CN112970671B - Manufacturing method and application of minimally invasive thrombolytic stroke model - Google Patents
Manufacturing method and application of minimally invasive thrombolytic stroke model Download PDFInfo
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Abstract
The invention provides a method for manufacturing a minimally invasive thrombolytic stroke model, which comprises the following steps: anaesthetizing a mouse, placing a constant-temperature blanket under the mouse, cutting a cut at the middle position of the neck of the mouse, retracting sternocleidomastoid muscle, exposing common carotid artery and neck bifurcation, placing a magnet under the common carotid artery, temporarily clamping external carotid artery by a titanium aneurysm clip, absorbing configured thrombin magnetic beads, injecting the thrombin magnetic beads into tail vein, forming carotid artery thrombus by the thrombin magnetic beads gathered at the common carotid artery and flowing blood, removing the magnet, slightly clamping the thrombus by forceps, moving upwards, suturing the neck skin, and forming a cerebral apoplexy model. The method is simple and convenient to operate, saves time, and can improve the success rate of model manufacturing. Compared with the traditional operation method of the cerebral apoplexy model, the invention reduces the operation time, improves the success rate of the operation and lays a solid foundation for the pathogenesis of cerebral ischemia and the screening of medicines.
Description
Technical Field
The invention relates to the technical field of medical models, in particular to a manufacturing method and application of a minimally invasive thrombolytic stroke model.
Background
The brain is one of the vital organs of human beings, and its weight is only 2% of the body weight, but its oxygen consumption accounts for 20% of the total oxygen consumption, and its blood supply accounts for 15% of the cardiac output, so the demand for oxygen and blood is particularly high, and it is very vulnerable to ischemic damage, among which cerebral ischemia caused by middle cerebral artery infarction is especially common. The statistical data provided by the official website of the Ministry of health of the people's republic of China shows that cerebrovascular diseases rank the second in the death causes of diseases in cities and rural areas, and are second to malignant tumors. The cerebrovascular diseases include ischemic cerebrovascular diseases and hemorrhagic cerebrovascular diseases, wherein the ischemic cerebrovascular diseases account for 80-85%. The middle cerebral artery is the site of a common hemorrhage or infarction. The preparation of the model for simulating the cerebral ischemia has important significance on the pathogenesis of the cerebral ischemia and the drug screening.
The most classical model of stroke at present is the middle cerebral artery embolus (MCAO), which is an abbreviated form of midle cerebral artery occlusion, in chinese meaning the middle cerebral artery embolism. The middle cerebral artery occlusion model is a cranial plug inserted through the internal carotid artery and into the middle cerebral artery, thereby occluding the blood supply to the middle cerebral artery supplying one hemisphere of the brain. Described briefly as inserting a fish line from the carotid artery all the way to the location of the middle cerebral artery. However, the model can not simulate real cerebral infarction thrombosis and thrombolytic therapy. Therefore, for the application of thrombolytic drug therapy, it is only an option to inject thrombin (promoting thrombosis) into the part of the middle cerebral artery bifurcation after craniotomy, or to form a blood clot in vitro, to be injected from the internal carotid artery, and to embolize with the blood stream at the middle cerebral artery. The two manufacturing methods are complex and have high animal mortality.
Disclosure of Invention
The invention aims to provide a method for manufacturing a minimally invasive thrombolytically-dissolvable stroke model, which is simple and convenient to operate, saves time, can improve the success rate of model manufacturing, and also provides application of the minimally invasive thrombolytically-dissolvable stroke model.
The invention provides a method for manufacturing a minimally invasive thrombolytic stroke model, which comprises the following steps:
anaesthetizing the mouse, placing a constant-temperature blanket under the mouse, cutting a cut at the middle position of the neck of the mouse, retracting sternocleidomastoid muscle, and exposing common carotid artery and neck bifurcation;
placing a magnet below a common carotid artery, temporarily clamping an external carotid artery by using a titanium aneurysm clip, sucking thrombin magnetic beads, injecting the thrombin magnetic beads into a tail vein, wherein the thrombin magnetic beads are magnetic beads with thrombin formed by the reaction of carboxylated magnetic beads and the thrombin, forming carotid thrombus with the thrombin magnetic beads gathered at the common carotid artery and flowing blood, removing the magnet, slightly clamping the thrombus by using forceps, and moving upwards;
and (5) sewing the neck skin to form a stroke model.
Preferably, when the mouse is anesthetized, the mouse is placed in an anesthesia induction box of an aeroanesthesia machine, and an anesthetic is introduced.
Preferably, the anesthetic is isoflurane, the flow rate is 1.5% -2%, the temperature of the thermostatic blanket is set to be 37 +/-0.2 ℃, the temperature influences the formation of thrombus, and the temperature needs to be controlled.
Preferably, the volume of the thrombin magnetic beads is 150ul-160ul.
Preferably, the time for the formation of the common carotid artery thrombosis is 3-5min.
Preferably, the knife edge is 1-1.5cm in length, and the magnet is circular and 2mm in diameter.
Preferably, the preparation method of the thrombin magnetic bead comprises the following steps:
s1, magnetic bead activation: mixing and activating carboxylated magnetic beads by a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride reagent and an N-hydroxysuccinimide reagent, then placing the mixture into a 2- (N-morpholine) ethanesulfonic acid buffer solution, and uniformly mixing to obtain activated magnetic beads;
s2, linking thrombin and activated magnetic beads: washing the activated magnetic beads obtained in the step S1 by using a 2- (N-morpholine) ethanesulfonic acid buffer solution, then adding thrombin, and uniformly mixing;
s3, sealing the unconnected carboxyl of the magnetic beads: placing the magnetic beads obtained in the step S2 on a magnetic frame, removing a supernatant by a liquid moving machine, and adding a 2- (N-morpholine) ethanesulfonic acid buffer solution and serum protein;
s4, cleaning and storing: and (4) placing the magnetic beads obtained in the step (S3) on a magnetic frame, discarding the supernatant, and adding phosphate buffer solution to obtain thrombin magnetic beads.
The carboxyl in the carboxylated magnetic beads reacts with the amino in the thrombin.
Preferably, the carboxylated magnetic beads are washed before the magnetic beads are activated, and the washing manner of the carboxylated magnetic beads is as follows: washing the carboxylated magnetic beads with 2- (N-morpholine) ethanesulfonic acid buffer solution, placing the washed carboxylated magnetic beads on a magnetic frame, gathering the carboxylated magnetic beads at the bottom of a test tube, and then sucking the supernatant by using a pipettor; the pH value of the 2- (N-morpholine) ethanesulfonic acid buffer solution is 6.5, the concentration is 0.05mol/L, and the volume ratio of the 2- (N-morpholine) ethanesulfonic acid buffer solution to the carboxylated magnetic beads in the washing process is 5.
Preferably, the particle size of the carboxylated magnetic beads in the step S1 is 100-120nm, and the concentration is 5mg/ml; the molar ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride reagent to the N-hydroxysuccinimide in step S1 is 1; can activate carboxyl efficiently.
Preferably, the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride reagent to the carboxylated magnetic beads in the step S1 is 1; the temperature for mixing in the step S1 is 20-25 ℃, and the time is 3-5h.
Preferably, the ratio of thrombin in the step S2 to carboxylated magnetic beads in the step S1 is 50-100, the thrombin is ug, and the carboxylated magnetic beads are uL; the temperature for uniformly mixing in the step S2 is 20-25 ℃, and the time is 6-8h.
Preferably, the mass ratio of the serum protein in the step S3 to the thrombin in the step S2 is 1000.
The invention also provides application of the minimally invasive thrombolytic stroke model prepared by the preparation method in preclinical research, treatment method and/or pathogenesis research of stroke treatment drugs. The cerebral apoplexy treatment drug is a commercially available thrombolytic drug.
The invention has the beneficial effects that:
(1) At present, the classic suture tying method needs to ligate blood vessels and insert a thread into the blood vessels; the magnet is arranged on the side of the blood vessel, and thrombin magnetic beads are gathered in the internal carotid artery by the magnet, so that the blood vessel is embolized upwards, the operation is convenient, ligation and insertion of a thread into the blood vessel are not needed, and the blood vessel is not damaged.
(2) Compared with the traditional operation method of the cerebral apoplexy model, the invention reduces the operation time, improves the success rate of the operation and lays a solid foundation for the pathogenesis of cerebral ischemia and the drug screening.
(3) The magnetic embolus can be dissolved by thrombolytic drug, and can better simulate physiological reaction brought by blood recanalization.
Drawings
Fig. 1 is a schematic diagram of an operation of the present invention for making a stroke model;
FIG. 2 is a laser speckle blood flow analysis chart of the brain before and after the operation of making a stroke model according to the present invention;
fig. 3 is a graph of stroke model behavior scores;
FIG. 4 is a graph comparing the operation time of the line-tying method and the stroke modeling method of the present invention;
FIG. 5 is an electron microscope image of magnetic thrombin beads used in the manufacturing method of the present invention;
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for manufacturing a minimally invasive thrombolytic stroke model comprises the following steps:
mouse anesthesia: placing the mouse in an anesthesia induction box of an aeroanesthesia machine, regulating the flow of isoflurane by 2 percent, and waiting for the mouse to be completely anesthetized;
mouse preoperative preparation: horizontally placing the anesthetized mouse on an operating table, placing a constant-temperature blanket at the temperature of 37 +/-0.2 ℃, removing neck hair, and continuously performing isoflurane inhalation anesthesia;
exposure of carotid artery: cutting a cut with the length of about 1cm from the middle of the neck of the mouse, retracting sternocleidomastoid muscle, and exposing common carotid artery and cervical bifurcation;
placing a magnet: placing a circular magnet with the diameter of 2mm below a common carotid artery, and temporarily clamping an external carotid artery by a titanium aneurysm clip;
injecting thrombin magnetic beads into tail vein, namely sucking 160ul thrombin magnetic beads (about 0.80 mg) and injecting the thrombin magnetic beads into tail vein;
thrombosis, namely forming thrombus by the magnetic beads gathered at the common carotid artery and flowing blood, and requiring 3min;
thrombus blocks the middle cerebral artery: removing the magnet, slightly clamping the thrombus by the forceps, moving upwards, and entering cerebral artery;
and (5) sewing the skin to form a stroke model.
The preparation process of the thrombin magnetic bead comprises the following steps:
s1, magnetic bead cleaning: washing 200uL of carboxylated magnetic beads with 1mL of 2- (N-morpholine) ethanesulfonic acid buffer solution for three times, treating the magnetic beads on a magnetic frame for 1min, gathering the magnetic beads at the bottom of a test tube, and removing impurities by sucking and removing supernatant with a pipettor; wherein the particle size of the carboxylated magnetic beads is 100nm, the concentration is 5mg/mL, and the carboxylated magnetic beads are a commercially available product, and the brand is Aladdin;
s2, magnetic bead activation: mixing and activating the washed carboxylated magnetic beads by using a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride reagent and an N-hydroxysuccinimide reagent, wherein the molar ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride reagent to the N-hydroxysuccinimide reagent is 1, the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride reagent to the carboxylated magnetic beads in the step S1 is 1;
s3, linking thrombin and activated magnetic beads: washing the activated magnetic beads once by using 2- (N-morpholine) ethanesulfonic acid buffer solution, then adding 80ug of thrombin, and adding into a mixing machine to mix uniformly at 25 ℃ for 8h, wherein the thrombin is a commercially available product and is called Solibao;
s4, blocking the unconnected carboxyl of the magnetic beads: and (3) placing the magnetic beads prepared in the S3 on a magnetic frame, removing a supernatant by a liquid moving machine, adding 2- (N-morpholine) ethanesulfonic acid buffer solution and 1ml of serum protein, and sealing the unbound active groups on the surfaces of the magnetic beads.
S5, cleaning and storing: and (3) placing the magnetic beads prepared in the step (S4) on a magnetic frame, removing the supernatant, adding phosphate buffer solution to obtain thrombin magnetic beads, and placing and storing the thrombin magnetic beads at 4 ℃ for use as soon as possible.
The pH of the buffer solution of 2- (N-morpholine) ethanesulfonic acid used in the preparation of the thrombin magnetic beads was 6.5, and the concentration was 0.05mol/L.
Example 2
A method for manufacturing a minimally invasive thrombolysis cerebral apoplexy model comprises the following steps:
anesthetizing the mouse: placing the mouse in an anesthesia induction box of an aeroanesthesia machine, regulating the flow of isoflurane by 1.5 percent, and waiting for the mouse to be completely anesthetized;
mouse preoperative preparation: horizontally placing the anesthetized mouse on an operating table, placing a constant temperature blanket at the temperature of 37 +/-0.2 ℃, removing the hair on the neck, and continuously performing isoflurane inhalation anesthesia;
exposure of carotid artery: cutting a cut with the length of about 1.5cm from the middle of the neck of the mouse, and retracting sternocleidomastoid muscle to expose common carotid artery and cervical bifurcation;
placing a magnet: placing a magnet with the diameter of 2mm below a common carotid artery, and temporarily clamping an external carotid artery by a titanium aneurysm clip;
injecting thrombin magnetic beads into the tail vein, namely sucking 150ul of prepared thrombin magnetic beads, about 0.75mg of prepared thrombin magnetic beads, and injecting the thrombin magnetic beads into the tail vein;
thrombosis, wherein the magnetic beads gathered at the common carotid artery and flowing blood form thrombus, and the thrombus needs 5min;
thrombus blocks the middle cerebral artery: removing the magnet, and lightly clamping the thrombus by the forceps to enter the cerebral artery;
and (5) sewing the skin to form a stroke model.
Wherein the preparation process of the thrombin magnetic bead comprises the following steps:
s1, magnetic bead cleaning: washing 200uL of carboxylated magnetic beads with 1mL of 2- (N-morpholine) ethanesulfonic acid buffer solution for three times, treating the magnetic beads on a magnetic frame for 1min, gathering the magnetic beads at the bottom of a test tube, and removing impurities by sucking and removing supernatant with a pipettor; wherein the particle size of the carboxylated magnetic beads is 100nm, the concentration is 5mg/mL, and the carboxylated magnetic beads are a commercially available product, and the brand is Aladdin;
s2, magnetic bead activation: mixing and activating the washed carboxylated magnetic beads by using a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride reagent and an N-hydroxysuccinimide reagent, wherein the molar ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride reagent to the N-hydroxysuccinimide reagent is 1, the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride reagent to the carboxylated magnetic beads in the step S1 is 1;
s3, linking thrombin and activated magnetic beads: washing the activated magnetic beads once by using 2- (N-morpholine) ethanesulfonic acid buffer solution, then adding 80ug of thrombin, and adding into a mixing machine to mix uniformly at 25 ℃ for 8h, wherein the thrombin is a commercially available product and is called Solibao;
s4, blocking the unconnected carboxyl of the magnetic beads: and (3) placing the magnetic beads prepared in the S3 on a magnetic frame, removing a supernatant by a liquid moving machine, adding 2- (N-morpholine) ethanesulfonic acid buffer solution and 1ml of serum protein, and sealing the unbound active groups on the surfaces of the magnetic beads.
S5, cleaning and storing: and (3) placing the magnetic beads prepared in the step (S4) on a magnetic frame, removing the supernatant, adding phosphate buffer solution to obtain thrombin magnetic beads, and placing and storing the thrombin magnetic beads at 4 ℃ for use as soon as possible.
The pH of the 2- (N-morpholine) ethanesulfonic acid buffer solution used in the preparation process of the thrombin magnetic beads is 6.5, and the concentration is 0.05mol/L.
Fig. 1 is a schematic diagram of an operation of making a stroke model according to the present invention.
Fig. 2 is a diagram of laser speckle blood flow analysis of the brain, and it is apparent that thrombus is successfully formed at cerebral arteries after the stroke modeling operation of the present invention, and blood supply to the brain portion is prevented.
As can be seen from fig. 3, after the stroke model is respectively manufactured by the conventional wire-embolism method and the method adopted by the present invention, the behavior of the mouse is scored, and after thrombolysis, the behavior of the mouse is scored, the lower the score is, the lighter the stroke disease is, the behavior score of the mouse of the wire-embolism stroke model is close to the behavior score of the mouse of the thrombin magnetic bead stroke model, and no statistical difference is generated, but after thrombolysis, the mouse of the thrombin magnetic bead stroke model has better recovery, and is more favorable for researching various physiological reactions after stroke and thrombolysis by drugs.
In fig. 4, the time required by the method for preparing the stroke model by the thrombin magnetic beads adopted by the invention is obviously shorter than the time required by preparing the stroke model by the thread-plug method, thereby reducing the operation time and laying a solid foundation for the pathogenesis of cerebral ischemia and drug screening.
FIG. 5 is an electron microscope image of magnetic beads of thrombin used in the manufacturing method of the present invention, and it can be seen from the image that the carboxylated magnetic beads are fully bonded with thrombin, and then dispersed and have uniform particle size.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1. A method for manufacturing a minimally invasive thrombolytic stroke model is characterized by comprising the following steps:
anaesthetizing the mouse, placing a constant temperature blanket under the mouse, retracting sternocleidomastoid muscle, and exposing common carotid artery and neck bifurcation;
placing a magnet below a common carotid artery, temporarily clamping an external carotid artery by using a titanium aneurysm clip, sucking thrombin magnetic beads, injecting the thrombin magnetic beads into a tail vein, wherein the thrombin magnetic beads are magnetic beads with thrombin formed by the reaction of carboxylated magnetic beads and thrombin, the thrombin magnetic beads are gathered at the common carotid artery close to the magnet, the thrombin magnetic beads gathered at the common carotid artery and flowing blood form carotid thrombus, and the magnet is removed, and the thrombus is lightly clamped by forceps and moves upwards; thereby forming a stroke model;
the volume of the thrombin magnetic beads is 150ul-160ul; the temperature of the constant temperature blanket is set to be 37 +/-0.2 ℃; the time for forming the thrombus of the common carotid artery is 3-5min; the magnet is circular, and the diameter is 2mm.
2. The method for preparing a stroke model of the minimally invasive thrombolytic of claim 1, wherein the method for preparing the thrombin magnetic beads comprises the following steps:
s1, magnetic bead activation: mixing and activating carboxylated magnetic beads by a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride reagent and an N-hydroxysuccinimide reagent, then placing the mixture into a 2- (N-morpholine) ethanesulfonic acid buffer solution, and uniformly mixing to obtain activated magnetic beads;
s2, linking thrombin and activated magnetic beads: washing the activated magnetic beads obtained in the step S1 by using a 2- (N-morpholine) ethanesulfonic acid buffer solution, then adding thrombin, and uniformly mixing;
s3, sealing the unconnected carboxyl of the magnetic beads: placing the magnetic beads obtained in the step S2 on a magnetic frame, removing a supernatant by a liquid moving machine, and adding a 2- (N-morpholine) ethanesulfonic acid buffer solution and serum protein;
s4, cleaning and storing: and (4) placing the magnetic beads obtained in the step (S3) on a magnetic frame, discarding the supernatant, and adding phosphate buffer solution to obtain thrombin magnetic beads.
3. The method for preparing stroke model of minimally invasive thrombolytic of claim 2, wherein the carboxylated magnetic beads are washed before the magnetic beads are activated, and the washing method of the carboxylated magnetic beads is as follows: washing the carboxylated magnetic beads with 2- (N-morpholine) ethanesulfonic acid buffer solution, placing the washed carboxylated magnetic beads on a magnetic frame, gathering the carboxylated magnetic beads at the bottom of a test tube, and then sucking the supernatant by using a pipettor; the pH value of the 2- (N-morpholine) ethanesulfonic acid buffer solution is 6.5, the concentration is 0.05mol/L, and the volume ratio of the 2- (N-morpholine) ethanesulfonic acid buffer solution to the carboxylated magnetic beads in the washing process is 5.
4. The method for preparing a stroke model with minimally invasive thrombolytic effect according to claim 2, wherein the carboxylated magnetic beads in step S1 have a particle size of 100-120nm and a concentration of 5mg/ml; the molar ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride reagent to the N-hydroxysuccinimide in step S1 is 1; the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride reagent to the carboxylated magnetic beads in the step S1 is 1; the temperature for mixing in the step S1 is 20-25 ℃, and the time is 3-5h.
5. The method for preparing the minimally invasive thrombolytic stroke model according to claim 2, wherein the ratio of the thrombin in step S2 to the carboxylated magnetic beads in step S1 is 50-100, 200, ug, uL; the temperature for uniformly mixing in the step S2 is 20-25 ℃, and the time is 6-8h.
6. The method for preparing a stroke model of minimally invasive thrombolytic according to claim 2, wherein the mass ratio of the serum protein in step S3 to the thrombin in step S2 is 1000 to 100.
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