D. Kulkarni1, P. Massie1, B. Coffman3, C. Pace1, N. Kanagy2, R. Clark1,2 1University Of New Mexico HSC, Surgery, Albuquerque, NM, USA 2University Of New Mexico HSC, Cell Biology And Physiology, Albuquerque, NM, USA 3Vetern’s Affairs Hospital, Pathology, Albuquerque, NM, USA
Introduction:
Peripheral arterial disease (PAD) affects millions of Americans and is responsible for significant detriments to mobility, with major limb loss possible at advanced stages. Ischemic myopathy associated with PAD is a recently recognized effect of chronic tissue malperfusion. Advanced age, characterized by defects in the sirtuin (SIRT1) histone deacetylase pathway, has been linked to impaired angiogenesis after ischemic insult. Hydrogen sulfide (H2S) has also recently been described as a key mediator of ischemic tissue recovery and neovascularization. Here we describe the development of reproducible ischemic myopathy in young mice as a comparison for mechanistic studies of tissue recovery in advanced age.
Methods:
Hindlimb ischemia was created in young (9-12 week) mice (C57Bl/6, n=9) under anesthesia. A 1 cm groin incision was created to ligate the femoral/external iliac artery. Animals were serially followed over a 21-day period by laser speckle contrast imaging (LSCI) and planimetric photography. After humane sacrifice, ischemic and contralateral non-ischemic gastrocnemius muscles were collected for histologic and molecular analysis. CD31 immunohistochemistry was performed to evaluate angiogenesis. RT-PCR investigated changes in H2S synthetic enzyme expression as well as age-related markers.
Results:
Mean pre-operative limb perfusion was 86.1 perfusion units (PU). After arterial ligation, flux was reduced to mean 40.1 PU, a 46% decrease from pre-operative values and mean 35% contralateral control limb perfusion. During the post-operative period, reperfusion was observed to increase predictably by post-operative day 21, achieving mean 91% baseline compared to the contralateral control. Histologic studies of myofiber diameter show a mean of 11.2 µm for the ischemic hindlimb and a mean diameter of 19.7µm for control muscle (P<0.01). CD31 immunohistochemistry reveals evidence of neovascularization
Conclusion:
Hindlimb ischemia secondary to arterial occlusion in young mice leads to reproducible perfusion deficits with subsequent tissue recovery. Histological changes consistent with ischemic myopathy and impairment in myofiber diameter mirrors the degree of ischemic injury. These findings have provided a comparison group whereby we can evaluate the effects of arterial occlusion on aging mice as well as the role of H2S and its interplay with SIRT1.