WO2021155866A1 - Preparation and application of animal model of neuronal dendritic dysplasia in extensive brain regions - Google Patents
Preparation and application of animal model of neuronal dendritic dysplasia in extensive brain regions Download PDFInfo
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K67/02—Breeding vertebrates
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
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- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Definitions
- the present invention relates to the field of biotechnology, in particular to the preparation and application of an animal model of neuronal dendritic development disorders in a wide range of brain regions.
- H/I Hypoxic/ischemic injury is one of the main causes of brain dysfunction in all ages. It has been extensively studied in clinical and experimental animal studies, including etiology, neuropathogenesis and pharmacological intervention. More and more studies have shown that H/I may have an adverse effect on rodent brain development.
- Blocking arterial blood vessels + hypoxia method-cerebral ischemia and hypoxia model Disadvantages: anesthesia has neuroprotective effect; surgery itself is traumatic; infarcts are highly variable and unstable; model making is troublesome and small in batches; success rate is low; The cycle is long.
- Middle cerebral artery occlusion model This model is often used in adult rats, and it is only a cerebral ischemia or cerebral ischemia-reperfusion injury model without hypoxia; it is more suitable for clinical cerebral ischemia. A focal lesion model. It is not suitable for the simulation of neonatal hypoxic ischemic encephalopathy.
- Clamping the trachea method the disadvantages are: anesthetic drugs have an impact on the nerves; the trauma caused by the operation itself; the technical requirements are high, the cycle is long; the batch size is small; because the mice used are generally older (too small, the operation is difficult), The consistency of pathological changes is poor.
- hypoxic brain injury animal model which can be used as an animal model for studying the pathogenesis of neuronal dendritic development disorders in a wide range of brain regions and a powerful tool for screening new drugs.
- the purpose of the present invention is to provide an animal model that can be used as a powerful tool for studying the pathogenesis of neuronal dendritic development disorders in a wide range of brain regions and for screening new drugs.
- the first aspect of the present invention provides a method for preparing a non-human mammalian model of neuronal dendritic development disorders in a wide range of brain regions, including the following steps:
- the oxygen concentration (volume ratio) drops from 10-20% to 1-5%.
- the neuron dendritic development disorder of the extensive brain region includes the neuron dendritic spine development disorder of the extensive brain region.
- the non-human mammals are rodents or primates, preferably including mice, rats, rabbits and/or monkeys.
- the non-human mammal is a newborn non-human mammal, preferably, a newborn non-human mammal (such as a rodent) within 24 hours of birth or a newborn non-human mammal in the perinatal period. Mammals) such as primates).
- an inert gas is added to reduce the oxygen concentration.
- the inert gas is selected from the following group: nitrogen, helium, or a combination thereof.
- the animal model of extensive brain neuron dendritic developmental disorder has one or more characteristics selected from the following group:
- the synaptic physiological activity includes: mEPSCs amplitude and frequency.
- the behavioral, emotional, and cognitive deficits include decreased spatial learning ability and memory ability, decreased active inquiry behavior, weaker fear of dangerous situations, and weaker natural fear of bright areas.
- the second aspect of the present invention provides the use of a non-human mammalian model prepared by the method of the first aspect of the present invention, which is used as an animal model for studying neuronal dendritic development disorders in a wide range of brain regions.
- the neuron dendritic development disorder of the extensive brain region includes the neuron dendritic spine development disorder of the extensive brain region.
- the third aspect of the present invention provides a use of the non-human mammalian model prepared by the method of the first aspect of the present invention to screen or identify substances that can alleviate or treat neuronal dendritic development disorders in a wide range of brain regions (treatment Agent).
- the neuron dendritic development disorder of the extensive brain region includes the neuron dendritic spine development disorder of the extensive brain region.
- the fourth aspect of the present invention provides a method for screening or identifying potential therapeutic agents for treating or relieving neuronal dendritic development disorders in a wide range of brain regions, including the following steps:
- test compound in the presence of the test compound, the test compound is applied to the non-human mammal model prepared by the method of the first aspect of the present invention, and the extensive brain area neurons of the animal model in the test group are detected
- the test compound is a treatment or alleviation of widespread A potential therapeutic agent for neuronal dendritic development disorders in the brain area.
- the detection of the severity of neuronal dendritic development disorders in a wide range of brain regions includes detecting changes in one or more indicators selected from the following group: synaptic electrophysiological activity; neuronal tree in each brain region The spine shape, size, number, distribution, and density of the spines and dendritic spines; the diameter, length, and number of the dendrites of neurons; the developmental status of the distribution of synapses and their constituent structures in each brain area; behavior, emotion, and cognition Function.
- the reduction in the severity of the neuronal dendritic development disorder in the extensive brain area is manifested as: a decrease in the degree of decrease in the electrophysiological activity of the synapses; the spine density, size, number, The decrease in density is reduced; the diameter, length, and number of dendrites of neurons are reduced; the development of the distribution of synapses and their constituent structures in each brain area is reduced; and/or the degree of deficits in behavior, emotion, and cognitive functions is reduced.
- the "significantly lower” means that the severity Q1 of the test group with biological duplication after administration of the test compound is lower than the severity Q2 of the control group with biological duplication after administration of the test compound, and After t test, the P value is less than 0.05.
- the "significantly lower” means that the ratio of severity Q1/severity Q2 is ⁇ 1/2, preferably ⁇ 1/3, more preferably, ⁇ 1/4.
- the method is non-diagnostic and non-therapeutic.
- the method includes step (c), applying the potential therapeutic agent screened or identified in step (b) to the non-human mammal model prepared by the method of the first aspect of the present invention, thereby determining its effect on The animal model is widely affected by the severity of neuronal dendritic development disorders in the brain area.
- the fifth aspect of the present invention provides a non-human mammal model, which is prepared by the method described in the first aspect of the present invention.
- Figure 1 shows that this model animal did not cause significant changes after undergoing hypoxia. Among them, A: No change in the shape of the brain was found; B: No change in body weight was caused.
- Figure 2 shows that the neuronal electrophysiological activities of the model rats are significantly affected after the hypoxic shock.
- AC in vitro electrophysiology shows the decrease of synaptic electrical activity between brain neurons after hypoxia
- DF in vivo electrophysiology shows changes in the electrophysiological function of the hippocampus with long-term depression (LYD) after hypoxia .
- Figure 3 shows the brain light microscope observation after hypoxia, no significant changes were found in neuron morphology, size, arrangement, number, distribution, and density (A-J).
- Figure 4 shows the brain light microscope observation after hypoxia, no significant changes were found in the morphology, size, arrangement, number, distribution, and density of microglia (A-F).
- FIG 5 shows that the dendritic diameter, size, number, and density (B) of neurons in the hippocampus area of the hippocampus in the early stage (7 days after birth) after hypoxia are significantly reduced compared to normal mice (A); this difference is in More significant in adulthood (C, D)
- Figure 6 shows other brain regions other than the hippocampus, such as the dentate gyrus (DG), the dendrites and dendritic spines of nerve cells have also seen hypoxia causing dendritic diameter reduction, dendritic length shortening, and dendritic spines Significant changes in density reduction (AF); in the CA3 area, the volume of the area composed of synapses composed of neuronal dendritic spines is reduced (GI).
- DG dentate gyrus
- GI neuronal dendritic spines
- Figure 7 shows that the length of the dendrites emitted by neurons cultured in a hypoxic environment using in vitro cell culture methods decreased significantly, markedly on the first day (A, B), and the difference was even greater on the seventh day (C ,D).
- Figure 8 shows the changes in the escape latency of the rats that have suffered hypoxia in the newborn period after adulthood, reflecting the significant decline in their spatial learning ability (A); the movement trajectory of the rats after being withdrawn from the platform (C) , Reflecting that its spatial memory ability is also significantly reduced (B, D).
- Figure 10 shows Nissl staining of hippocampal CA1 area of rat brain tissue showing pyramidal neurons. After 35 minutes of hypoxia, the newborn rats survived reoxygenation and respiration for 3 days. The brain paraffin sections were taken for Nissl staining, showing neurons and glial cells. The results showed that after 3 days of hypoxia, there were no significant abnormalities in nerve cells compared with the normal control group. There is no manifestation of cellular edema.
- the inventor unexpectedly discovered that exposure of non-human mammals to hypoxic conditions in which the oxygen concentration (volume ratio) is reduced from 10-20% to 1-5% for 30-40 minutes can be An animal model of neuronal dendritic development disorder in a wide range of brain regions is obtained.
- the animal model of the present invention is an effective animal model of neuronal dendritic development disorder in a wide range of brain regions, which can be used to study the development of neuronal dendrites in a wide range of brain regions. Barriers, and can be used for screening and testing of specific drugs. The present invention has been completed on this basis.
- the present invention establishes a relatively mild hypoxic condition (that is, mammals are exposed to a changing oxygen concentration environment, for example, the oxygen concentration (volume ratio) is reduced from 10-20% to 1-5% Under low oxygen conditions), significant neuron dendrites and their dendritic spines develop obstacles. Structural damage caused by such developmental obstacles persists, causing damage to learning, memory, and other cognitive behaviors.
- a very effective non-human mammalian model of neuronal dendritic development disorders in a wide range of brain regions is provided.
- non-human mammals include (but are not limited to): mice, rats, rabbits, monkeys, etc., more preferably rats and mice.
- the animal model of the present invention is prepared by the following method:
- the oxygen concentration (volume ratio) is reduced from 10-20% to 1-5%.
- the animal model obtained by the method of the invention is fertile and develops normally.
- a method for screening candidate drugs or therapeutic agents for the treatment of neuronal dendritic development disorders in a wide range of brain regions using the animal model of the present invention is also provided.
- a candidate drug or therapeutic agent refers to a substance that is known to have a certain pharmacological activity or is being tested that may have a certain pharmacological activity, including but not limited to nucleic acids, proteins, chemically synthesized small molecules or large molecules. Molecular compounds, cells, etc.
- the drug candidate or therapeutic agent can be administered orally, intravenously, intraperitoneally, subcutaneously, spinal canal, or direct intracerebral injection.
- the intervention treatment factor is single. It was only given a condition of hypoxia, and no surgery or medication was performed on the animal body.
- the model is uniform. Individual differences are small, the birth time of newborn rats is easy to grasp, and there is almost no external interference after birth.
- the function of the damage point is critical.
- the damage sites of the model are concentrated in the dendritic spines.
- the dendritic spines are the key nodes in the connection of the neurons that make up the network of the brain. They are the main channels for the transmission of signals that affect each other between neurons, and are the signal transmission efficiency of the central nervous system network system.
- the key point is the main structural basis for the level of brain function.
- the animal model of the present invention is phenotypically stable.
- the animal model obtained by the method of the present invention is fertile, and the general structure of the body is normally developed.
- the animal model of the present invention can be used to study the role and mechanism of TSH in early brain neuron dendritic spines development.
- the animal model of the present invention can be used to explore, develop, and verify potential drugs or methods and methods to interfere with TSH injury, block the developmental damage of dendritic spine, and improve the developmental obstacle of dendritic spine.
- the animal model of the present invention can be used to study the functional changes of the nervous system and its operating mechanism under the pathological conditions of dendritic spines developmental disorders.
- the animal model of the present invention is used as a reference for a nervous system model of mental retardation, for comparison and reference for learning and memory research under other neuropathological models (such as Alzheimer's disease).
- the hypoxic condition of the present invention is hypoxia under normal pressure.
- hypoxic conditions of the present invention have the characteristics of short duration and persistence.
- Pregnant female rats are kept in an animal room with 12 hours of light and 12 hours of darkness. They have free access to food and water until they give birth.
- the newborn rats were first placed in a hypoxic chamber with an oxygen concentration of 15%. Under the condition of 30°C, they were gradually adjusted to 3% by N2 within 30 minutes. After that, the oxygen concentration was maintained at 3%, and the newborn rats stayed in the box for another 5 minutes. Therefore, the total hypoxic injury lasted for 35 minutes, followed by normal breathing.
- the normoxia treatment group newborn rats were placed in the box for 35 minutes, and the normoxia concentration was 21%.
- hypoxia systemic reaction is obvious: at the end of the hypoxia treatment, the whole body color of the newborn rat is darker than the normal control group, and it recovers soon after the hypoxia treatment.
- the brain morphology remains unchanged: The researchers also carefully compared the brains of young mice that were treated with TSH and normoxia within 5 days and 7 days, respectively. There was no significant difference in brain morphology and brain size between the hypoxia treatment group and the normoxia treatment group (Figure 1A).
- TSH will not cause changes in the structure of brain nerve cells.
- microglia are not activated. TSH does not induce brain activation of microglia. Observe the morphology, number, distribution, density, etc. of hippocampal CA1 microglia. Compared with normal ( Figure 4, A, B, C), the hippocampal microglia of hypoxic rats showed no obvious activation ( Figure 4). , D, E, F).
- TSH causes the dendritic spine density of hippocampal neurons to decrease.
- the structure details of the dendrites of hippocampal CA1 neurons, dendritic spines and granular cells in the DG of the brain area were detected respectively.
- the dendritic spines of normal rat neurons are distributed in clusters along the dendrites.
- the TSH of the dendritic spines of the animals is significantly reduced, sparsely distributed, and only a few are distributed along the dendrites (Figure 5).
- the dendrites of neurons decrease in diameter.
- the neuronal dendrites of hippocampal CA1 especially the secondary branches.
- the average dendritic diameter of animals in the TSH group was roughly the same as that of the normal control group.
- the diameter of the secondary branches of neuronal dendrites in TSH-treated animals was statistically smaller than that of the normoxic control group. Similar results were also observed 90 days after hypoxia and normoxia treatments ( Figure 5 and Table 2).
- SH causes abnormal development of DG and CA3 regions.
- the observation of the hippocampal dentate gyrus further confirmed that the density of dendritic spines in the hippocampus CA1 was reduced and the secondary branches of dendrites became thinner ( Figure 6A, 6B, 6C).
- the dendritic spine density of DG granular cells in animals treated with TSH was 7.03 ⁇ 0.53/10um, which was lower than 14.8 ⁇ 0.60/10um in the normoxic control group, p ⁇ 0.0001 (Figure 6D).
- the length of dendrites in the TSH group was significantly shorter than that in the control group.
- the dendritic diameter of granular cells in the dentate gyrus of animals was 0.893 ⁇ 0.064m, which was significantly larger than 0.628 ⁇ 0.046m in the normal control group, p ⁇ 0.05 ( Figure 6E).
- the dendritic contraction length of the inner molecular layer (IML) granular cells in the hippocampus DG of animals 90 days after TSH treatment was 15.35 ⁇ 1.75m, which was significantly higher than 7.04 ⁇ 1.02m in the normoxic control group, p ⁇ 0.001 ( Figure 6F).
- TSH can cause long-term cognitive deficits.
- Morris water maze experiment was performed on rats treated with TSH and normoxia at 3 months old.
- rats in the control group and the hypoxia group showed similar improvements in the first 6 training sessions, and the latency of all animals in finding a platform was shortened.
- the latency of rats in the TSH group was 21.6 ⁇ 3.71, 16.6 ⁇ 1.79, 20.4 ⁇ 3.86, which were significantly longer than those of the control group 12.1 ⁇ 1.72, 7.99 ⁇ 1.03, 8.07 ⁇ 0.96, respectively (p ⁇ 0.05) (Figure 8A).
- Neonatal hypoxia injury is usually thought to be caused by umbilical cord around the neck, unlined head and pelvis, birth incarceration, poor blood supply to the uterus, and neonatal asphyxia.
- Perinatal hypoxic-ischemic brain injury causes higher mortality and chronic neurological morbidity in acute infants and children, and often sequelae symptoms.
- the present invention finds for the first time that TSH causes significant pathological changes, including the thinning and shortening of the tree of the central nervous system neurons, the decrease of dendritic spine density, the decrease of synaptic structure, and the decrease of synaptic electrophysiological function; in addition, in animals suffering from TSH Impaired cognitive function was observed in.
- the data of the present invention clearly shows that TSH can cause significant neurosynaptic and dendritic toxicity in the neonatal and adult stages. Therefore, the present invention provides a useful animal model that can be used to study the effects and mechanisms of sublethal hypoxia on early brain development, and to explore potential intervention drugs or methods.
- the study of the present invention shows that 6 hours after birth, newborn rats are given short sublethal hypoxia for 35 minutes alone, which is different from the previous animal model used for hypoxia/ischemia research.
- animals born 6 hours old are used.
- the animals used are about one Sunday old, which is closer to the clinical situation (umbilical cord around the neck, birth incarceration, uterine malformation, etc.).
- the shorter the time after birth the smaller the individual coefficient of variation of the animal after birth, and the better the uniformity of the nervous system among the animals.
- a single-factor gradient hypoxia supply is the second difference between the current study and previous animal models using carotid artery occlusion and hypoxia (two factors).
- TSH blocks the spontaneous synaptic activity of neurons. Background activity of electroencephalogram (EEG) is found in very early preterm infants and animals.
- EEG electroencephalogram
- TSH caused a decrease in the amplitude and frequency of mEPSCs in hippocampal CA1 neurons.
- TSH has a negative effect on the development of neuronal dendrites.
- the diameter of the animal's neuron dendrites is significantly reduced.
- the total dendritic length of the primary cultured hippocampal neurons was also significantly lower than that of the normal control group.
- TSH damage induces dendritic toxicity and affects the growth and development of neuronal dendrites. Insufficient energy supply caused by hypoxia may affect cytoskeletal dynamics including actin and microtubules. Actin and MT are the keys to conventional dendritic growth and development, and they are the targets of many molecular pathways that control the growth of neuronal dendrites. During the development of the brain, the structure of actin and MT cytoskeleton changes, resulting in neuronal processes. The present invention also found that TSH damage reduces the density and development of dendritic spines. More than 95% of excitatory synapses on these neurons occur on dendritic spines, and each spine head is usually connected to form a synapse.
- dendritic spines are very important for brain function.
- the development of dendritic spines is controlled by the oxygen sensor PHD2, targeting the actin cross-linking agent Filamin-A to regulate synaptic density and neuronal activity within the network.
- Dendritic spines-associated Rap-specific GDP enzyme activator protein is a post-synaptic protein that forms a complex with postsynaptic density (PSD)-95, which is subject to N-methyl-D-aspartic acid.
- NMDARs are involved in regulating the morphogenesis of dendritic spines.
- dendritic spines reflect the strength of synapses. In different brain diseases, including neurodegenerative diseases and mental diseases, the strength of synapses is usually severely affected. Dendritic spines can undergo several types of transitions, from growth to collapse, from elongation to shortening, and the time span for them to undergo this dynamic morphological activity is very short. Changes in the number and morphology of dendritic spines not only occur under pathological conditions such as excitotoxicity, but also occur in response to normal central nervous system development, hormone fluctuations, and neural activity under physiological environments.
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Abstract
A method for preparing a non-human mammalian model of neuronal dendritic dysplasia in extensive brain regions, comprising the following steps: exposing a non-human mammal in a low-oxygen condition for 30-40 min to obtain an animal model of neuronal dendritic dysplasia in extensive brain regions, wherein in the low-oxygen condition, the oxygen concentration (volume ratio) drops from 10-20% to 1-5%. The animal model can be applied to study neuronal dendritic dysplasia in extensive brain regions, and can be applied to screening and testing tests of particular drugs.
Description
本发明涉及生物技术领域，具体地，涉及一种广泛脑区神经元树突发育障碍动物模型的制备与应用。The present invention relates to the field of biotechnology, in particular to the preparation and application of an animal model of neuronal dendritic development disorders in a wide range of brain regions.
缺氧/缺血性(H/I)损伤是各年龄段脑功能缺陷的主要原因之一，在临床和实验动物研究中都有广泛的研究，包括病因学、神经发病机制和药理干预。越来越多的研究表明，H/I可能对啮齿动物大脑发育产生不利影响。Hypoxic/ischemic (H/I) injury is one of the main causes of brain dysfunction in all ages. It has been extensively studied in clinical and experimental animal studies, including etiology, neuropathogenesis and pharmacological intervention. More and more studies have shown that H/I may have an adverse effect on rodent brain development.
目前大多数低氧性脑损伤动物模型有：Currently, most animal models of hypoxic brain injury include:
1.阻断动脉血管+缺氧法—脑缺血缺氧模型：缺点：麻醉具有神经保护作用；手术本身具有创伤；梗死变异大，不稳定；模型制作麻烦，批量小；成功率低；制作周期长。1. Blocking arterial blood vessels + hypoxia method-cerebral ischemia and hypoxia model: Disadvantages: anesthesia has neuroprotective effect; surgery itself is traumatic; infarcts are highly variable and unstable; model making is troublesome and small in batches; success rate is low; The cycle is long.
2.大脑中动脉闭塞模型：这种模型，常常使用成年鼠，且只是一种脑缺血或脑缺血再灌注损伤模型，没有缺氧的因素；比较适合临床的脑缺血病症，是一种局灶性病变模型。不适合新生儿缺血缺氧性脑病的模拟。2. Middle cerebral artery occlusion model: This model is often used in adult rats, and it is only a cerebral ischemia or cerebral ischemia-reperfusion injury model without hypoxia; it is more suitable for clinical cerebral ischemia. A focal lesion model. It is not suitable for the simulation of neonatal hypoxic ischemic encephalopathy.
3.夹闭气管法：缺点是：麻醉药物存在对神经的影响；手术本身造成的创伤；技术要求高，周期长；批量小；由于使用的鼠一般日龄较大(太小手术困难)，带来病理变化的一致性不良。3. Clamping the trachea method: the disadvantages are: anesthetic drugs have an impact on the nerves; the trauma caused by the operation itself; the technical requirements are high, the cycle is long; the batch size is small; because the mice used are generally older (too small, the operation is difficult), The consistency of pathological changes is poor.
4.建立宫内缺氧模型：缺点：麻醉药物的数据影响；手术本身创伤；手术技术要求高；手术耗材消耗大；周期长，批量小；差异大，均一性差。4. Establish an intrauterine hypoxia model: Disadvantages: data impact of anesthetic drugs; surgery itself trauma; high surgical technical requirements; large consumption of surgical consumables; long cycle and small batches; large differences and poor uniformity.
因此，本领域迫切需要开发一种新的低氧性脑损伤动物模型，其可以作为研究广泛脑区神经元树突发育障碍的发病机理以及新药筛选的有力工具的动物模型。Therefore, there is an urgent need in this field to develop a new hypoxic brain injury animal model, which can be used as an animal model for studying the pathogenesis of neuronal dendritic development disorders in a wide range of brain regions and a powerful tool for screening new drugs.
发明内容Summary of the invention
本发明的目的在于提供一种可以作为研究广泛脑区神经元树突发育障碍的发病机理以及新药筛选的有力工具的动物模型。The purpose of the present invention is to provide an animal model that can be used as a powerful tool for studying the pathogenesis of neuronal dendritic development disorders in a wide range of brain regions and for screening new drugs.
本发明第一方面提供了一种广泛脑区神经元树突发育障碍的非人哺乳动物模型的制备方法，包括如下步骤：The first aspect of the present invention provides a method for preparing a non-human mammalian model of neuronal dendritic development disorders in a wide range of brain regions, including the following steps:
将非人哺乳动物暴露于低氧条件下30-40分钟，从而得到广泛脑区神经元树突发育障碍的动物模型，Expose non-human mammals to hypoxic conditions for 30-40 minutes to obtain an animal model of neuronal dendritic developmental disorders in a wide range of brain regions.
其中，在所述低氧条件下，氧浓度(体积比)从10-20％降到1-5％。Wherein, under the hypoxic condition, the oxygen concentration (volume ratio) drops from 10-20% to 1-5%.
在另一优选例中，所述广泛脑区神经元树突发育障碍包括广泛脑区神经元树突棘发育障碍。In another preferred embodiment, the neuron dendritic development disorder of the extensive brain region includes the neuron dendritic spine development disorder of the extensive brain region.
在另一优选例中，所述非人哺乳动物为啮齿动物或灵长目动物，较佳地包括小鼠、大鼠、兔和/或猴。In another preferred embodiment, the non-human mammals are rodents or primates, preferably including mice, rats, rabbits and/or monkeys.
在另一优选例中，所述非人哺乳动物为新生非人哺乳动物，较佳地，为出生24个小时内的新生非人哺乳动物(比如啮齿动物)或为围出生期的新生非人哺乳动物)比如灵长目动物)。In another preferred embodiment, the non-human mammal is a newborn non-human mammal, preferably, a newborn non-human mammal (such as a rodent) within 24 hours of birth or a newborn non-human mammal in the perinatal period. Mammals) such as primates).
在另一优选例中，在低氧条件下，加入惰性气体，从而降低氧浓度。In another preferred embodiment, under low oxygen conditions, an inert gas is added to reduce the oxygen concentration.
在另一优选例中，所述惰性气体选自下组：氮气、氦气、或其组合。In another preferred embodiment, the inert gas is selected from the following group: nitrogen, helium, or a combination thereof.
在另一优选例中，所述广泛脑区神经元树突发育障碍动物模型具选自下组的一种或多种特征：In another preferred embodiment, the animal model of extensive brain neuron dendritic developmental disorder has one or more characteristics selected from the following group:
(t1)脑形态和脑大小无显著变化；(t1) There is no significant change in brain shape and brain size;
(t2)动物的全身发育无显著影响；(t2) The animal's whole body development has no significant impact;
(t3)动物的体重无显著影响；(t3) The animal's body weight has no significant effect;
(t4)动物一般的生理活动，包括饮食、排泄、呼吸、心率、血压、视听觉、痛温觉、运动能力、反射能力等都没有显著的改变；(t4) The general physiological activities of animals, including diet, excretion, respiration, heart rate, blood pressure, vision and hearing, pain and temperature perception, exercise ability, reflex ability, etc. have no significant changes;
(t5)神经元光镜下的结构：神经元的形态、大小、极性、排列、数量、密度、分布没有发生显著变化；(t5) The structure of the neuron under the light microscope: the shape, size, polarity, arrangement, number, density, and distribution of the neuron have not changed significantly;
(t6)小胶质细胞在其胞体及其突起的形态、大小、形状、分布、数量、密度等没有显著的变化；(t6) There is no significant change in the morphology, size, shape, distribution, number, density, etc. of microglia in their cell bodies and their protrusions;
(t7)突触电生理活性显著下降；(t7) The electrophysiological activity of synapses is significantly decreased;
(t8)神经元树突和树突棘的棘形态、大小、数量、密度显著下降；(t8) The spine shape, size, number, and density of neuron dendrites and dendritic spines are significantly decreased;
(t9)神经元的树突直径和长度显著下降；(t9) The diameter and length of the dendrites of neurons decreased significantly;
(t10)神经元之间形成的突触的数量、分布、区域等显著减少；(t10) The number, distribution, area, etc. of synapses formed between neurons are significantly reduced;
(t11)行为、情绪、认知功能缺陷；(t11) Defects in behavior, emotion, and cognitive function;
(t12)脑组织的含水量无显著变化；(t12) There is no significant change in the water content of brain tissue;
(t13)神经细胞无显著异常。(t13) No significant abnormalities in nerve cells.
在另一优选例中，所述突触生理活性包括：mEPSCs振幅和频率。In another preferred embodiment, the synaptic physiological activity includes: mEPSCs amplitude and frequency.
在另一优选例中，所述行为、情绪、认知功能缺陷包括空间学习能力和记忆能力下降、主动探究行为降低、对危险境地的恐惧程度较弱、对明亮区域天然的 恐惧心理表现减弱。In another preferred example, the behavioral, emotional, and cognitive deficits include decreased spatial learning ability and memory ability, decreased active inquiry behavior, weaker fear of dangerous situations, and weaker natural fear of bright areas.
本发明第二方面提供了一种本发明第一方面所述方法制备的非人哺乳动物模型的用途，将该模型用作研究广泛脑区神经元树突发育障碍的动物模型。The second aspect of the present invention provides the use of a non-human mammalian model prepared by the method of the first aspect of the present invention, which is used as an animal model for studying neuronal dendritic development disorders in a wide range of brain regions.
在另一优选例中，所述广泛脑区神经元树突发育障碍包括广泛脑区神经元树突棘发育障碍。In another preferred embodiment, the neuron dendritic development disorder of the extensive brain region includes the neuron dendritic spine development disorder of the extensive brain region.
本发明第三方面提供了一种本发明第一方面所述方法制备的非人哺乳动物模型的用途，用于筛选或鉴定可减轻或治疗广泛脑区神经元树突发育障碍的物质(治疗剂)。The third aspect of the present invention provides a use of the non-human mammalian model prepared by the method of the first aspect of the present invention to screen or identify substances that can alleviate or treat neuronal dendritic development disorders in a wide range of brain regions (treatment Agent).
在另一优选例中，所述广泛脑区神经元树突发育障碍包括广泛脑区神经元树突棘发育障碍。In another preferred embodiment, the neuron dendritic development disorder of the extensive brain region includes the neuron dendritic spine development disorder of the extensive brain region.
本发明第四方面提供了一种筛选或鉴定治疗或缓解广泛脑区神经元树突发育障碍的潜在治疗剂的方法，包括以下步骤：The fourth aspect of the present invention provides a method for screening or identifying potential therapeutic agents for treating or relieving neuronal dendritic development disorders in a wide range of brain regions, including the following steps:
(a)在测试组中，在测试化合物的存在下，将测试化合物施用于本发明第一方面所述方法制备的非人哺乳动物模型，检测测试组中所述动物模型的广泛脑区神经元树突发育障碍的严重程度Q1；并且在不施用所述测试化合物且其他条件相同的对照组中，检测对照组中所述动物模型广泛脑区神经元树突发育障碍的严重程度Q2；(a) In the test group, in the presence of the test compound, the test compound is applied to the non-human mammal model prepared by the method of the first aspect of the present invention, and the extensive brain area neurons of the animal model in the test group are detected The severity of dendritic developmental disorder Q1; and in a control group where the test compound is not administered and other conditions are the same, the severity of neuronal dendritic developmental disorder in the extensive brain area of the animal model in the control group is tested Q2;
(b)将上一步骤所检测的严重程度Q1和严重程度Q2进行比较，从而确定所述测试化合物是否是治疗或缓解广泛脑区神经元树突发育障碍的潜在治疗剂；(b) Compare the severity Q1 and the severity Q2 detected in the previous step to determine whether the test compound is a potential therapeutic agent for the treatment or alleviation of neuronal dendritic development disorders in a wide range of brain regions;
其中，如果严重程度Q1显著低于严重程度Q2或如果施用了测试化合物的动物模型中的广泛脑区神经元树突发育障碍的严重程度降低时，则表示所述测试化合物为治疗或缓解广泛脑区神经元树突发育障碍的潜在治疗剂。Wherein, if the severity Q1 is significantly lower than the severity Q2 or if the severity of the extensive brain neuronal dendritic development disorder in the animal model to which the test compound has been administered is reduced, it means that the test compound is a treatment or alleviation of widespread A potential therapeutic agent for neuronal dendritic development disorders in the brain area.
在另一优选例中，所述的检测广泛脑区神经元树突发育障碍严重程度包括检测选自下组的一个或多个指标的变化：突触电生理活性；各脑区神经元树突和树突棘的棘形态、大小、数量、分布、密度；神经元的树突直径、长度、数量；各脑区突触及其构成的结构的分布的发育状况；行为、情绪、认知功能。In another preferred example, the detection of the severity of neuronal dendritic development disorders in a wide range of brain regions includes detecting changes in one or more indicators selected from the following group: synaptic electrophysiological activity; neuronal tree in each brain region The spine shape, size, number, distribution, and density of the spines and dendritic spines; the diameter, length, and number of the dendrites of neurons; the developmental status of the distribution of synapses and their constituent structures in each brain area; behavior, emotion, and cognition Function.
在另一优选例中，所述的广泛脑区神经元树突发育障碍的严重程度降低表现为：突触电生理活性的下降程度降低；神经元树突棘的棘密度、大小、数量、密度的下降程度降低；神经元的树突直径、长度、数量的降低；各脑区突触及其构成的结构的分布的发育程度降低；和/或行为、情绪、认知功能缺陷程度降低。In another preferred example, the reduction in the severity of the neuronal dendritic development disorder in the extensive brain area is manifested as: a decrease in the degree of decrease in the electrophysiological activity of the synapses; the spine density, size, number, The decrease in density is reduced; the diameter, length, and number of dendrites of neurons are reduced; the development of the distribution of synapses and their constituent structures in each brain area is reduced; and/or the degree of deficits in behavior, emotion, and cognitive functions is reduced.
在另一优选例中，所述“显著低于”指具有生物学重复的测试组在施用测试化合物后的严重程度Q1低于具有生物学重复的对照组在施用测试化合物后严重程度Q2，且经过t检验其P值小于0.05。In another preferred example, the "significantly lower" means that the severity Q1 of the test group with biological duplication after administration of the test compound is lower than the severity Q2 of the control group with biological duplication after administration of the test compound, and After t test, the P value is less than 0.05.
在另一优选例中，所述“显著低于”指严重程度Q1/严重程度Q2之比值≤1/2，较佳地≤1/3，更佳地，≤1/4。In another preferred example, the "significantly lower" means that the ratio of severity Q1/severity Q2 is ≤ 1/2, preferably ≤ 1/3, more preferably, ≤ 1/4.
在另一优选例中，所述的方法是非诊断性和非治疗性的。In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
在另一优选例中，所述方法包括步骤(c)，将步骤(b)筛选或鉴定的潜在治疗剂施用于本发明第一方面所述方法制备的非人哺乳动物模型，从而测定其对所述动物模型广泛脑区神经元树突发育障碍的严重程度的影响。In another preferred embodiment, the method includes step (c), applying the potential therapeutic agent screened or identified in step (b) to the non-human mammal model prepared by the method of the first aspect of the present invention, thereby determining its effect on The animal model is widely affected by the severity of neuronal dendritic development disorders in the brain area.
本发明第五方面提供了一种非人哺乳动物模型，用本发明第一方面所述方法制备。The fifth aspect of the present invention provides a non-human mammal model, which is prepared by the method described in the first aspect of the present invention.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them one by one here.
图1显示了本模型动物经受过缺氧打击后，没有引起大体上显著的变化。其中，A：没有发现脑的外形变化；B：没有引起体重的变化。Figure 1 shows that this model animal did not cause significant changes after undergoing hypoxia. Among them, A: No change in the shape of the brain was found; B: No change in body weight was caused.
图2显示了经历过缺氧打击后，模型鼠的神经元电生理活动受到显著的影响。其中，A-C：离体电生理显示缺氧后的脑神经元之间的突触电活性下降；D-F：在体电生理显示缺氧后的海马脑区长时程抑制(LYD)电生理功能变化。Figure 2 shows that the neuronal electrophysiological activities of the model rats are significantly affected after the hypoxic shock. Among them, AC: in vitro electrophysiology shows the decrease of synaptic electrical activity between brain neurons after hypoxia; DF: in vivo electrophysiology shows changes in the electrophysiological function of the hippocampus with long-term depression (LYD) after hypoxia .
图3显示了缺氧后的脑光镜下观察，神经元形态、大小、排列、数量、分布、密度没有发现显著的变化(A-J)。Figure 3 shows the brain light microscope observation after hypoxia, no significant changes were found in neuron morphology, size, arrangement, number, distribution, and density (A-J).
图4显示了缺氧后的脑光镜下观察，小胶质细胞形态、大小、排列、数量、分布、密度没有发现显著的变化(A-F)。Figure 4 shows the brain light microscope observation after hypoxia, no significant changes were found in the morphology, size, arrangement, number, distribution, and density of microglia (A-F).
图5显示了缺氧后在早期(生后7天)海马区域的神经元的树突直径、树突棘的大小、数量、密度(B)比较正常鼠(A)显著下降；这种差异在成年时期更为显著(C,D)Figure 5 shows that the dendritic diameter, size, number, and density (B) of neurons in the hippocampus area of the hippocampus in the early stage (7 days after birth) after hypoxia are significantly reduced compared to normal mice (A); this difference is in More significant in adulthood (C, D)
图6显示了除去海马之外的其它脑区，比如齿状回(DG)，神经细胞的树突和树突棘也见到了缺氧造成树突直径变细、树突长度缩短、树突棘密度降低的显著变化(A-F)；在CA3区，由神经元树突棘构成的突触所聚集构成的区域体积降低(G-I)。Figure 6 shows other brain regions other than the hippocampus, such as the dentate gyrus (DG), the dendrites and dendritic spines of nerve cells have also seen hypoxia causing dendritic diameter reduction, dendritic length shortening, and dendritic spines Significant changes in density reduction (AF); in the CA3 area, the volume of the area composed of synapses composed of neuronal dendritic spines is reduced (GI).
图7显示了利用体外细胞培养的方式，在低氧环境下培养的神经元，其发出的树突的长度显著下降，第一天就显著(A,B)，第7天差异更大(C,D)。Figure 7 shows that the length of the dendrites emitted by neurons cultured in a hypoxic environment using in vitro cell culture methods decreased significantly, markedly on the first day (A, B), and the difference was even greater on the seventh day (C ,D).
图8显示了成年以后，新生时期遭遇过缺氧打击的鼠，从逃避潜伏期的变化，反映出其在空间学习能力显著下降(A)；从撤出平台后，大鼠的运动轨迹(C)，反映出其空间记忆能力也同样显著下降(B,D)。Figure 8 shows the changes in the escape latency of the rats that have suffered hypoxia in the newborn period after adulthood, reflecting the significant decline in their spatial learning ability (A); the movement trajectory of the rats after being withdrawn from the platform (C) , Reflecting that its spatial memory ability is also significantly reduced (B, D).
图9显示了鼠脑组织含水量检测。新生大鼠缺氧35分钟后，再复氧呼吸存活1小时组(n＝10)、再复氧呼吸存活2小时组(n＝10)、再复氧呼吸存活3小时组(n＝10)，分别与未缺氧存活1小时对照组(n＝12)、未缺氧存活2小时对照组(n＝10)和未缺氧存活3小时对照组(n＝10)比较脑组织的净含水量，组间没有差异。Figure 9 shows the detection of water content in mouse brain tissue. After 35 minutes of hypoxia, neonatal rats survived reoxygenation for 1 hour group (n=10), reoxygenated respiration survived for 2 hours group (n=10), reoxygenated respiration survived for 3 hours group (n=10) , Compare the net content of brain tissue with the control group that survived for 1 hour without hypoxia (n=12), the control group survived for 2 hours without hypoxia (n=10), and the control group survived for 3 hours without hypoxia (n=10). There was no difference between the groups in the amount of water.
图10显示了鼠脑组织海马CA1区尼氏染色显示锥体神经元。新生大鼠缺氧35分钟后，再复氧呼吸存活3天，取脑石蜡切片尼氏染色，显示神经元与胶质细胞。结果显示，缺氧后再存活3天，与正常对照组比较，神经细胞未见显著异常。没有细胞水肿表现。Figure 10 shows Nissl staining of hippocampal CA1 area of rat brain tissue showing pyramidal neurons. After 35 minutes of hypoxia, the newborn rats survived reoxygenation and respiration for 3 days. The brain paraffin sections were taken for Nissl staining, showing neurons and glial cells. The results showed that after 3 days of hypoxia, there were no significant abnormalities in nerve cells compared with the normal control group. There is no manifestation of cellular edema.
发明人经过广泛而深入的研究，出乎意料地发现，将非人哺乳动物暴露于氧浓度(体积比)从10-20％降至1-5％的低氧条件下30-40分钟，可得到广泛脑区神经元树突发育障碍的动物模型，本发明的动物模型是一种有效的广泛脑区神经元树突发育障碍动物模型，可用于研究广泛脑区神经元树突发育障碍，并可以用于特定药物的筛选和测试试验。在此基础上完成了本发明。After extensive and in-depth research, the inventor unexpectedly discovered that exposure of non-human mammals to hypoxic conditions in which the oxygen concentration (volume ratio) is reduced from 10-20% to 1-5% for 30-40 minutes can be An animal model of neuronal dendritic development disorder in a wide range of brain regions is obtained. The animal model of the present invention is an effective animal model of neuronal dendritic development disorder in a wide range of brain regions, which can be used to study the development of neuronal dendrites in a wide range of brain regions. Barriers, and can be used for screening and testing of specific drugs. The present invention has been completed on this basis.
广泛脑区神经元树突发育障碍Disorders of neuronal dendritic development in a wide range of brain regions
在本发明中，本发明建立了一种相对缓和的缺氧条件(即哺乳动物暴露于浓度变化的氧浓度环境下，比如，氧浓度(体积比)从10-20％降至1-5％的低氧条件下)，造成了显著的神经元树突及其树突棘的发育障碍，这种发育障碍造成的结构性损伤持续存在，造成学习记忆甚至其他认知行为的损害。In the present invention, the present invention establishes a relatively mild hypoxic condition (that is, mammals are exposed to a changing oxygen concentration environment, for example, the oxygen concentration (volume ratio) is reduced from 10-20% to 1-5% Under low oxygen conditions), significant neuron dendrites and their dendritic spines develop obstacles. Structural damage caused by such developmental obstacles persists, causing damage to learning, memory, and other cognitive behaviors.
动物模型Animal model
在本发明中，提供了一种非常有效的广泛脑区神经元树突发育障碍的非人哺乳动物模型。In the present invention, a very effective non-human mammalian model of neuronal dendritic development disorders in a wide range of brain regions is provided.
在本发明中，非人哺乳动物的例子包括(但并不限于)：小鼠、大鼠、兔、猴等，更佳地是大鼠和小鼠。In the present invention, examples of non-human mammals include (but are not limited to): mice, rats, rabbits, monkeys, etc., more preferably rats and mice.
本发明的动物模型用如下方法制备：The animal model of the present invention is prepared by the following method:
将非人哺乳动物暴露于低氧条件下30-40分钟，从而得到广泛脑区神经元树突发育障碍的动物模型，Expose non-human mammals to hypoxic conditions for 30-40 minutes to obtain an animal model of neuronal dendritic developmental disorders in a wide range of brain regions.
其中，在所述低氧条件下，氧浓度(体积比)从10-20％降至1-5％。Wherein, under the low oxygen condition, the oxygen concentration (volume ratio) is reduced from 10-20% to 1-5%.
用本发明方法获得的动物模型可育，发育正常。The animal model obtained by the method of the invention is fertile and develops normally.
候选药物或治疗剂Candidate drug or therapeutic agent
在本发明中，还提供了一种利用本发明的动物模型，筛选治疗广泛脑区神经元树突发育障碍的候选药物或治疗剂的方法。In the present invention, a method for screening candidate drugs or therapeutic agents for the treatment of neuronal dendritic development disorders in a wide range of brain regions using the animal model of the present invention is also provided.
在本发明中，候选药物或治疗剂是指已知具有某种药理学活性或正在被检测的可能具有某种药理学活性的物质，包括但不限于核酸、蛋白、化学合成的小分子或大分子化合物、细胞等。候选药物或治疗剂的给药方式可以是口服、静脉注射、腹腔注射、皮下注射、椎管给药或直接脑内注射。In the present invention, a candidate drug or therapeutic agent refers to a substance that is known to have a certain pharmacological activity or is being tested that may have a certain pharmacological activity, including but not limited to nucleic acids, proteins, chemically synthesized small molecules or large molecules. Molecular compounds, cells, etc. The drug candidate or therapeutic agent can be administered orally, intravenously, intraperitoneally, subcutaneously, spinal canal, or direct intracerebral injection.
本发明的主要优点包括:The main advantages of the present invention include:
1.实验条件简单。1. The experimental conditions are simple.
2.制作过程简单。2. The production process is simple.
3.干预处理因素单一。只是给与了缺氧一个条件，没有对动物机体进行手术、给药等。3. The intervention treatment factor is single. It was only given a condition of hypoxia, and no surgery or medication was performed on the animal body.
4.模型均一。个体差异小，新生鼠出生时间容易掌握，出生后几乎没有受到外界干扰。4. The model is uniform. Individual differences are small, the birth time of newborn rats is easy to grasp, and there is almost no external interference after birth.
5.指标明确。形态学、电生理、脑功能变化指标确切，显著、稳定。5. Clear indicators. The changes in morphology, electrophysiology, and brain function were accurate, significant and stable.
6.损伤点聚焦。损伤位点集中在树突棘，明确、聚焦。细胞体、细胞形状、极向、排列、大小、数量、密度等，都不受影响。6. Focus on the damaged spot. The injury site is concentrated in the dendritic spines, clear and focused. The cell body, cell shape, polar orientation, arrangement, size, number, density, etc. are not affected.
7.损伤点功能关键。模型损伤的位点聚集在树突棘，树突棘是脑构成网络的神经元联系的关键节点，是神经元之间彼此影响的传递信号的主要通道，是中枢神经系统网络系统信号传递效率的关键点，是脑功能高低的主要结构基础。7. The function of the damage point is critical. The damage sites of the model are concentrated in the dendritic spines. The dendritic spines are the key nodes in the connection of the neurons that make up the network of the brain. They are the main channels for the transmission of signals that affect each other between neurons, and are the signal transmission efficiency of the central nervous system network system. The key point is the main structural basis for the level of brain function.
8.树突棘损伤机制清晰。缺氧造成了神经元代谢紊乱，影响到能量供应，导致树突棘的发育障碍，树突棘的发育畸形终生存在，并伴随脑功能的低下。8. The mechanism of dendritic spines injury is clear. Hypoxia causes neuronal metabolism disorders, affects energy supply, and leads to the development of dendritic spines. The developmental malformations of dendritic spines survive for life and are accompanied by low brain function.
9.成功率高。模型稳定，100％成功。9. High success rate. The model is stable and 100% successful.
10.可以批量生产。同时制作比较大量的同质模型，便于开展大规模的实验。10. Can be mass produced. At the same time, a relatively large number of homogeneous models are produced to facilitate large-scale experiments.
11.可以作为研究广泛脑区神经元树突发育障碍症的发病机理和新药筛选的有力工具。11. It can be used as a powerful tool to study the pathogenesis of neuronal dendritic dysplasia in a wide range of brain areas and to screen new drugs.
12.本发明动物模型表型稳定。12. The animal model of the present invention is phenotypically stable.
13.用本发明方法获得的动物模型可育，机体一般结构发育正常。13. The animal model obtained by the method of the present invention is fertile, and the general structure of the body is normally developed.
14.本发明的动物模型可以用来研究TSH在早期大脑神经元树突棘发育中的作用和机制。14. The animal model of the present invention can be used to study the role and mechanism of TSH in early brain neuron dendritic spines development.
15.本发明的动物模型可用来探索研究、开发、验证潜在的药物或手段、方法干预TSH损伤，阻断树突棘发育损伤；改善树突棘的发育障碍。15. The animal model of the present invention can be used to explore, develop, and verify potential drugs or methods and methods to interfere with TSH injury, block the developmental damage of dendritic spine, and improve the developmental obstacle of dendritic spine.
16.本发明的动物模型可用来研究树突棘发育障碍病理条件下，神经系统运行的功能性变化及其运行机制。16. The animal model of the present invention can be used to study the functional changes of the nervous system and its operating mechanism under the pathological conditions of dendritic spines developmental disorders.
17.本发明的动物模型作为一种智力障碍的神经系统模式参考，进行其他神经系统病理模式(比如老年痴呆)下的学习记忆研究对比参照。17. The animal model of the present invention is used as a reference for a nervous system model of mental retardation, for comparison and reference for learning and memory research under other neuropathological models (such as Alzheimer's disease).
18.本发明的低氧条件为常压下的低氧。18. The hypoxic condition of the present invention is hypoxia under normal pressure.
19.本发明的低氧条件具有短时长、持续性的特点。19. The hypoxic conditions of the present invention have the characteristics of short duration and persistence.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing The conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.
如无特别说明，实施例所用的材料均为市售产品。Unless otherwise specified, the materials used in the examples are all commercially available products.
制作过程Production process
1.怀孕的母鼠被关在一个动物房间里，有12小时的光照和12小时的黑暗周期，它们可以自由获取食物和水，直到它们分娩。1. Pregnant female rats are kept in an animal room with 12 hours of light and 12 hours of darkness. They have free access to food and water until they give birth.
2.在出生并由母亲喂养6小时后，这些新生鼠被随机分为两组。2. After 6 hours of birth and feeding by the mother, these newborn rats were randomly divided into two groups.
3.在TSH处理中，新生鼠首先被放置在低氧室中，氧气浓度为15％，在30℃条件下，30分钟内逐渐被N2调节到3％。此后，氧浓度维持在3％，新生鼠在箱内再呆5分钟。因此，总的缺氧损伤持续了35分钟，随后正常呼吸。对于正常氧 处理组，新生鼠被放入箱内35分钟，正常氧浓度为21％。3. In the TSH treatment, the newborn rats were first placed in a hypoxic chamber with an oxygen concentration of 15%. Under the condition of 30°C, they were gradually adjusted to 3% by N2 within 30 minutes. After that, the oxygen concentration was maintained at 3%, and the newborn rats stayed in the box for another 5 minutes. Therefore, the total hypoxic injury lasted for 35 minutes, followed by normal breathing. For the normoxia treatment group, newborn rats were placed in the box for 35 minutes, and the normoxia concentration was 21%.
4.缺氧过程结束后，所有的新生鼠，被送回笼子，与母鼠同处。4. After the hypoxia process is over, all the newborn rats are sent back to the cage and are in the same place with the mother rats.
所需制备条件Required preparation conditions
1.一台氧控细胞培养箱，或者专用的动物饲育箱1. An oxygen-controlled cell incubator, or a dedicated animal incubator
2.如果没有专用设备，可以使用一台普通细胞培养箱，外加一个能控制氧气浓度的密闭盒子。2. If there is no special equipment, you can use an ordinary cell incubator, plus a closed box that can control the oxygen concentration.
检测指标结果Test index results
1.成活率高：TSH处理后，50只新生鼠的脑大体形态及体重均恢复正常，全部成活。1. High survival rate: After TSH treatment, the general brain morphology and weight of 50 newborn mice returned to normal, and all of them survived.
2.缺氧全身反应明显：在低氧处理结束时，新生鼠的全身颜色比正常对照组更暗，并且在低氧处理结束后很快恢复。2. The hypoxia systemic reaction is obvious: at the end of the hypoxia treatment, the whole body color of the newborn rat is darker than the normal control group, and it recovers soon after the hypoxia treatment.
3.脑形态不变：研究人员还对分别在5天和7天内接受TSH和常氧处理的幼鼠的大脑进行了仔细的比较。低氧处理组和正常氧处理组在脑形态和脑大小上没有明显差异(图1A)。3. The brain morphology remains unchanged: The researchers also carefully compared the brains of young mice that were treated with TSH and normoxia within 5 days and 7 days, respectively. There was no significant difference in brain morphology and brain size between the hypoxia treatment group and the normoxia treatment group (Figure 1A).
4.体重不变：低氧处理的新生鼠和正常氧处理的新生鼠体重(n＝8,p>0.05)的生长速度相似(图1B)。这些观察结果表明，TSH对动物全身发育的影响不显著。4. The body weight is unchanged: the growth rate of the newborn rats under hypoxia treatment and the newborn rats under normal oxygen treatment (n=8, p>0.05) is similar (Figure 1B). These observations indicate that the effect of TSH on animal development is not significant.
5.突触电生理活性下降：为了探索是否TSH对神经功能的影响,利用膜片箝记录自发的突触活动，海马切片TSH(n＝27个神经元)和normoxia治疗大鼠神经元(n＝31),即谷氨酸受体介导微型兴奋性突触后电流(mEPSCs)记录在CA1神经元。TSH处理大鼠mEPSCs振幅(pA)为15.18±0.43(pA)，与对照组(p<0.001)的17.70±0.28(pA)相比，差异有统计学意义(图2A和图2B)。TSH处理的大鼠mEPSCs频率为3.98±0.55(Hz)，与对照组的6.35±0.49(Hz)相比，也显著降低(图2A和2C)(p<0.01)。此外，体内海马CA1突触活动记录显示，TSH损伤(n＝23个神经元)动物神经元放电率为8.74±0.63(spike/s)，明显高于正常运动对照组(n＝25个神经元)的5.06±0.45(spike/s)，p<0.001(图2D)。由于TSH大鼠海马CA1中mEPSCs的振幅和频率均显著降低，我们怀疑基础突触传递的减 少是否也会影响海马脑片的突触可塑性。TSH处理组海马CA1区细胞外长期电位(LTP)与正常对照组相同(图2E)。然而，缺氧显著抑制了长期抑郁(LTD)(图2F)。这些结果表明，TSH损伤后神经元突触活动中断。5. Decrease in synaptic electrophysiological activity: In order to explore whether TSH has an effect on nerve function, patch clamp was used to record spontaneous synaptic activity. Hippocampal slices of TSH (n=27 neurons) and normoxia were used to treat rat neurons (n =31), that is, glutamate receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) are recorded in CA1 neurons. The amplitude (pA) of mEPSCs in TSH-treated rats was 15.18±0.43 (pA), which was statistically significant compared with 17.70±0.28 (pA) in the control group (p<0.001) (Figure 2A and Figure 2B). The frequency of mEPSCs in rats treated with TSH was 3.98±0.55 (Hz), which was also significantly lower than the 6.35±0.49 (Hz) in the control group (Figures 2A and 2C) (p<0.01). In addition, in vivo hippocampal CA1 synaptic activity records showed that the firing rate of animal neurons with TSH injury (n=23 neurons) was 8.74±0.63 (spike/s), which was significantly higher than that of the normal exercise control group (n=25 neurons) ) Of 5.06±0.45 (spike/s), p<0.001 (Figure 2D). Since the amplitude and frequency of mEPSCs in hippocampal CA1 of TSH rats were significantly reduced, we doubted whether the reduction of basal synaptic transmission would also affect the synaptic plasticity of hippocampal slices. The extracellular long-term potential (LTP) of the hippocampal CA1 area of the TSH treatment group was the same as that of the normal control group (Figure 2E). However, hypoxia significantly suppressed long-term depression (LTD) (Figure 2F). These results indicate that synaptic activity of neurons is disrupted after TSH injury.
6.神经元形态及光镜下结构不变：TSH不会引起大脑神经细胞结构变化。我们检测了TSH损伤后海马神经元数量是否发生了改变。尼氏小染色显示(图3，A-J)。海马CA1神经元密度TSH和正常大鼠在P5,P7,P21和P90的观察,如表1所示。这些观察结果支持在TSH损伤后海马CA1在不同发育年龄没有发生明显的细胞丢失或整体结构改变。6. Neuron morphology and structure under light microscope are unchanged: TSH will not cause changes in the structure of brain nerve cells. We tested whether the number of hippocampal neurons has changed after TSH injury. Nissl small staining shows (Figure 3, A-J). The hippocampal CA1 neuron density TSH and normal rats were observed at P5, P7, P21 and P90, as shown in Table 1. These observations support that hippocampal CA1 did not undergo significant cell loss or overall structural changes at different developmental ages after TSH injury.
表1Table 1
7.小胶质细胞没有被激活。TSH不诱发脑激活小胶质。观察海马CA1小胶质细胞在的形态、数量、分布、密度等，与正常(图4，A,B,C)相比，缺氧大鼠的海马小胶质细胞表明没有明显激活(图4，D,E,F)。7. The microglia are not activated. TSH does not induce brain activation of microglia. Observe the morphology, number, distribution, density, etc. of hippocampal CA1 microglia. Compared with normal (Figure 4, A, B, C), the hippocampal microglia of hypoxic rats showed no obvious activation (Figure 4). , D, E, F).
8.TSH导致海马神经元树突棘密度下降。TSH处理后第7天和第90天分别检测海马CA1神经元树突、树突棘和脑区DG中颗粒细胞的结构细节。正常大鼠神经元树突棘沿树突成簇状分布,缺氧后动物的树突棘TSH显著下降,稀疏分布,只有少量沿着树突分布(图5)。生后第7天，海马CA1神经元的顶树突的树突棘密度是每10um,8.5±4.0，与正常对照组15.1±2.9/10m比较，差异显著of,<0.001(图5A,B)。TSH损伤后3个月，树突棘密度的降低为14.0±3.9/10um，而正常运动组动物的棘密度为23.7±4.6/10um(<0.05)(图5C,D)。8. TSH causes the dendritic spine density of hippocampal neurons to decrease. On the 7th and 90th day after TSH treatment, the structure details of the dendrites of hippocampal CA1 neurons, dendritic spines and granular cells in the DG of the brain area were detected respectively. The dendritic spines of normal rat neurons are distributed in clusters along the dendrites. After hypoxia, the TSH of the dendritic spines of the animals is significantly reduced, sparsely distributed, and only a few are distributed along the dendrites (Figure 5). On the 7th day after birth, the dendritic spine density of the apical dendrites of hippocampal CA1 neurons was per 10um, 8.5±4.0, compared with the normal control group 15.1±2.9/10m, the difference was significant, <0.001 (Figure 5A,B) . Three months after TSH injury, the dendritic spine density decreased to 14.0±3.9/10um, while the spine density of animals in the normal exercise group was 23.7±4.6/10um (<0.05) (Figure 5C, D).
9.神经元的树突直径下降。除树突棘密度外，在TSH处理或常氧处理后第7天和90天，我们还对海马CA1的神经元树突，尤其是次级分支进行了检测。在 P7时，TSH组动物的树突平均直径与正常对照组大体相同。然而，经TSH处理的动物的神经元树突二级分枝的直径在统计学上要比常氧对照组的小。在低氧和常氧处理后90天也观察到类似的结果(图5和表2)。9. The dendrites of neurons decrease in diameter. In addition to the dendritic spine density, on the 7th and 90th day after TSH treatment or normoxia treatment, we also examined the neuronal dendrites of hippocampal CA1, especially the secondary branches. At P7, the average dendritic diameter of animals in the TSH group was roughly the same as that of the normal control group. However, the diameter of the secondary branches of neuronal dendrites in TSH-treated animals was statistically smaller than that of the normoxic control group. Similar results were also observed 90 days after hypoxia and normoxia treatments (Figure 5 and Table 2).
表2Table 2
10.SH导致DG和CA3区域异常发育。海马齿状回的观察进一步证实了海马CA1中树突棘密度降低和树突二级分支变细(图6A、6B、6C)。在P90时，TSH处理后动物的DG颗粒细胞树突棘密度为7.03±0.53/10um，低于正常氧对照组的14.8±0.60/10um,p<0.0001(图6D)。TSH组树突长度较对照组明显缩短。TSH处理后90天，动物齿状回颗粒细胞的树突直径为0.893±0.064m，明显大于正常对照组的0.628±0.046m,p<0.05(图6E)。TSH处理后90d动物海马DG内分子层(IML)颗粒细胞的树突状收缩长度为15.35±1.75m，显著高于常氧对照组7.04±1.02m,p<0.001(图6F)。最后，在90天龄时对动物进行Timm染色的大脑切片显示，与对照组相比，TSH组海马CA3区域的Timm阳性颗粒明显减少(图6G和6H)。TSH损伤组海马CA3平均透明层宽度为117.3±4.63um，与正常对照组146.0±5.82um相比显著降低，p<0.005(图6I)。10. SH causes abnormal development of DG and CA3 regions. The observation of the hippocampal dentate gyrus further confirmed that the density of dendritic spines in the hippocampus CA1 was reduced and the secondary branches of dendrites became thinner (Figure 6A, 6B, 6C). At P90, the dendritic spine density of DG granular cells in animals treated with TSH was 7.03±0.53/10um, which was lower than 14.8±0.60/10um in the normoxic control group, p<0.0001 (Figure 6D). The length of dendrites in the TSH group was significantly shorter than that in the control group. 90 days after TSH treatment, the dendritic diameter of granular cells in the dentate gyrus of animals was 0.893±0.064m, which was significantly larger than 0.628±0.046m in the normal control group, p<0.05 (Figure 6E). The dendritic contraction length of the inner molecular layer (IML) granular cells in the hippocampus DG of animals 90 days after TSH treatment was 15.35±1.75m, which was significantly higher than 7.04±1.02m in the normoxic control group, p<0.001 (Figure 6F). Finally, Timm-stained brain sections of animals at 90 days of age showed that compared with the control group, Timm-positive particles in the hippocampal CA3 area of the TSH group were significantly reduced (Figures 6G and 6H). The average transparent layer width of hippocampal CA3 in the TSH injury group was 117.3±4.63um, which was significantly lower than that of the normal control group of 146.0±5.82um, p<0.005 (Figure 6I).
11.低氧神经细胞培养神经元树突生长受损。利用细胞培养的方法进行体外实验，进一步验证缺氧对神经元树突的生长发育的影响。实验结果显示，在低氧(5％O2)条件下连续培养7天，从第一天开始，就观察到低氧条件下的神经元树突生长缓慢，突起变短，分支减少(图7A,B)；并一直持续存在，在观察期的第7天，比较常氧状态下的神经元形态(图7C)，缺氧的神经元树突长度和密度都显著降低(图7D)。11. Hypoxic nerve cell cultured neuron dendrites growth is impaired. Using cell culture methods to conduct in vitro experiments to further verify the effect of hypoxia on the growth and development of neuronal dendrites. The experimental results showed that under hypoxic (5% O2) conditions for 7 days of continuous culture, from the first day, it was observed that the dendrites of neurons under hypoxic conditions grew slowly, the processes became shorter, and the branches decreased (Figure 7A, B); and persisted. On the 7th day of the observation period, comparing the neuron morphology under normoxia (Figure 7C), the length and density of the dendrites of hypoxic neurons were significantly reduced (Figure 7D).
12.TSH会导致长期的认知功能缺陷。对3个月大的TSH和常氧处理后的大鼠进行Morris水迷宫实验。在为期4.5天的空间导航训练中，对照组和缺氧组的大鼠在前6次训练中都表现出了相似的改善，并且所有的动物在找到平台方 面的潜伏期都有所缩短。然而，从第7、8和9个训练结果，TSH损伤和正常缺氧处理的动物之间存在显著差异。TSH组大鼠的潜伏期分别是21.6±3.71,16.6±1.79,20.4±3.86，显著长于对照组的12.1±1.72,7.99±1.03,8.07±0.96,分别(p<0.05)(图8A)。在9次训练试验后，所有大鼠在没有MWM平台的情况下进行空间探索测试。游泳的痕迹用数字记录下来了。TSH的平台网站穿越次数，第5日，9.43±1.29次和2.00±1.15，正常鼠的穿越次数分别是16.75±2.18，6.29±2.36，差异显著(p<0.05,图8B)。此外，TSH组动物在平台所在象限游泳距离为26475±2412mm，与对照组35862±3371mm相比，p<0.05(图8C和8D)。这些结果有力地表明，新生大鼠TSH损伤会导致大脑发育障碍，导致大脑功能缺陷，包括空间学习和记忆。12. TSH can cause long-term cognitive deficits. Morris water maze experiment was performed on rats treated with TSH and normoxia at 3 months old. During the 4.5-day space navigation training, rats in the control group and the hypoxia group showed similar improvements in the first 6 training sessions, and the latency of all animals in finding a platform was shortened. However, from the 7th, 8th and 9th training results, there are significant differences between the TSH injured and normal hypoxic treated animals. The latency of rats in the TSH group was 21.6±3.71, 16.6±1.79, 20.4±3.86, which were significantly longer than those of the control group 12.1±1.72, 7.99±1.03, 8.07±0.96, respectively (p<0.05) (Figure 8A). After 9 training trials, all rats were tested for space exploration without the MWM platform. The traces of the swimming are recorded in numbers. The number of crossings of TSH's platform website was 9.43±1.29 and 2.00±1.15 on the 5th day. The crossing times of normal mice were 16.75±2.18 and 6.29±2.36, respectively, and the difference was significant (p<0.05, Figure 8B). In addition, the swimming distance of animals in the TSH group in the platform quadrant was 26475±2412mm, which was p<0.05 compared with 35862±3371mm in the control group (Figure 8C and 8D). These results strongly indicate that TSH damage in neonatal rats can lead to brain development disorders, leading to defects in brain function, including spatial learning and memory.
13.缺氧后没有脑组织水肿和神经细胞水肿。新生大鼠1d内经历35分钟的渐次降低低氧损伤后，经历1-3小时的短时间复氧呼吸再存活后，脑组织没有发生显著的水肿(图9)；经历3天的短时间复氧呼吸再存活后，脑组织切片显示神经细胞基本正常，未见神经细胞水肿(图10)。结果表明，本发明的大鼠脑缺氧造成的神经元损伤的机制，不是基于脑组织及或细胞内水肿。13. There is no brain tissue edema and nerve cell edema after hypoxia. After the neonatal rats experienced 35 minutes of gradual reduction of hypoxic injury within 1 day, after experiencing a short reoxygenation respiration for 1-3 hours and then surviving, there was no significant edema in the brain tissue (Figure 9); after a short period of 3 days After oxygen respiration survived, the brain tissue sections showed that the nerve cells were basically normal, and no nerve cell edema was seen (Figure 10). The results show that the mechanism of neuronal damage caused by hypoxia in the rat brain of the present invention is not based on brain tissue and or intracellular edema.
讨论discuss
TSH围产期新生儿的动物模型研究。大脑是对缺氧最敏感的器官之一，长时间缺氧会导致昏迷、癫痫、认知障碍和其他神经功能障碍，甚至脑死亡。新生儿缺氧损伤通常被认为由于脐带绕颈、头盆不衬、出生嵌顿、子宫供血不良、新生儿窒息所致。围产期缺氧缺血性脑损伤造成急性婴儿和儿童较高的死亡率和慢性神经发病率，常常后遗症状。本发明首次发现TSH导致显著的病理变化包括中枢神经系统神经元的树突变细、短缩、树突棘密度降低、突触构造减少、突触电生理功能下降；此外，在患有TSH的动物中观察到认知功能受损。本发明的数据清楚地表明，TSH可以在新生期乃至成年期引起显著的神经突触和树突状毒性。因此，本发明提供了一个有用的动物模型，可以用于研究亚致死缺氧对早期大脑发育的作用和机制，并探索潜在的干预药物或方法。Study on the animal model of neonates in the perinatal period of TSH. The brain is one of the most sensitive organs to hypoxia. Prolonged hypoxia can lead to coma, epilepsy, cognitive impairment and other neurological dysfunctions, and even brain death. Neonatal hypoxia injury is usually thought to be caused by umbilical cord around the neck, unlined head and pelvis, birth incarceration, poor blood supply to the uterus, and neonatal asphyxia. Perinatal hypoxic-ischemic brain injury causes higher mortality and chronic neurological morbidity in acute infants and children, and often sequelae symptoms. The present invention finds for the first time that TSH causes significant pathological changes, including the thinning and shortening of the tree of the central nervous system neurons, the decrease of dendritic spine density, the decrease of synaptic structure, and the decrease of synaptic electrophysiological function; in addition, in animals suffering from TSH Impaired cognitive function was observed in. The data of the present invention clearly shows that TSH can cause significant neurosynaptic and dendritic toxicity in the neonatal and adult stages. Therefore, the present invention provides a useful animal model that can be used to study the effects and mechanisms of sublethal hypoxia on early brain development, and to explore potential intervention drugs or methods.
本发明的研究表明，新生大鼠出生6小时后，单独给予短暂亚致死缺氧35分钟，这与之前用于缺氧/缺血研究的动物模型不同。在目前的研究中使用出 生6个小时的动物,这比以往许多缺氧/缺血研究,使用动物大约有一个星期日龄，更接近临床情况(脐带绕颈、出生嵌顿、子宫畸形等)，出生后的时间越短，动物生后的个体变异系数越小，动物个体间神经系统的均一性越好。一个单因素的梯度缺氧供应，是目前研究与以往的动物模型使用颈动脉闭塞和缺氧(两个因素)的第二个差异。最后，TSH损伤组未见明显的神经病理改变，无神经元丢失，无神经胶质激活，但观察到持久的脑功能缺陷，是一种比较适宜于模拟临床更为常见的中轻度缺血缺氧性脑病的模型。由于其缺氧严重程度较轻，病情难以确诊，缺乏适当的护理或治疗，最终导致严重的脑发育功能障碍。因此，本研究为瞬态亚致死缺氧单独损伤的临床实践提供了一个重要的动物模型和有用的数据。The study of the present invention shows that 6 hours after birth, newborn rats are given short sublethal hypoxia for 35 minutes alone, which is different from the previous animal model used for hypoxia/ischemia research. In the current study, animals born 6 hours old are used. Compared with many previous hypoxia/ischemia studies, the animals used are about one Sunday old, which is closer to the clinical situation (umbilical cord around the neck, birth incarceration, uterine malformation, etc.). The shorter the time after birth, the smaller the individual coefficient of variation of the animal after birth, and the better the uniformity of the nervous system among the animals. A single-factor gradient hypoxia supply is the second difference between the current study and previous animal models using carotid artery occlusion and hypoxia (two factors). Finally, there were no obvious neuropathological changes in the TSH injury group, no neuron loss, no glial activation, but long-lasting brain function defects were observed, which is more suitable for simulating clinically more common mild to moderate ischemia Model of hypoxic encephalopathy. Due to its low severity of hypoxia, the condition is difficult to diagnose, and the lack of proper care or treatment will eventually lead to severe brain development dysfunction. Therefore, this study provides an important animal model and useful data for the clinical practice of transient sublethal hypoxia alone injury.
TSH阻断神经元的自发突触活动。在非常早期的早产儿和动物中发现了脑电图(EEG)的背景活动。在本研究中，与正常对照组相比，TSH导致海马CA1神经元mEPSCs的振幅和频率下降。TSH对神经元树突发育有负面影响。在本发明中，与对照组相比，经TSH处理后，动物的神经元树突直径明显减小。5％氧处理后原代培养海马神经元的树突总长度也明显低于正常对照组。这些结果表明，TSH的损伤诱导了树突毒性，影响了神经元树突的生长发育。缺氧引起的能量供应不足可能影响包括肌动蛋白和微管在内的细胞骨架动力学。肌动蛋白和MT是常规树突生长发育的关键，它们是许多控制神经元树突生长的分子途径的靶标。在大脑发育过程中，肌动蛋白和MT细胞骨架的结构都会发生变化，从而产生神经元突起。本发明还发现，TSH损伤降低了树突棘的密度和发育。这些神经元上超过95％的兴奋性突触发生在树突棘上，每个棘头通常连接构成一个突触。树突棘的形成和可塑性对大脑功能非常重要。树突棘的发育由氧传感器PHD2控制，以肌动蛋白交联剂Filamin-A为目标，调节突触密度和网络范围内的神经元活动。树突棘相关Rap特异性GDP酶激活蛋白(SPAR)是一种突触后蛋白，与突触后密度(PSD)-95形成复合物，其与N-甲基-D-天冬氨酸受体(NMDARs)，都参与调节树突棘的形态发生。TSH blocks the spontaneous synaptic activity of neurons. Background activity of electroencephalogram (EEG) is found in very early preterm infants and animals. In this study, compared with the normal control group, TSH caused a decrease in the amplitude and frequency of mEPSCs in hippocampal CA1 neurons. TSH has a negative effect on the development of neuronal dendrites. In the present invention, compared with the control group, after TSH treatment, the diameter of the animal's neuron dendrites is significantly reduced. After 5% oxygen treatment, the total dendritic length of the primary cultured hippocampal neurons was also significantly lower than that of the normal control group. These results indicate that TSH damage induces dendritic toxicity and affects the growth and development of neuronal dendrites. Insufficient energy supply caused by hypoxia may affect cytoskeletal dynamics including actin and microtubules. Actin and MT are the keys to conventional dendritic growth and development, and they are the targets of many molecular pathways that control the growth of neuronal dendrites. During the development of the brain, the structure of actin and MT cytoskeleton changes, resulting in neuronal processes. The present invention also found that TSH damage reduces the density and development of dendritic spines. More than 95% of excitatory synapses on these neurons occur on dendritic spines, and each spine head is usually connected to form a synapse. The formation and plasticity of dendritic spines are very important for brain function. The development of dendritic spines is controlled by the oxygen sensor PHD2, targeting the actin cross-linking agent Filamin-A to regulate synaptic density and neuronal activity within the network. Dendritic spines-associated Rap-specific GDP enzyme activator protein (SPAR) is a post-synaptic protein that forms a complex with postsynaptic density (PSD)-95, which is subject to N-methyl-D-aspartic acid. NMDARs are involved in regulating the morphogenesis of dendritic spines.
树突棘的结构和动态反映了突触的强度，在不同的脑部疾病中，包括神经退行性疾病和精神疾病，突触的强度通常受到严重影响。树突棘可以经历几种类型的转变，从生长到崩溃，从伸长到缩短，它们经历这种动态形态活动的时 间跨度非常短。树突棘数目和形态的变化不仅发生在兴奋性中毒等病理条件下，也发生在正常的中枢神经系统发育、激素波动和生理环境下神经活动的反应过程中。The structure and dynamics of dendritic spines reflect the strength of synapses. In different brain diseases, including neurodegenerative diseases and mental diseases, the strength of synapses is usually severely affected. Dendritic spines can undergo several types of transitions, from growth to collapse, from elongation to shortening, and the time span for them to undergo this dynamic morphological activity is very short. Changes in the number and morphology of dendritic spines not only occur under pathological conditions such as excitotoxicity, but also occur in response to normal central nervous system development, hormone fluctuations, and neural activity under physiological environments.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
Claims (10)
- 一种广泛脑区神经元树突发育障碍的非人哺乳动物模型的制备方法，其特征在于，包括如下步骤：A method for preparing a non-human mammalian model of neuronal dendritic developmental disorders in a wide range of brain regions, which is characterized in that it comprises the following steps:将非人哺乳动物暴露于低氧条件下30-40分钟，从而得到广泛脑区神经元树突发育障碍的动物模型，Expose non-human mammals to hypoxic conditions for 30-40 minutes to obtain an animal model of neuronal dendritic developmental disorders in a wide range of brain regions.其中，在所述低氧条件下，氧浓度(体积比)从10-20％降到1-5％。Wherein, under the hypoxic condition, the oxygen concentration (volume ratio) drops from 10-20% to 1-5%.
- 如权利要求1所述的制备方法，其特征在于，所述广泛脑区神经元树突发育障碍包括广泛脑区神经元树突棘发育障碍。The preparation method according to claim 1, wherein the neuron dendritic development disorder of the extensive brain region comprises a neuron dendritic spine development disorder of the extensive brain region.
- 如权利要求1所述的制备方法，其特征在于，所述广泛脑区神经元树突发育障碍动物模型具选自下组的一种或多种特征：The preparation method according to claim 1, wherein the animal model of extensive brain area neuronal dendritic development disorder has one or more characteristics selected from the following group:(t1)脑形态和脑大小无显著变化；(t1) There is no significant change in brain shape and brain size;(t2)动物的全身发育无显著影响；(t2) The animal's whole body development has no significant impact;(t3)动物的体重无显著影响；(t3) The animal's body weight has no significant effect;(t4)动物一般的生理活动，包括饮食、排泄、呼吸、心率、血压、视听觉、痛温觉、运动能力、反射能力等都没有显著的改变；(t4) The general physiological activities of animals, including diet, excretion, respiration, heart rate, blood pressure, vision and hearing, pain and temperature perception, exercise ability, reflex ability, etc. have no significant changes;(t5)神经元光镜下的结构：神经元的形态、大小、极性、排列、数量、密度、分布没有发生显著变化；(t5) The structure of the neuron under the light microscope: the shape, size, polarity, arrangement, number, density, and distribution of the neuron have not changed significantly;(t6)小胶质细胞在其胞体及其突起的形态、大小、形状、分布、数量、密度等没有显著的变化；(t6) There is no significant change in the morphology, size, shape, distribution, number, density, etc. of microglia in their cell bodies and their protrusions;(t7)突触电生理活性显著下降；(t7) The electrophysiological activity of synapses is significantly decreased;(t8)神经元树突和树突棘的棘形态、大小、数量、密度显著下降；(t8) The spine shape, size, number, and density of neuron dendrites and dendritic spines are significantly decreased;(t9)神经元的树突直径和长度显著下降；(t9) The diameter and length of the dendrites of neurons decreased significantly;(t10)神经元之间形成的突触的数量、分布、区域等显著减少；(t10) The number, distribution, area, etc. of synapses formed between neurons are significantly reduced;(t11)行为、情绪、认知功能缺陷；(t11) Defects in behavior, emotion, and cognitive function;(t12)脑组织的含水量无显著变化；(t12) There is no significant change in the water content of brain tissue;(t13)神经细胞无显著异常。(t13) No significant abnormalities in nerve cells.
- 一种权利要求1所述方法制备的非人哺乳动物模型的用途，其特征在于，将该模型用作研究广泛脑区神经元树突发育障碍的动物模型。A use of the non-human mammalian model prepared by the method of claim 1, wherein the model is used as an animal model for studying neuronal dendritic development disorders in a wide range of brain regions.
- 如权利要求4所述的用途，其特征在于，所述广泛脑区神经元树突发育障碍包括广泛脑区神经元树突棘发育障碍。The use according to claim 4, wherein the neuronal dendritic development disorder of the extensive brain area comprises a neuron dendritic spine development disorder of the extensive brain area.
- 一种权利要求1所述方法制备的非人哺乳动物模型的用途，其特征在于，用于筛选或鉴定可减轻或治疗广泛脑区神经元树突发育障碍的物质(治疗剂)。A use of the non-human mammalian model prepared by the method of claim 1, characterized in that it is used to screen or identify substances (therapeutic agents) that can alleviate or treat neuronal dendritic development disorders in a wide range of brain regions.
- 如权利要求6所述的用途，其特征在于，所述广泛脑区神经元树突发育障碍包括广泛脑区神经元树突棘发育障碍。The use according to claim 6, characterized in that the neuronal dendritic development disorder of the extensive brain area comprises a neuron dendritic spine development disorder of the extensive brain area.
- 一种筛选或鉴定治疗或缓解广泛脑区神经元树突发育障碍的潜在治疗剂的方法，其特征在于，包括以下步骤：A method for screening or identifying potential therapeutic agents for treating or alleviating neuronal dendritic development disorders in a wide range of brain regions, which is characterized in that it comprises the following steps:(a)在测试组中，在测试化合物的存在下，将测试化合物施用于权利要求1所述方法制备的非人哺乳动物模型，检测测试组中所述动物模型的广泛脑区神经元树突发育障碍的严重程度Q1；并且在不施用所述测试化合物且其他条件相同的对照组中，检测对照组中所述动物模型广泛脑区神经元树突发育障碍的严重程度Q2；(a) In the test group, in the presence of the test compound, the test compound is applied to the non-human mammal model prepared by the method of claim 1, and the extensive brain area neuron dendrites of the animal model in the test group are detected The severity of the developmental disorder Q1; and in the control group where the test compound is not administered and other conditions are the same, the severity of the neuronal dendritic development disorder in the extensive brain area of the animal model in the control group is tested Q2;(b)将上一步骤所检测的严重程度Q1和严重程度Q2进行比较，从而确定所述测试化合物是否是治疗或缓解广泛脑区神经元树突发育障碍的潜在治疗剂；(b) Compare the severity Q1 and the severity Q2 detected in the previous step to determine whether the test compound is a potential therapeutic agent for the treatment or alleviation of neuronal dendritic development disorders in a wide range of brain regions;其中，如果严重程度Q1显著低于严重程度Q2或如果施用了测试化合物的动物模型中的广泛脑区神经元树突发育障碍的严重程度降低时，则表示所述测试化合物为治疗或缓解广泛脑区神经元树突发育障碍的潜在治疗剂。Wherein, if the severity Q1 is significantly lower than the severity Q2 or if the severity of the extensive brain neuronal dendritic development disorder in the animal model to which the test compound has been administered is reduced, it means that the test compound is a treatment or alleviation of widespread A potential therapeutic agent for neuronal dendritic development disorders in the brain area.
- 如权利要求8所述的方法，其特征在于，所述的检测广泛脑区神经元树突发育障碍严重程度包括检测选自下组的一个或多个指标的变化：突触电生理活性；各脑区神经元树突和树突棘的棘形态、大小、数量、分布、密度；神经元的树突直径、长度、数量；各脑区突触及其构成的结构的分布的发育状况；行为、情绪、认知功能。8. The method according to claim 8, wherein said detecting the severity of neuronal dendritic development disorders in a wide range of brain regions comprises detecting changes in one or more indicators selected from the group consisting of: synaptic electrophysiological activity; The spine shape, size, number, distribution, and density of neuron dendrites and dendritic spines in each brain area; diameter, length, and number of dendrites of neurons; development status of the distribution of synapses and their constituent structures in each brain area; Behavior, emotion, and cognitive function.
- 如权利要求8所述的方法，其特征在于，所述的广泛脑区神经元树突发育障碍的严重程度降低表现为：突触电生理活性的下降程度降低；神经元树突棘的棘密度、大小、数量、密度的下降程度降低；神经元的树突直径、长度、数量的降低；各脑区突触及其构成的结构的分布的发育程度降低；和/或行为、情绪、认知功能缺陷程度降低。The method of claim 8, wherein the reduction in the severity of the neuronal dendritic development disorder in the extensive brain area is manifested as: a reduction in the degree of decrease in the electrophysiological activity of the synapses; Density, size, number, and density decrease; the dendritic diameter, length, and number of neurons decrease; the development of the distribution of synapses and their constituent structures in each brain area decreases; and/or behavior, emotion, and recognition The degree of functional defects is reduced.
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