Nonhypotensive Dose of Telmisartan Attenuates Cognitive Impairment Partially Due to Peroxisome Proliferator-Activated Receptor-γ Activation in Mice With Chronic Cerebral Hypoperfusion
Background and Purpose— The effect of telmisartan, an angiotensin II Type 1 receptor blocker with peroxisome proliferator-activated receptor-γ-modulating activity, was investigated against spatial working memory disturbances in mice subjected to chronic cerebral hypoperfusion.
Methods— Adult C57BL/6J male mice were subjected to bilateral common carotid artery stenosis using external microcoils. Mice received a daily oral administration of low-dose telmisartan (1 mg/kg per day), high-dose telmisartan (10 mg/kg per day), or vehicle with or without peroxisome proliferator-activated receptor-γ antagonist GW9662 (1 mg/kg per day) for all treatments for 30 days after bilateral common carotid artery stenosis. Cerebral mRNA expression of monocyte chemoattractant protein-1 and tumor necrosis factor-α was measured 30 days after bilateral common carotid artery stenosis, and postmortem brains were analyzed for demyelinating change with Klüver-Barrera staining and immunostained for glial, oxidative stress, and vascular endothelial cell markers. Spatial working memory was assessed by the Y-maze test.
Results— Mean systolic blood pressure and cerebral blood flow did not decrease with low-dose telmisartan but significantly decreased with high-dose telmisartan. Low-dose telmisartan significantly attenuated, but high-dose telmisartan provoked, spatial working memory impairment with glial activation, oligodendrocyte loss, and demyelinating change in the white matter. Such positive effects of low-dose telmisartan were partially offset by cotreatment with GW9662. Consistent with this, low-dose telmisartan reduced the degree of oxidative stress of vascular endothelial cells and the mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-α compared with vehicle.
Conclusions— Anti-inflammatory and antioxidative effects of telmisartan that were exerted in part by peroxisome proliferator-activated receptor-γ activation, but not its blood pressure-lowering effect, have protective roles against cognitive impairment and white matter damage after chronic cerebral hypoperfusion.
Drugs that target the renin–angiotensin system seem to have particular potential for prevention of dementias, including Alzheimer disease and vascular dementia. The Perindopril Protection Against Recurrent Stroke Study (PROGRESS) has suggested a protective effect of angiotensin-converting enzyme inhibitors on cognitive function in patients with stroke.1 Moreover, the Study on Cognition and Prognosis in the Elderly (SCOPE) trial demonstrated a positive effect of the angiotensin II Type 1 receptor blocker (ARB), candesartan, in a subgroup of elderly hypertensive patients with mild cognitive impairment.2 Notably, a prospective cohort analysis of 819 491 participants suggested that ARBs are associated with a significant reduction in the incidence and progression of dementia, even compared with angiotensin-converting enzyme inhibitors.3
No benefit was found in cognitive performance after administration of the ARB, telmisartan, at the subacute stage (within 15 days) after stroke in the Prevention Regimen for Effectively Avoiding Second Strokes (PRoFESS) study.4 However, in vitro studies have suggested that telmisartan, the strongest peroxisome proliferator-activated receptor-γ (PPAR-γ) activator among ARBs,5 may protect oligodendrocytes and neurons through a reduction of brain inflammation through PPAR-γ activation and AT1 receptor blockade. Telmisartan has structural similarities to the PPAR-γ ligand pioglitazone and thus could act as a partial agonist of PPAR-γ.5 PPAR-γ, one of the nuclear receptors, plays a critical role in a variety of biological processes, including angiogenesis, inflammation, oxidative stress, glucose metabolism, and adipogenesis.5 Moreover, PPAR-γ activation in the brain has been suggested as a protective effect against Alzheimer disease through its multifaceted effects, including anti-inflammation and amyloid-β clearance.6 Therefore, in the PRoFESS study, excessive lowering of blood pressure (BP) in the period with cerebrovascular autoregulatory dysfunction may have affected the cerebral circulation and neuronal function, although other factors could also be involved.
The present study is therefore designed to explore the multifaceted effects of telmisartan on cognitive disturbances in a mouse model of vascular dementia by administering a hypotensive or a nonhypotensive dose of telmisartan. This model of chronic cerebral hypoperfusion, which is produced by placing microcoils bilaterally on the common carotid arteries, invariably exhibits glial activation, oxidative stress, inflammation, demyelinating change, and axonal loss in the white matter with resultant spatial working memory deficits.7,8 This in vivo system will help determine whether telmisartan affects vascular autoregulatory function and whether and how telmisartan exerts its protective effect against cognitive impairment related to white matter damage.
Materials and Methods
The experimental protocol is shown in Figure 1. Nine-week-old male C57BL/6J mice (weighing 24 to 29 g; CLEA, Tokyo, Japan) were fed with the pelleted chow (MF) containing low-dose telmisartan (1 mg/kg per day), high-dose telmisartan (10 mg/kg per day), or vehicle with or without PPAR-γ antagonist GW9662 (1 mg/kg per day; Sigma-Aldrich) for all treatments, beginning from 7 days before the bilateral common carotid artery stenosis (BCAS) surgery until 30 days post-BCAS. Immediately after spatial working memory was assessed by the Y-maze test, mice were euthanized for histological and real-time reverse transcriptase–polymerase chain reaction examination 30 days post-BCAS.
Surgical Procedure of BCAS
Under anesthesia with halothane (2%), both common carotid arteries were exposed through a midline cervical incision, and a microcoil with an inner diameter of 0.18 mm was applied to the bilateral common carotid arteries. See Supplemental Method I for details (available at http://stroke.ahajournals.org).7,8
Systolic BP and Cerebral Blood Flow Measurements
Various doses of telmisartan (0 to 100 mg/kg per day) were administered and the BP measured for determining nonhypotensive and hypotensive doses of telmisartan. Then, in mice receiving the predetermined nonhypotensive or hypotensive dose of telmisartan or vehicle, systolic BP and cerebral blood flow (CBF) were monitored at 7 days before BCAS (before starting telmisartan treatment), immediately before BCAS, and 2 hours, 1 day, 3 days, 7 days, 14 days, and 30 days after BCAS. See Supplemental Method II for details.
Histochemical Evaluation of White Matter Lesions, Glial Activation, and Oxidative Stress
The mouse brains were analyzed for demyelinating change with Klüver-Barrera staining and immunostained for glial fibrillary acidic protein (a marker of astrocyte), ionized calcium binding adaptor molecule-1 (Iba-1; microglia), glutathione S-transferase-π (GST-π; oligodendrocyte), 8-hydroxy-deoxyguanosine (8-OHdG; oxidative stress), and CD31 (vascular endothelial cell). See Supplemental Method III for details.7
Quantitative Real-Time Reverse Transcriptase–Polymerase Chain Reaction
Cerebral mRNA levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were assessed by quantitative real-time reverse transcriptase–polymerase chain reaction pre-BCAS and 30 days post-BCAS. Detailed procedures are described in Supplemental Method IV.
Y-Maze Test for Spatial Working Memory Assessment
Spatial working memory was assessed by the Y-maze test. The detail of the Y-maze test protocol is described in Supplemental Method V.
Blood Concentration of Telmisartan
See Supplemental Method VI for details.
All values are expressed as means±SEM in the text and figures. One-way analysis of variance was used to evaluate significant differences among groups except when otherwise stated. When a statistically significant effect was found, a post hoc Tukey test or Tukey-Kramer test was performed to detect the difference between the groups. Temporal profiles of systolic BP and CBF were analyzed by 2-way repeated-measures analysis of variance followed by a post hoc Tukey test. Differences with P<0.05 were considered statistically significant in all statistical analyses used.
Systolic BP and CBF After Telmisartan Administration
Treatment with telmisartan, ≤1 mg/kg per day, did not result in a significant reduction in BP, whereas treatment with telmisartan ≥3 mg/kg per day resulted in a significant reduction in BP (Figure 2A). CBF was not significantly reduced ≤1 mg/kg per day but began to decrease at 3 mg/kg per day (Figure 2B). A nonhypotensive dose of 1 mg/kg per day or a hypotensive dose of 10 mg/kg per day was subsequently administered. Temporal profiles of systolic BP and CBF were not affected by administration of a nonhypotensive dose of telmisartan or addition of GW9662 (Figure 2C–E). CBF gradually recovered after BCAS in mice with vehicle or a nonhypotensive dose of telmisartan but not in those with a hypotensive dose of telmisartan (Figure 2E).
The mortality rates were 10% at ≤1 mg/kg per day in the telmisartan-treated group after BCAS surgery. The mortality rate increased to 30% at 3 mg/kg per day in the telmisartan-treated group and 50% at 10 mg/kg per day in the telmisartan-treated group. Eighty percent at 50 or 100 mg/kg per day in the telmisartan-treated group died within 3 days post-BCAS.
Effects of Telmisartan on Glial Activation, Oligodendrocyte Restoration, and White Matter Lesion in Mouse Brain With Chronic Cerebral Hypoperfusion
Immunohistochemical analysis showed that in response to ischemic insults, resting astrocytes and microglia appeared to enter a reactive state due to apparent morphological changes characterized by thick dendritic formation. Such morphological changes, however, were attenuated by a nonhypotensive dose of telmisartan (Figure 3; compare 3A and 3C and compare 3I and 3K). Both the number of glial fibrillary acidic protein-positive astrocytes and Iba-1-positive microglia were significantly reduced in both the corpus callosum and anterior commissure from a nonhypotensive dose of telmisartan-treated BCAS mice compared with the vehicle-treated BCAS mice (Figure 3G–H, O–P). Such effects of low-dose telmisartan were partially offset by cotreatment with GW9662 (Figure 3; compare 3C and 3D and compare 3K and 3L).
Next, a hypotensive dose of telmisartan was examined to assess whether it ameliorated glial activation in BCAS-treated mice. In contrast to a nonhypotensive dose, a hypotensive dose of telmisartan caused substantial glial activation in the white matter (Figure 3; compare 3C and 3E and compare 3K and 3M). Cotreatment with GW9662 did not lead to additional glial changes in the white matter of mice with vehicle (Figure 3; compare 3A and 3B and compare 3I and 3J) or high-dose telmisartan (Figure 3; compare 3E and 3F and compare 3M and 3N).
Klüver-Barrera staining showed that white matter lesions were significantly attenuated in the nonhypotensive group compared with the vehicle group (Figure 4; compare 4A and 4C). Although patterns in oligodendrocytes arrangement could not be seen in the vehicle-treated mice (Figure 4A), alignment in a row formation could be seen in the group given a nonhypotensive dose of telmisartan (Figure 4C). Such effects of low-dose telmisartan were also partially offset by GW9662 (Figure 4; compare 4C and 4D) with significant differences (Figure 4G–H). In contrast, high-dose telmisartan did not attenuate white matter lesions (Figure 4; compare 4A and 4E). There were no significant histological differences between vehicle and high-dose telmisartan-treated mice with or without GW9662. In addition, administration of GW9662 had no effects on morphology of the white matter in sham-operated mice (data not shown), vehicle-treated, BCAS-operated mice (Figure 4; compare 4A and 4B), and high-dose telmisartan-treated, BCAS-operated mice (Figure 4; compare 4E and 4F). White matter lesion Grade 3 (disappearance of myelinated fibers) was only partially (approximately 10% of the white matter) observed in mice with vehicle or high-dose telmisartan.
In addition, the number of GST-π-positive oligodendrocytes of the vehicle-treated mice were significantly decreased in the white matter compared with that of mice treated with a nonhypotensive dose of telmisartan (Figure 4I–K).
Telmisartan Attenuates mRNA Expression of Inflammatory Cytokines in Mouse Brain With Chronic Cerebral Hypoperfusion
Cerebral mRNA expression of inflammatory cytokines such as MCP-1 and TNF-α was significantly increased after the BCAS but significantly attenuated by a nonhypotensive dose of telmisartan 30 days post-BCAS (Figure 4L–M).
Vascular Endothelial Oxidative Stress in Mouse Brain With Chronic Cerebral Hypoperfusion Was Ameliorated by Telmisartan
To further explore the antioxidative effect of telmisartan, 8-OHdG-positive vascular endothelial cells of the brain were assessed. The number of CD31-positive vascular endothelial cells positive for 8-OHdG was markedly reduced by a nonhypotensive dose of telmisartan (Figure 5; compare 5A and 5C). The difference was statistically significant as assessed by 8-OHdG/CD31-positive area (%; the percentage of 8-OHdG-positive area to CD31-positive area; Figure 5J). Such antioxidative effects of low-dose telmisartan were partially offset by cotreatment with GW9662 (Figure 5; compare 5C and 5D). By contrast, high-dose telmisartan showed an attenuated antioxidative effect in comparison to low-dose telmisartan (Figure 5; compare 5A, 5C, and 5E). Cotreatment with GW9662 did not lead to additional histological changes in mice with vehicle (Figure 5; compare 5A and 5B) or high-dose telmisartan (Figure 5; compare 5E and 5F).
Spatial Working Memory in Mice With Chronic Cerebral Hypoperfusion Was Restored by a Nonhypotensive Dose of Telmisartan
Finally, we analyzed spatial working memory of BCAS mice by the Y-maze test as the final functional output. The percentage of alternation behaviors significantly decreased in vehicle-treated BCAS mice compared with the sham-operated mice but significantly increased in a nonhypotensive dose of telmisartan-treated mice. Such effects of low-dose telmisartan were partially offset by cotreatment with GW9662. Hypotensive doses of telmisartan-treated mice manifested in further impaired working memory (Figure 6A). There were no significant differences in the number of entries to each arm, which was considered to reflect locomotor activity, among the 5 groups (Figure 6B). These results suggest that a nonhypotensive dose of telmisartan, but not a hypotensive dose, improved spatial working memory of BCAS-operated mice.
Blood Concentration of Telmisartan
Plasma concentration after 1 mg/kg per day administration of telmisartan for 7 days was 142.86±14.85 ng/mL (n=4, values are expressed as mean±SEM), a lower limit of BP-lowering effect in humans.
This study showed that (1) a nonhypotensive dose of telmisartan alleviated microglial/astroglial activation, endothelial oxidative stress, oligodendrocyte loss, and demyelinating changes in the white matter of the mice with chronic cerebral hypoperfusion; (2) such protective effects against the white matter lesions were at least partially mediated by anti-inflammatory and antioxidative effects that were exerted in part by PPAR-γ activation; (3) by contrast, a hypotensive dose of telmisartan did not induce such positive effects in BCAS-operated mice in the white matter; and (4) a nonhypotensive, but not a hypotensive, dose of telmisartan ameliorated cognitive decline of the BCAS-operated mice. Thus, the protective effects of telmisartan against white matter damage and cognitive impairment are exerted by its multifaceted effects, partially through PPAR-γ activation, but these are abolished by its BP-lowering effects when given at a higher dose. Thus, telmisartan should be considered for putative treatment for subcortical vascular dementia, although strict monitoring of BP is required.
Telmisartan is an ARB with a high degree of lipophilicity and is able to cross the blood–brain barrier. Telmisartan inhibits TNF-α-induced nuclear factor-κB activation, mainly through AT1 receptor blockade.9 In addition, telmisartan suppresses MCP-1 expression through AT1 receptor blockade and PPAR-γ activation.10 Genetic deletion of AT1 receptor protects against damage due to brain ischemia.11 With its synergistic effects of AT1 receptor blockade and PPAR-γ activation, telmisartan may exert multiple beneficial effects, including an antioxidative and anti-inflammatory effect, as shown in this study.
The nonhypotensive dose of telmisartan suppressed superoxide production from the vessel wall without lowering CBF. Telmisartan reduces NADPH oxidase activity10; therefore, administration of telmisartan in such a low dose seems to decelerate the free radical system in the chronically hypoperfused mouse brain. However, a hypotensive dose of telmisartan substantially increased the degree of endothelial oxidative stress together with glial activation, white matter lesions, and spatial working memory deficits. Because mice with severe CBF reduction (approximately lower than 50% of the baseline level) died and were subsequently excluded from the analysis, such detrimental effects of high-dose telmisartan may even be underestimated. ARBs protect against ischemic cerebral injury, independently of BP.12 The benefit of normotension over hypotension lies in the absence of hypotension-induced aggravation of ischemic change. In the PRoFESS study, lowering BP at the subacute phase after stroke (within 15 days) may have decreased CBF. The Acute Candesartan Cilexetil Therapy in Stroke Survivors (ACCESS) study indicated that candesartan treatment immediately after stroke, without significant lowering of BP, decreased the rate of recurrent stroke.13 Therefore, the beneficial pleiotropic effect of telmisartan seems to be overwhelmed by BP-lowering if given in a high dose. Caution should therefore be exercised when lowering BP at an acute to subacute stage when cerebrovascular autoregulation is damaged. In clinical practice, appropriate timing and dose of telmisartan should be considered.
The effect of telmisartan on cognitive function was also assessed. The Y-maze tests showed that spatial working memory was significantly disrupted by chronic cerebral hypoperfusion. Nonhypotensive doses of telmisartan significantly attenuated the deterioration of spatial working memory together with histological improvement in the white matter. By contrast, hypotensive doses of telmisartan further aggravated spatial working memory together with histological deterioration. In this mouse model, pathological changes were restricted in the white matter only. The cerebral cortex and hippocampus, which are associated with spatial working memory,14 were not damaged in a relatively short period (for example, 30 days post-BCAS). Thus, telmisartan may have restored frontal–subcortical circuitry function, which is also associated with spatial working memory.8,14 However, hippocampal changes appear at 3 months post-BCAS15; it would therefore be of interest to extend the observation period to investigate effect of telmisartan on the gray matter in a future study.
Recently, it has been shown that cerebral endothelial cells secrete trophic factors that support the survival and proliferation of oligodendrocyte precursor cells.16 Such oligodendrocyte precursor cell-supportive phenomena in endothelial cells are mediated by Akt and Src signaling pathways. Noncytotoxic levels of oxidative stress downregulate the production of trophic factors such as brain-derived neurotrophic factor and fibroblast growth factor and disrupt the ability of cerebral endothelial cells to support oligodendrocyte precursor cells. Furthermore, GST-π-positive oligodendrocytes were restored by low-dose telmisartan. These data suggest that a novel function of telmisartan is to maintain “oligovascular niche” by sustaining oligodendrocyte homeostasis in mammalian brain.
In conclusion, long-term AT1 receptor blockade and PPAR-γ activation with telmisartan should be considered as a novel therapeutic approach for protection from damage associated with chronic cerebral hypoperfusion or subcortical vascular dementia.
We thank Prof Kalaria for his excellent advice on this work and Dr Khundakar for his editorial assistance and comments. We are indebted to Ms Nakabayashi, Ms Gomibuchi, Ms Katsukawa, and Mr Kubota for their excellent technical assistance. Telmisartan was provided by Boehringer Ingelheim (Ingelheim, Germany). Boehringer Ingelheim provided no other support for this study, except supplying telmisartan.
Sources of Funding
This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Japanese Ministry of Education, Science and Culture (to J.T.) and a grant from the Suzuken Memorial Foundation (to M.I., J.T.).
- Received March 9, 2010.
- Revision received April 13, 2010.
- Accepted May 5, 2010.
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Supplemental Method I
The mice were anesthetized with halothane (2%). Both common carotid arteries were exposed through a midline cervical incision, and a microcoil with an inner diameter of 0.18 mm was applied to the bilateral common carotid arteries as described elsewhere.1 Rectal temperature was maintained between 36.5°C and 37.5°C, and body weight was monitored until the animals were euthanized. All procedures were performed in accordance with the guidelines for animal experimentation from the ethical committee of Kyoto University. The mice were given access to food and water ad libitum.
Supplemental Method II
Systolic BP was monitored in conscious mice by the tail-cuff method (MK-2000ST; Muromachi Co, Kyoto, Japan). Ten measurements of mean systolic BP were averaged for each mouse. CBF was recorded by laser speckle blood flow imager (Omega Zone; Omegawave) under anesthesia with halothane (2%). CBF was expressed as a percentage of baseline flow. Mice were held in a small plastic holder on a warming pad and thermostatically controlled at 37°C. The mean CBF of 10 measurements in each group was determined.
Supplemental Method III
The mouse brain was dissected and fixed as shown previously.1 Six-μm-thick sections were subjected to Klüver-Barrera staining. The severity of the white matter lesions was graded as normal (Grade 0), disarrangement of the nerve fibers (Grade 1), the formation of marked vacuoles (Grade 2), and the disappearance of myelinated fibers (Grade 3) in the corpus callosum and anterior commissure.1 For immunohistochemistry, the remaining frozen coronal blocks were cut into 20-μm-thick sections on a cryostat and incubated overnight with a rabbit antiglial fibrillary acidic protein antibody (a marker of astrocyte, diluted 1:10000; DAKO), a rabbit anti-Iba-1 antibody (microglia, 1:500; Wako), and a rabbit antiglutathione S-transferase-pi antibody (oligodendrocyte, 1:10 000; Assay Designs). Subsequently, these sections were treated with the appropriate biotinylated secondary antibodies (1:200; Vector Laboratories) and were visualized with 0.01% diaminobenzidine tetrahydrochloride and 0.005% H2O2 in 50 mmol/L Tris HCl (pH 7.6). We counted the numerical density of the glial cell nuclei with immunopositive perikarya (/0.125 mm2) in the white matter. For evaluating oxidative stress, we used primary antibody for mouse anti-8-OHdG (oxidative stress, 1:100; Nikken Seil), rat anti-CD31 (vascular endothelial cell, 1:500; BD Biosciences), and then as secondary antibody Alexa Fluor 546 goat anti-IgG (1:400; Molecular Probes) and Alexa Fluor 488 goat anti-IgG (1:400; Molecular Probes) were used for double immunofluorescent staining. We analyzed 8-OHdG/CD31-positive area (%) in the white matter (the percentage of 8-OHdG-positive area to CD31-positive area; Scion imaging software).
Antibodies used in this study were summarized in Supplemental Table I.
Supplemental Method IV
Cerebral mRNA expression of inflammatory cytokines such as MCP-1 and TNF-α were assessed by quantitative real-time reverse transcriptase–polymerase chain reaction pre-BCAS and 30 days post-BCAS. Real-time reverse transcriptase–polymerase chain reaction was performed using brain homogenates obtained from the right anterior hemisphere. Quantitative real-time reverse transcriptase–polymerase chain reaction was performed using the SYBR Premix Ex Taq Kit (Takara Bio) on a Thermal Cycler Dice™ Real Time System (Takara Bio). Primer sequences used in this study were as follows: MCP-1—forward primer: 5′-GCATCCACGTGTTGGCTCA-3′, reverse primer: 5′-CTCCAGCCTACTCATTGGGATCA-3′; TNF-α—forward primer: 5′-GCATGATCCGCGACGTGGAA-3′, reverse primer: 5′-AGATCCATGCCGTTGGCCAG-3′; 18S rRNA—forward primer; 5′-TTCTGGCCAACGGTCTAGACAAC-3′, reverse primer: 5′-CCAGTGGTCTTGGTGTGCTGA-3′.
The mRNA transcript for 18S rRNA was chosen as a control (housekeeping gene) because of relatively small variations over time following cerebral ischemia.2 Levels of MCP-1 and TNF-α mRNA were presented after normalization to the level of 18S rRNA mRNA.
Supplemental Method V
Spatial working memory was assessed by the Y-maze test, as described elsewhere.3 The 8-arm radial arm maze test that requires dietary restriction for several days was not used because appropriate control of peroral dose of telmisartan was necessary for this study. The Y-maze test was conducted during the dark period (7:00 to 11:00 pm) and was performed 30 days post-BCAS. This task is based on spontaneous alternation behavior and is used to measure spatial working memory. The maze consists of 3 arms (40 cm long, 9.5 cm high, and 4 cm wide, labeled A, B, or C) diverging at a 120° from the central point. The experiments were performed in a dimly illuminated room, and the floor of the maze was cleaned with super hypochlorous water-soaked paper for discrimination of smell to avoid olfactory cues after each mouse was tested. Each mouse was placed at the end of the start arm and allowed to move freely through the maze during an 8-minute session without reinforcers such as food, water, or electric foot shock. The sequence of arm entries was manually recorded. A mouse was considered to have entered an arm when all 4 paws were positioned in the arm runway. An actual alternation was defined as entities into all the 3 arms on consecutive occasions (eg, the sequence, ABCBCBCA is counted 2 alternation with the first consecutive ABC and the last consecutive BCA out of 6 consecutive occasions; 33% alternations). The maximum alternation was subsequently calculated by measuring the total number of arm entries minus 2 and the percentage of alternation was calculated as (actual alternation/maximum alternation)×100. The total number of arms entered during the sessions, which reflect locomotor activity, was also recorded. Mice that entered arms less than 8 times during the test were eliminated because the data obtained from those mice were not considered to reflect precise alternation. Indeed, 6 (6.3%) of 96 mice used in this study exhibited total arm entries <8 times.
Supplemental Method VI
Plasma concentration of telmisartan was measured by high-performance liquid chromatography (HPLC). All HPLC was performed with the HPLC equipment from Shimadzu (Shimadzu Co, Kyoto, Japan). The HPLC system was controlled by the software of Shimadzu’s LCsolution. For chromatographic separation, a YMC-Pack Pro C18 (5 μm, 12 nm) reverse phase column (150×4.6 mm; YMC Co, Ltd, Kyoto, Japan) with a guard column was used at a temperature of 40°C kept within a column oven (type CTO-20A). The eluent was 100 mmol/L ammonium acetate/methanol (Nacalai tesque, Kyoto, Japan; 30:70, v/v) at a constant flow rate of 1 mL/min through a solvent delivery unit (type LC-20A) equipped with a ultraviolet-visible detector (type SPD-20A). Samples were treated as follows: 90 μL of plasma together with 10 μL internal standard losartan (1 mg/mL; LKT Laboratories, St Paul, Minn) were well mixed and treated with 3 mL of acetyl acetate for 3 minutes vortex to precipitate the proteins. After centrifugation at 1600 g for 15 minutes, 2.4 mL of the supernatant was transferred into a 10-mL tube. A nitrogen gas fluid was used to evaporate solvent and the residue was reconstituted in 50 μL of the eluent. A 20-μL aliquot of the solution was injected by an autosampler (type SIL-20A) onto the HPLC system for analysis. Telmisartan was detected by ultraviolet absorption at 297 nm. Peak identity was confirmed by measuring retention time of the sample with standard substance telmisartan. A linear standard curve was obtained in the range of 0.032 to 2.000 μg/mL in our HPLC system for telmisartan determination.
A peak eluting with a retention time (7.333±0.008 minutes, n=4) identical to that of telmisartan (7.155±0.175 minutes) was detected by HPLC analysis in all samples obtained from telmisartan (1 mg/kg per day)-treated mice (Supplemental Figure I). The equation of the standard curve was given by Y=−34.55018+1334.526 (r2=0.9915).
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