Novel irradiation fresh-keeping method for brain tissues

Abstract

The invention provides a novel irradiation fresh-keeping method for brain tissues. The novel irradiation fresh-keeping method comprises the following steps: 1) providing irradiation equipment; 2) obtaining animal brain tissues; 3) the animal brain tissue is placed in a radiation source to be irradiated, the irradiation dose is 1 kGy to 100 kGy, and after irradiation, a fixing agent does not need to be used for fixation; (4) dyeing the treated brain tissue; 5) slicing the animal brain tissue; (6) dehydrating and sealing the sliced animal brain tissue; and 7) performing imaging observation on the brain tissue sample. According to the method, a traditional irradiation preservation technology is applied to fixed preservation of the brain tissue for the first time, so that brain tissue degradation and structural damage caused by enzyme in the brain tissue are inhibited. According to the technology, the structure of the brain tissue sample can still be kept complete without adding a chemical reagent, enough fresh brain tissue samples are expected to be provided for brain science research, especially human brain research, and technical support is provided for brain science research.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical imaging, and more particularly to a new method for irradiation preservation of brain tissue. BACKGROUND

[0002] It is well known in the art that perfusion fixation after deep anesthesia is one of the necessary steps to prevent enzyme degradation and loss of antigens for model animals. However, for the study of specific specimens such as human brain, perfusion fixation is not possible due to methodological or ethical factors. According to the restrictions of ethics and other conditions, human tissues can only be obtained several hours after death. In order to understand the human brain, experiments are usually conducted using model animals, but the most direct and effective method is to conduct anatomical studies on postmortem human tissues. The main difference between the two is the experimental bias caused by postmortem delay. The longer the postmortem delay, the greater the degree of tissue / protein degradation that can occur. Therefore, the postmortem delay time is the biggest bottleneck in the study of human brain.

[0003] At present, irradiation technology has been widely used in food preservation as a non-residual physical preservation method. By directly destroying the biomolecules of microorganisms or generating free radicals from water to destroy cell metabolic pathways or other ways, the production of unnecessary biological active substances is inhibited to achieve the purpose of food storage. Therefore, it is desired to use irradiation technology to process brain tissue with a death delay in order to maintain the integrity of the brain tissue structure after the death of the animal or several hours after death. Electron beam irradiation (EBI), Co gamma irradiation and other radiation sources are a new non-thermal processing technology. Its mechanism is mainly to decompose water molecules in food, feed and plant and animal materials by irradiation. The free radicals H and OH generated in the process participate in the chemical changes of biological macromolecules in the material, leading to chain breakage and recombination, thereby causing physical and chemical reactions inside the material and weakening the enzyme activity inside the organism. Electron beam irradiation process is usually carried out at normal temperature and pressure, and the process is simple, efficient and has good penetration, so it is widely used. 60 Co gamma irradiation and other radiation sources are a new non-thermal processing technology. Its mechanism is mainly to decompose water molecules in food, feed and plant and animal materials by irradiation. The free radicals H and OH generated in the process participate in the chemical changes of biological macromolecules in the material, leading to chain breakage and recombination, thereby causing physical and chemical reactions inside the material and weakening the enzyme activity inside the organism. Electron beam irradiation process is usually carried out at normal temperature and pressure, and the process is simple, efficient and has good penetration, so it is widely used.

[0004] However, so far, the prior art still lacks a staining imaging method that can be applied to brain tissue with a death delay, especially human brain tissue with a death delay. SUMMARY

[0005] The purpose of the present application is to provide a new method for irradiation preservation of brain tissue, thereby solving the deficiencies in the prior art in terms of staining imaging of brain tissue with a death delay, especially human brain tissue with a death delay.

[0006] In order to solve the above technical problems, the present application adopts the following technical solutions:

[0007] The application provides a new method for irradiation preservation of brain tissue, which comprises the following steps: 1) providing an irradiation device; 2) obtaining animal brain tissue; 3) placing the animal brain tissue in a radiation source for irradiation, and the irradiation dose is 1-100 kGy, and the treated brain tissue does not need to be fixed after irradiation; 4) staining the treated brain tissue; 5) slicing the animal brain tissue; 6) dehydrating and mounting the sliced animal brain tissue; and 7) imaging the brain tissue sample.

[0008] According to the staining method provided by the application, the working principle is that the irradiation of the electron beam, 60 The irradiation treatment of the radiation source such as Co irradiation can alleviate the possible tissue / protein degradation of the brain tissue after death, maximally preserves the integrity of neurons in the brain tissue, enables the brain tissue with a death delay time to realize relatively complete staining of neurons, improves the staining efficiency of nerve fibers, and thus enhances the signal-to-noise ratio of the brain tissue in microscopic imaging.

[0009] The radiation source in the step 1) is respectively an electron beam irradiation device, 60 A Co irradiation device, an X-ray irradiation device, a proton irradiation device and the like.

[0010] The animal brain tissue with different death delay times in the step 2) includes all animal brain tissues or brain tissue slices such as fruit fly brain tissue, zebra fish brain tissue, mouse brain tissue, rat brain tissue, rabbit brain tissue, cat brain tissue, dog brain tissue, monkey brain tissue and human brain tissue.

[0011] The method for obtaining the model animal brain tissue mainly adopts a method that after blood is removed by a heart perfusion buffer or a fixing solution, the complete brain tissue is dissected. The animal brain tissue in the step 2) includes fresh unfixed animal brain tissue and brain tissue removed after irradiation after anesthesia of a living body.

[0012] The irradiation dose in step 3) is preferably 1 kGy-100 kGy, more preferably 1 kGy-50 kGy. The optimal irradiation dose is selected according to the size of the brain tissue of different animals. Most preferably, the irradiation dose is 10 kGy, which only requires short irradiation and then placement for a corresponding death delay time before subsequent tissue staining treatment. It should be understood that the irradiation time mainly depends on the setting of the dose rate, and under the irradiation dose set in the present application, short irradiation is sufficient, so the effect of the irradiation time on the technical effect is not considered. The present application also selects the Golgi staining method which is simple to operate and has intuitive effect for verification. After irradiation, the brain tissue is placed for a corresponding death delay time and then immersed in Golgi staining solution for 4 days to immerse the complete morphological structure of neurons, while the brain tissue sample without irradiation treatment is placed for a corresponding death delay time and then subjected to Golgi staining, and the morphology of neurons is not complete. This method greatly preserves the integrity of the morphology of neurons as time increases.

[0013] The staining method in step 4) includes but is not limited to Golgi staining, Nissl staining, H&E staining, immunohistochemistry, immunofluorescence, myelin staining, osmium tetroxide staining and various tissue staining methods.

[0014] The embedding in step 5) uses embedding materials such as collodion, paraffin, epoxy resin and OCT embedding agent. The slicing equipment includes brain mold 1 mm slicing mold, frozen section and vibration sectioning machine and paraffin sectioning machine, and the thickness of the section is 50 nm-5000 μm. Among them, the optimal thickness for optical microscope observation is 100 μm, the optimal thickness for electron microscope observation is 70 nm, and X-ray microscopic imaging mainly uses 1 mm-2 mm thick section.

[0015] The dehydration in step 6) uses gradient alcohol (30%, 50%, 75%, 90%, 100%) to dehydrate the brain tissue, and the dehydration time of each concentration of alcohol is 10 min-48 h.

[0016] After dehydration using gradient alcohol in step 6), xylene or sodium thiosulfate immersion is used for transparency treatment.

[0017] The sealing in step 6) is performed using neutral gum, nail polish or rapid sealing drying agent.

[0018] The microscopic imaging observation in the step 7) includes: optical microscope imaging, electron microscope imaging, nuclear magnetic resonance imaging, and synchrotron X-ray imaging. The optical microscope refers to a microscope based on optical imaging principles, such as a Zeiss optical microscope, a laser scanning light focusing microscope, a light sheet microscope, a lattice layer light microscope, a multi-photon microscope, and an ultrahigh-resolution optical microscope; and the synchrotron X-ray imaging observation is related to an imaging platform based on X-ray imaging of a synchrotron device, such as X-ray absorption imaging, X-ray diffraction, wide-angle X-ray scattering, small-angle X-ray scattering, X-ray phase contrast imaging, and X-ray fluorescence imaging, and the imaging lines can perform X-ray two-dimensional or three-dimensional imaging on brain tissue, so that brain neural connection imaging data with a certain resolution are obtained, and related brain connection maps are obtained.

[0019] At present, irradiation technology is mainly applied to food preservation and material modification, and in the aspect of tissue staining, especially brain tissue staining and marking, there is no literature reported before. It should be understood that the staining of brain tissue is mainly limited by the acquisition and preservation of the tissue, and the acquisition of brain tissue of special model animals, especially human brain tissue samples, is also limited by ethics. For example, donated human brain tissue is limited by the delay time of death and cannot be immediately fixed. In the traditional biological tissue staining method, the tissue preservation method involves soaking in a fixative, and the effect is different according to the size of the tissue, and when the size is large, it may take several hours to soak the inside of the tissue. However, after the brain tissue is preserved by the fixative and then stained, the signal-to-noise ratio is reduced during subsequent microscopic imaging, which affects the subsequent data analysis.

[0020] The key point of the present application is that the "irradiation preservation" is used to replace the "fixative fixation" in the prior art, and the fixative soaking step is omitted after irradiation. The use of irradiation technology can fix and preserve the brain tissue to a certain extent without the participation of chemical reagents, which is helpful for the complete staining and marking of the brain tissue after being taken out of the body.

[0021] Compared with the prior art, the positive progress effect of the present application is that:

[0022] 1) The current method for brain tissue staining and marking mainly includes Golgi staining, specific immunohistochemical staining, and virus marking. Among them, the specific immunohistochemical staining and virus marking are usually limited by ethics and other uncontrollable factors when marking neurons in human brain tissue, and Golgi staining can be used to stain and mark neurons in human brain tissue after death. Golgi staining has achieved good staining effect in non-human primate brain tissue. The brain tissue irradiation preservation method provided by the present application adopts the Golgi staining method, and good imaging effect is obtained in optical microscope imaging and synchrotron X-ray imaging, and has good application prospect in human brain neuron staining.

[0023] 2) In the process of experiment, in order to draw the brain map, due to the ethical restrictions, only after death can human tissue be obtained for several hours. When analyzing the neurons of brain tissue, the most important consideration involves the acquisition of the tissue and the extent to which the tissue can degrade before fixation. The longer the death delay time, the greater the extent of possible tissue / protein degradation. When using a fixative to soak, the penetration is not instantaneous, and according to the size of the tissue, it can take several hours to fully penetrate the inside of the tissue. The irradiation preservation means provided in the present application can completely preserve the structure of the brain tissue for subsequent experiments, and provides a more rapid and effective fixation and preservation method.

[0024] 3) The traditional Golgi staining method has been developed for more than a hundred years, and is mainly applied to optical microscopy imaging research, and has made outstanding contributions in the study of neurology. However, in the current study of brain mapping, Golgi staining has limitations: Golgi staining is a non-specific staining of neurons, which is random; the staining efficiency is low, and only 2-5% of the neurons can be stained; the stained neurons are discontinuous, and some small nerve fibers are not stained obviously. The method provided by the present application combines irradiation preservation of brain tissue and traditional Golgi staining method. Through irradiation, the cell metabolism and enzyme activity (autolysis) can be reduced to maintain the integrity of the brain tissue structure, to ensure the penetration of the staining solution in the neural tissue in the later stage, to improve the labeling efficiency of neurons, to reduce the influence of death delay time on brain tissue staining, and to establish a new tissue preservation method which can be applied to Golgi staining and specific immunohistochemical staining and other staining labeling techniques.

[0025] In summary, the present application establishes a new method for efficient preservation of irradiated brain tissue, which is expected to provide sufficient "fresh" brain tissue samples for brain science research, especially human brain research, and to provide technical support for brain science research. In addition, the method provided by the present application is expected to have similar effects in the preservation of other organ tissues. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The optical microscope imaging diagram in Example 1; wherein, A is the optical microscope imaging diagram of the cerebral cortex region of the mouse brain tissue after being soaked in Golgi staining at normal temperature and normal pressure without fixation; B is the optical microscope imaging diagram of the cerebral cortex region of the mouse brain tissue after being soaked in Golgi staining at normal temperature and normal pressure using a chemical fixative; C is the optical microscope imaging diagram of the cerebral cortex region of the mouse brain tissue after being soaked in Golgi staining at normal temperature and normal pressure without fixation liquid after irradiation treatment;

[0027] Figure 2Figure 2A-D are optical microscope images of the cerebral cortex region of mouse brain tissue; Figure 2A is an optical microscope image of the cerebral cortex region of mouse brain tissue post 96h immersion in Golgi stain after 12h post mortem delay at normal temperature and pressure; Figure 2B is an optical microscope image of the cerebral cortex region of mouse brain tissue post 96h immersion in Golgi stain after 12h post mortem delay at normal temperature and pressure after lower dose (5kGy) irradiation of the mouse brain tissue; Figure 2C is an optical microscope image of the cerebral cortex region of mouse brain tissue post 96h immersion in Golgi stain after 12h post mortem delay at normal temperature and pressure after lower dose (10kGy) irradiation of the mouse brain tissue; Figure 2D is an optical microscope image of the cerebral cortex region of mouse brain tissue post 96h immersion in Golgi stain after 12h post mortem delay at normal temperature and pressure after higher dose (50kGy) irradiation of the mouse brain tissue;

[0028] Figure 3 Figure 3A-D are optical microscope magnified images of the cerebral cortex region of mouse brain tissue; Figure 3A is an optical microscope magnified image of the cerebral cortex region of mouse brain tissue post 96h immersion in Golgi stain after 12h post mortem delay at normal temperature and pressure; Figure 3B is an optical microscope magnified image of the cerebral cortex region of mouse brain tissue post 96h immersion in Golgi stain after 12h post mortem delay at normal temperature and pressure after lower dose (5kGy) irradiation of the mouse brain tissue; Figure 3C is an optical microscope magnified image of the cerebral cortex region of mouse brain tissue post 96h immersion in Golgi stain after 12h post mortem delay at normal temperature and pressure after lower dose (10kGy) irradiation of the mouse brain tissue; Figure 3D is an optical microscope magnified image of the cerebral cortex region of mouse brain tissue post 96h immersion in Golgi stain after 12h post mortem delay at normal temperature and pressure after higher dose (50kGy) irradiation of the mouse brain tissue;

[0029] Figure 4 Figure 4A-E are synchrotron radiation images of the cerebral cortex region of mouse brain tissue; Figure 4A is a synchrotron radiation image of the cerebral cortex region of fresh unfixed mouse brain tissue post 96h immersion in Golgi stain at normal temperature and pressure; Figure 4B is a synchrotron radiation image of the cerebral cortex region of mouse brain tissue post 96h immersion in Golgi stain after 12h post mortem delay at normal temperature and pressure, which is the non-irradiated post mortem control; Figure 4C is a synchrotron radiation image of the cerebral cortex region of mouse brain tissue post 96h immersion in Golgi stain after 12h post mortem delay at normal temperature and pressure after lower dose (5kGy) irradiation of the mouse brain tissue; Figure 4D is a synchrotron radiation image of the cerebral cortex region of mouse brain tissue post 96h immersion in Golgi stain after 12h post mortem delay at normal temperature and pressure after lower dose (10kGy) irradiation of the mouse brain tissue; Figure 4E is a synchrotron radiation image of the cerebral cortex region of mouse brain tissue post 96h immersion in Golgi stain after 12h post mortem delay at normal temperature and pressure after higher dose (50kGy) irradiation of the mouse brain tissue. DETAILED DESCRIPTION

[0030] The application will be further described in conjunction with specific examples. It should be understood that the following examples are used to illustrate but not to limit the scope of the present application. The experimental methods in the following examples, if not specified, are selected according to the conventional methods and conditions, or according to the instructions of the commercial products.

[0031] The present application takes mouse brain tissue as an example but not as a limitation, and combines Golgi staining as an example for verification. The irradiation dose ranges from 1 to 100 kGy to avoid damage to the internal structure of the brain tissue due to high radiation dose; the Golgi staining solution is configured with a mass-volume ratio of 5% for potassium dichromate (K2Cr2O7), mercury chloride (HgCl2), and potassium chromate (K2CrO4), and the staining time is mainly not more than 15 days; the slices are selected as frozen sections, and the thickness of the optical microscope slices is 100 μm, and the thickness of the synchrotron X-ray imaging slices is 1 mm. The irradiation fresh-keeping brain tissue technology is combined with the traditional Golgi staining to establish a fresh-keeping method suitable for the death-delayed brain tissue, and combined with Golgi staining, more effective data can be obtained in optical microscopy and synchrotron X-ray brain tissue imaging. The following examples specifically illustrate the implementation effect of the present application.

[0032] Example 1 proves that the staining effect of neurons in different brain regions of the sample after irradiation fresh-keeping combined with the Golgi staining method is better than that of the brain tissue preserved by using a fixative.

[0033] The mouse is deeply anesthetized with 1% pentobarbital, and the blood is removed by heart perfusion with normal saline according to the experimental routine. The mouse brain tissue is quickly taken out and immersed in the prepared Golgi staining solution (5% potassium dichromate (K2Cr2O7), 5% mercury chloride (HgCl2), 5% potassium chromate (K2CrO4)) for 96 h. After the staining is completed, the brain tissue block is taken out and placed in a 30% sucrose solution for dehydration at 4°C and in the dark for about 3 days, and the 30% sucrose solution is replaced every 24 h. In this embodiment, the slices are prepared by freezing, and the slices are sagittal with a thickness of 100 μm. The cut mouse brain tissue slices are reacted in 20% ammonia water for 15 minutes, washed with water and terminated, and then dehydrated in 30%, 50%, 70%, 80%, 90% and 100% series gradient alcohol for 5 min each. Then, xylene is used for transparency for at least 15 min, and finally, the slices are mounted using a quick mounting drying agent. After the xylene is completely volatilized, a Zeiss optical microscope is used for observation. Figure 1 Figure A in the figure shows the optical microscope imaging of the cerebral cortex region of the fresh and unfixed mouse brain tissue after immersion in Golgi staining under normal temperature and pressure.

[0034] The mouse was deeply anesthetized with 1% pentobarbital, and the blood was removed by heart perfusion with physiological saline according to the experimental routine. The mouse brain tissue was quickly removed and fixed in the same fixing solution for 1-2 days. Then, the brain tissue was immersed in the prepared Golgi staining solution (5% potassium dichromate (K2Cr2O7), 5% mercuric chloride (HgCl2), and 5% potassium chromate (K2CrO4)). The subsequent operation was the same as the above operation. Figure 1 Fig. 8B is an optical microscope imaging diagram of the cerebral cortex region of the mouse brain tissue fixed by a chemical fixative at normal temperature and pressure after immersion in Golgi staining. As can be seen from the figure, after the brain tissue fixed by the fixative is immersed in Golgi staining, a large number of glial cells are immersed, which reduces the signal-to-noise ratio and increases the difficulty of subsequent data analysis.

[0035] The mouse was deeply anesthetized with 1% pentobarbital, and the blood was removed by heart perfusion with physiological saline according to the experimental routine. The mouse brain tissue was quickly removed and fixed in the same fixing solution for 1-2 days. Then, the brain tissue was immersed in the prepared Golgi staining solution (5% potassium dichromate (K2Cr2O7), 5% mercuric chloride (HgCl2), and 5% potassium chromate (K2CrO4)). The subsequent operation was the same as the above operation. Figure 1 Fig. 8C is an optical microscope imaging diagram of the cerebral cortex region of the mouse brain tissue after irradiation treatment without fixation by a fixative after immersion in Golgi staining. As can be seen from the figure, the effect of Golgi staining after irradiation is close to that of Figure 1 Fig. 8A shows the staining result, and the glial cells are not immersed in Golgi staining, and the immersion of the neuron cells is relatively complete. Obviously, the effect of Golgi staining after irradiation preservation is obviously better than that of the brain tissue fixed by the fixative.

[0036] Example 2 proves that the immersion efficiency of the sample after irradiation preservation combined with the Golgi staining method for different brain regions of neurons is better than that of the brain tissue without irradiation preservation by the Golgi staining method.

[0037] The mouse was deeply anesthetized with 1% pentobarbital, and the blood was removed by heart perfusion with physiological saline according to the experimental routine. The mouse brain tissue was quickly removed and fixed in the same fixing solution for 1-2 days. Then, the brain tissue was immersed in the prepared Golgi staining solution (5% potassium dichromate (K2Cr2O7), 5% mercuric chloride (HgCl2), and 5% potassium chromate (K2CrO4)). The subsequent operation was the same as the above operation. 60The brain tissue was irradiated with Co and other radiation sources at doses of 0 kGy, 5 kGy, 10 kGy, and 50 kGy. After irradiation, the tissue was left at room temperature and pressure for 12 hours to simulate the time delay in death. Subsequently, the tissue was immersed in a prepared Golgi staining solution (5% potassium dichromate (K2Cr2O7), 5% mercuric chloride (HgCl2), and 5% potassium chromate (K2CrO4)) for 96 hours. After staining, the brain tissue blocks were removed and placed in a 30% sucrose solution at 4°C in the dark for approximately 3 days to dehydrate. The 30% sucrose solution was replaced every 24 hours. In this embodiment, the sections were cryosectioned in sagittal format with a thickness of 100 μm. The sectioned mouse brain tissue was reacted in 20% ammonia for 15 minutes, washed with water, and the reaction was terminated. Then, it was dehydrated using a series of gradient alcohols (30%, 50%, 70%, 80%, 90%, and 100%), with each gradient dehydration time being 5 minutes. Afterward, the sections were cleared with xylene for at least 15 minutes. Finally, the sections were mounted with a rapid mounting desiccant, and after the xylene had completely evaporated, they were observed using a Zeiss optical microscope. It should be understood that, except for the irradiation step, all other steps in this process are the same as in the prior art, and no fixative treatment is used.

[0038] like Figure 2 As shown in Figure A, the staining of neurons in the cerebral cortex of mice after 96 hours of Golgi staining of untreated brain tissue at room temperature and pressure without irradiation showed that due to the increased time of death delay, the number of stained neurons in the cerebral cortex was significantly reduced, and the staining of neuronal cell bodies and some nerve fibers was significantly reduced. The staining effect on nerve fibers was particularly obvious, with only a small number of larger branch structures being stained. This was used as a control group to compare with the treated staining. Figure 2 Images B through D show the staining patterns of mouse cerebral cortex neurons after 96 hours of immersion in Golgi staining solution in irradiated brain tissue. B, C, and D represent the results of Golgi staining of brain tissue irradiated at doses of 5 kGy, 10 kGy, and 50 kGy, respectively, after a simulated death delay of 12 hours. Figure 2 It was observed that no damage to the internal structure of the irradiated brain tissue was observed, but the number of stained neuronal cell bodies increased significantly, and the morphology of the neuronal fibers remained relatively intact. Therefore, irradiation technology can preserve brain tissue for subsequent staining, and the higher the irradiation dose, the more neuronal cell bodies are stained. Prolonged preservation of irradiated samples before Golgi staining can enhance the staining effect on nerve fibers and their fine structures.

[0039] Figure 3Images A through D are magnified optical microscopic images of the cerebral cortex region after 96 hours of Golgi staining, following a 12-hour delayed placement in a simulated death environment, and after treatment with different irradiation doses (B, C, and D) at room temperature and pressure, without irradiation treatment (A) and after immersion in Golgi staining. Figure 3 As shown in Figure A, mouse cerebral cortex neurons stained with Golgi staining 96 hours after being kept at room temperature and pressure without irradiation showed relatively simple morphology, with visible neuronal cell bodies and some large primary structures of nerve fibers. However, after irradiation treatment, as shown in Figure A, the morphology of neurons in the irradiated brain tissue was relatively simple. Figure 3 As shown in B to D, the neurons are morphologically intact, and most of the nerve fibers can be seen to be infiltrated by primary, secondary, and tertiary structures. The morphology of dendritic spines can even be clearly observed.

[0040] In Example 3, the sample was irradiated and then stored for a long time before being stained with Golgi. The neuronal structure in the brain tissue could be imaged and analyzed under synchrotron radiation X-ray imaging technology.

[0041] The specific steps are as follows: Irradiated brain tissue was removed after a 12-hour delayed placement simulating death and immersed in prepared Golgi staining solution for 4 days. Then, it was dehydrated with 30% sucrose at 4°C in the dark for 72 hours. A 1 μm thick tissue was then subjected to reduction blackening in 20% ammonia for 1 hour, followed by rinsing to terminate the reaction. Dehydration was then performed using a series of gradient alcohols (30%, 50%, 75%, 90%, and 100%), with each gradient dehydration lasting 2 hours. Afterward, the tissue was immersed three times in a 1:1 mixture of anhydrous ethanol and xylene for 25 minutes each time. Finally, it was cleared four times with xylene for 25 minutes each time. After clearing, the thick tissue was immersed in paraffin twice for 1 hour each time. Following paraffin embedding, the tissue was embedded in paraffin using a paraffin embedding machine for further synchrotron X-ray imaging observation.

[0042] Figure 4 Image A shows a synchrotron radiation image of the cerebral cortex region of fresh mouse brain tissue after 96 hours of Golgi staining at room temperature and pressure. Figure 4 As can be clearly seen in B, the number of neurons in the mouse brain tissue after 12 hours of simulated death delay was significantly reduced after Golgi staining. Figure 4 Images C through E in the figures show synchrotron radiation imaging of the cerebral cortex of mice irradiated with 5 kGy, 10 kGy, and 50 kGy for 12 hours after simulated death, followed by 96 hours of Golgi staining. A significant increase in the number of stained neuronal cell bodies is clearly visible. This demonstrates that high-contrast imaging of brain tissue can be observed under high magnification, revealing a large number of stained neurons. The results of synchrotron radiation imaging are consistent with those obtained under optical microscopy.

[0043] The above merely describes preferred embodiments of the present application, and is not intended to limit the scope of the present application. The above-described embodiments of the present application can be variously changed. Any simple, equivalent changes and modifications made according to the content of the claims and the specification of the present application are intended to fall within the scope of the present application. The present application is not limited by the above-described embodiments.

Claims

1. A new method for irradiation preservation of brain tissue, characterized in that, The method comprises the following steps: 1) providing an irradiation device; 2) obtaining animal brain tissue; 3) placing the animal brain tissue in the irradiation source for irradiation, the irradiation dose being 1 kGy-100 kGy, and no fixing agent is needed after irradiation; 4) staining the treated brain tissue; 5) slicing the animal brain tissue; 6) dehydrating and mounting the sliced animal brain tissue; 7) imaging the brain tissue sample.

2. The novel method of irradiation preservation for brain tissue according to claim 1, wherein, The irradiation apparatus in the step 1) includes: an electron beam irradiation device, 60 a Co gamma irradiation device, an X-ray irradiation device, a proton irradiation device.

3. The novel method of irradiation preservation for brain tissue according to claim 1, wherein, The animal brain tissue in step 2) includes all animal whole brain tissue and / or brain tissue slices, including fruit fly brain tissue, zebra fish brain tissue, mouse brain tissue, rat brain tissue, rabbit brain tissue, cat brain tissue, dog brain tissue, monkey brain tissue and human brain tissue.

4. The novel method of irradiation preservation for brain tissue according to claim 1, wherein, The irradiation mode in step 3) includes immediate irradiation after the brain tissue is taken out, irradiation after the animal is anesthetized and then the brain tissue is taken out, irradiation before, during or after the thawing of the brain tissue stored in liquid nitrogen for a period of time.

5. The novel method of irradiation preservation for brain tissue as claimed in claim 1, wherein, The irradiation dose in step 3) is 1 kGy-50 kGy.

6. The novel method of irradiation preservation for brain tissue as claimed in claim 1, wherein, The staining method in step 4) includes Golgi staining, Nissl staining, H&E staining, immunohistochemistry, immunofluorescence, myelin staining or osmium tetroxide staining.

7. The novel method of irradiation preservation for brain tissue as claimed in claim 1, wherein, The slicing device used in step 5) includes a freezing microtome, a vibrating microtome, an ultramicrotome and a paraffin microtome, and the thickness of the slice ranges from 5 nm to 5000 μm.

8. The novel method of irradiation preservation for brain tissue as claimed in claim 1, wherein, The dehydration in step 6) is performed in a gradient alcohol manner.

9. The novel method of irradiation preservation for brain tissue as claimed in claim 1, wherein, The mounting in step 6) is performed using neutral gum, nail polish or a quick mounting drying agent.

10. The novel method of irradiation preservation for brain tissue as claimed in claim 1, wherein, The imaging method in step 7) includes nuclear magnetic resonance imaging, optical microscope imaging, electron microscope imaging or synchrotron X-ray imaging.

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