Following IACUC approval, left patellar tendons (PT) of anesthetized (isoflurane, 2–3% by volume, 0.4 L/min) adult female retired breeder Sprague-Dawley rats (n =28) (Charles River Laboratories, Ltd., Wilmington, MA) were surgically exposed and setup per our previously described protocols for fatigue loading.4 (link),5 (link) Briefly, under aseptic conditions, the tibia was fixed with a clamp, securing the limb at ~30° knee flexion. The patella was clamped and connected in series to a 50-lb load cell and actuator of a servo-hydraulic loading system, allowing loading of the PT without damaging the tendon from clamping. Fatigue loading was applied by cycling between 1 and 40N for either 100 (n =13) or 7,200 (n =15) cycles at 1 Hz. Additional rats (n =6) were used as controls. Diagnostic tests (1–15N) were applied before (diag1: 420 cycles) and after (diag2: 120 cycles) fatigue loading. Hysteresis, stiffness of the loading and unloading load-displacement curves, and actuator position were calculated for the last 10 cycles of the pre-fatigue diag1 and the post-fatigue diag2. The change in these parameters between diag1 and diag2 reflects the effect of fatigue loading and is referred to as the damage parameters.5 (link) Previous studies showed no differences in initial parameters between diag2 and a third diagnostic that was applied 45 min after loading, suggesting that most of changes between diag1 and diag2 are non-recoverable and can serve as indicators of the induced damage.5 (link)Rats were euthanized 3 (n =6 and 8 for the 100 cycle and 7,200 cycle groups, respectively) or 7 (n =7 per cycle group) days after loading. The quadriceps-patella-PT-tibia complex was harvested and fixed in zinc buffered formalin under ~2N tension. Blocks were decalcified and embedded in paraffin, and 5 μm sagittal sections were cut. Antigen retrieval in deparaffinized sections was achieved using DeCal solution (BioGenex Inc., Biogenex, Freemont, CA). Endogenous peroxidase activity was quenched using 3% H2O2. Non-specific binding was blocked with Dako Protein block. Immunohistochemical staining for cleaved Caspase-3 (Cell Signaling Technologies; diluted 1:1,000) was used to identify apoptotic cells. Incubation in rabbit serum without primary antibody was used as the negative staining control. Sections were counterstained with methylene blue to highlight negative cells. Multiple sister sections were visually compared to confirm the expected similarity. One of the sections was then used for all subsequent blinded quantitative analysis.
Under 400X magnification, normal and apoptotic cells were counted at the insertion (tibial end), origin (patellar end), and midsubstance. A region at the insertion and the origin was defined for analysis by drawing an object that consisted of one side that outlined the border between the tendon and fibrocartilage (line 1), two sides at each end of line 1 that were perpendicular to line 1 with both ending at the bursal end of the tendon (lines 2 and 3), and a final line that traced the surface of the tendon and connected lines 2 and 3. The tendon length was measured, and the midpoint was defined. Images throughout the full thickness of the tendon were captured at the midpoint to define the midsubstance region. For each region, the combined number of apoptotic cells and total cells was used to calculate the percent apoptotic and the apoptotic, alive, and total cell densities (cells/mm2). The repeatability of two trained graders was confirmed through three trials on a subset of five images. Control tendon analyses were averaged from both graders to minimize inter-observer variability.
Under 400X magnification, normal and apoptotic cells were counted at the insertion (tibial end), origin (patellar end), and midsubstance. A region at the insertion and the origin was defined for analysis by drawing an object that consisted of one side that outlined the border between the tendon and fibrocartilage (line 1), two sides at each end of line 1 that were perpendicular to line 1 with both ending at the bursal end of the tendon (lines 2 and 3), and a final line that traced the surface of the tendon and connected lines 2 and 3. The tendon length was measured, and the midpoint was defined. Images throughout the full thickness of the tendon were captured at the midpoint to define the midsubstance region. For each region, the combined number of apoptotic cells and total cells was used to calculate the percent apoptotic and the apoptotic, alive, and total cell densities (cells/mm2). The repeatability of two trained graders was confirmed through three trials on a subset of five images. Control tendon analyses were averaged from both graders to minimize inter-observer variability.
