**5.1.5 Conditioning to restraint stress**

132 Modern Telemetry

stress is considered a psychogenic stress. In the rat, it induces a rapid increase in heart rate and blood pressure, and it has been shown that this increase is higher in SHR when compared to normotensive controls (Yamori et al., 1969; Irvine et al., 1997). Immobilisation also induces an increase in body temperature in the rat (Gollnick & Iannuzzo, 1968; Briese & De Quijada, 1970; Stewart & Eikelboom, 1979; Singer et al., 1986), in the mouse and in the rabbit (Snow & Horita, 1982). In humans, psychogenic stressors also induce a rise in body temperature in addition to the rise of blood pressure and heart rate (Marazziti et al., 1992). The rise in body temperature induced by immobilisation is also stronger in SHR than in normotensive WKY (Berkey et al., 1990, Morley et al., 1990). At room temperature, nonstressed body temperature has been shown to be identical (Berkey et al., 1990) or higher in SHR as compared to WKY (Price & Wilmoth 1990). Wilson at al. (1977) indeed suggested that the basal temperature threshold of SHR is modified, thus explaining its abnormal temperature response to stress. Price and Wilmoth (1990) reported a higher vascular sensitivity to norepinephrine in SHR and O'Leary and Wang (1994) showed a decreased vasodilation in the tail vessels in SHR. Together, these 2 mechanisms could explain the higher temperature reached during immobilisation stress and the inability to go back to baseline rapidly after the stress. Therefore, the stronger temperature response to immobilization in SHR could serve as a marker of stress susceptibility. Indeed, we have found the genetic determinants of this enhanced stress response in hypertensive rats and observed a strong sexual dimorphism in the SHR, the Y chromosome from hypertensive origin contributing significantly to this enhanced response (Dumas et al., 2000a). Because a similar abnormal response has also been reported in humans subjected to psychogenic stress, and because this abnormal response correlates with the future hypertensive status of these individuals, it is important 1) to characterize and to be able to recognize this stress response and 2) to employ methods of blood pressure determination devoid of this

In rodents, there are 3 veins and one artery in the tail. However, because its length usually equals that of the body in mouse and rats, most of the time, no blood is flowing through these vessels because of the heat-loss that would result. Hence, the tail serves mainly for thermoregulation. Therefore, when attempting to determine blood pressure with the tailcuff method, in addition to the restrainer, heating of the animal to temperatures between 30 and 37°C is mandatory in order to get a significant blood flow into the tail. This heat stress thus adds to the body temperature increase due to stress and some animals may die even

Schlager (1974) have reported a higher heat sensitivity in the spontaneously hypertensive mouse (SHM) mice as compared to controls. SHR also presents an increased thermosensitivity (Wilson et al., 1977; Wright et al., 1978; McMurtry & Wexler, 1983). This thermosensitivity persists in culture and is already present in neonatal cardiomyocytes, indicatives of a primary genetic defect not consecutive to high blood pressure (Hamet et al., 1985). Malo from our group (1989) has unveiled the existence of the thermosensitivity locus *tms* associated with hypertension in SHM. When anaesthetized SHM mice were immersed in a 44°C water bath, their body temperature increase was faster (1,74 +/- 0,04°C versus 1,13 +/- 0,03°C degree per minute, p<0,001) and their survival decreased when compared to control mice. This was

important confounder.

**5.1.2 Effect of heating on blood pressure determination** 

**5.1.3 Thermosensitivity and hypertension** 

with prior training to the experimental conditions (Gross & Luft, 2003).

It was recognized that restraining is stressful and it has been proposed that seven days of training to the procedure would alleviate the effect of stress and enable the measurements of 'usual' blood pressure in the animals. The idea is the animals would get used to the restrainer and to the manipulation of their tails and display less stress-induced changes in their cardiovascular parameters following conditioning. Gross and Luft (2003) have shown that this conditioning period had no effect in mice. With mice implanted with telemetry transmitters, they have shown that heart rate, systolic and diastolic blood pressure at day 1 was not different than after 10 days of 30-minutes conditioning in the restrainers. A similar absence of conditioning was previously reported for rats (Bazil et al., 1993; Irvine et al., 1997).

This clearly demonstrates that immobilization of rodents in order to measure blood pressure will induce significant artefacts, that training does not prevent these bias and can further modulate the blood pressure in ways not consistent with what is sought. Furthermore, because the stress sensitivity may differ between strains, subtle differences in blood pressure may be missed. Finally, as we have seen, the different thermosensitivity of various strains of rodents in addition to the blood pressure-modulating effect of heat are two significant confounders that could ruin an experimental protocol. Hence, we think that the 'tail-cuff blood pressure method' can only be used when large blood-pressure differences (>= 15-20 mm Hg) are expected in the experimental setting when the restrainer effects become minor as compared to the effects of the tested hypothesis.

#### **5.2 Direct non-invasive method: Radiotelemetry**

Radiotelemetry for blood pressure monitoring in animals requires the surgical implantation of a catheter in a suitable artery, usually the carotid or femoral artery. The transmitter itself

Radio-Telemetry in Biomedical Research - Radio-Telemetry Blood Pressure

scientifically.

**from our laboratory** 

**6.1 Diet and stress modulation of hypertension** 

minutes immobilisation stress was also performed at weeks 0 and 12.

Measurements in Animal Models of Hypertension, How It Revolutionized Hypertension Research 135

number of animals and/or when large blood pressure effects are expected. The reasons why these methods are not recommended are their cost and the technical skills that are required to perform them successfully and reproducibly. They are, however far superior

**6. Radiotelemetry to study gene × environment in hypertension – Experience** 

Hypertension is an important contributor of mortality and morbidity in humans. Our understanding of the aetiology of the disease is incomplete and the search for the genetic determinants is complicated by the fact that blood pressure is very labile, with important changes occurring within seconds and minutes. As we have shown, blood pressure is hard to assess reproducibly without bias when performing epidemiological studies or GWAS. These problems are in part due to the important environmental component modulating blood pressure. Among these environmental modulators, psychogenic stress exerts a major influence. In experimental research using rodents, we have shown that the usual tail-cuff method for blood pressure determination induces stress and increases body temperature, two major modulators of blood pressure and hypertension. Therefore, we think that telemetry is the only technique to ascertain blood pressure with the least amount of bias. It is also the only technique allowing the measurement of the stress component modulating blood pressure. The few examples below will try to illustrate the level of refinement and robustness that can be achieved in hypertension research when the environmental influences are understood and telemetry the method employed for assessing blood pressure.

The work by Šedová et al. (2004) from our group illustrates the various concepts that we have presented here and take advantage of telemetric measurement of blood pressure to draw conclusions that would have been impossible to obtain otherwise. The manuscript describes the effects of diet-induced obesity in the SHR. Because the cardiovascular response to stress is a significant predictor of hypertension, it looked more specifically at the effects of the diet on the stress response and the global impact on cardiovascular morbidity. In order to do so, adult male SHR were fed a high fat diet for 12 weeks. Blood pressure and heart rate were measured by telemetry at week 0 and week 12. In addition to basal blood pressure recorded for 3 consecutive days each time, blood pressure determination in response to a 30-

As expected, immobilisation stress in restrainers for 30 minutes was able to increase blood pressure and heart rate in both groups. While there was no difference in the blood pressure response to stress in both groups between week 0 and 12, there was a significant lag in the return to the baseline after stress in the high-fat diet group at week 12 as compared to week 0. This was significant for systolic and diastolic blood pressure as well as for heart rate. No difference was observed in the response to stress in the control animals in the stress or poststress periods. Furthermore, because it allowed the measurement of the circadian pattern of blood pressure, telemetry could reveal a blood pressure increase during the night in the animals receiving the high-fat diet as compared to the normal-chow fed controls. As we have seen from the immobilisation stress data, this would have been impossible to detect with a one-time recording in stressful conditions by the tail-cuff method. Needless to say, measuring blood pressure during stress and after stress with any other techniques could not

is inserted in the abdominal cavity (for rats) or under the skin (for mice). Interestingly, with the miniaturization of electronic components and probably because of the pressure of animal rights activists, there has been an impressive decrease in the number of publications using telemetry in large animals (cats, dogs, swine, monkeys) and a significant increase in those employing rodents (Kramer et al., 2001). Our own experience is with the transmitters from Data Sciences International (DSI, St-Paul, MN, USA), but other manufacturers are present on the market. For a review of the progress in radiotelemetry in small animals, please refer to Kramer et al. (2001). Similarly, the interested reader can consult the original publication by Mills et al. (2000) describing the characteristics of the mouse transmitters from DSI as well as the review article by Huetteman & Bogie (2009) that describes in details the surgical procedures to implant DSI transmitters in rats and mice.

Because of the surgery needed for the installation of the transmitters, radiotelemetry can be considered an invasive technique. But, at the same time, when recovery from surgery is optimal, it is the least invasive method since the measurements are achieved in the usual environment without external stressors such as the direct intervention of the technician. Furthermore, measurements are performed not only on undisturbed animals, but also in freely moving animals, which contrasts with all the other measurement techniques. Indwelling catheters attached to tethering devices is the only method allowing some movement of the conscious animals but generates noise and stress and does not come close to what can be achieved by telemetry. Blood pressure can be performed continuously on a beat-to-beat basis and during the night, a period not always practical for the experimenter. It allows the monitoring of the circadian rhythm impact and greatly reduces the variation in the mean blood pressure when data are averaged over several hours. For instance, Van Vliet (2003) has shown that the 95% confidence interval of the mean of 24 hours of blood pressure measurements in a group of 9 mice was 8 mm Hg as compared to 14 mm Hg for 30-minutes average and 22 mm Hg for a single time point. In SHR, systolic blood pressure is 20-40 mm Hg higher and heart rate 100 bpm faster when assessed by tail-cuff or directly by indwelling catheters (Bazil et al., 1993). In mice, while the tail cuff results are highly correlated with direct arterial pressure (r=0,86, p<0,01), the tail-cuff values are 20 mm Hg higher on average (Krege et al., 1995). For both species, the lowest blood pressure values are obtained with implantable radiotelemetry because the method is devoid of stress and performed in freely moving animals without anaesthesia (Irvine et al., 1997; Mills et al., 2000; Kuneš et al., 2008).


Measuring blood pressure in conscious unrestrained animals Measuring blood pressure continuously over time Measuring blood pressure variability Quantifying hypertension or changes in blood pressure Quantifying relationship between blood pressure and other variables Studying blood pressure-dependent and independent effects following interventions

Table 3. Recommendations for the use of telemetry for measuring blood pressure in animals (adapted from Kurtz et al., 2005).

Table 3 summarizes the recommended use and advantages that apply to direct blood pressure measurements methods and especially to telemetry. The only application for which the AHA indicates that direct methods are not recommended is for screening of large number of animals and/or when large blood pressure effects are expected. The reasons why these methods are not recommended are their cost and the technical skills that are required to perform them successfully and reproducibly. They are, however far superior scientifically.
