A method for constructing a rat severe spinal cord injury immediate decompression model

Abstract

The application belongs to the technical field of biomedical experimental animal model construction, and provides a rat severe spinal cord injury immediate decompression model construction method. The method establishes a standardized surgical procedure, including minimally invasive exposure through the paraspinal muscle gap, precise dura mater incision, warm saline low-pressure lavage debridement, and strict dura mater suture repair. The method realizes immediate and effective reduction of intrathecal pressure, removal of harmful substances in the injury area, and stabilization of the local microenvironment after severe spinal cord injury. The application solves the problems of instability, poor repeatability, and difficulty in simulating the clinical decompression effect caused by the non-standard dura mater incision, uncontrollable lavage, and high risk of secondary injury in the prior art. The model constructed by the method is highly standardized and has strong repeatability, and is suitable for in-depth study of the neuroprotective mechanism of dura mater incision decompression treatment for spinal cord injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical experimental animal model construction technology, and relates to spinal cord injury (SCI) research models, specifically to a method for constructing an immediate decompression model of severe spinal cord injury in rats. Background Technology

[0002] Spinal cord injury (SCI) is a devastating disease that can lead to severe neurological dysfunction. Its pathological process is complex, involving primary mechanical injury and secondary cascade reactions. Establishing stable, reproducible animal models that effectively mimic clinicopathological features is fundamental to in-depth research into the mechanisms of SCI and the exploration of effective treatment strategies. Rats, due to their anatomical structure and physiological responses being somewhat similar to humans, and their relatively low cost, have become one of the most commonly used experimental animals in SCI research.

[0003] Currently, the mainstream methods for constructing rat models of severe spinal cord injury mainly rely on laminectomy combined with mechanical impact (such as free fall, controlled impact devices, etc.). These methods have made significant progress in standardizing injury parameters and can generate relatively consistent initial injuries. However, these traditional models have significant limitations in simulating the crucial "dural decompression" step and related treatments after severe clinical SCI, thus limiting their application value in studying decompression treatment mechanisms and evaluating novel interventions (such as hydrogels, cell transplantation, etc.).

[0004] First, the advantages of dural endoscopic incision have not been fully realized. In clinical treatment, dural endoscopic decompression of severely swollen spinal cord is one of the key surgical procedures for salvaging neurological function, aiming to rapidly reduce intracapsular pressure and improve spinal cord blood supply. However, existing rat models typically only involve mechanical trauma, or even if the dura mater is incised, lack subsequent systemic treatment. This leads to a rapid re-establishment of high pressure at the injury site due to continuous bleeding and tissue edema after decompression, resulting in persistent internal environmental disturbances and ineffective drainage of inflammatory mediators. Therefore, existing models struggle to reliably reproduce the neuroprotective effects of clinical decompression surgery, hindering in-depth research into the molecular and cellular mechanisms of decompression therapy.

[0005] Secondly, the dura mater incision procedure itself lacks standardization and controllability. Because the rat dura mater is not thin and the spinal cord is small, existing research lacks standardized incision procedures specifically tailored to their anatomical characteristics. Operators often rely on experience, easily leading to incision misalignment, inconsistent lengths, edge tearing, and even direct damage to the underlying spinal cord tissue. This uncontrollable secondary injury severely affects the stability of the model and the reliability of experimental results.

[0006] Third, there is a lack of effective methods for removing harmful substances from the injured area. After mechanical injury, blood clots, necrotic cell fragments, and inflammatory exudates rapidly form locally. Traditional models typically do not treat these substances, allowing them to remain. These residues not only continue to produce a space-occupying effect, increasing local pressure and hindering spinal cord pulsation, but the toxic substances they release can also exacerbate secondary injuries and may physically block the effective contact between subsequent therapeutic drugs or biomaterials and the injured tissue.

[0007] Fourth, the methods of irrigation and debridement are crude and risky. The few studies that have attempted irrigation have mostly used relatively high-pressure flushing methods, which can easily cause direct fluid impact damage to the already damaged and fragile spinal cord tissue, violating the principles of minimally invasive surgery. There is a lack of a refined irrigation technique that uses suitable temperature and controllable pressure to effectively remove debris while maximizing the protection of spinal cord tissue.

[0008] Fifth, the minimally invasive nature of auxiliary procedures such as intraoperative hemostasis is insufficient. Commonly used electrocoagulation hemostasis or strong suction can easily cause thermal or traction damage to the delicate subdural structures, affecting the purity and consistency of the model.

[0009] Finally, the overall process lacks a standardized system under a microscope. From muscle separation and the extent of lamina exposure to the location of the dural incision, existing methods lack unified and detailed technical standards, resulting in large variability in models built by different laboratories and even different batches within the same laboratory. This seriously affects the comparability of data and the reliability of conclusions in multi-center studies.

[0010] In summary, existing rat models of severe spinal cord injury have systemic shortcomings in simulating key aspects of clinical decompression therapy. Therefore, there is an urgent need to develop a comprehensive surgical method integrating standardized dural incision, controlled low-pressure irrigation and debridement, and refined dural repair. This would allow for the construction of a highly reproducible, immediate rat decompression model that accurately reflects the therapeutic effect of decompression, maintains a clean internal environment, and provides a reliable research tool for in-depth research into the mechanisms of decompression therapy after spinal cord injury and for developing new combined treatment strategies. Summary of the Invention

[0011] The purpose of this invention is to provide a method for constructing an immediate decompression model of severe spinal cord injury in rats. By establishing a standardized comprehensive surgical procedure of dural incision, controllable low-pressure irrigation and debridement, and refined dural repair, this invention aims to solve the problems of ineffective release of intracapsular pressure, non-standard dural incision operation, uncontrollable irrigation process, easy secondary damage, and poor model repeatability in existing models.

[0012] To achieve the above objectives, the technical solution of the present invention is as follows: A method for constructing an immediate decompression model of severe spinal cord injury in rats, comprising the following steps:

[0013] S1. Animal anesthesia and localization: Anesthetize the rats and locate the T9 / 10 vertebral body as the central segment;

[0014] S2. Surgical exposure: Make a small longitudinal incision in the T13 vertebral body region and extend the incision from the midline of the back to the head. Bluntly dissect the paravertebral muscles to expose the T9 / 10 lamina region. Without removing the muscle tissue, bite off the lamina tissue layer by layer to expose the dorsal dura mater of the spinal cord.

[0015] S3. Mechanical injury: Using a striking device to apply a quantitative impact to the exposed dorsal side of the spinal cord to create a severe spinal cord injury;

[0016] S4. Controlled dural incision: After disinfection with 0.1% povidone-iodine, the dura mater is lifted in the midline of the dorsal side of the spinal cord using a tool with a bent tip, in coordination with the breathing rhythm, and cut along the longitudinal axis to form a regular incision through the injured segment, allowing the blood in the cyst to flow out naturally. Then, 8-0 absorbable sutures are used to pull and fix the edges of the dura mater on both sides of the incision.

[0017] S5. Subdural irrigation and debridement: Use warm saline to irrigate the damaged area after the dura mater is cut with low pressure without directly impacting the spinal cord tissue. Use absorbent materials to remove blood clots and tissue debris until clear spinal cord pulsation is observed.

[0018] S6. Dura mater repair: The dura mater incision is sutured, and repair material is applied to the outside of the sutured dura mater.

[0019] S7. Close the surgical area: Suture the muscles and skin layer by layer.

[0020] Preferably, the blunt dissection of the paravertebral muscles in step S2 specifically involves: performing blunt dissection along the natural gaps between the paravertebral muscles with the spinous process as the center, thereby exposing the lamina.

[0021] Preferably, the striking device in step S3 is the LISA precision striking device, with the following operating conditions: depth 1.2 mm, time 0.42 s, and speed 1.000 m / s.

[0022] Preferably, the tool with the bent tip in step S4 is a bent insulin injection needle or a microdural knife.

[0023] Preferably, the temperature of the warm saline solution in step S5 is 37°C.

[0024] Preferably, the low-pressure method in step S5 is as follows: after drawing warm saline with a syringe, the fluid is allowed to flow slowly along the edge of the dura mater incision or the side of the damaged area, avoiding vertical spraying of the fluid onto the surface of the spinal cord tissue.

[0025] Preferably, the adsorbent material in step S5 is a sterile cotton ball.

[0026] Preferably, in step S6, 8-0 or finer absorbable sutures are used to perform continuous or interrupted suturing of the dura mater incision.

[0027] Preferably, the repair material in step S6 is a biological dura mater patch or an artificial dura mater material.

[0028] The beneficial effects of this invention are:

[0029] 1. Technical Operation: Achieving Precise Minimally Invasive Procedures and Structural Preservation

[0030] By employing blunt dissection of the paraspinal intermuscular space instead of traditional muscle resection, the anatomical integrity and biomechanical function of the paraspinal muscles are maintained to the greatest extent, significantly reducing intraoperative bleeding and tissue trauma, and providing a stable surgical field for microsurgical operations. A precise dural incision technique, combined with pre-bent micro-instruments synchronized with the respiratory cycle, avoids mechanical damage to the spinal cord parenchyma and irregular tears of the dura mater. The established isothermal-low-pressure perfusion system can remove blood clots and inflammatory mediators from the injured area while avoiding secondary damage to nerve tissue caused by fluid shear forces, and achieves anatomical and functional reconstruction of the dura mater through a double-layer repair structure.

[0031] 2. Model Building: Establishing a Standardized Research Paradigm

[0032] By systematically integrating key steps such as injury induction, acute decompression, microenvironment cleanup, and dural repair, a reproducible and consistent animal model of spinal cord injury was constructed. This model provides a standardized experimental platform for exploring the molecular mechanisms of dural incision and decompression, assessing the correlation between intracapsular pressure and neurological prognosis, and systematically validating neural repair strategies in a controlled subdural environment, significantly improving the reliability and comparability of subsequent studies.

[0033] 3. Treatment mechanism: Achieving synergistic regulation of multiple pathways.

[0034] The dural incision combined with irrigation and debridement (DDL) technique provided by this invention achieves multi-target intervention for secondary injuries through the synergistic effect of physical decompression and biochemical debridement:

[0035] (1) Improve local hemodynamics: effectively reduce intracapsular pressure in the injured area, restore microcirculation perfusion, and alleviate ischemia-reperfusion injury;

[0036] (2) Regulation of iron homeostasis metabolism: Clearing metabolites such as hemosiderin, downregulating the expression of iron metabolism-related proteins such as transferrin receptor, and inhibiting the activation of ferroptosis pathway;

[0037] (3) Remodeling the inflammatory microenvironment: reducing the expression of pro-inflammatory factors and complement system genes, enhancing anti-inflammatory regulation, and reducing oxidative stress damage.

[0038] In summary, this invention achieves comprehensive regulation of iron load, ferroptosis pathways, and inflammatory microenvironment in the damaged area through the synergistic effect of "decompression + irrigation," thereby reducing secondary injury reactions and providing an effective technical means to promote spinal cord tissue repair. Attached Figure Description

[0039] Figure 1 This is a flowchart of the rat SCI modeling process to dural incision, debridement, and irrigation in this invention (A is anesthesia; B is exposure of the target spinal cord segment; C is the application of a LISA impactor to induce severe contusion (impact depth 1.2 mm, impact velocity 1.0 m / s, residence time 0.42 s); D is local disinfection of the surgical field with 0.1% iodine; E is dural incision; F is local irrigation and debridement; G is dural repair and suturing).

[0040] Figure 2 This is a schematic diagram of the key steps of the dural incision, irrigation, and debridement procedure in this invention, along with corresponding representative intraoperative photographs (A and E show the dorsal spinal cord exposed after laminectomy; B and F show the immediate local condition of the injured spinal cord after severe spinal cord injury; C and G show the local condition of the injured spinal cord after dural incision; D and H show the local condition of the injured spinal cord after the completion of the dural incision, irrigation, and debridement procedure; scale bar: 2 mm).

[0041] Figure 3 This is a schematic diagram of the mechanism of action and key steps of the dural incision and irrigation debridement technique described in the invention in spinal cord injury (A is the spinal cord injury state without the intervention of the present invention; B is the state after the dural incision and irrigation debridement technique described in the present invention).

[0042] Figure 4 This is the analysis result of RNA sequencing (RNA-seq) of spinal cord tissue on day 7 after injury according to the present invention (A is a heatmap of genes related to iron metabolism and erythrocyte metabolism; B is a heatmap of genes related to inflammation and complement; C is a pathway enrichment analysis from the Kyoto Encyclopedia of Genes and Genomes (KEGG); D is a gene ontology (GO) enrichment analysis; E is a ferroptosis gene set enrichment analysis (GSEA)).

[0043] Figure 5 This is the Western blot detection result and quantitative analysis of iron metabolism-related proteins on day 7 after injury according to the present invention (* in the figure means p<0.05, ** means p<0.01, *** means p<0.001, **** means p<0.0001, ns means no significance).

[0044] Figure 6 The results of Western blot detection and quantification of inflammation and anti-inflammatory related proteins in this invention are shown in the figure (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no significance).

[0045] Figure 7 This invention presents the dynamic changes in dorsal spinal cord blood perfusion following contusion spinal cord injury and DDL intervention with decreased intracapsular pressure (A is a pseudo-color image of laser speckle imaging (LSCI) showing the baseline state (before injury), immediately after injury, and at 5, 30, and 90 minutes after injury, with the top row representing the SCI group and the bottom row representing the SCI+DDL group; B is the quantitative analysis result of the blood perfusion index (BFI) in the SCI group; C is the quantitative analysis result of the blood perfusion index (BFI) in the SCI+DDL group; all images use a uniform color scale (0-700 perfusion units, PU); scale bar: 2 mm; in the figure, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no significance). Detailed Implementation

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0049] Example 1

[0050] 1. Model Building

[0051] This invention provides the entire process from rat SCI modeling to dural incision, irrigation, and debridement. Figure 1 ), including the following steps:

[0052] S1. Animal Anesthesia and Positioning:

[0053] Anesthesia was induced by gas anesthesia and maintained by intraperitoneal injection of sodium barbital. After anesthesia, the rat's back was routinely prepared, disinfected with povidone-iodine, and draped with sterile cloths. The T9 / 10 vertebrae were located based on the ribs and used as the central segment for subsequent surgical exposure and injury.

[0054] S2. Surgical Exposure: A small longitudinal incision is made in the T13 vertebral body region and extended cephalad along the dorsal midline. The paravertebral muscles are bluntly dissected to expose the T9 / 10 lamina region. Without removing the muscle tissue, the lamina tissue is removed layer by layer to expose the dorsal dura mater of the spinal cord. Specific procedures are as follows:

[0055] (1) Initial incision and exposure

[0056] Make a small longitudinal incision in the T13 vertebral region to expose the subcutaneous and musculofascial layers. Using sterile scissors, extend the incision along the midline of the back towards the head to expose a larger area of ​​dorsal subcutaneous tissue. Reconfirm the T10 vertebral body using anatomical landmarks such as rib morphology, muscle-fascial junctions, and spinous process positions, and apply pressure with sterile cotton balls to achieve hemostasis.

[0057] (2) Paravertebral muscle separation and lamina exposure

[0058] Using a technique similar to the clinical TLIF approach, blunt dissection is performed along the paravertebral intermuscular space, centered on the spinous process, to progressively expose the lamina region. No muscle tissue is removed throughout the procedure, and damage to the underlying rib structures is avoided. The same procedure is performed bilaterally to achieve a symmetrical surgical field.

[0059] (3) Laminectomy and spinal cord exposure

[0060] In the exposed T9 / 10 lamina region, use small bone forceps to cut the spinous process and gradually enlarge the bone window to expose the T9 / 10 lamina, superior articular process, and inferior articular process. Cut along the superior margin of the inferior articular process to open the articular capsule, and remove the lamina tissue layer by layer until the dorsal dura mater of the spinal cord is fully exposed (see section 2A for the exposed dorsal spinal cord). Figure 2 (E). During all procedures, use sterile cotton balls to apply gentle pressure for hemostasis.

[0061] (4) Refinement of surgical field

[0062] Under a microscope, further clean the bone edges and debris to ensure complete exposure of the dorsal dura mater of the spinal cord. Observe the shape of the dorsal median vein of the spinal cord to confirm that it has not been damaged during the operation. Continuously moisten the surface of the spinal cord with warm saline and keep the surgical field clean.

[0063] S3. Mechanical Injury: Using the LISA precision impact device, spinal cord impact was performed at a preset depth of 1.2 mm, a time of 0.42 s, and a velocity of 1.000 m / s to create a highly repeatable model of severe spinal cord injury (see [reference to the immediate state of the injured spinal cord]). Figure 2 B, Figure 2 (Middle F).

[0064] S4. Controlled dural incision:

[0065] (1) Controlled hemostasis and visual field clearing after injury

[0066] Immediately after the impact, apply pressure with sterile cotton balls to stop the bleeding. At this time, spinal cord swelling, ruptured dorsal veins, and blood pooling can be observed, indicating increased intracapsular pressure. Irrigate the surgical field with warm saline solution under low pressure, avoiding direct impact on the spinal cord tissue.

[0067] (2) Durotomy

[0068] Before incising the dura mater, disinfection is performed using 0.1% povidone-iodine. Then, using a curved sterile insulin needle or microdural scalpel, the dura mater is gently lifted and incised along the longitudinal axis, penetrating the T9 / 10 segment, in coordination with respiratory movements. After the dura mater is incised, a large amount of blood accumulated in the cyst drains out naturally, and the local pressure decreases accordingly.

[0069] (3) Dura mater edge retraction

[0070] Use 8-0 absorbable sutures to pull and fix the dura mater edges on both sides of the incision, so that the dura mater is slightly everted to ensure complete exposure of the injured spinal cord (see the section on local injury status after dural incision). Figure 2 C, Figure 2 (G). Maintain uniform dura mater tension during traction to avoid tearing.

[0071] S5. Subdural irrigation and debridement:

[0072] Using a sterile syringe, draw 37°C warm saline solution and perform low-pressure irrigation and debridement of the spinal cord injury area under a microscope. During irrigation, avoid direct impact of saline spray on the spinal cord; all exudate is absorbed with sterile cotton balls. Debridement continues until blood and debris are largely removed and spinal cord pulsation is clearly visible (see [link to article on local spinal cord condition after dural incision, irrigation, and debridement]). Figure 2 D, Figure 2 (H).

[0073] S6. Dura mater repair:

[0074] After irrigation, the area is disinfected again and draped. The dura mater incision is then sutured continuously or intermittently to ensure a tight closure. A biological patch or artificial dura mater material is then placed over the dura mater to enhance the sealing effect and reduce the risk of cerebrospinal fluid leakage.

[0075] S7. Close the surgical area:

[0076] After the operation, the surgical area was cleaned with warm saline, and the muscles, subcutaneous tissue and skin were sutured layer by layer. During the recovery period, the experimental animals were provided with warmth and support care.

[0077] 2. Mechanism Investigation

[0078] Following spinal cord contusion, local vascular structures are disrupted, and extravasation of erythrocytes and release of heme lead to increased iron load in the injured area. Free iron ions can participate in lipid peroxidation and induce iron-dependent cell damage, accompanied by the release of inflammatory factors and microcirculatory disturbances, forming a secondary damage amplification circuit.

[0079] The dural incision combined with irrigation and debridement technique described in this invention reduces intraspinal pressure by longitudinally incising the dura mater, thereby alleviating mechanical compression and ischemia; at the same time, irrigation removes blood stasis, hemoglobin, and inflammatory exudates, thereby reducing iron sources and inflammatory stimulation signals from the source.

[0080] Based on the above-described process, the present invention may function through the following mechanisms:

[0081] (1) Reduce the amount of erythrocytes and heme residues, and lower the level of tissue iron pools;

[0082] (2) Inhibits iron-dependent lipid peroxidation and activation of the ferroptosis signaling pathway;

[0083] (3) Improve the local inflammatory microenvironment and oxidative stress state;

[0084] (4) Restore the microcirculation perfusion state of the damaged area.

[0085] The synergistic effect of these multiple processes can block the amplification of secondary damage, thereby promoting the repair of nerve tissue.

[0086] 2.1 Method

[0087] To evaluate the effects of open dural debridement and irrigation (DDL) on iron homeostasis and local microenvironment after spinal cord injury, spinal cord tissue from the injured segment was collected on day 7 post-injury for the following tests.

[0088] 1. Transcriptome analysis

[0089] Total RNA was extracted from tissues and subjected to high-throughput sequencing. Differentially expressed genes were analyzed, and gene analysis was performed on iron metabolism and erythrocyte metabolism-related genes, inflammation-related genes, KEGG pathway enrichment analysis, GO functional enrichment analysis, and ferroptosis gene set enrichment analysis (GSEA) to assess the regulation of ferroptosis pathway and inflammatory signaling pathway by DDL.

[0090] 2. Protein expression detection

[0091] The expression levels of iron metabolism-related proteins, as well as inflammation and anti-inflammatory proteins, were detected and quantitatively compared using Western blotting.

[0092] 3. Blood perfusion detection

[0093] Laser speckle imaging was used to detect the dorsal spinal cord blood perfusion index of the injured segment, and quantitative analysis was performed in a fixed region of interest to evaluate the impact of DDL on local microcirculation.

[0094] 2.2 Results

[0095] This invention demonstrates the absence of intervention ( Figure 3(A) and intervention using the method described in this invention ( Figure 3 The state of (B).

[0096] Without intervention ( Figure 3 (Case A): Following severe thoracic contusion-related spinal cord injury, rupture of dorsal blood vessels at the injured segment leads to hematoma formation and increased pressure within the spinal cord capsule. Erythrocyte extravasation and heme release increase the local erythrocyte / heme load, subsequently triggering iron deposition and tissue iron pool expansion, accompanied by inflammatory factor accumulation and enhanced oxidative stress. These changes result in an imbalance in the microenvironment of the injured segment, exacerbating secondary tissue damage and neuronal death.

[0097] After intervention ( Figure 3 (Medium B): After spinal cord injury, a longitudinal incision of the dura mater is performed at the injured segment to achieve decompression, thereby reducing the pressure within the spinal cord capsule. Subsequently, low-pressure irrigation and debridement are performed through the dural incision to remove hematoma, residual red blood cells, heme, and inflammatory exudate in the injured area. After the above steps, the heme and iron ion load in the injured area can be reduced, the iron pool level in the tissue can be lowered, the abnormal inflammatory microenvironment can be alleviated, and the secondary compression and iron-dependent cell damage process can be reduced.

[0098] The above results indicate that this invention, through the aforementioned synergistic mechanism of "decompression + irrigation," achieves active regulation of iron homeostasis and the local microenvironment in the damaged area, thus providing a novel technical means for inhibiting secondary injury responses and promoting nerve tissue repair. Specific results are as follows ( Figures 4-7 ):

[0099] A heatmap of genes related to iron metabolism and erythrocyte metabolism shows that ( Figure 4 In the SCI+DDL group (A), compared with the SCI-only group, the expression of iron metabolism and erythrocyte-related genes was downregulated overall, including heme oxygenase 1 (Hmox1), ferritin light chain (Ftl1), haptoglobin (Hp), albumin (Alb), hemoglobin β chain (Hbb), transferrin (Tf), transferrin receptor (Tfrc), and CD163, suggesting that DDL treatment can reduce iron load and erythrocyte metabolism-related responses in the damaged area. Heatmap results of inflammation and complement-related genes showed that ( Figure 4 In the SCI+DDL group (group B), the expression levels of genes related to inflammation and complement system were reduced, indicating that DDL intervention can inhibit acute inflammation-related transcriptional responses. Pathway enrichment analysis results from the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that (…) Figure 4 In the middle C group, significant differences were observed between the two groups in signaling pathways such as ferroptosis. Gene ontology (GO) enrichment analysis results showed that ( Figure 4 In the middle D), differentially expressed genes were significantly enriched in acute inflammatory responses and stress-related biological processes. The results of ferroptosis gene set enrichment analysis (GSEA) showed that ( Figure 4 The enrichment of ferroptosis-related gene sets was reduced in the SCI+DDL group (E), suggesting that the ferroptosis signaling pathway was suppressed.

[0100] Western blot analysis and quantitative analysis of iron metabolism-related proteins on day 7 post-injury showed that ( Figure 5 Compared with the SCI group, the SCI+DDL group showed reduced expression of iron metabolism-related proteins, suggesting that the disorder of tissue iron homeostasis was improved.

[0101] Western blot analysis and quantification of inflammation and anti-inflammatory related proteins showed that ( Figure 6 The SCI+DDL group showed decreased expression of inflammation-related proteins and relatively increased expression of anti-inflammatory proteins, indicating that the local inflammatory microenvironment was regulated.

[0102] Laser speckle imaging (LSCI) pseudo-color maps show the blood perfusion index (BFI) at baseline (before injury), immediately after injury, and at 5, 30, and 90 minutes post-injury. The results indicate that ( Figure 7 In the T9 / 10 segment, three fixed regions of interest (ROIs) were set: the dorsal median vessel region (black circle) and the adjacent regions on both sides (yellow on the left and red on the right). Quantitative analysis of blood perfusion index (BFI) in the SCI group and the SCI+DDL group showed that ( Figure 7 B, Figure 7 (C) The degree of blood perfusion recovery in the damaged area was improved after DDL treatment.

[0103] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for constructing an immediate decompression model of severe spinal cord injury in rats, characterized in that, Includes the following steps: S1. Animal anesthesia and localization: Anesthetize the rats and locate the T9 / 10 vertebral body as the central segment; S2. Surgical exposure: Make a small longitudinal incision in the T13 vertebral body region and extend the incision from the midline of the back to the head. Bluntly dissect the paravertebral muscles to expose the T9 / 10 lamina region. Without removing the muscle tissue, bite off the lamina tissue layer by layer to expose the dorsal dura mater of the spinal cord. S3. Mechanical injury: Using a striking device to apply a quantitative impact to the exposed dorsal side of the spinal cord to create a severe spinal cord injury; S4. Controlled dural incision: After disinfection with 0.1% povidone-iodine, the dura mater is lifted in the midline of the dorsal side of the spinal cord using a tool with a bent tip, in coordination with the breathing rhythm, and cut along the longitudinal axis to form a regular incision through the injured segment, allowing the blood in the cyst to flow out naturally. Then, 8-0 absorbable sutures are used to pull and fix the edges of the dura mater on both sides of the incision. S5. Subdural irrigation and debridement: Use warm saline to irrigate the damaged area after the dura mater is cut with low pressure without directly impacting the spinal cord tissue. Use absorbent materials to remove blood clots and tissue debris until clear spinal cord pulsation is observed. S6. Dura mater repair: The dura mater incision is sutured, and repair material is applied to the outside of the sutured dura mater. S7. Close the surgical area: Suture the muscles and skin layer by layer.

2. The method for constructing a rat model of severe spinal cord injury with immediate decompression according to claim 1, characterized in that, The blunt dissection of the paravertebral muscles described in step S2 specifically involves: performing blunt dissection along the natural gaps between the paravertebral muscles, with the spinous process as the center, to expose the lamina.

3. The method for constructing a rat model of severe spinal cord injury with immediate decompression according to claim 1, characterized in that, The striking device mentioned in step S3 is the LISA precision striking device, and the operating conditions are: depth 1.2 mm, time 0.42 s, and speed 1.000 m / s.

4. The method for constructing a rat model of severe spinal cord injury with immediate decompression according to claim 1, characterized in that, The tool with the bent tip mentioned in step S4 is a bent insulin injection needle or a microdural knife.

5. The method for constructing a rat model of severe spinal cord injury with immediate decompression according to claim 1, characterized in that, The temperature of the warm saline solution mentioned in step S5 is 37 ℃.

6. The method for constructing a rat model of severe spinal cord injury with immediate decompression according to claim 1, characterized in that, The low-pressure method described in step S5 is as follows: after drawing warm saline with a syringe, allow the fluid to flow slowly along the edge of the dura mater incision or the side of the damaged area, avoiding vertical spraying of the fluid onto the surface of the spinal cord tissue.

7. The method for constructing a rat model of severe spinal cord injury with immediate decompression according to claim 1, characterized in that, The adsorbent material mentioned in step S5 is a sterile cotton ball.

8. The method for constructing a rat model of severe spinal cord injury with immediate decompression according to claim 1, characterized in that, In step S6, the dura mater incision is sutured continuously or interrupted using 8-0 or finer absorbable sutures.

9. The method for constructing a rat model of severe spinal cord injury with immediate decompression according to claim 1, characterized in that, The repair material mentioned in step S6 is a biological dura mater patch or an artificial dura mater material.

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