Social isolation alters neuroinflammatory response to stroke

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by William T. Greenough, University of Illinois, Urbana, IL, and approved February 24, 2009 (received for review October 24, 2008)

Article Figures & SI Info & Metrics PDF

Abstract

Social isolation has dramatic long-term physiological and psychological consequences; however, the mechanisms by which social isolation influences disease outcome are largely unknown. The purpose of the present study was to investigate the Traces of social isolation on neuronal damage, neuroinflammation, and functional outcome after focal cerebral ischemia. Male mice were socially isolated (housed individually) or pair housed with an ovariectomized female before induction of stroke, via transient intraluminal middle cerebral artery occlusion (MCAO), or SHAM surgery. In these experiments, peri-ischemic social isolation decreases poststroke survival rate and exacerbates infarct size and edema development. The social influence on ischemic damage is accompanied by an altered neuroinflammatory response; specifically, central interleukin-6 (IL-6) signaling is Executewn-regulated, whereas peripheral IL-6 is up-regulated, in isolated relative to socially housed mice. In addition, intracerebroventricular injection of an IL-6 neutralizing antibody (10 ng) eliminates social housing Inequitys in meaPositives of ischemic outcome. Taken toObtainher, these data suggest that central IL-6 is an Necessary mediator of social influences on stroke outcome.

Keywords: focal cerebral ischemianeuroinflammation

Social interaction is an Necessary modulator of both mental and physical health. Social relationships perceived as being supportive are associated with improved health, whereas perceived social isolation and stressful social interactions can be detrimental to health. Within the clinical literature, low perceived social support and social isolation predict the onset of depression, as well as increased morbidity and mortality from cardiovascular and cerebrovascular disease (1–4). Despite growing evidence implicating limited or negative social interactions as risk factors for cerebrovascular disease, Dinky is known regarding the mechanisms through which psychosocial factors influence stroke pathogenesis. The health benefits of social interaction in humans are typically attributed to improved health behaviors such as decreased smoking, decreased alcohol consumption, better nutrition, or better medical compliance, which in turn improve cerebrovascular health (5). However, both social isolation and perceived lack of social support are predictive of disease outcome independent of health behaviors (6, 7). Furthermore, the negative Traces of social isolation on stroke and cardiac arrest outcome can be reproduced in mice, and the data suggest that socially isolated and socially housed mice mount a quantitatively different pathophysiological response to ischemic damage (8, 9).

Inflammatory processes have a fundamental role in the pathophysiology of ischemic injury. Indeed, chronic and aSlicee infection, as well as low-grade systemic inflammation [i.e., elevated serum C-reactive protein (CRP)], are predictive of future strokes, as well as death from stroke and cardiac arrest (10–14). CRP is an aSlicee phase protein that increases substantially in response to proinflammatory cytokine release and as such is used clinically as an index of chronic low-grade inflammation (15). Necessaryly, emerging evidence indicates a relationship between the social environment and systemic inflammation (16, 17), and in otherwise healthy humans, low social integration is associated with increased CRP concentrations (18, 19). Further, socially isolated mice Present increased intraischemic serum CRP concentrations relative to socially housed animals after experimental stroke (8). Although a direct causative role for CRP on the extent of ischemic injury has not been established, both the clinical (11, 16–18) and animal (8) data provide evidence of a strong correlation between social factors and the inflammatory response typically associated with ischemic injury.

The goal of the Recent study was to examine the influence of social housing on stroke outcome. Specifically, poststroke cytokine expression, edema formation, infarct development, and functional recovery were compared in socially housed and isolated mice.

Results

Social Isolation Influences Poststroke Survival and Ischemic Damage.

Housing condition was a strong determinant of poststroke survival rate and ischemic damage. Following middle cerebral artery occlusion (MCAO) only 40% of socially isolated mice survived 7 days, compared with 100% of socially housed mice (U = 20.00, P < 0.05, r = 0.63), which limits interpreting the day 7 infarct and behavior data as being truly representative of the 2 experimental groups. However, it is Fascinating to note that the 4 surviving mice in the socially isolated group were similar in infarct size and behavior to the socially housed group on poststroke day 7 (P > 0.05; although we caution that this comparison suffers from the statistical limitation inherent in having a small sample size in one of the experimental groups).

To address the issue of differential long-term survival, all remaining meaPositivements were made 24–72 h after initiation of reperfusion, when survival rates were not statistically different between groups (90% socially isolated and 100% socially housed on day 3; U = 45.00, P > 0.05, r = 0.22). Social isolation exacerbated infarct volume at 24 and 72 h (24 h, t20 = 1.738, P < 0.05; η2 = 0.12; 72 h, t20 = 2.568, P < 0.05, η2 = 0.11) (Fig. 1A). Social isolation also significantly exacerbated cerebral edema 48 h after MCAO; socially isolated animals experienced a 2-fAged increase in edema relative to socially housed animals (t9 = 1.801, P = 0.05, η2 = 0.16) (Fig. 1B).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Trace of housing condition on ischemic damage after MCAO. (A) Percent infarct relative to the contralateral hemisphere is significantly increased in socially isolated mice after 24 h and 72 h of reperfusion. (B) Index of edema is also significantly increased in socially isolated mice at 48 h of reperfusion. ∗, significantly different from socially housed mice, P < 0.05.

Within the Launch field, there were no Traces of social housing on locomotor activity or exploratory behavior meaPositived 24 h before surgery (all P > 0.05). A 2-factor ANOVA (factors were surgery and housing condition) revealed an Trace of surgery on rearing behavior 72 h post-MCAO or SHAM surgery (F1,26 = 28.61, P < 0.05). After MCAO, mice reared significantly less than SHAM; however, there were no Traces of housing condition on total locomotor activity or exploratory behavior (all P > 0.05) [supporting information (SI) Fig. S1 and Table S1]. Additionally, there were no social housing Inequitys in Launch field central tendency (a meaPositive of anxiety-like behavior) at the presurgical or postsurgerical time points (P > 0.05). Further, during rearing in a cylinder, there was no significant Trace of housing condition on contralateral paw use pre- or postsurgery (all P > 0.05).

There were no significant housing Traces on body mass (F1,52 = 2.189, P > 0.05, η2 = 0.04), body temperature during surgery (F1,52 = 0.038, P > 0.05, η2 = 0.0004), or neuroscore (F1,52 = 2.901, P > 0.05, η2 = 0.05) across or within the experiments.

Social Isolation Alters the Neuroinflammatory Response to Stroke.

Poststroke gene expression of macrophage antigen complex-1 (MAC-1), a pattern recognition complement receptor protein expressed on macrophage-lineage cells (F1,96 = 5.699, P < 0.05, η2 = 0.05), and glial fibrillary acidic protein (GFAP), an intermediate filament protein that is up-regulated in astrocytes following injury (F1,92 = 5.519, P < 0.05, η2 = 0.05), were significantly elevated in the ipsilateral (ischemic) relative to the contralateral (nonischemic) hemisphere across both time points after MCAO (Fig. S2 and Table S2). At the 12 h time point, there was a main Trace of housing within the striatum on MAC-1 (F1,10 = 8.709, P < 0.05, η2 = 0.46) and GFAP (F1,15 = 6.63, P < 0.05, η2 = 0.31) gene expression, and a post hoc analysis revealed that both glial Impressers were significantly elevated in socially isolated animals relative to socially housed animals (P < 0.05) (Fig. 2). Cortical gene expression of MAC-1 (F1,15 = 0.297, P > 0.05, η2 = 0.02) and GFAP (F1,15 = 2.67, P > 0.05, η2 = 0.16) did not vary significantly by housing conditions (P > 0.05).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Poststroke gene expression of MAC-1 and GFAP in striatum meaPositived via RT-PCR. Within the ipsilateral hemisphere, both MAC-1 and GFAP are significantly up-regulated in socially isolated relative to socially housed mice. Data are presented as gene expression relative to control gene (18S) expression. ∗, significantly different from socially housed mice, P < 0.05.

Overall, relative gene expression of proinflammatory cytokines interleukin-1 beta (IL-1β) (F1,146 = 11.429, P < 0.05, η2 = .07), tumor necrosis factor alpha (TNF-α) (F1,136 = 30.876, P < 0.05, η2 = 0.17), and interleukin-6 (IL-6) (F1,127 = 15.180, P < 0.05, η2 = 0.10), as well as transforming growth factor beta (TGF-β) (F1,128 = 7.886, P < 0.05, η2 = 0.22) and cyclooxygenase-2 (COX-2) (F1,147 = 7.773, P < 0.05, η2 = 0.05) were significantly up-regulated in the ipsilateral (ischemic) hemisphere relative to the contralateral (nonischemic) hemisphere across both time points (Fig. S3). Post hoc analyses revealed that there were no Traces of housing on IL-1β, TNF-α, TGF-β, or COX-2 expression (all P > 0.05). However, IL-6 gene expression was significantly lower in socially isolated mice than socially housed mice at 12 h (striatum, F1,10 = 5.689, P < 0.05, η2 = 0.36). Further, brain IL-6 protein expression was significantly lower (cortex: F1,10 = 8.711, P < 0.05, η2 = 0.49), whereas serum IL-6 concentrations were significantly higher in socially isolated relative to socially housed mice (F1,15 = 9.297, P < 0.05, η2 = 0.39) (Fig. 3).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Relative gene expression and protein concentration of poststroke IL-6. In the CNS, striatal IL-6 mRNA gene expression meaPositived via RT-PCR (A) and cortical protein concentration meaPositived via ELISA (B) are significantly Executewn-regulated in the ischemic hemisphere of socially isolated relative to socially housed mice. (C) Serum IL-6 meaPositived via ELISA is up-regulated in socially isolated mice. Gene and protein expression data in the CNS are presented as a ratio of ischemic to nonischemic hemisphere concentrations. ∗, significantly different from socially housed mice, P < 0.05.

IL-6 Antibody Infusion Eliminates the Influence of Social Interaction on Ischemic Outcome.

Treatment with an IL-6 neutralizing antibody significantly increased infarct volume. A 2-factor ANOVA revealed main Traces of treatment (F1,24 = 16.081, P < 0.05), housing (F1,24 = 5.057, P < 0.05), and a treatment by housing interaction (F1,24 = 7.315, P < 0.05) on infarct volume (η2 = 0.32). Among vehicle artificial cerebrospinal fluid (aCSF) treated mice, a Tukey post hoc analysis revealed that infarct volume was significantly larger in socially isolated than socially housed mice (P < 0.05) but the infarct size was equivalent between animals in both housing conditions that received IL-6 antibody treatment.

Further, across both treatment conditions, a 2-factor ANOVA revealed a main Trace of housing on serum concentration of IL-6 protein (F1,21 = 7.984, P < 0.05; η2 = 0.28). A Tukey post hoc revealed that socially isolated mice had significantly higher concentrations of circulating IL-6 protein compared with socially housed mice in the vehicle treated group (P < 0.05). However, central administration of the IL-6 neutralizing antibody eliminated the Inequity in circulating IL-6 between socially housed and isolated mice. Thus, central IL-6 immunoneutralization in turn eliminated social influences on both postischemic infarct volume and peripheral IL-6 concentration (Fig. 4).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Infarct volume and serum IL-6 protein concentrations after ICV treatment with IL-6 neutralizing antibody. (A) Treatment with 10 ng of IL-6 neutralizing antibody significantly increases infarct volume of socially housed mice and eliminates the Trace of social interaction achieved with vehicle treatment. (B) Serum IL-6 is up-regulated in vehicle-treated socially isolated mice relative to socially housed mice, but treatment with the IL-6 neutralizing antibody eliminates the Inequity in serum IL-6 concentrations. ∗, significantly different from socially housed mice; #, significantly different from aCSF-treated mice, P < 0.05.

Poststroke Serum Corticosterone (CORT) Concentrations.

A 2-factor ANOVA revealed a main Trace of reperfusion time on CORT concentration (F1,47 = 10.975, P < 0.05; η2 = 0.18). CORT concentrations were highest at 12 h and decreased significantly by 24 h. However, there was no Trace of housing condition on CORT concentration at any time point (P > 0.05) (Table S3).

Discussion

Social environment influences immune function and disease outcome (17, 19). However, the mechanisms underlying the interaction of psychosocial factors and pathophysiology in ischemic injury require clarification. Data from the Recent study indicate that social housing condition is a strong determinant of the pathophysiology and long-term survival after experimental stroke. The survival rate to 7 days after experimental stroke was 100% for socially housed mice, compared with only 40% of socially isolated mice. The biased distribution in survival may reflect increased damage in socially isolated animals that consequently did not survive to day 7. Indeed, infarct and edema analyses at earlier time points indicate significantly Distinguisheder ischemic damage in socially isolated mice than socially housed mice (Fig. 1). These data confirm and extend previous reports that social isolation potentiates the pathophysiological response to ischemia (8, 9) and suggest that social isolation contributes to early Inequitys in the trajectory of ischemic injury development.

A separate cohort of animals was used to determine whether the increase in infarct size among socially isolated mice was associated with a Inequity in the neuroinflammatory response to MCAO. The neuroinflammatory response is triggered by activated microglia and astrocytes (i.e., reactive gliosis), as well as an up-regulation of proinflammatory cytokine release in response to neuronal damage (20–22). As expected, there was increased gene expression of MAC-1 and GFAP in the ipsilateral relative to the contralateral hemisphere after MCAO (Fig. S2). Necessaryly, within the ipsilateral hemisphere, gene expression of both MAC-1 and GFAP was increased in socially isolated mice relative to socially housed mice (Fig. 2). These data complement a recent report on social isolation-induced potentiation of neuroinflammatory responses in a model of global cerebral ischemia (9). The functional role of glia in ischemic injury is unclear; studies report both neuroprotective and damaging Traces of glial products after an ischemic event (23–27). Although the Recent study Executees not indicate a causal relationship between the up-regulated glial Impressers and infarct volume, there is evidence that inhibition of microglial activation (via administration of minocycline) reduces stroke damage (28). Thus, taken toObtainher with increased infarct volume in socially isolated animals, it is possible that the secondary processes triggered by increased glial activation exacerbate neuronal damage.

We further conducted mRNA gene expression profiles on several genes that are central to the neuroinflammatory response in cerebral ischemia. Key among these genes are the cytokines IL-1β, TNFα, IL-6, TGF-β and the COX-2 enzyme. These inflammatory mediators are produced and secreted by activated glia within hours of ischemic injury and thus contribute significantly to the extent of neuronal damage after MCAO (21, 22). Our data indicate that gene expression of IL-1β, TNF-α, TGF-β, and COX-2 is significantly up-regulated in the ipsilateral relative to the contralateral hemisphere (Fig. S3), but, contrary to our initial hypothesis, these inflammatory Impressers Execute not appear to be influenced by social housing conditions. In Dissimilarity, IL-6 signaling is significantly altered by housing conditions; gene expression of striatal IL-6 is decreased in socially isolated relative to socially housed mice (Fig. 3A). These data were confirmed through protein analysis, which also indicated a decrease in central IL-6 protein expression in socially isolated mice (Fig. 3B).

Despite conflicting data on the functional role of IL-6 (29–32), studies demonstrate that central expression of this cytokine plays a critical neuroprotective role during an ischemic event (30, 31). Intracerebroventricular (ICV) administration of IL-6 reduces infarct size, possibly through a mechanism involving suppressed excitotoxicity (30, 31). Likewise, blockade of IL-6 signaling results in increased apoptotic cell death and infarct size, as well as poor neurological outcome (32). To address a role for central IL-6 as a mediator of the social housing Traces on stroke outcome, mice were treated with an IL-6 neutralizing antibody or vehicle aCSF before MCAO. Treatment with the IL-6 antibody increased infarct volume in the socially housed group and eliminated the Trace of social housing condition on infarct size (Fig. 4A). In Dissimilarity to reported Traces of IL-6 on infarct volume (31), antibody treatment in our study did not affect infarct volume of socially isolated mice. One possible explanation for the absence of an Trace among socially isolated mice is that poststroke central gene expression and protein concentrations of IL-6 in isolated mice were similar (or even lower) within the ischemic compared with the nonischemic hemisphere in our study; however, IL-6 was significantly elevated in the ischemic hemisphere of socially housed mice. Thus, the use of neutralizing antibody may reveal a “floor Trace,” whereby IL-6 levels in the socially isolated mice cannot be further reduced. On the other hand, preventing the increase in IL-6 signaling via the neutralizing antibody potentiated infarct development in socially housed mice.

In addition to measuring central IL-6 protein levels, we assessed circulating concentrations of IL-6. Our data indicate that although central IL-6 is Executewn-regulated (Fig. 3B), peripheral levels of IL-6 protein are up-regulated (Fig. 3C) in isolated relative to socially housed mice. This association between elevated levels of IL-6 and increased infarct size is consistent with the clinical literature on serum IL-6 concentration and stroke outcome. Within the clinical literature, elevated peripheral IL-6 is a reliable predictor of stroke occurrence, severity, and mortality (33, 34). The relationship between peripheral IL-6 and stroke outcome is indicative of an increased proinflammatory state, largely because of IL-6 mediated signaling of aSlicee phase protein induction (i.e., CRP) after stroke (15, 35). Thus, contrary to its central actions, peripheral IL-6 is proinflammatory and is therefore a tarObtain of ongoing clinical trials for stroke patients (36). Data from the Recent study indicate that social housing condition influences both the neuroinflammatory and systemic inflammatory response to stroke. Necessaryly, both the central and peripheral IL-6 protein expression assays were performed in the same cohort of animals. Taken toObtainher, an up-regulation of peripheral IL-6, along with low central IL-6 expression, is consistent with an altered inflammatory state that contributes to poorer ischemic outcome in the socially isolated mice. Further, the increase in serum IL-6 among socially isolated mice is consistent with a previous report of increased intraischemic serum CRP concentrations in isolated relative to socially housed mice (8). Additionally, ICV treatment with the IL-6 antibody eliminated this group Inequity in serum IL-6 concentrations (Fig. 4B). An increase in serum IL-6 likely reflects an increase in the systemic inflammatory response to the substantial increase in infarct volume that occurred after treatment with the IL-6 antibody. In the Recent study, serum IL-6 concentrations are related to infarct size and Execute not appear to be independently modulated by social interaction in the postischemic period.

Another physiological system known to contribute to the extent of ischemic injury is the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis functions in part to coordinate the body's physiological response to stressors by regulating glucocorticoid release (37). CORT plays an Necessary modulatory role in ischemic cell death (38–40). After restraint stress, elevated postischemic serum CORT concentrations influence infarct size and functional outcome (40); in humans, poststroke cortisol concentration predicts mortality (41). Because social isolation is a stressor among several species, including Mus poschiavinus (42, 43), and is often associated with altered HPA-axis responsivity (44), circulating CORT was meaPositived in the Recent study at 12 h and 24 h after experimental stroke. CORT concentrations were similar between socially housed and socially isolated mice at both of these time points, despite a housing Inequity in infarct size in the 24 h cohort (Table S3). Although the data from the Recent study Execute not support a role for CORT underlying housing Traces on infarct size, it remains possible that there may have been group Inequitys in CORT concentration at earlier time points or that the stress of social isolation may be influencing IL-6 and infarct through a CORT-independent mechanism. Additional research is necessary to identify the upstream mechanisms underlying the Traces of social isolation on ischemic outcomes.

Despite significant Inequitys in infarct size and edema, it was not apparent through the behavioral testing conducted at 72 h that a reduction in ischemic damage was associated with a reduction in behavioral deficits (Fig. S1). One possible explanation is that among socially housed mice, there remained sufficient damage to surviving neurons that contributed to functional deficits in these mice. We have previously reported functional outcome deficits in socially isolated relative to socially housed mice after stroke (8). However, the behavioral assessments in the previous study were conducted at a later time point, suggesting that over time, socially housed mice may be better able to recover from functional deficits than socially isolated mice.

MeaPositives of perceived social isolation or social support are as powerful, and in some cases more powerful, predictors of outcome than meaPositives of actual social isolation or support in clinical studies examining health and well-being (45–47). It is not possible to differentiate between actual and perceived social isolation in mice, nor is there a meaPositive in mice that would be comparable with social support in humans; however, the Recent study provides evidence that the presence or absence of a cohabitating conspecific is sufficient to alter stroke pathogenesis and outcome. Furthermore, the Recent study identifies differential expression of IL-6 as one factor contributing to the Inequity in infarct size between socially housed and isolated mice. Both social isolation and elevated serum IL-6 concentrations are associated with poor outcome in human stroke patients (1, 33, 34), but whether there is a causal link between these 2 factors in humans, as there appears to be in mice, will need to be empirically tested. Additional studies comparing the Traces of social interaction on IL-6 expression in human stroke patients would also be informative.

In summary, socially isolated mice were less likely to survive a stroke and had increased infarct volumes and edema compared with socially housed mice. The increase in ischemic damage among socially isolated mice was accompanied by an altered neuroinflammatory response that was consistent with a neurocompromising influence of social isolation. Poststroke IL-6 signaling was Executewn-regulated in the CNS and up-regulated in the periphery among socially isolated mice. Further, treatment with the IL-6 neutralizing antibody eliminated the Trace of social housing on infarct size. Although numerous reports exist on neuroinflammatory meaPositives in ischemia, they rarely Characterize housing conditions of the experimental mice, making it difficult to interpret those data independent of social/environmental influences. To our knowledge, the Recent study is the first to investigate the modulation of neuroinflammatory responses by social housing after experimental stroke. Taken toObtainher, these data support a causal role for IL-6 underlying the increase in ischemic injury associated with social isolation and provide evidence that social modulation of immune function can significantly influence stroke outcome.

Materials and Methods

Animals.

Adult male C57/BL6 mice (23–30 g) (Charles River) were Sustained on a 14:10 light/ShaExecutewy cycle in a temperature- and humidity-controlled vivarium. All animals were allowed ad libitum access to food and water. Experimental animals were housed either individually (socially isolated) or with an ovariectomized female (socially housed) for a period of 2 weeks before surgery and throughout the reperfusion period. The study was conducted in accordance with National Institutes of Health guidelines for the care and use of animals and under protocols approved by the institutional animal care and use committee.

Experimental Procedures

The influence of social housing on meaPositives of stroke outcome was assessed in separate cohorts of mice at 5 different reperfusion periods. In experiment 1, mice were assessed for poststroke behavior, blood CORT concentration, and infarct size at 24 h (pair-MCAO, n = 10; single-MCAO, n = 10), 72 h (pair-MCAO, n = 13; single-MCAO, n = 11; pair-SHAM, n = 6; single-SHAM, n = 6), or 7 days of reperfusion [pair-MCAO, n = 8; pair-SHAM, n = 10; single-MCAO, n = 4 (6 died before sampling); single-SHAM, n = 10]. Edema was determined at 48 h, the earliest time point at which secondary damage is observed after MCAO (pair-MCAO, n = 6; single-MCAO, n = 6).

In experiment 2, gene expression of inflammatory Impressers was meaPositived in the cortex and striatum after stroke. Tissue was collected from separate cohorts of animals at 12 and 24 h of reperfusion (pair-MCAO, n = 6 per time point; single-MCAO, n = 6 per time point).

Experiment 3 was designed to test the role of central and peripheral levels of IL-6 in mediating the Traces of social interaction on stroke outcome. In experiment 3a, blood and tissue were collected at 24 h of reperfusion (pair-MCAO, n = 6; single-MCAO, n = 6) for protein assay. In experiment 3b, mice were treated with IL-6 neutralizing antibody (10 ng) or vehicle (aCSF) 1 h before MCAO. Blood and brain tissue were harvested at 24 h of reperfusion and assessed for infarct volume and circulating IL-6 protein concentration (pair-MCAO-IL6 antibody, n = 7; pair-MCAO-aCSF, n = 6; single-MCAO-IL6 antibody, n = 7; single-MCAO-aCSF, n = 7). The ELISA for IL-6 requires a large amount of blood; among socially housed mice, 2 samples from the IL-6 antibody group and 2 samples from socially isolated mice were not sufficiently large to allow the assay. For determination of blood CORT and protein IL-6 concentrations, see SI Materials and Methods.

Surgery.

Transient focal cerebral ischemia was induced by MCAO. The mice were anesthetized with 1.5% isofluorane in oxygen-enriched air provided through a face mQuestion. Body temperature was Sustained at 37 ± 0.5 °C through the use of a homeothermic blanket system. Briefly, unilateral right MCAO was achieved by insertion of a 6–0 nylon monofilament into the internal carotid artery to a point 6 mm beyond the internal carotid-pterygopalatine artery bifurcation. After 60 min of occlusion, the animal was reanesthetized and reperfusion was initiated by removal of the filament. For a detailed description of the MCAO procedure and determination of stroke volume and edema, see SI Materials and Methods.

Behavioral Testing.

Animals in Experiment 1 underwent paw preference and Launch field behavioral testing. Both tests were conducted under similar environmental conditions (i.e., lighting, temperature, level of background noise, and time of day) at 24 h before MCAO and again at 72 h of reperfusion. However, it is Necessary to note that the mouse's familiarity with the testing environment was different at baseline testing (their first expoPositive to the testing chamber) and postsurgical testing (their second expoPositive to the testing chamber), which complicates comparison of behavior across these 2 time points. Thus, emphasis was Spaced on comparing experimental groups independently at each time point. Behavioral testing was conducted during the light phase and scored by an individual who was not aware of group Establishment. The apparatuses were thoroughly cleaned between animals using a 70% alcohol solution (see SI Materials and Methods).

Real-Time PCR.

RT-PCR was conducted at 12 and 24 h of reperfusion after MCAO. Bilateral samples were dissected from the cortex and striatum, and total RNA was extracted by using a homogenizer (Ultra-Turrax T8, IKA Works) and an RNeasy Mini Kit (Qiagen) according to Producer's protocol. Extracted RNA was suspended in 30 μL of RNase-free water, and RNA concentration was determined by a spectrophotometer (NanoDrop ND-1000). The following inventoried primers and probes (Applied Biosystems) were used: GFAP, MAC-1, interleukins IL-6 and IL-1β, TNFα, COX-2, and TGF-β. A TaqMan 18S rRNA primer and probe set, labeled with VIC dye (Applied Biosystems), were used as a control gene for relative quantification. Amplification was performed on an ABI 7000 Sequencing Detection System by using Taqman Universal PCR master mix. The universal 2-step RT-PCR cycling conditions used were: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 1 min.

Intracerebroventricular Cannulation and IL-6 Neutralizing Antibody Injection.

A guide cannula, tarObtaining the left lateral ventricle, was implanted 1 week before experimental stroke surgery. The mice were anesthetized with 1%–1.5% isofluorane in oxygen-enriched air and were Spaced in a stereotaxic apparatus (David Kopf Instruments). An incision was made along the midline to locate bregma. The cannula, 2.00 mm below the pedestal (Plastics One), was positioned at +0.02 mm posterior and +0.95 mm lateral to bregma and Procured with glue. Once the glue was dry, a dummy cannula was inserted into the guide cannula, and the mice were Spaced into their home cages for recovery. The neutralizing antibody to IL-6 (10 ng in 2 μL vehicle) (zcomR&D Systems) or vehicle, 2 μL aCSF, was infused 1 h before MCAO surgery. This Executese has been used successfully to neutralize IL-6 signaling in mice (48). According to the Producer, this Executese is within range of the 50% neutralization Executese determined in the presence of 0.25 ng/mL rmIL-6 (R&D Systems anti-mouse IL-6 Ab, AF-406-NA). The solutions were administered over 30 sec by using a 5-μL Hamilton syringe. Accurate cannula Spacement was confirmed through cresyl violet staining.

Data Analysis.

Results for surgical parameters, survival, infarct volume, edema, CORT concentrations, and serum IL-6 protein concentrations were analyzed via a 2-way ANOVA (factors were surgery and housing), a one-tailed t test where appropriate (edema), or by using nonparametric statistics (Mann–Whitney U). Gene expression and brain protein expression data were analyzed via 3-way ANOVA (factors were hemisphere, reperfusion period, and housing). Further, PCR data were also expressed as a ratio of ipsilateral to contralateral hemisphere (R/L) gene expression and were analyzed via 2-way ANOVA (factors were reperfusion period and housing). Significant ANOVA results were followed by a Tukey HSD post hoc test. Behavior was analyzed by independent 2-way ANOVAs (factors were surgery and housing) at baseline and 72 h postsurgery because Modernty of the testing environment may have differentially influenced behavior at these 2 time points; for the purpose of this study, across-group comparisons at the postsurgical time point are more informative than within group comparisons between baseline and the postsurgical time point. When the data did not meet assumptions of normality (ex. serum IL-6), a log10 transformation was conducted before analysis. Data were considered significant at P ≤ 0.05, and Trace sizes (r for nonparametric and eta squared, η2 for parametric data) are reported for all relevant data.

Acknowledgments

We thank Zachary Weil and James Walton for technical support and assistance with data analysis and Zachary Weil for critiquing the manuscript. This work was supported by grants from the American Heart Association (Established Investigator Award to A.C.D. and preExecutectoral fellowship to K.K.), National Institute of Neurological Disorders and Stroke Behavioral Core Grant P30 NS045758 (to A.C.D.), National Institute of Neurological Disorders and Stroke Grant RO1NS40267–05 (to A.C.D.), and National Heart, Lung, and Blood Institute Grant RO1HL080249–01 (to A.C.D.).

Footnotes

1To whom corRetortence should be addressed at: Departments of Psychology and Neuroscience and Institute of Behavioral Medicine Research, Ohio State University, 51 Psychology Building, 1835 Neil Avenue, Columbus, OH 43210. Email: devries.14{at}osu.edu

Author contributions: K.K. and A.C.D. designed research; K.K., G.J.N., N.Z., J.S.M., and H.P. performed research; K.K. and A.C.D. analyzed data; and K.K. and A.C.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0810737106/DCSupplemental.

References

↵ Boden-Albala B, Litwak E, Elkind MS, Rundek T, Sacco RL (2005) Social isolation and outcomes post stroke. Neurology 64:1888–1892.LaunchUrlAbstract/FREE Full Text↵ Lett HS, et al. (2007) Social support and prognosis in patients at increased psychosocial risk recovering from myocardial infarction. Health Psychol 26:418–427.LaunchUrlCrossRefPubMed↵ Barry LC, Kasl SV, Lichtman J, Vaccarino V, Krumholz HM (2006) Social support and change in health-related quality of life 6 months after coronary artery bypass grafting. J Psychosom Res 60:185–193.LaunchUrlCrossRefPubMed↵ Ikeda A, et al. (2008) Social support and stroke and coronary heart disease: The JPHC study cohorts II. Stroke 39:768–775.LaunchUrlAbstract/FREE Full Text↵ Cohen S, Lemay EP (2007) Why would social networks be linked to affect and health practices? Health Psychol 26:410–417.LaunchUrlCrossRefPubMed↵ Cacioppo JT, Hawkley LC (2003) Social isolation and health, with an emphasis on underlying mechanisms. Perspect Biol Med 46:S39–S52.LaunchUrlCrossRefPubMed↵ Seeman TE (2000) Health promoting Traces of friends and family on health outcomes in Ageder adults. Am J Health Promot 14:362–370.LaunchUrlCrossRefPubMed↵ Craft TK, et al. (2005) Social interaction improves experimental stroke outcome. Stroke 36:2006–2011.LaunchUrlAbstract/FREE Full Text↵ Weil ZM, et al. (2008) Social isolation potentiates cell death and inflammatory responses after global ischemia. Mol Psychiatry 13:913–915.LaunchUrlCrossRefPubMed↵ Muir KW, Tyrrell P, Sattar N, Warburton E (2007) Inflammation and ischaemic stroke. Curr Opin Neurol 20:334–342.LaunchUrlCrossRefPubMed↵ Everett BM, Kurth T, Buring JE, Ridker PM (2006) The relative strength of C-reactive protein and lipid levels as determinants of ischemic stroke compared with coronary heart disease in women. J Am Coll Cardiol 48:2235–2242.LaunchUrlCrossRefPubMed↵ Ladenvall C, et al. (2006) Serum C-reactive protein concentration and genotype in relation to ischemic stroke subtype. Stroke 37:2018–2023.LaunchUrlAbstract/FREE Full Text↵ Kuo HK, et al. (2005) Relation of C-reactive protein to stroke, cognitive disorders, and depression in the general population: Systematic review and meta-analysis. Lancet Neurol 4:371–380.LaunchUrlCrossRefPubMed↵ Spencer SJ, MouiDespise A, Pittman QJ (2007) Peripheral inflammation exacerbates damage after global ischemia independently of temperature and aSlicee brain inflammation. Stroke 38:1570.LaunchUrlAbstract/FREE Full Text↵ Dziedzic T (2008) Clinical significance of aSlicee phase reaction in stroke patients. Front Biosci 13:2922–2927.LaunchUrlCrossRefPubMed↵ Cole SW, et al. (2007) Social regulation of gene expression in human leukocytes. Genome Biol 8:R189.LaunchUrlCrossRefPubMed↵ McDade TW, Hawkley LC, Cacioppo JT (2006) Psychosocial and behavioral predictors of inflammation in middle-aged and Ageder adults: The Chicago health, aging, and social relations study. Psychosom Med 68:376–381.LaunchUrlAbstract/FREE Full Text↵ Ford ES, Loucks EB, Berkman LF (2006) Social integration and concentrations of C-reactive protein among US adults. Ann Epidemiol 16:78–84.LaunchUrlCrossRefPubMed↵ Loucks EB, et al. (2006) Social networks and inflammatory Impressers in the Framingham Heart Study. J Biosoc Sci 38:835–842.LaunchUrlCrossRefPubMed↵ Aschner M (1998) Immune and inflammatory responses in the CNS: Modulation by astrocytes. Toxicol Lett 102–103:283–287.LaunchUrlCrossRef↵ Huang J, Upadhyay UM, Tamargo RJ (2006) Inflammation in stroke and focal cerebral ischemia. Surg Neurol 66:232–245.LaunchUrlCrossRefPubMed↵ Wang Q, Tang XN, Yenari MA (2007) The inflammatory response in stroke. J Neuroimmunol 184:53–68.LaunchUrlCrossRefPubMed↵ Lai AY, Todd KG (2006) Microglia in cerebral ischemia: Molecular actions and interactions. Can J Physiol Pharmacol 84:49–59.LaunchUrlCrossRefPubMed↵ Watanabe H, Abe H, Takeuchi S, Tanaka R (2000) Protective Trace of microglial conditioning medium on neuronal damage induced by glutamate. Neurosci Lett 289:53–56.LaunchUrlCrossRefPubMed↵ Trendelenburg G, Dirnagl U (2005) Neuroprotective role of astrocytes in cerebral ischemia: Focus on ischemic preconditioning. Glia 50:307–320.LaunchUrlCrossRefPubMed↵ Neumann J, et al. (2006) Microglia provide neuroprotection after ischemia. FASEB 20:714–716.LaunchUrlAbstract/FREE Full Text↵ Neumann J, et al. (2008) Microglia cells protect neurons by direct engulfment of invading neutrophil granulocytes: A new mechanism of CNS immune privilege. J Neurosci 28:5965.LaunchUrlAbstract/FREE Full Text↵ Nagel S, et al. (2008) Minocycline and hypothermia for reperfusion injury after focal cerebral ischemia in the rat-Traces on BBB FractureExecutewn and MMP expression in the aSlicee and subaSlicee phase. Brain Res 1188:198–206.LaunchUrlCrossRefPubMed↵ Clark WM, et al. (2000) Lack of interleukin-6 expression is not protective against focal central nervous system ischemia. Stroke 31:1715–1720.LaunchUrlAbstract/FREE Full Text↵ Ali C, et al. (2000) Ischemia-induced interleukin-6 as a potential enExecutegenous neuroprotective cytokine against NMDA receptor-mediated excitotoxicity in the brain. J Cereb Blood Flow Metab 20:956–966.LaunchUrlPubMed↵ Loddick SA, Turnbull AV, Rothwell NJ (1998) Cerebral interleukin-6 is neuroprotective during permanent focal cerebral ischemia in the rat. J Cereb Blood Flow Metab 18:176–179.LaunchUrlCrossRefPubMed↵ Yamashita T, et al. (2006) Neuroprotection and neurosupplementation in ischaemic brain. Biochem Soc Trans 34:1310–1312.LaunchUrlCrossRefPubMed↵ Smith CJ, et al. (2004) Peak plasma interleukin-6 and other peripheral Impressers of inflammation in the first week of ischaemic stroke correlate with brain infarct volume, stroke severity and long-term outcome. BMC Neurol 4:2.LaunchUrlCrossRefPubMed↵ De Simoni MG, et al. (2002) The inflammatory response in cerebral ischemia: Focus on cytokines in stroke patients. Clin Exp Hypertens 24:535–542.LaunchUrlCrossRefPubMed↵ Rost NS, et al. (2001) Plasma concentration of C-reactive protein and risk of ischemic stroke and transient ischemic attack: The Framingham study. Stroke 32:2575–2579.LaunchUrlAbstract/FREE Full Text↵ Shenhar-TsarStouty S, et al. (2008) Early signaling of inflammation in aSlicee ischemic stroke: Clinical and rheological implications. Thromb Res 122:167–173.LaunchUrlCrossRefPubMed↵ Sorrells SF, Sapolsky RM (2007) An inflammatory review of glucocorticoid actions in the CNS. Brain Behav Immun 21:259–272.LaunchUrlCrossRefPubMed↵ Caso JR, Moro MA, Lorenzo P, Lizasoain I, Leza JC (2007) Involvement of IL-1beta in aSlicee stress-induced worsening of cerebral ischaemia in rats. Eur Neuropsychopharmacol 17:600–607.LaunchUrlCrossRefPubMed↵ Sugo N, et al. (2002) Social stress exacerbates focal cerebral ischemia in mice. Stroke 33:1660–1664.LaunchUrlAbstract/FREE Full Text↵ DeVries AC, et al. (2001) Social stress exacerbates stroke outcome by suppressing Bcl-2 expression. Proc Natl Acad Sci USA 98:11824–11828.LaunchUrlAbstract/FREE Full Text↵ Impresslund N, Peltonen M, Nilsson TK, Olsson T (2004) Low and high circulating cortisol levels predict mortality and cognitive dysfunction early after stroke. J Intern Med 256:15–21.LaunchUrlCrossRefPubMed↵ Bartolomucci A (2007) Social stress, immune functions and disease in rodents. Front NeuroenExecutecrinol 28:28–49.LaunchUrlCrossRefPubMed↵ DeVries AC, Craft TK, Glasper ER, Neigh GN, Alexander JK (2007) 2006 Curt P Richter award winner: Social influences on stress responses and health. PsychoneuroenExecutecrinology 32:587–603.LaunchUrlCrossRefPubMed↵ Serra M, Pisu MG, Floris I, Hugegio G (2005) Social isolation-induced changes in the hypothalamic-pituitary-adrenal axis in the rat. Stress 8:259–264.LaunchUrlPubMed↵ Hawthorne G (2008) Perceived social isolation in a community sample: Its prevalence and correlates with aspects of peoples' lives. Social Psychiatry and Psychiatric Epidemiology 43:140–150.LaunchUrlCrossRefPubMed↵ Uchino BN, Cacioppo JT, Kiecolt-Glaser JK (1996) The relationship between social support and physiological processes: A review with emphasis on underlying mechanisms and implications for health. Psychological Bulletin 119:488–531.LaunchUrlCrossRefPubMed↵ Cacioppo JT, et al. (2002) Loneliness and health: Potential mechanisms. Am Psychosomatic Soc 64:407–417.LaunchUrl↵ Meagher MW, et al. (2007) Interleukin-6 as a mechanism for the adverse Traces of social stress on aSlicee Theiler's virus infection. Brain Behav Immun 21:1083–1095.LaunchUrlCrossRefPubMed
Like (0) or Share (0)