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Early evidence of a regulated response to hypoxaemia in sheep that preserves the brain cortex
Neuroscience Letters 394 (2006) 174–178
Early evidence of a regulated response to hypoxaemia in sheep that preserves the brain cortex Des Gorman a,b,∗ , Huang Yi Lin a , Chris Williams b a
Department of Medicine, University of Auckland, New Zealand b Liggins Institute, University of Auckland, New Zealand
Received 16 September 2005; received in revised form 12 October 2005; accepted 12 October 2005
Abstract Hypoxaemia consequent to inspired carbon monoxide (CO), and to other causes, often does not injure the brain cortex. At least five types of brain and heart protective cardiovascular response to hypoxaemia have been reported. The underlying mechanism is unknown. The present study was designed to test the hypothesis that the reaction to inspired CO involves the amygdala as this structure is thought to be central to stress responsivity; involvement would support the additional hypothesis that the somatic response to CO-hypoxaemia is regulated. Eighteen ewes were randomly allocated to control and two CO groups. The CO groups were exposed to 1% CO for 120 min and killed either 5 or 15 days later. This exposure caused isolated white matter brain injury and a transient increase in protein-kinase C (gamma) activity in the pyramidal neurons in the nuclei of the central and basal–lateral amygdala and in the neurons of the audio-cortex (p < 0.05). This was associated with evidence of a sympathetic response. It would seem reasonable to hypothesise both that the amygdala is important in the processes by which the hypoxaemic effects of CO on the brain are prevented, delayed and/or mitigated and that these processes are regulated. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Amygdala; Carbon monoxide; Circadian rhythm; Hypoxaemia; Hypoxia; Protein-kinase C (gamma)
Hypoxaemia induces circulatory responses that could delay and/or reduce any hypoxic effect on the brain, and especially the cortex, and the heart. If concurrent anaesthesia is avoided and cerebrovascular behaviour is preserved, then even prolonged severe carbon monoxide (CO)-induced hypoxaemia only causes isolated brain white matter injury [9–13]. These putatively protective responses do not seem to be either initiated or maintained by hypoxia per se, but may be dependent on nitric oxide synthetase (NOS) and/or haeme oxygenase (HO) function [11,13,19–21]. First, brain and coronary artery blood flow is increased during hypoxaemia such that oxygen (O2 ) delivery and uptake is maintained [15,17,29]. This requires increased heart rate and work and has a finite limit, perhaps at a oxyhaemoglobin (OHb) concentration of less than 20–30%, which when exhausted will result in sudden circulatory failure and onset of marked brain and heart hypoxia [15,17]. ∗ Corresponding author at: Department of Medicine, University of Auckland, Private Bag 92019, Auckland, New Zealand. Tel.: +64 9 3737599x86261; fax: +64 9 3082379. E-mail address: [email protected] (D. Gorman).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.10.075
Second, there is evidence of a sympathetic response to COhypoxaemia [9–13], evidenced by tachycardia, tachypnoea and peripheral vasoconstriction (concurrent systemic hypertension and a fall in skin temperature), and hyperglycaemia, which would also facilitate preferential perfusion of the brain and heart. Third, brain blood flow may well be redirected from the white matter to the cortex during CO-hypoxaemia and under the influence of HO and NOS, which are certainly induced, such that the cortex is preserved at the expense of the white matter [11,13]. Fourth, neurons may hibernate when confronted with sudden CO- and inert diluent-hypoxaemia; adenosine may be a trigger in this process [6,7]. Fifth, red blood cells are released into the blood, presumably from the spleen, to increase O2 carriage in response to a COinduced fall in blood O2 content [9–13]. The question here is the extent to which these cardiovascular reactions to CO and hypoxic hypoxaemia exist as a regulated stress response [10,11,13,15,17,22,25]. The rationale for this preliminary study is best expressed by analogy; the hypothesis that an orchestra exists, proposed on the basis of finding some musicians playing instruments, has greater merit if an active conductor is discovered. The conductor we chose to observe is
D. Gorman et al. / Neuroscience Letters 394 (2006) 174–178
the amygdala. This is the most well-recognised brain regulatory centre for stress responses [25,2,4,16,23]. It is reasonable to hypothesise then that the amygdala should be active if there is a regulated body reaction to inhaled CO. We have previously shown that NOS and HO are induced in many areas of the brain by CO inhalation [11,13]. Secondary enzyme systems such as protein-kinase C (PKC) also appear to be increasingly active; PKC is a family of protein serine/threonine kinases consisting of multiple isoforms that are involved in various cellular functions; they play a fundamental role in signalling mechanisms leading to mitogenesis and proliferation of cells, apoptosis, platelet activation, remodelling of the actin cytoskeleton and modulation of ion channels [3,5,8,14,18,27]. The aim of this preliminary study then was to determine if the somatic response to CO-hypoxaemia is associated with NOS, HO or PKC induction in the amygdala. Eighteen 3- to 4-year-old Romney ewes, which weighed 50–60 kg, were obtained from the Animal Research Unit of the Faculty of Medical and Health Sciences, University of Auckland and randomly allocated to equal sized experimental Control, CO-1, and CO-2 groups. The experiment was approved by the University of Auckland Animal Ethics Committee. The surgical procedure was as described previously [10,15,28]. In brief, the sheep were anaesthetised with 3% halothane in O2 . First, electrodes for EEG recording were installed onto the surface of the dura overlying the fronto-parietal cortex. Second, the right common carotid artery and tarsal artery and vein were catheterised for blood pressure tension monitoring and temperature electrodes were installed subcutaneously. Postoperative care of the sheep was also as we have described before [10,15]. Three days later our cannulation technique was used to administer CO at a flow rate of 50 ml kg−1 min−1 for 120 min [15]. Control sheep underwent tracheal cannulation, but, were not subject to CO. The electrophysiological parameters were continuously monitored, processed on line, averaged over 1 min intervals and recorded using a custom-built acquisition program. Either 5 or 15 days after the CO exposure, sheep were killed with a barbiturate overdose. Both common carotid arteries were cannulated for in situ brain perfusion fixation with 1000 ml chilled heparinised saline followed by 1000 ml chilled 10% phosphate-buffered formalin. The brain was removed and cut into 50 mm coronal tissue blocks and post-fixed for 7 days in a 10% phosphate-buffered formalin solution. Following gradual dehydration, tissue blocks were paraffin embedded and 6 m thick sections were cut for histological examination. Sections at the stratum level, with amygdala included, were used for histological (H&E staining) and immunochemistry study. To identify the amygdala accurately, the sections were examined microscopically (NIKON ECLIPSE E800) and the outline of the amygdala, caudate nucleus and putamen were engraved on the back of the slides. Positively stained cells in the amygdala were counted in four 0.05 mm2 areas of the coronal section at the stratum level of the right hemisphere using an image analyser (Carl Zeiss Vision GmbH system, Axiovision 3.1, Germany) at 200× magnification. Only well-stained cells were counted and the positive
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counts were averaged for each slide. The slides were codified such that the outcome assessor was unaware of whether or not the sheep had been subject to a CO exposure. The ABC immunochemistry technique was used for both single and double-labeling HO and NOS as described before [11,13]; Vector VIP Substrate (Vector VIP Substrate Kit For Peroxidase, SK 4600, Vector Laboratories, CA, USA) was used instead of DAB for the secondary antigen labeling. For all enzymes, the antibody dilution was 1:200. To determine the integrity of the staining technique, immunohistochemical controls were obtained by replacing the primary antibodies to HO and NOS with buffer, using normal serum at equivalent dilutions. Pre-absorption studies were also performed using the corresponding antigens in cell lysate (Transduction Laboratories, Lexington, KY, USA); 1 g of primary antibody was incubated with 10 g of corresponding cell lysate in the same incubation solution as described above, for 3 days at 4 ◦ C. The antibody/antigen mix was then used instead of the primary antibody solution. In the double staining studies, incompatible antibodies were substituted for the species-specific secondary antibodies and/or one of the two primary antibodies was omitted. No positively stained cells were seen in those sections where the primary antibodies were eliminated from the incubation solution. When the secondary antibody was eliminated or one of the primary antibodies was omitted, there was no positive staining of the antibody-devoid antigen. Finally, when the primary antibodies against HO and NOS were pre-absorbed with their corresponding antigens, staining was either absent or very reduced. To detect PKC activities, mouse monoclonal antibody to PKC alpha (␣), PKC beta (), PKC gamma (␥) and PKC iota () (Transduction Laboratories, Lexington, KY, USA) were used according to the ABC method (ABC elite kit, Vectastain Universal, PK-6200, Vector laboratories, CA, USA) [10–13]. To classify the nature of PKC␥ positive cells in the amygdala and in the audio-cortex, double-labelling for antibody to PKC␥ with antibodies to Glial Fibrillary Acidic Protein (GFAP) (mouse IgG, diluted 1:200, cats. no. 814369. Boehringer Mannheim Biochemica, Germany), GABA a1, a3 (rabbit IgG, diluted 1:300, product no. G4416, G4291, SIGMA, USA), tyrosine hydroxylase (TH, rabbit IgG, diluted 1:150, CA-101, Protos Biotech corporation, NY, USA), and, NOS-1 and HO-1 (rabbit IgG, diluted 1:250, mouse IgG, diluted 1:200, Transduction Laboratories, USA) was performed using the ABC method and Vector VIP substrate (Vector VIP substrate kit for peroxidase, SK-4600, Vector laboratories, CA, USA), instead of DAB for the second antigen labeling. Again, stain integrity was established as no positively stained cells were seen when the primary or secondary antibodies for the PKC isoforms were replaced with buffer and normal serum at equivalent dilutions and the primary and secondary steps were omitted. In the double labeling studies for PKC␥ and GFAP, GABA and TH, omission of one of the primary antibodies also did not result in positive staining of the antibody-devoid antigen. When the primary antibody of PKC␥ was pre-absorbed with its corresponding antigen, immuno-staining was absent or considerably decreased.
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Data were analysed using one-way analyses of variance (Sigmastat Statistical Analysis System, version 2.03, and Sigmaplot 2000, Jandel Scientific, Ekrath, Germany). A significance level of 0.05 was chosen and all the data were recorded as mean ± S.E.M. As we have previously reported [9–13,15]: an inspired admixture of 1% CO in air caused agitation and then narcosis in sheep; and, subsequent histological and immunohistochemical changes were limited to periventricular white matter infarcts and a related glial reaction, axonal dysfunction, and a widespread induction of neuronal HO and NOS. Similarly, as before, the narcosis was accompanied by hyperpnoea and an increase in arterial carboxyhaemoglobin (COHb) level (60–70%), red blood cell mass, Hb concentration, blood lactate and glucose, heart rate, mean arterial blood pressure and EEG intensity (power). By contrast, the O2 Hb concentration fell, as did the arterial O2 content, blood pH, body surface temperature and EEG frequency. Following the CO exposure, the sheep again recovered very quickly in all respects but for the circadian rhythm of heart rate and EEG activity, which could not be identified until 4 days later (3.9 ± 0.8 days) in 9 of the 11 CO exposed sheep for the former and for about 2 days after the exposure (1.8 ± 0.3 days) in 6 of the 11 for the latter. The physiological changes cited above were associated with a similarly transient increase in PKC␥ activity, but not NOS or HO, in the neurons of the basal–lateral amygdala (BLA) and audio-cortex (Fig. 1). The activity of PKC␥ in the cells of the nucleus of the central and BLA and in the cells of the audio-cortex was increased in those sheep killed 5 days after the CO exposure (p < 0.05, Fig. 1); whereas in those sheep killed 15 days post-CO, the PKC␥ activity was not significantly different from control levels. There was no difference in the activity of the other PKC isoforms in either of the experimental or the control groups. The double labeling studies demonstrated that the PKC␥ positive cells were pyramidal neurons and the co-localisation of both GABA a1 and a3 receptors on these PKC␥ positive neurons further demonstrates that these are GABAergic neurons (Fig. 2). There was no colocalisation of TH, HO-1 and NOS-1 activity on these PKC␥ positive cells. This preliminary study shows again that there is evidence of a stress reaction during and immediately after the CO exposure and that this coincides with a transient increase of PKC␥ activity in the pyramidal GABAergic (a1 and a3 receptor bearing) neurons of the amygdala. As cited, the stress-response regulatory role of the amygdala is reasonably well established [25,4,23]. The BLA is normally under tonal inhibition by GABAergic neurons and functions as a major integrator and relay centre for anxiety and panic responses; it has been associated with the regulation of stressinduced cardiovascular response [25,2,16]. Protein-kinase C exists in multiple isoforms that are involved in various cellular functions [3,5,8,14,18,27]. The PKC␥ isoform is mainly expressed in the CNS and appears to be localised in the dendrites and cell bodies of neurons [24,26]; the physiological role of PKC␥ is still uncertain. Our study suggests that it may be involved in the response to CO-induced hypoxaemia.
Fig. 1. Loss of apparent normal day–night (24 h) rhythms in EEG and heart rate, and, the counts of PKC␥ positive neurons in the BLA of sheep brain postCO exposure. (Upper panel): the distribution of sheep that do not have a clear 24 h rhythm of EEG and heart rate showing recovery within 5 days of the CO exposure. (Middle panel): the EEG and heart rate for an individual sheep showing a suppressed rhythmicity for EEG frequency (2 days) and heart rate (5 days) postCO. (Lower panel): a bar graph showing the number of PKC␥ positive cells in the amygdala and audio-cortex of control sheep and those CO-exposed sheep killed 5 and 15 days after the exposure respectively; the 5-day-activity levels were significantly increased in comparison to that of control sheep and those killed after 15 days (p < 0.05, one-way ANOVA).
D. Gorman et al. / Neuroscience Letters 394 (2006) 174–178
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The exact way in which CO increases PKC␥ activity, and, if or how PKC␥ in the pyramidal neurons of the BLA affects the physiological response to CO-hypoxaemia, is unknown. Of note, protein NPAS2, a transcription factor that regulates circadian rhythms, is a haemoprotein for which the DNA binding is selectively regulated by CO [1]. However, we have clearly found an active conductor (the amygdala) and are in a better position to argue that the musicians (at least five types of cardiovascular response to hypoxaemia) we have found are part of an orchestra (the response is regulated and perhaps coordinated). A definitive study involving different mechanisms of hypoxaemia (inert-diluent and CO), differential grey and white matter blood flow (LASER Doppler) and selective enzyme (NOS and HO) blockade is consequently planned. Acknowledgements The study was supported by the University of Auckland School of Medicine Foundation, the Maurice and Phyllis Paykel Trust and the New Zealand Lotteries Commission. References
Fig. 2. Representative photomicrographs of PKC␥ immunochemistry and double labeling (GABA a1, a3, and GFAP) of the central and basal–lateral nuclei of the amygdala of sheep brain 5 days post-CO (A–D). (A) Arrows point to PKC␥ positive cells (pyramidal neurons in brown) while arrowheads indicate glia cells with GFAP positive staining (purple). (B) Double labeling for PKC␥ (brown) and GABA a1 (purple) showing co-location of PKC␥ and GABA a1 on some of the pyramidal neurons. (C) Pyramidal neurons (arrows) showing a co-location of PKC␥ positive staining (brown) and GABA a3 staining (purple); some round GABA a3 positive neurons (purple) can be seen around the pyramidal cells. (D) Double labeling of GABA a1 (brown) and GABA a3 (purple) showing the co-location of the two receptors on some of the pyramidal neurons (arrows). This indicates that the PKC␥ positive pyramidal neurons are GABAergic. Bar = 50 m (A–D).
[1] D. Boehning, S.H. Snyder, Circadian rhythms. Carbon monoxide and clocks, Science 298 (5602) (2002) 2339–2340 (comment). [2] S. Campeau, M. Davis, Involvement of the central nucleus and basolateral complex of the amygdala in fear conditioning measured with fear-potentiated startle in rats trained concurrently with auditory and visual conditioned stimuli, J. Neurosci. 15 (3, Pt 2) (1995) 2301–2311. [3] C. Chapline, J. Cottom, H. Tobin, J. Hulmes, J. Crabb, S. Jaken, A major, transformation-sensitive PKC-binding protein is also a PKC substrate involved in cytoskeletal remodelling, J. Biol. Chem. 273 (31) (1998) 19482–19489. [4] M. Davis, The role of the amygdala in fear-potentiated startle: implications for animal models of anxiety, Trends Pharmacol. Sci. 13 (1) (1992) 35–41. [5] D. de Chaffoy de Courcelles, The role of protein-kinase C in the human platelet, Blood Coagul. Fibrinolysis 1 (4,5) (1990) 401–413. [6] D.J. Doolette, D.I.B. Kerr, Comparison of carbon monoxide and nitrogen-induced hypoxia in the rat hippocampal slice, Neurosci. Lett. 138 (1992) 9–13. [7] D.J. Doolette, D.I.B. Kerr, Hyperexcitability in CA1 of the rat hippocampal slice following hypoxia or adenosine, Brain Res. 677 (1995) 127–137. [8] M. Geanacopoulos, J. Turner, K.E. Bowling, S.R. Vandenberg, A.R. Gear, The role of protein-kinase C in the initial events of platelet activation by thrombin assessed with a selective inhibitor, Thromb. Res. 69 (1) (1993) 113–124. [9] D.F. Gorman, A. Drewry, Y.L. Huang, C. Sames, The clinical toxicology of carbon monoxide, Toxicology 187 (2003) 25–38. [10] D.F. Gorman, Y.L. Huang, C. Williams, Prolonged exposure to one percent carbon monoxide causes a leucoencephalopathy in un-anaesthetised sheep, Toxicology 165 (2001) 97–107. [11] D.F. Gorman, Y.L. Huang, C. Williams, A narcotic dose of carbon monoxide induces neuronal haeme oxygenase and nitric oxide synthetase in sheep, Toxicology 179 (2002) 79–84. [12] D.F. Gorman, Y.L. Huang, C. Williams, A lignocaine infusion worsens the leukoencephalopathy due to a carbon monoxide exposure in sheep, Toxicology 186 (1,2) (2003) 143–150. [13] D.F. Gorman, Y.L. Huang, C. Williams, Blockade of haeme oxygenase and nitric oxide synthetase causes cortical dysfunction in sheep exposed to carbon monoxide, Toxicology 209 (2005) 237–243.
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[14] H. Hama, C. Hara, K. Yamaguchi, A. Miyawaki, PKC signalling mediates global enhancement of excitatory synaptogenesis in neurons triggered by local contact with astrocytes, Neuron 41 (3) (2004) 405–415. [15] P. Langston, D. Gorman, W. Runciman, R. Upton, The effect of carbon monoxide on oxygen metabolism in the brains of awake sheep, Toxicology 114 (3) (1996) 223–232. [16] J.E. LeDoux, C. Farb, D.A. Ruggiero, Topographic organization of neurons in the acoustic thalamus that project to the amygdala, J. Neurosci. 10 (4) (1990) 1043–1054. [17] G.L. Ludbrook, S.C. Helps, D.F. Gorman, P.L. Reilly, J.B. North, C. Grant, The relative effects of hypoxic-hypoxia and carbon monoxide on brain function in rabbits, Toxicology 75 (1) (1992) 71–80. [18] H.K. Majewski, I.F. Musgrave, Second messenger pathways in the modulation of neurotransmitter release, Aust. N Z J. Med. 25 (6) (1995) 817–821. [19] A. Mayesky, S. Meilin, G.G. Rogatsky, N. Zarchin, S.R. Thom, Multiparametric monitoring of the awake brain exposed to carbon monoxide, J. Appl. Physiol. 78 (1995) 1188–1196. [20] S. Meilin, G. Rogatsky, S.R. Thom, N. Zarchin, E. GuggenheimerFurman, A. Mayevsky, Effects of carbon monoxide on the brain may be mediated by nitric oxide, J. Appl. Physiol. 81 (1996) 1078– 1083. [21] A. Mendelman, N. Zarchin, S. Meilin, E. Guggenheimer-Furman, S.R. Thom, A. Mayevsky, Blood flow and ionic responses in the awake brain due to carbon monoxide, Neurol. Res. 24 (2002) 765–772.
[22] M. Meyer-Witting, S. Helps, D.F. Gorman, Acute carbon monoxide exposure and cerebral blood flow in rabbits, Anaesth. Intensive Care 19 (3) (1991) 373–377. [23] J.S. Morris, C.D. Frith, D.I. Perrett, D. Rowland, A.W. Young, A.J. Calder, R.J. Dolan, A differential neural response in the human amygdala to fearful and happy facial expressions, Nature 383 (6603) (1996) 812–815. [24] N. Saito, Y. Shirai, Protein-kinase C gamma (PKC gamma): function of neuron specific isotype, J. Biochem. 132 (5) (2002) 683–687. [25] A. Shekhar, T.J. Sajdyk, D. Gehlert, D.G. Rainnie, The amygdala, panic disorder, and cardiovascular responses, Ann. N Y Acad. Sci. 985 (2003) 308–325. [26] E.A. Van der Zee, M.A. Kronforst-Collins, E.T. Maizels, M. HunzickerDunn, J.F. Disterhoft, gamma Isoform-selective changes in PKC immunoreactivity after trace eyeblink conditioning in the rabbit hippocampus, Hippocampus 7 (3) (1997) 271–285. [27] S.G. Weller, I.K. Klein, R.C. Penington, W.E. Karnes Jr., Distinct protein-kinase C isozymes signal mitogenesis and apoptosis in human colon cancer cells, Gastroenterology 117 (4) (1999) 848–857. [28] C.E. Williams, A. Gunn, P.D. Gluckman, Time course of intracellular edema and epileptiform activity following prenatal cerebral ischemia in sheep, Stroke 22 (4) (1991) 516–521. [29] N. Zhu, H.R. Weiss, Effect of hypoxic and carbon monoxide-induced hypoxia on regional myocardial segment work and oxygen consumption, Res. Exp. Med. 194 (1994) 97–107.
Early evidence of a regulated response to hypoxaemia in sheep that preserves the brain cortex Des Gorman a,b,∗ , Huang Yi Lin a , Chris Williams b a
Department of Medicine, University of Auckland, New Zealand b Liggins Institute, University of Auckland, New Zealand
Received 16 September 2005; received in revised form 12 October 2005; accepted 12 October 2005
Abstract Hypoxaemia consequent to inspired carbon monoxide (CO), and to other causes, often does not injure the brain cortex. At least five types of brain and heart protective cardiovascular response to hypoxaemia have been reported. The underlying mechanism is unknown. The present study was designed to test the hypothesis that the reaction to inspired CO involves the amygdala as this structure is thought to be central to stress responsivity; involvement would support the additional hypothesis that the somatic response to CO-hypoxaemia is regulated. Eighteen ewes were randomly allocated to control and two CO groups. The CO groups were exposed to 1% CO for 120 min and killed either 5 or 15 days later. This exposure caused isolated white matter brain injury and a transient increase in protein-kinase C (gamma) activity in the pyramidal neurons in the nuclei of the central and basal–lateral amygdala and in the neurons of the audio-cortex (p < 0.05). This was associated with evidence of a sympathetic response. It would seem reasonable to hypothesise both that the amygdala is important in the processes by which the hypoxaemic effects of CO on the brain are prevented, delayed and/or mitigated and that these processes are regulated. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Amygdala; Carbon monoxide; Circadian rhythm; Hypoxaemia; Hypoxia; Protein-kinase C (gamma)
Hypoxaemia induces circulatory responses that could delay and/or reduce any hypoxic effect on the brain, and especially the cortex, and the heart. If concurrent anaesthesia is avoided and cerebrovascular behaviour is preserved, then even prolonged severe carbon monoxide (CO)-induced hypoxaemia only causes isolated brain white matter injury [9–13]. These putatively protective responses do not seem to be either initiated or maintained by hypoxia per se, but may be dependent on nitric oxide synthetase (NOS) and/or haeme oxygenase (HO) function [11,13,19–21]. First, brain and coronary artery blood flow is increased during hypoxaemia such that oxygen (O2 ) delivery and uptake is maintained [15,17,29]. This requires increased heart rate and work and has a finite limit, perhaps at a oxyhaemoglobin (OHb) concentration of less than 20–30%, which when exhausted will result in sudden circulatory failure and onset of marked brain and heart hypoxia [15,17]. ∗ Corresponding author at: Department of Medicine, University of Auckland, Private Bag 92019, Auckland, New Zealand. Tel.: +64 9 3737599x86261; fax: +64 9 3082379. E-mail address: [email protected] (D. Gorman).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.10.075
Second, there is evidence of a sympathetic response to COhypoxaemia [9–13], evidenced by tachycardia, tachypnoea and peripheral vasoconstriction (concurrent systemic hypertension and a fall in skin temperature), and hyperglycaemia, which would also facilitate preferential perfusion of the brain and heart. Third, brain blood flow may well be redirected from the white matter to the cortex during CO-hypoxaemia and under the influence of HO and NOS, which are certainly induced, such that the cortex is preserved at the expense of the white matter [11,13]. Fourth, neurons may hibernate when confronted with sudden CO- and inert diluent-hypoxaemia; adenosine may be a trigger in this process [6,7]. Fifth, red blood cells are released into the blood, presumably from the spleen, to increase O2 carriage in response to a COinduced fall in blood O2 content [9–13]. The question here is the extent to which these cardiovascular reactions to CO and hypoxic hypoxaemia exist as a regulated stress response [10,11,13,15,17,22,25]. The rationale for this preliminary study is best expressed by analogy; the hypothesis that an orchestra exists, proposed on the basis of finding some musicians playing instruments, has greater merit if an active conductor is discovered. The conductor we chose to observe is
D. Gorman et al. / Neuroscience Letters 394 (2006) 174–178
the amygdala. This is the most well-recognised brain regulatory centre for stress responses [25,2,4,16,23]. It is reasonable to hypothesise then that the amygdala should be active if there is a regulated body reaction to inhaled CO. We have previously shown that NOS and HO are induced in many areas of the brain by CO inhalation [11,13]. Secondary enzyme systems such as protein-kinase C (PKC) also appear to be increasingly active; PKC is a family of protein serine/threonine kinases consisting of multiple isoforms that are involved in various cellular functions; they play a fundamental role in signalling mechanisms leading to mitogenesis and proliferation of cells, apoptosis, platelet activation, remodelling of the actin cytoskeleton and modulation of ion channels [3,5,8,14,18,27]. The aim of this preliminary study then was to determine if the somatic response to CO-hypoxaemia is associated with NOS, HO or PKC induction in the amygdala. Eighteen 3- to 4-year-old Romney ewes, which weighed 50–60 kg, were obtained from the Animal Research Unit of the Faculty of Medical and Health Sciences, University of Auckland and randomly allocated to equal sized experimental Control, CO-1, and CO-2 groups. The experiment was approved by the University of Auckland Animal Ethics Committee. The surgical procedure was as described previously [10,15,28]. In brief, the sheep were anaesthetised with 3% halothane in O2 . First, electrodes for EEG recording were installed onto the surface of the dura overlying the fronto-parietal cortex. Second, the right common carotid artery and tarsal artery and vein were catheterised for blood pressure tension monitoring and temperature electrodes were installed subcutaneously. Postoperative care of the sheep was also as we have described before [10,15]. Three days later our cannulation technique was used to administer CO at a flow rate of 50 ml kg−1 min−1 for 120 min [15]. Control sheep underwent tracheal cannulation, but, were not subject to CO. The electrophysiological parameters were continuously monitored, processed on line, averaged over 1 min intervals and recorded using a custom-built acquisition program. Either 5 or 15 days after the CO exposure, sheep were killed with a barbiturate overdose. Both common carotid arteries were cannulated for in situ brain perfusion fixation with 1000 ml chilled heparinised saline followed by 1000 ml chilled 10% phosphate-buffered formalin. The brain was removed and cut into 50 mm coronal tissue blocks and post-fixed for 7 days in a 10% phosphate-buffered formalin solution. Following gradual dehydration, tissue blocks were paraffin embedded and 6 m thick sections were cut for histological examination. Sections at the stratum level, with amygdala included, were used for histological (H&E staining) and immunochemistry study. To identify the amygdala accurately, the sections were examined microscopically (NIKON ECLIPSE E800) and the outline of the amygdala, caudate nucleus and putamen were engraved on the back of the slides. Positively stained cells in the amygdala were counted in four 0.05 mm2 areas of the coronal section at the stratum level of the right hemisphere using an image analyser (Carl Zeiss Vision GmbH system, Axiovision 3.1, Germany) at 200× magnification. Only well-stained cells were counted and the positive
175
counts were averaged for each slide. The slides were codified such that the outcome assessor was unaware of whether or not the sheep had been subject to a CO exposure. The ABC immunochemistry technique was used for both single and double-labeling HO and NOS as described before [11,13]; Vector VIP Substrate (Vector VIP Substrate Kit For Peroxidase, SK 4600, Vector Laboratories, CA, USA) was used instead of DAB for the secondary antigen labeling. For all enzymes, the antibody dilution was 1:200. To determine the integrity of the staining technique, immunohistochemical controls were obtained by replacing the primary antibodies to HO and NOS with buffer, using normal serum at equivalent dilutions. Pre-absorption studies were also performed using the corresponding antigens in cell lysate (Transduction Laboratories, Lexington, KY, USA); 1 g of primary antibody was incubated with 10 g of corresponding cell lysate in the same incubation solution as described above, for 3 days at 4 ◦ C. The antibody/antigen mix was then used instead of the primary antibody solution. In the double staining studies, incompatible antibodies were substituted for the species-specific secondary antibodies and/or one of the two primary antibodies was omitted. No positively stained cells were seen in those sections where the primary antibodies were eliminated from the incubation solution. When the secondary antibody was eliminated or one of the primary antibodies was omitted, there was no positive staining of the antibody-devoid antigen. Finally, when the primary antibodies against HO and NOS were pre-absorbed with their corresponding antigens, staining was either absent or very reduced. To detect PKC activities, mouse monoclonal antibody to PKC alpha (␣), PKC beta (), PKC gamma (␥) and PKC iota () (Transduction Laboratories, Lexington, KY, USA) were used according to the ABC method (ABC elite kit, Vectastain Universal, PK-6200, Vector laboratories, CA, USA) [10–13]. To classify the nature of PKC␥ positive cells in the amygdala and in the audio-cortex, double-labelling for antibody to PKC␥ with antibodies to Glial Fibrillary Acidic Protein (GFAP) (mouse IgG, diluted 1:200, cats. no. 814369. Boehringer Mannheim Biochemica, Germany), GABA a1, a3 (rabbit IgG, diluted 1:300, product no. G4416, G4291, SIGMA, USA), tyrosine hydroxylase (TH, rabbit IgG, diluted 1:150, CA-101, Protos Biotech corporation, NY, USA), and, NOS-1 and HO-1 (rabbit IgG, diluted 1:250, mouse IgG, diluted 1:200, Transduction Laboratories, USA) was performed using the ABC method and Vector VIP substrate (Vector VIP substrate kit for peroxidase, SK-4600, Vector laboratories, CA, USA), instead of DAB for the second antigen labeling. Again, stain integrity was established as no positively stained cells were seen when the primary or secondary antibodies for the PKC isoforms were replaced with buffer and normal serum at equivalent dilutions and the primary and secondary steps were omitted. In the double labeling studies for PKC␥ and GFAP, GABA and TH, omission of one of the primary antibodies also did not result in positive staining of the antibody-devoid antigen. When the primary antibody of PKC␥ was pre-absorbed with its corresponding antigen, immuno-staining was absent or considerably decreased.
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Data were analysed using one-way analyses of variance (Sigmastat Statistical Analysis System, version 2.03, and Sigmaplot 2000, Jandel Scientific, Ekrath, Germany). A significance level of 0.05 was chosen and all the data were recorded as mean ± S.E.M. As we have previously reported [9–13,15]: an inspired admixture of 1% CO in air caused agitation and then narcosis in sheep; and, subsequent histological and immunohistochemical changes were limited to periventricular white matter infarcts and a related glial reaction, axonal dysfunction, and a widespread induction of neuronal HO and NOS. Similarly, as before, the narcosis was accompanied by hyperpnoea and an increase in arterial carboxyhaemoglobin (COHb) level (60–70%), red blood cell mass, Hb concentration, blood lactate and glucose, heart rate, mean arterial blood pressure and EEG intensity (power). By contrast, the O2 Hb concentration fell, as did the arterial O2 content, blood pH, body surface temperature and EEG frequency. Following the CO exposure, the sheep again recovered very quickly in all respects but for the circadian rhythm of heart rate and EEG activity, which could not be identified until 4 days later (3.9 ± 0.8 days) in 9 of the 11 CO exposed sheep for the former and for about 2 days after the exposure (1.8 ± 0.3 days) in 6 of the 11 for the latter. The physiological changes cited above were associated with a similarly transient increase in PKC␥ activity, but not NOS or HO, in the neurons of the basal–lateral amygdala (BLA) and audio-cortex (Fig. 1). The activity of PKC␥ in the cells of the nucleus of the central and BLA and in the cells of the audio-cortex was increased in those sheep killed 5 days after the CO exposure (p < 0.05, Fig. 1); whereas in those sheep killed 15 days post-CO, the PKC␥ activity was not significantly different from control levels. There was no difference in the activity of the other PKC isoforms in either of the experimental or the control groups. The double labeling studies demonstrated that the PKC␥ positive cells were pyramidal neurons and the co-localisation of both GABA a1 and a3 receptors on these PKC␥ positive neurons further demonstrates that these are GABAergic neurons (Fig. 2). There was no colocalisation of TH, HO-1 and NOS-1 activity on these PKC␥ positive cells. This preliminary study shows again that there is evidence of a stress reaction during and immediately after the CO exposure and that this coincides with a transient increase of PKC␥ activity in the pyramidal GABAergic (a1 and a3 receptor bearing) neurons of the amygdala. As cited, the stress-response regulatory role of the amygdala is reasonably well established [25,4,23]. The BLA is normally under tonal inhibition by GABAergic neurons and functions as a major integrator and relay centre for anxiety and panic responses; it has been associated with the regulation of stressinduced cardiovascular response [25,2,16]. Protein-kinase C exists in multiple isoforms that are involved in various cellular functions [3,5,8,14,18,27]. The PKC␥ isoform is mainly expressed in the CNS and appears to be localised in the dendrites and cell bodies of neurons [24,26]; the physiological role of PKC␥ is still uncertain. Our study suggests that it may be involved in the response to CO-induced hypoxaemia.
Fig. 1. Loss of apparent normal day–night (24 h) rhythms in EEG and heart rate, and, the counts of PKC␥ positive neurons in the BLA of sheep brain postCO exposure. (Upper panel): the distribution of sheep that do not have a clear 24 h rhythm of EEG and heart rate showing recovery within 5 days of the CO exposure. (Middle panel): the EEG and heart rate for an individual sheep showing a suppressed rhythmicity for EEG frequency (2 days) and heart rate (5 days) postCO. (Lower panel): a bar graph showing the number of PKC␥ positive cells in the amygdala and audio-cortex of control sheep and those CO-exposed sheep killed 5 and 15 days after the exposure respectively; the 5-day-activity levels were significantly increased in comparison to that of control sheep and those killed after 15 days (p < 0.05, one-way ANOVA).
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The exact way in which CO increases PKC␥ activity, and, if or how PKC␥ in the pyramidal neurons of the BLA affects the physiological response to CO-hypoxaemia, is unknown. Of note, protein NPAS2, a transcription factor that regulates circadian rhythms, is a haemoprotein for which the DNA binding is selectively regulated by CO [1]. However, we have clearly found an active conductor (the amygdala) and are in a better position to argue that the musicians (at least five types of cardiovascular response to hypoxaemia) we have found are part of an orchestra (the response is regulated and perhaps coordinated). A definitive study involving different mechanisms of hypoxaemia (inert-diluent and CO), differential grey and white matter blood flow (LASER Doppler) and selective enzyme (NOS and HO) blockade is consequently planned. Acknowledgements The study was supported by the University of Auckland School of Medicine Foundation, the Maurice and Phyllis Paykel Trust and the New Zealand Lotteries Commission. References
Fig. 2. Representative photomicrographs of PKC␥ immunochemistry and double labeling (GABA a1, a3, and GFAP) of the central and basal–lateral nuclei of the amygdala of sheep brain 5 days post-CO (A–D). (A) Arrows point to PKC␥ positive cells (pyramidal neurons in brown) while arrowheads indicate glia cells with GFAP positive staining (purple). (B) Double labeling for PKC␥ (brown) and GABA a1 (purple) showing co-location of PKC␥ and GABA a1 on some of the pyramidal neurons. (C) Pyramidal neurons (arrows) showing a co-location of PKC␥ positive staining (brown) and GABA a3 staining (purple); some round GABA a3 positive neurons (purple) can be seen around the pyramidal cells. (D) Double labeling of GABA a1 (brown) and GABA a3 (purple) showing the co-location of the two receptors on some of the pyramidal neurons (arrows). This indicates that the PKC␥ positive pyramidal neurons are GABAergic. Bar = 50 m (A–D).
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