Linda J. Müller

Corneal nerves: structure, contents and function

This review provides a comprehensive analysis of the structure, neurochemical content, and functions of corneal nerves, with special emphasis on human corneal nerves. A revised interpretation of human corneal nerve architecture is presented based on recent observations obtained by in vivo confocal microscopy (IVCM), immunohistochemistry, and ultrastructural analyses of serial-sectioned human corneas. Current data on the neurotransmitter and neuropeptide contents of corneal nerves are discussed, as are the mechanisms by which corneal neurochemicals and associated neurotrophins modulate corneal physiology, homeostasis and wound healing. The results of recent clinical studies of topically applied neuropeptides and neurotrophins to treat neurotrophic keratitis are reviewed. Recommendations for using IVCM to evaluate corneal nerves in health and disease are presented.

In vivo confocal microscopy after herpes keratitis.

Purpose. To describe the confocal microscopic findings, with special reference to corneal subbasal nerves, after herpes simplex virus (HSV) keratitis. Methods In this study, 16 HSV eyes and 14 contralateral eyes of 16 patients, diagnosed with unilateral HSV keratitis 1–12 months earlier by the presence of dendritic corneal ulceration or microbiologic confirmation, were examined by in vivo confocal microscopy for evaluation of corneal morphology. Results. Herpes simplex virus eyes: In 2 eyes the surface epithelial cells appeared large, and no abnormalities were observed in the basal epithelial cells. In 2 eyes subbasal nerve fiber bundles were completely absent, in 3 eyes there was a reduced number of long nerve fiber bundles, and in 11 eyes the subbasal nerve plexus appeared normal. In 10 corneas, highly reflective dendritic structures were found at the level of the basal epithelial cells. Frequently these structures were found in the vicinity of stromal fibrosis. Areas with increased abnormal extracellular matrix were found in 11 eyes. Stromal nerves were not visualized in all corneas, but appeared normal when observed. Contralateral eyes: No abnormalities were observed in the epithelium. All corneas presented with a normal subbasal nerve plexus, but in 2 eyes dendritic particles were observed. Three corneas presented with activated keratocytes and increased amounts of abnormal extracellular matrix. Conclusions. When visualized by confocal microscopy, the subbasal nerve plexus appears relatively unaffected in cases with resolved HSV keratitis. Unidentified dendritic structures, presumably Langerhans cells, are frequently seen at the level of the basal epithelium in corneas with a history of herpetic disease.

A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma

The purpose of the present study was to re-evaluate the three-dimensional organization of collagen fibrils and proteoglycans (PGs) in the human corneal stroma using an improved ultrastructural approach. After a short aldehyde prefixation, one half of seven fresh corneal buttons was stained for PGs with Quinolinic Phtalocyanin (QP) or Cupromeronic Blue (CB). Strips of 1 mm width were cut, subsequently treated with aqueous phosphotungstic acid (PTA) and further processed for light and electron microscopy. The other half of the corneas served as control and was routinely processed with OsO4. Embedding was as such that ultrathin sections could be cut precisely parallel (frontal sections) or perpendicular (cross sections) to the corneal surface. The mutual connections between collagen fibrils and PGs were studied and the length of PGs and their mutual distance were measured manually at a calibrated final magnification of 70,000 x. Prefixed fresh corneal tissue treated with QP and CB shows no signs of swelling and exhibits well contrasted PGs. In cross sections PGs form a repeating network of ring-like structures (approximately 45 nm) around the collagen fibrils. In frontal sections PGs are aligned orthogonal to the collagen fibrils, are equidistant (approximately 42 nm) attached to the collagen fibrils along their full length and have a thickness of approximately 11 nm and a length of approximately 54 nm. The observed maximal length of the PGs and the occurrence of ring-like structures enwrapping the collagen fibrils urged us to revisit the prevailing model of maurice (1962) on the organization of the corneal stroma. In the new model hexagonal arranged collagen fibrils are interconnected at regular distances with their next-nearest neighbours by groups of six PGs, attached orthogonal to the circumference of the fibrils. In this way a regular meshwork of ring-like structures enwrapping the collagen fibrils is formed. It is discussed that this new model more convincingly explains corneal resistance to compression and stretching and further rationalizes corneal transparency because of the low refractive index difference between the regularly arranged collagen fibrils and their inter-space filled with PGs.

The effects of organ-culture on the density of keratocytes and collagen fibers in human corneas.

Purpose. Keratocytes are important in regaining corneal transparency during wound healing after surgery or trauma. Hitherto, there are still controversies concerning the effects of organ culture on the density and integrity of keratocytes and collagen fibers. The current study aimed at a systematic analysis of the effects of organ-culture on the morphology and density of keratocytes and collagen fibers. Methods. Human corneas were organ-cultured in MEM for 7 (n=17, 3 pairs), 14 (n=18, 9 pairs) and 21days (n=18, 9 pairs). Of the pairs one cornea was processed in swollen condition and the fellow cornea after reversal of swelling in MEM plus Dextran. Eleven post-mortem corneas (PM) and 11 fresh corneas obtained from melanoma patients were used as controls. Stromal thickness, number of keratocyte profiles (corrected for swelling), number and diameter of collagen fibers were measured in light microscopical sections and electron micrographs. Results. Stromal swelling due to organ-culture resulted in large keratocyte profiles with many vacuoles and large distances between collagen fibers in the posterior stroma. In contrast both keratocytes and distances between collagen fibers were not affected in the anterior stroma. After reversed-swelling the posterior corneal stroma was similar to that in fresh controls, indicating that the swelling process is largely reversible. The initial decrease in keratocyte density (18%) in the early post-mortem period did not progress during 21 days of organ culture. Conclusion. With respect to the morphology and density of keratocytes and collagen fibers it can be concluded that donor corneas remain suitable for transplantation up to at least 21days after organ-culture.

Ultrastructural neuropathologic effects of Taxol on neurons of the freshwater snailLymnaea stagnalis

SummaryCerebral ganglia of the freshwater snailLymnaea stagnalis were incubatedin vitro in 10 μM Taxol for 8 and 24 h. Cremophor EL (0.1%) was used as a diluant. The tissue was processed for electron microscopy. Various ultrastructural parameters were assessed quantitatively. Cremophor EL appeared to seriously affect the cell somata of the multipeptidergic caudodorsal cells. In the Cremophor-controls the mean area of Golgi zones, the percentage dense material (neuropeptides) in these zones, the number of large electron dense granules (these are involved in neuropeptide processing) and the mean nuclear heterochromatin clump size, were significantly smaller than in the Ringer-controls, whereas the number of lipid droplets was higher. All these parameters, except for the lipid droplets, were not different in the Cremophor-controls and the Taxol-treated specimens. After 24 h treatment, but not after 8 h, Cremophor EL furthermore induced an increase in the number of axonal microtubules. It is argued that the results might signify activation of the neurons by Cremophor EL. Taxol induced a significant increase in the number of microtubules in axons and cell somata. Furthermore an increase in the number of Golgi zones was observed, suggesting activated neuropeptide synthesis. In all groups immunostaining with antibodies to neuropeptides produced by the caudodorsal cells was normal. Release of neuropeptide (exocytosis) from axon endings was elevated after Taxol treatment, and exceptionally high in specimens cotreated with Taxol and Org 2766 (incubation time 22 h). The effect of Org 2766 and Taxol on the number of microtubules was cumulative. It is argued that transport of neuropeptide granules from the cell somata to the axon terminals was not affected by Taxol. It is concluded that Taxol neurotoxicity is probably not due to impeded microtubular axonal transport.

The ACTH/MSH(4–9) analogue ORG 2766 stimulates microtubule formation in axons of central neurons of the snail Lymnaea stagnalis

Central nervous systems of the pond snail Lymnaea stagnalis were incubated in vitro in different concentrations of ORG 2766 (10(-9)-2.5 x 10(-4) M) for 10 and 20 h. Quantitative ultrastructural study of cross sections of the cerebral commissure showed that the number of microtubules in large axons had increased after 10 h of incubation by approximately 50% (Experiment 1) and 30% (Experiment 2), respectively. No further increase was observed after 20 h of incubation. (The higher concentrations were studied.) Maximal stimulation was already found at a concentration of 10(-8) M. At a concentration of 10(-9) M control levels were observed. It is concluded that ORG 2766 stimulates microtubule formation already at very low concentrations. It is not clear whether the compound stimulates synthesis of tubulin, induces assembly of microtubules, or causes an increase in stability of microtubules. Nevertheless, ultrastructural data on the morphology of the glial cells indicate that these cells are activated by ORG 2766 treatment, which suggest that ORG 2766 has general trophic effects.

Differential trophic effects of ORG 2766, an ACTH(4-9)/MSH(4-9) analogue, on peptidergic neurons and glial cells in the snail Lymnaea stagnalis.

Cerebral ganglia of the pond snail Lymnaea stagnalis were incubated in vitro in 10(-6) M ORG 2766 for 10 and 20 h, with or without regular refreshment of the medium. Quantitative ultrastructural study of cross sections of the cerebral commissure showed that the number of microtubules in large axons of all ORG 2766-treated groups had increased after 10 h by approximately 40%. In a separate experiment, central nervous systems were incubated in ORG 2766 for only 15 min and then kept in Ringers for 9 h and 45 min. Maximal stimulation (40% increase of microtubules) in these specimens was also observed. The results would seem to support the hypothesis that ORG 2766 binds to a receptor and initiates a long-lasting effect. It is argued that ORG 2766 stimulates novel synthesis of tubulin rather than being involved in the assembly of microtubules. Also, glial cells were found to be activated by ORG 2766. This was concluded from the fact that the number of heterochromatin clumps and the size of the clumps in these cells had decreased and the amount of glial tissue surrounding the axons had increased (approximately 50%). In contrast to the activating effects of ORG 2766 on glial tissue, this drug did not affect nucleoli, number, and size of the heterochromatin clumps and the Golgi apparatus in the neuropeptidergic caudodorsal cells. The data indicate that ORG 2766 exerts differential trophic effects.

3311 Ultrastructure of human corneal nerves

w The human wrnea is a densely innervated struchxe. However, data on nerve fiber (NF) distribution in tbe human cornea are scarce possibly due to fast post mortem degeneration. Therefore, moat descriptions on comeal innervation are based on studies in rats and rabbits. Tbe present study is focussed on the ulaastruaure of NF’s in the central and peripheral human cornea. Methods: Tissue samples of five corneas obtained from melanoma eyes were processed for light and electron microscopy (EM). Both frontal and cross-sections of stroma and epitbelium were studied. Ultrathin stromal and ultrathin serial sections between Bowman’s Membrane (BM) and the basal &helium were cut. Stmmai and subepitbeliai axon diameters were n&wed on EM micrographs. Egulls; Unmyelinated NF’s containing both clear and dense cored vesicles run parallel to tbe collagen fibers in the upper third of the central stroma. They consist of up to 30 fibers (diam. 0.5-2.5 pm) which are ensheathed by thin rims of Schwann cell protrusions and amorphic matrix. Some of these NF’s, containing glycogen particles, invaginate the cytoplasm of the stmmal keratocytes. After passing through BM, central stromal NF’s (diam. 0.05-0.5 @II), run parallel between BM and epitbelium. At the location of the variwsities, fibers measure up to 2 pm due to the presence of many mitochondria. These fibers turn upwards and protrude mainly into the basal cells and sometimes into the wing cells. Reconstruction of NF’s in semithin sections revealed that subepitbelial fibers, having one or more bifurcations, run in a 12h-6h direction in the centre and in a 3h-9h direction in the periphery. In contrast to the central cornea, myelinated fibers were only found in the peripheral limbal stroma. Conclusion: NF’s invaginating epitbelial cells and keratocytes suggest that both epitbelium and stromal keratocytes are directly innervated. Tbe numemua mitochondria present in tbe varicosities point at fibers from sensory nerves. Therefore, the conclusion seems to be justified that the presence of clear and dense cored vesicles in NF’s of sSroma and subepithelium point at either an adrenergic or an cholinergic input from sensory origin.