We used a non-surgical, minimally invasive technique to perform balloon injury in rabbit thoracic aortas by percutaneous catheterization of the auricular artery. In experimental restenosis models, catheterizations are traditionally performed after surgical cutdown of the femoral or carotid arteries and followed by subsequent balloon injury in femoral, iliac, coronary, carotid arteries and abdominal or thoracic aorta, respectively. The peripheral vessels of the rabbit are fragile, while surgical cut-down and catheterization are associated with long procedural time periods and deep general anesthesia with intubation . Moreover, a surgically accessed vessel is finally ligated, which frequently renders it vulnerable to iatrogenic trauma or even thrombosis. On the contrary, our alternative catheterization method is simple, rapid, safe and easily reproducible, as it is takes advantage of favourable auricular vascular anatomy of the rabbit. The major benefits of transauricular catheterization technique comprise the acceleration of endovascular access, the avoidance of surgical wounds, and, significantly, the preservation of valuable femoral and cervical vessels. In addition, animals experience less pain, bleeding complications, wound infections, while dissociative anaesthesia is sufficient.
Generally, the technique offers the advantages of percutaneous minimally invasive procedures, characterized by reduced morbidity and mortality as well as minimal distortion of normal anatomy and physiology. A minor disadvantage of transauricular catheterization method constitutes the peripheral auricular artery impairment that could be attributed to the cutting of the dermis along the course of the guide wire and further multiple dilatations so as to be achieved the insertion of sheaths.
In our model of induced atherosclerosis, angioplasty –induced atherosclerotic lesions appeared to be significantly more severe and advanced (type V) compared to spontaneous (type III or type IV) lesions, confirming studies with balloon injury through other approaches. It has been noticed that in the absence of hypercholesterolemia, angioplasty- induced lesions regress and resolve spontaneously , while, in the presence of hypercholesterolemia these lesions not only sustain but progress to larger lesions. Also, plaque size at sites of spontaneous lesions has been reported to increase with increasing blood cholesterol levels . Similarly, in our study, both spontaneous and angioplasty induced atherosclerotic lesions revealed significant intimal hyperplasia in uninjured and balloon injured rabbits respectively, after 9 weeks of 4% high cholesterol diet.
Another difference in pathogenesis between spontaneous and angioplasty-induced atherosclerotic lesions consists in cell type population. Especially, SMCs have been found to be abundant in the intima of balloon- injured aortas, whereas foam cell-derived macrophages have been recognized as the predominant cell type in the intima of undamaged aortas . In fact, we demonstrated that the percentage of macrophages (RAM-11 positive cells) revealed no difference between spontaneous and angioplasty- induced lesions and was in accordance with the histological stage of the respective lesions. Whereas, the percentage of SMCs (HHF-35 positive cells) revealed a statistically significant increase in angioplasty-induced lesions compared to spontaneous lesions of uninjured aortas, similarly to previous studies [11, 12].
Additionally, angioplasty induced lesions demonstrated a significant increase in intima/media ratio compared to spontaneous atherosclerotic lesions, despite the similar degree of intimal hyperplasia. Furthermore, the increased intima/media ratio was accompanied with significant lumen deterioration and total vessel reduction in angioplasty induced lesions, reflecting the artery’s shrinkage or constrictive remodeling. Indeed, the accumulation of SMCs in combination with ECM reorganization that characterized by increased collagen deposition, contributes to intimal thickening and negative remodeling, leading in restenosis after balloon angioplasty.
It should be noticed the safety of both transauricular balloon angioplasty and atherogenic diet with regard to the induction and progression of atherosclerosis. Especially, we used a non-commercial and easy manufactured atherogenic diet consisting of standard rabbit chow enriched with 4% cholesterol , without any additional atherogenic components, such as palm oil , peanut oil , high fat coconut oil , high fat corn oil , and lard combined with either yolk powder or peanut oil [18, 19]. Interestingly, the normal renal and liver function of balloon injured hyperlipidaemic animals confirms the absence of complications with percutaneous transauricular angioplasty, suggesting a safe alternative non-commercial atherogenic diet together with a minimally invasive restenosis model.
Generally, hypercholesterolemia has been known to induce the development of atherosclerotic lesions both in humans and animal models, while the cellular basis for this action has been largely attributed to the formation of oxidized LDL (oxLDL) and oxidative injury to endothelium . It has been reported that the oxidative modification of LDL correlated with reduction of endothelial nitric oxide synthase (eNOS) expression  or modulation of eNOS activity and free radical bioavailability , resulting in peroxynitrite and 3′-nitrotyrosine formation. Interestingly, application of the transauricular balloon injury model did not further increase protein nitration observed in hyperchoresterolemic animals, suggesting that balloon angioplasty does not cause peroxynitrite-mediated oxidative stress.