Devices, methods and uses for modulating adenosine release in vivo

Abstract

The present invention relates to an apparatus, method and use for regulating the release of adenosine in a living body. The apparatus includes a stimulus generator for generating a stimulus of a specific frequency. By applying the apparatus of the present invention to a living body, the adenosine concentration in the brain of the living body can be effectively increased, thereby providing an effective method for improving sleep disorders.

Description

[Technical field]

[0001] The present invention relates to the field of adenosine modulation, and in particular to devices, methods and uses for modulating the release of adenosine in vivo. [Background technology]

[0002] Adenosine and its derivatives are widely present in nature and play an essential role in regulating various biological behaviors. In 1997, McCharty's team first reported in Science that extracellular adenosine concentrations change with differences in the sleep and wakefulness states of the organism, and that accumulation of adenosine concentrations increases sleep pressure, leading to insomnia. However, until now, the development of adenosine-based antiinsomnia drugs has not progressed substantially because adenosine receptors are highly expressed in tissues and organs throughout the body, with significant side effects, low specificity, and agonist desensitization and resistance.

[0003] As revealed by the 2021 survey report of the China Sleep Research Society, currently, about 40% of the total population of China suffers from sleep disorders to different degrees. Chronic and severe insomnia significantly impairs patients' work efficiency and mental and physical health, and the losses caused by various accidents and incidents due to sleep disorders reach hundreds of billions of yuan each year. Chronic insomnia is also an important trigger of many neuropsychiatric diseases. With the rapid development of the Chinese economy, the occurrence of sleep disorders is becoming more common and younger.

[0004] In current clinical practice, insomnia is mainly treated with medication, but there are serious problems such as persistent drug use, drug dependency and peripheral side effects. In addition to medication, insomnia can also be treated with cognitive behavioral therapy and physical therapy using strong light therapy. Cognitive behavioral therapy has limitations, takes a long time to be effective, and strong light therapy is less effective. Summary of the Invention [Problem to be solved by the invention]

[0005] The technical problem that the present invention is intended to solve is that there is currently no safe and effective method for increasing adenosine levels in the body or for precisely improving sleep disorders. [Means for solving the problem]

[0006] In order to solve the above technical problems, the present inventors have discovered that flash stimulation efficiently increases the adenosine concentration in the brain, thereby providing an effective measure for improving sleep disorders.

[0007] Specifically, the present invention proposes the following technical solutions. In one aspect of the invention there is provided an apparatus for modulating the release of adenosine in vivo comprising a stimulus generator for generating audio at 1-100 Hz and / or a stimulus generator for generating flashing and / or full screen visual stimuli at 1-100 Hz.

[0008] The apparatus according to the present invention, wherein the stimulus generator comprises a visual stimulus generator, such as a flash, and / or an audio stimulus generator.

[0009] Optionally, said visual generator emits visual stimuli at 20-80 Hz.

[0010] Optionally, the frequency of the visual stimulus includes one or more of: 20 Hz, 25 Hz, 30 Hz, 35 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz, 60 Hz, 65 Hz, 70 Hz, 75 Hz, 80 Hz.

[0011] Optionally, the frequency of the visual stimulus comprises 30-60 Hz.

[0012] Optionally, the frequency of the visual stimulus includes one or more of: 30 Hz, 35 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz, 60 Hz.

[0013] Optionally, the frequency of the visual stimulus comprises 35-45 Hz.

[0014] Optionally, the frequency of the visual stimulus includes one or more of 35 Hz, 40 Hz and 45 Hz.

[0015] Optionally, the frequency of said visual stimulus is 40 Hz.

[0016] Optionally, the illuminance of the white light of said visual stimulus is 2000 lux or more.

[0017] Optionally, the wavelength of the monochromatic light of the visual stimulus is between 480 and 530 nm.

[0018] Optionally, the duty ratio of the visual stimulation is 20% to 80%, preferably 30% to 70%, alternatively 40% to 70%, alternatively 45% to 55%.

[0019] Optionally, the duty ratio of the visual stimulation is one or more selected from 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% and 80%.

[0020] Optionally, the visual stimulus has a duty cycle of 50%.

[0021] The apparatus of the present invention, wherein the visual stimulus generator comprises a lamp and / or a display screen.

[0022] Optionally, the lamp comprises a CCFL lamp, an LED lamp and / or an OLED lamp.

[0023] Optionally, said display screen comprises a CCFL display screen, an LED display screen and / or an OLED display screen.

[0024] Optionally, said lamp emits white and / or monochromatic light.

[0025] Optionally, the monochromatic light comprises one or more of ultraviolet light, blue light, green light, red light and near infrared light, preferably blue light and / or green light.

[0026] Optionally, the central wavelength of the ultraviolet light is 315 to 400 nm, for example 320 to 395 nm, or 325 to 390 nm, or 330 to 385 nm, preferably 380 nm.

[0027] Optionally, the central wavelength of the blue light is 450 to 490 nm, for example 455 to 488 nm, or 460 to 485 nm, preferably 480 nm.

[0028] Optionally, the central wavelength of the green light is 500 to 560 nm, for example, 505 nm to 555 nm, alternatively 510 nm to 550 nm, alternatively 515 nm to 545 nm, alternatively 520 nm to 540 nm, alternatively 525 nm to 545 nm, alternatively 530 nm to 535 nm, preferably 530 nm.

[0029] Optionally, the central wavelength of the red light is 600 to 680 nm, for example, 595 to 675 nm, or 590 to 670 nm, or 585 to 665 nm, or 580 to 650 nm, preferably 650 nm.

[0030] Optionally, the central wavelength of the near infrared radiation is 780 to 1100 nm, for example 790 to 1090 nm, alternatively 800 to 1080 nm, alternatively 810 to 1070 nm, alternatively 820 to 1060 nm, alternatively 830 to 1050 nm, alternatively 840 to 1040 nm, alternatively 850 to 1030 nm, and preferably 850 nm.

[0031] Optionally, the illuminance of the white light is 2 to 6000 lux.

[0032] Optionally, the monochromatic light has an illuminance of 2-500 lux.

[0033] Optionally, the illuminance of the white light emitted by the lamp is 500-3000 lux.

[0034] 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.

[0035] Optionally, said display screen displays a diffraction grating image.

[0036] Optionally, the contrast of the diffraction grating image is between 10% and 100%.

[0037] Optionally, the grating image comprises one or more of a moving grating image, a blinking grating image, a blinking raster image, and an inverted grating image.

[0038] Optionally, the contrast of the moving grating is between 10% and 100%.

[0039] Optionally, the orientation of the moving grating is between 0 and 180°.

[0040] Optionally, the spatial frequency of the moving grating image is 0.05 to 30 cycles / deg, for example, 0.05 to 10 cycles / deg.

[0041] Optionally, the moving rate of the moving grating image is between 1 and 10 Hz.

[0042] Optionally, the contrast of said inverse grating is between 10 and 100%.

[0043] Optionally, the inversion frequency of the inversion grating is between 20 and 60 Hz, or between 30 and 50 Hz, preferably 40 Hz.

[0044] Optionally, the spatial frequency of the inverse grating is between 0.1 and 30 cpd, or 0.1 cpd.

[0045] Optionally, the contrast of the inverse grating is 10%-100%, 70-90%, preferably 80%.

[0046] Optionally, the average luminance of the inverted grating is 100 to 500 cd / m 2 , or 140~180cd / m 2 , or 150~170cd / m 2 , or 160 cd / m 2 It is.

[0047] Optionally, the contrast of the blinking raster image is between 10% and 100%.

[0048] Optionally, the blinking raster image has a frequency between 1 and 100 Hz.

[0049] Optionally, the flashing raster image has a raster size of 0.5 to 10 deg.

[0050] A device according to the invention, including a mobile phone, a tablet, or a portable wearable device.

[0051] Optionally, the portable wearable device is selected from glasses, an eye mask, a face mask, a hat, a headband, a helmet, a bracelet, or a watch, and optionally, the glasses comprise VR glasses.

[0052] The device described herein, wherein the portable wearable device includes a frame, and optionally, the portable wearable device further includes a mirror leg or a headband.

[0053] Optionally, a lamp is provided at an edge of a lens surrounded by said frame, and optionally said lamp comprises a CCFL lamp, an LED lamp and / or an OLED lamp.

[0054] Optionally, a display screen is located at the center of the lens surrounded by said frame, and optionally said display screen comprises a CCFL display screen, an LED display screen and / or an OLED display screen.

[0055] The apparatus according to the present invention, wherein the lamps include monochromatic lamps and white light lamps.

[0056] Optionally, the illuminance of the monochromatic lamp is 2-500 lux.

[0057] Optionally, the illuminance of the white light lamp is 2-6000 lux.

[0058] Optionally, the lamps include a blue light lamp, a green light lamp, a red light lamp and a white light lamp.

[0059] The apparatus of the present invention, wherein the display screen displays a grating image, the grating image including a vertical motion grating, a horizontal motion grating, and / or a blinking raster.

[0060] Optionally, the parameters of the vertical motion grating are 0° orientation, optionally a spatial frequency of the vertical motion grating is 0.2 cpd, optionally a temporal frequency of the vertical motion grating is 1 Hz, and optionally a contrast of the vertical motion grating is 50%.

[0061] Optionally, the parameters of the horizontal motion grating are oriented at 90 degrees, optionally a spatial frequency of the horizontal motion grating is 0.2 cpd, optionally a temporal frequency of the horizontal motion grating is 1 Hz, and optionally a contrast of the horizontal motion grating is 50%.

[0062] In another aspect of the invention there is further provided the use of any of the above devices, including for promoting the release of adenosine in vivo.

[0063] Optionally, said use comprises use to increase the concentration of adenosine in an organism.

[0064] Optionally, said use comprises use for increasing extracellular adenosine concentration in neurons.

[0065] Optionally, the use includes use for treating a neurological disease, a cardiovascular disease and / or an ophthalmic disease.

[0066] Optionally, said nervous system disease comprises Parkinson's disease, depression, anxiety, dementia, Alzheimer's disease, post-traumatic stress disorder, hyperactivity disorder, autism and / or attention deficit in children.

[0067] Optionally, said cardiovascular disease comprises coronary heart disease and / or arrhythmia.

[0068] Optionally, said ophthalmic disease comprises myopia, strabismus and amblyopia, and age-related retinopathy.

[0069] Optionally, said use includes use for improving a sleep disorder. Optionally, improving a sleep disorder with visual stimulation of 20-80 Hz. Optionally, improving a sleep disorder with visual stimulation of 30-60 Hz. Optionally, improving a sleep disorder with visual stimulation of 35-45 Hz. Optionally, improving a sleep disorder with visual stimulation of 40 Hz.

[0070] In another aspect of the present invention, there is also provided a method of modulating the release of adenosine in vivo, comprising the steps of: Any of the above devices of the present invention are used to deliver 1-100 Hz stimulation to a subject, said delivery being non-invasive. Optionally, said stimulation comprises visual and / or audio stimulation.

[0071] Optionally, the frequency of the visual stimulus is between 1 and 100 Hz, and optionally, the frequency of the visual stimulus is between 20 and 80 Hz.

[0072] Optionally, the frequency of the visual stimulus includes one or more of: 20 Hz, 25 Hz, 30 Hz, 35 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz, 60 Hz, 65 Hz, 70 Hz, 75 Hz and 80 Hz.

[0073] Optionally, the frequency of the visual stimulus comprises 30-60 Hz.

[0074] Optionally, the frequency of the visual stimulus includes one or more of: 30 Hz, 35 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz and 60 Hz.

[0075] Optionally, the frequency of the visual stimulus comprises 35-45 Hz.

[0076] Optionally, the frequency of the visual stimulus includes one or more of 35 Hz, 40 Hz and 45 Hz.

[0077] Optionally, the frequency of said visual stimulus is 40 Hz.

[0078] Optionally, the illuminance of the white light of said visual stimulus is 2000 lux or more.

[0079] Optionally, the wavelength of the monochromatic light of the visual stimulus is between 480 and 530 nm.

[0080] Optionally, the duty ratio of the visual stimulation is 20% to 80%, preferably 30% to 70%, alternatively 40% to 70%, alternatively 45% to 55%.

[0081] Optionally, the duty ratio of the visual stimulation is one or more selected from 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% and 80%.

[0082] Optionally, the visual stimulus has a duty cycle of 50%.

[0083] The method according to the present invention, wherein the visual stimulus comprises a flash stimulus and / or an image stimulus.

[0084] Optionally, said flash comprises white light and / or monochromatic light.

[0085] Optionally, the image comprises a diffraction grating image.

[0086] The method according to the present invention, wherein the monochromatic light comprises one or more of ultraviolet light, blue light, green light, red light, and near-infrared light, and is preferably blue light and / or green light.

[0087] Optionally, the central wavelength of the ultraviolet light is 315-400 nm, optionally, the central wavelength of the blue light is 450-490 nm, optionally, the central wavelength of the green light is 500 nm-560 nm, optionally, the central wavelength of the red light is 600-680 nm, and optionally, the central wavelength of the near-infrared light is 780-1100 nm.

[0088] Optionally, the central wavelength of the ultraviolet light is 320-395 nm, alternatively 325-390 nm, alternatively 330-385 nm.

[0089] Optionally, the central wavelength of the blue light is 455 to 488 nm, or 460 to 485 nm.

[0090] Optionally, the central wavelength of the green light is between 505nm and 555nm, alternatively between 510nm and 550nm, alternatively between 515nm and 545nm, alternatively between 520nm and 540nm, alternatively between 525nm and 545nm, alternatively between 530nm and 535nm.

[0091] Optionally, the central wavelength of the red light is 595-675nm, alternatively 590-670nm, alternatively 585-665nm, alternatively 580-650nm.

[0092] Optionally, the central wavelength of the near infrared radiation is 790 to 1090 nm, alternatively 800 to 1080 nm, alternatively 810 to 1070 nm, alternatively 820 to 1060 nm, alternatively 830 to 1050 nm, alternatively 840 to 1040 nm, or alternatively 850 to 1030 nm.

[0093] Optionally, the ultraviolet light has a central wavelength of 380 nm, the blue light has a central wavelength of 480 nm, the green light has a central wavelength of 530 nm, the red light has a central wavelength of 650 nm, and the near infrared light has a central wavelength of 850 nm.

[0094] Optionally, the illuminance of the white light is 2 to 6000 lux.

[0095] Optionally, the monochromatic light has an illuminance of 2-500 lux.

[0096] Optionally, the illuminance of the white light emitted by the lamp is 500-3000 lux, and 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.

[0097] The method according to the present invention, wherein the contrast of the raster image is between 10% and 100%.

[0098] Optionally, the raster image comprises one or more of a moving grating image, a blinking grating image, a blinking raster image, and an inverted grating image.

[0099] Optionally, the contrast of the moving grating is between 10% and 100%.

[0100] Optionally, the orientation of the moving grating is between 0 and 180°.

[0101] Optionally, the spatial frequency of the moving grating image is between 0.05 and 30 cycles / degree.

[0102] Optionally, the moving rate of the moving grating image is between 1 and 10 Hz.

[0103] Optionally, the contrast of the inverse grating is between 10% and 100%.

[0104] Optionally, the frequency of the inverse grating is 20-60 Hz or 30-50 Hz, preferably 40 Hz.

[0105] Optionally, the spatial frequency of the inverse grating is between 0.1 and 30 cpd, or 0.1 cpd.

[0106] Optionally, the contrast of the inverse grating is 10%-100%, 70-90%, preferably 80%.

[0107] Optionally, the average luminance of the inverted grating is between 100 and 500 cd / m 2 , or 140~180cd / m 2 , or 150~170cd / m 2 , or 160 cd / m 2 It is.

[0108] Optionally, the contrast of the blinking raster image is between 10% and 100%.

[0109] Optionally, the blinking raster image has a frequency between 1 and 100 Hz.

[0110] Optionally, the grid size of the blinking raster image is 0.5 to 10 degrees.

[0111] Another aspect of the invention provides a method for assessing the therapeutic effect of a drug or medical device, comprising analyzing changes in adenosine concentration in an organism.

[0112] The evaluation method according to the present invention, wherein the drug or medical device is used for treating a nervous system disease, a cardiovascular disease, and / or an improvement in a sleep disorder.

[0113] Optionally, said nervous system disease comprises Parkinson's disease, depression, anxiety, dementia, Alzheimer's disease, post-traumatic stress disorder, hyperactivity disorder, autism and / or attention deficit in children.

[0114] Optionally, said cardiovascular disease comprises coronary heart disease and / or arrhythmia.

[0115] Optionally, the evaluation method according to the present invention comprises analyzing changes in extracellular adenosine concentration of neurons.

[0116] Beneficial effects of the present invention include the following: (1) the present invention provides a novel strategy for modulating the release of adenosine, which has the potential advantages of being non-invasive, having fewer side effects, being better tolerated, and being more effective; (2) the present invention provides a novel strategy for improving sleep disorders, in which adenosine-induced sleep promotion has a relatively small range of action on the brain nerve center, is mild in effect, is relatively safe, and has a low rate of central side effects; and (3) The present invention provides a portable, wearable device that is convenient for subjects to use anytime, anywhere. [Brief description of the drawings]

[0117] [Figure 1] 1 shows the facilitatory effect of 40 Hz flash illumination on the total concentration of adenosine in the primary visual cortex (V1) in the visual cortex, as analyzed by ultra-performance liquid chromatography (UPLC) in Example 1. [Diagram 2] In Example 1, the effect of different frequencies of flash stimulation on adenosine release from the V1 region is shown. A is a schematic diagram of an experiment to detect adenosine signals in the visual cortex V1 region after flash stimulation, B shows that a 40Hz flash can effectively induce gamma oscillations in the visual cortex V1 region, C shows that positive neurons in the visual cortex V1 region (Parvalbumin, PV) by optogenetic activation (40Hz) effectively increase the release of adenosine, and D shows a curve corresponding to the time-adenosine release amount in the visual cortex V1 region under flash stimulation of different frequencies (20Hz, 40Hz, 80Hz) (n=3-5 / group). [Diagram 3]Example 2 shows the effect of flash stimulation on the concentrations of the neuromodulators serotonin and acetylcholine in the primary visual cortex (V1) brain region. [Figure 4] 1 shows that the increase in adenosine release by 40 Hz flash is brain region specific in Example 3. A shows time-adenosine concentration change curves of eight brain regions including the basal forebrain (BF), visual cortex V1 region, nucleus accumbens (NAc), ventral tegmental nucleus (VTA), superior colliculus (SC), medial prefrontal cortex (mPFC), hippocampus (HPC), and dorsomedial striatum (DMS) after 30 minutes of stimulation with 40 Hz flash. [Diagram 5] In Example 4, we show the increase in extracellular adenosine specificity in the visual cortex by different modes of visual stimulation. A is a time-concentration curve of adenosine release in the visual cortex V1 region for flash stimulation at different wavelengths at 40 Hz; B shows that visual stimulation with a flashing light rake (inverted grating) significantly increases adenosine release in the visual cortex V1 region (n=3-5 for each group A-C); C is a time-concentration curve of adenosine release in the visual cortex V1 region for different motion grating visual stimulation modes. The example of the circular graph shows that adenosine release in the visual cortex V1 region increases rapidly with stimulation in the vertical orthogonal inhibitory motion grating stimulation mode; the example of the square graph shows that adenosine release in the visual cortex V1 region increases slowly with stimulation in the full-screen 90° azimuth motion grating stimulation mode; and Random shows that adenosine release does not increase in the full-screen random azimuth motion grating stimulation mode (shaded area is visual stimulation time, n=3 for each group). [Figure 6] Example 5 shows the effect of 40 Hz strobe flashes of different duty ratios on extracellular adenosine in the visual cortex. [Figure 7]Example 6 shows the effect of 40 Hz strobe flashes of different light intensities on extracellular adenosine in the visual cortex. [Figure 8] In Example 6, the effects of 40 Hz strobe flashes of different light wavelengths on extracellular adenosine in the visual cortex are shown. [Figure 9] In Example 7, 40 Hz significantly reduces nighttime locomotor activity in mice via adenosine signals. A to C are time-locomotor activity curves of mice at night after 30 minutes of flashing with normal white light, 20 Hz, 40 Hz, and 80 Hz, D to F are statistical analysis diagrams of mouse locomotor activity in the first 7 hours at night after 30 minutes of flashing with normal white light, 20 Hz, 40 Hz, and 80 Hz, G is the intraperitoneal injection of the equilibrate nucleotide transporter (ENT) inhibitor dipyridamole (dipyr, 15 mg / kg) to block the locomotor inhibitory effect of 40 Hz flash in CD73-KO mice, and H is the intraperitoneal injection of the adenosine A1 receptor activity antagonist DPCPX (6 mg / kg) to block the locomotor inhibitory effect of 40 Hz flash (n=8 / group, *p<0.05, **p<0.01 vs. normal white light group). [Figure 10] This shows that 40Hz flash significantly increased the amount of nighttime sleep in mice in Example 8. A-B are schematic diagrams of the electroencephalogram and electromyogram spectra of mice at night after receiving light treatment in the last 30 minutes of the daytime, C is a time-sleep amount correspondence curve showing that 40Hz flash significantly increased the amount of nighttime sleep in mice, D is a statistical diagram of the amount of sleep in the first 3 hours of the nighttime phase after receiving light treatment, E is the distribution of nighttime sleep energy spectral density of mice after receiving light treatment, showing that 40Hz flash increased the amount of sleep in mice and did not affect the spectral energy density (n=7, ***p<0.01, ***p<0.001, vs. normal white light group), and F-G show that 20Hz and 80Hz flash treatments did not significantly increase the amount of sleep in mice. [Figure 11]In Example 8, it is shown that adenosine injection into the visual cortex concentration-dependently increases total sleep in mice. [Figure 12] In Example 9, a distribution diagram of resistance scores is shown. [Figure 13] In Example 10, 40 Hz flashes show improvement on multiple sleep parameters including sleep latency, total sleep time, and wake time after sleep onset in patients with chronic insomnia. [Figure 14] In Example 11, it was shown that after plantar electric shock, astrocyte reactivity was significantly reduced by 40% compared to the control side (AAV2 / 9-GfaABC1D-mCherry), indicating that the AAV2 / 9-GfaABC1D-mCherry-hPMCA2w / b virus can reliably reduce astrocyte activity. [Figure 15] Example 11 shows the effect of 40 Hz flash stimulation on the extracellular adenosine concentration in astrocytes. [Figure 16] Example 11 shows the effect of 40 Hz flash stimulation on neuronal extracellular adenosine concentration. [Figure 17] 13 shows a schematic diagram of a portable visual stimulation device in Example 12. [Figure 18] 1 is a schematic diagram of the moving grating stimulation mode. The grating is made of alternating black and white stripes that move in the direction of the arrow in the figure. [Figure 19] Schematic diagram of inverse grating image change: the local light and dark of the grating (shown by the dotted lines) alternate with a frequency of 40 Hz, and the screen refresh rate is 120 Hz. [Figure 20] Schematic diagram of a blinking diffraction grating. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0118] As described above, the object of the present invention is to provide a device for regulating the release of adenosine in vivo, in order to solve the technical problem that there is still no safe and effective method for increasing adenosine concentration in vivo or precisely improving sleep disorders.

[0119] The technical solution of the present invention is described below clearly and completely. Obviously, based on the specific embodiments in the present invention, all other embodiments that can be obtained by those skilled in the art without creative labor belong to the protection scope of the present invention.

[0120] It should be noted that the terminology used herein is not intended to limit the exemplary embodiments according to the present invention, but is used only to describe particular embodiments.

[0121] Cold Cathode Fluorescent Lamps (CCFLs) have the advantages of high output and high brightness and are widely used in the fields of monitors and lighting.

[0122] A light emitting diode (LED) is a commonly used light emitting device.

[0123] Organic light emitting diodes (OLEDs) belong to the category of current-type organic light emitting devices.

[0124] The present invention provides a device for regulating the release of adenosine in vivo, comprising a stimulus generator for generating an audio signal of 1-100 Hz and / or for generating a flash stimulus and / or a full-screen visual stimulus of 1-100 Hz. The stimulus generator may, for example, comprise a flash stimulus (which may be emitted by a strobe flash or a grating device) generator, a visual stimulus generator and / or an audio stimulus generator. Optionally, the visual stimulus generator emits a visual stimulus of 20-80 Hz, for example, may emit a visual stimulus of 30-60 Hz, for example, may emit a visual stimulus of 35-45 Hz, for example, may emit a visual stimulus including one or more of 20 Hz, 25 Hz, 30 Hz, 35 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz, 60 Hz, 65 Hz, 70 Hz, 75 Hz and 80 Hz. Optionally, the frequency of the visual stimulus is 40 Hz. Optionally, the illuminance of the white light of the visual stimulus is 2000 lux or more, optionally, the wavelength of the monochromatic light of the visual stimulus is 480-530 nm, and optionally, the duty ratio of the visual stimulus is 20%-80%, preferably 30%-70%, or 40%-70%, or 45-55%. Optionally, the duty ratio of the visual stimulus is one or more selected from 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, and 80%, and optionally, the duty ratio of the visual stimulus is 50%.

[0125] The generator for providing the visual stimulus includes a lamp and / or a display screen, optionally wherein the lamp includes a CCFL lamp, an LED lamp and / or an OLED lamp, and optionally wherein the display screen includes a CCFL display screen, an LED display screen and / or an OLED display screen.

[0126] According to the device provided by the present invention, the stimulus generator can emit white light and / or monochromatic light, and optionally, the monochromatic light includes one or more of ultraviolet light, blue light, green light, red light and near infrared light, and optionally, the central wavelength of the ultraviolet light is 315-400 nm, or 320-395 nm, or 325-390 nm, or 330-385 nm, preferably 380 nm, and the blue light is 100 nm or more. The central wavelength of the green light is 500 to 560 nm, 505 to 555 nm, 510 to 550 nm, 515 to 545 nm, 520 to 540 nm, 525 to 545 nm, 530 to 535 nm, and preferably 530 nm. The wavelength is 600 to 680 nm, or 595 to 675 nm, or 590 to 670 nm, or 585 to 665 nm, or 580 to 650 nm, and preferably 650 nm. The central wavelength of the near infrared ray is 780 to 1100 nm, or 790 to 1090 nm, or 800 to 1080 nm, or 810 to 1070 nm, or 820 to 1060 nm, or 830 to 1050 nm, or 840 to 1100 nm. Optionally, the illuminance of the white light is 2-6000 lux, and the illuminance of the monochromatic light is 2-500 lux. Optionally, the brightness of the white light emitted by the lamp is 500-3000 lux. Optionally, the brightness of the white light emitted by the lamp is one or more of 500 lux, 1000 lux, 2000 lux and 3000 lux.

[0127] Different colors and wavelengths of light provide different stimulating effects and can be used to treat different diseases by adjusting parameters such as the color, wavelength, and frequency of the light emitted by the device.

[0128] According to the apparatus provided by the present invention, the stimulus generator is capable of generating a grating image stimulus; Optionally, the contrast of the diffraction grating image is between 10% and 100%; Optionally, the grating image comprises one or more of a moving grating image, a blinking grating image, a blinking raster image, and an inverted grating image; Optionally, the contrast of the moving grating image is between 10% and 100%; Optionally, the orientation of the moving grating is between 0 and 180°; Optionally, the spatial frequency of the moving grating image is 0.05 to 30 cycles / deg, for example, 0.05 to 10 cycles / deg; Optionally, the moving speed of the moving grating image is 1-10 Hz.

[0129] Optionally, the motion grating may include a vertical motion grating or a horizontal motion grating, the parameters of the vertical motion grating are 0° orientation, 0.2cpd spatial frequency, 1Hz temporal frequency, and 50% contrast, and the parameters of the horizontal motion grating are 90° orientation, 0.2cpd spatial frequency, 1Hz temporal frequency, and 50% contrast. Optionally, the blinking raster image contrast is 10%-100%, optionally, the blinking raster image frequency is 1-100Hz, and optionally, the blinking raster image grid size is 0.5-10deg.

[0130] As shown in Figures 17 to 19, the present invention provides different gratings. Figure 17 is a schematic diagram of the moving grating stimulation mode provided by the present invention, and the variable parameters are 100 to 500 candelas per square meter (cd / m 2 ), orientation (0–180°), spatial frequency (0.2–30 cycles / degree, i.e., cpd, width of a single grating), temporal frequency (1–10 Hz, speed of grating movement), contrast (1–100%), size of the stimulus field (0–30° or full-screen stimulation), and orthogonal stimulation (central receptive field 5°, peripheral receptive field 15°).

[0131] FIG. 18 is a schematic diagram of the inverted grating stimulation mode provided by the present invention, and the variable parameters are: 100-500 candela / meter squared (cd / m 2 ), orientation (0–180°), spatial frequency (0.1–30 cycles / degree, i.e., cpd, the width of a single grating), blinking frequency (1–100 Hz), contrast (1–100%), and size of the stimulus field (0–30° or full-screen stimulation).

[0132] FIG. 19 is a schematic diagram of the blinking raster stimulation mode provided by the present invention, in which the display screen appears in checkerboard raster mode, and the variable parameters include the length and width of a single black / white grid (1°-5°), contrast (1%-100%), and blinking frequency (1-100Hz). Among them, contrast is a measurement of the different luminance levels between the brightest white and the darkest black in the light and dark areas of an image, and the greater the range of difference, the higher the contrast (e.g., contrast 100%), and the smaller the range of difference, the lower the contrast (e.g., contrast 10%). The higher the contrast, the easier it is for our eyes to perceive.

[0133] The present invention provides a device for regulating the release of adenosine in vivo, including a mobile phone, a tablet, or a portable wearable device, where the portable wearable device can be, for example, glasses, eye masks, face masks, hats, headbands, helmets, bracelets, or watches, and optionally, the glasses include VR glasses. Optionally, the portable wearable device includes a frame, and optionally, the portable wearable device further includes a mirror leg or a headband, and optionally, a lamp is disposed at the edge position of a lens surrounded by the frame, and optionally, the lamp includes a CCFL lamp, an LED lamp, and / or an OLED lamp, and optionally, a display screen is disposed at the center position of a lens surrounded by the frame, and optionally, the display screen includes a CCFL display screen, an LED display screen, and / or an OLED display screen.

[0134] The devices provided by the present invention can be used to enhance adenosine release in vivo, for example to increase adenosine concentrations in vivo, and in particular to increase extracellular adenosine concentrations in neurons.

[0135] Adenosine and its receptors can be used to treat many diseases in the nervous system and cardiovascular diseases, and have been widely studied and known. Among them, the nervous system diseases include Parkinson's disease, depression, anxiety, dementia, Alzheimer's disease, post-traumatic stress disorder, hyperactivity disorder, autism and attention deficit in children, and the cardiovascular diseases include coronary heart disease and arrhythmia. Since the device according to the present invention can increase the adenosine concentration in the brain of a living body, it is expected that the device according to the present invention can be used to treat many diseases in the nervous system and cardiovascular diseases in the same way.

[0136] The device according to the present invention can be used to treat neurological, cardiovascular and / or ophthalmological diseases, optionally wherein the neurological diseases include Parkinson's disease, depression, anxiety, dementia, Alzheimer's disease, post traumatic stress disorder, hyperactivity disorder, autism and / or attention deficit in children, optionally wherein the cardiovascular diseases include coronary heart disease and / or arrhythmia, and optionally wherein the ophthalmological diseases include myopia, strabismus and amblyopia, age-related retinopathy, etc. The device provided by the present invention can be used to improve sleep disorders, optionally by visual stimulation of 20-80 Hz, optionally by visual stimulation of 30-60 Hz, optionally by visual stimulation of 35-45 Hz, optionally by visual stimulation of 40 Hz.

[0137] The present invention provides a method for modulating adenosine release in vivo, comprising the steps of: The device provided by the present invention is used to deliver a stimulus of 1-100 Hz to a subject, said delivery being non-invasive, and optionally said stimulus comprising a visual stimulus and / or an audio stimulus. Optionally, said visual stimulus has a frequency of 1-100 Hz, and optionally said visual stimulus has a frequency of 20-80 Hz, such as 30-60 Hz, e.g. 35-45 Hz, and said visual stimulus has a frequency comprising one or more of 20 Hz, 25 Hz, 30 Hz, 35 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz, 60 Hz, 65 Hz, 70 Hz, 75 Hz and 80 Hz. Optionally, said visual stimulus has a frequency of 40 Hz. Optionally, the illuminance of the white light of the visual stimulus is 2000 lux or more; optionally, the wavelength of the monochromatic light of the visual stimulus is 480 to 530 nm; optionally, the duty ratio of the visual stimulus is 10% or more; optionally, the duty ratio of the visual stimulus is 20% to 80%, preferably 30% to 70%, or 40% to 70%, or 45% to 55%; optionally, the duty ratio of the visual stimulus is one or more selected from 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% and 80%, and optionally, the duty ratio of the visual stimulus is 50%.

[0138] A method of regulating adenosine release in vivo according to the present invention, optionally wherein the visual stimulus comprises a flash stimulus and / or an image stimulus, optionally wherein the flash comprises white light and / or monochromatic light, and optionally wherein the image comprises a diffraction grating image. Optionally, the monochromatic light includes one or more of ultraviolet light, blue light, green light, red light, and near infrared light, and optionally, the ultraviolet light has a central wavelength of 315-400 nm, and optionally, the ultraviolet light has a central wavelength of 320-395 nm, or 325-390 nm, or 330-385 nm, and optionally, the blue light has a central wavelength of 450-490 nm, and optionally, the blue light has a central wavelength of 455-488 nm, or 460-485 nm, and the green light has a central wavelength of 500-560 nm, and optionally, the green light has a central wavelength of 505 nm-555 nm, or 510 nm-550 nm, or 515 nm-54 5 nm, or 520 nm to 540 nm, or 525 nm to 545 nm, or 530 nm to 535 nm; the central wavelength of the red light is 600 to 680 nm, and optionally the central wavelength of the red light is 595 to 675 nm, or 590 to 670 nm, or 585 to 665 nm, or 580 to 650 nm; the central wavelength of the near-infrared light is 780 to 1100 nm, and optionally the central wavelength of the near-infrared light is 790 to 1090 nm, or 800 to 1080 nm, or 810 to 1070 nm, or 820 to 1060 nm, or 830 to 1050 nm, or 840 to 1040 nm, or 850 to 1030 nm. Optionally, the central wavelength of the ultraviolet light is 380 nm, the central wavelength of the blue light is 480 nm, the central wavelength of the green light is 530 nm, the central wavelength of the red light is 650 nm, and the central wavelength of the near-infrared light is 850 nm, and optionally, the illuminance of the white light is 2-6000 lux, and the illuminance of the monochromatic light is 2-500 lux. Optionally, the illuminance of the white light of the light emitted by the lamp is 500-3000 lux, and optionally, the illuminance of the white light of the light emitted by the lamp includes one or more of 500 lux, 1000 lux, 2000 lux, and 3000 lux.Optionally, the contrast of the diffraction grating image is between 10% and 100%, and optionally, the diffraction grating image includes one or more of a moving diffraction grating image, a blinking diffraction grating image, a blinking raster image, and an inverted diffraction grating image. Optionally, the contrast of the moving diffraction grating is 10%-100%, optionally the orientation of the moving diffraction grating is 0-180°, optionally the spatial frequency of the moving diffraction grating image is 0.05-30 cycles / degree, and optionally the moving speed of the moving diffraction grating image is 1-10 Hz; Optionally, the flashing raster image contrast is 10%-100%, optionally, the flashing raster image frequency is 1-100Hz, optionally, the flashing raster image grid size is 0.5-10 degrees, and optionally, the method of regulating adenosine release in vivo of the present invention can increase extracellular adenosine concentration in neurons.

[0139] The present invention provides a method for evaluating the therapeutic effect of a drug or medical device, comprising analyzing changes in adenosine concentration in vivo (e.g., in brain regions such as the visual cortex (V1), basal forebrain (BF), superior colliculus (SC), and / or in cells or in neurons), the drug or medical device being used for treating a nervous system disease, for treating a cardiovascular disease, and / or for improving a sleep disorder, optionally wherein the nervous system disease comprises Parkinson's disease, depression, anxiety, dementia, Alzheimer's disease, post-traumatic stress disorder, hyperactivity disorder, autism and / or attention deficit in children, and optionally wherein the cardiovascular disease comprises coronary heart disease and / or cardiac arrhythmia.

[0140] The adenosine regulator and method for improving sleep disorders according to the present invention will be described below with reference to specific examples.

[0141] Working Example Example 1: Frequency-dependent increase in V1 adenosine concentration by 40 Hz flash stimulation

[0142] 1) Ultra-high performance liquid chromatography analysis demonstrated that exposure to a 40 Hz light flash significantly increased total adenosine concentration in V1. The total adenosine concentration (reported in the literature to contain 95% extracellular adenosine and 5% intracellular adenosine) in the visual cortex of mice was examined by ultra-high performance liquid chromatography. 6 mg / kg of dipyridamole (Dipyr), an adenosine transporter inhibitor, dissolved in 0.5% methylcellulose was intraperitoneally injected 30 minutes before light exposure, and 0.5% methylcellulose was administered to the control group. 30 minutes after injection, the blank control group and the dipyridamole group were irradiated with light of 3000 lux, duty ratio 50%, and strobe flash 40 Hz (hereinafter referred to as "40 Hz flash stimulus") for 30 minutes. Immediately after irradiation, samples were taken, weighed, and flash frozen in liquid nitrogen. The samples were processed, and a sharp peak was detected at about 2.0 by referring to the parameters of the standard substance, and the area under the curve at this point was taken as the adenosine concentration of the sample, and the adenosine concentration was finally obtained by correcting the quality of the sample.

[0143] The results, as shown in Figure 1, showed that the adenosine concentration increased significantly after flash stimulation (p=0.0245, n=7), dipyridamole could effectively suppress the total adenosine concentration inside and outside the cells, and the suppression of the total adenosine concentration inside and outside the cells by dipyridamole was effectively reversed after 30 minutes of flash irradiation at 40 Hz (p=0.0431, n=7). This indicated that 30 minutes of exposure to 40 Hz flash light could significantly increase the adenosine concentration in the V1 region, and the adenosine concentration could also be significantly increased in the dipyridamole administration group.

[0144] 2) Frequency Dependence In this experiment, a novel genetically encoded adenosine probe with high specificity and high time resolution was used to study adenosine dynamics changes in different brain regions at different frequencies. The working principle of this novel adenosine probe is to measure the change in extracellular adenosine concentration by the fluorescence intensity of green fluorescent protein when the cellular adenosine G protein-coupled receptor is activated. As shown in Figure 2A, the adenosine probe (AAV2 / 9-hsyn-GRABAdo1.0, Wuhan Privilege Brain Science and Technology Co., Ltd., PT-1348) and adenosine probe control group mutant (AAV2 / 9hsyn-GRABAdo-mut, Wuhan Privilege Brain Science and Technology Co., Ltd., PT-1348) virus control brains were stereotaxically injected into the brain region of the primary visual cortex (V1) of mice (bregma 3.6 μm, paragregma ±2.4 μm, depth -0.6 μm). The mice were returned to their cages for three weeks and then exposed to flash environments of 20 Hz, 40 Hz, and 80 Hz (lights on for 12.5 ms, lights off for 12.5 ms, i.e., duty ratio 50%, 3,000 lux, 60 watts) for 30 minutes, and the changes in adenosine concentrations before and after exposure to the flashes were compared.

[0145] The results are shown in Figure 2. As shown in Figure 2B, 40 Hz flash effectively induced gamma oscillations in the V1 region. As shown in Figure 2C, the change in adenosine concentration significantly increased after 40 Hz flash stimulation light irradiation. Figure 2D shows the time-adenosine release curves (n = 3-5 / group) in the V1 region under flash stimulation of different frequencies (20 Hz, 40 Hz, 80 Hz). The mice showed a slight increase in adenosine signals under 80 Hz flash stimulation, but no change in adenosine concentration was observed under 20 Hz. As a result, it was clear that the 40 Hz flash activated the release of adenosine from cells in the V1 region in a frequency-dependent manner. From the above, the release of extracellular adenosine may be related to a specific light frequency, which will be an important neurochemical basis in exploring visual stimulation to improve sleep disorders, cognitive disorders, etc.

[0146] Example 2: Flash stimulation specifically alters adenosine in the primary visual cortex (V1) brain region, but not the neuromodulators serotonin and acetylcholine

[0147] Neurotransmitters and neuromodulators are important intermediary molecules in the communication between neurons for effective information transmission and integration. To verify whether 40 Hz flash stimulation specifically alters adenosine in the primary visual cortex (V1) brain region, we combined a genetically encoded fluorescent probe with a fiber optic recording system to investigate the dynamic changes of serotonin (5-HT) and acetylcholine (ACh) under 40 Hz flash stimulation, respectively.

[0148] AAV9-hsyn-GRAB 5-HT3.5 and AAV9-hsyn-GRAB 5HT2.1-mut , AAV9-hsyn-GRAB ACh3.0 and AAV9-hSyn-GRAB ACh3.0-mut were injected into the left and right side of the mouse visual cortex, respectively (Figure 3A-B). After expressing the virus for 2 weeks, 40 Hz flash intervention was performed and the concentration changes of serotonin and acetylcholine were recorded in real time. As shown in Figure 3C-D, no significant changes were observed in 5-HT and Ach in the visual cortex (V1) after stimulation with 40 Hz flash.

[0149] Example 3: 40Hz 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.

[0150] In this experiment, a novel genetically encoded adenosine probe was used to detect adenosine release levels in brain regions such as the visual cortex (V1), basal forebrain (BF), nucleus accumbens (NAc), ventral tegmental area (VTA), superior colliculus (SC), medial prefrontal cortex (mPFC), hippocampus (HPC), and dorsal medial tegmental striatum (DMS). As shown in Figure 4, after 30 minutes of 40 Hz flash stimulation, adenosine signals were recorded, and the adenosine release levels in brain regions such as V1, SC, BF, mPFC, and HPC were significantly increased, whereas no increase in adenosine signals was detected in the control group injected with AAV2 / 9-hsyn-Mut virus (Wuhan Pivotal Brain Science and Technology Co., Ltd., PT-1348). In the brain regions where adenosine was elevated, the adenosine signal was strongest in brain regions related to visual information encoding, such as the V1 and SC brain regions, the adenosine signal in the BF brain region related to sleep-wakefulness was also significantly elevated, and the adenosine signal in brain regions related to cognition, such as the mPFC and HPC, was slightly elevated. These results clearly indicated that this 40Hz non-invasive flash stimulation mode can be used to enhance adenosine levels in sleep-related brain regions and regulate sleep.

[0151] Example 4: Increase in V1 adenosine release levels by different visual stimulation modes

[0152] 1) 40Hz flash stimulation As shown in Figure 5A, regarding the difference in adenosine release levels in area V1 under 40 Hz flash stimulation conditions with different wavelengths, only blue light with a wavelength of 480 nm and green light with a wavelength of 530 nm could significantly increase the adenosine concentration in area V1, while flash stimulation with other wavelengths (e.g., 380 nm, 650 nm, 850 nm) did not increase the adenosine concentration in area V1.

[0153] 2) Effect of moving grating As shown in Figure 5C, a horizontal motion grating stimulus was used (left side of Figure 18, arrows indicate the motion direction), and the stimulus parameters were: the grating was displayed full screen with high contrast (80%) so that the mouse could see the stimulus throughout the experiment (average luminance of the screen was 160 cd / m2). The horizontal motion grating had a spatial frequency of 0.2 cpd and a temporal frequency of 1-10 Hz (i.e., the motion speed of the grating). At the same time, we detected changes in adenosine concentration in the V1 area of ​​the mouse under the combined stimulation of a horizontal motion grating at about 5° in the center and a vertical motion grating at 15° in the periphery (center-periphery vertical motion grating, Figure 18).

[0154] The results showed that both types of motion gratings significantly increased V1 adenosine, but the increase was smaller for the horizontal motion grating than for the center-circumference vertical motion grating (Figure 5C, ΔF / F values ​​were 5% for the horizontal motion grating and 8% for the center-circumference vertical motion grating). This indicates that the wide-range grating stimulus has a significant effect on the production of extracellular adenosine.

[0155] 3) Effect of vertically inverted diffraction grating As shown in Figure 5B, a vertical inversion grating stimulus with a frequency of 40 Hz was used while keeping the light intensity constant. The stimulus parameters were a vertical sinusoidal inversion grating with a spatial frequency of 0.1 cpd, a grating with an inversion frequency of 40 Hz (i.e., alternating black and white gratings flashing, Figure 19), and the average light intensity did not change during a specific time period (gamma-corrected to remove screen luminance nonlinearity). The grating was a high-contrast (80%) full-screen (average screen luminance 160 cd / m 2 ) to ensure that the mouse could see the stimuli throughout the experiment.

[0156] As a result, the vertical inverted grating could significantly increase V1 adenosine, but the degree was lower than that of the 40 Hz flash stimulation (Figure 5B, ΔF / F value was 5% for the inverted raster, whereas it was 15-20% for the 40 Hz flash stimulation). This revealed that light intensity has an important effect on the production of extracellular adenosine.

[0157] Example 5. Effects of 40 Hz flash stimulation with different duty ratios on extracellular adenosine in the visual cortex

[0158] Considering the effect of the illuminance (total energy) of white flash stimulation on extracellular adenosine concentration, the total energy produced by a 40 Hz strobe flash (same illuminance 3000 lux) with different duty ratios (1%, 10%, 50%) is different. Therefore, we performed a 40 Hz strobe flash (same illuminance 3000 lux) stimulation for 30 minutes under conditions of different duty ratios (1%, 10%, 50%) and detected the effect on the adenosine level in the V1 region.

[0159] The results are shown in Figure 6. The peak ΔF / F of the increase in V1 adenosine level was 5 and 10 under the conditions of 1% and 10% duty cycles, respectively, which was lower than that of the stimulation with a duty cycle of 50% (peak ΔF / F was 25), indicating that the white flash stimulation with a duty cycle of 50% significantly increased the V1 adenosine level. Therefore, at the same illuminance (3000 lux), the flash stimulation with a frequency of 40 Hz increased the extracellular adenosine concentration in a duty cycle (1%, 10%, 50%)-dependent (specific) manner, and the 40 Hz strobe flash with a duty cycle of 50% was the most significant in increasing the V1 adenosine concentration.

[0160] Example 6: Effects of 40Hz strobe flashes with different light intensities and wavelengths on extracellular adenosine in the visual cortex

[0161] (1) Effect of light illuminance Using 40Hz white flash stimuli with different light illuminances (50Lux, 500Lux, 1000Lux, 2000Lux and 3000Lux), we studied the effect of different light illuminances on adenosine production in the visual cortex. As shown in Figure 7, the adenosine level induced by 40Hz flashing with low light illuminance (500Lux) was low, and the peak ΔF / F of the adenosine level increased by 40Hz white flash stimuli with high light illuminance (3000Lux) was 20, indicating that all 40Hz flash stimuli with light illuminances of 2000Lux or higher significantly increased the adenosine concentration in the V1 region. From the above, the finding that white light from different light illuminances and vertical rasters increased adenosine in the V1 region clearly indicated that light illumination intensity is an important factor in generating extracellular adenosine concentration in the primary visual cortex of the brain.

[0162] (2) Effect of light wavelength As shown in Figure 8, under 40Hz monochromatic flash stimulation with a light illuminance of 500 Lux, blue light with a wavelength of 480 nm and green light with a wavelength of 530 nm could significantly increase the adenosine concentration in the V1 region, but flash stimulation with other wavelengths (380 nm, 650 nm, 850 nm, etc.) did not increase the adenosine concentration in the V1 region. Notably, the kinetics of the increase in V1 adenosine concentration by this single wavelength was basically similar (increasing and decreasing from 10 min, reaching a maximum concentration at 30 min, and lasting for 2 to 3 h).

[0163] Example 7: Suppression of nocturnal locomotor activity in mice by 40 Hz flash stimulation

[0164] In the pre-workout study, we found that 40 Hz flash stimulation could significantly increase the adenosine concentration in the BF area, and BF plays an important role in regulating sleep and wakefulness, so we speculated that 40 Hz flash could regulate the sleep rhythm. First, wild-type C57BL / 6J mice were irradiated with flashes of different frequencies for the last 30 minutes of daytime (19:30-20:00), and nighttime locomotor activity was monitored as an indirect reflection of the mouse's sleep-wake state. As shown in Figure 9, 30 minutes after 40 Hz flash stimulation, the mouse's locomotor activity was suppressed for up to 8 hours, while neither 20 Hz nor 80 Hz flash stimulation had a significant effect on the mouse's locomotor activity, indicating that flash stimulation frequency-specifically suppresses the mouse's nighttime locomotor activity. To further clarify the source of adenosine produced by 40 Hz flash stimulation, we first performed a 40 Hz light irradiation experiment using CD73-KO mice. As a result, 40 Hz flash stimulation still significantly reduced nocturnal locomotor activity in CD73-KO mice, indicating that the extracellular ATP metabolic pathway is not the main source of adenosine. Next, we found that when dipyridamole (Dipyr) 15 mg / kg, a nucleotide transporter inhibitor, was intraperitoneally injected into CD73-KO mice, the locomotor suppression effect of 40 Hz flash stimulation was completely abolished, suggesting that intracellular adenosine is transported out of cells via nucleotide transporters as the main mechanism of adenosine release by 40 Hz flash stimulation in this phenotype. Similarly, we found that 8-cyclopentyl-1,3-dipropyl-1H-purine-2,6(3H,7H)-dione (DPCPX), an adenosine A1 receptor antagonist, was also able to inhibit the locomotor suppression effect of 40 Hz flash stimulation by intraperitoneal injection of 6 mg / kg into CD73-KO mice.

[0165] Example 8: 30 minutes of 40 Hz flash stimulation significantly increases the amount of sleep in mice by enhancing extracellular adenosine signals in the cortex.

[0166] (1) 30 minutes of stimulation with 40 Hz flashes significantly increased the amount of sleep in mice. To verify the sleep regulation effect of 40Hz flash in mice, a polysomnography recording system was used to record the electroencephalogram and electromyogram of mice, and the sleep-wake status was accurately determined and statistically analyzed based on the characteristics of different sleep-wake phases. In this experiment, a self-control method was used. On the first day, mice were given normal white light for the last 30 minutes of the daytime, and then the amount of sleep at night was recorded and analyzed. On the second day, mice were given 40Hz flash light for the last 30 minutes of the daytime, and then the amount of sleep at night was recorded and analyzed. As shown in Figure 10A-E, by comparing the amount of sleep of mice at night for two days, we found that the amount of NREM in mice at night could be significantly increased after 40Hz flash, and the slow wave activity during NREM sleep was not affected. These results suggest that 40Hz flash is a non-invasive and excellent means of promoting physiological sleep.

[0167] (2) Flash stimulation significantly increased the amount of sleep in mice and was frequency-specific. We further demonstrated that the sleep-promoting effect of flash is frequency-specific. First, we recorded baseline EEG / EMG of mice exposed to normal light during the dark phase (7:00 pm to 7:00 am) on day 1, and then exposed mice to three different frequencies of flash stimulation (20 Hz, 40 Hz, 80 Hz; 3000 lux; duty ratio 50%) for 30 minutes during the last 30 minutes of the light exposure staircase (6:30 pm to 7:00 pm during the light exposure staircase from 7:00 am to 7:00 pm) on days 2, 9, and 16, and recorded EEG / EMG during the dark phase.

[0168] By comparing the amount of sleep during the dark period after each treatment, we found that 30 minutes of exposure to 40 Hz flashes produced a strong sleep-promoting effect in mice. This significant sleep-promoting effect occurred mainly during the first 2 hours. Importantly, 20 Hz and 80 Hz flash treatments did not significantly increase the amount of sleep (Figure 10F-G), demonstrating that the sleep-promoting effect of flashes is frequency-specific.

[0169] (3) Injection of adenosine into the visual cortex increased total sleep time in mice in a concentration-dependent manner. To elucidate whether increasing adenosine concentration in the visual cortex increases the amount of sleep in mice, adenosine was locally injected into the V1 cortex of mice. Administration cannulas were implanted into the bilateral V1 brain regions of C57BL / 6J mice, and after approximately 10 days of recovery from surgery, the mice were placed in sleep recording cages. After 3 days of habituation, artificial cerebrospinal fluid (ACSF), adenosine 1.5 nmol / side, or adenosine 4.5 nmol / side (administration order was random, injection volume was 2 μl / side) was microinjected into the V1 visual cortex at the onset of the dark phase on days 1, 4, and 7, respectively, and EEG / EMG were recorded.

[0170] When comparing the amount of sleep in mice after different treatments, as shown in Figure 11, the group administered 4.5 nmol adenosine / side showed a significant increase in non-rapid eye movement sleep during the dark phase, reaching 3 hours, compared to the control group. On the other hand, the group administered 1.5 nmol adenosine / side showed a tendency to promote sleep compared to the control group, but no significant difference was observed. This clearly shows that the sleep-promoting effect of local administration of adenosine to the visual cortex depends on the dosage or concentration, and the experiment also showed that in order to increase the total amount of sleep in mice, it is necessary to increase the local adenosine concentration in the visual cortex to a certain threshold.

[0171] These frequency-specific effects on adenosine production and sleep quantity confirmed that 40 Hz flash stimulation promotes sleep by enhancing extracellular adenosine signals in the cortex.

[0172] Example 9: 40 Hz flash stimulation is well tolerated and safe in healthy subjects

[0173] To advance the use of 40Hz flash stimulation in clinical trials, we first recruited 15 healthy volunteers to test the tolerance and safety of the 40Hz portable flash device in healthy subjects. In this test, blood pressure and electrocardiogram were measured at 8am the previous day without breakfast, and blood samples were also taken, and blood routine and liver and kidney function indicators were chemically tested. At 7:30am the next morning, the subjects were given a 30-minute flash with the 40Hz portable flash device, blood pressure and electrocardiogram were measured, and blood samples were also taken, and blood routine and liver and kidney function indicators were chemically tested, and a side effect questionnaire was completed. As shown in Figure 12, compared with the case of no flash, the 40Hz flash stimulation did not cause any significant adverse reactions in the sensory system such as the eyes and nose or the nerve center of the brain, indicating that the tolerance of the 40Hz flash stimulation and the portable device was good. No significant abnormal changes were observed in the related indicators in blood pressure and blood tests. These results showed that the 40Hz flash and the portable device have good tolerance and safety in healthy subjects. [Table 1] [Table 2] [Table 3]

[0174] Example 10: 40Hz flash stimulation improves several sleep parameters, including sleep latency, total sleep time, and wake time after sleep onset, in patients with chronic insomnia.

[0175] Purpose of the study: This study recruited patients with chronic insomnia and evaluated the therapeutic effect of 40 Hz flash stimulation on patients with chronic insomnia.

[0176] Study Protocol: In this study, patients who met the diagnostic criteria for chronic insomnia of the International Classification of Sleep Disorders (ICSD3) were recruited from the Second Affiliated Hospital of Wenzhou Medical University from February 2022 to December 2022, and the patients' sleep was recorded by polysomnography monitoring (PSG). This study was a self-controlled design, with baseline sleep recorded on the first night, 40 Hz flash stimulation (3000 lux, duty ratio 50%) was administered for 30 minutes on the second night, and sleep was recorded again afterwards. The primary endpoints were a significant shortening of sleep latency and a significant increase in sleep efficiency. Secondary endpoints were a significant extension of total sleep time and a significant reduction in wakefulness time and number of awakenings after sleep onset.

[0177] Results: Age 9.4±3.1 years, BMI 15.40 (14.50-17.90) kg / m 2 A total of 27 patients, including 14 women (51.85%), were included in the study.

[0178] As a result, 40 Hz flash stimulation treatment significantly reduced sleep latency (min) compared to the baseline period (18.5 (10-29.5) vs 32.50 (15-49.5), p = 0.0233) (Figure 13A). On the other hand, 40 Hz significantly prolonged total sleep time (min) (528 (502-560) vs. 459 (411.5-497), p = 0.0002) (Figure 13B) and wakefulness time after sleep onset (min) (35.5 (28-51) vs. 57.5 (40-128.5), p = 0.0012) (Figure 13C), and significantly increased sleep efficiency (%) (89.3 (84.8-82.5) vs. 79.6 (73.2-87.5), p = 0.0003) (Figure 13D). Rapid eye movement sleep (REM) sleep latency (min) was significantly decreased (139.5 (107-182.5) vs 199 (156.5-239.5), p=0.0004) (Figure 13F) and the percentage of REM sleep (20.2 (17-21.3) vs 17.4 (11-20.3), p=0.0038) (Figure 13I). There was no significant effect on the number of awakenings or the percentage of light and deep sleep phases in total sleep time (all p>0.05) (Figure 13E,G,H).

[0179] Study conclusion: In this self-controlled clinical study of patients with chronic insomnia, 40 Hz flash stimulation 30 minutes before going to bed significantly shortened sleep latency, significantly extended total sleep time, and significantly reduced the duration and number of awakenings after sleep onset. Therefore, 40 Hz flash stimulation 30 minutes before going to bed can significantly improve sleep onset and sleep maintenance in patients with chronic insomnia.

[0180] Example 11: Investigation of the main cellular source of extracellular adenosine production induced by 40 Hz flash stimulation

[0181] Under normal conditions, extracellular adenosine production may originate from astrocytes or neurons. First, astrocyte plasma membrane Ca 2+ We virally knocked down the PMCA2 pump to determine whether astrocytes were the primary cellular source of adenosine release stimulated by 40 Hz flash stimulation. hPMCA2w / b induced plasma membrane Ca 2+ A subtype of PMCA pump that constitutively transports cytoplasmic Ca 2+ It functions to push out Ca into astrocytes. 2+ We injected a 1:1 mixture of AAV2 / 9-GfaABC1D-mCherry-hPMCA2w / b and AAV2 / 9-ShortGFAP-Gcamp6 into the V1 region of mice and expressed the virus for 2 weeks. We then used an electric foot shock experiment (Figure 14B) to investigate the virus-mediated astrocyte membrane Ca2+ signaling. 2+ We verified the feasibility of knocking down the PMCA2 pump. The results showed that after plantar shock, astrocyte reactivity was significantly reduced by 40% compared to the control side (AAV2 / 9-GfaABC1D-mCherry), indicating that this virus can indeed reduce astrocyte activity (Figure 14C).

[0182] To investigate whether astrocytes promote the release of adenosine in 40Hz flashes, mice injected with AAV2 / 9-GfaABC1D-mCherry-hPMCA2w / b virus were co-injected with AAV2 / 9-GfaABC1D-mCherry-hPMCA2w / b and AAV2 / 9-hsyn-GRABAdo1.0 (1:1 mixture, Figure 15A) in the V1 region, and after expressing the virus for 2 weeks, 40Hz flash stimulation was performed and the change in adenosine concentration was recorded in real time. The experimental results showed that after 40Hz flash stimulation, the astrocyte activity in the V1 region of the mice was reduced, and the increase and change in adenosine concentration were characterized by the experimental group (cell membrane Ca 2+ PMCA2 knockout mice) and control groups (plasma membrane Ca 2+ The results were almost the same in both PMCA2-pumping and normal mice (Figure 15B-C), indicating that the increased extracellular adenosine by 40 Hz flash stimulation was not primarily derived from astrocytes.

[0183] To clarify that neurons are the main cellular source of extracellular adenosine levels induced by 40 Hz flash stimulation, neurons in the V1 region were specifically damaged with caspase-3 and changes in adenosine concentration during 40 Hz flash stimulation were measured. AAV2 / 9-hsyn-GRABAdo1.0 and rAAV-hSyn-taCasp3-T2A-TEVp were mixed 1:1 and injected into the V1 region of mice. After expressing the virus for 2 weeks, a significant decrease in the number of neurons in the V1 region was observed by fluorescent staining. Then, 40 Hz flash stimulation was performed and changes in adenosine concentration were recorded in real time. As a result, a significant increase in extracellular adenosine was observed during 40 Hz flash stimulation in control mice (with normal neurons in the V1 region), but no increase in extracellular adenosine was observed during 40 Hz flash stimulation in mice lacking neurons in the V1 region (Figure 16A-B). This demonstrated that neurons in the V1 region play an important regulatory role in increasing the concentration of extracellular adenosine under 40 Hz flash stimulation.

[0184] Example 12. Adenosine release regulating glasses

[0185] As shown in Figure 17, the main device components include a micro LED display screen, a housing, a flexible circuit board, an inner cover, and left and right side clips. The device is generally in the shape of glasses and mainly achieves the following two functions: (A) A set of LED lamps with different wavelengths is fixedly sandwiched between the housing and the inner cover of the glasses, and a flexible circuit board is used to distribute the periphery of the display screen along the edge of the frame, so that various combinations of flash stimuli such as different wavelengths, frequencies, light intensities, and times can be freely adjusted. (B) A micro LED display screen is arranged to cover the lens position of the glasses and is used to display optimal visual stimulation images based on visual stimulation-adenosine concentration-sleep change screening. These two modes are superior to commercially available visual stimulation devices with fixed flash intensity and frequency, greatly reducing the discomfort caused by simple flash stimulation, and for the first time opening up a new direction in the treatment of sleep disorders with visual stimulation images.

Claims

1. A device for modulating the release of adenosine in vivo comprising a stimulus generator for generating audio at 1-100 Hz and / or for generating flashing and / or full screen visual stimuli at 1-100 Hz.

2. the stimulus generator includes a visual stimulus generator and / or an audio stimulus generator; Optionally, said visual generator emits visual stimuli at 20-80 Hz; Optionally, a frequency of the visual stimulus comprises one or more of: 20 Hz, 25 Hz, 30 Hz, 35 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz, 60 Hz, 65 Hz, 70 Hz, 75 Hz, 80 Hz; Optionally, the frequency of the visual stimulus comprises 30-60 Hz; optionally, the frequency of the visual stimulus comprises one or more of 30 Hz, 35 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz, 60 Hz; Optionally, the frequency of the visual stimulus comprises 35-45 Hz; optionally, the frequency of the visual stimulus comprises one or more of 35 Hz, 40 Hz, and 45 Hz; Optionally, the frequency of the visual stimulus is 40 Hz; Optionally, the visual stimulus has a white light illuminance of 2000 lux or more; Optionally, the wavelength of the monochromatic light of the visual stimulus is between 480 and 530 nm; Optionally, the duty ratio of the visual stimulation is 20% to 80%, preferably 30% to 70%, alternatively 40% to 70%, alternatively 45% to 55%; Optionally, the duty ratio of the visual stimulation is one or more selected from the group consisting of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% and 80%; Optionally, a duty cycle of the visual stimulus is 50%.

3. the visual stimulus generator includes a lamp and / or a display screen; Optionally, the lamp comprises a CCFL lamp, an LED lamp and / or an OLED lamp; 3. The apparatus of claim 2, optionally wherein the display screen comprises a CCFL display screen, an LED display screen and / or an OLED display screen.

4. said lamp emits white and / or monochromatic light, Optionally, the monochromatic light comprises one or more of ultraviolet light, blue light, green light, red light and near infrared light, preferably blue light and / or green light; Optionally, the central wavelength of the ultraviolet light is 315-400 nm, alternatively 320-395 nm, alternatively 325-390 nm, alternatively 330-385 nm, preferably 380 nm; Optionally, the central wavelength of the blue light is 450-490 nm, alternatively 455-488 nm, alternatively 460-485 nm, preferably 480 nm; Optionally, the central wavelength of the green light is 500 to 560 nm, alternatively 505 nm to 555 nm, alternatively 510 nm to 550 nm, alternatively 515 nm to 545 nm, alternatively 520 nm to 540 nm, alternatively 525 nm to 545 nm, alternatively 530 nm to 535 nm, preferably 530 nm; Optionally, the central wavelength of the red light is 600-680 nm, alternatively 595-675 nm, alternatively 590-670 nm, alternatively 585-665 nm, alternatively 580-650 nm, preferably 650 nm; Optionally, the central wavelength of the near infrared radiation is 780 to 1100 nm, alternatively 790 to 1090 nm, alternatively 800 to 1080 nm, alternatively 810 to 1070 nm, alternatively 820 to 1060 nm, alternatively 830 to 1050 nm, alternatively 840 to 1040 nm, alternatively 850 to 1030 nm, preferably 850 nm; Optionally, the white light has an illuminance of 2 to 6000 lux, and the monochromatic light has an illuminance of 2 to 500 lux; Optionally, the illuminance of the white light emitted by the lamp is between 500 and 3000 lux; 4. The apparatus of claim 3, wherein optionally, the illuminance of the white light emitted by the lamp comprises one or more of 500 lux, 1000 lux, 2000 lux, and 3000 lux.

5. the display screen displays a diffraction grating image, and optionally, a contrast of the diffraction grating image is between 10% and 100%; Optionally, the grating image comprises one or more of a moving grating image, a blinking grating image, a blinking raster image, and an inverted grating image; Optionally, the contrast of the moving grating is between 10% and 100%, optionally the orientation of the moving grating is between 0 and 180°, optionally the spatial frequency of the moving grating image is between 0.05 and 30 cycle / deg, for example between 0.05 and 10 cycle / deg, and optionally the moving speed of the moving grating image is between 1 and 10 Hz; Optionally, the contrast of the inverse diffraction grating is 10-100%, optionally the inversion frequency of the inverse diffraction grating is 20-60 Hz or 30-50 Hz, preferably 40 Hz, optionally the spatial frequency of the inverse diffraction grating is 0.1-30 cpd or 0.1 cpd, optionally the contrast of the inverse diffraction grating is 10%-100%, 70-90%, preferably 80%, and optionally the average brightness of the inverse diffraction grating is 100-500 cd / m ２ , or 140 to 180 cd / m ２ , or 150 to 170 cd / m ２ , or 160 cd / m ２ and 5. The apparatus of claim 3 or 4, optionally, a contrast of the blinking raster image is 10-100%, optionally, a frequency of the blinking raster image is 1-100 Hz, and optionally, a raster size of the blinking raster image is 0.5-10 deg.

6. A device according to any one of claims 1 to 5, comprising a mobile phone, a tablet or a portable wearable device.

7. 7. The device of claim 6, wherein the portable wearable device is selected from glasses, an eye mask, a face mask, a hat, a headband, a helmet, a bracelet or a watch, and optionally, the glasses include VR glasses.

8. The portable wearable device includes a frame, and optionally, the portable wearable device further includes a mirror leg or a headband; Optionally, a lamp is provided at an edge of the lens surrounded by the frame, and optionally, the lamp comprises a CCFL lamp, an LED lamp, and / or an OLED lamp; 8. The apparatus of claim 7, optionally comprising a display screen disposed at the center of the framed lens, optionally comprising a CCFL display screen, an LED display screen and / or an OLED display screen.

9. The lamps include monochromatic lamps and white light lamps; Optionally, the monochromatic light lamp has an illuminance of 2-500 lux; and optionally, the white light lamp has an illuminance of 2-6000 lux; 10. The apparatus of claim 8, optionally wherein the lamps include a blue light lamp, a green light lamp, a red light lamp and a white light lamp.

10. the display screen displays a grating image, the grating image including a vertical motion grating, a horizontal motion grating, and / or a blinking raster; Optionally, parameters of the vertical motion grating are 0° orientation, optionally a spatial frequency of the vertical motion grating is 0.2 cpd, optionally a temporal frequency of the vertical motion grating is 1 Hz, and optionally a contrast of the vertical motion grating is 50%; 10. The apparatus of claim 8 or 9, optionally with parameters of the horizontal motion grating: 90° orientation; optionally with a spatial frequency of the horizontal motion grating of 0.2 cpd; optionally with a temporal frequency of the horizontal motion grating of 1 Hz; and optionally with a contrast of the horizontal motion grating of 50%.

11. It is used to promote the release of adenosine in the body, Optionally, said use comprises use for increasing adenosine concentration in an organism; Optionally, the use comprises use for increasing the concentration of neuronal extracellular adenosine.

12. including for use in treating neurological disorders, cardiovascular disorders and / or ophthalmological disorders; Optionally, said nervous system disease comprises Parkinson's disease, depression, anxiety, dementia, Alzheimer's disease, post-traumatic stress disorder, hyperactivity disorder, autism and / or attention deficit in children; Optionally, the cardiovascular disease comprises coronary heart disease and / or arrhythmia; Optionally, the ophthalmic disease comprises myopia, strabismus and amblyopia, and age-related retinopathy.

13. 12. The use of claim 11 including use for improving a sleep disorder, optionally improving a sleep disorder with visual stimulation at 20-80 Hz, optionally improving a sleep disorder with visual stimulation at 30-60 Hz, optionally improving a sleep disorder with visual stimulation at 35-45 Hz, and optionally improving a sleep disorder with visual stimulation at 40 Hz.

14. 11. A method of modulating the release of adenosine in vivo comprising the steps of: delivering 1-100 Hz stimulation to a subject using a device according to any one of claims 1 to 10, said delivery being non-invasive, and optionally said stimulation comprising visual and / or audio stimulation.

15. The frequency of the visual stimulus is 1-100 Hz, and optionally the frequency of the visual stimulus is 20-80 Hz, the frequency of the visual stimulus including one or more of 20 Hz, 25 Hz, 30 Hz, 35 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz, 60 Hz, 65 Hz, 70 Hz, 75 Hz, and 80 Hz; Optionally, the frequency of the visual stimulus is between 30 and 60 Hz; optionally, the frequency of the visual stimulus includes one or more of 30 Hz, 35 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz, and 60 Hz; Optionally, the frequency of the visual stimulus is 40 Hz; Optionally, the visual stimulus has a white light illuminance of 2000 lux or more; Optionally, the wavelength of the monochromatic light of the visual stimulus is between 480 and 530 nm; Optionally, the duty ratio of the visual stimulation is 20% to 80%, preferably 30% to 70%, alternatively 40% to 70%, alternatively 45% to 55%; Optionally, the duty ratio of the visual stimulation is one or more selected from the group consisting of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% and 80%; 15. The method of claim 14, optionally wherein a duty cycle of the visual stimulus is 50%.

16. The visual stimulus includes a flash stimulus and / or an image stimulus; Optionally, said flash comprises white light and / or monochromatic light; Optionally, the image comprises a diffraction grating image.

17. the monochromatic light includes one or more of ultraviolet light, blue light, green light, red light, and near-infrared light, and is preferably blue light and / or green light; Optionally, the central wavelength of the ultraviolet light is 315-400 nm, the central wavelength of the blue light is 450-490 nm, the central wavelength of the green light is 500 nm-560 nm, the central wavelength of the red light is 600-680 nm, and the central wavelength of the near-infrared light is 780-1100 nm; Optionally, the central wavelength of the ultraviolet light is between 320 and 395 nm, alternatively between 325 and 390 nm, alternatively between 330 and 385 nm; Optionally, the central wavelength of the blue light is 455 to 488 nm, or 460 to 485 nm; Optionally, a central wavelength of the green light is between 505 nm and 555 nm, alternatively between 510 nm and 550 nm, alternatively between 515 nm and 545 nm, alternatively between 520 nm and 540 nm, alternatively between 525 nm and 545 nm, alternatively between 530 nm and 535 nm; Optionally, the central wavelength of the red light is 595-675 nm, alternatively 590-670 nm, alternatively 585-665 nm, alternatively 580-650 nm; Optionally, the central wavelength of the near infrared light is 790-1090 nm, alternatively 800-1080 nm, alternatively 810-1070 nm, alternatively 820-1060 nm, alternatively 830-1050 nm, alternatively 840-1040 nm, alternatively 850-1030 nm; optionally, the central wavelength of the ultraviolet light is 380 nm, the central wavelength of the blue light is 480 nm, the central wavelength of the green light is 530 nm, the central wavelength of the red light is 650 nm, and the central wavelength of the near infrared light is 850 nm; Optionally, the white light has an illuminance of 2 to 6000 lux, and the monochromatic light has an illuminance of 2 to 500 lux; 17. The method of claim 16, wherein optionally the white light illuminance of the light emitted by the lamp is between 500 and 3000 lux, and optionally the white light illuminance of the light emitted by the lamp includes one or more of 500 lux, 1000 lux, 2000 lux, and 3000 lux.

18. the contrast of the raster image is between 10% and 100%, Optionally, the raster image comprises one or more of a moving grating image, a blinking grating image, a blinking raster image, and an inverted grating image; Optionally, a contrast of the moving grating is between 10% and 100%, optionally an orientation of said moving grating is between 0 and 180°, optionally a spatial frequency of said moving grating image is between 0.05 and 30 cycles / degree, and optionally a moving speed of said moving grating image is between 1 and 10 Hz; Optionally, the contrast of the inverse grating is between 10% and 100%, optionally the frequency of the inverse grating is between 20 and 60 Hz or between 30 and 50 Hz, preferably 40 Hz, optionally the spatial frequency of the inverse grating is between 0.1 and 30 cpd or 0.1 cpd, optionally the contrast of the inverse grating is between 10% and 100%, between 70 and 90%, preferably 80%, and optionally the average brightness of the inverse grating is between 100 and 500 cd / m ２ , or 140 to 180 cd / m ２ , or 150 to 170 cd / m ２ , or 160 cd / m ２ and 18. The method of claim 16 or 17, optionally wherein the contrast of the blinking raster image is between 10% and 100%, optionally wherein the frequency of the blinking raster image is between 1 and 100 Hz, and optionally wherein the raster size of the blinking raster image is between 0.5 and 10 degrees.

19. A method for evaluating the therapeutic effect of a drug or medical device, comprising analyzing changes in adenosine concentration in a living body.

20. The drug or medical device is used for treating a nervous system disease, a cardiovascular disease, and / or an improvement in a sleep disorder, Optionally, said nervous system disease comprises Parkinson's disease, depression, anxiety, dementia, Alzheimer's disease, post-traumatic stress disorder, hyperactivity disorder, autism and / or attention deficit in children; 20. The method of claim 19, optionally wherein the cardiovascular disease comprises coronary heart disease and / or arrhythmia.

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