Brain, Behavior, and Immunity 40 (2014) 110–120

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Brain, Behavior, and Immunity

journal homepage: www.elsevier .com/locate /ybrbi

Neonatal experience interacts with adult social stress to alter acuteand chronic Theiler’s virus infection

http://dx.doi.org/10.1016/j.bbi.2014.03.0020889-1591/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Address: Department of Psychology, Texas A&M Uni-versity, College Station, TX 77843-4235, United States. Tel.: +1 (979) 845 2564; fax:+1 (979) 845 4727.

E-mail address: M-Meagher@tamu.edu (M.W. Meagher).

R.R. Johnson a, S. Maldonado Bouchard b,e, T.W. Prentice b, P. Bridegam b, F. Rassu b, C.R. Young c,A.J. Steelman c, T.H. Welsh Jr. d, C.J. Welsh c, M.W. Meagher b,⇑a Advanced brain Monitoring, Inc, Carlsbad, CA 92008, United Statesb Department of Psychology, College of Liberal Arts, Texas A&M University, United Statesc Departments of Veterinary Integrative Biosciences and Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, United Statesd Department of Animal Science, College of Agriculture and Life Sciences, Texas A&M University, United Statese Texas A&M Institute for Neuroscience, Texas A&M University, College Station, TX 77843, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 20 November 2013Received in revised form 19 February 2014Accepted 3 March 2014Available online 12 March 2014

Keywords:HandlingMaternal separationSocial disruptionStressTheiler’s virusMultiple sclerosisIL-6Resilience

Previous research has shown that neonatal handling has prolonged protective effects associated withstress resilience and aging, yet little is known about its effect on stress-induced modulation of infectiousdisease. We have previously demonstrated that social disruption stress exacerbates the acute and chronicphases of the disease when applied prior to Theiler’s virus infection (PRE-SDR) whereas it attenuates dis-ease severity when applied concurrently with infection (CON-SDR). Here, we asked whether neonatalhandling would protect adult mice from the detrimental effects of PRE-SDR and attenuate the protectiveeffects of CON-SDR on Theiler’s virus infection. As expected, handling alone decreased IL-6 and cortico-sterone levels, protected the non-stressed adult mice from motor impairment throughout infectionand reduced antibodies to myelin components (PLP, MBP) during the autoimmune phase of disease. Incontrast, neonatal handling X PRE/CON-SDR elevated IL-6 and reduced corticosterone as well as increasedmotor impairment during the acute phase of the infection. Neonatal handling X PRE/CON-SDR continuedto exacerbate motor impairment during the chronic phase, whereas only neonatal handling X PRE-SDRincreased in antibodies to PLP, MOG, MBP and TMEV. Together, these results imply that while handlingreduced the severity of later Theiler’s virus infection in non-stressed mice, brief handling may not be pro-tective when paired with later social stress.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

An enriched early environment influences developmental pro-gramming and has protective effects later in life (Fenoglio et al.,2005; Flanigan and Cook, 2011; Francis et al., 1999; Liu et al.,1997; McEwen, 2003; Parfitt et al., 2004). For example, in rats, fos-tering mothers that provide high amounts of arch-backed nursing(ABN) and licking & grooming (LG) beget offspring that exhibit de-creased physiological and behavioral responses to stressors andanxiety (Caldji et al., 1998; Fish et al., 2004). This high qualitymaternal care is linked to enhanced hippocampal synaptogenesisand reduced hypothalamic–pituitary-adrenal axis (HPA) activation

in response to stress as adults, compared to offspring from lowABN/LG fostering mothers (Francis et al., 1999; Liu et al., 1997).

Neonatal handling, a short maternal separation paradigm, hasbeen shown in rats to have prolonged protective effects associatedwith stress resilience and aging by modifying the functioning ofthe neuroendocrine and immune systems (Barnett and Burn,1967; Bilbo et al., 2007; Lee and Williams, 1974; Levine andMullins, 1967). Handling results in increased levels of maternalABN/LG. It involves removing rodent pups from their mothersand home cage, placing them in a small climate controlled container,and returning them back to their cage after 15–20 min. This is doneon a daily basis during the first few weeks of life. The procedure re-sults in robust and long-lasting behavioral and neuroendocrine ef-fects later in life, including reduced behavioral and HPA axisresponses to stress (Liu et al., 1997, 2000). Adult rats handled asneonates show lower secretion of corticosterone after exposureto immobilization stress, and they take less time to recover to basalcorticosterone levels, which results in lower stress hormone

R.R. Johnson et al. / Brain, Behavior, and Immunity 40 (2014) 110–120 111

exposure, decreased loss of hippocampal neurons, and better per-formance on spatial learning tests (Liu et al., 2000).

Less is known about the effects of neonatal maternal separationin mice. Parfitt et al. (2004) showed that 10-min neonatal handling(short maternal separation) leads to a blunted adult corticosteroneresponse in C57BL/6 mice, decreasing anxiety and adrenal reactiv-ity. In turn, Romeo et al. (2003) have shown that long maternalseparation (3 h) has a sex-dependent effect, increasing adult anxi-ety-like behavior in male mice, but decreasing adult anxiety-likebehavior in female mice in the diestrous phase of their estrouscycle.

Furthermore, relatively little is known about the effects of neo-natal handling on stress-induced modulation of infectious diseasein mice (Bilbo et al., 2007; Columba-Cabezas et al., 2009; Parfittet al., 2004). TMEV infection in mice induces a biphasic diseasecharacterized by an acute CNS inflammatory phase followed by achronic neuroinflammatory/autoimmune demyelination phasesimilar to multiple sclerosis (MS) (Lipton, 1975; Oleszak et al.,2004). We have previously shown that social stress in adulthoodin the form of social disruption stress (SDR) can modulate thecourse of Theiler’s virus (TMEV) infection in mice. Our previouswork has demonstrated that social disruption stress applied priorto TMEV infection (PRE-SDR) exacerbates the acute and chronicphases of the disease. In contrast, social disruption stress appliedconcurrent (CON-SDR) with infection attenuated the severity ofTMEV infection during both the acute and chronic phase (Johnsonet al., 2004, 2006). We therefore suspected that the protectiveeffects of CON-SDR were due to its action as an acute stressor.By increasing corticosterone levels, CON-SDR reduces CNSinflammation, and attenuates behavioral signs of infection in theacute phase, and reduced symptoms in the acute phase areassociated with less severe symptoms in the chronic phase as well(Meagher et al., 2006a,b). Although SDR has been shown tomodulate the severity of infection, it remains to be seen whetherthe protective effects of handling during the neonatal period willalter vulnerability to this stressor and its interaction with infectionlater in life.

If CON-SDR exerts its protective effects by increasing corticoste-rone levels and therefore reducing CNS inflammation, we can pre-dict that neonatal handling, which blunts HPA function in adultmice (Parfitt et al., 2004), may attenuate increases in corticoste-rone levels following CON-SDR, thereby disrupting the protectiveeffects of SDR (Meagher et al., 2006a,b). In the present study, weasked whether neonatal handling in mice would protect adult sub-jects from the detrimental effects of social disruption stress priorto infection (PRE-SDR) and attenuate the protective effects ofSDR concurrent with TMEV infection (CON-SDR). Balb/cJ male pupswere exposed to handling or an undisturbed control condition be-tween postnatal day 2 and 14. To examine the impact of handlingon stress-induced modulation of TMEV infection, during adoles-cence mice were exposed to SDR either before (beginning postnatalday 28) or concurrent with infection (postnatal day 35) or were leftundisturbed. Mice were intracranially infected with TMEV on

Table 1Experimental timeline.

postnatal day 35, which corresponds to the onset of puberty inmice. Mice were then examined regularly for behavioral andimmunological signs of the acute and chronic disease in order todetermine how handling in early life would interact with laterstress/disease exposure.

2. Methods

2.1. Subjects

Male Balb/cJ mice (N = 36) were bred in-house. They wereweaned at post-natal day 21 (pnd 21). Mice were housed threeper cage to limit aggression within the cage over the 6–8 mo re-quired for a chronic phase study of TMEV infection in Balb/cJ mice.They were maintained on a 12 h light/dark cycle (0500/1700 h)with access to food and water ad libitum. Each cage was assignedto one of three SDR conditions (PRE-SDR, CON-SDR, NON-SDR),counterbalancing by handling condition (H-15 or H-0), weight,and dam, for n = 6 per group. To reduce possible littermate con-founds, litters from the same dam were not placed into the sameexperimental groups; rather each dam contributed 2–3 pups fromeach litter to 2–3 of the experimental groups, and each dam con-tributed at least 2 l. Additional cages were left uninfected and un-stressed to provide reference data for several assays (e.g. footprintstride length, IL-6 ELISA, RIAs). This reference group began asn = 12 (half were H-0, half H-15) for acute measures. Howeverdue to intra-cage aggression that occurs over time, even with lit-termates, only n = 9 were available for early chronic phase compar-isons, and n = 3 by the end of the study. The n = 9 subset included 3cages of 2 and one cage of 3 animals. The final 3 survivors werefrom 3 separate cages, and living in isolation after aggression leadto separation of the littermates. SDR intruders were selected from apopulation of active male breeders based on latency to attack apeer (30s) and adolescents (2 min) during three separate assess-ments. For a timeline of experimental procedures, refer to Table 1.All animal protocols were in accordance with NIH Guidelines forCare and Use of Laboratory Animals and were approved by theTexas A&M Laboratory Animal Care and Use Committee.

2.2. Handling

Mice were either exposed to handling condition (H) of 15 min(H-15) or left undisturbed (H-0). H-15 pups were separated fromthe dams from pnd 2–15 at 0800 h for 15 min per day. During H-15, the dam was first removed from the home cage and placed ina separate cage with food and water. The pups were then removedfrom the home cage and placed in a warm (78–82 �C), humid (80%)chamber, with minimal manipulation by the experimenter. After15 min, the pups were replaced in the home cage, followed bythe replacement of the dam. H-0 pups were left undisturbed inthe home cage with the dam.

112 R.R. Johnson et al. / Brain, Behavior, and Immunity 40 (2014) 110–120

2.3. Social disruption (SDR) stress

Cages of 3 mice were assigned to the PRE-SDR, CON-SDR, or noSDR (NON-SDR), in a counterbalanced manner according to weightand handling condition. SDR mice experienced six SDR events overthe course of 1 wk: three consecutive cycles, one rest, three con-secutive cycles. Each cycle of SDR consisted in relocating the homecage to a procedure room at 1700, and introducing an aggressiveintruder for 2 h (Avitsur et al., 2001; Stark et al., 2001). Fig. 1 pro-vides the experimental timeline. SDR sessions were monitored toensure that the intruder attacked the residents within the first10 min and that the residents demonstrated submissive behavior.If it was not the case, the intruder was replaced, and the sessioncontinued for the remaining 2-h. Unique intruders were used foreach SDR cycle.

The SDR cycles were conducted the week prior to infection (i.e.pre-stressed) for the PRE-SDR group or the week following

Fig. 1. The effects of handling and SDR on (A) hind limb impairment, (B) stridelength and (C) body weight, during acute Theiler’s virus infection. Non-handled,Non-SDR mice showed significantly greater high limb impairment compared totheir handled counterparts. In contrast, handled mice in the PRE-SDR and CON-SDRgroups have significantly greater hind limb impairment than non-handled mice (A).Only the PRE-SDR/H-15 group had significantly reduced stride length compared tothe PRE-SDR/H-0 group (B). The PRE-SDR, non-handled mice showed decreasedbody weight in comparison to the CON-SDR non-handled mice (C). Asterisksindicate significant differences (⁄).

infection (i.e. concurrently stressed) for the CON-SDR group.NON-SDR cages were left undisturbed in the colony room.

2.4. Virus/Infection

The BeAn strain of Theiler’s virus (obtained from Dr. H.L. Lipton,Department of Microbiology and Immunology, University of Illi-nois, Chicago, IL.) was propagated and amplified in L-cells. The cul-ture supernatant containing infectious virus was aliquoted, titratedand stored at �70� C before use (Welsh et al., 1987). Mice wereanesthetized on day 0 pi/pnd 35 with isoflurane inhalation (VedcoInc., St. Joseph, MO) and injected intracranially (ic) into the rightmid-parietal cortex (depth approximately 1.5 mm) with5 � 104 pfu of the BeAn strain of Theiler’s virus in a 20 ll volume(Campbell et al., 2001; McGavern et al.,1999, 2000; Rose et al.,1998; Theil et al., 2000). Inoculation for all subjects occurred at2100 h. The procedure was conducted either following the lastSDR session (PRE-SDR), the first SDR session (CON-SDR), or inde-pendent of SDR (NON-SDR).

2.5. Behavioral assessment of motoric impairment and illness

Multiple measures of psychomotor behavior including hind limbimpairment ratings, locomotor activity (in the vertical and horizon-tal planes), and footprint stride length were examined in both theacute and chronic phase. The analysis conducted for stride lengthwas similar to that of McGavern, and colleagues (McGavern et al.,1999). The first 6 step lengths were averaged, and subtracted fromaveraged age-matched reference data (n = 12). This has been shownto be a sensitive measure of impairment at day 20 pi (Johnson et al.,2004, 2006). Acute phase behavioral assessments were conductedup to day 28 pi. Day 28 pi is generally when maximal viral clearancehas occurred across strains (Oleszak et al., 2004), followed by astrain dependent refractory period before chronic symptoms ap-pear. During the refractory period, mice were monitored weeklyand compared to non-infected age-matched controls, to determinethe onset of behavioral signs of recovery. Full behavioral recovery(based on comparison to age-matched controls) occurred by day77 pi for all mice, and lasted approximately 7–8 weeks (�day126 pi). Chronic phase was determined to have begun when ani-mals began to diverge from the age-matched controls (n = 9) onany measure. This occurred first in the PRE-SDR animals, aroundday 126 pi. The NON-SDR group began to diverge after day 140 pi.Thus we report chronic phase measures taken just prior to theNON-SDR group’s divergence to demonstrate the development ofthe disease, beginning around day 135 pi. (Note: CON-SDR symp-toms did not diverge until significantly later in the disease process.)From this point forward, behavioral measures were collected twiceweekly for chronic phase assessment. HLI is reported beginning atday 138 pi. Stride length was assessed at day 140 pi, due to thedevelopment of HLI around day 138 pi in the PRE-SDR group.

2.5.1. Behavioral ratings of illnessAs in previous studies, we collected a battery of behavioral and

immunological measures throughout both the acute and chronicphases of the disease. Our first measure of impairment is the hindlimb impairment scale, shown to be sensitive to the developmentof motor impairment in both the acute and chronic phases of TMEVinfection (Johnson et al., 2004, 2006). During the acute phase, micewere examined every other day for signs of encephalitis and hindlimb impairment (HLI). During the refractory period and chronicphase, these measures were taken weekly (see Fig 1). Illness raterswere blind to the subjects’ experimental conditions, and weretrained to an inter-rater reliability criterion of r = 90. All subjectsunderwent the exact same scoring procedure independent ofsymptoms or experimental condition. A separate numeric score

R.R. Johnson et al. / Brain, Behavior, and Immunity 40 (2014) 110–120 113

was given for each hind limb individually, based on the symptomsof impairment the mice displayed (0 = healthy; 1 = slight weaknessin grip; 2 = clear weakness in grip; 3 = slight paralysis; 4 = moder-ate paralysis, 5 = complete paralysis with muscle tone, 6 = com-plete paralysis with no muscle tone (Johnson et al., 2004). Theindividual leg scores were then added together and the combinedscore analyzed.

2.5.2. Open field activityWeekly 20 min sessions were used to measure horizontal and

vertical locomotion activity for the duration of the study. Six Ver-samax open field chambers (Omnitech), equipped with two banksof eight photocells on each wall were used to measure horizontaland vertical locomotion. These open field boxes are interfaced witha digital multiplexor (Coulbourn E61–58) located in an adjacentroom. Testing was conducted in the dark between 1500 and1700 h. White noise (64 dB) was used to mask extraneous distur-bances. Analyses of open field activity are separated into two setsof variables. First, anxiety related variables (center time and dis-tance). Second, motor impairment measures (horizontal and verti-cal activity, with vertical activity being the most indicative of hindlimb impairment). The anxiety-related measures were examinedbecause H-15 mice typically have reduced anxiety, possibly relatedthe hypo-responsive corticosteroid response (Hofer et al., 1999;Maciag et al., 2002; Zaharia et al.,1996).

2.5.3. Stride lengthFootprint stride length and spread were assessed following a

procedure similar to the method of McGavern et al. (1999, 2000).Briefly, hind limbs were painted with blue finger paint and fore-limbs with red. Mice then walked down a 2.500 by 3600 runway linedwith paper to record limb placement. This has been shown to pro-vide a reliable and valid measure of virally-mediated nerve damageand demyelination (McGavern et al., 1999, 2000). Due to the sim-ilarity of the hind limb and forelimb data, only the former ispresented.

2.5.4. Body WeightBody weight was collected weekly after weaning, as an assess-

ment of illness. Failure to gain weight normally (compared to non-infected age-matched controls or other experimental groups) wastaken as indication of illness and/or failure to thrive.

2.6. Serum Assays

2.6.1. Blood CollectionFor assays that occurred throughout the study (CORT, IL-6, and

antibody responses to Theiler’s virus and myelin components atdays 42 pi and 140 pi), blood collection order was counterbalancedacross conditions and age-matched non-infected controls werealso bled for comparison. For the CORT assessment associated withsocial stress, the PRE-SDR group was compared to the age matchedcontrols, as this metric was taken prior to infection for the PRE-SDRanimals. The NON-SDR group was bled for comparison of the CON-SDR group, to control for the effect of infection (both groups wereinfected the same length of time at the time of the bleed). For allother bleeds, the age-matched controls were bled at the sameapproximate age as the infected mice. Each mouse’s leg was shaved12 h prior to blood collection to minimize stress (Young et al.,2008). Mice were individually transported (in a small plastic con-tainer with air holes) to a separate procedure room and bled viathe saphenous vein within 2 min of cage disturbance to minimizestress associated with the handling for the bleed. After the bleedingprocedure, mice were placed in a recovery cage in the procedureroom, until all of the mice had been bled. For the day 170 pi anti-body responses to Theiler’s virus and myelin components assays,

blood was collected during the sacrifice procedure. Immediatelyfollowing collection, all blood was placed on ice and allowed tocoagulate, then centrifuged at 2000 rpm for 15 min and serum col-lected and stored at �20 �C until the time of assay.

2.6.2. Corticosteroid radioimmunoassay (RIA)Sera for corticosteroid assessment were collected following the

third session of SDR. The comparison for the PRE-SDR group werethe age matched controls (n = 12 at this pre-infection time point,half were H-15), and the NON-SDR animals are the comparisongroup for the CON-SDR data collection. The same control groupswere not possible to use, as this would have resulted in bleedingthe NON-SDR animals twice as often as the other groups. Thesedata were analyzed in two separate ANOVAs (PRE-SDR vs referencegroup of age matched controls; and CON-SDR vs NON-SDR). Theplasma CORT concentration was determined using a 125I-RIA kit(ICN Biomedicals, Inc., Costa Mesa, California) as in past studiesfrom this laboratory (Johnson et al., 2004, 2006; Meagher et al.,2007; Meagher et al., 2006a,b; Sieve et al., 2004; Young et al.,2008).

2.6.3. Il-6 elisaIL-6 levels, in sera collected at day 9 pi, were determined using

an ELISA assay (R & D Systems Madison, WI), following the manu-facturer’s instructions.

2.6.4. Antibody responses to Theiler’s virus and myelin componentsRIAs were used to measure sera antibodies against Theiler’s

virus (whole virus, identical to that used for infection), myelin ba-sic protein (recombinant bovine, MBP, Sigma, St. Louis, MO), mye-lin oligodendrocyte glycoprotein peptide (synthetic purified, MOG,Sigma, St. Louis, MO) and proteolipid protein peptide (syntheticpurified, PLP, AnaSpec Inc., CA) using previously described proce-dures (Dolimbek et al., 2002; Sieve et al., 2004; Young et al.,1983). The RIA was developed using radiolabeled protein A, whichbinds to the Fc portion of immunoglobulin. Therefore, the level ofradioactivity (counts per minute, CPM) measured is equated withantibody level (Dolimbek et al., 2002; Young et al., 1983). Antibodylevels were assessed at days 42, 140 and 170 pi (Sacrifice day).

2.6.5. Spleen weights/sacrificeMice were deeply anesthetized with ketamine (100 mg/kg)/

xylazine (5 mg/kg), bled from the brachial artery, and transcardial-ly perfused with PBS. Spleen were dissected out and weighed, todetermine if the experimental manipulations conducted herein al-tered splenic weights, as had previously been reported in bothacute and chronic Theiler’s virus infection (Johnson et al., 2006;Mi et al., 2006; Steelman et al., 2009).

2.7. Statistical analysis

Data are presented as mean + SEM. Analysis of variance (ANO-VA) was used to evaluate differences across SDR and H conditions.Repeated measures ANOVAs were used for analysis over time asappropriate. Generally the repeated measures ANOVA revealedno interaction of time with neonatal handling and SDR, (i.e. Adultstress) therefore the collapsed data is reported. Analyses were fol-lowed by Post hoc mean comparisons using Duncan’s New MultipleRange Test.

It is important to note that this study examined only TMEV in-fected animals; therefore basal levels of several measures were notavailable for ANOVA analysis (e.g. stride length and antibodies). Toaddress these issues, we ran a small number (n = 12) of age-matched, non-infected control reference mice. The mean of theage-matched, non-infected reference measurements were sub-tracted from individual experimental measurements prior to

114 R.R. Johnson et al. / Brain, Behavior, and Immunity 40 (2014) 110–120

analysis (carried out in order to ensure only the effect of the SDRand H-15, not infection, were considered). This was done for RIAand IL-6 levels. Pearson correlation coefficients (r) were used todetermine inter-rater reliability.

3. Results

Results are reported for the acute and chronic phase of the ill-ness separately. Only effects relevant to the adult stress X neonatalhandling interaction will be discussed in detail, given that animalsin the non-handled maternal condition (H-0) have been previouslyreported (Johnson et al., 2006). Table 2 reports signs of disease as afunction of neonatal handling within each adult stress group, inboth the acute and chronic phase.

3.1. Neonatal handling increases acute phase impairments (day 0–28pi) in SDR groups

3.1.1. Hind limb impairmentANOVA revealed a significant interaction between neonatal

handling and adult stress (SDR) on hind limb impairment, F(2,30) = 6.642, p < .0001, and a main effect of adult stress (SDR), F(2,30) = 33.155, p < .0001, but no main effect of neonatal handlingalone. Post hoc mean comparisons indicated that neonatal handlingled to greater impairment in both the PRE-SDR and CON-SDR ani-mals (p < .05) but reduced impairment in the NON-SDR animals(p < .05) (Fig. 1A).

3.1.2. Stride lengthANOVA conducted on stride length found a interaction between

neonatal handling and adult stress, F (2,30) = 5.555, p < .01, as wellas a main effect for adult stress, F (2,30) = 129.517, p < .0001, butno main effect for neonatal handling alone (Fig. 1B). Post hoc meancomparisons indicated that only the PRE-SDR/H-15 group had sig-nificantly reduced stride length compared to the PRE-SDR/H-0group. Furthermore, mean comparisons between the H-15 and H-0 groups within the NON-SDR and CON-SDR conditions failed toreach significance (p < .08 and .07, respectively).

3.1.3. Body weightsANOVA revealed a significant interaction between neonatal

handling and adult stress on body weight, F (2,30) = 5.734,p < .01, but no main effects of adult stress or neonatal handlingwere found (p > .05, Fig 1C). Consistent with prior work (Johnsonet al., 2006), post hoc analyses confirmed that the PRE-SDR non-handled mice had decreased body weights in comparison to theCON-SDR non-handled mice (p > .05). Furthermore, it was revealedthat neonatal handling led to marginally increased weights in thePRE-SDR condition (p = .10), and weight gain in the NON-SDR

Table 2Summary of experimental results per neonatal handling and SDR conditions during the acsigns expected to increase with disease, and signs expected to decrease with disease. Arrowdisease decreased. A grey arrow indicates a trend; a black arrow indicates a statistically signx H condition related to the control condition.

NON-SDR

ACUTE CHRONIC

H-15 H-0 H-15 H-0

Increase with disease HLI + +IL-6 +Spleen +TMEV loadMyelin antibodies +

Decrease with disease Stride length +Open field + +Center open field +

condition (p < .05), while the CON-SDR condition lost weight whenexposed to neonatal handling (p < .05).

3.1.4. Open field activityFig. 2 depicts open field activity center time and center distance

traveled. ANOVA found a significant interaction between neonatalhandling and adult stress, both Fs (2,30) P 10.777, p < .001, and amain effect for adult stress, both Fs (2,30) P 7.88, ps < .01. More-over, a main effect of neonatal handling was observed for centertime, F (1,30) = 7,51, p < .05, suggesting that handling reduced anx-iety, but it did not alter center distance (p > .05) (Fig 2A and B,respectively). Post hoc mean comparisons revealed that PRE-SDRreduced center time spent and motion compared to NON-SDR(p < .05). It was also found that neonatal handling significantly re-duced time and motion in the center area for the CON-SDR group(p < .001), and significantly increased it for the NON-SDR group(p < .05). For the PRE-SDR mice, neonatal handling lead to signifi-cantly less time spent in the center, but no change in center motion.

Horizontal and vertical motor function variables are shown inFig 3. These include horizontal activity, horizontal movement num-ber, horizontal movement time, vertical activity, vertical move-ment number and vertical movement time (Fig 3 A, B, C, D, E, F,respectively). ANOVA confirmed that significant interactions be-tween neonatal handling and adult stress occurred in five of thesix measures (all but vertical movement time), Fs (2,30) P 5.258,ps < .01. Adult stress (SDR) main effects were also found for thesame measures, Fs (2,30) P 3.661, ps < .05, except vertical move-ment numbers. Only horizontal activity and horizontal movementtime showed a significant neonatal handling main effect, Fs(2,30) P 5.45, ps < .05.

Post hoc mean comparisons revealed that PRE-SDR significantlyreduced horizontal activity, horizontal movement time, and verti-cal activity compared to the NON-SDR group (p < .05). Mean com-parisons of the H-15 versus H-0 within each SDR conditionrevealed that neonatal handling significantly reduced activity inthe PRE-SDR group on two horizontal measures (horizontal activityand movement number, p < .05) and on two vertical measures(vertical activity and movement number, p < .01). In contrast, inthe CON-SDR group, neonatal handling significantly reduced activ-ity on the horizontal measures only (horizontal activity, movementnumber and movement time, p < .05). Neonatal handling was asso-ciated with greater activity in the NON-SDR group, but was signif-icant only for horizontal activity, horizontal movement number,vertical activity and vertical movement number (p < .05).

3.1.5. ImmunomodulatorsCorticosteroids and IL-6 were assessed as immunomodulators.

As expected, neonatal handling reduced corticosteroid responsesduring both PRE-SDR and CON-SDR (Fig 4). ANOVA confirmed a

ute and chronic phases of disease. Signs of disease are separated into two categories:s up (*) indicate that signs of disease increased, and downward arrows (+) that signs ofificant difference. Two arrows up indicate worsened disease state for a particular SDR

PRE-SDR CON-SDR

ACUTE CHRONIC ACUTE CHRONIC

H-15 H-0 H-15 H-0 H-15 H-0 H-15 H-0

** * * ** *

* ***** *

** * * ** +** ** * *** * * ** + ** +

Fig. 2. The impact of handling and SDR on open field measures associated withanxiety in acute Theiler’s virus infection. Time spent in the center (A) and distancetraveled in the center of the space (B) are shown collapsed across the acute phasetime points. Handled mice spent less time in the center compared to non-handledmice in the PRE and CON-SDR groups. Handled mice in the CON-SDR group also hadsignificantly reduced motion in the center area. Conversely, handled mice in theNON-SDR group spent more time and showed increased motion in the center areacompared to non-handled mice. Significant effects are denoted with asterisks (⁄).

R.R. Johnson et al. / Brain, Behavior, and Immunity 40 (2014) 110–120 115

significant interaction between neonatal handling and adult stress,Fs (1,20) P 5.609, p < .05, and main effects for both adult stress andneonatal handling, Fs (1,20) P 9.458, ps < .01, (Fig 4). Post hoc anal-yses revealed that PRE-SDR and CON-SDR both led to a significantincrease in corticosterone response (p < .001). PRE-SDR led to ahigher increase in corticosterone response than CON-SDR andNON-SDR (p < .01). CON-SDR also led to a higher increase in corti-costerone. Furthermore, as expected, neonatal handling led to a re-duced corticosterone response in both the PRE-SDR and CON-SDRgroups (p < .001). The NON-SDR and age matched reference groupshowed no significant differences across neonatal handling.

For IL-6, ANOVA again found interaction between neonatal han-dling and adult stress, F (2,30) = 17.534, p < .0001, as well as main ef-fects for both neonatal handling and adult stress on acute phase IL-6levels, Fs (2,30) P 15.599, p < .001 (Fig 5). Post hoc mean compari-sons indicated that PRE-SDR significantly elevated IL-6 levels com-pared to both NON-SDR (p < .001) and CON-SDR (p < .001).Furthermore, neonatal handling significantly elevated IL-6 in bothSDR conditions (ps < .001), and reduced it in the NON-SDR group(p < .05).

3.2. Neonatal handling interacts with adult stress to exacerbatechronic phase impairments in SDR groups (day 135pi forward)

The same metrics were collected for the chronic phase, onlywith a reduced frequency of HLI data collection. In addition, oneanimal in the NON-SDR/H-15 group had to be euthanized due toinjuries resulting from fighting within the home cage. Thus thisgroup has an n = 5, versus n = 6 in all other groups.

3.2.1. Hind limb impairmentANOVA revealed an interaction between neonatal handling and

adult stress, F (2,30) = 8,75, p < .05, and a main effect for adultstress, F (2,30) = 43,32, p < .0001, but no main effect for neonatal

handling (Fig 6A). Post hoc mean comparisons revealed that PRE-SDR significantly increased impairment (p < .001), compared bothto CON-SDR and NON-SDR (p < .001). Furthermore, neonatal han-dling significantly increased impairment within PRE-SDR andCON-SDR conditions, whereas it reduced it within the NON-SDRcondition (ps < .05).

3.2.2. Stride lengthAgain, an ANOVA found significant interaction between neona-

tal handling and adult stress, F (2,30) = 10.23, p < .001, as well as amain effect of adult stress, F (2,30) = 22.88, p < .0001, and a maineffect of neonatal handling, F(1,30) = 7.96, p < .01. Post hoc meancomparisons revealed that stride length in the PRE-SDR groupwas reduced in comparison to stride length in the NON-SDR andthe CON-SDR groups (p < .001). Furthermore, we found that inthe CON-SDR group, neonatal handling reduced stride length,p < .001. In contrast, in the NON-SDR group, the H-15 group hada significantly smaller reduction in stride length. The post hoc com-parison for the PRE-SDR was marginal (p < .07) and in the expecteddirection, with neonatal handling reducing stride length (Fig 6B).

3.2.3. Open field activityHorizontal and vertical motor function variables are shown in

Fig 7. These include horizontal activity, horizontal movementnumber, horizontal movement time, vertical activity, verticalmovement number and vertical movement time (Fig 7 A, B, C, D,E, F, respectively). Deficits are apparent in both horizontal andvertical activity levels when neonatal handling is combined witheither SDR condition. An ANOVA confirmed interactions betweenneonatal handling and adult stress, F (2,30) P 7,348, p < .05, onall measures except horizontal movement numbers. Main effectsfor adult stress, F (2,30) P 12,25, p < .0001 in horizontal activity,horizontal movement time, vertical activity, vertical movementnumber, and vertical time were also shown. Main effects forneonatal handling, F (1, 30) = 8.48, p < .05, were also found forhorizontal activity, horizontal movement time, vertical activity,and vertical movement number.

Post hoc comparisons revealed that PRE-SDR reduced horizontalactivity (in comparison to both NON-SDR and CON-SDR groups),horizontal time (in comparison to the NON-SDR group), verticalactivity (in comparison to both NON-SDR and CON-SDR groups),vertical movement number (in comparison to both NON-SDR andCON-SDR groups) and vertical movement time (in comparison toboth NON-SDR and CON-SDR groups), ps < .05. Furthermore, neo-natal handling led to significant reductions in activity in the PRE-SDR animals on vertical activity, vertical movement number andvertical time (p < .05). Neonatal handling also led to significant de-creases in horizontal activity, horizontal movement time, verticalactivity, vertical movement and vertical time in the CON-SDRgroup (p < .05). The NON-SDR animals had significantly greatervertical activity when combined with neonatal handling on all 3vertical measures: overall activity, time spent, and movementnumber (p < .05).

3.3. Neonatal handling interacts with adult stress to increase immunefunction in chronic phase

Previously, we have shown that PRE-SDR resulted in enlargedspleen and increased antibodies to TMEV and myelin (Johnsonet al., 2004, 2006). The following analysis examined how neonatalhandling interacted with adult stress to potentially alter theseeffects.

3.3.1. Spleen weightsANOVA revealed a significant interaction between neonatal

handling and adult stress, F (2,29) = 6.906, p < .01. Post hoc analyses

Fig. 3. The effect of handling and SDR on open field measures associated with motor function and activity in the acute phase, including overall horizontal activity (A), numberof movements (B), time spent moving (C), overall vertical activity (D), number of vertical movements (E), and time spent vertical (F). Significant effects are denoted withasterisks (⁄).

Fig. 4. The impact of handling and SDR on corticosterone levels. PRE-SDR and CON-SDR both led to a significant increase in corticosterone response. Furthermore,neonatal handling led to a reduced corticosterone response in both the PRE-SDRand CON-SDR groups. Significant effects are denoted with asterisks (⁄).

Fig. 5. Effect of handling and SDR on IL-6 levels at day 9 pi. PRE-SDR elevated IL-6levels compared to both NON-SDR and CON-SDR. Handling led to elevated IL-6 inboth SDR conditions and decreased IL-6 in the NON-SDR group. Significant effectsare denoted with asterisks (⁄).

116 R.R. Johnson et al. / Brain, Behavior, and Immunity 40 (2014) 110–120

could not detect any significant differences between the groups(Fig 8A).

3.3.2. Antibodies to Theiler’s virusAveraged age-matched reference data were subtracted prior to

analysis (X = 211.6 CPM, n = 3), and the data were collapsed overtime. ANOVA indicated that neonatal handling interacted withadult stress, F (2,29) = 9.749, p < .001, and main effects for both

neonatal handling, F (1,29) = 7.592, p < .01, and adult stress, F(2,29) = 37.216 p < .0001 were found (Fig 8B). Post hoc analysesrevealed that antibodies to Theiler’s virus were significantlyincreased in the PRE-SDR group (p < .001) compared to theCON-SDR and NON-SDR group (p < .05). However, viral antibodieswere significantly decreased in the CON-SDR group comparedto the NON-SDR group (p < .05). Moreover, neonatal handling

Fig. 6. Effect of handling and SDR on motor impairment measures during thechronic phase, including hind limb impairment (A) and hind limb stride length (B).Handled mice in the PRE and CON-SDR groups showed greater impairment thannon-handled mice, whereas the opposite was true for handled mice in the NON-SDRgroup. Additionally, PRE-SDR mice showed more impairment than the CON-SDRand NON-SDR groups (A). Stride length in the PRE-SDR group was reduced incomparison to stride length in the NON-SDR and the CON-SDR groups. Neonatalhandling reduced stride length in the CON-SDR group (B). Significant effects aredenoted with asterisks (⁄).

Fig. 7. The effect of handling and SDR on open field measures associated with motor funmovements (B), time spent moving (C), overall vertical activity (D), number of verticaasterisks (⁄).

R.R. Johnson et al. / Brain, Behavior, and Immunity 40 (2014) 110–120 117

significantly increased antibodies to Theiler’s virus in the PRE-SDRgroup, p < .001, which accounts for the interaction betweenneonatal handling and adult stress.

3.3.3. Autoimmune response to myelin epitopesFig. 9 presents the results of the analysis of antibody levels to

myelin (MOG, MBP, and PLP, Fig 9 A, B, C, respectively), as indica-tors of autoimmune responses. As with the TMEV antibodies, in or-der to control for basal levels, the averages of age-matchedreference data were subtracted prior to analysis (Means for MOG,MBP, and PLP were, respectively, 218.4 CPM, 214.2 CPM, and210.6 CPM, n = 3).

A series of ANOVAs revealed significant interactions betweenneonatal handling and adult stress across all three measures, Fs(2,29) = 3.911, 18.028, 18.117 (MOG, MBP, and PLP, respectively),ps < .05. Main effects were also found for both adult stress, Fs(2,29) = 19.323, 143.434, 123.575 (MOG, MBP, and PLP, respec-tively), ps < .0001, and neonatal handling, Fs (1,29) = 7.030,10.282 (MBP and PLP, respectively), ps < .01. Post hoc means com-parisons found that MOG, MBP, and PLP antibodies were signifi-cantly increased in the PRE-SDR group compared to both theNON-SDR and CON-SDR groups (p < .001). They were also signifi-cantly increased in the NON-SDR group when compared to theCON-SDR group (p < .05). Furthermore, neonatal handling signifi-cantly increased antibodies to all three proteins in the PRE-SDRgroup (p < .001).

4. Discussion

The present study investigated the interaction between briefneonatal handling and SDR on neuroimmune, endocrine, and

ction and activity in the chronic phase, including horizontal activity (A), number ofl movements (E), and time spent vertical (F). Significant effects are denoted with

Fig. 8. Effect of handling and SDR during the chronic phase on spleen weights (A),and antibodies to TMEV (B). Handling in the PRE-SDR group led to a marginalincrease in spleen weight, but handling in both the PRE and CON-SDR led to reducedspleen weight compared to the non-handled mice. PRE-SDR group showed higherlevels of antibodies (CPM) to virus compared to the CON-SDR and NON-SDR group.Handling increased antibodies to Theiler’s virus in the PRE-SDR group. Significanteffects are denoted with asterisks (⁄).

Fig. 9. Effect of handling and SDR during the chronic phase on autoimmunemeasures of antibodies to MOG (A), MBP (B) and PLP (C) were analyzed in sera. PRE-SDR showed significantly higher levels of MOG, MBP and PLP compared to NON-SDR and CON-SDR groups. Handled mice in the PRE-SDR group displayed higherlevels of each myelin epitope compared to non-handled mice. Significant effects aredenoted with asterisks (⁄).

118 R.R. Johnson et al. / Brain, Behavior, and Immunity 40 (2014) 110–120

behavioral responses to TMEV infection in mice. Consistent withprior work in our lab demonstrating that brief neonatal handlingin mice increases the rate of viral clearance, our handling alonecondition with NON-SDR mice showed protection against laterinfection (Meagher et al., 2010). However, the interaction effectfound in mice exposed to both handling and SDR was unexpected.We predicted that neonatal handling would protect adult subjectsfrom the detrimental effects of social disruption stress prior toinfection and attenuate the protective effects of concurrent SDRon TMEV infection. Moreover, our past results have shown thatwhen SDR occurs prior to TMEV infection, the outcome is detri-mental, while SDR concurrent with infection reduces diseaseseverity (Johnson et al., 2004, 2006). Surprisingly, in the currentstudy, we found that brief handling may not be protective whenpaired with later social stress.

Normally, PRE-SDR in mice leads to higher corticosterone levels,which is linked to the development of glucocorticoid resistanceand a more severe disease course (Avitsur et al., 2001; Starket al., 2001). The present results indicate that handling reducedcorticosterone levels in PRE-SDR and CON-SDR groups, but it didnot ameliorate the disease course, at either the behavioral or cellu-lar level. Moreover, handling led to increased levels of IL-6 in boththe PRE-SDR and CON-SDR groups. An increase in IL-6 in the CON-SDR group was expected given that handling decreases the sub-jects’ stress response to SDR. However, we would have also ex-pected the PRE-SDR group to show lower levels of IL-6, ifhandling served a protective role in preventing glucocorticoidresistance resulting from PRE-SDR (and the increased pro-inflam-matory release that ensues).

In the chronic phase of the disease, handling was again found tobe detrimental in both PRE and CON-SDR groups. Handling x SDRincreased indicators of autoimmunity. The PRE-SDR group showedincreased levels of antibodies to myelin and virus (as compared to

the NON-SDR group, which showed decreased levels of these anti-bodies). Behaviorally, handling exacerbated the chronic phaseimpairments in both SDR groups. These results indicate thatchanges in the neuroendocrine system don’t fully explain thechanges observed in the neuroimmunological response.

We observed blunted corticosteroid responses in the handledmice subjected to SDR before or concurrently with infection.Blunted HPA-axis function as a result of handling and CON-SDRmay create a permissive environment, which increases acute CNSinflammation. This in turn may alter the immune response to thevirus and the development of glucocorticoid resistance that is typ-ical following SDR. Consistent with this theory, we found that han-dling prior to or concurrent with SDR, significantly elevated IL-6and reduced corticosterone in the acute phase as well as increasedhind limb impairment during the acute phase of the infection(when compared to controls). During the chronic phase, it led toa significant reduction in stride length for the CON-SDR, close to

R.R. Johnson et al. / Brain, Behavior, and Immunity 40 (2014) 110–120 119

significant for PRE-SDR. Significant increases in antibodies to PLPwhile a significant increase in antibodies to MOG, MBP and TMEVwas found in the handling X PRE-SDR mice. Together, these resultsimply handling may not be protective against disease when pairedwith later stressors.

It is possible that handling, which blunts the HPA-axis, de-creases the stress response in adulthood when exposed to SDR.CON-SDR alone normally has beneficial effects, as it enhances theimmune response to the virus (Johnson et al., 2004, 2006). Witha dampened stress response, the beneficial effect of CON-SDRmay be decreased, and this may explain the current results unveil-ing a more severe disease course in mice exposed to both handlingand CON-SDR as compared to mice exposed to CON-SDR alone.Similarly, HPA axis blunting due to handling may decrease the det-rimental role of PRE-SDR in the development of glucocorticoidresistance following infection, but this effect may be negligible.That is, the detrimental effect of PRE-SDR and infection may signif-icantly outweigh the beneficial effects of handling. However, thisonly explains part of the scenario.

It is increasingly clear that microglia and macrophages also playan important role in stress (Curry et al., 2010; Frank et al., 2007,2012; Vichaya et al., 2011). In a previous study examining the ef-fects of SDR on TMEV disease course, we observed a trend for in-creases in hippocampal CD11b + mRNA expression, a marker formicroglia/macrophages, in subjects exposed to repeated (six ses-sions) SDR (Vichaya et al., 2011). Curry et al., 2010 also found thatSDR alone led to an increase in the percentage of neutrophilsexpressing CD11b in the lungs. Furthermore, Wohleb et al., 2011showed that repeated social defeat stress increases reactivity ofmicroglia to an LPS challenge. This pro-inflammatory response tostress is mediated by glucocorticoids and leads to higher circulat-ing IL-6 and TNF-a (Frank et al., 2012).

Based on past findings, suggesting that handling decreasesmicroglia reactivity to LPS introduced in adulthood (Bilbo et al.,2007), we expected handling to decrease microglia reactivity andsubsequent IL-6 production following stress exposure and infec-tion. However, we instead found decreased corticosterone in boththe PRE-SDR and CON-SDR groups along with increased IL-6 re-sponse in the acute phase and increased viral titers in the chronicphase, denoting a poorer outcome. Whereas handling may play adampening role in the stress response, we suspect it may act as ajarring stimulus for microglia, activating them to maintain homeo-stasis. Once primed, the microglia may become more responsive tosubsequent stressors, such as the SDR condition and TMEV infec-tion. Consistent with this hypothesis, Frank et al. (2012) found thatrats introduced to an inescapable tail shock and then challengedwith LPS 24 h later had more reactive microglia. Future studiesshould examine the effect of timing of infection with respect to astressor, as this may be crucial in determining a positive or nega-tive outcome.

Our study is the first to demonstrate that early-life handlinginteracts with adult life social stress to alter vulnerability to CNSinfection. It must be noted that in both rats and mice, handlingalone has been found to lead to a more severe disease course inexperimental allergic encephalomyelitis (EAE), another model ofMS, which is comparable to the chronic MS-like phase of TMEVinfection (Columba-Cabezas et al., 2009; Laban et al., 1995; Ste-phan et al., 2002; Teunis et al., 2002). To our knowledge, however,the effect of handling and later social stress has not been examinedin the EAE model. Our findings may also point to another dimen-sion for the role of early life experience on microglia and infection.

One limitation of this study is the small sample size (n = 6/group), which limited our ability to distribute mice from differentlitters across experimental conditions. In the future, it would beimportant to attempt to replicate this experiment with a larger co-hort (n = 12/group).

5. Conclusion

Collectively, our results suggest that while brief handling bluntscorticosteroid responses to stress and attenuates the severity of la-ter Theiler’s virus infection in non-stressed mice, it increases dis-ease severity when paired with later social stress. These findingsare interesting in light of the growing data in the human literatureindicating that a blunted corticosteroid response is associated withseveral disease syndromes such as the chronic fatigue syndrome,fibromyalgia, hypothyroidism, asthma and in extreme cases, Addi-son’s and Cushing’s disease (Esposito et al., 2006; Griep et al.,1998; Kamilaris et al., 1987; McKenzie et al., 1998; Mileva andMaleeva, 1987; Ozbey et al., 1994; Saraclar et al., 1993). Animalstudies have predominantly shown that handling can act to ame-liorate negative effects of stress and infection (Anisman et al.,1998; Ladd et al., 2005; Shanks and Lightman, 2001; Zahariaet al., 1996); however few studies have looked at how handlingmodulates the effect of social stress prior to or concurrent withinfection. The current data demonstrate the potential for the inter-action of early life events that lead to blunted corticosterone re-sponses with later life events (as demonstrated in this studywith SDR) to be detrimental in the context of infectious diseases.Because many negative associations exist with the hypo-respon-sive HPA axes in the human literature, the positive effects foundin animal studies should be interpreted with caution.

6. Funding sources

NRSA 5F31NS50476-2 and NSF Fellowship to RRJ, and NMSSRG3128, NINDS RO1 NS39569 and NIH/NINDS R01-NS060822 toCJRW and MWM, and NINDS RO1NS060822 to MWM and CJW.

7. Conflict of interest

The authors have no conflicts of interest to declare.

Acknowledgments

We would like to thank Katie Hare, Marilyn Connor, E. AshleyHardin, Jessica Harrison and Jordan Teraski for their help and sup-port throughout this study. This research was supported in part byfellowships to RRJ (National Science Foundation and F31NS504762). Additional support was provided by National MultipleSclerosis Society RG3128 and National Institute of NeurologicalDisorders and Stroke RO1 NS39569 to CJRW and MWM, and by Na-tional Institute of Neurological Disorders and Stroke RO1NS060822to MWM and CJW.

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