6-1. Source of experimental animals
8-week-old male SHR (Spontaneously hypertensive rats / Charles River Laboratories, SHR / NCrlNarl) rats and 8-week-old male WKY rats (Wistar-Kyoto, WKY / CrlBltw) were purchased from the National Science Council of the National Laboratory Animal Center. The rats housed at a controlled ambient temperature of 21-23 with 50 %℃ -60 % relative humidity and a 12 hr/12 hr light (9:00-21:00)/ dark (21:00-9:00) cycle. The rats were fed with pelleted rat chow and RO water at libitum. After the adaptation for 1-2 weeks, we measured their tail pressure separately to make sure that blood pressure of the SHRs was significantly higher than WKY rats, and then went on the next steps to group them for the follow-up experiment.
6-2 Experimental animal groups
1. WKY: WKY male rats, as the negative control group with normal blood pressure
2. SHR: SHR male rats with sp ontaneous hypertension as a positive control group
3. EAS: SHR male rats for acupuncture treatment on sham-points 4. EAT: SHR male rats for acupuncture treatment on Taichong Point
5. CES: SHR male rats for catgut embedding treatment on the sham-points 6. CET: SHR male rats for catgut embedding treatment on Taichong points
6-3 the animal experiments
After adaption for 1-2 weeks, we randomized them into the six groups including the WKY group (WKY), the SHR untreated group (SHR), electro-acupuncture sham-point group (EAS), electro-acupuncture treatment group (EAT), catgut-embedding sham-point group (CES), and catgut-embedding treatment group (CET). For treating the rats, they were
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-anesthetized with isoflurane, and then we punctured them in the pseudo-points or Taichong point (LR-3) with electrical/catgut stimulation.
The EA groups were treated once per day and the CE groups were treated at the 1st and 4th day. We measured their tail pressure at the 0, 3rd and 7th day.
After the treatment for a week, all the rats were sacrificed, and their hearts were harvested. One of each group was put in 10 % formalin and the others were stored in -80 . ℃
6-4 The treatment
6-4-1. Locations of treatment points
A. Taichong point: It locates on the dorsum of the foot, between the first and second metatarsal bones, in the depression anterior to the combining site of the base of metatarsal bones. Depth of about 2-4 mm
B. Sham-Point: We chose the point was on the foot dorsal, between the third and fourth metatarsal depression in front of the bottom junction, depth of about 2-4 mm.
6-4-2. Electro-acupuncture parameters
The rats were quickly anesthetized with 5 % isoflurane mixed with oxygen, and then changed to 2-2.5 % to maintain anesthesia. Before puncture, we sterilized the local region with 75 % alcohol, inserted the needle of 32 G * 0.5-inch size respectively, and then connected to the positive and negative electrodes, and followed the parameter settings: 2 Hz / 1 mA/150 ms, 30 minutes has been set for every treatment per day.
6-4-3. Catgut-embedding parameters
We used 24G * 1 inch injection needle be a outer needle I and 30G * 1.5 inch acupuncture needle be an inner needle, and about 0.5 cm 4/0 catgut immersed in 75% alcohol for use. The rats were quickly anesthetized with 5
% isoflurane mixed with oxygen, and then changed to 2-2.5 % to maintain
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-anesthesia. We embedded the absorbable catgut on the Taichong point (LR-3) or sham-point of the rats. The method of embedding as following: insert the acupuncture needle into the matched injector then stuff with matched size of the catgut, and then puncture the needle points to the muscle layer, and make sure the catgut was completely embedded. The CE groups were treated at the 1st day and 4th day.
6-5 stained tissue sections
After sacrificed, we washed out the blood with cold PBS and got their heart tissue, then soaked in 10 % (w/v) formaldehyde at least 24 hours. The heart samples were sent to Changhua Christian Hospital to have embedded, sliced, and analyzed with Hematoxylin and Eosin stain (H & E stain) and Masson's Trichrome stain. H & E stain and Trichrome stain of the biopsies were observed and recorded with 400 times of the microscope (Olympus).
6-6. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay)
Sections were incubated in the hybridization incubator at 58 for overnight. ℃ Next day, sections were put into 100% Xylene for 5 min (repeat thee times), and then put in 100 %, 95 %, 90 %, 85 % and 75 % alcohol each for 5 minutes sequentially, and finally washed into ddH2O repeatedly twice for 5 minutes. This process was for dewaxing and rehydration. Removed the slices and wipe the slices around with lens tissue around carefully (don’t let the slices dry out). Use the DaKo pen to fence the scope of the sections. We covered the sections with proteinase K for 30 min in dark (range of coverage can be sliced off), then washed in PBS for 5 minutes twice. Added 3 % H2O2 for 10 minutes and washed with PBS for 5 minutes twice. Reacted with the permeabilisation solution (0.1 % sodium citrate) for 8 minutes and repeated washing step. Reacted with the blocking buffer for 1 hour, and washed with PBS for 5 minutes twice. Reacted with the mixture of the
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-Enzyme Solution and Label solution (in 1:9 ratio) (In Situ Cell Death Detection Kit, 11684817910, Roche, Mannheim, Germany) in dark for 1 hour at 37 , and then washed with PBS for 5 minutes twice. Reacted with ℃ DAPI stain (D9564, Sigma, USA) diluted to 20,000 times by PBS in dark for 5 min, and then washed with PBS for 5 minutes twice. Finally, the sections were observed and recorded with the fluorescent microscope.
6-7. process of disengaging for the left ventricle
After the animal models sacrificed, took their heart tissues and soaked in the icy PBS and then washed out blood, moved off the blood vessels which attached to the organs, also clipped the fat tissues and other connective tissues and then made them in dried condition. Cut away the left and right atrium and right ventricle. Finally, divided the disengaged left ventricle into equal portions, the weight for each part was about 0.1 g. All of them were put into centrifuge tubes and stored in -80 .℃
6-8. extracting the protein of LV
Got 0.1 grams of left ventricular tissue, added 1 mL homogenization buffer (20mM Tris, 2mM EDTA, 10 % glycerol, 50 mM 2-mercaptoethanol, protease inhibitor, 1/1000 phosphatase inhibitor, pH7.4), put on the ice, ground by tissue homogenizer, Centrifuged and rolled for 12,000 rpm at 4 ℃ for 40 minutes and repeated above for wice, then stored in -80 for ℃ overnight, Repeated above once again and then got supernatant fluid. This is the tissue protein solution.
6-9. Bradford protein assay
The quantitative concentration of protein we used Bradford protein assay.
The basic principle is according to Coomassie Brilliant Blue G-250 can easily bind protein as a feature. After binding , its color can be changed from brown to blue, the maximum absorbance from 470 nm into 595 nm.
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The advantages of this method is simple and quick, and has highly sensitive, the disadvantage is vulnerable to the interference of salts.
First, took 50μl Bio-Rad protein assay reagent and added 200μl of protein samples, with 0.5 mg / ml BSA and ddH2O preparation for the final concentration 0、0.01、0.02、0.03、0.04、0.05 mg/ml be a standard solution (Table 3),
Samples of protein solution diluted 200 times with ddH2O, after average mixing, took 200μl to the 96 96 MicroWell™ Plates, then determined with 595 nm.
Table.6-1. Standard solution( part2 ) BSA (0.5
mg/ml)
0μl 5μl 10μl 15μl 20μl 25μl
ddH2O 200μl 195μl 190μl 185μl 180μl 175μl Bio-Rad 50μl 50μl 50μl 50μl 50μl 50μl Total 250μl 250μl 250μl 250μl 250μl 250μl Final
sample protein 1.25 ddH2O 198.75μl Bio-Rad 50μl Total 250μl diluted factor 200
6-10 Western blotting
Taking 30-50μg protein samples to mix with 5x protein loading dye, with 95 heating for 5 min to process the SDS℃ -PAGE electrophoresis.
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-3.75% of the upper stacking gel, 10% or 12% separating gel at the lower part.
Loading the sample protein to the wells of the gel and running with 75 volts for 2.5hr, after finished electrophoresis, took out the gel to process the proteins translation. From the positive to negative in order to place Whatmam 3M filter paper, PVDF membrane, gel and Whatmam 3M filter paper. PVDF membranes to be pre-soaked in methanol, air bubbles should be out before it was fitted into the transfer holder. Placed into the transfer tank (Bio-Rad) and filling the transfer buffer, on ice for 2.5 hours with 100 volts to transfer for getting the PVDF membranes. And then mixed the blocking buffer (5% skim milk in TBST) at room temperature for 1 hour. the primary antibody was diluted with TBST to 1:1000 and hybridized with the protein on PVDF membrane at 4 for overnight. Wash the membran℃ e with TBST for 10 min for three times. The secondary antibody was diluted with TBST to 1:5000 and hybridized with the protein at room temperature for 1 hour, add cold reagent was observed after the machine recorded. Another work to do was to dilute the secondary antibody with TBST to 1:2000~5000 mixed at the room temperature for one hour and then added in the luminescence detection for observation and recordation under the machine.
6-11 Statistical analysis
Used the one way ANOVA to calculate the differences between groups, and post-tested with Neuman-Keuls test, when the P value of less than 0.05, as a statistically significant difference
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