Labial mucosal epithelium grafting in an ex vivo human donor cornea model.

The purpose of the study was to establish a simple ex vivo corneal re-epithelization model and study the labial mucosal epithelium grafting as a potential approach for ocular surface reconstruction. Four human donor corneal buttons were overstored in a corneal cold storage solution at 4 °C for 32-52 days. Four labial oral mucosa strips were dissected from four patients during fornix reconstruction after they signed informed consent. The substantia propria was trimmed off, and the resulting graft was sutured near the corneal limbus with running sutures (thus forming the tissue construct). Constructs were cultured under the standard conditions with the anterior corneal side outwards. After 3 weeks of culture, constructs were removed, washed, and fixed. Sections were stained with hematoxylin and eosin (HE), anti-keratins 4, 13, 19, and p63. Nuclei were counterstained with Hoechst. After the cultivation, all constructs were integral with the attached graft and non-loosened sutures. The native cells were absent in all donor corneas. Histological evaluation demonstrated that the labial mucosal grafts were attached to the Bowman's membrane (BM), and its cellular outgrowths were found to be transit from the graft to the BM over the anterior surface in all constructs. Cells expressed mucosal epithelial keratins 4, 13, and 19, and several were p63-positive in nuclei. In the study, a simple ex vivo corneal re-epithelization model was successfully established. The model was potent in studying the labial mucosal epithelium grafting as an option for autologous ocular surface reconstruction in patients with bilateral limbal stem cell deficiency.

Fluorescence characteristics of lipofuscin fluorophores from human retinal pigment epithelium.

Lipofuscin granules accumulate in the retinal pigment epithelium (RPE) with age, especially in patients with visual diseases, including progressive age-related macular degeneration (AMD). Bisretinoids and their photooxidation and photodegradation products are major sources of lipofuscin granule fluorescence. The present study focused on examining the fluorescence decay characteristics of bisretinoid photooxidation and photodegradation products to evaluate the connection between fluorescence lifetime and spectral characteristics of target fluorophore groups. The primary objective of the study was to apply experimental spectral analysis results of lipofuscin granule fluorescence properties to interpretation of fluorescence lifetime imaging ophthalmoscopy data. Fluorescence analysis of the lipofuscin granule fluorophores in RPE collected from cadaver eyes was performed. The fluorescence lifetimes were measured by picosecond-resolved time correlated single photon counting technique. A global analytical method was applied to analyze data sets. The photooxidation and photodegradation products of bisretinoids exhibited a longer fluorescence lifetime (average value approximately 6 ns) and a shorter wavelength maximum (530-580 nm). Further, these products significantly contributed (more than 30%), to total fluorescence compared to the other fluorophores in lipofuscin granules. Thus, the contribution of oxidized lipofuscin bisretinoids to autofluorescence decay kinetics is an important characteristic for fluorescence lifetime imaging microscopy data analysis. The higher average fluorescence lifetime in AMD eyes was likely due to the higher abundance of oxidized bisretinoids compared with non-oxidized bisretinoids. Because higher level of oxidized bisretinoids is indicative of pathological processes in the retina and RPE, the present findings have the potential to improve fluorescence lifetime imaging approaches for early diagnosis of degenerative processes in the retina and RPE.

Molecular genetic mechanisms of influence of laser radiation with 577 nm wavelength in a microimpulse mode on the condition of the retina.

THE AIM OF THE STUDY: was to investigate the molecular genetic mechanisms of the influence of laser radiation with 577 nm wavelength in a microimpulse mode on the retina in the experimental conditions after the intravitreal injection of VEGF. MATERIALS AND METHODS: The study was performed on 4-5 week-old male mice of the line C57BL/6J. The animals were divided into 4 groups of 5 mice in each group, one eye was excremental, the contralateral eye remained intact. In the first group, intravitreal injection of PBS was performed; in the second group, intravitreal injection of 50 ng/ml of recombinant VEGF165 in 2 µL of phosphate-buffered saline (PBS) was performed; in the third and fourth groups, a day after the intravitreal injection of recombinant VEGF165, laser radiation with wavelength 577 nm was applied in the micropulse and continuous modes, respectively. Tissue samples (neuroepithelium, pigment epithelium) for the microarray transcription analysis in the animals from group 1 and 2 were taken 2 days after the injection of PBS and VEGF, in the animals from group 3 and 4 - a day after the retina was exposed to laser radiation. RESULTS AND CONCLUSION: Molecular genetic mechanisms of the influence of laser radiation with wavelength 577 nm in a microimpulse mode on the retina in experimental conditions were studied and the genes that significantly changed the level of expression (the genes that take part in the regulation of neoangiogenesis, structural cell functions, processes of cells proliferation, transcription, differentiation, transmembrane transport, signaling, synaptic transmission, etc.) were identified.

Time-of-flight secondary ion mass spectrometry to assess spatial distribution of A2E and its oxidized forms within lipofuscin granules isolated from human retinal pigment epithelium.

Lipofuscin granules accumulate in the cells of retinal pigment epithelium with age, particularly in patients with hereditary diseases. These granules are heterogeneous, being composed of mixtures of proteins and lipids, including more than 21 different fluorescent compounds. Bisretinoids and their photo-oxidation and photodegradation products represent the main source of lipofuscin fluorescence and exhibit phototoxic properties. This study used time-of-flight secondary ion mass spectrometry (ToF-SIMS) with in-depth probing to assess the depth distribution of N-retinylidene-N-retinylethanolamine (A2E) and its singly and doubly oxidized forms (A2E-ox and A2E-2ox, respectively) within lipofuscin granules and in their surface layer (lipid membrane). ToF-SIMS showed that A2E and its oxidized forms were uniformly distributed throughout lipofuscin granules but were not present at the membrane surface layer. This finding is important for understanding the process involved in the formation of lipofuscin granules and in their toxicity.

Changes in spectral properties and composition of lipofuscin fluorophores from human-retinal-pigment epithelium with age and pathology.

Fundus autofluorescence mostly originates from bisretinoid fluorophores in lipofuscin granules, which accumulate in retinal-pigment-epithelium cells with age. The dynamics of accumulation, photo-oxidation, and photodegradation of bisretinoids during aging or in the presence of pathology have been insufficiently investigated. Changes in spectral properties and composition of human lipofuscin-granule fluorophores with age and pathology have now been investigated by a high-performance liquid chromatography method using spectrophotometric and fluorescent detectors connected in series. It was found that: (i) N-retinylidene-N-retinylethanolamine (A2E) fluorescence intensity is not predominant in the chloroform extract of human-cadaver-eye retinal pigment epithelium studied; bisretinoid photo-oxidation and photodegradation products have much higher fluorescent properties; (ii) the relative emission maximum in the fluorescence spectrum of suspended retinal-pigment-epithelium cells obtained from an individual human-cadaver eye without pathology is irrespective of donor age and falls within the range 575 ± 15 nm; in two cadaver eyes with signs of age-related macular degeneration, emission maxima were shifted by 23-36 nm towards the shortwave region; and (iii) the ratio of bisretinoid photo-oxidation and photodegradation products to unoxidized bisretinoids in the chloroform extract of cadaver-eye retinal pigment epithelium increases with donor age, from 0.69 ± 0.03 to 1.32 ± 0.04. The differences in fluorescence properties between chloroform extracts obtained from cadaver eyes with and without signs of age-related macular degeneration could be used to increase the potential of fundus autofluorescence imaging as a noninvasive diagnostic method.

Glueless Simple Limbal Epithelial Transplantation: The Report of the First 2 Cases.

PURPOSE: To assess the anatomic and visual results of the modified glueless simple limbal epithelial transplantation (G-SLET) in the treatment of unilateral limbal stem cell deficiency (LSCD). METHODS: This is a retrospective analysis of 2 patients who received G-SLET for corneal reepithelization after unilateral eye burn. After the recipient bed preparation on the eye with LSCD, radial symmetrical superficial incisions were applied to the corneal periphery. Next, short (1 mm) slightly oblique or horizontal tunnels were formed in every incision. The precut limbal pieces from the healthy eye were inserted into the tunnels with a scleral portion forward. At the end of surgery, the amniotic membrane was sutured to the sclera outside the corneal limbus with a single running suture. RESULTS: Slit lamp examination in the early postoperative period revealed that transplanted limbal pieces remained in place and were visible through the semitransparent amniotic membrane. The donor's eye had a small scar and light subconjunctival hemorrhage. Early and late postoperative periods were uneventful. Twelve months after surgery, the LSCD-affected cornea was entirely covered with tight and semitransparent epithelium. The donor's eye exhibited a small scar on the site of the biopsy. Visual improvement was achieved in case 2, but the vision did not improve due to the presence of a mature cataract in case 1. CONCLUSIONS: Modified G-SLET technique could be an option for LSCD treatment in patients with unilateral eye disease in cases when fibrin glue is not available for the surgeon.

Computed exophthalmometry is an accurate and reproducible method for the measuring of eyeballs' protrusion.

PURPOSE: The aim of this study was to assess reproducibility of the repeated measurements from proposed computed exophthalmometry and to make a comparison with the Hertel exophthalmometer. METHODS: Computed tomography scans of patients with pathological (group 1) and intact orbits (group 2) were included in this retrospective study. In both groups, a single investigator measured a difference of eyeballs' protrusion using the proposed method of computed exophthalmometry. Briefly, the distances from the corneal apices of the left and right eyeballs to the line placed through the styloid processes of the temporal bones were measured and compared to each other three times independently. RESULTS: In some patients with intact lateral orbital rims the results of computed exophthalmometry correlated with the measurements from the Hetrel exophthalmometer. The analysis of the triple measurements with computed exophthalmometry revealed no significant difference in the value of standard deviation of the results in patients with intact and pathological orbits. In comparison with the Hertel-type exophthalmometry, the proposed method demonstrated very low variability and high repeatability of the measurements. The difference of 0.10-0.87 mm in the eyeballs protrusion should be considered as normal. Computed exophthalmometry is an accurate and reproducible method, which can be used for the measurements of eyeballs' protrusion.

Spectral analysis of fundus autofluorescence pattern as a tool to detect early stages of degeneration in the retina and retinal pigment epithelium.

PURPOSE: The aim of this work is the determination of quantitative diagnostic criteria based on the spectral characteristics of fundus autofluorescence to detect early stages of degeneration in the retina and retinal pigment epithelium (RPE). METHODS: RPE cell suspension samples were obtained from the cadaver eyes with and without signs of age-related macular degeneration (AMD). Fluorescence analysis at an excitation wavelength of 488 nm was performed. The fluorescence lifetimes of lipofuscin-granule fluorophores were measured by counting time-correlated photon method. RESULTS: Comparative analysis of fluorescence spectra of RPE cell suspensions from the cadaver eyes with and without signs of AMD showed a significant difference in fluorescence intensity at 530-580 nm in response to fluorescence excitation at 488 nm. It was notably higher in eyes with visual pathology than in normal eyes regardless of the age of the eye donor. Measurements of fluorescence lifetimes of lipofuscin fluorophores showed that the contribution of photooxidation and photodegradation products of bisretinoids to the total fluorescence at 530-580 nm of RPE cell suspensions was greater in eyes with visual pathology than in normal eyes. CONCLUSION: Because photooxidation and photodegradation products of bisretinoids are markers of photodestructive processes, which can cause RPE cell death and initiate degenerative processes in the retina, quantitative determination of increases in these bisretinoid products in lipofuscin granules may be used to establish quantitative diagnostic criteria for degenerative processes in the retina and RPE.