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

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

A multi-sinus combined cerebral venous thrombosis animal model and its construction method

technical field

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

Background technique

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 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.

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).

Table 1. Advantages and disadvantages of various methods for making cerebral venous thrombosis models

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.

Contents of the invention

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.

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

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

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.

Further, in step (1), half ligation is performed on the cranial and caudal sides of the superior sagittal sinus respectively.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

The beneficial effects of the present invention are:

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.

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.

Description of 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 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.

Fig. 1 is the anesthesia stage in the process of constructing an animal model;

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;

Fig. 3 is the SSS semi-ligation operation in the process of building an animal model;

Fig. 4 is the operation of ferric chloride sticking in the process of building an animal model;

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;

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;

Fig. 7 is after confirming the thrombus formation in the experimental animal, suturing the incision;

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;

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;

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;

Fig. 11 is the variation of cerebral blood flow in the modeling process in the repeat experiment of the present invention;

Fig. 12 is the situation of cerebral venous thrombosis in the animal model in the stability test of the present invention.

Detailed ways

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.

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.

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.

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

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 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. 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. 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. 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. 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. 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. Suture the incision, disinfect the wound, put it back into the cage, and eat and drink normally (Figure 7).

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.

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.

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.

Example 2

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.

Table 2. Changes in cerebral blood flow at different stages during modeling

Example 3

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.

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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