Jan P.G. Bergmanson

Ultrastructural analysis of corneal exposure to UV radiation

Abstract. The primate cornea was exposed to 300 nm UVR with five levels of radiant exposure from 0.08 to 0.6 Jcm−2. All cellular layers of the cornea were damaged at the 0.08 Jcm−2 exposure, and damage became more severe as the exposure level was increased. The corneal cells showed variable response in that essentially normal cells were found among damaged cells. Eight days post‐exposure using the 0.6 Jcm−2 level, the epithelium had regained its normal thickness and was populated largely by normal appearing cells; however, the stroma showed damaged keratocytes and the loss of keratocytes. The corneal basement membranes (the epithelial basement membrane and the posterior limiting lamina) and the anterior limiting lamina were not damaged at any exposure level except for an isolated area along the epithelial basement membrane in one cornea. Therefore, one is lead to conclude that basement membranes are unaffected by UVR. The endothelium continued to demonstrate the loss of mitochondria, endoplasmic reticulum and some vacuoles at 8 days after exposure. However, the endothelium appeared to have resumed its physiological function as demonstrated by the reduced stromal oedema. This research gives the first complete description of UV‐B induced corneal damage and repair of the full, in‐depth cornea of the primate using the EM.

Dimensions and Morphology of the Cornea in Three Strains of Mice

PURPOSE To use a histologic approach to obtain dimensional and morphologic information on the cornea in three commonly used strains of mice. METHODS Adult mice (three each of 129/SVJ, C57BL/6, and BALB/c) were euthanatized, and the eyes were enucleated, immersed in 2% glutaraldehyde fixative, and prepared for light and transmission electron microscopy. The full corneal, epithelial, stromal, and posterior limiting lamina (PLL) with endothelium thicknesses were measured at the same location centrally and peripherally. RESULTS All three strains showed a statistically significant (P < 0.001) decrease in overall thickness in the peripheral compared with the central cornea. The decrease was due to a reduced thickness of both the epithelium and the stroma. The stroma and epithelium contributed to approximately two thirds and one third of the total corneal thickness, respectively. The epithelium had the classic stratified layout and consisted of 13.00 +/- 1.41 layers centrally versus 10.33 +/- 1.37 peripherally. Some adaptation of stromal tissue was found immediately adjacent to the epithelial basement membrane, but a clearly defined anterior limiting lamina did not exist. The stroma was organized into lamellae but lacked the anterior branching and interweaving reported in humans and had unmyelinated nerve fibers within micrometers of the endothelium. The PLL was 2.17 +/- 0.3 microm thick and was divided into pre- and postnatal layers, with striated bodies in the postnatal portion. CONCLUSIONS This study demonstrated that in the three strains of mice examined, the cornea becomes significantly thinner toward the periphery. Dimensionally, proportionally, and anatomically the three strains used appeared to be similar. However, morphologic differences were observed compared with other mammals, and awareness of these differences is important when using the mouse as an animal model applicable to the human.

Shrinkage and distortion of the rabbit corneal endothelial cell mosaic caused by a high osmolality glutaraldehyde-formaldehyde fixative compared to glutaraldehyde

The purpose of this study was to quantitatively compare cell dimensions and cell layer organization of the corneal endothelium after chemical fixation. Rabbit corneas (9-10 weeks of age) were prepared immediately postmortem for transmission electron microscopy (TEM) and scanning electron microscopy (SEM) using either a widely used high osmolality fixative (glutaraldehyde-formaldehyde, after Karnovsky; 1% formaldehyde, 2.5% glutaraldehyde, 0.1 M cacodylate, pH 7.6, 850 mOsm/kg) or a glutaraldehyde fixative (2% glutaraldehyde in 80 mM cacodylate, pH 7.4, 330 mOsm/kg). With the glutaraldehyde-formaldehyde fixative, TEM revealed gross shrinkage (up to 40%) and distortion of the cytoplasm and organelles, while the regions of the cell-cell junctions were not attenuated but included dilated extracellular space. With the glutaraldehyde fixative, TEM also revealed shrinkage but the cytoplasm was less compact than with the high osmolality fixative. The overall cell shrinkage and relative accentuation of the cell-cell borders was confirmed by SEM, which also revealed that the 11 to 20% area shrinkage was also related to the number of cell sides.

Quantified Histopathology of the Keratoconic Cornea

Purpose. This study systematically investigated and quantified histopathological changes in a series of keratoconic (Kc) corneas using a physiologically formulated fixative to not further distort the already distorted diseased corneas. Methods. Twelve surgically removed Kc corneal buttons were immediately preserved and processed for light and transmission electron microscopy using an established corneal protocol. Measurements were taken from the central cone and peripheral regions of the host button. The sample size examined ranged in length from 390 to 2608 &mgr;m centrally and 439 to 2242 &mgr;m peripherally. Results. The average corneal thickness was 437 &mgr;m centrally and 559 &mgr;m peripherally. Epithelial thickness varied centrally from 14 to 92 &mgr;m and peripherally from 30 to 91 &mgr;m. A marked thickening of the epithelial basement membrane was noted in 58% of corneas. Centrally, anterior limiting lamina (ALL) was thinned or lost over 60% of the area examined, whereas peripheral cornea was also affected but to a lesser extent. Histopathologically, posterior cornea remained undisturbed by the disease. Anteriorly in the stroma, an increased number of cells and tissue debris were encountered, and some of these cells were clearly not keratocytes. Conclusions. It is concluded that Kc pathology, at least initially, has a distinct anterior focus involving the epithelium, ALL, and anterior stroma. The epithelium had lost its cellular uniformity and was compromised by the loss or damage to the ALL. The activity of the hitherto unreported recruited stromal cells may be to break down and remove ALL and anterior stromal lamellae, leading to the overall thinning that accompanies this disease.

Entrapment of Conjunctival Goblet Cells by Desiccation-Induced Cornification

PURPOSE To evaluate the effects of desiccating stress on conjunctival goblet cell density and morphology and the expression of cornified envelope precursors by the ocular surface epithelia. METHODS Experimental dry eye (EDE) was created in C57BL/6 mice. Real-time PCR evaluated the expression of cornified envelope (CE) precursor proteins (involucrin and small proline-rich [Sprr] -1a, -1b, -2a, -2b, -2f, and -2g proteins), the cross-linking transglutaminase 1 enzyme (Tg-1) and Muc5AC mRNA transcripts by the ocular surface epithelia. Laser scanning confocal microscopy evaluated the expression of the CE precursor proteins Tg-1 and Muc5AC in cryosections. Tg-1 activity was measured by a fluorescein cadaverine assay. Muc5AC concentration was measured by ELISA. RESULTS Levels of involucrin; Sprr-1a, -1b, -2a, -2b, -2f, and -2g; and Tg1-1 mRNA transcripts in ocular surface tissues increased in response to desiccating stress. Expression and activity of Tg in the conjunctiva markedly increased after EDE. Desiccating stress caused progressive loss of mucin-filled goblet cells. The apical portion of the remaining conjunctival goblet cells became entrapped by adjacent stratified apical epithelia expressing increased levels of cornified envelope precursors. CONCLUSIONS Exposure to desiccating stress stimulates ocular surface epithelia to produce cornified envelope precursors and the tissue transglutaminase enzyme that cross-links them. This effect is accompanied by loss of mucin-filled goblet cells and entrapment of mucin contents in the remaining ones by cornifying cells that block the egress of mucin contents to the ocular surface. This mechanism may contribute to the conjunctival mucin deficiency that develops in dry eye.

The efficacy of a UV‐blocking soft contact lens in protecting cornea against UV radiation

Abstract. Recently, it has been shown that UV keratitis is more serious than previously thought because it is not limited to the corneal epithelium but also involves the stroma and the endothelium. It is, therefore, very important to avoid ultraviolet radiation (UVR) damage, and the purpose of the present study was to examine the ability of a UV absorbing hydrogel lens to filter such wavelengths. Ultrastructural observations of rabbit corneas showed that a regular soft (Vistamarc normal) contact lens offered no protection, and since wearing one adds stress to the cornea it seems possible that the cornea may be more vulnerable to UVR trauma. A UV absorbing soft contact lens (Vistakon UV‐BLOC) provided complete protection to all corneal layers. Subepithelial nerve fibres in the traumatized corneas were mostly of normal morphology and may explain why UV keratitis is a very painful experience. It was concluded that a UV absorbing soft contact lenses with acceptable clinical performance may be prescribed on a routine basis.

The diagnosis and characteristics of moderate dry eye in non-contact lens wearers

Purpose. To identify and characterize moderate dry eye in non–contact lens wearers with a new scoring system-based dry eye questionnaire and to determine which objective tests better differentiate patients with moderate dry eye from healthy patients. Methods. Fifty-two healthy subjects (21 women and 31 men with a mean age of 27.8 ± 9.2 years) and 37 subjects with moderate dry eye (33 women and 4 men with a mean age of 36.4 ± 12.9 years) completed a 42-item dry eye questionnaire. Seventeen healthy subjects (11 women and 6 men with a mean age of 30.5 ± 9.7 years) and 28 subjects with moderate dry eye (24 women and 4 men with a mean age of 38.50 ± 3.8 years) underwent additional objective assessment of ocular surface health, tear osmolality, tear stability, and tear volume. Results. Subjects with moderate dry eye scored significantly higher (49.8 ± 20.3, P<0.0001) on the dry eye questionnaire than did normal subjects (11.7 ± 10.3). Ocular irritation symptoms worsened with progression of time of day in both groups of subjects. Internal reliability (0.95 Cronbach α) was excellent, and concurrent validity (Spearman ρ 0.507) was acceptable when compared to the McMonnies and Ho dry eye questionnaire. Significant differences in tear osmolality (P<0.00001), invasive tear breakup time (P<0.034), and corneal vital dye staining (P<0.0001) were detected between the two groups of subjects. A stepwise linear regression on objective clinical tests, however, did not account for 77% of the total variance in the questionnaire scores. Conclusions. A unique scoring system-based dry eye questionnaire was validated to separate non–contact lens wearers with moderate dry eye from healthy subjects. Objective tests of tear osmolality and stability and ocular surface integrity were better than other clinical measures at identifying differences between the two subject groups. The results strongly support the evidence that the diagnosis and treatment of moderate dry eye requires a detailed assessment of self-perceived symptoms and that objective clinical testing alone may be insufficient.

Corneal damage in photokeratitis--why is it so painful?

The primate cornea was examined ultrastructurally subsequent to ultraviolet radiation (UVR) (300 nm) exposures of 0.08 and 0.225 J/cm2. The lower exposure caused significant, but incomplete, destruction of the epithelium and mild edema of the posterior stroma. The higher exposure resulted in complete destruction of the epithelium, significant stromal swelling, and some decomposition of endothelial cells. Widespread intracellular edema of the endothelium was also noted with the higher exposure. The ultrastructural examination subsequent to these UVR exposures also helps to explain why UV-induced keratitis is so painful. The 0.08 J/cm2 exposure left the epithelial and subepithelial-stromal nerve plexuses intact, whereas the 0.225 J/cm2 exposure destroyed the epithelial axons but spared the subepithelial ones. These results suggest that the pain associated with UV keratitis results from the combined effect of epithelial cell loss and sparing of the subepithelial axons.

Quantification of the ultraviolet radiation (UVR) field in the human eye in vivo using novel instrumentation and the potential benefits of UVR blocking hydrogel contact lens.

BACKGROUND/AIMS Certain degenerative eye conditions occur predominantly nasally, at the limbal region, and are associated with solar ultraviolet radiation (UVR) induced damage. The relative contribution to the in vivo ocular flux of (a) the reflection of UVR incident on the skin of the nose onto the nasal limbus, and (b) the focusing of UVR incident on the temporal side of the cornea onto the nasal limbus were examined. METHODS A novel photodiode sensor array was used to measure the UVR field across the eye. In addition, a novel spectrometer set-up was used to measure the spectrum of radiation refracted across the cornea. The efficacy of UVR blocking hydrogel contact lenses in filtering incident UVR was assessed in vivo. RESULTS Qualitative and quantitative data indicated an increase nasally of UVR. Photodiode readings showed a net UVR increase from the temporal to the nasal side. Transmission curves showed that most UVR incident on the limbal region is either absorbed by, or transmitted through, the ocular tissues. This radiation is filtered by UVR blocking soft contact lens. CONCLUSIONS An increased UVR flux on the nasal side of the eye, due to reflection off the nasal skin, was identified in vivo. Any UVR passing through the cornea is either absorbed by the conjunctiva and/or transmitted through it onto the sclera where it is absorbed. UVR blocking hydrogel contact lenses can eliminate these sources of UVR.

Histopathological analysis of corneal endothelial polymegethism.

The corneal endothelium from six humans (three contact lens wearers and three non-contact lens wearers) was examined. Scanning electron microscopy confirmed endothelial polymegethism. Observations with the transmission electron microscope showed that an oblique reorientation of the lateral walls of the endothelial cells had occurred in the contact-lens-wearing group of corneas. The endothelium of the contact lens wearers also showed some inter- and intracellular edema but were otherwise of a healthy appearance containing normal organelles. The oblique reorientation of the lateral wall of the endothelial cell allows for the possibility that a cell with a large anterior surface area may have a small posterior surface area or vice versa. Thus, polymegethous cells may not vary in cell volume. This hypothetical alternative to current theories of contact lens-induced polymegethism should be explored in future research.