Acupuncture is an effective method for treating asthma. Although the underlying mechanisms are not clear. This protocol mimics acupuncture in the clinic to study these mechanics in an animal model.
The main advantage of this technique is that the high platform can be used to fix the rats without the restriction, allowing full exposure of the dorsal acupoints. Beginners should have basic rat handling skills, and should practice inserting the acupuncture needles into a piece of cardboard before attempting to treat the animals. Visualization of the technique will help beginners become familiar with the skills necessary to successfully perform the experiment.
The length and width of the supporting device should be 86 by 17 centimeters. Vertically affix a 30 centimeter long by 14 centimeter wide connecting device to the supporting device, and fix a five by five centimeter horizontal pedal to the connecting device. The suspended height at the bottom of the horizontal pedal should be greater than or equal to 50 centimeters.
For OVA sensitization, grab and lift the tail of an adult rat and place the animal on a rough surface while gently pulling back on the tail. Firmly but gently grasp the skin of both ears and neck with the left thumb and index finger while tightly holding the back skin with the remaining three fingers and palm and use the right hand to intraperitoneally inject one milliliter of freshly prepared sensitization solution into the rat abdomen. On the 14th day after sensitization, drill a small hole in the side of a 39 by 28 by 23 centimeter box and use a rubber tube to connect the box to a nebulizing cup and compression nebulizer.
Place the rat into the box and place the box in a biosafety cabinet. Turn on the ultrasonic nebulizer and load 30 milliliters of freshly prepared 300 milligram OVA in saline challenge solution into the nebulizing cup. The nebulization can be repeated several times in one day or according to the severity of the asthma symptoms to be induced.
For acupuncture treatment of the asthmatic animal, select acupoints, including GV 14, bilateral BL 12, and bilateral BL 13, according to the theory of traditional Chinese medicine for treating asthma, and administer manual acupuncture on day two and every other day for a total of seven sessions after asthma establishment. For needle placement, transfer the rat to the high platform and grasping the handle of a disposable stainless steel needle with the thumb and index finger of the dominant hand, firmly press the back of the rat with the palm of the hand. For GV 14 needle placement, touch the depression in the middle of the upper edge of the scapula between the seventh cervical vertebra and the first thoracic vertebra on the midline of the back and gently prick the skin with the needle point to locate the acupoint.
Then quickly insert the needle and rapidly remove the right hand place. Place the BL 13 needles below the third thoracic vertebra on both sides of the intercostal spaces and place the BL 12 needles below the second thoracic vertebra on both sides of the intercostal spaces as demonstrated. When all of the needles have been placed, twist each needle 360 degrees, 60 times over a period of 10 to 20 seconds, every five minutes for 20 to 30 minutes before removing the needles.
To measure Penh in the acupuncture treated animals. First turn on the signal amplifier and click calibrate to return the course adjustment baseline to zero. Switch to the direct current position and fine tune the baseline to zero.
Use a syringe to inject five milliliters of air before switching to the alternating current position. After checking the channels, ensure that the error value is less than 0.5%Next, turn on the aerosol distribution and rodent bias flow supply systems and adjust the flow rate of 4.5 milliliters according to the liquid level on the mercury ball. Under Create Study Options"enter test for the study and select rat for the species.
Confirm that simple dose list is selected, and set the number of doses to six. To select the Penh and area parameters, click next and set the animal adaptation time to five minutes. The nebulization time to two minutes, the response time to three minutes, and the recovery time to two minutes.
Then add 200 microliters of each freshly prepared concentration of methacholine in PBS to the nebulizer in sequence as indicated. Real time changes in Penh should be observed. When all six solutions with different concentrations have been delivered and assessed, click file and end session.
With acupuncture at the GV 14, bilateral BL 12, and bilateral BL 13 points, the rat maintains a prone position and can twist its head to the outside only, as one direction is blocked with a wooden board. As illustrated in the graph, after asthma induction, as the methacholine concentration increases, the Penh concentration increases, promoting a gradual increase in the growth rate. Western blot, and the real time PCR can also be used to to start a specific protein and a change after acupuncture treatment.
This technique can help researchers to explore specific changes in key factors and a signal pathway using asthma after acupuncture.
