Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia.

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Therapeutic angiogenesis induced by human umbilical cord tissue-derived mesenchymal stromal cells in a murine model of hindlimb ischemia.

BACKGROUND: Mesenchymal stem cells derived from human umbilical cord tissue, termed UCX®, have the potential to promote a full range of events leading to tissue regeneration and homeostasis. The main goal of this work was to investigate UCX® action in experimentally induced hindlimb ischemia (HLI). METHODS: UCX®, obtained by using a proprietary technology developed by ECBio (Amadora, Portugal), were delivered via intramuscular injection to C57BL/6 females after unilateral HLI induction. Perfusion recovery, capillary and collateral density increase were evaluated by laser doppler, CD31 immunohistochemistry and diaphonisation, respectively. The activation state of endothelial cells (ECs) was analysed after EC isolation by laser capture microdissection microscopy followed by RNA extraction, cDNA synthesis and quantitative RT-PCR analysis. The UCX®-conditioned medium was analysed on Gallios flow cytometer. The capacity of UCX® in promoting tubulogenesis and EC migration was assessed by matrigel tubule formation and wound-healing assay, respectively. RESULTS: We demonstrated that UCX® enhance angiogenesis in vitro via a paracrine effect. Importantly, after HLI induction, UCX® improve blood perfusion by stimulating angiogenesis and arteriogenesis. This is achieved through a new mechanism in which durable and simultaneous upregulation of transforming growth factor ß2, angiopoietin 2, fibroblast growth factor 2, and hepatocyte growth factor, in endothelial cells is induced by UCX®. CONCLUSIONS: In conclusion, our data demonstrate that UCX® improve the angiogenic potency of endothelial cells in the murine ischemic limb suggesting the potential of UCX® as a new therapeutic tool for critical limb ischemia.

Ultrastructural aspects of the microvasculature of the cochlea: the internal spiral network.

We performed a study of the microvasculature of the cochlea with scanning electron microscopy on corrosion casts in the guinea pig. This study was performed in 140 cochleas from healthy adult guinea pigs. Different microvascular techniques were used, including injection-microdissection in 53 cases, injection-diaphanization in 27 cases, a histologic technique in 34 cases, injection-microdissection-diaphanization in 32 cases, and scanning electron microscopy on corrosion casts in 14 cases. The internal radiating arterioles branch off as collateral branches of the spiral modiolar artery, as first-order collateral branches. We detail the morphology, caliber, trajectory, and collateral branches. Among their collateral branches, are third-order arterioles, the arterioles to the spiral ganglia, and the arterioles of the tympanic lip. The arterioles of the tympanic lip form, through their anastomosis, a rich capillary network at the edge of the spiral lamina, called the internal spiral network. We emphasize the glomeruli of Schwalbe, which arise near the scala tympani as third-order arterioles with a medium caliber of 14 microm. The upper glomeruli, situated in the bony wall, and the lower glomeruli, situated in the width of the osseous spiral lamina, form vascular loops made of anastomosed capillaries with a caliber of less than 10 microm. These loops play an important role as efficient devices, or "relay stations," for regulation of cochlear blood flow. The comparison of results obtained with each technique gave the perspective of the cochlear microvasculature with great accuracy.

[Diaphanization: modifications of organ weight and volume]. / La diaphanisation: modifications du poids et du volume des organes.

The diaphanization of organs previously injected with coloured substances is an excellent auxiliary method of numerous morphological studies. The authors studied 52 organs, among 15 were taken from human beings and 37 from dogs, with the aim of knowing theirs weight and volume modifications after diaphanization. The organs were diaphanizated with several impregnations in 10% formaldehyde, perydrol, acetone and at least embedding in a mixture of methylsalicylate and benzyl benzoate by vacuum process. After each embedding, weight and volume of each organ were measured. The variations that the organs suffered were statistically considered, under a few aspects: 1-global organs' variations after each embedding 2-different organs' variations 3-comparison between human being and experimental animals. In all cases, the authors noticed a great decrease of weight and volume, especially after dehydration steps with acetone and clearing with the mixture of methylsalicylate and benzyl benzoate. We also didn't find any significant difference between human organs and those taken from dogs.

[Various aspects of the microvascularization of the thyroid follicles in the rabbit]. / Quelques aspects de la microvascularisation des follicules thyroidïens chez le lapin.

The authors, use histology and diaphanization after the injection of Micropaque 25% with gelatin 10% in 35 rabbits, 25 female and 10 male, with race, weight and age variable, to show some aspects of the thyroid follicles microvascularization. In this experimental animal we observe a follicle vascularization like the one described by other authors.