CN113243332A

CN113243332A - Preparation and application of extensive brain region neuron dendritic developmental disorder animal model

Info

Publication number: CN113243332A
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: 2021-08-13
Anticipated expiration: 2040-02-07
Also published as: CN113243332B; WO2021155866A1

Abstract

The invention relates to preparation and application of an extensive brain region neuron dendritic developmental disorder animal model. Specifically, the invention provides a preparation method of a non-human mammal model with extensive brain region neuron dendritic development disorder, which comprises the following steps: exposing a non-human mammal to hypoxic conditions in which the oxygen concentration (by volume) is reduced from 10-20% to 1-5% for 30-40 minutes, thereby obtaining an animal model of a wide brain area neuronal dendritic development disorder. The animal model can be used for researching extensive brain region neuron dendritic development disorder and can be used for screening and testing specific drugs.

Description

Preparation and application of extensive brain region neuron dendritic developmental disorder animal model

Technical Field

The invention relates to the technical field of biology, in particular to preparation and application of an extensive brain region neuron dendritic developmental disorder animal model.

Background

Hypoxic/ischemic (H/I) injury is one of the major causes of brain functional deficits at all ages, and has been extensively studied in both clinical and experimental animal studies, including etiology, neurological pathogenesis, and pharmacological intervention. More and more studies have shown that H/I may adversely affect rodent brain development.

At present, most animal models of hypoxic brain injury are:

1. blocking arterial vessels + hypoxia method-cerebral ischemia hypoxia model: the disadvantages are as follows: anesthesia has neuroprotective effect; the surgery itself is traumatic; the infarction has large variation and is unstable; 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: the model is usually an adult mouse, is only a cerebral ischemia or cerebral ischemia reperfusion injury model and has no hypoxia factor; is suitable for clinical cerebral ischemia diseases and is a focal lesion model. Is not suitable for simulating the ischemic and hypoxic encephalopathy of the newborn.

3. The method of clamping and closing the trachea: the disadvantages are that: the effects of the presence of anesthetic drugs on nerves; trauma from the operation itself; the technical requirement is high, and the period is long; the batch size is small; the mice used are generally older in the day (too little surgery difficult) and have a poor consistency of pathological changes.

4. Establishing an intrauterine hypoxia model: the disadvantages are as follows: data impact of anesthetic drugs; trauma to the operation itself; the surgical technical requirement is high; the consumption of surgical consumables is large; the period is long and the batch size 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 in the research of the pathogenesis of neuronal dendritic development disorders in a wide brain region and the screening of new drugs.

Disclosure of Invention

The invention aims to provide an animal model which can be used as a powerful tool for researching pathogenesis of extensive brain region neuron dendritic development disorder and screening new drugs.

The invention provides a preparation method of a non-human mammal model of extensive brain region neuron dendritic development disorder, which comprises the following steps:

exposing the non-human mammal to hypoxic conditions for 30-40 minutes, thereby obtaining an animal model of extensive brain area neuronal dendritic development disorder,

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

In another preferred example, the wide brain region neuronal dendritic development disorder comprises a wide brain region neuronal dendritic spine development disorder.

In another preferred embodiment, the non-human mammal is a rodent or primate, preferably comprising a mouse, rat, rabbit and/or monkey.

In another preferred example, 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 in perinatal period) such as a primate).

In another preferred embodiment, under low oxygen conditions, an inert gas is added 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 animal model of extensive brain area neuronal dendritic development disorder has one or more characteristics selected from the group consisting of:

(t1) no significant change in brain morphology and brain size;

(t2) no significant effect on the development of the whole body of the animal;

(t3) the body weight of the animal had no significant effect;

(t4) the general physiological activities of the animals including diet, excretion, respiration, heart rate, blood pressure, visual and auditory sensation, pain and temperature sensation, motor ability, reflex ability, etc. are not significantly changed;

(t5) neuron structure under microscope: the form, size, polarity, arrangement, number, density and distribution of the neurons are not changed obviously;

(t6) there is no significant change in the morphology, size, shape, distribution, number, density, etc. of microglia in their cell bodies and their processes;

(t7) a significant decrease in synaptic electrophysiological activity;

(t8) the morphology, size, number, density of spines of the dendrites and spines of the neurons are significantly reduced;

(t9) the dendrite diameter and length of the neuron are significantly reduced;

(t10) the number, distribution, area, etc. of synapses formed between neurons is significantly reduced;

(t11) behavioral, emotional, cognitive deficits.

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

In another preferred embodiment, the behavioral, emotional, and cognitive deficits include decreased spatial learning and memory, decreased active exploration behavior, reduced fear of critical situations, and diminished psychological performance of natural fear of bright areas.

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

In a third aspect, the invention provides the use of a non-human mammalian model prepared by a method according to the first aspect of the invention to screen or identify substances (therapeutic agents) that can reduce or treat a wide brain area neuronal dendritic development disorder.

In a fourth aspect, the present invention provides a method of screening for or identifying a potential therapeutic agent for the treatment or alleviation of dendrite development disorders of neurons in a broad brain region, comprising the steps of:

(a) administering a test compound to a non-human mammalian animal model prepared by a method according to the first aspect of the invention in the presence of the test compound in a test group, and testing said animal model in the test group for the severity of a wide brain region neuronal dendritic development disorder Q1; and in a control group not administered the test compound and otherwise identical, determining the severity of the neuronal dendritic development disorder in the extensive brain region of the animal model in the control group Q2;

(b) comparing the severity Q1 and the severity Q2 detected in the previous step to determine whether the test compound is a potential therapeutic agent for treating or ameliorating a wide brain area neuronal dendritic development disorder;

wherein a test compound is indicative of a potential therapeutic agent for treating or ameliorating a wide brain area neuronal dendritic development disorder if the severity degree of Q1 is significantly lower than the severity degree of Q2 or if the severity degree of the wide brain area neuronal dendritic development disorder in the animal model to which the test compound is administered is reduced.

In another preferred embodiment, said detecting the severity of said neuronal dendritic development disorder in the general brain region comprises detecting a change in one or more of the following: sudden electrical shock physiological activity; the form, size, number, distribution and density of the spines of the neuron dendrites and the dendrite spines in each brain area; dendritic diameter, length, number of neurons; the developmental status of the distribution of synapses and their constituent structures in each brain region; behavior, mood, cognitive function.

In another preferred embodiment, said reduced severity of said neuronal dendritic development disorder in the general brain region is characterized by: a reduced degree of decline in synaptic electrophysiological activity; the decrease degree of the density, size, number and density of the neuron dendritic spines is reduced; reduction in diameter, length, number of dendrites of neurons; a decrease in the degree of development of the distribution of synapses and their constituent structures in each brain region; and/or a reduced degree of behavioral, emotional, cognitive function deficits.

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

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

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 dendritic development disorders in the general brain region of said animal model.

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

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows that the model animals did not cause a substantially significant change after being subjected to hypoxic challenge. Wherein, A: no changes in the appearance of the brain were found; b: no weight change was induced.

Figure 2 shows that the neuronal electrophysiological activity of the model mice was significantly affected after a hypoxic challenge. Wherein, A-C: ex vivo electrophysiology shows a decrease in synaptic electrical activity between cerebral neurons after hypoxia; D-F: long-term inhibition (LYD) electrophysiological functional changes in the hippocampal brain region after hypoxia are shown in vivo electrophysiology.

Figure 3 shows that no significant changes in neuronal morphology, size, arrangement, number, distribution, density were observed under a cerebroscopic examination after hypoxia (a-J).

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

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

Figure 6 shows that in other brain areas except the hippocampus, such as the Dentate Gyrus (DG), dendrites and dendritic spines of nerve cells also see significant changes (a-F) in hypoxia resulting in a reduction in dendrite diameter, length of dendrites, and density of dendritic spines; in the CA3 region, the volume of the area formed by the aggregation of synapses formed by dendrite spines of neurons is reduced (G-I).

Figure 7 shows that neurons cultured in hypoxic environments using in vitro cell culture showed a significant decrease in the length of dendrites that emanated from them, with a significant difference on day one (a, B) and a greater difference on day 7 (C, D).

FIG. 8 shows that after adulthood, mice that have been subjected to hypoxic assault during the neonatal period, reflect a significant decline in spatial learning capacity from the change in escape latency (A); from the withdrawal of the platform, the rat's trajectory of motion (C), reflects a similarly significant decrease in spatial memory (B, D).

Detailed Description

The inventor has extensively and deeply studied and unexpectedly found that an animal model of wide brain area neuronal dendritic developmental disorder can be obtained by exposing a non-human mammal to hypoxic conditions in which the oxygen concentration (volume ratio) is reduced from 10-20% to 1-5% for 30-40 minutes. The present invention has been completed based on this finding.

Neuronal dendritic developmental disorder of extensive brain region

In the present invention, a relatively mild hypoxic condition (i.e., the exposure of the mammal to a changing oxygen concentration environment, e.g., a hypoxic condition in which the oxygen concentration (volume ratio) is reduced from 10-20% to 1-5%) is established, which causes significant developmental disorders of the dendrites and dendrites thereof, and the structural damage caused by the developmental disorders persists, causing impairment of learning and memory and even other cognitive behaviors.

Animal model

In the present invention, a very effective non-human mammalian model of neuronal dendritic development disorder in a wide brain region is provided.

In the present invention, examples of non-human mammals include (but are not limited to): mouse, rat, rabbit, monkey, etc., more preferably rat and mouse.

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

The animal model obtained by the method of the invention can be fertile and normally develops.

Drug candidate or therapeutic agent

In the invention, a method for screening candidate drugs or therapeutic agents for treating the neuron dendritic developmental disorder in the extensive brain region by using the animal model is also provided.

In the present invention, a drug candidate or therapeutic agent refers to a substance known to have a certain pharmacological activity or being tested, which may have a certain pharmacological activity, including but not limited to nucleic acids, proteins, chemically synthesized small or large molecular compounds, cells, and the like. The candidate drug or therapeutic agent may be administered orally, intravenously, intraperitoneally, subcutaneously, intradermally, 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 factor is single. Only one condition of hypoxia is given, and the animal body is not subjected to operation, administration and the like.

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

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

6. The damage point is focused. The site of injury is concentrated in the dendritic spine, well defined, focused. The cell body, cell shape, orientation, arrangement, size, number, density, etc. are not affected.

7. The function of the damage point is critical. The damaged sites of the model are gathered in the dendritic spines, the dendritic spines are key nodes of the neuron connection of the brain forming network, are main channels of transmission signals influenced by the neurons, are key points of signal transmission efficiency of a central nervous system network system, and are main structural foundations of brain function height.

8. The mechanism of dendritic spine injury is clear. Hypoxia causes a metabolic disorder of neurons, affects energy supply, causes a developmental disorder of dendritic spines, and developmental malformations of dendritic spines exist for a lifetime with accompanying hypofunction of the brain.

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

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

11. Can be used as a powerful tool for researching pathogenesis of wide brain area neuron dendritic development disturbance and screening new drugs.

12. The animal model has stable phenotype.

13. The animal model obtained by the method of the invention can be fertile, and the general structure of the organism develops normally.

14. The animal model of the invention can be used for researching the function and mechanism of TSH in the development of early cerebral neuron dendritic spines.

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

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

17. The animal model of the invention is used as a nervous system mode reference of 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 the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The materials used in the examples are all commercially available products unless otherwise specified.

Manufacturing process

1. Pregnant dams were housed in an animal room with 12 hours of light and 12 hours of dark cycles, and they had free access to food and water until they were delivered.

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

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

4. After the hypoxic process was completed, all newborn mice, together with the mother mice, were returned to their cages.

Required preparation conditions

1. An oxygen-controlled cell incubator or a special animal incubator

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

Result of detection index

1. The survival rate is high: after TSH treatment, the brain gross morphology and body weight of 50 newborn mice recovered normal and all survived.

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

3. Unchanged brain morphology: the researchers also performed careful comparisons of the brains of young mice receiving TSH and normoxic treatment within 5 days and 7 days, respectively. The hypoxic-treated group and normoxic-treated group did not differ significantly in brain morphology and brain size (fig. 1A).

4. The weight is not changed: the growth rates of the hypoxic treated neonatal mice and normoxic treated neonatal mice were similar in body weight (n ═ 8, p >0.05) (fig. 1B). These observations indicate that TSH has no significant effect on the systemic development of the animals.

5. Decrease in synaptic electrophysiological activity: to explore whether TSH affects neural function, spontaneous synaptic activity was recorded using patch clamp, hippocampal slices TSH (n ═ 27 neurons) and noroxaia treated rat neurons (n ═ 31), i.e. glutamate receptor mediated mini excitatory post-synaptic currents (mEPSCs) were recorded on CA1 neurons. The amplitude (pA) of mEPSCs from TSH-treated rats was 15.18. + -. 0.43(pA), which was statistically different from 17.70. + -. 0.28(pA) from the control group (p <0.001) (FIGS. 2A and 2B). The frequency of mEPSCs in TSH-treated rats was 3.98 ± 0.55(Hz), and also significantly decreased (fig. 2A and 2C) (p <0.01) compared to 6.35 ± 0.49(Hz) in the control group. In addition, in vivo hippocampal CA1 synaptic activity recordings showed that animals with TSH damage (n ═ 23 neurons) had neuronal firing rates of 8.74 ± 0.63(spike/s), significantly higher than 5.06 ± 0.45(spike/s) in the normal motor control group (n ═ 25 neurons), with p <0.001 (fig. 2D). Since both amplitude and frequency of mEPSCs were significantly reduced in the hippocampal CA1 of TSH rats, we suspect whether a reduction in basal synaptic transmission would also affect synaptic plasticity in hippocampal brain slices. Extracellular long-term potentials (LTPs) in CA1 region of hippocampus of TSH-treated group were the same as those of normal control group (fig. 2E). However, hypoxia significantly inhibited long-term depression (LTD) (fig. 2F). These results indicate that neuronal synaptic activity is interrupted after TSH injury.

6. The neuron form and the structure under the light lens are unchanged: TSH does not cause structural changes in brain nerve cells. We examined whether the number of hippocampal neurons has changed following TSH injury. Nissler small staining was shown (FIG. 3, A-J). Hippocampal CA1 neuronal density TSH and observations of 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 of hippocampal CA1 occurred at different developmental ages after TSH injury.

TABLE 1

7. Microglia were not activated. TSH does not induce brain activation of microglia. By observing the morphology, number, distribution, density, etc. of the hippocampal CA1 microglia, the hippocampal microglia of hypoxic rats showed no significant activation (fig. 4, D, E, F) compared to normal (fig. 4, a, B, C).

TSH caused a decrease in density of hippocampal dendritic spines. Structural details of granulosa cells in hippocampal CA1 neuronal dendrites, dendritic spines, and brain area DG were examined on days 7 and 90, respectively, after TSH treatment. The normal rat neuron dendritic spines are clustered along the dendrites, the dendritic spines TSH of the animals after hypoxia are remarkably reduced and sparsely distributed, and only a small amount of dendritic spines are distributed along the dendrites (figure 5). At postnatal day 7, the dendritic spine density of the apical dendrites of hippocampal CA1 neurons was 8.5 ± 4.0 per 10um, with a significant difference of <0.001 compared to the normal control group of 15.1 ± 2.9/10m (fig. 5A, B). The decrease in dendritic spine density was 14.0 ± 3.9/10um 3 months after TSH injury, while the spine density in normal sports animals was 23.7 ± 4.6/10um (<0.05) (fig. 5C, D).

9. The dendrite diameter of the neuron decreases. In addition to dendritic spine density, we also tested neuronal dendrites, especially secondary branches, of hippocampal CA1 on days 7 and 90 after TSH treatment or normoxic treatment. At P7, the average diameter of dendrites in TSH animals was approximately the same as that in normal controls. However, the diameter of the secondary branch of the neuronal dendrites in TSH-treated animals was statistically smaller than that of the normoxic control group. Similar results were also observed 90 days after hypoxic and normoxic treatments (figure 5 and table 2).

TABLE 2

SH causes abnormal development in the DG and CA3 regions. Observation of the hippocampal dentate gyrus further confirmed the decreased density of dendritic spines and the thinning of the secondary branches of the dendrites in hippocampal CA1 (fig. 6A, 6B, 6C). At P90, the density of DG granule cell dendritic spines in animals after TSH treatment was 7.03 + -0.53/10 um, lower than 14.8 + -0.60/10 um in the normoxic control group, P <0.0001 (FIG. 6D). The tress length of the TSH group is obviously shortened compared with that of the control group. The dendrite diameter of the odontoid granulosa cells in animals was 0.893 + -0.064 m 90 days after TSH treatment, which is significantly larger than 0.628 + -0.046 m in the normal control group, with p <0.05 (FIG. 6E). Dendritic contractile length of 90d animal hippocampal DG Inner Molecular Layer (IML) granular cells after TSH treatment was 15.35 + -1.75 m, significantly higher than that of normoxic control group 7.04 + -1.02 m, p <0.001 (FIG. 6F). Finally, timmm-stained brain sections from animals at 90 days of age showed a significant reduction of timmm-positive particles in the CA3 area of hippocampus of TSH group compared to the control group (fig. 6G and 6H). The average width of the stratum lucidum of the hippocampus CA3 in the TSH injured group was 117.3 + -4.63 um, which is significantly reduced compared with 146.0 + -5.82 um in the normal control group, and p is <0.005 (FIG. 6I).

11. Hypoxic neural cell cultures suffer from impaired neuronal dendritic 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. Experimental results showed that after 7 days of continuous culture under hypoxic (5% O2), slow growth of the dendrites of the neurons under hypoxic conditions, a shortened process and reduced branching were observed from the first day (fig. 7A, B); and persisting, at day 7 of the observation period, both the length and density of hypoxic neuronal dendrites were significantly reduced compared to neuronal morphology in the normoxic state (fig. 7C) (fig. 7D).

TSH can lead to long-term cognitive function deficits. The Morris water maze experiment was performed on rats after 3 months of TSH and normoxic treatment. Rats in both the control and hypoxic groups showed similar improvement in the first 6 trains during the 4.5 day spatial navigation training, and all animals had reduced latency in finding the platform. However, from the 7 th, 8 th and 9 th training results, there were significant differences between TSH-injured and normal hypoxia-treated animals. The latency of rats in the TSH group was 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, respectively, in the control group (p <0.05) (fig. 8A). After 9 training trials, all rats were tested for spatial exploration without the MWM platform. The trace of swimming was digitally recorded. The platform website crossing times of TSH, day 5, 9.43 +/-1.29 times and 2.00 +/-1.15 times, the crossing times of normal mice are respectively 16.75 +/-2.18 and 6.29 +/-2.36, and the difference is significant (p is less than 0.05, and figure 8B). In addition, animals in the TSH group swim at a distance of 26475. + -. 2412mm in the quadrant of the platform, with p <0.05 compared to control 35862. + -. 3371mm (FIGS. 8C and 8D). These results strongly suggest that TSH damage in neonatal rats leads to brain developmental disorders leading to defects in brain function, including spatial learning and memory.

Discussion of the related Art

Animal model studies of TSH perinatal neonates. The brain is one of the most sensitive organs to hypoxia, which over time can lead to coma, epilepsy, cognitive and other neurological disorders, and even brain death. Hypoxic injury in neonates is generally thought to be due to umbilical cord tortuosity, head basin unlined, birth incarceration, poor blood supply to the uterus, and asphyxia in the neonate. Perinatal hypoxic ischemic brain injury results in higher mortality and chronic neurological morbidity, often as sequelae, in acute infants and children. The invention discovers that TSH causes remarkable pathological changes including dendrite thinning, shortening, dendrite density reduction, synapse structure reduction and synapse electrophysiological 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 show that TSH can cause significant neurosynaptic and dendritic toxicity during neonatal as well as adult life. Thus, the present invention provides a useful animal model for studying the effects and mechanisms of sub-lethal hypoxia on early brain development and for exploring potential intervention drugs or methods.

The present study shows that neonatal rats are given transient sublethal hypoxia for 35 minutes 6 hours after birth, unlike previous animal models used for hypoxia/ischemia studies. In the current study, animals born for 6 hours are used, which is more similar to clinical conditions (umbilical cord constriction, birth incarceration, uterine malformation, etc.) in about one week of the day than in many previous hypoxia/ischemia studies, and the shorter the time after birth, the smaller the individual variation coefficient after birth, and the better the uniformity of the nervous system among the individual animals. A one-factor gradient hypoxia supply is the second difference between current studies and previous animal models using carotid occlusion and hypoxia (two factors). Finally, no obvious neuropathological change, no neuron loss and no glial activation are seen in the TSH injury group, but a lasting brain function defect is observed, so that the model is more suitable for simulating clinical common mild-moderate ischemic-hypoxic encephalopathy. Due to its mild degree of hypoxia, the condition is difficult to diagnose and lack of proper care or treatment, ultimately leading to severe brain development dysfunction. Therefore, the study provides an important animal model and useful data for clinical practice of transient sublethal hypoxia-independent injury.

TSH blocks spontaneous synaptic activity of neurons. Background activity of the electroencephalogram (EEG) is found in very early preterm infants and animals. In this study, TSH caused a decrease in amplitude and frequency of hippocampal CA1 neurons mEPSCs compared to normal controls. TSH negatively affects neuronal dendritic development. In the present invention, the neuron dendrite diameter of the animals was significantly reduced after the 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 controls. These results indicate that injury 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 dendritic growth, and are targets of many molecular pathways that control neuronal dendritic growth. During brain development, both actin and MT cytoskeleton structures change, resulting in neuronal processes. It has also been found in the present invention that TSH damage reduces the density and development of dendritic spines. More than 95% of the excitatory synapses on these neurons occur on dendritic spines, each of which is usually connected at the head to form a synapse. The formation and plasticity of dendritic spines is very important for brain function. The development of dendritic spines is controlled by an oxygen sensor PHD2, targeting actin cross-linking agent Filamin-A, and regulating neuronal activity within synaptic density and network range. Dendritic spine-associated Rap-specific GDP enzyme activator protein (SPAR) is a postsynaptic protein that forms complexes with postsynaptic density (PSD) -95, which is involved in modulating dendritic spine morphogenesis 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 transitions, from growth to collapse, from elongation to shortening, with very short time spans over which they undergo this dynamic morphological activity. The change of the number and the shape of the dendritic spines not only occurs under the pathological conditions of excitotoxicity and the like, but also occurs in the reaction process of normal central nervous system development, hormone fluctuation and nervous activity under the physiological environment.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A method for preparing a non-human mammal model with extensive brain area neuron dendritic development disturbance, which is characterized by comprising the following steps:

2. The method of claim 1, wherein the pervasive brain region neuronal dendritic development disorder comprises a pervasive brain region neuronal dendritic spine development disorder.

3. The method of claim 1, wherein the extensive brain area neuronal dendritic development disorder animal model has one or more characteristics selected from the group consisting of:

(t1) no significant change in brain morphology and brain size;

(t2) no significant effect on the development of the whole body of the animal;

(t3) the body weight of the animal had no significant effect;

(t7) a significant decrease in synaptic electrophysiological activity;

(t9) the dendrite diameter and length of the neuron are significantly reduced;

(t11) behavioral, emotional, cognitive deficits.

4. Use of a non-human mammalian animal model prepared by the method of claim 1 as an animal model for studying a wide brain region for neuronal dendritic development disorders.

5. The use of claim 4, wherein the extensive brain region neuronal dendritic development disorder comprises an extensive brain region neuronal dendritic spine development disorder.

6. Use of a non-human mammalian model prepared by the method of claim 1 to screen or identify agents (therapeutics) that reduce or treat a wide brain area neuronal dendritic development disorder.

7. The use of claim 6, wherein the extensive brain region neuronal dendritic development disorder comprises an extensive brain region neuronal dendritic spine development disorder.

8. A method of screening for or identifying a potential therapeutic agent for treating or ameliorating a wide range of brain region neuronal dendritic development disorders comprising the steps of:

(a) administering a test compound to the non-human mammalian model prepared by the method of claim 1 in the presence of the test compound in a test group, and determining the severity of the extensive brain region neuronal dendritic development disorder of said animal model in the test group Q1; and in a control group not administered the test compound and otherwise identical, determining the severity of the neuronal dendritic development disorder in the extensive brain region of the animal model in the control group Q2;

9. The method of claim 8, wherein detecting the severity of the dendrite development disorder of neurons of the extensive brain region comprises detecting a change in one or more of the following: sudden electrical shock physiological activity; the form, size, number, distribution and density of the spines of the neuron dendrites and the dendrite spines in each brain area; dendritic diameter, length, number of neurons; the developmental status of the distribution of synapses and their constituent structures in each brain region; behavior, mood, cognitive function.

10. The method of claim 8, wherein said reduced severity of said neuronal dendritic development disorder in the general brain region is characterized by: a reduced degree of decline in synaptic electrophysiological activity; the decrease degree of the density, size, number and density of the neuron dendritic spines is reduced; reduction in diameter, length, number of dendrites of neurons; a decrease in the degree of development of the distribution of synapses and their constituent structures in each brain region; and/or a reduced degree of behavioral, emotional, cognitive function deficits.