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Do hyperbaric oxygen-induced seizures cause brain damage?
Epilepsy Research (2012) 100, 37—41
journal homepage: www.elsevier.com/locate/epilepsyres
Do hyperbaric oxygen-induced seizures cause brain damage? Liran Domachevsky a,∗, Chaim G. Pick b, Yehuda Arieli a, Nitzan Krinsky a, Amir Abramovich a, Mirit Eynan a a b
Israel Naval Medical Institute, IDF Medical Corps, Haifa, Israel Department of Anatomy and Anthropology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
Received 6 June 2011; received in revised form 4 January 2012; accepted 7 January 2012 Available online 30 January 2012
KEYWORDS Oxygen; High pressure; Central nervous system; Toxicity; Convulsions; Apoptosis
Summary It is commonly accepted that hyperbaric oxygen-induced seizures, the most severe manifestation of central nervous system oxygen toxicity, are harmless. However, this hypothesis has not been investigated in depth. We used apoptotic markers to determine whether cells in the cortex and hippocampus were damaged by hyperbaric oxygen-induced seizures in mice. Experimental animals were exposed to a pressure of 6 atmospheres absolute breathing oxygen, and were randomly assigned to two groups sacrificed 1 h after the appearance of seizures or 7 days later. Control groups were not exposed to hyperbaric oxygen. Caspase 9, caspase 3, and cytochrome c were used as apoptotic markers. These were measured in the cortex and the hippocampus, and compared between the groups. Levels of caspase 3, cytochrome c, and caspase 9 in the hippocampus were significantly higher in the hyperbaric oxygenexposed groups compared with the control groups 1 week after seizures (p < 0.01). The levels of two fragments of caspase 9 in the cortex were higher in the control group compared with the hyperbaric oxygen-exposed group 1 h after seizures (p < 0.01). Hyperbaric oxygen-induced seizures activate apoptosis in the mouse hippocampus. The reason for the changes in the cortex is not understood. Further investigation is necessary to elucidate the mechanism underlying these findings and their significance. © 2012 Elsevier B.V. All rights reserved.
Introduction
∗ Corresponding author at: Israel Naval Medical Institute, P.O. Box 8040, 31080, Haifa, Israel. Tel.: +972 4 8693040; fax: +972 4 8693240. E-mail address: [email protected] (L. Domachevsky).
The central nervous system (CNS) is a major site of oxygen toxicity when breathing oxygen at high pressure. Prolonged exposure to 100% oxygen at a pressure of more than 3 atmospheres absolute (ATA) will result in CNS oxygen toxicity (Lambertsen, 1965). Also referred to as hyperbaric oxygen (HBO)-induced seizures, these are the most disturbing sign of CNS oxygen toxicity, although they are reversible,
0920-1211/$ — see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2012.01.004
38 disappearing on reduction of the partial pressure of the inspired oxygen. They are believed to cause no residual neurologic damage (Lambertsen, 1965), a belief that has led most researchers to focus on the mechanisms of CNS oxygen toxicity and to abandon the question of its long-term effect on the brain. One way of evaluating whether an insult such as HBOinduced seizures can cause neurologic damage may be to determine whether apoptosis has occurred. Apoptosis is recognized as a mode of ‘‘programmed cell death’’. The process is energy dependent, and has unique biochemical and morphologic features such as plasma and nuclear membrane blebbing, cell shrinkage, and the activation of proteases and endonucleases (Kerr et al., 1972; Häcker, 2000). In principle, there are two well established apoptotic pathways, the extrinsic and the intrinsic. The extrinsic pathway is initiated by binding of a ligand with a transmembrane death receptor (Ashkenazi and Dixit, 1998). Upon ligand binding, cytoplasmic adaptor proteins are recruited along with procaspase 8 and form a death-inducing signaling complex. This complex activates procaspase 8, which propagates into the executive pathway (Kischkel et al., 1995). The intrinsic pathway is mediated by intracellular signals that lead to increased permeability of the outer mitochondrial membrane, resulting in the massive release of proteins into the cytosol from the mitochondrial intermembrane space (Saelens et al., 2004). Cytochrome c, which is one of the proteins released, binds with and activates Apaf1. These recruit procaspase 9 to form an ‘‘apoptosome’’ (Chinnaiyan, 1999). The apoptosome is responsible for the activation of procaspase 9, which propagates into the executive pathway. When the intrinsic and extrinsic pathways converge at the executive pathway, caspase 3, caspase 6, and caspase 7 function as effector or ‘‘executioner’’ caspases, resulting in the activation of endonucleases and proteases (Slee et al., 2001). This results in cleavage of nuclear and cytoplasmic substrates, some of which control DNA repair and nuclear integrity (Datta et al., 1997; Enari et al., 1998; Sakahira et al., 1998). The final step of apoptosis is phagocytosis of the apoptotic cells. In the present study, we examined whether HBO-induced seizures initiate the process of apoptosis in an animal model at two time intervals: immediately after seizures and one week later. Apoptosis was evaluated by measuring levels of caspase 3, caspase 9, and cytochrome c in the cortex and the hippocampus. This model, in which the outcome is generalized convulsions, might perhaps serve to investigate epilepsy, which manifests as repeated generalized seizures.
Materials and methods Animals The experimental protocol was approved by the ethics committee of the Sackler School of Medicine, in compliance with the guidelines for animal experimentation of the National Institutes of Health (DHEW publication 85-23, revised 1995). The minimum possible number of animals was used, and every effort was made to minimize their suffering. Male ICR mice weighing 25—30 g were kept five per cage
L. Domachevsky et al. in a constant 12-h light/dark cycle at room temperature (23 ◦ C). Food (Purina rodent chow) and water were available ad libitum.
Study groups Sixteen mice were used in the study. Eight experimental mice were exposed to a pressure of 6ATA breathing oxygen to induce seizures. They were randomly assigned to two groups sacrificed 1 h after the appearance of HBO-induced seizures (compressed: immediate), or 7 days later (compressed: 1-week), four animals in each. The remaining eight mice, which were not exposed to HBO, served as control groups (control: immediate and control: 1-week). After the animals were sacrificed, the entire brain was quickly removed and the hippocampus was separated from the cortex. Both were kept at a temperature of −80 ◦ C for later biochemical analysis.
Hyperbaric exposure Mice were placed in a double-walled metal exposure cage measuring 13 cm × 25 cm × 25 cm. One side of the cage is made of Perspex, enabling continuous observation of the animal. The mouse was able to move about freely inside the cage. The exposure cage was closed and placed inside a 150-l hyperbaric chamber (Roberto Galeazzi, La Spezia, Italy), which was then sealed. At this stage, the pressure was raised to 6 ATA at a rate of 1 ATA per minute with oxygen flowing through the experimental cage. The mouse has no problem with pressure equalization of the middle ear, and any changes in its behavior can be seen through the Perspex wall. During the exposure to high pressure oxygen, the mouse was observed until the appearance of the first clinical seizures. The exposure was terminated immediately on appearance of the first convulsions, as determined by the observer. The pressure was reduced at 180 kPa/min, and the mouse was removed from the chamber and sacrificed. The hippocampus and cortex were removed on ice as soon as possible after sacrifice (2 ± 1 min), washed in 10 mM phosphate buffer saline (PBS, pH 7.4), placed in liquid nitrogen, and stored at −80 ◦ C for biochemical analysis.
Biochemical assays: Western blot analysis of cytochrome c, caspase 3, and caspase 9 The hippocampus and the cortex from the four animals in each group were thawed and homogenized with SDS buffer (20% glycerol and 6% SDS in 0.12 M Tris buffer, pH 6.8), centrifuged at 13,000 × g for 20 min at 4 ◦ C, and boiled for 10 min. The protein concentration of the brain specimens was quantified by the Bradford method (BioRad Laboratories, Richmond, CA, U.S.A.). Fifty micrograms total proteins were loaded in each gel well. After blotting, the membranes were incubated for 1 h in blocking solution containing 5% skimmed milk in tris-buffered saline Tween-20 (TBST). The membranes were then washed briefly in TBST and incubated overnight at 4 ◦ C with the polyclonal cytochrome c, caspase 3, and caspase 9 antibody diluted 1:1000 (Cell Signaling Technology, Beverly, MA, U.S.A.). Caspase 9 antibody detects levels of full length mouse caspase 9 (49 kDa) and two fragments of mouse caspase 9 (37 kDa and 39 kDa). After 3 repeated washings in TBST, the membranes were incubated at room temperature for 1 h with a secondary antibody (horseradish peroxidaseconjugated goat anti-rabbit IgG) in a 1:2000 dilution (Cell Signaling Technology, Beverly, MA, U.S.A.). After three repeated washings in TBST, the membranes were then developed to enhance detection by chemiluminescence (Pierce Biotechnology, Inc., Rockford, IL, U.S.A.) and exposed to X-ray film. Levels of cytochrome c, caspase 3, and caspase 9 were measured by scanning the films, and were evaluated with densitometry using Tina 2.0 software (Raytest, Straubenhardt, Germany). The value of each band was normalized to the level of the positive control for cytochrome
Do hyperbaric oxygen-induced seizures cause brain damage?
39
Table 1 Caspase 9 levels in the cortex in the immediate groups (sacrificed 1 h after the appearance of HBO-induced seizures). Caspase 9 fragment 37 kDa Compressed (n = 4) Control (n = 4) 39 kDa Compressed (n = 4) Control (n = 4)
Mean ± SD
p value
0.38 ± 0.16 2.26 ± 0.31
<0.01
0.48 ± 0.23 1.41 ± 0.15
<0.01
c, caspase 3, and caspase 9 loaded in each gel well (15 g). Each band density was measured five separate times and averaged.
Statistical analysis
Figure 1 Levels of caspase 3 in the hippocampus in the control group (n = 4) and compressed group (n = 4) 1 week after seizures. Results are presented as mean ± SD. *p < 0.01. P.C., positive control.
Statistical significance was evaluated by the Student t test. All data are expressed as mean ± SD. The level of significance was p < 0.01.
Results Caspase 3, caspase 9, and cytochrome c immunoblotting Cortex—–immediate evaluation There were no significant differences in the levels of caspase 3 and cytochrome c between the compressed and control groups. However, caspase 9 levels were higher in the control group compared with the compressed group in the 37 kDa and 39 kDa fragments (p < 0.01; Table 1), but not in the full length 49 kDa segment. Cortex—–evaluation after 1 week No significant differences were found between the compressed and control groups. Hippocampus—–immediate evaluation No significant differences were found between the compressed and control groups.
Figure 2 Levels of caspase 9 (39 kDa) in the hippocampus in the control group (n = 4) and compressed group (n = 4) 1 week after seizures. Results are presented as mean ± SD. *p < 0.01. P.C., positive control.
Hippocampus—–evaluation after 1 week Levels of caspase 3, cytochrome c, and caspase 9 (the 39 kDa fragment) in the compressed groups were significantly higher compared with the control groups (p < 0.01; Figs. 1—3).
Discussion CNS oxygen toxicity was first described by Paul Bert in 1878 (Bert, 1978), since when it has been extensively investigated. Lambertsen et al. (1987) established a dose-response curve that relates the appearance of CNS oxygen toxicity to the partial pressure of the inspired oxygen. Prolonged exposure to 100% oxygen at a pressure of more than 3 ATA will result in CNS oxygen toxicity (Lambertsen, 1965). The greater the partial pressure of the inspired oxygen, the earlier the symptoms and signs will appear. The manifestations of CNS oxygen toxicity include a variety of
Figure 3 Levels of cytochrome c in the hippocampus in the control group (n = 4) and compressed group (n = 4) 1 week after seizures. Results are presented as mean ± SD. *p < 0.01. P.C., positive control.
40 symptoms and signs (Butler and Thalmann, 1986; Donald, 1992). Non-specific symptoms include nausea, dizziness, abnormal sensations, headache, disorientation, lightheadedness, and a feeling of apprehension. Specific symptoms include blurred vision, tunnel vision, and tinnitus. Signs of CNS oxygen toxicity include respiratory disturbances, eye twitching, twitching of the lips, mouth and forehead, and seizures. There is no consistency in the order of appearance of symptoms and signs prior to the development of seizures. The two main populations that may be affected by this kind of seizure are professional divers, who use dive profiles in which an elevated partial pressure of oxygen exposes them to the risk of HBO-induced seizures, or patients being treated in the hyperbaric chamber for diseases that require the delivery of high pressure oxygen to tissues. In both cases oxygen is the trigger for seizures, and the supply of pure or elevated oxygen is interrupted immediately seizures appear. Seizures are the most disturbing sign of CNS oxygen toxicity, although they are reversible, disappearing on reduction of the oxygen partial pressure. The mechanism of CNS oxygen toxicity is yet to be fully understood. However, the increased production of reactive oxygen and nitrogen species seems to play a pivotal role by oxidizing lipids and proteins (Fridovich, 1998). The hippocampus is influenced by CNS oxygen toxicity, and may be responsible for the initiation of seizures. Chavko and Harabin (1996) have shown that lipid and protein peroxidation occurs in the hippocampus of rats exposed to oxygen at 5 ATA. A study conducted by Gutsaeva et al. (2006) revealed mitochondrial DNA damage in the hippocampus of rats exposed to the same pressure. In another study (Wang et al., 1998), hyperbaric oxygen exposure in rats led to the accumulation of intrasynaptosomal free calcium in the hippocampus. Increased calcium levels stimulate the synthesis of nitric oxide, which is also involved in the pathogenesis of HBO-induced seizures. The question of whether epileptic seizures cause permanent brain damage is one that has been investigated over the years. The leading theory in the past was that only status epilepticus or prolonged seizures result in damage to the brain. This was supported by a number of studies which demonstrated neuronal death both in the hippocampus itself and in extra-hippocampal regions. Tissue damage was caused via apoptotic or necrotic pathways, depending mainly on the time course of injury and the specific cell population (Kubová et al., 2002; Chen et al., 2010). However, over the last three decades there has been a growing body of evidence suggesting that even brief seizures may damage the brain. Cavazos and Sutula (1990) demonstrated that brief sporadic seizures caused by kindling stimulation of limbic structures can induce neuronal loss in the hippocampal formation. Zhang et al. (1998) reported that even a single seizure evoked by kindling induced neuronal apoptosis in the hippocampus. A study conducted by Kotloski et al. (2002) showed that repeated brief seizures in rats induced subfield specific hippocampal neuronal loss, resulting in characteristic deficits in spatial memory function. The pattern of neuronal loss resembled that seen in hippocampal sclerosis in cases of temporal lobe epilepsy, in which CA3c, CA1a, CA1c, and the hilus of the dentate gyrus are involved. It therefore seems clear that even a single
L. Domachevsky et al. seizure may cause some damage to brain tissue, mainly in the hippocampus which is the most vulnerable region. In the present study, we wished to elucidate whether HBO-induced seizures behave in a similar fashion, and whether breathing increased partial pressures of oxygen may thus cause neurologic damage. The commonly held belief that one or more brief seizures have no adverse effect on the brain has for many years been used to support the contention that HBO-induced seizures cause no harm. Indeed, in spite of its toxic effect at higher pressures, oxygen was considered to play a protective role in seizure outcome. Of the small number of studies that have discussed this question, one made the observation that schizophrenic patients treated with hyperbaric oxygen as a mode of therapy did not exhibit any clinical deterioration (Lambertsen, 1965). Another study conducted by Donald (1947) stated that no changes in neurologic integrity, intellectual ability or personality were noted in a three-year follow-up of experimental divers. However, there was no description of the methods used to evaluate patients. We used an animal model to examine whether HBO-induced seizures result in damage to brain tissue. Our criterion was apoptosis, which can appear after stressful events and represents active, programmed cascades by which irreversibly damaged cells are destroyed and removed. The time interval for the appearance of apoptotic markers is variable, and is influenced by a large number of factors (DiPietrantonio et al., 1999; Carambula et al., 2002). We therefore measured the levels of apoptotic markers at two points in time to increase the probability of detection. We examined the hippocampus, because it is known to be involved in both conventional seizures and CNS oxygen toxicity. We also evaluated the cortex to see whether an extra-hippocampal region may be affected. Our results have shown that all three caspase levels were increased in the hippocampus 1 week after seizures, implying that apoptosis occurred in the hippocampus. The elevated levels of caspase 9 and cytochrome c indicate the role of mitochondria in that process. It must be emphasized that the absence of markers at two separate points in time does not rule out apoptosis. The kinetics of apoptosis in the cortex might be quite different from that in the hippocampus, and only strict follow-up can determine whether apoptosis has occurred or not. The finding of decreased apoptotic markers in the cortex immediately after convulsions warrants further investigation, with a repeat examination as the first step. The mechanism by which HBO-induced seizures cause apoptosis remains to be determined. One possibility is via reactive oxygen species, which are known to induce apoptosis (Simon et al., 2000) and are elevated in HBO-induced seizures (Jamieson, 1989). Of the two components of HBO, high atmospheric pressure and oxygen breathing, either or both might be responsible for the initiation of apoptosis, although it may also be attributable to the same factors involved in other types of seizure. It was not the purpose of the present study to determine which of the aforementioned parameters plays a more important role in seizure pathogenesis, but rather to determine whether HBO-induced seizures as such cause postictal CNS changes. In conclusion, HBO-induced seizures activate apoptosis in the mouse hippocampus. Further investigation is necessary
Do hyperbaric oxygen-induced seizures cause brain damage? to elucidate the mechanism underlying this finding and its significance. The findings of the present study must also be interpreted very carefully due to a number of unresolved issues. We do not know exactly which cells are affected, glial or neuronal. We do not know whether the damage is reversible, or whether it is accompanied by structural change or neurologic impairment. Further investigation will be required to correlate our findings with structural changes in the brain, and with comprehensive neurologic and cognitive assessment.
Acknowledgements This study was supported by research grant no. 4440162662 from the Israel Defense Forces Medical Corps. The opinions and assertions contained herein are the private ones of the authors, and are not to be construed as official or as reflecting the views of the Israel Naval Medical Institute.
References Ashkenazi, A., Dixit, V.M., 1998. Death receptors: signaling and modulation. Science 281, 1305—1308. Bert, P., 1978. Barometric Pressure: Researches in Experimental Physiology. Undersea Medical Society, Inc., Bethesda, MD (Translation of: Bert, P., 1878. La Pression Barométrique. Recherches de Physiologie Expérimentale. Masson, Paris.). Butler Jr., F.K., Thalmann, E.D., 1986. Central nervous system oxygen toxicity in closed circuit scuba divers II. Undersea. Biomed. Res. 13, 193—223. Carambula, S.F., Matikainen, T., Lynch, M.P., Flavell, R.A., Dias Gonc ¸alves, P.B., Tilly, J.L., Rueda, B.R., 2002. Caspase-3 is a pivotal mediator of apoptosis during regression of the ovarian corpus luteum. Endocrinology 143, 1495—1501. Cavazos, J.E., Sutula, T.P., 1990. Progressive neuronal loss induced by kindling: a possible mechanism for mossy fiber synaptic reorganization and hippocampal sclerosis. Brain Res. 527, 1—6 (Erratum in Brain Res. 541, 179, 1991). Chavko, M., Harabin, A.L., 1996. Regional lipid peroxidation and protein oxidation in rat brain after hyperbaric oxygen exposure. Free Radic. Biol. Med. 20, 973—978. Chen, S., Fujita, S., Koshikawa, N., Kobayashi, M., 2010. Pilocarpine-induced status epilepticus causes acute interneuron loss and hyper-excitatory propagation in rat insular cortex. Neuroscience 166, 341—353. Chinnaiyan, A.M., 1999. The apoptosome: heart and soul of the cell death machine. Neoplasia 1, 5—15. Datta, R., Kojima, H., Yoshida, K., Kufe, D., 1997. Caspase3-mediated cleavage of protein kinase C Â in induction of apoptosis. J. Biol. Chem. 272, 20317—20320. DiPietrantonio, A.M., Hsieh, T., Wu, J.M., 1999. Activation of caspase 3 in HL-60 cells exposed to hydrogen peroxide. Biochem. Biophys. Res. Commun. 255, 477—482. Donald, K., 1992. Oxygen and the Diver. The SPA Ltd, Hanley Swan, UK. Donald, K.W., 1947. Oxygen poisoning in man: signs and symptoms of oxygen poisoning. Br. Med. J. 1, 712—717.
41 Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., Nagata, S., 1998. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43—50 (Erratum 1998. Nature 393, 396). Fridovich, I., 1998. Oxygen toxicity: a radical explanation. J. Exp. Biol. 201, 1203—1209. Gutsaeva, D.R., Suliman, H.B., Carraway, M.S., Demchenko, I.T., Piantadosi, C.A., 2006. Oxygen-induced mitochondrial biogenesis in the rat hippocampus. Neuroscience 137, 493—504. Häcker, G., 2000. The morphology of apoptosis. Cell Tissue Res. 301, 5—17. Jamieson, D., 1989. Oxygen toxicity and reactive oxygen metabolites in mammals. Free Radic. Biol. Med. 7, 87—108. Kerr, J.F.R., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239—257. Kischkel, F.C., Hellbardt, S., Behrmann, I., Germer, M., Pawlita, M., Krammer, P.H., Peter, M.E., 1995. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 14, 5579—5588. Kotloski, R., Lynch, M., Lauersdorf, S., Sutula, T., 2002. Repeated brief seizures induce progressive hippocampal neuron loss and memory deficits. Prog. Brain Res. 135, 95—110. Kubová, H., Druga, R., Haugvicová, R., Suchomelová, L., Pitkanen, A., 2002. Dynamic changes of status epilepticus-induced neuronal degeneration in the mediodorsal nucleus of the thalamus during postnatal development of the rat. Epilepsia 43 (Suppl. 5), 54—60. Lambertsen, C.J., 1965. Effects of oxygen at high partial pressure. In: Fenn, W.O., Rahn, H. (Eds.), Handbook of Physiology. Section 3: Respiration, II. American Physiological Society, Washington, DC, pp. 1027—1046. Lambertsen, C.J., Clark, J.M., Gelfand, R., Pisarello, J.B., Cobbs, W.H., Bevilacqua, J.E., Schwartz, D.M., Montabana, D.J., Leach, C.S., Johnson, P.C., Fletcher, D.E., 1987. Definition of tolerance to continuous hyperoxia in man: an abstract report of predictive studies V. In: Bove, A.A., Bachrach, A.J., Greenbaum Jr., L.J. (Eds.), Underwater and Hyperbaric Physiology IX. Proceedings of the Ninth International Symposium on Underwater and Hyperbaric Physiology. Undersea and Hyperbaric Medical Society, Inc., Bethesda, MD, pp. 717—735. Saelens, X., Festjens, N., Vande Walle, L., van Gurp, M., van Loo, G., Vandenabeele, P., 2004. Toxic proteins released from mitochondria in cell death. Oncogene 23, 2861—2874. Sakahira, H., Enari, M., Nagata, S., 1998. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391, 96—99. Simon, H.-U., Haj-Yehia, A., Levi-Schaffer, F., 2000. Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 5, 415—418. Slee, E.A., Adrain, C., Martin, S.J., 2001. Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J. Biol. Chem. 276, 7320—7326. Wang, W.J., Ho, X.P., Yan, Y.L., Yan, T.H., Li, C.L., 1998. Intrasynaptosomal free calcium and nitric oxide metabolism in central nervous system oxygen toxicity. Aviat. Space Environ. Med. 69, 551—555. Zhang, L.-X., Smith, M.A., Li, X.-L., Weiss, S.R.B., Post, R.M., 1998. Apoptosis of hippocampal neurons after amygdala kindled seizures. Brain Res. Mol. Brain Res. 55, 198—208.
journal homepage: www.elsevier.com/locate/epilepsyres
Do hyperbaric oxygen-induced seizures cause brain damage? Liran Domachevsky a,∗, Chaim G. Pick b, Yehuda Arieli a, Nitzan Krinsky a, Amir Abramovich a, Mirit Eynan a a b
Israel Naval Medical Institute, IDF Medical Corps, Haifa, Israel Department of Anatomy and Anthropology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
Received 6 June 2011; received in revised form 4 January 2012; accepted 7 January 2012 Available online 30 January 2012
KEYWORDS Oxygen; High pressure; Central nervous system; Toxicity; Convulsions; Apoptosis
Summary It is commonly accepted that hyperbaric oxygen-induced seizures, the most severe manifestation of central nervous system oxygen toxicity, are harmless. However, this hypothesis has not been investigated in depth. We used apoptotic markers to determine whether cells in the cortex and hippocampus were damaged by hyperbaric oxygen-induced seizures in mice. Experimental animals were exposed to a pressure of 6 atmospheres absolute breathing oxygen, and were randomly assigned to two groups sacrificed 1 h after the appearance of seizures or 7 days later. Control groups were not exposed to hyperbaric oxygen. Caspase 9, caspase 3, and cytochrome c were used as apoptotic markers. These were measured in the cortex and the hippocampus, and compared between the groups. Levels of caspase 3, cytochrome c, and caspase 9 in the hippocampus were significantly higher in the hyperbaric oxygenexposed groups compared with the control groups 1 week after seizures (p < 0.01). The levels of two fragments of caspase 9 in the cortex were higher in the control group compared with the hyperbaric oxygen-exposed group 1 h after seizures (p < 0.01). Hyperbaric oxygen-induced seizures activate apoptosis in the mouse hippocampus. The reason for the changes in the cortex is not understood. Further investigation is necessary to elucidate the mechanism underlying these findings and their significance. © 2012 Elsevier B.V. All rights reserved.
Introduction
∗ Corresponding author at: Israel Naval Medical Institute, P.O. Box 8040, 31080, Haifa, Israel. Tel.: +972 4 8693040; fax: +972 4 8693240. E-mail address: [email protected] (L. Domachevsky).
The central nervous system (CNS) is a major site of oxygen toxicity when breathing oxygen at high pressure. Prolonged exposure to 100% oxygen at a pressure of more than 3 atmospheres absolute (ATA) will result in CNS oxygen toxicity (Lambertsen, 1965). Also referred to as hyperbaric oxygen (HBO)-induced seizures, these are the most disturbing sign of CNS oxygen toxicity, although they are reversible,
0920-1211/$ — see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2012.01.004
38 disappearing on reduction of the partial pressure of the inspired oxygen. They are believed to cause no residual neurologic damage (Lambertsen, 1965), a belief that has led most researchers to focus on the mechanisms of CNS oxygen toxicity and to abandon the question of its long-term effect on the brain. One way of evaluating whether an insult such as HBOinduced seizures can cause neurologic damage may be to determine whether apoptosis has occurred. Apoptosis is recognized as a mode of ‘‘programmed cell death’’. The process is energy dependent, and has unique biochemical and morphologic features such as plasma and nuclear membrane blebbing, cell shrinkage, and the activation of proteases and endonucleases (Kerr et al., 1972; Häcker, 2000). In principle, there are two well established apoptotic pathways, the extrinsic and the intrinsic. The extrinsic pathway is initiated by binding of a ligand with a transmembrane death receptor (Ashkenazi and Dixit, 1998). Upon ligand binding, cytoplasmic adaptor proteins are recruited along with procaspase 8 and form a death-inducing signaling complex. This complex activates procaspase 8, which propagates into the executive pathway (Kischkel et al., 1995). The intrinsic pathway is mediated by intracellular signals that lead to increased permeability of the outer mitochondrial membrane, resulting in the massive release of proteins into the cytosol from the mitochondrial intermembrane space (Saelens et al., 2004). Cytochrome c, which is one of the proteins released, binds with and activates Apaf1. These recruit procaspase 9 to form an ‘‘apoptosome’’ (Chinnaiyan, 1999). The apoptosome is responsible for the activation of procaspase 9, which propagates into the executive pathway. When the intrinsic and extrinsic pathways converge at the executive pathway, caspase 3, caspase 6, and caspase 7 function as effector or ‘‘executioner’’ caspases, resulting in the activation of endonucleases and proteases (Slee et al., 2001). This results in cleavage of nuclear and cytoplasmic substrates, some of which control DNA repair and nuclear integrity (Datta et al., 1997; Enari et al., 1998; Sakahira et al., 1998). The final step of apoptosis is phagocytosis of the apoptotic cells. In the present study, we examined whether HBO-induced seizures initiate the process of apoptosis in an animal model at two time intervals: immediately after seizures and one week later. Apoptosis was evaluated by measuring levels of caspase 3, caspase 9, and cytochrome c in the cortex and the hippocampus. This model, in which the outcome is generalized convulsions, might perhaps serve to investigate epilepsy, which manifests as repeated generalized seizures.
Materials and methods Animals The experimental protocol was approved by the ethics committee of the Sackler School of Medicine, in compliance with the guidelines for animal experimentation of the National Institutes of Health (DHEW publication 85-23, revised 1995). The minimum possible number of animals was used, and every effort was made to minimize their suffering. Male ICR mice weighing 25—30 g were kept five per cage
L. Domachevsky et al. in a constant 12-h light/dark cycle at room temperature (23 ◦ C). Food (Purina rodent chow) and water were available ad libitum.
Study groups Sixteen mice were used in the study. Eight experimental mice were exposed to a pressure of 6ATA breathing oxygen to induce seizures. They were randomly assigned to two groups sacrificed 1 h after the appearance of HBO-induced seizures (compressed: immediate), or 7 days later (compressed: 1-week), four animals in each. The remaining eight mice, which were not exposed to HBO, served as control groups (control: immediate and control: 1-week). After the animals were sacrificed, the entire brain was quickly removed and the hippocampus was separated from the cortex. Both were kept at a temperature of −80 ◦ C for later biochemical analysis.
Hyperbaric exposure Mice were placed in a double-walled metal exposure cage measuring 13 cm × 25 cm × 25 cm. One side of the cage is made of Perspex, enabling continuous observation of the animal. The mouse was able to move about freely inside the cage. The exposure cage was closed and placed inside a 150-l hyperbaric chamber (Roberto Galeazzi, La Spezia, Italy), which was then sealed. At this stage, the pressure was raised to 6 ATA at a rate of 1 ATA per minute with oxygen flowing through the experimental cage. The mouse has no problem with pressure equalization of the middle ear, and any changes in its behavior can be seen through the Perspex wall. During the exposure to high pressure oxygen, the mouse was observed until the appearance of the first clinical seizures. The exposure was terminated immediately on appearance of the first convulsions, as determined by the observer. The pressure was reduced at 180 kPa/min, and the mouse was removed from the chamber and sacrificed. The hippocampus and cortex were removed on ice as soon as possible after sacrifice (2 ± 1 min), washed in 10 mM phosphate buffer saline (PBS, pH 7.4), placed in liquid nitrogen, and stored at −80 ◦ C for biochemical analysis.
Biochemical assays: Western blot analysis of cytochrome c, caspase 3, and caspase 9 The hippocampus and the cortex from the four animals in each group were thawed and homogenized with SDS buffer (20% glycerol and 6% SDS in 0.12 M Tris buffer, pH 6.8), centrifuged at 13,000 × g for 20 min at 4 ◦ C, and boiled for 10 min. The protein concentration of the brain specimens was quantified by the Bradford method (BioRad Laboratories, Richmond, CA, U.S.A.). Fifty micrograms total proteins were loaded in each gel well. After blotting, the membranes were incubated for 1 h in blocking solution containing 5% skimmed milk in tris-buffered saline Tween-20 (TBST). The membranes were then washed briefly in TBST and incubated overnight at 4 ◦ C with the polyclonal cytochrome c, caspase 3, and caspase 9 antibody diluted 1:1000 (Cell Signaling Technology, Beverly, MA, U.S.A.). Caspase 9 antibody detects levels of full length mouse caspase 9 (49 kDa) and two fragments of mouse caspase 9 (37 kDa and 39 kDa). After 3 repeated washings in TBST, the membranes were incubated at room temperature for 1 h with a secondary antibody (horseradish peroxidaseconjugated goat anti-rabbit IgG) in a 1:2000 dilution (Cell Signaling Technology, Beverly, MA, U.S.A.). After three repeated washings in TBST, the membranes were then developed to enhance detection by chemiluminescence (Pierce Biotechnology, Inc., Rockford, IL, U.S.A.) and exposed to X-ray film. Levels of cytochrome c, caspase 3, and caspase 9 were measured by scanning the films, and were evaluated with densitometry using Tina 2.0 software (Raytest, Straubenhardt, Germany). The value of each band was normalized to the level of the positive control for cytochrome
Do hyperbaric oxygen-induced seizures cause brain damage?
39
Table 1 Caspase 9 levels in the cortex in the immediate groups (sacrificed 1 h after the appearance of HBO-induced seizures). Caspase 9 fragment 37 kDa Compressed (n = 4) Control (n = 4) 39 kDa Compressed (n = 4) Control (n = 4)
Mean ± SD
p value
0.38 ± 0.16 2.26 ± 0.31
<0.01
0.48 ± 0.23 1.41 ± 0.15
<0.01
c, caspase 3, and caspase 9 loaded in each gel well (15 g). Each band density was measured five separate times and averaged.
Statistical analysis
Figure 1 Levels of caspase 3 in the hippocampus in the control group (n = 4) and compressed group (n = 4) 1 week after seizures. Results are presented as mean ± SD. *p < 0.01. P.C., positive control.
Statistical significance was evaluated by the Student t test. All data are expressed as mean ± SD. The level of significance was p < 0.01.
Results Caspase 3, caspase 9, and cytochrome c immunoblotting Cortex—–immediate evaluation There were no significant differences in the levels of caspase 3 and cytochrome c between the compressed and control groups. However, caspase 9 levels were higher in the control group compared with the compressed group in the 37 kDa and 39 kDa fragments (p < 0.01; Table 1), but not in the full length 49 kDa segment. Cortex—–evaluation after 1 week No significant differences were found between the compressed and control groups. Hippocampus—–immediate evaluation No significant differences were found between the compressed and control groups.
Figure 2 Levels of caspase 9 (39 kDa) in the hippocampus in the control group (n = 4) and compressed group (n = 4) 1 week after seizures. Results are presented as mean ± SD. *p < 0.01. P.C., positive control.
Hippocampus—–evaluation after 1 week Levels of caspase 3, cytochrome c, and caspase 9 (the 39 kDa fragment) in the compressed groups were significantly higher compared with the control groups (p < 0.01; Figs. 1—3).
Discussion CNS oxygen toxicity was first described by Paul Bert in 1878 (Bert, 1978), since when it has been extensively investigated. Lambertsen et al. (1987) established a dose-response curve that relates the appearance of CNS oxygen toxicity to the partial pressure of the inspired oxygen. Prolonged exposure to 100% oxygen at a pressure of more than 3 ATA will result in CNS oxygen toxicity (Lambertsen, 1965). The greater the partial pressure of the inspired oxygen, the earlier the symptoms and signs will appear. The manifestations of CNS oxygen toxicity include a variety of
Figure 3 Levels of cytochrome c in the hippocampus in the control group (n = 4) and compressed group (n = 4) 1 week after seizures. Results are presented as mean ± SD. *p < 0.01. P.C., positive control.
40 symptoms and signs (Butler and Thalmann, 1986; Donald, 1992). Non-specific symptoms include nausea, dizziness, abnormal sensations, headache, disorientation, lightheadedness, and a feeling of apprehension. Specific symptoms include blurred vision, tunnel vision, and tinnitus. Signs of CNS oxygen toxicity include respiratory disturbances, eye twitching, twitching of the lips, mouth and forehead, and seizures. There is no consistency in the order of appearance of symptoms and signs prior to the development of seizures. The two main populations that may be affected by this kind of seizure are professional divers, who use dive profiles in which an elevated partial pressure of oxygen exposes them to the risk of HBO-induced seizures, or patients being treated in the hyperbaric chamber for diseases that require the delivery of high pressure oxygen to tissues. In both cases oxygen is the trigger for seizures, and the supply of pure or elevated oxygen is interrupted immediately seizures appear. Seizures are the most disturbing sign of CNS oxygen toxicity, although they are reversible, disappearing on reduction of the oxygen partial pressure. The mechanism of CNS oxygen toxicity is yet to be fully understood. However, the increased production of reactive oxygen and nitrogen species seems to play a pivotal role by oxidizing lipids and proteins (Fridovich, 1998). The hippocampus is influenced by CNS oxygen toxicity, and may be responsible for the initiation of seizures. Chavko and Harabin (1996) have shown that lipid and protein peroxidation occurs in the hippocampus of rats exposed to oxygen at 5 ATA. A study conducted by Gutsaeva et al. (2006) revealed mitochondrial DNA damage in the hippocampus of rats exposed to the same pressure. In another study (Wang et al., 1998), hyperbaric oxygen exposure in rats led to the accumulation of intrasynaptosomal free calcium in the hippocampus. Increased calcium levels stimulate the synthesis of nitric oxide, which is also involved in the pathogenesis of HBO-induced seizures. The question of whether epileptic seizures cause permanent brain damage is one that has been investigated over the years. The leading theory in the past was that only status epilepticus or prolonged seizures result in damage to the brain. This was supported by a number of studies which demonstrated neuronal death both in the hippocampus itself and in extra-hippocampal regions. Tissue damage was caused via apoptotic or necrotic pathways, depending mainly on the time course of injury and the specific cell population (Kubová et al., 2002; Chen et al., 2010). However, over the last three decades there has been a growing body of evidence suggesting that even brief seizures may damage the brain. Cavazos and Sutula (1990) demonstrated that brief sporadic seizures caused by kindling stimulation of limbic structures can induce neuronal loss in the hippocampal formation. Zhang et al. (1998) reported that even a single seizure evoked by kindling induced neuronal apoptosis in the hippocampus. A study conducted by Kotloski et al. (2002) showed that repeated brief seizures in rats induced subfield specific hippocampal neuronal loss, resulting in characteristic deficits in spatial memory function. The pattern of neuronal loss resembled that seen in hippocampal sclerosis in cases of temporal lobe epilepsy, in which CA3c, CA1a, CA1c, and the hilus of the dentate gyrus are involved. It therefore seems clear that even a single
L. Domachevsky et al. seizure may cause some damage to brain tissue, mainly in the hippocampus which is the most vulnerable region. In the present study, we wished to elucidate whether HBO-induced seizures behave in a similar fashion, and whether breathing increased partial pressures of oxygen may thus cause neurologic damage. The commonly held belief that one or more brief seizures have no adverse effect on the brain has for many years been used to support the contention that HBO-induced seizures cause no harm. Indeed, in spite of its toxic effect at higher pressures, oxygen was considered to play a protective role in seizure outcome. Of the small number of studies that have discussed this question, one made the observation that schizophrenic patients treated with hyperbaric oxygen as a mode of therapy did not exhibit any clinical deterioration (Lambertsen, 1965). Another study conducted by Donald (1947) stated that no changes in neurologic integrity, intellectual ability or personality were noted in a three-year follow-up of experimental divers. However, there was no description of the methods used to evaluate patients. We used an animal model to examine whether HBO-induced seizures result in damage to brain tissue. Our criterion was apoptosis, which can appear after stressful events and represents active, programmed cascades by which irreversibly damaged cells are destroyed and removed. The time interval for the appearance of apoptotic markers is variable, and is influenced by a large number of factors (DiPietrantonio et al., 1999; Carambula et al., 2002). We therefore measured the levels of apoptotic markers at two points in time to increase the probability of detection. We examined the hippocampus, because it is known to be involved in both conventional seizures and CNS oxygen toxicity. We also evaluated the cortex to see whether an extra-hippocampal region may be affected. Our results have shown that all three caspase levels were increased in the hippocampus 1 week after seizures, implying that apoptosis occurred in the hippocampus. The elevated levels of caspase 9 and cytochrome c indicate the role of mitochondria in that process. It must be emphasized that the absence of markers at two separate points in time does not rule out apoptosis. The kinetics of apoptosis in the cortex might be quite different from that in the hippocampus, and only strict follow-up can determine whether apoptosis has occurred or not. The finding of decreased apoptotic markers in the cortex immediately after convulsions warrants further investigation, with a repeat examination as the first step. The mechanism by which HBO-induced seizures cause apoptosis remains to be determined. One possibility is via reactive oxygen species, which are known to induce apoptosis (Simon et al., 2000) and are elevated in HBO-induced seizures (Jamieson, 1989). Of the two components of HBO, high atmospheric pressure and oxygen breathing, either or both might be responsible for the initiation of apoptosis, although it may also be attributable to the same factors involved in other types of seizure. It was not the purpose of the present study to determine which of the aforementioned parameters plays a more important role in seizure pathogenesis, but rather to determine whether HBO-induced seizures as such cause postictal CNS changes. In conclusion, HBO-induced seizures activate apoptosis in the mouse hippocampus. Further investigation is necessary
Do hyperbaric oxygen-induced seizures cause brain damage? to elucidate the mechanism underlying this finding and its significance. The findings of the present study must also be interpreted very carefully due to a number of unresolved issues. We do not know exactly which cells are affected, glial or neuronal. We do not know whether the damage is reversible, or whether it is accompanied by structural change or neurologic impairment. Further investigation will be required to correlate our findings with structural changes in the brain, and with comprehensive neurologic and cognitive assessment.
Acknowledgements This study was supported by research grant no. 4440162662 from the Israel Defense Forces Medical Corps. The opinions and assertions contained herein are the private ones of the authors, and are not to be construed as official or as reflecting the views of the Israel Naval Medical Institute.
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