The overall goal of the following experiment is to produce a murn model of asthma. First mice are sensitized to volin by intraperitoneal injection as a second step. The mice are challenged with volin, either intratracheally or by nebulization.
This induces an allergic airway reaction similar to a human asthmatic exacerbation and can be measured by methylcholine challenge. In addition, cellular infiltration in the airways and serum levels of IgE E can be quantified to assess the severity of these typical asthma features. Ultimately, the mice develop asthma like manifestations, whose severity may be affected by further experimental manipulations.
This animal model can help answer important questions in the asthma field, such as effects of treatment with specific investigational drugs or of modulating specific signaling pathways. Demonstrating this procedure today will be Irvin and Somia graduate students in my laboratory. Prepare the sensitization solution by emulsifying one milligram per milliliter of volin in sterile PBS containing 10 milligrams per milliliter of aluminum hydroxide sensitized mice by injecting 200 microliters of the sensitization inoculum, or a control solution lacking volin intraperitoneal for this protocol.
Sensitize the mice as described on day zero and seven. Then perform challenge on alternative days, starting on day 14 to 22 for the intra tracheal challenge. Place the anesthetized mouse on an incline surface.
Hook a thread under the mouse's front incisors and hold the head back. Then level the paw to straighten the trachea and tape the hind legs to the work surface. To secure the position, soak the mouse's neck and chest area with 70%ethanol and swab with chlorhexidine to disinfect the surgical site using forceps, nip the skin on the throat and gently pull it forward with sterile surgical scissors.
Make a small vertical incision. Load a one milliliter syringe with 50 microliters of 0.1%Volin in PBS. Fit it with a 27 gauge needle and insert it into a repetitive pipette with one hand.
Use sterile forceps to expose the trachea with the other hand. Hold the pipette parallel to the trachea. Carefully insert the needle and inject the solution.
Gently close the wound and seal it with a suture. Taking care to maintain sterility. Place the mouse on a heating pad and allow it to recover for the challenge by nebulization.
Use the method described earlier to sensitize mice on day zero and 14. Then challenge the mice daily between from day 21 to day 25. To perform the challenge, place the mice in the main chamber of a whole body plethysmography system and allow them to acclimatize for at least 10 minutes.
Then place one milliliter of 0.1%of albumin in sterile PBS in the nebulizer cup and nebulize for 30 minutes. To perform non-invasive airway hyperresponsiveness measurements, acclimatize mice to the chamber as shown previously. Then record baseline readings for three minutes.
Next, place one milliliter of PBS in the nebulizer cup, nebulize for two minutes and monitor the respiratory variables for an additional six minutes during the drying phase after nebulization. This example illustrates the increased airway responsiveness developed by ovalbumin treated mice to perform invasive airway hyperresponsiveness measurements. Place the anesthetized mouse on its back, disinfect the neck with ethanol and chlorhexidine, and expose the trachea as described earlier in this video.
Once the trachea is visible, use fine sterile scissors to make a small incision. Taking care not to sever the trachea and gently insert the tracheal tube. Tie a suture around the trachea to prevent an air leak.
Place the mouse on its back and connect the tracheal tube to the ventilator. Start the mechanical ventilation for a 20 gram mouse. Set the respiratory rate to 150 strokes per minute and the tidal volume to 200 microliters cord baseline respiratory measurements.
Then repeat the test after delivery of PBS or increasing doses of methylcholine through an ultrasonic nebulizer. Using the invasive measurement, both airway resistance and lung elastin can be measured as expected. Volin treated mice developed increased resistance and elastin following the challenge.
Following euthanasia, expose the abdominal cavity of the mouse, then use scissors to carefully puncture the diaphragm while doing so. Take care not to puncture the lungs or heart. Cut away the rib cage.
Then puncture the heart using a one milliliter syringe fitted with a 27 gauge needle, and slowly collect the blood, save the blood for serum separation and analysis of ova specific IgE antibodies. Next, expose the trachea and used curved scissors to clear a path underneath it. Pass the tip of a curved forceps under the trachea, and then tie a piece of suture thread in a loose half knot around the trachea at a low position in the throat.
Once the thread is in place, make a small incision in the trachea above the knot and carefully insert a cannula in the hole. Then gently press it forward until it reaches the entrance to the lungs. Tighten and complete the knot.
Then connect a one milliliter syringe containing PBS to the cannula, and gently inject the fluid into the lungs. Observing the lungs inflate. Take care not to overfill and rupture the lungs.
Withdraw the lavage fluid, gently rotating or gently pressing the cannula a little deeper if resistance is encountered. Once the fluid has been collected, detach the syringe, transfer the fluid to a tube on ice. Repeat the procedure twice with fresh PBS.
After this procedure, the airway cells can be isolated by centrifugation as shown here. The airways of mice sensitized and challenged with volin contain many more total cells than those of control mice. Using diff quick stains, eosinophils, neutrophils, alveolar macrophages, and lymphocytes can be distinguished and counted.
In addition, treated mice can be seen to develop higher levels of volin specific IgE antibodies in the serum and bronchoalveolar lavage fluid After its development. This animal model has paved the way for the researchers in the field of asthma to explore the mechanisms underlying this important disease in a convenient marine model.
