Method for human brain tissue dyeing and synchrotron radiation X-ray imaging at near freezing point temperature

Abstract

The invention discloses a method for human brain tissue staining and synchrotron radiation X-ray imaging at a near freezing point temperature. The method comprises the following steps: 1) preparing a Golgi staining solution; (2) unfreezing the death delayed frozen human brain tissue which is subjected to cryopreservation; 3) dip-dyeing the human brain tissue obtained in the step 2) in the Golgi staining solution, and staining at a temperature close to a freezing point; 4) developing neurons in the human brain tissue obtained in the step 3); 5) performing dehydration, transparentizing and tissue embedding on the human brain tissue obtained in the step 4); and 6) imaging and observing under a synchrotron radiation X-ray microscope. According to the method, the Golgi staining method is carried out at the near freezing point temperature for the first time, efficient staining of neurons of the dead delayed frozen human brain tissue is achieved, the neuron marking efficiency is improved, synchrotron radiation X-ray imaging of the neuron form and connection of the large-size human brain tissue is achieved, and the imaging accuracy is improved. The method is expected to be applied to imaging of a fine structure of a large-volume human brain tissue sample.

Description

Technical Field

[0001] The present invention relates to the field of brain imaging, and more particularly to a method for staining human brain tissue at near-freezing temperature and synchrotron radiation X-ray imaging. Background Art

[0002] The brain is the most complex organ in the human body. The research on it has important scientific significance for analyzing the brain function mechanism, revealing the law of brain development, and promoting the diagnosis and treatment of brain diseases. Currently, the technologies for brain tissue structure imaging mainly include optical microscopes and electron microscopes. However, the slow imaging speed and frequent tissue sectioning still limit the further application of the above technologies. Synchrotron radiation X-ray imaging technology has the advantages of high penetrability and fast imaging speed, combined with its multi-resolution imaging ability from micron to sub-micron or even nano-scale resolution, providing an important technical means for rapid and non-destructive three-dimensional imaging of high-thickness and large-volume samples. Based on this, synchrotron radiation X-ray imaging technology can provide important imaging technical support for the fine structure analysis of large-scale brain neural networks.

[0003] Currently, the bottleneck problem of synchrotron radiation X-ray imaging for human brain imaging lies in the low natural contrast of human brain neurons. There is an urgent need to develop efficient brain neuron staining or labeling technologies. Golgi staining technology uses elements such as mercury and silver to randomly label neurons, which can generate contrast under an optical microscope or X-ray, so it has become an important tool for studying neuron morphology. Although Golgi staining technology can achieve the labeling of neuron structures in human brain samples, there is still a contradiction between its staining efficiency and tissue autolysis. Fresh brain tissue is difficult to obtain and will undergo rapid autolysis after being excised, resulting in the difficulty of retaining brain structure information. Conventional Golgi staining methods are carried out at 37°C, but the rapid autolysis of brain tissue at this temperature will cause significant damage to the fine structure of the tissue. Therefore, it is of great research significance to develop an efficient staining and labeling method applicable to the neuron structure of dead-delayed frozen human brain tissue.

[0004] Chinese Patent Application (CN202310340808.3) discloses a staining method for synchrotron radiation X-ray imaging of human brain tissue. However, when implementing the staining, this method does not overcome problems such as tissue autolysis and neuron degradation during the staining process, and does not achieve efficient staining and labeling of brain tissue neuron structures and high-resolution imaging, thus affecting the further research on human brain neuron morphology. Summary of the Invention

[0005] The object of the present invention is to provide a method for staining human brain tissue at near-freezing temperature and synchrotron radiation X-ray imaging, so as to solve the problem that existing staining methods cannot yet achieve efficient neuron staining of dead-delayed frozen human brain samples.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for staining human brain tissue and synchrotron radiation X-ray imaging at near-freezing temperatures is provided, including the following steps: 1) Prepare Golgi staining solution; 2) Thaw the cryopreserved human brain tissue with delayed death; 3) Immerse the human brain tissue obtained in step 2) in the Golgi staining solution prepared in step 1) and stain at near-freezing temperatures; 4) Visualize neurons in the human brain tissue obtained in step 3); 5) Dehydrate, clear, and embed the human brain tissue obtained in step 4); 6) Image and observe under a synchrotron radiation X-ray microscope.

[0008] According to the method for staining human brain tissue and synchrotron radiation X-ray imaging at near-freezing temperatures provided by the present invention, its working principle is as follows: Inspired by the application of near-freezing temperatures to organ preservation, which reduces metabolism and can inhibit the growth of microorganisms and the activity of oxidase without damaging tissues, the present invention proposes to use the Golgi staining method to stain neurons in brain tissue at near-freezing temperatures, combined with the effective slowdown of autolysis and neuronal structure degradation in brain tissue at near-freezing temperatures, thereby achieving efficient staining of neuronal cells in brain tissue.

[0009] There are four types of Golgi staining solutions with different components in step 1), namely: 1) Potassium dichromate and silver nitrate; 2) Potassium dichromate, mercury chloride, and potassium chromate; 3) Glutaraldehyde, potassium dichromate, and silver nitrate; 4) Osmic acid, potassium dichromate, and silver nitrate. Golgi staining solutions with different components and concentrations can all achieve staining. Among them, the optimal concentrations are respectively: 1) 1.25% dichromic acid and 1% silver nitrate; 2) 1% potassium dichromate, 1% mercury chloride, and 1% potassium chromate; 3) 5% glutaraldehyde, 4% potassium dichromate, and 1% silver nitrate; 4) 0.16% osmotic acid, 2% potassium dichromate, and 1% silver nitrate.

[0010] More preferably, the Golgi staining solution composed of 1% potassium dichromate, 1% mercury chloride, and 1% potassium chromate in step 1) can achieve more efficient neuronal staining.

[0011] The human brain tissue in step 2) is not selective and can be any brain region of the human brain.

[0012] The human brain tissue in step 2) is cryopreserved human brain tissue with delayed death, and the environmental temperature range for cryopreservation is -20°C to -196°C, and the best is -80°C.

[0013] In step 3), the human brain tissue is immersed in the Golgi staining solution, and the near-freezing temperature environment is an ice-water bath. The ice content in the ice-water bath is 10% - 90%, preferably 60% - 80%, and optimally 60%. The immersion time is 4 - 180 days, and optimally 30 days.

[0014] The developing solution in step 4) can be a lithium hydroxide solution, an ammonia water solution, a sodium thiosulfate solution, and an ammonium carbonate solution. Among them, the optimal one is the ammonia water solution.

[0015] The developing time of the brain tissue in step 4) is 1 - 1440 minutes. For different thicknesses of human brain tissue, the developing time of its neurons is different. For 100-micron-thick brain tissue, the optimal time is 10 minutes, and for millimeter-thick brain tissue, the optimal time is 120 minutes.

[0016] In step 5), dehydration is carried out by using gradient alcohol. Among them, the optimal concentration gradient is 30%, 50%, 70%, 80%, 90%, and 100%.

[0017] The tissue embedding technique in step 5) can be a paraffin embedding technique, a resin embedding technique, and a hydrogel embedding technique. Among them, the optimal one is the paraffin embedding technique.

[0018] In step 6), the energy for imaging observation by the synchrotron X-ray microscope is 280 eV - 40 keV. Among them, the optimal imaging energy is 14 keV.

[0019] It should be understood that although the Chinese patent application (CN202310340808.3) discloses a staining method for synchrotron X-ray imaging of human brain tissue, which belongs to the same category of staining methods for human brain tissue as the present invention, however, when implementing the staining, this method does not overcome problems such as tissue autolysis and neuron degradation during the staining process, and fails to achieve efficient staining and labeling of the neuron structure of the brain tissue and high-resolution imaging, thus affecting the further research on the morphology of human brain neurons. Based on this, we provide a method for staining neurons in a delayed-frozen human brain sample after death. By performing the Golgi staining method at a near-freezing temperature, efficient staining of neurons in the delayed-frozen human brain tissue after death is achieved, significantly inhibiting the autolysis of the human brain tissue, thereby solving the problem that the existing staining methods cannot achieve efficient neuron staining of the delayed-frozen human brain sample.

[0020] The improvement of the present invention over the prior art mainly lies in that the degradation of human brain tissue is significantly inhibited at near-freezing temperatures. On the one hand, the present invention defines that Golgi staining is carried out in an environment of near-freezing temperature. On the other hand, the present invention also optimizes the composition of the Golgi staining solution under this method. Through the optimized Golgi staining environmental temperature and the composition of the Golgi staining solution, the limitation that the existing staining method cannot achieve efficient neuron staining of dead delayed-frozen human brain samples is broken through.

[0021] The positive and progressive effects of the present invention are as follows: By performing Golgi staining in an environment of near-freezing temperature, the metabolism of tissues can be reduced. Without damaging the tissues, the growth of microorganisms and the activity of oxidase can be inhibited, and tissue autolysis and the degradation of neuron structures can be slowed down, enabling the Golgi staining solution to stain neuron structures more efficiently and meeting the further research on the morphology of human brain neurons. In short, a Golgi neuron staining method at near-freezing temperature provided by the present invention for X-ray imaging of the human brain has good application prospects in the research on the neuron morphology of dead delayed-frozen human brain tissues and synchrotron X-ray imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a diagram of an ice-water bath device for performing Golgi staining at near-freezing temperature (left figure) and an optical microscope result diagram of Golgi staining at near-freezing temperature (right figure);

[0023] Figure 2 is an optical microscope imaging diagram obtained by performing Golgi staining on brain tissues at near-freezing temperature and freezing temperature for 96 hours;

[0024] Figure 3 is a statistical chart of the number of neuron cells obtained by performing H&E staining at near-freezing temperature, 4°C, 26°C, and 37°C;

[0025] Figure 4 is a statistical chart of the number of neuron cells obtained by performing staining at near-freezing temperature, 4°C, 26°C, and 37°C;

[0026] Figure 5 is a statistical chart of the number of neuron cells obtained by performing staining in Golgi staining solutions with different components, where A: 1.25% potassium dichromate, 1% silver nitrate; B: 0.16% osmium acid, 2% potassium dichromate, 1% silver nitrate; C: 5% glutaraldehyde, 4% potassium dichromate, 1% silver nitrate; D: 1% potassium dichromate, 1% mercury chloride, 1% potassium chromate.

[0027] Figure 6 is a synchrotron X-ray imaging result diagram. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following embodiments are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0029] The present invention takes human brain tissue as an example rather than a limitation. The sample storage environment is selected as a freezer at -80 °C for long-term storage; the Golgi staining solution is prepared with potassium dichromate, mercuric chloride, potassium chromate, silver nitrate, and osmium tetroxide as the main components, and the staining time is mainly no more than 30 days. The near-freezing temperature is applied to the dead-delayed frozen human brain samples for neuron staining, and a staining method suitable for imaging dead-delayed frozen human brain tissue is established. The following examples specifically illustrate the implementation effects of the present invention.

[0030] Example 1 Establishment of a Golgi neuron staining method for dead-delayed frozen human brain tissue at near-freezing temperature.

[0031] The dead-delayed frozen human brain tissue is thawed at room temperature and the tissue thickness is trimmed to 3 mm. Subsequently, the human brain tissue is immediately immersed in a staining solution composed of 1% potassium dichromate, 1% mercuric chloride, and 1% potassium chromate, and stained in the dark at near-freezing temperature. Fresh staining solution is replaced on the 1st, 8th, 15th, 22nd, and 28th days of staining, and the staining duration is 30 days. After the staining is completed, the human brain tissue undergoes sucrose dehydration, OCT embedding, cryosectioning, overnight air drying, reduction with ammonia water solution, gradient alcohol dehydration (30%, 50%, 70%, 80%, 90%, 100%), clearing, and mounting. Finally, imaging is performed under an optical microscope.

[0032] Figure 1 The left figure in [Figure] is a diagram of an ice-water bath device for Golgi staining at near-freezing temperature. In this device, there are human brain tissues being stained, a temperature monitor, and ice-water mixture, realizing a staining environment at near-freezing temperature. The right figure is the optical microscope result diagram of staining at near-freezing temperature. The image shows that effective staining of the human cerebellum is achieved using this method, the molecular layer and granular layer can be distinguished, and typical Purkinje cells of the cerebellum can be observed.

[0033] Example 2 Comparison of Golgi staining results of brain tissue at near-freezing temperature and under freezing conditions.

[0034] The brain was taken to obtain fresh brain tissue. An ice-water bath group and a freezing group (-2°C) were set up respectively. The brain tissues of both groups were stained in the dark for 4 days in a staining solution composed of 1% potassium dichromate, 1% mercuric chloride, and 1% potassium chromate. After the staining was completed, the tissues were dehydrated with sucrose, embedded in OCT, frozen-sectioned, air-dried overnight, reduced with ammonia water solution, dehydrated with gradient ethanol (30%, 50%, 70%, 80%, 90%, 100%), cleared, and mounted. Finally, images were taken under an optical microscope.

[0035] As Figure 2 shown, under near-freezing temperature conditions, after only 4 days of Golgi staining treatment, stained cell bodies and short dendrite structures can be seen. However, when the tissue is stained under freezing conditions, only a very small number of cell body structures can be stained and labeled. These labels may originate from the initial stage of staining when the staining solution has not frozen, and part of the staining solution penetrates and labels within the brain tissue. Once the staining solution freezes, further penetration and labeling become difficult. On the contrary, staining at near-freezing temperature effectively balances the penetration and labeling process of the staining solution and can stain and label neurons in human brain tissue.

[0036] Example 3 Comparison of the staining effects of thawed brain tissue, near-freezing temperature, and human brain H&E staining after being placed at 37°C for 24 hours.

[0037] The human brain tissue with delayed death by freezing was thawed at room temperature. The brain tissue was divided, and a control group, a near-freezing temperature group, and a 37°C group were set up respectively. For the control group, the brain tissue was directly fixed in 4% paraformaldehyde solution. For the near-freezing temperature group and the 37°C group, the brain tissues were placed in physiological saline for 24 hours respectively, and then fixed and preserved with 4% paraformaldehyde solution. Subsequently, the brain tissue was dehydrated, cleared, infiltrated with wax, embedded, sectioned, and baked. After dewaxing the sections, the sections were immersed in Harris hematoxylin staining solution for 5 - 7 minutes, rinsed with tap water to turn blue, then the sections were immersed in 1% hydrochloric acid alcohol for differentiation for 2 - 5 seconds, rinsed with tap water to turn blue, the sections were immersed in 1% water-soluble eosin staining solution for 2 minutes, rinsed with tap water for 30 seconds, and finally the sections were rinsed, dehydrated with absolute ethanol, cleared with xylene, air-dried, and sealed with neutral gum, and the number of cell nuclei was counted under the microscope.

[0038] As Figure 3As shown, the average number of cell nuclei in the control group, near-freezing temperature group, and 37°C group were 121, 97, and 41, respectively. Compared with the control group, the number of cells in the near-freezing temperature group and 37°C group both decreased. The average number of cell nuclei in the 37°C group decreased by 66%. In contrast, the average number of cell nuclei in the near-freezing temperature group only decreased by 20%. This result may be attributed to the fact that the near-freezing temperature condition can significantly inhibit the autolysis of brain tissue and effectively retain the organizational structure information. An increase in temperature (room temperature) will cause rapid autolysis of brain tissue. The above results indicate that compared with the 37°C condition, staining at near-freezing temperature balances the penetration of the staining solution, labeling, and tissue autolysis, and can efficiently label the brain tissue structure.

[0039] Example 4 Comparison of the Golgi neuron staining effects of human brain tissue with delayed death and frozen at different temperatures.

[0040] The human brain tissue with delayed death and frozen was thawed at room temperature and trimmed to a thickness of 3 mm. The human brain tissue was immediately immersed in a staining solution composed of 1% potassium dichromate, 1% potassium chromate, and 1% mercury chloride solution. The temperature during staining was set at near-freezing temperature, 4°C, 26°C, and 37°C respectively, and protected from light. Fresh staining solution was replaced on the 1st, 8th, 15th, 22nd, and 28th days of staining, and the staining duration was 30 days. After the staining was completed, the human brain tissue was dehydrated with sucrose, embedded in OCT, frozen sectioned, air-dried overnight, reduced with ammonia water solution, dehydrated with gradient alcohol (30%, 50%, 70%, 80%, 90%, 100%), cleared, and mounted. Finally, images were taken under an optical microscope.

[0041] As Figure 4 shown, staining and labeling of the neuron structure of human brain tissue with delayed death and frozen can be achieved at near-freezing temperature, 4°C, 26°C, and 37°C. As the temperature increases, the number of neuron cells in the same imaging field of view gradually decreases. The average number of neuron cell bodies in the near-freezing temperature group, 4°C group, 26°C group, and 37°C group were 22, 18, 6, and 4 respectively. Compared with the 37°C group, the number of neuron cell bodies in the near-freezing temperature group increased by 5.5 times. This shows that low-temperature staining conditions (such as the near-freezing temperature group) have significant advantages in maintaining the integrity of the brain tissue structure. Low temperature can slow down tissue autolysis, retain the neuron structure, and can achieve efficient labeling of the neuron structure of brain tissue.

[0042] Example 5 Comparison of the neuron staining effects of staining solutions containing different components on human brain tissue with delayed death.

[0043] The human brain tissue with delayed death was frozen and thawed at room temperature and trimmed to a thickness of 3 mm. The human brain tissue was immediately immersed in staining solutions A, B, C, and D respectively, in the dark at a temperature near the freezing point. After staining, the human brain tissue was dehydrated with sucrose, embedded in OCT, cryosectioned, air-dried overnight, dehydrated with gradient alcohol (30%, 50%, 70%, 80%, 90%, 100%), cleared, mounted, and finally imaged under an optical microscope. Staining solution A consists of potassium dichromate and silver nitrate solution. The human brain tissue was immersed in 1.25% potassium dichromate for 10 days, and after the immersion, it was finally immersed in 1% silver nitrate solution for 20 days; Staining solution B consists of potassium dichromate, silver nitrate, and osmium tetroxide solution. The human brain tissue was immersed in a mixed solution of 2% potassium dichromate and 0.16% osmium tetroxide for 10 days, and finally immersed in 1% silver nitrate solution for 20 days; Staining solution C consists of glutaraldehyde, potassium dichromate, and silver nitrate solution. The human brain tissue was immersed in a mixed solution of 5% glutaraldehyde and 4% potassium dichromate for 10 days, and finally immersed in 1% silver nitrate solution for 20 days; Staining solution D consists of potassium dichromate, potassium chromate, and mercury chloride solution. The human brain tissue was immersed in 1% potassium dichromate, 1% potassium chromate, and 1% mercury chloride solution for 30 days, and fresh staining solutions were replaced on the 1st, 8th, 15th, 22nd, and 28th days of staining respectively. After staining, the human brain tissue was dehydrated with sucrose, embedded in OCT, cryosectioned, air-dried overnight, reduced with ammonia water solution (only used for staining solution D), dehydrated with gradient alcohol (30%, 50%, 70%, 80%, 90%, 100%), cleared, mounted, and finally imaged under an optical microscope.

[0044] As Figure 5 shown, the use of components A, B, C, and D can all achieve the staining and labeling of the neuronal structure of the human brain tissue with delayed death. However, under the same imaging field of view, the average number of cell bodies in groups A, B, and C is 4, 3, and 5 respectively, while the average number of cells in component D is as high as 22. This shows that under the condition of a temperature near the freezing point, using component D (potassium dichromate, potassium chromate, and mercury chloride solution) to stain the human brain can significantly increase the number of stained neurons.

[0045] Example 6 Golgi neuron staining method at a temperature near the freezing point and its application in X-ray imaging of human brain tissue.

[0046] The synchrotron radiation X-ray imaging of human brain tissue was carried out on the brain imaging beamline of the photon source. For large-field two-dimensional imaging, the paraffin-embedded tissue sample was placed on the sample stage and scanned with an imaging energy of 14 keV. The scanning boundaries were set according to the length and width of the tissue before imaging, and systematic automatic sequential imaging from left to right and from top to bottom was achieved to ensure full coverage of the tissue block. Then the obtained image data was stitched using Fiji ImageJ software.

[0047] AsFigure 6 As shown, two-dimensional large-field scanning imaging of near-freezing-point temperature Golgi-stained human brain tissue with dimensions of 1.5 cm × 1 cm in length and width was achieved using synchrotron radiation X-ray imaging technology. From the imaging results, we can observe that the cerebellar tissue section has a complete structure, and there are a large number of neuron cells and nerve fibers in the tissue. At the same time, different brain regions can be further divided and distinguished.

[0048] The above are only the preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the content of the specification of the present invention application fall within the scope of the claims of the present invention patent. Those not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for staining human brain tissue and synchrotron radiation X-ray imaging at near-freezing temperatures, characterized in that, It includes the following steps: 1) Prepare Golgi staining solution; 2) Thaw the cryopreserved human brain tissue with delayed death; 3) Immerse the human brain tissue obtained in step 2) in the Golgi staining solution prepared in step 1) and stain at a temperature near the freezing point; 4) Visualize the neurons in the human brain tissue obtained in step 3); 5) Dehydrate, clear, and embed the human brain tissue obtained in step 4); 6) Image and observe under a synchrotron radiation X-ray microscope.

2. The method according to claim 1, characterized in that, There are four types of Golgi staining solutions in step 1), namely: 1) 1.25% dichromic acid and 1% silver nitrate; 2) 1% potassium dichromate, 1% mercury chloride, and 1% potassium chromate; 3) 5% glutaraldehyde, 4% potassium dichromate, and 1% silver nitrate; 4) 0.16% osmium acid, 2% potassium dichromate, and 1% silver nitrate.

3. The method according to claim 1, wherein The human brain tissue in step 2) includes any brain region of the human brain.

4. The method according to claim 1, wherein The human brain tissue in step 2) is cryopreserved human brain tissue with delayed death, and the environmental temperature range for cryopreservation is -20°C to -196°C.

5. The method according to claim 1, wherein In step 3), the human brain tissue is immersed in the Golgi staining solution at a temperature near the freezing point, and the temperature environment near the freezing point is provided by an ice-water bath with an ice content of 10% to 90%, and the immersion time is 4 - 180 days.

6. The method according to claim 1, wherein The developing solution used in step 4) is selected from: lithium hydroxide solution, ammonia water solution, sodium thiosulfate solution, or ammonium carbonate solution.

7. The method according to claim 1, characterized in that, The developing time of the brain tissue in step 4) is 1 - 1440 minutes, and for human brain tissues with different thicknesses, the developing time of their neurons is different.

8. The method according to claim 1, characterized in that, The dehydration in step 5) is carried out by using gradient alcohol.

9. The method according to claim 1, wherein The tissue embedding technique in step 5) includes: paraffin embedding technique, resin embedding technique, or hydrogel embedding technique.

10. The method according to claim 1, characterized in that, The energy for imaging and observing with the synchrotron radiation X-ray microscope in step 5) is 280 eV to 40 keV.

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