HINDERED DIFFUSION OF HIGH-MOLECULAR-WEIGHT COMPOUNDS IN BRAIN EXTRACELLULAR MICROENVIRONMENT MEASURED WITH INTEGRATIVE OPTICAL IMAGING
HINDERED DIFFUSION OF HIGH-MOLECULAR-WEIGHT COMPOUNDS IN BRAIN EXTRACELLULAR MICROENVIRONMENT MEASURED WITH INTEGRATIVE OPTICAL IMAGING
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DOI：
10.1016/s0006-3495(93)81324-9
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发表时间：
1993-12-01
影响因子：
3.4
通讯作者：
TAO, L
中科院分区：
文献类型：
--
作者：
NICHOLSON, C;TAO, L
本文介绍了一体化光学成像系统的原理及其应用分析的3-，10-，40-和70-kDa的荧光葡聚糖分子在琼脂糖凝胶和脑细胞外微环境中的扩散。该方法使用一个精确定义的荧光分子的压力从微量移液器喷出的源，和一个详细的理论的强度贡献从一个三维介质中的焦点外的分子的二维图像。将用四甲基罗丹明或德克萨斯红标记的葡聚糖喷射到0.3%琼脂糖凝胶或保持在34 ℃灌注室中的大鼠皮质切片中，并使用具有10 x水浸物镜的复合落射荧光显微镜成像。以2-10-s的间隔拍摄约20张图像，用冷却的CCD相机记录，然后转移到486 PC进行定量分析。琼脂糖凝胶中的扩散系数D和脑组织中的表观扩散系数D* 通过拟合将测量的二维图像强度与理论三维葡聚糖浓度相关的积分表达式来确定。在稀释琼脂糖凝胶中的测量提供了D的参考值，并验证了该方法。的曲折度，λ =（D/D*）1/2，为3-和10-kDa的葡聚糖的值分别为1.70和1.63，这是一致的，与以前的值来自皮质中的四甲基铵测量。40-和70-kDa葡聚糖的弯曲度分别为2.16和2.25。这表明，细胞外空间可能有局部收缩，阻碍分子的扩散超过一个临界尺寸，位于许多神经营养化合物的范围。
This paper describes the theory of an integrative optical imaging system and its application to the analysis of the diffusion of 3-, 10-, 40-, and 70-kDa fluorescent dextran molecules in agarose gel and brain extracellular microenvironment. The method uses a precisely defined source of fluorescent molecules pressure ejected from a micropipette, and a detailed theory of the intensity contributions from out-of-focus molecules in a three-dimensional medium to a two-dimensional image. Dextrans tagged with either tetramethylrhodamine or Texas Red were ejected into 0.3% agarose gel or rat cortical slices maintained in a perfused chamber at 34-degrees-C and imaged using a compound epifluorescent microscope with a 10 x water-immersion objective. About 20 images were taken at 2-10-s intervals, recorded with a cooled CCD camera, then transferred to a 486 PC for quantitative analysis. The diffusion coefficient in agarose gel, D, and the apparent diffusion coefficient, D*, in brain tissue were determined by fitting an integral expression relating the measured two-dimensional image intensity to the theoretical three-dimensional dextran concentration. The measurements in dilute agarose gel provided a reference value of D and validated the method. Values of the tortuosity, lambda = (D/D*)1/2, for the 3- and 10-kDa dextrans were 1.70 and 1.63, respectively, which were consistent with previous values derived from tetramethylammonium measurements in cortex. Tortuosities for the 40- and 70-kDa dextrans had significantly larger values of 2.16 and 2.25, respectively. This suggests that the extracellular space may have local constrictions that hinder the diffusion of molecules above a critical size that lies in the range of many neurotrophic compounds.