Construction method and application of acute rodent cognitive disorder model

Abstract

The invention discloses a construction method and application of an acute rodent cognitive disorder model. According to the model construction, the cognitive impairment of rodents can be quickly caused by injecting formaldehyde into the dorsal sides of brain sea horses of rats and mice, and behavioral experimental verification of a Y labyrinth and a multifunctional closed labyrinth is carried out; the method can be used for constructing a rat and mouse animal model with cognitive impairment, has the beneficial effects that the modeling method is simple and easy to implement, the modeling is stable, the success rate is high, the economic cost is relatively low, part of defects of a cognitive impairment model in the modeling aspect in the prior art can be overcome, and meanwhile, the method can be used for screening medicines for treating or relieving cognitive impairment.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for constructing an acute rodent cognitive impairment model and an application thereof. Background Art

[0002] Cognitive dysfunction refers to symptoms of impaired learning and memory, language ability, attention and other functions, mainly including Alzheimer's disease (AD), cerebral apoplexy (CVA) and other diseases, and its pathogenic factors are relatively complex. As the global population ages, the incidence rate is also increasing year by year, and cognitive dysfunction has become a new challenge facing human public health issues.

[0003] As the main clinical manifestation of common neuropsychiatric diseases such as AD, studies have shown that cognitive dysfunction is related to Aβ toxicity, tau protein phosphorylation, gene mutation, synaptic damage, and oxidative stress response, which may involve multiple theories and molecular signaling pathways. Formaldehyde (FA) is the earliest small molecule on earth, and endogenous formaldehyde exists in the cytoplasm, nucleus and subcellular organelles of all organisms. Studies have shown that it is involved in the formation of memory, and the concentration accumulated in the hippocampus increases with age. When the concentration of formaldehyde accumulated in the brain exceeds the pathological concentration (about 0.5mmol / L), it exceeds the degradation capacity of the brain, inducing neuronal death and leading to memory loss.

[0004] Disease animal models play an important role in the research and verification of molecular and cellular mechanisms of pathology, clinical biomarkers, etc., and rodent models (mouse models, rat models) are widely used in the field of neurological diseases due to their high genetic homology and relatively low price. At present, the rodent models used to study cognitive dysfunction include transgenic models, aging models, and exogenous harmful substance injection models. The amyloid hypothesis has always been considered the most accepted hypothesis about AD, which believes that Aβ accumulated in the brain produces neurotoxicity and leads to cognitive impairment. However, the current Aβ injection modeling is affected by many factors, including the solubility state of Aβ, the purity level, etc., and the neurotoxicity produced is specific and age-related. The remaining animal models also have certain limitations, such as the unstable exogenous gene expression of the APP / PSl transgenic model, the inability of tau protein-based models and the injection of cholinergic inhibitors such as scopolamine to form Aβ deposition, and the high economic cost. Therefore, it is of great practical and innovative significance to establish an animal model that is simple to operate, has low economic cost, and quickly develops cognitive impairment.

[0005] Therefore, in order to solve the above problems, this paper proposes a method for constructing an acute rodent cognitive impairment model and its application. Summary of the invention

[0006] The purpose of the present invention is to design a method for constructing an acute rodent cognitive impairment model with low economic cost, effectiveness, reliability, ease of operation and strong repeatability.

[0007] In order to achieve the above technical effects, the present invention is implemented by the following technical scheme: A method for constructing an acute rodent cognitive impairment model, characterized in that it comprises the following steps:

[0008] S1. Anesthesia of experimental animals: Use small animal anesthetics for inhalation anesthesia, and then fix the anesthetized animals on a brain stereotaxic apparatus;

[0009] S2. Construction of animal cognitive impairment model: 5mM and 10mM formaldehyde were injected into the dorsal hippocampus of mice and rats through a stereotaxic apparatus to obtain the pre-validation model;

[0010] S3. Use a maze behavioral experiment to verify the prepared pre-verification model, and after verification, the desired animal cognitive impairment model can be obtained.

[0011] Furthermore, in S1, inhalation anesthesia is specifically as follows: placing the atomized anesthetic liquid into the inlet of the air pump, then connecting the air pump power supply, turning on the air pump switch, and rotating and adjusting the air source valve at the front end of the oxygen flow meter to make the output gas reach the required flow rate.

[0012] Furthermore, the required flow rate is determined by the type, weight and condition of the animal; specifically, it is adjusted to 500-700 ml / min for rats and 300-500 ml / min for mice.

[0013] Furthermore, in S2, the construction of the animal cognitive impairment model specifically includes the following steps:

[0014] S2.1. Shave the hair on the animal's head, disinfect it with iodine, make a midsagittal incision, cut the skin and subcutaneous tissue of the brain until the skull is exposed, and use a cotton swab dipped in hydrogen peroxide to remove excess blood;

[0015] S2.2. Determine the coordinates of the dorsal injection site in the hippocampus according to the stereotaxic atlas of the brain of rats and mice. The injection site coordinates for rats are 3.5 mm behind the anterior fontanelle, 2.0 mm to the left and right of the sagittal suture, and 3.5 mm below the brain surface; the injection site coordinates for mice are 2.0 mm behind the anterior fontanelle, 1.7 mm to the left and right of the sagittal suture, and 1.6 mm below the brain surface.

[0016] S2.3. Use a dental drill to drill a small hole in the corresponding skull, and use a needle to pierce and remove the meninges at the hole;

[0017] S2.4, using a microsyringe, slowly and evenly inject 5mM and 10mM formaldehyde into the dorsal hippocampus of mice and rats at the above injection sites at an injection speed of 0.5μl / min. The injection dose for rats is 2ul and the dose for mice is 1.5ul. After the injection, keep the needle in place for 3-8min to prevent liquid overflow, then slowly withdraw the microsyringe to prepare the experimental group;

[0018] S2.5. After suturing the skin incision and disinfecting it with iodine and alcohol, the two groups of experimental animals were placed in a comfortable environment and given food and water after they woke up; this way, the animal cognitive impairment model before verification was obtained.

[0019] Furthermore, in S3, the maze used was a Y-maze, a multifunctional closed maze, a Morris water maze, and a novel object discrimination test.

[0020] Another object of the present invention is to provide an application of an acute rodent cognitive impairment model, characterized in that the acute rodent cognitive impairment model is used to screen drugs for treating or alleviating cognitive impairment.

[0021] The beneficial effects of the present invention are:

[0022] The modeling method of the present invention is simple and easy, the modeling is stable, and the occurrence of cognitive disorders in experimental animals is verified by behavioral experiments and molecular biology experiments. The success rate is high and the economic cost is low. Some shortcomings of cognitive disorder models in the prior art in modeling can be overcome; at the same time, the model can be used to screen drugs for treating or alleviating cognitive disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0024] Figure 1 This is a schematic diagram of the results of the animal cognitive impairment model (rat) constructed in Example 1 of the present invention being verified in a Y-maze;

[0025] Figure 2 It is a schematic diagram of the results of verifying the animal cognitive impairment model (rat) constructed in Example 1 of the present invention in a multifunctional closed maze.

[0026] Figure 3 This is a schematic diagram of the results of validating the animal cognitive impairment model (mice) constructed in Example 1 of the present invention in the Morris water maze;

[0027] Figure 4 This is the result of verifying the animal cognitive impairment model (mice) constructed in Example 1 of the present invention in a new object discrimination experiment. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1

[0030] A method for constructing an acute rodent cognitive impairment model, the specific steps are as follows:

[0031] Step 1: Anesthesia of experimental animals:

[0032] Use small animal anesthetics for inhalation anesthesia; connect the air pump power supply, turn on the air pump switch, rotate and adjust the gas source valve at the front end of the oxygen flowmeter to make the output gas reach the required flow rate (generally adjusted to 500-700ml / min for rats, and generally adjusted to 300-500ml / min for mice). The required gas flow rate is mainly determined by the type, weight and condition of the animal. The rat (mice) inhales isoflurane, and then the anesthetized animal is fixed on the brain stereotaxic instrument.

[0033] Step 2: Construction of animal cognitive impairment model and control group:

[0034] The hair on the animal's head was shaved, disinfected with iodine, and a midsagittal incision was made to cut open the skin and subcutaneous tissue of the brain until the skull was exposed. A cotton swab dipped in hydrogen peroxide was used to remove excess blood.

[0035] S2.2. Determine the coordinates of the dorsal injection site in the hippocampus of the brain according to the stereotaxic atlas of the brain of rats and mice. The injection site coordinates selected for rats are 3.5 mm behind the anterior fontanelle, 2.0 mm to the left and right of the sagittal suture, and 3.5 mm below the brain surface; the injection site coordinates selected for mice are 2.0 mm behind the anterior fontanelle, 1.7 mm to the left and right of the sagittal suture, and 1.6 mm below the brain surface; use a dental drill to drill a small hole in the corresponding skull, and use a needle to prick and remove the meninges at the small hole;

[0036] Use a microsyringe to slowly and evenly inject 5mM and 10mM formaldehyde into the dorsal hippocampus of mice and rats at the injection site at an injection speed of 0.5μl / min. The injection dose for rats is 2ul and the dose for mice is 1.5ul. After the injection, keep the needle in place for 3-8min to prevent liquid overflow, then slowly withdraw the microsyringe to prepare the experimental group (animal cognitive impairment model);

[0037] Use a microsyringe to slowly and evenly inject formaldehyde into the dorsal hippocampus of mice and rats at the injection site at a speed of 0.5 μl / min. The injection dose for rats is 2ul, and the dose for mice is 1.5ul. After the injection, keep the needle in place for 8 to 12 minutes to prevent liquid overflow, and then slowly withdraw the microsyringe to prepare a control group.

[0038] After suturing the skin incisions of the two groups and disinfecting them with iodine and alcohol, the two groups of experimental animals were placed in a comfortable environment and given food and water after they woke up; thus, the animal cognitive impairment model before verification was obtained.

[0039] Step 3: Behavioral testing

[0040] 1. Y-maze detection: Figure 1 Figure A shows the number of arm entries in the Y-maze; Figure B shows the spontaneous alternation response rate in the Y-maze; data are expressed as Mean±SEM (n=10); Note: # P<0.05, ## P<0.01, ### P<0.001.

[0041] Compared with the sham operation group, the spontaneous alternation rate of FA5mM and FA10mM was significantly reduced, while the number of wall entries in the control group was basically the same as that in the FA5mM and FA10mM groups. It was concluded that under the premise that surgical administration does not affect the motor ability of experimental animals, the spatial memory learning ability of the model group was significantly reduced.

[0042] 2. Multifunctional closed maze detection: Figure 2 A is the change of active frequency of the multifunctional closed maze; B is the change of escape latency of the multifunctional closed maze; C is the trajectory map of the multifunctional closed maze; D is the heat map of the multifunctional closed maze; data are expressed as Mean±SEM (n=10). Note: # P<0.05, ## P<0.01, ### P<0.001.

[0043] Compared with the control group, the FA5mM group and the FA10mM group showed a smaller trend of decreasing escape latency with increasing experimental days; the density of the heat zone map and trajectory map of the FA5mM group and the FAmM group was significantly increased compared with the sham operation group; the active frequency of each group remained basically unchanged, indicating that the spatial memory learning ability of the FA5mM group and the FA10mM group was significantly reduced compared with the sham operation group.

[0044] 3. Morris water maze test: Figure 3 Figure A shows the swimming speed of mice in the directional cruising stage; Figure B shows the escape latency of mice in the directional cruising stage; Figure C shows the number of times mice cross the platform in the spatial exploration stage; data are expressed as Mean±SEM (n=10). Note: * P<0.05, ** P<0.01, *** P<0.001.

[0045] Compared with the sham operation group, the escape latency in the directional cruising stage was significantly increased in the low-dose formaldehyde (FA5mM) and high-dose formaldehyde (FA10mM) groups, and the number of times the mice crossed the platform in the spatial exploration stage was significantly reduced. However, the swimming speed of the sham operation group, the low-dose formaldehyde group, and the high-dose formaldehyde group was basically the same. It was concluded that under the premise that surgical administration does not affect the motor ability of experimental animals, the spatial memory learning ability of the low-dose formaldehyde group and the high-dose formaldehyde group was significantly reduced compared with the sham operation group.

[0046] 4. New object identification experiment: Figure 4 The figure shows the effect of formaldehyde injection into the hippocampus on the preference index (1h, 24h) of mice in the new object discrimination test. The data are expressed as Mean±SEM (n=10). Note: # P<0.05, ## P<0.01, ### P<0.001.

[0047] Depend on Figure 4 It can be seen that compared with the sham operation group, the priority index of the low-dose formaldehyde group and the high-dose formaldehyde group in the test phase was significantly reduced. The results suggested that surgical administration affected the learning and memory ability of mice.

Claims

1. A method for constructing an acute rodent cognitive impairment model, characterized in that: The following steps are involved: S1. Anesthesia of experimental animals: Use small animal anesthetics for inhalation anesthesia, and then fix the anesthetized animals on a brain stereotaxic apparatus; S2. Construction of animal cognitive impairment model: 5mM and 10mM formaldehyde were injected into the dorsal hippocampus of mice and rats through a stereotaxic apparatus to obtain the pre-validation model; S3. Use a maze behavioral experiment to verify the prepared pre-validation model, and after verification, the desired animal cognitive impairment model can be obtained.

2. The method for constructing an acute rodent cognitive impairment model according to claim 1, characterized in that: In S1, inhalation anesthesia is specifically as follows: placing the atomized anesthetic liquid into the inlet of the air pump, then connecting the air pump power supply, turning on the air pump switch, and rotating and adjusting the air source valve at the front end of the oxygen flow meter to make the output gas reach the required flow rate.

3. The method for constructing an acute rodent cognitive impairment model according to claim 2, characterized in that: The required flow rate is determined by the type, weight and condition of the animal; specifically, it is adjusted to 500-700 ml / min for rats and 300-500 ml / min for mice.

4. The method for constructing an acute rodent cognitive impairment model according to claim 1, characterized in that: In S2, the construction of the animal cognitive impairment model specifically includes the following steps: S2.

1. Shave the hair on the animal's head, disinfect it with iodine, make a midsagittal incision, cut the skin and subcutaneous tissue of the brain until the skull is exposed, and use a cotton swab dipped in hydrogen peroxide to remove excess blood; S2.

2. Determine the coordinates of the dorsal injection site in the hippocampus according to the stereotaxic atlas of the brain of rats and mice. The injection site coordinates for rats are 3.5 mm behind the anterior fontanelle, 2.0 mm to the left and right of the sagittal suture, and 3.5 mm below the brain surface; the injection site coordinates for mice are 2.0 mm behind the anterior fontanelle, 1.7 mm to the left and right of the sagittal suture, and 1.6 mm below the brain surface. S2.

3. Use a dental drill to drill a small hole in the corresponding skull, and use a needle to pierce and remove the meninges at the hole; S2.4, using a microsyringe, slowly and evenly inject 5mM and 10mM formaldehyde into the dorsal hippocampus of mice and rats at the above injection sites at an injection speed of 0.5μl / min. The injection dose for rats is 2ul and the dose for mice is 1.5ul. After the injection, keep the needle in place for 3-8min to prevent liquid overflow, then slowly withdraw the microsyringe to prepare the experimental group; S2.

5. After suturing the skin incision and disinfecting it with iodine and alcohol, the two groups of experimental animals were placed in a comfortable environment and given food and water after they woke up; this way, the animal cognitive impairment model before verification was obtained.

5. The method for constructing an acute rodent cognitive impairment model according to claim 1, characterized in that: In S3, the mazes used were a Y-maze, a multifunctional closed maze, a Morris water maze, and a novel object discrimination test.

6. Use of an acute rodent cognitive impairment model according to any one of claims 1 to 5, characterized in that: The acute rodent cognitive impairment model is used to screen drugs for treating or alleviating cognitive impairment.

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