Obese Mice with Dyslipidemia Exhibit Meibomian Gland Hypertrophy and Alterations in Meibum Composition and Aqueous Tear Production.

BACKGROUND: Dyslipidemia may be linked to meibomian gland dysfunction (MGD) and altered meibum lipid composition. The purpose was to determine if plasma and meibum cholesteryl esters (CE), triglycerides (TG), ceramides (Cer) and sphingomyelins (SM) change in a mouse model of diet-induced obesity where mice develop dyslipidemia. METHODS: Male C57/BL6 mice (8/group, age = 6 wks) were fed a normal (ND; 15% kcal fat) or an obesogenic high-fat diet (HFD; 42% kcal fat) for 10 wks. Tear production was measured and meibography was performed. Body and epididymal adipose tissue (eAT) weights were determined. Nano-ESI-MS/MS and LC-ESI-MS/MS were used to detect CE, TG, Cer and SM species. Data were analyzed by principal component analysis, Pearson's correlation and unpaired t-tests adjusted for multiple comparisons; significance set at p ≤ 0.05. RESULTS: Compared to ND mice, HFD mice gained more weight and showed heavier eAT and dyslipidemia with higher levels of plasma CE, TG, Cer and SM. HFD mice had hypertrophic meibomian glands, increased levels of lipid species acylated by saturated fatty acids in plasma and meibum and excessive tear production. CONCLUSIONS: The majority of meibum lipid species with saturated fatty acids increased with HFD feeding with evidence of meibomian gland hypertrophy and excessive tearing. The dyslipidemia is associated with altered meibum composition, a key feature of MGD.

Structure, development, and functional morphology of the cement gland of the giant danio, Devario malabaricus.

BACKGROUND: Aquatic species in several clades possess cement glands producing adhesive secretions of various strengths. In vertebrates, transient adhesive organs have been extensively studied in Xenopus laevis, other anurans, and in several fish species. However, the development of these structures is not fully understood. RESULTS: Here, we report on the development and functional morphology of the adhesive gland of a giant danio species, Devario malabaricus. We found that the gland is localized on the larval head, is composed of goblet-like secretory cells framed by basal, bordering, and intercalated apical epithelial cells, and is innervated by the trigeminal ganglion. The gland allows nonswimming larvae to adhere to various substrates. Its secretory cells differentiate by 12 hours postfertilization and begin to disappear in the second week of life. Exogenous retinoic acid disrupts the gland's patterning. More importantly, the single mature gland emerges from fusion of two differentiated secretory cells fields; this fusion is dependent on nonmuscle myosin II function. CONCLUSIONS: Taken together, our studies provide the first documentation of the embryonic development, structure, and function of the adhesive apparatus of a danioninae. To our knowledge, this is also the first report of a cement gland arising from convergence of two bilateral fields.

Dyslipidemia and Meibomian Gland Dysfunction: Utility of Lipidomics and Experimental Prospects with a Diet-Induced Obesity Mouse Model.

Meibomian gland dysfunction (MGD) is the leading cause of dry eye disease and loss of ocular surface homeostasis. Increasingly, several observational clinical studies suggest that dyslipidemia (elevated blood cholesterol, triglyceride or lipoprotein levels) can initiate the development of MGD. However, conclusive evidence is lacking, and an experimental approach using a suitable model is necessary to interrogate the relationship between dyslipidemia and MGD. This systematic review discusses current knowledge on the associations between dyslipidemia and MGD. We briefly introduce a diet-induced obesity model where mice develop dyslipidemia, which can serve as a potential tool for investigating the effects of dyslipidemia on the meibomian gland. Finally, the utility of lipidomics to examine the link between dyslipidemia and MGD is considered.

Corneal stroma microfibrils.

Elastic tissue was first described well over a hundred years ago and has since been identified in nearly every part of the body. In this review, we examine elastic tissue in the corneal stroma with some mention of other ocular structures which have been more thoroughly described in the past. True elastic fibers consist of an elastin core surrounded by fibrillin microfibrils. However, the presence of elastin fibers is not a requirement and some elastic tissue is comprised of non-elastin-containing bundles of microfibrils. Fibers containing a higher relative amount of elastin are associated with greater elasticity and those without elastin, with structural support. Recently it has been shown that the microfibrils, not only serve mechanical roles, but are also involved in cell signaling through force transduction and the release of TGF-ß. A well characterized example of elastin-free microfibril bundles (EFMBs) is found in the ciliary zonules which suspend the crystalline lens in the eye. Through contraction of the ciliary muscle they exert enough force to reshape the lens and thereby change its focal point. It is believed that the molecules comprising these fibers do not turn-over and yet retain their tensile strength for the life of the animal. The mechanical properties of the cornea (strength, elasticity, resiliency) would suggest that EFMBs are present there as well. However, many authors have reported that, although present during embryonic and early postnatal development, EFMBs are generally not present in adults. Serial-block-face imaging with a scanning electron microscope enabled 3D reconstruction of elements in murine corneas. Among these elements were found fibers that formed an extensive network throughout the cornea. In single sections these fibers appeared as electron dense patches. Transmission electron microscopy provided additional detail of these patches and showed them to be composed of fibrils (∼10 nm diameter). Immunogold evidence clearly identified these fibrils as fibrillin EFMBs and EFMBs were also observed with TEM (without immunogold) in adult mammals of several species. Evidence of the presence of EFMBs in adult corneas will hopefully pique an interest in further studies that will ultimately improve our understanding of the cornea's biomechanical properties and its capacity to repair.

A unique retinal epitheliopathy is associated with amyotrophic lateral sclerosis/Parkinsonism-Dementia complex of Guam.

BACKGROUND: The aim of this work was to examine whether a linear retinal pigment epitheliopathy is associated with the amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam. METHODS: A total of 918 Guamanian Chamorros, with and without amyotrophic lateral sclerosis/parkinsonism-dementia complex, were examined cross-sectionally for linear retinal pigment epitheliopathy (LRPE). Overall, 239 Guamanians, who were neurologically asymptomatic, were followed for up to 20 years to determine the risk of developing amyotrophic lateral sclerosis/parkinsonism-dementia complex. RESULTS: The epitheliopathy was present in 59.7% (117 of 196) patients with amyotrophic lateral sclerosis/parkinsonism-dementia complex, but in only 24.7% (178 of 722) of subjects who were neurologically asymptomatic (age- and sex-adjusted risk difference: 35.0%; 95% confidence interval [CI]: 27.5-42.6; p < 0.0001). Prospectively, 15 of 50 cases with epitheliopathy developed amyotrophic lateral sclerosis/parkinsonism-dementia complex, compared to 4 of 189 cases without epitheliopathy (age- and sex-adjusted hazard ratio: 13.1; 95% CI: 4.0-43.1; P < 0.0001). CONCLUSION: Amyotrophic lateral sclerosis/parkinsonism-dementia complex is associated with an LRPE and predicts future neurological disease. Identifying the cause of this retinopathy could provide an understanding about the pathogenesis of amyotrophic lateral sclerosis/parkinsonism-dementia complex and related diseases.

Integrin-dependent neutrophil migration in the injured mouse cornea.

As an early responder to an inflammatory stimulus, neutrophils (PMNs) must exit the vasculature and migrate through the extravascular tissue to the site of insult, which is often remote from the point of extravasation. Following a central epithelial corneal abrasion, PMNs recruited from the peripheral limbal vasculature migrate into the avascular corneal stroma. In vitro studies suggest PMN locomotion over 2-D surfaces is dependent on integrin binding while migration within 3-D matrices can be integrin-independent. Electron micrographs of injured mouse corneas show migrating PMNs make extensive surface contact not only with collagen fibrils in the extracellular matrix (ECM), but also keratocytes. Evidence supporting involvement of integrins in corneal inflammation has prompted research and development of integrin blocking agents for use as anti-inflammatory therapies. However, the role of integrin binding (cell-cell; cell-ECM) during stromal migration in the inflamed cornea has previously not been clearly defined. In this study in vivo time lapse imaging sequences provided the means to quantify cell motility while observing PMN interactions with keratocytes and other stromal components in the living eye. The relative contribution of ß1, ß2 and ß3 integrins to PMN locomotion in the inflamed mouse cornea was investigated using blocking antibodies against the respective integrins. Of the 3 integrin families (ß1, ß2 and ß3) investigated for their potential role in PMN migration, only ß1 antibody blockade produced a significant, but partial, reduction in PMN motility. The preferential migration of PMNs along the keratocyte network was not affected by integrin blockade. Hence, the dominant mechanism for PMN motility within the corneal stroma appears to be integrin-independent as does the restriction of PMN migration paths to the keratocyte network.

Protective role of murine ß-defensins 3 and 4 and cathelin-related antimicrobial peptide in Fusarium solani keratitis.

Antimicrobial peptides (AMPs), such as ß-defensins and cathelicidins, are essential components of innate and adaptive immunity owing to their extensive multifunctional activities. However, their role in fungal infection in vivo remains elusive. In this study, we investigated the protective effect of murine ß-defensin 3 (mBD3), mBD4, and the cathelicidin cathelin-related antimicrobial peptide (CRAMP) in a murine model of Fusarium solani keratitis. C57BL/6 mice showed significant corneal disease 1 and 3 days after infection, which was accompanied by enhanced expression of ß-defensins and CRAMP. Disease severity was significantly improved 7 days after infection, at which time AMP expression was returning to baseline. Mice deficient in mBD3 (genetic knockout), mBD4 (short interfering RNA knockdown), or CRAMP (genetic knockout) exhibited enhanced disease severity and progression, increased neutrophil recruitment, and delayed pathogen elimination compared to controls. Taken together, these data suggest a vital role for AMPs in defense against F. solani keratitis, a potentially blinding corneal disease.

Assessment of postnatal corneal development in the C57BL/6 mouse using spectral domain optical coherence tomography and microwave-assisted histology.

The eyes of newborn mice are relatively underdeveloped and the lids remain closed for the first 2 weeks after birth. There after the eyes undergo a period of rapid growth for several weeks. Eventually the eyes reach an age at which many ocular structures stabilize for the remainder of the animal's life, or for others, growth is significantly slowed. The central corneal thickness (CCT) is a parameter commonly reported in corneal studies. However there is a large discrepancy in values reported for adult mice as well as a lack of comprehensive values covering the time from birth through adulthood. In this study we report, for the first time, the use of spectral domain optical coherence tomography (SD-OCT) for in situ and in vivo determination of CCT from P0 to P250 for C57BL/6 mice. SD-OCT provided a reliable measure of CCT and we fit the data to an exponential rise to maximum growth curve resulting in a value of 49 µm for P0 and a maximum adult value of 106 µm. By comparison, corneas processed for conventional histology produced CCT values approximately 30-35% thicker and with greater variability. Ex vivo real-time imaging during fixation revealed swelling and gross distortion of the cornea beginning after only 10-15 min in fixative. The fixation artifacts were not observed when the cornea was processed using an optimized microwave fixation protocol. CCT values measured in corneas fixed with the microwave process compared favorably with values obtained with SD-OCT. We conclude that for corneal research, mice younger than 8 weeks of age should not be considered as adults since they are still in a rapid phase of growth up until that time. In addition we report the first use of microwave processed histological specimens for visualizing the murine cornea. Tissue processed in this manner has minimal artifacts, a CCT equivalent to that measured in vivo by SD-OCT and ultrastructural detail comparable to conventional fixation methods.

Designed Host Defense Peptides for the Treatment of Bacterial Keratitis.

Purpose: To limit corneal damage and potential loss of vision, bacterial keratitis must be treated aggressively. Innovation in antimicrobials is required due to the need for empirical treatment and the rapid emergence of bacterial resistance. Designed host defense peptides (dHDPs) are synthetic analogues of naturally occurring HDPs, which provide defense against invading pathogens. This study investigates the use of novel dHDPs for the treatment of bacterial keratitis. Methods: The minimum inhibitory concentrations (MICs) were determined for dHDPs on both Gram-positive and -negative bacteria. The minimum biofilm eradication concentrations (MBEC) and in vitro time-kill assays were determined. The most active dHDP, RP444, was evaluated for propensity to induce drug resistance and therapeutic benefit in a murine Pseudomonas aeruginosa keratitis model. Results: Designed HDPs were bactericidal with MICs ranging from 2 to >64 µg/mL and MBEC ranging from 6 to 750 µg/mL. In time-kill assays, dHDPs were able to rapidly reduce bacterial counts upon contact with as little as 2 µg/mL. RP444 did not induce resistance after repeated exposure of P. aeruginosa to subinhibitory concentrations. RP444 demonstrated significant efficacy in a murine model of bacterial keratitis as evidenced by a significant dose-dependent decrease in ocular clinical scores, a significantly reduced bacterial load, and substantially decreased inflammatory cell infiltrates. Conclusions: Innovative dHDPs demonstrated potent antimicrobial activity, possess a limited potential for development of resistance, and reduced the severity of murine P. aeruginosa keratitis. These studies demonstrate that a novel dHDP may have potential to treat patients with sight-threatening bacterial keratitis.

Effects of Topically Applied Vitamin D during Corneal Wound Healing.

Vitamin D is an important regulator of immune function and largely acts to dampen chronic inflammatory events in a variety of tissues. There is also accumulating evidence that vitamin D acts to enhance initial inflammation, beneficial during both infection and wound healing, and then promotes resolution and prevention of chronic, damaging inflammation. The current study examines the effect of topical vitamin D in a mouse of model of corneal epithelial wound healing, where acute inflammation is necessary for efficient wound closure. At 12 and 18 hours post-wounding, vitamin D treatment significantly delayed wound closure by ~17% and increased infiltration of neutrophils into the central cornea. Basal epithelial cell division, corneal nerve density, and levels of VEGF, TGFß, IL-1ß, and TNFα were unchanged. However, vitamin D increased the production of the anti-microbial peptide CRAMP 12 hours after wounding. These data suggest a possible role for vitamin D in modulating corneal wound healing and have important implications for therapeutic use of vitamin D at the ocular surface.

Toll-like receptor expression and activation in mice with experimental dry eye.

PURPOSE: To investigate the expression and/or function of toll-like receptors (TLRs) and antimicrobial peptides (AMPs) in dry eye inflammation. METHODS: Experimental dry eye (EDE) was induced in C57BL/6 mice and TLR mRNA and protein expression were determined at the ocular surface and lacrimal gland. TLR agonist cocktail was applied to the ocular surface in untreated (UT), corneal scratched, and EDE mice. The corneal expression of cathelin-related antimicrobial peptide (CRAMP; human LL-37 orthologue), and mouse beta defensin (mBD)-3 and -4 (human BD-2 orthologue) was compared. LL-37, hBD-2, TLR4, 5, and TLR9 mRNA expression was examined in patients with dysfunctional tear syndrome (DTS) via conjunctival impression cytology. Murine central corneal thickness (CCT) and inflammatory cell recruitment into the stroma was determined by in vivo imaging. RESULTS: EDE upregulated TLR2-4 and 9 mRNA expression in the palpebral conjunctiva and with the exception of TLR4, a similar expression, occurred in the corneal epithelium. TLR2 and 5 were upregulated in lacrimal gland and overall, there was a corresponding change in TLR protein. EDE decreased CRAMP mRNA and protein. hBD-2 and TLR9 expression were modulated in DTS subjects. Topical TLR agonist increased inflammatory cells recruitment and CCT in mice with a cornea scratch. In EDE, TLR agonist treatment downregulated corneal mBD-4 protein caused corneal epithelial loss, and stromal ulceration resulting in decreased CCT. CONCLUSIONS: DTS modulates the expression of TLR and CRAMP and topical application of TLR agonists in EDE mice resulted in corneal epithelial loss and thinning. These results suggest that TLRs are involved in DTS inflammation.