**6.1 Diet and stress modulation of hypertension**

134 Modern Telemetry

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

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

Quantifying relationship between blood pressure and other variables Studying blood pressure-dependent and independent effects following

Measuring blood pressure continuously over time

Quantifying hypertension or changes in blood pressure

Table 3. Recommendations for the use of telemetry for measuring blood pressure in animals

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

Measuring blood pressure variability

interventions

(adapted from Kurtz et al., 2005).

the surgical procedures to implant DSI transmitters in rats and mice.

**Recommended for:** 

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

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

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

telemetry is essential for quality phenotyping in genetic research.

displaying genetic heterogeneities that dilute the loci effects.

**7. General conclusion** 

**pressure** 

Measurements in Animal Models of Hypertension, How It Revolutionized Hypertension Research 137

markers, good phenotyping is essential if one wants to rely on the results. Given the important consequences if one wants to pursue the study of a candidate locus, we think that

**6.3 Dissection of hypertension: Positional cloning of quantitative trait loci for blood** 

Finally, we should not forget to mention the work from Alan Deng who dissects hypertension in the rat and was able to find several significant blood pressure quantitative trait loci (Deng, 2007). His systematic approach uses telemetry as the sole method for blood pressure determination. The blood pressure values are obtained from adult rats from inbred strains. He does, however, perform several genetic manipulations in order to be able to reveal the loci. This approach allowed him to report several loci, the chromosomal position of which are later refined by producing sub-congenics animals (Chauvet et al., 2008). He was also able to reveal some gene-diet interactions and gene-gene interactions. For instance, he was able to demonstrate that a minor effect locus (named C18QTL1) could exert a major effect when it was transferred onto another genetic background. The normotensive Lewis rat genetic background was preventing this locus from exerting its full effect (Charron et al., 2005). This indicates that the normotensives also possess high blood pressure genes, but their effects are counterbalanced by 'normotensive' genes. This demonstrates the genetic buffering capacity of the genome and probably explains why the GWAS performed in humans do not yield the desired major high blood pressure loci: the population are

In this review, we have tried to demonstrate the importance of the environmental component of hypertension. When recognizing this importance, we think that it becomes easier to select the right tools to measure blood pressure in animal models as well as in humans. Human studies tend to indicate a decrease of the prevalence of hypertension whereas it may only be the reflection of the new standardized methods for the measurement of blood pressure devoid of stress. We have shown that rodents used for hypertension research are also stress sensitive and that the most commonly used method for blood pressure determination exacerbates the stress perceived by the animals: it increases blood pressure and body temperature. Since we have presented evidences indicating that stress and temperature can in turn be involved in the development of hypertension, it seems essential to subtract their influence when studying hypertension. Therefore, we think that telemetry is the sole method to perform blood pressure measurement in animal models of hypertension because it enables to subtract the stress artefacts or, conversely, to study them. Furthermore, no genetic studies should be performed in animals if telemetry is not used to characterize the blood pressure levels. Some differences are small and inadequate phenotyping could ruin a GWAS by revealing spurious statistical associations and diverting researchers to wrong candidate genes. On the other side, in an effort to standardize the techniques and eliminating human interference and bias, human epidemiological and genetic studies tend to eliminate the stress component from the measurement of blood pressure. As we have shown, this approach is desired in animal studies especially because we are dealing with inbred strains and can test our hypothesis is several backgrounds. Small effect blood pressure loci have more chance of being 'true' and can be verified in congenics

have yielded such results. We think that telemetry is the most appropriate mean for measuring blood pressure in this kind of work involving the modulation of blood pressure by environmental stressors. We also want to underline the fact that stress is always present when dealing with blood pressure measurements in rodents. Given its impact on blood pressure, it has to be taken into consideration even when it is not specifically under study.

This example is a good illustration of the study of the gene × environment interactions: We had to increase the genetic variance in order to be able to monitor changes that would not be visible otherwise. The first challenge was the use of an obesity-inducing diet that revealed differences in blood pressure response to stress and in the night period of the circadian pattern. The second challenge was the stress test where we could observe a delay in the return to the baseline in the high-fat diet treated rats. This is very interesting in the light of another finding: an augmentation of the cardiac mass in the rats fed a high fat diet. With the telemetric data, we can hypothesize that the increase in cardiovascular reactivity observed in the night (active period of the rats) and following a stress could explain an increased workload for the heart and the hypertrophy after only 12 weeks of high-fat diet. The effect of the diet would thus be more important when the animals are awake, active and experiencing daily stressors. This interpretation would be impossible without the telemetric data.

#### **6.2 Genetic determinants of emotionality and stress response in mice**

Some strains of mice such as the A/J strain display high anxiety levels. The A/J and C57Bl6 strains are the progenitors of the recombinant congenic strains AcB/BcA. With the help of this recombinant congenic strains panel, Thifault et al. (2008) unveiled quantitative trait loci of the stress response and of emotionality in mice. For the stress response, a 30-minute immobilisation stress was carried out. In these mice, it is characterized by two phases: initial hypothermia followed by thermogenesis. For emotionality, open-field test (number of fecal boli) and elevated plus maze (enclosed arm duration and emergence latencies) were employed. The results unveiled several genomic loci associated with the differential response in the recombinant congenic strains panel. Of major interest, stress loci overlapped with candidate loci for cardiovascular diseases. For instance, the BcA70 strain showed a down regulation of the *Atp1a2* gene in the heart and brain as compared with the parental C57Bl6 strain (p<0,001 and p<0,05, respectively), and this is in accordance with the high emotionality of the knock-outs for that gene and its involvement in salt-sensitive hypertension.

These remarkable data, resulting from complex analysis of genomic and phenotypic results would not have been possible without telemetry. The body temperature recordings from the stress test were performed with the telemetry implants and a strain distribution pattern over 2,5°C was obtained and could be used to unveil significant loci associated. This temperature difference could not have been monitored with precision with any other means. As for blood pressure, telemetry allowed the monitoring of body temperature continuously and the circadian pattern was obtained. It showed a significant strain x diet effect with the AcB strains (mostly A/J background) displaying a lower body temperature under high-salt diet, and the strain differences were steeper during the dark phase of the circadian cycle. Again, these subtle observations would not have been possible without continuous monitoring by telemetry.

This study unveiled genetic loci responsible for the differential responses to many of the stressors and tests performed. Since it is easy to get spurious association with genetic markers, good phenotyping is essential if one wants to rely on the results. Given the important consequences if one wants to pursue the study of a candidate locus, we think that telemetry is essential for quality phenotyping in genetic research.

#### **6.3 Dissection of hypertension: Positional cloning of quantitative trait loci for blood pressure**

Finally, we should not forget to mention the work from Alan Deng who dissects hypertension in the rat and was able to find several significant blood pressure quantitative trait loci (Deng, 2007). His systematic approach uses telemetry as the sole method for blood pressure determination. The blood pressure values are obtained from adult rats from inbred strains. He does, however, perform several genetic manipulations in order to be able to reveal the loci. This approach allowed him to report several loci, the chromosomal position of which are later refined by producing sub-congenics animals (Chauvet et al., 2008). He was also able to reveal some gene-diet interactions and gene-gene interactions. For instance, he was able to demonstrate that a minor effect locus (named C18QTL1) could exert a major effect when it was transferred onto another genetic background. The normotensive Lewis rat genetic background was preventing this locus from exerting its full effect (Charron et al., 2005). This indicates that the normotensives also possess high blood pressure genes, but their effects are counterbalanced by 'normotensive' genes. This demonstrates the genetic buffering capacity of the genome and probably explains why the GWAS performed in humans do not yield the desired major high blood pressure loci: the population are displaying genetic heterogeneities that dilute the loci effects.
