Method for fixing brain skull of tree shrew and application thereof in nuclear magnetic resonance imaging

Abstract

The application belongs to the technical field of animal experiments, and particularly relates to a tree shrew brain skull fixing method and application thereof in nuclear magnetic resonance imaging. The tree shrew brain skull fixing method comprises the following steps: fixing the head of the anesthetized tree shrew on a brain stereotaxic apparatus, adjusting left and right ear rods to the same height and centering, making the gap between the second and third lateral teeth of the tree shrew clamped into the incisor rod clamping groove, and accurately adjusting the incisor rod vernier caliper to the reading of 5.00+ / -0.02 mm, at this time, the tree shrew cranium and brain are in a horizontal position. The application also provides application of the fixing method in tree shrew whole brain nuclear magnetic resonance imaging, and a callosum genu rostral connecting point-genu of the corpus callosum rostral connecting point horizontal zero plane reference coordinate system. The application can complete leveling in one step, and is fast, non-invasive, high in precision, and capable of eliminating individual differences, and can be used as an effective alternative to the existing tree shrew brain stereotaxic method.

Description

Technical Field

[0001] This invention belongs to the field of animal experimental technology, specifically relating to a method for fixing the skull of a tree shrew and its application in magnetic resonance imaging. Background Technology

[0002] Stereotactic brain imaging is a classic and routine technique in the field of brain science research. With the rapid development of brain science and neuroscience in recent years, the demand for techniques such as in vivo magnetic resonance imaging (MRI), deep brain imaging, in vivo electrophysiology, optogenetics, and neural circuit tracing has increased significantly. These research techniques are all based on the precise localization of brain nuclei in animals. Only by accurately locating the target nucleus can we conduct studies such as deep brain imaging, intracranial targeted drug delivery, in vivo electrophysiological recording, optogenetic control, and neural circuit tracing. Stereotactic fixation of the skull is a prerequisite for achieving precise navigation and localization of brain nuclei in animals, while horizontal adjustment methods of the skull are key to stereotactic brain imaging.

[0003] Currently, there is a lack of a unified standard for the stereotaxic brain atlas of the tree shrew (Tupaia glis). Tigges et al. (1969) used the Horsley-Clark coordinate system in their atlas, *A stereotaxic brain atlas of the tree shrew (Tupaia glis)*: the zero-point plane is defined by a plane passing through the auricle and the lower edge of the orbit; the vertical zero-point plane passes through the interauricular line (the line connecting the auricles) and is perpendicular to the horizontal zero-point plane. Yang Wenguang et al. (1990) used the following coordinate system in their book, *Stereotaxic Brain Atlas of the Tree Shrew (Tupaia glis)*: the horizontal zero-point plane is parallel to the plane passing through the auricle and the incisor bar; the vertical zero-point plane passes through the interauricular line (the line connecting the auricles) and is perpendicular to the horizontal zero-point plane. The inventors of this invention, in their 2016 publication, *Stereotaxic Brain Atlas of the Tree Shrew*, used the following coordinate system: the horizontal zero-point plane is parallel to the plane passing through the anterior and posterior fontanelles.

[0004] The coordinate system of these tree shrews, like the stereotactic coordinate system of other model animals, has some drawbacks in practical applications. For example, it requires cutting open the animal's scalp and exposing the skull, causing significant trauma to the animal, and it is not suitable for non-invasive brain localization surgery and imaging techniques.

[0005] The existing technologies described above have the following shortcomings: (1) Existing techniques for fixing and stereotactic positioning of the tree shrew's skull mainly employ: ① the plane of the auricle and the lower edge of the orbit, ② the plane of the incisor bar and the auricle, and ③ the plane of the anterior and posterior fontanelles on the skull surface. These three methods differ greatly and are not consistent in actual operation. Methods ① and ② are the simplest to operate; method ③ has the highest positioning accuracy, but it requires cutting open the scalp on the skull surface, and the positioning steps are time-consuming and cumbersome.

[0006] (2) The existing tree shrew cranial fixation technology is mainly developed for brain localization surgery, not for non-invasive brain localization surgery and in vivo brain imaging technology, and cannot meet the needs of different application scenarios in the current brain science field.

[0007] (3) Existing tree shrew brain fixation technology cannot simultaneously achieve rapid operation and precise control, and cannot meet the current practical needs of high precision, high throughput and rapid experimental operation.

[0008] Therefore, there is an urgent need in this field to develop a more convenient and precise method for fixing the brain of tree shrews, so as to facilitate further experimental research and data analysis. Summary of the Invention

[0009] To address the problems of existing technologies, this invention provides a method for fixing the skull of a tree shrew and its application in magnetic resonance imaging.

[0010] This invention provides a method for fixing the skull of a tree shrew, comprising the following steps: After anesthetizing, fix the tree shrew's head on the stereotaxic device and align the two ear rods in the left and right slots with the bone sutures next to the tree shrew's external auditory canal. Adjust the left and right ear rods to keep them at the same height and centered. With the ear rod height as the zero point, align the tree shrew's incisors with the incisor rod slots. Adjust the distance between the front and back of the incisor bar so that the gap between the second and third lateral teeth of the tree shrew fits into the groove of the incisor bar. Pull the incisor bar so that it hooks onto the inside of the second tooth and fix the incisor bar above the maxilla. Adjust the incisor bar vernier caliper on the stereotaxic instrument to a reading of -5.00 ± 0.02 mm. At this point, the tree shrew's brain is in a horizontal position.

[0011] Preferably, the adjustment reading of the vernier caliper on the incisor bar is -5.00 mm.

[0012] Preferably, the tree shrew is an adult tree shrew weighing 100-170 grams and aged 6-15 months.

[0013] The present invention also provides a magnetic resonance imaging method for non-diagnostic purposes, comprising the following steps: The tree shrew's skull was fixed according to the above-described method for fixing the skull. The tree shrew's head, which had been fixed by the aforementioned method, was placed in a magnetic resonance coil for whole-brain MRI scanning.

[0014] The present invention also provides a method for analyzing whole-brain magnetic resonance imaging data of tree shrews, characterized in that the horizontal zero plane coordinate system of the genu rostrum-beak lateral junction point of the corpus callosum-beak lateral junction point of the breech commissure is used as a reference system to level the horizontal zero plane during whole-brain magnetic resonance imaging of tree shrews. The leveling of the coordinate system is based on the above-mentioned fixing method and is applied to the adjustment of the initial state during the acquisition of tree shrew magnetic resonance imaging data.

[0015] Preferably, the horizontal zero-plane reference coordinate system of the beak-side connection point of the genu callosum-needle fusion beak-side connection point is convertible with the horizontal zero-plane reference coordinate system of the anterior and posterior fontanelles of the tree shrew skull.

[0016] Preferably, the horizontal zero-plane reference coordinate system of the genu rostrum-hymenal rostrum connection point refers to a coordinate system constructed by using the genu rostrum and hymenal rostrum connection points of the corpus callosum in the brain as two reference points and passing through the horizontal zero plane of these two reference points.

[0017] Preferably, the data analysis includes tree shrew brain stereotactic navigation or three-dimensional brain imaging.

[0018] The beneficial effects and advantages of the technical solution of this invention are as follows: (1) This invention adopts a new technology for fixing the skull of tree shrew brains. Through correction with multiple animals, individual differences are eliminated, and the fixation accuracy reaches 0.02 mm. This method provides a new high-precision skull fixation method for tree shrew brains, lays the foundation for tree shrew brain stereo positioning navigation and three-dimensional brain imaging, provides a standardized alternative, and solves the problems of difficult operation and inconsistent positioning of existing tree shrew brain structure navigation systems.

[0019] (2) The present invention can level the skull in just one step, which is simple and quick to operate and does not require exposure of the skull, causing no trauma to the animal and avoiding the potential impact of trauma on the animal. It is especially suitable for non-invasive brain localization surgery and live brain imaging technology.

[0020] (3) This invention is easy to operate and takes very little time, which greatly saves the preparation time for brain localization and imaging, and can meet the needs of high-throughput animal in vivo brain experiments.

[0021] (4) This invention provides a new application scenario for obtaining in situ images of the whole brain of tree shrews using magnetic resonance imaging. The method of this invention is more accurate and reliable, makes more efficient use of each tree shrew, reduces the experimental cycle and research cost, and meets animal welfare requirements.

[0022] (5) The method for fixing the skull of a tree shrew provided by the present invention can be used as an effective alternative to the most commonly used animal brain stereotactic method in the world, which is conducive to the exchange, sharing and reference of relevant research results.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 This is a side view of the skull of a tree shrew.

[0026] Figure 2 The image shows a coronal section of the whole brain of a tree shrew, with a spacing of 250 micrometers and a scale bar of 20 millimeters.

[0027] Figure 3 This diagram shows the zero-plane reference coordinate system for the rostral junction points of the genu of the corpus callosum (G) – rostral junction point of the habenular commissure (H), the zero-plane reference coordinate system for the anterior fontanelle (B) – posterior fontanelle (L), and the structural localization diagram for the incisor shank – interaural line (I). (a) and (b) are MRI brain imaging images; (c)–(f) are pathological section identification images; and (g) is a schematic diagram showing the relationship between skull fixation techniques and the two coordinate systems. Detailed Implementation

[0028] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0029] Example 1: Method for Fixing the Cranium of a Tree Shrew The method for fixing the skull of a tree shrew described in this invention and its application include the following specific steps: Step 1: Select 21 adult tree shrews weighing 100-170 grams and aged 6-15 months. Anesthetize the animals until they are in a deep anesthetized state to avoid stress.

[0030] Step 2: Secure the tree shrew's head to the stereotaxic apparatus. First, align the two ear rods in the left and right slots with the bone sutures next to the animal's external auditory canal. Adjust the left and right ear rods to maintain the same height and center (ear rod height is zero point) so that the animal's incisors are precisely aligned with the slots of the front incisor rods. Adjust the front-to-back distance of the incisor rods to ensure that the gap between the second and third lateral teeth of the tree shrew is precisely engaged in the slots of the incisor rods. Gently pull the incisor rods to ensure that they hook onto the inside of the second tooth. Secure the incisor rod pressure bar and gently press it against the upper part of the maxilla to prevent slippage (see...). Figure 1 ).

[0031] Step 3: Use the incisor bar vernier caliper on the stereotaxic instrument to make precise adjustments. Adjust the reading to -5.00 mm (the calculation basis is shown in Table 1). At this time, the animal's skull will be in a horizontal position.

[0032] Table 1. Calculation results of vertical distance between the cranial bones (incisor bar and interauricular line) of the tree shrew (based on the incisor bar height when the anterior and posterior fontanelles are horizontal, unit: mm) Example 2: Whole-brain MRI and analysis method of tree shrews After fixation using the method of Example 1, the tree shrew head with the skull fixed as described above can be further placed in a magnetic resonance coil for whole-brain magnetic resonance imaging (MRI) of the tree shrew. The scan results are shown in [Figure 1]. Figure 2 .

[0033] In MRI applications, a brain-level zero-plane reference coordinate system compatible with this invention is proposed (see...). Figure 3 The coordinate system refers to the beak-side connection point of the genu of the corpus callosum and the beak-side connection point of the ligament commissure. This coordinate system does not conflict with the existing skull reference coordinate system (anterior fontanelle-posterior fontanelle coordinate system), and they can be freely converted (conversion values ​​are shown in Table 2), which can meet different data analysis scenarios and application scenarios.

[0034] Table 2. Calculation results of horizontal and vertical distances between tree shrew brain structures (rostral genu beak-side junction, breech commissure beak-side junction, anterior fontanelle, posterior fontanelle, interauricular line) (unit: mm) Note: GH, genu of corpus callosum, beak-side junction - nebula commissure, beak-side junction; GB, genu of corpus callosum, beak-side junction - anterior fontanelle; BL, anterior fontanelle - posterior fontanelle; BI, anterior fontanelle - interauricular line.

[0035] As can be seen from the above embodiments, one of the objectives of this invention is to provide a method for fixing the skull of a tree shrew with the incisor bar 5.00 mm (5.00 ± 0.02 mm) below the interauricular line. The method uses a vernier caliper on a stereotaxic instrument to precisely adjust the incisor bar with an accuracy of 0.02 mm. The operation requires only one step and takes only a few seconds. The animal does not need to repeat the adjustment of the skull level afterward, which greatly saves experimental operation time.

[0036] The second objective of this invention is to provide the application of the above-mentioned fixation method in whole-brain magnetic resonance imaging of tree shrews.

[0037] A third objective of this invention is to provide a horizontal zero-plane reference coordinate system for the tree shrew brain, specifically a coordinate system for the genu callosum-buccal junction-buccal junction coordinate system, which can replace the existing skull surface reference coordinate system for data analysis of whole-brain MRI in tree shrews.

[0038] This invention aims to solve the technical problems of existing tree shrew cranial fixation methods, such as inconsistent operation, cumbersome steps, trauma caused by skull exposure, and inability to balance rapid operation and high precision. It proposes a new method for tree shrew cranial fixation with great application prospects.

Claims

1. A method for fixing the skull of a tree shrew, characterized in that, Includes the following steps: After anesthetizing, fix the tree shrew's head on the stereotaxic device and align the two ear rods in the left and right slots with the bone sutures next to the tree shrew's external auditory canal. Adjust the left and right ear rods to keep them at the same height and centered. With the ear rod height as the zero point, align the tree shrew's incisors with the incisor rod slots. Adjust the distance between the front and back of the incisor bar so that the gap between the second and third lateral teeth of the tree shrew fits into the groove of the incisor bar. Pull the incisor bar so that it hooks onto the inside of the second tooth and fix the incisor bar above the maxilla. Adjust the incisor bar vernier caliper on the stereotaxic instrument to a reading of -5.00 ± 0.02 mm. At this point, the tree shrew's brain is in a horizontal position.

2. The method for fixing the skull of a tree shrew according to claim 1, characterized in that, The adjustment reading of the vernier caliper on the incisor bar is -5.00 mm.

3. The method for fixing the skull of a tree shrew according to claim 1, characterized in that, The tree shrews referred to are adult tree shrews weighing 100-170 grams and aged 6-15 months.

4. A magnetic resonance imaging method for non-diagnostic purposes, characterized in that, Includes the following steps: Tree shrew cranial fixation method according to any one of claims 1 to 3 is used for tree shrew cranial fixation; The tree shrew's head, which had been fixed by the aforementioned method, was placed in a magnetic resonance coil for whole-brain MRI scanning.

5. A method for analyzing whole-brain magnetic resonance imaging data of tree shrews, characterized in that, Using the horizontal zero-plane reference coordinate system of the genu rostrum-beak lateral junction point and the breech commissure rostrum-beak lateral junction point as a reference system, the horizontal zero plane is leveled during whole-brain MRI of tree shrews. The leveling of the coordinate system is based on the above-described fixing method and is applied to the adjustment of the initial state during the acquisition of tree shrew MRI data. The coordinate system is established in combination with MRI data according to the fixing method described in any one of claims 1 to 3.

6. The method for analyzing whole-brain magnetic resonance imaging data of tree shrews according to claim 5, characterized in that, The coordinate system of the beak-side junction point of the genu callosum-needle junction is convertible with the horizontal zero plane reference coordinate system of the anterior and posterior fontanelles on the surface of the tree shrew skull.

7. The method for analyzing whole-brain magnetic resonance imaging data of tree shrews according to claim 5, characterized in that, The horizontal zero-plane reference coordinate system of the genu rostrum-hymenal rostrum refers to a coordinate system constructed by using the genu rostrum and hymenal rostrum as two reference points and passing through the horizontal zero plane of these two reference points.

8. The method for analyzing whole-brain magnetic resonance imaging data of tree shrews according to claim 5, characterized in that, The data analysis includes tree shrew brain stereotactic navigation or three-dimensional brain imaging.

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