APOPTOSIS – THE CAUSE OF ACHILLES TENDINOSIS?

Abstract

Introduction: The pathogenesis of chronic tendinopathy is unclear. The role of the increased apoptosis of tenocytes has been suggested by high intratendinous levels of glutamate being demonstrated in patients with tendinosis. Nitric oxide is a known mediator of apoptosis and nitric oxide synthase (NOS) isoforms have been shown to be upregulated in rotator cuff tendons as a result of chronic overuse. We found, the same upregulation of NOS in the Achilles tendon in non-insertional Achilles tendinopathy in a previous study.

The purpose of this study was to investigate whether apoptotic cells were present in these tissues with raised eNOS and iNOS levels.

Methods: Samples were obtained from the Achilles Tendons of patients with in non-insertional Achilles tendinopathy who had failed conservative treatment for at least six months and were undergoing a surgical procedure. Consent was obtained preoperatively from all patients and ethical approval was granted by the research ethics committee.

Several biopsies were taken of the visibly abnormal tendon tissue. Control samples were taken from macroscopically normal tendon correlating with areas of normal tissue on MRI.

Standard immunohistochemical techniques were used to identify the expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS).

Apoptotic cells were identified using terminal deoxynucleotidyl transferase-mediated dUTP neck end labelling (TUNEL reaction) with TdT-FragEL and the demonstration of Caspase-3 activation.

A power calculation was performed which showed that 14 patients in each group would be required to show a 50% difference between the two groups using a level of significance of 5%.

Results: Significant differences were found between the diseased tendon and the controls for all of the parameters measured. The mean Caspase-3 cell count for diseased tendon was 51.9 compared to 28.3 for the controls (p=0.000001). The mean TUNEL cell count for diseased tendon was 24.1 compared to 14.8 (p=0.00014). iNOS densitometry revealed a mean of 26.1 for the diseased tissue verses 15.0 for the controls (p=0.000009) and the values for eNOS were 48.3 and 23.7 respectively (p=0.015).

Conclusions: Apoptosis clearly plays a role in the development of non-insertional Achilles tendinopathy and appears to be related to the presence of raised eNOS and iNOS levels.

It is possible that, by blocking the apoptotic pathway, the tendinopathic process could be halted. This may lead to the development of treatments strategies for early Achilles tendinopathy.