An in vivo method for visualizing flow dynamics of cells within corneal lymphatics.

BACKGROUND: Monitoring the trafficking of specific cell populations within lymphatics could improve our understanding of processes such as transplant rejection and cancer metastasis. Current methods, however, lack appropriate image resolution for single-cell analysis or are incompatible with in vivo and longitudinal monitoring of lymphatics in their native state. We therefore sought to achieve high-resolution live imaging of the dynamic behavior of cells within lymph vessels in the rat cornea. METHODS/RESULTS: Inflammatory angiogenesis was induced by suture placement in corneas of Wistar rats. Pre- and up to 3 weeks post-induction, corneas were noninvasively examined by laser-scanning in vivo corneal confocal microscopy (IVCM) using only endogenous contrast. Lymph vessels and the cells harbored therein were documented by still images, real-time video, and 3D confocal stack reconstruction of live tissue. In vivo, conjunctival and corneal lymphatics were morphologically distinct, those with corneal location being one-quarter the diameter of those in the conjunctiva (p<0.001). Cells were recruited to initially empty pre-existing lymph vessels during the first day of inflammation and maintained a dense occupation of vessels for up to 7 days. A diverse population of cells (diameter range: 1.5-27.5 µm) with varying morphology was observed, and exhibited variable flow patterns and were transported singly and in clusters of at least 2-9 adherent cells. CONCLUSIONS: The in vivo microscopic technique presented enables lymph vessels and cell trafficking to be studied in high resolution in a minimally-perturbed physiologic milieu.

QALY weights for diabetic retinopathy--a comparison of health state valuations with HUI-3, EQ-5D, EQ-VAS, and TTO.

OBJECTIVE: To estimate quality-adjusted life-year weights for patients with diabetic retinopathy by using various methods and to investigate the empirical validity of the different measures. METHODS: The study population comprised 152 patients with diabetes in Östergötland County, Sweden. Participants were interviewed by telephone by using the time trade-off (TTO) method and a visual analogue scale (EQ-VAS) (direct valuations) as well as the EuroQol five-dimensional questionnaire (EQ-5D) and the health utilities index mark 3 (HUI-3) (indirect valuations). The quality-adjusted life-year weights were adjusted for potential confounders by using analysis of covariance. The empirical validity of the measures was examined by testing their ability to detect hypothetical differences between severity levels of diabetic retinopathy and by investigating the correlation between the measures and the 25-item National Eye Institute Visual Function Questionnaire (NEI VFQ-25). RESULTS: All measures detected significant differences in scores between patient groups classified according to visual impairment in the better eye (analysis of covariance, P < 0.05), but only HUI-3 and EQ-VAS detected significant differences between patient groups classified according to visual impairment or pathological progression in the worse eye. HUI-3 recorded a difference of 0.43 in values between normal vision and blindness in the better eye, which was more than twice the differences captured by the other measures (0.15-0.20). In addition, HUI-3 showed the highest correlation with NEI VFQ-25 (r = 0.54; P < 0.001). CONCLUSIONS: In cost-utility analyses, the choice of quality-adjusted life-year measure may affect whether an intervention is considered cost-effective. Furthermore, if decisions are to be based on values from the general public, HUI-3 can be recommended for cost-utility analyses of interventions directed at diabetic retinopathy.

Cellular level characterization of capillary regression in inflammatory angiogenesis using an in vivo corneal model.

In this study, we introduce a technique for repeated, microscopic observation of single regressing capillaries in vivo in inflamed murine corneas. Natural capillary regression was initiated by removal of inflammatory stimulus during an active pro-angiogenic phase, while the additional impact of anti-angiogenic treatment with triamcinolone or bevazicumab was investigated. Capillaries regressed naturally within 1 week and treatments did not further enhance the natural regression. Morphologically, early-phase regression was characterized by significant lumen narrowing and a significant reduction in CD11b+ myeloid cell infiltration of the extracellular matrix. By 1 week, vascular remodeling occurred concomitant with CD11b+CD68+KiM2R+ mature macrophage localization on capillary walls. Empty conduits without blood flow, positive for collagen IV and devoid of vascular endothelium and pericytes, were apparent in vivo and by 3 weeks were more numerous than perfused capillaries. By 3 weeks, macrophages aggregated around remaining perfused capillaries and were observed in apposition with degrading capillary segments. Abrupt termination of capillary sprouting in our regression model further revealed vascular endothelial abandonment of sprout tips and perfused capillary loop formation within a single angiogenic sprout, possibly as an intussusceptive response to cessation of the stimulus. Finally, we observed lumen constriction and macrophage localization on capillary walls in vivo in a clinical case of corneal capillary regression that paralleled findings in our murine model.

Transient anterior corneal deposits in a human immunodeficiency virus-positive patient.

PURPOSE: To report findings of pigmented anterior corneal deposits in a human immunodeficiency virus-positive patient. METHODS: Case report. A 49-year-old human immunodeficiency virus-positive patient was examined after the appearance of pigmented corneal deposits. Slit-lamp biomicroscopy, fundus photography, and laser-scanning in vivo confocal microscopy were performed to visually document the ocular condition. RESULTS: The patient had a history of Mycobacterium avium infection and was suspected to have recovery uveitis from a cytomegalovirus infection. Small, rounded, light brown-colored deposits were distributed across the anterior cornea from limbus to limbus, bilaterally. In vivo confocal microscopy revealed the deposits to be confined to the basal epithelium and Bowman layer, whereas the posterior stroma, Descemet membrane, and the endothelium appeared normal. Systemic steroid treatment was administered, and 2 weeks later, the deposits had vanished on slit-lamp examination, whereas remnants were observed at the microscopic level. CONCLUSIONS: The deposits were unusual for their anterior corneal location and pancorneal distribution. The response to systemic steroid treatment remains unexplained and illustrates the complexity of the underlying conditions, their treatment, and the associated pathways of ocular manifestation.

Cellular-level characterization of lymph vessels in live, unlabeled corneas by in vivo confocal microscopy.

PURPOSE: To determine whether in vivo confocal microscopy (IVCM) of the cornea can be used for the label-free detection and monitoring of lymph vessels in live corneas. METHODS: Parallel corneal hemangiogenesis and lymphangiogenesis was induced by the placement of a single suture in one cornea of male Wistar rats. Fourteen days after suture placement and under general anesthesia, laser-scanning IVCM was performed in the vascularized region. Corneas were subsequently excised for flat-mount double immunofluorescence with a pan-endothelial marker (PECAM-1/CD31) and a lymphatic endothelial specific marker (LYVE-1). Using the suture area and prominent blood vessels as points of reference, the identical microscopic region was located in both fluorescent and archived in vivo images. Additionally, vessel diameter, lumen contrast, and cell diameter and velocity within vessels were quantified from in vivo images. RESULTS: Comparison of identical corneal regions in fluorescence and in vivo revealed prominent CD31(+)/LYVE-1(3+) lymph vessels that were visible in vivo. In vivo, corneal lymph vessels were located in the vascularized area in the same focal plane as blood vessels but had a darker lumen (P < 0.001) sparsely populated by highly reflective cells with diameters similar to those of leukocytes in blood vessels (P = 0.61). Cell velocity in lymph vessels was significantly reduced compared with blood particle velocity (P < 0.001). Morphologic characteristics enabled subsequent identification of corneal lymphatics in live, vascularized rat corneas before immunofluorescence labeling. CONCLUSIONS: IVCM enabled the nondestructive, label-free, in vivo detection of corneal lymphatics. IVCM provides the possibility of observing lymphatic activity in the same live corneas longitudinally and, as a clinical instrument, of monitoring corneal lymphatics in live human subjects.

Relapsing polychondritis: a rare disease with varying symptoms.

PURPOSE: Relapsing polychondritis (RPC) is a rare systemic disease affecting primarily cartilaginous and proteoglycan-rich structures. It is a potentially fatal disease with unknown aetiology. There are no specific tests for RPC. The diagnosis is dependent on clinical criteria, which include chondritis of both auricles, non-erosive inflammatory polyarthritis, nasal chondritis, ocular inflammation, respiratory tract chondritis and cochlear and/or vestibular damage. Ocular symptoms will occur in approximately 60% of RPC patients. As an example, a patient with signs of RPC is described. METHODS/RESULT: A 30-year-old woman was referred to our department for evaluation of a central corneal ulcer in the left eye. She had a history of recurrent pain in both her auricles and was also found to have a nasal septum perforation. Relapsing polychondritis was suspected. CONCLUSION: Non-healing corneal ulcers should alert the ophthalmologist to look for unusual reasons for this condition. RPC is one possible cause.