Perfusion Apparatus to Avoid Shrinkage of Soft Tissue

描述(由申请人提供)： 细胞和组织必须经过广泛的处理，才能在显微镜下看到和分析。为显微镜准备动物组织最常见的方法是灌注法。通过灌流，研究人员利用动物的循环系统将固定剂输送到组织细胞，从而去除麻烦的红细胞。固定剂是将组织锁定在稳定状态，防止自然发生的自溶和变质的试剂。固定剂穿透组织的速度越快，保存的组织就越接近活着的状态。红细胞在许多重要的组织反应中产生背景标记；它们的去除改善了感兴趣细胞的对比度和可见度。 由于用于牺牲灌注的标准仪器和方案尚未商业化，实验室必须组装自己的仪器。这导致了仪器和协议的广泛变化，而这些往往没有在已发表的论文的方法部分进行描述。尽管灌注质量对组织分析有很大影响，但灌注技术却鲜有人关注。 最常见的灌注过程的一个缺点是它们会产生收缩--大约20%的脑组织收缩--从而改变大体解剖和显微形态。活着的大脑有20%的细胞外空间。使用普通方案灌流和固定的脑组织切片不存在这种空间，导致切片比来自新鲜、未固定组织的可比切片小得多。向内的塌陷扭曲了许多大脑结构的形状和位置。此外，由于细胞外空间的丧失，神经元元素可能在电子显微镜水平上错误地出现在对位。 然而，收缩并不是固定的必然结果。一种改进的灌注固定程序已经公布，需要更复杂的设备。这一过程不会导致整个器官收缩，不会导致细胞外空间的丧失，而且在去除红细胞方面更有效。这一过程产生了一致的可重复性、高组织质量。它所需要的仪器可以设计成便于复制灌流参数。 这项拟议的研究旨在开发和测试一种新的商业灌流设备，该设备将提高用于生物医学研究，特别是神经科学研究的动物组织的组织学效率和重复性。

DESCRIPTION (provided by applicant): Cells and tissues must undergo extensive processing before they can be seen and analyzed under a microscope. The most common method for preparing animal tissue for microscopy is known as perfusion. With perfusion, a researcher uses an animal's circulatory system to deliver a fixative to tissue cells and thereby remove troublesome red blood cells. Fixatives are agents that lock tissue in a stable state, preventing naturally occurring autolysis and deterioration. The faster a fixative penetrates a tissue, the more closely the preserved tissue resembles the living state. Red blood cells create background labeling in many important histological reactions; their removal improves the contrast and visibility of cells of interest. Because standard instrumentation and protocols for sacrifice perfusion have not been commercialized, laboratories must assemble their own apparatus. This has led to wide variations in apparatus and protocols, which are frequently not described in the methods section of published papers. Although perfusion quality can substantially impact tissue analysis, perfusion techniques have received remarkably little attention. A drawback in the most common perfusion procedures is that they produce shrinkage --about 20% shrinkage for brain tissue -- and thereby alter the gross anatomical and microscopic morphology. The living brain is 20% extracellular space. This space is absent from tissue sections of brain perfused and fixed using common protocols, resulting in sections that are substantially smaller than comparable sections from fresh, unfixed tissue. The inward collapse distorts shape and position of many brain structures. Moreover, neuronal elements may erroneously appear in apposition on an electron microscopic level, due to loss of extracellular space. Shrinkage, however, is not an inevitable result of fixation. An improved perfusion fixation procedure has been published requiring more sophisticated apparatus. This procedure does not cause whole organ shrinkage, does not result in loss of extracellular space, and is more effective in removing red blood cells. This procedure yields consistently reproducible, high tissue quality. The instrumentation it requires can be designed to facilitate replication of perfusion parameters. The proposed research is to develop and test a new commercial perfusion apparatus that will improve the efficiency and reproducibility of histology on animal tissues used in biomedical research, especially neuroscience.