Myocardial ischemia reperfusion injury animal model construction method and application thereof

Abstract

The invention discloses a myocardial ischemia reperfusion injury animal model construction method and application thereof in the technical field of model animal preparation. The method comprises the steps of hypertension and hyperlipidemia pretreatment, standardized ischemia-reperfusion operation, immune microenvironment regulation and control and model evaluation and verification. According to the invention, model construction is carried out on an integrated model operation table, so that a standardized ischemia-reperfusion process is realized, reproducibility of a myocardial injury degree and an immune cell infiltration rule of the model is realized, and suitability of the model and nano-drug research is enhanced; and a reliable tool is provided for research and development of targeted drugs such as immune cell membrane coated nanoparticles.

Description

Technical Field

[0001] This invention belongs to the field of animal model preparation technology, specifically a method for constructing an animal model of myocardial ischemia-reperfusion injury and its application. Background Technology

[0002] Myocardial ischemia / reperfusion injury (MIRI) is a common complication in patients with acute myocardial infarction (MI) after thrombolytic therapy or percutaneous coronary intervention. It is characterized by cardiomyocyte death, inflammatory response, and cardiac dysfunction following the restoration of blood flow to the ischemic myocardium, and is a significant cause of poor prognosis. Clinical studies have shown that the occurrence of MIRI is closely related to multiple mechanisms, including inflammatory response, oxidative stress, mitochondrial dysfunction, and intracellular calcium overload. Among these, the release of inflammatory factors (such as tumor necrosis factor-α and interleukin-8) and the infiltration of immune cells (such as neutrophils, monocytes, and macrophages) in the ischemic area are the core pathological processes.

[0003] Currently, there is still a lack of effective treatments for microvascular myocardial infarction (MIRI), mainly because traditional drugs are difficult to precisely target ischemic myocardial regions and are easily cleared by the body's immune system, resulting in low treatment efficiency and significant side effects. In recent years, nanomedicine delivery systems have received widespread attention in MIRI treatment research due to their advantages such as improved drug stability and prolonged circulation time. Among them, nanoparticles encapsulated in immune cell membranes can significantly enhance the targeting of nanoparticles to ischemic myocardium and reduce accumulation in non-target organs by inheriting the surface functional molecules of the source cells, making them a research hotspot for targeted therapy of MIRI.

[0004] Animal models are crucial tools for evaluating the efficacy of nanoparticle-targeted drugs. However, existing methods for constructing MIRI animal models have significant limitations, making it difficult to meet the needs of nanoparticle research: 1. Insufficient model standardization: Different studies show significant differences in ischemia time and reperfusion methods, leading to instability in the degree of myocardial injury and the immune microenvironment (such as immune cell infiltration patterns and inflammatory factor levels), which cannot accurately reflect the targeting accumulation efficiency of nanoparticles; 2. Low adaptability to nanoparticle targeting mechanisms: Existing models do not specifically simulate the recruitment characteristics of immune cells to ischemic areas in MIRI. The targeting of nanoparticles encapsulated in immune cell membranes depends on the specific interaction between source cells and ischemic myocardium, making it difficult to effectively verify the targeting mechanism of nanoparticles in the model; 3. Incomplete evaluation system: Existing models mostly use traditional indicators such as myocardial infarction area and myocardial enzyme levels to assess the degree of injury, lacking monitoring of key parameters such as the distribution dynamics of nanoparticles in the myocardium and their interaction with target cells (such as vascular endothelial cells and cardiomyocytes), which cannot provide accurate data for optimizing the targeting efficiency of nanoparticles.

[0005] Therefore, it is necessary to propose a standardized method for constructing an animal model of myocardial ischemia-reperfusion injury that can accurately simulate the MIRI immune microenvironment and is suitable for evaluation of nano-targeted drugs, and to propose its application. Summary of the Invention

[0006] To address the aforementioned issues, the present invention aims to provide a method for constructing an animal model of myocardial ischemia-reperfusion injury and its application. Through an integrated model operating platform, a standardized ischemia-reperfusion process is achieved, enabling the reproducibility of the degree of myocardial injury and the pattern of immune cell infiltration in the model. Pretreatment of experimental animals with comorbidities enhances the validation value of the targeting efficiency of nanomedicines in complex pathological environments. By regulating the immune microenvironment, the targeting mechanism of nanoparticles encapsulated in immune cell membranes is matched, achieving targeted regulation of nanomedicine target sites and enhancing the adaptability of the model to nanomedicine research. This provides a reliable tool for the development of targeted drugs such as nanoparticles encapsulated in immune cell membranes.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: A method for constructing an animal model of myocardial ischemia-reperfusion injury, comprising the following steps:

[0008] Step 1: Pretreatment. Adult mammals were selected, and a hypertension comorbidity model was constructed by combining a high-salt diet with angiotensin II injection, or a hyperlipidemia model was constructed by combining a high-fat diet, in order to simulate the basic pathological state of clinical MIRI patients.

[0009] Step 2: Standardize ischemia-reperfusion operation. On the model operating table, the left anterior descending coronary artery of the pretreated animal is ligated. The ischemia duration is set to 1 hour, and the reperfusion duration is set to a time gradient of 3 hours, 24 hours or 72 hours. During the reperfusion phase, the blood flow recovery rate is precisely controlled to 0.5 ml / min to avoid secondary injury.

[0010] Step 3: Regulation of the immune microenvironment. Monocyte chemoattractant protein-1 (MCP-1) at a concentration of 10-50 ng / ml is injected locally into the ischemic area to target and recruit Ly6C cells. + Mononuclear cells enhance the targeted binding sites of nanoparticles encapsulated in the ischemic areas of myocardium and immune cell membranes;

[0011] Step 4: Model evaluation and validation. At each time point of reperfusion, the targeted accumulation efficiency of nanomedicine in myocardial tissue is monitored by fluorescence imaging. The degree of myocardial injury is assessed by combining myocardial enzyme level detection, Masson staining and echocardiography to complete the model construction.

[0012] The basic principle of this approach is as follows: Based on the pathophysiological characteristics of clinical MIRI patients, this model construction method accurately simulates the occurrence and development of MIRI through the synergistic effects of multiple steps and factors. The pretreatment step simulates the patient's baseline pathological state, while the standardized ischemia-reperfusion procedure precisely simulates the core hemodynamic changes in MIRI. The immune microenvironment regulation step directionally shapes the immune environment of the ischemic myocardial region, providing a suitable biological background for targeted research of nanomedicines. Finally, comprehensive model evaluation and validation methods are used to comprehensively and dynamically monitor the model's construction effectiveness.

[0013] Each step is closely interconnected and interacts with the others. The pathological state induced by pretreatment provides the basis for the subsequent development of ischemia-reperfusion injury, influencing the distribution and activity of immune cells. Standardized ischemia-reperfusion procedures directly induce myocardial tissue damage and immune responses, providing targets for nanomedicines. Regulation of the immune microenvironment, by increasing specific immune cells and molecules, further enhances the interaction between the ischemic myocardial region and nanomedicines, making the targeting effect of nanomedicines more significant.

[0014] This method integrates multiple factors and mechanisms, systematically simulating the complex biological processes of MIRI from multiple perspectives, including physiological, pathological, and molecular levels, providing an ideal model basis for studying the pathogenesis of MIRI, drug development, and the exploration of treatment methods.

[0015] The beneficial effects of the basic scheme are: 1. By fixing the ischemia-reperfusion time gradient and blood flow recovery rate, experimental variables are reduced, and the degree of myocardial injury and immune microenvironment of different batches of models are consistent, thus solving the problem of poor reproducibility of traditional models.

[0016] 2. By constructing a comorbidity model, the impact of patients' underlying diseases on MIRI can be simulated, making the targeting efficiency research of nanomedicines closer to the actual clinical scenario, providing a more reliable basis for clinical research, and facilitating the comparison and verification of research results between different laboratories.

[0017] 3. By targeting and regulating the immune microenvironment, the targeting binding sites of nanoparticles encapsulated in the ischemic region and immune cell membrane are enhanced, providing a specific platform for verifying the targeting mechanism of nanomedicines, improving the accumulation efficiency of nanomedicines in the ischemic region of myocardium, and contributing to in-depth research on the targeting mechanism and therapeutic effect of nanomedicines.

[0018] 4. Combining fluorescence imaging to quantify nanodrug accumulation with traditional myocardial injury indicators comprehensively reflects the targeting efficiency and therapeutic effect of nanodrugs, providing more comprehensive and in-depth information for the research and optimization of nanodrugs and accelerating the clinical translation of nanodrugs.

[0019] Furthermore, the adult mammals mentioned in step one include SD rats and C57BL / 6 mice, and the experimental animals are 8-12 weeks old and weigh 250-300g to match the metabolic kinetics of nanoparticles in vivo.

[0020] The benefits of the basic protocol are as follows: SD rats and C57BL / 6 mice aged 8-12 weeks and weighing 250-300g were selected, exhibiting stable physiological functions and good tolerance to experimental procedures. These mice could withstand various interventions during model construction, reducing the risk of experimental failure due to individual animal differences. Furthermore, the metabolic kinetics of animals in this age and weight range closely resemble the metabolic processes of nanoparticles in humans, ensuring that the distribution, metabolism, and excretion of nanoparticles in the animals more closely match actual clinical conditions. This improves the accuracy of model construction, enabling it to more realistically reflect the occurrence and development of MIRI and the effects of nanomedicines.

[0021] Furthermore, step three also includes using gene editing to induce high expression of ICAM-1 in ischemic myocardial endothelial cells and fuse it with GFP fluorescent protein, in order to visualize the targeted binding process of nanoparticles to vascular endothelium.

[0022] The beneficial effects of the basic approach are: by using gene editing to induce high expression of ICAM-1 in endothelial cells within the ischemic myocardial region and fusing it with GFP fluorescent protein, more targeted binding sites are provided for nanoparticles encapsulated in immune cell membranes, enhancing the targeting efficiency of nanoparticles in the ischemic myocardial region. ICAM-1 is an important cell adhesion molecule, highly expressed on endothelial cells in the ischemic myocardial region, and can specifically bind to ligands on the surface of nanoparticles, enabling nanoparticles to more precisely target the lesion site.

[0023] Furthermore, in step four, the targeting accumulation efficiency of the nanomedicine is quantified by the ratio of fluorescence intensity in the ischemic area to the normal area (T / N ratio), where a T / N ratio ≥ 3 is considered as a qualified model targeting validation.

[0024] The beneficial effects of the basic approach are: 1. By using clear quantitative indicators, the influence of subjective factors on experimental results is reduced, improving the reliability and reproducibility of model construction and evaluation. In different laboratories or research teams, as long as the same evaluation criteria are followed, the targeting effects of nanomedicines can be evaluated more consistently.

[0025] 2. This quantitative standard helps researchers to more accurately screen and optimize the targeting performance of nanomedicines. Only when the accumulation efficiency of nanomedicines in ischemic areas reaches a certain level (i.e., T / N ratio ≥ 3) can it be considered to have good targeting properties, thus providing a reliable basis for subsequent drug development and clinical application.

[0026] Furthermore, the model operation platform described in step two includes a fixed platform, which is equipped with a constant temperature heater and a display controller. Several fixing clips are fixedly connected to the fixed platform. The fixed platform is equipped with a cardiac monitoring component, a blood flow control component, a multimodal imaging component, and a drug delivery component. The constant temperature heater, cardiac monitoring component, blood flow control component, multimodal imaging component, and drug delivery component are all connected to the controller signal.

[0027] Cardiac monitoring component, used to monitor changes in blood pressure and heart rate in animal models during the construction of cardiac reperfusion injury models;

[0028] The blood flow regulation component is used to perform cardiac reperfusion at regular intervals and in a quantitative manner, and to monitor blood flow pressure in real time to regulate the reperfusion blood flow rate.

[0029] A multimodal imaging component for dynamically acquiring fluorescence signals and distribution images of nanomedicines in myocardial tissue;

[0030] A drug delivery component is used in conjunction with a blood flow control component to inject drugs during reperfusion.

[0031] The beneficial effects of the basic scheme are: the cardiac monitoring component can acquire real-time data on changes in blood pressure and heart rate, reflecting the occurrence and development of myocardial ischemia-reperfusion injury; the multimodal imaging component can dynamically acquire fluorescence signals and distribution images of nanomedicines in myocardial tissue, providing intuitive and rich data support for studying the targeting and mechanism of action of nanomedicines, and helping to analyze the behavior and effects of drugs in vivo.

[0032] The model operating table integrates multiple functions such as fixation, monitoring, control, imaging and delivery, and can perform one-stop model construction operations on the model operating table, avoiding stress and accidents caused by the movement of model organisms.

[0033] Furthermore, the cardiac monitoring component includes a non-invasive blood pressure monitoring belt, which is fixedly connected to a mounting platform. The mounting platform is also fixedly connected to an electrocardiogram (ECG) monitoring electrode. Both the non-invasive blood pressure monitoring belt and the ECG monitoring electrode are connected to the display controller for signal transmission.

[0034] The beneficial effects of the basic scheme are: the combination of non-invasive blood pressure monitoring belt and electrocardiogram monitoring electrodes can monitor changes in blood pressure and heart rate in experimental animals in real time and non-invasively, comprehensively reflect the physiological state of the cardiovascular system, provide key physiological data for the construction of the MIRI model, ensure the precise execution of ischemia-reperfusion operations, and improve the stability and accuracy of model construction.

[0035] Furthermore, the blood flow control component includes an arterial clamp with a fiber optic pressure sensor. An injection tube is fixedly connected inside the arterial clamp, and the other end of the injection tube is connected to an injection pump. The injection pump is equipped with a flow sensor. The arterial clamp is used to block the left anterior descending coronary artery. The fiber optic pressure sensor is used to monitor blood flow pressure in real time, and the flow sensor is used to monitor the reperfusion blood flow rate. The fiber optic pressure sensor, injection pump, and flow sensor are all connected to the display controller signal.

[0036] The advantages of the basic approach are: the fiber optic pressure sensor can monitor blood flow pressure in real time, providing immediate data support for blood flow regulation; and the infusion pump, combined with a flow sensor, can precisely control the reperfusion blood flow rate, avoiding secondary damage and ensuring the accuracy and reliability of the experiment.

[0037] Furthermore, the multimodal imaging component includes an annular guide rail, which is fixedly sleeved to the side wall of the fixed stage. A slide block is slidably fitted on the annular guide rail, and a circumferential motor is fixedly connected to the slide block. A roller is coaxially fixedly connected to the output shaft of the circumferential motor, and the roller is rotatably fitted to the side wall of the fixed stage. A support bar is fixedly connected to the fixed stage, and a groove is opened at the top of the support bar. A slide strip is slidably connected in the groove. A telescopic motor is fixedly connected to the support bar, and a first bevel gear is coaxially fixedly connected to the output shaft of the telescopic motor. A drive gear is rotatably connected to the support bar, and a second bevel gear is coaxially fixedly connected to one side of the drive gear. The second bevel gear meshes with the first bevel gear. A pressing gear is rotatably connected to the top of the support bar. The slide strip has symmetrically opened toothed grooves at its top and bottom, which mesh with the pressing gear and the drive gear, respectively. A fluorescence imaging sensor and a photoacoustic imaging sensor are fixedly connected to the end of the slide strip away from the support bar. Both the fluorescence imaging sensor and the photoacoustic imaging sensor are signal connected to the display controller.

[0038] The beneficial effects of the basic scheme are: 1. The ring-shaped guide rail, together with the slide and the surrounding motor, allows the imaging sensor to move flexibly around the animal model, realize multi-angle imaging, capture the distribution of nano-drugs in myocardial tissue from all angles, and avoid information loss due to the limitation of imaging angle.

[0039] 2. The drive system, consisting of a telescopic motor, bevel gear, drive gear, and pressing gear, can precisely control the displacement of the fluorescence imaging sensor and photoacoustic imaging sensor, enabling high-precision positioning and monitoring of nanomedicines and improving the stability and clarity of imaging.

[0040] 3. Equipped with both fluorescence imaging and photoacoustic imaging sensors, it enables multimodal imaging. Fluorescence imaging can directly reflect the distribution and accumulation of nanomedicines, while photoacoustic imaging can provide structural information of myocardial tissue. The combination of the two provides more comprehensive and in-depth information for studying the targeting of nanomedicines and the repair process of myocardial injury.

[0041] Furthermore, the drug delivery component includes a syringe connected to an injection tube, which contains an injection pump and a drug for precise drug injection at the start of reperfusion. The injection pump is connected to a display controller.

[0042] The basic approach offers the following advantages: The syringe pump, connected to the display controller, allows for precise control of drug injection timing and dosage according to a preset program, ensuring accurate drug delivery at the start of reperfusion, avoiding human error, and improving experimental accuracy and reliability. The syringe pump monitors the drug injection rate and flow rate in real time and feeds the data back to the display controller, enabling researchers to monitor drug delivery and adjust injection parameters promptly to ensure successful experiment execution. The entire drug delivery process is automated by the syringe pump, reducing human intervention, improving experimental repeatability and stability, and facilitating comparison and verification of different experimental results. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the method for constructing an animal model of myocardial ischemia-reperfusion injury in an embodiment of the present invention.

[0044] Figure 2 This is an isometric view of the model operating platform in an embodiment of the present invention.

[0045] Figure 3 This is a top view of the model operation platform in an embodiment of the present invention.

[0046] Figure 4 This is a front sectional view of the model operation table in an embodiment of the present invention.

[0047] The reference numerals in the accompanying drawings include: 1. Display controller; 2. Fixing platform; 3. Circular guide rail; 4. Slide; 5. Roller; 6. Circular motor; 7. Support bar; 8. Telescopic motor; 9. Slide bar; 10. Gear groove; 11. First bevel gear; 12. Pressing gear; 13. Injection tube; 14. Artery clamp; 15. Fixing clamp; 16. Non-invasive blood pressure monitoring band; 17. Electrocardiogram monitoring electrode; 18. Syringe; 19. Thermostatic heater; 20. Second bevel gear; 21. Drive gear; 22. Fluorescence imaging sensor; 23. Photoacoustic imaging sensor. Detailed Implementation

[0048] The following detailed description illustrates the specific implementation method:

[0049] Example 1

[0050] The basics are as follows: Figure 1 As shown: A method for constructing an animal model of myocardial ischemia-reperfusion injury, comprising the following steps:

[0051] Step 1: Pretreatment. Adult mammals were selected, and a hypertension comorbidity model was constructed by combining a high-salt diet with angiotensin II injection, or a hyperlipidemia model was constructed by a high-fat diet, in order to simulate the basic pathological state of clinical MIRI patients. Adult mammals included SD rats and C57BL / 6 mice, and the experimental animals were 8-12 weeks old and weighed 250-300g to match the metabolic kinetics of nanoparticles in vivo.

[0052] Step 2: Standardize ischemia-reperfusion operation. On the model operating table, the left anterior descending coronary artery of the pretreated animal is ligated. The ischemia duration is set to 1 hour, and the reperfusion duration is set to a time gradient of 3 hours, 24 hours or 72 hours. During the reperfusion phase, the blood flow recovery rate is precisely controlled to 0.5 ml / min to avoid secondary injury.

[0053] Step 3: Regulation of the immune microenvironment. Monocyte chemoattractant protein-1 (MCP-1) at a concentration of 10-50 ng / ml is injected locally into the ischemic area to target and recruit Ly6C cells. + Mononuclear cells enhance the targeted binding sites of nanoparticles to the ischemic area of ​​myocardium and immune cell membranes. Through gene editing, the endothelial cells in the ischemic area of ​​myocardium express ICAM-1 and fuse it with GFP fluorescent protein to visualize the targeted binding process of nanoparticles to vascular endothelium.

[0054] Step 4: Model evaluation and validation. At each time point of reperfusion, the targeting accumulation efficiency of nanomedicine in myocardial tissue is monitored by fluorescence imaging. The degree of myocardial injury is assessed by combining myocardial enzyme level detection, Masson staining and echocardiography to complete the model construction. The targeting accumulation efficiency of nanomedicine is quantified by the ratio of fluorescence intensity in the ischemic area to the normal area. When the T / N ratio is ≥3, the model is considered to have passed the targeting validation.

[0055] The specific experimental procedure is as follows: I. Experimental Design

[0056] This experiment established three groups for comparison:

[0057] 1. Sham surgery group: No disease pretreatment was performed, and patients underwent open-chest surgery (without ligation of the coronary arteries) on a routine operating table without any immune intervention (n=8).

[0058] 2. Conventional model group (IR): A hypertension model was established by combining a high-salt diet with angiotensin II injection. Coronary artery ligation and reperfusion were performed on a conventional operating table without immune regulation intervention (n=8 for each group at reperfusion time points of 3h / 24h / 72h).

[0059] 3. Optimized model group (Opt-IR): The hypertension model is the same as the IR group, but the surgery is performed on the model operating table, and immune microenvironment regulation is implemented (MCP-1+ injection into the ischemic area to induce ICAM-1 / GFP expression) (the reperfusion time point of each group is the same as the IR group, n=8 for each group).

[0060] II. Experimental Procedure

[0061] 1. Preoperative preparation

[0062] Ten-week-old SD rats (280±20g) were induced to have hypertension by a high-salt diet (4% NaCl) and angiotensin II subcutaneous pump (500ng / kg / min) for 4 weeks.

[0063] 2. Intraoperative procedures

[0064] Animal fixation and monitoring: Anesthetized rats were fixed on the model operating table, and their blood pressure and heart rate were tracked in real time through the cardiac monitoring component (non-invasive blood pressure monitoring band 16 + electrocardiogram monitoring electrode 17).

[0065] Ischemia induction: Open the chest to expose the heart, and use the blood flow control component (arterial clamp 14 with integrated fiber optic pressure sensor) on the operating table to block the left anterior descending coronary artery (LAD) to confirm that the blood flow pressure is zero; the thermostatic heater 19 maintains the body temperature at 37±0.3℃.

[0066] Reperfusion and Intervention:

[0067] After 60 minutes of ischemia, blood flow was restored by precisely controlling the reperfusion rate (0.5±0.05ml / min) with an injection pump.

[0068] The drug delivery component simultaneously injects MCP-1 (30 ng / ml, 50 μl) into the ischemic marginal zone.

[0069] Visualization of the targeting process:

[0070] The multimodal imaging component (fluorescence / photoacoustic imaging sensor 23) automatically scans the heart along the annular guide rail 3 to dynamically capture the binding signal of GFP-labeled nanoparticles to ICAM-1-overexpressing vascular endothelium.

[0071] 3. Postoperative assessment

[0072] Animals were euthanized at three time points: 3h, 24h, and 72h after reperfusion.

[0073] Targeting efficiency: The ratio of fluorescence intensity (T / N) between the ischemic area (T) and the non-ischemic area (N) of myocardial tissue was calculated.

[0074] Damage indicators: detection of serum myocardial enzymes (cTnI), echocardiography to measure cardiac function (EF%), and Masson staining to quantify fibrosis area.

[0075] Core grouping variables: equipment type (conventional operating table vs. model operating table) and immune intervention (present or absent).

[0076] III. Experimental Results

[0077] As shown in the table below:

[0078] Table 1. Comparison of myocardial injury and nano-targeting efficiency (mean ± SD)

[0079]

[0080]

[0081] Note: P < 0.01 compared with the IR group at the same time point (t test); fibrosis was assessed only at 24h / 72h; there was no distinction in reperfusion time in the Sham group.

[0082] Table 2. Performance advantages of the control panel

[0083]

[0084] IV. Experimental Conclusions

[0085] The optimized model group (Opt-IR) achieved T / N ≥ 4.2 at all time points, which was significantly higher than that of the IR group (maximum 1.8), confirming that high expression of ICAM-1 combined with MCP-1 recruitment of monocytes can enhance the targeting of nanoparticles (P < 0.01).

[0086] The Opt-IR group showed a 35%-45% decrease in cTnI, a 12%-15% increase in EF%, and a 36%-45% reduction in fibrosis area, demonstrating that immune microenvironment regulation mitigates reperfusion injury (P < 0.01).

[0087] The model operating table significantly improves model stability and operational efficiency (reducing time by 25%) through precise blood flow control (0.5±0.05ml / min), isothermal maintenance, and multimodal intraoperative imaging, avoiding secondary damage and ensuring the visualization and verification of targeted interventions. This integrated approach successfully simulates the pathological process of MIRI combined with hypertension, providing a standardized platform for nanomedicine targeted delivery research, and is particularly suitable for dynamically evaluating the spatiotemporal distribution of drugs at different stages of reperfusion.

[0088] Example 2

[0089] The difference from the above embodiments is that, as shown in the appendix Figures 1 to 4 As shown: The model operation platform mentioned in step two includes a fixed platform 2. The fixed platform 2 is equipped with a constant temperature heater 19 and a display controller 1. Several fixing clips 15 are welded on the fixed platform 2. The fixed platform 2 is equipped with a cardiac blood monitoring component, a blood flow control component, a multimodal imaging component and a drug delivery component. The constant temperature heater 19, the cardiac blood monitoring component, the blood flow control component, the multimodal imaging component and the drug delivery component are all connected to the controller signal.

[0090] The cardiac blood monitoring component is used to monitor blood pressure and heart rate changes in animal models during the construction of a cardiac reperfusion injury model. The cardiac blood monitoring component includes a non-invasive blood pressure monitoring belt 16, which is bonded to a fixed platform 2. An electrocardiogram monitoring electrode 17 is also bonded to the fixed platform 2. Both the non-invasive blood pressure monitoring belt 16 and the electrocardiogram monitoring electrode 17 are connected to the display controller 1 for signal transmission.

[0091] The blood flow control component is used for timed and quantitative cardiac reperfusion and real-time monitoring of blood flow pressure to regulate the reperfusion blood flow rate. The blood flow control component includes an arterial clamp 14 with a fiber optic pressure sensor. An injection tube 13 is bonded inside the arterial clamp 14. The other end of the injection tube 13 is connected to an injection pump. A flow sensor is installed inside the injection pump. The arterial clamp 14 is used to block the left anterior descending coronary artery. The fiber optic pressure sensor is used to monitor blood flow pressure in real time, and the flow sensor is used to monitor the reperfusion blood flow rate. The fiber optic pressure sensor, injection pump, and flow sensor are all connected to the display controller 1.

[0092] A multimodal imaging component is used to dynamically acquire fluorescence signals and distribution images of nanomedicines in myocardial tissue. The component includes an annular guide rail 3, which is fixedly sleeved to the side wall of a fixed platform 2. A slide block 4 is slidably fitted onto the annular guide rail 3, and a circumferential motor 6 is welded onto the slide block 4. A roller 5 is coaxially welded to the output shaft of the circumferential motor 6, and the roller 5 rotatably engages with the side wall of the fixed platform 2. A support bar 7 is welded to the fixed platform 2, and a groove is formed at the top of the support bar 7. A slide strip 9 is slidably connected within the groove. A telescopic motor 8 is welded to the support bar 7, and the output shaft of the telescopic motor 8 is coaxially fitted. A first bevel gear 11 is welded on, a drive gear 21 is rotatably connected to the support bar 7, a second bevel gear 20 is coaxially welded to one side of the drive gear 21, the second bevel gear 20 meshes with the first bevel gear 11, a pressing gear 12 is rotatably connected to the top of the support bar 7, and the slide bar 9 has symmetrically opened toothed grooves 10, which mesh with the pressing gear 12 and the drive gear 21 respectively. A fluorescence imaging sensor 22 and a photoacoustic imaging sensor 23 are glued to the end of the slide bar 9 away from the support bar 7, and both the fluorescence imaging sensor 22 and the photoacoustic imaging sensor 23 are signal connected to the display controller 1.

[0093] The drug delivery component is used in conjunction with the blood flow control component to inject drugs during reperfusion. The drug delivery component includes a syringe 18 connected to the injection tube 13. The syringe 18 is equipped with an injection pump and contains drugs for precise drug injection at the start of reperfusion. The injection pump is connected to the display controller 1.

[0094] The specific implementation process is as follows: When using this device to construct an animal model of myocardial ischemia-reperfusion injury, firstly, pre-treated SD rats or C57BL / 6 mice (hypertension or hyperlipidemia models) are placed on the fixation platform 2. The animal's limbs are stabilized by the fixation clamps 15 to prevent positional displacement due to struggling during operation, laying the foundation for subsequent precise operation. At this time, the thermostatic heater 19 inside the fixation platform 2 is activated. The thermostatic heater 19 is an electric heating wire or electric heating plate. The display controller 1 sets the temperature to about 37°C to maintain the animal's body temperature stability, ensuring that its physiological metabolic state is not disturbed by low temperature and ensuring the stability of the animal's vital signs during the model construction process.

[0095] Next, the cardiac monitoring component is activated. The non-invasive blood pressure monitoring strap 16 is fixed to the animal's limbs, and the electrocardiogram monitoring electrodes 17 are attached to the corresponding positions on the chest. Both are connected to the display controller 1 to collect and display the animal's blood pressure and heart rate data in real time. During this process, if the animal experiences a sudden drop in blood pressure or arrhythmia, the display controller 1 will issue an alarm in a timely manner. The operator can then adjust the operating rhythm accordingly to avoid affecting the stability of the model due to fluctuations in the animal's physiological state, thus ensuring the safe conduct of the experiment.

[0096] The standardized ischemia-reperfusion procedure then commences. Precise control is achieved through a blood flow regulation component. The operator clamps the left anterior descending coronary artery of the animal using arterial clamp 14, equipped with a fiber optic pressure sensor. The fiber optic pressure sensor monitors blood flow pressure in real time and transmits the data to the display controller 1, ensuring the blood flow pressure remains stable at ≤20 mmHg during the ischemic phase, strictly maintaining the ischemic state for 1 hour. After ischemia ends, the display controller 1 automatically triggers a command to start the injection pump, restoring blood flow to the vessel through injection tube 13. A flow sensor monitors the reperfusion blood flow rate in real time and feeds the data back to the display controller 1. The controller adjusts the injection pump accordingly, stabilizing the blood flow recovery rate at 0.5 ml / min to avoid secondary myocardial injury caused by a sudden increase in blood flow, ensuring the standardization of the reperfusion process and guaranteeing the reproducibility of experiments across different batches. Figure 1 and Figure 2 As shown.

[0097] Simultaneously with the initiation of reperfusion, the drug delivery component operates synchronously. A quantitative amount of nano-targeted drug (such as nanoparticles wrapped in immune cell membranes) is pre-stored in the syringe 18 connected to the injection tube 13. The display controller 1 triggers the injection pump, so that the drug is accurately injected into the body as blood flow is restored, simulating the drug delivery scenario in clinical interventional treatment, ensuring that the nano-drug can reach the ischemic area in a timely manner with blood flow, creating conditions for subsequent targeted accumulation monitoring.

[0098] The multimodal imaging component is activated after reperfusion begins, enabling dynamic monitoring of the nanodrug targeting process. A circular motor 6 drives roller 5 to rotate along an annular guide rail 3, causing slide 4 and connected support bar 7 to move around the animal's thoracic cavity. Simultaneously, a telescopic motor 8, through a first bevel gear 11 meshing with a second bevel gear 20, drives drive gear 21 to rotate. This, in conjunction with a pressing gear 12, causes slide 9 to extend and retract within the groove of support bar 7, flexibly adjusting the spatial positions of fluorescence imaging sensor 22 and photoacoustic imaging sensor 23 to precisely align them with the ischemic area of ​​the myocardium. Fluorescence imaging sensor 22 captures the fluorescence signal of the nanodrug (such as fluorescence binding to GFP-labeled ICAM-1), while photoacoustic imaging sensor 23 acquires structural images of the myocardial tissue. Both data are transmitted in real-time to display controller 1, dynamically presenting the accumulation, distribution, and concentration changes of the nanodrug in the ischemic area, intuitively reflecting its targeting efficiency.

[0099] Throughout the process, the display controller 1 acts as the core hub, integrating vital sign data from the cardiac monitoring component, pressure and flow parameters from the blood flow regulation component, and image data from the multimodal imaging component. Operators can control the operational status of each component in real time to ensure precise execution of time gradients for ischemia (1 hour), reperfusion (3 hours / 24 hours / 72 hours), and ischemia-reperfusion. At each time point, by combining blood pressure and heart rate changes from cardiac monitoring, blood flow regulation parameter recordings, nanodrug accumulation images from multimodal imaging, and subsequent myocardial enzyme detection and Masson staining results, the degree of myocardial injury and the targeting effect of the nanodrugs in the model can be comprehensively assessed. Figure 3 and Figure 4 As shown.

[0100] Through the synergistic effect of its components, the device integrates the maintenance of vital signs, standardized control of ischemia-reperfusion, precise delivery of nanomedicines, and dynamic monitoring of targeting efficiency in animal model construction. This effectively improves the stability and reproducibility of the model and provides a reliable experimental platform for exploring the mechanism and evaluating the efficacy of nano-targeted drugs in myocardial ischemia-reperfusion injury research.

[0101] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0102] The above descriptions are merely embodiments of the present invention. Commonly known structures and characteristics are not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the application date or priority date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical known structures or methods should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing an animal model of myocardial ischemia-reperfusion injury, characterized in that: Includes the following steps: Step 1: Pretreatment. Adult mammals were selected, and a hypertension comorbidity model was constructed by combining a high-salt diet with angiotensin II injection, or a hyperlipidemia model was constructed by combining a high-fat diet, in order to simulate the basic pathological state of clinical MIRI patients. Step 2: Standardize ischemia-reperfusion operation. On the model operating table, the left anterior descending coronary artery of the pretreated animal is ligated. The ischemia duration is set to 1 hour, and the reperfusion duration is set to a time gradient of 3 hours, 24 hours or 72 hours. During the reperfusion phase, the blood flow recovery rate is precisely controlled to 0.5 ml / min to avoid secondary injury. Step 3: Regulation of the immune microenvironment. Monocyte chemoattractant protein-1 (MCP-1) at a concentration of 10-50 ng / ml is injected locally into the ischemic area to target and recruit Ly6C cells. + Mononuclear cells enhance the targeted binding sites of nanoparticles encapsulated in the ischemic areas of myocardium and immune cell membranes; Step 4: Model evaluation and validation. At each time point of reperfusion, the targeted accumulation efficiency of nanomedicine in myocardial tissue is monitored by fluorescence imaging. The degree of myocardial damage is assessed by combining myocardial enzyme level detection, Masson staining and echocardiography to complete the model construction.

2. The method for constructing an animal model of myocardial ischemia-reperfusion injury according to claim 1, characterized in that: The adult mammals mentioned in step one include SD rats and C57BL / 6 mice, and the experimental animals are 8-12 weeks old and weigh 250-300g to match the metabolic kinetics of nanoparticles in vivo.

3. The method for constructing an animal model of myocardial ischemia-reperfusion injury according to claim 1, characterized in that: Step three also includes using gene editing to induce high expression of ICAM-1 in ischemic myocardial endothelial cells and fuse it with GFP fluorescent protein to visualize the targeted binding process of nanoparticles to vascular endothelium.

4. The method for constructing an animal model of myocardial ischemia-reperfusion injury according to claim 1, characterized in that: In step four, the targeting accumulation efficiency of nanomedicines is quantified by the fluorescence intensity ratio between the ischemic area and the normal area. A T / N ratio ≥ 3 indicates that the model targeting validation is qualified.

5. The method for constructing an animal model of myocardial ischemia-reperfusion injury according to claim 1, characterized in that: The model operation platform mentioned in step two includes a fixed platform (2), which is equipped with a constant temperature heater (19) and a display controller (1) inside. Several fixing clips (15) are fixedly connected to the fixed platform (2). The fixed platform (2) is equipped with a cardiac monitoring component, a blood flow control component, a multimodal imaging component and a drug delivery component. The constant temperature heater (19), the cardiac monitoring component, the blood flow control component, the multimodal imaging component and the drug delivery component are all connected to the controller signal. Cardiac monitoring component, used to monitor changes in blood pressure and heart rate in animal models during the construction of cardiac reperfusion injury models; The blood flow regulation component is used to perform cardiac reperfusion at regular intervals and in a quantitative manner, and to monitor blood flow pressure in real time to regulate the reperfusion blood flow rate. A multimodal imaging component for dynamically acquiring fluorescence signals and distribution images of nanomedicines in myocardial tissue; A drug delivery component is used in conjunction with a blood flow control component to inject drugs during reperfusion.

6. The method for constructing an animal model of myocardial ischemia-reperfusion injury according to claim 5, characterized in that: The cardiac monitoring component includes a non-invasive blood pressure monitoring belt (16), which is fixedly connected to a fixed platform (2). The fixed platform (2) is also fixedly connected to an electrocardiogram monitoring electrode (17). Both the non-invasive blood pressure monitoring belt (16) and the electrocardiogram monitoring electrode (17) are signal-connected to the display controller (1).

7. The method for constructing an animal model of myocardial ischemia-reperfusion injury according to claim 5, characterized in that: The blood flow control component includes an arterial clamp (14) with a fiber optic pressure sensor. An injection tube (13) is fixedly connected inside the arterial clamp (14). The other end of the injection tube (13) is connected to an injection pump. A flow sensor is installed inside the injection pump. The arterial clamp (14) is used to block the left anterior descending coronary artery. The fiber optic pressure sensor is used to monitor blood flow pressure in real time. The flow sensor is used to monitor the reperfusion blood flow rate. The fiber optic pressure sensor, the injection pump and the flow sensor are all connected to the display controller (1) via signal.

8. The method for constructing an animal model of myocardial ischemia-reperfusion injury according to claim 5, characterized in that: The multimodal imaging component includes an annular guide rail (3), which is fixedly sleeved to the side wall of the fixed stage (2). A slide block (4) is slidably fitted on the annular guide rail (3). A circumferential motor (6) is fixedly connected to the slide block (4). A roller (5) is coaxially fixedly connected to the output shaft of the circumferential motor (6). The roller (5) is rotatably fitted to the side wall of the fixed stage (2). A support bar (7) is fixedly connected to the fixed stage (2). A groove is opened at the top of the support bar (7). A slide strip (9) is slidably connected in the groove. A telescopic motor (8) is fixedly connected to the support bar (7). A first bevel gear (11) is coaxially fixedly connected to the output shaft of the telescopic motor (8). 7) A drive gear (21) is rotatably connected to the top. A second bevel gear (20) is coaxially fixedly connected to one side of the drive gear (21). The second bevel gear (20) meshes with the first bevel gear (11). A pressing gear (12) is rotatably connected to the top of the support bar (7). The slide bar (9) has symmetrical toothed grooves (10) on its upper and lower sides. The toothed grooves (10) mesh with the pressing gear (12) and the drive gear (21) respectively. A fluorescence imaging sensor (22) and a photoacoustic imaging sensor (23) are fixedly connected to the end of the slide bar (9) away from the support bar (7). Both the fluorescence imaging sensor (22) and the photoacoustic imaging sensor (23) are connected to the display controller (1) via signals.

9. The method for constructing an animal model of myocardial ischemia-reperfusion injury according to claim 5, characterized in that: The drug delivery assembly includes a syringe (18) connected to an injection tube (13), the syringe (18) having an injection pump and containing a drug for precise injection of the drug at the start of reperfusion, the injection pump being signal-connected to a display controller (1).

10. The application of the animal model of myocardial ischemia-reperfusion injury obtained by the construction method according to any one of claims 1-9 in the study of the pathogenesis of myocardial ischemia-reperfusion injury and nanomedicine targeted therapy.

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