Direct inhibition by a statin of TNFα-induced leukocyte recruitment in rat pial venules — in vivo confocal microscopic study

Pathophysiology 11 (2004) 121–128

Direct inhibition by a statin of TNF␣-induced leukocyte recruitment in rat pial venules — in vivo confocal microscopic study Ruriko Obamaa,∗ , Hideyuki Ishidab , Shunya Takizawaa , Chizuko Tsujib , Hiroe Nakazawab , Yukito Shinoharaa a b

Department of Neurology, School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan Department of Physiology, School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan Received 5 April 2004; received in revised form 14 June 2004; accepted 1 July 2004

Abstract 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been shown to block leukocyte–endothelial interaction independently of their cholesterol-lowering properties. The effects of statins are generally attributed to a decrease in mevalonate caused by inhibition of HMG-CoA reductase, which results in an increase of nitric oxide (NO). However, a recent in vitro study demonstrated a novel effect which depended on the lipophilicity of statin and appeared to be unrelated to HMG-CoA reductase inhibition. The purpose of this study is to investigate whether the proposed mechanism actually operates in vivo. We examined the effects of simvastatin (lipophilic) and pravastatin (hydrophilic) on leukocyte behavior in a tumor necrosis factor ␣ (TNF␣)-induced leukocyte recruitment model. Leukocyte adhesion and rolling were examined in pial venules of rat brain by using confocal laser scanning microscopy after labeling leukocytes with rhodamine 6G. Experiments were conducted 4 h after TNF␣ injection (0.5 ␮g) in six groups: control, TNF␣ alone, TNF␣ + vehicle of simvastatin, TNF␣ + simvastatin (20 mg/kg, 2 ml/kg), TNF␣ + vehicle of pravastatin, and TNF␣ + pravastatin (40 mg/kg, 2 ml/kg). Statins and vehicles were injected subcutaneously for 3 days. TNF␣ caused a marked increase in rolling and adhered leukocytes. The number of adhered leukocytes in the simvastatin group was significantly less than in the vehicle group (276 ± 38 cells/mm2 versus 1155 ± 89 cells/mm2 , P < 0.01), whereas pravastatin had little effect. Both simvastatin and pravastatin showed a tendency to decrease the number of rolling leukocytes, but there were no significant differences among TNF␣-treated groups. Up-regulation of endothelial nitric oxide synthase (eNOS) mRNA or increased expression of P-selectin or intercellular adhesion molecule-1 (ICAM-1) was not observed, and therefore cannot account for the simvastatin-induced reduction of adhered leukocytes. Markedly different effect on leukocyte adhesion between simvastatin and pravastatin under comparable level of HMG-CoA reductase inhibitor was demonstrated in in vivo as was shown in in vitro study. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Statin; Leukocyte–endothelial interaction; eNOS; P-selectin; ICAM-1

1. Introduction Recent large clinical trials have demonstrated that 3hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, statins, decrease the incidence of cerebral ∗ Corresponding author. Tel.: +81 463 93 1121/2240; fax: +81 463 94 8764. E-mail address: [email protected] (R. Obama).

0928-4680/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2004.07.001

infarction in the subgroup of patients with a history of coronary heart disease and normal cholesterol level [1,2]. This suggested that statins have additional benefits beyond their cholesterol-lowering effects. As statin administration has been shown to improve endothelial dysfunction in patients and animal models [3], the target of their beneficial effects was considered to be the vascular endothelium. It was shown that statins up-regulate endothelial nitric oxide synthase (eNOS) by prolonging the half-life of eNOS mRNA [4],

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and inhibit leukocyte–endothelial interaction through downregulation of adhesion molecules. These effects have been attributed to a decrease in mevalonate via inhibition of HMGCoA reductase [5,6,7,8]. However, a novel property of statins which is entirely unrelated to HMG-CoA reductase inhibition was demonstrated in in vitro experiments by Weitz-Schmidt et al. [9]. They showed that statins inhibit leukocyte adhesion by directly interfering with leukocyte function antigen-1 (LFA-1), the counterpart molecule of leukocyte to intercellular adhesion molecule-1 (ICAM-1) on the endothelium. Using the crystal structure of LFA-1, as a basis, they designed a small compound which does not inhibit HMG-CoA reductase, but can bind to LFA-1. This compound had a potent inhibitory effect on the LFA-1–ICAM-1 interaction. Furthermore, it did not affect Mac-1-induced leukocyte–endothelial interaction, supporting the specificity of the compound to LFA-1. They also found that lovastatin, simvastatin and mevastatin bind specifically to LFA-1, but pravastatin does not. Although the mechanism underlying the lack of inhibition by pravastatin remains to be clarified, it was suggested that the hydroxyl group of pravastatin may inhibit binding to the hydrophobic environment of LFA-1. Thus, it is of interest to know whether a difference in hydrophobicity of statins can influence the adhesion of leukocytes to the vascular wall in vivo as well. The effect of statins on the brain microvasculature in vivo has not been examined, but confocal laser scanning microscopy enables the observation of leukocyte behavior in the vessels. Tumor necrosis factor ␣ (TNF␣) rapidly induces expression of P-selectin [10] and ICAM-1 [11] on endothelial cells, and brings about leukocyte rolling, followed by adhesion to the vascular wall. The rolling is mediated by P-selectin independently of LFA-1–ICAM-1 interaction, and adhesion is mediated by the LFA-1–ICAM-1 interaction. Thus, we performed this study to see whether or not the degree of hydrophobicity of statins influences leukocyte–endothelial cell interaction by directly observing rolling and adhesion of leukocytes in the pial venules of rats pretreated with TNF␣. We used simvastatin as a representative lipophilic statin and pravastatin as a hydrophilic statin. Leukocyte movement in the pial venules was evaluated using confocal laser scanning microscopy. The effects of these statins on the level of eNOS mRNA and on the induction of P-selectin and ICAM-1 expression were also examined.

2. Materials and methods 2.1. Animals All aspects of this study were approved by the Tokai University Animal Care and Use Committee. Male Wistar rats (8–11 weeks old, 214–360 g, Clea Japan Co. Ltd., Tokyo, Japan) were used. Animals were fasted overnight but allowed free access to water before the surgical procedure.

2.2. Drugs Simvastatin was kindly provided by Merck Research Laboratories (Rahway, NJ, USA). It was chemically activated by alkaline hydrolysis before subcutaneous injection. Pravastatin was kindly provided by Sankyo Co. Ltd. (Tokyo, Japan). 2.3. Experimental protocol Three series of experiments were performed: (1) observation of leukocyte behavior by confocal laser scanning microscopy (n = 51); (2) evaluation of expression of P-selectin and ICAM-1 on the vessels by immunohistochemistry (n = 24); and (3) evaluation of eNOS mRNA in the cerebral tissue by reverse transcription-polymerase chain reaction (RT-PCR) (n = 36). In all experiments, simvastatin, pravastatin or a corresponding volume (2 ml/kg) of the respective vehicle was administered subcutaneously once daily for 3 days till the day of the experiment. To induce adhesion molecules, 0.5 ␮g/animal of recombinant rat TNF␣ (R&D Systems, Minneapolis, MN, USA) was administered 4 h prior to the experiment. In the leukocyte behavior experiment, the injection time of TNF␣ was critically defined to be 4 h prior to observation by pre-selecting the time for surgical exposure of the brain microvasculature. In the eNOS mRNA and immunohistological experiments, cerebral hemispheres were obtained 4 h after TNF␣ injection. Rats were divided into six experimental groups: group 1; control, group 2; rats with TNF␣ alone, group 3; rats given simvastatin (20 mg/kg, 2 ml/kg in the mixture of ethanol, H2 O, NaOH and HCl for 3 days, subcutaneously) with TNF␣, group 4; rats given vehicle of simvastatin (mixture of ethanol, H2 O, NaOH and HCl, pH 7.4) with TNF␣, group 5; rats given pravastatin (40 mg/kg, 2 ml/kg in the phosphate buffer solution (PBS) for 3 days, subcutaneously) with TNF␣, group 6; rats given vehicle of pravastatin (PBS, pH 7.4) with TNF␣. 2.4. Observation of leukocyte behavior in the pial venules Rats were initially anesthetized with 3% halothane mixed with 30% oxygen and 70% nitrous oxide and connected to ventilator (SN-480-7 Shinano, Tokyo, Japan) through a tracheostomy. A maintenance dose of 1% halothane was used during the procedure. Polyethylene catheters (PE50) were inserted into the femoral artery for measurement of arterial blood pressure and blood sampling, and into the femoral vein for application of fluorescent dye. The skull was fixed in a stereotactic frame for preparation of the cranial window over the left parietal hemisphere. After a midsagital skin incision from the forehead to the neck, a rectangular 5 mm × 4 mm window was made with the dura mater left intact. The dura mater was flushed continuously with normal saline at 37.0 ◦ C. The body temperature was continuously maintained at 36.2–37.3 ◦ C by using a rectal probe connected to a feedback-controlled heating pad (Natsume

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Co. Ltd., Tokyo, Japan). Arterial blood gas was also monitored by using a blood gas analyzer (IL1304; Instrumentation Laboratory, Kirchheim, Germany). Confocal laser scanning microscopy (CSU 10, Yokogawa, Japan) with 10× lens through the cranial window was focused on post-capillary venules with a diameter of 50–120 ␮m. Leukocytes were labeled by injecting 0.1% rhodamine 6G intravenously (1 ml bolus followed by 1 ml/h infusion) (Sigma, St. Louis, MO, USA) and visualized with emission at 520 nm and excitation at 488 nm. Images were projected on a monitor through an intensified CCD camera (SR UBGEN III1, Solamere, Salt Lake City, UT, USA) and recorded using digital video. The motion of labeled leukocytes in one venule was recorded for 30 s and 5–11 venules in each rat were observed. We limited the total observation time to a maximum of 20 min. Rhodamine 6G was selected because it stained circulating leukocytes, but not red blood cells or endothelial cells [12,13]. 2.5. Analysis of leukocyte behavior Leukocyte behavior was analyzed in video playback. Rolling leukocytes were defined in terms of multiple intermittent contacts with the vascular wall, thereby advancing distinctly more slowly than freely moving leukocytes. Adherent leukocytes were defined as those attached to the vascular wall for more than 30 s. The numbers of rolling and adherent leukocytes in each venule were counted and results were expressed as total numbers of cells/mm2 of the venules in each rat.

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lowed by incubation for 2 min in paraformaldehyde. It was then incubated with a purified rabbit anti-human CD62P (Pselectin) polyclonal antibody at 1000-fold dilution 4 ◦ C for 12 h, and further with biotinylated rat anti-rabbit IgG and with 3,3 -diaminobenzidine–H2 O2 . All vessels were identified based on the presence of alkaline phosphatase. We observed five fields per one cerebral hemisphere and counted P-selectin-positive or ICAM-1-positive vessels by using a computer-assisted image analysis system (ZEISS KS 400 version 3.0, Carl Zeiss Vision, Oberkochen, Germany). Results were expressed as the ratio of vessels positive for Pselectin or ICAM-1 in total vessels after summing the data of five fields in each rat. 2.7. Measurement of eNOS mRNA The frozen tissue was homogenized and RNA was extracted. The expression levels of eNOS mRNA were measured with the RT-PCR method as previously described [14]. Briefly, equal amounts of RNA were reverse-transcribed into cDNA. The RT products were amplified with primers designed for eNOS from the rat gene sequence, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a standard. After amplification, each PCR mixture was electrophoresed through 1% agarose gel, and bands were stained with ethidium bromide. Each gel was photographed under ultraviolet light with the same exposure and development time. The bands on the positive film were scanned, and the density of each PCR product was evaluated by using National Institutes of Health (NIH) Image software. Results are expressed as eNOS/GAPDH ratios.

2.6. Immunohistochemistry 2.8. Data analysis Rats were sacrificed by peritoneal administration of an overdose of pentobarbital sodium. Cerebral hemispheres were rapidly removed, embedded in OCT compound, frozen in 2-methylbutane cooled in liquid N2 , and stored at −80 ◦ C. Coronal brain sections (10-␮m thick) were cut on a cryostat and thaw-mounted onto silane-coated slides. Immunohistochemical staining for the ICAM-1 antibody was performed by the indirect method. Tissues were fixed in pure acetone for 5 min and in PBS containing 10% normal sheep serum for 5 min, at 4 ◦ C. The sections were first incubated with monoclonal anti-rat ICAM-1 antibody at 50-fold dilution (CHEMICON, Temecula, CA, USA) for 12 h. After having been washed thoroughly with 0.01 mmol/l PBS (pH 7.2), the sections were incubated with horseradish peroxidase-labeled sheep anti-mouse IgG (Amersham, Buckinghamshire, UK) at 100-fold dilution for 1 h. The bound antibodies were visualized with 3,3 -diaminobenzidine and hydrogen peroxidase. In the control study, the same procedure as mentioned earlier was done, except that PBS alone was used in place of the first antibody. Immunohistochemical staining for the P-selectin antibody was performed with an Elite ABC Kit (Vector, Burlingame, CA, USA). Tissue was fixed for 10 min in pure acetone, fol-

Data are presented as mean ± S.E. One-way ANOVA followed by Newman–Keuls PLSD was used for multiple comparison. The criterion of statistical significance was set at P < 0.05.

3. Results 3.1. Physiological parameters All physiological parameters including arterial blood pressure, pH, PaO2 , PaCO2 , and hematocrit (Ht), were within normal ranges and were stable during the experiment (Table 1). Rhodamine 6G injection caused a slight decrease in blood pressure, but this returned to the baseline within a few minutes. 3.2. Leukocyte behavior in the pial venules Pial venules and leukocyte motion were clearly observable in 48 rats (3 rats were excluded from the analysis because of poor labeling of leukocytes). Counts of rolling leukocytes in

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Table 1 Physiological parameters at the end of the study

MABP (mmHg) pH PaO2 (mmHg) PaCO2 (mmHg) Ht (%)

Control (n = 7)

TNF␣ alone (n = 9)

Vehicle of simvastatin (n = 6)

Simvastatin (n = 6)

Vehicle of pravastatin (n = 7)

Pravastatin (n = 10)

88 ± 9 7.39 ± 0.03 135 ± 68 41 ± 5 45.8 ± 3.1

91 ± 9 7.41 ± 0.06 131 ± 17 37 ± 4 45.6 ± 2.0

87 ± 10 7.42 ± 0.03 112 ± 24 40 ± 3 47.3 ± 1.9

85 ± 10 7.46 ± 0.04 110 ± 23 36 ± 4 45.6 ± 2.0

81 ± 11 7.42 ± 0.02 114 ± 10 36 ± 4 47.8 ± 3.5

88 ± 7 7.44 ± 0.04 130 ± 21 36 ± 2 45.5 ± 3.3

Values are means ± S.E. MABP: mean arterial blood pressure. MABP, arterial gas tensions and hematocrit during experiments, all physiological parameters were within normal ranges.

the six groups are shown in Fig. 1. In the control group, which was not given TNF␣, we observed essentially no rolling in any of the venules. All groups with TNF␣ injection showed a marked increase in the number of rolling leukocytes compared with the control group (control: 152 ± 31 cells/mm2 ; TNF␣ alone: 598 ± 71 cells/mm2 ; vehicle of simvastatin: 643 ± 76 cells/mm2 ; simvastatin: 526 ± 45 cells/mm2 ; vehicle of pravastatin: 605 ± 60 cells/mm2 ; pravastatin: 528 ± 34 cells/mm2 ). Simvastatin and pravastatin showed a tendency to decrease the number of rolling leukocytes, but there was no statistically significant difference. Fig. 2 shows representative images of adhered leukocytes in the six groups. No adhered leukocytes were observed in the control, but a marked increase in adhered leukocytes occurred in all TNF␣-treated groups. Fig. 3 shows of the data for adhered leukocytes in all groups (control: 54 ± 13 cells/mm2 ; TNF␣: 870 ± 100 cells/mm2 ; vehicle of simvastatin: 1155 ± 89 cells/mm2 ; simvastatin: 276 ± 38 cells/mm2 ; vehicle of pravastatin: 1137 ± 147 cells/mm2 ; pravastatin: 828 ± 61 cells/mm2 ). It is noteworthy that simvastatin significantly inhibited leukocyte adhesion compared with that in the vehicle group (P < 0.01). Pravastatin showed a tendency to decrease the number of adhered leukocytes compared with the corresponding vehicle group, but the difference did not reach statistical significance.

3.3. Immunohistochemistry Fig. 4 shows the result of immunohistochemical staining of P-selectin (Fig. 4A) and ICAM-1 (Fig. 4B); double staining with alkaline phosphatase was used to identify the vessels. The expression of both P-selectin and ICAM-1 is seen in nearly half of the vessels. Quantification of P-selectin (Fig. 5A) revealed that P-selectin-positive vessels tended to be more frequent in the groups treated with TNF␣, but there was no statistically significant difference among the groups. As regards ICAM-1 expression, neither TNF␣ nor statin pretreatment increased it significantly (Fig. 5B). 3.4. RT-PCR Density scanning of bands after RT-PCR revealed that brain eNOS mRNA levels remained at comparable levels in all groups (Fig. 6). Namely, neither statin pretreatment nor TNF␣ injection influenced the eNOS mRNA level. Plasma nitrite and nitrate levels were not increased in statin groups (control: 6.81 ± 1.04 ␮M; vehicle of simvastatin: 7.13 ± 1.36 ␮M; simvastatin: 6.67 ± 1.80 ␮M; vehicle of pravastatin: 8.90 ± 1.77 ␮M; pravastatin: 5.78 ± 0.45 ␮M (mean ± S.E.)).

4. Discussion

Fig. 1. Number of rolling leukocytes in the six groups. Rolling leukocytes were counted and expressed as the number of cells/mm2 . Both simvastatin and pravastatin treatments tended to decrease the number of rolling leukocytes, but there was no statistically significant difference between each statin and the corresponding vehicle group. An asterisk denotes P < 0.01 as compared with the control group.

The present study showed that the TNF␣-induced leukocyte rolling and adhesion in rat pial venules in vivo could be quantified by using confocal laser scanning microscopy. Observation of leukocyte behavior revealed that simvastatin (lipophilic statin) markedly inhibited adhesion of leukocytes, while pravastatin (hydrophilic statin) was not significantly effective. There was no difference in inhibition of rolling between these statins. The attenuation of P-selectin and ICAM1 expressions on vessels by both statins could not explain the marked inhibition of adhesion seen only with simvastatin. These results are consistent with the previous in vitro study [9], which demonstrated that simvastatin, but not pravastatin, blocks adhesion of leukocytes to endothelial cells through inhibiting ICAM-1–LFA-1 interaction, leading to the suggestion that the lipophilicity of simvastatin may be more important for this action than its effect as an HMG-CoA reductase inhibitor.

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Fig. 2. Representative images of venules with adhered leukocytes in the six groups: (A) control; (B) TNF␣ alone; (C) vehicle of simvastatin with TNF␣; (D) simvastatin with TNF␣; (E) vehicle of pravastatin with TNF␣; (F) pravastatin with TNF␣. In the control group, there were essentially no adhered leukocytes (A). Abundant adhered leukocytes were observed in all groups with TNF␣ treatment (B–F). The number of adhered leukocytes in the simvastatin group was scant compared with that in the vehicle group (D). The calibration bar represents 100 ␮m.

Extensive studies have been performed to clarify the mechanisms underlying the pleiotropic effects of statins and a decrease in isoprenoids is thought to be important. This is because statins inhibit the conversion of HMG-CoA to mevalonate, a precursor of cholesterol, and mevalonate is also a precursor of isoprenoids [3,15–17]. Since isoprenoids serve as important lipid attachments to a variety of proteins, including Rho (the small guanosine-5 -triphosphate binding (GTPbinding) protein), and inhibition of Rho increases eNOS expression [4], most effects of statins are considered to be

attributable to increased bioavailability of NO [18–27]. As NO can inhibit expression of adhesion molecules [28,29] the present results may be interpreted as being due to the effect of NO. However, the plasma concentrations of NO metabolites, nitrite and nitrate, were similar in both groups and the eNOS mRNA level was not altered by either of the drugs. Thus, eNOS induction cannot be a major mechanism for the observed reduction in adhered leukocytes. Similarly, the small and comparable levels of attenuation in ICAM-1 expression by simvastain and pravastatin in the immunohistochemical

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Fig. 3. The number of adhered leukocytes in the six groups. Adhered leukocytes were counted and expressed as the number of cells/mm2 . All values are means ± S.E. A single asterisk denotes P < 0.01 as compared with the control group and double asterisks denotes P < 0.01 as compared with the corresponding vehicle group. The number of adhered leukocytes was significantly fewer in the simvastatin group than in the corresponding vehicle group. The number of adhered leukocytes appeared to be fewer in the pravastatin group than in the corresponding vehicle group, but without statistical significance.

study are not enough to explain the marked decrease in adhered leukocytes induced only by simvastatin. Thus, we consider that the major mechanism for the observed inhibition of leukocyte adhesion by simvastatin but not by pravastatin involves the hydrophobicity, since we selected the doses of simvastatin and pravastatin to give comparable levels of HMGCoA reductase inhibition [1,30]. The fact that simvastatin and pravastatin did not decrease P-selectin and ICAM-1 expression significantly in this study apparently conflicts with many previous studies in which significant attenuation of adhesion molecules by statins was observed [31,32]. However, the effects of statins differ markedly among various vessels or experimental conditions, as was shown in a recent study [23], which demonstrated a twoto three-fold increase in E-selectin and ICAM-1 by lovastatin in TNF␣-activated human vascular endothelial cells. The lack of increase in expression of eNOS mRNA in this

Fig. 5. Ratio of P-selectin-positive or ICAM-1-positive vessels in the six groups: (A) ratio of P-selectin-positive vessels in the brain; (B) ratio of ICAM-1-positive vessels in the brain. In the brain hemisphere of each rat, five fields were observed and P-selectin-positive or ICAM-1-positive vessels were counted. Results were expressed as the ratio of numbers of positive vessels (PV)/total vessels (TV). P-selectin-positive vessels tended to be more frequent in the groups treated with TNF␣, but there was no statistically significant difference among groups. Neither TNF␣ nor statin pretreatment significantly increased ICAM-1-positive vessels.

study may be explained partly by the short duration (3 days) of statin administration in the present study, since the study by Endres and co-workers showed that up-regulation was observed in the brain after statin treatment for 14 days [14,27].

Fig. 4. Representative staining of P-selectin and ICAM-1 in rat brain: (A) double staining of P-selectin and endothelial cells; (B) double staining of ICAM-1 and endothelial cells. P-selectin was visualized as brown staining, while vessels were shown in blue by alkaline phosphatase staining, a marker of vessels. ICAM-1 and P-selectin were visualized as brown staining in the whole vessel wall, including endothelial cells. The calibration bar represents 50 ␮m.

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Fig. 6. eNOS mRNA levels in the six groups. The eNOS mRNA levels were similar in all groups.

Differences in in vivo and in vitro conditions or experimental protocols, such as animal species [14,27], target organs (heart [28,29], lung [20], aorta [23] or brain) or experimental models (ischemia [18,20,27] or inflammation), may be another reason. Further studies are needed to clarify the mechanisms responsible for the effects observed here. For example, mevalonate could be added to confirm that the inhibition of ICAM1–LFA-1 interaction is not due to inhibition of HMG-CoA reductase. Moreover, inhibition of ICAM-1–LFA-1 interaction by adding anti-ICAM-1 antibody or anti-LFA-1 antibody should be examined. In conclusion, markedly different effect on leukocyte adhesion between simvastatin and pravastatin under comparable level of HMG-CoA reductase inhibitor was demonstrated in in vivo as was shown in in vitro study demonstrated by Weitz-Schmidt et al. [9].

Acknowledgements The skillful technical assistance of Kiyoshi Niwa, Saori Kohara (Department of Neurology), Yoko Takahari, Joubu Ito, and Tamaki Saso (Laboratories for Experimental Animals and Physiologic Research) in Tokai University School of Medicine are gratefully acknowledged. This investigation was supported in part by a grant from Tokai University School of Medicine Research Aid in 2001.

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