A study of neovascularization in the rat ischemic hindlimb using Araldite casting and Spalteholtz tissue clearing

Abstract

Introduction: Understanding neovascularization is an important prerequisite for therapeutic advances aimed at the salvation of ischemic tissues. We explored an alternative strategy to corrosion casting for visualizing neovascularization in a rat hindlimb ischemia model. Methods: Rats were subjected to hindlimb ischemia by femoral artery ligation. Directly after femoral artery occlusion and at 7, 14, 21 and 28 days postsurgery, rats were sacrificed, and neovascularization was evaluated by vascular casting with Araldite plastic and subsequent Spalteholtz tissue clearing. Results: Semitransparent preparations were obtained, in which the casted arteries could be directly and three-dimensionally visualized in detail and in relation to the surrounding tissue. In the vascular casts, collateral formation and recanalization of previously thrombosed arteries were demonstrated. Conclusions: We describe an alternative approach to study neovascularization in animal models. This method, which combines Araldite plastic vascular casting with Spalteholtz tissue clearing, preserves all vasculature as well as the surrounding tissue. In a small time series in rat ischemic hindlimbs, we show that restoration of blood flow after ischemia not only involves newly formed collaterals, but also recanalization of previously thrombosed arterial segments. D

MR Angiography, MR Imaging and Proton MR Spectroscopy In-Vivo Assessment of Skeletal Muscle Ischemia in Diabetic Rats

“…Before surgery, rats were anesthetized with a mixture of oxygen and halothane (2.5%), sodium pentobarbital (intra-peritoneally, i.p., 50 mg/Kg) and sodium heparin (1000 U/Kg). The common right iliac-femoral artery was ligated according to reported methods [2], [24]. The overlying skin was closed.…”

Nitric Oxide-Dependent Neovascularization Role in the Lower Extremity Disease

“…Under these conditions animals have a noticeable limping in the ischemic limb immediately after the surgery; however, after one to two weeks, animals exhibit mild limping or functionally normal limb [14]. We visualized the lower limb vasculature by vascular casting with araldite plastic [15] followed by the lower limb blood filling after femoral extirpation at the following times points: prior to artery resection, immediately after artery resection, and 3 and 14 days after femoral artery resection. In the control rat, the normal arterial distribution to the lower limb was observed (Fig.…”

Casting Materials and their Application in Research and Teaching

“…9) with the observation that collateral vessel formation and recanalization of previously thrombosed arteries occurred (Westerweel et al, 2005) to restore blood circulation in the limb. The latter authors combined epoxy resin casting with the Spalteholz technique, which entails dehydrating tissues in ascending concentrations of ethanol and clearing with methyl benzoate (Spalteholtz, 1914; Westerweel et al, 2005).

Figure 9 Series of araldite preparations photographed from the ventral side, made in a control rat ( a ) and after induction of ischemia by ligation of the left femoral artery.

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“…Epoxy resin (aradite) was used to study ischemia and neovascularization in the rat hind limb (Fig. 9) with the observation that collateral vessel formation and recanalization of previously thrombosed arteries occurred (Westerweel et al, 2005) to restore blood circulation in the limb. The latter authors combined epoxy resin casting with the Spalteholz technique, which entails dehydrating tissues in ascending concentrations of ethanol and clearing with methyl benzoate (Spalteholtz, 1914; Westerweel et al, 2005).…”

“…Total recovery has taken place after 21 days ( d ). Modified from Westerweel et al (2005), reproduced with permission from Cardiovascular Pathology , 14, 294–297. Copyright © 2011 Elsevier Limited.…”