Batch processing system and processing method for brain-spinal cord samples

Abstract

The invention belongs to the technical field of experimental devices, and provides a batch processing system and method for brain-spinal cord samples. The system comprises a brain-spinal cord sample fixing device, an internal processing box and an external processing box, the fixing device comprises a base, a brain fixing groove and a spinal cord fixing groove, wherein the brain fixing groove and the spinal cord fixing groove are formed in the base and communicate with each other. A frame is arranged in the internal processing box and comprises a plurality of guide rails for embedding the fixing device; and the external processing box is used for accommodating the internal processing box so as to perform unified reagent processing on the whole sample group. The invention also discloses a method for batch processing of brain-spinal cord samples by using the system. According to the invention, by designing the modularized sample bearing unit and the reagent treatment outer box, synchronous and standardized treatment of a plurality of samples is realized. A plurality of samples can be processed at the same time, and the processing efficiency is greatly improved; the height consistency between samples is ensured, batch errors are eliminated to the maximum extent, and the data comparability is ensured; and human intervention and injury are reduced to the greatest extent.

Description

Technical Field

[0001] This disclosure relates to the field of experimental apparatus technology, and in particular to a batch processing system and method for brain-spinal cord samples. Background Technology

[0002] In neuroscience research, to obtain statistically significant findings, it is often necessary to perform three-dimensional imaging and circuit reconstruction of brain-spinal cord tissue from a large number of experimental animals to compare differences between different groups, such as normal groups, disease model groups, and treatment groups. Therefore, high-throughput, high-quality preparation of multiple brain-spinal cord samples is a prerequisite for quantitative neuroanatomy research. Currently, methods for whole-segment embedding of long segments of brain and spinal cord tissue are basically based on a single-sample manual operation mode. The typical process is as follows: 1. Individual processing: The operator needs to fix, dehydrate, clear, and embed each isolated brain-spinal cord sample individually; 2. Sequential processing: Due to the lack of suitable batch carrying tools, these steps can only be performed on samples one by one, or large containers are used for mixed placement, resulting in samples being squeezed together and labeled incorrectly; 3. Manual transfer: At each processing step, such as when changing to different concentrations of fixative, individual samples need to be transferred from one reagent bottle to another by hand or tweezers. The above operating procedure has several problems: 1. Extremely low processing efficiency: The time required to process N samples sequentially is approximately N times the time required to process a single sample, which cannot meet the needs of large-scale studies and severely restricts the experimental progress. 2. Inconsistent processing conditions between samples: Even when using the same reagents and procedures, because samples are processed sequentially, the soaking time and concentration of reagents for each sample will differ, introducing batch effects. 3. Samples are easily damaged during operation: Frequent manual handling and transfer of slender spinal cord tissue can easily lead to physical damage to the tissue, label detachment, or sample confusion. 4. High reagent consumption: Frequent reagent changes to process a small number of samples, or the use of excessively large containers to hold multiple samples, both result in reagent waste.

[0003] Therefore, there is an urgent need for an integrated system that can standardize and process brain-spinal cord samples in batches, including fixation, dehydration and embedding, to solve problems such as low efficiency, poor consistency and easy damage. Summary of the Invention

[0004] This disclosure provides a batch processing system and method for brain-spinal cord samples to at least solve the above-mentioned technical problems existing in the prior art.

[0005] According to a first aspect of this disclosure, a batch processing system for brain-spinal cord samples is provided, including a fixation device for brain-spinal cord samples, an internal processing box, and an external processing box. The fixation device includes a base, a brain fixation groove and a spinal cord fixation groove disposed on the base, wherein the brain fixation groove and the spinal cord fixation groove are in communication; the brain fixation groove is a biomimetic groove adapted to the lateral anatomical contour of the brain, used to accommodate the side of the brain and expose the brainstem terminal; the spinal cord fixation groove is a spiral biomimetic groove adapted to the lateral anatomical contour of the spinal cord, used to accommodate the side of the spinal cord and maintain a natural connection angle between the spinal cord and the brainstem terminal. The internal processing box is provided with a frame, which includes several guide rails for mounting several of the fixing devices. The external processing box is used to house the internal processing box for uniform reagent processing of the entire sample group.

[0006] Specifically, the integrated connectivity and anatomically matched design of the brain fixation slot and spinal cord fixation slot disclosed herein ensures that the brain and spinal cord maintain a natural connection angle immediately after detachment, allowing for overall fixation without severance and ensuring the continuity of long-range neural circuits. The internal processing box integrates and carries multiple samples fixed by the fixation device, while the external processing box performs unified reagent processing on the entire sample group. When changing reagents, simply lift the entire internal processing box, discard the old reagent, and add the new reagent; a single operation completes reagent replacement for all samples, greatly improving efficiency and reducing human error. This architecture enables high-throughput processing without sacrificing the morphological preservation of individual samples.

[0007] Specifically, the brain-spinal cord samples mentioned above are brain-spinal cord samples from experimental animals, and are not limited to a single type of animal, such as brain-spinal cord samples from mice, monkeys, rabbits, dogs, etc. Correspondingly, the shape and size of the fixation device for the brain-spinal cord samples are designed according to the anatomical morphology and size of the brain and spinal cord of the selected animal, and are not specifically limited here.

[0008] In one embodiment, a transition arc is provided at the connection between the brain fixation groove and the spinal cord fixation groove. The radius of the transition arc is 2-3 mm, which is used to smoothly connect the end of the brainstem of the brain with the beginning of the spinal cord, so as to avoid the bending of nerve fibers at the connection site.

[0009] In one embodiment, the base, the brain fixation groove, and the spinal cord fixation groove are integrally molded, and the integral molding method includes any one of precision injection molding and CNC milling.

[0010] Specifically, if precision injection molding is chosen, the shape of the brain fixation groove and spinal cord fixation groove is designed as a mold cavity, and the grooved base is directly injection molded, with no splicing gap between the groove and the base body. If CNC milling is chosen, the overall base is first machined, and then grooves matching the anatomical contours of the brain and spinal cord are milled on the surface of the base using a precision milling cutter.

[0011] Specifically, the integrated molding process is used because: (1) the brain fixation slot and the spinal cord fixation slot are directly molded on the base, and their relative positions are guaranteed by the processing precision, avoiding the brain-spinal cord misalignment caused by assembly errors in traditional split-joint slots. (2) The seamless integrated structure can prevent the fixation slot from falling off or shifting during sample processing, and at the same time prevent reagent residues in the joints from causing sample contamination, meeting the standardization and cleanliness requirements of experimental operations. (3) No additional installation / calibration of the fixation slot is required. Experimenters can directly place brain and spinal cord samples into the preset grooves, reducing operational errors that rely on experience in traditional manual placement and improving the degree of standardization.

[0012] In one embodiment, the base is a mesh structure with square or regular hexagonal mesh holes.

[0013] In one embodiment, the fixation device further includes a snap-fit ​​fixation system, which is arranged along the trajectory of the spinal cord fixation groove to confine the spinal cord within the spinal cord fixation groove; the snap-fit ​​fixation system includes a plurality of snaps.

[0014] Specifically, the snap-fit ​​fixation system is designed to stably confine the spinal cord sample within the fixation groove using restraint pressure, preventing it from floating, entangled, rotating, or structurally damaged during fixation, dehydration, and other processes. It also adapts to the anatomical morphology of the spinal cord sample. The number of snaps depends on the specific spinal cord sample; longer samples require more snaps, while shorter samples require fewer, offering flexibility.

[0015] In one embodiment, the snap-fit ​​system is arranged along the radial trajectory of the spinal cord fixation groove.

[0016] Specifically, the radial trajectory of the spinal cord fixation groove refers to the ray direction extending outward from the center of the spiral. Each spiral groove has its corresponding radial ray, and these rays together constitute the radial trajectory system of the spiral groove. For the spinal cord fixation groove (spiral groove) in this disclosure, the movement tendency of the spinal cord is to slide around the spiral axis and move laterally along the spiral radial direction. Therefore, the snap-fit ​​fixation system is set along the radial trajectory (restraining lateral movement), which, together with the spiral shape of the groove itself (restraining around the sliding), perfectly forms a complete constraint on the spiral trajectory.

[0017] In one embodiment, the buckle includes a positioning support unit, an elastic constraint unit, and an adapter connection unit; the positioning support unit is connected to the base and is used to fix the buckle on the base; the elastic constraint unit is used to provide elastic pressure to confine the spinal cord within the spinal cord fixation groove; the adapter connection unit is used to connect the positioning support unit and the elastic constraint unit.

[0018] Specifically, the buckle includes any one of the following: a press-type elastic buckle, a flexible silicone strip buckle, and a magnetic elastic buckle.

[0019] Specifically, the structure of the snap fastener is not limited to one type, as long as it meets the following characteristics: (1) The constraint range of the snap fastener must strictly match the trajectory of the spinal cord fixation groove to ensure that the spinal cord is confined within the groove throughout the entire process, with no space to escape from the groove. (2) The constraint pressure on the spinal cord must be controlled within a range that does not damage the spinal cord structure or compress the axons, and the pressure distribution must be uniform with no local excessive compression. (3) Throughout the entire process of tissue fixation, dehydration, and clearing, the snap fastener must maintain its constraint effect to prevent the spinal cord from shifting due to reagent buoyancy or operational vibration. (4) The snap fastener structure must be compatible with the grid design of the base, not obstructing the grid pores, and not affecting reagent penetration.

[0020] Specifically, when the buckle is a press-type elastic buckle, the positioning support unit includes a sliding block disposed at the bottom of the buckle body and a sliding track disposed on the base; the elastic constraint unit includes a pressure adjusting spring and a pressure head disposed inside the buckle body; the adapter connection unit includes a knob, which is mechanically coupled to the pressure adjusting spring and is used to change the compression of the pressure adjusting spring to adjust the pressure applied to the spinal cord.

[0021] The aforementioned buckle body is a rigid, integrally molded component. All functional parts are directly mounted on its surface or internally, without undergoing elastic deformation; it only serves as a support and force transmission mechanism. Through the cooperation of the sliding block and sliding track, the buckle can move flexibly along the track while ensuring precise relative positioning between the buckle and the spinal cord fixation groove. The pressure adjusting spring is the core source of elastic force; the elastic force generated by its deformation is transmitted to the pressure head through the buckle body, forming constraint pressure on the spinal cord. It is the core functional component for achieving gentle fixation and preventing entanglement. The mechanical coupling between the knob and the pressure adjusting spring can be a threaded connection. Rotating the knob changes the compression of the pressure adjusting spring, thereby adjusting the pressure applied to the spinal cord without affecting the movement of the sliding block along the track.

[0022] Specifically, when the buckle is a flexible silicone strip buckle, the elastic constraint unit is a flexible silicone strip; the positioning support unit includes buckle connectors disposed at both ends of the flexible silicone strip and buckle grooves disposed on the base; the adapter connection unit is a detachable connection structure between the flexible silicone strip and the buckle connectors.

[0023] The aforementioned flexible silicone strip, due to its inherent softness, generates elasticity, conforming to the spinal cord surface and applying uniform pressure to confine the spinal cord within the groove. The snap-fit ​​connector engages with the groove, fixing the silicone strip above the spinal cord fixation groove and ensuring the strip is distributed along the groove's trajectory. The detachable connection structure can be the interlocking parts at both ends of the silicone strip, allowing for the replacement of silicone strips of different widths / thicknesses (adapting to different animal spinal cord diameters). Simultaneously, the quick disassembly of the snap-fit ​​connector and base enables portable installation and adjustment of the strip.

[0024] Specifically, when the buckle is a magnetic elastic buckle, the positioning support unit includes a magnetic base; the elastic constraint unit includes a metal spring and an arc-shaped pressure head; and the adapter connection unit is a buckle connection structure between the metal spring and the magnetic base.

[0025] The base includes a non-magnetic adsorption surface, and the magnetic base is fixed to the base by strong magnetic adsorption, allowing it to move and reposition freely along the spinal cord fixation groove. The elastic deformation of the metal spring itself generates pressure, which is transmitted to the spinal cord through an arc-shaped pressure head, the arc design of which adapts to the cylindrical surface of the spinal cord. The snap-fit ​​connection structure allows for the replacement of metal springs with different elastic coefficients, while the detachable connection between the spring and the magnetic base facilitates cleaning and maintenance, achieving pressure adaptation and ease of operation.

[0026] In one embodiment, the materials of the fixing device, the inner processing box, and the outer processing box are all selected from at least one of PTFE, PMMA, and specific organic reagent-resistant polymers.

[0027] Specifically, all of the above materials are resistant to chemical corrosion and can ensure that they do not swell, deform or degrade under long-term immersion in high-concentration organic reagents such as ethanol and resin.

[0028] In one embodiment, each guide rail is a groove structure arranged parallel to the length direction of the internal processing box, and a fixing device corresponds to a pair of groove structures, and the two side edges of the corresponding surface of the fixing device in the width direction are fitted and fitted with the groove wall of the groove structure.

[0029] Specifically, the two sides of the fixing device corresponding to the width direction are inserted into the groove structure of the guide rail. Specifically, the two sides of each side form a fitting fit with the groove wall of the groove structure. This allows the fixing device to be smoothly inserted and limited along the length direction of the groove, preventing the fixing device from shaking.

[0030] In one embodiment, the frame further includes a limiting structure, which is a stop or slot disposed at the end of the guide rail.

[0031] Specifically, the stop is a solid structure (such as a block-shaped protrusion, rib, etc.) protruding from the end surface of the guide rail, with a height or thickness greater than the fit clearance between the guide rail and the fixing device. When the fixing device is installed along the length of the guide rail, it will be physically blocked by the stop when it reaches the end of the guide rail, thus limiting the maximum embedding depth of the fixing device and ensuring that all fixing devices are in a uniform termination position in the internal processing box (avoiding uneven sample stress or reagent contact area differences due to varying embedding depths). The slot is a concave groove or notch structure set at the end of the guide rail, the shape of which matches the end protrusion / fitting part of the fixing device in the width direction. When the fixing device is installed to the end of the guide rail, the end of the fixing device will form a snap-fit ​​with the slot (such as a protrusion locking into a groove), achieving a dual function through structural interlocking: restricting the axial movement of the fixing device (along the length of the guide rail), preventing the fixing device from slipping out of the guide rail due to vibration during processing; and assisting in the positioning of the fixing device, ensuring that it will not undergo slight displacement after installation, further improving the stability of the sample morphology.

[0032] Specifically, the number of fixing devices that can be installed in the internal processing box is set according to requirements, such as 6 or 8, and there is no specific limitation here. The installation direction of each fixing device within the frame of the internal processing box is consistent.

[0033] In one embodiment, the bottom of the external processing box is provided with a flow guide groove and a bayonet adapted to the shaker.

[0034] Specifically, the bayonet allows the external processing box to be stably fixed on the shaker. When the shaker is oscillating, the guide groove can promote the formation of directional circulation of reagents, ensuring that the reagents around each sample can be fully and uniformly exchanged, avoiding local concentration gradients, and making the dehydration and transparency effects of all samples highly consistent.

[0035] In one embodiment, the lid of the external processing box is provided with a sealing ring to prevent reagent evaporation.

[0036] According to a second aspect of this disclosure, a method for processing brain-spinal cord samples using the aforementioned batch processing system is provided, comprising the following steps: S1: Fix several brain-spinal cord samples onto a brain-spinal cord sample fixation device; S2: Mount the sample-loading device onto the guide rail of the internal processing box; S3: Place the entire internal processing box of the embedding and fixing device from step S2 into the external processing box, add chemical reagents to the external processing box, and process multiple brain-spinal cord samples simultaneously; S4: After processing, multiple brain-spinal cord samples are embedded, sliced, stained, or subjected to three-dimensional imaging.

[0037] In one possible implementation, the brain-spinal cord sample in step S1 is a sample that has been fixed with formaldehyde.

[0038] In one possible implementation, step S2 can be numbered as needed after the assembly.

[0039] In one embodiment, step S3 involves placing the external treatment box containing the added chemical reagents on a shaker for vibration treatment.

[0040] In one embodiment, the treatment in step S3 includes at least one of dehydration, clearing, and permeation.

[0041] Specifically, in step S3, the corresponding chemical reagents are added according to the processing steps. There are no specific limitations here, such as phosphate buffer, ethanol, xylene, resin-based penetrants, etc.

[0042] In one embodiment, when changing chemical reagents for different treatments in step S3, the entire internal treatment box is removed, the old reagent is discarded, the new reagent is directly added to the external treatment box, and then the internal treatment box is put back in.

[0043] According to one possible implementation of this disclosure, at least the following beneficial effects are achieved: This disclosure constructs a batch workflow system integrating standardized fixation, secure carrying, and synchronous processing. It features a three-tiered modular architecture of fixation-carrying-processing, integrating the precise fixation of individual samples (Level 1: fixation device for brain-spinal cord samples), the integrated carrying of multiple samples (Level 2: internal processing cassette), and the unified reagent processing of the entire sample group (Level 3: external processing cassette). This architecture enables high-throughput processing without sacrificing the morphological preservation of individual samples.

[0044] Designed for batch damage prevention of long strip tissues: To address the issue of maintaining the integrity of soft tissues such as brain and spinal cord, which have a large aspect ratio and are prone to entanglement, in a batch oscillation environment, a unique fixation device is adopted to fix brain and spinal cord samples in brain fixation slots and spinal cord fixation slots. This ensures that the samples remain in place throughout the entire processing flow, avoiding physical damage and entanglement caused by manual transfer and contact between samples.

[0045] Standardization and Efficiency Improvement: The system disclosed herein achieves standardized processing procedures. Its modular design allows researchers to flexibly combine internal frames according to the number of samples in each experiment, enabling standardized batch processing from a few to dozens of samples. When the entire box is placed on a shaker, the flow channels promote directional reagent circulation, ensuring sufficient and uniform exchange of reagents around each sample, avoiding local concentration gradients, and resulting in highly consistent dehydration and clearing effects across all samples. Furthermore, when changing reagents, simply lift the entire internal processing box, discard the old reagents, and add the new ones. Reagent replacement for all samples can be completed in a single operation, eliminating the time lag and operational errors inherent in sequential single-sample processing, significantly improving efficiency, and providing crucial assurance for obtaining repeatable and comparable high-throughput 3D imaging data.

[0046] The materials used in the system disclosed herein are selected to be biocompatible with the reagents, ensuring the long-term stability and reliability of the entire system in harsh chemical processing environments and avoiding sample contamination or impact on processing results due to material deterioration.

[0047] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this disclosure, nor is it intended to limit the scope of this disclosure. Other features of this disclosure will become readily apparent from the following description. Attached Figure Description

[0048] The above and other objects, features, and advantages of this disclosure will become readily apparent from the following detailed description of exemplary embodiments, taken in conjunction with the accompanying drawings. Several embodiments of this disclosure are illustrated in the drawings by way of example and not limitation, in which: In the accompanying drawings, the same or corresponding reference numerals indicate the same or corresponding parts.

[0049] Figure 1 A schematic diagram of the structure of the fixation device for a mouse brain-spinal cord sample in Embodiment 1 of this disclosure is shown; Figure 2 A top view of the first and second latches in Embodiment 1 of this disclosure is shown; Figure 3 A schematic diagram of the internal processing box and the external processing box in Embodiment 1 of this disclosure is shown; Figure 4 A schematic diagram of the placement of a batch of mouse brain-spinal cord samples in Embodiment 2 of this disclosure is shown; Figure 5 The coronal imaging results of mouse brain-spinal cord samples No. 1 and No. 2 obtained after batch processing in Embodiment 2 of this disclosure are shown. Figure 6The diagram shows magnified views of the coronal plane and spinal cord of mouse brain-spinal cord samples No. 1 and No. 2 obtained after batch processing in Embodiment 2 of this disclosure.

[0050] Figure label: 1-Base; 2-Brain fixation groove; 3-Spinal cord fixation groove; 4-First buckle; 41-First buckle body; 42-First sliding block; 43-First sliding track; 44-First pressure head; 5-Second buckle; 51-Second buckle body; 52-Second sliding block; 53-Second sliding track; 54-Second pressure head; 6-Guide rail; 7-Bottom of external treatment box; 8-Lid of external treatment box. Detailed Implementation

[0051] To make the objectives, features, and advantages of this disclosure more apparent and understandable, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0052] This disclosure provides a batch processing system specifically for brain and spinal cord tissues. The system is compatible with a single fixed mold and aims to change the existing inefficient and volatile manual processing mode of single samples. By designing a modular sample carrying unit and reagent processing outer box, the synchronous and standardized processing of multiple samples is achieved. Its technical effects are mainly reflected in: (1) significantly improving processing efficiency: it can process up to dozens of samples at the same time, shortening the total processing time by several to several times, and achieving high-throughput preparation. (2) ensuring high consistency between samples: all samples complete all steps under the same container, the same batch of reagents, and the same time conditions, minimizing batch error and ensuring data comparability. (3) minimizing human intervention and damage: the samples are securely locked on the fixing device, and there is no need to pick them up one by one during the processing. The liquid can be changed by simply transferring the internal processing box as a whole, avoiding physical damage and sample confusion.

[0053] The following examples illustrate this in detail.

[0054] Example 1 This embodiment provides a batch processing system for brain-spinal cord samples. The brain-spinal cord samples here refer to those from experimental animals, and are not limited to a single animal, such as mice, monkeys, rabbits, or dogs. This embodiment selects mouse brain-spinal cord samples. The system includes a brain-spinal cord sample fixation device, an internal processing box, and an external processing box.

[0055] In this embodiment, the fixation device for the brain-spinal cord sample is a fixation device for a mouse brain-spinal cord sample, and its structural schematic diagram is shown below. Figure 1 As shown. The fixation device includes a base 1, a brain fixation groove 2 and a spinal cord fixation groove 3 disposed on the base 1, with the brain fixation groove 2 and the spinal cord fixation groove 3 connected. The brain fixation groove 2 is a biomimetic groove adapted to the lateral anatomical contour of the mouse brain, used to accommodate the side of the mouse brain and expose the brainstem terminal end of the mouse brain. The length of the brain fixation groove 2 is 20 mm, the width is 8 mm (at the maximum diameter), and the depth is 5 mm (these dimensions are the dimensions of the mouse brain fixation groove 2). The spinal cord fixation groove 3 is a spiral biomimetic groove adapted to the lateral anatomical contour of the mouse spinal cord, used to accommodate the side of the mouse spinal cord and maintain a natural connection angle between the mouse spinal cord and the brainstem terminal end. The total length of the spinal cord fixation groove 3 is 60 mm, the width is 1 mm, and the depth is 1 mm (these dimensions are the dimensions of the mouse spinal cord fixation groove 3).

[0056] A transition arc with a radius of 2-3 mm is provided at the connection between the brain fixation groove 2 and the spinal cord fixation groove 3. This transition arc is used to smoothly connect the end of the mouse brain's brainstem with the beginning of the mouse spinal cord, avoiding bending of nerve fibers at the connection site. In this embodiment, the radius of the transition arc is 3 mm.

[0057] The base 1, brain fixation groove 2, and spinal cord fixation groove 3 are integrally molded. The integral molding method includes either precision injection molding or CNC milling. Specifically, if precision injection molding is chosen, the shape of the brain fixation groove 2 and spinal cord fixation groove 3 is designed as a mold cavity, and the grooved base is directly injection molded, with no seam between the groove and the base body. If CNC milling is chosen, the integral base is first machined, and then grooves matching the anatomical contours of the brain and spinal cord are milled on the base surface using a precision milling cutter. This embodiment uses precision injection molding. The brain fixation groove 2 and spinal cord fixation groove 3 are directly molded onto the base 1, and their relative positions are ensured by machining precision, avoiding misalignment of the brain-spinal cord connection caused by assembly errors in traditional separate spliced ​​grooves. The seamless integral structure prevents the fixation groove from falling off or shifting during sample processing, and also prevents reagent residues in the splicing gaps from causing sample contamination, meeting the standardization and cleanliness requirements of experimental operations.

[0058] Base 1 has a grid-like structure, from Figure 1 As can be seen, the base 1 is a cube with a high porosity mesh structure. The mesh holes are square. The high porosity design allows the tissue fixed on the base 1 to be fully penetrated by the reagent from all directions when the reagent is immersed in it, ensuring that the brain and spinal cord samples can be processed uniformly and thoroughly, thus improving the quality of slicing and imaging.

[0059] like Figure 1As shown, the fixation device also includes a snap-fit ​​fixation system. This system is positioned along the trajectory of the spinal cord fixation groove 3 to confine the mouse spinal cord within it. Specifically, it is positioned along the radial trajectory of the spinal cord fixation groove 3 (referring to the ray direction extending outward from the spiral center; each spiral groove has its corresponding radial ray, and these rays together constitute the radial trajectory system of the spiral groove). This configuration complements the spiral shape of the groove itself, perfectly constraining the spiral trajectory. The snap-fit ​​fixation system includes several snaps. The snap-fit ​​fixation system is designed to stably confine the mouse spinal cord sample within the spinal cord fixation groove 3 through constraint pressure, preventing it from floating, entangled, rotating, or structurally damaged during fixation, dehydration, and other processes. It also adapts to the anatomical morphology of the mouse spinal cord sample. In this embodiment, the snap-fit ​​fixation system includes two snaps: a first snap 4 and a second snap 5. Both snaps extend along the radial rays of the spiral groove. One end of each snap points towards the central region of the spiral groove (i.e., the starting point of the radial ray), and the other end extends outward along the radial ray, covering one or two spiral groove loops. Each snap fastener includes a positioning support unit, an elastic constraint unit, and an adapter connection unit. The positioning support unit is connected to the base 1 and is used to fix the snap fastener to the base 1. The elastic constraint unit is used to provide elastic pressure to confine the mouse spinal cord within the spinal cord fixation groove 3. The adapter connection unit is used to connect the positioning support unit and the elastic constraint unit. The snap fastener may include any one of a press-type elastic snap fastener, a flexible silicone strip snap fastener, or a magnetic elastic snap fastener. In this embodiment, both the first snap fastener 4 and the second snap fastener 5 are press-type elastic snap fasteners, with the specific structure as follows: Figure 2 As shown.

[0060] The positioning support unit of the first buckle 4 includes a first sliding block 42 disposed at the bottom of the first buckle body 41 and a first sliding track 43 disposed on the base 1. The first buckle body 41 is a rigid component formed integrally, and all functional components are directly installed on its surface or inside, without undergoing elastic deformation, only serving as support and force transmission. Through the cooperation of the sliding block and the sliding track, the first buckle 4 can move flexibly along the track, while ensuring the precise relative position of the first buckle 4 and the spinal cord fixation groove 3. The elastic constraint unit includes a first pressure adjusting spring (not shown in the figure) and a first pressure head 44 disposed inside the first buckle body 41. The first pressure adjusting spring is the core source of elastic force. The elastic force generated by its deformation is transmitted to the first pressure head 44 through the first buckle body 41, forming constraint pressure on the rat spinal cord, which is the core functional component for achieving gentle fixation and preventing entanglement. The adapter connection unit includes a first knob (not shown in the figure), which is mechanically coupled to a first pressure adjusting spring. In this embodiment, the mechanical coupling adopts a threaded connection to change the compression of the first pressure adjusting spring in order to adjust the pressure applied to the rat spinal cord.

[0061] The positioning support unit of the second buckle 5 includes a second sliding block 52 disposed at the bottom of the second buckle body 51 and a second sliding track 53 disposed on the base 1. The second buckle body 51 is a rigid component formed as a whole, and all functional components are directly installed on its surface or inside, without undergoing elastic deformation, only serving as a support and force transmission function. Through the cooperation of the sliding block and the sliding track, the second buckle 5 can move flexibly along the track, while ensuring the precise relative position of the second buckle 5 and the spinal cord fixation groove 3. The elastic constraint unit includes a second pressure adjusting spring (not shown in the figure) and a second pressure head 54 disposed inside the second buckle body 51. The second pressure adjusting spring is the core source of elastic force. The elastic force generated by its deformation is transmitted to the second pressure head 54 through the second buckle body 51, forming a constraint pressure on the rat spinal cord, which is the core functional component for achieving gentle fixation and preventing entanglement. The adapter connection unit includes a second knob (not shown in the figure), which is mechanically coupled to a second pressure adjusting spring. In this embodiment, the mechanical coupling is a threaded connection used to change the compression of the second pressure adjusting spring in order to adjust the pressure applied to the rat spinal cord.

[0062] Figure 1 and Figure 2 As shown, the second clip 5 is used to restrict the mouse spinal cord in two loops of the spinal cord fixation groove 3, while the first clip 4 is used to restrict the mouse spinal cord in one loop of the spinal cord fixation groove 3. Therefore, the size of the second clip 5 is correspondingly larger than the size of the first clip 4, and the specific size is determined according to actual needs. The aforementioned first clip 4 and second clip 5 completely cover the slender spinal cord in the spinal cord fixation groove 3, effectively preventing the axonal fibers from rotating and twisting due to floating and entanglement in the reagent, thus ensuring the continuity of neural circuit tracking.

[0063] The materials used for the fixture, internal treatment box, and external treatment box are all selected from at least one of PTFE, PMMA, and specific organic reagent-resistant polymers. These materials are chosen for their resistance to chemical corrosion, ensuring that no swelling, deformation, or degradation occurs during prolonged immersion in high-concentration organic reagents such as ethanol and resins.

[0064] The structures of the internal processing box and the external processing box are as follows: Figure 3 As shown, the internal processing box ( Figure 3The internal structure (indicated by the dashed line) contains a frame, which includes several guide rails 6 for mounting several fixing devices. The number of guide rails 6 is set as needed; in this embodiment, it is set to 8 pairs to accommodate 8 fixing devices. Each fixing device is mounted in the same direction within the frame of the internal processing box. Each guide rail 6 is a groove structure arranged parallel to the length of the internal processing box. One fixing device corresponds to one pair of groove structures, and the two sides of the corresponding surface of the fixing device in the width direction form a fitting fit with the groove wall of the groove structure. This allows the fixing device to be smoothly inserted and positioned along the length of the groove, preventing the fixing device from shaking. The frame also includes a limiting structure (not shown in the figure), which is a stop or slot provided at the end of the guide rail 6. Specifically, the stop is a solid structure (such as a block protrusion, rib, etc.) protruding from the end surface of the guide rail 6, and its height or thickness is greater than the mating gap between the guide rail 6 and the fixing device. When the fixing device is installed along the length of guide rail 6, it will be physically blocked by a stop at the end of guide rail 6, preventing further movement. This limits the maximum embedding depth of the fixing device and ensures that all fixing devices are in the same termination position within the internal processing box (avoiding uneven sample stress or reagent contact area differences due to varying embedding depths). The slot is a concave groove or notch structure provided at the end of guide rail 6, the shape of which matches the end protrusion / fitting part of the fixing device in the width direction. When the fixing device is installed to the end of guide rail 6, the end of the fixing device will form a snap-fit ​​with the slot (such as a protrusion locking into a groove), achieving a dual function through structural interlocking: restricting the axial movement of the fixing device (along the length of guide rail 6), preventing the fixing device from slipping out of guide rail 6 due to vibration during processing; and assisting in the positioning of the fixing device, ensuring that it will not undergo slight displacement after installation, further improving the stability of the sample morphology. In this embodiment, the limiting structure is a stop, specifically a block-shaped protrusion.

[0065] External processing box (i.e.) Figure 3 The outermost box (the outermost compartment) houses the inner processing box for uniform reagent treatment of the entire sample group. The bottom 7 of the outer processing box has a flow channel and a locking mechanism (not shown in the figure) adapted to the shaker. The locking mechanism allows the outer processing box to be stably fixed to the shaker. When shaken, the flow channel promotes directional reagent circulation, ensuring sufficient and uniform reagent exchange around each sample, avoiding local concentration gradients, and resulting in highly consistent dehydration and clearing effects for all samples. The lid 8 of the outer processing box has a sealing ring (not shown in the figure) to prevent reagent evaporation.

[0066] Example 2 This embodiment uses the batch processing system of Example 1 to process mouse brain-spinal cord samples, as follows: (1) Take eight mouse brain and spinal cord samples fixed with paraformaldehyde and fix them sequentially on the brain-spinal cord sample fixation device; sequentially embed the eight sample-loading fixation devices into the guide rails of the internal processing box and number them; place the entire internal processing box containing the above-mentioned fixation devices into the external processing box, as shown. Figure 4 As shown.

[0067] (2) Pour phosphate buffer into the external processing box and rinse the mouse brain and spinal cord samples; then use gradient dehydration reagents, including 70%, 85%, and 95% ethanol, to dehydrate the mouse brain and spinal cord samples sequentially, for 60 min at each step; pour 50%, 70%, 85%, and 100% HM20 resin into the external processing box sequentially to perform gradient permeation on the mouse brain and spinal cord, for 2 h at each step, and finally permeate in 100% resin for 3 days. When changing reagents at each step, remove the entire internal processing box, discard the old reagents, add the new reagents directly to the external processing box, and then put the internal processing box back in.

[0068] (3) After processing, mouse brain and spinal cord samples were removed and placed in a 42℃ oven for 24 hours to polymerize, obtaining embedded samples. High-resolution three-dimensional imaging was performed on the polymerized intact mouse brain-spinal cord samples, and the results are as follows: Figure 5 As shown. Figure 5 The results show that images of the mouse brain and spinal cord can be obtained in the same image, and similar coronal images of the mouse brain and spinal cord can be obtained simultaneously between different samples, facilitating image comparison and quantitative analysis between different types of samples. Figure 5 A magnified view of a portion of the middle spinal cord shows the following results: Figure 6 As shown, the cell images of mouse brain and spinal cord stained with nucleic acid dye-PI are clear, and the morphology of spinal cord cells is well preserved. This indicates that the mouse brain and spinal cord samples prepared by the batch processing method disclosed herein can maintain good morphological contours and cell morphological structure information, which is beneficial for simultaneous observation and analysis of cell composition and circuit information between different mouse brain and spinal cord samples.

[0069] In summary, this disclosure constructs a batch workflow system that integrates standardized fixation, secure carrying, and synchronous processing. Its key features are: The three-tiered modular architecture of fixation-support-processing integrates the precise fixation of individual samples (Level 1: fixation device for brain-spinal cord samples), the integrated support of multiple samples (Level 2: internal processing box), and the unified reagent processing of the entire sample group (Level 3: external processing box). This architecture enables high-throughput processing without sacrificing the morphological preservation of individual samples.

[0070] Designed for batch damage prevention of long strip tissues: The unique fixation method is specially designed to maintain the integrity of soft tissues such as the brain and spinal cord, which have a large aspect ratio and are easily entangled, in a batch oscillation environment. It solves the most difficult problems of inter-sample interference and self-deformation in batch processing.

[0071] Standardization and Efficiency Improvement: This system achieves standardization of the processing workflow. All samples undergo the same processing environment, which not only greatly improves efficiency, but more importantly, it eliminates the time lag and operational errors inherent in single-sample processing, providing a key guarantee for obtaining repeatable and comparable high-throughput 3D imaging data.

[0072] It should be understood that the various forms of processes shown above can be used to rearrange, add, or delete steps. For example, the steps described in this disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution of this disclosure can be achieved, and this is not limited herein.

[0073] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this disclosure, "a plurality of" means two or more, unless otherwise explicitly specified.

[0074] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. A batch processing system for brain-spinal cord samples, characterized in that, The batch processing system includes a fixation device for brain-spinal cord samples, an internal processing box, and an external processing box; The fixation device includes a base, a brain fixation groove and a spinal cord fixation groove disposed on the base, wherein the brain fixation groove and the spinal cord fixation groove are in communication; the brain fixation groove is a biomimetic groove adapted to the lateral anatomical contour of the brain, used to accommodate the side of the brain and expose the brainstem terminal; the spinal cord fixation groove is a spiral biomimetic groove adapted to the lateral anatomical contour of the spinal cord, used to accommodate the side of the spinal cord and maintain a natural connection angle between the spinal cord and the brainstem terminal. The internal processing box is provided with a frame, which includes several guide rails for mounting several of the fixing devices. The external processing box is used to house the internal processing box for uniform reagent processing of the entire sample group.

2. The batch processing system according to claim 1, characterized in that, The connection between the brain fixation groove and the spinal cord fixation groove is provided with a transition arc with a radius of 2-3 mm, which is used to smoothly connect the end of the brainstem of the brain with the beginning of the spinal cord, and avoid the bending of nerve fibers at the connection site.

3. The batch processing system according to claim 1, characterized in that, The base, the brain fixation groove, and the spinal cord fixation groove are integrally molded, and the integral molding method includes any one of precision injection molding and CNC milling.

4. The batch processing system according to claim 1, characterized in that, The fixation device further includes a snap-fit ​​fixation system, which is arranged along the trajectory of the spinal cord fixation groove to restrict the spinal cord within the spinal cord fixation groove; the snap-fit ​​fixation system includes a plurality of snaps.

5. The batch processing system according to claim 4, characterized in that, The buckle includes a positioning support unit, an elastic constraint unit, and an adapter connection unit; the positioning support unit is connected to the base and is used to fix the buckle on the base; the elastic constraint unit is used to provide elastic pressure to confine the spinal cord within the spinal cord fixation groove; the adapter connection unit is used to connect the positioning support unit and the elastic constraint unit.

6. The batch processing system according to claim 1, characterized in that, Each guide rail is a groove structure arranged parallel to the length direction of the internal processing box. One fixing device corresponds to a pair of groove structures, and the two sides of the corresponding surface of the fixing device in the width direction are fitted and fitted with the groove wall of the groove structure.

7. The batch processing system according to claim 1, characterized in that, The frame also includes a limiting structure, which is a stop or slot provided at the end of the guide rail.

8. The batch processing system according to claim 1, characterized in that, The bottom of the external processing box is provided with a flow guide groove and a snap-fit ​​that is compatible with the shaker; the lid of the external processing box is provided with a sealing ring to prevent reagent evaporation.

9. A method for processing brain-spinal cord samples using the batch processing system according to any one of claims 1 to 8, characterized in that, Includes the following steps: S1: Fix several brain-spinal cord samples onto a brain-spinal cord sample fixation device; S2: Mount the sample-loading device onto the guide rail of the internal processing box; S3: Place the entire internal processing box of the embedding and fixing device from step S2 into the external processing box, add chemical reagents to the external processing box, and process multiple brain-spinal cord samples simultaneously. S4: After processing, multiple brain-spinal cord samples are embedded, sliced, stained, or subjected to three-dimensional imaging.

10. The method according to claim 9, characterized in that, In step S3, when changing chemical reagents for different treatments, the entire internal treatment box is removed, the old reagents are discarded, and the new reagents are added directly to the external reagent kit before the internal treatment box is put back in.

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