Johanna Tukler Henriksson

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.

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.

IL-13 Stimulates Proliferation and Expression of Mucin and Immunomodulatory Genes in Cultured Conjunctival Goblet Cells.

PURPOSE To investigate the effects of IL-13 on goblet cell proliferation, differentiation, and expression of mucin and immunomodulatory genes. METHODS Explants were excised from the conjunctiva of young C57BL/6 mice. Cultures received 200 μL per week of either Keratinocyte media (KSFM) or KSFM supplemented with 10 ng/mL IL-13 and were incubated for 3 (D3), 7 (D7), or 14 (D14) days. Subsequently, cell proliferation was assessed or cultures were immunostained, collected for dot blot, or for reverse transcription (RT) and quantitative real-time PCR (qPCR) or for RT-PCR gene array. RESULTS The cultured conjunctival epithelium expressed goblet cell associated keratin 7 and mucins MUC5AC and MUC2 and when stimulated with IL-13 showed increased proliferation at D3 and D7 (P < 0.05) compared with control. MUC5AC expression was increased in the IL-13-treated group at D3 and D14 (P < 0.05). IL-13-treated cultures showed increased chemokine ligand 26 (CCL26), chloride channel calcium activated channel 3 (CLCA3), fas ligand (FasL), and Relm-β at D7. All conjunctival cultures expressed MUC2, and its expression was decreased at D3 (P < 0.05) and increased at D14 (P < 0.05) with IL-13 treatment. CONCLUSIONS This study demonstrated that conjunctival goblet cells are IL-13 responsive cells that produce factors known to maintain epithelial barrier, stimulate mucin production, and modulate immune response in nonocular mucosa when treated with IL-13. The functional significance of IL-13-stimulated factors remains to be determined.

Dexamethasone nanowafer as an effective therapy for dry eye disease

Dry eye disease is a major public health problem that affects millions of people worldwide. It is presently treated with artificial tear and anti-inflammatory eye drops that are generally administered several times a day and may have limited therapeutic efficacy. To improve convenience and efficacy, a dexamethasone (Dex) loaded nanowafer (Dex-NW) has been developed that can release the drug on the ocular surface for a longer duration of time than drops, during which it slowly dissolves. The Dex-NW was fabricated using carboxymethyl cellulose polymer and contains arrays of 500 nm square drug reservoirs filled with Dex. The in vivo efficacy of the Dex-NW was evaluated using an experimental mouse dry eye model. These studies demonstrated that once a day Dex-NW treatment on alternate days during a five-day treatment period was able to restore a healthy ocular surface and corneal barrier function with comparable efficacy to twice a day topically applied dexamethasone eye drop treatment. The Dex-NW was also very effective in down regulating expression of inflammatory cytokines (TNF-α, and IFN-γ), chemokines (CXCL-10 and CCL-5), and MMP-3, that are stimulated by dry eye. Despite less frequent dosing, the Dex-NW has comparable therapeutic efficacy to topically applied Dex eye drops in experimental mouse dry eye model, and these results provide a strong rationale for translation to human clinical trials for dry eye.

Ultraviolet radiation transmittance of the mouse eye and its individual media components

Recently, the mouse has become the preferred animal model in ophthalmic research. Therefore, there is a need for enhanced understanding of the mouse eye to validate its use in different experimental setting. The purpose of this study was to determine the ocular transmittance of the whole mouse eye, the cornea and the crystalline lens, particularly in the ultraviolet radiation (UVR) wavebands. This was carried out using a non-cuvette based fiber optic spectrometer system and the resulting transmittance curves were compared with published cone spectral response curves and mouse ocular transmittance data. First, transmittance curves of the whole mouse eye were measured by removing a small disc of sclera from the posterior pole to provide an anterior to posterior optical path. No statistical difference was found between left and right eye in each of the four mice sampled, therefore, all eight eyes were included in the final statistical analysis. The average of five test measurements from each left and right eye for the four test mice showed a transmittance cut off at approximately 310 nm. Secondly, the cornea with a scleral rim was excised and transmittance curves obtained for all eight eyes. This data showed an average transmittance cut off at 280 nm for the cornea. Similarly measured data for the excised crystalline lens showed UVR transmittance down to 310 nm. The good correlation between total ocular UVR transmittance and the sum of the individually measured components (i.e. the cornea and the crystalline lens) supported the validity of our method and its findings. This experiment demonstrated that the mouse cornea transmits more UV-B (280-315 nm) than the rabbit and the human corneal transmittance. The mouse crystalline lens on the other hand showed a cut off in the UV-B at 310 nm, which is at a much lower UV-B wavelength than the approximate UV-A (315-400 nm) cut off for the human crystalline lens at around 390 nm. The increased transmittance of UVR in the mouse eye serves its vision, since the mouse has a cone photopigment peaking at approximately 350 nm. Due to the above stated differences between the mouse and the human it is concluded that the mouse is not an ideal model for the human eye in experiments involving UVR.

Morphologic alterations of the palpebral conjunctival epithelium in a dry eye model.

Purpose: To investigate the normal palpebral conjunctival histology in C57BL/6 mice and the structural changes that occur in a dry eye model. Methods: Twenty-four male and female C57BL/6 mice, 8 untreated and 16 exposed to experimental ocular surface desiccating stress (DS). Ocular dryness was induced by administration of scopolamine hydrobromide (0.5 mg/0.2 mL) four times a day for 5 days (DS5) or 10 days (DS10). Counts and measurements were obtained using anatomical reference points, and goblet cell density was investigated with a variety of stains. Results: Near the junction between the lid margin and the normal palpebral conjunctiva, the epithelium had an average thickness of 45.6 ± 10.5 &mgr;m, 8.8 ± 2.0 cell layers, versus 37.7 ± 5.6 &mgr;m, 7.4 ± 1.3 layers in DS10 (P < 0.05). In the goblet cell–populated palpebral region, the normal epithelium was thicker (P < 0.05) than on DS5 and DS10. In the control, 43% of the goblet cells were covered by squamous epithelium compared with 58% (DS5) and 63% (DS10) (P < 0.05). A decreased number of periodic acid–Schiff (PAS)–stained goblet cells and Alcian blue–stained goblet cells were observed in the dry eye. Not all goblet cells were stained with PAS and Alcian blue. Conclusion: The mouse palpebral conjunctival epithelium was structurally similar to the human. After DS, the palpebral conjunctival epithelium decreased in thickness and goblet cell access to the surface seemed to be inhibited by surrounding epithelial cells, potentially slowing down their migration to the surface. Differential staining with PAS and Alcian blue suggests that there may be different subtypes of conjunctival goblet cells.

An explanation for the central to peripheral thickness variation in the mouse cornea

Background:  The mouse corneal stroma varies in thickness across its diameter. The purpose of the present study was to explain this variation and to advance our understanding of stromal lamellar architecture in the mammalian cornea.

Interferon-gamma deficiency protects against aging-related goblet cell loss

Aging is a well-recognized risk factor for dry eye. Interferon-gamma (IFN-γ) has been implicated in conjunctival keratinization and goblet cell loss in dry eye. We investigated the role of IFN-γ in age-related dry eye by evaluating young (8 weeks) and aged (15 months; 15M) C57BL/6 (B6) and IFN-γKO mice. Age effects on the conjunctiva and cornea epithelium were assessed with PAS staining and corneal staining, respectively. Expression of T cell-related cytokines (IL-17A, IFN-γ), chemokines (CXCL10 and CCL20), in the ocular surface epithelium was evaluated by real time PCR. A significant decrease in filled goblet cells was noted in 15M B6 mice and this was significantly lower than age and sex-matched IFN-γKO mice. Aged male B6 had significantly higher IFN-γ, and CXCL10 mRNA in their conjunctiva than female B6 mice. Aged IFN-γKO females had significantly higher IL-17A mRNA in conjunctiva than IFN-γKO males and B6 mice. Corneal barrier dysfunction was observed in 15M female B6 and aged IFN-γKO mice of both sexes; however it was significantly higher in IFN-γKO compared to B6 mice. While there was a significant increase in IL 17A, and CCL20 in corneas of aged female B6 and IFN-γKO mice compared to males, these changes were more evident in aged female IFN-γKO group. Partial resistance of IFN-γKO mice to aging-induced goblet cell loss indicates IFN-γ is involved in the age-related decline in conjunctival goblet cells. Increased corneal IL-17A expression paralleled corneal barrier disruption in aging female of both strains. IFN-γ appears to suppress IL-17A on the ocular surface.