A method for constructing an animal model of obstructive sleep apnea

Abstract

The present invention relates to the field of biotechnology, and more particularly to a method for constructing an animal model of obstructive sleep apnea. The present invention provides a method for constructing an OSA animal model, comprising injecting 50% glycerol or LPS bilaterally into the palatopharyngeal muscles of a rabbit to construct an OSA animal model. This method can not only fully simulate OSA from the perspective of anatomical and pathophysiological characteristics, but also has the advantages of high safety, a high success rate, and the absence of high-end equipment and special operations. This method is consistent with the clinical practice of OSA animal models, greatly promoting the revelation of the pathogenesis of OSA and the development of new drugs, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing an obstructive sleep apnea animal model. Background Art

[0002] Obstructive sleep apnea (OSA) is a common sleep disorder characterized by snoring, recurrent awakenings, intermittent hypoxia, and excessive daytime sleepiness. OSA is associated with a variety of clinical diseases, including an increased risk of cardiovascular disease, metabolic disorders, and cognitive impairment. It is estimated that approximately 936 million people suffer from OSA worldwide, and this number is still growing. OSA has become an extremely important public health issue, but its pathophysiological mechanisms are not yet fully understood. As the basis for studying OSA, the establishment of OSA model animals has greatly promoted the revelation of the pathogenesis of the disease and the development of new drugs.

[0003] In the past few decades, due to the limitations of animal models, the research on the pathophysiology of OSA has progressed relatively slowly. Currently, a variety of OSA animal models have been developed, which are mainly divided into natural OSA animal models, direct OSA animal models, and indirect OSA animal models. Among them, the chemically induced OSA animal model is a direct OSA animal model. It directly simulates the typical characteristics of OSA by inducing upper airway obstruction in animals, reflecting multiple aspects of OSA more comprehensively, and has the obvious advantages of low invasiveness and high reproducibility. However, the chemically induced OSA animal model still has certain problems, such as the need for high-end experimental equipment, the effectiveness and safety of the model need to be improved, and it cannot fully simulate the anatomical and pathophysiological characteristics of OSA patients.

[0004] In summary, it is difficult to establish an OSA animal model that fully simulates the anatomical and pathophysiological characteristics, does not require high-end equipment and special operations, and is safe and effective with existing technology. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a method for constructing an obstructive sleep apnea animal model.

[0006] The present invention provides a method for constructing an obstructive sleep apnea animal model, which comprises the following steps:

[0007] Take the animals, inject 30-50% glycerol or lipopolysaccharide into the palatopharyngeal muscles on both sides, and continue to raise them.

[0008] Preferably, the animal is taken, 30-50% glycerol is injected into the palatopharyngeal muscles on both sides, and the animal is continued to be raised.

[0009] Preferably, the injection dose is 50-150 μL / mouse.

[0010] Preferably, the continued feeding period is 4-12 weeks.

[0011] Preferably, the animal is taken, lipopolysaccharide is injected into the palatopharyngeal muscles on both sides, and the animal is continued to be raised.

[0012] Preferably, the injection dose is 50-150 μg / kg, and the concentration of lipopolysaccharide is 1.8-2.2 mg / mL.

[0013] Preferably, the continued feeding period is 5-10 weeks.

[0014] Preferably, the animal is selected from monkeys, pigs, dogs, and rabbits.

[0015] Preferably, the animal is a New Zealand rabbit.

[0016] Preferably, the specific method of injection is: using an opener, using a 30G, 25mm needle, under the guidance of a visual laryngoscope, the needle reaches the root of the tonsil, the needle depth is 2~3mm, and the angle between the needle and the rabbit's incisors is 50-70 degrees.

[0017] The present invention provides a method for constructing an OSA animal model, wherein 50% glycerol or LPS is injected bilaterally into the palatopharyngeal muscles of rabbits to construct an OSA animal model. This method can not only simulate OSA in all aspects of anatomical and pathophysiological characteristics, but also solve the problem that existing OSA animal models cannot simultaneously simulate the multi-factor pathogenic factors such as fatty infiltration of the upper airway muscles, inflammatory response, and neurological dysfunction in OSA patients, but also has the advantages of simple operation, stable model phenotype, high modeling success rate, and good reproducibility; especially in the New Zealand rabbit model, this method can maintain high AHI and ODI levels for a relatively long period of time, and the pathological characteristics are highly consistent with the muscle manifestations of OSA patients. This method is consistent with the actual clinical OSA animal model, has a great promoting effect on revealing the pathogenesis of OSA and developing new drugs, and has good application prospects.

[0018] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0019] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A model diagram of injecting chemicals into animals.

[0021] Figure 2 The figures are polysomnographic images of model animals constructed by treating the palatopharyngeal muscle (Pal) with botulinum toxin type A (BTTA) or normal saline; A is the polysomnographic image of OSA model animals constructed by treating Pal with BTTA; B is the polysomnographic image of control group model animals constructed by treating Pal with corresponding isotonic normal saline.

[0022] Figure 3 These are polysomnographic images of model animals constructed by treating the genioglossus muscle (GG) with BTTA or normal saline; A is the polysomnographic image of the OSA model animal constructed by treating the GG with BTTA; B is the polysomnographic image of the control group model animal constructed by treating the GG with the corresponding isotonic normal saline.

[0023] Figure 4 Figures 1 and 2 show the AHI and ODI results of the OSA model animals constructed with low-dose BTTA and the control group; Figure 2 shows the AHI and ODI results of the OSA model animals constructed with BTTA-treated Pal and the control group; Figure 3 shows the ODI results of the OSA model animals constructed with BTTA-treated Pal and the control group; Figure 4 shows the AHI and ODI results of the OSA model animals constructed with BTTA-treated GG and the control group; Figure 5 shows the ODI results of the OSA model animals constructed with BTTA-treated GG and the control group.

[0024] Figure 5 These are polysomnographic images of model animals constructed by treating Pal with 50% glycerol or normal saline; A is the polysomnographic image of OSA model animals constructed by treating Pal with 50% glycerol; B is the polysomnographic image of control group model animals constructed by treating Pal with the corresponding isotonic normal saline.

[0025] Figure 6 Figure 2 is the polysomnographic monitoring diagram of the model animals constructed by treating GG with 50% glycerol or normal saline; Figure 2 is the polysomnographic monitoring diagram of the OSA model animals constructed by treating GG with 50% glycerol; Figure 2 is the polysomnographic monitoring diagram of the control group model animals constructed by treating GG with the corresponding isotonic normal saline.

[0026] Figure 7 Figure 3 is a graph showing the AHI and ODI results of the OSA model animals constructed with 50% glycerol and the control group; Figure A is a graph showing the AHI results of the OSA model animals constructed with Pal treated with 50% glycerol and the control group; Figure B is a graph showing the ODI results of the OSA model animals constructed with Pal treated with 50% glycerol and the control group; Figure C is a graph showing the AHI results of the OSA model animals constructed with GG treated with 50% glycerol and the control group; Figure D is a graph showing the ODI results of the OSA model animals constructed with GG treated with 50% glycerol and the control group.

[0027] Figure 8 These are polysomnographic images of model animals constructed by treating Pal with lipopolysaccharide (LPS) or normal saline; A is the polysomnographic image of OSA model animals constructed by treating Pal with LPS; B is the polysomnographic image of control group model animals constructed by treating Pal with corresponding isotonic normal saline.

[0028] Figure 9 These are polysomnographic images of model animals constructed by treating GG with LPS or saline; A is the polysomnographic image of OSA model animals constructed by treating GG with LPS; B is the polysomnographic image of control group model animals constructed by treating GG with corresponding isotonic saline.

[0029] Figure 10 Figure 3 is a graph showing the AHI and ODI results of the OSA model animals constructed with LPS and the control group; Figure A is a graph showing the AHI results of the OSA model animals constructed with LPS-treated Pal and the control group; Figure B is a graph showing the ODI results of the OSA model animals constructed with LPS-treated Pal and the control group; Figure C is a graph showing the AHI results of the OSA model animals constructed with LPS-treated GG and the control group; Figure D is a graph showing the ODI results of the OSA model animals constructed with LPS-treated GG and the control group.

[0030] Figure 11 Figures A and B show the DISE results of OSA model animals; Figure A shows the DISE results of model animals constructed with BTTA or normal saline; Figure B shows the DISE results of model animals constructed with 50% glycerol or normal saline; Figure C shows the DISE results of model animals constructed with LPS or normal saline.

[0031] Figure 12 The diagrams are the root mean square (RMS) results of the electromyogram of the model animals constructed by BTTA; A is the RMS result diagram of the electromyogram of the model animals constructed by treating Pal with BTTA; B is the RMS result diagram of the electromyogram of the model animals constructed by treating GG with BTTA.

[0032] Figure 13 Figure 5 is the result diagram of the electromyogram RMS of the model animal constructed with 50% glycerol; Figure A is the result diagram of the electromyogram RMS of the model animal constructed with 50% glycerol treatment of Pal; Figure B is the result diagram of the electromyogram RMS of the model animal constructed with 50% glycerol treatment of GG.

[0033] Figure 14 A is the result diagram of the electromyogram RMS of the model animal constructed by LPS treatment; A is the result diagram of the electromyogram RMS of the model animal constructed by LPS treatment of Pal; B is the result diagram of the electromyogram RMS of the model animal constructed by LPS treatment of GG.

[0034] Figure 15 Figure 5 is a graph showing the peripheral blood routine and blood lipid level results of OSA model animals constructed by BTTA; Figure A is a graph showing the peripheral blood routine and blood lipid level results of OSA model animals constructed by treating Pal with BTTA; Figure B is a graph showing the peripheral blood routine and blood lipid level results of OSA model animals constructed by treating GG with BTTA.

[0035] Figure 16 Figure 1 is a graph showing the peripheral blood routine and blood lipid level results of OSA model animals constructed with 50% glycerol; Figure 1 is a graph showing the peripheral blood routine and blood lipid level results of OSA model animals constructed with Pal treated with 50% glycerol; Figure 1 is a graph showing the peripheral blood routine and blood lipid level results of OSA model animals constructed with GG treated with 50% glycerol.

[0036] Figure 17 Figure 5 is a graph showing the peripheral blood routine and blood lipid level results of OSA model animals constructed by LPS treatment; Figure A is a graph showing the peripheral blood routine and blood lipid level results of OSA model animals constructed by LPS treatment of Pal; Figure B is a graph showing the peripheral blood routine and blood lipid level results of OSA model animals constructed by LPS treatment of GG.

[0037] Figure 18 The figures are the results of success rate and mortality of OSA model animals; A is the results of success rate and mortality of OSA model animals constructed by BTTA-treated Pal; B is the results of success rate and mortality of OSA model animals constructed by BTTA-treated GG; C is the results of success rate and mortality of OSA model animals constructed by 50% glycerol-treated Pal; D is the results of success rate and mortality of OSA model animals constructed by 50% glycerol-treated GG; E is the results of success rate and mortality of OSA model animals constructed by LPS-treated Pal; F is the results of success rate and mortality of OSA model animals constructed by LPS-treated GG.

[0038] Figure 19 Figure 1 is a result graph of the area between the curve of success rate and mortality rate of OSA model animals; Figure 1 is a result graph of the area between the curve of success rate and mortality rate of OSA model animals constructed by treating Pal with BTTA, 50% glycerol and LPS respectively; Figure 1 is a result graph of the area between the curve of success rate and mortality rate of OSA model animals constructed by treating GG with BTTA, 50% glycerol and LPS respectively. DETAILED DESCRIPTION

[0039] In the following examples and experimental examples, reagents and materials not otherwise specified are commercially available.

[0040] Male New Zealand rabbits, weighing 2.2-2.7 kg and 3 months old, were housed in a standardized laboratory environment in Chengdu, China, maintained at approximately 25°C with a 12-hour light and 12-hour dark cycle. Prior to modeling, all animals had no clinical or experimental history that could affect breathing and sleep or increase disease susceptibility. Pre-modeling, they were fed an adaptive diet for 1 week, during which time their weight and growth were observed, and any abnormalities were promptly culled.

[0041] Example 1 Construction of OSA Animal Model

[0042] (1) Preparation of solution

[0043] 50% glycerol: Mix pure glycerol with an equal volume of 0.9% saline and stir thoroughly.

[0044] (2) Construction of animal models

[0045] A New Zealand rabbit was injected with 150 μL of 50% glycerol bilaterally into the palatopharyngeal muscles. A mouthpiece was used to fully expose the field of view, and the injection process was guided by a video laryngoscope. The specific injection method used a 30-gauge, 25-mm needle. Under the guidance of a video laryngoscope, the needle was inserted directly into the base of the tonsil to a depth of 2-3 mm, with the needle angle between the rabbit and the incisor being 50-70 degrees. Injections were performed once a week for 2 consecutive weeks.

[0046] After the injection is completed, the rats are kept for 6 weeks with normal feeding during the feeding period to obtain an OSA animal model.

[0047] In other embodiments, the duration of continued feeding after completion of the injection can be adjusted to 4-12 weeks.

[0048] Example 2 Construction of OSA Animal Model

[0049] (1) Preparation of solution

[0050] Lipopolysaccharide (LPS) solution: Weigh 10 mg of LPS (E. coli O55:B5, Sigma-Aldrich, L2880) and dissolve it in 5 mL of 0.9% saline to obtain a 2 mg / mL solution. Mix thoroughly and store in the dark.

[0051] (2) Construction of animal models

[0052] The animal model was constructed according to the method described in Example 1, except that 150 μL of 50% glycerol was replaced with a 150 μg / kg LPS solution. Feeding was continued for 8 weeks. The volume of LPS solution injected into each New Zealand rabbit was approximately 100 μL, adjusted according to the rabbit's weight.

[0053] In other embodiments, the duration of continued feeding can be adjusted to 5-10 weeks.

[0054] Example 3 Construction of OSA Animal Model

[0055] The animal model was constructed according to the method described in Example 2, except that the dose of LPS solution was adjusted from 150 μg / kg animal to 50 μg / kg animal.

[0056] The beneficial effects of the present invention are further illustrated by the following test examples:

[0057] Experimental Example 1: Research on OSA model construction method

[0058] This study was approved by the Ethics Review Committee of West China Hospital, Sichuan University (approval number: 2021(1785)). All participants were informed of the nature of the study and provided written informed consent upon admission. All experimental procedures adhered to the principles of the Declaration of Helsinki. Animal use and care were approved by the Animal Experimentation Ethics Committee of West China Hospital, Sichuan University (approval number: 20230811001).

[0059] 1. Model Construction

[0060] 1 Experimental animals

[0061] Male New Zealand rabbits, weighing 2.2-2.7 kg and 3 months old, were housed in a standardized laboratory environment in Chengdu, China, maintained at approximately 25°C with a 12-hour light and 12-hour dark cycle. All animals had no clinical or experimental history of conditions that could affect breathing and sleep or increase disease susceptibility. They were fed a pre-experimental acclimatization program for one week, during which time their weight and growth were observed, and any abnormalities were promptly culled.

[0062] 2 Experimental reagents

[0063] Botulinum toxin A (BTTA) stock solution: Dissolve 100 U of BTTA (Allergan Inc) in 4 ml of 0.9% saline for injection. Use within 24 hours and store at 2-8°C.

[0064] 50% glycerol: Mix pure glycerol with an equal volume of 0.9% saline and stir thoroughly.

[0065] Lipopolysaccharide (LPS) solution: Dissolve LPS in 0.9% saline, mix thoroughly, and store in the dark.

[0066] 3 Experimental Grouping

[0067] Eighty male New Zealand rabbits were divided into experimental groups and injected into the palatopharyngeal (Pal) and genioglossus (GG) muscles. Each intervention drug was injected into the palatopharyngeal or genioglossus muscles to induce muscle paralysis, fatty infiltration, and inflammation.

[0068] 4 Experimental methods

[0069] (1) Injection method of palatopharyngeus and genioglossus muscles

[0070] like Figure 1 For palatopharyngeal injection, a mouthpiece is used to fully expose the visual field, and the injection process is guided by a video laryngoscope. The specific injection method is to use a 30-gauge, 25-mm needle. Under the guidance of a video laryngoscope, the needle is inserted directly into the base of the tonsil to a depth of 2-3 mm, with the angle between the needle and the rabbit's incisor being 50-70 degrees. The drug is injected slowly (25-30 seconds), and the needle is then quickly withdrawn.

[0071] For genioglossus injection, oral administration was performed using a 30-gauge, 25-mm needle, following the method described by Lee et al. (Lee, Myung-Chul et al. “Establishment of a rabbit model of obstructive sleep apnea by paralyzing thegenioglossus.” JAMA Otolaryngology-- Head & Neck Surgery vol. 139, 8 (2013): 834-40. doi:10.1001 / jamaoto.2013.4001).

[0072] (2) Anesthesia preparation

[0073] Rabbits were fasted for at least 8 hours before surgery and deprived of water for at least 4 hours before injection to reduce the risk of aspiration during surgery. Before injection, rabbits were fully anesthetized using 3% sodium pentobarbital solution (1 ml / kg) via the marginal ear vein. Food and water were withheld for at least 4 hours after the injection.

[0074] (3) OSA rabbit model established by BTTA

[0075] The OSA rabbit model was established by injection into the palatopharyngeal muscle: 100 μL (high dose), 80 μL (medium dose), and 60 μL (low dose) of BTTA stock solution were injected bilaterally into the palatopharyngeal muscle, and the corresponding volume of isotonic saline was used as a control.

[0076] The OSA rabbit model was established by injection into the genioglossus muscle: 100 μL of BTTA stock solution was injected into the genioglossus muscle, and the corresponding volume of isotonic saline was used as a control.

[0077] (4) OSA rabbit model established with 50% glycerol

[0078] The OSA rabbit model was established by injection into the palatopharyngeal muscle: 150 μL of 50% glycerol was injected into the palatopharyngeal muscle of the rabbit.

[0079] The OSA rabbit model was established by injection into the genioglossus muscle: 600 μL of 50% glycerol was injected into the genioglossus muscle of the rabbit.

[0080] Each model group was equipped with corresponding isotonic saline as a control group.

[0081] (5) OSA rabbit model established with lipopolysaccharide (LPS)

[0082] The OSA rabbit model was established by injection into the palatopharyngeal muscle: LPS solution was injected bilaterally into the palatopharyngeal muscle of the rabbit at a dose of 150 μg / kg.

[0083] The OSA rabbit model was established by injection into the genioglossus muscle: LPS solution was injected into the genioglossus muscle of the rabbit at a dose of 400 μg / kg.

[0084] Each model group was equipped with corresponding isotonic saline as a control group.

[0085] 2. Model Evaluation

[0086] 1 Evaluation method

[0087] (1) Sleep induction

[0088] Sleep was induced by simultaneous intramuscular injection of 0.3 mL / kg of Zoletil (fluthixene hydrochloride and zolazepam; Virbac) and 0.2 mL / kg of Rompun (fluoxetine; Bayer).

[0089] (2) Polysomnography (PSG)

[0090] Respiratory events were recorded using a portable polysomnographic device (Embletta MPR PG ST; Embla Systems). Monitoring primarily included nasal airflow or pressure, ear oxygen saturation, respiratory movements, and pulse. Nasal airflow was monitored using an infant-sized catheter. To monitor ear oxygen saturation and pulse, the ears were shaved with a mild depilatory cream, and a modified pulse oximeter clip was attached to the ear vessels to monitor oxygen saturation and pulse. Respiratory movements were monitored using two chest straps and one abdominal strap. The chest strap was placed 1 cm below the axilla, and the abdominal strap was placed at the midpoint of the body. Throughout monitoring, careful attention was paid to maintaining sensor position. Rabbits were placed in the supine position for sleep monitoring. Each rabbit was monitored for at least 30 minutes. Baseline PSG monitoring was performed after one week of adaptive feeding, followed by biweekly sleep monitoring. Because rabbit physiology differs from that of humans, an AHI > 0 on PSG was considered indicative of OSA.

[0091] (3) Drug-induced sleep endoscopy (DISE)

[0092] After waiting 10-15 minutes for the rabbit to fall asleep and its respiration and heart rate to stabilize, DISE was performed using a 2.8 mm diameter laryngoscope (Seesheen, BR-E11). During the examination, 1% flucasone hydrochloride solution (Changjiang Pharmaceutical) was applied to the rabbit's nasal vestibule and the first 10 cm of the endoscope. Examination focused on the palatopharyngeus, oropharynx, and epiglottis. The endoscope was held in each plane for 10 respiratory cycles. For the BTTA-treated Pal group, a low dose was used.

[0093] (4) Electromyography (EMG) and data processing

[0094] The EMG signals were recorded by a BL-420N biological signal acquisition and analysis system (Taimeng, BL-420N) with a low-pass filter of 1 kHz, a sweep speed of 0.5 s, a range of 500 μV, a time constant of 200 ms, and a sampling frequency of 5 kHz.

[0095] At week 8, EMG assessments were performed on the rabbits. After waiting 10-15 minutes for the rabbits to fall asleep, their respiration and heart rate gradually stabilized. Because the rabbits' palatal muscles are very small and difficult to perform EMG studies on, EMG studies were performed on the genioglossus muscle in the palatopharyngeal injection group. For the BTTA-treated palatal group, a low dose was used.

[0096] The rabbit was placed in the supine position with all four limbs immobilized. The equipment was connected to the system, and parameters were set as needed. A ground electrode was inserted subcutaneously. The rabbit's mouth was opened approximately 30 degrees using an opener. An oval clamp with a small gauze pad was used to grasp the tip of the tongue and slightly pulled out to expose the area beneath the tongue base. This facilitated insertion of two needle electrodes into the genioglossus muscle to initiate recording. Statistical analysis of the EMG data was performed using MATLAB R2023a (Mathworks, Natick, MA, USA) software to analyze the raw data.

[0097] (5) Blood collection and analysis

[0098] Blood samples were collected from the rabbit's ear vein. The rabbits were properly secured, and blood was drawn using a sterile syringe and transferred to EDTA-coated tubes for complete blood count (CBC) analysis and to plain tubes for lipid profile analysis. For the BTTA-treated Pal group, a low dose was used.

[0099] For CBC, blood samples were analyzed using an automated hematology analyzer (BC-2800Vet). Parameters measured included white blood cell count (WBC), neutrophil count, and neutrophil percentage.

[0100] For lipid profile testing, serum was separated by centrifugation at 3000 rpm for 10 minutes and then analyzed using an automated biochemical analyzer (BS-240VET). Lipid profile parameters measured included total cholesterol (TC) and low-density lipoprotein (LDL).

[0101] (6) Modeling success rate and mortality analysis

[0102] The modeling success rate was calculated by dividing the number of rabbits successfully established by the number of rabbits surviving every two weeks. A successful modeling was considered if the PSG results showed an AHI > 0. Mortality was calculated by dividing the cumulative number of rabbits that died every two weeks by the total number of models. Modeling success and mortality rates were calculated every two weeks. The area between the success rate and mortality rate curves was analyzed using Origin (2019b) software.

[0103] 2 Evaluation results

[0104] (1) PSG results

[0105] The results of the BTTA model are as follows Figure 2-4Figure 2: Injection of BTTA into both the Pal and GG follicles successfully induced muscle paralysis and OSA in rabbits with OSA. Apnea and hypopnea events of varying severity began to occur at week 6 in the Pal group and week 2 in the GG group. Apnea-hypopnea index (AHI) peaked at week 10 (Pal) and week 8 (GG), respectively. The AHI in the Pal and GG groups was significantly higher than that in the control group (Pal control group: p = 0.038, Student's t-test; GG control group: p = 0.030, Student's t-test). The impact of these apnea and hypopnea events on oxygenation was revealed by the oxygen desaturation index (ODI). The AHI and ODI in the Pal group continued to increase, reaching a significantly higher ODI than that in the control group at week 10 (Student's t-test, p < 0.05). The trends in the AHI and ODI in the GG group were not completely consistent with those in the Pal group. The AHI showed a decreasing trend from weeks 4 to 6, while the ODI showed a decreasing trend from weeks 2 to 4. The AHI of the GG group was significantly higher than that of the control group at week 4, while the ODI at weeks 4, 6, and 10 were also significantly higher than those of the control group (Student's t test, p < 0.05).

[0106] The results of the 50% glycerol model are as follows Figure 5-7 As shown: In a rabbit model of OSA established with 50% glycerol, apnea and hypopnea events were observed in the Pal and GG muscles, whereas these events were absent in the control group (Pal control group: p = 0.030, Student's t-test; GG control group: p = 0.020, Student's t-test). The AHI and ODI in the Pal group treated with 50% glycerol continued to increase until week 6, then decreased, likely due to the clearance of fatty infiltration from the muscle. The ODI was significantly increased at weeks 4 and 10 (Student's t-test, p < 0.05). The AHI in the GG group was significantly higher than that in the control group at week 6 (Student's t-test, p < 0.01), and the ODI was also significantly increased at weeks 6 and 8 (Student's t-test, p < 0.05).

[0107] The results of the LPS model are as follows Figure 8-10As shown: In the LPS-induced OSA rabbit model, apnea and hypopnea events were also observed in the Pal group, with slightly different trends and peak times compared to the BTTA model (Pal control group: p = 0.020, Student's t-test; GG control group: p = 0.081, Student's t-test). The AHI in the Pal group was significantly different at week 8 (Student's t-test, p < 0.05). The ODI trend in the LPS-treated group was similar to that of the AHI, with a strong correlation between the two, but the nonlinear relationship resulted in inconsistent changes. Significant differences were observed at weeks 6 and 8 (Pal) and 4 and 8 (GG) (Student's t-test, p < 0.05).

[0108] The above results show that the rabbit models constructed by treating the palatopharyngeus and genioglossus muscles of rabbits with BTTA, 50% glycerol, and LPS all have apnea and hypopnea conditions, and the apnea and hypopnea conditions can be maintained for a long time.

[0109] (2) DISE results

[0110] DISE results are as follows Figure 11 As shown, the rabbit models constructed with BTTA, 50% glycerol, or LPS all showed partial or complete airway collapse during sleep.

[0111] (3) EMG experimental results

[0112] The EMG experimental results are as follows Figure 12-14 As shown in Tables 1-3, the EMG root mean square (RMS) levels in the Pal groups treated with BTTA, 50% glycerol, and LPS were higher than those in the control group. The raw EMG signals from the rabbits demonstrate muscle activity in each group. These results indicate that the rabbit model established with BTTA, 50% glycerol, or LPS induces global fatigue of the upper airway muscles and induces OSA.

[0113] Table 1 EMG RMS results of BTTA model

[0114]

[0115] Table 2 EMG RMS results of 50% glycerol model

[0116]

[0117] Table 3 EMG RMS results of LPS model

[0118]

[0119] (4) Peripheral blood test results

[0120] Peripheral blood tests such as Figure 15-17 As shown in Tables 4-6: For the model constructed with BTTA, compared with the control group, there was no significant difference in the blood routine and blood lipid levels of the model constructed after BTTA treated Pal. Among the blood routine and blood lipid levels of the model constructed after BTTA treated Pal, only the low-density lipoprotein (LDL) was significantly increased; for the model constructed with 50% glycerol, compared with the control group, the neutrophil, total cholesterol and LDL in the blood routine and blood lipid levels of the model constructed after 50% glycerol treated Pal were significantly increased, and the total cholesterol and LDL in the blood routine and blood lipid levels of the model constructed after 50% glycerol treated GG were significantly increased; for the model constructed with LPS, compared with the control group, the neutrophil level in the blood routine and blood lipid levels of the model constructed after LPS treated Pal was significantly increased, and the total cholesterol and LDL in the blood routine and blood lipid levels of the model constructed after LPS treated GG were significantly increased.

[0121] In particular, in the model constructed after treating Pal with 50% glycerol, the neutrophil level, total cholesterol content and LDL content in the peripheral blood were significantly increased, indicating that inflammation was aggravated and lipid metabolism was significantly affected. This aggravated inflammation and lipid metabolism disorder are closely related to OSA.

[0122] Table 4 Peripheral blood routine and blood lipid level results of BTTA model

[0123]

[0124] Table 5 Peripheral blood routine and blood lipid level results of 50% glycerol model

[0125]

[0126] Table 6 Peripheral blood routine and blood lipid level results of LPS model

[0127]

[0128] (5) Modeling success rate and mortality results

[0129] The success rate results are as follows Figure 18 As shown in Table 7: For the model animals constructed by BTTA-treated Pal, the success rates of the medium-dose and low-dose groups were higher.

[0130] For the Pal group, the success rates of BTTA and LPS treatments peaked at week 8 (BTTA: 80%, LPS: 100%), while the peak for 50% glycerol treatment occurred at week 6 (50% glycerol: 100%). For the GG group, the success rate of BTTA treatment peaked at week 4 (BTTA: 100%), while the peaks for 50% glycerol and LPS treatments occurred at week 8 (50% glycerol: 100%, LPS: 66.7%).

[0131] Table 7 Success rate and mortality rate of BTTA treatment group

[0132]

[0133] The power results showed that for the Pal group, the animal model established with 50% glycerol took less time, and the animal model established with 50% glycerol and LPS had a high success rate of 100%; for the GG group, the BTTA group took less time, and the animal model established with BTTA and 50% glycerol had a high success rate of 100%.

[0134] Mortality results as Figure 18 As shown in Table 7: For the model animals constructed by BTTA-treated Pal, the mortality rate of the low-dose group was the lowest.

[0135] For the Pal group, the mortality rate was 25% after BTTA treatment, and 0% after glycerol and LPS treatment; for the GG group, the mortality rate was 25% after BTTA treatment, 20% after glycerol treatment, and 0% after LPS treatment.

[0136] The mortality results showed that the animal models established by 50% glycerol-treated Pal, LPS-treated Pal or GG were safe.

[0137] By calculating the area between the success rate and mortality rate curves, the results are as follows Figure 19 As shown in Tables 7 and 8, for model animals constructed with BTTA-treated Pal, the low-dose group had the highest area between the success rate and mortality curves, but this value was significantly lower than that for models constructed with 50% glycerol and LPS-treated Pal. Therefore, treating Pal with 50% glycerol and LPS and GG with BTTA is an effective strategy for constructing OSA models with high success rates.

[0138] Table 8 Success rate and mortality rate of LPS treatment group

[0139]

[0140] The above results are summarized in Tables 9 and 10 below. A comprehensive evaluation of the three OSA models was conducted using polysomnography (PSG), drug-induced sleep endoscopy (DISE), electromyography (EMG), and peripheral blood tests. Combined with success rate and mortality, the results showed that the OSA models constructed with 50% glycerol and LPS in Pal can fully simulate OSA from the structural, functional, and phenotypic aspects, and have the advantages of high safety, high success rate, and no need for high-end equipment or special operations. Among them, the OSA model constructed with 50% glycerol in Pal takes less time, has more significant pathophysiological characteristics, and is more relevant to the specific characteristics of OSA patients.

[0141] Table 9 Phenotypic characteristics of Pal in three OSA models

[0142]

[0143] Note: * p<0.05; ** p<0.01; *** p<0.001, “ns” means no statistical difference.

[0144] Table 10 Phenotypic characteristics of GG treated with three OSA models

[0145]

[0146] Note: * p < 0.05; ** p < 0.01; *** p < 0.001; "ns" indicates no statistically significant difference.

[0147] As can be seen from the aforementioned implementation cases and experimental examples, the present invention provides a method for constructing an OSA animal model by bilaterally injecting 50% glycerol or LPS into the palatopharyngeal muscles of rabbits. This method not only comprehensively simulates OSA from the structural, functional, and phenotypic perspectives, but also offers advantages such as high safety, a high success rate, and the absence of advanced equipment and specialized procedures. This method, consistent with clinically practical OSA animal models, significantly contributes to the elucidation of OSA pathogenesis and the development of new drugs, and holds great promise for future application.

Claims

1. A method for constructing an animal model of obstructive sleep apnea, characterized by: The steps include: The animals were taken and 30-50% glycerol was injected into the palatopharyngeal muscles on both sides respectively, and the animals were kept continuously; the injection dose was 50-150 μL / animal, and the animals were selected from rabbits.

2. The construction method according to claim 1, wherein: The time of continuing feeding is 4-12 weeks.

3. The construction method according to claim 1, wherein: The animal is a New Zealand rabbit.

4. The construction method according to claim 3, characterized in that The specific method of the injection is: using an opener, using a 30G, 25mm needle, under the guidance of a visual laryngoscope, the needle reaches the root of the tonsil, the needle depth is 2 to 3mm, and the angle between the needle and the rabbit's incisors is 50-70 degrees.

Images