ラット間膜の血管新生アッセイ

Welcome to the Department of Pathology at GaN University Hospital in Guttenburg, Sweden. I'm Dr.Klaus Norby, and the method that you're about to see has been developed in my laboratory, Clinically relevant angiogenesis, which occurs in connection with hypoxia, solid tumors, or any other angiogenesis disease takes place almost exclusively in natively vascularized tissue In adult small rodents, the mesentery, which connects the small intestine to the rear wall of the body, contains a number of thin membranous parts In rats, there are some 40 to 50 of these mesenteric windows. They're virtually transparent and thus window like to the naked eye, each window is surrounded by fatty tissue in adult male rats.

The mesenteric windows are natively vascularized and lack measurable physiological angiogenesis. They thus provide an opportunity to study de novo angiogenesis in adult natively vascularized mammalian tissue. IE, the initiation of a full angiogenic response from scratch, overcoming the angiogenesis inhibitors that operate endogenously in the test tissue as an all normal tissues in mice, native micro vessels that can act as parent micro vessels are absent in many windows, which unfortunately makes the assay unpredictable in mice in native form.

The mesenteric windows in rats normally contain a relatively small number of little blood vessels located on the periphery in adult rats. These mesenteric windows are natively only between five and 10 micrometers thick. This enables intact tissue to be spread on slides and observed microscopically, thereby enabling all the vessels in every window to be observed and giving the vessels what is essentially a two dimensional extension.

The method offers an unrivaled opportunity to make quantitative measurements of the spatial extension, density, pattern formation, and size of the blood microvessels. The subcutaneous site is popular for angiogenesis studies, however, due to endothelial cell heterogeneity and tissue specific properties, control of angiogenesis and subcutaneous tissues and subcutaneously, growing tumors may not predict control of angiogenesis and metastatic visceral organs such as lung, liver, bone, and brain. This is noteworthy as visceral metastases are usually ultimately responsible for cancer's lethality.

The fact that the mesenteric window is visceral should be advantageous in this regard. The aim of our project is to study aspects of de novo angiogenesis quantitatively. We therefore inject an established pro angiogenic factor or a test substance intraperitoneal.

We normally inject five milliliters at a time into adult non anesthetized rats. A volume of this kind quickly exposes the entire mesentery to the injected fluid. Any solution which is injected intraperitoneal provided that its volume is not diminutive, should affect all the cells in the test tissue, especially as the mesothelial layer of cells that covers the extremely thin mesenteric window is highly permeable to solutes as no surgery is involved, the angiogenesis to be studied in the mesenteric window is not affected by wound healing induced angiogenesis, so the angiogenic treatment is given in the form of intraperitoneal injections.

For practical reasons, we use a treatment protocol comprising two daily injections during a period of five days Monday morning to Friday morning, as standard when studying the angiogenic effects of defined proteins such as VEGF 1 65, 1 64, and basic FGF, for example. Usually Pharmacia infusion solution is used as vehicle. The number of treatments may appear to be large, but it has been demonstrated that it is not always possible to induce statistically significant angiogenesis even when using such potent pro angiogenic factors as VEGF and basic FGF when they're administered at approximately physiological levels in a single dose or few spaced out doses, it should be remembered that potent endogenous angiogenesis inhibitors operate in the tissue.

The administration of angiogenesis modulating therapeutic modalities imitates a clinical therapeutic situation. One or more angiogenesis modulating factors can be administered systemically, subcutaneously intravenously, or orally. For example.

This mimics a clinical treatment situation in that the effect not only of the parent molecule but also of its metabolites can be studied. The system permits a certain degree of control of the toxic effects of systemically administered angiogenesis modulating agents. As adult rats grow physiologically and can increase in weight by some 40%during an experiment lasting two or three weeks, it is possible by weighing the animals to obtain an understanding of the degree to which the treatment has slowed the increase in body weight, which can be taken as an expression of toxic effects.

This enables toxic doses of angiogenesis modulating tests, substances to be avoided. An important benefit as it may be impossible to interpret the results in terms of specific anti-angiogenic effects. If a toxic effect has been involved as adult rats grow considerably faster than adult mice, the effect of toxic agents on body weight gain should be a more sensitive indicator of general toxic effects in rats than in mice.

We would now like to show you how mesenteric window specimens are made. These animals were killed using CO2. The mesenteric windows are spread onto ordinary plus slides three by one inch starting distally in the mesentery.

Angiogenesis cannot be observed with a naked eye in the mesenteric window, starting from the ileocecal valve and moving in a retrograde direction. The four windows that are collected from the most distal part of the mesentery are used. The specimen on the slide is dried at room temperature for 20 minutes.

The intestinal stump is cut off. The specimens are then kept in a freezer at minus 20 centigrade. In this way, the specimens can be kept for several months, perhaps even years without any pronounced deterioration in quality.

Staining is done using a monoclonal antibody against rat endothelium, Allow the specimen to dry in air, do not mount with a covered glass. The quantification of the incidents of blood vessels in individual mesenteric windows is done using computer aided morphometric microscopy using a drawing microscope with a total optical enlargement of between 14 and 34. The entire mesenteric window is visualized.

The enlargement is chosen in fixed steps so that as much of the view field as possible is covered by the specimen. The whole area of the window is easily depicted as the border between the window and the surrounding fatty tissue is distinct. All the vascularized parts are then drawn according to carefully defined criteria.

Even the smallest newly formed blood vessels whose diameter at higher enlargements has been found to be around 2.5 to 3.0 micrometers can be clearly discerned. The fact that the mean diameter of the rat erythrocyte is equal or greater to 6.3 micrometers shows just how small these vessels are. The area of all the vascularized parts in a window as a percentage of the area of the whole window is known as the relative vascularized area va.

It is to all intents and purposes a measurement of the spatial extension of the vessels as they have what is essentially a two dimensional extension, the VA can easily be measured using computer aided telemetry, for example. The next step involves recording the micro vessels density and pattern formation within three central parts of the vascularized areas of every window. These parts are systemically chosen at random at an optical enlargement of 80.

Each vessel is visualized as a single line using a drawing microscope. The image of each view field is transferred to a digitalized image and an image analyzer, and every vessel in the digitalized image is then reduced to a thickness of one pixel.Okay. The machine then measures the number of pixels, which represents the vessels and are expressed as a fraction of all the pixels in the view field.

This percentage figure, the microvascular length or MVL represents the length of microvessels in each unit of the vascularized area, and is a composite measurement of the micro vessels density. By multiplying the VA mean of four windows per animal by the mean MVL for the corresponding treatment group, the total microvascular length per animal, the TMVL is computed. We have furthermore developed techniques for detailed quantitative analysis of micro vessels, density and composition using image analysis.

These include measuring the actual distance between two successive micro vessels, branching points, IE, the length of individual micro vessels, segments LEMS, the number of micro vessels, segments per unit tissue volume, NOMS, and the number of micro vessel branching points per unit tissue volume.NOBP. The degree of tortuosity in the microvessels MVT is given by the difference between the shortest distance and the actual distance between two successive branching points, the occurrence of micro vessels crossing over the index of intersection INIS, and of interconnecting loop formation. The index of loop formation INLF can also be measured accurately Using similar techniques at the periphery of the micro vessels network.

We are able also to quantify corresponding variables for sprouts, including length of sprouts, LESP, and number of sprouts per unit tissue volume, NOSP designing trials as there can be significant differences between animals in the same treatment group and even between mesenteric windows in one and the same animal. We have found that the following design may prove practical the number of animals per treatment group in time of observation, eight to 12, the number of windows per animal that are analyzed in terms of VA for the number of view fields for each window that is analyzed in terms of microvascular length, MVL length of micro vessels, segments, LEMS number of micro vessels segments NOMS, number of micro vessels, branching points, NOBP micro vessel tortuosity M VT index of intersection I index of loop formation INF three animals treated with a vehicle are used as controls. The mesenteric window angiogenesis assay is well-suited for dose effect studies and for angiogenesis kinetic analyses.

This can be done by treating groups of animals with different doses of pro angiogenic factor and sacrificing these animals at different time points during the angiogenic response. The expansive phase, the peak, and the subsequent angio regressive phase can thereby be discerned in quantitative terms. Studies of this kind have revealed that the de novo angiogenic response to any growth factor cytokine tested is specifically doses dependent in terms of velocity, potency, and duration.

Moreover, the pattern of the new vessels in terms of the spatial extension and changes in the micro vessels, density is often distinctly dose and time dependent compared with angiogenesis mediated by VEGF or basic FGF at approximately physiologic doses. Mast cell mediated angiogenesis, MCMA induced via selective activation of mast cells in situ features considerably higher potency and remarkably longer duration. Actually, mast cell mediated angiogenesis was first described using this assay because of the ability to quantify objective angiogenesis variables, the high degree of biological relevance of the system and the possibility to check for toxic treatment.

The methodology is uniquely suited to structure activity and dose response analyses. The rat MWAA features a model for studying angiogenesis in an adult natively vascularized visceral tissue that lacks physiologic angiogenesis and which is not affected by surgery, thus excluding any influence of wound healing induced angiogenesis. The assay is truly quantitative and assesses accurately a number of biologically relevant objective variables describing microvessels spatial extension, density, and pattern formation.

The assay is well suited for studying the systemic effects of angiogenesis, modulating factors, and for structure activity and dose response studies. The methodology described here has been developed at Dr.Kloss Carl Norby laboratory at the Department of Pathology, Reka University Hospital, jute Borg University Gothenburg in Sweden. The Swedish Medical Research Council, project number 5 9 4 2 has supported all the studies in this project.

The skillful technical assistance of Mrs. Gun Effer who has been involved since the start of the project, Ms.Monica Anderson, not present, and Mrs. Ann Nemar Lindgren is gratefully acknowledged.

I hope that this short presentation has given you an idea of the potential of the assay. If you use this assay at your laboratory, I would be happy to assist you if you run into any technical problem. I would moreover, very much appreciate any suggestion that you may have.

Thank you.