Anoxic depolarization of rat hippocampal slices is prevented by thiopental but not by propofol or isoflurane

R. Sasaki1, K. Hirota1,*, S. H. Roth2 and M. Yamazaki1

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
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. There is strong evidence to suggest that anoxic depolarization (AD) is an important factor in hypoxia/ischaemia-induced neural damage. Treatments that prevent the occurrence of AD may be useful in providing neuronal protection against hypoxia. The current study was designed to determine whether general anaesthetics which have been suggested to ‘induce prophylaxis’ against hypoxia can attenuate the incidence of AD.

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 (2x10–4 M) significantly reduced the incidence of AD (16%, P=0.0006). In comparison, propofol (2x10–4 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
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The phenomenon of cortical spreading depression, which is characterized by a propagating wave of cell membrane depolarization in neural tissue, was originally described by Leao.1 It consists of a synchronous, sudden and profound depolarization of a population of neurons accompanied by dramatic changes of electrical, biochemical and morphological properties.2 These changes include loss of neuronal membrane resistance, transmembrane redistribution of ions and neuronal swelling. A similar response occurs a few minutes after brain deprivation of oxygen or interruption of blood flow, and is termed ‘anoxic depolarization’ (AD).3 Bures and colleagues2 were the first to suggest an association between the generation of AD and irreversible neuronal damage. Subsequently, several authors have proposed that the initiation of AD is one of the most important factors in brain damage caused by hypoxia/ischaemia in vivo46 and in vitro.2 3 7

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
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Approval for the study was obtained from the Animal Research Committee of Toyama Medical and Pharmaceutical University. The hippocampal slice preparation was prepared as previously described.13 Briefly, male Wistar rats (100–200 g) were anaesthetized with sevoflurane in oxygen and then decapitated. The brain was rapidly removed and the dissected hippocampus was sliced in cold artificial cerebrospinal fluid (ACSF) transversely to its longitudinal axis (400 µm thick) using a Rotorslicer DTY–7700 (DSK, Osaka, Japan). The composition of the ACSF was as follows: NaCl 124 mM, KCl 5 mM, CaCl2 2 mM, NaH2PO4 1.25 mM, MgSO4 2 mM, NaHCO3 26 mM and glucose10 mM. The ACSF was precooled (8–10°C) and saturated with a 95% oxygen–5% carbon dioxide gas mixture before use (pH 7.1–7.3).

The slices were placed on a nylon mesh at a liquid–gas interface in a recording chamber maintained at 37°C. A humidified gas mixture (95% oxygen–5% carbon dioxide) was applied to the chamber at a flow rate of 1 litre min–1. ACSF was continuously perfused at a rate of 90 ml h–1. 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 (3–5 M{Omega} filled with 2 M NaCl) were placed in the CA1 cell body region to record extracellular field population spikes (PS). The minimum stimulus intensity (5–10 V) that elicited maximal PS amplitude was used to evoke a response. Square-wave stimuli (5–10 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% oxygen–5% carbon dioxide to 95% nitrogen–5% 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 10–30 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 (10–1 M) were prepared in pure dimethyl sulphoxide (DMSO) and then diluted in ACSF before perfusion into the chamber. The final concentration of DMSO (1.4x10–5 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% oxygen–5% 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 kg–1 and from 10 to 24 mg kg–1, respectively.14 Since i.v. anaesthetics are diluted by extracellular fluid (20–30% of the total body weight), the maximal concentrations of thiopental and propofol in the extracellular fluid are estimated to be in the ranges (3–5)x10–4 M and (2–6)x10–4 M, respectively. On the basis of these calculations, the concentration–response 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 2x10–4 M, propofol 2x10–4 M and isoflurane 1.5 vol%. Propofol is used clinically in humans at concentrations of 1.5x10–4 M,15 and burst suppression is associated with concentrations of (1.0–2.0)x10–4 M.16,17 However, taking into account 99% protein binding, the effect site concentration is estimated to be ~(1.0–2.0)x10–6 M.18 Therefore the concentration employed in the brain slice experiments (2x10–4 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 Mann–Whitney U-test, Student's t-test and Welch's t-test. Statistical differences among multiple groups were determined by the Kruskal–Wallis 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
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Figure 1 shows representative recordings of the effects of 3 min anoxia on field potential and PS waveform. In the absence of anaesthetics, PS amplitude gradually decreased and disappeared within 60 s after oxygen withdrawal. Subsequently the field potential showed a large negative shift (–30 mV), which is the hallmark of AD. Following re-oxygenation, the field potential rapidly returned to baseline (0 mV) and the PS waveform slowly recovered. In the presence of propofol or isoflurane, PS diminished within 100 s and AD was observed. The field potential fluctuated compared with the non-anaesthetic preparation. PS and field potential recovered following re-oxygenation. In contrast, thiopental prevented AD during anoxia. Although the PS amplitude disappeared by 120 s, it gradually recovered to the pre-anoxic value after re-oxygenation.



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Fig 1 Base-line trace of the field potential in the stratum pyramidale of CA1 before, during and after 3 min anoxia in the absence of anaesthetics and in the presence of thiopental 2x10–4 M, propofol 2x10–4 M and isoflurane 1.5 vol%. Representative examples show population spikes recorded in CA1 region evoked by stimulation of the Schaffer collateral before, during and after anoxia at each condition. The arrow indicates anoxic depolarization.

 
Figure 2 shows a summary of the occurrence of AD induced by 3 min anoxia in the four groups. In the absence of anaesthetics, AD occurred in 81% of the preparations. Thiopental 2x10–4 M significantly reduced the incidence of AD (16%, P=0.0006 vs untreated group), whereas propofol 2x10–4 M and isoflurane 1.5 vol% failed to significantly prevent AD (69%, P=0.9, and 60%, P=0.8, respectively). To evaluate whether prevention of AD by thiopental was concentration dependent, the effects of the anaesthetic at concentrations of 10–4 M, 2x10–4 M and 5x10–4 M were examined. Figure 3 shows that the incidence of AD was negatively correlated with the concentration of thiopental. In the presence of propofol and isoflurane, however, the incidence of AD was independent of anaesthetic concentrations, suggesting that higher concentrations of propofol and isoflurane could not prevent AD. Since previous studies in vivo suggested that blockade of the N-methyl-D-aspartate (NMDA) receptor provides neuronal protection,10 19 the effects of the NMDA receptor antagonist AP-5 (10–4 M) on the generation of AD were examined. However, the incidence of AD during anoxia was 100% (n=3) in the presence of AP-5 (data not shown).



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Fig 2 Incidence of AD induced by 3 min anoxia in each of the experimental groups. The incidence of AD was significantly lower in the group treated with thiopental 2x10–4 M (n=19) than in the groups treated with propofol 2x10–4 M (n=13) and isoflurane 1.5 vol% (n=10). *P=0.0001 vs untreated group (n=21) (Kruskal–Wallis test followed by Scheffé's method).

 


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Fig 3 Relationship between the incidence of AD and anaesthetic concentration. Thiopental reduced the incidence of AD with increasing concentration (A), whereas all concentrations of propofol (B) and isoflurane (C) tested showed no change in the incidence of AD. *P<0.05 vs concentration of 10–4 M thiopental-treated group (Kruskal–Wallis test followed by Scheffé's method).

 
Table 1 summarizes the recovery response of PS following anoxia in each series of experiments. In the absence of AD, PS amplitudes recovered in all groups. In contrast, the development of AD significantly reduced the degree of recovery; however, in the presence of thiopental, the recovery response was the same with or without AD. In the presence of isoflurane, the occurrence of AD markedly altered recovery responses. In order to investigate the relationships between AD and the degree of recovery, we analysed the time to AD onset, and the duration and magnitude of AD (Table 2), and compared this with the recovery rate.


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Table 1 Recovery of PS amplitude from 3 min anoxia with or without anoxic depolarization. Recovery response was determined 15 min after re-oxygenation. Results are expressed as a percentage of control in each experiment.

 

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Table 2 The effects of 3 min anoxia in untreated and anaesthetic-treated slices.

 
Figure 4 shows the relationships between the duration of AD and the degree of recovery in the untreated group. The degree of recovery was negatively correlated with AD duration (R=0.55, P=0.03).



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Fig 4 Relationship between the degree of recovery from 3 min anoxia and the duration of AD in the untreated group. The degree of recovery is expressed as a percentage of the pre-anoxic control value. The straight line was fitted by the equation y=98.7–0.22x; R=0.55, P=0.03 (n=17).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
AD is a phenomenon of the central nervous system that occurs soon after the onset of severe hypoxia or ischaemia. The present study demonstrates that thiopental can prevent AD induced by 3 min anoxia in rat hippocampal slices in a concentration-dependent manner. However, neither propofol nor isoflurane at high concentrations prevented AD. During cerebral anoxia/ischaemia or AD, glutamate accumulates in the brain at levels that hyperexcite glutamate receptors20 21 and causes uncontrolled calcium influx via the NMDA receptor.22 It has been shown that volatile and i.v. anaesthetics can decrease anoxia-induced glutamate release,23 and that thiopental and propofol can reduce anoxia-mediated calcium influx in rat hippocampal slices.17 2325 Therefore it has been postulated that these anaesthetics can prevent AD by suppressing calcium influx or reducing glutamate release. Although it has been reported that blockade of the NMDA receptor can provide neural protection,10 19 AP-5 failed to prevent AD in the present study. This result agrees with previous in vitro studies,7 26 27 showing a lack of protective effect of NMDA receptor antagonists during hypoxia. Simon and colleagues19 reported that NMDA receptor antagonists protect against post-ischaemic reperfusion disturbance, but do not prevent the extensive ionic changes caused by hypoxia. In contrast, a recent report suggests that a combination of sodium channel blocker, calcium channel blocker, NMDA and non-NMDA receptor antagonists can effectively prevent AD.28 Isoflurane fails to attenuate changes in potassium and sodium influx29 whereas propofol does not reduce the potassium efflux of hippocampal neurons during anoxia.17 In addition, Wang and colleagues25 reported that thiopental blocks sodium, potassium and calcium fluxes during anoxia, thus preventing ionic imbalance.

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
 
This work was carried out at the Department of Anaesthesiology, Toyama Medical and Pharmaceutical University School of Medicine, Japan.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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