sustained macrophage infiltration upon multiple intra articular injections an improved rat model of...

Research ArticleSustained Macrophage Infiltration uponMultiple Intra-Articular Injections An Improved Rat Model ofRheumatoid Arthritis for PET Guided Therapy Evaluation

Durga M S H Chandrupatla1 Karin Weijers1

Yoony Y J Gent1 Inge de Greeuw2 Adriaan A Lammertsma2 Gerrit Jansen1

Conny J van der Laken1 and Carla F M Molthoff2

1 Department of Rheumatology VU University Medical Center De Boelelaan 1117 1081 HV Amsterdam The Netherlands2Department of Radiology amp Nuclear Medicine VU University Medical Center De Boelelaan 1117 PO Box 7057 1007 MB1081 HV Amsterdam The Netherlands

Correspondence should be addressed to Carla F M Molthoff cfmmolthoffvumcnl

Received 6 June 2014 Revised 27 August 2014 Accepted 7 September 2014

Academic Editor Filippo Galli

Copyright copy 2015 Durga M S H Chandrupatla et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

To widen the therapeutic window for PET guided evaluation of novel anti-RA agents modifications were made in a rat modelof rheumatoid arthritis (RA) Arthritis was induced in the right knee of Wistar rats with repeated boosting to prolong articularinflammation The contralateral knee served as control After immunization with methylated bovine serum albumin (mBSA) incomplete Freundrsquos adjuvant and custom Bordetella pertussis antigen one or more intra-articular (ia) mBSA injections were givenover time in the right knee Serum anti-mBSA antibodies DTH response knee thickness motion and synovial macrophageswere analyzed and [18F]FDG(-general inflammation) and (R)-[11C]PK11195 (macrophages-)PET was performed followed by exvivo tissue distribution Significant anti-mBSA levels DTH swelling of arthritic knee and sustained and prolonged macrophageinfiltration in synovial tissue were found especially using multiple ia injections Increased [18F]FDG and (R)-[11C]PK11195accumulation was demonstrated in arthritic knees as compared to contralateral knees which was confirmed in ex vivo tissuedistribution studies Boosting proved advantageous for achieving a chronic model without remissionThemodel will offer excellentopportunities for repeated PET studies tomonitor progression of disease and efficacy of novel therapeutic agents for RA in the sameanimal

1 Introduction

Rheumatoid arthritis (RA) is an autoimmune disease thatresults in chronic and systemic inflammation of the jointsaffecting approximately 05ndash1 of the adult population [1]It is characterized by inflammation of the joints resultingin synovial hyperplasia by infiltration of immune cells fur-ther leading to cartilage and bone destruction [2] Timelyrecognition of RA will allow for earlier start of therapypreventing more severe expansion of the disease Moreoverseveral studies have shown that tight control as a treatmentstrategy in individual RA patients seems promising in achiev-ing predefined level of low disease activity or preferably

remission within a reasonable period of time [3 4] To thisend noninvasive imaging modalities may serve as sensitiveand accurate tools for assessment and monitoring of diseaseactivity during therapy to evaluate therapeutic efficacy

Positron emission tomography (PET) is a promisingnoninvasive imaging modality that can be used to visu-alize active arthritis at a molecular level in RA [5] viatargeting macrophages [6 7] Most human studies target-ing macrophages by PET have been performed with themacrophage tracer (119877)-[11C]PK11195 in various inflamma-tory diseases [8] (119877)-[11C]PK11195 targets the 18-kd translo-cator protein (formerly known as peripheral benzodiazepine

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 509295 11 pageshttpdxdoiorg1011552015509295

2 BioMed Research International

Table 1 Arthritis induction in rat variations of the original model to the modified groups

Code group Original Group A Group B Group C Group D Group E(control)

Group F(control)

Number of boost No No No 3 5 No NoSacrificed (days post) 6 6 28 19 28 6 28

1st immunization (mBSACFACBP antigen)lowastNo of rats 11 3 4 3 6 4 4Volume (120583L) 200 200 200 200 200 200 200Administration route sc sc sc sc sc sc scAdministration location Tail base Tail base Tail base Tail base Tail base Tail base Tail base

2nd immunization (mBSACFACBP antigen)lowastVolume (120583L) 200 100 100 100 100 100 100Administration route sc sc sc sc sc sc sc

Administration location Tail base Neck andupper flank

Neck andupper flank

Neck andupper flank

Neck andupper flank

Neck andupper flank

Neck andupper flank

Delayed time hypersensitivity (DTH) CBP antigenVolume (120583L)lowastlowast 25 25 25 25 25 25Route sc sc sc sc sc sc scAdministration site Right ear Right ear Right ear Right ear Right ear Right ear Right ear

Local (ia) arthritis induction (mBSA) Salineia injection (mBSAlowastlowastlowast or saline) 1 1 1 1 1 1 1mBSA boost ia injections 0 0 0 3 5 0 0Injected volume (120583L) 20 60 60 60 60 60 60Route ia ia ia ia ia ia iaAdministration site knee joint knee joint knee joint knee joint knee joint knee joint knee jointlowast50mgmBSA in 1mL 09NaCl emulsified with an equal volume of complete Freundrsquos adjuvant antigen (CFA) and customBordetella pertussis (CBP) antigen(1 times 1011 cellsmL) lowastlowastCBP antigen (27 times 1010 cellsmL 09 NaCl) lowastlowastlowast10mgmBSA in 1mL 09 NaCl and 1mgmBSA in 1mL 09 NaCl

receptor) a mitochondrial membrane protein that is upreg-ulated in activated macrophages [8] Histological studieshave shown that macrophages are an important biomarkerfor prediction and monitoring of therapeutic effects of awide range of disease modifying antirheumatic drugs andbiologics [9 10] Jahangier et al demonstrated a clear positiveclinical effect in RA patients after intra-articular treatmentwith Yttrium-90 and glucocorticoids correlating effect witha decrease in total numbers of macrophages [11]

Animal models can be applied for in vivo evaluationof efficacy of new therapeutic agents for RA [12] As ittakes some time for most antirheumatic drugs to read outtheir mode of action on arthritis activity with macrophageinfiltration as a biomarker a chronic RA animal model isrequired with sustained arthritis activity characterized bymacrophage infiltration in synovial tissue As currently nosuitable rat model is available that would allow noninvasivemacrophage PET guided evaluation of the therapeutic agentswe have optimized an antigen induced model with persistentarthritis in rats offering sufficiently sized inflamed joints toenable quantitative measurements of PET tracer uptake ininflamed joints as well as the opportunity for comparisonto contralateral noninflamed control joints within the sameanimals

2 Materials and Methods

21 Animals Wistar rats (male 150ndash200 grams CharlesRiver International Inc Sulzfeld Germany) were providedwith standard food (16 protein rodent diet Harlan Lab-oratories Inc Madison WI USA) and water ad libitum

Rats were housed in groups of three or four in conventionalcages and kept in a room with a 12-hour lightdark cycle andconstant room temperature (21∘C) and humidity level (50)All animal experiments were carried out in accordancewith the Dutch law on animal experimentation and wereapproved by the VUMedical Center Institutional Committeeon Animal Experimentation

22 Antigen Induced Rat Model As reference in this paperthe methylated bovine serum albumin (mBSA) induced ratmodel as described by Van De Putte et al [13] and Dijkstra etal [14] was applied (Table 1) indicated hereafter as ldquooriginalmodelrdquo In short according to the descriptions of the originalmodel rats were immunized subcutaneously (sc) twice atdays 0 and 7 with an emulsion containing mBSA (Sigma-Aldrich Chemie BV ZwijndrechtTheNetherlands) dissolvedwith complete Freundrsquos adjuvant (CFA) (Sigma AldrichSteinheim Germany) and custom Bordetella pertussis (CBP)antigen (Becton Dickinson Breda The Netherlands) [14]Rats were immunized with two administrations of 200 uLsolution containing 50mgmBSA in 1mL 09 NaCl emul-sified with an equal volume of complete Freundrsquos adjuvantantigen (CFA) and custom Bordetella pertussis (CBP) antigen(1 times 1011 cellsmL) Both the first and the second immuniza-tion were performed in the tail base At day 21 local arthritiswas induced by injecting 20 120583L mBSA solution containing10mgmBSA [15] in 1mL 09 NaCl intra-articular (ia) inthe right knee (RA knee) the contralateral left knee served asan internal control (Con-RA) The ia injection was situatedbetween femur and tibia and behind the patella tendon

BioMed Research International 3

5 10 15 20 25 30 35 4540 48Day (s)

F (saline 28d)

B (28d)

C (19d)

D (28d)

A (6d)

E (saline 6d)

Figure 1 Time line of RA rat model 1st (◼) and 2nd (◼) immu-nization DTH (Q) ia injection (q) boost ia injections (998771) PETandor ex vivo tissue distribution and (immuno-) histopathologyof groups A (6 d) B (28 d) C (19 d) D (28 d) E (saline 6 d) and F(saline 28 d) (998787)

23 Modifications of Original Rat Model Three modifica-tions were performed as compared to the original model(Table 1) At first the second immunization (initially 200 uLin the original model) step was adapted To minimize animaldiscomfort bymultiple immunizations at a single location (aswas performed in the original model) of second immuniza-tion was divided into two injections with one in the neckand one in the upper flank (away from the knees) with eachinjection consisting a volume of 100 uL Secondly the iainjection volume of mBSA was increased to 60 uL while inthe original it was 20 uL Both modifications were applied ingroups A B C and D

The last modification comprised repeated ia injections(resp 3x (group C) and 5x (group D)) while in group Aand B no boosts were appliedThe difference between groupsA and B was the sacrificing day 6 and 28 days after iainjection for groups A and B respectively (Figure 1) Controlrats received an ia injection in the right knee with sterilephysiological saline instead of mBSA Subgroups of controlrats were sacrificed at 6 (group E) and 28 (group F) daysrespectively

24 Validation Experiments

(i) Examination of Immunization Status

Serum Levels of Anti-MBSA Blood samples before and afterthe immunization procedure were obtained from the tailvein with a Microvette (cb300 Sarstedt BV Etten-LeurThe Netherlands) After centrifugation at room tempera-ture (5min2500timesg) serum samples were stored at minus80∘Cuntil use Anti-mBSA levels were determined by ELISA[16] Briefly 96-well microplates (Greiner bio-one Alphenad Rijn The Netherlands) were precoated overnight with100 120583Lwell of 5120583g mBSAmL phosphate buffered saline(PBS) at 37∘C After washing with PBS (100120583Lwell 5 times)wells were blocked with 01 gelatin (Baker Chemical Co

Austin Texas USA) in PBS for 30 minutes at 37∘C Subse-quently 1 100 1 200 and 1 400 diluted serum samples wereadded and incubated for 1 hour at room temperature Afterwashing with PBS horseradish peroxidase (HRP) labeled torabbit-anti-rat (R120572R) IgG1 antibody 1 1000 (Invitrogen NYUSA) was added to the wells and incubated for 2 hrs at roomtemperature Enzyme reaction was visualized with 08mgaminosalicylic acid (Sigma-Aldrich Chemie BV ZwijndrechtThe Netherlands) and 05120583L of 30 hydrogen peroxide(H2O2) dissolved in 1mL distilled water Absorption at

450 nmwasmeasured using an ELISA reader (Tecan SpectraFluor MTX Labsystems Inc Vienna USA)

Delayed Type Hypersensitivity Test (DTH) At day 19 immu-nization status was examined by DTH [17 18] response CBPantigen (25 120583L 27 times 1010 cellsmL 09 NaCl) was injectedsc in the right ear of the ratThe left ear served as an internalcontrol A control group of rats was injected with sterilephysiological saline in the left ear Subsequently ear thicknesswas measured at 0 6 24 and 48 hours after injection using adigital micrometer

(ii) Macroscopic Evaluation of Arthritis Activity Macroscopicevaluation of the severity of arthritis was assessed by kneemeasurements prior to (at the same day) every ia injectionIn between knees were measured 3 times a week until ratswere sacrificed Knees were measured by caliper measure-ment of knee thickness in mediolateral direction

25 Histopathology and Immunohistochemistry Both kneeswere dissected in toto and fixed for 7 days at 4∘C in 10freshly made paraformaldehyde in PBS with 2 sucrose (pH= 73) prior to decalcification in 123mM sodium ethylene-diaminetetraacetic acid (Na

2-EDTAsdot2H

2O) (Merck Darm-

stadt Germany) and 113mM sodium hydroxide (NaOH)(Sigma-Aldrich Chemie BV Zwijndrecht The Netherlands)(pH = 72) for sim55 weeks at 4∘C Decalcified knees wererinsed for 24 hours in 2 sucrose (Sigma-Aldrich ChemieBV Zwijndrecht The Netherlands) in PBS (pH = 72) and24 hours in 2 sucrose in PBS and 50mM NH

4Cl (Sigma-

Aldrich Chemie BV Zwijndrecht The Netherlands) (pH =71) Thereafter knees were embedded in paraffin Sections of5 120583m were cut through the center of the joint in longitudinaldirection and stained with haematoxylin and eosin to assessthe degree of inflammation in synovial tissue

Immunohistochemical localization of rat macrophageswas determined by a mouse anti-rat monoclonal antibodyED1 (HM3029 Hycult PA USA) a lysosomal membranerelated antigen on rat macrophages and by a mouse anti-rat monoclonal antibody ED2 (MCBIVUUniversityMedicalCenter Amsterdam) cell surface glycoprotein related antigenon rat macrophages [19] An IgG1 isotype antibody (HI1016Hycult PA USA) was used as negative control antibodyBriefly after antigen retrieval with a solution of 01 pepsin(Sigma-Aldrich Chemie BV Zwijndrecht The Netherlands)with 01 of HCl 37 in PBS per slide for 30min at 37∘Csections were incubated for 1 h with 1 100 diluted ED1 ED2or isotype control antibody in 01 BSAPBS for a periodof 1 h The detection EnVision kit (K4063 dual-link-HRP


rabbitmouse DAKO Glostrup UK) was used according toinstructions of the manufacturer for a period of 30min Afterwashing with PBS slides were stained for peroxidase activ-ity with 331015840-diaminobenzidine tetrahydrochloride (DAB)containing 001 H

2O2 Subsequently sections were coun-

terstained with haematoxylin dehydrated and mountedNegative controls were included by replacement of theprimary antibody with an isotype specific control antibodyImages were captured using a Leica 4000B microscope andLeica digital camera DC500 (Microsystems BV RijswijkTheNetherlands)

26 PET and Ex Vivo Tissue Distribution Studies [18F]FDGwith a radiochemical purity of gt97 was purchased fromBV Cyclotron VU (Amsterdam The Netherlands) (R)-[11C]PK11195 was synthesized as described previously [20]with radiochemical purity of gt98 and a mean specificactivity of 957 plusmn 284GBq120583mol Rats were anesthetizedusing inhalation anesthetics (isoflurane 2ndash25 and oxygen045 volume ) The jugular vein was cannulated with apolyurethane 3 French cannula During all procedures vitalbody signs like body temperature heartbeat respiratory rateand blood oxygen saturation were monitored continuouslyusing a rectal temperature probe and pulse oxygen meterwith SpO

2sensor Anesthetized rats were positioned in

a double-layer LSO high resolution research tomograph(HRRT) (SiemensCTI Knoxville TN USA) a small animaland human brain 3D scanner with high spatial resolution(23ndash34mm full width at halfmaximum) and high sensitivity[21] First a 6-minute transmission scan was acquired using a740MBq 137Cs rotating point source Next [18F]FDG (211plusmn51MBq) or (R)-[11C]PK11195 (105plusmn29MBq)was adminis-tered iv through the cannula and a dynamic emission scan of1 hourwas acquired PETdatawere normalized and correctedfor scatter random attenuation decay and dead time Datawere acquired in 64-bit list mode and converted into 16sinograms with frame durations increasing from 15 up to300 seconds Images were reconstructed using an iterative 3Dordinary Poisson ordered-subsets expectation-maximization(OSEM) algorithm with 8 iterations and 16 subsets and amatrix size of 256 times 256 times 207 resulting in a cubic voxelsize of 121 times 121 times 121mm3 PET images were madeof rats from the original model and of rats in group ASixty minutes after PET scanning rats were sacrificed andknees blood and various tissues were excised and weighedand the amount of radioactivity was determined using anLKB 1282 Compugamma CS gamma counter (LKB WallacTurku Finland) Rats without PET scanning (groups BndashE)were sacrificed 60 minutes after tracer injection followedby ex vivo tissue biodistribution Results were expressed aspercentage of the injected dose per gram tissue (IDg)PET images were analyzed using AMIDE software (AmidersquosMedical Image Data Examiner version 092) [22] Fixedsize ellipsoidal shaped regions of interest (ROI) (dimensions60 times 177 times 74mm3) were manually drawn over the areaof the left and right knees in the last frame of the imageROIs were projected onto the dynamic image sequence andtime-activity curve (TAC) data were extracted TACs were

expressed as standardized uptake values (SUV) mean ROIradioactivity concentration normalized for injected dose andbody weight [7]

27 Statistical Analysis Statistical tests were performed usingIBM SPSS version 20 A one-sample Kolmogorov-Smirnovtest was to test for normal distribution taking (119875 value)criteria for 119905-test A Wilcoxon signed rank (exact) test wasused to determine differences between paired observations(eg tracer uptake in right versus Con-RA knee thicknessof the antigen-induced versus control ear and mediolateralthickness of RA knee versus Con-RA knee) A Mann-Whitney (exact) test was used to determine differencesin absorbance before and after immunization A 119875 valuelt005 was considered statistically significant A Bonferronicorrection was applied when necessary

3 Results

During the entire study no major change in body weightwas observed and knee functionality was never dramaticallyimpaired during the course of the induction of arthritis in theRA knee of the rats

31 Immunization Status All rats showed a significant incr-ease (119875 lt 0001) in the level of mBSA antibody titers as compared with mBSA levels before immunization (Figure 2(a))

In addition a DTH test was executed and all rats showeda good DTH response with a significant (119875 = 0001) increasein ear thickness of the right ear at 6 24 and 48 hours afterinjection compared with the control left ear (Figure 2(b)) andcompared to control rat earrsquos injected with saline (data notshown)

32 Arthritis Evaluation of No-Boost Model As negativecontrol healthy rat knee sections stained with the ED1 andED2 rat macrophage specific antibodies showed no signs ofinflammation in the synovial tissue (Figure 3 left panels)Some macrophages were found in the single layered synoviallining In contrast the RA knees of the rats in the firstlyadapted no-boost group (group A 0x boost panels) showed amoderate influx of inflammatory cells in the synovium witha hyperplasia of synovial tissue consisting of 3-4 layers

PET tracer [18F]FDGuptake in the RA knees of rats fromgroup A was also low and showed no obvious difference ascompared to the Con-RA knees as shown in Figure 5(a)

33 Arthritis Evaluation of the Modifications in the Rat Model

331 Macroscopic Evaluation of Arthritis Activity In the no-boost group A a significant difference was observed in kneethickness between the RA knees compared to the Con-RA knees (119875 = 001) which persisted until day 6 afteria injection (119875 = 001) (Figure 2(c)) In the no-boostgroup followed for 28 days (group B Figure 2(c)) differencesbetween the RA versus Con-RA knee were significant (119875 =0001) at day 4 but gradually decreased (119875 = 01) in 28 daysContinued observation without boosting demonstrated thatthe RA knee thickness decreased significantly (119875 lt 00001)between 6 and 28 days (group A versus group B) Although


0

02

04

06

08

1

12

14

Before immunisationAfter immunisation

Abso

rban

ce (A

450

)plusmnSD

A (6d) B (28d) C (19d) D (28d)

(a)

00

05

10

15

20

25

0 12 24 36 48Time (h)

Ear t

hick

ness

(mm

plusmnSD

)

(b)

8

10

12

14

16

18

0 3 6 9 12 15 18 21 24 27 30Time (days)

Knee

thic

knes

s (m

mplusmn

SD)

(c)

Figure 2 (a) Measurement of anti-mBSA in serum in rats before immunization (left) and after immunization (right) (119875 lt 0001) (b) Calipermeasurement of right ear swelling of (◼) A (6 d) (Q) B (28 d) (e) C (19 d) (998771) D (28 d) compared to the control ear of (◻) A (6 d) (loz) B(28 d) (I) C (19 d) (998779) D (28 d) as a response to sc injection of antigen (119875 lt 0001) (c) Knee thickness of arthritic knee of (◻) A (6 d)(loz) B (28 d) (I) C (19 d) (998779) D (28 d) compared to control Con-RA knee of (◼) A (6 d) (Q) B (28 d) (e) C (19 d) (998771) D (28 d) All resultsdepicted represent mean plusmn SD

ED1

ED2

Healthy rat A (6d) B (28d) C (19d) D (28d)

Figure 3 Immunohistochemistry Images (200x) of ED1 (upper panel) and ED2 (lower panel) staining in knees of healthy rats (left panellimited amounts of macrophages) knees from rats in the no boost groups (A (6 d) and B (28 d)) showing clear influx of positively stainedED1 and ED2 macrophages in the inflamed synovium knees from rats in the boost groups C (19 d) and D (28 d) showing strong influx ofpositively stained ED1 and ED2 macrophages in the multilayered synovial linings


RA kneeCon-RA knee

0

40

80

120

160

200

240To

tal n

umbe

r of m

acro

phag

es (plusmn

SD)

A (6d) B (28d)

(a)

RA kneeCon-RA knee

0

40

80

120

160

200

240

Tota

l num

ber o

f mac

roph

ages

(plusmnSD

)

F (saline 28d) B (28d) C (19d) D (28d)

(b)

RA kneeCon-RA knee

0

100

200

300

400

500

Tota

l num

ber o

f mac

roph

ages

(plusmnSD

)

F (saline 28d) B (28d) C (19d) D (28d)

(c)

Figure 4Macrophage counting in histological knee sections (A)Total number (plusmnSD) of ED1 positivemacrophages in the lining and subliningof the knee synovial tissue from the no boost rats (A (6 d) and B (28 d)) RA knee light grey bars Con-RA knee black bars (B) Total number(plusmnSD) of ED1 positive macrophages in the lining and sublining of the knee synovial tissue from the groups B (28 d) C (19 d) and D (28 d)are added next to saline control rats group F (28 d) (C (19 d)) Total number (plusmnSD) of ED2 positive macrophages in the lining and subliningof the knee synovial tissue from the groups B (28 d) C (19 d) and D (28 d) are added next to saline control rats group F (28 d)

significantly decreased in size the knee thickness did notnormalize completely at 28 days when compared to the Con-RA knees of the same rats

When the rats were dosed with 3 boost injections (groupC) significant differences were found in knee thickness of theRA versus Con-RA knee (119875 = 0002 and 119875 = 0003 at day4 as well as at the sacrificing day resp) In the group of ratsdosed with 5 boost injections (group D) also significant dif-ferences were observed between the RA and Con-RA knees(119875 = 00001 and 119875 lt 00001 at days 4 and 28 resp)

Comparing the boost groups C and group D (3x and 5xboosts) on the day those rats were sacrificed a significantdifference in RA knee thickness was observed with groupC lt group D (119875 = 0024) Comparing group A (no boost)

with both boost groups (C and D) with respect to RA kneemeasurements significant differences were observed (groupA versus group C and group A versus groupD 119875 = 0003 and119875 lt 00001 resp)

332 Immunohistochemistry As shown in Figure 3 synovialtissue of healthy rat knees showed no signs of inflammation(Figure 3 left panel) and RA knees in group A (no boost)showed a moderate influx of inflammatory cells in thesynovium with a hyperplasia of synovial tissue consistingof 3-4 layers In contrast the representative images of theknees from rats in group C and groupD demonstrated a clearincrease in ED1 and ED2 positively stained macrophages andmultilayered synovial tissue (gt5 see also next paragraph)


SUV

1

2

0

(a)

0

1

2

SUV

(b)

SUV

3

15

0

(c)

Figure 5 Representative coronal PET images of (a) [18F]FDG and (b) (R)-[11C]PK11195 in arthritic rats group A (6 d) Uptake of both tracersis clearly shown in the right arthritic knee (arrow) compared with the contralateral knee (arrowhead) (c) PET images of [18F]FDG uptake inarthritic rats original model (left) and group A (6 d) (right)

The infiltration of macrophages was quantified in thedifferent groups of rats (between 2 and 4 rats per group)In Figure 4(a) the results for ED1 positive macrophages ingroup A and group B are shown In comparison to groupA group B showed significantly lower numbers of ED1+macrophages (119875 = 0028) (but still significantly higher thanthe control group F (119875 lt 00001)) In Figure 4(b) (ED1) andFigure 4(c) (ED2) the ED1 and ED2 positive macrophagesin knees of rats in all groups are represented includingrats injected with saline (showing very low numbers ofmacrophages) On comparing those numbers in group C andgroup B RA knees from rats in group C clearly displayedmore ED1 and ED2 positive macrophages For those in groupD an even higher amount of ED2+macrophages was found ascompared to the no-boost groups (Figure 4(c)) In Figure 3representative images of ED1 and ED2 staining sections ofdifferent groups are shown

PET and Ex Vivo Tissue Distribution Studies The feasibilityof PET evaluation of arthritis activity in the rats of group A(0x boost) as compared to the original model was assessedFigures 5(a) and 5(b) show representative [18F]FDG and(R)-[11C]PK11195 images The uptake of [18F]FDG in theRA knee of the group A was clearly higher than that inthe original model (Figure 5(c) left original model) Time-activity curves (SUV versus time after tracer injection) of thetracers up to 6 days are depicted in Figure 6 The SUV in theRA knee as compared to the Con-RA knee for [18F]FDG ingroup A rats was significantly increased (278 plusmn 0366 versus138 plusmn 0189 119875 = 0001 Figure 6(a)) and the ratio of uptakein the RA knee was 2 times higher than the Con-RA knee(Figure 6(c)) The SUV for (R)-[11C]PK11195 (Figure 6(b))also increased in the RA knee (200 plusmn 055 versus 111 plusmn 045)and the ratio in the RA knee uptake is 18 timesmore than theCon-RA knee in group A (Figure 6(c))

After PET ex vivo tissue distribution of group A ratswas performed and both [18F]FDG and (R)-[11C]PK11195(Figure 7) showed increased accumulation in the RA knee(16 and 14 times higher resp) compared to uptake inCon-RA knee [18F]FDG and (R)-[11C]PK11195 (052 plusmn 006and 079 plusmn 005 in RA knees 033 plusmn 004 and 057 plusmn005 resp) Furthermore both tracers showed increaseduptake in macrophage rich tissues such as spleen liver andbone (marrow) Moreover increased uptake of (R)-[11C]PK11195 in the heart could be related to TSPO expression onmyocardial cells Both tracers showed accumulation in theintestinal system and renal system (Figure 7) due to excretionof the tracers via hepatobiliary and renal route Physiologicaluptake of [18F]FDG was noted in heart and brain tissue

Results from the ex vivo tissue distribution one hour afterinjection of (R)-[11C] PK11195 in the no-boost groups (A andB) and the boost groups C andD are depicted in Figure 8 Forall groups extra-articular tracer uptake was again observedin macrophage rich tissue and physiological uptake in theintestines

No-boost groups (A and B) (Figure 8(a)) a 145 timeshigher uptake of (R)-[11C] PK11195 was measured in groupA between the RA and Con-RA knee In group B a 12times higher uptake in the RA knee (046 plusmn 005) was foundcompared to Con-RA knee (039 plusmn 003) (119875 = 005)Comparing group A with B it was observed that the traceruptake in the RA knee from rats in group B was a little bit less(although not significantly different from that of group A)

Multiple boost group C versus group D (Figure 8(b)) theuptake of (R)-[11C]PK11195 in RA knees was higher in ratsfrom group C and group D as compared to that in groupB although a level of significance was not reached betweenthe no-boost B and group C However comparing no-boost(group B) with the boost group (group D) a significantdifference in tracer uptake was found (119875 = 001)


0

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0 1000 2000 3000 4000Time (s)

RACON-RA

SUV

(plusmnSD

)

(a)

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SUV

(plusmnSD

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(b)

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RACON-RA

[18F]FDG

Ratio

SU

V (plusmn

SD)

Knee

[11C]PK11195

(c)

Figure 6 Time-activity-curves of tracer uptake expressed as SUV (plusmn SD) in RA knee (∙) and Con-RA knee (I) of (a) [18F]FDG and (b)(R)-[11C]PK11195 in rats of group A (6 d) (c) SUV ratios for RACon-RA are depicted

Zooming in on the rats sacrificed at day 28 ((groupsB D E and F) and for group C at day 19) after the lastia injection a linear correlation was found between uptakeand total number of ED1 positive macrophages In contrastno correlation could be demonstrated with respect to ED2positive macrophages

4 Discussion

In this study an mBSA-induced RA rat model was describedwith sustained and prolonged RA condition Adaptationswith respect to the original model described by Van De Putteet al [13] and Dijkstra et al [14] resulted in a model thatallows evaluation of arthritis activity and therapeutic efficacy

of novel antirheumatic drugs with PET The modificationsresulted in significantly higher influx of macrophages insynovial tissue corresponding to improved visualization ofarthritis with PET

So far no animal model was available for PET guidedimaging and monitoring of therapeutic efficacy with a sus-tained and prolonged arthritic condition long enough to testnew drugs as well as showing some level of systemic diseaseWith our focus on detection of (sub)clinical arthritis versusnoninflamed joints animal models with predominant bonedestruction and those with polyarticular distribution are notfavored Various rodent models of RA (acute and chronic)have been described where the very acute models are notvery useful for monitoring therapeutic efficacy [23] Chronic


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[18F]FDG[11C]PK11195

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nee

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rt

Live

r

Sple

en

Skin

Bone

Brai

n

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cle

Kidn

ey

(ID

gplusmn

SD)

Figure 7 Ex vivo tissue distribution of [18F]FDG (119899 = 6) light greybars and (119877)-[11C]PK11195 (119899 = 5 black bars) at 1 h after injectionin rats of group A (6 d) Results are expressed as percentage of theinjected dose per gram (IDg plusmn SD)

models such as collagen induced arthritis (CIA) bacterial(streptococcal) cell wall contents induced arthritis (SCW)and adjuvant induced arthritis (AIA) have their specificcharacteristics [24] In AIA rats develop polyarthritis withprominent bone destruction In CIA [24] and SCW [25]swelling of paws and limbs appears with periods of remis-sion dampening the readouts for therapeutic interventionwhereas SCW shows low significant systemic effect

Several advantages of our present RA model are pre-sented it is monoarthritic hence the contralateral knee couldbe used as an internal control its robustness and afterthe immunization period the relative rapid development ofarthritis within a week characterized by clear macrophageinfiltration in the synovium of one joint resembling humanRA and with the other joints as internal control Usingthe boosting procedure the therapeutic window was largelyenhanced Macrophages play an important role in earlyRA and may therefore be exploited as potential targets forthe development of new treatment and imaging agents [2627] This model might also be particularly useful for PETpurposes since arthritis is induced in a relatively large kneejoint which is an advantage as detection of arthritis in smallerjoints could be hampered by limited spatial resolution of PETscanners Also injection of larger volumes associated withlow specific activity of some PET tracers is more problematicin mice than in rats For monitoring response with PETimaging quantification is essential To this end assessment oftracer levels in bloodwill be needed at any time after injectionof the PET tracer which poses a problem with respect tofrequent blood sampling in mice

Limitations of the mBSA induced RA rat model arepossibly the longer total time period (immunizations ia andboost injections frequent knee measurements) Also skilled

biotechnicians are needed to perform the precise intra-articular injection in the knee joint Although the modelconsists of only one macroscopically inflamed knee joint thecontralateral knee also showed a low level of microscopicabnormalities related to the infiltration ofmacrophages in thesynovium being indicative for some systemic inflammatoryeffects as also observed in a study by Meyer et al [28]Nevertheless the clear difference of macrophage infiltrationin the arthritic versus the noninflamed knee allowed internalcomparison on PET

PET studies showed increased uptake of both [18F]FDGand (R)-[11C]PK11195 in RA compared to that in Con-RAknees Both tracers however have limitations for clinicalimaging of arthritis activity [18F]FDG has high sensitivitybut low specificity for imaging of arthritis In a previousclinical study absolute uptake of RA joints and osteoarthriticjoints was comparable [29] Although (R)-[11C]PK11195 is amore specific tracer targeting mainly macrophages a limita-tion of this tracer is the background uptake in periarticulartissues [30] The background binding of the PET tracerto both the noninflamed knee joint and periarticular bone(marrow) (in both knees and other physiological locations)could lead to underestimation of targeting properties of theapplied PET tracer since PET imaging of a target relieson the contrast between the target and its backgroundObtaining high arthritic to background ratios is of particularclinical relevance to detect very early (sub)clinical synovitisas it is often only subtly present at this stage of the dis-ease Therefore further research of new specific targets onactivated macrophages in the early phase of RA remainswarranted An alternative candidate target in this respect isthe folate receptor being selectively expressed on activatedmacrophages [31 32] Based on nanomolar binding affinitiesfor folate and folate-conjugated ligands this receptor mightbe an attractive target for both imaging and therapeuticapplications with folate linked therapeutic agents showinglittle or no collateral toxicity to normal tissue [33] andspecifically target the activated macrophages rather than thenonactivated macrophages [34]

5 Conclusions

In conclusion in this study a rat arthritis model suitable forPET guided evaluation of antirheumatic drugs was optimizedand validated The boost regimen proved advantageous forachieving a chronic model with sustained arthritis activityThismodel is excellent for in vivo testing of novel PET tracersfor their suitability for imaging of arthritis (activity) as well asfor PETmonitoring of efficacy of novel therapeutic agents forRA

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank R Bergstra and M Verlaan for excellentassistance in surgical procedures tissue biodistribution and


0

05

1

15

2

25

3

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

enA (6d)B (28d)

(ID

gplusmn

SD)

(a)

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

en

0

05

1

15

2

25

3

(ID

gplusmn

SD)

B (28d)

D (28d)C (19d)

(b)

Figure 8 Ex vivo tissue distribution of (R)-[11C] PK11195 at 1 h after injection in rats of (a) no boost groups A (6 d) and B (28 d) (b) no boostgroup B (28 d) boost groups C (19 d) and D (28 d) Results are expressed as percentage of the injected dose per gram (IDg plusmn SD)

PET studies and analysis M Mooijer for tracer planningMCM Al for help with DTH measurement and tissue dis-tribution Dr G Scheffer for help with ELISA and ProfessorDr P van der Valk and Professor Dr CJF van Noordenfor histopathological advice and helpful discussions Fundingwas received from the Dutch Arthritis Association (NRF-09-01-404)

References

[1] M Feldmann F M Brennan and R N Maini ldquoRheumatoidarthritisrdquo Cell vol 85 no 3 pp 307ndash310 1996

[2] I B McInnes and G Schett ldquoThe pathogenesis of rheumatoidarthritisrdquoTheNew England Journal of Medicine vol 365 no 23pp 2205ndash2219 2011

[3] Y P M Goekoop-Ruiterman J K de Vries-Bouwstra C FAllaart et al ldquoClinical and radiographic outcomes of fourdifferent treatment strategies in patients with early rheumatoidarthritis (the best study) a randomized controlled trialrdquoArthri-tis and Rheumatism vol 52 no 11 pp 3381ndash3390 2005

[4] M F Bakker J W G Jacobs S M M Verstappen and J W JBijlsma ldquoTight control in the treatment of rheumatoid arthritisefficacy and feasibilityrdquo Annals of the Rheumatic Diseases vol66 no 3 pp iii56ndashiii60 2007

[5] C Beckers C Ribbens B Andre et al ldquoAssessment of diseaseactivity in rheumatoid arthritis with 18F-FDG PETrdquo Journal ofNuclear Medicine vol 45 no 6 pp 956ndash964 2004

[6] K Kubota K Ito M Morooka et al ldquoWhole-body FDG-PETCT on rheumatoid arthritis of large jointsrdquo Annals ofNuclear Medicine vol 23 no 9 pp 783ndash791 2009

[7] Y Y J Gent K Weijers C F M Molthoff et al ldquoEvaluationof the novel folate receptor ligand [18F]fluoro-PEG-folate for

macrophage targeting in a rat model of arthritisrdquo ArthritisResearch andTherapy vol 15 no 2 article R37 2013

[8] R B Banati J Newcombe R N Gunn et al ldquoThe peripheralbenzodiazepine binding site in the brain in multiple sclerosisQuantitative in vivo imaging of microglia as a measure ofdisease activityrdquo Brain vol 123 no 11 pp 2321ndash2337 2000

[9] J J Haringman D M Gerlag A H Zwinderman et al ldquoSyn-ovial tissue macrophages a sensitive biomarker for response totreatment in patients with rheumatoid arthritisrdquo Annals of theRheumatic Diseases vol 64 no 6 pp 834ndash838 2005

[10] M D Smith M C Kraan J Slavotinek et al ldquoTreatment-induced remission in rheumatoid arthritis patients is character-ized by a reduction inmacrophage content of synovial biopsiesrdquoRheumatology vol 40 no 4 pp 367ndash374 2001

[11] Z N Jahangier JWG JacobsM C Kraan et al ldquoPretreatmentmacrophage infiltration of the synovium predicts the clinicaleffect of both radiation synovectomy and intra-articular gluco-corticoidsrdquoAnnals of the Rheumatic Diseases vol 65 no 10 pp1286ndash1292 2006

[12] B Bolon M Stolina C King et al ldquoRodent preclinical modelsfor developing novel antiarthritic molecules comparative biol-ogy and preferred methods for evaluating efficacyrdquo Journal ofBiomedicine and Biotechnology vol 2011 Article ID 569068 21pages 2011

[13] L B A Van De Putte J W Lens W B Van Den Berg and MWMKruijsen ldquoExacerbation of antigen-induced arthritis afterchallenge with intravenous antigenrdquo Immunology vol 49 no 1pp 161ndash167 1983

[14] C D Dijkstra E A Dopp I M C Vogels and C J F vanNoorden ldquoMacrophages and dendritic cells in antigen-inducedarthritis An immunohistochemical study using cryostat sec-tions of the whole knee joint of ratrdquo Scandinavian Journal ofImmunology vol 26 no 5 pp 513ndash523 1987


[15] R J Griffiths ldquoCharacterisation and pharmacological sensitiv-ity of antigen arthritis induced by methylated bovine serumalbumin in the ratrdquo Agents and Actions vol 35 no 1-2 pp 88ndash95 1992

[16] P L E M van Lent W B van Den Berg J Schalkwijk LB van de Putte and L van den Bersselaar ldquoAllergic arthritisinduced by cationic antigens relationship of chronicity withantigen retention and T-cell reactivityrdquo Immunology vol 62 no2 pp 265ndash272 1987

[17] S M Atkinson P A Usher P H Kvist H Markholst CHaase and A Nansen ldquoEstablishment and characterization ofa sustained delayed-type hypersensitivity model with arthriticmanifestations in C57BL6J micerdquo Arthritis Research and Ther-apy vol 14 no 3 article R134 2012

[18] D Tanaka T Kagari H Doi and T Shimozato ldquoAdministrationof anti-type II collagen antibody sustains footpad swelling ofmice caused by a delayed-type hypersensitivity reaction andinduces severe arthritisrdquo Clinical and Experimental Immunol-ogy vol 148 no 2 pp 360ndash367 2007

[19] C D Dijkstra E A Dopp P Joling and G Kraal ldquoTheheterogeneity of mononuclear phagocytes in lymphoid organsdistinct macrophage subpopulations in the rat recognized bymonoclonal antibodies ED1 ED2 and ED3rdquo Immunology vol54 no 3 pp 589ndash599 1985

[20] H Folkersma J C Foster Dingley B N M van Berckel etal ldquoIncreased cerebral (R)-[11C]PK11195 uptake and glutamaterelease in a rat model of traumatic brain injury a longitudinalpilot studyrdquo Journal ofNeuroinflammation vol 8 article 67 2011

[21] F E Froklage S Syvanen N H Hendrikse et alldquo[11C]Flumazenil brain uptake is influenced by the blood-brain barrier efflux transporter P-glycoproteinrdquo EJNMMIResearch vol 2 article 12 2012

[22] A M Loening and S S Gambhir ldquoAMIDE a free software toolfor multimodality medical image analysisrdquo Molecular Imagingvol 2 no 3 pp 131ndash137 2003

[23] D L Asquith A M Miller I B McInnes and F Y LiewldquoAnimal models of rheumatoid arthritisrdquo European Journal ofImmunology vol 39 no 8 pp 2040ndash2044 2009

[24] L Bevaart M J Vervoordeldonk and P P Tak ldquoEvaluation oftherapeutic targets in animal models of arthritis how does itrelate to rheumatoid arthritisrdquo Arthritis and Rheumatism vol62 no 8 pp 2192ndash2205 2010

[25] R C Schimmer D J Schrier C M Flory et al ldquoStreptococcalcell wall-induced arthritis requirements for IL-4 IL- 10 IFN-120574and monocyte chemoattractant protein-1rdquo Journal of Immunol-ogy vol 160 no 3 pp 1466ndash1471 1998

[26] F Chauveau N van Camp F Dolle et al ldquoComparativeevaluation of the translocator protein radioligands 11C-DPA-713 18F-DPA-714 and 11C-PK11195 in a rat model of acuteneuroinflammationrdquo Journal of Nuclear Medicine vol 50 no3 pp 468ndash476 2009

[27] C R Fischer C Muller J Reber et al ldquo[18F]fluoro-deoxy-glucose folate a novel PET radiotracer with improved in vivoproperties for folate receptor targetingrdquo Bioconjugate Chem-istry vol 23 no 4 pp 805ndash813 2012

[28] P Meyer H Burkhardt E Palombo-Kinne et al ldquo123I-antileukoproteinase scintigraphy reveals microscopic carti-lage alterations in the contralateral knee joint of rats withldquomonarticularrdquo antigen-induced arthritisrdquoArthritis amp Rheuma-tology vol 43 pp 298ndash310 2000

[29] E H Elzinga C J van der Laken E F I Comans A ALammertsma B A C Dijkmans and A E Voskuyl ldquo2-Deoxy-2-[F-18]fluoro-D-glucose joint uptake on positron emissiontomography images rheumatoid arthritis versus osteoarthritisrdquoMolecular Imaging and Biology vol 9 no 6 pp 357ndash360 2007

[30] Y Y J Gent A E Voskuyl R W Kloet et al ldquoMacrophagepositron emission tomography imaging as a biomarker forpreclinical rheumatoid arthritis findings of a prospective pilotstudyrdquoArthritis and Rheumatism vol 64 no 1 pp 62ndash66 2012

[31] J W van der Heijden R Oerlemans B A C Dijkmans et alldquoFolate receptor 120573 as a potential delivery route for novel folateantagonists tomacrophages in the synovial tissue of rheumatoidarthritis patientsrdquo Arthritis and Rheumatism vol 60 no 1 pp12ndash21 2009

[32] E L Matteson V J Lowe F G Prendergast et al ldquoAssessmentof disease activity in rheumatoid arthritis using a novel folatetargeted radiopharmaceutical Folatescan TMrdquo Clinical andExperimental Rheumatology vol 27 no 2 pp 253ndash259 2009

[33] C M Paulos M J Turk G J Breur and P S Low ldquoFolatereceptor-mediated targeting of therapeutic and imaging agentsto activated macrophages in rheumatoid arthritisrdquo AdvancedDrug Delivery Reviews vol 56 no 8 pp 1205ndash1217 2004

[34] W Xia A R Hilgenbrink E L Matteson M B Lockwood J-X Cheng and P S Low ldquoA functional folate receptor is inducedduring macrophage activation and can be used to target drugsto activated macrophagesrdquo Blood vol 113 no 2 pp 438ndash4462009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Arthritis induction in rat variations of the original model to the modified groups

Code group Original Group A Group B Group C Group D Group E(control)

Group F(control)

Number of boost No No No 3 5 No NoSacrificed (days post) 6 6 28 19 28 6 28

1st immunization (mBSACFACBP antigen)lowastNo of rats 11 3 4 3 6 4 4Volume (120583L) 200 200 200 200 200 200 200Administration route sc sc sc sc sc sc scAdministration location Tail base Tail base Tail base Tail base Tail base Tail base Tail base

2nd immunization (mBSACFACBP antigen)lowastVolume (120583L) 200 100 100 100 100 100 100Administration route sc sc sc sc sc sc sc

Administration location Tail base Neck andupper flank

Neck andupper flank

Neck andupper flank

Neck andupper flank

Neck andupper flank

Neck andupper flank

Delayed time hypersensitivity (DTH) CBP antigenVolume (120583L)lowastlowast 25 25 25 25 25 25Route sc sc sc sc sc sc scAdministration site Right ear Right ear Right ear Right ear Right ear Right ear Right ear

Local (ia) arthritis induction (mBSA) Salineia injection (mBSAlowastlowastlowast or saline) 1 1 1 1 1 1 1mBSA boost ia injections 0 0 0 3 5 0 0Injected volume (120583L) 20 60 60 60 60 60 60Route ia ia ia ia ia ia iaAdministration site knee joint knee joint knee joint knee joint knee joint knee joint knee jointlowast50mgmBSA in 1mL 09NaCl emulsified with an equal volume of complete Freundrsquos adjuvant antigen (CFA) and customBordetella pertussis (CBP) antigen(1 times 1011 cellsmL) lowastlowastCBP antigen (27 times 1010 cellsmL 09 NaCl) lowastlowastlowast10mgmBSA in 1mL 09 NaCl and 1mgmBSA in 1mL 09 NaCl

receptor) a mitochondrial membrane protein that is upreg-ulated in activated macrophages [8] Histological studieshave shown that macrophages are an important biomarkerfor prediction and monitoring of therapeutic effects of awide range of disease modifying antirheumatic drugs andbiologics [9 10] Jahangier et al demonstrated a clear positiveclinical effect in RA patients after intra-articular treatmentwith Yttrium-90 and glucocorticoids correlating effect witha decrease in total numbers of macrophages [11]

Animal models can be applied for in vivo evaluationof efficacy of new therapeutic agents for RA [12] As ittakes some time for most antirheumatic drugs to read outtheir mode of action on arthritis activity with macrophageinfiltration as a biomarker a chronic RA animal model isrequired with sustained arthritis activity characterized bymacrophage infiltration in synovial tissue As currently nosuitable rat model is available that would allow noninvasivemacrophage PET guided evaluation of the therapeutic agentswe have optimized an antigen induced model with persistentarthritis in rats offering sufficiently sized inflamed joints toenable quantitative measurements of PET tracer uptake ininflamed joints as well as the opportunity for comparisonto contralateral noninflamed control joints within the sameanimals

2 Materials and Methods

21 Animals Wistar rats (male 150ndash200 grams CharlesRiver International Inc Sulzfeld Germany) were providedwith standard food (16 protein rodent diet Harlan Lab-oratories Inc Madison WI USA) and water ad libitum

Rats were housed in groups of three or four in conventionalcages and kept in a room with a 12-hour lightdark cycle andconstant room temperature (21∘C) and humidity level (50)All animal experiments were carried out in accordancewith the Dutch law on animal experimentation and wereapproved by the VUMedical Center Institutional Committeeon Animal Experimentation

22 Antigen Induced Rat Model As reference in this paperthe methylated bovine serum albumin (mBSA) induced ratmodel as described by Van De Putte et al [13] and Dijkstra etal [14] was applied (Table 1) indicated hereafter as ldquooriginalmodelrdquo In short according to the descriptions of the originalmodel rats were immunized subcutaneously (sc) twice atdays 0 and 7 with an emulsion containing mBSA (Sigma-Aldrich Chemie BV ZwijndrechtTheNetherlands) dissolvedwith complete Freundrsquos adjuvant (CFA) (Sigma AldrichSteinheim Germany) and custom Bordetella pertussis (CBP)antigen (Becton Dickinson Breda The Netherlands) [14]Rats were immunized with two administrations of 200 uLsolution containing 50mgmBSA in 1mL 09 NaCl emul-sified with an equal volume of complete Freundrsquos adjuvantantigen (CFA) and custom Bordetella pertussis (CBP) antigen(1 times 1011 cellsmL) Both the first and the second immuniza-tion were performed in the tail base At day 21 local arthritiswas induced by injecting 20 120583L mBSA solution containing10mgmBSA [15] in 1mL 09 NaCl intra-articular (ia) inthe right knee (RA knee) the contralateral left knee served asan internal control (Con-RA) The ia injection was situatedbetween femur and tibia and behind the patella tendon


5 10 15 20 25 30 35 4540 48Day (s)

F (saline 28d)

B (28d)

C (19d)

D (28d)

A (6d)

E (saline 6d)












2-EDTAsdot2H

2O) (Merck Darm-


4Cl (Sigma-












3 Results









0

02

04

06

08

1

12

14


Abso

rban

ce (A

450

)plusmnSD

A (6d) B (28d) C (19d) D (28d)

(a)

00

05

10

15

20

25

0 12 24 36 48Time (h)

Ear t

hick

ness

(mm

plusmnSD

)

(b)

8

10

12

14

16

18

0 3 6 9 12 15 18 21 24 27 30Time (days)

Knee

thic

knes

s (m

mplusmn

SD)

(c)


ED1

ED2




RA kneeCon-RA knee

0

40

80

120

160

200

240To

tal n

umbe

r of m

acro

phag

es (plusmn

SD)

A (6d) B (28d)

(a)

RA kneeCon-RA knee

0

40

80

120

160

200

240

Tota

l num

ber o

f mac

roph

ages

(plusmnSD

)

F (saline 28d) B (28d) C (19d) D (28d)

(b)

RA kneeCon-RA knee

0

100

200

300

400

500

Tota

l num

ber o

f mac

roph

ages

(plusmnSD

)

F (saline 28d) B (28d) C (19d) D (28d)

(c)








SUV

1

2

0

(a)

0

1

2

SUV

(b)

SUV

3

15

0

(c)









0

1

2

3

4

0 1000 2000 3000 4000Time (s)

RACON-RA

SUV

(plusmnSD

)

(a)

RACON-RA

0

1

2

3

4

0 1000 2000 3000 4000Time (s)

SUV

(plusmnSD

)

(b)

0

05

1

15

2

25

RACON-RA

[18F]FDG

Ratio

SU

V (plusmn

SD)

Knee

[11C]PK11195

(c)



4 Discussion





0

05

1

15

2

25

3

35

4

[18F]FDG[11C]PK11195

RA k

nee

Con

-RA

kne

e

Bloo

d

Lung

Hea

rt

Live

r

Sple

en

Skin

Bone

Brai

n

Mus

cle

Kidn

ey

(ID

gplusmn

SD)







5 Conclusions




Acknowledgments



0

05

1

15

2

25

3

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

enA (6d)B (28d)

(ID

gplusmn

SD)

(a)

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

en

0

05

1

15

2

25

3

(ID

gplusmn

SD)

B (28d)

D (28d)C (19d)

(b)



References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5 10 15 20 25 30 35 4540 48Day (s)

F (saline 28d)

B (28d)

C (19d)

D (28d)

A (6d)

E (saline 6d)












2-EDTAsdot2H

2O) (Merck Darm-


4Cl (Sigma-












3 Results









0

02

04

06

08

1

12

14


Abso

rban

ce (A

450

)plusmnSD

A (6d) B (28d) C (19d) D (28d)

(a)

00

05

10

15

20

25

0 12 24 36 48Time (h)

Ear t

hick

ness

(mm

plusmnSD

)

(b)

8

10

12

14

16

18

0 3 6 9 12 15 18 21 24 27 30Time (days)

Knee

thic

knes

s (m

mplusmn

SD)

(c)


ED1

ED2




RA kneeCon-RA knee

0

40

80

120

160

200

240To

tal n

umbe

r of m

acro

phag

es (plusmn

SD)

A (6d) B (28d)

(a)

RA kneeCon-RA knee

0

40

80

120

160

200

240

Tota

l num

ber o

f mac

roph

ages

(plusmnSD

)

F (saline 28d) B (28d) C (19d) D (28d)

(b)

RA kneeCon-RA knee

0

100

200

300

400

500

Tota

l num

ber o

f mac

roph

ages

(plusmnSD

)

F (saline 28d) B (28d) C (19d) D (28d)

(c)








SUV

1

2

0

(a)

0

1

2

SUV

(b)

SUV

3

15

0

(c)









0

1

2

3

4

0 1000 2000 3000 4000Time (s)

RACON-RA

SUV

(plusmnSD

)

(a)

RACON-RA

0

1

2

3

4

0 1000 2000 3000 4000Time (s)

SUV

(plusmnSD

)

(b)

0

05

1

15

2

25

RACON-RA

[18F]FDG

Ratio

SU

V (plusmn

SD)

Knee

[11C]PK11195

(c)



4 Discussion





0

05

1

15

2

25

3

35

4

[18F]FDG[11C]PK11195

RA k

nee

Con

-RA

kne

e

Bloo

d

Lung

Hea

rt

Live

r

Sple

en

Skin

Bone

Brai

n

Mus

cle

Kidn

ey

(ID

gplusmn

SD)







5 Conclusions




Acknowledgments



0

05

1

15

2

25

3

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

enA (6d)B (28d)

(ID

gplusmn

SD)

(a)

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

en

0

05

1

15

2

25

3

(ID

gplusmn

SD)

B (28d)

D (28d)C (19d)

(b)



References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























3 Results









0

02

04

06

08

1

12

14


Abso

rban

ce (A

450

)plusmnSD

A (6d) B (28d) C (19d) D (28d)

(a)

00

05

10

15

20

25

0 12 24 36 48Time (h)

Ear t

hick

ness

(mm

plusmnSD

)

(b)

8

10

12

14

16

18

0 3 6 9 12 15 18 21 24 27 30Time (days)

Knee

thic

knes

s (m

mplusmn

SD)

(c)


ED1

ED2




RA kneeCon-RA knee

0

40

80

120

160

200

240To

tal n

umbe

r of m

acro

phag

es (plusmn

SD)

A (6d) B (28d)

(a)

RA kneeCon-RA knee

0

40

80

120

160

200

240

Tota

l num

ber o

f mac

roph

ages

(plusmnSD

)

F (saline 28d) B (28d) C (19d) D (28d)

(b)

RA kneeCon-RA knee

0

100

200

300

400

500

Tota

l num

ber o

f mac

roph

ages

(plusmnSD

)

F (saline 28d) B (28d) C (19d) D (28d)

(c)








SUV

1

2

0

(a)

0

1

2

SUV

(b)

SUV

3

15

0

(c)









0

1

2

3

4

0 1000 2000 3000 4000Time (s)

RACON-RA

SUV

(plusmnSD

)

(a)

RACON-RA

0

1

2

3

4

0 1000 2000 3000 4000Time (s)

SUV

(plusmnSD

)

(b)

0

05

1

15

2

25

RACON-RA

[18F]FDG

Ratio

SU

V (plusmn

SD)

Knee

[11C]PK11195

(c)



4 Discussion





0

05

1

15

2

25

3

35

4

[18F]FDG[11C]PK11195

RA k

nee

Con

-RA

kne

e

Bloo

d

Lung

Hea

rt

Live

r

Sple

en

Skin

Bone

Brai

n

Mus

cle

Kidn

ey

(ID

gplusmn

SD)







5 Conclusions




Acknowledgments



0

05

1

15

2

25

3

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

enA (6d)B (28d)

(ID

gplusmn

SD)

(a)

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

en

0

05

1

15

2

25

3

(ID

gplusmn

SD)

B (28d)

D (28d)C (19d)

(b)



References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

02

04

06

08

1

12

14


Abso

rban

ce (A

450

)plusmnSD

A (6d) B (28d) C (19d) D (28d)

(a)

00

05

10

15

20

25

0 12 24 36 48Time (h)

Ear t

hick

ness

(mm

plusmnSD

)

(b)

8

10

12

14

16

18

0 3 6 9 12 15 18 21 24 27 30Time (days)

Knee

thic

knes

s (m

mplusmn

SD)

(c)


ED1

ED2




RA kneeCon-RA knee

0

40

80

120

160

200

240To

tal n

umbe

r of m

acro

phag

es (plusmn

SD)

A (6d) B (28d)

(a)

RA kneeCon-RA knee

0

40

80

120

160

200

240

Tota

l num

ber o

f mac

roph

ages

(plusmnSD

)

F (saline 28d) B (28d) C (19d) D (28d)

(b)

RA kneeCon-RA knee

0

100

200

300

400

500

Tota

l num

ber o

f mac

roph

ages

(plusmnSD

)

F (saline 28d) B (28d) C (19d) D (28d)

(c)








SUV

1

2

0

(a)

0

1

2

SUV

(b)

SUV

3

15

0

(c)









0

1

2

3

4

0 1000 2000 3000 4000Time (s)

RACON-RA

SUV

(plusmnSD

)

(a)

RACON-RA

0

1

2

3

4

0 1000 2000 3000 4000Time (s)

SUV

(plusmnSD

)

(b)

0

05

1

15

2

25

RACON-RA

[18F]FDG

Ratio

SU

V (plusmn

SD)

Knee

[11C]PK11195

(c)



4 Discussion





0

05

1

15

2

25

3

35

4

[18F]FDG[11C]PK11195

RA k

nee

Con

-RA

kne

e

Bloo

d

Lung

Hea

rt

Live

r

Sple

en

Skin

Bone

Brai

n

Mus

cle

Kidn

ey

(ID

gplusmn

SD)







5 Conclusions




Acknowledgments



0

05

1

15

2

25

3

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

enA (6d)B (28d)

(ID

gplusmn

SD)

(a)

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

en

0

05

1

15

2

25

3

(ID

gplusmn

SD)

B (28d)

D (28d)C (19d)

(b)



References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















RA kneeCon-RA knee

0

40

80

120

160

200

240To

tal n

umbe

r of m

acro

phag

es (plusmn

SD)

A (6d) B (28d)

(a)

RA kneeCon-RA knee

0

40

80

120

160

200

240

Tota

l num

ber o

f mac

roph

ages

(plusmnSD

)

F (saline 28d) B (28d) C (19d) D (28d)

(b)

RA kneeCon-RA knee

0

100

200

300

400

500

Tota

l num

ber o

f mac

roph

ages

(plusmnSD

)

F (saline 28d) B (28d) C (19d) D (28d)

(c)








SUV

1

2

0

(a)

0

1

2

SUV

(b)

SUV

3

15

0

(c)









0

1

2

3

4

0 1000 2000 3000 4000Time (s)

RACON-RA

SUV

(plusmnSD

)

(a)

RACON-RA

0

1

2

3

4

0 1000 2000 3000 4000Time (s)

SUV

(plusmnSD

)

(b)

0

05

1

15

2

25

RACON-RA

[18F]FDG

Ratio

SU

V (plusmn

SD)

Knee

[11C]PK11195

(c)



4 Discussion





0

05

1

15

2

25

3

35

4

[18F]FDG[11C]PK11195

RA k

nee

Con

-RA

kne

e

Bloo

d

Lung

Hea

rt

Live

r

Sple

en

Skin

Bone

Brai

n

Mus

cle

Kidn

ey

(ID

gplusmn

SD)







5 Conclusions




Acknowledgments



0

05

1

15

2

25

3

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

enA (6d)B (28d)

(ID

gplusmn

SD)

(a)

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

en

0

05

1

15

2

25

3

(ID

gplusmn

SD)

B (28d)

D (28d)C (19d)

(b)



References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SUV

1

2

0

(a)

0

1

2

SUV

(b)

SUV

3

15

0

(c)









0

1

2

3

4

0 1000 2000 3000 4000Time (s)

RACON-RA

SUV

(plusmnSD

)

(a)

RACON-RA

0

1

2

3

4

0 1000 2000 3000 4000Time (s)

SUV

(plusmnSD

)

(b)

0

05

1

15

2

25

RACON-RA

[18F]FDG

Ratio

SU

V (plusmn

SD)

Knee

[11C]PK11195

(c)



4 Discussion





0

05

1

15

2

25

3

35

4

[18F]FDG[11C]PK11195

RA k

nee

Con

-RA

kne

e

Bloo

d

Lung

Hea

rt

Live

r

Sple

en

Skin

Bone

Brai

n

Mus

cle

Kidn

ey

(ID

gplusmn

SD)







5 Conclusions




Acknowledgments



0

05

1

15

2

25

3

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

enA (6d)B (28d)

(ID

gplusmn

SD)

(a)

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

en

0

05

1

15

2

25

3

(ID

gplusmn

SD)

B (28d)

D (28d)C (19d)

(b)



References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

0 1000 2000 3000 4000Time (s)

RACON-RA

SUV

(plusmnSD

)

(a)

RACON-RA

0

1

2

3

4

0 1000 2000 3000 4000Time (s)

SUV

(plusmnSD

)

(b)

0

05

1

15

2

25

RACON-RA

[18F]FDG

Ratio

SU

V (plusmn

SD)

Knee

[11C]PK11195

(c)



4 Discussion





0

05

1

15

2

25

3

35

4

[18F]FDG[11C]PK11195

RA k

nee

Con

-RA

kne

e

Bloo

d

Lung

Hea

rt

Live

r

Sple

en

Skin

Bone

Brai

n

Mus

cle

Kidn

ey

(ID

gplusmn

SD)







5 Conclusions




Acknowledgments



0

05

1

15

2

25

3

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

enA (6d)B (28d)

(ID

gplusmn

SD)

(a)

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

en

0

05

1

15

2

25

3

(ID

gplusmn

SD)

B (28d)

D (28d)C (19d)

(b)



References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

25

3

35

4

[18F]FDG[11C]PK11195

RA k

nee

Con

-RA

kne

e

Bloo

d

Lung

Hea

rt

Live

r

Sple

en

Skin

Bone

Brai

n

Mus

cle

Kidn

ey

(ID

gplusmn

SD)







5 Conclusions




Acknowledgments



0

05

1

15

2

25

3

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

enA (6d)B (28d)

(ID

gplusmn

SD)

(a)

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

en

0

05

1

15

2

25

3

(ID

gplusmn

SD)

B (28d)

D (28d)C (19d)

(b)



References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

25

3

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

enA (6d)B (28d)

(ID

gplusmn

SD)

(a)

RA k

nee

Con

-RA

kne

e

Bone

(leg

RA

)

Ster

num

Skin Fa

t

Live

r

Sple

en

0

05

1

15

2

25

3

(ID

gplusmn

SD)

B (28d)

D (28d)C (19d)

(b)



References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















sustained macrophage infiltration upon multiple intra articular injections an improved rat model of...

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