This protocol facilities the preparation of a working solution of granules for in vivo studies, as well as the preparation of granule-containing serum for in vitro experiments. One advantage of this technique is that the method can be easily repeated due to the low cost of the materials and the animals you create for the experiments. Begin by adding eight, 16, and 24 grams of Gushukang, or GSK, granules to individual 50-milliliter tubes.
Add 48 milliliters of water to each tube, and shake the tubes to completely dissolve the granules. Then, place 1.6 tablets of calcium carbonate supplemented with vitamin D3 into another 50-milliliter tube, and dissolve the tablets in 48 milliliters of saline with shaking. For intragastric administration of the solutions, first gently but firmly manually restrain a one-month-old, female, C57 Black 6 mouse for two to three minutes.
When the mouse has calmed, load a 40-millimeter, number 12 gavage needle with 0.24 milliliters of one working solution of GSK granules, and place the gavage needle into the mouse through one side of its mouth until the needle resistance is felt, indicating the needle has reached the stomach. Inject the entire volume of working solution into the animal. Then, retract the gavage needle, and return the mouse to its cage while the other mice are being administered GSK before injecting 0.24 milliliters of calcium carbonate and vitamin D3 solution into each mouse in the same manner.
For GSK-containing blood serum collection, after delivering the appropriate concentrations of GSK to a rat by gavage as just demonstrated, confirm a lack of response to toe pinch one hour after the last GSK granule administration. Make an abdominal skin and peritoneal incision, and expose the abdomen to the bottom of the thorax. Use tissue paper to remove the connective tissue of the abdominal aorta to expose the vessel clearly, and use a 10-milliliter syringe equipped with a 22-gauge needle to draw blood from the aorta.
Transfer the blood to a sterile 15-milliliter tube, and place the tube upright at room temperature for 30 to 60 minutes. When the blood has clotted, centrifuge the tube to separate out the serum, and transfer all of the serum for up to six rats into a new 50-milliliter tube. Shake the tube to mix the sera before inactivating the serum components in a 56-degree Celsius water bath for 30 minutes.
Then, filter the serum through a 0.22-micrometer-pore-size hydrophilic polyethersulfone syringe filter for storage at minus 80 degrees Celsius for up to a year. For in vitro osteoclastogenesis, dilute the three dosages of GSK-containing serum at a one-to-four ratio with minimum Eagle's medium supplemented with L-glutamine, ribonucleosides, and deoxyribonucleosides. Add 200 microliters of each dilution to each well of a bone marrow macrophage culture in a 96-well plate.
Then, initiate osteoclast generation by stimulating each well with macrophage colony-stimulating factor and receptor activator for nuclear factor-kappa B ligand. For in vitro osteoblastogenesis, add two milliliters of diluted GSK-containing serum to each well of a 24-well plate bone mesenchymal stem cell culture. Micro-computed tomography scanning results indicate that ovariectomized mice demonstrate significant bone loss compared to saline control mice.
Intervention with 90 days of GSK granule treatment greatly increases the bone marrow density, particularly in medium dose GSK-treated animals. GSK granule treatment also leads to an increased trabecular bone number, bone volume over total volume, and trabecular bone thickness, but a decreased bone-trabecular bone separation. Tartrate-resistant acid phosphatase, or TRAP, staining reveals an increase in the number of osteoclasts in ovariectomized mice compared to control animals, while GSK granule treatments decrease TRAP-positive osteoclast differentiation compared to the ovariectomized animals.
GSK granule-containing serum also reduces the number of TRAP-positive osteoclasts compared to serum collected from control animals. The successful penetration of the needle into the abdominal aorta is critical to guarantee that a high enough volume blood is harvested from each rat. A successful execution of this method should allot the accumulation of 15 to 20 millimeters of blood, enough material to be used for in vitro experiments.
