Detection method for influence of PGRN regulation on cerebellar synaptic development of ASD rat

Abstract

The invention discloses a detection method for influence of PGRN regulation on cerebellar synaptic development of ASD rats, and relates to the technical field of autism spectrum disorders. The method specifically comprises the following steps: step 1, breeding an experimental rat; 60 Sprague-Dawley rats in the childbearing age are purchased, the female-male ratio is 2: 1, the rats freely eat and drink water, and the rats are fed for three to five weeks; step 2, preparing a VPA induced ASD rat model; experimental rats are subjected to fertilization culture, female rats are randomly divided into two groups after being fertilized for 12.5 days, a VPA solution is injected into the VPA group through the abdominal cavity, and sterile normal saline with the same volume is injected into the control group. The detection method shows that the PGRN can obviously relieve VPA-induced dyskinesia and autism-like behaviors of ASD rats and relieve cerebellar Purkinje cell synaptic dysplasia, and the mechanism of the PGRN is possibly related to inhibition of the PGRN on lysosomal functions and autophagy activities in cerebells of the ASD rats.

Description

Technical Field

[0001] The present invention relates to the technical field of autism spectrum disorders, and in particular to a method for detecting the effect of PGRN regulation on cerebellar synapse development in ASD rats. Background Art

[0002] Autism spectrum disorder (ASD) is a neurodevelopmental disorder that usually develops in early childhood and lasts a lifetime. It is characterized by language development disorders, social impairments, stereotyped behaviors, and narrow interests. Progranulin (PGRN) is a multifunctional growth factor that contains 7.5 repeats of cysteine-rich motifs and plays a key role in a variety of physiological and disease processes such as cartilage formation, wound healing, inflammation, and tumorigenesis. PGRN has neurotrophic properties and can promote the survival and differentiation of neurons, the formation and maturation of synapses, and its abnormal expression is associated with a variety of neurodevelopmental diseases. Low PGRN levels may be a biomarker for autism spectrum disorder and cognitive developmental delay.

[0003] It has been confirmed that PGRN expression levels are abnormally altered in the prefrontal cortex, temporal cortex, hippocampus, and cerebellum of VPA-induced ASD model rats. Injection of r-PGRN protein into the cerebellum can alleviate autistic-like behaviors and improve the developmental disorders of synapses and dendrites, but its specific mechanism is still unclear. Synapses are highly dynamic components of neurons. During development, synapse elimination and synapse formation are in a state of balance to maintain the normal function of neuronal circuits. Dendritic spines are the main site for the formation of synapses between neurons.

[0004] The autophagy-lysosome pathway is the main mechanism for the degradation of intracellular macromolecules. Autophagy is a process in which the cell engulfs its own organelles or cytoplasmic proteins, encapsulates them into vesicles, and fuses with lysosomes to form autophagosomes, degrading the contents contained therein, thereby achieving the cell's own metabolic needs and the renewal of organelles.

[0005] During neural growth and development, autophagy of neurons and glial cells plays an important role in synaptic pruning. Dendritic spine pruning in the temporal cortex of ASD patients is significantly reduced, and these changes are negatively correlated with the level of autophagy marker LC3-Ⅱ, indicating that autophagy defects may lead to reduced synaptic pruning in ASD brain tissue. It has been found that PGRN plays an important role in maintaining lysosomal homeostasis. The loss of PGRN can lead to lysosomal dysfunction and autophagy defects by impairing lysosomal transport or affecting the activity of lysosomal proteases. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a detection method for the effect of PGRN regulation on cerebellar synaptic development in ASD rats.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for detecting the effect of PGRN regulation on cerebellar synaptic development in ASD rats, comprising the following steps:

[0009] Step 1: Breeding experimental rats; purchasing 60 Sprague-Dawley rats of reproductive age, with a male-female ratio of 2:1, and feeding and drinking water freely for three to five weeks;

[0010] Step 2: Prepare VPA-induced ASD rat model; perform pregnancy culture on the experimental rats, and randomly divide the female rats into two groups at 12.5 days of pregnancy. The VPA group is injected with VPA solution via intraperitoneal injection, and the control group is injected with an equal volume of sterile saline;

[0011] Step 3: Implant the microinjector in the cerebellum of ASD rats and administer the drug; divide the VPA offspring rats into VPA group, VPA+r-PGRN group, VPA+Vehicle group, and the control group is CON group; prepare r-PGRN reagent, perform experimental treatment on rats, and finally implant the microinjector and administer the drug to the rats;

[0012] Step 4: Conduct behavioral experiments on ASD rats, including cerebellar behavioral tests and autism-like behavioral tests;

[0013] Step 5: Extract brain tissue protein from ASD rats; anesthetize the rats, perfuse with saline, and finally take out the rat brains to extract tissue protein;

[0014] Step 6: BCA method protein concentration determination: The BCA method is used to determine the concentration of the extracted tissue protein;

[0015] Step 7: Western blot; specifically including: SDS-PAGE gel, electrophoresis, membrane transfer, blocking, incubation with primary antibody, washing, incubation with secondary antibody, washing, development, and band analysis;

[0016] Step 8: Golgi staining of tissue: peel off the rat brain tissue and process it, then make brain tissue slices and perform staining experiments under light-proof conditions;

[0017] Step 9: Analysis of dendritic branches of Purkinje cells and counting of dendritic spines: The stained brain tissue sections were photographed under a microscope, and the dendritic branches and dendritic spines of Purkinje cells were counted;

[0018] Step 10: Statistical methods: The obtained data were analyzed and statistically analyzed using GraphPad Prism software, and the results were expressed as mean ± standard error.

[0019] Preferably: in the step 3, 2.5 μl of r-PGRN solution is injected into each cerebellum of the experimental rats in the VPA+r-PGRN group, and 2.5 μl of sterile saline is injected into each cerebellum of the experimental rats in the VPA+Vehicle group.

[0020] Furthermore: the cerebellar behavioral tests include a balance beam test, a plane righting test, a gait analysis test, and a hanging test; the autism-like behavioral tests include an open field test and a self-stroking test, a three-box social test, and a youth play test.

[0021] As a preferred embodiment of the present invention, the tissue protein extraction in step 5 specifically includes the following steps:

[0022] S541: Place the isolated fresh cerebellum tissue on a tin foil and weigh it on a balance;

[0023] S542: According to the instructions of the Bio-Tech RIPA tissue lysis buffer, add 7 μl of RIPA lysis buffer mixture for every 1 mg of sample;

[0024] S543: Precool the homogenizer sample fixing tray to -20°C, place the sample in it and start grinding; after grinding, place the sample on ice for 30 minutes and centrifuge at low temperature for 20 minutes;

[0025] S544: The supernatant after centrifugation was collected into a new EP tube and stored in a sealed environment at -80°C.

[0026] On the basis of the above scheme: the rat brain tissue peeled in step eight is immersed in a mixture of solution 1 and solution 2 with a volume 5 times that of the brain tissue. After the rat brain tissue is immersed for 14 days, it is transferred to an equal volume of solution 3 and placed at 4°C for 12-24 hours. Then, solution 3 is replaced and placed at 4°C for 24-72 hours. Finally, the moisture of the rat brain tissue is removed and the rat brain tissue is sealed and stored at -80°C.

[0027] Preferably: the staining experiment in step eight is carried out under light-proof conditions, specifically including washing, staining, washing, dehydration, decolorization, and sealing.

[0028] Further: the Purkinje cell dendritic branch analysis uses Image J to open the image to be analyzed and sets the scale; uses the Neuron J plug-in to track neurons, draw the Purkinje cell dendritic branch diagram, and generate a two-dimensional distribution map of neurons; adjusts the image format to 8 bits and then performs Sholl analysis.

[0029] Based on the above scheme: the Purkinje cell dendritic spine number statistics use Neuron J to count the number of dendritic spines, draw the length of the counted dendritic spines and calculate the dendritic spine density.

[0030] The beneficial effects of the present invention are:

[0031] This detection method showed that PGRN can significantly reduce the motor dysfunction and autism-like behavior induced by VPA in ASD rats, and alleviate the abnormal synaptic development of cerebellar Purkinje cells. The mechanism may be related to the fact that PGRN can inhibit lysosomal function and autophagy activity in the cerebellum of ASD rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a flow chart of the method for detecting the effect of PGRN regulation on cerebellar synaptic development in ASD rats of the present invention. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0034] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0035] A method for detecting the effect of PGRN regulation on cerebellar synaptic development in ASD rats, such as Figure 1 As shown, the specific steps include:

[0036] Step 1: Breeding experimental rats: A total of 60 Sprague-Dawley (SD) rats of reproductive age were purchased from the Experimental Animal Center of Chongqing Medical University, with a male-female ratio of 2:1. The rats were allowed to eat and drink freely and were raised for three to five weeks at 23-25°C and a relative humidity of 50%-60%.

[0037] Step 2: Prepare a VPA (valproic acid)-induced ASD (autism spectrum disorder) rat model; the experimental rats were conceived and cultured, and the female rats were randomly divided into two groups at 12.5 days after conception. The VPA group was intraperitoneally injected with VPA solution, and the control group was injected with an equal volume of sterile saline.

[0038] The specific steps are as follows:

[0039] S21: Cages were combined at 19:30 every evening in a 1:1 ratio of male to female, and vaginal plugs and vaginal smears were observed at 7:30 the next morning;

[0040] S22: Female rats with vaginal plugs or spermatozoa in vaginal smears were marked and recorded as pregnant for 1 day;

[0041] S23: Female rats were randomly divided into two groups at 12.5 days of pregnancy. The VPA group was injected with VPA solution (500 mg / kg) intraperitoneally, and the control group (Con) was injected with an equal volume of sterile saline. The pregnant rats were housed individually after intraperitoneal injection, and the male offspring were kept for subsequent experiments after giving birth.

[0042] Step 3: Implant the microinjector tube in the cerebellum of ASD rats and administer the drug; divide the VPA offspring rats into VPA group, VPA+r-PGRN (recombinant progranulin) group, VPA+Vehicle group, and the control group is CON group; prepare r-PGRN reagent, perform experimental treatment on rats, and finally implant the tube and administer the drug to the rats.

[0043] The specific steps are as follows:

[0044] S31: Grouping: VPA offspring rats (PND17, postnatal day 17) were randomly divided into the following 3 groups: VPA group, VPA+r-PGRN group, VPA+Vehicle group; the control group was CON group.

[0045] S32: Reagent preparation: r-PGRN: Dissolve the r-PGRN dry powder in sterile saline to a concentration of 50 ng / μl, then dispense into 50 μl / tubes and store at -80°C for later use.

[0046] S33: Anesthesia: Each rat was weighed, ear-tagged, and intraperitoneally injected with 40 mg / kg pentobarbital.

[0047] S34: Fixation: The head of the anesthetized rat was fixed on a stereotaxic apparatus so that the top of the head was parallel to the horizontal plane.

[0048] S35: Positioning: trim the hair on the rat's head, wipe the scalp with iodine to disinfect it, cut the scalp along the middle suture of the skull to an appropriate length, wipe with hydrogen peroxide to remove the fascia on the skull to expose the skull; find the lambdoid suture and use it as the origin, 3.0 mm backward, and 1.5 mm to the left and right of the midline to mark the punching position.

[0049] S36: Drilling: After positioning, use a skull drill to drill holes at the marked locations.

[0050] S37: Implantation of tube: Gently insert the needle of the microinjector into the hole and fix it to the surface of the skull with dental cement.

[0051] S38: Drug administration: Drug administration began 3 days after postoperative recovery; anesthetized the rat and fixed its head, used a microsyringe to draw up the drug and connected the hose and the drug administration needle, unscrewed the cap of the buried tube, removed the plug, inserted the drug administration needle into the drug administration sleeve, and slowly injected the following reagents into the bilateral cerebellum at 0.5μl / min.

[0052] VPA+r-PGRN group: 2.5 μl r-PGRN solution was injected into each cerebellum; VPA+Vehicle group: 2.5 μl sterile saline was injected into each cerebellum.

[0053] After each administration, the needle was withdrawn for 10 minutes and the plug was inserted and the cap was screwed on to prevent foreign matter from entering the administration sleeve. During the entire administration process, the rats were placed on a heating blanket to keep warm until they woke up from anesthesia, and then returned to the cage for individual feeding. This was done once a day for 7 days.

[0054] Step 4: Conduct behavioral experiments on ASD rats, including cerebellar behavioral tests and autism-like behavioral tests.

[0055] The specific steps are as follows:

[0056] S41: Cerebellar behavioral test (motor coordination and muscle tone);

[0057] S411: Static beam test: A wooden stick 61 cm long and 2 cm in diameter was placed about 46 cm from the ground. The rats were trained and adapted to the equipment one day before the formal test. The next day, each rat was tested on the static beam three times, and the time required to completely pass the balance beam and the number of times the hind paws slipped were recorded. If the rat was motionless or fell from the balance beam or failed to pass the balance beam within the maximum recorded time (40 s), the maximum passing time of 40 s was recorded as the result of this experiment, and the whole process was recorded by a camera. After the three tests, the proportion of each rat passing the balance beam was calculated.

[0058] S412: Righting reflex test: The rat was placed 12.5 cm away from the table. The rat began to let go from the supine position for 3 times and the whole process was recorded by camera. The video speed was reduced by PotPlayer video software for frame-by-frame analysis to count the time when the four paws touched the ground at the same time.

[0059] S413: Gait analysis test: The hind paws and front paws of each rat were painted with non-toxic and odorless blue and ink red, respectively, and the rats were allowed to pass through a corridor with a 60×10 cm white paper on the bottom. Each rat was tested 3 times. After the rats passed, the paper was taken out and dried naturally, and the length of the footprints left by the rats was measured. The average value of the 3 experiments for each rat was taken.

[0060] S414: Traction test: Build a steel wire (2mm in diameter, 200cm in length) about 46cm above the ground. At the beginning of the test, let the rats actively grab the steel wire with their two front paws, and start timing when they let go until they fall off. The rats' hanging postures are scored, and the scoring criteria are: 3 points, both hind paws grab the steel wire; 2 points, one hind paw grabs the steel wire; 1 point, neither hind paw grabs the steel wire; each individual is tested twice, and rest for at least 35min to recover physical strength after each test. The statistical results are the average of the two experiments.

[0061] S42: Autism-like behavior test;

[0062] S421: Open field test and self-grooming test: This experiment uses an open opaque acrylic box (100×100×46.7 cm) to record the animal's movement activities and exploration.

[0063] The Smart behavioral detection software was used to divide the bottom of the box into 25 uniform square grids, with the 9 middle grids as the middle grid and the 16 outer grids as the outer grids. Before the experiment, each rat was given 10 minutes to enter the box to familiarize itself with the environment. After the experiment started, the timer was set for 10 minutes, during which the rats could explore freely.

[0064] The Smart behavioral software was used to record the time of the rats' intermediate and peripheral grids, and the time of the rats' hair stroking (repetitive stereotyped behavior) and the number of defecation times (anxiety status) were manually recorded.

[0065] After each rat was tested, the excrement was cleaned and the bottom of the box was wiped with 75% alcohol to remove residual odor.

[0066] S422: Three chamber test: A rectangular transparent acrylic box (120×45×46 cm) was divided into three chambers of equal size, which were marked from left to right as Chamber A, Chamber B, and Chamber C using Smart behavioral monitoring software.

[0067] This experiment consists of three stages:

[0068] Adaptation phase: Place iron cages of the same size, shape, color, and smell in chambers A and C, respectively labeled as circle a and circle b. Place the experimental rats from chamber B and allow them to move freely for 10 min.

[0069] Social test phase (0-10 min): Place an object (Object) in circle a and a strange male rat of the same age (Stranger 1) in circle b; put the test rat in chamber B, and the Smart software will automatically time for 10 min and monitor the time the rat stays in chambers A, B, and C, and the time the rat spends in olfactory contact with the Object and Stranger 1.

[0070] Social preference test phase (10-20 min): temporarily remove the test rat, replace the item in circle a with a new stranger male rat of the same age (Stranger 2), and keep circle b unchanged; then put the test rat into chamber B, and continue to use Smart software to record the time the rat stays in each chamber and the time the rat communicates with Stranger 1 and Stranger 2 through olfactory contact.

[0071] After all tests are completed, the time the rats stay in each chamber, the olfactory contact time between the rats and the objects, Stranger 1, and Stranger 2 are exported to Excel for data analysis.

[0072] S423: Juvenile social play test: The rats were placed in a square acrylic box (50×50 cm) covered with 5 cm thick clean bedding; the rats were allowed to move freely in the box for 5 min.

[0073] After resting for 5 minutes, the tested rats and unfamiliar rats (unfamiliar rats of the same age and gender as the tested rats) were placed in the test field for 10 minutes; during the test, the experimental rats' interaction time (following, sniffing), attack time of unfamiliar rats of the same age, and digging time of bedding were recorded.

[0074] Step 5: Extract brain tissue protein from ASD rats; after anesthetizing the rats, perfuse with saline, and finally remove the rat brains for tissue protein extraction.

[0075] The specific steps are as follows:

[0076] S51: Anesthesia: After weighing, 4% chloral hydrate (0.01 ml / g) was injected intraperitoneally, and subsequent experimental operations were performed after complete anesthesia.

[0077] S52: perfusion;

[0078] S521: Place the anesthetized rat in a supine position on a tray, cut the abdominal cavity 1-2 cm below the xiphoid process with a scissor, separate the diaphragm, and expose the heart;

[0079] S522: Insert the syringe needle into the left ventricle, cut the right atrial appendage, and inject normal saline into the heart at a constant speed; stop the perfusion when the color of the liver changes from red to white or light pink.

[0080] S53: Sampling: After perfusion, the neck was cut, and the skull was carefully removed using forceps and scissors to expose the entire brain. The brain tissue was quickly partitioned on an ice box.

[0081] S54: tissue protein extraction;

[0082] S541: Place the isolated fresh cerebellum tissue on a tin foil and weigh it on a balance;

[0083] S542: According to the instructions of the Bio-Tech RIPA tissue lysis buffer, 7 μl of RIPA lysis buffer mixture was added to each 1 mg sample (RIPA lysis buffer mixture was prepared and used immediately according to RIPA:PMSF=100:1) (RIPA: radioimmunoprecipitation assay; PMSF: phenylmethylsulfonyl fluoride);

[0084] S543: Precool the homogenizer sample fixing tray to -20°C, place the sample in it and start grinding; after grinding, place the sample on ice for 30 minutes, and centrifuge at low temperature for 20 minutes (4°C, 10,000 g);

[0085] S544: The supernatant was collected into a new EP tube and stored in a -80°C refrigerator.

[0086] Step 6: Determine the protein concentration by BCA (bicinchoninic acid) method; use the BCA method to determine the concentration of the extracted tissue protein; follow the instructions of the Bio-Tech BCA protein concentration determination kit.

[0087] Step 7: Western blot; specifically including: SDS-PAGE gel, electrophoresis, membrane transfer, blocking, incubation with primary antibody, washing, incubation with secondary antibody, washing, development, and band analysis.

[0088] The specific steps are as follows:

[0089] S71: Prepare SDS-PAGE gel:

[0090] S711: Separation gel: Prepare a separation gel of appropriate concentration according to the molecular weight of the target protein. After stirring evenly, slowly and evenly add the separation gel between the glass plates. Then add a small amount of anhydrous ethanol to make the surface of the separation gel flat and let it stand at room temperature for 30 minutes.

[0091] S712: Concentrated gel: When a clear dividing line appears between the separation gel and the anhydrous ethanol, gently pour out the anhydrous ethanol and absorb the remaining liquid. Prepare the concentrated gel and mix it evenly, slowly and evenly add it between the glass plates, and gently insert the sample comb, and let it stand at room temperature until it solidifies and then use it.

[0092] S72: electrophoresis and membrane transfer;

[0093] S721: Sample loading: Take out the glass plate and fix it in the electrophoresis tank, add 5 μl of protein marker to the first lane, and then add the samples to each lane in the order of samples, with 20-40 μg of protein loaded in each lane;

[0094] S722: Electrophoresis: Adjust the electrophoresis instrument to constant voltage mode (90V). The electrophoresis time needs to be determined according to the different gel concentrations and the different molecular weights of the target proteins.

[0095] S723: Determine the position of the target protein according to the position of the protein marker, cut the target protein and the internal reference protein at the same time and place them on the filter paper of the "sandwich" model, cut the PVDF membrane (0.45μm) of the corresponding size of the gel and mark it, activate it with methanol for 15-30s and then put it into the electrotransfer solution for cleaning, and cover the corresponding marked PVDF membrane on the corresponding gel to avoid bubbles;

[0096] S724: Transfer: After loading the "sandwich", fix it in the electrotransfer tank, add the electrotransfer solution, close the lid and start electrotransfer. Pay attention to distinguish the positive and negative poles. Add ice-water mixture to the outside of the electrotransfer box to offset the large amount of heat energy generated during the electrotransfer process. The electrophoresis condition is constant current mode (250mA). The electrotransfer time is determined according to the molecular weight of the target protein.

[0097] S73: Blocking: Prepare 5% skimmed milk powder. After the transfer, place the PVDF membrane in the skimmed milk powder for blocking. Place the membrane on an orbital shaker (50 rpm) at room temperature and shake slowly for 2 hours.

[0098] S74: Incubate with primary antibody: dilute the antibody with primary antibody diluent according to the antibody instructions. After blocking, completely immerse the PVDF membrane in the antibody solution and incubate overnight at 4°C.

[0099] S75: Washing: The next day, the PVDF membrane was taken out, placed in TBST (Tris-HCl buffered saline), and washed on an orbital shaker (120 rpm) for 10 min x 3 times.

[0100] S76: Incubation with secondary antibody: Use secondary antibody of corresponding species according to the source species of primary antibody, dilute to 1:5000, immerse the PVDF membrane in it, and incubate on orbital shaker (50 rpm) at room temperature for 1 hour.

[0101] S77: Washing: Repeat S75 after the secondary antibody incubation.

[0102] S78: Development: In a dark environment, according to the instructions of the ECL (enhanced chemiluminescence) luminescence kit, mix solution A and solution B in equal proportions, drop them onto the PVDF membrane, and use a chemiluminescence imaging system for exposure. The exposure time is set to automatic.

[0103] S79: Band analysis: Use Image J image analysis software to perform grayscale value analysis on the obtained target protein and internal reference protein images.

[0104] Step 8: Tissue Golgi staining: The rat brain tissue is peeled off and processed, and then brain tissue sections are made and staining experiments are performed under light-proof conditions.

[0105] The specific steps are as follows:

[0106] S81: Sampling: After anesthesia, use scissors to directly cut the neck, cut the scalp, carefully peel off the brain tissue, and soak the cerebellum directly in a mixture of solution 1 and solution 2 prepared one day in advance with 5 times the volume of brain tissue, and store it at room temperature away from light.

[0107] S82: After the brain tissue has been immersed in the mixture of solution 1 and solution 2 for 14 days, the tissue is removed and placed in an equal volume of solution 3 at 4°C for 12-24 hours.

[0108] S83: Replace solution 3 once and let stand at 4°C for 24-72h;

[0109] S84: Remove the brain tissue away from light and use filter paper to absorb moisture. Wrap the brain tissue with aluminum foil, mark it and store it in a -80℃ refrigerator until needed.

[0110] S85: The slides were embedded with gelatin.

[0111] S86: Take out the sample at -80℃ and put it into a freezing microtome, fix the sample in a mold with OCT embedding medium; use the freezing microtome to cut the sample into 100μm slices and stick them on the gelatin-embedded slide with solution 3.

[0112] S87: Drain excess liquid and place the slide in a 37°C dry incubator for more than 24 hours until the sections are dry;

[0113] S88: Perform the following staining process under light-proof conditions:

[0114] S881: Washing: Place the slices in a staining jar, add distilled water along the jar wall until the sample is submerged, and soak for 3 min × 2 times;

[0115] S882: Staining: Prepare the staining solution (2 ml solution 4 + 2 ml solution 5 + 6 ml distilled water), pour it into the staining jar, and transfer the sections into it to stain for 15 minutes;

[0116] S883: Washing: Repeat S881, soaking for 4 min × 2 times;

[0117] S884: Dehydration: The sections were placed in 50% (5 min), 70% (5 min), 95% (5 min) and anhydrous ethanol (20 min) for gradient dehydration;

[0118] S885: Decolorization: After dehydration, soak the sample in xylene for 10 min to decolorize;

[0119] S886: Sealing: Take out the sample slide and wipe off the liquid, add high concentration neutral resin on the sample, and seal it with a coverslip.

[0120] Step 9: Analysis of dendritic branches of Purkinje cells and counting of dendritic spines: The stained brain tissue sections were photographed under a microscope, and the dendritic branches and dendritic spines of Purkinje cells were counted.

[0121] The specific steps are as follows:

[0122] S91: Analysis of dendritic branching in Purkinje cells;

[0123] S911: Use Image J to open the image to be analyzed and set the scale;

[0124] S912: Use Neuron J plugin to trace neurons, draw Purkinje cell dendrite branching diagram, and generate two-dimensional distribution map of neurons;

[0125] S913: Adjust the image format to 8 bits and then perform Sholl analysis.

[0126] S92: Purkinje cell dendritic spine count Neuron J was used to count the number of dendritic spines, plot the length of the counted dendritic branches and calculate the dendritic spine density.

[0127] Step 10: Statistical methods; GraphPad Prism software was used for data analysis and statistics, and the results were expressed as mean ± standard error (mean ± SEM); t-test was used for statistical analysis for comparison between two groups; two-way ANOVA was used for comparison between multiple groups, and Sidak method was used as post hoc test; P < 0.05 indicated statistical significance.

[0128] The experimental results are as follows:

[0129] 1. VPA induced increased expression of lysosomal proteases in the cerebellum of ASD rats. Western blot was used to detect the protein expression levels of lysosomal proteases {CTSD (cathepsin D), CTSB (cathepsin B), CTSL (cathepsin L)} in the cerebellum tissue of the two groups of rats 35 days after birth. The results showed that the expression levels of CTSD, CTSB, and CTSL in the VPA group were significantly higher than those in the CON group (P < 0.05), indicating that the lysosomal function in the cerebellum of ASD rats is enhanced.

[0130] 2. VPA-induced enhanced autophagy in the cerebellum of ASD rats. The expression levels of autophagy-related proteins ULK1, LC3 (microtubule-associated protein light chain 3), and P62 (chelate 1) were detected by Western blot. The ratio of ULK1, an autophagy initiator, and LC3Ⅱ / Ⅰ, a marker of autophagy, were significantly increased in the VPA group (P<0.01), while the expression of P62, an autophagy substrate degraded by autophagy, was decreased (P<0.01). The above results indicate that the autophagy process is enhanced in the cerebellum of ASD rats.

[0131] 3. Intracerebellar injection of r-PGRN improved the motor coordination ability of ASD rats. The results of the balance beam test showed that compared with the VPA group, the rats in the VPA+r-PGRN group had a shorter balance beam passing time (P<0.001), a significantly reduced number of hind paw slips (P<0.001), and a significantly increased balance beam passing rate (P<0.0001). The results of the plane righting test showed that the time required for the rats in the VPA+r-PGRN group to go from supine position to full landing of all four paws was significantly shorter than that in the VPA group (P<0.0001). The results of the gait analysis test showed that the forepaw step length (P<0.001), hind paw step length (P<0.0001), and stride width (P<0.001) of the VPA+r-PGRN group were significantly increased compared with those of the VPA group. The hanging test showed that the hanging time (P<0.05) and hanging score (P<0.001) of the rats in the VPA+r-PGRN group were significantly increased compared with those of the VPA group.

[0132] 4. Intracerebellar injection of r-PGRN improved autistic-like behaviors in ASD rats. The open field test data showed that the time spent in the middle grid by rats after r-PGRN treatment was significantly increased (P<0.0001), indicating that the autonomous exploration ability and curiosity of the surrounding environment of the r-PGRN group rats increased, but there was no significant difference in the total movement distance between the groups. The self-stroking time of the VPA+r-PGRN group was significantly lower than that of the VPA group (P<0.01).

[0133] The first stage (0-10 min) of the three-box social test mainly tested the social ability of rats. Compared with the VPA group, the rats in the VPA+r-PGRN group spent significantly less time in chamber A where the object was located (P<0.0001), spent longer time in chamber C where stranger mouse 1 was located (P<0.05), spent significantly less time sniffing and touching the object (P<0.001), and spent significantly longer time communicating with stranger mouse 1 (P<0.001). The second stage (10-20 min) mainly tested the social preference of rats. Compared with the VPA group, the rats in the VPA+r-PGRN group spent longer time in chamber A where stranger mouse 2 was located (P<0.01), spent shorter time in chamber C where stranger mouse 1 was located (P<0.001), spent significantly more time sniffing and touching stranger mouse 2 (P<0.001), and spent significantly less time sniffing and touching stranger mouse 1 (P<0.01), showing a preference for communicating with the new stranger mouse 2.

[0134] The results of the youth play test showed that compared with the VPA group, the VPA+r-PGRN group had significantly increased contact time (P<0.001) and digging time (P<0.05) with social mice, but no difference in aggression.

[0135] 5. Intracerebellar injection of r-PGRN improved the dendritic development of cerebellar Purkinje cells in ASD rats. SHOLL analysis was performed after tissue Golgi staining. The results showed that the dendritic radius of Purkinje cells in the VPA+r-PGRN group (with the cell body as the center and the farthest dendritic branch as the radius) was significantly larger than that in the VPA group (P<0.01). The number of intersections between the dendritic branches and concentric circles of Purkinje cells in the VPA+r-PGRN group was significantly more than that in the VPA group at around 150μm (P<0.0001). Statistical analysis was performed on the dendritic spine density of cerebellar Purkinje cells. The results showed that the dendritic spine density in the VPA+r-PGRN group was significantly higher than that in the VPA group (P<0.001).

[0136] 6. Intracerebellar injection of r-PGRN reduces the expression level of lysosomal proteases in the cerebellum of ASD rats; the expression of lysosomal proteases in the cerebellum tissue of rats in each group was detected by Western blot. The results showed that intracerebellar injection of r-PGRN can downregulate the expression levels of CTSD (P<0.05), CTSB (P<0.05), and CTSL (P<0.01) in VPA-induced ASD rats.

[0137] 7. Intracerebellar injection of r-PGRN reduces cerebellar autophagy in ASD rats. Western blot was used to detect the expression of autophagy-related proteins ULK1, LC3Ⅱ / Ⅰ, and P62. Intracerebellar injection of r-PGRN can significantly downregulate the expression level of autophagy initiator ULK1 (P<0.01) and the ratio of autophagy marker LC3Ⅱ / Ⅰ (P<0.01) in the cerebellum tissue of ASD rats induced by VPA, and upregulate the expression of autophagy degradation substrate P62 (P<0.001). The above data indicate that PGRN can reduce the autophagy process in the cerebellum tissue of ASD rats.

[0138] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for detecting the effect of PGRN regulation on cerebellar synaptic development in ASD rats, characterized in that: The specific steps include: Step 1: Breeding experimental rats; purchasing 60 Sprague-Dawley rats of reproductive age, with a male-female ratio of 2:1, and feeding and drinking water freely for three to five weeks; Step 2: Prepare VPA-induced ASD rat model; perform pregnancy culture on the experimental rats, and randomly divide the female rats into two groups at 12.5 days of pregnancy. The VPA group is injected with VPA solution via intraperitoneal injection, and the control group is injected with an equal volume of sterile saline; Step 3: Implant the microinjector in the cerebellum of ASD rats and administer the drug; divide the VPA offspring rats into VPA group, VPA+r-PGRN group, VPA+Vehicle group, and the control group is CON group; prepare r-PGRN reagent, perform experimental treatment on rats, and finally implant the microinjector and administer the drug to the rats; Step 4: Conduct behavioral experiments on ASD rats; Including cerebellar behavioral tests and autism-like behavioral tests; Step 5: Extract brain tissue protein from ASD rats; anesthetize the rats, perfuse with saline, and finally take out the rat brains to extract tissue protein; Step 6: BCA method protein concentration determination; The concentration of extracted tissue protein was determined by BCA method; Step 7: Western blot; specifically including: SDS-PAGE gel, electrophoresis, membrane transfer, blocking, incubation with primary antibody, washing, incubation with secondary antibody, washing, development, and band analysis; Step 8: Tissue Golgi staining: The rat brain tissue is peeled off and processed, and then brain tissue sections are made and staining experiments are performed under light-proof conditions; Step 9: Analysis of dendritic branches of Purkinje cells and counting of dendritic spines: The stained brain tissue sections were photographed under a microscope, and the dendritic branches and dendritic spines of Purkinje cells were counted; Step 10: Statistical methods: The obtained data were analyzed and statistically analyzed using GraphPad Prism software, and the results were expressed as mean ± standard error.

2. The method for detecting the effect of PGRN regulation on cerebellar synaptic development in ASD rats according to claim 1, characterized in that: In the step 3, 2.5 μl of r-PGRN solution was injected into each cerebellum of the experimental rats in the VPA+r-PGRN group, and 2.5 μl of sterile saline was injected into each cerebellum of the experimental rats in the VPA+Vehicle group.

3. The method for detecting the effect of PGRN regulation on cerebellar synaptic development in ASD rats according to claim 1, characterized in that: The cerebellar behavioral tests include a balance beam test, a flat righting test, a gait analysis test, and a hanging test; the autism-like behavioral tests include an open field test and a self-stroking test, a three-box social test, and a youth play test.

4. The method for detecting the effect of PGRN regulation on cerebellar synaptic development in ASD rats according to claim 1, characterized in that: The tissue protein extraction in step 5 specifically includes the following steps: S541: Place the isolated fresh cerebellum tissue on a tin foil and weigh it on a balance; S542: According to the instructions of the Bio-Tech RIPA tissue lysis buffer, add 7 μl of RIPA lysis buffer mixture for every 1 mg of sample; S543: Precool the homogenizer sample fixing tray to -20°C, place the sample in it and start grinding; after grinding, place the sample on ice for 30 minutes and centrifuge at low temperature for 20 minutes; S544: The supernatant after centrifugation was collected into a new EP tube and stored in a sealed environment at -80°C.

5. The method for detecting the effect of PGRN regulation on cerebellar synaptic development in ASD rats according to claim 1, characterized in that: The rat brain tissue peeled in step eight is immersed in a mixture of solution 1 and solution 2 with a volume 5 times that of the brain tissue. After the rat brain tissue is immersed for 14 days, it is transferred to an equal volume of solution 3 and placed at 4° C. for 12-24 hours. Then, solution 3 is replaced and placed at 4° C. for 24-72 hours. Finally, the moisture of the rat brain tissue is removed and the rat brain tissue is sealed and stored at -80° C.

6. The method for detecting the effect of PGRN regulation on cerebellar synaptic development in ASD rats according to claim 1, characterized in that: The staining experiment in the step eight is carried out under light-proof conditions, specifically including washing, staining, washing, dehydration, decolorization, and sealing.

7. The method for detecting the effect of PGRN regulation on cerebellar synaptic development in ASD rats according to claim 1, characterized in that: The Purkinje cell dendritic branch analysis uses Image J to open the image to be analyzed and set the scale; uses the Neuron J plug-in to track neurons, draw the Purkinje cell dendritic branch diagram, and generate a two-dimensional distribution map of neurons; and adjusts the image format to 8 bits before performing Sholl analysis.

8. The method for detecting the effect of PGRN regulation on cerebellar synaptic development in ASD rats according to claim 1, characterized in that: The Purkinje cell dendritic spine number statistics were performed using Neuron J to count the number of dendritic spines, draw the length of the counted dendritic spines and calculate the dendritic spine density.

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