Brant L. Jarrett

Peripheral and Central Effects of Repeated Social Defeat Stress: Monocyte Trafficking, Microglial Activation, and Anxiety

The development and exacerbation of depression and anxiety are associated with exposure to repeated psychosocial stress. Stress is known to affect the bidirectional communication between the nervous and immune systems leading to elevated levels of stress mediators including glucocorticoids (GCs) and catecholamines and increased trafficking of proinflammatory immune cells. Animal models, like the repeated social defeat (RSD) paradigm, were developed to explore this connection between stress and affective disorders. RSD induces activation of the sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal (HPA) axis activation, increases bone marrow production and egress of primed, GC-insensitive monocytes, and stimulates the trafficking of these cells to tissues including the spleen, lung, and brain. Recently, the observation that these monocytes have the ability to traffic to the brain perivascular spaces and parenchyma have provided mechanisms by which these peripheral cells may contribute to the prolonged anxiety-like behavior associated with RSD. The data that have been amassed from the RSD paradigm and others recapitulate many of the behavioral and immunological phenotypes associated with human anxiety disorders and may serve to elucidate potential avenues of treatment for these disorders. Here, we will discuss novel and key data that will present an overview of the neuroendocrine, immunological and behavioral responses to social stressors.

Oxytocin Mediates Social Neuroprotection After Cerebral Ischemia

Background and Purpose— The reduced incidence, morbidity, and mortality of stroke among humans with strong social support have been well-documented; however, the mechanisms underlying these socially mediated phenomena remain unknown, but may involve oxytocin (OT), a hormone that modulates some aspects of social behavior in humans and other animals. Methods— In the present study, adult male mice were socially isolated (housed individually) or socially paired (housed with an ovariectomized female); social pairing increased hypothalamic OT gene expression. To determine whether a causal relationship exists between increased OT and improved stroke outcome, mice were treated with exogenous OT or OT receptor antagonist beginning 1 week before induction of experimental stroke via middle cerebral artery occlusion. Results— Relative to social isolation, social housing attenuated infarct size, neuroinflammation, and oxidative stress following experimental stroke; the neuroprotective effect of social housing was eliminated by receptor antagonist treatment. In contrast, administration of OT to socially isolated mice reproduced the neuroprotection conferred by social housing. We further report evidence for a direct suppressive action of OT on cultured microglia, which is a key instigator in the development of neuroinflammation after cerebral ischemia. Conclusions— These findings support the hypothesis that OT mediates the neuroprotective effect of social interaction on stroke outcome.

Stress and social isolation increase vulnerability to stroke

Stress is a universal experience for living organisms. Under many circumstances activation of the hypothalamic-pituitary adrenal (HPA) axis is an adaptive response to stress. However, when stress or HPA activation is prolonged or its timing immediately precedes or coincides with an ongoing neurodegenerative process, the results can be deleterious. A causal relationship among stress, HPA axis activity, and stroke outcome exists. Stress is one of many potential triggers of ischemic stroke and sustained elevations in glucocorticoids compromise neuronal survival following an ischemic attack. Indeed, glucocorticoid exposure is a critical determinant of stroke outcome; prior exposure to stress and elevated peri-ischemic glucocorticoid concentrations are associated with poor outcome among stroke patients and in rodent models of cerebral ischemia. Likely, stress and glucocorticoid exposure exacerbate stroke by sensitizing the neuroimmune response to ischemia; stroke induces an upregulation of pro-inflammatory cytokines which contributes to migration of leukocytes into cerebral tissue and neuronal death. Social isolation also appears to compromise stroke outcome through priming of the neuroimmune system. Among individuals who survive the stroke, residual inflammation is apt to further compromise quality of life via its effect on cognitive function and affect. A better understanding of the mechanisms through which stress and social environment modulate neuroimmune function could lead to improved treatment of stroke and other neurodegenerative diseases.

Sympathetic Release of Splenic Monocytes Promotes Recurring Anxiety Following Repeated Social Defeat.

BACKGROUND Neuroinflammatory signaling may contribute to the pathophysiology of chronic anxiety disorders. Previous work showed that repeated social defeat (RSD) in mice promoted stress-sensitization that was characterized by the recurrence of anxiety following subthreshold stress 24 days after RSD. Furthermore, splenectomy following RSD prevented the recurrence of anxiety in stress-sensitized mice. We hypothesize that the spleen of RSD-exposed mice became a reservoir of primed monocytes that were released following neuroendocrine activation by subthreshold stress. METHODS Mice were subjected to subthreshold stress (i.e., single cycle of social defeat) 24 days after RSD, and immune and behavioral measures were taken. RESULTS Subthreshold stress 24 days after RSD re-established anxiety-like behavior that was associated with egress of Ly6C(hi) monocytes from the spleen. Moreover, splenectomy before RSD blocked monocyte trafficking to the brain and prevented anxiety-like behavior following subthreshold stress. Splenectomy, however, had no effect on monocyte accumulation or anxiety when determined 14 hours after RSD. In addition, splenocytes cultured 24 days after RSD exhibited a primed inflammatory phenotype. Peripheral sympathetic inhibition before subthreshold stress blocked monocyte trafficking from the spleen to the brain and prevented the re-establishment of anxiety in RSD-sensitized mice. Last, β-adrenergic antagonism also prevented splenic monocyte egress after acute stress. CONCLUSIONS The spleen served as a unique reservoir of primed monocytes that were readily released following sympathetic activation by subthreshold stress that promoted the re-establishment of anxiety. Collectively, the long-term storage of primed monocytes in the spleen may have a profound influence on recurring anxiety disorders.

Social defeat promotes a reactive endothelium in a brain region-dependent manner with increased expression of key adhesion molecules, selectins and chemokines associated with the recruitment of myeloid cells to the brain

Repeated social defeat (RSD) in mice causes myeloid cell trafficking to the brain that contributes to the development of prolonged anxiety-like behavior. Myeloid cell recruitment following RSD occurs in regions where neuronal and microglia activation is observed. Thus, we hypothesized that crosstalk between neurons, microglia, and endothelial cells contributes to brain myeloid cell trafficking via chemokine signaling and vascular adhesion molecules. Here we show that social defeat caused an exposure- and brain region-dependent increase in several key adhesion molecules and chemokines involved in the recruitment of myeloid cells. For example, RSD induced distinct patterns of adhesion molecule expression that may explain brain region-dependent myeloid cell trafficking. VCAM-1 and ICAM-1 mRNA expression were increased in an exposure-dependent manner. Furthermore, RSD-induced VCAM-1 and ICAM-1 protein expression were localized to the vasculature of brain regions implicated in fear and anxiety responses, which spatially corresponded to previously reported patterns of myeloid cell trafficking. Next, mRNA expression of additional adhesion molecules (E- and P-selectin, PECAM-1) and chemokines (CXCL1, CXCL2, CXCL12, CCL2) were determined in the brain. Social defeat induced an exposure-dependent increase in mRNA levels of E-selectin, CXCL1, and CXCL2 that increased with additional days of social defeat. While CXCL12 was unaffected by RSD, CCL2 expression was increased by six days of social defeat. Last, comparison between enriched CD11b(+) cells (microglia/macrophages) and enriched GLAST-1(+)/CD11b(-) cells (astrocytes) revealed RSD increased mRNA expression of IL-1β, CCL2, and CXCL2 in microglia/macrophages but not in astrocytes. Collectively, these data indicate that key mediators of leukocyte recruitment were increased in the brain vasculature following RSD in an exposure- and brain region-dependent manner.

Ropivacaine and Bupivacaine prevent increased pain sensitivity without altering neuroimmune activation following repeated social defeat stress

OBJECTIVE Mounting evidence indicates that stress influences the experience of pain. Exposure to psychosocial stress disrupts bi-directional communication pathways between the central nervous system and peripheral immune system, and can exacerbate the frequency and severity of pain experienced by stressed subjects. Repeated social defeat (RSD) is a murine model of psychosocial stress that recapitulates the immune and behavioral responses to stress observed in humans, including activation of stress-reactive neurocircuitry and increased pro-inflammatory cytokine production. It is unclear, however, how these stress-induced neuroimmune responses contribute to increased pain sensitivity in mice exposed to RSD. Here we used a technique of regional analgesia with local anesthetics in mice to block the development of mechanical allodynia during RSD. We next investigated the degree to which pain blockade altered stress-induced neuroimmune activation and depressive-like behavior. METHODS Following development of a mouse model of regional analgesia with discrete sensory blockade over the dorsal-caudal aspect of the spine, C57BL/6 mice were divided into experimental groups and treated with Ropivacaine (0.08%), Liposomal Bupivacaine (0.08%), or Vehicle (0.9% NaCl) prior to exposure to stress. This specific region was selected for analgesia because it is the most frequent location for aggression-associated pain due to biting during RSD. Mechanical allodynia was assessed 12 h after the first, third, and sixth day of RSD after resolution of the sensory blockade. In a separate experiment, social avoidance behavior was determined after the sixth day of RSD. Blood, bone marrow, brain, and spinal cord were collected for immunological analyses after the last day of RSD in both experiments following behavioral assessments. RESULTS RSD increased mechanical allodynia in an exposure-dependent manner that persisted for at least one week following cessation of the stressor. Mice treated with either Ropivacaine or Liposomal Bupivacaine did not develop mechanical allodynia following exposure to stress, but did develop social avoidance behavior. Neither drug affected stress-induced activation of monocytes in the bone marrow, blood, or brain. Neuroinflammatory responses developed in all treatment groups, as evidenced by elevated IL-1β mRNA levels in the brain and spinal cord after RSD. CONCLUSIONS In this study, psychosocial stress was associated with increased pain sensitivity in mice. Development of mechanical allodynia with RSD was blocked by regional analgesia with local anesthetics, Ropivacaine or Liposomal Bupivacaine. Despite blocking mechanical allodynia, these anesthetic interventions did not prevent neuroimmune activation or social avoidance associated with RSD. These data suggest that stress-induced neuroinflammatory changes are not associated with increased sensitivity to pain following RSD. Thus, blocking peripheral nociception was effective in inhibiting enhanced pain signaling without altering stress-induced immune or behavioral responses.

The development of a reactive brain endothelium after psychosocial stress

Psychosocial stress is associated with increased inflammation and higher prevalence of mental health disorders like anxiety and depression. Through the activation of several neuroendocrine pathways, psychological stress leads to significant physiological, immunological, and behavioral changes. Repeated social defeat (RSD), a murine model of psychosocial stress, recapitulates many of the behavioral and immunological effects observed in humans, including increased circulating cytokines, immune cell recruitment, and prolonged anxiety-like behavior. The aim of this study was to elucidate the mechanisms underlying stress-induced immune cell trafficking to the brain that leads to the development of a reactive endothelium and behavioral changes. We show that RSD caused an exposure-dependent increase in the gene expression of ICAM1, VCAM1, E-selectin, CXCL1, and CXCL2 in the brain that increased with additional days of stress. RSD-induced ICAM1 and VCAM1 protein expression were localized to the vasculature of brain regions implicated in fear and anxiety responses, which spatially corresponded to previously reported patterns of myeloid cell trafficking. Comparison between enriched CD11b+ cells (microglia/macrophages) and enriched GLAST-1+/CD11b- cells (astrocytes) revealed that RSD increased the gene expression of IL-1 beta, CCL2, and CXCL2 in microglia/macrophages, but not astrocytes. Collectively, these data indicate that critical adhesion mediators are increased in the brain vasculature following RSD. This study begins to establish a mechanism by which the brain facilitates stress-induced immune cell recruitment that may underlie anxiety and mood disorders.

80. Minocycline attenuates stress-induced changes in leukocyte activation and anxiety-like behavior

Repeated social defeat (RSD) is a murine stress model that recapitulates many key behavioral, immunological, and physiological changes observed in humans exposed to psychological stressors. Exposure to RSD increases circulating monocytes, macrophage recruitment to the brain, and activation of microglia, resulting in an increase in anxiety-like behavior. Minocycline is an antibiotic that readily crosses the blood–brain-barrier and has anti-inflammatory properties, down regulating microglial pro-inflammatory cytokine output within the central nervous system. Thus, the objective of this study was to determine if suppressing microglia activation with minocycline prevents the RSD-induced alterations in immune function and anxiety-like behavior. In these experiments, daily oral administration of minocycline was given to C57bl/6 mice beginning two days prior to RSD. Here we show that minocycline attenuated RSD-induced alterations in myeloid cell trafficking, macrophage re-distribution and anxiety-like behavior. For example, minocycline prevented stress-induced increases in macrophage trafficking to the brain. In addition, mice treated with minocycline did not have stress-induced increases in circulating leukocytes or splenomegaly. Activation of circulating monocytes and granulocytes by RSD was also attenuated by minocycline. Furthermore, minocycline pretreatment blocked the induction of anxiety-like behavior after RSD. Collectively these data indicate that minocycline ameliorates stress-induced anxiety-like behavior by attenuating cellular redistribution and activation in the periphery and macrophage trafficking to the brain. Grants: R01MH097243-02 R01MH093473-03.

Sympathetic release of splenic monocytes promotes recurring anxiety following repeated social defeat

Neuroinflammatory signaling may contribute to the pathophysiology of chronic anxiety disorders. Previous work showed that repeated social defeat (RSD) in mice promoted stress-sensitization that was characterized by the recurrence of anxiety following sub-threshold stress 24 days after RSD. Furthermore, splenectomy following RSD prevented the recurrence of anxiety in stress-sensitized (SS) mice. We hypothesize that the spleen of RSD-exposed mice became a reservoir of primed monocytes that were released following neuroendocrine activation by sub-threshold stress. Sub-threshold stress 24 days after RSD re-established anxiety-like behavior that was associated with egress of Ly6Chi monocytes from the spleen. Moreover, splenectomy prior to RSD blocked monocyte trafficking and prevented the recurrence of anxiety-like behavior in sensitized mice. Splenectomy, however, had no effect on monocyte accumulation or anxiety when determined 14 hours after RSD. In addition, splenocytes cultured 24 days after RSD exhibited a primed inflammatory phenotype. Next, treatment with a peripheral sympathetic inhibitor prior to sub-threshold stress blocked monocyte redistribution and prevented the re-establishment of anxiety in RSD-sensitized mice. Additionally, increased availability of releasable monocyte was associated with monocyte-progenitor proliferation within the spleen. Collectively, these data show that the spleen is capable of producing and storing primed monocytes that promote exaggerated behavioral responses to acute stress, even many days after a sensitizing event.

110. Induction of leukocyte adhesion mediators in the neurovascular unit after repeated social defeat

Repeated social defeat (RSD) is a model of psychosocial stress that activates several neuroendocrine pathways resulting in significant physiological, immunological, and behavioral changes. For instance, RSD promotes microglia activation, anxiety-like behavior, and macrophage trafficking to the brain. Although RSD promotes myeloid cell trafficking to brain-specific regions associated with threat and appraisal (i.e., prefrontal cortex, amygdala), the process by which this occurs is unknown. Because microglia and neuronal activation following RSD is region-specific, we hypothesize that adhesion molecule expression on the neurovascular unit facilitates the recruitment of myeloid cells to the brain with stress. Here we show that RSD caused an exposure- and brain region-dependent increase in mRNA of ICAM1 and VCAM1, two key cell adhesion molecules involved in leukocyte trafficking, especially in brain regions associated with fear and anxiety responses. IHC analysis from WT and BM-chimera mice confirmed increases in ICAM1 and VCAM1 protein in the brain after RSD. Next, the mRNA expression of several chemokines, selectins, and MMPs were determined in specific brain areas and revealed robust differences in distinct selectins and chemokines in stressed mice. For example, CXCR2 and its ligands CXCL1 and CXCL2 showed an exposure- and brain-region dependent induction after RSD. Collectively, these data indicate that key adhesion mediators are increased in the brain vasculature following RSD and that the pattern of chemokine expression may underlie the mechanism of macrophage trafficking.