Monkey brain nucleus sampling device and application thereof

Abstract

The invention discloses a monkey brain nucleus sampling device and application thereof. The monkey brain nucleus sampling device comprises an inner tray, an outer tray and a sampling positioning template. When the monkey brain nucleus sampling device is used for sampling materials, expensive instruments and equipment are not needed, tools are cheap and easy to obtain, the repeatability is high, the consistency of sampling positions can be ensured, and synchronous sampling of multiple animals can be carried out by simple training of experimenters.

Description

Technical Field

[0001] The invention belongs to the field of animal experiment mechanical devices, and in particular relates to a monkey brain nucleus sampling device and an application thereof. Background Art

[0002] The brain is the most important component of the central nervous system of all animals. Deep in the brain tissue under the cerebral cortex, there are many different types of neuronal cell bodies (also known as neural nuclei), which are responsible for processing the neuropsychological activities of animals from basic sensations and motor functions to advanced cognition and memory. Abnormalities in their structure and function often lead to serious neuropsychiatric diseases, such as Huntington's disease, Parkinson's disease, schizophrenia, and depression. Therefore, in drug research and development, research on brain nuclei is of key significance: on the one hand, some drugs that are locally administered to the brain or act on the central nervous system require the separation of brain nuclei to confirm the safety and effectiveness of the drugs on the relevant nuclei; on the other hand, in some drugs that can pass through the blood-brain barrier or may have systemic distribution, tissue distribution tests are also required to determine whether the relevant brain nuclei are affected. In both cases, scientific researchers are required to quickly and accurately remove the target neural nuclei for subsequent research.

[0003] At present, the extraction and fixation of brain nuclei in experimental animals is a challenging problem for scientists at home and abroad. In rodents, one way is to cut the brain tissue into 10-100μm brain slices, and then perform laser capture microdissection or surgical cutting under a stereo microscope for extraction. This method requires high-end instruments and equipment, and it takes a long time, which cannot meet the requirements of the experiment type (such as PCR experiment, etc.) that requires extraction time; another way is to put the brain into a positioning cutting instrument for positioning cutting and extraction, but this method still takes a long time, and most of the brain nuclei are irregular in shape, and the positioning instrument can only perform sheet cutting, and cannot completely separate specific nuclei; in addition, there is a micro-sampling method, which is to put the brain into a fixed groove and use a specific syringe to absorb the target nucleus, but this method has a small sampling volume, and the brain is a three-dimensional structure, and the syringe will absorb the brain tissue of the entire needle channel, the tissue is shaped like tofu, and the morphology cannot be guaranteed to be complete in the future, and it cannot be used for specific experimental types (such as transmission electron microscopy, qPCR, etc.).

[0004] At present, there is no mature technology for extracting brain nuclei from non-human primates at home and abroad. The preliminary operation generally refers to the extraction method of rodents. The brain is cut into slices of a certain thickness at equal distances using a non-human primate brain mold (such as M5189), and then the nuclei are extracted. However, the defects of this method are also obvious: first, compared with rodents, the brain structure of non-human primates is more complex and delicate, and multiple nuclei will appear on different brain slices at the same time. When the nuclei are extracted in an in vitro state, the precision and accuracy of the extraction of nuclei are insufficient, and it takes more time and the tissue autolysis is obvious; secondly, the mold can only cut brain tissue with a fixed unit thickness (such as 2μm, 4μm, 6μm), resulting in some nuclei that cannot be fully exposed on the brain slices, and the brain tissues of animals of different ages and genders are slightly different, which will lead to differences in the positioning of the nuclei and make it impossible to extract them accurately; in addition, the brain mold is expensive and not suitable for promotion and use.

[0005] Therefore, there is an urgent need to develop a new method that is different from the above-mentioned extraction method to quickly, accurately and cost-effectively extract relevant nuclei from the brains of non-human primates, which can be conveniently used in subsequent drug development research or tissue distribution experiments. The animal tissues involved are from crab-eating macaques / rhesus monkeys (can also be used for other non-human primates). Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a monkey brain nucleus sampling device and its application, so as to achieve accurate positioning and extraction of neural nuclei, and obtain target neural nuclei with complete inherent morphology, so as to better provide samples for subsequent experiments, so as to effectively ensure the accuracy and timeliness of the required tissue distribution samples.

[0007] In order to solve the above technical problems, the first aspect of the present invention provides a monkey brain nucleus sampling device, the monkey brain nucleus sampling device comprising:

[0008] An inner tray, wherein the surface of the inner tray has a monkey brain model groove A and a monkey brain model groove B for placing a monkey brain, and a hollow cavity is provided in the inner tray as a cooling cavity, and the cooling cavity is used to cool the monkey brain located in the monkey brain model groove A and / or the monkey brain model groove B;

[0009] An outer tray, the inner tray is arranged inside the outer tray, the outer tray is used to hold a coolant and to cool the inner tray;

[0010] A material collection positioning template, the material collection positioning template comprises a material collection positioning template A and a material collection positioning template B, the material collection positioning template A and the material collection positioning template B have hollow positioning holes for locating the positions of different brain nuclei; the material collection positioning template A is connected to the surface of the inner tray and used in conjunction with the monkey brain model groove A, for locating the monkey brain placed in the monkey brain model groove A; the material collection positioning template B is connected to the surface of the inner tray and used in conjunction with the monkey brain model groove B, for locating the monkey brain placed in the monkey brain model groove B; and / or, the hollow positioning holes on the material collection positioning template A include parietal lobe hollow positioning holes, frontal lobe hollow positioning holes, occipital lobe hollow positioning holes and temporal lobe hollow positioning holes; the hollow positioning holes on the material collection positioning template B include hypothalamus hollow positioning holes, midbrain hollow positioning holes, thalamus hollow positioning holes, corpus callosum hollow positioning holes and cingulate gyrus hollow positioning holes.

[0011] In some embodiments, the surface of the inner tray has snap holes, and the snap holes are distributed around the monkey brain model groove A and the monkey brain model groove B. The material collection positioning template A and the material collection positioning template B are respectively connected to the monkey brain model groove A and the monkey brain model groove B through the snap holes.

[0012] In some embodiments, the number of the snap holes is 4, 5 or 6.

[0013] In some embodiments, a water injection hole is provided on a side of the inner tray, and the water injection hole is connected to the cooling chamber; a coolant is contained in the cooling chamber; and the monkey brain is a cynomolgus monkey brain.

[0014] In some embodiments, the sampling and positioning template B also includes a caudate nucleus head identification point.

[0015] In some embodiments, the coolant is an ice-water mixture.

[0016] In some embodiments, the monkey brain model groove A and the monkey brain model groove B are rectangular parallelepipeds.

[0017] In some embodiments, the dimensions of the monkey brain model groove A are 8 cm in length, 5 cm in width, and 5 cm in depth; the dimensions of the monkey brain model groove B are 8 cm in length, 5 cm in width, and 5 cm in depth.

[0018] In some embodiments, the material collection positioning template A and the material collection positioning template B are rectangular.

[0019] In some embodiments, the size of the material collection positioning template A is 8.5 cm in length and 5.5 cm in width; the size of the material collection positioning template B is 8.5 cm in length and 5.5 cm in width.

[0020] The shapes and sizes of the monkey brain groove A, monkey brain groove B, material collection positioning template A and material collection positioning template B are not limited to those described above and can be modified according to the actual size of the monkey brain.

[0021] In some embodiments, the midpoint of the right side of the sampling positioning template A is used as the coordinate origin (0, 0), and the horizontal coordinate axis is established with the right as the positive direction. The horizontal coordinate axis is rotated 90° counterclockwise with the coordinate origin as the center to obtain the vertical coordinate axis; the center coordinates of the parietal lobe hollow positioning hole are: (-4.4cm, 2.0cm), and the radius of the circle is 0.4cm; the center coordinates of the frontal lobe hollow positioning hole are: (-0.9cm, 0.9cm), and the radius of the circle is 0.4cm; the center coordinates of the occipital lobe hollow positioning hole are: (-6.6cm, 0.9cm), and the radius of the circle is 0.4cm; the center coordinates of the temporal lobe hollow positioning hole are: (-4.4cm, -0.8cm), and the radius of the circle is 0.3cm.

[0022] In some embodiments, the midpoint of the right side of the sampling positioning template B is used as the coordinate origin (0, 0), and the horizontal coordinate axis is established with the right as the positive, and the horizontal coordinate axis is rotated 90° counterclockwise with the coordinate origin as the center to obtain the vertical coordinate axis; the center coordinates of the hypothalamus hollow positioning hole are: (-3.0 cm, -0.2 cm), and the circle radius is 0.2 cm; the center coordinates of the midbrain hollow positioning hole are: (-3.9 cm, -0.2 cm), and the circle radius is 0.2 cm; the center coordinates of the thalamus hollow positioning hole are: (-3.2 cm, 0.2 cm), and the circle radius is 0. 2cm; the hollow positioning hole of the corpus callosum is an ellipse, and the coordinates of the ellipse center are: (-2.5cm, 0.8cm), the major axis of the ellipse is parallel to the horizontal axis, the radius of the major axis is 0.4cm, the minor axis of the ellipse is parallel to the vertical axis, and the radius of the minor axis is 0.2cm; the hollow positioning hole of the cingulate gyrus is an ellipse, and the coordinates of the ellipse center are: (-2.5cm, 1.4cm), the major axis of the ellipse is parallel to the horizontal axis, the radius of the major axis is 0.4cm, the minor axis of the ellipse is parallel to the vertical axis, and the radius of the minor axis is 0.2cm.

[0023] In some embodiments, the center coordinates of the circle marking point of the caudate nucleus head are: (-1.5 cm, 0.8 cm), and the radius of the circle is 0.2 cm.

[0024] In some embodiments, the monkey brain nucleus sampling device further comprises a stainless steel semicircular shovel; the stainless steel semicircular shovel comprises a shovel handle and a shovel head connected to each other; the length of the shovel handle is 30 to 70 mm.

[0025] In some embodiments, the limit depth of the shovel head is 1.0-8.0 mm, the shape of the shovel head is a semicircular arc, the diameter of the semicircular arc is 1.0-4.0 mm, and the central angle of the semicircular arc is 180°.

[0026] In some preferred embodiments, the connection is a detachable connection.

[0027] In some preferred embodiments, the length of the shovel handle is 40-60 mm.

[0028] In some preferred embodiments, the ultimate depth of the shovel head is 2.0-7.0 mm.

[0029] In some preferred embodiments, the semicircular arc has a diameter of 1.5 to 3.0 mm.

[0030] In some more preferred embodiments, the length of the shovel handle is 50 mm.

[0031] In some more preferred embodiments, the ultimate depth of the shovel head is 2.0 mm, 3.5 mm, 4.0 mm or 7.0 mm.

[0032] In some more preferred embodiments, the diameter of the semicircular arc is 2.0 mm.

[0033] In some embodiments, when sampling different monkey brain nuclei, shovel heads with different extreme depths can be selected according to the sampling depth.

[0034] In some embodiments, a spatula with a limit depth of 2.0 mm is used when sampling the cerebral cortex.

[0035] In some embodiments, a spatula with a limit depth of 3.5 mm is used when sampling the midbrain.

[0036] In some embodiments, a spatula with a limit depth of 4.0 mm is used when sampling the caudate nucleus and hypothalamus.

[0037] In some embodiments, a spatula with a limit depth of 7.0 mm is used when sampling the thalamus.

[0038] The second aspect of the present invention provides a use of the monkey brain nucleus sampling device as described in the first aspect of the present invention in extracting monkey brain nerve nuclei.

[0039] In some embodiments, the using comprises the following steps:

[0040] Collecting fresh monkey brains from monkey corpses, placing the monkey brains in the monkey brain nucleus sampling device, first sampling the cerebellum, pons and medulla oblongata, then sampling the cerebral cortex, midbrain, corpus callosum and cingulate cortex, caudate nucleus, thalamus and hypothalamus, and finally sampling the hippocampus;

[0041] The method of obtaining the cerebellum, pons and medulla oblongata includes: cutting the monkey brain in half longitudinally along the longitudinal fissure of the brain with a blade, cutting off one side of the cerebellum, finding the medullary pontine groove and the pontine enlargement, and separating the pons and medulla oblongata;

[0042] The method of sampling the hippocampus includes: opening a cross section of the hippocampus vertically at the temporal lobe position corresponding to the tail end of the caudate nucleus of the monkey brain and exposing the lateral ventricle between the hippocampus and the temporal lobe; entering the lateral ventricle, opening the temporal lobe along the long axis of the temporal lobe, and exposing the hippocampus from the opening; and removing the hippocampus from the direction parallel to the long axis of the hippocampus.

[0043] In some preferred embodiments, the cerebral cortex includes the frontal lobe, parietal lobe, temporal lobe and occipital lobe.

[0044] In some preferred embodiments, the opening completely exposes the temporal lobe.

[0045] In some more preferred embodiments, the monkey brain is a cynomolgus monkey brain.

[0046] In some embodiments, after the monkey brain is placed in the monkey brain nucleus sampling device, crushed ice is filled around the monkey brain.

[0047] In some embodiments, before placing the monkey brain in the monkey brain nucleus sampling device, the method further comprises:

[0048] Add dry ice and anhydrous ethanol to the outer tray; fill the inner tray with coolant.

[0049] In some preferred embodiments, the coolant is an ice-water mixture.

[0050] In some embodiments, after the monkey brain is placed in the monkey brain nucleus sampling device, the buckle hole is used to fix the monkey brain.

[0051] In some preferred embodiments, the amount of anhydrous ethanol added is 400-600 mL; the amount of dry ice added is 1.6-2.4 kg; and the amount of ice-water mixture added is 30%-70% of the volume of the cooling chamber.

[0052] In some more preferred embodiments, the amount of anhydrous ethanol added is 500 mL; the amount of dry ice added is 2 kg; and the amount of ice-water mixture added is 40% to 60% of the volume of the cooling chamber.

[0053] In some embodiments, the sampling of the cerebral cortex comprises the following steps:

[0054] ① Place the monkey brain into the groove A of the monkey brain model with the cut surface facing downward and the frontal lobe facing right;

[0055] ② Covering the material collection positioning template A with the hollow positioning hole on the monkey brain, and connecting the material collection positioning template A with the monkey brain groove A through the buckle hole;

[0056] ③ Use the stainless steel semicircular shovel described in the first aspect of the present invention to sample the required cerebral cortex in the hollow positioning hole of the sampling positioning template A; the maximum depth of the shovel head of the stainless steel semicircular shovel is 2.0mm, and the sampling depth is 2.0mm.

[0057] In some embodiments, the midbrain sampling comprises the following steps:

[0058] ① Remove the sampling positioning template 1, take out the monkey brain, and place the monkey brain into the groove B of the monkey brain model with the cut surface facing upward and the frontal lobe facing right, cover the sampling positioning template B with the hollow positioning hole on the monkey brain, and connect the sampling positioning template B with the monkey brain groove B through the buckle hole;

[0059] ② Use the stainless steel semicircular shovel described in the first aspect of the present invention to sample the midbrain in the hollow positioning hole of the positioning template 2. The maximum depth of the shovel head of the stainless steel semicircular shovel is 3.5mm, and the sampling depth is 3.5mm.

[0060] In some embodiments, the sampling of the corpus callosum and the cingulate cortex comprises the following steps:

[0061] ① Use the stainless steel semicircular shovel described in the first aspect of the present invention to shovel out the cingulate cortex in the hollow positioning hole of the cingulate gyrus of the sampling positioning template B; the limit depth of the shovel head of the stainless steel semicircular shovel is 2mm, and the sampling depth is 2mm;

[0062] ② Remove the corpus callosum along the edge with a tool, wherein the angle between the tool and the cutting surface is 30°-35°, and the tool is, for example, scissors and / or a scalpel.

[0063] In some embodiments, the sampling of the caudate nucleus comprises the following steps:

[0064] The stainless steel semicircular shovel as described in the first aspect of the present invention is used to take samples at the caudate nucleus head marking point on the sampling positioning template B; the limit depth of the shovel head of the stainless steel semicircular shovel is 4.0 mm, and the sampling depth is 4.0 mm.

[0065] In some embodiments, the sampling of the thalamus comprises the following steps:

[0066] The thalamus is sampled from the thalamus hollow positioning hole of the sampling positioning template B using the stainless steel semicircular shovel as described in the first aspect of the present invention; the maximum depth of the shovel head of the stainless steel semicircular shovel is 7.0 mm, and the sampling depth is 7.0 mm.

[0067] In some embodiments, the sampling of the hypothalamus comprises the following steps:

[0068] The hypothalamus is sampled from the hypothalamus hollow positioning hole of the sampling positioning template B using the stainless steel semicircular shovel as described in the first aspect of the present invention; the maximum depth of the shovel head of the stainless steel semicircular shovel is 4.0 mm, and the sampling depth is 4.0 mm.

[0069] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0070] The reagents and raw materials used in the present invention are commercially available.

[0071] The positive and progressive effects of the present invention are:

[0072] ① This operation does not require expensive auxiliary instruments and equipment (such as monkey brain molds). It only requires basic instruments such as a homemade pre-cooling device, a stainless steel semicircular shovel and positioning template, dissecting scissors and tweezers. The tools are cheap and easy to obtain, and are easy to operate and use.

[0073] ② Only one operator is needed to complete the entire sampling process, and the process can be controlled within half an hour; it has high repeatability, and personnel can perform simultaneous sampling of multiple animals with simple training.

[0074] ③ The sampled nuclei have complete morphology, clear structure and intact cells when observed under a microscope, with little damage to the three-dimensional structure of the brain tissue; sampling within the area specified by the template can ensure the consistency of the sampling position.

[0075] ④ Half of the brain tissue can be sampled separately, and the other half can be used for other operations, saving animal resources and being more in line with the 3R principle of animal use. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 It is a three-dimensional diagram of a monkey brain nucleus sampling device according to an embodiment of the present invention.

[0077] Figure 2 It is a side cross-sectional view of a monkey brain nucleus sampling device according to an embodiment of the present invention.

[0078] Figure 3 It is a three-dimensional diagram of the inner tray of an embodiment of the present invention; wherein, the inner tray has a monkey brain model groove A and a monkey brain model groove B.

[0079] Figure 4 2 is a three-dimensional diagram of an outer tray according to an embodiment of the present invention.

[0080] Figure 5 Schematic diagram of the structure of the material positioning template A according to an embodiment of the present invention.

[0081] Figure 6Schematic diagram of the structure of the material positioning template B according to an embodiment of the present invention.

[0082] Figure 7 It is a schematic structural diagram of a semicircular shovel according to an embodiment of the present invention.

[0083] Figure 8 The left and right cerebral hemispheres of a cut monkey brain, including the cerebrum, cerebellum and brain stem.

[0084] Fig. 9 Schematic diagram of the process of removing the cerebellum, pons and medulla oblongata.

[0085] Fig.10 Schematic diagram of the process of removing the corpus callosum.

[0086] Fig.11 Schematic diagram of the process of removing the hippocampus.

[0087] Description of reference numerals:

[0088] Monkey brain nucleus sampling device 100

[0089] Outer tray 1

[0090] Inner tray 2

[0091] Water injection hole 21

[0092] Monkey brain model groove A22

[0093] Monkey brain model groove B23

[0094] Buckle hole 24

[0095] Ice water mixture 25

[0096] Dry ice ethanol mixture 26

[0097] Material positioning template A3

[0098] Frontal lobe hollow hole 31

[0099] Temporal lobe hollow hole 32

[0100] Occipital lobe hollow hole 33

[0101] Top leaf hollow hole 34

[0102] Material positioning template B4

[0103] Caudate nucleus head landmark 41

[0104] Thalamic hollow hole 42

[0105] Hypothalamic Hollow Foramen 43

[0106] Buckle back hollow hole 44

[0107] Midbrain hollow hole 45

[0108] Corpus callosum hollow hole 46

[0109] Buckle 5

[0110] Semicircular shovel 6

[0111] Shovel head 61

[0112] Shovel handle 62 DETAILED DESCRIPTION

[0113] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0114] Example 1 Method for extracting brain nerve nuclei (and cerebellum and medulla oblongata) from non-human primates

[0115] Taking cynomolgus monkeys as an example, the method for extracting the brain nerve nuclei of non-human primates includes the following steps:

[0116] (1) Collection of fresh brain tissue from cynomolgus monkeys;

[0117] (2) Prepare a precooling device;

[0118] (3) Materials were collected from the cerebellum, pons, and medulla oblongata;

[0119] (4) Sampling from the cerebral cortex (frontal lobe, parietal lobe, temporal lobe, and occipital lobe);

[0120] (5) From the midbrain;

[0121] (6) Sampling the corpus callosum and cingulate cortex;

[0122] (7) Take the caudate nucleus;

[0123] (8) Taking materials from the thalamus;

[0124] (9) Hypothalamus sampling;

[0125] (10) Samples from seahorses;

[0126] In the step (1), the specific steps are as follows:

[0127] ① According to the animal euthanasia requirements specified in the standard operating procedures, the cynomolgus monkeys were deeply anesthetized and euthanized by inguinal vein bleeding;

[0128] ② Confirmation of death of cynomolgus macaque;

[0129] ③ Place the cynomolgus monkey horizontally on the dissecting table, cut the skin on the dorsal side of the monkey's brain, use a skull saw to carefully cut a circle along the skull, and remove the skull;

[0130] ④ Use surgical scissors to cut the dura mater to expose the brain tissue, then carefully separate the brain tissue from the skull. After cutting the spinal cord and optic chiasm, remove the brain tissue, rinse the surface with pre-cooled saline, and prepare for subsequent operations.

[0131] In the step (2), the specific steps are as follows:

[0132] ① Use a self-made monkey brain nucleus sampling device. The schematic diagram of the monkey brain nucleus sampling device is shown in Figure 1 ; Side cross-sectional view of the monkey brain nucleus sampling device Figure 2 shown.

[0133] ② The monkey brain nucleus sampling device includes an outer tray ( Figure 4 )、Inner tray( Figure 3 )、Material positioning template A( Figure 5 )、Material positioning template B( Figure 6 ) and stainless steel half round shovel ( Figure 7 );

[0134] ③ Add 2kg of dry ice into the outer tray and inject 500mL of anhydrous ethanol to mix. Dry ice can be added appropriately during the operation to maintain a low temperature environment;

[0135] ④ The inner tray is a hollow device. When in use, open the water injection hole on the side and inject the pre-cooled ice-water mixture into it. The amount of ice-water mixture added is 30% to 70% of the cooling chamber volume;

[0136] ⑤ The inner tray surface has monkey brain model groove A and monkey brain model groove B, in which the brain tissue can be placed for sampling. There are buckle holes around the monkey brain model groove A and monkey brain model groove B for fixing the sampling positioning template.

[0137] The parameters of the monkey brain nucleus sampling device are as follows:

[0138] The monkey brain model groove A and the monkey brain model groove B are rectangular parallelepipeds, and the size of the monkey brain model groove A is 8 cm long, 5 cm wide, and 5 cm deep; the size of the monkey brain model groove B is 8 cm long, 5 cm wide, and 5 cm deep; the material positioning template A and the material positioning template B are rectangular; the size of the material positioning template A is 8.5 cm long and 5.5 cm wide; the size of the material positioning template B is 8.5 cm long and 5.5 cm wide; the midpoint of the right side of the material positioning template A is taken as the coordinate origin (0,0), and the horizontal coordinate is established with the right side as positive Axis, the horizontal axis is rotated 90° counterclockwise with the origin of the coordinate as the center to obtain the longitudinal axis; the center coordinates of the parietal hollow positioning hole are: (-4.4cm, 2.0cm), and the radius of the circle is 0.4cm; the center coordinates of the frontal hollow positioning hole are: (-0.9cm, 0.9cm), and the radius of the circle is 0.4cm; the center coordinates of the occipital hollow positioning hole are: (-6.6cm, 0.9cm), and the radius of the circle is 0.4cm; the center coordinates of the temporal hollow positioning hole are: (-4.4cm, -0.8cm), and the radius of the circle is 0.3cm; The midpoint of the right side of the sampling positioning template B is taken as the coordinate origin (0,0), and the horizontal coordinate axis is established with the right as the positive, and the horizontal coordinate axis is rotated 90° counterclockwise with the coordinate origin as the center to obtain the vertical coordinate axis; the center coordinates of the hypothalamus hollow positioning hole are: (-3.0cm, -0.2cm), and the circle radius is 0.2cm; the center coordinates of the midbrain hollow positioning hole are: (-3.9cm, -0.2cm), and the circle radius is 0.2cm; the center coordinates of the thalamus hollow positioning hole are: (-3.2cm, 0.2cm), and the circle radius is 0.2cm; The hollow positioning hole of the corpus callosum is an ellipse, and the coordinates of the ellipse center are: (-2.5cm, 0.8cm), the major axis of the ellipse is parallel to the horizontal axis, the radius of the major axis is 0.4cm, the minor axis of the ellipse is parallel to the vertical axis, and the radius of the minor axis is 0.2cm; the hollow positioning hole of the cingulate gyrus is an ellipse, and the coordinates of the ellipse center are: (-2.5cm, 1.4cm), the major axis of the ellipse is parallel to the horizontal axis, the radius of the major axis is 0.4cm, the minor axis of the ellipse is parallel to the vertical axis, and the radius of the minor axis is 0.2cm.

[0139] In the step (3), the specific steps are as follows:

[0140] Place the monkey brain on the inner tray and cut it in half longitudinally along the longitudinal fissure of the brain with a blade (see Figure 8 ), one side of the monkey brain can be used for histopathological examination, cut the cerebellum of the other side of the monkey brain with scissors, find the medullary pontine groove and the pontine enlargement, separate the cerebellum, pons and medulla oblongata (see Fig. 9 );

[0141] In the step (4), the specific steps are as follows:

[0142] ① After removing the cerebellum and other structures, place the monkey brain into groove A of the monkey brain model with the cut surface facing downward and the frontal lobe to the right, for removing the cerebral cortex.

[0143] ② Cover the sampling positioning template A with hollow positioning holes on the brain tissue, buckle the buckles into the buckle holes around the groove, and confirm whether the positioning position is accurate after fixing it;

[0144] ③ After confirming that the positioning is accurate, use a special stainless steel semicircular shovel to shovel out the required cerebral cortex in the hollow positioning hole. The maximum depth of the stainless steel semicircular shovel head is 2.0mm, and the sampling depth is 2.0mm; when sampling, the stainless steel semicircular shovel should be operated parallel to the template as much as possible, and the shovel head should be replaced in time to prevent cross contamination.

[0145] In the step (5), the specific steps are as follows:

[0146] ① Remove the sampling positioning template 1, take out the monkey brain, and place it into the groove B of the monkey brain model with the cut surface facing upward and the frontal lobe facing right, exposing the structure to be sampled on the cut surface; cover the sampling positioning template B with hollow positioning holes on the brain tissue, fix it through the buckle holes, and confirm whether the positioning position is accurate after fixation;

[0147] ② After confirming that the positioning is accurate, use a special stainless steel semicircular shovel to make a hole in the midbrain to take a sample, and then use tweezers to push out the sample in the shovel. The maximum depth of the special shovel head of the stainless steel semicircular shovel is 3.5mm, and the sampling depth is 3.5mm; in order to avoid drilling too deep and touching other structures, a certain space can be left at the tail of the shovel head to facilitate the tweezers to reach in and push out the sample.

[0148] In the step (6), the specific steps are as follows:

[0149] ① Use a stainless steel semicircular shovel to shovel out the cingulate cortex in the hollow positioning hole of the cingulate gyrus of the sampling positioning template B, and try to operate the shovel parallel to the template;

[0150] ② Use forceps to gently lift the head of the corpus callosum and cut off the corpus callosum along the edge with scissors (see Fig.10 ), the angle between the scissors and the cutting surface is 30°-35° to avoid damaging the caudate nucleus below.

[0151] In the step (7), the specific steps are as follows:

[0152] Use a stainless steel semicircular shovel to punch holes at the marking point of the caudate nucleus head on the sampling positioning template B to take samples; the maximum depth of the stainless steel semicircular shovel is 4.0 mm, and the sampling depth is about 4.0 mm. Then use tweezers to push out the sample. If the sampling amount is large, you can punch holes and take samples in sequence.

[0153] In the step (8), the specific steps are as follows:

[0154] Use a stainless steel semicircular shovel to scoop the thalamus sample in the center of the thalamus hollow positioning hole of the sampling positioning template B. The limit depth of the stainless steel semicircular shovel is 7.0mm, and the sampling depth is 7.0mm. Then use tweezers to push out the sample. If the sampling amount is large, you can punch holes continuously around the edge of the central hole until enough samples are taken out.

[0155] In the step (9), the specific steps are as follows:

[0156] Use a stainless steel semicircular shovel to take samples from the hollow positioning hole of the hypothalamus of the sampling positioning template B. The maximum depth of the special shovel head of the stainless steel semicircular shovel is 4.0mm, and the sampling depth is 4.0mm. Then use tweezers to push out the sample.

[0157] In the step (10), the specific steps are as follows:

[0158] Remove the sample positioning template 2, take out the monkey brain, and use scissors to cut a vertical opening at the temporal lobe position corresponding to the tail end of the caudate nucleus. Note that the opening should cut through the entire temporal lobe. The egg roll-like cross section of the hippocampus and the lateral ventricle between the hippocampus and the temporal lobe can be seen at the opening;

[0159] Insert the scissors into the lateral ventricle, cut the temporal lobe along its long axis, and push the temporal lobe to both sides of the opening to expose the hippocampus. A white membrane structure can be seen on its surface. Make the long axis of the forceps parallel to the long axis of the hippocampus and push out the hippocampus (see Fig.11 ), being careful not to poke with the pointed end.

[0160] Note:

[0161] ① Before sampling, the operating instruments must be cleaned and sterilized under high pressure to remove residual DNA / RNA. During the operation, pay attention to the timely replacement of instruments and prevent cross contamination;

[0162] ②Prepare homemade pre-cooling device in advance;

[0163] ③ Wash the removed nuclei with physiological saline, drain them slightly, and immediately put them into an EP tube containing protective solution, place them in an ice box, and then carry out subsequent storage operations.

Claims

1. A monkey brain nucleus sampling device, characterized in that: The monkey brain nucleus sampling device comprises: An inner tray, wherein the surface of the inner tray has a monkey brain model groove A and a monkey brain model groove B for placing a monkey brain, and a hollow cavity is provided in the inner tray as a cooling cavity, and the cooling cavity is used to cool the monkey brain placed in the monkey brain model groove A and / or the monkey brain model groove B; An outer tray, the inner tray is arranged inside the outer tray, the outer tray is used to hold a coolant and to cool the inner tray; A material collection positioning template, the material collection positioning template comprises a material collection positioning template A and a material collection positioning template B, the material collection positioning template A and the material collection positioning template B have hollow positioning holes for locating the positions of different brain nuclei; the material collection positioning template A is connected to the surface of the inner tray and used in conjunction with the monkey brain model groove A, for locating the monkey brain placed in the monkey brain model groove A; the material collection positioning template B is connected to the surface of the inner tray and used in conjunction with the monkey brain model groove B, for locating the monkey brain placed in the monkey brain model groove B; and / or, the hollow positioning holes on the material collection positioning template A include parietal lobe hollow positioning holes, frontal lobe hollow positioning holes, occipital lobe hollow positioning holes and temporal lobe hollow positioning holes; the hollow positioning holes on the material collection positioning template B include hypothalamus hollow positioning holes, midbrain hollow positioning holes, thalamus hollow positioning holes, corpus callosum hollow positioning holes and cingulate gyrus hollow positioning holes.

2. The monkey brain nucleus sampling device according to claim 1, characterized in that: The surface of the inner tray has snap holes, and the snap holes are distributed around the monkey brain model groove A and the monkey brain model groove B. The material collection positioning template A and the material collection positioning template B are respectively connected to the monkey brain model groove A and the monkey brain model groove B through the snap holes; and / or, the side of the inner tray has a water injection hole, and the water injection hole is connected to the cooling cavity; the cooling cavity contains a coolant; the monkey brain is a crab-eating macaque brain.

3. The monkey brain nucleus sampling device according to claim 2, characterized in that: The sampling and positioning template B also includes a marking point of the head of the caudate nucleus; and / or the coolant is an ice-water mixture.

4. The monkey brain nucleus sampling device according to any one of claims 1 to 3, characterized in that: The monkey brain nucleus sampling device also includes a stainless steel semicircular shovel; the stainless steel semicircular shovel includes a shovel handle and a shovel head that are connected to each other; the length of the shovel handle is 30 to 70 mm; and / or the maximum depth of the shovel head is 1.0 to 8.0 mm, the shape of the shovel head is a semicircular arc, the diameter of the semicircular arc is 1.0 to 4.0 mm, and the central angle of the semicircular arc is 180°.

5. The monkey brain nucleus sampling device according to claim 4, characterized in that: The connection is a detachable connection; and / or the length of the shovel handle is 40 to 60 mm; and / or the limit depth of the shovel head is 2.0 to 7.0 mm, and the semicircular arc diameter is 1.5 to 3.0 mm; Preferably, the length of the shovel handle is 50 mm; and / or the limit depth of the shovel head is 2.0 mm, 3.5 mm, 4.0 mm or 7.0 mm, and the diameter of the semicircular arc is 2.0 mm.

6. Use of the monkey brain nucleus sampling device as described in claim 1 in extracting monkey brain nerve nuclei.

7. A method for removing monkey brain nerve nuclei, characterized in that: The method comprises the following steps: Collecting fresh monkey brain from a monkey corpse, placing the monkey brain in the monkey brain nucleus sampling device as described in any one of claims 1 to 5, first sampling the cerebellum, pons and medulla oblongata, then sampling the cerebral cortex, midbrain, corpus callosum and cingulate cortex, caudate nucleus, thalamus and hypothalamus, and finally sampling the hippocampus; The method of obtaining the cerebellum, pons and medulla oblongata includes: splitting the monkey brain in half along the longitudinal fissure of the brain, removing the cerebellum on one side, and separating the pons and medulla oblongata at the medullary pontine groove and the pontine enlargement; The sampling of the hippocampus comprises: vertically opening a cross section of the hippocampus at the temporal lobe position corresponding to the tail end of the caudate nucleus of the monkey brain and exposing the lateral ventricle between the hippocampus and the temporal lobe; entering the lateral ventricle, opening the temporal lobe along the long axis of the temporal lobe, and exposing the hippocampus from the opening; and removing the hippocampus from the direction parallel to the long axis of the hippocampus; Preferably, the cerebral cortex includes the frontal lobe, parietal lobe, temporal lobe and occipital lobe; the opening needs to completely expose the temporal lobe; More preferably, the monkey brain is cynomolgus monkey brain.

8. The method according to claim 7, characterized in that Before placing the monkey brain in the monkey brain nucleus sampling device, the method further comprises: Add dry ice and anhydrous ethanol to the outer tray; inject a coolant, preferably an ice-water mixture, into the inner tray; and / or, after placing the monkey brain in the monkey brain nucleus sampling device, fixing the monkey brain with the buckle hole; Preferably, the amount of anhydrous ethanol added is 400-600 mL; the amount of dry ice added is 1.6-2.4 kg; the amount of ice-water mixture added is 30%-70% of the volume of the cooling chamber; More preferably, the amount of anhydrous ethanol added is 500 mL; the amount of dry ice added is 2 kg; and the amount of ice-water mixture added is 40% to 60% of the volume of the cooling chamber.

9. The method according to claim 7, characterized in that The sampling of the cerebral cortex comprises the following steps: ① Place the monkey brain into the groove A of the monkey brain model with the cut surface facing downward and the frontal lobe facing right; ② Covering the material collection positioning template A with the hollow positioning hole on the monkey brain, and connecting the material collection positioning template A with the monkey brain groove A through the buckle hole; ③ Use the stainless steel semicircular shovel as described in claim 3 to sample the required cerebral cortex in the hollow positioning hole of the sampling positioning template A; the maximum depth of the shovel head of the stainless steel semicircular shovel is 2.0mm, and the sampling depth is 2.0mm.

10. The method according to claim 7, characterized in that The midbrain sampling comprises the following steps: ① Remove the sampling positioning template 1, take out the monkey brain, and place the monkey brain into the groove B of the monkey brain model with the cut surface facing upward and the frontal lobe facing right, cover the sampling positioning template B with the hollow positioning hole on the monkey brain, and connect the sampling positioning template B with the monkey brain groove B through the buckle hole; ② Using the stainless steel semicircular shovel as described in claim 3 to sample the midbrain in the hollow positioning hole of the positioning template 2, the limit depth of the shovel head of the stainless steel semicircular shovel is 3.5mm, and the sampling depth is 3.5mm; and / or, The sampling of the corpus callosum and the cingulate cortex comprises the following steps: ① Use the stainless steel semicircular shovel as described in claim 3 to remove the cingulate cortex in the cingulate hollow positioning hole of the sampling positioning template B; the limit depth of the shovel head of the stainless steel semicircular shovel is 2mm, and the sampling depth is 2mm; ② Remove the corpus callosum along the edge with a tool, wherein the angle between the tool and the cutting surface is 30°-35°, and the tool is, for example, scissors and / or a scalpel; and / or, The sampling of the caudate nucleus comprises the following steps: Use the stainless steel semicircular shovel as claimed in claim 3 to sample the caudate nucleus head identification point on the sampling positioning template B; the limit depth of the shovel head of the stainless steel semicircular shovel is 4.0 mm, and the sampling depth is 4.0 mm; and / or, The sampling of the thalamus comprises the following steps: The thalamus is sampled from the thalamus hollow positioning hole of the sampling positioning template B using the stainless steel semicircular shovel as described in claim 3; the limit depth of the shovel head of the stainless steel semicircular shovel is 7.0 mm, and the sampling depth is 7.0 mm; and / or, The hypothalamus sampling comprises the following steps: The hypothalamus is sampled by using the stainless steel semicircular shovel as described in claim 3 in the hypothalamus hollow positioning hole of the sampling positioning template B; the maximum depth of the shovel head of the stainless steel semicircular shovel is 4.0 mm, and the sampling depth is 4.0 mm.

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