Method for collecting hypothalamus and / or pituitary of animal

Abstract

The invention relates to a method for collecting animal hypothalamus and / or pituitary, which comprises the following steps of: (1) taking a slaughtered animal head, longitudinally cutting to the occipital condyle along the median line behind the cranium, transversely cutting at the occipital condyle to expose an occipital bone macropore, transversely cutting at the connecting line of two ears of the cranium behind the brain to the rear of two lateral ears, horizontally cutting at the rear lower parts of the two lateral ears to the occipital bone macropore, and opening a rear cranial cavity; (2) taking the hypothalamus and / or pituitary according to the following mode: taking out the complete brain from the posterior opening of the cranium in the step (1), enabling the hypothalamus to be positioned at the triangular area behind the optic nerve intersection at the bottom of the brain, and cutting and separating inwards to obtain the hypothalamus; and taking out the tissue in the pituitary fossa below the hypothalamus at the bottom of the cranial cavity, and separating to obtain the pituitary. According to the method disclosed by the invention, the complete brain tissue of the animal can be quickly obtained in a labor-saving manner by using fewer tools, an operator can accurately find the hypothalamus and the pituitary, the accuracy is high, and the damage to a sample is small.

Description

Technical Field

[0001] The invention relates to a method for taking animal hypothalamus and / or pituitary, belonging to the technical field of biological science. Background Art

[0002] The brain of an animal is the center that controls its behavior and dominates all life activities. The brain generally includes the cerebrum, brainstem, cerebellum, hypothalamus, pituitary gland and other tissues. Among them, the hypothalamus is a component of the diencephalon and is the center for regulating visceral and endocrine activities. It regulates important physiological functions such as body temperature, food intake, water balance, blood sugar and endocrine gland activities; the pituitary gland, located on the ventral side of the hypothalamus, is an oval body and is the most complex endocrine gland in the body. It mainly secretes gonadotropin, growth hormone, oxytocin, prolactin, thyroid-stimulating hormone, adrenocorticotropic hormone, etc. It can also receive gonadotropin-releasing hormone, thyrotropin-releasing hormone, adrenocorticotropic hormone-releasing hormone, etc. secreted by the hypothalamus. These hormones play an important role in the metabolism, growth and development, reproductive physiology, stress response, etc. of animals, and form a series of reaction axes with the hypothalamus. Therefore, in order to study the endocrine regulation of animal growth, production, reproduction and metabolism, it is necessary to collect endocrine tissues such as the hypothalamus and pituitary gland of experimental animals for molecular biology research (studying the mRNA and protein expression of related endocrine cell genes). However, due to the location of the hypothalamus and pituitary gland, it is difficult to collect the hypothalamus and pituitary gland.

[0003] The Chinese invention patent application document with application publication number CN104655448A discloses a method for collecting the pituitary gland and hypothalamus of pigs and goats, which specifically discloses: 1) Take the head of a slaughtered goat, use an electric saw or an axe to split the goat's skull horizontally at the frontal bone position perpendicular to the direction of the goat's nose, 2 to 4 cm in front of the ear and away from the occipital condyle, split into the bone 0.8 to 1.5 cm, and the brain tissue in the head can be seen; 2) Then split the bones on both sides along the split, separate the split parts, and directly expose the brain tissue; 3) Use ophthalmic forceps to push aside the cerebellum, expose the junction of the brainstem and the brain, so that the pituitary gland and hypothalamus of pigs and goats can be seen; 4) Use ophthalmic scissors to cut along the connection between the pituitary gland and the infundibulum, that is, to obtain a complete pig pituitary gland; then use ophthalmic scissors to cut the hypothalamus along the root. This collection method mainly uses an electric saw or an axe to split the skull horizontally, and then looks for brain tissue such as hypothalamus, pituitary and other brain tissue samples.

[0004] The Chinese invention patent application document with application publication number CN109612767A discloses a method for collecting the pituitary gland and hypothalamus of pigs, which specifically discloses: 1) Take the pig head, cut the skin in the middle position parallel to the nasal bone direction, peel off the skin from the front edge of the nasal bone to the back edge of the skull to both sides, and completely expose the skull; 2) Use a tool to cross-cut perpendicularly and parallel to the nasal bone direction, split into the bone, just enough to see the brain tissue in the head, and do not damage the cerebral cortex; 3) Then split the bones on both sides along the split, so that the skull cover is completely reversed 180°, separate the split parts, and directly expose the brain tissue; 4) Push aside the cerebellum, expose the junction of the brainstem and the brain, make the pig hypothalamus visible, and cut the hypothalamus along the root; 5) Cut along the connection between the pituitary gland and the funnel, that is, the position where the skull is connected, to obtain a complete pig pituitary gland. This collection method mainly uses an electric saw or a hacksaw to cross-cut perpendicularly and parallel to the nasal bone direction, and the cutting length is from the front edge of the nasal bone to the back edge of the skull.

[0005] The above two methods of collecting hypothalamus and pituitary gland are to open most of the cranial cavity from the forehead or the top of the head, remove the brain from the depth, and then search for the pituitary gland from the bottom. The work procedures are complicated, the labor intensity is high, and many tools are required (electric saw or cutting machine, 2 crowbars, bone forceps, etc.). During the collection process, the positioning of the hypothalamus and pituitary gland is not accurate, and the collected pituitary gland and hypothalamus are easily damaged by mechanical damage and have poor integrity; and it is very easy to mix the broken brain tissues together during the splitting process (such as fragile tissues such as brain, hypothalamus, cerebellum, pituitary gland, etc.), and the hypothalamus and pituitary gland cannot be quickly and accurately removed. The requirements for operators are high and they need to be familiar with the structure of brain tissue. It may also occur that the process of splitting the sample is time-consuming and the sample is exposed to the air for a long time, resulting in serious contamination. At the same time, the sample is exposed to the air for a long time, causing RNA (ribonucleic acid) degradation, which has a great impact on the subsequent extraction and identification of various RNA. Summary of the invention

[0006] The purpose of the present invention is to provide a method for collecting the hypothalamus and / or pituitary of an animal, so as to solve the problems in the prior art of collecting the hypothalamus and pituitary, such as complicated procedures, inaccurate positioning of the hypothalamus and pituitary, easy destruction of samples, and long time consumption.

[0007] In order to achieve the above object, a technical solution of the method of taking the hypothalamus and / or pituitary of an animal in the present invention is:

[0008] A method for taking the hypothalamus and / or pituitary of an animal, comprising the following steps:

[0009] (1) Take the head of a slaughtered animal, make a longitudinal incision along the midline of the back of the skull to the occipital condyle, make a transverse incision at the occipital condyle to expose the foramen magnum, make a transverse incision at the line connecting the two ears of the skull behind the brain to the back of the ears on both sides, make a horizontal incision below the back of the ears on both sides to the foramen magnum, and open the posterior cranial cavity;

[0010] (2) Take the hypothalamus and / or pituitary gland as follows:

[0011] The whole brain is removed from the posterior cranial opening in step (1). The hypothalamus is located in the triangular area behind the optic chiasm at the base of the brain. The hypothalamus can be obtained by cutting inward and separating it. The tissue in the pituitary fossa below the hypothalamus at the base of the cranial cavity is removed and separated to obtain the pituitary gland.

[0012] The beneficial effect of the above technical solution is that the present invention, based on the structure and position of animal brain tissue, explores a method for taking animal hypothalamus and / or pituitary gland, which is to open the skull from the back of the brain, first longitudinally cut along the midline of the back of the skull to the occipital condyle, then make a transverse cut at the occipital condyle and the line connecting the two ears of the skull from the back of the brain, and finally cut horizontally to the occipital bone below the ears on both sides, so as to quickly and effectively open the posterior cranial cavity. The method of the present invention only requires fewer tools (cutters or hacksaws), and the animal's posterior cranial cavity is opened simply, quickly and labor-savingly, and the complete brain tissue can be quickly taken out from the posterior cranial cavity without the phenomenon of brain tissue damage and mixing together. Therefore, the operator can quickly and accurately find the hypothalamus and pituitary gland, with a high accuracy rate, and the sample will not be difficult to find, and will not be exposed to the air for a long time, resulting in serious problems of pollution and RNA degradation. Taking sheep as an example, the method of taking animal hypothalamus and / or pituitary gland of the present invention only takes 10 to 15 minutes to obtain the hypothalamus and pituitary gland.

[0013] Specifically, the hypothalamus is located at the base of the brain in the triangular area behind the optic chiasm, that is, at the mastoid process.

[0014] As a further improvement, step (1) includes peeling the skin of the slaughtered animal's head from the back of the skull along the midline upward to the top of the head, and peeling the skin to both sides to the lower edge of the ear, without completely cutting it off, keeping the skin and bones connected at the lower edge of the ear.

[0015] The beneficial effects of the above technical solution are: peeling the skin of the animal's head from the back of the head along the midline upwards to the top of the head helps to accurately determine the craniotomy position during the subsequent craniotomy process, and can reduce pollution caused by the animal's fur. Moreover, peeling the skin to both sides to the lower edge of the ear helps to fix the animal's head.

[0016] As a further improvement, the method of removing the tissue in the pituitary fossa below the hypothalamus at the bottom of the cranial cavity in step (2) is to cut the covering cartilage on the pituitary fossa at the bottom of the cranial cavity, cut the surrounding fascia and blood vessels deep into the wall, and remove the connected tissue.

[0017] The beneficial effect of the above technical solution is that: according to the above method, the pituitary tissue can be quickly, effectively and completely removed without repeated searching and identification, which reduces the operator's requirements for the degree of understanding of brain tissue.

[0018] As a further improvement, the separation in step (2) is to peel off the fascia and blood vessels connected to the pituitary gland.

[0019] The beneficial effect of the above technical solution is that stripping off the fascia and blood vessels connected to the pituitary gland helps to obtain an independent and complete pituitary gland, which is conducive to the smooth progress of subsequent experiments.

[0020] As a further improvement, the animals include pigs, cattle and sheep.

[0021] As a further improvement, the method includes removing blood stains from the obtained hypothalamus and pituitary gland and preserving them at low temperature.

[0022] The beneficial effects of the above technical solution are: after taking the animal's hypothalamus and pituitary, removing the blood stains on the surface helps reduce the contamination of the sample; storing it at low temperature helps reduce the degradation of various RNAs in the sample cells, which is conducive to ensuring the smooth progress of subsequent testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A schematic diagram (rear view) of cutting the posterior skull of a goat head in the method for taking the hypothalamus and pituitary of a goat in Example 1 of the present invention;

[0024] Figure 2 This is an actual cutting diagram of longitudinal cutting along the posterior midline of the skull to the occipital condyle in the method for taking the hypothalamus and pituitary of a goat in Example 1 of the present invention;

[0025] Figure 3 This is an actual cutting diagram of the method for taking the hypothalamus and pituitary of a goat in Example 1 of the present invention, from the line connecting the two ears behind the skull to the back of the ears on both sides;

[0026] Figure 4 This is an actual cutting diagram of the cross section at the occipital condyle in the method for taking the hypothalamus and pituitary of a goat in Example 1 of the present invention;

[0027] Figure 5 This is an actual cutting diagram of horizontally cutting from the right side behind the ear to the foramen magnum in the method for taking the hypothalamus and pituitary of a goat in Example 1 of the present invention;

[0028] Figure 6 This is an actual cutting diagram of horizontally cutting from the left ear to the foramen magnum in the method for taking the hypothalamus and pituitary of a goat in Example 1 of the present invention;

[0029] Among them, 1-longitudinal section of the midline of the back of the skull, 2-transverse section at the line connecting the two ears of the back of the skull, 3-transverse section at the occipital condyle, 4-horizontal tangent behind the right ear, 5-horizontal tangent behind the left ear, 6-foramen magnum, 7-right ear, 8-left ear. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto; however, these embodiments are only examples and do not constitute any limitation on the scope of the present invention. The details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention. The test methods in the following embodiments are conventional methods unless otherwise specified. The test materials used in the following embodiments are purchased from ordinary hardware stores or medical equipment stores unless otherwise specified.

[0031] Example 1 A method for taking animal hypothalamus and pituitary

[0032] The present invention provides a method for taking the hypothalamus and pituitary of sheep (goats), and the specific implementation operation is as follows:

[0033] 1. Skinning

[0034] After slaughtering the sheep, obtain the sheep's head and peel the skin from the back of the head along the midline upwards to the base of the horns on the top of the head, and peel the skin to both sides to the cheeks below the ears, leaving a small part of the skin connected so that it can be pulled down and fixed by hand.

[0035] 1. Craniotomy

[0036] Use a cutting machine to make a craniotomy from the back of the skull:

[0037] ① Make a longitudinal incision along the posterior midline of the skull to the occipital condyle ( Figure 1 The longitudinal section of the midcranial posterior midline 1) is actually cut as Figure 2 As shown; ② Cut horizontally from the line connecting the two ears behind the skull to the back of the ears on both sides ( Figure 1 The cross section 2 at the midbrain posterior skull connecting the two ears, the figure shows both the left ear 8 and the right ear 7), the actual cutting is as follows Figure 3 ③ Make a transverse cut at the occipital condyle to expose the foramen magnum ( Figure 1 The transverse line at the middle occipital condyle 3), the actual cutting is as follows Figure 4 ④ Make a horizontal cut behind the ears on both sides to the foramen magnum ( Figure 1 The horizontal tangent line 4 behind the right ear and the horizontal tangent line 5 behind the left ear) are actually cut as follows Figure 5 , Figure 6 As shown, the posterior cranial cavity can be opened after all the sutures are opened.

[0038] In other practical cutting situations, the order of cutting can be adjusted.

[0039] 2. Remove the hypothalamus

[0040] Remove the cut bone slices, open the posterior cranial cavity, take out the complete brain from the cranial incision backwards, turn the brain tissue upwards, find the hypothalamus in the posterior triangle area (mastoid process) behind the optic chiasm at the bottom of the brain, remove it with ophthalmic scissors and ophthalmic tweezers, clean the blood stains with saline, put it into a prepared cryopreservation tube, and freeze it.

[0041] 3. Remove the pituitary gland

[0042] Cut and insert the fossa of the pituitary gland below the original hypothalamus (normal position) at the bottom of the cranial cavity, cut the cartilage along the wall, and cut the connected fascia and blood vessels deep into the wall with ophthalmic scissors. Take out the connected tissues and find the pituitary tissue of the size of soybean. Use ophthalmic forceps and ophthalmic scissors to peel and cut the surrounding fascia and blood vessels, separate the pituitary gland, clean the blood stains with saline, put it into the prepared cryopreservation tube, and freeze it.

[0043] The time from skinning to taking out the hypothalamus and pituitary gland is about 10 to 15 minutes, and the complete brain tissue can be taken out.

[0044] Comparative Example 1

[0045] This comparative example adopts the hypothalamus and pituitary of sheep (goat) according to the traditional method, and the specific implementation operation is as follows:

[0046] After slaughtering the sheep, obtain the sheep's head, use a cutting machine to saw open the cranial cavity from the front of the head or the forehead, use a crowbar to split the sheep's brain in half from the middle, take out the brain, find the hypothalamus behind the optic chiasm at the bottom of the brain, cut off the hypothalamus with scissors, take it out, and freeze it.

[0047] It takes at least 30 minutes to reach the hypothalamus, and there is often damage to the brain tissue. It is difficult to find the pituitary tissue during the search.

[0048] Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for collecting hypothalamus and / or pituitary gland of an animal, characterized in that: The following steps are involved: (1) Take the head of a slaughtered animal, make a longitudinal incision along the midline of the back of the skull to the occipital condyle, make a transverse incision at the occipital condyle to expose the foramen magnum, make a transverse incision at the line connecting the two ears of the skull behind the brain to the back of the ears on both sides, make a horizontal incision below the back of the ears on both sides to the foramen magnum, and open the posterior cranial cavity; (2) Take the hypothalamus and / or pituitary gland as follows: The whole brain is removed from the posterior cranial opening in step (1). The hypothalamus is located in the triangular area behind the optic chiasm at the base of the brain. The hypothalamus can be obtained by cutting inward and separating it. The tissue in the pituitary fossa below the hypothalamus at the base of the cranial cavity is removed and separated to obtain the pituitary gland.

2. The method for collecting animal hypothalamus and / or pituitary according to claim 1, characterized in that: Step (1) comprises peeling the skin of the head of the slaughtered animal from the back of the skull along the midline upward to the top of the head, peeling the skin to the sides to the lower edge of the ear, not completely cutting off, and keeping the skin and bones connected at the lower edge of the ear.

3. The method for collecting animal hypothalamus and / or pituitary according to claim 1, characterized in that: The method of removing the tissue in the pituitary fossa below the hypothalamus at the bottom of the cranial cavity in step (2) is to cut the cartilage covering the pituitary fossa at the bottom of the cranial cavity, cut the surrounding fascia and blood vessels deep into the wall, and remove the connected tissue.

4. The method for collecting animal hypothalamus and / or pituitary according to claim 1, characterized in that: The separation in step (2) is to peel off the fascia and blood vessels surrounding the pituitary gland.

5. The method for collecting hypothalamus and / or pituitary of an animal according to any one of claims 1 to 4, characterized in that: The animals include pigs, cattle and sheep.

6. The method for collecting hypothalamus and / or pituitary of an animal according to any one of claims 1 to 4, characterized in that: The method comprises removing blood from the obtained hypothalamus and pituitary gland and storing them at low temperature.

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