T.R.A.P.T. Methodology Analysis Informational Report

Table of Contents

1. Rodent​ ​Body​ ​Temperature​ ​Telemetry
2. Possible​ ​Sensor​ ​Methods
3. Rodent​ ​Respiration​ ​Telemetry
4. Non​ ​Invasive​ ​Blood​ ​Pressure​ ​Telemetry
5. Photoplethysmography
6. Piezoplethysmography
7. Volume​ ​Pressure​ ​Recording
8. Effects​ ​of​ ​Rodent​ ​Holders​ ​on​ ​BP
9. Animal​ ​Body​ ​Temperature​ ​in​ ​Relation​ ​with​ ​BP
10. Environmental​ ​Temperature
11. Animal​ ​Preparation

Rodent Body Temperature Telemetry

There are few ways to accurately measure body temperature in a rat without causing the animal stress and thus increasing its body temperature. Most body temperature measurements are conducted by inserting a thermometer, thermocouple, or other measurement device into the rectal cavity of esophagus of the rat, but these methods cause the rat stress and induce a higher baseline body temperature. Non-invasive methods typically include holding a thermometer or other measurement device against the skin of the rat. Skin temperature does not directly correlate to internal body temperature, making this method rather lackluster in the scheme of our project. All of these methods, however, are time consuming and require special attention from the investigator handling the animal.

Possible Sensor Methods

Due to the resounding lack of external sensor measuring equipment, it may be best to consider using a thermometer with a restraint that makes it easy to position and read to take continual manual measurements. If this is not preferable, however, it may be possible to incorporate a sensor designed for humans into the restraint such that it can be easily applied to the surface of the rat’s body. The sensor found at the following website is typically used for taking body temperature of humans by placing it on their head, but it has been shown to give very accurate and continual readings. This may be feasible for placing on the body of the rat if it is restrained so that it cannot shift away from the sensor.

https://www.draeger.com/en_uk/Hospital/Products/Accessories-and-Consumables/Adult-Patient-Monitoring-Accessories/Monitoring-Accessories/Tcore

One other idea could be to create a meshwork within the restraint, especially if it were one that inflated, which could measure the body temperature of the rat all over or at several different points. A sensor like this could be used:

https://www.cooking-hacks.com/body-temperature-sensor-mysignals-ehealth-medical

Several of these sensors could be incorporated into the restraint to take measurements along the sides and belly of the rat, giving an average skin temperature between all of the points.

Rodent Respiration Telemetry

Respiration is the process of inhalation and exhalation in order to obtain the necessary oxygen that cells require to grow and function. Respiration, in a laboratory setting, can be measured visually by analyzing the degree to which the chest moves, with respiratory sensors, or through the analysis of bicarbonate and carbon dioxide content in the blood. One sensor that can be used to measure respiration is a capnograph, which uses infrared spectroscopy to measure carbon dioxide levels in the respired air. It does this by comparing two other values: the concentration of carbon dioxide in venous blood and the degree of efficiency at which breathing occurs. Though the capnograph would be ideal as it is a very sensitive equipment, it is currently not within our budget (Tremoleda et al., 2012). Due to this, we have investigated other possible sensors that can measure respiration rate.

Another method of measuring respiration is through the use of a whole body plethysmograph. The plethysmograph measures multiple variables, including: breathing frequency, times of inhalation and exhalation, tidal volume, and potentially body temperature. Prices range; some are feasible and some are too expensive for our budget (“Whole Body Plethysmography”).

A relatively new technique for measuring respiration is accelerometry-based inductive plethysmography. This technique involves the use of an accelerometer on the surface of the rat’s chest to measure the expansion and contraction (rising and falling) of the rat’s chest, thus enabling this procedure to be non-invasive. This system, unlike the capnograph, does not require a tracheotomy or any intubation and will be relatively safe to use in a laboratory setting on live rats (Cleary et al., 2012). This method would be extremely useful for us as it would not be as expensive as other methods and would be easier to implement in the restraint.

Non Invasive Blood Pressure Telemetry

The non-invasive blood pressure methodology consists of placing a cuff on the animal’s tail to occlude the blood flow. Upon deflation, one of several types of non-invasive blood pressure sensors, placed distal to the occlusion cuff, can be used to monitor the blood pressure. There are three types of non-invasive blood pressure sensor technologies: photoplethysmography, piezoplethysmography and Volume Pressure Recording. Each method uses an occlusion tail-cuff as part of the procedure.

Photoplethysmography

The first and oldest method is Photoplethysmography (PPG), a light-based technology. It records the first appearance of the pulse while deflating the occlusion cuff, and the disappearance of pulses upon inflation of the occlusion cuff. PPG uses an incandescent or LED light source to record the pulse signal wave. The light source illuminates a small spot on the tail and attempts to record the pulse.

PPG is relatively inaccurate since the readings are based solely on the amplitude of a single pulse and cannot precisely measure the systolic blood pressure or the heart beat. There are many limitations to a light-based technology, such as over-saturation of the blood pressure signal by ambient light, extreme sensitivity to the rodent’s movement (motion artifact) and difficulty obtaining adequate 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 PPG since the technology records only the first appearance of the pulse. If the diastolic blood pressure is displayed on the PPG instrumentation, it is an estimation only, calculated by a software algorithm, and not a true measurement.

Additional variability and inaccuracy occurs in PPG devices that rely on obtaining blood pressure readings during occlusion cuffination. Occlusion cuff length, another source of variability and inaccuracy, is inversely related to the accuracy of the blood pressure measurements in PPG systems. Long cuffs, predominant in PPG devices, record pressures as lower than the actual blood pressure measurements. These limitations severely compromise the consistency, dependability, and accuracy of the non-invasive blood pressure measurements obtained by devices that use light- based/LED PPG technology. The PPG method correlates poorly with direct blood pressure measurements and is the least recommended sensor technology for non-invasive blood pressure in rodents, especially in mice.

Piezoplethysmography

The second non-invasive blood pressure sensor technology is piezoplethysmography. Piezoplethysmography and PPG require the same first appearance of a pulse in the tail to record the systolic blood pressure and heart rate. The two methods have similar clinical limitations.

Piezoplethysmography uses piezoelectric ceramic crystals to attempt to record a pulse signal. From a technical point of view, piezoplethysmography is far more sensitive than PPG since the signal from the sensor is the rate of change of the pulse rather than just the pulse amplitude. Therefore, even extremely small mice with high pulse rates will generate a sufficient signal to be detected with simple amplifiers.

Piezoelectric sensors are more accurate than light-based/ LED sensors, but the same PPG limitations continue to produce inaccuracies in blood pressure measurements. However, the skin pigment of the rodent is not a measurement issue with piezoplethysmography as it is with PPG. Although piezoplethysmography is better than PPG, both these non-invasive tail-cuff blood pressure technologies correlate poorly with direct blood pressure measurements.

Volume Pressure Recording

The third sensor technology is Volume Pressure Recording (VPR). VPR uses a specially designed differential pressure transducer to measure the blood volume in the tail non-invasively. VPR will actually measure six parameters simultaneously: systolic blood pressure, diastolic blood pressure, mean blood pressure, heart pulse rate, tail blood volume, and tail blood flow.

Since VPR uses a volumetric method to measure blood flow and blood volume in the tail, there are no measurement artifacts related to ambient light. Movement artifact is also greatly reduced. In addition, VPR is not dependent on the animal’s skin pigmentation, so dark skin has no negative effect on VPR measurements. Special attention is afforded to the length of the occlusion cuff with VPR in order to derive the most accurate blood pressure readings. VPR is the most reliable, consistent, and accurate method to measure the blood pressure non-invasively in rodents.

Effects of Rodent Holders on BP

The ideal animal holder should comfortably restrain the animal, create a low-stress environment and allow the researcher to observe the animal’s behavior constantly. A trained rat or mouse can comfortably and quietly remain in the holder for several hours.

It is very beneficial to incorporate a darkened nose cone into the rodent holder to limit the animal’s view and reduce its stress level. The animal’s nose should protrude through the front of the nose cone allowing for comfortable breathing. The tail of the animal should be fully extended and exit through the rear hatch opening of the holder.

The proper size animal holder is essential for proper blood pressure measurements. If the holder is too small, the limited lateral space will not allow the animal to breathe in a relaxed fashion. The animal will compensate by elongating its body, creating a breathing artifact that will cause excessive tail motion and undesirable blood pressure readings.

Animal Body Temperature in Relation with BP

A non-invasive blood pressure system should be designed to warm the animal comfortably, reduce the animal’s stress, and enhance blood flow to its tail. The rodent’s core body temperature is very important for accurate and consistent blood pressure measurements. The animal must have adequate blood flow in the tail to produce a blood pressure signal. Thermo-regulation is the method by which the animal reduces its core body temperature, dissipates heat through its tail and generates tail blood ow.

Anesthetized animals may have lower body temperatures than awake animals so additional care must be taken to maintain the animal’s proper core body temperature. An infrared warming blanket or a re-circulating water pump with a warm water blanket are the preferred methods for maintaining the animal’s proper core body temperature. The animal should be warm and comfortable but never hot. Extreme care must be exercised never to 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 for maintaining the animal’s core body temperature. These heating devices will overheat the animal and increase the animal’s respiratory rate and stress level. Such conditions will elicit poor thermoregulatory responses and create inconsistent and inaccurate blood pressure readings.

Environmental Temperature

Room temperature at or above 26°C is essential for accurate blood pressure measurements. If the room is too cool, below 22°C for example, the animal will not thermo-regulate, tail blood flow will be reduced and it may be difficult to obtain blood pressure signals. A cold steel table or a table near an air conditioning duct are undesirable for use during animal testing.

Animal Preparation

The animal should be in the holder at least 10 to 15 minutes before pressure measurements begin. Acclimated animals should provide faster BP measurements than non-acclimated animals. Proper animal handling is critical to consistent and accurate blood pressure measurements. A nervous, stressed animal may have diminished circulation in the tail. Most rodents will quickly adapt to new conditions and feel comfortable in small, dark, and confined spaces. Animal training is not necessary to obtain 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 the experiment.

Additionally, the animal should be allowed to enter 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 may be monitored throughout the experiment.

In summary, the tail-cuff non-invasive blood pressure measurements:

• can be accurate, consistent, and reproducible in studies of awake or anesthetized mice and rats
• make multiple animal testing very cost-effective for large scale, high throughput screening
• require that care be taken to handle the animals properly
• can benefit from training test animals and monitoring their’ temperatures

The VPR method:

• provides the highest degree of correlation with telemetry and direct blood pressure
• is the preferred tail-cuff sensor technology

Non-invasive blood pressure devices that use VPR are valuable tools in research and will continue to be bene cial in many study protocols. The main advantages are that VPR devices:

• require no surgery
• are significantly less expensive than other blood pressure equipment, such as telemetry
• can screen for systolic and diastolic BP changes over time in large numbers of animals
• provide the researcher with the ability to obtain accurate and consistent blood pressure measurements over time in long-term studies

References

• “Animal Handling and Restraint.” Course Materials. Animal Research Facility Rajiv Gandhi Centre for Biotechnology, n.d. http://210.212.254.115:198/arf/p1.php. Accessed 12 Sept. 2017.
• Cleary, Daniel R., et al. (2012). A novel, non-invasive method of respiratory monitoring for use with stereotactic procedures. Journal of Neuroscience Methods, 209(2), 337–343. doi:10.1016/j.jneumeth.2012.06.029.
• Tremoleda, Jordi L, et al. (2012). Anaesthesia and physiological monitoring during in vivo imaging of laboratory rodents: considerations on experimental outcomes and animal welfare. EJNMMI Research, 2:44. doi:10.1186/2191-219X-2-44.
• Whole Body Plethysmography. (n.d.). EMMS Supporting Science. Retrieved from http://www.electromedsys.com/wbp.html.
• Kemp, Robert W. “Handling and Restraint.” The Laboratory Rat (Handbook of Experimental Animals). N.p., n.d.
• Machholz, Elton, et al. “Manual Restraint and Common Compound Administration Routes in Mice and Rats.” Journal of Visualized Experiments, 2012, DOI: 10.3791/2771. Accessed 17 Sept. 2017.
• Stewart, Kay, and Valerie A. Schroeder. “Rodent Handling and Restraint Techniques.” Journal of Visualized Experiments, 2017.
• http://www.usp.br/bioterio/Artigos/Procedimentos%20experimentais/HandlingThe_Laboratory_Rat-By_George_J_Krinke.pdf. Accessed 17 Sept. 2017.
• https://www.jove.com/pdf/10221/jove-science-education-10221-rodent-handling-and-restraint-techniques. Accessed 17 Sept. 2017.
• https://www.karger.com/Article/PDF/135828
• https://www.ncbi.nlm.nih.gov/pubmed/1409790