A method of cochlear tissue transparency based on hydration

Abstract

The application discloses a cochlea tissue transparentization method based on hydration, relates to the field of biological tissue transparentization, and comprises the following steps: fixing a cochlea sample by using a paraformaldehyde solution; carrying out decalcification treatment on the fixed cochlea sample by using a disodium ethylenediaminetetraacetate solution; carrying out defatting treatment on the decalcified cochlea sample by using a mixed solution of a detergent and an amino alcohol; carrying out hydration and transparentization treatment on the defatted cochlea sample by using a urea solution; and carrying out oscillation and washing by using a phosphate buffered saline solution, and then sequentially adding a phosphate Tween buffer, a blocking solution, a primary antibody and a secondary antibody, carrying out permeation treatment, blocking treatment, incubation and light-proof incubation, carrying out immunofluorescence labeling and staining, and finally carrying out refractive index matching by using a refractive index matching liquid to obtain transparentized cochlea tissue. The original three-dimensional anatomical structure of the cochlea is perfectly reserved, and the optical permeability of the cochlea tissue is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of biological tissue transparency, and more particularly to a method and system for cochlear tissue transparency based on hydration. Background Technology

[0002] Globally, more than 460 million people suffer from hearing loss due to irreversible damage to inner ear hair cells. As the core unit of auditory transduction, the spatial arrangement and distribution of hair cells and their connection patterns with nerve fibers are of paramount scientific importance for studying the mechanisms of hearing loss and for drug screening.

[0003] However, due to its enclosed position within the rigid temporal bone and its intricate internal structure and high lipid content, large-scale in-situ three-dimensional imaging of the cochlear tissue is extremely challenging. Currently, characterization of cochlear hair cells faces the following major bottlenecks: First, traditional cochlear implantation requires mechanically stripping bone and cutting the cochlear duct, a process that inevitably damages the spiral topology of the cochlea, resulting in the loss of spatial arrangement information of hair cells.

[0004] Second, while traditional tissue sectioning techniques can observe local microstructures, they are difficult to reconstruct the three-dimensional distribution of a complete hair cell array.

[0005] Third, current tissue clearing techniques have significant drawbacks when treating the cochlea. For example, while organic solvent clearing techniques offer high transparency, they can easily cause severe tissue shrinkage and rapid quenching of fluorescence signals. Conventional hydrophilic clearing reagents, due to limitations in osmotic pressure and molecular diffusion, are inefficient at clearing dense ossified tissues, have long processing cycles, and often require the preparation of high-concentration refractive index matching solutions, resulting in high costs and complex operations. Summary of the Invention

[0006] The purpose of this invention is to provide a method for cochlear tissue transparency based on hydration, which solves the problems of tissue topological structure destruction and mechanical damage caused by traditional smearing techniques, as well as the problems of long processing cycles, easy quenching of fluorescence signals, and severe tissue deformation in existing transparency techniques.

[0007] To achieve the above objectives, the present invention provides a method for cochlear tissue transparency based on hydration, comprising the following steps: S1. Obtain a cochlear sample and fix the cochlear sample with paraformaldehyde solution; S2. Decalcification of the fixed cochlear samples was performed using disodium ethylenediaminetetraacetate solution. S3. A mixed solution of detergent and amino alcohol was used to degrease the decalcified cochlear sample. S4. Use urea solution to perform hydration and transparency treatment on the degreased cochlear sample, and use phosphate buffer solution to shake and wash the hydration and transparency treatment cochlear sample. S5. Add phosphate Tween buffer, blocking solution, primary antibody and secondary antibody in sequence. Perform immunofluorescence staining on the cochlear sample after shaking and washing by permeation treatment, blocking treatment, incubation and incubation in the dark. S6. The refractive index of the immunofluorescence-labeled cochlear samples was matched using a refractive index matching solution to obtain transparent cochlear tissue.

[0008] Preferably, the equivalent concentration of formaldehyde in the paraformaldehyde (PFA) solution is 4%; the fixation treatment is performed at a temperature of 3℃-5℃ for 12h-24h.

[0009] This application uses PFA solution to cross-link tissue proteins, locking the original spatial positions of hair cells, nerve fibers, and supporting cells in the cochlea, ensuring that the tissue structure does not disintegrate or deform during subsequent drastic chemical treatment.

[0010] Preferably, the concentration of the ethylenediaminetetraacetic acid (EDTA) solution is 0.3M-0.7M; the decalcification treatment is performed by shaking for 12-24 hours.

[0011] The cochlea is surrounded by a hard temporal bone (high in calcium). EDTA solution softens the bone by chelating calcium ions, thereby eliminating severe light scattering caused by bone salts and establishing a physical channel for subsequent clearing reagents and antibodies to enter the cochlear duct.

[0012] Preferably, the mixed solution of detergent and amino alcohol is a hydrophilic tissue clearing agent, the volume percentage concentration of the detergent is 5%-20%, and the mass-volume percentage concentration of the amino alcohol is 10%-35%; the degreasing treatment includes two consecutive shaking soaking treatments, each shaking soaking treatment lasting 12h-36h.

[0013] This application utilizes a two-cycle treatment with a mixed solution of detergent and amino alcohol to deeply remove lipids from cell membranes and myelin sheaths. Lipids are the largest source of scattering in tissues; degreasing not only improves transparency but also significantly increases tissue porosity, facilitating the subsequent penetration of macromolecular antibodies.

[0014] Preferably, the detergent is at least one of polyethylene glycol octylphenyl ether, polysorbate-20, and sodium dodecyl sulfate; and the amino alcohol is at least one of N,N,N',N'-tetra(2-hydroxypropyl)ethylenediamine, N-butyldiethanolamine, and triethanolamine.

[0015] Preferably, the concentration of urea in the urea solution is 4M-8M; the hydration and transparency treatment time is 12h-36h; and the shaking and washing is performed 3 times, with each shaking and washing lasting 2h.

[0016] This application utilizes urea solution to induce hyperhydration, causing partial breakage of hydrogen bonds in protein molecules within the tissue and the binding of a large amount of water. This significantly reduces the refractive index (RI) of the proteins, bringing them closer to an aqueous environment (RI≈1.33), achieving "inside-out" transparency. The highly permeable treatment solution is then removed by agitation and washing, restoring the tissue to a physiological pH environment suitable for immune response.

[0017] Preferably, the volume percentage concentration of polyethylene glycol octylphenyl ether in the phosphate Tween buffer is 0.3%-2%.

[0018] Preferably, the blocking solution comprises bovine serum albumin and polyethylene glycol octylphenyl ether; the mass-volume percentage concentration of bovine serum albumin in the blocking solution is 1%-10%, and the volume percentage concentration of polyethylene glycol octylphenyl ether is 0.1%-1%.

[0019] Preferably, the primary antibody is an antibody capable of specifically binding to the target antigen in the cochlear tissue sample; the secondary antibody is an antibody capable of specifically binding to the primary antibody and labeled with a fluorescent dye. The antibody can overcome the barrier of the cochlear duct wall and uniformly penetrate into the hair cell layer, achieving highly specific in-situ labeling of the target antigen in the cochlear tissue sample.

[0020] Preferably, the refractive index matching fluid comprises: a high-refractive-index sugar or a polyol; the high-refractive-index sugar is at least one selected from sucrose, fructose, and trehalose; the polyol is at least one selected from glycerol, sorbitol, and xylitol. By performing final refractive index matching with the refractive index matching fluid, the refractive index difference between the residual tissue background and the medium is eliminated, making the cochlea almost completely transparent, thereby enabling deep scanning at the hundreds of micrometers or even millimeters level.

[0021] In summary, the cochlear tissue transparency method based on hydration provided by this invention has the following advantages compared to traditional techniques: (1) This application eliminates the need for mechanical dissection of the cochlear duct or manual flattening of the spiral structure, thus avoiding mechanical damage to fragile hair cells caused by physical manipulation. It solves the problems of tissue topological structure destruction and mechanical damage caused by traditional smearing techniques, as well as the long processing cycle, easy quenching of fluorescence signals, and severe tissue deformation of existing transparentization techniques. Experimental results show that the processed cochlear samples completely retain the spiral topological structure from the base to the apex of the cochlea, perfectly preserving the original three-dimensional anatomical structure of the cochlea. It achieves in-situ spatial characterization of hair cells, spiral ganglia, and basilar membrane, significantly reducing the rate of missed diagnosis and misdiagnosis.

[0022] (2) The present invention utilizes the hydration effect induced by urea solution to actively reduce the refractive index of proteins in the tissue and match them with the aqueous environment. Combined with the final matching of the refractive index matching solution, the optical permeability of the cochlear tissue is significantly improved.

[0023] (3) Traditional fixed cochlear imaging depth is usually less than 50 μm, while the transparent cochlear tissue obtained in this application can achieve clear imaging of the complete cochlear duct (depth can reach 500 μm-1000 μm or more) through confocal microscopy, and can acquire continuous three-dimensional images of hair cell array in one stop; and the scattering of background light is reduced by more than 80%, which greatly improves the contrast and resolution of deep tissue imaging.

[0024] (4) This application shortens the whole cochlear transparency cycle from 2 weeks to 4-5 days, which is easy to promote in standardized laboratories. The chemical reagents used are low-cost, and there is no need to purchase expensive electrophoresis transparency equipment, which greatly reduces the research threshold and funding expenditure for whole organ transparency imaging.

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0026] Figure 1 This is a flowchart of a method for making cochlear tissue transparent based on hydration in this invention; Figure 2 This is a diagram of the transparent cochlear sample in Embodiment 1 of the present invention under a confocal microscope; Figure 3 This is a diagram of the cochlea under a confocal microscope in Comparative Example 1 of the present invention, showing the condition of a conventionally treated cochlea. Detailed Implementation

[0027] The technical method of the present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specifically stated, the relative arrangement and numerical values ​​of the steps in these embodiments do not limit the scope of this application.

[0028] The following description of at least one exemplary embodiment is merely illustrative and is not intended to limit the scope of this application or its application or use.

[0029] In all the examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0030] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0031] This invention provides a method for making cochlear tissue transparent based on hydration, such as... Figure 1 As shown, it includes the following steps: S1. Obtain cochlear samples and fix them with PFA solution at 3℃-5℃ for 12h-24h. The equivalent concentration of formaldehyde in the PFA solution is 4%.

[0032] S2. Decalcification of the fixed cochlear samples was performed using disodium EDTA solution. The concentration of the EDTA solution was 0.3M-0.7M. Decalcification was performed by shaking for 12-24 hours.

[0033] S3. A mixed solution of detergent and amino alcohol was used to degrease the decalcified cochlear samples. The mixed solution of detergent and amino alcohol was a hydrophilic tissue clearing agent, with the detergent concentration at 5%-20% by volume and the amino alcohol concentration at 10%-35% by mass. The detergent was at least one of polyethylene glycol octylphenyl ether, polysorbate-20, and sodium dodecyl sulfate; the amino alcohol was at least one of N,N,N',N'-tetra(2-hydroxypropyl)ethylenediamine, N-butyldiethanolamine, and triethanolamine. The degreasing treatment included two consecutive shaking soaking treatments, each lasting 12-36 hours.

[0034] S4. The degreased cochlear samples were hydrated and cleared using a urea solution, and then washed with a phosphate buffer solution using agitation. The concentration of urea in the urea solution was 4M-8M. The hydration and clearing treatment time was 12h-36h. The samples were washed three times, with each washing session lasting 2h.

[0035] S5. Phosphate-buffered solution with Tween 20 (PBST) buffer, blocking solution, primary antibody, and secondary antibody were added sequentially. The cochlear samples were then subjected to immunofluorescence staining after osmosis, blocking, incubation, and incubation in the dark.

[0036] The PBST buffer contains 0.3%-2% (v / v) polyethylene glycol octylphenyl ether (Triton X-100). The blocking solution consists of bovine serum albumin (BSAL) and PEG-octylphenyl ether, with BSAL at 1%-10% (v / v) and PEG-octylphenyl ether at 0.1%-1% (v / v). The primary antibody is an antibody that specifically binds to the target antigen in the cochlear tissue sample. The secondary antibody is an antibody specifically binding to the primary antibody and labeled with a fluorescent dye.

[0037] S6. The refractive index of the immunofluorescence-labeled cochlear sample was matched using a refractive index matching solution to obtain transparent cochlear tissue. The refractive index matching solution included: a high-refractive-index sugar or a polyol. The high-refractive-index sugar was at least one of sucrose, fructose, and trehalose. The polyol was at least one of glycerol, sorbitol, and xylitol.

[0038] The specific embodiments provided by this invention are as follows: Example 1 A method for making cochlear tissue transparent based on hydration includes the following steps: S1. Obtain cochlea samples from 4-week-old C57BL / 6 mice, place them in a PFA solution with an equivalent concentration of 4% formaldehyde, and fix them by standing in a refrigerator at 4°C. After 16 hours, remove the samples and rinse them with PBS buffer to remove excess PFA solution.

[0039] S2. Place the rinsed cochlear sample into a 0.5M EDTA disodium solution (the pH has been adjusted to 8.0 with NaOH), and shake it on a horizontal shaker at room temperature at 60 rpm. Observe it every 4 hours for 12 hours until the bone of the cochlear sample is completely softened. It can be removed by gently pressing it with the tip of tweezers until it does not feel stiff. Use PBS buffer to shake and wash the sample. Change the washing solution every 2 hours for a total of 3 times to completely remove the residual EDTA disodium solution.

[0040] S3. Place the cleaned cochlear sample into the hydrophilic tissue clearing reagent in the Nuohai hydrophilic tissue clearing kit. The hydrophilic tissue clearing reagent includes 15% polyethylene glycol octylphenyl ether (V / V) and 25% N,N,N',N'-tetra(2-hydroxypropyl)ethylenediamine (W / V). Then, continuously shake at room temperature for 24 hours. After observing that the tissue is slightly transparent and some lipids have been washed away, replace with fresh hydrophilic tissue clearing reagent and shake again for 24 hours to ensure that the complex membrane labyrinth lipids inside the cochlea are completely extracted, and obtain a degreased cochlear sample.

[0041] S4. Immerse the degreased cochlear sample in a 6M urea solution and shake at room temperature for 24 hours. During this process, urea molecules enter the protein molecules and break hydrogen bonds. The proteins absorb water and swell, causing their refractive index to decrease. Then, remove the cochlear sample and wash it three times with PBS buffer for 2 hours each time to remove the high concentration of denaturing agent (urea solution) and restore the physiological environment.

[0042] S5. First, place the washed cochlear sample in PBST buffer (PBST buffer contains 1% Triton X-100 by volume) and shake for 1 hour to increase tissue permeability. Then, block with a blocking solution containing bovine serum albumin and Triton X-100 (bovine serum albumin is 5% by volume and Triton X-100 is 0.3% by volume) at room temperature for 2 hours. Next, add a primary antibody targeting hair cell-specific proteins (such as Myosin VIIa), and incubate at 4°C with shaking for 24 hours. Then, wash three times with PBS buffer for 2 hours each time. Next, add the corresponding fluorescent secondary antibody (which can specifically bind to the primary antibody, such as Alexa Fluor 488), and incubate at room temperature with shaking in the dark for 12 hours. Then, wash three times with PBS buffer for 2 hours each time to achieve immunofluorescence staining.

[0043] S6. The cochlear samples stained with immunofluorescence were placed in a refractive index matching solution and shaken in the dark for 24 hours to achieve refractive index equilibration. The refractive index matching solution was a sucrose solution with a mass-volume percentage concentration of 60%. The treated cochlea was in a nearly glassy, ​​completely transparent state, and a transparent cochlear sample was obtained.

[0044] Comparative Example 1 Four-week-old C57BL / 6 mouse cochleas were used as cochlear samples. After fixation and decalcification treatment according to S1 and S2 in Example 1, the hydrophilic tissue clearing and degreasing treatment in S3, the urea hydration and clearing treatment in S4, and the refractive index matching treatment in S6 were not performed. Immunofluorescence staining under the same conditions as in S5 of Example 1 was performed to obtain ordinary treated cochleas.

[0045] The transparent cochlear sample from Example 1, along with the refractive index matching solution, was placed in a confocal culture dish and subjected to three-dimensional stacking scanning using a confocal microscope. Figure 2 As shown, the following phenomena can be observed under a confocal microscope: In Example 1, the transparent cochlear sample showed that the entire cochlear spiral tube structure was intact, and the hair cells on the basilar membrane were arranged in a clear "three outer and one inner" array without any mechanical or physical damage. Furthermore, the laser could easily penetrate deep into the cochlear tube, and without slicing, the imaging depth could cover the entire diameter of the cochlear tube (approximately 400-600 μm). The signal attenuation rate was reduced by 75% compared to the untreated sample (cochlea of ​​4-week-old C57BL / 6 mice). At the same time, the spatial orientation of the cilia above the hair cells could be clearly distinguished, the image background was clean, and the signal-to-noise ratio was extremely high.

[0046] Three-dimensional stacked scanning of the conventionally treated cochlea in Comparative Example 1 was performed using a confocal microscope, such as... Figure 3 As shown. From Figure 3 As observed, the cochlea remains noticeably opaque or semi-transparent under conventional treatment, with strong background scattering from the bony labyrinth and membranous labyrinth structures, making it difficult for the laser to effectively penetrate deep into the cochlear duct. Only localized hair cell signals can be obtained on the sample surface, with rapid decay of fluorescence signals in deeper regions. The continuity of the basilar membrane and the complete "three-outer-one-inner" array of hair cells are difficult to track. Compared to Example 1, Comparative Example 1 cannot achieve clear and continuous three-dimensional imaging at the scale of the entire cochlea, indicating that the hydrophilic translucency, hydration transparency, and refractive index matching steps of this invention can significantly improve the optical translucency and deep imaging quality of cochlear tissue.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for making cochlear tissue transparent based on hydration, characterized in that, Includes the following steps: S1. Obtain a cochlear sample and fix the cochlear sample with paraformaldehyde solution; S2. Decalcification of the fixed cochlear samples was performed using disodium ethylenediaminetetraacetate solution. S3. A mixed solution of detergent and amino alcohol was used to degrease the decalcified cochlear sample. S4. Use urea solution to perform hydration and transparency treatment on the degreased cochlear sample, and use phosphate buffer solution to shake and wash the hydration and transparency treatment cochlear sample. S5. Add phosphate Tween buffer, blocking solution, primary antibody and secondary antibody in sequence. Perform immunofluorescence staining on the cochlear sample after shaking and washing by permeation treatment, blocking treatment, incubation and incubation in the dark. S6. The refractive index of the immunofluorescence-labeled cochlear samples was matched using a refractive index matching solution to obtain transparent cochlear tissue.

2. The method for cochlear tissue transparency based on hydration according to claim 1, characterized in that, The equivalent concentration of formaldehyde in the paraformaldehyde solution is 4%; the fixation treatment is performed at a temperature of 3℃-5℃ for 12h-24h.

3. The method for cochlear tissue transparency based on hydration according to claim 1, characterized in that, The concentration of the ethylenediaminetetraacetic acid solution is 0.3M-0.7M; the decalcification treatment is carried out by shaking for 12-24 hours.

4. The method for cochlear tissue transparency based on hydration according to claim 1, characterized in that, The mixture of detergent and amino alcohol is a hydrophilic tissue clearing agent. The volume percentage concentration of the detergent is 5%-20%, and the mass-volume percentage concentration of the amino alcohol is 10%-35%. The degreasing treatment includes two consecutive shaking and soaking treatments, each lasting 12-36 hours.

5. The method for cochlear tissue transparency based on hydration according to claim 4, characterized in that, The detergent is at least one of polyethylene glycol octylphenyl ether, polysorbate-20, and sodium dodecyl sulfate; the amino alcohol is at least one of N,N,N',N'-tetra(2-hydroxypropyl)ethylenediamine, N-butyldiethanolamine, and triethanolamine.

6. The method for cochlear tissue transparency based on hydration according to claim 1, characterized in that, The concentration of urea in the urea solution is 4M-8M; the hydration and transparency treatment time is 12h-36h; the shaking and washing is performed 3 times, with each shaking and washing lasting 2h.

7. The method for cochlear tissue transparency based on hydration according to claim 1, characterized in that, The volume percentage concentration of polyethylene glycol octylphenyl ether in the phosphate Tween buffer is 0.3%-2%.

8. The method for cochlear tissue transparency based on hydration according to claim 1, characterized in that, The blocking solution comprises bovine serum albumin and polyethylene glycol octylphenyl ether; the mass-volume percentage concentration of bovine serum albumin in the blocking solution is 1%-10%, and the volume percentage concentration of polyethylene glycol octylphenyl ether is 0.1%-1%.

9. The method for cochlear tissue transparency based on hydration according to claim 1, characterized in that, The primary antibody is an antibody that can specifically bind to the target antigen in the cochlear tissue sample; the secondary antibody is an antibody that can specifically bind to the primary antibody and is labeled with a fluorescent dye.

10. The method for cochlear tissue transparency based on hydration according to claim 1, characterized in that, The refractive index matching liquid includes: high refractive index sugars or polyols; the high refractive index sugars are at least one of sucrose, fructose, and trehalose; the polyols are at least one of glycerol, sorbitol, and xylitol.

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