Anandacoomaraswamy K S, Dutton N, Rajan G P, Eikelboom R H, Atlas M D, Robertson T
School of Surgery and Pathology, The University of Western Australia, Australia.
Acta Otolaryngol. 2006 Feb;126(2):149-53. doi: 10.1080/00016480500312596.
The results of this study have demonstrated for the first time that tympanic membrane (TM) structure is preserved following removal of fresh, normal tissue from patients undergoing surgery. Greater clarity has been demonstrated using resin sections than in previous studies on paraffin sections. Of particular note, cytokeratin (CK) immunocytochemistry was successfully performed on resin sections, which has not been previously reported. This may have potential applications for future work involving tissues that express CKs.
To analyse the structure of normal, fresh human TM specimens after surgical removal and to evaluate their CK immunocytochemistry using resin techniques, neither of which have been demonstrated previously.
Seven TM specimens were removed during surgery and then preserved in a modified Karnovsky's fixative. Semi-thin and thin sections were examined by means of light and electron microscopy, respectively. For comparison purposes, paraffin block-embedded specimens were also sectioned. CK immunocytochemistry was performed on semi-thin sections using standard immunoperoxidase techniques, with expression being demonstrated using light microscopy.
The three-layer architecture of the TM was preserved. The morphology of the TM was vastly superior in the semi-thin resin sections than in the thicker paraffin sections. The outer, middle and inner layers were clearly demonstrated. The integrity of the outer epithelial layer was maintained, with an outer keratinizing stratum corneum and underlying stratum granulosum, stratum spinosum and stratum basale layers resting on the basal lamina. The thin inner mucosal layer was also viable, consisting of simple squamous or cuboidal cells. Preservation of the middle lamina propria was achieved, with demonstration of the outer radial and inner circular fibres. CK immunocytochemistry utilizing resin techniques provided excellent staining of CK 7 and 8 in the inner layer, with positive staining of CK 5 and 10 in the outer layer.
本研究结果首次证明，在接受手术的患者中，鼓膜（TM）结构在新鲜正常组织切除后得以保留。与以往石蜡切片研究相比，树脂切片显示出更高的清晰度。特别值得注意的是，细胞角蛋白（CK)免疫细胞化学在树脂切片上成功进行，此前未见报道。这可能对未来涉及表达CK的组织的研究有潜在应用价值。
分析手术切除后新鲜人正常TM标本的结构，并使用树脂技术评估其CK免疫细胞化学，这两者此前均未得到证实。
手术中取出7个TM标本，然后保存在改良的卡诺夫斯基固定液中。分别通过光学显微镜和电子显微镜检查半薄切片和超薄切片。为了进行比较，还对石蜡包埋标本进行了切片。使用标准免疫过氧化物酶技术在半薄切片上进行CK免疫细胞化学，通过光学显微镜显示其表达。
TM的三层结构得以保留。TM在半薄树脂切片中的形态比在较厚的石蜡切片中要好得多。外层、中层和内层清晰可见。外层上皮层的完整性得以维持，外层有角质化的角质层，其下方的颗粒层、棘层和基底层位于基膜上。薄的内层粘膜层也存活，由单层扁平或立方细胞组成。中层固有层得以保留，显示出外层的放射状纤维和内层的环形纤维。利用树脂技术进行的CK免疫细胞化学在内层对CK 7和8染色良好，外层CK 5和10呈阳性染色。
