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On the use of anaesthesia in rodents in studies with isolated mitochondria.

Anatoly Starkov.

      Some researchers new to the field of isolated mitochondria studies question the validity of “no anesthesia prior decapitation” approach. A typical question sounds thus:

“…I've got to renew my animal care protocol and noted that in your mitochondria prep protocol you have dire warnings about the consequences of using anesthetics. Our animal care protocol reviewers are getting more and more picky and I was wondering if there is a general reference that documents the anesthetic effect that I could use to assuage the concerns of our compliance guys.”

    This is in fact a very valid concern as ethical and practical aspects of “no anesthesia” approach are not appealing to the public and its scientific value is not immediately evident. I therefore decided to write a few words and point a few references clarifying the issue.
    A long-standing consensus in the field of isolated mitochondria research is that chemical anesthesia of rodents prior to their decapitation for tissue harvesting for mitochondria preparation should be avoided. Numerous studies performed from as early as 1935 [1] till the present time specifically addressed the effect of many and various anesthetics on various mitochondrial functions. The invariable conclusion of the overwhelming majority of these studies is that anesthetics DO ALTER the functioning and the structure of mitochondria, whether they are given to animals prior to tissue harvesting or added to isolated mitochondria at physiologically realistic concentrations. The use of anesthesia prior to decapitation has been shown to affect important physiological parameters such as release of free fatty acids in the tissue and levels of insulin in the serum which in turn can result in alterations of mitochondrial functions and therefore must be avoided [2]. Anesthetics induce mitochondria-mediated apoptosis in human T lymphocytes in vitro [3]. In vivo, life threatening complications due to potential effects of many anesthetics on mitochondria are well known to anesthesiologists [4]. General anesthetic procedures in humans results in an inhibition of the oxidation of several tricarboxylic acid cycle substrates in skeletal muscle mitochondria [5]. Others have conclusively shown that ultrastructure of isolated mitochondria does depend on the way animals were sacrificed, with or without the use of anesthetics [6], and that the latter may destroy the normal mitochondrial ultrastructure [7], change the lipid composition of the inner membrane [8], its physical structure and lipid-protein interactions [9,10]. At the functional level, anesthetics are known to uncouple mitochondria [11,12] or to inhibit their respiration [1,13] and transport of metabolites across their inner membrane [14], and to interfere with the Ca2+ handling [15].
    This short list is far from being complete. It simply illustrates various artefacts that use of anesthetics can potentially cause in studies with isolated mitochondria; more information can be found in PubMed by performing a search for a specific anesthetic of interest (e.g., “sefoflurane + mitochondria”, “isoflurane + mitochondria”, “volatile anesthetics + mitochondria”, “ketamine + mitochondria”, “phenobarbital + mitochondria”, “local anesthetics + mitochondria”, et cetera et cetera).
    So what is the conclusion and recommendations? The consensus “avoid them” opinion is well founded and should be followed whenever the onus of the study is on mitochondrial functions that depend on the integrity of their inner membrane and the functioning of their respiratory chain (their “bioenergetics”) or their Ca2+ handling. Use of anesthesia is prone to creating artificial changes in mitochondria.
    However, a conscious use of anesthesia is also quite acceptable in many studies. Of course, one’s using it must be aware of its effect on mitochondria and must understand how it may change the outcome of the study. For example, if one studies the mitochondrial proteome or changes in mtDNA, anesthesia-induced uncoupling of mitochondria, inhibition of their respiration, or changes in lipid composition should not be much of concern as it unlikely would change the outcome of the study. In this case, decapitating animals without anesthesia is unnecessary and therefore unethical, cruel, and barbaric.

[2]. Bhathena SJ. Comparison of effects of decapitation and anesthesia on metabolic and hormonal parameters in Sprague-Dawley rats. Life Sci. 1992 50(21):1649.
[3]. Loop T, Dovi-Akue D, Frick M, Roesslein M, Egger L, Humar M, Hoetzel A, Schmidt R, Borner C, Pahl HL, Geiger KK, Pannen BH. Volatile anesthetics induce caspase-dependent, mitochondria-mediated apoptosis in human T lymphocytes in vitro. Anesthesiology. 2005 102(6):1147.
[4]. Driessen, Jacques J Neuromuscular and mitochondrial disorders: what is relevant to the anaesthesiologist?. Current Opinion in Anaesthesiology. 2008 21(3):350.
[5]. O Miró, A Barrientos, J R Alonso, J Casademont, D Jarreta, A Urbano-Márquez, F Cardellach Effects of general anaesthetic procedures on mitochondrial function of human skeletal muscle. Eur J Clin Pharmacol. 1999 55 (1):35-41
[6]. Hertsens R, Jacob W, Van Bogaert A. Effect of hypnorm, chloralosane and pentobarbital on the ultrastructure of the inner membrane of rat heart mitochondria. Biochim Biophys Acta. 1984 769(2):411-8.
[7]. Abidova SS, Khoroshaev VA, Leonov FV. [Morphofunctional characteristics of the heart mitochondria during adrenergic stimulation and blockade under ketamine anesthesia] Anesteziol Reanimatol. 1990 (6):12-5.
[8]. Dekutovich GV, Kargapolov AV. [Characteristic effect of local anesthetics on the phospholipid composition of mitochondria] Vopr Med Khim. 1986 32(6):38-41.
[9]. Mazzanti L, Curatola G, Zolese G, Bertoli E, Lenaz G Lipid protein interactions in mitochondria. VIII. Effect of general anesthetics on the mobility of spin labels in lipid vesicles and mitochondrial membranes. J Bioenerg Biomembr. 1979 11(1-2):17-32.
[10]. Lenaz G, Curatola G, Mazzanti L, Parenti-Castelli G, Bertoli E.Effects of general anesthetics on lipid protein interactions and ATPase activity in mitochondria. Biochem Pharmacol. 1978 27(24):2835-44.
[11]. Dabadie P, Bendriss P, Erny P, Mazat JP. Uncoupling effects of local anesthetics on rat liver mitochondria. FEBS Lett. 1987 226(1):77-82.
[12]. Rottenberg H.Uncoupling of oxidative phosphorylation in rat liver mitochondria by general anesthetics. Proc Natl Acad Sci U S A. 1983 80(11):3313-7.
[13]. Tsyganiĭ AA, Medvinskaia NA. [Characteristics of the change in oxidative phosphorylation in the mitochondria of various organs under nitrous oxide anesthesia] Farmakol Toksikol. 1984 47(4):30-3.
[14]. Rafałowska U. Transport of malate and citrate into rat brain mitochondria under hypoxia and anesthesia. Neurochem Res. 1979 4(3):355-64.
[15]. Branca D, Varotto ML, Vincenti E, Scutari G. The inhibition of calcium efflux from rat liver mitochondria by halogenated anesthetics. Biochem Biophys Res Commun. 1988 155(2):978-83.

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