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Social stress alters the severity of acute Theiler's virus infection
Journal of Neuroimmunology 148 (2004) 74 – 85 www.elsevier.com/locate/jneuroim
Social stress alters the severity of acute Theiler’s virus infection R.R. Johnson a, R. Storts b, T.H. Welsh Jr. c,d, C.J.R. Welsh b,c, M.W. Meagher a,* a
Department of Psychology, College of Liberal Arts, Texas A&M University, Mailstop 4328, College Station, TX 77843, USA Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, USA c Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, USA d Department of Animal Science, College of Agriculture and Life Sciences, Texas A&M University, College Station, TX 77843, USA b
Received 5 September 2003; received in revised form 7 November 2003; accepted 10 November 2003
Abstract Our laboratory has previously shown that restraint stress resulted in decreased Theiler’s virus-induced CNS inflammation, while exacerbating illness behaviors during the acute phase of disease. In contrast, social disruption stress (SDR) applied prior to infection led to the development of glucocorticoid (GC) resistance, and these animals developed more severe disease course, with increased inflammation. However, when SDR was applied concurrent with infection, GC resistance fails to develop, disease course is less severe and inflammation was moderate. These results suggest that the effects of SDR on Theiler’s virus infection are dependent upon the timing of SDR application in relation to infection. D 2004 Elsevier B.V. All rights reserved. Keywords: Social disruption stress; CNS inflammation; Viral infection; Glucocorticoid resistance; Multiple sclerosis; Neurotropic virus; Corticosterone
1. Introduction Social stress models in rodents have been shown to induce powerful neuroendocrine responses and commensurate immune function alterations (De Groot et al., 1999; Fleshner et al., 1989; Stefanski and Engler, 1999). Such models have a well-documented history of elevating the primary stress hormone, corticosterone (Koolhaas et al., 1997a,b; Korte et al., 1995). Corticosteroids act directly to suppress proinflammatory cytokines, such as TNF-a and IL1 (Angeli et al., 1999; Lew et al., 1988), and indirectly in synergy with the anti-inflammatory cytokines, IL-10 and IL4 to further inhibit proinflammatory cytokines (Franchimont et al., 1999; Hart et al., 1990). Thus, corticosteroids generally suppress immune function through the suppression of inflammatory cytokines and other inflammatory factors. However, this is not always the case. Several studies have demonstrated increases in inflammation despite the presence of high corticosteroid levels (Koehler, 1985; Quan et al., 2001). This counter-intuitive increase in inflammation in the presence of elevated corticosteroids has recently been as* Corresponding author. Tel.: +1-979-845-2564; fax: +1-979-8454727. E-mail address: [email protected] (M.W. Meagher). 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2003.11.009
cribed to the development of glucocorticoid (GC) resistance (when immune cells fail to down regulate in the presence of GCs). The social stress model of social disruption (SDR) has been shown to induce GC resistance (Avitsur et al., 2001). In the presence of GC resistance, SDR animals have elevated systemic inflammatory responses to an LPS challenge (Quan et al., 2001). The current study examined the effects of a social stressor, SDR, in acute Theiler’s virus infection. Theiler’s virus infection in mice is a biphasic disease that causes CNS inflammation as a result of the infection of central neurons and glia in the acute phase followed by a neuroinflammatory demyelinating chronic phase (Lipton, 1975; Njenga et al., 1997a). In the acute phase, mice appear asymptomatic; however, under certain conditions, they exhibit signs of encephalitis and hind limb impairment similar to polio (Campbell et al., 2001; Welsh et al., in press). The chronic phase of Theiler’s virus infection has been used as a model for multiple sclerosis (MS)-like demyelination, as it has many similarities to the chronic progressive type of MS, including lesion type and behavioral manifestations (Lipton, 1975; McGavern et al., 1999, 2000). In Theiler’s virus infection, the virus must persist in the CNS beyond the resolution of acute phase symptoms in order to cause the later demyelinating phase (Aubert et al., 1987). The current study is the first in a series examining the
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effects of stress applied in the early phase of the disease on the neuropathogenesis of acute and chronic Theiler’s virus infection. As inflammatory processes are a key component of both the early and late disease, the SDR model with the associated GC resistance and elevations in inflammation is of particular interest. Our laboratory has previously examined the impact of chronic restraint stress on the course of Theiler’s virus infection (Campbell et al., 2001). These studies indicated that restraint stress administered beginning within 14 h of inoculation led to a decrease in CNS inflammation compared to non-restrained mice at day 14 post-infection (pi). This decrease in inflammation was associated with an increase in behavioral manifestations of encephalitis, higher mortality and higher viral titers. Restraint stress has also been associated with lower levels of inflammation in other models such as LPS-induced endotoxemia (Quan et al., 2001). In contrast, research using the recently developed social stress model of SDR (Avitsur et al., 2001; Stark et al., 2001) has found increased systemic inflammation and mortality following the LPS challenge (Quan et al., 2001). The underlying mechanism for the increased inflammation has been attributed to the development of functional GC resistance in the SDR animals (Avitsur et al., 2001; Stark et al., 2001). In contrast to the SDR animals, restraint stressed animals failed to develop GC resistance, leading to the hypothesis that GC resistance may be the underlying mechanism for the differential development of inflammation (Quan et al., 2001). The current studies were designed to examine the role of SDR on the acute phase of Theiler’s virus infection. This is an important question in light of the differential development of inflammation and mortality found between restraint stress and SDR using LPS. We expected that SDR applied prior to infection (Quan et al., 2001) would lead to an increase in inflammation compared to infected/non-stressed mice. In addition, we were interested in examining how SDR applied concurrent with infection (similar to the restraint schedule in Campbell et al., 2001) would affect the disease course. When we found that the two timing schedules resulted in divergent effects on the disease course, these two schedules were directly compared.
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or active male breeders, 6– 8 months of age. The intruders were selected based on latency to attack both peers and adolescents. All intruders consistently attacked peers within 30 s and adolescents within 2 min on three separate occasions. All animal care protocols were in accordance with NIH Guidelines for Care and Use of Laboratory Animals and were approved by the Texas A&M University Laboratory Animal Care and Use Committee (ULACC). 2.2. Social disruption stress Experimental mice were allowed to acclimate to the colony for at least 1 week during which time baseline measures were taken. Cages were then randomly assigned to either home cage control or social disruption condition. Three experiments were performed using separate groups of mice. Fig. 1 depicts the timing of experimental procedures. Experiment 1 evaluated the impact of SDR administered the week prior to Theiler’s virus infection (PRESDR) on hind limb impairment, corticosterone levels and GC sensitivity for infected (I) and non-infected (NI) mice. Experiment 2 examined the effects of SDR administered concurrent with infection (CON-SDR) on hind limb impairment and GC resistance in infected mice. Experiment 3 provided a replication of the first two experiments by directly comparing the PRE-SDR, CON-SDR and nonstressed (NON-SDR) conditions in infected mice. Additionally, this study examined the effects of PRE-SDR and CON-SDR on CNS inflammation and viral clearance. For the stressed mice, intruders were introduced into the cage of resident mice at dark cycle onset (1700 h) for a period of 2 h for a total of six SDR sessions. SDR occurred for three consecutive sessions, then one night off, followed by three additional consecutive sessions (Stark et al., 2001), in a separate procedure room. NON-SDR mice remained in the colony room, undisturbed for the duration of SDR. SDR sessions were monitored to ensure that the intruder attacked the residents and that the residents demonstrated submissive behaviors. Intruders that did not attack within 10 min of session initiation were replaced and the session continued for the remaining 2 h. 2.3. Corticosterone determination
2. Materials and methods 2.1. Animals Male BALBc/J mice were either bred in house (Experiment 1) or acquired from Jackson Labs (Bar Harbor, ME) (Experiments 2 and 3). Mice were weaned or arrived from the breeder at post-natal day (pnd) 21 and were assigned to cages of 3, counterbalanced by body weight. Mice were maintained on a 12-h light/dark cycle (0500/1700 h) with ad libitum access to food and water, with the exception of the 2-h SDR sessions. Intruders for the SDR were retired
The effect of social stress on plasma corticosterone levels was assessed after the third SDR session (Experiment 1). Mice were shaved on the thigh the morning prior to the bleed (minimum of 10 h in advance). A local anesthetic (EMLA, St. Joseph, MO) was applied and, 10 min later, the mice were transported to a nearby room where blood from the saphaneous vein was collected into EDTA vacutubes. Plasma was stored at 80 jC until concentrations of corticosterone were determined by radioimmunoassay (ICN Biomedicals, Costa, Mesa, CA) (Keith et al., 1978).
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Fig. 1. Application of SDR schedule across experiments. Experiment 1 applies SDR prior to infection (P), N = 48, n = 12 (by condition), n = 6 (condition by day of sacrifice). Experiment 2 applies SDR concurrent with infection (C), N = 12, n = 6. Experiment 3 applies SDR both prior to (P) and concurrent with (C) infection, N = 72, n = 12 (by condition), n = 6 (condition by sacrifice day). Animals in the PRE-SDR conditions were administered social disruption 1 week prior to infection beginning on day 7 pi/pnd 28. The CON-SDR animals were administered social disruption concurrently with infection, beginning at day 0 pi/pnd 35.
2.4. Infection Mice were anesthetized on day 0 pi with isoflurane (IsoSol from Vedco, St. Joseph, MO) and injected intracranially (i.c.) into the right cerebral hemisphere with 5 104 pfu of the BeAn strain of Theiler’s virus in a 20-Al volume as previously described (Campbell et al., 2001). Noninfected mice (Experiment 1) were mock infected by i.c. injection of 20-Al sterile PBS. Inoculation for all subjects occurred at 9 p.m. (2 h following the final social disruption session). 2.5. Psychomotor and illness behavior assessments Theiler’s virus induces hind limb impairment in the BALBc/J strain of mice during the acute phase of disease. Additionally, behavioral signs of illness and encephalitis have been observed in infected mice subjected to restraint stress in a previous study (Campbell et al., 2001). For this reason, several behavioral measures were taken to determine the impact of SDR on infection-related psychomotor deficits and illness behaviors. SDR-related wounding was also evaluated during the behavioral sessions (though very little was observed and no animals were removed due to excessive wounding). 2.5.1. Hind limb impairment In all experiments, blind ratings of hind limb impairment were conducted every 2– 3 days pi. The hind limb impairment scale was developed by observing the natural progression of impairment across time in BALBc/J mice. This scale consists of two subscales, weakness and coordination. Both subscales ranged from 0 to 6 and are based on 30– 45 s of observation. The progression for the weakness scale was as
follows: 0 = no weakness; 1 = over or under grasping 10– 50% of time; 2 = the same as 1, but more than 50% of time; 3 = began dangling off the grid, but upper thigh muscles appeared flexed, more than 10% of time; 4 = same as 3, except dangled lower and thigh muscles appeared extended; 5 = the entire hind end of animal slipped off grid at least two times and animal had difficulty in reattaching; 6 = no constructive use of limb. The progression was as follows for the coordination subscale: 0 = no coordination problems; 1 = repeated, but successful attempts to place foot (10 – 20% of time), middle toes may have bent against grid; 2 = repeated, but successful attempts to place foot with toes closed (20 – 100% of time), sliding instead of stepping for distances greater than 1 cm with toes closed; 3 = similar to 2, but with an open rather than closed toes; 4 = repeated, directed but unsuccessful attempts to place foot with toes open (20 – 100% of time), may have seen undirected opening and closing of grip, but not on grid; 5 = no directed movements, but thigh muscles had tone when tested by lightly pulling away from body when not on grid, dragged limb to side or behind when ambulating in home cage; 6 = as in 5, but with no muscle tone. The overall score was based on the progression through the subscales. Lower overall scores were associated with only weakness subscale observations, while at an overall score of 3, the coordination subscale dominated observations. An overall score of 6 was complete paralysis with no muscle tone. Each hind limb was assessed independently and reported as a combined score. This scale has inter-rater reliability of r = 0.9 between trained raters blind to condition. 2.5.2. Encephalitis scale In order to compare to our previous work with restraint stress, Experiment 3 also assessed the encephalitis-like
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symptoms. The severity of encephalitis signs of infection was scored on a 0 –4 scale that assessed the degree of encephalitis symptoms on several dimensions including hunched posture, piloerection, grooming, lethargy and sunken eyes. For each dimension, the animal received a rating from 0 to 6. This scale was derived by observing the natural progression of behavioral signs of illness and encephalitis in Theiler’s virus infected mice subjected to chronic restraint stress (Campbell et al., 2001; Welsh et al., in press). The animals in this study did not develop the entire range of symptoms observed following restraint and thus the overall scale (0– 4) did not discriminate between groups. However, the hunching subscale did discriminate between groups and thus this subscale is reported here. 2.5.3. Stride length Footprint stride length and spread were assessed similar to the method of McGavern et al. (1999, 2000). Briefly, hind limbs were painted with black tempura paint, and mice were allowed to walk down a 2.5 by 36 in. runway lined with paper to record the hind limb placement. This has been shown to provide a reliable and valid measure of virally mediated nerve damage and demyelination (McGavern et al., 1999, 2000). 2.6. Corticosteroid sensitivity assay On the sacrifice day, spleens were harvested to determine sensitivity of splenocytes to GC regulation (as per Stark, 2001). Briefly, spleens were placed in ice cold Hank’s balanced salt solution and mashed to obtain a single cell solution. Red blood cells were then lysed (Sigma, St. Louis, MO), followed by a wash of HBSS + 10% heat inactivated fetal bovine serum (FBS-HI, Equitech, Kerrville, TX). Viable cells were then counted using trypan blue dye exclusion and re-suspended at 2.5 106 cells/ml in supplemented RPMI (Sigma) + 10% FBS-HI, (supplementation: 0.75% sodium bicarbonate, 10 mM Hepes buffer, 100 U/ml penicillin, 100 Ag/ml streptomycin sulfate, 1.5 mM L-glutamine and 0.00035% 2-mercaptoethanol). LPS (Sigma #L6529) in 2% ethanol was added at a concentration of 1 Ag/ml for mitogen stimulation. GC resistance was tested by exposing aliquots of each suspension to dilutions of corticosteroid (0 – 5 AM, Sigma #C2505) dissolved in 2% ethanol and supplemented RPMI. Cell suspensions were placed in triplicate in flat-bottomed 96-well microtiter plates in 100-Al aliquots and incubated for 48 – 72 h at 37 jC and 5% CO2. After incubation, the cell proliferation assay was performed. Cell proliferation was assessed following the manufacturer’s instructions with the CellTiter 96 Aqueous nonradioactive proliferation assay kit from Promega (Madison, WI). Color changes were quantified by optical density readings at 490 nm from an EMAX ELISA plate reader (Molecular Devices, Sunnyvale, CA). Mean optical density values for the three replications for each sample were used, and the percentage of the corticosteroid exposed cells
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versus the LPS-stimulated cells was determined for statistical analysis. 2.7. Histological evaluation of CNS inflammation Mice were perfused with formalin, and brains and vertebral columns (containing intact spinal cord) were then removed and sectioned transversely into 4 and 12 pieces, respectively (see Campbell et al., 2001 for further detail). Tissues were then processed for routine hematoxylin and eosin (H&E) staining. Lesions of both the brain and spinal cord were scored in a similar manner by a rater blind to condition. Lesions evaluated included perivascular cuffing (accumulation of lymphocytes and macrophages around blood vessels), meningitis (accumulation of lymphocytes and macrophages in the meninges) and microgliosis (presence of increased microglia/macrophages within the parenchyma of the brain and spinal cord). 2.8. Viral load and clearance Prior studies have shown that viral titers in CNS are maximal at 1 – 2 weeks pi and by 3 –4 weeks pi, the virus is apparently cleared to non-detectable levels (Welsh et al., 1987, 1989). To evaluate the impact of SDR on CNS viral clearance, subsets of mice were sacrificed by an i.p. overdose of pentobarbital at days 7 and 21 pi. Brains and spinal cords were removed, homogenized and stored at 80 jC. The viral content of the supernatant from the homogenized brains and spinal cords were determined by plaque assay on L2 cells (Welsh et al., 1987). The plates were incubated at 37 jC at 5% CO2 for 72 h, and then stained with crystal violet to visualize the plaques that were formed. Assays were scored based on the number of plaques formed on the L2 cells and calculated per gram of tissue harvested (Welsh et al., 1987). 2.9. Statistical analysis Data are presented as mean F S.E.M. Analysis of variance (ANOVA) was used to evaluate differences across SDR and infection conditions; and repeated measure ANOVA were used for analysis over time as appropriate. These analyses were followed by post-hoc mean comparisons using Duncan’s new multiple range test. Trend analyses were performed to explore interactions over time. Pearson correlation coefficients (r) were used to determine inter-rater reliability, when noted.
3. Results 3.1. Neuroendocrine effects of SDR in BALBc/J mice Previous research indicated that SDR administered prior to LPS challenge not only induced an increase in circulating
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Fig. 2. The effect of SDR on mean plasma corticosteroid levels ( F S.E.M.) immediately following the third SDR session from Experiment 1. Corticosteroid response in the SDR-treated animals was double that of controls (*p < 0.0001). The animals were not infected at the time of sample collection for the corticosteroid assay (see Fig. 1).
GC levels, it also induced glucocorticoid resistance in C57BL/6 mice (Avitsur et al., 2001; Stark et al., 2001). Thus, we sought to evaluate whether BALBc/J mice would exhibit the same pattern of neuroendocrine changes in Experiments 1 and 2. 3.1.1. Effect of social disruption on circulating corticosteroid levels In Experiment 1 (PRE-SDR), higher concentrations of circulating corticosteroids were observed in SDR mice immediately following their third SDR session compared to NON-SDR mice, F(1,19) = 17.598, p < 0.0001, indicating that SDR was a physiologically efficacious stressor (Fig. 2). None of the mice were infected at this point in this study; thus, data was pooled across SDR condition, regardless of later infected status.
3.1.2. Effect of PRE-SDR on splenocyte sensitivity to corticosterone As shown in Fig. 3, the effect of PRE-SDR and CONSDR on GC resistance was also evaluated in Experiments 1 and 2. Slope analysis ANOVAs were performed, with the slope of the line formed across increasing concentrations of corticosteroids for individual subjects used as each data point. An ANOVA conducted on the slopes of non-infected mice (Experiment 1, PRE-SDR) revealed that the NI/PRE-SDR animals developed resistance to GC regulation of lymphocyte proliferation, F(1,10) = 4.89, p < 0.05 (Fig. 3A); however, in the infected animals (Experiment 1, PRE-SDR), GC resistance did not develop (Fig. 3B). Post-hoc comparison revealed that the slopes for the NI/PRE-SDR animals were significantly lower than those of the controls ( p < 0.05). In contrast, neither of the infected groups developed resistance ( p>0.05). In Experiment 2 (CON-SDR), all animals were infected at pnd 35 and sacrificed at day 14 pi, corresponding with SDR + 7 days, as in Experiment 1. Similar to Experiment 1, GC resistance was not apparent in either group following infection (Fig. 3C). Thus, PRE-SDR appears to induce GC resistance in non-infected BALBc/J mice, but not in infected BALBc/J mice. These findings are consistent with prior studies demonstrating that SDR induces GC resistance prior to LPS challenge (Quan et al., 2001). 3.2. Effect of SDR on motor function 3.2.1. Effect of the timing of SDR on hind limb impairment The effect of SDR administered prior to and concurrent with infection was investigated in Experiments 1 –3. Experiment 1 examined the effects of SDR administered 1 week prior to infection on hind limb motor impairment in infected and uninfected mice (Fig. 4A). Infection alone resulted in a
Fig. 3. The effect of PRE-SDR (3A), PRE-SDR plus infection (3B) and CON-SDR plus infection (3C) on mean proliferation of splenocytes to indicate GC resistance, from Experiments 1 (A, B) and 2 (C). GC resistance development was dependent upon infection status. GC resistance developed only in the noninfected/SDR animals (A), when SDR was applied prior to infection. All infected groups failed to develop GC resistance, whether PRE-SDR (B) or CON-SDR (C).
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F(8,160) = 3.742, p < 0.001, indicated that the effect of social disruption and infection on hind limb impairment changed over time. Post-hoc means comparison revealed that the I/PRE-SDR group exhibited greater impairment than the I/NON-SDR group at day 11 pi and for the duration of the experiment. In Experiment 2, the effect of SDR administered concurrent with infection on hind limb motor impairment was assessed in infected mice only (Fig. 4B). In contrast to the first experiment, significantly less hind limb impairment developed in the CON-SDR group compared to NON-SDR mice. An ANOVA confirmed a significant main effect for SDR, F(1,10) = 13.493, p < 0.005, as well as a significant interaction between SDR day PI, F(1,60) = 10.565, p < 0.0001. Mean comparisons revealed that the CON-SDR group had significantly less impairment beginning at day 3 pi and remained so for the remainder of the experiment. Experiment 3 directly compared the PRE-SDR and CON-SDR treatments (between subjects design) in infected mice within the same study, providing a replication of the results of Experiments 1 and 2 (Fig. 4C). Similar to Experiment 1, PRE-SDR exacerbated infectionrelated hind limb impairment, whereas CON-SDR reduced impairment as observed in Experiment 2. A main effect was found for SDR condition, F(2,32) = 16.532, p < 0.0001, and the interaction between SDR day PI was significant, F(18,288) = 4.099, p < 0.0001. Mean comparisons indicated that at day 3 pi the PRE-SDR animals had greater hind limb impairment than the other two groups; this difference was maintained throughout the experiment. In addition, by day 3 pi, the CON-SDR animals had less impairment than the NON-SDR animals, and this remained so through day 9 pi, when the difference between these two groups dissipated. This corresponds with the period of stress from days 0 to 7 pi for the CON-SDR condition (Fig. 1). Taken together, the results from Experiments 1– 3 demonstrate that PRESDR condition exacerbates infection-related hind limb paralysis, whereas CON-SDR condition results in a temporary reduction in impairment. Fig. 4. The effect PRE-SDR (A, Experiment 1), CON-SDR (B, Experiment 2) or comparing both (C, Experiment 3) on mean hind limb impairment across days pi ( F S.E.M.). Hind limb impairment developed with infection. In Experiment 1, PRE-SDR exacerbated development (A), while in Experiment 2, CON-SDR suppressed development (B). These effects were replicated in Experiment 3 (C).
progressive increase in hind limb impairment and PRE-SDR further exacerbated the level of impairment over time. An ANOVA confirmed a significant main effect for infection, F(1,20) = 116.129, p < 0.001, and SDR, F(1,20) = 18.212, p < 0.001, as well as a significant interaction between infection SDR, F(1,20) = 19.546, p < 0.001. A significant three-way interaction between infection SDR day PI,
3.2.2. Stride length As shown in Fig. 5A, the impact of SDR on stride length was evaluated in infected mice in Experiment 3. A main effect was found for SDR condition, F(2,32) = 8.478, p < 0.01. Mean comparisons indicated that PRE-SDR resulted in decreased stride length relative to the NONSDR and CON-SDR conditions ( p < 0.05). 3.2.3. Grid hang latencies Fig. 5B depicts grid hang latencies for the three stress conditions over time in Experiment 3. As expected, grid hang time decreased over the course of infection and PRESDR appears to reduce hang time even further (Fig. 5B). Although the main effect of SDR condition and interaction
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of encephalitis and high rates of mortality were not observed following exposure to social stress (all p’s>0.05). Because analysis of total scores on the encephalitis scale derived from our prior study did not reveal differences across groups (data not shown), exploratory analyses were conducted on individual subscales. The hunching subscale revealed group differences. 3.3.1. Hunching Hunching developed in all the animals, but the CONSDR animals had delayed onset of hunching, compared to the NON-SDR and PRE-SDR groups (Fig. 6). A main effect was found for both SDR condition, F(2,33) = 4.214, p < 0.05, and day pi, F(8,16) = 174.478, p < 0.0001, but condition did not interact with day pi [ F(18,297) = 0.871, p>0.05]. Post-hoc analysis revealed that the CON-SDR group developed hunching at a later time than the other groups and less hunching overall ( p < 0.05). In summary, the behavioral measures of hind limb impairment, hunching, grid hang latency and stride length all indicate that PRE-SDR resulted in a more severe disease course, while CON-SDR had either a beneficial or null effect compared to NON-SDR mice. 3.4. Effect of social disruption on CNS inflammation Application of SDR prior to infection resulted in an overall pattern of increased inflammation in spinal cord tissue (Fig. 7) and brain (data not shown). An ANOVA confirmed this with separate analysis of each measure at each sacrifice date, demonstrating a significant main effect for SDR at day 7 pi for microgliosis, F(2,159) = 4.328, p < 0.05, and meningitis ( p < 0.05) (Fig. 7B) in the spinal
Fig. 5. The effect of PRE-SDR and CON-SDR on mean stride length (A) and mean grid hang time (B) in Experiment 3. PRE-SDR had a detrimental effect on both stride length and hang time. Stride length was significantly reduced in the PRE-SDR animals at day 20 pi (A, *p < 0.01). In addition, grid hang time analysis revealed a linear trend for PRE-SDR animals to have reduced hang time (B). All data shown F S.E.M.
terms failed to reach significance, a linear trend was observed ( p < 0.05), indicating that PRE-SDR resulted in the shortest hang times. 3.3. Effect of the timing of SDR on behavioral measures of illness In contrast to the effects of chronic restraint stress on acute TMEV infection (Campbell et al., 2001), severe signs
Fig. 6. Mean hunching scores for infected mice exposed to PRE-SDR, CON-SDR or control non-stressed animals across days pi ( F S.E.M., Experiment 3). Hunching developed at a similar rate in all groups. However, the CON-SDR group began showing signs at a significantly later day pi and the PRE-SDR group developed the most severe signs.
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Fig. 7. Mean lesion scores in spinal cord of infected subjects in the PRE-SDR, CON-SDR and NON-SDR conditions ( F S.E.M., Experiment 3). Mice were sacrificed at day 7 pi and day 21 pi, and vertebral columns removed for histological assessment. Significant differences are denoted by asterisks (*p < 0.05) In the spinal cord, PRE-SDR resulted in significantly greater inflammation at day 7 pi on meningitis (A) and microgliosis (B). By day 21 pi, PRE-SDR resulted in greater inflammation as measured by meningitis (D), microgliosis (E) and perivascular cuffing (F).
cord. Post-hoc comparisons indicated that the PRE-SDR groups had greater microgliosis and meningitis compared to the NON-SDR group (Fig. 7A). Perivascular cuffing was not significantly different between the groups at day 7 pi ( p>0.05) (Fig. 7C). At day 21 pi, main effects for SDR on meningitis, F(2,159) = 4.327, p < 0.05, microgliosis, F(2,159) = 4.171, p < 0.05, and perivascular cuffing, F(2,159) = 3.002, p < 0.05, were revealed by ANOVA (Fig. 7D – F, respectively) in the spinal cord. Post-hoc means comparisons confirmed that PRE-SDR resulted in greater inflammation compared to NON-SDR and CON-SDR animals at day 21 pi. In the brain, the PRE-SDR animals had non-significant elevations in inflammation ( F’s < 2.3, p’s>0.05) at days 7 and 21 pi on the measures of microgliosis and perivascular cuffing. In addition, meningitis in the brain was not significantly different between groups at either sacrifice day ( p’s>0.05). In summary, PRE-SDR resulted in an overall pattern of elevated CNS inflammation
indicators at both days 7 and 21 pi, particularly in the spinal cord. 3.5. Effect of social disruption on viral titers and clearance Fig. 8 depicts log transformed viral titers in brain and spinal cord tissue measured at days 7 and 21 pi. In the brain, all conditions had high levels of virus at day 7 pi and significantly reduced levels at day 21 pi (Fig. 8A). Although viral titers decreased significantly over time ( p < 0.0001), no group differences were observed in brain. In contrast, group differences in viral clearance were observed in spinal cord. The PRE-SDR group had significantly lower titers than the other two groups at day 7 pi, F(2,15) = 5.474, p < 0.05 (Fig. 8B). In contrast, by day 21 pi, the CON-SDR animals had significantly lower titers, F(2,15) = 3.993, p < 0.05, compared to the other groups at day 21 pi. In addition, both the NON-SDR and CON-SDR
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Fig. 8. The effect of PRE-SDR, CON-SDR, and NON-SDR (Experiment 3) on mean viral titers ( F S.E.M.) and clearance in brain (A) and spinal cord (B). All significant differences are denoted by asterisks (*p < 0.05). In the brain, viral titers were uniformly elevated at day 7 pi and significantly decreased by day 21 pi (A), indicating uniform clearance as well. In contrast, the timing of the SDR affected both viral titers and clearance in the spinal cord. At day 7 pi, the PRESDR group has significantly lower titers compared to the other two groups, while at day 21 pi, the CON-SDR group has significantly lower titers compared to the other two groups. In addition, both the NON-SDR and CON-SDR groups have significantly reduced titers across time, while the PRE-SDR group failed to alter titers across time, indicating a failure of clearance in these animals (B).
groups had significantly lower titers over time, while the PRE-SDR group’s titer level did not change, F(2,28) = 6.259, p < 0.01, indicating a failure to clear the virus efficiently in these animals.
4. Discussion The current studies demonstrate that SDR, like restraint stress can exacerbate the course of Theiler’s virus infection. SDR altered behavioral signs of illness and motor function associated with the illness, including hunching, hind limb impairment, stride length and grid hang time. The behavioral signs were accompanied by alterations in both histological evidence for CNS inflammation and viral titers and clearance across time. Thus, SDR is effective in altering the course of Theiler’s virus infection. The direction of the effects of SDR on the course of Theiler’s virus infection was further dependent upon the timing of SDR. Specifically, SDR applied prior to infection (PRESDR) consistently exacerbated both behavioral and physiological measures of infection. In contrast, SDR applied concurrent with infection resulted in delayed hind limb impairment and hunching development, while other behavioral measures did not differ from NON-SDR mice. These animals (CON-SDR) also did not significantly differ from NON-SDR mice on measures of inflammation, but did demonstrate the most effective viral clearance competency.
4.1. Relation to prior empirical studies These findings are intriguing when compared to previous work from our laboratory using restraint stress (Campbell et al., 2001). Restraint stress was shown to exacerbate behavioral signs of illness and reduce viral clearance, much like PRE-SDR. However, PRE-SDR differs from restraint stress in two important ways. First, PRE-SDR increased inflammation, while restraint decreased it. Second, PRE-SDR is applied prior to infection, while restraint stress was applied concurrent with infection, much like CON-SDR. However, the current findings show that CONSDR actually reduced behavioral signs of infection and increased viral clearance, exactly the opposite of the effects found with restraint stress. 4.2. Potential neuroendocrine mechanisms One possible reason for the difference in behavioral sign development, inflammation and viral clearance may be the differential development of GC resistance. Normal elevations of corticosteroids were found both in the previous SDR studies (Avitsur et al., 2001) and in the current study. Thus, based on the majority of research showing that glucocorticoids reduce inflammation (Angeli et al., 1999; Lew et al., 1988), we would have expected a decrease in inflammation. However, prior studies characterizing the SDR model noted that this stressor results in GC resistance (Avitsur et al., 2001; Stark et al., 2001). In the current
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studies, GC resistance developed in the PRE-SDR animals, as indicated by the persistence of resistance in the NI/PRESDR animals, 7 days post-stress. Infected animals consistently failed to demonstrate GC resistance and this was true for the I/PRE-SDR animals, 7 days post-stress and infection. However, it is very likely that GC resistance was present in these animals at the time of infection. Both the I/PRE-SDR and NI/PRE-SDR groups were identical at the time of infection, and thus if the I/PRE-SDR group had not been infected, it is logical to assume that they too would have maintained GC resistance as the NI/PRE-SDR animals demonstrated. The presence of GC resistance at the time of infection would result in a more robust inflammatory response, as was found in the PRE-SDR animals (Quan et al., 2001). However, GC resistance dissipates and/or fails to develop in the presence of an immune challenge in these studies. Thus, the CON-SDR animals never developed GC resistance. Therefore, the CON-SDR animals did not have the same systemic environment that allowed for excess inflammation at the time of infection as the PRE-SDR animals did. GC resistance has previously been highly correlated with the degree of wounding (Avitsur et al., 2002). In the current studies, however, wounding was very mild and rare, yet GC resistance did develop. One explanation for this finding is the well-documented hyper-responsive nature of BALBc mice (genetically very similar to the BALBc/J mice used in the current study), and the relatively stress resilient nature of C57BL/6 mice (Shanks and Anisman, 1988; Shanks et al., 1994a; Shanks et al., 1994b; Shanks et al., 1990; Zacharko et al., 1990; Zaharia et al., 1996), used in the previous SDR studies (Avitsur et al., 2002; Avitsur et al., 2001; Quan et al., 2001; Stark et al., 2002; Stark et al., 2001). The initial SDR studies utilize C57BL/6 mice at 8 weeks of age or older at the time of SDR. The current study utilized younger (4– 5 weeks of age) BALBc/J mice due to constrictions of the Theiler’s virus model. (Theiler’s virus infection does not cause the chronic phase in C57BL/6 mice and susceptible strains such as the BALBc/J, must be infected during early adolescence.) The antagonistic confrontations between subject mice and the intruder were generally less severe in the current study, possibly due to this age difference, and resulted in the less severe wounding. However, due to the stress hyper-responsivity of the BALBc/J strain, it is possible that the threshold of defeat was simply lower than that found previously, and thus the antagonistic interactions resulting in severe wounding were not required for the development of GC resistance.
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animals at this time point (Biron et al., 1998; Fiette et al., 1995; Njenga et al., 1997b). However, at day 21 pi, the PRE-SDR group viral titers had not decreased, which may indicate an interruption or alteration of specific immune responses (CD4+, CD8+, B cell), which are known to be important in viral clearance in the acute phase Theiler’s virus infection (Borrow et al., 1992; Borrow et al., 1993; Murray et al., 1998; Welsh et al., 1987). In contrast, at day 21 pi, the CON-SDR group viral titers were significantly lower then the other two groups. As viral persistence in the CNS during the acute phase is essential for the development of the chronic demyelinating phase of Theiler’s virus (Brahic et al., 1981; Rodriguez et al., 1996), the PRESDR animals are at greatest risk for developing the later disease. The later demyelinating phase of Theiler’s virus is often used as a model for MS. The findings in this study are important when we examine the epidemiological evidence that MS may be caused by a viral infection in adolescence or early adulthood (Acheson, 1977; Kurtzke, 1993). A viral etiology is implicated by these studies, yet no single viral culprit has been found. Many viruses may be capable of initiating the autoimmune processes leading to MS. It may be that the systemic conditions at the time of infection are a factor in this development, such as the presence of a stressor that induces GC resistance. Further studies of the innate and acquired immune responses to Theiler’s virus in mice subjected to SDR will aid in our understanding of the mechanisms involved in the results reported herein. Based on prior studies using SDR (Quan et al., 2001), we anticipate that pro-inflammatory cytokines may be expressed in the CNS of PRE-SDR mice following infection with Theiler’s virus. Indeed, the observed increased histopathology following PRE-SDR and infection with Theiler’s virus in the present study is likely due to the increased production of pro-inflammatory cytokines. Finally, additional investigation will be needed to determine if the higher viral load and/or inability to clear virus in the PRE-SDR animals in comparison to the CONSDR animals will alter the development of the chronic demyelinating phase of the disease. In summary, SDR alters Theiler’s virus infection course, the alteration is dependent upon the timing of SDR application. These effects are apparent both behaviorally and physiologically, with PRE-SDR leading to the most severe disease course behaviorally, with greater inflammation and reduced viral clearance efficacy across time. In contrast, CON-SDR differs very little across time from non-stressed controls; however, they demonstrate the most effective viral clearance across time.
4.3. Potential immune mechanisms The PRE-SDR animals had both elevated inflammation and lower viral titers at day 7 pi. This indicates that innate immune processes associated with inflammation, such as increased type 1 IFNs may be aiding viral clearance in these
Acknowledgements This research was supported by an NSF Fellowship to RRJ and NMSS RG3128 and NINDS RO1 NS39569 to
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CJRW and MWM. In addition, we would like to thank David Padgett for his patient help, Jim Grau for insight into the final design of these experiments, Heath McCullough, Gabby Oroza, Amy Sieve and Lin Bustamante.
References Acheson, E.D., 1977. Epidemiology of multiple sclerosis. Br. Med. Bull. 33, 9 – 14. Angeli, A., Masera, R.G., Sartori, M.L., Fortunati, N., Racca, S., Dovio, A., Staurenghi, A., Frairia, R., 1999. Modulation by cytokines of glucocorticoid action. Ann. N.Y. Acad. Sci. 876, 210 – 220. Aubert, C., Chamorro, M., Brahic, M., 1987. Identification of Theiler’s virus infected cells in the central nervous system of the mouse during demyelinating disease. Microb. Pathog. 3, 319 – 326. Avitsur, R., Stark, J.L., Sheridan, J.F., 2001. Social stress induces glucocorticoid resistance in subordinate animals. Horm. Behav. 39, 247 – 257. Avitsur, R., Stark, J.L., Dhabhar, F.S., Sheridan, J.F., 2002. Social stress alters splenocyte phenotype and function. J. Neuroimmunol. 132, 66 – 71. Biron, C.A., Cousens, L.P., Ruzek, M.C., Su, H.C., Salazar-Mather, T.P., 1998. Early cytokine responses to viral infections and their roles in shaping endogenous cellular immunity. Adv. Exp. Med. Biol. 452, 143 – 149. Borrow, P., Tonks, P., Welsh, C.J., Nash, A.A., 1992. The role of CD8+ T cells in the acute and chronic phases of Theiler’s murine encephalomyelitis virus-induced disease in mice. J. Gen. Virol. 73 (7), 1861 – 1865. Borrow, P., Welsh, C.J., Nash, A.A., 1993. Study of the mechanisms by which CD4 + T cells contribute to protection in Theiler’s murine encephalomyelitis. Immunology 80, 502 – 506. Brahic, M., Stroop, W.G., Baringer, J.R., 1981. Theiler’s virus persists in glial cells during demyelinating disease. Cell 26, 123 – 128. Campbell, T., Meagher, M.W., Sieve, A., Scott, B., Storts, R., Welsh, T.H., Welsh, C.J., 2001. The effects of restraint stress on the neuropathogenesis of Theiler’s virus infection: I. Acute disease. Brain Behav. Immun. 15, 235 – 254. de Groot, J., van Milligen, F.J., Moonen-Leusen, B.W., Thomas, G., Koolhaas, J.M., 1999. A single social defeat transiently suppresses the antiviral immune response in mice. J. Neuroimmunol. 95, 143 – 151. Fiette, L., Aubert, C., Muller, U., Huang, S., Aguet, M., Brahic, M., Bureau, J.F., 1995. Theiler’s virus infection of 129Sv mice that lack the interferon alpha/beta or interferon gamma receptors. J. Exp. Med. 181, 2069 – 2076. Fleshner, M., Laudenslager, M.L., Simons, L., Maier, S.F., 1989. Reduced serum antibodies associated with social defeat in rats. Physiol. Behav. 45, 1183 – 1187. Franchimont, D., Martens, H., Hagelstein, M.T., Louis, E., Dewe, W., Chrousos, G.P., Belaiche, J., Geenen, V., 1999. Tumor necrosis factor alpha decreases, and interleukin-10 increases, the sensitivity of human monocytes to dexamethasone: potential regulation of the glucocorticoid receptor. J. Clin. Endocrinol. Metab. 84, 2834 – 2839. Hart, P.H., Whitty, G.A., Burgess, D.R., Croatto, M., Hamilton, J.A., 1990. Augmentation of glucocorticoid action on human monocytes by interleukin-4. Lymphokine Res. 9, 147 – 153. Keith, L.D., Winslow, J.R., Reynolds, R.W., 1978. A general procedure for estimation of corticosteroid response in individual rats. Steroids 31, 523 – 531. Koehler, T., 1985. Stress and rheumatoid arthritis: a survey of empirical evidence in human and animal studies. J. Psychosom. Res. 29, 655 – 663. Koolhaas, J.M., De Boer, S.F., De Rutter, A.J., Meerlo, P., Sgoifo, A., 1997a. Social stress in rats and mice. Acta Physiol. Scand., Suppl. 640, 69 – 72. Koolhaas, J.M., Meerlo, P., De Boer, S.F., Strubbe, J.H., Bohus, B., 1997b.
The temporal dynamics of the stress response. Neurosci. Biobehav. Rev. 21, 775 – 782. Korte, S.M., Buwalda, B., Meijer, O., De Kloet, E.R., Bohus, B., 1995. Socially defeated male rats display a blunted adrenocortical response to a low dose of 8-OH-DPAT. Eur. J. Pharmacol. 272, 45 – 50. Kurtzke, J.F., 1993. Epidemiologic evidence for multiple sclerosis as an infection. Clin. Microbiol. Rev. 6, 382 – 427. Lew, W., Oppenheim, J.J., Matsushima, K., 1988. Analysis of the suppression of IL-1 alpha and IL-1 beta production in human peripheral blood mononuclear adherent cells by a glucocorticoid hormone. J. Immunol. 140, 1895 – 1902. Lipton, H.L., 1975. Theiler’s virus infection in mice: an unusual biphasic disease process leading to demyelination. Infect. Immun. 11, 1147 – 1155. McGavern, D.B., Zoecklein, L., Drescher, K.M., Rodriguez, M., 1999. Quantitative assessment of neurologic deficits in a chronic progressive murine model of CNS demyelination. Exp. Neurol. 158, 171 – 181. McGavern, D.B., Zoecklein, L., Sathornsumetee, S., Rodriguez, M., 2000. Assessment of hind limb gait as a powerful indicator of axonal loss in a murine model of progressive CNS demyelination. Brain Res. 877, 396 – 400. Murray, P.D., Pavelko, K.D., Leibowitz, J., Lin, X., Rodriguez, M., 1998. CD4 (+) and CD8 (+) T cells make discrete contributions to demyelination and neurologic disease in a viral model of multiple sclerosis. J. Virol. 72, 7320 – 7329. Njenga, M.K., Asakura, K., Hunter, S.F., Wettstein, P., Pease, L.R., Rodriguez, M., 1997a. The immune system preferentially clears Theiler’s virus from the gray matter of the central nervous system. J. Virol. 71, 8592 – 8601. Njenga, M.K., Pease, L.R., Wettstein, P., Mak, T., Rodriguez, M., 1997b. Interferon alpha/beta mediates early virus-induced expression of H-2D and H-2K in the central nervous system. Lab. Invest. 77, 71 – 84. Quan, N., Avitsur, R., Stark, J.L., He, L., Shah, M., Caligiuri, M., Padgett, D.A., Marucha, P.T., Sheridan, J.F., 2001. Social stress increases the susceptibility to endotoxic shock. J. Neuroimmunol. 115, 36 – 45. Rodriguez, M., Pavelko, K.D., Njenga, M.K., Logan, W.C., Wettstein, P.J., 1996. The balance between persistent virus infection and immune cells determines demyelination. J. Immunol. 157, 5699 – 5709. Shanks, N., Anisman, H., 1988. Stressor-provoked behavioral changes in six strains of mice. Behav. Neurosci. 102, 894 – 905. Shanks, N., Griffiths, J., Zalcman, S., Zacharko, R.M., Anisman, H., 1990. Mouse strain differences in plasma corticosterone following uncontrollable footshock. Pharmacol. Biochem. Behav. 36, 515 – 519. Shanks, N., Griffiths, J., Anisman, H., 1994a. Central catecholamine alterations induced by stressor exposure: analyses in recombinant inbred strains of mice. Behav. Brain Res. 63, 25 – 33. Shanks, N., Griffiths, J., Anisman, H., 1994b. Norepinephrine and serotonin alterations following chronic stressor exposure: mouse strain differences. Pharmacol. Biochem. Behav. 49, 57 – 65. Stark, J.L., Avitsur, R., Padgett, D.A., Campbell, K.A., Beck, F.M., Sheridan, J.F., 2001. Social stress induces glucocorticoid resistance in macrophages. Am. J. Physiol., Regul. Integr. Comp. Physiol. 280, R1799 – R1805. Stark, J.L., Avitsur, R., Hunzeker, J., Padgett, D.A., Sheridan, J.F., 2002. Interleukin-6 and the development of social disruption-induced glucocorticoid resistance. J. Neuroimmunol. 124, 9 – 15. Stefanski, V., Engler, H., 1999. Social stress, dominance and blood cellular immunity. J. Neuroimmunol. 94, 144 – 152. Welsh, C.J., Tonks, P., Nash, A.A., Blakemore, W.F., 1987. The effect of L3T4 T cell depletion on the pathogenesis of Theiler’s murine encephalomyelitis virus infection in CBA mice. J. Gen. Virol. 68 (Pt 6), 1659 – 1667. Welsh, C.J.R., Blakemore, W.F., Tonks, P., Borrow, P., Nash, A.A., 1989. Theiler’s murine encephalomyelitis virus infection in mice: a persistent viral infection of the central nervous system which induces demyelination. In: Dimmock, N. (Ed.), Immune responses, Virus Infection and Disease, Oxford University Press, pp. 125 – 147.
R.R. Johnson et al. / Journal of Neuroimmunology 148 (2004) 74–85 Welsh, C.J.R., Bustamante, L., Nayak, M., Welsh, T.H., Dean, D.D., Meagher, M.W., 2004. The effects of restraint stress on the neuropathogenesis of Theiler’s virus infection: II. NK cell function and cytokine levels in acute disease. Brain Behav. Immun. (In press). Zacharko, R.M., Gilmore, W., MacNeil, G., Kasian, M., Anisman, H., 1990. Stressor induced variations of intracranial self-stimulation
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from the mesocortex in several strains of mice. Brain Res. 533, 353 – 357. Zaharia, M.D., Kulczycki, J., Shanks, N., Meaney, M.J., Anisman, H., 1996. The effects of early postnatal stimulation on Morris water-maze acquisition in adult mice: genetic and maternal factors. Psychopharmacology (Berl.) 128, 227 – 239.
Social stress alters the severity of acute Theiler’s virus infection R.R. Johnson a, R. Storts b, T.H. Welsh Jr. c,d, C.J.R. Welsh b,c, M.W. Meagher a,* a
Department of Psychology, College of Liberal Arts, Texas A&M University, Mailstop 4328, College Station, TX 77843, USA Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, USA c Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, USA d Department of Animal Science, College of Agriculture and Life Sciences, Texas A&M University, College Station, TX 77843, USA b
Received 5 September 2003; received in revised form 7 November 2003; accepted 10 November 2003
Abstract Our laboratory has previously shown that restraint stress resulted in decreased Theiler’s virus-induced CNS inflammation, while exacerbating illness behaviors during the acute phase of disease. In contrast, social disruption stress (SDR) applied prior to infection led to the development of glucocorticoid (GC) resistance, and these animals developed more severe disease course, with increased inflammation. However, when SDR was applied concurrent with infection, GC resistance fails to develop, disease course is less severe and inflammation was moderate. These results suggest that the effects of SDR on Theiler’s virus infection are dependent upon the timing of SDR application in relation to infection. D 2004 Elsevier B.V. All rights reserved. Keywords: Social disruption stress; CNS inflammation; Viral infection; Glucocorticoid resistance; Multiple sclerosis; Neurotropic virus; Corticosterone
1. Introduction Social stress models in rodents have been shown to induce powerful neuroendocrine responses and commensurate immune function alterations (De Groot et al., 1999; Fleshner et al., 1989; Stefanski and Engler, 1999). Such models have a well-documented history of elevating the primary stress hormone, corticosterone (Koolhaas et al., 1997a,b; Korte et al., 1995). Corticosteroids act directly to suppress proinflammatory cytokines, such as TNF-a and IL1 (Angeli et al., 1999; Lew et al., 1988), and indirectly in synergy with the anti-inflammatory cytokines, IL-10 and IL4 to further inhibit proinflammatory cytokines (Franchimont et al., 1999; Hart et al., 1990). Thus, corticosteroids generally suppress immune function through the suppression of inflammatory cytokines and other inflammatory factors. However, this is not always the case. Several studies have demonstrated increases in inflammation despite the presence of high corticosteroid levels (Koehler, 1985; Quan et al., 2001). This counter-intuitive increase in inflammation in the presence of elevated corticosteroids has recently been as* Corresponding author. Tel.: +1-979-845-2564; fax: +1-979-8454727. E-mail address: [email protected] (M.W. Meagher). 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2003.11.009
cribed to the development of glucocorticoid (GC) resistance (when immune cells fail to down regulate in the presence of GCs). The social stress model of social disruption (SDR) has been shown to induce GC resistance (Avitsur et al., 2001). In the presence of GC resistance, SDR animals have elevated systemic inflammatory responses to an LPS challenge (Quan et al., 2001). The current study examined the effects of a social stressor, SDR, in acute Theiler’s virus infection. Theiler’s virus infection in mice is a biphasic disease that causes CNS inflammation as a result of the infection of central neurons and glia in the acute phase followed by a neuroinflammatory demyelinating chronic phase (Lipton, 1975; Njenga et al., 1997a). In the acute phase, mice appear asymptomatic; however, under certain conditions, they exhibit signs of encephalitis and hind limb impairment similar to polio (Campbell et al., 2001; Welsh et al., in press). The chronic phase of Theiler’s virus infection has been used as a model for multiple sclerosis (MS)-like demyelination, as it has many similarities to the chronic progressive type of MS, including lesion type and behavioral manifestations (Lipton, 1975; McGavern et al., 1999, 2000). In Theiler’s virus infection, the virus must persist in the CNS beyond the resolution of acute phase symptoms in order to cause the later demyelinating phase (Aubert et al., 1987). The current study is the first in a series examining the
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effects of stress applied in the early phase of the disease on the neuropathogenesis of acute and chronic Theiler’s virus infection. As inflammatory processes are a key component of both the early and late disease, the SDR model with the associated GC resistance and elevations in inflammation is of particular interest. Our laboratory has previously examined the impact of chronic restraint stress on the course of Theiler’s virus infection (Campbell et al., 2001). These studies indicated that restraint stress administered beginning within 14 h of inoculation led to a decrease in CNS inflammation compared to non-restrained mice at day 14 post-infection (pi). This decrease in inflammation was associated with an increase in behavioral manifestations of encephalitis, higher mortality and higher viral titers. Restraint stress has also been associated with lower levels of inflammation in other models such as LPS-induced endotoxemia (Quan et al., 2001). In contrast, research using the recently developed social stress model of SDR (Avitsur et al., 2001; Stark et al., 2001) has found increased systemic inflammation and mortality following the LPS challenge (Quan et al., 2001). The underlying mechanism for the increased inflammation has been attributed to the development of functional GC resistance in the SDR animals (Avitsur et al., 2001; Stark et al., 2001). In contrast to the SDR animals, restraint stressed animals failed to develop GC resistance, leading to the hypothesis that GC resistance may be the underlying mechanism for the differential development of inflammation (Quan et al., 2001). The current studies were designed to examine the role of SDR on the acute phase of Theiler’s virus infection. This is an important question in light of the differential development of inflammation and mortality found between restraint stress and SDR using LPS. We expected that SDR applied prior to infection (Quan et al., 2001) would lead to an increase in inflammation compared to infected/non-stressed mice. In addition, we were interested in examining how SDR applied concurrent with infection (similar to the restraint schedule in Campbell et al., 2001) would affect the disease course. When we found that the two timing schedules resulted in divergent effects on the disease course, these two schedules were directly compared.
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or active male breeders, 6– 8 months of age. The intruders were selected based on latency to attack both peers and adolescents. All intruders consistently attacked peers within 30 s and adolescents within 2 min on three separate occasions. All animal care protocols were in accordance with NIH Guidelines for Care and Use of Laboratory Animals and were approved by the Texas A&M University Laboratory Animal Care and Use Committee (ULACC). 2.2. Social disruption stress Experimental mice were allowed to acclimate to the colony for at least 1 week during which time baseline measures were taken. Cages were then randomly assigned to either home cage control or social disruption condition. Three experiments were performed using separate groups of mice. Fig. 1 depicts the timing of experimental procedures. Experiment 1 evaluated the impact of SDR administered the week prior to Theiler’s virus infection (PRESDR) on hind limb impairment, corticosterone levels and GC sensitivity for infected (I) and non-infected (NI) mice. Experiment 2 examined the effects of SDR administered concurrent with infection (CON-SDR) on hind limb impairment and GC resistance in infected mice. Experiment 3 provided a replication of the first two experiments by directly comparing the PRE-SDR, CON-SDR and nonstressed (NON-SDR) conditions in infected mice. Additionally, this study examined the effects of PRE-SDR and CON-SDR on CNS inflammation and viral clearance. For the stressed mice, intruders were introduced into the cage of resident mice at dark cycle onset (1700 h) for a period of 2 h for a total of six SDR sessions. SDR occurred for three consecutive sessions, then one night off, followed by three additional consecutive sessions (Stark et al., 2001), in a separate procedure room. NON-SDR mice remained in the colony room, undisturbed for the duration of SDR. SDR sessions were monitored to ensure that the intruder attacked the residents and that the residents demonstrated submissive behaviors. Intruders that did not attack within 10 min of session initiation were replaced and the session continued for the remaining 2 h. 2.3. Corticosterone determination
2. Materials and methods 2.1. Animals Male BALBc/J mice were either bred in house (Experiment 1) or acquired from Jackson Labs (Bar Harbor, ME) (Experiments 2 and 3). Mice were weaned or arrived from the breeder at post-natal day (pnd) 21 and were assigned to cages of 3, counterbalanced by body weight. Mice were maintained on a 12-h light/dark cycle (0500/1700 h) with ad libitum access to food and water, with the exception of the 2-h SDR sessions. Intruders for the SDR were retired
The effect of social stress on plasma corticosterone levels was assessed after the third SDR session (Experiment 1). Mice were shaved on the thigh the morning prior to the bleed (minimum of 10 h in advance). A local anesthetic (EMLA, St. Joseph, MO) was applied and, 10 min later, the mice were transported to a nearby room where blood from the saphaneous vein was collected into EDTA vacutubes. Plasma was stored at 80 jC until concentrations of corticosterone were determined by radioimmunoassay (ICN Biomedicals, Costa, Mesa, CA) (Keith et al., 1978).
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Fig. 1. Application of SDR schedule across experiments. Experiment 1 applies SDR prior to infection (P), N = 48, n = 12 (by condition), n = 6 (condition by day of sacrifice). Experiment 2 applies SDR concurrent with infection (C), N = 12, n = 6. Experiment 3 applies SDR both prior to (P) and concurrent with (C) infection, N = 72, n = 12 (by condition), n = 6 (condition by sacrifice day). Animals in the PRE-SDR conditions were administered social disruption 1 week prior to infection beginning on day 7 pi/pnd 28. The CON-SDR animals were administered social disruption concurrently with infection, beginning at day 0 pi/pnd 35.
2.4. Infection Mice were anesthetized on day 0 pi with isoflurane (IsoSol from Vedco, St. Joseph, MO) and injected intracranially (i.c.) into the right cerebral hemisphere with 5 104 pfu of the BeAn strain of Theiler’s virus in a 20-Al volume as previously described (Campbell et al., 2001). Noninfected mice (Experiment 1) were mock infected by i.c. injection of 20-Al sterile PBS. Inoculation for all subjects occurred at 9 p.m. (2 h following the final social disruption session). 2.5. Psychomotor and illness behavior assessments Theiler’s virus induces hind limb impairment in the BALBc/J strain of mice during the acute phase of disease. Additionally, behavioral signs of illness and encephalitis have been observed in infected mice subjected to restraint stress in a previous study (Campbell et al., 2001). For this reason, several behavioral measures were taken to determine the impact of SDR on infection-related psychomotor deficits and illness behaviors. SDR-related wounding was also evaluated during the behavioral sessions (though very little was observed and no animals were removed due to excessive wounding). 2.5.1. Hind limb impairment In all experiments, blind ratings of hind limb impairment were conducted every 2– 3 days pi. The hind limb impairment scale was developed by observing the natural progression of impairment across time in BALBc/J mice. This scale consists of two subscales, weakness and coordination. Both subscales ranged from 0 to 6 and are based on 30– 45 s of observation. The progression for the weakness scale was as
follows: 0 = no weakness; 1 = over or under grasping 10– 50% of time; 2 = the same as 1, but more than 50% of time; 3 = began dangling off the grid, but upper thigh muscles appeared flexed, more than 10% of time; 4 = same as 3, except dangled lower and thigh muscles appeared extended; 5 = the entire hind end of animal slipped off grid at least two times and animal had difficulty in reattaching; 6 = no constructive use of limb. The progression was as follows for the coordination subscale: 0 = no coordination problems; 1 = repeated, but successful attempts to place foot (10 – 20% of time), middle toes may have bent against grid; 2 = repeated, but successful attempts to place foot with toes closed (20 – 100% of time), sliding instead of stepping for distances greater than 1 cm with toes closed; 3 = similar to 2, but with an open rather than closed toes; 4 = repeated, directed but unsuccessful attempts to place foot with toes open (20 – 100% of time), may have seen undirected opening and closing of grip, but not on grid; 5 = no directed movements, but thigh muscles had tone when tested by lightly pulling away from body when not on grid, dragged limb to side or behind when ambulating in home cage; 6 = as in 5, but with no muscle tone. The overall score was based on the progression through the subscales. Lower overall scores were associated with only weakness subscale observations, while at an overall score of 3, the coordination subscale dominated observations. An overall score of 6 was complete paralysis with no muscle tone. Each hind limb was assessed independently and reported as a combined score. This scale has inter-rater reliability of r = 0.9 between trained raters blind to condition. 2.5.2. Encephalitis scale In order to compare to our previous work with restraint stress, Experiment 3 also assessed the encephalitis-like
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symptoms. The severity of encephalitis signs of infection was scored on a 0 –4 scale that assessed the degree of encephalitis symptoms on several dimensions including hunched posture, piloerection, grooming, lethargy and sunken eyes. For each dimension, the animal received a rating from 0 to 6. This scale was derived by observing the natural progression of behavioral signs of illness and encephalitis in Theiler’s virus infected mice subjected to chronic restraint stress (Campbell et al., 2001; Welsh et al., in press). The animals in this study did not develop the entire range of symptoms observed following restraint and thus the overall scale (0– 4) did not discriminate between groups. However, the hunching subscale did discriminate between groups and thus this subscale is reported here. 2.5.3. Stride length Footprint stride length and spread were assessed similar to the method of McGavern et al. (1999, 2000). Briefly, hind limbs were painted with black tempura paint, and mice were allowed to walk down a 2.5 by 36 in. runway lined with paper to record the hind limb placement. This has been shown to provide a reliable and valid measure of virally mediated nerve damage and demyelination (McGavern et al., 1999, 2000). 2.6. Corticosteroid sensitivity assay On the sacrifice day, spleens were harvested to determine sensitivity of splenocytes to GC regulation (as per Stark, 2001). Briefly, spleens were placed in ice cold Hank’s balanced salt solution and mashed to obtain a single cell solution. Red blood cells were then lysed (Sigma, St. Louis, MO), followed by a wash of HBSS + 10% heat inactivated fetal bovine serum (FBS-HI, Equitech, Kerrville, TX). Viable cells were then counted using trypan blue dye exclusion and re-suspended at 2.5 106 cells/ml in supplemented RPMI (Sigma) + 10% FBS-HI, (supplementation: 0.75% sodium bicarbonate, 10 mM Hepes buffer, 100 U/ml penicillin, 100 Ag/ml streptomycin sulfate, 1.5 mM L-glutamine and 0.00035% 2-mercaptoethanol). LPS (Sigma #L6529) in 2% ethanol was added at a concentration of 1 Ag/ml for mitogen stimulation. GC resistance was tested by exposing aliquots of each suspension to dilutions of corticosteroid (0 – 5 AM, Sigma #C2505) dissolved in 2% ethanol and supplemented RPMI. Cell suspensions were placed in triplicate in flat-bottomed 96-well microtiter plates in 100-Al aliquots and incubated for 48 – 72 h at 37 jC and 5% CO2. After incubation, the cell proliferation assay was performed. Cell proliferation was assessed following the manufacturer’s instructions with the CellTiter 96 Aqueous nonradioactive proliferation assay kit from Promega (Madison, WI). Color changes were quantified by optical density readings at 490 nm from an EMAX ELISA plate reader (Molecular Devices, Sunnyvale, CA). Mean optical density values for the three replications for each sample were used, and the percentage of the corticosteroid exposed cells
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versus the LPS-stimulated cells was determined for statistical analysis. 2.7. Histological evaluation of CNS inflammation Mice were perfused with formalin, and brains and vertebral columns (containing intact spinal cord) were then removed and sectioned transversely into 4 and 12 pieces, respectively (see Campbell et al., 2001 for further detail). Tissues were then processed for routine hematoxylin and eosin (H&E) staining. Lesions of both the brain and spinal cord were scored in a similar manner by a rater blind to condition. Lesions evaluated included perivascular cuffing (accumulation of lymphocytes and macrophages around blood vessels), meningitis (accumulation of lymphocytes and macrophages in the meninges) and microgliosis (presence of increased microglia/macrophages within the parenchyma of the brain and spinal cord). 2.8. Viral load and clearance Prior studies have shown that viral titers in CNS are maximal at 1 – 2 weeks pi and by 3 –4 weeks pi, the virus is apparently cleared to non-detectable levels (Welsh et al., 1987, 1989). To evaluate the impact of SDR on CNS viral clearance, subsets of mice were sacrificed by an i.p. overdose of pentobarbital at days 7 and 21 pi. Brains and spinal cords were removed, homogenized and stored at 80 jC. The viral content of the supernatant from the homogenized brains and spinal cords were determined by plaque assay on L2 cells (Welsh et al., 1987). The plates were incubated at 37 jC at 5% CO2 for 72 h, and then stained with crystal violet to visualize the plaques that were formed. Assays were scored based on the number of plaques formed on the L2 cells and calculated per gram of tissue harvested (Welsh et al., 1987). 2.9. Statistical analysis Data are presented as mean F S.E.M. Analysis of variance (ANOVA) was used to evaluate differences across SDR and infection conditions; and repeated measure ANOVA were used for analysis over time as appropriate. These analyses were followed by post-hoc mean comparisons using Duncan’s new multiple range test. Trend analyses were performed to explore interactions over time. Pearson correlation coefficients (r) were used to determine inter-rater reliability, when noted.
3. Results 3.1. Neuroendocrine effects of SDR in BALBc/J mice Previous research indicated that SDR administered prior to LPS challenge not only induced an increase in circulating
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Fig. 2. The effect of SDR on mean plasma corticosteroid levels ( F S.E.M.) immediately following the third SDR session from Experiment 1. Corticosteroid response in the SDR-treated animals was double that of controls (*p < 0.0001). The animals were not infected at the time of sample collection for the corticosteroid assay (see Fig. 1).
GC levels, it also induced glucocorticoid resistance in C57BL/6 mice (Avitsur et al., 2001; Stark et al., 2001). Thus, we sought to evaluate whether BALBc/J mice would exhibit the same pattern of neuroendocrine changes in Experiments 1 and 2. 3.1.1. Effect of social disruption on circulating corticosteroid levels In Experiment 1 (PRE-SDR), higher concentrations of circulating corticosteroids were observed in SDR mice immediately following their third SDR session compared to NON-SDR mice, F(1,19) = 17.598, p < 0.0001, indicating that SDR was a physiologically efficacious stressor (Fig. 2). None of the mice were infected at this point in this study; thus, data was pooled across SDR condition, regardless of later infected status.
3.1.2. Effect of PRE-SDR on splenocyte sensitivity to corticosterone As shown in Fig. 3, the effect of PRE-SDR and CONSDR on GC resistance was also evaluated in Experiments 1 and 2. Slope analysis ANOVAs were performed, with the slope of the line formed across increasing concentrations of corticosteroids for individual subjects used as each data point. An ANOVA conducted on the slopes of non-infected mice (Experiment 1, PRE-SDR) revealed that the NI/PRE-SDR animals developed resistance to GC regulation of lymphocyte proliferation, F(1,10) = 4.89, p < 0.05 (Fig. 3A); however, in the infected animals (Experiment 1, PRE-SDR), GC resistance did not develop (Fig. 3B). Post-hoc comparison revealed that the slopes for the NI/PRE-SDR animals were significantly lower than those of the controls ( p < 0.05). In contrast, neither of the infected groups developed resistance ( p>0.05). In Experiment 2 (CON-SDR), all animals were infected at pnd 35 and sacrificed at day 14 pi, corresponding with SDR + 7 days, as in Experiment 1. Similar to Experiment 1, GC resistance was not apparent in either group following infection (Fig. 3C). Thus, PRE-SDR appears to induce GC resistance in non-infected BALBc/J mice, but not in infected BALBc/J mice. These findings are consistent with prior studies demonstrating that SDR induces GC resistance prior to LPS challenge (Quan et al., 2001). 3.2. Effect of SDR on motor function 3.2.1. Effect of the timing of SDR on hind limb impairment The effect of SDR administered prior to and concurrent with infection was investigated in Experiments 1 –3. Experiment 1 examined the effects of SDR administered 1 week prior to infection on hind limb motor impairment in infected and uninfected mice (Fig. 4A). Infection alone resulted in a
Fig. 3. The effect of PRE-SDR (3A), PRE-SDR plus infection (3B) and CON-SDR plus infection (3C) on mean proliferation of splenocytes to indicate GC resistance, from Experiments 1 (A, B) and 2 (C). GC resistance development was dependent upon infection status. GC resistance developed only in the noninfected/SDR animals (A), when SDR was applied prior to infection. All infected groups failed to develop GC resistance, whether PRE-SDR (B) or CON-SDR (C).
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F(8,160) = 3.742, p < 0.001, indicated that the effect of social disruption and infection on hind limb impairment changed over time. Post-hoc means comparison revealed that the I/PRE-SDR group exhibited greater impairment than the I/NON-SDR group at day 11 pi and for the duration of the experiment. In Experiment 2, the effect of SDR administered concurrent with infection on hind limb motor impairment was assessed in infected mice only (Fig. 4B). In contrast to the first experiment, significantly less hind limb impairment developed in the CON-SDR group compared to NON-SDR mice. An ANOVA confirmed a significant main effect for SDR, F(1,10) = 13.493, p < 0.005, as well as a significant interaction between SDR day PI, F(1,60) = 10.565, p < 0.0001. Mean comparisons revealed that the CON-SDR group had significantly less impairment beginning at day 3 pi and remained so for the remainder of the experiment. Experiment 3 directly compared the PRE-SDR and CON-SDR treatments (between subjects design) in infected mice within the same study, providing a replication of the results of Experiments 1 and 2 (Fig. 4C). Similar to Experiment 1, PRE-SDR exacerbated infectionrelated hind limb impairment, whereas CON-SDR reduced impairment as observed in Experiment 2. A main effect was found for SDR condition, F(2,32) = 16.532, p < 0.0001, and the interaction between SDR day PI was significant, F(18,288) = 4.099, p < 0.0001. Mean comparisons indicated that at day 3 pi the PRE-SDR animals had greater hind limb impairment than the other two groups; this difference was maintained throughout the experiment. In addition, by day 3 pi, the CON-SDR animals had less impairment than the NON-SDR animals, and this remained so through day 9 pi, when the difference between these two groups dissipated. This corresponds with the period of stress from days 0 to 7 pi for the CON-SDR condition (Fig. 1). Taken together, the results from Experiments 1– 3 demonstrate that PRESDR condition exacerbates infection-related hind limb paralysis, whereas CON-SDR condition results in a temporary reduction in impairment. Fig. 4. The effect PRE-SDR (A, Experiment 1), CON-SDR (B, Experiment 2) or comparing both (C, Experiment 3) on mean hind limb impairment across days pi ( F S.E.M.). Hind limb impairment developed with infection. In Experiment 1, PRE-SDR exacerbated development (A), while in Experiment 2, CON-SDR suppressed development (B). These effects were replicated in Experiment 3 (C).
progressive increase in hind limb impairment and PRE-SDR further exacerbated the level of impairment over time. An ANOVA confirmed a significant main effect for infection, F(1,20) = 116.129, p < 0.001, and SDR, F(1,20) = 18.212, p < 0.001, as well as a significant interaction between infection SDR, F(1,20) = 19.546, p < 0.001. A significant three-way interaction between infection SDR day PI,
3.2.2. Stride length As shown in Fig. 5A, the impact of SDR on stride length was evaluated in infected mice in Experiment 3. A main effect was found for SDR condition, F(2,32) = 8.478, p < 0.01. Mean comparisons indicated that PRE-SDR resulted in decreased stride length relative to the NONSDR and CON-SDR conditions ( p < 0.05). 3.2.3. Grid hang latencies Fig. 5B depicts grid hang latencies for the three stress conditions over time in Experiment 3. As expected, grid hang time decreased over the course of infection and PRESDR appears to reduce hang time even further (Fig. 5B). Although the main effect of SDR condition and interaction
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of encephalitis and high rates of mortality were not observed following exposure to social stress (all p’s>0.05). Because analysis of total scores on the encephalitis scale derived from our prior study did not reveal differences across groups (data not shown), exploratory analyses were conducted on individual subscales. The hunching subscale revealed group differences. 3.3.1. Hunching Hunching developed in all the animals, but the CONSDR animals had delayed onset of hunching, compared to the NON-SDR and PRE-SDR groups (Fig. 6). A main effect was found for both SDR condition, F(2,33) = 4.214, p < 0.05, and day pi, F(8,16) = 174.478, p < 0.0001, but condition did not interact with day pi [ F(18,297) = 0.871, p>0.05]. Post-hoc analysis revealed that the CON-SDR group developed hunching at a later time than the other groups and less hunching overall ( p < 0.05). In summary, the behavioral measures of hind limb impairment, hunching, grid hang latency and stride length all indicate that PRE-SDR resulted in a more severe disease course, while CON-SDR had either a beneficial or null effect compared to NON-SDR mice. 3.4. Effect of social disruption on CNS inflammation Application of SDR prior to infection resulted in an overall pattern of increased inflammation in spinal cord tissue (Fig. 7) and brain (data not shown). An ANOVA confirmed this with separate analysis of each measure at each sacrifice date, demonstrating a significant main effect for SDR at day 7 pi for microgliosis, F(2,159) = 4.328, p < 0.05, and meningitis ( p < 0.05) (Fig. 7B) in the spinal
Fig. 5. The effect of PRE-SDR and CON-SDR on mean stride length (A) and mean grid hang time (B) in Experiment 3. PRE-SDR had a detrimental effect on both stride length and hang time. Stride length was significantly reduced in the PRE-SDR animals at day 20 pi (A, *p < 0.01). In addition, grid hang time analysis revealed a linear trend for PRE-SDR animals to have reduced hang time (B). All data shown F S.E.M.
terms failed to reach significance, a linear trend was observed ( p < 0.05), indicating that PRE-SDR resulted in the shortest hang times. 3.3. Effect of the timing of SDR on behavioral measures of illness In contrast to the effects of chronic restraint stress on acute TMEV infection (Campbell et al., 2001), severe signs
Fig. 6. Mean hunching scores for infected mice exposed to PRE-SDR, CON-SDR or control non-stressed animals across days pi ( F S.E.M., Experiment 3). Hunching developed at a similar rate in all groups. However, the CON-SDR group began showing signs at a significantly later day pi and the PRE-SDR group developed the most severe signs.
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Fig. 7. Mean lesion scores in spinal cord of infected subjects in the PRE-SDR, CON-SDR and NON-SDR conditions ( F S.E.M., Experiment 3). Mice were sacrificed at day 7 pi and day 21 pi, and vertebral columns removed for histological assessment. Significant differences are denoted by asterisks (*p < 0.05) In the spinal cord, PRE-SDR resulted in significantly greater inflammation at day 7 pi on meningitis (A) and microgliosis (B). By day 21 pi, PRE-SDR resulted in greater inflammation as measured by meningitis (D), microgliosis (E) and perivascular cuffing (F).
cord. Post-hoc comparisons indicated that the PRE-SDR groups had greater microgliosis and meningitis compared to the NON-SDR group (Fig. 7A). Perivascular cuffing was not significantly different between the groups at day 7 pi ( p>0.05) (Fig. 7C). At day 21 pi, main effects for SDR on meningitis, F(2,159) = 4.327, p < 0.05, microgliosis, F(2,159) = 4.171, p < 0.05, and perivascular cuffing, F(2,159) = 3.002, p < 0.05, were revealed by ANOVA (Fig. 7D – F, respectively) in the spinal cord. Post-hoc means comparisons confirmed that PRE-SDR resulted in greater inflammation compared to NON-SDR and CON-SDR animals at day 21 pi. In the brain, the PRE-SDR animals had non-significant elevations in inflammation ( F’s < 2.3, p’s>0.05) at days 7 and 21 pi on the measures of microgliosis and perivascular cuffing. In addition, meningitis in the brain was not significantly different between groups at either sacrifice day ( p’s>0.05). In summary, PRE-SDR resulted in an overall pattern of elevated CNS inflammation
indicators at both days 7 and 21 pi, particularly in the spinal cord. 3.5. Effect of social disruption on viral titers and clearance Fig. 8 depicts log transformed viral titers in brain and spinal cord tissue measured at days 7 and 21 pi. In the brain, all conditions had high levels of virus at day 7 pi and significantly reduced levels at day 21 pi (Fig. 8A). Although viral titers decreased significantly over time ( p < 0.0001), no group differences were observed in brain. In contrast, group differences in viral clearance were observed in spinal cord. The PRE-SDR group had significantly lower titers than the other two groups at day 7 pi, F(2,15) = 5.474, p < 0.05 (Fig. 8B). In contrast, by day 21 pi, the CON-SDR animals had significantly lower titers, F(2,15) = 3.993, p < 0.05, compared to the other groups at day 21 pi. In addition, both the NON-SDR and CON-SDR
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Fig. 8. The effect of PRE-SDR, CON-SDR, and NON-SDR (Experiment 3) on mean viral titers ( F S.E.M.) and clearance in brain (A) and spinal cord (B). All significant differences are denoted by asterisks (*p < 0.05). In the brain, viral titers were uniformly elevated at day 7 pi and significantly decreased by day 21 pi (A), indicating uniform clearance as well. In contrast, the timing of the SDR affected both viral titers and clearance in the spinal cord. At day 7 pi, the PRESDR group has significantly lower titers compared to the other two groups, while at day 21 pi, the CON-SDR group has significantly lower titers compared to the other two groups. In addition, both the NON-SDR and CON-SDR groups have significantly reduced titers across time, while the PRE-SDR group failed to alter titers across time, indicating a failure of clearance in these animals (B).
groups had significantly lower titers over time, while the PRE-SDR group’s titer level did not change, F(2,28) = 6.259, p < 0.01, indicating a failure to clear the virus efficiently in these animals.
4. Discussion The current studies demonstrate that SDR, like restraint stress can exacerbate the course of Theiler’s virus infection. SDR altered behavioral signs of illness and motor function associated with the illness, including hunching, hind limb impairment, stride length and grid hang time. The behavioral signs were accompanied by alterations in both histological evidence for CNS inflammation and viral titers and clearance across time. Thus, SDR is effective in altering the course of Theiler’s virus infection. The direction of the effects of SDR on the course of Theiler’s virus infection was further dependent upon the timing of SDR. Specifically, SDR applied prior to infection (PRESDR) consistently exacerbated both behavioral and physiological measures of infection. In contrast, SDR applied concurrent with infection resulted in delayed hind limb impairment and hunching development, while other behavioral measures did not differ from NON-SDR mice. These animals (CON-SDR) also did not significantly differ from NON-SDR mice on measures of inflammation, but did demonstrate the most effective viral clearance competency.
4.1. Relation to prior empirical studies These findings are intriguing when compared to previous work from our laboratory using restraint stress (Campbell et al., 2001). Restraint stress was shown to exacerbate behavioral signs of illness and reduce viral clearance, much like PRE-SDR. However, PRE-SDR differs from restraint stress in two important ways. First, PRE-SDR increased inflammation, while restraint decreased it. Second, PRE-SDR is applied prior to infection, while restraint stress was applied concurrent with infection, much like CON-SDR. However, the current findings show that CONSDR actually reduced behavioral signs of infection and increased viral clearance, exactly the opposite of the effects found with restraint stress. 4.2. Potential neuroendocrine mechanisms One possible reason for the difference in behavioral sign development, inflammation and viral clearance may be the differential development of GC resistance. Normal elevations of corticosteroids were found both in the previous SDR studies (Avitsur et al., 2001) and in the current study. Thus, based on the majority of research showing that glucocorticoids reduce inflammation (Angeli et al., 1999; Lew et al., 1988), we would have expected a decrease in inflammation. However, prior studies characterizing the SDR model noted that this stressor results in GC resistance (Avitsur et al., 2001; Stark et al., 2001). In the current
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studies, GC resistance developed in the PRE-SDR animals, as indicated by the persistence of resistance in the NI/PRESDR animals, 7 days post-stress. Infected animals consistently failed to demonstrate GC resistance and this was true for the I/PRE-SDR animals, 7 days post-stress and infection. However, it is very likely that GC resistance was present in these animals at the time of infection. Both the I/PRE-SDR and NI/PRE-SDR groups were identical at the time of infection, and thus if the I/PRE-SDR group had not been infected, it is logical to assume that they too would have maintained GC resistance as the NI/PRE-SDR animals demonstrated. The presence of GC resistance at the time of infection would result in a more robust inflammatory response, as was found in the PRE-SDR animals (Quan et al., 2001). However, GC resistance dissipates and/or fails to develop in the presence of an immune challenge in these studies. Thus, the CON-SDR animals never developed GC resistance. Therefore, the CON-SDR animals did not have the same systemic environment that allowed for excess inflammation at the time of infection as the PRE-SDR animals did. GC resistance has previously been highly correlated with the degree of wounding (Avitsur et al., 2002). In the current studies, however, wounding was very mild and rare, yet GC resistance did develop. One explanation for this finding is the well-documented hyper-responsive nature of BALBc mice (genetically very similar to the BALBc/J mice used in the current study), and the relatively stress resilient nature of C57BL/6 mice (Shanks and Anisman, 1988; Shanks et al., 1994a; Shanks et al., 1994b; Shanks et al., 1990; Zacharko et al., 1990; Zaharia et al., 1996), used in the previous SDR studies (Avitsur et al., 2002; Avitsur et al., 2001; Quan et al., 2001; Stark et al., 2002; Stark et al., 2001). The initial SDR studies utilize C57BL/6 mice at 8 weeks of age or older at the time of SDR. The current study utilized younger (4– 5 weeks of age) BALBc/J mice due to constrictions of the Theiler’s virus model. (Theiler’s virus infection does not cause the chronic phase in C57BL/6 mice and susceptible strains such as the BALBc/J, must be infected during early adolescence.) The antagonistic confrontations between subject mice and the intruder were generally less severe in the current study, possibly due to this age difference, and resulted in the less severe wounding. However, due to the stress hyper-responsivity of the BALBc/J strain, it is possible that the threshold of defeat was simply lower than that found previously, and thus the antagonistic interactions resulting in severe wounding were not required for the development of GC resistance.
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animals at this time point (Biron et al., 1998; Fiette et al., 1995; Njenga et al., 1997b). However, at day 21 pi, the PRE-SDR group viral titers had not decreased, which may indicate an interruption or alteration of specific immune responses (CD4+, CD8+, B cell), which are known to be important in viral clearance in the acute phase Theiler’s virus infection (Borrow et al., 1992; Borrow et al., 1993; Murray et al., 1998; Welsh et al., 1987). In contrast, at day 21 pi, the CON-SDR group viral titers were significantly lower then the other two groups. As viral persistence in the CNS during the acute phase is essential for the development of the chronic demyelinating phase of Theiler’s virus (Brahic et al., 1981; Rodriguez et al., 1996), the PRESDR animals are at greatest risk for developing the later disease. The later demyelinating phase of Theiler’s virus is often used as a model for MS. The findings in this study are important when we examine the epidemiological evidence that MS may be caused by a viral infection in adolescence or early adulthood (Acheson, 1977; Kurtzke, 1993). A viral etiology is implicated by these studies, yet no single viral culprit has been found. Many viruses may be capable of initiating the autoimmune processes leading to MS. It may be that the systemic conditions at the time of infection are a factor in this development, such as the presence of a stressor that induces GC resistance. Further studies of the innate and acquired immune responses to Theiler’s virus in mice subjected to SDR will aid in our understanding of the mechanisms involved in the results reported herein. Based on prior studies using SDR (Quan et al., 2001), we anticipate that pro-inflammatory cytokines may be expressed in the CNS of PRE-SDR mice following infection with Theiler’s virus. Indeed, the observed increased histopathology following PRE-SDR and infection with Theiler’s virus in the present study is likely due to the increased production of pro-inflammatory cytokines. Finally, additional investigation will be needed to determine if the higher viral load and/or inability to clear virus in the PRE-SDR animals in comparison to the CONSDR animals will alter the development of the chronic demyelinating phase of the disease. In summary, SDR alters Theiler’s virus infection course, the alteration is dependent upon the timing of SDR application. These effects are apparent both behaviorally and physiologically, with PRE-SDR leading to the most severe disease course behaviorally, with greater inflammation and reduced viral clearance efficacy across time. In contrast, CON-SDR differs very little across time from non-stressed controls; however, they demonstrate the most effective viral clearance across time.
4.3. Potential immune mechanisms The PRE-SDR animals had both elevated inflammation and lower viral titers at day 7 pi. This indicates that innate immune processes associated with inflammation, such as increased type 1 IFNs may be aiding viral clearance in these
Acknowledgements This research was supported by an NSF Fellowship to RRJ and NMSS RG3128 and NINDS RO1 NS39569 to
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CJRW and MWM. In addition, we would like to thank David Padgett for his patient help, Jim Grau for insight into the final design of these experiments, Heath McCullough, Gabby Oroza, Amy Sieve and Lin Bustamante.
References Acheson, E.D., 1977. Epidemiology of multiple sclerosis. Br. Med. Bull. 33, 9 – 14. Angeli, A., Masera, R.G., Sartori, M.L., Fortunati, N., Racca, S., Dovio, A., Staurenghi, A., Frairia, R., 1999. Modulation by cytokines of glucocorticoid action. Ann. N.Y. Acad. Sci. 876, 210 – 220. Aubert, C., Chamorro, M., Brahic, M., 1987. Identification of Theiler’s virus infected cells in the central nervous system of the mouse during demyelinating disease. Microb. Pathog. 3, 319 – 326. Avitsur, R., Stark, J.L., Sheridan, J.F., 2001. Social stress induces glucocorticoid resistance in subordinate animals. Horm. Behav. 39, 247 – 257. Avitsur, R., Stark, J.L., Dhabhar, F.S., Sheridan, J.F., 2002. Social stress alters splenocyte phenotype and function. J. Neuroimmunol. 132, 66 – 71. Biron, C.A., Cousens, L.P., Ruzek, M.C., Su, H.C., Salazar-Mather, T.P., 1998. Early cytokine responses to viral infections and their roles in shaping endogenous cellular immunity. Adv. Exp. Med. Biol. 452, 143 – 149. Borrow, P., Tonks, P., Welsh, C.J., Nash, A.A., 1992. The role of CD8+ T cells in the acute and chronic phases of Theiler’s murine encephalomyelitis virus-induced disease in mice. J. Gen. Virol. 73 (7), 1861 – 1865. Borrow, P., Welsh, C.J., Nash, A.A., 1993. Study of the mechanisms by which CD4 + T cells contribute to protection in Theiler’s murine encephalomyelitis. Immunology 80, 502 – 506. Brahic, M., Stroop, W.G., Baringer, J.R., 1981. Theiler’s virus persists in glial cells during demyelinating disease. Cell 26, 123 – 128. Campbell, T., Meagher, M.W., Sieve, A., Scott, B., Storts, R., Welsh, T.H., Welsh, C.J., 2001. The effects of restraint stress on the neuropathogenesis of Theiler’s virus infection: I. Acute disease. Brain Behav. Immun. 15, 235 – 254. de Groot, J., van Milligen, F.J., Moonen-Leusen, B.W., Thomas, G., Koolhaas, J.M., 1999. A single social defeat transiently suppresses the antiviral immune response in mice. J. Neuroimmunol. 95, 143 – 151. Fiette, L., Aubert, C., Muller, U., Huang, S., Aguet, M., Brahic, M., Bureau, J.F., 1995. Theiler’s virus infection of 129Sv mice that lack the interferon alpha/beta or interferon gamma receptors. J. Exp. Med. 181, 2069 – 2076. Fleshner, M., Laudenslager, M.L., Simons, L., Maier, S.F., 1989. Reduced serum antibodies associated with social defeat in rats. Physiol. Behav. 45, 1183 – 1187. Franchimont, D., Martens, H., Hagelstein, M.T., Louis, E., Dewe, W., Chrousos, G.P., Belaiche, J., Geenen, V., 1999. Tumor necrosis factor alpha decreases, and interleukin-10 increases, the sensitivity of human monocytes to dexamethasone: potential regulation of the glucocorticoid receptor. J. Clin. Endocrinol. Metab. 84, 2834 – 2839. Hart, P.H., Whitty, G.A., Burgess, D.R., Croatto, M., Hamilton, J.A., 1990. Augmentation of glucocorticoid action on human monocytes by interleukin-4. Lymphokine Res. 9, 147 – 153. Keith, L.D., Winslow, J.R., Reynolds, R.W., 1978. A general procedure for estimation of corticosteroid response in individual rats. Steroids 31, 523 – 531. Koehler, T., 1985. Stress and rheumatoid arthritis: a survey of empirical evidence in human and animal studies. J. Psychosom. Res. 29, 655 – 663. Koolhaas, J.M., De Boer, S.F., De Rutter, A.J., Meerlo, P., Sgoifo, A., 1997a. Social stress in rats and mice. Acta Physiol. Scand., Suppl. 640, 69 – 72. Koolhaas, J.M., Meerlo, P., De Boer, S.F., Strubbe, J.H., Bohus, B., 1997b.
The temporal dynamics of the stress response. Neurosci. Biobehav. Rev. 21, 775 – 782. Korte, S.M., Buwalda, B., Meijer, O., De Kloet, E.R., Bohus, B., 1995. Socially defeated male rats display a blunted adrenocortical response to a low dose of 8-OH-DPAT. Eur. J. Pharmacol. 272, 45 – 50. Kurtzke, J.F., 1993. Epidemiologic evidence for multiple sclerosis as an infection. Clin. Microbiol. Rev. 6, 382 – 427. Lew, W., Oppenheim, J.J., Matsushima, K., 1988. Analysis of the suppression of IL-1 alpha and IL-1 beta production in human peripheral blood mononuclear adherent cells by a glucocorticoid hormone. J. Immunol. 140, 1895 – 1902. Lipton, H.L., 1975. Theiler’s virus infection in mice: an unusual biphasic disease process leading to demyelination. Infect. Immun. 11, 1147 – 1155. McGavern, D.B., Zoecklein, L., Drescher, K.M., Rodriguez, M., 1999. Quantitative assessment of neurologic deficits in a chronic progressive murine model of CNS demyelination. Exp. Neurol. 158, 171 – 181. McGavern, D.B., Zoecklein, L., Sathornsumetee, S., Rodriguez, M., 2000. Assessment of hind limb gait as a powerful indicator of axonal loss in a murine model of progressive CNS demyelination. Brain Res. 877, 396 – 400. Murray, P.D., Pavelko, K.D., Leibowitz, J., Lin, X., Rodriguez, M., 1998. CD4 (+) and CD8 (+) T cells make discrete contributions to demyelination and neurologic disease in a viral model of multiple sclerosis. J. Virol. 72, 7320 – 7329. Njenga, M.K., Asakura, K., Hunter, S.F., Wettstein, P., Pease, L.R., Rodriguez, M., 1997a. The immune system preferentially clears Theiler’s virus from the gray matter of the central nervous system. J. Virol. 71, 8592 – 8601. Njenga, M.K., Pease, L.R., Wettstein, P., Mak, T., Rodriguez, M., 1997b. Interferon alpha/beta mediates early virus-induced expression of H-2D and H-2K in the central nervous system. Lab. Invest. 77, 71 – 84. Quan, N., Avitsur, R., Stark, J.L., He, L., Shah, M., Caligiuri, M., Padgett, D.A., Marucha, P.T., Sheridan, J.F., 2001. Social stress increases the susceptibility to endotoxic shock. J. Neuroimmunol. 115, 36 – 45. Rodriguez, M., Pavelko, K.D., Njenga, M.K., Logan, W.C., Wettstein, P.J., 1996. The balance between persistent virus infection and immune cells determines demyelination. J. Immunol. 157, 5699 – 5709. Shanks, N., Anisman, H., 1988. Stressor-provoked behavioral changes in six strains of mice. Behav. Neurosci. 102, 894 – 905. Shanks, N., Griffiths, J., Zalcman, S., Zacharko, R.M., Anisman, H., 1990. Mouse strain differences in plasma corticosterone following uncontrollable footshock. Pharmacol. Biochem. Behav. 36, 515 – 519. Shanks, N., Griffiths, J., Anisman, H., 1994a. Central catecholamine alterations induced by stressor exposure: analyses in recombinant inbred strains of mice. Behav. Brain Res. 63, 25 – 33. Shanks, N., Griffiths, J., Anisman, H., 1994b. Norepinephrine and serotonin alterations following chronic stressor exposure: mouse strain differences. Pharmacol. Biochem. Behav. 49, 57 – 65. Stark, J.L., Avitsur, R., Padgett, D.A., Campbell, K.A., Beck, F.M., Sheridan, J.F., 2001. Social stress induces glucocorticoid resistance in macrophages. Am. J. Physiol., Regul. Integr. Comp. Physiol. 280, R1799 – R1805. Stark, J.L., Avitsur, R., Hunzeker, J., Padgett, D.A., Sheridan, J.F., 2002. Interleukin-6 and the development of social disruption-induced glucocorticoid resistance. J. Neuroimmunol. 124, 9 – 15. Stefanski, V., Engler, H., 1999. Social stress, dominance and blood cellular immunity. J. Neuroimmunol. 94, 144 – 152. Welsh, C.J., Tonks, P., Nash, A.A., Blakemore, W.F., 1987. The effect of L3T4 T cell depletion on the pathogenesis of Theiler’s murine encephalomyelitis virus infection in CBA mice. J. Gen. Virol. 68 (Pt 6), 1659 – 1667. Welsh, C.J.R., Blakemore, W.F., Tonks, P., Borrow, P., Nash, A.A., 1989. Theiler’s murine encephalomyelitis virus infection in mice: a persistent viral infection of the central nervous system which induces demyelination. In: Dimmock, N. (Ed.), Immune responses, Virus Infection and Disease, Oxford University Press, pp. 125 – 147.
R.R. Johnson et al. / Journal of Neuroimmunology 148 (2004) 74–85 Welsh, C.J.R., Bustamante, L., Nayak, M., Welsh, T.H., Dean, D.D., Meagher, M.W., 2004. The effects of restraint stress on the neuropathogenesis of Theiler’s virus infection: II. NK cell function and cytokine levels in acute disease. Brain Behav. Immun. (In press). Zacharko, R.M., Gilmore, W., MacNeil, G., Kasian, M., Anisman, H., 1990. Stressor induced variations of intracranial self-stimulation
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from the mesocortex in several strains of mice. Brain Res. 533, 353 – 357. Zaharia, M.D., Kulczycki, J., Shanks, N., Meaney, M.J., Anisman, H., 1996. The effects of early postnatal stimulation on Morris water-maze acquisition in adult mice: genetic and maternal factors. Psychopharmacology (Berl.) 128, 227 – 239.