CONFOCAL MICROSCOPY

Confocal high-resolution biomicroscopy is finding growing utility as a clinical tool for in vivo description and quantification of corneal nerves implicated in degenerative nerve diseases such as diabetic neuropathy, according to Rudolf F Guthoff MD. “In degenerative nerve disease, the subepithelial nerve plexus can be evaluated and hopefully quantified much better than before thanks to the latest advances in confocal microscopy. By using 3-D mapping and reconstruction in diabetic neuropathy, alterations can be noticed earlier by in vivo confocal microscopy than by measurements of corneal sensitivity, peripheral nerve dysfunction and skin biopsies,†he said.

Prof Guthoff, professor of ophthalmology at Rostock University Eye Department, Rostock, Germany, told delegates attending the World Ophthalmology Congress that high-resolution biomicroscopy (Heidelberg Retinal Tomograph II in conjunction with the Rostock Cornea Module) will enable degeneration and repair mechanisms under various conditions to be examined so that the findings can be correlated with those from conventional slit-lamp biomicroscopy. “In vivo confocal microscopy is still quite a challenge for us. It was developed 50 years ago but it is still not an instrument in everyday clinical use. However, we believe that looking at nerves and trying to quantify them in the cornea might be something that we can all benefit from in our daily practices,†he said. Prof Guthoff noted that the cornea is innervated primarily by sensory fibres arising from the ophthalmic nerve, a terminal division of the trigeminal nerve. Human corneal nerves are non-myelinated and vary in thickness between 0.2mm and 10mm. The nerve fibre bundles, which enter the anterior and central stroma in the corneal periphery, run parallel to the corneal surface in a radial pattern before making an abrupt 90-degree turn in the direction of Bowman’s membrane.

The laser scanning confocal microscope uses a 670 nm red wavelength diode laser and offers up to 400 times magnification with an axial resolution of approximately one micrometre. To create an image, a beam of light scans the cornea, creating a 384 x 384 point image in a 400 micron square at a magnification of 63X. Most anatomical layers and cell types may be viewed easily including superficial, intermediate and basal epithelial cells, nerve plexi, stromal layers with keratocytes, Descemet’s membrane, endothelial cells and immune response cells. Cross-sectional views may also be seen in oblique scans. Initial research efforts have been directed at monitoring and quantifying nerve fibres in diabetic neuropathy, explained Prof Guthoff. “Several papers have shown that there is a reduction in nerve fibre pattern in the subepithelial plexus in diabetic patients. As a model for degenerative changes, diabetic neuropathy is clinically manifest subclinical disease of the peripheral nerve that can affect both the somatic and the autonomic nerve system. The signs are varied, but it is very difficult to quantify them,†he said. Focusing on the subepithelial nerve plexus, the goal has been to use the Heidelberg Retinal Tomograph II in conjunction with the Rostock Cornea Module to try to quantify subepithelial nerve fibres using clinically relevant parameters such as length, density and tortuosity, said Prof Guthoff.

“We do have numerical elements to describe nerve fibre patterns. Neuropathic components in ocular surface disease can be identified by pattern analysis of the subepithelial nerve plexus. We have shown that it works nicely in the lab in a small series of patients. There is also evidence from animal experiments that confocal in vivo microscopy can quantify the effect of neurotrophic growth factors for nerve fibre degeneration in vivo,†he concluded.