Comparative study of functional and structural muscle changes in peripheral artery disease: rubidium-82 positron emission tomography and histological correlation
Authors: Alashi A , Vermillion B , Callegari S , Burns R , Guo L , Moulton E , Guerrera N , Depino A , Papademetris X , Zeiss C J , Thorn S L , Liu C , Sinusas A J 
Published: June 2024

Background

Elucidating the functional and structural alterations in skeletal muscle perfusion and perfusion reserve is pivotal for the effective management of peripheral artery disease (PAD). This investigation correlates functional imaging metrics with structural histological modifications in a rabbit hindlimb ischemia model (HLI).

Methods

HLI was created in rabbits (n=8) by right femoral ligations and Rb-82 PET/CT imaging performed on digital PET scanner (Vision600, Siemens) at either 2 or 4 weeks post-ligation. Rabbits were injected with Rb-82 (1.5 mCi) with constant activity infusion over 30 sec at rest and 30 sec following reactive hyperemia (RH) induced by 3 min cuff occlusion of thighs bilaterally. PET images were acquired with shuttling scanner from heart to feet using 8 passes over 7 min. Summed images were created excluding the first 90 sec post injection. Calf muscles were segmented based on CT images. Mean Rb-82 standardized uptake values (SUVs) were calculated for calf muscles - gastrocnemius (GC), soleus (SOL) for both ischemic (IS) and non-ischemic (NI) limbs. CT angiography was performed, and tissues analyzed for fiber composition using Fast myosin (FM) that stains type 2 muscle fibers, collagen area percentages as an indicator of fibrosis using Mason trichrome (MT), and capillary densities using CD31 in the two distinct classical oxidative Type 1 (SOL) and glycolytic Type 2 (GC) muscles.

Results

PET imaging demonstrated differential perfusion and perfusion reserve among calf muscles (Fig. 1A, 1B). While no marked difference was observed between GC or SOL (IS vs. NI) in either rest or stress conditions, the SOL exhibited a higher Rb-82 uptake than GC during both conditions (P&lt;0.05). IS to NI uptake ratios during RH were lower than at rest, suggesting RH may identify subtle perfusion deficits in IS limbs (P&lt;0.05). Histologically, both GC and SOL showed an increase in capillary densities in IS limb (GC: NI 1.8 ± 0.3, IS 2.2 ± 0.2 , p=0.01; SOL: NI 2.7 ± 0.2, IS 3.1 ± 0.4 , p=0.03), while SOL had a significantly higher capillary density compared to GC (p&lt;0.05). FM staining revealed consistent Type 2 myofiber prevalence in GC across IS and NI conditions (NI 84.6% ± 10.2%, IS 85.0% ± 9.3%, ns) with minimal Type 2 staining in SOL (NI 2.1% ± 4.1%, IS 1.9% ± 5.2%, ns); SOL vs GC (P&lt;0.01). MT staining indicated stable collagen levels (GC NI 1.2% ± 0.6%, IS 1.3% ± 1.0%, ns; SOL: NI 2.7% ± 1.0%, IS 2.4% ± 0.7%, ns) (Fig. 1 C-E).

Conclusion

PET imaging of the lower extremities demonstrated increased perfusion in SOL versus GC in association with an increased capillary to muscle fiber ratio. A reduction in IS/NI perfusion ratio was not detected at rest although could be detected during RH. The integration of histological findings with skeletal muscle perfusion provides useful pathophysiological insight in our clinical relevant pre-clinical model of PAD, offering a valuable approach to evaluate treatment strategies in PAD.