चूहा अन्त्रपेशी Angiogenesis परख
Summary
सामान्य वयस्क vascularized स्तनधारी ऊतक कि शारीरिक angiogenesis और उस का अभाव शल्य चिकित्सा का अध्ययन किया है हस्तक्षेप करने के लिए उजागर नहीं किया गया: (i) दीक्षा और परीक्षण एजेंटों के intraperitoneal प्रशासन निम्नलिखित angiogenesis के विकास, और (ii) angiogenesis के प्रणालीगत प्रशासन के बाद संशोधन परीक्षण एजेंट चुना गया.
Abstract
The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; (iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).
Protocol
Discussion
परख तीन +१९८६ में पेश किया गया था और क्रमिक आगे और विकसित किया गया है हमारे 7,10-12 प्रयोगशाला में परिष्कृत. समीक्षा कागजात (आमंत्रित) 1, 2 में चर्चा की, अन्य स्तनधारी परख के साथ अच्छी तरह से vivo angiogenesis परख में संबंध में तुलना natively विशेष रूप से वयस्क परीक्षण के ऊतकों जैसे महत्वपूर्ण सुविधाओं है vascularized और शारीरिक angiogenesis का अभाव है , कम से कम आघात है, यदि कोई हो, ऊतक पर दिए गए, वास्तव में मात्रात्मक चर मापा जो ध्वनि सांख्यिकीय खुराक – प्रतिक्रिया और आणविक गतिविधि अध्ययन के बारे में विश्लेषण की अनुमति देता है, अंकुरण angiogenesis होता है, जो सामान्य ऊतकों और ट्यूमर में predominating है, और विषाक्तता डेटा चूहों कि robustly बढ़ने में आसानी से जमा कर रहे हैं physiologically वयस्कता में. हानिकर सुविधाओं के तथ्य यह है कि परख अपेक्षाकृत समय लेने वाली है, खासकर जब और व्यक्तिगत microvessel क्षेत्रों और microvessel अंकुरित की संख्या की लंबाई का आकलन शामिल हो सकते हैं, कि यह real-time/serial टिप्पणियों की अनुमति नहीं है, और है कि यह शायद ही चूहों के लिए उपयुक्त है के बाद चूहों में कई mesenteric खिड़कियों संभावित माता पिता microvessels कमी से नया capillaries पैदा कर सकते हैं.
वास्तविक समय टिप्पणियों, लेकिन कर रहे हैं, जहां exteriorized लेकिन बरकरार mesenteric खिड़कियां एक खुर्दबीन मंच पर बाहर splayed हैं, जबकि पशु anesthetized है, के रूप में 1 में चर्चा intravital माइक्रोस्कोपी द्वारा सक्षम है.
Corneal micropocket, लड़की chorioallantoic झिल्ली, और सुप्रीम कोर्ट Matrigel प्लग assays है, जो उपयोगी है, लेकिन अंततः सीमित उपकरण हैं जैसे मॉडल का उपयोग कर पिछले दशकों में angiogenesis अनुसंधान के क्षेत्र में उल्लेखनीय प्रगति हासिल किया गया है. विरोधी angiogenic और समर्थक angiogenic चिकित्सा के भविष्य के नैदानिक अनुप्रयोगों स्थापना और अधिक संवेदनशील और biologically प्रासंगिक है, वास्तव में मात्रात्मक वर्तमान रिपोर्ट में वर्णित के रूप में पूर्व नैदानिक मॉडल, के सत्यापन की जरूरत है.
Disclosures
The authors have nothing to disclose.
Acknowledgements
पढ़ाई के अधिकांश के लिए वित्तीय सहायता स्वीडिश चिकित्सा अनुसंधान परिषद (5942 अनुदान) द्वारा प्रदान किया गया.
Materials
Buffers and reagents
0.5 M Tris-HCl (pH 7.8) stock solution
Tris (hydroxymethyl) aminomethan (60.57 g) is dissolved in ~900 ml aqua dest and ~45 ml of 25% HCl (concentrated) is added to achieve pH 7.8 followed by dilution to 1 liter with aqua dest.
Tris-buffer saline (TBS; pH 7.8)
To 200 ml of the stock solution is added 18 g NaCl dissolved in ~1800 ml aqua dest with adjustment of pH to 7.8 using the stock solution, followed by dilution to 2 liter with aqua dest.
TBS (pH 7.4)
To 200 ml of the stock solution is added 18 g NaCl dissolved in dissolved in ~1800 ml aqua dest with adjustment of pH to 7.4 using the stock solution, followed by dilution to 2 liter with aqua dest.
Trypsin solution (0.05% trypsin, 0.1% CaCl2)
Contains 0.1 g trypsin and 0.2 g CaCl2 in 200 ml TBS (pH 7.8).
Sucrose solution 2.5%
Contains 5 g sucrose in 200 ml TBS (pH 7.8).
Stock solution of 1% Sodium azide in TBS (pH 7.4)
Contains 1 g Na-azide in 100 ml with TBS (pH 7.4).
Diluting buffer: 0.1% BSA, 0.01% Na-azide
(Used to dilute antibodies in the Vectastain ABC kit). Contains 0.1 g bovine serum albumin (BSA) and 1 ml of 1% Na-azide, diluted to 100 ml with TBS (pH 7.4).
ABC kit Vectastain PK 4002, ABC peroxidase mouse IgG
Normal horse serum; blocking serum; yellow solution: 3 drops/10 ml dilution buffer.
Biotinylated antibody; anti-mouse IgG; blue solution: 1 drop/10 ml dilution buffer.
ABC-reagent: orange solution, 2 drops plus brown solution 2 drops/10 ml TBS (pH 7.4).
Mix these solutions 20 minutes before use!
Mouse (monoclonal) anti-rat endothelium(Biosource International, Camarillo, CA, U.S.A.)
| Catalog # | ARU0181 |
| Clone # | MRC OX-43 |
This antibody is directed against all vascular endothelial cells in the rat, with the exception of those in the brain.
Vectastain peroxidase Avidin Biotin Complex (ABC) kit
The kit contains primary antibody, biotinylated secondary antibody, avidin-biotinylated enzyme complex (ABC), and enzyme substrate. See www.vectorlabs.com or www.immunkemi.se for protocols.
Bovine serum albumin(BSA) (Sigma-Aldrich, catalog # A-4503).
DAB= 3,3′-Diaminobenzidine Isopac (Sigma-Aldrich, catalog # D 9015).
Trypsinfrom Bovine Pancreas (Sigma-Aldrich, catalog # T 4665).
Vectastain peroxidase ABC-kit, Mouse IgG (Immunkemi F & D AB, Jarfalla, Sweden)
References
- Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
- Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).
- Norrby, K., Jakobsson, A., Sorbo, J. Mast-cell-mediated angiogenesis: a novel experimental model using the rat mesentery. Virchows Arch. B Cell. Pathol. Incl. Mol. Pathol. 52, 195-206 (1986).
- Norrby, K. Mast cells and angiogenesis. APMIS. 110, 355-371 (2002).
- Albertsson, P., Lennernas, B., Norrby, K. On meteronomic chemotherapy: modulation of angiogenesis mediated by VEGF-A. Acta Oncol. 45, 144-155 (2006).
- Norrby, K. Human apo-lactoferrin enhances angiogenesis mediated by vascular endothelial growth factor A in vivo. J. Vasc. Res. 41, 293-304 (2004).
- Norrby, K., Mattsby-Baltzer, I., Innocenti, M., &, T. u. n. e. b. e. r. g., S, . Orally administered bovine lactoferrin systemically inhibits VEGF(165)-mediated angiogenesis in the rat. Int. J. Cancer. 91, 236-240 (2001).
- Norrby, K. Low-molecular-weight heparins and angiogenesis. APMIS. 114, 79-102 (2006).
- Norrby, K., Nordenhem, A. Dalteparin, a low-molecular-weight heparin, promotes angiogenesis mediated by heparin-binding VEGF-A in vivo. APMIS. 118, 949-957 (2010).
- Norrby, K. Basic fibroblast growth factor and de novo mammalian angiogenesis. Microvasc. Res.. 48, 96-113 (1994).
- Norrby, K. Vascular endothelial growth factor and de novo mammalian angiogenesis. Microvasc. Res. 51, 153-163 (1996).
- Norrby, K. Microvascular density in terms of number and length of microvessel segments per unit tissue volume in mammalian angiogenesis. Microvasc. Res. 55, 43-53 (1998).
