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Differential severity of anxiogenic effects resulting from a brief swim or underwater trauma in adolescent male rats
Pharmacology, Biochemistry and Behavior 102 (2012) 264–268
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Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Differential severity of anxiogenic effects resulting from a brief swim or underwater trauma in adolescent male rats☆ Nicole L.T. Moore ⁎, Sangeeta Gauchan, Raymond F. Genovese Center for Military Psychiatry and Neuroscience, Behavioral Biology Branch, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, MD 20910‐7500, USA
a r t i c l e
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Article history: Received 7 February 2012 Received in revised form 25 April 2012 Accepted 5 May 2012 Available online 11 May 2012 Keywords: Adolescent Stress Underwater trauma Anxiety Glucocorticoids
a b s t r a c t Clinical studies have shown a link between early-life adversity and severity of adulthood responses to a traumatic stress event (post-traumatic stress disorder, PTSD). Despite a need for basic research, few rodent models are available to test the lasting impacts of early-life traumatic stressors. Underwater trauma (UWT) has been used previously to model traumatic stress; however, effects of this procedure have only been characterized in adulthood. Susceptibility of younger animals to physiological or psychological damage from a forced submersion procedure is unknown. A procedure involving swimming may be a stressful stimulus outside of the underwater component of the experience, as well. The acute effects of a 1-minute sham exposure (empty water tank), swim‐only, and UWT (40 s swim followed by 20 s underwater) were compared in adolescent rats at postnatal day 37. No effects on blood oxygenation or lung tissue were observed. Stepwise decreases in open arm behavior were observed on the elevated plus maze (EPM) in swim‐only rats, while UWT rats showed an immediate, lasting decrease in open arm behavior. UWT rats showed a significant decrease in basal corticosterone one week after trauma. These results show that while water immersion is a stressor, UWT causes a distinct syndrome of traumatic stress response in adolescent rats. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Post-traumatic stress disorder (PTSD) is a potential long-term outcome of exposure to intense traumatic stimuli, characterized by the hallmarks of intrusive recollection, hyperarousal, and avoidant behaviors (APA, 2000). Despite an alarming prevalence of PTSD in selected populations, such as first responders (Benedek et al., 2007) and military veterans (Milliken et al., 2007), the condition remains poorly understood. A further complication of present understanding of PTSD is the finding of an interaction effect between early-life adversity and combat exposure in post-deployment surveys of military personnel (Cabrera et al., 2007). Additionally, several studies and
Abbreviations: UWT, Underwater trauma; EPM, Elevated plus maze. ☆ Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Research was conducted in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 and/or 2011 edition. All procedures were reviewed and approved by the WRAIR Institutional Animal Care and Use Committee, and performed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. ⁎ Corresponding author at: Center for Military Psychiatry and Neuroscience, Behavioral Biology Branch, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, MD 20910, USA. Tel.: +1 301 319 9297; fax: +1 301 319 9979. E-mail address: [email protected] (N.L.T. Moore). 0091-3057/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.pbb.2012.05.002
meta-analyses of military and civilian populations have found exposure to early adverse events to be a risk factor for developing PTSD following adulthood traumatic stress exposure (Brewin et al., 2000; Iversen et al., 2008; McLaughlin et al., 2010; Ozer et al., 2003). Early-life stress has been hypothesized to alter key physiological developmental outcomes relevant to stress response, including epigenetics and neuroendocrine signaling (reviewed in Yehuda et al., 2010). While early-life stress has been surveyed in these studies, little distinction is made between juvenile stress and adolescent stress. Adolescence is a unique phase of neurobiological and behavioral development, with characteristics that are distinct from both juvenile development and adulthood (Spear, 2000). Acute and lasting outcomes of stressors delivered across differing phases of development should not be assumed to be identical. Lasting consequences of adolescent traumatic stress remain poorly understood. Preclinical models are needed to further define the underlying neurochemical mechanisms, as well as test for factors of vulnerability or resilience resulting from adolescent traumatic stress exposure. One rodent model of traumatic stress with potential uses in furthering understanding of developmental stress in lifelong PTSD vulnerability is the underwater trauma (UWT) paradigm. In this model, the rat is placed into a water tank and allowed to swim, then is briefly submerged (Richter-Levin, 1998). The UWT represents a direct uncontrollable threat: a key factor in the traumatic stress experience. The brief nature of the UWT avoids catastrophic physiological results such as drowning or airway trauma while allowing neurobehavioral effects of traumatic stress to take place. Previous UWT work in adult rats has shown acute
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and lasting decreases in open arm activity on the elevated plus maze (EPM) and impaired spatial memory in the Morris water maze (Richter-Levin, 1998), as well as acutely reduced long-term potentiation (LTP) in the dentate gyrus of hippocampus (Wang et al., 2000) and impaired contextual odor discrimination (Cohen et al., 2009), after a 30-s UWT exposure. As this is a relatively new model, no studies have yet evaluated the developmental effects of UWT using an early-life stage such as adolescence, when alterations of developmental outcomes may result from a traumatic experience. However, exposure to the water and brief swimming aspects of the UWT procedure may themselves represent a mildly aversive experience beyond the submersion aspect — particularly in adolescence, a phase of ontogeny in which temperature and respiratory mechanisms have gained some maturity, but may not yet be fully developed (Gomes et al., 2001; Hill, 1947). This potentially stressful experience during development may also result in some changes in behavior. The effects of the water exposure aspect of the UWT experience have yet to be differentiated from the effects of the underwater submersion aspect. The present study works to establish UWT as a potential model for adolescent traumatic stress by addressing the effects of UWT and swim experience during adolescence, focusing on acute and subacute effects on anxiety. Additionally, acute and subacute physiological effects resulting from brief physical submersion are addressed. 2. Materials and methods 2.1. Animals Research was conducted in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 and/or 2011 edition. All procedures were reviewed and approved by the WRAIR Institutional Animal Care and Use Committee, and performed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. Adolescent male Sprague–Dawley rats (postnatal day, P22–24) were received from Charles River Laboratories (Germantown, MD) and housed under a 12:12 light–dark cycle (lights on at 0600) and with ad libitum access to food and water. Rats were pair-housed on arrival, and allowed one week to acclimate to the facility. Before any experimental manipulations took place, rats were acclimated to daily (Monday–Friday) handling and weighing procedures for at least four additional days. Handling and weighing took place daily until the end of the experiment. All experimental exposures took place on P37. This age was chosen to reflect mid-adolescence. 2.2. Stressors Treatment groups were counterbalanced based on open arm distances travelled in the baseline behavioral session. Rats were placed into a 10-gallon Plexiglas aquarium containing approximately 12 L of normal saline at 22–23 °C. For pulsoximetry and pathology studies, UWT rats swam for 30 s in the saline, and were then gently submerged using an inverted colander. They were allowed to struggle completely under the surface for 30 s, then were removed from the tank and briefly dried. Of the seven UWT rats in this study, one rat died of undetermined causes approximately 15 min after the 30‐s submersion. Consequently, in the second experiment, the UWT procedure was changed to 40 s of swim time followed by 20 s of submersion. Swimonly rats swam for the entire 60‐s time window, then were removed from the tank and briefly dried. Water-naive rats were placed into a separate empty tank for 60 s. All rats recovered rapidly after the procedure, regardless of treatment.
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2.3. Pulsoximetry UWT and swim-only rats were briefly restrained in a Plexiglas restraint tube, and a veterinary pulsoximeter (Heska, Loveland CO) was used to detect percent saturation of peripheral oxygen (% SpO2) at the tail artery, with the sensor placed at the base of the tail. 2.4. Corticosterone Rats were briefly restrained in a Plexiglas restraint tube, and blood was collected by tail vein nick. All collections were completed between 0900 and 1100 h. Blood was stored on ice for 30 min, and then centrifuged at 3000 ×g for 30 min at 4 °C. Serum was removed and stored at −80 °C until use. Corticosterone (ng/mL) was quantified using RIA (Siemens Healthcare Diagnostics, Deerfield, IL). 2.5. Elevated plus maze Exploratory behavior was observed using a Kinder Scientific plus maze (Poway, CA). The maze consisted of four arms in a “plus” configuration and was made of tinted plastic. Each arm was 50 cm long and 10 cm wide. Closed arms (east and west) had walls 40 cm in height. Open arms (north and south) had no walls. The maze was elevated 80 cm above the floor. Testing on the EPM apparatus was conducted over five-minute sessions in dim lighting conditions (open arms 7 lx, closed arms 1–2 lx and intersection 2 lx). Photobeam tracking was used to quantify distance, time, and entries in each zone of the maze. 2.6. Lung tissue evaluation UWT and swim-only rats were sacrificed with an i.p. injection of 100 mg/kg pentobarbital. After cessation of respiration and heartbeat, rats were transported to Pathology. Following gross examination, all five lobes of lung were collected for histopathologic evaluation and fixed in 10% neutral buffered formalin. Paraffin-embedded sections (5 μm) were processed with hematoxylin and eosin (H&E) stain and evaluated microscopically for evidence of lung injury. 2.7. Statistical analyses All analyses were carried out using SAS software (SAS Institute, Cary, NC). Pulsoximetry results were compared across swim and UWT groups using a test for equivalence (TOST, two one sided t-test (Schuirmann, 1987)) with a lower bound of 30% change considered to reflect significant disruption of physiological function. Alpha of 0.025 was considered significant in the TOST. Corticosterone values and behavioral results were analyzed using mixed model repeated measures ANOVA with contrasts. Factors of group and time were analyzed, with time as the repeated measure. Alpha of 0.05 was considered to be statistically significant. 3. Results 3.1. Experiment 1 — physiological function and tissue pathology Rats were randomly assigned to one of two groups, UWT (n = 7) and Swim (n = 3). Pulsoximetry was used immediately before and after experimental manipulation and preceding euthanasia on day seven. Rats were euthanized three or seven days after swim or UWT exposure, in order to determine the presence of early and delayed lung tissue changes resulting from the exposures. Immediately following cessation of heartbeat and respiration, rats were transported to the pathology lab for evaluation. A conventional postmortem exam of organs in the abdominal, thoracic, and cranial cavities revealed no abnormalities in either swim
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or UWT rats. One rat died unexpectedly after the UWT procedure; a thorough pathological survey found no abnormalities in this subject and cause of death was not determined. Tissue was collected from each lobe of lung in the remaining rats, and stained with H&E. Minor alveolar interstitial congestion and low numbers of alveolar macrophages were found in all rats. Moderate congestion throughout the lung lobes was observed in three rats, of which two were assigned to the swim group and one to UWT. All lesions observed in lung tissue were determined to be background or incidental and not related to study conditions. Pulsoximetry was measured at the base of the tail at several time points: at baseline, immediately after experimental manipulation, 3 h after manipulation, and then seven days later. Fig. 1 shows saturation of peripheral oxygen (SpO2) values at each timepoint, with UWT rats normalized to the Swim group (percent of swim) at each measurement. Observed values remained stable across all timepoints measured in both swim and UWT groups. Test for equivalence showed that swim and UWT exposure had the same overall effects on peripheral blood oxygenation (t[6,30] = −4.65, p b 0.01). 3.2. Experiment 2 — EPM behavior and endocrine signaling effects Rats were counterbalanced into Naïve, Swim, and UWT groups (n = 12 per group) based upon baseline open arm distances measured in the EPM. Blood samples were taken to measure circulating corticosterone at baseline, immediately after experimental manipulation, and then again under low-stress conditions seven days later. In every instance, blood samples were collected prior to any behavioral testing. Serum corticosterone was measured using RIA (Fig. 2). A main effect of time was found (F[2,62]= 3.21, p b 0.05). ANOVA contrasts indicated that only in the UWT group, corticosterone was significantly lower at day seven than the baseline (baseline vs. 7 d, F[1,62] = 5.20, p b 0.05) or immediately post-UWT (post vs. 7 d, F[1,62] = 8.00, p b 0.01) timepoints. Rats were allowed to explore the EPM at baseline, 1–3 h after experimental manipulation, and again seven days later. A decreasing trend was found in basic movements across repeated EPM sessions regardless of treatment group (main effect of time, F[2,66] = 59.23, p b 0.001) (Fig. 3). Open arm entries also decreased over repeated EPM sessions (main effect of time, F[2,66] = 32.93, p b 0.0001) (Fig. 4). However, contrasts showed the UWT group exhibited an immediate and lasting decrease in open arm entries immediately after experimental manipulation (baseline vs. post: F[1,66]= 12.10, p b 0.001, baseline vs. 7 d: F[1,66]= 17.34, p b 0.0001, post vs. 7 d not significantly different) while the Swim group exhibited continued decreases at each
Fig. 1. Underwater exposure does not affect peripheral blood oxygen saturation compared to a swim-only control group at any timepoint measured. Values presented are normalized to the swim-only control group at each timepoint. Swim n = 3, UWT n = 7.
Fig. 2. Serum corticosterone changes over time in naïve, swim and UWT rats. Compared to baseline, CORT is significantly lower only in the UWT group and only at the 7 days timepoint. n = 12 per group. * indicates significantly lower than baseline, p b 0.05.
timepoint (baseline vs. post: F[1,66]= 6.93, p b 0.05, post vs. 7 d: F[1,66] = 6.41, p b 0.05). Only at day 7 did the Naïve group show significantly decreased open arm entries compared to baseline (F[1,66] = 22.41, p b 0.0001). Open arm distances additionally showed a decrease over repeated sessions (main effect of time, F[2,66] = 21.53, p b 0.0001) (Fig. 5). ANOVA contrasts showed open arm distances were significantly lower than baseline at the post and 7-day timepoints in Swim and UWT rats (swim baseline vs. post: F[1,66] = 6.22, p b 0.05, swim baseline vs. 7 d: F[1,66]= 21.98, p b 0.0001, UWT baseline vs. post: F[1,66] = 4.19, p b 0.05, UWT baseline vs. 7 d: F[1,66] = 9.24, p b 0.01). In a further step down in open arm exploration, only the Naïve and Swim groups showed a decrease in open arm distance between the post and 7-day timepoints (naïve post vs. 7 d: F[1,66] = 4.50, p b 0.05, swim post vs. 7 d: F[1,66] = 4.81, p b 0.05). The UWT group displayed maximal, lasting effect immediately with no significant change between post and 7-day timepoints. 4. Discussion Underwater trauma has been used as a traumatic stress experience in adult rats with several measures of success (Cohen et al., 2009; Richter-Levin, 1998; Wang et al., 2000). The purpose of the present
Fig. 3. Basic movements on the EPM decrease over time in Naïve, Swim, and UWT adolescent rats. Rats make significantly fewer movements in the EPM at the 7 days (non-stress conditions) timepoint compared to baseline. n = 12 per group. *** indicates significantly lower than baseline, p b 0.001.
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Fig. 4. Open arm entries on the EPM decrease over time in Naïve, Swim, and UWT adolescent rats. A stepwise decrease pattern is seen in Swim rats, with an immediate and lasting drop in UWT rats. Naïve rats showed a significant decrease only at the 7 days timepoint. n = 12 per group. * indicates significantly lower than baseline, p b 0.05, ** p b 0.01, *** p b 0.001. + indicates significantly lower than post, p b 0.05, ++ p b 0.01.
study was to characterize and adapt the UWT procedure for use as an adolescent stressor, in order to establish a model for future use in understanding the lasting effects of adolescent traumatic stress exposure. We investigated the acute and subacute effects of UWT on measures of physiologic function, lung tissue pathology, stress response, and EPM behavior. We found that the UWT procedure does not inflict lung damage or hypoxia in adolescent rats. Our results show decreased open arm exploration behaviors on the EPM 2–3 h after rats were exposed to either water condition (UWT or Swim), but not in rats exposed only to the empty water tank. Open arm exploration on day 7, however, was significantly decreased in all groups. This finding may have been related to the fact that all rats decreased total basic movements over repeated exposures in the EPM. Additionally, we found a significant decrease in circulating serum CORT under non-stressed conditions one week after UWT, but not after any other treatment. We conclude that in adolescent rats, the 20‐s submersion UWT procedure does not induce effects deleterious to basic physiological function, and does cause significant changes in EPM exploratory behavior and stress hormone signaling compared to Swim and Naïve conditions. Few studies have examined the effects of UWT in rats to date, and this is the first study to our knowledge to conduct UWT during the mid-adolescent developmental phase. Adolescence does not reflect
Fig. 5. Open arm distances on the EPM decrease over time in Naïve, Swim, and UWT adolescent rats. A stepwise decrease pattern is seen in Swim rats, with an immediate and lasting drop in UWT rats. Naïve rats showed a significant decrease only at the 7 days timepoint. n = 12 per group. * indicates significantly lower than baseline, p b 0.05, *** p b 0.001. + indicates significantly lower than post, p b 0.05.
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the completed product of development; many maturational processes remain ongoing throughout this phase of life. Thermoregulatory mechanisms are largely in place by P37, but continued improvement of temperature control occurs until P60 (Hill, 1947). Additionally, while the lungs have largely matured functionally by mid-adolescence, total lung air space surface area continues to increase from P30 to P90 even when normalized to lung volume or lung air space volume (Gomes et al., 2001). This may result in comparably greater homeostatic drive for breath, and therefore greater drive to escape an underwater situation where air is not available, as well as greater susceptibility to adverse events relative to adults. As such, an underwater stressor may represent a distinct stress in adolescents compared to adult rats. A previous study employing the EPM in adult rats (Richter-Levin, 1998) found significantly decreased open arm behavior 1 h after UWT compared to swimming control rats. This significant effect was also observed in a separate cohort, three weeks after UWT. Additionally, while previous work with UWT has shown resulting behavioral changes, this is the first study to examine tissue pathology after UWT. The lack of damage is important to note, as these findings indicate that UWT effects are not confounded by physiological damage to the subject. Water immersion is considered to be aversive to rodents, and the Morris water maze and Forced Swim Test are two commonly used behavioral measures which rely on the premise that the task is driven by motivation to escape. In adult male and female rats, serum CORT is increased over baseline after one or twelve water maze training trials, with a maximum time of 120 s per trial at 29 °C (Beiko et al., 2004). Employing a six-minute Forced Swim Test in 24 °C water prior to testing on the EPM also results in an anxiogenic effect in adult mice (Andreatini and Bacellar, 1999). However, an earlier study found that placing 6-week old rats into a bucket containing 5 cm of water at 30 °C for 7 min did not significantly change open arm behavior on the first EPM exposure 15 min later (Falter et al., 1992). The present results suggest that a 1-minute immersion in saline at 22–23 °C may have anxiogenic properties in adolescent rats. Both Swim and UWT groups demonstrated a significant decrease in open arm exploratory behavior 2–3 h after immersion or submersion. However, by the seventh day post-exposure, all groups decreased open arm exploration and no differences were noted between water-exposed and naive rats. A decreasing trend of open arm exploration over time in naïve controls suggests that repeated exposures to the EPM are anxiogenic in this age group. The experimental design incorporated three exposures to the EPM: one baseline, another after the procedure (one day after baseline), and a third exposure one week after the stressor. Increased plasma corticosterone after an EPM session suggests that the plus maze is itself a stressful experience (File et al., 1994). Additionally, EPM experience may become more anxiogenic with increasing number of exposures, so that sequential trials in the EPM may reflect differing psychological phenomena (reviewed in Carobrez and Bertoglio, 2005). In light of the finding that all rats decreased open arm exploration at day 7, as well as basic movements in general, further studies will be needed to determine the specific effects of multiple EPM trials in adolescent rats exposed to water or UWT. PTSD may be precipitated by widely variable traumatic events in humans. Accordingly, multiple differing modes of traumatic stress exposure in rodents produce lasting behavioral and physiological alterations. Numerous stressors of varying duration and intensity have been employed in rodent models of traumatic stress to date. UWT is a brief, high-intensity exposure to stress. Commonly used models such as single prolonged stress (SPS) and chronic stress exposure involve qualitatively different, longer-lasting stress exposures. Reports of chronic stress effects upon EPM behaviors show mixed results, to include increased open arm behavior (Kompagne et al., 2008; D'Aquila et al., 1994), as well as a delayed-onset decrease in open arm behavior (McGuire et al., 2010) or no effect at all (Matuszewich et al.,
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2007). SPS has been shown to produce decreased EPM open arm behavior at seven (Imanaka et al., 2006) and 14 days (Wang et al., 2010) after stressor exposure. Meanwhile, a single predator species (cat) exposure produces anxiogenic-like effects on the EPM lasting at least one week in rats and mice (Adamec et al., 2004; Adamec and Shallow, 1993). Studies describing rodent models of PTSD often interpret findings of acutely or persistently elevated circulating corticosterone as validation of the model. However, the human studies literature does not uniformly report elevated cortisol in subjects with PTSD. In fact, a range of responses are reported in human studies, and decreases in basal cortisol are commonly reported (reviewed in Raison and Miller, 2003 and in Yehuda, 2006). Meanwhile, rodent studies predominantly report increased corticosterone, with rare exceptions (Adamec, 1997). This discrepancy between preclinical and clinical studies is one example of the difficulty of using rodents to model the effects of a severe, traumatic stressor upon humans. The present study found no change in corticosterone immediately after Swim or UWT exposures and decreased basal corticosterone one week after UWT treatment, indicating a lasting depression of basal hypothalamic-pituitary axis (HPA) signaling following the experimental trauma. While this result may seem paradoxical in comparison to the existing rodent literature, it parallels results of numerous human studies, demonstrating that UWT delivers a unique traumatic stress that merits further investigation as a rodent model of PTSD. In summary, the 20‐s UWT procedure delivers a traumatic experience to adolescent rats, without causing lung damage or altering respiratory function. Decreased EPM open arm behavior and a latent decrease in circulating CORT are indicators of an effective traumatic experience. Future studies will address lasting effects of adolescent UWT exposure upon adulthood behaviors and adulthood reactivity to a distinct stressor event. Role of the Funding Source This study was supported by the Military Operational Medicine Research Program, US Army Medical Research and Materiel Command. N.L.T.M is a National Research Council Postdoctoral Fellow. Acknowledgments We thank Neel Aziz, DVM for pathology support, Christina Johnson and Christine Tobin for laboratory support, and Cynthia Kuhn, PhD for support with running the corticosterone radioimmunoassay. References Adamec R. Transmitter systems involved in neural plasticity underlying increased anxiety and defense–implications for understanding anxiety following traumatic stress. Neurosci Biobehav Rev 1997;21:755–65. Adamec RE, Shallow T. Lasting effects on rodent anxiety of a single exposure to a cat. Physiol Behav 1993;54:101–9. Adamec R, Walling S, Burton P. Long-lasting, selective, anxiogenic effects of feline predator stress in mice. Physiol Behav 2004;83:401–10. Andreatini R, Bacellar LF. The relationship between anxiety and depression in animal models: a study using the forced swimming test and elevated plus-maze. Braz J Med Biol Res 1999;32:1121–6. APA. Diagnostic and Statistical Manual of Mental Disorders DSM-IV-TR. 4th ed. Washington, DC: American Psychiatric Association; 2000.
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Differential severity of anxiogenic effects resulting from a brief swim or underwater trauma in adolescent male rats☆ Nicole L.T. Moore ⁎, Sangeeta Gauchan, Raymond F. Genovese Center for Military Psychiatry and Neuroscience, Behavioral Biology Branch, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, MD 20910‐7500, USA
a r t i c l e
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Article history: Received 7 February 2012 Received in revised form 25 April 2012 Accepted 5 May 2012 Available online 11 May 2012 Keywords: Adolescent Stress Underwater trauma Anxiety Glucocorticoids
a b s t r a c t Clinical studies have shown a link between early-life adversity and severity of adulthood responses to a traumatic stress event (post-traumatic stress disorder, PTSD). Despite a need for basic research, few rodent models are available to test the lasting impacts of early-life traumatic stressors. Underwater trauma (UWT) has been used previously to model traumatic stress; however, effects of this procedure have only been characterized in adulthood. Susceptibility of younger animals to physiological or psychological damage from a forced submersion procedure is unknown. A procedure involving swimming may be a stressful stimulus outside of the underwater component of the experience, as well. The acute effects of a 1-minute sham exposure (empty water tank), swim‐only, and UWT (40 s swim followed by 20 s underwater) were compared in adolescent rats at postnatal day 37. No effects on blood oxygenation or lung tissue were observed. Stepwise decreases in open arm behavior were observed on the elevated plus maze (EPM) in swim‐only rats, while UWT rats showed an immediate, lasting decrease in open arm behavior. UWT rats showed a significant decrease in basal corticosterone one week after trauma. These results show that while water immersion is a stressor, UWT causes a distinct syndrome of traumatic stress response in adolescent rats. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Post-traumatic stress disorder (PTSD) is a potential long-term outcome of exposure to intense traumatic stimuli, characterized by the hallmarks of intrusive recollection, hyperarousal, and avoidant behaviors (APA, 2000). Despite an alarming prevalence of PTSD in selected populations, such as first responders (Benedek et al., 2007) and military veterans (Milliken et al., 2007), the condition remains poorly understood. A further complication of present understanding of PTSD is the finding of an interaction effect between early-life adversity and combat exposure in post-deployment surveys of military personnel (Cabrera et al., 2007). Additionally, several studies and
Abbreviations: UWT, Underwater trauma; EPM, Elevated plus maze. ☆ Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Research was conducted in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 and/or 2011 edition. All procedures were reviewed and approved by the WRAIR Institutional Animal Care and Use Committee, and performed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. ⁎ Corresponding author at: Center for Military Psychiatry and Neuroscience, Behavioral Biology Branch, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, MD 20910, USA. Tel.: +1 301 319 9297; fax: +1 301 319 9979. E-mail address: [email protected] (N.L.T. Moore). 0091-3057/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.pbb.2012.05.002
meta-analyses of military and civilian populations have found exposure to early adverse events to be a risk factor for developing PTSD following adulthood traumatic stress exposure (Brewin et al., 2000; Iversen et al., 2008; McLaughlin et al., 2010; Ozer et al., 2003). Early-life stress has been hypothesized to alter key physiological developmental outcomes relevant to stress response, including epigenetics and neuroendocrine signaling (reviewed in Yehuda et al., 2010). While early-life stress has been surveyed in these studies, little distinction is made between juvenile stress and adolescent stress. Adolescence is a unique phase of neurobiological and behavioral development, with characteristics that are distinct from both juvenile development and adulthood (Spear, 2000). Acute and lasting outcomes of stressors delivered across differing phases of development should not be assumed to be identical. Lasting consequences of adolescent traumatic stress remain poorly understood. Preclinical models are needed to further define the underlying neurochemical mechanisms, as well as test for factors of vulnerability or resilience resulting from adolescent traumatic stress exposure. One rodent model of traumatic stress with potential uses in furthering understanding of developmental stress in lifelong PTSD vulnerability is the underwater trauma (UWT) paradigm. In this model, the rat is placed into a water tank and allowed to swim, then is briefly submerged (Richter-Levin, 1998). The UWT represents a direct uncontrollable threat: a key factor in the traumatic stress experience. The brief nature of the UWT avoids catastrophic physiological results such as drowning or airway trauma while allowing neurobehavioral effects of traumatic stress to take place. Previous UWT work in adult rats has shown acute
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and lasting decreases in open arm activity on the elevated plus maze (EPM) and impaired spatial memory in the Morris water maze (Richter-Levin, 1998), as well as acutely reduced long-term potentiation (LTP) in the dentate gyrus of hippocampus (Wang et al., 2000) and impaired contextual odor discrimination (Cohen et al., 2009), after a 30-s UWT exposure. As this is a relatively new model, no studies have yet evaluated the developmental effects of UWT using an early-life stage such as adolescence, when alterations of developmental outcomes may result from a traumatic experience. However, exposure to the water and brief swimming aspects of the UWT procedure may themselves represent a mildly aversive experience beyond the submersion aspect — particularly in adolescence, a phase of ontogeny in which temperature and respiratory mechanisms have gained some maturity, but may not yet be fully developed (Gomes et al., 2001; Hill, 1947). This potentially stressful experience during development may also result in some changes in behavior. The effects of the water exposure aspect of the UWT experience have yet to be differentiated from the effects of the underwater submersion aspect. The present study works to establish UWT as a potential model for adolescent traumatic stress by addressing the effects of UWT and swim experience during adolescence, focusing on acute and subacute effects on anxiety. Additionally, acute and subacute physiological effects resulting from brief physical submersion are addressed. 2. Materials and methods 2.1. Animals Research was conducted in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 and/or 2011 edition. All procedures were reviewed and approved by the WRAIR Institutional Animal Care and Use Committee, and performed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. Adolescent male Sprague–Dawley rats (postnatal day, P22–24) were received from Charles River Laboratories (Germantown, MD) and housed under a 12:12 light–dark cycle (lights on at 0600) and with ad libitum access to food and water. Rats were pair-housed on arrival, and allowed one week to acclimate to the facility. Before any experimental manipulations took place, rats were acclimated to daily (Monday–Friday) handling and weighing procedures for at least four additional days. Handling and weighing took place daily until the end of the experiment. All experimental exposures took place on P37. This age was chosen to reflect mid-adolescence. 2.2. Stressors Treatment groups were counterbalanced based on open arm distances travelled in the baseline behavioral session. Rats were placed into a 10-gallon Plexiglas aquarium containing approximately 12 L of normal saline at 22–23 °C. For pulsoximetry and pathology studies, UWT rats swam for 30 s in the saline, and were then gently submerged using an inverted colander. They were allowed to struggle completely under the surface for 30 s, then were removed from the tank and briefly dried. Of the seven UWT rats in this study, one rat died of undetermined causes approximately 15 min after the 30‐s submersion. Consequently, in the second experiment, the UWT procedure was changed to 40 s of swim time followed by 20 s of submersion. Swimonly rats swam for the entire 60‐s time window, then were removed from the tank and briefly dried. Water-naive rats were placed into a separate empty tank for 60 s. All rats recovered rapidly after the procedure, regardless of treatment.
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2.3. Pulsoximetry UWT and swim-only rats were briefly restrained in a Plexiglas restraint tube, and a veterinary pulsoximeter (Heska, Loveland CO) was used to detect percent saturation of peripheral oxygen (% SpO2) at the tail artery, with the sensor placed at the base of the tail. 2.4. Corticosterone Rats were briefly restrained in a Plexiglas restraint tube, and blood was collected by tail vein nick. All collections were completed between 0900 and 1100 h. Blood was stored on ice for 30 min, and then centrifuged at 3000 ×g for 30 min at 4 °C. Serum was removed and stored at −80 °C until use. Corticosterone (ng/mL) was quantified using RIA (Siemens Healthcare Diagnostics, Deerfield, IL). 2.5. Elevated plus maze Exploratory behavior was observed using a Kinder Scientific plus maze (Poway, CA). The maze consisted of four arms in a “plus” configuration and was made of tinted plastic. Each arm was 50 cm long and 10 cm wide. Closed arms (east and west) had walls 40 cm in height. Open arms (north and south) had no walls. The maze was elevated 80 cm above the floor. Testing on the EPM apparatus was conducted over five-minute sessions in dim lighting conditions (open arms 7 lx, closed arms 1–2 lx and intersection 2 lx). Photobeam tracking was used to quantify distance, time, and entries in each zone of the maze. 2.6. Lung tissue evaluation UWT and swim-only rats were sacrificed with an i.p. injection of 100 mg/kg pentobarbital. After cessation of respiration and heartbeat, rats were transported to Pathology. Following gross examination, all five lobes of lung were collected for histopathologic evaluation and fixed in 10% neutral buffered formalin. Paraffin-embedded sections (5 μm) were processed with hematoxylin and eosin (H&E) stain and evaluated microscopically for evidence of lung injury. 2.7. Statistical analyses All analyses were carried out using SAS software (SAS Institute, Cary, NC). Pulsoximetry results were compared across swim and UWT groups using a test for equivalence (TOST, two one sided t-test (Schuirmann, 1987)) with a lower bound of 30% change considered to reflect significant disruption of physiological function. Alpha of 0.025 was considered significant in the TOST. Corticosterone values and behavioral results were analyzed using mixed model repeated measures ANOVA with contrasts. Factors of group and time were analyzed, with time as the repeated measure. Alpha of 0.05 was considered to be statistically significant. 3. Results 3.1. Experiment 1 — physiological function and tissue pathology Rats were randomly assigned to one of two groups, UWT (n = 7) and Swim (n = 3). Pulsoximetry was used immediately before and after experimental manipulation and preceding euthanasia on day seven. Rats were euthanized three or seven days after swim or UWT exposure, in order to determine the presence of early and delayed lung tissue changes resulting from the exposures. Immediately following cessation of heartbeat and respiration, rats were transported to the pathology lab for evaluation. A conventional postmortem exam of organs in the abdominal, thoracic, and cranial cavities revealed no abnormalities in either swim
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or UWT rats. One rat died unexpectedly after the UWT procedure; a thorough pathological survey found no abnormalities in this subject and cause of death was not determined. Tissue was collected from each lobe of lung in the remaining rats, and stained with H&E. Minor alveolar interstitial congestion and low numbers of alveolar macrophages were found in all rats. Moderate congestion throughout the lung lobes was observed in three rats, of which two were assigned to the swim group and one to UWT. All lesions observed in lung tissue were determined to be background or incidental and not related to study conditions. Pulsoximetry was measured at the base of the tail at several time points: at baseline, immediately after experimental manipulation, 3 h after manipulation, and then seven days later. Fig. 1 shows saturation of peripheral oxygen (SpO2) values at each timepoint, with UWT rats normalized to the Swim group (percent of swim) at each measurement. Observed values remained stable across all timepoints measured in both swim and UWT groups. Test for equivalence showed that swim and UWT exposure had the same overall effects on peripheral blood oxygenation (t[6,30] = −4.65, p b 0.01). 3.2. Experiment 2 — EPM behavior and endocrine signaling effects Rats were counterbalanced into Naïve, Swim, and UWT groups (n = 12 per group) based upon baseline open arm distances measured in the EPM. Blood samples were taken to measure circulating corticosterone at baseline, immediately after experimental manipulation, and then again under low-stress conditions seven days later. In every instance, blood samples were collected prior to any behavioral testing. Serum corticosterone was measured using RIA (Fig. 2). A main effect of time was found (F[2,62]= 3.21, p b 0.05). ANOVA contrasts indicated that only in the UWT group, corticosterone was significantly lower at day seven than the baseline (baseline vs. 7 d, F[1,62] = 5.20, p b 0.05) or immediately post-UWT (post vs. 7 d, F[1,62] = 8.00, p b 0.01) timepoints. Rats were allowed to explore the EPM at baseline, 1–3 h after experimental manipulation, and again seven days later. A decreasing trend was found in basic movements across repeated EPM sessions regardless of treatment group (main effect of time, F[2,66] = 59.23, p b 0.001) (Fig. 3). Open arm entries also decreased over repeated EPM sessions (main effect of time, F[2,66] = 32.93, p b 0.0001) (Fig. 4). However, contrasts showed the UWT group exhibited an immediate and lasting decrease in open arm entries immediately after experimental manipulation (baseline vs. post: F[1,66]= 12.10, p b 0.001, baseline vs. 7 d: F[1,66]= 17.34, p b 0.0001, post vs. 7 d not significantly different) while the Swim group exhibited continued decreases at each
Fig. 1. Underwater exposure does not affect peripheral blood oxygen saturation compared to a swim-only control group at any timepoint measured. Values presented are normalized to the swim-only control group at each timepoint. Swim n = 3, UWT n = 7.
Fig. 2. Serum corticosterone changes over time in naïve, swim and UWT rats. Compared to baseline, CORT is significantly lower only in the UWT group and only at the 7 days timepoint. n = 12 per group. * indicates significantly lower than baseline, p b 0.05.
timepoint (baseline vs. post: F[1,66]= 6.93, p b 0.05, post vs. 7 d: F[1,66] = 6.41, p b 0.05). Only at day 7 did the Naïve group show significantly decreased open arm entries compared to baseline (F[1,66] = 22.41, p b 0.0001). Open arm distances additionally showed a decrease over repeated sessions (main effect of time, F[2,66] = 21.53, p b 0.0001) (Fig. 5). ANOVA contrasts showed open arm distances were significantly lower than baseline at the post and 7-day timepoints in Swim and UWT rats (swim baseline vs. post: F[1,66] = 6.22, p b 0.05, swim baseline vs. 7 d: F[1,66]= 21.98, p b 0.0001, UWT baseline vs. post: F[1,66] = 4.19, p b 0.05, UWT baseline vs. 7 d: F[1,66] = 9.24, p b 0.01). In a further step down in open arm exploration, only the Naïve and Swim groups showed a decrease in open arm distance between the post and 7-day timepoints (naïve post vs. 7 d: F[1,66] = 4.50, p b 0.05, swim post vs. 7 d: F[1,66] = 4.81, p b 0.05). The UWT group displayed maximal, lasting effect immediately with no significant change between post and 7-day timepoints. 4. Discussion Underwater trauma has been used as a traumatic stress experience in adult rats with several measures of success (Cohen et al., 2009; Richter-Levin, 1998; Wang et al., 2000). The purpose of the present
Fig. 3. Basic movements on the EPM decrease over time in Naïve, Swim, and UWT adolescent rats. Rats make significantly fewer movements in the EPM at the 7 days (non-stress conditions) timepoint compared to baseline. n = 12 per group. *** indicates significantly lower than baseline, p b 0.001.
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Fig. 4. Open arm entries on the EPM decrease over time in Naïve, Swim, and UWT adolescent rats. A stepwise decrease pattern is seen in Swim rats, with an immediate and lasting drop in UWT rats. Naïve rats showed a significant decrease only at the 7 days timepoint. n = 12 per group. * indicates significantly lower than baseline, p b 0.05, ** p b 0.01, *** p b 0.001. + indicates significantly lower than post, p b 0.05, ++ p b 0.01.
study was to characterize and adapt the UWT procedure for use as an adolescent stressor, in order to establish a model for future use in understanding the lasting effects of adolescent traumatic stress exposure. We investigated the acute and subacute effects of UWT on measures of physiologic function, lung tissue pathology, stress response, and EPM behavior. We found that the UWT procedure does not inflict lung damage or hypoxia in adolescent rats. Our results show decreased open arm exploration behaviors on the EPM 2–3 h after rats were exposed to either water condition (UWT or Swim), but not in rats exposed only to the empty water tank. Open arm exploration on day 7, however, was significantly decreased in all groups. This finding may have been related to the fact that all rats decreased total basic movements over repeated exposures in the EPM. Additionally, we found a significant decrease in circulating serum CORT under non-stressed conditions one week after UWT, but not after any other treatment. We conclude that in adolescent rats, the 20‐s submersion UWT procedure does not induce effects deleterious to basic physiological function, and does cause significant changes in EPM exploratory behavior and stress hormone signaling compared to Swim and Naïve conditions. Few studies have examined the effects of UWT in rats to date, and this is the first study to our knowledge to conduct UWT during the mid-adolescent developmental phase. Adolescence does not reflect
Fig. 5. Open arm distances on the EPM decrease over time in Naïve, Swim, and UWT adolescent rats. A stepwise decrease pattern is seen in Swim rats, with an immediate and lasting drop in UWT rats. Naïve rats showed a significant decrease only at the 7 days timepoint. n = 12 per group. * indicates significantly lower than baseline, p b 0.05, *** p b 0.001. + indicates significantly lower than post, p b 0.05.
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the completed product of development; many maturational processes remain ongoing throughout this phase of life. Thermoregulatory mechanisms are largely in place by P37, but continued improvement of temperature control occurs until P60 (Hill, 1947). Additionally, while the lungs have largely matured functionally by mid-adolescence, total lung air space surface area continues to increase from P30 to P90 even when normalized to lung volume or lung air space volume (Gomes et al., 2001). This may result in comparably greater homeostatic drive for breath, and therefore greater drive to escape an underwater situation where air is not available, as well as greater susceptibility to adverse events relative to adults. As such, an underwater stressor may represent a distinct stress in adolescents compared to adult rats. A previous study employing the EPM in adult rats (Richter-Levin, 1998) found significantly decreased open arm behavior 1 h after UWT compared to swimming control rats. This significant effect was also observed in a separate cohort, three weeks after UWT. Additionally, while previous work with UWT has shown resulting behavioral changes, this is the first study to examine tissue pathology after UWT. The lack of damage is important to note, as these findings indicate that UWT effects are not confounded by physiological damage to the subject. Water immersion is considered to be aversive to rodents, and the Morris water maze and Forced Swim Test are two commonly used behavioral measures which rely on the premise that the task is driven by motivation to escape. In adult male and female rats, serum CORT is increased over baseline after one or twelve water maze training trials, with a maximum time of 120 s per trial at 29 °C (Beiko et al., 2004). Employing a six-minute Forced Swim Test in 24 °C water prior to testing on the EPM also results in an anxiogenic effect in adult mice (Andreatini and Bacellar, 1999). However, an earlier study found that placing 6-week old rats into a bucket containing 5 cm of water at 30 °C for 7 min did not significantly change open arm behavior on the first EPM exposure 15 min later (Falter et al., 1992). The present results suggest that a 1-minute immersion in saline at 22–23 °C may have anxiogenic properties in adolescent rats. Both Swim and UWT groups demonstrated a significant decrease in open arm exploratory behavior 2–3 h after immersion or submersion. However, by the seventh day post-exposure, all groups decreased open arm exploration and no differences were noted between water-exposed and naive rats. A decreasing trend of open arm exploration over time in naïve controls suggests that repeated exposures to the EPM are anxiogenic in this age group. The experimental design incorporated three exposures to the EPM: one baseline, another after the procedure (one day after baseline), and a third exposure one week after the stressor. Increased plasma corticosterone after an EPM session suggests that the plus maze is itself a stressful experience (File et al., 1994). Additionally, EPM experience may become more anxiogenic with increasing number of exposures, so that sequential trials in the EPM may reflect differing psychological phenomena (reviewed in Carobrez and Bertoglio, 2005). In light of the finding that all rats decreased open arm exploration at day 7, as well as basic movements in general, further studies will be needed to determine the specific effects of multiple EPM trials in adolescent rats exposed to water or UWT. PTSD may be precipitated by widely variable traumatic events in humans. Accordingly, multiple differing modes of traumatic stress exposure in rodents produce lasting behavioral and physiological alterations. Numerous stressors of varying duration and intensity have been employed in rodent models of traumatic stress to date. UWT is a brief, high-intensity exposure to stress. Commonly used models such as single prolonged stress (SPS) and chronic stress exposure involve qualitatively different, longer-lasting stress exposures. Reports of chronic stress effects upon EPM behaviors show mixed results, to include increased open arm behavior (Kompagne et al., 2008; D'Aquila et al., 1994), as well as a delayed-onset decrease in open arm behavior (McGuire et al., 2010) or no effect at all (Matuszewich et al.,
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2007). SPS has been shown to produce decreased EPM open arm behavior at seven (Imanaka et al., 2006) and 14 days (Wang et al., 2010) after stressor exposure. Meanwhile, a single predator species (cat) exposure produces anxiogenic-like effects on the EPM lasting at least one week in rats and mice (Adamec et al., 2004; Adamec and Shallow, 1993). Studies describing rodent models of PTSD often interpret findings of acutely or persistently elevated circulating corticosterone as validation of the model. However, the human studies literature does not uniformly report elevated cortisol in subjects with PTSD. In fact, a range of responses are reported in human studies, and decreases in basal cortisol are commonly reported (reviewed in Raison and Miller, 2003 and in Yehuda, 2006). Meanwhile, rodent studies predominantly report increased corticosterone, with rare exceptions (Adamec, 1997). This discrepancy between preclinical and clinical studies is one example of the difficulty of using rodents to model the effects of a severe, traumatic stressor upon humans. The present study found no change in corticosterone immediately after Swim or UWT exposures and decreased basal corticosterone one week after UWT treatment, indicating a lasting depression of basal hypothalamic-pituitary axis (HPA) signaling following the experimental trauma. While this result may seem paradoxical in comparison to the existing rodent literature, it parallels results of numerous human studies, demonstrating that UWT delivers a unique traumatic stress that merits further investigation as a rodent model of PTSD. In summary, the 20‐s UWT procedure delivers a traumatic experience to adolescent rats, without causing lung damage or altering respiratory function. Decreased EPM open arm behavior and a latent decrease in circulating CORT are indicators of an effective traumatic experience. Future studies will address lasting effects of adolescent UWT exposure upon adulthood behaviors and adulthood reactivity to a distinct stressor event. Role of the Funding Source This study was supported by the Military Operational Medicine Research Program, US Army Medical Research and Materiel Command. N.L.T.M is a National Research Council Postdoctoral Fellow. Acknowledgments We thank Neel Aziz, DVM for pathology support, Christina Johnson and Christine Tobin for laboratory support, and Cynthia Kuhn, PhD for support with running the corticosterone radioimmunoassay. References Adamec R. Transmitter systems involved in neural plasticity underlying increased anxiety and defense–implications for understanding anxiety following traumatic stress. Neurosci Biobehav Rev 1997;21:755–65. Adamec RE, Shallow T. Lasting effects on rodent anxiety of a single exposure to a cat. Physiol Behav 1993;54:101–9. Adamec R, Walling S, Burton P. Long-lasting, selective, anxiogenic effects of feline predator stress in mice. Physiol Behav 2004;83:401–10. Andreatini R, Bacellar LF. The relationship between anxiety and depression in animal models: a study using the forced swimming test and elevated plus-maze. Braz J Med Biol Res 1999;32:1121–6. APA. Diagnostic and Statistical Manual of Mental Disorders DSM-IV-TR. 4th ed. Washington, DC: American Psychiatric Association; 2000.
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