Construction method of cerebral artery ischemia reperfusion model

Abstract

The invention discloses a construction method of a cerebral artery ischemia reperfusion model, and belongs to the technical field of animal models. Three-dimensional positioning coordinates of the middle cerebral artery are obtained through magnetic resonance scanning, the far end of the middle cerebral artery can be accurately positioned, the middle cerebral artery is induced to be blocked after irradiation by setting the blood vessel irradiation position, the rose red dosage and the laser irradiation intensity and time, blood vessel recanalization is caused along with blood impact within 4-5 hours, and finally an ischemia reperfusion model is formed. Compared with a traditional craniotomy electrocoagulation middle cerebral artery (permanent occlusion) and a traditional balloon embolism middle cerebral artery (ischemia reperfusion), the construction method has the advantages that wounds on animals are smaller, the animals are easy to survive, and compared with traditional photochemical induction which can only form infarction lesions on the surface layer of the brain, the method can induce deep brain tissue to form infarction, so that the method has the advantages of being simple and convenient to operate and the like. The method can also be expanded to other brain areas to induce deep vascular occlusion. Meanwhile, the method is also suitable for carrying out model construction on old animals, and the modeling success rate is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models, and more particularly to a method for constructing a cerebral artery ischemia-reperfusion model. Background Art

[0002] The effects of ischemic stroke on brain function and other systems are complex and varied, making it impossible to simulate this disease in vitro. Therefore, common experimental animals represented by rodents, canines, rabbits, pigs, and non-human primates are used to establish animal models of cerebral ischemia. Among them, rodents are widely used in animal models of cerebral thrombosis due to their low cost, abundant sources, and simple operation. However, there are still certain differences between rodents and non-human primates in terms of body size, hemodynamics, brain structure and function, inflammatory response, etc., which leads to the failure of many therapies that are effective in rodents in clinical trials. The brain of non-human primates is usually composed of multiple gyri, with a similar cortical functional column structure to that of humans and a complete cerebral artery circuit similar to that of humans. The distribution of its internal carotid artery and vertebral artery is also very similar to that of humans. Therefore, the establishment of a non-human primate cerebral ischemia animal model and the monitoring of cerebrovascular and brain functional networks in the ischemic penumbra area have very important clinical translational application significance.

[0003] At present, the methods reported at home and abroad for constructing local cerebral ischemia models in non-human primates can generally be divided into three categories: surgical method, intravascular intervention method and photochemical method. Among them, ① surgical method includes the commonly used electrocoagulation method, endothelin-1 induced occlusion method and microvascular clip occlusion method. The above modeling methods can accurately locate the infarction site with high repeatability, but excessive surgical trauma has a great impact on the physiological and psychological images of animals, which is not conducive to the subsequent behavioral evaluation. At the same time, the risk of surgical failure is high and it is easy to cause animal death. It is not suitable for constructing models in elderly animals. In addition, due to the large individual differences in animal cerebral vascular collateral circulation, it is difficult to control the range and volume of the infarction focus; ② intravascular intervention method includes autologous thromboembolism method, balloon Embolization method, microcatheter embolization method, etc. The above modeling methods are less traumatic to animals, and the modeling mechanism is similar to clinical vascular embolism, but the location and volume of the infarction are difficult to determine, and the surgical techniques and equipment are high and difficult to carry out; ③ Photochemical induction method is a commonly used ischemic stroke modeling technology in rodents. In recent years, it has also gradually begun to be used in non-human primates. The advantage of photochemical induction method is that it uses non-invasive operation, minimal animal damage, precise and stable infarction range, and the process of photothrombosis formation is similar to cerebral embolism. However, its biggest disadvantage is that the traditional photochemical method can only induce the formation of photothrombosis in the terminal blood vessels of the brain (brain surface), and the infarction focus can only be formed on the brain surface, and the model cannot simulate the common clinical cerebral ischemia-reperfusion characteristics.

[0004] Therefore, how to provide a cerebral ischemia-reperfusion model for inducing infarction in deep brain tissue is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In response to the above problems, the present invention provides a method for constructing a cerebral artery ischemia-reperfusion model, which can induce infarction in deep brain tissue. At the same time, it can be expanded to other brain regions to induce deep vascular occlusion, which not only improves the success rate of modeling, but is also suitable for model construction in elderly animals.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for constructing a cerebral artery ischemia-reperfusion model comprises the following steps:

[0008] (1) Three-dimensional coordinate construction: Obtain medical images of the horizontal, sagittal, and coronal planes of brain magnetic resonance imaging, perform three-dimensional segmentation, and determine the X, Y, and Z three-dimensional coordinates of the blood vessels;

[0009] (2) Construction of cerebral artery ischemia-reperfusion model: The sample is fixed on a brain stereotaxic apparatus, and the sample is processed according to the X, Y, and Z three-dimensional coordinates of the blood vessels in step (1) before laser irradiation;

[0010] (3) After step (2) was completed, the sample was processed. Three days after modeling, magnetic resonance T2WI sequence scanning imaging showed high signals around the laser irradiation position, indicating that the modeling was successful.

[0011] Preferably, the scanning sequence used in the magnetic resonance imaging in step (1) is t2 mx3d sag, and the parameters are: TR=3400ms, TE=398ms, and layer thickness=0.5mm.

[0012] Preferably, the method for determining the X coordinate of the blood vessel in step (1) is as follows: in the horizontal section view, a reference line between the glabella and the internal occipital protuberance is parallel to the sagittal suture of the skull, and the plane formed by the two lines is the median horizontal plane of the brain, and the above-mentioned reference line is the midline of the brain, and the horizontal distance from the blood vessel to the midline of the brain is the X coordinate.

[0013] Preferably, the method for determining the Y coordinate of the blood vessel in step (1) is as follows: after determining the position of the blood vessel in the sagittal view, the blood vessel is located on the midline of the brain, and the vertical distance from the lowest point between the frontal bone and the eyebrow arch to the cerebral blood vessel is taken as the Y coordinate.

[0014] Preferably, the method for determining the Z coordinate of the blood vessel in step (1) is as follows: in the coronal image, after the blood vessel position is determined, the vertical distance from the dura mater to the cerebral blood vessel is the Z coordinate.

[0015] Preferably, the treatment in step (2) is injection of Rose Bengal.

[0016] Preferably, the treatment in step (2) is injection of antibiotics and mannitol to maintain intracranial pressure.

[0017] Through the above technical solutions, it can be known that the present invention discloses a method for constructing a cerebral artery ischemia-reperfusion model. The three-dimensional positioning coordinates of the middle cerebral artery of the experimental sample are obtained by magnetic resonance scanning, and the distal end of the middle cerebral artery can be accurately located. By setting the vascular irradiation position, rose bengal dose, laser irradiation intensity and time, the middle cerebral artery is induced to be occluded after irradiation, and the blood vessel is recanalized with blood shock within 4-5 hours, and finally an ischemia-reperfusion model is formed.

[0018] Compared with the traditional craniotomy electrocoagulation of the middle cerebral artery (permanent occlusion) and balloon embolization of the middle cerebral artery (ischemia-reperfusion), the construction method of the present invention is less traumatic to animals and is easy for animals to survive. Compared with the traditional photochemical induction that can only form infarction foci on the surface of the brain, the method of the present invention can induce infarction in deep brain tissues and can also be extended to other brain regions to induce deep vascular occlusion. At the same time, it is also suitable for model construction for elderly animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Horizontal section of the monkey brain magnetic resonance image with the X coordinate determined;

[0020] Figure 2 : Sagittal MRI image of the monkey brain to determine the Y coordinate;

[0021] Figure 3 : Coronal MRI images of the monkey brain to determine the Z coordinate;

[0022] Figure 4 : MRI scans of the successful establishment of the deep tissue ischemia-reperfusion model in the monkey brain, a: T2WI sequence scanning brain three-dimensional reconstruction, b: T2WI sequence scanning brain cross-section, c: T2WI sequence scanning brain sagittal plane, d: T2WI sequence scanning brain coronal plane;

[0023] Figure 5: Time-flight magnetic resonance imaging images of target blood vessel blood flow from before modeling to 28 days after modeling, A: Vascular imaging before modeling, 1h, 5h and 3d after modeling, B: Vascular imaging 7d, 14d, 21d, 28d after modeling, red arrows indicate the location of blood vessels;

[0024] Figure 6 : Analysis of brain functional network connectivity and white matter nerve fiber conditions before modeling, 4 hours after modeling, and 3 days after modeling;

[0025] Figure 7: Magnetic resonance imaging was used to evaluate the phenotype of cerebral infarction before modeling, and at 5 hours, 3 days, 7 days, and 14 days after modeling. The red arrows indicate high signal, the blue arrows indicate low signal, and the purple circle indicates the position of the laser-irradiated blood vessel. The purple circle was used as the ROI, and the average signal intensity within the circle was measured.

[0026] Figure 8 : Weighted imaging of magnetic resonance T2WI sequence scans 14 days after modeling, where red: damaged area;

[0027] Fig. 9A : Anatomical image of the experimental monkey brain 90 days after modeling, red circle: infarct focus;

[0028] Fig. 9B :Magnetic resonance T2WI sequence structural scan, red circle position: infarction focus;

[0029] Fig.10 : Comparison of HE staining, GFAP staining and IBA1 staining results of brain tissue sections of experimental monkeys 90 days after modeling (×100um), red dotted line: dividing line, the left side is the infarct focus, and the right side is normal,

[0030] A: HE staining, green arrows: neurons, blue arrows: glial cells,

[0031] B: GFAP staining, red arrows: activated astrocytes,

[0032] C: IBA1 staining, red arrows: activated microglia. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The reagents involved in the embodiments of the present invention are all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0035] A method for constructing a cerebral artery ischemia-reperfusion model comprises the following steps:

[0036] (1) Three-dimensional coordinate construction: Obtain medical images of the horizontal, sagittal, and coronal planes of brain magnetic resonance imaging, perform three-dimensional segmentation, and determine the X, Y, and Z three-dimensional coordinates of the blood vessels;

[0037] (2) Construction of cerebral artery ischemia-reperfusion model: The sample is fixed on a brain stereotaxic apparatus, and the sample is processed according to the X, Y, and Z three-dimensional coordinates of the blood vessels in step (1) before laser irradiation;

[0038] (3) After step (2) was completed, the sample was injected with antibiotics and mannitol for treatment. Three days after modeling, magnetic resonance T2WI sequence scanning imaging showed high signals around the laser irradiation position, indicating that the modeling was successful.

[0039] Preferably, the scanning sequence used in the magnetic resonance imaging in step (1) is t2 mx3d sag, and the parameters are: TR=3400ms, TE=398ms, and layer thickness=0.5mm.

[0040] Preferably, the method for determining the X coordinate of the blood vessel in step (1) is as follows: in the horizontal section view, a reference line between the glabella and the internal occipital protuberance is parallel to the sagittal suture of the skull, and the plane formed by the two lines is the median horizontal plane of the brain, and the above-mentioned reference line is the midline of the brain, and the horizontal distance from the blood vessel to the midline of the brain is the X coordinate.

[0041] Preferably, the method for determining the Y coordinate of the blood vessel in step (1) is as follows: after determining the position of the blood vessel in the sagittal view, the blood vessel is located on the midline of the brain, and the vertical distance from the lowest point between the frontal bone and the eyebrow arch to the cerebral blood vessel is taken as the Y coordinate.

[0042] Preferably, the method for determining the Z coordinate of the blood vessel in step (1) is as follows: in the coronal image, after the blood vessel position is determined, the vertical distance from the dura mater to the cerebral blood vessel is the Z coordinate.

[0043] Preferably, the sample in step (2) is an adult monkey or an elderly monkey.

[0044] Preferably, the treatment in step (2) is injection of Rose Bengal.

[0045] Preferably, the injection amount of Rose Bengal is: 35 mg of Rose Bengal per kilogram of experimental monkey.

[0046] Preferably, the laser irradiation in step (2) is: using a laser with a power of 15 mW for 30 minutes.

[0047] Example 1 Construction of cerebral artery ischemia-reperfusion model

[0048] Twelve-year-old female cynomolgus monkeys were selected as experimental animals.

[0049] (1) After the monkey was anesthetized, it was made to lie flat on the MRI machine. The monkey's head was raised with a pad so that its face was facing upwards and the monkey's lower eye socket was flush with the middle of the ear bar. The 3.0T MRI of the monkey was selected using the United Imaging UMR 790. The t2 mx3d sag scanning sequence parameters were set to TR = 3400ms, TE = 398ms, and the slice thickness = 0.5mm. MRI scans were performed to obtain horizontal, sagittal, and coronal MRI images of the monkey's brain structure.

[0050] The measurement tools of UIH Viewer software were used to perform three-dimensional medical image segmentation on the above-mentioned magnetic resonance images to determine the X, Y, and Z coordinates. The specific process was as follows:

[0051] Determination of X coordinate: In the horizontal section of the monkey brain MRI, the reference line between the glabella and the internal occipital protuberance is parallel to the sagittal suture of the monkey's skull. The plane formed by the two lines is the median horizontal plane of the brain. The above reference line is the brain midline, and the horizontal distance from the blood vessel to the brain midline is the X coordinate, see Figure 1 ;

[0052] Determination of Y coordinate: In the sagittal MRI image of the monkey brain, after determining the position of the blood vessel, make the blood vessel located on the midline of the brain, and take the vertical distance from the lowest point between the frontal bone and the eyebrow arch to the cerebral blood vessel as the Y coordinate. Figure 2 ;

[0053] Determination of Z coordinate: In the coronal image of the monkey brain, after the blood vessel position is determined, the vertical distance from the dura mater to the cerebral blood vessels is the Z coordinate, see Figure 3 .

[0054] (2) After the above-mentioned experimental monkeys were allowed free access to water and fasted for 12 hours, 20 mg of ketamine, 2 mg of sulfanilamide and 5 mg of atropine were injected per kg of experimental animal body weight to induce anesthesia. After that, the monkeys were fixed on a stereotaxic apparatus. During this period, a respiratory anesthesia machine was used to maintain anesthesia (0.6%) and body temperature at 35-38°C. The skin of the skull top was prepared and the skin was routinely disinfected.

[0055] According to the X, Y, and Z three-dimensional coordinates of the blood vessels that have been obtained, an incision about 1 to 2 cm long is made on the scalp just above the coordinate position, and the subcutaneous tissue, tendons, muscles, periosteum, etc. are separated layer by layer. The soft tissue is fully expanded with an expander to expose the skull, and a portable cranial drill is used to drill a hole in the skull until the skull is penetrated and the dura mater is reached.

[0056] The laser generator was connected to the optical fiber and fixed on the brain stereotaxic instrument. According to the three-dimensional coordinates, the front end of the optical fiber was inserted into the located blood vessel through the small hole. The wound was covered with sterile gauze soaked in saline around the small hole. Rose Bengal was injected intravenously (the injection volume was 35 mg / kg experimental monkey). Three minutes after the injection, a laser with a power of 15 mW was used for 30 minutes of irradiation. After the irradiation was completed, the light source was removed and the periosteum and skin were sutured.

[0057] (3) The animals need to be injected with ampicillin sodium (1g / day) for 3-5 days, given liquid food, and intravenous injection of mannitol (3g / 15ml / day) to reduce intracranial pressure. MRI scans were performed 3 days after surgery to determine the condition of cerebral infarction. 3 days after modeling, magnetic resonance T2WI sequence scanning imaging showed high signals around the laser irradiation position, indicating successful modeling. See the schematic diagram Figure 4 .

[0058] Example 2 Imaging Evaluation of Cerebral Artery Ischemia-Reperfusion Model

[0059] After modeling in Example 1, the blood flow of the target blood vessels was dynamically monitored using time-of-flight magnetic resonance angiography (TOF-MRA), and the results are shown in FIG5 .

[0060] Depend on Figure 5A It can be seen that compared with before modeling, vascular imaging 1 hour after modeling showed that blood supply was interrupted in the M1 segment of the blood vessel, indicating that 1 hour after injection of rose bengal laser irradiation, the M1 segment of the middle cerebral artery could be occluded; vascular imaging 5 hours after modeling showed that blood flow in the M1 segment of the blood vessel gradually recovered, indicating that 5 hours after occlusion, blood flow in the M1 segment was restored, indicating that the model showed ischemia-reperfusion characteristics, and Figure 5B It can be seen that after 3 days, the blood vessels are all open and there is no difference.

[0061] At the same time, blood oxygen level-dependent functional magnetic resonance imaging (BOLD-fMRI) and diffusion tensor imaging (DTI) were used to evaluate brain functional network connectivity and white matter nerve fiber analysis (see Figure 6 ), the results showed that the arrangement of brain nerve fiber bundles began to become disordered 4 hours after modeling, and the disorder of brain nerve fiber bundles became more serious 3 days after modeling, indicating that the connection of brain fiber bundles of animals was abnormal in the early stage of modeling.

[0062] Further combined with clinical stroke staging and MRI scan changes, the brain pathological changes of the above models were dynamically monitored using MRI T1WI, T2WI and DWI scan sequences at different stages before modeling, 5 hours, 3 days, 7 days and 14 days after modeling. The results are shown in Figure 7 .Depend on Figure 7It can be seen that 5 hours after modeling (hyperacute phase, 0-6 hours), T1WI showed low signal at the infarct focus, T2WI showed high signal, and DWI showed enhanced signal at the infarct focus than before modeling. The magnetic resonance imaging results showed that the animals had cytotoxic brain edema. 3 days after modeling (acute phase, 6-72 hours), T1WI showed low signal at the infarct focus and surrounding areas, T2WI showed high signal in a large area, and DWI showed high signal in the infarct focus area, indicating that the animal model had cytotoxic brain edema. Edema characteristics: 7 days after modeling (3-10 days in the subacute stage), T1WI showed low signal at the infarction focus, T2WI showed high signal, and DWI still maintained high signal in the infarction focus area, indicating that the animal model still showed characteristics of cytotoxic cerebral edema; 14 days after modeling (chronic stage, 10 days later), the lesions gradually liquefied, the T1WI signal was not obvious, T2WI showed high signal at the infarction focus, and DWI signal decreased in the infarction focus area, indicating that the animal model showed characteristics of vasogenic cerebral edema.

[0063] Furthermore, the volume of cerebral infarction was analyzed 14 days after modeling (see Figure 8 ), showing that 14 days after modeling, the position of T2WI in the whole brain was basically stable, and the volume of cerebral infarction was 1.21%.

[0064] In summary, according to the imaging evaluation of the above-mentioned animal model of cerebral artery ischemia-reperfusion, the modeling method of the present invention can successfully induce ischemic reperfusion in the deep brain blood vessels of elderly experimental monkeys. The animals survived well after modeling, showed typical clinical cerebral edema characteristics in various stages of stroke, and formed stable infarction foci.

[0065] Example 3 Pathological evaluation of cerebral artery ischemia-reperfusion model

[0066] The experimental monkeys were euthanized 90 days after modeling in the example, and the intact brain was obtained by dissection. The brain structure was scanned in vitro by magnetic resonance imaging to obtain the location of the infarct focus. The brain was dissected and sampled by positioning the structure scan. The gross dissection showed that tissue necrosis occurred at the location of the animal's cerebral infarction focus (see Fig. 9A ), which is consistent with the results of magnetic resonance T2WI sequence structure scanning (see Fig. 9B ).

[0067] The brain tissue was further embedded and sectioned, and HE staining, GFAP staining and IBA1 staining were performed. The results are shown in Fig.10 .

[0068] Depend on Fig.10 It was shown that 90 days after local cerebral ischemia, the infarct lesion formed liquefactive necrosis and was surrounded by glial cells (mainly astrocytes and microglia) ( Fig.10 A) A large number of necrotic neurons can be seen in the infarct focus ( Fig.10A, green arrows), a large number of activated glial cells can be seen in and around the infarct ( Fig.10 A, blue arrow). Immunofluorescence staining showed that activated astrocytes were mainly distributed around the infarct focus ( Fig.10 B, red arrows), activated microglia are mainly distributed in the infarct lesions ( Fig.10 C, red arrow).

[0069] In summary, according to the pathological evaluation of the above-mentioned animal model of cerebral artery ischemia-reperfusion, the modeling method of the present invention can successfully induce the formation of stable infarct foci in the brain, and the infarct foci can show typical pathological characteristics of clinical stroke such as neuronal necrosis and glial cell activation.

[0070] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0071] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one 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 present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a cerebral artery ischemia-reperfusion model, characterized in that: The following steps are involved: (1) Three-dimensional coordinate construction: Obtain medical images of the horizontal, sagittal, and coronal planes of brain magnetic resonance imaging, perform three-dimensional segmentation, and determine the X, Y, and Z three-dimensional coordinates of the blood vessels; (2) Construction of cerebral artery ischemia-reperfusion model: The sample is fixed on a brain stereotaxic apparatus, and the sample is processed according to the X, Y, and Z three-dimensional coordinates of the blood vessels in step (1) before laser irradiation; (3) After step (2) was completed, the sample was processed. Three days after modeling, magnetic resonance T2WI sequence scanning imaging showed high signals around the laser irradiation position, indicating that the modeling was successful.

2. The method for constructing a cerebral artery ischemia-reperfusion model according to claim 1, characterized in that: The scanning sequence used in the magnetic resonance imaging in step (1) is t2 mx3d sag, and the parameters are: TR=3400ms, TE=398ms, and layer thickness=0.5mm.

3. The method for constructing a cerebral artery ischemia-reperfusion model according to claim 1, characterized in that: The method for determining the X coordinate of the blood vessel in step (1) is as follows: in the horizontal section view, a reference line between the glabella and the internal occipital protuberance is parallel to the sagittal suture of the skull, and the plane formed by the two lines is the median horizontal plane of the brain. The above-mentioned reference line is the midline of the brain, and the horizontal distance from the blood vessel to the midline of the brain is the X coordinate.

4. The method for constructing a cerebral artery ischemia-reperfusion model according to claim 1, characterized in that: The method for determining the Y coordinate of the blood vessel in step (1) is as follows: after determining the position of the blood vessel in the sagittal view, the blood vessel is located on the midline of the brain, and the vertical distance from the lowest point between the frontal bone and the eyebrow arch to the cerebral blood vessel is taken as the Y coordinate.

5. The method for constructing a cerebral artery ischemia-reperfusion model according to claim 1, characterized in that: The method for determining the Z coordinate of the blood vessel in step (1) is as follows: in the coronal image, after the blood vessel position is determined, the vertical distance from the dura mater to the cerebral blood vessel is the Z coordinate.

6. The method for constructing a cerebral artery ischemia-reperfusion model according to claim 1, characterized in that: The treatment in step (2) is injection of rose bengal.

7. The method for constructing a cerebral artery ischemia-reperfusion model according to claim 1, characterized in that: The treatment in step (3) is injection of antibiotics and mannitol to maintain intracranial pressure.

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