Protective Effect of Telmisartan Against Progressive Oxidative Brain Damage and Synuclein Phosphorylation in Stroke-resistant Spontaneously Hypertensive Rats Yusuke Fukui, BS, Toru Yamashita, MD, PhD, Tomoko Kurata, MD, Kota Sato, MD, Violeta Lukic, MD, Nozomi Hishikawa, MD, Kentaro Deguchi, MD, and Koji Abe, MD, PhD
Previously, we reported that reactive oxygen species and signaling molecules of angiotensin II produced lipid peroxides, degenerated proteins, and injured DNA after cerebral ischemia in normotensive Wistar rats. Here, we investigated the longterm effect of the angiotensin II type I receptor blocker telmisartan on oxidative stress and hyperphosphorylated a-synuclein accumulation in stroke-resistant spontaneously hypertensive rats (SHR-SR). At the age of 3 months, SHR-SR were divided into 3 treatment groups: SHR-SR vehicle (SHR/Ve), SHR-SR low-dose telmisartan (.3 mg/kg/day) (SHR/low), and SHR-SR high-dose telmisartan (3 mg/kg/day) (SHR/high). Immunohistologic analyses were conducted in these groups and Wistar rats at the age of 6, 12, and 18 months. The SHR/Ve group demonstrated more progressive increase in advanced glycation end product (AGE)-, 4-hydroxy-2nonenal (4-HNE)-, and phosphorylated a-synuclein (pSyn)-positive cells in the cerebral cortex and hippocampus compared with the Wistar group at 18 months. These expressions were reduced in the SHR/low group even without lowering blood pressure (BP), and expressions were dramatically suppressed in the SHR/high group with lowering of BP. These data suggest that persistent hypertension in SHR-SR strongly potentiate the markers of oxidative damage (AGEs and 4-HNE) and abnormal accumulation of pSyn, which were greatly suppressed by telmisartan in a dose-dependent manner without and with lowering of BP. Key Words: Strokeresistant spontaneously hypertensive rat—telmisartan—oxidative stress— phosphorylated a-synuclein—peroxisome proliferator–activated receptor gamma. Ó 2014 by National Stroke Association
Introduction We have reported that reactive oxygen species (ROS) and the signaling molecules of angiotensin II produce lipid peroxides, degenerate proteins, and injure DNA after cerebral ischemia in normotensive Wistar rats.1,2 Antihypertensive drugs, such as angiotensin II type I From the Department of Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Received November 22, 2013; revision received December 23, 2013; accepted December 25, 2013. This work was partly supported by Grant-in-Aid for Scientific Research (B) 21390267 from the Ministry of Education, Science, Culture, and Sports of Japan, Grants-in-Aid from the Research Committee of CNS Degenerative Diseases (I. Nakano), and grants (H.
receptor blockers (ARBs), inhibit such ROS production and prevent cerebrovascular disorders.3-5 ARBs also show protective effects against stroke without lowering blood pressure (BP).6,7 Telmisartan is an ARB with high lipid solubility8,9 and has been expected to prevent atherosclerosis and metabolic disorders.10
Mizusawa, M. Nishizawa, H.Sasaki, G. Sobue) from the Ministry of Health, Labour, and Welfare of Japan. Address correspondence to Koji Abe, MD, PhD, Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmacy, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.12.052
Journal of Stroke and Cerebrovascular Diseases, Vol. -, No. - (---), 2014: pp 1-9
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Figure 1. Representative photomicrographs of AGE-positive cell staining (A) and the number of AGE-positive cells per square millimeter of cerebral cortex (B) at 6, 12, and 18 months. Note the BP- and age-dependent increase in the number of AGE-positive cells (arrowheads). (The order of staining strength in the brain was SHR/ Ve . SHR/low . SHR/high . Wistar rat [scale bar 5 100 mm]. **P , .01 versus Wistar group, ##P , .01 versus SHR/Ve group.) Abbreviations: AGE, advanced glycation end product; BP, blood pressure; high, high-dose telmisartan group; low, low-dose telmisartan group; M, months; SHRSR, stroke-resistant spontaneously hypertensive rats; Ve, vehicle; Wis, Wistar rat.
Spontaneously hypertensive rats (SHR) and strokeresistant spontaneously hypertensive rats (SHR-SR) are genetic models that develop hypertension, followed by progressive neurobehavioral impairment,11 Alzheimer’s disease,12 and immune alterations.13 In SHR, telmisartan has been shown to improve autoregulation of cerebral blood flow and reduce generation of superoxide.14 Studies have also demonstrated that oxidative stress is induced in SHR-SR15 and stroke-prone spontaneously hypertensive rats,16 with the accumulation of hyperphosphorylated a-synuclein (pSyn).17 However, oxidative stress and pSyn accumulation under chronic hypertension have not been studied. SHR-SR live much longer than stroke-prone spontaneously hypertensive rats, so we investigated the long-term effect of telmisartan on oxidative stress and pSyn accumulation in SHR-SR.
Materials and Methods All experimental procedures were approved by the Animal Committee of the Graduate School of Medicine and Dentistry, Okayama University (Okayama, Japan).
Animals and Drug Preparation Seven-week-old male Wistar rats and SHR-SR were provided by the Disease Model Cooperative Re-
search Association (Kyoto, Japan) and placed on a basal diet. At the age of 3 months, the Wistar rats mentioned previously (n 5 20) were started on a daily dose of .5% methyl cellulose (MC) in .1 mL of water by oral gavage as the normotensive control group for 3, 9, and 15 months until killed. The SHR-SR mentioned previously (n 5 54) were divided into 3 treatment groups: the SHR-SR vehicle group (SHR/Ve, n 5 17), SHR-SR low-dose telmisartan group in which the BP did not fall significantly (SHR/ low, n 5 19), and SHR-SR high-dose telmisartan group in which the BP fell by30 mm Hg or more (SHR/high, n 5 18). Rats received daily oral doses of .5% MC only (SHR-SR/Ve), .5% MC plus low-dose telmisartan (.3 mg/kg/day), or .5% MC plus high-dose telmisartan (3 mg/kg/day) by oral gavage for 3, 9, and 15 months until killed. The telmisartan dose was determined as described previously.14,18 Telmisartan was provided by Boehringer Ingelheim (Ingelheim am Rhein, Germany) and was given to the 2 rat groups as a suspension with .5% MC in .1 mL of water every day. At the age of 6, 12, or 18 months, rats were perfused transcardially with chilled heparinized saline (5 U/mL), followed by 4% paraformaldehyde in phosphate buffer (PB) under deep anesthesia (pentobarbital 20 mg/250 g body weight). After decapitation, brains were removed
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Figure 2. Representative photomicrographs 4HNE-positive cell staining (A) and the number of 4-HNE-positive cells per square millimeter of the cerebral cortex (B) at 6, 12, and 18 months. Note the BP- and age-dependent increase in 4HNE staining, and dose-dependent reduction by telmisartan (scale bar 5 100 mm) (**P , .01 versus Wistar group, ##P , .01 versus SHR/Ve group). Abbreviations: BP, blood pressure; 4HNE, 4-hydroxy-2-nonenal; M, months; SHR, spontaneously hypertensive rats; Ve, vehicle; Wis, Wistar rat.
and brain weights measured. The present study focused on the oxidative stress and pSyn in a whole project in the comparison of Wistar rats and SHR-SR and the effect of telmisartan on SHR-SR.
Immunohistochemical Analyses Brains were immersed and fixed in 4% paraformaldehyde with .1 M of PB (pH 7.6) for 8 hours, embedded in paraffin, and 5-mm thick sections were prepared for immunostaining. For immunostainings for advanced glycation end products (AGEs), 4-hydroxy-2-nonenal (4-HNE), and pSyn, brain sections were pretreated by heating thrice in a 500-W microwave for 5 min in 10 mM (pH 6.0) citric acid buffer. These pretreated sections were then immersed in .5% periodic acid to block intrinsic peroxidase and treated with 5% normal horse serum in 50 mM phosphate-buffered saline (pH 7.4) containing .05% Tween 20 to block nonspecific antibody responses. Finally, they were incubated overnight with each primary antibody. The following primary antibodies were used: mouse antiAGE monoclonal antibody (1:100 dilution; TRANCE GENIC, Inc, Kumamoto, Japan), mouse anti-4-HNE monoclonal antibody (1:50; JaICA, Shizuoka, Japan), and mouse anti-pSyn monoclonal antibody (1:500; Wako Pure Chemical Industries, Ltd, Osaka, Japan). A biotinylated antimouse antibody was used as the secondary antibody.
Specific labeling was visualized using a Vectastain Elite ABC kit (Vector, Burlingame, CA). To guarantee specificstaining primary antibodies, brain sections were also stained without primary antibodies.
Detection Stained sections were digitized with a digital microscope camera (BX-51; Olympus Optical Co, Japan). The numbers of AGE-positive cells, 4-HNE-positive cells, and pSyn-positive cells per each 1-mm2 cerebral cortex in hemispheric coronal sections were counted at 3 levels (frontal cortex, basal ganglia, and posterior hippocampus). Three serial sections were used for each level, and all cell counts in the cerebral cortex per animal were determined and added. Furthermore, AGE-, 4-HNE-, and pSyn-positive cells were quantified by counting stained cells in the CA1 and CA3 regions of the bilateral hippocampus and expressed as the percentage of AGE-, 4-HNE-, and pSyn-positive cells over the total number of cells in these regions.
Statistical Analyses Data are expressed as the mean 6 standard deviation. Statistical analyses were conducted using an analysis of variance with repeated measures (multiple comparisons).
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Figure 3. Representative photomicrographs of pSyn-positive cell staining (A) and the number of pSyn-positive cells per square millimeter of cerebral cortex (B) at 6, 12, and 18 months. pSyn was progressively labeled with age in SHR/Ve, which was greatly reduced in the other groups (arrowheads) (scale bar 5 100 mm) (**P , .01 versus Wistar group, ##P , .01 versus SHR/Ve group). Abbreviations: M, months; pSyn, phosphorylated a-synuclein; SHR, spontaneously hypertensive rats; Ve, vehicle; Wis, Wistar rat.
Planned comparisons were used for Tukey–Kramer post hoc analyses. A value of P less than .05 was considered significant. All statistical analyses were undertaken with Statcelver2 (OMS Inc, Tokorozawa, Japan).
Results Immunohistochemical Analyses in the Cerebral Cortex AGE Staining in the Cerebral Cortex In Wistar rats, AGE was scarcely labeled in cerebrocortical cells at 6 months (Fig 1A, a), but became evident gradually in the cytoplasm at 12 and 18 months (Fig 1A, e, i). The mean numbers of AGE-positive cells in the cerebral cortex were (per square millimeter) 18.2 6 13.0 at 6 months, 28.5 6 8.5 at 12 months, and 32.1 6 23.4 at 18 months (Fig 1B, open bars). In the SHR/Ve group, AGE-positive cells were already evident at 6 months, and the numbers in the cerebral cortex increased with age (Fig 1A, b, f, j), that is, they were (per square millimeter) 59.7 6 35.4 at 6 months, 72.4 6 44.4 at 12 months, and 123.3 6 41.6 (**P , .01 versus Wistar group) at 18 months (Fig 1B). In the 2 telmisartan groups, the numbers of AGEpositive cells in the cerebral cortex were (per square milli-
meter) 27.7 6 16.3 at 6 months, 33.6 6 19.2 at 12 months, and 44.1 6 40.4 (##P , .01 versus SHR/Ve group) at 18 months in the SHR/low group. In the SHR/high group, the numbers of AGE-positive cells in the cerebral cortex were (per square millimeter) 15.7 6 6.2 at 6 months, 31.1 6 16.2 at 12 months, and 32.5 6 17.4 (##P ,.01 versus SHR/Ve group) at 18 months (Fig 1, B). 4-HNE Staining in the Cerebral Cortex In Wistar rats, 4-HNE was scarcely labeled in cerebrocortical cells at 6 months (Fig 2A, a), but became evident gradually in the cytoplasm from 12-18 months (Fig 2A, e, i). The mean numbers of 4-HNE-positive cells in the cerebral cortex were (per square millimeter) 82.8 6 5.8 at 6 months, 84.2 6 18.7 at 12 months, and 94.1 6 30.0 at 18 months (Fig 2B, open bars). 4-HNE was already clearly labeled in the cytoplasm of cerebrocortical cells at 6 months in the SHR/Ve group, which became much stronger at 12 and 18 months (Fig 2A, b, f, j). The numbers of 4-HNE-positive cells in the cerebral cortex were (per square millimeter) 221.1 6 31.2 (**P , .01 versus Wistar group) at 6 months, 288.1 6 67.7 (**P , .01 versus Wistar group) at 12 months, and 311.7 6 109.1 (**P ,.01 versus Wistar group) at 18 months (Fig 2B).
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Figure 4. Representative photomicrographs of AGE-positive cell staining (A) and the ratio of AGE-positive cells of CA1 and CA3 in the hippocampus (B) at 6, 12, and 18 months. Note the BP- and age-dependent increase in AGE staining (arrowheads), and dose-dependent reduction by telmisartan (scale bar 5 100 mm) (**P , .01 versus Wistar group, ##P , .01 versus SHR/Ve group). Abbreviations: AGE, advanced glycation end product; BP, blood pressure; M, months; SHR, spontaneously hypertensive rats; Ve, vehicle; Wis, Wistar rat.
In the 2 telmisartan groups, 4-HNEwas already labeled in the cytoplasm of cerebrocortical cells (Fig 2A, c, d) and increased slightly at 12 and 18 months (Fig 2A, g, h, k, l). The numbers of 4-HNE-positive cells in the cerebral cortex in the SHR/low group were (per square millimeter) 120.3 6 12.1 (##P ,.01 versus SHR/Ve group) at 6 months, 126.4 6 13.0 (##P , .01 versus SHR/Ve group) at 12 months, and 126.8 6 12.1 (##P , .01 versus SHR/Ve group) at 18 months, and those in the SHR/high group were 91.9 6 17.3 (##P , .01 versus SHR/Ve group) at 6 months, 96.7 6 58.3 (##P , .01 versus SHR/Ve group) at 12 months, and 103.8 6 34.9 (##P , .01 versus SHR/ Ve group) at 18 months (Fig 2B). pSyn Staining in the Cerebral Cortex pSyn was scarcely labeled in cerebrocortical cells at 6 months (Fig 3A, a), but became evident gradually in the cytoplasm at 12 and 18 months (Fig 3A, e, i). The mean numbers of pSyn-positive cells in the cerebral cortex were (per square millimeter) 1.3 6 1.1 at 6 months, 2.9 6 2.0 at 12 months, and 5.8 6 3.6 at 18 months (Fig 3B, open bars). pSyn was already weakly labeled in cerebrocortical cells at 6 months and became evident in the cytoplasm at 12 and 18 months in the SHR/Ve group (Fig 3A, b, f, j). The numbers of pSyn-positive cells in the cerebral cortex were (per square millimeter) 17.8 6 2.0 (**P , .01 versus Wistar group) at 6 months, 20.6 6 4.8 (**P , .01
versus Wistar group) at 12 months, and 22.0 6 11.1 (**P , .01 versus Wistar group) at 18 months (Fig 3B). In the 2 telmisartan groups, pSyn was scarcely labeled in cerebrocortical cells at 6 months (Fig 3A, c, d) and increased only slightly in the cytoplasm at 12 and 18 months (Fig 3A, g, h, k, l). The numbers of pSynpositive cells in the cerebral cortex in the SHR/low group were (per square millimeter) 5.4 6 5.7 (##P , .01 versus SHR/Ve group) at 6 months, 7.5 6 6.7 (##P , .01 versus SHR/Ve group) at 12 months, and 8.6 6 6.0 (##P , .01 versus SHR/Ve group) at 18 months (Fig 3B), and those in the SHR/high group were 4.0 6 5.1 (##P , .01 versus SHR/Ve group) at 6 months, 4.4 6 3.2 (##P , .01 versus SHR/Ve group) at 12 months, and 5.4 6 4.5 (##P , .01 versus SHR/Ve group) at 18 months (Fig 3B).
Immunohistochemical Analyses in the Hippocampus AGE Staining in the Hippocampus In Wistar rats, AGE was scarcely labeled in the cytoplasm of CA1 and CA3 cells throughout 6-18 months (Fig 4A, a, e, i). The ratios of AGE-positive cells in CA1 and CA3 were .03 6 .01 at 6 months, .09 6 .10 at 12 months, and .10 6 .09 at 18 months, and no change was detected with age in the Wistar group (Fig 4B, open bars). In the SHR/Ve group, AGE was slightly labeled in CA1 and CA3 cells at 6 months, and this staining became more
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Figure 5. Representative photomicrographs of 4-HNE-positive cell staining (A) and the ratio of 4-HNE-positive cells of CA1 and CA3 in the hippocampus (B) at 6, 12, and 18 months. Note the BP- and age-dependent increase in the number of 4-HNE-positive cells. (The order of staining strength in the brain was SHR/Ve . SHR/ low . SHR/high . Wistar rat [scale bar 5 100 mm]. *P , .05 versus Wistar group, **P , .01 versus Wistar group, #P , .05 versus SHR/Ve group, ##P ,.01 versus SHR/Ve group.) Abbreviations: BP, blood pressure; 4-HNE, 4hydroxy-2-nonenal; M, months; SHR, spontaneously hypertensive rats; Ve, vehicle; Wis, Wistar rat.
evident in the cytoplasm at 12 and 18 months (Fig 4A, b, f, j). The ratios of AGE-positive cells of CA1 and CA3 were .29 6 .07 (**P , .01 versus Wistar group) at 6 months, .30 6 .07 (**P , .01 versus Wistar group) at 12 months, and .51 6 .11 (**P ,.01 versus Wistar group) at 18 months (Fig 4B). Between the 2 telmisartan groups, AGE was labeled less in the cytoplasm of CA1 and CA3 cells (Fig 4A, c, d). The ratios of AGE-positive cells of CA1 and CA3 in the SHR/low group were .09 6 .04 (##P , .01 versus SHR/Ve group) at 6 months, .11 6 .06 (##P , .01 versus SHR/Ve group) at 12 months, and .16 6 .09 (##P , .01 versus SHR/Ve group) at 18 months (Fig 4B), and those in the SHR/high group were .09 6 .06 (#P , .05 versus SHR/Ve group) at 6 months, .10 6 .06 (##P , .01 versus SHR/Ve group) at 12 months, and .14 6 .16 (##P , .01 versus SHR/Ve group) at 18 months (Fig 4B). 4-HNE Staining in the Hippocampus In Wistar rats, 4-HNE was scarcely labeled in CA1 and CA3 cells throughout 6-18 months, with no agedependent change in the cytoplasm (Fig 5A, a, e, i). The ratios of 4-HNE-positive cells in CA1 and CA3 were .07 6 .02 at 6 months, .12 6 .02 at 12 months, and .14 6 .04 at 18 months (Fig 5B, open bars). 4-HNE was already labeled in CA1 and CA3 cells at 6 months in the SHR/Ve group, but almost no change
in the cytoplasm was observed at 12 and 18 months (Fig 5A, b, f, j). The ratios of 4-HNE-positive cells of CA1 and CA3 were .48 6 .2 (**P , .01 versus Wistar group) at 6 months, .49 6 .26 (*P , .05 versus Wistar group) at 12 months, and .51 6 .2 (**P , .01 versus Wistar group) at 18 months (Fig 5B). In the 2 telmisartan groups, 4-HNE was weakly labeled in the cytoplasm of CA1 and CA3 cells at 6 months (Fig 5A, c, d). The ratios of 4-HNE-positive cells of CA1 and CA3 in the SHR/low group were .19 6 .05 (##P ,.01 versus SHR/ Ve group) at 6 months, .21 6 .12 at 12 months, and .21 6 .1 (##P , .01 versus SHR/Ve group) at 18 months, and those in the SHR/high group were .15 6 .02 (##P , .01 versus SHR/Ve group) at 6 months, .17 6 .18 (#P , .05 versus SHR/Ve group) at 12 months, and .18 6 .08 (##P , .01 versus SHR/Ve group) at 18 months (Fig 5B). pSyn Staining in the Hippocampus In Wistar rats, pSyn was scarcely labeled in CA1 and CA3 cells throughout 6-18 months with no agedependent change in the cytoplasm (Fig 6A, a, e, i). The ratios of pSyn-positive cells in CA1 and CA3 were .02 6 .02 at 6 months, .02 6 .02 at 12 months, and .02 6 .01 at 18 months (Fig 6B, open bars). pSyn was already clearly labeled in CA1 and CA3 cells at 6 months in the SHR/Ve group and became evident gradually in the cytoplasm at 12 and 18 months (Fig 6A,
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Figure 6. Representative photomicrographs of pSyn-positive cell staining (A) and the ratio of pSyn-positive cells of CA1 and CA3 in the hippocampus (B) at 6, 12, and 18 months. Note that pSyn was labeled progressively with age in the SHR/Ve group, which was greatly reduced in the other groups (scale bar 5 100 mm) (**P , .01 versus Wistar group, ##P , .01 versus SHR/Ve group). Abbreviations: M, months; pSyn, phosphorylated a-synuclein; SHR, spontaneously hypertensive rats; Ve, vehicle; Wis, Wistar rat.
b, f, j). The ratios of pSyn-positive cells of CA1 and CA3 were .13 6 .03 (**P ,.01 versus Wistar group) at 6 months, .14 6 .04 (**P , .01 versus Wistar group) at 12 months, and .15 6 .06 (**P , .01 versus Wistar group) at 18 months (Fig 6B). In the 2 telmisartan groups, pSyn was weakly labeled in the cytoplasm of CA1 and CA3 cells at 6 months (Fig 5A, c, d) and increased slightly in the cytoplasm at 12 and 18 months (Fig 6A, g, h, k, l). The ratios of pSynpositive cells of CA1 and CA3 in the SHR/low group were .02 6 .02 (##P , .01 versus SHR/Ve group) at 6 months, .04 6 .05 (##P , .01 versus SHR/Ve group) at 12 months, and .05 6 .02 (##P , .01 versus SHR/Ve group) at 18 months, and those in the SHR/high group were .02 6 .01 (##P , .01 versus SHR/Ve group) at 6 months, .03 6 .02 (##P , .01 versus SHR/Ve group) at 12 months, and .03 6 .05 (##P , .01 versus SHR/Ve group) at 18 months (Fig 6B).
Discussion The SHR/Ve group, with mean blood pressure (MBP) 175.6 6 22.6 mm Hg at 12 months, demonstrated progressive increases in AGE-, 4-HNE-, and pSyn-positive cells in the cerebral cortex and hippocampus with aging (Figs 1-6). However, telmisartan reduced these changes even without lowering BP (SHR/low; MBP 168.9 6 24.0 mm Hg at
12 months), and high-dose telmisartan showed further improvement by lowering of BP (SHR/high; MBP 149.0 6 15.0 mm Hg at 12 months) (Figs 1-6). Previously, we reported that ROS produce lipid peroxides, degenerate proteins, and injure DNA after cerebral ischemia in normotensive Wistar rats.1,2 ROS is produced by nicotinamide adenine dinucleotide phosphate (NAD [P]H) oxidase that is activated by morbid stimulation such as hypertension. ROS inactivates nitric oxide causing functional disorder and cell death of vascular endothelium and smooth muscle.19,20 In addition, angiotensin II is also an activator of NAD(P)H oxidase, produces ROS,21 and increases oxidative stress.22 To investigate the long-term effect of hypertension, we studied oxidative stress in SHR-SR for 18 months. We found that persistent hypertension in SHR-SR demonstrated a progressive increase in oxidative stress (AGE and 4-HNE) and pSyn accumulation compared with Wistar rats (Figs 1-6). These expressions were suppressed by telmisartan in a dose-dependent manner (Figs 1-6). Of note, low-dose telmisartan ameliorated these changes without lowering BP, indicating an antioxidative effect independent of BP. The results speculated that telmisartan decreased ROS production through inhibiting angiotensin II. a-Synuclein is accumulated in the presynaptic terminals of the cerebral cortex, hippocampus, substantia
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nigra, thalamus, and cerebellum. Therefore, it is involved in neurotransmission. a-Synuclein is phosphorylated only at z4% in normal rat brains, but pSyn in patients with synucleinopathy, such as Parkinson’s disease and dementia with Lewy bodies, have greater than 90% pSyn in the postmortem brain.23,24 In addition, oxidative stress hyperphosphorylates a-synuclein, resulting in its accumulation.17 Abnormal accumulation of pSyn can lead to the death of neuronal cells.25 Persistent hypertension induced abnormal accumulation of pSyn in the cerebral cortex and hippocampus (Figs 3, 6), demonstrating that long-lasting hypertension and oxidative stress are correlated with pSyn accumulation. The dose-dependent effect of telmisartan on pSyn reduction suggests a protective effect probably through antioxidative mechanisms with further protection by a BPlowering mechanism (Figs 3, 6). ARBs inhibit ROS production,4,5 and telmisartan shows antihypertensive effects along with activation of peroxisome proliferator-activated receptor (PPAR)-g.26 Recent evidence suggests a novel role for PPAR-g in protecting against age-dependent oxidative stress and endothelial dysfunction.27 Furthermore, telmisartan demonstrates neuroprotective effects by reducing angiotensin type 1 receptor signaling by upregulating type 2 signaling28 and increasing levels of a brain-derived neurotrophic factor.29 AR blockade with PPAR-g activation by telmisartan can help improve cognitive decline in type 2 diabetic mice.30 In the present study, we speculated that the protective effects of low-dose telmisartan were through PPAR-g activation. In conclusion, telmisartan reduced oxidative stress and pSyn accumulation in SHR-SR. PPAR-g activation and synergistic BP lowering may protect against oxidative brain damage because of hypertension.
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Supplementary Data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.12. 052.
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fect of angiotensin II type 1 receptor blockade in patients with coronary artery disease. Arterioscler Thromb Vasc Biol 2002;22:1845-1851. Tsuda M, Iwai M, Li JM, et al. Inhibitory effects of AT1 receptor blocker, olmesartan, and estrogen on atherosclerosis via anti-oxidative stress. Hypertension 2005; 45:545-551. Schrader J, Luders S, Kulschewski A, et al. The ACCESS Study: evaluation of Acute Candesartan Cilexetil Therapy in Stroke Survivors. Stroke 2003;34:1699-1703. Schrader J, Luders S, Kulschewski A, et al. Morbidity and mortality after stroke, eprosartan compared with nitrendipine for secondary prevention: principal results of a prospective randomized controlled study (MOSES). Stroke 2005;36:1218-1226. Maillard MP, Perregaux C, Centeno C, et al. In vitro and in vivo characterization of the activity of telmisartan: an insurmountable angiotensin II receptor antagonist. J Pharmacol Exp Ther 2002;302:1089-1095. Gohlke P, Weiss S, Jansen A, et al. AT1 receptor antagonist telmisartan administered peripherally inhibits central responses to angiotensin II in conscious rats. J Pharmacol Exp Ther 2001;298:62-70. Mogi M, Li JM, Tsukuda K, et al. Telmisartan prevented cognitive decline partly due to PPAR-gamma activation. Biochem Biophys Res Commun 2008;375:446-449. Wultz B, Sagvolden T, Moser EI, et al. The spontaneously hypertensive rat as an animal model of attention-deficit hyperactivity disorder: effects of methylphenidate on exploratory behavior. Behav Neural Biol 1990;53:88-102. Sabbatini M, Catalani A, Consoli C, et al. The hippocampus in spontaneously hypertensive rats: an animal model of vascular dementia? Mech Ageing Dev 2002;123:547-559. Sanz-Rosa D, Oubina MP, Cediel E, et al. Eplerenone reduces oxidative stress and enhances eNOS in SHR: vascular functional and structural consequences. Antioxid Redox Signal 2005;7:1294-1301. Kumai Y, Ooboshi H, Ago T, et al. Protective effects of angiotensin II type 1 receptor blocker on cerebral circulation independent of blood pressure. Exp Neurol 2008; 210:441-448. Suematsu M, Suzuki H, Delano FA, et al. The inflammatory aspect of the microcirculation in hypertension: oxidative stress, leukocytes/endothelial interaction, apoptosis. Microcirculation 2002;9:259-276. Ikeda K, Kitamura A, Machida H, et al. Effect of Undaria pinnatifida (Wakame) on the development of cerebrovascular diseases in stroke-prone spontaneously hypertensive rats. Clin Exp Pharmacol Physiol 2003;30:44-48. Chau KY, Ching HL, Schapira AH, et al. Relationship between alpha synuclein phosphorylation, proteasomal inhibition and cell death: relevance to Parkinson’s disease pathogenesis. J Neurochem 2009;110:1005-1013. Wienen W, Schierok HJ. Effects of telmisartan, hydrochlorothiazide and their combination on blood pressure and renal excretory parameters in spontaneously hypertensive rats. J Renin Angiotensin Aldosterone Syst 2001; 2:123-128. Burlacu A, Jinga V, Gafencu AV, et al. Severity of oxidative stress generates different mechanisms of endothelial cell death. Cell Tissue Res 2001;306:409-416. Didion SP, Faraci FM. Angiotensin II produces superoxide-mediated impairment of endothelial function in cerebral arterioles. Stroke 2003;34:2038-2042. Hanna IR, Taniyama Y, Szocs K, et al. NAD(P)H oxidasederived reactive oxygen species as mediators of angiotensin II signaling. Antioxid Redox Signal 2002;4:899-914.
TELMISARTAN PROTECTS OXIDATIVE BRAIN DAMAGE WITH PPAR-G ACTIVATION 22. Rajagopalan S, Kurz S, Munzel T, et al. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/ NADPH oxidase activation. Contribution to alterations of vasomotor tone. J Clin Invest 1996;97: 1916-1923. 23. Giasson BI, Duda JE, Murray IV, et al. Oxidative damage linked to neurodegeneration by selective alpha-synuclein nitration in synucleinopathy lesions. Science 2000;290: 985-989. 24. Fujiwara H, Hasegawa M, Dohmae N, et al. alpha-Synuclein is phosphorylated in synucleinopathy lesions. Nat Cell Biol 2002;4:160-164. 25. Sugeno N, Takeda A, Hasegawa T, et al. Serine 129 phosphorylation of alpha-synuclein induces unfolded protein response-mediated cell death. J Biol Chem 2008; 283:23179-23188. 26. Benson SC, Pershadsingh HA, Ho CI, et al. Identification of telmisartan as a unique angiotensin II receptor antago-
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nist with selective PPARgamma-modulating activity. Hypertension 2004;43:993-1002. Modrick ML, Kinzenbaw DA, Chu Y, et al. Peroxisome proliferator-activated receptor-gamma protects against vascular aging. Am J Physiol Regul Integr Comp Physiol 2012;302:R1184-R1190. Kurihara T, Ozawa Y, Shinoda K, et al. Neuroprotective effects of angiotensin II type 1 receptor (AT1R) blocker, telmisartan, via modulating AT1R and AT2R signaling in retinal inflammation. Invest Ophthalmol Vis Sci 2006;47:5545-5552. Ola MS, Ahmed MM, Abuohashish HM, et al. Telmisartan ameliorates neurotrophic support and oxidative stress in the retina of streptozotocin-induced diabetic rats. Neurochem Res 2013;38:1572-1579. Min LJ, Mogi M, Shudou M, et al. Peroxisome proliferatoractivated receptor-gamma activation with angiotensin II type 1 receptor blockade is pivotal for the prevention of blood-brain barrier impairment and cognitive decline in type 2 diabetic mice. Hypertension 2012;59:1079-1088.
Previously, we reported that reactive oxygen species and signaling molecules of angiotensin II produced lipid peroxides, degenerated proteins, and injured DNA after cerebral ischemia in normotensive Wistar rats. Here, we investigated the longterm effect of the angiotensin II type I receptor blocker telmisartan on oxidative stress and hyperphosphorylated a-synuclein accumulation in stroke-resistant spontaneously hypertensive rats (SHR-SR). At the age of 3 months, SHR-SR were divided into 3 treatment groups: SHR-SR vehicle (SHR/Ve), SHR-SR low-dose telmisartan (.3 mg/kg/day) (SHR/low), and SHR-SR high-dose telmisartan (3 mg/kg/day) (SHR/high). Immunohistologic analyses were conducted in these groups and Wistar rats at the age of 6, 12, and 18 months. The SHR/Ve group demonstrated more progressive increase in advanced glycation end product (AGE)-, 4-hydroxy-2nonenal (4-HNE)-, and phosphorylated a-synuclein (pSyn)-positive cells in the cerebral cortex and hippocampus compared with the Wistar group at 18 months. These expressions were reduced in the SHR/low group even without lowering blood pressure (BP), and expressions were dramatically suppressed in the SHR/high group with lowering of BP. These data suggest that persistent hypertension in SHR-SR strongly potentiate the markers of oxidative damage (AGEs and 4-HNE) and abnormal accumulation of pSyn, which were greatly suppressed by telmisartan in a dose-dependent manner without and with lowering of BP. Key Words: Strokeresistant spontaneously hypertensive rat—telmisartan—oxidative stress— phosphorylated a-synuclein—peroxisome proliferator–activated receptor gamma. Ó 2014 by National Stroke Association
Introduction We have reported that reactive oxygen species (ROS) and the signaling molecules of angiotensin II produce lipid peroxides, degenerate proteins, and injure DNA after cerebral ischemia in normotensive Wistar rats.1,2 Antihypertensive drugs, such as angiotensin II type I From the Department of Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. Received November 22, 2013; revision received December 23, 2013; accepted December 25, 2013. This work was partly supported by Grant-in-Aid for Scientific Research (B) 21390267 from the Ministry of Education, Science, Culture, and Sports of Japan, Grants-in-Aid from the Research Committee of CNS Degenerative Diseases (I. Nakano), and grants (H.
receptor blockers (ARBs), inhibit such ROS production and prevent cerebrovascular disorders.3-5 ARBs also show protective effects against stroke without lowering blood pressure (BP).6,7 Telmisartan is an ARB with high lipid solubility8,9 and has been expected to prevent atherosclerosis and metabolic disorders.10
Mizusawa, M. Nishizawa, H.Sasaki, G. Sobue) from the Ministry of Health, Labour, and Welfare of Japan. Address correspondence to Koji Abe, MD, PhD, Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmacy, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.12.052
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Figure 1. Representative photomicrographs of AGE-positive cell staining (A) and the number of AGE-positive cells per square millimeter of cerebral cortex (B) at 6, 12, and 18 months. Note the BP- and age-dependent increase in the number of AGE-positive cells (arrowheads). (The order of staining strength in the brain was SHR/ Ve . SHR/low . SHR/high . Wistar rat [scale bar 5 100 mm]. **P , .01 versus Wistar group, ##P , .01 versus SHR/Ve group.) Abbreviations: AGE, advanced glycation end product; BP, blood pressure; high, high-dose telmisartan group; low, low-dose telmisartan group; M, months; SHRSR, stroke-resistant spontaneously hypertensive rats; Ve, vehicle; Wis, Wistar rat.
Spontaneously hypertensive rats (SHR) and strokeresistant spontaneously hypertensive rats (SHR-SR) are genetic models that develop hypertension, followed by progressive neurobehavioral impairment,11 Alzheimer’s disease,12 and immune alterations.13 In SHR, telmisartan has been shown to improve autoregulation of cerebral blood flow and reduce generation of superoxide.14 Studies have also demonstrated that oxidative stress is induced in SHR-SR15 and stroke-prone spontaneously hypertensive rats,16 with the accumulation of hyperphosphorylated a-synuclein (pSyn).17 However, oxidative stress and pSyn accumulation under chronic hypertension have not been studied. SHR-SR live much longer than stroke-prone spontaneously hypertensive rats, so we investigated the long-term effect of telmisartan on oxidative stress and pSyn accumulation in SHR-SR.
Materials and Methods All experimental procedures were approved by the Animal Committee of the Graduate School of Medicine and Dentistry, Okayama University (Okayama, Japan).
Animals and Drug Preparation Seven-week-old male Wistar rats and SHR-SR were provided by the Disease Model Cooperative Re-
search Association (Kyoto, Japan) and placed on a basal diet. At the age of 3 months, the Wistar rats mentioned previously (n 5 20) were started on a daily dose of .5% methyl cellulose (MC) in .1 mL of water by oral gavage as the normotensive control group for 3, 9, and 15 months until killed. The SHR-SR mentioned previously (n 5 54) were divided into 3 treatment groups: the SHR-SR vehicle group (SHR/Ve, n 5 17), SHR-SR low-dose telmisartan group in which the BP did not fall significantly (SHR/ low, n 5 19), and SHR-SR high-dose telmisartan group in which the BP fell by30 mm Hg or more (SHR/high, n 5 18). Rats received daily oral doses of .5% MC only (SHR-SR/Ve), .5% MC plus low-dose telmisartan (.3 mg/kg/day), or .5% MC plus high-dose telmisartan (3 mg/kg/day) by oral gavage for 3, 9, and 15 months until killed. The telmisartan dose was determined as described previously.14,18 Telmisartan was provided by Boehringer Ingelheim (Ingelheim am Rhein, Germany) and was given to the 2 rat groups as a suspension with .5% MC in .1 mL of water every day. At the age of 6, 12, or 18 months, rats were perfused transcardially with chilled heparinized saline (5 U/mL), followed by 4% paraformaldehyde in phosphate buffer (PB) under deep anesthesia (pentobarbital 20 mg/250 g body weight). After decapitation, brains were removed
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Figure 2. Representative photomicrographs 4HNE-positive cell staining (A) and the number of 4-HNE-positive cells per square millimeter of the cerebral cortex (B) at 6, 12, and 18 months. Note the BP- and age-dependent increase in 4HNE staining, and dose-dependent reduction by telmisartan (scale bar 5 100 mm) (**P , .01 versus Wistar group, ##P , .01 versus SHR/Ve group). Abbreviations: BP, blood pressure; 4HNE, 4-hydroxy-2-nonenal; M, months; SHR, spontaneously hypertensive rats; Ve, vehicle; Wis, Wistar rat.
and brain weights measured. The present study focused on the oxidative stress and pSyn in a whole project in the comparison of Wistar rats and SHR-SR and the effect of telmisartan on SHR-SR.
Immunohistochemical Analyses Brains were immersed and fixed in 4% paraformaldehyde with .1 M of PB (pH 7.6) for 8 hours, embedded in paraffin, and 5-mm thick sections were prepared for immunostaining. For immunostainings for advanced glycation end products (AGEs), 4-hydroxy-2-nonenal (4-HNE), and pSyn, brain sections were pretreated by heating thrice in a 500-W microwave for 5 min in 10 mM (pH 6.0) citric acid buffer. These pretreated sections were then immersed in .5% periodic acid to block intrinsic peroxidase and treated with 5% normal horse serum in 50 mM phosphate-buffered saline (pH 7.4) containing .05% Tween 20 to block nonspecific antibody responses. Finally, they were incubated overnight with each primary antibody. The following primary antibodies were used: mouse antiAGE monoclonal antibody (1:100 dilution; TRANCE GENIC, Inc, Kumamoto, Japan), mouse anti-4-HNE monoclonal antibody (1:50; JaICA, Shizuoka, Japan), and mouse anti-pSyn monoclonal antibody (1:500; Wako Pure Chemical Industries, Ltd, Osaka, Japan). A biotinylated antimouse antibody was used as the secondary antibody.
Specific labeling was visualized using a Vectastain Elite ABC kit (Vector, Burlingame, CA). To guarantee specificstaining primary antibodies, brain sections were also stained without primary antibodies.
Detection Stained sections were digitized with a digital microscope camera (BX-51; Olympus Optical Co, Japan). The numbers of AGE-positive cells, 4-HNE-positive cells, and pSyn-positive cells per each 1-mm2 cerebral cortex in hemispheric coronal sections were counted at 3 levels (frontal cortex, basal ganglia, and posterior hippocampus). Three serial sections were used for each level, and all cell counts in the cerebral cortex per animal were determined and added. Furthermore, AGE-, 4-HNE-, and pSyn-positive cells were quantified by counting stained cells in the CA1 and CA3 regions of the bilateral hippocampus and expressed as the percentage of AGE-, 4-HNE-, and pSyn-positive cells over the total number of cells in these regions.
Statistical Analyses Data are expressed as the mean 6 standard deviation. Statistical analyses were conducted using an analysis of variance with repeated measures (multiple comparisons).
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Figure 3. Representative photomicrographs of pSyn-positive cell staining (A) and the number of pSyn-positive cells per square millimeter of cerebral cortex (B) at 6, 12, and 18 months. pSyn was progressively labeled with age in SHR/Ve, which was greatly reduced in the other groups (arrowheads) (scale bar 5 100 mm) (**P , .01 versus Wistar group, ##P , .01 versus SHR/Ve group). Abbreviations: M, months; pSyn, phosphorylated a-synuclein; SHR, spontaneously hypertensive rats; Ve, vehicle; Wis, Wistar rat.
Planned comparisons were used for Tukey–Kramer post hoc analyses. A value of P less than .05 was considered significant. All statistical analyses were undertaken with Statcelver2 (OMS Inc, Tokorozawa, Japan).
Results Immunohistochemical Analyses in the Cerebral Cortex AGE Staining in the Cerebral Cortex In Wistar rats, AGE was scarcely labeled in cerebrocortical cells at 6 months (Fig 1A, a), but became evident gradually in the cytoplasm at 12 and 18 months (Fig 1A, e, i). The mean numbers of AGE-positive cells in the cerebral cortex were (per square millimeter) 18.2 6 13.0 at 6 months, 28.5 6 8.5 at 12 months, and 32.1 6 23.4 at 18 months (Fig 1B, open bars). In the SHR/Ve group, AGE-positive cells were already evident at 6 months, and the numbers in the cerebral cortex increased with age (Fig 1A, b, f, j), that is, they were (per square millimeter) 59.7 6 35.4 at 6 months, 72.4 6 44.4 at 12 months, and 123.3 6 41.6 (**P , .01 versus Wistar group) at 18 months (Fig 1B). In the 2 telmisartan groups, the numbers of AGEpositive cells in the cerebral cortex were (per square milli-
meter) 27.7 6 16.3 at 6 months, 33.6 6 19.2 at 12 months, and 44.1 6 40.4 (##P , .01 versus SHR/Ve group) at 18 months in the SHR/low group. In the SHR/high group, the numbers of AGE-positive cells in the cerebral cortex were (per square millimeter) 15.7 6 6.2 at 6 months, 31.1 6 16.2 at 12 months, and 32.5 6 17.4 (##P ,.01 versus SHR/Ve group) at 18 months (Fig 1, B). 4-HNE Staining in the Cerebral Cortex In Wistar rats, 4-HNE was scarcely labeled in cerebrocortical cells at 6 months (Fig 2A, a), but became evident gradually in the cytoplasm from 12-18 months (Fig 2A, e, i). The mean numbers of 4-HNE-positive cells in the cerebral cortex were (per square millimeter) 82.8 6 5.8 at 6 months, 84.2 6 18.7 at 12 months, and 94.1 6 30.0 at 18 months (Fig 2B, open bars). 4-HNE was already clearly labeled in the cytoplasm of cerebrocortical cells at 6 months in the SHR/Ve group, which became much stronger at 12 and 18 months (Fig 2A, b, f, j). The numbers of 4-HNE-positive cells in the cerebral cortex were (per square millimeter) 221.1 6 31.2 (**P , .01 versus Wistar group) at 6 months, 288.1 6 67.7 (**P , .01 versus Wistar group) at 12 months, and 311.7 6 109.1 (**P ,.01 versus Wistar group) at 18 months (Fig 2B).
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Figure 4. Representative photomicrographs of AGE-positive cell staining (A) and the ratio of AGE-positive cells of CA1 and CA3 in the hippocampus (B) at 6, 12, and 18 months. Note the BP- and age-dependent increase in AGE staining (arrowheads), and dose-dependent reduction by telmisartan (scale bar 5 100 mm) (**P , .01 versus Wistar group, ##P , .01 versus SHR/Ve group). Abbreviations: AGE, advanced glycation end product; BP, blood pressure; M, months; SHR, spontaneously hypertensive rats; Ve, vehicle; Wis, Wistar rat.
In the 2 telmisartan groups, 4-HNEwas already labeled in the cytoplasm of cerebrocortical cells (Fig 2A, c, d) and increased slightly at 12 and 18 months (Fig 2A, g, h, k, l). The numbers of 4-HNE-positive cells in the cerebral cortex in the SHR/low group were (per square millimeter) 120.3 6 12.1 (##P ,.01 versus SHR/Ve group) at 6 months, 126.4 6 13.0 (##P , .01 versus SHR/Ve group) at 12 months, and 126.8 6 12.1 (##P , .01 versus SHR/Ve group) at 18 months, and those in the SHR/high group were 91.9 6 17.3 (##P , .01 versus SHR/Ve group) at 6 months, 96.7 6 58.3 (##P , .01 versus SHR/Ve group) at 12 months, and 103.8 6 34.9 (##P , .01 versus SHR/ Ve group) at 18 months (Fig 2B). pSyn Staining in the Cerebral Cortex pSyn was scarcely labeled in cerebrocortical cells at 6 months (Fig 3A, a), but became evident gradually in the cytoplasm at 12 and 18 months (Fig 3A, e, i). The mean numbers of pSyn-positive cells in the cerebral cortex were (per square millimeter) 1.3 6 1.1 at 6 months, 2.9 6 2.0 at 12 months, and 5.8 6 3.6 at 18 months (Fig 3B, open bars). pSyn was already weakly labeled in cerebrocortical cells at 6 months and became evident in the cytoplasm at 12 and 18 months in the SHR/Ve group (Fig 3A, b, f, j). The numbers of pSyn-positive cells in the cerebral cortex were (per square millimeter) 17.8 6 2.0 (**P , .01 versus Wistar group) at 6 months, 20.6 6 4.8 (**P , .01
versus Wistar group) at 12 months, and 22.0 6 11.1 (**P , .01 versus Wistar group) at 18 months (Fig 3B). In the 2 telmisartan groups, pSyn was scarcely labeled in cerebrocortical cells at 6 months (Fig 3A, c, d) and increased only slightly in the cytoplasm at 12 and 18 months (Fig 3A, g, h, k, l). The numbers of pSynpositive cells in the cerebral cortex in the SHR/low group were (per square millimeter) 5.4 6 5.7 (##P , .01 versus SHR/Ve group) at 6 months, 7.5 6 6.7 (##P , .01 versus SHR/Ve group) at 12 months, and 8.6 6 6.0 (##P , .01 versus SHR/Ve group) at 18 months (Fig 3B), and those in the SHR/high group were 4.0 6 5.1 (##P , .01 versus SHR/Ve group) at 6 months, 4.4 6 3.2 (##P , .01 versus SHR/Ve group) at 12 months, and 5.4 6 4.5 (##P , .01 versus SHR/Ve group) at 18 months (Fig 3B).
Immunohistochemical Analyses in the Hippocampus AGE Staining in the Hippocampus In Wistar rats, AGE was scarcely labeled in the cytoplasm of CA1 and CA3 cells throughout 6-18 months (Fig 4A, a, e, i). The ratios of AGE-positive cells in CA1 and CA3 were .03 6 .01 at 6 months, .09 6 .10 at 12 months, and .10 6 .09 at 18 months, and no change was detected with age in the Wistar group (Fig 4B, open bars). In the SHR/Ve group, AGE was slightly labeled in CA1 and CA3 cells at 6 months, and this staining became more
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Figure 5. Representative photomicrographs of 4-HNE-positive cell staining (A) and the ratio of 4-HNE-positive cells of CA1 and CA3 in the hippocampus (B) at 6, 12, and 18 months. Note the BP- and age-dependent increase in the number of 4-HNE-positive cells. (The order of staining strength in the brain was SHR/Ve . SHR/ low . SHR/high . Wistar rat [scale bar 5 100 mm]. *P , .05 versus Wistar group, **P , .01 versus Wistar group, #P , .05 versus SHR/Ve group, ##P ,.01 versus SHR/Ve group.) Abbreviations: BP, blood pressure; 4-HNE, 4hydroxy-2-nonenal; M, months; SHR, spontaneously hypertensive rats; Ve, vehicle; Wis, Wistar rat.
evident in the cytoplasm at 12 and 18 months (Fig 4A, b, f, j). The ratios of AGE-positive cells of CA1 and CA3 were .29 6 .07 (**P , .01 versus Wistar group) at 6 months, .30 6 .07 (**P , .01 versus Wistar group) at 12 months, and .51 6 .11 (**P ,.01 versus Wistar group) at 18 months (Fig 4B). Between the 2 telmisartan groups, AGE was labeled less in the cytoplasm of CA1 and CA3 cells (Fig 4A, c, d). The ratios of AGE-positive cells of CA1 and CA3 in the SHR/low group were .09 6 .04 (##P , .01 versus SHR/Ve group) at 6 months, .11 6 .06 (##P , .01 versus SHR/Ve group) at 12 months, and .16 6 .09 (##P , .01 versus SHR/Ve group) at 18 months (Fig 4B), and those in the SHR/high group were .09 6 .06 (#P , .05 versus SHR/Ve group) at 6 months, .10 6 .06 (##P , .01 versus SHR/Ve group) at 12 months, and .14 6 .16 (##P , .01 versus SHR/Ve group) at 18 months (Fig 4B). 4-HNE Staining in the Hippocampus In Wistar rats, 4-HNE was scarcely labeled in CA1 and CA3 cells throughout 6-18 months, with no agedependent change in the cytoplasm (Fig 5A, a, e, i). The ratios of 4-HNE-positive cells in CA1 and CA3 were .07 6 .02 at 6 months, .12 6 .02 at 12 months, and .14 6 .04 at 18 months (Fig 5B, open bars). 4-HNE was already labeled in CA1 and CA3 cells at 6 months in the SHR/Ve group, but almost no change
in the cytoplasm was observed at 12 and 18 months (Fig 5A, b, f, j). The ratios of 4-HNE-positive cells of CA1 and CA3 were .48 6 .2 (**P , .01 versus Wistar group) at 6 months, .49 6 .26 (*P , .05 versus Wistar group) at 12 months, and .51 6 .2 (**P , .01 versus Wistar group) at 18 months (Fig 5B). In the 2 telmisartan groups, 4-HNE was weakly labeled in the cytoplasm of CA1 and CA3 cells at 6 months (Fig 5A, c, d). The ratios of 4-HNE-positive cells of CA1 and CA3 in the SHR/low group were .19 6 .05 (##P ,.01 versus SHR/ Ve group) at 6 months, .21 6 .12 at 12 months, and .21 6 .1 (##P , .01 versus SHR/Ve group) at 18 months, and those in the SHR/high group were .15 6 .02 (##P , .01 versus SHR/Ve group) at 6 months, .17 6 .18 (#P , .05 versus SHR/Ve group) at 12 months, and .18 6 .08 (##P , .01 versus SHR/Ve group) at 18 months (Fig 5B). pSyn Staining in the Hippocampus In Wistar rats, pSyn was scarcely labeled in CA1 and CA3 cells throughout 6-18 months with no agedependent change in the cytoplasm (Fig 6A, a, e, i). The ratios of pSyn-positive cells in CA1 and CA3 were .02 6 .02 at 6 months, .02 6 .02 at 12 months, and .02 6 .01 at 18 months (Fig 6B, open bars). pSyn was already clearly labeled in CA1 and CA3 cells at 6 months in the SHR/Ve group and became evident gradually in the cytoplasm at 12 and 18 months (Fig 6A,
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Figure 6. Representative photomicrographs of pSyn-positive cell staining (A) and the ratio of pSyn-positive cells of CA1 and CA3 in the hippocampus (B) at 6, 12, and 18 months. Note that pSyn was labeled progressively with age in the SHR/Ve group, which was greatly reduced in the other groups (scale bar 5 100 mm) (**P , .01 versus Wistar group, ##P , .01 versus SHR/Ve group). Abbreviations: M, months; pSyn, phosphorylated a-synuclein; SHR, spontaneously hypertensive rats; Ve, vehicle; Wis, Wistar rat.
b, f, j). The ratios of pSyn-positive cells of CA1 and CA3 were .13 6 .03 (**P ,.01 versus Wistar group) at 6 months, .14 6 .04 (**P , .01 versus Wistar group) at 12 months, and .15 6 .06 (**P , .01 versus Wistar group) at 18 months (Fig 6B). In the 2 telmisartan groups, pSyn was weakly labeled in the cytoplasm of CA1 and CA3 cells at 6 months (Fig 5A, c, d) and increased slightly in the cytoplasm at 12 and 18 months (Fig 6A, g, h, k, l). The ratios of pSynpositive cells of CA1 and CA3 in the SHR/low group were .02 6 .02 (##P , .01 versus SHR/Ve group) at 6 months, .04 6 .05 (##P , .01 versus SHR/Ve group) at 12 months, and .05 6 .02 (##P , .01 versus SHR/Ve group) at 18 months, and those in the SHR/high group were .02 6 .01 (##P , .01 versus SHR/Ve group) at 6 months, .03 6 .02 (##P , .01 versus SHR/Ve group) at 12 months, and .03 6 .05 (##P , .01 versus SHR/Ve group) at 18 months (Fig 6B).
Discussion The SHR/Ve group, with mean blood pressure (MBP) 175.6 6 22.6 mm Hg at 12 months, demonstrated progressive increases in AGE-, 4-HNE-, and pSyn-positive cells in the cerebral cortex and hippocampus with aging (Figs 1-6). However, telmisartan reduced these changes even without lowering BP (SHR/low; MBP 168.9 6 24.0 mm Hg at
12 months), and high-dose telmisartan showed further improvement by lowering of BP (SHR/high; MBP 149.0 6 15.0 mm Hg at 12 months) (Figs 1-6). Previously, we reported that ROS produce lipid peroxides, degenerate proteins, and injure DNA after cerebral ischemia in normotensive Wistar rats.1,2 ROS is produced by nicotinamide adenine dinucleotide phosphate (NAD [P]H) oxidase that is activated by morbid stimulation such as hypertension. ROS inactivates nitric oxide causing functional disorder and cell death of vascular endothelium and smooth muscle.19,20 In addition, angiotensin II is also an activator of NAD(P)H oxidase, produces ROS,21 and increases oxidative stress.22 To investigate the long-term effect of hypertension, we studied oxidative stress in SHR-SR for 18 months. We found that persistent hypertension in SHR-SR demonstrated a progressive increase in oxidative stress (AGE and 4-HNE) and pSyn accumulation compared with Wistar rats (Figs 1-6). These expressions were suppressed by telmisartan in a dose-dependent manner (Figs 1-6). Of note, low-dose telmisartan ameliorated these changes without lowering BP, indicating an antioxidative effect independent of BP. The results speculated that telmisartan decreased ROS production through inhibiting angiotensin II. a-Synuclein is accumulated in the presynaptic terminals of the cerebral cortex, hippocampus, substantia
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nigra, thalamus, and cerebellum. Therefore, it is involved in neurotransmission. a-Synuclein is phosphorylated only at z4% in normal rat brains, but pSyn in patients with synucleinopathy, such as Parkinson’s disease and dementia with Lewy bodies, have greater than 90% pSyn in the postmortem brain.23,24 In addition, oxidative stress hyperphosphorylates a-synuclein, resulting in its accumulation.17 Abnormal accumulation of pSyn can lead to the death of neuronal cells.25 Persistent hypertension induced abnormal accumulation of pSyn in the cerebral cortex and hippocampus (Figs 3, 6), demonstrating that long-lasting hypertension and oxidative stress are correlated with pSyn accumulation. The dose-dependent effect of telmisartan on pSyn reduction suggests a protective effect probably through antioxidative mechanisms with further protection by a BPlowering mechanism (Figs 3, 6). ARBs inhibit ROS production,4,5 and telmisartan shows antihypertensive effects along with activation of peroxisome proliferator-activated receptor (PPAR)-g.26 Recent evidence suggests a novel role for PPAR-g in protecting against age-dependent oxidative stress and endothelial dysfunction.27 Furthermore, telmisartan demonstrates neuroprotective effects by reducing angiotensin type 1 receptor signaling by upregulating type 2 signaling28 and increasing levels of a brain-derived neurotrophic factor.29 AR blockade with PPAR-g activation by telmisartan can help improve cognitive decline in type 2 diabetic mice.30 In the present study, we speculated that the protective effects of low-dose telmisartan were through PPAR-g activation. In conclusion, telmisartan reduced oxidative stress and pSyn accumulation in SHR-SR. PPAR-g activation and synergistic BP lowering may protect against oxidative brain damage because of hypertension.
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Supplementary Data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.12. 052.
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