Comparison of techniques for determination of chondrocyte viability after thermal injury.
OBJECTIVE
To compare 2 methods of quantitating chondrocyte viability and to determine chondrocyte response to thermal injury over time.


METHODS
108 stifle joints from 54 adult rats.


METHODS
Cartilage from the distal aspect of the femur was treated ex vivo with radiofrequency energy at a probe setting that would result in immediate partial-thickness chondrocyte death; untreated sections served as controls. Explants were cultured, and cell viability was compared by use of lactate dehydrogenase (LDH) histochemical staining and calcein AM and ethidium homodimer-1 confocal laser microscopy (CLM) cell viability staining. Terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling (TUNEL) was used to detect apoptosis. All labeling studies were performed 0, 1, 3, 7, 14, and 21 days after treatment.


RESULTS
In the treated tissues, a greater percentage of viable cells were found with CLM, compared with LDH staining. This result contrasted that of control tissues in which LDH staining indicated a greater percentage of live cells than CLM. The greatest number of TUNEL-positive chondrocytes was present at day 3, declining at later time intervals.


CONCLUSIONS
CLM and LDH histochemistry techniques yield different absolute numbers of live and dead cells, resulting in differing percentages of live or dead cells with each technique. These differences may be related to the enzymes responsible for activation in each technique and the susceptibility of these enzymes to thermal injury. Results of TUNEL indicate that apoptosis contributes to chondrocyte death after thermal injury, with a peak signal identified 3 days after insult.
Rauch
Beth
B
Comparative Orthopaedic Research Laboratory, Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 53706-1102, USA.
Edwards
Ryland B
RB
Lu
Yan
Y
Hao
Zhengling
Z
Muir
Peter
P
Markel
Mark D
MD
D000818:Animals; D016923:Cell Death; D002470:Cell Survival; D019902:Chondrocytes / Q000166:cytology* / Q000473:pathology; D004996:Ethidium / Q000031:analogs & derivatives; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006358:Hot Temperature; D007770:L-Lactate Dehydrogenase; D008297:Male; D018613:Microscopy, Confocal; D051381:Rats; D013194:Staining and Labeling / Q000379:methods*
D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM; C018533:ethidium homodimer; D007770:L-Lactate Dehydrogenase; D004996:Ethidium