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The influence of trait anxiety on the elevation of arterial pressure induced by l -NAME in rats
Neuroscience Letters 583 (2014) 11–15
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
The influence of trait anxiety on the elevation of arterial pressure induced by l-NAME in rats Flávia Barreto Garcez, Fábio Ursulino Reis Carvalho, Ana Paula dos Santos Soares, Tiago Costa Goes, Márcio Roberto Viana dos Santos, Flavia Teixeira-Silva ∗ Departamento de Fisiologia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Sergipe, 49100-000 São Cristóvão, SE, Brazil
h i g h l i g h t s • • • •
The relationship between anxiety and elevated blood pressure was investigated in rats. l-NAME (20 mg/kg/day – 7 days) increases blood pressure only in high trait anxiety rats. Blood pressure elevation (short term) does not affect trait anxiety. The higher the trait anxiety, the higher the blood pressure peak in response to l-NAME.
a r t i c l e
i n f o
Article history: Received 17 April 2014 Received in revised form 5 August 2014 Accepted 2 September 2014 Available online 16 September 2014 Keywords: Trait anxiety Hypertension l-NAME Rats Free-exploratory paradigm Blood pressure
a b s t r a c t Due to the high prevalence of anxiety disorders and hypertension comorbidity in the general population, the establishment of anxiety as a risk factor for elevated blood pressure, or the reverse, is of great relevance. In this context, animal models can be of great scientific value, as they permit the control of several variables. Bearing this in mind, the influence of anxiety, not as a state, but as a personality trait (trait anxiety), on blood pressure elevation and vice versa were investigated for the first time in rats, using the free-exploratory paradigm (FEP). Sixty adult male Wistar rats were evaluated on FEP and categorized according to their levels of anxiety. From this sample, 24 animals with high (n = 12) and low (n = 12) trait anxiety were allocated to two treatment groups: (1) l-NAME (NG -nitro-l-arginine methyl ester, 20 mg/kg, p.o., for 7 days to increase blood pressure; n = 6/anxiety category); (2) CTRL (tap water, p.o., for 7 days; n = 6/anxiety category). During treatment, measurements of systolic blood pressure (SBP) were taken daily. After treatment, the animals were again tested on FEP. SBP and trait anxiety levels were compared pre- and post-treatment. Additionally, correlations between trait anxiety levels and SBP increases (l-NAME group) were analyzed. The results showed that l-NAME was able to induce significant SBP elevation, but only for the high-anxious animals, while SBP elevation did not significantly interfere with anxiety levels. A significant correlation between anxiety levels and SBP peaks in response to l-NAME was also shown. No differences were observed between the levels of anxiety before and after treatment. These findings suggest that individuals with high trait anxiety are more susceptible to increases in blood pressure, but that high blood pressure does not affect the levels of trait anxiety. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
∗ Corresponding author at: Departamento de Fisiologia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Sergipe, Campus São Cristóvão 49100-000, SE, Brazil. Tel.: +55 79 2105 6645; fax: +55 79 2105 6414. E-mail addresses: fl[email protected] (F.B. Garcez), fabioursu [email protected] (F.U.R. Carvalho), [email protected] (A.P.d.S. Soares), [email protected] (T.C. Goes), [email protected] (M.R.V. dos Santos), teixeira [email protected], prof-fl[email protected] (F. Teixeira-Silva). http://dx.doi.org/10.1016/j.neulet.2014.09.011 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Several studies have shown that anxious people have a higher risk of developing hypertension [1–4].However, the nature of the association between anxiety and blood pressure is not yet understood, in part because only a few of these studies have actually used methods which are capable of establishing a causal relationship. In this context, animal models can be of great scientific value, as they permit the control of several variables, such as genetic, environmental and nutritional factors, as well as allowing the study of the interaction among these variables. This kind of approach is
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fundamental, since both anxiety and hypertension are multifactor conditions. Despite this, studies involving animals on this subject are scarce, and the existing ones either investigate the relationship between hypertension and state anxiety (anxiety a subject experiences at a particular moment in time, when facing threat) [5], taking no consideration of the anxious trait of the individuals; or do not aim to reveal the causal relationship between the conditions, only investigating their common physiological or pharmacological characteristics [6,7]. Bearing in mind the high prevalence of anxiety disorders, hypertension and their comorbidity in the general population [8], the establishment of anxiety as a risk factor for elevated blood pressure or vice versa is of great relevance, certainly having an impact on the prevention of theses illnesses and on the choice among all the possible therapies that can be applied to anxious and/or hypertensive patients. Taking all this into consideration, the aim of the present study was to evaluate, in rats, the relationship between trait anxiety (enduring feature of an individual, relatively stable over time) and elevated blood pressure. To achieve this, animals were tested in the free-exploratory paradigm (FEP) and had their blood pressure monitored, before and after treatment with l-NAME (NG -nitro-larginine methyl ester). FEP is, to the best of our knowledge, the only test proposed as a model of trait anxiety. In this situation, in which there is no change in state anxiety, animals are given the opportunity to move around freely within an environment containing both familiar and novel parts. This approach allows the evaluation of neophobic responses. As the animals have a choice between novelty and familiarity, it is expected that individuals with low trait anxiety would exhibit a preference for novelty, whereas high trait anxiety subjects would prefer familiarity [9–12]. l-NAME inhibits the biosynthesis of nitric oxide (NO), which increases cyclic GMP and cause vasorelaxation on the microcirculation [13,14]. Therefore, chronic treatment with l-NAME results in hypertension in rats [13,15] and, for this reason, was used in the present study.
2. Animals, materials and methods 2.1. Animals Sixty (2–3 months) male Wistar rats, from our own colony, were used. The animals were kept five per cage (41 cm × 34 cm × 18 cm), in a temperature (22–24 ◦ C) and light (12 h/12 h light/dark cycle, lights on at 06:00 a.m.) controlled room, with water and food ad libitum. All procedures were in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC), as well as the Brazilian National Council on the Control of Animal Experimentation (Conselho Nacional de Controle de Experimentac¸ão Animal – CONCEA, Brazil) and were conducted with approval from the Ethics Committee in Research with Animals of Universidade Federal de Sergipe, Brazil.
2.3. Blood pressure elevation Blood pressure elevation was induced by nitric oxide synthase inhibition with NG -nitro-l-arginine methyl ester (l-NAME, Sigma, USA) [13,15]. In comparison to the original l-NAME-induced hypertension protocol, a lower dose of l-NAME (20 mg/kg for 7 days) was chosen, as it has been shown that lower doses are able to elevate blood pressure without causing lesions in target organs as do high doses or long term administration of l-NAME [15,17]. As a result, it is possible to get a sample of “purely” hypertensive subjects. The systolic blood pressure (SBP) of rats was measured using a non-invasive tail-cuff method [18]. Data were obtained with a data-acquisition system (LE5002, Letica, Spain). Measurements were made once a day for 8 days, starting 1 day before the 7-day treatment period. In order to reduce external interferences to the measurements, they were all performed by the same researcher and around the same time of day (07:00 p.m.). Furthermore, to facilitate data acquisition, before every measurement session, each animal was individually placed in a heated chamber (40 ◦ C) for 10 min. Warming the animal up comfortably is necessary for most non-invasive blood pressure systems, in order to enhance blood flow to the tail and consequently produce a blood pressure signal [19]. Although it has been demonstrated that SBP measurements obtained by the heated-animal tail-cuff method are higher than those obtained by non-heated-animal methods [20], the method chosen here produces consistent results [19] and therefore it should not interfere with comparative results, when all the animals go through the same procedure. 2.4. Procedure The animals were first tested on FEP. The obtained results were used to classify them according to the %TNS, as presenting high (%TNS < 51), medium (51 ≤ %TNS ≤ 80) or low (%TNS > 80) levels of anxiety, according to a previous categorization performed in our lab [21]. A week later, rats with high (n = 12) and low anxiety (n = 12) were submitted to 5 days of adaptation to the SBP measuring apparatus, described above. After this adaptation period, the pre-treatment SBP was assessed, 1 day before the beginning of the treatment. Subsequently, the animals were administered either l-NAME (20 mg/kg/day; n = 6/anxiety category), dissolved in their drinking water, or just tap water for 7 days (n = 6/anxiety category). In order to assure that the animals had the target intake of l-NAME, one week before the treatment began, the water ingestion for each home cage was monitored every day, so an average consumption for each rat, per day, was determined. This information and measured body weights were used to calculate the concentration of l-NAME in the drinking water, which was prepared daily and offered to the animals in two bottles per cage, in order to guarantee that no animals would be impeded from drinking by an alpha rat. During this treatment period, SBP was measured daily. On each of the eight SBP evaluations, five consecutive SBP measurements were taken, in order to obtain a mean value. All records started only after heart rate stabilization, in order to minimize the effects of initial stress due to animal handling. After this 7-day period, the animals were again tested on FEP.
2.2. Animal model of trait anxiety 2.5. Statistical analysis FEP was automated by a computerized system for animal tracking (Anymaze® , Stoelting Co., USA) and set up as described by Antunes et al. [16]. The following parameters were measured: total distance travelled (TDT), and the time spent in each compartment, from which the percentage of time in the novel side (%TNS) was calculated.
The obtained data were first analyzed using Kolmogorov–Smirnov’s test for normal distribution and Bartlett’s test for the homogeneity of variances. No impediments to the use of parametric tests were found. For the analysis of SBP data, pre-treatment (SBPpre ) and post-treatment (SBPpost ) evaluations were compared. In order to
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minimize the effects of compensatory mechanisms that could mask the results if only the last day of treatment was considered, SBPpost corresponded to the mean values of all 7 days of treatment. Due to the small sample size and to the impossibility of testing animals belonging to all the different treatment and anxiety groups on each day of evaluation, the data were then analyzed using a two-factor analysis of variance (ANOVA) for repeated measures (factor 1: treatment group; factor 2: time) for each anxiety level, in replacement to a three-factor ANOVA. When the interaction between factors was significant, the analyses were followed by one-way, repeated measures ANOVA, conducted on the time variable for each treatment group. A correlational study was also performed, using Pearson’s correlation coefficient. This way, %TNS from the first FEP evaluation was confronted with the mean percent variation in SBP [SBP = (SBPpost − SBPpre )/SBPpre × 100] and with the highest percent variation in SBP, achieved during treatment (MaxSBP), for all the animals (high and low trait anxiety) receiving l-NAME. All significance tests were two-tailed and were performed at the 5% significance level. 3. Results The means and standard deviations of the absolute values of all evaluated parameters are presented in Table 1. 3.1. SBP evaluation (Fig. 1)
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changes induced by the treatment, according to this formula: SBP = [(SBPpost − SBPpre )/SBPpre ] × 100. For the high trait anxiety animals, the interaction between treatment group and time was significant (F(1,10) = 11.703; p = 0.007). Analysis of time as a single factor for the each treatment group revealed a significantly higher SBP for the l-NAME group (F(1,5) = 79.498; p = 0.001), but not for the CTRL group (F(1,5) = 63.021; p = 0.468). For the low trait anxiety animals, the interaction between treatment group and time was not significant (F(1,10) = 3.695; p = 0.084), therefore the two main effects were analyzed. Neither time (F(1,10) = 1.667; p = 0.226) or treatment (F(1,10) = 3.600; p = 0.087) changed SBP significantly. 3.2. Trait-anxiety evaluation (Table 1) For the high trait anxiety animals, the interaction between treatment group and time was not significant (F(1,10) = 0.035; p = 0.856), therefore the two main effects were analyzed. The time effect was significant (F(1,10) = 11.468; p = 0.007), increasing %TNS. The treatment group effect was not significant (F(1,10) = 0.360; p = 0.562). For the low trait anxiety animals, the interaction between treatment group and time was not significant (F(1,10) = 2.945; p = 0.117), therefore the two main effects were analyzed. Neither time (F(1,10) = 3.795; p = 0.080) or treatment (F(1,10) = 1.449; p = 0.256) changed %TNS significantly. 3.3. %TNS × SBP correlation
To aid visualization of these results, the graphs shown represent the profile of the responses presented by the animals, with the pre-treatment being considered as the “zero” point for the
Pearson’s R test revealed a lack of significant correlation (r = −0.476, p = 0.118) between %TNS and SBP.
Table 1 Data obtained on the free-exploratory paradigm and absolute values of systolic blood pressure, pre- and post-treatment. Anxiety
Treatment
%TNSpre
%TNSpost
High
l-NAME CTRL l-NAME CTRL
36.16 ± 18.41 27.35 ± 29.40 84.33 ± 3.57 83.81 ± 4.13
55.93 ± 33.97 45.06 ± 35.19 66.72 ± 21.73 82.70 ± 16.42
Low
*
SBPpre
SBPpost
127.44 ± 2.95 125.33 ± 18.75 130.73 ± 8.20 129.56 ± 15.22
143.76 ± 5.73* 120.75 ± 15.41 141.93 ± 4.91 127.36 ± 5.39
Data are presented by mean ± SD. l-NAME: NG -nitro-l-arginine methyl ester; CTRL: control; %TNS: percentage of time in the novel side; SBP: systolic blood pressure; Pre: pre-treatment; Post: post-treatment. * Significantly different from pre-treatment (p < 0.05).
High Anxiety SBP (Difference from Pre-treatment)
14%
Low Anxiety
*
12% 10% 8% 6% 4%
L-NAME
2%
CTRL
0% -2% -4% -6% Pre-treatment
Post-treatment
Pre-treatment
Post-treatment
Fig. 1. Profile of systolic blood pressure (SBP) of high-anxious (left panel) and low-anxious (right panel) animals, in response to pharmacological treatment. Data are presented as mean ± SD. l-NAME: NG -nitro-l-arginine methyl ester; CTRL: control. *Significantly different from pre-treatment (p = 0.001).
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3.4. %TNS × MaxSBP Pearson’s R test revealed a significant negative correlation (r = −0.605, p = 0.037) between %TNS and MaxSBP. Here it is worth remembering that the greater the %TNS, the lower the anxiety.
4. Discussion The present study aimed to investigate the effect of trait anxiety on SBP elevation, as well as the influence of elevated SBP on anxious trait. The obtained results showed that: (1) l-NAME, in the administered dose, was able to induce significant SBP elevation, but only for the high trait anxiety animals; and (2) SBP elevation did not significantly interfere with trait anxiety levels. To the best of our knowledge, this is the first animal study performed to explore the relationship between trait anxiety and hypertension. The data presented here suggest that individuals with high levels of trait anxiety are more susceptible to the development of hypertension, since high-anxious trait rats presented a significant SBP elevation, in response to the administration of 20 mg/kg/day of l-NAME for 7 days, which did not occur for lowanxious trait rats submitted to the same protocol. On the other hand, elevated SBP does not seem to influence trait anxiety, as the animals did not significantly change their performance on FEP after the treatment with l-NAME, that is to say individuals do not tend to see the world as more threatening because they have elevated blood pressure. This specific result must be interpreted with care, though, as SBP was only elevated for a short period of time. The possible influence of long term hypertension on trait anxiety cannot yet be discarded. It is worth commenting here on the fact that high trait anxiety animals, either treated with l-NAME or water, presented an increase in %TNS on the second exposition to FEP. This apparent anxiety reduction has been reported before and was probably due to the animals having had a previous “out of cage experience” [12], to which low trait anxiety animals seem to be less sensitive. Maybe the most interesting result was provided by the correlational study, which showed that the higher the trait anxiety, the higher the blood pressure peak in response to l-NAME treatment. Naturally, it could be argued that statistical correlation tests provide information on association rather than a cause-and-effect relationship between variables, so it is possible that high trait anxiety leads to SBP peaks, but it is also possible that a third uninvestigated variable is causing both effects. At least, due to our experimental design, the possibility that the increase in blood pressure would be the cause of high trait anxiety can be ruled out. In any case, this result suggests that high-anxious subjects are more prone to present peaks in blood pressure, and this is particularly important because it is the peaks, not mean blood pressure, that are most closely correlated with stroke and intracranial bleeding risk [22]. In general, all these results are congruent with previous clinical studies that investigated the connection of emotional factors, such as anxiety, to initiation and progression of hypertension [3,23,24]. Such studies showed that anxiety, as well as other psychosocial conditions, could be associated with the development of hypertension. However, these findings are somewhat vulnerable as, in clinical studies, it is difficult to control for genetic and/or environmental factors, and it is often impossible to be sure that the blood pressure elevation followed, rather than preceded, a supposed psychological risk factor [25]. In this context, the present work, as a preclinical study, contributes a lot to the understanding of the complex relationship between anxiety and blood pressure. After all, the use of animal models presents important advantages in comparison to clinical
studies, such as better control of environmental factors, a smaller number of confounding variables and quicker completion [26]. Furthermore, it was possible to explore the temporal relationship between the two studied conditions; i.e., it was possible to know the levels of trait anxiety of the subjects before and after the increase in blood pressure, which allowed the investigation of the influence of trait anxiety on blood pressure elevation and vice versa. As a result, there is now better support for anxiety as a risk factor to hypertension, but no more support for the contrary. Several mechanisms to explain the possible influence of psychological factors on the regulation of blood pressure have been proposed: (1) cardiovascular reactivity to stress, which would lead to a hyperactivity of the sympathetic nervous system, keeping the blood pressure levels increased over time [27]; (2) neurohormonal models, with speculations about alterations in neurotransmitters and impaired baroreflex control [28]; and (3) tendency of psychiatric patients to present predisposing behaviours to hypertension, such as poor diet, obesity, sedentarism, smoking and alcohol abuse [29]. Investigating the physiopathological mechanisms relating anxiety to hypertension was never the aim of the present work. However, some previous animal studies have demonstrated an autonomic dysfunction in high-anxious trait animals [25], in which there could be sympathetic dominance, reduced vagal activity and baroreceptor alterations [30–33]. Such increased sympathetic activity seems to occur independently of environmental stressors, which affect the anxious state, being inherent to anxious individuals [28,34]. Enhancement of sympathetic modulation has been shown to induce endothelial dysfunction [35], leading, over time, to decreased brachial artery flow-mediated dilation, which is in fact associated to high trait anxiety in elderly people [36]. Considering all this, and the fact that one of the hallmarks of a dysfunctional endothelium is diminished levels of bioavailable NO [37], it is tempting to speculate that enhanced sympathetic activity is responsible for the increased susceptibility of high-anxious trait animals to SBP elevation, as observed here. Nevertheless, due to the multifactor aetiology of hypertension, it is difficult to represent it in a single animal model [38], therefore more studies will be necessary in order to better understand the physiopathological relationship between anxiety and hypertension. The fact that the data presented here seem to contradict a supposed influence of blood pressure on trait anxiety disagrees with clinical studies in which increase in anxiety levels were observed in hypertensive patients [39–42]. Again, none of those studies had a prospective design, which would be more suitable in order to investigate causality. Conversely, they were cross-sectional studies, which described an association between anxiety and hypertension, only demonstrating a high frequency of anxiety disorders in hypertensive patients [40,42]. Regarding previous animal studies, the few investigations conducted until now seem to agree with the present data, despite the fact that they did not evaluate anxiety as a personality trait, but as a transitory reaction to threat (state anxiety). For example, Faria at al. [5] investigated the effect of the inhibition of NO synthesis and the elevation of blood pressure on the anxious state, using the Elevated Plus-maze Test. Their results showed an anxiolytic effect for the acute administration of l-NAME (10 or 60 mg/kg), which was no longer observed after chronic treatment (7 days), whereas twokidney one-clip hypertension had no effect on animals’ anxiety-like behaviour. Consequently, the authors concluded that the anxiolysis observed following acute l-NAME treatment was due to the inhibition of NO synthesis in the central nervous system and not due to hypertension. Another indication that hypertension per se does not influence anxiety comes from the fact that pharmacological manipulation of the blood pressure of SHR rats (spontaneously
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hypertensive rats) does not necessarily change their anxiety-like behaviour on the Elevated Plus-maze Test [43]. In summary, despite the small sample size and the necessity of further research, the present study shows, for the first time in animals, the influence of anxiety as a personality trait on hypertension development. This will certainly lead to future pre-clinical studies, which will permit the investigation of the biological mechanisms underlying the complex relationship between anxiety and blood pressure. Finally, if one chooses to extrapolate from animals to humans, the data presented here support the idea that, in patients with hypertension and comorbid anxiety disorders, an anxious profile is the precursor of a hypertensive state and not the opposite. Acknowledgment This work was supported by grant to Flávia Barreto Garcez from Programa Institucional de Bolsas de Iniciac¸ão Científica (PIBIC/CNPq). References [1] A.T. Ginty, D. Carroll, T.J. Roseboom, A.C. Philips, S.R. de Rooij, Depression and anxiety are associated with a diagnosis of hypertension 5 years later in a cohort of late middle-aged men and women, J. Hum. Hypertens. 27 (2013) 187–190. [2] B.S. Jonas, J.F. Lando, Negative affect as a prospective risk factor for hypertension, Psychosom. Med. 62 (2000) 188–196. [3] B.S. Jonas, P. Franks, D.D. Ingram, Are symptoms of anxiety and depression risk factors for hypertension? Longitudinal evidence from the National Health and Nutrition Examination Survey I epidemiologic follow-up study, Arch. Fam. Med. 6 (1997) 43–49. [4] P.P. Roy-Byrne, K.W. Davidson, R.C. Kessler, G.J.G. Asmundson, R.D. Goodwin, L. Kubzansky, R.B. Lydiard, M.J. Massie, W. Katon, S.K. Laden, M.B. Stein, Anxiety disorders and comorbid medical illness, Focus 6 (2008) 467–485. [5] M.S. Faria, M.N. Muscará, H. Moreno Júnior, S.A. Teixeira, H.B. Dias, B. De Oliveira, F.G. Graeff, G. De Nucci, Acute inhibition of nitric oxide synthesis induces anxiolysis in the plus maze test, Eur. J. Pharmacol. 323 (1997) 37–43. [6] S. Salim, M. Asghar, G. Chugh, M. Taneja, Z. Xia, K. Saha, Oxidative stress: a potential recipe for anxiety, hypertension and insulin resistance, Brain Res. 1359 (2010) 178–185. [7] J. Srinivasan, B. Suresh, M. Ramanathan, Differential anxiolytic effect of enalapril and losartan in normotensive and renal hypertensive rats, Physiol. Behav. 78 (2003) 585–591. [8] A. Grimsrud, D.J. Stein, S. Seedat, D. Williams, L. Myer, The association between hypertension and depression and anxiety disorders: results from a nationallyrepresentative sample of South African adults, PLoS ONE 4 (2009) e5552. [9] T.C. Goes, F.D. Antunes, F. Teixeira-Silva, Trait and state anxiety in animal models: is there correlation? Neurosci. Lett. 450 (2009) 266–269. [10] G. Griebel, C. Belzung, R. Misslin, E. Vogel, The free-exploratory paradigm: an effective method for measuring neophobic behaviour in mice and testing potential neophobia-reducing drugs, Behav. Pharmacol. 4 (1993) 637–644. [11] R.N. Hughes, Behaviour of male and female rats with free choice of two environments differing in novelty, Anim. Behav. 16 (1968) 92–96. [12] F. Teixeira-Silva, F.D. Antunes, P.R.S. Silva, T.C. Goes, E.C. Dantas, M.F. Santiago, R.M. Andrade, The free-exploratory paradigm as a model of trait anxiety in rats: test–retest reliability, Physiol. Behav. 96 (2009) 729–734. [13] M.O. Ribeiro, E. Antunes, G. De-Nucci, S.M. Lovisolo, R. Zatz, Chronic inhibition of nitric oxide synthesis: a new model of arterial hypertension, Hypertension 20 (1992) 298–303. [14] J. Bartunek, E.O. Weinberg, M. Tajima, S. Rohrbach, S.E. Katz, P.S. Douglas, B.H. Lorell, Chronic N(G)-nitro-l-arginine methyl ester-induced hypertension: novel molecular adaptation to systolic load in absence of hypertrophy, Circulation 101 (February (4)) (2000) 423–429. [15] C. Baylis, B. Mitruka, A. Deng, Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage, J. Clin. Invest. 90 (1992) 278–281.
15
[16] F.D. Antunes, T.C. Goes, M.G. Vígaro, F. Teixeira-Silva, Automation of the freeexploratory paradigm, J. Neurosci. Methods 97 (2011) 216–220. [17] K. Numaguchi, K. Egashira, M. Takemoto, T. Kadokami, H. Shimokawa, K. Sueishi, A. Takeshita, Chronic inhibition of nitric oxide synthesis causes coronary microvascular remodeling in rats, Hypertension 26 (1995) 957–962. [18] A. Rowberg, D. Franklin, R.L. Van Citters, Nontraumatic method for measurement of blood pressure in animals with tails, J. Appl. Physiol. 27 (1969) 301–302. [19] J. Malkoff, Non-invasive Blood Pressure for Mice and Rats, Animal Lab News, Kent Scientific Corporation, Torrington, Connecticut, USA, March 2011, pp. 1–12. [20] Y. Kubota, K. Umegaki, S. Kagota, N. Tanaka, K. Nakamura, M. Kunitomo, K. Shinozuka, Evaluation of blood pressure measured by tail-cuff methods (without heating) in spontaneously hypertensive rats, Biol. Pharm. Bull. 29 (2006) 1756–1758. [21] T.C. Goes, F.D. Antunes, T.H. Almeida-Souza, F.R.C. Ursulino, F.B. Garcez, A.L.L. Melo, F. Teixeira-Silva, Comportamento de ratos Wistar no Paradigma da Explorac¸ão Livre, Scientia Plena 9 (2013) 1–7. [22] E.C. Leira, A.B. Acharva, S. Cruz-Flores, O.J. Albaker, V.K. Rao, S.I. Abdulrauf, Blood pressure surge precedes intracerebral hemorrhage, Neurocrit. Care 2 (2005) 43–45. [23] F.C.A. Fonseca, R.Z. Coelho, R. Nicolato, L.F. Malloy-Diniz, H.C.S. Filho, A influência de fatores emocionais sobre a hipertensão arterial, J. Bras. Psiquiatr. 58 (2009) 128–134. [24] T. Rutledge, E. Brenda, M.A. Hogan, A quantitative review of prospective evidence linking psychological factors with hypertension development, Psychosom. Med. 64 (2002) 758–766. [25] G.G. Berntson, M. Sarter, J.T. Cacioppo, Anxiety and cardiovascular reactivity: the basal forebrain cholinergic link, Behav. Brain Res. 94 (1998) 225–248. [26] R. Andreatini, A importância dos modelos animais em psiquiatria, Rev. Bras. Psiquiatr. 24 (2002) 164. [27] K.A. Matthews, K.L. Woodall, M.T. Allen, Cardiovascular reactivity to stress predicts future blood pressure status, Hypertension 22 (1993) 479–485. [28] L.L. Watkins, P. Grossman, R. Krishnan, A. Sherwood, Anxiety and vagal control of heart rate, Psychosom. Med. 60 (1998) 498–502. [29] S.A. James, Psychosocial precursors of hypertension: a review of the epidemiologic evidence, Circulation 76 (1987) I60–I66. [30] B.H. Friedman, J.F. Thayer, Autonomic balance revisited: panic anxiety and heart rate variability, J. Psychosom. Res. 44 (1998) 133–151. [31] S. Gaburro, O. Stiedl, P. Sartori, S.B. Landgraf, R. Singewald, A mouse model of high trait anxiety shows reduced heart rate variability that can be reversed by anxiolytic drug treatment, Int. J. Clin. Exp. Neuropsychol. 14 (2011) 1341–1355. [32] C. Sévoz-Couche, C. Brouillard, F. Camus, D. Laude, S.F. De Boer, C. Becker, J.J. Benoliel, Involvement of the dorsomedial hypothalamus and the nucleus tractus solitarii in chronic cardiovascular changes associated with anxiety in rats, J. Physiol. 591 (2013) 1871–1887. [33] V.K. Yeragani, R. Pohl, K. Srinivasan, R. Balon, C. Ramesh, R. Berchou, Effects of isoproterenol infusions on heart rate variability in patients with panic disorder, Psychiatry Res. 56 (1995) 289–293. [34] B.F. Fuller, The effects of stress-anxiety and coping styles on heart rate variability, Int. J. Psychophysiol. 12 (1992) 81–86. [35] L. Ghiadoni, A.E. Donald, M. Cropley, M.J. Mullen, G. Oakley, M. Taylor, G. O’Connor, J. Betteridge, N. Klein, A. Steptoe, J.E. Deanfield, Mental stress induces transient endothelial dysfunction in humans, Circulation 102 (2000) 2473–2478. [36] K. Narita, T. Murata, T. Hamada, T. Takahashi, H. Kosaka, H. Yoshida, Y. Wada, Association between trait anxiety and endothelial function observed in elderly males but not in young males, Int. Psychogeriatr. 19 (2007) 947–954. [37] C. Napoli, F. de Nigris, S. Williams-Ignarro, O. Pignalosa, V. Sica, L.J. Ignarro, Nitric oxide and atherosclerosis: an update, Nitric Oxide 15 (2006) 265–279. [38] L.O. Lerman, A.R. Chade, V. Sica, C. Napoli, Animal models of hypertension: an overview, J. Lab. Clin. Med. 146 (2005) 160–173. [39] R.C. Boutelle, S. Epstein, M.C. Ruddy, The relation of essential hypertension to feelings of anxiety, depression and anger, Psychiatry 50 (1987) 206–217. [40] A. Grimsrud, D.J. Stein, S. Seedat, D. Williams, L. Myer, The association between hypertension and depression and anxiety disorders: results from a nationallyrepresentative sample of South African adults, PLoS ONE 4 (2009) 1–9. [41] N.D. Vlachakis, R. Schiavi, M. Mendlowitz, D. De Guia, R.L. Wolf, Hypertension and anxiety, Am. Heart J. 87 (1974) 518–526. [42] T.M. Wei, L. Wang, Anxiety symptoms in patients with hypertension: a community-based study, Int. J. Psychiatry Med. 36 (2006) 315–322. [43] W.C. Rosa, G.M. Oliveira, E.M. Nakamura-Palacios., Effects of the antihypertensive drugs alpha-methyldopa and hydralazine on the performance of spontaneously hypertensive rats in the elevated plus-maze, Braz. J. Med. Biol. Res. 27 (1994) 55–59.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
The influence of trait anxiety on the elevation of arterial pressure induced by l-NAME in rats Flávia Barreto Garcez, Fábio Ursulino Reis Carvalho, Ana Paula dos Santos Soares, Tiago Costa Goes, Márcio Roberto Viana dos Santos, Flavia Teixeira-Silva ∗ Departamento de Fisiologia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Sergipe, 49100-000 São Cristóvão, SE, Brazil
h i g h l i g h t s • • • •
The relationship between anxiety and elevated blood pressure was investigated in rats. l-NAME (20 mg/kg/day – 7 days) increases blood pressure only in high trait anxiety rats. Blood pressure elevation (short term) does not affect trait anxiety. The higher the trait anxiety, the higher the blood pressure peak in response to l-NAME.
a r t i c l e
i n f o
Article history: Received 17 April 2014 Received in revised form 5 August 2014 Accepted 2 September 2014 Available online 16 September 2014 Keywords: Trait anxiety Hypertension l-NAME Rats Free-exploratory paradigm Blood pressure
a b s t r a c t Due to the high prevalence of anxiety disorders and hypertension comorbidity in the general population, the establishment of anxiety as a risk factor for elevated blood pressure, or the reverse, is of great relevance. In this context, animal models can be of great scientific value, as they permit the control of several variables. Bearing this in mind, the influence of anxiety, not as a state, but as a personality trait (trait anxiety), on blood pressure elevation and vice versa were investigated for the first time in rats, using the free-exploratory paradigm (FEP). Sixty adult male Wistar rats were evaluated on FEP and categorized according to their levels of anxiety. From this sample, 24 animals with high (n = 12) and low (n = 12) trait anxiety were allocated to two treatment groups: (1) l-NAME (NG -nitro-l-arginine methyl ester, 20 mg/kg, p.o., for 7 days to increase blood pressure; n = 6/anxiety category); (2) CTRL (tap water, p.o., for 7 days; n = 6/anxiety category). During treatment, measurements of systolic blood pressure (SBP) were taken daily. After treatment, the animals were again tested on FEP. SBP and trait anxiety levels were compared pre- and post-treatment. Additionally, correlations between trait anxiety levels and SBP increases (l-NAME group) were analyzed. The results showed that l-NAME was able to induce significant SBP elevation, but only for the high-anxious animals, while SBP elevation did not significantly interfere with anxiety levels. A significant correlation between anxiety levels and SBP peaks in response to l-NAME was also shown. No differences were observed between the levels of anxiety before and after treatment. These findings suggest that individuals with high trait anxiety are more susceptible to increases in blood pressure, but that high blood pressure does not affect the levels of trait anxiety. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
∗ Corresponding author at: Departamento de Fisiologia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Sergipe, Campus São Cristóvão 49100-000, SE, Brazil. Tel.: +55 79 2105 6645; fax: +55 79 2105 6414. E-mail addresses: fl[email protected] (F.B. Garcez), fabioursu [email protected] (F.U.R. Carvalho), [email protected] (A.P.d.S. Soares), [email protected] (T.C. Goes), [email protected] (M.R.V. dos Santos), teixeira [email protected], prof-fl[email protected] (F. Teixeira-Silva). http://dx.doi.org/10.1016/j.neulet.2014.09.011 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Several studies have shown that anxious people have a higher risk of developing hypertension [1–4].However, the nature of the association between anxiety and blood pressure is not yet understood, in part because only a few of these studies have actually used methods which are capable of establishing a causal relationship. In this context, animal models can be of great scientific value, as they permit the control of several variables, such as genetic, environmental and nutritional factors, as well as allowing the study of the interaction among these variables. This kind of approach is
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fundamental, since both anxiety and hypertension are multifactor conditions. Despite this, studies involving animals on this subject are scarce, and the existing ones either investigate the relationship between hypertension and state anxiety (anxiety a subject experiences at a particular moment in time, when facing threat) [5], taking no consideration of the anxious trait of the individuals; or do not aim to reveal the causal relationship between the conditions, only investigating their common physiological or pharmacological characteristics [6,7]. Bearing in mind the high prevalence of anxiety disorders, hypertension and their comorbidity in the general population [8], the establishment of anxiety as a risk factor for elevated blood pressure or vice versa is of great relevance, certainly having an impact on the prevention of theses illnesses and on the choice among all the possible therapies that can be applied to anxious and/or hypertensive patients. Taking all this into consideration, the aim of the present study was to evaluate, in rats, the relationship between trait anxiety (enduring feature of an individual, relatively stable over time) and elevated blood pressure. To achieve this, animals were tested in the free-exploratory paradigm (FEP) and had their blood pressure monitored, before and after treatment with l-NAME (NG -nitro-larginine methyl ester). FEP is, to the best of our knowledge, the only test proposed as a model of trait anxiety. In this situation, in which there is no change in state anxiety, animals are given the opportunity to move around freely within an environment containing both familiar and novel parts. This approach allows the evaluation of neophobic responses. As the animals have a choice between novelty and familiarity, it is expected that individuals with low trait anxiety would exhibit a preference for novelty, whereas high trait anxiety subjects would prefer familiarity [9–12]. l-NAME inhibits the biosynthesis of nitric oxide (NO), which increases cyclic GMP and cause vasorelaxation on the microcirculation [13,14]. Therefore, chronic treatment with l-NAME results in hypertension in rats [13,15] and, for this reason, was used in the present study.
2. Animals, materials and methods 2.1. Animals Sixty (2–3 months) male Wistar rats, from our own colony, were used. The animals were kept five per cage (41 cm × 34 cm × 18 cm), in a temperature (22–24 ◦ C) and light (12 h/12 h light/dark cycle, lights on at 06:00 a.m.) controlled room, with water and food ad libitum. All procedures were in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC), as well as the Brazilian National Council on the Control of Animal Experimentation (Conselho Nacional de Controle de Experimentac¸ão Animal – CONCEA, Brazil) and were conducted with approval from the Ethics Committee in Research with Animals of Universidade Federal de Sergipe, Brazil.
2.3. Blood pressure elevation Blood pressure elevation was induced by nitric oxide synthase inhibition with NG -nitro-l-arginine methyl ester (l-NAME, Sigma, USA) [13,15]. In comparison to the original l-NAME-induced hypertension protocol, a lower dose of l-NAME (20 mg/kg for 7 days) was chosen, as it has been shown that lower doses are able to elevate blood pressure without causing lesions in target organs as do high doses or long term administration of l-NAME [15,17]. As a result, it is possible to get a sample of “purely” hypertensive subjects. The systolic blood pressure (SBP) of rats was measured using a non-invasive tail-cuff method [18]. Data were obtained with a data-acquisition system (LE5002, Letica, Spain). Measurements were made once a day for 8 days, starting 1 day before the 7-day treatment period. In order to reduce external interferences to the measurements, they were all performed by the same researcher and around the same time of day (07:00 p.m.). Furthermore, to facilitate data acquisition, before every measurement session, each animal was individually placed in a heated chamber (40 ◦ C) for 10 min. Warming the animal up comfortably is necessary for most non-invasive blood pressure systems, in order to enhance blood flow to the tail and consequently produce a blood pressure signal [19]. Although it has been demonstrated that SBP measurements obtained by the heated-animal tail-cuff method are higher than those obtained by non-heated-animal methods [20], the method chosen here produces consistent results [19] and therefore it should not interfere with comparative results, when all the animals go through the same procedure. 2.4. Procedure The animals were first tested on FEP. The obtained results were used to classify them according to the %TNS, as presenting high (%TNS < 51), medium (51 ≤ %TNS ≤ 80) or low (%TNS > 80) levels of anxiety, according to a previous categorization performed in our lab [21]. A week later, rats with high (n = 12) and low anxiety (n = 12) were submitted to 5 days of adaptation to the SBP measuring apparatus, described above. After this adaptation period, the pre-treatment SBP was assessed, 1 day before the beginning of the treatment. Subsequently, the animals were administered either l-NAME (20 mg/kg/day; n = 6/anxiety category), dissolved in their drinking water, or just tap water for 7 days (n = 6/anxiety category). In order to assure that the animals had the target intake of l-NAME, one week before the treatment began, the water ingestion for each home cage was monitored every day, so an average consumption for each rat, per day, was determined. This information and measured body weights were used to calculate the concentration of l-NAME in the drinking water, which was prepared daily and offered to the animals in two bottles per cage, in order to guarantee that no animals would be impeded from drinking by an alpha rat. During this treatment period, SBP was measured daily. On each of the eight SBP evaluations, five consecutive SBP measurements were taken, in order to obtain a mean value. All records started only after heart rate stabilization, in order to minimize the effects of initial stress due to animal handling. After this 7-day period, the animals were again tested on FEP.
2.2. Animal model of trait anxiety 2.5. Statistical analysis FEP was automated by a computerized system for animal tracking (Anymaze® , Stoelting Co., USA) and set up as described by Antunes et al. [16]. The following parameters were measured: total distance travelled (TDT), and the time spent in each compartment, from which the percentage of time in the novel side (%TNS) was calculated.
The obtained data were first analyzed using Kolmogorov–Smirnov’s test for normal distribution and Bartlett’s test for the homogeneity of variances. No impediments to the use of parametric tests were found. For the analysis of SBP data, pre-treatment (SBPpre ) and post-treatment (SBPpost ) evaluations were compared. In order to
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minimize the effects of compensatory mechanisms that could mask the results if only the last day of treatment was considered, SBPpost corresponded to the mean values of all 7 days of treatment. Due to the small sample size and to the impossibility of testing animals belonging to all the different treatment and anxiety groups on each day of evaluation, the data were then analyzed using a two-factor analysis of variance (ANOVA) for repeated measures (factor 1: treatment group; factor 2: time) for each anxiety level, in replacement to a three-factor ANOVA. When the interaction between factors was significant, the analyses were followed by one-way, repeated measures ANOVA, conducted on the time variable for each treatment group. A correlational study was also performed, using Pearson’s correlation coefficient. This way, %TNS from the first FEP evaluation was confronted with the mean percent variation in SBP [SBP = (SBPpost − SBPpre )/SBPpre × 100] and with the highest percent variation in SBP, achieved during treatment (MaxSBP), for all the animals (high and low trait anxiety) receiving l-NAME. All significance tests were two-tailed and were performed at the 5% significance level. 3. Results The means and standard deviations of the absolute values of all evaluated parameters are presented in Table 1. 3.1. SBP evaluation (Fig. 1)
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changes induced by the treatment, according to this formula: SBP = [(SBPpost − SBPpre )/SBPpre ] × 100. For the high trait anxiety animals, the interaction between treatment group and time was significant (F(1,10) = 11.703; p = 0.007). Analysis of time as a single factor for the each treatment group revealed a significantly higher SBP for the l-NAME group (F(1,5) = 79.498; p = 0.001), but not for the CTRL group (F(1,5) = 63.021; p = 0.468). For the low trait anxiety animals, the interaction between treatment group and time was not significant (F(1,10) = 3.695; p = 0.084), therefore the two main effects were analyzed. Neither time (F(1,10) = 1.667; p = 0.226) or treatment (F(1,10) = 3.600; p = 0.087) changed SBP significantly. 3.2. Trait-anxiety evaluation (Table 1) For the high trait anxiety animals, the interaction between treatment group and time was not significant (F(1,10) = 0.035; p = 0.856), therefore the two main effects were analyzed. The time effect was significant (F(1,10) = 11.468; p = 0.007), increasing %TNS. The treatment group effect was not significant (F(1,10) = 0.360; p = 0.562). For the low trait anxiety animals, the interaction between treatment group and time was not significant (F(1,10) = 2.945; p = 0.117), therefore the two main effects were analyzed. Neither time (F(1,10) = 3.795; p = 0.080) or treatment (F(1,10) = 1.449; p = 0.256) changed %TNS significantly. 3.3. %TNS × SBP correlation
To aid visualization of these results, the graphs shown represent the profile of the responses presented by the animals, with the pre-treatment being considered as the “zero” point for the
Pearson’s R test revealed a lack of significant correlation (r = −0.476, p = 0.118) between %TNS and SBP.
Table 1 Data obtained on the free-exploratory paradigm and absolute values of systolic blood pressure, pre- and post-treatment. Anxiety
Treatment
%TNSpre
%TNSpost
High
l-NAME CTRL l-NAME CTRL
36.16 ± 18.41 27.35 ± 29.40 84.33 ± 3.57 83.81 ± 4.13
55.93 ± 33.97 45.06 ± 35.19 66.72 ± 21.73 82.70 ± 16.42
Low
*
SBPpre
SBPpost
127.44 ± 2.95 125.33 ± 18.75 130.73 ± 8.20 129.56 ± 15.22
143.76 ± 5.73* 120.75 ± 15.41 141.93 ± 4.91 127.36 ± 5.39
Data are presented by mean ± SD. l-NAME: NG -nitro-l-arginine methyl ester; CTRL: control; %TNS: percentage of time in the novel side; SBP: systolic blood pressure; Pre: pre-treatment; Post: post-treatment. * Significantly different from pre-treatment (p < 0.05).
High Anxiety SBP (Difference from Pre-treatment)
14%
Low Anxiety
*
12% 10% 8% 6% 4%
L-NAME
2%
CTRL
0% -2% -4% -6% Pre-treatment
Post-treatment
Pre-treatment
Post-treatment
Fig. 1. Profile of systolic blood pressure (SBP) of high-anxious (left panel) and low-anxious (right panel) animals, in response to pharmacological treatment. Data are presented as mean ± SD. l-NAME: NG -nitro-l-arginine methyl ester; CTRL: control. *Significantly different from pre-treatment (p = 0.001).
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3.4. %TNS × MaxSBP Pearson’s R test revealed a significant negative correlation (r = −0.605, p = 0.037) between %TNS and MaxSBP. Here it is worth remembering that the greater the %TNS, the lower the anxiety.
4. Discussion The present study aimed to investigate the effect of trait anxiety on SBP elevation, as well as the influence of elevated SBP on anxious trait. The obtained results showed that: (1) l-NAME, in the administered dose, was able to induce significant SBP elevation, but only for the high trait anxiety animals; and (2) SBP elevation did not significantly interfere with trait anxiety levels. To the best of our knowledge, this is the first animal study performed to explore the relationship between trait anxiety and hypertension. The data presented here suggest that individuals with high levels of trait anxiety are more susceptible to the development of hypertension, since high-anxious trait rats presented a significant SBP elevation, in response to the administration of 20 mg/kg/day of l-NAME for 7 days, which did not occur for lowanxious trait rats submitted to the same protocol. On the other hand, elevated SBP does not seem to influence trait anxiety, as the animals did not significantly change their performance on FEP after the treatment with l-NAME, that is to say individuals do not tend to see the world as more threatening because they have elevated blood pressure. This specific result must be interpreted with care, though, as SBP was only elevated for a short period of time. The possible influence of long term hypertension on trait anxiety cannot yet be discarded. It is worth commenting here on the fact that high trait anxiety animals, either treated with l-NAME or water, presented an increase in %TNS on the second exposition to FEP. This apparent anxiety reduction has been reported before and was probably due to the animals having had a previous “out of cage experience” [12], to which low trait anxiety animals seem to be less sensitive. Maybe the most interesting result was provided by the correlational study, which showed that the higher the trait anxiety, the higher the blood pressure peak in response to l-NAME treatment. Naturally, it could be argued that statistical correlation tests provide information on association rather than a cause-and-effect relationship between variables, so it is possible that high trait anxiety leads to SBP peaks, but it is also possible that a third uninvestigated variable is causing both effects. At least, due to our experimental design, the possibility that the increase in blood pressure would be the cause of high trait anxiety can be ruled out. In any case, this result suggests that high-anxious subjects are more prone to present peaks in blood pressure, and this is particularly important because it is the peaks, not mean blood pressure, that are most closely correlated with stroke and intracranial bleeding risk [22]. In general, all these results are congruent with previous clinical studies that investigated the connection of emotional factors, such as anxiety, to initiation and progression of hypertension [3,23,24]. Such studies showed that anxiety, as well as other psychosocial conditions, could be associated with the development of hypertension. However, these findings are somewhat vulnerable as, in clinical studies, it is difficult to control for genetic and/or environmental factors, and it is often impossible to be sure that the blood pressure elevation followed, rather than preceded, a supposed psychological risk factor [25]. In this context, the present work, as a preclinical study, contributes a lot to the understanding of the complex relationship between anxiety and blood pressure. After all, the use of animal models presents important advantages in comparison to clinical
studies, such as better control of environmental factors, a smaller number of confounding variables and quicker completion [26]. Furthermore, it was possible to explore the temporal relationship between the two studied conditions; i.e., it was possible to know the levels of trait anxiety of the subjects before and after the increase in blood pressure, which allowed the investigation of the influence of trait anxiety on blood pressure elevation and vice versa. As a result, there is now better support for anxiety as a risk factor to hypertension, but no more support for the contrary. Several mechanisms to explain the possible influence of psychological factors on the regulation of blood pressure have been proposed: (1) cardiovascular reactivity to stress, which would lead to a hyperactivity of the sympathetic nervous system, keeping the blood pressure levels increased over time [27]; (2) neurohormonal models, with speculations about alterations in neurotransmitters and impaired baroreflex control [28]; and (3) tendency of psychiatric patients to present predisposing behaviours to hypertension, such as poor diet, obesity, sedentarism, smoking and alcohol abuse [29]. Investigating the physiopathological mechanisms relating anxiety to hypertension was never the aim of the present work. However, some previous animal studies have demonstrated an autonomic dysfunction in high-anxious trait animals [25], in which there could be sympathetic dominance, reduced vagal activity and baroreceptor alterations [30–33]. Such increased sympathetic activity seems to occur independently of environmental stressors, which affect the anxious state, being inherent to anxious individuals [28,34]. Enhancement of sympathetic modulation has been shown to induce endothelial dysfunction [35], leading, over time, to decreased brachial artery flow-mediated dilation, which is in fact associated to high trait anxiety in elderly people [36]. Considering all this, and the fact that one of the hallmarks of a dysfunctional endothelium is diminished levels of bioavailable NO [37], it is tempting to speculate that enhanced sympathetic activity is responsible for the increased susceptibility of high-anxious trait animals to SBP elevation, as observed here. Nevertheless, due to the multifactor aetiology of hypertension, it is difficult to represent it in a single animal model [38], therefore more studies will be necessary in order to better understand the physiopathological relationship between anxiety and hypertension. The fact that the data presented here seem to contradict a supposed influence of blood pressure on trait anxiety disagrees with clinical studies in which increase in anxiety levels were observed in hypertensive patients [39–42]. Again, none of those studies had a prospective design, which would be more suitable in order to investigate causality. Conversely, they were cross-sectional studies, which described an association between anxiety and hypertension, only demonstrating a high frequency of anxiety disorders in hypertensive patients [40,42]. Regarding previous animal studies, the few investigations conducted until now seem to agree with the present data, despite the fact that they did not evaluate anxiety as a personality trait, but as a transitory reaction to threat (state anxiety). For example, Faria at al. [5] investigated the effect of the inhibition of NO synthesis and the elevation of blood pressure on the anxious state, using the Elevated Plus-maze Test. Their results showed an anxiolytic effect for the acute administration of l-NAME (10 or 60 mg/kg), which was no longer observed after chronic treatment (7 days), whereas twokidney one-clip hypertension had no effect on animals’ anxiety-like behaviour. Consequently, the authors concluded that the anxiolysis observed following acute l-NAME treatment was due to the inhibition of NO synthesis in the central nervous system and not due to hypertension. Another indication that hypertension per se does not influence anxiety comes from the fact that pharmacological manipulation of the blood pressure of SHR rats (spontaneously
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hypertensive rats) does not necessarily change their anxiety-like behaviour on the Elevated Plus-maze Test [43]. In summary, despite the small sample size and the necessity of further research, the present study shows, for the first time in animals, the influence of anxiety as a personality trait on hypertension development. This will certainly lead to future pre-clinical studies, which will permit the investigation of the biological mechanisms underlying the complex relationship between anxiety and blood pressure. Finally, if one chooses to extrapolate from animals to humans, the data presented here support the idea that, in patients with hypertension and comorbid anxiety disorders, an anxious profile is the precursor of a hypertensive state and not the opposite. Acknowledgment This work was supported by grant to Flávia Barreto Garcez from Programa Institucional de Bolsas de Iniciac¸ão Científica (PIBIC/CNPq). References [1] A.T. Ginty, D. Carroll, T.J. Roseboom, A.C. Philips, S.R. de Rooij, Depression and anxiety are associated with a diagnosis of hypertension 5 years later in a cohort of late middle-aged men and women, J. Hum. Hypertens. 27 (2013) 187–190. [2] B.S. Jonas, J.F. Lando, Negative affect as a prospective risk factor for hypertension, Psychosom. Med. 62 (2000) 188–196. [3] B.S. Jonas, P. Franks, D.D. Ingram, Are symptoms of anxiety and depression risk factors for hypertension? Longitudinal evidence from the National Health and Nutrition Examination Survey I epidemiologic follow-up study, Arch. Fam. Med. 6 (1997) 43–49. [4] P.P. Roy-Byrne, K.W. Davidson, R.C. Kessler, G.J.G. Asmundson, R.D. Goodwin, L. Kubzansky, R.B. Lydiard, M.J. Massie, W. Katon, S.K. Laden, M.B. Stein, Anxiety disorders and comorbid medical illness, Focus 6 (2008) 467–485. [5] M.S. Faria, M.N. Muscará, H. Moreno Júnior, S.A. Teixeira, H.B. Dias, B. De Oliveira, F.G. Graeff, G. De Nucci, Acute inhibition of nitric oxide synthesis induces anxiolysis in the plus maze test, Eur. J. Pharmacol. 323 (1997) 37–43. [6] S. Salim, M. Asghar, G. Chugh, M. Taneja, Z. Xia, K. Saha, Oxidative stress: a potential recipe for anxiety, hypertension and insulin resistance, Brain Res. 1359 (2010) 178–185. [7] J. Srinivasan, B. Suresh, M. Ramanathan, Differential anxiolytic effect of enalapril and losartan in normotensive and renal hypertensive rats, Physiol. Behav. 78 (2003) 585–591. [8] A. Grimsrud, D.J. Stein, S. Seedat, D. Williams, L. Myer, The association between hypertension and depression and anxiety disorders: results from a nationallyrepresentative sample of South African adults, PLoS ONE 4 (2009) e5552. [9] T.C. Goes, F.D. Antunes, F. Teixeira-Silva, Trait and state anxiety in animal models: is there correlation? Neurosci. Lett. 450 (2009) 266–269. [10] G. Griebel, C. Belzung, R. Misslin, E. Vogel, The free-exploratory paradigm: an effective method for measuring neophobic behaviour in mice and testing potential neophobia-reducing drugs, Behav. Pharmacol. 4 (1993) 637–644. [11] R.N. Hughes, Behaviour of male and female rats with free choice of two environments differing in novelty, Anim. Behav. 16 (1968) 92–96. [12] F. Teixeira-Silva, F.D. Antunes, P.R.S. Silva, T.C. Goes, E.C. Dantas, M.F. Santiago, R.M. Andrade, The free-exploratory paradigm as a model of trait anxiety in rats: test–retest reliability, Physiol. Behav. 96 (2009) 729–734. [13] M.O. Ribeiro, E. Antunes, G. De-Nucci, S.M. Lovisolo, R. Zatz, Chronic inhibition of nitric oxide synthesis: a new model of arterial hypertension, Hypertension 20 (1992) 298–303. [14] J. Bartunek, E.O. Weinberg, M. Tajima, S. Rohrbach, S.E. Katz, P.S. Douglas, B.H. Lorell, Chronic N(G)-nitro-l-arginine methyl ester-induced hypertension: novel molecular adaptation to systolic load in absence of hypertrophy, Circulation 101 (February (4)) (2000) 423–429. [15] C. Baylis, B. Mitruka, A. Deng, Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage, J. Clin. Invest. 90 (1992) 278–281.
15
[16] F.D. Antunes, T.C. Goes, M.G. Vígaro, F. Teixeira-Silva, Automation of the freeexploratory paradigm, J. Neurosci. Methods 97 (2011) 216–220. [17] K. Numaguchi, K. Egashira, M. Takemoto, T. Kadokami, H. Shimokawa, K. Sueishi, A. Takeshita, Chronic inhibition of nitric oxide synthesis causes coronary microvascular remodeling in rats, Hypertension 26 (1995) 957–962. [18] A. Rowberg, D. Franklin, R.L. Van Citters, Nontraumatic method for measurement of blood pressure in animals with tails, J. Appl. Physiol. 27 (1969) 301–302. [19] J. Malkoff, Non-invasive Blood Pressure for Mice and Rats, Animal Lab News, Kent Scientific Corporation, Torrington, Connecticut, USA, March 2011, pp. 1–12. [20] Y. Kubota, K. Umegaki, S. Kagota, N. Tanaka, K. Nakamura, M. Kunitomo, K. Shinozuka, Evaluation of blood pressure measured by tail-cuff methods (without heating) in spontaneously hypertensive rats, Biol. Pharm. Bull. 29 (2006) 1756–1758. [21] T.C. Goes, F.D. Antunes, T.H. Almeida-Souza, F.R.C. Ursulino, F.B. Garcez, A.L.L. Melo, F. Teixeira-Silva, Comportamento de ratos Wistar no Paradigma da Explorac¸ão Livre, Scientia Plena 9 (2013) 1–7. [22] E.C. Leira, A.B. Acharva, S. Cruz-Flores, O.J. Albaker, V.K. Rao, S.I. Abdulrauf, Blood pressure surge precedes intracerebral hemorrhage, Neurocrit. Care 2 (2005) 43–45. [23] F.C.A. Fonseca, R.Z. Coelho, R. Nicolato, L.F. Malloy-Diniz, H.C.S. Filho, A influência de fatores emocionais sobre a hipertensão arterial, J. Bras. Psiquiatr. 58 (2009) 128–134. [24] T. Rutledge, E. Brenda, M.A. Hogan, A quantitative review of prospective evidence linking psychological factors with hypertension development, Psychosom. Med. 64 (2002) 758–766. [25] G.G. Berntson, M. Sarter, J.T. Cacioppo, Anxiety and cardiovascular reactivity: the basal forebrain cholinergic link, Behav. Brain Res. 94 (1998) 225–248. [26] R. Andreatini, A importância dos modelos animais em psiquiatria, Rev. Bras. Psiquiatr. 24 (2002) 164. [27] K.A. Matthews, K.L. Woodall, M.T. Allen, Cardiovascular reactivity to stress predicts future blood pressure status, Hypertension 22 (1993) 479–485. [28] L.L. Watkins, P. Grossman, R. Krishnan, A. Sherwood, Anxiety and vagal control of heart rate, Psychosom. Med. 60 (1998) 498–502. [29] S.A. James, Psychosocial precursors of hypertension: a review of the epidemiologic evidence, Circulation 76 (1987) I60–I66. [30] B.H. Friedman, J.F. Thayer, Autonomic balance revisited: panic anxiety and heart rate variability, J. Psychosom. Res. 44 (1998) 133–151. [31] S. Gaburro, O. Stiedl, P. Sartori, S.B. Landgraf, R. Singewald, A mouse model of high trait anxiety shows reduced heart rate variability that can be reversed by anxiolytic drug treatment, Int. J. Clin. Exp. Neuropsychol. 14 (2011) 1341–1355. [32] C. Sévoz-Couche, C. Brouillard, F. Camus, D. Laude, S.F. De Boer, C. Becker, J.J. Benoliel, Involvement of the dorsomedial hypothalamus and the nucleus tractus solitarii in chronic cardiovascular changes associated with anxiety in rats, J. Physiol. 591 (2013) 1871–1887. [33] V.K. Yeragani, R. Pohl, K. Srinivasan, R. Balon, C. Ramesh, R. Berchou, Effects of isoproterenol infusions on heart rate variability in patients with panic disorder, Psychiatry Res. 56 (1995) 289–293. [34] B.F. Fuller, The effects of stress-anxiety and coping styles on heart rate variability, Int. J. Psychophysiol. 12 (1992) 81–86. [35] L. Ghiadoni, A.E. Donald, M. Cropley, M.J. Mullen, G. Oakley, M. Taylor, G. O’Connor, J. Betteridge, N. Klein, A. Steptoe, J.E. Deanfield, Mental stress induces transient endothelial dysfunction in humans, Circulation 102 (2000) 2473–2478. [36] K. Narita, T. Murata, T. Hamada, T. Takahashi, H. Kosaka, H. Yoshida, Y. Wada, Association between trait anxiety and endothelial function observed in elderly males but not in young males, Int. Psychogeriatr. 19 (2007) 947–954. [37] C. Napoli, F. de Nigris, S. Williams-Ignarro, O. Pignalosa, V. Sica, L.J. Ignarro, Nitric oxide and atherosclerosis: an update, Nitric Oxide 15 (2006) 265–279. [38] L.O. Lerman, A.R. Chade, V. Sica, C. Napoli, Animal models of hypertension: an overview, J. Lab. Clin. Med. 146 (2005) 160–173. [39] R.C. Boutelle, S. Epstein, M.C. Ruddy, The relation of essential hypertension to feelings of anxiety, depression and anger, Psychiatry 50 (1987) 206–217. [40] A. Grimsrud, D.J. Stein, S. Seedat, D. Williams, L. Myer, The association between hypertension and depression and anxiety disorders: results from a nationallyrepresentative sample of South African adults, PLoS ONE 4 (2009) 1–9. [41] N.D. Vlachakis, R. Schiavi, M. Mendlowitz, D. De Guia, R.L. Wolf, Hypertension and anxiety, Am. Heart J. 87 (1974) 518–526. [42] T.M. Wei, L. Wang, Anxiety symptoms in patients with hypertension: a community-based study, Int. J. Psychiatry Med. 36 (2006) 315–322. [43] W.C. Rosa, G.M. Oliveira, E.M. Nakamura-Palacios., Effects of the antihypertensive drugs alpha-methyldopa and hydralazine on the performance of spontaneously hypertensive rats in the elevated plus-maze, Braz. J. Med. Biol. Res. 27 (1994) 55–59.