- Email: [email protected]
Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model
Brain Hemorrhages xxx (xxxx) xxx
Contents lists available at ScienceDirect
Brain Hemorrhages
CHINESE ROOTS GLOBAL IMPACT
journal homepage: www.keaipublishing.com/en/journals/brain-hemorrhages/
Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model Masato Naraoka ⇑, Yuchen Li, Takeshi Katagai, Hiroki Ohkuma Department of Neurosurgery, Hirosaki University School of Medicine & Hospital, Hirosaki, Japan
a r t i c l e
i n f o
Article history: Received 19 December 2019 Accepted 19 December 2019 Available online xxxx Keywords: Subarachnoid hemorrhage Cerebral vasospasm Oxidative stress Apple polyphenols
a b s t r a c t Subarachnoid hemorrhage (SAH) is almost always caused by ruptured cerebral aneurysms, and the most serious complication after SAH is cerebral vasospasm. Oxidative stress due to free radicals released from subarachnoid hemorrhage clots causes cerebral vasospasm, as proven by animal experiments. Apple polyphenols have already been demonstrated to strongly suppress oxidized low-density lipoprotein (ox-LDL) and lectin-like oxidized LDL receptor-1 (LOX-1) derived from reactive oxygen species (ROS), and to improve cerebral vasospasm. However, it is unclear how apple polyphenol acts downstream of ROS. The purpose of this study is to reveal a more detailed mechanism of functioning for apple polyphenols in suppressing oxidative stress and cerebral vasospasm. Using the double-hemorrhage rabbit subarachnoid hemorrhage (SAH) model, we investigated the effect of apple polyphenols by measuring the basilar artery diameter, endothelial NO synthase (eNOS) expression, diacron reactive oxygen metabolites (d-ROM), biological antioxidant potential (BAP), and malondialdehyde (MDA). The mean diameter of the basilar artery in the apple polyphenol treatment group was significantly larger than in the SAH group (p < 0.01). The expression of endothelial nitric oxide synthase (eNOS), evaluated by immunohistochemistry in the apple polyphenol treatment group, was higher than in the SAH group (p < 0.05). The BAP test in the apple polyphenol treatment group showed a significant difference, while there was no significant difference in the values of d-ROM and MDA. The results of the present study revealed that apple polyphenol significantly improved cerebral vasospasm and eNOS. In spite of a significant increase in antioxidant power expressed by increased BAP, oxidative stress measured by the d-ROM value was not significantly suppressed. Either the number of experimental animals was not sufficient to reach statistical significance, or the eNOS increase due to apple polyphenols and suppression of vasospasm are attributable to pathways other than lipid oxidation detected by MDA. Ó 2020 Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Subarachnoid hemorrhage (SAH) is almost always caused by a ruptured cerebral aneurysm, with cerebral vasospasm being one of the serious, subsequent complications.1–4 Cerebral vasospasm is thought to be caused by the continuous contraction of smooth muscle in main cerebral arteries at 4- to 14 days after the onset of SAH. This contraction results in cerebral ischemia and cerebral infarction, which can cause severe neurological deficits.4,5 Many vasoactive substances derived from subarachnoid blood clots have been implicated, including free radicals, histamine, norepinephrine, oxidized hemoglobin, prostaglandins, thrombin, IL-6 ⇑ Corresponding author at: Department of Neurosurgery, Hirosaki University School of Medicine, 5-Zaihuchou, Hirosaki Aomori pref 036-8562, Japan. Fax: +81 172 39 5116. E-mail address: [email protected] (M. Naraoka).
etc.6 Among these, free radicals are considered to be the main substances causing cerebral vasospasm. In this regard, free-radical scavengers and antioxidants have been used in an attempt to prevent and treat cerebral vasospasm after SAH. We also recently administered apple polyphenols as an antioxidant to an experimental subarachnoid hemorrhage model.7 In particular, we focused on the procyanidins, the main active antioxidant components in apple polyphenols, which inhibit oxidized low-density lipoprotein (ox-LDL) and lectin-like oxidized LDL receptor-1 (LOX-1) from reactive oxygen species (ROS). As a result, we have demonstrated that apple polyphenols suppresses ox-LDL and LOX-1, improve endothelial NO synthase (eNOS) expression and cerebral vasospasm. Therefore, the local antioxidant effect of apple polyphenols on cerebral arteries has been demonstrated through a specific pathway. However, the antioxidative effects and the suppression of oxidative stress and lipid
https://doi.org/10.1016/j.hest.2019.12.006 2589-238X/Ó 2020 Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: M. Naraoka, Y. Li, T. Katagai et al., Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model, Brain Hemorrhages, https://doi.org/10.1016/j.hest.2019.12.006
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peroxidation on a systemic basis that includes the mechanisms have not been clarified. In the present study, in order to evaluate the actions of apple polyphenols, the marker diacron reactive oxygen metabolites (dROM), the biological antioxidant potential (BAP), and malondialdehyde (MDA) were measured using a rabbit SAH model. 2. Materials and methods This experimentation was approved by the Animal Experimentation Ethics Committee of Hirosaki University School of Medicine. Japanese white rabbits were used. All rabbits were maintained under standard conditions (temperature of 25 ± 2 °C, 12-hour light/dark cycle), and were given ad libitum access to food and water. The animals were assigned randomly to two groups; the SAH group (n = 10) and the apple polyphenol treatment group (n = 10). Experimental SAH was produced according to the twohemorrhage method as previously described.8 In the apple polyphenol treatment group, after the SAH was produced by the same procedure as in the SAH group, apple polyphenol (50 mg/ kg) was administered orally once a day from day 0 to day 3. The peripheral blood samples were collected from the auricular vein on day 0 and day 4 for measuring d-ROM and for the BAP test. On day 4, perfusion-fixation was performed under deep anesthesia for immunohistochemical and histological evaluation of the basilar arteries. The other five rabbits in both group were euthanized under deep anesthesia without perfusion-fixation and the basilar arteries were removed for protein analysis of eNOS and MDA by ELISA. The d-ROM test provides a measure for oxidative stress in peripheral blood by evaluating the level of reactive oxygen metabolites, mainly measured as hydroperoxidase. It proved to be a reliable and effective tool for readily evaluating oxidative stress in both humans and animals.9–11 The BAP test assesses antioxidant power.12,13 This test evaluates the blood concentration of antioxidants as agents that can reduce the ferric form (Fe3+) to the ferrous (Fe2+). MDA is one of the lipid peroxidative degradation products that is widely used as a major marker of lipid peroxidation, because polyunsaturated fatty acids (PUFA) are oxidized by reactive oxygen species (ROS) and free radicals to form MDA as the final product (Fig. 1).11,14–16 2.1. Induction of SAH Experimental SAH was produced according to the two hemorrhage method as previously described.8 On the first day (day 0), the rabbits were anesthetized with an intravenous injection of pentobarbital (30 mg/kg) and an intramuscular injection of ketamine (20 mg/kg). After anesthesia and under spontaneous breathing a 23-gauge butterfly needle was inserted percutaneously into
the cisterna magna. After aspiration of 1.0 ml cerebrospinal fluid, the same amount of nonheparinized arterial blood from the femoral artery was slowly injected into the cisterna magna for 1 min under aseptic technique. Rabbits were then placed in a 30° head-down position for 30 min. After recovery from anesthesia, they were returned to the feeding room. Forty-eight hours afterwards (day 2), the second experimental SAH was performed in the same manner as the first. 2.2. Administration of apple polyphenols In the apple polyphenol treatment group, apple polyphenols (50 mg/kg, Asahi Food and Healthcare Co., LTD) were orally administered once a day for 4 days from day 0 to day 3. Polyphenols contain 63.8% procyanidins, which are widely used as an antioxidant.17 2.3. Perfusion fixation Perfusion fixation was performed on day 4 under deep anesthesia with high-dose intravenous pentobarbital injection (300 mg/ kg) and an intramuscular ketamine injection (100 mg). The thoracotomy was performed and a cannula was immediately inserted through the left ventricle into the ascending aorta. The descending aorta was then clamped and the right atrium opened. Perfusionfixation started with 500 ml saline injected with heparin, followed by 500 ml 4% paraformaldehyde (pH 7.4) under a perfusion pressure of 75 mm Hg. After perfusion fixation, the whole brain was excised with the basilar artery. 2.4. Measurement of basilar artery cross-sectional area A tissue specimen was prepared by hematoxylin-eosin (HE) staining of the fixed basilar artery. The degree of cerebral vasospasm was evaluated by measuring the basilar artery lumen cross-sectional area and converting it to a diameter. Paraffinembedded basilar artery sections (thickness 6 lm) were HE stained and the basilar artery cross-sectional area was calculated using ImageJÒ version 1.45 (NIH). The cross-sectional area was calculated from the perimeter of the luminal border, while the area included in the boundary of the inner elastic plate was ignored. For each basilar artery, three sequential sections were calculated proximally, intermediately, and distal, and then converted into the vessel diameter. Diameter mean ± SEM values obtained for each artery were used for analysis. 2.5. Immunostaining of eNOS Immunostaining of eNOS was performed on the fixed basilar artery. eNOS expressed in the cerebral vascular epithelium was thereby evaluated.18 Immunohistochemistry was performed on paraffin-embedded sections to determine the immunoreactivity of eNOS. Sections were deparaffinized and rehydrated in graded concentrations of ethanol to distilled water. Sections were incubated with primary anti-eNOS antibody (1:200 dilution, BD Biosciences) for 1 h at room temperature, followed by a 15-min wash in PBS. Sections were incubated with goat anti-rabbit IgG (1:500 dilution) for 60 min at room temperature. Diaminobenzidine was used as the chromogen and counterstaining was performed with hematoxylin. 2.6. Measurement of d-ROM and the BAP test
Fig. 1. Outline of oxidative stress factors and measurement markers.
The d-ROM measurement and the BAP test were done with venous blood at day 0 and day 4 using a free radical analyzer (Free carrio duo, Wismerll Co., LTD).9,19,20
Please cite this article as: M. Naraoka, Y. Li, T. Katagai et al., Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model, Brain Hemorrhages, https://doi.org/10.1016/j.hest.2019.12.006
M. Naraoka et al. / Brain Hemorrhages xxx (xxxx) xxx
The d-ROM test was measured by mixing 20 ml of collected venous blood with an acetic acid buffer solution (pH 4.8) to stabilize the hydrogen ion concentration. In the acid buffer solution, bivalent and trivalent iron from blood ionized to form a catalyst that breaks down the hydroperoxidase groups in the blood to become free radicals. The blood samples were then transferred into a cuvette containing colorless chromogen (N. N. diethylparaphenylenediamine), which is oxidized by free radicals and changes into a radical cation with a magenta color. The density of the magenta color, which was measured using a photometer (505 nm), is proportional to the quantity of ROMs. The d-ROM test measured the concentrations of various hydroperoxidase in the blood, and the results were expressed in arbitrary units called ‘‘Carratelli units” (U.CARR). The BAP test was performed at the same time. The antioxidant potential of blood plasma was evaluated by measuring the degree of reduction of the ferric ion (Fe3+) to the ferrous form (Fe2+) as a result of antioxidant action. In practice, 100 ml of blood are centrifuged for 90 s, after which the amount of trivalent iron that is deoxidized in 5 min is measured in units of mmol/l.
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Fig. 2. Histological findings for the basilar artery. Left: Photomicrographs of representative sections from the SAH group, Right: the apple polyphenol treatment group. The both sections show the enlarged section of the vessel wall below. Scale bar = 200 lm.
2.7. Measurement of eNOS and MDA by ELISA After removing the subarachnoid hematoma on the surface of the extracted basilar artery, the vessel was ultrasonically disrupted using a buffer. Centrifugation was performed at 2000g for 10 min, and the separated supernatant was further centrifuged at 15,000g at 4 °C for 15 min. Protein concentration was measured by a standard microplate assay using a protein assay kit (5000002JA, BIO-RAD). After correcting to the protein concentration of each group, the concentration was adjusted to a constant value and measured again for confirmation. eNOS was quantitatively evaluated by ELISA (ser1176, Tebu-bio CO., LTD). MDA was performed according to the protocol using an ELISA kit manufactured by Cell Biolabs, Inc.
2.8. Statistical analysis Analysis of results was performed for the SAH group and the treatment group for all measurements. Statistical analyses were performed using the JMPÒ (Version 11; SAS Institute Inc., Cary, NC). Data are expressed as mean ± SEM. In order to compare the two groups expressed as the mean ± SEM, normal distribution is not paired; we used the Mann–Whitney U test, significance determined at p < 0.05.
Fig. 3. Immunohistochemical findings of eNOS for the basilar artery. Left: Photomicrographs of representative sections from the SAH group, Right: the apple polyphenol treatment group. The area enclosed by the square on the top is enlarged below. Arrowheads indicate eNOS staining. Scale bar = 50 lm.
3. Results 3.1. Evaluation of cerebral vasospasm The cross sections of the basilar artery in both groups, determined by staining with HE staining and observed under the light microscope, are shown in Fig. 2. The mean diameter of the basilar artery was 0.53 ± 0.13 mm for the SAH group and 1.55 ± 0.27 mm for the apple polyphenol group (p < 0.01).
3.2. Evaluation of eNOS on the basilar artery The results of immunostaining of eNOS on the tissue of the basilar artery of both groups are shown in Fig. 3. It was confirmed that more eNOS was expressed in the apple polyphenol group. Furthermore, a significant difference could be confirmed quantitatively by ELISA (p < 0.05, Fig. 4).
Fig. 4. Expression of eNOS was significantly improved in the vascular smooth muscle cells by ELISA.
Please cite this article as: M. Naraoka, Y. Li, T. Katagai et al., Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model, Brain Hemorrhages, https://doi.org/10.1016/j.hest.2019.12.006
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Fig. 5. The result of the BAP test on day 0 and day 4. There was no significant difference between the SAH group and the polyphenol group, but there was a significant difference between the two groups on day 4.
Fig. 6. The result of the d-ROM test on day 0 and day 4. Significant differences were observed within each group of SAH group and the polyphenol group. 1 CARRU = 0.08 mg H2O2/dL.
3.3. Evaluation of chemical markers associated with oxidative stress The result of the BAP test is shown in Fig. 5. On Day 4, a significant difference was observed in the apple polyphenol group (3138 ± 172.3 lmol/L) compared to the SAH group (2845 ± 193.6 lmol/L). There was no significant difference between Day 0 and Day 4 within each group. On the other hand, the value of d-ROM increased significantly in both groups after SAH production (Fig. 6). The result of the d-ROM test in the apple polyphenol group was suppressed (412 ± 102.6 CARRU) compared to that of the SAH group (492 ± 82.3 CARRU); however, there was no significant difference between these groups. The results of MDA are shown in Fig. 7. Although MDA was lower in the treatment group than in the SAH group, there was no significant difference. 4. Discussion The results of the present study revealed that apple polyphenols significantly improved cerebral vasospasm and eNOS, which was comparable to the result of our previous study. The effect was considered to be attributable to the antioxidant action of apple
Fig. 7. Expression of MDA was significantly improved in the vascular smooth muscle cells by ELISA.
Please cite this article as: M. Naraoka, Y. Li, T. Katagai et al., Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model, Brain Hemorrhages, https://doi.org/10.1016/j.hest.2019.12.006
M. Naraoka et al. / Brain Hemorrhages xxx (xxxx) xxx
polyphenol, because the BAP test, which is an index of antioxidant power, was significantly increased in the apple polyphenol treatment group. In another report, it was proven that a BAP increase in neonatal blood was induced by the administration of lutein, which is a type of carotenoid that exhibits antioxidant activity.21 In spite of a significant increase in antioxidant power expressed by increased BAP, oxidative stress measured by d-ROM values was not significantly suppressed. One of the possibilities may be insufficient dosage of apple polyphenols, which can increase antioxidant action but not decrease oxidative stress significantly. Another possibilities is that oxidative stress in the blood may be influenced by not only a local event such as SAH, but also by a systemic event such as anesthesia. Suppressed oxidative stress at the local SAH by apple polyphenol could therefore be masked by the systemic generation of oxidative stress. In our previous experimentation, and in this study, the decrease in eNOS by the blood vessel wall was suppressed, which suggested lipid peroxidation of the blood vessel wall was suppressed, but MDA changes in the present study could not entirely support this view, as MDA, an index of local lipid peroxidation, showed a mild decrease from the administration of apple polyphenols without statistically significance compared to the SAH group. The number of experimental animals was not sufficient to reach statistical significance, or the eNOS increase due to apple polyphenols and suppression of vasospasm are attributable to pathways other than lipid oxidation detected by MDA. Other markers of oxidative stress are known, including 8-OHdG, which is an indicator of DNA damage, and nitrosothiols and 3-NT, which are indicators of protein damage. In the future, we plan to elucidate the effects and mechanisms of antioxidants, including apple polyphenols, by adding these marker measurements and increasing the number of animals. Conflict of interest The authors declare no competing financial interests. References 1. Ecker A, Riemenschneiderr PA. Arteriographic demonstration of spasm of the intracranial arteries, with special reference to saccular arterial aneurysms. J Neurosurg. 1951;8:660–667.
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2. King JT. Epidemiology of aneurysmal subarachnoid hemorrhage. Neuroimaging Clin N Am. 1997;7:659–668. 3. Pluta RM, Hansen-Schwartz J, Dreier J, et al.. Cerebral vasospasm following subarachnoid hemorrhage: time for a new world of thought. Neurol Res. 2009;31:151–158. 4. Vergouwen MDI, Vermeulen M, van Gijn J, et al.. Definition of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage as an outcome event in clinical trials and observational studies: proposal of a multidisciplinary research group. Stroke. 2010;41:2391–2395. 5. Naraoka M, Munakata A, Matsuda N, Shimamura N, Ohkuma H. Suppression of the Rho/Rho-kinase pathway and prevention of cerebral vasospasm by combination treatment with statin and fasudil after subarachnoid hemorrhage in rabbit. Transl Stroke Res. 2013;4:368–374. 6. Suzuki H. What is early brain injury?. Transl Stroke Res. 2015;1–3. 7. Matsuda N, Ohkuma H, Naraoka M, Munakata a, Shimamura N, Asano K. Role of oxidized LDL and lectin-like oxidized LDL receptor-1 in cerebral vasospasm after subarachnoid hemorrhage. J Neurosurg. 2014;121:621–630. 8. Foley PL, Caner HH, Kassell NF, Lee KS. Reversal of subarachnoid hemorrhageinduced vasoconstriction with an endothelin receptor antagonist. Neurosurgery. 1994;34:108–112. discussion 112-3. 9. Cesarone MR, Belcaro G, Carratelli M, et al.. A simple test to monitor oxidative stress. Int Angiol. 1999;18:127–130. 10. Trotti R, Carratelli M, Barbieri M, et al.. Oxidative stress and a thrombophilic condition in alcoholics without severe liver disease. Haematologica. 2001;86:85–91. 11. Gerardi G, Usberti M, Martini G, et al.. Plasma total antioxidant capacity in hemodialyzed patients and its relationships to other biomarkers of oxidative stress and lipid peroxidation. Clin Chem Lab Med. 2002;40:104–110. 12. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power”: the FRAP assay. Anal Biochem. 1996;239:70–76. 13. Faienza MF, Francavilla R, Goffredo R, et al.. Oxidative stress in obesity and metabolic syndrome in children and adolescents. Horm Res Paediatr. 2012;78:158–164. 14. Antus B. Oxidative stress markers in sputum. Oxid Med Cell Longev. 2016;2016. 15. Ho E, Karimi Galougahi K, Liu CC, Bhindi R, Figtree GA. Biological markers of oxidative stress: applications to cardiovascular research and practice. Redox Biol Elsevier. 2013;1:483–491. 16. Pompella A, Corti A. Editorial: the changing faces of glutathione, a cellular protagonist. Front Pharmacol. 2015;6:4–7. 17. Shoji T, Akazome Y, Kanda T, Ikeda M. The toxicology and safety of apple polyphenol extract. Food Chem Toxicol. 2004;42:959–967. 18. Sabri M, Ai J, Knight B, et al.. Uncoupling of endothelial nitric oxide synthase after experimental subarachnoid hemorrhage. J Cereb Blood Flow Metab. 2011;31:190–199. 19. Kanaoka Y, Inagaki EI, Hamanaka S, Masaki H, Tanemoto K. Analysis of reactive oxygen metabolites (ROMs) after cardiovascular surgery as a marker of oxidative stress. Acta Med Okayama. 2010;64:323–330. 20. Dohi K, Satoh K, Ohtaki H, Shioda S, Miyake Y, Shindo M, et al.. Elevated plasma levels of bilirubin in patients with neurotrauma reflect its pathophysiological role in free radical scavenging. In Vivo. 2005;19:855–860. 21. Yoshida A, Watanabe K, Iwasaki A, Kimura C, Matsushita H, Wakatsuki A. Placental oxidative stress and maternal endothelial function in pregnant women with normotensive fetal growth restriction. J Matern Neonatal Med. 2018;31:1051–1057.
Please cite this article as: M. Naraoka, Y. Li, T. Katagai et al., Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model, Brain Hemorrhages, https://doi.org/10.1016/j.hest.2019.12.006
Contents lists available at ScienceDirect
Brain Hemorrhages
CHINESE ROOTS GLOBAL IMPACT
journal homepage: www.keaipublishing.com/en/journals/brain-hemorrhages/
Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model Masato Naraoka ⇑, Yuchen Li, Takeshi Katagai, Hiroki Ohkuma Department of Neurosurgery, Hirosaki University School of Medicine & Hospital, Hirosaki, Japan
a r t i c l e
i n f o
Article history: Received 19 December 2019 Accepted 19 December 2019 Available online xxxx Keywords: Subarachnoid hemorrhage Cerebral vasospasm Oxidative stress Apple polyphenols
a b s t r a c t Subarachnoid hemorrhage (SAH) is almost always caused by ruptured cerebral aneurysms, and the most serious complication after SAH is cerebral vasospasm. Oxidative stress due to free radicals released from subarachnoid hemorrhage clots causes cerebral vasospasm, as proven by animal experiments. Apple polyphenols have already been demonstrated to strongly suppress oxidized low-density lipoprotein (ox-LDL) and lectin-like oxidized LDL receptor-1 (LOX-1) derived from reactive oxygen species (ROS), and to improve cerebral vasospasm. However, it is unclear how apple polyphenol acts downstream of ROS. The purpose of this study is to reveal a more detailed mechanism of functioning for apple polyphenols in suppressing oxidative stress and cerebral vasospasm. Using the double-hemorrhage rabbit subarachnoid hemorrhage (SAH) model, we investigated the effect of apple polyphenols by measuring the basilar artery diameter, endothelial NO synthase (eNOS) expression, diacron reactive oxygen metabolites (d-ROM), biological antioxidant potential (BAP), and malondialdehyde (MDA). The mean diameter of the basilar artery in the apple polyphenol treatment group was significantly larger than in the SAH group (p < 0.01). The expression of endothelial nitric oxide synthase (eNOS), evaluated by immunohistochemistry in the apple polyphenol treatment group, was higher than in the SAH group (p < 0.05). The BAP test in the apple polyphenol treatment group showed a significant difference, while there was no significant difference in the values of d-ROM and MDA. The results of the present study revealed that apple polyphenol significantly improved cerebral vasospasm and eNOS. In spite of a significant increase in antioxidant power expressed by increased BAP, oxidative stress measured by the d-ROM value was not significantly suppressed. Either the number of experimental animals was not sufficient to reach statistical significance, or the eNOS increase due to apple polyphenols and suppression of vasospasm are attributable to pathways other than lipid oxidation detected by MDA. Ó 2020 Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Subarachnoid hemorrhage (SAH) is almost always caused by a ruptured cerebral aneurysm, with cerebral vasospasm being one of the serious, subsequent complications.1–4 Cerebral vasospasm is thought to be caused by the continuous contraction of smooth muscle in main cerebral arteries at 4- to 14 days after the onset of SAH. This contraction results in cerebral ischemia and cerebral infarction, which can cause severe neurological deficits.4,5 Many vasoactive substances derived from subarachnoid blood clots have been implicated, including free radicals, histamine, norepinephrine, oxidized hemoglobin, prostaglandins, thrombin, IL-6 ⇑ Corresponding author at: Department of Neurosurgery, Hirosaki University School of Medicine, 5-Zaihuchou, Hirosaki Aomori pref 036-8562, Japan. Fax: +81 172 39 5116. E-mail address: [email protected] (M. Naraoka).
etc.6 Among these, free radicals are considered to be the main substances causing cerebral vasospasm. In this regard, free-radical scavengers and antioxidants have been used in an attempt to prevent and treat cerebral vasospasm after SAH. We also recently administered apple polyphenols as an antioxidant to an experimental subarachnoid hemorrhage model.7 In particular, we focused on the procyanidins, the main active antioxidant components in apple polyphenols, which inhibit oxidized low-density lipoprotein (ox-LDL) and lectin-like oxidized LDL receptor-1 (LOX-1) from reactive oxygen species (ROS). As a result, we have demonstrated that apple polyphenols suppresses ox-LDL and LOX-1, improve endothelial NO synthase (eNOS) expression and cerebral vasospasm. Therefore, the local antioxidant effect of apple polyphenols on cerebral arteries has been demonstrated through a specific pathway. However, the antioxidative effects and the suppression of oxidative stress and lipid
https://doi.org/10.1016/j.hest.2019.12.006 2589-238X/Ó 2020 Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: M. Naraoka, Y. Li, T. Katagai et al., Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model, Brain Hemorrhages, https://doi.org/10.1016/j.hest.2019.12.006
2
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peroxidation on a systemic basis that includes the mechanisms have not been clarified. In the present study, in order to evaluate the actions of apple polyphenols, the marker diacron reactive oxygen metabolites (dROM), the biological antioxidant potential (BAP), and malondialdehyde (MDA) were measured using a rabbit SAH model. 2. Materials and methods This experimentation was approved by the Animal Experimentation Ethics Committee of Hirosaki University School of Medicine. Japanese white rabbits were used. All rabbits were maintained under standard conditions (temperature of 25 ± 2 °C, 12-hour light/dark cycle), and were given ad libitum access to food and water. The animals were assigned randomly to two groups; the SAH group (n = 10) and the apple polyphenol treatment group (n = 10). Experimental SAH was produced according to the twohemorrhage method as previously described.8 In the apple polyphenol treatment group, after the SAH was produced by the same procedure as in the SAH group, apple polyphenol (50 mg/ kg) was administered orally once a day from day 0 to day 3. The peripheral blood samples were collected from the auricular vein on day 0 and day 4 for measuring d-ROM and for the BAP test. On day 4, perfusion-fixation was performed under deep anesthesia for immunohistochemical and histological evaluation of the basilar arteries. The other five rabbits in both group were euthanized under deep anesthesia without perfusion-fixation and the basilar arteries were removed for protein analysis of eNOS and MDA by ELISA. The d-ROM test provides a measure for oxidative stress in peripheral blood by evaluating the level of reactive oxygen metabolites, mainly measured as hydroperoxidase. It proved to be a reliable and effective tool for readily evaluating oxidative stress in both humans and animals.9–11 The BAP test assesses antioxidant power.12,13 This test evaluates the blood concentration of antioxidants as agents that can reduce the ferric form (Fe3+) to the ferrous (Fe2+). MDA is one of the lipid peroxidative degradation products that is widely used as a major marker of lipid peroxidation, because polyunsaturated fatty acids (PUFA) are oxidized by reactive oxygen species (ROS) and free radicals to form MDA as the final product (Fig. 1).11,14–16 2.1. Induction of SAH Experimental SAH was produced according to the two hemorrhage method as previously described.8 On the first day (day 0), the rabbits were anesthetized with an intravenous injection of pentobarbital (30 mg/kg) and an intramuscular injection of ketamine (20 mg/kg). After anesthesia and under spontaneous breathing a 23-gauge butterfly needle was inserted percutaneously into
the cisterna magna. After aspiration of 1.0 ml cerebrospinal fluid, the same amount of nonheparinized arterial blood from the femoral artery was slowly injected into the cisterna magna for 1 min under aseptic technique. Rabbits were then placed in a 30° head-down position for 30 min. After recovery from anesthesia, they were returned to the feeding room. Forty-eight hours afterwards (day 2), the second experimental SAH was performed in the same manner as the first. 2.2. Administration of apple polyphenols In the apple polyphenol treatment group, apple polyphenols (50 mg/kg, Asahi Food and Healthcare Co., LTD) were orally administered once a day for 4 days from day 0 to day 3. Polyphenols contain 63.8% procyanidins, which are widely used as an antioxidant.17 2.3. Perfusion fixation Perfusion fixation was performed on day 4 under deep anesthesia with high-dose intravenous pentobarbital injection (300 mg/ kg) and an intramuscular ketamine injection (100 mg). The thoracotomy was performed and a cannula was immediately inserted through the left ventricle into the ascending aorta. The descending aorta was then clamped and the right atrium opened. Perfusionfixation started with 500 ml saline injected with heparin, followed by 500 ml 4% paraformaldehyde (pH 7.4) under a perfusion pressure of 75 mm Hg. After perfusion fixation, the whole brain was excised with the basilar artery. 2.4. Measurement of basilar artery cross-sectional area A tissue specimen was prepared by hematoxylin-eosin (HE) staining of the fixed basilar artery. The degree of cerebral vasospasm was evaluated by measuring the basilar artery lumen cross-sectional area and converting it to a diameter. Paraffinembedded basilar artery sections (thickness 6 lm) were HE stained and the basilar artery cross-sectional area was calculated using ImageJÒ version 1.45 (NIH). The cross-sectional area was calculated from the perimeter of the luminal border, while the area included in the boundary of the inner elastic plate was ignored. For each basilar artery, three sequential sections were calculated proximally, intermediately, and distal, and then converted into the vessel diameter. Diameter mean ± SEM values obtained for each artery were used for analysis. 2.5. Immunostaining of eNOS Immunostaining of eNOS was performed on the fixed basilar artery. eNOS expressed in the cerebral vascular epithelium was thereby evaluated.18 Immunohistochemistry was performed on paraffin-embedded sections to determine the immunoreactivity of eNOS. Sections were deparaffinized and rehydrated in graded concentrations of ethanol to distilled water. Sections were incubated with primary anti-eNOS antibody (1:200 dilution, BD Biosciences) for 1 h at room temperature, followed by a 15-min wash in PBS. Sections were incubated with goat anti-rabbit IgG (1:500 dilution) for 60 min at room temperature. Diaminobenzidine was used as the chromogen and counterstaining was performed with hematoxylin. 2.6. Measurement of d-ROM and the BAP test
Fig. 1. Outline of oxidative stress factors and measurement markers.
The d-ROM measurement and the BAP test were done with venous blood at day 0 and day 4 using a free radical analyzer (Free carrio duo, Wismerll Co., LTD).9,19,20
Please cite this article as: M. Naraoka, Y. Li, T. Katagai et al., Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model, Brain Hemorrhages, https://doi.org/10.1016/j.hest.2019.12.006
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The d-ROM test was measured by mixing 20 ml of collected venous blood with an acetic acid buffer solution (pH 4.8) to stabilize the hydrogen ion concentration. In the acid buffer solution, bivalent and trivalent iron from blood ionized to form a catalyst that breaks down the hydroperoxidase groups in the blood to become free radicals. The blood samples were then transferred into a cuvette containing colorless chromogen (N. N. diethylparaphenylenediamine), which is oxidized by free radicals and changes into a radical cation with a magenta color. The density of the magenta color, which was measured using a photometer (505 nm), is proportional to the quantity of ROMs. The d-ROM test measured the concentrations of various hydroperoxidase in the blood, and the results were expressed in arbitrary units called ‘‘Carratelli units” (U.CARR). The BAP test was performed at the same time. The antioxidant potential of blood plasma was evaluated by measuring the degree of reduction of the ferric ion (Fe3+) to the ferrous form (Fe2+) as a result of antioxidant action. In practice, 100 ml of blood are centrifuged for 90 s, after which the amount of trivalent iron that is deoxidized in 5 min is measured in units of mmol/l.
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Fig. 2. Histological findings for the basilar artery. Left: Photomicrographs of representative sections from the SAH group, Right: the apple polyphenol treatment group. The both sections show the enlarged section of the vessel wall below. Scale bar = 200 lm.
2.7. Measurement of eNOS and MDA by ELISA After removing the subarachnoid hematoma on the surface of the extracted basilar artery, the vessel was ultrasonically disrupted using a buffer. Centrifugation was performed at 2000g for 10 min, and the separated supernatant was further centrifuged at 15,000g at 4 °C for 15 min. Protein concentration was measured by a standard microplate assay using a protein assay kit (5000002JA, BIO-RAD). After correcting to the protein concentration of each group, the concentration was adjusted to a constant value and measured again for confirmation. eNOS was quantitatively evaluated by ELISA (ser1176, Tebu-bio CO., LTD). MDA was performed according to the protocol using an ELISA kit manufactured by Cell Biolabs, Inc.
2.8. Statistical analysis Analysis of results was performed for the SAH group and the treatment group for all measurements. Statistical analyses were performed using the JMPÒ (Version 11; SAS Institute Inc., Cary, NC). Data are expressed as mean ± SEM. In order to compare the two groups expressed as the mean ± SEM, normal distribution is not paired; we used the Mann–Whitney U test, significance determined at p < 0.05.
Fig. 3. Immunohistochemical findings of eNOS for the basilar artery. Left: Photomicrographs of representative sections from the SAH group, Right: the apple polyphenol treatment group. The area enclosed by the square on the top is enlarged below. Arrowheads indicate eNOS staining. Scale bar = 50 lm.
3. Results 3.1. Evaluation of cerebral vasospasm The cross sections of the basilar artery in both groups, determined by staining with HE staining and observed under the light microscope, are shown in Fig. 2. The mean diameter of the basilar artery was 0.53 ± 0.13 mm for the SAH group and 1.55 ± 0.27 mm for the apple polyphenol group (p < 0.01).
3.2. Evaluation of eNOS on the basilar artery The results of immunostaining of eNOS on the tissue of the basilar artery of both groups are shown in Fig. 3. It was confirmed that more eNOS was expressed in the apple polyphenol group. Furthermore, a significant difference could be confirmed quantitatively by ELISA (p < 0.05, Fig. 4).
Fig. 4. Expression of eNOS was significantly improved in the vascular smooth muscle cells by ELISA.
Please cite this article as: M. Naraoka, Y. Li, T. Katagai et al., Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model, Brain Hemorrhages, https://doi.org/10.1016/j.hest.2019.12.006
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Fig. 5. The result of the BAP test on day 0 and day 4. There was no significant difference between the SAH group and the polyphenol group, but there was a significant difference between the two groups on day 4.
Fig. 6. The result of the d-ROM test on day 0 and day 4. Significant differences were observed within each group of SAH group and the polyphenol group. 1 CARRU = 0.08 mg H2O2/dL.
3.3. Evaluation of chemical markers associated with oxidative stress The result of the BAP test is shown in Fig. 5. On Day 4, a significant difference was observed in the apple polyphenol group (3138 ± 172.3 lmol/L) compared to the SAH group (2845 ± 193.6 lmol/L). There was no significant difference between Day 0 and Day 4 within each group. On the other hand, the value of d-ROM increased significantly in both groups after SAH production (Fig. 6). The result of the d-ROM test in the apple polyphenol group was suppressed (412 ± 102.6 CARRU) compared to that of the SAH group (492 ± 82.3 CARRU); however, there was no significant difference between these groups. The results of MDA are shown in Fig. 7. Although MDA was lower in the treatment group than in the SAH group, there was no significant difference. 4. Discussion The results of the present study revealed that apple polyphenols significantly improved cerebral vasospasm and eNOS, which was comparable to the result of our previous study. The effect was considered to be attributable to the antioxidant action of apple
Fig. 7. Expression of MDA was significantly improved in the vascular smooth muscle cells by ELISA.
Please cite this article as: M. Naraoka, Y. Li, T. Katagai et al., Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model, Brain Hemorrhages, https://doi.org/10.1016/j.hest.2019.12.006
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polyphenol, because the BAP test, which is an index of antioxidant power, was significantly increased in the apple polyphenol treatment group. In another report, it was proven that a BAP increase in neonatal blood was induced by the administration of lutein, which is a type of carotenoid that exhibits antioxidant activity.21 In spite of a significant increase in antioxidant power expressed by increased BAP, oxidative stress measured by d-ROM values was not significantly suppressed. One of the possibilities may be insufficient dosage of apple polyphenols, which can increase antioxidant action but not decrease oxidative stress significantly. Another possibilities is that oxidative stress in the blood may be influenced by not only a local event such as SAH, but also by a systemic event such as anesthesia. Suppressed oxidative stress at the local SAH by apple polyphenol could therefore be masked by the systemic generation of oxidative stress. In our previous experimentation, and in this study, the decrease in eNOS by the blood vessel wall was suppressed, which suggested lipid peroxidation of the blood vessel wall was suppressed, but MDA changes in the present study could not entirely support this view, as MDA, an index of local lipid peroxidation, showed a mild decrease from the administration of apple polyphenols without statistically significance compared to the SAH group. The number of experimental animals was not sufficient to reach statistical significance, or the eNOS increase due to apple polyphenols and suppression of vasospasm are attributable to pathways other than lipid oxidation detected by MDA. Other markers of oxidative stress are known, including 8-OHdG, which is an indicator of DNA damage, and nitrosothiols and 3-NT, which are indicators of protein damage. In the future, we plan to elucidate the effects and mechanisms of antioxidants, including apple polyphenols, by adding these marker measurements and increasing the number of animals. Conflict of interest The authors declare no competing financial interests. References 1. Ecker A, Riemenschneiderr PA. Arteriographic demonstration of spasm of the intracranial arteries, with special reference to saccular arterial aneurysms. J Neurosurg. 1951;8:660–667.
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Please cite this article as: M. Naraoka, Y. Li, T. Katagai et al., Effects of apple polyphenols on oxidative stress and cerebral vasospasm after subarachnoid hemorrhage in a rabbit double hemorrhage model, Brain Hemorrhages, https://doi.org/10.1016/j.hest.2019.12.006