Avaliação do potencial de leucócitos marcados com 99mTC-ECD na identificação de processo inflamatório na pele íntegra de membros posteriores isquêmicos em modelo experimental de Diabetes tipo 2

Abstract

Diabetes mellitus (DM) is a chronic disease considered worldwide epidemic and associated with numerous complications. One of the vascular complications, Peripheral Arterial Disease (PAD), is the narrowing of the lumen of lower arteries and consequent loos of blood supply. Ischemia stimulates vascular and inflammatory response both in the limb muscle and skin, which can lead to ulcers and possible progress to amputation of the affected limb. The objective of this study was to evaluate the potential of 99mTc-ECD-labeled leukocytes in the identification of inflammatory processes in the skin of ischemic hindlimbs in a type 2 DM model induced by a hyperlipid diet. C57BL/6 mice (n = 45) 8 weeks old were divided into two groups: diabetic (D) (n = 25) and non-diabetic (ND) (n = 25 ou 20??). Group D received a hyperlipid diet containing 60% of fat, while the ND group received standard diet. After 12 weeks of diet, insulin sensitivity and glucose tolerance tests were performed, and the posterior limb ischemia was induced by permanent unilateral occlusion of the femoral artery (OAF) and confirmed by laser Doppler perfusion imaging. Ischemia was maintained throughout the experimental period, with a slight increase in perfusion on day 3 (p <0.05). The OAF stimulated the expression of HIF1 in the skin of ischemic hindlimbs and this expression was reduced in diabetic animals compared to nondiabetic (p <0.01). The animals of group D presented insulin resistance and glucose intolerance, as well as obesity (observed by exacerbated weight gain and greater adiposity when compared to the ND control group). In diabetic animals, higher total leukocyte was observed in peripheral blood (p 0.001 vs. ND before OAF). There was no number of circulating leukocytes 1 day after OAF (p <0.001 vs. ND before OAF), which was maintained until the 3rd day. Initially, we evaluated an inflammatory activity in the intact skin of hind limbs 1 and 3 days after ischemia induction. In the skin of the ischemic limbs of diabetic animals, higher levels of the proinflammatory cytokine TNF (assessed by ELISA) were observed 1 day after an OAF (p <0.05 vs. ND). The content of cutaneous neutrophils and macrophages was evaluated indirectly by measurement of enzyme activity MPO and NAG, respectively. Non-diabetic animals showed significant non-infiltrated cutaneous neutrophils only on day 3 after OAF (p <0.05 vs. ND 1 day). The diabetic animals, however, presented less neutrophilic infiltrate both on 1st (p <0.05 vs. ND) and no 3rd (p <0.001 vs. ND) day after an OAF. In contrast, the content of macrophages in the skin remained unchanged in all groups throughout the experimental period. For an evaluation of the diagnostic potential of radiolabeled leukocytes, total leukocytes were isolated from the peripheral blood, labeled with 99mTc-ECD and injected into the tail vein of the animals 1 or 3 days after an OAF. On day 1, there was lower uptake of radioactivity without ischemic limbs of both diabetic (p <0.05) and non-diabetic animals (p <0.01), whereas on day 3, in the ischemic limbs of both groups (p <0.01). Interestingly and corroborating the data of neutrophil inflammatory infiltrate, the uptake of radioactivity was lower in the skin of the diabetic animals, both at 1st (p <0.05 vs. ND), and 3rd (p <0.01 vs. ND ) days after an OAF. Thus, our data suggest the ability of radiolabeled leukocytes to identify the inflammatory processes in the skin of ischemic hindlimbs of obese mice with type 2 diabetes.