RU2642215C2 - Method and device for endogenous substances microsampling from one structure of brain and subsequent introduction into another structure of brain of animals in free behaviour - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: microsampling method is performed in non-fixed animals under conditions of their free behaviour. At that, according to stereotaxic coordinates, at least 2 cannulas with mandrins are implanted into the brain structures and fixed on the head, with an external diameter of a cannula no more than 1 mm and a length equal to the depth of immersion in the brain. Then, to take the substances from the cannula, the mandrin is removed and replaced by a capillary for the time necessary to fill it with endogenous substances under the action of capillary forces of surface tension. Further, this capillary is removed from the cannula and the mandrin is inserted therein. The external opening of the removed capillary that had no contact with the brain is sealed. Then, to remove the substances contained in capillary, the mandrin is removed from the cannula located in another brain structure of the same individual, or in another individual brain structure, and a capillary with endogenous substances is inserted therein. A laser beam remotely located within 1m, with a power of 500-3000 mW, is directed to the external hermetic end of the capillary to heat the capillary cavity to 40-50 degrees for thermal expansion of air inside it, pressure increase and complete removal of the substances. Then the capillary is removed and a mandrin is inserted into this cannula instead. A kit for method implementation is also provided. It includes at least 2 guiding fine cannulas with external diameter of not more than 1 mm and length equal to the depth of their immersion in a particular brain structure. It also includes mandrins corresponding to the cannulae in terms of length and inner diameter, to be placed in each cannula with a possibility of longitudinal movement therein. The kit also includes capillaries open at both ends, with diameter equal to the mandrins and replacing them in cannulas. The kit contains a laser with power of 500-3000 mW.

EFFECT: inventions allow to study the roles of different brain structures and to identify endogenous substances involved in formation of various emotional-motivational states and pathological processes in unfixed animals under conditions of their free behaviour.

2 cl, 2 dwg

Description

The invention relates to medicine, in particular to neurophysiology and psychophysiology, for studying the mechanisms of brain activity in the formation of various physiological conditions of the body, for identifying structures and endogenous substances involved in various brain activities. The invention can be used in neurophysiological, psychophysiological, pharmacological, biochemical studies and in molecular biology.

In modern neurophysiology and neurosurgery, two separate, independent from each other methods of introducing and extracting substances from the brain exist and are widely used.

The first method is intended only for introducing various biologically active substances into individual brain structures with a further assessment of their physiological effects. Using stereotactic coordinates, a thin cannula is inserted into a certain brain structure filled with a biologically active substance, which is brought into the brain structure under the influence of hydrostatic pressure. This method of injecting microscopic amounts of solutions into a tissue under pressure using a thin needle or cannula is called microinjection (1). During the introduction of substances, the animal is fixed or limited in movement, since pressure for microinjection is transmitted from the syringe through a thin tube to the cannula fixed on the animal's head.

Along with this method, there is a method of microionophoresis (2, 3, 4), in which biologically active substances, such as acetylcholine, norepinephrine, etc., which are in an ionized form in a multi-channel microelectrode, are removed to the brain cells using direct current.

These methods are focused only on the introduction of substances into the structure of the brain. They do not allow to extract endogenous substances from the brain structure and introduce them into another brain structure of the same or another individual.

The second method, called tissue microdialysis, is designed to collect endogenous substances from intercellular fluid in vivo and analyze their content. Delgado J.M. et al. (5) in 1972 described the use of glass catheters for sampling extracellular fluid, which served as the prototype of modern microdialysis. Urgenstedt U. and Pycock C. (6) published a similar technique, which was further developed and led to the creation of tissue microdialysis in its modern form (7-10).

Thin cannulas are used to extract various endogenous substances from the brain structures, which are introduced by the stereotactic method into the studied brain structure. To take tissue substances, a catheter is inserted inside the cannula, at the end of which there is a semi-impermeable membrane, through which from the tissue, due to the concentration gradient, endogenous substances diffuse into the dialysis fluid circulating inside the catheter. Continuous renewal of the solution in the membrane area supports the transmembrane concentration gradient.

The microdialysis catheter is a two-sheet concentric polyurethane tube with an outer diameter of 1 mm, the end section of which is represented by a semi-impermeable membrane (standard length 10 mm). A special infusion pump with a solution similar in electrolyte composition to tissue fluid is connected to the catheter.

The permeability of a membrane for most substances is determined by its pore size (membrane cut-off size). Typical catheters have pores of 20 kilodaltons (CMA70 catheter to detect small molecules, ions, amino acids) and 100 kilodaltons (CMA71 catheter allows additional extraction of small proteins and cytokines). The solution containing the recovered substances accumulates in a 200 microliter micro ampoule.

In the collected dialysis fluid, the content and concentration of various substances are determined using an analyzer (liquid chromatography, mass spectroscopy, etc.).

The concentration of the substance in the dialysate never corresponds to its concentration in the tissue fluid. The recoverable amount is part of the true extracellular concentration of a substance in the tissue.

With standard membrane lengths (10 mm) and perfusion rates (0.3 microliters / min), the relative recovery is 70% of the true concentration (11).

Thus, the concentration of substances contained in the dialysate is not equal to the true content of substances in the tissue fluid. It depends on the perfusion rate of the dialysis fluid and the properties of semipermeable membranes, which, in accordance with permeability, limit the release of substances into the dialysis fluid, and therefore, all substances present in the brain tissue are not represented in the dialysis fluid.

The method of microdialysis is focused only on the collection of endogenous substances from the tissue and does not allow the substance extracted from the tissue to be removed into another brain structure.

During microdialysis, the animal is fixed or restricted in movement since the microdialysis catheter fixed on the head is connected through a thin tube to the infusion pump.

These methods have different purposes and do not allow:

- extract endogenous substances from the studied structure of the brain and introduce them into another structure of the brain;

- conduct research in the free behavior of animals, because there are connections of cannulas located in the brain with external devices in the form of wires, tubes, etc .;

- to extract and determine the true content of endogenous substances in the structures of the brain under various physiological conditions.

Thus, none of the existing methods makes it possible to extract endogenous substances from brain structures and transfer them to other brain structures of one or more individuals. Each of these methods is not a prototype of the claimed invention.

The goal is to develop and create a fundamentally new technology for the extraction of endogenous substances from individual brain microstructures and their subsequent excretion into other brain structures under various conditions of experimental animals, in conditions of free behavior, to determine the physiological role of endogenous substances in the activity of the brain and their chemical analysis.

A method for collecting endogenous substances from one brain structure and for their subsequent introduction into another brain structure

The claimed method for the collection of endogenous substances from individual microstructures of the brain and their subsequent introduction into other microstructures is based on the fact that the (single) one and the same universal capillary is first used to extract, due to existing capillary forces, endogenous substances located in a certain brain structure, and then the same capillary is used to remove the collected substances from it into another structure of the brain by increasing the pressure in the capillary when the laser beam heats its closed cavity in sleep guns.

A device for collecting endogenous substances from one brain structure and for their subsequent introduction into another brain structure

The proposed device (Fig. 1, 2) consists of:

- a thin guide cannula, with a diameter of not more than 1 mm, along the length corresponding to its immersion depth in the brain (1), made of a biologically inert polymer or metal;

- mandraine corresponding to the cannula in length and inner diameter and placed in each cannula with the possibility of longitudinal movement in it (2), made of a biologically inert polymer or metal wire;

- a single universal capillary (3) for micro-sampling and excretion of substances, equal in diameter to the mandrin and replacing it in a cannula made of a biologically inert polymer, which has openings at both ends (4) for communication with the atmosphere during the intake of substances and a closed external hole (4) in the excretion of substances;

- a laser with a power of 500-3000 mW, for remote heating within 1 m of the cavity in the outer part of the capillary (5).

The method of micro-sampling of endogenous substances from one structure and their removal into another brain structure in non-fixed (free) animals is characterized by the fact that they are previously implanted into the brain structures by stereotactic coordinates and a certain number of cannulas with mandrains are fixed on the head, the length of which is equal to the depth of immersion in the brain ; then, for micro-sampling of substances, the mandrin is removed from the cannula and a capillary is placed in its place for the necessary time to fill it with endogenous substances under the action of capillary forces; then this capillary is removed from the cannula and a mandrin is inserted into it; in the extracted capillary, an external opening is sealed that has no contact with the brain; then, to remove the substances contained in the capillary from the cannula, which is in another structure of the brain, the mandrin is removed and a capillary with endogenous substances is inserted into it; direct a laser beam with a power of 500-3000 mW to the external sealed end of the capillary for thermal expansion of the air inside it, pressure increase and complete removal of substances; then the capillary is removed and mandrin is inserted into this cannula instead.

A device for micro-sampling of endogenous substances from one brain structure and their subsequent introduction into another animal brain structure in free behavior is characterized by the fact that it includes at least 2 (several) guide thin cannulas with an external diameter of not more than 1 mm and a length equal to the immersion depth them into a specific brain structure; mandrils corresponding to the cannula in length and inner diameter and placed in each cannula with the possibility of longitudinal movement in them; capillaries open at both ends, equal in diameter to the mandrins and replacing them in cannulas, as well as a laser with a power of 500-3000 mW.

The method and device is illustrated by the following drawings.

FIG. 1. The layout of the cannula and mandrin, which are in the structure of the brain, before the endogenous substances are taken from the brain into the capillary and the prepared capillary is inserted into the cannula, where the numbers indicate the following positions: 1 - cannula, 2 - mandren, 3 - capillary, 4 - the outer part of the capillary 5 is a laser. The hole in the outside of the capillary is open.

FIG. 2. The layout of the cannula and capillary located in the structure of the brain during the intake into the capillary of endogenous substances from the brain, and the mandrin removed from the cannula (numbers indicate the same positions). The hole of the external, not facing the brain part of the capillary is closed.

The method is as follows.

To conduct the study, two identical cannulas are made in advance, the corresponding mandrils and capillaries of the required length and diameter, for immersion in certain structures of the brain, one of which is selected for the intake of substances, the other for excretion of substances.

Cannulas, mandrins and capillaries can be made of glass or a biologically inert polymer by stretching to the desired diameter when heated. The outer end of the capillary has an extension ending in a hole. The inner end of the capillary - the open tip facing the brain, can be stretched to the required micron sizes in diameter.

Previously, using stereotactic technique, the prepared cannulas with mandrin are implanted into the brain structures under anesthesia, from which substances will be extracted and into which these substances will be removed.

The essence of the method is as follows: through a thin cannula inserted into the brain, with a diameter of 0.3-1 mm, and a microcapillary inside the cannula, corresponding to its internal diameter, the endogenous substances are sampled into the microcapillary from a certain brain structure. The entry of fluid with endogenous substances into the microcapillary occurs due to capillary forces of surface tension.

The height of the capillary rise is determined by the formula of Juren:

,

,

where N is the height of the rise of water, mm;

A is the coefficient of surface tension;

r is the radius of the capillary, mm;

g - acceleration of gravity, m / s;

d is the density of water at a given temperature, g / cm ³ .

It follows from the formula that the liquid rises in the capillaries the higher, the greater its surface tension, the smaller the diameter of the capillary and the lower the density. The surface tension of tissue fluid, plasma is close to the surface tension of water.

It is important to note that the concentration of all substances in the microcapillary is equivalent to their content in the studied brain structure.

Then the microcapillary with its contents is removed from the cannula and inserted to remove substances into another cannula located in the brain structure of the same individual or another individual. To remove substances from the capillary into the brain structure, a hole in the upper, outward-facing part of the capillary is pre-sealed (sealed). The removal of substances into the structure of the brain from the microcapillary occurs due to the thermal expansion of air in the sealed upper cavity of the capillary when it is heated to 40-50 degrees using remote laser action. The capillaries free of the cannula are closed with mandrins and these cannulas are kept closed for repeated repeated experiments.

Knowing the volume of the microcapillary, it is possible to calculate and see with a microscope the volume of liquid in the microcapillary before and after its introduction into the selected brain structure.

During the excretion of substances and after the microdose of endogenous substances is withdrawn into the selected brain structure, the researcher observes and records various manifestations of the individual’s behavioral, vegetative, and other reactions in various experimental models.

Depending on the purpose of the experiment, cannulas are implanted into different structures of the brain - some for taking substances, others for removing substances into the brain, from one animal or several animals. The transfer of substances can be from one brain structure to another brain structure of the same animal or from the brain structure of one animal to the brain structure of another animal.

During the experiment, all animals can be in free behavior, while any connection with wires, tubes, etc. with external devices is completely excluded.

In each particular experiment, depending on the purpose and tasks, cannulas and capillaries of different diameters are used. By changing the diameter of the tip of the capillary inside the brain tissue, more or less large endogenous substances can be selectively taken from the brain structure.

Having collected the required amount of endogenous substances contained in microcapillaries, it is possible to carry out chemical analysis using liquid chromatography, mass spectroscopy, etc., as is done during microdialysis.

The advantages of the method and device:

- allow you to extract endogenous substances from one brain structure and transfer them to another brain structure;

- allow us to study the role of endogenous substances in individual brain structures in the formation of emotional-motivational states, memory, and other forms of brain activity.

- allow the study of endogenous substances in individual brain structures in conditions of free animal behavior;

- allow you to determine the true content of endogenous substances using modern analytical methods in the structures of the brain under various physiological conditions of animals. The developed method and device for microexchange of endogenous substances between separate brain structures can be used to study the role of different brain structures and to reveal the participation of biologically active substances in the formation of various emotional and motivational states and pathological processes, to determine the chemical structure of endogenous substances.

Literature

1. Poland, R.S., Bull, S., Syed, W.A., Bowers, M.S. Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior. 2015. J. Vis. Exp. (103), e53018, doi: 10.3791 / 53018 http://www.jove.com/video/53018/-?language=English

2. Yumatov E.A., Fadeev Yu.A. Microinophoresis. 1971. BME Yearbook, p. 511-516.

3. Yumatov EA Methods of fixation of non-anesthetized rats for microelectrode and microionophoretic studies of the brain. 1980. J. Higher. Nerve. Figures., T. 30, No. 6, p. 429-430.

4. Yumatov EA, Kiyatkin EA Methods of microionophoretic study of neurons in rats under conditions of free behavior. 1981. J. Higher. Nerve. Figures., T. 31, No. 4, p. 878-882.

5. Delgado JM, DeFeudis FV, Roth RH, Ryugo DK, Mitruka BM. Dialytrode for long term intracerebral perfusion in awake monkeys. 1972. Arch Int. Pharmacodyn Ther. 198 (1), p. 9-21.

6. Ungerstedt U, Pycock C. 1974. Functional correlates of dopamine neurotransmission. Bull Schweiz. Akad. Med. Wiss. 30 (1-3), p. 44-55.

7. Microdialysis. http://www.science-pribor.ru/Upload/files/CMA_Booklet2012.pdf

8. Lonnroth P, Jansson PA, Smith U. A microdialysis method allowing characterization of intercellular water space in humans. 1987. Am. J. Physiol. 253 (2 Pt 1), p. E228-31.

9. Tissue microdialysis, http://www.neuromonitoring.ru/tkanevoie-mikrodializ.html

10. Sadykov A.M., Korabaev R.S., Adilbekov E.B. The use of brain microdialysis in neurosurgery. J. Neurosurgery and neurology of Kazakhstan. 2013, No. 4 (33), p. 31-35.

11. Hutchinson PJ, O'Connell MT, al-Rawi PG, Kett-White R, Gupta AK, Kirkpatrick PJ, Pickard JD. Clinical cerebral microdialysis-determining the true extracellular concentration. 2002. Acta Neurochir. Suppl. No. 81, p. 359-62.

Claims (2)

1. The method of micro-sampling of endogenous substances from one structure and introducing them into another brain structure in non-fixed animals under conditions of their free behavior, characterized in that at least 2 cannulas with mandrains are fixed on the head using stereotactic coordinates and are fixed on the head the outer diameter of the cannulas is not more than 1 mm and a length equal to the depth of their immersion in the brain; then, for micro-sampling of substances, the mandrin is removed from the cannula and a capillary is placed in its place for the necessary time to fill it with endogenous substances under the influence of capillary forces of surface tension; then this capillary is removed from the cannula and a mandrin is inserted into it; in the extracted capillary, an external opening is sealed that has no contact with the brain; then, to remove the substances contained in the capillary from the cannula located in another brain structure of the same individual or in the brain structure of another individual, the mandrin is removed and a capillary with endogenous substances is inserted into it; direct a laser beam, remotely located within 1 m, with a power of 500-3000 mW to the outer sealed end of the capillary to heat the cavity of the capillary to 40-50 degrees with the aim of thermal expansion of the air inside it, increase pressure and completely remove substances; then the capillary is removed and mandrin is inserted into this cannula instead. 2. A kit for implementing the method according to claim 1, characterized in that it includes at least 2 guide thin cannulas with an outer diameter of not more than 1 mm and a length equal to the depth of immersion in a particular brain structure; mandrels corresponding to the cannula in length and inner diameter for placement in each cannula with the possibility of longitudinal movement in them; capillaries open at both ends, equal in diameter to the mandrins and replacing them in cannulas, as well as a laser with a power of 500-3000 mW.

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