Ginsberg M D, Zhao W, Singer J T, Alonso O F, Loor-Estades Y, Dietrich W D, Globus M Y, Busto R
Department of Neurology (D4-5), University of Miami School of Medicine, FL 33101, USA.
J Neurosci Methods. 1996 Oct;68(2):225-33. doi: 10.1016/0165-0270(96)00084-2.
We report the application of a computer-based image-averaging strategy to the quantitative topographic analysis of in situ hybridization autoradiographs, based upon a disparity-analysis algorithm. We illustrate this approach for a representative antisense riboprobe-the astrocytic marker, glial fibrillary acid protein (GFAP)-in the setting of fluid-percussion brain injury in rats. Sequential coronal autoradiographs in individual animals are first digitized and aligned by disparity analysis. Next, coronal sections of all brains of a given experimental group are placed in register with one another, using a common anatomic reference level. One brain of the series serves as a template, and corresponding sections of other brains are mapped into its contour at each level. In this manner, average and standard deviation image data sets may be generated. With thresholding techniques, individual data sets can be dichotomized with respect to a chosen threshold, and frequency maps can be generated at each coronal level, displaying numbers of brains showing supra-threshold levels of mRNA at each pixel location. Pixel-by-pixel statistical comparison of data sets obtained under two different conditions (e.g., 30 min vs. 24 h following brain trauma) is then feasible. A digitized functional-anatomic brain atlas may be fitted to the images to assist analysis. Computer-based image analysis of in situ hybridization autoradiographs greatly extends the utility and applicability of this technique.
我们报告了一种基于计算机的图像平均策略在原位杂交放射自显影片定量地形分析中的应用，该策略基于差异分析算法。我们以大鼠液压脑损伤模型中一种代表性的反义核糖探针——星形胶质细胞标记物胶质纤维酸性蛋白（GFAP）为例来说明这种方法。首先，通过差异分析对单个动物的连续冠状放射自显影片进行数字化和对齐。接下来，使用共同的解剖参考水平，将给定实验组所有大脑的冠状切片相互对齐。该系列中的一个大脑作为模板，其他大脑的相应切片在每个层面映射到其轮廓中。通过这种方式，可以生成平均和标准差图像数据集。利用阈值技术，可以根据选定的阈值将单个数据集进行二分，并在每个冠状层面生成频率图，显示在每个像素位置显示mRNA超阈值水平的大脑数量。然后可以对在两种不同条件下（例如，脑外伤后30分钟与24小时）获得的数据集进行逐像素统计比较。可以将数字化的功能解剖脑图谱与图像拟合以辅助分析。基于计算机的原位杂交放射自显影片图像分析极大地扩展了该技术的实用性和适用性。
