CN112243898A - Zebra fish cerebral arterial thrombosis model, construction method and application - Google Patents
Zebra fish cerebral arterial thrombosis model, construction method and application Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/10—Animals modified by protein administration, for non-therapeutic purpose
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/40—Fish
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0375—Animal model for cardiovascular diseases
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Farming Of Fish And Shellfish (AREA)
Abstract
The invention belongs to the technical field of biomedicine, and particularly relates to a zebra fish model with cerebral arterial thrombosis, a construction method and application. Injecting beta-amyloid solution with concentration of 0.01-0.05 mug/ml into the head of a normally developed adult zebra fish, wherein the injection amount is 1 muL/kg body weight, placing the zebra fish in physiological saline for 20-40min after injection, and then transferring the zebra fish into a feeding solution containing an inducer for feeding for 4-7 d; the inducer is a soluble lanthanum compound or a soluble lead compound. The invention successfully constructs the cerebral arterial thrombosis model of cerebellar obstruction and lays a foundation for the research and development of medicaments related to cognitive function nerve injury.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a zebra fish model with cerebral arterial thrombosis, a construction method and application.
Background
Cerebral apoplexy is a vascular disease directing to the brain to transport blood, and ischemic cerebral apoplexy is the blood supply disorder of local brain tissues as the name suggests, so that the function of cerebral nerves is damaged or lost. After the stroke occurs, serious consequences of influencing life activities and motor dysfunction can exist, so that the life of a human body cannot be self-managed, and the problem concerned by the medical field is solved. The stroke may occur in different areas of human brain, which may result in different physiological function impairment, and the types of drugs for clinically treating stroke at present are very limited, such as butylphthalide and the like, although having certain clinical efficacy, there are also side effects of hepatotoxicity and nephrotoxicity, and the drugs cannot be repaired for specific brain areas. Because stroke recovery is slow and disability rate is high, if more nerve repair drugs can be developed or nerve repair drugs for a certain specific brain area can be developed, the treatment efficiency of stroke is greatly improved.
In the prior art, when the curative effect of a medicament is researched, various animal models such as cerebral apoplexy models of mice, rats, rabbits, monkeys, zebra fishes and the like are usually constructed, and the animals are easy to feed and have wide sources and can be used for medicament toxicology and pathological research. Chinese patent CN108739556A discloses a construction method of a photochemically induced zebra fish model with cerebral arterial thrombosis, which comprises the steps of injecting rose bengal solution into the abdomen of zebra fish, transferring the zebra fish into the zebra fish containing tricaine aminobenzoate ethyl methyl sulfonate, finally exposing the head cap region under a light source for irradiation, inducing and recovering for 24 hours after the light source is irradiated, and finally obtaining the model with cerebral arterial thrombosis. Through further research, the method is mainly found to cause the phenomena of loose and infarct at the telencephalon part of the brain tissue. However, the brain tissue also includes parts such as cerebellum, which is mainly related to the motor behavior of zebrafish, and the cerebellum is mainly related to cognitive functions. The human cerebral apoplexy may occur in different parts of cerebellum, brain, etc. when the medicine related to cognitive function nerve damage needs to be developed, a cerebral apoplexy model of the cerebellum part needs to be constructed.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for constructing a zebra fish model with cerebral arterial thrombosis.
The invention aims to provide a construction method of a zebra fish cerebral arterial thrombosis model, which comprises the steps of injecting beta-amyloid liquid with the concentration of 0.01-0.05 mu g/ml into the head of a normally developed adult zebra fish, wherein the injection amount is 1 mu L/kg body weight, placing the zebra fish into physiological saline for 20-40min after injection, and then transferring the zebra fish into a feeding liquid containing an inducer for feeding for 4-7 d;
the inducer is a soluble lanthanum compound or a soluble lead compound.
Preferably, in the method for constructing the zebra fish model of ischemic stroke, the soluble lanthanum compound is lanthanum nitrate or lanthanum chloride.
Preferably, in the method for constructing the zebra fish model of ischemic stroke, the soluble lead compound is lead nitrate or lead acetate.
Preferably, in the method for constructing the zebra fish model of ischemic stroke, the concentration of the inducer in the feeding liquid is 0.5-2mg/100 ml.
Preferably, in the method for constructing the zebra fish model of ischemic stroke, the concentration of the inducer fed in the feeding liquid for 0-2 days is 0.5-1mg/100ml, and the concentration of the inducer after 2 days is 1.5-2mg/100 ml.
Preferably, in the method for constructing the zebra fish model with cerebral arterial thrombosis, the feeding liquid is tap water added with fish feed.
Preferably, in the construction method of the zebra fish model for cerebral arterial thrombosis, the adult zebra fish is an AB or TU strain wild type adult zebra fish.
Preferably, the temperature of the normal saline and the temperature of the feeding liquid are the same and are both 22-28 ℃ in the construction method of the zebra fish model for cerebral arterial thrombosis.
The invention also provides a zebra fish cerebral ischemic stroke model constructed by any one of the methods.
The invention also provides application of the inducer in constructing an animal ischemic stroke model.
Compared with the prior art, the invention has the following beneficial effects:
in the prior art, beta-amyloid liquid is commonly used for constructing animal Alzheimer disease models, and the influence of the beta-amyloid liquid on the pathological changes of brain hippocampal regions is concerned. The invention firstly adopts the combined action of two factors of beta-amyloid liquid and an inducer (a soluble lanthanum compound or a soluble lead compound) to construct the zebra fish model with cerebral arterial thrombosis. The pertinence of the brain injection beta-amyloid protein liquid is strong, and the induction agent is added into the feeding liquid to ensure that the zebra fish lives in a toxic environment to induce the generation of a model. Furthermore, the invention also adopts a mode of inducer concentration change, and the toxicity is gradually enhanced to generate a progressive induction model.
The result shows that the motor behavior ability of the zebra fish is reduced, the cerebellum tissue is obstructed, and the model is successfully constructed. The zebra fish has high gene similarity with human, and the cerebellar region is mainly related to cognitive function, so that after an ischemic stroke model of cerebellar obstruction is constructed, a foundation is laid for the research and development of medicines related to cognitive function nerve injury, and the zebra fish has good application prospect.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present invention to practice, the present invention will be further described with reference to the following specific examples.
The normal saline in the invention is 0.85g/100mL sodium chloride solution, the feeding liquid is tap water added with commercial fish feed, and the feeding liquid adopted in all experiments is the same. 15 adult zebra fish of 6 months age were taken for each experimental group, and the test results were averaged over 3 measurements.
Example 1
A construction method of a zebra fish cerebral arterial thrombosis model comprises the steps of selecting normally developed AB strain wild type adult zebra fish, injecting beta-amyloid liquid with the concentration of 0.01 mu g/ml into the head of the normally developed adult zebra fish, wherein the injection amount is 1 mu L/kg of body weight, immediately placing the adult zebra fish into physiological saline at 25 ℃ for 20min after injection, and then transferring the zebra fish into a feeding liquid containing lead nitrate at 25 ℃ for feeding for 4 d; the concentration of lead nitrate in the feed solution was 1mg/100 ml.
Example 2
A construction method of a zebra fish cerebral arterial thrombosis model comprises the steps of selecting normally developed AB strain wild type adult zebra fish, injecting beta-amyloid liquid with the concentration of 0.01 mu g/ml into the head of the normally developed adult zebra fish, wherein the injection amount is 1 mu L/kg of body weight, immediately placing the adult zebra fish into physiological saline at 25 ℃ for 40min after injection, and then transferring the zebra fish into a feeding liquid containing lanthanum nitrate at 25 ℃ for feeding for 4 d; the concentration of lanthanum nitrate in the feed solution was 1mg/100 ml.
Example 3
A construction method of zebra fish cerebral arterial thrombosis model comprises the steps of selecting normally developed AB strain wild type adult zebra fish, injecting beta-amyloid liquid with the concentration of 0.01 mu g/ml into the head of the normally developed adult zebra fish, wherein the injection amount is 1 mu L/kg body weight, immediately placing the adult zebra fish into physiological saline at 25 ℃ for 20min after injection, and then transferring the zebra fish into a feeding liquid containing lead acetate at 25 ℃ for feeding for 5 d; the concentration of lead acetate in the feed solution was 2mg/100 ml.
Example 4
A construction method of a zebra fish cerebral arterial thrombosis model comprises the steps of selecting normally developed TU strain wild type adult zebra fish, injecting beta-amyloid solution with the concentration of 0.05 mu g/ml into the head of the normally developed adult zebra fish, wherein the injection amount is 1 mu L/kg of body weight, immediately placing the adult zebra fish into physiological saline with the temperature of 22 ℃ for 20min after injection, and then transferring the zebra fish into a feeding solution with the temperature of 22 ℃ containing lanthanum chloride for feeding for 6 d; the concentration of lanthanum chloride in the feed solution was 0.5mg/100 ml.
Example 5
A construction method of a zebra fish cerebral arterial thrombosis model comprises the steps of selecting normally developed AB strain wild type adult zebra fish, injecting beta-amyloid liquid with the concentration of 0.01 mu g/ml into the head of the normally developed adult zebra fish, wherein the injection amount is 1 mu L/kg of body weight, immediately placing the adult zebra fish into physiological saline at 25 ℃ for 20min after injection, and then transferring the zebra fish into a feeding liquid containing lead nitrate at 25 ℃ for feeding for 4 d; the concentration of lead nitrate in the feeding liquid for feeding for 0-2 days is 0.5mg/100ml, and the concentration of lead nitrate for feeding for 3-4 days is 1.8mg/100 ml.
The models constructed in examples 1-5 were evaluated to include in particular the following experiments:
control group: the procedure of example 1 was followed except that the normal developed AB strain wild type adult zebrafish was not injected with a beta-amyloid solution and was not treated with lead nitrate.
Treatment group 1: the normal developing AB strain wild type adult zebrafish were treated in the same manner as in example 1 without injection of the beta-amyloid solution. Aims to investigate the influence of the inducer lead nitrate on the zebra fish ischemic stroke model.
Treatment group 2: the normal developing AB strain wild type adult zebrafish were not treated with lead nitrate and the procedure was as in example 1. Aims to investigate the influence of the beta-amyloid liquid on the zebra fish ischemic stroke model.
Model group 1: the procedure is as in example 1.
Model group 2: the procedure is as in example 2. The aim was to demonstrate that the metallic element lanthanum can also be used for modeling.
Model group 3: the procedure is as in example 5. The purpose is to investigate the modeling effect of adopting different concentrations of the inducer in different periods.
Experiment-spontaneous movement experiment
Refer to Shihui Du, Zhangjun, Penghui, etc. Neurotoxic effects of chlorpromazine during development of zebrafish embryos and early juvenile fish [ J ]. 150-156 in the journal of Chinese medicine, 2011, 21 (Z1). "spontaneous motility test of young fish" in "and the 3min moving distance and moving speed of adult zebra fish were recorded. The results in table 1 show that compared with the control group, the movement speed and the movement distance in 3min of the zebra fish in other groups are reduced, the reduction range of the model group 1-3 is the largest, and the difference with the control group is significant (P is less than 0.05).
TABLE 1 spontaneous movement test results
Experiment two-panic escape response test
Refer to Shihui Du, Zhangjun, Penghui, etc. Neurotoxic effects of chlorpromazine during development of zebrafish embryos and early juvenile fish [ J ]. 150-156 in the journal of Chinese medicine, 2011, 21 (Z1). The ' middle ' juvenile fish evades the reflex test in clear sky under the stimulation of strong light ', and the motion acceleration of the adult zebra fish under the condition of alternating dark light and light is recorded. The results in Table 2 show that the decrease of the acceleration of the model groups 1-3 is the largest compared with the control group, and the difference is significant (P < 0.05).
TABLE 2 results of the flight avoidance response test
| Grouping | Acceleration of motion (mm/s)2) |
| Control group | 0.24±0.03 |
| Treatment group 1 | 0.18±0.03 |
| Treatment group 2 | 0.17±0.01 |
| Model set 1 | 0.11±0.02 |
| Model group 2 | 0.09±0.04 |
| Model group 3 | 0.08±0.01 |
Experimental three T maze test
Refer to "Zhu Xiao Qiao, Tang Tian le, Peng Xiang, et al. Bisphenol AF exposure reduced the learning and memory ability of zebrafish and affected nervous system related gene expression [ J ]. The biological toxicology report, 2017, 12(001): 119-126. "adult zebrafish are trained for 15 days before treatment of each group, and the time to reach the right arm (i.e., latency into the designated area) of the zebrafish is determined. The results are shown in table 3 and show that the latency time for model groups 1-3 to enter the defined zone is significantly longer (P <0.05) compared to the control group.
TABLE 3T maze test results
| Grouping | Latency(s) |
| Control group | 25±1 |
| Treatment group 1 | 35±2 |
| Treatment group 2 | 32±3 |
| Model set 1 | 39±4 |
| Model group 2 | 40±2 |
| Model group 3 | 46±3 |
Experiment four-brain tissue staining experiment
After the observation of the behavioral experiment is finished, selecting three fish at random for sacrifice, taking the whole brain tissue, dyeing by using 2% TTC, transferring the dyed whole brain tissue to 4% paraformaldehyde for fixation overnight, and then photographing for imaging, wherein the tissue of an ischemic cerebral infarction area is white, and the cerebral infarction area of each group is observed, and the contrast group has no infarction phenomenon; the other groups showed obstruction of different degrees, nerve cells disappeared of different degrees, and cell tissues were loosened of different degrees. In the constructed zebra fish model with cerebral arterial thrombosis, infarction changes mainly occur in a small brain area, and infarction phenomena at other parts are not obvious. The percentage of infarct area in cerebellar tissue was calculated according to the following formula (average of 3 calculations):
the percentage of the infarcted area of the brain tissue (white stained area of cerebellum tissue/total surface area of cerebellum tissue) is 100%
The results showed that the infarct size percentages of cerebellum tissues of the treatment groups 1-2 and the model groups 1-3 were 23.6%, 21.7%, 31.5%, 32.4%, and 36.7%, respectively.
It should be noted that, when the present invention relates to a numerical range, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A construction method of a zebra fish cerebral arterial thrombosis model is characterized in that a normal adult zebra fish head is injected with beta-amyloid liquid with the concentration of 0.01-0.05 mu g/ml, the injection amount is 1 mu L/kg body weight, the zebra fish is placed in physiological saline for 20-40min after being injected, and then the zebra fish is transferred to a feeding liquid containing an inducer to be fed for 4-7 d;
the inducer is a soluble lanthanum compound or a soluble lead compound.
2. The method for constructing the zebrafish ischemic stroke model as recited in claim 1, wherein the soluble lanthanum compound is lanthanum nitrate or lanthanum chloride.
3. The method for constructing the zebrafish model of ischemic stroke according to claim 1, wherein the soluble lead compound is lead nitrate or lead acetate.
4. The method for constructing a zebrafish model of ischemic stroke according to claim 1, wherein the concentration of the inducer in the feeding solution is 0.5-2mg/100 ml.
5. The method for constructing the zebrafish model of ischemic stroke according to claim 4, wherein the concentration of the inducer fed in the feeding liquid for 0-2 days is 0.5-1mg/100ml, and the concentration of the inducer after 2 days is 1.5-2mg/100 ml.
6. The method for constructing the zebrafish model of ischemic stroke according to claim 1, wherein the feeding liquid is tap water added with fish feed.
7. The method for constructing the zebrafish model for cerebral arterial thrombosis according to claim 1, wherein the adult zebrafish is a wild-type adult zebrafish of AB or TU strain.
8. The method for constructing the zebrafish model of ischemic stroke according to claim 1, wherein the physiological saline and the feeding solution have the same temperature, and are both 22-28 ℃.
9. A zebrafish model of ischemic stroke constructed according to the method of any one of claims 1-8.
10. Use of the inducer of claim 1 for constructing an animal model of ischemic stroke.
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