Jorge L. Jacot

Computer analysis of corneal innervation density using a novel double stain in rat corneal whole mounts

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 Descemets 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°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.

Improved Gold Chloride Procedure for Nerve Staining in Whole Mounts of Rat Corneas

The purpose of this study was to modify the gold chloride procedure for studies of total innervation in corneal whole mounts to provide a decrease in nonspecific background staining and to eliminate the progressively deteriorating stain quality of standard gold chloride techniques. Modifications included use of cryoprotective agents, mechanical removal of Descemets membrane-endothelium complex prior to fixation, treatment with alpha amylase, and halting the reduction of gold chloride to metallic gold using Kodak rapid fixer with hardener. Rat corneas were stored at -70 C in O.C.T. compound. The Descemets membrane-endothelium complex was removed after thawing, and corneas were fixed in 4% NaPO4-buffered paraformaldehyde with 8% sucrose. Fixed corneas were incubated in NaPO4-buffered saline containing alpha amylase, placed in 100% lemon juice, then in 1% gold chloride solution, transferred to glacial acidic acid, placed in rapid fixer, rinsed in NaPO4-buffered saline, and dehydrated in graded alcohols. Flat mounts of whole corneas were examined using contralateral corneas as controls. Freezing corneas in O.C.T. compound, removal of the Descemets membrane-endothelium complex, and treatment with alpha amylase reduced non-specific background staining compared to controls. Treatment with Kodak rapid fixer prevented the deterioration of staining quality for at least 8 months. These improvements allow the gold chloride technique to be used with immunohistochemical procedures where the reaction products would be obscured by background staining.

Diabetic Retinopathy: Unraveling the Paradoxical Effects of Intensive Insulin Treatment

Abstract Background: Several major clinical trials have demonstrated that the tight glycemic control achieved with intensive insulin treatment may lead to an early worsening of diabetic retinopathy but that intensive long-term insulin treatment slows the progression of diabetic retinopathy. Objective: The goal of this article was to review developments in understanding both the pathophysiologic pathways involved in diabetic retinopathy and the biologic effects of insulin to provide a possible explanation why insulin treatment may have deleterious short-term effects yet provide long-term benefits. Methods: The content of this article was based on a search of MEDLINE from 1984 through 2006. English-language articles were chosen using the search terms diabetic retinopathy , insulin effects , and clinical trials . Results: Early worsening of diabetic retinopathy from acute intensive insulin treatment may result from upregulation of retinal vascular endothelial growth factor (VEGF) expression through increased binding of hypoxia-inducible factorla to the VEGF promoter. This results in increased gene transcription and VEGF levels. Increased VEGF levels also promote activation of protein kinase C; both events contribute to increased vasopermeability and a breakdown of the blood-retinal barrier. However, long-term insulin treatment reduces inflammatory and oxidative stress components that may contribute to progressive diabetic retinopathy. Both inflammatory effects and oxidative stress may play a role in the etiology of diabetic retinopathy. The beneficial biologic effects of insulin may thus supercede the acute deleterious effects of the VEGF-mediated blood-retinal-barrier breakdown. We speculate that the long-term beneficial effects of intensive insulin therapy on the progression of diabetic retinopathy arise from insulins recently realized potent anti-inflammatory effects and ability to modulate redox-sensitive signaling pathways. Conclusions: Insulin treatment introduced before irreversible pathologic changes develop in the retina can impart beneficial effects to the vascular cells, may help suppress future deleterious events, and may slow the development or progression of retinal microangiopathic changes.