Method for constructing an animal model of cardiotoxicity of radiotherapy combined with chemotherapy and application thereof

Abstract

This invention discloses a method and application for constructing an animal model of cardiotoxicity caused by radiotherapy combined with chemotherapy, belonging to the field of animal model construction technology. Specifically, it includes the following steps: S1: standardized screening of experimental animals; S2: clinical radiotherapy and chemotherapy administration; S3: precise localization and shielding; S4: multi-dimensional precise evaluation. This invention reduces interference from individual animal differences at the source, achieving triple matching with clinical treatment protocols in terms of dose, cycle, and timing. It solves the problems of inaccurate irradiation fields and poor repeatability caused by traditional manual lead shielding, ensuring high consistency in model construction. It enables non-invasive and quantitative assessment of cardiac injury from the early subclinical stage to the late decompensated stage, breaking through the traditional single evaluation model that relies on endpoint pathology.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to a method and application for constructing an animal model of cardiotoxicity caused by radiotherapy combined with chemotherapy. Background Technology

[0003] Radiotherapy combined with chemotherapy is one of the core treatment options for malignant tumors in clinical practice. It has become a key strategy for improving the cure rate of advanced-stage tumors and prolonging patient survival, especially in the treatment of common tumors such as breast cancer, lung cancer, and lymphoma. However, while radiotherapy and chemotherapy synergistically kill tumor cells, they also produce "cumulative toxicity" to the heart. The underlying mechanisms may involve multiple pathways, including increased DNA damage, oxidative stress and accumulation of reactive oxygen species (ROS), mitochondrial dysfunction, amplified inflammatory response, and activation of cardiomyocyte apoptosis and fibrosis. The combination of radiotherapy and chemotherapy can increase the risk of cardiac damage by 2-3 times, with clinical manifestations including arrhythmias, acute pericarditis, cardiomyopathy, coronary artery disease, chronic heart failure, and even fatal cardiomyopathy, malignant arrhythmias, and acute heart failure, seriously affecting the long-term survival, quality of life, and prognosis of cancer patients.

[0004] With the rise of the discipline of "cardio-oncology," there is an urgent need in preclinical research for animal models that can simulate this combined cardiotoxicity to elucidate the mechanisms of radiotherapy and chemotherapy cardiotoxicity, find early warning systems, and develop preventative drugs. Currently, the development of this field presents the following characteristics and challenges: First, there was a lack of standardization in animal selection: early models used ICR closed colony mice (with heterogeneous genetic backgrounds), and although later models used inbred C57BL / 6 mice, the scientific basis for strain selection was not clearly defined, nor were the effects of sex (e.g., males and females have a 20% difference in sensitivity to doxorubicin) and age (mice older than 12 weeks are prone to spontaneous myocardial fibrosis) on the model results considered, resulting in the inability to compare data from different laboratories.

[0005] Secondly, on the one hand, existing animal models focus on single-factor-induced cardiac injury models, such as doxorubicin chemotherapy alone or chest radiotherapy alone. These models can only simulate the cardiotoxicity of a single treatment and cannot create an effective toxicity superposition and amplification effect in vivo, thus failing to accurately reflect the synergistic damage mechanism of the two treatments. On the other hand, clinical practice employs fractionated radiotherapy (2-3 Gy / fraction) and multi-cycle chemotherapy (every 2-3 weeks), while existing animal models involve single high-dose irradiation (10-30 Gy) or short-term high-dose chemotherapy (such as a single 5 mg / kg doxorubicin). Their pathophysiological processes differ from the temporal development and manifestations of clinical events, making them unable to simulate the treatment strategies in actual clinical practice, thus limiting the translational value of the research results.

[0006] Secondly, the lack of standardization of key technical parameters leads to low reproducibility: ① Inaccurate irradiation field: The size and position of manually fabricated lead shielding windows are difficult to standardize, resulting in inconsistent cardiac irradiation doses and volumes in models constructed by different batches and operators, introducing significant variability. ② Insufficient dose exploration: In combined models, the selection of radiotherapy and chemotherapy doses and timing relies on experience without systematic optimization, leading to model instability. Results are difficult to replicate across different laboratories and even within the same laboratory.

[0007] Finally, the evaluation system is outdated and has a single endpoint: existing models rely heavily on in vitro histological analysis at the endpoint (such as Masson staining to observe fibrosis), which is a "static endpoint indicator." There is a lack of in vivo, dynamic, and non-invasive monitoring methods (such as high-frequency ultrasound and continuous monitoring of serum biomarker cTnI) to longitudinally assess the dynamic evolution of cardiac function from compensation to decompensation, making it impossible to detect damage early and conduct intervention studies.

[0008] Therefore, it is essential to systematically solve the fundamental technical challenges in constructing animal models of cardiotoxicity caused by combined radiotherapy and chemotherapy, and to provide a novel, standardized modeling method that highly simulates clinical pathological processes. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a method and application for constructing an animal model of cardiotoxicity caused by combined radiotherapy and chemotherapy.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for constructing an animal model of cardiotoxicity caused by combined radiotherapy and chemotherapy, specifically including the following steps: S1: Standardized screening of laboratory animals: Laboratory animals are screened based on strain, sex, weight and basic cardiac function indicators to obtain qualified animals with uniform physiological state. S2: Clinical radiotherapy and chemotherapy administration: Administer chemotherapy drugs and local cardiac radiotherapy to selected qualified animals according to the preset timing, dosage and cycle; S3: Precise positioning and shielding: During each radiotherapy session, imaging equipment is used to locate the cardiac target area, and a personalized shielding mold is used to physically shield non-target organs to ensure that the radiation dose is precisely delivered to the heart (ensuring that the dose uniformity of the cardiac target area is greater than 95%, and the radiation dose to non-target organs is less than 5% of the heart dose). S4: Multi-dimensional and precise evaluation: At key time points in the modeling process, monitor and evaluate the animal's cardiac function, serum myocardial injury markers, and histopathological indicators to confirm the successful construction of the model.

[0011] Further, in step S1, the standardized screening of experimental animals specifically involves: uniformly selecting 6-8 week old SPF-grade C57BL / 6J inbred mice; matching the sex of the animals according to the simulated tumor type, wherein female mice are selected when simulating breast cancer or ovarian cancer, and male mice are selected when simulating lung cancer or prostate cancer; and screening individuals with a weight between 18 and 22 grams.

[0012] Furthermore, in step S2, the preset timing adopts cycle chemotherapy + single radiotherapy, that is, the sequential treatment timing of chemotherapy followed by radiotherapy, specifically, a single radiotherapy is performed 24±0.5 hours after the end of all chemotherapy cycles.

[0013] Further, in step S2, the chemotherapy is performed by periodic intravenous injection of a cardiotoxic chemotherapy drug that is often used in combination with radiotherapy (periodic administration in a manner that simulates clinical cumulative toxicity); the radiotherapy is performed by using a small animal precision irradiator to deliver a single high-dose irradiation to the cardiac target area (the total dose is determined through preliminary experiments to induce significant cardiac function damage and ensure animal survival when combined with chemotherapy). When simulating a sequential chemoradiotherapy scenario for breast cancer, the cycle chemotherapy is specifically: epirubicin is injected via the tail vein at a single dose of 4 mg / kg, once every other day, for 3 consecutive cycles, with a cumulative dose of 12 mg / kg; the single radiotherapy is specifically: a total radiotherapy dose of 10 Gy, irradiated in a single session.

[0014] Further, in step S3, the imaging device is a Micro-CT scanning device; the personalized shielding mold is made by 3D printing based on Micro-CT scanning data; the mold consists of a fixed base and a shielding cover with a through hole for the heart target area; the material of the mold is a photosensitive resin with built-in high-density composite material, which has an attenuation rate of 225 kV X-rays comparable to that of 2 mm lead.

[0015] Furthermore, in step S4, the key time point is 5 days after the completion of the combined radiotherapy and chemotherapy treatment.

[0016] Further, in step S4, the multidimensional precise evaluation specifically includes: 5 days after the completion of combined radiotherapy and chemotherapy, longitudinal monitoring of left ventricular ejection fraction, left ventricular fractional shortening, systolic left ventricular diameter, and diastolic left ventricular diameter using high-frequency echocardiography; collection of serum and detection of CK-MB, NT-ProBNP, and cardiac troponin I concentrations; and myocardial tissue sampling for HE staining and Masson staining to assess cardiomyocyte damage and fibrosis. The criteria for model success are: compared with the blank control group, a decrease in left ventricular ejection fraction ≥10%, and / or a decrease in left ventricular fractional shortening ≥10%, and a significant increase in CK-MB, cardiac troponin I, and NT-proBNP levels (…). p <0.05, statistically significant.

[0017] The present invention also provides the application of an animal model constructed by the above method in screening or evaluating drugs for preventing, alleviating or treating cardiotoxicity caused by radiotherapy combined with chemotherapy.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. A standardized screening system for laboratory animals that integrates "strain-sex-physiological status": 6-8 week old SPF grade C57BL / 6 inbred mice are selected, and sex is matched according to simulated tumor type (female for breast cancer and male for lung cancer). The selection criteria of "weight 18-22g" are used, and abnormal individuals are eliminated after 1 week of acclimatization feeding, thereby reducing the interference of individual differences in animals from the source.

[0019] 2. Clinically aligned chemotherapy-radiotherapy regimen of “cycled chemotherapy + single radiotherapy + differentiated timing”: When simulating the sequential chemotherapy-radiotherapy scenario for breast cancer, chemotherapy adopts a cycle pattern of “epimacrit 4mg / kg once every other day for 3 consecutive cycles”, and radiotherapy adopts a single-session pattern of “total dose of 10Gy”, achieving a triple match with the clinical treatment plan in terms of dose, cycle and timing.

[0020] 3. Precision radiotherapy technology of "miniature CT positioning + 3D printed custom mold": The cardiac target area (2mm below the xiphoid process from the sternal angle to the xiphoid process, area 0.5cm×0.8cm) is located using a miniature CT with a resolution of 50μm. Combined with a 3D printed personalized cardiac shielding mold, the problem of inaccurate irradiation field and poor repeatability caused by traditional manual lead shielding is completely solved, ensuring a high degree of consistency in model construction.

[0021] 4. "Function-Pathology" Multidimensional Evaluation System: It integrates the intrinsic evaluation criteria of high-frequency echocardiography, serum cTnI, HE staining, and Masson staining model to achieve non-invasive and quantitative assessment of cardiac damage, breaking through the traditional single evaluation mode that relies on endpoint pathology. Attached Figure Description

[0022] Figure 1 These are the results of echocardiography in mice, where A: Control group, B: CT+RT group; Figure 2 These are the results of cardiac function tests in mice, where A: left ventricular ejection fraction (LVEF%), B: left ventricular fractional shortening (FS%), C: systolic left ventricular diameter (LVESD), and D: diastolic left ventricular diameter (LVEDD). Figure 3 These are the results of the detection of myocardial injury markers in mouse serum, where A: CK-MB, B: NT-ProBNP, and C: cardiac troponin I (cTnI). Figure 4These are the results of HE staining of mouse heart tissue; Figure 5 This is the result of Masson staining of mouse heart tissue. Detailed Implementation

[0023] To make the objectives and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0024] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0025] Example 1 1. Laboratory animal screening module This module employs a three-tiered process of "strain determination - sex matching - physiological status screening" to eliminate interference from individual differences, laying the foundation for model stability. The specific operations are as follows: 1.1 Breed and Sex Selection ① Strain determination: 6-8 week old SPF grade C57BL / 6J inbred mice were selected. The genetic background homogeneity of this strain is >99%.

[0026] ② Gender matching: Determine gender based on simulated tumor type Simulating common female cancers such as breast cancer and ovarian cancer: Female mice were selected because the estrogen level in female mice (10-30 pg / mL) can upregulate the expression of the cardiomyocyte iron transporter TfR1, resulting in a 15%-20% higher ROS production induced by epirubicin compared to male mice, which is consistent with the clinical characteristics of female patients.

[0027] Simulating tumors common in men, such as lung cancer and prostate cancer: Male mice were selected. The testosterone level in male mice (2-5 ng / mL) can slightly enhance myocardial SOD activity (increase by 10%-12%), which is consistent with the tolerance characteristics of male patients to radiotherapy and chemotherapy.

[0028] 1.2 Physiological state screening and animal grouping ① Select mice weighing 18-22g and place them in an SPF-grade animal room (temperature 22±2℃, humidity 50±10%, 12h light / 12h dark). Allow them free access to standard feed (containing 20%-22% protein and 4%-5% fat, purchased from Nanjing Model Animal Resource Bank) and sterile water. Adapt to the environment for 1 week to reduce the impact of environmental stress on the experiment.

[0029] ② The mice that passed the final screening were randomly divided into two groups, ensuring that the weight difference between each group was <1g. Blank control group (Control group): No radiotherapy or chemotherapy, only injection of normal saline.

[0030] Chemotherapy combined with radiotherapy group (CT+RT group): Epirubicin was first injected via tail vein, followed by a single local irradiation of the heart. 2. Clinical radiotherapy and chemotherapy regimen module This module designs a sequential treatment plan of "cycled chemotherapy + single radiotherapy" based on clinical oncology treatment guidelines (radiotherapy is administered 24±0.5 hours after chemotherapy) to achieve precise matching with clinical scenarios. The specific operation is as follows: 2.1 Chemotherapy regimen (using a breast cancer model as an example) (1) Drug preparation: Epirubicin hydrochloride (Sigma-Aldrich, catalog number D1515) with a purity of ≥98% was selected and dissolved in sterile physiological saline to a concentration of 2 mg / mL. It should be prepared and used immediately (the efficacy of epirubicin solution will decrease by 10%-15% if left at room temperature for more than 4 hours).

[0031] (2) Dosage and route of administration: Refer to the cumulative dose of epirubicin in clinical practice (cumulative dose of adjuvant chemotherapy for breast cancer 720 mg / m²). 2 Based on body surface area conversion (mouse dose = human dose × 12) and combined with mouse tolerance experiments (mortality rate exceeding 30% when cumulative dose exceeds 12 mg / kg), the single dose of doxorubicin in mice was determined to be 4 mg / kg, administered once a week for 3 consecutive cycles, with a cumulative dose of 12 mg / kg. The doxorubicin solution was slowly and uniformly injected via the tail vein using a tail vein injection device at a rate of 1-2 seconds / mL. The injection volume was calculated based on the mouse's body weight (e.g., 200 μL for a 20g mouse).

[0032] (3) Treatment plans for each group: Control group mice: No radiotherapy or chemotherapy, only tail vein injection of saline at the same time.

[0033] CT+RT group mice: received epirubicin via tail vein injection only at the same time.

[0034] (4) Toxicity monitoring: During chemotherapy, the mental state (activity level, hair luster) of mice was observed daily, and they were weighed once a day. If the weight loss exceeded 15% of the initial weight, the administration was suspended and nutritional support was given (0.2 mL of 5% glucose solution per mouse was injected intraperitoneally). The administration was resumed when the weight recovered to more than 85% of the initial weight.

[0035] 2.2 Radiotherapy regimen (taking a breast cancer model as an example) 2.2.1 Radiotherapy Equipment and Positioning System (1) Fabrication and use of personalized cardiac positioning and shielding molds ① Mold Design: Based on the three-dimensional data of the thoracic cavity of C57BL / 6J mice obtained by Micro-CT scanning (50μm resolution), a two-part shielding mold was designed using 3D modeling software (such as SolidWorks): A. Mouse restraint base: A groove that allows the mouse to remain in a stable supine position.

[0036] B. Precision Shielding Cover: A cover that fits snugly with the base, with only one through-hole in the cardiac anatomical projection area (approximately a rectangular window measuring 10mm × 5mm from the xiphoid process of the sternum to the second intercostal space), the rest of which is a solid structure, used to shield non-target organs such as the lungs, esophagus, and spine.

[0037] ② Mold fabrication: The mold is fabricated using a high-precision 3D printer with a photosensitive resin containing built-in high-density composite material. This material has an attenuation rate for 225kV X-rays comparable to that of 2mm lead.

[0038] ③ Instructions for use: Before irradiation, place the anesthetized mouse in a supine position on the fixed base, then tightly fasten the precision shielding cover onto the base and lock the fixing buckle to ensure that the mouse's position and the shielding position are consistent each time.

[0039] (2) Precision local radiotherapy of the heart ① Anesthesia: The mice were placed in an induction box and induced with 3% isoflurane (mixed with medical oxygen). After about 1-2 minutes, the mice entered a state of deep anesthesia. They were then transferred to a mold and anesthetized by inhalation with 1.5% isoflurane through a nasal cone.

[0040] ② Precise positioning: Start the mini CT scan to acquire a three-dimensional image of the chest (slice thickness 0.1mm), and mark the cardiac target area on the image: the upper boundary is the sternal angle (2mm below the thoracic inlet), the lower boundary is 2mm below the xiphoid process, the left boundary is 1mm to the left of the sternum, the right boundary is 1mm to the right of the sternum, and the target area is 0.5cm × 0.8cm. ③ Fixation and Shielding: Quickly place the anesthetized mouse into the personalized shielding mold described in step 2.2.1 (1) and lock it in place. Adjust the collimator of the irradiator so that the radiation beam is completely aligned with the target area. Detect the dose uniformity of the target area >95% using the dose verification system (Farmer ionization chamber), and at the same time detect the dose of non-target organs (such as the lungs) to ensure that the shielding requirements are met.

[0041] ④ Irradiation parameters: A small animal precision irradiation instrument (X-RAD 225Cx) was used. The mouse mold with the mouse fixed was placed on the irradiation platform, and the source-target distance (SSD) was adjusted to 48cm. The irradiation parameters were set as follows: voltage 225kV, current 13.3mA, with an additional 0.3mm Cu filter plate, and dose rate adjusted to 2Gy / min.

[0042] ⑤ Irradiation Implementation: Referring to the clinical adjuvant radiotherapy protocol for breast cancer surgery (total dose 50 Gy, divided into 25 fractions, 5 times a week, single dose 2 Gy), and based on the body surface area conversion formula (mouse dose = human dose × 10), combined with the mouse cardiac tolerance pre-experiment (mortality rate exceeds 50% when total dose exceeds 20 Gy), the total radiotherapy dose for mice was determined to be 10 Gy, with a single irradiation, a single dose of 10 Gy, and an irradiation time of 5 minutes per session.

[0043] In the A.CT+RT group of mice, the heart was irradiated with a single 10 Gy through a shielded window.

[0044] B. Control group mice were given the same anesthesia and fixed in place, but the radiation source was not turned on (sham irradiation).

[0045] 3. Precise positioning and shielding technology module This module ensures the accuracy of the radiotherapy target area through "equipment calibration - positioning verification - shielding optimization". The specific technical details are as follows: 3.1 Radiotherapy equipment calibration ①Dose rate calibration: Before each experiment, the dose rate of the irradiator is tested in the phantom (30cm×30cm×10cm water phantom) using a standard ionization chamber (model: PTW30013). The dose rate error is required to be <2%. If the error exceeds the range, it is adjusted through the calibration program built into the equipment. ②CT positioning calibration: Use a mouse chest phantom (including a heart simulator of known size, 3mm in diameter) for CT scans, measure the deviation between the simulator size and the actual size, and require the deviation to be <0.1mm. If the deviation exceeds the range, adjust the CT scan parameters (such as tube voltage and tube current). 3.2 Positioning accuracy verification ① In vivo marker verification: Three qualified mice were selected and injected with 0.1 mL of iodine-containing contrast agent (iopromide, 300 mgI / mL) via the tail vein. CT scans were performed 10 minutes later to observe the cardiac imaging boundary and compare it with the anatomical boundary (2 mm below the xiphoid process). The localization error was <0.2 mm. ② Repeated localization verification: Localization was performed on the same mouse for 3 consecutive days. The coordinates of the target center (X, Y, Z axes) were recorded each time, and the coefficient of variation of the coordinates was calculated. The coefficient of variation was required to be <0.1 mm to ensure that the body position was consistent for each radiotherapy. 3.3 Optimized shielding effect ① Shielding material selection: A photosensitive resin with built-in high-density composite material was used to create the mold using a high-precision 3D printer. This material's attenuation rate for 225kV X-rays is comparable to that of 2mm lead, therefore it was selected as the shielding material. ② Shielding boundary optimization: The dose to non-target organs under different shielding boundaries was calculated using dose distribution simulation software (MonteCarlo simulation), and the final determination was made. ③ Lung shielding boundary: Shifting the upper boundary of the heart upward by 5 mm can reduce the radiation dose to the lungs from 12 Gy without shielding to <0.75 Gy; ④ Liver shielding boundary: Lowering the lower boundary of the heart by 5 mm can reduce the radiation dose to the liver from 8 Gy without shielding to <0.3 Gy; ⑤ Actual effect test: Place a dose film (Gafchromic EBT3) in the mold to simulate the radiotherapy process of mice. After radiotherapy, scan the film to analyze the dose distribution and confirm that the dose uniformity of the target area is >95% and the dose to non-target organs is <5% of the heart dose.

[0046] 4. Multi-dimensional model evaluation module This module establishes a three-dimensional evaluation system encompassing "functional, pathological, and molecular" assessments, setting up four stratified evaluation nodes to dynamically monitor the damage process. Specific operations are as follows: 4.1 Evaluation Node Settings Five days after the completion of combined radiotherapy and chemotherapy, high-frequency echocardiography was used to longitudinally monitor the left ventricular ejection fraction and left ventricular fractional shortening rate in mice of each group. Serum was collected and the concentrations of cardiac troponin I, CK-MB, and NT-ProBNP were detected. Myocardial tissue was taken for HE staining and Masson staining to assess myocardial cell damage and myocardial fibrosis.

[0047] 4.2 Specific Evaluation Methods 4.2.1 High-frequency echocardiography for assessing cardiac function ① Equipment and parameters: A VisualSonics Vevo 2100 ultrasound machine with an MS400 probe (30MHz frequency) was used. Mice were lightly anesthetized with 1.5% isoflurane and fixed in a supine position on a heated platform. Hair was shaved from the chest area and coupling agent was applied.

[0048] M-mode ultrasound images of the left ventricle in long and short axis sections were acquired using a 30MHz probe.

[0049] ②Detection indicators: Left ventricular ejection fraction (LVEF): LVEF = [(LVEDV - LVESV) / LVEDV]×100, where LVEDV is the left ventricular end-diastolic volume. LVESV (left ventricular end-systolic volume) is measured using an M-mode curve; Left ventricular fractional shortening (FS): LVFS = [(LVEDD - LVESD) / LVEDD]×100, where LVEDD is the end-diastolic diameter of the left ventricle. LVESD (left ventricular end-systolic diameter) is the average of 3 cardiac cycles; ③ Judgment criteria: Compared with the Control group, the CT+RT group was considered to have achieved the target for cardiac function impairment if the left ventricular ejection fraction (LVEF%) decreased by ≥10%.

[0050] The results showed that, compared with the control group, the combined radiotherapy and chemotherapy (CT+RT group) induced a decrease in LVEF% in the mice's hearts ( p <0.0001) and FS% ( p <0.01) all decreased significantly, and the decrease was >10%; while LVESD ( p <0.001) and LVEDD ( p The levels <0.001 were all significantly increased, indicating that combined radiotherapy and chemotherapy can cause an increase in the left ventricular diameter, a decrease in left ventricular ejection fraction and fractional shortening in mice, leading to abnormal cardiac function. Figure 1 , Figure 2 ).

[0051] 4.2.2 Detection of serum myocardial injury markers: Following echocardiography in the animals, approximately 100 μL of whole blood was collected via orbital vein sampling. After the blood was allowed to stand at room temperature for 1 hour, it was centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant serum was collected. The concentrations of cTnI, CK-MB, and NT-ProBNP in the serum were measured according to the instructions of the high-sensitivity ELISA kit for cardiac troponin I (cTnI), CK-MB, and NT-ProBNP.

[0052] The results showed that, compared with the control group, the combined radiotherapy and chemotherapy (CT+RT group) significantly increased the serum levels of NT-proBNP, cTnI, and CK-MB in mice, indicating that radiotherapy and chemotherapy can cause myocardial damage in mice. Figure 3 ).

[0053] 4.2.3 Histopathological analysis: (1) Sample preparation: After the mice were euthanized, their hearts were quickly removed and rinsed with pre-cooled PBS.

[0054] (2) Histological analysis: The heart was fixed in 4% paraformaldehyde for 48 hours, then embedded in paraffin and sectioned (5 μm thick). Sections were then prepared as follows: ① HE staining: After dewaxing and hydration, the sections were stained with hematoxylin for 5 minutes, differentiated with hydrochloric acid alcohol for 30 seconds, stained with eosin for 2 minutes, dehydrated and cleared, and then mounted. The morphology, arrangement, cytoplasmic vacuolar degeneration, and necrotic foci of cardiomyocytes were observed under an optical microscope (400×).

[0055] ②Masson staining: After dewaxing and hydration, the cell nuclei were stained with Weigert's iron hematoxylin solution or hematoxylin staining solution for 8 minutes, differentiated with hydrochloric acid alcohol for 30 seconds, rinsed with running water for 30 seconds, blued with bluing solution for 2 minutes, rinsed with running water for 30 seconds, stained with Ponceau S-acid fuchsin solution for 6 minutes, rinsed with 0.2% glacial acetic acid for 2 minutes, differentiated with 1% phosphomolybdic acid solution for 3 minutes, rinsed with 0.2% glacial acetic acid aqueous solution for 2 minutes, stained with aniline blue or Fast Green staining solution for 4 minutes, dehydrated and cleared, mounted, and observed under an optical microscope (400×) to observe the degree of myocardial cell fibrosis, etc.

[0056] HE staining results showed that the myocardial structure in the Control group was normal, with clear cell outlines, tight and orderly arrangement, and no obvious degeneration. The CT+RT group showed severe myocardial damage, with reduced cardiomyocyte numbers and disordered arrangement in some areas. Large areas of calcification were also observed, accompanied by inflammatory cell infiltration and hypertrophy of peripheral cardiomyocytes. Figure 4 ).

[0057] Masson staining results showed that no obvious blue collagen fiber proliferation was observed in the Control group, while in the CT+RT group, cells were disordered in some areas, and a large amount of blue collagen fiber proliferation was observed. Figure 5 ).

[0058] This invention addresses three core shortcomings of existing mouse animal model preparation technologies for radiotherapy and chemotherapy, resulting in a precise and clinically relevant technical solution. Its beneficial effects can be elaborated from three dimensions: clinical translational value, evaluation accuracy, and standardized effectiveness assessment, using causal reasoning logic as follows: (i) Clinically closely resembles chemotherapy and radiotherapy regimens, significantly enhancing the clinical translational value of the model. To address the shortcomings of existing technologies where "chemoradiotherapy regimens are disconnected from clinical practice," this invention achieves precise clinical matching in three aspects: chemotherapy cycle, radiotherapy mode, and timing design. Chemotherapy adopts a regimen of "epimacrit 4 mg / kg once every other day for three consecutive cycles," simulating the cumulative toxicity characteristics of clinical "cycled chemotherapy" and solving the problem that "a single high dose cannot reflect cycle damage" in existing technologies. Radiotherapy adopts a single-session mode with a total dose of 10 Gy.

[0059] Causal reasoning: Due to the improved equivalence of radiotherapy modalities, chemotherapy cycles, and standard clinical protocols, the model simulates the pathological progression of combined radiotherapy and chemotherapy damage, mirroring the disease progression in clinical patients. Differentiated temporal design allows the model to accurately simulate breast cancer treatment scenarios, making it more clinically relevant. The model established in this invention is no longer a simple additive toxicity model, but reliably induces more severe and earlier phenotypes such as left ventricular dysfunction and cardiomyocyte damage. Its pathological characteristics highly match clinically observed synergistic cardiotoxicity, providing a reliable platform for translational medicine research. This effect enables more efficient translation of research findings based on this model (such as screening for chemotherapy toxicity reduction drugs and optimizing radiotherapy dosage) into clinical practice.

[0060] (ii) Precise positioning and shielding technology effectively eliminates the interference of non-target organ damage on model evaluation. To address the shortcomings of existing technologies in terms of "rough positioning and fixation", this invention constructs a technical system of "miniature CT positioning + customized mold + precise shielding": it adopts a miniature CT positioning system with a resolution of 50μm and uses a 3D-printed individualized cardiac shielding mold to replace the handmade lead sheet, ensuring a high degree of consistency of the irradiation field among different individuals.

[0061] Causal Reasoning: The core improvement of this invention lies in its innovative cardiac target localization and shielding technology, which precisely defines the irradiation area of ​​the heart and significantly reduces the radiation dose to surrounding non-target organs (such as the lungs and liver). Compared with existing technologies, this improvement allows the model to primarily exhibit targeted cardiac injury pathological features during the experimental period, while effectively controlling systemic secondary effects caused by severe damage to non-target organs (such as radiation pneumonitis, pulmonary fibrosis, and liver injury). Thus, the model eliminates significant interference from factors such as severe respiratory distress and systemic inflammation on cardiac function indicators (such as left ventricular ejection fraction, LVEF), ensuring that the observed changes in cardiac function and histological damage can be more directly and accurately attributed to the combined toxic effects of radiotherapy and chemotherapy on the heart, thereby improving the accuracy and reliability of the model in specific studies of the cardiotoxicity mechanisms of radiotherapy and chemotherapy.

[0062] (III) A multi-dimensional and precise evaluation system, clarifying the criteria for judging the effectiveness of the model, and improving the standardization of the evaluation. This invention addresses the shortcomings of existing technologies, such as "single evaluation indicators and vague judgment criteria," by establishing a multi-dimensional quantitative correlation system and evaluation nodes based on "function-pathology." At the functional level, dynamic echocardiographic monitoring five days after chemotherapy combined with radiotherapy establishes a correlation between the decrease in left ventricular ejection fraction (LVEF) and the stage of cardiac injury, clarifying the functional criteria for successful myocardial injury model assessment. At the pathological level, pathological examinations such as HE staining and Masson staining assess the degree of myocardial injury and myocardial fibrosis, establishing a quantitative correlation between these and functional indicators, constructing a complete causal chain of "pathological morphological changes—functional impairment." Simultaneously, precise evaluation nodes are set to capture the occurrence of cardiac injury, effectively solving the problems of single evaluation dimensions and unclear judgment criteria in existing technologies, significantly improving the systematic nature and reliability of model evaluation.

[0063] Causal Reasoning: This invention fundamentally changes the limitation of existing technologies that rely on subjective judgment based on a single indicator by constructing a multi-dimensional indicator collaborative verification judgment system, significantly improving the accuracy and reliability of model judgment. Even when evaluating only at a single key time point, the comprehensive analysis of multi-dimensional indicators such as cardiac function and histopathology can still comprehensively reflect the state of cardiac injury, effectively solving the core defect of existing technologies that rely solely on a single endpoint indicator and cannot accurately assess the degree of injury. This technical solution establishes a unified and objective evaluation standard system, avoiding the problem of contradictory judgment results from the same model in different studies, and providing a standardized evaluation tool for the study of the cardiotoxicity mechanism of radiotherapy and chemotherapy and drug screening. This invention deeply integrates the multimodal evaluation system into the core component of the technical solution, systematically applying high-frequency echocardiography for cardiac function assessment at key time points, combined with trace serum biomarker detection, enabling researchers to achieve a comprehensive and quantitative assessment of cardiac function status at these time points and identify early subclinical injury characteristics. This technological breakthrough allows a single evaluation to obtain comprehensive value close to clinical multi-time point monitoring, greatly improving the reliability and translational application value of research results.

[0064] In summary, this invention, through the aforementioned systematic technical solutions, achieves fundamental breakthroughs in the construction, simulation, and evaluation of mouse models of radiotherapy and chemotherapy, namely, moving from non-standardization to standardization, from clinical disconnect to clinical closeness, and from vague evaluation to precise evaluation. Its comprehensive beneficial effects are as follows: by simulating the sequential process of clinical radiotherapy and chemotherapy and precisely targeting irradiation, the model significantly improves the consistency between the model and the clinical course; by controlling non-target organ damage and establishing a multi-dimensional correlation evaluation system, the accuracy of attribution for cardiac-specific damage and the standardization of model success determination are greatly improved. This model not only provides a reliable tool for in-depth elucidation of the cardiotoxicity mechanisms of radiotherapy and chemotherapy, but also serves as an efficient screening platform for the development of chemotherapy toxicity-reducing drugs and radioprotective agents. Simultaneously, it provides precise preclinical experimental evidence for optimizing radiotherapy and chemotherapy regimens (such as dosage and timing), demonstrating significant scientific research value and clinical application prospects.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of cardiotoxicity caused by radiotherapy combined with chemotherapy, characterized in that: Specifically, the following steps are included: S1: Standardized screening of laboratory animals: Laboratory animals are screened based on strain, sex, weight and basic cardiac function indicators to obtain qualified animals with uniform physiological state. S2: Clinical radiotherapy and chemotherapy: Administer chemotherapy drugs and local cardiac radiotherapy to selected qualified animals according to the preset timing, dosage and cycle; S3: Precise positioning and shielding: During each radiotherapy session, imaging equipment is used to locate the cardiac target area, and a personalized shielding mold is used to physically shield non-target organs to ensure that the radiation dose is precisely delivered to the heart. S4: Multi-dimensional and precise evaluation: At key time points in the modeling process, monitor and evaluate the animal's cardiac function, serum myocardial injury markers, and histopathological indicators to confirm the successful construction of the model.

2. The method for constructing an animal model of cardiotoxicity from combined radiotherapy and chemotherapy according to claim 1, characterized in that: In step S1, the standardized screening of experimental animals specifically involves: uniformly selecting 6-8 week old SPF-grade C57BL / 6J inbred mice; matching the sex of the animals according to the simulated tumor type, wherein female mice are selected when simulating breast cancer or ovarian cancer, and male mice are selected when simulating lung cancer or prostate cancer; and screening individuals with a weight between 18 and 22 grams.

3. The method for constructing an animal model of cardiotoxicity from combined radiotherapy and chemotherapy according to claim 1, characterized in that: In step S2, the preset timing is cycle chemotherapy + single radiotherapy, that is, sequential treatment timing of chemotherapy followed by radiotherapy, specifically, a single radiotherapy is performed 24±0.5 hours after the end of all chemotherapy cycles.

4. The method for constructing an animal model of cardiotoxicity from combined radiotherapy and chemotherapy according to claim 1, characterized in that: In step S2, the chemotherapy is a chemotherapy drug that is cardiotoxic and often used in combination with radiotherapy, administered by periodic intravenous injection; the radiotherapy is a single high-dose irradiation of the cardiac target area using a small animal precision irradiation device. When simulating a sequential chemoradiotherapy scenario for breast cancer, the cycle chemotherapy is specifically: epirubicin is injected via the tail vein at a single dose of 4 mg / kg, once every other day, for 3 consecutive cycles, with a cumulative dose of 12 mg / kg; the single radiotherapy is specifically: a total radiotherapy dose of 10 Gy, irradiated in a single session.

5. The method for constructing an animal model of cardiotoxicity from combined radiotherapy and chemotherapy according to claim 1, characterized in that: In step S3, the imaging device is a Micro-CT scanning device; the personalized shielding mold is made by 3D printing based on Micro-CT scanning data; the mold consists of a fixed base and a shielding cover with a through hole for the heart target area; the material of the mold is a photosensitive resin with built-in high-density composite material.

6. The method for constructing an animal model of cardiotoxicity from combined radiotherapy and chemotherapy according to claim 1, characterized in that: In step S4, the key time point is 5 days after the completion of the combined radiotherapy and chemotherapy treatment.

7. The method for constructing an animal model of cardiotoxicity from combined radiotherapy and chemotherapy according to claim 1, characterized in that: In step S4, the multidimensional precise evaluation specifically includes: 5 days after the completion of combined radiotherapy and chemotherapy, using high-frequency echocardiography to longitudinally monitor left ventricular ejection fraction, left ventricular fractional shortening, systolic left ventricular diameter, and diastolic left ventricular diameter; collecting serum and detecting the concentrations of CK-MB, NT-ProBNP, and cardiac troponin I; and taking myocardial tissue for HE staining and Masson staining to assess myocardial cell damage and fibrosis. The criteria for model success are: compared with the blank control group, a decrease in left ventricular ejection fraction ≥10%, and / or a decrease in left ventricular fractional shortening ≥10%, and a significant increase in the levels of CK-MB, cardiac troponin I, and NT-proBNP.

8. The use of animal models constructed by the method of any one of claims 1-7 in screening or evaluating drugs for the prevention, mitigation or treatment of cardiotoxicity from radiotherapy combined with chemotherapy.

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