CN114342879A - Construction method of animal model of neurodegenerative disease - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K67/027—New or modified breeds of vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/30—Animals modified by surgical methods
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
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- Life Sciences & Earth Sciences (AREA)
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- Animal Behavior & Ethology (AREA)
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a construction method of a neurodegenerative disease animal model. The method for constructing the animal model of the neurodegenerative disease comprises the following steps: selecting animals 1-10 days after birth; and injecting a sodium glutamate solution into the animals according to the dose of 3-5 g/kg, and continuously injecting for 3-12 days. The animal model of the invention directly intervenes on the hypothalamus-pituitary-adrenal axis when the blood brain barrier is not closed after birth to cause direct nerve cell damage, and has the characteristics of progressive neurodegenerative disease along with the growth of animals.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a construction method of a neurodegenerative disease animal model.
Background
Neurodegenerative diseases (NDD) are caused by the loss of neuronal and neural myelin, which may progressively worsen over time, leading to a range of dysfunction-related diseases including cerebral ischemia, cognitive disorders, vascular dementia, parkinson's Disease, glaucoma, alzheimer's Disease and the like.
NDD occurs mainly by four etiological factors, oxidative stress, mitochondrial dysfunction, excitotoxins, and immune inflammation. Animal models currently on the market for such diseases include natural aging animal models, the SAMP8 (rapid aging) mouse model, the abeta injection-induced model, the lateral ventricle injection streptozotocin-induced model, the chronic hypoxia animal model, the APP transgenic mouse model, the Apoe knockout mouse model, and the like. The animal model uses methods including cerebral artery ischemia caused by fish line embolism, simulation of senile dementia and Parkinson's disease by using transgenic or gene knockout animals, or induction of neurodegenerative diseases by raising animals to the elderly, and induction of intracranial nervous system injury by using other neuro-excitant preparations. The method mostly adopts physical compulsion to cause cerebral ischemia, or adopts a complex gene editing or knocking-out method to cause neuropathy, and the modeling principle is mostly limited to (1) aging expression caused by natural time progress; (2) direct destruction of the adult animal ventricles by physical injection, resulting in irreversible injury; (3) influencing oxidative stress pathways by physical means such as oxygen deficiency; (4) an animal model with physiological defects is obtained by utilizing a transgenic technology. None of the above model animals has a progressive progression from normal to disease state, and neurodegenerative disease is precisely a chronic, dynamic, progressive disease, rather than sudden overt injury in some way.
Therefore, the characteristics of neuropathy generated by the animal models are greatly different from clinical morbidity characteristics, and do not belong to progressive or degenerative changes strictly, and the models cannot simulate clinical degenerative processes, for example, the cerebral tissue ischemia characteristics are that acute cerebral ischemia is caused by directly blocking blood vessels by adopting a physical method, which is greatly different from the degenerative progress of the nervous system of a clinical cerebral apoplexy patient; since neuropathy is not degenerative, early cognitive impairment features are also difficult to produce; it is difficult to see obvious changes in organs or tissues which typically show the occurrence of neurodegenerative diseases.
Disclosure of Invention
The invention aims to provide a method for constructing an animal disease model with neurodegenerative characteristics.
The method for constructing an animal model of neurodegenerative disease according to an embodiment of the present invention comprises the steps of: selecting animals 1-10 days after birth; and injecting a sodium glutamate solution into the animals according to the dose of 3-5 g/kg, and continuously injecting for 3-12 days.
In the invention, an animal which is just born is taken as a modeling object, and the blood-brain barrier (BBB) of the animal is not completely closed at the moment, so that the animal has certain permeability, macromolecular substances can freely enter the brain, and sodium glutamate is continuously administrated intracranially/subcutaneously within one week after birth and can smoothly pass through the BBB, thereby causing the neuroexcitotoxicity. The sodium glutamate is a salt formed by glutamic acid and sodium ions, and can directly damage intracranial nerve cells when the blood brain barrier is not closed, so that the progressive nerve damage condition gradually appears along with the growth of a life body, and the characteristic of the sodium glutamate is very close to the long nerve degeneration phenomenon of clinical sick personnel.
The specific mechanism by which sodium glutamate causes nerve damage is as follows: glutamate receptors, such as the n-methyl-d-aspartate (NMDA) receptor, the alpha-amino-3 hydroxy-5 methyl-4 isoxazole receptor (AMPAR) and the metabotropic receptors (mGluRs), are strongly involved in the activation of neurotoxicity as excitatory neurotransmitters, i.e., when glutamate receptors are over-activated, they induce neurotoxicity, which leads to neuronal cell death. In the present invention, excessive ingestion of sodium glutamate by a newborn animal leads to excessive activation of glutamate receptors, thereby causing Ca2+、Na+Excessive internal flow, the ability to inhibit the binding of intracellular calcium ions, causes high calcium load in cells, increases the synthesis of nitric oxide, thereby affecting the normal synthesis and repair of DNA, causing excitation of neurons, generating excitotoxicity, and causing damage to nerve cells. The process fully reflects the characteristic of progressive loss of specific cells in brain tissues and related tissues of neurodegenerative diseases, and proves that the sodium glutamate can induceThe inducer for the neurodegenerative disease selects the time of about one week after the birth of the newborn animal to intervene with sodium glutamate, so as to obtain the animal with progressive neurodegenerative disease characteristics.
According to the construction method of the animal model of the neurodegenerative disease, animals selected in the construction of the animal model are mammals; preferably, the animal of the present invention is a rodent, an object of the class mammalia, a small-and medium-sized mammal having a pair of chisel-shaped incisors, commonly known as a mouse, including big and small mice, yellow rats, gerbils, golden hamsters, guinea pigs, and the like.
According to a method for constructing an animal model of neurodegenerative disease in accordance with a specific embodiment of the present invention, the animal is rat or mouse.
Wherein the rat is white rat (Rattus norvegicus) belonging to the class of mammalia, Rodentia, and Muidae. The experimental rat (Laboratory Rats) is a variety of brown Rats, adult Rats generally have the body length of 16-21 cm and the body weight of about 250-450 g, male Rats are large, the hair color is pure white, the adult Rats are densely covered with hair and are glossy, the heads and the faces of the adult Rats are pointed, harder tentacles are arranged at the front parts of the mouths and the faces of the adult Rats, the eyes are large and bright, and the irises are pink. The tail length is generally 16-18 cm, and short hair and annular cutin squamae are grown on the tail.
Mouse refers to the white mouse (Mus musculus) which is a variety of wild trabs, the mouse species, the adult mouse body length is not more than 15.5 cm, the face is pointed, palpus is grown on the mouse body, the ear stands vertically and is semicircular, the eyes are large, the nose tip, the tail length is similar to the body length, and the tail part has a transverse scale under the skin.
The mouse strains which can be selected by the construction method of the animal model comprise an ICR mouse, a BALB/C mouse, a KM mouse, an HIN mouse, a C57 mouse, a KK mouse, an SD rat, a Wistar rat, a Fisher344 rat, an SHR rat and the like.
According to the construction method of the animal model of the neurodegenerative disease, a certain dosage of sodium glutamate solution is injected into the test animal subcutaneously, the concentration of the sodium glutamate solution is 5-15%, and preferably, the concentration of the sodium glutamate solution is 10%.
According to the construction method of the animal model of neurodegenerative disease of the present embodiment, the animal is injected with the sodium glutamate solution at a dose of 4g/kg for 7 days.
A method of constructing an animal model of neurodegenerative disease according to an embodiment of the present invention, the neurodegenerative disease including Alzheimer's Disease (AD), vascular dementia, cognitive impairment, glaucoma, Parkinson's Disease (PD), Huntington's Disease (HD), Amyotrophic Lateral Sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease, and the like.
According to the construction method of the animal model of the neurodegenerative disease, rodent animals 1-10 days after birth are selected; and injecting a sodium glutamate solution into the rodent according to the dose of 3-5 g/kg, and continuously injecting for 3-12 days.
The invention has the beneficial effects that:
compared with the traditional model, the animal model has the characteristics of low construction cost and high molding rate, the molding success rate can reach 80-90%, and the molding method is different from the traditional method on the market, and progressive nerve injury development occurs in the later growth process of animals by directly giving sodium glutamate inducer to the intracranial nerve cells of the animals during the period that the blood brain barrier of the animals is not closed, so that the neurodegenerative pathological changes are reflected, the pathological changes of the neurodegenerative disease better accord with the clinical characteristics, the diseases are progressive, chronic, free of obvious surgical trauma and free of physical intervention, the animal model is very suitable for evaluating the neurodegenerative disease and the drug curative effect thereof, and the pathogenesis of the neurodegenerative disease can be better understood.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the body weight profile of a mouse in an animal model constructed in accordance with the present invention;
FIG. 2 shows the result of the positive rate calculation of the mouse brain slice hippocampal immunohistochemistry;
FIG. 3 shows the mouse brain slice hippocampal immunohistochemical results of the present invention;
FIG. 4 shows multi-target fluorescence imaging of hippocampal region of mouse brain slices of the present invention;
FIG. 5 shows the body weight profile of rats in an animal model constructed in accordance with the present invention;
FIG. 6 shows the result of the immunohistochemical positive rate calculation of hippocampal region of rat brain slices of the present invention;
FIG. 7 shows the immunohistochemical results of hippocampal regions of rat brain slices of the present invention;
FIG. 8 shows multi-target fluorescence imaging of hippocampal region of rat brain slices of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention. Example 1 construction of animal models of neurodegenerative diseases
ICR suckling mice (purchased from Beijing Huafukang Biotechnology GmbH, license number SCXK (Jing) 2019-:
group one (5% sodium glutamate): subcutaneously injecting 5% sodium glutamate with 3g/kg dose from the second day of birth of ICR suckling mice for 3 days;
second group (10% sodium glutamate): subcutaneously injecting 10% sodium glutamate with the dose of 4g/kg from the second day of birth of ICR suckling mice for 7 days;
third group (15% sodium glutamate): from the second day of birth of ICR suckling mice, 15% sodium glutamate is injected subcutaneously at a dose of 5g/kg, and molding is carried out by continuous injection for 12 days.
Meanwhile, several suckling mice on the same day without sodium glutamate injection were used as a control group.
After feeding the suckling mice for 21 days, the young mice are weaned and separated into male and female parts, and are raised in an SPF-level animal room, the temperature is controlled at 22 +/-2 ℃, the relative humidity is 50 +/-5%, the light and dark circulation is carried out for 12 hours, and the young mice are free to eat and drink water.
Example 2 verification of the Effect of animal model construction
2.1 body weights were measured at 4W, 8W, 12W, 16W, 18W weeks of age in the first, second and third groups of mice, respectively. Specific results are shown in table 1.
TABLE 1 weight change of mice in the sodium glutamate group and the Normal group
Note: p <0.05 compared to Control group; p < 0.01; p < 0.001.
As shown in the above table, the body weight of the suckling mice injected with sodium glutamate continuously increased compared to the normal control group from 4 weeks of age (see fig. 1).
Brain HE sections were image-acquired with an optical microscope (Olympus BX51, japan) in the format of TIF, using 1360 × 1024 and 4608 × 3456 pixels, respectively.
Immunohistochemistry was performed on the same sample brain section, and staining was performed using a BCA method using a 1:500 dilution of glutamate receptor (NMDAR), neuregulin (NRG-1), and glutamate (Glu) primary antibody, followed by image acquisition using the same optical microscope after staining, and the positive rate was calculated.
And the positive rate calculation is to read the pixels of the stained brain section image by adopting a histogram function in a Photoshop 2019 version to obtain pixel values, and calculate according to a formula.
Model positive ratio (%). Model brain section pixel value × 100/Control brain section pixel value.
The results are shown in table 2 and fig. 2.
TABLE 2 comparison of Positive rates of immune combination indexes of sodium glutamate group and Normal group mice
The presence of metabotropic glutamate receptor (GluR) and ionotropic glutamate receptor subtype 2A (NMDAR2A) in the hippocampal region of the brain; NRG1 is a neuregulin, which is mainly expressed in synapses and glial cells of neurons centrally in brain, and influences the actions of neuronal synapses, neuronal migration and neuronal growth and development by modulating the expression of neuronal receptors (NMDAR and the like), while loss of NRG-1 expression leads to reduced neurotransmission.
The results are shown in the table above, it can be seen that sodium glutamate with different concentrations causes different degrees of damage to the mouse brain in the animal model of neurodegenerative disease over time compared with the blank mice, and the expression levels of the three proteins NMDAR2A, GluR2 and NRG1 in the hippocampal region are gradually decreased. Statistical analysis and processing are carried out on the data by using SPSS 20.0 software, after a model group is compared with a blank control group, the lower positive rate indicates that nerve cells are more seriously damaged, and as shown in figure 2, the three proteins have extremely remarkable statistical differences, and the sodium glutamate is further proved to down regulate the expression of the three proteins, so that the brain nerve cells are lost or die, and the neurodegenerative diseases are caused. The higher the concentration of sodium glutamate, the lower the positive rate of the three indicators of immunohistochemistry, indicating that the more nerve cells in the hippocampal region of the brain die, and thus the lower the positive rate.
(P < 0.05;. P < 0.01;. P < 0.001; statistics are obtained by significance testing methods, typically with P <0.05 as a statistical difference, P <0.01 as a significant statistical difference, and P <0.001 as a very significant statistical difference.) compared to the blank control group.)
Immunohistochemistry of hippocampal tissues was first performed using Fosb for the same sample brain sections at different time points, and the immunohistochemistry results for model groups of 4W (see FIG. 3-B), 12W (see FIG. 3-C), and 18W (see FIG. 3-D) were found to be significantly increased in Fosb compared with the normal group (see FIG. 3-A) and to be statistically different.
Secondly, the brain slices at different time points are marked by multiple markers with fluorescence, and the positive rate of the target protein marked by each fluorescence is counted, 5 percent of the total positive rate is calculated (as shown in figure 4-B)1/B2/B3) 10% (see FIG. 4-C)1/C2/C3) 15% (see FIG. 4-D)1/D2/D3) Modeling sodium glutamate with three concentrations and normal group (see FIG. 4-A)1/A2/A3) A comparison is made.
The results show that the expression level of NRG1 protein in the model group is significantly changed compared with that in the normal control group, the expression level is statistically different, and the higher the concentration of sodium glutamate is, the more nerve cells in the brain hippocampus die.
Example 3 construction of animal model (rat) for neurodegenerative disease
SD rats were selected and randomly divided into 4 groups, 15 animals for neurodegenerative diseases and normal control groups, respectively, and molded.
Group one (5% sodium glutamate): subcutaneously injecting 5% sodium glutamate with a dose of 3g/kg from the second day of birth of SD rat for 3 days;
second group (10% sodium glutamate): subcutaneously injecting 10% sodium glutamate with a dose of 4g/kg from the second day of birth of SD rat for 7 days;
third group (15% sodium glutamate): from the second day of birth of SD rats, 15% sodium glutamate was subcutaneously injected at a dose of 5g/kg for 12 days continuously for molding.
Meanwhile, several suckling mice on the same day without sodium glutamate injection were used as a control group.
After feeding the suckling mice for 21 days, the young mice are weaned and separated into male and female parts, and are raised in an SPF-level animal room, the temperature is controlled at 22 +/-2 ℃, the relative humidity is 50 +/-5%, the light and dark circulation is carried out for 12 hours, and the young mice are free to eat and drink water.
Body weights were measured at 4W, 8W, 12W, 16W, 18W week old rats, respectively, as shown in fig. 5.
TABLE 3 weight change of rats in the sodium glutamate group and the Normal group
Note: p <0.05 compared to Control group; p < 0.01; p < 0.001.
Brain HE sections were image-acquired with an optical microscope (Olympus BX51, japan) in the format of TIF, using 1360 × 1024 and 4608 × 3456 pixels, respectively.
Immunohistochemistry was performed on the same sample brain section, and staining was performed using a BCA method using a 1:500 dilution of glutamate receptor (NMDAR), neuregulin (NRG-1), and glutamate (Glu) primary antibody, followed by image acquisition using the same optical microscope after staining, and the positive rate was calculated.
And the positive rate calculation is to read the pixels of the stained brain section image by adopting a histogram function in a Photoshop 2019 version to obtain pixel values, and calculate according to a formula.
Model positive ratio (%). Model brain section pixel value × 100/Control brain section pixel value.
The results are shown in table 4 and fig. 6.
TABLE 4 comparison of Positive rates of immunological combination indexes of sodium glutamate group and Normal group rats
The results are shown in the table above, it can be seen that sodium glutamate with different concentrations causes different degrees of damage to the mouse brain in the animal model of neurodegenerative disease over time compared with the blank group of rats, and the expression levels of the three proteins NMDAR2A, GluR2 and NRG1 in the hippocampal region are gradually decreased. Statistical analysis and processing are carried out on the data by using SPSS 20.0 software, after a model group is compared with a blank control group, the lower positive rate indicates that nerve cells are more seriously damaged, as shown in figure 6, the three protein expression are extremely remarkably statistically different, and the sodium glutamate is further proved to down regulate the expression of the three proteins, so that the brain nerve cells are lost or die, and the neurodegenerative diseases are caused. The higher the concentration of sodium glutamate, the lower the positive rate of the three indicators of immunohistochemistry, indicating that the more nerve cells in the hippocampal region of the brain die, and thus the lower the positive rate.
(P < 0.05;. P < 0.01;. P < 0.001; statistics are obtained by significance testing methods, typically with P <0.05 as a statistical difference, P <0.01 as a significant statistical difference, and P <0.001 as a very significant statistical difference.) compared to the blank control group.)
Immunohistochemistry of hippocampal tissues was first performed using Fosb for the same sample brain sections at different time points, and the immunohistochemistry results of model groups of 4W (see FIG. 7-B), 12W (see FIG. 7-C) and 18W (see FIG. 7-D) were found to be significantly increased and statistically different from the normal group (see FIG. 7-A).
Secondly, the brain slices at different time points are marked by multiple markers with fluorescence, and the positive rate of the target protein marked by each fluorescence is counted, 5 percent of the total positive rate is calculated (as shown in figure 8-B)1/B2/B3) 10% (see FIG. 8-C)1/C2/C3) 15% (see FIG. 8-D)1/D2/D3) Modeling sodium glutamate with three concentrations and normal group (see FIG. 8-A)1/A2/A3) A comparison is made.
The results show that the expression level of NRG1 protein in the model group is significantly changed compared with that in the normal control group, the expression level is statistically different, and the higher the concentration of sodium glutamate is, the more nerve cells in the brain hippocampus die.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (9)
1. A construction method of a neurodegenerative disease animal model is characterized by comprising the following steps: selecting animals 1-10 days after birth; and injecting a sodium glutamate solution into the animals according to the dose of 3-5 g/kg, and continuously injecting for 3-12 days.
2. The method of claim 1, wherein the animal model is a rodent.
3. The method for constructing an animal model of neurodegenerative disease according to claim 1 or 2, wherein the animal is a rat or a mouse.
4. The method of claim 1, wherein the concentration of the sodium glutamate solution is 5% to 15%.
5. The method for constructing an animal model of neurodegenerative disease according to claim 1 or 4, wherein the concentration of the sodium glutamate solution is 10%.
6. The method of claim 1, wherein the animal is injected with 4g/kg of sodium glutamate solution for 7 days.
7. The method of claim 1, wherein the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.
8. The method of constructing an animal model of neurodegenerative disease according to claim 1 or 7, wherein the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, Huntington's disease, cognitive disorders, vascular dementia, glaucoma and amyotrophic lateral sclerosis.
9. The method of claim 1, wherein the rodent is selected 1 to 10 days after birth, and the rodent is injected with 10% sodium glutamate solution at a dose of 3 to 5g/kg for 3 to 12 days.
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