Cortical changes during deep anaesthesia Romanian Journal of Anaesthesia and Intensive Care 2015 Vol 22 No 2, 83-88
ORIGINAL PAPER
Changes of cortical connectivity during deep anaesthesia Bogdan Pavel1, Camelia Alexandra Acatrinei1, Fabien Menardy2, Carmen Mihaela Denise Zahiu1, Daniela Popa2, Ana-Maria Zagrean1, Leon Zagrean1
1 2
“Carol Davila” University of Medicine and Pharmacy, Division of Physiology and Fundamental Neurosciences, Bucharest, Romania Institut de Biologie de l’Ecole Normale Supérieure, Paris, France
Abstract Background and Aims. The aim of this study was to evaluate the frontal intracortical connectivity during deep anaesthesia (burst-suppression). Methods. Experiments were carried out on 5 adult Sprague Dawley rats. The anaesthesia was induced and maintained with isoflurane. Following the induction of anaesthesia, rats were placed in a stereotactic instrument. A hole was drilled in the skull over the frontal cortex and electrodes were inserted in order to record the local field potentials. Rats were maintained in deep level anaesthesia (burst-suppression). The cortical connectivity was assessed by computing the coherence spectra. The frontal intracortical connectivity was calculated during burst, suppression (non-burst) and slow wave anaesthesia periods. Results. The global cortical connectivity (0.5-100 Hz) was 0.61 ± 0.078 during the burst periods compared to 0.55 ± 0.032 (p < 0.05) during the suppression periods and 0.55 ± 0.015 (p < 0.05) during slow wave anaesthesia. Conclusions. The global cortical connectivity increased during the burst periods compared to the suppression periods and slow wave anaesthesia. This increase in the cortical synchronization might be due to the subcortical origin of the bursts. Keywords: cortical connectivity, anaesthesia, burst suppression Rom J Anaesth Int Care 2015; 22: 83-88
Introduction The cortical connectivity analyses the level of communication between cortical areas. Various methods of evaluation such as electroencephalogram (EEG), electrocorticogram (ECoG), local field potentials (LFP), cerebral blood flow (fMRI, functional MRI) and cerebral oxygen use (PET, Positron Emission Tomography) are utilized to estimate the cortical connectivity, which increases with the similarity Adress for correspondence:
Bogdan Pavel, MD, PhD Physiology and Fundamental Neurosciences Division, “Carol Davila” University of Medicine and Pharmacy 8 Eroii Sanitari Blvd 050474, Bucharest, Romania E-mail: [email protected]
between the signals recorded in different cortical areas [1-4]. In order to evaluate the functional cortical connectivity, mathematical algorithms based on the analysis of the statistical correlation between signals from different cortical areas are used: the correlation coefficient, cross approximate entropy (XAppEn), and the magnitude square coherence (mscohere) [5, 6]. There is a correlation between the depth of anaesthesia and the EEG pattern. Thus, theta and delta slow waves are characteristic of anaesthesia performed for surgical procedures, whereas a specific pattern of the EEG consisting of bursts of activity on a suppressed background, called “burst-suppression” (BS), can be noticed during profound anaesthesia [7]. A decrease of cortical connectivity is observed during loss of consciousness conditions such as sleep, traumatic brain injury and anaesthesia [8-10]. Thus, slow-wave sleep leads to decreased cortical connectivity, whereas sleep deprivation leads to loss of cortical connectivity in the
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frontal cortex [11, 12]. The frontal-parietal cortical connectivity decreases during slow-wave anaesthesia performed with isoflurane, sevoflurane and propofol [13] and an increase in depth of anaesthesia leads to a decrease in cortical connectivity. However, the cortical connectivity during BS is difficult to analyse through electrophysiology methods because the connectivity during the suppression periods cannot be correctly estimated. Thus, by evaluating the connectivity between two periods of suppression (seen as isoelectric line on the EEG recording) based merely on the similarity of the electrical signals, the connectivity would be equal to one. Nevertheless, this result is inconsistent with the imagistic studies which have revealed that cortical connectivity decreases during BS [14-16]. The aim of this study was to analyse the cortical connectivity during slow-wave anaesthesia, as well as BS pattern profound anaesthesia, by recording the cortical local field potentials.
Material and method Experiments were carried out in Sprague Dawley rats, weighing between 250 and 300 g, with water and food ad libitum. This study was conducted with the approval of the local Committee for Animal Research of “Carol Davila” University of Medicine and Pharmacy (Bucharest, Romania) and of the Institute de Biologie de l’Ecole Normale Supérieure – IBENS (Paris, France) and in accordance with the European Communities Council Directive 86/609/EEC on the protection of animals used for scientific purposes. In order to minimize the number of animals, while obtaining the appropriate data for statistical analysis, we analysed 4 different epochs of recordings from each rat, for the 5 rats used. Anaesthetic protocol The anaesthesia was induced in the animal induction chamber using 2% isoflurane, with a delivery rate 0.8 l/min for 5 minutes, using an anaesthesia apparatus (Classical T3 Vaporizer, SurgiVet, USA). The rat was subsequently placed in the stereotaxic instrument (Kopf: model 942, Kopf, USA) and coupled to the facemask of the anaesthesia apparatus in an open circuit. The anaesthesia was maintained using 2.5% isoflurane in air (FiO2 = 0.21), with spontaneous respiration. The respiratory frequency and the amplitude of the respiratory movements were monitored during anaesthesia. Surgical technique The hair of the cervical region was removed and the scalp was infiltrated with 0.2 ml 0.5% bupivacaine. Five minutes afterwards, a median line incision of 1.5
cm was performed and the scalp was carefully dissected and removed, using an electrocautery to maintain haemostasis. A bone window was made in the area of the frontal cortex using an electric mill and the dura mater was subsequently removed. Electrode placement Sixteen linear electrodes (electrodes NeuroNexusTechnologies, Ann Arbor, MI, USA) were used in order to register the cortical local field potentials. These were placed in the frontal lobe using a stereotaxic instrument (3 mm anterior to the bregma and 3 mm to the left side of the medial line, at 1 mm depth), in the area corresponding to the motor cortex. The electrodes were subsequently coupled to the acquisition system (setup TDT, Tucker-Davis Technologies, Alachua, FL, USA). 10-minute recordings at various anaesthetic depths (ranging from slow-wave to burst-suppression of different depths) were performed, by varying the isoflurane concentration. Data analyses and statistics The recordings obtained were exported as .mat files. The cortical connectivity was evaluated using the mscohere MATLAB function, which calculates the functional connectivity between superficial and deep cortical layers using epochs of 3 seconds selected from the slow-wave, suppression and burst recordings. In order to analyse the cortical connectivity during the slow-wave anaesthesia, one recording was selected for each rat. 4 epochs of 3 seconds corresponding to these specific recordings were analysed and the resulted values were subsequently averaged. For the analysis of cortical connectivity during profound anaesthesia (bursting frequency of 9 bursts/min), 4 epochs of suppression and the following bursts were selected for each rat. Data were presented as mean value +/- standard deviation. The mean values were compared using the one way ANOVA test with Dunnet T3 posthoc test and the SPSS 20.0 statistical program.
Results The local field potentials recorded during profound anaesthesia (characterized by burst-suppression activity) revealed high amplitude waves (400-800 µV) during the burst periods and low amplitude waves during the suppression periods (50-100 µV) (figure 1). Furthermore, high frequency waves were noticed only at the beginning of long bursts (duration of 2 seconds or more) compared to shorter bursts (duration of 1 second) (figure 2). Following the analysis of the frontal intracortical local field potentials, an increase in the cortical connectivity during the burst periods (0.61 ± 0.078) compared to the suppression periods (0.55 ± 0.032) was noticed
Cortical changes during deep anaesthesia
Fig. 1. Local field potentials during burst and non-burst activity, showing slow wave oscillation during non-burst activity
Fig. 2. Burst aspects according with its length. The first burst lasts almost 4 seconds and presents high frequency waves at the beginning followed by slow waves. The second burst is short (almost 1 second) and presents slow waves
(p < 0.05). Furthermore, the cortical connectivity during the slow-wave activity was 0.55 ± 0.015 (p < 0.05) (figure 3).
Discussion The burst-suppression pattern is noticed in various states of cortical suppression such as general anaesthesia, traumatic brain injury, following resuscitated cardio-respiratory arrest or Ohtahara syndrome [17-19]. Nevertheless, this phenomenon is not thoroughly understood and two theories have attempted to explain it. On one hand, the metabolic theory of Brown et al. suggests that the BS pattern is a consequence of the decreased cerebral metabolism, the link between the cerebral metabolism and the electrical activity being made through the ATPdependent potassium channel [20]. On the other hand,
Fig. 3. Coherence between superficial and deep layers of the frontal cortex during profound anaesthesia (burst suppression) and slow waves anaesthesia. Error bars represent standard deviation. Star indicates statistical significance (p < 0.05)
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Amzica et al. suggest that this phenomenon is caused by variations in the calcium ion concentration at the synaptic level [21]. Burst-suppression is also a cortical phenomenon modulated by internal and external subcortical stimuli [22, 23]. The increase in cortical connectivity during the burst compared to the non-burst periods (seen as suppression on the EEG or ECoG recording) noticed in this study may be the consequence of a subcortical stimulus that induces synchronization of the electrical activity at the cortical level. The magnitude squared coherence values range from 0 to 1, perfect cortical synchronization leading to coherence equal to 1. In our study, the coherence between the frontal superficial and profound LFP is equal to 0.7. This situation can be explained by the different modulation of the subcortical excitation by the cortical layers. The intracortical electrical activity is similar during the slow-wave and non-burst periods as far as the frequency of the waves is concerned (the amplitude remains however, different). Nevertheless, this similarity cannot explain why the values of the cortical connectivity between these two states are nearly equal. Further investigation is needed in order to explain these observations. These changes in cortical connectivity are noticed under basal conditions, without external stimulation. In a recently published study, we noticed that nociceptive stimulation during anaesthesia leads to an increase in frontal-parietal cortical connectivity during the slowwave periods [24]. As far as the BS periods are concerned, it is known that the number of bursts of activity determined by the intra-anaesthetic stimulation increases with the depth of anaesthesia [22, 23]. Nevertheless, no study evaluates the effect of nociceptive stimulation on cortical connectivity during these burst suppression periods, further studies being required. The cortical local field potentials recorded during profound anaesthesia (depth of anaesthesia equal to 9 bursts/min) revealed an electrical activity characterized by waves of 50-100 µV amplitude during the so-called suppression period. Similar observations were made by Amzica et al. in recordings in the hippocampus, during profound anaesthesia (characterized by burstsuppression activity) induced by isoflurane [25]. Furthermore, the frequency of the waves decreases as the depth of anaesthesia is increased (from 15 Hz during BS to 3 Hz during the so called isoelectric line). These changes were also noticed during the anaesthesia induced by propofol or etomidate [26, 27]. These electrical phenomena have different aspects on EEG/ ECoG recordings compared to local field potentials because the local field potentials can record the dendro-somatic synaptic currents for 50-350 µm around the recording electrode [28]. By contrast, synchronous activity of 108 pyramidal neurons is
required for a 6 cm2 surface around an electrode in order to obtain an EEG recording [29]. Understanding the BS phenomenon at the level of synaptic currents could lead to a deeper understanding of the correlations between electrophysiological aspects and clinical observations. For instance, it is known that in neonates with hypoxic brain injuries there is a correlation between the BS activity and cortical reactivity during the BS state and the neurological outcomes [30-32]. We noticed a different aspect of the burst morphology, according to the length of the burst. Thus, the bursts of 2 seconds include high frequency waves at the beginning of the burst, followed by low frequency waves. These changes are in accordance with the ECoG recordings performed in our laboratory (unpublished results).
Conclusions Our data showed that the intracortical connectivity increased during the burst periods compared to the non-burst or slow-wave periods. This increase in cortical synchronization can be explained through the subcortical origin of the bursts. Furthermore, a different morphology of the bursts was noticed depending on the length of the bursts. The short bursts are composed of small frequency waves, while the long bursts are composed of high frequency followed by slow frequency waves. Further studies are required in order to explain this difference in the burst morphology. Conflict of interest Nothing to declare
References 1. Liu X, Pillay S, Li R, Vizuete JA, Pechman KR, Schmainda KM, et al. Multiphasic modification of intrinsic functional connectivity of the rat brain during increasing levels of propofol. Neuroimage 2013; 83: 581-592 2. Tu Y, Yu T, Fu XY, Xie P, Lu S, Huang XQ, et al. Altered thalamocortical functional connectivity by propofol anaesthesia in rats. Pharmacology 2011; 88: 322-326 3. Popa D, Spolidoro M, Proville RD, Guyon N, Belliveau L, Léna C. Functional role of the cerebellum in gamma-band synchronization of the sensory and motor cortices. J Neurosci 2013; 33: 6552-6556 4. Passow S, Specht K, Adamsen TC, Biermann M, Brekke N, Craven AR, Ersland L, Grüner R, Kleven-Madsen N, Kvernenes OH, Schwarzlmüller T, Olesen RA, Hugdahl K. Default-mode network functional connectivity is closely related to metabolic activity. Hum Brain Mapp 2015; 36: 2027-2038 5. Bonita JD, Albano AM, Ambolode LCC, Rapp PE. Study of Correlations of Multichannel Human EEG. In: Proceedings of
Cortical changes during deep anaesthesia the 11 th SPVM National Physics Conference and Workshop. s.l.: s.n., 2009: 31-33; ISSN 1655-4620 6. Kreuzer M, Hentschke H, Antkowiak B, Schwarz C, Kochs EF, Schneider G. Cross-approximate entropy of cortical local field potentials quantifies effects of anaesthesia – a pilot study in rats. BMC Neurosci 2010; 11: 122 7. Schmidt GN, Müller J, Bischoff P. Measurement of the depth of anaesthesia. Anaesthesist 2008; 57: 9-30, 32-36 8. Massimini M, Ferrarelli F, Murphy M, Huber R, Riedner B, Casarotto S, et al. Cortical reactivity and effective connectivity during REM sleep in humans. Cogn Neurosci 2010; 1: 176-183 9. Cao C, Slobounov S. Alteration of cortical functional connectivity as a result of traumatic brain injury revealed by graph theory, ICA, and sLORETA analyses of EEG signals. IEEE Trans Neural Syst Rehabil Eng 2010; 18: 11-19 10. Ku SW, Lee U, Noh GJ, Jun IG, Mashour GA. Preferential inhibition of frontal-to-parietal feedback connectivity is a neurophysiologic correlate of general anaesthesia in surgical patients. PLoS One 2011; 6: e25155 11. Massimini M, Ferrarelli F, Huber R, Esser SK, Singh H, Tononi G. Breakdown of cortical effective connectivity during sleep. Science 2005; 309: 2228-2232 12. Verweij IM, Romeijn N, Smit DJ, Piantoni G, Van Someren EJ, van der Werf YD. Sleep deprivation leads to a loss of functional connectivity in frontal brain regions. BMC Neurosci 2014; 15: 88 13. Lee U, Ku S, Noh G, Baek S, Choi B, Mashour GA. Disruption of frontal-parietal communication by ketamine, propofol, and sevoflurane. Anesthesiology 2013; 118: 1264-1275 14. Scheller MS, Tateishi A, Drummond JC, Zornow MH. The effects of sevoflurane on cerebral blood flow, cerebral metabolic rate for oxygen, intracranial pressure, and the electroencephalogram are similar to those of isoflurane in the rabbit. Anesthesiology 1988; 68: 548-551 15. Laureys S, Goldman S, Phillips C, Van Bogaert P, Aerts J, Luxen A, et al. Impaired effective cortical connectivity in vegetative state: preliminary investigation using PET. Neuroimage 1999; 9: 377-382 16. Laureys S, Lemaire C, Maquet P, Phillips C, Franck G. Cerebral metabolism during vegetative state and after recovery to consciousness. J Neurol Neurosurg Psychiatry 1999; 67: 121 17. Brown EN, Lydic R, Schiff ND. General anaesthesia, sleep, and coma. N Engl J Med 2010; 363: 2638-2650 18. Young GB. The EEG in coma. J Clin Neurophysiol 2000; 17: 473-485 19. Ohtahara S, Yamatogi Y. Ohtahara syndrome: with special reference to its developmental aspects for differentiating from early myoclonic encephalopathy. Epilepsy Res 2006; 70 Suppl 1: S58-67 20. Ching S, Purdon PL, Vijayan S, Kopell NJ, Brown EN. A neurophysiological-metabolic model for burst suppression. Proc Natl Acad Sci U S A 2012; 109: 3095-3100 21. Amzica F. Basic physiology of burst-suppression. Epilepsia 2009; 50 Suppl 12: 38-39 22. Călin A, Kumaraswamy VM, Braver D, Nair DG, Moldovan M, Simon MV. Intraoperative somatosensory evoked potential monitoring decreases EEG burst suppression ratio during deep general anaesthesia. J Clin Neurophysiol 2014; 31: 133-137 23. Land R, Engler G, Kral A, Engel AK. Auditory evoked bursts in mouse visual cortex during isoflurane anesthesia. PLoS One 2012; 7(11): e49855
24. Pavel B, Daneasa A, Rosca AE, Calin A, Zahiu D, Panaitescu A, et al. Fronto-parietal connectivity changes following noxious stimulation during anaesthesia. J Med Life 2014; 7: 387-390 25. Kroeger D, Florea B, Amzica F. Human brain activity patterns beyond the isoelectric line of extreme deep coma. PLoS One 2013 Sep 18; 8: e75257 26. Butovas S, Rudolph U, Jurd R, Schwarz C, Antkowiak B. Activity patterns in the prefrontal cortex and hippocampus during and after awakening from etomidate anesthesia. Anesthesiology 2010; 113: 48-57 27. Hanrahan SJ, Greger B, Parker RA, Ogura T, Obara S, Egan TD, et al. The effects of propofol on local field potential spectra, action potential firing rate, and their temporal relationship in humans and felines. Front Hum Neurosci 2013; 7: 136 28. Legatt AD, Arezzo J, Vaughan HG Jr. Averaged multiple unit activity as an estimate of phasic changes in local neuronal activity: effects of volume-conducted potentials. J Neurosci Methods 1980; 2: 203-217 29. Olejniczak P. Neurophysiologic basis of EEG. J Clin Neurophysiol 2006; 23: 186-189 30. Douglass LM, Wu JY, Rosman NP, Stafstrom CE. Burst suppression electroencephalogram pattern in the newborn: predicting the outcome. J Child Neurol 2002; 17: 403-408 31. Jose A, Matthai J, Paul S. Correlation of EEG, CT, and MRI brain with neurological outcome at 12 months in term newborns with hypoxic ischemic encephalopathy. J Clin Neonatol 2013; 2: 125-130 32. Iyer KK, Roberts JA, Hellström-Westas L, Wikström S, Hansen Pupp I, Ley D, et al. Cortical burst dynamics predict clinical outcome early in extremely preterm infants. Brain 2015; 138(Pt 8): 2206-2218
Modificări ale conectivităţii corticale în timpul anesteziei profunde Rezumat Obiectiv. În acest studiu ne-am propus să evaluăm conectivitatea frontală intracorticală în timpul anesteziei profunde (burst suppression). Metoda. Experimentele s-au desfăşurat pe şobolani adulţi Sprague Dawlei, în număr de 5. Anestezia a fost indusă şi menţinută cu izofluran. După inducţia anesteziei şobolanul a fost plasat într-un stereotax şi s-a efectuat o gaură de trepan deasupra cortexului frontal prin care au fost introduşi electrozii pentru înregistrarea potenţialelor de câmp. Şobolanii au fost menţinuţi în anestezie profundă (burst suppression). Pentru evaluarea conectivităţii corticale am folosit coerenţa spectrală. Conectivitatea frontală intracorticală a fost calculată în timpul perioadelor de burst, a perioadelor de „supresie” (non-burst) şi în timpul anesteziei cu unde lente. Rezultate. Conectivitatea corticală globală (0,5100 Hz) a fost de 0,61 ± 0,078 în timpul perioadelor de
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burst în comparaţie cu 0,55 ± 0,032 în timpul perioadelor de „supresie” (p < 0,05) şi 0,55 ± 0,015 (p < 0,05) în timpul anesteziei cu unde lente. Concluzii. Conectivitatea corticală a crescut în timpul perioadelor de burst în comparaţie cu perioadele
de non-burst şi a anesteziei cu unde lente. Această creştere a sincronizării corticale ar putea fi explicată prin originea subcorticală a burst-urilor. Cuvinte cheie: conectivitate corticală, anestezie, burst suppression
ORIGINAL PAPER
Changes of cortical connectivity during deep anaesthesia Bogdan Pavel1, Camelia Alexandra Acatrinei1, Fabien Menardy2, Carmen Mihaela Denise Zahiu1, Daniela Popa2, Ana-Maria Zagrean1, Leon Zagrean1
1 2
“Carol Davila” University of Medicine and Pharmacy, Division of Physiology and Fundamental Neurosciences, Bucharest, Romania Institut de Biologie de l’Ecole Normale Supérieure, Paris, France
Abstract Background and Aims. The aim of this study was to evaluate the frontal intracortical connectivity during deep anaesthesia (burst-suppression). Methods. Experiments were carried out on 5 adult Sprague Dawley rats. The anaesthesia was induced and maintained with isoflurane. Following the induction of anaesthesia, rats were placed in a stereotactic instrument. A hole was drilled in the skull over the frontal cortex and electrodes were inserted in order to record the local field potentials. Rats were maintained in deep level anaesthesia (burst-suppression). The cortical connectivity was assessed by computing the coherence spectra. The frontal intracortical connectivity was calculated during burst, suppression (non-burst) and slow wave anaesthesia periods. Results. The global cortical connectivity (0.5-100 Hz) was 0.61 ± 0.078 during the burst periods compared to 0.55 ± 0.032 (p < 0.05) during the suppression periods and 0.55 ± 0.015 (p < 0.05) during slow wave anaesthesia. Conclusions. The global cortical connectivity increased during the burst periods compared to the suppression periods and slow wave anaesthesia. This increase in the cortical synchronization might be due to the subcortical origin of the bursts. Keywords: cortical connectivity, anaesthesia, burst suppression Rom J Anaesth Int Care 2015; 22: 83-88
Introduction The cortical connectivity analyses the level of communication between cortical areas. Various methods of evaluation such as electroencephalogram (EEG), electrocorticogram (ECoG), local field potentials (LFP), cerebral blood flow (fMRI, functional MRI) and cerebral oxygen use (PET, Positron Emission Tomography) are utilized to estimate the cortical connectivity, which increases with the similarity Adress for correspondence:
Bogdan Pavel, MD, PhD Physiology and Fundamental Neurosciences Division, “Carol Davila” University of Medicine and Pharmacy 8 Eroii Sanitari Blvd 050474, Bucharest, Romania E-mail: [email protected]
between the signals recorded in different cortical areas [1-4]. In order to evaluate the functional cortical connectivity, mathematical algorithms based on the analysis of the statistical correlation between signals from different cortical areas are used: the correlation coefficient, cross approximate entropy (XAppEn), and the magnitude square coherence (mscohere) [5, 6]. There is a correlation between the depth of anaesthesia and the EEG pattern. Thus, theta and delta slow waves are characteristic of anaesthesia performed for surgical procedures, whereas a specific pattern of the EEG consisting of bursts of activity on a suppressed background, called “burst-suppression” (BS), can be noticed during profound anaesthesia [7]. A decrease of cortical connectivity is observed during loss of consciousness conditions such as sleep, traumatic brain injury and anaesthesia [8-10]. Thus, slow-wave sleep leads to decreased cortical connectivity, whereas sleep deprivation leads to loss of cortical connectivity in the
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frontal cortex [11, 12]. The frontal-parietal cortical connectivity decreases during slow-wave anaesthesia performed with isoflurane, sevoflurane and propofol [13] and an increase in depth of anaesthesia leads to a decrease in cortical connectivity. However, the cortical connectivity during BS is difficult to analyse through electrophysiology methods because the connectivity during the suppression periods cannot be correctly estimated. Thus, by evaluating the connectivity between two periods of suppression (seen as isoelectric line on the EEG recording) based merely on the similarity of the electrical signals, the connectivity would be equal to one. Nevertheless, this result is inconsistent with the imagistic studies which have revealed that cortical connectivity decreases during BS [14-16]. The aim of this study was to analyse the cortical connectivity during slow-wave anaesthesia, as well as BS pattern profound anaesthesia, by recording the cortical local field potentials.
Material and method Experiments were carried out in Sprague Dawley rats, weighing between 250 and 300 g, with water and food ad libitum. This study was conducted with the approval of the local Committee for Animal Research of “Carol Davila” University of Medicine and Pharmacy (Bucharest, Romania) and of the Institute de Biologie de l’Ecole Normale Supérieure – IBENS (Paris, France) and in accordance with the European Communities Council Directive 86/609/EEC on the protection of animals used for scientific purposes. In order to minimize the number of animals, while obtaining the appropriate data for statistical analysis, we analysed 4 different epochs of recordings from each rat, for the 5 rats used. Anaesthetic protocol The anaesthesia was induced in the animal induction chamber using 2% isoflurane, with a delivery rate 0.8 l/min for 5 minutes, using an anaesthesia apparatus (Classical T3 Vaporizer, SurgiVet, USA). The rat was subsequently placed in the stereotaxic instrument (Kopf: model 942, Kopf, USA) and coupled to the facemask of the anaesthesia apparatus in an open circuit. The anaesthesia was maintained using 2.5% isoflurane in air (FiO2 = 0.21), with spontaneous respiration. The respiratory frequency and the amplitude of the respiratory movements were monitored during anaesthesia. Surgical technique The hair of the cervical region was removed and the scalp was infiltrated with 0.2 ml 0.5% bupivacaine. Five minutes afterwards, a median line incision of 1.5
cm was performed and the scalp was carefully dissected and removed, using an electrocautery to maintain haemostasis. A bone window was made in the area of the frontal cortex using an electric mill and the dura mater was subsequently removed. Electrode placement Sixteen linear electrodes (electrodes NeuroNexusTechnologies, Ann Arbor, MI, USA) were used in order to register the cortical local field potentials. These were placed in the frontal lobe using a stereotaxic instrument (3 mm anterior to the bregma and 3 mm to the left side of the medial line, at 1 mm depth), in the area corresponding to the motor cortex. The electrodes were subsequently coupled to the acquisition system (setup TDT, Tucker-Davis Technologies, Alachua, FL, USA). 10-minute recordings at various anaesthetic depths (ranging from slow-wave to burst-suppression of different depths) were performed, by varying the isoflurane concentration. Data analyses and statistics The recordings obtained were exported as .mat files. The cortical connectivity was evaluated using the mscohere MATLAB function, which calculates the functional connectivity between superficial and deep cortical layers using epochs of 3 seconds selected from the slow-wave, suppression and burst recordings. In order to analyse the cortical connectivity during the slow-wave anaesthesia, one recording was selected for each rat. 4 epochs of 3 seconds corresponding to these specific recordings were analysed and the resulted values were subsequently averaged. For the analysis of cortical connectivity during profound anaesthesia (bursting frequency of 9 bursts/min), 4 epochs of suppression and the following bursts were selected for each rat. Data were presented as mean value +/- standard deviation. The mean values were compared using the one way ANOVA test with Dunnet T3 posthoc test and the SPSS 20.0 statistical program.
Results The local field potentials recorded during profound anaesthesia (characterized by burst-suppression activity) revealed high amplitude waves (400-800 µV) during the burst periods and low amplitude waves during the suppression periods (50-100 µV) (figure 1). Furthermore, high frequency waves were noticed only at the beginning of long bursts (duration of 2 seconds or more) compared to shorter bursts (duration of 1 second) (figure 2). Following the analysis of the frontal intracortical local field potentials, an increase in the cortical connectivity during the burst periods (0.61 ± 0.078) compared to the suppression periods (0.55 ± 0.032) was noticed
Cortical changes during deep anaesthesia
Fig. 1. Local field potentials during burst and non-burst activity, showing slow wave oscillation during non-burst activity
Fig. 2. Burst aspects according with its length. The first burst lasts almost 4 seconds and presents high frequency waves at the beginning followed by slow waves. The second burst is short (almost 1 second) and presents slow waves
(p < 0.05). Furthermore, the cortical connectivity during the slow-wave activity was 0.55 ± 0.015 (p < 0.05) (figure 3).
Discussion The burst-suppression pattern is noticed in various states of cortical suppression such as general anaesthesia, traumatic brain injury, following resuscitated cardio-respiratory arrest or Ohtahara syndrome [17-19]. Nevertheless, this phenomenon is not thoroughly understood and two theories have attempted to explain it. On one hand, the metabolic theory of Brown et al. suggests that the BS pattern is a consequence of the decreased cerebral metabolism, the link between the cerebral metabolism and the electrical activity being made through the ATPdependent potassium channel [20]. On the other hand,
Fig. 3. Coherence between superficial and deep layers of the frontal cortex during profound anaesthesia (burst suppression) and slow waves anaesthesia. Error bars represent standard deviation. Star indicates statistical significance (p < 0.05)
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Amzica et al. suggest that this phenomenon is caused by variations in the calcium ion concentration at the synaptic level [21]. Burst-suppression is also a cortical phenomenon modulated by internal and external subcortical stimuli [22, 23]. The increase in cortical connectivity during the burst compared to the non-burst periods (seen as suppression on the EEG or ECoG recording) noticed in this study may be the consequence of a subcortical stimulus that induces synchronization of the electrical activity at the cortical level. The magnitude squared coherence values range from 0 to 1, perfect cortical synchronization leading to coherence equal to 1. In our study, the coherence between the frontal superficial and profound LFP is equal to 0.7. This situation can be explained by the different modulation of the subcortical excitation by the cortical layers. The intracortical electrical activity is similar during the slow-wave and non-burst periods as far as the frequency of the waves is concerned (the amplitude remains however, different). Nevertheless, this similarity cannot explain why the values of the cortical connectivity between these two states are nearly equal. Further investigation is needed in order to explain these observations. These changes in cortical connectivity are noticed under basal conditions, without external stimulation. In a recently published study, we noticed that nociceptive stimulation during anaesthesia leads to an increase in frontal-parietal cortical connectivity during the slowwave periods [24]. As far as the BS periods are concerned, it is known that the number of bursts of activity determined by the intra-anaesthetic stimulation increases with the depth of anaesthesia [22, 23]. Nevertheless, no study evaluates the effect of nociceptive stimulation on cortical connectivity during these burst suppression periods, further studies being required. The cortical local field potentials recorded during profound anaesthesia (depth of anaesthesia equal to 9 bursts/min) revealed an electrical activity characterized by waves of 50-100 µV amplitude during the so-called suppression period. Similar observations were made by Amzica et al. in recordings in the hippocampus, during profound anaesthesia (characterized by burstsuppression activity) induced by isoflurane [25]. Furthermore, the frequency of the waves decreases as the depth of anaesthesia is increased (from 15 Hz during BS to 3 Hz during the so called isoelectric line). These changes were also noticed during the anaesthesia induced by propofol or etomidate [26, 27]. These electrical phenomena have different aspects on EEG/ ECoG recordings compared to local field potentials because the local field potentials can record the dendro-somatic synaptic currents for 50-350 µm around the recording electrode [28]. By contrast, synchronous activity of 108 pyramidal neurons is
required for a 6 cm2 surface around an electrode in order to obtain an EEG recording [29]. Understanding the BS phenomenon at the level of synaptic currents could lead to a deeper understanding of the correlations between electrophysiological aspects and clinical observations. For instance, it is known that in neonates with hypoxic brain injuries there is a correlation between the BS activity and cortical reactivity during the BS state and the neurological outcomes [30-32]. We noticed a different aspect of the burst morphology, according to the length of the burst. Thus, the bursts of 2 seconds include high frequency waves at the beginning of the burst, followed by low frequency waves. These changes are in accordance with the ECoG recordings performed in our laboratory (unpublished results).
Conclusions Our data showed that the intracortical connectivity increased during the burst periods compared to the non-burst or slow-wave periods. This increase in cortical synchronization can be explained through the subcortical origin of the bursts. Furthermore, a different morphology of the bursts was noticed depending on the length of the bursts. The short bursts are composed of small frequency waves, while the long bursts are composed of high frequency followed by slow frequency waves. Further studies are required in order to explain this difference in the burst morphology. Conflict of interest Nothing to declare
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Modificări ale conectivităţii corticale în timpul anesteziei profunde Rezumat Obiectiv. În acest studiu ne-am propus să evaluăm conectivitatea frontală intracorticală în timpul anesteziei profunde (burst suppression). Metoda. Experimentele s-au desfăşurat pe şobolani adulţi Sprague Dawlei, în număr de 5. Anestezia a fost indusă şi menţinută cu izofluran. După inducţia anesteziei şobolanul a fost plasat într-un stereotax şi s-a efectuat o gaură de trepan deasupra cortexului frontal prin care au fost introduşi electrozii pentru înregistrarea potenţialelor de câmp. Şobolanii au fost menţinuţi în anestezie profundă (burst suppression). Pentru evaluarea conectivităţii corticale am folosit coerenţa spectrală. Conectivitatea frontală intracorticală a fost calculată în timpul perioadelor de burst, a perioadelor de „supresie” (non-burst) şi în timpul anesteziei cu unde lente. Rezultate. Conectivitatea corticală globală (0,5100 Hz) a fost de 0,61 ± 0,078 în timpul perioadelor de
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burst în comparaţie cu 0,55 ± 0,032 în timpul perioadelor de „supresie” (p < 0,05) şi 0,55 ± 0,015 (p < 0,05) în timpul anesteziei cu unde lente. Concluzii. Conectivitatea corticală a crescut în timpul perioadelor de burst în comparaţie cu perioadele
de non-burst şi a anesteziei cu unde lente. Această creştere a sincronizării corticale ar putea fi explicată prin originea subcorticală a burst-urilor. Cuvinte cheie: conectivitate corticală, anestezie, burst suppression
