Rho-Kinase inhibition decreases focal cerebral ischemia-induced glial activation in rats

Focal cerebral ischemia model

All animals were used according to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines, and guidelines of the Institute of Experimental Animals of Shimane University. The experimental protocols and procedures were approved by the Ethical Committee of Shimane University (approval code: IZ30-3). Adult male Sprague-Dawley rats (CLEA Japan, Inc Tokyo, Japan) of 8-10 weeks weighing between 250-280 g were used to generate the middle cerebral artery occlusion (MCAO) model as previously described. ¹⁴ Briefly, the animals were anesthetized with 4.0% halothane, and the right common carotid, external carotid, and internal carotid arteries were exposed via a ventral midline incision. A 4-0 monofilament nylon suture (Nescosuture, Tokyo, Japan), having a tip rounded by silicon (Xantopren L blue, Germany) coating, was inserted through the right external carotid artery, and moved into the internal carotid artery to block the origin of the right middle cerebral artery for 60 minutes Then the rats were re-anesthetized, and the filament was removed. After recovery from anesthesia, rats were returned to their cages. Rectal temperature was maintained at 37°C throughout the surgical procedure utilizing a feedback-regulated heating system. A total of 42 rats were used to generate the MCAO model. Among them, 4 rats died within the study period (mortality rate of about 9.5%), and 8 rats were excluded (5 rats due to insufficient neurological deficit after 1 day, and 3 rats due to intracerebral hemorrhage) from the study. The schematic representation of the study design is shown in the supplemental Figure 1.

Evaluation of neurological performance

Neurological performance was evaluated before and 1, 2, 3, and 4 days after MCAO using a modified neurological severity scoring (mNSS) system. ³⁷ The mNSS is a composite of the motor, sensory, balance tests, and reflexes, and graded on a scale of 0 to 22, where the points were counted for the inability to perform a test, or alteration of a tested reflex. Therefore, an increasing score indicates the severity of the neurological deficit.

Intraperitoneal injection of Rho-kinase inhibitor (Fasudil)

A Rho-kinase inhibitor, Fasudil (Asahi Kasei Pharma Corporation, Japan), was dissolved in PBS at a 4 mg/ml concentration. After MCAO, rats were randomly divided into 1) Fasudil or 2) vehicle group (n = 15 in each group), and received the drug (5 mg/kg) or vehicle (PBS) intraperitoneally. Each group was further subdivided into 2 subgroups, a) Day 2 (total n = 20, PBS = 10 and Fasudil = 10) and b) Day 4 (total n = 10; PBS = 5 and Fasudil = 5). The administration of the drug started 1 hour after reperfusion and continued as a single daily injection till the day of sacrifice.

MRI

A T2-weighted MRI image of the whole brain (n = 5/group) was acquired with a 1.5 T MRI system (Mrmini SA; DS Pharma Biomedical, Osaka, Japan) on Day 4 after MCAO. The area of the infarct region in 5 slices of the MRI image was measured using ImageJ (NIH), expressed as % of the contralateral side, averaged, and considered as the lesion size of the animal.

Tissue preparation and immunohistochemical analysis

At 2 and 4 days after MCAO, the rats were deeply anesthetized with 5% isoflurane and the brains were fixed by transcardial perfusion of normal saline, followed by 4% paraformaldehyde (PFA) in PBS. The rat brains were removed, cryoprotected, sectioned, and frozen on dry ice. Coronal tissue sections 10 μm thick were cut with a cryostat. After quenching endogenous peroxidase activity, the sections were incubated in a blocking solution containing 10% normal goat or horse serum. The sections were incubated with a primary antibody against a microglia-specific ionized calcium-binding molecule-1 (Iba-1, 1:500, rabbit, Abcam, Waltham, MA, USA), glial fibrillary acidic protein (GFAP, rabbit, DAKO, Carpinteria, CA, USA), rat CD68 (ED-1, 1:100, GeneTex, Hsinchu, Taiwan, R.O.C) inducible nitric oxide synthase (iNOS, 1:100, rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA), aquaporin 4 (AQP4, 1:100, rabbit, Santa Cruz), NF-κB (p65, 1:100, rabbit, Santa Cruz), tumor necrosis factor α (TNF-α; 1:100, rabbit, Santa Cruz), CREB-1 (1:100, rabbit, Santa Cruz) or interleukin-1β (IL-1β; 1:100, rabbit, Santa Cruz). After incubation with primary antibody, sections were incubated with Texas red- or FITC-conjugated species-specific IgG (1:100, Santa Cruz). The immunoreactive proteins were analyzed with a fluorescence microscopy system (Nikon, ECLIPSE, E600, Tokyo, Japan). Immune reaction positive cells in the coronal brain sections at similar positions were counted in 5 random microscopic fields of the cortical core and the IBZ area at ×400 magnification in a blinded manner. The average of such immune reaction positive cells was represented as the cell population of that rat.

Cell culture

A human microglial cell line, HMO6, was established by isolating microglia from human fetal telencephalon tissue and immortalizing it using a retroviral vector encoding v-myc. ³⁸ HMO6 cells were cultured in DMEM supplemented with antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin), L-glutamine, and 10% FCS. During in vitro stimulations, HMO6 was cultured in DMEM containing 1% FCS, L-glutamine, and antibiotics. DMEM, FCS, antibiotics, and L-glutamine were purchased from Invitrogen (ThermoFisher, Waltham, MA, USA)

Immunocytochemistry of myosin light chain phosphorylation

HMO6 cells were cultured on a sterile glass coverslip in a complete cell culture medium. After appropriate treatment, cells on the coverslip were washed and fixed with 4% paraformaldehyde. The cells were incubated in a blocking solution containing 10% normal goat serum and .2% Triton-X in PBS for 30 minutes. Then the cells were incubated for 60 minutes with polyclonal goat anti-MLC2 or polyclonal goat anti-pMLC2 antibody (1:100, Santa Cruz), followed by biotinylated anti-goat IgG (1:200, Santa Cruz). Then the coverslips were incubated with an avidin-biotin-peroxidase complex (ABC, Vector Laboratories, Burlingame, CA, USA), and the immunoreaction products were visualized with 3,30-diaminobenzidine (DAB, Sigma, St Louis, MO, USA). The immunoreactive cells were analyzed by light microscopy.

Migration assay

Migration assay was performed using a 48-well micro-chemotaxis chamber (Neuroprobe, Cabin John, MD, USA) and a 5-μm pore membrane (Costar, High Wycombe, England) coated with fibronectin (6.5 μg/mL, Sigma) as described previously. ³⁹ Briefly, MIP-1α was diluted in DMEM containing 1% FCS, and 28 μL of each dilution was applied in triplicate into the lower wells of the chemotaxis chamber. The filled lower chamber was then overlaid with the membrane with the coated surface facing downwards, and the top chamber was assembled to form wells. Cell suspension (5 × 10⁵/mL) in DMEM containing 1% FCS was applied (50 μL) in the upper wells. After incubation, the migrated cells on the fibronectin-coated lower surface of the membrane were fixed with methanol and stained with Harris’ hematoxylin (Sigma). Migrated cells were counted in 5 microscopic fields per well at ×400 magnification.

Total RNA isolation and real-time PCR

The mRNA expression of proinflammatory mediators was analyzed by quantitative real-time PCR. ²³ After appropriate stimulation, total RNA was isolated from HMO6 cells using RNA STAT reagent (Tel-Test, Friendswood, Tx, USA) according to the manufacturer’s instructions. Two μg of total RNA was reverse transcribed using reverse transcriptase enzyme (ReverTraAce, Toyobo, Osaka, Japan), and quantitative real-time PCR was done with SyBr green real-time PCR system (Applied Biosystem, Warrington WA1 4SR, UK) and gene-specific primers, using an ABI Prism 7300 Sequence Detection System (Applied Biosystem).

Tissue preparation for western blot analysis

Two days after MCAO, rats were deeply anesthetized and transcardially perfused with normal saline. ²³ The brains were removed, and 5 mm coronal sections were made starting from 2 mm anterior to bregma to 3 mm posterior to bregma. The first 2 mm of brain tissue was fixed with 4% paraformaldehyde (PFA), cryoprotected, and used to assess the infarction size by Hematoxylin-Eosin staining. The cortical portion of remaining 3 mm tissue sections then subdivided into core, IBZ and contralateral parts, homogenized in ice-cold RIPA buffer (PBS, pH 7.4, 1% Nonidet p-40, .5% sodium deoxycholate, .1%SDS, 10 mg/ml PMSF, and 1 mg/ml aprotinin) (1:10 wt-vol) and stored at -70^oC for further use.

Preparation of nuclear extract

After appropriate treatment, nuclear extracts were prepared, as described previously. ⁴⁰ In a brief, cells were harvested by scraping in ice-cold PBS and centrifuge at 1500 rpm for 5 minutes at 4°C. The cells were incubated in 2 packed cell volume of hypotonic buffer A (10 mM HEPES [pH 8.0], 1.5 mM MgCl₂, 10 mM KCl, .5 mM dithiothreitol [DTT], 200 mM sucrose, .5 mm phenylmethanesulfonyl fluoride [PMSF], 1 mg/ml each of leupeptin and aprotinin, .5% Nonidet P-40) for 5 minutes at 4°C. Then the nuclei were pelleted by centrifuging the samples at 10,000 rpm for 5 seconds. The nuclei were dissolved in 2/3^rd packed cell volume of extraction buffer C (20 mm HEPES [pH 7.9], 1.5 mM MgCl₂, 420 mm NaCl, .2 mM EDTA, .5 mM PMSF, 1.0 mM DTT, 1.0 mg/ml each of leupeptin and aprotinin) and incubated on a rocking platform for 30 minutes at 4°C. Then the samples were centrifuged at 15,000 rpm for 10 minutes, the supernatants were collected and stored at -70°C until further use.

Western blot analysis

Forty to eighty μg of nuclear extracts or brain tissue samples were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The target proteins were detected with the following antibodies: anti-AQP-4 rabbit polyclonal IgG, anti-CREB-1 rabbit polyclonal IgG, anti-NF-κB (p65) rabbit polyclonal IgG, anti-IRF-1 mouse monoclonal IgG and anti-TNFα rabbit polyclonal IgG. All antibodies were purchased from Santa Cruz. After stripping, the same membrane was used to detect β-Actin (mouse monoclonal IgG, Santa Cruz). Immunoreactive proteins were detected using an enhanced chemiluminescence system (Amersham, Buckinghamshire, UK) according to the manufacturer’s protocol.

Statistical analysis

The numerical data are expressed as mean ± SD. To determine the statistical significance of the results, student’s t-test, one-way ANOVA followed by Student-Newman-Keuls multiple comparisons, or Scheffe post hoc test was employed (SPSS). The statistical significance level was set at P < .05.

Assessment of neurological performance, infarct volume, and cellular apoptosis

All animals showed normal neurological functions before the generation of MCAO. One day after MCAO all animals, irrespective of groups, showed similar neurological deficits (mNSS= 14-18). As shown in Figure 1A, neurological performances were improved in the Fasudil groups from Day 2 onwards after MCAO compared to the PBS group, which became statistically significant on Day 3 and Day 4 (Figure 1A).OPEN IN VIEWER

Next, the effect of Fasudil on infarction size was evaluated. The MRI results demonstrated that in the Fasudil group, infarct volume was significantly decreased at Day 4 (infarct size- PBS group 79.1 ± 8.3 vs Fasudil group 48.1 ± 7.8% of contralateral hemisphere, P < .01) after MCAO (Figure 1B). Moreover, T2 weighted images of PBS groups were brighter, suggesting that the edema was decreased in the Fasudil group (Figure 1C).

Fasudil treatment decreased water channel protein Aquaporin 4 levels in MCAO brains.

Water channel protein Aquaporin 4 (AQP4) plays an important role in the development of cerebral edema in stroke condition.^41,42 Hence, we decided to check its regulation in MCAO conditions. Our immunostaining results showed that AQP4 was expressed around the vessel-like structures, which was decreased in the core and ischemic boundary zone (IBZ) areas in the Fasudil group (Figure 2A). Then, we quantified AQP4 protein levels by Western blotting. The results showed that the M1 isoform of AQP4 was significantly decreased in the core and IBZ regions in the Fasudil group compared to the PBS group, whereas the M23 isoform was not changed much (Figure 2B and C).OPEN IN VIEWER

Accumulation of microglia/macrophage and astrocytes was inhibited by Fasudil

Macrophage/microglial accumulation in the MCAO rat brain was evaluated by Iba-1 immunostaining. On Day 2 and Day 4 after MCAO, Iba-1⁺ cells were detected in the core and the IBZ (Figure 3A and B). Fasudil significantly decreased Iba1⁺ macrophage/microglial accumulation both in the core and the IBZ on Day 4 after MCAO (Figure 3C).OPEN IN VIEWER

Accumulation of the astrocytes was evaluated by GFAP immunofluorescence staining. Very few GFAP⁺ cells were detected in the core area both on Day 2 and Day 4 after MCAO (data not shown), which were mainly found in the IBZ. GFAP⁺ cells in IBZ were significantly decreased in the Fasudil group on Day 4 after MCAO (Figure 3D and E).

Effects of Fasudil on transcription factors expression in MCAO rats

To investigate the effects of Fasudil on inflammatory gene transcription in MCAO conditions, we analyzed the protein level of several transcription factors in the MCAO rat brain. Our Western blotting results showed that on Day 2 after MCAO, NF-κB protein levels were decreased in the core region of the Fasudil group compared to their PBS counterparts (Figure 4A and B). Conversely, CREB-1 and IFR-1 levels were not decreased in the same region of the Fasudil-treated rats (Figure 4A and B). Double immunofluorescence staining revealed that both NF-κB and CREB-1 were expressed in the ED-1⁺ macrophage/microglia in the core region (Figure 4C).OPEN IN VIEWER

Effects of Fasudil on the levels of proinflammatory mediators in MCAO rats

To investigate the effects of Fasudil on inflammatory mediators in MCAO conditions, we analyzed the protein levels of IL-1β, TNF-α, and iNOS in the MCAO rat brain by immunofluorescence staining. The immunostaining results demonstrated that on Day 4 after MCAO, IL-1β, TNF-α, and iNOS positive cell number was decreased in the lesion areas of the Fasudil group compared to the PBS group (Figure 5A). Double immunofluorescence results showed that ED-1⁺ macrophage/microglia expressed IL-1β, TNF-α, and iNOS in the core areas (Figure 5B). Western blotting results showed that on Day 2 after MCAO, TNF-α levels were significantly decreased in the core and IBZ regions of the Fasudil group (Figure 5C and D).OPEN IN VIEWER

Microglial cell migration was inhibited by Fasudil

To assess whether decreased Iba-1⁺ macrophage/microglia accumulation in the lesion areas of the Fasudil group was due to decreased migration, we investigated the effect of Fasudil on macrophage inflammatory protein 1α (MIP-1α)-induced migration of an immortalized human microglial cell line HMO6. Preliminary time course experiments at 1, 2, 4, and 6 hours showed a time-dependent increase in migrated cell number. Further experiments were performed at a single time point of 4 hours. Dose-response experiments demonstrated that migrated cell number was increased up to 40 ng/ml of MIP-1α, making a typical bell-shaped migration curve (Figure 6A). Fasudil significantly decreased MIP-1α-induced HMO6 cell migration in a dose-dependent manner (Figure 6B).OPEN IN VIEWER

Myosin light chain (MLC) phosphorylation is essential for cytoskeleton rearrangement and cell migration. ²⁹ To check whether Rho-kinase affects these events during microglia migration, MIP-1α-induced MLC phosphorylation in HMO6 was investigated by immunocytochemical analysis. MIP-1α treatment with or without Fasudil for 1 hour did not change the total MLC level in HMO6 cells (Figure 6C-a, b and c). MIP-1α treatment markedly increased the phosphorylated form of MLC (pMLC) level in HMO6. However, when pretreated with Fasudil for 30 minutes, MIP-1α failed to increase the pMLC level in HMO6 (Figure 6C- d, e, and f).

Cytokine-induced transcription factors activation and proinflammatory gene expression were modulated by Rho-kinase inhibition in a microglial cell line

The inhibitory effect of Fasudil on microglial migration raises the possibility that decreased NF-κB protein levels in the Fasudil group might be due to decreased number of macrophage/microglia cells in the lesion areas. Moreover, changing the protein level might not be sufficient, because activation and nuclear translocation of NF-κB are necessary to regulate NF-κB-dependent gene expression. As NF-κB and other transcription factors translocate to the nucleus after activation, we investigated whether Fasudil modulates the cytokine-induced nuclear accumulation of transcription factors in HMO6. The Western blotting results of HMO6 nuclear proteins revealed that combined IL-1β and IFNγ treatment resulted in increased nuclear translocation of NF-κB, which was inhibited by Fasudil (Figure 7A and B). Conversely, combined IL-1β and IFNγ treatment did not increase CREB-1 translocation. However, the addition of Fasudil significantly increased its translocation to the nucleus (Figure 7A and B). Similarly, real-time quantitative PCR results revealed that Rho-kinase inhibition by Fasudil transiently inhibited IL-1β and IFNγ-induced mRNA expression of NF-κB-dependent genes including IL-1β, TNF-α, and iNOS after 4 hours (Figure 7C and Table 1). After 24 hours stimulation, only TNF-α and iNOS were inhibited by Fasudil (Figure 7E and Table 1).OPEN IN VIEWER

OPEN IN VIEWER

Table 1.

Effects of Fasudil on the mRNA expression of pro-inflammatory molecules.

mRNA	4 h		8 h		24 h
	IL-1β + IFNγ	IL-1β+ IFNγ + Fasudil	IL-1β + IFNγ	IL-1β + IFNγ+Fasudil	IL-1β + IFNγ	IL-1β + IFNγ + Fasudil
IL-1β	78.7 ± 2.2	49.2 ± 12.9 b	454.5 ± 160.5	408.6 ± 97.3	2759 ± 1986	1893 ± 1316
TNF-α	23.2 ± 3.3	13.6 ± 2.7 b	29.5 ± 7.6	25.8 ± 8.4	52.7 ± 25.8	14.4 ± 6.3 a
iNOS	105 ± 22.2	29.2 ± 20.8 b	490.6 ± 295.6	478.6 ± 54.3	7909 ± 4217	964.2 ± 774.3 a

A human microglial cell line (HMO6) was stimulated with IL-1β and IFNγ (10 ng/ml each) with or without Fasudil (10 μM) for indicated times and the mRNA expression of IL-1β, TNFα, and iNOS were measured by quantitative real-time PCR. Results were normalized with corresponding GAPDH mRNA and calculated as fold induction relative to unstimulated conditions and expressed as mean ± SD of 3 independent experiments. The statistical significance was denoted as follows.

^ap < .05.

^bp < .01 vs IL-1β and IFNγ condition.