Jacot J L, Glover J P, Robison W G
Section on Pathophysiology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892-2740, USA.
J Anat. 1997 Aug;191 ( Pt 2)(Pt 2):191-9. doi: 10.1046/j.1469-7580.1997.19120191.x.
Computerised morphometry and a double stain technique were utilised to examine the corneal nerves in whole mounts. This novel stain combines the nonspecific acetylcholinesterase (NsAchE) and gold chloride (AuCl) procedures to enhance staining contrast and facilitate computerised detection of corneal nerves. Fresh rat corneas were dissected, and the Descemet's membrane-endothelium complex was removed. Then the corneas were fixed in 4% paraformaldehyde with 50 mM Na-K phosphate buffer (pH 7.2) and 8% sucrose for 30 min. They were rinsed and stained singly with NsAchE or AuCl, or were double stained using NsAchE followed by AuCl. Between NsAchE and AuCl staining the corneas were stored frozen in OCT compound at -70 degrees C. Flat mounts of whole corneas were photographed before and after the second staining. Measurable stromal innervation density (mean +/- S.D.) in age-matched corneas stained with AuCl (3.90 +/- 0.36 mm/mm2) was not significantly different from that of NsAchE stained corneas. However, double staining compared with NsAchE staining of the same corneas revealed a 48 +/- 27% increase in demonstrable innervation density of the subepithelial nerve plexus (7.95 +/- 0.86 mm/mm2 vs 5.52 +/- 1.31 mm/mm2, respectively). Improved visualisation of epithelial nerves and their fine ramifications (leashes) was also obtained by double staining. This novel combination of 2 procedures enhances the detection of corneal nerves for analysis by computerised morphometry and provides a more representative estimate of total corneal innervation density.
利用计算机形态测量法和双重染色技术对全层角膜神经进行检查。这种新型染色法结合了非特异性乙酰胆碱酯酶（NsAchE）和氯化金（AuCl）程序，以增强染色对比度并便于计算机检测角膜神经。解剖新鲜大鼠角膜，去除后弹力层 - 内皮细胞复合体。然后将角膜固定在含有50 mM Na-K磷酸盐缓冲液（pH 7.2）和8%蔗糖的4%多聚甲醛中30分钟。将它们冲洗后，单独用NsAchE或AuCl染色，或者先用NsAchE染色，然后再用AuCl进行双重染色。在NsAchE和AuCl染色之间，将角膜冷冻保存在OCT复合物中，温度为-70℃。在第二次染色前后对全层角膜的平铺标本进行拍照。用AuCl染色的年龄匹配角膜的可测量基质神经支配密度（平均值±标准差）（3.90±0.36 mm/mm²）与用NsAchE染色的角膜相比无显著差异。然而，与同一角膜的NsAchE染色相比，双重染色显示上皮下神经丛的可显示神经支配密度增加了48±27%（分别为7.95±0.86 mm/mm²和5.52±1.31 mm/mm²）。通过双重染色还能更好地观察上皮神经及其细微分支（束状分支）。这两种程序的这种新型组合增强了角膜神经的检测，便于通过计算机形态测量法进行分析，并能更具代表性地估计角膜总神经支配密度。
