The roots of S. miltiorrhiza Bunge (Labiatae) were purchased in March 2005 at the University Oriental Herbal Drugstore, Iksan, Korea, and their identity was verified by Dr. Kyu-Kwan Jang of the Botanical Garden, Wonkwang University. A voucher specimen (no. WP05-87) was deposited at the herbarium of the College of Pharmacy, Wonkwang University (Korea). Dried and pulverized roots of S. miltiorrhiza (2 kg) were soaked in 1.6 l distilled water for 12 hours at room temperature, extracted with hot ethanol for 2 hours, and filtered with filter paper. The filtrate was the evaporated in a vacuole to produce an ethanol extract (277 g). The ethanol extract was suspended in distilled water (500 ml) and then filtered. The residue derived from the filtration was dissolved in hot ethanol and filtered again. The filtrate was then evaporated in a vacuole to obtain a standardized fraction of S. miltiorrhiza (PF2401-SF, 20 g, 1.0 w/w%). High performance liquid chromatography (HPLC) was used to determine the content of TsI in the standardized fraction as follows (Fig. 1). In a 10-ml volumetric flask, the standard compound (~4 mg) was accurately weighed and dissolved in HPLC-grade methanol to prepare a stock solution. A working calibration solution was prepared with a range from 25 to 400 µg/ml by successive 2-fold serial dilution of the stock solution with methanol. A Sykam 2100 series HPLC system (Sykam, Gilching, Germany) equipped with a column oven, a binary pump, and a degasser was used. A 10-µl volume of standard or sample solution was injected directly into an Inertsil ODS-3 column (4.6 mm×150 mm, 5 µm, GL Sciences, Inc., Tokyo, Japan) using a mixture of acetonitrile-water (65:35, v/v). The flow rate was 1.0 ml/min, and detection was carried out at UV 254 nm.

Eight-week-old male C57BL/6 mice (body weight, 20-25 g) obtained from the Experimental Animal Center, Kangwon International University, Chunchon, South Korea were used. The animals were housed in a conventional cage under adequate temperature (23℃) and humidity (60%) conditions, a 12-hour light/12-hour dark cycle, and unlimited access to water and food. Animal handling and care was in line with international laws and policies (National Institutes of Health [NIH] Guide for the Care and Use of Laboratory Animals, NIH Publication no. 85-23, 1985, revised 1996), and the study was approved (approval no. Hallym-1-35) by the Hallym Medical Center Institutional Animal Care and Use Committee (IACUC). We aimed to minimize the number of animals used and avoid animal suffering.

To elucidate the neuroprotective effects of TsI against ischemic damage, the mice were divided into 3 groups: a sham-operated group (sham group); a vehicle-treated ischemia group; and a TsI-treated (10 mg/kg) ischemia group. Vehicle and TsI were administered intraperitoneally 30 minutes before ischemic surgery. TsI was dissolved in dimethyl sulfoxide (DMSO) and diluted to the desired concentration with saline (final DMSO concentration, 1%), and the same dose of DMSO was administered to animals in the vehicle-treated group. TsI dose was selected based on the findings of a previous study [15]. In this experiment, the dose was the minimum required for neuroprotection in the ischemic brain [14].

We used 21 male mice (n=7 per group) in this study. The experimental animals were anesthetized with a mixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. A midline ventral incision was then made in the neck, and the left common carotid artery was isolated, freed of nerve fibers, and permanently ligated with 5-0 surgical silk. Following a 2-hour recovery and feeding period, the animals were exposed to a 30-minute period of hypoxia (92% N₂, 8% O₂) by placing them in airtight containers partially submerged in a 37℃ water bath to maintain a constant thermal environment.

Neurological deficits in the mice at 1 day after the onset of hypoxia/ischemia were assessed and scored as described in a previous paper [16]. The following scoring was used: 0, no observable neurological deficit (normal); 1, failure to extend left forepaw on the lifting of the body by the tail (mild); 2, circling to the contralateral side (moderate); and 3, leaning to the contralateral side at rest or no spontaneous motor activity (severe). Animals that did not show neurological deficits were excluded from the study.

The mice were euthanized 24 hours after hypoxia-ischemia and perfused transcardially with 0.1-M phosphate-buffered saline (PBS, pH 7.4). Brains were removed, and 1-mm coronal sections were dissected from the frontal pole using a mouse brain slicer (brain Matrix, ASI Instruments, Houston, TX, USA). Six slices were selected according to the mouse brain atlas [17], including the main portion of the infarct. The slices were incubated for 30 minutes in 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) at 37℃, and fixed by immersion in 4% paraformaldehyde solution in PBS (pH 7.4) for 6 hours. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. To evaluate infarct volume, images of TTC-stained brain sections (n=7 per group) were obtained using a digital camera (Sony, Tokyo, Japan). Measurements were obtained by manually outlining the margins of infarcted areas and were then analyzed using NIH Image software. The infarct volumes were calculated by multiplying the size of the infarcted area by the slice thickness and summing the volume of the 6 slices. Because post-ischemic brain edema would increase brain volume in the infarcted area, the corrected infarct volume was calculated to compensate for the effect of brain edema. An edema index was calculated by dividing the total volume of the hemisphere ipsilateral to hypoxia-ischemia by the total volume of the contralateral hemisphere.

Brain tissues were embedded in paraffin and were then sectioned on a microtome (Leica, Heidelburg, Germany) into 5-µm coronal sections. The sections were mounted on slides and were stained with H&E following standard histochemical procedures. After dehydration, the sections were mounted with Canada Balsam (Kanto, Tokyo, Japan). Images were obtained using a light microscope (BX53, Olympus, Hamburg, Germany) equipped with a digital camera (DP72, Olympus) connected to a PC monitor.

To confirm neuronal death in the brain after hypoxia-ischemia in sham- and ischemia-operated animals, F-J B histofluorescence-a staining procedure using a high-affinity fluorescent marker for localization of neuronal degeneration-was conducted 1 day after hypoxic-ischemic surgery under the same conditions for all the animals. F-J B histofluorescence staining procedures were accomplished according to the methods described in a previous study [18]. In brief, the sections were immersed in a solution containing 1% sodium hydroxide in 80% alcohol, followed by immersion in a solution of 70% alcohol. Next, the sections were transferred to a solution of 0.06% potassium permanganate and then to a 0.0004% F-J C (Histochem, Jefferson, AR, USA) staining solution. After washing, the sections were placed on a slide warmer (approximately 50℃) and examined under an epifluorescent microscope (Carl Zeiss, Oberkochen, Germany) with blue excitation light (450-490 nm) and a barrier filter. In this method, neurons that undergo degeneration fluoresce brightly in comparison with the background [19].

All measurements were obtained to ensure objectivity in blind conditions (2 observers for each experiment), and control and experimental samples were assayed under the same conditions. The number of H&E- and F-J B-positive neurons in a 250×250-µm square were counted in a location around the penumbra and core region of the cerebral cortex using an image analyzing system (Optimas 6.5, CyberMetrics, Scottsdale, AZ, USA). According to anatomical landmarks corresponding to AP +1.1 to -0.6 mm of the mouse brain atlas [17], the studied tissue sections were selected with 300-µm intervals. Cell counts were obtained by averaging the total number of H&E- and F-J B-positive neurons from each animal per group, and a ratio of the count was calibrated as a percentage.

Data are expressed as mean±SEM. Data were evaluated using a one-way ANOVA SPSS program (SPSS Inc., Chicago, IL, USA), and the means were assessed using Duncan's multiple-range test. A P<0.05 was considered statistically significant.

Compared with vehicle treatment, treatment with TsI (10 mg/kg) significantly reduced neurological deficits and increased functional recovery from hypoxia-ischemia at 1 day after hypoxia (Fig. 2).

F-J B histofluorescence staining was also performed to confirm the neuronal degeneration in the infarct region one day after the induction of hypoxia-ischemia. F-J B-positive neurons were not detected in any brain regions of the sham group (Fig. 5A, B, G) or in the contralateral region of vehicle- and TsI-treated hypoxia-ischemia groups (Fig. 5C, E). In contrast, in the vehicle-treated hypoxia-ischemia group, many F-J B-positive neurons were observed in the ipsilateral region, (Fig. 5D, G). However, in the TsI-treated hypoxia-ischemia group, the number of F-J B-positive neurons was 34.2% lower than that in the vehicle-treated hypoxia-ischemia group (Fig. 5F, G).