Jaxwest1 project protocol

Multi-system analysis of physiology on 7 inbred strains of mice   (2003)

Jackson Laboratory
With: Yang Y, Kitten A, Griffey S, Chang F, Dixon K, Browne C, Clary D




This high-throughput, non-invasive physiological assessment involves the following protocols: electrocardiogram, blood pressure and pulse, lung function, grip strength, thermal pain response, and body weight, composition, and bone densitometry.


Testing schedule by strain


Mice were tested according to the following schedule. Numbers indicate age (in wks) at time of testing.
Strain   ECG     BP pulse   Lung
function		 Grip
strength		Thermal
response		Body/bone
densitometry
129S1/SvImJ101010121212
A/J101010101112
BALB/cJ9911111112
C57BL/6J910,1111111113
DBA/2J91012111112
NOD/ShiLtJ1011,1410121211
SJL/J8,109,109,109,109,1011


Electrocardiogram (ECG)


ECGs were recorded using a non-invasive AnonyMOUSE recording platform from Mouse Specifics, Inc. (Boston, MA) according to the method of Chu et al., 2001 and described in detail in the protocol section of Hampton TG, Paigen B, Seburn KL - Cardiac characterization.

Mice were removed from their cages and placed on the recording platform containing 3 gel-coated electrodes embedded in the floor. Each mouse was allowed to acclimatize 10 min prior to collection of ECG waveform data. At the end of the acclimatization period, mice were positioned so that both hind paws and the right front paw each came into contact with an electrode. Positioning was accomplished by gently moving the mouse and/or the paws with a cotton-tipped swab. The electrodes were connected to an amplifier by a shielded 3-electrode lead set. Electrical signals were digitized with 16-bit precision at a sampling rate of 2000 samples/second. In most cases, data records containing at least 12 consecutive ECG waveforms were used for analyses (in two cases, data records containing fewer than 12 consecutive waveforms were used; the numbers of waveforms used for each calculation are available for each animal in the raw data set only). Data were sent to the Mouse Specifics, Inc. website (www.mousespecifics.com) using standard file-transfer protocols for analyses.

ECG analyses. ECG waveforms were analyzed using e-MOUSE, an internet-based analysis portal (see Chu et al., 2002 and Hampton1 protocol for complete details). Briefly, e-MOUSE software uses a peak detection algorithm to find the peaks of the R-waves and to calculate heart rate (HR). The software plots its interpretation of P, Q, R, S, and T for each beat, and the mean of the ECG time intervals for each set of waveforms is calculated. Heart rate variability (HR_var) is calculated as the standard deviation of all R-R intervals for each set of ECG signals and the coefficient of variance (HR_cv) is calculated as a percentage of mean HR_var to mean HR. QT interval is calculated by using the end of the T-wave of each signal, defined as the point where the signal returns to the isoelectric line (the mean voltage between the preceding P-wave and QRS interval), and includes the inverted and/or biphasic portions of the T-wave (Mitchell et al., 1998; Wang et al., 2001). QT intervals are rate-corrected (QTc) according to the equation described by Mitchell et al. 1998.



Blood pressure and heart rate


Systolic blood pressure and heart rate were measured in conscious mice using the BP-2000 Blood Pressure Analysis tail cuff system (Visitech Systems, Apex, NC, USA). Prior to testing, the inflation pressure was calibrated using a mercury manometer and the heated platform was checked with a surface thermometer to verify uniform heating to 34 ± 0.5 °C.

Mice were brought to the testing room, in which the lights were dimmed, and allowed to acclimatize for 20 min. Mice were placed on the blood pressure platform and held in place in an opaque restraining chamber open at the front with a notch at the back for the tail. Tails were threaded through balloon cuffs and placed in a groove containing a light source and photoresistor for measuring tail blood flow.

Blood pressure was measured during 30 consecutive, computer-automated inflation/deflation cycles of the balloon cuff (10 preliminary measurements and 20 test measurements). Data from the preliminary measurements were discarded and data from the 20 test measurements were averaged. The mean systolic blood pressure (mm Hg) and pulse rate (beats per minute) from the 20 test measurements were computed and recorded as the blood pressure and pulse rate for each mouse.

Investigator Notes:

  • In our experience, heating the platform to 38 °C (as in Krege et al., 1995; Sugiyama et al., 2002) increased mouse movement, resulting in fewer valid blood pressure measurements.
  • If a test session yielded fewer than 10 valid blood pressure measurements (out of 20 measurements total) or if the standard deviation of valid test measurements was greater than 20% of the mean, we elected to omit the data from the dataset rather than repeat the blood pressure measurement since all mice in this study were tested only one time.
  • Blood pressure values below 60 mm Hg on individual test measurements were not included in the average since they indicated machine failure to detect the pulse of the mouse (probably due to excessive mouse movement; see Sugiyama et al., 2002; Lorenz, 2002).
  • Variability in testing conditions was minimized by measuring all mice within a strain at the same time (i.e., in groups of four, one after the other until all mice were tested) and by having the same technician measure all mice within a strain.



Lung function


Respiratory function was measured in conscious, freely-moving mice using whole body plethysmographs (WBP) from BUXCO Electronics, Inc. (Troy, NY). WPB chambers allow animals to move freely within the chamber while respiratory function is measured (see Hamelmann et al., 1997; Petak et al. 2001; Albertine et al., 2002; Bates and Irvin, 2003; Irvin and Bates 2003). Six chambers were used simultaneously so that for each strain, six mice of each sex were measured at the same time.

Each WBP chamber is connected to a bias flow regulator to supply a smooth, constant flow of fresh air during testing. A transducer attached to each chamber detects pressure changes that occur as the animal breathes. Pressure signals are amplified by a MAX II Strain Gauge preamplifier and analyzed by the Biosystem XA software supplied with the system (BUXCO Electronics, Inc.). Pressure changes within each chamber are calibrated prior to testing by injecting exactly 1 ml of air through the injection port and adjusting the computer signal accordingly.

Mice were placed in the WPB chambers and allowed to acclimate for 10 min. Testing was conducted by allowing animals to move and breathe freely for 15 min while lung volume and respiratory rate parameters were measured. Raw data for each parameter were captured and averaged once per min to give a total of 15 data points per parameter, except for accumulated tidal volume, which is cumulative and represents the sum of all tidal volumes for the entire 15-min test session.



Grip strength


Mice were brought to the testing room and allowed to acclimatize for 10 min. A Grip Strength Meter from Columbus Instruments (Columbus, OH) was used to measure forelimb grip strength as an indicator of neuromuscular function as previously described (Costa et al., 1999; Van Damme et al., 2003; Whittemore et al., 2003). The grip strength meter was positioned horizontally, and mice were held by the tail and lowered toward the apparatus. Mice were allowed to grasp the smooth, metal, triangular pull bar (forelimbs only) and were then pulled backward in the horizontal plane. The force applied to the bar at the moment the grasp is released is recorded as the peak tension. The test was repeated 5 consecutive times within the same session (data for individual trials are available in the raw data set only), and the mean of all trials and the highest value from all trials were recorded for the grip strength for that animal. Mice are not trained prior to testing and each mouse is tested once (5 trials equal one test session).



Thermal pain response


Mice were brought to the testing room and allowed to acclimatize for 10 min. Pain reflexes in response to a thermal stimulus were measured using a Cold/Hot Plate Analgesia Meter from Columbus Instruments (cat #1445, Columbus, OH) as previously described (Malmberg and Bannon, 1999; Mogil et al., 1999; Wilson et al. 2001). Briefly, the The surface of the hot plate was heated to a constant temperature of 55 °C, as measured by a built-in digital thermometer with an accuracy of 0.1 °C and verified by a surface thermometer. Mice were placed on the hot plate (25.4 cm x 25.4 cm), which is surrounded by a clear acrylic cage (19 cm tall, open top). The latency to respond with either a hindpaw lick, hindpaw flick, or jump (which ever comes first) was measured to the nearest 0.1 s. The mouse was immediately removed from the hot plate and returned to its home cage.

Investigator Notes:

  • If a mouse did not respond within 30 s, the test was terminated and the mouse removed from the hot plate.
  • Animals were tested one at a time and were not habituated to the apparatus prior to testing.
  • Each animal was tested only once.


Body weight, body composition, and bone densitometry


Body composition and bone densitometry were assessed in anesthetized mice using a Lunar PIXImus II Mouse Densitometer (GE Medical Systems, distributed by Faxitron X-Ray Corporation, Wheeling, IL) as previously described (Beamer et al., 1996; Pietrobelli et al., 1996; Pietrobelli et al., 1998, Nagy and Clair, 2000). Each test day, the PIXImus was calibrated for bone mineral density and percent fat content using the "phantom mouse" unit supplied with the machine. This calibration procedure is part of the quality control process. Measurements for bone mineral density and percent fat must be within 2% of the expected value (0.0647g/cm2 and 11.9%, respectively) in order to proceed with animal scans.

Body weight was measured on a scale and recorded. Mice were anesthetized with tribromoethanol (250 mg/kg body weight) and placed ventral side down on disposable plastic trays so that the entire body and tail were measured in the scan. Trays were positioned so that the head was always oriented toward the left from the investigator's point of view. After the scan was completed, mice were removed from the plastic tray and returned to their home cage to recover from anesthesia.

PIXImus scans were analyzed using the PIXImus2 software (version 1.46.007), excluding the head from the analysis. Values for whole body (without the head), both femurs, and spine were obtained. Left femur data are not available for MPD analysis but are provided in the project data set.