The mouse model employed in this study effectively simulates the effect of chronic nicotine ingestion and repeated exposure to silica on lung fibroses through epithelial mesenchymal transition in human beings. The jury portion model was achieved by subcutaneous injection of nicotine and the nasal drip of silica in a simple and a reliable way. Suspend sterile crystalline silica in saline to prepare a 20 milligrams per liter suspension.
Oscillate it in an ultrasonic shaking water bath for 25 minutes. Place the silica suspension with a vortex oscillator for three minutes. Aspirate the suspension to mix it, and then use 50 microliters for the nasal drip.
To begin silica exposure, first, place the anesthetized mouse on the palm of one hand and expose the nasal cavity of the mouse. Inject 50 microliters of the silica suspension from one nostril within four to eight seconds. To allow for quick lung penetration of the suspension, gently press the mouse's chest with the index finger for three to five seconds, repeating the press three to five times each second to maintain its respiratory rate.
Once the mouse displays uniform respiration, place it under observation in a cage for three minutes. Prepare nicotine syringes by drawing air until the mark of 0.2 milliliter into a one milliliter syringe. Then, draw up 125 microliters of nicotine.
Tap the syringe to fill the needle and the front end with nicotine, and then place it in a tray protected from light. With the right hand, grasp the mouse's tail. When the mouse has relaxed, Use the left thumb and forefinger to apply moderate pressure on the skin from the tail to the edge of the ear.
Now, release the right hand and use the left, small, and ring fingers to pinch the tail and hind limbs to immobilize the mouse. Hold the injection syringe in the right hand and puncture the skin on the back of the neck in a head to tail direction. Inject the nicotine at a uniform rate.
Fix the anesthetized mouse on a foam test plate and spray the fur with 75%alcohol. Make an incision along the midline of the abdomen exposing the chest cavity. Make a small cut at the apex of the right side of the heart.
Inject 20 milliliters of pre-cooled PBS slowly and uniformly from the apex of the left heart, causing blood to flow out from the opening created at the apex of the right heart. Remove the entire lung lobe and refrigerate it at minus 80 degrees Celsius. Perfuse the remaining mice with 10 milliliters of 4%para-formaldehyde.
After the PBS perfusion, preserve the lungs in 30 milliliters of the para-formaldehyde solution. After 72 hours, immerse the lungs in the prepared fixative and place them in an ultrasonic water bath. Tempered at 40 degrees Celsius for 30 minutes.
Next, perform dehydration by placing the tissue in 75%ethanol, then in 95%ethanol, and then in anhydrous ethanol for 50 minutes each. Immerse the lung tissue in xylene for 50 minutes twice to wash the tissue. Place the tissue in melted paraffin wax for two to three hours at 55 to 60 degrees Celsius.
Once the wax has hardened, use a sectioning machine to obtain the lung slices of four to five micrometer thickness. Heat the sections at 45 degrees Celsius in ultrapure water. Place the lung sections onto an adherent microscope slide once the wax melts.
Hematoxylin and eosin staining studies showed that mice exposed to nicotine combined with silica had significantly more severe lung damage than those exposed to either nicotine or silica alone. Masson's staining revealed increased collagen fiber deposition in the lungs exposed to nicotine silica combination relative to the other groups. Alveolar structures were destroyed in the silica-exposed mice.
Immunohistochemical studies revealed increased CD two zero six positive macrophages and the pro-fibrotic factor, TGF beta one positive cells in the mice exposed to the combination of nicotine and silica. Vimentin expression was higher in mice subjected to the dual expression relative to the other groups. Immunohistochemical studies and protein quantification suggest severe epithelial mesenchymal transformation.
To ensure a successful nicotine injection, the operator has to become familiar with grasping the mice as grasping the skin at the back of the neck could be painful for them.
