CN112889746B

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

Info

Publication number: CN112889746B
Application number: CN202110056611.8A
Authority: CN
Inventors: 段建钢; 肖立坡; 吉训明
Original assignee: Xuanwu Hospital
Current assignee: Xuanwu Hospital
Priority date: 2021-01-15
Filing date: 2021-01-15
Publication date: 2023-01-20
Anticipated expiration: 2041-01-15
Also published as: CN112889746A

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

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

Technical Field

The invention relates to the technical field of medicines, in particular to a cerebral venous thrombosis animal model.

Background

Intracranial venous thrombosis (CVT) is a special type of cerebrovascular disease different from arterial stroke, mainly affects young people and children, is an important cause of young and middle-aged stroke, the incidence rate tends to increase year by year, an epidemiological survey from australia shows that the annual incidence rate of CVT is 1.57/10 ten thousand people/year, and is higher than 1.3/10 ten thousand people reported before, and the lesion sites are multiple venous sinuses [57.14% ], superior sagittal sinus [16.19% ], transverse sinus [11.43% ] and sigmoid sinus [8.57% ]insequence. About 60% of CVT patients develop venous cerebral infarction or hemorrhage, i.e. local edema, hemorrhage of the brain tissue caused by the occluded sinus or cortical vein of the cerebral vein. CVTs with venous cerebral infarction or hemorrhage fall into the category of severe CVTs, with patients often having more severe clinical manifestations and poorer prognosis. It is reported that although the mortality rate of intracranial venous thrombosis is reduced along with the development of diagnosis and treatment technology, the mortality rate of serious intracranial venous thrombosis patients is still as high as 34.2%, which may not be clearly related to the pathophysiological mechanism of the severe CVT at present. However, animal models of cerebral venous thrombosis have important value for discussing the pathogenesis, pathophysiological processes and treatment strategies thereof.

The CVT animal model can be used for simulating human intracranial venous sinus thrombosis, and the research on animal disease models facilitates the deeper research and discussion on the etiology and the pathophysiology process of CVT diseases, so that the research on new treatment targets is facilitated, the good outcome of the diseases is promoted, and the research value of the CVT animal model is huge.

The CVT animal models at present are of a permanent ligation type, a temporary blocking type, a chemical induction type, an intervention type or a cannula type, an implantation self-made graft type and a bipolar coagulation type. The permanent ligation model can induce pathological changes of superior sagittal sinus thrombus, venous cerebral infarction and cerebral hemorrhage, but the permanent ligation limits the research of treatment strategies; temporary block type, simple operation, but unstable thrombus; the chemical induction type is also simple to operate, but the thrombus is unstable and cannot last for one week; the insertion or intubation type can induce superior sagittal sinus thrombus and venous cerebral infarction, but the operation is more complicated and the material consumption is high; the self-made graft type is implanted, the operation is simple, the superior sagittal sinus thrombus is simulated, but the thrombus is not formed in the true sense, and the self-made graft type can not be used for the research of treatment strategies; the bipolar coagulation type is suitable for researching the cortical venous thrombosis, but has larger trauma. The advantages and disadvantages of each type of model are shown in the following table (table 1 is introduced from the journal of modern neurological diseases in china).

TABLE 1 advantages and disadvantages of various cerebral venous thrombosis models

The severe CVT model of the animal model, which mainly induces simple ascending sagittal sinus thrombus and multi-sinus combined thrombus and combines large-area venous cerebral infarction, is not reported at home and abroad at present. In clinical work, patients with combined multiple sinuses and thrombus are the most common, so an animal model capable of simulating combined multiple sinuses and severe human CVT is urgently needed.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a multi-sinus combined animal model of cerebral venous thrombosis and a construction method thereof.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

in a first aspect, the invention provides a method for preparing a multi-sinus combined animal model of cerebral venous thrombosis, which comprises the following steps:

(1) Using a non-human animal as an animal model construction object, performing half ligation on superior sagittal sinus (sss) by using a non-absorbable surgical suture after anesthesia;

(2) After ligation, applying 20-40% ferric chloride solution on the surface of the superior sagittal sinus for 5-10 minutes; preferably, 40% ferric chloride solution is adopted for 5 minutes;

(3) The thrombin solution was injected into the sinus cavity of the superior sagittal sinus, and the total amount of thrombin injected was 150u.

According to the method, the ferric chloride solution is firstly pasted to cause the injury of the endothelial cells of the venous sinuses, small thrombus can be formed locally, and then the thrombin is injected, so that the formation and the spread of the thrombus are facilitated. If thrombin is injected firstly, bleeding at the needle insertion part is easy to cause, and thrombin can rapidly enter pulmonary circulation of a rat to easily cause pulmonary embolism to cause death of the rat.

Further, in step (1), the cephalic side and caudal side of the superior sagittal sinus are half-ligated, respectively.

Furthermore, the step (2) is carried out in the dark, the ferric chloride solution has strong oxidizing property, and is more favorable for damaging the endothelial cells of the venous sinus under the condition of keeping out of light, and the oxidizing effect of the endothelial cells of the venous sinus can be influenced under the condition of not keeping out of light.

Further, the step (3) is completed within 1 minute, so that thrombus formation is facilitated, a large amount of thrombin easily enters the pulmonary circulation of the rat due to too high speed, the rat is dead due to the secondary pulmonary embolism, and the thrombus formation is not facilitated due to too low speed.

Further, in step (3), the thrombin solution was injected 3 times into the sinus cavity of the superior sagittal sinus using a micro-syringe, each time 0.1mL of the thrombin solution was injected, and the thrombin content was 50u.

In actual operation, in order to ensure the realization of the operation purpose, after the step (1) is finished, detecting a blood flow signal in the superior sagittal sinus ligation segment by using a laser speckle instrument, and verifying that the half ligation operation is finished when the blood flow signal is reduced to 50 +/-5% before ligation; after the step (3) is finished, detecting a blood flow signal in the upper sagittal sinus ligation segment by using a laser speckle pattern instrument, and determining that the blood flow signal is further reduced compared with the blood flow signal after the hemiligation (fig. 10).

In a particular embodiment of the invention, the animal model is, by way of illustration, a rat. However, in practical applications, it is not limited thereto.

In a second aspect, the invention provides a multi-sinus combined animal model of cerebral venous thrombosis, which is constructed by the preparation method.

The animal model has multiple venous sinus combination thrombi, at least including upper sagittal sinus thrombi and transverse sinus thrombi, and also including internal jugular vein thrombi.

The invention can cause the blood stasis of the superior sagittal sinus by half-binding the superior sagittal sinus, the ferric chloride line segment is stuck on the surface of the superior sagittal sinus to cause the damage of the endothelium of the superior sagittal sinus, and the injection of thrombin can cause the high blood coagulation state, namely three factors of the simulation of the formation of thrombus and can induce the thrombus of a plurality of venous sinuses, thereby being more close to the pathological process of the severe CVT of human beings. The animal model provided by the invention can simulate the severe CVT of human, can be used as a novel severe CVT animal model, can be used for researching the pathophysiological mechanism of the severe CVT and evaluating a treatment strategy, and has a good application prospect.

In a third aspect, the invention further provides application of the animal model in researching or screening a cerebral venous thrombosis treatment target or a treatment medicament, and application of the animal model in evaluating the cerebral venous thrombosis treatment effect.

The animal model provided by the invention can be used for researching and discussing pathophysiological mechanism of the severe CVT of human, researching and exploring a novel treatment target point, researching and developing a novel medicament for treating the severe CVT, and possibly being beneficial to treatment and outcome of severe CVT patients.

The raw materials or reagents involved in the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified.

The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.

The invention has the beneficial effects that:

the invention constructs a multi-sinus combined cerebral venous thrombosis animal model by a method of combining hemiligation with ferric chloride and thrombin, can induce pathological changes of 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 the like, is a severe CVT animal model with more serious neurological function defect and longer thrombus duration, can be used for researching and evaluating a pathological physiological mechanism of the severe CVT and a treatment strategy, and is possibly more beneficial to treatment of severe CVT patients, promotion of good prognosis, search of new targets and research and development of novel medicaments so as to reduce the death rate of the severe CVT patients.

The construction method of the animal model is simple to operate and low in material consumption. The animal model has good stability, the thrombus can be maintained for more than one week, and the combined thrombus of a plurality of venous sinuses and large-area venous cerebral infarction can be induced, so that the severe CVT of human can be well simulated.

Drawings

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.

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive exercise.

FIG. 1 shows the anesthesia phase during the construction of an animal model;

FIG. 2 is a cranial drilling operation during the construction of an animal model, exposing the superior sagittal sinus and bilateral cortex, leaving the dura intact;

FIG. 3 illustrates SSS semi-ligation in the construction of an animal model;

FIG. 4 illustrates the ferric chloride application operation during the construction of an animal model;

FIG. 5 is a black upper sagittal sinus color observed following injection of thrombin solution during construction of an animal model;

FIG. 6 is a schematic diagram showing the detection of blood flow signals in the ligation section by a laser speckle analyzer during the construction of an animal model;

FIG. 7 is a view showing that after confirming thrombosis in the experimental animal, the incision is sutured;

FIG. 8 is a graph of an animal model that has induced multiple venous sinus thrombosis, and large area venous cerebral infarction; wherein yellow arrow indicates a superior sagittal sinus thrombus, red arrow indicates a transverse sinus thrombus, and blue arrow indicates an internal jugular vein thrombus;

FIG. 9 is a TTC staining of animal model brain tissue showing large area venous cerebral infarction of the lateral cortex of the superior sagittal sinus;

FIG. 10 is a schematic flow diagram of an animal model for constructing a multi-sinus combined cerebral venous thrombosis by combining semi-ligation with ferric chloride thrombin according to the present invention;

FIG. 11 shows the change of cerebral blood flow during the modeling process in the repetitive experiments of the present invention;

FIG. 12 is a graph of cerebral venous thrombosis in an animal model in a stability assay of the invention.

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, a solution of the present invention will be further described below. It should be noted that the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the invention may be practiced otherwise than as described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

1. Anesthesia was induced using enflurane, delivered through an anesthesia mask with 70% nitrous oxide and 30% oxygen, and the entire procedure and monitoring was maintained. The anesthetized rat was mounted in the prone position on a stereotaxic frame and the body temperature was maintained at 37 ℃ with a thermostatic blanket throughout the procedure (fig. 1).

2. After preparing the skin, a 1.5cm long surgical incision was made in the center of the head, the subcutaneous tissue was separated, the skull was exposed, a burr drill was used to drill a hole on one side between bregma and the point of the herringbone, the hole was operated under a microscope and gradually expanded to form a 10mm x 3mm bone window, sss and bilateral cortex were exposed, the dura was kept intact, and the drill was continuously cooled with physiological saline during the craniotomy to avoid burning the dura and cortex (fig. 2).

3. And after the SSS is exposed, detecting blood flow signals in the SSS by using a laser scatterometer, and recording a measured value. Under a microscope, the SSS is half-ligated on the head side and the tail side of the exposed SSS by using an 8-0 polypropylene suture, blood flow signals in the SSS ligation section are detected by using a laser speckle pattern instrument, the blood flow signals are determined to be reduced to about 50% of the blood flow signals before ligation, and the blood flow signals are recorded to ensure that half ligation is performed but not complete ligation (fig. 3).

4. The segments were removed by applying 3-0 segments dipped in 40% ferric chloride to the SSS surface for 5min in the dark (FIG. 4).

5. 0.1ml (50 u) of thrombin solution was withdrawn by a micro-syringe and injected into the sinus cavity in 1 minute three consecutive times for a total of 150u, and the upper sagittal sinus was observed to turn black (FIG. 5), and the blood flow signal in the ligated section was again detected by a laser speckle analyzer to determine a further decrease in blood flow indicative of thrombosis (FIG. 6).

6. As shown in FIG. 6, the cerebral blood flow in the superior sagittal sinus before ligation was 300pu, which became 150pu after half ligation (50% before ligation), and 50pu after thrombin injection with ferric chloride, indicating that a thrombus had formed.

7. The incision was sutured closed, the wound was disinfected, and returned to the cage with normal water intake (fig. 7).

8. The combination of the semi-ligation and the iron chloride thrombin can induce the thrombosis of a plurality of venous sinuses and the large-area venous cerebral infarction, as shown in figure 8 and figure 9.

As shown in FIG. 8, yellow arrows indicate ascending sagittal sinus thrombus, red arrows indicate transverse sinus thrombus, and blue arrows indicate internal jugular vein thrombus.

As shown in fig. 9, TTC staining of brain tissue showed extensive venous cerebral infarction of the cortex on both sides of the superior sagittal sinus, indicated by white arrows.

Example 2

To illustrate the repeatability of the animal model construction method of the invention, table 2 lists the change of cerebral blood flow of 20 rats before and after half ligation and after application of ferric chloride thrombin, we expressed the cerebral blood flow of 20 rats before and after half ligation in terms of mean ± standard deviation, namely 359.42 ± 72.61pu,189.98 ± 31.12pu and 95.93 ± 28.31PU, and calculated F =151.90 and P <0.0001 (table 3) by one-way variance analysis, which indicates that the three groups of data have significant statistical difference, and compared with each other, P <0.0001, which indicates that all the groups have thrombosis (see fig. 11), indicating that the construction method has good repeatability.

TABLE 2 cerebral blood flow changes at different stages of the modeling process

Example 3

For the 20 rat cerebral venous thrombosis animal models constructed in example 2, one of the animals died due to epidural hematoma 24 hours after modeling, the survival rate 24 hours after operation was 95%, two animals died due to severe cerebral tissue ischemia 48 hours and 1 week after modeling, and the death rate 1 week after operation was 25% (5/20), wherein the death rate due to severe cerebral tissue ischemia was 21.1% (4/19), which is closer to the death rate of human severe CVT. After 12 hours of operation, after taking 5 rats to perform deep anesthesia, the rats are perfused with saline heart, and it can be found that 5 rats all have thrombus formation in the upper sagittal sinus, transverse sinus and internal jugular vein, even cortical vein thrombus (see fig. 12A, yellow arrow shows the thrombus in the upper sagittal sinus, green arrow head shows the thrombus in the transverse sinus, blue arrow shows the thrombus in the internal jugular vein, black arrow shows the thrombus in the cortical vein), and after 24 hours and 48 hours of operation, 5 rats are taken respectively, and upper sagittal sinus and transverse sinus thrombus are found by the same method (see fig. 12B and 12C, yellow arrow shows the thrombus in the upper sagittal sinus, green arrow shows the thrombus in the transverse sinus), and similarly, after 1 week of operation, 5 rats are found to have thrombus in the upper sagittal sinus (see fig. 12D, yellow arrow shows the thrombus in the upper sagittal sinus), which all indicate that the stability of the new model is good.

The above description is merely illustrative of particular embodiments of the invention that enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

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.