RU2555136C1 - Method of monitoring microvessel of mesentery in laboratory animals using biomicroscopy - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: animal is placed on the movable thermostated preparation microscope stage. Median laparotomy is carried out. Bleeding is stopped by thermocoagulation, fixing the wound edges using ligatures. Paramedial incision is made in the right at the level of lower third of abdomen, through which the removable hollow light guide of cylindrical shape is inserted into the abdominal cavity and it is filled with indifferent oils or normal saline. The mesentery of small intestine is thrown on the upper end of the light guide and biomicroscopy is carried out using a fixed camera for a microscope, connected to the personal computer, the results are recorded at various stages of research, followed by treatment of the data obtained, using a computer program.

EFFECT: method eliminates drying and adhesion of the area under study, as well as bending the vessels, which enables to perform long-term studies.

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Description

The invention relates to experimental medicine and biology and can be used in vivo study of microcirculation of microvessels in the abdominal cavity of laboratory animals.

One of the main ways to study microcirculation, i.e. microvascular circulation, is biomicroscopy. Any type of biomicroscopy requires the fulfillment of the following basic conditions: immobilization of the animal (anesthesia), reliable immobilization of the microscopic area, its maintenance under the maximum possible physiological conditions, and registration of the observed phenomena.

To study the microcirculation of the microvessels of the mesentery of laboratory animals, a technique is used that includes the extraction of the intestinal loop, for example, through a small paramedial incision on the right at the level of the lower third of the abdomen, followed by the placement of the mesenteric segment in the chambers for the intestinal loop of various designs or on the light guide of thermostated tables. Prevention of drying of the investigated area is carried out by irrigation with physiological solutions (A.M. Chernukh, PN Aleksandrov, OV Alekseev. Microcirculation / edited by A.M. Chernukha. - M .: Medicine, 12-12-13, 1975).

There is a method for studying the state of the mesentery of small and medium animals (mouse, rat), which consists in the fact that the zanarcotized experimental animal is placed on a table whose temperature is maintained at a physiological level. Then, a median laparotomy is performed, a part of the small intestine with the mesentery is removed, which are straightened on the table and irrigated with a warm solution using special supply and heating systems for the irrigated solution. Next, a microcirculation study technique is used to determine the rate of lymphatic drainage. Using a monitoring system, which is a microscope mounted on a tripod with a video camera, they monitor the development of the process in real time and save this information in the computer's memory (RU 2465833, АВВ 10/00, 2012).

A known method of biomicroscopy of capillaries of the mesentery of small laboratory animals (SU 1391638, A61D 3/00, 1988). The method consists in the fact that the animal is fixed on the table and the abdominal cavity is opened. A loop of the intestine is carried out through an opening in the chamber and fixed on a ring with spikes; the abdominal cavity is hermetically connected to the chamber into which the necessary solution is poured. The device is mounted on a microscope stage and biomicroscopy is performed as usual.

There is a known method of intravital microscopy of the mesentery, for the implementation of which a thermostatic chamber, a table and a tray for physiological saline are used, in the bottom of which a rectangular quartz glass fiber is built-in and covered with a hemisphere made of transparent polyethylene or rubber forming a chamber. The device is mounted on a microscope stage (SU 745498, A61B 1/06, 1980). The method consists in placing an animal under anesthesia on a stand. Through an incision in the anterior abdominal wall, the studied internal organ (for example, the mesentery of the small intestine) is removed, which is placed on a special platform of a transparent elastic chamber filled with warm circulating fluid. To prevent drying of the test organ, it is additionally irrigated with saline or lubricated with liquid paraffin.

In another embodiment of this method, the test organ is placed on a light guide consisting of a threaded rod and a set of truncated cones made of optically transparent material, the large bases of which form a platform for the microscopic organ (SU 980687, А61В 1/06, 1982).

These methods have a number of limitations that are associated with the need to remove the intestinal loop from the abdominal cavity and place it in a special chamber, in which it is necessary to maintain a constant temperature and humidity by irrigation with physiological solutions, which dramatically reduces the observation time and increases the risk of infection of the intestinal loop. In addition, mechanical damage to the investigated area during manipulations is not ruled out due to the possibility of adhesion to the surface of the chamber and the optical fiber of the object of study, as well as kinking of the vessels. These factors can contribute to biased research results.

The closest (prototype) is the method described in the patent (RU 2465833, AB 10/00, 2012).

The objective of the invention is to improve this technique with the aim of conducting research in more physiological conditions.

The technical result consists in preventing microcirculation disorders in the intestinal loop and nearby segments of the mesentery, as well as increasing the purity of the experiment and bringing the conditions closer to physiological.

The technical result is achieved by the fact that in the method of monitoring the microvessels of the mesentery in laboratory animals, comprising anesthesia, median laparotomy, biomicroscopy and monitoring using a video camera mounted on a microscope and connected to a computing unit, according to the invention, the animal is placed on a movable thermostatically controlled preparation table attached to a laparotomy to the microscope instead of the stage, bleeding is stopped using thermocoagulation, the edges of the wound are fixed and Using ligatures, a paramedial incision is made on the right at the level of the lower third of the abdomen, biomicroscopy of the mesenteric microvessels is carried out using a removable hollow fiber guide of a cylindrical shape, inserted directly into the abdominal cavity through a paramedial incision on the right side, followed by the mesentery of the terminal ileum loop looped onto a pre-filled indifferent oil or indifferent physiological solutions, the hollow upper end of the fiber, the lower blind end of which is passed through the hole in paratsionnom table beyond, the operative field closes swab wetted with physiological sodium chloride solution, table, the thus prepared animal is moved to the coaxial alignment of the fiber and the lens of the microscope with transmitted light beam output from the capacitor. The light source is turned on and using a fixed video camera for a microscope connected to a personal computer, the results are recorded at various stages of the study, followed by processing of the obtained data using a computer program designed to store, view and process videos.

Figure 1 presents a diagram of a biomicroscopy of anesthetized laboratory animals in the abdominal cavity; figure 2 presents a phased execution of the method (fixing the edges of the wound and the introduction of the light guide into the abdominal cavity of anesthetized laboratory animals); figure 3 presents the design of the used fiber.

Designations in figures 1, 2 and 3: 1 — hollow cylindrical fiber, 2 — mesentery, 3 — thermostatically controlled preparation stage, 4 — lens, 5 — eyepiece, 6 — condenser, 7 — median laparatomy, 8 — ligatures, 9 — paramedial section on the right, 10 - the lower end of the fiber, 11 - the upper hollow end of the fiber, 12 - the limiter of the fiber, 13 - gauze swabs, 14 - the light source.

Method Description

Anesthesia is achieved by intraperitoneal administration of a solution of chloralose (40 mg / kg) and urethane (6 mg / kg). Before the study, in order to exclude contamination in the abdominal cavity, as well as the lower third of the abdomen, wool is removed from the right side of the body. After the onset of deep anesthesia, the laboratory animal is placed on a movable thermostatically controlled (37-37.5 ° C) preparation table 3 in the supine position with the front and hind legs spread out to the sides. Depending on the height of the anterior abdominal wall, a glass fiber 1 is selected. Median laparotomy is performed 7. Bleeding from small vessels of the skin and the anterior wall of the peritoneum is stopped by thermocoagulation. The edges of the wound are fixed using ligatures 8 (figa). On the right, at the level of the lower third of the abdomen, a small paramedial incision 9 is made through which the upper open end 11 of the removable glass hollow fiber 1 is inserted, the lower end 10 (Fig. 1a, b) is brought out through the hole in the plate of the preparation table 3 and fixed with limiter 12 of the fiber. Through the upper end 11 of the fiber, the cavity of the fiber is filled to the upper boundary with liquid paraffin, which is indifferent, which eliminates the possibility of the object of study sticking to the walls of the fiber 1, falling into the fiber and the kink of the vessels. The mesentery 2 with the intestinal loop is located on the upper end 11 of the fiber. The surgical field is closed with tampons 13 moistened with physiological saline of sodium chloride (t = 37-37.5 ° C) (Fig.2g). As necessary, the test area is irrigated with an isotonic sodium chloride solution. Table 3, with the animal thus prepared, is moved until the optical fiber 1 and lens 4 of the microscope are aligned coaxially with the output of the transmitted light beam from the capacitor 6. The light source 14 is turned on. Using a fixed camera for the microscope (not shown in the figures) connected to the computing unit , namely, a personal computer (not shown in the figures), the research results are recorded at various stages, followed by processing of the obtained data using a computer program, for example, the Tou program pView, designed for storing, viewing and processing videos obtained from Levenhuk microscope cameras.

Thus, the application of our proposed method for monitoring microvessels in the abdominal cavity increases the purity of the experiment, eliminates drying and adhesion of the test site, as well as the failure and kinking of vessels, which allows for long-term studies. The presence of a video camera allows real-time recording with subsequent detailed study of changes in microvessels and blood flow velocity.

Claims (1)

A method for monitoring mesenteric microvessels in laboratory animals using biomicroscopy, comprising anesthesia, median laparotomy, biomicroscopy and monitoring using a video camera mounted on a microscope and connected to a computing unit, characterized in that the animal is placed on a movable thermostatically controlled preparation stage of the microscope before laparotomy, bleeding is stopped using thermocoagulation, fix the edges of the wound using ligatures, make a paramedial incision on the right outside the lower third of the abdomen, the biomicroscopy of the mesenteric microvessels is carried out using a removable hollow fiber of a cylindrical shape, inserted directly into the abdominal cavity through a paramedial incision on the right side, followed by the injection of the small intestine mesentery onto the hollow upper end of the optical fiber, which is filled with indifferent oils or physiological solutions, lower end which is passed through an opening in the preparation stage beyond, the surgical field is closed with tampons, moistened With a physiological solution of sodium chloride, the table, with the animal thus prepared, is moved until the fiber and the microscope lens are aligned coaxially with the output of the transmitted light beam from the capacitor, the light source is turned on, and using a fixed microscope camera connected to a personal computer, the results are recorded on various stages of the study, followed by processing of the obtained data using a computer program designed to save, view and process the form Ozapisey.

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