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GLIBENCLAMIDE REDUCES SECONDARY BRAIN DAMAGE AFTER EXPERIMENTAL TRAUMATIC BRAIN INJURY
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Q1 K. ZWECKBERGER, * K. HACKENBERG C. S. JUNG,
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Department of Neurosurgery, University Heidelberg, Germany
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Abstract—Following traumatic brain injury (TBI) SUR1-regulated NCCa-ATP (SUR1/TRPM4) channels are transcriptionally up-regulated in ischemic astrocytes, neurons, and capillaries. ATP depletion results in depolarization and opening of the channel leading to cytotoxic edema. Glibenclamide is an inhibitor of SUR-1 and, thus, might prevent cytotoxic edema and secondary brain damage following TBI. Anesthetized adult Sprague–Dawley rats underwent parietal craniotomy and were subjected to controlled cortical impact injury (CCI). Glibenclamide was administered as a bolus injection 15 min after CCI injury and continuously via osmotic pumps throughout 7 days. In an acute trial (180 min) mean arterial blood pressure, heart rate, intracranial pressure, encephalographic activity, and cerebral metabolism were monitored. Brain water content was assessed gravimetrically 24 h after CCI injury and contusion volumes were measured by MRI scanning technique at 8 h, 24 h, 72 h, and 7 d post injury. Throughout the entire time of observation neurological function was quantified using the ‘‘beam-walking’’ test. Glibenclamide-treated animals showed a significant reduction in the development of brain tissue water content(80.47% ± 0.37% (glibenclamide) vs. 80.83% ± 0.44% (control); p < 0.05; n = 14). Contusion sizes increased continuously within 72 h following CCI injury, but glibenclamide-treated animals had significantly smaller volumes at any time-points, like 172.53 ± 38.74 mm3 (glibenclamide) vs. 299.20 ± 64.02 mm3 (control) (p < 0.01; n = 10; 24 h) or 211.10 ± 41.03 mm3 (glibenclamide) vs. 309.76 ± 19.45 mm3 (control) (p < 0.05; n = 10; 72 h), respectively. An effect on acute parameters, however, could not be detected, most likely because of the up-regulation of the channel within 3–6 h after injury. Furthermore, there was no significant effect on motor function assessed by the beam-walking test throughout 7 days. In accordance to these results and the available literature, glibenclamide seems to have promising potency in the treatment of TBI. Ó 2014 Published by Elsevier Ltd. on behalf of IBRO.
Key words: traumatic brain injury, glibenclamide, secondary brain damage, brain edema, cerebral metabolism, epileptic seizures.
D. N. HERTLE, K. L. KIENING, A. W. UNTERBERG AND O. W. SAKOWITZ
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INTRODUCTION Despite significant improvements in intensive care Q4 management, severe traumatic brain injury (TBI) still remains the leading cause of death and long-term disability and dependency in young patients. Brain edema, leading to uncontrollable increased intracranial pressure (ICP), is predominantly responsible for the development of secondary brain damage characterized by enlarged contusion volumes, and hence, deteriorated neurological outcome. Treatment guidelines, thus currently focus on the control and the reduction of brain swelling (Brain Trauma Foundation, 2007). Osmotherapy has been the mainstay of pharmacological therapy and is typically administered as an escalating treatment scheme (Brain Trauma Foundation, 2007; Walcott et al., 2012). Among others, a novel treatment target for cerebral edema might be the SUR1-regulated NCCa-ATP (SUR1/TRPM4) channel (Simard et al., 2006; Walcott et al., 2012). Recently, this non-specification channel has been described in ischemic astrocytes, neurons, and capillaries regulated by sulfonylurea receptor 1 (SUR1). Depletion of ATP, as it can frequently be observed in the peri-contusional area following trauma, causes depolarization and opening of this channel. This finally leads to an uncontrolled influx of cations and, according to the osmotic pressure, of water resulting in cell ‘‘blebbing’’ that is characteristic of cytotoxic edema (Simard et al., 2006). In situations of mechanical stress, inflammation, and hypoxia an up-regulation of the ABcc8 gene that encodes for SUR1 receptors could be observed (Simard et al., 2012a). SUR-1 receptors can specifically be blocked by the administration of glibenclamide, explaining the fact, that patients with diabetes type II receiving glibenclamide seem to be in favor to reach a better neurological outcome after stroke quantified by the Modified Ranking Scale and the National Institute of Health Stroke Scale in comparison to controls (Kunte et al., 2007). Furthermore, in a rodent model of cerebral stroke, blocking of SUR-1 receptors with low-dose glibenclamide has significantly reduced cerebral edema, infarct volume, and mortality rate (Simard et al., 2006, 2009a; Zhou et al., 2009). In experimental models of subarachnoid hemorrhage glibenclamide seems to have positive effects on inflamma-
*Corresponding author. Address: Department of Neurosurgery, University of Heidelberg, ImNeuenheimer Feld 400, 69120 Q2 Heidelberg, Germany. Tel.: +49 62215636314. E-mail address: [email protected] (K. Zweckberger). Q3 Abbreviations: CCI, controlled cortical impact injury; EEG, electroencephalography; HF, heart rate; ICP, intracranial pressure; MANOVA, multivariate analysis of variance; MAP, mean arterial blood pressure; MD, microdialysis; TBI, traumatic brain injury. http://dx.doi.org/10.1016/j.neuroscience.2014.04.040 0306-4522/Ó 2014 Published by Elsevier Ltd. on behalf of IBRO. 1
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tion and edema formation (Simard et al., 2009b). In experimental spinal cord injury, glibenclamide diminishes the development of hemorrhagic necrosis, leading to improved neurological outcome (Simard et al., 2007a, 2012b,c; Popovich et al., 2012). The inhibition of the upregulated SUR1 receptor by low-dose glibenclamide displays furthermore a key role in the prevention of developing a progressive secondary hemorrhage following TBI (Simard et al., 2009c). In this current study the effect of glibenclamide on the development of secondary brain damage after experimental TBI is investigated emphasizing the influence on brain edema, contusion volume, cortical activity, and cerebral metabolism.
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EXPERIMENTAL PROCEDURES
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Animals
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Q5 A total of 68 Sprague–Dawley rats (Charles River,
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Germany, 350–380 g) were subjected to this study. Guidelines for laboratory animal care were strictly followed. Animals had free access to food and water throughout the study and all experiments were performed under deep anesthesia with isoflurane.
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Experimental groups
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A total of three experimental sections were investigated: In sections 1 (n = 20), acute (patho-) physiological changes, encompassing mean arterial blood pressure (MAP), heart rate (HF), body temperature, ICP, encephalographic activity, and cerebral metabolism (microdialysis (MD)) were monitored continuously over 180 min following trauma. In section 2 (n = 28), brain water content was assessed gravimetrically 24 h after controlled cortical impact injury (CCI), and, in section 3 (n = 20), contusion volumes by MRI scanning and neurological assessment were quantified 8 h, 24 h, 72 h, and 7 d following trauma, respectively. In all sections animals were randomized either to a treatment-, receiving glibenclamide, or to a control group that received placebo. In section 1, furthermore a shamoperated group was added. In this group animals were not traumatized, but only received a craniotomy.
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Anesthesia and trauma application
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All experiments were performed under deep anesthesia using 1.8–2.2% isoflurane, 68% N2O and 30% O2. During the entire time of anesthesia a catheter in the tail artery digitally monitored systemic MAP and HF. Furthermore, blood gases were controlled in regular intervals. In order to control and maintain body temperature at 37.0 ± 0.5 °C, rats were positioned on a feedback-controlled heating pad. As previously described, experimental TBI was performed using a ‘‘Controlled Cortical Impact’’ injury (CCI) device (Zweckberger et al., 2003, 2006). Briefly, after induction of anesthesia, the head was fixed in a stereotactic frame and, using a micro-drill under permanent cooling with saline, a temporo-parietal craniotomy was performed.
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Thereby special attention was paid to leave the dura intact. The trauma was performed perpendicular to the surface of the brain (90 degrees) by an impactor that was adjusted at an angle of 35 degrees. The diameter of the flat impactor (with rounded edges) was 5 mm, the velocity 7.5 m/s, the impact depth 1.5 mm, and the impact duration 300 ms. In order to avoid any decompressive effects created by the craniotomy itself, the bone-flap was replaced immediately after CCI and fixed with dental cement.
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Drug administration
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15 min after CCI, a loading dose of glibenclamide (10 lg/kg) was administered subcutaneously, and additionally, osmotic pumps (AlzetÒ, Model 2ML1; ALZET Osmotic Pumps, DURECT Corporation, USA) Q6 were implanted subcutaneously in the left flank region for continuous delivery of the drug throughout 7 days (10 ll/h). All rats, randomized either to the treatment or Q7 the control group, have received the same total volume. According to Simard et al. [13], a stock solution of 25 mg of glibenclamide (Sigma, St. Louis, MO, USA) in 10 ml dimethylsulfoxide (DMSO) was prepared. The infusion/injection solution was made by mixing 200 ll stock in 25 ml un-buffered normal saline (0.9%NaCl), thus, administering a total concentration of 200 ng/h.
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ICP and electro-encephalography (EEG)
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ICP was measured using an intraparenchymal micro-ICP probe (CodmanÒ MicroSensor Basic Kit; Raynham, MA 02767) that was inserted in the cortex via a borehole 2 mm rostral from the anterior edge of the craniotomy. Monitoring started 10 min prior to CCI receiving a physiological baseline, and was continued throughout the entire time of observation following CCI injury (180 min). In order to monitor encephalographic activity (EEG) two stainless screws were inserted in two boreholes fronto-lateral and parieto-occipital referred to the craniotomy. Bipolar recording was amplified using a Bio Potential Amplifier Module type 675 (Hugo Sachs Electronic-Harvard Apparatus GmbH, March-Hugstetten, Germany). Filter limits were set to 0.003–100 Hz. To avoid electrical interference at 50 Hz a notch filter was applied. These records were screened for epileptiform activity by one of the investigators who was blinded to the entire treatment procedure. Quantitative measurements of the EEG signals, such as root mean square values, delta (1–3 Hz), theta- (4–7 Hz), alpha(8–15 Hz), and beta (15–30 Hz) power, as well as the mean frequency, were derived from the artifact-free recordings 10 min before and up to 180 min after CCI (Sakowitz et al., 2002).
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MD
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Following CCI, the MD catheter (CMA12: length 14 mm; length of the permeable membrane 2 mm; diameter
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0.5 mm; molecular cut-off 100,000 Dalton) was inserted 2 mm rostral from the contusion, in the peri-contusional area representing the most active metabolic region of the traumatized brain. The perfusion rate was 1.2 ll/min. Using the MD technique extracellular concentrations of glutamate, lactate, pyruvate, and glucose were assessed in the acute stage up to 180 min following CCI injury. Dialysates were collected at regular 30-min intervals and stored immediately at 70 °C for further spectral photometric analyses.
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Brain water content
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In order to assess brain water content, animals were sacrificed in deep isoflurane anesthesia 24 h after CCI. After careful removal of the brains, hemispheres were divided subsequently. Finally, brain water content was assessed gravimetrically, as already described (Zweckberger et al., 2003, 2006).
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Contusion volume
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To quantify posttraumatic contusion volumes, repeated MRI scans were performed with a 2.35-T animal Ò scanner (Biospect 24/40, BRUKER Medizintechnik, Ettlingen, Germany) 8 h, 24 h, 72 h, and 7 days after trauma, offering both the advantage to re-investigate the same animals at different time-points and therefore, to reduce the total number of needed animals. Contusion volumes were estimated using 2-mm slices of T2-weighted spin echo (TSE) sequences and an Image J software package (ImageJ v.1.36, US National Institutes of Health, Bethesda, MD).
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Neurological outcome
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In order to assess neurological function and impairment the ‘‘beam-walking test’’ representing motor-function and -coordination was performed. Tests started 3 days prior surgery receiving reliable baseline scores, were repeated daily, and performed throughout the 7 days of observation.
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Data analysis
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Statistical analyses were performed using a standard statistic software package (IBM SPSS Statistics 18; SPSS Inc., an IBM Company, Chicago, IL, SAD). All averaged data are given as mean ± standard deviation of the mean (SD) if not indicated otherwise. A threshold value for significance (p-value) of less than 0.05 was presumed. Student’s t-test was used for comparison of two independent normal distributed samples, repeatedmeasures analysis of variance (ANOVA) with Bonferroni comparison for analysis within a series of measurements and multivariate ANOVA (MANOVA) for measurement series between two groups. If significance was proven in MANOVA, individual group comparisons were conducted using post hoc tests.
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RESULTS
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Physiological parameters
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In the present study physiological parameters were neither impaired by anesthesia nor by the administration of glibenclamide. MAP remained constant throughout the entire time of acute monitoring (104.62 ± 12.31 mmHg, 30 min after CCI; 102.00 ± 15.23 mmHg, 180 min after CCI, respectively). None of the animals showed any hypotension characterized by a MAP less than 80 mmHg. Furthermore, there was no influence on HF that ranged within physiological levels at any time (in average from 393.26 ± 37.04/min, 120 min after CCI to 403.32 ± 33.89/min, 120 min after CCI, respectively, with a maximal range from 326/min to 476/min). Body temperature was kept constant by using a feedback control heating pad and ranged in average at 37.47 ± 0.07 °C excluding hypo- or hyperthermia. Blood gas analyses that were assessed both in the acute and the chronic trial ruled out glibenclamide-induced hypoglycemia (serum) and showed physiological concentrations of serum electrolytes. Continuous administration of low-dose glibenclamide for 7 days led not to significant differences between the treatment and control groups that ranged within physiological levels (glibenclamide: 167.72 ± 30.24 mg/dl vs. control: 156.22 ± 56.28 mg/dl; p = 0.57; n = 10).
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ICP
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Ten minutes prior to CCI injury baseline ICP averaged about 8 mmHg (glibenclamide: 8.30 ± 1.10 mmHg vs. control: 8.08 ± 1.79 mmHg; p > 0.05; n = 10) and did not show any significant difference between the treatment and the control groups. Following CCI injury, ICP increased significantly (p < 0.05) and reached 14.80 ± 2.90 mmHg in the treatment group vs. 13.80 ± 2.15 mmHg in controls (p > 0.05; n = 10) 120 min after trauma. A significant difference between the treatment and control groups, however, could not be observed at this early stage after trauma. (Fig. 1).
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Cerebral MD
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In parallel to ICP measurements, concentrations of extracellular metabolic parameters were assessed in the peri-contusional region in the acute trial up to 180 min after CCI injury. Instantaneously after trauma, glucose concentrations dropped to a ‘‘close to zero’’ level (0.036 ± 0.022 mmol/l (glibenclamide) vs. 0.023 ± 0.019 mmol/l (control); p > 0.05) and recovered within 120 min (0.143 ± 0.110 mmol/l (glibenclamide) vs. 0.115 ± 0.080 mmol/l (control); p > 0.05). As a sign of anaerobic metabolism and cell death the concentrations of lactate and glutamate increased between 30 and 60 min following trauma in both groups. (glutamate 30 min. after CCI: 1.487 ± 1.387 lmol/l (glibenclamide) vs. 0.904 ± 0.326 lmol/l (control), p > 0.05, and glutamate 60 min. after CCI: 12.857 ± 8.742 lmol/l (glibenclamide) vs. 11.879 ± 10.151 lmol/l (control), p > 0.05, respectively). At later time-points of observation both, glutamate and lactate concentrations
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Fig. 1. Continuous measurement of the intracranial pressure (ICP) from 10 min before to 180 min after controlled cortical impact injury (CCI): Following stable baseline conditions, ICP has increased significantly within 30 min after trauma and has stabilized at a moderate level without showing any significant difference between the treatment (glibenclamide) and the control groups.
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declined continuously. Following a non-significant peak of the lactate/pyruvate ratio 60 min after CCI, its level was kept constant in both groups (Fig. 2).
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EEG
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Both, in the control and treatment groups average spectral frequency was reduced after CCI injury and showed a significant difference in comparison to shamoperated animals throughout all time-points. In Q8 comparison to baseline levels, in the glibenclamide group a-, c-, and d-power kept significantly increasing 90 min after CCI injury, but there was no significant difference between the treatment and control groups. Glibenclamide-treated animals, however, showed, in comparison to controls, a trend for increased EEGc-, and d-power 90 min after CCI injury (c-power: 508.77 ± 274.34 lV2 (Glibenclamide) vs. 346.36 ± 338.20 lV2 (Control); d-power: 107.53 ± 60.65 lV2 (Glibenclamide) vs. 81.11 ± 80.53 lV2 (Control)). Neither during baseline monitoring, nor in sham-operated animals any epileptic seizures could be detected. In summary, in nine traumatized animals seizures occurred (four in the control-, and five in the treatment group). Animals, treated with glibenclamide showed on the average 2.8 ± 6.2 seizures with a total duration of 35.7 ± 94.7 s and an average duration of 4.3 ± 5.7s. In control animals, both, number (4.1 ± 7.2) and duration were increased (total duration: 64.1 ± 103.4 s; average duration: 6.8 ± 8.9 s), but have not reached significance. In comparison between EEG and MD data, furthermore, there was a positive correlation between e.c. glutamate concentrations and the number (r = 0.54; p < 0.01) and duration of epileptic seizures (r = 0.46; p < 0.01) at 90 and 120 min after trauma.
Brain water content
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24 h following CCI injury brain water content of the traumatized and non-traumatized hemispheres were separately assessed gravimetrically. Brain water content Q9 of the non-injured hemisphere was 78.52 ± 0.19% in the Glibenclamide group, and 78.48 ± 0.29% in the control group and thus, represented physiological levels. In the injured hemisphere, however, a significant increase of brain water content could be observed. In comparison to the control group, rats having been randomized to the treatment group showed a significant reduction in brain water content (80.47 ± 0.37% vs. 80.83 ± 0,44%; p < 0.05; n = 14). Treatment with Glibenclamide, thus, could reduce the post-traumatic increase of brain water content by 15.3% (Fig. 3).
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Contusion volume
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Treatment with glibenclamide had a significant beneficial effect on the development of contusion volumes. Using MRI scanning technique, a reliable and non-invasive method quantifying contusion sizes, significant effects of glibenclamide on contusion volume could be detected 8 h, 24 h, 72 h, and 7 days following trauma(Fig. 4). Within the first 72 h a continuous increase of contusion size from158.00 ± 36.82 mm3 (glibenclamide) vs. 264.11 ± 44.37 mm3 (control) (n = 10; p < 0.05) 8 h, to 172.53 ± 38.74 mm3 (glibenclamide) vs. 299.20 ± 64.02 mm3 (control) (p < 0.01; n = 10) 24 h and, finally, to 211.10 ± 41.03 mm3 (glibenclamide) vs. 309.76 ± 64.38 mm3 (control) (p < 0.01; n = 10) 72 h after CCI injury could be measured. Contusion volumes were reduced 7 days after trauma, but still, a significant difference between the treatment and the control groups
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Q12 Fig. 2. Concentration of extracellular metabolic parameters, such as glutamate, lactate, pyruvate and the lactate/pyruvate ratio between 30 and 180 min after CCI injury: Not showing any differences between the treatment and control groups, the post-traumatic increase of e.c. lactate and glutamate concentrations represent an anaerobic metabolism and cell death. (Very low concentrations, e.g. lactate and pyruvate at 30 min are not detectable). 337 338
was evident: 107.13 ± 21.33 mm3 (glibenclamide) vs. 163.96 ± 28.99 mm3 (control) (p < 0.01; n = 10).
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Neurological function
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Motor function and coordination were assessed by performing the ‘‘beam-walking’’ test. Prior CCI injury animals showed 0.15 ± 0.36 misplacements referring to the corresponding contralateral hind paw. After trauma the number of misplacements increased significantly and reached 10.89 ± 2.83 (glibenclamide) and 10.24 ± 4.20 (control) (p < 0.01; n = 10) followed by continuous recovery in both groups. A significant difference between the treatment and control groups, however, could not be observed within 7 days of observation.
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DISCUSSION
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In this study it was found that treatment with glibenclamide can reduce brain edema development and contusion volume following experimental TBI.
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Despite several attempts to influence inflammation, excitotoxicity, and vascularization, clinical treatment of post-traumatic brain edema is still a challenge, and osmotherapy has been the mainstay for years. Both, mannitol and hyper-oncotic saline are well-established (Berger et al., 1995; Brain Trauma Foundation, 2007; Finke, 2012; Marks et al., 2012), although, there is no treatment available that already prevents the development of brain edema. Among others, the currently described SUR1regulated NCCa-ATP (SUR1/TRPM4) channel might offer a new treatment target (Simard et al., 2006; Walcott et al., 2012). This channel is not constitutively expressed, but is transcriptionally up-regulated de novo in all cells of the neurovascular unit in many forms of central nervous system injury, including cerebral ischemia, TBI, spinal cord injury, and subarachnoid hemorrhage (Simard et al., 2012a). In comparison and unlike the well-known KATP-channel, SUR-1, but not Kir6.1 or Kir6.2 is transcriptionally up-regulated in ischemic astrocytes, neurons, and capillaries representing the regulatory subunit of this
Please cite this article in press as: Zweckberger K et al. Glibenclamide reduces secondary brain damage after experimental traumatic brain injury. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.04.040
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Fig. 3. Gravimetrical assessment of brain water content 24 h following controlled cortical impact injury (CCI): Animals treated with glibenclamide showed significant reduced brain edema (white). In reference to physiological levels of the contralateral hemisphere, the post-traumatic increase of brain edema could be reduced by 15.3%.
Fig. 4. Post-traumatic contusion volume quantified by MRI scanning technique at 8 h, 24 h, 72 h, and 7 d post injury. Animals treated with glibenclamide showed significant reduced contusion sizes at any time-points (white).
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channel (Simard et al., 2006). On the other hand, Trpm4 is considered to be the pore-forming unit (Simard et al., 2007b; Woo et al., 2013). Ischemia-induced increase in SUR-1 was corroborated by measurements of Abcc8 mRNA, encoding SUR-1, which increased two- to threefold in the core center of the infarct in a MCA stroke model 3 h after the insult (Deeley et al., 2006; Simard et al., 2006). In the peri-infarct region the ascent of SUR-1 was delayed and a peak could sharply be demarcated
8–16 h post stroke (Simard et al., 2006, 2012a). The NCCa-ATP channel is highly dependent on ATP. Depletion of ATP, as it can be observed frequently in the peri-contusional area following trauma or in the so-called penumbra region after stroke causes depolarization and, thus, opening of the pore-unit. As a consequence cations, accompanied by water following the osmotic gradient, are floating into cells resulting in ‘‘cell blebbing’’ that is characteristic of cytotoxic edema (Simard et al., 2006).
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Administration of low-dose glibenclamidecan inhibits the regulatory sub-unit SUR-1 of the NCCa-ATP channel. 395Q10 As a rare fluke patients with stroke that were on 396 glibenclamide treatment having diabetes mellitus seem 397 to be in favor to reach a better neurological 398 outcome(Kunte et al., 2007). The group of Simard et al. 399 could further convincingly show in a rodent model of cere400 bral MCA stroke that administration of glibenclamide can 401 significantly reduce cerebral edema, infarct volume, and 402 mortality rate (Simard et al., 2006, 2009a; Zhou et al., 403 2009). 404 Following experimental TBI using the Feeney device, 405 Simard et al. (2009c) could also demonstrate that SUR406 1 is up-regulated, especially in capillaries, within 3 h in 407 the tissue beneath the impact. Furthermore a prominent 408 peak could be shown in deeper structures, like the hippo409 campus, 24 h after trauma. By treating rats with glibencla410 mide fragmentation of capillaries and, thus, hemorrhagic 411 necrosis measured by Drabkin’s reagent and contusion 412 size (Nissl sections) could be reduced resulting in a better 413 neurological outcome quantified by ‘‘spontaneous vertical 414 exploration’’ (Simard et al., 2009c). 415 Despite these promising results, experimental data 416 addressing post-traumatic pathologies, like ICP, 417 metabolic changes, and post-traumatic brain edema 418 have been lacking, so far. In the current study using a 419 CCI model treatment with low-dose glibenclamide has 420 resulted in a highly significant reduction of brain edema 421 assessed gravimetrically 24 h post CCI injury. In MRI 422 scans a continuous increase of contusion sizes could be 423 depicted at 8, 24, and 72 h following CCI injury. In 424 comparison to controls, animals that were treated with 425 glibenclamide furthermore showed significant smaller 426 contusion sizes at any time. Despite those results, ICP 427 and extracellular metabolism were not significantly 428 influenced by glibenclamide in the acute stage within 429 180 min following trauma. This could be due to the fact 430 that SUR-1 is transcriptionally up-regulated de novo 431 within 3–6 h following trauma, as mentioned above. 432 Thus, treatment in the very acute stage seems to be 433 less effective, although, in accordance with the results 434 of Simard et al. (2006, 2012a), we could show that contu435 sion sizes were already reduced 8 h after injury. Espe436 cially with reference to the MD data, the intensity of the 437 trauma can be regarded as moderate. After an acute drop 438 of the e.c. glucose concentration (‘‘close to zero level’’) 439 that is typical following trauma and representing an acute 440 energy failure (Hutchinson et al., 1991, 2000; Persson 441 et al., 1996; Goodman et al., 1999; Kett-White et al., 442 2002), e.c. glutamate and lactate concentrations have 443 significantly increased within 60 min, but continuously 444 recovered throughout the further time of observation. 445 The incline of glutamate and lactate in the peri446 contusional region implies a shift toward anaerobic 447 metabolism leading to cell death that only occurred in 448 the very acute stage (60 min), but did not continue or 449 aggravate throughout the remaining time of observation 450 as it would be expected in severe TBI (Obrenovitch, 451 1999; Alessandri et al., 2003; Hillered et al., 2005; 452 Chamoun et al., 2010). This could potentially explain the 453 lack of difference between the treatment and the control 393 394
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454 groups in the ‘‘beam-walking’’ test assessing misplace455 ments of the contralateral hind paw. Post CCI injury rats 456 of the treatment and the control groups showed signifi457 cantly more misplacements, but recovered very fast 458 within a few days and did not show long-lasting impair459 ments. More specifically, glibenclamide seems to improve 460 ‘‘behavioral abnormalities’’ quantified by ‘‘spontaneous 461 vertical exploration’’ (Simard et al., 2009c) and to reduce 462 hippocampal injury resulting in improved spatial learning 463 (Patel et al., 2010). 464 Impairment of neurotransmitter metabolism leads to 465 an imbalance in neurotransmitter receptor expression 466 (Cremer et al., 2009). Changes in the membrane distribu467 tion of N-methyl-D-aspartate (NMDA) glutamate recep468 tors, e.g., seem to produce sweeping modifications in 469 neuronal excitability (Va`zquez-Lo`pez et al., 2005). In vitro 470 experiments could show that induced astrocyte glutamate 471 release triggers transient depolarization and epileptic 472 form discharges (Kang et al., 2005). Buckingham et al. (2011) could further demonstrate that the release of gluta- Q11473 474 mate after the transplantation of glioma cells into mice 475 have led to epileptiform hyper-excitability that spreads 476 over the adjacent brain tissue, explaining the observation 477 that seizures are a common comorbidity in primary brain 478 tumors. Therefore, the correlation between e.c. glutamate 479 concentrations and the number and duration of seizures, 480 as mentioned above, are well explained and might be an 481 interesting point for pursuing investigations. 482 In summary and in accordance with the available 483 literature, we could demonstrate that treatment with low484 dose glibenclamide reduces post-traumatic brain edema 485 and contusion volume following CCI injury in rats. 486 Possibly owing to the delayed transcriptional up487 regulation of the regulatory sub-unit of the NCCa-ATP 488 channel, no effect on ICP and cerebral metabolism in 489 the very acute stage following CCI injury could be 490 detected. Although glibenclamide seems to have high 491 therapeutic potency in the treatment of traumatic brain 492 and spinal cord injury, as well as stroke, further 493 experimental and clinical studies are necessary to 494 further delineate the role of glibenclamide in the 495 treatment of TBI.
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(Accepted 17 April 2014) (Available online xxxx)
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GLIBENCLAMIDE REDUCES SECONDARY BRAIN DAMAGE AFTER EXPERIMENTAL TRAUMATIC BRAIN INJURY
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Q1 K. ZWECKBERGER, * K. HACKENBERG C. S. JUNG,
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Department of Neurosurgery, University Heidelberg, Germany
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Abstract—Following traumatic brain injury (TBI) SUR1-regulated NCCa-ATP (SUR1/TRPM4) channels are transcriptionally up-regulated in ischemic astrocytes, neurons, and capillaries. ATP depletion results in depolarization and opening of the channel leading to cytotoxic edema. Glibenclamide is an inhibitor of SUR-1 and, thus, might prevent cytotoxic edema and secondary brain damage following TBI. Anesthetized adult Sprague–Dawley rats underwent parietal craniotomy and were subjected to controlled cortical impact injury (CCI). Glibenclamide was administered as a bolus injection 15 min after CCI injury and continuously via osmotic pumps throughout 7 days. In an acute trial (180 min) mean arterial blood pressure, heart rate, intracranial pressure, encephalographic activity, and cerebral metabolism were monitored. Brain water content was assessed gravimetrically 24 h after CCI injury and contusion volumes were measured by MRI scanning technique at 8 h, 24 h, 72 h, and 7 d post injury. Throughout the entire time of observation neurological function was quantified using the ‘‘beam-walking’’ test. Glibenclamide-treated animals showed a significant reduction in the development of brain tissue water content(80.47% ± 0.37% (glibenclamide) vs. 80.83% ± 0.44% (control); p < 0.05; n = 14). Contusion sizes increased continuously within 72 h following CCI injury, but glibenclamide-treated animals had significantly smaller volumes at any time-points, like 172.53 ± 38.74 mm3 (glibenclamide) vs. 299.20 ± 64.02 mm3 (control) (p < 0.01; n = 10; 24 h) or 211.10 ± 41.03 mm3 (glibenclamide) vs. 309.76 ± 19.45 mm3 (control) (p < 0.05; n = 10; 72 h), respectively. An effect on acute parameters, however, could not be detected, most likely because of the up-regulation of the channel within 3–6 h after injury. Furthermore, there was no significant effect on motor function assessed by the beam-walking test throughout 7 days. In accordance to these results and the available literature, glibenclamide seems to have promising potency in the treatment of TBI. Ó 2014 Published by Elsevier Ltd. on behalf of IBRO.
Key words: traumatic brain injury, glibenclamide, secondary brain damage, brain edema, cerebral metabolism, epileptic seizures.
D. N. HERTLE, K. L. KIENING, A. W. UNTERBERG AND O. W. SAKOWITZ
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INTRODUCTION Despite significant improvements in intensive care Q4 management, severe traumatic brain injury (TBI) still remains the leading cause of death and long-term disability and dependency in young patients. Brain edema, leading to uncontrollable increased intracranial pressure (ICP), is predominantly responsible for the development of secondary brain damage characterized by enlarged contusion volumes, and hence, deteriorated neurological outcome. Treatment guidelines, thus currently focus on the control and the reduction of brain swelling (Brain Trauma Foundation, 2007). Osmotherapy has been the mainstay of pharmacological therapy and is typically administered as an escalating treatment scheme (Brain Trauma Foundation, 2007; Walcott et al., 2012). Among others, a novel treatment target for cerebral edema might be the SUR1-regulated NCCa-ATP (SUR1/TRPM4) channel (Simard et al., 2006; Walcott et al., 2012). Recently, this non-specification channel has been described in ischemic astrocytes, neurons, and capillaries regulated by sulfonylurea receptor 1 (SUR1). Depletion of ATP, as it can frequently be observed in the peri-contusional area following trauma, causes depolarization and opening of this channel. This finally leads to an uncontrolled influx of cations and, according to the osmotic pressure, of water resulting in cell ‘‘blebbing’’ that is characteristic of cytotoxic edema (Simard et al., 2006). In situations of mechanical stress, inflammation, and hypoxia an up-regulation of the ABcc8 gene that encodes for SUR1 receptors could be observed (Simard et al., 2012a). SUR-1 receptors can specifically be blocked by the administration of glibenclamide, explaining the fact, that patients with diabetes type II receiving glibenclamide seem to be in favor to reach a better neurological outcome after stroke quantified by the Modified Ranking Scale and the National Institute of Health Stroke Scale in comparison to controls (Kunte et al., 2007). Furthermore, in a rodent model of cerebral stroke, blocking of SUR-1 receptors with low-dose glibenclamide has significantly reduced cerebral edema, infarct volume, and mortality rate (Simard et al., 2006, 2009a; Zhou et al., 2009). In experimental models of subarachnoid hemorrhage glibenclamide seems to have positive effects on inflamma-
*Corresponding author. Address: Department of Neurosurgery, University of Heidelberg, ImNeuenheimer Feld 400, 69120 Q2 Heidelberg, Germany. Tel.: +49 62215636314. E-mail address: [email protected] (K. Zweckberger). Q3 Abbreviations: CCI, controlled cortical impact injury; EEG, electroencephalography; HF, heart rate; ICP, intracranial pressure; MANOVA, multivariate analysis of variance; MAP, mean arterial blood pressure; MD, microdialysis; TBI, traumatic brain injury. http://dx.doi.org/10.1016/j.neuroscience.2014.04.040 0306-4522/Ó 2014 Published by Elsevier Ltd. on behalf of IBRO. 1
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tion and edema formation (Simard et al., 2009b). In experimental spinal cord injury, glibenclamide diminishes the development of hemorrhagic necrosis, leading to improved neurological outcome (Simard et al., 2007a, 2012b,c; Popovich et al., 2012). The inhibition of the upregulated SUR1 receptor by low-dose glibenclamide displays furthermore a key role in the prevention of developing a progressive secondary hemorrhage following TBI (Simard et al., 2009c). In this current study the effect of glibenclamide on the development of secondary brain damage after experimental TBI is investigated emphasizing the influence on brain edema, contusion volume, cortical activity, and cerebral metabolism.
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EXPERIMENTAL PROCEDURES
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Animals
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Q5 A total of 68 Sprague–Dawley rats (Charles River,
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Germany, 350–380 g) were subjected to this study. Guidelines for laboratory animal care were strictly followed. Animals had free access to food and water throughout the study and all experiments were performed under deep anesthesia with isoflurane.
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Experimental groups
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A total of three experimental sections were investigated: In sections 1 (n = 20), acute (patho-) physiological changes, encompassing mean arterial blood pressure (MAP), heart rate (HF), body temperature, ICP, encephalographic activity, and cerebral metabolism (microdialysis (MD)) were monitored continuously over 180 min following trauma. In section 2 (n = 28), brain water content was assessed gravimetrically 24 h after controlled cortical impact injury (CCI), and, in section 3 (n = 20), contusion volumes by MRI scanning and neurological assessment were quantified 8 h, 24 h, 72 h, and 7 d following trauma, respectively. In all sections animals were randomized either to a treatment-, receiving glibenclamide, or to a control group that received placebo. In section 1, furthermore a shamoperated group was added. In this group animals were not traumatized, but only received a craniotomy.
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Anesthesia and trauma application
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All experiments were performed under deep anesthesia using 1.8–2.2% isoflurane, 68% N2O and 30% O2. During the entire time of anesthesia a catheter in the tail artery digitally monitored systemic MAP and HF. Furthermore, blood gases were controlled in regular intervals. In order to control and maintain body temperature at 37.0 ± 0.5 °C, rats were positioned on a feedback-controlled heating pad. As previously described, experimental TBI was performed using a ‘‘Controlled Cortical Impact’’ injury (CCI) device (Zweckberger et al., 2003, 2006). Briefly, after induction of anesthesia, the head was fixed in a stereotactic frame and, using a micro-drill under permanent cooling with saline, a temporo-parietal craniotomy was performed.
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Thereby special attention was paid to leave the dura intact. The trauma was performed perpendicular to the surface of the brain (90 degrees) by an impactor that was adjusted at an angle of 35 degrees. The diameter of the flat impactor (with rounded edges) was 5 mm, the velocity 7.5 m/s, the impact depth 1.5 mm, and the impact duration 300 ms. In order to avoid any decompressive effects created by the craniotomy itself, the bone-flap was replaced immediately after CCI and fixed with dental cement.
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Drug administration
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15 min after CCI, a loading dose of glibenclamide (10 lg/kg) was administered subcutaneously, and additionally, osmotic pumps (AlzetÒ, Model 2ML1; ALZET Osmotic Pumps, DURECT Corporation, USA) Q6 were implanted subcutaneously in the left flank region for continuous delivery of the drug throughout 7 days (10 ll/h). All rats, randomized either to the treatment or Q7 the control group, have received the same total volume. According to Simard et al. [13], a stock solution of 25 mg of glibenclamide (Sigma, St. Louis, MO, USA) in 10 ml dimethylsulfoxide (DMSO) was prepared. The infusion/injection solution was made by mixing 200 ll stock in 25 ml un-buffered normal saline (0.9%NaCl), thus, administering a total concentration of 200 ng/h.
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ICP and electro-encephalography (EEG)
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ICP was measured using an intraparenchymal micro-ICP probe (CodmanÒ MicroSensor Basic Kit; Raynham, MA 02767) that was inserted in the cortex via a borehole 2 mm rostral from the anterior edge of the craniotomy. Monitoring started 10 min prior to CCI receiving a physiological baseline, and was continued throughout the entire time of observation following CCI injury (180 min). In order to monitor encephalographic activity (EEG) two stainless screws were inserted in two boreholes fronto-lateral and parieto-occipital referred to the craniotomy. Bipolar recording was amplified using a Bio Potential Amplifier Module type 675 (Hugo Sachs Electronic-Harvard Apparatus GmbH, March-Hugstetten, Germany). Filter limits were set to 0.003–100 Hz. To avoid electrical interference at 50 Hz a notch filter was applied. These records were screened for epileptiform activity by one of the investigators who was blinded to the entire treatment procedure. Quantitative measurements of the EEG signals, such as root mean square values, delta (1–3 Hz), theta- (4–7 Hz), alpha(8–15 Hz), and beta (15–30 Hz) power, as well as the mean frequency, were derived from the artifact-free recordings 10 min before and up to 180 min after CCI (Sakowitz et al., 2002).
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MD
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Following CCI, the MD catheter (CMA12: length 14 mm; length of the permeable membrane 2 mm; diameter
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0.5 mm; molecular cut-off 100,000 Dalton) was inserted 2 mm rostral from the contusion, in the peri-contusional area representing the most active metabolic region of the traumatized brain. The perfusion rate was 1.2 ll/min. Using the MD technique extracellular concentrations of glutamate, lactate, pyruvate, and glucose were assessed in the acute stage up to 180 min following CCI injury. Dialysates were collected at regular 30-min intervals and stored immediately at 70 °C for further spectral photometric analyses.
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Brain water content
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In order to assess brain water content, animals were sacrificed in deep isoflurane anesthesia 24 h after CCI. After careful removal of the brains, hemispheres were divided subsequently. Finally, brain water content was assessed gravimetrically, as already described (Zweckberger et al., 2003, 2006).
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Contusion volume
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To quantify posttraumatic contusion volumes, repeated MRI scans were performed with a 2.35-T animal Ò scanner (Biospect 24/40, BRUKER Medizintechnik, Ettlingen, Germany) 8 h, 24 h, 72 h, and 7 days after trauma, offering both the advantage to re-investigate the same animals at different time-points and therefore, to reduce the total number of needed animals. Contusion volumes were estimated using 2-mm slices of T2-weighted spin echo (TSE) sequences and an Image J software package (ImageJ v.1.36, US National Institutes of Health, Bethesda, MD).
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Neurological outcome
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In order to assess neurological function and impairment the ‘‘beam-walking test’’ representing motor-function and -coordination was performed. Tests started 3 days prior surgery receiving reliable baseline scores, were repeated daily, and performed throughout the 7 days of observation.
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Data analysis
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Statistical analyses were performed using a standard statistic software package (IBM SPSS Statistics 18; SPSS Inc., an IBM Company, Chicago, IL, SAD). All averaged data are given as mean ± standard deviation of the mean (SD) if not indicated otherwise. A threshold value for significance (p-value) of less than 0.05 was presumed. Student’s t-test was used for comparison of two independent normal distributed samples, repeatedmeasures analysis of variance (ANOVA) with Bonferroni comparison for analysis within a series of measurements and multivariate ANOVA (MANOVA) for measurement series between two groups. If significance was proven in MANOVA, individual group comparisons were conducted using post hoc tests.
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RESULTS
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Physiological parameters
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In the present study physiological parameters were neither impaired by anesthesia nor by the administration of glibenclamide. MAP remained constant throughout the entire time of acute monitoring (104.62 ± 12.31 mmHg, 30 min after CCI; 102.00 ± 15.23 mmHg, 180 min after CCI, respectively). None of the animals showed any hypotension characterized by a MAP less than 80 mmHg. Furthermore, there was no influence on HF that ranged within physiological levels at any time (in average from 393.26 ± 37.04/min, 120 min after CCI to 403.32 ± 33.89/min, 120 min after CCI, respectively, with a maximal range from 326/min to 476/min). Body temperature was kept constant by using a feedback control heating pad and ranged in average at 37.47 ± 0.07 °C excluding hypo- or hyperthermia. Blood gas analyses that were assessed both in the acute and the chronic trial ruled out glibenclamide-induced hypoglycemia (serum) and showed physiological concentrations of serum electrolytes. Continuous administration of low-dose glibenclamide for 7 days led not to significant differences between the treatment and control groups that ranged within physiological levels (glibenclamide: 167.72 ± 30.24 mg/dl vs. control: 156.22 ± 56.28 mg/dl; p = 0.57; n = 10).
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ICP
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Ten minutes prior to CCI injury baseline ICP averaged about 8 mmHg (glibenclamide: 8.30 ± 1.10 mmHg vs. control: 8.08 ± 1.79 mmHg; p > 0.05; n = 10) and did not show any significant difference between the treatment and the control groups. Following CCI injury, ICP increased significantly (p < 0.05) and reached 14.80 ± 2.90 mmHg in the treatment group vs. 13.80 ± 2.15 mmHg in controls (p > 0.05; n = 10) 120 min after trauma. A significant difference between the treatment and control groups, however, could not be observed at this early stage after trauma. (Fig. 1).
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Cerebral MD
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In parallel to ICP measurements, concentrations of extracellular metabolic parameters were assessed in the peri-contusional region in the acute trial up to 180 min after CCI injury. Instantaneously after trauma, glucose concentrations dropped to a ‘‘close to zero’’ level (0.036 ± 0.022 mmol/l (glibenclamide) vs. 0.023 ± 0.019 mmol/l (control); p > 0.05) and recovered within 120 min (0.143 ± 0.110 mmol/l (glibenclamide) vs. 0.115 ± 0.080 mmol/l (control); p > 0.05). As a sign of anaerobic metabolism and cell death the concentrations of lactate and glutamate increased between 30 and 60 min following trauma in both groups. (glutamate 30 min. after CCI: 1.487 ± 1.387 lmol/l (glibenclamide) vs. 0.904 ± 0.326 lmol/l (control), p > 0.05, and glutamate 60 min. after CCI: 12.857 ± 8.742 lmol/l (glibenclamide) vs. 11.879 ± 10.151 lmol/l (control), p > 0.05, respectively). At later time-points of observation both, glutamate and lactate concentrations
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Fig. 1. Continuous measurement of the intracranial pressure (ICP) from 10 min before to 180 min after controlled cortical impact injury (CCI): Following stable baseline conditions, ICP has increased significantly within 30 min after trauma and has stabilized at a moderate level without showing any significant difference between the treatment (glibenclamide) and the control groups.
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declined continuously. Following a non-significant peak of the lactate/pyruvate ratio 60 min after CCI, its level was kept constant in both groups (Fig. 2).
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EEG
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Both, in the control and treatment groups average spectral frequency was reduced after CCI injury and showed a significant difference in comparison to shamoperated animals throughout all time-points. In Q8 comparison to baseline levels, in the glibenclamide group a-, c-, and d-power kept significantly increasing 90 min after CCI injury, but there was no significant difference between the treatment and control groups. Glibenclamide-treated animals, however, showed, in comparison to controls, a trend for increased EEGc-, and d-power 90 min after CCI injury (c-power: 508.77 ± 274.34 lV2 (Glibenclamide) vs. 346.36 ± 338.20 lV2 (Control); d-power: 107.53 ± 60.65 lV2 (Glibenclamide) vs. 81.11 ± 80.53 lV2 (Control)). Neither during baseline monitoring, nor in sham-operated animals any epileptic seizures could be detected. In summary, in nine traumatized animals seizures occurred (four in the control-, and five in the treatment group). Animals, treated with glibenclamide showed on the average 2.8 ± 6.2 seizures with a total duration of 35.7 ± 94.7 s and an average duration of 4.3 ± 5.7s. In control animals, both, number (4.1 ± 7.2) and duration were increased (total duration: 64.1 ± 103.4 s; average duration: 6.8 ± 8.9 s), but have not reached significance. In comparison between EEG and MD data, furthermore, there was a positive correlation between e.c. glutamate concentrations and the number (r = 0.54; p < 0.01) and duration of epileptic seizures (r = 0.46; p < 0.01) at 90 and 120 min after trauma.
Brain water content
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24 h following CCI injury brain water content of the traumatized and non-traumatized hemispheres were separately assessed gravimetrically. Brain water content Q9 of the non-injured hemisphere was 78.52 ± 0.19% in the Glibenclamide group, and 78.48 ± 0.29% in the control group and thus, represented physiological levels. In the injured hemisphere, however, a significant increase of brain water content could be observed. In comparison to the control group, rats having been randomized to the treatment group showed a significant reduction in brain water content (80.47 ± 0.37% vs. 80.83 ± 0,44%; p < 0.05; n = 14). Treatment with Glibenclamide, thus, could reduce the post-traumatic increase of brain water content by 15.3% (Fig. 3).
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Contusion volume
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Treatment with glibenclamide had a significant beneficial effect on the development of contusion volumes. Using MRI scanning technique, a reliable and non-invasive method quantifying contusion sizes, significant effects of glibenclamide on contusion volume could be detected 8 h, 24 h, 72 h, and 7 days following trauma(Fig. 4). Within the first 72 h a continuous increase of contusion size from158.00 ± 36.82 mm3 (glibenclamide) vs. 264.11 ± 44.37 mm3 (control) (n = 10; p < 0.05) 8 h, to 172.53 ± 38.74 mm3 (glibenclamide) vs. 299.20 ± 64.02 mm3 (control) (p < 0.01; n = 10) 24 h and, finally, to 211.10 ± 41.03 mm3 (glibenclamide) vs. 309.76 ± 64.38 mm3 (control) (p < 0.01; n = 10) 72 h after CCI injury could be measured. Contusion volumes were reduced 7 days after trauma, but still, a significant difference between the treatment and the control groups
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Q12 Fig. 2. Concentration of extracellular metabolic parameters, such as glutamate, lactate, pyruvate and the lactate/pyruvate ratio between 30 and 180 min after CCI injury: Not showing any differences between the treatment and control groups, the post-traumatic increase of e.c. lactate and glutamate concentrations represent an anaerobic metabolism and cell death. (Very low concentrations, e.g. lactate and pyruvate at 30 min are not detectable). 337 338
was evident: 107.13 ± 21.33 mm3 (glibenclamide) vs. 163.96 ± 28.99 mm3 (control) (p < 0.01; n = 10).
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Neurological function
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Motor function and coordination were assessed by performing the ‘‘beam-walking’’ test. Prior CCI injury animals showed 0.15 ± 0.36 misplacements referring to the corresponding contralateral hind paw. After trauma the number of misplacements increased significantly and reached 10.89 ± 2.83 (glibenclamide) and 10.24 ± 4.20 (control) (p < 0.01; n = 10) followed by continuous recovery in both groups. A significant difference between the treatment and control groups, however, could not be observed within 7 days of observation.
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DISCUSSION
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In this study it was found that treatment with glibenclamide can reduce brain edema development and contusion volume following experimental TBI.
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Despite several attempts to influence inflammation, excitotoxicity, and vascularization, clinical treatment of post-traumatic brain edema is still a challenge, and osmotherapy has been the mainstay for years. Both, mannitol and hyper-oncotic saline are well-established (Berger et al., 1995; Brain Trauma Foundation, 2007; Finke, 2012; Marks et al., 2012), although, there is no treatment available that already prevents the development of brain edema. Among others, the currently described SUR1regulated NCCa-ATP (SUR1/TRPM4) channel might offer a new treatment target (Simard et al., 2006; Walcott et al., 2012). This channel is not constitutively expressed, but is transcriptionally up-regulated de novo in all cells of the neurovascular unit in many forms of central nervous system injury, including cerebral ischemia, TBI, spinal cord injury, and subarachnoid hemorrhage (Simard et al., 2012a). In comparison and unlike the well-known KATP-channel, SUR-1, but not Kir6.1 or Kir6.2 is transcriptionally up-regulated in ischemic astrocytes, neurons, and capillaries representing the regulatory subunit of this
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Fig. 3. Gravimetrical assessment of brain water content 24 h following controlled cortical impact injury (CCI): Animals treated with glibenclamide showed significant reduced brain edema (white). In reference to physiological levels of the contralateral hemisphere, the post-traumatic increase of brain edema could be reduced by 15.3%.
Fig. 4. Post-traumatic contusion volume quantified by MRI scanning technique at 8 h, 24 h, 72 h, and 7 d post injury. Animals treated with glibenclamide showed significant reduced contusion sizes at any time-points (white).
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channel (Simard et al., 2006). On the other hand, Trpm4 is considered to be the pore-forming unit (Simard et al., 2007b; Woo et al., 2013). Ischemia-induced increase in SUR-1 was corroborated by measurements of Abcc8 mRNA, encoding SUR-1, which increased two- to threefold in the core center of the infarct in a MCA stroke model 3 h after the insult (Deeley et al., 2006; Simard et al., 2006). In the peri-infarct region the ascent of SUR-1 was delayed and a peak could sharply be demarcated
8–16 h post stroke (Simard et al., 2006, 2012a). The NCCa-ATP channel is highly dependent on ATP. Depletion of ATP, as it can be observed frequently in the peri-contusional area following trauma or in the so-called penumbra region after stroke causes depolarization and, thus, opening of the pore-unit. As a consequence cations, accompanied by water following the osmotic gradient, are floating into cells resulting in ‘‘cell blebbing’’ that is characteristic of cytotoxic edema (Simard et al., 2006).
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Administration of low-dose glibenclamidecan inhibits the regulatory sub-unit SUR-1 of the NCCa-ATP channel. 395Q10 As a rare fluke patients with stroke that were on 396 glibenclamide treatment having diabetes mellitus seem 397 to be in favor to reach a better neurological 398 outcome(Kunte et al., 2007). The group of Simard et al. 399 could further convincingly show in a rodent model of cere400 bral MCA stroke that administration of glibenclamide can 401 significantly reduce cerebral edema, infarct volume, and 402 mortality rate (Simard et al., 2006, 2009a; Zhou et al., 403 2009). 404 Following experimental TBI using the Feeney device, 405 Simard et al. (2009c) could also demonstrate that SUR406 1 is up-regulated, especially in capillaries, within 3 h in 407 the tissue beneath the impact. Furthermore a prominent 408 peak could be shown in deeper structures, like the hippo409 campus, 24 h after trauma. By treating rats with glibencla410 mide fragmentation of capillaries and, thus, hemorrhagic 411 necrosis measured by Drabkin’s reagent and contusion 412 size (Nissl sections) could be reduced resulting in a better 413 neurological outcome quantified by ‘‘spontaneous vertical 414 exploration’’ (Simard et al., 2009c). 415 Despite these promising results, experimental data 416 addressing post-traumatic pathologies, like ICP, 417 metabolic changes, and post-traumatic brain edema 418 have been lacking, so far. In the current study using a 419 CCI model treatment with low-dose glibenclamide has 420 resulted in a highly significant reduction of brain edema 421 assessed gravimetrically 24 h post CCI injury. In MRI 422 scans a continuous increase of contusion sizes could be 423 depicted at 8, 24, and 72 h following CCI injury. In 424 comparison to controls, animals that were treated with 425 glibenclamide furthermore showed significant smaller 426 contusion sizes at any time. Despite those results, ICP 427 and extracellular metabolism were not significantly 428 influenced by glibenclamide in the acute stage within 429 180 min following trauma. This could be due to the fact 430 that SUR-1 is transcriptionally up-regulated de novo 431 within 3–6 h following trauma, as mentioned above. 432 Thus, treatment in the very acute stage seems to be 433 less effective, although, in accordance with the results 434 of Simard et al. (2006, 2012a), we could show that contu435 sion sizes were already reduced 8 h after injury. Espe436 cially with reference to the MD data, the intensity of the 437 trauma can be regarded as moderate. After an acute drop 438 of the e.c. glucose concentration (‘‘close to zero level’’) 439 that is typical following trauma and representing an acute 440 energy failure (Hutchinson et al., 1991, 2000; Persson 441 et al., 1996; Goodman et al., 1999; Kett-White et al., 442 2002), e.c. glutamate and lactate concentrations have 443 significantly increased within 60 min, but continuously 444 recovered throughout the further time of observation. 445 The incline of glutamate and lactate in the peri446 contusional region implies a shift toward anaerobic 447 metabolism leading to cell death that only occurred in 448 the very acute stage (60 min), but did not continue or 449 aggravate throughout the remaining time of observation 450 as it would be expected in severe TBI (Obrenovitch, 451 1999; Alessandri et al., 2003; Hillered et al., 2005; 452 Chamoun et al., 2010). This could potentially explain the 453 lack of difference between the treatment and the control 393 394
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454 groups in the ‘‘beam-walking’’ test assessing misplace455 ments of the contralateral hind paw. Post CCI injury rats 456 of the treatment and the control groups showed signifi457 cantly more misplacements, but recovered very fast 458 within a few days and did not show long-lasting impair459 ments. More specifically, glibenclamide seems to improve 460 ‘‘behavioral abnormalities’’ quantified by ‘‘spontaneous 461 vertical exploration’’ (Simard et al., 2009c) and to reduce 462 hippocampal injury resulting in improved spatial learning 463 (Patel et al., 2010). 464 Impairment of neurotransmitter metabolism leads to 465 an imbalance in neurotransmitter receptor expression 466 (Cremer et al., 2009). Changes in the membrane distribu467 tion of N-methyl-D-aspartate (NMDA) glutamate recep468 tors, e.g., seem to produce sweeping modifications in 469 neuronal excitability (Va`zquez-Lo`pez et al., 2005). In vitro 470 experiments could show that induced astrocyte glutamate 471 release triggers transient depolarization and epileptic 472 form discharges (Kang et al., 2005). Buckingham et al. (2011) could further demonstrate that the release of gluta- Q11473 474 mate after the transplantation of glioma cells into mice 475 have led to epileptiform hyper-excitability that spreads 476 over the adjacent brain tissue, explaining the observation 477 that seizures are a common comorbidity in primary brain 478 tumors. Therefore, the correlation between e.c. glutamate 479 concentrations and the number and duration of seizures, 480 as mentioned above, are well explained and might be an 481 interesting point for pursuing investigations. 482 In summary and in accordance with the available 483 literature, we could demonstrate that treatment with low484 dose glibenclamide reduces post-traumatic brain edema 485 and contusion volume following CCI injury in rats. 486 Possibly owing to the delayed transcriptional up487 regulation of the regulatory sub-unit of the NCCa-ATP 488 channel, no effect on ICP and cerebral metabolism in 489 the very acute stage following CCI injury could be 490 detected. Although glibenclamide seems to have high 491 therapeutic potency in the treatment of traumatic brain 492 and spinal cord injury, as well as stroke, further 493 experimental and clinical studies are necessary to 494 further delineate the role of glibenclamide in the 495 treatment of TBI.
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(Accepted 17 April 2014) (Available online xxxx)
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