RU2759892C1 - Perfusion chamber, system and method for studying the brain activity in vivo - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, namely to a perfusion chamber, a system and a method for studying the brain activity in vivo. The perfusion chamber for studying the brain activity in vivo constitutes a monolithic structure. The structure is made of plastic exhibiting dielectric properties, applying a technology based on a three-dimensional digital model. The structure consists of a load-bearing body. The body comprises an input channel for the perfusion solution, a channel for introducing the reference electrode, a channel for introducing the input perfusion solution temperature sensor, a preliminary chamber, an input spiral chute between the preliminary and main chambers, a main chamber, an output spiral chute between the main chamber and the perfusion solution output hole, a perfusion solution output hole, mounting grooves, a point for attaching the skull of an animal. The input channels of the chamber are made in the form of through ducts. The through ducts exit into the preliminary chamber. The preliminary chamber is connected with the main chamber by means of an inlet spiral chute. The exit from the preliminary chamber is located above the bottom of the main chamber and is configured to supply a liquid gravitationally. The main chamber has the shape of a truncated paraboloid of rotation and is connected by an outlet spiral chute with the perfusion solution output hole in the lower part of the perfusion chamber, configured to accommodate a second temperature sensor for controlling the temperature of the output solution. Located along the perimeter of the perfusion chamber are three mounting grooves for attachment to the stereotactic apparatus. The point of attachment of the skull of an animal is located at the bottom of the main chamber and is made in the form of a truncated cone adapted to the size and curvature of the skull of the studied animal. The system for studying the brain activity in vivo consists of a perfusion chamber and a brain surface clamp. The brain surface clamp is made of plastic with application of the technology based on a three-dimensional digital model. The clamp constitutes a plastic disk with a smooth surface, perforated with a set of periodic round holes. The clamp is equipped with a technical rim along the perimeter of the disk, configured to reinforce the structure. The reinforced plastic disk is connected with a fastener by means of two handles. The fastener is inserted into the clamp holder. The brain surface clamp is therein configured to be replaced in case of failure thereof, applying the technology based on a three-dimensional digital model. The clamp holder is installed in a micromanipulator, configured for adjusting the position and degree of pressure on the brain tissues. The brain surface clamp is placed directly on the surface of the brain in the main chamber of the perfusion chamber after attachment thereof to the animal and removal of a section of the skull and the dura mater. In another variant, the system consists of a perfusion chamber, a brain surface clamp, a system for attaching to a stereotactic apparatus, and a flow perfusion system. The system for attaching to a stereotactic apparatus constitutes support columns whereon the perfusion chamber rests at the points of the mounting grooves. Bolts are passed through the mounting grooves of the perfusion chamber and the support columns. The bolts are secured in the stereotactic apparatus. The perfusion chamber is attached to the animal in the provided point of attachment to the skull of the animal. The flow perfusion system constitutes elements of a perfusion system located consecutively. Each subsequent element is therein located below the previous one to ensure gravitational supply of a liquid, including a buffer tank, a connecting tube, a controller dropper, a connecting tube, a heating linear flow element, a perfusion chamber, a flexible connection, a perfusion solution level adjuster, a vacuum suction channel connected to a vacuum suction system. The flexible connection is made of two fittings connected by a flexible tube. The buffer tank is connected with the controller dropper via a connecting tube, configured to control the flow rate of the perfusion solution and ensure galvanic isolation of the perfusion solution in the perfusion chamber and the buffer tank. The controller dropper, in turn, is connected by means of a tube with the heating linear flow element, configured to set the required temperature of the perfusion solution. The outlet of the heating linear flow element is connected with the perfusion solution input channel of the perfusion chamber by means of a tube. The perfusion solution outlet of the perfusion chamber is connected by means of a flexible connection and is coupled with the perfusion solution level adjuster, configured to perform reciprocating vertical movement thereof and thus adjust the level of perfusion solution in the main chamber due to the effect of communicating vessels, configured for excess perfusion solution in the perfusion solution level adjuster to be removed through the vacuum suction channel connected to the vacuum suction system. In implementation of the method, a preliminary preparatory operation for removing the scalp and the cover tissues is performed on the warmed animal under anesthesia. The skull of the animal is attached to the perfusion chamber at the provided attachment point using first cyanoacrylate glue, then dental cement. The perfusion chamber is attached in the stereotactic apparatus using the mounting grooves. Bolts are inserted into the grooves, passing through the support columns, whereon the claimed perfusion chamber rests, in turn. The perfusion chamber is connected to the flow perfusion system. For this purpose, the outlet of the heating linear flow element is connected with the perfusion solution input channel of the perfusion chamber by means of a tube. The perfusion solution outlet of the perfusion chamber is connected by means of a flexible connection to the perfusion solution level adjuster. The adjuster performs reciprocating vertical movement and thus adjusts the level of perfusion solution in the main chamber due to the effect of communicating vessels. Excess perfusion solution in the perfusion solution level adjuster is removed through the vacuum suction channel connected to the vacuum suction system. The temperature sensors are placed through the perfusion solution input channel in the preliminary chamber and the holes for the perfusion solution outlets to monitor the actual temperature of the input and output solution. A section of the skull and the dura mater is removed. The brain surface clamp is placed directly on the brain surface in the main chamber of the perfusion chamber. Electrophysiological studies of the brain activity are conducted using a reference electrode in the form of silver chlorinated wire. The wire is therein placed in the channel for introducing the reference electrode and recording electrodes located in the brain through the holes of the perforated reinforced plastic disk. Optical studies of the brain activity are conducted by means of recording the electromagnetic emission of the visible and infrared ranges reflected from the brain tissues in the areas of holes of the perforated reinforced plastic disk by video surveillance systems.

EFFECT: apparatus and method for studying the brain activity in vivo are provided for conducting electrophysiological and optical studies of the brain activity, such as studies of the effect of medicinal products and biologically active substances on the brain activity in vivo.

4 cl, 7 dwg

Description

The invention relates to medicine and veterinary medicine, in particular - to objects of medical technology, namely, to devices for studying the activity of the brain in vivo and methods of their work. The claimed technical solution can also be used to study the effect of drugs and biologically active substances on the activity of the brain in vivo, because the main stages of the formation of the central nervous system of mammals, including early rhythms of activity, which, for example, in rats and humans are similar, while the critical period of development of the somatosensory system in rats is also comparable in terms of its functional significance and the level of development of the cortex with the human fetus during the last trimester of gestation.

Further in the text, the applicant provides the terms that are necessary to facilitate an unambiguous understanding of the essence of the declared materials and to exclude contradictions and / or controversial interpretations when performing an examination on the merits.

In vivo - means conducting research on (or inside) living tissue in a living organism [https://ru.wikipedia.org/wiki/In_vivo].

Neurotransmitter is a biologically active chemical substance through which an electrochemical impulse is transmitted from a nerve cell through the synaptic space between neurons, and also, for example, from neurons to muscle tissue or glandular cells [https://ru.wikipedia.org/wiki/Neurotransmitter] ...

Gestation is the period of time of bearing a child (pregnancy), due to the number of full weeks [https://vlanamed.com/gestatsiya/].

Dielectric is a substance (material) that conducts electric current relatively poorly [https://ru.wikipedia.org/wiki/Dielectric].

Perfusion is an artificial passage of a solution through biological tissues [https://ru.wikipedia.org/wiki/Perfusion].

Perfusion system - a system of devices for providing, directing and regulating perfusion.

Perfusion artifacts are a short-term violation of the stationarity and laminarity of the flow of the perfusion solution in the perfusion system due to endogenous processes, such as cavitation [https://ru.wikipedia.org/wiki/Cavitation], temperature fluctuations in the density of the perfusion solution, etc.

Adhesion - adhesion of surfaces of dissimilar solids and / or liquids [https://ru.wikipedia.org/wiki/Adhesion].

Stereotaxic apparatus - a device attached to the head during surgery and designed to accurately position the target object in space for the introduction of electrodes, cannulas or an instrument at a given point in the brain according to pre-calculated spatial coordinates [https://dic.academic.ru/dic.nsf / medic2 / 2948].

Vibrissae are tactile mechanosensitive long coarse hair of many mammals protruding above the surface of the coat [https://ru.wikipedia.org/wiki/Vibrissy].

Neuroimaging is the general name for several methods that allow visualizing the structure, function, and biochemical characteristics of the brain. Includes computed tomography, magnetic resonance imaging, etc. This is a relatively new discipline, which is a branch of medicine, and more specifically neurology, neurosurgery and psychiatry [https://ru.wikipedia.org/wiki Neuroimaging].

The first week after birth is a critical period for the formation of cortical maps in the somatosensory cortex of rats [Rakic P. / Prenatal genesis of connections subserving ocular dom- inance in the rhesus monkey // Nature (1976). 261: 467-471. DOI: 10.1038 / 261467a0; Katz LC, Shatz CJ. / Synaptic activity and the construction of cor- tical circuits // Science (1996) 274: 1133-1138 DOI: 10.1126 / science.274.5290.1133; Lopez-Bendito G, Lujan R, Shigemoto R, Ganter P, Paulsen O, Molnar Z. / Blockade of GABA (B) receptors alters the tangential migration of cortical neurons // Cereb Cortex (2003) 13: 932–942 DOI: 10.1093 /cercor/13.9.932; O'Leary DD, Sahara S. / Genetic regulation of arealization of the neocortex // Curr Opinion Neurobiol. (2008) 18: 90–100 DOI: 10.1016 / j.conb.2008.05.011; Rakic P, Ayoub AE, Breunig JJ, Dominguez MH. / Decision by div- ision: making cortical maps // Trends Neurosci. (2009) 32: 291–301 DOI: 10.1016 / j.tins.2009.01.007; Erzurumlu RS, Gaspar P. / Development and critical period plasti- city of the barrel cortex // Eur J Neurosci. (2012) 35: 1540-1553].

During the first week after birth, in the barrel bark of rats, rhythms of oscillatory electrical activity, unique for this period of development, are observed, such as early gamma oscillation, as well as spindle-shaped oscillations [Khazipov R, Sirota A, Leinekugel X, Holmes GL, Ben Ari Y, Buzsaki G / Early motor activity drives spindle bursts in the developing somatosensory cortex // Nature (2004) 432: 758–761. DOI: 10.1038 / nature03132; Yang JW, Hanganu-Opatz IL, Sun JJ, Luhmann HJ. / Three patterns of oscillatory activity differentially synchronize developing neocortical networks in vivo // J Neurosci. (2009) 29: 9011–9025 DOI: 10.1523 / jneurosci.5646-08.2009; Minlebaev M, Colonnese M, Tsintsadze T, Sirota A, Khazipov R. /. Early gamma oscillations synchronize developing thalamus and cortex // Science (2011) 334: 226-229 DOI: 10.1126 / science.1210574; Minlebaev M, Ben-Ari Y, Khazipov R. / Network mechanisms of spindle-burst oscillations in the neonatal rat barrel cortex in vivo // J Neurophysiol. (2007) 97: 692-700].

Both early rhythms described above can be caused both by stimulation of the sensory periphery, and can be observed during registration of spontaneous activity.

In addition, these early rhythms of activity are actively involved in the formation of somatosensory cortical maps and disappear without a trace after the end of the critical period of development of the somatosensory system [Feldman, D. E. /. Map Plasticity in Somatosensory Cortex // Science (2005) 310 (5749) 810-815. DOI: 10.1126 / science.1115807; Fox K. / Barrel Cortex // Cambridge University Press (2008) DOI: 10.1017 / CBO9780511541636; Feldman, D. E. / Synaptic Mechanisms for Plasticity in Neocortex // Annual Review of Neuroscience (2009), 32 (1), 33–55. DOI: 10.1146 / annurev.neuro.051508.135516].

However, despite the vital importance of early rhythms in the formation of a highly organized structure of the central nervous system, the mechanisms of early rhythms still remain unexplored.

The question of understanding these mechanisms is also relevant because the main stages of the formation of the central nervous system of mammals, including the early rhythms of activity, are similar. The critical period of development of the somatosensory system in rats is comparable in terms of its functional significance and the level of development of the cortex with the human fetus during the last trimester of gestation.

In the cortical activity of a premature baby at this time, one can also observe rhythms similar to early gamma oscillations and spindle-shaped oscillations in rats [Milh, M., Kaminska, A., Huon, C., Lapillonne, A., Ben-Ari, Y., & Khazipov, R./. Rapid Cortical Oscillations and Early Motor Activity in Premature Human Neonate // Cerebral Cortex ((2006)) 17 (7), 1582-1594. Doi: 10.1093 / cercor / bhl069; Vecchierini, M.-F., André, M., & d 'Allest, A. M. /. Normal EEG of premature infants born between 24 and 30 weeks gestational age: Terminology, definitions and maturation aspects // Neurophysiologie Clinique / Clinical Neurophysiology (2007), 37 (5), 311–323. DOI: 10.1016 / j.neucli.2007.10.008; Koolen, N., Dereymaeker, A., Räsänen, O., Jansen, K., Vervisch, J., Matic, V., Vanhatalo, S. / Early development of synchrony in cortical activations in the human // Neuroscience (2016 ). 322, 298-307. DOI: 10.1016 / j.neuroscience.2016.02.017; Kaminska, A., Delattre, V., Laschet, J., Dubois, J., Labidurie, M., Duval, A., ... Chiron, C. / Cortical Auditory-Evoked Responses in Preterm Neonates: Revisited by Spectral and Temporal Analyzes // Cerebral Cortex (2017), 1–16. DOI: 10.1093 / cercor / bhx206].

Since these rhythms are a hallmark of the electrical activity of the cortex during this prenatal period, knowledge of the mechanisms of their generation will make it possible to diagnose and, possibly, prevent the presence of cognitive and neurological disorders in humans.

The study of the mechanisms of neuronal activity consists in the study of the underlying formation of electrical activity of certain membrane ion channels of neurons.

For this, it is necessary to be able to forcibly activate or deactivate chemosensitive membrane ion channels by means of appropriate neurotransmitters.

This can be achieved in a variety of ways, but each of them presents specific technical challenges. Indeed, the classical application of neurotransmitters by placing them directly on the brain tissue has serious drawbacks, since the process of washing out the neurotransmitters in this case is impossible. This entails an irrational waste of animal material, when, in order to test one possible mechanism, it is necessary to dispose of the whole animal. At the same time, intracerebral injections of neurotransmitters can affect brain activity by increasing internal pressure on brain tissue due to the formation of an injection inclusion [Greig, NH / Optimizing drug delivery to brain tumors // Cancer Treatment Reviews (1987), 14 (1), 1-28. doi: 10.1016 / 0305-7372 (87) 90048-x]. The use of light-released neurotransmitters requires genetically modified animals and additional light sources [https://en.wikipedia.org/wiki/Photostimulation].

Thus, in modern neurobiological studies of the brain in vivo, the use of the perfusion technique of washing substances onto the surface of the brain using perfusion chambers has a number of advantages over conventional methods of studying the brain as a whole.

Further, the applicant lists factors detailing the advantages of the claimed technical solution described above as a whole.

The claimed technical solution based on a perfusion chamber:

- firstly, it allows the study of neuronal activity in vivo under long-term controlled pharmacological conditions.

-secondly, it provides unlimited access to a vast area of the brain for electrophysiological studies [Khazipov R, Holmes GL. / Synchronization of kainate-induced epileptic activity via GABAergic inhibition in the superfused rat hippocampus in vivo // J Neurosci. (2003) 23: 5337-53411, DOI: 10.1523 / jneurosci.23-12-05337.2003].

- thirdly, it provides the ability to regulate the temperature of the brain to eliminate the problem of uncontrolled loss of heat in the brain.

- fourthly, it provides the possibility of optical research of brain activity using the optical range of visible light and near infrared radiation.

Thus, a number of serious requirements are imposed on perfusion chambers, especially in optical and electrophysiological studies of activity in the brain.

As you know, in modern research, the electrical activity of the brain is usually recorded using special differential amplifiers, for example, the Neuralynx amplifier with a sampling rate of up to 40 kHz and an input range of -121 to 121 mV, where the recording electrode is located in the studied area of the brain, and the reference electrode is outside of it.

Registration of electrophysiological activity of brain neurons in a perfusion chamber has limitations on the use of metal parts in the construction of the chamber. Metals have high electrical conductivity and are good receivers of electromagnetic waves, which leads to the presence of noise radio signals in the immediate vicinity of the electrophysiological registration zone.

Such parts should be grounded or isolated from the perfusion solution. Thus, in the manufacture of the perfusion chamber, it is preferable to use dielectric materials such as plastic.

It should be borne in mind that the pharmacological application requires direct access of the perfusion solution to the intact brain without the dura mater, which leads to brain pulsations and / or hernia of the brain caused by internal blood pressure and cerebral pressure, respectively. [Khazipov R, Holmes GL. / Synchronization of kainate-induced epileptic activity via GABAergic inhibition in the superfused rat hippocampus in vivo // J Neurosci. (2003) 23: 5337-53411, DOI: 10.1523 / jneurosci.23-12-05337.2003].

As a result of the above, in order to resolve the issue of neutralizing the influence of such a negative consequence as pulsations and / or hernia of the brain, in the claimed design of the perfusion chamber, a neutralizing additional structure is provided in the form of a perforated plate, fixed on an adjustable holder, which is mounted (installed) on the experimental table.

Moreover, according to the applicant, attention should be paid to the presence in the claimed technical solution of the technological ability to regulate the temperature of the solution covering the brain to the required value, which also eliminates the problem of uncontrolled loss of brain heat.

Based on the foregoing, it is possible to make a logical conclusion that the claimed technical solution, namely, the design features of the claimed perfusion chamber, the system with its use and the method of its operation are the optimal technical solution for brain research and provide the opportunity to use all research methods available at the date of submission of the application materials, along with the ability to control the brain temperature to regulate to the required temperature and eliminate the problem of uncontrolled brain heat loss, which ensures the achievement of the maximum possible positive effect of brain research as a whole.

The perfusion chamber can also potentially be used in brain studies using neuroimaging technology.

The study of brain activity using the optical range of visible light and near infrared radiation assumes the presence of a wide open area of the brain surface and a perfusion solution with low absorption of light radiation in this range (500-900 nm).

In addition, the optical medium of the solution should be as homogeneous as possible, which means it should not contain foreign inclusions, turbulent vortices, etc.

Therefore, when using neuroimaging methods, conditions are necessary for creating a stationary flow of the perfusion solution, a stable depth of coverage of the brain with a perfusion solution, as well as leveling of perfusion artifacts.

Since any displacement of the perfusion chamber can lead to damage to the brain and / or recording electrodes, in vivo experiments also require a firm attachment of the perfusion chamber to the skull.

In this case, the reusability of the perfusion chamber is reduced, since the most widely used fastener materials with high adhesion, such as dental cement, do not allow the perfusion chamber to be disconnected from the skull.

Thus, it is necessary either to use fastening materials that can be easily cleaned, or to use cheap materials and high-speed technologies for the manufacture of perfusion chambers for the purpose of their one-time use.

The first option requires additional tests of the bonding agent for strength and toxicity.

In the second case, when designing a perfusion chamber, it is necessary to take into account both the required technical characteristics and the simplicity and speed of its production.

Manufacturing of parts of complex shape with internal cavities can be realized by such methods as casting [https://ru.wikipedia.org/wiki/Moulding], milling [https://ru.wikipedia.org/wiki/Milling] and turning [ https://ru.wikipedia.org/wiki/Turning_machining], laser stereolithography [https://ru.wikipedia.org/wiki/Laser_stereolithography] and additive 3D printing technology [https://ru.wikipedia.org/wiki / Additive_technology].

Each of them has its own advantages and disadvantages, however, any can be used as a working method for the manufacture of monolithic structures.

Moreover, modern automated equipment in each of the methods has the ability to use three-dimensional digital models of the product as a basis for the corresponding processing.

Also in this case, one of the important criteria when choosing a manufacturing method is to take into account the possibility of simplicity of product modification, since the solution of scientific problems often requires additional technical solutions.

Therefore, the manufacturing technology must take into account the balance between mass production and obsolescence of the relevance of specific studies.

Currently, additive 3D printing technology is becoming an increasingly popular method of manufacturing complex parts, especially for scientific research [C. Zhang, N.C. Anzalone, R.P. Faria, J.M. Pearce / Open-source 3D-printable optics equipment // PLoS ONE, 8 (2013), Article e59840, DOI: 10.1371 / journal.pone.0059840; M.S. Sulkin, E. Widder, C. Shao, K.M. Holzem, C. Gloschat, S.R. Gutbrod, et al. / Three-dimensional printing physiology laboratory technology // Am. J. Physiol.-Heart Circulatory Physiol., 305 (2013), pp. H1569-H1573, DOI: 10.1152 / ajpheart.00599.2013; T. Baden, A.M. Chagas, G. Gage, T. Marzullo, L.L. Prieto-Godino, T. Euler / Open Labware: 3-D printing your own lab equipment // PLoS Biol., 13 (2015), Article e1002086, DOI: 10.1371 / journal.pbio.1002086; J. Bravo-Martinez / Open source 3D-printed 1000mL micropump // HardwareX, 3 (2018), pp. 110-116, DOI: 10.1016 / j.ohx.2017.08.002; B.J. Winters, D. Shepler / 3D printable optomechanical cage system with enclosure // HardwareX, 3 (2018), pp. 62-81, DOI: 10.1016 / j.ohx.2017.12.001; B.C. Gross, J.L. Erkal, S.Y. Lockwood, C. Chen, D.M. Spence / Evaluation of 3D printing and its potential impact on biotechnology and the chemical sciences // Anal Chem, 86 (2014), pp. 3240-3253, DOI: 10.1021 / ac403397r]. This technology allows researchers to create functional devices for solving specific scientific and applied problems in the shortest possible time and at minimal cost and cost.

The variety of plastics used in 3D printing allows materials with different properties (elasticity, transparency, strength, flexibility, etc.) to be reproduced. Taken together, the benefits of 3D printing form an attractive and inexpensive method of plant design. Fused deposition 3D printing is an additive technology widely used in prototyping and industrial production. This technology has minimal post-processing procedures and the ability to create objects up to tenths of a millimeter in size, which allows you to quickly and easily create, including miniature devices.

In the table in FIG. 1 shows the parameters of the types of plastics most widely used in 3D printing by the method of layer-by-layer fusion (https://www.simplify3d.com/support/materials-guide/properties-table/), namely:

• ABS - acrylonitrile butadiene styrene,

• PLA - polylactide,

• HIPS - high strength polystyrene,

• PETG - polyethylene terephthalate glycol,

• Nylon - polyamide.

The characteristics of all presented plastics satisfy the conditions of use of the declared perfusion chamber, since they all have the properties of dielectrics and do not react with perfusion saline solutions.

In this case, the operation of the declared perfusion chamber is carried out by the applicant at a temperature of 30-40 ° C, low dynamic loads and for a short period of time, since studies of biological objects in vivo, according to ethical requirements, are carried out during the day, under physiological conditions and with anesthesia of the animal ...

Therefore, the applicant does not limit the scope of patent claims on the material of the claimed perfusion chamber only to the polylactide PLA given in the implementation section of the claimed technical solution of the present description, but expands the scope of patent claims to any plastics with dielectric properties, for example, the most widely used plastics in 3D printing. presented in Fig. 1.

Based on the foregoing, it can be concluded that additive technologies are the most acceptable for the manufacture of the claimed perfusion chamber and system, however, they do not limit the scope of the applicant's patent claims, and the choice of the method (technology) for manufacturing the perfusion chamber and system using it remains within the competence researcher / manufacturer and depends on many factors, such as mass production, the possibility and / or feasibility of reuse, the cost of the final product and demand in a competitive market.

From the investigated prior art, the applicant identified an invention under the patent RU No. 2175348 "A chamber for electrophysiological studies of receptive epithelia". The essence of the proposed device is a Chamber for electrophysiological studies of the receptive epithelium, containing a housing with a base and a cavity for saline solution, a protrusion located in it with a cuff for fixing the studied epithelium, electrodes and a housing cover with a light window, characterized in that one of the electrodes is a liquid a glass microelectrode mounted in a holder on the lid with the possibility of angular turns and movement along its axis to introduce this electrode into the epithelial cells, while the protrusion is made with the possibility of its vertical movement to regulate the gap between it and the body and is provided with a channel in which the light guide is placed.

The invention enables the use of various methods of potential withdrawal from any receptive epithelium, the fixation of the epithelium in the chamber without damage, and a simple and reliable supply of a light pulse.

The disadvantage of the known technical solution, in relation to the design in comparison with the claimed technical solution, is the lack of the possibility of studying the brain in vivo, namely, the lack of the possibility of attachment directly to the animal's skull, because is intended for the study of receptor epithelia in biology, biophysics, medicine, as well as for the study of the effect of drugs and biologically active substances on the epithelium and individual cells. In this case, the known device does not coincide with the claimed perfusion chamber in terms of structural features included in the claims of the claimed invention.

The disadvantages of the known technical solution with respect to the method of operation and intended use is that it cannot be used for its intended purpose due to the non-coinciding field of application as intended.

From the investigated prior art, the applicant has identified a useful model under the patent RU No. 81199 "Perfusion chamber for the surviving drug". The essence of the proposed device is a perfusion chamber for the surviving drug, containing a tube for supplying saline solution passing through the wall of the water bath, its filling and its cover with an opening above the working chamber, as well as a discharge tube for the spent saline solution, characterized in that the tube for supplying saline solution is made made of polyethylene and equipped at the outlet with a glass microcapillary placed above the hole in the water bath lid, the outlet tube connects the hole in the bottom of the working area through the filling and the wall of the water bath with the reservoir for the spent saline solution.

The advantages of the proposed device for the perfusion chamber are that mechanical movements of the microscopic surviving drug in the perfusion chamber are excluded, and water hammer of the drug from the peristaltic pump is excluded.

The disadvantage of the known technical solution, with respect to the design in comparison with the claimed technical solution, is the lack of the possibility of studying the brain in vivo, namely, the lack of the possibility of attachment directly to the animal's skull. In this case, the known device does not coincide with the claimed perfusion chamber in terms of structural features included in the claims of the claimed invention.

The disadvantages of the known technical solution with regard to the method of operation and intended use is that it also cannot be used for its intended purpose due to the non-matching field of application as intended.

From the investigated prior art, the applicant identified the invention "Device for microelectrode research of biological objects" according to patent SU No. 1017722. The essence of the prototype is a device for microelectrode research of biological objects, containing a housing with interconnected preliminary chamber, a suction chamber, a working perfusion chamber made in the form a hollow cylinder, the upper base of which is flush with the body, and the center of symmetry lies at an equal distance from the center of symmetry of the preliminary chamber and the suction chamber, is additionally equipped with a device for maintaining a stable temperature, made in the form of a hollow cylinder with a channel for introducing a temperature sensor into the working chamber, installed inside the working perfusion chamber. In this case, the outer wall of the cylinder is made in the form of a spiral-shaped rectangular groove, equipped with channels for supplying and removing the coolant, and the suction chamber is made in the form of a hollow elliptical cylinder and is equipped with channels for introducing indifferent electrodes.

The disadvantage of the known technical solution, with respect to the design in comparison with the claimed technical solution, is the lack of the possibility of studying the brain in vivo, namely, the lack of the possibility of attachment directly to the animal's skull. In this case, the known device does not coincide with the claimed perfusion chamber in terms of structural features included in the claims of the claimed invention.

The disadvantages of the known technical solution with regard to the method of operation and intended use is that it also cannot be used for its intended purpose due to the non-matching field of application as intended.

From the investigated prior art, the applicant identified an analogue that coincides with the claimed technical solution for the purpose of "Synchronization of kainate-induced epileptic activity by GABAergic inhibition in the perfused rat hippocampus in vivo" [Khazipov R, Holmes GL. / Synchronization of kainate-induced epileptic activity via GABAergic inhibition in the superfused rat hippocampus in vivo // J Neurosci. (2003) 23: 5337-53411, DOI: 10.1523 / jneurosci.23-12-05337.2003]. The essence is a cylindrical cut 2 mm long from a 1 ml plastic syringe, to which a lycra mesh is glued from below using cyanoacrylate glue.

The use of the known device consisted in the fact that it was placed in the cavity formed after the removal of a part of the cortex in the brain, so that the mesh weakly pressed the hippocampus to prevent pulsation of the brain. Previously, the skull of the anesthetized animal was cleared of the scalp and a round hole was made in the cranial bone over the proposed area of study. Part of the cerebral cortex above the study area was removed by vacuum suction until the hippocampus surface was exposed and a cavity was formed. The attachment of the device to the animal was carried out by means of dental cement placed between the outer wall of the prototype and the cranial bone surrounding the upper edge of the prototype. During the fixation of the prototype, the degree of pressing of the hippocampus surface was controlled only visually. In this case, the rat was fixed in a stereotaxic apparatus by fixing the skull to the technical beam of the apparatus in the nasal part of the skull, as well as in the occipital part of the skull to conical rods using dental cement. The supply and intake of the perfusion solution was carried out by means of two metal hollow needles, where at each one of the ends was placed in the working volume of the perfusion chamber, and the other was included in the perfusion system.

The disadvantages of the known device, in comparison with the claimed technical solution, are:

- lack of structural features to control the temperature of the perfusion solution;

- the use of a static system of pressing the open surface of the brain in the perfusion chamber, which leads to a lack of regulation of the degree of pressure on the brain tissue, limits the use only on flat areas of the animal's skull, and also makes it impossible to change the study area locally;

- lack of structural features for leveling perfusion artifacts;

- the method of attaching the perfusion chamber to the animal's skull does not allow subsequent control of the relative position of the perfusion chamber;

- the lack of a wide viewing angle of the investigated area, which limits the possibilities of carrying out optical studies;

- the presence of several components.

In this case, the known device does not coincide with the declared perfusion chamber in terms of structural features, but coincides with it in terms of purpose.

Due to the fact that the analysis of the studied prior art did not allow identifying analogs that are the closest in terms of the set of coinciding features in relation to the perfusion chamber, the independent claims are drawn up without a limiting part.

The aim of the claimed invention is to develop a perfusion chamber for studying the activity of the brain in vivo, a system with its use and a method of operating the system. The claimed invention makes it possible to use the principles of a perfusion chamber to study the activity of the brain in vivo.

The technical result of the claimed invention is the development of a monolithic design of a perfusion chamber, a system with its use and a method of operation of the system, providing:

- the ability to control the temperature of the inlet and outlet perfusion solution due to a specially provided channel for entering the temperature sensor of the inlet perfusion solution 4 and the feasibility of placing the temperature sensor in a specially located opening for the outlet of the perfusion solution 9;

- the use of an adjustable system of pressing the perfusion chamber to the open surface of the brain due to external structures with the possibility of conducting electrophysiological and optical studies of the brain, as well as the absence of the need for preliminary connection with the clamping devices of the brain tissue, which makes it possible to regulate the degree of pressure on the brain tissue, to use areas of the animal's skull with any curvature, and also makes it possible to local change the study area;

- the presence of structural features for leveling the artifacts of perfusion, namely, the preliminary chamber 5, the input spiral groove 6 and the output spiral groove 8;

- the presence of an adjustable system of the relative position of the perfusion chamber in space due to the use of specially provided mounting slots 10 for attachment to the stereotaxic apparatus;

- the ability of the perfusion camera to provide a wide viewing angle of the investigated area, which in turn provides the possibility of conducting optical studies as well;

- lack of components in the perfusion chamber, which simplifies operation.

The essence of the claimed group of inventions is a perfusion chamber for studying the activity of the brain in vivo, which is a monolithic structure made of plastic with dielectric properties, using a technology based on a three-dimensional digital model, consisting of a supporting body containing a channel for entering a perfusion solution, channel for inserting the reference electrode, channel for entering the temperature sensor of the inlet perfusion solution, preliminary chamber, inlet spiral groove between the preliminary and main chambers, main chamber, outlet spiral groove between the main chamber and the outlet for perfusion solution, outlet for perfusion solution, mounting grooves, a place for attaching the animal's skull; while the inlet channels of the perfusion chamber are made in the form of through ducts that go into the preliminary chamber, the preliminary chamber is connected to the main chamber using an inlet spiral groove; the outlet from the preliminary chamber is located above the bottom of the main chamber with the possibility of gravitational fluid supply, the main chamber has the shape of a truncated paraboloid of revolution, while the main chamber is connected by an outlet spiral chute with an opening for the outlet of the perfusion solution in the lower part of the perfusion chamber with the possibility of accommodating a second temperature control temperature sensor output solution; and along the perimeter of the perfusion chamber there are three mounting slots for attaching to the stereotaxic apparatus; the place of attachment of the animal's skull is located below the main chamber and is made in the form of a truncated cone, adapted to the size and curvature of the skull of the animal under study. A system for the study of brain activity in vivo using a perfusion chamber, consisting of a perfusion chamber and a pressure of the cerebral surface, while the perfusion chamber is made in the form of a monolithic structure of plastic with dielectric properties, using technology based on a three-dimensional digital model, consisting of a carrier body containing a channel for the inlet of a perfusion solution, a channel for an input of a reference electrode, a channel for an input of a temperature sensor of an inlet perfusion solution, a preliminary chamber, an inlet spiral groove between the preliminary and main chambers, a main chamber, an outlet spiral groove between the main chamber and an outlet opening perfusion solution, opening for the outlet of the perfusion solution, mounting grooves, a place for attaching the animal's skull; while the inlet channels of the perfusion chamber are made in the form of through ducts that go into the preliminary chamber, the preliminary chamber is connected to the main chamber using an inlet spiral groove; the outlet from the preliminary chamber is located above the bottom of the main chamber with the possibility of gravitational fluid supply, the main chamber has the shape of a truncated paraboloid of revolution, while the main chamber is connected by an outlet spiral chute with an opening for the outlet of the perfusion solution in the lower part of the perfusion chamber with the possibility of placing a second sensor

temperature control of the temperature of the output solution; and along the perimeter of the perfusion chamber there are three mounting slots for attaching to the stereotaxic apparatus; the place of attachment of the animal's skull is located at the bottom of the main chamber and is made in the form of a truncated cone, adapted to the size and curvature of the skull of the animal under study; at the same time, the pressure of the cerebral surface is made of plastic using a technology based on a three-dimensional digital model and is a plastic disc with a smooth surface and perforated with a set of periodic circular holes, equipped with a technical board along the perimeter of the disc with the possibility of reinforcing the structure, while the reinforced plastic disc is connected with a fastening element using two handles, and the fastening element is inserted into the clamp holder, while the brain surface clamp is replaceable in case of breakage using technology based on a three-dimensional digital model, while the clamp holder is installed in the micromanipulator with the ability to adjust both the position and the degree of pressure on the brain tissue; the clamp of the cerebral surface is placed directly on the surface of the brain in the main chamber of the perfusion chamber after attachment to the animal and removal of a portion of the skull and dura mater. A system for studying brain activity in vivo using a perfusion chamber, consisting of a perfusion chamber, pressing the cerebral surface, an attachment system to a stereotaxic apparatus and a flow-through perfusion system, while the perfusion chamber is a monolithic structure made of plastic with dielectric properties, using technology based on a three-dimensional digital model, consisting of a supporting body containing a channel for the inlet of a perfusion solution, a channel for a reference electrode, a channel for a temperature sensor of an inlet perfusion solution, a preliminary chamber, an inlet spiral groove between the preliminary and main chambers, a main chamber , an outlet spiral groove between the main chamber and the opening for the outlet of the perfusion solution, the opening for the outlet of the perfusion solution, mounting grooves, a place for attaching the animal's skull; the inlet channels of the perfusion chamber are made in the form of through ducts opening into the preliminary chamber, the preliminary chamber is connected to the main chamber using an inlet spiral groove; the outlet from the preliminary chamber is located above the bottom of the main chamber with the possibility of gravitational fluid supply, the main chamber has the shape of a truncated paraboloid of revolution, while the main chamber is connected by an outlet spiral chute with an opening for the outlet of the perfusion solution in the lower part of the perfusion chamber with the possibility of accommodating a second temperature control temperature sensor output solution; and along the perimeter of the perfusion chamber there are three mounting slots for attaching to the stereotaxic apparatus; the place of attachment of the animal's skull is located below the main chamber and is made in the form of a truncated cone, adapted to the size and curvature of the skull

the investigated animal; at the same time, the pressure of the cerebral surface is made of plastic using a technology based on a three-dimensional digital model and is a plastic disc with a smooth surface and perforated with a set of periodic circular holes, equipped with a technical board along the perimeter of the disc with the possibility of reinforcing the structure, while the reinforced plastic disc is connected with a fastening element using two handles, and the fastening element is inserted into the clamp holder, while the brain surface clamp is replaceable in case of breakage using technology based on a three-dimensional digital model, while the clamp holder is installed in the micromanipulator with the ability to adjust both the position and the degree of pressure on the brain tissue; the clamp of the cerebral surface is placed directly on the surface of the brain in the main chamber of the perfusion chamber after attachment to the animal and removal of a portion of the skull and dura mater; in this case, the system of attachment to the stereotaxic apparatus is represented by supporting columns on which the perfusion chamber rests in the places of the mounting slots, while bolts fixed in the stereotaxic apparatus pass through the mounting slots of the perfusion chamber and the supporting columns, and the declared perfusion chamber is attached to the animal in the designated place attachments to the skull of an animal; in this case, the flow-through perfusion system is a series of elements of the perfusion system, where each next element is lower than the previous one to ensure gravitational fluid supply, including a buffer tank, a connecting tube, a dropper-regulator, a connecting tube, a heating linear flow element, a perfusion chamber, a flexible connection made of two fittings connected by a flexible tube, a regulator of the perfusion solution level, a vacuum suction channel connected to a vacuum suction system; in this case, the buffer tank is connected through a connecting tube with a dropper-regulator with the ability to regulate the flow rate of the perfusion solution, as well as with the possibility of galvanic isolation of the perfusion solution in the perfusion chamber and the buffer tank, while the dropper-regulator, in turn, is connected through a tube with a heating linear flow element with the ability to set the required temperature of the perfusion solution, while the outlet of the heating linear flow element is connected through a tube to the channel for the inlet of the perfusion solution of the perfusion chamber by means of a tube, while the opening for the outlet of the perfusion solution of the perfusion chamber is connected by means of a flexible connection made of two fittings connected flexible tube, and is connected with the regulator of the level of the perfusion solution with the possibility of its reciprocating vertical movement and the possibility of thereby regulating the level of the perfusion solution in the main chamber due to the effect of communicating vessels, with the possibility of removing excess perfusion

solution in the level regulator of the perfusion solution through the vacuum suction channel connected to the vacuum suction system. A method for studying the activity of the brain in vivo using the system according to claim 3, consisting in the fact that a preliminary preparatory operation is carried out to remove the scalp and integumentary tissues over the warmed animal under anesthesia, then the animal's skull is attached to the perfusion chamber at the intended attachment point using first cyanoacrylate glue, then dental cement, then the perfusion chamber is attached to the stereotaxic apparatus using mounting slots, into which bolts are then inserted passing through the support columns, on which the claimed perfusion chamber rests; then the perfusion chamber is connected to the flow-through perfusion system, for which the outlet of the heating linear flow-through element is connected to the channel for the inlet of the perfusion solution of the perfusion chamber by means of a tube, then the opening for the outlet of the perfusion solution of the perfusion chamber is connected by means of a flexible connection made of two fittings connected by a flexible tube, to the perfusion solution level regulator, which makes a reciprocating vertical movement and thus regulates the perfusion solution level in the main chamber due to the effect of communicating vessels, while the excess perfusion solution in the perfusion solution level regulator is removed through a vacuum suction channel connected to a vacuum system suction; then, temperature sensors are placed through the channel for the input of the input perfusion solution in the preliminary chamber and the holes for the output of the perfusion solution to control the actual temperature of the input and output solution; then a section of the skull and dura mater is removed, after which a clamp is placed on the brain surface directly on the brain surface in the main chamber of the perfusion chamber; then, electrophysiological studies of brain activity are carried out using a reference electrode in the form of a silver chlorinated wire placed in the channel for the input of the reference electrode and recording electrodes located in the brain through the holes of the perforated reinforced plastic disc; then, optical studies of brain activity are carried out using video surveillance systems of electromagnetic radiation of the visible and infrared ranges reflected from the brain tissue in the areas of the holes of the perforated reinforced plastic disk.

The claimed technical solution is illustrated in Fig. 1 - Fig. 7.

Figure 1 shows Table 1, which presents the parameters of the types of plastics most widely used in 3D printing by layer-by-layer deposition and having dielectric properties.

Figure 2 shows a 3D model of the claimed perfusion chamber,

2A is a top view,

2B - bottom view, where:

1 - carrying body,

2 - channel for the inlet of the perfusion solution,

3 - channel for entering the reference electrode,

4 - channel for entering the temperature sensor of the inlet perfusion solution,

5 - preliminary chamber,

6 - inlet spiral groove,

7 - main camera,

8 - outlet spiral chute,

9 - hole for the outlet of the perfusion solution,

10 - mounting grooves,

11 - the place of attachment of the animal's skull.

2B - 3D model of the clamp of the brain surface and 3D model of the clamp holder, where:

25 - pressing the surface of the brain,

26 - clamp holder.

Figure 3 shows a diagram of a possible attachment of the claimed perfusion chamber to the skull of an animal and mounting on a stereotaxic apparatus,

3A is a left side view,

3B - right side view, where

12 is a schematic representation of an animal,

13 is a schematic representation of the moving part of the stereotaxic apparatus,

14 - mounting bolts, e.g. M4,

15 - support columns for creating a support for the claimed perfusion chamber.

Figure 4 shows a diagram of a possible connection of a flow-through perfusion system using the claimed perfusion chamber, where:

2 - channel for the inlet of the perfusion solution,

5 - preliminary chamber,

6 - inlet spiral groove,

7 - main camera,

8 - outlet spiral chute,

9 - opening for the outlet of the perfusion solution,

11 - the place of attachment of the animal's skull,

12 is a schematic representation of an animal,

16 - buffer tank,

17 - dropper-regulator,

18 - heating linear flow element,

19 - flexible connection, consisting of two fittings connected by a flexible tube, for example, silicone,

20 - regulator of the level of the perfusion solution,

21 - vacuum suction channel.

Figure 5 shows:

5A is a diagram of the use of the claimed perfusion chamber for multichannel recording of electrical signals from the rat brain at different brain temperatures and during stimulation of the vibrissa;

5B is a diagram of the use of the claimed perfusion chamber for multichannel recording of electrical signals from the rat brain at different brain temperatures and during stimulation of the vibrissa, where:

22 - temperature sensor,

23 - reference electrode,

24 - recording multichannel electrode,

25 - pressing the surface of the brain,

26 - clamp holder.

5B are graphs illustrating the results of using the claimed perfusion chamber for multichannel recording of electrical signals of the rat somatosensory cortex at different brain temperatures and during stimulation of the vibrissa, where the black solid curves are the evoked local field potential (LPP), and the red vertical lines are multicellular activity (MCA) in the representation of vibrissa in the somatosensory cortex of the rat brain.

Figure 6 shows a possible use of the claimed perfusion chamber for the simultaneous registration of the evoked internal optical signal (BOC) and electrical signals of the somatosensory cortex of the rat brain during stimulation of vibrissae.

6A is a diagram for recording an internal optical signal, where:

7 - main camera,

12 is a schematic representation of an animal,

25 - pressing the surface of the brain,

27 - CCD camera

28 - optical system, for example, a microscope,

29 - infrared LED,

30 - green / red LED.

The dashed black line represents the approximate optical path of reflected light rays through the optical system 28 to the CCD camera 27, the approximate cone of illumination is indicated by maroon and green / red lines for infrared diode 29 and green / red LED 30, respectively.

6B is an illustration of VOS visualization, where 24 is a recording multichannel electrode, 25 is a clamping of the brain surface, orange is the area around the recording multichannel electrode 24, not covered by the clamp of the brain surface 25, black is an arbitrary area covered by a clamp of the brain surface 25.

6B are graphs illustrating the dependence of the change in hemoglobin concentration during a series of stimuli, where orange indicates a change in the area around the recording multichannel electrode 24, not covered by the pressure of the brain surface 25, black indicates a change in an arbitrary area covered by the pressure of the brain surface 25.

6D - graphs illustrating the dependence of the change in the concentration of the pseudochromophore corresponding to light scattering, where orange indicates a change in the area around the recording multichannel electrode 24, not covered by the clamp of the brain surface 25, black indicates the change in an arbitrary area covered by the clamp of the brain surface 25.

6D - graphs illustrating the registration of evoked electrical signals of the somatosensory cortex of the rat brain using a recording multichannel electrode 24 and during stimulation of vibrissae, where the black solid curve is the evoked local field potential (LPP), and the red vertical lines are multicellular activity (MCA) in the representative vibrissae in the somatosensory cortex of the rat brain. In all illustrations, the period of vibrissa stimulation is indicated by a gray translucent area.

Figure 7 shows Table 2, which shows the purpose of the elements of the claimed perfusion chamber and the system with its use and the signs of the method of its operation (to simplify the formation of the features of the formula of the claimed technical solution and the selection of essential features from them).

The set goals and the claimed technical result are achieved by the fact that with the help of additive manufacturing technologies, a monolithic structure of the declared perfusion chamber is created , made of plastic using technology based on a three-dimensional digital model with dielectric properties (Fig. 2), then a system is created using the declared perfusion chamber .

The claimed perfusion chamber is a monolithic product made using additive 3D printing technology and represents the following design (Fig. 2).

The carrier body 1 of the perfusion chamber contains three inlet channels 2, 3, 4, two open chambers: a preliminary chamber 5 and a main chamber 7, connected by an inlet spiral chute 6, as well as an opening for the outlet of the perfusion solution 9, connected to the main chamber 7 by an outlet spiral chute 8. Three input channels 2, 3, 4 are used to enter the perfusion solution, place the reference electrode (Fig. 4, pos. 22) and the temperature sensor (Fig. 4, pos. 23) of the perfusion solution, respectively. The inlet channels 2, 3, 4 are made in the form of through ducts that exit into the preliminary chamber 5.

The applicant explains that the presence of a preliminary chamber 5 is necessary to protect the main chamber 7 from air bubbles and water hammer, which are possible with artifacts in the flow-through perfusion system.

The preliminary chamber 5 is connected to the main chamber 7 by means of an inlet spiral groove 6, the shape and inclination of which is used both against direct water hammer and to ensure gravitational fluid supply. In this case, the outlet from the preliminary chamber 5 is located above the bottom of the main chamber 7, to ensure gravitational fluid supply.

The spiral shape of the inlet spiral groove 6 is due to:

firstly, the difference in heights of the outlet of the preliminary chamber 5 and the entrance of the main chamber 7;

second, by avoiding a straight path of perfusion solution delivery;

thirdly, by ensuring laminarity and stationarity of the perfusion solution flow.

The main camera 7 has the shape of a truncated paraboloid of rotation to provide a larger viewing angle for optical research, as well as to provide free space for placement of research equipment.

The main chamber 7 is also connected with an outlet spiral chute 8 with an outlet for the perfusion solution 9 located in the lower part of the claimed perfusion chamber.

The spiral shape of the outlet spiral groove 8 is due to:

- firstly, the difference in the heights of the outlet of the main chamber 7 and the opening for the outlet of the perfusion solution 9;

- secondly, by avoiding the straight path of the perfusion solution supply;

- thirdly, ensuring the laminarity and stationarity of the flow of the perfusion solution.

In the opening for the outlet of the perfusion solution 9, if necessary, a second temperature sensor (not shown in the figure) can be placed to control the temperature of the outlet perfusion solution.

At the bottom of the main chamber 7, an attachment point for the skull of the animal 11 is provided, made in the form of a truncated cone and adapted, for example, to the size and curvature of the skull of a rat.

Along the perimeter of the perfusion chamber, there are three mounting slots 10 for attachment to the stereotaxic apparatus.

The size of the perfusion chamber for the experiment shown in Examples 3-5 (with rats) is at most 30 x 49 x 3 mm.

At the same time, the applicant explains that the size of the perfusion chamber by the applicant is not indicated in the claims, since the sample of the perfusion chamber shown in the present description is designed to work specifically with rats, but can be scaled to the size of any studied animal, for example, a rabbit, ferret, cat, etc. dr.

The claimed perfusion chamber has the technical ability to be attached to the skull of an animal by means of, for example, dental cement and in compliance with the stipulated ethical standards. In this case, the perfusion chamber itself is a supporting structure and can, in turn, be attached both to the stereotaxic apparatus, thus being simultaneously a support for the animal and, at the user's choice, to other necessary positioning elements, if necessary.

All elements for ensuring stationary perfusion are made directly in the carrier body to create conditions for stationary and stable perfusion. This can be achieved through either casting, milling and turning, laser stereolithography, additive 3D printing technology, or any other technology based on a 3D digital model. As an example, the claimed perfusion chamber is shown, manufactured using additive 3D printing technology, which ensures fast reproducibility and ease of manufacture and also eliminates the time and material costs for production preparation.

Next, the applicant describes a clamp for the brain.

To avoid herniation of the brain outward after removal of a sufficiently large fragment of the skull and dura mater, a brain clamp (Fig. 2B) made using additive 3D printing technologies is used.

The clamp is a thin (~ 0.2 mm) plastic disc with a smooth surface and perforated with a set of periodic round holes ~ 1 mm.

For ease of use, the clamp for the brain is divided into two parts - the clamp of the brain surface 25 and the clamp holder 26 (Fig. 2B). This allows you to quickly and easily remove a broken clamp and significantly reduce re-printing times.

The strength of the structure has been increased by adding a technical board around the perimeter of the disc.

The brain surface clamp in the form of a reinforced plastic disk is connected to the fastening element by means of two handles, while the fastening element is inserted into the clamp holder, making it possible to easily and quickly replace the brain surface clamp in the event of a breakdown using technology based on a three-dimensional digital model ...

The clamp holder is inserted into a manipulator, for example, a three-position Narishige M-152 micromanipulator, for precise adjustment of the position and degree of pressure on the brain tissue. The cerebral surface clamp is placed directly on the brain surface in the main chamber of the perfusion chamber after attachment to the animal and removal of a portion of the skull and dura mater, while the cerebral surface clamp is made of plastic using technology based on a three-dimensional digital model.

In this experiment, a brain clamp was placed directly above the brain surface immediately after removing part of the skull bone (~ 4 mm ² ) and part of the dura mater.

In this case, the applicant points out that the claimed perfusion chamber can be used without pressing the cerebral surface, for example, in the case of optical studies.

Thus, the claimed technical solution retains all the advantages of research on objects in vivo, but at the same time there is no need for preliminary connection with the pressure devices of the brain tissue, which potentially allows to regulate the degree of pressure on the brain tissue, to use parts of the animal's skull with any curvature, and also makes it possible local change of the study area due to external structures.

At the same time, the design of the claimed perfusion chamber contains features that contribute to the leveling of perfusion artifacts, namely, the presence of a preliminary chamber 5, a spiral inlet groove 6 and a spiral outlet groove 8.

Next, the applicant describes a system for attaching to a stereotaxic apparatus.

The system of attachment to the stereotaxic apparatus is represented by support columns 15, on which the declared perfusion chamber rests, in the places of the mounting slots 10, while bolts 14 pass through the mounting slots 10 and support columns, for example, M4 bolts, fixed in the moving part of the stereotaxic apparatus 13, and the claimed perfusion chamber is attached to the animal at the intended attachment point to the skull of the animal 11. The attachment system to the stereotaxic apparatus facilitates the regulation of the relative position of the perfusion chamber in space.

Also, the design of the claimed perfusion chamber provides the ability to control the temperature of the inlet and outlet perfusion solution due to a specially provided channel 4 for entering a temperature sensor and the feasibility of placing a temperature sensor in a specially located opening 9 for withdrawing the perfusion solution.

The three-dimensional model of the claimed perfusion chamber was developed in the free design environment Tinkercad (Autodesk), which is also applicable to all the technologies described above.

In the examples presented, additive 3D printing technology was used as a manufacturing method, and as a material for the perfusion chamber, plastics for 3D printing by layer-by-layer fused technology were used (see the table in Fig. 1), for example, polylactide (PLA), which are dielectrics and not reacting with perfusion saline solutions. The operation of the declared perfusion chamber occurs at a temperature of 30-40 ° C, low dynamic loads and for a short period of time. Therefore, all the characteristics of the examples of materials presented in the Table in Fig. 1 satisfy the expected operating conditions. At the same time, the applicant does not limit the scope of patent claims on the material of the claimed perfusion chamber only with polylactide PLA, given in the implementation section of the claimed technical solution of the present description, but expands the scope of patent claims to any plastic with dielectric properties, for example, the most widely used plastics in 3D printing. presented in Fig. 1.

Tubes such as silicone tubes are used to connect the perfusion chamber to the perfusion system.

Next, the applicant describes a flow-through perfusion system.

A flow-through perfusion system is a series of elements of a perfusion system, where each next element is lower than the previous one to ensure gravitational fluid supply, including a buffer tank 16, a connecting tube, a dropper-regulator 17, a connecting tube, a heating linear flow element 18, the declared perfusion chamber. a flexible connection 19 made of two fittings connected by a flexible tube, a perfusion solution level regulator 20, a vacuum suction channel 21 connected to a vacuum suction system; while the buffer reservoir 16 is connected through a connecting tube to the dropper-regulator 17 with the possibility of adjusting the flow rate of the perfusion solution, as well as with the possibility of galvanic isolation of the perfusion solution in the claimed perfusion chamber and the buffer reservoir 16, while the dropper-regulator 17 is in turn connected by a connecting tube with a heating linear flow element 18 with the possibility of setting the required temperature of the perfusion solution, while the outlet of the heating linear flow element 18 is connected by means of a tube with a channel for the inlet of the perfusion solution 2 of the declared perfusion chamber through a tube, while the opening for the outlet of the perfusion solution 9 of the declared perfusion the chamber is connected by means of a flexible connection made of two fittings connected by a flexible tube, for example, a silicone one, and connected to the regulator of the level of the perfusion solution 20 with the possibility of its return translational vertical movement and the possibility of thereby regulating the level of the perfusion solution in the main chamber due to the effect of communicating vessels, with the possibility of removing excess perfusion solution in the level regulator of the perfusion solution 20 through the vacuum suction channel 21 connected to the vacuum suction system.

Below are examples of specific implementation of the use of the claimed technical solution.

Example 1. Attachment of the claimed perfusion chamber to an animal and mounting on a stereotaxic apparatus (Fig. 3).

To perform the experiments, male and female Wistar rats from postnatal days [P] 4-7 (3 rats) were used.

The operation was performed under isoflurane anesthesia, for example, 5% during induction and 1.5% during surgery.

First, a preliminary preparatory operation is performed to remove the scalp and integumentary tissues over the warmed animal under anesthesia.

After removal of the scalp, the skull is cleaned and covered with cyanoacrylate glue, except for a window of ~ 50 mm ² above the cortex for placing the electrodes.

The animals were warmed to recover from anesthesia.

During the recording, the head is attached to the perfusion chamber at the intended attachment point with cyanoacrylate glue (for pre-fixation) and then with dental cement (for secure fixation).

The animals are surrounded with cotton wool and heated with a thermal mat, for example, at 35–37 ° C.

During the experiments, the rats were anesthetized by intraperitoneal injection of urethane at a concentration of, for example, 1.5 g / kg.

The claimed perfusion chamber is attached to the animal 12 at the intended attachment point to the skull of the animal 11 (Fig. 2), as well as to the stereotaxic apparatus 13, for example, in the manner shown in Fig. 3: bolts 14 are inserted into the mounting grooves 10, for example, bolts M4, which pass through the support columns 15, on which the claimed perfusion chamber rests, and are fixed in the stereotaxic apparatus 13 by means of, for example, M4 nuts.

Example 2. Connecting a flow-through perfusion system (Fig. 4).

After connecting the claimed perfusion chamber to the animal's skull and fixing it on a stereotaxic apparatus, for example, as shown in Example 1, connect the claimed perfusion chamber to a flow-through perfusion system, for example, as shown in Fig. 4.

As a perfusion solution, for example, artificial cerebrospinal fluid of the following composition is used: 126 mM NaCl, 3.5 mM KCl, 1.2 mM NaH ₂ PO ₄ , 2 mM CaCl ₂ , 13 mM MgCl ₂ ).

The flow-through perfusion system is organized as follows.

Each next element of the perfusion system is located below the previous one to provide gravitational fluid delivery.

The perfusion solution flows from the buffer reservoir 16 to the dropper-regulator 17 through a connecting tube, for example, silicone.

The dropper-regulator 17 is used for galvanic isolation of the perfusion solution in the declared perfusion chamber and the buffer tank 16, as well as for regulating the flow rate of the perfusion solution.

Next, the dropper-regulator 17 is connected using a tube, for example, a silicone one, to the heating linear flow element 18 to achieve the required temperature of the perfusion solution.

The outlet of the heating linear flow element 18 is connected to the channel for the inlet of the perfusion solution 2 using a tube, for example, a silicone one.

After passing through the system of channels and chambers of the claimed perfusion chamber, the perfusion solution leaves it through the opening for the outlet of the perfusion solution 9. The opening for the outlet of the perfusion solution 9 by means of a flexible connection 19, consisting of two fittings connected by a flexible tube, for example, a silicone one, is connected to a level regulator perfusion solution 20, having the ability to move up and down and thus regulate the level of the perfusion solution in the main chamber 7 due to the effect of communicating vessels.

Excess perfusion solution in the perfusion solution level regulator 20 is removed through the vacuum suction channel 21 connected to the vacuum suction system.

Example 3. Use of the claimed perfusion chamber for multichannel recording of electrical signals from the rat brain under different temperature conditions during stimulation of the vibrissa (Fig. 5).

The applicant has verified the possibility of using the claimed perfusion chamber for electrophysiological studies of brain activity at different temperatures.

For this, the claimed perfusion chamber is attached to the animal and the stereotaxic apparatus according to Example 1.

Then the claimed perfusion chamber is connected to a flow-through perfusion system according to Example 2.

Example 3 uses the flow-through perfusion system shown in FIG. 3. The change in brain temperature is made by changing the temperature of the perfusion fluid. Temperature sensors are placed through the channel for the input of the temperature sensor of the inlet perfusion solution 4 in the preliminary chamber 5 and the opening for the outlet of the perfusion solution 9, then the actual temperature of the inlet and outlet solution is monitored.

If there is no need to control the actual temperature of the inlet and outlet solution, temperature sensors are not placed.

In order to avoid herniation of the brain outward after removal of a sufficiently large fragment of the skull and dura mater, the applicant, prior to the experiment, uses a brain clamp 25 (Fig. 5A), made using additive 3D printing technologies, which (the brain clamp) is placed after removal section of the skull and dura mater directly on the surface of the brain in the main chamber 7 of the claimed perfusion chamber.

The clamp is a thin (~ 0.2 mm) perforated plastic disc with a smooth surface and a set of periodic round holes (perforations) with a diameter of ~ 1 mm. The strength of the clamping structure is increased by the applicant by adding a bead around the perimeter of the disc.

A reinforced perforated plastic disc with a fastener is connected by two handles. For ease of use, the brain clamp is divided by the applicant into two parts - the actual clamp of the brain surface 25 and the clamp holder 26. This allows you to easily and quickly replace the clamp in case of breakage, with a significant reduction in time through the use of reprint.

The clamp holder is inserted into the micromanipulator for fine adjustment of the position and degree of pressure on the brain tissue. In this experiment, a brain clamp was placed directly above the brain surface immediately after preliminary removal of a part of the skull bone (~ 4 mm ² ).

The claimed system in the form of a combination of the claimed perfusion chamber and a clamping device for the brain, made using 3D printing, provides a successful study of the effect of brain temperature on neuronal activity in the representation of one vibrissa in the rat cerebral cortex under perfusion.

Then, before the experiment, the vibrissae on the animal's face were cut to a length of 3 mm. The vibrissae were stimulated by the piezoelectric deflection method.

A piece of sponge was glued to the end of the piezoelectric deflector (Noliac) and a vibrissa was inserted into the sponge to stimulate all vibrissae.

To induce a deflection of the piezoelectric deflectors, rectangular pulses with an amplitude of 80–90 V and a duration of 10 ms were used.

To avoid suppression of the evoked response, vibrissae were stimulated every 50 s.

An in-line solution heater (Warner instruments) was used to control the temperature of the solution.

Temperature sensors 23 (Fig. 5A) (thermistors, Warner instruments) were placed through the channel for the input of the temperature sensor of the inlet perfusion solution 4 in the preliminary chamber 5 and the outlet for the perfusion solution 9 to control the actual temperature of the inlet and outlet solution.

The data from temperature sensors 23 were processed using a Digidata 1440A electrical signal amplifier (Axon Instruments).

The results obtained were used to estimate the temperature of the solution in the main chamber 7.

At the end of the experiment, the temperature of the body and brain was also measured using temperature sensors, placing them rectally and directly in the brain, respectively.

All protocols used an additional rectal temperature sensor to monitor the body temperature of the animal.

The reference electrode 22 (Fig. 5A) in the form of a silver chlorinated wire was placed in a special channel for the introduction of the reference electrode 3.

Extracellular recordings of evoked cortical activity were performed using, for example, a silicon single-rod 16-channel electrode (Neuronexus Technologies, USA with a distance of 100 μm between the electrodes). The recording multichannel electrode 24 (Fig. 5A) was positioned vertically and perpendicular to the brain surface at a depth of 1500 μm from the surface. Recording multichannel electrode 24 was placed in the rat somatosensory cortex for simultaneous recording of evoked extracellular electrical activity - local field potential (LPP) and multicellular activity (MCA).

Analysis of electrophysiological data was performed on the basis of records of 15 cortical responses.

The raw data was previously prepared using specially designed functions in MATLAB (MathWorks).

The initial data were examined to detect MCA, followed by downsampling of the initial data to 1000 Hz for further spectral analysis of the LPP. The analysis was performed at 500 ms windows after the stimulus to determine the evoked activity [Suchkov, D., Sintsov, M., Sharipzyanova, L., Khazipov, R., & Minlebaev, M. /. Attenuation of the Early Gamma Oscillations During the Sensory-Evoked Response in the Neonatal Rat Barrel Cortex // BioNanoScience (2016), 6 (4), 575–577. doi: 10.1007 / s12668-016-0289-7]. MUA was detected with a bandpass signal (> 200 and <4000 Hz), when all negative events exceeding 3.5 SD were considered peaks (> 99.9% confidence) [Mitrukhina, O., Suchkov, D., Khazipov, R ., & Minlebaev, M. / Imprecise Whisker Map in the Neonatal Rat Barrel Cortex // Cerebral Cortex (2014), 25 (10), 3458–3467. doi: 10.1093 / cercor / bhu169].

Figure 5B shows graphs of the dependence of the nature of the evoked sensory activity of the brain on the temperature of the brain, illustrating the effect of brain temperature on neuronal activity in the representation of one vibrissa in the cerebral cortex of a rat under perfusion.

These data demonstrate a pronounced dependence of sensory activity on brain temperature - the duration of LPP-induced sustained MCA increases with decreasing brain temperature. In addition, as the temperature decreases, the evoked response shows a gradual dominance of slow-wave oscillations. At the same time, the return of the brain temperature to the initial values restores the control parameters of the evoked electrical activity, which indicates the presence of a temperature dependence.

Example 4. The use of the claimed system for the simultaneous registration of the evoked internal optical signal and electrical signals of the somatosensory cortex of the rat brain during stimulation of vibrissae (Fig. 6).

The applicant has verified the possibility of using the claimed system for electrophysiological studies of brain activity at various temperatures.

The combination of a perfusion chamber and a pressure device for the brain, such as in Example 3, allows the successful use of neuroimaging techniques for preliminary determination of the position of the somatosensory cortex [Minlebaev, M., Colonnese, M., Tsintsadze, T., Sirota, A., and Khazipov , R. / Early gamma oscillations synchronize developing thalamus and cortex // Science (2011) 334, 226-229. DOI: 10.1126 / science.1210574].

Among the many approaches used in neuroimaging, recording and analysis of the internal optical signal (IOS) makes it possible to characterize the properties of active neuronal tissue.

The applicant used the parameters of the components of light scattering and blood filling of neuronal tissue reconstructed with the help of BOC in order to find and characterize the active areas of the cortex (Fig. 5B).

To do this, create the declared system.

For this, the claimed perfusion chamber is attached to the animal and the stereotaxic apparatus according to Example 1.

Then the claimed perfusion chamber is connected to the flow-through perfusion system according to Example 2. In Example 4, the flow-through perfusion system shown in FIG. 2 is used.

The change in brain temperature is produced by changing the temperature of the perfusion fluid.

Preparation of animal vibrissae and electrophysiological registration using a brain clamp was carried out according to Example 3. A recording multichannel electrode 24 (Figs. 5A, 5B) was placed in the somatosensory cortex of a rat for simultaneous recording of evoked extracellular electrical activity - local field potential (LPP) and multicellular activity (MCA).

An internal optical signal (BOC) was recorded using a video recording system as shown in FIG. 6A. CCD camera 27 was positioned perpendicular to the skull above the proposed location of the somatosensory cortex. To detect FOS, the CCD camera 27 (abbreviated from “charge-coupled device” [https://ru.wikipedia.org/wiki/CCD- matrix]) was focused through the optical system 28, for example, a microscope at 400-1200 microns ( depending on the age of the animal) below the surface of the skull to the expected depth of the thalamo-recipient fourth layer of the somatosensory cortex.

To assess the scattering components of nerve tissue and blood flow, the brain surface was illuminated with an infrared diode 29 (LED Engin, LZ1-00R400, 850 nm) and green / red 30 (OSRAM, LRT GFTM-ST7-1 + VV9-29-0-A-R33 -ZB, 572/610 nm) diodes. The reflected light was collected by a CCD camera 27 (QICAM Fast 1394, resolution 130x174, 1 pixel = 35 μm). Optical image stabilization was calculated based on the average of twenty video re-shoots. Each video recording had a duration of 100 seconds, including: 5 seconds of the period before stimulation, 10 seconds of a series of vibrissae deviations with a frequency of 2 Hz and a duration of 10 ms each, 85 seconds of a period after stimulation. During preprocessing, the recorded video was subjected to a change in the sampling rate up to 10 Hz, followed by the detection of VOC, when the averaged video frames during stimulation were compared with the averaged video frames before stimulation. The coordinates of the vibrissa cortical representations of interest were determined using the VOC imaging described above.

The results of the simultaneously recorded electrical activity of the brain and the internal optical signal are presented in Figs. 6B, 6D. Also in Fig.6B, 6D are shown restored using a previously developed method [Sintsov M, Suchkov D, Khazipov R, Minlebaev M. / Developmental changes in sensory-evoked optical intrinsic signals in the rat barrel cortex // Front Cell Neurosci. (2017) 11: 392. DOI: 10.3389 / fncel.2017.00392] changes in light scattering and blood filling of neuronal tissue during sensory activation of the area of the somatosensory cortex, which is under perfusion in the claimed perfusion chamber.

Based on the obtained experimental results shown in Fig. 6B, it is possible to draw the following conclusions:

- in the area where the extracellular electrode is located, the presence of VOS is observed, which is indicated by darkening in the visualization of VOS in Fig. 6B.

- on one of the channels of the recording multichannel electrode 24, located at the depth of the thalamoreceptive layer of the somatosensory cortex, there is an evoked electrical activity in the form of high-amplitude oscillations of LPP, supported by MCA.

- the claimed perfusion chamber can be used both for conducting joint electrophysiological and optical studies of the brain, and for conducting separate electrophysiological or optical studies of the brain.

This suggests that the neuronal tissue of the brain was indeed activated in the area under consideration, contributing to the appearance of evoked VOS and demonstrating the presence of evoked electrical activity. In turn, the activity of neuronal tissue has a two-component nature [Hillman, E. M. C. // Optical brain imaging in vivo: techniques and applications from animal to man // J. Biomed. Opt. (2008). 12: 051402. DOI: 10.1117 / 1.2789693], which manifests itself in light scattering (tissue component) and changes in blood circulation (hemodynamic component). Thanks to the previously developed method [Sintsov M, Suchkov D, Khazipov R, Minlebaev M. / Developmental changes in sensory-evoked optical intrinsic signals in the rat barrel cortex // Front Cell Neurosci. (2017) 11: 392. DOI: 10.3389 / fncel.2017.00392] and using VOS from different illumination wavelengths, the applicant was able to restore the relative changes in these components, reflected in Fig. 6B (hemodynamic) and Fig. 6D (tissue). Changes in these components showed the presence of a change in the physiological state of the studied area, which can be further studied in detail.

Thus, the claimed perfusion chamber, the claimed system with its use and the claimed method of its operation made it possible to control the temperature of the perfusion solution simultaneously with conducting electrophysiological and optical studies of the activity of the rat somatosensory cortex in vivo. With the aid of the claimed perfusion chamber, the claimed system with its use and the claimed method of its operation, the applicant has shown for the first time that temperature has a significant effect on the parameters of evoked cortical activity, for example, in rats. Thus, the claimed technical solution provides conditions for both separate and simultaneous electrophysiological and optical recording of brain activity in vivo with the possibility of constant monitoring of the temperature of the brain surface.

From the above, we can conclude that the applicant has achieved the goals and the claimed technical result , namely , a perfusion chamber has been developed for studying the activity of the brain in vivo, a system using it and a method of operating the system. The claimed invention makes it possible to use the principles of a perfusion chamber to study the activity of the brain in vivo.

At the same time, a monolithic design of a perfusion chamber has been developed, consisting of a supporting body with technological channels, grooves and chambers to ensure stationary regulated perfusion, as well as the provided possibility of attachment to the animal's skull and stereotaxic apparatus, a system with its use and a method of operation of the system, providing:

- the ability to control the temperature of the inlet and outlet perfusion solution due to a specially provided channel for entering the temperature sensor of the inlet perfusion solution 4 and the feasibility of placing the temperature sensor in a specially located opening for the outlet of the perfusion solution 9;

- the use of an adjustable system of pressing the perfusion chamber to the open surface of the brain due to external structures with the possibility of conducting electrophysiological and optical studies of the brain, as well as the absence of the need for preliminary connection with the clamping devices of the brain tissue, which makes it possible to regulate the degree of pressure on the brain tissue, to use areas of the animal's skull with any curvature, and also makes it possible to local change the study area;

- the presence of structural features for leveling the artifacts of perfusion, namely, the preliminary chamber 5, the input spiral groove 6 and the output spiral groove 8;

- the presence of an adjustable system of the relative position of the perfusion chamber in space due to the use of specially provided mounting slots 10 for attachment to the stereotaxic apparatus;

- the ability of the perfusion camera to provide a wide viewing angle of the investigated area, which in turn provides the possibility of conducting optical studies as well;

- the absence of component parts, along with the use of a clamping of the brain surface with a holder, which simplified operation.

Moreover, in the above examples, it is demonstrated that the declared perfusion chamber, the system using it and the way the system works:

- allows the study of neuronal activity in vivo under long-term controlled pharmacological conditions.

- provides unlimited access to a vast area of the brain for electrophysiological studies.

- provides the ability to regulate the temperature of the brain to eliminate the problem of uncontrolled loss of heat in the brain.

- provides the possibility of optical research of brain activity using the optical range of visible light and near infrared radiation.

At the same time, the claimed technical solution was shown on the example of manufacturing using additive technology using 3D printing from plastic with dielectric properties.

The claimed technical solution allows for both electrophysiological studies of brain activity in vivo and optical studies, and has been successfully used to study the activity of the brain in vivo.

The claimed technical solution, if necessary, can be used to attach the animal's head to a stereotaxic apparatus for conducting other studies in vivo, which does not discredit the stated goals and the achieved technical result.

The claimed technical solution complies with the "novelty" criterion for inventions, since the applicant did not identify technical solutions from the investigated state of the art that possess the claimed set of essential features.

The claimed technical solution meets the criterion of "inventive step" for inventions, since no technical solutions have been identified that have features that coincide with the distinctive features of the claimed invention, and the influence of the distinctive features on the claimed technical result is not known.

The claimed technical solution meets the criterion of "industrial applicability" for inventions, since it can be manufactured on standard equipment using known materials and parts.

Claims (16)

1. A perfusion chamber for studying the activity of the brain in vivo, which is a monolithic structure made of plastic with dielectric properties, using technology based on a three-dimensional digital model, consisting of a supporting body containing a channel for entering a perfusion solution, a channel for reference electrode input, channel for input of the temperature sensor of the inlet perfusion solution, preliminary chamber, inlet spiral groove between the preliminary and main chambers, main chamber, outlet spiral groove between the main chamber and outlet for perfusion solution, outlet for perfusion solution, mounting grooves, a place for attaching the animal's skull; the inlet channels of the perfusion chamber are made in the form of through ducts opening into the preliminary chamber, the preliminary chamber is connected to the main chamber using an inlet spiral groove; the outlet from the preliminary chamber is located above the bottom of the main chamber with the possibility of gravitational fluid supply, the main chamber has the shape of a truncated paraboloid of revolution, while the main chamber is connected by an outlet spiral chute with an opening for the outlet of the perfusion solution in the lower part of the perfusion chamber with the possibility of placing a second temperature control temperature sensor output solution; and along the perimeter of the perfusion chamber there are three mounting slots for attaching to the stereotaxic apparatus; the place of attachment of the animal's skull is located below the main chamber and is made in the form of a truncated cone, adapted to the size and curvature of the skull of the animal under study. 2. A system for studying the activity of the brain in vivo using a perfusion chamber, consisting of a perfusion chamber and a pressure of the cerebral surface, in this case, the perfusion chamber is made in the form of a monolithic structure made of plastic with dielectric properties, using a technology based on a three-dimensional digital model, consisting of a carrier body containing a channel for entering a perfusion solution, a channel for introducing a reference electrode, a channel for introducing a temperature sensor inlet perfusion solution, preliminary chamber, inlet spiral groove between the preliminary and main chambers, main chamber, outlet spiral groove between the main chamber and the outlet for perfusion solution, outlet for perfusion solution, mounting grooves, place for fastening the animal's skull; the inlet channels of the perfusion chamber are made in the form of through ducts opening into the preliminary chamber, the preliminary chamber is connected to the main chamber using an inlet spiral groove; the outlet from the preliminary chamber is located above the bottom of the main chamber with the possibility of gravitational fluid supply, the main chamber has the shape of a truncated paraboloid of revolution, while the main chamber is connected by an outlet spiral chute with an opening for the outlet of the perfusion solution in the lower part of the perfusion chamber with the possibility of accommodating a second temperature control temperature sensor output solution; and along the perimeter of the perfusion chamber there are three mounting slots for attaching to the stereotaxic apparatus; the place of attachment of the animal's skull is located at the bottom of the main chamber and is made in the form of a truncated cone, adapted to the size and curvature of the skull of the animal under study; at the same time, the pressure of the cerebral surface is made of plastic using technology based on a three-dimensional digital model, and is a plastic disc with a smooth surface and perforated with a set of periodic circular holes, equipped with a technical board along the perimeter of the disc with the possibility of reinforcing the structure, while a reinforced plastic disc is connected to the fastening element using two handles, and the fastening element is inserted into the clamp holder, while the cerebral surface clamp is made replaceable in case of breakage using technology based on a three-dimensional digital model, while the clamp holder is installed in the micromanipulator with the ability adjusting both the position and the degree of pressure on the brain tissue; the clamp of the cerebral surface is placed directly on the surface of the brain in the main chamber of the perfusion chamber after attachment to the animal and removal of a portion of the skull and dura mater. 3. A system for studying the activity of the brain in vivo using a perfusion chamber, consisting of a perfusion chamber, pressing the cerebral surface, an attachment system to a stereotaxic apparatus and a flow-through perfusion system, in this case, the perfusion chamber is a monolithic structure made of plastic with dielectric properties, using a technology based on a three-dimensional digital model, consisting of a carrier body containing a channel for entering a perfusion solution, a channel for introducing a reference electrode, a channel for introducing a sensor the temperature of the inlet perfusion solution, the preliminary chamber, the inlet spiral groove between the preliminary and main chambers, the main chamber, the outlet spiral groove between the main chamber and the outlet for the perfusion solution, the outlet for the perfusion solution, mounting grooves, a place for attaching the animal's skull; the inlet channels of the perfusion chamber are made in the form of through ducts opening into the preliminary chamber, the preliminary chamber is connected to the main chamber using an inlet spiral groove; the outlet from the preliminary chamber is located above the bottom of the main chamber with the possibility of gravitational fluid supply, the main chamber has the shape of a truncated paraboloid of revolution, while the main chamber is connected by an outlet spiral chute with an opening for the outlet of the perfusion solution in the lower part of the perfusion chamber with the possibility of placing a second temperature control temperature sensor output solution; and along the perimeter of the perfusion chamber there are three mounting slots for attaching to the stereotaxic apparatus; the place of attachment of the animal's skull is located at the bottom of the main chamber and is made in the form of a truncated cone, adapted to the size and curvature of the skull of the animal under study; at the same time, the pressure of the cerebral surface is made of plastic using technology based on a three-dimensional digital model, and is a plastic disc with a smooth surface and perforated with a set of periodic circular holes, equipped with a technical board along the perimeter of the disc with the possibility of reinforcing the structure, while a reinforced plastic disc is connected to the fastening element using two handles, and the fastening element is inserted into the clamp holder, while the cerebral surface clamp is made replaceable in case of breakage using technology based on a three-dimensional digital model, while the clamp holder is installed in the micromanipulator with the ability adjusting both the position and the degree of pressure on the brain tissue; the clamp of the cerebral surface is placed directly on the surface of the brain in the main chamber of the perfusion chamber after attachment to the animal and removal of a portion of the skull and dura mater; in this case, the system of attachment to the stereotaxic apparatus is represented by supporting columns on which the perfusion chamber rests in the places of the mounting slots, while bolts fixed in the stereotaxic apparatus pass through the mounting slots of the perfusion chamber and the supporting columns, and the declared perfusion chamber is attached to the animal in the designated place attachments to the skull of an animal; in this case, the flow-through perfusion system is a series of elements of the perfusion system, where each next element is lower than the previous one to ensure gravitational fluid supply, including a buffer tank, a connecting tube, a dropper-regulator, a connecting tube, a heating linear flow element, a perfusion chamber, a flexible connection made of two fittings connected by a flexible tube, a regulator of the perfusion solution level, a vacuum suction channel connected to a vacuum suction system; in this case, the buffer tank is connected through a connecting tube with a dropper-regulator with the ability to regulate the flow rate of the perfusion solution, as well as with the possibility of galvanic isolation of the perfusion solution in the perfusion chamber and the buffer tank, while the dropper-regulator, in turn, is connected through a tube with a heating linear flow element with the ability to set the required temperature of the perfusion solution, while the outlet of the heating linear flow element is connected through a tube to the channel for the inlet of the perfusion solution of the perfusion chamber by means of a tube, while the opening for the outlet of the perfusion solution of the perfusion chamber is connected by means of a flexible connection made of two fittings connected flexible tube, and is connected with the regulator of the level of the perfusion solution with the possibility of its reciprocating vertical movement and the possibility of thereby regulating the level of the perfusion solution in the main chamber due to the effect of communicating vessels, with the possibility of removing excess perfusion solution in the perfusion solution level regulator through the vacuum suction channel connected to the vacuum suction system. 4. A method for studying the activity of the brain in vivo using the system according to claim 3, which consists in the fact that a preliminary preparatory operation is carried out to remove the scalp and integumentary tissues over the warmed animal under anesthesia, then the animal's skull is attached to the perfusion chamber at the intended attachment point using first cyanoacrylate glue, then dental cement, then the perfusion chamber is attached in a stereotaxic apparatus using mounting grooves, into which bolts are then inserted, passing through the support columns, on which, in turn, the declared perfusion chamber rests; then the perfusion chamber is connected to the flow-through perfusion system, for which the outlet of the heating linear flow-through element is connected to the channel for the inlet of the perfusion solution of the perfusion chamber by means of a tube, then the opening for the outlet of the perfusion solution of the perfusion chamber is connected by means of a flexible connection made of two fittings connected by a flexible tube, to the perfusion solution level regulator, which makes a reciprocating vertical movement and thus regulates the perfusion solution level in the main chamber due to the effect of communicating vessels, while the excess perfusion solution in the perfusion solution level regulator is removed through a vacuum suction channel connected to a vacuum system suction; then, temperature sensors are placed through the channel for the input of the input perfusion solution in the preliminary chamber and the holes for the output of the perfusion solution to control the actual temperature of the input and output solution; then a section of the skull and dura mater is removed, after which the pressure of the brain surface is placed directly on the brain surface in the main chamber of the perfusion chamber; then, electrophysiological studies of brain activity are carried out using a reference electrode in the form of a silver chlorinated wire placed in the channel for the input of the reference electrode and recording electrodes located in the brain through the holes of the perforated reinforced plastic disc; then, optical studies of brain activity are carried out using video surveillance systems of electromagnetic radiation of the visible and infrared ranges reflected from the brain tissue in the areas of the holes of the perforated reinforced plastic disk.

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