Radiofrequency energy is commonly used for thermal modification of joint capsular and ligamentous instability and thermal chondroplasty in human sports medicine.1–5 Increasing reports exist on its use in veterinary medicine as well.6 In small animal surgery, RFE is used for meniscectomy, biceps tenotomies, and capsulorrhaphy procedures.a In equine surgery, it is used for synovectomy, chondroplasty, and other soft tissue debridement procedures during arthroscopy and tenoscopy.b Radiofrequency energy contours the cartilage surface through the application of heat in the form of electromagnetic energy by use of a generator. Application of RFE can smooth and reshape articular surfaces, anneal chondral fractures, remove delaminated regions, and create a smooth transition between treated and adjacent untreated regions. Results of previous studies7–9 indicate that treatment of the cartilage with RFE causes discrete regions of chondrocyte death.
Use of the fluorochromes calcein AM to label live cells and EthD-1 to label dead cells is common.10–13 Calcein AM is an uncharged nonfluorescent substrate that freely diffuses into live cells and is enzymatically converted to the intensely fluorescent calcein by a cytoplasmic esterase. The polyanionic calcein is charged and only retained in live cells, producing green fluorescence on excitation. Ethidium homodimer-1 is excluded by the intact plasma membrane of live cells. However, EthD-1 readily enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding nucleic acids, producing a strong red fluorescence in nonviable cells. Detection of the presence of these fluorochromes is best accomplished through the use of CLM. Activity of LDH has also been used as an indicator of cell viability.14–16 Lactate dehydrogenase is an enzyme that catalyzes the reaction of pyruvate and nicotinamide-adenine dinucleotide to form lactate and nicotinamide-adenine dinucleotide. Viable chondrocytes will actively metabolize the substrate and can be identified by the presence of blue formazan granules in their cellular cytoplasm generated by the reaction, whereas devitalized cells are not able to catalyze the reaction and therefore lack the presence of these granules.14–16
Two types of cell death exist, necrosis and apoptosis. Necrosis is the death of cells through accidental or toxic insult that results in a passive catabolic process.11 Apoptosis is an active process producing programmed cell death, a mechanism that regulates cell numbers in tissues and eliminates cells that threaten the survival of an animal.17–23 Detection of cells undergoing apoptosis can be achieved through the use of modified nucleotides and enzymes to label DNA fragments. Enzymes will bind to 3′-OH termini of broken strands through the use of a modified nucleotide such as X-dUTP. Terminal deoxynucleotidyl transferase is then incorporated to label the blunt ends of the DNA fragment. This end process is called TUNEL.19,24–27 The TUNEL method for detection of apoptosis has been widely used.18,21,25,26,28 The TUNEL method is often used in conjunction with electron microscopy to verify the structural changes of the TUNEL-positive cells, and TUNEL has been found to be an accurate assessment of cells undergoing apoptosis.11
Some controversy exists regarding the application of calcein AM and EthD-1 accompanied by CLM to determine chondrocyte viability in cartilage explants,29 despite its use and presentation in peer-reviewed work.10,12,13 The purpose of the study reported here was to compare vital cell staining of chondrocytes with 2 accepted methods for determining cell viability in tissues by use of an articular cartilage thermal injury model. In addition, we wished to determine the contribution of apoptosis to the loss of chondrocytes over time. We hypothesized that CLM and LDH techniques would provide similar results and that apoptosis would contribute to chondrocyte loss over the first 7 days of culture.
Calcein acetoxymethyl ester
Confocal laser microscopy
Terminal deoxynucleotidyl transferase
TdT-mediated X-dUTP nick end labeling
Sams AE, The Andrew Sams Clinic, Mill Valley, Calif: Personal communication, 2002.
Honnas CM, College of Veterinary Medicine, Texas A&M University, College Station, Tex: Personal communication, 2002.
SASCO Sprague Dawley rats, colony K62, Charles River Laboratories Inc, Wilmington, Mass.
Beuthanasia-D Special, Schering-Plough Animal Health Corp, Kenilworth, NJ.
Vulcan EAS, Smith & Nephew Endoscopy, Andover, Mass.
Whittaker HL-1 serum-free culture media, Bio-Whittaker, Walkersville, Me.
Gentocin, Sigma Chemical Co, St Louis, Mo.
Gryphon diamond band saw model C-40, Gryphon Corp, Sylmar, Calif.
MRC-1024, LSCM, Bio-Rad Laboratories, Hercules, Calif.
NIH Image, National Institutes of Health, Bethesda, Md.
TACS 2 TdT-DAB in situ apoptosis detection kit, Trevigen Inc, Gaithersburg, Md.
Nikon Eclipse E600, Nikon, Melville, NY.
Sony DSP 3CCD ExWave HAD, Sony Corp, Tokyo, Japan.
Nikon Plan Fluor, 10X/0/0.30 DIC L, Nikon, Melville, NY.
SAS Windows, version 9.1.2, SAS Institute Inc, Cary, NC.
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