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Analyses of collateral vessel growth in a rat cell therapy model of ischemia hindlimb: a computer tomography based research about angiogenesis and arteriogenesis assessment.
840 Bio-Plex (bead-based immunoassay) procedures. The rate of protein synthesis was determined by averaging the daily rate of protein synthesis over a 3-day period. The production of genetically modified fibroblasts was standardised in order to fulfil the requirements of good manufacturing practice. The study-design is completed to the application of genetically modified fibroblasts to the animals and optimized according to current EMEA’s practice for preclinical evaluation. The results of in-vitro experiments have showed the stability of noneviral transfection process. Therefore, the in-vitro results correlate with effect of genetically modified fibroblasts in the rat hindlimb ischemia model. Evaluation of non-viral cell transfection and validation of check-up methods in compliance with current EMEA’s guidelines build stable basis for development of new technique to induce angiogenic reaction in ischemic tissue.
Analyses of collateral vessel growth in a rat cell therapy model of ischemia hindlimb: a computer tomography based research about angiogenesis and arteriogenesis assessment. ¨fera, J. Frenza, Z. Zhanga, W. Itoa, A. Slobodianskia, A. Katho b H.G. Machens a University Hospital of Schleswig-Holstein, Campus Lu¨beck, Lu¨beck, Germany, b Clinical Centre rechts der Isar, Technische Universitaet Muenchen, Germany Cell and gene based therapy are proved to be promising in angiogenesis and arteriogenesis research. In rat hindlimb ischemia models, angiography is the most used technique. However analysis of vessels growth is always limited by the flat image visualization. The micro computer tomography system makes three dimensional visualization and quantification of the new vascular net possible. In the present study, we developed several methods based on computer tomography technology for the assessment of angiogenesis and arteriogenesis in the rat hindlimb ischemia model. Hindlimb ischemia was induced by acute femoral artery ligation. Rats were divided in to two groups: (1) - gene therapy group: intramuscular injection of genetic modified fibroblasts. (2) - control group: intramuscular injection of non transfected fibroblasts.7 days after femoral artery ligation, rats were anesthetized, the aorta cannulated, the legs rinsed with saline and then filled with contrast medium. Animals were immediately placed on crushed ice. The legs were then fixed in 4% formalin and analyzed with a micro-CT (SkyScan).The collateral artery growth was visualized by a high resolution micro computer tomography system. After reconstruction of the original projections, the total number of the vessels crossing the ischemia zone and the corkscrew vessels were analyzed separately. The volume quantification data of the collateral growth zone were obtained by computer-assisted voxel measurement. Three kinds of analysis techniques employed were proved to be useful. An increase of the vessel number and volume value could be observed in all the experimental rats compare as to control. Results of computer tomography were approved by using of several other methods, which were established as an extension for monitoring of research data New medical techniques provide us better ways in the angiogenesis and arteriogenesis research. With the help of computer technology, our in vivo three dimensional vessel analyze techniques can be used for analysis of collateral growth in rat hindlimb models.
Hemodynamic changes related to arteriogenesis and angiogenesis in view of flap survival J. Plock, K. Merz, S. Schlosser, A. Banic, D. Erni Department of Hand and Plastic Surgery, Inselspital, University of Berne, Bern, Switzerland
Abstracts Department of Clinical Research, University of Berne, Bern, Switzerland Angiogenesis and arteriogenesis have been reported as two different processes of vascular regeneration aiming to encounter critical ischemia. We have recently established a model that allowed us to assess both morphological and hemodynamic changes in the microvasculature of a critically ischemic mouse skin flap over time. The purpose of this study was to elucidate the orchestration of vascular regeneration and hemodynamic changes in terms of a temporal profile in order to question which of the two e arteriogenesis or angiogenesis e may be more effective in improving microcirculation and survival in critically ischemic flaps. To this end, a dorsal skin flap was mounted into a window chamber in mice for both morphological and hemodynamic assessment. The flap model consisted of a well-vascularized proximal part and a collateralized distal part. Tissue partial oxygen tension was measured with Clark type microprobes. Ischemia-related changes in the oxidative energy metabolism were assessed with microdialysis. Morphological and microhemodynamic investigations were performed using intravital microscopy. In the distal part of the flap, partial oxygen tension was 5 2 mmHg, whereas it was 12 4 mmHg in the proximal part and 30 5 mmHg in the healthy abdominal skin at the first postoperative day. The severity of ischemic hypoxia in the distal flap tissue was confirmed by a 4.4-fold increase in lactate/pyruvate ratio. Glycerol, a marker of cell membrane desintegration, was not significantly elevated. During the first seven days morphological signs of arteriogenesis were detectable like arteriolar dilation (150% of baseline, p < 0.01), increase in relative arteriolar length (350% of baseline, p < 0.01) and arteriolar corkscrew formation. The microvascular changes related to angiogenesis, such as sprouting and capillary reorganization, were visible from the 7th day on only. Functional capillary density was significantly different between the proximal and the distal part of the flap at baseline (240 vs 143 cm/cm2, p < 0.01). In the collateralized part, functional capillary density was increased to 190% of the baseline value after the 10th day (p < 0.01). According to Poiseuille’s law, arteriogenesis resulted in a drastic reduction in peripheral vascular resistance that was already present very few days after surgery. Angiogenesis, on the other hand, resulted in relevant hemodynamic changes only after 10 days postoperatively. Therefore, our results suggest that arteriogenesis may be more effective in improving flap survival. Improved Regeneration of Autologous Nerve Transplants by Means of VEGF-Gene Therapy T. Holzbacha, R. Milojcica, M. Antonb, T. Brillb, M.A. Konerdingc, ¨nsbacherb, H.G. Machensa, R.E. Giuntaa B. Ga a Department of Plastic Surgery and Hand Surgery, Klinikum rechts der Isar, Technische Universita¨t Mu¨nchen, Munich, Germany b Institute for Experimental Oncology and Therapy Research, Klinikum rechts der Isar, Technische Universita¨t Mu¨nchen, Munich, Germany c Department of Anatomy, Johannes Gutenberg Universita¨t, Mainz, Germany The impact of the Vascular Endothelial Growth Factor (VEGF) on the angiogenic cascade is proven. Recently its neuroprotective effect after peripheral nerve injuries on a-motoneurons in the spinal cord was shown. The biological effect is mainly mediated by the binding of two tyrosine kinase receptors (VEGFR1&VEGFR2), but in addition an effect via Neuropilin (NP)-1 and NP-2, receptors essential for the development of the nervous system, was reported. Thus a sprouting of axons could be monitored as well as an improved survival of neurons and glial cells. Experiments on
Analyses of collateral vessel growth in a rat cell therapy model of ischemia hindlimb: a computer tomography based research about angiogenesis and arteriogenesis assessment. ¨fera, J. Frenza, Z. Zhanga, W. Itoa, A. Slobodianskia, A. Katho b H.G. Machens a University Hospital of Schleswig-Holstein, Campus Lu¨beck, Lu¨beck, Germany, b Clinical Centre rechts der Isar, Technische Universitaet Muenchen, Germany Cell and gene based therapy are proved to be promising in angiogenesis and arteriogenesis research. In rat hindlimb ischemia models, angiography is the most used technique. However analysis of vessels growth is always limited by the flat image visualization. The micro computer tomography system makes three dimensional visualization and quantification of the new vascular net possible. In the present study, we developed several methods based on computer tomography technology for the assessment of angiogenesis and arteriogenesis in the rat hindlimb ischemia model. Hindlimb ischemia was induced by acute femoral artery ligation. Rats were divided in to two groups: (1) - gene therapy group: intramuscular injection of genetic modified fibroblasts. (2) - control group: intramuscular injection of non transfected fibroblasts.7 days after femoral artery ligation, rats were anesthetized, the aorta cannulated, the legs rinsed with saline and then filled with contrast medium. Animals were immediately placed on crushed ice. The legs were then fixed in 4% formalin and analyzed with a micro-CT (SkyScan).The collateral artery growth was visualized by a high resolution micro computer tomography system. After reconstruction of the original projections, the total number of the vessels crossing the ischemia zone and the corkscrew vessels were analyzed separately. The volume quantification data of the collateral growth zone were obtained by computer-assisted voxel measurement. Three kinds of analysis techniques employed were proved to be useful. An increase of the vessel number and volume value could be observed in all the experimental rats compare as to control. Results of computer tomography were approved by using of several other methods, which were established as an extension for monitoring of research data New medical techniques provide us better ways in the angiogenesis and arteriogenesis research. With the help of computer technology, our in vivo three dimensional vessel analyze techniques can be used for analysis of collateral growth in rat hindlimb models.
Hemodynamic changes related to arteriogenesis and angiogenesis in view of flap survival J. Plock, K. Merz, S. Schlosser, A. Banic, D. Erni Department of Hand and Plastic Surgery, Inselspital, University of Berne, Bern, Switzerland
Abstracts Department of Clinical Research, University of Berne, Bern, Switzerland Angiogenesis and arteriogenesis have been reported as two different processes of vascular regeneration aiming to encounter critical ischemia. We have recently established a model that allowed us to assess both morphological and hemodynamic changes in the microvasculature of a critically ischemic mouse skin flap over time. The purpose of this study was to elucidate the orchestration of vascular regeneration and hemodynamic changes in terms of a temporal profile in order to question which of the two e arteriogenesis or angiogenesis e may be more effective in improving microcirculation and survival in critically ischemic flaps. To this end, a dorsal skin flap was mounted into a window chamber in mice for both morphological and hemodynamic assessment. The flap model consisted of a well-vascularized proximal part and a collateralized distal part. Tissue partial oxygen tension was measured with Clark type microprobes. Ischemia-related changes in the oxidative energy metabolism were assessed with microdialysis. Morphological and microhemodynamic investigations were performed using intravital microscopy. In the distal part of the flap, partial oxygen tension was 5 2 mmHg, whereas it was 12 4 mmHg in the proximal part and 30 5 mmHg in the healthy abdominal skin at the first postoperative day. The severity of ischemic hypoxia in the distal flap tissue was confirmed by a 4.4-fold increase in lactate/pyruvate ratio. Glycerol, a marker of cell membrane desintegration, was not significantly elevated. During the first seven days morphological signs of arteriogenesis were detectable like arteriolar dilation (150% of baseline, p < 0.01), increase in relative arteriolar length (350% of baseline, p < 0.01) and arteriolar corkscrew formation. The microvascular changes related to angiogenesis, such as sprouting and capillary reorganization, were visible from the 7th day on only. Functional capillary density was significantly different between the proximal and the distal part of the flap at baseline (240 vs 143 cm/cm2, p < 0.01). In the collateralized part, functional capillary density was increased to 190% of the baseline value after the 10th day (p < 0.01). According to Poiseuille’s law, arteriogenesis resulted in a drastic reduction in peripheral vascular resistance that was already present very few days after surgery. Angiogenesis, on the other hand, resulted in relevant hemodynamic changes only after 10 days postoperatively. Therefore, our results suggest that arteriogenesis may be more effective in improving flap survival. Improved Regeneration of Autologous Nerve Transplants by Means of VEGF-Gene Therapy T. Holzbacha, R. Milojcica, M. Antonb, T. Brillb, M.A. Konerdingc, ¨nsbacherb, H.G. Machensa, R.E. Giuntaa B. Ga a Department of Plastic Surgery and Hand Surgery, Klinikum rechts der Isar, Technische Universita¨t Mu¨nchen, Munich, Germany b Institute for Experimental Oncology and Therapy Research, Klinikum rechts der Isar, Technische Universita¨t Mu¨nchen, Munich, Germany c Department of Anatomy, Johannes Gutenberg Universita¨t, Mainz, Germany The impact of the Vascular Endothelial Growth Factor (VEGF) on the angiogenic cascade is proven. Recently its neuroprotective effect after peripheral nerve injuries on a-motoneurons in the spinal cord was shown. The biological effect is mainly mediated by the binding of two tyrosine kinase receptors (VEGFR1&VEGFR2), but in addition an effect via Neuropilin (NP)-1 and NP-2, receptors essential for the development of the nervous system, was reported. Thus a sprouting of axons could be monitored as well as an improved survival of neurons and glial cells. Experiments on