1 Department of Anaesthesiology, Toyama Medical and Pharmaceutical University of Medicine, 2630 Sugitani, Toyama, 930-0194, Japan and 2 Departments of Pharmacology and Therapeutics and Anaesthesia, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada
* Corresponding author. E-mail: koki{at}ms.toyama-mpu.ac.jp
Accepted for publication November 10, 2004.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. The effects of anoxia (3 min) on evoked extracellularly recorded field potentials of CA1 neurons in rat hippocampal slices were assessed in the absence and presence of the i.v. general anaesthetics thiopental and propofol and the volatile anaesthetic isoflurane.
Results. In the absence of anaesthetics, AD occurred in 81% of the preparations tested. Thiopental (2x104 M) significantly reduced the incidence of AD (16%, P=0.0006). In comparison, propofol (2x104 M) and isoflurane (1.5 vol%) were ineffective (69% and 60%, respectively). Furthermore, in the presence of thiopental, the population spike amplitude recovered with and without AD (90% and 94% of pre-anoxic value, respectively) following 3 min anoxia.
Conclusion. The prophylactic effect of thiopental against hypoxia might be induced, in part, by preventing the generation of AD.
Keywords: anaesthetics, i.v., thiopental ; anaesthetics, i.v., propofol ; anaesthetics, volatile, isoflurane ; measurement techniques, electrophysiology ; model brain slice, hippocampus
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been suggested that general anaesthetics can provide prophylaxis against hypoxia and thus attenuate neuronal damage.812 In an in vivo study, Patel and colleagues10 demonstrated that isoflurane and pentobarbital could reduce the frequency of cortical AD and infarct volume following focal cerebral ischaemia in rats. However, it may be difficult to induce identical hypoxic brain damage in control and anaesthetic-applied groups, since the extent of ischaemia is modified as a result of collateral circulation. Also, cerebral blood flow could be altered as a result of anaesthetic-induced cardiovascular depression. In the present study we were able to induce an identical degree of anoxia in preparations without the changes in extracellular fluid perfusion rate and carbon dioxide concentration that could occur in in vivo models. The aim of this study was to determine, using an in vitro hippocampal slice preparation, whether general anaesthetics (thiopental, propofol and isoflurane) could prevent the generation of AD during anoxia.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The slices were placed on a nylon mesh at a liquidgas interface in a recording chamber maintained at 37°C. A humidified gas mixture (95% oxygen5% carbon dioxide) was applied to the chamber at a flow rate of 1 litre min1. ACSF was continuously perfused at a rate of 90 ml h1. Slices were incubated for 90 min without electrical stimulation. Bipolar nichrome stimulating electrodes were placed in the region of the stratum radiatum to activate Schaffer-collateral inputs to CA1 pyramidal neurons. Glass microelectrodes (35 M filled with 2 M NaCl) were placed in the CA1 cell body region to record extracellular field population spikes (PS). The minimum stimulus intensity (510 V) that elicited maximal PS amplitude was used to evoke a response. Square-wave stimuli (510 V, 0.05 ms, 0.1 Hz) were delivered using an SEN-3301 stimulator (Nihon Kohden, Tokyo, Japan). Field potentials were amplified (Nihon Kohden MEZ-8301), filtered (1 Hz to 10 kHz) and digitally converted (100 kHz) using an iNet system (GWI, Somerville, MA, USA) and stored on a Macintosh computer (Apple, Cupertino, CA, USA) for later analysis. PS amplitudes were measured from peak positive to peak negative. Anoxia (0% oxygen) was induced in the slices by switching the gas mixture from 95% oxygen5% carbon dioxide to 95% nitrogen5% carbon dioxide for 3 min. This was performed in the absence and presence of general anaesthetics. Recovery response was determined 15 min after re-oxygenation. AD was identified as a sudden negative shift of 1030 mV in the extracellular potential.
All preparations used in this study exhibited control variability <5% during the initial data acquisition period. The i.v. anaesthetic thiopental was directly dissolved in ACSF. Stock solutions of propofol (101 M) were prepared in pure dimethyl sulphoxide (DMSO) and then diluted in ACSF before perfusion into the chamber. The final concentration of DMSO (1.4x105 M) did not affect field potentials. The volatile anaesthetic isoflurane was applied as a vapour to the tissue chamber via the prewarmed and humidified 95% oxygen5% carbon dioxide gas stream above the slices using an appropriate vaporizer (Forawick, Muraco, Tokyo, Japan). Concentrations of isoflurane, expressed as volume per cent (vol%), refer to dial settings on the vaporizer. Concentrations of isoflurane in the perfusate of the recording chamber were determined using gas chromatography (Shimazu, Kyoto, Japan). The concentrations of isoflurane in solution were found to be linear (0.55 mM per 1.0 vol%) up to 5.0 vol%. The doses of thiopental and propofol required to anaesthetize experimental animals ranged from 20 to 30 mg kg1 and from 10 to 24 mg kg1, respectively.14 Since i.v. anaesthetics are diluted by extracellular fluid (2030% of the total body weight), the maximal concentrations of thiopental and propofol in the extracellular fluid are estimated to be in the ranges (35)x104 M and (26)x104 M, respectively. On the basis of these calculations, the concentrationresponse curves generated in preliminary experiments and the calculated 50% effective dose (ED50 values), the following concentrations of anaesthetics were tested in the current study: thiopental 2x104 M, propofol 2x104 M and isoflurane 1.5 vol%. Propofol is used clinically in humans at concentrations of 1.5x104 M,15 and burst suppression is associated with concentrations of (1.02.0)x104 M.16,17 However, taking into account 99% protein binding, the effect site concentration is estimated to be (1.02.0)x106 M.18 Therefore the concentration employed in the brain slice experiments (2x104 M) is higher than physiologically relevant. To test the effects of anaesthetics on anoxia, thiopental and propofol were pre-applied for 20 min, and isoflurane was pre-applied for 10 min before the start of recording. Thiopental, propofol and isoflurane were purchased from Tanabe (Osaka, Japan), Aldrich Chemical Co. (Milwaukee, USA) and Dinabot (Osaka, Japan), respectively.
A total of 101 hippocampal slices prepared from 22 rats were used in the study. Ninety-eight slices were divided into four groups (untreated, and thiopental treated, propofol treated and isoflurane treated). Three slices were used for the experiments in the presence of DL-2-amino-5-phosphonovaleric acid (AP-5). Rat weights [138 (SD 61) g] and dissection times [356 (31) s] were similar in the four groups.
Statistical analysis
Data are expressed as mean (SD). The statistical difference between two groups was determined by the MannWhitney U-test, Student's t-test and Welch's t-test. Statistical differences among multiple groups were determined by the KruskalWallis test. The post hoc Scheffé test was used for multiple comparisons. A P-value <0.05 was considered significant. Linear and quadratic functions were fitted to the data using SigmaPlot (Jandel Scientific, San Rafael, CA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the absence of anaesthetics, the PS waveform decreased during anoxia but recovered following re-oxygenation, suggesting that a 3 min period of anoxia did not produce irreversible neurological effects in our preparation. Pretreatment with general anaesthetics did not alter these effects unless AD occurred. Recovery deteriorated with AD in the untreated, propofol-treated and isoflurane-treated groups, but not in the thiopental-treated group. This suggests that some general anaesthetics may modify AD and intracellular homeostasis.
Our results indicate that the duration of AD is negatively correlated with the degree of PS amplitude recovery following anoxia. This finding is in agreement with the results of Bures and colleagues,2 who proposed that delay of onset of AD could improve the chance of recovery of brain function following ischaemia, and of Mies and colleagues,4 who demonstrated a linear relationship between the frequency of AD and infarct volume. Moreover, Joshi and Andrew7 reported that hypothermia protected hemi-brain slices from oxygen/glucose deprivation damage by inhibiting AD onset. Since isoflurane prolonged the duration of AD, the degree of PS recovery was much lower in the isoflurane-treated group (Table 1).
Pretreatment with barbiturates has been shown to significantly reduce the area of infarction in permanent cerebral vascular occlusion models in vivo.9 12 Varathan and colleagues30 reported that barbiturates could improve the survival rate of rat cortical neurons following 24 h of hypoxia. A randomized prospective study in humans demonstrated that thiopental could decrease the neuropsychiatric complications of open-ventricle operations requiring cardiopulmonary bypass.8 Thus our results could explain the cerebral protective effects of barbiturates against hypoxia/ischaemia.
In summary, we have investigated the effects of thiopental, propofol and isoflurane on AD during anoxia using a rat hippocampal slice preparation. Our results demonstrate that thiopental (but not propofol or isoflurane) was able to suppress AD in a concentration-dependent manner. These results support clinical observations that barbiturates induce prophylactic effects against ischaemia.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 Bures J, Buresova O, Krivanek J. The Mechanism and Applications of Leao's Spreading Depression of Electroencephalographic Activity. New York: Academic Press, 1974
3 Balestrino M. Pathophysiology of anoxic depolarization: new findings and a working hypothesis. J Neurosci Methods 1995; 59: 99103[CrossRef][ISI][Medline]
4 Mies G, Iijima T, Hossmann K-A. Correlation between peri-infarct DC shifts and ischaemic neuronal damage in rats. Neuroreport 1993; 4: 70911[ISI][Medline]
5 Nedergaard M. Spreading depression as a contributor to ischemic brain damage. Adv Neurol 1996; 71: 7584[ISI][Medline]
6 Kaminogo M, Suyama K, Ichikura A, Onizuka M, Shibata S. Anoxic depolarization determines ischemic brain injury. Neurol Res 1998; 20: 3438[ISI][Medline]
7 Joshi I, Andrew RD. Imaging anoxic depolarization during ischemia-like conditions in the mouse hemi-brain slice. J Neurophysiol 2001; 85: 41424
8 Nussmeier NA, Arlund C, Slogoff S. Neuropsychiatric complications after cardiopulmonary bypass: cerebral protection by a barbiturate. Anesthesiology 1986; 64: 16570[ISI][Medline]
9 Cheng MA, Theard A, Tempelhoff R. Intravenous agents and intraoperative neuroprotection. Crit Care Clin 1997; 13: 18599[ISI][Medline]
10 Patel PM, Drummond JC, Cole DJ, Kelly PJ, Watson M. Isoflurane and pentobarbital reduce the frequency of transient ischemic depolarizations during focal ischemia in rats. Anesth Analg 1998; 86: 77380[Abstract]
11 Popovic R, Liniger R, Bickler PE. Anesthetics and mild hypothermia similarly prevent hippocampal neuron death in an in vitro model of cerebral ischemia. Anesthesiology 2000; 92: 13439[ISI][Medline]
12 Cole DJ, Cross LM, Drummond JC, Patel PM, Jacobsen WK. Thiopentone and methohexital, but not pentobarbitone, reduce early focal cerebral ischemic injury in rats. Can J Anaesth 2001; 48: 80714
13 Hirota K, Roth SH. The effects of sevoflurane on population spikes in CA1 and dentate gyrus of the rat hippocampus in vitro. Anesth Analg 1997; 85: 42630[Abstract]
14 Wakasugi M, Hirota K, Roth SH, Ito Y. The effects of general anesthetics on excitatory and inhibitory synaptic transmission in area CA1 of the rat hippocampus in vitro. Anesth Analg 1999; 88: 67680
15 Smith C, McEwan AI, Jhaveri R, et al. The interaction of fentanyl on the CP50 of propofol for loss of consciousness and skin incision. Anesthesiology 1994; 81: 8208[ISI][Medline]
16 Shyr M-H, Yang C-H, Kuo TBJ, et al. Power spectral analysis of the electroencephalographic and hemodynamic correlates of propofol anesthesia in the rat: Intravenous bolus administration. Neurosci Lett 1993; 153: 1614[CrossRef][ISI][Medline]
17 Amorim P, Chambers G, Cottrell J, Kass IS. Propofol reduces neuronal transmission damage and attenuates the changes in calcium, potassium, and sodium during hyperthermic anoxia in the rat hippocampal slice. Anesthesiology 1995; 83: 125465[CrossRef][ISI][Medline]
18 Hara M, Kai Y, Ikemoto Y. Propofol activates GABAA receptor-chloride ionophore complex in dissociated hippocampal pyramidal neurons of the rat. Anesthesiology 1993; 79: 7818[ISI][Medline]
19 Simon RP, Swan JH, Griffiths T, Meldrum BS. Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain. Science 1984; 226: 8502[ISI][Medline]
20 Rossi DJ, Oshima T, Attwell D. Glutamate release in severe brain ischaemia is mainly by reversed uptake. Nature 2000; 403: 31621[CrossRef][ISI][Medline]
21 Satoh M, Asai S, Katayama Y, Kohno T, Ishikawa K. Real-time monitoring of glutamate transmitter release with anoxic depolarization during anoxic insult in rat striatum. Brain Res 1999; 822: 1428[CrossRef][ISI][Medline]
22 Szatkowski M, Attwell D. Triggering and execution of neuronal death in brain ischaemia: two phases of glutamate release by different mechanisms. Trends Neurosci 1994; 17: 35965[CrossRef][ISI][Medline]
23 Bickler PE, Buck LT, Feiner JR. Volatile and intravenous anesthetics decrease glutamate release from cortical brain slices during anoxia. Anesthesiology 1995; 83: 123340[CrossRef][ISI][Medline]
24 Zhan R-Z, Fujiwara N, Endoh H, et al. Thiopental inhibits increases in [Ca2+]i induced by membrane depolarization, NMDA receptor activation, and ischemia in rat hippocampal and cortical slices. Anesthesiology 1998; 89: 45666[CrossRef][ISI][Medline]
25 Wang T, Raley-Susman KM, Wang J, et al. Thiopental attenuates hypoxic changes of electrophysiology, biochemistry, and morphology in rat hippocampal slice CA1 pyramidal cells. Stroke 1999; 30: 24007
26 Aitken PG, Balestrino M, Somjen GG. NMDA antagonists: lack of protective effect against hypoxic damage in CA1 region of hippocampal slices. Neurosci Lett 1988; 89: 18792[CrossRef][ISI][Medline]
27 Jarvis CR, Anderson TR, Andrew RD. Anoxic depolarization mediates acute damage independent of glutamate in neocortical brain slices. Cereb Cortex 2001; 11: 24959
28 Somjen GG. Mechanisms of spreading depression and hypoxic spreading depression-like depolarization. Physiol Rev 2001; 81: 106596
29 Kass IS, Amorim P, Chambers G, Austin D, Cottrell JE. The effect of isoflurane on biochemical changes during and electrophysiological recovery after anoxia in rat hippocampal slices. J Neurosurg Anesthesiol 1997; 9: 2806[ISI][Medline]
30 Varathan S, Shibuta S, Shimizu T, Varathan V, Mashimo T. Hypothermia and thiopentone sodium: individual and combined neuroprotective effects on cortical cultures exposed to prolonged hypoxic episodes. J Neurosci Res 2002; 68: 35262[CrossRef][ISI][Medline]