Establishment of animal models and grouping
Total of 200 adult healthy male Wistar rats (weighted 230-250 g, SPF grade) were supplied by the Experiment Animal Center of Qingdao Drug Inspection Institute (SCXK (LU) 20100100). This experiment was approved by the Ethics Committee of Qingdao University Medical College (QUMC 2011–09). The local legislation for ethics of experiment on animals and guidelines for the care and use of laboratory animals were followed in all animal procedures. All animals were acclimatized for 7d humidity-controlled housing with natural illumination and allowed to eat and drink freely at room temperature (23 ± 2°C). Fifteen (5 × 3) rats were randomly selected for control group, and the rest 185 rats were anesthetized by injecting intraperitoneally 10% chloral hydrate (3 ml/kg) after fasting for 12 h and fixed in supine position to conduct aseptic operation strictly. The bilateral common carotid arteries were colligated to establish forebrain ischemic models (BCCAO)
[36, 37]. The rats of control group (5 × 3) were operated as the same experimental procedures besides BCCAO. Core body temperature was keeping with a rectal probe and maintained at 36-37°C using a homeothermic blanket control unit during and after the surgery operation. Twenty-six rats un-awakened or died after 2 h of operation were rejected out, while 159 successful models whose cerebral blood flow curve (PeriFlux 5000, Sweden) dropped to 30% were brought into the experiment and were randomly divided into model group (5 × 3 cases) and treatment group (16 × 3 × 3 case).
Orthogonal experimental design and intervention
The treatment group rats (16 × 3 × 3 case) were sub-grouped according to the principle of orthogonal experimental design of [L
16(45)] consisting of two impact factors with four impact levels. The impact factor A is the therapeutic time widow designed four levels as 1.0 h, 1.5 h, 2.0 h, 2.5 h after ischemia. The impact factor B is the therapeutic drug dose which has four levels as following 5 mg/kg, 10 mg/kg, 20 mg/kg and 40 mg/kg body weight. The orthogonal experimental test was repeated 3 times.
Picroside II (CAS No: 39012-20-9) which the purity exceed 98% and molecular formula is C23H28O13, supplied by Tianjin Kuiqing Medical Technology Co. Ltd. According to the weight of rats, corresponding dose of picroside II powder was taken and diluted into 1 ml solution by isotonic saline solution and injected intraperitoneally according to the corresponding designed doses at designed time in the orthogonal layout [L
16(45)]. Rats in control group and model group were intraperitoneally injected the same dose saline after cerebral ischemia 2 h. The brain tissue was took out to evaluate the therapeutic effect of picroside II after treatment 24 h.
Fast green staining(FGS)
The rats from control group (5 cases), model group (5 cases) and treatment group (16 × 3 cases) were randomly chosen and anesthetized by injecting intraperitoneally 10% chloral hydrate (3 ml/kg), and perfused by normal saline 200 ml and fixed by 4% paraformaldehyde solution 200 ml successively via heart. Then the whole brain was taken out and post-fixed in 4% formaldehyde solution for 2 h and soaked in distilled water for 4 h. After conventional gradient ethanol dehydration, xylene transparent, paraffin embedding, coronal sections with a thickness of 5 μm were continuously cut forward from the posterior of optic chiasma by a microtome (Leica CM2027, Germany) and then adhered on the slices processed with poly-lysine.
The paraffin sections were dewaxed by dimethyl benzene and washed routinely, dyed 1 h in fast green alcohol solution at 37°C after dealing with 95% alcohol 1 min, and then washed by 95% alcohol (10 s × 2 times) and distilled water (15 s × 3 times), putted in 0.3% lithium carbonate to separate color, re-stained 90s by nuclear fast red after washing by distilled water (15 s × 3 times), finally washed by distilled water and conventionally dehydrated by gradient ethanol, cleared by xylene and sealed with neutral balsam. Normal myelin showed cord-like, dark green, tightly packed, and cells were red under light microscope. Five non-overlap visual fields at ischemic area were randomly observed under 400-fold light microscope. Quantity One software was used to analyse the gray value and took the mean. The change of myelin was presented by myelin gray value (MGV = MBP gray value – background gray value).
Transmission electron microscopy (TEM)
Ultrathin sections: Took some fresh brain tissue from the ischemic area and cut into pieces of 1 mm × 1 mm × 1 mm, fixed with 2.5% glutaraldehyde for 24 h and 1% osmium tetroxide for 2 h and dehydrated by graded series of acetone. Then soaked with the mixture of acetone and embedding solution (1:1) for 1.5 h and pure embedding solution overnight at 37°C respectively. The samples were put into the embedding plate filled with epoxy resin Epon812 to form embedding blocks eventually. The 50 nm ultrathin sections were cut by the ultramicrotome (Leica EM UC6, Germany) and placed on the nets prepared with polyvinyl formal, stored at 4°C.
TEM: Dripped a drop of 3% uranyl acetate-alcohol saturated solution (pH = 3.5) in a petri dish, covered the nets of ultrathin sections to contact with the dye liquor to stain for 30 min, and rinsed with double-distilled water for 10 min × 3 times to suck up water. Then, covered the nets of ultrathin sections to a drop of 6% lead citrate dye liquor (pH = 12) in another petri dish to stain for 5 min, rinsed with non-carbon dioxide double-distilled water for 10 min × 3 times, dried at room temperature. The ultrastructure of myelin was observed under TEM (JEM-1200EX, Japan).
Immunohistochemical (IHC) assay
Paraffin sections prepared as above were dewaxed and washed routinely, operated by the specification of SABC kit, developed by DAB chromogenic reagent kit and re-stained by hematoxylin (all kits were provided by Wuhan Boster Biotech Co. Ltd). Under light microscope myelin showed claybank streak and positive cells’ cytoplasm presented uneven coloring, vacuolization in cells. Negative control slices were dyed with 0.01 mol/L PBS instead of rabbit anti-rat MBP primary antibody and no positive reaction appeared. Five non-overlap visual fields randomly at ischemic area were chose under 400-fold light microscope. The gray value was analysed by the software of Quantity One and last took the mean. The expressing intensity of MBP was presented by gray value of myelin (GVM = MBP gray value – background gray value).
Western blot (WB)
Extraction of total protein: After treatment 24 h, we randomly chose 5 rats from control group, 5 from model group and (16 × 3) from treatment group to perfuse from heart with normal saline 200 ml following anesthetizing by 10% chloral hydrate. Took 200 mg ischemic brain tissue and put it into 1.5 ml EP tubes, added cell lysis buffer as the proportion of 1:4 (No. P0013, Biyuntian Biotech Co. Ltd., China), then grinded fully and homogenized by ultrasonic wave at −4°C ice bath, and collected the supernatant in another EP tube after centrifuging with 10,949 g for 10 min at 4°C (Eppendorf 5801, Germany). The BCA-100 protein quantitative kit (Shenneng Biotech. Co. Ltd., China) was used to determine the protein content, then stored at −20°C.
Western blot: MBP proteins (18, 23 kD) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (12% separating gel on 75 V and 5% concentrated gel on 120 V successively) and transferred onto a polyvinylidene difluoride membranes (40 min with 360 mA). Phosphate buffered saline with Tween-10 (PBST) was used to wash the gel films 5 min by 3 times, then the films were added rabbit anti-rat MBP primary antibodies (1:450, Abcam Company, Ab40390) to incubate 2 h and washed by PBST for 10 min by 3 times, then incubated 1 h in horseradish peroxidase goat anti-rabbit antibodies (1:10000, Beijing Golden Bridge Biotech. Co. Ltd., ZB-2301), finally washed with PBST and PBS successively for 5 min by 3 times. The gel film images was developed in A-B mixed developing agent and scanned with Bio-Rad-2000 gel-imaging system to analysed gray value of strap by Quantity One software. In the same specimen, the gray value of β-action (42 kD), as an internal parameter, was also detected to calibrate the content of each target protein. The relative content of protein (RCP) = the gray value of MBP/the gray value β-action. The experiment was repeated 3 times and the results presented with mean ± standard deviation.
Reverse transcription polymerase chain reaction (RT-PCR)
Extraction of total RNA: Chose 5 rats from control group and model group respectively and (16 × 3) rats in treatment group randomly and anesthetized by chloral hydrate after treatment 24 h. Took 200 mg ischemic brain tissue and put into 1.5 ml EP tube. Added RNA-Solv reagent 1 ml, minced and grinded, oscillated ultrasonically for 30 s and placed 5 min at room temperature, and centrifuged (4°C 12,000 g) for 15 min. Took the supernatant into another EP tube and added chloroform 0.2 ml, shocked and mixed 15 s, placed on ice for 10 min and centrifuged (4°C 12,000 g) for 15 min. Then, collected supernatant into another EP tube and join isopropyl alcohol 0.5 ml, blended gently, then placed on the ice for 10 min, centrifuged (4°C 12,000 g) for 15 min and discarded supernatant. Washed precipitation using 1 ml 75% alcohol, mixed and centrifuged (4°C 7,500 g) for 5 min, then abandoned supernatant carefully, dried 30 min (precipitation changed from white to transparent) in fume hood, and put in 57°C water bath for 10 min after adding 0.1% DEPC-H2O 30 μl. The purity and abundance of RNA were determined by ultraviolet spectrophotometer (Bekamann DU640, USA) and stored at −20°C.
RT-PCR: (1) Primers were designed with Premier 5.0 software and synthesized by Shanghai Invitrogen Co. Ltd. Target gene NSE (103 bp), sense primer: 5'–CCC ATT GGT GCA CAC TAA CCT-3', antisense: 5'-CGA CTT GAT TCA GCG ACA GGA-3'; GAPDH (110 bp) as an internal parameter, sense primer: 5'-CGT TGA CAT CCG TAA AGA CCT C-3', antisense: 5'-TAG GAG CCA GGG CAG TAA TCT-3'. (2) Reverse transcription synthesis system of cDNA (25 μl in total): Oligod T 2 μl and RNA 2 μg with DEPC-H2O added to 13.4 μl, and the mixed liquid was placed at 70°C for 5 min, then ice-bath for 5 min. Plused M-MLV RT 5 × 5 μl, dNTP mixture 5 μl, RNase inhibitor 0.62 μl and M-MLV RTase 1 μl. Then the mixed liquid was placed at 42°C for 1 h, reacted at 70°C for 15 min, and finally preserved at −20°C. (3) PCR system (50 μl): 5 μl 10 × PCR buffer, 1 μl dNTP (10 mmol/L), 1 μl cDNA, 1 μl primer1 (10 um), 1 μl primer2 (10 um), 0.4 μl Taq polymerase, with 0.1% DEPC-H2O added to 50 μl. PCR condition: The cDNA of MBP and GAPDH were amplified for 30 cycles, at 95°C for 3 min, 94°C for 30 s, 58°C for 30 s, and 72°C for 40 s, and finally extension at 72°C for 3 min. (4) Electrophoresis: 50 μl RT-PCR system with 10 μl 6 × DNA loading buffer added into, was shocked and blended, and then centrifuged for 5 s, followed with 10 μl sample loaded. Appropriate DNA Marker was selected to load 2 μl, and followed with 2% agarose gel electrophoresis (120 V/100 mA) for 30 min and ethidium bromide (EB) staining. Quantity One software was used to analyse the gray value after scanning by Bio-Rad-2000 gel-imaging system. The results were presented as relative abundance of mRNA (RAM): the gray values of MBP mRNA/GAPDH mRNA. The results were repeated 3 times and expressed with mean ± standard deviation.
Determination of statistical significance was carried out with Student’s t-test between two groups. One-way analysis of variance (One-way ANOVA) was used for the comparison of multiple sets of data, then further study was made by Least significant differences (LSD) to compare multiple data. All datum statistically analysed by SPSS 17.0 software. Values were considered to be significant when P was less than 0.05.