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The first study stood as a preliminary proof-of-principle study of the MMPSense 680 probe. The second study was initiated to test the applicability of an alternative fluorescent probe, MMPSense750 FAST. Two female DBA-1 mice were immunised at day 0, and again at day 21, as described in section 4.2.2. A third DBA-1 mouse served as a naïve control. Clinical signs of arthritis were first observed in one mouse at day 24, but the other animal remained unaffected until day 34, when it presented with swelling in the proximal interphalangeal (PIP) joint (figure 4.3.7A). Clinical scores and paw diameters increased in the arthritic animal from day 24 (figure 4.3.7B). Paw diameter increased in comparison to the naïve control, with swelling peaking at day 29 before falling slightly in subsequent observations (figure 4.3.7C). As with the initial study, disease severity was accompanied by a loss in weight (figure 4.3.7D). Weight loss did not exceed the 20% limit specified in the project licence.

Figure 4.3.7 Experimental arthritis in the MMPSense750 evaluation study

The two CIA animals (labelled CIA 1 and CIA 2) and the naïve control were observed at least twice weekly from the day of the second i.d. (day 21). Animals were monitored for: A) arthritis incidence, B) clinical score, C) paw diameter and D) body weight. i.d., intradermal booster injection of collagen; i.v., intravenous injections of MMPSense750 probe; CIA, Collagen-induced arthritis animal.

4.3.2.1 MMPSense750 fluorescence was enhanced in arthritis compared with a naïve control

An i.v. injection of MMPSense750 was to be administered in animals at the first clinical sign of arthritis; in one CIA immunised animal, this occurred day 27. The entire volume (70 μl) of the probe was successfully injected into the tail vein of this mouse and the naïve control. The second CIA mouse was not injected with the probe until day 34, as there were no signs of disease until this time (explained further in section 4.3.2.3). Imaging was carried out immediately upon injection of the probe (0h) and every 24 hours thereafter, using the Xenogen IVIS 200, as detailed in section 2.2.2.3. Analysis of whole animal fluorescent overlays showed that the limbs fluoresced strongly, particularly the hind paws, up to 120h post-injection (figure 4.3.8). MMP fluorescence was considerably more intense in the arthritic mouse than in the naïve mouse, as evident from the pseudocolour display. MMPSense fluorescence was apparent at the injection site in the base of the tail for all both of the mice injected with the probe.

Figure 4.3.8 Detection of MMPSense750 Fast

Panel shows photographic images of the three mice, with fluorescent intensity shown as a pseudocolour overlay using the Xenogen IVIS 200 imaging system. Two mice were administered MMPSense750 FAST, the naïve control (CON) and the one mouse with clinical signs of arthritis (middle column). The other CIA mouse did not receive MMPSense750 FAST (no probe). Whole animal images were taken on day 27 (immediately after probe administration and every 24 hours thereafter up to 120h post- injection (time course continued overleaf). Boxes within each image depict high power hind paw images. Red boxes show the ROIs used in the analysis. Intensity scale (minimum=5 x 10-6_{,maximum=1.0 x10}-5 _units).

4.3.2.2 MMPSense750 fluorescence was still detectable five days post-injection

Fluorescence efficiency was monitored daily from day 27 to 32 to determine signal drop-off. Although the MMPSense750 probe was not completely cleared by day 32 (120h post-injection), fluorescence in the arthritic mouse had fallen close to those of the naïve (non-arthritic) control in both front paws (figure 4.3.9A) and hind paws (figure 4.3.9B). In contrast to hind paws, there was negligible difference in front paw fluorescence between naïve and CIA mice (mean efficiency values 2.75 x 10-6 _{and 2.93} x 10-6_{, naïve and CIA, respectively), indicating that fluorescence was not as enhanced in} these tissues compared with hind paws.

The results obtained from experiments with MMPSense680 were compared against the data acquired for MMPSense750. The drop in MMPSense750 versus MMPSense680 fluorescence efficiency over the first 72 hours post injection was plotted (figure 4.3.10). These data show the half-life of the MMPSense750 probe was 57.2 hours (front paws) and 61.3 hours (hind paws) compared against 1.3 hours (front paws) and 3.6 hours (hind paws) for MMPSense680. These data contradict the manufacturer’s guide, which claim a half-life of 5 hours for the MMPSense750 probe (Groves et al.,

2010).

Figure 4.3.9 MMPSense750 FAST signal drop off

Mice (n=2 limbs per animal) were imaged on day 27 (0h), prior to injection of MMPSense750 using the XENOGEN IVIS 200. Mice were then imaged 24 hours post- injection, and every 24 hours thereafter, up until 120 hours post-injection (day 32). The no probe mouse was CIA immunised but with no clinical signs of disease. Raw fluorescence values (four per animal) were determined at every time point using ROI analysis. A) Drop in efficiency values in front paws by 120 hours. B) Hind paw efficiency values from 0-120 hours. Data points were graphically presented with mean ± standard error of the mean (S.E.M). Values here are raw values obtained from ROI software, not corrected for background autofluorescence.

Figure 4.3.10 Comparison of MMPSense680 and MMPSense750 probe clearance Raw fluorescence values from arthritic mice were taken from each time point up until 72 hours post-injection to determine which probe had the fastest rate of clearance. From these raw values a percentage drop/rise in fluorescence relative to the first imaging time point (24 hours p.i.) was determined for each subsequent time point. A) MMPsense680 free-form ROI net intensity values using KODAK MI software. Rate of decay from 24 hours p.i. was calculated using one-phase exponential decay (Prism) and determined using non-linear regression analyses (three mice, n=6 front and hind limbs). Front paws: half-life=1.26 hours, K=0.06 units/h, hind paws: half-life=3.59 hours, K=0.19 units/h. B) MMPSense750 efficiency values using the IVIS 200 system with ROI analysis of a fixed dimension around the limb. Fluorescence from the first imaging time point 24 hours p.i. (one mouse, n=2 front and hind limbs). The plateau values taken from the no-probe control were used to accurately determine half-life. Front paws: half-life=57.24 hours, K=0.012 units/h, hind paws: half-life=61.3 hours, K=0.011 units/h. F= half-life of front paws, H= half-life of hind paws.

4.3.2.3 MMP fluorescence signal differed in early and advanced disease

On day 34, the second CIA immunised mouse had begun to exhibit very early clinical signs of arthritis (i.e. a swollen PIP joint, and clinical score of 1). This mouse was injected with MMPSense probe, as well as the first CIA mouse and the naïve control. For this part of the experiment, this former ‘no probe control’ was subsequently referred to as an early arthritis (EA) animal. The other CIA mouse, whose clinical score on day 34 was 14, was referred to as the advanced arthritis (AA) animal. Six hours post-injection, the early arthritis animal exhibited a small area of enhanced fluorescence, which was localised to the single proximal interphalangeal (PIP) joint in the left hind paw (figure 4.3.11). The front paws remained unaffected, and showed no evidence of fluorescence. In contrast, the mouse with advanced arthritis demonstrated a large degree of fluorescence in all limbs (figure 4.3.11). Although MMPSense750 fluorescence had reduced in intensity in the naïve and advance arthritis animal from the previous injection administered on day 27, a small amount of residual fluorescence remained, which was taken into account in the analysis. Overall, these data reiterate the sensitivity of the MMPSense probes, and how in situ MMP activity is a good reflection of the disease state of the animal.

Figure 4.3.11 MMPSense750 FAST fluorescence images on day 34

Panel shows photographic images of the three mice, with fluorescent intensity shown as a pseudocolour overlay using the Xenogen IVIS 200 imaging system. All animals were administered MMPSense750 FAST: the naive control (CON), the mouse with advanced arthritis (AA), and the mouse with early arthritis (EA). Whole animal images were all taken on days 34 and 35, immediately before probe administration (top panel) and 24 hours post-injection (bottom panel). Boxes within each image depict high power hind paw images. i) High power magnification of early arthritic hind paw, showing enhanced fluorescence in one of the proximal interphalangeal joints (PIPJ). Red boxes show the ROIs used in the analysis. Intensity scale (minimum=5.0 x 10-6_{,maximum=1.0 x10}-5 units.

4.3.2.4 Early signs of arthritis were detectible in radiological and histological examination

The three experimental animals (described above) were sacrificed at day 34.Their hind paws were X-rayed and scored (section 2.2.1.2). The hind paws from the naïve and early arthritis mice (CIA onset day 34) exhibited no signs of radiological damage. In contrast, the hind paws of the mouse with advanced arthritis (CIA onset day 24) showed signs of osteopenia and erosions around the cortical bone (figure 4.3.12). This was reflected in the radiology scores obtained (figure 4.3.12D). Due to the small sample size, no statistical analyses were performed on these data.

Hind paws were then processed for histological analyses (section 2.3.1). The advanced arthritis animal had evidence of inflammatory infiltrate and signs of cartilage erosion (figure 4.3.13). This was in contrast to the naïve control (figure 4.3.13A), which showed no evidence of damage. One paw from the mouse with early arthritis showed some evidence of disease, with a high number of inflammatory cells localised around one of the proximal interphalangeal (PIP) joints and no other pathology (figure 4.3.13C). These differences were reflected in the histological (figure 4.3.13D) and cartilage erosion scores (figure 4.3.13E).

Figure 4.3.12 Comparison of radiographic images of hind paws

Representative hind paw images taken at time of sacrifice (day 34), from A) naive control mouse, B) mouse with advanced arthritis (AA) and C) mouse in the early stages of disease (EA). D) Radiology scores obtained from each mouse (n=2 limbs per mouse).

Figure 4.3.13 Histological assessment of disease

A) A representative hind paw of the naïve control mouse (CON). Boxed area: High power magnification of the hind-foot region from a normal tissue section showing i) normal adipose tissue, ii) no synovial exudate, and iii) normal healthy cartilage. B) Histological image of a mouse with advanced arthritis (AA) Boxed area: High power magnification of the diseased hind-foot region contains substantial iv) synovial infiltrate, v) exudate and vi) some loss of continuity of Safranin O staining, indicating early signs of cartilage erosion. C) High paw from early arthritis (EA) mouse. Boxed area: swollen proximal interphalangeal joint. D) Histology and E) Cartilage erosion scores taken from each mouse (n=2 limbs for each).