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Anesthetic methods in rats determine outcome after experimental focal cerebral ischemia: mechanical ventilation is required to obtain controlled experimental conditions

https://doi.org/10.1016/S1385-299X(02)00138-1Get rights and content

Abstract

Objective: Anesthetic agents, pH, blood gases and blood pressure have all been found to influence the pathophysiology of experimental stroke. In experimental research, rats are predominantly used to investigate the effects of focal cerebral ischemia. Chloral hydrate, applied intraperitoneally (i.p.), and halothane, applied via face-mask in spontaneously breathing animals or via endotracheal tube in mechanically ventilated animals are popular methods of anesthesia. We investigated the potential of these anesthetic methods to maintain physiologic conditions during focal cerebral ischemia and their influence on postischemic mortality and histological outcome. Methods: Thirty male Sprague–Dawley rats were subjected to 90 min of middle cerebral artery occlusion by insertion of an intraluminal thread and assigned to one of three groups (n=10 each): (A) chloral hydrate i.p./spontaneously breathing; (B) halothane in 70:30 (%) N2O/O2 via face-mask/spontaneously breathing; and (C) halothane in 70:30 (%) N2O/O2 via endotracheal tube/mechanically ventilated. Physiologic parameters were measured before, during, and after ischemia. Infarct volume was histologically assessed after 7 days. Results: All anesthetic techniques except mechanical ventilation via an endotracheal tube resulted in considerably fluctuating blood gases levels, hypercapnia, acidosis and low blood pressure. All spontaneously breathing animals (groups A and B) exhibited a higher postischemic mortality and significantly larger infarct volumes than group C with intubated and ventilated animals. Conclusions: Intra- and postischemic physiologic parameters such as blood pressure, pH, and blood gases critically determine outcome after focal cerebral ischemia. Although anesthesia by halothane via face-mask allowed better control of depth of anesthesia than chloral hydrate, we have found this method to be unsatisfactory due to insufficient control of ventilation and waste of anesthetic gases. Experiments with rats requiring normal physiologic parameters should be performed under conditions of controlled mechanical ventilation and sufficient analgesia.

Section snippets

Type of research

The rat is one of the most widely used small animals in neuroscience research in general and in research in cerebral ischemia especially today [15], [34]. Most models of cerebral ischemia are surgically invasive and thus require anesthetics during the ischemic insult. Modern anesthesia in man combines volatile anesthetic gases, intravenous agents, and neuromuscular blocking agents to maintain unconsciousness while maintaining cardiovascular stability. In this context, a variety of commonly used

Time required

All described anesthetic procedures, intraperitoneal injection of chloral hydrate, or application of halothane via face-mask or an orotracheal tube, can be managed within a few minutes. After initial immobilization of the rat by restraining in a box or pharmacologically in a container with 4% halothane, all procedures lead to unconsciousness and immobility of the animals within 1–5 min. In experienced hands with adequate optical instruments, e.g. operational microscope, intubation can be made

Materials

A total of 35 male Sprague–Dawley rats (250–300 g body weight) were used for this study. Animals were purchased from Charles River Laboratory (Sulzfeld, Germany) and were cared for before and at all stages of the experiment in compliance with applicable institutional guidelines and regulations of the government of Bavaria.

Animal preparation and monitoring

Rats were fasted overnight before surgery with free access to water. After induction of anesthesia according to the experimental groups, all rats were subjected to 90 min of MCA occlusion by insertion of a silicone-coated 4-0 nylon monofilament via the external carotid artery as described by Koizumi et al. [25]. Reperfusion was achieved by withdrawing the filament into the external carotid artery after 90 min. Five animals with subarachnoid hemorrhage as confirmed by autopsy were excluded from

Physiological parameters

In group A, anesthesia with chloral hydrate within all subgroups resulted in hypercapnia with mean values between 52.1±6.9 and 57.3±6.3 mmHg without significant differences over time. All animals of group A showed respiratory acidosis during anesthesia with mean values between 7.32±0.02 and 7.37±0.05 without significant differences between groups or over time. Mean blood pressure ranged between 68.3±7.9 and 75.1±12.0 mmHg.

Anesthesia with halothane via face-mask (group B) also resulted in

Discussion

Anesthetic agents, blood pressure, arterial pH and blood gases have all been implicated in the pathophysiology of experimental stroke [7], [9], [47], [55], [56]. Despite their widespread usage, comparative studies on appropriate methods of rat anesthesia are scarce. We investigated the influence of various common anesthetic techniques on intra- and postischemic physiological variables, mortality and histological outcome in a model of transient focal cerebral ischemia.

In our study all anesthetic

Conclusion

Intra- and postischemic physiologic parameters such as blood pressure, pH and blood gases critically determine outcome after focal cerebral ischemia. All spontaneously breathing animals, whether anesthetized by chloral hydrate i.p. or by halothane via face-mask, exhibited hypercapnia, acidosis, and decreased blood pressure during anesthesia, resulting in high mortality and a significantly increased infarct volume compared to mechanically ventilated animals with halothane anesthesia. Although

Quick procedure

  • 1.

    Rats are anesthetized in a container with 4% halothane.

  • 2.

    Rats are then placed in supine position on an inclined plane and the vocal cords are visualized under the operating microscope by lifting the jaw and the tongue with a small surgical clamp, bent at the tip.

  • 3.

    The tube (A 16 G venous catheter, diameter 1.8 mm, with a 2.0-cm long conical silicone cuff), supplied with a blunted steel mandrin, is placed intratracheally and pushed forward until an increasing resistance due the silicone cuff is felt.

Essential literature references

Original papers: [7], [9], [17], [22], [29], [49], [54], [67]

Book chapters: [37]

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (Schm1067/2-1) and Friedrich Baur Stiftung. Special thanks to Professor Lawrence M. Shuer, MD, Stanford University, CA for permission to reproduce the drawing of the face mask.

References (67)

  • M. Ben et al.

    Anesthesia in the rat

    Fed. Proc.

    (1969)
  • A. Bhardwaj et al.

    Hypertonic saline worsens infarct volume after transient focal ischemia in rats

    Stroke

    (2000)
  • D.D. Breimer

    Clinical pharmacokinetics of hypnotics

    Clin. Pharmacokinet.

    (1977)
  • J.L. Browning et al.

    Effects of halothane, alpha-chloralose, and pCO2 on injury volume and CSF beta-endorphin in focal cerebral ischemia

    Mol. Chem. Neuropathol.

    (1997)
  • H. Chen et al.

    Anti-Cd11B monoclonal antibody reduces ischemic cell damage after transient focal cerebral ischemia in rat

    Ann. Neurol.

    (1994)
  • D.J. Cole et al.

    Focal cerebral ischemia in rats—effect of phenylephrine-induced hypertension during reperfusion

    J. Neurosurg. Anesthesiol.

    (1992)
  • J.C. Drummond et al.

    Phenylephrine-induced hypertension reduces ischemia following middle cerebral artery occlusion in rats

    Stroke

    (1989)
  • D. Duverger et al.

    The quantification of cerebral infarction following focal cerebral ischemia in the rat: Influence of strain, arterial pressure, blood glucose concentration, and age

    J. Cereb. Blood Flow Metab.

    (1988)
  • W. Erhardt et al.

    Anaesthesiologische Erfahrungen in der experimentellen Chirurgie

    Anaesthesist

    (1979)
  • M.D. Ginsberg et al.

    Rodent models of cerebral ischemia

    Stroke

    (1989)
  • J.J. Grome et al.

    The effects of apomorphine upon local cerebral glucose utilization in conscious rats and in rats anesthetized with chloral hydrate

    J. Neurochem.

    (1983)
  • N.F. Jensen et al.

    A comparison of the vasodilating effects of halothane and isoflurane on the isolated rabbit basilar artery with and without intact endothelium

    Anesthesiology

    (1992)
  • E. Kagstrom et al.

    Cerebral circulatory responses to hypercapnia and hypoxia in the recovery period following complete and incomplete cerebral ischemia in the rat

    Acta Physiol. Scand.

    (1983)
  • B. Kaplan et al.

    Temporal thresholds for neocortical infarction in rats subjected to reversible focal cerebral ischemia

    Stroke

    (1991)
  • K. Katsura et al.

    Acidosis induced by hypercapnia exaggerates ischemic brain damage

    J. Cereb. Blood Flow Metab.

    (1994)
  • J.R. Kirsch et al.

    Anesthetics and cerebroprotection: experimental aspects

    Int. Anesthesiol. Clin.

    (1996)
  • E.D. Kirson et al.

    Presynaptic and postsynaptic actions of halothane at glutamatergic synapses in the mouse hippocampus

    Br. J. Pharmacol.

    (1998)
  • K. Kogure et al.

    Effects of changes in carbon dioxide pressure and arterial pressure on blood flow in ischemic regions of the brain in dogs

    Circ. Res.

    (1969)
  • J. Koizumi et al.

    Experimental studies of ischemic brain edema. 1. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area

    Jpn. J. Stroke

    (1986)
  • R. Koorn et al.

    Effect of etomidate on in vivo ischemia-induced dopamine release in the corpus striatum of the rat: a study using cerebral microdialysis

    Anesth. Analg.

    (1994)
  • J. Krieglstein et al.

    Comparative study of the effects of chloral hydrate and trichloroethanol on cerebral metabolism

    Naunyn Schmiedebergs Arch. Pharmacol.

    (1973)
  • R.E. Lewis et al.

    Error of intraperitoneal injections in rats

    Lab. Anim. Care

    (1966)
  • P.A. Li et al.

    Role of hyperglycaemia-related acidosis in ischaemic brain damage

    Acta Physiol. Scand.

    (1997)
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