CN112889746B - Multi-sinus combined cerebral venous thrombosis animal model and construction method thereof - Google Patents

Multi-sinus combined cerebral venous thrombosis animal model and construction method thereof Download PDF

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CN112889746B
CN112889746B CN202110056611.8A CN202110056611A CN112889746B CN 112889746 B CN112889746 B CN 112889746B CN 202110056611 A CN202110056611 A CN 202110056611A CN 112889746 B CN112889746 B CN 112889746B
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段建钢
肖立坡
吉训明
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Xuanwu Hospital
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Abstract

The invention relates to the technical field of medicines, and particularly discloses a multi-sinus combined cerebral venous thrombosis animal model and a construction method thereof. According to the invention, a plurality of venous sinus combined thrombi (such as upper sagittal sinus + transverse sinus, upper sagittal sinus + transverse sinus + internal jugular vein), large-area venous cerebral infarction, cerebral hemorrhage and other pathological changes are induced by a method of combining hemiligation with ferric chloride and thrombin, and the severe CVT animal model with more serious neurological defect and longer thrombus duration can be used for researching and evaluating the pathophysiological mechanism of the severe CVT and treating strategies, so that the treatment of severe CVT patients can be facilitated, good prognosis can be promoted, targets can be searched, novel medicines can be developed, and the death rate of the severe CVT patients can be reduced.

Description

一种多窦联合的脑静脉血栓动物模型及其构建方法A multi-sinus combined cerebral venous thrombosis animal model and its construction method

技术领域technical field

本发明涉及医药技术领域,尤其涉及一种脑静脉血栓动物模型。The invention relates to the technical field of medicine, in particular to an animal model of cerebral venous thrombosis.

背景技术Background technique

颅内静脉系统血栓形成(cerebral venous thrombosis,CVT)是一种不同于动脉性卒中的特殊类型的脑血管疾病,主要影响年轻人和儿童,是青年和中年卒中的重要原因,发病率有逐年增加的趋势,一项来自澳大利亚的流行病学调查显示,CVT年发病率为1.57/10万人/年,比以前报道的1.3/10万人更高,病变部位依次为多静脉窦[57.14%]、上矢状窦[16.19%]、横窦[11.43%]和乙状窦[8.57%]。约60%的CVT患者发生静脉性脑梗死或出血,即由闭塞的脑静脉窦或皮层静脉引起的脑组织局部水肿、出血。有静脉性脑梗死或出血的CVT属于重症CVT范畴,患者常有更严重的临床表现和更差的预后。有报道称,尽管颅内静脉血栓死亡率随着诊疗技术的发展在降低,但是严重的颅内静脉血栓患者死亡率仍高达34.2%,这可能与重症CVT的病理生理学机制目前仍然不是很清楚有关系。然而,脑静脉血栓动物模型对探讨其发病机制、病理生理学过程和治疗策略具有重要价值。Cerebral venous thrombosis (CVT) is a special type of cerebrovascular disease different from arterial stroke. It mainly affects young people and children. It is an important cause of stroke in young and middle-aged people. An increasing trend, an epidemiological survey from Australia showed that the annual incidence of CVT was 1.57/100,000 people/year, which was higher than the previously reported 1.3/100,000 people, and the lesion sites were multiple venous sinuses [57.14% ], superior sagittal sinus [16.19%], transverse sinus [11.43%] and sigmoid sinus [8.57%]. Venous cerebral infarction or hemorrhage occurs in about 60% of CVT patients, that is, local edema and hemorrhage of brain tissue caused by occluded cerebral venous sinuses or cortical veins. CVT with venous cerebral infarction or hemorrhage belongs to the category of severe CVT, and patients often have more serious clinical manifestations and poorer prognosis. It has been reported that although the mortality rate of intracranial venous thrombosis is decreasing with the development of diagnosis and treatment technology, the mortality rate of patients with severe intracranial venous thrombosis is still as high as 34.2%, which may be related to the fact that the pathophysiological mechanism of severe CVT is still not very clear. relation. However, animal models of cerebral venous thrombosis are of great value for exploring its pathogenesis, pathophysiological process and therapeutic strategies.

CVT动物模型能用来模拟人类的颅内静脉窦血栓形成,通过研究动物疾病模型,以便于对CVT这种疾病的病因及病理生理过程进行更深入的研究和探讨,从而有利于研发新的治疗靶点,促进疾病良好转归,所以CVT动物模型的科研价值是巨大的。CVT animal models can be used to simulate human intracranial venous sinus thrombosis. By studying animal disease models, we can conduct more in-depth research and discussion on the etiology and pathophysiological process of CVT, which is conducive to the development of new treatments. target and promote a good outcome of the disease, so the scientific research value of CVT animal models is huge.

目前的CVT动物模型有永久性结扎型、临时阻断型、化学诱导型、介入型或插管型、植入自制移植物型、双极电凝型。永久性结扎型模型,能诱导上矢状窦血栓、静脉性脑梗死、脑出血病理变化,但是永久性结扎限制了治疗策略的研究;临时阻断型,操作简单,但是血栓不稳定;化学诱导型,同样也是操作简单,但是血栓不稳定,不能持续一周;介入或插管型,能诱导出上矢状窦血栓、静脉性脑梗死,但是操作比较复杂,耗材高昂;植入自制移植物型,操作简单,模拟上矢状窦血栓,但不是真正意义上的血栓形成,且不能用于治疗策略的研究;双极电凝型,适合研究皮层静脉血栓,但是创伤比较大。各类型模型优缺点见下表(表1引自中国现代神经疾病杂志)。The current animal models of CVT include permanent ligation, temporary blockage, chemical induction, intervention or intubation, implantation of self-made grafts, and bipolar coagulation. Permanent ligation model can induce pathological changes of superior sagittal sinus thrombosis, venous cerebral infarction, and cerebral hemorrhage, but permanent ligation limits the research of treatment strategies; temporary blocking model, easy to operate, but unstable thrombus; chemical induction The type is also simple to operate, but the thrombus is unstable and cannot last for a week; the interventional or intubation type can induce superior sagittal sinus thrombosis and venous cerebral infarction, but the operation is more complicated and the consumables are high; the self-made graft type is implanted , easy to operate, simulates superior sagittal sinus thrombosis, but not a real thrombus, and cannot be used for research on treatment strategies; bipolar coagulation type, suitable for research on cortical venous thrombosis, but the trauma is relatively large. The advantages and disadvantages of each type of model are shown in the table below (Table 1 is quoted from the Chinese Journal of Modern Neurological Diseases).

表1.各种脑静脉血栓模型制作方法优缺点Table 1. Advantages and disadvantages of various methods for making cerebral venous thrombosis models

Figure BDA0002901049740000021
Figure BDA0002901049740000021

以上动物模型大部分诱导的单纯上矢状窦血栓,多窦联合血栓并且合并大面积静脉性脑梗死的重症CVT模型,目前国内外尚未见报道。而临床工作中以多窦联合血栓患者最为多见,所以,目前急需一种能模拟多窦联合的、人类重症CVT的动物模型。The simple superior sagittal sinus thrombosis induced by most of the above animal models, and the severe CVT model with multiple sinus combined with thrombosis and large area venous cerebral infarction have not been reported at home and abroad. In clinical work, patients with multiple sinuses combined with thrombosis are the most common, so there is an urgent need for an animal model of severe CVT in humans that can simulate multiple sinuses combined.

发明内容Contents of the invention

为了解决现有技术中存在的问题,本发明的目的是提供一种多窦联合的脑静脉血栓动物模型及其构建方法。In order to solve the problems in the prior art, the object of the present invention is to provide an animal model of cerebral venous thrombosis combined with multiple sinuses and its construction method.

为了实现本发明目的,本发明的技术方案如下:In order to realize the object of the invention, the technical scheme of the present invention is as follows:

第一方面,本发明提供了一种多窦联合的脑静脉血栓动物模型的制备方法,包括如下步骤:In a first aspect, the present invention provides a method for preparing an animal model of cerebral venous thrombosis with multiple sinuses, comprising the following steps:

(1)以非人动物为动物模型构建对象,麻醉后采用非吸收性外科缝线对上矢状窦(sss)进行半结扎;(1) Non-human animals were used as animal models to construct objects, and the superior sagittal sinus (sss) was semi-ligated with non-absorbable surgical sutures after anesthesia;

(2)结扎后,在上矢状窦表面贴敷20~40%氯化铁溶液,贴敷时间为5~10分钟;优选采用40%氯化铁溶液贴敷5分钟;(2) After ligation, apply 20-40% ferric chloride solution on the surface of the superior sagittal sinus for 5-10 minutes; preferably 40% ferric chloride solution for 5 minutes;

(3)向上矢状窦的窦腔内注射凝血酶溶液,凝血酶的注射总量为150u。(3) Thrombin solution was injected into the sinus cavity of the superior sagittal sinus, and the total amount of thrombin injected was 150u.

所述方法先贴敷氯化铁溶液可造成静脉窦内皮细胞的损伤,局部会形成小的血栓,后注射凝血酶,更有利于血栓的形成及蔓延。若先注射凝血酶,容易造成进针部位的出血,同时凝血酶会迅速进入大鼠的肺循环容易造成肺栓塞致大鼠死亡。In the method, applying the ferric chloride solution first can cause damage to the endothelial cells of the venous sinus, and small thrombi will form locally, and then inject thrombin, which is more conducive to the formation and spread of the thrombus. If the thrombin is injected first, it is easy to cause bleeding at the needle insertion site, and at the same time, the thrombin will quickly enter the pulmonary circulation of the rat and easily cause pulmonary embolism and death of the rat.

进一步地,步骤(1)中,分别对上矢状窦的头侧和尾侧进行半结扎。Further, in step (1), half ligation is performed on the cranial and caudal sides of the superior sagittal sinus respectively.

进一步地,所述步骤(2)在黑暗条件下进行,氯化铁溶液具有强氧化性,避光更有利于损伤静脉窦内皮细胞,不避光的情况下,可能会影响它的氧化作用。Further, the step (2) is carried out under dark conditions. The ferric chloride solution has strong oxidative properties, and avoiding light is more conducive to damaging the venous sinus endothelial cells. If it is not protected from light, its oxidation may be affected.

进一步地,所述步骤(3)在1分钟之内完成,这样更有利于血栓的形成,速度太快容易造成大量的凝血酶进入大鼠肺循环,继发肺栓塞而致大鼠死亡,速度太慢也不利于血栓形成。Further, the step (3) is completed within 1 minute, which is more conducive to the formation of thrombus. If the speed is too fast, it will easily cause a large amount of thrombin to enter the pulmonary circulation of rats, which will lead to the death of rats due to pulmonary embolism. Slow is also not conducive to thrombosis.

更进一步地,步骤(3)中,利用微量注射器向上矢状窦的窦腔内分3次注射凝血酶溶液,每次注射凝血酶溶液0.1mL,凝血酶含量50u。Furthermore, in step (3), the thrombin solution was injected into the sinus cavity of the upper sagittal sinus three times with a micro-syringe, 0.1 mL of the thrombin solution was injected each time, and the thrombin content was 50 u.

在实际操作中,为确保操作目的的实现,在步骤(1)完成后,利用激光散斑仪探测上矢状窦结扎段内血流信号,血流信号下降到结扎前的50%±5%,验证半结扎操作完成;在步骤(3)完成后,利用激光散斑仪探测上矢状窦结扎段内血流信号,确定血流信号相较半结扎后进一步下降(图10)。In actual operation, in order to ensure the realization of the operation purpose, after the completion of step (1), the blood flow signal in the ligated segment of the superior sagittal sinus is detected by a laser speckle meter, and the blood flow signal drops to 50%±5% of that before ligation , to verify the completion of the half ligation operation; after the completion of step (3), the laser speckle meter was used to detect the blood flow signal in the ligated segment of the superior sagittal sinus, and it was confirmed that the blood flow signal further decreased compared with that after the half ligation (Figure 10).

在本发明的具体实施方式中,作为示例性说明,所述动物模型为大鼠。然而在实际应用中,并不限于此。In a specific embodiment of the present invention, as an illustration, the animal model is a rat. However, in practical application, it is not limited to this.

第二方面,本发明提供一种多窦联合的脑静脉血栓动物模型,所述动物模型由前述制备方法构建而成。In a second aspect, the present invention provides an animal model of multiple sinus combined cerebral venous thrombosis, which is constructed by the aforementioned preparation method.

所述动物模型具有多个静脉窦联合血栓,至少包括上矢状窦血栓和横窦血栓,还可包括颈内静脉血栓。The animal model has multiple combined venous sinus thrombi, at least including superior sagittal sinus thrombus and transverse sinus thrombus, and may also include internal jugular vein thrombus.

本发明通过半结扎上矢状窦,可造成上矢状窦血流淤滞,上矢状窦表面贴氯化铁线段,可造成上矢状窦内皮的损伤,注射凝血酶可造成血液高凝状态,即模拟了血栓形成的三要素,并且能诱导出多个静脉窦的血栓,所以更加接近人类重症CVT的病理过程。本发明所提供的动物模型可以模拟人类重症CVT,可作为新型重症CVT动物模型,可用于重症CVT病理生理机制的研究和治疗策略的评估,具有很好的应用前景。The present invention can cause blood flow stasis in the superior sagittal sinus by semi-ligating the superior sagittal sinus. The surface of the superior sagittal sinus is pasted with ferric chloride line, which can cause damage to the endothelium of the superior sagittal sinus. The injection of thrombin can cause blood hypercoagulability. , which simulates the three elements of thrombosis, and can induce multiple venous sinus thrombus, so it is closer to the pathological process of human severe CVT. The animal model provided by the present invention can simulate severe CVT in humans, can be used as a new animal model of severe CVT, can be used for research on the pathophysiological mechanism of severe CVT and evaluation of treatment strategies, and has a good application prospect.

第三方面,本发明进一步提供所述动物模型在研究或筛选脑静脉血栓治疗靶点或治疗药物方面的应用,以及所述动物模型在评价脑静脉血栓治疗效果方面的应用。In the third aspect, the present invention further provides the application of the animal model in researching or screening therapeutic targets or therapeutic drugs for cerebral venous thrombosis, and the application of the animal model in evaluating the therapeutic effect of cerebral venous thrombosis.

本发明提供的动物模型,可用于人类重症CVT病理生理机制的研究和探讨,以及研究和探索新型治疗靶点,并用于治疗重症CVT的新型药物的研发,可能有利于重症CVT患者的治疗及转归。The animal model provided by the present invention can be used for the research and exploration of the pathophysiological mechanism of human severe CVT, as well as for the research and exploration of new therapeutic targets, and for the research and development of new drugs for the treatment of severe CVT, which may be beneficial to the treatment and transformation of severe CVT patients. return.

本发明涉及到的原料或试剂均为普通市售产品,涉及到的操作如无特殊说明均为本领域常规操作。The raw materials or reagents involved in the present invention are all commercially available products, and the operations involved are conventional operations in the field unless otherwise specified.

在符合本领域常识的基础上,上述各优选条件,可以相互组合,得到具体实施方式。On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined with each other to obtain specific implementation modes.

本发明的有益效果在于:The beneficial effects of the present invention are:

本发明通过半结扎联合氯化铁和凝血酶的方法,构建得到一种多窦联合的脑静脉血栓动物模型,该方法能诱导出多个静脉窦联合血栓(如上矢状窦+横窦,上矢状窦+横窦+颈内静脉),大面积静脉性脑梗死,脑出血等病理变化,且神经功能缺损更加严重、血栓持续时间更长的重症CVT动物模型,可用于重症CVT病理生理机制的研究和治疗策略的研究及评估,从而可能更有利于重症CVT患者的治疗和促进良好的预后、探寻新靶点和研发新型药物,以降低重症CVT患者的死亡率。The present invention constructs and obtains a kind of multi-sinus combined cerebral venous thrombosis animal model through the method of semi-ligation combined ferric chloride and thrombin, and this method can induce multiple venous sinus combined thrombus (such as superior sagittal sinus+transverse sinus, superior sagittal sinus+transverse sinus, superior Sagittal sinus + transverse sinus + internal jugular vein), large area of venous cerebral infarction, cerebral hemorrhage and other pathological changes, and severe CVT animal models with more serious neurological deficits and longer thrombus duration can be used for the pathophysiological mechanism of severe CVT The study and evaluation of advanced research and treatment strategies may be more conducive to the treatment of severe CVT patients and promote good prognosis, explore new targets and develop new drugs to reduce the mortality of severe CVT patients.

本发明动物模型的构建方法操作简单,耗材低廉。动物模型稳定性好,血栓能维持超过一周,且能诱导出多个静脉窦联合血栓,大面积静脉性脑梗死,能很好的模拟人类重症CVT。The method for constructing the animal model of the present invention is simple to operate and has low cost of consumables. The animal model has good stability, and the thrombus can last for more than a week, and can induce multiple venous sinus combined with thrombus, large area of venous cerebral infarction, and can well simulate severe CVT in humans.

附图说明Description of drawings

此处的附图被并入说明书中并构成本说明书的一部分,示出了符合本发明的实施例,并与说明书一起用于解释本发明的原理。The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description serve to explain the principles of the invention.

为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,对于本领域普通技术人员而言,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, In other words, other drawings can also be obtained from these drawings without paying creative labor.

图1为构建动物模型过程中的麻醉阶段;Fig. 1 is the anesthesia stage in the process of constructing an animal model;

图2为构建动物模型过程中的颅骨钻孔操作,暴露上矢状窦和两侧皮质,保持硬脑膜完整;Figure 2 shows the skull drilling operation during the construction of the animal model, exposing the superior sagittal sinus and both sides of the cortex, and keeping the dura mater intact;

图3为构建动物模型过程中的SSS半结扎操作;Fig. 3 is the SSS semi-ligation operation in the process of building an animal model;

图4为构建动物模型过程中的氯化铁贴敷操作;Fig. 4 is the operation of ferric chloride sticking in the process of building an animal model;

图5为构建动物模型过程中注射凝血酶溶液后,观察到上矢状窦变为黑色;Figure 5 shows that the superior sagittal sinus was observed to turn black after the thrombin solution was injected during the construction of the animal model;

图6为构建动物模型过程中,利用激光散斑仪检测结扎段内血流信号;Figure 6 shows the detection of blood flow signals in the ligated segment by using a laser speckle meter during the process of building an animal model;

图7为确认实验动物血栓形成后,缝合切口;Fig. 7 is after confirming the thrombus formation in the experimental animal, suturing the incision;

图8为动物模型已诱导多个静脉窦血栓形成,及大面积静脉性脑梗死;其中,黄色箭头所示为上矢状窦血栓,红色箭头所示为横窦血栓,蓝色箭头所示为颈内静脉血栓;Figure 8 shows that the animal model has induced multiple venous sinus thrombosis and large area venous cerebral infarction; among them, the yellow arrow shows the superior sagittal sinus thrombosis, the red arrow shows the transverse sinus thrombosis, and the blue arrow shows the Internal jugular vein thrombosis;

图9为动物模型脑组织TTC染色图,显示上矢状窦两侧皮层大面积静脉性脑梗死;Figure 9 is a TTC staining diagram of the brain tissue of the animal model, showing large-area venous cerebral infarction in the cortex on both sides of the superior sagittal sinus;

图10为本发明半结扎联合氯化铁凝血酶构建多窦联合的脑静脉血栓动物模型的流程示意图;Fig. 10 is a schematic flow diagram of constructing an animal model of cerebral venous thrombosis with multiple sinuses combined with semi-ligation and ferric chloride thrombin in the present invention;

图11为本发明重复性实验中造模过程中脑血流量的变化情况;Fig. 11 is the variation of cerebral blood flow in the modeling process in the repeat experiment of the present invention;

图12为本发明稳定性试验中动物模型的脑静脉血栓形成情况。Fig. 12 is the situation of cerebral venous thrombosis in the animal model in the stability test of the present invention.

具体实施方式Detailed ways

为了能够更清楚地理解本发明的上述目的、特征和优点,下面将对本发明的方案进行进一步描述。需要说明的是,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。In order to understand the above-mentioned purpose, features and advantages of the present invention more clearly, the solutions of the present invention will be further described below. It should be noted that, in the case of no conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

在下面的描述中阐述了很多具体细节以便于充分理解本发明,但本发明还可以采用其他不同于在此描述的方式来实施;显然,说明书中的实施例只是本发明的一部分实施例,而不是全部的实施例。In the following description, many specific details have been set forth in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described here; obviously, the embodiments in the description are only some embodiments of the present invention, and Not all examples.

下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的,并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下,可以对本发明进行各种修改和替换。Preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are given for the purpose of illustration only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

下述实施例中所使用的实验方法如无特殊说明,均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.

下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

实施例1Example 1

1、使用恩氟烷诱导麻醉,通过麻醉面罩用70%的一氧化二氮和30%的氧气送出,维持整个手术和监测过程。将麻醉好的大鼠俯卧位固定于立体定位架上,用恒温毯维持体温37℃于整个手术过程(图1)。1. Enflurane was used to induce anesthesia, and 70% nitrous oxide and 30% oxygen were sent through an anesthesia mask to maintain the entire operation and monitoring process. The anesthetized rats were fixed in a prone position on a stereotaxic frame, and the body temperature was maintained at 37°C with a constant temperature blanket throughout the operation (Figure 1).

2、备皮后于头部正中做一长约1.5cm手术切口,分离皮下组织,暴露颅骨,用打磨钻先于前囟前和人字点之间一侧钻孔,显微镜下操作,逐渐扩展为形成一10mm×3mm骨窗,暴露sss和两侧皮质,保持硬脑膜完整,钻颅过程中用生理盐水持续冷却钻头,避免灼伤硬脑膜及皮质(图2)。2. After skin preparation, make a surgical incision about 1.5cm in the middle of the head, separate the subcutaneous tissue, expose the skull, drill a hole with a grinding drill on the side between the anterior bregma and the herringbone point, operate under a microscope, and gradually expand In order to form a 10mm×3mm bone window, expose the sss and both sides of the cortex, and keep the dura mater intact, the drill bit was continuously cooled with saline during drilling to avoid burning the dura mater and cortex (Figure 2).

3、暴露SSS后,用激光散点仪探测SSS内血流信号,记录测量值。显微镜下用8-0聚丙烯缝线于暴露的SSS头侧和尾侧分别半结扎SSS,再用激光散斑仪探测SSS结扎段内血流信号,确定血流信号下降到结扎前的50%左右,并记录,以保证是半结扎而不是完全性结扎(图3)。3. After the SSS was exposed, a laser scatterer was used to detect the blood flow signal in the SSS, and the measured value was recorded. Under the microscope, 8-0 polypropylene suture was used to half-ligate the SSS on the head and tail sides of the exposed SSS, and then the laser speckle meter was used to detect the blood flow signal in the ligated segment of the SSS, and it was confirmed that the blood flow signal dropped to 50% of that before ligation Left and right, and record to ensure that it is a half ligation rather than a complete ligation (Figure 3).

4、在黑暗条件下用蘸有40%氯化铁的3-0线段贴敷在SSS表面5min,取下线段(图4)。4. Apply a 3-0 line segment dipped in 40% ferric chloride on the SSS surface for 5 minutes in the dark, and remove the line segment (Figure 4).

5、用微量注射器抽取凝血酶溶液0.1ml(50u),1分钟内注射到窦腔内,连续三次,总量150u,可以观察到上矢状窦变为黑色(图5),再次用激光散斑仪检测结扎段内血流信号,确定血流量进一步下降说明血栓形成(图6)。5. Use a micro-syringe to extract 0.1ml (50u) of thrombin solution, and inject it into the sinus cavity within 1 minute, three times in a row, with a total of 150u. It can be observed that the superior sagittal sinus turns black (Fig. 5). The blood flow signal in the ligated segment was detected by a spot meter, and a further decrease in blood flow was determined to indicate thrombus formation (Fig. 6).

6、如图6所示,结扎前上矢状窦内脑血流量为300pu,半结扎后变为150pu(为结扎前的50%),贴氯化铁注射凝血酶后变为50pu,说明已经形成血栓。6. As shown in Figure 6, the cerebral blood flow in the superior sagittal sinus was 300pu before ligation, and changed to 150pu after half ligation (50% of that before ligation), and changed to 50pu after ferric chloride injection and thrombin injection, indicating that the Formation of blood clots.

7、缝合切口,消毒伤口,放回笼中,正常进食水(图7)。7. Suture the incision, disinfect the wound, put it back into the cage, and eat and drink normally (Figure 7).

8、半结扎联合氯化铁凝血酶能诱导多个静脉窦血栓形成,大面积静脉性脑梗死,见图8、图9。8. Semi-ligation combined with ferric chloride thrombin can induce multiple venous sinus thrombosis and large-area venous cerebral infarction, as shown in Figure 8 and Figure 9.

如图8所示,黄色箭头所示为上矢状窦血栓,红色箭头所示为横窦血栓,蓝色箭头所示为颈内静脉血栓。As shown in Figure 8, the yellow arrow indicates the superior sagittal sinus thrombus, the red arrow indicates the transverse sinus thrombus, and the blue arrow indicates the internal jugular vein thrombus.

如图9所示,脑组织TTC染色显示上矢状窦两侧皮层大面积静脉性脑梗死,白色箭头所示。As shown in Figure 9, TTC staining of the brain tissue showed large areas of venous cerebral infarction in the cortex on both sides of the superior sagittal sinus, as indicated by the white arrows.

实施例2Example 2

为说明本发明动物模型构建方法的重复性,表2列出了20只大鼠在半结扎前、半结扎后和应用氯化铁凝血酶后的脑血流量变化情况,我们将半结扎前、半结扎后和应用氯化铁凝血酶后的脑血流量用均数±标准差的形式来表示,即359.42±72.61PU,189.98±31.12PU,95.93±28.31PU,通过单因素方差分析计算出F=151.90,P<0.0001(表3),说明三组数据存在显著的统计学差异,并且两两比较,P<0.0001,均存在显著的统计学差异(见图11),提示均有血栓形成,说明构建方法重复性良好。In order to illustrate the repeatability of the animal model construction method of the present invention, table 2 has listed the cerebral blood flow change situation of 20 rats before half ligation, after half ligation and application ferric chloride thrombin, we will half ligation, before half ligation, The cerebral blood flow after semi-ligation and application of ferric chloride thrombin is expressed in the form of mean ± standard deviation, that is, 359.42±72.61PU, 189.98±31.12PU, 95.93±28.31PU, calculated by one-way analysis of variance F =151.90, P<0.0001 (Table 3), indicating that there is a significant statistical difference in the three groups of data, and comparing each other, P<0.0001, there is a significant statistical difference (see Figure 11), suggesting that there is thrombus formation, It shows that the construction method has good repeatability.

表2.造模过程中不同阶段脑血流量的变化Table 2. Changes in cerebral blood flow at different stages during modeling

Figure BDA0002901049740000081
Figure BDA0002901049740000081

实施例3Example 3

针对实施例2所构建的20只大鼠脑静脉血栓动物模型,造模后24小时,有一只因硬膜外血肿死亡,术后24小时生存率是95%,造模后48小时、1周各有两只死于严重的脑组织缺血,术后1周死亡率为25%(5/20),其中因严重脑组织缺血死亡率为21.1%(4/19),这与人类重症CVT死亡率比较接近。术后12小时,取5只大鼠深度麻醉后,用盐水心脏灌注,可发现5只大鼠均有上矢状窦、横窦及颈内静脉血栓形成,甚至有皮层静脉血栓形成(见图12A,黄色箭头所示为上矢状窦血栓,绿色剪头所示为横窦血栓,蓝色箭头所示为颈内静脉血栓,黑色箭头所示为皮层静脉血栓),术后24、48小时,各取5只大鼠,用同样方法均发现有上矢状窦及横窦血栓(见图12B、12C,黄色箭头所示为上矢状窦血栓,绿色剪头所示为横窦血栓),同样,术后1周,发现5只大鼠上矢状窦内仍有血栓(见图12D,黄色箭头所示为上矢状窦血栓),这些均说明新模型稳定性是良好的。For the 20 rat cerebral venous thrombosis animal models constructed in Example 2, 24 hours after modeling, one died of epidural hematoma, and the 24-hour survival rate after operation was 95%. Two each died of severe cerebral ischemia, and the mortality rate at 1 week after operation was 25% (5/20), among which the mortality rate due to severe cerebral ischemia was 21.1% (4/19), which is similar to that of human severe cerebral ischemia. CVT mortality rates are relatively similar. Twelve hours after the operation, 5 rats were deeply anesthetized and perfused with saline heart. It was found that all 5 rats had thrombosis in the superior sagittal sinus, transverse sinus and internal jugular vein, and even cortical vein thrombosis (see Fig. 12A, the yellow arrow indicates superior sagittal sinus thrombosis, the green clipped head indicates transverse sinus thrombosis, the blue arrow indicates internal jugular vein thrombosis, and the black arrow indicates cortical vein thrombosis), 24 and 48 hours after operation , each of 5 rats was found to have thrombus in the superior sagittal sinus and transverse sinus by the same method (see Figure 12B, 12C, the yellow arrow indicates the superior sagittal sinus thrombus, and the green clipped head indicates the transverse sinus thrombus) , similarly, 1 week after the operation, thrombus was still found in the superior sagittal sinus of 5 rats (see Figure 12D, the yellow arrow shows the superior sagittal sinus thrombus), all of which indicated that the stability of the new model was good.

以上所述仅是本发明的具体实施方式,使本领域技术人员能够理解或实现本发明。对这些实施例的多种修改对本领域的技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所述的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。The above descriptions are only specific embodiments of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (3)

1. A preparation method of a multi-sinus combined cerebral venous thrombosis animal model is characterized by comprising the following steps:
(1) A rat is used as an animal model construction object, and after anesthesia, a non-absorbable surgical suture is adopted to semi-ligate the superior sagittal sinus; respectively performing half ligation on the cephalic side and the caudal side of the superior sagittal sinus; after the ligation, detecting a blood flow signal in the upper sagittal sinus ligation section by using a laser speckle instrument, and verifying that the blood flow signal is reduced to 50 +/-5% before ligation, so that the half ligation operation is completed;
(2) After ligation, applying 40% ferric chloride solution on the surface of the superior sagittal sinus for 5 minutes under dark condition;
(3) Injecting thrombin solution into the sinus cavity of the superior sagittal sinus for 3 times, wherein the total injection amount of thrombin is 150u, and the injection is completed within 1 minute; 0.1mL of thrombin solution is injected each time, and the thrombin content is 50u; after the ligation, a blood flow signal in the upper sagittal sinus ligation segment is detected by using a laser speckle pattern instrument, and the blood flow signal is determined to be further reduced compared with the blood flow signal in the semi-ligation segment.
2. The application of the animal model constructed by the preparation method of claim 1 in researching or screening the therapeutic target of cerebral venous thrombosis or therapeutic drugs.
3. The use of an animal model constructed by the method of claim 1 for evaluating the therapeutic effect of cerebral venous thrombosis.
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