CN112931398A - Construction method and application of lupus encephalopathy animal model - Google Patents
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Abstract
The invention relates to a construction method of an animal disease model, in particular to a construction method of a lupus encephalopathy animal model and application thereof. The invention constructs an animal model for inducing lupus encephalopathy by using pristine, the model adopts 8-week-old female BALB/c mice, and the model is made by injecting pristine into the abdominal cavity, and the invention has the advantages of simple operation, high molding rate and low cost. The invention researches the lupus encephalopathy induced by environmental factors for the first time, is suitable for the whole disease cycle research of the lupus encephalopathy, is convenient for being used for the research of searching the specific treatment method of the lupus encephalopathy in large quantities, and lays a solid foundation for the research of the lupus encephalopathy.
Description
Technical Field
The invention relates to a construction method of an animal disease model, in particular to a construction method of a lupus encephalopathy animal model and application thereof.
Background
Lupus encephalopathy (Lupus encephalopathy) is a neuropsychiatric complication that is more common in patients with Systemic Lupus Erythematosus (SLE). Since tissue materials of human lupus encephalopathy are difficult to obtain and are easily influenced by social ethics and medical environments, animal models become necessary means for systematically researching pathophysiological mechanisms of the disease.
Because the neural immune regulation mechanism of the mouse is similar to that of human beings to a great extent and has the characteristic of convenience in gene regulation, the mouse becomes the most widely applied animal model for the research of lupus encephalopathy. The previous research aiming at the lupus encephalopathy mainly uses two spontaneous animal models of NZB/W F1 and MRL/lpr which are modified by specific genes, but the clinical manifestations of the two animal models are slight and can not simulate the important influence of environmental factors on the SLE, so that the two animal models can not comprehensively reflect the characteristics of the lupus encephalopathy.
Although pristane (with the molecular formula of C19H40) induced mouse model is applied to SLE research, the model is applied to peripheral systems such as kidney and the like, and the simulation effect and the effect on lupus encephalopathy are not systematically evaluated.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to provide a construction method and application of a lupus encephalopathy animal model, the invention constructs the animal model for inducing the lupus encephalopathy by pristane, the model adopts 8-week-old female BALB/c mice, and the pristane is injected in the abdominal cavity for molding, and the invention has the advantages of simple operation, high molding rate and low cost. The invention completely monitors the behavioral changes (including olfactory experiment, open field experiment and forced swimming) of the mouse 1 month, 2 months, 4 months and 8 months after the model is molded for the first time and the dynamic changes of the microglia, the astrocyte, the IgG and the complement C3 of the brain tissue, simultaneously evaluates the co-localization expression of the choroid microglia and the IgG in the brain and the co-localization expression of the microglia, the excitatory presynaptic substance and the postsynaptic substance, and completely discloses the occurrence and development process of the lupus encephalopathy symptoms of the model and the corresponding pathophysiological mechanism. The model provided by the invention lays a solid foundation for the research of lupus encephalopathy.
In order to achieve the purpose, the invention adopts the following technical scheme.
An animal model of lupus encephalopathy, which is an animal model of the lupus encephalopathy induced by pristine, and the model is made by injecting pristine into the abdominal cavity of an 8-week-old female BALB/c mouse.
The construction method of the lupus encephalopathy model specifically comprises the following steps.
And 2, removing mouse hair on the abdomen of the mouse by using a clipper, and disinfecting the abdomen skin by using alcohol.
Step 3, selecting a 1ml medical sterilization injector, extracting 0.5ml pristine, smearing the needle head obliquely upwards, slowly inserting the needle at a point of 0.5ml beside the midline of the middle and lower abdomen, slowly pushing the injector after blood-free withdrawal, slowly enabling the medicine to enter the abdominal cavity, facilitating medicine absorption, and immediately pulling out the needle after injection; disinfecting the skin near the needle eye with alcohol again; the mice were returned to the cages and observed for 30 min.
The lupus encephalopathy animal model is applied to the mechanism of the lupus encephalopathy.
Further, the lupus encephalopathy animal model is applied to the mechanism of lupus encephalopathy induced by environmental factors.
Further, the lupus encephalopathy animal model is applied to screening of lupus encephalopathy treatment drugs induced by environmental factors.
Compared with the prior art, the invention has the following beneficial effects.
The invention constructs an animal model for inducing lupus encephalopathy by using pristine, the model adopts 8-week-old female BALB/c mice, and the model is made by injecting pristine into the abdominal cavity, and the invention has the advantages of simple operation, high molding rate and low cost. The invention completely monitors the behavioral changes (including olfactory experiment, open field experiment and forced swimming) of the mouse 1 month, 2 months, 4 months and 8 months after the model is molded for the first time and the dynamic changes of the microglia, the astrocyte, the IgG and the complement C3 of the brain tissue, simultaneously evaluates the co-localization expression of the choroid microglia and the IgG in the brain and the co-localization expression of the microglia, the excitatory presynaptic substance and the postsynaptic substance, and completely discloses the occurrence and development process of the lupus encephalopathy symptoms of the model and the corresponding pathophysiological mechanism. The model provided by the invention lays a solid foundation for the research of lupus encephalopathy.
Compared with the prior spontaneous animal model, the lupus encephalopathy animal model provided by the invention has the following advantages: 1) can be used for researching the mechanism of lupus encephalopathy induced by environmental factors; 2) behavioral changes occur early and with obvious symptoms.
The invention researches lupus encephalopathy induced by environmental factors for the first time, is suitable for the whole disease cycle research of lupus encephalopathy, and is convenient for being widely used for the research of searching a specific treatment method of lupus encephalopathy.
Drawings
FIGS. 1A-1D show the results of the behavioral studies of forced swimming (A), olfaction (B) and open field (C, D) performed on mice before and after molding, 1 month, 2 months, 4 months and 8 months, respectively. Wherein A is the abnormal behavior of pristane mice gradually showing along with the extension of the molding time, including the extension of the immobility time of forced swimming representing depression-like behavior; b is the decrease in olfactory ability to address biological odors, C and D are decreases in total distance to movement and time to central movement in the open field for anxiety-like behavior. P <0.05, P < 0.01.
FIG. 2 is a Pristane-induced change in microglia and neurons in the hippocampal region of a model of lupus encephalopathy.
FIG. 3 shows the presence of astrocyte morphological changes in the hippocampal region of Pristane-induced lupus encephalopathy model.
FIG. 4 shows the presence of IgG deposits in the vessel wall and lateral ventricles of the hippocampal region of Pristane-induced lupus encephalopathy model.
FIG. 5 shows the presence of complement C3 deposition in the blood vessel wall and lateral ventricles in the hippocampal region of Pristane-induced lupus encephalopathy model.
FIGS. 6A-6E are the presence of choroidal Iba1 and IgG colocalization in the Pristane-induced lupus encephalopathy model.
FIG. 7 shows Pristane-induced phagocytosis of neuronal excitatory presynaptic and postsynaptic substances by cortical microglia in a model of lupus encephalopathy.
FIGS. 8A-8D are Pristane-induced upregulation of cytokine expression in brain tissue in a model of lupus encephalopathy.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 lupus encephalopathy animal model construction method.
An animal model of lupus encephalopathy, which is an animal model of the lupus encephalopathy induced by pristine, and the model is made by injecting pristine into the abdominal cavity of an 8-week-old female BALB/c mouse.
A construction method of a lupus encephalopathy model specifically comprises the following steps.
And 2, removing mouse hair on the abdomen of the mouse by using a clipper, and disinfecting the abdomen skin by using alcohol.
And 3, selecting a 1ml medical sterilization injector, extracting 0.5ml pristine, smearing the needle obliquely upwards, slowly inserting the needle at a point of 0.5ml beside the midline of the middle and lower abdomen, slowly pushing the injector after blood is not drawn back, slowly introducing the medicine into the abdominal cavity, facilitating medicine absorption, and immediately pulling out the needle after injection. The skin near the needle eye was again alcohol-sterilized. The mice were returned to the cages and observed for 30 min.
Example 2 the invention provides animal models that exhibit lupus encephalopathy characteristics.
1. Behavioral abnormalities exist in the Pristane-induced lupus encephalopathy model.
The invention respectively carries out the ethological research of forced swimming (A), olfaction experiment (B), open field experiment (C, D) and the like on the mouse before the model building and 1 month, 2 months, 4 months and 8 months after the model building.
1) Study of forced swimming.
The experimental method comprises the following steps: forced swimming is an experimental study used to evaluate depression-like behavior in mice. The experimental mice were placed in glass beakers having an inner diameter of 10cm and a height of 25 cm. The beaker is filled with deionized water at a temperature of 24 ℃. The whole experimental process is independently completed by the same experimenter. The experiment was divided into two parts, totaling 6 min: the first part is an adaptation phase, which lasts for 2 min; lifting the tail of the mouse, and gently placing the mouse in a beaker to ensure that the mouse fully adapts to the temperature and the environment of water in the beaker; the second phase is a recording phase for 4 min. Mice struggled in water and the time to crawl and swim was not recorded. The time recorded is the immobility time of the mice in the beaker, specifically defined as: the mice were in an upright position with the head above the water surface and no displacement. After the experiment was completed, the mice were immediately removed from the water surface, excess water on the mouse fur was removed with absorbent paper, and the mice were returned to a clean cage. The mice were warmed with a heating blanket to help recover their body temperature. The above experimental procedures were performed only once per mouse.
2) And (4) performing olfactory test.
The experimental method comprises the following steps: the time taken for mice to smell different odors was tested: alcohol, vinegar, male and female feces to test olfactory ability. A clean cage was prepared and mice were allowed to acclimate in the cage for 2 minutes; respectively preparing 10% ethanol, vinegar, male feces and female feces solutions, dipping appropriate amount of the solutions with clean cotton swab, placing above the cage, recording each time for 2 minutes, and resting for 1 minute at intervals for 3 times; the time for the mouse to smell the swab was recorded each time. Each mouse only completes the operation once, and after the experiment of each mouse is finished, 95% alcohol is applied to clean the cage and replace padding.
3) Open field experiments.
The experimental method comprises the following steps: the apparatus for the open field experiment consisted of a rectangular field (40 x 28 cm) with walls 40 cm high made of black polyvinyl chloride plastic (Muromachi Kikai, tokyo, japan). The test procedure was recorded by a video camera placed 80 cm above the field and the video was analyzed using the home-made matlab program. The software identifies the position of the mouse based on the contrast difference between the animal's body and the surrounding background color. The mice were then located by the point where the body contrast of the mice was the strongest. The open field is divided into a central region (20cm × 14cm square) and a peripheral region (including an open field peripheral region (less than 10cm from the long side and less than 7cm from the short side) and a wall region). In the test process, the self-made matlab programming is utilized to realize the automatic measurement of the total distance and the central movement time. All animals received only one open field test. The open field was thoroughly cleaned with 95% ethanol before testing on each animal.
As shown in FIGS. 1A-1D, pristine mice gradually exhibited behavioral abnormalities with the extended molding time, including extended periods of forced swim immobility, which represents depressive-like behavior, as shown in FIG. 1A; the decrease in olfactory capacity for biological odors is shown in 1B, and the decrease in total motor distance and motor time in the center of the field, which represent anxiety-like behavior, is shown in 1C and 1D. (wherein P <0.05, P < 0.01).
2. Pristane-induced changes in microglia and neurons were present in the hippocampal region of lupus encephalopathy models.
The experimental method comprises the following steps: after anesthetizing mice of Control groups for 1 month, 2 months, 4 months and 8 months respectively, removing blood by using PBS in a heart perfusion mode, then perfusing paraformaldehyde to fix tissues, taking brains, soaking the brains in 4% paraformaldehyde solution for overnight at 4 ℃, placing the brains in 20% -30% sucrose solution for sugar precipitation the next day, and preparing sections when the brains are precipitated at the bottom of the solution. And (3) placing the specimen in an embedding medium, and cutting a frozen section with the thickness of 10um by using a freezing microtome. The excised part was the hippocampal region. After soaking the sections in PBS solution for 10min, 10% goat serum was incubated to seal the sections. Goat serum was discarded after 1 hour and anti-Iba 1 antibody (1:200, Abcam) and anti-Neun antibody (1:500, Abcam) were incubated overnight in a refrigerator at 4 ℃. The next day, after the sections were taken out of the refrigerator and returned to room temperature, the sections were washed 3 times with PBS solution, and a goat anti-rabbit secondary antibody (1:200, proteintech) and a goat anti-mouse secondary antibody (1:300, proteintech) were incubated at room temperature in the dark. After 2 hours, the sections were washed 3 times with PBS solution, and then mounted with anti-quenching mounting solution, and the change of microglia and neurons in the CA3 region of hippocampus of each mouse brain tissue was observed under a microscope.
As shown in FIG. 2, microglia (Iba1) in hippocampal CA3 region were gradually increased in 1 month (B) and 2 months (C) and decreased in hippocampal CA3 in 4 months (D) and 8 months (E) compared to control group (A). There was no significant change in neurons in the CA3 region of the hippocampus (Neun) in 1 month (B) and 2 months (C) groups, and there was a reduction in the number of neurons in the CA3 region of the hippocampus in 4 months (D) and 8 months (E) groups, as compared to control group (A).
3. There was morphological alteration of astrocytes in the hippocampal region of the Pristane-induced lupus encephalopathy model.
The experimental method comprises the following steps: after anesthetizing mice of Control groups for 1 month, 2 months, 4 months and 8 months respectively, removing blood by using PBS in a heart perfusion mode, then perfusing paraformaldehyde to fix tissues, taking brains, soaking the brains in 4% paraformaldehyde solution for overnight at 4 ℃, placing the brains in 20% -30% sucrose solution for sugar precipitation the next day, and preparing sections when the brains are precipitated at the bottom of the solution. And (3) placing the specimen in an embedding medium, and cutting a frozen section with the thickness of 10um by using a freezing microtome. The excised part was the hippocampal region. After soaking the sections in PBS solution for 10min, 10% goat serum was incubated to seal the sections. Goat serum was discarded after 1 hour and anti-GFAP antibody (1:500, Abcam) was incubated overnight in a refrigerator at 4 ℃. The next day, the sections were taken out of the refrigerator and returned to room temperature, and then washed with PBS solution 3 times, and goat anti-rabbit secondary antibody (1:200, proteintech) was incubated at room temperature in the dark. After 2 hours, the sections were washed 3 times with PBS solution, and then mounted with anti-quenching mounting solution, and the change of astrocytes in the hippocampal CA3 region of each mouse brain tissue group was observed under a microscope.
As shown in FIG. 3, the numbers of astrocytes (GFAP) in the CA3 region of hippocampus were gradually increased in 1 month (B), 2 months (C), 4 months (D) and 8 months (E) compared with the control group (A), and the activation state was exhibited.
4. Pristane-induced lupus encephalopathy model IgG deposits were present in the vessel wall and lateral ventricles of the hippocampal region.
The experimental method comprises the following steps: after anesthetizing mice of Control groups for 1 month, 2 months, 4 months and 8 months respectively, removing blood by using PBS in a heart perfusion mode, then perfusing paraformaldehyde to fix tissues, taking brains, soaking the brains in 4% paraformaldehyde solution for overnight at 4 ℃, placing the brains in 20% -30% sucrose solution for sugar precipitation the next day, and preparing sections when the brains are precipitated at the bottom of the solution. And (3) placing the specimen in an embedding medium, and cutting a frozen section with the thickness of 10um by using a freezing microtome. The excised part was the hippocampal region. After the sections were soaked in PBS solution for 10min, goat anti-mouse IgG antibody (1:200, proteintech) was incubated at room temperature in the dark. After 2 hours, the slices are washed 3 times by using PBS solution, and then the slices are sealed by using anti-quenching sealing liquid, and the IgG deposition condition of the hippocampal vessel wall and the lateral ventricle of each group of mouse brain tissues is observed under a microscope.
The results are shown in FIG. 4, in which the hippocampal vascular wall and lateral ventricle IgG (green) deposition increased gradually in the 1 month (B), 2 month (C), 4 month (D) and 8 month (E) groups compared to the control group (A).
5. Pristane-induced lupus encephalopathy model complement C3 deposition was present in the vessel wall and lateral ventricles of the hippocampal region.
The experimental method comprises the following steps: after anesthetizing mice of Control groups for 1 month, 2 months, 4 months and 8 months respectively, removing blood by using PBS in a heart perfusion mode, then perfusing paraformaldehyde to fix tissues, taking brains, soaking the brains in 4% paraformaldehyde solution for overnight at 4 ℃, placing the brains in 20% -30% sucrose solution for sugar precipitation the next day, and preparing sections when the brains are precipitated at the bottom of the solution. And (3) placing the specimen in an embedding medium, and cutting a frozen section with the thickness of 10um by using a freezing microtome. The excised part was the hippocampal region. After soaking the sections in PBS solution for 10min, 10% goat serum was incubated to seal the sections. Goat serum was discarded after 1 hour and anti-complement C3 antibody (1:50, Santa Cruz) was incubated overnight in a refrigerator at 4 ℃. The next day, after the sections were taken out of the refrigerator and returned to room temperature, the sections were washed 3 times with PBS solution and incubated with goat anti-mouse secondary antibody (1:300, proteintech) at room temperature in the dark. After 2 hours, the sections were washed 3 times with PBS solution, and then mounted with anti-quenching mounting solution, and the tissue deposits of hippocampal vessel wall and lateral ventricle C3 in each group of mice were observed under a microscope.
As shown in FIG. 5, the deposition of C3 (green) was gradually increased in the hippocampal vascular wall and lateral ventricular complement in the groups at 1 month (B), 2 months (C), 4 months (D) and 8 months (E) as compared to the control group (A).
6. Pristane-induced lupus encephalopathy model the brain tissue choroid presents a co-localization of Iba1 and IgG.
The experimental method comprises the following steps: after anesthetizing mice of Control groups for 1 month, 2 months, 4 months and 8 months respectively, removing blood by using PBS in a heart perfusion mode, then perfusing paraformaldehyde to fix tissues, taking brains, soaking the brains in 4% paraformaldehyde solution for overnight at 4 ℃, placing the brains in 20% -30% sucrose solution for sugar precipitation the next day, and preparing sections when the brains are precipitated at the bottom of the solution. And (3) placing the specimen in an embedding medium, and cutting a frozen section with the thickness of 10um by using a freezing microtome. The excised part was the hippocampal region. After soaking the sections in PBS solution for 10min, 10% goat serum was incubated to seal the sections. Goat serum was discarded after 1 hour and anti-Iba 1 antibody (1:200, Abcam) was incubated overnight in a refrigerator at 4 ℃. The next day, after the sections were taken out of the refrigerator and returned to room temperature, the sections were washed 3 times with PBS solution, and goat anti-rabbit secondary antibody (1:200, proteintech) and goat anti-mouse IgG (1:300, proteintech) were incubated at room temperature in the dark. After 2 hours sections were washed 3 times with PBS solution, followed by anti-quenching mounting fluid mounting, and co-localization of Iba1 and IgG on the ventriculo-cerebro-choroidal choroid on the brain tissue side of each group of mice was observed microscopically.
As shown in FIGS. 6A-6E, the number of co-localization of Iba1 and IgG was gradually increased in brain tissue choroid at 1 month (B), 2 months (C), 4 months (D) and 8 months (E) compared to control group (A).
7. Pristane-induced lupus encephalopathy model cortical microglia phagocytize neuronal excitatory presynaptic and postsynaptic substances.
The experimental method comprises the following steps: after anesthetizing mice of Control groups for 1 month, 2 months, 4 months and 8 months respectively, removing blood by using PBS in a heart perfusion mode, then perfusing paraformaldehyde to fix tissues, taking brains, soaking the brains in 4% paraformaldehyde solution for overnight at 4 ℃, placing the brains in 20% -30% sucrose solution for sugar precipitation the next day, and preparing sections when the brains are precipitated at the bottom of the solution. And (3) placing the specimen in an embedding medium, and cutting a frozen section with the thickness of 10um by using a freezing microtome. The excised part was the hippocampal region. After soaking the sections in PBS solution for 10min, 10% goat serum was incubated to seal the sections. Goat serum was discarded after 1 hour and anti-Iba 1 antibody (1:200, Abcam), anti-PSD 95 antibody (1:500, Thermo), anti-vglu 1 antibody (1:500, SYSY) were incubated overnight in a refrigerator at 4 ℃. After the section was taken out from the refrigerator the next day and returned to room temperature, the section was washed 3 times with PBS solution, and goat anti-rabbit secondary antibody (1:200, proteintech), goat anti-mouse IgG (1:300, proteintech) and goat anti-guinea pig (1:500, Abcam) were incubated at room temperature in the dark. After 2 hours, the sections were washed 3 times with PBS solution, and then the co-localization of each group of mice brain tissue cortex Iba1, PSD95 and VGlut1 was observed under a confocal microscope using anti-quenching mounting liquid mounting.
The results are shown in fig. 7, comparing to control group (a), the Pristane-induced lupus encephalopathy model (B) cortical microglia Iba1 phagocytosed neuronal excitatory presynaptic (VGluT1) and postsynaptic material (PSD 95).
8. Pristane-induced lupus encephalopathy model brain tissue has up-regulation of cytokine expression.
The experimental method comprises the following steps: after anesthetizing the mice in the Control group for 1 month, 2 months, 4 months and 8 months, the mice were allowed to remove blood from circulation by heart perfusion with PBS, and then the brains were collected. Preparing 10% brain tissue homogenate by using PBS solution containing protease inhibitor, adding 1% Triton-100, breaking membrane, freezing in-80 refrigerator, dissolving specimen after 1 hr, centrifuging at 4 deg.C 10000g for 5min, collecting supernatant 100ul, operating according to corresponding elisa kit operation flow, and determining brain tissue IL-6, IL-10, TNF alpha and IL-1 beta expression.
As shown in FIGS. 8A to 8D, the Pristane-induced lupus encephalopathy models were increased in the expression of the brain tissue cytokines IL-6(A), IL-10(B), TNF α (C) and IL-1 β (D) gradually at 1 month, 2 months, 4 months and 8 months as compared with the control group. P <0.05, P < 0.01.
Claims (5)
1. An animal model of lupus encephalopathy, which is an animal model of lupus encephalopathy induced by pristine, and is prepared by injecting pristine into 8-week-old female BALB/c mice.
2. The method for constructing a lupus encephalopathy model according to claim 1, specifically comprising the steps of:
step 1, an experimental subject is a healthy female BALB/c mouse with the age of 8 weeks, the skin of the back of the mouse is lightly grasped, the mouse is fixed in a palm, and the abdomen of the mouse is fully exposed;
step 2, removing mouse hair on the abdomen of the mouse by using a clipper, and disinfecting the abdomen skin by using alcohol;
step 3, selecting a 1ml medical sterilization injector, extracting 0.5ml pristine, smearing the needle head obliquely upwards, slowly inserting the needle at a point of 0.5ml beside the midline of the middle and lower abdomen, slowly pushing the injector after blood-free withdrawal, slowly enabling the medicine to enter the abdominal cavity, facilitating medicine absorption, and immediately pulling out the needle after injection; disinfecting the skin near the needle eye with alcohol again; the mice were returned to the cages and observed for 30 min.
3. Use of the lupus encephalopathy animal model of claim 1 in the mechanism of lupus encephalopathy.
4. Use of the lupus encephalopathy animal model of claim 1 in environmental factor-induced lupus encephalopathy mechanisms.
5. The use of the lupus encephalopathy animal model of claim 1 in screening drugs for treating lupus encephalopathy induced by environmental factors.
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