Mies G, Bodsch W, Paschen W, Hossmann K A
J Cereb Blood Flow Metab. 1986 Feb;6(1):59-70. doi: 10.1038/jcbfm.1986.8.
A triple-tracer autoradiographic technique is described that permits the simultaneous measurement of cerebral blood flow, glucose consumption, and protein synthesis using 131I-iodoantipyrine (131I-IAP), [14C]deoxyglucose ([14C]DG), and 3H-amino acids as radioactive tracers. Autoradiographic differentiation between isotopes was performed by taking advantage of different half-lives, solubility of labeled tracers in a wash solution, and sensitivity of the photographic material to disintegrations of the radionuclides. Blood flow autoradiograms using 131I-IAP were obtained by immediate exposure of brain sections to Kodak NMB film for 24 h. During 131I autoradiography contamination by 3H was absent and by 14C was negligible at tissue concentrations of less than 0.45 microCi/g brain tissue. After complete decay of 131I, reexposure of brain sections to Kodak NMB film for 2 weeks provided autoradiograms that stemmed exclusively from 14C disintegrations without contamination by either 131I or 3H and that represented regional glucose utilization. Brain sections were then wash-incubated for 12 h to remove [14C]DG, [14C]DG-6-phosphate, and free 3H-amino acids from the tissue, and exposed to 3H-sensitive LKB Ultrofilm for 2 weeks for autoradiography of 3H-amino acid incorporation into proteins. 14C radioactivity remaining in the tissue section after wash-incubation was determined by exposing sections again for 2 weeks to Kodak NMB film; the resulting contribution to the blackening of 3H-autoradiograms was corrected for by means of digital subtraction using an image-processing system. The triple-tracer autoradiographic technique was validated in rats under various physiological and pathophysiological conditions. In intact animals extinction correction was necessary only for 3H-autoradiograms. Under pathophysiological conditions, however, significant contamination of 131I by 14C occurred in regions with low blood flow and increased glucose utilization rate; this also required correction by digital subtraction. The interpretation of triple-tracer autoradiographic results is limited by the same restrictions as single-tracer autoradiography, but the simultaneous assessment of the three parameters considerably facilitates the interpretation of the flow/metabolic relationship, particularly under pathological conditions.
本文描述了一种三重示踪放射自显影技术，该技术允许使用131I-碘安替比林（131I-IAP）、[14C]脱氧葡萄糖（[14C]DG）和3H-氨基酸作为放射性示踪剂，同时测量脑血流量、葡萄糖消耗和蛋白质合成。利用不同的半衰期、标记示踪剂在洗涤溶液中的溶解度以及感光材料对放射性核素衰变的敏感性，对同位素进行放射自显影鉴别。使用131I-IAP的血流量放射自显影片是通过将脑切片立即暴露于柯达NMB胶片24小时获得的。在131I放射自显影过程中，在脑组织浓度低于0.45微居里/克时，不存在3H污染，14C污染可忽略不计。131I完全衰变后，将脑切片再次暴露于柯达NMB胶片2周，得到的放射自显影片完全来自14C衰变，无131I或3H污染，代表区域葡萄糖利用情况。然后将脑切片洗涤孵育12小时，以从组织中去除[14C]DG、[14C]DG-6-磷酸和游离3H-氨基酸，并将其暴露于对3H敏感的LKB Ultrofilm 2周，以进行3H-氨基酸掺入蛋白质的放射自显影。洗涤孵育后组织切片中剩余的14C放射性通过将切片再次暴露于柯达NMB胶片2周来测定；使用图像处理系统通过数字减法校正对3H放射自显影片变黑的影响。三重示踪放射自显影技术在各种生理和病理生理条件下的大鼠中得到验证。在完整动物中，仅对3H放射自显影片需要进行消光校正。然而，在病理生理条件下，在血流量低且葡萄糖利用率增加的区域，14C对131I有明显污染；这也需要通过数字减法进行校正。三重示踪放射自显影结果的解释受到与单示踪放射自显影相同的限制，但同时评估这三个参数极大地促进了对血流/代谢关系的解释，特别是在病理条件下。
