2.1.

Animals

Animals used in this study were maintained in accordance with the guide for the care and use of laboratory animals published by the U.S. National Institutes of Health (NIH Publication NO. 85-23, revised 1996). All experiments were approved by the Local Animal Welfare Committee.

The arthritis studies were performed in female Lewis rats with a body weight of $(175\pm 15)\mathrm{g}$ (Charles River Laboratories, Sulzfeld, Germany) fed a normal diet.

2.2.

Animal Model

RA was induced in 11 female Lewis rats by intradermal collagen injections. Three animals served as controls. CIA was induced as described elsewhere.¹⁴ Control injections were performed in the same way as the collagen injections.¹⁴ After 1 week, the procedure was repeated to boost the immune response. From previous experiments, it is known that arthritis develops 13 to 15 days after the first collagen injection, but is highly variable in severity. Animals may show different degrees of arthritis in the right- and left-ankle joint, or there may be animals without signs of clinical or histological changes in one or both joints.

Based on macroscopic and microscopic patterns, CIA progression was divided into three stages: (1) preclinical (from first collagen injection to clinically evident disease onset), (2) acute clinical [from disease onset (day 0) to day 14], and (3) chronic clinical (after day 14), where clinical (hind and fore paw swelling) and structural (inflammation and articular erosions in hind paws) evidence of joint involvement plateaus.¹⁵

Skeletal erosion begins 1 to 2 days after the onset of paw swelling and is associated with acute synovitis. Left untreated, cartilage matrix degeneration, and bone attrition progress rapidly. The widespread formation of osteophytes along the periosteal surface may eventually result in fusion (ankylosis) of the affected joints with joint deformities resulting in a significant reduction of mobility.¹⁶ Hence, the study duration was restricted to 3 weeks to include the acute and chronic phases of rat CIA. Symptomatic rats received analgesic treatment. As the study aimed at monitoring the response to treatment, only rats with significant arthritis symptoms were assigned to the “meloxicam” group, while presymptomatic or mildly to moderately affected animals were selected for the “no-therapy control” group, permitting monitoring of disease progression.

2.3.

Arthritis Evaluation

Animals were observed daily for the onset of arthritis with respect to swelling, erythema, gait analysis, and functional impairment of the distal joints, in particular the tibiotarsal joints. A 4-point arthritis scale was used for grading the clinical symptoms of each hind paw as follows: “without arthritis signs” (no clinical symptoms), “low-grade arthritis” (mild swelling, erythema, and functional deficits), “moderate arthritis” (moderate swelling, erythema, and functional deficits), or “high-grade arthritis” (severe swelling with erythema and reduced mobility).¹⁴

2.4.

Treatment

Tramadol hydrochloride is a centrally acting synthetic opioid analgesic used to treat moderate to moderately severe pain with no anti-inflammatory effect. Tramadol hydrochloride (Tramal®, Grünenthal GmbH, Aachen, Germany) was administered to rats subcutaneously at a dose of $10\mathrm{mg}/\mathrm{kg}$ body weight/day upon onset of clinical symptoms.

Meloxicam is a nonsteroidal anti-inflammatory drug with analgesic effects and is especially suitable for the treatment of arthritis in animals.¹⁷ Meloxicam (Metacam^®, Boehringer Ingelheim Vetmedica GmbH, Ingelheim, Germany) was administered to rats subcutaneously at a dose of $0.5\mathrm{mg}/\mathrm{kg}$ body weight/day after assignment to the meloxicam treatment group.

2.5.

Histology

For histological workup, the hind legs of the animals were removed and fixed in 4% buffered formaldehyde solution (Mallinckrodt Baker, Deventer, The Netherlands).

Subsequently, the specimens were placed in ethylenediamine tetra-acetic acid decalcifying solution (Herbeta Arzneimittel, Berlin, Germany) for 5 weeks at 60°C. The solution was changed every week. The decalcified hind legs were embedded in paraffin; thereafter, about 4-μm thick histological sections were cut and stained with hematoxylin and eosin.

The histopathological specimens were assessed for signs of arthritis using the following criteria: synovitis, periarthritis, tenosynovitis, periostitis, and cartilage/bone destruction. Each criterion was graded semiquantitatively by one reader blinded to NIRF values as well as clinical scores. Each joint was graded as follows: 0, no arthritis; 1 to 5, low-grade arthritis; 6 to 10, moderate arthritis; and 11 to 15, high-grade arthritis.^14,18

2.6.

Fluorescent Dye

The nonspecific NIRF dye is a TSC dye based on an indotricarbocyanine (ITCC) chromophore. TSC is a low-molecular weight dye ($836.9\mathrm{g}/\mathrm{mol}$) with both anionic and hydrophilic properties.^19,20 In phosphate-buffered saline, TSC shows an absorption maximum at ${\lambda }_{\mathrm{abs}}=755\mathrm{nm}$ and a fluorescence emission maximum at ${\lambda }_{\mathrm{em}}=778\mathrm{nm}$. The dye was dissolved in phosphate-buffered saline and was diluted with physiological saline solution. TSC was administered intravenously at a dose of $1\mu \mathrm{mol}/\mathrm{kg}$ body weight in a volume of 0.2 ml per 100 g body weight.

2.7.

In Vivo NIRF Imaging

The NIRF laser imaging system was described previously.¹⁴ Rats were anesthetized prior to and during the imaging procedure as described before.¹⁴ Imaging was performed before and 30 min after TSC administration. A solid, polymeric cube containing the TSC-related NIR dye ITCC was placed next to each animal as reference. Acquired NIRF intensity data were digitally stored. Each animal was subjected to NIRF imaging prior to and after 2 and 3 weeks of therapy. Upon completion of the final imaging, the anesthetized rats were euthanized by an intracardiac injection of 0.5 ml of embutramide/mebezonium/tetracaine (T61® Intervet Deutschland GmbH, Unterschleissheim, Germany).

2.8.

Data Analysis and Statistics

Four circular regions of interest (ROIs) with a constant diameter of 20 pixels were defined (see circles in Fig. 1): the left- and right-ankle joints with the center of the ROI positioned over the external malleolus, the tail base, and the reference cube on the left side.

Fig. 1

Location of ROIs (white circles) for measurement of fluorescence intensities in ankle joints, tail, and reference cube.

Mean fluorescence intensities $I_{\mathrm{F}}$ were the average of all the fluorescence intensity values of pixels covered by the ROI of a particular region under investigation divided by the number of pixels. For quantitative analysis, the fluorescence intensities of the ankle joints were normalized to the background ($I_{\mathrm{NF}}$), i.e., to the fluorescence intensity of the tail, to compensate for laser fluctuations and the decay of dye concentration. This ratio was calculated as

Descriptive statistics tests were used to compare different groups.

Differences between any two groups were tested for significance using the nonparametric Mann-Whitney $U$-test. Significance was assumed at $p<0.05$.

Associations were tested by calculating Spearman correlation coefficients ($r_{\mathrm{s}}$) (very high correlation: 0.9 to 1.0, high correlation: 0.7 to 0.89, moderate correlation: 0.5 to 0.69, low correlation: 0.3 to 0.49, and little or no correlation: 0.00 to 0.29). Significance was assumed at $p<0.05$.

As graphic representation of the data, box plots were drawn, displaying the first and third quartiles and the median (second quartile). The ends of the whiskers mark the minimum and maximum of all data.

Statistical tests were performed using SPSS version 13.0 (SPSS Inc., Chicago).

3.1.

Arthritis Severity and NIRF Imaging before Therapy

The joints of the animals were assessed and graded according to the clinical severity of arthritis. Only four joints showed low-grade or moderate arthritis at this time (known model-related variability in the development of arthritis severity at disease onset)¹⁶ and were pooled for further statistical analysis (Fig. 2). The results revealed a statistically significant differentiation of control animals and animals with clinically apparent arthritis with respect to $I_{\mathrm{NF}}$ values (median $I_{\mathrm{NF}}$: control 1.03, low to moderate 1.59, high 1.81) and a high correlation of the $I_{\mathrm{NF}}$ values with the clinical arthritis scores ($r_{\mathrm{s}}=0.860$). These data are in accordance with our previous findings in humans and experimental arthritis.^11,14,21,22

Fig. 2

Illustration of normalized fluorescence intensities ($I_{\mathrm{NF}}$) of ankle joints 30 min after intravenous administration of TSC before therapy. Animals are grouped according to clinical arthritis scores.

3.2.

Group Allocation for Therapy

Due to the intrinsic variability in disease severity at onset, animals were assigned to the different groups based on the above-described arthritis grading at day 15:

Six healthy joints from three animals without collagen injections comprised the control group (Table 1). Four CIA rats with clinical arthritis in seven joints were assigned to the meloxicam group. Another four CIA rats with clinical symptoms of arthritis in seven joints comprised the no-therapy control group. At the beginning of the treatment period, two animals of this group showed arthritis symptoms in two joints. During the study period, another five joints became symptomatic. Finally, a total of four animals with seven affected joints received analgesic treatment with tramadol hydrochloride. We excluded eight ankle joints from CIA rats that did not develop clinical arthritis during the study period from further analysis (bold numbers in circles, Table 1).

Table 1

The numbers of joints with and without clinical arthritis in each group before and after therapy are shown. Eight ankle joints, from the meloxicam group (n=1) and no-therapy control group (n=7), which did not develop clinical arthritis throughout the treatment period, were excluded from further analysis (bold numbers in circles).

3.3.

Arthritis Severity and NIRF Imaging after Therapy

Clinical scoring and in vivo NIRF imaging of all animals were repeated after 2 and 3 weeks of therapy. The $I_{\mathrm{NF}}$ values for the ankle joints were calculated from the fluorescence intensity data 30 min after TSC injection.

A statistically significant improvement of clinical arthritis symptoms from high- or moderate-grade to moderate- or low-grade scores became obvious in the meloxicam group ($n=7$ joints) already after 2 weeks of treatment, while all ankle joints ($n=7$) in the no-therapy control group (tramadol hydrochloride) showed statistically significant disease progression to high-grade scores with severe erythema and tissue swelling, which persisted throughout the study period (Table 2). The therapeutic success of meloxicam treatment, reflected by decreasing clinical scores, translated into decreasing $I_{\mathrm{NF}}$ values in NIRF imaging (median $I_{\mathrm{NF}}$: pre-1.81, after 2 weeks of therapy 1.32, after 3 weeks of therapy 1.23). Moreover, disease progression, seen in the no-therapy control group, accordingly translated into increasing $I_{\mathrm{NF}}$ values (median $I_{\mathrm{NF}}$: pre-1.04, after 2 weeks of therapy 1.63, after 3 weeks of therapy 1.66) (Fig. 3). Even more important, the correlation between $I_{\mathrm{NF}}$ values and clinical scores was high or moderate, respectively, for each time point (before therapy: $r_{\mathrm{s}}=0.860$, after 2 weeks of therapy: $r_{\mathrm{s}}=0.825$, after 3 weeks of therapy: $r_{\mathrm{s}}=0.634$) as well as throughout the whole study ($r_{\mathrm{s}}=0.758$).

Table 2

Number of joints and their respective clinical arthritis score before and after 2 and 3 weeks of therapy. Eight ankle joints from the meloxicam group (n=1) and no-therapy control group (n=7), which did not develop clinical arthritis throughout the treatment period, were excluded from further analysis (bold numerals in circles).

Fig. 3

Illustration of normalized fluorescence intensities ($I_{\mathrm{NF}}$) of ankle joints by treatment group 30 min after intravenous TSC administration. The results after 2 and 3 weeks of therapy are shown in comparison to controls. The circle indicates an outlier. Eight ankle joints from the meloxicam group ($n=1$) and no-therapy control group ($n=7$), which did not develop clinical arthritis throughout the treatment period, were excluded from this analysis.

3.4.

Treatment Effect

The clinical manifestation of arthritis and the histologically detectable destruction of the joints were statistically significantly reduced by the anti-inflammatory meloxicam therapy within 3 weeks. This therapeutic success could reliably be visualized by NIRF imaging using TSC.

3.5.

Histology

After 3 weeks of treatment, all ankle joints were removed and investigated by histology to assess the presence and extent of arthritis symptoms such as synovial membrane hyperplasia, subsynovial fibrosis of the surrounding loose connective tissue, inflammatory cell infiltration, and pannus formation (organized inflammatory exudate within the joint space).

Already at low magnification, the different grades of destruction in the ankle joints between treated animals and controls became visible (Fig. 4). The ankle joints of control animals showed unchanged healthy joint structures. In contrast, the inflamed joints of animals after 3 weeks of meloxicam treatment revealed histological signs of moderate arthritis with synovial proliferation, synovial fibrosis, inflammatory cell infiltration, and pannus formation. All signs of chronic inflammatory arthritis were highly pronounced in animals with pain treatment only (Fig. 4).

Fig. 4

Representative histological sections of ankle joints with corresponding NIRF images and $I_{\mathrm{NF}}$ values. Fluorescence images are shown in false colors (range 0 to 100,000 for all color bars) (a) Control animal without arthritis showing faint fluorescence in both ankle joints and normal histological structures (left joint); (b) animal after 3 weeks of meloxicam therapy with prominent fluorescence in both ankle joints revealing histological medium grade destruction of the joints (right joint); (c) ankle joints with persisting high-grade arthritis during analgesic tramadol hydrochloride treatment only, revealing severe histological joint destruction (right joint) and bright fluorescence. Fluorescence at the tail base is due to local inflammation at the site of intradermal collagen injection. The reference cube is visible in the right upper corner.