هیپرترمی ناشی از تعلیق دم : اندازه گیری جدیدی واکنش پذیری استرسی

Abstract The tail suspension test (TST), an antidepressant screening paradigm, uses the uncontrollable, inescapable stressor of tail suspension to elicit immobility. As hyperthermia occurs following numerous stressors, hyperthermia might exist following the TST. We tested whether tail suspension induced hyperthermia (TSIH) was a distinct variable for TST. Hyperthermia was measured by two methods: a rectal probe and a subcutaneously implanted microchip (ELAMS™). In outbred ICR male mice, TSIH was robustly demonstrated compared to control (No-TST) mice. TSIH peaked after TST and remained elevated at 120 min. Among five (129/SvEvTac, A/J, C57BL/6J, NMRI and ICR) strains examined for TSIH, significant strain variations were detected. NMRI showed the highest temperature rise (2.3 °C) and A/J mice showed the lowest (0.6 °C). Sex differences were found for the C57BL/6J and NMRI strains on TSIH. TSIH and duration of immobility were not significantly correlated (r=0.22, P=0.17) in outbred mice. Both duration of TST immobility and TSIH were measured when ICR male mice were administered diazepam, imipramine (a TCA antidepressant), venlafaxine (a SNRI antidepressant), sertraline and paroxetine (SSRI antidepressants), propranolol and nadolol (β-adrenergic receptor blockers), CP-154,526 (a CRF1 receptor antagonist), and indomethacin (a cyclo-oxygenase inhibitor). Diazepam dose-dependently increased immobility and decreased TSIH. Propranolol blocked TSIH, but nadolol had no effect. Antidepressants showed more complex patterns of effects with venlafaxine, sertraline, and paroxetine inhibiting TSIH. TSIH demonstrated inter-strain variability, sex differences and a distinct pharmacology, suggesting that TSIH provides an independent, robust physiologic parameter to supplement the TST paradigm. This TSIH method may prove useful for pharmacologic, transgenic, and mechanistic studies.

. Introduction The regulation of body temperature is a highly evolved, physiologically regulated, and easily measurable trait with a complex network of homeostatic regulators (Boulant, 1997). While studies of fever have documented the roles of thermosensitive neurons in the hypothalamus, pyrogenic cytokines, and the pharmacology of anti-pyretic drugs (Mackowiak, 1997), the phenomena and etiology of “psychogenic fever” or emotional hyperthermia remain less well understood [reviewed (Oka et al., 2001)]. Emotional or stress induced hyperthermia (SIH) is the rise of body temperature following exposure to psychological stress and has been demonstrated across species (mice, rats, pigs and humans) (Borsini et al., 1989, Briese, 1995, Briese and De Quijada, 1970, Briese et al., 1991b, Groenink et al., 1994, Hajos and Engberg, 1986, Hasan and White, 1979, Kleitman, 1945, Lecci et al., 1990b, Renburn, 1960, Van der Heyden et al., 1997 and Zethof et al., 1995). In humans, anticipatory anxiety seems sufficient to induce hyperthermia (Briese, 1995 and Renburn, 1960). This rise in temperature provides one dimension of an acute stress response that varies among individuals. In rodents, mild psychological stressors inducing hyperthermia include placing animals in novel environments (e.g. an open field arena), restricting an animal's activity (e.g. restraint tube), noise, and handling animals in a variety of ways (e.g. cage change). In the “stress-induced hyperthermia” (SIH) paradigm, group-housed mice (e.g. 10–15 mice per cage) are sequentially removed at 1-min intervals and rectal temperatures are measured immediately upon removal (Lecci et al., 1990b; Zethof et al., 1994). While the first three mice show little change in temperature, by the 10th animal (10 min), there is a robust increase in temperature, which is maintained for 60-min after the procedure. Also, a singly housed mouse version of this technique has been developed with repeated disturbance as the stressor, offering an advantage when mice are a limiting resource (Van der Heyden et al., 1997). These robust SIH phenomenon in mice have been validated pharmacologically and interpreted as a stress reaction. While studying the tail suspension test (TST), a well-validated antidepressant screening test (Porsolt et al., 1987 and Steru et al., 1987), we hypothesized that hyperthermia might be induced in response to this uncontrollable, inescapable stressor. The rise in rectal temperature following tail suspension (i.e. tail suspension induced hyperthermia, TSIH) might also provide a simple method for assessing individual differences in acute stress responses in mice. The TST paradigm hangs a mouse by its tail for 6-min. A typical response in this paradigm is struggling alternating with passive immobility. The duration of immobility is accumulated throughout the 6-min period and temperature is taken after tail suspension. This duration of immobility has been the principal measure in the TST and this immobility is interpreted as a measure of “behavioral despair”. Traditionally, antidepressants decrease the duration of immobility. These initial experiments tested the validity and utility of an acute rise of body temperature following the TST as a measure of stress reactivity. Further experiments attempted to validate the paradigm by (1) demonstrating genetic and sex variation in TSIH responses and (2) characterizing TSIH pharmacologically, distinguishing this hyperthermic measure as a unique aspect of the TST response.

. Results The time course of TSIH (expressed as ΔT above pre-TST temperature) was measured with ELAMS™ microchips in outbred ICR male mice ( Fig. 1). TST markedly increased body temperature (about 2 °C) in the TST group, shown as 0-min interval. During the subsequent 120-min observation window, the TST group showed a decline in temperature but did not fully recover to pre-TST baseline. One way repeated measures ANOVA revealed a significant main effect of time interval [F (6, 66)=33.09, P<0.001]. Simple contrasts indicated ΔT at 0, 15, 30 45, 60 and 120 min were significantly higher than pre-TST (baseline) (from P<0.001 at 0 min to P=0.013 at 120 min). The minor handling stress of the No-TST group increased temperature (about 0.9 °C) at 0 min [F (6, 60)=7.32, P<0.001], maintained this mild hyperthermia (simple comparisons did not show differences between 0 min and other intervals: 15, 30, 45, 60 min) with recovery to baseline 120 min later. The time course patterns for TST and No-TST groups were significantly different as indicated by a mixed two-way ANOVA, revealing a significant interaction effect between group and time interval [F (6, 126)=3.78, P<0.01]. The time course of TSIH measured by rectal probe showed a similar pattern as that measured by ELAMS™ [F (6, 63)=18.47, P<0.001], though ΔT was lower ( Fig. 1). Time course of tail suspension induced hyperthermia (TSIH) in outbred ICR male ... Fig. 1. Time course of tail suspension induced hyperthermia (TSIH) in outbred ICR male mice. ΔT was obtained by subtracting the pre-TST (baseline) temperature from temperatures at other time intervals (0, 15, 30, 45, 60 and 120 min). Pre-TST temperature was taken immediately before TST (TST group) or No-TST (No-TST group). Temperatures at subsequent time intervals (min) were taken at 0, 15, 30, 45, 60 and 120 min after TST / No-TST. TST-ELAMS™ (N=12) and No TST-ELAMS™ (N=11) groups were measured with ELAMS™ microchips, while TST-Rectal (total N=40) were measured with a rectal probe. Figure options F1(129×NMRI) mice (n=61) were simultaneously measured via ELAMS™ and rectal probes. The two methods demonstrated excellent reliability with no significant difference in means (ΔTrectal=1.73 °C, S.D.=0.92 °C and ΔTELAMS=1.80 °C, S.D.=0.98 °C) and with a correlation coefficient of 0.88 (P<0.0001) between methods. To validate the paradigm's sensitivity in detecting variation by strain and gender, we examined baseline temperature and TSIH of five strains divided by sex (Appendix). Baseline temperatures did not show variations among strains tested by one-way ANOVAs. Fig. 2 displays ΔT (the differences between TSIH and baseline temperature) for five strains split by sex. TSIH demonstrated marked variation across strains [F (4, 109)=73.24, P<0.001]. The Swiss derived NMRI and ICR strains showed the greatest increases in ΔT, while A/J strain displayed the least. A significant sex difference was observed in C57BL/6J (t (20)=3.75, P<0.01) and NMRI (t (18)=2.78, P=0.01) strains only. In housing four mice per cage, the potential confound of SIH in sequentially removing mice was examined for TSIH. In the strains tested, the effect of removal order on TSIH was not significant by ANOVAs. Among the outbred ICR male mice (n=40), the correlation coefficient between ΔT (difference between pre-TST and post-TST temperatures) and the duration of immobility was not significant (r=0.22, P=0.17). Strain and sex variations of tail suspension induced hyperthermia (TSIH). ΔT was ... Fig. 2. Strain and sex variations of tail suspension induced hyperthermia (TSIH). ΔT was obtained by subtracting the mean temperature of the No-TST group from the mean temperature of the TST mice. Asterisks indicate significant differences between genders. **P<0.01, *P<0.05. Figure options Distinct classes of drugs probed the mechanism of tail suspension induced hyperthermia (TSIH) and interrelationships between TST immobility and TSIH in ICR male mice (Table 1). Specifically, we tested the ability of drugs to inhibit the ΔT rise of TSIH. Diazepam, an anxiolytic agent via GABAA receptors, significantly lengthened the duration of TST immobility, producing a significant dose effect on immobility in a one-way ANOVA [F (3, 42)=14.24, P<0.01]. Post hoc testing showed immobility at three doses (2.0, 3.5 and 5.0 mg/kg) were significantly longer than that for vehicle (0 mg/kg; P<0.01). In contrast, diazepam decreased ΔT dose-dependently for the TST group, while it did not affect ΔT for the No-TST control group, indicated by a significant two-way interaction effect between TST/No-TST and dose [F (2, 65)=6.31, P<0.01]. Simple contrasts showed ΔT at 3.5 and 5.0 mg/kg of diazepam were significantly lower than that at 0 mg/kg in the TST group (P<0.01). Table 1. Drug effects on TST immobility and tail-suspension induced hyperthermia (TSIH) in ICR male micea Drug Dose (mg/kg) TST immobility ΔT c(°C) Immobility(s) % from 0 mg/kgb One-way ANOVA No-TST TST ANOVA Diazepam 0 163 (31) 100 F (3, 42)=14.24, 0.21 (0.94) 2.08 (0.48) Interaction: 2.0 226 (46)** 139 P<0.01 – 1.96 (0.73) F (2, 65)=6.31, 3.5 256 (31)** 157 0.11 (0.57) 1.03 (0.56)** P<0.01 5.0 257 (42)** 158 0.14 (0.45) 0.49 (0.76)** Imipramine 0 189 (53) 100 F (5, 42)=11.69, −0.10 (0.45) 1.81 (0.85) Interaction: 3.75 170 (52) 90 P<0.01 – 1.45 (0.46) F (2, 62)=0.17, 7.5 166 (86) 88 – 1.68 (0.76) P=0.84 15 141 (40)* 75 – 1.24 (0.49) 30 133 (33)* 70 −1.53 (0.78)** 0.14 (1.69)** 60 139 (50)* 74 −1.80 (0.83)** −0.28 (1.30)** Venlafaxine 0 231 (29) 100 F (5, 55 )=11.45, 0.54 (0.74) 2.05 (0.65) One-way for TST group: 4 220 (37) 95 P<0.01 0.30 (0.37) 1.04 (1.1)** F (5, 46)=3.38, 8 198 (30)* 86 0.49 (0.61) 1.22 (0.40)* P<0.05 16 166 (31)** 72 0.29 (0.78) 1.53 (1.06 ) 24 155 (33)** 67 – 1.00 (0.52)* 32 153 (36)** 66 0.14 (0.79) 0.86 (0.83)** Sertraline 0 207 (43) 100 F (3, 35)=1.53, 0.05 (0.59) 2.23 (0.32) Interaction: 8 217 (41) 105 P=0.22 – 1.50 (1.07)* F (2, 56)=4.46, 16 203 (47) 98 −0.27 (0.94) 0.98 (0.86)** P<0.05 24 241 (44) 116 −1.35 (0.44) 1.43 (0.77)* Paroxetine 0 223 (29) 100 F (3, 27)=0.37, – 0.8 (1.1) One-way: 4 215 (38) 96 P=0.77 – 1.36 (1.24) F (3, 27)=1.93, 8 223 (30) 100 – 1.63 (0.80) P=0.15 16 233 (36) 105 – 0.30 (1.53) Propranolol 0 182 (28) 100 F (3, 41 )=0.87, 0.46 (0.45 ) 2.54 (0.76) Interaction: 5 195 (40) 107 P=0.46 – 2.00 (0.78) F (2, 68)=7.14, 10 171 (45) 94 −0.98 (1.11) 1.93 (0.50) P<0.01 20 175 (41) 96 −0.33 (0.70) −0.33 (1.49)** Nadolol 0 207 (27) 100 F (2, 28 )=1.28, – 1.93 (0.68) One-way: 10 188 (35) 91 P=0.30 – 1.95 (0.73) F (2, 27)=0.04, 20 207 (30) 100 – 1.86 (0.78) P=0.96 CP-154,526 0 190 (30) 100 F (3, 94)=1.16, – 1.97 (0.50) One-way: 10 198 (39) 104 P=0.33 – 2.01 (0.60) F (3, 94)=1.22, 20 201 (43) 106 – 1.77 (0.38) P=0.31 40 211 (44) 111 – 1.91 (0.40) Indomethacin 0 165 (36) 100 F (1, 31)=0.26, – 2.20 (0.52) F (1, 31)=8.23, 10 172 (33) 104 P=0.62 – 2.77 (0.47)** P<0.01 a Values represent means (S.D.). Each group consisted of 8–12 mice/ dose. All drugs were administered i.p. 30 min prior to TST, except for paroxetine which was administered via gavage. The 0 mg/kg dose corresponds to vehicle injection. b % from 0 mg/kg was calculated over the immobility at 0 mg/kg dose level for each drug. One-way ANOVA tested the variations of immobility across dose levels. Asterisks in the column of Immobility (s) indicate the significance levels when compared to the control group (0 mg/kg), by using the Bonferroni post hoc tests (*P<0.05, **P<0.01). c ΔT was calculated by subtracting the mean of pre-TST or pre-No-TST temperature from post-TST or post-No-TST temperature. Two-way ANOVA tested interaction effects on ΔT between the factor of TST/No-TST groups and the factor of dose levels where a No-TST group was included. One-way ANOVA tested variations of ΔT for TST group where only TST group was included. Asterisks in the last column indicate the significance levels when compared to the control group (0 mg/kg) by applying simple contrasts (*P<0.05, ** P<0.01). Table options As expected for a tricyclic antidepressant, imipramine significantly shortened the duration of TST immobility [F (5, 42)=11.69, P<0.01]. Imipramine had similar hypothermic effects in both No-TST and TST mice, shown by a main dose effect [F (5, 62)=9.75, P<0.01]. The interaction between dose and ΔT was not significant [F (2, 62)=0.17, P=0.84]. Simple contrasts indicated ΔT at 30 and 60 mg/kg in both No-TST and TST groups were lower than those of controls (0 mg/kg; P<0.01). Venlafaxine, a serotonin and norepinephrine reuptake inhibitor (SNRI), devoid of histaminergic and cholinergic antagonism, dose-dependently shortened immobility [F (5, 55)=11.45, P<0.01] at doses of 8 mg/kg (P<0.05), and 16, 24 and 32 mg/kg (P<0.01). Venlafaxine was not hypothermic as it did not affect ΔT in the No-TST group. Venlafaxine did decrease ΔT in the TST group [F (5, 46)=3.38, P<0.05] at 4 mg/kg (P<0.01), 8 mg/kg (P<0.05), 24 mg/kg (P<0.05) and 32 mg/kg (P<0.01), but not at 16 mg/kg. Sertraline, a selective serotonin reuptake inhibitor (SSRI), did not affect immobility. Nonetheless, sertraline decreased ΔT in the TST group, but not in the No-TST group, shown by a significant two-way interaction effect [F (2, 56)=4.46, P<0.05). Simple contrasts indicated ΔT at 8, 16 and 24 mg/kg were lower than that at 0 mg/kg (P<0.05 or P<0.01). Surprisingly, paroxetine, another SSRI, affected neither immobility nor ΔT in ICR male mice. However, in inbred NMRI male mice, paroxetine (16 mg/kg p.o.) significantly blocked TSIH [F (1,18)=7.36, P=0.01) without significant effect on TST mobility (data not shown). Propranolol, a lipophilic, β1- and β2-adrenergic receptor antagonist, did not affect immobility. Propranolol decreased ΔT in the TST mice, but did not affect ΔT in the No-TST mice, shown by a significant two-way interaction effects [F (2, 68)=7.14, P<0.01]. Simple contrasts indicated ΔT at 20 mg/kg of propranolol in the TST mice was significantly lower than that at 0 mg/kg (p<0.01). Nadolol, the peripherally active, hydrophilic non-specific β-blocker, showed no significant effect on either immobility or ΔT. CP-154,526, a non-peptide corticotropin-releasing factor-1 (CRF1) receptor antagonist, affected neither immobility nor ΔT. Indomethacin, a cyclo-oxygenase inhibitor, at a dosage (10 mg/kg) known to block fever mediated by prostaglandins, did not affect immobility, while showing an increased effect on ΔT [F (1, 31)=8.23, P<0.01].