In the described mouse silicosis model generated by intra laser infusion of crystal silica dust, all CSD suspension several times simulated the occurrence and development of human silicosis to a large extent. This method is time saving and level saving, as well as practical and effective. Further, causes no mechanical injury to the upper respiratory tract due to operation.
This method could also be used to prepare disease models of viruses, bacteria, et cetera. Demonstrating the procedure will be Hangbing Cao, a master grade student from my laboratory, skilled in preparing the silicosis mouse model. Begin with preparing the crystalline silica dust or CSD at least one day before administering the nasal drips.
To do so, grind silica in an agate mortar for half an hour. To check the particle size and shape, prepare the sample for scanning electron microscopy or SEM using conductive tape to bind the silicon particles. Using a hair dryer, gently blow away the particles not firmly bonded before proceeding to capture the image.
Next, mix the ground silica and sterile saline at a concentration of 20 milligrams per milliliter using an ultrasonic shaker. Finally, vortex the prepared CSD suspension for 10 seconds. Administer 50 microliters of CSD to the anesthetized mouse via a nasal drip for four to eight seconds.
Treat the control mouse with an equal amount of saline, then holding the mouse's body with the palm, pinch the skin on the back of the neck with the thumb and index finger while fixing the mouse's hind limbs with the other fingers. Press the mouse's heart beating area gently with the other index finger five to 10 times for five seconds. Heat the paraffin embedded lung tissue on a hot plate at 60 degrees Celsius for over four hours.
To dewax and hydrate the paraffin section, soak the slide in xylene for 30 minutes twice, then sequentially dip the slide for five minutes each in anhydrous 95%85%and 75%ethanol, and finally, in deionized water, then place the slide in the hematoxylin staining bucket for 10 minutes before rinsing gently under running water for five minutes. Dip it in the eosin staining bucket for 10 seconds. After dehydrating the sample in 75%85%95%and anhydrous ethanol for five minutes each, clear the tissue section by immersing it in xylene for five minutes, and seal it with approximately 60 microliters of neutral resin drops.
Carefully place the cover slide over the section. For Masson staining, stain the dewaxed and hydrated paraffin samples with freshly prepared 50%Weigert's hematoxylin for 10 minutes, then after soaking the tissue in acidic ethanol liquifaction for 10 seconds, rinse the slide gently with running water for nuclei bluing. Next, stain the sample with drops of red staining solution for seven minutes, and wash with 30%hydrochloric acid working solution for one minute.
After dipping the slide in 95%alcohol for 20 seconds, dehydrate the sample with anhydrous ethanol for one to three seconds twice, and as demonstrated before, clear and seal the sample. For Sirius red staining, infiltrate the dewaxed and hydrated section for one hour with Sirius red staining solution, then stain the cell nuclei for eight to 10 minutes with Mayer hematoxlyn staining solution. Gently rinse it under running water for 10 minutes before dehydrating, clearing, and sealing the slide.
Infiltrate the dewaxed and hydrated paraffin sample with 30 milliliters of a three milligrams per milliliter EDTA antigen retrieval solution. Boil the sample for 20 to 30 minutes before washing it with deionized water and incubating it in PBST for five minutes. Soak the sample for 15 minutes with freshly prepared 0.3%hydrogen peroxide solution, and wash it three times for five minutes each with PBST.
Permeabilize the membrane of the specimen for 15 minutes with 0.3%Triton 100 solution, and block with 30 to 40 microliters of 5%bovine serum albumin or BSA for one hour. After removing the blocking solution, add appropriately diluted primary antibodies NF-kappa B and CD-68, and incubate the specimen overnight at two to eight degrees Celsius in a microscope slide IHC wet box. Next day, after transferring the sample to room temperature for one hour, wash it three times for five minutes each with PBST.
Incubate the sample for one hour in rabbit anti-mouse horseradish peroxidase labeled secondary antibodies at room temperature. After incubation, wash the samples again three times for five minutes each with PBST, then incubate the sample with the DAB substrate corresponding to the enzyme labeled antibody for five to 20 minutes. After quenching the reaction with deionized water, counterstain the sample for 30 seconds with Weigert's hematoxylin.
Rinse it under running water for one minute, dehydrate, clear, and seal the tissue. Before proceeding to western blot analysis, homogenize the tissue with RIPA working solution on ice for five minutes using a handheld electric grinder. Incubate for one hour on ice with shaking, and centrifuge the homogenate at 14, 800 G for 15 minutes at four degrees Celsius.
Collect the supernatant, and determine the protein concentration with a BCA protein assay kit. Add 20 microliters of 5X loading buffer to the supernatant, then heat the six micrograms per microliter protein storage solution prepared with RIPA in a metal bath at 100 degrees Celsius for 20 minutes. Store 100 microliter aliquots of the protein solution in each tube at minus 80 degrees Celsius.
For electrophoresis, dilute the aliquots to two to three micrograms per microliter with RIPA lysate, then add 20 micrograms of sample per well to run the gel. Transfer the protein to a PVDF membrane pre-activated with methanol for 20 seconds using the wet transfer method with 400 milliampere current for one to two hours. After washing the membrane five times for five minutes each with PBST solution, block with 5%BSA or skimmed milk for one hour.
Submerge the strips in the primary antibody solution containing NF-kappa B and beta actin diluted with 5%BSA. Shake the strips gently overnight at two to eight degrees Celsius before washing them with PBST. Next, incubate the strips, first in the diluted secondary antibody for one hour at room temperature with gentle shaking, and then with enhanced chemiluminescence developer for three minutes.
Expose the strip to a gel imager for 20 seconds and measure the gray value of the strip to assess the protein level by system software. The SEM images of CSD showed that the particles were irregularly shaped. Sirius red staining performed to measure collagen deposition showed that the progression of pulmonary fibrosis in mice was significantly accelerated upon CSD exposure for one month.
Polarizing microscopy revealed three different types of collagen fibers, of which type one collagen fibers shown in red are a risk factor for silicosis, however, no significant fibrosis was found in the vehicle group. This was also indicated by the respective fibrosis scores. HE staining of CSD treated mouse lung tissue showed typical silica nodules characterized by liquified necrosis after phagocytosis of CSD in the center surrounded by macrophages in the periphery.
Masson staining also showed that the nodules were enriched with CSD accompanied by fibrosis. The immunohistochemistry staining of CD-68 further revealed the wide presence of macrophages in lung tissue. Polarized light microscopy showed that these macrophages had ingested CSD, causing severe lung injury.
Immunohistochemistry staining showed higher NF-kappa B staining, indicating higher inflammatory response in the CSD treated group than in the vehicle group. Representative western blot also showed that the CSD treated mice had higher NF-kappa B expression in the lung, and the difference in expression between the two groups was significant. To avoid respiratory obstruction and promote respiratory recovery, the heart area of the mouse must be gently massaged five to 10 times as demonstrated after a five second period.
This model can explore some underlying mechanism of silicosis and aid therapeutic drug screening, along with establishing crucial parameters like dosage and duration. This technology offers a convenient, effective, and economic means to expose mouse models to particulate matter whether this add bacteria. Monitoring mice behavior can aid in determining the potential effects of inhaling particulate matter.
