J Mol Neurosci (2009) 39:59–68 DOI 10.1007/s12031-009-9175-x

Correspondence Between Neurological Deficit, Cerebral Infarct Size, and Rho-Kinase Activity in a Rat Cerebral Thrombosis Model Koh Kawasaki & Kazuo Yano & Kunie Sasaki & Shunsuke Tawara & Ichiro Ikegaki & Shin-ichi Satoh & Yuji Ohtsuka & Yuki Yoshino & Hiroshi Kuriyama & Toshio Asano & Minoru Seto

Received: 29 October 2008 / Accepted: 7 January 2009 / Published online: 23 January 2009 # Humana Press 2009

Abstract Whether Rho-kinase activity is really associated with the pathogenesis of cerebral infarction remains unclear. To consider this question, we investigated correspondences between severity of neurological deficit, infarct size, amount of various marker proteins, and Rho-kinase activity in a rat cerebral infarction model. Sodium laurate was injected into the left internal carotid artery, inducing cerebral infarction in the ipsilateral hemisphere in rats. We prepared rats with various severities of neurological deficit (mild to severe) 3 days after injection of laurate, then measured infarct size and amount of various marker proteins, phosphorylation of substrates of Rho-kinase, myosin-binding subunit (MBS), myosin light chain (MLC), ezrin/radixin/moesin (ERM), and adducin using Western blot methods. First, infarct size increased corresponding to the severity of neurological deficit. Second, amounts of activating transcription factor 3, nestin, CD68, proliferating cell nuclear antigen, and heat shock protein 70 were increased, whereas neurofilament and myelinassociated glycoprotein were decreased corresponding to the severity of neurological deficit and infarct size. Finally, Rho-kinase activity (phospho-MBS/MBS, phospho-MLC/ MLC, phospho-ERM/ERM, and phospho-adducin/adducin) was increased corresponding to the severity of neurological deficit and infarct size. Rho-kinase thus appears to play a crucial role in the pathogenesis of cerebral infarction. K. Kawasaki (*) : K. Yano : K. Sasaki : S. Tawara : I. Ikegaki : S.-i. Satoh : Y. Ohtsuka : Y. Yoshino : H. Kuriyama : T. Asano : M. Seto Laboratory for Pharmacology, Asahi Kasei Pharma, 632-1 Mifuku, Izunokuni-shi, Shizuoka 410-2321, Japan e-mail: [email protected]

Keywords Rho-kinase (ROCK) . Cerebral infarction . Rats

Introduction Cerebral infarction results from a reduction in cerebral blood flow and is frequently caused by occlusion of cerebral arteries by either embolus or local thrombosis. In cerebral infarction, the brain is damaged by both the primary insult of impaired cerebral blood flow and secondary injury such as inflammation by neutrophils and macrophages migrating into the ischemic brain (Dirnagl et al. 1999). Recent evidence has suggested that Rho-kinase is involved in processes of primary insult and secondary injury after cerebral ischemia. However, whether Rhokinase activity is really associated with the pathogenesis of cerebral infarction remains uncertain. Rho-kinase, one of the downstream effectors of Rho, is a serine/threonine kinase that is activated by binding to the active GTP-bound form of Rho. Rho-kinase exists as two isomers, Rho-kinase α/ROK α/ROCK2 and Rho-kinase β/ ROK β/ROCK1 (Wettschureck and Offermanns 2002), which are known to phosphorylate various substrates, including the myosin-binding subunit (MBS) of myosin phosphatase, ezrin/radixin/moesin family proteins (ERM), myosin light chain (MLC), LIM kinase, and adducin (Fukata et al. 2001). We have reported that administration of fasudil and hydroxy fasudil, both of which are Rhokinase inhibitors, improves neurological deficits and reduces the infarct volume in models of cerebral infarction (Satoh et al. 1999, 2001; Toshima et al. 2000). These findings suggest that Rho-kinase contributes to cerebral infarction. Recent studies have demonstrated that Rho-

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kinase plays important roles in various cellular functions (Amano et al. 2000; Riento and Ridley 2003; Shimokawa and Rashid 2007), including cell motility and mobility, actin cytoskeleton organization, contraction of vascular vessels, and retraction of neurites, which may be involved in the pathogenesis of cerebral infarction. Levels of Rho have been found to be upregulated in the brains of patients who have died following focal cerebral infarction (Brabeck et al. 2003) and in mouse brains after middle cerebral artery occlusion (Trapp et al. 2001), suggesting the upregulation of Rho/Rho-kinase signaling. However, direct evidence of whether Rho-kinase is activated after cerebral ischemia is lacking. Rho-kinase activity is generally estimated by measuring phosphorylation levels of Rho-kinase substrates. Some investigators have recently reported that Rho-kinase activity is elevated in models of cerebral infarction. Phosphorylation of MBS is increased after middle cerebral artery occlusion (MCAO) in mice (Rikitake et al. 2005; Shin et al. 2007), and phosphorylation of adducin is increased after MCAO in mice (Yamashita et al. 2007) and rats (Yagita et al. 2007). Furthermore, Rho-kinase in brain extracts after cerebral ischemia was activated in a rat stroke model created by injecting sodium laurate into the left internal carotid artery (Yano et al. 2008). However, information regarding correspondences between Rho-kinase activity, severity of neurological deficits, and infarct size remains fragmentary. We have previously reported a rat stroke model created by injecting sodium laurate into the left internal carotid artery. This model is useful for investigating correspondences between indices and severity of neurological deficits, as some variations in neurological score can be observed in this model. The present study assessed whether Rho-kinase activity is activated in the ipsilateral hemisphere and whether Rho-kinase activity corresponds to the severity of neurological deficits, infarct size, and changes in cell populations due to the migration of inflammatory cells and neural degeneration.

Materials and Methods Induction of Cerebral Microthrombosis All animal protocols were approved by the Committee on Ethics in Animal Experiments at Asahi Kasei Pharma (Shizuoka, Japan) and were performed in accordance with the Guidelines for Animal Experiments of Asahi Kasei Pharma and the Care and Use of Laboratory Animals as adopted and promulgated by the United States National Institutes of Health (NIH). Sprague–Dawley male rats (age, 7–9 weeks; Japan SLC, Shizuoka, Japan) were used for the present study. Cerebral microthrombosis was induced by injecting sodium laurate into the left internal carotid artery

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as described previously (Toshima et al. 2000) with some modifications. In brief, rats were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg). After exposure of the left common, external and internal carotid arteries, the left external carotid, occipital, and pterygopalatine were ligated using 6-0 silk braid. Common and internal carotid arteries were temporarily closed with hemostatic forceps. A polyethylene catheter was inserted into the left external carotid artery. Internal carotid artery was opened and sodium laurate (120 μg/200 μL/body) dissolved in saline was injected into the internal carotid artery. The internal external carotid artery was ligated at a position slightly distal to the site of injection. The catheter and the hemostatic forceps at common carotid artery were removed. Neurological Examination Neurological deficits of rats were scored on the basis of severity of the following symptoms: truncal curvature, circling behavior, and rolling fit. Each symptom was scored as: 2, severe; 1, mild; and 0, normal. Neurological deficit of each rat was expressed as a total score of each symptom. Some variations were apparent in the cerebral infarction model induced by injection of laurate. To investigate correspondences to severity of neurological deficit, rats with various severity of neurological deficit were prepared. Rats were divided into four groups (n=6 each): group N, normal rats; group C, control (sham-operated) rats; group M, mild symptom rats (neurological score 0–3 after sodium laurate injection); and group S, severe symptom rats (neurological score 4–6 after sodium laurate injection). Estimation of Infarct Size Infarct area was evaluated using the 2,3,5-triphenyltetrazolium chloride (TTC) staining method (Bederson et al. 1986). Briefly, 3 days after sodium laurate injection, brains were quickly removed and cut transversely at −3 mm from the optic chiasm to produce rostral and caudal sections. Rostral sections were placed in 1% TTC solution for 45 min at 37°C. Areas of infarct and areas of both hemispheres were measured on an image of the cut surface of TTC-stained brain by tracing these areas using the Image J 1.41 software (NIH, Bethesda, USA). The area of infarction in each section was expressed as the percentage of the infarct area to the area of the contralateral hemisphere. Preparation of Cerebral Extracts Three days after sodium injection of laurate, brains were quickly removed and cut transversely at −3 mm the optic chiasm to produce the rostral and caudal sections. Caudal sections were separated into left and right cerebral hemi-

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spheres, and then homogenized in fixative solution (10% trichloroacetic acid). After the sample was centrifuged for 10 min at 15,000 rpm, the pellet was resuspended in extraction buffer containing 8 M urea, 2% sodium dodecyl sulfate (SDS), and 62.5 mM Tris (pH 6.8). Protein concentrations of extracts were measured using a BCA Protein Assay kit (Pierce, Rockford, USA).

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QGVTLT” corresponding to the region around Thr696 of human phospho-MBS (Ito et al. 2003). Mouse antiphospho-MLC antibody was raised against a synthetic phospho-MLC peptide “KKRPQRATS(PO3H2)NVFC” corresponding to the region around Ser19 of MLC (Sakurada et al. 1998). Statistical Analysis

Western Blot Analysis Equal amounts of cerebral extracts (10 μg of protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, USA). Blots were incubated with the following primary antibodies: rabbit anti-RhoA (Santa Cruz, Santa Cruz, USA), mouse anti-ROCK1 (Rho-kinase β; BD Bioscience, Flankrin Lakes, USA), mouse anti-ROCK2 (Rhokinase α; BD Bioscience), mouse anti-MBS (BD Biosciences Pharmingen, San Diego, USA), rabbit anti-phospho-MBS, mouse anti-adducin (Santa Cruz), rabbit anti-phospho-adducin (Santa Cruz), rabbit anti-ERM (Cell Signaling, Danvers, USA), rabbit anti-phospho-ERM (Cell Signaling), rabbit antiMLC (Santa Cruz), mouse anti-phospho-MLC, mouse antinestin (Chemicon International, Temecula, USA), rabbit anti-myelin-associated glycoprotein (MAG) (Santa Cruz), mouse anti-CD68 (ED1) (AbD Serotec, Oxford, UK), rabbit anti-heat shock protein 70 (HSP70) (Cell Signaling), mouse anti-neurofilament light chain (NF-L) (Cell Signaling), mouse anti-glial fibrillary acidic protein (GFAP) (Cell Signaling), mouse anti-proliferating cell nuclear antigen (PCNA) (Cell Signaling), rabbit anti-Olig2 (Chemicon International), mouse anti-myelin basic protein (MBP) (Calbiochem, California, USA), rabbit anti-ATF3 (Santa Cruz), mouse anti-tubulin (Molecular Probes, Eugene, USA), and mouse anti-actin (Sigma). Incubation was then performed using HRP-conjugated sheep anti-mouse immunoglobulin G (IgG) or HRP-conjugated donkey anti-rabbit IgG (GE Healthcare Bio-Science, Uppsala, Sweden) as secondary antibody. The band of each protein was detected using a chemiluminescent method using SuperSignal WestDura (Pierce). Images of bands were detected with an instant camera (GE Healthcare Bio-Science) and the instant photographs were scanned in a scanner (CanoScan 5000F; Canon, Tokyo, Japan). Relative intensities of bands were analyzed using the Image J software (NIH). Values were represented as the difference between relative intensities of bands from the contralateral and ipsilateral hemispheres. Materials Rabbit anti-phospho-MBS antibody was raised against a synthetic phospho-MBS peptide “RQSRRST(PO 3 H 2 )

Values represent the mean±standard error of the mean (SEM). Correspondences between protein levels (relative intensity) by Western blot, infarct size, and neurological score were investigated by calculating Spearman’s rank correlation coefficient. Protein levels by Western blot and infarct size in groups N, C, M, and S were analyzed by Dunnett’s type multiple comparison test. Values of P<0.05 were considered significant.

Results Correspondence Between Neurological Deficit and Infarct Size We tested neurological deficit and infarct size 3 days after laurate injection. Rats with various severities of neurological deficit were prepared. Representative pictures for group N (Fig. 1a; neurological score, 0; infarct size, 0%), group C (Fig. 1b; neurological score, 0; infarct size, 0%), group M (Fig. 1c; neurological score, 3; infarct size, 12%), and group S (Fig. 1d; neurological score, 6; infarct size, 54%) are shown. Infarct size was significantly increased in group S compared to group C (Fig. 1e). In addition, infarct size corresponded strongly to the severity of neurological deficits (rs =0.90) (Fig. 1f). Western Blot Analysis of Various Marker Proteins Amounts of actin (control protein), tubulin (control protein), NF-L (a marker of neurofilament), GFAP (a marker of glia), CD68 (a marker of macrophages/microglia), MBP and MAG (a marker of myelin), Olig2 (a marker of oligodendrocytes), PCNA (a marker of cell proliferations), nestin (a marker of reactive astrocytes), ATF3 (a marker of neural cell damage), and HSP70 (a marker of cell damage) were estimated by Western blot analysis in cerebral hemispheres 3 days after injection of laurate (Fig. 2). The difference between the relative intensity of the band from the contralateral hemisphere and that of the ipsilateral hemisphere was calculated in each rat. No significant difference was seen between groups N and C in all indices. Relative amounts of actin and tubulin as control protein were not significantly changed in rats 3 days after injection of

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A

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laurate (i.e., groups M and S) compared to group C (Figs. 2 and 3). The relative amounts of CD68, nestin, and ATF3 were significantly increased in group S compared to group C. In addition, the relative amounts of PCNA and HSP70 were significantly increased in groups M and S compared to group C. In contrast, the relative amounts of NF-L and MAG were significantly decreased

S

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Infact size (%)

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Figure 1 Correspondence between severity of neurological deficits and infarct size in a rat model of cerebral infarction. Representative pictures show TTC staining and images (mesh area shows infarct area) of brain sections 3 days after injection of saline or laurate into the left internal carotid artery. Images are from normal rat (a neurological score, 0; infarct size, 0%), control (sham-operated) rat (b neurological score, 0; infarct size, 0%), mild symptom rat (c neurological score, 3; infarct size, 12%), and severe symptom rat (d neurological score, 6; infarct size, 54%). Rats were divided into four groups (n= 6 each): normal rats (group N), control (sham-operated) rats (group C), mild symptom rats (group M; neurological score 0–3 after sodium laurate injection), and severe symptom rats (group S; neurological score 4–6 after sodium laurate injection). Infarct sizes for group S were significantly increased compared to group C (e). No significant difference was apparent between groups C and N. Results are expressed as the mean±SEM. *P<0.05, compared with group C. Correspondence was investigated by calculating Spearman’s rank correlation coefficient in groups M and S (total, n= 12). Infarct size positively corresponded to severity of neurological deficits with rs =0.90 according to Spearman’s rank correlation coefficient (f)

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rs = 0.90

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in group S compared to group C. Regarding correspondences, the correlation coefficient (rs) between neurological scores and relative amounts of actin, NF-L, GFAP, CD68, MAG, PCNA, nestin, ATF3, and HSP70 were −0.32, −0.41, 0.20, 0.67, −0.70, 0.72, 0.65, 0.68, and 0.13, respectively. In addition, correlation coefficients (rs) between infarct size and relative amounts of actin, NF-L,

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Actin Tubulin NF-L GFAP CD68 MBP MAG Olig2 PCNA Nestin ATF3 HSP70 Hemisphere L R L R L R L R L R L R L R L R Rat No.

N1 N2 C1 C2 C3 C4 C5 C6

L R L R L R L R L R L R L R L R

L R L R L R L R L R L R L R L R

N3 N4 M1 M2 M3 M4 M5 M6

N5 N6 S1 S2 S3 S4 S5 S6

Neurological score Infarct size (%) Figure 2 Amount of various marker proteins estimated by Western blot analysis in a rat model of cerebral infarction. Amounts of actin, tubulin, NF-L, GFAP, CD68, MBP, MAG, Olig2, PCNA, nestin, ATF3, and HSP70 were measured by Western blot method. Rats were divided into four groups: group N, N1–N6; group C, C1–C6; group M,

M1–M6; and group S, S1–S6. Western blot analysis was performed with extracts from ipsilateral (L) and contralateral (R) hemispheres in rats. Neurological score and infarct size of each rat are shown at the bottom

GFAP, CD68, MAG, PCNA, nestin, ATF3, and HSP70 were −0.25, −0.61, 0.08, 0.79, −0.79, 0.89, 0.84, 0.80, and 0.18, respectively.

MLC, ERM, MBS, and adducin are known substrates of Rho-kinase. To estimate Rho-kinase activity, phosphorylation levels of these substrates were measured by Western blot analysis, then phospho-MLC/MLC, phospho-ERM/ ERM, phospho-MBS/MBS, and phospho-adducin/adducin were calculated. Phospho-MLC/MLC, phospho-ERM/ ERM, phospho-MBS/MBS, and phospho-adducin/adducin were significantly increased in group S compared to group C (Fig. 6, first and second steps). Correlation coefficients (rs) between neurological scores and Rho-kinase activity including phospho-MLC/MLC, phospho-ERM/ERM, phospho-MBS/MBS, and phospho-adducin/adducin were 0.69, 0.59, 0.77, and 0.38, respectively. In addition, correlation coefficient (rs) between infarct size and Rho-kinase activity including phospho-MLC/MLC, phospho-ERM/ ERM, phospho-MBS/MBS, and phospho-adducin/adducin was 0.81, 0.80, 0.89, and 0.64, respectively (Fig. 6, third step

Evaluation of the Rho/Rho-kinase Pathway Three days after the injection of laurate, relative amounts of RhoA, ROCK1, ROCK2, MLC, phospho-MLC, ERM, phospho-ERM, MBS, phospho-MBS, adducin, and phospho-adducin in extracts of the cerebral hemispheres were estimated by Western blot analysis (Fig. 4). Differences between relative intensities of bands from the contralateral and ipsilateral hemispheres were calculated in each rat. No significant difference was identified between groups N and C in any indices. Relative amounts of RhoA, ROCK1, and ROCK2 were unchanged in rats with injection of laurate compared to group C (Fig. 5).

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Figure 3 Quantitative analysis of amounts of various marker proteins in a rat model of cerebral infarction. Quantitative results obtained for Western blot analysis in Fig. 2 are shown. Rats were divided into four groups: groups N, C, M, and S. Results for actin, NF-L, GFAP, CD68, MAG, PCNA, nestin, ATF3, and HSP70 are shown. Amounts of CD68, nestin, and ATF3 in group S and amount of PCNA and HSP70 in groups M and S were increased compared to group C. Amounts of NF-L and MAG in group S were decreased compared to group C. No significant difference was seen between groups N and C. Values in each rat were obtained from differences between relative intensity of bands from the ipsilateral and contralateral hemispheres. Results are expressed as the mean±SEM (n=6 in each group). *P<0.05, compared with group C

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and bottom). Rho-kinase activity appeared to be elevated in rats 3 days after injection of laurate corresponding to severity of neurological deficits and infarct size.

Discussion Recent studies have indicated that Rho-kinase inhibitors such as fasudil, hydroxy fasudil, and Y27632 exert neuroprotective effects in models of cerebral infarction in rats and mice (Rikitake et al. 2005; Satoh et al. 1999, 2001; Toshima et al. 2000). However, the physiological importance of Rho-kinase in the pathogenesis of cerebral infarction remains unclear. We, therefore, investigated whether Rho-kinase activity and specific marker proteins correspond to the severity of neurological deficits and infarct size in the laurate injection model in rats to consider the roles of Rho-kinase in the pathogenesis of cerebral infarction. We first prepared rats with various severities of neurological deficits and then investigated correspondences between severity of neurological deficits and infarct size. Second, several marker proteins were measured using Western blot methods to reveal the pathophysiological characteristics of the laurate injection model. Finally, we investigated Rho-kinase activity and correspondences to severity of neurological deficits and infarct size in the brain in a rat model of cerebral infarction.

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2.5 2 1.5 1 0.5 0 -0.5 2.5 2 1.5 1 0.5 0 -0.5 3 2.5 2 1.5 1 0.5 0 -0.5 -1

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The present study revealed several novel findings. First, infarct size corresponds to the severity of neurological deficit. Second, CD68, PCNA, nestin, ATF3, and HSP70 increase, while NF-L and MAG decrease corresponding to the severity of neurological deficits and infarct size. Third, Rho-kinase activity represented as phospho-MBS/MBS, phospho-ERM/ERM, phospho-MLC/MLC, and phosphoadducin/adducin is elevated corresponding to the severity of neurological deficits and infarct size at 3 days after injection of laurate in the cerebral ischemia model. The cerebral ischemia model we used displayed some variations in the severity of neurological deficits. However, this is useful for investigating correspondences between severity of neurological deficits and target indices. ATF3 and HSP70 are known to be upregulated in response to cerebral ischemia (Ohba et al. 2003; Hata et al. 2000). In the present study, amounts of ATF3 and HSP70 were increased in rat brains 3 days after laurate injection compared to control rats. The data support the notion that infarct sizes increase corresponding to neurological deficits. CD68 is a marker of macrophage/microglia, which are key players in early stage inflammation. Amounts of CD68 were increased in rat brains 3 days after injection of laurate corresponding to the severity of neurological deficits, suggesting that macrophage/microglia accelerate infarct formation. Yagita et al. (2002) demonstrated that nestin, a marker of reactive astrocytes, was increased in rat brains in

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RhoA ROCK1 ROCK2 MLC p-MLC ERM p-ERM MBS p-MBS Adducin p-Adducin Hemisphere L R L R L R L R L R L R L R L R

L R L R L R L R L R L R L R L R

L R L R L R L R L R L R L R L R

Rat No. Neurological score Infarct size (%)

a model of cerebral ischemia model. Likewise, amounts of nestin were increased in rat brain 3 days after injection of laurate in the present study. PCNA is a marker of cell proliferation. Increased levels of PCNA 3 days after laurate injection should contribute to the activation of macrophages, microglia, and reactive astrocytes in response to

ischemia. By contrast, MAG, a marker of myelin, and NF-L, a marker of neurofilaments, were decreased in rat brains 3 days after laurate injection compared to control rats, indicating degeneration of neuronal fibers. These data suggest that important and drastic changes in the pathogenesis of infarct occur at 3 days after laurate injection.

(ipsilateral–contralateral)

into four groups: group N (normal, N1–N6); group C (control; C1– C6); group M (mild, M1–M6); and group S (severe, S1–S6). Western blot analysis was performed with extracts from ipsilateral (L) and contralateral (R) hemispheres in rats. Neurological score and infarct size of each rat are shown at the bottom

Relative intensity

Figure 4 Evaluation of Rho/Rho-kinase pathway by Western blot analysis in a rat model of cerebral infarction. Amount of RhoA, Rhokinase β (ROCK1), Rho-kinase α (ROCK2), MLC, phospho-MLC, ERM, phospho-ERM, MBS, phospho-MBS, adducin, and phosphoadducin were measured by Western blot method. Rats were divided

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Figure 5 Quantitative analysis of amount of RhoA, ROCK1 (Rhokinase β), and ROCK2 (Rho-kinase α) in a rat model of cerebral infarction. Quantitative results obtained for Western blot analysis in Fig. 4 are shown. Rats were divided into four groups: groups N, C, M, and S. Results of RhoA, ROCK1, and ROCK2 are shown. No

C

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significant differences were seen in amounts of RhoA, ROCK1, and ROCK2 among groups C, M, and S. No significant differences were seen between groups N and C. Values for each rat were obtained from differences between relative intensity of bands from ipsilateral and contralateral hemispheres. Results are expressed as the mean±SEM (n=6)

Relative intensity

(ipsilateral – contralateral )

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Figure 6 Quantitative and correspondence analysis of Rho-kinase activity in a rat model of cerebral infarction. Quantitative results obtained for Western blot analysis in Fig. 4 are shown. Rats were divided into four groups: groups N, C, M, and S. Rho-kinase activity was represented as phospho-MLC/MLC (p-MLC/MLC), phospho-ERM/ ERM (p-ERM/ERM), phosphoMBS/MBS (p-MBS/MBS), and phospho-adducin/adducin (p-adducin/adducin), all of which were increased in group S compared to group C (first and second steps). No significant difference was seen between groups N and C. Results are expressed as the mean±SEM (n=6 in each group). *P<0.05, compared with control group. Correspondences between Rho-kinase activity and infarct size were investigated by calculating Spearman’s rank correlation coefficient in rats of groups M and S (total, n=12) (third step and bottom). Values for p-MLC/MLC, p-ERM/ERM, p-MBS/MBS, and p-adducin/ adducin display strong correspondence to infarct sizes with rs =0.81, 0.80, 0.89, and 0.64, respectively (Spearman’s rank correlation coefficient)

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So far, increases in Rho-kinase activity have been demonstrated by estimating levels of MBS and adducin phosphorylation in cerebral ischemia models (Rikitake et al. 2005; Shin et al. 2007; Yamashita et al. 2007; Yagita et al. 2007). We tried to use four kinds of substrates, such as MBS, MLC, ERM, and adducin, to obtain more confident evidence of whether Rho-kinase is activated. We demonstrated that phospho-MBS/MBS, phospho-MLC/MLC, phosphor-ERM/ERM, and phospho-adducin/adducin are increased 3 days after injection of laurate, suggesting the

Infarct size (%)

activation of Rho-kinase. Substrates of Rho-kinase, such as MBS, MLC, ERM, and adducin, are known to play important roles in cell motility, membrane ruffling, smooth muscle contraction, and cell aggregation. For example, in terms of MBS and MLC, Rho-kinase is able to regulate the phosphorylation of MLC by direct phosphorylation of MLC and by inactivation of myosin phosphatase through the phosphorylation of MBS. Rho-kinase and myosin phosphatase thus coordinate to regulate the phosphorylation state of MLC, which is thought to induce smooth muscle

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contraction and stress fiber formation in nonmuscle cells (Kawano et al. 1999). In addition, adducin is a membrane skeletal protein that promotes the binding of spectrin to actin filaments and is concentrated at cell–cell contact sites in epithelial cells (Kimura et al. 1998). ERM family proteins are thought to function as general cross-linkers between the plasma membrane and actin filaments. Suppression of ERM protein expression with antisense oligonucleotides destroys microvilli, cell–cell, and cell–matrix adhesion sites (Takeuchi et al. 1994). This evidence leads to the speculation that activation of Rho-kinase and subsequent phosphorylation of substrates including MBS, MLC, ERM, and adducin would be involved in the pathogenesis of decreased blood flow, recruitment and migration of inflammatory cells, generation of reactive astrocytes, and neural degeneration after cerebral ischemia. Some crucial points, such as improvement of blood flow, inhibition for activation of inflammatory cells, and beneficial effects for neurons, could act to protect against infarct formation. Fasudil, a Rho-kinase inhibitor, improves blood flow in a cerebral ischemic model (Satoh et al. 2008) and inhibits macrophage accumulation and migration and coronary lesion formation in a porcine model (Miyata et al. 2000). Rho-kinase inhibitors protect against neural degeneration and activate neuronal regeneration (Yamagishi et al. 2005; Mueller et al. 2005). The mechanism by which Rho-kinase inhibitors exert beneficial effects on cerebral ischemic models would be explained by improvement of blood flow, inhibition of inflammatory cell migration, protection against neural degeneration, and activation of neural regeneration. Elevation of Rho-kinase activity in neural and vascular cells may be associated with the pathogenesis of cerebral infarction. For example, overexpression of the constitutively active form of RhoA (Bito et al. 2000; Kozma et al. 1997) and Rho-kinase (Amano et al. 1998) induces neurite retraction. Prostaglandin F2α induces the contraction of arterial smooth muscle cells through the activation of Rho-kinase (Ito et al. 2003). Rho-kinase is activated in the basilar artery in cerebral vasospasm (Sato et al. 2000). These findings suggest the possibility that activation of Rho-kinase in neuronal cells may induce disturbance of neuronal networks and result in neurological deficits and that, in vascular cells, this may induce contraction of blood vessels and reduction of cerebral blood flow, causing progression of infarction. These hypotheses are consistent with the improved outcomes of cerebral infarction seen with the use of Rho-kinase inhibitors. In the present study, amounts of Rho-kinase and Rho, an upstream factor of Rho-kinase, did not change significantly despite enhancement of Rho-kinase activity. Quantitative changes in Rho and Rho-kinase thus do not appear responsible for the activation of Rho-kinase in the present

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model of cerebral infarction. Rho is upregulated in the brains of patients who die following focal cerebral infarction (Brabeck et al. 2003) and in a mouse cerebral infarction model (Trapp et al. 2001). This discrepancy in findings appears attributable to differences in state of the ischemic brain and duration between cerebral infarction and examination in patients and model animals. In conclusion, the present findings suggest that Rhokinase is activated corresponding to the severity of neurological deficit and infarct size in our cerebral infarction model and that Rho-kinase could be an important target of treatment for cerebral infarction.

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