Cellular Response to in Vivo Overload of the Achilles Tendon in Young and Old Rats

Tendinopathy is a highly debilitating condition increasing in incidence with ageing. Treatment options are limited, mainly due to a poor understanding of disease development. Repetitive loading and accumulation of matrix damage are implicated in tendinopathy pathogenesis 1–2 , however, the initial events leading to matrix damage accumulation, before the initiation of pain and clinical symptoms, remain unclear, particularly in aged tendons. Here, we use a previously evidenced model of in vivo cyclical loading of rat Achilles tendon (AT) to interrogate the initial subclinical cellular response to overload in aged tendons contrasting it to that of young tendons. The AT of young (24 weeks) and old rats (86 weeks) was subjected to a single bout of overload through controlled dorsiflexion of the ankle joint (10,000 cycles, 3Hz) 3 . The AT was subsequently collected either immediately following overload, or at 48 h, one week, or four weeks post-loading along with the contralateral control AT. Signalling events post-loading were investigated using qPCR and immunohistochemistry. Gait analysis was also performed before overload and 48 h, one, two, three, and four weeks post-loading to assess the presence of pain post-overload. Gait analysis revealed no gait alterations following a single bout of overload, confirming absence of pain following the delivered overload. Baseline gene expression between young and old control ATs did not differ except for of Mmp2 which was significantly lower in old ATs. Overload in young tendons was followed by a temporal downregulation in the gene expression of inflammatory mediators Il8 (p=0.082) and Il33 (p=0.037), and upregulation and downregulation, respectively, in macrophage markers Cd86 (p=0.015) and Cd163 (p=0.054). Aged tendons showed greater inter-animal variability and an absence of Cd86 gene expression upregulation following overload in contrast to young tendons. Immunohistochemistry of inflammatory mediator IL6 revealed staining specific to the vasculature of the inter-subtendon matrix of the ATs. IL6 staining post-loading showed an age-specific temporal response with delayed upregulation in aged tendons compared to young (p=0.037). Chronic low-grade inflammation termed “inflamm-aging” was not noted in the gene expression of ATs from old rats. Instead, the blunted response of Cd86 to overload in aged rats (fold change young/old 1.56) and the delayed upregulation in IL6 abundance points to a less robust response to the loading, which may restrict rapid repair. Further exploration of early overload-induced signalling events is of great importance to understand tendinopathy aetiopathogenesis and in particular the cellular response of aged tendons to overload and age-related tendinopathy.