A construction method for a novel animal model of brain metastases by carotid artery injection

Abstract

The present invention discloses a method for constructing a novel animal model of brain metastases by carotid artery injection, which relates to the field of animal model construction methods. The method comprises the following steps: 1) permanently ligating the external carotid artery; 2) temporarily blocking the proximal end of the common carotid artery; 3) injecting tumor cells into the common carotid artery; 4) after completion, temporarily blocking the distal end of the common carotid artery to prevent blood reflux; 5) repairing the injection puncture site with hydrogel; 6) sequentially releasing the blockades of the distal end and the proximal end of the common carotid artery. The modeling method of the present application takes a short time and has a low technical difficulty. By ligating the external carotid artery, the internal carotid artery is kept unobstructed, and there is no obvious change in the intracranial hemodynamics, greatly reducing the mortality rate and the postoperative cerebral infarction rate caused by the traditional modeling process, and being closer to the process of peripheral tumors entering the intracranial and colonizing under natural conditions.

Description

Technical Field

[0001] The present invention relates to the field of animal model construction, and particularly to a method for constructing a new animal model of brain metastases by carotid artery injection. Background Art

[0002] Compared with primary tumors, the biological mechanism of brain metastases is still unclear, and the research on brain metastases remains challenging. One of the important reasons is the lack of a suitable experimental model to accurately simulate the process of brain metastases. Currently, the widely used preclinical animal models of brain metastases mainly include tail vein injection, cardiac injection, intracranial in-situ injection, and carotid artery injection, etc. Each model has its own advantages and disadvantages, and the method of model production should be selected according to the main research questions. The characteristics of tail vein injection and cardiac injection are simple operation, but they have the characteristics of low intracranial tumor formation rate, and many mice die before the formation of intracranial tumors because metastases form in organs outside the central nervous system. The method of carotid artery injection has the highest brain metastases formation rate. Since it does not directly enter the brain, it can be included in the study of the blood-brain barrier, but the disadvantage is that researchers need good microsurgical techniques.

[0003] Generally, it is considered that the distribution of brain tumor lesions is closely related to cerebral blood perfusion, and the previous intracarotid injection brain metastases model requires permanent ligation of the common carotid artery. Due to the existence of the Willis arterial circle, permanent occlusion of one internal carotid artery does not necessarily lead to cerebral infarction, but chronic occlusion of the internal carotid artery will cause changes in intracranial hemodynamics, thereby affecting the distribution of tumor cells in the brain. At the same time, it may cause activation of some astrocytes in the brain, or lead to changes in the brain microenvironment, thus affecting the colonization of brain metastases in the brain to a certain extent.

[0004] In addition, studies have found that changes in brain microvessels have a protective effect on the development of lung cancer brain metastases. Once peripheral cancer cells enter the cerebral circulation, they may stay in the slow-flowing parts of the capillary bed at the vascular branch points, which is a necessary step for the formation of brain metastases. Then the cancer cells enter the microenvironment of the brain through the blood-brain barrier. In the previous intracarotid injection model, due to the occlusion of the internal carotid artery, it may increase the formation of tumor emboli. Even without severe ischemia, changes in intracranial hemodynamics may lead to abnormal distribution of peripheral tumor cells after entry. Here, peripheral tumor cells will encounter various cells and cytokines in the microenvironment. Studies have also found that astrocytes, microglia, neuronal cells, and immune cells in the microenvironment may affect tumor cells. Therefore, theoretically, changes in microenvironmental cells or cytokines may lead to changes in the formation of brain metastases.

[0005] So far, most studies on brain metastasis using the internal carotid artery injection model have ignored a series of problems caused by ligation of the internal carotid artery. Chronic occlusion of the internal carotid artery can cause changes in intracranial hemodynamics, thereby affecting the distribution of tumor cells in the brain. At the same time, it may cause activation of some astrocytes in the brain or lead to changes in the brain microenvironment, thus affecting the colonization of brain metastases in the brain to a certain extent. Summary of the Invention

[0006] The present invention provides a method for constructing a novel animal model of brain metastasis by carotid artery injection. Through improvement, the common carotid artery is repaired to maintain the patency of the internal carotid artery blood flow, so as to better simulate the process of peripheral tumor cells entering the intracranial cavity and colonizing under natural conditions.

[0007] To solve the above technical problems, an embodiment of the present invention provides a method for constructing a novel animal model of brain metastasis by carotid artery injection, including the following steps:

[0008] 1) Permanently ligate the external carotid artery;

[0009] 2) Temporarily block the proximal end of the common carotid artery;

[0010] 3) Inject tumor cells into the common carotid artery;

[0011] 4) After completion, temporarily block the distal end of the common carotid artery to prevent blood reflux;

[0012] 5) Repair the injection puncture site with hydrogel;

[0013] 6) Release the blockages at the distal and proximal ends of the common carotid artery in sequence.

[0014] By adopting the above scheme, the time required for model establishment in this application is short. The patency of the internal carotid artery is maintained by ligating the external carotid artery, and there is no obvious change in intracranial hemodynamics, greatly reducing the mortality rate and postoperative cerebral infarction rate caused by the traditional model establishment process, and being closer to the process of peripheral tumors entering the intracranial cavity and colonizing through the internal carotid artery under natural conditions; moreover, the technology of this application does not require high-difficulty microsurgical techniques such as suturing techniques. Utilizing the compression and repair effects of hydrogel, the damaged blood vessel is quickly repaired, which is easy to replicate and promote among researchers, thereby improving the success rate and being closer to the real natural hematogenous metastasis state.

[0015] As a preferred scheme, in the step 3), the injection volume of the tumor cells is 100 ul, the concentration is 2*10 5 / mL, and the injection time is more than 60 s.

[0016] As a preferred solution, in step 2), it also includes loosely tying a suture near the bifurcation at the distal end of the common carotid artery; in step 4), lift the suture at the distal end of the common carotid artery and tie a knot to temporarily block the blood vessel to prevent blood reflux at the distal end of the common carotid artery.

[0017] As a preferred solution, in step 5), drop 1 - 2 drops of the prepared hydrogel onto the puncture site, and after irradiating with a blue - light flashlight, determine that the hydrogel has solidified and adhered to the puncture site.

[0018] As a preferred solution, the hydrogel is a photocurable methacrylated hydrogel.

[0019] As a preferred solution, in step 6), first release the block at the distal end of the common carotid artery. If no blood back - flow is visible at the distal end, then carefully release the block at the proximal end of the common carotid artery. If no blood oozes out at this time, it indicates that the repair is successful, and clean the useless hydrogel around the blood vessel.

[0020] As a preferred solution, in step 2), temporarily block the proximal end of the common carotid artery with a vascular clamp.

[0021] As a preferred solution, before step 1), it also includes anesthesia with isoflurane gas.

[0022] As a preferred solution, before step 1) and after anesthesia, it also includes exposing the common carotid artery and the bifurcation of the common carotid artery.

[0023] As a preferred solution, exposing the common carotid artery and the bifurcation of the common carotid artery includes depilating the animal's neck, disinfecting, making a longitudinal midline incision, retracting the skin, exposing the salivary gland, retracting the salivary gland, exposing the trachea, incising the muscle on the surface of the internal carotid artery on the left side of the trachea, exposing the common carotid artery and the bifurcation of the common carotid artery, and sharply separating the vagus nerve beside the common carotid artery.

[0024] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0025] The time required for model establishment in this application is short, the technical means is of low difficulty. By ligating the external carotid artery, the patency of the internal carotid artery is maintained, and there is no obvious change in the intracranial hemodynamics. It greatly reduces the mortality rate and the postoperative cerebral infarction rate caused by the traditional model - establishment process. Utilizing the compression and repair effects of the hydrogel, it quickly repairs the damaged blood vessel, is easy to replicate and promote the technology among researchers, and is closer to the process of peripheral tumors entering the intracranial cavity and colonizing through the internal carotid artery under natural conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 - It is an operation schematic diagram of a method for constructing a novel animal model of brain metastases by carotid artery injection in the embodiments of the present invention;

[0027] Figure 2 -In vivo imaging results of the mouse brain for the construction method of a novel animal model of brain metastases by carotid artery injection in the embodiments of the present invention (Note: Fluorescent signals can be seen in the left brain of the mouse, indicating the formation of brain metastases);

[0028] Figure 3 -Results of fluorescence microscopy detection of the mouse brain for the construction method of a novel animal model of brain metastases by carotid artery injection in the embodiments of the present invention (Note: Since the tumor cells express GFP green fluorescent protein, the green fluorescent lesions can be seen under the stereoscopic fluorescence microscope as tumor metastasis foci, thereby judging the size, quantity, and distribution of brain metastasis foci in the brain);

[0029] Figure 4 -Results of IF immunofluorescence staining of the mouse brain for the construction method of a novel animal model of brain metastases by carotid artery injection in the embodiments of the present invention (Note: In the immunofluorescence results, the green fluorescent part is the tumorigenic focus of brain metastasis);

[0030] Figure 5 -Statistical results of mortality rates for the construction method of a novel animal model of brain metastases by carotid artery injection in the embodiments of the present invention and the existing traditional model construction method (Note: Left - existing traditional model construction method, upper right and lower right - embodiments of the present application);

[0031] Figure 6 -Statistical results of tumorigenesis rates for the construction method of a novel animal model of brain metastases by carotid artery injection in the embodiments of the present invention and the existing traditional model construction method (Note: Upper left and lower left - existing traditional model construction method, upper right and lower right - embodiments of the present application). Detailed implementation manners

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0033] Embodiment 1

[0034] A construction method of a novel animal model of brain metastases by carotid artery injection, as Figure 1 shown, includes the following steps:

[0035] 1. Research object: Select nod-scid mice at 6 - 10 weeks old.

[0036] 2. Tumor cell preparation: Construct the PC90GFP-Luc or A549-GFP-Luc cell line in advance. After resuscitation, passage the cells 2 - 3 times. On the day of tumor inoculation, digest the tumor cells with trypsin, count the cells, and dilute the tumor cells to 2*10 5 / mL with Hanks buffer, and place them on ice for later use.

[0037] 3. Anesthesia: Anesthetize the mice with isoflurane gas.

[0038] 4. Position and incision:

[0039] A) Expose the common carotid artery and the bifurcation of the common carotid artery in the mouse neck:

[0040] Depilate the mouse neck, disinfect it with iodophor. Make a longitudinal midline incision about 1 cm long. Retract the skin to expose the salivary gland, then retract the salivary gland to expose the trachea. Incise the muscle on the surface of the internal carotid artery on the left side of the trachea to expose the common carotid artery and its bifurcation. Sharply dissect the vagus nerve beside the common carotid artery;

[0041] B) Permanently ligate the external carotid artery:

[0042] Free the fascia between the bifurcations of the common carotid artery and ligate the external carotid artery with an 8-0 suture;

[0043] C) Temporarily block the proximal end of the common carotid artery:

[0044] Loosely tie the distal end of the common carotid artery near the bifurcation with an 8-0 suture to keep the arterial diameter unchanged. Temporarily block the proximal end of the common carotid artery 8 mm away from the bifurcation with a vascular clamp;

[0045] D) Inject tumor cells into the common carotid artery:

[0046] Prepare the tumor cells. After resuspension, aspirate them into a 1 ml syringe, expel the air bubbles, change the syringe needle to a 34G bevel needle. Elevate the common carotid artery by placing a wet cotton ball under it. Insert the syringe into the common carotid artery at an angle of about 15 degrees and slowly inject 100 ul of tumor cells resuspended in Hanks solution. The injection time should not be less than 60 s;

[0047] E) Temporarily block the distal end of the common carotid artery after injection:

[0048] After successful injection, withdraw the syringe needle. At the same time, lift the 8-0 suture at the distal end of the common carotid artery and tie a knot with a pair of microsurgical forceps held in both hands to temporarily block the blood vessel and prevent blood reflux from the distal end of the common carotid artery;

[0049] F) Repair the injection puncture site with hydrogel:

[0050] Immediately drop 1 - 2 drops of the pre - configured photo - curable methacrylated hydrogel (HAMA) aspirated into a 0.3 ml micro - syringe onto the puncture site. After irradiating with a blue - light flashlight for 15 s, judge that the hydrogel has solidified and adhered to the puncture site;

[0051] D) Release the occlusions at the distal and proximal ends of the common carotid artery in sequence:

[0052] Then, first loosen the ligature at the distal end of the common carotid artery. It can be seen that there is no blood return at the distal end of the common carotid artery. Then carefully release the vascular clamp at the proximal end of the common carotid artery. If there is no blood leakage at this time, it indicates successful repair. Clean the useless hydrogel around the blood vessel to avoid compressing the common carotid artery and causing vascular stenosis and reduced blood flow. It can be seen that there is blood flow through the common carotid artery and the pulsation is good, indicating successful repair. Reduce the routine suture of the skin.

[0053] 5. Post - operative treatment: Place the mouse on a warming pad to re - warm until the mouse wakes up. After the operation, place the mouse in an SPF barrier environment, feed and water it routinely, and closely observe the mental state, activity ability, and body weight of the mouse, etc. If there are situations such as a decrease in mental state, a weakening of activity ability, or a weight loss, perform in - vivo imaging to observe whether a tumor has formed. Generally, start in - vivo imaging observation about 1 month after modeling. As the modeling time extends, the tumor gradually grows. Determine the end - point of modeling according to the experimental purpose. Depending on whether the experimental purpose is to observe the size of the tumor formation or the survival period after brain metastasis of the mouse, the results are as Figure 2 shown; if a tumor forms, dislocate and sacrifice the mouse, perfuse PBS and PFA through the heart, take the brain, and take pictures under a fluorescence microscope to see the fluorescent part of the tumor in the brain. The results are as Figure 3 shown. Observe the distribution of the tumor, then dehydrate, fix, and embed the mouse brain with OCT, and perform IF immunofluorescence staining. The results are as Figure 4 shown.

[0054] 6. Result analysis:

[0055] Using the existing traditional model - building method as a control, perform permanent ligation on the common carotid artery. The specific operation is to inject tumor cells into the common carotid artery and then ligate the common carotid artery without repair. The time required for modeling in this application is short. After being proficient in the technique, it takes about 20 minutes or so. Moreover, the internal carotid artery remains unobstructed, and there is no obvious change in the intracranial hemodynamics, greatly reducing the mortality rate and the postoperative cerebral infarction rate caused by the traditional modeling process. As Figure 5 shown, compared with the mortality rate of 37.14% of the previous modeling methods, the mortality rate of modeling in this application is reduced to 5.71%, improving the success rate of modeling. At the same time, it improves the tumor formation rate of brain metastases. As Figure 6As shown, PC90GFP-Luc and A549-GFP-Luc were used as tumor cell samples respectively. The tumor formation rate of PC9 lung cancer cells increased from 50% of the traditional modeling method to 69.23%, and the tumor formation rate of A549 lung cancer cells increased from 40% of the traditional modeling method to 57.14%, which is closer to the process of peripheral tumors entering the intracranial cavity and colonizing through the internal carotid artery under natural conditions.

[0056] Moreover, the technology of this application does not require highly difficult microscopic operation techniques such as suturing techniques. By utilizing the compression and repair effects of the hydrogel, the damaged blood vessels can be rapidly repaired. It is easy to replicate and promote the technology among researchers, thereby improving the success rate of the internal carotid artery injection model for mouse brain metastases and being closer to the real natural hematogenous metastasis state.

[0057] In addition, this technology can also be applied to the process of injecting other tumor cells, neural stem cells or various drugs that require internal carotid artery injection through the internal carotid artery without sacrificing the internal carotid artery, thereby protecting the blood supply of the ipsilateral internal carotid artery to the greatest extent and minimizing the interference to scientific research caused by insufficient blood supply of the internal carotid artery.

[0058] The specific embodiments described above further elaborate on the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. It is particularly pointed out that for those skilled in the art, any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing an animal model of brain metastases by carotid artery injection, characterized in that, It includes the following steps: Step 1) Permanently ligate the external carotid artery; Step 2) Temporarily block the proximal end of the common carotid artery, and loosely tie it with a suture near the bifurcation at the distal end of the common carotid artery; Step 3) Inject tumor cells into the common carotid artery; Step 4) Lift the suture at the distal end of the common carotid artery to tie a knot to temporarily block the blood vessel and prevent blood reflux at the distal end of the common carotid artery; Step 5) Repair the injection puncture site with hydrogel, and the hydrogel is a photocurable methacrylated hyaluronic acid hydrogel; Step 6) First release the block at the distal end of the common carotid artery. If there is no blood return at the distal end, then carefully release the block at the proximal end of the common carotid artery. If there is no blood leakage at this time, it means the repair is successful, and clean the useless hydrogel around the blood vessel.

2. The construction method of an animal model of brain metastases by carotid artery injection according to claim 1, characterized in that, In step 3), the injection volume of the tumor cells is 100 μl, the concentration is 2×10 5 cells / mL, and the injection time is more than 60 s.

3. The construction method of an animal model of brain metastases by carotid artery injection according to claim 1, characterized in that, In the said Step 5), drop 1 - 2 drops of the prepared hydrogel onto the injection puncture site. After irradiating with a blue light flashlight, judge that the hydrogel has solidified and adhered to the injection puncture site, so as to repair the injection puncture site.

4. The construction method of an animal model of brain metastases by carotid artery injection according to claim 1, wherein, In the said Step 2), use a vascular clamp to temporarily block the proximal end of the common carotid artery.

5. The construction method of an animal model of brain metastases by carotid artery injection according to claim 1, characterized in that, Before the said Step 1), it also includes anesthesia with isoflurane gas.

6. The construction method of an animal model of brain metastases by carotid artery injection according to claim 1, wherein Before the said Step 1) and after anesthesia, it also includes exposing the common carotid artery and the bifurcation of the common carotid artery.

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