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Eicosapentaenoic Acid Prevents Memory Impairment After Ischemia by Inhibiting Inflammatory Response and Oxidative Damage
Eicosapentaenoic Acid Prevents Memory Impairment After Ischemia by Inhibiting Inflammatory Response and Oxidative Damage Naohiko Okabe, MD,* Takehiro Nakamura, MD, PhD,* Tetsuhiko Toyoshima, PhD,* Osamu Miyamoto, MD, PhD,† Feng Lu, MD,* and Toshifumi Itano, PhD*
Previous studies have demonstrated that the generation of reactive oxygen species and an excessive inflammatory reaction are involved in the progression of neural damage following brain ischemia. In this study, we focused on the anti-inflammatory and antioxidant properties of eicosapentaenoic acid (EPA). Gerbils were treated intraperitoneally with 500 mg/kg of EPA ethyl for 4 weeks until the day of forebrain ischemia, which was induced by occluding the bilateral common carotid artery for 5 minutes. In the first part of the 2-part experiment, the effect of EPA treatment was evaluated using hematoxylin and eosin staining and deoxynucleotidyl transferase–mediated dUTP nick-end labeling as a marker of cell death (n 5 3 per group). The inflammatory reaction was evaluated using anti-Iba1 immunohistochemistry, a marker of microglial activation (n 5 3 per group), and detection of 8-hydroxyl-20 -deoxyguanosine, a marker of oxidative DNA damage (n 5 4 per group). In the second part of the experiment, the effect of EPA treatment on memory function was examined using an 8-arm radial maze (n 5 6 per group). EPA treatment significantly inhibited DNA oxidative damage (P ,.05) and accumulation of Iba1-positive cells in the CA1 area at 12 and 72 hours after the induction of ischemia, and also decreased apoptotic neurons and neuronal death (P , .001) at 72 hours after ischemia. EPA treatment also significantly improved memory function (P ,.05). These findings suggest that EPA inhibits the inflammatory reaction and oxidative damage occurring after ischemic brain injury, and also may contribute to the prevention of neural damage and memory impairment following such injury. Key Words: Eicosapentaenoic acid—memory function—forebrain ischemia— inflammation—oxidation—gerbil. Ó 2011 by National Stroke Association
Cerebral ischemia or stroke results in irreversible brain damage and subsequent neuropsychological dysfunction, including learning and memory impairment.1,2 A series From the *Department of Neurobiology, Kagawa University Faculty of Medicine, Kagawa, Japan; and †Department of Physiology, Kawasaki Medical University, Kurashiki, Japan. Received February 3, 2009; accepted July 14, 2009. Supported by a Grant-in-Aid for Scientific Research from the Japanese Society for Promotion of Science. Address correspondence to Takehiro Nakamura, MD, PhD, Department of Neurobiology, Kagawa University Faculty of Medicine, 17501 Ikenobe, Miki, Kagawa 761-0793, Japan. E-mail: [email protected]. jp. 1052-3057/$ - see front matter Ó 2011 by National Stroke Association doi:10.1016/j.jstrokecerebrovasdis.2009.11.016
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of events, including glutamate receptor activation, intracellular calcium overload, and reactive oxygen species (ROS) formation, are known to be involved in the pathogenesis of neuronal injury after brain ischemia. In addition, edema and the inflammatory response are known to exacerbate tissue destruction.3 Approaches to ameliorating ischemia-induced brain damage that have been investigated include interfering with the excitatory action of glutamate,4 preventing intracellular calcium overload,5 and scavenging ROS.6 Successful therapies have not yet been established, however.3 In this study, we focused on eicosapentaenoic acid (EPA), an n-3 essential fatty acid (EFA). Previous studies have shown that docosahexaenoic acid (DHA)7,8 and an oil mixture containing n-3 EFA9 provide protective
Journal of Stroke and Cerebrovascular Diseases, Vol. 20, No. 3 (May-June), 2011: pp 188-195
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effects following brain ischemia. Some studies also have suggested that EPA has antioxidant10 and antiinflammatory11,12 properties. In Japan, EPA has been used to treat hypercholesterolemia, a major risk factor for stroke.13 Furthermore, long-term administration of EPA has been demonstrated to improve cerebral blood flow and decrease mortality in stroke-prone spontaneously hypertensive rats.14 Thus, elucidation of the putative protective effects of EPA after brain ischemia may demonstrate a further advantage of EPA treatment. The purposes of the current study were to examine whether treatment with EPA ethyl (EPADEL S; Mochida Pharmaceutical, Tokyo, Japan) can reduce neuronal damage and behavioral deficits, and to investigate how EPA works on the brain after transient global ischemia in gerbils.
Materials and Methods Animal Preparation and Transient Forebrain Ischemia Animal protocols were approved by Kagawa University’s Animal Use Committee. A total of 48 male Mongolian gerbils (age 13-15 weeks; Kyudo, Saga, Japan), with a body weight of 60-80 g, were used for all experiments. The gerbils were given free access to food and water before the experiments. Transient forebrain ischemia was induced by a 5-minute bilateral common carotid artery occlusion, according to the following protocol. Gerbils were anesthetized with sodium pentobarbital (30 mg/kg intraperitoneally [IP]). After a medial neck incision, both common carotid arteries were isolated and occluded for 5 minutes using microaneurysm clips (Sugita Clip; Mizuho, Nagoya, Japan).15 Sham operated animals underwent the same surgical procedure except for the common carotid artery occlusion. During surgery, rectal temperature was controlled at 37-38 C with a feedback-controlled heating pad (CMA, Stockholm, Sweden) to prevent hypothermia. The rectal temperature was maintained for 30 minutes after recirculation, after which the gerbils was housed in a room at 24 C.
Experimental Groups The study was performed in 2 parts. Part 1 examined the effect of EPA on histological changes after ischemia and evaluated DNA oxidative damage using enzymelinked immunosorbent assay (ELISA). Thirty gerbils were divided randomly into 3 groups: the sham group, the Ischemia group, and the EPA4w 1 Ischemia group (n 5 10 per group). The EPA4w 1 Ischemia group was treated with undiluted EPA 500 mg/kg (0.55 mL/kg) IP once a day for 4 weeks until the day before induction of ischemia. The dose of EPA was determined based on the results of a preliminary study showing a maximal effect on a memory test (data not shown). The sham group and the Ischemia group were treated with a equivalent volume of saline solution (0.55 mL/kg) for 4 weeks to equalize the effect of IP injection.16,17
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Part 2 investigated the effect of EPA treatment on memory function. Eighteen gerbils were divided into 3 groups: the sham group, the Ischemia group, and the EPA4w 1 Ischemia group (n 5 6 per group). EPA and placebo control were administered as in part 1 of the study.
Histological Examination We examined neuronal damage using hematoxylin and eosin (HE) staining and deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) as a marker of cell death.18 We also evaluated the inflammatory response by anti-Iba1 immunohistochemistry.19,20 The gerbils (n 5 3 per group) were anesthetized with sodium pentobarbital (50 mg/kg IP) at 12 or 72 hours after induction of ischemia, then transcardially perfused with 4% phosphate-buffered paraformaldehyde after flushing with 10% phosphate-buffered saline (PBS). The brains were removed and placed in the same fixative overnight, and then immersed in 25% sucrose for 2 days. The brains were then placed in OCT embedding compound (Sakura Finetek USA, Torrance, CA) and sectioned (14 mm) on a cryostat. Sections in the CA1 area at 21.7 mm from the bregma (including the dorsal hippocampal area) were analyzed. In HE-stained sections, the number of viable neurons (cell/mm) was counted in the CA1 area.2 Immunohistochemistry was performed using the avidin-biotin complex method.20 After blocking of nonspecific binding with 1% albumin solution and washing in 0.01 M PBS, a monoclonal antibody against Iba1 (1:100 dilution; Wako, Osaka, Japan) was applied to sections overnight at 4 C. Sections were incubated with biotinylated goat anti-rabbit IgG (1:1000 dilution; Vector Laboratories, Burlingame, CA) for 90 minutes and then incubated with avidin-biotin horseradish peroxidase (Vector Laboratories) for 90 minutes.21 A molecular histochemical system (Apoptosis In Situ Detection Kit; Wako) was used for specific staining of DNA fragmentation (ie, TUNEL staining).22 This method is based on the specific binding of terminal deoxynucleotidyl transferase (TdT) to the 30 -OH ends of DNA. In brief, sections were permeabilized with 0.1% sodium citrate and 0.1% Triton X-100, the 30 DNA ends were labeled with TdT reaction solution, endogenous peroxidase activity was inactivated with 3% H2O2 in PBS, sections were labeled with peroxidase-conjugated antibody, and peroxidase was detected with diaminobenzidine. The labeling targets of this kit were the new 30 -OH DNA ends generated by DNA fragmentation.23
Assays for DNA Oxidative Damage DNA oxidative damage was investigated using an 8-hydroxyl- 20 -deoxyguanosine (8-OHdG) assay.24 Gerbils (n 5 4 per group) were reanaesthetized (50 mg/kg pentobarbital IP) and decapitated 12 hours after induction of ischemia. Then the brains were removed, and the
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Figure 1. (A) HE staining in the hippocampal CA1 area. Representative sections from the Ischemia group (top) and the EPA4w 1 Ischemia group (4 weeks of treatment) (bottom) 72 hours after indiction of ischemia. The left panels show lower magnification (scale bar: 500 mm); the right panels show higher magnification (scale bar: 50 mm). Neurons with shrunken cell bodies were rarely observed in the EPA 4w 1 Ischemia group. (B) Viable neurons per millimeter of the hippocampal CA1 area. n 5 3 per group. *P , .05 compared with the sham group; **P , .001 compared with the sham group; ##P , .001 compared with the Ischemia group. Viable neurons were significantly increased in the EPA 4w 1 Ischemia group compared with the Ischemia group.
hippocampal complex was preserved. DNA extraction was performed with a DNA isolation kit (Dojindo Molecular Technologies, Kumamoto, Japan). This method, in which purified DNA is isolated from brain tissue using the guanidine method and RNase A and proteinase K, avoids the use of phenol and heating that might induce background.25 8-OHdG levels were evaluated by ELISA (Japan Institute for the Control of Aging, Shizuoka, Japan), which can measure extremely low levels of 8-OHdG. The specificity of the monoclonal antibody was established. The optical density of the wells was measured at 450 nm. The data, expressed as nanograms of 8-OHdG per microgram of DNA, were calculated on the basis of a linear calibration curve generated for each experiment using 8-OHdG standard solutions.25
Memory Test The 8-arm radial maze was used to assess memory function. We used a technique similar to that reported by
Miyoshi et al.26 For the spatial memory task, the central part of the maze was 22 cm in diameter, and the arms were 25 cm long, 6 cm high, and 6 cm wide, made of transparent Plexiglass. The maze was always oriented in the same direction in space. The test was started at 7 days after the operation. Gerbils (n 5 6 per group) were tested on 4 consecutive days, with 1 trial conducted per day. A small food pellet was placed at the end of each arm, and the animal was placed in the central part of the maze. An error was noted if the animal entered an arm that it had visited previously. The number of errors was recorded. The trial ended when all of the pellets had been eaten.
Statistics Data are expressed as mean 6 standard deviation. The differences in the histological data and ELISA data were analyzed for significance by one-way ANOVA followed by Scheffe’s post hoc test. Two-way repeated ANOVA
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Figure 2. Immunohistochemical staining for Iba1 in the hippocampal CA1 area in representative sections. The left panels show lower magnification (scale bar: 500 mm); the right panels show higher magnification (scale bar: 100 mm). The intensity of Iba1 immunoreactivity was induced at 12 hours and increased until 72 hours after induction of ischemia in each group. Iba1-positive cells were more weakly stained in the EPA 4w 1 Ischemia group compared with the Ischemia group.
was used to compute the behavioral data, and the differences among the groups were evaluated for significance using Scheffe’s post hoc test.2
Results Administration of EPA or placebo control caused no medical problems in the animals. Activity and appetite was maintained in all groups, and body weight did not differ significantly among the groups. Body temperature during the surgical procedure was well controlled in all groups.
Effects of EPA Treatment on Histological Changes Examination of HE-stained sections revealed no significant changes in the Ischemia group or the EPA4w 1 Ischemia group at 12 hours after ischemia. However, in the Ischemia group, marked neuronal damage, along with pyknosis and eosinophilia in CA1 pyramidal neurons, were seen at 72 hours after ischemia (Fig 1A). In contrast, sections from the EPA4w 1 Ischemia group showed some eosinophilic cells, but very few karyopyknotic cells. EPA treatment significantly reduced hippocampal neuronal loss (P , .001) (Fig 1B).
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Figure 3. TUNEL in the hippocampal CA1 area. Representative sections from the Ischemia group 12 hours (upper left) and 72 hours (lower left) after ischemia and from the EPA4w 1 Ischemia group 12 hours (upper right) and 72 hours (lower right) after ischemia. (Scale bar: 50 mm.) Apoptotic neurons were observed only in the Ischemia group at 72 h after ischemia.
Figure 2 shows photomicrographs of Iba1 immunohistochemical staining in the hippocampus of the Ischemia group and the EPA4w 1 Ischemia group at 12 hours and 72 hours after ischemia. Both the Ischemia group and the EPA4w 1 Ischemia group showed an increased number of Iba1-positive cells over time, which exhibited marked hypertrophy. Iba1-positive cells tended to gather in and around the CA1 area, especially in a gerbil from the Ischemia group at 72 hours after ischemia. The EPA4w 1 Ischemia group demonstrated less immunostaining than the Ischemia group at each time point examined. Using the TUNEL method, we observed the staining of many hippocampal neurons in tissue sections from animals in the Ischemia group 72 hours after ischemia. Tissue sections from the other groups showed no TUNEL-positive neurons (Fig 3).
groups, and transient forebrain ischemia significantly increased the number of errors (P , .01). The number of errors was significantly lower in the EPA4w 1 Ischemia group compared with the Ischemia group (P , .05).
Discussion Several previous studies have demonstrated the protective effect of DHA and an oil mixture containing n-3 EFAs after brain ischemia, resulting in improved neuronal function. In this study, we investigated the effect of pure EPA, rather than DHA or the n-3 EFA-containing oil. EPA is a well-known antithrombotic drug27 and cholesterol-lowering agent,28 but it has been less well investigated than DHA7,8 or the n-3 EFA oil mixture9 for use
Effect of EPA Treatment on Oxidative DNA Damage Figure 4 shows the level of the DNA oxidation marker 8-OHdG at 12 hours after induction of ischemia. The Ischemia group had a significantly higher 8-OHdG level compared with the sham group (P , .01) and a higher level compared with the EPA4 W 1 Ischemia group (P , .05).
Effect of EPA on Memory Function Figure 5 shows the number of errors made in the 8-arm radial maze. Two-way repeated ANOVA revealed significant major effects of groups (F2,45 5 8.093; P , .01) and trials (F3,45 5 44.895; P , .0001). There was no interaction between the groups and trials (F6,60 5 1.225; P . .05). The number of errors tended to decrease with time in all
Figure 4. 8-OHdG levels in the hippocampus measured by ELISA; n 5 3 per group. **P ,.01 compared with the sham group; #P ,.05 compared with the Ischemia group. The EPA 4w 1 Ischemia group had a significantly lower 8-OHdG level than the Ischemia group.
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oxide, and additional ROS, which disrupt the blood-brain barrier and induce brain edema and additional cell death.32 EPA also modulate arteriolar reactivity, which is normally controlled by cyclooxygenase (COX) metabolism and formation of oxygen free radicals,33 improving cerebral blood flow following transient forebrain ischemia.34 Actually, it has been shown that reduction of PG or LT levels by COX or lipoxygenase inhibitors led to reduced brain damage, including lipid oxidation, cell death, and brain edema.35,36 These findings suggest that EPA treatment may reduce deleterious inflammation and attenuate further inflammatory reaction, tissue damage, and subsequent apoptosis and cell death by inhibiting eicosanoid production.
Effect of EPA Treatment on Oxidative DNA Damage
Figure 5. Performance in the 8-arm radial maze trial (number of errors); n 5 6 per group. **P , .01 compared with the sham group; #P , .05 compared with the Ischemia group. The EPA4w 1 Ischemia group demonstrated significantly improved memory function compared with the Ischemia group.
after brain ischemia. Our findings suggest that EPA decreases oxidative damage, the inflammatory response, and subsequent apoptosis and cell death and also may improve memory function, similar to the previously reported effects of DHA.
Effect of EPA Treatment on Cell Death and the Inflammatory Response Histological investigation revealed that EPA treatment significantly decreased the number of microglia at both 12 hours and 72 hours, the number of dead or damaged neurons, and the number of apoptotic cells at 72 hours in the CA1 area after the induction of ischemia. These results suggest that EPA treatment inhibited cell death and apoptosis and acutely inhibited inflammation in the CA1 area. It is important to note that EPA treatment inhibited the pathology associated with forebrain ischemia at 12 hour, at which point cell death and apoptosis had not yet occurred. Previous studies have found that omega-3 fatty acids, including EPA, have immunomodulative effects on the synthesis of the arachidonic acid–derived eicosanoid mediators prostaglandins (PGs) and leukotrienes (LTs).29 A recent study showed that DHA reduced eicosanoid production following transient forebrain ischemia in gerbils.30 Eicosanoids are potent chemoattractants for inflammatory cells, including microglia,31 and inflammatory cells can release various cytotoxic agents, including cytokines, matrix metalloproteinases, nitric
TUNEL staining demonstrated that EPA treatment completely prevented DNA fragmentation in the hippocampus at 72 hours after ischemia. However, HE staining at the same time showed a 70% decrease in viable neurons in the CA1 area compared with the sham group. These findings suggest that EPA treatment may affect apoptosis, but not necrosis. The 8-OHdG assay at 12 hours after ischemia revealed a dramatically increased 8-OHdG level in the Ischemia group, but a reduced level in the EPA4w 1 Ischemia group to near the level seen in the sham group. Because the 8-OHdG level reveals DNA strand breaks produced by interactions between DNA and ROS,24 a decreased 8-OHdG level suggests that EPA treatment decreases oxidative DNA damage. Previous studies have shown n-3 EFA treatment enhances the activity of antioxidant enzymes such as glutathione peroxidase, superoxide dismutase, and catalase after transient brain ischemia,7-9 and that augmenting the activity of these antioxidant enzymes by EPA might play a pivotal role in protection against ROS. But because ROS levels are determined by a balance between the amount of antioxidant enzymes and the generation of ROS, EPA’s antiinflammatory properties may be due to a reduction in free oxygen radical generation, leading to attenuation of DNA oxidation.
Effect of EPA Treatment on Memory Function and Treatment Duration In all groups, the number of errors made in the 8-arm radial maze was highest on day 1 and declined sharply on day 2, followed by a gradual decrease thereafter. This 2-stage reduction in the number of errors likely reflects 2 forms of brain function. Because the gerbils did not undergo a training period in this study, our findings may reflect task acquisition or learning in addition to working memory.1 This result also suggests that learning ability was maintained in the Ischemia group. However, 2-way repeated ANOVA revealed a significant increase
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in the number of errors in the Ischemia group compared with the sham group or the EPA4w 1 Ischemia group, suggesting significant memory impairment in the Ischemia group and improvement in the EPA4w 1 Ischemia group. Although gerbils treated with EPA for 4 weeks exhibited improved memory function, gerbils treated for 1 week did not demonstrate a significant decrease in the number of errors in the preliminary experiment (data not shown). This suggests that EPA treatment provides benefits in a time-dependent manner. Pharmacokinetic studies have shown that EPA requires a long time to reach a steady-state concentration in the brain, necessitating repeated administration.37 Thus, the tissue concentration may have been insufficient after just 1 week of EPA treatment, and this delayed distribution to the tissues may explain the ineffectiveness of short-term preischemic or postischemic n-3 EFA treatment.38,39 These findings suggest that EPA may not be suitable for emergency care; however, because EPA has a long half-life,37 intermittent administration after achievement of steady-state may be effective. In conclusion, our findings suggest that EPA treatment may reduce oxidative DNA damage and subsequent apoptosis and cell death, resulting in improved memory function. These protective effects may be due to EPA’s anti-inflammatory properties. Thus, EPA may be useful not only in reducing the risk of brain ischemic events by lowering cholesterol, but also in preventing neuronal damage after brain ischemia.
References 1. Amano M, Hasegawa M, Hasegawa T, et al. Characteristics of transient cerebral ischemia–induced deficits on various learning and memory tasks in male Mongolian gerbils. Jpn J Pharmacol 1993;63:469-477. 2. Nakamura T, Miyamoto O, Yamagami S, et al. The chronic cell death with DNA fragmentation after post-ischaemic hypothermia in the gerbil hippocampus. Acta Neurochir 1999;141:407-412. 3. Juurlink BH, Sweeney MI. Mechanisms that result in damage during and following cerebral ischemia. Neurosci Biobehav Rev 1997;21:121-128. 4. McCulloch J. Excitatory amino acid antagonists and their potential for the treatment of ischaemic brain damage in man. Br J Clin Pharmacol 1992;34:106-114. 5. Barone FC, Price WJ, Jakobsen P, et al. Pharmacological profile of a novel neuronal calcium channel blocker includes reduced cerebral damage and neurological deficits in rat focal ischemia. Pharmacol Biochem Behav 1994;48:77-85. 6. Slemmer JE, Shacka JJ, Sweeney MI, et al. Antioxidants and free radical scavengers for the treatment of stroke, traumatic brain injury and aging. Curr Med Chem 2008; 15:404-414. 7. Pan HC, Kao TK, Ou YC, et al. Protective effect of docosahexaenoic acid against brain injury in ischemic rats. J Nutr Biochem 2009;20:715-725. 8. Cao DH, Xu JF, Xue RH, et al. Protective effect of chronic ethyl docosahexaenoate administration on brain injury in ischemic gerbils. Pharmacol Biochem Behav 2004; 79:651-659.
N. OKABE ET AL. 9. Bas O, Songur A, Sahin O, et al. The protective effect of fish n-3 fatty acids on cerebral ischemia in rat hippocampus. Neurochem Int 2007;50:548-554. 10. Nishida T, Morita N, Yano Y, et al. The antioxidative function of eicosapentaenoic acid in a marine bacterium, Shewanella marinintestina IK-1. FEBS Lett 2007;581:4212-4216. 11. Choi-Kwon S, Park KA, Lee HJ, et al. Temporal changes in cerebral antioxidant enzyme activities after ischemia and reperfusion in a rat focal brain ischemia model: Effect of dietary fish oil. Dev Brain Res 2004;152:11-18. 12. Rees D, Miles EA, Banerjee T, et al. Dose-related effects of eicosapentaenoic acid on innate immune function in healthy humans: A comparison of young and older men. Am J Clin Nutr 2006;83:331-342. 13. Leys D, Deplanque D, Lucas C, et al. Hypolipemic agents for stroke prevention. Clin Exp Hypertens 2002;24:573-594. 14. Kashiwagi F, Katayama Y, Memezawa H, et al. Effect of long-term administration of eicosapentaenoic acid (EPA) on survival, lipid metabolism, endothelium of middle cerebral artery (MCA), and experimental cerebral ischemia in stroke-prone spontaneously hypertensive rats (SHRSP). Jpn J Stroke 1990;12:326-333 (in Japanese). 15. Toyoshima T, Yamagami S, Ahmed BY, et al. Expression of calbindin-D28K by reactive astrocytes in gerbil hippocampus after ischaemia. Neuroreport 1996;7:2087-2091. 16. Black KL, Hsu S, Radin NS, et al. Effect of intravenous eicosapentaenoic acid on cerebral blood flow, edema and brain prostaglandins in ischemic gerbils. Prostaglandins 1984;28:545-556. 17. Katayama Y, Katsumata T, Muramatsu H, et al. Effect of long-term administration of ethyl eicosapentate (EPA-E) on local cerebral blood flow and glucose utilization in stroke-prone spontaneously hypertensive rats (SHRSP). Brain Res 1997;761:300-305. 18. Snider BJ, Gottron FJ, Choi DW. Apoptosis and necrosis in cerebrovascular disease. Ann NY Acad Sci 1999; 893:243-253. 19. Ito D, Imai Y, Ohsawa K, et al. Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res Mol Brain Res 1998;57:1-9. 20. Stoll G, Jander S, Schroeter M. Inflammation and glial responses in ischemic brain lesions. Prog Neurobiol 1998; 56:149-171. 21. Wu D, Miyamoto O, Shibuya S, et al. Co-expression of radial glial marker in madrophages/microglia in rat spinal cord contusion injury model. Brain Res 2005;1051:183-188. 22. Xu RX, Nakamura T, Nagao S, et al. Specific inhibition of apoptosis after cold-induced brain injury by moderate postinjury hypothermia. Neurosurgery 1998;43:107-114. 23. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119:493-501. 24. Liu PK, Hsu CY, Dizdaroglu M, et al. Damage, repair, and mutagenesis in nuclear genes after mouse forebrain ischemia-reperfusion. J Neurosci 1996;16:6795-6806. 25. Nakamura T, Kuroda Y, Yamashita S, et al. Edaravone attenuates brain edema and neurologic deficits in a rat model of acute intracerebral hemorrhage. Stroke 2008; 39:463-469. 26. Miyoshi K, Itano T, Enomoto S, et al. The role of corpus callosum in spatial memory. Neuroscience 1995;21:1-5. 27. Wojenski CM, Silver MJ, Walker J. Eicosapentaenoic acid ethyl ester as an antithrombotic agent: Comparison to an extract of fish oil. Biochim Biophys Acta 1991;1081:33-38. 28. Mizuguchi K, Yano T, Tanaka Y, et al. Mechanism of the lipid-lowering effect of ethyl all-cis-5,8,11,14,17-icosapentaenoate. Eur J Pharmacol 1993;231:121-127.
EPA FOR FOREBRAIN ISCHEMIA 29. Calder PC. Immunomodulation by omega-3 fatty acids. Prostaglandins Leukot Essent Fatty Acids 2007; 77:327-335. 30. Cao D, Zhou C, Sun L, et al. Chronic administration of ethyl docosahexaenoate reduces gerbil brain eicosanoid productions following ischemia and reperfusion. J Nutr Biochem 2006;17:234-241. 31. Cimino PJ, Keene CD, Breyer RM, et al. Therapeutic targets in prostaglandin E2 signaling for neurologic disease. Curr Med Chem 2008;15:1863-1869. 32. Wang Q, Tang XN, Yenari MA. The inflammatory response in stroke. J Neuroimmunol 2007;184:53-68. 33. Ellis EF, Police RJ, Dodson LY, et al. Effect of dietary n-3 fatty acids on cerebral microcirculation. Am J Physiol 1992;262:H1379-H1386. 34. Black KL, Hoff JT, Radin NS, et al. Eicosapentaenoic acid: Effect on brain prostaglandins, cerebral blood flow and edema in ischemic gerbils. Stroke 1984;15:65-69.
195 35. Candelario-Jalil E, Gonza´lez-Falco´n A, Garcı´a-Cabrera M, et al. Assessment of the relative contribution of COX-1 and COX-2 isoforms to ischemia-induced oxidative damage and neurodegeneration following transient global cerebral ischemia. J Neurochem 2003;86:545-555. 36. Baskaya MK, Hu Y, Donaldson D, et al. Protective effect of the 5-lipoxygenase inhibitor AA-861 on cerebral edema after transient ischemia. J Neurosurg 1996;85:112-116. 37. Ishiguro J, Tada T, Ogihara T, et al. Metabolic disposition of ethyl eicosapentaenoate and its metabolites in rats and dogs. J Pharmacobiodyn 1988;11:251-261. 38. Juliana SF, Marco AM, Roberta E, et al. Long-term treatment with fish oil prevents memory impairment but not hippocampal damage in rats subjected to transient, global cerebral ischemia. Nutr Res 2008;28:798-808. 39. Yang DY, Pan HC, Yen YJ, et al. Detrimental effects of posttreatment with fatty acids on brain injury in ischemic rats. Neurotoxicology 2007;28:1220-1229.
Previous studies have demonstrated that the generation of reactive oxygen species and an excessive inflammatory reaction are involved in the progression of neural damage following brain ischemia. In this study, we focused on the anti-inflammatory and antioxidant properties of eicosapentaenoic acid (EPA). Gerbils were treated intraperitoneally with 500 mg/kg of EPA ethyl for 4 weeks until the day of forebrain ischemia, which was induced by occluding the bilateral common carotid artery for 5 minutes. In the first part of the 2-part experiment, the effect of EPA treatment was evaluated using hematoxylin and eosin staining and deoxynucleotidyl transferase–mediated dUTP nick-end labeling as a marker of cell death (n 5 3 per group). The inflammatory reaction was evaluated using anti-Iba1 immunohistochemistry, a marker of microglial activation (n 5 3 per group), and detection of 8-hydroxyl-20 -deoxyguanosine, a marker of oxidative DNA damage (n 5 4 per group). In the second part of the experiment, the effect of EPA treatment on memory function was examined using an 8-arm radial maze (n 5 6 per group). EPA treatment significantly inhibited DNA oxidative damage (P ,.05) and accumulation of Iba1-positive cells in the CA1 area at 12 and 72 hours after the induction of ischemia, and also decreased apoptotic neurons and neuronal death (P , .001) at 72 hours after ischemia. EPA treatment also significantly improved memory function (P ,.05). These findings suggest that EPA inhibits the inflammatory reaction and oxidative damage occurring after ischemic brain injury, and also may contribute to the prevention of neural damage and memory impairment following such injury. Key Words: Eicosapentaenoic acid—memory function—forebrain ischemia— inflammation—oxidation—gerbil. Ó 2011 by National Stroke Association
Cerebral ischemia or stroke results in irreversible brain damage and subsequent neuropsychological dysfunction, including learning and memory impairment.1,2 A series From the *Department of Neurobiology, Kagawa University Faculty of Medicine, Kagawa, Japan; and †Department of Physiology, Kawasaki Medical University, Kurashiki, Japan. Received February 3, 2009; accepted July 14, 2009. Supported by a Grant-in-Aid for Scientific Research from the Japanese Society for Promotion of Science. Address correspondence to Takehiro Nakamura, MD, PhD, Department of Neurobiology, Kagawa University Faculty of Medicine, 17501 Ikenobe, Miki, Kagawa 761-0793, Japan. E-mail: [email protected]. jp. 1052-3057/$ - see front matter Ó 2011 by National Stroke Association doi:10.1016/j.jstrokecerebrovasdis.2009.11.016
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of events, including glutamate receptor activation, intracellular calcium overload, and reactive oxygen species (ROS) formation, are known to be involved in the pathogenesis of neuronal injury after brain ischemia. In addition, edema and the inflammatory response are known to exacerbate tissue destruction.3 Approaches to ameliorating ischemia-induced brain damage that have been investigated include interfering with the excitatory action of glutamate,4 preventing intracellular calcium overload,5 and scavenging ROS.6 Successful therapies have not yet been established, however.3 In this study, we focused on eicosapentaenoic acid (EPA), an n-3 essential fatty acid (EFA). Previous studies have shown that docosahexaenoic acid (DHA)7,8 and an oil mixture containing n-3 EFA9 provide protective
Journal of Stroke and Cerebrovascular Diseases, Vol. 20, No. 3 (May-June), 2011: pp 188-195
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effects following brain ischemia. Some studies also have suggested that EPA has antioxidant10 and antiinflammatory11,12 properties. In Japan, EPA has been used to treat hypercholesterolemia, a major risk factor for stroke.13 Furthermore, long-term administration of EPA has been demonstrated to improve cerebral blood flow and decrease mortality in stroke-prone spontaneously hypertensive rats.14 Thus, elucidation of the putative protective effects of EPA after brain ischemia may demonstrate a further advantage of EPA treatment. The purposes of the current study were to examine whether treatment with EPA ethyl (EPADEL S; Mochida Pharmaceutical, Tokyo, Japan) can reduce neuronal damage and behavioral deficits, and to investigate how EPA works on the brain after transient global ischemia in gerbils.
Materials and Methods Animal Preparation and Transient Forebrain Ischemia Animal protocols were approved by Kagawa University’s Animal Use Committee. A total of 48 male Mongolian gerbils (age 13-15 weeks; Kyudo, Saga, Japan), with a body weight of 60-80 g, were used for all experiments. The gerbils were given free access to food and water before the experiments. Transient forebrain ischemia was induced by a 5-minute bilateral common carotid artery occlusion, according to the following protocol. Gerbils were anesthetized with sodium pentobarbital (30 mg/kg intraperitoneally [IP]). After a medial neck incision, both common carotid arteries were isolated and occluded for 5 minutes using microaneurysm clips (Sugita Clip; Mizuho, Nagoya, Japan).15 Sham operated animals underwent the same surgical procedure except for the common carotid artery occlusion. During surgery, rectal temperature was controlled at 37-38 C with a feedback-controlled heating pad (CMA, Stockholm, Sweden) to prevent hypothermia. The rectal temperature was maintained for 30 minutes after recirculation, after which the gerbils was housed in a room at 24 C.
Experimental Groups The study was performed in 2 parts. Part 1 examined the effect of EPA on histological changes after ischemia and evaluated DNA oxidative damage using enzymelinked immunosorbent assay (ELISA). Thirty gerbils were divided randomly into 3 groups: the sham group, the Ischemia group, and the EPA4w 1 Ischemia group (n 5 10 per group). The EPA4w 1 Ischemia group was treated with undiluted EPA 500 mg/kg (0.55 mL/kg) IP once a day for 4 weeks until the day before induction of ischemia. The dose of EPA was determined based on the results of a preliminary study showing a maximal effect on a memory test (data not shown). The sham group and the Ischemia group were treated with a equivalent volume of saline solution (0.55 mL/kg) for 4 weeks to equalize the effect of IP injection.16,17
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Part 2 investigated the effect of EPA treatment on memory function. Eighteen gerbils were divided into 3 groups: the sham group, the Ischemia group, and the EPA4w 1 Ischemia group (n 5 6 per group). EPA and placebo control were administered as in part 1 of the study.
Histological Examination We examined neuronal damage using hematoxylin and eosin (HE) staining and deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) as a marker of cell death.18 We also evaluated the inflammatory response by anti-Iba1 immunohistochemistry.19,20 The gerbils (n 5 3 per group) were anesthetized with sodium pentobarbital (50 mg/kg IP) at 12 or 72 hours after induction of ischemia, then transcardially perfused with 4% phosphate-buffered paraformaldehyde after flushing with 10% phosphate-buffered saline (PBS). The brains were removed and placed in the same fixative overnight, and then immersed in 25% sucrose for 2 days. The brains were then placed in OCT embedding compound (Sakura Finetek USA, Torrance, CA) and sectioned (14 mm) on a cryostat. Sections in the CA1 area at 21.7 mm from the bregma (including the dorsal hippocampal area) were analyzed. In HE-stained sections, the number of viable neurons (cell/mm) was counted in the CA1 area.2 Immunohistochemistry was performed using the avidin-biotin complex method.20 After blocking of nonspecific binding with 1% albumin solution and washing in 0.01 M PBS, a monoclonal antibody against Iba1 (1:100 dilution; Wako, Osaka, Japan) was applied to sections overnight at 4 C. Sections were incubated with biotinylated goat anti-rabbit IgG (1:1000 dilution; Vector Laboratories, Burlingame, CA) for 90 minutes and then incubated with avidin-biotin horseradish peroxidase (Vector Laboratories) for 90 minutes.21 A molecular histochemical system (Apoptosis In Situ Detection Kit; Wako) was used for specific staining of DNA fragmentation (ie, TUNEL staining).22 This method is based on the specific binding of terminal deoxynucleotidyl transferase (TdT) to the 30 -OH ends of DNA. In brief, sections were permeabilized with 0.1% sodium citrate and 0.1% Triton X-100, the 30 DNA ends were labeled with TdT reaction solution, endogenous peroxidase activity was inactivated with 3% H2O2 in PBS, sections were labeled with peroxidase-conjugated antibody, and peroxidase was detected with diaminobenzidine. The labeling targets of this kit were the new 30 -OH DNA ends generated by DNA fragmentation.23
Assays for DNA Oxidative Damage DNA oxidative damage was investigated using an 8-hydroxyl- 20 -deoxyguanosine (8-OHdG) assay.24 Gerbils (n 5 4 per group) were reanaesthetized (50 mg/kg pentobarbital IP) and decapitated 12 hours after induction of ischemia. Then the brains were removed, and the
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Figure 1. (A) HE staining in the hippocampal CA1 area. Representative sections from the Ischemia group (top) and the EPA4w 1 Ischemia group (4 weeks of treatment) (bottom) 72 hours after indiction of ischemia. The left panels show lower magnification (scale bar: 500 mm); the right panels show higher magnification (scale bar: 50 mm). Neurons with shrunken cell bodies were rarely observed in the EPA 4w 1 Ischemia group. (B) Viable neurons per millimeter of the hippocampal CA1 area. n 5 3 per group. *P , .05 compared with the sham group; **P , .001 compared with the sham group; ##P , .001 compared with the Ischemia group. Viable neurons were significantly increased in the EPA 4w 1 Ischemia group compared with the Ischemia group.
hippocampal complex was preserved. DNA extraction was performed with a DNA isolation kit (Dojindo Molecular Technologies, Kumamoto, Japan). This method, in which purified DNA is isolated from brain tissue using the guanidine method and RNase A and proteinase K, avoids the use of phenol and heating that might induce background.25 8-OHdG levels were evaluated by ELISA (Japan Institute for the Control of Aging, Shizuoka, Japan), which can measure extremely low levels of 8-OHdG. The specificity of the monoclonal antibody was established. The optical density of the wells was measured at 450 nm. The data, expressed as nanograms of 8-OHdG per microgram of DNA, were calculated on the basis of a linear calibration curve generated for each experiment using 8-OHdG standard solutions.25
Memory Test The 8-arm radial maze was used to assess memory function. We used a technique similar to that reported by
Miyoshi et al.26 For the spatial memory task, the central part of the maze was 22 cm in diameter, and the arms were 25 cm long, 6 cm high, and 6 cm wide, made of transparent Plexiglass. The maze was always oriented in the same direction in space. The test was started at 7 days after the operation. Gerbils (n 5 6 per group) were tested on 4 consecutive days, with 1 trial conducted per day. A small food pellet was placed at the end of each arm, and the animal was placed in the central part of the maze. An error was noted if the animal entered an arm that it had visited previously. The number of errors was recorded. The trial ended when all of the pellets had been eaten.
Statistics Data are expressed as mean 6 standard deviation. The differences in the histological data and ELISA data were analyzed for significance by one-way ANOVA followed by Scheffe’s post hoc test. Two-way repeated ANOVA
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Figure 2. Immunohistochemical staining for Iba1 in the hippocampal CA1 area in representative sections. The left panels show lower magnification (scale bar: 500 mm); the right panels show higher magnification (scale bar: 100 mm). The intensity of Iba1 immunoreactivity was induced at 12 hours and increased until 72 hours after induction of ischemia in each group. Iba1-positive cells were more weakly stained in the EPA 4w 1 Ischemia group compared with the Ischemia group.
was used to compute the behavioral data, and the differences among the groups were evaluated for significance using Scheffe’s post hoc test.2
Results Administration of EPA or placebo control caused no medical problems in the animals. Activity and appetite was maintained in all groups, and body weight did not differ significantly among the groups. Body temperature during the surgical procedure was well controlled in all groups.
Effects of EPA Treatment on Histological Changes Examination of HE-stained sections revealed no significant changes in the Ischemia group or the EPA4w 1 Ischemia group at 12 hours after ischemia. However, in the Ischemia group, marked neuronal damage, along with pyknosis and eosinophilia in CA1 pyramidal neurons, were seen at 72 hours after ischemia (Fig 1A). In contrast, sections from the EPA4w 1 Ischemia group showed some eosinophilic cells, but very few karyopyknotic cells. EPA treatment significantly reduced hippocampal neuronal loss (P , .001) (Fig 1B).
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Figure 3. TUNEL in the hippocampal CA1 area. Representative sections from the Ischemia group 12 hours (upper left) and 72 hours (lower left) after ischemia and from the EPA4w 1 Ischemia group 12 hours (upper right) and 72 hours (lower right) after ischemia. (Scale bar: 50 mm.) Apoptotic neurons were observed only in the Ischemia group at 72 h after ischemia.
Figure 2 shows photomicrographs of Iba1 immunohistochemical staining in the hippocampus of the Ischemia group and the EPA4w 1 Ischemia group at 12 hours and 72 hours after ischemia. Both the Ischemia group and the EPA4w 1 Ischemia group showed an increased number of Iba1-positive cells over time, which exhibited marked hypertrophy. Iba1-positive cells tended to gather in and around the CA1 area, especially in a gerbil from the Ischemia group at 72 hours after ischemia. The EPA4w 1 Ischemia group demonstrated less immunostaining than the Ischemia group at each time point examined. Using the TUNEL method, we observed the staining of many hippocampal neurons in tissue sections from animals in the Ischemia group 72 hours after ischemia. Tissue sections from the other groups showed no TUNEL-positive neurons (Fig 3).
groups, and transient forebrain ischemia significantly increased the number of errors (P , .01). The number of errors was significantly lower in the EPA4w 1 Ischemia group compared with the Ischemia group (P , .05).
Discussion Several previous studies have demonstrated the protective effect of DHA and an oil mixture containing n-3 EFAs after brain ischemia, resulting in improved neuronal function. In this study, we investigated the effect of pure EPA, rather than DHA or the n-3 EFA-containing oil. EPA is a well-known antithrombotic drug27 and cholesterol-lowering agent,28 but it has been less well investigated than DHA7,8 or the n-3 EFA oil mixture9 for use
Effect of EPA Treatment on Oxidative DNA Damage Figure 4 shows the level of the DNA oxidation marker 8-OHdG at 12 hours after induction of ischemia. The Ischemia group had a significantly higher 8-OHdG level compared with the sham group (P , .01) and a higher level compared with the EPA4 W 1 Ischemia group (P , .05).
Effect of EPA on Memory Function Figure 5 shows the number of errors made in the 8-arm radial maze. Two-way repeated ANOVA revealed significant major effects of groups (F2,45 5 8.093; P , .01) and trials (F3,45 5 44.895; P , .0001). There was no interaction between the groups and trials (F6,60 5 1.225; P . .05). The number of errors tended to decrease with time in all
Figure 4. 8-OHdG levels in the hippocampus measured by ELISA; n 5 3 per group. **P ,.01 compared with the sham group; #P ,.05 compared with the Ischemia group. The EPA 4w 1 Ischemia group had a significantly lower 8-OHdG level than the Ischemia group.
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oxide, and additional ROS, which disrupt the blood-brain barrier and induce brain edema and additional cell death.32 EPA also modulate arteriolar reactivity, which is normally controlled by cyclooxygenase (COX) metabolism and formation of oxygen free radicals,33 improving cerebral blood flow following transient forebrain ischemia.34 Actually, it has been shown that reduction of PG or LT levels by COX or lipoxygenase inhibitors led to reduced brain damage, including lipid oxidation, cell death, and brain edema.35,36 These findings suggest that EPA treatment may reduce deleterious inflammation and attenuate further inflammatory reaction, tissue damage, and subsequent apoptosis and cell death by inhibiting eicosanoid production.
Effect of EPA Treatment on Oxidative DNA Damage
Figure 5. Performance in the 8-arm radial maze trial (number of errors); n 5 6 per group. **P , .01 compared with the sham group; #P , .05 compared with the Ischemia group. The EPA4w 1 Ischemia group demonstrated significantly improved memory function compared with the Ischemia group.
after brain ischemia. Our findings suggest that EPA decreases oxidative damage, the inflammatory response, and subsequent apoptosis and cell death and also may improve memory function, similar to the previously reported effects of DHA.
Effect of EPA Treatment on Cell Death and the Inflammatory Response Histological investigation revealed that EPA treatment significantly decreased the number of microglia at both 12 hours and 72 hours, the number of dead or damaged neurons, and the number of apoptotic cells at 72 hours in the CA1 area after the induction of ischemia. These results suggest that EPA treatment inhibited cell death and apoptosis and acutely inhibited inflammation in the CA1 area. It is important to note that EPA treatment inhibited the pathology associated with forebrain ischemia at 12 hour, at which point cell death and apoptosis had not yet occurred. Previous studies have found that omega-3 fatty acids, including EPA, have immunomodulative effects on the synthesis of the arachidonic acid–derived eicosanoid mediators prostaglandins (PGs) and leukotrienes (LTs).29 A recent study showed that DHA reduced eicosanoid production following transient forebrain ischemia in gerbils.30 Eicosanoids are potent chemoattractants for inflammatory cells, including microglia,31 and inflammatory cells can release various cytotoxic agents, including cytokines, matrix metalloproteinases, nitric
TUNEL staining demonstrated that EPA treatment completely prevented DNA fragmentation in the hippocampus at 72 hours after ischemia. However, HE staining at the same time showed a 70% decrease in viable neurons in the CA1 area compared with the sham group. These findings suggest that EPA treatment may affect apoptosis, but not necrosis. The 8-OHdG assay at 12 hours after ischemia revealed a dramatically increased 8-OHdG level in the Ischemia group, but a reduced level in the EPA4w 1 Ischemia group to near the level seen in the sham group. Because the 8-OHdG level reveals DNA strand breaks produced by interactions between DNA and ROS,24 a decreased 8-OHdG level suggests that EPA treatment decreases oxidative DNA damage. Previous studies have shown n-3 EFA treatment enhances the activity of antioxidant enzymes such as glutathione peroxidase, superoxide dismutase, and catalase after transient brain ischemia,7-9 and that augmenting the activity of these antioxidant enzymes by EPA might play a pivotal role in protection against ROS. But because ROS levels are determined by a balance between the amount of antioxidant enzymes and the generation of ROS, EPA’s antiinflammatory properties may be due to a reduction in free oxygen radical generation, leading to attenuation of DNA oxidation.
Effect of EPA Treatment on Memory Function and Treatment Duration In all groups, the number of errors made in the 8-arm radial maze was highest on day 1 and declined sharply on day 2, followed by a gradual decrease thereafter. This 2-stage reduction in the number of errors likely reflects 2 forms of brain function. Because the gerbils did not undergo a training period in this study, our findings may reflect task acquisition or learning in addition to working memory.1 This result also suggests that learning ability was maintained in the Ischemia group. However, 2-way repeated ANOVA revealed a significant increase
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in the number of errors in the Ischemia group compared with the sham group or the EPA4w 1 Ischemia group, suggesting significant memory impairment in the Ischemia group and improvement in the EPA4w 1 Ischemia group. Although gerbils treated with EPA for 4 weeks exhibited improved memory function, gerbils treated for 1 week did not demonstrate a significant decrease in the number of errors in the preliminary experiment (data not shown). This suggests that EPA treatment provides benefits in a time-dependent manner. Pharmacokinetic studies have shown that EPA requires a long time to reach a steady-state concentration in the brain, necessitating repeated administration.37 Thus, the tissue concentration may have been insufficient after just 1 week of EPA treatment, and this delayed distribution to the tissues may explain the ineffectiveness of short-term preischemic or postischemic n-3 EFA treatment.38,39 These findings suggest that EPA may not be suitable for emergency care; however, because EPA has a long half-life,37 intermittent administration after achievement of steady-state may be effective. In conclusion, our findings suggest that EPA treatment may reduce oxidative DNA damage and subsequent apoptosis and cell death, resulting in improved memory function. These protective effects may be due to EPA’s anti-inflammatory properties. Thus, EPA may be useful not only in reducing the risk of brain ischemic events by lowering cholesterol, but also in preventing neuronal damage after brain ischemia.
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