Immunohistochemical localization of cathepsin D in ocular tissues

Cathepsin D has been believed to play an important role in the catabolism of protein in various tissues. In retinal pigment epithelium, cathepsin D degrades rod outer segments and rhodopsin into glycopeptides. To our knowledge, no reports have described the immunohistochemical localization of cathepsin D in whole ocular tissues. We investigated the reaction of bovine, rat, and human eyes with a polyclonal antibody to cathepsin D from bovine spleen. Cathepsin D immunoreactivity was observed in the cytoplasm of the following cells: epithelium and endothelium of the cornea; keratocytes; pigmented and nonpigmented epithelium of the ciliary body; epithelium and cortex of the lens; epithelium and sphincter and dilator muscles of the iris; Müller cells; ganglion cells and pigment epithelium of the retina; and endothelium of various vessels. Positively stained ocular tissues were believed to have a high activity of protein catabolism. Since cathepsin D was closely associated with phagosomes in retinal pigment epithelium, we concluded that cathepsin D probably contributes to the physiologic degradation of rod outer segments.     

Studies of gamma-glutamyl transpeptidase in human ocular tissues.

γ-Glutamyl transpeptidase was found to be in relatively much higher concentrations in human retina and iris than in the lens. In the lens, the enzyme was localized to a major extent in the epithelium plus capsule fraction. The lens cortex and nucleus had very little enzyme activity.γ-Glutamyl transpeptidase was purified about 200-fold to a specific activity of 897 mU per mg protein from cultured human lens epithelium. The purification steps included heating at 37° for 2 hr, deoxycholate extraction, DE-52 column chromatography, Sephadex G-200 gel filtration and affinity chromatography. The enzyme preparation was found to have a K_m of about 0·7 mm for γ-glutamyl p-nitroanilide and a pH optimum of 8·2. Magnesium had no significant effect on the enzyme activity, whereas, sodium and potassium inhibited the enzyme slightly. The competitive inhibition of γ-glutamyl transpeptidase by GSH and GSSG when γ-glutamyl p-nitroanilide was used as substrate indicates that the artificial substrate binds to the protein at the same site as the natural substrate, i.e. GSH. The enzyme was not inhibited by sulfhydryl blocking reagents up to a concentration of 1 mm indicating that the enzyme does not require the presence of sulfhydryl groups for its activity.Antibodies raised against an apparently homogeneous γ-glutamyl transpeptidase from human kidney precipitated the enzyme from ocular tissues and the enzyme purified from the cultured lens epithelial cells. This indicates that the enzyme of the ocular tissues and the kidney are of similar genetic origin.     

Immunocytochemical localization of ornithine aminotransferase in rat ocular tissues

Gyrate atrophy of the choroid and retina is a rare inherited form of chorioretinal degeneration due to a deficiency of ornithine aminotransferase (OAT). We localized the enzyme in rat ocular tissues using immunocytochemical procedures. Immunoreactivity was observed in the epithelia of ciliary body, iris, and lens. Retinal pigment epithelium and Müller cells were immunoreactive in the retina. A little immunoreactive product was found in the choroid. Our findings suggested that OAT plays an important role in ornithine metabolism in these ocular tissues.     

Peroxiredoxin in bovine ocular tissues: immunohistochemical localization and in situ hybridization.

Peroxiredoxins are widely distributed in nature and constitute a molecular family of antioxidant enzymes which decompose hydrogen peroxide and alkyl hydroperoxides. We have previously characterized a peroxiredoxin from bovine ciliary body and deduced its amino acid sequence from analysis of cDNA clones encoding the protein. In this work, we investigated the immunolocalization of this novel antioxidant enzyme and its mRNA expression in bovine eye tissues. High levels of immunoreactivity and mRNA for the enzyme were detected in corneal epithelium. Distinct immunoreactivity and mRNA expression for peroxiredoxin were also detected in uveal tissues, some of the retinal cell layers and ocular vasculature.     

Freezing-thawing hysteresis. II. Investigation of human ocular tissues

The freezing-thawing behavior of cataractous eye lenses and corneas was investigated by 1H-NMR. The corneas have shown hysteresis similarly to the normal lens. No hysteresis could be demonstrated in cataractous lenses. The transparency of lens seems to be correlated with the existence of the freezing-thawing hysteresis.     

Immunohistochemical localization of angiotensin-converting enzyme, angiotensin II and AT1 receptor in human ocular tissues

We investigated the immunohistochemical distribution of 3 components of the renin-angiotensin system (RAS), angiotensin-converting enzyme (ACE), angiotensin II (AngII) and AT1 receptor (AT1), in the human eye. ACE and AngII were localized to nonpigmented epithelial cells of the ciliary body, to endothelial and epithelial cells of the cornea, to epithelial cells of the conjunctiva and to trabecular meshwork cells in the anterior part of the eye. In the posterior part of the eye, ACE and AngII were localized to ganglion cells, some cells in the inner nuclear layer, photoreceptor cells and to endothelial cells of the retinal and choroidal vessels. The overall intensity of AT1 immunoreactivity was weak in all ocular tissues, but the main localization was in ganglion cells. As a preliminary investigation, we were able to include 2 Alzheimer's disease (AD) cases. In AD, no differences from controls were found in the cellular distribution and staining intensity of all 3 antigens. The manifold localization sites of the observed antigens point to rather generalized functions of the RAS in human ocular tissues, such as regulatory effects on neuronal cells, vessels and vitreous humor homeostasis.     

Acquired ocular toxoplasmosis. A fluorescein angiography study.

A 31-year-old man exhibited a bilateral deterioration of vision over the course of 1 week. The right fundus showed the picture of a central vein occlusion, and the left of a disseminated choroiditis. Indirect immunofluorescent testing indicated a titer of 1:2048 for toxoplasmosis. Fluorescein angiography revealed a hitherto unique picture of isolated choroidal occlusions but neither a central vein occlusion nor a disseminated choroiditis. Using only specific antitoxoplasmotic therapy, the patient regained full visual acuity on both sides. The original findings and the results of a 14-year follow-up are presented.     

Presence of ornithine ketoacid aminotransferase in human ocular tissues

Ornithine ketoacid aminotransferase activity in the human ocular tissues was biochemically studied. The choroid plus retina, ciliary body, and iris showed high enzyme activity. The enzyme had a pH optimum at near 8.0–8.1, and the K_m value for pyridoxal phosphate was 8 M. The possible role of ornithine ketoacid aminotransferase in the human ocular tissues was discussed.     

Prevalence of SARS-CoV-2 in human post-mortem ocular tissues

BackgroundSARS-CoV-2 is found in conjunctival swabs and tears of COVID-19 patients. However, the presence of SARS-CoV-2 has not been detected in the human eye to date. We undertook this study to analyze the prevalence of SARS-CoV-2 in human post-mortem ocular tissues.MethodsThe expression of SARS-CoV-2 RNA was assessed by RT-PCR in corneal and scleral tissues from 33 surgical-intended donors who were eliminated from a surgical use per Eye Bank Association of America (EBAA) donor screening guidelines or medical director review or positive COVID-19 test. Ocular levels of SARS-CoV-2 RNA (RT-PCR), Envelope and Spike proteins (immunohistochemistry) and anti-SARS-CoV-2 IgG and IgM antibodies (ELISA) in blood were evaluated in additional 10 research-intent COVID-19 positive donors.FindingsOf 132 ocular tissues from 33 surgical-intended donors, the positivity rate for SARS-CoV-2 RNA was ~13% (17/132). Of 10 COVID-19 donors, six had PCR positive post-mortem nasopharyngeal swabs whereas eight exhibited positive post-mortem anti-SARS-CoV-2 IgG levels. Among 20 eyes recovered from 10 COVID-19 donors: three conjunctival, one anterior corneal, five posterior corneal, and three vitreous swabs tested positive for SARS-CoV-2 RNA. SARS-CoV-2 spike and envelope proteins were detected in epithelial layer of the corneas that were procured without Povidone-Iodine (PVP–I) disinfection.InterpretationsOur study showed a small but noteworthy prevalence of SARS-CoV-2 in ocular tissues from COVID-19 donors. These findings underscore the criticality of donor screening guidelines, post-mortem nasopharyngeal PCR testing and PVP-I disinfection protocol to eliminate any tissue harboring SARS-CoV-2 being used for corneal transplantation.     

Mitomycin concentration in rabbit and human ocular tissues after topical administration.

The authors measured mitomycin C (MMC) concentrations in ocular tissues with high-performance liquid chromatography. Mitomycin C concentration after a single subconjunctival injection of the drug in rabbit eyes showed a rapid decrease with a half-life of 0.18 to 0.30 hours for the conjunctiva and 0.20 to 0.45 hours for the sclera at the injection site. Irrigating the ocular surface with 200 ml of saline after MMC application reduced the initial drug concentration to one fifth in the sclera and to one fifteenth in the conjunctiva but did not change the half-life. The MMC concentration in human trabeculectomy specimens obtained immediately after MMC application (0.2 mg/0.5 ml) and irrigation was 5.4 to 12.0 micrograms/g with a mean of 8.4 micrograms/g, a level similar to that in the rabbit sclera immediately after the irrigation after administration of the same MMC dose. These results indicate that MMC disappears rapidly from the ocular tissues and that irrigating the tissues significantly reduces the tissue concentration of MMC.     

Enhanced documentation of slit-lamp images of the human vitreous stained with fluorescein sodium.

BACKGROUND AND OBJECTIVE: To develop a new method to document the slit-lamp image of the vitreous body as stained with fluorescein sodium. PATIENTS AND METHODS: The system consists of a Goldmann-type slit-lamp biomicroscope, a highly sensitive monochromatic charge-coupled device camera, and a video attachment for real-time observation and recording. As a standard procedure, observation of the vitreous was conducted 60 minutes after intravenous administration of 500 mg of fluorescein sodium. This method was applied to 19 eyes with various eye diseases. RESULTS: Fine details of the vitreous were observed in all of the eyes. Posterior vitreous detachment could be recorded in 5 of the 19 eyes examined. Vitreoschisis and large lacunae were documented in 11 eyes. CONCLUSION: This method promises to be of value in detecting early age-related and pathological changes of the vitreous such as posterior vitreous detachment and vitreoschisis in clinics.