Journal of Neuroimmunology 230 (2011) 85–94

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Journal of Neuroimmunology

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Adjuvant-induced arthritis induces c-Fos chronically in neurons in the hippocampus

Jeffrey L. Carter a, Cheri Lubahn b, Dianne Lorton b,c, Tracy Osredkar b, T.C. Der b, Jill Schaller b,Stephen Evelsizer a, Schari Flowers a, Natalie Ruff a, Bethany Reese a, Denise L. Bellinger a,⁎a Department of Human Anatomy and Pathology, Loma Linda University School of Medicine, Loma Linda, CA, United Statesb Hoover Arthritis Research Center, Banner Sun Health Research Institute, Sun City, AZ, United Statesc Department of Psychology, Kent State University and Summa Health Systems, Kent-Summa Institute for Clinical and Translational Research, Akron, OH, United States

⁎ Corresponding author. Dept. of Pathology and HUniversity School of Medicine, Alumni Hall for the BaCampus Street, Loma Linda, CA 92352, United States. Te909 558 0432.

E-mail address: dbellinger@llu.edu (D.L. Bellinger).

0165-5728/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.jneuroim.2010.09.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 18 June 2010Received in revised form 1 September 2010Accepted 14 September 2010

Keywords:c-FosNeural-immuneRheumatoid arthritisHippocampusImmunohistochemistry

Chronic pain, sickness behaviors, and cognitive decline are symptoms in rheumatoid arthritis. In the adjuvant-induced arthritis Lewis rat model, we examined the dynamics of c-Fos expression in the hippocampus, abrain region important for these symptoms. Brain sections were stained for c-Fos using immunohistochem-istry. c-Fos-positive nuclei were counted in CA1, CA2, CA3 and the dentate gyrus of the dorsal hippocampifrom rats receiving no treatment or base-of-the-tail injections of (1 or 2) incomplete or complete Freund'sadjuvant (low- or high-dose), (3), Mycobacterium butyricum cell wall suspended in saline, or (4) saline, andsacrificed 4, 14, 21, or 126 days post-immunization. Disease severity was evaluated by dorsoplantar foot padwidths and X-ray analysis. We report sustained dose- and subfield-dependent c-Fos expression with arthritis,but transient expression in nonarthritic groups, suggesting long-term genomic changes in rheumatoidarthritis that may be causal for behavioral changes, adaptation to chronic pain and/or cognitive declineassociated with disease.

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1. Introduction

A major challenge to the medical community is managing thephysical and psychological sequelae of patients with rheumatoidarthritis that diminish their quality of life and reduce their lifeexpectancy. Patients with rheumatoid arthritis and juvenile chronicarthritis develop a number of sickness behaviors, including low-gradefever, anorexia, fatigue, weight loss, and depression, which aremanifested as reduced activity, hypersomnia, social withdrawal,depressed mood, anhedonia, apathy, lack of concentration, and lossof interest (reviewed in Lorton et al., 2008). Chronic inflammation inpatients with rheumatoid arthritis also can be accompanied by adecline in cognition.

Sickness behaviors are induced via circulating inflammatorycytokines after peripheral immune challenge, which then activateneural and humoral pathways, and secondarily induce centrallyproduced cytokines in discrete brain regions (Konsman et al., 2002;Dantzer, 2004; Buttini and Boddeke, 1995; Quan et al., 1998; Maieret al., 1998; Rivest, 2003). The specific brain regions activated, andtiming and degree of activation, are dependent upon the type ofantigen challenge and load, and the route of administration. Immune-

to-brain signaling, particularly after challenge with lipopolysaccha-ride, has been studied by mapping the expression of early immediategene products, especially c-Fos, andmore recently other Fos- and Jun-related proteins (Frenois et al., 2007; reviewed in Elmquist et al.,1996). c-Fos and other Fos-related antigens (FRA) are proteins thatbind with other proteins of the Jun, ATF, and JDP families to formheterodimeric activator protein-1 (AP-1) (reviewed in Shaulian andKarin, 2001). The transcriptional regulation of c-Fos is mediated byextracellular signal-regulated kinases (ERKs) (Sgambato et al., 1998),and AP-1 transcriptional activity is under the influence of mitogen-activated protein kinase (MAPK)/ERK signaling pathways (reviewedin Murphy and Blenis, 2006). AP-1 is induced by cytokines,neurotransmitters and growth factors, and is involved in diverseprocesses, such as cell differentiation, proliferation, apoptosis, andtransformation (reviewed in Shaulian and Karin, 2001; Herdegen andWaetzig, 2001; Hess et al., 2004; Shiozawa and Tsumiyama, 2009).

Few studies have investigated neuronal activation after immunechallenge with antigens used to induce inflammatory arthritis inrodent models for rheumatoid arthritis. Therefore, the purpose of thisstudy was to examine the dynamics of c-Fos expression in the hip-pocampus of male Lewis rats with adjuvant-induced arthritis (AA).The specific goals of this studywere to: 1) evaluate c-Fos expression inthe different hippocampal regions of arthritic rats across time afterantigen challenge (i.e., time of antigen processing, disease onset, acuteand severe disease, and resolution of inflammation); 2) determinedisease-specific changes in neuronal activation in each hippocampalregion by assessing how individual components of complete Freund's

86 J.L. Carter et al. / Journal of Neuroimmunology 230 (2011) 85–94

adjuvant (CFA), which do not induce disease, affect c-Fos expression;and 3) investigate the effect of bacterial cell wall load in the CFA onhippocampal neuronal activation. We report for the first time that AAin male Lewis rats induced robust expression of c-Fos in all regions ofthe hippocampus that persisted through 18 weeks after immuniza-tion with CFA. In contrast, challenge with components of CFA thatalone do not produce AA induced transient c-Fos staining in specifichippocampal regions that were CFA component- and time-dependent.Sustained neuronal activation in the hippocampus in AA suggests achronic increase in the c-Fos-c-Jun class of AP-1 and greater activationof the MAPK/ERK signaling pathway, possibly due to epigeneticphenomena. Functionally, chronically altered signalling in thehippocampus may be important for mediating sustained sicknessbehaviors and cognitive changes, and/or adaptive coping of chronicpain or pain perception in rheumatoid arthritis.

2. Methods and materials

2.1. Chemicals and adjuvant

CFA (0.015 or 0.03 mg dried, heat-killed Mycobacterium butyricum(M. butyricum, Difco, Detroit, MI) emulsified in 10 ml sterile mineraloil) was prepared by grinding the M. butyricum with a mortar andpestle until the bacterial cell wall turned from light beige to aneggshell white powder. The mineral oil then was slowly worked intothe bacterial cell wall powder using a mortar and pestle. The sus-pension was treated with a sonic dismembraner for 5 min to ensurethat the bacterial cell wall remained suspended in the mineral oilduring the injection of the animals. While there is variability in theseverity of disease development between the preparations of CFA,there is very little variability in the severity of disease developmentwithin individual batches. In this study, all animals in the AA groups ateach time point were challenged with the same preparation ofadjuvant. In this study, 100% of the animals developed arthritis, withonset around 11 to 12 days post-challenge. M. butyricum (0.03 mg)emulsified in 10 ml sterile, endotoxin-free saline (SMB), was preparedas described for CFA as a control for the effects of a bacterial antigenchallenge in the absence of adjuvant (mineral oil).

2.2. Animals and experimental design

Young adult male Lewis rats (200–250 g) were purchased fromCharles River Laboratories (Raleigh, NC). The ratswere housed two percage in plastic-bottom cages with CareFresh bedding and maintainedon a 12-h off/12-h on light/dark schedule. Rats were fed standardlaboratory rat chow andwater ad libitum under standard conditions oflight, temperature, and humidity. Rats were allowed to adjust to theconditions of the vivarium for 1–2 weeks prior to the start of theexperiment. Cages used to house arthritic rats were equipped withwater bottles topped with long sipper tubes, and food pellets wereplaced on the bottom of the cage for easy access. All animals wereobserved daily to eat and drink during the experimental period, andwere weighed to ensure adequate weight gain. Dorsoplantar foot padwidths were measured daily for 2 weeks before beginning theexperiment to acclimate the animals to the handling and stress ofthis manipulation, and to obtain baseline data. Every attempt wasmade to handle the animals within and between each experiment inthe same manner and by the same personnel. Other than thedevelopment of arthritis, good health was maintained throughoutthe experiment.

Rats were sacrificed 4, 14, 21 or 126 days after inoculation withCFA or vehicle by an overdose of chloral hydrate (1.0 ml of 8% chloralhydrate in sterile physiological saline) administered intraperitoneally.Day 4 represents preclinical expression of arthritis and a time duringantigen processing. Days 14 and 21 represent the onset of AA, andsevere disease, respectively. Day 126 represents a time point at which

the disease has been resolved, based on a lack of inflammation in theaffected joints. After anesthesia, radiographs were taken of the hindlimbs to assess disease severity, and then rats were perfusedwith fixative in preparation for immunohistochemistry for c-Fos, asdescribed later. All protocols for the use and care of the animals in thisstudy were approved by the Banner Sun Health Research Institute andLoma Linda University Animal Use and Care Committees prior tobeginning the experiments, and complied with NIH guidelines for thehumane use and care of research animals.

Male Lewis rats were randomly assigned to 1 of 6 treatment groupswith an n of 6–10 animals per group. Rats were immunized with either100 μl of a low or high concentration of CFA (0.015 or 0.03 mgM. butyricum suspended in 10 ml of sterile mineral oil, respectively)by an intradermal injection into the base of the tail. These treatmentgroups were designated L-CFA or H-CFA, respectively. Vehicle controlgroups received an intradermal injection into the base of the tail of100 μl (1)M. butyricum in saline (SMB; 0.03 mg); (2) 0.9% physiological,sterile, endotoxin-free saline (Sal), or (3) mineral oil (incompleteFreund's adjuvant or IFA). Animals were challenged with SMB, arelevant immunogen that does not produce AA, to dissociate disease-relatedfindings from those resulting fromantigen challengewith gram-positive cell wall components. The IFA groupwas used to control for theeffects of adjuvant vehicle of the cellwall suspension; challengewith IFAdoes not induce AA. Saline-treated (Sal) and non-treated rats (NoTx)served as controls for the stress of handling,manipulations for assessingfoot pad widths, and injections.

2.3. Assessment of arthritis

The inflammatory response in arthritic ratswas assessed by routinemethods, as previously described (Lorton et al., 1999). Briefly, using aMitutoyo Corporation dial thickness gauge, dorsoplantar widths of thehind feet were measured daily beginning 2 weeks prior to immuni-zation with CFA or control injections, and every other day afterimmunization until sacrifice. For time points at, or after, disease onset,radiographs using an X-OMAT processor at 40 cm were taken prior tosacrifice using the following settings: 400 nN, 50 kvp, and 0.4 sexposure time. X-rays were evaluated using a grading scale modifiedfrom Ackerman et al. (1979). In brief, the radiographs were coded toobscure the treatment groups. Next, 2 independent observerssubjectively rated each of the radiographs using the scale: 0 (normal),1 (slight), 2 (mild), 3 (moderate), and 4 (severe) abnormalities in thetissue for each of 5 characteristic features of AA. Radiographs werescored for: (1) soft tissue swelling, as indicated by the widths of softtissue shadows and alterations in the normal configuration of the softtissue planes; (2) osteoporosis, as measured by bone density(recognized by increases in radiolucency relative to uninvolvedadjacent bone); (3) loss of cartilage, shown by narrowing of the jointspaces; (4) heterotopic ossification, defined as proliferation of newbone tissue (fine ossified line paralleling normal bone but notcontiguous with calcified area of the bone itself); and (5) boneerosions. Radiographs were scored for each category, and then thesescores were summed for both hind limbs giving a maximum score of40.

2.4. Tissue preparation

After overdose anesthesia (as described previously), rats wereperfused transcardially with approximately 75 ml of Hartman'ssodium phosphate buffer (phosphate buffer; 0.15 M, pH 7.4) contain-ing 150 ml of 1% sodium nitrite and 15,000 IU/l heparin followed by250 ml of 4% paraformaldehyde plus 0.2% picric acid. Brains weredissected, blocked coronally into 3 pieces, and immersed in the samefixative for 48 h at 4 °C. Next, brains were placed sequentially into 10,20, and 30% sucrose in phosphate buffer for 24 h per sucrose con-centration at 4 °C, frozen on dry ice, and stored at −80 °C until tissue

87J.L. Carter et al. / Journal of Neuroimmunology 230 (2011) 85–94

sectioning. Brains were mounted onto the freezing stage of a slidingmicrotome, and coronal sections were cut at 30 μm. Sections werecollected in phosphate buffer, and then transferred to a cryoprotectantsolution for storage at −20 °C, as described by Watson et al. (1986).

2.5. Immunohistochemistry for c-Fos

Immunohistochemistry for c-Fos in brain tissue sections wasperformed using the antiserum, sc-52 (Santa Cruz Biotechnology, CA).The sc-52 antiserum was raised in rabbit against a synthetic peptidecorresponding to amino acids 3–16 at the N-terminal of human andmouse c-Fos, and cross-reacts with rat c-Fos. This antibody recognizesa 62-kDa protein, inducible by phorbol ester application, on Westernblot, corresponding to the expected molecular weight of c-Fos, butdoes not cross-react with other members of the Fos or Jun proteinfamilies, according to the manufacturer's Western blot analyses.

For immunohistochemical staining, all washes were performedwith phosphate buffer (3×10 min) and all steps were performed atroom temperature with gentle agitation, unless otherwise specified.Free-floating sections were washed, placed in 10% normal goat serumfor 30 min, and then incubated overnight at 4 °C in the primaryantibody (1:1000) containing 0.4% Triton-X, 1% bovine serum al-bumin, and 4% normal goat serum for 24 h at 4 °C. Some tissuesections from each animal were incubated under the same conditionsin the same solution minus the primary antibody to serve as negativecontrol tissue. Tissue sections then were washed, transferred into 10%normal goat serum for 30 min, and incubated in a goat anti-rabbitbiotinylated IgG secondary antibody (1:600; Vector Elite Kit; VectorLaboratories, Inc., Burlingame, CA) in 0.4% Triton-X, 1% bovine serumalbumin and 0.15 M phosphate buffer. After washing, sections wereplaced in phosphate buffer containing 2.5% methanol and 5% hy-drogen peroxide for 30 min to remove endogenous peroxidases. Thesections were washed, incubated in phosphate buffer containingavidin–biotin–peroxidase complex (1:600; Vector Elite Kit; VectorLaboratories, Inc.) for 90 min, and rinsed in 0.05 M sodium acetatebuffer containing 0.03 M imidazole (pH 7.4; acetate-imidazole buffer)(3×10 min). The reaction product was developed by placing brainsections in acetate-imidazole buffer containing 0.25% nickel (II)sulfate, 0.04% 3,3′-diaminobenzidine plus 0.005% hydrogen peroxidefor 5 min. Next, brain sections were washed first in acetate-imidazolebuffer (3×10 min), and then in phosphate buffer (3×10 min). Finally,the stained sections were mounted onto Vectabond-coated slides(Vector Laboratories, Inc.), dehydrated with a graded series of eth-anol, cleared in xylene, and coverslipped with CC/Mount, an aqueousmounting media (Sigma Aldrich, St. Louis, MO). Some tissue sectionswere counterstainedwith 5% cresyl violet (AmericanMaster Tech, CA)prior to dehydration with ethanol.

Fig. 1. Schematic diagram of 4 coronally-sectioned brain slices corresponding to −3.80 mmWatson (1986). These schematic sections illustrate the rostral to caudal extent of, and subfisections from each treatment group and time point after treatment. Four regions or subfieldthe left-most schematic section. The area sampled in each subfield is illustrated in the seco

2.6. Morphometric analysis of c-Fos expressing neurons

Brain sectionswerevisualizedusingaZeissAxiomatphotomicroscope,and RGB color images were captured and digitized using an Olympusimage high-resolution CCD video capture system at a resolution of300 pixels per inch. Images of sections through the CA1, CA2, CA3and the dentate gyrus (DG) of the dorsal hippocampus that arerepresentative of the regions shown in Plates 33–36 (Bregma −3.80to −4.52 mm) of the atlas of the rat brain by Paxinos and Watson(1986), were captured (see Fig. 1). Initially, both the dorsal andventral hippocampi were examined, but no significant differences inthe expression of c-Fos immunoreactivity were found and so thepresent study is focused on the dorsal hippocampus. Similarly, theright and left side were examined for differences, but there were nodiscernible differences. Nuclei expressing c-Fos-immunoreactivity ineach region of the hippocampus from 4 different tissue sections (2each from the left and right side) per rat per treatment group werecounted within a rectangular area of 200 μm×100 μm or 20 mm2 insize using Image-Pro Plus imaging software (version 4.1). The longedge of the rectangular area was positioned on each hippocampalregion parallel to the dorsal surface of the hippocampus for CA1 andCA2, the ventral surface of hippocampus for CA3 and the dorsomedialsurface of the DG, as far as possible. c-Fos-positive nuclei wereselected by the Image-Pro Plus software based upon their RGB colorvalue and intensity. A standard color from the section was chosen toindicate positive staining and the software then marked nucleiexpressing this color as positive.

2.7. Statistical analysis

2.7.1. Expression of c-FosThe mean number of c-Fos-positive nuclei in each hippocampal

region for each rat was averaged, and the data expressed as the meannumber of nuclei per region±standard error of themean (SEM).Meannuclear counts taken from the right and left sides of each region werecompared using a Student's t-test to discern whether c-Fos expressionexhibited significant hemispheric differences in any of the treatmentgroups (pb0.05). Since no statistical differences were found (data notshown), data from right and left hemispheres were collapsed. c-Fosdata were initially analyzed using a two-way analysis of variance(ANOVA) (pb0.05) with subsequent Bonferroni post-hoc testing todetermine whether there were significant treatment×region ortreatment×post-immunization day effects. Two-way ANOVArevealed no significant interactions between treatment and region ortreatment and post-immunization day, so c-Fos data were analyzedusing a one-way ANOVA and Bonferroni's post-hoc testing for sig-nificant ANOVAs (pb0.05). Only data showing significant differences

to −4.52 mm Bregma based on and adapted from the rat brain atlas by Paxinos andelds in, the dorsal hippocampus that were analyzed for c-Fos staining in representatives were examined: CA1=1; CA2=2; CA3=3, and the dentate gyrus (DG)=4 shown innd section from the left by shaded boxes. The ventricular system is shown in black.

Table 1Summary table showing semi-quantitation of c-Fos expression in different regions of the hippocampus.

D4 H-CFA L-CFA IFA SMB Sal NoTx D14 H-CFA L-CFA IFA SMB Sal NoTx

CA1 +++ ++ ± + 0 0 CA1 +++ ++ ± ++ 0 0CA2 +++ + 0 0 0 0 CA2 ++ ++ + + 0 0CA3 +++ ++ 0 ± 0 0 CA3 ++ ++ + ++ 0 0DG ++++ ++ 0 ± ± 0 DG ++ ++ ++ ++ ± ±

D21 H-CFA L-CFA IFA SMB Sal NoTx D126 H-CFA L-CFA IFA SMB Sal NoTx

CA1 +++ ++ + + ± 0 CA1 ++ ++ ± + ± ±CA2 ++ ++ + + ± 0 CA2 ++ ++ + + + +CA3 ++ ++ ± 0 ± 0 CA3 ++ ++ + + + +DG ++ ++ + + + ± DG ++ ++ ± + + +

Semi-quantitation of c-Fos immunoreactivity in the CA1, CA2, CA3, and the dentate gyrus (DG) of the dorsal hippocampus from rats receiving no treatment (NoTx) or treatment withsaline (Sal), saline-M. butyricum (SMB), mineral oil (IFA), or low- or high-dose complete Freund's adjuvant (L- or H-CFA, respectively). Brains from rats in each treatment group weresectioned and immunocytochemistry for c-Fos was performed. Brain sections between Bregma−3.80 to−4.52 mm based on the rat brain atlas by Paxinos andWatson (1986) wereused for scoring by two scorers blinded to the treatment groups. c-Fos immunoreactivity within each region of interest was scored according to the number of positive nuclei asfollows: 0 (virtually no labeling), ± (very few labeling, 1–5), + (few labeling, 6–25), ++ (intense labeling, 26–50), +++ (very intense labeling, 51–85), ++++ (extremelyintense labeling, 86–100). H-CFA, high-dose complete Freund's adjuvant; low-dose complete Freund's adjuvant, L-CFA; IFA, incomplete Freund's adjuvant; SMB, saline-M. butyricum;Sal, saline; NoTx, no treatment.

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are described in the results. Additionally, c-Fos immunostaining wassemi-quantitatively scored in the regions and structures indicated inFig. 1, and were scored according to the number of positive nuclei as0 (virtually no labeling), ± (very few labeling, 1–5), + (few labeling,6–25), ++ (intense labeling, 26–50), +++ (very intense labeling,51–85), and ++++ (extremely intense labeling, 85–100) (Table 1.)

2.7.2. Assessment of arthritisThe right and left foot pads from each animal were averaged to-

gether. The individual means of both foot pad widths from animalswithin each treatment group then were averaged, and wereexpressed as mean foot pad width in cm±SEM. Foot pad widthswere analyzed using one-way ANOVA (pb0.05), with dorsoplantarwidth as a repeated measure. ANOVAs reaching significance weresubjected to the Bonferroni post-hoc test (pb0.05). Mean X-ray scoresfor animals in each treatment groupwere averaged and expressed as amean±SEM, and then subjected to Kruskal–Wallis statistical analysis(non-parametric statistic equivalent to an ANOVA; pb0.05) followedby Dunn post-hoc testing (pb0.05).

3. Results

3.1. Temporal pattern of c-Fos expression in the dorsal hippocampus

Fig. 2 shows representative sections of the DG from the dorsalhippocampus stained immunocytochemically for c-Fos from eachtreatment group at day 21 to demonstrate the general pattern of c-Fosexpression. Table 1 semi-quantitatively summarizes c-Fos expressionwithin the 4 regions of the hippocampus between 4 and 126 days afterimmunization. Immunoreactive nuclei (see arrows, Fig. 2A) appearedas small, round, bluish-black circles of similar size (approximately10 μm in diameter) scattered randomly throughout the pyramidal celllayer of the hippocampus (Fig. 2). Brain sections from arthritic rats(Fig. 2B–C) displayed strikingly greater numbers of c-Fos-positivenuclei compared with nonarthritic controls (Fig. 2D–F; Table 1),regardless of time after challenge or region of the hippocampus ex-amined (CA1, CA2, CA3 or the DG). Furthermore, rats treated with ahigher concentration of CFA (H-CFA) (Fig. 2B; Table 1) displayedqualitatively greater c-Fos immunoreactivity compared with L-CFA(Fig. 2C; Table 1).

Basal levels, as indicated by c-Fos immunostaining in non-treatedrats (NoTx) were very low in all regions of the dorsal hippocampus.There was no apparent qualitative difference between the basal(Fig. 2G) density of c-Fos-immunoreactive nuclei compared withsaline (Sal) (Fig. 2F),M. butyricum suspended in saline (SMB) (Fig. 2D),or mineral oil (IFA) (Fig. 2E) (see Table 1). Brain sections processed for

immunohistochemistry in the absence of primary antibody had lowbackground (Fig. 2H), and confirmed specific nuclear stainingwith theprimary antibody. Counter-staining with cresyl violet confirmed thatc-Fos immunostaining was localized within pyramidal neuronslocated in the pyramidal cell layer of the hippocampus (Fig. 2A).

Quantitative analysis of c-Fos immunoreactivity using two-wayANOVAs revealed significant treatment effects (pb0.0001), but nosignificant treatment×region interaction (pN0.05), or treatment×dayinteractions (pN0.05). At day 4 post-immunization (Fig. 3A), immuno-reactivity for c-Fos in the hippocampus from H-CFA-treated rats wasdifferentially expressed depending on the hippocampal region, with ageneral ranking from high to low as follows: DGNCA1NCA2=CA3,whereas the high-to-low expression rankingwith L-CFA treatment wasCA1NDGNCA3NCA2. One-way ANOVA revealed a significant dose-dependent increase in the mean number of c-Fos-immunoreactivenuclei in all hippocampal regions from H- or L-CFA-treated ratscompared with all other treatment groups (*, pb0.0001, exceptfor CA2: L-CFA vs. IFA, pb0.01; L-CFA vs. SMB or Sal, pb0.001). Nuclearc-Fos immunoreactivity in CA1 from IFA-treated rats, but not otherhippocampal regions, wasmodestly increased comparedwith the othercontrol groups (IFA vs. SMB, pb0.01; IFA vs. Sal or NoTx, pb0.0001).Similarly, the number of c-Fos-positive nuclei in CA1 only from SMB-treated rats was significantly greater (pb0.0001 or pb0.001, respec-tively) than in rats receiving saline or no treatment.

Fourteen days after challenge (Fig. 3B), a time point coincidingwith disease onset, nuclear c-Fos immunoreactivity in CA1 and CA2from CFA-treated arthritic rats remained dose-dependent, and H- orL-CFA was significantly greater than the other treatment groups, withthe exception that in CA2, L-CFA treatment did not differ from IFAtreatment. In CA1, IFA treatment increased (pb0.0001) the mean c-Fos-positive nuclear counts compared with untreated and SMB- andSal-treated rats. In CA2, c-Fos-immunoreactive nuclear counts weresimilar with IFA and SMB treatment, and both of these groups hadhigher counts than in Sal-treated (pb0.0001 and pb0.01, respective-ly) and untreated (pb0.0001 and pb0.001, respectively) controls. InCA3 and DG, the number of c-Fos-positive nuclei from H-CFA-, L-CFA-,and IFA-treated rats were comparable. In CA3, but not the DG, thenumber of c-Fos-stained nuclei was greater (pb0.001, pb0.01, andpb0.01, respectively) in H-CFA-, L-CFA-, and IFA-treated rats than inSMB-treated rats. The mean c-Fos nuclear counts in both CA3 and DGwere significantly greater in H-CFA-, L-CFA- and IFA-treated rats thanin NoTx or Sal-treated rats, and were also higher (pb0.01) in DG fromSMB-treated rats than untreated controls (CA3: H- and L-CFA vs. Sal orNoTx, pb0.001; IFA vs. Sal or NoTx, pb0.01; DG: H-CFA vs. Sal or NoTx,and L-CFA vs. NoTx, pb0.0001; L-CFA vs. Sal and IFA vs. NoTx,pb0.001; IFA vs. Sal, pb0.01).

Fig. 2. Photomicrographs AQF demonstrate c-Fos immunoreactivity (arrows shown in A) in the dentate gyrus (DG) of the dorsal hippocampus 21 days after treatment with (A) high-dose complete Freund's adjuvant (H-CFA) counterstained with cresyl violet, (B) H-CFA, (C) low-dose complete Freund's adjuvant (L-CFA), (D) M. butyricum in saline (SMB), (E)incomplete Freund's adjuvant (IFA), (F) saline (Sal), (G) no treatment (NoTx), and (H) negative control (Neg Ctrl). In CFA-treated rats, c-Fos-immunoreactive nuclei are abundantand scattered within the pyramidal cell layer in the DG (AQC), particularly with H-CFA treatment. Cresyl violet counterstaining reveals c-Fos nuclear staining in pyramidal cells (A).Very few c-Fos-positive nuclei were present in control groups (DQG). Negative control tissue has low background and confirmed the specificity of the c-Fos antibody (H). Calibrationbars (A)=200 µm, Calibration bar (BQF)=100 µm.

89J.L. Carter et al. / Journal of Neuroimmunology 230 (2011) 85–94

At 21 days post-challenge with CFA (Fig. 3C), a time of severeinflammatory disease, a significant dose-dependent increase in meanc-Fos-positive nuclear counts was observed in CA1 (pb0.0001), CA3(pb0.0001) and the DG (pb0.001) from CFA-treated rats, such thatthe higher dose gave the greater response. In all hippocampal regions,nuclear c-Fos expression in H- or L-CFA-treated rats was significantlygreater (pb0.0001) than the other treatment groups. In the DG, IFA,SMB and Sal treatment raised (pb0.01) the mean nuclear c-Fos ex-pression over non-treated controls.

To examine post-inflammatory disease effects on neuronalactivation, c-Fos immunoreactivity was examined at 126 days post-challenge (Fig. 3D). In all hippocampal subfields, the dose-dependenteffects of CFA on mean nuclear c-Fos expression were lost at thistime point, but c-Fos expression remained significantly higher with H-or L-CFA treatment than all control groups (CA1 and CA3, H- or L-CFA

vs. control groups, pb0.0001; CA2 and DG: H-CFA vs. all controlgroups, pb0.0001; CA2: L-CFA vs. IFA, SMB, or NoTx, pb0.01; CA2 andDG: L-CFA vs. Sal, pb0.001; DG: L-CFA vs. IFA or SMB, pb0.0001; DG:L-CFA vs. NoTx, pb0.001).

3.2. Assessment of arthritis

3.2.1. Dorsoplantar foot pad widthsIn each set of experiments with 14-, 21-, and 126-day time points

(Fig. 4A, B, and C, respectively), both L- and H-CFA treatments pro-gressively increased the mean dorsoplantar foot pad widths inarthritic rats compared with nonarthritic controls. In general, ratstreated with H-CFA displayed an early onset of disease, and there wasa trend for foot pad widths to be greater than in L-CFA-treated rats ineach study period (significantly different 21-day study). In the 14-day

Fig. 3. Themean number of c-Fos-immunoreactive cells±SEM in the CA1, CA2, CA3, and dentate gyrus (DG) of the dorsal hippocampus from rats receiving no treatment (NoTx, openbars), saline (Sal, gross cross-hatched bars), saline-M. butyricum (SMB, fine cross-hatched bars), incomplete Freund's adjuvant (IFA, vertically lined bars); low-dose completeFreund's adjuvant (L-CFA, solid bars); high-dose complete Freund's adjuvant (H-CFA, horizontally lined bars) at (A) 4, (B)14, (C) 21, or (D) 126 days post-treatment. Regardless oftime post-treatment, the mean number of c-Fos-immunoreactive nuclei was low in all control groups compared with rat that received L- or H-CFA (A-D). The effects of adjuvanttreatment were dose-dependent for all hippocampal regions at day 4 (A) and day 21 (C), but not at day 14 (B). Dose response effects were demonstrated only in CA1 and CA2 at day21, and at day 126 (D) dose response effects were not observed. Significant differences between treatment groups at each time point are indicated with asterisks (*); refer to the textfor specific p values.

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study (Fig. 4A), a significant rise in mean dorsoplantar in H- or L-CFAwas observed by day 11 or 12, respectively, and continued throughday 14 (days 11–14: H-CFA vs. all control groups, pb0.001; days 12–14, L-CFA vs. all control groups, pb0.001). Similar findings were seenin the 21-day study (Fig. 4B), with significant differences in meandorsoplantar widths between arthritic groups and nonarthriticgroups found from 11 to 21 days post-challenge in rats receiving H-CFA (H-CFA vs. all control groups, pb0.001) and from 15 to 21 daysafter L-CFA treatment (L-CFA vs. all control groups, pb0.001).Additionally, mean foot pad widths were significantly greater(pb0.05) in H-CFA compared with L-CFA treatment between 15 and21 days post-challenge. In the 126-day study (Fig. 4C), a rise in meandorsoplantar foot pad widths was evident between 10 and 14 daysafter immunization with L- or H-CFA. In these two groups, foot padwidths continued to rise through day 28, and then remained elevatedthrough day 126 compared with nonarthritic controls whose foot padwidths remained at baseline levels and unchanged throughout thestudy period. Two-way ANOVA with repeated measures revealedsignificant differences (pb0.0001) in dorsoplantar foot pad widthsbetween treatment groups starting at day 18 and continuingthrough day 126, and there was a significant treatment×days post-immunization interaction (pb0.0001). Post-hoc analysis showed that

mean dorsoplantar foot pad widths from L- and H-CFA-treated ratswere significantly greater than all nonarthritic control groups from 18to 126 days post-immunization (days 18–126: H-CFA vs. all controlgroups, pb0.0001; days 18–21: L-CFA vs. all control group, pb0.01;days 21–126: L-CFA vs. all control groups, pb0.0001). Between days21 and 126, treatment with L-CFA was consistently lower thantreatment with H-CFA; however, statistically these treatment groupswere not different. Mean dorsoplantar foot pad widths from allnonarthritic control groups remained at baseline levels and therewere no significances differences between these groups (pN0.05).

3.2.2. Assessment of radiographsThe radiographs shown in Fig. 5 are representative of the hind

limbs for each treatment group at 14 (5A–5E), 21 (5F–5J), and 126(5K–5O) days post-challenge. Radiographs of the hind limbs revealedprogressive joint destruction and swelling of the soft tissues in ratsimmunized with L- or H-CFA (Fig. 5D, I, N or E, J, O, respectively)across time, an effect that appears to be dose-dependent. At each timepoint, joint destruction and soft tissue swelling was most severe inrats treated with H-CFA compared with L-CFA (day 14: Fig. 5D vs. E;day 21: Fig. 5I vs. J; and day 126: Fig. 5N vs. O). In arthritic groups,there was bone loss, soft tissue swelling and periosteal bone

Fig. 4. Mean dorsoplantar foot pad widths from male Lewis rats are plotted over timeafter treatment with high-dose complete Freund's adjuvant (H-CFA, ), low-dose complete Freund's adjuvant (L-CFA, ), mineral oil (IFA, ), saline-M.butyricum (SMB, ), saline (Sal, ), or no treatment (NoTx, ). No changein mean dorsoplantar foot pad widths was observed across time in the NoTx, Sal, SMBor IFA treatment groups (A–C). In rats treated with L- or H-CFA, mean dorsoplantarfoot pad widths progressively increased beginning around 11–12 through 21 days thenplateaued from day 21 through 126 (A–C). Significant differences between treatmentgroups at each time point are indicated with asterisks (*); refer to the text for specific pvalues.

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formation coupled with narrowing of the spaces between themetatarsals, and a decrease in bone radiolucency compared withcontrol animals at each time point. There was no discernible effect ofSal (Fig. 5A, F, and K), IFA (Fig. 5B, G, and L) or SMB (Fig. 5C, H, and M)treatments on joint integrity or the soft tissue of the hind limbsregardless of time after treatment.

Radiographic scoring of the hind limbs at 14, 21 and 126 days post-treatment (Fig. 6A, B and C, respectively) was consistent with qual-itative observations (Fig. 5), and demonstrated significant dose-and time-dependent destructive joint changes in both CFA-challengedgroups compared with controls on day 14 (H-CFA or L-CFA vs. allcontrol groups, pb0.05), day 21 (H-CFA vs. all control groups,pb0.001, L-CFA vs. all control groups, pb0.01) and day 126 (H-CFAvs. IFA, pb0.001; H-CFA vs. SMB or Sal; pb0.0001; L-CFA vs. IFA,pb0.01; L-CFA vs. SMB, pb0.001; L-CFA vs. Sal, pb0.0001).

4. Discussion

The present study demonstrates that AA induced a persistentdose-dependent expression of c-Fos in dorsal hippocampal pyramidalneurons of all 4 regions up to 126 days after immunization (high-doseinduced greater activation). Furthermore, c-Fos expression in AA ratswas disease-specific based on our findings with immunogenic controlgroups (IFA and SMB). Across time, the greatest neuronal recruitmentoccurred in AA rats in all regions compared with IFA and SMBtreatment, except at disease onset (day 14). At disease onset, onlydisease-specific effects occurred in CA1, whereas c-Fos expression inCA2, CA3 and the DG may be explained, at least in part, by IFA and/orSMB challenge. Additionally, the effects of immunogenic controls onneuronal activation were seen only in CA1 at day 4 and all regions atday 14. The individual components of CFA have unique activationalprofiles in the hippocampus that differ from CFA challenge, suggestingthey are neither additive nor synergistic. Collectively, these findingssuggest that the hippocampus can discern, and differentially respondto, qualitative and quantitative differences in the individual CFAcomponents and CFA, i.e., each antigen or antigen combination appearto have its own unique activational signature.

Wewereable tofindonepreliminary longitudinal reportwith annof2 per treatment group per timepoint that investigated the expression ofFRA in thehippocampus fromadifferent rat strainwithAA. In this study,two antibodies were used with immunohistochemistry to localize FRAand c-Fos, an anti-FRA primary antibody that cross-reacts with c-Fos,FRA-1, FRA-2, and FosB, and an anti-c-Fos primary antibody, respec-tively. Omura et al. (1998) reported a chronically increased expressionof FRA, but not c-Fos, in the hippocampus of arthriticWistar–Lewismalerats compared with untreated or IFA-treated controls. The inability todetect c-Fos expression in the hippocampus conflicts with our studyeven though the same source of anti-c-Fos antibody was used forstaining in both studies. It is possible that this discrepancymaybedue tostrain differences and/or other limitations of their study. For example, itis not clear whether the conditions for c-Fos staining were optimized,becausenobasal c-Fos levelswere reported in any of the central nervoussystem sites examined, and the use of a positive control tissue is notevident. The FRA immunoreactivity in the hippocampus reported byOmura et al. (1998) may be due, in part, to the presence of c-Fos. Wefounda similar pattern of c-Fos staining in thehippocampus asOmura etal. (1998) report for FRA expression, but with lower numbers of c-Fos-positive nuclei. Given the broader specificity of the FRA antibody, thehigher number of immunoreactive hippocampal neurons suggest thatFRA, in addition to c-Fos, is also expressed. This may also explain thesecond rise in FRA expression inCA3 and theDGat later stages of diseasethat we did not observe with c-Fos staining.

The discrepancy in c-Fos expression between these studies mayalso be attributed to differences in the type of adjuvant used toprepare the CFA (paraffin oil vs. mineral oil), or the preparation of theCFA, which determines disease severity. It is not possible to comparedisease outcomes in the two studies, because disease severity was notevaluated by Omura et al. (1998). In contrast, disease outcomes weredetermined in rats receiving one of two concentrations of CFA usingfoot pad widths and X-ray analysis in our study. Despite a 2-folddifference in theM. butyricum concentration of the L- and H-CFA, footpad widths and X-ray analysis of the hind limbs did not reveal asignificant dose-related difference, although after disease onset therewas a trend for lower outcomemeasures with L-CFA. This findingmayin part reflect the significant well known contribution of the vehicle(IFA) for disease expression, whose volume did not differ betweenthese treatment groups. Persistent c-Fos expression in hippocampalpyramidal neurons is consistent with our findings of progressivedegenerative changes in the hind limbs, and with reports of be-havioral and cognitive changes in rheumatoid arthritis patients andanimal models for rheumatoid arthritis (reviewed in Shiozawa andTsumiyama, 2009; Shiozawa et al., 1997). Collectively, these data

Fig. 5. Hind limb radiographs taken on days 14, 21 and 126 (upper, middle and lower panels, respectively) of animals treated with saline (Sal), incomplete Freund's adjuvant (IFA),saline-M. butyricum (SMB), low-dose complete Freund's adjuvant (L-CFA; 1.5 mg/ml); and high-dose (complete Freund’s adjuvant (H-CFA; 3.0 mg/ml) (panels from left to right,respectively). There were no changes in bone integrity or soft tissue measurements in the limbs from animals given Sal (A, F, K), SMB (C, H, M), or IFA (B, G, L) at any of the timepoints, however, there was a decrease in bone density (loss of bone integrity and bone shape), and an increase in soft tissue swelling, osteoporosis (bone luminescence), periostealbone formation (laying down of bone in inappropriate areas), and narrowing of the joint spaces between the small bones of the feet in both the H- and L-CFA-treated animals acrossall time points.

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indicate that the hippocampus is more sensitive in detecting antigenicload in the presence of IFA than the disease outcomemeasures chosenfor this study indicate, and may reflect ceiling effects of foot padwidths or the lack of sensitivity of the non-parametric scoring for X-ray analysis.

The mechanism(s) responsible for c-Fos expression in thehippocampus was not determined in this study. However, there is arelatively large literature demonstrating that immunization with avariety of immunogens (particularly lipopolysaccharide), andalso proinflammatory cytokines like tumor necrosis factor-α orinterleukin-1 (Rivest and Laflamme, 1995), can induce c-Fos immu-noreactivity in hypothalamic, autonomic and limbic brain regions,which typically return to basal levels within 24–48 h after treatment(Lacroix and Rivest, 1997). In studies that immunize rats with lipo-polysaccharide, a gram-negative bacterial cell wall component, themechanisms responsible for c-Fos expression in these sites include arise in circulating proinflammatory cytokines or peripheral afferent

nerve stimulation that secondarily induces proinflammatory cyto-kines centrally. Few studies have examined the effect of immunizationwith gram-positive bacterial cell wall components like SMB. Incontrast to SMB and CFA, Frenois et al. (2007) have reported anincrease in FosB/deltaFosB (but not c-Fos) expression in thehippocampus 24 h after immunization with lipopolysaccharide, atime consistent with the timing of depressive-like behavior. Similarly,Ceccarelli et al. (1999) reported c-Fos expression in CA3 and the DG2 h after subcutaneous injection of 50 μl of 10% formalin into the hindpaw that returned to baseline 24 h later. In a similar pain model, they(Ceccarelli et al., 2003) later reported no effect of repetitive sub-cutaneous injection of 5% formalin (1 time per week) into alternatehind paws on c-Fos expression in the DG 1 week later. They attributedthe effects of formalin to pain, but did not consider that formalinwould induce an immune response thatmay also indirectly contributeto the neural activation. The transient c-Fos expression with theseimmune stimuli is similar to our findings with SMB and IFA

Fig. 6. Radiographic scores from the hind limbs of Lewis rats treated with Saline (Sal),M. butyricum in saline (SMB), mineral oil (IFA), low-dose complete Freund's adjuvant(L-CFA, 1.5 mg/ml), or high-dose complete Freund's adjuvant (H-CFA, 3.0 mg/ml) onday 14 (A), 21 (B) and 126 (C) post-immunization. L-CFA- and H-CFA-treated animalsshowed a significant increase in radiographic score 14 (A), 21 (B), and 126 (C) daysafter challenge compared with all control groups. Significant differences betweentreatment groups at each time point are indicated with asterisks (*); refer to the text forspecific p values.

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treatments, but contrasts to the chronic expression of c-Fos in thehippocampus after CFA treatment. These differences may reflectphasic immune stimuli in the former cases and persistent/recurrentimmune stimuli of antigens released slowly from oil-based adjuvantsover preclinical, acute, and chronic phases of the disease in the lattercase. Each of these aversive immune stimuli appears to induce aunique pattern of central nervous system reactivity that leaves long-lasting traces; for CFA the hippocampus is at least one of these sites.Collectively, our findings, as well as the literature, support thatimmunogen-specific profiles of immune activation, which induceunique cytokine profiles, may explain differences in the regional,magnitude and temporal expression of c-Fos, and possibly othermembers of the Fos family, in the hippocampus.

The significance of the persistent increase in c-Fos expression inpyramidal neurons of the hippocampus in AA is not clear. c-Fos hasa half-life of about 30 min, and accumulates only if its C-terminus

is phosphorylated under conditions of sustained ERK activation,suggesting a chronic increase in ERK activity in hippocampalpyramidal cells in AA. Increased stability of c-Fos by phosphorylation,in turn, enhances ERK phosphorylation at its C-terminus (Murphyet al., 2002), which is required for cell transformation (Murphy et al.,2002). Therefore, by its ability to exist in unstable and stabilized states,the c-Fos transcription factor enables cells to distinguish between thelarge numbers of mediators that induce different activation kinetics,and thus serves as a ‘sensor’ for ERK activation dynamics (Murphy andBlenis, 2006). Alteredbehavior of c-Fos after sustained ERK signaling inAA is likely to chronically alter pyramidal cell functional activity,whichin turn, may contribute to disease development and/or alter be-havior and cognition in AA. Other conditions, such as seizure activity(Popoviciu et al., 1988), convulsive drug-induced seizures (Dragunowand Robertson, 1987), hypoxia (Taniguchi et al., 1994), and electricalstimulation of hind limb motor/sensory cortex in adult rats (Sagaret al., 1988), induce long-lasting c-Fos expression in the hippocampus,with similar interpretations by investigators that c-Fos and otherimmediate early gene products in the brain regulate synaptic activity,and may represent the basis of learning and memory, which is es-sential for adaptation and survival.

Expression of the Jun:c-Fos class of AP-1 is also involved in mo-lecular mechanisms underlying learning and memory. Transgenicmice that lack c-Fos in the brain have impaired spatial reference andcontextual learning and exhibit a reduced long-term potentiation ofsynaptic transmission at CA3 to CA1 synapses (Fleischmann et al.,2003). In contrast, transgenicmice inwhich fra-1was knocked into thec-fos locus showed impaired spatial, but regular contextual, learningand normal long-term potentiation responses (Gass et al., 2004). Theredundancy of functions regulated by c-Fos and Fra-1 may result fromc-Fos activation of fra-1 (Bergers et al., 1995; Matsuo et al., 2000). Aswitch from c-Fos to Fra-1 containing AP-1 transcription factors hasalso been implicated in other behavioral processes, such as addictionbehaviors from chronic cocaine use (Hope et al., 1994). These studiessupport distinct temporal expression, and functions, ofmembers of theFos family by inducing the expression of different AP-1 target genes.

Since, c-Fos is an important component of the AP-1 transcriptionfactor, increased stability leads to a greater occupancy of the promoterand consequently a greater target gene expression (Murphy et al.,2002) and cellular transformation (Chen et al., 1996; Okazaki andSagata, 1995). Additionally, the expression of the late-response gene,fra-1, is a target of c-Fos (Schreiber et al., 1997) that is expressed onlyafter prolonged c-Fos expression (Murphy et al., 2004), suggestingthat sustained c-Fos expression in the hippocampus of AA rats maytrigger the prolonged expression of other FRA. This hypothesis isconsistent with the findings by Omura et al. (1998), and wouldexplain the greater numbers of FRA-positive cells compared with ourc-Fos staining. Since different genes in the Fos family induceexpression of different AP-1 target genes and therefore distinct geneproducts, these findings suggest that chronic expression of c-Fos inhippocampal pyramidal cells of AA rats could affect many differentcellular functions, including neurotransmitters, electrical activity, andsynaptogenesis.

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