Primate model of central motor function impairment and method for producing same

Abstract

The present invention relates to a primate model of central motor function impairment. More specifically, the present invention provides: a primate model of central motor function impairment, in which the use of the hand is lost due to damage to a brain region associated with manual dexterity, from immediately after the damage through the acute and chronic periods, while functions of other regions are preserved; and a method for producing the model.

Description

Primate model of central motor dysfunction and method for producing the same

[0001] The present invention relates to a primate model of central motor dysfunction, and more particularly, to a primate model of central cortical motor dysfunction stroke without damage to other brain regions.

[0002] Stroke is a neurological condition caused by damage to a portion of the brain due to a blocked or ruptured blood vessel. It is classified as ischemic stroke or hemorrhagic stroke. The main symptom is sudden paralysis or weakness of a body part, especially on one side. Speech and speech disorders may occur, along with decreased vision in one or both eyes and double vision. Symptoms may also include sudden dizziness, difficulty walking, or sudden, unexplained, severe headaches. To understand the complex mechanisms of stroke and develop effective treatments, research using animal models is essential. Ischemic stroke models, including focal and global cerebral ischemia, are essential for studying stroke treatment using animal models. However, the existing middle cerebral artery ischemic model has a low survival rate of 30-40% in primates, and has limitations in that motor function decline occurs non-specifically. In addition, in the case of the existing embedded injury stroke model, white matter is mainly damaged in the process of inducing damage to the deep brain through a stereotactic functional method, and there are limitations in that it causes damage to undesired areas depending on the differences between individuals or does not induce direct damage to gray matter because the motor cortex of the cerebrum is preserved. In this regard, Korean Patent No. 2601270 describes a primate stroke model and a method for manufacturing the same.

[0003] However, in the case of the above prior art, there is a problem that not only motor dysfunction occurs by irradiating the internal capsule area of ​​the animal model with a laser to induce local cerebral ischemia, but also lesions occur in other brain organs.

[0004] The present invention aims to solve various problems including the above-mentioned problems, and provides a primate model with central motor dysfunction in which the motor function of the central cerebral cortex is impaired without damage to other brain regions by photocoagulation targeting the brain region that assists movement and motor function among the cerebral cortex related to dexterity, and a method for producing the same. However, these tasks are exemplary and the scope of the present invention is not limited thereby.

[0005] According to one aspect of the present invention, there is provided a skull surgery step of administering an anesthetic to a primate animal model to anesthetize it, and then using a medical drill to open the skull to expose a targeted cerebral cortex area to the outside;

[0006] A light irradiation step in which light is irradiated to the motor cortex area of ​​the cerebral cortex to induce thrombus formation through a photocoagulation reaction;

[0007] A photoembolization induction step in which a photosensitizer is administered into the vein of the primate animal model to induce cerebral vascular occlusion; and

[0008] A method for producing a non-human primate model of acute ischemic stroke is provided, comprising the step of suturing the skull and dura mater.

[0009] According to another aspect of the present invention, a non-human primate stroke model animal manufactured by the above manufacturing method is provided.

[0010] According to another aspect of the present invention, there is provided a step of administering a stroke therapeutic agent candidate to the non-human primate stroke model animal;

[0011] A step of performing hand function evaluation in the above model animal; and

[0012] A method for screening a stroke treatment candidate substance is provided, including a step of selecting a stroke treatment candidate substance that restores hand function based on the above hand function evaluation results.

[0013] As described above, the primate model of the present invention with central motor function impairment is damaged in a brain region related to dexterity while preserving the functions of other regions. The gray matter and subcortical white matter of the cerebral cortex are damaged similarly to actual stroke patients, and the hand cannot be used for an acute or chronic period immediately after the injury. Therefore, it can be utilized in various fields such as drug efficacy evaluation for brain diseases, disease mechanism research, and promotion of brain plasticity. Of course, the scope of the present invention is not limited by these effects.

[0014] Figure 1 is a schematic diagram schematically illustrating the main experimental procedures, including photoembolization induction, MRI acquisition, and animal sacrifice dates, for producing a primate model of central motor dysfunction of the present invention.

[0015] Figure 2 is a photograph showing cortical changes before and after photoembolization of the surgical site after craniotomy in a primate model of the present invention.

[0016] Figure 3 is a drawing showing the infarct area by 3D MRI reconstruction performed on day 1 postoperatively (POD1).

[0017] Figure 4 is an MRI T2-weighted image showing infarction progression over time after photoembolization (PT) alone.

[0018] Figure 5 is a reconstructed diffusion tensor imaging (DTI) image of the corticospinal tract (CST) using fiber tract counting. In the PT-only group, the corticospinal tract bundles are observed to be reduced and not recover.

[0019] Figure 6 is a graph showing the results of a quantitative analysis of the increase in the rate of stroke scar formation in the damaged area and the contralateral normal area of ​​the brain following ischemic cerebral infarction in a primate model of the present invention. A relative increase in stroke scar was observed.

[0020] Figure 7 is a schematic diagram illustrating the evaluation of dexterity function in a primate model of the present invention. Functional decline due to cerebral infarction was objectively assessed.

[0021] Figure 8 is a photograph showing the results of immunohistochemical analysis using brain tissue of a primate model of the present invention. Through immunohistochemical staining, the area of ​​brain tissue damage due to ischemia ipsilateral to the photocoagulation reaction irradiation site is clearly observed in the cerebral cortex area including hand function. In the 8x magnification photograph, brain tissue necrosis is clearly observed, and densely packed cells and scar formation at the necrotic border are observed.

[0022] Figure 9 is a schematic diagram schematically showing the brain tissue structure of a human (A) and a primate model (B) of the present invention.

[0023] Definition of terms:

[0024] The term "stroke" as used in this document refers to a sudden, localized neurological deficit caused by cerebrovascular disease, i.e., abnormal cerebral blood flow. Stroke is divided into cerebral infarction or ischemic cerebrovascular disease, caused by blockage of a cerebral blood vessel, and cerebral hemorrhage or hemorrhagic cerebrovascular disease, caused by rupture of a cerebral blood vessel.

[0025] The term "photothrombosis," as used in this document, refers to the deliberate formation of blood clots within the brain of animals using specific photochemicals and light to induce ischemic stroke in the cerebral cortex. This technique is actively being used to establish animal models of ischemic stroke and to establish in vivo analyses of stroke diagnosis and treatment techniques.

[0026] As used in this document, the term "motor cortex" refers to the area of ​​the cerebral cortex, part of the central nervous system (CNS), involved in the planning, control, and execution of locomotion and voluntary movement, primarily located within the frontal lobe, specifically concentrated in the area known as the precentral gyrus, just in front of the central sulcus.

[0027] The term "gray matter" used in this document refers to the densely packed nerve cells of the central nervous system. A cross-section of the cerebrum or spinal cord can distinguish between gray matter and white matter. Gray matter contains a large number of nerve cell bodies, while white matter contains a large number of myelinated nerve fibers.

[0028] Detailed description of the invention:

[0029] According to one aspect of the present invention, there is provided a skull surgery step of administering an anesthetic to a primate animal model to anesthetize it, and then using a medical drill to open the skull to expose a targeted cerebral cortex area to the outside;

[0030] A light irradiation step in which light is irradiated to the motor cortex area of ​​the cerebral cortex to induce thrombus formation through a photocoagulation reaction;

[0031] A photoembolization induction step in which a photosensitizer is administered into the vein of the primate animal model to induce cerebral vascular occlusion; and

[0032] A method for producing a non-human primate model of acute ischemic stroke is provided, comprising the step of suturing the skull and dura mater.

[0033] The light source used in the present invention may be a semiconductor laser, a laser diode, or a light emitting diode (LED), and specifically, may be a green light (blue light), and the laser may be a light source that emits a peak wavelength of 510 to 580 nm with a maximum light output of 50 to 150 mW. Preferably, in the above manufacturing method, the light irradiation step may irradiate a light source that emits a peak wavelength of 510 to 530 nm with a maximum light output of 50 to 70 mW for 15 to 25 minutes.

[0034] In the above manufacturing method, the motor cortex may include the primary motor cortex, the premotor cortex, the supplementary motor cortex, and the prefrontal association cortex of the frontal lobe, and may be an area including the primary and secondary motor areas of the motor cortex spinal cord and the precentral corridor, which is a motor supplementary area, and the primary and secondary motor center functions of the premotor cortex and the prefrontal association area, and may induce a decline in contralateral dexterity motor function and a decline in ipsilateral dexterity fine motor function through light irradiation.

[0035] When light is irradiated to the desired area of ​​the brain where infarction is to occur, blood coagulation occurs locally in the blood vessels of that specific area, forming a thrombus. Rose Bengal, a photosensitizer, can be administered intravenously to induce vascular occlusion. After administration, when blue light is irradiated, it generates reactive oxygen species, selectively damaging vascular endothelial cells and causing brain tissue damage. Animal models in which thrombi have formed develop a stroke due to cerebral infarction in the specific area of ​​the brain that has been selectively irradiated with a laser.

[0036] In the above manufacturing method, the photosensitizer may be selected from the group consisting of rose bengal, indocyanine green (ICG), chlorin e6, porphyrin, eosin Y, and methylene blue, and the non-human primate may be selected from the group consisting of cynomolgus monkeys, macaques, spider monkeys, owl monkeys, baboons, rhesus monkeys, gorillas, and chimpanzees.

[0037] According to another aspect of the present invention, a non-human primate stroke model animal manufactured by the above manufacturing method is provided.

[0038] In the above model animals, the functions of other parts are preserved, but the hand may be permanently unusable immediately after the injury, and the gray matter and subcortical white matter of the motor cortex area in the cerebral cortex may be damaged.

[0039] According to another aspect of the present invention, there is provided a step of administering a stroke therapeutic agent candidate to the non-human primate stroke model animal;

[0040] A step of performing hand function evaluation in the above model animal; and

[0041] A method for screening a stroke treatment candidate substance is provided, including a step of selecting a stroke treatment candidate substance that restores hand function based on the above hand function evaluation results.

[0042] In the above screening method, the hand function evaluation may be a standardized reach and retrieve test.

[0043] The anesthesia performed in the present invention can be performed according to a method commonly performed by those skilled in the art, and for example, a method of administering ketamine / xylazine, a method of administering ketamine / medetomidine, a method of administering ketamine / xylazine / acepromazine, a method of administering sudium pentibarbital alone, or a method of administering isofurane can be used.

[0044] Ischemic stroke is a syndrome accompanied by sudden loss of consciousness and motor paralysis caused by cerebral blood circulation disorder. The area where the stroke occurs is largely divided into cerebral infarction, which is an area of ​​irreversible damage, and penumbra, which is an area surrounding the infarction and has the possibility of recovery. In the present invention, unless otherwise specified, the stroke includes both cerebral infarction and damage to the penumbra.

[0045] Stroke is a leading cause of death in adults worldwide, occurring when brain cells die due to insufficient blood supply. Ischemic stroke can be classified as "complete ischemia" or "partial ischemia" depending on the degree of circulatory impairment. In complete ischemia, blood flow to a localized area of ​​the brain is completely blocked, resulting in the death of a portion of the brain, or "cerebral infarction." The infarcted area cannot regain its function, and therefore, the resulting disability can be permanent and severe. Once a stroke occurs, it leaves irreversible neurological damage and is incurable. Therefore, analyzing the causes and preventing it is paramount, and active research is underway to identify and address numerous risk factors.

[0046] The test drug that is the target of the screening method using the model animal of the present invention is not particularly limited, and any drug that has the purpose of evaluating the therapeutic effect on ischemic stroke can be used. Typically, it refers to a pharmaceutical or quasi-drug, but it also includes supplementary foods such as specific health foods (functional foods), specific nutritional supplements, and health foods. The drug that is the target of evaluation can be screened for any route of administration and is not particularly limited. Drugs administered parenterally or orally can also be screened. The dosage of the drug should be determined appropriately and optimally based on the properties of the drug, the type of the administration subject, age, weight, and other conditions, and is not particularly limited. The administration conditions, such as the timing and frequency of administration, can be set to be optimal appropriately based on the properties of the drug, the purpose of the test and evaluation, and are not particularly limited.

[0047] The screening method for test drugs using a non-human primate model animal of the present invention may utilize any method that achieves the desired purpose. Preferably, the initial imaging examination for acute ischemic cerebral infarction using magnetic resonance imaging (MRI) may be utilized, but diffusion-weighted imaging (DWI), perfusion-weighted imaging (PWI), MR angiography (MRA), and gradient-echo imaging (GE) may also be utilized.

[0048] The stroke of the present invention can be classified into cerebral infarction or ischemic brain disease caused by blockage of a cerebral blood vessel, and cerebral hemorrhage or hemorrhagic cerebrovascular disease caused by rupture of a cerebral blood vessel. The cerebral infarction is classified into transient ischemic attack, cerebral infarction in large vessel disease, cerebral infarction in cardiogenic embolism, small vessel disease, and lacunar infarction, and depending on the location, it can be selected from the group consisting of cerebral hemorrhage or intracerebral hemorrhage, intraventricular hemorrhage, subarachnoid hemorrhage, epidural hemorrhage, and subdural hemorrhage.

[0049] The term "ischemia," as used in this document, also known as hypoxemia, hypoglycemia, anemia, or ischemia, refers to a condition in which the blood supply to tissues within the body is restricted, resulting in a lack of oxygen and glucose necessary for metabolism. Ischemia involves not only a lack of oxygen and glucose, but also reduced nutrient utilization and inadequate removal of metabolites.

[0050] The non-human primate of the present invention may include any non-human primate capable of inducing ischemic stroke, preferably selected from the group consisting of cynomolgus monkeys, macaques, spider monkeys, owl monkeys, baboons, rhesus monkeys, gorillas and chimpanzees, and more preferably, but not limited to, cynomolgus monkeys.

[0051] The present invention relates to a method for assessing hand function and fine motor skills in primates, characterized by the use of a monkey chair and a reach panel device specifically designed for primates. The monkey chair is designed to restrict the primate's trunk movement while allowing free movement of the dominant arm and hand (Figure 7). The reach panel consists of a black panel with spaces for grasping specific foods requiring various finger and hand movements. Prior to surgery, the primates were trained to reach and retrieve a designated food reward (e.g., a raisin or a small piece of fruit) from a designated panel using only the affected hand. Following stroke-induced surgery, the primates were tested in ten test sessions, recording the number of attempts and success rates. Each trial began when the food was placed on the panel and ended when the primate successfully retrieved the food or when a maximum 60-second trial period elapsed. The success or failure of each attempt was recorded, and the time taken to retrieve the food was measured using a stopwatch. The number of successful food retrievals and the average time taken were used as primary outcome measures for the hand function assessment. All of the above sessions were videotaped to ensure accuracy of subsequent analysis and data collection. Performance was assessed by comparing recovery times and success rates before and after surgery. The monkey chair used to assess hand function in the primate model consists of a padded restraint and an ergonomic seat to ensure the primate's safety and comfort.

[0052] The primate model of the present invention induces cerebral infarction by irradiating light to the motor area of ​​the cerebral cortex, an anatomical structure unique to primates. This method is advantageous for experiments in which recovery occurs later because it does not damage organs inside the brain (e.g., internal capsule). Although there is a prior art on a method for producing a stroke model using a mouse using a method similar to the present invention, there was a problem in that it was unsuitable for producing a stroke model because it self-recovered after 1-2 weeks. Accordingly, the present inventors recognized this problem and developed the primate model of the present invention and a method for producing the same to solve the problem. Specifically, for the production of a primate animal model, the motor cortex is located at the central sulcus, which forms the border between the frontal lobe and the parietal lobe in the brain. The premotor cortex is a motor cortex region within the frontal lobe located immediately in front of the primary motor cortex, and has been studied primarily in primates including monkeys and humans (Fig. 9). However, there was a problem in the past that even if the motor cortex was damaged, the premotor cortex promoted its recovery, leading to self-recovery. However, the present inventors damaged the motor cortex region, including the premotor cortex, which is a motor and motor function auxiliary brain region among the cerebral cortex related to dexterity, with a photocoagulation reaction, thereby causing a stroke lesion. It was confirmed that the gray matter and subcortical white matter of the cerebral cortex were damaged similarly to those of actual stroke patients, resulting in permanent hand loss due to central cortical motor function decline without damage to other brain regions.Therefore, the central motor function impairment primate model of the present invention is advantageous for experiments on recovery after stroke induction because the organs inside the brain are not damaged, and can be utilized in experiments on evaluating the efficacy of drugs for brain diseases including stroke.

[0053] Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the examples disclosed below, but can be implemented in various different forms. The following examples are provided to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention.

[0054] Example 1: System Configuration

[0055] The primates used in the present invention were cynomolgus macaques, and ischemic stroke was induced using a photochemical embolization system. The system consists of a light irradiation assembly for inducing photochemical thrombosis within a specific region of the cynomolgus macaque brain, and a precision positioning device equipped with a three-dimensional manual stage and a motorized stage (MTS50 / M-Z8, Torlabs) for precise control of lateral and axial probe placement. The light source was a single-channel, fiber-coupled laser diode (LDFLS_520 / 060, Doric Lenses, Inc., Quebec, Canada) emitting a peak wavelength of 520 nm with a maximum optical output of 60 mW, and a Zoom Beam Expander (ZBE4A, Torlabs) used to activate the photoembolization agent, Rose Bengal, and induce vascular occlusion in the target area within the brain.

[0056] Example 2: Animal preparation

[0057] In this study, two male and two female crab-eating macaques (3.1–5.5 kg, 3–4 years old) were used. The monkeys were individually caged and provided with food, water, and various fruits ad libitum. Furthermore, the rearing environment was maintained at 24±2°C (24±2°C), 50±5% relative humidity, 100% fresh air at ≥12 ventilation rates per hour, and a 12:12-h light:dark cycle.

[0058] Example 3: Photoembolization

[0059] The primate animal model of the present invention was randomly assigned to a treatment regimen after photoembolization. As a control, saline solution was orally administered with various concentrations of a H2O2-removing scavenger (10 mg / kg * 1 day, 0.1 mg / kg * 1 day, 0.03 mg / kg * 3 days). The treatments were administered orally in a randomized manner to ensure unbiased administration and to evaluate the dose-dependent efficacy and safety profile of the H2O2-removing scavenger in improving the effects of induced cerebral ischemic injury. To evaluate the therapeutic effects of the above-mentioned treatment, each monkey received only one of the assigned treatments after photoembolization, and the experimental animals were cared for in accordance with the guidelines established by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). This study was performed according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the Daegu-Gyeongbuk Medical Innovation Foundation (Approval Number: IACUC No. KMEDI-23080101-00), and all procedures were performed in accordance with the guidelines. Before photoembolization, cynomolgus monkeys were anesthetized with intramuscular injection of ketamine (8 mg / kg, YUHAN, Seoul, Republic of Korea) and medetomidine (0.05 mg / kg, Provet Veterinary Products Ltd, Seoul, Republic of Korea). Subsequent procedures were performed under 1.5% isoflurane anesthesia and maintained at 1.2%. After anesthesia, craniotomy was performed to expose the target brain regions, including the motor cortex and premotor cortex anterior to the central sulcus (Fig. 2). For photothrombosis, the monkey was positioned with its head on the table at a craniotomy position at an elevation of approximately 15 degrees, and the light irradiation probe was aligned 10 mm lateral to bregma. To induce photoembolism, laser light exposure at 520 nm was performed simultaneously with rose bengal (Sigma-Aldrich, St. Louis, MO) light exposure for 20 minutes.St. Louis, MO, United States) was diluted to 20 mg / mL and intravenously injected at a rate of 80 mg / kg over 3 minutes. After photoembolization, the monkeys were administered antibiotics (enrofloxacin 5 mg / kg, meloxicam 0.2 mg / kg) and analgesics (tramadol 4 mg / kg) and received total parenteral nutrition (TPN) before being transferred to their housing facility with free access to food and water. MRI data were then acquired according to experimental procedures, and histological evaluation was performed after euthanasia (Fig. 1). All procedures, including the surgical approach, were performed under sterile conditions by experienced neurosurgeons and technicians to ensure the highest level of experimental fidelity and animal welfare, including postoperative care.

[0060] Example 4: Tissue processing and histological evaluation

[0061] For histological evaluation, primate models of the present invention were deeply anesthetized with a lethal dose of sodium pentobarbital. After washing away blood with saline, transcardial perfusion was performed to fix brain tissue with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). To ensure thorough preservation of tissue architecture, the brain was carefully extracted and post-fixed overnight at 4°C in the same fixative. The fixed brain was transferred to a 30% sucrose solution for cryoprotection until it sank to the bottom, indicating sufficient penetration. The brain was then sectioned coronally at 40-micrometer thickness using a cryostat. Sections were serially collected and stored in cryoprotectant at -20°C until further processing. Hematoxylin and eosin (H&E) staining was used to provide a general overview of tissue architecture, highlight cellular and subcellular details, and identify pathologic changes such as neuronal loss and gliosis.

[0062] Example 5: Statistical Analysis

[0063] Each subject's performance was evaluated by comparing pre- and postoperative retrieval times and success rates. A paired t-test was used to determine the significance of observed differences, and a p-value less than 0.05 was considered statistically significant. Data are presented as the mean ± standard deviation.

[0064] Example 6: Magnetic Resonance Imaging (MRI)

[0065] To confirm the location of the infarct, we performed MRI scans on the primate model induced by photoembolism. Before MRI, cynomolgus monkeys were anesthetized by intramuscular administration of ketamine (8 mg / kg, YUHAN, Seoul, Republic of Korea) and medetomidine (0.05 mg / kg, Provet Veterinary products Ltd, Seoul, Republic of Korea). MRI data at the Neuroscience Research Institute of Daegu-Gyeongbuk Medical Innovation Foundation were acquired with a Siemens Skyra 3T scanner (Erlangen, Germany) equipped with a gradient coil insert (XQ gradient, maximum gradient strength 45 mT / m, maximum gradient strength 200 mT / m / m). A 15-channel TX / RX coil was used, and MR images were acquired in the following sequence: T1-weighted images (TR / TE=2010 / 4 ms, FOV=160x160 mm) 2 , matrix size=320x320, slice thickness=1 mm, 88 slices, flip angle=9°, NEX=2), T2-weighted image (TR / TE=4000 / 564 ms, FOV=160x160 mm 2 , matrix size=320x320, slice thickness=1 mm, 144 slices, flip angle=120°, NEX=2), DWI(TR=19530 ms, TE1 / TE2=72 / 119 ms, FOV=224x224 mm 2 , matrix size = 224x224, slice thickness = 1 mm, 88 slices, flip angle = 180°) is a RESOLE (readout segmentation of long-edge echo train) 4-scan sequence, b value = 1000 s / mm2 , EPI (Echo Planar Imaging) based DTI image (TR / TE = 9000 / 101 ms, FOV = 175x175 mm 2 , matrix size=118x118, slice thickness=1.5 mm, 56 slices, flip angle=90°, 64 directions, b value=1000 s / mm 2 ) and fMRI (functional MRI) images (TR / TE= 1500 / 28 ms, FOV= 192x192 mm 2, matrix size = 64x64, slice thickness = 3 mm, flip angle = 64, total 236 scans). In addition, the affected brain areas were delineated on MR images using a semi-automatic segmentation technique to quantify the extent and severity of damage caused by intracerebral photothrombosis. The 3D Slicer software (version 5.6.1, ref1) platform was used to calculate the areas with MRI T2 signal intensity of brain damage, including the edema area, and to evaluate the characteristics of the lesion. The acquired T2-weighted MRI data were carefully inspected for satisfactory image quality to quantify the changes of ischemic stroke from preoperative to postoperative day 1, 7, and 14. DSI Studio (Chen 2024 January version, ref. 2), a multipurpose software tool tailored for diffusion MRI analysis and tractography, was utilized for detailed examination and reconstruction of the corticospinal tract (CST). Using this platform, the inventors performed a comprehensive assessment of the corticospinal tract, which is central to motor function. Preprocessed Diffusion Tensor Imaging (DTI) data were imported into DSI Studio, and precise anatomical regions of interest (ROIs) within the internal capsule, motor cortex, and premotor cortex were established to perform tractography. These ROIs were strategically selected based on their importance to the corticospinal tract and their susceptibility to photoembolic damage. DSI Studio's tractography feature enabled the application of sophisticated algorithms that delineate white matter tracts along the directional diffusion of water molecules. This approach facilitated the visualization of the trajectories and connectivity of the corticospinal tract. To ensure the accuracy and reliability of the tractography results, several parameters, including seed point density, step size, and angular threshold, were adjusted.The analysis yielded both qualitative visualizations, such as fiber counts and fiber length distributions, and quantitative data. Longitudinal analyses were performed to compare tractography results at various time points, preoperatively and at 7 and 14 days postoperatively. As a result, it was confirmed that ischemic cerebral infarction was induced in the motor area of ​​the cerebral cortex of the primate model of the present invention (Figs. 3 to 5). In the experimental group of non-human primate animal models induced only by photoembolization, scar formation, neuronal death and atrophy, and motor function decline were induced, and astrocyte atrophy, border zone hyperactivity, and hypertrophic reactive astrocytes were observed (Fig. 6).

[0066] Example 7: Hand function evaluation in a primate model

[0067] To assess the fine motor skills and functional recovery of hand movements in a primate model of the present invention after stroke induction, a standardized reach and retrieval test was performed using a custom-designed monkey chair and reach panel (board) device. The monkey chair allowed free movement of the arms and hands while restricting body movement and was designed to comfortably secure the primate model while allowing unrestricted access to the reach box. Padded safety harnesses and an ergonomic seat ensured safety and comfort. The use of the monkey chair was crucial for standardizing positioning and eliminating extraneous body movements that could influence hand function assessment. The reach board was equipped with black panels containing graspable food items spaced apart to require distinct finger and hand movements for task completion. The non-human primate model of the present invention was trained before surgery to reach and retrieve a food reward (e.g., a raisin, a small piece of fruit, or a grape) from a designated well using only the affected hand (Figure 7). Postoperatively, the primate model was tested over 10 testing sessions, and the time taken to retrieve the food and the success rate were meticulously recorded. Each trial began with the placement of food in the well, and ended when the monkey successfully retrieved the food or the maximum trial time of 60 seconds. Success or failure of each trial was recorded, and the task completion time was measured using a stopwatch. The number of successful retrievals out of 10 trials and the average time taken to retrieve the food were used as the primary outcome measures to assess hand function. Short breaks were interspersed between trials to prevent fatigue. To ensure the accuracy of data collection, all sessions were videotaped for subsequent analyses. As a result, it was observed that paralysis persisted for more than two weeks in the ischemic infarction-induced group.

[0068] Example 8: Histological analysis of the cerebral cortex

[0069] To investigate the effects of neurological damage, such as stroke or neurodegenerative disease, on the cellular integrity of the cerebral cortex, the inventors performed histological analysis using brain tissue from a primate model of the present invention. Various degrees of cell density and morphology were also assessed to assess the severity of neuronal damage. As a result, as shown in Figure 8, the staining highlighted the cellular structure of the brain, revealing various layers and structures within the cerebral cortex and subcortical regions. The ipsilateral coronal section revealed the area of ​​damage caused by cerebral infarction, and the stained, dense nuclear structure at the border indicated the presence of nucleic acids within the cell bodies and the formation of a scar at the border of the brain damage.

[0070] While the present invention has been described with reference to the above-described embodiments, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible. Therefore, the true scope of technical protection of the present invention should be determined by the technical spirit of the appended claims.

[0071] (National Research and Development Project that supported this invention)

[0072] This study, which investigated the optimization of vagus nerve stimulation to promote neuroplasticity, was supported by the Sejong Science Fellowship from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT) (Project ID: 20240052). Furthermore, this study, which investigated the cognitive function of glial cells, was supported by the Institute for Basic Science Research Operational Expenses (IBS) funded by the Ministry of Science and ICT (MSIT) (Project ID: 1711196192).

Claims

1. A skull surgery step in which anesthesia is administered to a primate animal model, and then the skull is opened using a medical drill to expose the targeted cerebral cortex area to the outside; A light irradiation step in which light is irradiated to the motor cortex area of ​​the cerebral cortex to induce thrombus formation through a photocoagulation reaction; A photoembolization induction step in which a photosensitizer is administered into the vein of the primate animal model to induce cerebral vascular occlusion; and A method for producing a non-human primate model of acute ischemic stroke, comprising the step of suturing the skull and dura mater.

2. In paragraph 1, A manufacturing method in which the above light irradiation step is performed by irradiating a light source emitting a peak wavelength of 510 to 530 nm with a maximum light output of 50 to 70 mW for 15 to 25 minutes.

3. In paragraph 1, A manufacturing method, wherein the motor cortex includes a primary motor cortex, a premotor cortex, a supplementary motor cortex, and a prefrontal association cortex.

4. In paragraph 1, A manufacturing method, wherein the photosensitizer is selected from the group consisting of rose bengal, indocyanine green (ICG), chlorin e6, porphyrin, eosin Y, and methylene blue.

5. In paragraph 1, A method of manufacturing a non-human primate, wherein the non-human primate is selected from the group consisting of crab-eating monkeys, macaques, spider monkeys, owl monkeys, baboons, rhesus monkeys, gorillas and chimpanzees.

6. A non-human primate stroke model animal manufactured using any one of the manufacturing methods of clauses 1 to 5.

7. In paragraph 6, A nonhuman primate model of stroke that permanently loses use of the hand immediately after injury, while preserving the function of other parts of the body.

8. In paragraph 6, A nonhuman primate model of stroke with damage to the gray matter and subcortical white matter of the motor cortex in the cerebral cortex.

9. A step of administering a stroke treatment candidate to a non-human primate stroke model animal of Article 6; A step of performing hand function evaluation in the above model animal; and A method for screening a stroke treatment candidate, comprising a step of selecting a stroke treatment candidate that restores hand function based on the above hand function evaluation results.

10. In paragraph 9, The above hand function assessment is a screening method that is a standardized reach and retrieve test.

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