Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Augusto Ministro, Paula de Oliveira, Raquel J. Nunes, Andre dos Santos Rocha, Tiago Ferreira, J. Goyri-O'Neill, Susana Constantino Rosa Santos

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
JournalJournal of Visualized Experiments
Issue number148
DOIs
Publication statusPublished - Jun 2019

Keywords

  • Medicine
  • Issue 148
  • Angiogenesis
  • Therapeutic Angiogenesis
  • Critical Limb Ischemia
  • Peripheral Arterial Disease
  • Murine Hindlimb Ischemia model
  • CLI

Cite this

Ministro, Augusto ; de Oliveira, Paula ; Nunes, Raquel J. ; Rocha, Andre dos Santos ; Ferreira, Tiago ; Goyri-O'Neill, J. ; Rosa Santos, Susana Constantino. / Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia. In: Journal of Visualized Experiments. 2019 ; No. 148.
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abstract = "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.",
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Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia. / Ministro, Augusto; de Oliveira, Paula; Nunes, Raquel J.; Rocha, Andre dos Santos; Ferreira, Tiago; Goyri-O'Neill, J.; Rosa Santos, Susana Constantino.

In: Journal of Visualized Experiments, No. 148, 06.2019.

Research output: Contribution to journalArticle

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T1 - Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

AU - Ministro, Augusto

AU - de Oliveira, Paula

AU - Nunes, Raquel J.

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AU - Ferreira, Tiago

AU - Goyri-O'Neill, J.

AU - Rosa Santos, Susana Constantino

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