Sabine Aisenbrey

Polypoidal choroidal vasculopathy pattern in age-related macular degeneration - A clinicopathologic correlation.

Purpose: To report the histopathologic features of surgically removed submacular tissue from an elderly patient with a pattern of polypoidal choroidal vasculopathy on indocyanine green angiography. Methods: Clinical examination including fluorescein and indocyanine green angiography and light microscopy of surgical specimen. Results: A thick yellow proteinaceous subretinal fluid was seen in the right macula of an 81‐year‐old white man. Fluorescein angiography indicated progressive leakage from undetermined source apart from a few focal hyperfluorescent points. Indocyanine green angiography showed several polyps as well as dilated choroidal vessels in the macula and along the superior temporal arcade. A large plaque was visualized in the late phase. Microscopically, the specimen consisted of a thick fibrovascular membrane located on the choroidal side of the retinal pigment epithelium (RPE). The RPE layer was discontinuous whereas on its choroidal side an almost intact layer of diffuse drusen was observed. A group of dilated thin‐walled vessels were found that appeared to be saccular on serial sections. Some of these were located almost immediately under the diffuse drusen. Conclusion: Histologic examination of submacular tissue removed from an eye with polypoidal choroidal vasculopathy showed several aneurysmal dilatations located directly under diffuse drusen within a sub‐RPE, intra‐Bruchs fibrovascular membrane.

Vitreous Levels of Bevacizumab and Vascular Endothelial Growth Factor-A in Patients with Choroidal Neovascularization

PURPOSEnTo investigate the vitreous levels of bevacizumab and vascular endothelial growth factor-A (VEGF-A) after intravitreal injection of the drug in patients with choroidal neovascularization (CNV).nnnDESIGNnInterventional case series.nnnPARTICIPANTSnEleven eyes of 11 patients with submacular hemorrhage and CNV due to age-related macular degeneration (n = 10) or angioid streaks (n = 1).nnnMETHODSnAll patients were treatment naïve except for a single dose of intravitreal injection of bevacizumab (1.25 mg/50 muL dose) and subsequent vitrectomy after various intervals (1-101 days) because of active and progressive lesion. Intravitreal free bevacizumab and VEGF-A levels were measured using enzyme-linked immunosorbent assay and microsphere-based immunoassay, respectively. Vitreous VEGF-A isoforms were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting.nnnMAIN OUTCOME MEASURESnIntravitreal bevacizumab and VEGF-A levels were measured and pharmacokinetic parameters were calculated.nnnRESULTSnPharmacokinetics of intravitreal bevacizumab followed a 2-compartment model with initial and terminal half-lives of 0.5 and 6.7 days, respectively. Bevacizumab could be detected in all cases, ranging from 2.63 ng/ml to 165 microg/ml. The peak concentration was observed on the second day after intravitreal bevacizumab injection. Vitreous free VEGF-A levels ranged from 0.2 to 33.9 pg/ml and showed a negative correlation with the bevacizumab concentration (P<0.001; r = -0.955) and a positive correlation with time (P<0.001; r = 0.964). However, the percentage expression of VEGF-A(165) exhibited a positive correlation with the bevacizumab concentration (P = 0.032, r = 0.645) and a negative correlation with time (P = 0.007, r = -0.755). A time-dependent increase was found for the percentage expression of VEGF-A(189) (P = 0.023, r = 0.673). Neither bevacizumab- nor time-related alterations were found for VEGF-A(121).nnnCONCLUSIONSnBased on pharmacokinetics, the interval of 6-7 weeks would be appropriate for efficacy, although clinical trials should guide dosing recommendations. Vitreous levels of free VEGF-A showed a negative correlation with the bevacizumab concentration, which confirmed the in vivo binding affinity of bevacizumab to VEGF-A. The analysis of the VEGF-A isoforms suggests differences of interaction between bevacizumab and individual VEGF-A isoforms.

Clinicopathological correlation in exudative age related macular degeneration: histological differentiation between classic and occult choroidal neovascularisation.

AIMS To analyse the histopathology of classic and occult choroidal neovascular membrane surgical specimens in age related macular degeneration. METHODS 35 membranes, from a consecutive series of surgically removed choroidal neovascular membranes in age related macular degeneration, were classified as classic or occult following the guidelines of the Macular Photocoagulation Study. Membranes with classic as well as occult components were considered as mixed membranes. The membranes were serially sectioned and stained with haematoxylin and eosin, Masson trichrome, periodic acid-Schiff, and phosphotungstic acid haematoxylin stain. The correlation has been made in a masked fashion. RESULTS 31 membranes (19 classic, 10 occult, and two mixed membranes) could be analysed histologically. 18 classic choroidal neovascular membranes had a major subretinal fibrovascular component and 10 of these had an additional, minor fibrovascular component under the retinal pigment epithelium. The 10 occult membranes contained a fibrovascular component under the retinal pigment epithelium and the two mixed membranes contained fibrovascular tissue on both sides of the retinal pigment epithelium. Fibrin and remains of outer segments tended to occur at the lateral edges of classic membranes and to cover the inner surface of occult membranes. CONCLUSION Classic choroidal neovascularisation in age related macular degeneration is predominantly composed of subretinal fibrovascular tissue while occult choroidal neovascularisation is composed of fibrovascular tissue at the choroidal side of the retinal pigment epithelium.

SAFETY PROFILE OF BEVACIZUMAB ON CULTURED HUMAN CORNEAL CELLS

Purpose: To study the corneal biocompatibility of bevacizumab on various cultured human corneal cells. Methods: Cell cultures of corneal keratinocytes (CKs), corneal fibroblasts (CFs), and corneal endothelial cells (CECs) were harvested from human donor eyes and exposed to various concentrations of bevacizumab (0.25-5.0 mg/mL). Cell viability was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at days 1 and 4 after exposure. For cytotoxicity testing, confluent cells were cultured in serum-depleted medium, and the MTT test was performed after 24 hours of incubation. Expression of vascular endothelial growth factor (VEGF), VEGF receptors (VEGFR1 and VEGFR2), keratan sulphate (KS), and cytokeratin-3 (AE5) was studied by immunohistochemistry. Live/dead viability/cytotoxicity assay was performed and analyzed by fluorescence microscopy after 24 hours of incubation. Cell morphology was assessed with a phase-contrast microscope after 7 days of exposure with different concentrations of bevacizumab (0.25-5.0 mg/mL), and signs of cellular damage were assessed. Results: No cytotoxic effect of bevacizumab on CKs, CFs, and CECs could be observed when used at a concentration of 5.0 mg/mL or lower. Bevacizumab-treated cells showed no signs of cellular damage compared with the control. CKs, CFs, and CECs stained positively for VEGF, VEGFR1, and VEGFR2. CKs and CECs stained positively for AE5, whereas CFs were immunopositive for KS. Conclusions: Bevacizumab is not toxic to corneal cells of human origin in vitro at doses usually used for treatment of corneal neovascularization, which is 20-fold higher than that used for intravitreal application.

Clinicopathological correlation of deep retinal vascular anomalous complex in age related macular degeneration

AIMS To analyse the histopathology of “deep retinal vascular anomalous complex” or “chorioretinal anastomosis”. METHODS Six patients with a deep retinal vascular anomalous complex (age range 66–88 years) had fundus photography and fluorescein angiography not more than 14 days before foveal translocation surgery. Four patients were also documented with indocyanine green angiography. The surgical specimens were serially sectioned and stained in a stepped fashion with Masson trichrome, periodic acid Schiff, and phosphotungstic acid haematoxylin, a histochemical stain for fibrin. RESULTS A subretinal fibrovascular membrane was surrounded by a rim consisting of diffuse drusen (basal laminar deposits), retinal pigment epithelium, and amorphous, fibrinous material interspersed with remains of outer segments in all specimens. In two specimens vascular structures were identified that left the specimen towards the retina. Amorphous material with the remains of outer segments was not found on the retinal side of the fibrovascular tissue itself but in four specimens a small neuroretinal portion (outer nuclear layer) was adherent to the complex. In three specimens a thin fibrocellular membrane was seen at the choroidal side of the diffuse drusen. CONCLUSION Deep retinal vascular anomalous complex represents histologically neovascularisation growing out of the neuroretina, into the subretinal space, which mimics choroidal neovascularisation. The term therefore appears rightly chosen.

Efficacy of intravitreal bevacizumab in treating postoperative pseudophakic cystoid macular edema

PURPOSE: To describe the efficacy of intravitreal bevacizumab (Avastin) in patients with cystoid macular edema (CME) after cataract surgery (Irvine‐Gass syndrome). METHODS: This retrospective case series comprised 16 eyes of 16 patients with CME after cataract surgery refractory to current standard treatment who received an injection of 1.25 mg intravitreal Avastin. The main outcome measures were visual acuity, retinal thickness on optical coherence tomography (OCT), and complications related to treatment. RESULTS: The median duration of CME before treatment with intravitreal Avastin was 14 weeks (range 3 to 84 weeks). Although the mean retinal thickness decreased slightly after intravitreal Avastin, the mean visual acuity remained unchanged. Visual acuity improved by 2 Early Treatment Diabetic Retinopathy Study lines in 1 patient, remained unchanged in 12 patients, and decreased by 2 lines in 2 patients. Repeated Avastin injections did not result in a better outcome. Other than mild ocular irritation, there were no adverse effects of the intravitreal injections. CONCLUSIONS: Intravitreal injection of Avastin, although safe, did not result in improved visual function in patients with postoperative CME. In contrast to findings in a previous case report, the beneficial effect of vascular endothelial growth factor inhibition in Irvine‐Gass syndrome was not observed.

Z-suture: a new knotless technique for transscleral suture fixation of intraocular implants

The presented Z-suture is a simple, rapid and safe knotless technique that facilitates transscleral suture fixation of various intraocular implants in the ciliary sulcus, such as sutured intraocular lenses, artificial iris prostheses and iris diaphragms. As the knotless approach reliably avoids suture erosion, external fixation can be performed without any protecting scleral flaps or lamellar grooves. The needle is simply passed through the sulcus and the emerging polypropylene suture is secured in the sclera using a zigzag-shaped intrascleral suture (Z-suture). Each pass starts directly adjacent to the exiting site. Five passes are sufficient to reliably fix the suture so that it resists even maximum tractive forces. Once this procedure is done, the suture can be cut without any knot. By avoiding suture knots, and hence the need for intrascleral flaps, this knotless approach may help to reduce suture-related complications such as scleral atrophy, suture erosion and infections.

Sutureless amniotic membrane fixation using fibrin glue for ocular surface reconstruction in a rabbit model.

Purpose: Amniotic membrane transplantation has become an important treatment option for corneal surface reconstruction. However, suture fixation of the transplant has various disadvantages like corneal irritation, scarring, graft loss due to membrane shrinkage, and the need for subsequent suture removal. Replacement of sutures by bioadhesives might be an advantageous alternative. This controlled study was designed to evaluate a new sutureless technique for amniotic membrane fixation onto the corneal surface by using fibrin glue. Methods: Standardized disks of cryopreserved amniotic membranes were transplanted onto the deepithelialized cornea of 12 rabbits using either conventional suture fixation or a new fibrin glue technique. The rabbits were followed-up with slit-lamp examination and fluorescein staining until epithelialization was completed. Consecutively, the rabbits were killed and the eyes processed for histology and immunohistochemistry for cytokeratin-3. Results: All membranes of both groups stayed in place throughout the follow-up time and showed a progressive graft epithelialization that was completed after 12 days. Whereas suture-fixated membranes showed progressive tissue shrinkage, fibrin-glued sheets remained unaltered. In the bioadhesive group, histology revealed a smooth fibrin layer in the graft-host interface and a continuous, stratified layer of cytokeratin-3 expressing corneal epithelial cells on the membrane surface. In contrast, suture-fixated membranes showed contracted and prominent membrane edges with epithelial ingrowth into the submembrane interface. Conclusion: Our results demonstrate the general feasibility of reproducible and reliable sutureless amniotic membrane fixation onto the corneal surface in rabbits. Stable adherence is maintained until epithelialization is completed. The sutureless technique gives sufficient manipulation time for the sheet before the final cross-linking process is completed. Furthermore, several advantageous characteristics could be demonstrated as increased biocompatibility, better epithelialization pattern and the lack of membrane shrinkage.

Ganciclovir for the treatment of anterior uveitis

Abstractu2002Background: Ganciclovir, administered systemically or intraocularly, is effective in controlling cytomegalovirus (CMV) retinitis in immunocompromised patients. The efficacy of therapy with this antiviral substance was investigated in an immunocompetent patient with CMV uveitis causing secondary glaucoma. Methods: To identify the presence of an intraocular viral infection, anterior chamber taps to detect the intraocular synthesis of IgG antibodies and PCR testing were carried out. Clinically, the degree of intraocular inflammation and the intraocular pressure (IOP) values were monitored. During this time, the patient was treated systemically with ganciclovir administered orally and intravenously. Results: The intraocular synthesis of IgG antibodies specific for CMV was found in two samples of aqueous humor, but negative for other viruses. PCR testing was negative for HSV, VZV and CMV at each time. During this time, the patient was treated systemically with ganciclovir administered either intravenously or orally. As a response to therapy with ganciclovir, the elevated IOP values decreased to normal and the intraocular inflammation declined. After cessation of ganciclovir administration, the inflammation and secondary glaucoma recurred. Conclusion: In this case of anterior uveitis presumably caused by CMV inducing secondary glaucoma, treatment with ganciclovir led to a decrease of the inflammation and normalization of IOP. It appears that continuous administration may be required to control the infection in an immunocompetent patient.

Comparative toxicity and proliferation testing of aflibercept, bevacizumab and ranibizumab on different ocular cells

Background/aims Vascular endothelial growth factor (VEGF) is a key factor in the pathogenesis of neovascular retinal diseases including age-related macular degeneration. VEGF inhibitors including ranibizumab, pegaptanib or bevacizumab improve retinal morphology and vision in many patients. The recently approved drug aflibercept (VEGF Trap-Eye/Eyelea, Regeneron, Tarrytown, New York, USA) offers a new therapy modality. We therefore tested for toxic and anti-proliferating effects of aflibercept. Methods The effects of aflibercept (0.125, 0.5, 2u2005mg), ranibizumab (0.125u2005mg) and bevacizumab (0.3125u2005mg) after 1, 24, 48 and 72u2005h on cell morphology via phase contrast pictures, cell viability via MTS assay, total cell amount via crystal violet staining, apoptosis induction via caspase 3/7 assay and proliferation via BrdU assay were investigated. Three ocular cell lines were chosen for toxicology testing: ARPE19 cells, RGC-5 cells and 661W cells. Results Aflibercept did not cause changes in cell morphology, induce apoptosis or cause permanent decrease in cell viability, cell density or proliferation in any cell line or concentration investigated. In general, aflibercept had fewer effects (upregulation or downregulation) compared with controls than bevacizumab or ranibizumab. Conclusions In our experiments, aflibercept did not lead to any negative effects on retinal cell lines and might therefore be used safely in clinical applications.