Vorbrodt A W, Dobrogowska D H, Tarnawski M, Lossinsky A S
New York State Office of Mental Retardation and Developmental Disabilities, Institute for Basic Research in Developmental Disabilities, Staten Island 10314.
J Neurocytol. 1994 Dec;23(12):792-800. doi: 10.1007/BF01268091.
The time sequence of the blood-brain barrier opening to endogenous albumin in rat brain after intracarotid infusion of hyperosmolar L(+)arabinose was studied using quantitative immunocytochemistry. Brain samples obtained 1, 5, and 30 min after insult were immersion-fixed in formaldehyde-glutaraldehyde mixture and embedded at low temperature in Lowicryl K4M. Untreated rats or rats exposed only to Ringer's solution were used as a control. Ultrathin sections were exposed to anti-rat albumin antiserum followed by protein A-gold. The density of immunosignals (gold particles per square micrometre) was recorded over four compartments: vascular lumen, endothelium, subendothelial (perivascular) space including basement membrane, and brain parenchyma (neuropil). The labelling density of the vessel lumen, containing blood plasma, was considered to represent 100% of the circulating albumin. Morphometric and statistical analysis indicated that in control animals only 0.4-0.6% of circulating albumin appears in the subendothelial space and in the basement membrane. As soon as one minute after L(+)arabinose infusion, this value increased to 3%, followed by a further increase to 25% and 56% after 5 and 30 min, respectively. A slow increase of the labelling density in the adjacent neuropil suggests that the basement membrane represents an obstacle for escaping albumin, which apparently sticks to or is trapped by this membrane. The results indicate that the applied procedure, although based on morphometric analysis of static electron micrographs can also be used for studying dynamic processes such as transvascular passage of albumin after disruption of the brain-blood barrier.
采用定量免疫细胞化学方法，研究了经颈动脉注射高渗L(+)阿拉伯糖后，大鼠脑内血脑屏障对内源性白蛋白开放的时间序列。在损伤后1、5和30分钟获取脑样本，将其浸入甲醛-戊二醛混合液中固定，并在低温下包埋于Lowicryl K4M中。未处理的大鼠或仅暴露于林格氏液的大鼠用作对照。超薄切片先与抗大鼠白蛋白抗血清孵育，然后再与蛋白A-金结合。在四个区域记录免疫信号的密度（每平方微米的金颗粒数）：血管腔、内皮、包括基底膜在内的内皮下（血管周）间隙以及脑实质（神经毡）。含有血浆的血管腔的标记密度被认为代表循环白蛋白的100%。形态计量学和统计分析表明，在对照动物中，仅0.4 - 0.6%的循环白蛋白出现在内皮下间隙和基底膜中。在注射L(+)阿拉伯糖后一分钟内，该值就增加到3%，随后在5分钟和30分钟后分别进一步增加到25%和56%。相邻神经毡中标记密度的缓慢增加表明基底膜是白蛋白逸出的障碍，白蛋白显然会黏附于该膜或被其捕获。结果表明，尽管所采用的方法基于静态电子显微镜的形态计量分析，但也可用于研究诸如血脑屏障破坏后白蛋白的跨血管转运等动态过程。
