Construction method of obstructive sleep apnea animal model

Abstract

The invention relates to the technical field of biology, in particular to a construction method of an obstructive sleep apnea animal model. The invention provides a construction method of an OSA animal model, which comprises the following steps: respectively injecting 50% glycerol or LPS (lipopolysaccharide) on both sides of the palatopharyngeal muscle of a rabbit to construct the OSA animal model. The method not only can simulate the OSA comprehensively from dissection and pathophysiological features, but also has the advantages of high safety, high success rate and no need of high-end equipment and special operation. The method accords with a clinical practical OSA animal model, has a great promotion effect on revealing OSA pathogenesis, developing new drugs and the like, and has a good application prospect.

Description

Technical Field

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

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

[0003] In the past few decades, due to the limitations of animal models, the research progress on the pathophysiology of OSA has been relatively slow. Currently, a variety of OSA animal models have been developed, 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, which directly simulates the typical characteristics of OSA by triggering upper airway obstruction in animals, comprehensively reflects multiple aspects of OSA, and has the obvious advantages of less 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 effects 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 in the prior art that can fully simulate anatomical and pathophysiological characteristics, does not require high-end equipment and special operations, and is safe and effective. Summary of the Invention

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

[0006] The present invention provides a method for constructing an animal model of obstructive sleep apnea, which includes the following steps: Take an animal, inject 30 - 50% glycerol or lipopolysaccharide into both sides of the palatopharyngeal muscle respectively, and continue to breed to obtain the model.

[0007] Preferably, take an animal, inject 30 - 50% glycerol into both sides of the palatopharyngeal muscle respectively, and continue to breed to obtain the model.

[0008] Preferably, the injection dose is 50 - 150 μL / animal.

[0009] Preferably, the time for continued feeding is 4 - 12 weeks.

[0010] Preferably, take an animal, inject lipopolysaccharide into both sides of the palatopharyngeal muscle, and continue feeding to obtain it.

[0011] Preferably, the injection dose is 50 - 150 μg / kg, and the concentration of the lipopolysaccharide is 1.8 - 2.2 mg / mL.

[0012] Preferably, the time for continued feeding is 5 - 10 weeks.

[0013] Preferably, the animal is selected from monkeys, pigs, dogs, and rabbits.

[0014] Preferably, the animal is a New Zealand rabbit.

[0015] Preferably, the specific injection method is as follows: Under the use of a mouth gag, use a 30G, 25 - mm needle. Under the guidance of a video laryngoscope, the needle reaches the root of the tonsil directly, the needle depth is 2 - 3 mm, and the angle between the needle and the rabbit's front teeth is 50 - 70 degrees.

[0016] The present invention provides a method for constructing an OSA animal model, by injecting 50% glycerol or LPS into both sides of the palatopharyngeal muscle of a rabbit to construct an OSA animal model. This method can not only simulate OSA comprehensively from anatomical and pathophysiological characteristics, solve the problem that existing OSA animal models cannot simultaneously simulate multiple pathogenic factors such as upper airway muscle fat infiltration, inflammatory response, and nerve 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 time, and the pathological characteristics highly fit the muscle performance of OSA patients. This method conforms to the OSA animal model in clinical practice, and has a great promoting effect on revealing the pathogenesis of OSA and developing new drugs, etc., and has good application prospects.

[0017] Obviously, based on the above content of the present invention, according to the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions, or changes can be made.

[0018] The following is a further detailed description of the above content of the present invention 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 belong to the scope of the present invention. Brief Description of the Drawings

[0019] Figure 1 Model diagram of injecting chemical drugs into an animal.

[0020] Figure 2 Polysomnogram of model animals constructed by treating the palatopharyngeal muscle (Pal) with botulinum toxin type A (BTTA) or saline; where A is the polysomnogram of OSA model animals constructed by treating Pal with BTTA; where B is the polysomnogram of control model animals constructed by treating Pal with corresponding isotonic saline.

[0021] Figure 3 Polysomnogram of model animals constructed by treating the genioglossus muscle (GG) with BTTA or saline; where A is the polysomnogram of OSA model animals constructed by treating GG with BTTA; where B is the polysomnogram of control model animals constructed by treating GG with corresponding isotonic saline.

[0022] Figure 4 AHI and ODI result graphs of OSA model animals and control groups constructed with BTTA at low doses; where A is the AHI result graph of OSA model animals and control groups constructed by treating Pal with BTTA; where B is the ODI result graph of OSA model animals and control groups constructed by treating Pal with BTTA; where C is the AHI result graph of OSA model animals and control groups constructed by treating GG with BTTA; where D is the ODI result graph of OSA model animals and control groups constructed by treating GG with BTTA.

[0023] Figure 5 Polysomnogram of model animals constructed by treating Pal with 50% glycerol or saline; where A is the polysomnogram of OSA model animals constructed by treating Pal with 50% glycerol; where B is the polysomnogram of control model animals constructed by treating Pal with corresponding isotonic saline.

[0024] Figure 6 Polysomnogram of model animals constructed by treating GG with 50% glycerol or saline; where A is the polysomnogram of OSA model animals constructed by treating GG with 50% glycerol; where B is the polysomnogram of control model animals constructed by treating GG with corresponding isotonic saline.

[0025] Figure 7 AHI and ODI result graphs of OSA model animals and control groups constructed with 50% glycerol; where A is the AHI result graph of OSA model animals and control groups constructed by treating Pal with 50% glycerol; where B is the ODI result graph of OSA model animals and control groups constructed by treating Pal with 50% glycerol; where C is the AHI result graph of OSA model animals and control groups constructed by treating GG with 50% glycerol; where D is the ODI result graph of OSA model animals and control groups constructed by treating GG with 50% glycerol.

[0026] Figure 8 Polysomnogram of model animals constructed by treating Pal with lipopolysaccharide (LPS) or saline; among which, A is the polysomnogram of the OSA model animals constructed by treating Pal with LPS; among which, B is the polysomnogram of the control group model animals constructed by treating Pal with corresponding isotonic saline.

[0027] Figure 9 Polysomnogram of model animals constructed by treating GG with LPS or saline; among which, A is the polysomnogram of the OSA model animals constructed by treating GG with LPS; among which, B is the polysomnogram of the control group model animals constructed by treating GG with corresponding isotonic saline.

[0028] Figure 10 AHI and ODI result graphs of OSA model animals constructed by LPS and the control group; among which, A is the AHI result graph of the OSA model animals constructed by treating Pal with LPS and the control group; among which, B is the ODI result graph of the OSA model animals constructed by treating Pal with LPS and the control group; among which, C is the AHI result graph of the OSA model animals constructed by treating GG with LPS and the control group; among which, D is the ODI result graph of the OSA model animals constructed by treating GG with LPS and the control group.

[0029] Figure 11 DISE result graph of OSA model animals; among which, A is the DISE result graph of the model animals constructed by BTTA or saline; among which, B is the DISE result graph of the model animals constructed by 50% glycerol or saline; among which, C is the DISE result graph of the model animals constructed by LPS or saline.

[0030] Figure 12 Result graph of the root mean square value (RMS) of electromyogram of model animals constructed by BTTA; among which, A is the electromyogram RMS result graph of the model animals constructed by treating Pal with BTTA; among which, B is the electromyogram RMS result graph of the model animals constructed by treating GG with BTTA.

[0031] Figure 13 Result graph of the electromyogram RMS of model animals constructed by 50% glycerol; among which, A is the electromyogram RMS result graph of the model animals constructed by treating Pal with 50% glycerol; among which, B is the electromyogram RMS result graph of the model animals constructed by treating GG with 50% glycerol.

[0032] Figure 14 Result graph of the electromyogram RMS of model animals constructed by LPS; among which, A is the electromyogram RMS result graph of the model animals constructed by treating Pal with LPS; among which, B is the electromyogram RMS result graph of the model animals constructed by treating GG with LPS.

[0033] Figure 15 Results graphs of peripheral blood routine and blood lipid levels of OSA model animals constructed with BTTA; among them, A is the results graph of peripheral blood routine and blood lipid levels of OSA model animals constructed with Pal treated with BTTA; among them, B is the results graph of peripheral blood routine and blood lipid levels of OSA model animals constructed with GG treated with BTTA.

[0034] Figure 16 Results graphs of peripheral blood routine and blood lipid levels of OSA model animals constructed with 50% glycerol; among them, A is the results graph of peripheral blood routine and blood lipid levels of OSA model animals constructed with Pal treated with 50% glycerol; among them, B is the results graph of peripheral blood routine and blood lipid levels of OSA model animals constructed with GG treated with 50% glycerol.

[0035] Figure 17 Results graphs of peripheral blood routine and blood lipid levels of OSA model animals constructed with LPS; among them, A is the results graph of peripheral blood routine and blood lipid levels of OSA model animals constructed with Pal treated with LPS; among them, B is the results graph of peripheral blood routine and blood lipid levels of OSA model animals constructed with GG treated with LPS.

[0036] Figure 18 Results graphs of success rate and mortality rate of OSA model animals; among them, A is the results graph of success rate and mortality rate of OSA model animals constructed with Pal treated with BTTA; among them, B is the results graph of success rate and mortality rate of OSA model animals constructed with GG treated with BTTA; among them, C is the results graph of success rate and mortality rate of OSA model animals constructed with Pal treated with 50% glycerol; among them, D is the results graph of success rate and mortality rate of OSA model animals constructed with GG treated with 50% glycerol; among them, E is the results graph of success rate and mortality rate of OSA model animals constructed with Pal treated with LPS; among them, F is the results graph of success rate and mortality rate of OSA model animals constructed with GG treated with LPS.

[0037] Figure 19 Results graphs of the area under the curve between the success rate and mortality rate of OSA model animals; among them, A is the results graph of the area under the curve between the success rate and mortality rate of OSA model animals constructed with Pal treated with BTTA, 50% glycerol, and LPS respectively; among them, B is the results graph of the area under the curve between the success rate and mortality rate of OSA model animals constructed with GG treated with BTTA, 50% glycerol, and LPS respectively. Detailed implementation methods

[0038] In the following examples and experimental examples, reagents and materials not specifically mentioned are commercially available products.

[0039] New Zealand rabbits, male, 2.2 - 2.7 kg, 3 months old, were raised in a standardized laboratory environment in Chengdu, China. The temperature was maintained at about 25°C, with a 12-hour light and 12-hour dark cycle. Before animal modeling, all animals had no clinical or experimental history that could affect respiration, sleep, or increase disease susceptibility. One week of adaptive feeding was carried out before modeling. During this period, the body weight changes and growth status were observed, and any abnormalities were removed in a timely manner.

[0040] Example 1 Construction of an OSA animal model (1)Preparation of solutions 50% glycerol: Pure glycerol was mixed with an equal volume of 0.9% normal saline and stirred well.

[0041] (2)Construction of the animal model One New Zealand rabbit was taken, and 150 μL of 50% glycerol was injected into both sides of the palatopharyngeal muscle. During injection, an oral speculum was used to fully expose the field of view, and the injection process was guided by a videolaryngoscope. The specific injection method was to use a 30G, 25mm needle. Under the guidance of the videolaryngoscope, the needle reached the root of the tonsil, with a needle depth of 2 - 3 mm and an angle of 50 - 70 degrees between the needle and the rabbit's incisors. Injection was performed once a week for 2 consecutive weeks.

[0042] After the injection was completed, the rabbits were continued to be raised for 6 weeks. During the raising period, normal feeding was carried out, and an OSA animal model could be obtained.

[0043] In other examples, the time for continued raising after the injection was completed could be adjusted within 4 - 12 weeks.

[0044] Example 2 Construction of an OSA animal model (1)Preparation of solutions Lipopolysaccharide (LPS) solution: 10 mg of LPS (E. coli O55:B5, Sigma - Aldrich, L2880) was weighed and dissolved in 5 mL of 0.9% normal saline to obtain a 2mg / mL solution. After thorough mixing, it was stored in the dark.

[0045] (2)Construction of the animal model According to the method for constructing the animal model described in Example 1, the difference was that 150 μL of 50% glycerol was replaced with an LPS solution of 150 μg / kg animal; the rabbits were continued to be raised for 8 weeks. The volume of the LPS solution injected into each New Zealand rabbit was approximately 100 μL, and it was adjusted adaptively according to the body weight of the New Zealand rabbit.

[0046] In other examples, the time for continued raising could be adjusted within 5 - 10 weeks.

[0047] Example 3 Construction of an OSA animal model The construction method of the animal model described in Example 2 is different in that the dose of the LPS solution is adjusted from 150 μg / kg animal to 50 μg / kg animal.

[0048] The beneficial effects of the present invention are further illustrated by the following test examples: Test Example 1 Research on the OSA Model Construction Method This study was approved by the Ethics Review Committee of West China Hospital, Sichuan University (approval number: 2021(1785)). All participants were informed at the time of admission and signed a written informed consent form. All experimental procedures followed the principles in the Declaration of Helsinki. The use and care of animals were approved by the Animal Experiment Ethics Committee of West China Hospital, Sichuan University (approval number: 20230811001). I. Model Construction 1 Experimental Animals New Zealand rabbits, male, 2.2 - 2.7 kg, 3 months old, were raised in a standardized laboratory environment in Chengdu, China. The temperature was maintained at about 25°C, with a 12-hour light and 12-hour dark cycle. Before the experiment, all animals had no clinical or experimental history that could affect respiration, sleep, or increase disease susceptibility. They were fed adaptively for 1 week before the experiment, and their body weight changes and growth status were observed during this period. Any abnormalities were excluded in a timely manner.

[0049] 2 Experimental Reagents Botulinum toxin A (BTTA) stock solution: 100 U of BTTA (Allergan Inc) was dissolved in 4 ml of 0.9% saline for injection, and it needed to be used within 24 hours and stored between 2 - 8°C.

[0050] 50% glycerol: Pure glycerol was mixed with an equal volume of 0.9% saline and stirred well.

[0051] Lipopolysaccharide (LPS) solution: LPS was dissolved in 0.9% saline and stored in the dark after thorough mixing.

[0052] 3 Experimental Groups 80 male New Zealand rabbits were divided into experimental groups and injected into the palatopharyngeal muscle (Pal) and genioglossus muscle (GG) respectively. By injecting different concentrations of BTTA, 50% glycerol, or LPS, each intervention drug was injected into the palatopharyngeal muscle or genioglossus muscle to induce muscle paralysis, fat infiltration, and inflammation.

[0053] 4 Experimental Methods (1) Injection Methods for the Palatopharyngeal Muscle and Genioglossus Muscle As Figure 1As shown, for palatopharyngeal muscle injection, an oral speculum was used to fully expose the field of view, and the injection process was guided by a videolaryngoscope. The specific injection method was to use a 30G, 25-mm needle. Under the guidance of the videolaryngoscope, the needle was inserted directly to the root of the tonsil, with the needle depth of 2-3 mm and the angle between the needle and the rabbit's incisors of 50-70 degrees. The drug was slowly injected (25-30 seconds), and then the needle was quickly withdrawn.

[0054] For genioglossus muscle injection, the method described by Lee et al. (Lee, Myung-Chul et al. “Establishment of a rabbit model of obstructive sleep apnea by paralyzing the genioglossus.” JAMA otolaryngology-- head & neck surgery vol. 139,8 (2013):834-40. doi:10.1001 / jamaoto.2013.4001) was adopted, and oral administration was performed using a 30G, 25-mm needle.

[0055] (2)Anesthesia preparation The experimental rabbits were fasted for at least 8 hours before surgery and water was withheld for at least 4 hours before injection to reduce the risk of aspiration during the operation. Before injection, a 3% sodium pentobarbital solution (1 ml / kg) was used to induce sufficient anesthesia through the marginal ear vein. After the injection was completed, food and water intake were suspended for at least 4 hours.

[0056] (3)OSA rabbit models established by BTTA For the OSA rabbit model established by palatopharyngeal muscle injection: 100 μL (high dose), 80 μL (medium dose), and 60 μL (low dose) of the BTTA stock solution were taken respectively and injected bilaterally into the palatopharyngeal muscle, and the corresponding volume of isotonic saline was used as a control.

[0057] For the OSA rabbit model established by genioglossus muscle injection: 100 μL of the BTTA stock solution was taken and injected into the genioglossus muscle, and the corresponding volume of isotonic saline was used as a control.

[0058] (4)OSA rabbit models established by 50% glycerol For the OSA rabbit model established by palatopharyngeal muscle injection: 150 μL of 50% glycerol was injected into the palatopharyngeal muscle of the rabbit.

[0059] For the OSA rabbit model established by genioglossus muscle injection: 600 μL of 50% glycerol was injected into the genioglossus muscle of the rabbit.

[0060] An isotonic saline corresponding to each group of models was set as a control group.

[0061] (5)Osa rabbit model established by lipopolysaccharide (LPS) For the Osa rabbit model established by injecting into the palatopharyngeal muscle: Take the LPS solution and inject it bilaterally into the palatopharyngeal muscle of the rabbit at a dose of 150 μg / kg.

[0062] For the Osa rabbit model established by injecting into the genioglossus muscle: Take the LPS solution and inject it into the genioglossus muscle of the rabbit at a dose of 400 μg / kg.

[0063] An isotonic saline solution was set as the control group for each group of models.

[0064] II. Model evaluation 1 Evaluation method (1) Sleep induction Induce sleep by intramuscular injection of 0.3 mL / kg of Zoletil (flupenthixol hydrochloride and zolazepam; Virbac) and 0.2 mL / kg of Rompun (fluoxetine; Bayer) simultaneously.

[0065] (2) Polysomnography (PSG) Use a portable polysomnography device (Embletta MPR PG ST; Embla Systems) to record respiratory events. Monitoring mainly includes nasal airflow or pressure, ear oxygen saturation, respiratory movement, and pulse. Nasal airflow is monitored using an infant-sized catheter. To monitor ear oxygen saturation and pulse, use a mild depilatory cream to remove the ear hair, and fix a modified pulse oximeter clip on the ear blood vessel to monitor oxygen saturation and pulse. Respiratory movement is monitored by two chest belts and one abdominal belt. The chest belts are placed 1 cm below the armpits, and the abdominal belt is placed at the midpoint of the body. During the entire monitoring process, closely monitor the holding position of the sensors. The rabbits are placed in the supine position for sleep monitoring. Each rabbit is monitored for at least 30 minutes. Baseline PSG monitoring is performed after one week of adaptive feeding, and then sleep monitoring is performed every two weeks. Since the physiology of rabbits is different from that of humans, an AHI > 0 in the PSG results is regarded as an indication of OSA.

[0066] (3) Drug-induced sleep endoscopy (DISE) After waiting for 10 - 15 minutes for the rabbit to fall asleep and its breathing and heart rate to gradually stabilize, perform DISE using a laryngoscope with a diameter of 2.8 mm (Seesheen, BR-E11). During the examination, apply a 1% fluticasone hydrochloride solution (Yangtze River Pharmaceutical) to the rabbit's nasal vestibule and the first 10 cm part of the endoscope. The examination mainly focuses on the palatopharyngeal muscle, oropharynx, and epiglottis. The endoscope stays at each plane for 10 respiratory cycles. For the experimental group treated with BTTA for Pal, a low dose is used.

[0067] (4) Electromyogram (EMG) and data processing The EMG signals were recorded by a BL-420N biological signal acquisition and analysis system (Taimeng, BL-420N). The low-pass filter was set at 1 kHz, the sweep speed was 0.5 s, the range was 500 μV, the time constant was 200 ms, and the sampling frequency was 5 kHz.

[0068] At week 8, the rabbits were subjected to EMG evaluation. After waiting for the rabbits to fall asleep for 10 - 15 minutes, the respiration and heart rate of the rabbits gradually stabilized. Since the volume of the Pal muscle in rabbits is extremely small and it is difficult to perform EMG examination, the genioglossus muscle of the palatopharyngeal muscle injection group was used for the EMG experiment. For the experimental group treated with BTTA on Pal, a low dose was used.

[0069] The rabbits were placed in the supine position and their limbs were fixed. The device was connected to the system and the parameters were set as needed. The ground electrode was inserted subcutaneously. The rabbit's mouth was opened about 30 degrees using an oral speculum. The tongue tip was clamped with an oval clamp with a small gauze and slightly pulled out to expose the area under the root of the tongue, facilitating the insertion of two needle electrodes into the genioglossus muscle to start recording. The EMG data was statistically analyzed using MATLAB R2023a (Mathworks, Natick, MA, USA) software to analyze the raw data.

[0070] (5) Blood collection and analysis Blood samples were collected from the marginal ear vein of the rabbits. The rabbits were properly fixed, and blood was drawn using a sterile syringe and transferred to a tube coated with EDTA for complete blood count (CBC) analysis, and at the same time, it was transferred to a plain tube for lipid profile detection. For the experimental group treated with BTTA on Pal, a low dose was used.

[0071] For CBC, the blood samples were analyzed using an automated hematology analyzer (BC-2800Vet). The measured parameters included white blood cell count (WBC), neutrophils, neutrophil percentage, etc.

[0072] For lipid level detection, the serum was separated by centrifugation at 3000 rpm for 10 minutes and then analyzed using an automated biochemical analyzer (BS-240VET). The measured lipid level parameters included total cholesterol (TC), low-density lipoprotein (LDL), etc.

[0073] (6) Analysis of modeling success rate and mortality The modeling success rate was calculated by dividing the number of successfully established rabbits by the number of rabbits surviving every two weeks. Among them, an AHI > 0 in the PSG results was recorded as modeling success; the mortality rate was calculated by dividing the cumulative number of dead rabbits every two weeks by the total number of models. The modeling success rate and mortality rate were calculated every 2 weeks. The area between the success rate and mortality rate curves was analyzed using Origin (2019b) software.

[0074] 2 Evaluation Results (1)PSG Results The results of the BTTA model are as Figures 2 - 4 shown: For the OSA rabbit model established by BTTA, injection of BTTA into the Pal and GG both successfully caused muscle paralysis and OSA. For the model injected with Pal (Pal group) at week 6, and for the model injected with GG (GG group) at week 2, varying degrees of apnea or hypopnea events began to occur. The apnea hypopnea index (AHI) reached its peak at week 10 (Pal) and week 8 (GG) respectively. The AHI of the Pal group and GG group was significantly higher than that of 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 or hypopnea events on oxygenation was revealed by the oxygen desaturation index (ODI). The AHI and ODI of the Pal group continued to rise, and the ODI at week 10 was significantly higher than that of the control group (Student's t-test, p < 0.05). The trends of AHI and ODI in the GG group were not exactly the same as those in the Pal group. The AHI showed a downward trend from week 4 to week 6, while the ODI showed a downward trend from week 2 to week 4. The AHI of the GG group at week 4 was significantly higher than that of the control group, and the ODI at weeks 4, 6, and 10 was also significantly higher than that of the control group (Student's t-test, p < 0.05).

[0075] The results of the 50% glycerol model are as Figures 5 - 7 shown: For the OSA rabbit model established with 50% glycerol, apnea or hypopnea events were also observed in the Pal and GG muscles, while these events were not present 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 of the Pal group treated with 50% glycerol continued to increase before week 6 and then decreased, probably due to the clearance of fat infiltration in the muscle. The ODI at weeks 4 and 10 increased significantly (Student's t-test, p < 0.05). The AHI of the GG group at week 6 was significantly higher than that of the control group (Student's t-test, p < 0.01), and the ODI also increased significantly at weeks 6 and 8 (Student's t-test, p < 0.05).

[0076] The results of the LPS model are as Figures 8 - 10 shown: For the OSA rabbit model established by LPS, apnea and hypopnea events were also observed in the Pal group, and the change trends and peak times were slightly different from those of the BTTA model (Pal control group: p = 0.020, Student's t-test; GG control group: p = 0.081, Student's t-test). There was a significant difference in the AHI of the Pal group at week 8 (Student's t-test, p < 0.05). The ODI trend in the LPS treatment group was similar to that of the AHI, and there was a strong correlation between the two, but the non-linear relationship resulted in inconsistent changes between them. Significant differences were observed at weeks 6 and 8 (Pal), weeks 4 and 8 (GG) (Student's t-test, p < 0.05).

[0077] The above results indicate that for the rabbit models constructed by treating the palatopharyngeal and genioglossus muscles of rabbits with BTTA, 50% glycerol, and LPS, apnea and hypopnea exist, and the apnea and hypopnea can be maintained for a long time.

[0078] (2) DISE results The DISE results are as Figure 11 shown. In the rabbit models constructed by BTTA, 50% glycerol, or LPS, partial or complete collapse of the airway occurred during sleep.

[0079] (3) EMG experiment results The EMG experiment results are as Figures 12 - 14 shown in Tables 1 - 3: In the Pal groups treated with BTTA, 50% glycerol, and LPS, the root mean square (RMS) level of EMG was higher than that of the control group; the original EMG signals of the rabbits demonstrated the muscle activities of each group. These results indicate that the rabbit models constructed by BTTA, 50% glycerol, or LPS can cause overall fatigue of the upper airway muscles and induce OSA.

[0080] Table 1 Results of the root mean square value of electromyogram of the BTTA model Table 2 Results of the root mean square value of electromyogram of the 50% glycerol model Table 3 Results of the root mean square value of electromyogram of the LPS model (4) Peripheral blood test results The peripheral blood test is as Figures 15 - 17As shown in Table 4-6: For the model constructed with BTTA, compared with the control group, there were no significant differences in the blood routine and blood lipid levels of the model constructed after treating Pal with BTTA. Among the blood routine and blood lipid levels of the model constructed after treating Pal with BTTA, only the index of low-density lipoprotein (LDL) increased significantly; for the model constructed with 50% glycerol, compared with the control group, among the blood routine and blood lipid levels of the model constructed after treating Pal with 50% glycerol, the levels of neutrophils, total cholesterol, and LDL all increased significantly. Among the blood routine and blood lipid levels of the model constructed after treating GG with 50% glycerol, the levels of total cholesterol and LDL increased significantly; for the model constructed with LPS, compared with the control group, among the blood routine and blood lipid levels of the model constructed after treating Pal with LPS, the level of neutrophils increased significantly. Among the blood routine and blood lipid levels of the model constructed after treating GG with LPS, the levels of total cholesterol and LDL increased significantly.

[0081] Among them, particularly, in the model constructed after treating Pal with 50% glycerol, the levels of neutrophils, total cholesterol content, and LDL content in the peripheral blood all increased significantly, indicating that the inflammation was aggravated and lipid metabolism was significantly affected. This aggravation of inflammation and disorder of lipid metabolism are closely related to OSA.

[0082] Table 4 Results of Peripheral Blood Routine and Blood Lipid Levels in BTTA Model Table 5 Results of Peripheral Blood Routine and Blood Lipid Levels in 50% Glycerol Model Table 6 Results of Peripheral Blood Routine and Blood Lipid Levels in LPS Model (5)Results of Modeling Success Rate and Mortality The success rate results are as Figure 18 and shown in Table 7: For the model animals constructed by treating Pal with BTTA, the success rates of the medium-dose and low-dose groups were relatively high.

[0083] For the Pal group, the success rates of BTTA treatment and LPS treatment reached their peaks at the 8th week (BTTA: 80%, LPS: 100%), while the peak of 50% glycerol treatment appeared at the 6th week (50% glycerol: 100%). For the GG group, the success rate of BTTA treatment reached its peak at the 4th week (BTTA: 100%), while the peaks of 50% glycerol and LPS treatments appeared at the 8th week (50% glycerol: 100%, LPS: 66.7%).

[0084] Table 7 Results of Success Rate and Mortality in BTTA Treatment Group The power results showed that for the Pal group, the animal model established with 50% glycerol took less time, and the success rate of the animal model established with 50% glycerol and LPS was high, reaching 100%; for the GG group, the BTTA group took less time, and the success rate of the animal model established with BTTA and 50% glycerol was high, reaching 100%.

[0085] The mortality results were as Figure 18 shown in Table 6 and Table 7: For the model animals constructed by treating Pal with BTTA, the mortality rate of the low-dose group was the lowest.

[0086] For the Pal group, the mortality rate of the BTTA treatment was 25%, and that of the glycerol and LPS treatments was 0%; for the GG group, the mortality rate of the BTTA treatment was 25%, that of the glycerol treatment was 20%, and that of the LPS treatment was 0%.

[0087] The mortality results indicated that the animal models established by treating Pal with 50% glycerol, treating Pal with LPS, or treating GG had good safety.

[0088] By calculating the area between the success rate and mortality curves, the results were as Figure 19 shown in Table 6, Table 7 and Table 8: For the model animals constructed by treating Pal with BTTA, the area between the success rate and mortality curves of the low-dose group was the highest, but its value was significantly lower than that of the model animals constructed by treating Pal with 50% glycerol or treating Pal with LPS. Therefore, treating Pal with 50% glycerol or LPS and treating GG with BTTA are effective strategies for constructing an OSA model with a high success rate.

[0089] Table 8 Success rate and mortality results of the LPS treatment group The above results are summarized in Tables 9 and 10 as follows. The three OSA models were comprehensively evaluated by polysomnography (PSG), drug-induced sleep endoscopy (DISE), electromyogram (EMG), and peripheral blood tests. Combining the success rate and mortality rate, the results showed that the OSA models established by treating Pal with 50% glycerol or LPS could simulate OSA in terms of structure, function, and phenotypic characteristics in all aspects, and had the advantages of high safety, high success rate, no need for high-end equipment, and special operations. Among them, the OSA model established by treating Pal with 50% glycerol took less time, had more significant pathophysiological characteristics, and was more relevant to the specific characteristics of OSA patients.

[0090] Table 9 Phenotypic characteristics of the three OSA models treating Pal Note: * p<0.05; ** p<0.01; *** p<0.001, “ns” indicates no statistical difference.

[0091] Table 10 Phenotypic characteristics of three OSA models in dealing with GG Note: * p<0.05; ** p<0.01; *** p<0.001; "ns" indicates no statistical difference. As can be seen from the above-mentioned implementation cases and experimental examples, the present invention provides a method for constructing an OSA animal model by injecting 50% glycerol or LPS bilaterally into the palatopharyngeal muscles of rabbits to construct an OSA animal model. This method can not only simulate OSA in terms of structure, function, and phenotypic characteristics in an all-round way, but also has the advantages of high safety, high success rate, and no need for high-end equipment and special operations. This method conforms to the OSA animal model in clinical practice and has a great promoting effect on revealing the pathogenesis of OSA and developing new drugs, etc., and has a good application prospect.

Claims

1. A method for constructing an animal model of obstructive sleep apnea, characterized in that: It includes the following steps: Take an animal, inject 30 - 50% glycerol or lipopolysaccharide into both sides of the palatopharyngeal muscle respectively, and continue to raise it to obtain the desired result.

2. The construction method according to claim 1, wherein: It includes the following steps: Take an animal, inject 30 - 50% glycerol into both sides of the palatopharyngeal muscle respectively, and continue to raise it to obtain the desired result.

3. The construction method according to claim 2, characterized in that: The dosage of the injection is 50 - 150 μL per animal.

4. The construction method according to claim 2, wherein: The time for continued raising is 4 - 12 weeks.

5. The construction method according to claim 1, wherein: It includes the following steps: Take an animal, inject lipopolysaccharide into both sides of the palatopharyngeal muscle respectively, and continue to raise it to obtain the desired result.

6. The construction method according to claim 5, characterized in that: The dosage of the injection is 50 - 150 μg / kg of the animal, and the concentration of the lipopolysaccharide is 1.8 - 2.2 mg / mL.

7. The construction method according to claim 5, characterized in that: The time for continued raising is 5 - 10 weeks.

8. The construction method according to claim 1, wherein: The animal is selected from monkeys, pigs, dogs, and rabbits.

9. The construction method according to claim 8, wherein: The animal is a New Zealand rabbit.

10. The construction method according to claim 9, characterized in that, The specific method of the injection is as follows: Under the use of an oral speculum, use a 30G, 25 - mm needle. Under the guidance of a videolaryngoscope, the needle reaches directly to the root of the tonsil. The depth of the needle is 2 - 3 mm, and the angle between the needle and the rabbit's front teeth is 50 - 70 degrees.

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