Experimental animals
Adult male Sprague-Dawley rats (300–350 g) were purchased from Lasco Co. (Taiwan). All rats were housed under standard laboratory conditions and were maintained in a constant 12:12 h light–dark cycle, at a controlled temperature of 22–24 °C, and in relative humidity of 55%. All study procedures were performed following the guidelines approved by the Institutional Animal Care and Use Committee of China Medical University (Permit Number: 2018-075). All surgical procedures were performed under appropriate anesthesia to avoid or minimize discomfort, pain, and stress in the animals.
ASD extract preparation
ASD extracts, with ASD powders (Batch Number A1306801), were purchased from Chuang Song Zong Pharmaceutical Co., Ltd. (Kaohsiung, Taiwan). ASD extract powder was prepared as previously described [29]. In brief, the dried roots of ASD were extracted using boiling water for 1.5 h and then the aqueous extract and essential oils from ASD were collected. The concentrated aqueous extract, essential oils, and excipients including microcrystalline cellulose and corn starch were placed in the fluidized bed dryer and granulated using fluidized bed granulation techniques. Each gram of the ASD powder consists of 0.5 g of the DG extract, 0.4 g of microcrystalline cellulose, and 0.1 g of corn starch. In this study, 2 g of the ASD extract powder was dissolved in 8 mL of normal saline. The supernatant fraction of the ASD extract was subsequently obtained by centrifugation at 1000 × g at 4 ºC for 10 min. The final concentration of the ASD aqueous extract was 0.125 g/mL.
Assessment of ASD extract indicators using ultra-performance liquid chromatography
The indicator compounds of the ASD extract, including ferulic acid and ligustilide, were detected using an Agilent ultra-performance liquid chromatography (UPLC) system (Agilent 1260 Infinity II, Agilent Technologies, Inc.). Ferulic acid (purity > 99%, Sigma-Aldrich, St. Louis, MO, USA) and ligustilide (purity > 98%, Adooq BioScience, Irvine, CA, USA) were accurately weighted and subsequently dissolved in methanol to prepare the standard solutions. A series of standard solutions were prepared with concentrations of 1, 0.5, 0.25, 0.1, 0.05, 0.025, and 0.01 mg/mL. The aqueous extract of ASD (0.125 g/mL) was prepared as the test solution. One microliter of the standard solution or 3 μL of the test solution was injected using an autosampler (Agilent 1260 Infinity II Vialsampler). The UPLC profile of the ASD extract was performed using a RP-18 column (Poroshell 120 EC-C18, 3.0 × 100 mm. I.D., 2.7 μm). The mobile phase consisted of a mixture of acetonitrile (A) and 0.03% phosphoric acid (B) solution. Gradient elution was performed as follows: 0–12 min (phase A: 5%–70%), 12–13 min (phase A: 70%–80%), and 13–15 min (phase A: 80%–100%). The flow rate was 0.5 mL/min and the ultraviolet detection wavelength was set at 320 nm.
Transient GCI
GCI was induced in the rats using the 4-vessel occlusion (4-VO) method as previously described [30]. In brief, the rats were anesthetized by inhalation of 5% isoflurane, and the anesthesia was maintained using 2% isoflurane. A dorsal neck midline incision was performed to expose the alar foramina of the first cervical vertebrae, and the bilateral vertebral arteries were permanently occluded using electrocautery. Twenty-four hours later, another incision was performed on the ventral neck to expose the bilateral common carotid arteries (CCAs), which were occluded for 25 min using vascular clips. The rats that displayed the loss of righting reflex and whose pupils were dilated during the ischemic period were validated as successful 4-VO models and were included in this study.
Passive avoidance task
A passive avoidance task was performed to evaluate 4-VO-induced memory deficits; the task was a modification of previously described procedures [31]. The passive avoidance apparatus (San Diego Instruments, San Diego, CA, USA) consisted of a box with two equal-sized light/dark compartments (25 × 20 × 17 cm3 each), which were separated by a guillotine-type door (9 × 7 cm2). The floor of each compartment consisted of 14 stainless steel rods. The floor rods of the dark compartment administered a brief electric shock. The memory evaluation test consisted of training and retention protocols. The training protocol comprised two acquisition tests 1 h before surgery. In the first acquisition test, the rats were individually placed in the light compartment for a 30-s habituation period. The guillotine door was then raised; when the rat entered the dark compartment, the door was immediately closed and a brief electric foot shock (50 Hz, 0.5 mA, 3 s) was delivered. The training was completed when the rats no longer entered the dark compartment before the cut-off time (120 s). The retention protocol was performed without foot shock at 7, 14, 21, and 28 d after reperfusion. The latency to enter the dark compartment (step-through latency [STL]) was measured, and the maximum score was recorded as 300 s.
Experiment A
Grouping
The rats were randomly divided into seven groups (n = 4–5): Sham, Control, ASD-0.25 g, ASD-0.5 g, ASD-1 g, D+ASD-1 g, and SB+ASD-1 g groups. The rats in the ASD-0.25 g, ASD-0.5 g, and ASD-1 g groups were intragastrically administered 0.25 g/kg, 0.5 g/kg, and 1 g/kg of the ASD extract, respectively, at 1, 3, 7, 10, 14, 17, 21, and 24 d after transient GCI. Furthermore, to determine the rate of progenitor proliferation, the rats received daily intraperitoneal injections of BrdU (50 mg/kg, Sigma-Aldrich) from day 14 to day 27 after reperfusion. On day 28 after reperfusion, the rats underwent CO2 euthanasia until 1 min after breathing had stopped and their brains were quickly removed. The rats in the D+ASD-1 g group underwent the same experimental procedures as those in the ASD-1 g group, but they were intracerebroventricularly (ICV) administered 1% dimethyl sulfoxide (DMSO) before the bilateral CCA occlusion. The rats in the SB+ASD-1 g group underwent the same experimental procedures as those in the D+ASD-1 g group, but they were ICV administered SB203580, a selective inhibitor of p38 MAPK, instead of 1% DMSO. The rats in the Control group underwent the same experimental procedures as those in the ASD-1 g group, but the ASD extract was substituted by saline solution. The rats in the Sham group underwent the same experimental procedures as those in the Control groups, but their bilateral CCAs were only exposed instead of occluded.
ICV administration of SB203580 or 1% DMSO
The rats were ICV administered SB203580 or 1% DMSO after anesthesia maintenance through isoflurane inhalation, and two symmetrical burr holes were drilled on both sides of the parietal bone. The burr holes were 2 mm lateral, 3 mm posterior to the bregma, and 3.5 mm under the cortical surface. The rats were ICV administered 10 μL SB203580 (2 mM, #S1076 Selleckchem.com) or 1% DMSO solution through each burr hole. ICV administration of the solution was performed using a microliter syringe (10 μL, Hamilton Company, Reno, NV, USA).
Assessment of immunofluorescence staining
The rats were euthanized by inhalation of CO2 at 28 d after reperfusion, and their brains were rapidly removed. The brain tissue sections were prepared as previously described [29]. Coronal brain sections were fixed with 4% paraformaldehyde for 15 min at room temperature (RT). After the brain sections were washed thrice with phosphate-buffered saline/0.01% Tween 20 (PBST), the sections were incubated with 1% Triton X-100 for 30 min at RT. The sections were incubated with 1% bovine serum albumin for 1 h at RT and were subsequently exposed to rabbit (1:100 dilution) and mouse (1:100 dilution) primary antibodies (listed in Table 1) overnight at 4 ºC. After the sections were washed thrice with PBST, they were incubated with anti-rabbit (1:200 dilution) and anti-mouse (1:200 dilution) secondary antibodies for 1.5 h at 37 ºC. The sections were then counterstained with 4',6-diamidino-2-phenylindole (DAPI, ab4139 abcam) and mounted using an aqueous mounting medium. The immunopositive cells in the selected hippocampal SGZ or CA1 regions were detected in each of three 400× magnification fields under a fluorescence microscope (CKX53, Olympus, Tokyo, Japan).
Assessment of immunohistochemistry
The brain tissue sections were prepared, and subsequent immunohistochemistry (IHC) evaluations were performed as previously described [32]. Anti-ki67 antibody used in the IHC evaluation was listed in Table 1. Immunopositive cells in the hippocampal SGZ region were calculated in each of three 400× magnification fields using a light microscope (Axioskop 40, Zeiss, Oerzen, Germany). For the negative control, the brain section in the ASD-1 g group was incubated without Ki67 primary antibody.
Experiment B
Grouping
The rats were randomly divided into seven groups (n = 4): Sham, Control, ASD-0.25 g, ASD-0.5 g, ASD-1 g, D+ASD-1 g, and SB+ASD-1 g groups. These groups underwent the same procedures as those performed in Experiment A.
Evaluation of Western blot
The rats underwent CO2 euthanasia at 28 d after reperfusion, and their brains were immediately removed. The right and left hippocampal tissues were carefully separated, collected, and homogenized on ice. The protein concentrations of the hippocampal tissues were measured using a Bio-Rad protein assay. The proteins separated by gel electrophoresis, transfer onto nitrocellulose membranes, and densitometric analysis were performed as previously described [33]. The primary antibodies used in the Western blot analyses were listed in Table 1.
Statistical analysis
All the results are expressed as means ± standard deviations. Statistical analysis of the data obtained for the experiment groups from SLT, immunofluorescence (IF), IHC, and Western blot analyses was performed using one-way analysis of variance followed by Scheffe’s post-hoc test. Significant differences were accepted for P < 0.05.