Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-014-1626-8
INVITED REVIEW
The two-pore domain potassium channel KCNK5 deteriorates outcome in ischemic neurodegeneration Eva Göb & Stefan Bittner & Nicole Bobak & Peter Kraft & Kerstin Göbel & Friederike Langhauser & György A. Homola & Marc Brede & Thomas Budde & Sven G. Meuth & Christoph Kleinschnitz
Received: 1 September 2014 / Revised: 29 September 2014 / Accepted: 1 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Potassium channels can fulfill both beneficial and detrimental roles in neuronal damage during ischemic stroke. Earlier studies have characterized a neuroprotective role of the two-pore domain potassium channels KCNK2 (TREK1) and KCNK3 (TASK1). Protective neuronal hyperpolarization and prevention of intracellular Ca2+ overload and glutamate excitotoxicity were suggested to be the underlying mechanisms. We here identify an unexpected role for the related KCNK5 channel in a mouse model of transient middle cerebral artery occlusion (tMCAO). KCNK5 is strongly upregulated on neurons upon cerebral ischemia, where it is most likely involved in the induction of neuronal apoptosis. Hypoxic conditions elevated neuronal expression levels of
KCNK5 in acute brain slices and primary isolated neuronal cell cultures. In agreement, KCNK5 knockout mice had significantly reduced infarct volumes and improved neurologic function 24 h after 60 min of tMCAO and this protective effect was preserved at later stages of infarct development. KCNK5 deficiency resulted in a significantly reduced number of apoptotic neurons, a downregulation of pro-apoptotic and upregulation of anti-apoptotic factors. Results of adoptive transfer experiments of wild-type and Kcnk5−/− immune cells into Rag1−/− mice prior to tMCAO exclude a major role of KCNK5 in poststroke inflammatory reactions. In summary, KCNK5 expression is induced on neurons under ischemic conditions where it most likely exerts pro-apoptotic effects.
First authors Eva Göb and Stefan Bittner and senior authors Sven G. Meuth and Christoph Kleinschnitz made equal contributions. Electronic supplementary material The online version of this article (doi:10.1007/s00424-014-1626-8) contains supplementary material, which is available to authorized users. E. Göb : P. Kraft : F. Langhauser : C. Kleinschnitz Department of Neurology, University Clinics Würzburg, Würzburg, Germany S. Bittner : N. Bobak : K. Göbel : S. G. Meuth (*) Department of Neurology, University of Münster, Münster, Germany e-mail: [email protected] S. Bittner Interdisciplinary Center for Clinical Research (IZKF), Münster, Germany N. Bobak LabEx ICST, Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice-Sophia Antipolis, Valbonne, France P. Kraft Institute of Clinical Epidemiology and Biometry, Comprehensive Heart Failure Center, University of Würzburg, Würzburg, Germany
G. A. Homola Department of Neuroradiology, University Clinics Würzburg, Würzburg, Germany
M. Brede Department of Anesthesiology and Critical Care, University Clinics Würzburg, Würzburg, Germany
T. Budde Department of Physiology I, University of Münster, Münster, Germany
S. G. Meuth Department of Physiology I - Neuropathophysiology, University of Münster, Münster, Germany
Pflugers Arch - Eur J Physiol
Hence, pharmacological blockade of KCNK5 might have therapeutic potential in preventing ischemic neurodegeneration. Keywords Stroke . K2P channels . Potassium channels . Neuroprotection . KCNK5 . TASK2 Abbreviations ATP BBB CREB K2P channels MRI TASK2 tMCAO TTC TUNEL WT
Adenosine triphosphate Blood-brain barrier cAMP response element-binding protein Two-pore domain potassium channels Magnetic resonance imaging TWIK-related acid-sensitive potassium channel 2 Transient middle cerebral artery occlusion Triphenyltetrazolium chloride TdT-mediated dUTP-biotin nick end labeling wild-type
Introduction Ischemic stroke is one of the leading causes of death and longterm disability in the world [19]. To date, neuroprotective strategies have been shown to fail in clinical trials, elucidating the need for a better understanding of the underlying molecular mechanisms associated with neuronal cell death in cerebral ischemia [25, 62]. Important mechanistic insights into the complex pathophysiology of the ischemic cascade have identified major harmful events initiating and perpetuating neuronal death. Hypoxia and glucose deprivation lead to a lack of energy necessary to maintain adenosine triphosphate (ATP)driven ion pumps and ionic balance. Furthermore, a massive release of the excitatory neurotransmitter glutamate results in neuronal depolarization, intracellular calcium overload and excitotoxic death [46]. Neurons display a broad array of homeostatic protective mechanisms counterbalancing the hyperexcitability induced by hypoxic and ischemic periods. Hyperpolarizing potassium channels are especially suited to control neuronal excitability as has been demonstrated for KATP [5], Ca2+-activated big potassium (BK) channels [58], and voltage-gated potassium channels [50]. Furthermore, it is now well established that innate and adaptive immunity contributes significantly to central nervous system (CNS) inflammation, infarct size, and functional damage after stroke. Potassium channels have been shown to regulate migration and effector functions of immune cells [18]. In accordance, pharmacological blockade of the calcium-activated potassium channel KCa3.1, which is involved in microglial, macrophage, and T cell function, had beneficial effects in a rat model of ischemic stroke [20],
demonstrating the therapeutic potential of targeting inflammation in cerebral ischemia. Two-pore domain potassium channels (K2P channels) play an important role in setting the resting membrane potential, regulating neuronal excitability and integrating extracellular signals under pathophysiological conditions [34]. KCNK5 (TWIK-related acid-sensitive potassium channel 2, TASK-2) is a member of the K2P channel family displaying unique biophysical properties enabling regulation of channel activity by pathophysiological hallmarks of stroke development [21]. KCNK5 is sensitive to changes in extracellular pH values and is inhibited by extracellular acidification (10 % activity at pH 6.5 [57]). Furthermore, an activation of KCNK5 upon hypoxia has been described that might be due to a direct activation of KCNK5 by reactive oxygen species [30]. In peripheral organs, KCNK5 channels regulate HCO3- reabsorption in kidney proximal tubules and are involved in cell volume regulation [3, 15, 21]. On human T lymphocytes, KCNK5 regulates T cell effector functions and its expression levels on pathogenic T cells correlate with disease activity in autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis [9, 10]. Conflicting reports exist concerning the expression of KCNK5 within the CNS. While some found no expression of KCNK5 in the adult brain [1, 63], others describe KCNK5 channels in brainstem nuclei [30], hippocampus, and cerebellum [23, 29]. Furthermore, KCNK5 can be upregulated on neurons and astrocytes under pathophysiological conditions, as has been found in a rat model of epilepsy [35]. In summary, the regulatory mechanisms and expression patterns suggest an as yet unidentified involvement of KCNK5 in cerebral ischemia. Therefore, we here investigated the pathophysiological role of KCNK5 in a mouse model of acute ischemic stroke.
Material and methods Stroke model All animal experiments were approved by the local state authorities (Regierung von Unterfranken) and conducted in agreement with the German Animal Welfare Act (German Ministry of Agriculture, Health and Economic Cooperation). Focal cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in 6- to 8-week-old male C57BL/6 wild-type (WT) mice (Charles River) and Kcnk5−/− mice [6], weighing between 20 and 25 g, using the intraluminal filament technique [49]. Mice were anesthetized with 2.0 % isoflurane, and core body temperature was maintained at 37 °C throughout surgery. Following a midline cervical skin incision, the proximal common carotid artery and the external carotid artery were ligated and a standardized silicon rubber-coated nylon monofilament (6023910PK10,
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Doccol Corp.) was inserted and advanced via the right internal carotid artery to occlude the origin of the right middle cerebral artery. The intraluminal suture was left in situ for 60 min. After that, mice were re-anesthetized and the occluding monofilament was withdrawn to allow reperfusion. Operation time per animal did not exceed 10 min. For reconstitution of Rag1−/ − mice with CD4+ T cells, immune cells were isolated from spleen and lymph node cell suspensions of Kcnk5−/− and C57BL/6 WT mice in RPMI medium containing 1 % Lglutamine, 1 % FCS, and 1 % penicillin/streptomycin. After removal of red blood cells with ammonium chloride, T cells were enriched using the CD4+ T Cell Isolation Kit II (Miltenyi Biotec), resuspended to 7.5 × 10 5 cells per 100 μl in phosphate-buffered saline and subsequently injected intravenously into Rag1−/− mice. T cell-reconstituted Rag1−/− mice were subjected to tMCAO 24 h after injection [39]. Stroke study population and quality control All stroke experiments were conducted according to the recommendations for research in experimental stroke studies [26] and the current Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines (http://www.nc3rs.org/ ARRIVE). Animals were randomly assigned to the operators by independent persons not involved in data acquisition and analysis. Surgeries and evaluation of all readout parameters were performed while being blinded to the experimental groups. The following conditions excluded mice from end-point analyses (exclusion criteria): 1, death within 24 h after MCAO (except for survival studies); 2, subarachnoid hemorrhage or bleeding into the brain parenchyma (as macroscopically assessed during brain sampling or by magnetic resonance imaging [MRI]); 3, Bederson score = 0 (24 h after MCAO); and 4, operation time >10 min. In total, 159 mice (80 C57BL/ 6 WT, 52 Kcnk5−/−, 27 Rag1−/−) were used in this study. Of the 116 mice subjected to tMCAO, 11 mice (9.5 %) met at least one exclusion criterion after randomization and, therefore, were withdrawn from the study. The number of excluded animals (dropout rate) was evenly distributed between the groups (p>0.05).
onto string by one or both forepaws plus one or both hindpaws; 4, hangs onto string by forepaws and hindpaws plus tail wrapped around string; and 5, escape (to the supports). Determination of stroke size Mice were sacrificed 24 h after tMCAO, respectively. Brains were quickly removed and cut into 2-mm-thick coronal sections using a mouse brain slice matrix (Harvard Apparatus). The slices were stained with 2 % triphenyltetrazolium chloride (TTC, Sigma-Aldrich) to visualize the infarctions [7]. Planimetric measurements (ImageJ software, National Institutes of Health) were used to calculate lesion volumes, which were corrected for brain edema according to the following equation: Vindirect (mm3)=Vinfarct ×(1−(Vih −Vch)/Vch. The term (Vih −Vch) represents the difference in volumes between the ischemic hemisphere (ih) and the control hemisphere (ch), and (Vih −Vch)/Vch expresses the difference as a percentage of the control hemisphere. Determination of edema formation The extent of brain edema was calculated by planimetry from TTC-stained brain sections according to the following equation: Brain edema area (%)=[(AL+AI+AC)×100/(AC×2)]− 100, where AL represents the total area of TTC-negative (ischemic) brain tissue, AI represents the total area of viable tissue of the ipsilateral (ischemic) hemisphere, and AC represents the total area of the contralateral (healthy) hemisphere [33]. Laser Doppler flowmetry Laser Doppler flowmetry (Moore Instruments) was performed in C57BL/6 WT and Kcnk5−/− mice at baseline (before ischemia), 10 min after inserting the occluding filament (ischemia) and 10 min after removing the occluding filament (reperfusion) [22]. The regional cerebral blood flow was measured in the territory of the right middle cerebral artery (6 mm lateral and 2 mm posterior from bregma) (Fig. S1). Magnet resonance imaging
Assessment of functional outcome Global neurologic deficits were quantified according to the Bederson score [8]: 0, no deficit; 1, forelimb flexion; 2, as for 1, plus decreased resistance to lateral push; 3, unidirectional circling; 4, longitudinal spinning; and 5, no movement. The grip-test score was applied to monitor motor function and coordination [51]. For this purpose, the mouse was placed midway on a string between two supports and rated as follows: 0, mouse falls off; 1, hangs onto string by one or both forepaws; 2, as for 1, and attempts to climb onto string; 3, hangs
To analyze infarct dynamics and to scan for possible intracerebral bleeding, stroke assessment was performed by serial MRI on a 1.5-T MRI unit (MAGNETOM SymphonyVision, Siemens) on days 1 and 7 after tMCAO as described [43]. Assessment of the cerebral vasculature To assess the structure of the cerebral vasculature, we examined the circle of Willis and main arteries by ink perfusion. Mice were deeply anesthetized with CO2 and transcardially
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perfused with 4 % paraformaldehyde (PFA), followed by 3 ml of black ink diluted in 4 % PFA (1:5v/v). Brains were carefully removed, fixed in 4 % PFA overnight at 4 °C, and the circle of Willis was examined under a microscope, and the development of the posterior communicating arteries (PComA) was quantified using the PComA score [53] (Fig. S1 and Table S1). Blood gas analysis A 100 μl sample of arterial blood was drawn from the left cardiac ventricle of anesthetized mice using a heparinized syringe. We then determined PaO2, PaCO2, and pH using an ABL 77 automated blood gas analyzer (Radiometer) (Table S1). Invasive hemodynamics For the assessment of blood pressure and heart rate, mice were anesthetized with 2.0 % isoflurane and catheterized via the right carotid artery with a high-fidelity 1.4 F Millarmicrotip catheter (Millar Instruments) as described [17]. Hemodynamic data were digitized via a MacLabsystem (AD Instruments) connected to an Apple G4 PowerPC computer and analyzed (Table S1). Western blot Cortices or basal ganglia were dissected from native brains and homogenized in RIPA buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1 % NP-40) containing 0.1 % sodium dodecyl sulfate (SDS) and 4 % proteinase inhibitor (complete protease inhibitor cocktail, Roche). After sonication for 10 s, tissue lysates were centrifuged at 15,000×g for 30 min at 4 °C, and supernatants were used for bicinchoninic acid protein assay (BCA). Total lysates were diluted in 2× SDS sample buffer (120 mM Tris/HCl, 10 % SDS, 20 % glycerine, 20 % 2mercaptoethanol; pH 6.8) to a final concentration of 2 μg/μl and used for SDS-PAGE and Western blot analysis as previously described [33]. The following antibodies were used: pAb anti-TASK2 (P1106, Sigma-Aldrich, 1:1000), mAb anti-CREB (48H2; 9197, Cell Signaling, 1:500), mAb antiphospho-CREB (Ser133) (87G3; 9198, Cell Signaling, 1:500), pAb anti-AIF (ab1998; Abcam, 1:2000), pAb anticaspase-9 (9504, Cell Signaling, 1:500), pAb anti-occludin (ab31721; Abcam, 1:5000), anti-β-actin (A5441; SigmaAldrich, 1:250,000), donkey anti-rabbit IgG HRPconjugated (711-035-152; Dianova, 1:10,000), and donkey anti-mouse IgG HRP-conjugated (715-035-150; Dianova, 1:10,000). Protein bands were quantified by densitometric analysis using ImageJ software (National Institutes of Health) and normalized to the actin band, which served as loading control.
Quantitative real-time polymerase chain reaction Tissue homogenization, RNA isolation, and quantitative realtime polymerase chain reaction (RT-PCR) were performed as described [43]. For total RNA preparation, TRIzol reagent® (Invitrogen) was used and RNA concentration was quantified spectrophotometrically. RNA (1 μg) was reversely transcribed with Fermentas reagents according to the manufacturer’s protocol using random hexamers. Relative gene expression levels of TASK2 (assay ID Mm00498900_m1, Applied Biosystems) were quantified with the fluorescent TaqMan® technology using the StepOnePlus™ Real-time PCR System (Applied B i o s y s t e m s ) . V I C l a b e l e d 1 8 s R N A ( Ta q M a n ® Predeveloped Assay Reagents for gene expression, part number 4319413E, Applied Biosystems) was used as an endogenous control to normalize the amount of sample RNA. Water controls were included to ensure specificity. Each sample was measured in triplicate, and data points were examined for integrity by analysis of the amplification plot. The comparative CT method was used for relative quantification of gene expression as described [61].
Vital brain slices Vital brain slices from 14- to 22-day-old male C57BL/6 WT mouse brains (between −2 and −4 mm from bregma) were prepared as described previously [48]. In brief, coronal sections were cut on a vibratome (Vibratome®, Series 1000 Classic) in an ice-chilled solution (200 mM saccharose, 20 mM PIPES, 2.5 mM KCl, 1.25 mM NaH2PO4, 10 mM MgSO4, 0.5 mM CaCl2, 10 mM glucose, pH 7.35) adjusted with NaOH. Prior to the staining procedure, slices were kept submerged in artificial cerebrospinal fluid (ACSF, 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM HEPES, 2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, pH adjusted to 7.35 by bubbling with a mixture of 95 % O2 and 5 % CO2). For ischemic conditions, slices were incubated in ACSF with reduced glucose (5 mM) and pH 6.4 for 4 h at 37 °C in a humidified incubator with 5 % CO2, 5 % O2, and 90 % N2.
Hippocampal neuronal cell cultures Neuronal cell cultures were obtained from C57BL/6 WT mouse embryos (E18) as described previously [40]. Neuronal cultures were incubated at 37 °C and 5 % CO2 and maintained in culture for up to 5 to 7 days before performing the experiments. Cell viability was assessed by the In Situ Cell Death Detection Kit (TMR red, Roche) according to the manufacturer’s instructions. For ischemic conditions, O2 was restricted per incubation at 37 °C in a humidified incubator with 5 % CO2, 5 % O2, and 90 % N2 for 4 h.
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Isolation of mouse brain microvascular endothelial cells We prepared mouse brain microvascular endothelial cells (MBMECs) from brains of C57BL/6 WT mice as described [12] and used them for immunohistochemistry (see below). Purity and cell morphology were controlled regularly by flow cytometry and microscopy. For functional analysis, cells were cultured under normoxic or ischemic conditions for 4 h at 37 °C in a humidified incubator with 5 % CO2 or 5 % CO2, 5 % O2, and 90 % N2, respectively. Immunohistochemistry Immunohistochemistry of native mouse brain sections, vital brain slices, and cultured cells was performed according to standard procedures [12, 37, 43]. For specific staining, the following antibodies were used: pAb anti-TASK2 (P1106, Sigma-Aldrich, 1:100) and pAb anti-NeuN (MAB377, Merck Millipore, 1:1000). For immunofluorescence analysis, slices were co-incubated with Cy3-labeled goat anti-rabbit and Alexa-Fluor 488 anti-mouse secondary antibodies (Abcam, 1:200). DNA was counterstained using ProLong® Gold anti-fade reagent containing 4′,6-diamidino-2phenylindole (DAPI) (Life technologies). Negative controls included omission of the primary or secondary antibodies and produced no signal. TdT-mediated dUTP-biotin nick end labeling (TUNEL) was performed to visualize apoptotic cells on 10-μm-thick cryo-embedded brain sections 24 h after tMCAO using the TUNEL in situ cell death detection kit, TMR red (Roche). Co-staining with antibodies against neuronal marker NeuN was applied to detect apoptotic neurons. For quantification of TUNEL-positive cells, identical brain sections from the ischemic basal ganglia were selected, and cell counting was performed from five subsequent slices (distance 100 μm) as described [37]. All sections were analyzed and acquired with a Nikon Eclipse 50i microscope equipped with the DSU3 DS camera control unit and the NIS-Elements software (Nikon) and a Zeiss Axio Scope microscope (Zeiss) equipped with a UI-1410-C CMOS camera (UEye) and the AxioVision software (Zeiss). Digital images were processed using Adobe Photoshop (Adobe Systems).
Bioscience, 556925). Cells were counted on a FACS Calibur (Becton Dickinson). Neutrophils and macrophages were isolated from the bone marrow of C57BL/6 mice, extracted out of tibia and femur, and separated using MACS separation kits (Anti-Ly-6G MicroBead Kit, CD11b MicroBeads, Miltenyi Biotech) according to the manufacturer’s instructions. T cells were isolated from spleens of C57BL/6 mice using MACS separation kit (Pan T cell isolation Kit II, Miltenyi Biotech) according to the manufacturer’s instructions. Platelets were isolated as previously described [38] using two centrifugation steps with a HEPES-Tyrode buffer (129 mM NaCl, 8.9 mM NaHCO3, 2.8 mM KCl, 0.8 mM KH2PO4, 5.6 mM glucose, 10 mM HEPES, 0.8 mM MgCl2). Prior to RNA isolation and protein lysate preparation in order to perform RT-PCR or Western blot analysis, T cells were stimulated with Dynabeads® Mouse TActivator CD3/CD28 for 48 h (Life Technologies) and macrophages and neutrophils were activated with LPS overnight. Statistics All results are expressed as mean±standard error of the mean (SEM) except for ordinal functional outcome scales which were depicted as scatter plots, including median with the 25% percentile and the 75% percentile given in brackets in the text. The numbers of animals (n=10) necessary to detect a standardized effect size on infarct volumes ≥0.25 (Kcnk5−/− vs. C57BL/6 WT mice) was determined via a priori sample size calculation with the following assumptions: α=0.05, β=0.2 (power 80 %), and mean of 20 % standard deviation of the mean (GraphPad Stat Mate 2.0, GraphPad Software). For statistical analysis, the GraphPad Prism 5 software package was used. Data were tested for Gaussian distribution with the Kolmogorov-Smirnov normality test and then analyzed by one-way ANOVA, or in case of measuring the effects of two factors simultaneously by two-way ANOVA with post hoc Bonferroni adjustment for p values. Nonparametric functional outcome scores were compared by the Kruskal-Wallis test with post hoc Dunn’s corrections. If only two groups were compared, unpaired, two-tailed Student’s t test or the nonparametric Mann-Whitney test was applied. p
INVITED REVIEW
The two-pore domain potassium channel KCNK5 deteriorates outcome in ischemic neurodegeneration Eva Göb & Stefan Bittner & Nicole Bobak & Peter Kraft & Kerstin Göbel & Friederike Langhauser & György A. Homola & Marc Brede & Thomas Budde & Sven G. Meuth & Christoph Kleinschnitz
Received: 1 September 2014 / Revised: 29 September 2014 / Accepted: 1 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Potassium channels can fulfill both beneficial and detrimental roles in neuronal damage during ischemic stroke. Earlier studies have characterized a neuroprotective role of the two-pore domain potassium channels KCNK2 (TREK1) and KCNK3 (TASK1). Protective neuronal hyperpolarization and prevention of intracellular Ca2+ overload and glutamate excitotoxicity were suggested to be the underlying mechanisms. We here identify an unexpected role for the related KCNK5 channel in a mouse model of transient middle cerebral artery occlusion (tMCAO). KCNK5 is strongly upregulated on neurons upon cerebral ischemia, where it is most likely involved in the induction of neuronal apoptosis. Hypoxic conditions elevated neuronal expression levels of
KCNK5 in acute brain slices and primary isolated neuronal cell cultures. In agreement, KCNK5 knockout mice had significantly reduced infarct volumes and improved neurologic function 24 h after 60 min of tMCAO and this protective effect was preserved at later stages of infarct development. KCNK5 deficiency resulted in a significantly reduced number of apoptotic neurons, a downregulation of pro-apoptotic and upregulation of anti-apoptotic factors. Results of adoptive transfer experiments of wild-type and Kcnk5−/− immune cells into Rag1−/− mice prior to tMCAO exclude a major role of KCNK5 in poststroke inflammatory reactions. In summary, KCNK5 expression is induced on neurons under ischemic conditions where it most likely exerts pro-apoptotic effects.
First authors Eva Göb and Stefan Bittner and senior authors Sven G. Meuth and Christoph Kleinschnitz made equal contributions. Electronic supplementary material The online version of this article (doi:10.1007/s00424-014-1626-8) contains supplementary material, which is available to authorized users. E. Göb : P. Kraft : F. Langhauser : C. Kleinschnitz Department of Neurology, University Clinics Würzburg, Würzburg, Germany S. Bittner : N. Bobak : K. Göbel : S. G. Meuth (*) Department of Neurology, University of Münster, Münster, Germany e-mail: [email protected] S. Bittner Interdisciplinary Center for Clinical Research (IZKF), Münster, Germany N. Bobak LabEx ICST, Institut de Pharmacologie Moléculaire et Cellulaire, CNRS and Université de Nice-Sophia Antipolis, Valbonne, France P. Kraft Institute of Clinical Epidemiology and Biometry, Comprehensive Heart Failure Center, University of Würzburg, Würzburg, Germany
G. A. Homola Department of Neuroradiology, University Clinics Würzburg, Würzburg, Germany
M. Brede Department of Anesthesiology and Critical Care, University Clinics Würzburg, Würzburg, Germany
T. Budde Department of Physiology I, University of Münster, Münster, Germany
S. G. Meuth Department of Physiology I - Neuropathophysiology, University of Münster, Münster, Germany
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Hence, pharmacological blockade of KCNK5 might have therapeutic potential in preventing ischemic neurodegeneration. Keywords Stroke . K2P channels . Potassium channels . Neuroprotection . KCNK5 . TASK2 Abbreviations ATP BBB CREB K2P channels MRI TASK2 tMCAO TTC TUNEL WT
Adenosine triphosphate Blood-brain barrier cAMP response element-binding protein Two-pore domain potassium channels Magnetic resonance imaging TWIK-related acid-sensitive potassium channel 2 Transient middle cerebral artery occlusion Triphenyltetrazolium chloride TdT-mediated dUTP-biotin nick end labeling wild-type
Introduction Ischemic stroke is one of the leading causes of death and longterm disability in the world [19]. To date, neuroprotective strategies have been shown to fail in clinical trials, elucidating the need for a better understanding of the underlying molecular mechanisms associated with neuronal cell death in cerebral ischemia [25, 62]. Important mechanistic insights into the complex pathophysiology of the ischemic cascade have identified major harmful events initiating and perpetuating neuronal death. Hypoxia and glucose deprivation lead to a lack of energy necessary to maintain adenosine triphosphate (ATP)driven ion pumps and ionic balance. Furthermore, a massive release of the excitatory neurotransmitter glutamate results in neuronal depolarization, intracellular calcium overload and excitotoxic death [46]. Neurons display a broad array of homeostatic protective mechanisms counterbalancing the hyperexcitability induced by hypoxic and ischemic periods. Hyperpolarizing potassium channels are especially suited to control neuronal excitability as has been demonstrated for KATP [5], Ca2+-activated big potassium (BK) channels [58], and voltage-gated potassium channels [50]. Furthermore, it is now well established that innate and adaptive immunity contributes significantly to central nervous system (CNS) inflammation, infarct size, and functional damage after stroke. Potassium channels have been shown to regulate migration and effector functions of immune cells [18]. In accordance, pharmacological blockade of the calcium-activated potassium channel KCa3.1, which is involved in microglial, macrophage, and T cell function, had beneficial effects in a rat model of ischemic stroke [20],
demonstrating the therapeutic potential of targeting inflammation in cerebral ischemia. Two-pore domain potassium channels (K2P channels) play an important role in setting the resting membrane potential, regulating neuronal excitability and integrating extracellular signals under pathophysiological conditions [34]. KCNK5 (TWIK-related acid-sensitive potassium channel 2, TASK-2) is a member of the K2P channel family displaying unique biophysical properties enabling regulation of channel activity by pathophysiological hallmarks of stroke development [21]. KCNK5 is sensitive to changes in extracellular pH values and is inhibited by extracellular acidification (10 % activity at pH 6.5 [57]). Furthermore, an activation of KCNK5 upon hypoxia has been described that might be due to a direct activation of KCNK5 by reactive oxygen species [30]. In peripheral organs, KCNK5 channels regulate HCO3- reabsorption in kidney proximal tubules and are involved in cell volume regulation [3, 15, 21]. On human T lymphocytes, KCNK5 regulates T cell effector functions and its expression levels on pathogenic T cells correlate with disease activity in autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis [9, 10]. Conflicting reports exist concerning the expression of KCNK5 within the CNS. While some found no expression of KCNK5 in the adult brain [1, 63], others describe KCNK5 channels in brainstem nuclei [30], hippocampus, and cerebellum [23, 29]. Furthermore, KCNK5 can be upregulated on neurons and astrocytes under pathophysiological conditions, as has been found in a rat model of epilepsy [35]. In summary, the regulatory mechanisms and expression patterns suggest an as yet unidentified involvement of KCNK5 in cerebral ischemia. Therefore, we here investigated the pathophysiological role of KCNK5 in a mouse model of acute ischemic stroke.
Material and methods Stroke model All animal experiments were approved by the local state authorities (Regierung von Unterfranken) and conducted in agreement with the German Animal Welfare Act (German Ministry of Agriculture, Health and Economic Cooperation). Focal cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in 6- to 8-week-old male C57BL/6 wild-type (WT) mice (Charles River) and Kcnk5−/− mice [6], weighing between 20 and 25 g, using the intraluminal filament technique [49]. Mice were anesthetized with 2.0 % isoflurane, and core body temperature was maintained at 37 °C throughout surgery. Following a midline cervical skin incision, the proximal common carotid artery and the external carotid artery were ligated and a standardized silicon rubber-coated nylon monofilament (6023910PK10,
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Doccol Corp.) was inserted and advanced via the right internal carotid artery to occlude the origin of the right middle cerebral artery. The intraluminal suture was left in situ for 60 min. After that, mice were re-anesthetized and the occluding monofilament was withdrawn to allow reperfusion. Operation time per animal did not exceed 10 min. For reconstitution of Rag1−/ − mice with CD4+ T cells, immune cells were isolated from spleen and lymph node cell suspensions of Kcnk5−/− and C57BL/6 WT mice in RPMI medium containing 1 % Lglutamine, 1 % FCS, and 1 % penicillin/streptomycin. After removal of red blood cells with ammonium chloride, T cells were enriched using the CD4+ T Cell Isolation Kit II (Miltenyi Biotec), resuspended to 7.5 × 10 5 cells per 100 μl in phosphate-buffered saline and subsequently injected intravenously into Rag1−/− mice. T cell-reconstituted Rag1−/− mice were subjected to tMCAO 24 h after injection [39]. Stroke study population and quality control All stroke experiments were conducted according to the recommendations for research in experimental stroke studies [26] and the current Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines (http://www.nc3rs.org/ ARRIVE). Animals were randomly assigned to the operators by independent persons not involved in data acquisition and analysis. Surgeries and evaluation of all readout parameters were performed while being blinded to the experimental groups. The following conditions excluded mice from end-point analyses (exclusion criteria): 1, death within 24 h after MCAO (except for survival studies); 2, subarachnoid hemorrhage or bleeding into the brain parenchyma (as macroscopically assessed during brain sampling or by magnetic resonance imaging [MRI]); 3, Bederson score = 0 (24 h after MCAO); and 4, operation time >10 min. In total, 159 mice (80 C57BL/ 6 WT, 52 Kcnk5−/−, 27 Rag1−/−) were used in this study. Of the 116 mice subjected to tMCAO, 11 mice (9.5 %) met at least one exclusion criterion after randomization and, therefore, were withdrawn from the study. The number of excluded animals (dropout rate) was evenly distributed between the groups (p>0.05).
onto string by one or both forepaws plus one or both hindpaws; 4, hangs onto string by forepaws and hindpaws plus tail wrapped around string; and 5, escape (to the supports). Determination of stroke size Mice were sacrificed 24 h after tMCAO, respectively. Brains were quickly removed and cut into 2-mm-thick coronal sections using a mouse brain slice matrix (Harvard Apparatus). The slices were stained with 2 % triphenyltetrazolium chloride (TTC, Sigma-Aldrich) to visualize the infarctions [7]. Planimetric measurements (ImageJ software, National Institutes of Health) were used to calculate lesion volumes, which were corrected for brain edema according to the following equation: Vindirect (mm3)=Vinfarct ×(1−(Vih −Vch)/Vch. The term (Vih −Vch) represents the difference in volumes between the ischemic hemisphere (ih) and the control hemisphere (ch), and (Vih −Vch)/Vch expresses the difference as a percentage of the control hemisphere. Determination of edema formation The extent of brain edema was calculated by planimetry from TTC-stained brain sections according to the following equation: Brain edema area (%)=[(AL+AI+AC)×100/(AC×2)]− 100, where AL represents the total area of TTC-negative (ischemic) brain tissue, AI represents the total area of viable tissue of the ipsilateral (ischemic) hemisphere, and AC represents the total area of the contralateral (healthy) hemisphere [33]. Laser Doppler flowmetry Laser Doppler flowmetry (Moore Instruments) was performed in C57BL/6 WT and Kcnk5−/− mice at baseline (before ischemia), 10 min after inserting the occluding filament (ischemia) and 10 min after removing the occluding filament (reperfusion) [22]. The regional cerebral blood flow was measured in the territory of the right middle cerebral artery (6 mm lateral and 2 mm posterior from bregma) (Fig. S1). Magnet resonance imaging
Assessment of functional outcome Global neurologic deficits were quantified according to the Bederson score [8]: 0, no deficit; 1, forelimb flexion; 2, as for 1, plus decreased resistance to lateral push; 3, unidirectional circling; 4, longitudinal spinning; and 5, no movement. The grip-test score was applied to monitor motor function and coordination [51]. For this purpose, the mouse was placed midway on a string between two supports and rated as follows: 0, mouse falls off; 1, hangs onto string by one or both forepaws; 2, as for 1, and attempts to climb onto string; 3, hangs
To analyze infarct dynamics and to scan for possible intracerebral bleeding, stroke assessment was performed by serial MRI on a 1.5-T MRI unit (MAGNETOM SymphonyVision, Siemens) on days 1 and 7 after tMCAO as described [43]. Assessment of the cerebral vasculature To assess the structure of the cerebral vasculature, we examined the circle of Willis and main arteries by ink perfusion. Mice were deeply anesthetized with CO2 and transcardially
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perfused with 4 % paraformaldehyde (PFA), followed by 3 ml of black ink diluted in 4 % PFA (1:5v/v). Brains were carefully removed, fixed in 4 % PFA overnight at 4 °C, and the circle of Willis was examined under a microscope, and the development of the posterior communicating arteries (PComA) was quantified using the PComA score [53] (Fig. S1 and Table S1). Blood gas analysis A 100 μl sample of arterial blood was drawn from the left cardiac ventricle of anesthetized mice using a heparinized syringe. We then determined PaO2, PaCO2, and pH using an ABL 77 automated blood gas analyzer (Radiometer) (Table S1). Invasive hemodynamics For the assessment of blood pressure and heart rate, mice were anesthetized with 2.0 % isoflurane and catheterized via the right carotid artery with a high-fidelity 1.4 F Millarmicrotip catheter (Millar Instruments) as described [17]. Hemodynamic data were digitized via a MacLabsystem (AD Instruments) connected to an Apple G4 PowerPC computer and analyzed (Table S1). Western blot Cortices or basal ganglia were dissected from native brains and homogenized in RIPA buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1 % NP-40) containing 0.1 % sodium dodecyl sulfate (SDS) and 4 % proteinase inhibitor (complete protease inhibitor cocktail, Roche). After sonication for 10 s, tissue lysates were centrifuged at 15,000×g for 30 min at 4 °C, and supernatants were used for bicinchoninic acid protein assay (BCA). Total lysates were diluted in 2× SDS sample buffer (120 mM Tris/HCl, 10 % SDS, 20 % glycerine, 20 % 2mercaptoethanol; pH 6.8) to a final concentration of 2 μg/μl and used for SDS-PAGE and Western blot analysis as previously described [33]. The following antibodies were used: pAb anti-TASK2 (P1106, Sigma-Aldrich, 1:1000), mAb anti-CREB (48H2; 9197, Cell Signaling, 1:500), mAb antiphospho-CREB (Ser133) (87G3; 9198, Cell Signaling, 1:500), pAb anti-AIF (ab1998; Abcam, 1:2000), pAb anticaspase-9 (9504, Cell Signaling, 1:500), pAb anti-occludin (ab31721; Abcam, 1:5000), anti-β-actin (A5441; SigmaAldrich, 1:250,000), donkey anti-rabbit IgG HRPconjugated (711-035-152; Dianova, 1:10,000), and donkey anti-mouse IgG HRP-conjugated (715-035-150; Dianova, 1:10,000). Protein bands were quantified by densitometric analysis using ImageJ software (National Institutes of Health) and normalized to the actin band, which served as loading control.
Quantitative real-time polymerase chain reaction Tissue homogenization, RNA isolation, and quantitative realtime polymerase chain reaction (RT-PCR) were performed as described [43]. For total RNA preparation, TRIzol reagent® (Invitrogen) was used and RNA concentration was quantified spectrophotometrically. RNA (1 μg) was reversely transcribed with Fermentas reagents according to the manufacturer’s protocol using random hexamers. Relative gene expression levels of TASK2 (assay ID Mm00498900_m1, Applied Biosystems) were quantified with the fluorescent TaqMan® technology using the StepOnePlus™ Real-time PCR System (Applied B i o s y s t e m s ) . V I C l a b e l e d 1 8 s R N A ( Ta q M a n ® Predeveloped Assay Reagents for gene expression, part number 4319413E, Applied Biosystems) was used as an endogenous control to normalize the amount of sample RNA. Water controls were included to ensure specificity. Each sample was measured in triplicate, and data points were examined for integrity by analysis of the amplification plot. The comparative CT method was used for relative quantification of gene expression as described [61].
Vital brain slices Vital brain slices from 14- to 22-day-old male C57BL/6 WT mouse brains (between −2 and −4 mm from bregma) were prepared as described previously [48]. In brief, coronal sections were cut on a vibratome (Vibratome®, Series 1000 Classic) in an ice-chilled solution (200 mM saccharose, 20 mM PIPES, 2.5 mM KCl, 1.25 mM NaH2PO4, 10 mM MgSO4, 0.5 mM CaCl2, 10 mM glucose, pH 7.35) adjusted with NaOH. Prior to the staining procedure, slices were kept submerged in artificial cerebrospinal fluid (ACSF, 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM HEPES, 2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, pH adjusted to 7.35 by bubbling with a mixture of 95 % O2 and 5 % CO2). For ischemic conditions, slices were incubated in ACSF with reduced glucose (5 mM) and pH 6.4 for 4 h at 37 °C in a humidified incubator with 5 % CO2, 5 % O2, and 90 % N2.
Hippocampal neuronal cell cultures Neuronal cell cultures were obtained from C57BL/6 WT mouse embryos (E18) as described previously [40]. Neuronal cultures were incubated at 37 °C and 5 % CO2 and maintained in culture for up to 5 to 7 days before performing the experiments. Cell viability was assessed by the In Situ Cell Death Detection Kit (TMR red, Roche) according to the manufacturer’s instructions. For ischemic conditions, O2 was restricted per incubation at 37 °C in a humidified incubator with 5 % CO2, 5 % O2, and 90 % N2 for 4 h.
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Isolation of mouse brain microvascular endothelial cells We prepared mouse brain microvascular endothelial cells (MBMECs) from brains of C57BL/6 WT mice as described [12] and used them for immunohistochemistry (see below). Purity and cell morphology were controlled regularly by flow cytometry and microscopy. For functional analysis, cells were cultured under normoxic or ischemic conditions for 4 h at 37 °C in a humidified incubator with 5 % CO2 or 5 % CO2, 5 % O2, and 90 % N2, respectively. Immunohistochemistry Immunohistochemistry of native mouse brain sections, vital brain slices, and cultured cells was performed according to standard procedures [12, 37, 43]. For specific staining, the following antibodies were used: pAb anti-TASK2 (P1106, Sigma-Aldrich, 1:100) and pAb anti-NeuN (MAB377, Merck Millipore, 1:1000). For immunofluorescence analysis, slices were co-incubated with Cy3-labeled goat anti-rabbit and Alexa-Fluor 488 anti-mouse secondary antibodies (Abcam, 1:200). DNA was counterstained using ProLong® Gold anti-fade reagent containing 4′,6-diamidino-2phenylindole (DAPI) (Life technologies). Negative controls included omission of the primary or secondary antibodies and produced no signal. TdT-mediated dUTP-biotin nick end labeling (TUNEL) was performed to visualize apoptotic cells on 10-μm-thick cryo-embedded brain sections 24 h after tMCAO using the TUNEL in situ cell death detection kit, TMR red (Roche). Co-staining with antibodies against neuronal marker NeuN was applied to detect apoptotic neurons. For quantification of TUNEL-positive cells, identical brain sections from the ischemic basal ganglia were selected, and cell counting was performed from five subsequent slices (distance 100 μm) as described [37]. All sections were analyzed and acquired with a Nikon Eclipse 50i microscope equipped with the DSU3 DS camera control unit and the NIS-Elements software (Nikon) and a Zeiss Axio Scope microscope (Zeiss) equipped with a UI-1410-C CMOS camera (UEye) and the AxioVision software (Zeiss). Digital images were processed using Adobe Photoshop (Adobe Systems).
Bioscience, 556925). Cells were counted on a FACS Calibur (Becton Dickinson). Neutrophils and macrophages were isolated from the bone marrow of C57BL/6 mice, extracted out of tibia and femur, and separated using MACS separation kits (Anti-Ly-6G MicroBead Kit, CD11b MicroBeads, Miltenyi Biotech) according to the manufacturer’s instructions. T cells were isolated from spleens of C57BL/6 mice using MACS separation kit (Pan T cell isolation Kit II, Miltenyi Biotech) according to the manufacturer’s instructions. Platelets were isolated as previously described [38] using two centrifugation steps with a HEPES-Tyrode buffer (129 mM NaCl, 8.9 mM NaHCO3, 2.8 mM KCl, 0.8 mM KH2PO4, 5.6 mM glucose, 10 mM HEPES, 0.8 mM MgCl2). Prior to RNA isolation and protein lysate preparation in order to perform RT-PCR or Western blot analysis, T cells were stimulated with Dynabeads® Mouse TActivator CD3/CD28 for 48 h (Life Technologies) and macrophages and neutrophils were activated with LPS overnight. Statistics All results are expressed as mean±standard error of the mean (SEM) except for ordinal functional outcome scales which were depicted as scatter plots, including median with the 25% percentile and the 75% percentile given in brackets in the text. The numbers of animals (n=10) necessary to detect a standardized effect size on infarct volumes ≥0.25 (Kcnk5−/− vs. C57BL/6 WT mice) was determined via a priori sample size calculation with the following assumptions: α=0.05, β=0.2 (power 80 %), and mean of 20 % standard deviation of the mean (GraphPad Stat Mate 2.0, GraphPad Software). For statistical analysis, the GraphPad Prism 5 software package was used. Data were tested for Gaussian distribution with the Kolmogorov-Smirnov normality test and then analyzed by one-way ANOVA, or in case of measuring the effects of two factors simultaneously by two-way ANOVA with post hoc Bonferroni adjustment for p values. Nonparametric functional outcome scores were compared by the Kruskal-Wallis test with post hoc Dunn’s corrections. If only two groups were compared, unpaired, two-tailed Student’s t test or the nonparametric Mann-Whitney test was applied. p
