摘要
神经元损伤、神经变性和神经解剖学变化是癫痫、中风、脑外伤、帕金森病、自闭症和阿尔茨海默病等各种神经系统疾病的主要病理特征。准确量化不同脑区的神经元和中间神经元对于了解动物模型中神经退行性疾病的进展至关重要。传统的评分方法往往是肤浅的、有偏见的，而且在评估神经病理学方面也不可靠。立体学是一种利用细胞三维可视化的定量工具，它为评估神经元损伤和神经退行性变提供了一种可靠的方案。本文介绍了一种全面优化的立体学方案，用于对大鼠和小鼠模型中的神经元损伤、神经变性和神经发生进行无偏见的定量分析。该方案包括通过对脑切片进行免疫组化处理，精确计数损伤神经元、存活神经元和中间神经元，以检测NeuN（+）主神经元、parvalbumin（PV+）中间神经元、doublecortin（DCX+）新生神经元和Fluoro-Jade B（FJB+）染色的损伤细胞。使用 Visiopharm 立体学软件驱动的复合显微镜识别和分析预定义的海马和杏仁核区域。细胞密度和绝对细胞数采用光学分馏法测定。我们的立体学方案准确估计了大鼠海马中 150 万个 NeuN（+）主神经元和 05 万个 PV（+）中间神经元，以及小鼠海马中 120 万个主神经元和 0.25 万个中间神经元。FJB（+）计数提供了受损神经元的定量指标，DCX（+）神经元的立体结构显示了神经发生的程度。总之，这种立体学方案能对任何脑区的神经元总数进行精确、准确和无偏见的计数。这为研究各种急性脑损伤、神经毒性和慢性神经系统疾病模型中的神经元损伤和保护提供了可靠的定量工具。© 2024 Wiley Periodicals LLC.基本方案 1：大鼠大脑切片中海马区主要神经元、中间神经元和未成熟神经元的立体定量 基本方案 2：小鼠大脑切片中海马区主要神经元、中间神经元和未成熟神经元的立体定量 基本方案 3：大鼠海马区和杏仁核中用荧光玉 B 染色的损伤或坏死细胞的立体定量 基本方案 4：基本程序 5：大脑固定和组织学处理 基本程序 6：主要神经元、中间神经元和新生神经元的免疫化学基本程序 7：受伤神经元的荧光玉 B 染色。Neuronal injury, neurodegeneration, and neuroanatomical changes are key pathological features of various neurological disorders, including epilepsy, stroke, traumatic brain injury, Parkinson's disease, autism, and Alzheimer's disease. Accurate quantification of neurons and interneurons in different brain regions is critical for understanding the progression of neurodegenerative disorders in animal models. Traditional scoring methods are often superficial, biased, and unreliable for evaluating neuropathology. Stereology, a quantitative tool that uses 3-dimensional visualization of cells, provides a robust protocol for evaluating neuronal injury and neurodegeneration. This article presents a comprehensive and optimized stereology protocol for unbiased quantification of neuronal injury, neurodegeneration, and neurogenesis in rat and mouse models. This protocol involves precise counting of injured neurons, surviving neurons, and interneurons through immunohistochemical processing of brain sections for NeuN(+) principal neurons, parvalbumin (PV+) interneurons, doublecortin (DCX+) newborn neurons, and Fluoro-Jade B (FJB+)-stained injured cells. Predefined hippocampal and amygdala regions were identified and analyzed using a Visiopharm stereology software-driven compound microscope. Cell density and absolute cell numbers were determined using the optical fractionation method. Our stereology protocol accurately estimated 1.5 million total NeuN(+) principal neurons and 0.05 million PV(+) interneurons in the rat hippocampus, as well as 1.2 million total principal neurons and 0.025 million interneurons in the mouse hippocampus. FJB(+) counting provided a quantitative index of damaged neurons, and the stereology of DCX(+) neurons demonstrated the extent of neurogenesis. Overall, this stereology protocol enables precise, accurate, and unbiased counting of total neurons in any brain region. This offers a reliable quantitative tool for studying neuronal injury and protection in various models of acute brain injury, neurotoxicity, and chronic neurological disorders. © 2024 Wiley Periodicals LLC.
Basic Protocol 1: Stereological quantification of principal neurons, interneurons, and immature neurons in the hippocampus in rat brain sections
Basic Protocol 2: Stereological quantification of principal neurons, interneurons, and immature neurons in the hippocampus in mouse brain sections
Basic Protocol 3: Stereological quantification of injured or necrotized cells stained with Fluoro-Jade B in the hippocampus and amygdala in rats
Basic Protocol 4: Stereological quantification of injured or necrotized cells stained with Fluoro-Jade B in the hippocampus and amygdala regions in mice
Basic Protocol 5: Brain fixation and histology processing
Basic Protocol 6: Immunochemistry of principal neurons, interneurons, and newborn neurons
Basic Protocol 7: Fluoro-Jade B staining of injured neurons
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
