UVR-B-induced NKR-1 Expression in Ocular Tissues is blocked by Substance P Receptor Antagonist Fosaprepitant in the Exposed as well as Unexposed Partner Eye.

Purpose: To investigate the effect of NKR-1 antagonists in an established UVR-B-induced cataract mouse model. Furthermore, to examine the expression of pro-inflammatory cytokines/chemokines in mouse eyes following unilateral UVR-B exposure.Methods: Mice received intraperitoneally injections of Fosaprepitant and Spantide I, before and after unilateral exposure to UVR-B. After day 3 and 7 post-exposure, ocular tissues were extracted for the detection of NKR-1 protein level by ELISA.Results: Pretreatment with Fosaprepitant decreases NKR-1 expression in exposed ocular tissues as well as in the unexposed lens epithelium compared to the saline group. Spantide I treatment showed a tendency of NKR-1 overexpression in ocular tissues.Conclusion: The clinically approved NKR-1 receptor antagonist Fosaprepitant decreases NKR-1 protein expression effectively not only in the exposed but also in the unexposed partner eye in a UVR-B irradiation mouse model. No effect was seen on the protein concentration of pro-inflammatory cytokines/chemokines in either eye.

Ultraviolet Radiation Exposure of One Eye Stimulates Sympathizing Expression of Neurokinin-1 Receptor but Not Monocyte Chemoattractant Protein-1 in the Partner Eye.

PURPOSE: To investigate the influence of unilateral ultraviolet radiation (UVR) exposure on the unexposed, partner eye in vivo. To characterize the immunological cross-talk between the eyes and verify a sympathizing reaction of the partner eye via a neurokinin-dependent signaling pathway of substance P and its neurokinin-1 receptor (NKR-1) and/or monocyte chemoattractant protein-1 (MCP-1). METHODS: C57BL/6 mice were unilaterally exposed in vivo to UVR-B to a 5-fold cataract threshold equivalent dose of 14.5 kJ/m2 with a UV irradiation Bio-Spectra system. The unexposed contralateral eye was completely shielded during irradiation. After 3 and 7 days post exposure, eyes were stained with fluorescence-coupled antibody for substance P NKR-1. The same was performed in control animals receiving only anesthesia but no UVR-B exposure. NKR-1 and MCP-1 levels in ocular tissue lysates were quantified by enzyme-linked immunosorbent assay. RESULTS: UVR-B induces NKR-1 upregulation after 3 and 7 days in the exposed and in the unexposed, contralateral mouse eye. NKR-1 protein level was upregulated in the exposed and contralateral iris/ciliary body complex, choroidea and in the contralateral retina as well as in the exposed cornea. MCP-1 levels were elevated in the exposed cornea, iris/ciliary body complex, and aqueous humor but not in contralateral ocular tissues. CONCLUSIONS: UVR-B exposure triggers NKR-1 upregulation not only in the exposed but also in the unexposed, partner eye in various ocular tissues. Following UVR-B exposure, MCP-1 protein levels are upregulated in the exposed eye, but the contralateral side remains unaffected.

Ultraviolet radiation exposure triggers neurokinin-1 receptor upregulation in ocular tissues in vivo.

The purpose of this study was to investigate the neurokinin receptor-1 (NKR-1) protein expression in ocular tissues before and after supra-cataract threshold ultraviolet radiation (UVR-B peak at 312 nm) exposure in vivo in a mouse model. Six-week-old C57Bl/6 mice were unilaterally exposed to a single (2.9 kJ/m2) and an above 3-fold UVR-B cataract threshold dose (9.4 kJ/m2) of UVR. UVR-exposure (λpeak = 312 nm) was performed in mydriasis using a Bio-Spectra exposure system. After latency periods of 3 and 7 days, eyes were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and stained with fluorescence coupled antibody for NKR-1 and DAPI for cell nuclei staining. Control animals received only anesthesia but no UVR-exposure. Cataract development was documented with a Leica dark-field microscope and quantified as integrated optical density (IOD). NKR-1 is ubiquitously present in ocular tissues. An above 3-fold cataract threshold dose of UV-radiation induced NKR-1 upregulation after days 3 and 7 in the epithelium and endothelium of the cornea, the endothelial cells of the iris vessels, the pigmented epithelium/stroma of the ciliary body, the lens epithelium, pronounced in the nuclear bow region and the inner plexiform layer of the retina. A significant upregulation of NKR-1 could not be provoked with a single cataract threshold dose (2.9 kJ/m2 UVR-B) ultraviolet irradiation. All exposed eyes developed anterior subcapsular cataracts. Neurokinin-1 receptor is present ubiquitously in ocular tissues including the lens epithelium and the nuclear bow region of the lens. UV-radiation exposure to an above 3-fold UVR-B cataract threshold dose triggers NKR-1 upregulation in the eye in vivo. The involvement of inflammation in ultraviolet radiation induced cataract and the role of neuroinflammatory peptides such as substance P and its receptor, NKR-1, might have been underestimated to date.

Effect of Viscous Agents on Corneal Density in Dry Eye Disease.

PURPOSE: To investigate the effect of the viscous agents, hydroxypropyl methylcellulose (HPMC), carbomer, povidone, and a combination of HPMC and povidone on corneal density in patients with dry eye disease. METHODS: In total, 98 eyes of 49 patients suffering from dry eye and 65 eyes of 33 healthy age-matched individuals were included in this prospective, randomized study. Corneal morphology was documented with Scheimpflug photography and corneal density was analyzed in 5 anatomical layers (epithelium, bowman membrane, stroma, descemet's membrane, and endothelium). Corneal density was evaluated for the active ingredients HPMC, carbomer, povidone, and a combination of HPMC and povidone as the viscous agents contained in the artificial tear formulations used by the dry eye patients. Data were compared to the age-matched healthy control group without medication. RESULTS: Corneal density in dry eye patients was reduced in all 5 anatomical layers compared to controls. Corneal density was highest and very close to control in patients treated with HPMC containing ocular lubricants. Patients treated with lubricants, including carbomer as the viscous agent displayed a significant reduction of corneal density in layers 1 and 2 compared to control. CONCLUSION: HPMC containing ocular lubricants can help to maintain physiological corneal density and may be beneficial in the treatment of dry eye disease.

Ultrastructure of UVR-B-induced cataract and repair visualized with electron microscopy.

PURPOSE: The aim of the study is to investigate and visualize the ultrastructure of cataract morphology and repair, after in vivo exposure to double threshold dose UVR-B in the C57BL/6 mouse lens. METHODS: Twenty-six-week-old C57BL/6 mice received in vivo double threshold dose (6.4 kJ/m2) UVR-B for 15 min. The radiation output of the UVR-source had λMAX at 302.6 nm. After a latency period of 1, 2, 4 and 8 days following UVR-B exposure, the induced cataract was visualized with electron microscopy techniques. Induced, cataract was quantified as forward lens light scattering. Damage to the lens epithelium and the anterior cortex was investigated with light microscopy in toluidine blue-stained semi-thin sections, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and dark field illumination photography. RESULTS: UVR-B-exposed lenses developed anterior subcapsular and/or cortical and nuclear cataract after 1 day. Lens light scattering peaked 2 days after exposure. Lens epithelial cell damage was seen in TEM as apoptotic cells, apoptotic bodies, nuclear chromatin condensation, and swollen and disrupted anterior cortex fibres throughout the sections of the whole anterior lens surface. These morphologic changes were also visualized with SEM. Within 8 days, anterior subcapsular cataract was repaired towards the anterior sutures. CONCLUSION: UVR-B exposure of double cataract threshold dose induces a subtotal loss of epithelial cells across the whole anterior surface of the lens. This damage to the epithelium is repaired by epithelial cell movement from the equator towards the lens sutures, thus in retrograde direction to regular epithelial cell differentiation.

Localisation and significance of in vivo near-infrared autofluorescent signal in retinal imaging.

AIM: To evaluate near-infrared (NIR) autofluorescence (AF) in patients with geographic atrophy (GA) secondary to age-related macular degeneration and to investigate the origin of the signal by in vivo and histological analysis in rats and in a human donor eye. METHODS: Confocal scanning laser ophthalmoscopy in vivo imaging, including blue (excitation: 488 nm, emission 500-700 nm) and NIR (excitation: 790 nm, emission >810 nm) AF was performed in 21 eyes of 18 GA patients. Pigmented and albino rats underwent with the same device both in vivo and post-mortem imaging. For the latter, cryostat prepared retinal cross-sections were imaged using an additional customised magnification lens. Finally, cross-sections of a 49-year old human donor eye were recorded. RESULTS: Atrophic areas in GA were characterised by low NIR AF intensities. In the junctional zone of atrophy, focal areas of increased intensity were seen which appeared to seldom correlate to blue AF findings. Confocal live scanning in pigmented rats identified the maximum of the NIR AF signal in the outer retina, with histological confirmation of the signal origin localised to the retinal pigment epithelium and sclera in both animals and human donor eye. No NIR AF was found in the retina of young non-pigmented rats. DISCUSSION: This study further underscores the assumption that melanin is the main source of NIR AF in the healthy retina. Increased NIR AF intensities in the junctional zone in GA may represent accumulation of melanolipofuscin, which may reflect disease activity and thus may allow for early identification of patients at high-risk of GA enlargement.

Tryptophan deficiency arrests chromatin breakdown in secondary lens fibers of rats.

Tryptophan deficiency is known for long time to cause cataract in rats. However, up till now the underlying mechanism is still enigmatic. Histological studies showed an extended lens bow suggesting that the normal breakdown of nuclei in the lens fibres is arrested under these conditions. Using advanced ultrastructural techniques we aimed to clarify this aberrant final differentiation of lens fibres. Albino and pigmented rats were permanently or intermittently raised on a tryptophan deficient diet for 12 and 16 weeks, respectively. Rats of the same age raised on a normal diet served as controls. Lenses were treated for light and electron microscopy. For histology sections were stained for DNA and gamma-crystallins. In addition to routine transmission electron microscopy (TEM), ultrathin sections were subjected to electron tomography and energy dispersive X-ray microanalysis (EDX). Histology verified the extended lens bow for albino and pigmented rats and showed that in the intermittent period of normal diet the fibre nuclei are broken down as in controls. It was further shown that gamma-crystallins are co-localized with DNA in the nuclear domain. TEM revealed that during final differentiation nuclear chromatin becomes highly compacted in a chromosome-like manner and than rapidly evanesces in control rats. This compacted stage persists indefinitely in the tryptophan deficient rats. Electron tomography showed that during differentiation chromatin is first uncoiled to 30 nm solenoids, subsequently to highly compacted 10 nm beads-on-a-string fibrils and than is segregated from the nuclear proteins. EDX revealed that the late stage persisting nuclei consist of domains rich in DNA associated with histones and in domains with mainly proteins. This study corroborates previous findings on the final breakdown of nuclei of lens fibres. It further shows that the chromatin is ultimately uncoiled to beads-on-a-string fibrils and that as the last step chromatin is broken down at this unmasked stage. Except for this last step nuclear breakdown is identical in control and tryptophan deficient rats suggesting that it is not the availability of tryptophan for protein synthesis in general which causes the arrest. Two alternatives for this final arrest are discussed. A low tryptophan content, most pronounced in deeper cortical layers, may inhibit the late synthesis of the DNases and proteases necessary for chromatin breakdown. The radical scavenging by indoleamine 2,3-dioxygenase, which cleaves the pyrrole ring of tryptophan to form formylkynurenine using free oxygen radicals, is impaired by low levels of tryptophan. This decreased scavenging of oxygen radicals will expose the catalytic enzymes for chromatin breakdown, residing in the nucleus in an inactive form for quite a long period, to high levels of oxygen radicals and may affect the activity of these enzymes and therefore the execution of the chromatin breakdown.