Zhang Shu-Xin, Holmberg Eric G, Geddes James W
Spinal Cord and Brain Injury Research Center, University of Kentucky College of Medicine, Lexington, KY 40536, USA.
J Neurosci Methods. 2007 Jun 15;163(1):38-43. doi: 10.1016/j.jneumeth.2007.02.013. Epub 2007 Feb 22.
Extensive dendritic beading of MAP2 (microtubule-associated protein 2) immunoreactivity has previously been observed in the contused rat spinal cord. However, we have also observed dendritic beading in occasional uninjured animals. The purpose of this study was to examine the possibility that perfusion conditions contributed to the dendritic beading. Under deep anesthesia, uninjured rats (adult female Long-Evans, 200-225 g) were transcardially perfused with 0.9% saline solution followed by 4% paraformaldehyde at cold (4 degrees C) or warm (20 degrees C) temperature, and at a low (20 ml/min) or high (50 ml/min) flow rate. Dendrites were visualized by MAP2 immunoreactivity. The results demonstrate that perfusion with cold solutions at a high flow rate induces pronounced dendritic beading, and when perfused at a low flow rate, results in moderate dendritic beading. Warm perfusates did not induce dendritic beading when administered at a low flow rate, but occasional beading was observed with a high flow rate. Western blots revealed spectrin breakdown, but not MAP2 loss, in rats perfused with cold saline solution at a high flow rate, conditions that also resulted in dendritic beading. These findings demonstrate that dendritic morphology is sensitive to both temperature and flow rate of the perfusate. Warm fixative and a low perfusion flow rate minimized the perfusion-induced dendritic beading.
先前在挫伤的大鼠脊髓中观察到微管相关蛋白2（MAP2）免疫反应性广泛的树突串珠现象。然而，我们也在偶尔未受伤的动物中观察到了树突串珠。本研究的目的是检验灌注条件是否导致树突串珠的可能性。在深度麻醉下，对未受伤的大鼠（成年雌性Long-Evans，体重200 - 225克）经心脏灌注0.9%盐溶液，随后在低温（4℃）或高温（20℃）下，以低流速（20毫升/分钟）或高流速（50毫升/分钟）灌注4%多聚甲醛。通过MAP2免疫反应性使树突可视化。结果表明，在高流速下用冷溶液灌注会诱导明显的树突串珠，而在低流速下灌注则导致中度树突串珠。当以低流速灌注时，温灌注液不会诱导树突串珠，但在高流速下偶尔会观察到串珠现象。蛋白质免疫印迹显示，在以高流速用冷盐溶液灌注的大鼠中存在血影蛋白分解，但不存在MAP2丢失，这种情况也会导致树突串珠。这些发现表明，树突形态对灌注液的温度和流速都很敏感。温热固定剂和低灌注流速可将灌注诱导的树突串珠降至最低。
