Construction method and application of cerebral small vascular disease animal model

Abstract

The invention discloses a construction method and application of a cerebral small vascular disease animal model, and relates to the technical field of cerebral small vascular diseases. Injecting the microbubble mixed liquid into the animal to be modeled, and then performing ultrasonic action on the upper part of the brain of the animal to be modeled by using an ultrasonic probe; during ultrasonic action, the center frequency of the ultrasonic probe is 3-5 MHz, the mechanical index is 1.2-1.5, and the ultrasonic irradiation time is 900-1200 s. The animal constructed by the method can simulate the pathology and image features of cerebral small vascular diseases, can simulate the features of micro-bleeding, micro-infarction and the like, shows the features of white matter high signal, iron-containing hemeroflavin deposition, encephalatrophy and the like in imaging examination, and is obviously superior to a laser-induced micro-bleeding model. Compared with a traditional method, the animal model building method is simple, multi-generation breeding does not need to be conducted on the animal to be modeled, and the modeling method has the technical advantages of being short in time consumption, free of craniotomy and additional injury and economical and practical.

Description

Technical Field

[0001] The present invention relates to the technical field of cerebral small vessel disease, and in particular to a method for constructing a cerebral small vessel disease animal model and application thereof. Background Art

[0002] The animal disease models currently used in the study of cerebral small vessel disease include: transgenic mice, MCAO models, stereotactic injection of drugs, laser damage-induced microbleeding, etc.

[0003] Among them, the pathological feature simulated by the laser injury model is microbleeding, and it cannot simulate microinfarction, white matter high signal, hemosiderin deposition, brain atrophy, etc. The ultrasound blood-brain barrier opening technology UTMD can safely, non-destructively, and reversibly open the blood-brain barrier for drug delivery across the blood-brain barrier, but it cannot simulate the disease characteristics of cerebral small vessel disease (CSVD).

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing an animal model of cerebral small vessel disease and its application to provide a method for constructing an animal model of cerebral small vessel disease. The animal model of cerebral small vessel disease constructed by the present invention can simulate characteristics such as microbleeding and microinfarction, and in imaging examinations, it shows characteristics such as high signals in white matter, hemosiderin deposition, and brain atrophy, which is significantly better than the laser-induced microbleeding model. Compared with the ultrasound opening blood-brain barrier technology, the method for constructing an animal model of cerebral small vessel disease provided by the present invention focuses on destroying the blood-brain barrier and inducing characteristics such as cerebral microbleeding and microinfarction.

[0006] The present invention is achieved in that:

[0007] In the first aspect, the present invention provides a method for constructing an animal model of cerebral small vessel disease, which comprises the following steps: intravenously injecting a microbubble mixture into the animal to be modeled, and then using an ultrasound probe to perform ultrasound on the top of the brain of the animal to be modeled; during the ultrasound action, the center frequency of the ultrasound probe is 3-5 MHz, the ultrasound irradiation time is 900-1200 s, and the mechanical index is 1.2-1.5.

[0008] In a preferred embodiment of the present invention, the final injection concentration of the microbubbles in the microbubble mixture is 1*10 8 -10*10 8 / ml, the injection dose of microbubble mixture is 4ml / kg±0.5ml / kg.

[0009] In a preferred embodiment of the present invention, the average particle size of the microbubbles is 1-10 μm;

[0010] In a preferred embodiment of the present invention, the average particle size of the microbubbles is 1-5 μm.

[0011] In a preferred embodiment of the present invention, the microbubbles include gas microbubbles and a stabilizer; the gas of the gas microbubbles is selected from at least one of sulfur hexafluoride, perfluoropropane and perfluorobutane;

[0012] The stabilizer includes at least one of the following substances: propylene glycol and glycerol.

[0013] In a preferred embodiment of the present invention, the oil phase comprises partially or completely halogenated hydrocarbons or mixtures thereof;

[0014] In a preferred embodiment of the present invention, the animal to be modeled is selected from non-human mammals.

[0015] In a preferred embodiment of the present invention, the non-human mammal is selected from the group consisting of mice, rats, horses, sheep, cattle, deer, pigs, monkeys, dogs and apes.

[0016] In a second aspect, the present invention also provides the use of the cerebral small vessel disease animal model constructed by the above-mentioned method for constructing a cerebral small vessel disease animal model in screening drugs for preventing and / or treating cerebral small vessel disease.

[0017] The present invention has the following beneficial effects:

[0018] The present invention provides a method for constructing an animal model of cerebral small vessel disease. The animal constructed by the method can simulate the pathological and imaging characteristics of cerebral small vessel disease, can simulate characteristics such as microbleeding and microinfarction, and in imaging examinations, it shows characteristics such as high signals in the white matter, hemosiderin deposition, and brain atrophy, which is significantly better than the laser-induced microbleeding model. Compared with the ultrasound opening blood-brain barrier technology, the method for constructing an animal model of cerebral small vessel disease provided by the present invention focuses on destroying the blood-brain barrier, inducing characteristics such as cerebral microbleeding and microinfarction, and has a higher ultrasound intensity.

[0019] Compared with transgenic mice, MCAO, stereotactic injection, laser and other methods, the animal model construction method provided by the present invention is simple and does not require multiple generations of breeding of the animals to be modeled. The modeling method has the technical advantages of being short in time, requiring no craniotomy, requiring no additional damage, and being economical.

[0020] In addition, the cerebral small vessel disease model established by the present invention can control the damaged area of ​​the animal to be modeled, does not require complex surgical operations, and does not involve the safety issues of the in vivo application of rare earth-doped NaGdF4, BaGdF5 and other nanomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 The results of MRI examination and ULM after modeling (AC are the results of MRI examination after modeling, A shows microbleeding (low signal) in magnetic susceptibility imaging (SWI sequence), B shows subcortical infarction with high signal in diffusion weighted imaging (DWI sequence), C shows widening of perivascular space (high signal), D shows ULM with decreased vascular density in the modeling area, E shows ULM with decreased blood flow velocity in the modeling area, F shows a gross picture of brain tissue, GH shows Golgi staining with decreased number of neuronal connections);

[0023] Figure 2 HE pathological sections and electron microscopy results (A is the HE pathological section result, B is the result of the enlarged gap between endothelial cells and surrounding tissues, and CD are the electron microscopy results);

[0024] Figure 3 The figures are the results of Evens Blue (EB) leakage experiments conducted on the animal models of Example 1 and Comparative Examples 1-2, respectively. DETAILED DESCRIPTION

[0025] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0026] The term "microbubble" or "conventional contrast microbubble" is used herein to describe microbubbles with a diameter in the range of 0.2 to 10 microns, usually with an average diameter between 1 and 10 μm. Conventional contrast microbubbles include commercially available preparations such as Sonazoid (GE Healthcare), Optison (GE Healthcare), Sonovue (Bracco Spa.), Definity (Lantheus Medical Imagin), and preclinical preparations such as Micromarker (VisualSonics Inc.), Polyson L (Miltenyi Biotec GmbH) and (IMCOR Pharmaceuticals Inc., San Diego, CA, USA).

[0027] "Sonotherapy," also known as "US radiation," is a term used to describe exposure to ultrasound waves.

[0028] In the first aspect, the present invention provides a method for constructing an animal model of cerebral small vessel disease, which comprises the following steps: injecting a microbubble mixture into the animal to be modeled, and then using an ultrasound probe to perform ultrasound on the top of the brain of the animal to be modeled; during the ultrasound action, the center frequency of the ultrasound probe is 3-5 MHz, the ultrasound irradiation time is 900-1200 s, and the mechanical index is 1.2-1.5.

[0029] Different from conventional ultrasound microbubble therapy, the present invention sets the ultrasound probe with a higher central frequency, thereby destroying the blood-brain barrier with higher-intensity ultrasound, inducing cerebral microbleeding, microinfarction, etc., and simulating the disease characteristics of CSVD.

[0030] The animals constructed by the present invention can simulate the pathological and imaging characteristics of cerebral small vessel disease, can simulate characteristics such as microbleeding and microinfarction, and show characteristics such as high signal in white matter, hemosiderin deposition, and brain atrophy in imaging examination, which is significantly better than the laser-induced microbleeding model. Compared with the ultrasound opening blood-brain barrier technology, the method for constructing the animal model of cerebral small vessel disease provided by the present invention focuses on destroying the blood-brain barrier, inducing cerebral microbleeding, microinfarction and other characteristics, and the ultrasound intensity is higher.

[0031] Compared with transgenic mice, MCAO, stereotactic injection, laser and other methods, the animal model construction method provided by the present invention is simple and does not require multiple generations of breeding of the animals to be modeled. The modeling method has the technical advantages of being short in time, requiring no craniotomy, requiring no additional damage, and being economical.

[0032] In addition, the cerebral small vessel disease model established by the present invention can control the damaged area of ​​the animal to be modeled, does not require complex surgical operations, and does not involve the safety issues of the in vivo application of rare earth-doped NaGdF4, BaGdF5 and other nanomaterials.

[0033] The above-mentioned ultrasound equipment includes but is not limited to low-frequency focused ultrasound equipment.

[0034] The mechanical index is, for example, 1.2, 1.3, 1.4 or 1.5. The mechanical index can be adjusted according to the type of animal model.

[0035] The center frequency of the ultrasound probe is 3 MHz, 4 MHz or 5 MHz, and the ultrasound irradiation time is 900 s, 950 s, 1000 s, 1100 s or 1200 s.

[0036] The pulse repetition frequency of the ultrasound probe is 20-200 Hz, for example 20 Hz, 30 Hz, 40 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz, 100 Hz, 110 Hz, 120 Hz, 130 Hz, 140 Hz, 150 Hz, 160 Hz, 170 Hz or 180 Hz.

[0037] In a preferred embodiment of the present invention, the final injection concentration of the microbubbles in the microbubble mixture is 1*10 8 -10*10 8 The injection dose of the microbubble mixture is 3ml / kg±6ml / kg. Under the above injection dose, the ultrasound can play a good effect of opening the blood-brain barrier.

[0038] The final injection concentration of microbubbles is 1*10 8 , 2*10 8 , 3*10 8 , 4*10 8 , 5*10 8 , 6*10 8 ,8*10 8 or 10*10 8 The injection dose of the microbubble mixture is 3ml / kg, 4ml / kg, 5ml / kg or 6ml / kg.

[0039] In a preferred embodiment of the present invention, the average particle size of the microbubbles is 1-10 μm, for example, the average particle size of the microbubbles is 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm or 10 μm.

[0040] In a preferred embodiment of the present invention, the average particle size of the microbubbles is 1-5 μm.

[0041] In a preferred embodiment of the present invention, the microbubbles include gas microbubbles and a stabilizer; the gas of the gas microbubbles is selected from sulfur hexafluoride, perfluoropropane and perfluorobutane. The gas of the gas microbubbles can oscillate in volume under the action of ultrasonic sound waves, and be excited under the action of ultrasound to cause a biomechanical effect, thereby opening the blood-brain barrier.

[0042] Any biocompatible gas may be present in the microbubble, and the term "gas" as used herein includes any substance (including mixtures) that is at least partially, for example substantially or completely, in a gaseous state (including vapor) at a normal human body temperature of 37° C. Thus, the gas may, for example, include: air; nitrogen; oxygen; carbon dioxide; hydrogen; an inert gas such as helium, argon, xenon or krypton; a sulfur fluoride such as sulfur hexafluoride, disulfur decafluoride or trifluoromethylsulfur pentafluoride; selenium hexafluoride; an optionally halogenated silane such as methylsilane or dimethylsilane; a low molecular weight hydrocarbon (e.g. containing up to 7 carbon atoms), such as an alkane such as methane, ethane, propane, butane or pentane, a cycloalkane such as cyclopropane, cyclobutane or cyclopentane, an olefin such as ethylene, propylene, propadiene or butene, or an alkyne such as acetylene or propyne; an ether such as dimethyl ether; a ketone; an ester; a halogenated low molecular weight hydrocarbon (e.g. containing up to 7 carbon atoms); or a mixture of any of the above. Advantageously, the gas is a halogenated gas, such as a perfluorinated gas. Advantageously, at least some of the halogen atoms in the halogenated gas are fluorine atoms; thus, the biocompatible halogenated hydrocarbon gas can be, for example, selected from bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, vinyl fluoride, ethyl fluoride, 1,1-difluoroethane and perfluorocarbons. Representative perfluorocarbons include: perfluoroalkanes, such as perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane (e.g., perfluoro-n-butane, optionally mixed with other isomers such as perfluoroisobutane); perfluoroolefins, such as perfluoropropylene, perfluorobutene (e.g., perfluorobut-2-ene), perfluorobutadiene, perfluoropentene (e.g., perfluoropent-1-ene) or perfluoro-4-methylpent-2-ene; perfluoroalkynyl groups such as perfluorobut-2-yne; and perfluorocycloalkanes, such as perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodifluoromethylcyclobutane, perfluorotrimethylcyclobutane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentane, perfluorocyclohexane, perfluoromethylcyclohexane or perfluorocycloheptane. Other halogenated gases include methyl chloride, fluorinated (e.g., perfluorinated) ketones such as perfluoroacetone and fluorinated (e.g., perfluorinated) ethers such as perfluorodiethyl ether.

[0043] It is particularly advantageous to use perfluorinated gases, such as sulfur hexafluoride and perfluorocarbons (such as perfluoropropane, perfluorobutane, perfluoropentane and perfluorohexane), because microbubbles containing such gases have a recognized high stability in the bloodstream. Other gases having physicochemical characteristics that allow them to form highly stable microbubbles in the bloodstream are also useful. Preferably, the gas of the gas microbubbles comprises sulfur hexafluoride, perfluoropropane, perfluorobutane, nitrogen, air or any mixture thereof, and preferably comprises sulfur hexafluoride.

[0044] The stabilizer includes at least one of the following substances: propylene glycol and glycerol. The stabilizer is used to maintain the stability of gas microbubbles.

[0045] In a preferred embodiment of the present invention, the animal to be modeled is selected from non-human mammals.

[0046] In a preferred embodiment of the present invention, the non-human mammal is selected from the group consisting of mice, rats, horses, sheep, cattle, deer, pigs, monkeys, dogs and apes.

[0047] In a second aspect, the present invention also provides the use of the cerebral small vessel disease animal model constructed by the above-mentioned method for constructing a cerebral small vessel disease animal model in screening drugs for preventing and / or treating cerebral small vessel disease.

[0048] The screening application of the drug includes at least one of the following application methods:

[0049] i: Administer the test drug to the animal model of cerebral small vessel disease constructed above, and examine the pathological characteristics and imaging manifestations of the animals after drug administration. Compared with before drug administration, if the animal examination results after drug administration show at least one of the following characteristics: elimination of microbleeding foci, reduction or elimination of subcortical infarction area, reduction of perivascular space, increase in vascular density in the modeling area, increase in blood flow velocity in the modeling area, increase in the number of neuronal connections, repair of microvascular endothelial cell damage, and reduction in NSS score of neurological function improvement, it indicates that the test drug has a good effect in treating cerebral small vessel disease;

[0050] ii: Administer the test drug to the above-constructed cerebral small vessel disease animal model, and examine the pathological characteristics and imaging manifestations of the animals after drug administration. Compared with before drug administration, if the animal examination results after drug administration show at least one of the following characteristics: enlarged microbleeding foci, enlarged subcortical infarct area, enlarged perivascular space, reduced vascular density in the modeling area, reduced blood flow velocity in the modeling area, reduced number of neuronal connections, aggravated microvascular endothelial cell damage, and increased neurological function improvement NSS score, it indicates that the test drug has no effect on treating cerebral small vessel disease.

[0051] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0052] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0053] Example 1

[0054] This embodiment provides a method for constructing an animal model of cerebral small vessel disease, and the model construction includes the following steps:

[0055] (1) Select 200-250 g SPF Wistar rats, remove the hair on the head after anesthesia, and fix them on a brain stereotaxic instrument;

[0056] (2) The ultrasonic equipment was selected and the ultrasonic blasting parameters were set. The center frequency of the ultrasonic probe was 3 MHz, the ultrasonic irradiation time was 900 s, and the mechanical index was 1.2. The pulse repetition frequency of the ultrasonic probe was 100 Hz. The ultrasonic probe was fixed above the rat's brain and ultrasonic microbubbles (Sonovue) were continuously injected through the tail vein. The average particle size of the microbubbles was 2-3 μm, and the injection dose of the microbubbles was 1*10 8 / ml, the injection dose of microbubble mixture is 5ml / kg, and ultrasonic blasting starts at the same time;

[0057] (3) After the ultrasound, the mice were returned to the cage and waited to wake up. After the model was established, MRI and ULM examinations were performed on them, and samples were taken for histopathological sections and tissue electron microscopy.

[0058] Comparative Example 1

[0059] Compared with Example 1, the only difference is that the mechanical index is different. This comparative example adopts a mechanical index of 1.0 for modeling, and other conditions and methods are the same as Example 1.

[0060] Comparative Example 2

[0061] Compared with Example 1, the only difference is that the ultrasonic irradiation time is different. The ultrasonic irradiation time of this comparative example is 600s. The modeling is carried out, and the other conditions and methods are the same as in Example 1.

[0062] Experimental Example 1

[0063] The rats were subjected to MRI and ULM examinations after modeling. The results of MRI examinations were referred to Figure 1As shown in Figure 5, the magnetic susceptibility imaging (SWI sequence) in Figure A shows microbleeding (low signal), the diffusion weighted imaging (DWI sequence) in Figure B shows high signal subcortical infarction, and the T2-weighted imaging of nuclear magnetic resonance in Figure C shows widening of the perivascular space (high signal). ULM shows that the vascular density in the modeling area is reduced ( Figure 1 D in the figure), ULM showed that the blood flow velocity in the modeling area decreased ( Figure 1 Figure E in the figure), general diagram of brain tissue ( Figure 1 Figure D), Golgi staining showed a decrease in the number of neuronal connections ( Figure 1 GH diagram in Figure 3).

[0064] Experimental Example 2

[0065] The rats were stained with HE after modeling, and the tissues were sliced ​​and electron micrographed after pathological section.

[0066] Results reference Figure 2 As shown, Figure 2 HE pathological section in A shows microinfarction, while Figure 2 The results in B show that the gap between endothelial cells and surrounding tissues is enlarged; Figure 2 The CD electron microscopic results in the figure show damage to the microvascular endothelial cells in the brain.

[0067] Experimental Example 3

[0068] The rats were evaluated for neurological function using the modified NSS score after model establishment.

[0069] Table 1 shows the modified NSS scores of neurological function in five rats after modeling

[0070]

[0071] The results in Table 1 show that the rats had mild neurological impairment after modeling, which is consistent with the expected modeling results.

[0072] Experimental Example 4

[0073] The animal models of Example 1 and Comparative Examples 1-2 were respectively subjected to Evens Blue (EB) leakage test to detect whether the BBB was damaged.

[0074] After the model was established, 2% EB (2 ml / kg) was injected through the tail vein. After 12 hours, the mice were systemically perfused. The chest cavity was opened and 0.9% sodium chloride solution (containing heparin) was slowly injected from the apex of the heart. The right atrium was cut open until the fluid flowing out of the right atrium became clear. The perfusion was stopped and the brain was taken for photographs. The blue part represented BBB destruction.

[0075] Evans Blue CAS 314-13-6 is a non-membrane permeable dye. When the plasma membrane is damaged, the dye can enter the cytoplasm and nucleus, thereby dyeing them blue, which can be used to detect cell viability.

[0076] Results reference Figure 3 As shown, the results show that after completing transcardiac systemic blood perfusion, EB dye is deposited in the brain tissue of the animal model successfully constructed according to the method of Example 1 (ie, the optimal parameters).

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing an animal model of cerebral small vessel disease, characterized in that: The method comprises the following steps: injecting a microbubble mixture into an animal to be modeled, and then using an ultrasonic probe to perform ultrasonic action on the upper part of the skull of the animal to be modeled; during the ultrasonic action, the central frequency of the ultrasonic probe is 3-5 MHz, the ultrasonic irradiation time is 900-1200 s, and the mechanical index is 1.2-1.

5.

2. The method for constructing an animal model of cerebral small vessel disease according to claim 1, characterized in that: The pulse repetition frequency of the ultrasonic probe is 20-200 Hz.

3. The method for constructing an animal model of cerebral small vessel disease according to claim 1, characterized in that: The final injection concentration of the microbubbles in the microbubble mixture is 1*10 8 -10*10 8 The injection dose of the microbubble mixture is 3ml / kg-6ml / kg.

4. The method for constructing an animal model of cerebral small vessel disease according to claim 3, characterized in that: The average particle size of the microbubbles is 1-10 μm.

5. The method for constructing an animal model of cerebral small vessel disease according to claim 4, characterized in that: The average particle size of the microbubbles is 1-5 μm.

6. The method for constructing an animal model of cerebral small vessel disease according to claim 4, characterized in that: The microbubbles include gas microbubbles and a stabilizer; the gas of the gas microbubbles is selected from at least one of sulfur hexafluoride, perfluoropropane and perfluorobutane.

7. The method for constructing an animal model of cerebral small vessel disease according to claim 6, characterized in that: The stabilizer includes the following substances: propylene glycol and glycerol.

8. The method for constructing an animal model of cerebral small vessel disease according to claim 1, characterized in that: The animal to be modeled is selected from non-human mammals.

9. The method for constructing an animal model of cerebral small vessel disease according to claim 8, characterized in that: The non-human mammal is selected from the group consisting of mice, rats, horses, sheep, cows, deer, pigs, monkeys, dogs, or apes.

10. Use of the cerebral small vessel disease animal model constructed by the method for constructing a cerebral small vessel disease animal model according to any one of claims 1 to 9 in screening drugs for preventing and / or treating cerebral small vessel disease.

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