Specific pathogen-free female BALB/c mice (4 to 6 weeks of age, 18 to 20 g) were obtained from Charles River (Wilmington, MA, USA) and were housed in a controlled environment (72°F; 12 h:12 h light-dark cycle) under specific-pathogen free conditions with water and food provided ad libitum. All experiments were performed in accordance with VisEn IACUC guidelines for ethical animal care and use.
Therapeutic studies with the collagen antibody-induced arthritis animal model
BALB/c mice were injected intravenously with 4 mg arthrogen-collagen-induced arthritis monoclonal antibody cocktail (Clones D1, F10, A2 and D8 to collagen type II; Chemicon, Temecula, CA, USA), according to the manufacturer's instructions. Measurable morphological changes were determined by paw thickness measurement using a digital Vernier caliper (VWR, West Chester, PA, USA) on days 4, 6, and 8. Observational clinical scores (scale from 0 to 3) were also made based upon the following criteria of redness and swelling: 0 = no swelling or redness (normal paws), 1 = swelling and/or redness in one digit or in the ankle, 2 = swelling and/or redness in one or two digits and ankle, and 3 = entire paw is swollen or red.
Beginning on day 3 post antibody cocktail injection (prior to signs of disease), cohorts of CAIA mice (n = 12 per group) were treated daily (8 or 15 days) with either prednisolone (10 mg/kg per oral, twice daily), a p38 MAPK inhibitor (SD0006; 15 mg/kg per oral, twice daily), and celecoxib (15 mg/kg per oral, twice daily). Two additional groups, healthy mice (n = 12) and arthritic mice (n = 12), received vehicle treatment only (0.5% aqueous methyl cellulose and 0.025% Tween-80) and served as controls.
Fluorescent agents for the detection of inflammation
Three commercially available imaging agents (VisEn Medical Inc., Bedford, MA, USA) were used to measure disease and therapeutic efficacy in CAIA. For assessing the inflammatory infiltrate, two NIR protease-activatable agents were used, one activated by cathepsins (ProSense750) and the other activated by a family of MMPs (MMPSense680), including MMP-3, MMP-9, and MMP-13. These agents were administered via intravenous route (2 nmol (fluorophore) in 150 μl saline) in all imaging studies. A third NIR imaging agent that detects changes in bone associated with disease (OsteoSense680) was used to image and quantify bone loss. For MMPSense680 and OsteoSense680, the 2 nmol dose of fluorophore corresponds to 2 nmol substrate or pamidronate, respectively. For ProSense750, the 2 nmol dose of fluorophore corresponds to ~0.1 nmol substrate.
Imaging arthritis disease progression
CAIA and control mice were injected intravenously with ProSense750 or MMPSense680 on day 7 following injection of collagen antibody cocktail. OsteoSense680 was injected in additional studies on both day 7 and day 14. At the time of imaging (24 h post agent injection), mice were anesthetized using an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (20 mg/kg). CAIA and control mice were then imaged with the FMT 2500™ fluorescence tomography in vivo
imaging system (VisEn Medical) using fluorescence tomographic scanning capabilities as described previously [37
]. Briefly, the anesthetized mice were carefully positioned in a prone position in the imaging cassette. Both hind paws were elevated on a resin block (designed to mimic optical scattering and absorption properties of the mouse's body) to allow larger tomographic scanning fields for simultaneous imaging of both paws. A NIR laser diode transilluminated the hindpaws, with signal detection occurring via a thermoelectrically cooled charge-coupled device camera placed on the opposite side of the imaged animal. Appropriate optical filters allowed collection both of fluorescence and excitation datasets, the entire imaging acquisition requiring 4 to 5 minutes per mouse.
Fluorescence molecular tomographic reconstruction and analysis
The collected fluorescence data were reconstructed by FMT 2500 system software (TruQuant™; VisEn Medical) for the quantification of the fluorescence signal within the paws. Three-dimensional regions of interest were drawn to encompass each foot and subregions of the foot. A threshold was applied identically to all animals equal to twice the mean paw fluorescence (nanomolar) of the control, nonarthritic mice to minimize low-intensity, background fluorescence. The total amount of ankle, midfoot, toes or total paw fluorescence (in picomoles) was automatically calculated relative to internal standards generated with known concentrations of appropriate NIR dyes. For visualization and analysis purposes, the FMT 2500 system software provided three-dimensional images and tomographic slices.
The right ankle from each animal was fixed in 10% neutral buffered formalin for 24 hours at 20°C, followed by decalcification in Immunocal™ (Decal Chemical Corporation, Tallman, NY, USA) for 7 days at 20°C. Decalcified joints were then paraffin embedded, sectioned twice (4 μm each), and stained with H & E for general evaluation or toluidine blue for specific assessment of cartilage changes. The ankles were evaluated via histopathology and scored for inflammation, cartilage damage, pannus and bone resorption according to previously published criteria [38
For inflammation, scores were as follows: 0 = normal, 1 = minimal infiltration of inflammatory cells in the synovial and/or periarticular tissues, 2 = mild infiltration with mild edema, 3 = moderate infiltration (including joint space) with moderate edema, 4 = marked infiltration with marked edema, and 5 = severe infiltration with severe edema.
For cartilage damage, scores were as follows: 0 = normal, 1 = loss of toluidine blue staining with no chondrocyte degeneration/loss and/or matrix disruption, 2 = loss of toluidine blue staining with minimal chondrocyte degeneration/loss and/or mild matrix disruption in some affected joints, 3 = loss of toluidine blue staining with moderate chondrocyte loss and obvious (depth to deep zone) matrix loss in affected joints, 4 = loss of toluidine blue staining with marked (depth to tide mark) chondrocyte and matrix loss, and 5 = loss of toluidine blue staining with severe (depth to subchondral bone) chondrocyte loss and matrix loss in affected joints.
For bone resorption, scores were as follows: 0 = normal, 1 = minimal (small areas of resorption in the medullary trabecular or cortical bone, not readily apparent on low magnification, and rare osteoclasts), 2 = mild (increasing areas of resorption in medullary trabecular or cortical bone, not readily apparent on low magnification, with osteoclasts more numerous), 3 = moderate (obvious resorption of the medullary trabecular and cortical bone, without full-thickness defects, lesion apparent on low magnification, and osteoclasts more numerous), 4 = marked (full-thickness defects in the cortical bone, marked loss of medullary trabecular bone, numerous osteoclasts), and 5 = severe (full-thickness defects in the cortical bone, severe loss of medullary trabecular bone).
Immunoassay analysis of plasma biomarkers
Plasma MMP-3, a soluble marker for joint pathology, was quantified by the R&D System (Minneapolis, MN, USA) Quantikine mouse MMP-3 (total) Immunoassay (catalog number MMP300) according to the manufacturer's instructions. Plasma cytokines and chemokines - eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, GRO/KC, IFNγ, leptin, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17, IL-18, IP-10, MCP-1, MIP-1β, RANTES, TNFα and vascular endothelial growth factor - were assessed using a multiplex Luminex-based assay from Millipore (catalog number MPXMCYTO-70K-PMX24; Billerica, MA USA) with the addition of 1× Complete® protease inhibitor cocktail (catalog number 11697498001; Roche, Indianapolis, IN, USA).
Data are presented as the mean ± standard error of the mean. Significance analysis of in vivo paw fluorescence was conducted using a two-tailed unpaired Student t test when two groups were analyzed or a one-tailed analysis of variance Scheffe multiple-comparison post test. P < 0.05 was considered significant.