Photocoagulation thymus ischemia animal model, method and application of photocoagulation thymus ischemia animal model in rapid simulation of thymus degeneration and immune aging

Abstract

The invention belongs to the technical field of experimental animal models, biomedical research and immunology, and discloses a photocoagulation thymus ischemia animal model, a method and application of the photocoagulation thymus ischemia animal model in rapid simulation of thymus degeneration and immune senescence, and the model shows at least one or more characteristics 1-7 days after induction: thymus atrophy, thymus weight or thymus index is significantly reduced; the cortical-medullary structure of thymus is disordered or disappears; the expression distribution of the Keratin 5 / Keratin 8 is abnormal; thymic cell apoptosis is increased, and lipid deposition is enhanced; the proportion of CD4 + CD8 + double positive T cells is reduced, and the proportion of DN2 / DN3 stage T cells is increased; the initial T cell output is reduced; and the immune function is reduced. The pathological mechanism is clear; the modeling period is short, and the immune senescence phenotype can be stably obtained within several days; the injury focus is controllable, and systemic stress or hormone interference is avoided; the model has high repeatability and is suitable for mechanism research and drug screening.

Description

Technical Field

[0001] This invention belongs to the fields of experimental animal models, biomedical research and immunology, and in particular, a photocoagulation animal model of thymic ischemia, a method and its application in rapidly simulating thymic degeneration and immune aging. Background Technology

[0002] The thymus is a core organ in the mammalian immune system responsible for the development and differentiation of T cells. Its functional integrity is crucial for maintaining peripheral immune homeostasis, resisting infection, and preventing tumor development. With age, the thymus undergoes progressive involution, characterized by cortical-medullian structural disorder, a decrease in the number of thymocytes, increased fatty infiltration, and reduced output of new T cells. Ultimately, this leads to a shrinking peripheral pool of naive T cells and a decline in immune response. This process is considered one of the core drivers of immunosensenescence and is closely associated with various age-related diseases, including post-stroke immunosuppression and susceptibility to infection, vascular dementia, metabolic diseases, tumor immune escape, and neurodegenerative diseases.

[0003] Currently, animal models used to study thymic degeneration and immune aging mainly include: 1) Natural aging model: This model obtains a natural thymic atrophy phenotype by feeding animals for a long period of time. Although it can realistically reflect age-related physiological processes, the experimental cycle is long (usually requiring mice ≥18 months old), costly, and with large individual differences, making it difficult to meet the needs of rapid research. 2) Glucocorticoid-induced model: Exogenous glucocorticoids can induce thymic atrophy in a short time, but their systemic effects are significant, accompanied by bone marrow suppression, peripheral immune changes, and hormone-dependent damage, making it difficult to distinguish between local thymic effects and systemic stress responses. 3) Surgical resection model: Surgical removal of the thymus directly leads to thymic dysfunction. Although it can directly verify the contribution of the thymus to immune homeostasis, this method is highly invasive and irreversible, easily leading to severe changes in the overall immune environment of the body, and cannot simulate the natural pathological process of progressive thymic degeneration.

[0004] In summary, existing thymic involution models all have limitations: natural aging models have excessively long cycles, glucocorticoid-induced models are affected by systemic effects, and surgical resection models are highly invasive and lack reversibility. These limitations make it difficult for researchers to independently, rapidly, and specifically assess the mechanisms by which thymic involution affects immunosenescence and the development of related diseases. Therefore, there is an urgent need to develop a rapid, reproducible, highly specific animal model of thymic involution that does not affect systemic hormone levels, providing an innovative tool for in-depth analysis of immunosenescence mechanisms and the development of intervention strategies. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a photocoagulation animal model of thymic ischemia, a method thereof, and its application in rapidly simulating thymic degeneration and immune aging.

[0006] The technical solution adopted by this invention to solve its technical problem is: A photocoagulation animal model of thymic ischemia, wherein the thymic ischemia animal model exhibits at least one or more of the following typical thymic degeneration and immune aging characteristics within 1–7 days after induction: (1) Thymus atrophy, with a significant decrease in thymus weight or thymus index; (2) Disorder or disappearance of the thymic cortex-medullary structure; (3) Abnormal expression distribution of Keratin 5 / Keratin 8; (4) Increased thymocyte apoptosis and enhanced lipid deposition; (5) CD4 + CD8 + The proportion of double-positive T cells decreased, while the proportion of T cells in the DN2 / DN3 stage increased, suggesting T cell developmental arrest. (6) Decreased initial T cell output and decreased immune function.

[0007] Furthermore, the thymic ischemia animal model can continuously induce cognitive dysfunction and abnormal peripheral immune response for at least 28 days after induction, manifesting as stable pathological changes and functional defects. The animal model of thymic ischemia exhibited thymic degeneration and immunosenescence phenotypes within 1–7 days.

[0008] The method for constructing the photocoagulation animal model of thymic ischemia, as described above, involves administering a photosensitizer to experimental animals and applying light of a specific wavelength to the projection area of ​​the thymus surface within a predetermined time window. This induces thrombosis in local blood vessels of the thymus, thereby causing focal ischemic injury. This method achieves specific ischemic intervention of the thymus without significantly affecting systemic hormone levels and systemic physiological state. The thymic ischemic degeneration animal model (TI) established using the above method can rapidly and stably reproduce the thymic degeneration process within a short period and further induce immune aging-related phenotypes.

[0009] Furthermore, it includes the following steps: (1) A photosensitizer is administered to the experimental animal, wherein the photosensitizer is a compound that can induce intravascular thrombosis under irradiation with light of a specific wavelength; (2) After the photosensitizer is administered, the surface projection area of ​​the animal's thymus is irradiated at a fixed point within a predetermined time window to induce thrombosis in the local blood vessels of the thymus, thereby inducing focal ischemic injury of the thymus. (3) After exposure to light, animals were fed a recovery diet to obtain an animal model with thymic degeneration and immune aging-related phenotypes.

[0010] Furthermore, the photosensitizer is selected from Rose Bengal, Erythrosin B, or their functional equivalents; Alternatively, the photosensitizer may be administered via intraperitoneal injection, intravenous injection, or oral administration. Alternatively, the photosensitizer dose is 10–100 μg / g body weight; Alternatively, the illumination wavelength is 500–580 nm, and the illumination time is 5–30 minutes; Alternatively, the radius of the irradiated area corresponding to the surface projection region of the thymus is 0.2–1.0 cm; Alternatively, the light exposure may begin 1–15 minutes after administration; Alternatively, the experimental animals may be fully developed adult experimental animals. Age selection note: In various embodiments of the present invention, the experimental animals are preferably fully developed adult animals. The inventors have found that, compared to juvenile animals, adult animals are more likely to exhibit stable and persistent thymic degeneration and immunosenescence-related phenotypes after thymic ischemia; however, the technical solution of the present invention is not limited to adult animals within a specific age range.

[0011] The application of the photocoagulation animal model of thymic ischemia, as described above, in screening interventions to improve thymic degeneration or immune aging.

[0012] The application of the photocoagulation thymic ischemia animal model, as described above, in the study of diseases related to the brain-thymus-immune axis.

[0013] The application of the photocoagulation thymic ischemia animal model described above in the study of the mechanisms of thymic degeneration and immune aging.

[0014] The applications of the photocoagulation thymic ischemia animal model, as described above, in studies of immune remodeling and cognitive impairment following cerebrovascular diseases such as stroke and vascular dementia, and / or in studies of metabolic syndrome, infection susceptibility, and tumor immune escape mechanisms, and / or in studies of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The application of the photocoagulation thymic ischemia animal model described above in the screening and efficacy evaluation of drugs, interventions or cell products that delay thymic degeneration or improve immune aging.

[0015] The advantages and positive effects of this invention are as follows: 1. Compared with natural aging models (long modeling period), glucocorticoid-induced models (significant systemic effects), and thymectomy models (highly invasive and irreversible), this invention has the following significant advantages: 1) Rapid induction: Thymic involution and immunosenescence phenotypes can be stably reproduced within 7–28 days; 2) High specificity: It induces thymic ischemia through local vascular photocoagulation, independent of changes in systemic hormone levels; 3) The method is simple: it is highly controllable, has good repeatability, and is suitable for a variety of laboratory animals; 4) Wide range of applications: It can be used for basic mechanism research, as well as as a disease model and intervention evaluation tool.

[0016] 2. This invention establishes for the first time an animal model of thymic degeneration based on ischemia mechanism, with a clear pathological mechanism; the modeling cycle is short, and the immune aging phenotype can be stably obtained within a few days; the damage is focal and controllable, avoiding systemic stress or hormonal interference; the model has high reproducibility and is suitable for mechanism research and drug screening; it provides a new experimental tool for studying the brain-thymus-immune axis and related diseases.

[0017] 3. The animal model of thymic ischemia established in this invention and its applications can be widely used for: research on the mechanisms of thymic degeneration and immune aging; research on immune remodeling and cognitive dysfunction after cerebrovascular diseases such as stroke and vascular dementia; research on metabolic syndrome, infection susceptibility and tumor immune escape-related mechanisms; research on neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease; and screening and efficacy evaluation of drugs, interventions or cell products that delay thymic degeneration or improve immune aging.

[0018] 4. The model of this invention is an animal model that induces focal ischemic injury in the thymus of animals through photosensitizer-mediated site-directed photocoagulation technology, thereby rapidly and stably constructing an animal model of thymic degeneration and immune aging phenotype. This model can be applied to immune aging, brain-immune axis, disease mechanism research and drug screening.

[0019] 5. This invention overcomes the shortcomings of existing thymic degeneration models, such as long modeling cycles, susceptibility to systemic effects, or significant operational trauma. It provides a simple, controllable, and highly reproducible method for constructing an animal model of thymic ischemia, which can be used to rapidly and specifically simulate thymic degeneration and immune aging phenotypes, and can be further applied to the study of immune aging-related mechanisms and the immunological basis of disease development.

[0020] 6. This invention provides a rapid, specific, and reproducible animal model of thymic degeneration and its applications. A thymic ischemic degeneration model is established by inducing local ischemia in the mouse thymus using photocoagulation thrombosis technology. Within 1–7 days, this model exhibits typical degenerative features such as thymic atrophy, cortical-medullary structural disorder, increased lipid deposition, accumulation of senescence-related cells, and T-cell developmental arrest. Within 28 days after induction, it further induces cognitive impairment and systemic peripheral immune responses, manifesting as stable pathological and functional deficiencies. Compared with existing natural aging models (long cycle), glucocorticoid-induced models (with systemic effects), and thymectomy models (highly invasive and affecting systemic immunity), this invention has advantages such as rapid induction, simple method, high specificity, and independence from systemic hormone levels. The model and method of this invention can be widely applied to the study of thymic degeneration, immunosenescence, and the occurrence, development, and intervention of related diseases. Attached Figure Description

[0021] Figure 1 This is a TI modeling flowchart of the present invention; Figure 2 This is a diagram showing the changes in tissue structure and keratin expression after thymic ischemia-injury in this invention. Figure 3 This is a diagram showing the detection results of changes in senescent cells and lipid deposition after thymic ischemia-induced injury in this invention. Figure 4 This is a flow cytometry result of the number of thymocytes and the differentiation of T cell subsets after thymic ischemia injury in this invention. Figure 5 This is a figure showing the results of differential gene expression and GO enrichment analysis after thymic ischemia injury in this invention; Figure 6 This diagram illustrates the cognitive impairment and myelin damage caused by thymic ischemia-induced injury in this invention. Detailed Implementation

[0022] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0023] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0024] A photocoagulation animal model of thymic ischemia, wherein the thymic ischemia animal model exhibits at least one or more of the following typical thymic degeneration and immune aging characteristics within 1–7 days after induction: (1) Thymus atrophy, with a significant decrease in thymus weight or thymus index; (2) Disorder or disappearance of the thymic cortex-medullary structure; (3) Abnormal expression distribution of Keratin 5 / Keratin 8; (4) Increased thymocyte apoptosis and enhanced lipid deposition; (5) CD4 + CD8 + The proportion of double-positive T cells decreased, while the proportion of T cells in the DN2 / DN3 stage increased, suggesting T cell developmental arrest. (6) Decreased initial T cell output and decreased immune function.

[0025] Furthermore, the thymic ischemia animal model can continuously induce cognitive dysfunction and abnormal peripheral immune response for at least 28 days after induction, manifesting as stable pathological changes and functional defects. The animal model of thymic ischemia exhibited thymic degeneration and immunosenescence phenotypes within 1–7 days.

[0026] The method for constructing the photocoagulation animal model of thymic ischemia, as described above, involves administering a photosensitizer to experimental animals and applying light of a specific wavelength to the projection area of ​​the thymus surface within a predetermined time window. This induces thrombosis in local blood vessels of the thymus, thereby causing focal ischemic injury. This method achieves specific ischemic intervention of the thymus without significantly affecting systemic hormone levels and systemic physiological state. The thymic ischemic degeneration animal model (TI) established using the above method can rapidly and stably reproduce the thymic degeneration process within a short period and further induce immune aging-related phenotypes.

[0027] Furthermore, it includes the following steps: (1) A photosensitizer is administered to the experimental animal, wherein the photosensitizer is a compound that can induce intravascular thrombosis under irradiation with light of a specific wavelength; (2) After the photosensitizer is administered, the surface projection area of ​​the animal's thymus is irradiated at a fixed point within a predetermined time window to induce thrombosis in the local blood vessels of the thymus, thereby inducing focal ischemic injury of the thymus. (3) After exposure to light, animals were fed a recovery diet to obtain an animal model with thymic degeneration and immune aging-related phenotypes.

[0028] Furthermore, the photosensitizer is selected from Rose Bengal, Erythrosin B, or their functional equivalents; Alternatively, the photosensitizer may be administered via intraperitoneal injection, intravenous injection, or oral administration. Alternatively, the photosensitizer dose is 10–100 μg / g body weight; Alternatively, the illumination wavelength is 500–580 nm, and the illumination time is 5–30 minutes; Alternatively, the radius of the irradiated area corresponding to the surface projection region of the thymus is 0.2–1.0 cm; Alternatively, the light exposure may begin 1–15 minutes after administration; Alternatively, the experimental animals may be fully developed adult experimental animals. Age selection note: In various embodiments of the present invention, the experimental animals are preferably fully developed adult animals. The inventors have found that, compared to juvenile animals, adult animals are more likely to exhibit stable and persistent thymic degeneration and immunosenescence-related phenotypes after thymic ischemia; however, the technical solution of the present invention is not limited to adult animals within a specific age range.

[0029] The application of the photocoagulation animal model of thymic ischemia, as described above, in screening interventions to improve thymic degeneration or immune aging.

[0030] The application of the photocoagulation thymic ischemia animal model, as described above, in the study of diseases related to the brain-thymus-immune axis.

[0031] The application of the photocoagulation thymic ischemia animal model described above in the study of the mechanisms of thymic degeneration and immune aging.

[0032] The applications of the photocoagulation thymic ischemia animal model, as described above, in studies of immune remodeling and cognitive impairment following cerebrovascular diseases such as stroke and vascular dementia, and / or in studies of metabolic syndrome, infection susceptibility, and tumor immune escape mechanisms, and / or in studies of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The application of the photocoagulation thymic ischemia animal model described above in the screening and efficacy evaluation of drugs, interventions or cell products that delay thymic degeneration or improve immune aging.

[0033] Specifically, the relevant preparation and testing methods are as follows: 1. Method for establishing the thymic ischemia (TI) model: Healthy male C57BL / 6 mice (30-35g, SPF grade) aged 6-8 months were used for the experiment. The mice were anesthetized by isoflurane inhalation (anesthetic volume concentration 2%-3%, maintenance concentration 1.5%). After the mice were completely anesthetized, the surgical area (the projection of the thymus surface) was prepared, disinfected with povidone-iodine, and treated with alcohol to remove iodine. The mice were fixed in a supine position on a constant temperature operating table (maintaining body temperature 37±0.5℃).

[0034] Precisely locate the projection area of ​​the thymus body: Using the intersection of the anterior midline and the manubrium of the sternum as the center, delineate the circular irradiation area with a radius of 0.5 cm using a sterile marker pen. Subsequently, inject the photosensitizing dye rose red (dissolved in 0.9% physiological saline, dose 70 μg / g, injection volume not exceeding 0.2 ml / 10g body weight) intraperitoneally, and wait 7 minutes after injection to allow the dye to fully distribute.

[0035] A cold-light green laser (model KL 1600 LED SCHOTT Germany, wavelength 535±25nm) was used. The laser probe was adjusted to be vertically aligned with the marked area, and the light intensity was set to level 4.5. Irradiation was continued for 20 minutes to induce local stable coagulant formation. After the light exposure, the irradiated skin area was disinfected again with povidone-iodine. The skin incision was intermittently sutured with 4-0 absorbable sutures. The mice were placed in a constant-temperature resuscitation incubator until they spontaneously recovered. Postoperative care and observation were normal.

[0036] Figure 1 Modeling flowchart for TI: The thymus region of mice was irradiated with a 535±25 nm cold light source for 20 minutes via intraperitoneal injection of the photosensitizer rose red to induce local vascular occlusion and construct a thymus ischemia model. Olfactory tests and three-box social tests were conducted on days 7, 14 and 28 after modeling. Mice were sacrificed on day 28 after modeling, and tissue samples were collected for pathological staining, flow cytometry, RNA-Seq and other detection to analyze the pathological and molecular characteristics of the model.

[0037] 2. Tissue Morphology Staining Method: Adult male C57BL / 6 mice were sacrificed 7 and 28 days after thymic ischemia modeling, and thymic tissue was collected. The thymic tissue was quickly fixed in 4% paraformaldehyde fixative at 4°C for 24-48 hours. A portion of the fixed thymic tissue was routinely dehydrated, cleared, paraffin-embedded, and prepared into paraffin blocks for HE staining. Another portion, if used for immunofluorescence experiments, could be used directly or dehydrated in a sucrose gradient to 30% sucrose solution, then embedded in OCT and prepared as 5-10 μm thick frozen sections. For HE paraffin staining, the paraffin sections were first dewaxed sequentially, then rehydrated in ethanol at various gradients, and finally washed twice with distilled water to complete the phase transition. The sections were then immersed in Harris hematoxylin staining solution for 3-5 minutes at room temperature, rinsed with tap water for 10 minutes, and then placed in 1% hematoxylin solution. Differentiate in hydrochloric acid-ethanol solution for 30-60 seconds, then immediately transfer to 0.5% ammonia solution for 3-5 minutes to re-blue, and rinse with tap water for 10 minutes. Next, stain with eosin for 30-60 seconds, then gently wash with tap water. Dehydrate sequentially with each grade of ethanol for 3 minutes each, and clear with xylene for 5 minutes. Finally, add neutral resin, cover with a coverslip, and air dry at room temperature before observation under an optical microscope. For immunofluorescence staining, the fixed thymus tissue sections are washed three times with 1×PBS buffer (pH 7.4), incubated at 37°C for 1 hour with blocking solution containing 5% bovine serum albumin and 0.3% Triton X-100 to block non-specific binding sites. After discarding the blocking solution, add a mixture of Keratin 5 and Keratin 8 diluted with the blocking solution at a ratio of 1:200-1:500. Incubate the sections overnight at 4°C in a humidified chamber. The next day, remove the sections and wash with 1×PBS for 3 minutes. Next, add fluorescently labeled secondary antibodies (Alexa Fluor 488-labeled anti-Keratin 5 and Alexa Fluor 594-labeled anti-Keratin 8) diluted at a volume ratio of 1:500. Incubate at room temperature in the dark for 1 hour. After rinsing three times with 1×PBS in the dark, add DAPI staining solution at a final concentration of 1 μg / mL and stain at room temperature in the dark for 5 minutes. Rinse three times with 1×PBS in the dark. After slightly drying the sections, add anti-fluorescence quenching mounting medium and cover with coverslips. Finally, observe and acquire images under a fluorescence microscope at the appropriate excitation wavelengths (DAPI: 350 nm, Alexa Fluor 488: 488 nm, Alexa Fluor 594: 594 nm).

[0038] like Figure 2As shown, this experiment included a sham surgery group (specifically, the sham surgery group in this invention is the same as the thymic ischemia (TI) model group except that the laser irradiation was not performed; all other procedures were the same, except that the cold light source green laser probe was vertically aligned with the marked area and held in position for 20 minutes, followed by routine postoperative treatment and normal feeding and observation), a thymic ischemia model sacrificial group 7 days after the model, and a thymic ischemia model sacrificial group 28 days after the model. The thymic tissue structure and keratin (Keratin5, Keratin8) expression levels were detected by hematoxylin-eosin staining and immunofluorescence staining. Figure 2 A. Changes in tissue structure (hematoxylin-eosin staining): The corticomena of the thymus were clearly defined in the sham-operated group (arrows indicated); 7 days after thymic ischemia, the corticomena disappeared completely, and the tissue arrangement was disordered; 28 days after thymic ischemia, the corticomena partially recovered, but the tissue atrophy was obvious, and the cortex / medulla ratio was significantly lower than that in the sham-operated group (4.0±0.3 in the sham-operated group and 2.0±0.2 in the 28-day group, p<0.001).

[0039] Figure 2 B. Changes in keratin expression (immunofluorescence staining): Medullary marker (cytokeratin 5): The mean fluorescence intensity in the sham-operated group was 120±5.0; it significantly decreased to 50±3.0 in the 7-day post-thymic ischemia group (p<0.001), and remained at 45±4.0 in the 28-day group (p<0.001); Cortical marker (cytokeratin 8): The mean fluorescence intensity in the sham-operated group was 200±8.0; it significantly decreased to 150±6.0 in the 7-day post-thymic ischemia group (p<0.05), and further decreased to 120±5.0 in the 28-day group (p<0.05). These results indicate that thymic ischemia-induced corticomedullary structural disorder occurred 7 days after injury, and although partial recovery occurred at 28 days, tissue atrophy was observed; simultaneously, the expression of medullary and cortical-specific keratins continued to decrease significantly, suggesting a progressive damage to thymic structure and function.

[0040] 3. Oil Red O staining, SA-β-Gal staining, and TUNEL apoptosis staining methods: Male C57BL / 6 mice aged 6-8 months were euthanized at 7 and 28 days post-modeling for thymic ischemia, and thymic tissue was harvested. The thymic tissue was fixed in 4% paraformaldehyde at 4°C for 24-36 hours. After fixation, the thymic tissue was subjected to a gradient dehydration process, first by immersion in 20% sucrose solution at 4°C until set, then in 30% sucrose solution at 4°C until set. Subsequently, it was embedded in OCT embedding medium and cryostated at -20°C to prepare 8-10 μm thick sections for Oil Red O staining. For SA-β-Gal staining, the fixed thymic tissue was first fixed in 4% paraformaldehyde at room temperature for 15-20 minutes, then gently washed three times with 1×PBS buffer (pH 7.4) for 5 minutes each time. After discarding the 1×PBS, SA-β-Gal staining working solution (containing 1 mg / mL X-Gal and 40 mmol / L) was added. For the staining of tissues or sections, use a citrate-disodium hydrogen phosphate buffer (pH 6.0), 5 mmol / L potassium ferricyanide, 5 mmol / L potassium ferrocyanide, 150 mmol / L sodium chloride, and 2 mmol / L magnesium chloride. Place the tissues or sections in a humidified chamber and incubate at 37°C in the dark for 8-16 hours. After incubation, rinse three times with 1×PBS buffer for 5 minutes each time to terminate the reaction. Then, counterstain with 0.1% nucleoside O staining solution at room temperature for 5-8 minutes. Gently rinse with tap water for 2-3 minutes to remove excess staining solution. Add glycerol gelatin mounting medium, cover with a coverslip, and allow to air dry at room temperature before observing under an optical microscope. For Oil Red O staining, place the frozen sections on a slide, allow to air dry at room temperature, and fix in 4% paraformaldehyde fixative for 10 minutes. Rinse three times with distilled water for 2 minutes each time. Then, add Oil Red O working solution (Oil Red O stock solution and distilled water mixed at a 3:2 volume ratio, filtered, and used) and incubate at room temperature for 10 minutes. Differentiate the sections with isopropanol solution for 30 seconds until the background is colorless. Rinse twice with distilled water for 2 minutes each time. Then add hematoxylin staining solution to stain the nuclei at room temperature for 3 minutes. After rinsing with distilled water, use 0.5% ammonia solution to blue back for 30 seconds. Rinse three times with distilled water for 2 minutes each time. Finally, add glycerol gelatin mounting medium, cover with a coverslip, and observe the lipid staining under an optical microscope.

[0041] TUNEL apoptosis staining (frozen sections, 488 fluorescent labeling): Frozen sections of the thymus (8-10 μm thick) 7 days and 28 days after thymic ischemia modeling were placed on glass slides, air-dried at room temperature, and then fixed in 4% paraformaldehyde fixative at room temperature for 15 min. The slides were rinsed three times with distilled water for 2 min each time. Subsequently, 0.3% Triton X-ray solution was added. X-100 was permeabilized with 1×PBS solution at room temperature for 15 min, then gently rinsed three times with 1×PBS buffer for 5 min each time. After discarding the 1×PBS, TUNEL apoptosis detection working solution (containing terminal deoxynucleotidyl transferase, FITC-labeled dUTP, and reaction buffer) was added, and the slides were placed in a humidified chamber and incubated at 37°C in the dark for 60 min. After incubation, the slides were rinsed three times with 1×PBS buffer in the dark for 5 min each time to terminate the reaction. DAPI staining solution with a final concentration of 1 μg / mL was added, and the slides were stained at room temperature in the dark for 5 min, then rinsed three times with 1×PBS in the dark for 2 min each time. After the slides were slightly dried, anti-fluorescence quenching mounting medium was added, and coverslips were placed on top. After drying at room temperature, the slides were observed under a fluorescence microscope, and fluorescence images of apoptotic cells were acquired at the corresponding excitation wavelengths (DAPI: 350 nm, Alexa Fluor 488: 488 nm).

[0042] pass Figure 3 It can be seen that β-galactosidase staining, Oil Red O staining, and TUNEL staining can be used to detect senescent cells, lipid deposition, and apoptotic cell status in thymic tissue.

[0043] Figure 3 A. β-Galactosidase staining (detection of senescent cells): The percentage of positive areas in the sham-operated group was at the baseline level; the percentage of positive areas in the group 7 days after thymic ischemia showed no significant change compared to the sham-operated group. p =ns); 28 days after thymic ischemia, the proportion of positive areas significantly increased to 1.5±0.3% in the group (compared to 0.8±0.2% in the sham surgery group). p <0.05), indicating a significant increase in the number of senescent thymic cells at 28 days.

[0044] Figure 3 B. Oil Red O staining (lipid deposition detection): The percentage of positive areas in the sham-operated group was at the baseline level; 7 days after thymic ischemia, the percentage of positive areas significantly increased to 4.2±0.5% (compared to 2.0±0.3% in the sham-operated group). p <0.05); 28 days after thymic ischemia, the proportion of positive areas in the group decreased to 3.0±0.4%, which was still higher than that in the sham surgery group but not significantly different from that in the 7-day group. p =ns), indicating a significant increase in thymic lipid deposition 7 days after ischemia, which showed a downward trend at 28 days.

[0045] Figure 3C. Increased thymocyte apoptosis level (TUNEL apoptosis staining): The mean fluorescence intensity of apoptotic thymocytes in the sham-operated group was 2.2±0.3; 7 days after thymic ischemia-injury, the fluorescence intensity of apoptotic cells significantly increased to 4.5±0.4 (p<0.05), an increase of about 105% compared with the sham-operated group; 28 days after surgery, the fluorescence intensity of apoptotic cells further increased to 5.8±0.5 (p<0.05), an increase of about 164% compared with the sham-operated group.

[0046] The above results indicate that after thymic ischemia-induced injury, senescent cells significantly accumulated at 28 days, while lipid deposition peaked at 7 days; both factors jointly contribute to the pathological damage process following thymic ischemia. Thymic ischemia-induced injury can induce a significant increase in thymocyte apoptosis levels, and this apoptosis activation effect persists.

[0047] 4. Flow cytometry method: Adult male C57BL / 6 mice aged 6-8 months were sacrificed on days 1, 3, and 7 after thymic ischemia modeling. Thymic tissue was aseptically removed and immediately rinsed three times gently in pre-chilled pH 7.4 1×PBS buffer (containing 1% penicillin-streptomycin mixture) to remove residual blood and connective tissue. The washed thymic tissue was then transferred to a sterile culture dish containing 2 mL of pre-chilled 1×PBS and minced to 1 mm using sterile ophthalmic scissors. 3 The resulting homogenate was then filtered through a 200-mesh nylon cell strainer into a 15 mL centrifuge tube. The strainer was rinsed twice with 5 mL of pre-cooled 1×PBS, and all filtrate was collected to obtain a thymus single-cell suspension. The single-cell suspension was centrifuged at 1200 rpm for 5 min at 4°C. The supernatant was discarded, and 1 mL of 1×PBS containing 10% fetal bovine serum (FBS) was added for resuscitation. The cells were gently pipetted until no obvious precipitation occurred. 20 μL of the cell suspension was used for cell counting using a hemocytometer. Based on the counting results, the cell concentration was adjusted to 1×10⁻⁶ cells / mL using 1×PBS containing 10% FBS. 6Cells / mL; Take 100 μL of the adjusted cell suspension and add it to a flow cytometry tube. Add fluorescently labeled surface molecules (anti-mouse CD3-PE antibody, anti-mouse CD4-FITC antibody, anti-mouse CD8-APC antibody, anti-mouse CD44-PE-Cy7 antibody, and anti-mouse CD62L-APC-Cy7 antibody; each antibody is diluted 1:100 with 1×PBS containing 10% FBS, 5 μL per tube). Gently mix and incubate at 4°C in the dark for 30 min. During incubation, gently invert the tube once every 10 min to ensure sufficient antibody binding to cells. After incubation, add 3 mL of pre-chilled 1×PBS to each tube, centrifuge at 1200 rpm for 5 min at 4°C, discard the supernatant, and repeat this washing step twice. Finally, add 300 μL of [unspecified ingredient] to each tube. Pre-cool 1×PBS and gently pipette to resuspend cells, ensuring they are single-celled and not aggregated. Place the processed cell sample on the flow cytometer sample holder, set the excitation wavelength (FITC: 488nm, PE: 488nm, APC: 633nm, PE-Cy7: 488nm, APC-Cy7: 633nm) and detection channels. Use a blank cell suspension without antibody as a negative control and known positive cells as a positive control for instrument calibration. Then analyze the samples, select the target cell population using flow cytometry software (such as FlowJo), and calculate CD3. + CD4 in T cells + CD8 - CD4 - CD8 + Subgroup ratios and CD4 + CD8 + CD44 in T cells + CD62L - (Effective memory T cells), CD44 - CD62L + Phenotypic distribution of (naive T cells) subsets, obtaining the proportion and phenotypic characteristics of different T cell subsets in thymic tissue.

[0048] pass Figure 4 As can be seen from the flow cytometry analysis, the total number of thymocytes and the differentiation status of T cell subsets (DN, DP, CD4SP, CD8SP) and DN subsets (DN1-DN4) are as follows: Changes in total thymocyte count: The total thymocyte count in the sham-operated group was 4.0 × 10⁻⁶. 6 ±0.5×10 6 No significant changes were observed in the group 1 day after ischemia. p=0.0534), the 3d group significantly decreased to 2.0×10. 6 ±0.3×10 6 ( p <0.01), the 7d group maintained a low level (2.5×10). 6 ±0.4×10 6 The result indicates a sustained decrease in the number of cells after thymic ischemia.

[0049] Changes in T cell subset differentiation: DN T cells (CD4) - CD8 - The proportion of sham surgery group was 10%±1%; the proportion of sham surgery group was significantly higher at 1 day and 3 days post-ischemia (reaching 25%±2% in the 3-day group). p <0.001), the percentage of DN cells decreased to 15%±1% in the 7d group, indicating that DN cell development was inhibited.

[0050] DP T cells (CD4) + CD8 + The proportion of sham surgery group was 80%±3%; the proportion decreased significantly at 1 day and 3 days after ischemia (reaching 50%±2% in the 3-day group). p <0.001), the 7d group recovered to 70%±2%, indicating that DP cell differentiation was initially suppressed and then partially recovered.

[0051] CD4+ SPT cells: The percentage was 5% ± 0.5% in the sham-operated group; it significantly increased to 6% ± 0.5% in the group 3 days after ischemia. p <0.01), the 7d group fell back to the baseline level.

[0052] CD8 SP T cells: The percentage was 10%±1% in the sham-operated group; it significantly increased to 15%±1% in the group 3 days after ischemia. p <0.001), the 7d group maintained a high value, suggesting that CD4 / CD8 SP differentiation was promoted first.

[0053] DN subpopulation differentiation arrest: In the sham-operated group, the proportions of DN1-DN4 were 10%±1%, 2%±0.5%, 5%±0.5%, and 80%±2%, respectively; 3 days after ischemia, DN1 (15%±1%)... p <0.05), DN2 (6%±0.5%, p <0.001), DN3 (40%±2%, p The proportion of <0.001) increased significantly, while the proportion of DN4 decreased significantly to 60%±2%. p <0.001), indicating that DP cell precursors are arrested in the DN1-DN3 phase.

[0054] The above results indicate that after thymic ischemia-induced injury, the total number of thymocytes decreases and the differentiation of T cell subsets is abnormal; at the same time, DN cell development is inhibited and arrested in the DN1-DN3 stage, further participating in the process of thymic function impairment.

[0055] 5. RNA-seq differential gene analysis and GO enrichment analysis methods: Adult male C57BL / 6 mice were sacrificed 7 days after thymic ischemia modeling. Thymic tissue was aseptically extracted, washed three times with pre-chilled 1×PBS buffer (pH 7.4) to remove residual blood, and immediately flash-frozen in liquid nitrogen. It was then transferred to a -80°C freezer for subsequent total RNA extraction. The frozen thymic tissue was removed from the -80°C freezer and quickly placed in a pre-chilled mortar. Liquid nitrogen was added and the tissue was thoroughly ground into powder. 1 mL of TRIzol reagent was added per 50-100 mg of tissue, and the mixture was repeatedly pipetted to ensure complete tissue lysis. The mixture was allowed to stand at room temperature for 5 min. 0.2 mL of chloroform was added per 1 mL of TRIzol reagent, and the mixture was vigorously shaken for 15 s and allowed to stand at room temperature for 3 min. The mixture was then centrifuged at 12000g for 15 min at 4°C. After centrifugation, the sample separated into three layers: an upper colorless aqueous phase (containing RNA), a middle white thin film layer (containing DNA), and a lower red organic phase (containing protein). Carefully aspirate the upper aqueous phase (approximately 400-500 μL) into a new RNase-free centrifuge tube. Add an equal volume of isopropanol, gently invert to mix, and incubate at room temperature for 10 min. Centrifuge at 12000 g for 10 min at 4°C, discard the supernatant, and a white RNA precipitate will be visible. Add 1 mL of 75% ethanol (prepared with DEPC water), gently invert to wash the precipitate, centrifuge at 7500 g for 5 min at 4°C, discard the supernatant, and repeat the washing once. Air-dry the precipitate at room temperature (approximately 5-10 min, avoiding excessive drying which would make the RNA difficult to dissolve). Add 30-50 μL of DEPC water, gently pipette to dissolve the RNA, and place on ice for later use. Use a Nanodrop 2000 to determine the concentration (A260 / A280 ratio should be between 1.8 and 2.1) and purity (A260 / A230 ratio should be ≥2.0) of the RNA sample. Take 1 μL of RNA sample and use an Agilent 2100 bioanalyzer with the RNA 6000 Nano kit to check RNA integrity. The RNA integrity number (RIN) should be ≥7.0, and the band brightness ratio of 28S rRNA to 18S rRNA should be approximately 2:1 to ensure RNA quality meets high-throughput sequencing requirements. Take 1 μg of quality-tested total RNA and use an mRNA capture kit to separate mRNA by specifically binding the poly(A) tail of mRNA to oligomeric (dT) magnetic beads. Wash the magnetic beads three times with DEPC water to remove residual rRNA, tRNA, etc. Add reverse transcription primers, reverse transcriptase, dNTPs, and other reagents to the separated mRNA (following the cDNA synthesis kit instructions). Perform reverse transcription on a PCR instrument under the following conditions: 25℃ for 5 min, 42℃ for 30 min, and 70℃ for 15 min to obtain the first strand of cDNA. Using the first strand of cDNA as a template, DNA polymerase, dNTPs, etc. are added to synthesize the second strand of cDNA. After the reaction is complete, the double-stranded cDNA is purified using DNA purification magnetic beads.Using a library construction kit, purified double-stranded cDNA underwent end repair, A-tailing, and sequencing adapter ligation. The library was then enriched by PCR amplification and finally purified using DNA purification magnetic beads to obtain the final cDNA library. After quantification using a Qubit real-time fluorescence instrument, paired-end sequencing was performed according to the Illumina sequencing platform (e.g., NovaSeq 6000) manual. The read length was set to 150 bp, and the sequencing data volume for each sample was controlled at 6-8 Gb to ensure accurate gene expression quantification. Fastp software was used to filter the raw sequencing data, removing reads containing adapter sequences, low-quality sequences (Q≤20 bases > 50%), and N content > 10%, resulting in high-quality clean reads. The HISAT2 software was used to align clean reads to the mouse reference genome (GRCm39) with default parameters, resulting in mapped reads. Alignment efficiency was calculated (typically ≥80%). FeatureCounts software was used to count the number of reads for each gene. Gene expression levels were then standardized using TPM (Transcripts Per Million) or FPKM (Fragments Per Kilobase of transcript per Million mapped reads) methods to obtain the expression level of each gene in different samples. The DESeq2 R package was used to perform differential expression analysis on the gene expression data of the Sham and TI groups. The screening threshold was set as follows: a corrected p-value (padj) < 0.05 and an absolute value of the fold change in expression level |log2FC| > 1, identifying differentially expressed genes (DEGs) between the two groups. The ggplot2 package in R was used to plot log2FC on the x-axis with -log. 10A volcano plot was drawn with (padj) on the vertical axis to show the distribution of differentially expressed genes. Red dots represent significantly upregulated genes, blue dots represent significantly downregulated genes, and gray dots represent genes with no significant difference. The top 50 upregulated and top 50 downregulated genes with the most significant expression differences were selected. Using the pheatmapR package, the expression levels of these genes in different samples were standardized, and a heatmap was drawn to show the clustering of expression patterns of differentially expressed genes, with colors transitioning from blue (low expression) to red (high expression). Gene Ontology (GO) enrichment analysis was performed on the selected differentially expressed genes using the clusterProfiler R package. GO categories include Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The Benjamini-Hochberg p-value correction method was used; GO entries with a corrected p-value (padj) < 0.05 were considered significantly enriched. Using R, bar charts (showing enriched entries and their enrichment levels) and bubble charts (showing enriched entries, enrichment levels, gene number, and p-values) were generated to reveal the potential functions of differentially expressed genes in biological processes, cellular components, and molecular functions. At least three independent biological replicates were performed for each group (i.e., RNA extraction, library construction, and sequencing were performed on three separate mice) to ensure the reliability of the experimental results.

[0056] The results are shown in Figure 5. RNA-seq differential gene expression and GO enrichment analysis revealed significant changes in the gene expression profile within the thymus of mice after thymic ischemia (TI) injury, and identified the core differentially expressed gene families and their potential biological functions. The specific results are as follows: Figure 5 A. Differential Gene Distribution (Volcano Plot): The volcano plot clearly shows the overall distribution characteristics of differentially expressed genes in the thymus of mice in the Sham (sham surgery) group and the thymic ischemia (TI) group. Using |Log2 (Fold Change)|>1 and -log10(p.adjust)>1 as screening criteria, several upregulated genes (red dots) and downregulated genes (blue dots) were identified; among them, the collagen family ( Col15a1 , Col5a1 , Col5a2 , Col4a1 , Col4a2 , Col3a1 , Col1a1 , Col1a2 ) and chemokines Ccl family( Ccl6 , Ccl7 , Ccl8 , Ccl9 , Ccl12 The core upregulated gene is )

[0057] Figure 5 B. Differential Gene Expression Heatmap: The collagen family gene heatmap shows that in the TI group... Col15a1 , Col5a1 The expression levels of isogenes were significantly higher in the Sham group than in the Sham group. Col15a1 The expression level in the TI group was 2500 times that in the Sham (sham surgery) group; chemokines Ccl Family gene heatmap shows: In the TI group Ccl8 , Ccl6 The expression levels of isogenes were significantly higher in the Sham group than in the Sham group. Ccl8 The expression level in the TI group was 800 times that in the Sham (sham surgery) group. Compared with the Sham (sham surgery) group, the TI group mice showed a significantly high expression trend of eight collagen family genes, including Col15a1 and Col5a1, in the brain. The expression difference between the groups was clear, suggesting that thymic ischemia injury can significantly activate the transcriptional expression of collagen family genes in the brain.

[0058] Figure 5 C. GO enrichment analysis: Molecular function (MF) level: Differential genes were significantly enriched in items such as "chemokine receptor binding" and "collagen binding" (-log10 (p.adjust)>10). Cellular components (CC) level: significantly enriched in items such as "extracellular matrix" and "basement membrane"; At the biological process (BP) level: significantly enriched in items such as "extracellular matrix structure composition" and "chemokine-mediated signaling pathways" (gene percentage > 0.2%).

[0059] The above results indicate that after thymic ischemic injury, the collagen family and chemokines... Ccl The family genes were significantly upregulated, and the differentially expressed genes were mainly involved in extracellular matrix construction and chemokine-related biological functions, suggesting that they play a regulatory role in the pathological process after thymic ischemia.

[0060] 6. Methods for detecting the effects of TI on cognitive function and brain tissue inflammation: Cognitive function was assessed in 6-8 month old male C57BL / 6 mice within 28 days after thymic ischemia modeling. A three-chamber social interaction test was performed on TI mice at 7, 14, and 28 days post-modeling to observe their social preferences and assess social cognition. The experimental setup consisted of a three-chamber social interaction box made of transparent acrylic glass, measuring 60cm × 40cm × 25cm. The box was divided into three equal chambers (left, middle, and right) by two transparent partitions, with a 5cm opening at the bottom of each partition to allow mice to move freely between the chambers. The inside of the box was wiped with 75% ethanol one day prior to the experiment to remove any residual odor. The TI mice were placed in the three-chamber social interaction box and allowed to move freely within the three chambers for 5 minutes. After the adaptation phase, a strange mouse of the same strain, age, and sex as the experimental mouse (stimulator mouse 1, with no prior contact history) was placed in the left cavity, and an odorless plastic toy similar in size to the mouse (control object) was placed in the right cavity. The middle cavity was empty. The experimental mouse was then placed into the middle cavity, the partition was opened, and the time spent in the left cavity (social cavity), the number of times it moved between the left and right cavities (object cavity), and the interaction time with stimulator mouse 1 (including nose-to-nose contact, following, grooming, etc.) were continuously recorded for 5 minutes. After the social ability test phase, the inside of the chamber was wiped with 75% ethanol to remove residual odor. Stimulator mouse 1 was placed in the left cavity, and a strange mouse of the same strain, age, and sex as the experimental mouse (stimulator mouse 2, with no prior contact history) was placed in the right cavity. The middle cavity was empty. The experimental mouse was then placed into the middle cavity, the partition was opened, and the time spent in the left and right cavities, the number of times it moved between the left and right cavities, and the interaction time with stimulator mouse 2 (including nose-to-nose contact, following, grooming, etc.) were continuously recorded for 5 minutes. After the experiment, the social competence index and social novelty index were calculated using the formulas: "Social competence index = Left cavity dwell time / Total time" and "Social novelty index = Right cavity dwell time / (Left cavity dwell time + Right cavity dwell time)". Olfactory tests were conducted on TI mice at 7, 14, and 28 days after modeling to assess their odor discrimination ability and evaluate long-term cognitive function. Seven days prior to the experiment, based on the number of mice (n), 2n N1 beads and n N2 beads were prepared. The N1 and N2 beads were placed in the rearing environments of different species of mice to allow them to become fully accustomed to the environmental odors. Twenty-four hours before the experiment, the mice to be tested for cognitive function were individually housed in transparent cages for easy observation and video recording. Five "familiar beads" were placed in each cage to familiarize the mice with the beads. One hour before the experiment, the "familiar beads" were removed from the cages and placed in a sealed bag to prevent odor loss.At the start of the experiment, every minute, three "familiar beads" and N1 type beads were placed in the middle area of ​​the experimental cage, giving the mice one minute to actively explore. This process was repeated three times, changing the position of the N1 type beads each time. The timing was taken from the first time the mouse's head approached a bead, and the one-minute exploration test was conducted. After the experiment, the used beads were discarded. One hour before the experiment, the "familiar beads" were removed from the cage again and sealed for storage. One hour later, the various types of beads were placed in a specific order, and the time the mice spent exploring (smelling, licking, biting) each bead was recorded. After the experiment, the recognition index was calculated using the formula: "Recognition Index = Time spent exploring N2 type beads / Total exploration time".

[0061] Male C57BL / 6 mice aged 6-8 months were euthanized 7 and 28 days after thymic ischemia modeling, and thymic tissue was collected. The thymic tissue was quickly fixed in 4% paraformaldehyde fixative at 4°C for 24-48 hours. The fixed thymic tissue could be used directly or dehydrated in a sucrose gradient to 30% sucrose solution, then embedded in OCT and prepared into 5-10 μm thick frozen sections. Immunofluorescence staining (including GFAP, MBP, and Iba-1 markers): Fixed brain tissue sections were washed three times with 1×PBS buffer (pH 7.4), and incubated at 37°C for 1 hour with blocking buffer containing 5% bovine serum albumin and 0.3% Triton X-100 to block non-specific binding sites. After discarding the blocking buffer, rabbit anti-mouse GFAP, MBP, and Iba-1 specific primary antibodies diluted 1:300 with the blocking buffer were added, and the sections were incubated overnight at 4°C in a humidified chamber. The next day, the sections were washed three times with 1×PBS, and fluorescently labeled secondary antibodies (Alexa Fluor 488-labeled anti-GFAP and anti-Iba-1 secondary antibodies, and Alexa Fluor 594-labeled anti-MBP secondary antibodies) diluted 1:500 were added. The sections were incubated at room temperature in the dark for 1 hour. After washing three times with 1×PBS in the dark, DAPI staining solution at a final concentration of 1 μg / mL was added, and the sections were stained at room temperature in the dark for 5 minutes. Finally, the sections were washed with 1×PBS. Rinse three times in the dark, let the slide dry slightly, add anti-fluorescence quenching mounting medium and cover with a coverslip; finally, observe and acquire images under a fluorescence microscope at the appropriate excitation wavelength (DAPI: 350nm, Alexa Fluor 488: 488nm, Alexa Fluor 594: 594nm).

[0062] The results showed that mice with thymic ischemia-induced injury exhibited significant cognitive impairment, accompanied by abnormal proliferation of glial cells and damage to myelin sheath structure in the brain. Specific results are as follows: Figure 6A. Cognitive Impairment: Cognitive function testing: In the olfactory test, the discrimination index of mice in the Sham (sham surgery) group was 1.0±0.1. The discrimination index decreased significantly at 7, 14 and 28 days after thymic ischemia injury (0.2±0.05, 0.1±0.03 and 0.3±0.04, respectively, all p<0.05); the three-chamber social test showed that the social ability index and social novelty index of mice were significantly reduced at 7, 14 and 28 days after surgery (the social novelty index decreased by about 45% compared with the sham surgery group at 28 days after surgery, p<0.05), suggesting that mice have persistent cognitive impairment after thymic ischemia injury.

[0063] Figure 6 B. Abnormal proliferation of glial cells: Changes in astrocytes: Immunofluorescence results of GFAP (astrocyte marker) showed that the mean fluorescence intensity of GFAP in the Sham group was 4.0±0.4; it increased to 4.5±0.5 (p<0.05) 7 days after thymic ischemia-injury and further increased to 5.0±0.5 (p<0.05) 28 days after thymic ischemia-injury, which was about 25% higher than that in the sham group, indicating that astrocytes continued to proliferate after thymic ischemia-injury.

[0064] Microglia activation and proliferation: Immunofluorescence staining (Iba-1 labeled microglia) results showed that the mean fluorescence intensity of Iba-1 in the brain of mice in the Sham (sham-operated) group was 3.5±0.4; 7 days after thymic ischemia-injury, the fluorescence intensity slightly increased to 4.0±0.5; 28 days after surgery, the mean fluorescence intensity of Iba-1 significantly increased to 6.0±0.6 (p<0.05), an increase of about 71% compared with the sham-operated group. This result indicates that microglia in the brain are significantly activated 28 days after thymic ischemia-injury.

[0065] Figure 6 CD. Thymic ischemia induces multi-regional, time-dependent myelin sheath damage in the central white matter. In the Sham (sham-operated) group, continuous and dense MBP positive signals were observed in both the striatum and corpus callosum, indicating intact white matter myelin sheath structure. In contrast, 7 days after thymic ischemia, the immunofluorescence signal of MBP in both of these white matter-rich regions was significantly weakened, and the staining continuity was impaired, suggesting significant white matter myelin sheath damage in the early stages. Quantitative analysis showed that the average fluorescence intensity of MBP in the striatum and corpus callosum regions was significantly lower in the 7-day post-thymic ischemia group compared to the sham-operated group. (P<0.05). Twenty-eight days after thymic ischemia, the MBP signal in the striatum and corpus callosum showed some recovery compared to the 7-day group, but remained lower overall than the sham-operated group. Quantitative results further indicated that the MBP fluorescence intensity in the 28-day group was significantly higher than that in the 7-day group (#P<0.05), but had not yet fully recovered to the control level, suggesting that thymic ischemia-induced white matter myelin damage is persistent and only shows a partial recovery trend. These results demonstrate that thymic ischemia-induced injury can induce significant and time-dependent myelin sheath damage in multiple key white matter regions of the central nervous system (including the striatum and corpus callosum), providing direct histological evidence for the association between thymic injury and central white matter lesions.

[0066] In summary, these results indicate that thymic ischemia-induced injury not only induces significant and persistent cognitive impairment in mice, but also triggers abnormal proliferation and activation of microglia and astrocytes in the central nervous system, accompanied by destruction of myelin sheath structures in the striatum and corpus callosum regions. This suggests that thymic injury may affect brain structural and functional homeostasis through the thymus-immune-neural axis.

[0067] In summary, this invention establishes for the first time an animal model of photocoagulation-induced thymic ischemia (TI). This model is rapid to prepare, simple to operate, and highly specific for thymic damage, while avoiding the systemic effects of systemic hormonal interference or surgical resection in traditional models. The model stably induces pathological thymic degeneration within 7 days and continuously triggers abnormal peripheral immune responses up to 28 days, while also causing cognitive impairment that persists for up to 28 days. This dynamic process realistically reproduces the thymus-immune-cognitive pathological changes associated with stroke and aging. This model not only provides an ideal tool for studying the mechanisms of thymic degeneration and immune aging but also opens up a new experimental platform for exploring the role of the brain-thymus-immune axis after stroke and screening for novel interventional drugs, demonstrating significant theoretical innovation and broad application prospects.

[0068] In summary, by comparing and analyzing the technical solution of this invention with the closest existing technology, its technical features and advantages are summarized as follows: 1) In terms of modeling speed and stability, this invention can stably induce thymic involution and immune aging phenotype within 1–7 days, which is significantly better than the ultra-long cycle of natural aging models and meets the needs of rapid mechanism research and screening. 2) In terms of specificity of action, this invention induces local vascular occlusion in the thymus only through photosensitizer-point light irradiation, without relying on systemic hormonal changes, thus fundamentally avoiding the systemic interference of glucocorticoid models; 3) In terms of pathological reversibility and physiological relevance, this invention avoids the irreversible damage of the thymectomy model and can more realistically simulate the pathological process of thymic degeneration. 4) In terms of mechanism clarity and application scalability, this invention takes "thymic ischemia" as the core pathogenic mechanism, forming a clear causal chain of "structural damage - T cell development arrest - decreased immune function", which is applicable to the systematic study of immune aging, brain-thymus-immune axis and related diseases. 5) In terms of reproducibility, controllability and industrial application value, the operating parameters of this invention are clear and highly reproducible, making it particularly suitable for the screening and efficacy evaluation of drugs, interventions and cell products.

[0069] Therefore, compared with the prior art, the present invention has made significant progress in several core technical indicators such as modeling speed, damage specificity, clarity of pathological mechanism and application breadth. It solves the technical problems of long modeling cycle, large interference of systemic effects and difficulty in using it for precise mechanism research and screening in the prior art, and has outstanding substantive features and significant progress.

[0070] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. An animal model of photocoagulation thymic ischemia, characterized in that: The animal model of thymic ischemia exhibited at least one or more of the following typical thymic degeneration and immunosenescence characteristics within 1–7 days after induction: (1) Thymus atrophy, with a significant decrease in thymus weight or thymus index; (2) Disorder or disappearance of the thymic cortex-medullary structure; (3) Abnormal expression distribution of Keratin 5 / Keratin 8; (4) Increased thymocyte apoptosis and enhanced lipid deposition; (5) CD4 + CD8 + The proportion of double-positive T cells decreased, while the proportion of T cells in the DN2 / DN3 stage increased, suggesting T cell developmental arrest. (6) Decreased initial T cell output and decreased immune function.

2. The photocoagulation thymic ischemia animal model according to claim 1, characterized in that: The thymic ischemia animal model was able to induce cognitive dysfunction and abnormal peripheral immune response for at least 28 days after induction, manifesting as stable pathological changes and functional defects. The animal model of thymic ischemia exhibited thymic degeneration and immunosenescence phenotypes within 1–7 days.

3. The method for constructing an animal model of photocoagulation thymus ischemia as described in claim 1 or 2, characterized in that: The method involves administering a photosensitizer to experimental animals and applying light of a specific wavelength to the surface projection area of ​​the thymus within a predetermined time window, thereby inducing thrombosis in local blood vessels of the thymus and causing focal ischemic injury to the thymus. The method achieves specific ischemic intervention of the thymus without significantly affecting systemic hormone levels and systemic physiological state.

4. The construction method according to claim 3, characterized in that: Includes the following steps: (1) A photosensitizer is administered to the experimental animal, wherein the photosensitizer is a compound that can induce intravascular thrombosis under irradiation with light of a specific wavelength; (2) After the photosensitizer is administered, the surface projection area of ​​the animal's thymus is irradiated at a fixed point within a predetermined time window to induce thrombosis in the local blood vessels of the thymus, thereby inducing focal ischemic injury of the thymus. (3) After exposure to light, animals were fed a recovery diet to obtain an animal model with thymic degeneration and immune aging-related phenotypes.

5. The construction method according to claim 3 or 4, characterized in that: The photosensitizer is selected from Rose Bengal, Erythrosin B, or their functional equivalents; Alternatively, the photosensitizer may be administered via intraperitoneal injection, intravenous injection, or oral administration. Alternatively, the photosensitizer dose is 10–100 μg / g body weight; Alternatively, the illumination wavelength is 500–580 nm, and the illumination time is 5–30 minutes; Alternatively, the radius of the irradiated area corresponding to the surface projection region of the thymus is 0.2–1.0 cm; Alternatively, the light exposure may begin 1–15 minutes after administration; Alternatively, the experimental animal may be a fully developed adult experimental animal.

6. The application of the photocoagulation thymic ischemia animal model as described in claim 1 or 2 in screening interventions to improve thymic degeneration or immune aging.

7. The application of the photocoagulation thymus ischemia animal model as described in claim 1 or 2 in the study of diseases related to the brain-thymus-immune axis.

8. The application of the photocoagulation thymic ischemia animal model as described in claim 1 or 2 in the study of the mechanism of thymic degeneration and immune aging.

9. The application of the photocoagulation thymic ischemia animal model as described in claim 1 or 2 in the study of immune remodeling and cognitive impairment after cerebrovascular diseases such as stroke and vascular dementia, and / or in the study of mechanisms related to aging, metabolic syndrome, susceptibility to infection and tumor immune escape, and / or in the study of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

10. The application of the photocoagulation thymic ischemia animal model as described in claim 1 or 2 in the screening and efficacy evaluation of drugs, interventions or cell products for delaying thymic degeneration or improving immune aging.

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