CN113243332B

CN113243332B - Preparation and application of extensive brain area neuron tree sudden-breeding obstacle animal model

Info

Publication number: CN113243332B
Application number: CN202010082845.5A
Authority: CN
Inventors: 魏佑震; 唐文洁
Original assignee: Shanghai East Hospital Tongji University Affiliated East Hospital
Current assignee: Shanghai East Hospital Tongji University Affiliated East Hospital
Priority date: 2020-02-07
Filing date: 2020-02-07
Publication date: 2023-07-28
Anticipated expiration: 2040-02-07
Also published as: CN113243332A; WO2021155866A1

Abstract

The invention relates to preparation and application of a neuronal tree sudden-breeding obstacle animal model in a wide brain area. Specifically, the invention provides a preparation method of a non-human mammal model for treating sudden abortion of a neuron tree in a wide brain region, which comprises the following steps: exposing the non-human mammal to hypoxia conditions for 30-40 minutes to obtain an animal model of a wide range of neuronal tree sudden growth disorder in the brain region, wherein the oxygen concentration (volume ratio) decreases from 10-20% to 1-5%. The animal model of the invention can be used for researching the burst breeding disorder of the neuron tree in the wide brain area, and can be used for screening and testing specific medicines.

Description

Preparation and application of extensive brain area neuron tree sudden-breeding obstacle animal model

Technical Field

The invention relates to the technical field of biology, in particular to preparation and application of an animal model for treating sudden-breeding disorder of a neuron tree in a wide brain area.

Background

Hypoxia/ischemic (H/I) injury is one of the main causes of brain functional deficits at all ages, and has been widely studied in both clinical and experimental animal studies, including etiology, neuropathogenesis, and pharmacological intervention. More and more studies have shown that H/I may adversely affect brain development in rodents.

Most current animal models of hypoxic brain injury are:

1. blocking arterial vessel + hypoxia-cerebral ischemia hypoxia model: disadvantages: anesthesia has neuroprotective effect; surgery itself has trauma; large infarct variation and instability; the model is troublesome to manufacture and small in batch; the success rate is low; the manufacturing period is long.

2. Middle cerebral artery occlusion model: this model, which is often used in adult mice, is only a model of cerebral ischemia or cerebral ischemia reperfusion injury, and has no hypoxia factor; is more suitable for clinical cerebral ischemia diseases and is a focal lesion model. Is not suitable for the simulation of ischemic and anoxic encephalopathy of newborns.

3. The method for clamping the air pipe comprises the following steps: the disadvantages are: the effects of anesthetic drugs on nerves; trauma caused by the operation itself; the technical requirement is high, and the period is long; the batch is small; since mice used are generally older (too little surgery difficult), they have poor consistency of pathological changes.

4. Establishing an intrauterine hypoxia model: disadvantages: data effects of anesthetic drugs; the operation itself is wounded; the surgical technical requirements are high; the consumption of surgical consumables is high; the period is long, and the batch is small; large difference and poor uniformity.

Therefore, there is an urgent need in the art to develop a new hypoxic brain injury animal model that can be used as a powerful tool for studying the pathogenesis of neuronal tree outbreak disorders in the wide brain area and for new drug screening.

Disclosure of Invention

The invention aims to provide an animal model which can be used as a powerful tool for researching pathogenesis of neuronal tree sudden growth disorder in a wide brain area and screening new medicines.

The first aspect of the invention provides a method for preparing a non-human mammal model for treating sudden dystocia of a neuronal tree in a wide brain region, comprising the following steps:

exposing the non-human mammal to hypoxia for 30-40 minutes, thereby obtaining an animal model of the neuronal tree sudden-growth disorder in the wide brain region,

wherein the oxygen concentration (volume ratio) is reduced from 10-20% to 1-5% under the low oxygen condition.

In another preferred embodiment, the extensive brain region neuronal tree sudden-growth disorder comprises an extensive brain region neuronal dendritic spine development disorder.

In another preferred embodiment, the non-human mammal is a rodent or primate, preferably comprising mice, rats, rabbits and/or monkeys.

In another preferred embodiment, the non-human mammal is a neonatal non-human mammal, preferably a neonatal non-human mammal (such as a rodent) within 24 hours of birth or a neonatal non-human mammal such as a primate at a perinatal period.

In another preferred embodiment, the inert gas is added under low oxygen conditions to reduce the oxygen concentration.

In another preferred embodiment, the inert gas is selected from the group consisting of: nitrogen, helium, or a combination thereof.

In another preferred embodiment, the extensive brain region neuronal tree sudden-growth disorder animal model has one or more characteristics selected from the group consisting of:

(t 1) no significant changes in brain morphology and brain size;

(t 2) no significant effect on systemic development of the animal;

(t 3) no significant effect on body weight of animals;

(t 4) no significant changes in the animal's general physiological activities including diet, excretion, respiration, heart rate, blood pressure, visual and auditory, pain and warmth, motor ability, reflex ability, etc.;

(t 5) structure under neuronal light mirror: the morphology, size, polarity, arrangement, number, density and distribution of the neurons are not changed significantly;

(t 6) microglial cells have no significant changes in the morphology, size, shape, distribution, number, density, etc. of their cell bodies and their projections;

(t 7) the synaptic physiological activity is significantly reduced;

(t 8) the morphology, size, number, density of the neuronal dendrites and dendritic spines is significantly reduced;

(t 9) the dendrite diameter and length of neurons decrease significantly;

(t 10) the number, distribution, area, etc. of synapses formed between neurons is significantly reduced;

(t 11) behavioral, emotional, cognitive functional deficits.

In another preferred embodiment, the synaptic physiological activity comprises: the mEPSCs amplitude and frequency.

In another preferred example, the behavioral, emotional, and cognitive functional deficits include reduced spatial learning and memory, reduced active exploring behavior, weaker fear of dangerous situations, and reduced natural mental performance of fear of bright areas.

In a second aspect, the invention provides the use of a non-human mammalian model prepared by a method according to the first aspect of the invention as an animal model for studying neuronal tree sudden growth disorders in the wide brain area.

In a third aspect, the invention provides the use of a non-human mammalian model prepared by the method of the first aspect of the invention for screening or identifying substances (therapeutic agents) which reduce or treat neuronal tree sudden growth disorders in the broad brain region.

In a fourth aspect, the invention provides a method of screening or identifying a potential therapeutic agent for treating or alleviating a neuronal tree emergency disorder in a broad brain region, comprising the steps of:

(a) In a test group, administering a test compound to a non-human mammalian model prepared by the method of the first aspect of the invention in the presence of the test compound, and detecting the severity Q1 of a neuronal tree burst disorder in the brain region of the animal model in the test group; and detecting the severity of neuronal tree burst disorder in the extensive brain area of the animal model in a control group without the test compound administered and under otherwise identical conditions Q2;

(b) Comparing the severity Q1 and severity Q2 detected in the previous step to determine whether the test compound is a potential therapeutic agent for treating or alleviating neuronal tree emergency disorders in the extensive brain area;

wherein a test compound is indicated as a potential therapeutic agent for treating or alleviating a broad brain area neuronal tree sudden growth disorder if the severity Q1 is significantly lower than the severity Q2 or if the severity of the broad brain area neuronal tree sudden growth disorder is reduced in an animal model to which the test compound is administered.

In another preferred embodiment, said detecting the severity of a neuronal tree sudden-breeding disorder in the brain region comprises detecting a change in one or more indicators selected from the group consisting of: sudden electric shock physiological activity; the shape, size, number, distribution and density of the spines of the nerve cells dendrites and dendritic spines in each brain region; the dendrite diameter, length, number of neurons; the development status of the distribution of the structures formed by the brain area synapses; behavior, emotion, cognitive function.

In another preferred embodiment, the decrease in severity of the wide brain area neuronal tree sudden growth disorder is manifested as: the degree of decrease in the physiological activity of the sudden electric shock is reduced; the density, size, number and degree of density decrease of the neuron dendritic spines are reduced; a reduction in the dendrite diameter, length, number of neurons; the extent of development of the distribution of the brain region processes and the structures they constitute is reduced; and/or reduced levels of behavioral, emotional, and cognitive deficits.

In another preferred embodiment, the "significantly lower" means that the test group with biological replicates has a lower severity Q1 after administration of the test compound than the control group with biological replicates has a severity Q2 after administration of the test compound, and has a P-value of less than 0.05 upon t-test.

In another preferred embodiment, the term "significantly lower" means that the ratio of severity Q1/severity Q2 is 1/2 or less, preferably 1/3 or less, more preferably 1/4 or less.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred embodiment, the method comprises the step of (c) administering the potential therapeutic agent screened or identified in step (b) to a non-human mammalian model prepared by the method of the first aspect of the invention, thereby determining its effect on the severity of neuronal tree burst disorder in the extensive brain region of said animal model.

In a fifth aspect the invention provides a non-human mammalian model prepared by the method of the first aspect of the invention.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Figure 1 shows that the present model animals did not cause substantial significant changes after being subjected to hypoxia challenge. Wherein A: no change in the appearance of the brain was found; b: no change in body weight was caused.

Figure 2 shows that the neuronal electrophysiological activity of the model mice was significantly affected after experiencing hypoxia stroke. Wherein, A-C: in vitro electrophysiology showed a decrease in synaptic activity between brain neurons after hypoxia; D-F: changes in electrophysiological function in the hippocampal brain region after hypoxia are shown to be inhibited over a long period of time (LYD).

Fig. 3 shows that no significant changes in neuronal morphology, size, arrangement, number, distribution, density were observed under the brain mirror after hypoxia (a-J).

Figure 4 shows that no significant changes in microglial morphology, size, arrangement, number, distribution, density were observed under the brain microscope after hypoxia (a-F).

Figure 5 shows that the dendritic diameter, size, number, density of neurons in the hippocampal region (B) decreased significantly in early stages (7 days post-natal) after hypoxia compared to normal mice (a); this difference is more pronounced in adulthood (C, D)

FIG. 6 shows that other brain regions besides the hippocampus, such as Dentate Gyrus (DG), the dendrites and dendritic spines of nerve cells also see significant changes (A-F) in the reduction of dendrite diameter, shortening of dendrite length, and reduction of dendritic spine density due to hypoxia; in the CA3 region, the volume of the region formed by the aggregation of synapses formed by the neuronal dendrites is reduced (G-I).

Fig. 7 shows that neurons cultured in a hypoxic environment, which give significantly reduced length of dendrites, were significantly reduced on day 7 (a, B) and significantly different on day 7 (C, D) by means of in vitro cell culture.

FIG. 8 shows that after adulthood, mice that had been subjected to hypoxia strikes during the neonatal period, from the change in escape latency, reflected a significant decline in spatial learning (A); after withdrawal from the platform, the movement trace (C) of the rat reflects the same significant decline in spatial memory (B, D).

Detailed Description

The inventor has conducted extensive and intensive studies, and has unexpectedly found that an animal model of a wide brain region neuronal tree sudden-growth disorder can be obtained by exposing a non-human mammal to a low oxygen condition in which the oxygen concentration (volume ratio) is reduced from 10 to 20% to 1 to 5% for 30 to 40 minutes. The present invention has been completed on the basis of this finding.

Burst dysgenesis of neuronal tree in extensive brain area

In the present invention, a relatively mild hypoxic condition (i.e., a mammal exposed to an oxygen concentration environment of varying concentration, e.g., a reduced oxygen concentration (volume ratio) from 10-20% to 1-5%) is established, resulting in significant developmental disorders of neuronal dendrites and their dendritic spines, which result in sustained structural damage that can lead to impairment of learning and memory or even other cognitive activities.

Animal model

In the present invention, a very effective non-human mammalian model of a wide brain area neuronal tree sudden growth disorder is provided.

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.

The animal model of the invention is prepared by the following method:

The animal model obtained by the method is fertile and has normal development.

Candidate drugs or therapeutic agents

In the present invention, there is also provided a method for screening candidate drugs or therapeutic agents for treating a neuronal tree emergency disorder in a wide brain area using the animal model of the present invention.

In the present invention, a candidate drug or therapeutic agent refers to a substance known to have a certain pharmacological activity or being tested for a potential to have a certain pharmacological activity, including but not limited to nucleic acids, proteins, chemically synthesized small or large molecular compounds, cells, etc. The drug candidate or therapeutic agent may be administered orally, intravenously, intraperitoneally, subcutaneously, intraspinal, or by direct intracerebral injection.

The main advantages of the invention include:

1. the experimental conditions are simple.

2. The manufacturing process is simple.

3. The intervention treatment factors are single. Only one condition of hypoxia is given, and no operation, administration and the like are performed on animal bodies.

4. The model is uniform. The individual difference is small, the birth time of the newborn is easy to grasp, and the newborn is hardly interfered by the outside after birth.

5. The index is clear. The morphological, electrophysiological and brain function change indexes are definite, obvious and stable.

6. The lesion point is focused. The injury sites are concentrated in the dendritic spines, and are clear and focused. Cell body, cell shape, polar orientation, arrangement, size, number, density, etc. are not affected.

7. The function of the damage point is critical. The sites of model damage are gathered on dendritic spines, the dendritic spines are key nodes of the neuron connection of the brain forming network, are main channels for transmitting signals, which are mutually influenced, among the neurons, are key points of the signal transmission efficiency of the central nervous system network system, and are main structural bases of brain functions.

8. The damage mechanism of the dendritic spines is clear. Hypoxia causes a disturbance of neuronal metabolism, affects energy supply, causes dysplasia of the dendritic spine, and the developmental deformity of the dendritic spine exists for life and accompanies a decrease in brain function.

9. The success rate is high. The model was stable, 100% successful.

10. Can be produced in batch. Meanwhile, a large number of homogeneous models are manufactured, so that large-scale experiments can be conveniently carried out.

11. Can be used as a powerful tool for researching pathogenesis of the neuronal tree sudden-growth disorder in the wide brain area and screening new drugs.

12. The animal model of the invention has stable phenotype.

13. The animal model obtained by the method is fertile, and the general structure of the organism is normal.

14. The animal model of the invention can be used to study the role and mechanism of TSH in early brain neuron dendritic spine development.

15. The animal model of the invention can be used for exploring, researching, developing and verifying potential drugs or means and methods to intervene TSH injury and block dendritic spine development injury; improving development disorder of dendritic spines.

16. The animal model of the invention can be used for researching the functional change of the nervous system operation and the operation mechanism thereof under the pathological condition of dendritic spine development disorder.

17. The animal model of the invention is used as a nervous system model reference for mental disorder, and is used for study and memory comparison reference under other nervous system pathological modes (such as senile dementia).

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Unless otherwise indicated, the materials used in the examples were all commercially available products.

Manufacturing process

1. Pregnant mice were kept in an animal room with 12 hours of light and a 12 hour dark cycle, which allowed free access to food and water until they were born.

2. After birth and 6 hours of maternal feeding, these neonates were randomly divided into two groups.

3. In TSH treatment, the neonates were first placed in a hypoxic chamber with an oxygen concentration of 15% and gradually adjusted to 3% by N2 over 30 minutes at 30 ℃. Thereafter, the oxygen concentration was maintained at 3%, and the neonate rat was left in the tank for another 5 minutes. Thus, the total hypoxic injury lasted 35 minutes. For the normoxic group, the neonates were placed in the box for 35 minutes with a normoxic concentration of 21%.

4. After the hypoxia process is completed, all the neonatal mice are returned to the cage, and the neonatal mice are the same as the mother mice.

Desired preparation conditions

1. An oxygen control cell incubator, or a special animal raising box

2. If no special equipment is provided, a common cell incubator can be used, with the addition of a closed box capable of controlling the oxygen concentration.

Detecting index results

1. The survival rate is high: after TSH treatment, the brain general morphology and body weight of 50 newborn mice are recovered to be normal, and the total survival is achieved.

2. The hypoxia systemic reaction is obvious: at the end of hypoxia treatment, the whole body color of the neonate mice was darker than the normal control group and recovered soon after the end of hypoxia treatment.

3. Brain morphology is unchanged: the brains of young rats that received TSH and normoxic treatment within 5 and 7 days, respectively, were also carefully compared by the investigator. There were no significant differences in brain morphology and brain size between the hypoxia-treated group and the normoxic group (fig. 1A).

4. Body weight is unchanged: the growth rates of hypoxia-treated and normoxic-treated neonates were similar (n=8, p > 0.05) weights (fig. 1B). These observations indicate that TSH has no significant effect on the overall development of the animal.

5. The physiological activity of the sudden electric shock is reduced: to explore whether TSH effects on neurological function, spontaneous synaptic activity was recorded using patch clamp, hippocampal slices TSH (n=27 neurons) and normoxica treated rat neurons (n=31), i.e. glutamate receptor mediated mini excitatory postsynaptic currents (mEPSCs) were recorded in CA1 neurons. TSH treated rats had a mpescs amplitude (pA) of 15.18±0.43 (pA), and the difference was statistically significant compared to 17.70±0.28 (pA) of the control group (p < 0.001) (fig. 2A and 2B). TSH treated rats had a frequency of 3.98±0.55 (Hz) and were also significantly reduced (figures 2A and 2C) compared to 6.35±0.49 (Hz) for the control group (p < 0.01). In addition, in vivo hippocampal CA1 synaptic activity recordings showed that TSH injured (n=23 neurons) animals had a neuronal firing rate of 8.74±0.63 (spike/s), significantly higher than 5.06±0.45 (spike/s), p <0.001 (fig. 2D) for the normal motor control group (n=25 neurons). Since both amplitude and frequency of mpescs were significantly reduced in TSH rat hippocampal CA1, we suspected if the reduction of basal synaptic transmission also affected synaptic plasticity of hippocampal brain slices. The extracellular long-term potential (LTP) of the hippocampal CA1 region was the same as that of the normal control group in the TSH treated group (fig. 2E). However, hypoxia significantly inhibited long-term depression (LTD) (fig. 2F). These results indicate that neuronal synaptic activity is disrupted after TSH injury.

6. The morphology and the optical lens structure of the neuron are unchanged: TSH does not cause structural changes in brain nerve cells. We examined whether the number of hippocampal neurons was altered following TSH injury. Nile staining was shown (FIG. 3, A-J). Observations of hippocampal CA1 neuron density TSH and normal rats at P5, P7, P21 and P90 are shown in table 1. These observations support that no significant cell loss or overall structural changes in hippocampal CA1 occurred at different developmental ages after TSH injury.

TABLE 1

7. Microglial cells were not activated. TSH does not induce brain activation of microglia. Observation of the morphology, number, distribution, density, etc. of hippocampal CA1 microglial cells in rats with hypoxia, compared to normal (FIG. 4, A, B, C), showed no apparent activation (FIG. 4, D, E, F).

Tsh results in a decrease in density of hippocampal neurons dendritic spines. Structural details of granulosa cells in hippocampal CA1 neurons dendrites, dendritic spines and brain region DG were examined at day 7 and day 90 after TSH treatment, respectively. Normal rat neurons had dendritic spines distributed in clusters along dendrites, and the dendritic spines TSH of animals were significantly reduced after hypoxia, sparsely distributed, and only a small amount distributed along dendrites (fig. 5). On day 7 post-natal, the density of dendritic spines per 10um,8.5±4.0, was significant of, <0.001 compared to the normal control group 15.1±2.9/10m (fig. 5a, b). 3 months after TSH injury, the reduction in dendritic spine density was 14.0.+ -. 3.9/10um, while the normal sports group animals had a spine density of 23.7.+ -. 4.6/10um (< 0.05) (FIGS. 5C, D).

9. The dendrite diameter of neurons decreases. In addition to dendritic spine density, we examined neuronal dendrites of hippocampal CA1, especially secondary branches, on days 7 and 90 after TSH treatment or normoxic treatment. At P7, the mean dendrite diameter of TSH animals was approximately the same as that of the normal control group. However, the diameter of the secondary branches of the neuronal dendrites of TSH treated animals was statistically smaller than that of normoxic control. Similar results were also observed 90 days after hypoxia and normoxic treatment (fig. 5 and table 2).

TABLE 2

Sh causes abnormal development of DG and CA3 regions. Observations of the dentate gyrus of the hippocampus further confirmed the reduced density of dendritic spines and the secondary branch attenuation of dendrites in hippocampus CA1 (fig. 6A, 6B, 6C). At P90, the DG granulosa cells of the TSH treated animals had a dendritic spine density of 7.03.+ -. 0.53/10um, which was lower than 14.8.+ -. 0.60/10um, P <0.0001 for the normoxic control group (FIG. 6D). The length of the TSH group dendrite is obviously shortened compared with that of the control group. 90 days after TSH treatment, the dendritic diameter of the animal dentate gyrate granulosa cells was 0.893.+ -. 0.064m, significantly greater than 0.628.+ -. 0.046m, p <0.05 for the normal control group (FIG. 6E). Dendritic shrinkage length of the 90d animals hippocampal DG molecular layer (IML) granulosa cells after TSH treatment was 15.35±1.75m, significantly higher than Yu Chang oxygen control group 7.04±1.02m, p <0.001 (fig. 6F). Finally, brain sections of animals stained for tim at 90 days of age showed a significant decrease in tim positive particles in the CA3 region of the hippocampus of TSH group compared to the control group (fig. 6G and 6H). The mean transparent layer width of hippocampal CA3 in TSH-injured group was 117.3±4.63um, significantly reduced compared to 146.0±5.82um in normal control group, p <0.005 (fig. 6I).

11. Hypoxia nerve cell culture neurons are impaired in dendrite growth. In vitro experiments are carried out by using a cell culture method, and the influence of hypoxia on the growth and development of the neuron dendrites is further verified. The experimental results showed that the growth of the neuronal dendrites was slow, the protrusion was shortened, and the branching was reduced under hypoxia (5% o 2) conditions for 7 days continuously, starting from the first day; and persisted, and on day 7 of the observation period, neuronal morphology in normoxic conditions was compared (fig. 7C), with a significant decrease in both hypoxic neuronal dendrite length and density (fig. 7D).

Tsh can lead to long-term cognitive dysfunction. Morris water maze experiments were performed on 3 months old TSH and normoxic treated rats. In spatial navigational training over 4.5 days, rats in the control group and the hypoxic group showed similar improvement in the first 6 exercises and all animals had a shortened latency in finding the platform. However, from training results 7, 8 and 9, there was a significant difference between TSH injury and normal hypoxia treated animals. The incubation periods of the TSH group rats were 21.6± 3.71,16.6 ± 1.79,20.4 ±3.86, respectively, significantly longer than 12.1± 1.72,7.99 ± 1.03,8.07 ±0.96 of the control group, respectively (p < 0.05) (fig. 8A). After 9 training trials, all rats were subjected to space exploration testing without MWM platform. The trace of swimming was recorded digitally. The number of platform website traversals of TSH, on day 5, 9.43+ -1.29 and 2.00+ -1.15, was 16.75+ -2.18,6.29 + -2.36 for normal mice, respectively, with significant differences (p <0.05, FIG. 8B). In addition, TSH animals swim a distance of 26475±2412mm in the quadrant where the platform was located, p <0.05 compared to control 35862±3371mm (fig. 8C and 8D). These results strongly suggest that neonatal rat TSH injury can lead to brain developmental disorders, leading to brain functional deficits, including spatial learning and memory.

Discussion of the invention

Animal model study of TSH perinatal neonates. The brain is one of the most sensitive organs to hypoxia, which can lead to coma, epilepsy, cognitive dysfunction and other neurological dysfunction, and even brain death, over time. Neonatal hypoxia injury is generally thought to be due to umbilical cord cervical winding, head basin unlevel, birth incarceration, uterine ischemia, and neonatal asphyxia. Perinatal hypoxic ischemic brain injury causes higher mortality and chronic neurological morbidity in acute infants and children, often with sequelae. The invention discovers that TSH causes obvious pathological changes including dendritic thinning, shortening, dendritic spine density reduction, synaptic structure reduction and synaptic physiological function reduction of central nervous system neurons for the first time; furthermore, impaired cognitive function was observed in animals with TSH. The data of the present invention clearly demonstrate that TSH can cause significant neurotensin and dendritic toxicity in the neonatal as well as in the adult stage. Thus, the present invention provides a useful animal model for studying the effects and mechanisms of sublethal hypoxia on early brain development and for exploring potential intervention drugs or methods.

The study of the present invention shows that neonatal rats were given a short sublethal hypoxia for 35 minutes alone 6 hours after birth, unlike animal models previously used for hypoxia/ischemia studies. Animals born for 6 hours are used in the current study, which is more similar to clinical situations (umbilical cord neck winding, birth incarceration, uterine deformity, etc.) than many previous hypoxia/ischemia studies, wherein animals are used for about one day old, the shorter the time after birth, the smaller the individual variation coefficient of the animals after birth, and the better the uniformity of the nervous system among the animals. One single factor gradient hypoxia supply is the second difference between current studies and past animal models using carotid occlusion and hypoxia (two factors). Finally, the TSH injury group does not have obvious neuropathological changes, does not have neuron loss and glial activation, but has durable brain function defects observed, and is a model which is more suitable for simulating more common clinical moderate and mild ischemic anoxic encephalopathy. Because of its low severity of hypoxia, the condition is difficult to diagnose, lacks adequate care or treatment, and ultimately leads to severe brain developmental dysfunction. Thus, the present study provides an important animal model and useful data for clinical practice of transient sublethal hypoxia single injury.

TSH blocks spontaneous synaptic activity of neurons. Background activity of electroencephalogram (EEG) was found in very early premature infants and animals. In this study, TSH resulted in a decrease in amplitude and frequency of hippocampal CA1 neurons, mpescs, compared to the normal control group. TSH has a negative impact on neuronal dendrite development. In the present invention, the neuronal dendrite diameter of animals was significantly reduced after TSH treatment compared to the control group. The total length of dendrites of primary cultured hippocampal neurons after 5% oxygen treatment was also significantly lower than that of normal control group. These results indicate that damage to TSH induces dendritic toxicity, affecting the growth and development of neuronal dendrites. Hypoxia-induced energy deficit may affect cytoskeletal dynamics including actin and microtubules. Actin and MT are key to the development of conventional dendrites and are targets for many molecular pathways that control neuronal dendrite growth. During brain development, both actin and MT cytoskeletal structures change, producing neuronal projections. The present invention also finds that TSH damage reduces the density and development of dendritic spines. More than 95% of excitatory synapses on these neurons occur on dendritic spines, with each spiny head typically connected to form a synapse. The formation and plasticity of dendritic spines is important for brain function. Development of dendritic spines is controlled by oxygen sensor PHD2, targeting actin crosslinker Filamin-a, regulating synaptic density and neuronal activity within the network. Dendritic spine associated Rap-specific gdpase activator protein (SPAR) is a postsynaptic protein that forms a complex with postsynaptic density (PSD) -95, which is involved in regulating the morphogenesis of dendritic spine with N-methyl-D-aspartate receptors (NMDARs).

The structure and dynamics of dendritic spines reflect the strength of synapses, which are often severely affected in different brain diseases, including neurodegenerative and psychiatric diseases. Dendritic spines can undergo several types of transformations, from growth to collapse, from elongation to shortening, with very short spans of time that they undergo such dynamic morphological activities. The change of the number and morphology of dendritic spines occurs not only in pathological conditions such as excitotoxicity, but also in the normal central nervous system development, hormonal fluctuations and the response of neural activity in physiological environments.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. A method for preparing a non-human mammal model for treating sudden dystocia of a neuron tree in a wide brain region, which is characterized by comprising the following steps:

wherein the oxygen concentration volume ratio is reduced from 10-20% to 1-5% under the hypoxia condition, and the extensive brain region neuron tree sudden-growth disorder animal model has a plurality of characteristics selected from the group consisting of:

the physiological activity of the sudden electric shock is obviously reduced;

the morphology, size, number and density of the dendrites of the neurons and the dendrites are remarkably reduced;

the dendrite diameter and length of neurons decrease significantly;

the number, distribution, and area of synapses formed between neurons are significantly reduced, and the extensive brain region neuronal tree sudden growth disorder includes extensive brain region neuronal dendritic spine development disorder.

2. The method of claim 1, wherein the non-human mammal is a rodent or primate.

3. The method of claim 2, wherein the non-human mammal comprises a mouse, a rat, a rabbit, and/or a monkey.

4. The method of claim 1, wherein the non-human mammal is a neonatal non-human mammal.

5. The method of claim 4, wherein the non-human mammal is a neonatal non-human mammal within 24 hours of birth or a neonatal non-human mammal in the perinatal period.

6. Use of a non-human mammalian model prepared by the method of claim 1 as an animal model for studying a wide brain area neuronal tree sudden growth disorder, including a wide brain area neuronal dendritic spine development disorder.

7. Use of a non-human mammalian model prepared by the method of claim 1, for screening or identifying substances that reduce or treat a wide brain area neuronal tree emergency disorder, including a wide brain area neuronal dendritic spine development disorder.

8. A method of screening or identifying a potential therapeutic agent for treating or alleviating a neuronal tree emergency disorder in a broad brain region, comprising the steps of:

(a) In a test group, administering a test compound to a non-human mammalian model prepared by the method of claim 1 in the presence of the test compound, and detecting the severity Q1 of a neuronal tree burst disorder in the extensive brain area of said animal model in the test group; and detecting the severity of neuronal tree burst disorder in the extensive brain area of the animal model in a control group without the test compound administered and under otherwise identical conditions Q2;

wherein, if the severity Q1 is significantly lower than the severity Q2 or if the severity of a wide brain area neuronal tree sudden-growth disorder in an animal model to which a test compound is administered is reduced, the test compound is indicated to be a potential therapeutic agent for treating or alleviating a wide brain area neuronal tree sudden-growth disorder, including a wide brain area neuronal dendritic spine development disorder.

9. The method of claim 8, wherein detecting the severity of the extensive brain region neuronal tree sudden-growth disorder comprises detecting a change in a plurality of indicators selected from the group consisting of: sudden electric shock physiological activity; the shape, size, number, distribution and density of the spines of the nerve cells dendrites and dendritic spines in each brain region; the dendrite diameter, length, number of neurons; each brain region protrudes and develops in the distribution of its constituent structures.

10. The method of claim 8, wherein the decrease in severity of the extensive brain region neuronal tree sudden growth disorder is manifested by: the degree of decrease in the physiological activity of the sudden electric shock is reduced; the density, size, number and degree of density decrease of the neuron dendritic spines are reduced; a reduction in the dendrite diameter, length, number of neurons; and the extent of development of the distribution of the brain region processes and the structures they constitute is reduced.