Application of adenosine signal blocker in preparation of medicine for treating amblyopia and improving visual plasticity

Abstract

The invention relates to application of an adenosine signal blocker in preparation of a medicine for treating amblyopia and improving visual plasticity. The blocker is used for blocking an adenosine A1 receptor. The invention finds that inhibition of adenosine signal transduction can enhance the visual plasticity of adults, particularly adenosine signal inhibition resets excitability inhibition balance in a visual cortex, and a potential treatment method is provided for adult amblyopia; limiting adenosine signal transduction to enhance visual plasticity is a novel and safe method for clinical use, and the mechanism is deeply understood; the caffeine is a safe molecule suitable for clinical use, and has the advantages of small side effect and easy acceptance as a non-specific adenosine receptor antagonist.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of an adenosine signal blocker in preparing a medicine for treating amblyopia and improving visual plasticity. Background Art

[0002] Amblyopia is a common eye disease with an incidence rate of 1-5%. Amblyopia leads to a decrease in the best corrected visual acuity of one or both eyes, a decrease in contrast sensitivity, and also causes abnormalities in binocular vision such as stereopsis. Amblyopia not only causes damage to multiple visual functions, but also causes abnormalities in higher-level functions such as attention deficit, leading to a decrease in children's self-perception and social skills, affecting the quality of life of patients and their families.

[0003] Amblyopia can be divided into childhood amblyopia and adult amblyopia according to age. Clinically, childhood amblyopia is considered to be curable. However, adult amblyopia is considered difficult to cure. In the clinical treatment of amblyopia, classic occlusion therapy and a variety of visual training methods have certain effects on adult amblyopia, but they cannot cure adult amblyopia. Finding an effective method to cure adult amblyopia is the current difficulty and hot spot. Adult amblyopia is difficult to cure because of the extremely low visual plasticity in adulthood. The reason for the low visual plasticity in adulthood is that the neuronal circuits in the cortex are more stable, which is manifested as the neural circuits mediated by excitatory neurons and inhibitory neurons are in a stable fixed state. Therefore, breaking the stability of excitatory and inhibitory neuronal circuits can reshape visual plasticity in adulthood.

[0004] Based on the theory of excitation-inhibition balance, previous studies have found that taking drugs to enhance excitability may improve plasticity. Previous studies have shown that contrast sensitivity and inhibition of central scotomas in adult amblyopia are improved after taking levodopa. Clinical studies have shown that the use of occlusion therapy plus oral supplementation of levodopa improves the vision of amblyopic patients more than occlusion therapy alone. The therapeutic effect of levodopa is not very significant and there is still some controversy. In another study on residual vision defects after amblyopia treatment, it was found that there was no significant difference in the treatment effect between the occlusion therapy plus oral supplementation of levodopa group and the occlusion therapy plus oral placebo group.

[0005] After passing through the blood-brain barrier, citicoline can increase the levels of dopamine, acetylcholine, and norepinephrine in the central nervous system. Many previous articles have shown that the combined treatment effect or stability of citicoline and occlusion therapy is higher than that of occlusion therapy, indicating that citicoline can improve visual plasticity. Fluoxetine is a selective serotonin reuptake inhibitor that can increase extracellular serotonin and norepinephrine and is often used in the treatment of depression. In many studies, fluoxetine has been found to enhance visual plasticity or improve visual function. Donepezil is a centrally active cholinesterase inhibitor that enhances cholinergic function by increasing acetylcholine levels in synapses and is often used in the treatment of Alzheimer's and other neurological diseases. Many studies have shown that donepezil can enhance visual plasticity in patients with amblyopia and normal adults.

[0006] In summary, a variety of drugs can enhance adult visual plasticity to a certain extent. However, these methods are far from sufficient to cure adult amblyopia, so it is necessary to find new molecular targets to treat adult amblyopia. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an application of an adenosine signal blocker in the preparation of a drug for treating amblyopia and improving visual plasticity in view of the current status of the prior art.

[0008] The technical solution adopted by the present invention to solve the above technical problem is: use of an adenosine signal blocker in the preparation of a drug for treating amblyopia and improving visual plasticity, wherein the blocker is used to block adenosine A1 receptors.

[0009] Preferably, the blocker comprises at least one of an adenosine A1 receptor antagonist, an adenosine A1 receptor inhibitor, and a down-regulator that reduces the expression of adenosine A1 receptor.

[0010] Preferably, the adenosine A1 receptor antagonist comprises DPCPX.

[0011] Preferably, the DPCPX improves visual plasticity by changing the excitation-inhibition balance in the visual cortex.

[0012] Preferably, the DPCPX changes the excitation-inhibition balance of the visual cortex by enhancing excitatory neurons, thereby improving visual plasticity.

[0013] Preferably, the adenosine A1 receptor antagonist comprises caffeine. Caffeine is a plant alkaloid, which exists in many plants and is widely accepted by people.

[0014] Preferably, the down-regulator comprises at least one of active peptides, proteins, oligonucleotides and small molecule compounds that inhibit the expression of adenosine A1 receptors.

[0015] Preferably, the down-regulator is an interfering molecule, and the interfering molecule is shRNA.

[0016] Compared with the prior art, the advantages of the present invention are:

[0017] (1) It was found that inhibiting adenosine signaling can enhance visual plasticity in adults. Specifically, adenosine signaling inhibition resets the excitatory-inhibitory balance in the visual cortex, providing a potential treatment for adult amblyopia. Limiting adenosine signaling to enhance visual plasticity is not only a new, safe method for clinical use, but its mechanism is also well understood.

[0018] (2) Caffeine is clinically used to treat primary apnea of ​​premature newborns, which shows that caffeine is a safe molecule suitable for clinical use. As a nonspecific adenosine receptor antagonist, it has the advantages of few side effects and easy acceptance. Previous studies have identified many molecules and cells that affect visual plasticity, such as γ-aminobutyric acid (GABA) transmission, parvalbumin (PV)-positive interneurons, astrocytes, and genes such as clock genes and BDNF. However, regulating these molecules or cells may cause serious clinical side effects and affect normal physiological functions. In addition, visual disorders such as amblyopia are not life-threatening, which increases the difficulty of convincing patients to accept these important molecules or cells for treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a graph showing the relationship between the adenosine content in the visual cortex and the age of mice in Example 1 of the present invention, wherein: Figure 1 a Bar graph showing adenosine content in the visual cortex of mice at different ages (16 days, 19 days, 26 days, 37 days, around 48 days, and 150 days after birth); Figure 1 b is a line graph showing the changing trend of adenosine content in the visual cortex of mice of different ages;

[0020] Figure 1 c represents the daily increase of adenosine in the visual cortex with age; Figure 1 d represents the correlation between adenosine content and age before and after the critical period of visual development;

[0021] Figure 2 This is a graph showing the effect of inhibiting different adenosine receptors on visual plasticity in mice in Example 2 of the present invention, wherein pre-MD represents the binocular visual evoked potential ratio before three days of monocular covering; MD3d represents the binocular visual evoked potential ratio after three days of monocular covering; red represents intraperitoneal injection of saline containing DPCPX; blue represents intraperitoneal injection of saline containing KW6002; black represents intraperitoneal injection of saline containing DMSO (as a control group);

[0022] Figure 3 This is a graph showing the effect of adenosine A1 receptor siRNA interference virus on visual plasticity in mice in Example 2 of the present invention, wherein pre-MD represents the binocular visual evoked potential ratio before three days of monocular covering; MD3d represents the binocular visual evoked potential ratio after three days of monocular covering; red represents the adenosine A1 receptor siRNA virus group injected into the visual cortex; black represents the control group injected with the control siRNA virus into the visual cortex;

[0023] Figure 4 Schematic diagram of binocular rivalry paradigm in Example 3 of the present invention;

[0024] Figure 5 3 is a graph showing the effects of different concentrations of caffeine on visual plasticity in mice in Example 3 of the present invention, wherein pre-MD represents the binocular visual evoked potential ratio before three days of monocular covering; MD3d represents the binocular visual evoked potential ratio after three days of monocular covering; red represents the adult mouse group injected with 60 mg / kg caffeine intraperitoneally; green represents the adult mouse group injected with 30 mg / kg caffeine intraperitoneally; blue represents the adult mouse group injected with 15 mg / kg caffeine intraperitoneally; and black represents the adult mouse control group injected with normal saline intraperitoneally;

[0025] Figure 6 This is a graph showing the ratio of binocular rivalry between the dominant eye and the non-dominant eye after the monocular deprivation is released for different time periods in Example 3 of the present invention, wherein Caffeine is the experimental group, Placebo is the control group, the abscissa is the time after the monocular deprivation is released, and the ordinate is the ratio of binocular rivalry between the dominant eye and the non-dominant eye;

[0026] Figure 7 The figure is an area under the curve of ocular dominance plasticity induced by short-time covering after oral administration of two different coffees in Example 3 of the present invention at 0 to 9 minutes; the abscissa is the experimental group (n=15), the ordinate is the control group (n=15), and each point represents the area under the curve of ocular dominance plasticity induced by short-time covering at 0 to 9 minutes; the larger the value, the stronger the plasticity;

[0027] Figure 8 This is a graph of the number of spikes released by visual cortical neurons after different times of drug injection in Example 4 of the present invention, the horizontal axis is the detection time (with intraperitoneal injection as 0 point), the vertical axis represents the ratio of the number of electrical releases after drug injection to the baseline, and the baseline is the average value of electrical signal release 15 minutes before intraperitoneal injection of the drug; red represents the adult mouse group injected with 60 mg / kg DPCPX intraperitoneally; green represents the adult mouse group injected with 60 mg / kg caffeine intraperitoneally; blue represents the adult mouse group injected with 30 mg / kg caffeine intraperitoneally; black represents the adult mouse control group injected with normal saline intraperitoneally;

[0028] Fig. 9 The transcriptomic results of the adult mice with simple monocular occlusion for 3 days and the adult mice with adenosine signal inhibition combined with monocular occlusion for 3 days in Example 4 of the present invention are shown;

[0029] Fig.10 This is a graph of ARC protein content in the adenosine signal inhibition combined with masking group and the simple masking group in Example 4 of the present invention, wherein GAPDH is an internal reference protein;

[0030] Fig.11 for Fig.10 Statistical graph of ARC protein content in ;

[0031] Fig.12 This is a graph showing the effect of intraperitoneal injection of physiological saline containing DPCPX on the excitation-inhibition balance of the visual cortex in Example 5 of the present invention; wherein Exc represents excitatory neurons, Inb represents inhibitory neurons; Exc / Inb represents the ratio of excitatory and inhibitory neurons; red represents the experimental group, and black represents the control group; the ordinate represents the ratio of the excitation-inhibition balance 30 minutes after intraperitoneal injection of the drug to the excitation-inhibition balance before intraperitoneal injection;

[0032] Fig.13 This is a graph showing the effect of intraperitoneal injection of physiological saline containing DPCPX on the activity of visual cortical neurons in Example 5 of the present invention; wherein Exc represents excitatory neurons, Inb represents inhibitory neurons; red represents the experimental group, and black represents the control group; the ordinate represents the ratio of neuronal activity 30 minutes after intraperitoneal injection of the drug to neuronal activity before intraperitoneal injection. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below with reference to the accompanying drawings.

[0034] Example 1 The adenosine content in the visual cortex of mice before and after the critical period of visual plasticity development is positively correlated with age

[0035] An experimental method

[0036] First, the changes in adenosine in the visual cortex of mice before and after the critical period of visual plasticity development were detected. Mice at 16 days, 19 days, 26 days, 37 days, about 48 days and 150 days after birth were selected (16 days, 19 days, 26 days, 37 days, about 48 days and 150 days after birth corresponded to the mice before the critical period, the beginning of the critical period, the critical period, the end of the critical period, the post-critical period and adulthood, respectively). The mice were quickly killed by cervical dislocation, and the visual cortex brain tissue of the mice was removed under a microscope and the adenosine content was measured by high performance liquid chromatography. Male C57BL / 6 mice were used in this application, and the mice were older than 16 days and provided by Weitong Lihua Company; the mice involved in the experiment were approved by the ethics committee and were raised in standard breeding laboratory conditions that met the requirements. The living environment temperature of the mice was 22±0.5℃, the relative humidity was 60±2%, and the light / dark cycle was strictly controlled for 12 hours.

[0037] The steps of measuring the adenosine content in the visual cortex of mice of different ages by high performance liquid chromatography are as follows:

[0038] (1) Sample preparation: mice of different ages were taken and quickly killed by cervical dislocation. The visual cortex tissue of the mice was removed and weighed under a microscope. Then, the visual cortex tissue of each sample was stored in a -80°C refrigerator together with 100 ul of adenosine inhibitor (the adenosine inhibitor in this embodiment is racemic-9-(2-hydroxy-3-nonyl)adenine hydrochloride, EHNA) and three grinding beads to prevent adenosine degradation;

[0039] (2) Sample processing: Take out the sample from the -80°C refrigerator, homogenize the sample at 4°C for one minute, add 400 μl of acetonitrile, vortex for 30 seconds, and then let it stand for 10 minutes; centrifuge at 13,000 r / min for 5 minutes, and take 200 μl of the supernatant;

[0040] (3) Preparation of standard substances: Prepare adenosine standards with different solubility gradients so as to draw the standard substance curve later;

[0041] (4) Determining adenosine content: The supernatant obtained in step (2) and adenosine standards with different solubility gradients are analyzed in an HPLC system, and finally the adenosine content in the visual cortex is obtained based on the measurement results and the corresponding sample weight; the specific operation of HPLC is as follows: adenosine is separated using a Polaris 5C18-a column at a temperature of 25°C, and the detection wavelength is 254 nm; isothermal elution is performed using a mobile phase composed of sodium dihydrogen phosphate and methanol (80:20) at a flow rate of 0.4 mL / min, wherein the mobile phase is adjusted to pH 7.0 with triethylamine, and the adenosine content in the visual cortex is calculated based on the measurement results and the sample weight.

[0042] 2. Experimental Results

[0043] The relationship between the adenosine content in the visual cortex and the age of mice Figure 1 As shown, specifically, Figure 1 As shown in a and 1b, the adenosine content in the visual cortex increases with age; Figure 1 As shown in Figure c, adenosine changes the most before and at the beginning of the critical period, with daily changes of more than 10 ng / mg. At the end of the critical period and in adulthood, daily changes in adenosine are less than 2 ng / mg. The trend of changes in visual plasticity is also stronger in the early stages, and plasticity decreases with age. The daily changes in adenosine content have the same trend as changes in visual plasticity, suggesting that there may be a certain correlation between adenosine content and plasticity. Therefore, we further analyzed the correlation between adenosine content and age before and after the critical period, as shown in Figure 5. Figure 1 As shown in Figure d, the adenosine levels at 16, 19, 26, 37, 48 and 150 days after birth were 240.354±39.106, 273.830±73.113, 312.175±52.284, 340.036±85.920, 352.627±107.003 and 377.770±87.990 ng / mg, respectively. The results showed that the older the age, the more adenosine content in the visual cortex (r=0.4781, p<0.0001), and the older the age, the worse the visual plasticity, which suggests that adenosine content and plasticity are negatively correlated.

[0044] Example 2 Inhibition of adenosine A1 receptor improves visual plasticity, while inhibition of adenosine A2a receptor does not improve plasticity

[0045] An experimental method

[0046] Gold-plated screw electrodes were fixed on the surface of the visual cortex of adult mice over 8 weeks old, and the visual evoked potentials of the left and right eyes were recorded by recording electrodes. Three days of monocular light deprivation were caused by covering one eye. The mice in the experimental group were injected with specific adenosine A1 receptor antagonists DPCPX and adenosine A2a receptor antagonists KW6002 intraperitoneally during the three-day monocular light deprivation; while the mice in the control group were injected with saline containing only DMSO intraperitoneally. After three days of monocular light deprivation, the visual evoked potentials of the left and right eyes were measured. The strength of the visual plasticity of the mice was determined by observing the changes in the ratio of the visual evoked potentials of the left and right eyes. If the visual plasticity of the mice is strong, the ratio of the visual evoked potentials of the left and right eyes will change; while the plasticity of adult mice is low, and the three-day covering will not cause changes in the ratio of the visual evoked potentials of the left and right eyes.

[0047] The experimental steps for electrode implantation and visual evoked potential measurement in adult mice visual cortex are as follows:

[0048] 1. Inclusion criteria: Male C57BL / 6 mice aged 8 to 12 weeks, weighing 22 to 28 grams, were selected for surgery;

[0049] 2. Anesthesia: After intraperitoneal injection of 1.2% aflototin (0.1 ml / 10 g), test whether the mouse has a rolling reflex. If the rolling reflex disappears, the subsequent experiment can be started;

[0050] 3. Skin preparation: shave the hair on the top of the mouse's head and apply erythromycin ointment to both eyes of the mouse;

[0051] 4. Fix the mouse: fix the mouse's temporomandibular joint on the clamp of the stereotaxic apparatus, and fix the mouse's upper teeth on the front fixator;

[0052] 5. Level the skull plane: disinfect the skin on the top of the mouse head with alcohol, cut along the midline of the mouse brain from the ear to the back of the eyes, fully expose the skull field of vision, and ensure that the anterior and posterior fontanelles are clearly visible; wipe off the connective tissue on the top of the skull with an alcohol cotton ball; fix the cranial drill bit of the stereotaxic instrument to the frame, perpendicular to the skull surface; start from the anterior fontanelle, and space 2.0mm to the left and right, and adjust the left and right height errors within 0.05mm; adjust the vertical level with the height of the anterior fontanelle and the posterior fontanelle as the standard, and the height difference between the anterior fontanelle and the posterior fontanelle should be controlled within 0.05mm;

[0053] 6. Drilling: Starting from the bregma, drill 3.6mm backward and 2.9mm to the left / right. Use a 0.5mm diameter drill to drill the skull and remove the bone fragments with ophthalmic forceps.

[0054] 7. Lower electrode: Drill a gold-plated screw electrode into the skull to make it close to the surface of the brain tissue, drill a hole at the rear end of the posterior fontanelle and insert a common screw to fix it; use super glue to glue the screw and fix it with dental cement; remove the mouse only after the dental cement has hardened; let the mouse rest for at least seven days before measuring the visual evoked potential;

[0055] 8. Measurement of visual evoked potential: turn on the electrophysiological instrument and anesthetize the mouse with 1.2% afloatin solution; place the mouse on the operating table after it has no turning over reflex; insert the ground electrode into the subcutaneous space in front of the mouse's tail; insert the reference electrode into the subcutaneous space of the mouse's cheek; clamp the experimental electrode to the gold-plated screw on the top of the mouse's skull; use a full-field flash VEP module to record the visual evoked potential of one eye of the mouse; when measuring one eye, cover the other eye with an eyepatch.

[0056] The experimental steps of monocular light deprivation are as follows:

[0057] 1. Experimental preparation: First, cut the highly transparent plastic plate to fit the shape of the mouse neck collar, and then stick tape on the inner circle of the collar to prevent the plastic collar from cutting the mouse neck in subsequent experiments;

[0058] 2. Wearing eye masks: After the mice are anesthetized with the volatilized gas of isoflurane, a plastic collar is put on the mice and the collar is fixed with a stapler (the neck collar should not be fixed too tightly, as it will affect the mice's eating; the collar does not fall off); then glue is applied to the edge of the mouse eye mask and the right eye of the mouse is covered; during the experiment, it is important to be careful not to apply glue to the center of the eye mask to prevent the glue from sticking to the mouse's eyes;

[0059] 3. Subsequent observation: The mice wore the eye patch for 72 hours. During this period, it was necessary to pay attention to whether the eye patch fell off. If the eye patch fell off, the mouse would not be included in the subsequent experiments.

[0060] In order to further explore the role of adenosine A1 receptors in visual plasticity in the visual cortex, siRNA interference virus for adenosine A1 receptors was injected into the visual cortex (siRNA interference virus for adenosine A1 receptors was purchased commercially) to specifically knock down adenosine A1 receptors in the visual cortex to block the effects of adenosine A1 receptors. At the same time, control siRNA interference virus was injected into the visual cortex of mice in the control group. After 3 weeks, the binocular visual evoked potential ratio was measured and used as the baseline, and the binocular visual evoked potential ratio was re-measured after 3 days of monocular cover.

[0061] The experimental steps for injecting adenosine A1 receptor siRNA interference virus and control siRNA interference virus into the visual cortex are as follows:

[0062] 1. Make a syringe: turn on the hot melt glue gun in advance to preheat; use the syringe to extract a certain amount of glycerin so that the micro syringe is filled with glycerin and no bubbles are generated; use the syringe to push glycerin from the end of the glass electrode so that the glass electrode is filled with glycerin and no bubbles are generated; connect the glass electrode and the micro syringe, seal it with a hot melt glue gun, push the syringe after cooling and observe whether there is glycerin discharged from the tip to confirm that it is well sealed;

[0063] 2. Inclusion criteria: Male C57BL / 6 mice aged 8 to 12 weeks, weighing 22 to 28 grams, were selected for surgery;

[0064] 3. Anesthesia: After intraperitoneal injection of 1.2% aflototin (0.1 ml / 10 g), test whether the mouse has a rolling reflex. If the rolling reflex disappears, the subsequent experiment can be started;

[0065] 4. Skin preparation: Shave the hair on the top of the mouse's head and apply erythromycin eye ointment to both eyes of the mouse.

[0066] 5. Fix the mouse: Fix the temporomandibular joint of the mouse on the clamp of the stereotaxic apparatus, and fix the upper teeth of the mouse on the front fixator.

[0067] 6. Level the skull: Disinfect the skin on the top of the mouse's head with alcohol, cut along the midline of the mouse's brain from the ear to the back of the eyes, fully expose the skull field of vision, and ensure that the anterior and posterior fontanelles are clearly visible. Wipe off the connective tissue on the top of the skull with an alcohol cotton ball. Fix the skull drill of the stereotaxic instrument to the frame, perpendicular to the skull surface. Starting from the anterior fontanelle, 2.0mm is spaced left and right, and the left and right height errors are adjusted to within 0.05mm. Adjust the vertical level based on the height of the anterior fontanelle and the posterior fontanelle; the height difference between the anterior fontanelle and the posterior fontanelle must be controlled within 0.05mm.

[0068] 7. Drilling: Starting from the anterior fontanelle, drill 3.6 mm backward and 2.9 mm to the left. Use a 0.5 mm diameter drill bit to drill open the skull, and then use ophthalmic forceps to pick out the bone fragments.

[0069] 8. Virus injection: Use a microsyringe to absorb the corresponding virus, take the dura mater as the zero point, and then use ophthalmic forceps to pierce the dura mater; position the syringe 0.6mm below the dura mater and inject the corresponding virus. After the injection is completed, keep the needle in place for 15 minutes, and then slowly pull out the syringe; drill a gold-plated screw electrode into the skull to make it close to the surface of the brain tissue, drill a hole at the rear end of the posterior fontanelle and insert an ordinary screw to fix it; use super glue to glue the screws and fix them with dental cement. The mice can be removed only after the dental cement has hardened; let the mice rest for at least seven days before measuring visual evoked potentials.

[0070] 2. Experimental Results

[0071] like Figure 2 As shown, intraperitoneal injection of saline containing only DMSO in the control group mice did not cause changes in the ratio of left and right eye visual evoked potentials (n=7, p=0.782, paired t-test); this is consistent with previous research results. We found that specific inhibition of adenosine A1 receptor antagonist DPCPX can change the ratio of left and right eye visual evoked potentials (n=10, p=0.048, paired t-test), indicating that adenosine A1 receptor antagonist DPCPX can increase visual plasticity in adult mice; while specific inhibition of adenosine A2a receptors cannot change the ratio of left and right eye visual evoked potentials (n=7, p=0.775, paired t-test), indicating that adenosine A2a receptor antagonists cannot increase visual plasticity in adult mice.

[0072] In addition, the experimental results of specific knockdown of adenosine A1 receptors in the visual cortex are as follows Figure 3As shown: the ratio of left and right eye visual evoked potentials of control mice did not change (2.876±0.524 vs. 3.012±0.639, mean±SEM, paired t-test, t(8)=-0.741, p=0.480); while the ratio of left and right eye visual evoked potentials of mice injected with adenosine A1 receptor miRNA interference virus changed (3.185±0.767 vs. 2.624±0.673, mean±SEM, one-tailed, t(7)=2.170, P=0.034). This indicates that specific inhibition of adenosine A1 receptors in the visual cortex can change visual plasticity.

[0073] In previous studies, we found that the specific adenosine A1 receptor blocker DPCPX can enhance visual plasticity in mice, but this drug cannot be used clinically to truly solve clinical problems. Caffeine, as a non-specific adenosine antagonist widely used in clinical practice, can antagonize adenosine receptors and may be expected to enhance visual plasticity.

[0074] Example 3 High concentrations of adenosine receptor nonspecific antagonist caffeine can improve visual plasticity in mice; drinking coffee at a specific concentration can improve short-term ocular dominance plasticity in adults

[0075] An experimental method

[0076] Three different concentrations of caffeine were selected: 15mg / kg, 30mg / kg and 60mg / kg, to explore whether caffeine can mimic the effects of the specific adenosine A1 receptor inhibitor DPCPX, thereby enhancing visual plasticity. The visual evoked potentials of the left and right eyes were recorded by recording electrodes, and the changes in the ratio of the visual evoked potentials of the left and right eyes were measured after 3 days of monocular light deprivation by covering one eye.

[0077] In order to further verify the effect of caffeine on improving visual plasticity in adults, both the experimental group and the control group drank coffee to control the different caffeine concentrations; the caffeine content of the coffee consumed by the experimental group was 258.292±10.782mg, n=10, and the caffeine content of the coffee consumed by the control group was 1.910±0.403mg, n=10. One hour after oral coffee, the dominant eye was covered for one hour, and then the changes in eye dominance plasticity were measured at 0, 3, 6, 9 and 30 minutes respectively.

[0078] Competitive ocular dominance plasticity in adults was measured as follows:

[0079] (1) Subject information

[0080] A total of 15 normal adult subjects were included in the rapid contrast sensitivity experiment, with an average age of 23.45±1.37 years old. Among them, 6 were male and 9 were female. All subjects drank coffee ≤2 times a week. The refractive power of all subjects was ≤40; the refractive power of both eyes was ≤100° and the astigmatism was ≤100°. All subjects had normal or correctable binocular vision (≤0.00logMAR); and there was no severe dry eye and severe visual fatigue, no other eye diseases and surgical history. Before the experiment, all subjects underwent vision correction to ensure that the subjects were under the best vision. The subjects had to get enough sleep the night before the experiment, and did not drink alcohol or eat spicy food. The coffee machine was a NESPRESSO Pixie imported fully automatic coffee machine. Each person in the experimental group drank two Kazzar caffeine capsules at a time; each person in the control group used two Arpeggio Decaffeinato coffee capsules at a time. After putting the coffee capsule into the coffee machine, select the button with the large cup logo (the small cup button is not used during the whole process). The liquid volume of two coffee capsules is greater than 200ml, which is placed in a 330ml disposable paper cup; the volume can be adjusted to 330ml with milk according to the needs of the subjects. Each subject drinks high-caffeinated coffee and low-caffeinated coffee experiments on different days; that is, each person must complete two experiments.

[0081] This study complies with the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of the Affiliated Eye and Optometry Hospital of Wenzhou Medical University. The subjects were fully informed of the possible effects of this experiment and their consent was obtained. All subjects were unaware of the purpose of this experiment.

[0082] (2) Experimental equipment

[0083] The experimental programs were written in Matlab and Psychtoolbox extension and run on a MacBook Pro computer (Apple Inc, CA, USA). Visual stimuli were presented on a gamma-corrected head-mounted GOOVIS display. During the experiment, the subjects' heads were fixed with a forehead / chin rest to ensure the accuracy of the test distance.

[0084] (3) Experimental sight mark

[0085] The visual stimuli used in this experiment were all grating sight marks, such as Figure 4 The subject's left and right eyes saw two gratings with opposite tilt angles (with the vertical direction as the 0 axis facing +45° and -45°), respectively, for 90 seconds and repeated twice.

[0086] (4) Experimental design

[0087] In this experiment, the subject's left and right eyes saw two different gratings, and the two gratings were oriented in opposite directions. Therefore, the visual cortex will receive two competing gratings. From the subject's subjective experience, the subject sometimes perceives the grating to the left and sometimes to the right, depending on the competition of the gratings in the visual cortex. The subject presses the button corresponding to the perceived direction. Change the button immediately when the perceived direction changes. The dominant eye and the dominance ratio (Ocular Dominance Ratio) are calculated by the duration of the grating direction recorded by the program.

[0088] 2. Experimental Results

[0089] like Figure 5 As shown, when monocular covering was performed for 3 days, caffeine was injected intraperitoneally. We found that intraperitoneal injection of 60 mg / kg of caffeine changed the ratio of visual evoked potentials of the left and right eyes (2.811±0.491 vs. 2.169±0.698, paired t-test, t(13)=5.141, p<0.001), indicating that its plasticity was enhanced, while intraperitoneal injection of 15 mg / kg and 30 mg / kg of caffeine did not significantly change the ratio of visual evoked potentials of the left and right eyes (Ps>0.05), indicating that its plasticity was not improved. These results indicate that high concentrations of caffeine can enhance the visual plasticity of mice, providing evidence that caffeine can improve the visual plasticity of the adult population.

[0090] like Figure 6 As shown in the study, after an hour of brief occlusion, the dominant eye's ability to perceive visual stimuli in adults is enhanced, as shown by the increase in the ratio of binocular competition between the dominant eye and the non-dominant eye. The larger the ratio, the stronger the short-term plasticity induced by monocular occlusion. We found that the competitive eye advantage plasticity of the normal caffeine group from 0 to 9 minutes after short-term occlusion was significantly greater than that of the control low caffeine group (A repeated-measures ANOVA, F(1,28)=4.484, p=0.043). Figure 7 As shown, further analysis of the area under the curve of eye dominance plasticity from 0 to 9 minutes showed that the normal caffeine group was significantly higher than the control low-dose caffeine group (11.682±2.181 vs. 10.210±1.425, paired t-test, t(14)=2.618, p=0.020).

[0091] These results indicate that oral caffeine can enhance short-term ocular dominance plasticity in adults, providing new evidence for the clinical application of caffeine in enhancing visual plasticity in adults.

[0092] Example 4 Both electrical activity and protein levels of visual cortical neurons suggest that inhibiting adenosine signals can activate visual cortical neuronal activity and enhance plasticity

[0093] An experimental method

[0094] A 16-channel array electrode was implanted in the visual cortex of mice to record the firing rate of the electrical activity of the visual cortex neurons. The mouse's head was fixed so that the mouse could not move, eliminating the interference of head movement on the signal recording process. First, the firing rate of the electrical activity of the visual cortex neurons was recorded for 15 minutes as a baseline, and then different solutions were intraperitoneally injected: DPCPX 6mg / kg, Coffeine 60mg / kg, Coffeine 30mg / kg and saline; then the firing rate of the neuronal electrical activity was recorded for one hour, and the ratio of the firing rate of the electrical activity after injection to the firing rate of the electrical activity before injection represented the change in neuronal activity.

[0095] The transcriptome of adult mice with single eye occlusion for 3 days and those with adenosine signal inhibition combined with single eye occlusion for 3 days were further analyzed to explore whether inhibition of adenosine signaling enhances plasticity. The changes in ARC protein between the adenosine signal inhibition combined with occlusion group and the simple occlusion group were measured to further verify that inhibition of adenosine signaling can increase ARC protein.

[0096] Three days after intraperitoneal injection of the specific adenosine A1 receptor antagonist DPCPX, the content of activity regulated cytoskeletal protein (ARC) was detected. The specific steps for the determination of ARC protein are as follows:

[0097] 1. Extraction of visual cortex protein: The mice were quickly killed by cervical dislocation, and the visual cortex brain tissue of the mice was removed under a microscope and weighed; 100-200ul of cell tissue lysis solution containing protease inhibitors was added to the visual cortex tissue (the specific amount depends on the size of the tissue); after being fully ground by a fully automatic sample cold grinder, the fully ground sample was placed in a 4°C centrifuge, centrifuged at 12000rpm for 5 minutes, and the supernatant was taken; the supernatant was heated in a 100°C metal bath for 15 minutes, and stored at -80°C after cooling;

[0098] 2. Detect protein concentration (BCA method): Add PBS (15ul), the cell lysate protein sample (5ul) in step 1, and 200ul of BCA working solution (50 volumes of BCA reagent A plus 1 volume of BCA reagent B) to the wells of a 96-well plate in sequence; incubate the well plate in a 37°C oven for 30 minutes, and measure the OD value (at a wavelength of 562nm) in an enzyme labeling instrument. Draw a standard curve based on the results and calculate the protein concentration of each sample;

[0099] 3. Preparation of protein electrophoresis loading solution: add buffer solution to the determined protein sample (the ratio of sample protein to buffer solution is 4:1), then vortex and centrifuge; heat in a metal bath for 5 minutes and then cool to obtain protein electrophoresis loading solution, and store it at -80 for later use;

[0100] 4. SDS-PAGE gel preparation: After preparing 12% separation gel and 5% concentration gel, fix the two gels on the electrode plate, fill the inner tank with electrophoresis solution and make sure there is no electrophoresis solution overflow, then add electrophoresis solution to the outer tank to the specified scale;

[0101] 5. Sample loading: Add the protein electrophoresis sample solution obtained in step 3 to the sample wells in the order of the control group and the experimental group, and load according to the protein concentration (about 40ug total protein); electrophoresis conditions: after running at a constant voltage of 60V for 40 minutes, adjust the voltage to 120V and run for 80 minutes;

[0102] 6. Transfer: Prepare the transfer solution; prepare a PVDF membrane of appropriate size and activate it in methanol in advance; Transfer conditions: put ice cubes of appropriate size in the electrophoresis tank to maintain low temperature, 300mA constant current, and transfer for 70 minutes;

[0103] 7. Blocking and antibody incubation: After the transfer and gel running are completed, you can start blocking, use 5% skim milk powder, block at room temperature for 1 hour; dilute the primary antibody according to the antibody instructions, and shake the incubated sample in a 4 degree Celsius refrigerator overnight (14-18 hours); the next day, recover the primary antibody incubation solution and store it in a -20 degree Celsius refrigerator for next use; wash the PVDF membrane with 1% TTBS on a shaker at room temperature for 4 times, each time for 15 minutes; then incubate the secondary antibody at a ratio of 1:2000 (CST, Cat.7074&7076) on a shaker at room temperature for 1 hour;

[0104] 8. Color development: The chemiluminescence method was used to expose the sample for 30 seconds in the Bio-Rod gel imaging system, and the positions of the bands were determined by protein markers on the membrane to detect Arc and GAPDH proteins ( Fig.10 ).

[0105] 2. Experimental Results

[0106] like Figure 8As shown, we found that there was no statistical difference between the Coffeine 30mg / kg group and the control group (Arepeated-measures ANOVA, F(1,154)=0.024, p=0.878), indicating that a moderate dose of caffeine cannot activate the activity of visual cortical neurons. However, there was a statistical difference between the Coffeine 60mg / kg group and the control group; indicating that a high dose of caffeine can activate the activity of visual cortical neurons (A repeated-measures ANOVA, F(1,48)=10.03p=0.002). There was a statistical difference between the 60mg / kg DPCPX group and the control group (A repeated-measures ANOVA, F(1,151)=25.58, p<0.001); indicating that specific antagonism of adenosine A1 receptors can enhance neuronal activity. In vivo electrophysiology found that inhibiting adenosine signals can enhance the number of spikes in visual cortical neurons.

[0107] We have shown that inhibiting adenosine signaling can enhance the activity of individual neurons and thus increase plasticity. Fig. 9 As shown in the figure, we found that the inhibition of adenosine can activate multiple pathways related to plasticity, such as the stabilization of membrane potential and maintenance of synapse structure. In these pathways, we found that the transcription level of cytoskeletonaassociated protein (ARC) was reduced. We further used western blot to detect the effect of adenosine inhibitor DPCPX on the expression level of Arc protein in the visual cortex. Fig.10 , 11 As shown in the figure, compared with the control group, there was no statistical difference in ARC protein in the left visual cortex of the adenosine inhibition group (two-sample t-test, t(16)=3.002, P=0.865); while the ARC protein in the right visual cortex of the adenosine inhibition group increased significantly (two-sample t-test, t(16)=3.002, P=0.008). This means that inhibiting adenosine signals may induce enhanced ARC protein activity, and ARC protein is closely related to enhanced plasticity caused by enhanced neuronal activity.

[0108] Example 5 Inhibition of adenosine signals changes the excitation-inhibition balance in the visual cortex, mainly by enhancing excitatory neurons

[0109] An experimental method

[0110] Previous experiments have shown that inhibiting adenosine signals can enhance visual plasticity, and its mechanism is related to enhanced neuronal activity and enhanced ARC protein function. To further explore its mechanism, we further explored the excitation-inhibition balance of the visual cortex and investigated the changes in the excitation-inhibition balance of the visual cortex 30 minutes after intraperitoneal injection of saline containing DPCPX.

[0111] To further explore how inhibiting adenosine signals affects the balance of excitation and inhibition, excitatory neurons and inhibitory neurons were studied separately.

[0112] 2. Experimental Results

[0113] like Fig.12 As shown, compared with the DMSO control group, the excitation-inhibition balance of the DPCPX experimental group control mice was significantly changed 30 minutes after drug injection (two-sample t-test, t(10)=3.289, P=0.008).

[0114] like Fig.13 As shown in the figure, compared with the control group, the excitatory neurons in the experimental group were significantly enhanced (two-sample t-test, one-tailed, t(10) = 2.029, P = 0.035); while there was no statistical difference in inhibitory neurons in the experimental group (two-sample t-test, t(10) = -1.747, P = 0.111). In summary, inhibiting adenosine signals changes the excitation-inhibition balance in the visual cortex, mainly by enhancing excitatory neurons.

[0115] In general, the adenosine content in the visual cortex is positively correlated with the age of mice, and the older the age, the lower the plasticity, suggesting that the adenosine content in the visual cortex may be negatively correlated with visual plasticity. Specific inhibition of adenosine A1 receptors can improve visual plasticity through pharmacological experiments, while specific inhibition of adenosine A2a receptors does not affect visual plasticity; specific knockout of cortical adenosine A1 receptors can also improve visual plasticity, indicating that adenosine A1 receptors in the visual cortex play an important role in this. High doses of caffeine, a nonspecific antagonist of adenosine, can also improve visual plasticity, while low doses of caffeine cannot, suggesting that adenosine receptors need to be antagonized to a certain extent to improve visual plasticity; we also verified this effect in adults, and oral coffee can improve short-term eye dominance plasticity in adults. In vivo electrophysiology found that inhibiting adenosine signals can enhance the number of spikes in visual cortical neurons, and the ARC protein level and transcription level, which are closely related to enhanced plasticity caused by enhanced neuronal activity, are increased. Both in vivo electrophysiology and protein levels suggest that inhibiting adenosine signals can activate the activity of visual cortical neurons and enhance plasticity. Inhibition of adenosine can change the excitation-inhibition balance in the visual cortex, mainly enhancing the activity of excitatory neurons rather than changing the activity of inhibitory neurons

[0116] The advantage of the present invention over the prior art is that inhibiting adenosine signaling can enhance visual plasticity in adults, which provides a potential treatment for adult amblyopia. Previous studies have identified many molecules and cells that affect visual plasticity, such as γ-aminobutyric acid (GABA)-ergic transmission, parvalbumin (PV)-positive interneurons, astrocytes, and genes such as clock genes and BDNF. However, regulating these molecules or cells may cause serious clinical side effects and affect normal physiological functions. In addition, visual disorders such as amblyopia are not life-threatening, which increases the difficulty of persuading patients to accept these important molecules or cell treatments. Therefore, it is crucial to find a safe molecule suitable for clinical use to enhance visual plasticity. In this context, caffeine, as a nonspecific adenosine receptor antagonist, may become an ideal choice. The key difference between this study and previous studies is the discovery of a new, clinically safe method to improve visual plasticity in adults. Limiting adenosine signaling to enhance visual plasticity is not only a safe method for clinical use, but its mechanism has also been deeply understood. Studies have shown that adenosine signal inhibition resets the excitatory-inhibitory balance in the visual cortex. Previous in vitro single-cell studies have also shown that adenosine signals regulate this balance. In clinical practice, adult amblyopia is generally considered difficult to cure because neural plasticity decreases after adulthood, and new methods need to be found to enhance visual plasticity in adults for therapeutic intervention. Studies have found that inhibiting adenosine A1 receptors can enhance visual plasticity in adult mice, and this mechanism is related to changes in the excitation-inhibition balance in the visual cortex.

[0117] To further evaluate the clinical feasibility of this approach, the effects of caffeine, a nonspecific adenosine receptor antagonist, on ocular dominance plasticity in normal adults were investigated. The observation that oral caffeine improved short-term ocular dominance plasticity in adults provides a new perspective for the treatment of adult amblyopia. Overall, limiting adenosine signaling is a well-understood and clinically applicable approach to enhance visual plasticity. Future studies should investigate whether caffeine can enhance the efficacy of occlusion therapy or visual training in adults with amblyopia.

Claims

1. Use of an adenosine signal blocker in the preparation of a drug for treating amblyopia and improving visual plasticity, characterized in that: The blocker is used to block adenosine A1 receptors.

2. The use according to claim 1, characterized in that: The blocker includes at least one of an adenosine A1 receptor antagonist, an adenosine A1 receptor inhibitor, and a down-regulator that reduces the expression of adenosine A1 receptor.

3. The use according to claim 2, characterized in that: The adenosine A1 receptor antagonists include DPCPX.

4. The use according to claim 3, characterized in that: The DPCPX enhances visual plasticity by altering the excitation-inhibition balance in the visual cortex.

5. The use according to claim 4, characterized in that: The DPCPX changes the excitation-inhibition balance in the visual cortex by enhancing excitatory neurons, thereby improving visual plasticity.

6. The use according to claim 2, characterized in that: The adenosine A1 receptor antagonists include caffeine.

7. The use according to claim 2, characterized in that: The down-regulator includes at least one of active peptides, proteins, oligonucleotides and small molecule compounds that inhibit the expression of adenosine A1 receptors.

8. The use according to claim 7, characterized in that: The down-regulator is an interfering molecule, and the interfering molecule is siRNA.

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