval (30 min to 16 h) were examined upon the intensity of TTC staining. (+)-MK 801 prevented the ischaemia-induced reduction in TTC staining, consistent with cerebroprotection. These data suggest that TTC staining of brain slices may be used to quantify ischaemic injury and cerebroprotection.

Effects of metabolic alterations on dopamine release in an in vitro model of neostriatal ischaemia.

Release of neurotransmitters, including dopamine (DA), plays a central role in neuronal death during cerebral ischaemia. We investigated the effects of changes in energy demand and supply on DA release in cerebral ischaemia in vitro. Rat striatal slices were superfused (400 ml/h) with an artificial cerebrospinal fluid at 34 degrees C, unless otherwise stated. Ischaemia were mimicked by removal of O2 and reduction in glucose concentration from 4 to 2 mM. DA release was monitored by voltammetry. The profile of ischaemia-induced DA release was temperature-dependent. Hypothermia (to 24 degrees C) delayed, slowed, and reduced ischaemia-induced DA release relative to 34 degrees C. Pretreatment of the slices for 3 h with creatine (25 mM) delayed and slowed ischaemia-induced DA release. Conversely, blockade of Na+/K+ ATPase with ouabain induced an anoxic depolarisation and rapid DA release similar to ischaemia. In summary, the onset of DA release in this model is controlled by the balance between energy supply and utilisation. Strategies that increase availability of energy substrates for the membrane sodium pump (i.e., pre-incubation with creatine) or decrease their utilisation (hypothermia) slow and delay DA release. Hypothermia may owe part of its neuroprotective effect to a delay and slowing of ischaemia-induced release of DA and/or other neurotransmitters.

Sodium channel blockade unmasks two temporally distinct mechanisms of striatal dopamine release during hypoxia/hypoglycaemia in vitro.

Massive striatal dopamine release during cerebral ischaemia has been implicated in the resulting neuronal damage. Sodium influx is an early event in the biochemical cascade during ischaemia and blockade of sodium channels may increase resistance to ischaemia by reducing energy demand involved in compensation for sodium and potassium fluxes. In this study, we have determined the effects of opening and blockade of voltage-gated sodium channels on hypoxia/hypoglycaemia-induced dopamine release. Slices of rat caudate nucleus were maintained in a slice chamber superfused by an oxygenated artificial cerebrospinal fluid containing 4 mM glucose. Ischaemia (hypoxia/hypoglycaemia) was mimicked by a switch to a deoxygenated artificial cerebrospinal fluid containing 2 mM glucose and dopamine release was measured using fast cyclic voltammetry. In drug-free (control) slices, there was a 2-3 min delay after the onset of hypoxia/hypoglycaemia followed by a rapid dopamine release event which was associated with anoxic depolarization. In slices treated with the Na+ channel opener, veratridine (1 microM), the time to onset of dopamine release was shortened (101 +/- 20 s, compared with 171 +/- 8 s in controls, P < 0.05). Conversely, phenytoin (100 microM), lignocaine (200 microM) and the highly selective sodium channel blocker, tetrodotoxin (1 microM) markedly delayed and slowed dopamine release vs paired controls. In the majority of cases, dopamine release was biphasic after sodium channel blockade: a slow phase preceded a more rapid dopamine release event. The latter was associated with anoxic depolarization. Neither the fast nor the slow release events were affected by pretreatment with the selective dopamine uptake blocker GBR 12935 (0.2 microM), suggesting that uptake carrier reversal did not contribute to these events. In conclusion, sodium channel antagonism delays and slows hypoxia/hypoglycaemia-induced dopamine release in vitro. Furthermore, sodium channel blockade delays anoxic depolarization and its associated neurotransmitter release, revealing an earlier dopamine release event that does not result from reversal of the uptake carrier.

Double-blind, randomized study of the effect of cisapride on gastric emptying in critically ill patients.

OBJECTIVES: To investigate the absorption of the gastrokinetic drug, cisapride, and effect of cisapride on gastric emptying in critically ill patients; and to assess the usefulness of clinical signs of gastric emptying. DESIGN: Prospective, randomized, controlled study. SETTING: Medical/surgical/trauma intensive care unit (ICU) in a university hospital. PATIENTS: Twenty-seven consecutively enrolled patients, aged 18 to 65 yrs, with normal hepatic and renal biochemistry who were not receiving enteral nutrition and who had no contraindications to enteral nutrition. These patients were expected to stay in the ICU for at least 4 days. INTERVENTIONS: Patients were randomized to receive either placebo or rectal cisapride, 60 mg initially followed by two doses of 30 mg at 8-hr intervals. MEASUREMENTS AND MAIN RESULTS: Gastric emptying was estimated, using acetaminophen absorption on day 1 of the study. Placebo or cisapride was administered and a second acetaminophen absorption test for gastric emptying was carried out on day 2,24 hrs after the first test. Four patients were excluded because of incomplete data. Statistical analysis was performed, using the area under the acetaminophen absorption curve from 0 to 60 mins as the primary measure of gastric emptying. There was no significant change in the area under the acetaminophen absorption curve from 0 to 60 mins from day 1 to day 2 in patients who received placebo or cisapride. Using the combination of the time to maximum acetaminophen concentration (< or = 30 mins) with a maximum concentration (> 12 mg/L) to define "normal" emptying, on day 1, four of the 11 placebo patients had the "normal" gastric emptying, and by day 2, five patients fulfilled this criterion. Before administration of cisapride, four of the 12 patients fulfilled this criterion, whereas nine fulfilled the criterion after receiving cisapride. There was a large variation in gastric emptying from day 1 to day 2; a power calculation suggests that approximately 150 patients would have to be studied to determine the effect of cisapride. There was no correlation between gastric emptying and the volume of gastric aspirate or the presence of bowel sounds. Plasma cisapride concentrations 4 hrs after the third dose, during the second acetaminophen absorption test, averaged 53 ng/mL (range 20 to 111). CONCLUSIONS: Rectal cisapride in the dose given achieved average plasma concentrations similar to those concentrations achieved in healthy subjects after 30 mg of cisapride rectally. There is a large variation in gastric emptying from one day to the next and large numbers of patients are required to determine if cisapride administration improves early gastric emptying in critically ill patients. The volume of gastric aspirate and the presence of bowel sounds do not correlate with gastric emptying.

A model of gastric emptying using paracetamol absorption in intensive care patients.

OBJECTIVES: To describe the range and factors which may affect gastric emptying in the ICU patient. DESIGN: Validation sample. SETTING: The adult Intensive Care Unit (ICU) of a teaching hospital. PATIENTS: Twenty-seven ICU patients, aged 18-65 years were studied within 3 days of their ICU admission. All patients had normal hepatic and renal chemistry and had no contraindications to enteral feeding. MEASUREMENTS AND MAIN RESULTS: The area under the concentration curve from 0-60 min (AUC60) of a paracetamol absorption test was used as the measure of gastric emptying. The variables of the presence or absence of bowel sounds, volume of gastric aspirate ( > 50 ml or < 50 ml), an estimated risk of death (ROD), an APACHE II score calculated 24 h before the study, a pHi measurement, the use of dopamine (2.5-5 microg/kg, yes or no) and of opioids were included in a multiple regression analysis. Using Pearson correlation, AUC60 was positively correlated with the estimated ROD (r = 0.50, p < 0.05). There was a statistically significant difference in the mean AUC60 between those patients who did, and those who did not, receive dopamine (t = 3.06, p < 0.005). On multiple regression analysis the only variable which was significantly associated with AUC60 was estimated ROD, which accounted for 25% of the variance in AUC60. CONCLUSION: The results suggest that there is a wide range in gastric emptying in critically ill patients. The results may be due to the case mix of the patients. The use of dopamine may adversely affect gastric emptying and requires further investigation in the ICU patient. Prediction of gastric emptying is difficult in these patients and further investigation is necessary in order to improve our understanding of this process.

Involvement of N- and P/Q- but not L- or T-type voltage-gated calcium channels in ischaemia-induced striatal dopamine release in vitro.

Calcium influx and transmitter efflux are central events in the neuropathological cascade that occurs during and following cerebral ischaemia. This study explored the role of voltage-gated calcium channels (VGCCs) in ischaemia-induced striatal dopamine (DA) release in vitro. Slices (350 microm thickness) of rat neostriatum were superfused (400 ml/h) with an artificial cerebrospinal fluid (aCSF) at 34 degrees C and subjected to episodes of 'ischaemia' by reduction of the glucose concentration from 4 to 2 mM and gassing with 95% N2/5% CO2. DA release was monitored with fast cyclic voltammetry at implanted carbon fibre microelectrodes. The time to onset, time to peak, rate and magnitude of DA release were measured. Non-selective blockade of VGCCs with a high concentration of Ni2+ (2.5 mM), markedly delayed (P < 0.01) and slowed (P < 0.05) DA release but preferential blockade of T-type VGCCs with a lower concentration (200 microM) had no effect. DA release was also unaffected by selective antagonism of L-type VGCCs with nimodipine and nicardipine (10 microM each). Selective blockade of N-type VGCCs with omega-conotoxin GVIA (100 nM) delayed DA release (P < 0.05) but did not affect its rate or magnitude. Blockade of P- and possibly Q-type VGCCs with omega-agatoxin IVA (up to 200 nM) both delayed (P < 0.05) and slowed (P < 0.05) DA release. Preferential blockade of P- type VGCCs with neomycin (500 microM) also delayed (P < 0.05) and slowed (P < 0.05) DA release. These findings suggest that N-, P- and possibly Q- but not L- or T-type VGCCs mediate ischaemia-induced DA release. Although it is not possible to say, on the basis of these results, that the effects are directly upon the dopamine terminals, these calcium channels nevertheless constitute promising targets for therapeutic intervention.

Characteristics of the NMDA receptor modulating hypoxia/hypoglycaemia-induced rat striatal dopamine release in vitro.

We investigated the functional characteristics of the NMDA receptor that modulates hypoxia/hypoglycaemia-induced striatal dopamine release. Dopamine release was detected by fast cyclic voltammetry in rat neostriatal slices. Four variables were measured: T(on) -- time from initiation of hypoxia/hypoglycaemia to the onset of dopamine release, Tpk -- time from onset to maximum, deltaDA/delta(t) -- rate of dopamine release and DAmax -- maximum extracellular dopamine concentration. In controls, T(on) = 164.9 +/- 1.7 s, Tpk = 20.9 +/- 0.9 s, deltaDA/delta(t) = 5.31 +/- 0.44 microM/s and DAmax = 79.1 +/- 2.5 microM (means +/- S.E.M., n = 203). Cis-4-(phosphonomethyl)piperidine-2-carboxylic acid (CGS 19755, 20 microM) lengthened, while N-methyl-D-aspartate (NMDA) (100 microM) shortened T(on). (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine hydrogen maleate (MK 801, 1 and 10 microM) and dextromethorphan (10 and 100 microM) increased Tpk and decreased DAmax. Neither glycine (100 microM), 7-chlorokynurenic acid (50 microM) nor 5-nitro-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione (ACEA 1021, 100 microM) had any effect although 7-chlorokynurenic acid blocked the effect of NMDA. Increasing [Mg2+] from 1.3 to 3.7 mM, increased Tpk and decreased deltaDA/delta(t). Dithiothreitol (1 mM) accelerated T(on) while 5.5-dithio-bis-(2-nitrobenzoic acid) (1 mM) delayed T(on). Neither drug affected Tpk, DAmax or deltaDA/delta(t). Neither spermidine (100 microM) nor arcaine (100 microM) affected T(on), Tpk or deltaDA/delta(t) although arcaine decreased DAmax. In conclusion, hypoxia/hypoglycaemia-induced dopamine release was influenced by an NMDA receptor although modulation of the glycine recognition site of the receptor was ineffective, as were agents acting at polyamine modulatory zones. These findings highlight differences between recombinant and native NMDA receptors and suggest caution in extrapolating molecular biology to functional studies.

'Real time' measurement of dopamine release in an in vitro model of neostriatal ischaemia.

Dopamine (DA) is released in large quantities during ischaemia and may be neurotoxic. For instance, reduction of DA release is associated with a better histological outcome following experimental stroke. In the present study, we report the measurement of transmitter release in an in vitro model of cerebral ischaemia using brain slices. Striatal slices were subjected to 'ischaemia' by reducing the O2 and glucose content of the superfusate in a controlled fashion. The resultant monoamine release, measured in real time by fast cyclic voltammetry at carbon fibre microelectrodes, was shown to be DA by electrochemical criteria. Upon imposition of an ischaemic episode, there was a lag period (159 +/- 2 s, mean +/- S.E.M., n = 99) followed by a sudden release of DA, reaching a maximum extracellular concentration of 95 +/- 4 microM in 25 +/- 2 s. This overall profile of DA release was qualitatively similar to that measured in the striatum in vivo following cardiac arrest. The DA uptake inhibitor GBR 12935 (1.0 microM) had no effect on any DA release variable. We conclude that this model mimics many of the features of cerebral ischaemia in vivo and may be a suitable vehicle for the investigation of neuroprotective drugs.

Prediction of postoperative nausea and vomiting using a logistic regression model.

In a previous study, logistic regression analysis was used to determine the association of independent fixed patient factors with the incidence of postoperative nausea and vomiting (PONV). Female sex, previous history of PONV, use of postoperative opioids, previous history of motion sickness and an interaction between male sex and previous history of PONV were combined in an equation from which risk of PONV could be estimated. The present study was designed to test this equation in a group of patients with wide selection criteria. Data on 400 patients were collected in relation to pre-, per- and postoperative factors which may influence the incidence of PONV. The equation was used to predict PONV, and actual outcome was compared with that predicted. The overall incidence of PONV was 36%. The equation predicted an overall probability of PONV of 27.4%. If the model was used to define individual patients as predicted to have or not to have PONV, it was correct only 71% of the time. However, there was good agreement between the actual incidences of PONV and those predicted among the 16 risk groups created by the model.

The cerebral function monitor (CFM) is a useful addition to a bilateral carotid artery, vein graft model.

Following human coronary artery bypass surgery, vein graft occlusion is a major cause of morbidity and mortality. An agent is required which will reduce the incidence of early graft thrombosis without causing systemic bleeding. To assess the efficacy of such agents a suitable experimental vein-graft model is required. A porcine, unilateral saphenous vein-carotid artery bypass graft model has been described previously, although to assess the effect of locally applied anticoagulant drugs, insertion of grafts bilaterally would be advantageous, allowing treated and control grafts to be implanted in the same animal which would then act as its own control. Pigs are reported as having an excellent collateral cerebral circulation and hence in theory, would be suitable animals to use as a bilateral carotid vein-graft model. This paper describes the occurrence of serious neurological complications during the development of such a model and suggests that by using a cerebral function monitor, detection of critical reductions in cerebral perfusion can be made early and remedial action take.

Evaluation of neuromuscular effects and antagonism of rocuronium bromide: a preliminary report.

Twenty ASA I-II patients received either 2 or 3 x ED95 doses of rocuronium bromide during nitrous oxide, oxygen, propofol, fentanyl-based anaesthesia. The mean times to maximum block were 98 s and 74 s and the mean duration of clinical relaxation (recovery to 25% T1) was 35 min and 46 min following 620 micrograms kg-1 and 930 micrograms kg-1, respectively. Neuromuscular blockade was antagonized with either neostigmine or edrophonium from a twitch height of 25%. Although there was no significant difference between the recovery times neostigmine appeared to give more consistent antagonism of rocuronium-induced blockade.