Page 151 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

SPECT imaging of pro-inflammatory macrophages
Osteoarthritis induction
Experimental OA was induced, during light time of the day/night cycle, by destabilization of the medial meniscus (DMM) (31). The right knees of 40 male C57BL/6 mice of 12-14 weeks old (Harlan Laboratories/Envigo, Cambridgeshire, UK) underwent dissection of the medial meniscotibial ligament (MMTL). As control, the contralateral knee underwent a sham procedure, which entailed no transection of the MMTL.
Immunohistochemical analysis
At 1, 3, 7, 14, 28, and 56 days after OA induction both DMM and control knees were excised and fixed for 10 days in 4% formaldehyde (BoomLab, Meppel, The Netherlands). After decalcification the knees were dehydrated, embedded in paraffin and completely sectioned in the coronal plane. For the CD64 histochemical (IHC) staining, a CD64 antibody (Sino-Biological, 50086-R001, 5 ug/ml) was incubated on the slides for 60min, and subsequently incubated with link and label (Concentrated MultiLink® and Concentrated AP Label; BioGenex, Fremont, California, USA); New Fuchsine was used as substrate (32). The extent of positive CD64 staining in the sections was scored by bright-field microscopy and ranked. Ranking means that all knees were arranged from least intensely stained to most intensely stained using bright field microscopy. The staining intensity of the markers was ranked amongst all time points.
To confirm that OA was induced after 8 weeks, three consecutive thionin-stained sections of the medial femoral condyle and medial tibial plateau were evaluated. The structural cartilage damage was scored by the OARSI scoring system by Glasson et al. (1, 33). The score of the quadrants of three consecutive sections was summed and used for analysis. Histological images were acquired with a NanoZoomer 2.0-HT slide scanner (Hamamatsu, Hamamatsu City, Japan).
In vivo imaging
To evaluate the influx of pro-inflammatory macrophages during OA development, OA was induced in mice (n=6) as described above. Because macrophage polarization is a dynamic process, we wanted to scan the knee as soon as possible after tracer injection. The mice were imaged 2 hours after i.v. injection of radiolabeled tracer (60 MBq/200pmol [111In]In-DTPA-
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