Manufacturing method of late onset AD mouse experimental model

Abstract

The invention provides a manufacturing method of a late onset AD mouse experimental model, which is characterized in that a late onset AD model with decline of gonad axis function and Abeta hippocampus deposition of old people is simulated through a method of combining castration with Abeta 1-42 oligomer injection, and the model is closer to pathophysiology characteristics of late onset AD; and the molding cost is low, the period is short and the success rate is high.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical laboratory animal modeling, and in particular to a method for preparing a late-onset AD mouse experimental model. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease with pathological features including loss of synapses and neurons, formation of neurofibrillary tangles, and senile plaques. The clinical manifestations of this disease are characterized by progressive decline in memory and other cognitive functions. It is crucial to establish a reliable animal model for studying the pathogenesis, drug prevention, and treatment of AD.

[0003] From a histopathological point of view, Alzheimer's disease is distinguished from other dementias by the presence of a large amount of β-amyloid (Aβ) deposits outside its neurons. A large number of studies have shown that Aβ plays a key role in the occurrence and development of Alzheimer's disease. Aβ is derived from the processing of amyloid precursor protein (APP). Studies have shown that Aβ and its deposits have a wide range of neurotoxicity, from inhibiting neuronal plasticity to inducing neuronal cell death, including necrosis and apoptosis. Many experiments have confirmed that Aβ-induced neuronal apoptosis is the main cause of the chronic loss of a large number of neurons in the development of Alzheimer's disease. The hippocampal dentate gyrus (DG)-CA3 system plays a central role in the encoding, storage, and retrieval of memory. Aβ infiltration in the dentate gyrus of the hippocampus of animals is a rapid and effective method to establish an Alzheimer's disease model.

[0004] Aging is the biggest risk factor for late-onset Alzheimer's disease, which suggests that certain factors in the aging process may trigger or promote the occurrence of Alzheimer's disease. Aging is closely related to changes in the hypothalamic-pituitary-gonadal (HPG) axis and the gradual decline of gonadal function. In recent years, many studies have shown that androgens or estrogens play a neuroprotective role by reducing the production of Aβ, improving synaptic signaling, and combating neuronal death, and their deficiency is associated with the occurrence and development of elderly Alzheimer's disease. Therefore, the physiological state of reduced sex hormone levels in patients with Alzheimer's disease, especially those with late-onset disease, cannot be ignored.

[0005] Based on the main pathological and physiological characteristics of Alzheimer's disease patients, we established and evaluated a modified mouse Alzheimer's disease model by castration combined with bilateral injection of Aβ into the dentate gyrus of the hippocampus. 1-42 Oligomers were constructed. Summary of the invention

[0006] The present invention provides a method for preparing a late-onset AD mouse experimental model by combining "castration combined with hippocampal dentate gyrus Aβ 1-42The "oligomer injection" method was used to simulate the late-onset AD model in which the gonadal axis function declined in the elderly and Aβ was deposited in the hippocampus.

[0007] To achieve the above object, the technical solution adopted by the present invention is: A method for preparing a late-onset AD mouse experimental model, characterized in that it comprises the following steps: S1, castration, i.e., removal of bilateral gonads of adult mice; S2. Inject Aβ into the bilateral dentate gyrus of the hippocampus of adult mice 1-42 Oligomers.

[0008] As a further preferred embodiment of the present invention, the method for removing bilateral gonads of adult mice comprises: Removal of bilateral testicles in male mice: Use 1% pentobarbital (50 mg / kg) to anesthetize adult mice, prepare the skin 1.5 cm above the pubic symphysis of the mice, expose the ventral surface and disinfect, cut a small incision of about 1.5 cm along the midline of the abdomen, cut the muscle layer and peritoneum in turn, use blunt forceps to clamp the fat pad on the left side, and bring out the testicles and vas deferens, peel and ligate the vas deferens, separate the epididymal ligament and fascia, cut the blood vessels, cut off the testicles, and finally use blunt forceps to clamp the fat pad and return it to the abdominal cavity; the right side is treated in the same way as the left side. After the treatment is completed, the muscles and skin are sutured in turn. The sham operation process of the control group is the same as above. To prevent infection, intramuscular injection of penicillin is started on the same day after the gonad (testis) removal surgery or sham surgery of mice, 6×10 4 U / kg·d for 7 consecutive days.

[0009] Removal of bilateral ovaries in female mice: Use 1% pentobarbital (50 mg / kg) to anesthetize adult mice, prepare the skin along the umbilicus on the midline of the mouse abdomen, expose the ventral surface and disinfect, and make a 1-2 cm longitudinal incision; after cutting the skin, use small tweezers to separate the skin and abdominal wall muscles on the left side to expose the abdominal cavity, and use surgical scissors to cut the connection between the ovary and the ovarian ligament and blood vessels, and gradually separate the ovary, paying attention to hemostasis. Then suture the separated abdominal wall and skin layers one by one; the right side is treated the same as the left side. After the treatment is completed, suture the muscles and skin in turn. The sham operation process of the control group is the same as above. To prevent infection, intramuscular injection of penicillin was started on the same day after the gonad (ovary) removal surgery or sham surgery of the mice, 6×10 4 U / kg·d for 7 consecutive days.

[0010] As a further preferred embodiment of the present invention, the Aβ 1-42 The injection method of oligomers is: 10 days after gonadectomy, mice were anesthetized with 1% pentobarbital and fixed on a stereotaxic apparatus. The skin was prepared and the surgical area was disinfected with alcohol. The skin above the mouse's head was cut along the midline, with an opening of about 1 cm. The incision was propped open with an eyelid opener and the mucosa on the surface of the skull was pushed aside with scissors to expose the skull. The bregma site was determined, and the skull drilling site was determined 2 mm backward (toward the occipital bone) and 1.4 mm to the side. A small hole with a diameter of about 0.7 mm was polished at the drilling site with a dental grinder. After drilling the skull, the microinjector fixed on the stereotaxic apparatus was inserted vertically 2.5 mm from the skull surface to reach the dentate gyrus of the mouse's hippocampus. 200 μM Aβ was injected into each hippocampus 1-42 1 μl of oligomers was injected into each mouse with 2 μl, and the injection was carried out on each side of the hippocampus for 5 minutes, and the needle was retained for 5 minutes. After the injection, the needle was withdrawn, the wound was cleaned, sutured, and a small amount of penicillin powder was applied to prevent infection.

[0011] Aβ in the dentate gyrus of the hippocampus was combined with castration 1-42 The oligomer injection method simulates the pathophysiological characteristics of late-onset AD. Compared with the traditional AD transgenic and long-term D-galactose intake methods to induce AD ​​animal models, this method has the following advantages: 1. It is closer to the pathophysiological characteristics of late-onset AD; 2. The modeling cost is low, the cycle is short, and the success rate is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of Aβ injection in mouse hippocampus; Figure 2 Schematic diagram of Morris water maze experiment to evaluate the learning and memory ability of mice; Figure 2 In the figure, A is the changing trend of the escape latency of mice in each group; B is the comparison of the escape latency of mice in each group on the 6th day of the positioning navigation experiment; C is the comparison of the percentage of activity time of mice in the quadrant of the platform area; Figure 3 This is a Nissl body staining picture; Figure 3 In the figure, A is a schematic diagram of the Nissl staining results of mice in the sham injury group; B is a schematic diagram of the Nissl staining results of mice in the castration group; C is the Nissl staining results of mice in the castration + Aβ group; D is a comparison of the Nissl staining results of mice in each group; Figure 4 This is a schematic diagram of the results of TUNEL labeling of apoptotic neurons in the CA1 region of the mouse hippocampus; Figure 4 In the figure, A is a schematic diagram of the TUNEL experimental results of mice in the sham injury group; B is a schematic diagram of the TUNEL experimental results of mice in the Aβ group; C is a schematic diagram of the TUNEL experimental results of mice in the castration + Aβ group; D is a comparison of the TUNEL experimental results of mice in each group. DETAILED DESCRIPTION

[0013] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0014] The present invention provides a method for preparing a late-onset AD mouse experimental model, wherein the model is prepared by castration combined with bilateral hippocampal dentate gyrus injection of Aβ 1-42 The oligomers (Aβ refers to the ability to aggregate into oligomers or fibers, and 1-42 refers to the presence of 42 amino acids) are constructed in the following steps: 1. Castration of mice: (1) Removal of bilateral testicles in male mice: Use 1% pentobarbital (50 mg / kg) to anesthetize adult mice, prepare the skin 1.5 cm above the pubic symphysis of the mice, expose the ventral surface and disinfect, cut a small incision of about 1.5 cm along the midline of the abdomen, cut the muscle layer and peritoneum in turn, use blunt forceps to clamp the fat pad on the left side, and bring out the testicles and vas deferens, peel and ligate the vas deferens, separate the epididymal ligament and fascia, cut the blood vessels, cut off the testicles, and finally use blunt forceps to clamp the fat pad and return it to the abdominal cavity; the right side is treated in the same way as the left side. After the treatment is completed, the muscles and skin are sutured in turn. The sham operation process of the control group is the same as above. To prevent infection, penicillin is injected intramuscularly on the same day after the gonad (testis) removal surgery or sham surgery of the mice, 6×10 4 U / kg·d for 7 consecutive days.

[0015] (2) Removal of both ovaries of female mice: Use 1% pentobarbital (50 mg / kg) to anesthetize adult mice, prepare the skin along the umbilicus on the midline of the mouse abdomen, expose the ventral surface and disinfect it, and make a 1-2 cm longitudinal incision; after cutting the skin, use small forceps to separate the skin and abdominal wall muscles on the left side to expose the abdominal cavity, and use surgical scissors to cut the connection between the ovary and the ovarian ligament and blood vessels, and gradually separate the ovary, paying attention to hemostasis. Then suture the separated abdominal wall and skin layers one by one; the right side is treated in the same way as the left side. After the treatment is completed, suture the muscles and skin in turn. The sham operation process of the control group is the same as above. To prevent infection, intramuscular injection of penicillin is started on the same day after the gonad (ovary) removal surgery or sham surgery of the mice, 6×10 4 U / kg·d for 7 consecutive days.

[0016] 2. Bilateral hippocampal dentate gyrus Aβ 1-42 Injection of oligomers: 10 days after gonadectomy, mice were anesthetized with 1% pentobarbital and fixed on a stereotaxic apparatus. The skin was prepared and the surgical area was disinfected with alcohol. The skin above the mouse's head was cut along the midline, with an opening of about 1 cm. The incision was propped open with an eyelid opener and the mucosa on the surface of the skull was pushed aside with scissors to expose the skull. The bregma site was determined, and the skull drilling site was determined 2 mm backward (toward the occipital bone) and 1.4 mm to the side. A small hole with a diameter of about 0.7 mm was polished at the drilling site with a dental grinder. After drilling the skull, the microinjector fixed on the stereotaxic apparatus was inserted vertically 2.5 mm from the skull surface to reach the dentate gyrus of the mouse's hippocampus. 200 μM Aβ was injected into each hippocampus 1-42 1 μl of oligomers, 2 μl per mouse, injected into each hippocampus for 5 min, and the needle was left in place for 5 min (e.g. Figure 1 A and B). After the injection, the needle is removed, the wound is cleaned, sutured, and a small amount of penicillin powder is applied to prevent infection.

[0017] The late-onset AD model was simulated using the above method, and the following evaluations were performed on the model: Eight-week-old ICR mice were randomly divided into sham injury group, Aβ group and castration + Aβ group, with 12 mice in each group. Mice in the castration + Aβ group underwent gonadectomy and bilateral hippocampal Aβ 1-42 Oligomer injection; Aβ group mice were only injected with bilateral hippocampal Aβ 1-42 Oligomer injection: The sham injury group mice were injected with an equal volume of sterile saline into the bilateral hippocampus.

[0018] 1. Morris water maze test to evaluate the spatial learning and memory ability of mouse hippocampus: Morris water maze experiment was performed 2-7 days after the injection of the hippocampus. 1. Positioning navigation experiment: The pool was divided into four quadrants, and a platform was placed in the center of the fourth quadrant. The water temperature was (25±1)℃. Each mouse studied for 6 consecutive days, and studied at the same time every day. Each time, it entered the water at four different locations, and the four locations for each mouse were the same. Each learning time was 60s, and the time required to find the platform (escape latency) was recorded. Those who did not find the platform within 60s were counted as 60s, and the mouse was placed on the platform for 20s. 2. Spatial exploration experiment: 2h after the positioning navigation test on the 6th day, the platform in the first quadrant was removed, and a spatial search experiment was performed. Each mouse was tested only once, the time was 60s, and the water locations for different mice were the same. Parameters such as the mouse escape latency, swimming time in each quadrant, etc. were recorded.

[0019] Experimental results: The curves of the escape latency of the three groups of mice in the navigation experiment are as follows: Figure 2 As shown in A. Compared with the sham injury group, on the 6th day of the navigation experiment, the latency of the mice in the sham injury group to find the platform was significantly shorter than that in the Aβ group and the castration + Aβ group ( Figure 2B), while in the spatial search experiment, the percentage of swimming time in the platform quadrant of the sham injury group mice was significantly higher than that in the Aβ group and the castration + Aβ group ( Figure 2 C), indicating that Aβ and castration combined with Aβ intervention both led to a decline in cognitive function in mice. However, in terms of the degree of difference, the difference between the castration + Aβ group and the sham injury group in terms of escape latency and percentage of useful swimming time in the platform quadrant was more significant, with P values ​​less than 0.01 and 0.05, respectively, while the P values ​​in the comparison of escape latency and percentage of useful swimming time in the platform quadrant between the Aβ group and the sham injury group were both less than 0.05, indicating that castration combined with Aβ injection had a more obvious effect on cognitive impairment in mice.

[0020] Figure 2 In the experiment, the escape latency of mice in the Aβ group (33.27±7.56s, n=12) was significantly different from that in the sham injury group (23.77±7.79s, n=12) (P<0.05); the escape latency of mice in the castration+Aβ group (38.75±8.29s, n=12) was significantly different from that in the sham injury group (P<0.001). (C) Comparison of the percentage of activity time of mice in the platform area quadrant. In the spatial search experiment, the percentage of swimming time in the platform area quadrant of mice in the sham injury group (34.72±6.21%, n=12) was significantly higher than that in the Aβ group (26.79±6.38%, n=12, P<0.05) and the castration+Aβ group (25.35±6.11%, n=12, P<0.01). The data of each group are expressed as mean±standard deviation (`X±SD). One-way analysis of variance was used to test the differences in multiple groups of data, and Tukey's test was used to compare the means between groups. P < 0.05 indicated statistically significant differences.

[0021] 2. Nissl body staining to evaluate the neuronal function in the mouse hippocampus: After the behavioral experiment, the mice were anesthetized and killed, and the brain tissue was collected on ice. The mouse tissue was made into paraffin sections using conventional methods, and then Nissl body staining was performed after dewaxing and hydration, toluidine blue staining solution, etc. The staining results were analyzed by image J software, and the Nissl staining gray value of the hippocampus of each group of mice was compared (the ratio of the total gray value of the Nissl staining image of the sagittal section of the hippocampus to the total pixel value of the image).

[0022] Observation results: The results are as follows Figure 3 As shown in the figure, both Aβ injection and castration combined with Aβ injection intervention resulted in a decrease in Nissl bodies in the hippocampus of mice and impaired neuronal function. The average grayscale value of Nissl staining in the hippocampus of mice in the castration combined with Aβ injection group was significantly lower than that in the Aβ group, indicating that castration combined with intracerebral injection of Aβ caused more significant damage to the neurons in the hippocampus of mice.

[0023] Figure 3 The gray value of Nissl staining in the Aβ group (23.52±5.56, n=6) was significantly different from that in the sham injury group (36.59±6.38, n=6) (P<0.01); the gray value of Nissl staining in the castration+Aβ group (14.26±5.45, n=6) was significantly different from that in the sham injury group (P<0.001); the gray value of Nissl staining in the castration+Aβ group was significantly different from that in the Aβ group (P<0.05). The data of each group are expressed as mean ± standard deviation (`X±SD). One-way analysis of variance was used to test the difference of multiple groups of data, and Tukey test was used to compare the means between groups. P<0.05 represented statistically significant differences. Scale bar: 100μm.

[0024] 3. TUNEL method to mark apoptotic neurons: The paraffin sections of mice in each group were labeled with apoptotic neurons by TUNEL method and counterstained with hematoxylin. The total number of apoptotic neurons labeled in the hippocampal CA1 region in three sections of each mouse was counted as a sample data.

[0025] The results are as follows Figure 4 As shown in the figure, Aβ intracerebral injection leads to a large number of neuronal loss in the CA1 region of the mouse hippocampus, and a large number of neuronal nuclei are darkly stained with hematoxylin, while the number of apoptotic neuronal markers does not increase significantly. In mice that have undergone castration combined with Aβ intracerebral injection, a large number of neuronal nuclei are also observed to be darkly stained with hematoxylin, and the number of apoptotic neuronal markers increases significantly.

[0026] Figure 4 In the experiment, the number of apoptotic neurons in the CA1 region of mice in the Aβ group (27.43±15.15, n=6) was not significantly different from that in the sham injury group (14.33±8.40, n=6) (P>0.05); the number of apoptotic neurons in the CA1 region of mice in the castration+Aβ group (55.49±20.39, n=6) was significantly different from that in the sham injury group (P<0.001); the number of apoptotic neurons in the CA1 region of mice in the castration+Aβ group was significantly different from that in the Aβ group (P<0.001). The data of each group are expressed as mean±standard deviation (`X±SD). One-way analysis of variance was used to test the differences of multiple groups of data, and Tukey test was used to compare the means between groups. P<0.05 represented statistically significant differences. Scale bar: 50μm.

[0027] The above behavioral experiments and pathological tissue morphology observations have shown that castration combined with Aβ 1-42 The oligomer injection model is more effective than the bilateral hippocampal dentate gyrus Aβ 1-42 Oligomer injection caused more obvious damage to the neuronal function and cognitive function of animals.1-42 The AD animal model created by the oligomer injection method is consistent with the pathophysiological characteristics of late-onset AD.

[0028] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a late-onset AD mouse experimental model, characterized in that: The following steps are involved: S1, castration, i.e., removal of bilateral gonads of adult mice; S2. Inject Aβ into the bilateral dentate gyrus of the hippocampus of adult mice 1-42 Oligomers.

2. The method for preparing a late-onset AD mouse experimental model according to claim 1, characterized in that: The method for removing bilateral gonads of adult mice comprises: Removal of bilateral testicles in male mice: anesthetize adult mice with anesthetics, prepare the skin 1.5 cm above the pubic symphysis of the mice, expose the ventral surface and disinfect, make a 1-2 cm incision along the midline of the abdomen, cut the muscle layer and peritoneum in turn, clamp the left fat pad, take out the testicle and vas deferens, peel and ligate the vas deferens, separate the epididymal ligament and fascia, cut the blood vessels, cut off the testicles, and finally clamp the fat pad and put it back into the abdominal cavity; the right side is treated in the same way as the left side. After the treatment is completed, the muscles and skin are sutured in turn, and anti-infection treatment is performed; Removal of bilateral ovaries in female mice: Use anesthetic to anesthetize adult mice, prepare the skin along the umbilicus on the midline of the mouse abdomen, expose the ventral surface and disinfect it, and make a 1-2 cm longitudinal incision; after cutting the skin, separate the skin and abdominal wall muscles on the left side to expose the abdominal cavity, cut off the connection between the ovary and the ovarian ligament and blood vessels, thereby separating the ovary, and then suture the separated abdominal wall and skin layers one by one; the right side is treated in the same way as the left side. After the treatment is completed, the muscles and skin are sutured in turn, and the incision is treated to prevent infection.

3. The method for preparing a late-onset AD mouse experimental model according to claim 2, characterized in that: The anesthetic used was 1% sodium pentobarbital, with a dosage of 50 mg / kg.

4. The method for preparing a late-onset AD mouse experimental model according to claim 2, characterized in that: The method for preventing infection of the incision is as follows: after the operation, intramuscular injection of penicillin is started on the same day, 6×10 4 U / kg·d for 7 consecutive days.

5. The method for preparing a late-onset AD mouse experimental model according to claim 1, characterized in that: The Aβ 1-42 The injection method of oligomers is: Ten days after gonadectomy, mice were anesthetized with anesthetics and fixed on a stereotaxic instrument. The skin was prepared and the surgical area was disinfected with alcohol. The skin above the mouse's head was cut along the midline, the incision was stretched open, and the mucosa on the surface of the skull was pushed aside to expose the skull. The skull drilling site was determined by the anterior fontanelle site, and a small hole was polished at the drilling site. After drilling the skull, the microinjector fixed on the stereotaxic instrument was inserted vertically from the skull surface to reach the dentate gyrus of the mouse's hippocampus. Aβ was injected into each hippocampus with the microinjector. 1-42 Oligomer; after the injection, the needle is removed, the wound is cleaned, sutured and treated to prevent infection.

6. The method for preparing a late-onset AD mouse experimental model according to claim 5, characterized in that: The method for determining the skull drilling site through the anterior fontanelle site is as follows: determine the anterior fontanelle site, 2 mm posterior to the anterior fontanelle site and 1.4 mm lateral to determine the skull drilling site.

7. The method for preparing a late-onset AD mouse experimental model according to claim 5, characterized in that: 200 μM Aβ was injected into each hippocampus 1-42 1 μl of oligomers was injected into each mouse, 2 μl was injected into each hippocampus for 5 min, and the needle was left in place for 5 min.

8. The method for preparing a late-onset AD mouse experimental model according to claim 5, characterized in that: The wound infection prevention treatment method is: applying penicillin powder on the wound.

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