Optical tomographic imaging discriminates between disease-modifying anti-rheumatic drug (DMARD) and non-DMARD efficacy in collagen antibody-induced arthritis

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.

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.

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.

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).

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.

To assess whether mouse CAIA can be used to effectively discriminate between DMARD and non-DMARD treatments, BALB/c mice were injected with a cocktail of anti-collagen type II antibodies, boosted with lipopolysaccharide on day 3, and treatments were initiated on day 4. Prednisolone (as a steroid), a p38 MAPK inhibitor [8] (as a DMARD treatment), and the non-DMARD COX-2 inhibitor celecoxib (an NSAID) were used to characterize different classes of treatments. Animals were treated throughout the study until the peak of disease on day 8. Nondiseased controls, vehicle, and treated mice were assessed for changes in paw thickness and clinical score on days 4, 6, and 8.

The disease incidence (Figure 1a) was 92% in the vehicle group, with 90% of individual arthritic paws showing a clinical score ≥1 by day 8. Treatment groups showed comparable kinetics and incidence of disease. Prednisolone treatment, as a strong positive control, effectively ablated the clinical score (Figure 1b) and paw swelling (Figure 1c) endpoints as early as day 6 post disease induction, maintaining this effect through to the end of the study. The p38 MAPK inhibitor showed mild, but significant, inhibition of clinical score at day 6, with an increase in efficacy by day 8; and celecoxib showed a similar trend, albeit with less overall effect by day 8. All treatments showed highly significant inhibition of paw swelling (Figure 1c). The clinical scoring and paw swelling readouts thus poorly discriminated between the different types of treatments.

Histopathologic assessment of vehicle-treated mice revealed edema and inflammatory cell influx (general inflammation) in the synovial tissues, joint spaces and extra-articular soft tissues, in addition to mild articular cartilage damage, mild osteoclast-mediated bone resorption, and extraarticular fibroplasia; no appreciable pannus formation was observed in this model (Figure 2a). These results were as expected for this acute model of antibody-induced arthritis. Prednisolone treatment ablated all microscopic evidence of disease (Figure 2b), whereas celecoxib showed no reduction in edema, inflammation or bone resorption (Figure 2c). The p38 MAPK inhibitor decreased edema and inflammatory cell infiltration, particularly within the joint space, compared with vehicle-treated and celecoxib-treated groups (Figure 2d).

There was a statistically significant decrease in general inflammation histopathology scores in both the prednisolone and p38 MAPK inhibitor-treated groups, but not in the celecoxib-treated group (Figure 3a). Cartilage damage and osteoclast-mediated bone resorption was mild in the vehicle-treated group (Figure 3b and 3c, respectively), as expected in this acute and rapid model. Most notably, statistically significant decreases were only observed in prednisolone-treated mice.

Plasma samples from control, vehicle, and treated mice were analyzed for levels of MMP-3 as well as a variety of cytokines and chemokines (eotaxin, 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). The majority of the plasma cytokine/chemokine panel showed either very low levels or no appreciable pattern of change (data not shown). We detected significant plasma elevations of only IL-6, G-CSF, and MMP-3 at day 8 (peak disease) in the mouse CAIA model when compared with naïve animals (Figure 4b to 4d). Eotaxin plasma levels, although showing no increase in vehicle-treated animals, decreased significantly with celecoxib and p38 MAPK inhibitor treatments, but not with prednisolone treatment (Figure 4a). IL-6 and G-CSF biomarkers increased in CAIA and were significantly decreased by all treatments to a similar extent (Figures 4b, c). In contrast, disease-related increases in plasma MMP-3 were decreased ~50% by all treatments with minimal statistical significance. Plasma levels of IL-6, G-CSF, and MMP-3 at day 8 did not discriminate between DMARD and non-DMARD treatments (Figure 4b to 4d), and no disease-related elevations of these biomarkers were observed on day 15 of disease (data not shown).

To assess the relative benefits of optical imaging of CAIA, we imaged untreated and control mice at the peak of inflammatory disease (day 8) using a three-dimensional FMT imaging approach. In this study, intravenous injection of a cathepsin-activatable agent, ProSense750, allowed the detection of activated inflammatory cells (for example, monocytes, lymphocytes) within the joints and paws of arthritic mice, confirming the findings of other researchers using only semiquantitative two-dimensional surface FRI imaging [33, 36, 39]. We built upon these earlier observations by assessing three-dimensional FMT imaging and quantification of arthritic mouse paws (in units of picomoles rather than relative fluorescence units), revealing quantitative 30-fold to 40-fold increases in the level of fluorescent signal as compared with control mice (Figure 5a, b).

FMT imaging offered clear advantages in the depth of detection throughout the paw and ankle, as shown by tomographic slices and the ability to clearly establish a pattern of disease with a significantly larger region of inflammation in the ankles than in the rest of the arthritic paw (Figure 5b). Importantly, non-invasive tomographic fluorescence imaging not only detected the inflammation based on increased protease activity, generating three-dimensional tissue images and tomographic slices, but also provided accurate quantification of this fluorescence (Figure 5c). FMT measured a >40-fold increase in ProSense750 fluorescence in the ankles and midfoot of arthritic mice (~100 pmol/ankle) as compared with those of control mice (<5 pmol/ankle) (Figure 5c). In the toe regions, disease was localized to a smaller anatomical area, showing less overall signal and only ~20-fold increased over controls in the toe regions of the hindpaws.

As clinical scoring, paw swelling, and plasma biomarkers were not very effective at detecting the differences between DMARD and non-DMARD therapeutics when compared with the histological assessment of inflammation, we used our NIR imaging agents to detect, image, and quantify the protease activity associated with the inflammatory cells in the affected tissue. Such an approach, by virtue of direct labeling of the inflammatory cells, should provide a superior means of assessing therapeutic efficacy comparable with that obtained by histopathology scoring. To achieve this, the study subjects described in Figure 1 were injected intravenously with ProSense750 and MMPSense680 on day 7 for imaging on day 8.

Tomographic slices of paw imaging acquired by FMT showed a high fluorescence signal in vehicle-treated mice as compared with those of controls (Figure 6a, b), and, in support of our contention, the different classes of therapeutics showed differential effects on overall paw fluorescent signal. Prednisolone treatment, as a positive control, showed a clear ablation of both cathepsin and MMP signals across all tomographic slices through the ankle, midfoot, and toe regions, suggesting the complete absence of disease. Similarly, the p38 MAPK inhibitor dramatically decreased the signal throughout most of the paw, with higher apparent effects on the ankle and midfoot than in the toes. In contrast to the effects of the p38 MAPK inhibitor, celecoxib treatment had no obvious effect on the cathepsin and MMP signals in any paw region. Tomographic imaging - in contrast to clinical scoring, paw swelling and plasma biomarker measures - thus discriminated effectively between p38 MAPK inhibitor and celecoxib treatments, and revealed the paw subregions showing predominant therapeutic impact.

Tomographic quantification of ankle, midfoot, and toe fluorescence in treated and untreated animals showed clear and statistically significant differences in the ProSense750 signal (Figure 7a) and the MMPSense680 signal (Figure 7b) in the ankle and midfoot regions upon p38 MAPK inhibitor treatment, confirming the visual differences in tomographic slices (Figure 6). Only prednisolone showed efficacy in the mild inflammation of the toe region of the arthritic mice. Quantification of imaging datasets revealed an excellent ability to distinguish between a p38 MAPK inhibitor and celecoxib treatments when compared with histopathology (Figure 3a).

To better understand which CAIA readouts align best with the underlying inflammation, the readouts for individual paws from animals in each treatment were clustered according to their histopathology inflammation scores (from 0 to 4). For each cluster, the average values from optical tomography (cathepsin, MMP), clinical scores, and paw swelling were determined and graphed in comparison with inflammation scores (Figure 8a to 8d). Excellent linear relationships with inflammation scoring were seen with total paw cathepsin (Figure 8a) and MMP (Figure 8b) activity quantification, suggesting that tomographic imaging truly detected and quantified underlying inflammation. Indeed, the non-invasive FMT results (with either ProSense750 or MMPSense680) were able to accurately predict the histology scores determined by the pathologist (Figure 8e).

Neither clinical score (Figure 8c) nor paw swelling (Figure 8d) correlations showed adequate alignment with the inflammation scores, with the pattern of deviation from linearity providing clear evidence that these readouts grossly overestimate drug efficacy regardless of therapeutic class. In addition, it is not surprising that plasma biomarkers showed very poor, nonlinear relationships to inflammation scoring, underestimating or overestimating drug efficacy depending on the biomarker assessed (data not shown).

To assess whether quantitative FMT imaging can detect subtle bone changes in this acute model of arthritis, we imaged mice using OsteoSense680, a NIR-labeled bisphosphonate agent that localizes in vivo to hydroxyapatite that is exposed during bone turnover processes. This agent has been used to detect and quantify bone morphogenic protein-2-induced bone growth and fracture repair [40], as well as bone loss in ovariectomized mice [41]. Vehicle-treated animals showed approximately twofold higher OsteoSense680 incorporation into bone as compared with nondiseased controls on day 8 (Figure 9). By day 15, OsteoSense680 incorporation further increased to achieve a ratio of fourfold over normal control animals, suggesting that this imaging agent can detect and quantify arthritis progression. Prednisolone treatment decreased OsteoSense680 incorporation into bone relative to vehicle controls on day 8, maintaining this differential profile on day 15 but showing a higher signal. The p38 MAPK inhibitor treatment showed a minimal, statistically significant decrease in OsteoSense680 signal on day 8, with improved efficacy seen by day 15. In contrast, celecoxib showed no signs of activity by this readout at either timepoint. The OsteoSense680 imaging results on day 15 were thus in strong agreement with ProSense750/MMPSense680 imaging on day 7 as regards discriminating between celecoxib and p38 MAPK inhibitor therapeutic efficacy.