Chondrocyte Apoptosis Correlated with Progression of Osteoarthritis in Dmm Mice

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Ryan Anderson and Dr. Robert E Seegmiller, Physiology and Developmental Biology

Osteoarthritis (OA) is a progressive disease resulting in the degeneration of synovial joint cartilage and is the leading cause of chronic disability in the elderly1. Despite the prevalence of this disease, its pathogenesis is poorly understood. There is need for low cost, short generation animal model to study this disease. For this, we used a proven mouse model as the means for researching OA, the Disproportionate micromellia (Dmm) mouse which has been shown to develop early onset OA. One mechanism involved in OA that we are specifically researching is programmed cell death, apoptosis, of cartilage cells, chondrocytes and how it is directly correlated to the progression of OA. Mason et al reported apoptosis to be linearly correlated with the severity of osteoarthritis in the STR/ort mouse model using the terminal deoxynucleotidal transferase biotin-dUTP nick end labeling (TUNEL) assay2. An increase in apoptotic cells compared with normal cells has also been demonstrated in human osteoarthritis also using TUNEL3. Our study has involved a relatively new technique of confirming the presence of apoptotic cells in chondrocytes, In Situ Oligoligation (ISOL).

Recently, the accuracy of the TUNEL assay has been questioned with the suggestion that it may report elevated concentrations of apoptotic cells due to false positives arising from bone autofluorescence4. To confirm our preliminary findings are representative of a genuine correlation between increasing apoptosis and severity of osteoarthritis, we performed another assays. We planned to repeat our analysis and expand upon our preliminary data using a Peroxidase In Situ Oligo Ligation (ISOL) assay, a newer method of detecting apoptosis that reports decreased levels of false positives compared to TUNEL 5.

Apoptosis, programmed cell death, is characterized by breakdown of the DNA backbone leading to DNA fragmentation. Apoptosis derived DNA fragmentation tends to result in a concentration of blunt end and one base overhang breaks in the strands. ISOL discriminately ligates a synthetic oligonucletide to these fragments, linked to a streptavidin conjugate that functions as a reporter and labeled with biotin. The conjugate is reacted with peroxidase causing the apoptotic DNA to fall out of solution which can then be seen using bright-field microscopy as brown spots against a methyl green colored background of the bone.

Our study included a total of four, 6 months old mice consisting of 2 wild type (+/+) and 2 Dmm (D/+) mice. Knees were fixed in 10% buffered formalin (NBF), decalcified with 14% EDTA, dehydrated, and then embedded in paraffin wax. On two of the blocks, samples were oriented in the blocks so that the tibia is parallel with the block face with the femur facing back. The two other tissues were oriented in the blocks with the medial side of the fibula, knee and femor on the block face. The tissues were sectioned in the lab 6 micrometers (µm) thick using a Heidelberg microtome and mounted on slides treated with saline. In the last step, sections were stained according to the ApopTag® Peroxidase In Situ Oligo Ligation (ISOL) kit S7200 protocol (Chemicon International, Tramecula, CA) that they may be visible under a light microscope.

Assays were conducted and tissues were evaluated using light microscopy in April and then in December of 2007. A total of 4 wild type mice and 4 Dmm mice were used between the two studies. In April, the first 4 tissues were prepared strictly using our protocol from our lab and Dr. Seegmiller’s help. We followed the protocol strictly, but to our disappointment, the sectioning of the tissues was not done very well on my part. The tissues were wrinkled which made it very difficult to orient ourselves on the slide and distinguish between the different tissue types that were on the slide. The staining of the slides was also something that was unexpected though not quite due to operator error like the sectioning. The tissues were stained and revealed that there was little or no apoptosis occurring within the knee joints. No cells were identified as apoptotic on the wild type knee joints. Very few cells were identified as apoptotic on the Dmm mice which do have osteoarthritis. Apoptotic cells were identified in the bone marrow within the tissues of both Dmm mice knee sections.

This Fall 2007 semester, we were able to conduct the same assay again, now using 6 month old mice again in order to confirm or disavow our previous results. In order to make sure that the tissues were sectioned correctly, we had them sectioned in a lab at Utah Valley Regional Medical Center were one my fellow lab members works. The tissues came back sectioned and were done correctly, easily allowing us to orient ourselves on the slides. We used the ISOL protocol once again to assay for apoptotic cells and came up with the same results as the previous time back in the spring.

Our hypothesis, increasing apoptosis is correlated with severity of osteoarthritis in (Dmm) mice, is not confirmed using this latest ISOL protocol. Several possible reasons why this might happen were found. First of all, the tissues were decalcified unlike heart or mammary tissue were ISOL had been used to detect Apoptosis suggesting the 14% EDTA or other decalcifiers which could alter the DNA in a way that does not allow the label to attach any DNA5. Another possibility is that apoptosis is really not a principal player in the progression of apoptosis and that other cell death processes are actually occurring. Necrosis could be an option or even a relatively new process called Entosis which involves one cell engulfing another to kill it might be an option6. A final conclusion from this project is that apoptosis occurs at earlier or later than 6 months of age in Dmm mice. In the future, we will assay younger mice to see if apoptosis occurs at an earlier stage in life to actually start the process of osteoarthritic degradation as opposed to being the mechanism of its progression.

References

  • Issa SN (2006) Epidemiology of osteoarthritis: an update. Curr Rheumatol and Rep 8:7-15.
  • Mason R, Chambers M, Flannelly J, Gaffen J, Dudhia J, Bayliss M (2001) The STR/ort mouse and its use as a model of osteoarthritis. Osteoarthr Cartil 9:85-91.
  • Blanco FJ, Guitian R, Vázquez-Martul E, DeToro FJ, Galdo F (1998) Osteoarthritis chondrocytes die by apoptosis. Arthr Rheum 41:284-289.
  • Aigner T (2003) Chondrocyte apoptosis in osteoarthritis: Comment on the letter by Kouri and Abbud Losoya. Arthritis and Rheumatism 48:1166-1167.
  • Lesauskaite V, Epistolato M, Ivanoviene L, Tanganelli P (2004) Apoptosis of cardiomyocytes in explanted and transplanted hearts. Am J Clin Pathol 121:108-116.
  • Overholtzter, M. et al. (2007). A Nonapoptotic Cell Death Process: Entosis, that Occurs by cell to cell invasion. Cell 131:5.