A method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation and a sleep box

Abstract

The application discloses a kind of based on intestinal flora transplantation intestinal sleep disorder mouse model construction method and sleep box, based on flora separation technology, preparation insomnia patient intestinal flora;Using sterile animal flora transplantation technology, insomnia patient intestinal flora is implanted in sterile mouse in vivo;The sterile mouse of implantation insomnia patient intestinal flora is fed in sterile isolation bag and carries out flora colonization;The behavior, physiology and microbiome of mouse after flora colonization are evaluated and analyzed, and the construction of intestinal sleep disorder mouse phenotype is verified.By flora transplantation technology, insomnia patient flora is colonized in sterile animal intestine, and the behavior, physiology and microbiome are evaluated and analyzed, and intestinal sleep disorder mouse model is verified and constructed, which provides model support for sleep pathogenic bacteria exploration and sleep probiotics research and development.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation and a sleep chamber. Background Technology

[0002] Insomnia is a common sleep disorder characterized by dissatisfaction with sleep duration, quality, or overall well-being, affecting the health and quality of life of a large population. Insomnia is not limited to difficulty falling asleep or interrupted sleep; it also includes waking up early and having difficulty falling back asleep. Chronic insomnia is closely linked to health risks such as cardiovascular disease and metabolic syndrome. Therefore, researchers are exploring the relationship between insomnia and other chronic diseases and attempting to find prevention and intervention strategies.

[0003] Currently, treatment options for sleep-related disorders are limited, and common medications often have significant side effects and only treat the symptoms, not the root cause. Basic research using animal models of sleep disorders can provide more comprehensive tools for diagnosis and treatment. Establishing stable and reliable animal models of insomnia can provide crucial scientific evidence for the clinical translation of insomnia. A deeper understanding of the physiological basis of insomnia will enable the development of more personalized treatment methods and interventions to improve patient outcomes and quality of life.

[0004] Currently, researchers typically use one or more of the following animal models:

[0005] Sleep deprivation: By restricting an animal's sleep time or altering its sleep cycle, animals may exhibit symptoms similar to insomnia, such as difficulty falling asleep or shallow sleep.

[0006] Drug-induced insomnia: Administering specific drugs, such as caffeine or sleeping pills, to animals to mimic the symptoms of insomnia in humans. Genetic alteration: Using gene editing technology or selective breeding, altering the genes or genetic background of animals to make them more susceptible to exhibiting insomnia-related traits. Environmental stimulation: Exposing animals to different environmental stimuli, such as light, noise, or social isolation, to induce insomnia-like responses.

[0007] However, these models cannot fully reflect the complexity and multifactorial influence of human insomnia. For example, drug-induced insomnia involves the use of specific drugs or chemical substances, such as hypnotics, anti-anxiety drugs, or steroids. It is difficult to remove the effects of the drugs themselves on mice from the models.

[0008] Environmental changes involve altering the mice's environmental conditions, such as irregular light cycles or noise exposure. However, environmental changes can easily cause stress in mice, leading to emotional problems.

[0009] It can be seen that these models are all passively induced by external stimuli and cannot effectively simulate the behavior and pathological changes of insomnia. In the past, gut-derived disease models mostly used antibiotics to clear animal flora for modeling. Since the current antibiotic modeling method cannot completely clear all the gut flora of the recipient mice, it will affect the colonization efficiency after the flora is transplanted, and thus affect the success rate of modeling.

[0010] This shows that there is currently a lack of animal models of sleep disorders in basic scientific research, and the existing models are insufficient to meet the needs of studying the pathogenesis of insomnia and developing drugs. Summary of the Invention

[0011] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0012] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0013] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for constructing a mouse model of gut-derived sleep disorder based on gut microbiota transplantation.

[0014] To solve the above-mentioned technical problems, the present invention provides the following technical solution: including,

[0015] Based on microbial isolation technology, the gut microbiota of insomnia patients was prepared.

[0016] Using sterile animal microbiota transplantation technology, the gut microbiota of insomnia patients was transplanted into sterile mice;

[0017] Germ-free mice implanted with gut microbiota from insomnia patients were housed in sterile isolation packs to allow for microbiota colonization.

[0018] The construction of the mouse phenotype of gut-derived sleep disorder was verified by evaluating and analyzing the behavior, physiology, and microbiome of mice after colonization of the gut microbiota.

[0019] As a preferred embodiment of the method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation according to the present invention, the method for preparing the intestinal flora of the insomnia patient includes: collecting fresh fecal samples from the insomnia patient under sterile conditions, diluting and uniformly mixing them with sterile physiological saline to form a coarse fecal suspension, filtering to remove large particles, ensuring the suspension is uniform, and obtaining the fecal suspension of the insomnia patient, that is, the fecal suspension containing the intestinal flora of the insomnia patient.

[0020] As a preferred embodiment of the method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation according to the present invention, the coarse fecal suspension is subjected to centrifugation, filtration, and density gradient centrifugation.

[0021] As a preferred embodiment of the method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation according to the present invention, the germ-free mouse breeding process is not subject to antibiotic treatment.

[0022] As a preferred embodiment of the method for constructing a mouse model of gut-derived sleep disorder based on gut microbiota transplantation according to the present invention, the behavioral assessment includes the assessment of activity level and sleep pattern.

[0023] As a preferred embodiment of the method for constructing a mouse model of enterogenic sleep disorder based on gut microbiota transplantation according to the present invention, the sleep pattern includes sleep / wake cycles, rapid eye movement (REM) sleep, and non-rapid eye movement (NREM) sleep.

[0024] As a preferred embodiment of the method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation according to the present invention, the physiological assessment includes brain electrical activity, muscle activity, and stress hormone levels.

[0025] The beneficial effects of this construction method are:

[0026] Currently, there is a lack of animal models for sleep disorders in basic scientific research. To address this, this invention discloses a modeling method for a mouse model of gut-derived sleep disorders based on gut microbiota transplantation. This method utilizes the gut microbiota of insomnia patients and germ-free mice raised in a completely sterile environment. Through microbiota transplantation technology, the gut microbiota of insomnia patients is colonized into the gut of germ-free animals. Through behavioral, physiological, and microbiome evaluation and analysis, a mouse model of gut-derived sleep disorders is verified and constructed, providing model support for the exploration of sleep-causing pathogens and the development of sleep-promoting bacteria.

[0027] This invention also proposes a sleep chamber for use in the above-mentioned method for constructing a mouse model of intestinal sleep disorder based on gut microbiota transplantation. The chamber includes: a body for providing a feeding space for the mice; a water dispenser mounted on the body, comprising a device mounted on the body, a feeding tube communicating with the device and extending into the inner side of the body, and a medicine compartment mounted on the device; a floating mechanism movably disposed inside the water dispenser; and a driving mechanism capable of moving the floating mechanism relative to the inner side of the water dispenser. The device is used to hold drinking water for feeding the mice, the medicine compartment is used to hold medicine, and the floating mechanism, when submerged in the drinking water, can raise the level of the drinking water inside the device.

[0028] In a preferred embodiment of the sleep chamber of the present invention, the water dispenser has an inner shaft; the floating mechanism includes a sliding ring located outside the inner shaft and an expansion body mounted on the sliding ring; the driving mechanism includes a power component mounted on the chamber and a magnetic attractor connected to the power component; the sliding ring can be attracted by the magnetic attractor.

[0029] As a preferred embodiment of the sleep box of the present invention, it further includes a switching mechanism; which includes a lifting core installed on the inner side of the inner shaft; a plug extending to the outer side of the inner shaft and capable of sealing the connection between the device body and the feeding tube; and an extension body disposed at the end of the lifting core and extending to the outer side of the inner shaft.

[0030] When the floating mechanism moves upward, it can push the extension body, and the lifting core can cause the plug to disengage from the connection between the device body and the feeding tube.

[0031] The beneficial effects of this sleep chamber are: by setting up a floating mechanism and a drive mechanism, it can mix the water and medicine stored inside the water dispenser, thereby ensuring that the medicine in the drinking water of mice meets the experimental requirements. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0033] Figure 1 This is a schematic diagram showing the colonization and distribution of the intestinal flora in recipient mice after microbiota transplantation according to the present invention.

[0034] Figure 2 This diagram illustrates the sleep monitoring results of recipient mice after microbial transplantation, as well as the activity of Orexin neurons in the brain region and the serum short-chain fatty acid content.

[0035] Figure 3 This is a schematic diagram of the overall structure of the sleep chamber in this invention.

[0036] Figure 4 This is a schematic diagram of the internal structure of the sleep chamber in this invention.

[0037] Figure 5 This is a schematic diagram of the water dispenser and drive mechanism in this invention.

[0038] Figure 6 This is a schematic diagram of the internal structure of the water dispenser in this invention.

[0039] Figure 7 This is a schematic diagram of the floating mechanism structure in this invention.

[0040] Figure 8 For the present invention Figure 7 Enlarged view of point A in the middle.

[0041] Figure 9 This is a schematic diagram of the switching mechanism in this invention.

[0042] Figure 10 For the present invention Figure 9 Enlarged view of section B in the middle.

[0043] Figure 11 This is a schematic diagram of the slip ring structure in this invention. Detailed Implementation

[0044] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0045] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0046] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single embodiment or an embodiment selectively excluded from other embodiments.

[0047] Materials and equipment:

[0048] Laboratory mice: germ-free mice

[0049] Stool samples: Stool samples from insomnia patients and healthy donors.

[0050] Aseptic processing equipment: such as laminar flow hoods.

[0051] Fecal transplantation equipment: gavage syringe, syringe.

[0052] Example 1: This example provides a method for constructing a mouse model of gut-derived sleep disorder based on gut microbiota transplantation. Specifically:

[0053] S1: Based on microbial isolation technology, prepare gut microbiota from insomnia patients and healthy donors;

[0054] Patients diagnosed with insomnia are included in the clinical category according to the diagnostic criteria for insomnia in the Diagnostic and Statistical Manual of Mental Disorders or the International Classification of Diseases.

[0055] Fresh fecal samples were collected from insomnia patients and healthy donors, diluted with sterile saline and mixed evenly to form a coarse fecal suspension. The suspension was then centrifuged, filtered, and subjected to density gradient centrifugation to remove large particles and ensure uniformity. Fecal suspensions from insomnia patients and healthy donors were obtained, representing the intestinal flora of insomnia patients and healthy donors, respectively. These suspensions were then stored at low temperatures for later use.

[0056] S2: Using sterile animal microbiota transplantation technology, gut microbiota are implanted into sterile mice;

[0057] The fecal suspension of insomnia patients obtained in step S1 was transplanted into sterile recipient SPF mice housed in sterile isolation packs via oral or rectal route using a gavage syringe or syringe to obtain the experimental group. The control group was transplanted with fecal suspension from healthy donors.

[0058] S3: Mice in the experimental and control groups were raised under sterile conditions for bacterial colonization;

[0059] After transplantation, the mice were housed in sterile isolation packs for two weeks to allow for bacterial colonization.

[0060] S4: Validate the construction of the mouse phenotype of gut-derived sleep disorder;

[0061] The study validated the findings by evaluating the behavior, physiology, and microbiome of mice after colonization with the microbial community.

[0062] A1: Behavioral and physiological monitoring;

[0063] Three weeks after transplantation, the mice underwent behavioral tests (such as the elevated cross maze experiment) and were assessed for sleep-wake cycles in recipient mice colonized with gut microbiota from insomnia patients using techniques such as electroencephalography (EEG), electromyography (EMG), and polysomnography (PSG).

[0064] Specifically, brain sleep monitoring and muscle testing require electrode implantation surgery. The surgical procedure is as follows:

[0065] Mice need to acclimatize to the experimental environment and treatment process beforehand.

[0066] Mice were anesthetized before surgery, and their body temperature and anesthesia were kept stable during the operation.

[0067] EEG electrode implantation:

[0068] EEG electrodes are precisely implanted into specific brain regions, such as the frontal, parietal, or temporal lobes, using micromanipulation devices to record brain electrical activity. The position and depth of the EEG electrodes need to be precisely controlled to ensure clear signal recording and avoid damage to brain tissue.

[0069] EMG electrode implantation:

[0070] EMG electrodes are implanted into the neck or other muscle areas of mice to record muscle electrical activity. These electrodes can be used to monitor muscle tone in mice at different sleep stages, especially muscle relaxation during rapid eye movement (REM) sleep.

[0071] Electrode fixation: After implantation, the electrodes are fixed to the skull or muscles of the mouse using specific fixation materials (such as clinical-grade medical adhesive or bone cement) to prevent electrode displacement or detachment.

[0072] Postoperative care: After surgery, it is necessary to monitor the recovery of mice and provide appropriate care and pain relief medication. There is usually an adaptation period during which the mice can return to normal behavior and sleep patterns, so that subsequent experimental records and analysis can be carried out.

[0073] A2: Collect mouse fecal samples and perform 16S sequencing on human donor samples and mouse fecal samples;

[0074] The gut microbiota of human donors and recipient mice were sequenced using 16S rRNA sequencing to verify the effectiveness of gut microbiota colonization after transplantation. Specific steps are as follows:

[0075] Sample collection: Microbial DNA was collected from mouse fecal samples. Before collection, the samples were kept at low temperature and dry conditions to prevent changes in the microbial community.

[0076] DNA extraction: Total microbial DNA is extracted from fecal samples using appropriate DNA extraction methods. This step requires special attention to ensure that the extracted DNA has sufficient purity and quality for subsequent sequencing analysis.

[0077] PCR amplification: Amplifying specific regions of the 16S rRNA gene using specific primers. These primers are usually designed to cover variable regions (such as V1-V9) in the 16S rRNA gene in order to obtain sufficient sequence information during sequencing.

[0078] Sequencing: High-throughput sequencing technology (such as the Illumina sequencing platform) is used to sequence the amplified 16S rRNA gene fragment. This method can sequence a large number of samples at the same time, providing high-resolution information on the microbial community structure.

[0079] Data analysis: Bioinformatics analysis of the sequencing data, including sequence processing, OTU (operational taxonomic unit) clustering, species diversity assessment, comparison of microbial community structure, and functional prediction.

[0080] This step can reveal the composition, diversity, and dynamic changes of the mouse gut microbiota, study the effects of different factors (such as diet, drugs, disease models, etc.) on the gut microbiota, and explore the relationship between gut microbiota and host health status (such as immune regulation, intestinal diseases, etc.).

[0081] A3: Data analysis and validation;

[0082] The data from sleep EEG monitoring were analyzed and presented to compare the sleep characteristics of the gut microbiota of insomnia patients and healthy mice, thus verifying the construction of the sleep disorder phenotype.

[0083] Example 2: Referring to Example 1, this example constructs a mouse model of gut-derived sleep disorder based on gut microbiota transplantation using scientific experimental methods. Specifically:

[0084] S1: Based on microbial isolation technology, prepare gut microbiota from insomnia patients and healthy donors;

[0085] Patients diagnosed with insomnia are included in the clinical category according to the diagnostic criteria for insomnia in the Diagnostic and Statistical Manual of Mental Disorders or the International Classification of Diseases.

[0086] Fresh fecal samples were collected from insomnia patients and healthy donors, diluted with sterile saline and mixed evenly to form a coarse fecal suspension. Large particles were removed by filtration to ensure the suspension was homogeneous, and fecal suspensions of insomnia patients and healthy donors were obtained, namely the intestinal flora of insomnia patients and healthy donors, which were stored at low temperature for later use.

[0087] S2: Using sterile animal microbiota transplantation technology, gut microbiota are implanted into sterile mice;

[0088] The fecal suspension of insomnia patients obtained in step S1 was transplanted into sterile recipient SPF mice housed in sterile isolation packs via oral or rectal route using a gavage syringe or syringe to obtain the experimental group. The control group was transplanted with fecal suspension from healthy donors.

[0089] S3: Mice in the experimental and control groups were raised under sterile conditions for bacterial colonization;

[0090] The transplanted mice were housed in sterile isolation packs for two weeks to prevent interference from external microorganisms.

[0091] S4: Validate the construction of the mouse phenotype of gut-derived sleep disorder;

[0092] The study validated the findings by evaluating the behavior, physiology, and microbiome of mice after colonization with the microbial community.

[0093] A1: Behavioral and physiological monitoring;

[0094] Three weeks after transplantation, the mice underwent behavioral tests (such as the elevated cross maze experiment) and were assessed for sleep-wake cycles in recipient mice colonized with gut microbiota from insomnia patients using techniques such as electroencephalography (EEG) and electromyography (EMG).

[0095] Specifically, brain sleep monitoring and muscle testing require electrode implantation surgery. The surgical procedure is as follows:

[0096] Mice need to acclimatize to the experimental environment and treatment process beforehand.

[0097] Mice were anesthetized before surgery, and their body temperature and anesthesia were kept stable during the operation.

[0098] EEG electrode implantation:

[0099] EEG electrodes are precisely implanted into specific brain regions, such as the frontal, parietal, or temporal lobes, using micromanipulation devices to record brain electrical activity. The position and depth of the EEG electrodes need to be precisely controlled to ensure clear signal recording and avoid damage to brain tissue.

[0100] EMG electrode implantation:

[0101] EMG electrodes are implanted into the neck or other muscle areas of mice to record muscle electrical activity. These electrodes can be used to monitor muscle tone in mice at different sleep stages, especially muscle relaxation during rapid eye movement (REM) sleep.

[0102] Electrode fixation: After implantation, the electrodes are fixed to the skull or muscles of the mouse using specific fixation materials (such as clinical-grade medical adhesive or bone cement) to prevent electrode displacement or detachment.

[0103] Postoperative care: After surgery, it is necessary to monitor the recovery of mice and provide appropriate care and pain relief medication. There is usually an adaptation period during which the mice can return to normal behavior and sleep patterns, so that subsequent experimental records and analysis can be carried out.

[0104] A2: Collect mouse fecal samples and perform 16S sequencing on human donor samples and mouse fecal samples;

[0105] The gut microbiota of human donors and recipient mice were sequenced using 16S rRNA sequencing to verify the effectiveness of gut microbiota colonization after transplantation. Specific steps are as follows:

[0106] Sample collection: Microbial DNA was collected from mouse fecal samples. Before collection, the samples were kept at low temperature and dry conditions to prevent changes in the microbial community.

[0107] DNA extraction: Total microbial DNA is extracted from fecal samples using appropriate DNA extraction methods. This step requires special attention to ensure that the extracted DNA has sufficient purity and quality for subsequent sequencing analysis.

[0108] PCR amplification: Amplifying specific regions of the 16S rRNA gene using specific primers. These primers are usually designed to cover variable regions (such as V1-V9) in the 16S rRNA gene in order to obtain sufficient sequence information during sequencing.

[0109] Sequencing: High-throughput sequencing technology (such as the Illumina sequencing platform) is used to sequence the amplified 16S rRNA gene fragment. This method can sequence a large number of samples at the same time, providing high-resolution information on the microbial community structure.

[0110] Data analysis: Bioinformatics analysis of the sequencing data, including sequence processing, OTU (operational taxonomic unit) clustering, species diversity assessment, comparison of microbial community structure, and functional prediction.

[0111] This step can reveal the composition, diversity, and dynamic changes of the mouse gut microbiota, study the effects of different factors (such as diet, drugs, disease models, etc.) on the gut microbiota, and explore the relationship between gut microbiota and host health status (such as immune regulation, intestinal diseases, etc.).

[0112] A3: Data analysis and validation;

[0113] The data from sleep EEG monitoring were analyzed and presented to compare whether there were differences in the sleep characteristics of the gut microbiota of mice with insomnia and healthy human mice, thus verifying the construction of the sleep disorder phenotype in germ-free animals (SPF).

[0114] As attached Figure 1 Appendix Figure 2 The image shows the verification of microbial colonization after microbial transplantation in this embodiment. Figure 1 The contribution of PCoA1 on the x-axis was 26.46%, and the contribution of PCoA2 on the y-axis was 10.62%, indicating a significant change in β-diversity after fecal microbiota transplantation. FMT_HE received fecal microbiota from healthy individuals; FMT_IN received fecal microbiota from insomnia patients (n=7-8).

[0115] The butyrate content in the blood of FMT_IN mice was significantly reduced after fecal microbiota transplantation. Figure 1(a) shows the co-labeling of c-Fos and orexin neurons in recipient mice after fecal microbiota transplantation (scale bar: 500µm, 50µm; green: c-Fos protein; red: orexinA neurons); (b) shows a decrease in the number of co-labeled c-Fos and orexin neurons in FMT_IN mice after fecal microbiota transplantation. FMT_HE received fecal microbiota from healthy individuals; FMT_IN received fecal microbiota from patients with insomnia. Data are expressed as mean ± standard error (n=5), ** indicates p<0.01.

[0116] Appendix Figure 2 (A) Schematic diagram of fecal microbiota transplantation (FMT); (B) Temporal distribution of recipient mice during Wake, NREM, and REM sleep over 24 hours after FMT; (C) Temporal distribution of recipient mice during Wake, NREM, and REM sleep during the 12-hour light period after FMT; (D) Temporal distribution of recipient mice during Wake, NREM, and REM sleep during the 12-hour dark period after FMT. FMT_HE received fecal microbiota from healthy individuals; FMT_IN received fecal microbiota from patients with insomnia. Data are expressed as mean ± standard error (n=12), ** represents p<0.01, *** represents p<0.001.

[0117] Comparative Example 1 differs from Example 2 in that the feeding conditions of the mice in step S3 are adjusted, specifically:

[0118] S3: Mice in the experimental and control groups were raised under sterile conditions for bacterial colonization;

[0119] After transplantation, the mice were housed in sterile isolation packs to prevent interference from external microorganisms, and were given a mixed antibiotic solution instead of drinking water for two consecutive weeks.

[0120] The remaining steps are the same as in Example 2, and the comparative example of an antibiotic-based (AIMD) sleep disorder model is constructed.

[0121] Example 3 differs from the above examples in that: this example also discloses a sleep box applied to the above construction method, including a box body 100 for providing a feeding space for mice.

[0122] The sleep chamber includes a water dispenser 200 mounted on a chamber body 100, a body 201 mounted on the body 100, a feeding tube 202 communicating with the body 201 and extending into the inside of the body 100, and a medicine compartment 203 located on the body 201. The water dispenser 200 can be mounted on the side wall of the body 100. One end of the feeding tube 202 communicates with the internal space of the body 201, allowing liquid inside the body 201 to enter the inside of the feeding tube 202. The feeding tube 202 is located within the chamber. A ball bearing is installed at the inner end of the body 100. The ball bearing blocks the feeding tube 202. The feeding tube 202 is located at the inner end of the body 100 and is a constricted opening, that is, the inner diameter gradually decreases. The ball bearing is located inside the constricted opening. Under normal conditions, the ball bearing can block the constricted opening under the action of gravity. When the mouse licks the ball bearing, the ball bearing moves upward inside the constricted opening, so that a gap is created between the ball bearing and the inner wall of the constricted opening. The liquid inside the body 201 can then flow out from the feeding tube 202 for the mouse to drink.

[0123] The medicine compartment 203 is located on the side wall of the body 201. One end of it is connected to the internal space of the body 201, and the other end has a cover. After removing the cover, medicines can be placed inside the medicine compartment 203. In order to improve the sealing effect, the cover is preferably made of rubber or silicone material. Multiple medicine compartments 203 are provided on the side wall of the same body 201 for loading different medicines, which can be loaded inside different medicine compartments 203.

[0124] The sleeping box includes a floating mechanism 300, which is movably located inside the water drinker 200; the sleeping box includes a drive mechanism 400, which can drive the floating mechanism 300 to move inside the water drinker 200; the body 201 is used to hold drinking water for feeding mice, the medicine compartment 203 is used to hold medicine, and when the floating mechanism 300 sinks into the drinking water, it can raise the liquid level of the drinking water inside the body 201.

[0125] Furthermore, the floating mechanism 300 can move up and down inside the container 201. Therefore, the floating mechanism 300 has two states: one is a high position that does not contact the water inside the container 201, and the other is a low position that can sink into the water inside the container 201. The driving mechanism 400 can adopt various powered structures, preferably an electric telescopic rod, which can drive the floating mechanism 300 to move up and down and adjust the position of the floating mechanism 300 inside the container 201.

[0126] In this embodiment, three water dispensers 200 are installed on the entire housing 100. During use, the medicine and water in one of the water dispensers 200 are mixed, while the water and medicine in the other two water dispensers 200 are separated. Over time, some of the medicine will become completely ineffective in the water. At this time, the drive mechanism 400 corresponding to the two spare water dispensers 200 drives the floating mechanism 300 to move downward and sink into the water. The water level rises, overflows the medicine compartment 203, and mixes with the medicine inside the medicine compartment 203.

[0127] Therefore, when using the above construction method, the experimenter can prepare the medicine and water in one go, and when the medicine inside the water dispenser 200 in use fails, the backup water dispenser 200 can quickly replace it.

[0128] Example 4 differs from Example 3 in that: the water dispenser 200 has an inner shaft 204 inside; the floating mechanism 300 includes a slip ring 301 that can slide on the outside of the inner shaft 204, and an expansion body 302 installed on the slip ring 301; the driving mechanism 400 includes a power component 401 installed on the housing 100, and a magnetic attractor 402 connected to the power component 401; the slip ring 301 can be attracted by the magnetic attractor 402.

[0129] The power component 401 is preferably an electric push rod, which can drive the magnetic body 402 to move up and down. Through the attraction force of the magnetic body 402 on the slip ring 301, the slip ring 301 is driven to move up and down on the outside of the inner shaft 204. Therefore, the slip ring 301 can drive the expansion body 302 to move up and down, thereby completing the drive of the entire floating mechanism 300. In this way, since the drive mechanism 400 and the floating mechanism 300 are not directly connected, the same drive mechanism 400 can be applied to different water dispensers 200 to drive the floating mechanism 300 inside different water dispensers 200.

[0130] For example, a slide rail and an electric slider structure are provided on the housing 100. The power component 401 is installed on the electric slider and can drive the power component 401 to move. The power component 401 can move to the corresponding position of any one of the three water dispensers 200 and drive the floating mechanism 300 inside the housing 201.

[0131] To improve the mixing effect of medicine and water, the floating mechanism 300 also includes a bottom tank 303 installed below the slip ring 301, and a buoyancy body 304 located inside the bottom tank 303. The buoyancy body 304 has a certain deformation capacity. The top of the bottom tank 303 is connected to the interior of the expansion body 302 through the slip ring 301. The top of the expansion body 302 is also provided with a water inlet groove 305. A water outlet groove 306 is provided on the radial outer wall of the bottom tank 303. The connection position between the top of the bottom tank 303 and the slip ring 301 has a flared opening 30. 7. Under normal conditions, the buoyancy body 304 is located inside the bottom tank 303 and can rise and fall. When the drive mechanism 400 drives the slip ring 301 to move downward, the bottom tank 303 will first come into contact with the liquid inside the vessel body 201. The buoyancy causes the buoyancy body 304 to move upward, blocking the water outlet 306 and the flared mouth 307, so that the liquid cannot directly enter the inside of the expansion body 302 from the bottom tank 303. Therefore, the ability of the expansion body 302 to raise the liquid level when it sinks into the water inside the vessel body 201 is maximized.

[0132] As the expansion body 302 descends, the water level inside the container 201 overflows the medicine tank 203, flushing the medicine into the water. The medicine then enters the water, and the water level reaches the inlet tank 305, entering the interior of the expansion body 302 through the inlet tank 305. Then, the drive mechanism 400 drives the slip ring 301, thereby causing the expansion body 302 to move upward. When it leaves the water surface, the gravity of the water presses down on the buoyancy body 304, causing the buoyancy body 304 to move downward inside the bottom tank 303. The mixture of water and medicine can flow out from the outlet tank 306, allowing the water and medicine to collide and mix, improving the mixing effect of water and medicine.

[0133] When the magnetic 402 drives the slip ring 301, in order to improve the adsorption force, the outer diameter of the slip ring 301 can be enlarged, so that the radial outer wall of the slip ring 301 is close to the inner wall of the device body 201, thereby improving the adsorption capacity of the magnetic 402 on the slip ring 301.

[0134] When three water dispensers 200 use the same drive mechanism 400, when the magnetic attractor 402 in the drive mechanism 400 attracts the slip ring 301 inside one of the water dispensers 200, it is also necessary to consider the position of the slip rings 301 inside the other two water dispensers 200, so as to avoid the floating mechanism 300 from falling into the water under the action of gravity, causing the medicine and water to come into contact too early.

[0135] Therefore, the inner radial wall of the slip ring 301 has an arc-shaped spring piece 308 and a positioning protrusion 309. The two ends of the arc-shaped spring piece 308 are connected to the inner radial wall of the slip ring 301, and the outer arc-shaped wall abuts against the inner shaft 204. A positioning groove 204a is provided on the outer radial wall of the inner shaft 204. When the slip ring 301 is not attracted by the magnetic body 402, the elastic force of the arc-shaped spring piece 308 will abut against the slip ring 301, so that the positioning protrusion 309 is located inside the positioning groove 204a. When there is no magnetic body 402 adsorption, the positioning protrusion 309 located inside the positioning groove 204a can maintain the position of the entire floating mechanism 300, and avoid the floating mechanism 300 from falling into the water under the action of gravity, causing the medicine and water to come into premature contact.

[0136] When the magnetic 402 attracts the slip ring 301 to the side close to the positioning protrusion 309, the slip ring 301 will move slightly towards the magnetic 402, the arc-shaped spring 308 will deform, and the positioning protrusion 309 will disengage from the positioning groove 204a, thus releasing the positioning of the slip ring 301 and allowing the slip ring 301 to move.

[0137] In order for the magnetic 402 to be able to attract the slip ring 301 from the side near the positioning protrusion 309 each time, it is necessary to prevent the floating mechanism 300 from rotating. This can be achieved by setting a guide block and a guide groove between the inner shaft 204 and the floating mechanism 300, which will not be described in detail here.

[0138] Example 5 differs from the above examples in that the sleep chamber further includes a switching mechanism 500; it includes a lifting core 501, which is installed on the inner side of the inner shaft 204; a plug 502, which extends to the outer side of the inner shaft 204 and can block the connection between the device body 201 and the feeding tube 202; an extension body 503, which is located at the end of the lifting core 501 and extends to the outer side of the inner shaft 204; when the floating mechanism 300 moves upward, it can push the extension body 503, and the lifting core 501 drives the plug 502 to disengage from the connection between the device body 201 and the feeding tube 202.

[0139] The inner shaft 204 has a cavity 204b inside, and the bottom of the cavity 204b is open. The end of the lifting core 501 with the plug 502 extends out from here. The plug 502 can block the connection port 205 between the device body 201 and the feeding tube 202. The top of the inner shaft 204 has a top cavity 204c with a diameter larger than that of the cavity 204b. The lifting core 501 is provided with a limiting plate 501a at the end of the top cavity 204c. The extension body 503 is installed on the limiting plate 501a. A through groove is opened at the bottom of the top cavity 204c. Under the action of gravity, the bottom end of the extension body 503 extends out from the inside of the top cavity 204c through the through groove.

[0140] Therefore, when the floating mechanism 300 does not abut against the extension body 503, under the influence of gravity, the plug 502 seals the connection between the device body 201 and the feeding tube 202. Thus, when one of the devices 201 is in use, the magnetic attractor 402 drives the slip ring 301, causing the entire floating mechanism 300 to reach the state of abutting against the extension body 503. At this time, the plug 502 disengages from the connection port 205 between the device body 201 and the feeding tube 202.

[0141] When the medicine inside the device 201 becomes ineffective and is no longer used, the magnetic 402 disengages from the floating mechanism 300, the floating mechanism 300 can no longer contact the extension body 503, the lifting core 501 moves downward, and the plug 502 can block the connection port 205 between the device 201 and the feeding tube 202, so that the mouse can only take the drinking water containing the medicine.

[0142] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for constructing a mouse model of intestinal sleep disorder based on gut microbiota transplantation, characterized by comprising the following steps: Comprising, ​ Under sterile conditions, a fresh fecal sample is collected from an insomnia patient, diluted with sterile normal saline and mixed evenly to form a coarse fecal suspension, large particles are removed by filtration, and the suspension is ensured to be uniform, to obtain a fecal suspension of the insomnia patient, i.e., a fecal suspension containing the intestinal flora of the insomnia patient, and the coarse fecal suspension is subjected to centrifugation, filtration, and density gradient centrifugation treatment; The obtained fecal suspension of the insomnia patient is transplanted into a sterile recipient mouse raised in a sterile isolation bag through an oral route or a rectal route using a gavage needle tube or a syringe; The sterile mouse implanted with the intestinal flora of the insomnia patient is raised in a sterile isolation bag for flora colonization; The construction of the intestinal sleep disorder mouse phenotype is verified by evaluating and analyzing the behavior, physiology, and microbiome of the mouse after flora colonization.

2. The method for constructing a mouse model of intestinal sleep disorder based on gut microbiota transplantation according to claim 1, wherein: The sterile mouse is raised without antibiotic treatment. 3.The method for constructing a mouse model of intestinal sleep disorder based on gut microbiota transplantation according to claim 1, wherein: The evaluation of the behavior includes the evaluation of activity level and sleep pattern. 4.The method for constructing a mouse model of intestinal sleep disorder based on gut microbiota transplantation according to claim 3, wherein: The sleep pattern includes sleep / wake cycle, rapid eye movement sleep, and non-rapid eye movement sleep. 5.The method for constructing a mouse model of intestinal sleep disorder based on gut microbiota transplantation according to claim 1, wherein: The evaluation of the physiology includes electroencephalographic activity, muscle activity, and stress hormone level.

Images