**3. Genes X environment interactions, impact on blood pressure and development of hypertension**

Up to 70% of blood pressure variance is attributed to the environment. The impact of the environment can be seen as additive or in interaction with genes. Figure 1 summarizes the possible contributions of genes and environment to hypertension.

In the first case (a), the genes and the environment exert their influence on blood pressure independently. Thus, a disease gene (Gd) and a deleterious environment (Ed) would act separately to cause hypertension in an additive manner. In the second case (b), the environment is not "deleterious" *per se*. Its influence will depend on environment-sensitive genes (Gs). These are called susceptibility genes. They are not disease genes by themselves, they only permit the environment to reveal or amplify its impact. This is the G × E

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

Measurements in Animal Models of Hypertension, How It Revolutionized Hypertension Research 125

Fig. 2. Gene × environment interactions. Phenotypic expression of 3 different genotypes (alleles) when measured in 2 different environments (1, 2); adapted from Lynch & Walsh, 1997. It is important at this point to distinguish the impact of the environment that needs to be assessed for its contribution to the disease from the environmental variance that is the variance due to the measurements and is represented by the error bars on the graphs. For instance, in Figure 2, the genetic variance varies depending on the environment whereas the environmental variance does not change. The ratio of the genetic variance over the total variance (genetic + environmental variance) is called 'heritability' and the higher the heritability, the higher the contribution of genes to the studied trait. Technically, this means that when searching for genetic determinants of a disease, one should work with traits displaying a high heritability. However, heritability is not static, especially when considering susceptibility genes. As can be seen from Figure 2, while the environmental variability does not change (error bars), the genetic variance can be modified depending on the environment. Therefore, the key to study the contribution of susceptibility genes is to find the conditions where the genetic variance is at its maximum (for instance, Figure 2.b, environment 2). Because such conditions are not necessarily seen *a priori* in an experimental set-up, it is essential to challenge the model by changing the environment in which the trait of interest is measured. In hypertension research, that can be translated by the use of different diets or drugs in order to blow-up the differences in blood pressure and find the

This understanding is fundamental for selecting the parameters that will allow appreciating the influence of the environment in an experimental setting. This is the area where, we think, radiotelemetry can be the most useful in hypertension research. We will demonstrate later how the currently accepted methods for measuring blood pressure in animal models

genes responsible for the differential effect.

interaction. The best example in the field of hypertension is salt-sensitivity where only saltsensitive individuals are displaying hypertension consecutive to a higher intake of salt in the diet (Weinberger, 1990).

Fig. 1. Gene and environment in hypertension.

#### **3.1 How to approach the genes X environment interactions – Concept of genetic susceptibility to the environment**

Thus, while a genetic polymorphism could be associated with an increased risk of developing a disease, a differential response to the environment could define a susceptibility to this environment. This environmental susceptibility being modulated by environmentsensitive genes, we proposed the concept of *genetic susceptibility to the environment* (Hamet, 1996; Hamet et al., 1998; Pausova et al., 1999). The hallmark of a susceptibility gene is that it cannot be coined normal or abnormal since its expression *per se* will not cause the disease. Environment-sensitive variants of these genes will reveal their impact only when the organism is subjected to the specific triggering environment. We will now present the basis of the genes × environment interactions (adapted from Lynch & Walsh, 1997). Figure 2 shows the phenotypic response of 3 different variants of an environment-sensitive gene (called genotypes or alleles) in the presence of 2 different environments.

In Figure 2.a, the phenotypic response is enhanced in the presence of the 2nd environment. This response, however, is identical for the 3 genotypes (the curves are parallel). In contrast, in figures 2.b, c and d, the genetic variance is modified by environment '2', which is translated by an increase (Figure 2.b and c) or a decrease (Figure 2.d) in genetic variance between the 3 genotypes. In addition, the rank can be modified with the most sensitive genotype in one environment becoming the least sensitive in another environment (Figure 2.c and d). In all these 3 cases, the differential impact of the environment is allele-dependent. The increase in genetic variance observed with the environment '2' for figures 2.b and c is essential if one wants to unveil genetic susceptibilities.

interaction. The best example in the field of hypertension is salt-sensitivity where only saltsensitive individuals are displaying hypertension consecutive to a higher intake of salt in the

**Gd** 

**Ed** 

**HYPERTENSION**

**3.1 How to approach the genes X environment interactions – Concept of genetic** 

**Normal Blood Pressure** 

**HYPERTENSION** 

(called genotypes or alleles) in the presence of 2 different environments.

essential if one wants to unveil genetic susceptibilities.

Thus, while a genetic polymorphism could be associated with an increased risk of developing a disease, a differential response to the environment could define a susceptibility to this environment. This environmental susceptibility being modulated by environmentsensitive genes, we proposed the concept of *genetic susceptibility to the environment* (Hamet, 1996; Hamet et al., 1998; Pausova et al., 1999). The hallmark of a susceptibility gene is that it cannot be coined normal or abnormal since its expression *per se* will not cause the disease. Environment-sensitive variants of these genes will reveal their impact only when the organism is subjected to the specific triggering environment. We will now present the basis of the genes × environment interactions (adapted from Lynch & Walsh, 1997). Figure 2 shows the phenotypic response of 3 different variants of an environment-sensitive gene

**E x G (interaction)** 

**E + G (Additivity)** 

In Figure 2.a, the phenotypic response is enhanced in the presence of the 2nd environment. This response, however, is identical for the 3 genotypes (the curves are parallel). In contrast, in figures 2.b, c and d, the genetic variance is modified by environment '2', which is translated by an increase (Figure 2.b and c) or a decrease (Figure 2.d) in genetic variance between the 3 genotypes. In addition, the rank can be modified with the most sensitive genotype in one environment becoming the least sensitive in another environment (Figure 2.c and d). In all these 3 cases, the differential impact of the environment is allele-dependent. The increase in genetic variance observed with the environment '2' for figures 2.b and c is

diet (Weinberger, 1990).

**G** 

*a* 

*b* 

**E** 

**E** 

Fig. 1. Gene and environment in hypertension.

**Normal Blood Pressure**

**susceptibility to the environment** 

**G** 

**Gs** 

Fig. 2. Gene × environment interactions. Phenotypic expression of 3 different genotypes (alleles) when measured in 2 different environments (1, 2); adapted from Lynch & Walsh, 1997.

It is important at this point to distinguish the impact of the environment that needs to be assessed for its contribution to the disease from the environmental variance that is the variance due to the measurements and is represented by the error bars on the graphs. For instance, in Figure 2, the genetic variance varies depending on the environment whereas the environmental variance does not change. The ratio of the genetic variance over the total variance (genetic + environmental variance) is called 'heritability' and the higher the heritability, the higher the contribution of genes to the studied trait. Technically, this means that when searching for genetic determinants of a disease, one should work with traits displaying a high heritability. However, heritability is not static, especially when considering susceptibility genes. As can be seen from Figure 2, while the environmental variability does not change (error bars), the genetic variance can be modified depending on the environment. Therefore, the key to study the contribution of susceptibility genes is to find the conditions where the genetic variance is at its maximum (for instance, Figure 2.b, environment 2). Because such conditions are not necessarily seen *a priori* in an experimental set-up, it is essential to challenge the model by changing the environment in which the trait of interest is measured. In hypertension research, that can be translated by the use of different diets or drugs in order to blow-up the differences in blood pressure and find the genes responsible for the differential effect.

This understanding is fundamental for selecting the parameters that will allow appreciating the influence of the environment in an experimental setting. This is the area where, we think, radiotelemetry can be the most useful in hypertension research. We will demonstrate later how the currently accepted methods for measuring blood pressure in animal models

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

stress-sensitive target organ in susceptible hypertensive subjects.

**susceptibility to stress** 

Measurements in Animal Models of Hypertension, How It Revolutionized Hypertension Research 127

development of hypertension. The blood pressure response of two standardized tests correlated highly (0,8) with the blood pressure at work (Morales-Ballejo et al., 1988). These standardized tests were a math test and a timed competitive video game test where subjects had to increase their score after each successfully reached levels. The strong correlation with

In addition to changes in blood pressure and heart rate, Anderson and colleagues (1987) showed a significant increase in blood flow in the arm of children from hypertensive parents during a math test, suggestive of changes in vascular resistance. Interestingly, physical exercise (isometric hand contractions) and math test in hypertensive were both able to increase circulating catecholamines and vasopressin but only the math test induced a rise in plasma renin and aldosterone levels (Sakamoto at al., 1992). For the authors, the activation of the renin-angiotensin-aldosterone system by this psychogenic test made the kidney a

"real-life" blood pressure made these tests good surrogates for everyday life stressors.

**3.5 Enhanced stress response is predictive of hypertension – Concept of genetic** 

The hemodynamic response to stressors is highly heritable (Hassan et al., 2009) and may help distinguish susceptible individuals. Falkner et al. (1979) observed an enhanced response of hemodynamic parameters in normotensive teens from hypertensive parents following a mental stress test. This enhanced response could predict who would become hypertensive later in life (Falkner et al., 1981). Normotensive men with a positive family history of hypertension showed an increased diastolic blood pressure after isometric handgrip testing when compared to controls without a family history of hypertension (Widgren et al., 1992). Similarly, the microcirculation response to local heat stress and skin blood flow is altered in normotensive with family history of hypertension (Gryglewska et al., 2010). In a cohort of adults and their children followed for six years, this increased response to stress was the strongest in the subjects found to display the highest diastolic and systolic blood pressure six years later. This was true in the adult and the boys, but not the girls of the cohort (Matthews et al., 1993). Maybe this sexual dimorphism reflected the young age of the children (20 years) at the end of the study. Presumably, the girls would be protected whereas their mothers (mean age, 48) would not be anymore because of menopause. Thus, the response to a psychogenic stress could be used as a tool to predict a future hypertension status in normotensive children with a positive family history of hypertension. In this case, blood pressure *per se* is not important; it is blood pressure in response to a specific change of environment (stress test) that proved to be the strongest marker. It comes back to what we tried to demonstrate earlier: the right question must be asked and the right measurements performed. Here, the change in environment (stress test) allowed revealing the genetic susceptibility (increase of genetic variance). If the enhanced response to stress can produce a transient or sustained increase in blood pressure as we have seen, it can also impact on the development of other cardiovascular pathologies. For instance, the hypersensitivity of the sympathetic nervous system has been proposed as a 'switch' responsible for the future decrease of cardiac function (Middlekauff et al., 1997), with endogenous catecholamines stimulating left ventricular hypertrophy. This has been shown in normotensive children of hypertensive parents (Trimarco et al., 1985). Thus, the continuous stimulation of the sympathetic nervous system by various stressors over a certain threshold could induce a rise in blood pressure and permanent changes in the cardiovascular system resulting in an irreversible hypertensive state (Capone et al., 2011;

are not only adding artefacts and biases to the measurements, but how they are creating them because of their nature and the standard protocols employed.

#### **3.2 Stress as an important environmental modulator of hypertension**

We will now describe the genetic susceptibility to stress and its effects on blood pressure and hypertension development since it is among the most important environmental factor impacting hypertension (Hamet & Tremblay, 2002). The concept of stress was first defined and described by Hans Selye (1956). He called it "the general adaptation syndrome" or G.A.S. It is defined by 3 phases: the alarm reaction, the resistance period and the exhaustion stage. He demonstrated the role of the sympathetic nervous system with the stimulation of the hypothalamic-pituitary-adrenal axis and the surge of the stress hormones cortisol and cathecholamines. While he was proposing that different stressors would always induce the same response, Henry (1992) showed that the neuroendocrine response is dependent on the perceived stress. For instance, when the stress is bearable, the response will be characterized by a secretion of noradrenalin. In contrast, when the stress persists and is overwhelming, a depressive state will appear. It is characterized by the strong stimulation of the pituitaryadrenal axis with intense secretion of cortisol and high levels of ACTH while the cathecholamines are unchanged. This, in turn, induces a sustained renin secretion, promoting a gradual and steady increase in blood pressure. When this increase is chronic, it leads to hypertension.

#### **3.3 Psychosocial stress and cardiovascular diseases**

Alexander (1939) suggested a link between emotional stress and the development of hypertension. For instance, people exposed to an ever changing environment see their blood pressure rise gradually (Gutmann & Benson, 1971). The relationship between the stress of modern life and common diseases started to be more systematically assessed (Cobb & Rose, 1973) and the Health Examination Survey (HES) and Health Examination and Nutrition Survey (HANES) allowed evaluating the link between the perceived stress at work with infarct prevalence. In 4,833 men enrolled in these studies, the results showed that job strain and absence of latitude and decisional power was perceived as a psychological stress that was significantly associated with an increased prevalence of infarct in this cohort (Karasek et al., 1988). A similar study on 2,556 men showed that the job strain was associated with increased prevalence of hypertension and left ventricular hypertrophy, a risk factor for infarct and cardiovascular diseases (Schnall et al., 1990). Data from the Framingham cohort showed that anxiety was predictive of a future hypertensive state in middle aged men followed for 20 years. This relationship was not verified in women however (Markovitz et al., 1993). There was therefore a need to better define psychosocial stress and standardize research protocols to draw reliable and reproducible results. More recent studies reported similar findings related to job stress with interactions with blood pressure-modulating genes (Ohlin B et al, 2008; Yu et al., 2008).

#### **3.4 Psychogenic stress**

It is well-recognized that stressful events such as wartime, natural disasters or more commonly hard living conditions are associated to a rise in arterial pressure in sufferers. A need for a test mimicking this perceived stress was necessary to characterize and understand the possible causal relationship between psychogenic stressors (i.e. from psychological origin) and the

are not only adding artefacts and biases to the measurements, but how they are creating

We will now describe the genetic susceptibility to stress and its effects on blood pressure and hypertension development since it is among the most important environmental factor impacting hypertension (Hamet & Tremblay, 2002). The concept of stress was first defined and described by Hans Selye (1956). He called it "the general adaptation syndrome" or G.A.S. It is defined by 3 phases: the alarm reaction, the resistance period and the exhaustion stage. He demonstrated the role of the sympathetic nervous system with the stimulation of the hypothalamic-pituitary-adrenal axis and the surge of the stress hormones cortisol and cathecholamines. While he was proposing that different stressors would always induce the same response, Henry (1992) showed that the neuroendocrine response is dependent on the perceived stress. For instance, when the stress is bearable, the response will be characterized by a secretion of noradrenalin. In contrast, when the stress persists and is overwhelming, a depressive state will appear. It is characterized by the strong stimulation of the pituitaryadrenal axis with intense secretion of cortisol and high levels of ACTH while the cathecholamines are unchanged. This, in turn, induces a sustained renin secretion, promoting a gradual and steady increase in blood pressure. When this increase is chronic, it

Alexander (1939) suggested a link between emotional stress and the development of hypertension. For instance, people exposed to an ever changing environment see their blood pressure rise gradually (Gutmann & Benson, 1971). The relationship between the stress of modern life and common diseases started to be more systematically assessed (Cobb & Rose, 1973) and the Health Examination Survey (HES) and Health Examination and Nutrition Survey (HANES) allowed evaluating the link between the perceived stress at work with infarct prevalence. In 4,833 men enrolled in these studies, the results showed that job strain and absence of latitude and decisional power was perceived as a psychological stress that was significantly associated with an increased prevalence of infarct in this cohort (Karasek et al., 1988). A similar study on 2,556 men showed that the job strain was associated with increased prevalence of hypertension and left ventricular hypertrophy, a risk factor for infarct and cardiovascular diseases (Schnall et al., 1990). Data from the Framingham cohort showed that anxiety was predictive of a future hypertensive state in middle aged men followed for 20 years. This relationship was not verified in women however (Markovitz et al., 1993). There was therefore a need to better define psychosocial stress and standardize research protocols to draw reliable and reproducible results. More recent studies reported similar findings related to job stress with interactions with blood pressure-modulating genes

It is well-recognized that stressful events such as wartime, natural disasters or more commonly hard living conditions are associated to a rise in arterial pressure in sufferers. A need for a test mimicking this perceived stress was necessary to characterize and understand the possible causal relationship between psychogenic stressors (i.e. from psychological origin) and the

them because of their nature and the standard protocols employed.

**3.3 Psychosocial stress and cardiovascular diseases** 

leads to hypertension.

(Ohlin B et al, 2008; Yu et al., 2008).

**3.4 Psychogenic stress** 

**3.2 Stress as an important environmental modulator of hypertension** 

development of hypertension. The blood pressure response of two standardized tests correlated highly (0,8) with the blood pressure at work (Morales-Ballejo et al., 1988). These standardized tests were a math test and a timed competitive video game test where subjects had to increase their score after each successfully reached levels. The strong correlation with "real-life" blood pressure made these tests good surrogates for everyday life stressors.

In addition to changes in blood pressure and heart rate, Anderson and colleagues (1987) showed a significant increase in blood flow in the arm of children from hypertensive parents during a math test, suggestive of changes in vascular resistance. Interestingly, physical exercise (isometric hand contractions) and math test in hypertensive were both able to increase circulating catecholamines and vasopressin but only the math test induced a rise in plasma renin and aldosterone levels (Sakamoto at al., 1992). For the authors, the activation of the renin-angiotensin-aldosterone system by this psychogenic test made the kidney a stress-sensitive target organ in susceptible hypertensive subjects.

#### **3.5 Enhanced stress response is predictive of hypertension – Concept of genetic susceptibility to stress**

The hemodynamic response to stressors is highly heritable (Hassan et al., 2009) and may help distinguish susceptible individuals. Falkner et al. (1979) observed an enhanced response of hemodynamic parameters in normotensive teens from hypertensive parents following a mental stress test. This enhanced response could predict who would become hypertensive later in life (Falkner et al., 1981). Normotensive men with a positive family history of hypertension showed an increased diastolic blood pressure after isometric handgrip testing when compared to controls without a family history of hypertension (Widgren et al., 1992). Similarly, the microcirculation response to local heat stress and skin blood flow is altered in normotensive with family history of hypertension (Gryglewska et al., 2010). In a cohort of adults and their children followed for six years, this increased response to stress was the strongest in the subjects found to display the highest diastolic and systolic blood pressure six years later. This was true in the adult and the boys, but not the girls of the cohort (Matthews et al., 1993). Maybe this sexual dimorphism reflected the young age of the children (20 years) at the end of the study. Presumably, the girls would be protected whereas their mothers (mean age, 48) would not be anymore because of menopause. Thus, the response to a psychogenic stress could be used as a tool to predict a future hypertension status in normotensive children with a positive family history of hypertension. In this case, blood pressure *per se* is not important; it is blood pressure in response to a specific change of environment (stress test) that proved to be the strongest marker. It comes back to what we tried to demonstrate earlier: the right question must be asked and the right measurements performed. Here, the change in environment (stress test) allowed revealing the genetic susceptibility (increase of genetic variance). If the enhanced response to stress can produce a transient or sustained increase in blood pressure as we have seen, it can also impact on the development of other cardiovascular pathologies. For instance, the hypersensitivity of the sympathetic nervous system has been proposed as a 'switch' responsible for the future decrease of cardiac function (Middlekauff et al., 1997), with endogenous catecholamines stimulating left ventricular hypertrophy. This has been shown in normotensive children of hypertensive parents (Trimarco et al., 1985). Thus, the continuous stimulation of the sympathetic nervous system by various stressors over a certain threshold could induce a rise in blood pressure and permanent changes in the cardiovascular system resulting in an irreversible hypertensive state (Capone et al., 2011;

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

usage of the synteny between rat, human and mouse genomes.

**4.1 Animal models to study the stress response** 

be involved in the development of hypertension.

telemetric measurement of blood pressure could be a very useful tool.

Measurements in Animal Models of Hypertension, How It Revolutionized Hypertension Research 129

salt or it may go unnoticed and create bias in analysis and results inconsistencies. Other rat inbred strains were developed for the study of hypertension. We will not go into details as it is not the goal of this chapter, but we should not forget to mention the Dahl salt-sensitive and salt-resistant rat and the Milan hypertensive rat among the most popular models. For review, please see Bader (2010). With the advent of these models and the sequencing of the rat genome, discoveries in the rat can be directly verified in humans, and several quantitative trait loci mapped on the rat genome were also described in humans. The review of Stoll and Jacob (2001) about rat models of hypertension and their relationship to human hypertension is suggested as well as the review of Kwitek et al. (2001) for a description and

Several animal models can be used to study the stress response and the choice is dependent on the stressor to be employed and the expected outcomes. For instance, chronic immobilisation of the arm for a period of 15 months induced a gradual rise in blood pressure in monkeys (Kuneš et al., 1990). This hypertensive state persisted even when the animals were freed from that restraint and was observed in conscious or anaesthetized monkeys. For short-term and more ethically-acceptable studies, rats are the model of choice. Several stressors can induce hypertension: introduction of an intruder into the cage (Mitra et al., 2011), conflictual situation like food consumption followed by an electric shock (Friedman & Dahl, 1977), cold stress applied to the floor of the cage (Kanayama et al., 1979) and a psychogenic stress, immobilisation (Kvetnansky et al., 1970). Interestingly, in the SHR, immobilisation will induce a rise in blood pressure to levels higher than what is observed in normotensive WKY rats (Grundt et al., 2009; Yamori et al., 1969). McMurtry and Wexler (1981) have shown that ether, heat and immobilisation induce an increase of several biochemical markers of the stress response, with the SHR being more sensitive than the Sprague-Dawley control. SHR was then considered a model of 'neurogenic' hypertension similar to what is seen in humans (Folkow, 1982). Therefore, this hypersensitivity to stress found in SHR and also in hypertensive mice (Davern et al., 2010) may be involved in the development of hypertension by lowering the threshold at which a stimulus is perceived as a real stress. The higher stress response resulting can affect several pathways and organs and will contribute to the development of hypertension. Of note, the stress gene expression is enhanced in SHR as compared to WKY and Brown-Norway rats after a 1-hour immobilisation stress, and the genetic difference points to the heat-shock transcription factor *hstf* (Dumas et al., 2000a & 2000b). Others have reported differences in blood flow or increase in sympathetic nerve activity associated with sodium retention in SHR following stress (Yamamoto et al., 1987; Koepke & DiBona, 1985). All these genes and mechanisms can

In conclusion, hypertension research is performed mostly on the SHR rat, an animal that was bred for its spontaneous hypertension. We now know that the various colonies kept by vendors or institutions around the world do present some important genetic differences, several of them impacting blood pressure and hypertension development. Furthermore, SHR is a stress-sensitive model that mimics neurogenic hypertension. Therefore, because this hypersensitivity to stress and genetic differences pertaining to salt-sensitivity may impact the development of hypertension in SHR, it seems essential to study these environmental susceptibilities when studying hypertension in SHR or, to the least, be aware of these confounders in blood pressure measurements and data analysis. This is where

Thayer et al., 2010; Kuneš et al., 1990; Markel, 1985). Furthermore, genetic polymorphisms of tyrosine hydroxylase, the rate-limiting enzyme in catecholamines synthesis, are directly linked to the levels of the stress response (Rao et al., 2008). Thus, this neurogenic hypertension would result from the various environmental and genetic influences on the cardiovascular system. Therefore, the differential response to stress would define sensitive or resistant individuals. **Because it is based on sensitivity genes, we call it the genetic susceptibility to stress**.

In the previous paragraphs, we wanted to introduce this concept of *genetic susceptibility to stress* and insist on the fact that it is an essential component of hypertension development. Therefore, we think that it is not possible to study hypertension and the genetic basis of hypertension and ignore the impact of this environmental component. The rest of our review will demonstrate the essential role of telemetry in hypertension research in monitoring and dissecting this component, a goal that was impossible to achieve before the advent of telemetric measurement of blood pressure.

## **4. Animal models in hypertension research**

Animals were first studied to understand normal physiology, especially when necropsy on human beings was forbidden by the Church. Soon, it became evident that animals could suffer from most of the same ailments as humans. It then became natural to study diseases in animals in order to draw parallels with the human disease counterpart and test treatments. With the discovery of the laws of genetics by Mendel and their re-discovery at the beginning of the 20th century, the breeding of animals for selection of desired traits had now a scientific basis. At that time, mice were very popular as pets and breeders rivalled to produce strains with unique and "desirable" aesthetic characteristics. In order to perpetuate these characteristics in the next generations, brother-sister mating was performed over several generations. This gave rise to the first inbred lines, many of them still available today. The reader can refer to Beck et al. (2000) for a complete review. This massive breeding of mice by amateur breeders and scientists gave also rise to surprising discoveries: animals that were plagued with genetic ailments. Because the strains were inbred, the study of the disease was then possible on multiple animals displaying the same phenotype and over time. It was the beginning of modern biological science. Mice are very good genetic models of diseases, but rats were always the preferred model for physiology because of their "practical size". For organ size, blood drawing and any kind of measurements, rats provide more "research material", are bigger, sturdier, and, not the least, less aggressive than mice. This explains why the rat and mostly the Spontaneously Hypertensive Rat (SHR) is *the* animal model for hypertension research. The SHR was developed by Okamoto and Aoki (1963) by selective breeding of hypertensive males and females from their Wistar colony. The original inbred strain was then distributed to the NIH and institutes around the world. Because of this world-wide distribution, the SHR became the most popular animal model for the study of hypertension. The drawback of this popularity and wide distribution is that several substrains were generated since 1962, either by breeding errors or simply due to genetic drift. For instance, a vendor, Taconic Farms, has a salt-sensitive substrain of SHR (SHR-S) available from its IBU-3 colony (Mozaffari et al., 1991). This sole example simply to illustrate that, even within the same strain, there can be significant genetic differences impacting the studied phenotype. Furthermore, this example of salt-sensitivity illustrates an environmental susceptibility not usually present in SHR. As a result, researchers should be aware of the heterozygosity of the strain and be able to monitor the differential impact of salt or it may go unnoticed and create bias in analysis and results inconsistencies. Other rat inbred strains were developed for the study of hypertension. We will not go into details as it is not the goal of this chapter, but we should not forget to mention the Dahl salt-sensitive and salt-resistant rat and the Milan hypertensive rat among the most popular models. For review, please see Bader (2010). With the advent of these models and the sequencing of the rat genome, discoveries in the rat can be directly verified in humans, and several quantitative trait loci mapped on the rat genome were also described in humans. The review of Stoll and Jacob (2001) about rat models of hypertension and their relationship to human hypertension is suggested as well as the review of Kwitek et al. (2001) for a description and usage of the synteny between rat, human and mouse genomes.

#### **4.1 Animal models to study the stress response**

128 Modern Telemetry

Thayer et al., 2010; Kuneš et al., 1990; Markel, 1985). Furthermore, genetic polymorphisms of tyrosine hydroxylase, the rate-limiting enzyme in catecholamines synthesis, are directly linked to the levels of the stress response (Rao et al., 2008). Thus, this neurogenic hypertension would result from the various environmental and genetic influences on the cardiovascular system. Therefore, the differential response to stress would define sensitive or resistant individuals. **Because it is based on sensitivity genes, we call it the genetic** 

In the previous paragraphs, we wanted to introduce this concept of *genetic susceptibility to stress* and insist on the fact that it is an essential component of hypertension development. Therefore, we think that it is not possible to study hypertension and the genetic basis of hypertension and ignore the impact of this environmental component. The rest of our review will demonstrate the essential role of telemetry in hypertension research in monitoring and dissecting this component, a goal that was impossible to achieve before the

Animals were first studied to understand normal physiology, especially when necropsy on human beings was forbidden by the Church. Soon, it became evident that animals could suffer from most of the same ailments as humans. It then became natural to study diseases in animals in order to draw parallels with the human disease counterpart and test treatments. With the discovery of the laws of genetics by Mendel and their re-discovery at the beginning of the 20th century, the breeding of animals for selection of desired traits had now a scientific basis. At that time, mice were very popular as pets and breeders rivalled to produce strains with unique and "desirable" aesthetic characteristics. In order to perpetuate these characteristics in the next generations, brother-sister mating was performed over several generations. This gave rise to the first inbred lines, many of them still available today. The reader can refer to Beck et al. (2000) for a complete review. This massive breeding of mice by amateur breeders and scientists gave also rise to surprising discoveries: animals that were plagued with genetic ailments. Because the strains were inbred, the study of the disease was then possible on multiple animals displaying the same phenotype and over time. It was the beginning of modern biological science. Mice are very good genetic models of diseases, but rats were always the preferred model for physiology because of their "practical size". For organ size, blood drawing and any kind of measurements, rats provide more "research material", are bigger, sturdier, and, not the least, less aggressive than mice. This explains why the rat and mostly the Spontaneously Hypertensive Rat (SHR) is *the* animal model for hypertension research. The SHR was developed by Okamoto and Aoki (1963) by selective breeding of hypertensive males and females from their Wistar colony. The original inbred strain was then distributed to the NIH and institutes around the world. Because of this world-wide distribution, the SHR became the most popular animal model for the study of hypertension. The drawback of this popularity and wide distribution is that several substrains were generated since 1962, either by breeding errors or simply due to genetic drift. For instance, a vendor, Taconic Farms, has a salt-sensitive substrain of SHR (SHR-S) available from its IBU-3 colony (Mozaffari et al., 1991). This sole example simply to illustrate that, even within the same strain, there can be significant genetic differences impacting the studied phenotype. Furthermore, this example of salt-sensitivity illustrates an environmental susceptibility not usually present in SHR. As a result, researchers should be aware of the heterozygosity of the strain and be able to monitor the differential impact of

**susceptibility to stress**.

advent of telemetric measurement of blood pressure.

**4. Animal models in hypertension research** 

Several animal models can be used to study the stress response and the choice is dependent on the stressor to be employed and the expected outcomes. For instance, chronic immobilisation of the arm for a period of 15 months induced a gradual rise in blood pressure in monkeys (Kuneš et al., 1990). This hypertensive state persisted even when the animals were freed from that restraint and was observed in conscious or anaesthetized monkeys. For short-term and more ethically-acceptable studies, rats are the model of choice. Several stressors can induce hypertension: introduction of an intruder into the cage (Mitra et al., 2011), conflictual situation like food consumption followed by an electric shock (Friedman & Dahl, 1977), cold stress applied to the floor of the cage (Kanayama et al., 1979) and a psychogenic stress, immobilisation (Kvetnansky et al., 1970). Interestingly, in the SHR, immobilisation will induce a rise in blood pressure to levels higher than what is observed in normotensive WKY rats (Grundt et al., 2009; Yamori et al., 1969). McMurtry and Wexler (1981) have shown that ether, heat and immobilisation induce an increase of several biochemical markers of the stress response, with the SHR being more sensitive than the Sprague-Dawley control. SHR was then considered a model of 'neurogenic' hypertension similar to what is seen in humans (Folkow, 1982). Therefore, this hypersensitivity to stress found in SHR and also in hypertensive mice (Davern et al., 2010) may be involved in the development of hypertension by lowering the threshold at which a stimulus is perceived as a real stress. The higher stress response resulting can affect several pathways and organs and will contribute to the development of hypertension. Of note, the stress gene expression is enhanced in SHR as compared to WKY and Brown-Norway rats after a 1-hour immobilisation stress, and the genetic difference points to the heat-shock transcription factor *hstf* (Dumas et al., 2000a & 2000b). Others have reported differences in blood flow or increase in sympathetic nerve activity associated with sodium retention in SHR following stress (Yamamoto et al., 1987; Koepke & DiBona, 1985). All these genes and mechanisms can be involved in the development of hypertension.

In conclusion, hypertension research is performed mostly on the SHR rat, an animal that was bred for its spontaneous hypertension. We now know that the various colonies kept by vendors or institutions around the world do present some important genetic differences, several of them impacting blood pressure and hypertension development. Furthermore, SHR is a stress-sensitive model that mimics neurogenic hypertension. Therefore, because this hypersensitivity to stress and genetic differences pertaining to salt-sensitivity may impact the development of hypertension in SHR, it seems essential to study these environmental susceptibilities when studying hypertension in SHR or, to the least, be aware of these confounders in blood pressure measurements and data analysis. This is where telemetric measurement of blood pressure could be a very useful tool.

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

tail-cuff method, please refer to Buñag (1983).

variables

animals (adapted from Kurtz et al., 2005)

**response and in body temperature** 

 Measuring blood pressure variability Measuring diastolic or pulse pressure

acting substances

**Not recommended for:**

**Recommended for non-invasive detection or screening of:**

Frank systolic hypertension

Assessing small differences in blood pressure

 Measuring blood pressure in conscious rodents Measuring blood pressure in stress-sensitive animals

Substantial group differences in systolic blood pressure

Substantial changes in systolic blood pressure over time

 High-throughput screening of large numbers of animals when large differences in blood pressure are expected

Quantifying relationships between blood pressure and other

Studying blood pressure-independent effects of any intervention

Monitoring possible hemodynamic effects from rapid and short-

Table 2. Recommendations for the use of indirect methods for measuring blood pressure in

**5.1.1 Effect of restraint on blood pressure determination: Increase in cardiovascular** 

In order to be able to install the cuff and record blood pressure, mice and rats are put in restrainers with the tail hanging out. As we have shown at paragraph 4.2, immobilisation

Measurements in Animal Models of Hypertension, How It Revolutionized Hypertension Research 131

which a cuff could be wrapped (limbs can be employed for bigger animals such as dogs), hence the name «tail-cuff method». In a mouse, the tail is irrigated by one ventral artery and 2 lateral caudal veins. The rats possess the same vascular architecture with the addition of a dorsal vein. Therefore, the cuff squeezes the tail to a pressure greater than systolic pressure, and a sensor distal to the cuff is placed to detect the return of the blood flow as the cuff is deflated. Several sensors are available on the market, with some more sensitive than others. In all cases however, only the systolic blood pressure can be perceived with this method. Furthermore, because the only tail artery is on the ventral side, positioning of the sensor is critical to get a good signal. The strength of the signal is also dependent on the position of the cuff and sensor on the tail: the closest to the animal (proximal), the better the signal. Therefore, one can imagine that the size of the cuff must be adapted to the size of the tail, and the position of the cuff must be standardized, at least within the same experiment. To our knowledge, these precautions are rarely mentioned as most authors will only say *"blood pressure was performed as previously mentioned"* without further details. But these are the least of the culprits that are impacting the measurement with the tail-cuff method. In the light of what we have presented, we will put the emphasis on a major issue responsible for most problems and artefacts: the mandatory use of restraint cages in order to be able to perform this fine tuning on conscious animals. For a detailed depiction of all the other pitfalls of the

#### **5. Blood pressure measurement in mouse and rat**

An essential requirement for hypertension research in animal models is the ability to reliably and accurately monitor blood pressure and its slight variations in unanaesthetized conscious animals. The choice of a research methodology and the techniques to be used will be dependent on the specific research goals. Thus, a given method for measuring blood pressure may be well suited for one type of study and not recommended or useful for another. Furthermore, the more expensive and technologically-advanced technique is not necessary the best to employ. For instance, if there is a need to monitor rapid changes in blood pressure consecutive to the intravenous injection of a test compound, telemetry is most suitable because it can record almost instantly the changes in blood pressure. This is particularly true for fast and short-acting substances. Inversely, if one wants to study the changes in target organs consecutive to a long-term treatment with a new vasoactive drug, a sole determination close to the end of the treatment with a non-invasive and 'coarse' method such as tail-cuff should be enough to assess if the test article behaved as expected in the treated animals. We will now review the methods for blood pressure measurement in rodents with an emphasis on the most commonly used technique, the indirect tail-cuff method, and the state-of the art non-invasive telemetric method. Again, the interested readers will be directed to the review by Kurtz et al. (2005), Zhao et al. (2011) or Feng & DiPetrillo (2009) for details and information on other methods.

Ruban D. Buñag started his 1983 review of blood pressure measurement techniques in rats by «A rat's tail is a slender appendage on which the weight of so much research in hypertension hangs, yet blood pressure measurements recorded from it are usually taken for granted, often abused, but seldom discussed» (Buñag RD, 1983). This is the commentary of someone who had first-hand experience of the available techniques for blood pressure determination in animal models of hypertension. At that time, the only available techniques were direct puncture of an artery under light anaesthesia (usually the carotid artery) or the indirect tail-cuff method.

#### **5.1 Indirect method in conscious rodents: The tail-cuff method (Williams et al., 1939)**

Often cited as «the tail-cuff method» without further details, this method is an adaptation of the indirect method used in humans. In humans, physicians wrap a cuff around the arm of the patient. With the stethoscope, they can hear the heartbeats while deflating the cuff (Korotkoff sounds). When done correctly, the beats are audible only when the cuff pressure is smaller than the systolic pressure or higher than the diastolic pressure. Thus, these limits represent respectively the systolic and diastolic values of blood pressure of the individual. In order to achieve the measurement, one needs an artery (here, the brachial artery), a cuff to squeeze the artery and interrupt the blood flow and a device to perceive and monitor the blood flow during inflation and deflation (the stethoscope). This key technique to many diagnostic procedures is performed everyday by physicians in their offices and in the hospitals around the world. Its ease of use and the knowledge it brings to assess the condition of the patients makes it probably one of the most important techniques available to physician, even in the era of computers and scanners. The ease of use of the indirect blood pressure determination in humans comes mostly from the fact that the blood pressure determination is performed in conscious subjects and uses a non-invasive procedure on the arm.

How can it be adapted to rodent models of hypertension? In a straightforward adaptation in the animals, researchers considered using the tail in rats and mice as the «organ» around which a cuff could be wrapped (limbs can be employed for bigger animals such as dogs), hence the name «tail-cuff method». In a mouse, the tail is irrigated by one ventral artery and 2 lateral caudal veins. The rats possess the same vascular architecture with the addition of a dorsal vein. Therefore, the cuff squeezes the tail to a pressure greater than systolic pressure, and a sensor distal to the cuff is placed to detect the return of the blood flow as the cuff is deflated. Several sensors are available on the market, with some more sensitive than others. In all cases however, only the systolic blood pressure can be perceived with this method. Furthermore, because the only tail artery is on the ventral side, positioning of the sensor is critical to get a good signal. The strength of the signal is also dependent on the position of the cuff and sensor on the tail: the closest to the animal (proximal), the better the signal. Therefore, one can imagine that the size of the cuff must be adapted to the size of the tail, and the position of the cuff must be standardized, at least within the same experiment. To our knowledge, these precautions are rarely mentioned as most authors will only say *"blood pressure was performed as previously mentioned"* without further details. But these are the least of the culprits that are impacting the measurement with the tail-cuff method. In the light of what we have presented, we will put the emphasis on a major issue responsible for most problems and artefacts: the mandatory use of restraint cages in order to be able to perform this fine tuning on conscious animals. For a detailed depiction of all the other pitfalls of the tail-cuff method, please refer to Buñag (1983).

#### **Recommended for non-invasive detection or screening of:**

Frank systolic hypertension Substantial group differences in systolic blood pressure Substantial changes in systolic blood pressure over time High-throughput screening of large numbers of animals when large differences in blood pressure are expected

#### **Not recommended for:**

130 Modern Telemetry

An essential requirement for hypertension research in animal models is the ability to reliably and accurately monitor blood pressure and its slight variations in unanaesthetized conscious animals. The choice of a research methodology and the techniques to be used will be dependent on the specific research goals. Thus, a given method for measuring blood pressure may be well suited for one type of study and not recommended or useful for another. Furthermore, the more expensive and technologically-advanced technique is not necessary the best to employ. For instance, if there is a need to monitor rapid changes in blood pressure consecutive to the intravenous injection of a test compound, telemetry is most suitable because it can record almost instantly the changes in blood pressure. This is particularly true for fast and short-acting substances. Inversely, if one wants to study the changes in target organs consecutive to a long-term treatment with a new vasoactive drug, a sole determination close to the end of the treatment with a non-invasive and 'coarse' method such as tail-cuff should be enough to assess if the test article behaved as expected in the treated animals. We will now review the methods for blood pressure measurement in rodents with an emphasis on the most commonly used technique, the indirect tail-cuff method, and the state-of the art non-invasive telemetric method. Again, the interested readers will be directed to the review by Kurtz et al. (2005), Zhao et al. (2011) or Feng &

Ruban D. Buñag started his 1983 review of blood pressure measurement techniques in rats by «A rat's tail is a slender appendage on which the weight of so much research in hypertension hangs, yet blood pressure measurements recorded from it are usually taken for granted, often abused, but seldom discussed» (Buñag RD, 1983). This is the commentary of someone who had first-hand experience of the available techniques for blood pressure determination in animal models of hypertension. At that time, the only available techniques were direct puncture of an artery under light anaesthesia (usually the carotid artery) or the

**5.1 Indirect method in conscious rodents: The tail-cuff method (Williams et al., 1939)**  Often cited as «the tail-cuff method» without further details, this method is an adaptation of the indirect method used in humans. In humans, physicians wrap a cuff around the arm of the patient. With the stethoscope, they can hear the heartbeats while deflating the cuff (Korotkoff sounds). When done correctly, the beats are audible only when the cuff pressure is smaller than the systolic pressure or higher than the diastolic pressure. Thus, these limits represent respectively the systolic and diastolic values of blood pressure of the individual. In order to achieve the measurement, one needs an artery (here, the brachial artery), a cuff to squeeze the artery and interrupt the blood flow and a device to perceive and monitor the blood flow during inflation and deflation (the stethoscope). This key technique to many diagnostic procedures is performed everyday by physicians in their offices and in the hospitals around the world. Its ease of use and the knowledge it brings to assess the condition of the patients makes it probably one of the most important techniques available to physician, even in the era of computers and scanners. The ease of use of the indirect blood pressure determination in humans comes mostly from the fact that the blood pressure determination is performed in

How can it be adapted to rodent models of hypertension? In a straightforward adaptation in the animals, researchers considered using the tail in rats and mice as the «organ» around

**5. Blood pressure measurement in mouse and rat** 

DiPetrillo (2009) for details and information on other methods.

conscious subjects and uses a non-invasive procedure on the arm.

indirect tail-cuff method.

 Assessing small differences in blood pressure Quantifying relationships between blood pressure and other variables Studying blood pressure-independent effects of any intervention Measuring blood pressure variability Measuring diastolic or pulse pressure Measuring blood pressure in conscious rodents Measuring blood pressure in stress-sensitive animals Monitoring possible hemodynamic effects from rapid and shortacting substances

Table 2. Recommendations for the use of indirect methods for measuring blood pressure in animals (adapted from Kurtz et al., 2005)

#### **5.1.1 Effect of restraint on blood pressure determination: Increase in cardiovascular response and in body temperature**

In order to be able to install the cuff and record blood pressure, mice and rats are put in restrainers with the tail hanging out. As we have shown at paragraph 4.2, immobilisation

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

reversed upon the interruption of this pre-conditioning (Malo et al., 1990).

manifestation must be favoured.

**5.1.5 Conditioning to restraint stress** 

as compared to the effects of the tested hypothesis.

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

**5.1.4 Seasonal temperature and the development of cardiovascular diseases** 

Measurements in Animal Models of Hypertension, How It Revolutionized Hypertension Research 133

indicative of a defect in temperature regulation in this strain. When subjected to a 5-min conditioning at a non-lethal 40°C temperature for 20 consecutive days, SHM mice exhibited a 20 mm Hg lower blood pressure as compared to non-treated SHM, and this effect was

It is interesting to note that seasonal temperature itself has an impact on mortality and incidence of cardiovascular diseases. For instance, it is known for a long time that there is an inverse relationship between outside average temperature and blood pressure levels (Rose, 1961; Hata et al., 1982). In Montreal (Canada), where outside temperature varies from -24°C to 27 °C on a yearly average, a study on 2,000 patients with a total of 42,813 blood pressure readings showed a significant inverse relationship between blood pressure and average outside temperature (Kuneš et al., 1991). More recently, a 1°C reduction in daily mean temperature was associated with a 2% cumulative increase in risk of myocardial infarction over the current and following month, with the strongest effects observed after lags of 1 and 2 weeks (Bhaskaran et al., 2010). In many countries, death rates in winter are 10-25% higher as compared to the rest of the year (Curwen, 1991). When corrected for confounders, only temperature showed a constant correlation strongly suggestive of causality. Therefore, stressors that are impacting body temperature should not be seen merely as a side effect of immobilisation. We do think that these must be taken into account when measuring blood pressure and techniques for blood pressure determination that are minimizing stress and its

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

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

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

conditioning was previously reported for rats (Bazil et al., 1993; Irvine et al., 1997).

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 important confounder.

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

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

#### **5.1.3 Thermosensitivity and hypertension**

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 indicative of a defect in temperature regulation in this strain. When subjected to a 5-min conditioning at a non-lethal 40°C temperature for 20 consecutive days, SHM mice exhibited a 20 mm Hg lower blood pressure as compared to non-treated SHM, and this effect was reversed upon the interruption of this pre-conditioning (Malo et al., 1990).
