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1 Department of Pharmacology, Monash University, Victoria 3800, Australia
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(Received 13 June 2004;
accepted after revision 20 October 2004; first published online 12 November 2004)
Corresponding author R. E. Widdop: Department of Pharmacology, Monash University, Victoria 3800, Australia. Email: robert.widdop{at}med.monash.edu.au
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Introduction |
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Tachycardic and pressor responses have been recorded in various rat strains subjected to a number of manipulations including common animal husbandry and intraperitoneal injections (Schnecko et al. 1998); and in various stressor protocols including novel environment (van den Buuse et al. 2001), acoustic arousal (Bao et al. 1999), conditioned fear (Carrive, 2000, 2002), immobilization, air-jet (Ely, 1995) and restraint (Chen & Herbert, 1995). Indeed many of these manipulations including handling, air-jet and restraint also cause hypothalamo-pituitary-adrenal axis activation as indicated by increases in plasma adrenocorticotropin and corticosterone concentrations (Dobrakovova et al. 1993; Engelmann et al. 1996; Chung et al. 2000).
We have previously shown that the cardiovascular component of the stress response in spontaneously hypertensive rats (SHRs) differs from that of Wistar-Kyoto rats (WKYs) when subjected to restraint stress (McDougall et al. 2000). Furthermore there was a difference in the adaptation of the cardiovascular response between the strains, where the duration of the restraint-induced tachycardia observed in SHRs normalized with repeated exposure to ‘WKY-like’ levels. In the present study, we determined whether the cardiovascular responses of WKYs and SHRs would differ in response to other psychological stressors as we had found with restraint stress (McDougall et al. 2000).
To this end, we have characterized the cardiovascular stress response to repeated stress in SHRs, utilizing three common experimental stressors: handling, air-jet and restraint (McDougall et al. 2000). In addition, a heterologous protocol was included, whereby rats experienced 10 days of air-jet stress followed immediately by 10 consecutive days of restraint stress. This protocol was designed to determine what effect repeated mild stress has upon the cardiovascular response to a subsequent more severe stressor (i.e. heterologous stress). We have compared these effects in SHRs with those in WKYs and Sprague-Dawley (SD) rats, representing inbred and outbred normotensive strains.
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Methods |
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Experimental protocol
Groups of adult male SD, WKY and SHR were implanted with telemetry probes (TA11-PAC40; Data Sciences International) under general anaesthesia (ketamine and xylazine, 67 mg kg–1 and 7 mg kg–1, respectively, I.P.) as previously described (McDougall et al. 2000). All rats were left to recover for a 10-day period.
Each group/strain was handled at the same time each day for 10 consecutive days; this involved the experimenter lifting and holding the rat by the tail and torso from its home cage, such that contact lasted less than 30 s. This protocol was based upon a routine that has been previously described (McDougall et al. 2000), and was performed so that all groups would be treated exactly the same before exposure to either air-jet or restraint protocols. Telemetry data were sampled for 10 s every minute for each rat over the daily 2-h experimental period.
For air-jet stress, the rats were exposed to a 2-ms burst of air (pressure regulated at 150 kPa) directed at the forehead, of sufficient force to part the fur, every minute for 1 h day–1 for 10 consecutive days. The rats remained in their home cage throughout the stress challenge. Data were sampled for 4 s every 30 s, with the air-jet burst timed to coincide with sampling on the minute interval.
The day after the final air-jet session, the three groups experienced restraint stress, which involved confinement within a Perspex tube (diameter, 6 cm; Plastic Laboratories) for 1 h day–1 for 10 days where data were sampled for 10 s every min for the 2-h experimental period. This portion of the regime we termed ‘heterologous restraint’. A separate group of SD rats were put through the restraint (only) protocol, as we described for WKY and SHR groups in our previous study (McDougall et al. 2000), in order for data comparisons to be made. All stress procedures were carried out between 08.00 and 12.00 h to minimize diurnal variation.
From these protocols, a total of 10 strain/stressor combinations were obtained: SD rats exposed to repeated handling (n = 8), air-jet (n = 7), restraint (n = 8) and the heterologous restraint (n = 7) stress protocols and WKYs and SHRs exposed to repeated handling (n = 5 per group), air-jet (n = 10 per group) and heterologous restraint (n = 5 and 6 for WKYs and SHRs, respectively) stress protocols. For comparison of ‘within strain’ and ‘between strain’ stressor effects, as summarized in Tables 2 and 3, data from our previous study describing the effect of repeated restraint on WKYs (n = 5) and SHRs (n = 5) rats were utilized (McDougall et al. 2000), therefore 12 strain/stressor combinations are discussed.
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All data are represented as mean ± S.E.M. except in Fig. 1 where only mean values are shown to better illustrate the actual response patterns. Baseline values were defined as the average of the 30 min immediately prior to a given stress session. Maximum change, % change and the duration of the heart rate (HR) and mean arterial pressure (MAP) responses were determined, taking the basal values into account, for each stress period for all stress protocols. To obtain an indication of the duration and intensity of the HR and MAP responses for the 1-h period after the beginning of stress, an area under curve (AUC) calculation was used (GraphPad Prism). AUC data were compared by one-way ANOVA with repeated measures for analysis within each of the stress protocols in each strain or by t-test as appropriate. A two-way ANOVA for analysis of the temporal responses was also employed for strain and stress protocol comparisons. Post hoc testing with a Dunnett's or Newman–Keuls test was performed as appropriate. Maximum change (as % change) in HR and MAP responses were analysed similarly. In all cases P < 0.05 was considered significant.
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Results |
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SHRs consistently exhibited elevated resting MAP as measured via telemetry (Figs 1 and 2). The SD group exhibited higher resting HR values than those of both the WKY and SHR groups on day 1 of the air-jet protocol (Fig. 1); however, this day seemed to be an exception because resting HR across the groups were not significantly different on days 2–10 (Fig. 2).
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Figure 1 illustrates the mean HR and MAP response patterns evoked within each strain as caused by acute (day 1) exposure to handling, air-jet and heterologous restraint. For all strains, handling generally evoked an increase in HR (30–121 beats min–1 or 12–38%, day 1) and MAP (11–24 mmHg or 11–19%, day 1) that initially peaked with contact; thereafter most of the resulting cardiovascular response was apparently due to the animal exploring its home cage as indicated by activity counts (movement across the horizontal plane; data not shown), during which time the HR and MAP fell back to basal levels (Fig. 1).
Air-jet stress (sampled twice per minute) caused a pulsatile-like change in HR in each strain. An overall rise in HR (61–78 beats min–1 or 17–28%, day 1) and MAP (19–41 mmHg or 19–31%, day 1), compared to basal levels, was measured as the air puffs were repeated over the hour period (Fig. 1).
The restraint (only) and heterologous restraint response patterns were similar in SD rats, where each stressor elicited the largest changes in HR (149–185 beats min–1 or 43–59%, day 1) and MAP (31–42 mmHg or 31–46%, day 1) compared to handling and air-jet stress exposure (Figs 1 and 4). Similarly in WKYs and SHRs, heterologous restraint caused the largest changes in HR (209–214 beats min–1, or 59–85% day 1) and MAP (47–53 mmHg or 44–50%, day1) as compared to the handling and air-jet protocols (Fig. 1, Table 1). During the restraint and heterologous restraint sessions, the duration of the tachycardia and pressor responses varied, as indicated by AUC measurements, and was dependent upon the strain and/or day of exposure.
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Sprague-Dawley rats. Acute exposure (day 1) to each of the stress protocols caused differential degrees of cardiovascular activation in SD rats. For example, upon exposure to each of the stressors for the first time (day 1), SD rats exhibited increases in HR and MAP of 61 ± 24 beats min–1 (17 ± 7%) and 19 ± 6 mmHg (19 ± 6%) to air-jet; 91 ± 12 beats min–1 (28 ± 4%) and 11 ± 1 mmHg (11 ± 1%) to handling; 149 ± 21 beats min–1 (43 ± 7%) and 31 ± 3 mmHg (31 ± 4%) to restraint; and 185 ± 11 beats min–1 (59 ± 4%) and 43 ± 3 mmHg (46 ± 4%) to heterologous restraint, respectively, (Figs 2 and 4, Table 1). Analysis of the maximum changes and duration of HR and MAP for the overall stress responses (days 1–10 combined, Figs 2–4) showed that heterologous restraint and then restraint (only) were more effective stressors in SD rats compared to handling and air-jet (Table 2). There were no differences in the cardiovascular responses to each of the stressors when repeated on days 2–10 as compared to the acute (day 1) response, indicating that SD rats did not adapt their cardiovascular responses to handling, air-jet or restraint stress protocols.
The heterologous restraint group (i.e. those SD rats that had previously experienced 10 days of air-jet stress), had a significantly heightened and a longer-lasting pressor response when first exposed to heterologous restraint compared to day 1 of the restraint (only) group (Fig. 4). Moreover, the overall HR and MAP effects (both change and duration over 10 days) in the heterologous restraint group were significantly greater than those of the restraint (only) group (Fig. 4 and Table 2).
Wistar-Kyoto rats. The overall change in HR and MAP and duration of tachycardic and pressor responses in WKYs showed that heterologous restraint and restraint (only) were equally effective followed by handling and air-jet (Figs 2 and 3, Table 2). There was no adaptation evident in the maximum cardiovascular responses (Fig. 2 and Table 1) to handling and air-jet, as there were no differences between cardiovascular responses of day 1 and subsequent days (2–10) in each of these protocols; a result similar to that demonstrated with the restraint (only) protocol (McDougall et al. 2000). However, the duration of tachycardia (AUC data) due to acute (day 1) heterologous restraint exposure was significantly greater than that of the acute restraint (only) (McDougall et al. 2000) response. This tachycardic response was not maintained on subsequent exposure to repeated heterologous restraint as the duration of the tachycardic response decreased, indicating adaptation within the WKY heterologous restraint group (Fig. 3). This adaptive response resulted in days 3–10 being significantly different from day 1 and similar to levels previously measured in a restraint (only) WKY group (McDougall et al. 2000).
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Stressor comparison between strains
Analysis of raw data indicates that generally SHRs are hyper-responsive with respect to changes in MAP in response to all stressors when compared to the two normotensive strains; that is, SHRs had significantly greater pressor responses that persisted longer than both WKYs and SD rats for all stressor protocols. Normalization of the maximum change data shows that these responses were not dissimilar from those in the normotensive strains (Table 1), although clearly peak pressor responses were greater in SHRs. In addition, the maximum change in, and duration of, tachycardic responses was lower in SHRs for handling and air-jet than that of WKYs and SD rats but this rank order of strains was reversed for the restraint-based protocols (Table 3). There were also a number of significant differences between the normotensive strains; for example the duration of the tachycardic response in SD rats was maintained after handling and during air-jet for longer than that of the WKY strain. In addition, WKYs and SD rats had similar changes in, and duration of, tachycardic responses to restraint alone; however, for the heterologous restraint protocol, the response of WKYs was significantly lower than that of the SD rats (and SHRs).
Of the stressors presented, the restraint-based protocols clearly evoke the greatest change in cardiovascular activity, yet submaximal responses were evoked in response to handling and air-jet. This difference, or grading, of the cardiovascular response based on the stressor presented was common to all three strains tested.
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Discussion |
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An initial aim of this study was to assess the effect of various stressor protocols on the cardiovascular component of the stress response and it was observed that each rat strain graded their cardiovascular response when exposed to each of the stressor protocols. For example, the maximum changes in HR on the first day of exposure to air-jet, handling and restraint in SD rats were 61 ± 24 (17 ± 7%), 91 ± 12 (28 ± 4%), 149 ± 21 (43 ± 7%) beats min–1, respectively. Such grading of the cardiovascular response to stressful stimuli is noteworthy in the context of patterned sympathetic responses, as recently reviewed (Morrison, 2001; Saper, 2002), where evidence for differential autonomic outflow in response to varying physiological demands is strong. However, there is limited evidence of different patterns of sympathetic activity with exposure to different stressors of a psychological nature. Indeed, the data presented here suggest a level of control with respect to the cardiovascular system such that tachycardic responses are graded according to stressor type. Such a notion would be consistent with the integration of information by higher centres of the CNS yielding a series of graded cardiovascular outputs.
A number of studies have shown central specificity between what may be broadly classified as ‘physical’ and ‘psychological’ stressors, such that the central circuitry involved in producing the final stress response is distinct between these categories of stressors (Thrivikraman et al. 2000; Dayas et al. 2001). Based on these and other studies, restraint, air-jet and handling can be classified as the psychological-type of stressors; however, we have clearly shown that each of these stressors caused a differential cardiovascular response. Thus, these results would suggest that a gating mechanism is operative in the CNS that subsequently determines the degree of cardiovascular activation, according to the perceived threat, although the precise anatomical and/or functional location(s) is not known. Restraint and immobilization stress, have previously been shown to cause differential levels of Fos immunoreactivity (a marker of neuronal activation) in the paraventricular nucleus of the hypothalamus, locus coeruleus and central and medial nuclei of the amygdala (Chowdhury et al. 2000), implicating those regions in such a gating mechanism within the ‘psychological stress’ circuitry. Other regions possibly involved may include the dorsomedial hypothalamus, as inhibition of this region with muscimol significantly attenuates ‘air stress’-induced cardiovascular responses (Stottpotter et al. 1996). Given that the aforementioned nuclei are known to be able to modulate central cardiovascular control (Dampney, 1994) it seems likely that such nuclei may at least be partly responsible for mediating the central processing that resulted in the differential cardiovascular responses to handling, air-jet and restraint observed in this study.
We have previously shown that the cardiovascular response to repeated restraint differed between WKYs and SHRs (McDougall et al. 2000), in that SHRs had heightened pressor responses that remained elevated for longer when compared to WKYs. In addition, SHRs had longer lasting tachycardic responses that decreased with repeated restraint. In the present study we have demonstrated that such differences occur with other psychological stressors as SHRs produced significantly greater peak pressor responses (both change in and duration) to handling, air-jet, restraint and heterologous restraint compared to those of the normotensive SD and WKY strains. Our raw data are consistent with other studies indicating that SHRs have significantly greater changes in blood pressure to open field (novel environment) (van den Buuse et al. 2001), 15-min immobilization and 30-s air-jet (Ely, 1995) as compared to WKYs. On the other hand, no difference was reported between SHRs and WKYs in peak MAP or pressor duration to an airpuff startle protocol (Palmer & Printz, 1999), although the pattern of the HR response to individual airpuff stimuli differed between the strains. However, normalization of the maximum change in HR and MAP data indicates negligible differences between the strains although duration of pressor responses (AUC) was prolonged in SHRs. Thus, as SHRs exhibited extreme absolute peak pressure for a prolonged period during exposure to each of the stressors as compared to the normotensive WKY and SD strains, this in turn may have an impact on their cardiovascular status particularly if the effects of the stressors are maintained.
In SHRs there was evidence of adaptation of the cardiovascular responses to repeated handling and air-jet, as we have seen previously with restraint (McDougall et al. 2000). The adaptation seen in the cardiovascular response of SHRs was associated with an exaggerated initial response compared with responses in either WKYs or SD rats. Subsequent repeated exposure results in the responses of SHRs being modified to levels similar (or even lower) to those measured in the normotensive SD and WKY strains. In the SHR restraint (only) group, this occurred by day 7 of the 10-day regimen with respect to the duration of the tachycardia response (McDougall et al. 2000), while in the present study the change in MAP due to air-jet reached ‘normotensive-like’ levels by day 6. With handling, the duration of the MAP response in SHRs was comparable to that of the responses in WKYs and SD rats by the second experience. The number of exposures it takes for SHRs to ‘normalise’ may in turn be related to the magnitude of the initial stress-induced cardiovascular response. On the other hand, WKYs and SD rats did not show any signs of cardiovascular adaptation to each of the stressors alone in the present study, although the tachycardic response to repeated restraint did adapt in a different normotensive rat strain (Listar) (Chen & Herbert, 1995; Chung et al. 2000).
Finally, the heterologous restraint protocol was included to elucidate what impact repeated air-jet exposure has on the responses of a subsequent heterologous stressor, in this instance restraint. There are few studies that have focused on the cardiovascular system with heterologous protocol exposure, and those usually present the heterologous stressor once (Bhatnagar & Dallman, 1998; Bhatnagar et al. 1998), not repeatedly, as in this study. This technique revealed specific differences between the strains. The blood pressure response (both change in and duration) in SD rats was increased in response to heterologous restraint as compared to the restraint (only) group on day 1. In addition, the HR and MAP responses over the 10-day period in the heterologous restraint group were significantly greater than that of the restraint (only) group. In WKYs, the duration of the tachycardic response was elevated on day 1 in the heterologous restraint group as compared to restraint alone; however, with repeated exposure to the heterologous stressor (restraint) a subsequent decrease in the tachycardic response in WKYs was recorded which returned to restraint-alone levels by day 3. In SHRs, there were minimal differences between the heterologous restraint and restraint (only) stress-evoked cardiovascular responses. Collectively, the heterologous stress protocol indicates that repeated mild stress causes rats to exhibit exaggerated cardiovascular reactivity, at least transiently in normotensive strains, when exposed to a secondary stressor.
In conclusion, the present study provides a comparison of the cardiovascular profiles obtained as a result of repeated exposure to differing stressors. SHRs exhibit prolonged cardiovascular responses to all stressor types tested but show an ability to adapt those responses to ‘normotensive-like’ levels with repeated exposure. Normotensive strains show some degree of sensitized cardiovascular responses to heterologous stress, where subsequent repeated exposure leads to strain-specific responses. The differential tachycardia responses induced within each of the strains as evoked by different stressors reflect specificity in the regulation of the cardiovascular system. These data clearly support the hypothesis that under different stressful scenarios higher brain centres can modulate the cardiovascular system to produce graded responses.
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) and duration of (AUC) tachycardic and pressor responses as evoked by each stressor protocol within each strain
), Wistar-Kyoto rats (WKYs) (
) and spontaneously hypertensive rats (SHRs) (
). Arrow indicates point in time when handling occurred (A and B); dashed bar represents air-jet period (C and D), continuous bar represents restraint period (E and F). SD handling, n = 8; SD air-jet, n = 7; SD heterologous restraint, n = 7; WKY handling, n = 5; WKY air-jet, n = 10; WKY heterologous restraint, n = 5; SHR handling, n = 5; SHR air-jet, n = 10; SHR heterologous restraint, n = 6.
P < 0.05 for individual day versus day 1 within same group (one-way ANOVA).
