RAPID EVALUATION OF CELL VIABILITY AND APOPTOSIS IN INTACT CARTILAGE VIA CONFOCAL MICROSCOPY

Introduction: In recent years an interest in cell viability and apoptosis has increased along with methods in attempting to detect this phenomenon. Methods routinely used to evaluate viability and to detect apoptosis utilise cells in culture or fixed and sectioned tissues. The major focus of this study has been to develop rapid methods to assess the viability of whole, live and unfixed cartilage tissue employing laser scanning confocal (LSM) microscopy. The live/dead (LD) assay (Molecular Probes) has been reported on live sectioned cartilage tissue (1,2), which employs calcein to register live cell esterase activity (green fluorescence) and ethidium homodimer-1 (red) that enters into dead cells with compromised cell walls. In addition to the live/dead technique, we have developed protocols for intact tissue assessment of caspase-3 activity and the loss of mitochondrial transmembrane potential; both associated with early apoptosis (3,4). The aims of this study were: i) To predict longer term tissue survivability of cartilage in an ex vivo model using the above fluorescence markers after Holmium laser (Ho:YAG) exposure; ii) To differentiate between cell death via necrosis or programmed cell death following laser exposure; iii) To verify an adapted direct TUNEL assay on whole-live sectioned tissue samples as opposed to paraffin embedded samples. Material and Methods Preparation for laser treatment: Bovine articular cartilage (AC) was derived from 3-6 month old cows, 2-3 days after slaughter. Pieces of AC from the Femoral condyle (15x15x6mm) were detached from the subchondral bone and maintained in sterile media until exposed to laser treatment. A multiwavelength HO:YAG laser light source was used that was operated in a free running mode, at a pulse repetition rate of 8 Hz and a pulse duration of 400 μs (FWHM). For each treatment, the laser energy was 780 mJ, where the laser fibre tip was positioned at a 90° angle to the cartilage surface at a constant distance of 1mm. A single line of laser shooting was performed across each treatment sample and 2 untreated control pieces were maintained for each time point. Subsequent to laser exposure, the cartilage pieces (n=12 for each time point) were maintained in media for 1 day, 3 days and 6 days before confocal assessment. Preparations for analyses: Pieces for confocal were sliced 150μm thick and incubated in: a) the LD buffer for calcein and ethidium homodimer staining; b) caspase buffer (Phiphilux, Calbiochem) with ethidium homodimer; c) caspase buffer with CMXRos (Molecular Probes); d) prepared for an adapted direct TUNEL assay (In Situ Death Detection Kit, TMR red, Boehringer). The remaining unsliced sample was divided for fixation in 5% gluteraldehyhe for transmission electron microscopy (TEM) and fixed in 4% buffered paraformaldehyde for paraffin embedding (for later histology and standard TUNEL assessment). Methods of confocal assessment: Measurements of defect size was made on a 10x objective using the LSM410 system (Zeiss). The boundaries of the visible live and dead demarcation zone were measured in terms of lesion width and depth from the surface for each time point using the LSM410 software. The visible boundary of TUNEL positive cells was measured in the same manner. Statistics: data were compared using a one-way ANOVA and subsequently assessed via a t-test when significant differences (P<0.05) were established. Linear regression was calculated on each variable over time. Results: The LD assessment clearly demarked the zone of laser damage (Fig.2A), which significantly (P<0.05) increased in depth by day 3, but did not significantly change after 6 days (Figure 1). Both direct and standard TUNEL assay tests were consistent and the area of TUNEL positive cells significantly (p<0.05) increased in width by 3 days and did not significantly change by day 6. However, the TUNEL signal significantly (P<0.02) increased between day 3 and 6. TUNEL measurements were always significantly smaller than those of LD (Fig.2B). Linear regression of both ethidium and TUNEL measurements over time reveal significant (P<0.05) trends towards the progression of lesion size, in both width and depth. Caspase-3 signal was strong within the sharply defined ethidium homodimer positive stained area and did not extend beyond at any time point. Mitochondrial activity (CMXRos signal) was evident in the calcein (live/green) area and also costained in boarder regions of the ethidium/caspase positive zone. Alcian blue staining reveal matrix depletion and the formation of vacuoles in the immediate impact site (Fig.2C). H&E and alcian staining show the presence of pycnotic cells beyond the site of matrix depletion. TEMs reveal that cells located outside the zone of ethidium positive cells (calcein positive) have the morphology consistent with healthy cells. However, cells located on the peripheral zone, but within the ethidium positive zone, show unusual morphology and are likely to be apoptotic. Necrosis is predominant especially closer to the mid-section of laser impact area (TUNEL positive) where cellular contents are forced into the immediate surrounding matrix (Figure 2D).