阅读说明：本技术 超声联合微泡建立大鼠脑出血模型的方法 (Method for establishing rat cerebral hemorrhage model by combining ultrasound and microbubbles ) 是由 范国峰 于 2020-06-02 设计创作，主要内容包括：本发明涉及超声联合微泡建立大鼠脑出血模型的方法,属于生物模型制备技术领域。该方法执行如下,1)SPF级雄性SD大鼠一只,体质量240-270g；2)准备工作；3)在辅助工具帮助下,从大鼠尾静脉注入微泡造影剂,注入量依照所述大鼠的体质量决定；4)超声干预；5)对大鼠行头CT平扫；6)以白炽灯照射大鼠至少2h,保持肛温在37±0.5℃；7)对大鼠神经功能评分,并饲养观察待用。本发明通过超声靶向作用于造影剂,借助微泡瞬间空化效应,对脑内目标区局部血管产生破坏作用,使得大鼠出现脑实质内出血现象,符合自发性脑出血的研究要求,具有无创、简便、易推广的优点,是合适的可再现的生物造模方法。同时,还引入了辅助设备,使得操作更为简便可行。(The invention relates to a method for establishing a rat cerebral hemorrhage model by combining ultrasound and microbubbles, belonging to the technical field of biological model preparation. The method is carried out as follows, 1) one SPF male SD rat with the body mass of 240-270 g; 2) preparing; 3) injecting a microbubble contrast agent from the tail vein of a rat with the help of an auxiliary tool, wherein the injection amount is determined according to the physique of the rat; 4) performing ultrasonic intervention; 5) performing horizontal scanning on the rat horizontal head CT; 6) irradiating the rat with incandescent lamp for at least 2h, and keeping the anal temperature at 37 + -0.5 deg.C; 7) rats were scored for neurological function and were fed for observation. The invention acts on the contrast agent through ultrasonic targeting, and generates destructive action on local blood vessels of a target area in the brain by virtue of the microbubble transient cavitation effect, so that the phenomenon of intracerebral parenchyma internal bleeding of a rat is generated, the research requirement of spontaneous cerebral hemorrhage is met, and the method has the advantages of no wound, simplicity, convenience and easy popularization, and is a proper reproducible biological modeling method. Meanwhile, auxiliary equipment is introduced, so that the operation is simpler, more convenient and more feasible.)

1. The method for establishing the rat cerebral hemorrhage model by combining ultrasound and microbubbles is characterized by comprising the following steps:

1) one SPF male SD rat with the body mass of 240-270 g;

2) preparing;

fixing the rat on a brain stereotaxic apparatus, and performing anesthesia; irradiating the rat with a 60w incandescent lamp at a vertical distance of 50cm, and keeping the anal temperature of the rat at 37 +/-0.5 ℃ all the time in the molding process; depilating on the cranial vertex of a rat;

3) injecting a microbubble contrast agent from the tail vein of a rat with the help of an auxiliary tool, wherein the injection amount is determined according to the physique of the rat;

4) ultrasonic intervention, starting ultrasonic equipment, tightly combining an ultrasonic probe with the skull of a rat by using an ultrasonic coupling agent, and implementing the ultrasonic intervention to enable the microbubble contrast agent to reach an instantaneous cavitation threshold;

5) performing parallel scanning on the rat through a CT (computed tomography) to determine the cerebral hemorrhage part and the amount of hemorrhage;

6) irradiating the rat with 60w of incandescent lamp at a vertical distance of 50cm for at least 2h, and keeping the anal temperature of the rat at 37 +/-0.5 ℃;

7) and (4) scoring the nerve function of the rat, wherein the rat meeting the scoring standard is the rat which is successfully modeled, and feeding and observing the rat for later use.

2. The method for establishing a rat cerebral hemorrhage model by combining ultrasound and microbubbles according to claim 1, wherein the model comprises the following steps: in step 3), the injection amount of the microbubble contrast agent is 0.1 mL/kg.

3. The method for establishing a rat cerebral hemorrhage model by combining ultrasound and microbubbles according to claim 1, wherein the model comprises the following steps: the microbubble contrast agent is sulfur hexafluoride microbubble.

4. The method for establishing a rat cerebral hemorrhage model by combining ultrasound and microbubbles according to claim 3, wherein the model comprises the following steps: in the step 4), the frequency of ultrasonic intervention is 2Mhz, the energy is 5-10W/cm2, and the action time is 120 s.

5. The method for establishing a rat cerebral hemorrhage model by combining ultrasound and microbubbles according to claim 1, wherein the model comprises the following steps: in the step 2), 10 percent of chloral hydrate by mass is used for carrying out intraperitoneal injection on anesthetized rats, and the concentration is 300-400 mg/kg.

6. The method for establishing a rat cerebral hemorrhage model by combining ultrasound and microbubbles according to claim 1, wherein the model comprises the following steps: step 7) the method for scoring the rat nerve function adopts one or more of a Longa scoring method, a Berderson scoring posture reflex test, a Climbing experiment, a screen test, a limb placement test, an open field test method, MNSS, a Rotation rod test, a Rotation test, a limb Symmetry test scoring method, a rotarod test, an adhesive-removable sensory test, a Spontaneous Activity, a Symmetry in the Movement of limbs, a Forepaw outletting, a Climbing, a Body propriority and a reactive tissue Touch.

7. The method for establishing a rat cerebral hemorrhage model by combining ultrasound and microbubbles according to claim 6, wherein the model comprises the following steps: the nerve function scoring process includes scoring and recording the tested rats in a single blind method by at least 3 persons participating in the test, and then carrying out statistical calculation on scores after averaging scoring results of at least 3 groups.

Technical Field

The invention relates to a method for establishing a rat cerebral hemorrhage model by combining ultrasound and microbubbles, belonging to the technical field of biological model preparation.

Background

Cerebral Hemorrhage (ICH) is a common cerebrovascular disease in neurology department, has the characteristics of high morbidity, disability rate, recurrence rate and lethality rate, and the morbidity rises year by year, and the onset age tends to be younger. Clinical treatment of cerebral hemorrhage mainly includes dehydration for lowering intracranial pressure, surgical decompression and rehabilitation exercise, but most patients still have different degrees of neurological dysfunction, which causes serious medical and economic burden to families and society and affects the quality of life of patients and families. Therefore, the establishment of a cerebral hemorrhage animal model with high repeatability and good stability, which is highly similar to that of human beings, can effectively and deeply research the aspects of pathogenesis, pathophysiological change, treatment and the like of cerebral hemorrhage.

The currently known cerebral hemorrhage modeling methods include a collagenase induced cerebral hemorrhage model, a cerebral hemorrhage model formed by injecting autologous blood, a spontaneous cerebral hemorrhage model, and a microballoon sac inflated cerebral hemorrhage model. The collagenase induced cerebral hemorrhage model is a diffuse type blood seepage model, the formation time of hematoma is long, acute space occupying effect is not available, collagenase has cytotoxicity effect, inflammatory reaction around hematoma is heavy, blood brain barrier can be directly destroyed, and brain tissue can be injured. The intracerebral hematoma formed by the cerebral hemorrhage model injected by autoblood is not caused by intracerebral vascular rupture, the hematoma amount has no direct relation with the hematoma form, the blood is easy to reflux and overflow when being injected, the blood is solidified in a needle too slowly, the pressure of the hematoma in the brain is difficult to fix, the hematoma is easy to expand and crack, and the brain tissue flows into the ventricle to form the intracerebral hematoma, so the formed hematoma has poor repeatability of form and size. The animal model of spontaneous cerebral hemorrhage has the disadvantages that the animal model is easy to have the conditions of seed breaking and variety, has limitations in heredity, is difficult to feed, has higher manufacturing cost and has less available sources. The micro-balloon inflation cerebral hemorrhage model can only simulate the space occupying effect of intracerebral hematoma after cerebral hemorrhage, and neglects the influence of other reasons on brain tissues after cerebral hemorrhage.

In addition, in order to avoid the disadvantages of the single method, there are also composite methods, such as collagenase induction + autologous blood injection induced cerebral hemorrhage model, collagenase + heparin injection induced cerebral hemorrhage model, autologous blood + heparin injection made cerebral hemorrhage model, genetic gene change mode + autologous blood injection method combination. Although the problems in the original method are solved to a certain extent, the defects existing in the method are not completely solved, and only the scheme of '1 +1= 2' is adopted, and the method does not exceed the scheme of the predecessor at all. Certainly, an interventional puncture type molding scheme is provided, but in actual operation, the success rate of molding is required to be ensured according to the proficiency of experimenters in the molding process, a large-scale application foundation is lacked, and the reappearance difficulty is high.

Spontaneous cerebral hemorrhage is intracerebral vascular rupture hemorrhage, the method cannot simulate the pathological process, and the problems of high technical requirement, wound, incapability of standardization and the like in the modeling process exist at the same time. Therefore, a method is needed to fundamentally solve the problems encountered in the current scheme of cerebral hemorrhage modeling.

Disclosure of Invention

In order to solve the technical problems, the technical scheme aims to solve the defects of the prior art and provide a method for establishing a rat cerebral hemorrhage model by combining ultrasound and microbubbles.

The method for establishing the rat cerebral hemorrhage model by combining the ultrasound and the microbubbles according to the technical scheme executes the following steps:

1) one SPF male SD rat with the body mass of 240-270 g;

2) preparing;

fixing the rat on a brain stereotaxic apparatus, and performing anesthesia; irradiating the rat with a 60w incandescent lamp at a vertical distance of 50cm, and keeping the anal temperature of the rat at 37 +/-0.5 ℃ all the time in the molding process; depilating on the cranial vertex of a rat;

3) injecting a microbubble contrast agent from the tail vein of a rat with the help of an auxiliary tool, wherein the injection amount is determined according to the physique of the rat;

4) ultrasonic intervention, starting ultrasonic equipment, tightly combining an ultrasonic probe with the skull of a rat by using an ultrasonic coupling agent, and implementing the ultrasonic intervention to enable the microbubble contrast agent to reach an instantaneous cavitation threshold;

5) performing parallel scanning on the rat through a CT (computed tomography) to determine the cerebral hemorrhage part and the amount of hemorrhage;

6) irradiating the rat with 60w of incandescent lamp at a vertical distance of 50cm for at least 2h, and keeping the anal temperature of the rat at 37 +/-0.5 ℃;

7) and (4) scoring the nerve function of the rat, wherein the rat meeting the scoring standard is the rat which is successfully modeled, and feeding and observing the rat for later use.

The further improvement of the scheme is as follows: in step 3), the injection amount of the microbubble contrast agent is 0.1 mL/kg.

The further improvement of the scheme is as follows: in the step 4), the frequency of ultrasonic intervention is 2Mhz, the energy is 5-10W/cm2, and the action time is 120 s.

The further improvement of the scheme is as follows: the microbubble contrast agent is sulfur hexafluoride microbubble.

The further improvement of the scheme is as follows: in the step 2), 10 percent of chloral hydrate by mass is used for carrying out intraperitoneal injection on anesthetized rats, and the concentration is 300-400 mg/kg.

The further improvement of the scheme is as follows: step 7) the method for scoring the rat neural function adopts one or more of a Longa scoring method, a Berderson scoring posture reflex test, a Climbing rope test, a screen test, a limb placing test, an open field test method, MNSS, a Rotation rod test, a Rotation test, a limb Symmetry test scoring method, rotadtest, an adhesive-removal sensory test, a Spontanocous Activity, Symmetry in the movement of Limbs, a Forepaw outletting, a Climbing, a Body propriority and a Response to visual contact.

The further improvement of the scheme is as follows: the nerve function scoring process includes scoring and recording the tested rats in a single blind method by at least 3 persons participating in the test, and then carrying out statistical calculation on scores after averaging scoring results of at least 3 groups.

The technical scheme of the invention has the following beneficial effects: the invention acts on the contrast agent through ultrasonic targeting, and generates destructive action on local blood vessels of a target area in the brain by virtue of the microbubble transient cavitation effect, so that the phenomenon of intracerebral parenchyma internal bleeding of a rat is generated, the research requirement of spontaneous cerebral hemorrhage is met, and the method has the advantages of no wound, simplicity, convenience and easy popularization, and is a proper reproducible biological modeling method. Meanwhile, auxiliary equipment is introduced, so that the implementation difficulty is reduced, and the operation is simpler, more convenient and more feasible.

Detailed Description

According to the method for establishing the rat cerebral hemorrhage model by combining the ultrasound and the microbubbles, the treatment on animals in the molding process meets the regulations of Ethical standards of related animals in Ethical esses in animal experiment 2009 in 2009; the following steps are carried out:

1) one SPF male SD rat with the body mass of 240-270 g;

rat breeding environment: keeping the temperature at 24 ℃, turning on the lamp at 7:00-19:00, turning off the lamp at night, and keeping ventilation. The rats can be fasted and forbidden to supply water 12 hours before the model building so as to concentrate the blood components of the animals, so that the blood is in a high coagulation state and hematoma is easy to form;

preparing equipment;

fluorescence microscopy: OLYMPUS BX51, japan;

fluorescent digital camera: penguin Camera 150CL, Pixera, usa;

image acquisition software:

VIEWFINDER 3.0.0, Pixera, usa;

IMAGE Pro-Plus4.5.0.19, Media Cybernetics, Inc, USA;

view software:

STUDIO 3.0, Media Cybernetics, Inc, usa;

image analysis software special image acquisition card: IMAGE Pro-Plus, Media Cybernetics, Inc, USA;

image acquisition card dongle: SN 41N45000-34584, Media Cybernetics, Inc, USA;

a computer: compatible computers with large memories;

an ultrasonic generating device: sequoia 512, Acuson, usa;

TS-2 ultrasonic generator, China, medical Instrument development institute of military medical university, Ming Jiefang military;

a constant temperature circulator: HX-1050 constant temperature circulator, Beijing Boyikang laboratory instruments Inc., China;

transparent water cushion tank: self-making;

a micro-injection pump: terumo Terufusion system pump TE311, TERUMO Corporation, Japan;

multifunctional voltage stabilizer: TM88, east phoenix, large and high electronic equipment factory, zhongshan city, guangdong, china;

microsurgical instruments: microsurgical forceps, microsurgical scissors, microsurgical needle holders, shanghai medical instruments ltd surgical instruments factory, china;

PE50, PE30 clear polyester catheter, usa;

5,10,20 and 50ml of injectors, common surgical instruments and China;

MP120-2 electronic balance: shanghai, second balance instrumentation plant, China.

Tail-assisted injection device: S4-RGDQ2 organic glass rat fixator, DN50, scotch biotechnology (beijing) ltd, china; CN201420106282.9 a tail-assisted injection device;

S3-TMQ rat shaver: cisco nordstock biotechnology (beijing) ltd, china;

stainless steel rat metabolic cage: cisco nordstock biotechnology (beijing) ltd, china;

2) preparing;

fixing the rat on a brain stereotaxic apparatus, and carrying out anesthesia by injecting 10% chloral hydrate into abdominal cavity of the anesthetized rat (300 mg/kg) by mass fraction; irradiating the rat with a 60w incandescent lamp at a vertical distance of 50cm, and keeping the anal temperature of the rat at 37 +/-0.5 ℃ all the time in the molding process; depilating on the cranial vertex of a rat;

3) continuously injecting a microbubble contrast agent from the tail vein of a rat at a constant speed by a micro-injection pump through a tail auxiliary injection device, wherein the microbubble contrast agent is sulfur hexafluoride microbubble (Bracco Suisse SA) in Switzerland, and the injection amount is determined according to the body mass of the rat and is specifically 0.1 mL/kg;

4) ultrasonic intervention, starting ultrasonic equipment, tightly combining an ultrasonic probe with the skull of a rat by using an ultrasonic coupling agent, and implementing the ultrasonic intervention to enable the ultrasonic probe to reach the instantaneous cavitation threshold of the sulfur hexafluoride micro-bubbles; the frequency of ultrasonic intervention is 2Mhz, and the energy is 10W/cm²Time of action 120 s;

5) performing parallel scanning on the rat through a CT (computed tomography) to determine the cerebral hemorrhage part and the amount of hemorrhage;

6) irradiating the rat with 60w of incandescent lamp at a vertical distance of 50cm for at least 2h, and keeping the anal temperature of the rat at 37 +/-0.5 ℃;

7) and (4) scoring the nerve function of the rat, wherein the rat meeting the scoring standard is the rat which is successfully modeled, and feeding and observing the rat for later use.

There are many methods for scoring rat neural function, such as Longa scoring method, Berderson scoring posture reflex test, Climbing experiment, screen test, limb placement test, open field test, MNSS, Rotation lever test, Rotation, limb Symmetry test scoring, rotarod test, adhesive-removal society of the animal, spinning Activity, Symmetry in the Movement of Limbs, ForepawOutstrolling, Climbing, Body pro recording, and Response to viral contact.

Taking the neuro-behavioral check Longa scoring method as an example, the criteria of the scoring method are as follows: score 0, no neurological deficit symptoms; 1 minute, the left forelimb can not be completely straightened; rotating leftwards for 2 minutes to generate a turn; 3 minutes, the body topples to the side of hemiparalysis when walking; 4 points, spontaneous walking and loss of consciousness. The molding success is judged when the score is 2-3.

In the neurological scoring process of this example, at least 3 persons who participate in the test score and record the rats in the test by a single blind method, and then the scores obtained by averaging the scoring results of at least 3 groups are statistically calculated.

The resulting model of the method of this example is a biological model ranging from a bleeding brain model to a bleeding brain model with acute placeholder effect. The energy control of ultrasonic intervention is 5-10W/cm²The sound pressure is 0.48-2.4 MPa, wherein 0.64MPa, 0.8MPa and 1.2MPa have outstanding effect. When the energy is close to 5w/cm²The obtained biological model is closer to the biological model of the bleeding brain. However, since each rat is not identical, the injected amount of contrast medium is different from one rat to another, and thus there is no clear relationship between energy and biological model. Therefore, the rat needs to be subjected to parallel head CT flat scanning to clearly determine the cerebral hemorrhage part and the amount of hemorrhage.

The microbubble contrast agent used in this example was sulfur hexafluoride microbubble (Bracco Suisse SA, switzerland), and the above data are given based on this microbubble contrast agent. If different microbubble contrast agents are adopted, corresponding parameters need to be changed along with the microbubble contrast agents, and the parameters can be changed according to the parameters provided by microbubble contrast agent manufacturers and the characteristics of the microbubble contrast agents, such as stability, gas type and the like. But the specific results and amounts thereof vary considerably from that which is contemplated. And need not be described in detail herein.

Except for the case where the chloral hydrate is used in an amount of 300mg/kg in mass fraction of 10% in step 2) of this example, the injection amount may be increased stepwise as required, but the total injection amount should not exceed 400 mg/kg.

The neurological function scoring method of this embodiment may be performed by using, in addition to the neurobehavioral examination Longa scoring method used in step 7), a method such as a bererson scoring posture reflex test, a Climbing rope test, a web screen test, a limb placement test, an open field test, an MNSS, a Rotation lever test, a Rotation, a limb Symmetry test scoring, a rotared test, an adhesive-removal sensory test, a span Activity, a Symmetry in the Movement of Four Limbs, a forward output scoring, a clinmbing, a body pro printing, a Response video testing Touch.

Finally, it should be noted that the described embodiment is only one specific application example of the present invention, and is not intended to limit the present invention, and for those skilled in the art, the order of the partial steps may be selected and adjusted as appropriate according to the specific situation in the practical application process, but the protection scope of the present invention is not limited at all, and any modification, equivalent replacement, improvement, etc. made should be included in the protection scope of the present invention.