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in vivo 22 : 435-440 (2008)

Corticosterone Concentrations in and Excretion in Faeces after ACTH Administration in Male Sprague-Dawley Rats HARRY SISWANTO 1, JANN HAU 1,2 , HANS-ERIK CARLSSON 1, RENÉE GOLDKUHL 1 and KLAS S.P. ABELSON 1

1Division of Comparative , Department of Neuroscience, Biomedical Centre, Uppsala University, Uppsala, Sweden; 2Department of Experimental Medicine, University of Copenhagen and National Hospital, Copenhagen, Denmark

Abstract. The aim of the present study was to analyse the homeostatic mechanisms of the body, including the immune, response to exogenous ACTH in the the endocrine, and the reproductive systems (3-5). is circulation of catheterised male rats and to investigate the therefore generally acknowledged not only as a confounding sensitivity of faecal corticosterone output as a measure of variable in experimental results, but also as a major cause of preceding elevated levels in the circulation. A total of 21 suffering in laboratory animals. Refinement of stressful adult male Sprague-Dawley rats permanently catheterised (v. procedures to which laboratory animals are subjected is jugularis externa for intravenous administration of ACTH therefore essential. In order to achieve this, development of and a. carotis communis for blood sampling), were used. adequate objective methods for the assessment and Administration of both 10 and 100 μg/kg ACTH resulted in a recognition of stress in laboratory animals is a major rapid and pronounced corticosterone increase three minutes challenge in biomedical research. Stress can be assessed by after injection (226 and 220 ng/ml, respectively), but the quantifying different endogenous stress markers, of which duration of the response was different. In the 10 μg/kg group, the most commonly investigated are . A corticosterone levels were significantly elevated for 3-90 min stressful stimulus results in an activation of the hypothalamic after injection, while in the 100 μg/kg group, the levels pituitary adrenal (HPA) axis which causes a release of remained elevated for 240 min after injection. In faeces, a corticosteroids from the (2, 6, 7). The significant increase during eight hours after ACTH injection biologically active corticosteroids are generally or was found in the group treated with 100 μg/kg, but not in the corticosterone depending on the species. In rats and mice, group treated with 10 μg/kg. In conclusion, quantification of the predominant is corticosterone (8). faecal excretion of corticosteroids is a useful non-invasive Non-invasive measures of stress may be obtained by measure of prior substantial stress (e.g. surgery), but not quantifying corticosteroid metabolites excreted in faeces. sufficiently sensitive to reveal minor stress or acute stress of This method has been shown to be useful to assess preceding short duration. stress in numerous species, including rats (9, 10) and mice (11, 12). Corticosteroids can also be quantified directly from Stress in laboratory animals significantly alters normal blood samples obtained via either manual or automated and , and thereby increases variation sampling. Manual sampling is associated with direct within and between individual animals. Combined with the interaction with the animal and causes an unwanted stress between-animal variation in stress perception, this makes response in itself (13). By contrast, automated blood stress a major source of experimental error (1, 2). Persistent sampling enables blood sampling without any interference stress is accompanied by several adverse effects on most with the animal during sampling, but this methodology requires preceding surgery associated with the insertion of catheters (14, 15). Using faecal excretion of corticosteroids as a measure of Correspondence to: Dr. Jann Hau, Department of Comparative preceding stress-associated changes in the concentration of Medicine, University of Copenhagen and National Hospital, 3 these molecules in blood is not unproblematic. Faeces is a Blegdamsvej, 2200 N Copenhagen, Denmark. Tel: +45 35 327363, Fax: +45 35 327399, e-mail: [email protected] heterogeneous material compared with blood and urine, rendering concentration measures somewhat unreliable Key Words: Non-invasive stress assessment, corticosterone, ACTH, resulting in the cumbersome practice of collecting and rats. homogenising all faeces excreted in the study period (16).

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An additional problem is the lag phase between route of administration (15). Rats were placed in an induction ® corticosteroid changes in the blood and these changes being chamber and anaesthesia was induced with 5% isoflurane (Forene ; manifested in excreted molecules in the faeces. The interval Abbot Scandinavia, Stockholm, Sweden) delivered in 100% oxygen. Once the paw withdrawal reflex was absent, the rats were shaved at between an increase in blood corticosterone and its excretion the incision sites and attached to a Simtec anaesthetic mask for into faeces may differ considerably with a range between 4 spontaneous respiration. Isoflurane was maintained at a level of 2.5- and 12 h after the stressful event (9-12, 15). Finally, the 3% to ensure adequate anaesthesia. The shaved parts were washed  magnitude and/or duration of a change in corticosteroid with iodine (Jodopax vet ; Pharmaxin AB, Helsingborg, Sweden). concentration in blood necessary to result in recordable An incision was made in the skin of the neck and a dual cannulation changes in faecal excretion are not known. was performed by catheterisation of v. jugularis externa (for The use of administration of exogenous ACTH to induce a intravenous administration of ACTH) and a. carotis communis (for blood sampling). Catheters were filled with heparinised saline to corticosterone secretion in rats is well established and has prevent blood clotting. The catheters were secured in the vessels and  been used in investigations of e.g. blood pressure during led subcutaneously through a DiLab Dacron button attached to the (17), animal models for hormonal research dorsal region of the neck. The catheters were led further through a  (18), cell physiology (19) and pain sensitivity (20). In metal spring and connected to an AccuSampler (DiLab, Lund, addition, the method is also common in clinical Sweden) for automated blood sampling. endocrinological research (21). The aim of the present study was to investigate the time Postoperative care. To ensure adequate recovery, the rats’ activity was observed regularly during the first hours after awakening and required from ACTH administration to a detectable increase daily water intake was recorded. The rats received an additional in corticosterone concentration in blood, the dynamics of the dose of buprenorphine 0.4 mg/kg in Nutella in the morning one and corticosterone response in the circulation and faeces, as well two days after surgery. as the magnitude of the response in the circulation required for detecting the subsequent changes in corticosterone ACTH administration and blood sampling. The experiments were excretion in the faeces. conducted three days after surgery, when corticosterone is known to have resumed normal cyclic rhythmicity in the circulation (15). Rats Materials and Methods were randomly divided into four treatment groups. One control group was left undisturbed during the entire experiment. The rats in Animals. All animal experiments in this study were approved by the a second control group were injected with 1 ml/kg vehicle [0.5% Uppsala Animal Ethics Committee in Uppsala, Sweden. Twenty-one bovine serum albumin (BSA) in 0.9% saline]. The rats in the two male Sprague-Dawley rats (Scanbur B&K, Sollentuna, Sweden) experimental groups were injected with 1 ml/kg ACTH (ACTH 1- with an average body weight of 368±9 g [mean±standard error of 24; PolyPeptide Laboratories Inc., Torrance, CA, USA), delivered the mean (SEM)] were used in the study. Male rats were chosen to in 0.5% BSA in 0.9% saline, in doses of 10 and 100 μg/kg, minimise confounding variables related to oestrus cycle-associated respectively. hormonal fluctuations in females. After arrival, the rats were kept Each experiment was started at 7.00 am in the morning, since the for seven days acclimatisation in animal rooms under standardised basal corticosterone levels are normally negligible at this time of conditions: Diurnal rhythm was regulated with a 12 h light 12 h day (15, 25). Three blood samples (100 μl each) were collected dark cycle with lights on from 06.00 to 18.00; temperature was kept every twentieth minute to determine the pre-experimental basal at 20±2˚C, relative humidity at 30-60%; the air was changed levels. At 8.00 am, the rats were injected with either vehicle or appro ximately 15 times per hour; and clean cages were provided ACTH. After injection, blood was collected during four hours, twice a week. Aspen chips (Finn Tapvei, Kortteinen, Finland) were followed by a blood sample every twelfth hour until the end of the used as bedding material. The animals had free access to food experiment at 8 am two days after injection. pellets (R36 Laktamin, Stockholm, Sweden) and tap water at all times. Food pellets were placed on the bedding to improve Faecal sampling. All rats were handled by the experimenter daily accessibility after surgery. Two days before surgery, the rats were after surgery to habituate them and thereby minimise stress response transferred to single housing in Macrolone type III cages and moved in connection to faecal sampling. At noon the day before ACTH to a designated laboratory with similar environmental conditions, administration, the rats were put into a clean cage. The day of where the experiment was conducted. After transferral, each rat was ACTH administration, after the first four hours of blood sampling, given Nutella ® hazelnut and chocolate for habituation to the all faecal pellets were collected. Corticosteroid metabolites from flavour to facilitate future oral administration of pre-emptive these faecal samples served as a basal excretion level to compare analgesia. All rats were handled regularly each day to habituate with levels induced by ACTH administration. After the first faecal them to the experimenter. collection (basal level) faeces were collected 8, 12, 24, 36 and 48 hours after injection. Surgery. All surgeries were commenced and completed before noon. After completion of faecal sampling, the rats were euthanized One hour before surgery, rats were treated for pre-emptive analgesia with an intravenous injection of pentobarbital (100 mg/ml in 1 ml). with buprenorphine (Temgesic ®; Schering-Plough Europe, Brussels, Belgium), 0.4 mg/kg dissolved in Nutella (2 g/kg body weight) for Corticosterone analysis. Blood samples were collected in cooled oral ad libitum administration. The dose was based on that tubes at 4˚C, after which they were centrifuged to remove blood recommended in the literature (22-24) and on our experience of this cells and obtain serum. Serum was stored at −20˚C until analysis.

436 Siswanto et al : Corticosterone in Blood and Faeces after ACTH Administration in Rats

Figure 1. Serum corticosterone concentration one hour prior to and four hours after exogenous administration of ACTH. The concentration is displayed as mean value±standard error of the mean (SEM). Significantly different from: *, pre-experimental basal level; #, non-injected control group; and ψ, vehicle-injected control group.

Faecal pellets from each collection point were combined and were stable at low levels during four hours after injection, as homogenised and faecal corticosterone was extracted as described expected. Animals in the vehicle control group did not elsewhere (10, 15, 26). Serum and faecal corticosterone was display any significant increase of corticosterone levels in quantified with -linked immunosorbent assay (ELISA) using direct connection to the injection. However, corticosterone a commercial Correlate-ELISA kit for corticosterone (Assay- Designs Inc., Ann Arbor, MI, USA) according to the manufacturer’s levels increased significantly, but not with a maximal instructions. It is unknown to which extent a combination of native response, between 60 and 120 min compared to basal levels corticosterone and immunoreactive corticosterone metabolites were as determined with ANOVA with Dunnett’s post hoc test quantified in the faecal samples. With respect to the faecal (F (13,69) =4.71; p< 0.0001), and compared to the non-injected corticosterone results, a more correct terminology would have been control group as determined with Tukey’s post hoc test “corticosterone and immunoreactive corticosterone metabolites”. (F (3,17) ≥11.97; p≤ 0.0001). However, for clarity, this nomenclature has not been used in the A rapid and pronounced corticosterone increase in serum present paper. The intra-assay coefficient of variation was 2.1% and the inter-assay coefficient was 6.3%. was observed as early as three minutes after injection in the ACTH-treated groups. The average maximum corticosterone Statistical analysis. The data are presented as mean±SEM. The level reached was similar (226 and 220 ng/ml for 10 and means and variances of all groups were compared with analysis of 100 μg/kg groups, respectively), but the duration of the variance (ANOVA) with Dunnett’s and Tukey’s post hoc tests, using response was different. In the group treated with 10 μg/kg, SPSS version 14.0. The statistics are presented as F (df1, df2) = x; the corticosterone levels were significantly higher than basal p=significance, where df1 and df2 are degrees of freedom between levels 3-90 min after injection (Dunnett’s post hoc groups and within groups respectively. P-values <0.05 were considered significant. F(13,51) =15.21; p< 0.0001), and than those of the non- injected control group 3-90 min (Tukey’s post hoc Results F(2,12) ≥8.79; p≤ 0.004). In the 100 μg/kg group, corticosterone levels were significantly higher than basal Serum corticosterone. The corticosterone levels in serum levels 10-240 min after injection (Dunnett’s post hoc after ACTH treatment are shown in Figure 1. The pre- F(13,55) =13.56; p< 0.0001) and higher than in the non- experimental basal corticosterone levels were low in all four injected control group 3-240 min (Tukey’s post hoc groups. In animals left undisturbed, the corticosterone levels F(2,12) ≥8.79; p≤ 0.004).

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Figure 3. Faecal corticosterone excretion 48 hours after ACTH Figure 2. Diurnal rhythm of serum corticosterone 12-48 hours following administration. The concentration is displayed as mean value±SEM. initiation of the experiment. The concentration is displayed as mean #Significantly different from the non-injected control group. value±SEM.

In addition, serum corticosterone levels in the ACTH- method using an AccuSampler ® system for automated blood treated groups were significantly higher compared to the sampling in the present investigation is a convenient tool to vehicle-injected control group at different occasions. The achieve stable basal corticosterone levels, making reliable group treated with 10 μg/kg ACTH had significantly higher studies of induced corticosterone levels possible. serum corticosterone levels at 10, 40 and 60 minutes It is well recognised that most human interactions with (Tukey’s post hoc F(2,13) ≥4.09; p≤ 0.044), while the group laboratory rats such as handling and restraint (13, 28), or treated with 100 μg/kg ACTH had higher levels at 180 and anaesthesia and surgery (14, 29) are more or less stressful to 240 minutes F (2,13) ≥5.02; p≤ 0.024. the animals and results in an activation of the HPA-axis. The A diurnal rhythm in corticosterone levels, with high levels present investigation demonstrates that corticosterone rapidly in the evening and low levels in the morning, was recorded increases in blood after ACTH stimulation, suggesting that in all groups from twelve hours after injection until the end the time required from a stressful event to a significant of the experiment (Figure 2). corticosterone increase is limited to a few minutes. This is supported by a previous study (28), which found that Faecal corticosteroid metabolites. The faecal corticosterone corticosterone was elevated five minutes after a stressful levels are shown in Figure 3. There was no significant event. Since corticosterone has an impact on most difference in pre-experimental basal corticosterone levels physiological functions in the body there is a significant risk between rats of the four groups. A significant increase at that the outcome of an experiment may be considerably eight hours after injection was found in the group treated biased when using blood sampling involving handling and with 100 μg/kg ACTH compared to the non-injected control restraint. In this context, automated blood sampling is (Tukey’s post hoc test F (2,11) =4.70; p=0.033), followed by a superior to manual blood sampling. This is supported by the normal diurnal rhythm with an inverted pattern compared to finding that corticosterone levels in the undisturbed control that of blood corticosterone. group were not affected during the sampling period (Figure 1), demonstrating that the blood sampling procedure per se Discussion had no effect on the HPA-axis. In addition, a previous study (13) has made similar conclusions since they found that Measuring corticosterone levels in blood is not corticosterone levels were lower in blood from catheterized uncomplicated. Corticosterone levels vary in both diurnal rats compared to levels in blood collected from rats that were and ultradian rhythms (15, 25, 27) and the blood sampling anaesthetized during each sampling occasion. procedure may cause a stress response (13). Therefore it may The maximal corticosterone serum levels recorded after the be difficult to establish reliable baseline corticosterone levels ACTH stimulation are in agreement with previous studies, to compare with induced corticosterone levels. However, the where serum corticosterone was measured in male Sprague-

438 Siswanto et al : Corticosterone in Blood and Faeces after ACTH Administration in Rats

Dawley immediately after surgery (14, 15). However, the significantly. When studying the curve of the vehicle control duration of the response is interesting. The increase in (Figure 1), a tendency for increased corticosterone secretion corticosterone lasted for 90 minutes after 10 μg/kg ACTH and can be observed during the first 40 minutes after injection. for at least four hours after 100 μg/kg ACTH. This suggests This is likely due to some animals in this group perceiving that the adrenal glands have the capacity to release significant the presence of the experimenter during the injection, or the amounts of corticosterone for a considerable time period after injection itself, as stressful, despite the absence of handling a stressful event, without being depleted. Taken into or restraint. This might have been avoided by pre-treatment consideration that increased corticosterone levels after a with in order to inhibit endogenous ACTH moderate stress situation, such as noise stress (25), or release (18, 21). However, this treatment was not chosen increased physiological parameters such as blood pressure since it was considered preferable to study intact animals and heart rate after intragastric feeding or transfer to a novel whose HPA-axis was unaffected by any . environment (30, 31) all return to normal levels within 30 In summary, the present study demonstrates that minutes, the increased corticosterone levels observed in the corticosterone is rapidly secreted within three minutes in the present study must be considered as equivalent to a blood as a response to ACTH stimulation. The maximal substantial stress response. This is important information with response in male Sprague-Dawley rats was approximately regard to the sensitivity of corticosterone quantification in 220 ng/ml serum. The adrenal glands have the capacity to faecal excretions. It is evident from the present data that an secrete large amounts of corticosterone after a stressful event increase in corticosterone excretion was detected in faeces for at least four hours. An increase in blood corticosterone eight hours after ACTH injection. However, the increase in can be detected in faeces eight hours after the event, but the faecal corticosterone was only observed after injection of 100 increase in blood must be substantial and of long duration in μg/kg ACTH, compared to the other groups. This indicates order to be detected in faecal samples using the ELISA kit that when using quantification of faecal corticosterone for employed in the present study. The use of faecal samples for stress assessment, the preceding stress response has to be stress assessment is appropriate primarily for the detection substantial in order to be detectable in faecal samples. This of prior substantial stress. finding is corroborated by previous observations where faecal corticosterone was significantly elevated after surgery (15) Acknowledgements but not after single-housing in metabolic cages (26). The present investigation was supported by generous grants from As shown in Figure s 2 and 3, both serum and faecal the Swedish Research Council. DiLab kindly donated two corticosterone levels displayed a diurnal rhythm after the AccuSampler ® systems. ACTH administration, with an expected inverted faecal pattern compared to the pattern of blood corticosterone due References to the lag phase between events in blood and these events being detectable in faeces (15). A return to normal levels 1 Hau J, Andersson E and Carlsson HE: Development and after an induced response or after a stressful event is validation of a sensitive ELISA for quantification of secretory important, since a flattening of the diurnal corticosterone IgA in rat saliva and faeces. Lab Anim 35 : 301-306, 2001. 2 Morton DB and Hau J: Welfare assessment and humane rhythm may indicate a neuroendocrine dysfunction (32, 33). endpoints. In : Handbook of Laboratory Animal Science, vol. 1, In the present study, ACTH was administered in 2nd edition. Hau J and Van Hoosier Jr L (eds.). Boca Raton, FL, physiological saline containing BSA. BSA was used to CRC Press, pp. 457-486, 2002. stabilise the ACTH peptide and to prevent adherence of 3 Glaser R, Rice J, Sheridan J, Fertel R, Stoot J, Speicher C, ACTH to syringes and tubings, as previously described (18). Pinsky D, Kotur M, Post A and Beck M: Stress-related immune However, considering the increase of corticosterone between suppression: health implications. Brain Behav Immun 5: 7-20, 60 and 120 minutes in the vehicle control, there might have 1987. 4 Klein F, Lemaire V, Sandi C, Vitiello S, Van der Logt J, Laurent been an unwanted physiological effect from the BSA. It has PE, Neveu P, Le Moal M and Mormede P: Prolonged increase been reported that BSA is pyrogenic in rabbits and that the of corticosterone secretion by chronic social stress does not fever is actually due to the albumin and not to contamination necessarily impair immune functions. Life Sci 50 : 723-731, (34). In addition, it has been reported that fever in rats is 1992. associated with corticosterone increase in the blood (35). 5 Moberg GP and Mench JA: The biology of animal stress. Basic Hence, it might have been more appropriate to choose rat principles and implications for animal welfare. Wallingford, serum as a vehicle. However, both ACTH treatments resulted Oxon, CABI, 2000. 6 O'Brien D, Tibi Opi P, Stodulski G, Saibaba P and Hau J: Stress in a rapid corticosterone response that reached maximal perception of surgical anaesthesia in rats. In : Proceedings of the levels, regardless of the dose used. This was not the case with Fifth FELASA Symposium: Welfare and Science. Bunyan J the vehicle control. It is therefore unlikely that the use of rat (ed.). London, Royal Society of Medicine Press, pp. 24-27, serum as vehicle would have changed the effects of ACTH 1995.

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