Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats

This protocol can intuitively experimental results such as bone phenotype, metabolic indicators, and so on. The main application of this experiment is micro-CT, which can perform a tissue 3D reconstruction of bone morphology analysis, it greater on their basis of skin samples. To begin the procedure, apply local analgesia with a topical injection of bupivacaine on a properly anesthetized rat.

Then use a sterile cotton ball to disinfect the shaved area with ethanol and povidone iodine three times. Using a scalpel, perform a two centimeter midline incision on the abdomen of the animal. Expose the intact ovary using forceps and ligate at the root of the ovary using a 4-0 surgical suture.

Remove the intact ovary using surgical scissors before suturing the midline incision. After intragastric administering the drug for four months and anesthetizing the animal, cut and peel the skin of the groin to expose the femoral artery using surgical scissors and tweezers. Collect a blood sample from the femoral artery using a vacuum blood collection tube.

Then apply pressure with a cotton ball to stop bleeding. Continue to cut and peel the skin of the groin in the lower limb to expose the intact tibia. Remove the tibia and wash it three times with saline.

Using tweezers and ophthalmic scissors, remove excess muscle tissue from the tibia placed in saline. Disinfect the back skin of the lumbar L4 three times with ethanol and povidone iodine using a sterile cotton ball. Use surgical scissors and tweezers to cut the back skin and expose the lumbar L4.Sever the upper and lower vertebrae of the lumbar L4 with a ronguer and cut off the sacrum around it with surgical scissors.

Remove excess muscle tissue from the washed lumbar L4 placed in saline using tweezers and micro scissors. Store the tibia and L4 vertebra in 10%formalin tissue fluid at room temperature for three days. After turning on the micro CT scanner, Start the software to heat the X-ray source.

Create a database by clicking on create new sample to generate a file to save the data before obtaining the next one. In the software control window, assign the data acquisition parameters. Set the X-ray tube voltage to 50 kilovolts, CT X-ray tube current and live X-ray tube current to 100 micro amperes, the field of view to 18 millimeters, no gating technique, and the scanning technique to high resolution in four minutes.

After washing the tibia with saline three times, blot out excess water with filter paper. Wrap the tibia tissue on the bed with plastic film to fix its position. Adjust the tibia tissue to the center of the imaging field by rotating the button in the instrument.

Close the instrument door and turn the live mode on. Press the capture button to view the tibial tissue. Start the scan by clicking the CT scan button.

Click yes to produce the image. Click on image save to save the image to the desired folder. Transfer the tibia and L4 vertebra samples previously stored in 10%formalin to 20%formic acid solution for a four day decalcification.

Post decalcification, wash the tibia and L4 vertebra placed in an embedding box with running water for over six hours. Dehydrate the samples with gradient concentration alcohol solutions in an automated tissue processor. Place the tissue specimens in xylene for two hours to make them translucent.

Then place the permeabalized samples in paraffin wax at 60 degrees Celsius for three hours before embedding them in an automatic processor. Obtain 4 micron tissue sections using a rotary slicer. Place the sections in hematoxylin stain for three to eight minutes and eosin stain for one to three minutes.

Transfer the stained sections sequentially to pure alcohol and xylene. Seal and fix the sections with neutral gum to view under an optical microscope. To perform heat-induced epitope retrieval on the tibia sections, place the carrier sheet in the appropriate antigen repair buffer in a microwaveable container.

For antigen repair, microwave the container for 20 minutes at 98 degrees Celsius. After removing the container, rinse its interior with cold tap water for 10 minutes. To block endogenous peroxidase activity, incubate the tibia slices with 3%hydrogen peroxide for 15 minutes at room temperature.

Wash the sections in PBS three times for five minutes each. Then incubate the samples with normal goat serum blocking solution in a moist chamber for 15 minutes at room temperature. Add 200 microliters each of RANKL, osteoprotegerin, or OPG, and sclerostin antibodies into the slice samples, and incubate the samples overnight at four degrees Celsius in the dark.

Next, incubate the sections with biotin-labeled goat anti-rabbit IgG at room temperature for 30 minutes. After rinsing the with PBS, use diaminobenzidine to stain those for three to five minutes at room temperature. Dye the sections with hematoxylin for one to two minutes before sealing the dehydrated and dried sections with neutral gum.

Add 25 microliters of controlled, standard, or sample to each of their respective wells. Then add 200 microliters of 17 beta-estradiol HRP conjugate to each well. Cover all the wells with foil and incubate the plate at 37 degrees Celsius for two hours.

After removing the liquid from each well, wash the wells three times with 300 microliters of 1X washing solution. Then add 100 microliters of TMB substrate solution to each well and incubate. Post incubation, add 100 microliters of stop solution to each well.

Gently shake the micro titer plate for 30 seconds before measuring the absorbance of the sample at 450 nanometers within 30 minutes. The reconstructed micro CT images of the right tibia revealed that the ovariectomy-induced trabecular structural changes seen in the model OVX group were significantly reduced in ovariectomized rats treated with the Bazi Bushen capsule or BZBS group. Compared to the SHAM group, the trabecular bone in the OVX group showed a significant decrease in bone mineral density.

A similar trend was seen for bone volume fraction, the number of bone trabeculae and bone trabecular thickness. An opposite trend was seen for the trabecular separation degree. Compared with the OVX groups, bone volume fraction, the number of bone trabeculae and bone trabecular thickness in the Bazi Bushen treatment groups were significantly increased and the trabecular separation degree was markedly decreased.

The hematoxylin and eosin staining results indicated that the histomorphological damage in the tibia in L4 vertebrae of ovariectomized rats was relieved after administering Bazi Bushen. The immunohistochemistry results showed that compared with the OVX groups, the Bazi Bushen treatment groups showed a decreased expression of RANKL, increased expression of OPG and reduced expression of SOST. Various biochemical markers of bone metabolism in ovariectomized rats were also improved by Bazi Bushen treatment.

The application of micro CT is highly reusable for bone research. This technology is very convenient for enlightening bone structure and can be operated according to step three.