|
Protocol Index
| Prepared by: |
APP |
| Date Prepared: |
5/17/01 |
| Code: |
MP-005 |
| Reviewed by: |
KLS |
| Revision #: |
2.5 |
| Date Revised: |
6/21/02 |
| 0.0 |
Abstract: Blood pressure and pulse rate are taken over a 5 day period. |
| 1.0 |
Equipment and Instrumentation: Visitech BP-2000, Cary, NC. |
| 2.0 |
Reagents and Expendables (Items Needed): Scissors, balloons, medical tape, paper towels, ethanol (70%), clamps |
| 3.0 |
Setup: Before you start the experiment, there are a few simple things that need to be done:
- Choose a time of day to test the mice. The experiment will need to be done at the same time every day to avoid normal daily variance in blood pressure. The experiment will run for 10 days (5 days of training and five days of measurements).
- Ear marking (punch) the mice. Ear marking (punch) allows a mouse ID number to be determined easily. Because the mice will be upset on the day you ear punch them, the procedure should be done a few days before you begin to test the mice. Mice can be identified by their color, and if there are no matching cage mates, this is the simple way to determine their number. On the cage card, simply write the color next to the mouse number. If some of the mice are the same color, you will need to ear mark (punch).
- Check the specimen platform temperature (VERY IMPORTANT) - it should be 38°C in the middle. If the temperature is not correct, adjust it with the black knob on the pressure unit; remember to allow enough time for it to stabilize. Now you are ready to install the balloons. Cut a long 5-6" balloon into 4 evenly sectioned 1 1/3" pieces. Thread the balloon through the tail-cuff and fold back the ends to wrap around the tail-cuff. Small plastic holders are then stuck on the end of the tail-cuff to hold the balloon in place (DO NOT STRETCH THE BALLOON). If you need more black end pieces, they can be made by cutting off a small portion of heat shrink tubing and sliding them onto some brass tubing. They will be too large, so you need to heat shrink them to fit by using a heat gun or a lighter
- Check balloon holes. Choose Calibrate Pressure from the Configuration menu. A screen will come up telling you to attach the manometer, but ignore it and enter OK. The next screen should tell you the pressure the transducer is reading. The value may or may not be correct, but do not worry about it. Now you are only checking the balloons for holes. Choose the pressurize button and watch the Current Measured Pressure closely. If the numbers jump up fast, only hitting two or three times in a section of ten numbers, the balloons are good. An example is to take the section of ten numbers between 130 to 140. If the balloons have no holes the pressure might read 131, 135, 139. However, if the pressure reads 131, 133, 135, 137, 139, be suspicious of holes in the balloons. To check which balloon has the hole, take a pair of clamps (use a smooth tubing clamp and not hemostat or forceps as the latter may damage the tubing) and clamp off the tubing leading to an individual balloon and restart the pressurizing. If the numbers now go up correctly you have found the balloon with the hole. Howeve,r if the problem stays the same, you need to switch to another tail-cuff to try to find the flawed one. It is very important not to let the pressure exceed 200 when you are testing for holes. Pressure this high can create holes. Testing up to 175 should be adequate. After testing for holes, enter Cancel. The computer will now prompt you to unhook the tubing from the manometer and back to the tail-cuff machine, but ignore it and enter OK.
- Calibration needs to be checked. Go back under Configuration and select Calibrate Pressure again. This time when the computer prompts you to hook the tubing up to the manometer, do it and then enter OK. This is done by detaching the tubing from the specimen platform and connecting this end to the manometer via a stopcock. Select Pressurize setting and watch the Current Measured Pressure on the screen increase. When the value reaches 200, enter stop. Now carefully check the manometer reading against the Current Measured Pressure (do not take a long time to do this as this pressure may produce holes). There should be no more than 0.5 mmHg difference between the two. Make a note of the two readings and then select Depressurize. When the Current measured Pressure reaches 150, select stop and check the two values again. Continue doing 50 mmHg increments until you are at 50. Now look over your numbers. If they all match closely, you are finished. Simply select Cancel and exit out of that screen. If your numbers do not match, you will have to re-calibrate the computer. To calibrate select Pressurize again and then Stop when the pressure reaches 200. Now select Set from the list of options. A mini screen will come up and ask you to enter a value. You enter the value that the manometer reads, whatever it is and press enter. Next Depressurize 50 mmHg more and do the same process all over again.
Do this until you get to zero. Now enter Finished. It is extremely important to enter finished when you are done because this is the only way the computer will recalibrate itself. By hitting Finished, the computer takes you to the screen that tells you to unhook the manometer. Ignore it and hit enter. You must now go back and check the calibration. Go back into configuration and select Calibrate pressure again. If the calibration is off, you must reset them again. Keep doing this until the calibration is good.
Typically, the balloons need to be checked for holes every day before the experiment begins. I usually put in completely fresh balloons on the first day of testing with the hope that they will last all five days. The calibration needs to be checked the first days of training and measurement, but I run the calibration everyday.
There are other parameters that only need to be set once. These should be present, but it is worth checking them if others have used the machine. If you go under Configuration into Settings a screen will come up which allows you to set the number of blood pressure measurement cycles. We usually set values as follows:
| Cycles |
Preliminary 10; Measurement 20 |
| Frequency Limits |
Lower: 3.0 Hz |
| |
Higher: 15.0 Hz |
| Timeouts |
Pulse timeout: 30 seconds |
| |
Systolic timeout: 30 seconds |
| Pulse Detection |
Number of consecutive peaks: 70 |
| |
Minimum average pulse (%): 15 |
| Systolic Detection Settings |
Threshold (%): 20 |
| |
Criterion (%): 70 |
| Time Interval |
0.30 |
| Systolic Validation |
Apply validation: Enabled |
| |
Validation 1.0 |
| Miscellaneous |
Time between cycles: 2.5 |
| |
Ignore peaks: 0.5 | |
| 4.0 |
Run: Loading the mice: Now that all the computer information is right, it is time to focus on the mice. First, plan which mice will run together. Make a list of all the mice and put them into groups of four. Typically, I use two machines at one time and rotate the mice through all channels in every machine to even out any discrepancies. For instance, mouse #1 will go into slot #1 on Machine A the first day. On the second day, mouse #1 will go into slot #2 on Machine A. On the third day, mouse #1 will go into slot #3 on Machine A, etc. Continue on with this pattern for all the mice.
When you've selected the mouse order, go under Analysis and choose Register Specimens. Enter experiment title, mouse ID, etc. To go from one box to the next use the tab key.
Now you are ready to add the mice. Look up at the screen and determine where each mouse goes. Now look down into the cage and determine which mouse is which. Do not pick up the mice to do this; use the ear punch method. Select a mouse and calmly pick it up by the tail. In a fluid movement, pick up the channel where the mouse will go with the other hand and gently set the mouse on the platform. Replace the cover over the mouse, but further up than where the magnets are. Thread the mouse's tail through the tail-cuff and pull the channel (not the mouse!) back to the correct position. Now pull the mouse back into the channel so that it's rump rests snugly against the back. Pulling the mouse back after the channel is already in place ensures that you do not pinch the mouse's feet between the magnets. This is painful for the mouse and will upset it. Now simply tape the mouse's tail into place. When the taping is not enough to restrain the mouse, first you put the tape around the tail and second tape the taped tail (you need to take off the tape from the tail after measurements every day). The entrance of the restrainer is covered with paper wall, but not tight. We have to wait for 5 to 10 min to get the mice warming before measurement.
Run the machines at the same time to avoid excess noise if possible. All the mice need to have their blood pressure taken on the same part of the tail every day. To start the run, go Analysis and select Analyze. If you don't get a good pulse, check the mice during the preliminary measurement.
When the mice are done, you save the data and after that gently take off tape from their tail, and let them come out of the channel. Pick them up and quietly put them back into their cage. The whole experience for the mice should be as relaxing as possible.
The specimen platform temperature must be checked every day (if you found sweaty mice during measurement, you should confirm the temperature of the platform). After cleaning the machine, tape two thermometers to the platform (one in the middle and one on the side) and cover the thermometers with restrainers. Rotate the thermometer through the different channels on different days to ensure that things are even. |
| 5.0 |
Clean Up: Throw out feces from the platform and wipe the platform with wet paper towel (baby wipes) and put the next mouse on the platform. At the end of the day, you should clean the platform with a wet towel containing hot water, after that with a baby wipe. |
| 6.0 |
Data Reduction: Exit the BP-2000 system. To save the data onto disk, you go to My Computer, open BP file, and export all the data files to a disk. Give the disk to Data Processing for analysis. The computer is turned off, but DO NOT TURN OFF the specimen platform. |
| 7.0 |
Safety: n/a |
| 8.0 |
Time and Capacity: One technician can process 8 mice per hour. |
| 9.0 |
Protocols: n/a |
| 10.0 |
Notes: n/a |
Protocol Index
Program for Genomic Applications (PGA)
Supported by the National Heart, Lung, and Blood Institute (NHLBI) (Grant # HL66611) |
