Animals
Six-week-old C57BL/6 male mice (Samtako Bio, Osan, Korea), weighing 20–22 g, were housed in polypropylene cages at 24 ± 4℃, under a 12 h light/dark cycle for 1 week prior to the experiment. The experimental animals were fed a standard pellet diet and water ad libitum. All experimental procedures performed in the present study were approved by the Ethics Committee for Animal Care and Use at Pusan National University (Approval No. PNU 2019–2485), certified by the Korean Association of Laboratory Animal Care.
Reagents
Cresyl violet (CV) and 2,3,5-triphenyl-tetrazolium chloride (TTC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was purchased from Bio Basic Inc. (Markham, Ontario, Canada). Saline was purchased from JW Pharmaceutical Co., Ltd. (Seoul, Korea). The optimal cutting temperature (OCT) compound cryostat embedding medium was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Methanol was obtained from SK Chemicals (Ulsan, Korea). The protein extraction solution was obtained from iNtRON (Seongnam-si, Gyeonggi-do, Korea). Primary antibodies against phospho-c-Jun N-terminal kinase (p-JNK), JNK, p-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (p-IκBα), IκBα, manganese superoxide dismutase (Mn-SOD), and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies for goat anti-rabbit IgG pAb, goat anti-mouse IgG, and pAb were purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The BCA reagent, bovine serum albumin (BSA) standard, and enhanced chemiluminescence (ECL) western blotting chemiluminescent substrate were obtained from Thermo Fisher Scientific (Waltham, MA, USA).
Preparation and high-pressure liquid chromatography (HPLC) analysis of WG and RG extracts
Standardized WG and RG, manufactured from a 5-year-old ginseng sample, were obtained from Gwangmyung Natural Pharmaceutical Co. (Busan, Korea), and voucher specimens (No. 201KWG and 201KRG) were deposited at the Herbarium of School of Korean Medicine, Pusan National University. For both WG and RG, 200 g of each were finely ground and extracted with 10 times the volume of 99% methanol at 25℃ for 3 days; this was repeated three times. After filtration using filter paper (Advantec, USA), methanol was removed using a vacuum evaporator (Eyela, Japan) at 45℃, and then stored at -20℃ until use. The final lyophilized WG extract (WGex) and RG extract (RGex) from WG and RG weighed 24.2 g and 25.3 g, yielding 12.1% and 12.7%, respectively.
For quality assurance of WGex and RGex, each extract was subjected to HPLC analysis. To obtain a fingerprint of extracts used in this experiment, we determined the presence of ginsenoside Rg1, a main component of ginseng, using an HPLC system (Shimadzu, Kyoto, Japan) consisting of an LC-20AD pump, a SIL-20A autosampler, an SPD-M20A detector, and a CTO-20A column oven. The samples were separated using a YMC-Triart C18 column. The mobile phase and elution systems are listed in Table 1.
Induction of transient middle cerebral artery occlusion (tMCAO)
In the present study, Koizumi’s method was modified at our laboratory, and the modified method was performed in mice; detailed protocols and materials have been previously reported [27]. In brief, for left hemispheric MCAO, anesthesia was induced by inhalation of 1.5-2% isoflurane in N2O/O2 (70%/30%) until mice showed no response to mechanical stimuli. Following the induction of anesthesia, the mice were placed on a heating pad (Harvard Instruments, Boston, MA, USA). During the tMCAO surgery, the body temperature of mice was monitored using a rectal temperature probe and maintained at 37 ± 1℃. The surgical site, as well as the chest area of mice, were disinfected with povidone and 70% ethyl alcohol. Then, the left side of the skull, near the ear, was exposed using surgical mesh, and an optic fiber was attached to the skull to monitor the relative cerebral blood flow (rCBF). A midline incision was made on the neck, and soft tissues over the trachea were gently retracted. The ECA, distal CCA (dCCA), and proximal CCA (pCCA) were loosely knotted with 4–0 suture silk, and the ECA and CCA were carefully isolated from the vagus nerve. The ECA and dCCA were ligated before clamping of the internal carotid artery (ICA). Then, an 8–0 monofilament suture line (Ethicon, CA, USA) coated with silicon was inserted into the MCA through the perforated CCA. For the tMCAO operation, the monofilament was retracted when the predetermined occlusion time ended in the present experiment (2 h tMCAO). To complete the surgical process, the incised region was sutured and disinfected, and the mice were maintained in a recovery cage for 30 min. All surgeries were performed using laser Doppler flowmetry (Moor Instruments Ltd., Devon, UK) to detect rCBF and confirm successful occlusion and reperfusion.
Pretreatment with WGex and RGex
Experimental mice were randomly divided into eight groups as follows: 100, 300, and 1000 mg/kg WGex-treated; 100, 300, and 1000 RGex-treated; PBS-treated control; sham-operated sham group. Each group consisted of 15 mice (Supplementary Fig. 1, Fig. S1). In Korean medicine, 30 mg/kg has been recommended as the daily dose of WG and RG extract in humans; in mice, the equivalent dose was determined as 400 mg/kg. Thus, the dose range varied between 100 and 1000 mg/kg. For each WGex and RGex concentration prepared in PBS, the solution was orally administered using an oral zonde 1 h before MCAO induction, with sham and control mice orally administered the same volume of PBS. After 24 h of MCAO induction, the mice were sacrificed to perform several assays. A schematic diagram of the experiment is shown in Fig. 1.
Infarct and edema area measurements
TTC staining can be assumed as a function of mitochondria and is reportedly an important marker of ischemic volume following ischemic stroke. The mice were anesthetized by CO2 respiration, and blood was collected to obtain serum (25℃ room temperature, 10 min, 13,000×g). Then, the mouse brain was harvested to measure the total infarct volume, and the olfactory bulb and cerebellum were isolated. Serial coronal sections (1 mm thickness) were stained with 2% TTC solution to assess infarct volume. Next, the sections were digitized using a digital camera and a tablet, and the ischemic area and non-infarct tissue were separated using an image analysis system (Digimizer, Ostend, Belgium).
Areas of brain edema were calculated by dividing the total infarction volumes in ipsilateral hemispheres by brain edema indices using TTC-stained brain sections. The formula used to calculate brain edema indices is as follows:
Brain edema index = total volume of ipsilateral hemisphere / total volume of the contralateral hemisphere
Neurological deficit score (NDS) measurement
In mice, the behavioral changes were measured using a 5-scale assessment after surgery, and the characterization of each score was described as follows: Score 0, mouse presenting behavior similar to a normal mouse; Score 1, spontaneous exercise could be performed, but the behavior was slower; Score 2, slightly reduced temperature and reduced (2–3 g) body weight, and mice moved toward the contralateral direction; Score 3: reduced body temperature and weight (3–4 g), and mice demonstrated sensitivity to mechanical stimuli, including pain, walking, or circling to the right; Score 4, reduced body temperature and weight (4–5 g), with mice failing to exhibit spontaneous behavior or demonstrate extreme sensitivity to mechanical stimuli.
Sacrifice and cardiac perfusion for harvesting the brain
The mouse abdomen was incised, and cardiac perfusion with PBS was performed. In brief, the pulmonary artery was blocked, the left ventricle was pierced with a 21 gauge needle; then, the needle was fixed in the ascending aorta. Immediately after perfusion, the right atrium was incised using scissors. PBS and 4% paraformaldehyde (PFA) solution were used for perfusion and fixation, respectively. Post-fixation, the brain was soaked in 10% PFA with 10-30% sucrose at 4℃ for 3 days, followed by cryosectioning for appropriate staining.
Frozen sections of mice brain
Mouse brains were sequentially placed in 10%, 20%, and 30% sucrose solutions before freezing (in OCT compound), and then stored at -80°C. For each mouse group, brain sections were obtained using a cryostat (Leica, Wetzlar, Germany) at a thickness of 30 μm, and the sections were then placed on glass slides for 12 h and stored at -80℃ until use.
Hematoxylin and eosin (H&E) staining
For each mouse group, the brain sections on slides were allowed to dry on a slide warmer; then, the slides were dipped into 80% ethyl alcohol. The slides were immersed in a hematoxylin solution for 5 min, followed by gently washing with distilled water. Next, the slides were soaked in acid alcohol (1%) two times, and immersed in a lithium carbonate solution. Then, the sections were washed and slides were dipped in eosin solution for 30 sec. The slides were immersed in graded ethyl alcohol concentrations (95% and 100% sequentially) for 1 min, placed in xylene, mounted, and then observed under a light microscope. The density of H&E-positive cells was measured in the cortical region of mouse brains using the ImageJ program (NIH, MD, USA).
Nissl staining
For each mouse group, the sections on glass slides were allowed to dry on a slide warmer. Then, the slides were immersed in an alcohol:chloroform solution overnight. The slides were incubated in 0.1% CV solution for 10 min at 40℃ in an incubator. Then, the slides were quickly washed with distilled water, placed in graded ethyl alcohol (95% and 100% sequentially) for 5 min, sealed with a coverslip and mounting solution, and then observed under a light microscope. The density of neuronal cells was measured in the cortical region of the mouse brains using the ImageJ program (NIH, MD, USA).
Western blot analysis
Proteins in the ipsilateral brain hemisphere were isolated using a protein extraction solution. Brain tissue lysates were obtained by centrifugation at 15,871×g for 10 min at 4℃. The total protein level in the supernatant was quantitated using the BSA method. In brief, 30 µm of the protein was developed and separated by SDS-PAGE, and then transferred onto PVDF membranes (Millipore, Darmstadt, Germany). The PVDF membranes were blocked with 5% skim milk in TBST buffer (Tris-buffered saline, 0.1% Tween 20) for 1 h at 25℃, and then incubated overnight at 4℃ with primary antibodies against inducible nitric oxide synthase (iNOS), p-IkBα, IkBα, p-JNK (1:1000), JNK (1:1000), Mn-SOD (1:1000), and β-actin (1:1000). After overnight incubation, secondary antibodies for HRP-conjugated goat anti-rabbit IgG, pAb (1:5000), and HRP-conjugated goat anti-mouse IgG pAb (1:3000) were added for 1 h at 25℃. After incubation, the membranes were treated with an ECL solution kit, and the expression levels of each protein were detected using a luminescent analyzer system (Amersham™ Imager 600, Buckinghamshire, UK). The densities of all detected bands were analyzed using the ImageJ program (NIH, MD, USA).
Statistical analysis
One-way analysis of variance (ANOVA) was performed, followed by the Holm-Sidak test to determine the significance of differences among groups using Sigmaplot 12.0 (Systat Software Inc., CA, USA). Data are expressed as the mean ± standard deviation (SD), and a p < 0.05 was deemed significant.