Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Brazil.
J Neurosci Methods. 2013 Jan 15;212(1):72-8. doi: 10.1016/j.jneumeth.2012.09.015. Epub 2012 Sep 24.
Isotropic fractionation is a quantitative technique that allows reliable estimates of absolute numbers of neuronal and non-neuronal brain cells. However, being fast for single small brains, it requires a long time for processing large brains or many small ones, if done manually. To solve this problem, we developed a machine to automate the method, and tested its efficiency, consistency, and reliability as compared with manual processing. The machine consists of a set of electronically controlled rotation and translation motors coupled to tissue grinders, which automatically transform fixed tissue into homogeneous nuclei suspensions. Speed and torque of the motors can be independently regulated by electronic circuits, according to the volume of tissue being processed and its mechanical resistance to fractionation. To test the machine, twelve paraformaldehyde-fixed rat brains and eight human cerebella were separated into two groups, respectively: one processed automatically and the other, manually. Both pairs of groups (rat and human tissue) followed the same, published protocol of the method. We compared the groups according to nuclei morphology, degree of clustering and number of cells. The machine proved superior for yielding faster results due to simultaneous processing in multiple grinders. Quantitative analysis of machine-processed tissue resulted in similar average numbers of total brain cells, neurons, and non-neuronal cells, statistically similar to the manually processed tissue and equivalent to previously published data. We concluded that the machine is more efficient because it utilizes many homogenizers simultaneously, equally consistent in producing high quality material for counting, and quantitatively reliable as compared to manual processing.
各向同性分离是一种定量技术，可可靠估计神经元和非神经元脑细胞的绝对数量。然而，对于单个小大脑来说，它的速度很快，但如果手动处理，处理大大脑或许多小大脑则需要很长时间。为了解决这个问题，我们开发了一种机器来自动化该方法，并测试了其与手动处理相比的效率、一致性和可靠性。该机器由一组电子控制的旋转和平移电机与组织研磨机耦合而成，可自动将固定组织转化为均质核悬浮液。电子电路可根据处理的组织体积及其对分离的机械阻力，独立调节电机的速度和扭矩。为了测试该机器，我们将十二只福尔马林固定的大鼠脑和八个人脑小脑分为两组，分别进行自动和手动处理。两组（大鼠和人脑组织）都遵循相同的、已发表的方法方案。我们根据核形态、聚类程度和细胞数量对两组进行了比较。由于在多个研磨机中同时进行处理，机器的处理速度更快，结果也更好。对机器处理的组织进行定量分析得出的总脑细胞、神经元和非神经元细胞的平均数量相似，与手动处理的组织在统计学上相似，与之前发表的数据相当。我们得出的结论是，该机器的效率更高，因为它同时使用了多个匀浆机，在产生用于计数的高质量材料方面同样一致，并且与手动处理相比在定量上是可靠的。
