鼻电位差量化小鼠跨上皮离子转运

The nasal potential difference is a valuable method to characterize the phenotype of transgenic mice modeling cystic fibrosis. It allows investigation for the efficacy of new types of therapies for cystic fibrosis. The main advantages of the technique are that it provides reliable measurements in continuously breeding mice.

It reduces related mortality and it allows repeating the test in the same animal. To prepare the double lumen nasal catheter, keep the middle of a 20-centimeter long polyethylene tube in the flame about 10 to 15 seconds until it is soft enough for pulling. Then, pull the two ends apart to obtain a very thin capillary tube of about 15 centimeters long and an outer diameter of 0.1 millimeters for the nasal probe.

Afterward, clean and degrease the two capillaries with pure ethanol. Join them with tape and glue them together using cyanoacrylate glue. Cut away the excess length of the probes with a razor blade to obtain the double lumen catheter of about eight centimeters long.

Then, inject water through both lumens to verify that they are permeable, and make a mark at a five millimeter distance from the tip. Subsequently, prepare a cream mix by mixing the electrolyte cream and saturated three molar potassium solution in a ratio of one to one, avoiding formation of tiny air bubbles. For the experimental setup, to prevent hypothermia during and after the test, use two heating pads, one for the measuring setup, and the other one for the box in which the animal will be placed for recovery at the end of the test.

Turn on the computer, which is connected to the data memory high input impedance and high resolution voltmeter. Then open the software for data capture. Connect the electrodes to the voltmeter.

Dip the tip of the electrodes together in the cream mix. Check the initial electrode offset value and reject the electrodes if the value is larger than plus or minus two millivolts. Then, switch on the peristaltic pump and insert the pump tube into the perfusing lumen of the nasal catheter.

Fill one lumen of the nasal catheter with electrolyte cream mix through a 10 microliter pipette tip. Adapt the catheter to a silicone tube connector, and fill the connector with cream mix. Afterward, insert the positive electrode in the connector.

Check the measuring electrode bridge offset value and reject the bridges if the value is larger than plus or minus two millivolts. Subsequently, connect the proximal outlet of the pump tube to the vial containing the selected solution for nasal perfusion. Then connect the distal outlet of the pump tube to the perfusion lumen of the nasal catheter.

Next, fill the second lumen of the nasal catheter with fresh solution A1.Dip the tip of the nasal catheter in the vial containing solution A.Reverse the direction of the peristaltic pump to fill the length of the nasal catheter corresponding to 10 seconds of perfusion. Subsequently, fold a sheet of absorbing tissue to make a three centimeter wide pillow that will be used as a support for the mouse head. Cover the heating pad with the sheet of absorbing tissue.

Then, lay the mouse on its back on the heating pad, and tape the limbs and the tail to the pad. Afterward, insert the reference intravenous catheter in the subcutaneous space of a hind limb. Remove the needle and adapt into the silicone tube connector.

Fill the catheter and the connector with cream mix. Next, connect the positive measuring electrode to the nasal catheter, and the negative reference electrode to the catheter inserted in the subcutaneous space in the hind limb of the animal. Then, fix the catheter and the electrode with tape as required to prevent any displacement.

After placing the reference electrode and bridges, verify the measuring electrode bridge offset value again by dipping the tip of the nasal catheter in the electrode cream mix inside the connector of the IV catheter. Record the stable, final offset value. Now, fix the mouse ears with tape on the heating pad in a free space between the two pieces of absorbing tissue.

Fix the vibrancy without touching the eyes. To absorb fluid from the oral cavity, move the tongue sideways and insert a pointed wick of filter paper about one centimeter into the mouth. To absorb fluid running out of the perfused nostril, place a second piece of filter paper at the toi of the nose.

Check the postive control value of the epithelial potential by placing the nasal catheter tip in the inside of the mouth. Holding it with fine forceps and under good direct illumination, delicately introduce the nasal catheter into one nostril, up to four to six millimeters from the nose tip. Five minutes after injecting the anesthetic drugs, gently tilt the heating pad by about 30 degrees with the animal head downwards.

Monitor the maximal basal PD value. When it is stable over 30 seconds, start perfusing the nasal mucosa at 10 microliters per minute with the four buffered solutions in succession. Perfuse the first solution for 10 seconds, and each following solution for five minutes, or until a stable voltage has been reached.

Stop perfusing Forskolin when the transient Forskolin response starts going down. After the assessment, release the mouse from the heating pad. Administer it with the anesthetic antagonist mix and transfer it to the recovery box with heating pad until full recovery.

Here are the representative nasal PD tracings from a homozygous, normal mouse and a mouse homozygous for the F508 deletion CFTR mutation, together with the individual values obtained for the nasal PD parameters. Amiloride response is the difference between the values of nasal PD at the end and at the beginning of perfusion of the nasal mucosa with basal buffered salt solution containing amiloride. Chloride free response is the difference between the values of nasal PD at the end and at the beginning of perfusion of the nasal mucosa with chloride-free buffered salt solution, plus amiloride.

Forskolin response is the difference between the values of nasal PD at the end and at the beginning of perfusion of the nasal mucosa with chloride free buffered salt solution, plus Forskolin and amiloride. Total chloride response is the sum of the last two parameters obtained under zero chloride perfusion. The project can be used to explore, provide the transport defectors in the mouse, but also in the brat, in the big, and in the ferret cystic fibrosis model.

And sodium transport defectors in the eunuch mouse model. All the transporters can be studied by adopting the protocol. Working in the nasal cavity of rodents is extremely delicate because of the essential wall of sensory inputs from the nasal facial region.

Adequate death of anesthesia is dosed particularly challenging when operating in the mouse nasal cavity.