Abdominal surgery prior to chronic psychosocial stress promotes spleen cell (re)activity and glucocorticoid resistance.

There is convincing evidence from different mouse models that chronic psychosocial stress promotes splenomegaly, basal and lipopolysaccharide (LPS)-induced in vitro splenocyte activation and insensitivity towards glucocorticoids (GC) in in vitro LPS-treated splenocytes. However, we just recently showed, employing the chronic subordinate colony housing (CSC) paradigm, that bite wounds received during stressor exposure drive these stress-induced spleen changes. As skin wounds induced by planned surgery or physical trauma are more adequately reflecting what chronically stressed humans are likely to experience, it was the objective of the present study to investigate whether abdominal surgery prior to stressor exposure also promotes respective stress-induced spleen effects in the absence of any bite wounds. In line with our hypothesis, abdominal surgery prior to CSC induced splenomegaly, increased in vitro cell viability under basal and LPS conditions as well as the delta response to LPS (LPS - basal), and promoted the inability of isolated splenocytes to respond with a decreased cell viability to increasing concentrations of corticosterone following LPS-stimulation in vitro. Together with previous data, these findings demonstrate that physical injury, either in form of received bite wounds during stressor exposure or in form of abdominal surgery prior to stressor exposure, promotes the development of splenic immune activation and GC resistance.

The role of physical trauma in social stress-induced immune activation.

It has been extensively studied in several mouse models how chronic, in particular chronic psychosocial, stressors facilitate the (re)activity of the innate immune system and, consequently, drive stress-associated pathologies. Here we first summarize the resulting concept and underlying mechanisms, proposing that social stress-induced bone marrow myelopoiesis, priming, emigration and activation of newly formed myeloid cells and accumulation of these cells in the spleen, gut, brain and fracture hematoma promote septic shock, colitis, anxiety and disturbed fracture healing, respectively. We further propose and discuss the hypothesis that it is not the social character of a particular stressor that promotes splenic invasion and subsequent full activation of stress-induced myeloid cells, but rather the occurrence of bite wounds as a result of direct physical interaction. Finally, we discuss the hypothesis that it is the combination of chronic stress, regardless of whether social or non-social in nature, and any kind of planned (i.e. surgery) or unplanned (i.e. bite wounds, injury) physical trauma that drives splenic invasion and subsequent full activation of stress-induced myeloid cells.

Sensory contact to the stressor prevents recovery from structural and functional heart damage following psychosocial trauma.

Cardiovascular disorders (CVD) and posttraumatic stress disorder (PTSD) are highly comorbid, but the underlying mechanisms are not fully understood. Chronic psychosocial stress was induced in male mice by chronic subordinate colony housing (CSC), a pre-clinically validated mouse model for PTSD. Cardiac structure and function were assessed on day 20 of the CSC paradigm. Following CSC, mice were kept in different sensory contact modalities to the last aggressor for 30 days, and development of cardiac function and behavioral aspects were determined. Here we show that psychosocial trauma affects heart structure by disturbing cell-to-cell integrity of cardiomyocytes, causes tachycardia, disturbance of diurnal heart rate rhythmicity and behavioral deficits in a mouse model for PTSD. Structural and functional alterations were also found in cardiomyocytes upon in vitro treatment with pro-inflammatory cytokines typically increased after psychosocial trauma. Interestingly, sensory contact to the aggressor subsequent to psychosocial trauma prohibits functional and structural heart recovery, while isolation was beneficial for cardiac but detrimental for mental health. These findings contribute to our understanding of potential mechanisms underlying the high comorbidity of CVD and PTSD.

Chronic psychosocial stress compromises the immune response and endochondral ossification during bone fracture healing via ß-AR signaling.

Chronic psychosocial stress/trauma represents an increasing burden in our modern society and a risk factor for the development of mental disorders, including posttraumatic stress disorder (PTSD). PTSD, in turn, is highly comorbid with a plethora of inflammatory disorders and has been associated with increased bone fracture risk. Since a balanced inflammatory response after fracture is crucial for successful bone healing, we hypothesize that stress/trauma alters the inflammatory response after fracture and, consequently, compromises fracture healing. Here we show, employing the chronic subordinate colony housing (CSC) paradigm as a clinically relevant mouse model for PTSD, that mice subjected to CSC displayed increased numbers of neutrophils in the early fracture hematoma, whereas T lymphocytes and markers for cartilage-to-bone transition and angiogenesis were reduced. At late stages of fracture healing, CSC mice were characterized by decreased bending stiffness and bony bridging of the fracture callus. Strikingly, a single systemic administration of the ß-adrenoreceptor (AR) blocker propranolol before femur osteotomy prevented bone marrow mobilization of neutrophils and invasion of neutrophils into the fracture hematoma, both seen in the early phase after fracture, as well as a compromised fracture healing in CSC mice. We conclude that chronic psychosocial stress leads to an imbalanced immune response after fracture via ß-AR signaling, accompanied by disturbed fracture healing. These findings offer possibilities for clinical translation in patients suffering from PTSD and fracture.

The Role of the Intestinal Microbiome in Chronic Psychosocial Stress-Induced Pathologies in Male Mice.

Chronic psychosocial stress is a risk factor for the development of physical and mental disorders accompanied or driven by an activated immune system. Given that chronic stress-induced systemic immune activation is lacking in germ-free and antibiotics-treated mice, a causal role of the gut microbiome in the development of stress-related disorders is likely. To address this hypothesis in the current study we employed the chronic subordinate colony housing (CSC, 19 days) paradigm, a pre-clinically validated mouse model for chronic psychosocial stress, known to alter the gut microbial signature and to induce systemic low-grade inflammation, as well as physical and mental abnormalities. In detail, we investigated if (i) CSC-induced alterations can be prevented by repeated transplantation of feces (FT) from non-stressed single-housed control (SHC) mice during CSC exposure, and (ii) if the transplantation of a "stressed" CSC microbiome is able to induce CSC effects in SHC mice. Therefore, we repeatedly infused SHC and CSC recipient mice rectally with SHC donor feces at days 4 and 11 of the CSC paradigm and assessed anxiety-related behavior on day 19 as well as physiological, immunological, and bone parameters on day 20. Furthermore, SHC and CSC recipient mice were infused with CSC donor feces at respective days. To exclude effects of rectal infusions per se, another set of SHC and CSC mice was infused with saline, respectively. Our results showed that transplantation of SHC feces had mild stress-protective effects, indicated by an amelioration of CSC-induced thymus atrophy, anxiety, systemic low-grade inflammation, and alterations in bone homeostasis. Moreover, transplantation of CSC feces slightly aggravated CSC-induced systemic low-grade inflammation and alterations in bone homeostasis in SHC and/or CSC animals. In conclusion, our data provide evidence for a role of the host's microbiome in many, but not all, adverse consequences of chronic psychosocial stress. Moreover, our data are consistent with the hypothesis that transplantation of healthy feces might be a useful tool to prevent/treat different adverse outcomes of chronic stress. Finally, our data suggests that stress effects can be transferred to a certain extend via FT, proposing therapeutic approaches using FT to carefully screen fecal donors for their stress/trauma history.

Biomarkers for classification and class prediction of stress in a murine model of chronic subordination stress.

Selye defined stress as the nonspecific response of the body to any demand and thus an inherent element of all diseases. He reported that rats show adrenal hypertrophy, thymicolymphatic atrophy, and gastrointestinal ulceration, referred to as the stress triad, upon repeated exposure to nocuous agents. However, Selye's stress triad as well as its extended version including reduced body weight gain, increased plasma glucocorticoid (GC) concentrations, and GC resistance of target cells do not represent reliable discriminatory biomarkers for chronic stress. To address this, we collected multivariate biological data from male mice exposed either to the preclinically validated chronic subordinate colony housing (CSC) paradigm or to single-housed control (SHC) condition. We then used principal component analysis (PCA), top scoring pairs (tsp) and support vector machines (SVM) analyses to identify markers that discriminate between chronically stressed and non-stressed mice. PCA segregated stressed and non-stressed mice, with high loading for some of Selye's stress triad parameters. The tsp analysis, a simple and highly interpretable statistical approach, identified left adrenal weight and relative thymus weight as the pair with the highest discrimination score and prediction accuracy validated by a blinded dataset (92% p-value < 0.0001; SVM model = 83% accuracy and p-value < 0.0001). This finding clearly shows that simultaneous consideration of these two parameters can be used as a reliable biomarker of chronic stress status. Furthermore, our analysis highlights that the tsp approach is a very powerful method whose application extends beyond what has previously been reported.

Splenic glucocorticoid resistance following psychosocial stress requires physical injury.

Mice exposed to chronic subordinate colony housing (CSC) stress show glucocorticoid (GC) resistance of in vitro lipopolysaccharide (LPS)-stimulated splenocytes, increased anxiety and colitis. Similar effects were reported in wounded mice exposed to social disruption (SDR). Here we show that CSC exposure induced GC resistance in isolated and in vitro LPS-stimulated, but not unstimulated, splenocytes, and these effects were absent when CD11b+ splenocytes were depleted. Moreover, re-active coping behaviour during CSC correlated with the attacks and bites received by the resident, which in turn highly correlated with the dimension of splenic GC resistance, as with basal and LPS-induced in vitro splenocyte viability. Importantly, social stress promoted spleen cell activation, independent of bite wounds or CD11b+/CD11b- cell phenotype, whereas GC resistance was dependent on both bite wounds and the presence of CD11b+ cells. Together, our findings indicate that the mechanisms underlying splenic immune activation and GC resistance following social stress in male mice are paradigm independent and, to a large extent, dependent on wounding, which, in turn, is associated with a re-active coping style.

Chronic psychosocial stress disturbs long-bone growth in adolescent mice.

Although a strong association between psychiatric and somatic disorders is generally accepted, little is known regarding the interrelationship between mental and skeletal health. Although depressive disorders have been shown to be strongly associated with osteoporosis and increased fracture risk, evidence from post-traumatic stress disorder (PTSD) patients is less consistent. Therefore, the present study investigated the influence of chronic psychosocial stress on bone using a well-established murine model for PTSD. C57BL/6N mice (7 weeks old) were subjected to chronic subordinate colony housing (CSC) for 19 days, whereas control mice were singly housed. Anxiety-related behavior was assessed in the open-field/novel-object test, after which the mice were euthanized to assess endocrine and bone parameters. CSC mice exhibited increased anxiety-related behavior in the open-field/novel-object test, increased adrenal and decreased thymus weights, and unaffected plasma morning corticosterone. Microcomputed tomography and histomorphometrical analyses revealed significantly reduced tibia and femur lengths, increased growth-plate thickness and reduced mineral deposition at the growth plate, suggesting disturbed endochondral ossification during long-bone growth. This was associated with reduced Runx2 expression in hypertrophic chondrocytes in the growth plate. Trabecular thicknesses and bone mineral density were significantly increased in CSC compared to singly housed mice. Tyrosine hydroxylase expression was increased in bone marrow cells located at the growth plates of CSC mice, implying that local adrenergic signaling might be involved in the effects of CSC on the skeletal phenotype. In conclusion, chronic psychosocial stress negatively impacts endochondral ossification in the growth plate, affecting both longitudinal and appositional bone growth in adolescent mice.

Light and water are not simple conditions: fine tuning of animal housing in male C57BL/6 mice.

While animal housing conditions are highly controlled and standardized between different laboratories, there are still many subtle differences that unavoidably influence the host organisms and, consequently, interlaboratory reproducibility. Here, we investigated the physiological and immunological consequences between two light/dark cycle (LDC) lengths (14-h/10-h vs. 12-h/12-h LDC) and two commonly used forms of drinking water (acidified drinking water (AW) versus normal tap water (NW)) in single-housed (SH) mice. Our results indicate that SH mice bred under a 12-h/12-h LDC and NW at the supplier's facility showed increased basal morning plasma corticosterone (CORT) levels even 4 weeks after arrival at our animal facility employing a 14-h/10-h LDC and AW. This effect was even more pronounced two weeks after arrival and had abated after 8 weeks. In agreement, increased plasma adrenocorticotropic hormone (ACTH), adrenal in vitro ACTH sensitivity, as well as relative and absolute adrenal weight normalized during this 8-week exposure to the novel and unfamiliar 14-h/10-h LDC and AW. Employment of a 12-h/12-h LDC in our facility completely abrogated the CORT-elevating effects of the 14-h/10-h LDC, despite these animals drinking AW. When both the water and light conditions were matched to those at the supplier's facility, we observed a further reduction in adrenal weight, increased thymus weight, and decreased pro-inflammatory cytokine secretion of isolated and anti-CD3/28-stimulated mesenteric lymph node cells. In summary, our results indicate that prolonged alteration of both the light phase and drinking water represent severe and long-lasting stressors for laboratory rodents. These findings are of general interest for all scientists obtaining their experimental animals from conventional suppliers.

Individual differences in stress vulnerability: The role of gut pathobionts in stress-induced colitis.

Chronic subordinate colony housing (CSC), an established mouse model for chronic psychosocial stress, promotes a microbial signature of gut inflammation, characterized by expansion of Proteobacteria, specifically Helicobacter spp., in association with colitis development. However, whether the presence of Helicobacter spp. during CSC is critically required for colitis development is unknown. Notably, during previous CSC studies performed at Regensburg University (University 1), male specific-pathogen-free (SPF) CSC mice lived in continuous subordination to a physically present and Helicobacter spp.-positive resident. Therefore, it is likely that CSC mice were colonized, during the CSC procedure, with Helicobacter spp. originating from the dominant resident. In the present study we show that employing SPF CSC mice and Helicobacter spp.-free SPF residents at Ulm University (University 2), results in physiological responses that are typical of chronic psychosocial stress, including increased adrenal and decreased thymus weights, decreased adrenal in vitro adrenocorticotropic hormone (ACTH) responsiveness, and increased anxiety-related behavior. However, in contrast to previous studies that used Helicobacter spp.-positive resident mice, use of Helicobacter spp.-negative resident mice failed to induce spontaneous colitis in SPF CSC mice. Consistent with the hypothesis that the latter is due to a lack of Helicobacter spp. transmission from dominant residents to subordinate mice during the CSC procedure, colonization of SPF residents with Helicobacter typhlonius at University 2, prior to the start of the CSC model, rescued the colitis-inducing potential of CSC exposure. Furthermore, using SPF CSC mice and H. typhlonius-free SPF residents at University 1 prevented CSC-induced colitis. In summary, our data support the hypothesis that the presence or absence of exposure to certain pathobionts contributes to individual variability in susceptibility to stress-/trauma-associated pathologies and to reproducibility of stress-related outcomes between laboratories.

Chronic subordinate colony housing paradigm: A mouse model for mechanisms of PTSD vulnerability, targeted prevention, and treatment-2016 Curt Richter Award Paper.

There is considerable individual variability in vulnerability for developing posttraumatic stress disorder (PTSD); evidence suggests that this variability is related in part to genetic and environmental factors, including adverse early life experience. Interestingly, recent studies indicate that induction of chronic low-grade inflammation may be a common mechanism underlying gene and environment interactions that increase the risk for development of PTSD symptoms, and, therefore, may be a target for novel interventions for prevention or treatment of PTSD. Development of murine models with face, construct, and predictive validity would provide opportunities to investigate in detail complex genetic, environmental, endocrine, and immunologic factors that determine vulnerability to PTSD-like syndromes, and furthermore may provide mechanistic insight leading to development of novel interventions for both prevention and treatment of PTSD symptoms. Here we describe the potential use of the chronic subordinate colony housing (CSC) paradigm in mice as an adequate animal model for development of a PTSD-like syndrome and describe recent studies that suggest novel interventions for the prevention and treatment of PTSD.