A method for modeling alzheimer's disease in tree shrews

Abstract

This invention discloses a method for establishing Alzheimer's disease in tree shrews. The specific steps involve mixing an ethanol solution of 0.5 μg / μl Microcystin-LR with a muscarine solution, and then injecting 2 μl into each of the bilateral Meynert basal ganglia of the tree shrew. After administration, the tree shrew is fed normally for two weeks to complete the Alzheimer's disease model. The muscarine solution is prepared under sterile conditions by adding 200 μl of sterile saline to a vial containing 1 mg of muscarine, repeatedly pipetting to ensure complete dissolution, resulting in a concentration of 5 μg / μl. The Microcystin-LR solution is a commercially available product. This invention involves injecting a mixture of 0.1 μg MC-LR and 3 μg IBO solution into the bilateral Meynert basal ganglia of tree shrews, establishing a stable tree shrew AD model after two weeks. Three days after administration, tree shrews exhibited AD behavioral manifestations, such as lethargy and reduced activity. Two weeks later, the tree shrew brains showed typical AD pathological manifestations, including Aβ deposition and elevated expression levels at multiple phosphorylation sites of Tau protein.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology for diseases, and particularly relates to a method for modeling Alzheimer's disease in tree shrews. Background Technology

[0002] Alzheimer's disease (AD), also known as senile dementia, is a common, progressive neurodegenerative disease. Its main clinical manifestations include progressive memory loss, cognitive impairment, behavioral abnormalities, and social difficulties, eventually leading to loss of the ability to live independently. Patients often die from complications within 10-20 years of onset. The prevalence increases significantly with age. This not only places immense pressure on patients and their families but also imposes a huge economic burden on society. Therefore, seeking methods to prevent AD, finding new treatment pathways, and developing new drugs for treating AD are both necessary and urgent.

[0003] Alzheimer's disease (AD) has an insidious and progressive course. Generally, before clinical manifestations are detected, the brain has already undergone a variety of subtle pathophysiological changes. These changes include the abnormal accumulation of Aβ toxic proteins produced by APP shearing, forming amyloid plaques; excessive phosphorylation of tubulin tau leading to numerous neurofibrillary tangles; reduced brain glucose metabolism; uncontrolled activation of microglia; chronic oxidative stress damage; and the secretion of neurotoxins and inflammatory factors. However, the specific pathogenesis remains unclear, with theories including the β-amyloid cascade theory, the tau protein theory, the cholinergic damage theory, the oxidative stress / mitochondrial cascade theory, the excitatory glutamate toxicity theory, and the gene mutation theory. Due to the complexity of AD pathogenesis and the numerous steps involved, there are currently no satisfactory therapeutic drugs. Establishing suitable animal models is a crucial step in exploring the pathogenesis and potential therapeutic drugs for AD. Existing AD animal models include aging, injection-induced, transgenic, and combined models. These four models are widely used in AD pathogenesis and drug treatment research, each with its own advantages and disadvantages. However, existing animal models of Alzheimer's disease (AD) are time-consuming and inefficient, severely impacting subsequent research progress. For example, D-galactose combined with IBO requires more than two months to establish an AD model. Therefore, developing animal models that are more closely related to humans, require less time, and can effectively simulate AD pathology is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a method for modeling Alzheimer's disease in tree shrews.

[0005] The objective of this invention is achieved as follows: a method for modeling Alzheimer's disease in tree shrews, the specific steps of which are as follows:

[0006] (1) Reagent preparation:

[0007] Microcystin-LR solution: This is an ethanolic solution of 0.5 μg / μl Microcystin-LR; Microcystin-LR is a highly effective protein phosphatase inhibitor, abbreviated as MC-LR, which is a microcystin or microcystin toxin, and is a commercially available product purchased from MCE.

[0008] Preparation of amatoxins solution: Under aseptic conditions, add 200 μl of sterile physiological saline to a vial containing 1 mg of amatoxins, and repeatedly pipette until completely dissolved to prepare a concentration of 5 μg / μl.

[0009] (2) After mixing the Microcystin-LR solution prepared in step (1) with the amatoxinsine solution, inject 2 μl into each of the bilateral Meynert basal nuclei of the tree shrew. The aforementioned mixed solution was obtained under sterile conditions by diluting it according to the ratio of Microcystin-LR: amatoxinsine solution: sterile physiological saline = 1:3:6.

[0010] (3) After the drug administration is completed, normal feeding is carried out for 2 weeks to complete the Alzheimer's disease model.

[0011] The beneficial effects of this invention are:

[0012] This invention involves injecting a mixture of MC-LR (0.1 μg) and IBO solution (3 μg) into the bilateral Meynert basal ganglia of tree shrews, establishing a stable tree shrew AD model after 2 weeks. Three days after administration, tree shrews exhibited AD behavioral manifestations, such as lethargy and reduced activity. Two weeks later, the tree shrew brains showed typical AD pathological manifestations, including Aβ deposition and elevated expression levels at multiple phosphorylation sites of Tau protein. Attached Figure Description

[0013] Figure 1 This is a comparative diagram showing the expression of Aβ protein in the hippocampus and cortex of tree shrews in five sets of comparative experiments in this application;

[0014] Figure 2 This is a comparative diagram showing the expression of Aβ protein in the hippocampus and cortex of tree shrews in five sets of comparative experiments in this application;

[0015] Figure 3 This is a comparison diagram of the changes in the expression of p-Tau (T231), p-Tau (S396), and Tau proteins in tree shrews during five comparative experiments in this application;

[0016] Figure 4 This is a comparison diagram of the changes in p-Tau(T231) protein expression in tree shrews in five groups of comparative experiments in this application;

[0017] Figure 5 This is a comparison diagram of the changes in p-Tau(S396) white blood cell expression in tree shrews during five comparative experiments in this application;

[0018] Figure 6 This is a comparison diagram of the changes in Tau protein expression in tree shrews during the five comparative experiments in this application. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments are within the protection scope of the present invention.

[0020] 1. Laboratory animals

[0021] Thirty ordinary-grade male Burmese tree shrews, weighing 120-150 g, were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK(Yunnan)K2023-0003] and housed in the ordinary-grade animal facility of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK(Yunnan)K2021-0003]. They were housed in stainless steel cages at a room temperature of (24±2)℃, relative humidity of (50±5)%, and a light / dark cycle of 12 h / 12 ​​h.

[0022] 2. Main reagents and consumables

[0023] Reagent Name company Microcystin-LR Mce Amanoic acid Mce β-Amyloid antibody Abcam Tau antibody Abcam p-Tau (T231) antibody Abcam p-Tau (S396) antibody Abcam GAPDH antibody Abcam Anti-rabbit IgG CST RIPA lysis buffer (strong) Azure Sky Prestained protein marker Thermo Fisher Bovine serum albumin (BSA) Sigma SurperECLPlus Superluminescent Prile Blocking sheep serum Zhongshan Jinqiao Universal Two-Step Detection Kit Zhongshan Jinqiao DAB colorimetric reagent kit Zhongshan Jinqiao PVDF membrane Millipore

[0024] 3. Main Instruments

[0025] Instrument Name company stereotaxic brain Rayward Micro-injection pump Lange Nanophotometer IMPLEN Protein electrophoresis tank, electroporation tank, power supply Bio-Rad ECL luminescence imaging system Tanon Automatic dehydrator Wuhan Junjie Paraffin embedding machine Leica, Germany Rotary slicer Leica, Germany microscope OLYMPUS BX53

[0026] 4. Preparation of reagents

[0027] Microcystin-LR (MC-LR): The purchased reagent was an ethanol solution with a concentration of 0.5 μg / μl MC-LR.

[0028] Amantadine (IBO): Under aseptic conditions, add 200 μl of sterile physiological saline to a vial containing 1 mg of IBO, and repeatedly pipette until completely dissolved to prepare a concentration of 5 mg / ml (5 μg / μl).

[0029] 5. Experimental Methods

[0030] 5.1 Animal Model Making

[0031] Tree shrews were randomly divided into the following five groups

[0032] ① Normal (NC) group: Tree shrews were fed normally without any treatment.

[0033] ② Sham surgery group: 2 μl of normal saline (containing 20% ​​ethanol) was injected bilaterally into the Meynert basal ganglia.

[0034] ③ MC-LR group: 2 μl of 0.05 μg / μl MC-LR solution was injected into each bilateral Meynert basal ganglia (under aseptic conditions, 0.5 μg / μl MC-LR was diluted with sterile physiological saline at a ratio of 1:9).

[0035] ④ IBO group: 2 μl of 1.5 μg / μl IBO solution was injected into each of the bilateral Meynert basal ganglia (under aseptic conditions, 5 μg / μl IBO solution was diluted with sterile physiological saline at a ratio of 3:7).

[0036] ⑤ MC-LR+IBO combined group: 2 μl of a mixture of MC-LR (0.05 μg / μl) and IBO solution (1.5 μg / μl) was injected into each Meynert basal ganglia bilaterally (under aseptic conditions, the solution was diluted with sterile saline at a ratio of 1:3:6 to 0.5 μg / μl MC-LR: 5 μg / μl IBO solution).

[0037] 5.2 Bilateral Meynert basal ganglia (BM) injection method

[0038] (1) Fixing the tree shrew: The tree shrew was anesthetized by intraperitoneal injection of 3% sodium pentobarbital at 2 μl / g body weight. After complete anesthesia, the hair on the cranial face of the tree shrew was removed with animal clippers and fixed on the stereotaxic device. The ear rod was inserted into the tree shrew's ear canal and the left and right ear rods were balanced so that the line connecting the two ears of the tree shrew was in the same straight line as the ear rod. The tree shrew's incisors were pried open with tweezers and the upper incisors were locked in the hole of the adapter. The nose rod locking screw was pressed down and the height of the incisor clamp was adjusted up and down, and the position of the adapter was adjusted back and forth to keep the cranial face of the tree shrew horizontal. The screw was locked so that the tree shrew's head could not move. According to the tree shrew's body size, the tree shrew's body was raised with foam on the base of the stereotaxic device so that the head and body were kept horizontal to prevent the tree shrew's breathing from being blocked.

[0039] (2) Micro-injection pump parameter settings: Turn on the power switch of the micro-injection pump, set the operation mode to first draw and then infuse, and set the drawing parameters: the volume of liquid drawn is 2μl, the drawing time is 0.5min; the interval between drawing and infusing is 0.5min; and the infusing time is 5min.

[0040] (3) Positioning reference point: After fixing the RN type injection needle (specification 10μL) on the holder, turn on the power switch of the digital positioning instrument, disinfect the skin surface of the tree shrew skull with iodine, make a longitudinal incision from the line connecting the two ears to the eyes to expose the skull, find the intersection of the interauricular line and the sagittal suture, i.e. the anterior fontanelle point, use this point as the reference point of the three-dimensional coordinate system, move the three-dimensional manipulator arm to align its positioning point with the anterior fontanelle point, and press the "CLR" key of the digital display base X, Y, Z axis to zero (desktop digital positioning instrument).

[0041] (4) Locating the target area: Determine the coordinate values ​​of the basal ganglia relative to the anterior fontanelle, namely ML value (X axis), AP value (Y axis), and DV value (Z axis). Starting from the anterior fontanelle reference point, set the coordinate point according to the position of the basal ganglia. Method 1: AP: 8.7mm anterior to the anterior fontanelle, ML: ±3mm of the sagittal suture. Move the operating arm the corresponding distance (absolute values ​​of AP and ML) to the target position (Z axis does not move). Then, use the positioning pin as a reference point and mark it with a marker. Remove the cross-shaped operating arm and drill a hole in the skull.

[0042] (5) Experiment: After the micro-injection pump is turned on to draw the prepared compound solution, within 0.5 min, according to the determined value DV (Z-axis coordinate of point 1): 10 mm below the dura mater, the operating arm is moved downward to the target depth. After the injection is completed, the needle is left in place for 5 min, and then the needle is slowly withdrawn at a speed of 1.0 mm / min. The coordinate value is set according to the method of locating the target area to carry out the injection experiment at another coordinate point. After the injection is completed, the RN-type injection needle is withdrawn, the injection hole is glued with bone glue, the wound is sutured and disinfected with povidone-iodine, and an appropriate amount of erythromycin ointment is applied to the wound. After the operation, penicillin (22000 IU / kg / d) is injected into the peritoneum for 3 consecutive days.

[0043] 5.3 Behavioral Observations of Tree Shrews

[0044] After the injection, the tree shrews in each group were observed daily for changes in their diet, exercise, coat color, and mental state.

[0045] 5.3 Immunohistochemical detection of Aβ expression

[0046] After the tree shrews were administered the drug, they were fed normally for two weeks before the tree shrews were harvested. After anesthesia, the tree shrews were perfused with physiological saline and then 4% paraformaldehyde solution through their hearts. The brains were then removed and placed in 4% paraformaldehyde to prepare paraffin sections for subsequent immunohistochemical staining.

[0047] Immunohistochemical staining: Paraffin sections were dewaxed to water, then immersed in an antigen retrieval box containing citrate buffer (pH 6.0) in a microwave oven for antigen retrieval. Blocked with 3% hydrogen peroxide for 30 min in the dark (to eliminate endogenous peroxidase activity); blocked with 5% sheep serum for 1 h, then incubated overnight at 4°C with primary antibody (Aβ1:400). After rinsing with PBST, a universal two-step assay reagent was added, and incubated at room temperature for 30 min; after rinsing with PBST, freshly prepared DAB chromogenic solution was added, and incubated at room temperature in the dark for 5-15 min. Staining was terminated with distilled water; counterstained with hematoxylin, differentiated with 1% hydrochloric acid ethanol, routinely dehydrated and cleared, mounted with neutral resin, and the staining results were observed and photographed under a regular optical microscope.

[0048] 5.4 Western bolt inspection

[0049] After the tree shrews were administered the drug, they were fed normally for two weeks before tissue collection. Following anesthesia, the thorax was opened, and the heart was perfused with physiological saline. After observing the liver turning white, the brain tissue was removed. The brain was isolated on ice and gently rinsed with 0.1 mol / L PBS. 500 μL of RIPA lysis buffer (containing 50 μL of protease inhibitor) was added per 100 mg of fresh tissue. The tissue was thoroughly minced with sterile ophthalmic scissors and homogenized using an ultrasonic cell disruptor for 20 seconds on ice. Lysis was then carried out on ice for 20 minutes. The samples were centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected. Approximately 360 μL of supernatant was aspirated from each tube, and 90 μL of 5× loading buffer (1 / 4 of the supernatant volume) was added. The mixture was thoroughly mixed, briefly centrifuged for 10 seconds, and then boiled in a metal bath at 100°C for 10 minutes to fully denature the proteins. Protein concentration was measured after the samples cooled. Then, SDS-PAGE protein electrophoresis was performed. Electrophoresis was performed at a constant voltage of 80V until the lower edge of the sample touched the separating gel (about 30 minutes). Electrophoresis was then continued at a constant voltage of 130V for nearly 2 hours until bromophenol blue just overflowed from the lower edge of the separating gel. The membrane was then transferred using a wet transfer at 280mA / 1h. 5% skim milk was added for blocking at room temperature for 2 hours. Primary antibodies (rabbit anti-Tau (phospho T231) antibody 1:1000, rabbit anti-Tau (phospho S396) antibody 1:1000, rabbit anti-Tau antibody 1:1000, GAPDH antibody 1:2000) were added and incubated overnight on a silent mixer at 4℃. The membrane was washed 3 times with TBST buffer and incubated at room temperature for 2 hours with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1000). The membrane was washed 3 times with TBST buffer and then developed using a gel imaging system with ECL reagent added by chemiluminescence.

[0050] 6. Experimental Results

[0051] 6.1 Behavioral Observations of Tree Shrews

[0052] Three days after the MC-LR+IBO combined group of tree shrews, the tree shrews became lethargic and less active. As time went on, the tree shrews' activity level decreased further, and the luster of their hair deteriorated. Seven days after the IBO group of tree shrews was injected with the drug, the tree shrews' activity level decreased slightly compared to the previous few days, but there were no other abnormalities. The tree shrews in the other three groups had normal activity levels and no abnormalities were observed.

[0053] 6.2 Immunohistochemical detection of Aβ expression

[0054] To investigate the damage caused by MC-LR and IBO to the tree shrew brain, we used immunohistochemistry to detect the expression of Aβ protein in the tree shrew brain. The results showed that, compared with the NC group, injection of MC-LR or (and) IBO increased the expression of Aβ in the hippocampus and cortex of the tree shrew brain, and the combined injection of MC-LR and IBO resulted in the most significant increase in Aβ expression.

[0055] 6.3 Western bolt inspection

[0056] Western blot analysis was performed to determine the protein levels of Tau, p-Tau(T231), and p-Tau(S396) in the brain tissue of tree shrews. The results showed that, compared with the NC group, the level of p-Tau(T231) in the tree shrew brain was significantly increased after injection of MC-LR or (and) IBO, with statistically significant differences in the IBO group and the MC-LR+IBO group (p < 0.05). Similarly, compared with the NC group, the level of p-Tau(S396) in the tree shrew brain was significantly increased after injection of MC-LR or (and) IBO, with statistically significant differences (p < 0.05). However, there was no significant difference in Tau protein levels (p > 0.05).

[0057] Table 1. Changes in p-Tau (T231), p-Tau (S396), and Tau protein expression in five groups of tree shrews during the experiment.

[0058] Experimental Groups p-Tau (T231) / GAPDH p-Tau (S396) / GAPDH Tau / GAPDH NC 0.478±0.123 0.696±0.168 0.947±0.049 sham 0.488±0.152 0.719±0.129 0.963±0.076 MC-LR 0.803±0.19 <![CDATA[0.999±0.072 a ]]> 0.956±0.113 IBO 1.183±0.287 <![CDATA[0.986±0.072 a ]]> <![CDATA[1.006±0.091 b ]]> MC-LR+IBO 1.458±0.171 <![CDATA[1.302±0.159 b,c,e ]]> <![CDATA[1.008±0.142 b,d ]]>

[0059] n = 3, x±s , aP <0.05, b P <0.01, compared with the NC group; c P <0.05, d P <0.01, compared with the MC-LR group; e P <0.05, f P <0.01 compared with the IBO group.

Claims

1. A method for modeling Alzheimer's disease in tree shrews, characterized in that: Includes the following steps: (1) Reagent preparation: Microcystin-LR solution: an ethanol solution with a concentration of 0.5 μg / μl Microcystin-LR; Preparation of amatoxins solution: Under aseptic conditions, add 200 μl of sterile physiological saline to a vial containing 1 mg of amatoxins, and repeatedly pipette until completely dissolved to prepare a concentration of 5 μg / μl. (2) After mixing the Microcystin-LR solution prepared in step (1) with the amatoxinsine solution, inject 2 μl into each of the bilateral Meynert basal nuclei of the tree shrew. The aforementioned mixed solution was obtained under sterile conditions by diluting it according to the ratio of Microcystin-LR: amatoxinsine solution: sterile physiological saline = 1:3:

6. (3) After the drug administration is completed, normal feeding is carried out for 2 weeks to complete the Alzheimer's disease model.

2. The tree shrew Alzheimer's disease modeling method according to claim 1, characterized in that, The bilateral Meynert basal ganglia injection method is as follows: (1) Fixing the tree shrew: The tree shrew was anesthetized by intraperitoneal injection of 3% sodium pentobarbital at 2 μl / g body weight. After complete anesthesia, the hair on the cranial face of the tree shrew was removed with animal clippers and fixed on the stereotaxic device. The ear rod was inserted into the tree shrew's ear canal and the left and right ear rods were balanced so that the line connecting the two ears of the tree shrew was on the same straight line as the ear rod. The tree shrew's incisors were pried open with tweezers and the upper incisors were locked in the hole of the adapter. The nose rod locking screw was pressed down and the height of the incisor clamp was adjusted up and down, and the position of the adapter was adjusted back and forth to keep the cranial face of the tree shrew horizontal. The screw was locked so that the tree shrew's head could not move. According to the tree shrew's body size, the tree shrew's body was raised with foam on the base of the stereotaxic device so that the head and body were kept horizontal to prevent the tree shrew's breathing from being blocked. (2) Micro-injection pump parameter settings: Turn on the power switch of the micro-injection pump, set the operation mode to first draw and then perfuse, and set the drawing parameters: the volume of liquid drawn is 2μl, the drawing time is 0.5min; the interval between drawing and perfuse is 0.5min; and the perfuse time is 5min. (3) Positioning reference point: After fixing the RN type injection needle on the holder, turn on the power switch of the digital positioning instrument, disinfect the skin surface of the tree shrew's skull with iodine, make a longitudinal incision from the line connecting the two ears to the eyes to expose the skull, find the intersection of the interauricular line and the sagittal suture, i.e. the anterior fontanelle point, use this point as the reference point of the three-dimensional coordinate system, move the three-dimensional manipulator arm to align its positioning point with the anterior fontanelle point, and press the "CLR" key of the digital display base X, Y, Z axes to zero; (4) Locating the target area: Determine the coordinates of the basal ganglia relative to the anterior fontanelle, i.e., ML value, AP value, and DV value. Starting from the anterior fontanelle reference point, set the coordinate points according to the position of the basal ganglia. Method 1: AP: 8.7mm anterior to the anterior fontanelle, ML: ±3mm of the sagittal suture. Move the operating arm the corresponding distance to the target position, and then use the positioning pin as a reference point and mark it with a marker pen; remove the "+" operating arm and drill a hole in the skull. (5) Complete the injection: After the micro-injection pump is turned on to draw the prepared compound solution, within 0.5 min, according to the determined value DV: 10 mm below the dura mater, move the operating arm down to the target depth. After the injection is completed, leave the needle in place for 5 min, and then slowly withdraw the needle at a speed of 1.0 mm / min. Set the coordinate value according to the method of locating the target area to carry out the injection experiment at another coordinate point. After the injection is completed, the RN-type injection needle is withdrawn, the injection hole is sealed with bone glue, the wound is sutured and disinfected with povidone-iodine, an appropriate amount of erythromycin ointment is applied to the wound, and penicillin is administered intraperitoneally for 3 consecutive days after the operation.

3. The tree shrew Alzheimer's disease modeling method according to claim 1, characterized in that, The mass of the tree shrew is 120-150g.

4. The tree shrew Alzheimer's disease modeling method according to claim 1, characterized in that, The tree shrews were raised at a room temperature of 24±2℃, a relative humidity of 50±5%, and a light / dark cycle of 12h / 12h.

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