Construction method and application of spinal cord injury animal model for maintaining integrity of subarachnoid space
By constructing a closed model by suturing the dura mater after spinal cord injury, the problem of spinal cord microenvironment instability caused by unsutured dura mater in spinal cord injury models was solved, achieving more efficient functional recovery and treatment results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI AIPOKANG BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-03-31
- Publication Date
- 2026-05-15
AI Technical Summary
In existing spinal cord injury models, the dura mater is not sutured, leading to an unstable spinal cord microenvironment, which affects the assessment of functional recovery and the simulation of the repair process, and makes it impossible to study the repair process under closed chamber conditions.
After spinal cord injury, the dura mater is tightly sutured, and biological glue is used to ensure its integrity, thus constructing a closed model to maintain the subarachnoid space.
It improves the repeatability and stability of the model, promotes the recovery of motor function and tissue morphology in animals, provides a more sensitive evaluation platform for neural repair therapy, simulates the clinical surgical process, and enhances treatment outcomes.
Smart Images
Figure CN122031132A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biology, specifically to a method for preparing an animal model, and more particularly to a method for constructing and applying an animal model of spinal cord injury that maintains the integrity of the subarachnoid space. Background Technology
[0002] Spinal cord injury is a severe central nervous system trauma that typically results in permanent loss of sensory and motor function below the level of injury. Currently, much research focuses on complex treatment strategies such as cell transplantation, drug intervention, and biomaterial filling. For example, some studies have used MgFe-LDH / NT3 nanomaterials to fill the injury cavity combined with ultrasound stimulation. Others have used PLGA / type I collagen / chitosan composite membranes as a substitute for the dura mater. While these methods have shown some effectiveness, they also present challenges such as high cost, technical complexity, potential immune rejection, and uncertain safety.
[0003] In the preparation of spinal cord injury models, the treatment of the dura mater is often considered the last step in the surgical procedure, and its importance has long been overlooked. The routine practice is to leave it unsutured, simply cover it, or use artificial materials as a substitute. However, an intact dura mater is a crucial structure for maintaining normal intracranial pressure in the spinal cord, providing physical protection, preventing scar tissue invasion, and maintaining local concentrations of neurotrophic factors.
[0004] Existing models of complete spinal cord transection have limitations in simulating clinical injury pathology and assessing repair strategies. The dura mater is continuously open, which disrupts the stability of the spinal cord microenvironment and introduces a large number of confounding variables. For example, the loss of cerebrospinal fluid can change the hydraulic environment around the spinal cord, which may lead to subarachnoid collapse, flushing away or diluting local inflammatory mediators, further inhibiting endogenous repair mechanisms, resulting in distorted assessment of functional recovery. It is also impossible to study the repair process under closed chamber conditions (which are closer to most clinical situations). Summary of the Invention
[0005] To address the aforementioned technical problems in the prior art, this invention provides a method for constructing and applying an animal model of spinal cord injury that maintains the integrity of the subarachnoid space. This method and application of an animal model of spinal cord injury that maintains the integrity of the subarachnoid space aims to solve the technical problem in the prior art where the dura mater is not sutured, making it impossible to study the repair process under closed chamber conditions.
[0006] This invention provides a method for constructing an animal model of spinal cord injury that maintains the integrity of the subarachnoid space, comprising the following steps: Using mammals with spinal cord injuries, the opening of the dura mater is clamped, and bio-adhesive is dripped into the clamped area. Once the bio-adhesive forms a frosty consistency, the dura mater is adhered together, and then sutured.
[0007] The present invention also provides a method for preparing the above-mentioned spinal cord injury mammal, comprising the following steps: 1) First, anesthetize the mammal, use a wound retractor to open the skin, cut open the muscles and fascia, bluntly dissect the muscles to expose the lamina, locate the T8-T10 position, and with the T9 lamina as the center, separate the T9-10 interlaminar space to expose the ligamentum flavum, clamp the T9 spinous process, and cut the ligamentum flavum along the middle of the T9-10 interlaminar space to expose the transparent dura mater and the underlying spinal cord tissue; 2) Locate the U-shaped blood vessel under the T9 vertebral lamina, cut open the dura mater along the side of the spinal cord, sever the spinal cord, cut the severed spinal cord into pieces, aspirate the fragmented spinal cord tissue, and remove the spinal cord tissue; thus, obtain the spinal cord injury of the mammal.
[0008] Furthermore, the mammal in question is a mouse.
[0009] Furthermore, the bio-adhesive is α-cyanoacrylate.
[0010] This invention also provides the application of the spinal cord injury animal model prepared by the above method, which maintains the integrity of the subarachnoid space, in the study of motor function, nerve tissue recovery, or tissue morphology recovery in animals after spinal cord injury.
[0011] This invention provides a method for constructing a spinal cord injury model that maintains the physiological integrity of the subarachnoid space (a method for modeling spinal cord injury with dura mater suturing in mice). After spinal cord injury modeling in experimental animals (such as mice), without using other complex biological materials or drug interventions, the cut dura mater is directly watertightly sutured to restore its integrity and continuity, which helps in the prognosis and recovery of spinal cord injury and improves the therapeutic efficiency of spinal cord injury drugs. This method can better simulate the surgical process and treatment methods for spinal cord injury in clinical practice, that is, improving the treatment effect of spinal cord injury and promoting nerve repair by suturing the dura mater. This invention adds a key step after completing a complete spinal cord transection: precisely suturing the combined dura mater, thereby constructing a spinal cord injury animal model that maintains the physiological integrity of the subarachnoid space with a closed dura mater. This invention shows that after induced experimental spinal cord injury, tight suturing of the dura mater can improve the motor function and tissue morphological recovery in mice.
[0012] Compared with existing technologies, the technical effects of this invention are positive and obvious.
[0013] 1. Superior Model: This model provides a more reproducible and stable experimental method, reducing evaluation variability caused by factors such as external tissue adhesion.
[0014] 2. Better functional recovery: Experiments have shown that the recovery speed of hindlimb motor function and sensory function in animals under this model is significantly better than that of traditional models, providing a more sensitive and better baseline platform for evaluating neurorepair therapy.
[0015] 3. Enhanced Mechanism Research Value: This model creates a unique, intradural repair microenvironment. Through this model, key processes such as endogenous neural stem cell activation, axonal regeneration, myelin regeneration, and neural circuit remodeling can be studied more efficiently and purely, without interference from external tissues. This model is a powerful tool for studying the mechanisms of "neural repair within a confined space."
[0016] 4. Significance for clinical translation: This model is closer to the principle of repairing the dura mater as much as possible to isolate it from the outside world during clinical surgery. Therefore, effective therapies screened based on this model have high potential for clinical translation.
[0017] Through innovative thinking and extensive experimental verification, the inventors discovered that meticulous watertight suturing of the dura mater after injury contributes far more to functional recovery than expected, even surpassing the effects of various complex interventions. This discovery provides a completely new research direction and model for the treatment of spinal cord injuries. Attached Figure Description
[0018] Figure 1 A schematic diagram of a complete spinal cord transection injury model designed to preserve the integrity of the dura mater. Figure 1 In this diagram, 'a' represents a schematic diagram of a mouse surgery. Figure 1 b in the figure represents: a simulated spinal cord diagram after T9 region modeling in the SCI group and the SCI+dure group; Figure 1 In the image, 'c' represents surgical photos of three different treatment methods: the Sham group, the SCI group, and the SCI+dura group.
[0019] Figure 2 The images show the results of body weight analysis, BMS score, and MRI analysis of the mice in the SCI+dura group, indicating that the mice recovered their motor function and spinal cord morphology better. Figure 2 In this context, 'a' represents the mouse weight record data. Figure 2 In this context, 'b' represents the mouse BMS score record data. Figure 2 In the image, 'c' represents the MRI images of the Sham group, SCI+dura group, and SCI group, with the arrow pointing to the location of spinal cord injury.
[0020] Figure 3 Footprint analysis and hindlimb and functional position results for the Sham group, SCI group, and SCI+dura group show that the motor function of mice in the SCI+dura group recovered better.
[0021] Figure 4The figure shows the RNA sequencing analysis results of this invention, which shows that the SCI+dura group mice highly express repair genes such as Sfrp4, Col3a1, and Mmp12. Figure 4 In the figure, 'a' represents the heatmap of inter-group correlation. The horizontal and vertical axes are groups (sham, SCI, SCI+dura), and the colors represent the Pearson correlation coefficients (the color bars range from 0.96 to 1.00, with darker red indicating higher correlation). Figure 4 b: Top differentially upregulated gene heatmap, showing the expression levels of the top 10 genes that were significantly upregulated in the SCI+dura and SCI groups in different samples; Figure 4 The c:GO enrichment analysis plot shows the GO functional enrichment analysis of upregulated genes in the SCI+dura group.
[0022] Figure 5 The figure shows the RNA sequencing analysis results of this invention, which shows that the SCI+dura group mice highly express five major genes: Fn1, Col1a2, Col3a1, Fbn1, and Mmp9. Detailed Implementation Example
[0023] This invention provides a method for constructing an animal model of spinal cord injury that maintains the integrity of the subarachnoid space, specifically including the following steps: 1. Spinal cord exposure stage: Mice (C57 / BL6 wild-type mice, 8 weeks old, female) were anesthetized by intraperitoneal injection of sodium pentobarbital. The hair on their backs was removed, and the surgical site was disinfected with povidone-iodine. The skin was incised along the middle of the back with a scalpel for about 1.5 cm. The skin was opened with a wound retractor, and the muscle layer and fascia were cut. The muscles were bluntly dissected to expose the lamina. The T8-T10 position was located. With the T9 lamina as the center, the T9-10 interlaminar space was separated to expose the ligamentum flavum. The T9 spinous process was clamped with hemostatic forceps, and the ligamentum flavum was cut along the middle of the T9-10 interlaminar space to expose the transparent dura mater and the underlying spinal cord tissue.
[0024] 2. Spinal cord resection stage: Using a spinal clamp, the spinal cord is positioned at the U-shaped blood vessel below the T9 lamina. The dura mater is incised along the side of the spinal cord with an ophthalmic scalpel. Fine forceps are used to carefully insert into the dura mater to clamp the spinal cord. The clamped spinal cord is gently cut into pieces with a vitreous microscissors. The fragmented spinal cord tissue is slowly aspirated with a suction device and a negative pressure suction device. Finally, 1.5 mm of spinal cord tissue is removed.
[0025] 3. Dura mater suturing stage: Gently clamp the opening of the dura mater with fine forceps, and slowly drip biological glue (a-cyanoacrylate) (medical commercial product) into the interface using a glass electrode. Wait 3-5 seconds and release the micro-forceps. When the glue forms a frosty state, the dura mater is adhered together. The suturing technique should be gentle, and the number of stitches should be maintained at 8-10 to ensure the airtightness of the wound. The muscles and skin are sutured sequentially using a discontinuous suturing method.
[0026] The animal models obtained above were also cared for: after suturing, iodine solution and erythromycin ointment were applied to the wound.
[0027] All procedures were performed in accordance with aseptic techniques. After the procedure, the mice were placed on a warm mat to wait for recovery until they regained consciousness. Two days after the procedure, cefuroxime was administered intraperitoneally to prevent infection.
[0028] Observe the mice's condition and weight daily, provide them with cotton for warmth, and house them individually.
[0029] The mice used in the specific control experiment were constructed using the method described above.
[0030] Sham group mice: lamina were removed and the dura mater was peeled off without severing the spinal cord tissue.
[0031] Blank control group mice (SCI group): 1.5 mm of spinal cord tissue was removed and the dura mater at the injury site was completely severed.
[0032] Experimental mice (SCI+dura group): 1.5 mm of spinal cord tissue was removed and the dura mater was sutured.
[0033] After the surgery, artificial urination was performed twice a day using the Crede technique until bladder function recovered.
[0034] Experimental verification: 1. Evaluation of animal locomotion function: Motor function in mice was assessed using BMS scores, weight analysis, and footprint analysis.
[0035] 1.1 The BMS scoring system was used to evaluate the joint movement and coordination of the hind limbs of mice, ranging from 0 to 9 points. Two observers conducted a double-blind scoring experiment on each mouse for 3-5 minutes at fixed time points each week.
[0036] 1.2 Weight analysis was used to perform basic assessments of the mice's postoperative condition. At a fixed time each week, one observer measured the weight of each mouse.
[0037] 1.3 Footprint analysis was used to observe the recovery of hind limb gait in mice. Seven weeks post-surgery, the forelimbs of mice were colored blue and the hind limbs were colored red. Mice walked on a fixed path covered with white paper, and their footprints were collected for subsequent analysis.
[0038] 2. Correlation analysis of magnetic resonance imaging (MRI) of spinal cord structures Forty-one days after spinal cord injury, mice were anesthetized and fixed in a prone position. Magnetic resonance imaging was performed using a United Imaging 9.4T ultra-high field animal magnetic resonance imaging system to analyze the structure and morphology of the spinal cord.
[0039] 3. Bulk RNA sequencing analysis of spinal cord tissue Fifty-seven days after modeling, both the experimental and control groups were euthanized by intraperitoneal injection of an excessive amount of sodium pentobarbital (100 mg / kg). After euthanasia, the mice were dissected, and the spinal cord tissue (approximately 5 mm), including the damaged area, was carefully removed using microforceps for RNA transcriptome sequencing and bioinformatics analysis.
[0040] 4. Results: 4.1 This invention developed a novel model—the dura mater suture model—using the T9 spinal cord transection model in C57 / BL6 mice. The advantages of this model were verified through the above experiments. (See details...) Figure 1 (a, b, c in the text).
[0041] 4.2 Within 8 weeks after modeling, the Basso Mouse Scale (BMS) scores and body weight of mice were analyzed. Compared with the SCI group, the SCI+dura group showed better recovery of motor function, improved Basso Mouse Scale scores, and improved body weight. (See details...) Figure 2 (a and b in the text).
[0042] 4.3 Further analysis using magnetic resonance imaging (MRI) revealed that the SCI+dura group showed better recovery compared to other groups. The signal within the spinal cord parenchyma was homogeneous, the spinal cord tissue appeared as a relatively uniform gray, and the cerebrospinal fluid within the spinal canal appeared as a uniform bright white surrounding the spinal cord, forming a good natural contrast and highlighting the normal contours of the spinal cord. In contrast, the imaging results of the SCI group showed abnormal signals diffusing from the central region to the periphery, with unclear boundaries from the normal spinal cord gray matter signal, consistent with typical manifestations of internal spinal cord lesions. (See details...) Figure 2 (c in the text)
[0043] 4.4 Gait analysis and photographic analysis of hind limb functional recovery in mice revealed that, compared to the SCI group, the SCI+dura group showed better recovery of motor function. Hind limb dragging was reduced, hind limbs were stronger, and the feet could be slightly lifted during walking, indicating enhanced coordination and significantly improved motor function. Figure 3 ) Finally, transcriptome sequencing revealed that in the SCI+dura group, the genes Sfrp4, Col3a1, Mmp12, Cd36, Acp5, Ctsk, Clec4d, Anxa1, Mmp13, and Mmp3 were significantly upregulated, while the genes Pcdhga4, Ndor1, Mid1, Erd1, Sgk1, Plin4, Hspa1b, Snhg4, Rasl11a, and Rbx1-ps were significantly downregulated. Considering the functional characteristics of these two gene groups and the pathological mechanisms of spinal cord injury (SCI), the first group of genes (Sfrp4, Col3a1, Mmp12, etc.) plays a more complex and crucial role in injury repair, while the second group of genes (Pcdhga4, Mid1, Hspa1b, etc.) mainly maintains cellular homeostasis. From the perspective of "contributing to repair," the expression of the first group of genes is an indispensable process in the repair process. Figure 4 ).
[0044] RNA transcriptome sequencing analysis revealed five significantly upregulated source genes in the SCI+dura group compared to those expressed in the SCI group: Fn1, Col1a2, Col3a1, Fbn1, and Mmp9. The upregulated Fn1, Col1a2, Col3a1, Mmp9, and Fbn1 genes collectively point to a core biological process—suture treatment promotes dynamic ECM remodeling during the chronic phase (7 weeks) of spinal cord injury, including the coupling of collagen deposition and degradation, and the synthesis of scaffold proteins, potentially leading to more structurally optimized fibrous scars and providing a better microenvironment for tissue repair and functional recovery. The presence of Mmp9 is crucial, indicating that suture-induced repair is not simply fibrosis, but an active and regulated matrix renewal process. Figure 5 ).
[0045] In summary, this invention constructs a spinal cord injury model with greater translational medical significance by simulating clinical dura mater repair surgery. This model reveals that simply 'closing the repair door' (suturing the dura mater) can create a superior 'construction environment' for the complex 'repair engineering' within the spinal cord. This provides a direct theoretical basis and validation model for developing novel treatment strategies aimed at optimizing the local microenvironment of the injury.
Claims
1. A method for constructing an animal model of spinal cord injury that maintains the integrity of the subarachnoid space, characterized in that... Includes the following steps: Using mammals with spinal cord injuries, the opening of the dura mater is clamped, and bio-adhesive is dripped into the clamped area. Once the bio-adhesive forms a frosty consistency, the dura mater is adhered together, and then sutured.
2. The method for constructing an animal model of spinal cord injury that maintains the integrity of the subarachnoid space according to claim 1, characterized in that, The preparation method for mammals with spinal cord injuries is as follows: 1) First, anesthetize the mammal, use a wound retractor to open the skin, cut open the muscles and fascia, bluntly dissect the muscles to expose the lamina, locate the T8-T10 position, and with the T9 lamina as the center, separate the T9-10 interlaminar space to expose the ligamentum flavum, clamp the T9 spinous process, and cut the ligamentum flavum along the middle of the T9-10 interlaminar space to expose the transparent dura mater and the underlying spinal cord tissue; 2) At the U-shaped blood vessel under the T9 vertebral lamina, cut open the dura mater along the side of the spinal cord, sever the spinal cord, cut the severed spinal cord into pieces, aspirate the fragmented spinal cord tissue, and remove the spinal cord tissue; thus, a mammal with spinal cord injury is obtained.
3. The method for constructing an animal model of spinal cord injury that maintains the integrity of the subarachnoid space according to claim 1, characterized in that, The mammal in question is the mouse.
4. The method for constructing an animal model of spinal cord injury that maintains the integrity of the subarachnoid space according to claim 1, characterized in that, The bio-adhesive is α-cyanoacrylate.
5. The application of the spinal cord injury animal model maintaining the integrity of the subarachnoid space prepared by the method of claim 1 in the study of motor function, neural tissue recovery or tissue morphology recovery in animals after spinal cord injury.