Histologic evaluation of pathologic changes in gastrocnemius muscle in the model of rodent hindlimb ischemia

Aim. Development of a model of hindlimb ischemia in rats to assess the effectiveness of gene and cell therapy.

Methods. In the first stage of this approach the external iliac artery was ligated immediately after bifurcation of the common iliac artery. Then the femoral artery was ligated before its branching into the descending genicular, popliteal and saphenous arteries with following dissection of arteries between ligatures. The second stage of this approach was performed seven days after the completion of the first stage: branches of popliteal artery to gastrocnemius muscle and formed collaterals were ligated and dissected. Hindlimb blood flow was assessed by laser Doppler flowmetry. Animals were euthanazied 14, 17, 21, 28, 35, 42 days after the first stage. The paraffin sections of gastrocnemius muscles were stained with Mallory trichrome and with antibodies to CD34.

Results. Histopathological analysis showed a continuous hindlimb ischemia of operated limb without any native skeletal muscle structure restoration and with significant interstitial fibrosis (11.89±5.53% vs 2.55±2.13% in intact limb by day 42, p <0.05). Polymorphic muscle fibers in operated limb with significantly smaller diameter and reduced capillary density (0.82±0.03 vs 1.91±0.06 in intact limb by day 42, p <0.05) were revealed. Laser Doppler flowmetry revealed reduction of blood flow in the operated limb (0.67±0.22 of intact limb blood flow by day 42).

Conclusion. Our developed two-stage model of hindlimb ischemia in rats leads to histologic changes characteristic for the same pathology in human.