Non-Invasive Blood Pressure in Mice and Rats
Over the past 20 years, research scientists have attempted to non-invasively measure mice and rat blood pressure (BP) with varying degrees of success.
The ability to precisely and non-invasively measure the systolic and diastolic blood pressure, in addition to the heart pulse rate and other blood flow parameters in rodents, is of great clinical value to the researcher.
Invasive Blood Pressure, Rat and Mouse Measurement
Direct blood pressure, an invasive surgical procedure, is the gold standard to compare the accuracy of non-invasive blood pressure (NIBP) technologies. Direct blood pressure should be obtained on the rodent’s carotid artery when comparing to NIBP. “Validation in Awake Rats of a Tail Cuff Method for Measuring Systolic Pressure”, Bunag, R.D., Journal of Applied Physiology, Vol 34, Pgs 279-282, 1973.
Radiotelemetry, a highly invasive surgical procedure, is a very reliable blood pressure technology and is also utilized to compare the accuracy of NIBP technologies. Telemetry involves the implantation of radio transmitters in the rodent’s body. This technique is well validated and has excellent correlation with direct blood pressure.
The advantage of implantable radio telemetry is the ability to continuously measure rat and mouse blood pressure in free moving laboratory animals.
The disadvantages of radiotelemetry are: (1) morbidity associated with the initial surgical implantation of the transmitter; (2) morbidity associated with surgery required to replace the battery, which has a short battery life; (3) increase in the animal’s level of stress, especially mice, in relationship to the large, heavy transmitters (2004, ATLA, 4th World Congress, Einstein, Billing, Singh and Chin); (4) abnormal behavior since the animal cannot have social interaction due to the current technology requiring the implanted animal to be secluded, one animal per cage; (5) inability to perform high throughput screening; (6) high cost of the initial equipment set-up and the expensive transmitters that require frequent factory maintenance; (7) cost of material and human resources relating to current surgeries; and (8) the without of a competitive market resulting in high product and servicing costs.
Non-Invasive Blood Pressure, Rat and Mouse Measurement
The NIBP methodology consists of employing a tail cuff placed on the tail to occlude the blood flow. Upon deflation, one of several types of NIBP sensors, placed distal to the occlusion cuff, can be utilized to monitor the rat BP. There are three (3) types of NIBP sensor technologies: photoplethysmography, piezoplethysmography and quantity Pressure Recording. Each method will utilize an occlusion tail-cuff as part of the procedure.
The first and oldest sensor kind is Photoplethysmography (PPG), a light-based technology. The purpose is to record the first turn up of the pulse while deflating the occlusion cuff or the disappearance of pulses upon inflation of the occlusion cuff. Photoplethysmography utilizes an incandescent or LED light source to record the pulse signal wave. As such, this light-based plethysmographic method uses the light source to illuminate a small identify on the tail and attempts to record the pulse.
Photoplethysmography (PPG) is comparatively inaccurate since the readings are based solely on the amplitude of a single pulse and can only imprecisely measure the systolic blood pressure and the heart beat. There are many limitations to a light-based technology, such as: (1) over-saturation of the BP signal by ambient light; (2) extreme sensitivity to the rodent’s movement (motion artifact); and (3) the difficulty in obtaining adequate mice blood pressure signals in dark skinned rodents (Pigmentation Differentiation). Light-based sensors also cause tail burns from close contact and prolonged exposure.
Diastolic blood pressure cannot be measured by photoplethysmography since the technology records only the first turn up of the pulse. If the diastolic BP is displayed on the photoplethysmographic instrumentation, it is only an estimation that is calculated by a software algorithm instead of a true measurement.
Additional tendency to change and inaccuracy occurs in PPG devices that rely on obtaining readings during occlusion cuff inflation.
Occlusion cuff length is also another source of tendency to change and inaccuracy. Occlusion cuff length is inversely related to the accuracy of the blood pressure. Long cuffs, predominantly in most photoplethysmographic devices, record lower than the actual blood pressure measurements.
These limitations severely compromise the consistency, dependability and accuracy of the NIBP measurements obtained by devices that utilize light-based/LED photoplethysmographic technology.
The photoplethysmography method correlates poorly with direct blood pressure measurements and is the least recommended sensor technology for NIBPe in rodents, especially mice.
The second NIBP sensor technology is piezoplethys-mography. Piezoplethysmography and photoplethysmography require the same first turn up of a pulse in the tail to record the systolic blood pressure and heart rate.
Both plethysmographic methods have similar clinical limitations. while photoplethysmography uses a light source to attempt to record the pulse signal, piezoplethysmography utilizes piezoelectric ceramic crystals to do the same. From a technical point of view, piezoplethysmography is far more sensitive than photoplethysmography since the signal from the sensor is the rate of change of the pulse instead of just the pulse amplitude. consequently, already extremely small mice with high velocity pulses will generate a sufficient signal to be detected with simple amplifiers.
Piezoelectric sensors are more accurate than light-based/LED sensors but the same plethysmographic limitations continue to produce inaccuracies in blood pressure measurements. On a positive observe, the skin pigment of the rodent is not a measurement issue with piezoplethysmography as with photoplethysmography.
Although piezoplethysmography is better than photoplethysmography, both non-invasive tail-cuff blood pressure technologies correlate poorly with direct blood pressure measurements.
3. quantity Pressure Recording
The third sensor technology is quantity Pressure Recording (VPR). The quantity Pressure Recording sensor utilizes a specially designed differential pressure transducer to non-invasively measure the blood quantity in the tail. quantity Pressure Recording will truly measure six (6) blood pressure parameters simultaneously: systolic, diastolic, average, heart pulse rate, tail blood quantity and tail blood flow.
Since quantity Pressure Recording utilizes a volumetric method to measure the blood flow and blood quantity in the tail, there are no measurement artifacts related to ambient light; movement artifact is also greatly reduced. In addition, quantity Pressure Recording is not dependent on the animal’s skin pigmentation. Dark-skinned animals have no negative effect on quantity Pressure Recording measurements. Very small, 10-gram C57/BL6 black mice are easily measured by the quantity Pressure Recording method.
Special attention is afforded to the length of the occlusion cuff with quantity Pressure Recording in order to origin the most accurate blood pressure readings.
quantity Pressure Recording is the most reliable, consistent and accurate method to non-invasively measure the blood pressure in mice as small as 10 grams to rats greater than 950 grams.
In an independent clinical validation study conducted in 2003 at Yale University, New Haven, Connecticut, quantity Pressure Recording correlated 99 percent with direct blood pressure:
“quantity Pressure Recording is excellent. It is very accurate and dependable. We performed experiments on temperature-controlled, adult rats and the non-invasive blood pressure measurements showed almost perfect correlation with invasive blood pressure measurements. We are very pleased with the results.”
Numerous published research papers are obtainable validating the accuracy, reliability and consistency of quantity Pressure Recording. See the Clinical Bibliography section.
Rodent Holders, Rat and Mouse
The ideal animal holder should comfortably restrain the animal, create a low-stress ecosystem and allow the researcher to regularly observe the animal’s behavior. A trained rat or mouse can comfortably and quietly keep in the holder for several hours.
It is very advantageous to incorporate a darkened nose cone into the rodent holder to limit the animal’s view and reduce the level of animal stress. The animal’s nose will extend by the front of the nose cone allowing for comfortable breathing. The tail of the animal should be fully extended and exit by the rear hatch opening of the holder.
The proper size animal holder is basic for proper blood pressure measurements. If the holder is too small for the animal, the limited lateral space will not allow the animal to breathe in a relaxed fact. The animal will compensate by elongating its body, thereby creating a breathing artifact. A breathing artifact will cause excessive tail motion and undesirable blood pressure readings.
Animal Body Temperature, Rat and Mouse
A NIBP system should intended to comfortably warm the animal, reduce the animal’s stress and enhance blood flow to the tail.
The rodent’s chief body temperature is very important for accurate and consistent blood pressure measurements. The animal must have adequate blood flow in the tail to acquire a blood pressure signal. Thermo-regulation is the method by which the animal reduces its chief body temperature, dissipates heat by its tail and generates tail blood flow.
Anesthetized animals may have a lower body temperature than awake animals so additional care must be administered to continue the animal’s proper chief body temperature. An infrared warming blanket or a re-circulating water pump with a warm water blanket is the preferred method to continue the animal’s proper chief body temperature. The animal should be warm and comfortable but never hot. Extreme care must be exercised to never overheat the animal.
Warming devices such as hot air heating chambers, heat lamps or heating platforms that apply direct heat to the animal’s feet are not advisable to continue the animal’s chief body temperature. These heating devices will overheat the animal and increase the animal’s respiratory rate, thereby increasing the animal’s stress level. These conditions will elicit poor thermo-regulatory responses and create inconsistent and inaccurate blood pressure readings.
The proper room temperature is basic for accurate blood pressure measurements. The room temperature should be at or above 26C. If the room temperature is too cool, such as below 22C, the animal will not thermo-control, tail blood flow will be reduced and it may be difficult to acquire blood pressure signals. A cold steel table or a nearby air conditioning duct are undesirable during animal testing.
The animal should be placed in the holder at the minimum 10 to 15 minutes prior to obtaining pressure measurements. Acclimated animals will provide faster BP measurements than non-acclimated animals. Proper animal handling is basic to consistent and accurate blood pressure measurements. A nervous, stressed animal may have reduced circulation in the tail.
Most rodents will quickly adapt to new conditions and feel comfortable in small, dark and confined spaces. Training is not necessary to acquire accurate blood pressure readings, however, some researchers prefer training sessions. Rodents can easily be trained in approximately three days, 15-minutes each day before beginning your experiment.
The animal should be allowed to go into the holder freely. After the animal is in the holder, adjust the nose cone so the animal is comfortable but not able to move excessively. The animal should never have its head bent sideways or its body compressed against the back hatch. The animal’s temperature should be observed throughout the experiment.
Tail-cuff NIBP measurements can be consistent, accurate and reproducible when studying awake and anesthetized mice and rats. In addition, multiple animal testing is very cost-effective for large extent, high throughput screening. Care must be exercised to properly manager the animals. Training the animals and monitoring the animal’s temperature may also be advantageous.
The volumetric pressure recording method provides the highest degree of correlation with telemetry and direct blood pressure and is clearly the preferred tail-cuff sensor technology.
NIBP devices that utilize quantity Pressure Recording are a valuable tool in research and will continue to be advantageous in many study protocols. The main advantages are: (1) they require no surgery; (2) they are considerably less expensive than other blood pressure equipment, such as telemetry; (3) they can screen for systolic and diastolic BP changes over time in large numbers of animals; and (4) they provide the researcher with the ability to acquire accurate and consistent blood pressure measurements over time in long-term studies.