Exposure to CO without isoflurane had variable effects on activated caspase-3 and the number of TUNEL positive nuclei compared with air-exposed controls (Figs. 2 and3). Differences trended toward significance between CO-exposed cohorts without isoflurane in the number of TUNEL positive nuclei in the hippocampus and hypothalamus/thalamus of 100 ppm CO exposed mice versus animals exposed to 5 ppm CO (Fig. 3).
Peroxidation of cardiolipin is an upstream event that is important for cytochrome c release and initiation of the mitochondrial apoptosis pathway.12 CO can bind to the cytochrome c-cardiolipin complex and inhibit cytochrome c peroxidase activity, thereby preventing oxidation of cardiolipin and mobilization and release of cytochrome c.12,19 Thus, we extracted cytochrome c from forebrain mitochondria immediately after 1-hour exposure to either 0 ppm CO (air), 5 ppm CO, or 100 ppm CO with or without isoflurane on P7 and measured peroxidase activity of cytochrome c using spectrophotometry. To assess for cytochrome c release, we measured the amount of heme c (the heme moiety of cytochrome c) in forebrain mitochondrial and cytosolic fractions immediately after exposure.
Isoflurane either significantly increased or trended toward a significant increase in forebrain cytochrome c peroxidase activity after 1-hour exposure in air compared with all nonisoflurane-exposed cohorts (Fig. 4). All isoflurane-exposed cohorts demonstrated significantly higher or a trend toward significantly higher cytochrome c peroxidase activity compared with nonisoflurane CO matched cohorts (Fig. 4). CO exposure significantly decreased forebrain cytochrome c peroxidase activity in isoflurane and nonisoflurane-exposed cohorts in a concentration-dependent manner (Fig. 4). CO-mediated inhibition of cytochrome c peroxidase resulted in enzyme activities that were below that of air-exposed control values in all CO-exposed cohorts (Fig. 4).
After isoflurane exposure for 1 hour in air, heme c levels decreased significantly in the mitochondrial fraction and increased significantly in the cytosolic fraction compared with nonisoflurane air-exposed controls, suggesting increased cytochrome c release in the forebrain after exposure (Fig. 5). CO exposure without isoflurane trended toward a significant increase in the amount of heme c in forebrain mitochondria in 5 ppm exposed animals compared with air-exposed controls (Fig. 5A). Cytosolic heme c content in forebrain decreased significantly in both cohorts exposed to CO without isoflurane compared with air-exposed controls, indicating inhibition of cytochrome c release (Fig. 5B). Exposure to CO with isoflurane resulted in significantly increased heme c levels within forebrain mitochondria of 100 ppm exposed mice and significantly decreased heme c in cytosol of both CO-exposed cohorts compared with animals exposed to isoflurane alone (Fig. 5). Decreases in cytosolic heme c after CO exposure with isoflurane were dose-dependent, and levels were at or below air-exposed control values (Fig. 5B). Mitochondrial heme c levels in animals exposed to isoflurane with 100 ppm CO were equivalent to levels seen in forebrain mitochondria of controls exposed to air alone (Fig. 5A). Taken together, the data suggest that CO inhibits forebrain cytochrome c peroxidase and, depending on concentration, can decrease isoflurane-induced cytochrome c release.
Our findings are consistent with previous work and support the concept that isoflurane causes neurotoxicity in the developing mammalian brain via activation of the intrinsic apoptosis pathway.6,11 In addition, we demonstrate for the first time that, upstream from cytochrome c release, isoflurane increases forebrain cytochrome c peroxidase activity. It is important to note that subclinical concentrations of CO inhibited isoflurane-induced cytochrome c peroxidase activation in a dose-dependent manner and decreased the release of cytochrome c, limiting apoptosis in the developing brain after exposure to isoflurane.
Because hydrogen peroxide is required for induction of cytochrome c peroxidase, increased peroxidase activity likely resulted from isoflurane-induced oxidative stress.38,39 Although we did not measure free radicals as part of this study, isoflurane and other anesthetic drugs generate reactive oxygen, nitrogen species, and hydrogen peroxide in developing neurons, hippocampus, subiculum, and thalamus.40–42 In addition, cytochrome c peroxidase activity has been shown to increase in the forebrain of mice during oxidative stress, and cytochrome c has been developed as a biosensor for hydrogen peroxide detection.43,44 Thus, isoflurane exposure likely increased hydrogen peroxide production that, in turn, enhanced the peroxidase activity of cytochrome c.38
Finding increased cytochrome c peroxidase activity after isoflurane exposure is significant because it uncovers a potential target for therapeutic intervention. Peroxidase activation is necessary and critical for mobilization and release of cytochrome c from mitochondria during apoptosis.12 Release of cytochrome c is often considered the “point of no return” in the pathway.45 Thus, targeting the peroxidase activity of cytochrome c is logical and inhibiting cytochrome c peroxidase could prevent cytochrome c release during proapoptotic stimuli such as with anesthetic exposure. In support of this concept, low concentrations of inhaled CO (50–500 ppm) have been shown to prevent apoptosis in endothelium, vascular smooth muscle, liver, lung tissue during hyperoxia, sepsis, and ischemia-reperfusion.14–18 In previous work, we demonstrated that brief exposure to low CO concentrations can inhibit cytochrome c peroxidase in vivo and impair programmed cell death in the developing forebrain of newborn mice.19 Here we demonstrate that CO, inspired at subclinical concentrations, inhibits activation of cytochrome c peroxidase during isoflurane exposure and impairs release of cytochrome c.
CO prevents apoptosis by binding to the cytochrome c-cardiolipin complex and inhibiting cytochrome c peroxidase activity.12 This prevents oxidation of cardiolipin and subsequent release of cytochrome c.12 Although it is possible that CO may exert its prosurvival response via a variety of other mechanisms, our data suggest that abrogation of isoflurane-induced apoptosis may be due to CO-mediated inhibition of cytochrome c peroxidase activation. This is supported by finding concentration-dependent responses to subclinical CO exposure within different aspects of the intrinsic apoptosis pathway that we assayed (cytochrome c peroxidase, cytochrome c release, activation of caspase-3, and DNA breakage [TUNEL]).
Although the data indicate that low concentrations of CO can inhibit apoptosis in the developing brain, we have not conclusively shown that CO directly prevents isoflurane-induced neuronal apoptosis. This is important because the CO effect could simply be a generalized, nonspecific phenomenon. Thus, it is possible that CO-mediated inhibition of apoptosis and anesthetic-induced apoptosis are 2 distinct processes that occur simultaneously and independently within the developing brain but not necessarily in the same cells. If these 2 processes are mutually exclusive, then certain cell populations would undergo apoptosis after exposure to isoflurane while totally different cell populations, destined to die via developmental programmed cell death, would survive due to CO-mediated inhibition of the intrinsic apoptosis pathway. Although the total number of neurons undergoing apoptosis in this scenario would be relatively decreased compared with isoflurane exposure alone, the impact on neurodevelopment could be devastating. This is because we have previously demonstrated that exposure to low concentrations of CO in the absence of an anesthetic prevents natural programmed cell death in the neocortex and hippocampus of 10-day-old mice and impairs neurocognitive development.19 Thus, although CO has antiapoptotic properties, its unchecked effects in the developing brain could be equally as deleterious as the effects of anesthetics alone.
Furthermore, brief, low concentration CO exposures can also cause oxidative stress.46 Such undesired effects could enhance apoptosis during development and could explain our findings regarding activated caspase-3 and TUNEL in the cohorts exposed to CO for 1 hour without isoflurane. So it is possible that CO has the potential to exert proapoptotic effects that could act synergistically with isoflurane. Thus, before implementing subclinical CO exposure in everyday clinical practice, its safety and efficacy must be established given the fact that its pro- and antiapoptotic effects could have adverse consequences in the developing brain.
The only way to prove that CO directly prevents the proapoptotic effect of isoflurane is to evaluate neurocognitive function and behavior after exposure. The fact that we have not included such an assessment in this work is a major limitation of the study. However, this will be the focus of future work. Thus, until we determine neurodevelopmental outcome after combined exposure to CO with isoflurane, we cannot draw definitive conclusions about the benefit of CO during an anesthetic.
Regarding clinical toxicity, elevated COHb levels are formed after exposure to high concentrations of CO and can interfere with tissue oxygen delivery by impairing oxygen binding to and dissociation from hemoglobin.37 Overt clinical toxicity manifests from tissue hypoxia when COHb levels are higher than 70%, and signs and symptoms first appear when COHb levels are higher than 10%.13,37 Acute exposure to CO concentrations larger than 800 ppm can rapidly cause brain injury, cerebral edema, coma, and death, while brief exposure to 220 ppm results in headache, dizziness, and impaired judgment.34 Exposure to concentrations <120 ppm does not elicit any appreciable clinical symptoms.34 Thus, short-term exposure to 5 or 100 ppm CO is a non–life-threatening subclinical exposure, and the resultant COHb levels are well below values that cause tissue hypoxia and clinical signs and symptoms.
CO is endogenously produced, and infants and children routinely inspire subclinical concentrations of CO when rebreathing is permitted during low-flow general anesthesia.20,21,47 In previous work, we found that children inspire an average of 2 to 3 ppm CO and as much as 18 ppm CO during low-flow anesthesia.20,21 This resulted in an increase in COHb by an average of 0.2% up to 0.5% from baseline after 1 hour of low-flow anesthesia.20 In the current work, we found a similar increase in COHb after 1-hour exposure to 5 ppm CO and a 3% to 4% increase after exposure to 100 ppm CO. Thus, exposing newborn mice to 5 ppm CO with isoflurane mimics a subclinical CO exposure during low-flow anesthesia at a time point in late infancy.
Here we found that 5 ppm CO limited apoptosis after isoflurane exposure while 100 ppm CO maintained levels of apoptosis at or below control values. These findings suggest that higher concentrations of subclinical CO may be necessary to completely offset the proapoptotic response to isoflurane. Thus, it is possible that levels of CO encountered with rebreathing during routine low-flow anesthesia may limit isoflurane-induced neurotoxicity but may not be adequate to completely prevent it. However, more investigation is necessary before such conclusions can be made.
Targeting cytochrome c peroxidase to offset the oxidative stress of an anesthetic exposure is a novel and intriguing concept. Given that CO is commonly rebreathed during low-flow anesthesia and readily diffuses across the blood-brain barrier to gain access to the mitochondrial inner membrane, it has the potential to be developed as an antiapoptotic agent to prevent anesthesia-induced neurotoxicity.48 However, before routine application of CO as such a therapeutic agent, neurodegeneration needs to be definitively shown to occur in children after anesthetic exposure, CO must be shown to directly inhibit isoflurane-induced apoptosis, and the safety of subclinical CO exposure during development needs to be established. With further work, however, it is possible that low-flow anesthesia, intended to result in low concentration CO exposure, may be established as a standard paradigm designed to protect the developing brain of the infants and children we care for.
1. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci. 2003;23:876–82
2. Stefovska VG, Uckermann O, Czuczwar M, Smitka M, Czuczwar P, Kis J, Kaindl AM, Turski L, Turski WA, Ikonomidou C. Sedative and anticonvulsant drugs suppress postnatal neurogenesis. Ann Neurol. 2008;64:434–45
3. Istaphanous GK, Loepke AW. General anesthetics and the developing brain. Curr Opin Anaesthesiol. 2009;22:368–73
4. Brambrink AM, Evers AS, Avidan MS, Farber NB, Smith DJ, Zhang X, Dissen GA, Creeley CE, Olney JW. Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain. Anesthesiology. 2010;112:834–41
5. Istaphanous GK, Howard J, Nan X, Hughes EA, McCann JC, McAuliffe JJ, Danzer SC, Loepke AW. Comparison of the neuroapoptotic properties of equipotent anesthetic concentrations of desflurane, isoflurane, or sevoflurane in neonatal mice. Anesthesiology. 2011;114:578–87
6. Olney JW, Young C, Wozniak DF, Ikonomidou C, Jevtovic-Todorovic V. Anesthesia-induced developmental neuroapoptosis. Does it happen in humans? Anesthesiology. 2004;101:273–5
7. Rizzi S, Ori C, Jevtovic-Todorovic V. Timing versus duration: determinants of anesthesia-induced developmental apoptosis in the young mammalian brain. Ann N Y Acad Sci. 2010;1199:43–51
8. Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, Gleich SJ, Schroeder DR, Weaver AL, Warner DO. Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiology. 2009;110:796–804
9. DiMaggio C, Sun LS, Kakavouli A, Byrne MW, Li G. A retrospective cohort study of the association of anesthesia and hernia repair surgery with behavioral and developmental disorders in young children. J Neurosurg Anesthesiol. 2009;21:286–91
10. Flick RP, Katusic SK, Colligan RC, Wilder RT, Voigt RG, Olson MD, Sprung J, Weaver AL, Schroeder DR, Warner DO. Cognitive and behavioral outcomes after early exposure to anesthesia and surgery. Pediatrics. 2011;128:e1053–61
11. Yon JH, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V. Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience. 2005;135:815–27
12. Kapetanaki SM, Silkstone G, Husu I, Liebl U, Wilson MT, Vos MH. Interaction of carbon monoxide with the apoptosis-inducing cytochrome c-cardiolipin complex. Biochemistry. 2009;48:1613–9
13. Kao LW, Nañagas KA. Carbon monoxide poisoning. Emerg Med Clin North Am. 2004;22:985–1018
14. Zhou H, Liu J, Pan P, Jin D, Ding W, Li W. Carbon monoxide inhalation decreased lung injury via anti-inflammatory and anti-apoptotic effects in brain death rats. Exp Biol Med (Maywood). 2010;235:1236–43
15. Song R, Kubo M, Morse D, Zhou Z, Zhang X, Dauber JH, Fabisiak J, Alber SM, Watkins SC, Zuckerbraun BS, Otterbein LE, Ning W, Oury TD, Lee PJ, McCurry KR, Choi AM. Carbon monoxide induces cytoprotection in rat orthotopic lung transplantation via anti-inflammatory and anti-apoptotic effects. Am J Pathol. 2003;163:231–42
16. Bernardini C, Zannoni A, Bacci ML, Forni M. Protective effect of carbon monoxide pre-conditioning on LPS-induced endothelial cell stress. Cell Stress Chaperones. 2010;15:219–24
17. Sarady JK, Zuckerbraun BS, Bilban M, Wagner O, Usheva A, Liu F, Ifedigbo E, Zamora R, Choi AM, Otterbein LE. Carbon monoxide protection against endotoxic shock involves reciprocal effects on iNOS in the lung and liver. FASEB J. 2004;18:854–6
18. Wang X, Wang Y, Kim HP, Nakahira K, Ryter SW, Choi AM. Carbon monoxide protects against hyperoxia-induced endothelial cell apoptosis by inhibiting reactive oxygen species formation. J Biol Chem. 2007;282:1718–26
19. Cheng Y, Thomas A, Mardini F, Bianchi SL, Tang JX, Peng J, Wei H, Eckenhoff MF, Eckenhoff RG, Levy RJ. Neurodevelopmental consequences of sub-clinical carbon monoxide exposure in newborn mice. PLoS One. 2012;7:e32029
20. Levy RJ, Nasr VG, Rivera O, Roberts R, Slack M, Kanter JP, Ratnayaka K, Kaplan RF, McGowan FX Jr. Detection of carbon monoxide during routine anesthetics in infants and children. Anesth Analg. 2010;110:747–53
21. Nasr V, Emmanuel J, Deutsch N, Slack M, Kanter J, Ratnayaka K, Levy R. Carbon monoxide re-breathing during low-flow anaesthesia in infants and children. Br J Anaesth. 2010;105:836–41
22. Woehlck HJ. Carbon monoxide rebreathing during low flow anesthesia. Anesth Analg. 2001;93:516–7
23. Farahani R, Kanaan A, Gavrialov O, Brunnert S, Douglas RM, Morcillo P, Haddad GG. Differential effects of chronic intermittent and chronic constant hypoxia on postnatal growth and development. Pediatr Pulmonol. 2008;43:20–8
24. Rice D, Barone S Jr. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ Health Perspect. 2000;108(Suppl 3):511–33
25. Sanno H, Shen X, Kuru N, Bormuth I, Bobsin K, Gardner HA, Komljenovic D, Tarabykin V, Erzurumlu RS, Tucker KL. Control of postnatal apoptosis in the neocortex by RhoA-subfamily GTPases determines neuronal density. J Neurosci. 2010;30:4221–31
26. Johnson SA, Young C, Olney JW. Isoflurane-induced neuroapoptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol. 2008;20:21–8
27. Franklin KBJ, Paxinos G The Mouse Brain in Stereotaxic Coordinates. 1997 San Diego, CA Academic Press
28. Paxinos G, Watson C Atlas of the Developing Mouse Brain: At E17.5, PO, and P6. 2007 San Diego, CA Academic Press
29. Jacobs EE, Sanadi DR. The reversible removal of cytochrome c from mitochondria. J Biol Chem. 1960;235:531–4
30. Kim NH, Jeong MS, Choi SY, Kang JH. Peroxidase activity of cytochrome c. bull. Korean Chem Soc. 2004;25:1889–92
31. Piel DA, Gruber PJ, Weinheimer CJ, Courtois MR, Robertson CM, Coopersmith CM, Deutschman CS, Levy RJ. Mitochondrial resuscitation with exogenous cytochrome c in the septic heart. Crit Care Med. 2007;35:2120–7
32. Ozawa T, Tanaka M, Shimomura Y. Crystallization of the middle part of the mitochondrial electron transfer chain: cytochrome bc1-cytochrome c complex. Proc Natl Acad Sci U S A. 1980;77:5084–6
33. Tanaka M, Ogawa N, Ihara K, Sugiyama Y, Mukohata Y. Cytochrome aa(3) in Haloferax volcanii. J Bacteriol. 2002;184:840–5
34. Winter PM, Miller JN. Carbon monoxide poisoning. JAMA. 1976;236:1502
35. Peterson JE, Stewart RD. Absorption and elimination of carbon monoxide by inactive young men. Arch Environ Health. 1970;21:165–71
36. Stewart RD, Peterson JE, Baretta ED, Bachand RT, Hosko MJ, Herrmann AA. Experimental human exposure to carbon monoxide. Arch Environ Health. 1970;21:154–64
37. Gorman D, Drewry A, Huang YL, Sames C. The clinical toxicology of carbon monoxide. Toxicology. 2003;187:25–38
38. Puchkov MN, Vassarais RA, Korepanova EA, Osipov AN. Cytochrome c produces pores in cardiolipin-containing planar bilayer lipid membranes in the presence of hydrogen peroxide. Biochim Biophys Acta. 2013;1828:208–12
39. Kim H, Oh E, Im H, Mun J, Yang M, Khim JY, Lee E, Lim SH, Kong MH, Lee M, Sul D. Oxidative damages in the DNA, lipids, and proteins of rats exposed to isofluranes and alcohols. Toxicology. 2006;220:169–78
40. Bai X, Yan Y, Canfield S, Muravyeva MY, Kikuchi C, Zaja I, Corbett JA, Bosnjak ZJ. Ketamine enhances human neural stem cell proliferation and induces neuronal apoptosis via reactive oxygen species-mediated mitochondrial pathway. Anesth Analg. 2013;116:869–80
41. Matsuoka H, Watanabe Y, Isshiki A, Quock RM. Increased production of nitric oxide metabolites in the hippocampus under isoflurane anaesthesia in rats. Eur J Anaesthesiol. 1999;16:216–24
42. Boscolo A, Milanovic D, Starr JA, Sanchez V, Oklopcic A, Moy L, Ori C C, Erisir A, Jevtovic-Todorovic V. Early exposure to general anesthesia disturbs mitochondrial fission and fusion in the developing rat brain. Anesthesiology. 2013;118:1086–97
43. . Programmed cell death is impaired in the developing brain of FMR1 mutants. Dev Neuroscience. 2013;35:347–58
44. De Wael K, Bashir Q, Van Vlierberghe S, Dubruel P, Heering HA, Adriaens A. Electrochemical determination of hydrogen peroxide with cytochrome c peroxidase and horse heart cytochrome c entrapped in a gelatin hydrogel. Bioelectrochemistry. 2012;83:15–8
45. Ferraro E, Pulicati A, Cencioni MT, Cozzolino M, Navoni F, di Martino S, Nardacci R, Carrì MT, Cecconi F. Apoptosome-deficient cells lose cytochrome c through proteasomal degradation but survive by autophagy-dependent glycolysis. Mol Biol Cell. 2008;19:3576–88
46. Thom SR, Ischiropoulos H. Mechanism of oxidative stress from low levels of carbon monoxide. Res Rep Health Eff Inst. 1997;80:1–19
47. Hayashi M, Takahashi T, Morimatsu H, Fujii H, Taga N, Mizobuchi S, Matsumi M, Katayama H, Yokoyama M, Taniguchi M, Morita K. Increased carbon monoxide concentration in exhaled air after surgery and anesthesia. Anesth Analg. 2004;99:444–8
48. Sutherland BA, Harrison JC, Nair SM, Sammut IA. Inhalation gases or gaseous mediators as neuroprotectants for cerebral ischaemia. Curr Drug Targets. 2013;14:56–73