Novaes Rômulo D, Cupertino Marli C, Sarandy Mariaurea M, Souza André, Soares Evelise A, Gonçalves Reggiani V
1Department of Structural Biology,Federal University of Alfenas,Rua Gabriel Monteiro da Silva, 700,Campus Universitário,Centro,Alfenas,MG 37130-000,Brazil.
2Department of General Biology,Federal University of Viçosa,Viçosa,MG 36570-000,Brazil.
Microsc Microanal. 2015 Dec;21(6):1482-1490. doi: 10.1017/S1431927615015366. Epub 2015 Nov 5.
Skin samples were used to compare microscopy methods used to quantify collagen with potential applicability to resolve time-dependent collagen deposition during skin wound healing in rats. Skin wounds by secondary intention were made in rats and tissue fragments were collected every 7 days for 21 days. Collagen content determined by biochemical analysis was compared with collagen measured by point counting (PC) on histological skin sections stained by Gomori's trichrome method (Trichrome/PC), Sirius red under polarized light (PL) microscopy (Sirius red/PL-PC), and computational color segmentation (CS) applied to sections stained with Sirius red (Sirius red/PL-CS). All microscopy methods investigated resolved the time-dependent dynamics of collagen deposition in scar tissue during skin wound healing in rats. Collagen content measured by Sirius red/PL-PC and Sirius red/PL-CS was significantly lower when compared with Trichrome/PC. The Trichrome/PC method provided overestimated values of collagen compared with biochemical analysis. In the early stages of wound healing, which shows high production of noncollagenous molecules, Sirius red/PL-CS and Sirius red/PL-PC methods were more suitable for quantification of collagen fibers. Trichrome staining did not allow clear separation between collagenous and noncollagenous elements in skin samples, introducing a marked bias in collagen quantification.
使用皮肤样本比较用于量化胶原蛋白的显微镜方法，这些方法具有潜在适用性，可用于解析大鼠皮肤伤口愈合过程中随时间变化的胶原蛋白沉积情况。对大鼠进行二期愈合的皮肤伤口造模，并在21天内每隔7天收集组织碎片。将通过生化分析测定的胶原蛋白含量与通过点计数（PC）在经Gomori三色染色法（三色/PC）染色的组织学皮肤切片上、在偏振光（PL）显微镜下对天狼星红染色切片（天狼星红/PL-PC）以及应用于天狼星红染色切片的计算颜色分割（CS）（天狼星红/PL-CS）所测量的胶原蛋白进行比较。所研究的所有显微镜方法都解析了大鼠皮肤伤口愈合过程中瘢痕组织中胶原蛋白沉积的时间依赖性动态变化。与三色/PC相比，通过天狼星红/PL-PC和天狼星红/PL-CS测量的胶原蛋白含量显著更低。与生化分析相比，三色/PC方法提供的胶原蛋白值被高估。在伤口愈合的早期阶段，此时非胶原蛋白分子产量较高，天狼星红/PL-CS和天狼星红/PL-PC方法更适合于定量胶原蛋白纤维。三色染色无法在皮肤样本中清晰区分胶原蛋白和非胶原蛋白成分，从而在胶原蛋白定量中引入了明显偏差。
