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Goreisan Prevents Brain Edema after Cerebral Ischemic Stroke by Inhibiting Aquaporin 4 Upregulation in Mice
ARTICLE IN PRESS
Goreisan Prevents Brain Edema after Cerebral Ischemic Stroke by Inhibiting Aquaporin 4 Upregulation in Mice Takafumi Nakano, PhD,*,†,‡ Chisa Nishigami, MS,† Keiichi Irie, PhD,† Yutaka Shigemori, MD, PhD,§ Kazunori Sano, PhD,† Yuta Yamashita, MS,† Takayuki Myose, MS,† Koji Tominaga, PhD,* Koichi Matsuo, PhD,* Yoshihiko Nakamura, MD, PhD,§ Hiroyasu Ishikura, MD, PhD,§ Hidetoshi Kamimura, PhD,*,‡ Takashi Egawa, PhD,* and Kenichi Mishima, PhD†
Background: Aquaporin 4 (AQP4) is a water-selective transport protein expressed in astrocytes throughout the central nervous system. AQP4 level increases after cerebral ischemia and results in ischemic brain edema. Brain edema markedly influences mortality and motor function by elevating intracranial pressure that leads to secondary brain damage. Therefore, AQP4 is an important target to improve brain edema after cerebral ischemia. The Japanese herbal Kampo medicine, goreisan, is known to inhibit AQP4 activity. Here, we investigated whether goreisan prevents induction of brain edema by cerebral ischemia via AQP4 using 4-hour middle cerebral artery occlusion (4h MCAO) mice. Methods: Goreisan was orally administered at a dose of 500 mg/kg twice a day for 5 days before MCAO. AQP4 expression and motor coordination were measured by Western blotting and rotarod test, respectively. Results: Brain water content of 4h MCAO mice was significantly increased at 24 hours after MCAO. Treatment with goreisan significantly decreased both brain water content and AQP4 expression in the ischemic brain at 24 hours after MCAO. In addition, treatment with goreisan alleviated motor coordination deficits at 24 hours after MCAO. Conclusions: The results of this study suggested that goreisan may be a useful new therapeutic option for ischemic brain edema. Key Words: Goreisan—brain edema—aquaporin 4—cerebral ischemia. © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.
Introduction From the *Department of Pharmaceutical and Health Care Management; †Department of Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Jyonan, Fukuoka, Japan; ‡Department of Pharmacy; and §Department of Emergency and Critical Care Medicine, Fukuoka University Hospital, Fukuoka, Japan. Received June 30, 2017; revision received September 29, 2017; accepted October 10, 2017. Address correspondence to Keiichi Irie, PhD, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma 8-19-1, Jyonan, Fukuoka 814-0180, Japan. E-mail: [email protected]. 1052-3057/$ - see front matter © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jstrokecerebrovasdis.2017.10.010
Although brain ischemic stroke is a leading cause of mortality and disability worldwide, limited therapeutic options are available.1 Brain ischemia is usually caused by embolic or thrombotic occlusion of a cerebral artery. The pathophysiology of ischemic brain damage is complex and multifactorial. Cerebral ischemia and reperfusion induce brain edema,2 which was shown to be caused by cellular dysfunction with Na+/K+ pump failure due to vascular injury resulting in abnormal increases in extracellular and intracellular water contents, thus leading to brain swelling.3 There are 2 patterns of brain edema: vasogenic edema, which is often observed in brain tumors,
Journal of Stroke and Cerebrovascular Diseases, Vol. ■■, No. ■■ (■■), 2017: pp ■■–■■
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freezing injury, and cerebral trauma; and cytotoxic edema, which is associated with intracellular ionic imbalance induced by cerebral ischemia.4 These types of brain edema induced by cerebral ischemia markedly affect mortality and motor function due to elevated intracranial pressure that leads to secondary brain damage.5 Therefore, brain edema plays an important role in ischemic stroke and represents a potential target for therapy. However, limited therapeutic options are available for brain edema. Aquaporins (AQPs) are known as a family of water channel proteins. Aquaporin (AQP) 1 was purified and identified in human erythrocytes as the first member of the AQP family.6 Currently, more than 10 AQPs have been discovered in mammals.7 AQP4 is also a water-selective transport protein expressed at the plasma membrane in astrocytes throughout the central nervous system.8 The expression of AQP4 increases after cerebral ischemia and results in ischemic brain edema.9,10 Therefore, the occurrence of brain edema is closely related to the expression of AQP4 in brain tissue after cerebral ischemia. Yao et al reported the improved outcome in AQP4 knockout mice following focal ischemia produced by middle cerebral artery occlusion (MCAO).11 In addition, several correlative investigations reported reduced brain edema after ischemia with reduced AQP4 expression.12-14 Thus, AQP4 is a potential therapeutic target in ischemic brain edema. Goreisan is a Japanese herbal Kampo medicine consisting of a mixture of 5 herbs (Alisma rhizome, Atractylodes rhizome or Atractylodes lancea rhizome, Polyporus sclerotium, Poria sclerotium, and Cinnamon bark), which has been used to treat watery diarrhea, edema, vertigo, and nausea. Hayashi reported the effects of goreisan on brain edema complicated by central nervous system disease.15 However, the clinical effectiveness of goreisan for treatment of brain edema remains unclear. As goreisan was recently reported to inhibit AQP4,16,17 we postulated that it may prevent brain edema induced by cerebral ischemia via AQP4. The present study was performed to investigate whether treatment with goreisan prevents brain edema via AQP4 using 4-hour middle cerebral artery occlusion (4h MCAO) mice. The results suggested that goreisan is a potential new therapeutic option for brain edema in ischemic stroke.
Materials and Methods Focal Cerebral Ischemia Focal cerebral ischemia was induced in male ddY mice (6-8 weeks old, 25-35 g; Kiwa Experimental Animal Laboratory, Wakayama, Japan) as described previously.18 The mice were reanesthetized with isoflurane (Escain; Pfizer, Osaka, Japan) 4 hours after occlusion, and reperfusion was established by withdrawal of the filament. Occlusion of the middle cerebral artery was confirmed by examining forelimb flexion after awakening from anesthesia. All procedures regarding animal care and use were
performed in compliance with the regulations established by the Experimental Animal Care and Use Committee of Fukuoka University.
Drug Administration Goreisan (Tsumura, Tokyo, Japan) was dissolved in water and orally administered at a dose of 500 mg/kg twice a day for 5 days at 24 hours before MCAO. Water (vehicle) was used as a control.
Physiological Parameters Conscious mice were weighed and their blood pressures were measured at 24 hours after 5 days of repeated treatment using a noninvasive, computerized tail-cuff system with BP Monitor for Rats & Mice model MK200 (Muromachi Kikai Co., Tokyo, Japan). Rectal temperature was measured at 24 hours after 5 days of drug treatment using a digital laboratory thermometer (BAT-12; Physitemp Instruments, Clifton, NJ) with a needletype thermometer, at room temperature (23°C ± 1°C). Blood samples were also collected at 24 hours after 5 days of drug treatment. Serum was obtained after centrifugation (1500 × g for 15 minutes at 4°C). Serum Na, K, and Cl levels were measured by the ion-selective electrode method (Oriental Yeast Co. Nagahama LSL, Shiga, Japan).
Brain Water Content at 24 Hours after MCAO The left cerebral hemispheres of the mice were collected at 24 hours after MCAO and weighed immediately after euthanasia (wet tissue weight). Brain samples were dried in an oven at 110°C for 48 hours. The dried samples were then weighed again (dry tissue weight), and the brain water content was calculated as [(wet tissue weight − dry tissue weight)/wet tissue weight] × 100%.
Immunoblotting The expression of AQP4 was evaluated by Western blotting. Tissue samples (striatum) were homogenized at 4°C for 1 minute in PRO-PREP protein extraction solution (iNtRON Biotechnology, Seongnam-Si, Korea). The tissue extract was centrifuged at 15,000 rpm at 4°C for 30 minutes. The supernatant was treated in the same way as the tissue extract. The total protein concentration of each tissue lysate was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Protein (20 µg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; 20% gel). Equivalent amounts of each protein were separated by SDS–PAGE and transferred onto polyvinylidene difluoride membranes according to standard procedures. After blocking with 5% skim milk in Tris-buffered saline containing .1% Tween 20 (TBS-T, Sigma-Aldrich, St. Louis, MO.), membranes were probed with primary antibodies (antiAQP4, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA;
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Table 1. Summary of physiological parameters after 5 days of drug treatment in intact mice
Control (n = 6) Vehicle (n = 6) Goreisan (n = 6)
BW (g)
sBP (mmHg)
RT (°C)
Na (mEq/L)
K (mEq/L)
Cl (mEq/L)
32.8 ± .6 33.5 ± .6 33.5 ± .6
88.3 ± 6.0 92.8 ± 6.7 88.3 ± 3.3
36.3 ± .1 36.5 ± .2 36.2 ± .1
149.0 ± .4 148.8 ± 1.0 148.7 ± .7
6.6 ± .2 6.7 ± .1 6.6 ± .2
104.7 ± .8 102.0 ± .4 105.2 ± .6
Abbreviations: BW, body weight; RT, rectal temperature; sBP, systolic blood pressure. All values are means ± standard error of the mean.
anti-β-actin, 1:1000; Abcam, Cambridge, MA) for 1 hour at room temperature. The membranes were washed with TBS-T and incubated with horseradish peroxidaseconjugated secondary antibody (1:5000; Santa Cruz Biotechnology) for 1 hour at room temperature. After washing 3 times with TBS-T, the bands were developed using an ECL Advance Western Blotting Detection Kit (GE Healthcare, Little Chalfont, UK) and detected using FluorChem8900 (Alpha Innotech, San Leandro, CA). The signal intensity on the blots was measured using NIH Image (version 1.63; National Institutes for Health, Bethesda, MD).
Rotarod Test Motor coordination was measured by the rotarod test at 24 hours after MCAO as described previously.19 Mice were placed on a rotating rod that is 3 cm in diameter (Neuroscience Inc., Tokyo, Japan) with a nonskid surface, and the latency to fall was measured for up to 2 minutes. The rotation speed was 10 rpm.
Figure 1. Effects of goreisan on brain water content after ischemic stroke in 4h MCAO mice. Quantitative analysis of brain water content in control (n = 8), vehicle (n = 10), and 500 mg/kg goreisan twice a day (n = 11) groups. **P < .01 versus sham, ††P < .01 versus vehicle. All values are means ± standard error of the mean. Abbreviation: 4h MCAO, 4-hour middle cerebral artery occlusion.
Statistical Analysis Data are presented as means ± standard error of the mean. Multiple comparisons were evaluated by Tukey test. In all analyses, P < .05 was taken to indicate statistical significance.
Results Effects of Goreisan on Physiological Parameters in Intact Mice There were no significant differences among control, vehicle, and goreisan in any of the physiological parameters measured (Table 1). Thus, repeated treatment with goreisan did not influence the physiological parameters at the onset of cerebral ischemia.
Effects of Goreisan on Brain Water Content after Ischemic Stroke in Mice Subjected to 4H MCAO Brain water content was significantly increased in 4h MCAO mice at 24 hours after MCAO. Treatment with goreisan (500 mg/kg twice a day) significantly decreased brain water content compared with vehicle alone at 24 hours after MCAO (Fig 1).
Effects of Goreisan on AQP4 after Ischemic Stroke in Mice Subjected to 4H MCAO Brain ischemia–reperfusion has been shown to upregulate AQP4 expression and induce brain edema. 2,9,10 The level of AQP4 expression was increased at 24 hours after MCAO. However, treatment with goreisan (500 mg/kg twice a day) significantly decreased the AQP4 expression level compared with vehicle alone at 24 hours after MCAO (Fig 2).
Effects of Goreisan on Motor Coordination Estimated by Rotarod Test and Survival Rate after Ischemic Stroke in Mice Subjected to 4H MCAO Treatment with goreisan (500 mg/kg twice a day) significantly alleviated motor coordination deficits compared with vehicle controls at 24 hours after MCAO (Fig 3). However, the survival rates of vehicle and goreisan groups at 24 hours after MCAO were not significantly different (90.3%, n = 27 and 93.2%, n = 33, respectively). The MCAO mouse model was shown previously not to have a high mortality rate at 24 hours after MCAO.19
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Figure 2. Effects of goreisan on AQP4 after ischemic stroke in 4h MCAO mice. Quantitative analysis of AQP4 expression in sham (n = 6), vehicle (n = 6) and 500 mg/kg goreisan twice a day (n = 6) groups. **P < .01 versus sham, †P < .05 versus vehicle. All values are means ± standard error of the mean. Abbreviations: 4h MCAO, 4-hour middle cerebral artery occlusion; AQP4, aquaporin 4.
Figure 3. Effects of goreisan on motor coordination estimated by rotarod test after ischemic stroke in 4h MCAO mice. Quantitative analysis of motor coordination estimated by rotarod test in sham (n = 6), vehicle (n = 10), and 500 mg/kg goreisan twice a day (n = 13) groups. *P < .05 versus sham, **P < .01 versus sham, †P < .05 versus vehicle. All values are means ± standard error of the mean. Abbreviation: 4h MCAO, 4-hour middle cerebral artery occlusion.
Discussion The present study was performed to evaluate the effects of goreisan on ischemic brain edema via AQP4. Our results indicated that treatment with goreisan decreased both the brain water content and the expression of AQP4 after
cerebral ischemic stroke. Moreover, goreisan treatment alleviated the motor dysfunction after cerebral ischemic stroke. These observations suggest that goreisan may be a novel option to prevent ischemic brain edema. Several studies reported that brain edema was upregulated 1-3 days after cerebral ischemia.4,20,21 We previously evaluated brain edema at 1-3 days after MCAO and showed that the peak of brain edema occurred 24 hours after MCAO in our ischemic model mice (data not shown). Therefore, we evaluated brain edema, infarct volume, rotarod test performance, and AQP4 expression at 24 hours after MCAO in the present study. Brain edema induced by ischemic stroke occurs due to cellular dysfunction with Na + /K + pump failure. 3 The consequences are lethal due to the increased intracranial pressure and the compromised cerebral blood flow. Moreover, AQP4 is involved in the deterioration of brain edema. AQP4 has been shown to be the primary water transport pathway across the blood–brain barrier (BBB).8 AQP4 is rapidly upregulated on astrocytic endfeet surrounding cerebral blood vessels in the peri-infarcted cortex in MCAO model mice, resulting in aggravation of brain edema.8,20,22,23 Thus, its inhibition prevents brain edema, making it a potential therapeutic target. In the present study, we examined whether goreisan can prevent brain edema induced by cerebral ischemia in mice exposed to MCAO for 4 hours. First, we examined the goreisan dose regimen. Goreisan was reported previously to require repeated administration for 3 days to influence AQP mRNA expression.24 Moreover, we reported that Kampo medicine required the repeated administration of around 1 week to treat the cerebrovascular disease.25-27 Therefore, we investigated the effects of repeated oral administration of goreisan on brain edema at a dose of 500 mg/kg once or twice a day for 5 days before MCAO. Treatment with goreisan at a dose of 500 mg/kg twice a day was shown to decrease brain edema at 24 hours after MCAO, confirming that this Kampo medicine is effective for ischemic brain edema. We also investigated the effects of a single oral administration of goreisan after reperfusion on brain edema in mice subjected to MCAO. However, single administration of goreisan did not affect brain edema at 24 hours after MCAO, indicating that repeated administration is required to influence ischemic brain edema. Next, we investigated the relationship between brain edema and AQP4 expression after cerebral ischemic stroke. AQP4 is known to be involved in the deterioration of brain edema. In the present study, AQP4 expression was shown to be upregulated at 24 hours after MCAO. Thus, inhibition of this upregulation of AQP4 expression may lead to improvement of ischemic brain edema. Goreisan is known to inhibit AQP4 expression in brain edema.16,17,24 Although the mechanism by which goreisan inhibits AQP4 expression remains unclear, Isohama reported that this agent inhibits water permeability in MLE-12 cells by
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5 2+
inhibiting AQP molecules and that Mn and Zn ions are involved in this process.28 In the present study, treatment with goreisan at a dose of 500 mg/kg twice a day inhibited the upregulation of AQP4 induced by cerebral ischemic stroke at 24 hours after MCAO. Therefore, goreisan may prevent formation of brain edema by affecting AQP4 expression after cerebral ischemia stroke. On the contrary, our results showed that goreisan tended to improve infarct volume at 24 hours after MCAO, but the differences between goreisan and vehicle groups were not significant (data not shown). Several studies indicated that deletion of AQP4 reduces brain edema and infarct volume in a model of MCAO.14,29,30 AQP4 is also known to affect the BBB in ischemic stroke. Disruption of the BBB is a critical event during brain ischemic injury followed by passive diffusion of water, leading to brain edema and secondary brain injury.31 In the present study, we evaluated brain edema in mice exposed to MCAO for 4 hours, which is a stronger ischemia–reperfusion injury model than in previous studies. Previously, we reported that our ischemic model mice show severe nerve injury.18 Therefore, goreisan may not be effective for infarct volumes induced by 4 hours of MCAO. Nevertheless, model mice treated with goreisan showed improvement of brain edema. The effect of goreisan on AQP4 may be expected to improve brain edema rather than have a neuroprotective effect. Cho et al also reported that repeated administration of mannitol, a therapeutic drug for brain edema, reduced the water content of brain edema without affecting the infarct volume after MCAO in rats.32 Therefore, improvement of brain edema appears to be useful for treatment of cerebral ischemic stroke without affecting the infarct lesions. Moreover, improvement of brain edema also has a marked impact on recovery of brain function. In fact, treatment with goreisan alleviated motor coordination deficits at 24 hours after MCAO. This result has important clinical implications because motor dysfunction is the greatest negative effect of cerebral ischemic stroke. Inoue reported that motor dysfunction occurred due to compression of the pyramidal system by brain edema.33 In addition, there have been previous reports regarding the relation between improvement of cerebral edema and motor function.34,35 Moreover, deletion of AQP4 was reported to alleviate intraparenchymal pressure and improve motor dysfunction due to neurological disease.36 Thus, the effect of goreisan on AQP4 expression can be expected to alleviate motor dysfunction by improving brain edema. Goreisan has been regarded as a useful medicine since ancient times, and has been shown to be efficacious in treatment of watery diarrhea, edema in multiple organs, vertigo, and nausea. We consider goreisan to be easy to use for prevention and treatment of brain edema in patients with ischemic stroke in clinical practice. Therefore, further studies are required to investigate the effects of postischemic administration of goreisan.
In conclusion, the results of the present study indicated that AQP4 expression is involved in ischemic brain edema and that goreisan prevents brain edema by preventing upregulation of AQP4 after cerebral ischemic stroke. Moreover, goreisan ameliorated motor dysfunction induced by ischemic brain edema. We consider that goreisan alleviated motor dysfunction after cerebral ischemic stroke by preventing brain edema via its preventive effect on AQP4 upregulation. The results presented here suggest that goreisan may be useful as a new additional therapeutic option for improvement of brain function after cerebral ischemic stroke.
References 1. Moskowitz MA, Lo EH, Iadecola C. The science of stroke: mechanisms in search of treatments. Neuron 2010;67:181198. 2. O’Brien MD. Ischemic cerebral edema. A review. Stroke 1979;10:623-628. 3. Simard JM, Kent TA, Chen M, et al. Brain oedema in focal ischaemia: molecular pathophysiology and theoretical implications. Lancet Neurol 2007;6:258-268. 4. Wang WW, Xie CL, Zhou LL, et al. The function of aquaporin4 in ischemic brain edema. Clin Neurol Neurosurg 2014;127:5-9. 5. Walberer M, Ritschel N, Nedelmann M, et al. Aggravation of infarct formation by brain swelling in a large territorial stroke: a target for neuroprotection? J Neurosurg 2008; 9:287-293. 6. Denker BM, Smith BL, Kuhajda FP, et al. Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules. J Biol Chem 1988;263:15634-15642. 7. Takata K, Matsuzaki T, Tajika Y. Aquaporins: water channel proteins of the cell membrane. Prog Histochem Cytochem 2004;39:1-83. 8. Badaut J, Ashwal S, Adami A, et al. Brain water mobility decreases after astrocytic aquaporin-4 inhibition using RNA interference. J Cereb Blood Flow Metab 2011;31:819831. 9. Taniguchi M, Yamashita T, Kumura E, et al. Induction of aquaporin-4 water channel mRNA after focal cerebral ischemia in rat. Brain Res Mol Brain Res 2000;78:131137. 10. Hirt L, Ternon B, Price M, et al. Protective role of early aquaporin 4 induction against postischemic edema formation. J Cereb Blood Flow Metab 2009;29:423433. 11. Yao X, Derugin N, Manley GT, et al. Reduced brain edema and infarct volume in aquaporin-4 deficient mice after transient focal cerebral ischemia. Neurosci Lett 2015;584:368-372. 12. Manley GT, Fujimura M, Ma T, et al. Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke. Nat Med 2000;6:159-163. 13. Hirt L, Fukuda AM, Ambadipudi K, et al. Improved long-term outcome after transient cerebral ischemia in aquaporin-4 knockout mice. J Cereb Blood Flow Metab 2017;37:277-290. 14. Li W, Tan C, Liu Y, et al. Resveratrol ameliorates oxidative stress and inhibits aquaporin 4 expression following rat cerebral ischemia-reperfusion injury. Mol Med Rep 2015;12:7756-7762.
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6 15. Hayashi A. Effectiveness of goreisan for eliminating brain edema due to intracranial malignant brain tumors. KAIM 2010;5:10-16. 16. Isohama Y. Risuizai “Goreisan” no sayo mekanizum. Kampo Igaku 2005;29:213-215. 17. Mitsuhashi T, Nagase M, Arai H. Efficacy of goreisan for asymptomatic bilateral and unilateral chronic subdural hematoma. Tradit kampo Med 2016;3:28-32. 18. Nakano T, Irie K, Hayakawa K, et al. Delayed treatment with ADAMTS13 ameliorates cerebral ischemic injury without hemorrhagic complication. Brain Res 2015; 1624:330-335. 19. Hayakawa K, Mishima K, Nozako M, et al. Delayed treatment with minocycline ameliorates neurologic impairment through activated microglia expressing a high-mobility group box1-inhibiting mechanism. Stroke 2008;39:951-958. 20. Li S, Hu X, Zhang M, et al. Remote ischemic postconditioning improves neurological function by AQP4 downregulation in astrocytes. Behav Brain Res 2015;289:18. 21. Dong Y, Bao C, Yu J, et al. Receptor-interacting protein kinase 3-mediated programmed cell necrosis in rats subjected to focal cerebral ischemia-reperfusion injury. Mol Med Rep 2016;14:728-736. 22. Thiagarajah JR, Papadopoulos MC, Verkman AS. Noninvasive early detection of brain edema in mice by near-infrared light scattering. J Neurosci Res 2005;80:293299. 23. Bonomini F, Rezzani R. Aquaporin and blood brain barrier. Curr Neuropharmacol 2010;8:92-96. 24. Kurita T, Nakamura K, Tabuchi M, et al. Effects of Gore-san: a traditional Japanese kampo medicine, on aquaporin 1, 2, 3, 4 and V2R mRNA expression in rat kidney and forebrain. J Med Sci 2011;11:30-38. 25. Nogami A, Sakata Y, Uchida N, et al. Effects of yokukansan on anxiety-like behavior in a rat model of cerebrovascular dementia. J Nat Med 2011;65:275-281. 26. Egashira N, Iba H, Kuwano H, et al. Kamishoyosan reduces conditioned fear-induced freezing behavior in
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Goreisan Prevents Brain Edema after Cerebral Ischemic Stroke by Inhibiting Aquaporin 4 Upregulation in Mice Takafumi Nakano, PhD,*,†,‡ Chisa Nishigami, MS,† Keiichi Irie, PhD,† Yutaka Shigemori, MD, PhD,§ Kazunori Sano, PhD,† Yuta Yamashita, MS,† Takayuki Myose, MS,† Koji Tominaga, PhD,* Koichi Matsuo, PhD,* Yoshihiko Nakamura, MD, PhD,§ Hiroyasu Ishikura, MD, PhD,§ Hidetoshi Kamimura, PhD,*,‡ Takashi Egawa, PhD,* and Kenichi Mishima, PhD†
Background: Aquaporin 4 (AQP4) is a water-selective transport protein expressed in astrocytes throughout the central nervous system. AQP4 level increases after cerebral ischemia and results in ischemic brain edema. Brain edema markedly influences mortality and motor function by elevating intracranial pressure that leads to secondary brain damage. Therefore, AQP4 is an important target to improve brain edema after cerebral ischemia. The Japanese herbal Kampo medicine, goreisan, is known to inhibit AQP4 activity. Here, we investigated whether goreisan prevents induction of brain edema by cerebral ischemia via AQP4 using 4-hour middle cerebral artery occlusion (4h MCAO) mice. Methods: Goreisan was orally administered at a dose of 500 mg/kg twice a day for 5 days before MCAO. AQP4 expression and motor coordination were measured by Western blotting and rotarod test, respectively. Results: Brain water content of 4h MCAO mice was significantly increased at 24 hours after MCAO. Treatment with goreisan significantly decreased both brain water content and AQP4 expression in the ischemic brain at 24 hours after MCAO. In addition, treatment with goreisan alleviated motor coordination deficits at 24 hours after MCAO. Conclusions: The results of this study suggested that goreisan may be a useful new therapeutic option for ischemic brain edema. Key Words: Goreisan—brain edema—aquaporin 4—cerebral ischemia. © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.
Introduction From the *Department of Pharmaceutical and Health Care Management; †Department of Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Jyonan, Fukuoka, Japan; ‡Department of Pharmacy; and §Department of Emergency and Critical Care Medicine, Fukuoka University Hospital, Fukuoka, Japan. Received June 30, 2017; revision received September 29, 2017; accepted October 10, 2017. Address correspondence to Keiichi Irie, PhD, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma 8-19-1, Jyonan, Fukuoka 814-0180, Japan. E-mail: [email protected]. 1052-3057/$ - see front matter © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jstrokecerebrovasdis.2017.10.010
Although brain ischemic stroke is a leading cause of mortality and disability worldwide, limited therapeutic options are available.1 Brain ischemia is usually caused by embolic or thrombotic occlusion of a cerebral artery. The pathophysiology of ischemic brain damage is complex and multifactorial. Cerebral ischemia and reperfusion induce brain edema,2 which was shown to be caused by cellular dysfunction with Na+/K+ pump failure due to vascular injury resulting in abnormal increases in extracellular and intracellular water contents, thus leading to brain swelling.3 There are 2 patterns of brain edema: vasogenic edema, which is often observed in brain tumors,
Journal of Stroke and Cerebrovascular Diseases, Vol. ■■, No. ■■ (■■), 2017: pp ■■–■■
1
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freezing injury, and cerebral trauma; and cytotoxic edema, which is associated with intracellular ionic imbalance induced by cerebral ischemia.4 These types of brain edema induced by cerebral ischemia markedly affect mortality and motor function due to elevated intracranial pressure that leads to secondary brain damage.5 Therefore, brain edema plays an important role in ischemic stroke and represents a potential target for therapy. However, limited therapeutic options are available for brain edema. Aquaporins (AQPs) are known as a family of water channel proteins. Aquaporin (AQP) 1 was purified and identified in human erythrocytes as the first member of the AQP family.6 Currently, more than 10 AQPs have been discovered in mammals.7 AQP4 is also a water-selective transport protein expressed at the plasma membrane in astrocytes throughout the central nervous system.8 The expression of AQP4 increases after cerebral ischemia and results in ischemic brain edema.9,10 Therefore, the occurrence of brain edema is closely related to the expression of AQP4 in brain tissue after cerebral ischemia. Yao et al reported the improved outcome in AQP4 knockout mice following focal ischemia produced by middle cerebral artery occlusion (MCAO).11 In addition, several correlative investigations reported reduced brain edema after ischemia with reduced AQP4 expression.12-14 Thus, AQP4 is a potential therapeutic target in ischemic brain edema. Goreisan is a Japanese herbal Kampo medicine consisting of a mixture of 5 herbs (Alisma rhizome, Atractylodes rhizome or Atractylodes lancea rhizome, Polyporus sclerotium, Poria sclerotium, and Cinnamon bark), which has been used to treat watery diarrhea, edema, vertigo, and nausea. Hayashi reported the effects of goreisan on brain edema complicated by central nervous system disease.15 However, the clinical effectiveness of goreisan for treatment of brain edema remains unclear. As goreisan was recently reported to inhibit AQP4,16,17 we postulated that it may prevent brain edema induced by cerebral ischemia via AQP4. The present study was performed to investigate whether treatment with goreisan prevents brain edema via AQP4 using 4-hour middle cerebral artery occlusion (4h MCAO) mice. The results suggested that goreisan is a potential new therapeutic option for brain edema in ischemic stroke.
Materials and Methods Focal Cerebral Ischemia Focal cerebral ischemia was induced in male ddY mice (6-8 weeks old, 25-35 g; Kiwa Experimental Animal Laboratory, Wakayama, Japan) as described previously.18 The mice were reanesthetized with isoflurane (Escain; Pfizer, Osaka, Japan) 4 hours after occlusion, and reperfusion was established by withdrawal of the filament. Occlusion of the middle cerebral artery was confirmed by examining forelimb flexion after awakening from anesthesia. All procedures regarding animal care and use were
performed in compliance with the regulations established by the Experimental Animal Care and Use Committee of Fukuoka University.
Drug Administration Goreisan (Tsumura, Tokyo, Japan) was dissolved in water and orally administered at a dose of 500 mg/kg twice a day for 5 days at 24 hours before MCAO. Water (vehicle) was used as a control.
Physiological Parameters Conscious mice were weighed and their blood pressures were measured at 24 hours after 5 days of repeated treatment using a noninvasive, computerized tail-cuff system with BP Monitor for Rats & Mice model MK200 (Muromachi Kikai Co., Tokyo, Japan). Rectal temperature was measured at 24 hours after 5 days of drug treatment using a digital laboratory thermometer (BAT-12; Physitemp Instruments, Clifton, NJ) with a needletype thermometer, at room temperature (23°C ± 1°C). Blood samples were also collected at 24 hours after 5 days of drug treatment. Serum was obtained after centrifugation (1500 × g for 15 minutes at 4°C). Serum Na, K, and Cl levels were measured by the ion-selective electrode method (Oriental Yeast Co. Nagahama LSL, Shiga, Japan).
Brain Water Content at 24 Hours after MCAO The left cerebral hemispheres of the mice were collected at 24 hours after MCAO and weighed immediately after euthanasia (wet tissue weight). Brain samples were dried in an oven at 110°C for 48 hours. The dried samples were then weighed again (dry tissue weight), and the brain water content was calculated as [(wet tissue weight − dry tissue weight)/wet tissue weight] × 100%.
Immunoblotting The expression of AQP4 was evaluated by Western blotting. Tissue samples (striatum) were homogenized at 4°C for 1 minute in PRO-PREP protein extraction solution (iNtRON Biotechnology, Seongnam-Si, Korea). The tissue extract was centrifuged at 15,000 rpm at 4°C for 30 minutes. The supernatant was treated in the same way as the tissue extract. The total protein concentration of each tissue lysate was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Protein (20 µg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; 20% gel). Equivalent amounts of each protein were separated by SDS–PAGE and transferred onto polyvinylidene difluoride membranes according to standard procedures. After blocking with 5% skim milk in Tris-buffered saline containing .1% Tween 20 (TBS-T, Sigma-Aldrich, St. Louis, MO.), membranes were probed with primary antibodies (antiAQP4, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA;
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Table 1. Summary of physiological parameters after 5 days of drug treatment in intact mice
Control (n = 6) Vehicle (n = 6) Goreisan (n = 6)
BW (g)
sBP (mmHg)
RT (°C)
Na (mEq/L)
K (mEq/L)
Cl (mEq/L)
32.8 ± .6 33.5 ± .6 33.5 ± .6
88.3 ± 6.0 92.8 ± 6.7 88.3 ± 3.3
36.3 ± .1 36.5 ± .2 36.2 ± .1
149.0 ± .4 148.8 ± 1.0 148.7 ± .7
6.6 ± .2 6.7 ± .1 6.6 ± .2
104.7 ± .8 102.0 ± .4 105.2 ± .6
Abbreviations: BW, body weight; RT, rectal temperature; sBP, systolic blood pressure. All values are means ± standard error of the mean.
anti-β-actin, 1:1000; Abcam, Cambridge, MA) for 1 hour at room temperature. The membranes were washed with TBS-T and incubated with horseradish peroxidaseconjugated secondary antibody (1:5000; Santa Cruz Biotechnology) for 1 hour at room temperature. After washing 3 times with TBS-T, the bands were developed using an ECL Advance Western Blotting Detection Kit (GE Healthcare, Little Chalfont, UK) and detected using FluorChem8900 (Alpha Innotech, San Leandro, CA). The signal intensity on the blots was measured using NIH Image (version 1.63; National Institutes for Health, Bethesda, MD).
Rotarod Test Motor coordination was measured by the rotarod test at 24 hours after MCAO as described previously.19 Mice were placed on a rotating rod that is 3 cm in diameter (Neuroscience Inc., Tokyo, Japan) with a nonskid surface, and the latency to fall was measured for up to 2 minutes. The rotation speed was 10 rpm.
Figure 1. Effects of goreisan on brain water content after ischemic stroke in 4h MCAO mice. Quantitative analysis of brain water content in control (n = 8), vehicle (n = 10), and 500 mg/kg goreisan twice a day (n = 11) groups. **P < .01 versus sham, ††P < .01 versus vehicle. All values are means ± standard error of the mean. Abbreviation: 4h MCAO, 4-hour middle cerebral artery occlusion.
Statistical Analysis Data are presented as means ± standard error of the mean. Multiple comparisons were evaluated by Tukey test. In all analyses, P < .05 was taken to indicate statistical significance.
Results Effects of Goreisan on Physiological Parameters in Intact Mice There were no significant differences among control, vehicle, and goreisan in any of the physiological parameters measured (Table 1). Thus, repeated treatment with goreisan did not influence the physiological parameters at the onset of cerebral ischemia.
Effects of Goreisan on Brain Water Content after Ischemic Stroke in Mice Subjected to 4H MCAO Brain water content was significantly increased in 4h MCAO mice at 24 hours after MCAO. Treatment with goreisan (500 mg/kg twice a day) significantly decreased brain water content compared with vehicle alone at 24 hours after MCAO (Fig 1).
Effects of Goreisan on AQP4 after Ischemic Stroke in Mice Subjected to 4H MCAO Brain ischemia–reperfusion has been shown to upregulate AQP4 expression and induce brain edema. 2,9,10 The level of AQP4 expression was increased at 24 hours after MCAO. However, treatment with goreisan (500 mg/kg twice a day) significantly decreased the AQP4 expression level compared with vehicle alone at 24 hours after MCAO (Fig 2).
Effects of Goreisan on Motor Coordination Estimated by Rotarod Test and Survival Rate after Ischemic Stroke in Mice Subjected to 4H MCAO Treatment with goreisan (500 mg/kg twice a day) significantly alleviated motor coordination deficits compared with vehicle controls at 24 hours after MCAO (Fig 3). However, the survival rates of vehicle and goreisan groups at 24 hours after MCAO were not significantly different (90.3%, n = 27 and 93.2%, n = 33, respectively). The MCAO mouse model was shown previously not to have a high mortality rate at 24 hours after MCAO.19
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Figure 2. Effects of goreisan on AQP4 after ischemic stroke in 4h MCAO mice. Quantitative analysis of AQP4 expression in sham (n = 6), vehicle (n = 6) and 500 mg/kg goreisan twice a day (n = 6) groups. **P < .01 versus sham, †P < .05 versus vehicle. All values are means ± standard error of the mean. Abbreviations: 4h MCAO, 4-hour middle cerebral artery occlusion; AQP4, aquaporin 4.
Figure 3. Effects of goreisan on motor coordination estimated by rotarod test after ischemic stroke in 4h MCAO mice. Quantitative analysis of motor coordination estimated by rotarod test in sham (n = 6), vehicle (n = 10), and 500 mg/kg goreisan twice a day (n = 13) groups. *P < .05 versus sham, **P < .01 versus sham, †P < .05 versus vehicle. All values are means ± standard error of the mean. Abbreviation: 4h MCAO, 4-hour middle cerebral artery occlusion.
Discussion The present study was performed to evaluate the effects of goreisan on ischemic brain edema via AQP4. Our results indicated that treatment with goreisan decreased both the brain water content and the expression of AQP4 after
cerebral ischemic stroke. Moreover, goreisan treatment alleviated the motor dysfunction after cerebral ischemic stroke. These observations suggest that goreisan may be a novel option to prevent ischemic brain edema. Several studies reported that brain edema was upregulated 1-3 days after cerebral ischemia.4,20,21 We previously evaluated brain edema at 1-3 days after MCAO and showed that the peak of brain edema occurred 24 hours after MCAO in our ischemic model mice (data not shown). Therefore, we evaluated brain edema, infarct volume, rotarod test performance, and AQP4 expression at 24 hours after MCAO in the present study. Brain edema induced by ischemic stroke occurs due to cellular dysfunction with Na + /K + pump failure. 3 The consequences are lethal due to the increased intracranial pressure and the compromised cerebral blood flow. Moreover, AQP4 is involved in the deterioration of brain edema. AQP4 has been shown to be the primary water transport pathway across the blood–brain barrier (BBB).8 AQP4 is rapidly upregulated on astrocytic endfeet surrounding cerebral blood vessels in the peri-infarcted cortex in MCAO model mice, resulting in aggravation of brain edema.8,20,22,23 Thus, its inhibition prevents brain edema, making it a potential therapeutic target. In the present study, we examined whether goreisan can prevent brain edema induced by cerebral ischemia in mice exposed to MCAO for 4 hours. First, we examined the goreisan dose regimen. Goreisan was reported previously to require repeated administration for 3 days to influence AQP mRNA expression.24 Moreover, we reported that Kampo medicine required the repeated administration of around 1 week to treat the cerebrovascular disease.25-27 Therefore, we investigated the effects of repeated oral administration of goreisan on brain edema at a dose of 500 mg/kg once or twice a day for 5 days before MCAO. Treatment with goreisan at a dose of 500 mg/kg twice a day was shown to decrease brain edema at 24 hours after MCAO, confirming that this Kampo medicine is effective for ischemic brain edema. We also investigated the effects of a single oral administration of goreisan after reperfusion on brain edema in mice subjected to MCAO. However, single administration of goreisan did not affect brain edema at 24 hours after MCAO, indicating that repeated administration is required to influence ischemic brain edema. Next, we investigated the relationship between brain edema and AQP4 expression after cerebral ischemic stroke. AQP4 is known to be involved in the deterioration of brain edema. In the present study, AQP4 expression was shown to be upregulated at 24 hours after MCAO. Thus, inhibition of this upregulation of AQP4 expression may lead to improvement of ischemic brain edema. Goreisan is known to inhibit AQP4 expression in brain edema.16,17,24 Although the mechanism by which goreisan inhibits AQP4 expression remains unclear, Isohama reported that this agent inhibits water permeability in MLE-12 cells by
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inhibiting AQP molecules and that Mn and Zn ions are involved in this process.28 In the present study, treatment with goreisan at a dose of 500 mg/kg twice a day inhibited the upregulation of AQP4 induced by cerebral ischemic stroke at 24 hours after MCAO. Therefore, goreisan may prevent formation of brain edema by affecting AQP4 expression after cerebral ischemia stroke. On the contrary, our results showed that goreisan tended to improve infarct volume at 24 hours after MCAO, but the differences between goreisan and vehicle groups were not significant (data not shown). Several studies indicated that deletion of AQP4 reduces brain edema and infarct volume in a model of MCAO.14,29,30 AQP4 is also known to affect the BBB in ischemic stroke. Disruption of the BBB is a critical event during brain ischemic injury followed by passive diffusion of water, leading to brain edema and secondary brain injury.31 In the present study, we evaluated brain edema in mice exposed to MCAO for 4 hours, which is a stronger ischemia–reperfusion injury model than in previous studies. Previously, we reported that our ischemic model mice show severe nerve injury.18 Therefore, goreisan may not be effective for infarct volumes induced by 4 hours of MCAO. Nevertheless, model mice treated with goreisan showed improvement of brain edema. The effect of goreisan on AQP4 may be expected to improve brain edema rather than have a neuroprotective effect. Cho et al also reported that repeated administration of mannitol, a therapeutic drug for brain edema, reduced the water content of brain edema without affecting the infarct volume after MCAO in rats.32 Therefore, improvement of brain edema appears to be useful for treatment of cerebral ischemic stroke without affecting the infarct lesions. Moreover, improvement of brain edema also has a marked impact on recovery of brain function. In fact, treatment with goreisan alleviated motor coordination deficits at 24 hours after MCAO. This result has important clinical implications because motor dysfunction is the greatest negative effect of cerebral ischemic stroke. Inoue reported that motor dysfunction occurred due to compression of the pyramidal system by brain edema.33 In addition, there have been previous reports regarding the relation between improvement of cerebral edema and motor function.34,35 Moreover, deletion of AQP4 was reported to alleviate intraparenchymal pressure and improve motor dysfunction due to neurological disease.36 Thus, the effect of goreisan on AQP4 expression can be expected to alleviate motor dysfunction by improving brain edema. Goreisan has been regarded as a useful medicine since ancient times, and has been shown to be efficacious in treatment of watery diarrhea, edema in multiple organs, vertigo, and nausea. We consider goreisan to be easy to use for prevention and treatment of brain edema in patients with ischemic stroke in clinical practice. Therefore, further studies are required to investigate the effects of postischemic administration of goreisan.
In conclusion, the results of the present study indicated that AQP4 expression is involved in ischemic brain edema and that goreisan prevents brain edema by preventing upregulation of AQP4 after cerebral ischemic stroke. Moreover, goreisan ameliorated motor dysfunction induced by ischemic brain edema. We consider that goreisan alleviated motor dysfunction after cerebral ischemic stroke by preventing brain edema via its preventive effect on AQP4 upregulation. The results presented here suggest that goreisan may be useful as a new additional therapeutic option for improvement of brain function after cerebral ischemic stroke.
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