Computer analysis of corneal innervation density using a novel double stain in rat corneal whole mounts
| Autor: | J P Glover, J. L. Jacot, W. Gerald Robison |
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| Rok vydání: | 1997 |
| Předmět: |
Male
Pathology medicine.medical_specialty Histology ALIZARIN RED Stain Cornea Rats Sprague-Dawley chemistry.chemical_compound Computer analysis medicine Image Processing Computer-Assisted Animals Paraformaldehyde Coloring Agents Molecular Biology Ecology Evolution Behavior and Systematics Whole mount Chemistry Endothelium Corneal Nerve plexus Cell Biology eye diseases Gold Compounds Staining Rats Double staining medicine.anatomical_structure Acetylcholinesterase Female sense organs Anatomy Developmental Biology Research Article |
| Zdroj: | Journal of anatomy. 191 |
| ISSN: | 0021-8782 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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