Depression of neuronal activity by sedatives is associated with adverse effects after brain injury

brain research 1510 (2013) 1–9

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Research Report

Depression of neuronal activity by sedatives is associated with adverse effects after brain injury D. Hertlen, L. Werhahn, C. Beynon, K. Zweckberger, B. Vienenko¨tter, C.S. Jung, A. Unterberg, K. Kiening, O. Sakowitz Department of Neurosurgery, University Hospital Heidelberg, 69120 Heidelberg, Germany

art i cle i nfo

ab st rac t

Article history:

Analgesics and sedatives are frequently used in the treatment of acute brain injury and

Accepted 12 March 2013

subsequent brain swelling. Most agents act on specific receptors to modulate neuronal

Available online 20 March 2013

activity, which is normally involved in feedback loops that direct system building and

Keywords:

maintenance. We investigated the neurodegenerative effects of midazolam and isoflurane

Traumatic brain injury

in a rat model of controlled cortical impact injury (CCII). Two hours prior to CCII, four experimental groups were treated with different agents

TBI Neuronal death Isoflurane

including a minimum alveolar concentration (MAC 1.0) of isoflurane. For additional sedation, isoflurane MAC 1.67, midazolam alone, or midazolam in combination with

Midazolam

flumazenil was used. Blood pressure and blood gas analysis were monitored to investigate

Sedation

systemic side effects. Two days after treatment, relative apoptotic cell counts were determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. With isoflurane and midazolam, electroencephalographic (EEG) recordings revealed a decrease in amplitude size and altered frequency distribution. Treatment using deep sedation with isoflurane MAC 1.67 or midazolam increased relative apoptotic cell count by 14.8% (95% CI 3.6 to 26.1, po0.01) and 18.0% (95% CI 6.8 to 29.3, po0.01), respectively. Co-treatment with flumazenil reversed the neurodegenerative effect of midazolam by −13.2% (95% CI −24.5 to −2.0, po0.05). Functional neurological outcome was worse after isoflurane MAC 1.67 (18.8 score points; po0.01) and midazolam (21.4 score points, po0.001). Flumazenil antagonized the neurodegenerative effects of midazolam. In conclusion, neuronal survival and functional recovery are reduced by sedative use in a rat model of acute brain injury. & 2013 Elsevier B.V. All rights reserved.

1.

Introduction

Sedation is often used in clinical settings after acute brain injury. Sedated patients demonstrate reduced responses to

stress stimuli, which facilitate diagnostic and therapeutic treatment. Furthermore, the administration of sedatives increases comfort and aids sleep. Due to reductions in energy metabolism, sedation was long thought to be beneficial for

n Correspondence to: Department of Neurosurgery, University Hospital Heidelberg, INF 400, D-69120 Heidelberg, Germany. Fax: þ49 6221 5636302. E-mail address: [email protected] (D. Hertle).

0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.03.009

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neurologic outcome. All randomized multicenter trials, however, failed to demonstrate a beneficial effect for benzodiazepines, barbiturates or N-methyl-D-aspartate (NMDA) receptor blockers (Roberts et al., 2011). This finding was surprising, given that the reduction of energy metabolism by sedatives can exceed 40% of baseline activity (Alkire et al., 1999; Shulman et al., 2009). At risk, malperfused brain tissue was thought to thrive under sedativereduced energy consumption. This hypothesis was supported by findings in rodent experiments where sedatives have been proven to protect neuronal tissue after injury (Culley et al., 2003, 2007; Kawaguchi et al., 2005; Kvolik et al., 2005; Wei et al., 2005; Loop et al., 2005; Sarissky et al., 2005; Statler et al., 2006a, 2006b; Kitano et al., 2007; Ochalski et al., 2010; Dong et al., 2009). Given that the hypothesized neuroprotection by sedatives could not be reproduced in patients suffering from traumatic brain injury, subarachnoid hemorrhage and stroke, answers are needed to explain this finding. In contrast to this, a relatively new concept involves elevated neuronal activity as a mechanism of neuroprotection. According to this, neuronal activity by itself can prevent neuronal death (Hardingham and Bading, 2003). Activitydependent neuroprotection is based on the influence of electrical brain activity on NMDA receptors. This receptor activates two separate pathways, depending on the position of the receptor on the cell surface (Hardingham et al., 2002). The position of individual NMDA receptors is variable and depends on neuronal activity. Reduction of neuronal activity causes a shift of synaptic NMDA receptors to the extrasynaptic membrane (Zhang et al., 2007). Synaptic and extrasynaptic NMDA receptors are linked to distinct intracellular pathways that activate a neuroprotective gene program or promote cell death, respectively (Zhang et al., 2009; Hardingham and Lipton, 2011). Given that the effect of sedation is based on a reduction of neuronal activity, sedatives were hypothesized to contribute to neuron loss and functional neurological impairment (Eckenhoff et al., 2004; Xie et al., 2006; Hertle et al., 2012). The goal of our study was to further investigate the effects of sedatives after acute brain injury (Hertle et al., 2012). We focused (a) on the concentration-dependent effects of isoflurane and (b) the reversal of midazolam-induced neurodegeneration by flumazenil. A previously established and reliable model for reproducible brain injury was used. Systemic side effects were closely monitored.

2.

Results

To monitor the effect of sedation, electrical brain activity was analyzed (Fig. 1A). For delta (1–3 Hz) and theta (4–7 Hz) frequency-bands, no significant difference between groups was found. Activity in the alpha (8–13 Hz) band was more pronounced in all experimental groups when compared to isoflurane MAC 1.0, which ranged from 3.09 mV2/Hz for midazolam (95% CI 2.14 to 4.04, po0.001) to 1.38 mV2/Hz for midazolam and flumazenil (95% CI 0.43 to 2.32, po0.01). A shift towards the fast beta band (14–25 Hz) was caused by midazolam alone (2.08 μV2/Hz. 95% CI 1.17 to 3, po0.001) and midazolam in combination with flumazenil (1.59 μV2/Hz, 95% CI 0.68 to 2.51, po0.001). Gamma frequency (26–50 Hz) power

was decreased after deepening of sedation with isoflurane MAC 1.67 (−3.22 μV2/Hz, 95% CI −3.94 to −2.5, po0.001) but increased by midazolam (2.83 μV2/Hz, 95% CI −2.12 to −3.55, po0.001) with and without addition of flumazenil (1.19 μV2/Hz, 95% CI −0.467 to −1.904 po0.001; Fig. 1B).These results suggest that cortical activity is distinctively altered by deepening of sedation with isoflurane or the use of midazolam alone or in combination with flumazenil. Increasing sedation slowed brain activity towards near isoelectric EEG. Burst suppression ratio was increased when isoflurane MAC 1.67 was administered (44.15 95% CI 35.83 to 52.48, po0.001). Midazolam infusion also induced a tendency towards more burst suppression. With addition of flumazenil, EEG amplitudes were slightly, but not significantly, elevated (Fig. 1C). Blood pressure and arterial blood gas analysis were monitored during surgery. All measured blood parameters remained within normal limits (Table 1). Deep sedation reduced arterial blood pressure (Fig. 2A). Mean arterial blood pressure was decreased with both increased sedation regimens; i.e., after applying isoflurane MAC 1.67 (−24.39 mmHg, 95% CI −36.28 to −12.51, po0.001) and midazolam (−20.53 mmHg, 95% CI −33.84 to −7.21, po0.01). While administration of flumazenil reversed the decrease in brain activity, it did not reverse the reduced arterial blood pressure (−29.44 mmHg, 95% CI −42.36 to −16.53, po0.001). We found a tendency towards lower blood pressures in animals that received midazolam and flumazenil when compared to animals that received midazolam alone (Fig. 2A). We used referenced cortical areas to determine the number of apoptotic cells. Areas were found one and two millimeters in the midline from the margo superior in a 901 angle on the cortical surface close to the trauma site, as previously reported. The total size of the coronal slices was examined (Fig. 2B). Changes in brain slice size due to hemispheric swelling and tissue fixation were very small in trauma groups (maximum difference 2/−2 mm2, p40.05 for all groups). This finding indicates that the cortical areas of interest were comparable between trauma groups. The aim of this study was to evaluate the potential neurodegenerative effects caused by deep sedation. Apoptotic cells were labeled two days after traumatic brain injury using the TUNEL method (Fig. 3). For calculation of relative density of TUNEL positive cells, coronal brain slices adjacent to TUNEL tissue sections were stained with methyl green. Labeled cells were counted in two referenced cortical areas per animal close to the site of brain injury. Compared to isoflurane (MAC 1.0), relative apoptotic cell count increased after deep sedation with isoflurane (MAC 1.67) with a mean difference of 14.82% (95% CI 3.56 to 26.09, po0.01). After addition of midazolam the mean difference was 18.04% (95% CI 6.78 to 29.31, po0.01). Flumazenil treatment reversed the neurodegenerative effect of midazolam with a mean difference of −13.22% (95% CI −24.48 to −1.96, po0.05). Sham-groups that were treated with midazolam (n¼ 3) and isoflurane MAC 1.67 (n¼ 3) did not exhibit elevated levels of apoptotic cells when compared to sham isoflurane MAC 1.0 (n¼2), although single TUNEL positive cells were found in referenced cortical areas. Extended examination of coronal sections from sham experiments revealed few

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Fig. 1 – Sedative drugs modulate brain activity measured with surface electrodes. (A) Example traces for each sedative. (B) Box plots EEG band power for isoflurane MAC 1.0 (blue, n¼ 12), isoflurane MAC 1.67 (red, n ¼ 9), midazolam (green, n ¼ 12) and midazolam & flumazenil (magenta, n¼ 9). (C) Burst suppression ratio was increased after deep sedation. Asterisks indicate a significant difference relative to isoflurane MAC 1.0.

TUNEL positive cells throughout the tissue with a focus closer to the site of surgery. Functional neurological outcome was determined in three behavioral tests. First, the response to a foot pinch was measured after sedation (Fig. 4). As expected, wake-up time was prolonged after sedation (12.39 min, 95% CI 3.89 to 20.88, po0.01) with isoflurane MAC 1.67 and after administration of midazolam (30.98 min, 95% CI 22.67 to 39.29, po0.001). In this test, flumazenil did not significantly reverse the effect of midazolam. Four hours after terminating sedation, we observed longlasting effects of the drug. According to the Kruskal–Wallis test and the Dunn's multiple comparison test, significantly worse climbing scores were observed after isoflurane MAC 1.67 (18.78 difference in rank sum points; po0.01) and midazolam (21.42, po0.001). After flumazenil reversal, no significant difference was detected. At 48 h, a functional neurological impairment after isoflurane MAC 1.67 (17.87, po0.01) and midazolam (22.17, po0.001) was still present. In this delayed climb test, flumazenil significantly antagonized the

effects of midazolam (16.39, po0.01). Reversal of midazolam sedation by flumazenil resulted in a functional neurologic outcome not different from that seen with the baseline sedation isoflurane MAC 1.0. In the neurological examination, similar trends were found but did not reach statistical significance (Fig. 4). Taken together deeper sedated animals had worse functional neurological outcomes. Administration of flumazenil was able to reverse the deleterious effect of midazolam.

3.

Discussion

Activity-dependent neuroprotection is a novel concept in the determination of neuronal survival and death. It is based on previous observations on neuronal survival during brain development (Catsicas et al., 1992; Sherrard and Bower, 1998; Ikonomidou et al., 1999; Rothstein et al., 2008). After brain injury and during development synapses exhibit responses similarly (Belousov and Fontes, 2012). In addition,

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125.1711.90 119.8712.41 100.272.28 99.3371.8 1.1570.21 1.170.19 5.3170.50 5.5570.43 140.871.4 141.171.46 14.1970.64 13.7370.46 45.0072.96 43.7872.22 2.1770.85 2.7271.09 28.0771.04 28.1470.59 39.1670.93 39.4071.12 7.4370.03 7.4170.03

89.5473.95 86.1975.8

125.8711.91

126.478.18 101.872.25

100.672.5 0.9170.09

0.9870.18 5.6870.59

5.93170.45 141.371.273

141.371.68 14.4 70.72

14.470.83 43.7572.77

43.2571.58 2.4671.07

2.3371.01 28.0971.03

27.3570.88 92.3977.34

39.2170.75 7.3970.04

Isoflurane (MAC 1.67) n¼ 8 Midazolam n¼ 9 Midazolam & flumazenil n¼ 9

39.3171.13 7.4370.03 Isoflurane (MAC 1.0) n¼ 8

90.7379.3

Naþ (mmol/L) HCO3 (mmol/L) pO2 (mmHg) pCO2 (mmHg) pH

Table 1 – Arterial blood gas analysis. Mean77standard deviation.

BE(B) (mmol/L)

Hct (%)

tHB (g/dL)

Kþ (mmol/L)

Ca2þ (mmol/L)

CL− (mmol/L)

Glu (mg/dL)

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regulation of developmental cytokines might double in a different function for neuronal recovery after injury of the brain (Bauer et al., 2007; Hertle et al., 2008; Bechstein et al., 2012). Given that repair after acute brain injury shares mechanisms with brain development, the involvement of activity based neuroprotection seems possible. This might explain to some extent why experiments in rodents led to incorrect assumptions that sedatives are purely neuroprotective. Whether neuronal recovery after brain injury and developmental physiology share a common mechanism influenced by sedatives has to be further investigated. The neurodegenerative properties of anesthetics have been described in animal experiments that closely mimic human scenarios. A correlation was found between deep sedation accompanied with isoflurane-induced burst suppression and neurologic deterioration after ischemic brain injury in both rats and primates (Nehls et al., 1987; Drummond et al., 1995). GABAergic drugs did not improve or occasionally worsened neurologic outcome in patients and animal models after ischemic stroke (Lyden et al., 2002; Chaulk et al., 2003; Stover et al., 2004; Lodder et al., 2006). In addition, depriving neuronal activity with anticonvulsants had adverse effects after brain injury (Temkin et al., 1999; Narayan et al., 2008). The exact timeframe for the experimental application of drugs that target neuronal survival is known to be critical. The same stimulus, depending on its temporal relationship to brain injury, can be neuroprotective or cause neurodegeneration. Hypoxia is a well-established example for such preconditioning (Murry et al., 1986; Schurr et al., 1986). While hypoxia before brain injury can be protective, it is known to cause severe additive injury when applied during the acute phase of brain injury (Ishige et al., 1987; Nangunoori et al., 2011). To investigate a neuroprotective effect based on neuronal activity, sedatives were administered for 2 h before injury. This is an artificial situation with no clinical counterpart. It models an ideal situation where the activity level at the time of injury or shortly before) is modulated. It did reveal strong activity dependent neurodegeneration, i.e., a preconditioning effect of deep sedation increases post-traumatic apoptotic cell injury. Application of deep sedation hours after the brain injury might not result in the same neurodegenerative effect. Secondary injuries, however, such as vasospasm after subarachnoid hemorrhage or expanding contusions after traumatic brain injury, patients can suffer significant injury days after initial ictus during their stay in intensive care, where sedatives are commonly used. In these cases any unnecessary pretreatment with sedatives could, according to our finding, increase apoptotic imaging mechanisms. We measured sedative-dependent neurological deterioration over a period of two days because early recovery after brain trauma is an important measure and can prevent secondary complications (Fakhry et al., 2004). After neurological testing, the brain tissue was perfused and cryodissected. The slices were used for TUNEL staining to obtain additional insight into cellular degeneration. Since neurologic function was improved in less sedated animals, it is unlikely that these animals displayed higher apoptosis rates at the same time. Investigating a single time point may have potentially lead us to an underestimation of apoptosis that occurs in the very early stages after the trauma or several days later.

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Fig. 2 – Drug preconditioning had a defined effect on blood pressure (A) but did not alter size of coronal section (B). Asterisks indicate a significant difference relative to isoflurane MAC 1.0 (A) and sham (B).

Fig. 3 – Apoptotic cell counts are increased after traumatic brain injury and preconditioning with deep sedation. (Left) Box plot graph of analyzed groups (n ¼6). (Right) Representative images from micrographs. Sham-group: post-midazolam preconditioning without CCII; positive control: DNAse pretreatment. Asterisks indicate a significant difference relative to isoflurane MAC 1.0.

The volatile anesthetic isoflurane and the intravenous benzodiazepine midazolam were used for our investigation. Both are commonly used anesthetic drugs. Although we

chose relatively high doses, we took special care to rule out systemic side effects as a cause of neurological deterioration. According to all monitoring modalities, including

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Fig. 4 – Neurological outcome was impaired after traumatic brain injury and preconditioning with deep sedation. Asterisks indicate significant results.

oxygenation and blood pressure measurements, basic systems of the rat organism remained within their physiological limits. Induction of burst-suppression was reached in deeply sedated animals, but not in animals with lower levels of sedation. This was especially true for deeper sedation with isoflurane. Deep sedation significantly reduced the mean arterial blood pressure (MAP). Although the recorded arterial pressure remained within physiological limits, lower MAP might influence brain perfusion and oxygen delivery to brain cells. Our study is limited, given that intracranial pressure and intracranial perfusion pressure were not monitored. The pharmacological reversal of midazolam-dependent apoptosis and functional decline by flumazenil, however, was not influenced by decreased blood pressure. Therefore, a MAPdependent effect is unlikely to be involved. An alternative approach would have been to introduce cardiovascular agents to elevate blood pressure. This approach was dismissed since the combination of sedatives with continuous administration of catecholamines can have additional unwanted side effects including elevated blood pressure and the exacerbation of brain edema by fluid excess (Kroppenstedt et al., 2002; Strover et al., 2003). Systemic sedative application resulting in unwanted side effects was one additional reason for application of the brain injury after sedation. Future experiments with focal drug application after brain injury will give more insight into this issue. Other models of brain injury might reveal additional information.

4.

Experimental procedures

Fifty-eight adult male Sprague Dawley rats (Charles River Germany; mean body weight: 392.5714.8 g) were used. Rats were under a 24 h light-dark cycle with food and water available ad libitum. All experimental procedures were consistent with the European Parliament guidelines 2010/63/EU ABl/L 276. Preparation and maintenance of animals during experiments were in accordance with the University of Heidelberg's animal protection representative and approved by the responsible agency of the German Government (Regierungspraesidium Karlsruhe Referat 35). For surgical procedures, all animals were deeply anaesthetized with an initial isoflurane (Draeger vaporizer) dose of

5 vol% in N2O/O2 (7:3) for 3 min and placed on a feedbackcontrolled heating pad to maintain body temperature at 37 1C. Sedation was maintained at regular levels, 1.0 Minimum Alveolar Concentration (MAC), whereas rats lacked their pedal reflexes. First, arterial and venous catheters were inserted via the femoral artery and vein of the left groin. In our setup, the arterial catheter was placed with its tip in the aorta to monitor central blood pressure. Heads were then placed in a stereotactic frame and kept in a flat scull position for the entire surgical procedure. A sagittal skin incision was made between the ears and the skull bone from bregma to the occipital region until the left squamosal bone was exposed. A left-sided craniotomy (10 mm diameter) was performed using a high speed drill. Next, two stainless steel screws (Plastics One, Roanoke, VA, USA) for surface electroencephalographic (EEG) recordings were placed frontal and caudal to the craniotomy. Depending on the experimental group, a preconditioning treatment with additional isoflurane (MAC 1.67), midazolam (30 mg/kg/h) or midazolam (30 mg/kg/h) with flumazenil (10 mg/kg/h) was administered for 2 h. At the end of each hour, MAP was calculated from blood pressure recordings that were displayed online. Arterial blood pressure was recorded with an electric transducer (Foehr Medical, Seeheim Ober-Beerbach, Germany) and subsequently amplified (Hugo Sachs Elektronik, March-Hugstetten, Germany). A bipolarreferenced setup including an onboard high-pass and low-pass filter with a cut off frequency of 0.3 Hz/0.3 kHz and 50 Hz hum elimination band-stop filter (Hugo Sachs Elektronik, MarchHugstetten, Germany) was used for surface recordings. Within the last 10 min of the experiment, a 20 s artifact-free surface recording was made, and a 0.6 ml blood sample was drawn for blood gas analysis. After 2 h of experimental sedation, a focal brain lesion was placed. For controlled cortical impact injury (CCII), a pneumatic driven bolt (tip size: 5 mm; light barrier measured speed: 7 m/s) was used. After the induction of trauma, all catheters were removed, and wounds were closed. For midazolam administration, intraperitoneal administrations of flumazenil were made, 10 mg/kg after the end of sedation and 5 mg/kg each for the next 2 h. The rats were placed in an oxygen-enriched cage, and wake-up time was monitored. Sham groups received the same treatment except CCII.

4.1.

Functional outcome assessment

Neurological outcome was assessed 4 h and 48 h after brain trauma with a standardized test sequence (adapted from Zweckberger et al., 2010). For a climb test, rats were placed three times in the center of an angulated board with a length of one meter and a maximal height of 65 cm, 60 cm, 55 cm, 45 cm and 40 cm. For each angle, the rat was evaluated three times. Scoring—0 points: sitting for 20 s or walking for 10 s; 1 point: sliding and braking for 10 s; 2 points sliding down. The score ranged from the best score with good performance at 0 to a maximal score of 30. Animals were placed in the center of the board with no angle and observed for 3 min. Each animal was scored for spontaneous movement and gently stimulated when necessary. A stick was used to check for possible paralysis. Scoring —0 points: no visible neurological deficit; 1 point: the animal

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is not using the contralateral front paw; 2 points: the contralateral font paw paralyzed; 3 points: movement in all directions; 4 points: spontaneous movement, contralateral movement only when stimulated; 5 points: contralateral movement; 6 points: no reaction to external stimuli; 7 dead.

4.2.

Apoptotic cell counts (TUNEL staining)

After neurological testing, animals were transcardially rinsed with saline solution 0.9% and perfused with freshly prepared 4% paraformaldehyde for 20 min in deep sedation. The brain was removed from the skull, cryoprotected with sucrose and rapidly frozen. Coronal sections of the trauma site were made with a cryotome and mounted on poly-L-lysine coated slides. The trauma site was identified as parietal cortex, area 1, between −3.14 mm and −4.8 mm from bregma (The Rat Brain, Paxinos & Watson 1998; Academic Press, Inc). Glass slides were frozen and stored at −80 1C until further use. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) protocol was adapted from Roche Applied Science “In Situ Cell Death Detection” protocol to fit the needs of this study. All washing and incubation steps were made in Tris–HClbuffered saline (TBS) at pH 7.4 and room temperature unless indicated otherwise. Tissue was washed three times and covered with proteinase K (50 μg/ml, Sigma-Aldrich, Schnelldorf, Germany) for 10 min. Proteinase was replaced with TBS and washed three times in 0.5% Triton-X/TBS for further permeabilization. Positive controls were incubated with 125 μg/ml DNAse (Roche, Mannheim, Germany) for 10 min. After three more washing steps, tissue sections were transferred to dilution buffer (Roche). TUNEL was started with TdT enzyme and Biotin-16-UTP (both 1:250, Roche, Mannheim, Germany) in dilution buffer at 37 1C for 1 h. Finally, peroxidase/DAB development was made, as previously reported, and tissue was mounted on glass slides for microscopy. Pictures of two referenced cortical areas were taken from each experiment and saved to disk for further analysis (Hertle et al., 2012).

4.3.

Contusion size

Coronal brain slices adjacent to TUNEL tissue sections were used for this analysis. All sections were labeled with methyl green (Sigma-Aldrich, Schnelldorf, Germany) and mounted on glass slides. Slides were then scanned at high resolution (HP 4300C scanner) and converted to binary images. ImageJ (Rasband, 1997–2009) was used to measure the total surface for each contusion and each complete coronal slice. An average from three measurements was used to compare coronal expansion. Methyl green stained slices were further used for calculation of relative density of TUNEL positive cells. Microscopic pictures of the same referenced cortical area were taken and cells manually counted using ImageJ.

4.4.

Data reduction, analysis and statistics

For signal transduction, a BCN Connector Block (Hugo Sachs Elektronic, March-Hugstetten, Germany) with a PC-based A/D converter was used. Recordings were made at 500 Hz with Scope Version 2.2.0.30 (Data Translation Inc). Surface

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electrode recordings were analyzed with a MATLAB-based (MathWorks, Ismaning, Germany) version of EEGLAB 9.0 (Delorme and Makeig, 2004). For burst suppression ratio, suppression was identified as periods longer than 0.2 s of isoelectric EEG75 mV (Rampil, 1998; Wallenborn et al., 2004). TUNEL positive cells and methyl green stainings were marked and counted on microscopic images using ImageJ (Rasband, 1997– 2009). The experimenter was blinded to the different groups while counting cells. Prism (GraphPad, La Jolla) was used for statistical analysis and the preparation of figures. Analysis of variance (one-way ANOVA) and the post-hoc Tukey's multiple comparison test were used to compare experimental groups. Outcome scores were compared with nonparametric statistics, Kruskal–Wallis test and Dunn's multiple comparison test. For figure layout and modifications, Illustrator and Photoshop (Adobe) were used. Significance was set at po0.05.

5.

Conclusion

Our findings indicate an acute and antagonizable neurodegenerative effect of sedative drugs when used prior to acute brain injury. Pretreatment with midazolam or isoflurane can lead to cell death and worsen outcomes in acutely braininjured rats. This observation needs further exploration with discrimination of apoptotic/necrotic cell death, and effects of post-injury sedation and focal sedation regimens after induction of brain injury to rule out systemic side effects.

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