Lymphatic-like system regulation and control method and application

Abstract

The invention relates to a lymphatic system regulation and control method and application. A lymphatic system modulator and / or a physical stimulus is provided to the subject to modulate the lymphatic system. The method provided by the invention is applied to organisms and can effectively improve the circulation function of a lymphatic-like system in a biological brain, so that an effective strategy is provided for treatment of brain function diseases.

Description

Technical Field

[0001] The present invention relates to the field of lymphatic system regulation, and in particular to a method and application for regulating the lymphatic system. Background Art

[0002] The cerebrospinal fluid (CSF) in the subarachnoid space can enter the brain parenchyma through the perivascular spaces (PVS) formed by the surface arteries, perforating arteries and veins of the brain, and exchange substances with the interstitial fluid (ISF). In particular, it can remove metabolic waste and toxic proteins in the interstitial spaces of brain cells, which plays an extremely important role in maintaining the normal working environment in the brain. This functional cerebrospinal fluid circulation pathway is called the glymphatic system. Aquaporin 4 (AQP4) is considered to be the "valve" of this system. Under normal circumstances, AQP4 is polarized and mainly distributed on the terminal ends of astrocytes next to blood vessels, mediating fluid exchange and substance clearance between CSF and ISF in the PVS.

[0003] In recent years, the clearance function of the glymphatic system has attracted extensive research interest, and a large number of literatures have revealed its association with the abnormal aggregation and clearance of pathogenic proteins in neurodegenerative diseases. These studies have clearly revealed that impaired glymphatic system function during the development of the disease has a significant acceleration and aggravation effect on neurodegenerative diseases. At the same time, this type of research also provides a new therapeutic strategy for alleviating the progression of neurodegenerative diseases by improving glymphatic clearance.

[0004] However, there is currently limited research on treatment methods that enhance the clearance function of the glymphatic system with limited side effects, and progress in this field is still relatively slow. For example, US18 / 124,388 discloses a method for improving the clearance efficiency of the glymphatic system, implanting a pump near the spine to provide pressure waves to the CSF to increase CSF flux. This method requires surgery to complete the implantation of the pump, and it is difficult to avoid the trauma and danger of surgery to the patient. Summary of the invention

[0005] The technical problem to be solved by the present invention is that there is currently no safe and effective strategy to improve the function of the lymphatic system.

[0006] In order to solve the above technical problems, the inventors found that light flash stimulation can effectively regulate the glymphatic system, thereby providing an effective strategy for improving brain function.

[0007] Specifically, the present invention proposes the following technical solutions:

[0008] 1. The present invention provides a method for regulating the lymphatic system, which provides a lymphatic system regulator and / or physical stimulation to a subject.

[0009] 2. Optionally, the present invention provides the method described in item 1 above, wherein the lymphatic system regulator is a non-gene therapy preparation or a gene therapy preparation.

[0010] 3. Optionally, the present invention provides the method described in item 2 above, wherein the non-gene therapy preparation comprises one or more of adenosine, adenosine analogs and adenosine receptor agonists; optionally, the adenosine is provided to the subject by injection or is released in the subject by inducing the physical stimulation; optionally, the adenosine receptor agonist is selected from one or more of CCPA, R-PIA, AMP579, IB-MECA and CGS21680; optionally, the adenosine analog is NECA.

[0011] 4. Optionally, the present invention provides the method described in item 2 above, wherein the gene therapy preparation is a vector that increases the expression of adenosine equilibrative transporter and / or a vector that increases the expression of aquaporin-4, and optionally, the adenosine equilibrative transporter is adenosine equilibrative transporter 2.

[0012] 5. Optionally, the present invention provides the method described in any one of 1-4 above, wherein the frequency of the physical stimulation includes 30-50 Hz; optionally, the frequency of the physical stimulation includes one or more of 30 Hz, 35 Hz, 40 Hz, 45 Hz and 50 Hz;

[0013] Optionally, the physical stimulation frequency includes 35-45 Hz; Optionally, the physical stimulation frequency includes one or more of 35 Hz, 40 Hz and 45 Hz;

[0014] Optionally, the physical stimulation frequency is 40 Hz.

[0015] 6. Optionally, the present invention provides the method described in any one of 1 to 5 above, wherein the physical stimulation is visual stimulation,

[0016] Optionally, the duty cycle of the visual stimulation is 20%-80%, preferably 30%-70%, or 40%-70%, or 45-55%;

[0017] Optionally, the duty cycle of the visual stimulation is selected from one or more of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% and 80%;

[0018] Optionally, the duty cycle of the visual stimulation is 50%.

[0019] 7. Optionally, the present invention provides the method described in item 6 above, wherein the visual stimulation includes flash stimulation and / or image stimulation;

[0020] Optionally, the flash comprises white light and / or monochromatic light;

[0021] Optionally, the image comprises a raster image.

[0022] 8. Optionally, the present invention provides the method described in item 7 above, wherein the monochromatic light comprises one or more of ultraviolet light, blue light, green light, red light and near infrared light;

[0023] Optionally, the central wavelength of the ultraviolet light is 315-400nm; the central wavelength of the blue light is 450-490nm; the central wavelength of the green light is 500nm-560nm; the central wavelength of the red light is 600-680nm; the central wavelength of the near infrared light is 780-1100nm;

[0024] Optionally, the central wavelength of the ultraviolet light is 380nm; the central wavelength of the blue light is 488nm; the central wavelength of the green light is 530nm; the central wavelength of the red light is 650nm; and the central wavelength of the near infrared light is 850nm.

[0025] Optionally, the white light illumination is 2-6000 Lux, and the monochromatic light illumination is 2-500 Lux.

[0026] 9. Optionally, the present invention provides the method described in item 7 or 8 above, wherein the contrast of the grating image is 10%-100%;

[0027] Optionally, the grating image includes one or more of a moving grating image, a flickering grating image, a flickering grid image, and a flipped grating image;

[0028] Optionally, the motion grating contrast is 10%-100%; Optionally, the motion grating orientation is 0-180°; Optionally, the motion grating image spatial frequency is 0.05-30cycle / degree; Optionally, the motion grating image movement speed is 1-10Hz;

[0029] Optionally, the contrast of the scintillation grid image is 10%-100%; optionally, the frequency of the scintillation grid image is 1-100 Hz; optionally, the grid size of the scintillation grid image is 0.5-10 degree.

[0030] 10. The present invention provides a method for treating lymphatic system-related diseases, wherein the lymphatic system is regulated and treated by any of the methods 1-9 above.

[0031] 11. Optionally, the present invention provides the method described in item 10 above, wherein the lymphatic system-related disease is a neurodegenerative disease, and optionally, the neurodegenerative disease includes Alzheimer's disease and / or Parkinson's disease.

[0032] 12. Optionally, the present invention provides the method described in item 10 above, wherein the lymphatic system-related disease is a sleep disorder or a mental illness, and optionally, the mental illness is anxiety and depression related to sleep disorders.

[0033] 13. Optionally, the present invention provides the method described in item 10 above, wherein the lymphatic system-related disease is brain injury or stroke.

[0034] 14. The present invention provides a method for maintaining brain health, by maintaining the regulation of the glymphatic system through any of the methods 1-9 above.

[0035] 15. The present invention provides a method for improving drug delivery efficiency, wherein the delivery efficiency is improved by regulating the lymphatic system through any one of the methods 1-9 above.

[0036] 16. The present invention provides a drug delivery system, comprising a drug and a lymphatic system regulating device, wherein the lymphatic system regulating device provides the lymphatic system regulating method described in any one of 1-9 above.

[0037] 17. The present invention provides a method for evaluating the therapeutic effect of a drug or medical device, comprising analyzing changes in lymphoid inflow or outflow in a living body.

[0038] 18. Optionally, the present invention provides the evaluation method described in item 17 above, wherein the drug or medical device is used to treat lymphatic system-related diseases or improve sleep disorders.

[0039] 19. Optionally, the present invention provides the evaluation method described in item 17 or 18 above, wherein the lymphatic inflow or outflow tracer is selected from fluorescent tracers, and optionally, the fluorescent tracer is selected from fluorescent peptides.

[0040] 20. Optionally, the present invention provides the evaluation method described in item 19 above, wherein the fluorescent peptide is selected from fluorescent group-modified α-synuclein peptides, optionally, the fluorescent group is selected from one or more of Fitc, Cy2, Cy3, FAM, EDANS, Glu(EDANS)NH2, MCA, and Abz, optionally, the fluorescent group is a FITC group, optionally, the fluorescent peptide is a Y39-1 fluorescent peptide, and the sequence is Fitc-GKTKEGVLYVGSKTK.

[0041] The beneficial effects of the present invention include:

[0042] (1) The present invention provides a new strategy for regulating the lymphatic system, which can be used to treat lymphatic system-related diseases and has the potential advantages of being non-invasive, having few side effects, being well tolerated and having good efficacy.

[0043] (2) The present invention provides a new strategy for improving brain function by improving the cleaning efficiency of the glymphatic system, thereby reducing the risk of neurological diseases and enhancing cognitive function.

[0044] (3) The present invention provides a drug delivery method that improves the efficiency of drug delivery by regulating the lymphatic system, and is expected to be used to treat brain tumors and other nervous system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : 40 Hz flash frequency-dependently increases glymphatic inflow in the brain. A, Functional flow chart for detecting glymphatic inflow after flash stimulation of different frequencies. 30 minutes after injection of CSF tracer into the cerebellomedullary cisterna, animals were perfusion-fixed and whole-slice fluorescence was assessed; B, Representative fluorescence images showing a significant acceleration of CSF tracer entry into the brain after 40 Hz flash; CD, Quantitative analysis of whole-slice fluorescence area and mean fluorescence intensity of brain slices stimulated by flashes of different frequencies.

[0046] Figure 2 : 40 Hz flash frequency-dependently increases the outflow of the brain glymphatic system. A, Flowchart for detecting glymphatic outflow after flash stimulation of different frequencies. One hour after intrastriatal injection of CSF tracer, the animals were perfused and fixed, and the whole-slice fluorescence was evaluated; B, Representative fluorescence images showing that the outflow of CSF tracer from the striatum was significantly accelerated after 40 Hz flash; CD, Quantitative analysis of the whole-slice fluorescence area of ​​brain slices exposed to flashes of different frequencies; E, Representative fluorescence images of deep cervical lymph node slices; F, Quantitative analysis of the fluorescence area of ​​deep cervical lymph node slices showed that the tracer distribution was significantly increased in the 40 Hz illumination group.

[0047] Figure 3 :The enhancement of glymphatic inflow by 40 Hz flash and its frequency dependence do not depend on the structural composition of the peptides. A, Sequences of fluorescent peptide Y39-1 and fluorescent control peptide Y39-1-mut; B, Representative fluorescence images showing that the CSF tracer control peptide enters the brain significantly faster after 40 Hz flash, C-D, Quantitative analysis of whole-slice fluorescence area and mean fluorescence intensity of brain slices subjected to flashes of different frequencies.

[0048] Figure 4: 40 Hz flash enhancement of glymphatic influx depends on AQP4 expression. A, Representative fluorescence images showing that the entry of CSF tracers into the brain of AQP4- / - mice was significantly slowed down, and 40 Hz light exposure failed to increase the influx of tracers in the brain of AQP4- / - mice. BC, Quantitative analysis of mean fluorescence intensity and fluorescence area of ​​whole brain slices.

[0049] Figure 5 : 40Hz flash significantly enhances the polar expression of AQP4 on the end feet of perivascular astrocytes. A, Changes in the protein level of AQP4 after 40Hz illumination B, Representative images of the 40Hz illumination and normal illumination groups, C, Polarization index analysis, the polarization index is equal to the peak fluorescence value of the vascular end feet minus the 10μm baseline. The adjusted peak fluorescence value was normalized for easy comparison and visualization, D, AQP4 staining centered on the blood vessels, indicating that AQP4 is concentrated in the vascular system.

[0050] Figure 6 :40Hz flash significantly increases adenosine content in cerebrospinal fluid and enhances brain blood flow. A, Ultra-high performance liquid chromatography analysis of adenosine concentration in cerebrospinal fluid after 40Hz flash; BC, Representative images of laser speckle blood flow imaging show that blood perfusion in the mouse brain is significantly accelerated after 40Hz flash.

[0051] Figure 7 :The increase in adenosine after the flash but not during the flash mediates the enhancement of glymphatic inflow by 40 Hz flash. A, Dipyridamole was injected intraperitoneally into mice before 40 Hz flash to detect the glymphatic inflow function. B, Representative fluorescence images indicate that the injection of dipyridamole before the flash blocked the enhancement of glymphatic inflow by 40 Hz flash. C, Quantitative analysis of mean fluorescence intensity and fluorescence area of ​​whole brain slices. E, Dipyridamole was injected intraperitoneally into mice before the end of 40 Hz flash to detect the glymphatic inflow function. F, Representative fluorescence images indicate that the injection of dipyridamole before the end of the flash blocked the enhancement of glymphatic inflow by 40 Hz flash. GH, Quantitative analysis of mean fluorescence intensity and fluorescence area of ​​whole brain slices.

[0052] Figure 8 : Adenosine signaling controlled by adenosine equilibrative transporter 2 (ENT2) mediates the enhancement of glymphatic influx by 40 Hz light flashes. A, Representative fluorescence images showing that 40 Hz light exposure failed to increase the influx of tracers in the brain of ENT2- / - mice, BC, Quantitative analysis of mean fluorescence intensity and fluorescence area in whole brain slices.

[0053] Fig. 9:40Hz flash increases the inflow of lymphoid tissue, which is not related to blood pressure and heart rate. A, There is no significant difference in the heart rate of mice before and after 40Hz flash using BP-2010A sphygmomanometer; BD, There is no significant difference in systolic blood pressure, mean arterial pressure analysis and diastolic blood pressure analysis of mice before and after 40Hz flash.

[0054] Fig.10 :40Hz flash increases adenosine release with brain region specificity. 30min after 40Hz flash stimulation, the time-adenosine concentration curves of 8 brain regions including basal forebrain (BF), visual cortex V1, nucleus accumbens (NAc), ventral tegmental nucleus (VTA), superior colliculus (SC), medial prefrontal cortex (mPFC), hippocampus (HPC), dorsomedialstriatum (DMS) were plotted.

[0055] Fig.11 :Different visual stimulation patterns increase the specificity of extracellular adenosine in the visual cortex. A, Time-concentration curves of adenosine release in the visual cortex V1 area under 40Hz flash stimulation of different wavelengths; B, Visual stimulation of flickering light rake (flipping grating) can significantly increase adenosine release in the visual cortex V1 area; AC, n=3-5 per group; C, Time-concentration curves of adenosine release in the visual cortex V1 area under different motion grating visual stimulation patterns, the circular legend indicates that adenosine release in the visual cortex V1 increases rapidly with stimulation under the vertical orthogonal inhibitory motion grating stimulation pattern, the square legend indicates that adenosine release in the visual cortex V1 area increases slowly under the full-screen 90° azimuth motion grating stimulation pattern, and Random indicates that adenosine release does not increase under the full-screen random azimuth motion grating stimulation pattern. The shaded part is the visual stimulation time, n=3 per group. .

[0056] Fig.12 :Effects of 40 Hz stroboscopic light with different duty cycles on extracellular adenosine in the visual cortex.

[0057] Fig.13 :Effects of 40Hz stroboscopic light with different illumination on extracellular adenosine in the visual cortex.

[0058] Fig.14 :Effects of 40Hz stroboscopic light with different wavelengths on extracellular adenosine in the visual cortex.

[0059] Fig.15 : Schematic diagram of the moving grating stimulation pattern. The grating is alternating black and white stripes and moves in the direction of the arrow in the figure.

[0060] Fig.16: Schematic diagram of the flipped grating image changes: the local brightness and darkness of the grating (taking the dotted line as an example) alternate at a frequency of 40Hz; the screen refresh rate is 120Hz.

[0061] Fig.17 : Schematic diagram of the flickering grid. DETAILED DESCRIPTION

[0062] As described above, the purpose of the present invention is to provide a method for regulating the lymphatic system to solve the technical problem that there is currently no safe and effective strategy to improve the function of the lymphatic system.

[0063] The technical solution of the present invention will be described clearly and completely below. Obviously, based on the specific implementations of the present invention, all other implementations obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0064] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention.

[0065] The present invention provides a method for regulating the lymphatic system, which comprises providing a lymphatic system regulator and / or physical stimulation to a subject.

[0066] The present invention finds that the release of adenosine in the brain can enhance the lymphatic function of the brain parenchyma, and the lymphatic system regulator can be adenosine or an adenosine analog or an adenosine receptor agonist. The content of adenosine in the body can be increased by injecting adenosine or an adenosine analog into the subject, or by inducing the release of adenosine in the subject through physical stimulation such as flash stimulation, or by injecting adenosine receptor agonists to increase the expression of adenosine equilibrium transporters. Optionally, the adenosine receptor agonist is selected from one or more of CCPA, R-PIA, AMP579, IB-MECA and CGS21680, preferably CGS21680; optionally, the adenosine analog is NECA.

[0067] The present invention also found that adenosine signals controlled by adenosine equilibrative transporter 2 enhance the influx of the lymphatic system, and the enhanced polar expression of AQP4 on the end feet of perivascular astrocytes can also enhance the influx of the lymphatic system. The lymphatic system regulator can be a gene therapy preparation, such as a vector that increases the expression of adenosine equilibrative transporter and / or a vector that increases the expression of aquaporin-4.

[0068] The present invention has found that some specific physical stimulations can enhance the inflow and outflow of the brain's glymphatic system. The physical stimulation provided by the present invention has a frequency of 30-50Hz; optionally, the physical stimulation frequency includes one or more of 30Hz, 35Hz, 40Hz, 45Hz and 50Hz; optionally, the physical stimulation frequency includes 35-45Hz; optionally, the physical stimulation frequency includes one or more of 35Hz, 40Hz and 45Hz; optionally, the physical stimulation frequency is 40Hz. The physical stimulation provided by the present invention is non-invasive stimulation, including visual stimulation and / or sound stimulation. Optionally, the duty cycle of the visual stimulation is 20%-80%, preferably 30%-70%, or 40%-70%, or 45-55%; optionally, the duty cycle of the visual stimulation is selected from one or more of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% and 80%; optionally, the duty cycle of the visual stimulation is 50%.

[0069] Visual stimulation can be in various forms, including flash stimulation and / or image stimulation; optionally, the flash includes white light and / or monochromatic light; optionally, the image includes a grating image.

[0070] Light of different colors and light of different wavelengths will produce different stimulation effects. By adjusting the color, wavelength, frequency and other parameters of the light, different control effects can be achieved. Optionally, the monochromatic light includes one or more of ultraviolet light, blue light, green light, red light and near infrared light; Optionally, the central wavelength of the ultraviolet light is 315-400nm, 320-395nm, or 325-390nm, or 330-385nm; preferably 380nm; the central wavelength of the blue light is 450-490nm, or 455-488nm, or 460-485nm; preferably 480nm; the central wavelength of the green light is 500nm-560nm, or 505nm-555nm, or 510nm-550nm, or 515nm-545nm, or 520nm-540nm, or 525nm-545nm, or 530nm-540nm nm~535nm; preferably 530nm; the central wavelength of the red light is 600~680nm, or 595~675nm, or 590~670nm, or 585~665nm, or 580~650nm; preferably, 650nm; the central wavelength of the near infrared light is 780~1100nm, or 790~1090nm, or 800~1080nm, or 810~1070nm, or 820~1060nm, or 830~1050nm, or 840~1040nm, or 850~1030nm; preferably 850nm; optionally, the white light illumination is 2-6000Lux, and the monochromatic light illumination is 2-500Lux. Optionally, the illuminance of the white light emitted by the lamp is 500-3000 Lux. Optionally, the illuminance of the white light emitted by the lamp includes one or more of 500 Lux, 1000 Lux, 2000 Lux and 3000 Lux.

[0071] like Figure 15-17 As shown, the present invention provides different gratings.

[0072] Fig.15 Schematic diagram of the motion grating stimulation mode provided by the present invention, the variable parameters include: 100 to 500 candela per square meter (cd / m 2 ), orientation (0-180°), spatial frequency (0.2-30cycle / degree, i.e., cpd, single grating width), temporal frequency (1-10Hz, grating movement speed), contrast (1-100%), stimulation range size (0-30° or full-screen stimulation), orthogonal stimulation (central receptive field 5°, peripheral receptive field 15°).

[0073] Fig.16 Schematic diagram of the flip grating stimulation mode provided by the present invention, the variable parameters include: 100 to 500 candela per square meter (cd / m 2), orientation (0-180°), spatial frequency (0.1-30 cycle / degree, i.e. cpd, single grating width), flicker frequency (1-100 Hz), contrast (1-100%), and stimulation range size (0-30° or full-screen stimulation).

[0074] Fig.17 The flickering grid stimulation mode provided by the present invention is intended to present the display screen in a checkerboard grating mode, with variable parameters: length and width of a single black / white grid (1° to 5°), contrast (1% to 100%), and flicker frequency (1 to 100 Hz). Among them, contrast refers to the measurement of different brightness levels between the brightest white and the darkest black in the light and dark areas of an image. The larger the difference range, the greater the contrast (such as a contrast of 100%), and the smaller the difference range, the smaller the contrast (such as a contrast of 10%). The higher the contrast, the easier it is for our naked eyes to distinguish.

[0075] There is a correlation between the clearance function of the glymphatic system and the aggregation and clearance of pathogenic proteins in neurodegenerative diseases. The present invention also provides a method for treating diseases related to the glymphatic system. The glymphatic system regulation method provided by the present invention improves the clearance function of the glymphatic system, thereby preventing and treating degenerative diseases. Preferably, by enhancing glymphatic clearance with 40Hz flash, it is expected to delay the progression of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease and reduce the severity of the disease.

[0076] Glymphatic function is enhanced during sleep, so sleep disorder-related diseases are often accompanied by glymphatic circulation dysfunction, and its malignancy is often related to the severity of the disease. The present invention also provides a method for treating sleep disorders and mental illnesses such as anxiety and depression related to sleep disorders. The glymphatic system regulation method provided by the present invention improves the clearance function of the glymphatic system, thereby improving sleep. Preferably, 40Hz flash enhances glymphatic function, which can compensate for the damage to glymphatic function caused by sleep disorders.

[0077] The clearance function of the glymphatic system may help reduce inflammation and waste accumulation after injury and promote the repair of neural tissue. The present invention also provides a method for treating brain trauma or injury. The glymphatic system regulation method provided by the present invention improves the clearance function of the glymphatic system, thereby helping brain recovery. Preferably, the clearance function of the glymphatic system is promoted by 40Hz flashes.

[0078] The glymphatic system helps to remove cellular metabolic waste and toxic proteins, which is essential for maintaining brain health. The present invention also provides a method for maintaining brain health. The glymphatic system regulation method provided by the present invention improves the clearance function of the glymphatic system and improves the efficiency of brain waste removal. Preferably, regulating the system through 40Hz flashes may help reduce the accumulation of brain waste, thereby reducing the risk of neurological diseases and enhancing cognitive function.

[0079] Regulating the glymphatic system can be used to improve the method of delivering drugs to the brain. By understanding the working principle of the glymphatic system, more effective drug delivery systems can be designed, which are expected to be used to treat brain tumors and other neurological diseases. The present invention provides a method for improving the efficiency of drug delivery and a drug delivery system, and improves the efficiency of drug delivery by improving the function of the glymphatic system through the glymphatic system regulation method provided by the present invention.

[0080] The present invention also provides a method for evaluating the therapeutic effect of a drug or medical device, including analyzing changes in lymphatic inflow or outflow in a living body. It can be used to evaluate the effect of treating neurological diseases and / or improving diseases such as sleep disorders. Changes in lymphatic inflow or outflow are analyzed by tracers. Preferably, fluorescent tracers such as fluorescent peptides can be used in the evaluation method of the present invention. The lymphatic system is mainly related to neurodegenerative diseases. The present invention designs fluorescent peptides based on the core structure of α-synuclein, a representative pathogenic protein of degenerative diseases. Preferably, the fluorescent peptide is selected from α-synuclein peptides modified with fluorescent groups, and optionally, the fluorescent groups are selected from one or more of Fic, Cy2, Cy3, FAM, EDANS, Glu (EDANS) NH2, MCA, and Abz. FITC (fluorescein isothiocyanate) is a commonly used fluorescent group with multiple advantages, which makes it widely used in biological and biochemical research: High brightness: The fluorescent signal generated by FITC is relatively bright, and this high brightness helps to improve the detection sensitivity of the experiment. Chemical resistance: FITC-labeled molecules are generally chemically and light-stable, meaning they do not easily fade or decompose under experimental conditions. This helps ensure the accuracy and reproducibility of experimental results. Relatively small molecular size: The relatively small molecular size of FITC means that it can be easily attached to most biological molecules, such as proteins, antibodies, nucleic acids, etc., without significantly changing the properties of these molecules. Multiple detection applications: The fluorescent signal of FITC can be used on different detection platforms, including fluorescence microscopy, flow cytometers, and spectrophotometers. This versatility makes it an important tool in biological research. Wide availability: FITC-labeled antibodies and probes, as well as other fluorescent molecules, are easily available, and suppliers usually offer a variety of labeling options, allowing researchers to choose according to their needs. In summary, FITC is a commonly used fluorescent labeling molecule with the advantages of high brightness, chemical resistance, and a variety of applications.

[0081] The lymphatic system regulation method of the present invention is described below through specific examples.

[0082] We conducted a series of experiments and found that a brief 30-minute 40Hz flash significantly enhanced the polarity of AQP4 around the blood vessels of mice and the inflow and outflow circulation of the glymphatic system, and this effect was frequency-dependent. In addition, in AQP4 transgenic knockout mice, 40Hz flash could not change the inflow of the glymphatic system, indicating that this effect depends on the function of AQP4 protein in the glymphatic system. In addition, the detection of adenosine content in cerebrospinal fluid by HPLC found that 40Hz flash significantly increased adenosine content and enhanced brain blood flow. At the same time, intraperitoneal injection of adenosine equilibrative transporter (ENT) inhibitor dipyridamole (15mg / kg) into mice completely eliminated the effect of 40Hz flash on the glymphatic system. After further using ENT2 transgenic knockout mice, we found that 40Hz flash could not improve the glymphatic circulation of ENT2-KO mice. Therefore, this series of experimental results suggest that 40Hz flash increases adenosine in the brain through ENT2 release and further regulates the circulation of the glymphatic system.

[0083] Experimental Materials and Methods

[0084] animal:

[0085] All experimental procedures were in accordance with the guidelines of the Institutional Ethics Committee for the Use of Animals in Research and Education of Wenzhou Medical University. C57BL / 6 wild-type mice were purchased from a supplier approved by the institute (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.). ENT1 knockout mice (ENT1-KO mice) and ENT2 knockout mice (ENT2-KO mice) were created by Beijing Biocytogen using CRISPR / Cas9 technology and provided by Professor Li Yulong's laboratory. AQP4 knockout mice (AQP4- / - mice) were created by Shanghai Model Organisms Center using CRISPR / Cas9 technology and provided by Professor Bai Ruiliang's laboratory. Mice were housed in a living environment with free access to food and water and followed a 12-h light / 12-h dark cycle.

[0086] Drugs and CSF tracers:

[0087] Dipyridamole was dissolved in 4% methylcellulose solution at a concentration of 1.5 mg / ml. The drug solution was prepared approximately 30 minutes before each injection.

[0088] CSF tracer is dissolved in artificial cerebrospinal fluid at 0.1% w / v. Y39-1 sequence is Fitc-GKTKEGVLYVGSKTK, molecular weight is 2kDa. Y39-1-mut sequence is Fitc-GKTKEGVGFVGTKSK, molecular weight is 2kDa. Synthesized by Xianna Microorganisms.

[0089] Cisterna magna (CM) injection:

[0090] Anesthetized mice were fixed in a stereotaxic apparatus (RWD), the CM was surgically exposed, and a 33-gauge needle (Hamilton, USA) was connected to a PE20 tube filled with tracer (2kDa Y39-1 or Y39-1-mut), which was then connected to a 10μL Hamilton syringe (80365, Hamilton, USA) and inserted into the CM. A syringe pump (RWD Life Science, China) was used to infuse 10μL CSF tracer at a rate of 2μL / min for 5 minutes. Thirty minutes after the start of the intracisternal infusion, the animals were killed by decapitation and the brain was removed (Note: the needle was left in place after the infusion to prevent cerebrospinal fluid reflux). The brain was fixed overnight in PBS containing 4% paraformaldehyde and then vibrated (100μm) to detect lymphoid inflow.

[0091] Intrastriatal injection:

[0092] Anesthetized mice were fixed in a brain stereotaxic apparatus (RWD), the skin was opened and the skull was exposed. Holes were made with a handheld drill at the coordinates: +0.5 mm anterior / posterior, +2.0 mm medial / lateral, and +3.25 mm dorsal / ventral from the anterior bregma. The pulled glass capillary was implanted into the brain parenchyma. A volume of 1 μl of 0.1% Y39-1 was then infused at a rate of 0.1 μl / min by a micropump. The microsyringe remained in place for 5 minutes and then was withdrawn very slowly to avoid any possible backflow. One hour after intrastriatal injection, the abdominal aorta of the mouse was bled, the brain and deep cervical lymph nodes (dcLN) were removed and post-fixed for 24 hours, followed by vibration sectioning (100 μm) to detect lymphoid outflow.

[0093] Immunofluorescence and quantification:

[0094] The protein expression pattern of AQP4 and glial fibrillary acidic protein (GFAP) was evaluated by free-floating immunofluorescence staining of 40 μm fixed coronal brain slices. The brain slices were blocked with 10% donkey serum for 1 hour at room temperature. The primary antibodies were rabbit anti-AQP4 (AB3594, Millipore, 1:500) and mouse anti-GFAP (G3893, Sigma, 1:200) and incubated overnight at room temperature. The secondary antibodies were donkey anti-rabbit Alexa Fluor 555 (1:500) and goat anti-mouse Alexa Flour 488 (1:500). The stained sections were visualized using a confocal microscope (LSM900, Zeiss) or a fluorescence microscope (DM6B, Leica).

[0095] To quantify AQP4 polarization, a 50 μm region centered on the vessel was analyzed using the line graph tool in ImageJ. For polarity calculations, the baseline was defined as the average intensity from -20 μm to -10 μm beyond 10 μm from the peak fluorescence. The baseline fluorescence intensity was subtracted from the peak fluorescence intensity. The adjusted peak fluorescence values ​​were normalized for easier comparison and visualization.

[0096] Determination of adenosine concentration in cerebrospinal fluid:

[0097] Ultra-high performance liquid chromatography (HPLC) was used to measure the changes in the concentration of adenosine in the cerebrospinal fluid of mice after 40Hz flash stimulation. After 30 minutes of 40Hz flash stimulation, the cerebrospinal fluid of mice was quickly collected and placed in a mixed solution of adenosine deaminase inhibitor (EHNA) and adenosine equilibrative transporter (ENT) inhibitor dipyridamole. After thorough mixing, the mixture was centrifuged at 4℃13000r / min for 10 minutes, and the supernatant was aspirated for examination. A standard solution of adenosine (Sigma-Aldrich, purity>98%) was prepared with a concentration range of 1 to 1000ng / mL, and a concentration calibration curve was established.

[0098] Blood flow measurement:

[0099] The RFLSI ZW laser speckle blood flow imaging system can monitor in real time and intuitively present the speed and changes of microcirculatory blood flow in living organ tissues. The light intensity is set to 80Mw laser intensity, 5ms camera exposure time, 3s filter constant, Sliding mode, 1.4x magnification, 0-700 pseudo-color threshold, and 8s recording per test.

[0100] Blood pressure and heart rate:

[0101] Blood pressure and heart rate of mice were measured indirectly and noninvasively using the tail pressure photoplethysmography method using a BP-2010A sphygmomanometer (Softron, Tokyo, Japan). The same time period was selected to test the blood pressure of each mouse (8-9 weeks old) to avoid normal daily fluctuations in blood pressure. Three consecutive days were used to accustom the mice to the device. In order to optimize cardiovascular circulation, the mice were wrapped in cotton cloth and kept at 37°C in a cylindrical heater except for the tail. After the pressurized sensor was put on the mouse tail and entered the pulse detection state, the blood flow pulse oscillation of the mouse tail was automatically detected, and the blood pressure was measured when the pulse wave was stable and regular. The software reads and records the blood pressure measurements. After three consecutive days of training, the blood pressure and heart rate of each mouse were measured before and after the flash. After the test, the mouse was gently grasped by the tail and gently returned to the cage.

[0102] Example 1 40 Hz flash frequency-dependently increases inflow and outflow of the glymphatic system in the brain of wild-type mice

[0103] First, we used the fluorescent tracer Y39-1 (2kDa) to conduct brain penetration and clearance experiments. Y39-1 is a fluorescent peptide constructed based on the core structure of α-synuclein and spliced ​​with a fluorescent FITC group. In this experiment, it was used to study the effects of flash stimulation of different frequencies (20Hz, 40Hz and 80Hz) on the glymphatic system after 30 minutes.

[0104] like Figure 1 As shown in AD, after the mice were exposed to light of different frequencies for 30 minutes, Y39-1 was injected into the cisterna magna (CM) with a total injection volume of 10 μL and a speed of 2 μL / min. 30 minutes after the injection, the brain was removed and fixed in PFA overnight. The next day, the sections were vibrated with a thickness of 100 μm, and brain slices at a distance of 1.2, 0.6, 0, -0.6, -1.2, and -1.8 from the bregma were collected and photographed under a fluorescence microscope. Quantitative analysis results showed that the parenchymal distribution of Y39-1 in mice under 40 Hz illumination was significantly higher than that in mice under normal illumination. It is worth noting that the tracer distribution in mice under 80 Hz illumination was not significantly different from that in mice under normal illumination. This indicates that the increase in glymphatic inflow in wild-type mice by 40 Hz flash frequency is frequency-dependent. Consistent with this, 40 Hz flash also promoted the clearance of molecules from the brain to the peripheral lymphatic system. As Figure 2As shown in AF, after 30 minutes of light exposure at different frequencies, Y39-1 was injected into the striatum, with a total injection volume of 1 μL and a speed of 0.1 μL / min. One hour after the injection, the brain and ipsilateral deep cervical lymph nodes were obtained. They were fixed in PFA overnight. The next day, the sections were vibrated with a thickness of 100 μm, and brain slices at 1.2, 0.6, 0, -0.6, and -1.2 from the bregma were collected and photographed under a fluorescence microscope. The results of quantitative analysis showed that the percentage of fluorescent areas in the brain of mice in the 40 Hz flash group was significantly reduced, and the percentage of fluorescent areas in the deep cervical lymph nodes (dcLN) was significantly increased. In contrast, there was no significant difference in the percentage of tracer distribution in the brain and deep cervical lymph nodes (dcLN) of mice in the 20 Hz and 80 Hz groups compared with the normal light group. In order to exclude the functional effects of the peptide, we also used the fluorescent control peptide Y39-1-mut to detect the effects of flashes of different frequencies on lymphatic inflow, such as Figure 3 As shown, the same method was used to evaluate the glymphatic function of mice after exposure to light of different frequencies. The results were consistent with the previous results, demonstrating that the enhancement effect of 40 Hz flash inflow on the glymphatic system and its frequency dependence do not depend on the structural composition of the peptide.

[0105] Example 2 40 Hz flash enhances lymphoid influx and is dependent on the expression of AQP4 and can significantly enhance the polar expression of AQP4 on perivascular astrocyte end feet

[0106] AQP4 is highly polarized and expressed on the end feet of perivascular astrocytes, which together with the ependymal cells of the vessels form the boundary between the para-arterial CSF inflow and the para-venous ISF clearance pathways.

[0107] like Figure 4 As shown in the figure, AQP4- / - mice and their littermates were randomly assigned to a flash group and a normal light group. The mice in the flash group were placed under 40Hz light for 30 minutes. Similarly, samples were taken 30 minutes after injection in the cerebellomedullary cisterna magna (CM), and the glymphatic inflow function was analyzed by fluorescence quantitative analysis. The results showed that in AQP4- / - mice, the fluorescence brightness and distribution in the brain parenchyma were significantly lower than those in wild-type mice. At the same time, 40Hz light exposure failed to increase the influx of tracers in the brain of AQP4- / - mice. Therefore, 40Hz flash enhancement of the influx of the glymphatic system depends on the expression of AQP4.

[0108] A variety of neurological diseases (such as Alzheimer's disease, traumatic brain injury, stroke, etc.) generally have a decline in glymphatic function, accompanied by a decrease in AQP4 polarity. Therefore, we explored whether 40Hz flashes would affect the polar expression of AQP4 in blood vessels. Figure 5As shown in the figure, by measuring the immunofluorescence intensity of AQP4 along the lumen of the blood vessel, we found that the AQP4 immunofluorescence in the tissue immediately surrounding the blood vessel increased significantly after the 40 Hz flash, proving that the 40 Hz flash can increase the polar expression of AQP4 on the end feet of perivascular astrocytes, thereby enhancing the influx of lymphocytes.

[0109] Example 3 40 Hz flash significantly increases adenosine content in cerebrospinal fluid and enhances brain blood flow

[0110] Previous studies in our laboratory found that 30 minutes of 40Hz flash significantly and continuously increased the extracellular adenosine concentration in the primary visual cortex, proposing for the first time the potential neurotransmitter / modulator basis for 40Hz in regulating brain function. Later, we used ultra-high performance liquid chromatography (HPLC) to measure the changes in adenosine concentration in the cerebrospinal fluid (CSF) of mice after 40Hz flash. 30 minutes after 40Hz flash stimulation, the cerebrospinal fluid of mice was quickly collected and placed in a mixed solution of adenosine deaminase inhibitor (EHNA) and adenosine equilibrative transporter (ENT) inhibitor dipyridamole. After thorough mixing, centrifugation was performed at 4°C and 13,000 r / min for 10 minutes, and the supernatant was aspirated for examination. A standard solution of adenosine (Sigma-Aldrich, purity >98%) was prepared, and the standard parameters were referred to. After the peak value of about 1.6 was detected on the machine, and the area under the curve at this moment was used as the sample adenosine concentration. Finally, the adenosine concentration was obtained by calibration. Figure 6 As shown in Figure 2, the adenosine concentration in CSF after 40Hz flash was significantly higher than that in the normal light group. In addition, through laser speckle blood flow imaging, we observed that the blood flow velocity in the mouse brain was significantly increased after 40Hz flash, but there was no difference in the blood flow velocity in the mouse brain after 20Hz and 80Hz flash compared with the baseline.

[0111] Example 4 Adenosine increase after, but not during, the flash mediates 40 Hz flash enhancement of lymphoid inflow

[0112] We next used the ENT1 / 2 inhibitor dipyridamole to investigate the contribution of the equilibrative adenosine transporter (ENT) to adenosine release after 40 Hz flashes. Figure 7 As shown in the figure, we injected dipyridamole (15 mg / kg) intraperitoneally into mice before or before the 40 Hz flash, and injected CSF tracer into the cerebellomedullary cisterna (CM) after the light ended. Quantitative analysis showed that the increase in tracer influx caused by 40 Hz light was eliminated in mice injected with dipyridamole before or before the flash stopped. This indicates that the increase in adenosine after the flash, rather than during the flash, mediates the enhancement of glymphatic influx by 40 Hz flash.

[0113] Example 5 Adenosine signaling controlled by adenosine equilibrative transporter 2 mediates enhancement of glymphatic system influx by 40 Hz flashes

[0114] The transmembrane transport of adenosine is accomplished by two different adenosine equilibrative transporters (ENTs), of which ENT1 has a higher affinity for adenosine but is present in lower quantities, while ENT2 has a higher quantity but lower affinity. Based on previous studies in our laboratory, we found that 40 Hz light induced intracellular adenosine production and that adenosine efflux mediated by equilibrative nucleoside transporter-2 (ENT2) was the molecular pathway responsible for extracellular adenosine production. Therefore, we used the recently developed ENT2-KO mice to determine their role in the enhancement of glymphatic influx after 40 Hz light flashes. Figure 8 As shown, in ENT2-KO mice, we observed a failure to increase tracer influx after 40 Hz light illumination. These findings highlight the critical role of ENT2 in controlling adenosine signaling-mediated enhancement of glymphatic influx in response to 40 Hz light flashes.

[0115] Example 6: 40 Hz flash increases lymphatic inflow independently of blood pressure and heart rate

[0116] Previous studies have shown that the cardiovascular system has a significant impact on the flow of CSF around blood vessels. The bulk flow of CSF around blood vessels is positively correlated with arterial pulsation, with the same frequency as the cardiac cycle and the same overall direction as blood flow. Studies have shown that hypertension significantly reduces the amplitude of arterial pulsation and impairs the inflow function of the glymphatic system. Therefore, we wanted to investigate whether the increase in glymphatic inflow by 40 Hz flashes is related to blood pressure and heart rate. We used a BP-2010A sphygmomanometer (Softron, Tokyo, Japan) to indirectly and noninvasively measure blood pressure and heart rate in mice using tail pressure photoplethysmography. The same time period was selected to test the blood pressure of each mouse (8-9 weeks old) to avoid the normal daily fluctuations in blood pressure. Three consecutive days were used to habituate the mice to the device. To optimize cardiovascular circulation, the mice were wrapped in cotton cloth and kept at 37°C in a cylindrical heater, except for the tail. After the pressurized sensor was put on the mouse tail and entered the pulse detection state, it automatically detected the blood flow pulse oscillation of the mouse tail and measured the blood pressure when the pulse wave was stable and regular. The software read and recorded the blood pressure measurement. After three days of continuous training, each mouse's blood pressure and heart rate were measured before and after the flash. After the test, the mouse was gently grasped by the tail and gently returned to the cage. Fig. 9 As shown in the figure, by comparing the blood pressure and heart rate of mice before and after 40Hz flash, we found that 40Hz flash had no significant effect on the heart rate, systolic blood pressure and diastolic blood pressure of mice. Therefore, the increase of glymphatic inflow by 40Hz flash is not related to blood pressure and heart rate.

[0117] Example 7 40 Hz flash stimulation can effectively increase adenosine release levels in brain regions such as V1, BF, mPFC and SC, and this phenomenon is brain region specific

[0118] In this experiment, adenosine release levels were detected in the visual cortex (V1), basal forebrain (BF), nucleus accumbens (NAc), ventral tegmental area (VTA), superior colliculus (SC), mesial prefrontal cortex (mPFC), hippocampus (HPC), dorsal medial striatum (DMS) and other brain regions using a new genetically encoded adenosine probe. Fig.10 As shown in the figure, after 40Hz flash stimulation for 30min, adenosine signals were recorded. We found that the adenosine release levels in brain regions such as V1, SC, BF, mPFC and HPC were significantly increased, while in the control group, the AAV2 / 9-hsyn-Mut virus (Wuhan Shumi Brain Science Technology Co., Ltd., PT-1348) injected did not show an increase in adenosine signals. Among the brain regions with elevated adenosine, the adenosine signals in brain regions related to visual information encoding, such as V1 and SC, were the strongest; the adenosine signals in the BF brain region related to sleep and wakefulness were also significantly enhanced; the adenosine signals in brain regions related to cognition, such as mPFC and HPC, were slightly enhanced.

[0119] Example 8 Different visual stimulation patterns increase V1 adenosine release levels

[0120] 1) 40 Hz flash stimulation

[0121] like Fig.11 As shown in A, the difference in adenosine release levels in the V1 area under different wavelengths under 40 Hz flash stimulation conditions. Only blue light with a wavelength of 480 nm and green light with a wavelength of 530 nm can significantly increase the adenosine concentration in the V1 area, while flash stimulation of other wavelengths (380 nm, 650 nm, 850 nm, etc.) did not increase the adenosine concentration in the V1 area.

[0122] 2) Influence of moving grating

[0123] like Fig.11 As shown in C, horizontal motion grating stimulation ( Fig.15 Left side, arrow indicates the direction of movement), the excitation parameters are: the grating is presented in full screen at high contrast (80%) (the average screen brightness is 160cd / m 2 ) to ensure that the mouse can see the stimulus throughout the experiment. The spatial frequency of the horizontal motion grating is 0.2 cpd, and the temporal frequency is 1-10 Hz (i.e., the grating movement speed). At the same time, a 5° horizontal motion grating is applied in the center and a 15° vertical motion grating is applied in the periphery (center-periphery vertical motion grating, Fig.15 The changes in adenosine levels in the V1 region of mice were detected under the combined stimulation formed by the stimuli (right).

[0124] The results showed that both types of motion gratings could significantly increase V1 adenosine, but the increase of horizontal motion gratings was smaller than that of center-periphery vertical motion gratings ( Fig.11 C, ΔF / F values ​​were 5% for horizontal motion gratings and 8% for center-periphery vertical motion gratings), indicating that large-scale grating stimulation has an important effect on extracellular adenosine production.

[0125] 3) The influence of vertically flipped grating

[0126] like Fig.11 As shown in B, while keeping the light intensity unchanged, a vertical flip grating stimulation at a frequency of 40 Hz was used. The stimulation parameters were: the spatial frequency of the vertical sinusoidal flip grating was 0.1 cpd, and the flip frequency was 40 Hz (i.e., a black grating and a white grating flashed alternately, Fig.16 ), and the average light intensity does not change during a specific time period (after gamma correction to eliminate the brightness nonlinearity of the screen). The grating is presented in full screen at high contrast (80%) (the average screen brightness is 160cd / m 2 ) to ensure that mice could see the stimulus throughout the experiment.

[0127] The results showed that vertical flip grating could significantly increase V1 adenosine, but the increase was smaller than that of 40 Hz flash stimulation ( Fig.11 B, ΔF / F values ​​were 5% for flipped gratings and 15-20% for 40 Hz flash stimulation), indicating that light intensity has an important effect on the production of extracellular adenosine.

[0128] Example 9 Effects of 40 Hz flash stimulation with different duty cycles on extracellular adenosine in the visual cortex

[0129] Considering the effect of the illuminance (total energy) of white flash stimulation on the extracellular adenosine concentration, and the total energy produced by different duty ratios (1%, 10%, 50%) of 40Hz strobe (same illuminance 3000lux) is different, we tested the effect of 40Hz strobe (same illuminance 3000lux) stimulation for 30 minutes under different duty ratios (1%, 10%, 50%) on the adenosine level in the V1 area.

[0130] The results are as follows Fig.12As shown in the figure, it can be seen that under the 1% duty cycle condition and the 10% duty cycle condition, the peak ΔF / F of the V1 adenosine increase level is 5 and 10 respectively, which is lower than that under the 50% duty cycle stimulation (peak ΔF / F is 25), indicating that white flash stimulation can significantly increase the V1 adenosine level at a 50% duty cycle. Therefore, at the same illumination (3000lux), 40Hz white flash stimulation increases the extracellular adenosine concentration in a duty cycle (1%, 10%, 50%) dependent (specific) manner, and 40Hz stroboscopic light at a 50% duty cycle has the most significant effect on increasing the V1 adenosine level.

[0131] Example 10 Effects of 40 Hz stroboscopic light with different light intensities and wavelengths on extracellular adenosine in the visual cortex

[0132] 1) Influence of light intensity

[0133] The effects of different light intensities (50 Lux, 500 Lux, 1000 Lux, 2000 Lux and 3000 Lux) on adenosine production in the visual cortex were studied by using 40 Hz white flash stimulation. Fig.13 As shown in the figure, we found that low-intensity (500 Lux) 40 Hz flicker induced low adenosine levels, high-intensity (3000 Lux) 40 Hz white flash stimulation increased adenosine levels with a peak ΔF / F of 20, and 40 Hz flash stimulation above 2000 Lux could significantly increase adenosine concentrations in the V1 region. In summary, these findings from white light with different illuminations and vertical gratings that increased adenosine in the V1 region indicate that light intensity is a key factor in producing extracellular adenosine levels in the primary visual cortex of the brain.

[0134] 2) Influence of light wavelength

[0135] like Fig.14 As shown in the figure, under the 40Hz monochromatic light flash stimulation with an illumination of 500Lux, the blue light with a wavelength of 480nm and the green light with a wavelength of 530nm can significantly increase the adenosine concentration in the V1 area under the 40Hz stroboscopic condition, while the flash stimulation of other wavelengths (380nm, 650nm, 850nm, etc.) did not increase the adenosine concentration in the V1 area. It is worth noting that the kinetics of this single wavelength to increase the adenosine concentration in V1 are basically similar (starting to increase and decrease from 10 minutes, reaching the highest concentration at 30 minutes, and lasting for 2-3 hours).

Claims

1. A method for regulating the lymphatic system, characterized in that: A glymphatic system modulator and / or physical stimulation is provided to the subject.

2. The method according to claim 1, wherein the lymphatic system regulator is a non-gene therapy preparation or a gene therapy preparation.

3. The method according to claim 2, wherein the non-gene therapy preparation comprises one or more of adenosine, adenosine analogs and adenosine receptor agonists; optionally, the adenosine or adenosine analogs are provided to the subject by injection or induced to be released in the subject by the physical stimulation; optionally, the adenosine receptor agonist is selected from CCPA, One or more of R-PIA, AMP579, IB-MECA and CGS21680; optionally, the adenosine analog is NECA.

4. The method according to claim 2, wherein: The gene therapy preparation is a vector for increasing the expression of adenosine equilibrative transporter and / or a vector for increasing the expression of aquaporin-4. Optionally, the adenosine equilibrative transporter is adenosine equilibrative transporter 2.

5. The method according to any one of claims 1 to 4, wherein: The frequency of the physical stimulation includes 30-50 Hz; optionally, the frequency of the physical stimulation includes one or more of 30 Hz, 35 Hz, 40 Hz, 45 Hz and 50 Hz; Optionally, the physical stimulation frequency includes 35-45 Hz; Optionally, the physical stimulation frequency includes one or more of 35 Hz, 40 Hz and 45 Hz; Optionally, the physical stimulation frequency is 40 Hz.

6. The method according to any one of claims 1 to 5, wherein: The physical stimulus is a visual stimulus, Optionally, the duty cycle of the visual stimulation is 20%-80%, preferably 30%-70%, or 40%-70%, or 45-55%; Optionally, the duty cycle of the visual stimulation is selected from one or more of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% and 80%; Optionally, the duty cycle of the visual stimulation is 50%.

7. The method according to claim 6, wherein: The visual stimulation includes flash stimulation and / or image stimulation; Optionally, the flash comprises white light and / or monochromatic light; Optionally, the image comprises a raster image.

8. The method according to claim 7, wherein: The monochromatic light includes one or more of ultraviolet light, blue light, green light, red light and near infrared light; Optionally, the central wavelength of the ultraviolet light is 315-400nm; the central wavelength of the blue light is 450-490nm; the central wavelength of the green light is 500nm-560nm; the central wavelength of the red light is 600-680nm; the central wavelength of the near infrared light is 780-1100nm; Optionally, the central wavelength of the ultraviolet light is 380nm; the central wavelength of the blue light is 488nm; the central wavelength of the green light is 530nm; the central wavelength of the red light is 650nm; and the central wavelength of the near infrared light is 850nm. Optionally, the white light illumination is 2-6000 Lux, and the monochromatic light illumination is 2-500 Lux.

9. The method according to claim 7 or 8, wherein: The grating image contrast is 10%-100%; Optionally, the grating image includes one or more of a moving grating image, a flickering grating image, a flickering grid image, and a flipped grating image; Optionally, the motion grating contrast is 10%-100%; Optionally, the motion grating orientation is 0-180°; Optionally, the motion grating image spatial frequency is 0.05-30cycle / degree; Optionally, the motion grating image movement speed is 1-10Hz; Optionally, the contrast of the scintillation grid image is 10%-100%; optionally, the frequency of the scintillation grid image is 1-100 Hz; optionally, the grid size of the scintillation grid image is 0.5-10 degree.

10. A method for treating diseases related to the lymphatic system, wherein the disease is treated by regulating the lymphatic system through the method according to any one of claims 1 to 9.

11. The method according to claim 10, wherein: The lymphatic system-related disease is a neurodegenerative disease. Optionally, the neurodegenerative disease includes Alzheimer's disease and / or Parkinson's disease.

12. The method according to claim 10, wherein: The lymphatic system-related disease is a sleep disorder or a mental illness. Optionally, the mental illness is anxiety and depression associated with sleep disorders.

13. The method according to claim 10, wherein: The lymphatic system-related disease is brain injury or stroke.

14. A method for maintaining brain health, comprising maintaining the regulation of the glymphatic system by the method of any one of claims 1 to 9.

15. A method for improving drug delivery efficiency, comprising regulating the lymphatic system by the method according to any one of claims 1 to 9 to improve the delivery efficiency.

16. A drug delivery system, comprising a drug and a lymphatic system regulating device, wherein the lymphatic system regulating device provides the lymphatic system regulating method according to any one of claims 1 to 9.

17. A method for evaluating the therapeutic effect of a drug or medical device, comprising analyzing changes in lymphoid inflow or outflow in a living body.

18. The evaluation method according to claim 17, wherein: The drug or medical device is used to treat lymphatic system-related diseases or improve sleep disorders.

19. The evaluation method according to claim 17 or 18, wherein: The lymphatic inflow or outflow tracer is selected from fluorescent tracers, and optionally, the fluorescent tracer is selected from fluorescent peptides.

20. The evaluation method according to claim 19, wherein: The fluorescent peptide is selected from fluorescent group-modified α-synuclein peptide, and optionally, the fluorescent group is selected from Fitc, Cy2, Cy3, FAM, EDANS, Glu(EDANS)NH2, One or more of MCA and Abz, optionally, the fluorescent group is a FITC group, optionally, the fluorescent peptide is a Y39-1 fluorescent peptide, and the sequence is Fitc-GKTKEGVLYVGSKTK.

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