JP5606713B2 - Method for stimulating and / or measuring central nerve and probe therefor - Google Patents

Description

  The present invention relates to a method for stimulating and / or measuring central nervous tissue and a probe therefor.

  As a method of examining the dynamics of the central nervous system at the cellular level, a probe is directly inserted into nerve tissue, and electrical and / or optical stimulation is applied, an electrical signal is detected, or an optical image is observed. The method is known.

  For example, Patent Literature 1 discloses a behavior analysis apparatus that can apply brain stimulation to a specimen such as a rat and observe the behavior.

  In Non-Patent Document 1, a fluorescent protein or the like is expressed in a nerve cell in a mouse brain tissue, and the nerve cell in a living mouse is observed with a microscope through a minute lens inserted in the brain tissue. A method is disclosed.

  In such a method, it is possible to observe the target organism in a state closer to the original form of conducting life activity. Therefore, it is possible to eliminate the influence caused by sectioning and partialization as much as possible, and to observe and / or measure the original shape of the target cell or tissue rather than the method using tissue section specimens or cultured cell specimens. it can.

  In the method of stimulating and / or measuring the central nervous system or the like of a target organism in such a living state, it is important to maintain as much as possible the state in which the organism performs normal life activities. However, an undesirable reaction occurs in the tissue due to puncture of the central nervous tissue or insertion of a probe or the like.

  One is the problem of nerve tissue damage. The cells to be examined are not necessarily located on the tissue surface, and may be located deep in the nerve tissue, such as the hippocampus located below the cerebrum. In such a case, it is necessary to make a deep hole in the nerve tissue as a route for the probe to reach the site. In the process of making a hole, many nerve cells and axons located in the path are damaged. When nerve cells and axons are damaged, some of the functions of the central nervous system that they carry out are impaired. In other words, this means that the function of the central nervous system is impaired. From the viewpoint of observing the original state, it is ideal that there is no damage to nerve cells or axons.

  Another problem is the formation of glial scars. When central nervous tissue is damaged, a tissue called glial scar is formed in the damaged site within a few days. A similar glial scar is also formed around the probe when a foreign object such as a probe is punctured, inserted or placed in the central nervous tissue. Since the glial scar has a high electric resistance, when it is formed around the electric probe, it is difficult to apply an electrical stimulus to the site of interest and to detect an electrical signal from the site. Therefore, it is desirable to suppress the formation of glial scars, particularly in experiments using electrical probes. However, for example, it is known that the formation of glial scars is not suppressed even when the material, shape, or puncture speed of the electric probe is changed (Non-Patent Document 2). In Non-Patent Document 2, the glial scar is called “sheath”, that is, “sheath”.

JP-A-62-222105

Karl Deisseroth et al., The Journal of Neuroscience, 2006, 26 (41): 10380-10386 Vadim S. et al., Journal of Neuroscience Methods, 2005, 148: 1-18

  The present invention particularly relates to a method for inhibiting nerve tissue damage and the formation of glial scars, thereby stimulating and / or measuring the central nerve in a more natural state of the organism of interest, and such a method It is an object of the present invention to provide a probe that can be used for the above.

  According to an embodiment of the present invention, there is provided a method for stimulating or measuring a central nervous tissue of a vertebrate other than a human using an energy transfer means, comprising a) puncturing the tissue, b) puncturing the puncture site Administration of an active ingredient having a central nerve repair effect, c) energy transfer between the energy transfer means and the central nervous tissue at the punctured site, wherein the energy transfer is the active ingredient The present invention provides a method characterized in that the method is executed in a state where a central nerve repair effect is obtained.

  According to another embodiment of the present invention, there is provided a probe for stimulating or measuring central nervous tissue of vertebrates other than humans for administering an active ingredient having a central nerve repair effect to a target site. There is provided a probe comprising an active ingredient administration means and an energy exchange means for exchanging energy with the central nervous tissue at the target site.

  ADVANTAGE OF THE INVENTION According to this invention, the damage | damage of a nerve tissue and formation of a glial scar can be suppressed, As a result, the method which can stimulate and / or measure a central nerve in the state nearer the target organism is provided.

2 is a flowchart showing an embodiment of a method according to the present invention. FIG. 3 shows an embodiment of the method according to the invention. The figure which shows the conventional method. The figure which shows the state by which the probe based on this invention was punctured by the structure | tissue. The figure which shows the example of the probe which concerns on this invention. The figure which shows the probe which concerns on this invention. The figure which shows a part of cross section of the probe which concerns on this invention. The figure which shows the probe which concerns on this invention. The figure which shows the probe which concerns on this invention. The figure and sectional drawing which show the probe which concerns on this invention. Sectional drawing which shows the probe which concerns on this invention. Sectional drawing which shows the probe which concerns on this invention. The figure which shows the apparatus which concerns on this invention.

  The method according to the present invention is a method for stimulating or measuring the central nervous tissue of vertebrates other than humans using an energy transfer means, which comprises a) puncturing the tissue, b) centralizing the puncture site. Administering an active ingredient having a nerve repairing effect, and c) performing energy transfer between the energy transfer means and the central nervous tissue at the punctured site.

Hereinafter, a method according to an embodiment of the present invention will be described with reference to the drawings.
FIG. 1 is a flow chart illustrating one embodiment of a method according to the present invention. In this method, the skin of the head of a target organism such as a rat or mouse is first peeled to expose the skull (1-1 in FIG. 1). Next, a hole is made in the skull to expose the brain (1-2). Next, the exposed brain tissue is punctured through a hole formed in the skull (1-3). Next, an active ingredient is administered to the puncture site (1-4). Then, energy is exchanged at the punctured site (1-5).

  The steps of exposing the skull and making a hole in the skull (1-1 and 1-2) can be performed by a method generally performed in the art. For example, by anesthetizing a mouse with an anesthetic such as ketamine and a sedative such as xylazine, the head is shaved, an incision is made about 1-1.5 cm along the midline, and the skin is expanded from the incision. The skull can be exposed. Thereafter, a hole can be drilled at a target position of the skull with a medical drill or the like.

  The step (1-3) of puncturing the exposed central nerve is mainly performed in order to make the probe or the like inserted thereafter reach the target site as much as possible without destroying the tissue. When the tip of a probe or the like to be used later is not sufficiently pointed with respect to the tissue, it is expected that the degree of destruction becomes more serious when the tissue is punctured. On the other hand, further tissue destruction can be suppressed by inserting the probe along a hole formed by puncturing in advance. As the means for puncturing or perforating the central nervous tissue used in this step (puncturing means), an object having a sufficiently sharp tip for puncturing the tissue can be used. For example, a hollow injection needle or tube or a center-filled needle can be used. As the material, a material having sufficient rigidity for puncturing a tissue, such as metal, plastic or glass, can be used. Furthermore, the length can be appropriately set so that the target part can be reached. Further, for example, a shape that can avoid a specific part that should not be damaged, for example, a curved needle-like shape may be used.

  The step (1-4) of administering an active ingredient having a CNS repair effect to the puncture site suppresses a biological reaction that is hindered in the subsequent application of stimulation to the tissue or detection of information from the tissue. Done for. Such suppression is performed by administering an active ingredient to tissues such as the central nerve. When studying the central nervous tissue, the active ingredient having such a central nervous repair effect is to prevent the damage from the nervous tissue and to avoid problems caused by the formation of glial scars. Any active ingredient that promotes recovery and / or suppresses the formation of glial scars may be used.

  Administration of such an active ingredient is performed using an active ingredient administration means. The “active ingredient administration means” in the present invention refers to a means for administering an active ingredient having a specific effect as described later to a tissue such as the central nerve. For example, an injection needle can be used as the active ingredient administration means. In this case, the injection needle is connected to a syringe or the like as an active ingredient supply means, and the active ingredient delivered from the active ingredient supply means is discharged to the punctured target site. In addition, as an active ingredient administration means, a needle or a rod coated with or holding an active ingredient can be used. In this case, the active ingredient is applied or held particularly at the tip of the needle or stick, and the active ingredient is administered by inserting the needle or stick into the part punctured by the puncture means and diffusing to the target site. . It is also possible to administer the active ingredient through the inside of the injection needle while simultaneously holding the active ingredient on the surface of the injection needle in contact with the tissue and administering it by diffusion.

  According to the administration in this step, the active ingredient may reach not only the directly administered site but also the surrounding site by diffusion and / or self-running. “Self-propelled” means that, for example, when the active ingredient is a cell, the cell moves from a directly administered site to a peripheral site or the like (details of the active ingredient and the cell will be described later). .

  Further, the “puncture site” referred to in this step is not limited to the portion punctured by the puncture means, but includes a site to be punctured. Therefore, “administering an active ingredient to the puncture site” may be performed prior to puncturing by the puncturing means. In this case, by applying or dripping the active ingredient onto the tissue surface at or near the site to be punctured, the active ingredient reaches the target site located deep in the tissue by diffusion and / or self-running. Therefore, the term “puncture” in the present invention means that the means for stimulation or measurement finally penetrates into a specific region of a cell or tissue as a target site so that it can be stimulated or measured with a minimum invasive volume. Widely means the process to do.

  In the step of transferring energy at the punctured site (1-5), the energy transfer means is used to stimulate the target site or cell and / or detect information on the target site or cell. In the present invention, “stimulate” mainly refers to giving an electrical stimulus or stimulating by light to a specific part of a cell or tissue, but is not limited thereto, and treatment By stimulation or ultrasound by supplying a specific drug (except for a drug having a repair effect for maintaining the original activity of the central nerve of the present invention) to the punctured site for the purpose of diagnosis, research, etc. It includes providing all stimuli, such as stimuli, performed on cells and tissues in the field. In the present invention, “measurement” means that information including medical information is finally obtained by stimulation or detection, and may be either qualitative measurement or quantitative measurement. In the present invention, “detecting” mainly means measuring an electrical signal generated in a cell or tissue, or detecting biological luminescence such as a fluorescent protein expressed in the cell or tissue. This includes, but is not limited to, any detection performed on cells or tissues in the art. Therefore, in the present invention, the term “detect” includes the meaning of “observing” a cell or tissue. In addition, “energy transfer means” refers to applying energy or signals by light or electricity to a target site such as tissue or cells, and detecting energy or signals by light or electricity from such sites. Or a means of doing both. For example, an optical fiber, a microscope objective lens, or an electrode corresponds to this. In this field, generally, each of the optical fiber, the microscope objective lens, and the electrode may be referred to as a “probe”, but the term “probe” used in the present invention refers to such general use. Are strictly different. That is, the “probe” used in the present invention includes at least an “energy transfer unit” such as an optical fiber, a microscope objective lens, or an electrode, and optionally includes a “puncture unit” and / or an “active ingredient administration unit”. Refers to things. Accordingly, the “probe” used in the present invention may include “energy transfer means” and may not include “puncture means” and “active ingredient administration means” or “energy transfer means”. And “active ingredient administration means” may be included and “puncture means” may not be included, or “energy transfer means”, “active ingredient administration means” and “puncture means” may be included. Good. In the present invention, the term “probe” is not used synonymously with an optical fiber, a microscope objective lens, or an electrode, and refers to an element that includes at least an “energy transfer means” such as an optical fiber, a microscope objective lens, or an electrode as a constituent component. Is done.

The effect of the present invention will be described with reference to FIGS.
FIG. 3 is a diagram showing a typical example of a method for stimulating and / or measuring a central nerve using a conventional probe. In the conventional method, the central nervous tissue 1 is punctured by the stimulation and / or measurement probe 10 as shown in FIG. 3 without puncturing the tissue with a needle or the like in advance before inserting the probe (see FIG. 3). 3-2). By the puncture, the axon 4 of the nerve cell 2 is cut, and an axon cutting part 7 is generated (3-2, a part surrounded by a broken line in the figure). When the probe 10 does not have a sufficiently sharp structure suitable for puncture, the degree of damage to the central nervous tissue 1 is increased. In response to such damage, the glial cells 5 are activated (3-3) and gather in a damaged portion including the probe 10 puncture portion and the axotomy section 7 (3-4). The aggregated glial cells 5 undergo morphological changes as shown in (3-5) (denatured glial cells 12), intercellular bridges are formed, the glial cells adhere to each other, and a glial scar 13 is formed ( 3-6). When the probe 10 is an electrode probe, electrical stimulation to the tissue and detection of electricity from the tissue are inhibited by the glial scar 13. When the probe 10 is an optical probe (that is, an optical fiber, a microscope objective lens, etc.), the observation itself is hindered by the intermediary of the glial scar 13, and the formation of the glial scar 13 and the cutting of the axon cuts the tissue. The environment has changed, and it is impossible to investigate what should be observed.

  FIG. 2 describes a method according to one embodiment of the present invention. In the present invention, as shown in (2-2), the central nervous tissue 1 is punctured using the puncture means 6. By this puncture, it is considered that the axon is cut and the axon cutting part 7 is formed (2-2). Thereafter, the puncture means 6 is extracted from the central nervous tissue 1 and the active ingredient administration means 8 is inserted (2-3). When the puncture means 6 and the active ingredient administration means 8 are integrated (for example, when an injection needle or the like is used), the puncture and the active ingredient administration may be performed in a series of steps. Next, the active ingredient 9 is administered from the active ingredient administration means 8 (2-3, in the figure, the active ingredient administration means 8 is represented as a cross-sectional view). As shown in the figure, the active ingredient 9 diffuses and / or self-propels from the administration site to the periphery thereof. After the administration, the active ingredient administration means 8 is extracted from the central nervous tissue 1 (2-4), and then the probe 10 is inserted (2-5). At this time, the action of the active ingredient suppresses the aggregation of glial cells 5 that occurs in the conventional method, and as a result, the formation of glial scars is suppressed. Moreover, the axon cut | disconnected by the effect | action of an active ingredient is repaired (repaired axon 11). Thereby, it is possible to apply or detect electrical stimulation, detect an optical image, or the like by the probe 10 in a state of the central nervous environment before puncturing, that is, a state closer to the original central nervous environment.

  In the method of the present invention, the target tissue is a central nervous tissue. That is, the central nervous tissue is brain and spinal cord tissue in vertebrates. Although the present invention can be used for any organism that has a central nervous tissue, it is preferred to use the central nervous tissue of a vertebrate. In particular, it is preferable to use mammalian central nervous tissue. Specifically, mice, rats, monkeys, dogs, cats, pigs and the like can be used.

  In the present invention, the active ingredient is particularly an active ingredient having a central nerve repair effect. Here, the “central nerve repair effect” refers to an effect of returning the central nervous tissue that has been changed by puncture or insertion of a probe or the like to its original state when the organism has survived normally. . For example, it has an effect of promoting repair of cut axons, suppression of activation of glial cells, particularly astrocytes (one type of glial cells) and / or suppression of formation of glial scars. Therefore, the “central nerve repair effect” refers to an axonal elongation promoting effect, an astrocyte activation inhibitory effect, a glial scar formation inhibitory effect, or a combination of any of these three effects.

  As the active ingredient exhibiting such an effect, those known in the art can be used. As the active ingredient, cells can be used, and substances other than cells can also be used.

  Examples of cells as active ingredients having a CNS repairing effect that can be used in the present invention include astrocyte-restricted progenitor cells (JP 2006-503543, JP-A 2008-289486), olfactory nerve sheath glial cells (SP) Table 2007-536901), postpartum-derived cells (placental tissue-derived cells, umbilical cord tissue-derived cells) (special table 2007-521793), glia-restricted progenitor cells (special table 2001-525164) or active immature astrocytes (special table 02 -50535) can be used. Not only these cells but also other cells described in the above-mentioned literature, and any cells having a CNS repair effect can be used.

  Examples of substances that can be used in the present invention as active ingredients having a CNS repair effect include heparin, heparin derivatives (JP 2005-068057), noggin (JP 2008-255071), fasudil hydrochloride hydrate ( JP2007-246466), IL-17F (special table 2008-513415), EphA4 receptor antagonist, antibody against EphA4 receptor, antisense RNA against EphA4 receptor, antisense DNA against EphA4 receptor, siRNA against EphA4 receptor , Ephrin antagonists, antibodies to ephrin, antisense RNA to ephrin, antisense DNA to ephrin, siRNA to ephrin (all related to EphA4 receptor and ephrin, 2008-5) 2394), a cyclic peptide containing the amino acid sequence of Arg-Gly-Glu (special table 2007-505145), p75 receptor binding agent (special table 2007-505145), Nogo inhibitor (special table 2006-526382) ), TNR inhibitor (special table 2006-526382), MAG inhibitor (special table 2006-526382), CD81 (special table 2005-537779, special table 2004-517917), phosphodiesterase type 4 inhibitor (special table 2004-532809) ), Axon forming factor 1 (Tokuhei Hei 10-505238), axon forming factor 2 (Tokuhyo Hei 10-505238) or osteogenic factor 1A type receptor inhibitor (WO06 / 137377). Not only these substances but also other substances described in the above-mentioned literature, and any substance having a central nerve repair effect can be used.

  As described above, the method according to the present invention includes a) puncturing a tissue, b) administering an active ingredient having a central nerve repair effect to the puncture site, c) energy transfer means in the puncture site. And a transfer of energy between the central nervous system and the central nervous tissue, a to c can be performed in independent processes. That is, a) puncture using some puncture means, and after removing the puncture means from the tissue, b) administering an active ingredient having a CNS repair effect using an active ingredient administration means different from the puncture means And c) a specific site of the central nervous tissue can be stimulated or measured by energy transfer using energy transfer means independent of the puncture means and the active ingredient administration means. For example, a needle can be used as the puncturing means, an injection needle as the active ingredient administration means, and a microscope objective lens as the energy transfer means.

  Furthermore, the above steps a to c can be performed in any combination as the same step or a series of steps. In this case, the terms “puncture means”, “active ingredient administration means”, and “energy transfer means” in the present invention indicate not only one independent object but also a part of a specific object. There is also. This means that one or more of these means can be combined in one object. This will be described in detail below.

  In the present invention, for example, steps a and b can be performed in the same or a series of steps. For example, after puncturing using an injection needle as the puncturing means in step a, the active ingredient can be administered from the injection needle as it is in step b. In this case, it does not matter whether the injection needle is connected from the beginning to a syringe or the like as an active ingredient supply means or is connected after administration after puncture. In such an embodiment, it can be said that the injection needle serves as both the puncture means and the active ingredient administration means. As another example, it is possible to use one in which an active ingredient is applied or held on the tip of the needle and its peripheral portion. At the same time as puncturing with such a needle, administration is performed by diffusing the active ingredient applied from the moment of puncturing.

  In the present invention, the steps b and c can be performed in the same or a series of steps. For example, a probe provided with an active ingredient administration means and an energy transfer means can be used. In this case, after inserting the probe into the hole that can be punctured by the puncture means, the active ingredient is first administered to the puncture site by the active ingredient administration means, and then the stimulus is given by the energy transfer means while the probe is inserted. And detection is done. In addition, as described above, energy transfer is not limited to the completion of administration of the active ingredient, and it is also possible to perform administration of the active ingredient and energy transfer at the same time. Also, considering that the administration of the active ingredient is a repair at the part where energy is to be transferred, after the probe is inserted, only the active ingredient administration is started from the active ingredient administration means, and the tissue is kept constant. After exerting the repair effect, it is preferable to start stimulation or detection by the energy transfer means while continuing the administration. As a specific example of a probe provided with an active ingredient administration means and an energy transfer means, an electrode, a microscope objective lens, an optical fiber or the like added with an injection needle or a tube for administering an active ingredient, or an electrode In addition, a microscope objective lens or a part of an optical fiber or the like on which an active ingredient is coated or held can be used. These probes will be described in detail later. In such a probe, a portion where an injection needle or an active ingredient is applied or held is referred to as an “active ingredient administration unit”, and a part that performs functions such as an electrode, a microscope objective lens, or an optical fiber is referred to as an “energy transfer unit”. be able to.

  Furthermore, in the present invention, the above steps a to c can be performed in the same or a series of steps. For example, puncturing, administration and energy transfer can be performed with a single probe. That is, the probe provided with the active ingredient administration means and the energy transfer means as described above, and further provided with the puncture means can be used. “Further provided with puncturing means” includes those provided with a needle or injection needle in parallel with the active ingredient administration means and the like, and those with a structure in which the tip of the probe is sufficiently sharpened for puncturing. Further, the structure may be such that the active ingredient administration means and the energy transfer means are housed inside the hollow injection needle-like puncture means. That is, a probe having a structure in which the active ingredient administration means and the energy transfer means are accommodated in the puncture means as a sheath may be used. In such a probe, after the tissue is punctured, the active ingredient administration means and the energy transfer means housed inside the puncture means are pushed out of the puncture means to reach the target site, and administration and energy transfer are performed. Can do. Alternatively, in such a probe, after the tissue is punctured, only the active ingredient administration means and the energy transfer means are left, and only the puncture means is partially or completely withdrawn from the tissue. As a result, the active ingredient administration means, etc. Can be exposed to tissue and perform specific functions.

  These methods are not limited to the case of puncturing and stimulating or measuring one place in the central nervous tissue. That is, the above-described methods can be performed at a plurality of locations in the central nervous tissue. For example, it can be performed for a plurality of locations apart from each other, or it can be performed for a peripheral portion around a specific portion. For example, when a probe having an optical lens is used as the energy transfer means, the size of the field of view is generally smaller than the diameter of the probe used. Accordingly, when a plurality of locations to be observed are located apart from each other in the central nervous tissue, it may be difficult to keep them within the field of view of one probe. Even in such a case, it is possible to observe simultaneously by applying the above method to each of a plurality of locations. Also, for example, when using a probe having an energy transfer means as an electrode, a stimulation electrode is installed at one location, a detection electrode is installed at another location, and stimulation to a specific region is performed on the other location. By examining the influence on the region, it is also possible to examine the relationship between a plurality of regions of the central nervous tissue. Furthermore, an optical lens and an electrode can be used in combination. When conventional methods are applied, accumulation of damage and excessive formation of glial scars occur, and it is difficult to perform appropriate detection or observation. Since it is recovered and the formation of glial scars is suppressed, even if it is applied to a plurality of locations, the problems in the conventional case are unlikely to occur, and good detection or observation can be performed. Further, analysis by the naked eye or image processing may be performed based on the observed image.

The present invention relates to a probe that can be used in the above method.
One embodiment of such a probe according to the present invention is a probe for stimulating or measuring central nervous tissue of vertebrates other than humans, and for administering an active ingredient having a central nerve repair effect to a target site. The active ingredient administration means and an energy exchange means for performing energy exchange with the central nervous tissue at the target site.

  That is, the probe includes an active ingredient administration unit and an energy transfer unit as an integral unit. When stimulating or measuring central nervous tissue, a hole is made in the tissue in advance by puncturing means, the probe is inserted into the hole, and the active ingredient is administered from the active ingredient administration means, or from the active ingredient administration means. While administering the active ingredient, the tissue is stimulated or the tissue is measured by the energy transfer means. In addition, the probe itself can be provided with a puncturing means. When such a probe is used, it is not necessary to puncture the tissue with an independent puncturing means in advance, and the probe is directly punctured into the tissue. Subsequent administration and stimulation and / or measurement can be performed.

  The energy transfer means in such a probe is to provide energy or signal by light or electricity to a target site such as tissue or cell, and detect energy or signal by light or electricity from such site. Or means for performing both the giving and the detection. For example, an optical fiber, a microscope objective lens, an electrode, or the like can be used.

  Moreover, the active ingredient administration means in such a probe refers to a means for administering an active ingredient that exhibits the above-described central nerve repair effect to tissues such as the central nerve. The active ingredient administration means can have any configuration as long as it can administer the active ingredient to the punctured site. For example, a structure in which the active ingredient is held so as to diffuse in the central nervous tissue, such as when the active ingredient is applied to the surface of the probe or the active ingredient is held in some structure, can be used. Or it can be set as the structure which has a flow path for allowing an active ingredient to pass through like an injection needle and a tube, and a discharge port for releasing an active ingredient from the said flow path.

  When the active ingredient administration means has a structure that holds the active ingredient so as to diffuse in the central nervous tissue, as shown in FIG. 4, a portion of the probe 10 that is inserted into the tissue 1 (insertion section 14). It is preferable that the active ingredient is applied or held in (1). In particular, when an active ingredient is applied, it is noted which part of the probe 10 is applied.

  As shown in FIG. 5A, when the probe is an electrode probe 15, at least the distal tip 16 has an active ingredient out of the conductive distal tip 16 and the non-conductive non-conductive portion 17. Applied. As a result, the formation of glial scars in the vicinity of the tip 16 that is the part that actually performs electrical stimulation to tissues and cells can be suppressed, and the electrical stimulation to tissues can be prevented from being disturbed. Preferably, the active ingredient is applied not only to the tip 16 but also to a part of the non-conductive portion 17, and most preferably, in addition to the tip 16, all surfaces that contact the tissue of the non-conductive portion 17 (ie, The active ingredient is applied to the insertion part 14) shown in FIG. Thereby, in addition to the inhibitory effect on glial scar formation, the repair of axons damaged by puncture is promoted, and the function of the entire central nervous tissue can be brought close to the state before puncture.

  Further, as shown in FIG. 5B, when the probe is an optical probe 18 (for example, a microscope objective lens or an optical fiber is provided to give an optical stimulus or detect an optical image of a tissue). The active component is applied to at least the measurement unit 20 among the measurement unit 20 for light emission or detection and the non-measurement unit 19 which is another part. Thereby, the formation of glial scars in the vicinity of the measurement unit 20 is suppressed, and light blocking between the measurement unit 20 and the target cell or tissue can be prevented. Preferably, the active ingredient is applied not only to the measurement unit 20 but also to a part of the non-measurement unit 19, and most preferably, in addition to the measurement unit 20, all surfaces that contact the tissue of the non-measurement unit 19 (that is, The active ingredient is applied to the insertion part 14) shown in FIG. Thereby, in addition to the inhibitory effect on glial scar formation, the repair of axons damaged by puncture is promoted, and the function of the entire central nervous tissue can be brought close to the state before puncture.

  In addition, as shown in FIG. 6, a structure that holds an active ingredient (that is, a holding structure 101) may be provided in a part of the probe 10. If such a structure is used, the active ingredient can be diffused into the tissue more continuously and at a more constant rate than when the active ingredient is applied to the surface. The holding structure 101 can be provided at any part of the probe 10, but is preferably provided at a part to be inserted into the tissue. Further, the holding structure 101 is preferably provided in the vicinity of a portion important for energy transfer, such as the tip 16 or the measurement unit 20 shown in FIG. As long as it does not interfere with energy transfer, it can also be provided in parts important for energy transfer. The holding structure 101 may be formed on the surface of the probe 10.

  FIG. 7 shows an example of such a holding structure 101 in a cross-sectional view. 7A to 7C are enlarged views of a part of the cross section of the probe 10 (portion surrounded by a square). In the case of the probe (a), the holding structure 101 is an adsorption layer 21 for adsorbing the active ingredient 9 formed around the probe interior 102. The adsorption layer may have a structure having biological affinity for the active ingredient 9, for example, and may have a structure in which an antibody or binding protein for the active ingredient 9 is fixed. The adsorption layer 21 may be a film of a substance (for example, gelatin or albumin) that can hold active ingredient molecules therein. The adsorption layer 21 may be applied evenly around the probe interior 102. In the case of (b), the holding structure 101 is a concavo-convex layer 22 having concavo-convex portions for holding the active ingredient 9 formed around the probe interior 102. By making it uneven, the area in contact with the active ingredient 9 increases, and the amount retained is considered to increase. Further, since the active ingredient 9 is held in the concave portion of the concavo-convex structure, the active ingredient 9 is prevented from falling off due to friction when the probe 10 is inserted, and the active ingredient 9 can be reliably administered to the target site. Become. The shape of the recess may be a hole or a groove. Its dimensions need to be greater than the dimensions of the active ingredient molecules or cells. For example, if the active ingredient is a cell having a size of 30 μm, the width and depth of the recess are preferably about 50 to 300 μm. The manufacturing method of the concavo-convex shape may be by mechanical means such as cutting or chemical means such as etching. In the case of (c), the holding structure 101 is a porous layer 23 having a large number of pores that hold the active ingredient 9 formed outside the probe interior 102. A larger amount of the active ingredient 9 can be held in the large number of pores, and the active ingredient 9 can be released more continuously. As a material of such a porous layer 23, a liposome membrane, a semipermeable film, or a porous resin can be used. These three structures may be different from the material of other parts of the probe 10 or may be the same. Alternatively, these structures may be formed on members on the surface of the probe 10. Further, the member on the surface of the probe 10 itself may be a member having these structures.

  When the active ingredient administration means in the probe has a structure having a flow path for passing the active ingredient and a discharge port for discharging the active ingredient from the flow path, for example, a probe as shown in FIG. 10 can be used. In FIG. 10, the active ingredient administration unit 24 is connected in parallel with the energy transfer unit 28. In this case, the active ingredient administration means 24 includes a flow path 25 for allowing the active ingredient 9 to pass therethrough and a discharge port 26 for releasing the active ingredient 9 from the flow path 25. For example, the active ingredient administration means 24 can be a tube or an injection needle. The end of the active ingredient administration means 24 opposite to the discharge port 26 is connected to, for example, an active ingredient supply means 27. The active ingredient supply means 27 is a syringe, for example, and stores a solution containing the active ingredient 9 therein. In this case, the active ingredient 9 can be discharged | emitted with respect to a structure | tissue from the discharge port 26 via the flow path 25 by pushing in the piston of a syringe manually or mechanically by arbitrary pressures and speeds. As shown in FIG. 8, the present invention is not limited to the case where one active ingredient administration unit 24 is provided for one energy transfer unit 28, and a plurality of active ingredient administrations are performed for one energy transfer unit 28. Means 24 may be provided. For example, the plurality of active ingredient administration means 24 may be provided at symmetrical positions around the energy transfer means 28 or at equally spaced positions around the energy transfer means 28.

  Further, the number of discharge ports 26 is not limited to one as in the case of FIG. 8, and a plurality of discharge ports 26 can be provided as shown in FIG. In FIG. 9, a plurality of discharge ports 26 are provided on the side surface of the probe 10. Moreover, the discharge port 26 can be provided in the downward direction as shown in FIG. 8, and the discharge port 26 can also be provided on the side surface as shown in FIG. In the tissue, it is preferable that the active ingredient 9 particularly acts on a region where energy is transferred to and from the energy transfer means 28. Therefore, it is preferable that there is a discharge port 26 so that it can be discharged in the direction of the region. In addition to the discharge port 26, it is preferable to further provide a discharge port 26 that does not directly release in the direction of the region but discharges the active ingredient 9 to the entire nearby tissue.

  FIG. 10 shows another example in which the active ingredient administration means has a structure including a flow path and a discharge port. FIG. 10A shows a schematic diagram of the probe 10. The probe 10 is provided with a plurality of discharge ports 26 so as to surround a side surface at a portion (lower portion in FIG. 10A) to be inserted into a tissue. In addition, a flow path 25 connected to the active ingredient supply unit is connected to a portion that is not inserted into the tissue (upper portion in FIG. 10A). FIG. 10B is a cross-sectional view of the probe 10. In this probe 10, the energy transfer means 28 is fixed so that the outer cylinder 29 surrounds it. As shown in the figure, this probe 10 has a space between the energy transfer means 28 and the outer cylinder 29, and this space functions as the flow path 25 of the active ingredient administration means. The active ingredient 9 supplied from the active ingredient supply unit passes through the flow path 25 and is discharged from the discharge port 26 to the outside. In the probe 10 shown in FIG. 10, in the energy transfer means 28, a portion that exchanges energy with the tissue (referred to as an energy transfer region 200) is a portion surrounded by a broken line in FIG. 10B. As illustrated, according to this example, the energy transfer region 200 is located on the outermost surface of the probe 10. For this reason, the probe 10 in this example is advantageous in that it does not have a configuration that obstructs any energy transfer. The outer cylinder 29 can be made of any known material as long as it is corrosion resistant to the solution containing the active ingredient 9 and is less harmful to tissues and cells. For example, stainless steel can be used as the metal, polyethylene resin or PEEK (polyetherethertone) resin can be used as the plastic, and metal that does not contain heavy metals such as lead-free glass can be used as the glass.

  Another example of the case where the active ingredient administration means is structured by a flow path and a discharge port is shown as a cross-sectional view in FIG. In the probe 10 according to FIG. 11, the energy transfer means 28 is accommodated in the outer cylinder 29. The outer cylinder 29 is closed at the target side end by the optical window 30. In FIG. 11, the target side end is the lower end of the probe 10. The space created by the energy transfer means 28, the outer cylinder 29, and the optical window 30 functions as the flow path 25a of the active ingredient administration means. The optical window 30 has a plurality of holes, which function as the discharge ports 26 of the active ingredient administration means. In the upper part of the outer cylinder 29, a flow path 25b extends from the input port 201 toward the active ingredient supply means. The flow path 25b is connected to, for example, the active ingredient supply means, and the active ingredient supplied from the active ingredient supply means passes through the flow path 25b, passes through the input port 201, enters the flow path 25a, and is discharged from the discharge port 26. In this probe 10, the energy transfer region 200 does not protrude from the outer surface of the probe 10. That is, the flow path 25 and the optical window 30 filled with the solution containing the active ingredient 9 exist between the energy transfer region 200 and the external tissue. The probe 10 in this example has the same surface for transmitting and receiving energy and the surface for releasing the active ingredient 9, and is capable of releasing the active ingredient 9 in a concentrated manner at a site where the formation of the glial scar is most desired to be avoided. It is advantageous. The probe 10 emits the active ingredient 9 from the optical window 30, and the energy transfer means 28 transmits and receives energy to and from tissues and cells via the optical window 30. At this time, in the probe 10, the energy transmission / reception means 28 may transmit / receive energy to / from tissues or cells via one or a plurality of discharge ports 26 that release the active ingredient 9, or the optical window 30. You may carry out through the optical material which comprises.

  FIG. 12 shows a case where the energy transfer means 28 is a microscope objective lens 33 in the probe 10 as shown in FIG. In this case, in order to perform good observation with the microscope objective lens 33, the relationship between the hole of the optical window 30 and the wavelength of light is important. As indicated by the optical path 32, the light passes through the lens 31 and further passes through the optical window 30 having a plurality of holes to the cell or region to be observed, and from the observation object to the microscope main body through the reverse path. Head to. Therefore, in order to suppress the scattering of light due to the holes existing in the optical window 30, it is preferable that the wavelength of the light used is larger than the diameter of the hole of the optical window. Also, in order for the active ingredient 9 to be released through the pores, the diameter of the pores naturally needs to be larger than the molecular size of the active ingredient 9. Accordingly, when the probe 10 as shown in FIG. 12 is used, it is preferable that [wavelength of light]> [diameter of hole of optical window]> [molecular size of active ingredient]. In the method of the present invention, the wavelength of light is generally 400 to 700 nm, and the size of a general protein molecule is about 1 to 20 nm. Therefore, the diameter of the hole of the optical window is about 2 to 70 μm. Preferably there is. Furthermore, it is more preferable to set it to 2 times or more for the molecular size of the active ingredient used and 1/10 or less for the wavelength of light used. Further, the discharge ports 26 provided in the optical window 30 do not necessarily have to have the same diameter, and only the holes in a specific region of the optical window 30 can be made larger or smaller. For example, the hole in the region that becomes the optical path 32 (for example, the center of the optical window 30) has a diameter that meets the above conditions, while the hole in the region that does not become the optical path 32 (for example, around the optical window 30) is larger than the diameter. It can be. The diameter of the animal cell is generally 10 to 30 μm and has a size several tens of times the wavelength of the light. Therefore, when cells are released through the hole of the optical window, the hole may be provided around the optical window 30, that is, at a position avoiding the optical path 32.

The present invention relates to an apparatus for stimulating or measuring vertebrate central nervous tissue having one or more probes as described above.
Such an apparatus includes at least one probe according to the present invention, and, in addition, if necessary, a part for controlling the energy transfer means, a part for controlling the active ingredient administration means, and a puncturing means. And a portion for positioning and fixing the probe and various means with respect to the target animal. The means for controlling the energy transfer means is, for example, a power source or an electric signal detector when the energy transfer means is an electrode, and if the energy transfer means is an optical fiber or a microscope objective lens, illumination is performed. A light source, a photodetector, a microscope body, and the like. For example, when the active ingredient supply means is a syringe, the portion for controlling the active ingredient administration means means that the amount or speed of the active ingredient to be administered is pushed by pushing the piston of the syringe at a constant pressure or speed. It is a part for adjusting the height. The part for controlling the puncturing means includes, for example, a control device that can puncture precisely at a depth aimed at a site where the needle is aimed when a puncture needle or the like is used independently of the probe. It is an important part.

  FIG. 13 shows an apparatus having a plurality of probes. In FIG. 13, two probes 10 are applied to the brain tissue of the mouse 35. In the probe 10, the flow path 25 is connected to the active ingredient administration means 27, and an image optical fiber 36 with a small objective lens (not shown) attached to the tip of the brain tissue side is connected to the microscope body 34. Optical images detected by the respective probes 10 are captured by the respective microscope bodies 34 to which they are connected. By setting it as such an apparatus, a specific site | part can be observed from a different angle, or a specific site | part and another site | part can be observed simultaneously, and those relations can be investigated. When the conventional method is applied to a plurality of places, damage accumulation and excessive glial scar formation occur, and it is difficult to carry out an appropriate study. However, if the method according to the present invention is applied, damage is caused in individual places. Is restored and the formation of glial scars is suppressed, so that stimulation and / or measurement (especially observation) can be performed continuously in a state where various electrical or optical obstacles caused by the glial scars are eliminated. Therefore, even if it is applied to a plurality of locations, problems in the conventional case are hardly caused, and good stimulation and / or measurement can be performed.

  DESCRIPTION OF SYMBOLS 1 ... Central nervous tissue, 2 ... Nerve cell, 3 ... Cell body, 4 ... Axon, 5 ... Glial cell, 6 ... Puncturing means, 7 ... Axonal cutting part, 8 ... Active ingredient administration means, 9 ... Active ingredient, DESCRIPTION OF SYMBOLS 10 ... Probe, 11 ... Repaired axon, 12 ... Degenerated glial cell, 13 ... Glia scar, 14 ... Insert part, 15 ... Electrode probe, 16 ... Tip tip, 17 ... Non-conductive part, 18 ... Optical probe, 19 ... non-measurement part, 20 ... measurement part, 21 ... adsorption layer, 22 ... uneven layer, 23 ... porous layer, 24 ... active ingredient administration means, 25 ... flow path, 26 ... discharge port, 27 ... syringe, 28 ... energy Transfer means, 29 ... outer cylinder, 30 ... optical window, 31 ... lens, 32 ... optical path, 33 ... microscope objective lens, 34 ... microscope main body, 35 ... mouse, 36 ... optical fiber, 101 ... holding structure, 102 ... inside probe, Energy transfer area ... 200, investment Mouth ... 201

Claims (4)

Using an energy transfer means is an optical fiber or a microscope objective lens, a method for measuring the central nervous system of vertebrates except humans as optically stimulate and optical image,
a) puncturing the tissue;
b) administering an active ingredient having a central nerve repair effect to the puncture site;
c) performing energy transfer between the energy transfer means and the central nervous tissue at the punctured site, wherein the energy transfer is performed in a state where the central nerve repair effect by the active ingredient is obtained. A method characterized by. The method according to claim 1, wherein the central nerve repair effect is an axonal elongation promoting effect, an astrocyte activation inhibitory effect, and / or a glial scar formation inhibitory effect. Wherein the active ingredient is a cell selected from the group consisting of astrocyte restricted precursor cells, olfactory ensheathing glia cells, placenta tissue-derived cells, umbilical cord tissue-derived cells, glial restricted precursor cells and activated immature astrocytes, The method according to claim 1 or 2 . The active ingredient is heparin, heparin derivative, noggin, fasudil hydrochloride hydrate, IL-17F, EphA4 receptor antagonist, antibody against EphA4 receptor, antisense RNA against EphA4 receptor, antisense DNA against EphA4 receptor, EphA4 SiRNA against receptor, ephrin antagonist, antibody against ephrin, antisense RNA against ephrin, antisense DNA against ephrin, siRNA against ephrin, cyclic peptide containing amino acid sequence Arg-Gly-Glu, p75 receptor binding agent, Nogo inhibition Selected from the group consisting of agents, TNR inhibitors, MAG inhibitors, CD81, phosphodiesterase type 4 inhibitors, axon forming factor 1, axon forming factor 2 and osteogenic factor 1A type receptor inhibitors Is a substance, the method according to claim 1 or 2.

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