Method for establishing rat bionic model for sudden deafness caused by inner ear microcirculation disturbance
By using exosomes derived from mesenchymal stem cells as carriers, combined with three-dimensional culture technology and intratympanic injection method, a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorder was constructed. This solved the problems of large trauma and poor targeting in existing technologies, and achieved a high degree of biomimetic effect and stable model establishment.
Patent Information
- Application Number
- CN202511205016.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-27
- Publication Date
- 2025-12-02
AI Technical Summary
Existing methods for establishing animal models of sudden deafness caused by inner ear microcirculation disorders have problems such as large trauma, poor targeting, and poor biomimetic effect. They cannot accurately reflect the true relationship between inner ear microcirculation disorders and sudden deafness, and can also cause damage to surrounding tissues and other organs throughout the body.
Using exosomes derived from mesenchymal stem cells as carriers, exosome production was increased through three-dimensional culture technology, and procoagulant factors were delivered to the inner ear of rats via intratympanic injection to induce local microthrombus formation, thus constructing a sudden deafness model.
This reduces damage to the rat inner ear tissue, improves the accuracy and stability of the model, and can reliably simulate the pathological process of sudden deafness in humans, providing a reliable experimental tool for related research.
Smart Images
Figure CN121041069A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of animal model construction technology, specifically a method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorders. Background Technology
[0002] Sudden deafness, a sensorineural hearing loss disorder that occurs abruptly and has no known cause, is often accompanied by symptoms such as tinnitus and vertigo, severely impacting patients' quality of life. Currently, its pathogenesis is not fully understood, but inner ear microcirculation disorders are considered a significant contributing factor. In the field of medical research, establishing animal models that mimic human pathological processes is crucial. These models provide a powerful tool for in-depth research into the pathogenesis of sudden deafness, lay the foundation for developing effective therapeutic drugs, and promote research progress and clinical treatment advancements in related fields.
[0003] Existing methods for establishing animal models of sudden deafness caused by inner ear microcirculatory disorders mainly include ligation of blood vessels and injection of chemoembolic agents. However, these methods have significant limitations. Ligation of blood vessels is a highly invasive procedure that can easily damage surrounding tissues, affecting their normal function and potentially interfering with research on the pathogenesis of inner ear microcirculatory disorders themselves. This can lead to biased experimental results and fail to accurately reflect the true relationship between inner ear microcirculatory disorders and sudden deafness. Injection of chemoembolic agents, on the other hand, lacks targeting. After entering the body, the chemoembolic agents are difficult to precisely target the local microvessels of the inner ear and may spread to other organs throughout the body via the bloodstream, causing unnecessary damage. Furthermore, because they cannot accurately simulate the pathological process of local inner ear microcirculatory disorders, the established animal models differ significantly from the actual pathological condition of sudden deafness in humans. This makes it difficult to provide reliable evidence for in-depth research on the pathogenesis of sudden deafness and also limits the accuracy and effectiveness of related therapeutic drug development. Therefore, traditional methods are insufficient to meet the current needs of sudden deafness research in terms of precision, targeting, and biomimetic effects. Summary of the Invention
[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorders. This method utilizes exosomes derived from mesenchymal stem cells as carriers. Mesenchymal stem cells are widely available and easily obtained, and their exosome production is stable and exhibits good biocompatibility, reducing damage to rat inner ear tissues. Furthermore, they possess natural targeted transport capabilities, specifically binding to inner ear vascular endothelial cells and increasing the concentration of procoagulant factors at the target site. By employing three-dimensional culture technology combined with an optimized cell culture system, exosome production is effectively increased. After loading procoagulant factors into exosomes, they are delivered to the rat inner ear via intratympanic injection through the external auditory canal. This allows the exosomes to specifically bind to inner ear vascular endothelial cells and release procoagulant factors, inducing local microthrombus formation, blocking inner ear microvessels, and causing microcirculatory disorders, thereby constructing a rat model of sudden deafness. This model demonstrates good biomimetic effects and stability, providing a reliable experimental model for related research.
[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorders, the method comprising:
[0006] Extraction and purification of exosomes: bone marrow was obtained from healthy rats, mesenchymal stem cells were extracted, and they were cultured in culture flasks. The cell culture supernatant was collected, and exosomes were extracted by ultracentrifugation.
[0007] Loading of procoagulant factors and exosomes: Prothrombin fragments were selected as procoagulant factors and processed. The resulting exosome suspension was mixed with the prothrombin fragment solution at a volume ratio of 1:1 and loaded using electroporation.
[0008] Tympanic cavity injection procedure: SPF-grade healthy male SD rats were selected. Before the experiment, the rats were anesthetized by injection. The tympanic membrane was exposed through the external auditory canal using an otoscope. The exosome suspension loaded with prothrombin fragments was injected into the tympanic cavity through the anterior and inferior quadrant of the tympanic membrane. After the injection, the entrance of the external auditory canal was gently pressed to prevent the fluid from flowing back.
[0009] Rat model testing and validation: Audiological testing, inner ear histopathological examination, and inner ear microcirculation testing were performed on the model rats to verify whether the model was successfully established.
[0010] Furthermore, in the exosome extraction and purification step, healthy SPF-grade rats weighing 220-280g, regardless of sex, are selected, and bone marrow is isolated and obtained under sterile conditions to extract rat bone marrow mesenchymal stem cells.
[0011] Furthermore, in the extraction and purification steps of exosomes, mesenchymal stem cells are cultured using three-dimensional culture technology. A three-dimensional culture system is constructed using PET membrane material. Cells are seeded on the PET membrane, and the cell seeding density is adjusted to 3×10^5 cells / mL. The cells are placed in DMEM medium containing 5% exosome-depleted fetal bovine serum and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reaches 70%-80%, the medium is replaced with serum-free DMEM medium and cultured for another 48-72 hours. The cell culture supernatant is collected, and exosomes are extracted using ultracentrifugation.
[0012] Furthermore, in the extraction and purification steps of exosomes, exosomes are extracted using ultracentrifugation. Specifically, the cell culture supernatant is centrifuged at 300g for 10 min at 4°C to remove cell debris. The supernatant is then centrifuged at 2000g for 20 min to further remove impurities. The supernatant is then centrifuged at 10000g for 30 min, the supernatant is discarded, the precipitate is resuspended in PBS, and centrifuged at 100000g for 70 min to obtain the exosome precipitate. Finally, the exosomes are resuspended in PBS and stored at -80°C for later use.
[0013] Furthermore, in the loading step of the procoagulant factor and exosomes, the procoagulant factor is a prothrombin fragment with a purity of ≥95%, which is dissolved in sterile PBS buffer to prepare a solution of 100-500 ng / mL, and then used after being sterilized by filtration through a 0.22 μm filter membrane.
[0014] Furthermore, in the loading step of the procoagulant factor and exosomes, electroporation is used for loading. The electroporation parameters are: voltage 100-200V, pulse time 5-10ms, and pulse count 3-5 times. After loading, the exosomes are washed with PBS 2-3 times to remove unbound prothrombin fragments, resulting in an exosome suspension loaded with prothrombin fragments.
[0015] Furthermore, in the tympanic cavity injection procedure, SPF-grade healthy male SD rats were selected and acclimatized for one week. Before the experiment, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, fixed on a rat board, and the tympanic membrane was exposed through the external auditory canal using an otoscope. Using a 10μL microsyringe, an exosome suspension loaded with prothrombin fragments was injected into the tympanic cavity through the anterior and inferior quadrant of the tympanic membrane. The injection dose was 20μL, and the injection rate was 2μL / s. After the injection, the needle was slowly withdrawn after 10 seconds. The entrance of the external auditory canal was gently pressed with a sterile cotton ball for 3-5 minutes. The rats were then returned to their cages and allowed to wake up naturally. During the recovery process, the rats were kept warm.
[0016] Furthermore, in the rat model detection and verification steps, the audiological detection uses the auditory brainstem response test to detect changes in hearing threshold at different time points after rat injection; the inner ear histopathological examination includes sectioning the rat inner ear to observe microthrombus formation and tissue damage; and the inner ear microcirculation detection uses a laser Doppler flowmeter to detect changes in inner ear blood flow.
[0017] Compared with existing technologies, this method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorders has the following advantages:
[0018] I. This invention utilizes exosomes derived from mesenchymal stem cells as carriers of procoagulant factors. These exosomes are widely available and easily obtained, have stable production, and good biocompatibility. They can reduce damage to rat inner ear tissues. Furthermore, exosomes have natural targeted transport capabilities, specifically binding to inner ear vascular endothelial cells, increasing the concentration of procoagulant factors at the target site, and enhancing the accuracy of model establishment. This provides a more precise model basis for in-depth research on the mechanisms related to inner ear microcirculation disorders.
[0019] Second, this invention delivers exosomes via intratympanic injection, effectively avoiding the side effects of systemic administration. This allows the exosomes to act directly on the inner ear, improving the efficiency and stability of model establishment. Furthermore, this method simulates the pathological process of sudden deafness in humans by inducing local microthrombus formation in the inner ear, demonstrating excellent biomimetic effects and providing a reliable experimental tool for the research and drug development of sudden deafness.
[0020] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description
[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.
[0022] Figure 1 A flowchart illustrating the method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorders;
[0023] Figure 2 A flowchart illustrating the extraction and purification of exosomes in a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorders.
[0024] Figure 3A flowchart illustrating the loading of procoagulant factors and exosomes in a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorders. Detailed Implementation
[0025] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.
[0026] The purpose of this invention is to overcome the shortcomings of existing methods for establishing animal models of sudden deafness caused by inner ear microcirculation disorders, such as large trauma, poor targeting, and unsatisfactory biomimetic effects. This invention provides a method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorders. This method utilizes exosomes derived from mesenchymal stem cells as carriers. Mesenchymal stem cells are widely available and easily obtained, and their exosome production is stable and has good biocompatibility, reducing damage to rat inner ear tissue. They also possess natural targeted transport capabilities, specifically binding to inner ear vascular endothelial cells, increasing the concentration of procoagulant factors at the target site. Furthermore, by employing three-dimensional culture technology combined with an optimized cell culture system, exosome production is effectively increased. After loading procoagulant factors into exosomes, they are delivered to the rat inner ear via intratympanic injection through the external auditory canal. The exosomes specifically bind to inner ear vascular endothelial cells and release procoagulant factors, inducing local microthrombus formation, blocking inner ear microvessels, causing microcirculatory disorders, and thus constructing a rat model of sudden deafness, improving the biomimetic effect and reliability of the model.
[0027] To achieve the above objectives, this invention provides a method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorders, such as... Figure 1 As shown, it includes the following steps:
[0028] Extraction and purification of exosomes:
[0029] Healthy SPF-grade rats, weighing 220-280g, regardless of sex, were selected. Bone marrow was isolated and mesenchymal stem cells were extracted from the rats. The obtained tissue was minced and added to a 0.25% trypsin-EDTA mixture. The mixture was digested in a 37°C water bath for 5-10 minutes, with gentle shaking every 2 minutes. After digestion, DMEM / F12 medium containing 10% exosome-depleted fetal bovine serum was added to terminate the digestion. The mixture was filtered through a 200-mesh cell sieve, and the filtrate was collected. The filtrate was centrifuged at 1000 rpm for 10 minutes at 4°C, and the supernatant was discarded to obtain the mesenchymal stem cell precipitate.
[0030] Mesenchymal stem cells were cultured using three-dimensional culture technology. A three-dimensional culture system was constructed using a gelatin-coated PET membrane with a pore size of 10 μm. Cells were seeded onto the PET membrane at a seeding density of 2 × 10⁻⁶ cells / cm².5 Cells were cultured at a density of 1 cell / mL in DMEM / F12 medium containing 5% exosome-depleted fetal bovine serum (prepared by centrifugation at 100,000g for 16 hours). The cells were incubated at 37°C in a 5% CO2 incubator. Cell growth was observed daily. When cell confluence reached 70%-80%, the medium was replaced with serum-free DMEM / F12 medium and cultured for another 48-72 hours to increase exosome yield.
[0031] Cell culture supernatant was collected, and exosomes were extracted using ultracentrifugation. The specific procedure was as follows: the cell culture supernatant was centrifuged at 300g for 10 min at 4℃ to remove cell debris; the supernatant was then centrifuged at 2000g for 20 min to further remove impurities; the supernatant was then centrifuged at 10000g for 30 min and discarded; the precipitate was resuspended in PBS and centrifuged at 100000g for 70 min to obtain the exosome precipitate; finally, the exosomes were resuspended in 1 mL of pre-chilled PBS and aliquoted into EP tubes. The extracted exosomes were identified as follows: their typical cup-shaped morphology was observed by transmission electron microscopy; the particle size distribution was detected using nanoparticle tracking analysis to ensure that the particle size was within the range of 30-150 nm; the expression of exosome-specific markers CD63 and CD9 was detected by Western blot. After successful identification, the exosomes were stored at -80℃ for later use, with a storage time not exceeding 3 months.
[0032] Loading of coagulant factors and exosomes:
[0033] like Figure 3 As shown, prothrombin fragments (purity ≥95%) were dissolved in sterile PBS buffer to prepare a solution with a concentration of 100-500 ng / mL. The solution was then sterilized by filtration through a 0.22 μm filter and stored at 4°C for later use. The stored exosome resuspended solution was centrifuged at 100,000 g for 70 min at 4°C, the supernatant was discarded, and the exosomes were resuspended in sterile PBS buffer, adjusting the exosome concentration to 2 × 10⁻⁶. 10 Take 1 mL of exosome suspension and mix it with 1 mL of prothrombin fragment solution. Transfer the mixture to an electroporation cuvette and place the cuvette in an electroporator. Set the electroporation parameters as follows: voltage 100-200V, pulse time 5-10ms, pulse count 3-5 times. Perform electroporation. After electroporation, transfer the mixture to a centrifuge tube and centrifuge at 100,000g for 70 min at 4°C. Discard the supernatant and resuspend the precipitate in sterile PBS buffer. Repeat the centrifugation and resuspension process 2-3 times to completely remove unbound prothrombin fragments, obtaining an exosome suspension loaded with prothrombin fragments. Detect the protein concentration in the suspension using the BCA method and adjust the exosome concentration to 1×10⁻⁶. 9 -1×10 11Quantity / mL, store in an ice box for later use.
[0034] Tympanic injection procedure:
[0035] SPF-grade healthy male SD rats, weighing 250-300g, were selected and acclimatized for one week in an environment with a temperature of 22-25℃, humidity of 50%-60%, and a 12-hour light-dark cycle. They were allowed free access to food and water, but were fasted for 12 hours before the experiment, although water was not restricted. They were anesthetized by intraperitoneal injection of 10% chloral hydrate solution at a dose of 3mL / kg body weight. After the corneal reflex disappeared and the muscles of the limbs relaxed, the rats were fixed on a constant-temperature operating table and their body temperature was maintained at 37±0.5℃.
[0036] Using an otoscope, gently expose the tympanic membrane through the external auditory canal, avoiding damage to the skin of the external auditory canal and the tympanic membrane. Use a 10μL microsyringe (30G needle) to draw up an exosome suspension loaded with prothrombin fragments. Insert the needle slowly into the anterior inferior quadrant of the tympanic membrane at a 30° angle to the tympanic membrane, to a depth of about 0.5mm. Slowly inject the exosome suspension at a dose of 20μL and an injection rate of 2μL / s. Closely observe the rat's response during the injection. After the injection, hold for 10 seconds and then slowly withdraw the needle. Gently press the entrance of the external auditory canal with a sterile cotton ball for 3-5 minutes to prevent fluid reflux. Keep the rat in a lateral head position for 3-5 minutes to allow the exosome suspension to fully contact the tympanic cavity mucosa. Return the rat to its cage and wait for it to wake up naturally. Keep it warm during the recovery process. After recovery, observe its activity and eating habits.
[0037] Rat model detection and validation:
[0038] Audiological testing: Tests were conducted 1 day before injection and 1, 3, 7, and 14 days after injection. Before testing, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate solution at 3 mL / kg body weight and fixed on a temperature-controlled operating table in a soundproof box. An auditory brainstem response testing system was used. The recording electrodes were stainless steel needle electrodes with a diameter of 0.5 mm, inserted approximately 2 mm subcutaneously in the midline of the skull. The reference electrode was inserted approximately 2 mm subcutaneously in the mastoid process of the test ear, and the ground electrode was inserted approximately 2 mm subcutaneously in the mastoid process of the contralateral ear. The impedance between the electrodes was kept below 5 kΩ. The stimulation sound was a short sound with a stimulation frequency of 10 Hz. The filtering range was 100-3000 Hz, the analysis time was 10 ms, and the number of superpositions was 1024. The stimulation intensity started at 90 dBSPL and decreased in 5 dB increments. Each intensity was stimulated 3 times. Repeatable waveforms of waves I, III, and V were recorded until the response waveform disappeared. The stimulation intensity at which the last repeatable waveform appeared was taken as the hearing threshold.
[0039] Histopathological examination of the inner ear tissue: 14 days after injection, three model rats were randomly selected and euthanized using an overdose anesthesia method. Bilateral inner ear tissue was quickly removed and fixed in 4% paraformaldehyde solution for 24 hours, then transferred to 10% EDTA solution for decalcification. Decalcification took 2-3 weeks, with the decalcification solution changed weekly until there was no resistance when a needle was inserted into the inner ear tissue. After decalcification, the inner ear tissue was dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and continuously sectioned along the cochlear axis with a section thickness of 5 μm. The sections were attached to poly-L-lysine-coated slides and baked at 60°C for 2 hours for further examination. HE staining: Sections were dewaxed to water with xylene, rehydrated with 95%, 80%, and 70% ethanol solutions in sequence, and rinsed three times with distilled water; stained with hematoxylin for 5-10 min, rinsed with tap water, differentiated with 1% hydrochloric acid ethanol for 30 s, rinsed with tap water for 10 min to regain blue color; stained with eosin for 30 s-1 min, and rinsed with distilled water; dehydrated with 70%, 80%, and 95% ethanol solutions in sequence, cleared with xylene, mounted with neutral resin, and observed under an optical microscope for morphological changes in the stria vascularis, spiral ligament, cochlear hair cells, and other tissue structures of the inner ear, with a focus on observing whether microthrombi formed in the microvessels of the inner ear.
[0040] Inner ear microcirculation detection: Detection was performed 1 day before injection and 1, 3, and 7 days after injection. The rats were anesthetized using the same method as in audiological testing, fixed on the operating table, and a skin incision of about 1 cm was made at the posterior edge of the auricle to expose the temporal bone and cochlea, avoiding damage to the cochlear structure and blood vessels. The probe of the laser Doppler flowmeter was fixed on the microsurgical arm, and the distance between the probe and the cochlear basilar membrane was adjusted to 0.5 mm to ensure that the probe was perpendicular to the detection site. Blood perfusion volume was recorded continuously for 5 minutes, and the average value was taken as the blood perfusion volume at that time point. Three model rats were selected at each detection time point, and three normal rats were selected as controls. The ratio of blood perfusion volume between the model rats and normal rats was calculated to assess the status of inner ear microcirculation.
[0041] The following example illustrates the establishment of an animal model of sudden deafness caused by inner ear microcirculatory disturbance:
[0042] Healthy SPF-grade rats, weighing 220-280g, regardless of sex, were selected. Bone marrow was isolated and mesenchymal stem cells were extracted from the rats. The obtained tissue was minced and added to a 0.25% trypsin-EDTA mixture. The mixture was digested in a 37°C water bath for 5-10 minutes, with gentle shaking every 2 minutes. After digestion, DMEM / F12 medium containing 10% exosome-depleted fetal bovine serum was added to terminate the digestion. The mixture was filtered through a 200-mesh cell sieve, and the filtrate was collected. The filtrate was centrifuged at 1000 rpm for 10 minutes at 4°C, and the supernatant was discarded to obtain the mesenchymal stem cell precipitate.
[0043] Mesenchymal stem cells were cultured using three-dimensional culture technology. A three-dimensional culture system was constructed using a gelatin-coated PET membrane with a pore size of 10 μm. Cells were seeded onto the PET membrane at a seeding density of 2 × 10⁻⁶ cells / cm². 5 Cells were cultured at a density of 1 cell / mL in DMEM / F12 medium containing 5% exosome-depleted fetal bovine serum (prepared by centrifugation at 100,000g for 16 hours). The cells were incubated at 37°C in a 5% CO2 incubator. Cell growth was observed daily. When cell confluence reached 70%-80%, the medium was replaced with serum-free DMEM / F12 medium and cultured for another 48-72 hours to increase exosome yield.
[0044] Cell culture supernatant was collected, and exosomes were extracted using ultracentrifugation. The specific procedure was as follows: the cell culture supernatant was centrifuged at 300g for 10 min at 4℃ to remove cell debris; the supernatant was then centrifuged at 2000g for 20 min to further remove impurities; the supernatant was then centrifuged at 10000g for 30 min and discarded; the precipitate was resuspended in PBS and centrifuged at 100000g for 70 min to obtain the exosome precipitate; finally, the exosomes were resuspended in 1 mL of pre-chilled PBS and aliquoted into EP tubes. The extracted exosomes were identified as follows: their typical cup-shaped morphology was observed by transmission electron microscopy; the particle size distribution was detected using nanoparticle tracking analysis to ensure that the particle size was within the range of 30-150 nm; the expression of exosome-specific markers CD63 and CD9 was detected by Western blot. After successful identification, the exosomes were stored at -80℃ for later use, with a storage time not exceeding 3 months.
[0045] Prothrombin fragments with a purity of 96% were dissolved in sterile PBS buffer to prepare a 300 ng / mL solution. After sterilization by filtration through a 0.22 μm filter, the solution was stored at 4°C for later use. The stored exosome resuspended solution was centrifuged at 100,000 g for 70 min at 4°C. The supernatant was discarded, and the exosomes were resuspended in sterile PBS buffer, adjusting the exosome concentration to 2 × 10⁻⁶. 10 per mL.
[0046] Mix 1 mL of exosome resuspension with 1 mL of prothrombin fragment solution, transfer to an electroporation cuvette, place the cuvette in an electroporator, and set the electroporation parameters to 150 V, 8 ms pulse time, and 4 pulses. After electroporation, transfer the mixture to a centrifuge tube and centrifuge at 100,000 g for 70 min at 4 °C. Discard the supernatant and resuspend the precipitate in sterile PBS buffer. Repeat the centrifugation and resuspension process three times to obtain an exosome suspension loaded with prothrombin fragments. Adjust the exosome concentration to 5 × 10⁻⁶ using the BCA method. 10 Quantity / mL, store in an ice box for later use.
[0047] SPF-grade healthy male SD rats weighing 280g were selected and acclimatized for one week in an environment with a temperature of 23℃, humidity of 55%, and a 12-hour light-dark cycle. They were allowed free access to food and water, but fasted for 12 hours before the experiment, although water was allowed. They were anesthetized by intraperitoneal injection of 10% chloral hydrate solution at a dose of 3mL / kg body weight. After the corneal reflex disappeared and the muscles of the limbs relaxed, the rats were fixed on a constant-temperature operating table and their body temperature was maintained at 37℃.
[0048] Using an otoscope, the tympanic membrane was gently exposed through the external auditory canal, avoiding damage to the skin of the external auditory canal and the tympanic membrane. A 10 μL microsyringe (30G needle) was used to draw an exosome suspension containing prothrombin fragments. The needle was slowly inserted into the anterior inferior quadrant of the tympanic membrane at a 30° angle to a depth of approximately 0.5 mm. The exosome suspension was slowly injected at a rate of 20 μL / s. After injection, the needle was withdrawn slowly after 10 seconds. A sterile cotton ball was gently pressed at the entrance of the external auditory canal for 4 minutes to prevent reflux. The rat was kept in a lateral position for 4 minutes to ensure sufficient contact between the exosome suspension and the tympanic membrane. The rat was then returned to its cage and allowed to recover naturally, keeping it warm during recovery. After recovery, its activity and eating habits were observed.
[0049] Based on the above test results, this embodiment successfully established a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorder. This model can stably simulate the pathological process of sudden deafness caused by inner ear microcirculation disorder.
[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. A method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorder, characterized in that, The method includes: Extraction and purification of exosomes: bone marrow from healthy rats was obtained, mesenchymal stem cells were extracted, and the cells were cultured in culture flasks. The cell culture supernatant was collected, and exosomes were extracted by ultracentrifugation. Loading of procoagulant factors and exosomes: Prothrombin fragments were selected as procoagulant factors and processed. The resulting exosome suspension was mixed with the prothrombin fragment solution at a volume ratio of 1:1 and loaded using electroporation. Tympanic cavity injection procedure: SPF-grade healthy male SD rats were selected. Before the experiment, the rats were anesthetized by injection. The tympanic membrane was exposed through the external auditory canal using an otoscope. The exosome suspension loaded with prothrombin fragments was injected into the tympanic cavity through the anterior and inferior quadrant of the tympanic membrane. After the injection, the entrance of the external auditory canal was gently pressed. Rat model testing and validation: Audiological testing, inner ear histopathological examination, and inner ear microcirculation testing were performed on the model rats to verify whether the model was successfully established.
2. The method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorder according to claim 1, characterized in that, In the exosome extraction and purification step, healthy SPF-grade rats weighing 220-280g, regardless of sex, were selected, and bone marrow was isolated and obtained under sterile conditions to extract rat bone marrow mesenchymal stem cells.
3. The method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorder according to claim 2, characterized in that, In the extraction and purification steps of exosomes, mesenchymal stem cells are cultured using three-dimensional culture technology. A three-dimensional culture system is constructed using PET membrane material. Cells are seeded on the PET membrane, and the cell seeding density is adjusted to 3×10^5 cells / mL. The cells are placed in DMEM medium containing 5% exosome-depleted fetal bovine serum and cultured in a 37℃, 5% CO2 incubator. When the cell confluence reaches 70%-80%, the medium is replaced with serum-free DMEM medium and cultured for another 48-72 hours. The cell culture supernatant is collected, and exosomes are extracted using ultracentrifugation.
4. The method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorder according to claim 3, characterized in that, In the extraction and purification steps of the exosomes, exosomes are extracted using ultracentrifugation. Specifically, the cell culture supernatant is centrifuged at 300g for 10 min at 4°C to remove cell debris. The supernatant is then centrifuged at 2000g for 20 min to further remove impurities. The supernatant is then centrifuged at 10000g for 30 min, the supernatant is discarded, the precipitate is resuspended in PBS, and centrifuged at 100000g for 70 min to obtain the exosome precipitate. Finally, the exosomes are resuspended in PBS and stored at -80°C for later use.
5. The method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorder according to claim 1, characterized in that, In the loading step of the procoagulant factor and exosomes, the procoagulant factor is a prothrombin fragment with a purity of ≥95%, which is dissolved in sterile PBS buffer to prepare a solution of 100-500 ng / mL, and then used after being filtered through a 0.22 μm filter membrane for sterilization.
6. The method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorder according to claim 1, characterized in that, In the loading step of the procoagulant factor and exosomes, the loading is carried out by electroporation. The electroporation parameters are: voltage 100-200V, pulse time 5-10ms, pulse number 3-5 times. After loading, the exosomes are washed with PBS 2-3 times to remove unbound prothrombin fragments, and an exosome suspension loaded with prothrombin fragments is obtained.
7. The method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorder according to claim 1, characterized in that, In the tympanic cavity injection procedure, SPF-grade healthy male SD rats were selected and acclimatized for 1 week. Before the experiment, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, fixed on a rat board, and the tympanic membrane was exposed through the external auditory canal using an otoscope. Using a 10μL microsyringe, an exosome suspension loaded with prothrombin fragments was injected into the tympanic cavity through the anterior and inferior quadrant of the tympanic membrane. The injection dose was 20μL, and the injection rate was 2μL / s. After the injection, the needle was slowly withdrawn after 10 seconds. The entrance of the external auditory canal was gently pressed with a sterile cotton ball for 3-5 minutes. The rats were then returned to their cages and allowed to wake up naturally.
8. The method for establishing a rat biomimetic model of sudden deafness caused by inner ear microcirculation disorder according to claim 1, characterized in that, In the rat model detection and verification steps, the audiological detection uses the auditory brainstem response test to detect changes in hearing threshold at different time points after rat injection; the inner ear histopathological examination includes sectioning the rat inner ear to observe microthrombus formation and tissue damage; and the inner ear microcirculation detection uses a laser Doppler flowmeter to detect changes in inner ear blood flow.