Small animal model for skull base high-flow cerebrospinal fluid leakage and skull base reconstruction, construction method and application

Abstract

The invention belongs to the technical field of medical experimental animal models, and discloses a small animal model for skull base high-flow cerebrospinal fluid leakage and skull base reconstruction as well as a construction method and application of the small animal model. The technical obstacle that due to the fact that the nasal cavity of a small animal is narrow and the turbinate structure is complex, surgical instruments cannot reach the skull base through the nose is overcome, meanwhile, a modeling and repairing area is arranged on the rear nasal meatus and the top wall of the nasopharynx, and the temperature and humidity conditions and environment of the nasal mucosa are reserved; and the microenvironment for tissue healing after the endoscopic transnasal skull base surgery is restored on the pathophysiological level. The dissected marking points are clear, and compared with narrow anterior skull base low-flow cerebrospinal fluid leakage of small animals, the standardized bone defect constructed in the middle skull base butterfly saddle area can cause continuous and high-flow cerebrospinal fluid leakage; autologous bone flap and free mucous membrane repair is used, the method is the same as current clinical practice, and a new platform can be provided for screening of skull base repair materials or operation evaluation and retrograde intracranial infection research after an operation.

Description

Technical Field

[0001] This invention belongs to the field of medical experimental animal model technology, and mainly relates to small animal models of high-flow cerebrospinal fluid leakage at the base of the skull and skull base reconstruction, as well as their construction methods and applications. Background Technology

[0002] Endoscopic extended transnasal approach for the resection of skull base tumors is currently the mainstream surgical procedure in neurosurgery. Postoperative high-flow cerebrospinal fluid leakage is its most significant and serious complication, which can easily lead to retrograde intracranial infection and endanger the patient's life. To verify the effectiveness of different skull base repair materials and reconstruction techniques, establishing highly realistic and reproducible animal models is fundamental to related research.

[0003] Current animal experimental models have two main limitations: First, while large animals (such as pigs and sheep) can simulate the transnasal route, the experimental costs are high, making it difficult to conduct large-scale statistical analysis. Second, the small animal models currently used for cerebrospinal fluid (CSF) leakage research mainly establish CSF leakage models by grinding away vertebral bone and opening the dura mater, or by grinding away parietal bone and cutting the dura mater (transcranial approach). Although these models can successfully replicate CSF leakage and can be used to evaluate the effectiveness of sealing materials, they fail to simulate the clinical reality of skull base defects after endoscopic extended transnasal skull base surgery. This is because the modeling sites of these small animal models are located in the spinal region or the parietal bone region, which cannot simulate the warm, moist environment and nasal microbiota of the nasal cavity. Furthermore, they do not fully consider the impact of intracranial pressure and CSF pulsation on the repaired tissue, thus limiting the clinical reference value of the experimental data.

[0004] To construct a more clinically accurate model of skull base cerebrospinal fluid (CSF) leakage, some researchers have attempted to simulate the transnasal approach in small animals. For example, using Sprague-Dawley rats, after removing the nasal and frontal bones, metal instruments were used to penetrate the cribriform plate separating the olfactory bulb from the anterior nasal mucosa, thus causing CSF leakage. Alternatively, in rabbit models, the nasal bones were opened, the cribriform plate removed, and the mucosa smoothed under a microscope. A 2mm diameter bone hole was then drilled in the frontal skull base with a diamond drill, and the dura mater was incised to simulate skull base defects and CSF leakage. Both approaches resulted in skull defects with CSF leakage in the experimental animals. However, due to the narrow nostrils and nasal passages and complex turbinate structures in small animals, these models suffer from limitations such as small skull base defects, low CSF leakage flow, difficulty in controlling defect size, and difficulty in placing repair materials. These limitations prevent them from becoming ideal animal models for simulating skull base defects with high intraoperative CSF leakage after endoscopic enlarged transnasal approach surgery.

[0005] In summary, the current technology lacks a model that can overcome the anatomical limitations of the nasal cavity in small animals and establish a standardized high-flow cerebrospinal fluid leakage model located in the physiological environment of the nasopharynx, which limits the in-depth development of skull base reconstruction-related research. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes a novel small animal model and construction method for high-flow cerebrospinal fluid leakage at the skull base and skull base reconstruction, along with its application.

[0007] The technical solution of this invention is as follows: A method for constructing an animal model of cerebrospinal fluid leakage and skull base reconstruction at the skull base includes the following steps: S1. After general anesthesia, the experimental rabbits were placed in a prone position and their oral cavity was opened to establish an artificial airway. S2. Make an incision and pull on the soft palate to expose the posterior nasal meatus and the mucosa of the roof of the nasopharynx; S3. Dissect the nasopharyngeal mucosa to expose the bone of the sella turcica base. Using the anatomical landmarks of the middle cranial base as the boundary, grind away and remove the bone flap to form a bone defect window at the cranial base. S4. Make an incision inside the skull base bone defect window as described in step S3 to create a dura mater defect until cerebrospinal fluid flows out, thus completing the construction of the skull base cerebrospinal fluid leakage animal model; S5. Use sealing material to fill the dura mater defect, then reimplant the bone flap removed in step S3 into the skull base bone defect window to restore the skull base bone structure. Finally, cover the reimplanted bone flap with the nasopharyngeal mucosa peeled off in step S3 to complete the skull base reconstruction of the skull base cerebrospinal fluid leakage animal model.

[0008] Furthermore, the specific operation of the soft palate incision in step S2 is as follows: a longitudinal incision is made at the anatomical midline of the soft palate behind the junction of the soft palate and hard palate.

[0009] Furthermore, the dissection range of the nasopharyngeal mucosa in step S3 is as follows: the mucosa including the sella turcica floor and the two sides of the skull base is removed with the line connecting the top of the pharyngeal openings of the two Eustachian tubes as the bottom edge.

[0010] Furthermore, the anatomical landmarks of the middle cranial base mentioned in step S3 are specifically as follows: the anterior boundary is the cartilaginous junction at the junction of the anterior and middle cranial bases, the posterior boundary is the middle of the sella turcica foramen, and the lateral boundaries are the two sides of the sella turcica bone.

[0011] Furthermore, the size of the dura mater defect described in step S4 is (3-5mm)×(1-1.5mm).

[0012] Furthermore, the sealing material mentioned in step S5 is gelatin sponge.

[0013] Animal models of cerebrospinal fluid leakage and skull base reconstruction were constructed according to the aforementioned construction method.

[0014] The application of the skull base cerebrospinal fluid leakage and skull base reconstruction animal model in screening skull base repair materials or evaluating skull base reconstruction surgery.

[0015] Application of the animal model of cerebrospinal fluid leakage and skull base reconstruction at the skull base in the study of retrograde intracranial infection after skull base surgery.

[0016] A system for constructing an animal model of cerebrospinal fluid leakage and skull base reconstruction at the skull base, the system comprising: Fixation unit: used to fix experimental animals in a prone head-tilted position, including an oral retractor, used to continuously expand the experimental animal's oral cavity and expose the surgical field; Airway management unit: includes endotracheal intubation, used to maintain airway patency in laboratory animals during surgery; Negative pressure suction unit: includes a suction device and suction tube, used to remove blood and cerebrospinal fluid from the surgical area of ​​laboratory animals; Endoscopic visualization unit: used to provide illumination and magnified images of the surgical area in laboratory animals; Skull base grinding unit: includes a grinding drill for grinding away bone in the sella turcica region of the middle skull base and cutting the dura mater to create a bone defect window at the skull base.

[0017] Compared with the prior art, the present invention has at least the following advantages: This invention relates to small animal models of high-flow cerebrospinal fluid (CSF) leakage at the skull base and methods for skull base reconstruction, as well as their applications. This method overcomes the technical obstacle of narrow nasal cavities and complex turbinate structures in small experimental animals, which prevent surgical instruments from reaching the middle skull base via the nose, by using a transoral soft palate incision path. Simultaneously, the modeling and repair area is placed in the posterior nasal meatus and the roof of the nasopharynx, preserving the temperature, humidity, and environment of the nasal mucosa, thus replicating the microenvironment of tissue healing after endoscopic transnasal skull base surgery in humans at the pathophysiological level. The anatomical landmarks of this invention are clearly defined. Compared to low-flow CSF leakage at the narrow anterior skull base in small animals, the standardized bone defect constructed in the sella turcica region of the middle skull base can induce persistent, high-flow CSF leakage. The animal model uses autologous bone flaps and free mucosa for repair, consistent with current clinical practice, providing a new platform for screening skull base repair materials, evaluating skull base reconstruction surgery, and studying retrograde intracranial infections after skull base surgery. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0019] Figure 1 This is an endoscopic diagram of the animal model creation process of the present invention; Figure 2 Images of the dissected free nasopharyngeal mucosal flap and the ground-down in situ bone flap in Embodiment 1 of the present invention; Figure 3 This is a diagram showing the state of the soft palate mucosa after suturing in Embodiment 1 of the present invention; Figure 4 This is a comparison image of the sagittal CT images and three-dimensional reconstructions of the skulls of the control rabbit and the experimental rabbit in this invention. Detailed Implementation

[0020] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0021] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0022] Example 1: Animal Model Construction The method for constructing an animal model of high-flow cerebrospinal fluid leakage and repair via endoscopic transoral approach to the skull base according to the present invention includes the following steps: S1. Animal selection and preoperative preparation: The experimental rabbits were ordinary New Zealand white rabbits, which were fed normally for 7 days, fasted for 12 hours and deprived of water for 4 hours before surgery, and then weighed.

[0023] S2. Anesthesia and preoperative medication: Prepare an anesthetic by mixing xylazine hydrochloride injection (Dunhua Shengda Animal Pharmaceutical Co., Ltd.) and Silta 50 (Victoire de France) in a 1:1 volume ratio, and administer intramuscularly (thigh) at a dose of 0.1 ml / kg; administer subcutaneous injection of meloxicam (Baoding Sunshine Herbal Medicine Co., Ltd., 0.6 mg / kg) and ceftiofur sodium (Qilu Animal Health Products Co., Ltd., 1 ml) for analgesia and anti-infection.

[0024] S3. Positioning and Airway Establishment: The experimental rabbits were placed on the operating table in a prone position with their limbs extended and fixed. The head was slightly tilted back in a hyperextended state to fully extend the neck angle. An oral retractor was used to open and lock the rabbit's oral cavity. The depth and angle of the oral retractor were adjusted until the hard and soft palate structures deep in the oral cavity could be clearly exposed. A sterile perforated drape was routinely laid, and the oral mucosa and perioral area were disinfected. Subsequently, under endoscopic visualization, a 2.5 mm diameter endotracheal tube was inserted through the glottis and properly secured to ensure airway patency during the operation.

[0025] S4. Surgical approach exposure: at the V-shaped boundary between the soft and hard palate (see attached...) Figure 1Make a longitudinal incision along the midline of the soft palate behind (A black dotted line). Use a 5-0 suture to laterally pull the soft palate mucosa to expose the posterior nasal meatus and the roof of the nasopharynx mucosa (attached). Figure 1 B), cotton ball packing of the posterior nasal meatus (attached) Figure 1 C).

[0026] S5. Obtaining a nasopharyngeal mucosal graft: Using the line connecting the tops of the pharyngeal openings of the two Eustachian tubes as the base, harvest a whole piece of nasopharyngeal mucosa including the sella turcica floor and both sides of the skull base for later use (see attached document). Figure 1 D, Appendix Figure 2 A and B show the autologous repair material to be reimplanted completely after the operation, with the right side showing the dissected nasopharyngeal mucosal flap.

[0027] S6. Bone Exposure and Hemostasis: Blunt dissection of the soft tissues at the base of the skull exposes the bone at the base of the sella turcica, where a mobile foramen turcica is visible (see attached image). Figure 1 E), use bone wax to pack and stop the bleeding.

[0028] S7. Preparation of in situ bone flap: Using a suction device connected to a suction tube (E3 Xinhua suction tube, 1.5mm in diameter), surrounding blood is aspirated. The bone window is positioned as follows: the anterior boundary of the bone window is the junction of the anterior and middle skull bases (i.e., the cartilaginous junction where white cartilage meets pale yellow bone); the posterior boundary is the middle of the exposed sella turcica bone foramen; the lateral boundaries are the two sides of the naturally raised edges of the sella turcica bone. A rectangular in situ bone flap is prepared using a 0.5mm diamond drill and removed for later use. In this embodiment, the in situ bone flap has a long diameter of 5mm and a wide diameter of 2mm, forming a skull base bone defect window (see attached diagram). Figure 1 F is a black dashed line, with... Figure 2 (In A and B, the left side shows the in-situ bone flap that was removed completely by micro-drilling). The long diameter of the in-situ bone flap described in this invention can be adaptively adjusted within the range of 4-6 mm and the wide diameter can be adjusted within the range of 2-3 mm.

[0029] S8. Creating a cerebrospinal fluid leak: Clearly expose and open the dura mater along the midline until a dura mater defect is created. In this embodiment, the size of the dura mater defect is approximately 4 × 1 mm. At this point, cerebrospinal fluid can be seen gushing out, and the high-flow-rate cerebrospinal fluid leak rabbit animal model is complete. (See attached diagram) Figure 1 GI); The size of the dura mater defect described in this invention can be adaptively adjusted within the range of (3-5mm)×(1-1.5mm).

[0030] S9. Repair and Reconstruction: The dural defect is filled with gelatin sponge, the in-situ bone flap is repositioned, ensuring that the bone flap completely covers the dural defect, and a free mucosal flap is placed on the outside of the bone flap; appropriately sized iodine-soaked cotton balls are used to cover the repair tissue, ensuring that it adheres tightly to and supports the repair tissue (see attached). Figure 1 JL).

[0031] S10. Postoperative recovery: Clean any remaining blood from the posterior nasal passages, ensure nasal patency, remove the endotracheal tube, and suture the soft palate mucosa (see attached image). Figure 3 The oral cavity was cleaned and moistened with saline solution. After an intramuscular injection of 1.5 times the volume of the anesthetic benzoyl hydrochloride injection (Changsha Byte Biotechnology Research Institute Co., Ltd.), the experimental rabbits were returned to their cages for rewarming and awaiting awakening. Following the experiment, the rabbits showed good appetite and no complications such as diarrhea, lethargy, or death, indicating successful modeling.

[0032] In some embodiments, a system for constructing an animal model of cerebrospinal fluid leakage and skull base reconstruction is also provided, the system comprising: Fixation unit: used to fix experimental animals in a prone head-tilted position, including an oral retractor, used to continuously expand the experimental animal's oral cavity and expose the surgical field; Airway management unit: includes endotracheal intubation, used to maintain airway patency in laboratory animals during surgery; Negative pressure suction unit: includes a suction device and suction tube, used to remove blood and cerebrospinal fluid from the surgical area of ​​laboratory animals; Endoscopic visualization unit: used to provide illumination and magnified images of the surgical area in laboratory animals; Skull base grinding unit: includes a grinding drill for grinding away bone in the sella region of the middle skull base and cutting the dura mater to create skull base bone defects; Processing unit: includes a needle holder for puncturing and pulling open the soft palate mucosa to expose the submucosal structures of the soft palate and for finally suturing the soft palate mucosa; microsurgical scissors for cutting open the soft palate mucosa, cutting free mucosal flaps, and cutting open the dura mater; and microforceps for grasping objects or tissues.

[0033] Example 2: Evaluation after animal model construction To verify the anatomical accuracy of the modeling method described in this invention and the effectiveness of the in situ bone flap repair technique in living organisms, head imaging examinations were performed on experimental animals one week after the surgery.

[0034] Sagittal CT scan results showed that the sella turcica bone structure of the control rabbits was continuous and intact. Figure 4 A); while the bone below the sella turcica in the surgical rabbits was missing due to modeling, a linear high-density bone structure was visible between the dorsal dorsum and the original tubercle of the sella turcica. Figure 4 (B) This structure is the in situ bone flap used for repair. Its position is relatively fixed, which confirms that the replanted bone flap can provide effective rigid support for the skull base defect without early displacement.

[0035] The 3D reconstruction diagram based on CT data shows the spatial location of the pituitary gland and sella turcica, confirming that the surgical modeling site was precisely located at the base of the skull, between the tubercle sellae and the bottom of the sella turcica in the experimental subject. Figure 4(C, D) indicates that the transoral approach model can highly reproduce the anatomical position of the middle skull base in clinical endoscopic transnasal surgery, achieving precise modeling and reconstruction of the target area.

[0036] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for constructing an animal model of cerebrospinal fluid leakage and skull base reconstruction, characterized in that, Includes the following steps: S1. After general anesthesia, the experimental rabbits were placed in a prone position and their oral cavity was opened to establish an artificial airway. S2. Make an incision and pull on the soft palate to expose the posterior nasal meatus and the mucosa of the roof of the nasopharynx; S3. Dissect the nasopharyngeal mucosa to expose the bone of the sella turcica base. Using the anatomical landmarks of the middle cranial base as the boundary, grind away and remove the bone flap to form a bone defect window at the cranial base. S4. Make an incision inside the skull base bone defect window as described in step S3 to create a dura mater defect until cerebrospinal fluid flows out, thus completing the construction of the skull base cerebrospinal fluid leakage animal model; S5. Use sealing material to fill the dura mater defect, then reimplant the bone flap removed in step S3 into the skull base bone defect window to restore the skull base bone structure. Finally, cover the reimplanted bone flap with the nasopharyngeal mucosa peeled off in step S3 to complete the skull base reconstruction of the skull base cerebrospinal fluid leakage animal model.

2. The construction method according to claim 1, characterized in that, The specific operation of the soft palate incision in step S2 is as follows: make a longitudinal incision at the anatomical midline of the soft palate behind the junction of the soft palate and hard palate.

3. The construction method according to claim 1, characterized in that, The nasopharyngeal mucosa dissection range in step S3 is as follows: the mucosa including the sella turcica floor and the two sides of the skull base is removed with the line connecting the top of the pharyngeal openings of the two Eustachian tubes as the bottom edge.

4. The construction method according to claim 1, characterized in that, The anatomical landmarks of the middle cranial base mentioned in step S3 are as follows: the anterior boundary is the cartilaginous junction at the junction of the anterior and middle cranial bases, the posterior boundary is the middle of the bone foramen in the middle of the sella turcica, and the lateral boundaries are the two sides of the sella turcica.

5. The construction method according to claim 1, characterized in that, The size of the dura mater defect described in step S4 is (3-5mm)×(1-1.5mm).

6. The construction method according to claim 1, characterized in that, The sealing material mentioned in step S5 is gelatin sponge.

7. The animal model of cerebrospinal fluid leakage and skull base reconstruction constructed by the construction method according to any one of claims 1-6.

8. The application of the skull base cerebrospinal fluid leakage and skull base reconstruction animal model as described in claim 7 in screening skull base repair materials or evaluating skull base reconstruction surgery.

9. The application of the animal model of cerebrospinal fluid leakage and skull base reconstruction as described in claim 7 in the study of retrograde intracranial infection after skull base surgery.

10. A system for constructing an animal model of cerebrospinal fluid leakage and skull base reconstruction at the skull base, characterized in that, The construction system includes: Fixation unit: used to fix experimental animals in a prone head-tilted position, including an oral retractor, used to continuously expand the experimental animal's oral cavity and expose the surgical field; Airway management unit: includes endotracheal intubation, used to maintain airway patency in laboratory animals during surgery; Negative pressure suction unit: includes a suction device and suction tube, used to remove blood and cerebrospinal fluid from the surgical area of ​​laboratory animals; Endoscopic visualization unit: used to provide illumination and magnified images of the surgical area in laboratory animals; Skull base grinding unit: includes a grinding drill for grinding away bone in the sella turcica region of the middle skull base and cutting the dura mater to create a bone defect window at the skull base.

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