A method for obtaining dorsal root ganglia of dead rats

Abstract

The application provides a method for obtaining dorsal root ganglion of dead rats, and belongs to the technical field of biological materials. The method for obtaining dorsal root ganglion of dead rats provided by the application has a high success rate of obtaining, can stably obtain complete DRG tissue, avoids the problems of tissue fragmentation or loss in traditional methods, the DRG tissue morphology is well preserved, the neuron cell body structure is complete, the background is clean and free of pollution, and the requirements of morphological detection and molecular biology experiments can be met at the same time, the operation process is simple and efficient, special equipment is not needed, the obtaining time is significantly shortened, and the operation difficulty and personnel work intensity are reduced. The obtaining method of the application realizes high-quality and repeatable standardized DRG obtaining, provides reliable experimental material guarantee for related fields such as pain mechanism research, nerve injury and regeneration, drug target screening and the like, and has a good scientific research application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a method for obtaining material from the dorsal root ganglion of a dead rat. Background Technology

[0002] The dorsal root ganglion is a collection of primary sensory neuron cell bodies distributed segmentally along both sides of the spinal cord. It consists of pseudounipolar neurons, satellite cells, and nerve fibers. Its function is to transmit sensory information generated by peripheral receptors to the central nervous system via the posterior roots of spinal nerves. It is a key structure in the pathways of pain, temperature, touch, and proprioception. Due to its unique anatomical location and important physiological function in sensory pathways, the dorsal root ganglion has received considerable attention in neuroscience, pain medicine, and pharmacology research. It is an important research subject for exploring chronic pain, neuropathic pain, mechanisms of nerve regeneration after injury, and screening targets for analgesics.

[0003] Currently, the commonly used methods for obtaining dorsal root ganglia are the laminectomy method and the nerve tracing method. The laminectomy method typically involves removing the animal's spine, then using scissors or bone forceps to open the vertebral lamina at each segment to expose the vertebral canal, and then extracting the dorsal root ganglia through the intervertebral foramen. The nerve tracing method utilizes the anatomical continuity between the sciatic nerve and the lumbar dorsal root ganglia, tracing and pulling out the nerve along its course. Both methods have been used in this field for many years.

[0004] Furthermore, dorsal root ganglion tissue is small in size, requiring high precision in sampling. The quality of samples obtained varies significantly between different operators, and the pretreatment methods used after sampling directly impact the reliability of subsequent experimental results. Therefore, establishing a stable method for obtaining high-quality dorsal root ganglion samples is of significant practical importance for related research fields. Summary of the Invention

[0005] The purpose of this invention is to provide a method for obtaining dorsal root ganglion samples from dead rats, which solves the technical problem of the difficulty in obtaining high-quality dorsal root ganglion samples in the prior art.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for obtaining tissue samples from the dorsal root ganglion of a dead rat, comprising the following steps: Take the dead rat, expose the spine, and remove the complete spine. Remove the soft tissue attached to the vertebrae to expose the bony landmarks of the vertebrae; Starting from the lumbar vertebrae, the spinous processes and lamina of each vertebra are separated segment by segment along the midline of the dorsal side of the spine, and the posterior wall of the spinal canal is lifted as a whole to expose the spinal cord and the dorsal root ganglia of each segment. Pre-fixation was performed by applying fixative to the exposed dorsal root ganglion area, followed by in-situ staining with hematoxylin staining solution to make the dorsal root ganglion appear in a different color from the surrounding tissue. Tracing the posterior root of the spinal nerve to the dorsal root ganglion, first cut the spinal nerve at the distal end of the dorsal root ganglion, then cut the posterior root at the proximal end of the dorsal root ganglion, and remove the dorsal root ganglion from its original position.

[0007] Preferably, the fixative is a lithium carbonate solution with a mass percentage concentration of 5% to 15%.

[0008] Preferably, the initial fixed processing time is 1 to 3 minutes.

[0009] Preferably, the staining time of the hematoxylin staining solution is 1 to 2 minutes.

[0010] Preferably, the method further includes a step of rinsing off excess dye after the in-situ staining.

[0011] Preferably, the location where the distal end of the spinal nerve is first cut is 0.5 to 2.5 mm from the distal end of the dorsal root ganglion, and the location where the proximal end of the posterior root is then cut is 0.5 to 2.5 mm from the proximal end of the dorsal root ganglion.

[0012] Preferably, the method further includes the steps of laying the removed dorsal root ganglion flat on filter paper and placing it into an embedding container, as well as the step of post-fixation with a fixative.

[0013] Preferably, the fixative used for post-fixation is a neutral buffered formalin with a mass percentage concentration of 5% to 15%.

[0014] Preferably, the fixing time after fixation is 24 to 72 hours.

[0015] Preferably, the specific operation of exposing the spine and removing the complete spine is as follows: take a dead rat, cut open the abdomen and sternum along the midline of the abdomen from the pubic symphysis upwards to expose the thoracic cavity, separate the thoracic organs, and then cut the connection between the cervical vertebrae and the skull transversely at the foramen magnum and remove the complete spine.

[0016] The beneficial effects of this invention are: The method for obtaining dorsal root ganglion (DRG) tissue from dead rats provided by this invention boasts a high success rate, consistently yielding intact DRG tissue and avoiding tissue fragmentation or loss issues common in traditional methods. Tissue identification is significantly enhanced, clearly distinguishing the DRG from surrounding connective tissue during sampling. This method is particularly suitable for beginners or large-scale sampling scenarios, effectively reducing missed samples and accidental cutting. The DRG tissue retains its morphology well, with intact neuronal cell structures and a clean, uncontaminated background, simultaneously meeting the requirements for morphological detection and molecular biology experiments. The operation is simple and efficient, requiring no special equipment, significantly shortening sampling time and reducing operational difficulty and workload. This sampling method achieves high-quality, reproducible, and standardized DRG sampling, providing reliable experimental material for pain mechanism research, nerve injury and regeneration, drug target screening, and other related fields, demonstrating promising research application prospects. Attached Figure Description

[0017] Figure 1 A gross photograph of the dorsal root ganglion region before anterior fixation and hematoxylin in situ staining; Figure 2 This is a gross photograph of the dorsal root ganglion region after pre-fixation with lithium carbonate solution and in situ staining with hematoxylin. Figure 3 Photographs of dorsal root ganglia under microscope (HE staining) before anterior fixation and in situ hematoxylin staining. Figure 4 This is a microscopic photograph of the dorsal root ganglion after pre-fixation with lithium carbonate solution and in situ staining with hematoxylin (HE staining). Detailed Implementation

[0018] This invention provides a method for obtaining tissue samples from the dorsal root ganglion of a dead rat, comprising the following steps: Take the dead rat, expose the spine, and remove the complete spine. Remove the soft tissue attached to the vertebrae to expose the bony landmarks of the vertebrae; Starting from the lumbar vertebrae, the spinous processes and lamina of each vertebra are separated segment by segment along the midline of the dorsal side of the spine, and the posterior wall of the spinal canal is lifted as a whole to expose the spinal cord and the dorsal root ganglia of each segment. Pre-fixation was performed by applying fixative to the exposed dorsal root ganglion area, followed by in-situ staining with hematoxylin staining solution to make the dorsal root ganglion appear in a different color from the surrounding tissue. Tracing the posterior root of the spinal nerve to the dorsal root ganglion, first cut the spinal nerve at the distal end of the dorsal root ganglion, then cut the posterior root at the proximal end of the dorsal root ganglion, and remove the dorsal root ganglion from its original position.

[0019] Take the dead rat, expose the spine, and remove the complete spine.

[0020] In this invention, the rat is one of the most commonly used experimental animals in medical and biological research. The rat's spine consists of cervical, thoracic, lumbar, sacral, and caudal vertebrae, with 7 cervical vertebrae, 13 thoracic vertebrae, 6 lumbar vertebrae, 4 sacral vertebrae, and the number of caudal vertebrae varying depending on the breed. The spine is located in the midline of the rat's dorsal trunk and is an important bony structure supporting the trunk and protecting the spinal cord. "Exposing the spine" refers to fully exposing the bony structure of the spine to the operating field by incising the skin and separating the subcutaneous tissue and muscle layer. "Removing the complete spine" refers to completely separating the spine from the rat's trunk, preserving all vertebrae from the cervical to the sacral vertebrae and their attached intraspinal tissues, ensuring that the dorsal root ganglia of each segment remain in their original anatomical position, avoiding nerve root traction or DRG displacement due to spinal fracture or vertebral misalignment.

[0021] In this invention, "soft tissue" refers to various non-osseous tissues attached to the surface of the vertebrae, including deep back muscles, intercostal muscles, paravertebral muscles, large blood vessels on the ventral side of the spine, esophagus, trachea, and connective tissue. These soft tissues cover the spinous processes, transverse processes, lamina, and intervertebral foramina of the vertebrae. If not removed, they will seriously affect the operator's identification of the bony structures of the vertebrae and the accuracy of subsequent operations. "Bone landmarks" refer to bony protrusions and structures on the vertebrae that have anatomical significance, mainly including the spinous process (the bony plate protruding posteriorly from the midline of the dorsal side of the vertebra), the transverse process (the bony protrusions laterally on both sides of the vertebral body), and the intervertebral foramen (the passage through which nerve roots emerge between adjacent vertebrae). The spinous processes of the rat cervical vertebrae are relatively short, the spinous processes of the thoracic vertebrae are relatively long and slender and tilted posteriorly, and the spinous processes of the lumbar vertebrae are broad and plate-like. The morphological differences between the segments can be used as a reference for localization. By thoroughly removing the soft tissue attached to the surface of the vertebrae, the aforementioned bony landmarks are clearly exposed. The operator can then accurately determine the segmental position of each vertebra and the specific location of the intervertebral foramen, thus providing a reliable anatomical reference for subsequent precise DRG localization.

[0022] In this invention, the spinous processes and lamina of each vertebra are sequentially severed along the dorsal midline of the spine, starting from the lumbar vertebrae, and the posterior wall of the vertebral canal is lifted as a whole, thus longitudinally opening the vertebral canal and exposing the spinal cord and dorsal root ganglia of each segment. The "dorsal midline of the spine" in this invention refers to the longitudinal midline along the dorsal side of the spine connecting the tips of the spinous processes, which is the most prominent anatomical landmark on the dorsal side of the rat spine. The "spinous process" refers to the bony protrusion protruding posteriorly from the midline of the dorsal side of the vertebral arch; the spinous processes of each vertebra are interconnected by the interspinous ligaments. The "lamina" refers to the plate-like bony structures located on both sides of the vertebral arch between the spinous processes and the vertebral bodies; the left and right lamina and spinous processes together form the dorsal wall of the vertebral canal. The "vertebral canal" is a bony canal formed longitudinally by the vertebral foramina of each vertebra, containing the spinal cord, spinal nerve roots, blood vessels, and meninges. "Severing" refers to cutting and separating the bony structures by shearing or biting, thus interrupting their original continuity. During the procedure, the spinal cord is operated segment by segment upwards from the lumbar vertebrae, severing the bony connection between the spinous process and the lamina, so that the posterior wall of the spinal canal is completely separated from the vertebral body. Then, the entire spinal canal is lifted up and the posterior wall of the spinal canal is removed from the lumbar vertebrae to the cervical vertebrae, so that the spinal canal is transformed from a closed bony canal into a longitudinally open state, and the entire length of the spinal cord and the DRGs attached to both sides are fully exposed.

[0023] In this invention, a fixative solution is first applied to the exposed dorsal root ganglion region for pre-fixation, followed by in-situ staining with hematoxylin to make the dorsal root ganglion appear a different color from the surrounding tissue. The "fixative solution" refers to a chemical solution that can denature or cross-link proteins in tissue cells, thereby rapidly terminating various intracellular enzymatic reactions, stabilizing cell structure, and preserving tissue antigenicity. If tissue is not fixed promptly after ex vivo, endogenous cellular enzymes will initiate autolysis, leading to cell structure destruction and antigen loss, severely affecting the accuracy of subsequent morphological and molecular biological detection results. The fixative solution, by forming cross-links between protein molecules, fixes intracellular proteins, nucleic acids, and other biomacromolecules in their physiological spatial positions, thereby maintaining the tissue's natural morphological structure. Tissue fixation is divided into pre-fixation and post-fixation. Pre-fixation refers to fixation treatment performed before or very shortly after tissue ex vivo. Its core purpose is to minimize the time interval between tissue ex vivo and fixation, rapidly terminate the tissue autolysis process, and protect the cell ultrastructure and antigenic activity. The term "anterior fixation" refers to applying fixation fluid in situ for fixation before the DRG is removed from the spine.

[0024] In this invention, the fixative is preferably a lithium carbonate solution. The weakly alkaline environment of the lithium carbonate solution can stabilize the nucleic acid and protein structures within cells to a certain extent, and its weak alkalinity also facilitates the binding of the subsequent hematoxylin staining solution to acidic substances in the cell nucleus. In this invention, the mass percentage concentration of the lithium carbonate solution can be 5%–15%, preferably 8%–12%, and more preferably 10%.

[0025] In this invention, "hematoxylin," also known as hematoxylin, is a natural staining agent extracted from the heartwood of the legume Caesalpinia sappan. After oxidation, it forms hematein trioxide, a basic dye. Hematoxylin is one of the most classic nuclear staining agents in histology. Its staining principle is based on the fact that the chromatin in the cell nucleus contains a large amount of deoxyribonucleic acid (DNA). The phosphate groups in DNA molecules carry a negative charge and have a high affinity for the positively charged basic dye hematoxylin. The two bind through ionic bonds, staining the cell nucleus blue-purple. After hematoxylin staining, details such as the morphology, size, chromatin distribution, and mitotic figures of the cell nucleus are clearly visible. In this invention, "in situ staining" refers to staining the DRG directly at its original anatomical location before it is removed from the spine. Through in situ staining, the neuronal nuclei within the DRG are stained a bright blue-purple by hematoxylin, while the surrounding nerve fibers, adipose tissue, and connective tissue, which contain little or no nuclei, are essentially unstained or only show a very light background color. This creates a significant color difference between the DRG and the surrounding tissue that is clearly visible to the naked eye. The operator can quickly and accurately identify the location and boundaries of the DRG based on this color difference. In this invention, the staining time of the hematoxylin staining solution can be 1–2 minutes, preferably 1.2–1.8 minutes, and more preferably 1.5 minutes.

[0026] In this invention, the posterior root of the spinal nerve is traced to the dorsal root ganglion. First, the distal end of the spinal nerve in the dorsal root ganglion is severed, then the proximal end of the posterior root is severed, and the dorsal root ganglion is removed from its original location. The "spinal nerve" refers to the peripheral nerves originating from the spinal cord. Each pair of spinal nerves connects to the spinal cord via anterior (ventral) and posterior (dorsal) roots. The anterior root is composed of axons from motor neurons, responsible for transmitting central motor commands to the periphery; the posterior root is composed of central processes from sensory neurons, responsible for transmitting peripheral sensory information to the spinal cord. The "posterior root of the spinal nerve," or dorsal root, is the pathway for sensory nerve fibers to enter the spinal cord. The "dorsal root ganglion" is the enlarged structure located on the posterior root of the spinal nerve, a collection of pseudounipolar primary sensory neuron cell bodies embedded in glial tissue and connective tissue matrix. Each dorsal root ganglion contains thousands of sensory neuron cell bodies. Its distinctive T-shaped structure divides into two branches: one (peripheral process) distributes to peripheral receptors via the spinal nerve, and the other (central process) forms the posterior root of the spinal nerve and enters the spinal cord. The dorsal root ganglion is the first-order neuron in the sensory conduction pathway, playing a crucial role in the transmission of pain, temperature, touch, and proprioception.

[0027] In this invention, the "distal end" refers to the end along the nerve's course, away from the spinal cord and closer to the periphery, with the dorsal root ganglion as a reference point. The "proximal end" refers to the end along the nerve's course, closer to the spinal cord and further away from the periphery, with the dorsal root ganglion as a reference point. The dorsal root ganglion is located on the posterior root of the spinal nerve, with its posterior root fibers extending proximally to connect to the spinal cord, and the spinal nerve extending distally to connect to the periphery. During the procedure, the posterior root of the spinal nerve is first traced to the dorsal root ganglion to confirm the accurate location of the DRG. Then, the spinal nerve is first cut at the distal end of the DRG, severing the connection between the DRG and the peripheral nerves. At this point, the DRG is only connected to the spinal cord through its proximal posterior root. Next, the posterior root is cut at the proximal end of the DRG, and the DRG is completely removed from its original position. This sequence of cutting the distal end first and then the proximal end ensures that the DRG body is not subjected to traction during the entire cutting process, avoiding neuronal cell body damage caused by traction.

[0028] In this invention, the location for first severing the distal spinal nerve can be 0.5–2.5 mm from the distal end of the dorsal root ganglion, preferably 1.0–2.0 mm from the distal end of the dorsal root ganglion, and more preferably 1.5 mm from the distal end of the dorsal root ganglion. The location for then severing the proximal posterior root can be 0.5–2.5 mm from the proximal end of the dorsal root ganglion, preferably 1.0–2.0 mm from the proximal end of the dorsal root ganglion, and more preferably 1.5 mm from the proximal end of the dorsal root ganglion. Retaining appropriate lengths of nerve stumps on both sides of the DRG serves two purposes: firstly, it acts as a "handle" for grasping the DRG, facilitating operation; secondly, it avoids mechanical damage to the DRG cell body caused by the severing operation itself.

[0029] In this invention, the mass percentage concentration of the lithium carbonate solution can be 5% to 15%. Further, the mass percentage concentration of the lithium carbonate solution is preferably 8% to 12%, more preferably 10%. Those skilled in the art can appropriately adjust the concentration of the lithium carbonate solution within the above range according to actual material availability, and all such adjustments are within the scope of protection of this invention.

[0030] In this invention, the pre-fixation treatment time refers to the time required from the contact of the fixative with the DRG tissue to the time the fixative takes effect and completes in situ fixation. The pre-fixation treatment time can be 1 to 3 minutes, preferably 1.5 to 2.5 minutes, and more preferably 2 minutes. Too short a pre-fixation time may lead to insufficient fixation and incomplete termination of cell autolysis; too long a pre-fixation time may result in over-fixation, affecting the subsequent hematoxylin staining effect. Within the above time range, cell structure can be sufficiently stabilized, autolysis prevented, and good subsequent staining results can be ensured.

[0031] In this invention, the staining time of the hematoxylin staining solution refers to the time it takes for the hematoxylin staining solution to come into contact with and stain the DRG tissue. The staining time can be 1 to 2 minutes, preferably 1.2 to 1.8 minutes, and more preferably 1.5 minutes. Too short a staining time may result in insufficient staining, with an indistinct color difference between the DRG and surrounding tissues; too long a staining time may result in excessively dark staining, obscuring the fine structures within the tissue. Within the above time range, the cell nuclei of the DRG are stained a bright blue-purple, forming an optimal color contrast with the surrounding unstained tissue, facilitating accurate identification by the operator.

[0032] In this invention, "rinsing off excess staining solution" refers to washing the stained area with an appropriate rinsing solution after in situ staining to remove free staining solution that has not bound to the tissue. If free hematoxylin staining solution remains on the tissue surface, it can interfere with the operator's judgment of the staining effect and may cause background staining. The rinsing solution can be phosphate-buffered saline (PBS), physiological saline, or deionized water. In this invention, PBS buffer is preferred. PBS buffer is one of the most commonly used buffer systems in biological experiments; its osmotic pressure and pH value are similar to those of mammalian body fluids, enabling effective rinsing off of excess staining solution without damaging tissue cells. After rinsing with PBS buffer, free staining solution is removed, DRG appears as a clear blue-purple color, the background is clean, and the tissue boundaries are distinct.

[0033] In this invention, "flattening" refers to gently flattening the removed DRG tissue on filter paper to maintain its natural anatomical shape and avoid curling, folding, or twisting. DRG tissue is small in size and, if not properly treated after removal, is prone to curling due to surface tension or mechanical manipulation, affecting the integrity of the tissue section during subsequent sectioning. The "filter paper" is a fibrous paper material with good absorbency and a certain mechanical strength, capable of absorbing residual liquid on the DRG surface while providing a flat support surface. The "embedding container" is generally an embedding cassette, a specialized container used in histological experiments to hold tissue samples for subsequent dehydration, clearing, paraffin infiltration, and embedding. It is typically made of organic solvent-resistant plastic or metal materials, and the cassette has perforations to facilitate the exchange of fixative and subsequent processing liquids. The DRG, flattened on the filter paper, is placed into the embedding container along with the filter paper; the filter paper maintains the flatness of the DRG during fixation. "Post-fixation" refers to the process of fully immersing the DRG in a fixative solution for thorough fixation after it has been removed from the spine. The purpose of post-fixation is to ensure that the tissue is fully and uniformly fixed, guaranteeing that intracellular proteins, nucleic acids, and other biomolecules are completely cross-linked and fixed to maintain the tissue's morphology and antigenicity long-term. Post-fixation typically takes longer than pre-fixation to ensure that the fixative solution fully penetrates the tissue.

[0034] Preferably, the fixative used for post-fixation is neutral buffered formalin with a mass percentage concentration of 5% to 15%. In this invention, "neutral buffered formalin," also known as neutral formalin or neutral buffered formaldehyde solution, is one of the most commonly used fixatives in histology. Neutral buffered formalin mainly consists of formaldehyde (the effective fixative component), a phosphate buffer system (to maintain pH), and deionized water. Its fixation principle is that formaldehyde molecules undergo a cross-linking reaction with the amino and thiol groups in protein molecules, forming methylene bridges. This results in stable covalent cross-linking between protein molecules, thereby fixing intracellular proteins and other biomolecules in their physiological spatial positions. The pH of neutral buffered formalin is usually maintained between 7.2 and 7.4, close to the physiological pH. Fixation under these pH conditions provides good preservation of tissue antigenicity. Neutral buffered formalin has a fast penetration speed, uniform fixation effect, and allows for long-term preservation of fixed tissues. It is the most commonly used fixative for optical microscopy and immunohistochemical detection. In this invention, the mass percentage concentration of the neutral buffered formalin can be 5% to 15%, preferably 8% to 12%, and more preferably 10%.

[0035] Preferably, the post-fixation time is 24–72 hours. In this invention, the post-fixation time refers to the duration during which the DRG tissue is completely immersed in the fixative for fixation. The post-fixation time can be 24–72 hours, preferably 36–60 hours, and more preferably 48 hours. Too short a fixation time may result in insufficient tissue fixation, incomplete stabilization of cell structures, and affect the morphological observation and immunohistochemical staining effects of subsequent sections; too long a fixation time may lead to excessive cross-linking, causing the antigenic determinants to be shielded, affecting the sensitivity of immunohistochemical detection. Within the above time range, the DRG tissue can be fully and uniformly fixed, with well-preserved cell morphology and effective preservation of antigenicity, and can be directly used for subsequent paraffin embedding, HE staining, immunohistochemical staining, and in situ hybridization.

[0036] Preferably, the specific operation of exposing and removing the complete spine is as follows: Take a dead rat, cut open the abdomen and sternum upwards along the midline of the abdomen from the pubic symphysis to expose the thoracic cavity, separate the thoracic organs, and then transversely cut the connection between the cervical vertebrae and the skull at the foramen magnum to remove the complete spine. In this invention, the "midline of the abdomen" refers to the longitudinal line running along the midline of the rat's abdomen from the pubic symphysis to the xiphoid process of the sternum. The skin in this area is thin and has fewer blood vessels; cutting along this line minimizes bleeding. The "pubic symphysis" is the fibrocartilaginous connection between the two pubic bones at the front of the pelvis, and is an easily identifiable bony landmark at the lowest point of the rat's abdomen. The "sternum" is a flat bony plate in the center of the ventral side of the rat's chest, connecting the two ribs. Cutting open the sternum fully exposes the thoracic cavity. The "foramen magnum" is a large foramen on the occipital bone at the base of the skull, serving as a passage between the cranial cavity and the vertebral canal; the spinal cord connects to the brain through this foramen. Transversely severing the connection between the cervical vertebrae and the skull at the foramen magnum allows for separation of the spine from the skull while preserving the complete spinal canal structure, avoiding damage to the spinal cord and nerve roots within the spinal canal. This procedure yields a complete spinal specimen from the cervical vertebrae to the sacrum, with each segment's DRG preserved in its original anatomical position.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example This embodiment provides a method for obtaining rat dorsal root ganglion (DRG): Step 1: Animal pretreatment and spinal exposure Take the dead rat, fix it in a supine position, and cut along the midline of the abdomen upwards from the pubic symphysis, cutting open the abdominal skin and abdominal wall muscles to fully expose the thoracic and abdominal cavities. After carefully separating the thoracic organs, locate the junction of the cervical vertebrae and skull at the top of the spine (around the foramen magnum), and use bone shears to transversely cut the cervical vertebrae and skull at this location.

[0039] Step 2: Remove soft tissue around the spine Place the separated spine on a pre-cooled dissection table, dorsally upward. Carefully remove the deep back muscles, intercostal muscles, and major blood vessels, esophagus, trachea, and other organs attached to the vertebral injury using curved forceps and dissecting scissors. Proceed from the cervical spine to the lumbar spine, taking care to preserve the thin layer of connective tissue around the intervertebral foramina.

[0040] Step 3: Longitudinal incision of the spinal canal With the spine facing upwards, use a pointed bone-biting forceps or laminar bone-biting forceps, starting from the lumbar vertebrae, to sequentially bite off the spinous processes and laminae of each vertebral body along the midline. First, make a small hole at the junction of the lamina and vertebral body on one side. Then, insert one side of the forceps into the spinal canal and carefully slide it upwards, peeling back the entire posterior wall (lamina) of the spinal canal segment by segment. Be careful to keep the forceps parallel to the surface of the spinal cord to avoid downward pressure.

[0041] Step 4: Dissect the intervertebral foramen and expose the nerve root and DRG. Under a stereomicroscope, the connective tissue at the intervertebral foramen is gently grasped with micro-forceps, and the dura mater and epineurium covering the DRG are torn away along the direction of the nerve root. From the lumbar spine to the cervical spine, the enlarged structures inside each intervertebral foramen are located sequentially. The annulus fibrosus around the DRG is cut with micro-scissors, completely freeing the DRG from the bony border of the intervertebral foramen.

[0042] Step 5: After fixation with 10% lithium carbonate solution, use hematoxylin and eosin for staining (in-situ marking before sampling). Gently rinse the exposed spinal cord and DRG surface with pre-cooled physiological saline to remove blood stains. Then, add 10% lithium carbonate solution to cover the entire exposed area and let it stand for 1 minute. Next, add hematoxylin staining solution to the DRG area and stain for 1-2 minutes, then quickly rinse off excess staining solution with PBS buffer.

[0043] Step Six: Separate the nerves from the spinal cord within the spinal canal and cut the nerves. Gently lift the anterior and posterior roots of the spinal nerve on one side of the spinal cord using micro-forceps, and trace the DRG distally along the posterior root. Cut the nerve root at the proximal end (closer to the spinal cord) and distal end (closer to the intervertebral foramen) of the DRG using micro-scissors. For each DRG, first cut the distal spinal nerve, then cut the proximal posterior root, and finally gently remove the DRG from its original position with forceps. Repeat this process until all target segments of the DRG are removed.

[0044] Step 7: Post-embedding and fixation treatment Gently transfer the extracted DRG to filter paper with tweezers, onto a small amount of 10% neutral buffered formalin (10% NFB). Under a stereoscope, use a fine needle to flatten the DRG. Then, fold the filter paper to wrap the DRG and place it in a perforated embedding cassette, labeling it with a number. Immerse the embedding cassette in a fixation bottle containing 5-8 times its volume of 10% NBF and fix at room temperature for 48 hours.

[0045] In this example, DRG samples were collected from rats (n=5, weighing 200-250g, half male and half female), and the results are as follows: All rats' cervical, thoracic, and lumbar vertebrae DRGs were completely removed, with an average of 7-8 DRGs obtained per animal. None of them were broken or lost due to operational errors.

[0046] Under a light microscope, HE-stained sections showed intact DRG neuron cell bodies, clear Nissl bodies, and tightly packed satellite cells, with no obvious vacuoles or nuclear pyknosis. After hematoxylin staining, the cell nuclei appeared blue, the cytoplasm appeared pink, and the background was clean with no blood cell contamination.

[0047] Using the traditional laminectomy method as a control, which involves opening each segment of the vertebral lamina after harvesting the animal's spine to expose the vertebral canal, and then removing the dorsal root ganglia through the intervertebral foramen, the sampling and statistical analysis were performed under the same conditions and methods. The sampling efficiency of the method of the present invention was compared with that of the traditional method. The results show that the method of the present invention significantly increases the number of complete DRGs obtained while shortening the sampling time for each DRG. Statistical analysis showed that the difference between the two groups was highly significant. The specific statistical methods and results are as follows: Data analysis was performed using SigmaPlot 12.0 software. Data are expressed as mean ± standard deviation (X ± S). The t-test was used to analyze the differences between the two groups, and P < 0.01 was considered statistically significant.

[0048] The results are shown in Table 1 below: Table 1. Effects of different sampling methods on the efficiency and number of intact rat DRG samples obtained. Traditional methods 5 6.80 ± 0.831 3.01 ± 0.600 This invention 5 5.30 ± 0.882 7.86 ± 1.022 The material extraction effect of the embodiment is as follows Figures 1-4 As shown: Figure 1 and Figure 2 These are gross photographs of the DRG region before and after fixation with hematoxylin in situ staining, respectively, without and after the application of this invention. Figure 3 and Figure 4 The images shown are corresponding microscopic observations. The results indicate that after lithium carbonate prefixation and hematoxylin staining, the DRG showed a significant color difference from the surrounding tissue, making it easy to locate and separate; under the microscope, the neuronal cell bodies were intact, Nissl bodies were clear, and the background was clean. In contrast, the untreated group showed blurred boundaries between the DRG and the surrounding tissue, and under the microscope, cellular structure damage and blood cell contamination were visible. This demonstrates that the prefixation and staining steps of this invention can effectively improve the accuracy of DRG identification and the quality of tissue sampling.

[0049] As demonstrated by the above embodiments, this invention provides a method for obtaining dorsal root ganglia from dead rats, enabling efficient and complete acquisition of dorsal root ganglion tissue from various segments of the cervical, thoracic, and lumbar vertebrae. The obtained dorsal root ganglion neurons exhibit intact cell body structures, clearly identifiable Nissl bodies, tightly surrounding satellite cells, a clean background free of blood cell contamination, and well-preserved tissue morphology, fully meeting the experimental requirements for subsequent detection such as paraffin sectioning, HE staining, immunohistochemical staining, and in situ hybridization. The sampling method of this invention features clear operational steps, a smooth process, high sampling success rate, and strong tissue identification accuracy, demonstrating good operability and reproducibility, and providing reliable dorsal root ganglion experimental materials for neuroscience research.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for obtaining tissue from the dorsal root ganglion of a dead rat, characterized in that, Includes the following steps: Take the dead rat, expose the spine, and remove the complete spine. Remove the soft tissue attached to the vertebrae to expose the bony landmarks of the vertebrae; Starting from the lumbar vertebrae, the spinous processes and lamina of each vertebra are separated segment by segment along the midline of the dorsal side of the spine, and the posterior wall of the spinal canal is lifted as a whole to expose the spinal cord and the dorsal root ganglia of each segment. Pre-fixation was performed by applying fixative to the exposed dorsal root ganglion area, followed by in-situ staining with hematoxylin staining solution to make the dorsal root ganglion appear in a different color from the surrounding tissue. Tracing the posterior root of the spinal nerve to the dorsal root ganglion, first cut the spinal nerve at the distal end of the dorsal root ganglion, then cut the posterior root at the proximal end of the dorsal root ganglion, and remove the dorsal root ganglion from its original position.

2. The material sampling method according to claim 1, characterized in that, The fixative is a lithium carbonate solution with a mass percentage concentration of 5% to 15%.

3. The material sampling method according to claim 1, characterized in that, The pre-fixed processing time is 1 to 3 minutes.

4. The material sampling method according to claim 1, characterized in that, The staining time for the hematoxylin staining solution is 1 to 2 minutes.

5. The material sampling method according to claim 1, characterized in that, It also includes the step of rinsing off excess dye after the in situ staining.

6. The material sampling method according to claim 1, characterized in that, The location where the distal end of the spinal nerve is first cut is 0.5–2.5 mm from the distal end of the dorsal root ganglion, and the location where the proximal end of the posterior root is then cut is 0.5–2.5 mm from the proximal end of the dorsal root ganglion.

7. The material sampling method according to claim 1, characterized in that, It also includes the steps of laying the removed dorsal root ganglion flat on filter paper and placing it into an embedding container, as well as the step of post-fixation with fixative.

8. The material extraction method according to claim 7, characterized in that, The fixative used for post-fixation is a neutral buffered formalin with a mass percentage concentration of 5% to 15%.

9. The material extraction method according to claim 7, characterized in that, The fixation time is 24 to 72 hours.

10. The material sampling method according to claim 1, characterized in that, The specific procedure for exposing and removing the complete spine is as follows: Take a dead rat, cut open the abdomen and sternum along the midline of the abdomen from the pubic symphysis upwards to expose the thoracic cavity, separate the thoracic organs, and then cut the connection between the cervical vertebrae and the skull transversely at the foramen magnum to remove the complete spine.

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