光化学诱导食蟹猴缺血性脑卒中模型
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基于光敏物质（玫瑰红 Rose Bengal），在激光照射下，释放自由基，损伤血管内皮，促使血小板聚集，激活凝血过程，形成血栓。本模型利用MRI定位脑中动脉血管，插入光纤，静脉注射玫瑰红，激光照射诱导血栓形成，显示典型缺血再灌注特征。 数据质量：a.实验动物和福利伦理 动物福利伦理：动物模型制备实验方案受广东省生物技术研究院（广东省实验动物监测中心）动物伦理委员会（IACUC）许可，实验过程受IACUC成员、设施兽医人员监督管理，伦理编号IACUC2024146。动物模型制备遵循3R原则，动物饲养符合动物福利伦理要求。 动物护理和饲养：动物护理和饲养依照广东省生物技术研究院（广东省实验动物监测中心） b.影像数据采集使用联影 uMR790 磁共振成像仪器，该设备质量符合国家相关标准，所有设备操作者均接受上岗培训，以确保获得合格的高清晰度图像。 c.模型评价 利用MRI TOF序列定位大脑中动脉血管，插入光纤，注射玫瑰红，激光照射形成血栓，3-5 h后闭塞血管再次恢复血流，模型具有缺血再灌注特征。 在建模前后，通过MRI（TOF、DWI、T1/T2、DTI）、运动行为（神经损伤评分、山和谷阶梯评价）和病理，对缺血性脑卒中模型进行评价。 数据来源：a.实验动物信息：食蟹猴、普通级、性别年龄不限、体重不限。动物来源于广东蓝岛生物技术有限公司（许可证号SCXK（粤）2024-0074），中科灵瑞（湛江）生物技术有限公司（许可证号SCXK（粤）2019-0046）、从化市华珍动物养殖场（普通合伙）（许可证号SCXK（粤）2020-0028）。动物试验在广东省生物技术研究院（广东省实验动物监测中心）开展（许可证号SYXK（粤）2021-0122）。 b.模型制作方法 利用MRI TOF序列定位大脑中动脉血管，插入光纤，注射玫瑰红，激光照射形成血栓，3-5 h后闭塞血管再次恢复血流，模型具有缺血再灌注特征。 c.影像学数据 扫描仪类型：联影 uMR790 线圈：12通道猴脑线圈。 麻醉方式：肌肉注射盐酸氯胺酮（20 mg/kg）、盐酸赛拉嗪（2 mg/kg）和硫酸阿托品（1 mg/kg）进行诱导麻醉，气管插管后连接呼吸麻醉机，使用异氟烷维持麻醉。 扫描体位：俯卧头先进 d.病理数据 TH 染色：猕猴双侧黑质的冠状切片用 3% H2O2（5 min）、3% triton X-100（4 min，中国北京索莱宝）和山羊血清（15 min，中国福州迈新）处理，然后兔抗 TH 抗体（1:1000，AB152，Millipore）4 °C 过夜。次日，切片与二抗（PV-9000，30 min，中国北京中杉金桥）37 °C 孵育。之后 DAB 显色 2 min（DAB-1031 Kit，20×，中国福州迈新），苏木精复染细胞核。 染色后，所有切片乙醇梯度脱水并用二甲苯透明，中性树脂封片，生物显微镜（Leica DM3000 LED）采集照片。 e.行为学数据 参照中国实验动物学会团体标准“实验动物 实验猴神经行为评价规范”利用神经损害评分表和山和谷阶梯任务装置进行运动感知行为评价，山和谷阶梯任务评价：测试前动物禁食12小时以内。山和谷装置的阶梯均为5层，从低到高每层阶梯对应的分数为1、2、3、4、5分，实验时，单次测试总分为15分。训练阶段，如果动物在3分钟内均能够顺利地从阶梯的每层中取食，则视为训练成功。评价阶段，每天山或谷阶梯任务的左右侧分别进行3次测试，并将一侧3次得分相加后得到测试分数，总分为45分，连续测定5 天，最终根据5天测定的平均分数评价动物单侧上肢的运动功能。 数据生产方式：a.影像学数据评价 TOF 通过3.0 T MRI系统从动物中获取头颅MRI数据，使用T1/T2加权涡轮自旋回波序列获得结构图像，通过TOF3D成像，可见术前M1/M2段血管畅通、术中M1/M2段血管闭塞后端血流中断、术后3-5 h闭塞血管再次恢复血流。 DWI 通过DWI扫描评价梗死灶体积，梗死区在DWI上呈现显著高信号，通过在ADC图上勾画显示低信号的病变区域，由软件自动计算其三维体积。血管闭塞后3h能清楚地观察到梗死灶，闭塞M1前段脑梗死体积偏大，闭塞M1后段、M2段脑梗死体积偏小。 T1/T2 利用T1WI和T2WI序列，获得脑梗死区域结构成像图。在超急性期和急性期，由于梗死区域有水肿，T1WI和T2WI敏感性低于DWI，在亚急性期后，核心梗死区在T1WI上呈低信号，在T2WI上呈明显高信号，边界变得清晰。对模型不同时间段T1WI和T2WI变化分析，术后2w梗死体积区域稳定。闭塞M2段，梗死位置主要集中在额叶、岛叶和颞叶交汇区；闭塞M1后段，梗死位置主要集中在额叶（眶前）、岛叶和颞叶交汇区，更靠近眶前额叶区；闭塞M1前段，梗死位置主要集中在深部核团和颞叶区， DTI 通过对模型的弥散张量成像（DTI）分析缺血性卒中特征，主要包括FA（各向异性分数）、MD（平均弥散率）、AD（轴向弥散系数）、RD（径向弥散系数）4个核心参数。 （1）急性期 FA值变化不定，MD显著下降，AD值降低，RD值也降低。 （2）亚急性期FA显著下降，MD由低转高，AD值从急性期的低值回升，并逐渐接近或超过正常值，RD值显著升高。 （3）慢性期FA维持低值，MD持续升高，AD值升高，RD值显著升高。 b.行为学数据评价 神经损伤评分 依据神经损害评分表，对模型进行神经损伤评分。由于模型梗死体积小，神经损伤评分变化主要在术后1w内，闭塞M1后段和M2段，2w后运动行为未出现明显异常。闭塞M1前段，动物早期出现偏瘫症状，术后2m后偏瘫症状也逐渐消失。 山和谷阶梯评价 通过“山和谷”阶梯试验评价运动和感知损伤，损伤对侧的山阶梯试验显示运动损伤，损伤侧的谷阶梯试验显示感知损伤。除了M1前段闭塞猴（M1908021）显示术后1.5m运动损伤，术后3m运动损伤有恢复趋势，感知功能在术后3m有损伤趋势，其他闭塞方式未见明显的运动和感知损伤。 c.病理评价 通过大体解剖和显微镜检，从急性期、亚急性期和慢性期可见病变组织从肿胀、软化、液化进一步发展。模型急性期病变已明显，梗死灶组织与正常组织边界清晰，可见病灶中心区神经元变性/坏死，细胞核固缩或溶解，细胞质嗜酸性增强、肿胀及空泡形成。亚急性期梗死灶组织边缘区新生毛细血管增生伴管腔淤血，血管周围间隙水肿明显，间质疏松水肿。慢性期梗死区边缘可见大量新生的毛细血管增生，血管周围及组织间隙显著增宽，大量巨噬细胞聚集，巨噬细胞胞浆内及组织间可见大量含铁血黄素沉积；可见胶质细胞增生，组织间质疏松水肿，形成胶质瘢痕。 时间范围：2023年11月20日-2025年9月1日
Based on the photosensitizer Rose Bengal, which releases free radicals under laser irradiation to damage vascular endothelium, promote platelet aggregation, activate the coagulation cascade, and ultimately form thrombi. This model uses MRI to locate the middle cerebral artery, inserts an optical fiber, intravenously injects Rose Bengal, and induces thrombus formation via laser irradiation, exhibiting typical ischemia-reperfusion characteristics. Data Quality: a. Experimental Animals and Welfare Ethics Animal Welfare Ethics: The experimental protocol for animal model preparation was approved by the Animal Ethics Committee (IACUC) of Guangdong Biotechnology Research Institute (Guangdong Laboratory Animal Monitoring Center), and the experimental process was supervised by IACUC members and facility veterinarians, with the ethics approval number IACUC2024146. The animal model preparation follows the 3R principles, and animal husbandry complies with animal welfare and ethical requirements. Animal Care and Husbandry: Animal care and husbandry are carried out in accordance with the regulations of Guangdong Biotechnology Research Institute (Guangdong Laboratory Animal Monitoring Center) b. Imaging Data Acquisition The United Imaging uMR790 magnetic resonance imaging system was used for data collection. This device meets relevant national quality standards, and all equipment operators have completed mandatory on-the-job training to ensure the acquisition of qualified high-definition images. c. Model Evaluation The middle cerebral artery was located using the MRI TOF sequence, an optical fiber was inserted, Rose Bengal was injected, and thrombus was formed via laser irradiation. Reperfusion of the occluded vessel was observed 3-5 hours later, confirming the model exhibits ischemia-reperfusion characteristics. Before and after model establishment, the ischemic stroke model was evaluated via MRI (TOF, DWI, T1/T2, DTI), motor behavior (neural injury score, hill-valley staircase test), and pathological examination. Data Sources: a. Experimental Animal Information The animals used were cynomolgus monkeys, of general grade, with no restrictions on gender, age, or body weight. The animals were sourced from Guangdong Landao Biotechnology Co., Ltd. (license number SCXK (Yue) 2024-0074), Zhongke Lingrui (Zhanjiang) Biotechnology Co., Ltd. (license number SCXK (Yue) 2019-0046), and Conghua Huazhen Animal Farm (General Partnership) (license number SCXK (Yue) 2020-0028). Animal experiments were conducted at Guangdong Biotechnology Research Institute (Guangdong Laboratory Animal Monitoring Center) (license number SYXK (Yue) 2021-0122). b. Model Preparation Method The middle cerebral artery was located using the MRI TOF sequence, an optical fiber was inserted, Rose Bengal was injected, and thrombus was formed via laser irradiation. Reperfusion of the occluded vessel was observed 3-5 hours later, confirming the model exhibits ischemia-reperfusion characteristics. c. Imaging Data Scanner Type: United Imaging uMR790 Coil: 12-channel monkey brain coil. Anesthesia Protocol: Anesthesia was induced via intramuscular injection of ketamine hydrochloride (20 mg/kg), xylazine hydrochloride (2 mg/kg), and atropine sulfate (1 mg/kg). After tracheal intubation, the animal was connected to a respiratory anesthesia machine, and anesthesia was maintained with isoflurane. Scanning Position: Prone, head first. d. Pathological Data TH Staining: Coronal sections of the bilateral substantia nigra of cynomolgus monkeys were treated with 3% H2O2 (5 min), 3% Triton X-100 (4 min, Solarbio, Beijing, China), and goat serum (15 min, Maxin Biotech, Fuzhou, China), then incubated with rabbit anti-TH antibody (1:1000, AB152, Millipore) overnight at 4 °C. The next day, sections were incubated with secondary antibody (PV-9000, 30 min, ZSGB-BIO, Beijing, China) at 37 °C. Subsequently, DAB chromogenic reaction was performed for 2 min (DAB-1031 Kit, 20×, Maxin Biotech, Fuzhou, China), and nuclei were counterstained with hematoxylin. After staining, all sections were dehydrated in graded ethanol, cleared with xylene, mounted with neutral balsam, and photographed using a biological microscope (Leica DM3000 LED). e. Behavioral Data Referring to the Chinese Association for Laboratory Animals group standard "Standard for Experimental Animal Neurobehavioral Evaluation of Experimental Monkeys", motor and perceptual behavior was evaluated using the neural injury score scale and the hill-valley staircase test device. Hill-valley staircase test evaluation: Animals were fasted for no more than 12 hours before testing. The hill-valley apparatus has 5 levels of stairs, with corresponding scores of 1, 2, 3, 4, and 5 points from low to high, with a total single test score of 15 points. During the training phase, the animal is considered successfully trained if it can successfully retrieve food from each stair level within 3 minutes. During the evaluation phase, each side (left and right) of the hill or valley staircase test was performed 3 times per day, and the scores of 3 tests on one side were summed to obtain the test score, with a total score of 45 points. Tests were conducted continuously for 5 days, and the motor function of the unilateral upper limb was evaluated based on the average score of the 5-day measurements. Data Production Methods: a. Imaging Data Evaluation TOF Cranial MRI data were acquired from animals using a 3.0 T MRI system. Structural images were obtained using T1/T2-weighted turbo spin-echo sequences, and 3D TOF imaging was performed. Preoperative patency of the M1/M2 segments of the middle cerebral artery, intraoperative interruption of blood flow distal to the occluded M1/M2 segments, and reperfusion of the occluded vessel 3-5 hours after surgery were observed. DWI Infarct volume was evaluated via DWI scanning. Infarcted areas showed significant hyperintensity on DWI, and hypointense lesion regions were delineated on ADC maps, with their 3D volume automatically calculated by software. Infarcts could be clearly observed 3 hours after vessel occlusion; infarct volume was larger when the proximal M1 segment was occluded, and smaller when the distal M1 segment or M2 segment was occluded. T1/T2 Brain infarct region structural images were obtained using T1WI and T2WI sequences. During the hyperacute and acute phases, due to edema in the infarcted region, the sensitivity of T1WI and T2WI is lower than that of DWI. After the subacute phase, the core infarcted region shows hypointensity on T1WI and significant hyperintensity on T2WI, with clear boundaries. Analysis of T1WI and T2WI changes at different time points after surgery showed that the infarct volume region stabilized at 2 weeks post-surgery. When the M2 segment was occluded, infarcts were mainly located in the junctional region of the frontal lobe, insula, and temporal lobe; when the distal M1 segment was occluded, infarcts were mainly located in the junctional region of the (orbital) frontal lobe, insula, and temporal lobe, closer to the prefrontal orbital region; when the proximal M1 segment was occluded, infarcts were mainly located in the deep nuclei and temporal lobe regions. DTI Diffusion tensor imaging (DTI) analysis was performed on the model to characterize ischemic stroke, including four core parameters: FA (fractional anisotropy), MD (mean diffusivity), AD (axial diffusivity), and RD (radial diffusivity). (1) Acute phase: FA values fluctuate, MD decreases significantly, AD and RD values decrease. (2) Subacute phase: FA decreases significantly, MD increases from low to high, AD rebounds from the acute low values and gradually approaches or exceeds normal values, RD increases significantly. (3) Chronic phase: FA remains low, MD continues to increase, AD increases, and RD increases significantly. b. Behavioral Data Evaluation Neural Injury Score The model was evaluated using the neural injury score scale. Due to the small infarct volume of the model, changes in neural injury score mainly occurred within 1 week post-surgery. For animals with occlusion of the distal M1 segment or M2 segment, no obvious abnormalities in motor behavior were observed 2 weeks after surgery. For animals with occlusion of the proximal M1 segment, hemiplegia symptoms appeared in the early stage, and the hemiplegia symptoms gradually disappeared 2 months after surgery. Hill-valley Staircase Evaluation Motor and perceptual impairments were evaluated via the "hill-valley" staircase test. The hill staircase test on the contralateral side of the injury showed motor impairment, while the valley staircase test on the injured side showed perceptual impairment. Except for the monkey with proximal M1 segment occlusion (M1908021) which showed motor impairment 1.5 months post-surgery, with a trend of recovery in motor impairment at 3 months post-surgery and a trend of perceptual impairment at 3 months post-surgery, no obvious motor or perceptual impairments were observed in other occlusion methods. c. Pathological Evaluation Gross anatomy and microscopic examination showed that the lesion tissue progressed from swelling, softening, and liquefaction across the acute, subacute, and chronic phases. Obvious lesions were observed in the acute phase of the model, with clear boundaries between infarct tissue and normal tissue. Neuronal degeneration/necrosis was observed in the lesion center, with nuclear pyknosis or lysis, enhanced eosinophilia of the cytoplasm, swelling, and vacuolization. In the subacute phase, neovascularization was observed in the marginal zone of the infarct, accompanied by vascular congestion, obvious perivascular space edema, and interstitial edema. In the chronic phase, extensive neovascularization was observed at the edge of the infarcted area, the perivascular and interstitial spaces were significantly widened, a large number of macrophages aggregated, and a large amount of hemosiderin deposition was observed in the cytoplasm of macrophages and between tissues; gliosis was observed, with interstitial edema and the formation of glial scars. Time Range: November 20, 2023 - September 1, 2025
