RU2726074C1 - Method of measuring concentration of reactive oxygen species (ros) in a subcutaneous tumor of living experimental animals - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to experimental medicine, and can be used for in-life observations of the level of reactive oxygen species (ROS) in organs and tissues. Method involves preliminary preparation of experimental animals with subcutaneously grafted tumor, for which animals are anaesthetized by intraperitoneal administration of zoletil solution in concentration 50–75 mg/kg with xylasine in concentration of 5–7.5 mg/kg, providing access to subcutaneous tumor of animal, forming a bath for examination from a skin fold and a subcutaneous back musculature of an animal, filling a bath with a phosphate-salt buffer with pH 7.4, preparing a measuring nanoelectrode, which is a nanopipette filled with pyrolytic carbon with platinum deposited thereon, with preliminary calibration of the nanoelectrode by hydrogen peroxide, placing the experimental animal on the microscope object table, installation of a measuring nanoelectrode in an intravital-electrochemical module (IVEC module) holder, mutual positioning of a tumor and a measuring nanoelectrode, placing the silver chloride reference electrode in a bath with a phosphate-salt buffer solution, connecting it to a measuring system, supplying a linear potential sweep from -800 mV to +800 mV, step-by-step introduction of the nanoelectrode into the tumor at a given depth at a given angle and measuring current strength at potential of +800 mV to determine the level of ROS at each step of immersing the nanoelectrode, determining the value of the ROS concentration in the tumor by the corresponding calibration curve, for construction of which a nanoelectrode and a comparison electrode are connected to instruments for removing current-voltage characteristics and successively lowered into aqueous solutions of hydrogen peroxide with a known concentration in range of 10to 10mol/l, potential sweep from -800 mV to +800 mV relative to silver chloride reference electrode is applied to nanoelectrode and current value is measured at +800 mV in each water solution of hydrogen peroxide and calibration curve is plotted, where concentration of hydrogen peroxide is given on one axis, and on other axis there is concentration of hydrogen peroxide, and on other axis – magnitude of current strength.EFFECT: use of the invention enables to determine concentration of ROS inside a tumor of a living experimental animal at a given point with high spatial and temporal resolution, which enables to obtain a complete picture of ROS distribution in the tumor volume.4 cl, 1 ex, 6 dwg

Description

Technical field

The claimed invention relates to the field of biomedicine and relates to methods for implementing intravital observations of the level of reactive oxygen species (ROS) in organs and tissues and can be used in screening to assess the properties of the tumor microenvironment, as well as the effect of antitumor drugs on tumor cells.

ROS are vital metabolites in numerous biological functions. Violation of cellular mechanisms can lead to the overproduction of ROS and cause oxidative damage to DNA, proteins, cells and tissues, which is associated with the pathogenesis of a number of neurodegenerative and inflammatory diseases.

State of the art

It is known that in almost all malignant neoplasms, ROS are present in high concentrations, which contribute to the development and progression of the tumor. However, tumor cells also express elevated levels of antioxidant proteins (enzymes). This fact indicates that for the normal functioning of tumor cells, a balance is necessary in the intracellular level of ROS. In addition, the type of ROS generated, the place of its formation in the tumor, and local concentration are of great importance. In this regard, a sensitive method is needed that would allow us to study the level of ROS at a specific point in the tumor in the animal. At the moment, this requirement is met by electrochemical methods for the determination of ROS.

Closest to the technical nature of the claimed invention is a method for determining the level of ROS disclosed in the patent of the Russian Federation No. 2647464 and used in the future to determine the cytotoxicity of substances by changing the level of intracellular ROS. In the known method, the determination of the level of ROS is carried out on cells located on a solid substrate. The determination of the ROS level is carried out by introducing into the cell a carbon-filled quartz nanocapillary containing platinum in the cavity of the tip of the nanocapillary having the shape of a truncated cone, followed by determining the signal change caused by the electrochemical reaction to the tip of the capillary with the participation of ROS.

The known method has such signs that coincide with the essential features of the proposed technical solution, such as measuring ROS using a carbon-filled quartz nanocapillary containing platinum in the cavity of the tip of a nanocapillary having the shape of a truncated cone, followed by determining the signal change caused by an electrochemical reaction to the tip of the capillary with the participation AFC.

The disadvantage of this method is the inability to measure ROS inside the subcutaneous tumor of an experimental animal.

Disclosure of the invention

The technical problem solved by the claimed invention lies in the need to overcome the disadvantages inherent in analogues, by creating a method for determining the concentration of ROS in the subcutaneous tumor of experimental animals, which allows measurement in a selected area of the tumor.

The technical result achieved by using the claimed invention consists in providing the ability to determine the concentration of ROS inside the tumor of a living experimental animal at a given point with high spatial and temporal resolution using a nanoelectrode at a potential of +800 mV relative to the silver chloride reference electrode, which will allow to obtain a complete picture of the distribution of ROS in the volume of the tumor.

The problem is solved in that the method of measuring the concentration of ROS in the subcutaneous tumor of experimental animals involves the sequential execution of the following steps:

1) preliminary preparation of live experimental animals with a subcutaneous inoculated tumor, for which the animals are anesthetized by intraperitoneal administration of a solution of zoletil (50-75 mg / kg) with xylazine (5-7.5 mg / kg),

2) providing access to the subcutaneous tumor of the animal, for which a skin incision is made along the spine and the skin fold with the tumor is separated from adjacent tissues, blood vessels are cauterized at the incision sites and the tumor is cleaned from the capsule by removing its upper layers,

3) the formation of the bath for research from the skin folds and subcutaneous muscles of the back of the animal, for which the edges of the skin folds are lifted and fixed with suture material,

4) filling the bath with phosphate-buffered saline with a pH of 7.4,

5) preparation of a measuring nanoelectrode, which is a nanopipette filled with pyrolytic carbon with platinum deposited on it, with preliminary calibration of the nanoelectrode with hydrogen peroxide,

6) placing the experimental animal on the stage of the microscope, installing the measuring nanoelectrode in the holder of the intravital electrochemical module (IVEC module), the relative positioning of the tumor and the measuring nanoelectrode, placing the silver chloride comparison electrode in a bath with a solution of phosphate-saline buffer, connecting it to the measuring system ,

7) linear potential sweep supply from -800 mV to +800 mV,

8) the step-by-step introduction of the nanoelectrode into the tumor at a given depth at a given angle and measuring the current strength at +800 mV to study the concentration of ROS at each step of immersion of the nanoelectrode

9) determination of the concentration of ROS in the tumor according to the corresponding calibration curve.

To implement the method, BALB / c mice with subcutaneously inoculated tumors of mouse mammary carcinoma 4T1 are used. The experimental animal is subjected to additional anesthesia through a catheter installed in the tail vein of the animal. With increasing concentration of ROS, an increase in current occurs, since near the working surface of the electrode there is an increase in the number of hydrogen peroxide molecules that are oxidized at a potential of +800 mV. The immersion depth of the nanoelectrode in the tumor, as a rule, is 1-3 mm.

Brief Description of the Drawings

The invention is illustrated by the following drawings and images.

In FIG. Figure 1 shows the location of the tumor, subcutaneously inoculated with the BALB / c mouse.

In FIG. 2 schematically presents an experimental animal prepared for measurement and a measuring system for implementing the inventive method.

In FIG. 3 shows an image of a nanoelectrode.

In FIG. 4 shows a micrograph of the tip of the nanoelectrode.

In FIG. Figure 5 shows an example of a calibration curve constructed to calculate the concentration of hydrogen peroxide by current.

In FIG. 6 presents the results of measurements of the concentration of ROS upon penetration into the tumor.

The positions in the drawings indicate:

1. Place of tumor implantation in an experimental animal (lateral to the midline)

2. The place of tumor implantation in an experimental animal (cranially from the base of the tail)

3. The tumor

4. Venous catheter

5. Skin fold with a tumor

6. Suture material

7. Silver chloride reference electrode

8. Nanoelectrode

9. Adhesive plaster

10. Current amplifier

11. The micromanipulator

12. Platform for creating a bath

The implementation of the invention

The following is a detailed description of the invention. The specialist is clear that the following description of the implementation of the present invention is purely explanatory and does not limit the scope of the claims claimed in the claims.

The inventive method is implemented in several stages.

1) preliminary preparation of experimental animals with subcutaneous inoculated tumor

Laboratory mice were used to measure ROS in the tumor. When conducting experimental work on the implementation of the proposed method were analyzed tumors of various sizes - from 15 to 80 mm ² - to identify the most optimal. It was found that tumors, the area of which exceeded 40 mm ² , are not suitable for this type of research, since such tumors have a very developed branched system of blood vessels, which complicates their preparation. As for tumors, the area of which did not exceed 25 mm ² , their use for work also seemed to be not optimal, because, due to their size, they had a limited number of places for introducing a nanoelectrode (1-2 measurements per tumor). In this regard, tumors with an area of 25–40 mm2 were used to conduct research on the measurement of ROS. For this, mice were injected subcutaneously with 0.5 × 10 ⁶ -1.5 × 10 ⁶ tumor cells and experiments were performed after 7-8 days. In order to ensure access of the nanoelectrode to the maximum surface area of the tumor, it should be implanted at a distance of 0.7-1 cm lateral to the midline (spine line) and 2.5-3.5 cm cranially from the base of the tail of the animal (Fig. 1) .

To ensure general anesthesia of experimental animals, the dose of 50-75 mg / kg of zoetil mixed with xylazine 5-7.5 mg / kg is used when implementing the proposed method. The specified dose provides adequate anesthesia of the animal for 40-90 minutes. The maintenance dose was 10% of the initial; its introduction every 30-60 minutes allowed to conduct research for 3-6 hours

2) providing access to the subcutaneous tumor of the animal

The animal is placed on its stomach on an operating table. Preferably, the table is equipped with a camera to maintain the body temperature of the animal, as well as movable LED lamps and a magnifying glass. A catheter is inserted into the tail vein of the animal, through which injections of an additional mixture of zoetil with xylazine are carried out during the experiment. The limbs of the animal are fixed on the surface of the table using a Blenderm skin patch. Surgical intervention is started, making sure that the animal is in deep anesthesia (slow breathing, inhibition of tendon and vibriss reflexes). Despite the fact that before the implantation of the tumor, the injection zone is shaved, at the time of the measurement, the hair grows back, in addition, the electric shaver cannot completely remove the hair. At the same time, even small hair getting on the surface of the preparation limits the working area for research. In this regard, before the cut, the skin surface is treated with mineral oil, which reduces the likelihood of wool getting on the tools and allows you to achieve the purity of the drug. The incision is carried out using scissors and tweezers along the line of the spine from the base of the tail upwards by 3-5 cm. Deviation from the midline can lead to bleeding. The bleeding stop in this and other cases is carried out by a coagulator.

3) the formation of the bath for research from the skin folds and subcutaneous muscles of the back of the animal

Around the tumor create a special bath with sufficiently long (3-5 cm long and 2-3 cm wide) sizes, 0.5-0.7 cm high. This is due to the fact that a small or narrow bath, as well as high the walls of the bath can impede the movement of the nanoelectrode fixed in the holder at an angle of 45 °, leading to large measurement errors. On the one hand, the muscles of the back of the mouse serve as the edge of the bath, and on the other, the skin fold.

To form a bath using two tweezers with a curved edge, a skin flap with a tumor is separated, cutting off the connective tissue films with scissors. In this case, direct contact of the instruments with the surface of the tumor, as well as damage to blood vessels, should be avoided. After making sure that the selected skin flap allows the tumor to be exposed, the edges of this flap are fixed with suture material with an atraumatic needle on both sides. To do this, the needle is captured by the needle holder, pierce the skin on the edge of the flap, avoiding damage to large vessels. Next, a needle with a thread is passed through the hole and, cutting off 7-10 cm of thread, fix one end of it to the skin using a double knot. The free end is attached to the surface of the surgical table using a Blenderm skin patch so as to ensure that the flap is smooth and evenly stretched with the tumor on the table. After the skin flap with the tumor is fixed and stretched, cauterize its edges (where the incision was made) to prevent bleeding, even if its visible signs are absent. Then, using special scissors and tweezers, under careful visual control using a magnifying glass, the connective tissue of the capsule is carefully removed from the surface of the tumor, avoiding damage to the vessels and, if possible, minimizing the number of contacts of the instruments with the intended area of study. After that, the tumor is washed with a solution of phosphate-buffered saline (PBS) to remove tissue debris and small debris, excess solution is removed with a napkin from the edge of the drug. The procedure is repeated twice. The bath is filled with PBS so that the entire tumor is completely immersed in the solution. Thus prepared animal is placed next to the measuring installation (Fig. 2).

4) preparation of a measuring nanoelectrode

The measurement is carried out using a nanoelectrode, the basis of which is a pipette made of quartz glass, the sharp end of which is filled with carbon. In the cavity of the deposited carbon contains platinum (Fig. 3, 4). The manufacturing technique of nanoelectrodes is described in detail in RF patent No. 2647464.

It is known that the concentration of ROS inside a tumor can vary. Therefore, when implementing the proposed method, it is necessary to preliminarily calibrate the measuring nanoelectrode introduced into the tumor according to one of the types of the most stable ROS, for example, according to hydrogen peroxide.

To build a calibration graph for ROS, the nanoelectrode and the reference electrode are connected to traditionally used devices for taking current-voltage characteristics and sequentially immersed in aqueous solutions of hydrogen peroxide with a known concentration in the range from 10 ^-7 to 10 ^-4 mol / l, a potential scan from - 800 mV to +800 mV relative to the silver chloride reference electrode on the nanoelectrode and the current values are measured at + 800 mV in each aqueous solution of hydrogen peroxide. This allows you to build a calibration curve, where on one axis shows the concentration of hydrogen peroxide, and on the other - the magnitude of the current, which will allow a quantitative assessment of the level of ROS (Fig. 5).

Hydrogen peroxide acts as the most stable form of ROS. Most unstable ROS are converted to hydrogen peroxide. Therefore, the construction of a calibration curve for hydrogen peroxide is justified for further measurements of the total concentration of ROS inside the tumor of an experimental animal.

5) placement of the experimental animal on the microscope stage, installation of the measuring nanoelectrode in the holder of the intravital electrochemical module (IVEH module), mutual positioning of the tumor and the measuring nanoelectrode, placing the silver chloride comparison electrode in a bath with a solution of phosphate-saline buffer, connecting it to the measuring system

The nanoelectrode is placed in the holder so that the silver wire has contact with a layer of pyrolytic graphite. The holder, located in the micromanipulator, is connected to a measuring system (amplifier), which allows recording the current strength and / or potential, as well as applying the potential difference to the nanoelectrode. The measuring system is connected to an analog-to-digital converter in order to further transmit the signal to a computer. Using a manipulator, the nanoelectrode is lowered into the buffer solution above the tumor. A salt bridge connected to a container containing a silver chloride electrode is placed there.

6) supply linear sweep potential to study the concentration of hydrogen peroxide

A linear potential sweep from -800 mV to +800 mV is applied to the nanoelectrode relative to the silver chloride electrode with a sweep speed of, for example, 400 mV / s, and a current-voltage characteristic is obtained. First, in phosphate-buffered saline above the tumor, then inside the tumor. To determine the concentration of ROS, the current is recorded at +800 mV and recalculated according to the calibration curve to the concentration of hydrogen peroxide.

7) the step-by-step introduction of the nanoelectrode into the tumor to a predetermined depth at a given angle and measurement of the current strength at each step of immersion of the nanoelectrode

Using a micromanipulator, the nanoelectrode is introduced into the tumor step by step. The step size can be different and is, for example, 100 microns. The angle at which the nanoelectrode is introduced can be different and range, for example, from 30 ° to 55 ° with respect to the plane on which the experimental animal is placed. The choice of the angle of the nanoelectrode supply depends on the placement of the experimental animal on the microscope stage and the choice of the location of entry into the tumor. Thus, the angle of entry of the nanoelectrode is selected so that the nanoelectrode penetrates normal to the selected area. At each step, 10 cycles of the current-voltage characteristic are recorded. The depth of entry into the tumor can be different and is, for example, from 1000 to 3000 microns.

8) determination of the concentration of ROS in the tumor by the corresponding calibration curve

Using the Origin software, the current values are determined at +800 mV for the obtained current-voltage characteristics at various depths. The obtained values using the previously obtained calibration curve are converted into the concentration of ROS (Fig. 6).

Case Studies

The advantages of the proposed method are illustrated by the following examples.

Example 1

Using the proposed method was measured the concentration of ROS in the surface and deep layers of breast carcinoma, subcutaneously inoculated with experimental mice of the BALB / c line. Animals were obtained from the Andreevsky animal center (Andreevka, Russia) aged 7–9 weeks and weighing 18–21 g. All animal experiments were approved by the bioethical committee of the N.I. Pirogov (protocol No. 25/2017, 26/2017). Mice were kept in 5 cells in cages with an individual ventilation system.

Installation for studying the concentration of ROS, with which the inventive method is implemented, which allows to measure ultra-low currents of the order of 1 pA, includes the following elements:

- Analog-to-digital converter AxonDigidata 1550 V (MolecularDevices, USA);

- Patch-clamp current amplifier Axon MultiClamp 700 V (Molecular Devices, USA);

- PatchStar micromanipulator (Scientifica, USA);

- A computer with installed WinWCP program and with installed software for managing and processing data;

- Digital microscope with a magnification of 200x;

- Table for suppression of extraneous vibration (SuperTech, Hungary).

ROS concentration in the surface and deep layers of the tumor was measured using nanoelectrodes based on nanopipettes filled with pyrolytic carbon with platinum deposited on them. The nanoelectrode was calibrated with hydrogen peroxide immediately before the study. For this, it was placed in hydrogen peroxide solutions with various concentrations from 10 ^-7 to 10 ^-4 mol / L and the current strength was determined at +800 mV. As a result, a calibration curve was obtained, based on which the concentration of hydrogen peroxide in the tumor was subsequently calculated, measured using the IVEC module. The nanoelectrode was installed in the holder and the latter was connected to the preamplifier head. Using the manipulator, the nanoelectrode was lowered into the PBS solution located in the tumor bath. A salt bridge from a silver chloride reference electrode was placed in the same solution. The potential was applied from -800 mV to +800 mV (sweep speed 400 mV / s) and the current values were noted at +800 mV. Using the joystick, the nanoelectrode was brought to the selected site of the tumor. Parameters were set for further profiling of the tumor by ROS in increments of 100 μm. After introducing the nanoelectrode into the tumor to a depth of 2 mm and stabilizing the current-voltage characteristics, the nanoelectrode was removed step by step. This procedure was repeated 5 times, while choosing different places of the tumor located at a distance of 3 mm from each other. As a result, data were obtained showing that at a depth of 250-400 μm, areas with a high ROS content are found relative to those at a depth of up to 100 μm (37 μM compared to 15 μM), as well as areas where the concentration of ROS is at the same level as in the surface layers of the tumor (about 10 μm).

The inventive method provides the ability to determine the concentration of ROS inside the tumor of a living experimental animal at a given point with high spatial and temporal resolution. The measurement of the concentration of ROS inside the tumor of an experimental animal is provided by special preparation of the experimental animal for the experiment. The ultra-small size of the nanoelectrode makes it possible to determine the ROS inside the tumor with high spatial resolution at various depths. The potential delivery program used allows recording the concentration of ROS inside the tumor with a good temporal resolution in real time.

Claims (4)

1. A method for measuring the concentration of reactive oxygen species (ROS) in a subcutaneous tumor of experimental animals, including pre-treatment of experimental animals with a subcutaneous inoculated tumor, for which the animals are anesthetized by intraperitoneal administration of a solution of zoletil at a concentration of 50-75 mg / kg with xylazine at a concentration of 5- 7.5 mg / kg, providing access to the subcutaneous tumor of the animal, for which a skin incision is made along the spine and the skin fold with the tumor is separated from adjacent tissues, blood vessels are cauterized at the incision sites and the tumor is cleaned from the capsule by removing its upper layers, the formation of a bath for research from the skin fold and subcutaneous muscles of the back of the animal, for which the edges of the skin fold are lifted and fixed with suture material, filling the bath with phosphate-buffered saline with a pH of 7.4, preparing a measuring nanoelectrode, which is a nanopipette filled with a pyrolytic carbon with platinum deposited on it, with preliminary calibration of the nanoelectrode using hydrogen peroxide, placement of the experimental animal on the microscope stage, installation of the measuring nanoelectrode in the holder of the intravital electrochemical module (IVEC module), relative positioning of the tumor and the measuring nanoelectrode, placing the silver chloride comparison electrode in a bath with a solution of phosphate-saline buffer, connecting it to the measuring system, applying a linear sweep of the potential from -800 mV to +800 mV, stepwise introducing the nanoelectrode into the tumor to a predetermined depth at a given angle and measuring the current at a potential of +800 mV to determine the level ROS at each step of immersion of the nanoelectrode, determination of the concentration of ROS in the tumor according to the corresponding calibration curve, for the construction of which the nanoelectrode and the reference electrode are connected to devices for measuring the current-voltage characteristics and successively immersed in water solutions hydrogen sulfide with a known concentration in the range from 10 ^-7 to 10 ^-4 mol / l, a potential scan of -800 mV to +800 mV relative to the silver chloride reference electrode is applied to the nanoelectrode and current values are measured at +800 mV in each aqueous peroxide solution hydrogen and build a calibration curve, where on one axis shows the concentration of hydrogen peroxide, and on the other - the magnitude of the current. 2. The method of claim 1, wherein BALB / c mice are used with subcutaneously vaccinated 4T1 mouse breast carcinoma tumors. 3. The method according to p. 1, in which when performing the steps of the method, the experimental animal is subjected to additional anesthesia through a catheter installed in the tail vein of the animal. 4. The method according to p. 1, in which the nanoelectrode is injected into the tumor to a depth of 1-3 mm

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