Transpupillary collagen photocrosslinking for targeted modulation of ocular biomechanics.

The central vision-threatening event in glaucoma is dysfunction and loss of retinal ganglion cells (RGCs), thought to be promoted by local tissue deformations. Here, we sought to reduce tissue deformation near the optic nerve head by selectively stiffening the peripapillary sclera, i.e. the scleral region immediately adjacent to the optic nerve head. Previous scleral stiffening studies to treat glaucoma or myopia have used either pan-scleral stiffening (not regionally selective) or regionally selective stiffening with limited access to the posterior globe. We present a method for selectively stiffening the peripapillary sclera using a transpupillary annular light beam to activate methylene blue administered by retrobulbar injection. Unlike prior approaches to photocrosslinking in the eye, this approach avoids the damaging effects of ultraviolet light by employing red light. This targeted photocrosslinking approach successfully stiffened the peripapillary sclera at 6 weeks post-treatment, as measured by whole globe inflation testing. Specifically, strain was reduced by 47% when comparing treated vs. untreated sclera within the same eye (n = 7, p=0.0064) and by 54% when comparing the peripapillary sclera of treated vs. untreated eyes (n = 7, p<0.0001). Post-treatment characterization of RGCs (optic nerve axon counts/density, and grading), retinal function (electroretinography), and retinal histology revealed that photocrosslinking was associated with some ocular toxicity. We conclude that a transpupillary photocrosslinking approach enables selective scleral stiffening targeted to the peripapillary region that may be useful in future treatments of glaucoma.

Analysis of Damage and Wound Healing in the Retinal Pigmented Epithelium.

Previous studies of retinal pigment epithelium (RPE) morphology found cell-level and spatial patterning differences in many quantitative metrics in comparing normal and disease conditions. However, most of these studies examined eyes from deceased animals. Here we sought to compare noninvasively imaged RPE cells from live mice to histopathology. We describe changes to improve noninvasive imaging of RPE in the live mouse. In retinal diseases, there can be invasion by Iba1-positive cells, which can be detected by noninvasive imaging techniques. Here we can detect potential Iba1-positive cells at the level of the RPE noninvasively.

[Lymphoma of Ocular and Periocular Tissues - Clinicopathological Correlations]. / Lymphome des peri- und intraokularen Gewebes - klinisch-pathologische Korrelationen.

Lymphomas of the ocular adnexa and intraocular tissue include a wide range of lymphoproliferative neoplastic disorders. They are predominantly extranodal non-Hodgkin lymphomas (NHL). The World Health Organization (WHO) classification of lymphoid neoplasm and individual morphological, immunophenotypical, and molecular genetic features, indicate that they may be divided into B-cell (approximately 80 % of all NHL) and T-cell lymphomas (approximately 10-20 % of all NHL). The most common forms of ocular NHL are extranodal marginal zone lymphoma (EMZL) of the mucosa-associated lymphoid tissue (MALT-type), follicular lymphoma (FL), diffuse large B-cell lymphoma, and mantel cell lymphoma. The clinical signs and symptoms are usually very unspecific and depend on the location, size, and extent of the underlying lymphoma subtype. Typical low grade lymphomas have an indolent clinical course and often remain unrecognized for many years. On the other hand, high grade NHLs, such as DLBCL or MCL, are frequently aggressive, with rapid tumour growth and poor prognosis, despite early detection. Histopathology is still the gold standard in the diagnosis of ocular lymphomas. Basic understanding of the principal pathophysiological and clinical aspects of the development and progression of orbital and ocular lymphomas seems to be mandatory for optimal diagnosis and treatment and for improving survival and prognosis. Both residents in training and board certified ophthalmologists should be aware of these problems.

Sustained reduction of intraocular pressure by supraciliary delivery of brimonidine-loaded poly(lactic acid) microspheres for the treatment of glaucoma.

Although effective drugs that lower intraocular pressure (IOP) in the management of glaucoma exist, their efficacy is limited by poor patient adherence to the prescribed eye drop regimen. To replace the need for eye drops, in this study we tested the hypothesis that IOP can be reduced for one month after a single targeted injection using a microneedle for administration of a glaucoma medication (i.e., brimonidine) formulated for sustained release in the supraciliary space of the eye adjacent to the drug's site of action at the ciliary body. To test this hypothesis, brimonidine-loaded microspheres were formulated using poly(lactic acid) (PLA) to release brimonidine at a constant rate for 35 days and microneedles were designed to penetrate through the sclera, without penetrating into the choroid/retina, in order to target injection into the supraciliary space. A single administration of these microspheres using a hollow microneedle was performed in the eye of New Zealand White rabbits and was found to reduce IOP initially by 6 mmHg and then by progressively smaller amounts for more than one month. All administrations were well tolerated without significant adverse events, although histological examination showed a foreign-body reaction to the microspheres. This study demonstrates, for the first time, that the highly-targeted delivery of brimonidine-loaded microspheres into the supraciliary space using a microneedle is able to reduce IOP for one month as an alternative to daily eye drops.

[Orbital invasion of a maxillary ameloblastoma]. / Orbitale Infiltration durch ein Ameloblastom der Maxilla.

A 66-year-old man with a history of repeated surgery, external radiation and brachytherapy for ameloblastoma presented with a recurrence of the tumor with sinus, intraorbital and skull base infiltration. Histopathologic examination of the resected orbital and sinus tissue confirmed the diagnosis of ameloblastoma. Immunohistochemical staining for CD56 was strongly positive in the tumor cells. Although ameloblastoma is usually a low-grade malignant tumor, it can be locally aggressive with invasion of the surrounding tissue. Maxillary ameloblastomas are more likely to infiltrate the orbit.

[Clinico-pathological correlations: posterior compartment of the eye and orbit]. / Klinisch-pathologische Korrelationen: Befunde des hinteren Augenabschnitts und der Orbita.

Diseases of the posterior compartment and the orbit are characterised by histological findings, most of which can be reproduced clinically. Examples are the examination of calcifications in retinoblastoma by ultrasonography. In the present review, histological findings of tumour and other diseases of the posterior ocular compartment and the orbit are presented and correlated with the clinical pictures and imaging techniques: uveal melanoma, choroidal nevus, choroidal metastases, choroidal hemangioma, retinoblastoma, Coat's disease, sympathetic ophthalmia, pleomorphic adenoma (benign mixed tumour) of the lacrimal gland, dacryoadenitis, lymphoma, rhabdomyosarcoma, Langerhans cell histiocytosis, orbital metastases, and phthisical eyes. Histopathology is usually the gold standard for a definitive diagnosis. It is very important for residents and those in training to become familiar with clinico-pathological correlations as these provide insight in pathophysiological processes. Regarding ophthalmic surgery, ophthalmic pathology offers the possibility to study wound healing and complications. A close collaboration between clinicians and ocular pathologists allows for an optimised processing of the submitted tissue and diagnosis. Thus, pre- and postoperative care can also be improved. This outstanding knowledge that ophthalmologists have gained over the last decades and beyond, should be preserved and passed on to the next generations in order to maintain a high standard in ophthalmological care.

[Diffuse anterior retinoblastoma]. / Diffuses anteriores Retinoblastom.

Retinoblastoma is the most common primary intraocular tumor in childhood. Diffuse anterior retinoblastoma is an uncommon variant and usually occurs in comparatively older children. Typically, there is an extensive infiltration of the anterior segment by tumor cells clinically mimicking anterior uveitis with pseudohypopyon. The actual retinal focus is often very small and may not be detected despite a thorough histological examination. In this case report the clinical and histological findings of a diffuse anterior retinoblastoma are described.

Intravenous transferrin, RGD peptide and dual-targeted nanoparticles enhance anti-VEGF intraceptor gene delivery to laser-induced CNV.

Choroidal neovascularization (CNV) leads to loss of vision in age-related macular degeneration (AMD), the leading cause of blindness in adult population over 50 years old. In this study, we developed intravenously administered, nanoparticulate, targeted nonviral retinal gene delivery systems for the management of CNV. CNV was induced in Brown Norway rats using a 532 nm laser. We engineered transferrin, arginine-glycine-aspartic acid (RGD) peptide or dual-functionalized poly-(lactide-co-glycolide) nanoparticles to target delivery of anti-vascular endothelial growth factor (VEGF) intraceptor plasmid to CNV lesions. Anti-VEGF intraceptor is the only intracellularly acting VEGF inhibitory modality. The results of the study show that nanoparticles allow targeted delivery to the neovascular eye but not the control eye on intravenous administration. Functionalizing the nanoparticle surface with transferrin, a linear RGD peptide or both increased the retinal delivery of nanoparticles and subsequently the intraceptor gene expression in retinal vascular endothelial cells, photoreceptor outer segments and retinal pigment epithelial cells when compared to nonfunctionalized nanoparticles. Most significantly, the CNV areas were significantly smaller in rats treated with functionalized nanoparticles as compared to the ones treated with vehicle or nonfunctionalized nanoparticles. Thus, surface-functionalized nanoparticles allow targeted gene delivery to the neovascular eye on intravenous administration and inhibit the progression of laser-induced CNV in a rodent model.

Formation and growth of lipofuscin in the retinal pigment epithelium cells.

The kinetics of lipofuscin growth in diseased retinal pigment epithelium cells is investigated using Monte Carlo simulations and scaling theory on a cluster aggregation model. The model captures the essential physics of lipofuscin growth in the cells. A remarkable feature is that small particles may be removed from the cells while the larger ones become fixed and grow by aggregation. Model simulations are compared to the number of lipofuscin granules in eyes with early age-related degeneration.

[Coats' disease. Important differential diagnosis for retinoblastoma]. / Morbus Coats : Wichtige Differenzialdiagnose zum Retinoblastom.

Advanced Coats' disease is one of the most difficult differential diagnoses of retinoblastoma in early childhood. We describe the clinical and histological findings in two boys, ages 9 months and 21 months, with unilateral leucocoria. Despite comprehensive diagnostics that included examination under general anaesthesia, magnetic resonance imaging, and ultrasound, retinoblastoma could not be excluded, and the eyes were enucleated. Histological diagnosis of Coats' disease was confirmed. Because differentiation between retinoblastoma and Coats' disease may be difficult, enucleation seems to be indicated in uncertain cases due to the reduced visual prognosis and the risk of secondary complications in advanced Coats' disease.

Histopathological findings in postmortem eyes after photodynamic therapy for choroidal neovascularisation in age-related macular degeneration: report of two cases.

BACKGROUND: To report the histopathological findings after photodynamic therapy (PDT) in eyes obtained postmortem with choroidal neovascularisation (CNV) secondary to age-related macular degeneration (AMD). METHODS: Two eyes were obtained postmortem from two patients with CNV secondary to AMD. Both of the patients had been treated with PDT. Serial sections through the posterior poles were obtained and stained with haematoxylin-eosin, periodic acid-Schiff, Masson trichrome or phosphotungstic acid haematoxylin (PTAH). Two-dimensional reconstructions were prepared and compared with fluorescein angiograms. RESULTS: The interval between PDT and death was 3 months and 17 months in each patient, respectively. Light-microscopic examination showed that CNV enveloped with retinal pigment epithelium (RPE) in both eyes. The average size of the CNV was 550 x 280 microm. One eye had combined (subRPE/subretinal) growth pattern CNV, and the other eye had both type I (subRPE) and combined growth pattern CNV. All specimens contained fibrous proliferation and patent vascular channels within the CNV, and there was no thrombus formation within the vascular channels. No apparent abnormalities in the choroid were observed by light microscopy. CONCLUSIONS: Although involution with fibrous tissue proliferation occurred, PDT did not result in permanent occlusion of the vascular channels in the CNV. Our findings indicate that PDT may accelerate involution of CNV, thus limiting its size and preserving photoreceptors.

Effects of celecoxib in human retinoblastoma cell lines and in a transgenic murine model of retinoblastoma.

BACKGROUND/AIM: Celecoxib, a cyclooxygenase-2 inhibitor and antiangiogenic agent, has demonstrated potent anticancer effects in preclinical studies and in human clinical trials. To evaluate the potential utility of this agent in the treatment of retinoblastoma, the authors investigated the effects of celecoxib in retinoblastoma cell lines and in a murine model of this disease. METHODS: Growth inhibitory effects of celecoxib were evaluated in Y79 and Weri-RB1 human retinoblastoma cell lines by WST-1 cell proliferation assay. For animal study, two groups of 24, 8 week old LHbeta-TAg transgenic mice were treated with celecoxib (250 mg/kg, orally once a day) or vehicle control, 5 days/week for 6 weeks. Mice were sacrificed on day 43. Enucleated eyes were serially sectioned and ocular tumour burden was quantified by histopathological analysis. RESULTS: Celecoxib did not inhibit proliferation of Y79 or Weri-RB1 cells, even at concentrations far exceeding clinically achievable levels. No significant difference in ocular tumour burden between celecoxib treated and control mice (p=0.73) was found. CONCLUSION: Celecoxib was ineffective at inhibiting proliferation of retinoblastoma cells in vitro and was ineffective at controlling retinoblastoma tumour growth in a murine model of this disease. On the basis of these findings, oral celecoxib therapy is unlikely to have clinical utility in the treatment of retinoblastoma.

Helicobacter pylori (H. pylori) molecular signature in conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma.

Conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma is an extranodal marginal zone B-cell lymphoma that is characterized by an exaggerated clonal expansion of B cells, which implicate a pathological proliferative response to antigen(s) including bacteria. Helicobacter pylori (H. pylori) infection is recognized as one of the causative agents of gastric MALT lymphoma; however, it has not been reported in extra gastric MALT lymphoma. We studied 5 patients (4 adults and 1 child) with salmon-colored conjunctival lesions. One patient also had a history of abnormal bone marrow biopsy a year earlier with lymphoid aggregates involving 5% of the overall bone marrow. The conjunctival lesions of the 5 patients were biopsied. Histopathological diagnoses were consistent with conjunctival MALT lymphoma. Lymphoma and normal conjunctival cells were microdissected using laser capture microscopy or manual techniques. DNA was extracted and subjected to PCR amplification using H. pylori gene-specific primers from the urease B and vac/m2 gene. Cells from chronic conjunctivitis (normal lymphocytes), conjunctival human T-cell lymphotropic virus type-1/adult T-cell leukemia/lymphoma (HTLV-1/ATL), and orbital B-cell lymphoma were also microdissected, processed and analyzed. PCR amplification and Southern blot hybridization demonstrated H. pylori DNA in the conjunctival MALT lymphoma cells of 4/5 cases. The negative case was the one with a history of abnormal bone marrow. In contrast, H. pylori gene was not detected in normal conjunctival cells from the cases of MALT lymphoma or the lymphocytes, ATL and orbital B-lymphoma cells from the controls. These data suggest that H. pylori may play a role in conjunctival MALT lymphoma.

[HLA class I antigen expression correlates with histological cell type in uveal melanoma]. / HLA-I-Antigenexpression korreliert mit dem histologischen Zelltyp uvealer Melanome.

PURPOSE: The prognosis of uveal melanoma is correlated with its histologic cell type. The epithelioid cell type is associated with a higher metastatic rate than the spindle cell type. The Human Leucocyte Antigen Class I (HLA-I) expression of the melanoma also correlates with the prognosis. In this study, we analyzed HLA-I antigen expression of uveal melanomas to determine whether a relationship exist between antigenic expression and melanoma cell type. METHODS: Formalin-fixed, paraffin-embedded spindle cell type (n = 11) and epithelioid cell type (n = 11) uveal melanomas were immunostained with the HC10 antibody (1:80) for HLA-I antigen expression with appropriate positive and negative controls. Sections were assessed semiquantitatively according to the percentage of stained cells. RESULTS: Among the spindle cell type melanomas, 2 out of 11 (18%) stained with HC10 antibodies. The staining intensity was less than 25% of the cells in these two melanomas. Among the epithelioid cell type melanomas, 9 out of 11 (82%) stained with HC10. The staining intensity was more than 25% of the cells in 5 of these 9 melanomas. CONCLUSIONS: It is unknown why spindle and epithelioid cell type uveal melanomas have different prognoses. Human uveal melanoma cell lines with low HLA-I expression are susceptible to NK cell-mediated lysis in vitro and in murine studies. The prognostically more favorable spindle cell type melanoma expresses less HLA-I than the epithelioid cell type melanoma. These results stress the role of NK cells in the rejection of uveal melanoma.

Murine high-fat diet and laser photochemical model of basal deposits in Bruch membrane.

OBJECTIVE: To examine the histologic, histochemical, and ultrastructural changes in Bruch membrane in mice on a high-fat diet with and without laser photochemical injury. METHODS: Five groups of C57BL/6 mice were studied. Group 1 included 2-month-old mice on a normal diet; group 2 included 8-month-old mice on a normal diet; group 3 included 8-month-old mice on a high-fat diet; groups 4 and 5 included 8-month-old mice on a normal diet or high-fat diet, respectively, that underwent laser application of one eye with argon blue laser (488 nm). The mice were killed and plasma lipid levels were measured. The eyes were examined by standard electron microscopy, filipin histochemistry for unesterified cholesterol (UC) and esterified cholesterol (EC), and the osmium-tannic acid-phenylenediamine method for preserving extracellular lipid particles. RESULTS: The plasma cholesterol level was significantly higher in the mice on the high-fat diet than the controls (P<.001). Bruch membrane was thicker in group 2 than group 1 (P =.04) and group 3 had a thicker Bruch membrane than group 2 (P =.003). All eyes in group 3 exhibited accumulation of electron-lucent debris. There was no histochemical and ultrastructural evidence that this material represented accumulated UC or EC. Seven of 9 laser-injured eyes in group 5 accumulated basal laminar deposit (BlamD)-like material in Bruch membrane (P =.02). CONCLUSIONS: Electron-lucent debris accumulates in murine Bruch membrane, and the amount correlates with age and high-fat diet. This debris has some similarities with basal linear deposits, although the debris does not form a discrete layer external to the basement membrane of the retinal pigment epithelium as occurs in basal linear deposits. These deposits do not appear to be UC or EC. Laser photochemical injury of the retinal pigment epithelium may result in the appearance of BlamD-like deposits in eyes with electron-lucent debris. The BlamD-like deposits in this model are similar to the basal laminar deposits that occur in age-related macular degeneration. CLINICAL RELEVANCE: This is an animal model of ultrastructural BlamD-like material in Bruch membrane that is very similar to the deposits that occur in age-related macular degeneration.

Keratolysis following excimer laser phototherapeutic keratectomy in a patient with keratoconus.

PURPOSE: We report a case of keratolysis following phototherapeutic keratectomy (PTK) for a subepithelial nodule in a patient with keratoconus. METHODS: A 29-year-old male with keratoconus who became contact lens intolerant because of a raised subepithelial nodule was treated with excimer laser phototherapeutic keratectomy (PTK). RESULTS: The epithelium failed to heal postoperatively, and progressive keratolysis led to a central descemetocele by the eighth postoperative day. The patient was managed with a penetrating keratoplasty. CONCLUSIONS: Excimer laser phototherapeutic keratectomy has been shown to be an effective treatment for subepithelial nodules in patients with keratoconus. Rapidly progressive keratolysis is a potential complication of this procedure.

Quantification of optic nerve axon loss associated with a relative afferent pupillary defect in the monkey.

OBJECTIVE: To quantify the amount of optic nerve axonal loss associated with the presence of a mild relative afferent pupillary defect (RAPD) in an experimental monkey model. METHODS: The right macula of 5 rhesus monkeys (Macaca mulatta) was treated with concentrically enlarging diode laser burns until an RAPD was detected using a transilluminator light and measured with neutral density filters. Intervals between treatments were 3 to 7 days over a period of 2 months. Pupillary responses to light stimulation were recorded with a monocular infrared television pupillometer. Two months after detection of an RAPD, 5 treated and 4 control monkeys underwent euthanasia and enucleation. Histopathologic analysis and quantification of optic nerve axon counts using an image analysis system were performed. RESULTS: No RAPD was observed despite an estimated ganglion cell loss of up to 26%. A 0.6 log unit RAPD was present in 5 monkeys when the laser scar incorporated the entire macula within the temporal vascular arcades. One eye had progressive vitreomacular traction with worsening of the RAPD to 1.8 log units without further laser treatment. Histopathologic evaluation disclosed complete loss of the normal retinal architecture within the macula. The average fiber loss for the 4 treated eyes with 0.6 log unit RAPDs compared with fellow eyes was 53.3% (95% confidence interval [CI], 45.0%-61.6%). The average difference in axon counts between untreated pairs of optic nerves was 12.8% (95% CI, 10.0%-15.6%). Optic nerve axon loss between pairs of experimental and control eyes was statistically significant (P<.001). CONCLUSION: In rhesus monkeys, an RAPD develops after an approximate unilateral loss between 25% and 50% of retinal ganglion cells. CLINICAL RELEVANCE: Owing to redundancy in the anterior visual pathways, unilateral retinal ganglion cell loss may occur prior to the observation of an RAPD. The presence of an RAPD measuring 0.6 log units implies that significant retinal ganglion cell injury has occurred.

[Metastatic uveal melanoma. Development of neoadjuvant treatment in animal experiments]. / Metastasierendes uveales Melanom. Tierexperimentelle Entwicklung neoadjuvanter Therapien.

BACKGROUND: Up to 50% of patients with uveal melanoma develop metastases but none of the existing treatments of the primary tumor has been able to reduce the metastatic rate. Probably, micrometatases have already developed before treatment of the uveal melanoma and dormant micrometastases can persist for years before they start growing. This long time-span provides the possibility to treat micrometastases. METHODS: In order to develop an animal model for metastatic uveal melanoma, B16 melanoma cells were injected into the posterior ocular compartment of C57BL6 mice. These cells grew and metastasised to the lungs and liver. Immunological factors for the metastatic process and possible neoadjuvant treatments were investigated. RESULTS: Natural killer cells (NK) are of significance in the rejection of metastases and HLA-I expression of uveal melanomas correlates with the melanoma cell type. Interferon-alpha-2b increases the activity of NK cells and reduces the metastatic rate in the animal model. CONCLUSION: Treatment with interferon-alpha-2b results in decreased metastases from intraocular melanoma in a murine model.

Indocyanine green effect on cultured human retinal pigment epithelial cells: implication for macular hole surgery.

PURPOSE: To evaluate potential toxic effects of indocyanine green dye on cultured human retinal pigment epithelial cells. METHODS: Controlled laboratory experiment. Cultured human retinal pigment epithelial cells were exposed to balanced saline solution, balanced saline solution with endoillumination, indocyanine green or indocyanine green with endoillumination. Cells were evaluated by light microscopy, electron microscopy, and a mitochondrial dehydrogenase assay. RESULTS: Retinal pigment epithelial cells exposed to indocyanine green showed no histologic or ultrastructural changes. Those exposed to indocyanine green alone or indocyanine green plus light demonstrated a significant decrease in mitochondrial enzyme activity (P = 0.0002 and 0.005, respectively). CONCLUSION: Brief exposure of cultured human retinal pigment epithelial cells to indocyanine green results in decreased mitochondrial enzyme activity but does not appear to influence cellular morphology or ultrastructure.