Method for establishing a magnetic field intensity induced non-invasive ischemic stroke model

Abstract

This application discloses a method for establishing a non-invasive ischemic stroke model induced by magnetic field strength, comprising: injecting magnetic nanoparticles into the tail veins of multiple groups of mice; placing a magnetic field strength controller at the internal carotid artery of each group of mice; adjusting the magnetic field strength at the internal carotid artery using the magnetic field strength controller; and moving the adjusted magnetic field strength controller along the internal carotid artery to allow the magnetic nanoparticles to form microthrombi with the flowing blood, thereby obtaining a non-invasive ischemic stroke model. This application applies magnetic field strengths of different magnitudes at specific locations, strongly attracting the injected magnetic nanoparticles to the internal carotid artery, where they form microthrombi with the flowing blood, thus creating a series of non-invasive ischemic stroke models of varying degrees. It has the advantages of being non-invasive, simple and convenient to operate, and the models are highly accurate and reliable, laying a solid foundation for understanding the pathogenesis of cerebral ischemia and drug screening.

Description

Technical Field

[0001] This application relates to the field of animal model technology, and in particular to a method for establishing a magnetic field strength-induced non-invasive ischemic stroke model. Background Technology

[0002] Animal disease models are animals established for various medical and scientific research that exhibit simulated human diseases. They are primarily used in experimental physiology, experimental pathology, and experimental therapeutics (including drug screening). The development of human diseases is highly complex. Using humans as experimental subjects to deeply explore disease mechanisms and advance medicine has been slow. Clinical experience is limited not only in time and space, but also in many ethical and methodological constraints. However, indirect research using animal models allows for the conscious alteration of factors that are impossible or difficult to eliminate under natural conditions. This enables more accurate observation of model results and comparative studies with human diseases, facilitating a more convenient and effective understanding of the patterns of human disease development and the research of prevention and treatment measures.

[0003] Ischemic stroke, a serious neurological disease, has become a leading cause of death and disability in my country. There are many clinical studies on ischemic stroke, but existing stroke models all require invasive surgery, which involves making an incision in the animal's neck to expose the internal carotid artery. The surgery itself is difficult and may have a certain impact on the animal's physiology, thus affecting the accuracy and reliability of the animal model and causing the established animal model to lose its reference value.

[0004] Therefore, there is a need for a method to establish a non-invasive ischemic stroke model induced by magnetic field strength, which can realize the establishment of a non-invasive ischemic stroke model, is simple and convenient to operate, and has high accuracy and reliability. Summary of the Invention

[0005] To address the problems existing in the prior art, this application provides a method for establishing a non-invasive ischemic stroke model induced by magnetic field strength. This method enables the establishment of a non-invasive ischemic stroke model, is simple and convenient to operate, and has high accuracy and reliability. The technical solution is as follows:

[0006] This application provides a method for establishing a magnetic field strength-induced noninvasive ischemic stroke model, including:

[0007] Magnetic nanoparticles were injected into the tail veins of multiple groups of mice.

[0008] The magnetic field strength controller was placed at the internal carotid artery of each group of mice.

[0009] The magnetic field strength at the internal carotid artery is adjusted using the magnetic field strength controller.

[0010] The adjusted magnetic field strength controller is moved along the internal carotid artery so that the magnetic nanoparticles form microthrombi with the flowing blood, thus obtaining a non-invasive ischemic stroke model.

[0011] Furthermore, the mice were healthy adult male C57 / B mice aged 3-4 months.

[0012] Furthermore, the particle size of the nanomagnetic particles is 100-120 nm, and the volume of the nanomagnetic particles injected into the tail vein is 130-150 μL.

[0013] Furthermore, the injection of magnetic nanoparticles into the tail veins of multiple groups of mice includes:

[0014] Different concentrations of the magnetic nanoparticles were injected into the tail veins of multiple groups of mice; or

[0015] The same concentration of the magnetic nanoparticles was injected into the tail vein of multiple groups of mice.

[0016] Furthermore, the concentrations of the multiple sets of magnetic nanoparticles include at least one of a first concentration, a second concentration, and a third concentration; the first concentration is greater than 0 mg / mL and not greater than 2 mg / mL, the second concentration is greater than 2 mg / mL and not greater than 4 mg / mL, and the third concentration is greater than 4 mg / mL and not greater than 6 mg / mL.

[0017] Furthermore, before or after injecting different concentrations of magnetic nanoparticles into the tail veins of multiple groups of mice, the method further includes:

[0018] The mice were anesthetized and placed on a temperature-controlled blanket;

[0019] Remove the neck hair from the mice and mark the locations of the common carotid artery, internal carotid artery, and external carotid artery.

[0020] Furthermore, the preset adjustment range of the magnetic field strength controller is 0 to 1T.

[0021] Furthermore, the formation time of microthrombi in the internal carotid artery is 4–5 minutes.

[0022] Furthermore, after moving the adjusted magnetic field strength controller along the internal carotid artery to cause the magnetic nanoparticles to form microthrombi with the flowing blood, thereby obtaining a non-invasive ischemic stroke model, the method further includes:

[0023] The treated mice were returned to their home environment and subjected to neurobehavioral assessments.

[0024] Furthermore, the neurobehavioral assessment is as follows:

[0025] In a single-blind setting, each group of mice underwent grid walking test, cylinder test, and spaghetti grasping test; wherein the neurobehavioral assessment was conducted within one week prior to injection of the magnetic nanoparticles and within 1 to 15 weeks after injection of the magnetic nanoparticles.

[0026] Implementing this application will have the following beneficial effects:

[0027] This application utilizes the hemodynamic characteristics of magnetic nanoparticles to apply magnetic fields of varying strengths at specific locations, strongly attracting the magnetic nanoparticles to specific internal carotid arteries, where they form microthrombi with flowing blood, thereby creating a series of non-invasive ischemic stroke models of different degrees. Compared with traditional surgical methods for stroke models, this application has the advantages of being non-invasive and simple to operate. Furthermore, the non-invasive ischemic stroke models of this application have high accuracy and reliability, facilitating the simulation and observation of the characteristics of different degrees of injury in human ischemic stroke, laying a solid foundation for the pathogenesis of cerebral ischemia and drug screening. Attached Figure Description

[0028] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a flowchart illustrating a possible embodiment of the present invention of a method for establishing a non-invasive ischemic stroke model induced by magnetic field strength. Detailed Implementation

[0030] The technical solutions of this application will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments, and therefore should not be construed as limiting this application. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0031] It should be noted that the terms "comprising" and "having" and any variations thereof in the specification and claims of this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or server that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such processes, methods, products, or devices.

[0032] Example 1

[0033] Given that existing mouse models of ischemic stroke cannot achieve non-invasive establishment, this application provides a method for establishing a magnetic field-induced non-invasive ischemic stroke model, as detailed in the attached specification. Figure 1 As shown, it includes:

[0034] S1, magnetic nanoparticles were injected into the tail veins of multiple groups of mice.

[0035] S2, Place the magnetic field strength controller at the internal carotid artery of each group of mice.

[0036] S3, the magnetic field strength at the internal carotid artery is adjusted by the magnetic field strength controller.

[0037] S4, the adjusted magnetic field strength controller is moved along the internal carotid artery so that the magnetic nanoparticles form microthrombi with the flowing blood, thereby obtaining a non-invasive ischemic stroke model.

[0038] Specifically, in one possible implementation of this specification, the mice used in step S1 are healthy adult male C57 / B mice aged 3 to 4 months.

[0039] Specifically, before or after step S1, the method for establishing the magnetic field strength-induced noninvasive ischemic stroke model also includes:

[0040] The mice were anesthetized and placed on a temperature-controlled blanket;

[0041] Remove the neck hair from the mice and mark the locations of the common carotid artery, internal carotid artery, and external carotid artery.

[0042] The temperature of the constant temperature blanket is set to 37±0.2℃ to closely match the body temperature of the mice, so as to avoid abnormal environments affecting the physiology of the mice and thus affecting the accuracy of the non-invasive ischemic stroke model. Removing the hair from the mouse's neck exposes the skin of the mouse's neck, making it easier to observe and mark the common carotid artery, internal carotid artery and external carotid artery. This makes it easier to place the magnetic field strength controller at the internal carotid artery in step S2, ensuring that microthrombi can form in the internal carotid artery, improving the convenience of operation and the accuracy of the model.

[0043] Specifically, in one possible implementation of this specification, depending on the level of sophistication required to establish the non-invasive ischemic stroke model, the injection of magnetic nanoparticles into the tail veins of multiple groups of mice in step S1 may include:

[0044] S101, injecting different concentrations of the magnetic nanoparticles into the tail veins of multiple groups of mice; or

[0045] S102, the same concentration of the magnetic nanoparticles was injected into the tail vein of multiple groups of mice.

[0046] When the required level of model precision is low, step S102 can be performed to establish a series of non-invasive ischemic stroke models of different degrees by adjusting the magnetic field strength in step S3 at the same injection concentration. However, when the magnetic nanoparticles injected into mice at a single concentration cannot meet the requirements for establishing a large number of non-invasive ischemic stroke models of different degrees, step S101 can be performed to expand the range of simulated non-invasive ischemic stroke models by adjusting the magnetic nanoparticles of different concentrations and magnetic field strengths of different sizes, thereby enriching the non-invasive ischemic stroke model while ensuring accuracy and reliability.

[0047] Specifically, in one possible embodiment of this specification, the magnetic nanoparticles can be selected as Fe3O4, which has moderate magnetism and can be stably attracted by the magnetic field strength controller, making it quick and convenient to form microthrombi; and in another possible embodiment of this specification, the particle size of the magnetic nanoparticles can be selected as 100-120 nm, which facilitates the movement of the magnetic nanoparticles in blood vessels.

[0048] Specifically, in one possible embodiment of this specification, in step S1, a certain volume of magnetic nanoparticles is aspirated and injected into the tail vein of a mouse. The volume of the aspirated and injected magnetic nanoparticles can be selected as 130–150 μL, and the concentration of the multiple sets of magnetic nanoparticles includes at least one of a first concentration c1, a second concentration c2, and a third concentration c3; wherein 0 < c1 ≤ 2 mg / mL, 2 mg / mL < c2 ≤ 4 mg / mL, and 4 mg / mL < c3 ≤ 6 mg / mL. The total number of magnetic nanoparticles injected into the mouse tail vein can be varied in more detail by adjusting the specific volume and concentration of the magnetic nanoparticles, thus facilitating subsequent steps in S3. Different magnetic field intensities in the process simulate more non-invasive ischemic stroke models of varying degrees. Furthermore, when the injection concentration is low, the total number of magnetic nanoparticles has a maximum threshold. Therefore, as the magnetic field strength increases, its ability to attract nanoparticles may far exceed the actual threshold of the injected nanoparticles, resulting in the inability to form a corresponding degree of non-invasive ischemic stroke model, leading to model distortion. Injecting high concentrations of magnetic nanoparticles, however, can match a larger magnetic field strength. That is, the total number of injected magnetic nanoparticles is always higher than the number of magnetic nanoparticles that the corresponding magnetic field strength can attract, ensuring accurate formation of a corresponding degree (severe) of non-invasive ischemic stroke model with high reliability.

[0049] For example, in this embodiment, the volume of the injected magnetic nanoparticles can be selected as 150 μL. The mice in step S1 are divided into three groups, with injection concentrations of 1 mg / ml, 3 mg / ml, and 5 mg / ml, respectively. Then, in step S3, different magnetic field strengths are applied to the three groups of mice. A smaller magnetic field strength is applied to the group with an injection concentration of 1 mg / ml, a medium magnetic field strength is applied to the group with an injection concentration of 3 mg / ml, and a larger magnetic field strength is applied to the group with an injection concentration of 5 mg / ml, so as to form non-invasive ischemic stroke models of different degrees. The operation is simple and convenient. Within each group, multiple mice can also be set up, and multiple mice in the same group are injected with the same concentration of magnetic nanoparticles and the same magnetic field strength is applied, so that the establishment method of this application has reproducibility and higher reliability.

[0050] In another possible implementation of this specification, different magnetic field strengths can be applied to multiple mice within each group (all mice in the group have been injected with the same concentration of magnetic nanoparticles). Due to the limitation of the injection concentration itself, the number of magnetic nanoparticles in the mouse body is limited. Assuming that injecting magnetic nanoparticles at a concentration of 1 mg / ml can meet the adjustment requirements of a small magnetic field strength range (let's call it interval T1), then within this small magnetic field strength range (interval T1), different small magnetic field strengths can be applied to multiple mice within the group. These different small magnetic field strengths are all within the interval T1, thus obtaining mice with different degrees of mild microthrombus formation. The mice in this group all exhibit different degrees of mildness, i.e., different degrees of non-invasive ischemic stroke models, further refining the model; then in this... In this case, mice are first grouped according to different concentrations, and then grouped according to different magnetic field strengths. The final total number of groups is equal to the number of groups in the first level multiplied by the number of groups in the second level. It should be noted that the concentration-based grouping in step S1 is not limited to three groups. Four, five or even more groups can be set according to actual needs, and the concentration intervals between adjacent groups are smaller, thereby simulating a more refined non-invasive ischemic stroke model. In addition, the grouping of mice in step S1 is only for descriptive convenience and is not limited to concentration-based grouping. They can also be grouped according to different magnetic field strengths, and then multiple mice in the same group (the same magnetic field strength will be applied in the subsequent step S3) are injected with different concentrations of magnetic nanoparticles, making the establishment method flexible and varied.

[0051] In the pathogenesis of stroke, microthrombi form in the internal carotid artery. Therefore, after the magnetic field strength is set in step S3, moving the magnetic field strength controller upward along the internal carotid artery can better guide the magnetic nanoparticles into the internal carotid artery, ensuring that the microthrombi are accurately formed in the mouse internal carotid artery and improving the reliability of the model.

[0052] Specifically, in one possible embodiment of this specification, in step S3, the preset adjustment range of the magnetic field strength controller is set to 0–1T, which is a wide adjustment range and allows for the formation of stroke models to a large extent. In this embodiment, according to the increase of injection concentration, with the injection concentrations of the three groups of mice being 1 mg / ml, 3 mg / ml, and 5 mg / ml respectively, the magnetic field strength applied to the three groups of mice can be set in the ranges of 0–10 mT (excluding 0), 20–30 mT, and 31–40 mT respectively, forming mild, moderate, and severe non-invasive ischemic stroke models. Similarly, in other possible embodiments of this specification, depending on the number of groups, the magnetic field strength range of each group can be reduced, and the concentration of the injected nanomagnetic particles can be adjusted according to the reduced magnetic field strength range to increase the number of non-invasive ischemic stroke models and further enrich the non-invasive ischemic stroke model.

[0053] Specifically, in this application, the formation time of microthrombi by attracting magnetic nanoparticles with magnetic field strength is 4-5 minutes, which is relatively short and can quickly and conveniently obtain a large number of non-invasive ischemic stroke models, saving time.

[0054] Specifically, after step S4, i.e., after obtaining the noninvasive ischemic stroke model, the method further includes:

[0055] The treated mice were returned to their home environment and subjected to neurobehavioral assessments.

[0056] Neurobehavioral assessment can specifically include:

[0057] In a single-blind setting, each group of mice underwent grid walking test, cylinder test, and spaghetti grasping test.

[0058] The assessment time for neurobehavioral evaluation includes one week before injection of magnetic nanoparticles (i.e., surgery to form microthrombi in the internal carotid artery of mice) and 1 to 15 weeks after injection of magnetic nanoparticles. The assessment results one week before surgery can be used as a control group to compare normal mice with non-invasive ischemic stroke models of different degrees. In this embodiment, the assessment time can be further set to one week before surgery and 1, 3, 5, 7, 11, and 15 weeks after surgery.

[0059] The grid walking test is an evaluation method for assessing an animal's ability to precisely control the placement of its hind paws after brain or spinal cord injury. It is applicable to small animals such as cats and mice. Depending on the angle of grid placement, it can be divided into the horizontal grid test and the inclined grid test. During the assessment, the mouse is placed on a horizontal or inclined grid (2.5 cm apart at both ends), and behavioral data such as the number of times the mouse's hind paws slips off the grid, the number of footsteps made, and the time taken to traverse the distance are recorded within a certain period of time.

[0060] The cylinder test is a research tool for asymmetrical central nervous system diseases. It mainly studies the asymmetrical use of limbs in rodents during wall-standing, also known as the spontaneous forelimb task assessment test. During the assessment, mice are placed in a cylinder with a diameter of 20 cm and a height of 40 cm. A camera in an automatic recording system records the number of times, the time, and the proportion of times the mouse stands with its left forelimb, right forelimb, and both forelimbs simultaneously against the wall within a certain period of time. The automatic recording system records and analyzes the data, which ensures the objectivity of the assessment results and saves assessment time and manpower.

[0061] The pasta grasping test involved recording video of the animal's forepaw movements as it grasped and ate uncooked 2.5 cm long Italian rice noodles. During the process, the camera's position and angle could be adjusted to obtain the best field of view of the forepaw movements. Slow-motion playback allowed for clear observation of the mouse's forepaw adjustments and confirmed the clear movement and alternation of any toe in the forepaw grasping the noodles after the mouse began eating. The number of times the mouse adjusted its forepaw on each noodle and the time taken to finish eating each noodle were recorded in each test. The test was repeated at least four times to ensure quantitative analysis of changes in forepaw sensitivity.

[0062] This application utilizes the direct proportionality between the number of aggregated magnetic nanoparticles and the magnetic field strength of a magnetic field controller. By adjusting the magnetic field strength of the controller to different magnitudes, the number of magnetic nanoparticles attracted to the internal carotid artery is controlled, resulting in the formation of microthrombi of varying sizes between different numbers of magnetic nanoparticles and flowing blood. This leads to a series of non-invasive ischemic stroke models of varying degrees. Taking three groups of mice as examples, in this embodiment, the establishment method can be specifically as follows:

[0063] Multiple mice were divided into three groups. 1 mg / mL of magnetic nanoparticles was injected into the tail vein of the first group of mice, 3 mg / mL of magnetic nanoparticles was injected into the tail vein of the second group of mice, and 5 mg / mL of magnetic nanoparticles was injected into the tail vein of the third group of mice. The volume of magnetic nanoparticles injected in all three groups was the same, 150 μL.

[0064] Mice were anesthetized with isoflurane and placed on a temperature-controlled blanket. The hair on the mice's necks was removed, and the locations of the common carotid artery, internal carotid artery, and external carotid artery were marked to facilitate subsequent operations and observations.

[0065] The magnetic field strength controller was placed at the internal carotid artery of the mice, and the setting value of the magnetic field strength controller was adjusted so that the magnetic field strength applied to the neck of the first group of mice was in the range of 0 to 10 mT (excluding 0), the magnetic field strength applied to the neck of the second group of mice was in the range of 20 to 30 mT, and the magnetic field strength applied to the neck of the third group of mice was in the range of 31 to 40 mT.

[0066] The adjusted magnetic field strength controller is moved upward along the internal carotid artery to guide the nanomagnetic particles into the internal carotid artery, where they form microthrombi with the flowing blood, resulting in three non-invasive ischemic stroke models. The corresponding relationship of their stroke severity should be shown in Table 1.

[0067] Table 1 shows the correspondence between injection concentration, magnetic field strength, and stroke severity in this embodiment.

[0068]

[0069] The treated mice were returned to their rearing state and subjected to neurobehavioral assessments, including grid walking test, cylinder test, and spaghetti grasping test. The severity of stroke shown in the assessment results was consistent with the predictions in Table 1. This indicates that the method for establishing the magnetic field strength-induced non-invasive ischemic stroke model is simple and convenient to operate, does not require surgery, and the established non-invasive ischemic stroke model has high accuracy and reliability, and high reference value, laying a solid foundation for the pathogenesis of cerebral ischemia and drug screening.

[0070] Example 2

[0071] The difference between this embodiment and Embodiment 1 is that the mice were divided into nine groups, and different magnetic field strengths were applied to the nine groups of mice. The corresponding relationship is shown in Table 2. The rest is the same as in Embodiment 1.

[0072] Table 2 shows the correspondence between injection concentration, magnetic field strength, and stroke severity in Example 2.

[0073]

[0074] It should be noted that for the cases in Table 2 where the injection concentration is 1 mg / mL and the magnetic field strength is 21 mT or higher, due to the limited total number of nanomagnetic particles in the mouse body, even if a higher magnetic field strength is applied, it is impossible to form microthrombi corresponding to the magnetic field strength. Instead, only microthrombi corresponding to the magnetic field threshold (i.e., the magnetic field strength that just attracts 1 mg / mL of nanomagnetic particles) can be formed, which is actually meaningless. Therefore, it is not listed in this embodiment.

[0075] The above description is merely some embodiments of this application and is not intended to limit this application. Those skilled in the art should understand that various changes and improvements may be made to this application, and any modifications, equivalent substitutions and improvements made in accordance with this application fall within the scope of protection claimed by this application.

Claims

1. A method for establishing a magnetic field strength-induced noninvasive ischemic stroke model, characterized in that, include: Magnetic nanoparticles were injected into the tail veins of multiple groups of mice; this included injecting different concentrations of the magnetic nanoparticles into the tail veins of multiple groups of mice, or injecting the same concentration of the magnetic nanoparticles into the tail veins of multiple groups of mice. The magnetic field strength controller was placed at the internal carotid artery of each group of mice. The magnetic field strength at the internal carotid artery is adjusted by the magnetic field strength controller; the total number of injected magnetic nanoparticles is higher than the number of magnetic nanoparticles that the corresponding magnetic field strength can attract. The adjusted magnetic field strength controller is moved along the internal carotid artery so that the magnetic nanoparticles form microthrombi with the flowing blood, thus obtaining a non-invasive ischemic stroke model.

2. The method for establishing a magnetic field strength-induced non-invasive ischemic stroke model according to claim 1, characterized in that, The mice were healthy adult male C57 / B mice aged 3-4 months.

3. The method for establishing a magnetic field strength-induced non-invasive ischemic stroke model according to claim 1, characterized in that, The nanomagnetic particles have a particle size of 100–120 nm, and the volume of the nanomagnetic particles injected into the tail vein is 130–150 μL.

4. The method for establishing a magnetic field strength-induced non-invasive ischemic stroke model according to claim 1, characterized in that, The concentrations of the magnetic nanoparticles in the plurality of groups include at least one of a first concentration, a second concentration, and a third concentration; the first concentration is greater than 0 mg / mL and not greater than 2 mg / mL, the second concentration is greater than 2 mg / mL and not greater than 4 mg / mL, and the third concentration is greater than 4 mg / mL and not greater than 6 mg / mL.

5. The method for establishing a magnetic field strength-induced non-invasive ischemic stroke model according to claim 1, characterized in that, Before or after injecting different concentrations of magnetic nanoparticles into the tail veins of multiple groups of mice, the method further includes: The mice were anesthetized and placed on a temperature-controlled blanket; Remove the neck hair from the mice and mark the locations of the common carotid artery, internal carotid artery, and external carotid artery.

6. The method for establishing a magnetic field strength-induced non-invasive ischemic stroke model according to claim 1, characterized in that, The preset adjustment range of the magnetic field strength controller is 0 to 1T.

7. The method for establishing a magnetic field strength-induced non-invasive ischemic stroke model according to claim 1, characterized in that, The formation time of microthrombi in the internal carotid artery is 4 to 5 minutes.

8. The method for establishing a magnetic field strength-induced non-invasive ischemic stroke model according to claim 1, characterized in that, After moving the adjusted magnetic field strength controller along the internal carotid artery to allow the magnetic nanoparticles to form microthrombi with the flowing blood, thus obtaining a non-invasive ischemic stroke model, the method further includes: The treated mice were returned to their home environment and subjected to neurobehavioral assessments.

9. The method for establishing a magnetic field strength-induced non-invasive ischemic stroke model according to claim 8, characterized in that, The neurobehavioral assessment is as follows: In a single-blind setting, each group of mice underwent grid walking test, cylinder test, and spaghetti grasping test; wherein the neurobehavioral assessment was conducted within one week prior to injection of the magnetic nanoparticles and within 1 to 15 weeks after injection of the magnetic nanoparticles.

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