Carmen Rummelt

Immunohistochemical localization of vascular endothelial growth factor, transforming growth factor alpha, and transforming growth factor beta1 in human corneas with neovascularization.

Purpose. To analyze presence and distribution of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)&agr;, and TGF&bgr;1 in human corneas with neovascularization due to different corneal diseases. Methods. Indirect immunohistochemistry for VEGF, TGF&agr;, and TGF&bgr;1 was performed on paraffin-embedded corneas obtained by keratoplasty. Corneas from each of the four main groups of histopathologic diagnoses associated with corneal neovascularization were analyzed (scarring after keratitis, graft rejection/insufficiency, acute necrotizing keratitis, scarring after mechanical/chemical injury). Subclassification of inflammatory infiltrates was done using immunohistochemistry for CD3 (T-lymphocytes) and CD68 (macrophages). Results. The analyzed angiogenic factors were detectable in corneas from all four histopathologic groups in a similar distribution; capillary endothelial cells, stromal and intravascular inflammatory cells (T-lymphocytes, macrophages), and basal corneal epithelial cells stained positive for the tested angiogenic factors. Conclusion. The angiogenic factors VEGF, TGF&agr;, and TGF&bgr;1 are detectable in human corneas with neovascularization. Their distribution is quite uniform in different corneal diseases, resulting in corneal angiogenesis. An antiangiogenic therapy inhibiting corneal neovascularization by antagonizing angiogenic factors would have to counteract several angiogenic factors.

Safety profile of topical VEGF neutralization at the cornea.

PURPOSE Bevacizumab eyedrops inhibit corneal neovascularization. The purpose of this study was to analyze the safety profile of VEGF-A neutralization at the ocular surface. METHODS Bevacizumab eyedrops (5 mg/mL) and an antimurine VEGF-A antibody (250 microg/mL) were applied to normal murine corneas five times a day for 7 and 14 days. Subsequently, corneas were analyzed for morphologic changes by light and electron microscopy. In a mouse model of corneal epithelial abrasion, the effects of topically applied anti-VEGF antibodies on epithelial wound healing were analyzed: the treatment group received bevacizumab (5 mg/mL) or the antimurine VEGF-A antibody (250 microg/mL) as eyedrops, and the control group received an equal volume of saline solution. After 12, 18, and 24 hours, corneas were photographed in vivo with and without fluorescein staining for morphometry. Afterwards the mice were killed, and eyes were removed for histology, immunohistochemistry with Ki67/DAPI, and electron microscopy. The effect of midterm anti-VEGF therapy on corneal nerve density was assessed by staining corneas treated with an FITC-conjugated anti-neurofilament antibody and morphometric analysis. RESULTS Murine corneas treated with two different types of anti-VEGF antibody eyedrops did not show obvious corneal morphologic changes at the light and electron microscopic levels. Furthermore, anti-VEGF antibody eyedrops had no significant impact on the wound healing process after corneal epithelial injury or on normal murine corneal nerve fiber density. CONCLUSIONS Topical neutralization of VEGF-A at the corneal surface does not have significant side effects on normal corneal epithelial wound healing, normal corneal integrity, or normal nerve fiber density. Therefore, anti-VEGF eyedrops seem to be a relatively safe option to treat corneal neovascularization.

Evaluation of corneal flap dimensions and cut quality using the Automated Corneal Shaper microkeratome.

PURPOSE To evaluate flap dimensions and cut deterioration with repeated blade use in an automated microkeratome. METHODS The Automated Corneal Shaper (Chiron-Adatomed, Munich, Germany), 160-microm plate attached, was used to make a corneal flap in 90 pig cadaver eyes, reusing blades up to five times. Flap diameter was measured by planimetry and thickness was calculated by ultrasound pachymetry. Scanning electron microscopy of stromal beds and blade cutting edges was performed to assess cut deterioration after repeated blade use. RESULTS Mean flap central thickness was 125 +/- 32 microm. Mean vertical flap diameter was 7.6 +/- 0.4 mm. No correlation was found between thickness and diameter (r = 0.15, P = .45). Progressive thinning of the flap was observed in the direction of the flap hinge. Smooth cuts (using new blades) with periodic chatter lines at the keratectomy edge and in the stromal bed were observed with scanning electron microscopy. Increasing tissue remnants on the stromal bed and decreasing cut quality occurred with repeated blade use. Blades showed larger tissue remnants, nicks, and even folds on the cutting edge proportional to the number of times blades were used. CONCLUSION Satisfactory cut quality and reproducibility were obtained after a single use of stainless steel blades in the Automated Corneal Shaper microkeratome. Cut quality was degraded dramatically by repeated use of blades.

Evaluation of corneal flap dimensions and cut quality using a manually guided microkeratome.

BACKGROUND To evaluate reproducibility of corneal flap dimensions and cut quality with repeated blade use with a manually guided microkeratome in pig eyes. METHODS Corneal flaps were created using a manually guided microkeratome (Model One, Moria) with an intended 130-microns cut depth in 130 enucleated pig eyes. Flap thickness was calculated by pachymetry and diameter was estimated by means of applanation lenses compared to planimetry. Histology and scanning electron microscopy of samples and blades were performed to evaluate the keratectomy surface and blade cutting edge after repeated use of the blades. RESULTS Mean flap central thickness was 135 microns (SD, 37 microns). The mean diameter of 8.4 mm (SD, 0.4 mm) correlated significantly (P < .001) to the intended diameter (r = .79). Mean difference from the intended diameter was 0.8 mm (SD, 0.3 mm; range, 0.04 to 1.4 mm). Scanning electron microscopy showed even and smooth cuts with chatter lines at the keratectomy edge using new blades. After repeated blade use, increasing cut irregularity, folds, and tissue remnants on the corneal bed surface, and nicks and tissue remnants at the cutting edge of the blades were observed. CONCLUSION Reproducible flap dimensions were obtained using the Moria One microkeratome on pig eyes. The cut surface was regular and smooth with a new blade, but surface quality deteriorated considerably after repeated use of the same blade.

Orientation teeth in non-mechanical laser corneal trephination for penetrating keratoplasty: 2.94 µm Er:YAG v 193 nm ArF excimer laser

BACKGROUND/AIMS “Orientation teeth” at the donor trephination margin and correspondent “notches” at the host margin facilitate graft orientation and avoid “horizontal torsion” induced by asymmetric suture placement. In this study the quality and reproducibility of these structures created by non-mechanical laser corneal trephination were compared using two laser emissions. METHODS The procedure was performed in 20 enucleated pigs’ eyes using open metal masks with eight “orientation teeth/notches” (0.3 × 0.15 mm, base × height), an automated globe rotation device, and either a 193 nm ArF excimer laser or a Q switched 2.94 μm Er:YAG laser. “Teeth/notches” were analysed by planimetry and scanning electron microscopy (SEM). RESULTS Mean size was 0.30 (0.027) × 0.16 (0.017) mm for “teeth” and 0.30 (0.035) × 0.15 (0.021) mm for “notches” (excimer), and 0.31 (0.022) × 0.16 (0.015) mm and 0.30 (0.031) × 0.14 (0.021) mm respectively (Er:YAG). Overall, variability of notches was higher than that of teeth. By SEM, comparable cut regularity and sustained ablation profile were observed with both lasers. However, the corneal surface at the cut edge appeared slightly elevated (⩽35 μm) in the Er:YAG group. CONCLUSION Orientation teeth/notches resembling those obtained with the excimer laser can be created using the Q switched Er:YAG laser, with potential advantages of lower costs, convenient equipment size, and solid state safety.

Absence of blood and lymphatic vessels in the developing human cornea.

Purpose: The normal human cornea is devoid of both blood and lymphatic vessels and actively maintains this avascularity (corneal angiogenic privilege). Whether and when corneal angiogenic privilege is achieved during development is unknown. Methods: This study analyzed whether the cornea is primarily devoid of both blood and lymphatic vessels during intrauterine development or whether secondary regression of pre-existing vessels occurs before delivery. Indirect double immunohistochemistry was performed on 4-μm serial pupil-optic disc sections of paraffin-embedded human eyes stillborn at gestational ages of 17 to 41 weeks with antibodies against von Willebrand factor (vWF; factor VIII-associated antigen) as a panendothelial marker and with antibodies against lymphatic vessel endothelial hyaluronate receptor 1 (LYVE1) as a marker specific for lymphatic vascular endothelium. Results: Human corneas were devoid of both vWF+++/LYVE-1− blood vessels and vWF+/LYVE-1+++ lymphatic vessels at all time-points analyzed. In contrast, there were numerous blood and lymphatic vessels detectable in the adjacent conjunctiva. Conclusion: The normal human cornea is primarily avascular and devoid of both blood and lymphatic vessels. Corneal angiogenic privilege is already achieved very early during fetal intrauterine development. This suggests early and strong expression of both antiangiogenic and antilymphangiogenic factors in the human cornea during development.

Detection of varicella zoster virus DNA and viral antigen in human cornea after herpes zoster ophthalmicus.

This article describes the histopathology, immunohistochemistry, and varicella zoster virus DNA in situ hybridization of 14 corneal buttons obtained from 14 patients (average age 69.0 years) after perforating keratoplasty (four patients) or surgical enucleation (10 patients) at different times after the clinical onset of herpes zoster ophthalmicus (average 58.7 months). The main histopathologic features were intense stromal vascular scarring (12 patients) and granulomatous reaction to Descemets membrane (nine patients). Using the peroxidase-antiperoxidase method, varicella zoster virus (VZV) antigen could be detected by immunohistochemistry in two patients within epithelial cells of the cornea and in the limbal episclera during the active phase of herpes zoster ophthalmicus. For in situ hybridization we used the 35Slabeled HindIll A and C fragment of VZV and identified viral DNA in five corneal buttons obtained 1 day to 8 years after the clinical onset of infection. Viral DNA was mainly found in mononuclear cells with eosinophilic intracytoplasmic inclusions within vascular stromal scars, in keratocytes, and in epithelial cells of the cornea. Our results show that VZV DNA is detectable in human cornea even 8 years after the clinical onset of herpes zoster ophthalmicus and may indicate VZV persistence in a latent form in corneal tissue or reactivation of the virus from an endogenous or exogenous source causing a severe and often recurrent keratitis in the progress of herpes zoster ophthalmicus.

Postoperative Kunstlinsen-Eintrübungen bei 12 Hydrogel-Intraokularlinsen (Hydroview®)

Background: To evaluate postoperative lens opacifications in foldable hydrophilic intraocular lenses. Patients and Methods: 12 patients (9 female; 3 male; mean age 77.5 ± 3 years) were referred from one ophthalmologic surgeon because of opacification of lOLs and markedly decreased visual acuity. Time between implantation and explantation varied from 8 month to 3 years. IOL explantation was performed in all 12 patients and IOL were examined by light-, transmission- and scanning electron microscopy. Results: IOL-explantation was uneventful in all 12 patients. The explanted IOLs showed crystalline deposits 0.5 to 2 μm in diameter immediately beneath the surface of the lens. Eight of 12 patients had elevated serum levels for glucose (6 patients with manifest diabetes mellitus, 2 patients with pathological elevated levels for glucose). Conclusions: Postoperative opacification of hydrogel foldable lenses (Hydroview®) are appearantly caused by formation of crystalline deposits beneath the lens surface. These deposits may be associated with metabolic disorders, e.g. diabetes mellitus.

Congenital Nonpigmented Epithelial Iris Cyst after Amniocentesis: Clinicopathologic Report on Two Children

BACKGROUND Congenital nonpigmented epithelial iris cysts are not common. They may arise spontaneously from developmental entrapment of surface ectodermal epithelium or from occult ocular trauma prenatally or at birth. PATIENTS AND METHODS Between 1989 and 1991, an 8-month-old child and a 6-year-old child presented with large, progressive congenital epithelial iris cysts. Both children had a maternal history of diagnostic amniocentesis after an ultrasound scan, and there was no history of postnatal ocular trauma. The cysts were successfully removed by a modified block excision and tectonic corneoscleral grafting. RESULTS A dense adherence of the cyst wall to Descemets membrane resembled old anterior synechiae after occult perforation of the globe in both patients. On histopathologic examination, the epithelial lining of the cysts consisted of non-keratinizing stratified squamous epithelium with goblet cells resembling conjunctival epithelium. A perforating limbal scar with a corresponding break in Descemets membrane could be detected in one eye. The long-term visual acuity of both children was encouraging, and there was no evidence of recurrence of the iris cyst during the follow-up period (average, 23 months). CONCLUSIONS The authors conclude that the clinical and histopathologic features of these congenital iris cysts may be consistent with an occult intrauterine limbal perforation of the anterior chamber with a needle during amniocentesis. Amniocentesis, when not guided by a real-time ultrasound scan, may be a risk factor for prenatal ocular trauma, which should be considered in the differential diagnosis of congenital ocular disorders.

Triple Retinal Infection with Human Immunodeficiency Virus Type 1, Cytomegalovirus, and Herpes Simplex Virus Type 1

PURPOSE This report describes the histopathologic and virologic findings of the retina from a 55-year-old bisexual patient with the acquired immune deficiency syndrome (AIDS), who had concurrent human immunodeficiency virus type 1 (HIV-1), cytomegalovirus (CMV), and herpes simplex virus type 1 (HSV-1) retinitis, and was treated with ganciclovir. METHODS The eyes were obtained at autopsy and processed for light microscopy and transmission electron microscopy. Immunohistochemical stains for HSV-1, CMV, HIV-1, varicella zoster virus, and glial fibrillary acidic protein were carried out using the peroxidase-antiperoxidase and streptavidin-biotin-alkaline phosphatase techniques. For in situ hybridization, a radiolabeled CMV DNA probe (Eco-RI-Y fragment of strain AD 169) was used. RESULTS Results of histopathologic examination showed a full-thickness necrotizing retinitis with cytomegalic and herpes viral intranuclear inclusions in cells of the neurosensory retina, retinal vascular endothelium, and the retinal pigment epithelium. Some areas of the retina were replaced by glial tissue. The choroid contained only a few chronic inflammatory cells. Immunoperoxidase studies disclosed CMV antigens diffusely distributed throughout all layers of the retina and the retinal pigment epithelium. Herpes simplex virus type 1 antigens were present in retinal cells and the retinal vascular endothelium. Human immunodeficiency virus type 1 antigens were found in mononuclear cells in all layers of the sensory retina. Dual infections with HIV-1 and CMV of individual multinucleated giant cells of glial origin were demonstrated immunohistochemically. Transmission electron microscopy showed herpes viral particles in the vascular endothelium of the retinal vessels and the choriocapillaris. Human immunodeficiency virus particles were identified in the endothelium of the choriocapillaris. CONCLUSIONS The possibility of multiple viral infections of the retina, mimicking classic CMV retinitis, should be considered in the clinical and histologic differential diagnosis of necrotizing retinitis in patients with AIDS.