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. 2002 Jan;7(1):36–46. doi: 10.1379/1466-1268(2002)007<0036:asitii>2.0.co;2

Acute stress-induced tissue injury in mice: differences between emotional and social stress

Olga Sánchez 1, Anna Arnau 1, Miguel Pareja 1, Enric Poch 1, Ignasi Ramírez 1, Maria Soley 1,1
PMCID: PMC514800  PMID: 11892986

Abstract

Emotional stress affects cellular integrity in many tissues including the heart. Much less is known about the effects of social stress. We studied the effect of emotional (immobilization with or without cold exposure) or social (intermale confrontation) stress in mice. Tissue injury was measured by means of the release of enzyme activities to blood plasma: lactate dehydrogenase (LDH), creatine kinase (CK), aspartate transaminase (AST), and alanine transaminase (ALT). Tape-immobilization increased all these activities in the plasma. AST-ALT ratio was also increased in these animals. Electrophoretic analysis of CK isoenzymes showed the appearance of CK-MB. These results indicate that the heart was injured in immobilized mice. Analysis of LDH isoenzymes and measurement of α-hydroxybutyrate dehydrogenase (HBDH) activity suggests that other tissues, in addition to the heart, contribute to the increase in plasma LDH activity. Restraint in small cylinders increased plasma LDH, CK, AST, and ALT activities, but to lower levels than in tape immobilization. Because the decrease in liver glycogen and the increase in plasma epidermal growth factor (EGF) were also smaller in restraint than in the tape-immobilization model of emotional stress, we conclude that the former is a less intense stressor than the latter. Cold exposure during the restraint period altered the early responses to stress (it enhanced liver glycogen decrease, but abolished the increase in plasma EGF concentration). Cold exposure during restraint enhanced heart injury, as revealed by the greater increase in CK and AST activities. Intermale confrontation progressively decreased liver glycogen content. Plasma EGF concentration increased (to near 100 nM from a resting value of 0.1 nM) until 60 minutes, and decreased thereafter. Confrontation also affected cellular integrity in some tissues, as indicated by the rise in plasma LDH activity. However, in this type of stress, the heart appeared to be specifically protected because there was no increase in plasma CK activity, and both AST and ALT increased, but the AST-ALT ratio remained constant. Habituation to restraint (1 h/d, 4 days) made mice resistant to restraint-induced tissue injury as indicated by the lack of an increase in plasma LDH, CK, AST, or ALT activities. Similar general protection against homotypic stress-induced injury was observed in mice habituated to intermale confrontation.

INTRODUCTION

The emergence of the stress concept in physiology was not linked to early endocrine or metabolic responses, but to some longer-term effects: adrenal enlargement, gastrointestinal ulcers, and thymolymphatic atrophy, which results in altered immune response (see Szabo 1998, for review). Later studies indicated that tissues, including the heart, are damaged because of short-term stress. Thus, emotional stress (immobilization or restraint, alone or in combination with other stimuli) may induce myocardial necrotic lesions in several animal species (Raab et al 1964; Jönsson and Johansson 1974; Downing and Chen 1985; Matsuoka et al 1998). Stress-induced injury results in the release of cytosolic enzyme activities to blood plasma (Meltzer 1971; Arakawa et al 1997).

Heart damage induced by emotional stress resembles that induced by catecholamine administration (Szakacs and Cannon 1958; Analóczy 1985; Downing and Chen 1985; Rona 1985). This suggests that catecholamines are responsible for the effect of emotional stress. In fact, β-blockers, but not cholinergic blockers, reduce the release of enzyme activities in the restraint-water immersion stress model (Arakawa et al 1997). In humans, it was shown that β-blockade reduced both tissue injury (measured by the increase in plasma activity of the creatine kinase–MB isoform) and supraventricular tachycardia induced by acute stress caused by severe head injury (Cruickshank et al 1987). In addition to the acute induction of necrotic lesions, studies in ventricular myocytes (Communal et al 1998) and in whole animals (Shizukuda et al 1998) indicate that sustained β-adrenergic stimulation may also result in apoptotic cell death. Therefore, hypertrophic response to sustained catecholamine stimulation should be understood as a compensatory adaptation (Yamazaki et al 1998; Engelhardt et al 1999; Wagner et al 1999). Hypertrophy leads to heart dysfunction and is, therefore, a cause of pathology.

Social stress is the result of conflicting interaction between individuals. In rodents, the most common model of social stress is the defeat experience obtained in the resident-intruder paradigm (Martínez et al 1998).This type of stress is used to study behavioral changes and may serve as a model of human diseases related to stress (eg, anxiety and depression) (Martínez et al 1998). Few studies compare emotional and social stress. Recently, it was shown that social stress resulted in a higher incidence of arrhythmia and enhanced heart rate acceleration when compared with nonsocial stress (Sgoifo et al 1998). These alterations may be because of a higher sympathetic activation and a lower parasympathetic antagonism in social than in nonsocial stress models (Sgoifo et al 1997). However, there is little information concerning the effect of social stress on tissue injury (Matte 1975). Therefore, we have studied the effect of both emotional and social stress on tissue injury in mice, measured by the release of cytosolic enzyme activities to blood plasma.

MATERIALS AND METHODS

Animals

Adult Swiss-CD1 mice were obtained from Interfauna (Barcelona, Spain). All animals were male (2.5–3 months old, 35–40 g), fed ad libitum, maintained under a constant 12-hour light and 12-hour dark cycle (lights on at 8 AM), and controlled conditions of humidity (45–55 %) and temperature (22 ± 1°C). All experimental procedures were approved by the Committee on Animal Care of the University of Barcelona.

Tape-immobilization stress

Upon arrival to our animalarium facilities, mice were grouped in cages (5 mice per cage), and allowed to adapt for 10 days before use. On the day of the experiment (starting at 9 AM), mice (under very light ether anaesthesia) were fixed with adhesive tape to a table in a supine position. In less than 1 minute, the effect of ether completely disappeared, and acute stress symptoms were observed. At indicated times, animals were sacrificed and processed, as indicated subsequently, or returned to individual cages at room temperature for recovery. During the recovery period, mice had free access to water, but not to food.

Restraint stress

On the day of the experiment (starting at 9 AM), mice were introduced into small flat-bottom cylinders (restrainers) with adjustable head and tail gates (Panlab, Barcelona, Spain). This procedure resulted in an almost complete immobilization of the animals. Animals were maintained either at room temperature or at 4°C for the indicated period of time. Then, they were immediately sacrificed (see later) or returned to individual cages at room temperature for recovery. During the recovery period, the mice had free access to water, but not to food.

Habituation to restraint stress

To induce habituation, mice were subjected to restraint stress (1 h/d, 9–10 AM) for 4 consecutive days. On the fifth day, the habituated animals were divided into 3 groups. In the first group, animals were sacrificed without restraint. In the second, animals were sacrificed 30 minutes after being introduced into restrainers. In the last group, mice were restrained for 1 hour and allowed to recover for 3 hours. Animals not exposed to previous restraint were used as controls.

Intermale confrontation stress

After the adaptation period to the animalarium, mice were maintained for 15 days in individual cages (14.02 dm2). On the day of the experiment (starting at 9 AM), each mouse (the intruder) was introduced into the cage of an older (and bigger) mouse (the resident) that had been isolated in a smaller cage (7.56 dm2) for at least 21 days. Residents had been tested for aggressiveness before the experiment. At the indicated time, intruders were taken from the residents' cages and sacrificed (see later).

Habituation to intermale confrontation stress

To induce habituation, intruders were exposed for 4 consecutive days to a 3-hour confrontation period per day, in the cage of daily changing residents. On the fifth day, the habituated animals were divided into 3 groups. In the first group, animals were sacrificed without confrontation. In the second, animals were sacrificed 60 minutes after being introduced into the resident's cage. In the last group, mice were exposed to the complete 3-hour confrontation period. Animals not exposed to previous confrontation were used as controls.

Sampling and analysis

To obtain samples, mice were anaesthetized (sodium pentobarbital 60 mg/kg). Blood was collected into heparinized syringes from the inferior vena cava. Blood plasma was obtained by centrifugation. A sample was deproteinized and neutralized, as indicated elsewhere (Grau et al 1996), and then used for glucose quantification (Trinder 1969). Another sample was processed as indicated (Grau et al 1994) for epidermal growth factor (EGF) quantification. Lactate dehydrogenase (LDH), α-hydroxybutyrate dehydrogenase (HBDH), creatine kinase (CK), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities in plasma were determined by standard procedures (Boehringer Mannheim, Mannheim, Germany; assay kits LDH MPR1, HBDH MRP1, CK MRP1, AST MRP1, and ALT MPR1, respectively). Plasma corticosterone was determined as indicated (Benavides et al 1998). Immediately after bleeding, liver and submandibular salivary glands were excised, frozen in liquid nitrogen, and stored at −80°C until further processed (in less than 1 week). Submandibular glands were homogenized in 10 mL phosphate-buffered saline. After centrifugation (100 000 × g for 60 minutes at 4°C), the supernatant was stored at −40°C for EGF quantification (Grau et al 1994). Liver glycogen was determined as indicated elsewhere (Grau et al 1996).

CK and LDH isoenzymes analysis

Plasma CK and LDH isoenzymes were analyzed using Sigma Diagnostics (St Louis, MO, USA) kits (715-AM and 705-A, respectively), following the manufacturer's instructions. Briefly, fresh plasma samples (0.05 mL) were diluted with an equal volume of PBS and supplemented with 0.01 mL mercaptoethanol solution. Three microlitres of the mixture was run in 1 % agarose gel at 100 V for 45 minutes, and immediately developed at 37°C for 30–60 minutes (until bands were clearly observed). Gels were then rinsed for 30 minutes in acetic acid-methanol-water (5:70:25, v/v/v), and finally in water for 15 minutes. Because band intensity declined progressively, gels were immediately scanned in an Epson GT-8500 scanner.

Statistical analysis

Results are shown as mean ± SE of 6–12 animals per group. Before statistical comparisons, homogeneity of variances was analyzed by Bartlett's test. When no homogeneity was observed (always in plasma corticosterone and EGF, and often in some plasma enzymes), logarithmic transformation was used. Depending on the particular experiment, 1-way or 2-way analysis of variance was used. When significant F value was obtained, post hoc comparisons between particular groups were made by Tukey's test.

RESULTS

In the first experiment, mice were subjected to adhesive tape immobilization, which was shown to be a potent stressor in mice (Tebar et al 2000). Plasma glucose was raised, and liver glycogen was decreased at 20 minutes immobilization (Table 1). Extending immobilization time to 180 minutes did not further decrease liver glycogen, and plasma glucose was slightly decreased (the difference between 20 minutes and 180 minutes was significant, P < 0.01), but was still significantly higher than in control mice. Plasma EGF concentration increased transiently in immobilized mice: it was significantly higher at 20 minutes immobilization, but returned to control values at 180 minutes. EGF in submandibular salivary glands was decreased at 20 minutes, and remained thus for at least 180 minutes.

Table 1.

 Effect of immobilization on stress-sensitive parameters

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After 20 minutes of immobilization, plasma LDH, CK, and AST activities had increased moderately (Fig 1). ALT activity was not significantly increased after 20 minutes of immobilization. Contrary to the parameters commented on earlier, all 4 enzyme activities continued to increase upon extending the immobilization time. Thus, after 180 minutes of immobilization, plasma LDH activity was increased by 9-fold, CK by 12-fold, AST by 7-fold, and ALT by near 3-fold. AST-ALT ratio is useful in clinical biochemistry (Pappas 1989; Rej 1989). In control mice, AST-ALT ratio was 1.72 ± 0.10. This ratio progressively increased with immobilization time (2.45 ± 0.25 after 20 minutes, P < 0.01; 4.31 ± 0.41 after 180 minutes, P < 0.001). To determine whether the continuous increase in enzyme activities was a delayed outcome of an earlier stimulus or whether it required the continued presence of the stressor, in this experiment, we also studied animals immobilized for 20 minutes and allowed to recover for 160 minutes. For no enzyme was there a significant difference between animals allowed to recover, when compared with those maintained immobilized (Fig 1).

Fig 1.

Fig 1.

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 Effect of immobilization stress on plasma enzyme activities. Mice were immobilized (IMMO) for the indicated period of time and immediately sacrificed or allowed to recover before sacrifice. Plasma was obtained to measure enzyme activities. Results are mean ± SE of 12 animals per group. Significance of the differences vs zero time (control) value is given by * P < 0.05, ** P < 0.01, *** P < 0.001. Differences between 180 minutes IMMO and 20 minutes IMMO + 160 minutes recovery were nonsignificant

There are 3 different isoenzymes of CK (namely CK-MM, CK-MB, and CK-BB). Mouse heart contains both MM and MB isoenzymes: skeletal muscles express the MM isoenzyme, and brain expresses the BB isoenzyme. Thus, the appearance of the MB isoenzyme in plasma clearly indicates cardiac damage, and it is used as a clinical marker of the magnitude of damage (Kragten et al 1996). Similarly, LDH exists in 5 different isoenzymes. LDH-1 release into plasma is also used as a clinical marker of heart injury (Ladi et al 1990). Therefore, to determine whether our results reflect heart damage, we analyzed the appearance of CK and LDH isoenzymes in plasma from control and 180-minute immobilized mice. Figure 2 shows the results for 1 control and 1 immobilized mouse. The experiment was repeated 3 times with comparable results. In control mouse plasma, only the CK-MM isoenzyme was visible. In immobilized mouse, the band corresponding to this isoenzyme was more intense, and the band corresponding to CK-MB isoenzyme was also clearly observed.

Fig 2.

Fig 2.

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 Isoenzyme analysis of creatine kinase (CK) and lactate dehydrogenase (LDH) in the plasma of immobilized mice. Plasma from a 180-minute immobilized mouse (I) and a control mouse (C) were immediately separated in 1 %-agarose–gel electrophoresis and processed to detect either CK or LDH isoenzymes. Position of isoenzymes was determined by using commercial standards and indicated at the margin of each gel

Concerning LDH isoenzymes, we observed only LDH-5 isoenzyme in control mouse plasma (Fig 2). In immobilized mouse plasma, all 5 isoenzymes were observed. The intensities of LDH isoenzyme bands were higher for LDH-5 and LDH-4 isoenzymes than for LDH-3, LDH-2, and LDH-1 isoenzymes. This pattern was the opposite of that observed in mouse heart homogenate (data not shown). This suggests that tissues other than heart tissue contributed more to LDH activity in plasma. To further explore this hypothesis, we measured HBDH activity in plasma. It is known that both LDH-1 and LDH-2, but not the other LDH isoenzymes, can oxidize α-hydroxybutyrate, and thus HBDH activity is used as a clinical marker of heart injury (Hooper and Bangert 1995). We measured both LDH and HBDH in the plasma of control and 180-minute immobilized mice. In control mice, LDH activity was 7 ± 1 nkat/mL, and HBDH activity was 2 ± 1 nkat/mL. In 180-minute immobilized mice, LDH activity was 40 ± 3 nkat/mL, and HBDH activity was 13 ± 2 nkat/mL. This indicates that LDH-1 + LDH-2 accounted for 33 % of the rise in LDH activity in the plasma of immobilized mice.

It was described that hypothermia increased CK release in restrained rats (Meltzer 1971), but it is not known whether hypothermia also affects the injury markers of other tissues. Therefore, we studied how low temperature affected the release of cytosolic enzymes to blood plasma. In this experiment, we substituted adhesive tape immobilization by restraint because this procedure did not require the continuous presence of the experimenter. In a preliminary experiment, we compared the effects of tape immobilization and restrainer immobilization. We observed that restraint was a weaker stressor than tape immobilization, judging by the smaller rise in plasma glucose and also by the smaller increase of LDH, CK, AST, and ALT activities (data not shown). Table 2 shows how restraint, combined or not combined with cold exposure, affected the plasma concentration of corticosterone, glucose, and EGF, and also of liver glycogen and submandibular glands EGF. The most remarkable differences were that in cold-exposed restrained mice, the decrease in liver glycogen content was significant and plasma EGF concentration did not increase. To determine whether cold exposure affected the release of enzyme activities to blood plasma, mice were restrained for 60 minutes either at room temperature or at 4°C. All of them were then allowed to recover for 180 minutes at room temperature. The increase in LDH activity was similar in both groups of animals (Fig 3). The increase in plasma CK and AST activities were significantly higher in cold-exposed than in room temperature restrained mice. The increase in ALT activity was moderate and similar in both groups of restrained animals. Therefore, the AST-ALT ratio was significantly higher in cold-exposed restrained mice (9.32 ± 1.22) than in room temperature restrained mice (4.81 ± 0.67) (P < 0.01). This ratio was 2.07 ± 0.74 in control mice. One-hour exposure to 4°C without restraint followed by 3 hours at room temperature had no effect on any enzyme activity (in nkat/mL: LDH, 2.65 ± 0.53; CK, 0.18 ± 0.07; AST, 0.83 ± 0.20; ALT, 0.38 ± 0.10).

Table 2.

 Effect of restraint combined or not with cold exposure on stress-sensitive parameters

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Fig 3.

Fig 3.

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 Effect of restraint stress, combined or not with cold exposure, on plasma enzyme activities. Mice were introduced into restrainers and kept either at room temperature (R) or at 4°C (C/R) during 1 hour. Control mice (CONT) were maintained in their cages at room temperature. All restrained animals were returned to their cages (all at room temperature) and sacrificed 3 hours later to determine enzyme activities in the plasma. Results are mean ± SE of 6 animals per group. Comparison vs control value is given by *, and comparison vs room temperature value by ▴. Significance of the differences is given by *,▴ (P < 0.05); **,▴▴ (P < 0.01); ***,▴▴▴ (P < 0.001)

To characterize stress response in the resident-intruder model of social stress (Martínez et al 1998), we determined changes in plasma parameters (corticosterone, glucose, and EGF), as well as in liver glycogen and submandibular glands EGF. A mouse (maintained in isolation for 15 days) was introduced into the cage of a resident mouse. Resident mice always attacked intruders after a brief exploratory approach. Intruders adopted defensive behavior (bipedal posture oriented to the resident) and showed clear symptoms of defeat: defensive behavior in every attack episode, escaping attitude, and sometimes a completely motionless prone position. Attacks were more frequent during the first hour, but continued evenly all through the experiment unless the intruders adopted the prone posture.

Intermale confrontation raised plasma corticosterone concentration, which reached a peak value at 30 minutes and decreased thereafter (Table 3). Contrary to immobilization or restraint stress, plasma glucose concentration did not increase in these animals, but liver glycogen decreased progressively. It is remarkable that after 180 minutes, glycogen content in liver decreased to 30 % of the initial value. Plasma EGF concentration experienced a large increase at 30 minutes, and continued increasing until 60 minutes after intrusion. After 180 minutes, plasma EGF concentration had returned to near-control value. EGF content in submandibular glands decreased to 56 % of control value at 30 minutes after intrusion, and remained at this level for at least until 180 minutes.

Table 3.

 Effect of intermale confrontation on stress-sensitive parameters

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This stress model affected cellular integrity in tissues, as indicated by the increase in plasma enzyme activities (Fig 4). The pattern obtained was, however, very different. LDH, AST, and ALT activities were increased after 180 minutes, but CK activity was not. None of the enzyme activities were increased at 30 minutes. It is also remarkable that the increase in ALT runs parallel to that in AST, and thus the AST-ALT ratio remained constant (2.14 ± 0.53 at 0 minute, 2.14 ± 0.77 at 30 minutes, and 2.24 ± 1.10 at 180 minutes).

Fig 4.

Fig 4.

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 Effect of intermale confrontation on plasma enzyme activities. Intruder mouse (isolated for 15 days before the experiment) was introduced in the cage of a resident mouse. At the indicated time, animals were sacrificed, and samples were obtained to determine enzyme activities in plasma. Results are mean ± SE of 6 animals per group. Significance of the differences vs zero time (control) value is given by ** (P < 0.01)

Habituation (progressive lose of responsiveness against a repeated stressor) is a characteristic of the stress response (McEwen 1998). Therefore, we studied the response to an acute emotional (restraint) or social (intermale confrontation) stress in mice previously exposed to the corresponding homotypic stressor. We first studied the effect of previous exposure to a 1-hour restraint for 4 consecutive days on the response to restraint on the fifth day. During the habituation period, mice did not gain, but actually lost, body weight (0.33 ± 0.07 and −0.23 ± 0.03 g/d for control and experimental groups, respectively; P < 0.001). These animals had a lower food intake (4.79 ± 0.11 g/d vs 5.41 ± 0.16 g/d for controls; P < 0.05) and a normal water intake (6.39 ± 0.33 mL/d vs 6.67 ± 0.29 mL/d for controls; nonsignificant). There were no significant differences between habituated and nonhabituated mice in the basal value of any parameter studied, except for plasma corticosterone, which was moderately increased in habituated mice (Fig 5). Plasma corticosterone concentration after 30 minutes restraint increased to a similar value in habituated mice as in nonhabituated mice. Glucose and EGF concentrations in the plasma of habituated mice did not increase. The effect of habituation on the release of enzyme activities to plasma was striking: the activity of any of the enzymes studied (LDH, CK, AST, and ALT) was not increased in the plasma of habituated mice upon a new restraint period.

Fig 5.

Fig 5.

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 Habituation to restraint stress. Nonhabituated (white bars) or habituated (black bars) mice were divided into 2 groups. Group A: animals were subjected to restraint stress for 30 minutes (RES) and immediately sacrificed to determine plasma corticosterone, glucose, and EGF concentrations. Control animals (C) were not restrained. Group B: to determine the effect of restraint on plasma enzyme activities, mice were subjected to restraint stress for 60 minutes, and returned to their cages until sacrifice (3 hours later). Control animals were maintained in their cages all the time. Results are mean ± SE of 8 animals per group. Comparison vs corresponding control value is given by *, and comparison vs corresponding nonhabituated value by ▴. Significance of the differences is given by *,▴ (P < 0.05); **,▴▴ (P < 0.01); ***,▴▴▴ (P < 0.001)

Finally, we studied the effect of habituation to intermale confrontation. Mice were exposed for 4 consecutive days to intermale confrontation (3 hours per day), and studied on the fifth day. Results are shown in Figure 6. As it happened in mice habituated to restraint, habituation to social stress did not affect the basal value of any parameter analyzed, except for plasma corticosterone, which was also moderately increased. Habituation affected the response to a new encounter. Plasma corticosterone levels rose, but to a significantly lower level in habituated than in nonhabituated mice. Plasma EGF concentration did not increase at all in habituated animals. Plasma glucose concentration was unaffected in habituated mice as it was in nonhabituated mice. Liver glycogen was similarly affected in both groups: it decreased to 191 ± 37 μmol glycosyl residues/g and 217 ± 17 μmol glycosyl residues/g in habituated and nonhabituated mice, respectively, after 1 hour of the new encounter (habituated and nonhabituated zero time controls had 381 ± 35 μmol glycosyl residues/g and 344 ± 38 μmol glycosyl residues/g, respectively). None of the cytosolic enzymes measured in the plasma (LDH, CK, AST, and ALT) were increased in habituated mice after 3 hours of the new encounter.

Fig 6.

Fig 6.

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 Habituation to intermale confrontation stress. Nonhabituated (white bars) or habituated (black bars) mice were divided into 2 groups. Group A: animals were subjected to intermale confrontation for 60 minutes (CONF) and immediately sacrificed to determine plasma corticosterone, glucose, and EGF concentrations. Control animals (C) were kept in their cages. Group B: to determine the effect of confrontation on plasma enzyme activities, intruder mice were maintained in the resident cage for 180 minutes, and then immediately sacrificed. Control animals were maintained in their cages all the time. Results are mean ± SE of 6 animals per group. Comparison vs corresponding control value is given by *, and comparison vs corresponding nonhabituated value by ▴. Significance of the differences is given by *,▴ (P < 0.05); **,▴▴ (P < 0.01); ***,▴▴▴ (P < 0.001)

DISCUSSION

Early response to acute stress

The early response to acute stress involves the activation of both catecholaminergic neurons of the locus ceruleus, and corticotropin-releasing hormone and arginine-vasopressin neurons of the paraventricular nuclei of the hypothalamus (Stratakis and Chrousos 1995). These general responses will lead to the activation of the efferent sympathetic-adrenomedullary system and the hypothalamic-pituitary-adrenal axis. The intensity of the response of each system depends on both the intensity and the nature of the stressor (Kopin 1995). Activation of the hypothalamic-pituitary-adrenal axis was evidenced by the rise in plasma corticosterone in both restraint (with and without cold exposure) and confrontation stress models.

We have no direct measurement of the sympathetic-adrenomedullary system activity, but we analyzed one of its more direct metabolic consequences: stimulation of glycogen breakdown in the liver (Gardemann et al 1992). All 3 emotional stress models (tape immobilization and restraint with or without cold exposure) induced a marked hyperglycemia, although the decrease in liver glycogen varied from one model to another. The higher decrease observed in tape-immobilized mice (28 %) than in restrained mice (14 %) suggests that the former is perceived as a more intense stressor than the latter, as previously described (Armario et al 1990). Intermale confrontation stress had different consequences on plasma glucose and glycogen breakdown when compared with immobilization or restraint. Although liver glycogen decreased progressively throughout the confrontation period, there was no hyperglycemia in this model, likely because of the enhanced skeletal muscle activity. It is conceivable that the absence of hyperglycemia allowed the sustained decrease in liver glycogen content in fighting animals, but not in immobilized or restrained mice, because it is known that glycogenolysis in the liver is sensitive to plasma glucose concentration (Bollen et al 1998).

Male mice accumulate a large amount of EGF in the submandibular salivary glands, which is released to the saliva (Byyny et al 1974) and to the plasma (Byyny et al 1974; Tuomela 1990; Grau et al 1994) upon adrenergic stimulation. We found that tape immobilization induced a similar rise in plasma EGF than adrenaline administration (Tebar et al 2000). In keeping with those results suggesting that restraint is perceived as a less intense stressor than tape immobilization, we have shown here that the decrease in the submandibular salivary glands EGF in restrained mice is smaller than in tape-immobilized mice, and the rise in plasma EGF was also smaller in restrained mice (2.5-fold) than in tape-immobilized mice (6.5-fold). Restraint in a cold environment did not raise the plasma EGF concentration. We have not further investigated the reasons for this reduced secretion of EGF, but we may suggest that it could be the result of hypothermia, which would in the first place affect subcutaneous organs like submandibular salivary glands.

One of the most remarkable results shown here is the huge increase (about 625-fold) in plasma EGF concentration in the intermale confrontation model of social stress. In these animals, plasma EGF reached a concentration that exceeds by 2 orders of magnitude that obtained with a single maximal dose of adrenaline (Tebar et al 2000) or with restraint stress. Using radioimmunoassay to determine EGF in plasma, Nexo et al (1981) found that after 20 minutes of aggressive intermale encounter, EGF concentration increased by more than 250-fold. It is remarkable that maximal concentration is not achieved until 60 minutes after the beginning of intermale encounter. Plasma EGF concentration reaches a maximum, 10 minutes after stimulation of the submandibular salivary glands with a single intravenous administration of an α1-adrenergic agonist (Grau et al 1994). Therefore, a continuous increase for 60 minutes may reflect repeated stimulation of these glands, as a result of attacks by the resident mouse. To our knowledge, this is the largest increase in plasma EGF concentration ever reported, and suggests that acute intermale confrontation may constitute the best model to study the endocrine function of EGF in mice.

Stress-induced tissue injury

Our results, in keeping with those in many previous reports on rats (Raab et al 1964; Preus et al 1988; Arakawa et al 1997), hamsters (Matsuoka et al 1998), and pigs (Thoren and Jonsson 1983), suggest that emotional stress causes heart damage. First, plasma CK activity increased, which was in part because of the appearance of the CK-MB isoenzyme. In agreement with others (Nicholson and Matheson 1981), we found both CK-MM and CK-MB isoenzymes in heart homogenates, whereas in brain and skeletal muscle homogenates we found only CK-BB and CK-MM isoenzymes, respectively (data not shown). Second, AST-ALT ratio increased in the 3 models studied. The rise in the AST-ALT ratio is in keeping with the occurrence of heart damage (Pappas 1989; Rej 1989). Finally, we observed the appearance of LDH-1 and LDH-2 isoenzymes, the most abundant LDH isoenzymes in the heart, in the plasma of immobilized mice. In agreement with this observation, we found increased HBDH activity in the plasma of immobilized mice. The fact that the increase in HBDH accounted for only a part of the total increase in LDH, and that the less abundant LDH isoenzymes in the heart are the most abundant in the plasma of immobilized mice, suggests that other tissues were also damaged in immobilized mice. Immobilization increases lipid peroxidation in the liver (Kovacs et al 1996), suggesting that this tissue may also be damaged in this acute stress model. In fact, Salas et al (1980) found rough endoplasmic fragmentation and dilatation, mitochondrial enlargement, and an increased number of autophagic vacuoles 48 hours after several stress regimens including restraint. In keeping with these observations, we have described recently the induction of c-Fos accumulation in the liver of immobilized mice (Fernández-Varó et al 2000).

Emotional stress-induced heart damage was attributed to catecholamines (Analóczy 1985; Opie and Thomas 1985; Arakawa et al 1997). It was suggested that restraint is a less intense stressor than tape immobilization (Armario et al 1990), and our results concerning early response to both models of emotional stress agree with this interpretation (see earlier). In keeping with the hypothesized lower stimulation of the sympathetic-adrenomedullary system in restraint stress, we observed that restraint induced a similar pattern of enzyme activities than tape immobilization, but the absolute value of each enzyme activity achieved in these mice was about half of that obtained in tape-immobilized mice. Based on the enhanced raise of CK and AST activities, and the AST-ALT ratio, we conclude that cold exposure potentiates specifically the effect of restraint, causing heart injury. This effect may be attributed either to an enhanced adrenergic action on the heart (but not on other tissues susceptible to catecholamine-induced injury) or to the lack of some specific protecting factor in this particular model (see later).

Another of the more remarkable observations reported here is that social stress also induces tissue damage. The pattern of enzyme activities released, however, suggests that the heart is protected in this type of stress. First, although LDH activity increased to a value similar to that in restraint stress, there was no increase in the plasma CK activity at all. Second, both AST and ALT increased, but the AST-ALT ratio remained constant. Protection of the heart may not be attributed to lower sympathetic-adrenomedullary system activation. Sgoifo et al (1997) found that both noradrenaline and adrenaline concentrations in the plasma increased to a higher level in defeat-stressed than in restrained rats. These authors have also reported that social challenge can be far more detrimental for cardiac electrical stability than other nonsocial aversive stimuli (Sgoifo et al 1999).

Hypothalamic-pituitary-adrenal axis stimulation influences heart function both through a direct action of corticosteroids in the heart and through effects of these hormones in the central nervous system (Bohus and Korte 2000). Whether, and how, these effects are involved in the protection against stress-induced injury is not yet understood, although recent studies by Valen et al (2000) suggest that glucocorticoids protect the heart against ischaemia-reperfusion injury through an increased expression of Hsp72. Nevertheless, we may rule out a role for corticosteroids in the specific protection of the heart in social stress or in the enhanced heart damage in cold-restraint stress because our results indicate that restraint (with and without cold exposure) and confrontation stress induce roughly similar increase in the plasma corticosterone concentration.

It is tempting to suggest that the large increase in the plasma EGF concentration in intruder mice may be involved in heart protection. A function of EGF as a cytoprotective factor has been described in other tissues (Olsen et al 1984; Ishikawa et al 1994; Rao et al 1997; Yoshinaga et al 1998; Gibson et al 1999). Furthermore, we have found that EGF interferes with the β-adrenergic–mediated cyclic adenosine monophosphate (AMP) rise in the main metabolic targets of catecholamines, namely adipocytes (Tebar et al 1993, 1996) and hepatocytes (Grau et al 1997). Furthermore, we found that EGF decreases the cyclic AMP signal generated by β-adrenergic agonists in cardiac myocytes, and that EGF deceases the inotropic and chronotropic effects of adrenaline in perfused heart (Lorita, Escalona, Faraudo, Pareja, Sánchez, Soley, and Ramírez, in preparation). This background gives support to the hypothesis enunciated earlier, and it is now under experimental study in our laboratory. This hypothesis would explain the enhanced heart damage in cold-restraint stress because in this model there was no increase in plasma EGF concentration.

Habituation to emotional and social stress

Repeated exposure of animals to a single stressor leads often to a diminished response to this stressor (habituation), but to an enhanced response to a different stressor (sensitization) (McCarty et al 1992). Although we observed partial or no habituation of corticosterone increase in restraint and confrontation stress, respectively, we found complete habituation in the response of plasma EGF in both models, which is a sympathetic-adrenomedullary system–linked response (Byyny et al 1974; Grau et al 1994). In addition, hyperglycemia, which is the result of adrenergic stimulation of liver glycogenolysis in restraint stress, also disappeared (glycemia, and even liver glycogen, in fighting animals is more difficult to correlate to the adrenergic control of glycogenolysis because liver glycogen metabolism is controlled primarily by glucose itself, and hence by the glucose requirements of active muscles [Bollen et al 1998]). These results suggest a substantial reduction of the sympathetic-adrenomedullary system activation in habituated mice to either emotional or social stress. This is in keeping with the observation that repeated exposure to homotypic stressors, including restraint, in a predictable sequence reduced substantially the norepinephrine and epinephrine release upon a new episode of stressor stimulus (McCarty et al 1992).

Habituation of neuroendocrine responses to homotypic stressors has been interpreted as a mechanism that permits conservation of energy (McCarty et al 1992). Our results agree with such an interpretation: hyperglycemia in restrained mice has no metabolic advantage because muscle mass is not able to use this excess of available fuel. Lack of hyperglycemia allows a reduced energy waste provided that none of the used glucose would return to the liver for storage. But our results further indicate that repeated exposure to homotypic stress, either from restraint or from fighting, protects mice against homotypic stress-induced injury.

This effect of habituation may be the result of decreased sympathetic-adrenomedullary system activation, but it may also be a consequence of the induction of a general protection mechanism. Heat shock proteins (Hsps) are induced by a variety of cellular stress insults, and their function in cell protection is well documented (Ellis and Van der Vies 1991; Hendrick and Hartl 1993). Both corticosteroids (Valen et al 2000) and catecholamines (White and White 1986; Paroo and Noble 1999) induce the expression of different Hsps in tissues including the heart. Although these hormonal effects suggest that both emotional and social stress might induce the expression of Hsp in mice, the reported effects of stressors, including restraint, are not conclusive. Thus, it was reported that a single restraint episode did not affect the expression of Hsp70 in rat heart, although it was enhanced in adrenal glands and aorta (Udelsman et al 1993). Others reported that restraint decreased the expression of Hsp90 in the liver and the spleen of rats (Vamvakopoulos et al 1993). However, habituation may have a different effect on Hsp expression: it was reported that repeated restraint induced the expression of several Hsp70 isoforms in rat heart (Meerson et al 1992). Therefore, the involvement of Hsp in the general protection against stress-induced tissue injury by habituation is an attractive hypothesis that deserves further investigation.

Taking all our results together, we conclude that acute stress, either emotional or social, has damaging effects on cellular integrity in tissues. There may be other differences between both types of stress studied here, but we can only conclude that the heart appears to be specifically protected in an intermale confrontation model of social stress. We suggest that the large increase in plasma EGF may be involved in such a protection. Repeated exposure to the same stress stimulus induced a rapid adaptative response that allows a general protection of cellular integrity. Therefore, several mechanisms exist that sequentially participate in a particular or general protection of cellular integrity against the damaging consequences of stress.

Acknowledgments

This work was supported by grant PB97-0936 from Dirección General de Enseñanza Superior e Investigación Científica, Ministerio de Educación y Ciencia, Spain.

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