Recovery of the locomotor function of the right side of body was evaluated by using the scale described later.

PMCAO resulted in moderate motor paralysis of the contralateral side (scale: 2) 0.5 h after the occlusion. Recovery from the motor paralysis was significantly enhanced by intraperitoneal administration of DAEE compared with the paralysis seen in the vehicle-treated group, when evaluated 48, 72, and 96 h after PMCAO. The neurological condition was thus improved in the DAEE-treated group. That is, improvement of the locomotor function from scale 2 to scale 1 occurred earlier in the DAEE-treated group than in the vehicle-treated group (Figure 2). The survival rate of 96 h after PMCAO was higher in the DAEE-treated group than that of the vehicle-treated group (91.7% vs. 84.5%, respectively), however, no significant difference was observed (P = 0.23, Chi-square test).

Next we examined the infarct volume by staining with 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich, Saint Louis, MO, USA) to assess the effect of DAEE treatment quantitatively 96 h after PMCAO. TTC reacts with intact mitochondrial respiratory enzymes to generate a bright-red color that stand in contrast with the pale color of an infarction [11,12]. As the borderlines between normal and infarct areas cannot be detected in microscopic view by TTC staining, Nissl staining is applicable to designate the ischemic region histochemically [13]. The infarction range detected by TTC is perceived to be the same as that detected by Nissl staining. So, we confirmed that the borderlines determined by Nissl staining almost corresponded to those by TTC staining at the microscopic level. Although neuronal death had occurred in the infarct area, no significant differences in the infarct area or volume were found between vehicle- and DAEE-treated groups (Figure 3).

Activation of ERK1/2 by PMCAO was assessed by Western blot analysis with specific antibody against ERK1/2 or phosphorylated ERK1/2 (pERK1/2). DAEE-treatment significantly raised the level of pERK1/2 4 days after PMCAO to one higher than that of the vehicle-treatment group (Figure 4-A).

Nissl staining was performed to determine the border between the normal and infarct areas. The Nissl-positive cells were defined as the cells with a diameter of 10 μm or more. We regarded the normal area as that where the density of Nissl-positive cells was over 5 cells/50 × 50 μm²; and the infarct area, as the region with a lower cell density. In this way, we could get polygonal lines. So, we drew curves through the midpoints of each straight line of them fitted by eye. Therefore, we defined the curve as the borderline between the normal and infarct areas. On each consecutive slice, the number of pERK1/2-positive cells was counted in the normal, infarct, and border areas. We counted the numbers in a square area 200 μm on a side with a focus on the borderline as the border area, in the same sized square just medial to the border area as the normal area, and in the square just lateral to it as the infarct area. These areas were counted in high-powered fields on a section through the anterior commissure. Each count was expressed as number of pERK1/2-positive cells/200 × 200 μm² (n = 3). In the DAEE-treated group, pERK1/2-positive cells were observed in the border area between the infarct and normal areas, namely, in the area containing Nissl stained neurons, in the cerebral cortex. On the other hand, in the vehicle-treated group, pERK1/2-positive cells in the border area were significantly fewer than those in the DAEE-treated group. The numbers of the pERK1/2-positive cells in the other areas, i.e., normal and infarct areas, were not significantly different between the two groups (Figure 4-B).

The mRNA expression of BDNF was investigated by reverse transcription-polymerase chain reaction (RT-PCR). DAEE-treatment significantly raised the expression of BDNF 96 h after PMCAO higher than that of the vehicle-treatment group (Figure 5).

The experiments were conducted in accordance with the Animal Care Guidance issued by the Animal Experiments Committee of the graduate school of Gifu University and Gifu Pharmaceutical University. All experiments were performed by using male ddY mice (5-week old; body weight 27 to 33 g) purchased from Japan SLC Ltd., (Shizuoka, Japan). Cerebral infarction was produced by permanent occlusion of the left middle cerebral artery. Anesthesia was induced using 2.0 to 3.0% isoflurane and maintained by using 1.5 to 2.0% isoflurane with 70% N₂O/30% O₂ delivered by means of an animal general anesthesia machine (Soft Lander, Sin-ei Industry Co Ltd., Saitama, Japan). The regional cerebral blood flow in each mouse was monitored by laser-Doppler flowmetry (Omega-Flow flo-N1; Omegawave Inc., Tokyo, Japan). A flexible probe was fixed to the skull (2 mm posterior and 6 mm lateral to bregma). After a midline skin incision had been made, the left common carotid artery was exposed and occluded. An 8-0 nylon monofilament (Eticon, Somerville, NJ, USA) coated with a mixture of silicone resin (Provil; Hereus Kulzer Inc., Hanau, Germany) was introduced into the left common carotid artery and advanced it along the internal carotid artery until the tip occluded the proximal stem of the middle cerebral artery, as described earlier [31,32].

DAEE was purchased from Sigma-Aldrich Co. LLC. DAEE was dissolved in 0.1% dimethylsulfoxide (DMSO; Wako Pure Chemical Industries Ltd., Osaka, Japan) and diluted with phosphate-buffered saline (PBS). It was administered intraperitoneally into the animals at a dose of 100 μg/kg at 0.5, 24, 48, and 72 h after PMCAO. Control animals received vehicle (PBS) containing 0.1% DMSO without DAEE.

Mice (vehicle; n = 58, DAEE; n = 60) were tested for neurological deficits, as described earlier [31] at 0.5, 24, 48, 72, and 96 h after PMCAO. Scoring was done by using the following scale: 0, no observable neurological deficits (normal); 1, failure to extend the right forepaw (mild); 2, circling to the contralateral side (moderate); 3, loss of walking or righting reflex (severe); 4, dead. The investigators were masked as to the group to which each mouse belonged.

The brains were removed immediately after the mice had been given an overdose of diethyl ether, at 96 h after PMCAO, and cut into 5 serial 2-mm-thick coronal block slices. These slices were immersed for 20 min in a 2% solution of TTC. Image J software was used to measure the unstained areas of the total infarctions, and the infarction volume was calculated as described previously [31].

The brains were dissected out as described in the previous section. The left hemisphere of the anterior 4th slice was used for Western blotting. Tissues were homogenized in lysis buffer (20 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 2 mM EDTA, 1% NP-40, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 50 mM NaVO4, 1 mM phenylmethylsulfonyl fluoride [PMSF], 0.1% sodium dodecyl sulfate [SDS] and 1% Na deoxycholate) and then centrifuged, after which the supernatant fluids were collected. Protein concentrations of the supernatants were determined with a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Each sample, 2.8 μg protein for ERK1/2 and 7.0 μg protein for pERK1/2 was subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes by electrophoresis. The membranes were blocked for 1 h at room temperature with 5% skim milk (Morinaga Milk Products, Tokyo, Japan) in Tris-buffered saline containing 0.1% Tween 20 (TBS/Tween 20) and then incubated with primary antibody against ERK1/2 or pERK1/2 (1:1000, Cell Signaling Technology, Beverly, MA, USA) for 2 h. After having been washed with TBS/Tween 20, the membranes were incubated with alkaline phosphatase-conjugated anti-rabbit IgG secondary antibody (1:5000 Promega, Madison, WI, USA) for 3 h. Finally the specific protein bands were developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indrylphosphate p-toluidine salt. The intensity of immunoreactive bands was measured by use of image-analysis software (NIH Image J).

Brains were removed after cardio-perfusion with 4% (w/v) paraformaldehyde solution prepared in 0.1 M phosphate buffer, pH 7.4 (fixative), 96 h after PMCAO, postfixed in the same fixative overnight, soaked in PBS containing 20% (w/v) sucrose, and frozen in embedding compound (Sakura Finetek, Tokyo, Japan). Coronal sections of 30-μm thickness were prepared with a cryostat (model CM 1800; Leica, Nussloch, Germany), attached to adhesive-coated slides (Matsunami, Osaka, Japan), dried, and soaked in the above fixative for 30 min.

The sections were stained for Nissl substance with 0.1% thionin, dehydrated by passage through an ascending series of ethanol, cleared in xylene, and coverslipped with Euikit (O. Kindler, Freiburg, Germany) [33]. For immunohistochemistry with the chromogen diaminobenzidine (DAB), the sections were treated with TBS/0.3% Triton X-100 containing 0.3% H₂O₂ for 3 h, rinsed for 1 h in the same solution, washed 3 times with TBS and blocked for 30 min with 2% (w/v) Block Ace (Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan) dissolved in TBS (blocking solution). Next, the sections were incubated overnight at 4 °C with antibody against pERK1/2 (1:1000; Chemicon, Temecula, CA, USA). After a wash with TBS, the sections were incubated for 3 h with anti-rabbit IgG goat antibody (1:1000; Vector Laboratories, Burlingame, CA, USA). The sections were then incubated in ABC solution (Elite ABC, Vector Laboratories) with gentle shaking at 4 °C overnight and further treated according to the glucose oxidase-DAB-nickel method. Finally the sections were stained with 3,3′-diaminobenzidine tetrahydrochloride (Dojindo Laboratories, Kumamoto, Japan) for 5–10 min.

The brains were dissected out as described in the previous section. The left hemisphere of the anterior 4th slice was used for RT-PCR experiment. RNA was prepared from the collected tissues by using TRIZOL (Invirtogen, Carlsbad, CA, USA) according to the manufacture’s instructuins. RT-PCR was performed with PowerScript^TM Reverse Transcriptase (Clontech, CA, USA) by following the manufacturer’s instructions. The synthesized cDNAs were amplified with each pair of primers by PCR. The following primer sets were used: β-actin, 5′-GTGGGCCGCTCTAGGCACCAA-3′ and 5′-CTCTTTGATATCACGCACGAT-3′ BDNF, 5′-GGAATTCGAGTGATGACCATCCTTTTCCTTAC-3′ and 5′-CGGATCCCTATCTTCCCCTTTTAATGGTCAGTG-3′. The amplification was carried out under the following conditions: denaturation, 94 °C for 45 s; annealing, 63 °C (for β-actin) or 65 °C (for BDNF) for 45 s; and extension, 72 °C for 45 s. The cycle was repeated 26 times for β-actin, 33 times for BDNF. β-actin was used as an internal control. PCR products were electrophoresed in 2% agarose gels and visualized by Ethidium Bromide staining. The intensity of the bands was analyzed by used of image-analysis software (NIH Image J).

Statistical comparisons were made using two-way analysis of variance followed by Mann-Whitney U test, Chi-square test, or Student’s t-test.