Live porcine thirty days delayed recovery surgery: Qualitative findings with the hypersonic vitrectomy.

INTRODUCTION: Qualitatively assess the possible delayed structural, macroscopic and microscopic changes in the neuro-retina, retinal vasculature, retinal pigment epithelium (RPE) and optic nerve head (ONH) after pars plana vitrectomy (PPV) surgery using a new hypersonic vitrector (HV). MATERIALS AND METHODS: Eight live porcine eyes underwent PPV using either the HV or a conventional pneumatic guillotine vitrector (GV). The un-operated fellow eye from each pig was used as an external control. The pigs were post-operatively kept alive for 30 days before termination and enucleation of the eyes. Prior to enucleation, all eyes underwent examination of the lens and indirect ophthalmoscopy. Enucleated eyes were fixed in formalin, examined macroscopically and processed for histological assessment. Microscopic analysis included assessment of neuro-retina, retinal vasculature, RPE and ONH, as well as observation for any morphological intraocular changes. Comparison was made between: (1) treated and untreated areas of the same eye (internal control) (2) different areas within the same eye operated on using different vitrector settings (3) eyes operated on with the HV and GV (4) eyes receiving surgery and the fellow un-operated eye (external control, same pig). RESULTS: All lenses had remained clear at 30 days into the postoperative period. On indirect ophthalmoscopy, the retina had remained attached in all eyes with no visible changes to the neuro-retina, retinal vasculature, RPE or ONH. Two eyes showed localized RPE depigmentation secondary to previously documented intraoperative retinal touch. The Morphological changes in the retinal layers above depigmented RPE were no different from retinal change elsewhere. There were mild and similar microscopic changes observed in the neuro-retina, retinal vasculature, RPE or ONH associated with either the HV or GV PPVs. Preliminary histological findings revealed no significant differences between eyes operated on with the HV and those operated on the GV. DISCUSSION: At 30 days into the postoperative period, there seemed to be similar morphological changes attributable to the use of HV and GV. Therefore, the HV promises to be a new alternative to the currently commercially available GV for PPV.

Ultrastructural and histopathologic findings after pars plana vitrectomy with a new hypersonic vitrector system. Qualitative preliminary assessment.

PURPOSE: Preliminary assessment of a new prototype ultrasound-based hypersonic vitrector (HV) by qualitatively examining the histopathological changes in the retina and vitreous body after pars plana vitrectomy (PPV) and its ability to fragment vitreous collagen. METHODS: Fourteen porcine cadaveric eyes, 20 eyes in live swine and six human cadaveric eyes underwent PPV using the HV or a pneumatic guillotine vitrector (GV). An additional 4 porcine crystalline lenses were touched with either the HV or GV for 1 minute. Following PPV, human vitreous was removed and processed for electron microscopy (EM). Eyes and lenses were fixed and sectioned for light microscopy (LM). RESULTS: There were no macroscopic retinal or optic nerve defects associated with either HV or GV PPVs. Cadaveric retinal specimens showed separation of the inner limiting membrane (ILM) and vacuolization and fragmentation at the nerve fiber layer (NFL) and the ganglion cell layer (GCL). ILM fragmentation and separation were found after PPV in live swine with both vitrectors. Small disruptions of the posterior capsule or structural lens defects were found after HV touch. The EM analysis revealed more fragmentation of human vitreous collagen fibrils after HV compared to GV PPV. CONCLUSIONS: LM and EM analysis of retina, vitreous, and crystalline lens after PPV showed similar morphological changes using the HV or the GV. Vitreous fragmentation appeared more effective with the HV. Overall this study suggests that the HV may be a promising new technology. More work is needed to quantitatively assess its safety and efficacy.

Bruch's membrane abnormalities in PRDM5-related brittle cornea syndrome.

BACKGROUND: Brittle cornea syndrome (BCS) is a rare, generalized connective tissue disorder associated with extreme corneal thinning and a high risk of corneal rupture. Recessive mutations in transcription factors ZNF469 and PRDM5 cause BCS. Both transcription factors are suggested to act on a common pathway regulating extracellular matrix genes, particularly fibrillar collagens. We identified bilateral myopic choroidal neovascularization as the presenting feature of BCS in a 26-year-old-woman carrying a novel PRDM5 mutation (p.Glu134*). We performed immunohistochemistry of anterior and posterior segment ocular tissues, as expression of PRDM5 in the eye has not been described, or the effects of PRDM5-associated disease on the retina, particularly the extracellular matrix composition of Bruch's membrane. METHODS: Immunohistochemistry using antibodies against PRDM5, collagens type I, III, and IV was performed on the eyes of two unaffected controls and two patients (both with Δ9-14 PRDM5). Expression of collagens, integrins, tenascin and fibronectin in skin fibroblasts of a BCS patient with a novel p.Glu134* PRDM5 mutation was assessed using immunofluorescence. RESULTS: PRDM5 is expressed in the corneal epithelium and retina. We observe reduced expression of major components of Bruch's membrane in the eyes of two BCS patients with a PRDM5 Δ9-14 mutation. Immunofluorescence performed on skin fibroblasts from a patient with p.Glu134* confirms the generalized nature of extracellular matrix abnormalities in BCS. CONCLUSIONS: PDRM5-related disease is known to affect the cornea, skin and joints. Here we demonstrate, to the best of our knowledge for the first time, that PRDM5 localizes not only in the human cornea, but is also widely expressed in the retina. Our findings suggest that ECM abnormalities in PRDM5-associated disease are more widespread than previously reported.

A role for repressive complexes and H3K9 di-methylation in PRDM5-associated brittle cornea syndrome.

Type 2 brittle cornea syndrome (BCS2) is an inherited connective tissue disease with a devastating ocular phenotype caused by mutations in the transcription factor PR domain containing 5 (PRDM5) hypothesized to exert epigenetic effects through histone and DNA methylation. Here we investigate clinical samples, including skin fibroblasts and retinal tissue from BCS2 patients, to elucidate the epigenetic role of PRDM5 and mechanisms of its dysregulation in disease. First we report abnormal retinal vascular morphology in the eyes of two cousins with BCS2 (PRDM5 Δ exons 9-14) using immunohistochemistry, and mine data from skin fibroblast expression microarrays from patients with PRDM5 mutations p.Arg590* and Δ exons 9-14, as well as from a PRDM5 ChIP-sequencing experiment. Gene ontology analysis of dysregulated PRDM5-target genes reveals enrichment for extracellular matrix (ECM) genes supporting vascular integrity and development. Q-PCR and ChIP-qPCR confirm upregulation of critical mediators of ECM stability in vascular structures (COL13A1, COL15A1, NTN1, CDH5) in patient fibroblasts. We identify H3K9 di-methylation (H3K9me2) at these PRDM5-target genes in fibroblasts, and demonstrate that the BCS2 mutation p.Arg83Cys diminishes interaction of PRDM5 with repressive complexes, including NuRD complex protein CHD4, and the repressive chromatin interactor HP1BP3, by co-immunoprecipitation combined with mass spectrometry. We observe reduced heterochromatin protein 1 binding protein 3 (HP1BP3) staining in the retinas of two cousins lacking exons 9-14 by immunohistochemistry, and dysregulated H3K9me2 in skin fibroblasts of three patients (p.Arg590*, p.Glu134* and Δ exons 9-14) by western blotting. These findings suggest that defective interaction of PRDM5 with repressive complexes, and dysregulation of H3K9me2, play a role in PRDM5-associated disease.

Cloudy corneas as an initial presentation of multiple myeloma.

SUMMARY: We report a case of previously unsuspected myeloma, presenting with cornea verticillata due to intracorneal paraprotein deposition. HISTORY: An 85-year-old female presented via her optician with a 4-month history of cloudy vision. She had undergone an uneventful bilateral phacoemulsification surgery 7 years earlier. Extensive spiraling corneal epithelial opacification was noted on slit-lamp examination. On further investigation, she was found to have a previously unsuspected low-grade multiple myeloma. We established the nature of the corneal deposits with corneal epithelial biopsy histopathology and electron microscopy. It is very rare for multiple myeloma to present in this fashion. Ophthalmologists should be aware that such a presentation may rarely be due to systemic multiple myeloma.

Histologically proven epithelial ingrowth in failed Descemet stripping automated endothelial keratoplasty (DSAEK) managed by repeat DSAEK.

PURPOSE: To report a case of corneal graft failure due to epithelial ingrowth after an uneventful combined Descemet stripping automated endothelial keratoplasty (DSAEK) and phacoemulsification cataract surgery with intraocular lens implant treated successfully with a repeat DSAEK. METHODS: A 77-year-old male patient underwent combined DSAEK and phacoemulsification with intraocular lens implant implantation for Fuchs' endothelial dystrophy plus cataract in the right eye. The donor cornea was cut on the Moria ALTK system and introduced using a suture pull-through technique. After an episode of endothelial rejection, the graft failed, with signs suggesting epithelial ingrowth. It was stripped from the host cornea using a Descemet's membrane stripper, and a Simcoe irrigation-aspiration cannula was used to remove all traces of interface material. The excised lenticule was examined histologically using a hematoxylin and eosin stain. RESULT: The patient regained and maintained excellent visual acuity with no sign of recurrence of epithelial ingrowth. Histopathological evaluation of the donor tissue of the first graft showed epithelial ingrowth on the stromal surface of the graft and very few endothelial cells, in keeping with the diagnosis of graft failure. CONCLUSION: Epithelial ingrowth is a possible cause of endothelial graft failure, but histologically proven cases are rare. Surgical intervention can achieve successful clearance, with the potential for cure and an excellent outcome.

Amyloidosis of the orbit and adnexae.

PURPOSE: To present a series of patients with orbital and adnexal amyloidosis and illustrate the diversity of disease and the challenges of managing such cases. METHODS: Descriptive case series of ten patients with biopsy proven amyloidosis involving the orbit, conjunctiva and eyelids. The presentation, clinical findings and management are discussed for each case. RESULTS: All patients had some form of eyelid abnormality or malposition. Presenting complaints included ptosis, epiphora and ocular discomfort. Other clinical findings included conjunctival lesions and proptosis. The majority of patients had localised amyloidosis and one patient had systemic disease. Conservative management included lubrication and the use of bandage contact lenses. Surgical management included debulking, ptosis or other lid surgery. CONCLUSION: Amyloidosis can present to an Occuloplastic clinic in a wide variety of ways. Definitive diagnosis is based on the histopathological findings. Management is often challenging. Multi-disciplinary team involvement is critical in view of its systemic associations.

An alpha 2 collagen VIII transgenic knock-in mouse model of Fuchs endothelial corneal dystrophy shows early endothelial cell unfolded protein response and apoptosis.

Fuchs endothelial corneal dystrophy (FECD) is a leading indication for corneal transplantation. FECD is characterized by progressive alterations in endothelial cell morphology, excrescences (guttae) and thickening of the endothelial basement membrane and cell death. Ultimately, these changes lead to corneal edema and vision loss. Due to the lack of vision loss in early disease stages and the decades long disease course, early pathophysiology in FECD is virtually unknown as studies of pathologic tissues have been limited to end-stage tissues obtained at transplant. The first genetic defect shown to cause FECD was a point mutation causing a glutamine to lysine substitution at amino acid position 455 (Q455K) in the alpha 2 collagen 8 gene (COL8A2) which results in an early onset form of the disease. Homozygous mutant knock-in mice with this mutation (Col8a2(Q455K/Q455K)) show features strikingly similar to human disease, including progressive alterations in endothelial cell morphology, cell loss and basement membrane guttae. Ultrastructural analysis shows the predominant defect as dilated endoplasmic reticulum (ER), suggesting ER stress and unfolded protein response (UPR) activation. Immunohistochemistry, western blotting, quantitative reverse transcriptase polymerase chain reaction and terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end-labeling analyses support UPR activation and UPR-associated apoptosis in the Col8a2(Q455K/Q455K) mutant corneal endothelium. This study confirms the Q455K substitution in the COL8A2 gene as being sufficient to cause FECD in the first mouse model of this disease and supports the role of the UPR and UPR-associated apoptosis in the pathogenesis of FECD caused by COL8A2 mutations.

Mutations in PRDM5 in brittle cornea syndrome identify a pathway regulating extracellular matrix development and maintenance.

Extreme corneal fragility and thinning, which have a high risk of catastrophic spontaneous rupture, are the cardinal features of brittle cornea syndrome (BCS), an autosomal-recessive generalized connective tissue disorder. Enucleation is frequently the only management option for this condition, resulting in blindness and psychosocial distress. Even when the cornea remains grossly intact, visual function could also be impaired by a high degree of myopia and keratoconus. Deafness is another common feature and results in combined sensory deprivation. Using autozygosity mapping, we identified mutations in PRDM5 in families with BCS. We demonstrate that regulation of expression of extracellular matrix components, particularly fibrillar collagens, by PRDM5 is a key molecular mechanism that underlies corneal fragility in BCS and controls normal corneal development and maintenance. ZNF469, encoding a zinc finger protein of hitherto undefined function, has been identified as a quantitative trait locus for central corneal thickness, and mutations in this gene have been demonstrated in Tunisian Jewish and Palestinian kindreds with BCS. We show that ZNF469 and PRDM5, two genes that when mutated cause BCS, participate in the same regulatory pathway.

Presumed early-onset sarcoidosis: a case of devastating ocular inflammation in an infant.

Involvement of the orbit and posterior segment of the eye in early-onset sarcoidosis may necessitate prompt and aggressive immunosuppressive treatment to prevent a poor visual outcome.

Comparison of lectin binding of drusen, RPE, Bruch's membrane, and photoreceptors.

PURPOSE: Drusen are deposits located between the retinal pigment epithelium and Bruch's membrane in age-related maculopathy. They are believed to be photoreceptor byproducts that are incompletely metabolized by the retinal pigment epithelium. This study therefore compares the lectin histochemistry of drusen, photoreceptors, retinal pigment epithelium, and Bruch's membrane. METHODS: Semithin sections of three eyes with age-related maculopathy were studied using 19 biotinylated lectins and an avidin-peroxidase-revealing system with and without neuraminidase pretreatment. RESULTS: High mannose, bi and tri-antennary nonbisected and bisected complex N-glycan, N-acetyl glucosamine and galactose were expressed by drusen, retinal pigment epithelium, Bruch's membrane, and photoreceptors while N-acetyl galactosamine and fucose were absent; treatment with neuraminidase exposed subterminal galactose in both sites and sparse N-acetyl galactosamine residues in drusen alone. Overall, there were striking similarities between the lectin binding of drusen, retinal pigment epithelium, and the photoreceptor outer segments, though cone outer segments were distinct in some features of their O-linked glycosylation. CONCLUSIONS: The results suggest that the pathogenesis of drusen is a combined mechanism, involving photoreceptors, Bruch's membrane, and the retinal pigment epithelium.

Oligosaccharide composition is similar in drusen and dense deposits in membranoproliferative glomerulonephritis type II.

Drusen are a feature of age-related macular degeneration (AMD). Lesions similar in appearance to drusen are also found in the fundi of patients with membranoproliferative glomerulonephritis type II (dense deposit disease, DDD). The lamina densa of the glomerular basement membrane, in DDD, is transformed into an electron-dense structure by deposition of microscopically homogeneous material. Our study sought to compare the saccharide composition of drusen and dense deposits in the formalin-fixed, paraffin-embedded tissue from the eye and kidney. Six eye specimens were obtained from patients diagnosed with AMD but another eye was obtained from a patient with partial lipodystrophy, who died after renal failure presumably because of DDD. The kidney specimens were from three biopsy-proven cases of DDD. Glycosylation patterns were measured by the binding of 19 biotinylated lectins before and after neuraminidase pre-treatment. High mannose, bi/tri-antennary non-bisected and bisected complex N-glycan, N-acetyl glucosamine, galactose, and sialic acid residues were found in both drusen and dense deposits. Treatment with neuraminidase exposed subterminal galactose in both sites and sparse N-acetyl galactosamine residues in drusen alone. Our study found similar pathologic oligosaccharide structures in the eye and kidney, suggesting that drusen may be a common end result of retinal and glomerular disease.

Infection and temporal arteritis: a PCR-based study to detect pathogens in temporal artery biopsy specimens.

The possibility of infectious triggers stimulating the development of inflammatory vascular diseases has generated much recent interest. This study uses PCR to detect the presence of Chlamydia pneumoniae, parvovirus B19 and all the human herpes viruses except HHV8 in temporal artery biopsy specimens. Samples from 37 temporal artery biopsies with histological evidence of arteritis and 66 samples from histologically negative temporal artery biopsies, all from different patients, were negative for C. pneumoniae, HSV, VZV, EBV, and HHV7 DNA. Two of the 37 histologically positive specimens were positive for HHV6, another two for CMV and a further two for parvovirus B19 DNA. Parvovirus B19 DNA was also detected in five histologically negative biopsies, one positive for HCMV DNA and a further one was positive for HHV6 DNA. There is no statistically significant difference to the presence of virus DNA in the two types of specimens (P = 0.538). This study does not support a role for C. pneumoniae, parvovirus B19 or human herpes viruses in the pathogenesis of temporal arteritis.

Glycoproteins of drusen and drusen-like lesions.

Drusen are a marker of age-related macular degeneration (AMD). Lesions similar to drusen, both in histology and their clinical appearance, are also seen in choroidal tumours, chronic inflammatory and degenerative conditions of the eye, and in mesangiocapillary glomerulonephritis type II (MCGN-II). This study aims to compare the saccharide composition of these drusen-like lesions in the various ocular pathological groups and in MCGN-II. Formalin fixed and paraffin wax embedded tissue from 21 eyes was studied. The histological diagnoses included AMD, retinal detachment, phthisis bulbi following failed retinal detachment surgery, malignant melanoma, long-standing uveitis, glaucoma and MCGN II. Glycosylation was examined using a panel of twenty biotinylated lectins and an avidin-peroxidase DAB-cobalt revealing system, with and without neuraminidase pre-treatment. High mannose, bi/tri-nonbisected and bisected complex N-glycan, N-acetyl glucosaminyl, galactosyl and sialyl residues were found to be expressed by drusen, while treatment with neuraminidase exposed subterminal N-acetyl galactosamine and galactosyl residues. Similar binding patterns were found in the various pathological groups studied. As there was no significant difference in the lectin-binding pattern in drusen in different pathologies, a common pathogenesis or at least a final common pathway for the elaboration of carbohydrate components of drusen is suggested.

Microsporidium stromal keratitis: in vivo confocal findings.

PURPOSE: To relate the clinical signs, histopathologic features, and in vivo confocal biomicroscopy findings of a case of stromal microsporidial keratitis and to describe the use of in vivo confocal microscopy to monitor treatment effect. METHODS: An immunocompetent male patient presented with unilateral indolent stromal keratitis. Stromal microsporidiosis was confirmed after corneal biopsy. He underwent examination that used in vivo confocal microscopy (Heidelberg Retina Tomograph II and Rostock Cornea Module) before and after treatment with topical fumagillin and oral albendazole. Clinicopathologic correlation of the confocal scan was performed. RESULTS: Corneal biopsy showed extracellular microsporidium spores aligned along keratocytes and corneal lamellae. In vivo confocal scans showed similar morphology, with bright dots aligned along keratocytes. Treatment with antimicrobials and topical steroid gave resolution of active keratitis, correlating with disappearance of the bright spores on repeat in vivo confocal scanning. CONCLUSIONS: The in vivo confocal microscopy appearance of microsporidial keratitis corresponds to the histologic features from biopsy material. Treatment response may be monitored by using this technique, although definitive diagnosis requires corneal biopsy.