Aggrecans

At sacrifice the operated knees (mid femur to mid tibia) of all mice, and non-operated knees from 3 animals, were harvested, and the skin and muscle removed. Specimens were fixed in 10% neutral buffered formalin for 24 hrs, decalcified for 3 days in 10% formic acid/5% formalin, and paraffin embedded. Serial 4μm sagittal sections were cut across the width of the medial femoro-tibial joint and mounted on superfrost plus glass slides (3 serial sections per slide) with heating at 85°C for 30 minutes then overnight at 55°C. Sections every 40μm were stained with 0.04% toluidine blue and counterstained with 0.1% fast green (12–15 slides per mouse).
Two observers (CBL, AB) blinded to genotype and post-operative time, scored cartilage aggrecan loss (0–3) and structural damage (0–7), with maximal and summed score (sum of all scores in all slides) recorded as previously described (7 (link)). Each slide received a single score for each parameter representing the maximal score in the three sections on the slide. The number of slides with scores for structural damage was recorded as a measure of the “stage” of OA (width of joint affected). The presence or absence of morphological chondrocyte hypertrophy (enlarged chondrocyte lacunae with lack of toluidine blue stain around a collapsed cell as typically observed in the growth plate or calcified cartilage) in the non-calcified articular cartilage was recorded. Osteophyte size (0 = none, 1 = small ~ the same thickness as the adjacent cartilage, 2 = medium ~ 1–3 × the thickness as the adjacent cartilage, 3 = large >3 × the thickness as the adjacent cartilage) and osteophyte maturity (0 = none, 1 = predominantly cartilaginous, 2 = mixed cartilage and bone with active vascular invasion and endochondral ossification, 3 = predominantly bone) were scored on coded digital images of the same location of the anterior-medial tibia in each animal.

The targeting vector plasmid for the Aggrecan knockin mice contains a 2.4 kb 5′ homologous arm, an IRES-CreER^T2 cassette followed by a Pgk-neomycin (neo) cassette flanked with FRT sites, a 3.7 kb 3′ homologous arm, and a MC1-thymidine kinase cassette outside of the homology (Indra et al., 1999 (link); Mansour et al., 1988 (link)). The homologous arms were generated by PCR amplification using BAC template DNA from a clone isolated by a screening of a RPCI-22 129S6 Mouse BAC library (BAC PAC Resources). The nucleotide sequence of the PCR products were sequenced, and compared to sequences deposited in the mouse genome database using the BLAT algorithm (Kent et al., 2002 (link)). In the targeting vector, the neo cassette is in the reverse transcriptional orientation to the IRES-CreER^T2 cassette. G4 ES cells (George et al., 2007 (link)) were electroporated with 25 micrograms of linearized targeting vector and placed under positive and negative selection according to standard procedures. Correctly targeted ES cell clones were screened by Southern blotting with flanking 5′ and 3′ probes. Chimeras generated by injection of C57BL/6 blastocysts were bred to C57BL/6 mice and all of the analysis was conducted in a mixed 129S6 × C57BL/6 genetic backround. Heterozygous Rosa26 reporter mice (Soriano, 1999b (link)) were bred to heterozygous aggrecan knockin mice (Agc1^{tm(IRES-creERT2)/+}) and tissues from resulting progeny containing one copy of the R26R allele and the Aggrecan knockin allele were stained with X-gal (Gold Bio Technology). We used heterozygous Agc1^{tm(IRES-creERT2)/+} mice for all experiments, and the Frt-flanked Neo cassette was not removed.

After terminal anesthesia by barbiturate overdose mice were perfused transcardially with 10% formalin (Sigma). Spinal cords were removed, post-fixed overnight, and cryoprotected in buffered 30% sucrose for 48 hours. Frozen sections (30μm horizontal) were prepared using a cryostat microtome (Leica) and processed for immunofluorescence as described^{16 (link)–18 (link)}. Primary antibodies were: rabbit anti-GFAP (1:1000; Dako, Carpinteria, CA); rat anti-GFAP (1:1000, Zymed Laboratories); goat anti-CTB (1:1000, List Biology Lab); rabbit anti-5HT (1:2000, Immunostar); goat anti-5HT (1:1000, Immunostar); mouse anti-CSPG^{22 (link)} (1:100, Sigma); rabbit-anti hemagglutinin (HA) (1:500 Sigma); mouse-anti HA (1:3000 Covance); sheep anti-BrdU (1:6000, Maine Biotechnology Services, Portland, ME); rabbit anti-laminin (1:80, Sigma, Saint Louis, MO); guinea pig anti-NG2 (CSPG4) (Drs. E.G. Hughes and D.W. Bergles^{57 (link)}, Baltimore, MA); goat anti-aggrecan (1:200, NOVUS); rabbit anti-brevican (1:300, NOVUS); mouse anti-neurocan (1:300, Milipore); mouse anti-phosphacan (1:500, Sigma); goat anti-versican (1:200, NOVUS); rabbit anti-neurglycan C (CSPG5) (1:200, NOVUS). Fluorescence secondary antibodies were conjugated to: Alexa 488 (green) or Alexa 350 (blue) (Molecular Probes), or to Cy3 (550, red) or Cy5 (649, far red) all from (Jackson Immunoresearch Laboratories). Mouse primary antibodies were visualized using the Mouse-on-Mouse detection kit (M.O.M. ®, Vector). BDA tract-tracing was visualized with streptavidin-HRP plus TSB Fluorescein geen or Tyr-Cy3 (Jackson Immunoresearch Laboratories). Nuclear stain: 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI; 2ng/ml; Molecular Probes). Sections were coverslipped using ProLong Gold anti-fade reagent (InVitrogen, Grand Island, NY). Sections were examined and photographed using deconvolution fluorescence microscopy and scanning confocal laser microscopy (Zeiss, Oberkochen, Germany).