Significance Stroke is one of the most prevalent types of acute brain injury, and rehabilitation poses globally a significant personal as well as socioeconomic burden. We here demonstrate a potent mitigation of stroke-induced brain damage by blocking adrenergic receptors. Stroke induces cortical spreading depolarizations which repeatedly increases extracellular potassium. We show that adrenergic receptor antagonism promotes the normalization of extracellular medium following stroke. From a translational standpoint, this approach may offer an affordable approach to current treatments such as tissue plasminogen activator administration and mechanical thrombectomy.

Abstract Spontaneous waves of cortical spreading depolarization (CSD) are induced in the setting of acute focal ischemia. CSD is linked to a sharp increase of extracellular K+ that induces a long-lasting suppression of neural activity. Furthermore, CSD induces secondary irreversible damage in the ischemic brain, suggesting that K+ homeostasis might constitute a therapeutic strategy in ischemic stroke. Here we report that adrenergic receptor (AdR) antagonism accelerates normalization of extracellular K+, resulting in faster recovery of neural activity after photothrombotic stroke. Remarkably, systemic adrenergic blockade before or after stroke facilitated functional motor recovery and reduced infarct volume, paralleling the preservation of the water channel aquaporin-4 in astrocytes. Our observations suggest that AdR blockers promote cerebrospinal fluid exchange and rapid extracellular K+ clearance, representing a potent potential intervention for acute stroke.

The ionic composition of the intracellular and extracellular environments in the central nervous system critically influences neuronal networks since action potentials and synaptic input rely on transmembrane electrochemical gradients. Acute stroke disrupts the ion homeostasis in the involving infarct and induces repeated spontaneous waves of cortical spreading depolarization (CSD) in patients (1, 2) and in experimental stroke models (3⇓–5). CSD is initiated as a slowly propagating wave of depolarization that travels across the cortical surface with a velocity of ∼4 mm/min (6). Increases in extracellular potassium (K+) and glutamate are mediators of CSD (7⇓⇓–10). During the peak of CSD, the extracellular potassium concentration [K+] e rises from a basal level of 3.5–4.5 to 30–60 mM. This massive rise of [K+] e depolarizes neurons and astrocytes, resulting in the release of neurotransmitters and the opening of voltage-dependent K+ channels and N-methyl-d-aspartate receptors (NMDARs, K+-permeable glutamate receptors), which in a positive feedforward trigger an autoregenerative elevation of [K+] e as CSD propagates across the cortex (11). The membrane depolarization inactivates voltage-dependent Na+ channels, resulting in prolonged electrical silence of neurons. Neurons will gradually regain responsiveness during the recovery period concurrently with normalization of ionic gradients including that of [K+] e . However, the factors determining [K+] e recovery are poorly understood, in part because there have been few attempts to monitor cortex-wide network activity linked to CSD following stroke incidents.

Astrocytes, a major glial cell type, play a key role in the uptake of K+ and the maintenance of ionic homeostasis through Na+-K+ pumps (12) and K+ channels (13). Additionally, K+ uptake is regulated by astrocytic Ca2+ signaling (14). In experimental animal models, the focal application of high K+ on the cortical surface reliably evokes CSD (7). While the mechanisms for initiation and propagation of CSD have been investigated in the past, the recovery phase and the basis of its molecular control have received relatively little attention. Propagation of high-K+–induced CSD in and of itself has been suggested to be benign to cortical tissue (15), or even beneficial, as it promotes development of ischemic tolerance (16). However, it is clear that spontaneously evoked waves of CSD in the setting of acute stroke enhance permanent cortical damage and compromise functional recovery (17⇓–19).

The glymphatic model proposes that extracellular fluid movement in the brain is regulated by aquaporin-4 (AQP4), the water channel expressed predominantly in astrocytes, and by noradrenaline levels (20, 21). We found that a systemic adrenergic receptor (AdR) blockade facilitates the normalization of [K+] e after acute ischemic stroke, improves motor recovery, and mitigates the resultant tissue damage. To test if AQP4 plays a role in this treatment mechanism, we evaluated the expression of AQP4 at various time points after stroke by histology and immunoblot. We found that AQP4 expression decreased in peripheral areas of the photothrombosis induction site, whereas pretreatment of the AdR blockers preserved AQP4 expressions after stroke. This result suggests that the positive effect of AdR blockade on the enhancement of recovery from the stroke could be due to AQP4-dependent clearance of the extracellular fluid and that AdR blockers constitute a potent therapeutic alternative for the treatment of acute ischemic stroke.

Discussion In this study, we report that noradrenergic antagonism reduced ischemic brain damage and accelerated motor recovery following photothrombotic stroke (Fig. 1). Moreover, AdR blockers spared the expression and polarization of AQP4, which is otherwise attenuated by photothrombotic stroke (Figs. 3 and 4). AdR blockers also facilitated normalization of [K+] e after stroke in a manner dependent on AQP4 (Fig. 5). The mechanism of systemic neuroprotection by adrenergic antagonism during stroke recovery likely involves a complex interaction of multiple systems and cell types. Recently, the Nedergaard group has proposed that the glymphatic system (20), a fluid exchange system between CSF and ISF, is negatively modulated by AdR activation (21). The neuroprotective effects in stroke mice seen with AdR antagonists could thus be viewed as an extension of glymphatic system function. Neurons in the locus coeruleus, the major source of noradrenergic projections to the cortex, show bursting activity during K+-induced cortical spreading depression (34), a phenomenon observed in pathological states of the brain including ischemic stroke (18, 19). Aberrant astrocytic activities have been reported within an hour after photothrombotic stroke (35), hinting at an active involvement of this cell type in the hyper-acute phase. Noradrenaline has been identified as one of the principal neuromodulators that trigger astrocytic Ca2+ activities in the cerebral cortex (36, 37). Indeed, massive noradrenaline releases by ischemic stroke have been reported in rats (38). These noradrenaline releases will activate AdRs, which in turn can restrict glymphatic flow. Blockade of AdRs would not only counteract the resultant glymphatic flow restriction, but also promote the influx of fresh CSF with normal ionic balance to the parenchyma. Such a mechanism would clear excess K+ to the venular perivascular space. Recently, cortical [K+] e levels were observed to be higher in awake states than in sleep, which is correlated with higher noradrenergic activity during waking (39). In support of this view, expression of AQP4, the astrocytic water channel protein critical for glymphatic flow (20), is markedly decreased 3 h after photothrombotic stroke, but was unaffected in AdR-blocker–treated mice (Fig. 3). The decrease of AQP4 after photothrombotic stroke provides additional mechanistic evidence for compromised glymphatic flow after CSD in mice (40). Indeed, tracer diffusion after injection to the cisterna magna suggested preservation of CSF/ISF flow in peripheral infarct areas by AdR blockers (Fig. 5 C–E). The effectiveness of AdR blockers was lost when administered 3 h after stroke, which coincided with the loss of AQP4 expression and polarity, suggesting that the main beneficial effect of AdR blockade is likely to be through preservation of AQP4. The current literature on the role of AQP4 in ischemic stroke appears contradictory. For example, Manley et al. (30) reported improved outcomes of MCAO infarct in AQP4 KO mice, while other studies reported little effect or worse outcomes in AQP4 KO mice (41⇓–43). In the present photothrombotic model, we found a trend (P = 0.09) toward reduced infarct area in AQP4 KO mice (Fig. 4F), agreeing with the results of Manley et al. (30). Furthermore, we find that AdR antagonism in AQP4 KO mice is ineffective in reducing infarct size, adding support for the involvement of AQP4 in stroke progression in WT mice. Meanwhile, there is a general consensus on compromised K+ clearance in AQP4 KO mice in physiological and pathophysiological conditions, including CSD (44⇓–46). Considering the above, deleterious poststroke edema formation could develop in the following phases. (i) Hyper-acute phase: osmotic water influx through AQP4 to the parenchyma due to elevated [K+] e contributes to swelling. (ii) Acute phase: astrocytic AQP4 expression decreases in an AdR-dependent manner. (iii) Postacute phase: water is trapped in the parenchyma, and chronic edema is established. In this model, it is conceivable that AQP4 KO (31) and pretreatment with an AQP4 blocker (29) improve outcomes of ischemia because they interfere with phase (i). On the other hand, AdR antagonism is effective in acute and postacute phases, when water efflux is required to mitigate against the edema formed during the hyper-acute phase. Thus, our observations can potentially explain the conflicting observation in the literature. Enhanced recovery from CSD by AdR antagonism may also reflect modulation of cerebral blood flow (CBF). Elevation of extracellular noradrenaline levels during the post-CSD period results in noradrenaline-dependent cerebral vessel constriction mediated by perivascular cells (47). Indeed, CBF is regulated in part by a fine balance between the alpha-1 AdR and alpha-2 AdR, which are linked to G q and G i signaling pathways, respectively. Since prazosin (alpha-1 AdR antagonist) can reverse the vessel constriction by atipamezole (alpha-2 AdR antagonist) (48), the AdR-blocker mixture used in this study has the net effect of reversing vasoconstriction. The AdR-blocker–induced decrease of vascular resistance would then lead to increased delivery of oxygen and glucose and possibly promote the exchange of ISF and blood, which is ultimately needed to clear K+ from the CNS. We measured CBF using laser Doppler flowmetry. We found that the CBF decrease was less pronounced in AdR blockade mice for the first 60 min after photothrombosis under isoflurane anesthesia, suggesting that poststroke preservation of CBF may contribute to the neuroprotective effect of AdR antagonists (SI Appendix, Fig. S6). However, the finding that TGN-020 application failed to affect AdR blocker suggests that the enhancement of CSF/ISF exchange after AdR blockade, possibly mediated by the glymphatic system, is a favorable precondition for the recovery of neuronal activity. Our observations show that the triple AdR-blocker combination tended to be more effective than prazosin or propranolol administered alone (both P < 0.1, Fig. 1F). Previous reports of drugs targeting alpha-2 AdRs for the treatment of ischemic brain damage have conflicting results. For example, pretreatment with alpha-2 AdR agonists is reported to be neuroprotective in some reports (49, 50), but not in others (51, 52). Moreover, postincident treatment with the alpha-2 AdR antagonist atipamezole reportedly enhanced recovery of some motor functions (53⇓⇓⇓–57) even though infarct size remained similar (58). The preincident agonist approach supposedly decreases noradrenaline levels due to the presence of presynaptic alpha-2 autoreceptors (59, 60), whereas the postincident antagonist approach would increase noradrenaline levels. Our approach of using an AdR-blocker combination is distinct from previous approaches in that we antagonize a broad spectrum of AdR subtypes, thereby enhancing systemic circulation in the glymphatic and/or vascular systems. This may favor greater effects in both infarct size and motor recovery than in some previous reports. Remarkably, we saw significant improvements in motor function recovery in AdR-blocker–treated mice exposed to somatosensory cortex ischemia. Our analysis shows that this systemic treatment can be effective in the hours after injury, suggesting a possible therapeutic potential. Of course, altered noradrenaline signaling throughout the sympathetic nervous system will interfere with autonomic body functions such as blood pressure, heart rate, and body temperature. Future preclinical studies should determine the optimal degree and nature of AdR antagonism in lissencephalic and gyrencephalic brains to minimize brain damage caused by prolonged depolarization in the setting of ischemia.

Materials and Methods All experimental protocols were approved by The Institute of Physical and Chemical Research (Japan) (RIKEN) Institutional Animal Care and Use Committee or the University Committee on Animal Resources at the University of Rochester. Efforts were taken to minimize the number of animals used. Surgical Procedures for Acute Experiments. Adult (older than 9 wk) male and female C57BL/6 wild type, G7NG817 (61), and AQP4-KO/GFP-KI (33) (CDB0758K, http://www2.clst.riken.jp/arg/mutant%20mice%20list.html) mice were used. G7NG817 and AQP4-KO/GFP-KI mice are available from RIKEN BioResource Center (RBRC09650 and RBRC04364, respectively). The background strain of these mice is C57BL/6. Mice were housed under a 12 h/12 h light/dark cycle and raised in groups of up to five. See SI Appendix, SI Materials and Methods, for details. Drug Administration. AdR blockers. The combination of AdR antagonists used throughout the article is as follows (except experiments for Fig. 1F): propranolol—10 mg/kg; prazosin—10 mg/kg; and atipamezole—1 mg/kg. These antagonists were prepared in 0.1% solution in saline and administered successively. For preemptive treatment, the drugs were applied intraperitoneally 30 min before stroke induction. For postincident treatment, drug administration was made 1–6 h after stroke induction. The control group is represented by naive mice. TGN-020. The AQP4-specific antagonist TGN-020 (Tocris, 5425) was applied intraperitoneally immediately after photothrombotic stroke induction (Fig. 4E and SI Appendix, Fig. S6D; dosage: ∼500 µg/kg; 0.2 mL of 300 µM solution in saline for a 25-g mouse). See SI Appendix, SI Materials and Methods for details. Photothrombosis Induction. Mice were anesthetized with isoflurane (induction: 4%; maintenance: 2%) and placed under a fluorescence stereomicroscope (Leica MZ10F). The skull was exposed via scalp incision and treated with a mixture of paraffin oil and Vaseline (1:1) to increase skull transparency. Rose bengal was administered 5 min before photothrombosis (330,000, intraperitoneal 110 mg/kg; Sigma). To induce photothrombosis, the target area (4-mm diameter, primary visual cortex) was illuminated by a green light for 15 min at the target area. See SI Appendix, SI Materials and Methods, for details. MCAO. The mice were anesthetized with 1.25–3% isoflurane for induction and 0.75–1.5% for maintenance (1.25 and 0.75% were used for the AdR antagonist group to avoid morbidity during surgery; 3 and 1.5% were used for control group). The right MCA was occluded by a 7–0 polypropylene monofilament (Ethicon) coated with silicon resin, which was inserted via the external carotid artery through the internal carotid artery. Cortical blood flow was continuously monitored by laser Doppler flowmetry (Perimed). Rectal temperature was maintained at 37 ± 0.5 °C using a feedback-controlled heating system (Harvard Apparatus). Forty-five minutes after occlusion, the inserted filament was removed, and MCA blood flow was restored, as confirmed by flowmetry. The animals were returned to their temperature-controlled home cage (ThermoCare) and allowed to recover for 4 h. Histology. TTC staining. Twenty-four hours after photothrombosis induction, anesthetized mice were perfused with ice-cold PBS, and extracted brains were soaked in 2% TTC (Sigma) for 15 min at 37 °C. See SI Appendix, SI Materials and Methods, for details. Immunohistochemistry. Mouse brains were perfusion-fixed with 4.0% paraformaldehyde (pH 7.4 in 0.1 M phosphate buffer). Following overnight postfixation in the same medium, coronal slices (60 μm thick) were prepared using a microslicer (PRO 7; Dosaka). For GFAP, AQP4, GLAST, GLT-1, and CX-43 staining, sections were incubated with primary antibodies (1:1,000 for GFAP, AQP4, GLAST, and GLT-1 antibodies and 1:2,000 for CX-43 antibody in Tris-buffered saline with 0.1% Triton X-1000) overnight. The sections were subsequently washed in phosphate buffer and incubated with Alexa 488-conjugated secondary antibodies (Invitrogen) for 2 h for fluorescent labeling. See SI Appendix, SI Materials and Methods, for details. Polarization index. Double-immunolabeled fluorescent images with anti-CD31 and anti-AQP4 antibodies were used to compute polarization indices. Pixels with intensity exceeding the mean + 1 SD of the CD31 positive area were regarded as blood vessels. The AQP4 high signal area at astrocytic endfeet (Area_high) was defined by those pixels overlapping with blood vessels having AQP4 pixel intensity exceeding the mean + 1 SD of the AQP4 positive area (Area_low). CD31- and AQP4-positive areas are defined as any pixels with intensity greater than the mean of the respective image. Polarization index is defined as Area_high/Area_low. Western Blot. Brains were removed from deeply anesthetized mice (3% isoflurane) and cortical areas of interest (surface area: ∼6 mm2) were immediately dissected and placed separately in Eppendorf tubes (1 mL), which were then frozen in liquid nitrogen. These samples were kept in −80 °C for later Western blot analysis. See SI Appendix, SI Materials and Methods, for details. Behavior Experiments. Open field test. Individual mice were placed in an empty plastic box that had a size of 40 × 40 cm. The mice explored the box for 9 min, and the entire session was recorded. The total traveled distance was computed from the video file using MATLAB. Cylinder test. The cylinder test was performed similarly to Li et al. (23). A transparent hollow acrylic cylinder (10 × 20 × 2 cm) was set behind two adjacently placed mirrors (26.8 × 20 cm) forming an angle of about 100°. For each test, a mouse was placed inside the cylinder for 10 min, and its behavior was recorded by a video camera (SONY HDR-XR550V). See SI Appendix, SI Materials and Methods, for details. Surgical Procedures for Virus Inoculation. pAAV-hGFAP-G-CaMP7 (a gift from Masanori Matsuzaki, University of Tokyo, Tokyo; ref. 62) was synthesized by modifying the pAAV-hSyn-EGFP vector (Addgene plasmid #50465, a gift from Bryan Roth, University of North Carolina School of Medicine, Chapel Hill, NC) with a human GFAP promoter (63) and G-CaMP7 cDNA. AAV9-hGFAP-G-CaMP7 was purified at a titer of 1.2 × 1014 vg ⋅ mL−1. AAV1-Syn.NES-jRGECO1a.WPRE.SV40 (3.44 × 1013 vg ⋅ mL−1) was purchased from Penn Vector Core. The viruses were diluted to 5–7 × 1012 vg ⋅ mL−1 with PBS for microinjection. Mice were anesthetized with isoflurane (1.5%) or ketamine-xylazine intraperitoneally (56 and 8 mg ⋅ kg−1, respectively), and fixed in a stereotaxic frame. A small craniotomy was made at the site of prospective imaging and a glass micropipette containing AAV was inserted to a depth of 250 μm below the surface of the cortex. Microinjection of 300 nL was made over 5 min using a Femtojet injector (Eppendorf). Imaging experiments were performed at least 2 wk later. In Vivo Two-Photon Imaging. Two-photon imaging was performed on urethane-anesthetized adult mice (as above) using a resonant-scanner–based B-Scope (Thorlabs) with a Chameleon Vision 2 laser (Coherent, wavelength 920 nm) and an Olympus objective lens (XLPlan N 25×). The B-Scope was equipped with a reverse dichroic mirror (ZT405/488/561/680–1100rpc; Chroma), and the emission light was separated by using a dichroic mirror (FF562-Di03; Semrock) with bandpass filters FF03-525/50 and FF01-607/70 (both from Semrock) for the green and red channels, respectively. Images were acquired using the ThorImage software with a frame rate of 30 Hz. Extracellular Potassium Recording. Ion-sensitive microelectrodes for measuring extracellular K+ were made from double-barreled glass pipettes (A-M Systems, Inc., 607000), which were pulled using a pipette puller (P-97; Sutter), and the tip was broken to have a diameter of <10 µm for each cavity. Pipettes were silanized by exposure to dimethylsilane vapor in a small container for 1 h at 200 °C. One or the other electrode tip was loaded with 2.5 µL of valinomycin-based K+ ion-exchange resin (potassium ionophore I–mixture B, front-loaded) and subsequently filled with 150 mM KCl. The other tip was filled with 150 mM NaCl and used as a reference for the LFP recording. K+-sensitive electrodes were calibrated before and after each experiment using a set of solutions with known K+ concentration (2.5, 3.5, 4.5, 10, 20, 50, 100, and 300 mM). Each electrode was calibrated by linear-regression least-squares fitting of the relationship between voltage and K+ concentration. The offset effect by 50 μM prazosin is documented in SI Appendix, Fig. S7. In vivo extracellular K+ recordings were made using a DC amplifier (MultiClamp 700B; Axon Instruments) from cortical layer 2/3 (250 µm below the pial surface) and were recorded at a 20-kHz sampling rate. Simultaneous recordings were made from K+-sensitive and reference LFP electrodes. The reference LFP was subtracted from the K+-sensitive electrode recording, and the resulting data were resampled to 10 Hz and converted to millimolars according to the calibration curve. Injection of Fluorophore-Tagged Tracer from Cisterna Magna. Neck muscles were surgically removed, and the cisterna magna membrane was carefully exposed under urethane (1.6 g/kg) anesthesia. A glass pipette (tip diameter: ∼10 μm) filled with 0.5% BDA (70 kDa) in PBS was inserted into the cisterna magna by piercing the dura mater. To avoid CSF leakage, the cisterna magna membrane was immediately sealed by cyanoacrylate adhesive around the tip of the inserted glass pipet. A syringe driver (KDS Legato Series; KD Scientific) was used to infuse the fluorescence tracer at a rate of 1 μL/min for 5 min (5 μL in total). The pipette tip was left in place until the experiments were terminated at 30 min. Recording of Physiological Parameters. Heart rate, respiratory rate, and arterial oxygen saturation level (SpO 2 ) were measured using a commercial pulse oximeter (MouseOx PLUS; Starr LifeSciences). In brief, the oximeter sensor was placed on a shaved area of neck above the carotid artery, and parameters were recorded in anesthetized mice (urethane, 1.7 g/kg). Laser Doppler flowmetry (Omegaflo, FLO-C1; Omegawave) was used for the measurement of CBF through the skull above the barrel area of the somatosensory cortex as previously described in Takata et al. (64) In Vivo Transcranial Fluorescence Imaging. Mice were fixed to a stereotaxic stage by clamping the head frame and placing them under a fluorescence stereomicroscope (MZ10F; Leica). A GFP3 filter set (excitation 470 ± 20 nm, emission 525 ± 25 nm; Leica) was used with an EL6000 light source (Leica). Images were acquired using the ORCA-Flash 4.0 CMOS camera (Hamamatsu Photonics) using HC Image software (Hamamatsu Photonics). The HC Image software also controlled a shutter unit to illuminate the skull only during imaging. Images were acquired with a size of 512 × 512 pixels and 16-bit resolution. Images were acquired at 10 Hz. Data Analysis. Edema compensation. Edema compensation for TTC-stained coronal slices was computed according to the previously published methods (65, 66). First, we measured infarct volume including edema at the photothrombosis induction site (V e ). We next measured the volume of the entire hemisphere of photothrombosis (V R ) and finally the volume of the contralateral hemisphere (V L ). Essentially, the infarct volume compensating for edema inflation (V i ) was estimated as V i = V L × V e /V R . Correlation. For each region of interest (ROI) of gliopils, a correlation coefficient of the 1-min bin was calculated. The average of the correlations was plotted to the distance between the center of gravity of the paired ROI (Fig. 2B). LFP power. LFP power for slow oscillations (Fig. 5B) was computed as follows: the spectrograms were calculated for LFP traces by fast Fourier transform of consecutive 10-s bins. The power of slow oscillations was then computed by averaging the amplitude of the 0.5- to 2-Hz frequency bin from the spectrogram for the period of interest. Tracer intensity quantification. Intensity of the fluorescence tracer was quantified along the orthogonal line against the pia mater. The analyzed position was set to fall 1.5 ∼ 2 mm anterior to bregma (1 mm away from the center of photothrombosis) and 2 mm from the midline. See SI Appendix, SI Materials and Methods, for details. Data and Materials Availability. All transgenic mice used in this article are available from RIKEN BioResource Center.

Acknowledgments We thank Drs. Shinichi Takahashi (Keio University), Elizabeth M. C. Hillman (Columbia University), K. C. Brennan (The University of Utah), Charles Yokoyama (RIKEN), and Paul Cumming for discussion and comments on the manuscript and the RIKEN Center for Brain Science–Olympus Collaboration Center for technical assistance with image acquisition. This work was supported by the RIKEN Center for Brain Science and Japan Society for the Promotion of Science KAKENHI Grants 18K14859 (to H. Monai), 26117520, 16H01888, and 18H05150 (to H.H.), 17K19637 and 16H05134 (to Y.A.), and 15H04688 and 18H02606 (to M.Y.). This work was also supported by Human Frontier Science Program Grant RGP0036/2014 (to H.H.); Japan Society for the Promotion of Science Core-to-Core Program Advanced Research Networks; the Adelson Medical Research Foundation (M.N.); the Lundbeck Foundation; the Novo Nordisk Foundation; and the US Department of Defense (M.N.). H.H. is a recipient of the Lundbeck Foundation Visiting Professorship.

Footnotes Author contributions: H. Monai, Y.A., M.Y., Y.I., M.N., and H.H. designed research; H. Monai, X.W., K.Y., N.L., H. Mestre, Q.X., Y.A., and M.Y. performed research; H. Monai contributed new reagents/analytic tools; H. Monai, X.W., and H.H. analyzed data; and H. Monai, Y.A., M.Y., Y.I., M.N., and H.H. wrote the paper.

Conflict of interest statement: A patent has been applied for in Japan for adrenergic blocker treatment for stroke (H.H., H. Monai, Y.I.).

This article is a PNAS Direct Submission.

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