JP7094551B2 - Buried device for small animals - Google Patents

Description

The present invention relates to a device implanted in the head of a small animal such as a mouse or rat.

Animal experiments using small animals such as mice and rats will be conducted to evaluate the safety and efficacy of drugs in new drug development. In recent years, in small animal experiments, there are cases where a monitoring device is attached to an individual to monitor the biological activity of the individual. As such a monitoring device, for example, there is a device equipped with Bluetooth (registered trademark) and an accelerometer (see, for example, Non-Patent Document 1).

In animal experiments, reagents may be administered or physical stimuli may be given to individuals to induce or suppress specific biological activities for the purpose of creating conditions for drug efficacy evaluation, but quantitative evaluation is repeated. There is a problem that it is difficult. As a technique for solving this problem, a method called optogenetics, which induces / suppresses biological activity by using light stimulation, has been developed in recent years. For example, small wireless optogenetic devices have been developed that are implanted in the head of a mouse and used (see, for example, Non-Patent Documents 2 and 3).

Pasquet MO et al., “Wireless inertial measurement of head kinematics in freely-moving rats,” SCIENTIFIC REPORTS, 6:35689, DOI: 10.1038 / srep35689, SPRINGER NATURE, 21 October 2016, pp.1-13 Park SI et al., “Soft, stretchable, fully implantable miniaturized optoelectronic systems for wireless optogenetics,” NATURE BIOTECHNOLOGY, Vol. 33, No. 12, Nature America, December 2015, pp. 1280-1286 Montgomery KL et al., “Wirelessly powered, fully internal optogenetics for brain, spinal and peripheral circuits in mice,” NATURE METHODS, Vol. 12, No. 10, Nature America, October 2015, pp. 969-974

Due to the large size of conventional monitoring devices, it is difficult to implant them in the heads of individuals, especially small mice. As a result, the device may adhere to the skin or may not be completely embedded and part of the device may be exposed outside the body, which may cause inflammation and infection and increase the stress on the body. It ends up. In addition, when a plurality of individuals are bred and tested in one place, the device may be recognized as a foreign substance during a fight or grooming, and the device may be destroyed by another individual or itself. As described above, there is a problem that the conventional monitoring device is not suitable for simultaneous monitoring of a plurality of individuals.

Furthermore, it is desirable to adopt optogenetics technology as a means for inducing or suppressing specific biological activities in individuals.

In view of the above problems, the present invention provides a small animal implant device capable of inducing and suppressing specific biological activity by light stimulation, completely implanting an individual, and simultaneously monitoring the biological activity of a plurality of individuals. The purpose is to provide.

According to one aspect of the present invention, it is a small animal implant device used by implanting it in the head of a small animal such as a mouse or a rat, and has a size that fits in the parietal region of the skull of the small animal in a plan view. The substrate is equipped with a light stimulating unit that gives a light stimulus to the brain of a small animal, a sensing unit that measures biological information and behavior of a small animal, a light stimulating unit, and wireless communication between the sensing unit and an external device. A small size that is equipped with a wireless communication part to perform and a power supply part that supplies power to each part, and the substrate has an alignment mark for aligning with at least one of the bregma and lambda of the skull of a small animal. Implantation devices for animals are provided.

The alignment mark is, for example, a hole penetrating the substrate. Alternatively, at least a portion of the alignment mark on the substrate may be formed of a transparent material, and the alignment mark may be printed or engraved on the substrate.

The plan view shape of the substrate may be tapered. Further, the substrate may be a curved substrate that matches the shape of the crown region of the skull of a small animal.

The power supply unit may have a secondary battery that supplies electric power to each unit and a wireless power receiving device that is wirelessly supplied with power from the outside to charge the secondary battery. Alternatively, the power supply unit may have a secondary battery that supplies electric power to each unit and a power generation device that generates power and charges the secondary battery.

According to the present invention, a specific biological activity can be induced / suppressed by light stimulation, and the biological activity of a plurality of individuals can be monitored at the same time by completely implanting the individual.

Top view of the planting device for small animals according to an embodiment of the present invention. A block diagram showing an example of the functional configuration of a planting device for small animals according to an embodiment of the present invention. A block diagram showing another example of the functional configuration of the planting device for small animals according to the embodiment of the present invention. Top view of mouse skull Figure showing an example of implanting a small animal implant device in the head of a mouse Sagittal view of the head of a mouse implanted with a small animal implantation device Schematic diagram of drug efficacy evaluation test (during disease induction by photostimulation) using an individual in which an implant device for small animals was implanted Schematic diagram of drug efficacy evaluation test (during monitoring) using an individual in which an implant device for small animals was implanted Plan view of a small animal implant device according to a modified example

Hereinafter, embodiments will be described in detail with reference to the drawings as appropriate. However, more detailed explanation than necessary may be omitted. For example, detailed explanations of already well-known matters and duplicate explanations for substantially the same configuration may be omitted. This is to avoid unnecessary redundancy of the following description and to facilitate the understanding of those skilled in the art.

It should be noted that the inventor intends to limit the subject matter described in the claims by those skilled in the art by providing the accompanying drawings and the following description in order to fully understand the present invention. do not have. In addition, the dimensions, thickness, detailed shape of details, etc. of each member drawn in the drawing may differ from the actual ones.

The planting device for small animals according to one embodiment of the present invention is a device that has been made smaller, thinner, and lighter so that it can be completely implanted in the head of a small animal such as a mouse or a rat. Hereinafter, for convenience, an example of an implantable device for a mouse will be described.

≪Structure≫
First, the structure of the planting device for small animals according to the embodiment of the present invention will be described. FIG. 1 is a plan view of a small animal planting device 100 according to an embodiment of the present invention. The planting device 100 for small animals is configured by mounting electronic components, a battery, and various sensors on a substrate 10. In consideration of complete implantation in a living body, the small animal implantation device 100 is protected by a thin film having high biocompatibility, for example, a thickness of about 1 to 2 microns containing a paraxylylene-based polymer as a main component. It is totally covered with a film. Complete implantation means that the entire small animal implantation device 100 is embedded in the mouse body without exposing a part of the small animal implantation device 100 to the outside of the mouse body.

The substrate 10 has a tapered or substantially oval shape in a plan view, and its size is such that the length from the bottom of the taper to the tip of the taper (horizontal direction in FIG. 1) is about 1 cm, and the width at the bottom of the taper (). In FIG. 1, the vertical direction) is about 1 cm, and the thickness is about 100 microns. As will be described later, when implanting the small animal implantation device 100 in the head of a mouse, the small animal has a tapered tip facing the front of the mouse and the bottom of the taper facing the back of the mouse. The implantable device 100 is fixed (adhered) in contact with the skull of the mouse. As described above, the substrate 10 has a size that fits in the parietal region of the skull of the mouse in a plan view. Further, the substrate 10 has a curved surface shape that matches the shape of the crown region of the skull of the mouse. The crown region referred to here refers to a region extending from the occipital bone to the frontal bone. Details will be described later.

In the substrate 10, alignment marks 11 and 12 are provided at substantially the center of the substrate 10 and near the bottom of the taper on the center line connecting the tip of the taper and the bottom of the taper, respectively. Specifically, the alignment marks 11 and 12 are holes having a diameter of 1 mm to several mm formed so as to penetrate the substrate 10. The alignment mark 11 is a mark for aligning the substrate 10 with the bregma of the skull of the mouse. The alignment mark 12 is a mark for aligning the substrate 10 with the lambda of the skull of the mouse. In FIG. 1, the alignment marks 11 and 12 are circular in consideration of ease of processing, but the present invention is not limited to this, and the alignment marks 11 and 12 may have other shapes, for example, a cross shape. The alignment of the small animal implant device 100 using the alignment marks 11 and 12 will be described later.

The substrate 10 can be realized by a general printed circuit board. The curved surface shape of the substrate 10 described above can be realized, for example, by scanning the skull region of the mouse skull sample with a 3D scanner and reproducing the shape of the skull region using a 3D printer.

An IC (Integrated Circuit) chip 13, a Bluetooth (registered trademark) module (BT module) 14, a power supply module 15, and various sensors 16 are mounted on the surface of the substrate 10. Further, an LED (Light Emitting Diode), which will be described later, is mounted on the back surface of the substrate 10. The power supply module 15 has a thin battery 151. Power lines are wired from the power supply module 15 to the IC chip 13 and the BT module 14, respectively. A communication line is wired between the IC chip 13 and the BT module 14. The various sensors 16 are wired and connected to the IC chip 13.

The size, arrangement position, and number of sensors 16 of these constituent members are merely examples. In particular, as will be described later, when an invasive type sensor for measuring the neural activity of a mouse is used for the sensor 16, the arrangement position of the sensor 16 may change depending on the target site in the brain to be measured. Further, in the case of an invasive type sensor, an electrode (probe) (not shown) protrudes from the back surface of the substrate 10.

As described above, the small animal implantation device 100 has been made smaller, thinner, and lighter so that it can be completely implanted in the head of a mouse. In particular, the weight is 1 g or less, which is 1/20 of the weight of a standard mouse of 20 g, so that the stress applied to the mouse is minimized.

≪Functional configuration≫
Next, the functional configuration of the small animal implantation device 100 having the above structure will be described. FIG. 2 is a block diagram showing an example of the functional configuration of the planting device 100 for small animals. The small animal implant device 10 has a light stimulating unit 101, a sensing unit 102, a wireless communication unit 103, and a power supply unit 104 as functional blocks.

The light stimulating unit 101 is a functional element that gives a light stimulus to the mouse brain. Nerve cells (target cells) at specific sites in the brain of mice in which the implant device 100 for small animals is implanted are genetically engineered in advance to express photosensitive proteins to impart photosensitivity and photostimulation. It is designed to ignite nerves when it receives it. Specifically, the light stimulating unit 101 has the above-mentioned LED 17 and an LED drive circuit 131 for driving the LED 17. In addition to blue, green or red can be used as the emission color of the LED 17. The emission color of the LED 17 may be an appropriate one depending on the purpose. The LED drive circuit 131 is integrated in the IC chip 13 by CMOS (Complementary Metal Oxide Semiconductor) technology. The light stimulating unit 101 gives a light stimulus to the mouse brain by causing the LED 17 to emit light. As described above, the photostimulator 101 can freely control the induction or suppression of a specific biological activity in the mouse by light using the technique of optogenetics.

The sensing unit 102 is a functional element for measuring biological information and behavior of a mouse. Specifically, the sensing unit 102 has various sensors 16 and an A / D conversion circuit 132 that converts electrical signals measured by the various sensors 16 into digital values. More specifically, the sensing unit 102 has a heart rate sensor, a body temperature sensor, and the like as sensors for measuring biological information. The sensing unit 102 may have an invasive type sensor that measures neural activity in a specific region of the brain by inserting a probe into a target site such as a motor area or a visual cortex in the brain of a mouse. Further, the sensing unit 102 has an acceleration sensor, a gyro sensor, and the like as sensors for measuring the behavior of the mouse. The A / D conversion circuit 132 is integrated in the IC chip 13 by CMOS technology.

The wireless communication unit 103 is a functional element that performs wireless communication between the optical stimulation unit 101 and the sensing unit 102 and an external device (a personal computer as a data collection / analysis device described later) (described later). Specifically, the wireless communication unit 103 is composed of the BT module 14. More specifically, the wireless communication unit 103 has a microcontroller and a timer (not shown). The microcontroller sends a signal to the light stimulating unit 101 by an instruction from an external device or a trigger from a timer to switch lighting / extinguishing of the LED 17. Further, the microcontroller sends a signal to the sensing unit 102 by an instruction from an external device or a trigger from a timer to cause each of the above sensors to perform a measurement operation, and receives the measurement data from the sensing unit 102 to the external device. Forward.

In addition to Bluetooth (registered trademark), WiFi (registered trademark), ZigBee (registered trademark), infrared communication, and the like can be used as wireless communication standards. Among various wireless communication standards, Bluetooth (registered trademark) supports sleep mode. It returns from sleep mode by giving a signal from the outside, transfers the measurement data of various sensors 16 to an external device, and then sleeps again. It is excellent in terms of low power consumption such as transitioning to a mode. In addition, Bluetooth (registered trademark) is suitable for the implantable device 100 for small animals in that it can simultaneously communicate with a plurality of devices and can simultaneously observe the biological information and behavior of a plurality of mice in real time.

The power supply unit 104 is a functional element that supplies electric power to each of the above units. Specifically, the power supply unit 104 includes a battery 151 as a secondary battery and a wireless power receiving device 152 that is wirelessly supplied from the outside to charge the battery 151. The wireless power supply method that can be used may be a small one that can wirelessly supply power at a short distance in consideration of an experimental environment using a mouse in which a small animal implant device 100 is implanted. For example, Qi (registered trademark), AirFuel (registered trademark), and the like can be used.

The power supply unit 104 may have a power generation device that generates power and charges the battery 151 instead of the wireless power receiving device 152. FIG. 3 is a block diagram showing another example of the functional configuration of the small animal planting device 100 according to the embodiment of the present invention. The block diagram of FIG. 3 differs from the block diagram of FIG. 2 in that the power supply unit 104 has the power generation device 153 instead of the wireless power receiving device 152. A dye-sensitized solar cell can be used as a device capable of generating electricity even when the planting device 100 for small animals is completely implanted in the head of a mouse. The solar cell can generate electricity with infrared rays, and since infrared rays pass through the scalp of a mouse, it is suitable as a power generation device for a small animal implant device 100. It is also possible to use a temperature difference power generation device as the power generation device 153.

It is also possible to use a primary battery as the battery 151 instead of the secondary battery. In this case, the planting device 100 for small animals cannot be reused and is disposable, but since the wireless power receiving device 152 and the power generation device 153 are not required, a primary battery having a large capacity can be arranged on the substrate 10. , The operable time of the small animal implantation device 100 can be secured as long as possible.

As described above, in the small animal implant device 100, the circuit that realizes the light stimulation function and the sensing function is integrated into the IC chip 13 by CMOS technology, which enables miniaturization, weight reduction, and low power consumption. I have to. A wireless communication function may be added to the IC chip 13, but various BT modules 13 having a small size, light weight, and low power consumption are already on the market at a relatively low price, so an appropriate one among the commercially available products is available. Just select.

≪Example of implantation in the head of a mouse≫
Next, an example of implanting the small animal implantation device 100 in the head of a mouse will be described. FIG. 4 is a plan view of the skull of a mouse. In the skull 200 of the mouse 20, the parietal region extending to the left and right frontal bones 201, the left and right parietal bones 202, and the occipital bone 203 is slightly convex but generally flat. The plan view shape of the parietal region is a tapered shape from the occipital bone 203 toward the frontal bone 201. The size of the crown region is about 1 cm in the sagittal direction and about 1 cm in the coronal direction.

In the parietal region of the skull 200 of the mouse 20, the frontal suture 204 forming the boundary between the left and right frontal bones 201, the coronal suture 205 forming the boundary between the frontal bone 201 and the skull 202, and the boundary between the left and right skulls 202 are formed. There is a sagittal suture 206 and a lambda suture 207 that borders the skull 206 and the occipital bone 203, the intersection of the frontal suture 204, the coronal suture 205 and the sagittal suture 206 is the bregma 208, and the sagittal suture and the lambda suture 207. The intersection with is Lambda 209. An incision in the scalp of the crown of the mouse 20 exposes the various bones and sutures that make up the crown region.

FIG. 5 is a diagram showing an example in which a small animal implanting device 100 is implanted in the head of a mouse 20. FIG. 6 is a sagittal view of the head of the mouse 20 in which the small animal implant device 100 is implanted. When implanting the small animal implant device 100 in the head of the mouse 20, the scalp of the mouse 20 is incised to expose the crown region of the skull 200, and a hole is made in a predetermined position of the skull 200 using a manipulator or the like. Open. The predetermined location is a location where the probe of the invasive type sensor 16 is inserted and a location where the LED 17 is embedded.

As described above, the small animal implant device 100 has a hole (alignment mark 11) for aligning the substrate 10 with the bregma 208 of the skull 200 of the mouse 20 and a hole (alignment) for aligning the substrate 10 with the lambda 209. The mark 12) has been opened. The substrate 10 is positioned so that the Bregma 208 and the Lambda 209 can be seen through these holes, and the substrate 10 is fixed to the skull 200 of the mouse 20 with an adhesive. This allows the probe of the invasive type sensor 16 and the LED 17 to be accurately positioned in place on the skull 200 of the mouse 20. After fixing the small animal implant device 100 to the skull 200 of the mouse 20, the scalp is sutured. As a result, the small animal implantation device 100 is completely implanted in the head of the mouse 20.

≪Example of animal experiment using a buried device≫
Next, an example of an animal experiment using the small animal implantation device 100 according to the present embodiment will be described. 7 and 8 are schematic views of a drug efficacy evaluation test using mice in which a small animal implant device 10 is implanted, and FIG. 7 shows a state in which a specific individual is light-stimulated to induce a disease. FIG. 8 shows how to monitor the biological information and behavior of each individual.

Photosensitivity is expressed by genetically manipulating nerve cells at specific sites in the brain of multiple individuals (6 mice A to F in the examples of FIGS. 7 and 8) used in the experiment. Is given. Then, the implanting device 100 for small animals is implanted in the head of each individual that has undergone such genetic manipulation. Mice A to F in which the small animal implantation device 100 is implanted are bred in the same breeding environment in the cage shown in the figure. A personal computer 30 as a data collection / analysis device is installed on the side of the cage. The personal computer 30 can operate as a master device of Bluetooth (registered trademark) and individually wirelessly communicate with a small animal implant device 100 as a slave device to acquire biological information and behavior sensing data of each individual. can. In this way, in animal experiments using the small animal implantation device 100, it is possible to monitor the biological activity of each individual group-reared so that they can freely act in real time.

With reference to FIG. 7, when inducing a specific disease in a specific individual (for example, mice B, C, E), the experimenter operates a personal computer 30 to identify the target individual. The personal computer 30 transmits a light stimulation command to the small animal implant device 100 embedded in the specified individual by wireless communication of Bluetooth (registered trademark). Upon receiving the command, the small animal implant device 100 causes the LED 17 to emit light. This makes it possible to give a light stimulus to the target individual anywhere in the cage to induce a disease.

With reference to FIG. 8, the drug to be evaluated for efficacy is administered to an individual (for example, mice B, C, E) that has induced a specific disease. Before and after administration of the drug, the personal computer 30 continuously acquires biological information and behavioral sensing data of each individual from the small animal implant device 100 implanted in each individual. Then, by analyzing the data, the effect of the administered drug can be evaluated.

≪Effect≫
According to this embodiment, the following effects are achieved.
1) Since the size and shape of the substrate 10 are matched to the parietal region of the skull 200 of the mouse 20, the small animal implant device 100 can be completely implanted in the head of the mouse 20. This makes it possible to eliminate the causes of inflammation and infectious diseases in an individual, and to accurately monitor the biological activity of each individual without stressing the individual and without restricting the behavior of the individual.
2) The alignment marks 11 and 12 can accurately and easily determine the mounting position of the small animal implantation device 100.
3) It is possible to induce or suppress a specific biological activity by giving a light stimulus to a specific individual by wireless communication with an external device (personal computer 30), and simultaneously monitor the biological activity of a plurality of individuals. can do. This makes it possible to carry out a group test while breeding a plurality of individuals in a natural state.

≪Variation example≫
The small animal implant device 100 according to the present embodiment can be modified as follows. FIG. 9 is a plan view of the planting device for small animals according to the modified example. The small animal implant device 100 according to the modification includes a substrate 10 made of a transparent material such as PET (polyethylene terephthalate) and PI (transparent polyimide). The alignment marks 11 and 12 are printed or engraved on the substrate 10 instead of the holes penetrating the substrate 10. Even if such a modification is applied to the small animal implantation device 100, the above effect does not change at all. In the example of FIG. 9, the alignment marks 11 and 12 are crosses, but they may be triangles, squares, stars, circles, or the like. Further, it is not necessary to form the entire substrate 10 with a transparent material, and at least the portions of the alignment marks 11 and 12 may be formed with a transparent material.

Further, although the two alignment marks 11 and 12 are provided on the substrate 10 in FIGS. 1 and 9, one of them may be omitted. That is, the number of alignment marks may be one. If there is at least one alignment mark, the taper shape of the substrate 10 is aligned with the sagittal direction of the skull 200 of the mouse 20, and the offset position in the sagittal direction is determined by Bregma 208 or Lambda 209. The small animal implantation device 100 can be placed in the correct position.

As described above, an embodiment has been described as an example of the technique in the present invention. To that end, the accompanying drawings and detailed description are provided.

Therefore, among the components described in the attached drawings and the detailed description, not only the components essential for problem solving but also the components not essential for problem solving in order to illustrate the above technique. Can also be included. Therefore, the fact that those non-essential components are described in the accompanying drawings or detailed description should not immediately determine that those non-essential components are essential.

Further, since the above-described embodiment is for exemplifying the technique of the present invention, various changes, replacements, additions, omissions, etc. can be made within the scope of claims or the equivalent thereof.

100 ... Small animal implant device, 10 ... Substrate, 11 ... Alignment mark, 12 ... Alignment mark, 101 ... Photostimulation unit, 102 ... Sensing unit, 103 ... Wireless communication unit, 104 ... Power supply unit, 151 ... Battery (2) Next battery example), 152 ... wireless power receiving device, 153 ... power generation device, 20 ... mouse (example of small animal), 200 ... skull, 208 ... bregma, 209 ... lambda, 30 ... personal computer (example of external device)

Claims (7)

It is an implant device for small animals that is used by implanting it in the head of small animals such as mice and rats.
A substrate sized to fit in the parietal region of the skull of the small animal in plan view.
On the substrate
A light stimulating unit that gives light stimulation to the brain of the small animal,
Sensing unit that measures biological information and behavior of the small animal,
A wireless communication unit that performs wireless communication between the optical stimulation unit and the sensing unit and an external device, and a power supply unit that supplies electric power to each unit are mounted.
An implantable device for small animals, characterized in that the substrate has an alignment mark for aligning with at least one of the bregma and lambda of the skull of the small animal. The small animal implant device according to claim 1, wherein the alignment mark is a hole penetrating the substrate. At least the portion of the alignment mark on the substrate is formed of a transparent material.
The small animal implant device according to claim 1, wherein the alignment mark is printed or engraved on the substrate. The planting device for small animals according to any one of claims 1 to 3, wherein the substrate has a tapered shape in a plan view. The implantable device for small animals according to any one of claims 1 to 4, wherein the substrate is a curved substrate having a curved shape that matches the shape of the skull region of the small animal. The small animal according to any one of claims 1 to 5, wherein the power supply unit has a secondary battery that supplies electric power to each unit and a wireless power receiving device that is wirelessly supplied from the outside to charge the secondary battery. Buried device for. The planted device for small animals according to any one of claims 1 to 5, wherein the power supply unit has a secondary battery for supplying electric power to each unit and a power generation device for generating electric power to charge the secondary battery. ..

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