H.-M. Helb

In Vivo Visualization of Dendritic Cells, Macrophages, and Microglial Cells Responding to Laser-Induced Damage in the Fundus of the Eye

PURPOSE To study the in vivo response of mononuclear phagocytes (i.e., dendritic cells [DCs] and macrophages [MPhis]) in the posterior eye segment after laser-induced injury, and to gain a better understanding of the role of these cells in inflammatory eye disease. METHODS CX(3)CR1(GFP/+) knockin mice were used, in which DCs, MPhis, and microglia cells (microGCs) are constitutively fluorescent. These reporter mice were examined by a confocal scanning laser ophthalmoscope (cSLO) after argon laser coagulation. cSLO was complemented by fluorescence microscopy of retinal flatmounts and eye cryosections, to study cell morphology and location, and by multicolor flow cytometry, to determine the number and identity of the fluorescent cells. RESULTS The retina of healthy reporter mice featured abundant fluorescent microGCs. After laser injury to the fundus, these cells accumulated and migrated laterally toward injury after 60 minutes. Distinctly shaped fluorescent cells accumulated within laser spots and were identified by flow cytometry and immunofluorescence microscopy as DCs and MPhis in the retina and choroid. The DCs rapidly disappeared from the retina, whereas the MPhis stayed longer. Choroidal infiltrates were detectable even 35 days after laser injury, in particular in larger spots resulting from higher laser intensity. In addition, nonfluorescent granulocytes were detected in the choroid. CONCLUSIONS The synergistic use of ophthalmoscopy, flow cytometry, and immunofluorescence microscopy allows detailed dissection of the in vivo response of mononuclear phagocytes to laser injury of the fundus. The number of microGCs increased in the retina. DCs and MPhis were present in the retina and choroid infiltrate. MPhis and granulocytes persisted in the choroid infiltrate longer than previously thought.

Incidence of rhegmatogenous retinal detachments after intravitreal antivascular endothelial factor injections

Acta Ophthalmol. 2011: 89: 70–75

INTRAVITREAL BEVACIZUMAB FOR NEOVASCULAR AGE-RELATED MACULAR DEGENERATION

ZusammenfassungDie Wirksamkeit des Anti-VEGF-Therapiekonzepts bei der neovaskulären altersabhängigen Makuladegeneration (AMD) konnte bereits für Pegaptanib und Ranibizumab gezeigt werden. Mit Bevacizumab steht ein Antikörper gegen den vaskulären endothelialen Wachstumsfaktor (VEGF) zur Verfügung, der ursprünglich für onkologische Anwendungsgebiete mit intravenöser Applikation entwickelt wurde. Im „Off-label use“ von Bevacizumab zeigte sich in ersten Fallberichten auch ein Therapieeffekt nach intravitrealer Applikation bei der neovaskulären AMD. Weltweit wurden mittlerweile auf diese Weise, auch aufgrund der mitunter erstaunlich positiven morphologischen und funktionellen Effekte, schon mehrere Tausend Patienten behandelt. Allerdings fehlen kontrollierte, prospektive Studien zur Sicherheit und Wirksamkeit von Bevacizumab bei der neovaskulären AMD. Hier sollen aktuelle Daten zusammengefasst und diskutiert werden. Mit dem Pooling von Daten und einer dafür eigens eingerichteten webbasierten Datenbank sollen bald aussagekräftigere Ergebnisse zur Verfügung stehen. Insbesondere gibt es noch offene Fragen hinsichtlich Dosierung, Applikationsintervall, Gesamtdauer der Applikation, biologischer Halbwertszeit und Langzeitsicherheit des Medikaments.AbstractThe efficacy and safety of the therapeutic anti-VEGF concept has already been demonstrated for pegaptanib and ranibizumab. Bevacizumab acts as an antibody against all VEGF-A isoforms and has been developed for oncological indications with intravenous application. Initial reports on intravitreal administration in patients with neovascular age-related macular disease (AMD) have shown beneficial morphological and functional effects. In the meantime, bevacizumab has been used off-label in thousands of patients with AMD. However, data from prospective, controlled, randomized trials on both safety and efficacy are lacking. Herein recent experiences with bevacizumab are summarized and discussed. Furthermore, a web-based platform for online data registration and pooled analyses is presented.

Intravitreales Bevacizumab bei der neovaskulären altersabhängigen Makuladegeneration

ZusammenfassungDie Wirksamkeit des Anti-VEGF-Therapiekonzepts bei der neovaskulären altersabhängigen Makuladegeneration (AMD) konnte bereits für Pegaptanib und Ranibizumab gezeigt werden. Mit Bevacizumab steht ein Antikörper gegen den vaskulären endothelialen Wachstumsfaktor (VEGF) zur Verfügung, der ursprünglich für onkologische Anwendungsgebiete mit intravenöser Applikation entwickelt wurde. Im „Off-label use“ von Bevacizumab zeigte sich in ersten Fallberichten auch ein Therapieeffekt nach intravitrealer Applikation bei der neovaskulären AMD. Weltweit wurden mittlerweile auf diese Weise, auch aufgrund der mitunter erstaunlich positiven morphologischen und funktionellen Effekte, schon mehrere Tausend Patienten behandelt. Allerdings fehlen kontrollierte, prospektive Studien zur Sicherheit und Wirksamkeit von Bevacizumab bei der neovaskulären AMD. Hier sollen aktuelle Daten zusammengefasst und diskutiert werden. Mit dem Pooling von Daten und einer dafür eigens eingerichteten webbasierten Datenbank sollen bald aussagekräftigere Ergebnisse zur Verfügung stehen. Insbesondere gibt es noch offene Fragen hinsichtlich Dosierung, Applikationsintervall, Gesamtdauer der Applikation, biologischer Halbwertszeit und Langzeitsicherheit des Medikaments.AbstractThe efficacy and safety of the therapeutic anti-VEGF concept has already been demonstrated for pegaptanib and ranibizumab. Bevacizumab acts as an antibody against all VEGF-A isoforms and has been developed for oncological indications with intravenous application. Initial reports on intravitreal administration in patients with neovascular age-related macular disease (AMD) have shown beneficial morphological and functional effects. In the meantime, bevacizumab has been used off-label in thousands of patients with AMD. However, data from prospective, controlled, randomized trials on both safety and efficacy are lacking. Herein recent experiences with bevacizumab are summarized and discussed. Furthermore, a web-based platform for online data registration and pooled analyses is presented.

Incidence of Damage to the Crystalline Lens During Intravitreal Injections

PURPOSE To report the incidence of traumatic lens injuries as a complication of intravitreal injection at 5 high-volume academic centers. METHODS We determined in a retrospective, interventional, multicenter case series the consecutive number of the injections between January 5, 2006 and December 22, 2008 from the injection log books. All injections were performed under sterile conditions in a laying position, 3.5-4.0 mm behind the limbus in an oblique fashion. The main outcome measure was the incidence of lens damage. RESULTS A total of 32,318 intravitreal injections were performed, and 3 cases of iatrogenic lens damage were reported during 36 consecutive months. All affected eyes were hyperopic. The overall incidence rate of lens injury was 0.006% (2/32,318) for intravitreal injections and 1 during a paracentesis 0.003 (1/32,318). The rate of phakic eyes determined was 67%, and thus, the incidence rate of lens damage in phakic eyes was 0.009% (2/21,653) (95% confidence interval, 0.00%-0.05%). CONCLUSIONS Although there is no agreement regarding the proper intravitreal injection technique, the incidence of traumatic injuries to the crystalline lens was very low in a large series of injected patients in a community setting. The incidence compares favorably with that reported in clinical trials in which much more extensive preinjection preparation was mandated. A good preparation of the surgical incision with proper anesthesia and detailed information of the patient, as well as good anatomical skills of the treating physician, are mandatory to prevent this rare adverse event.

Discrete arcs of increased fundus autofluorescence in retinal dystrophies and functional correlate on microperimetry.

PurposeTo describe the occurrence of discrete arcs of increased fundus autofluorescence (FAF) associated with various retinal dystrophies and to assess their functional significance by fundus-controlled microperimetry.MethodsSeven patients, three with pigmented paravenous retinochoroidal atrophy (PPRCA), one with sector retinitis pigmentosa (RP), one with typical RP, and two with macular dystrophy were assessed by retinal imaging including FAF imaging. Serial images were obtained within a review period of 6 and 10 years in a patient with PPRCA and macular dystrophy, respectively. Fundus-controlled microperimetry was performed in eight eyes of five patients to determine light increment sensitivity.ResultsA discrete arched line of increased FAF was observed without obvious correlate on fundus biomicroscopy. The orientation of this line differed from ring shape in RP and macular dystrophy, a semi-circle structure in sector RP to crescent shape with tiplike extensions towards branching retinal veins in PPRCA. Longitudinal investigation revealed slight migration of the arc in PPRCA and peripheral extension of the ring diameter in macular dystrophy. Microperimetry revealed that the arc of increased FAF sharply delineated areas of severely impaired retinal sensitivity.ConclusionsThe findings indicate that arcs of increased FAF in PPRCA and other retinal dystrophies demarcate areas of impaired retinal function and may migrate over time. FAF imaging may therefore reveal the exact extent of neurosensory dysfunction that may exceed the dimensions anticipated by conventional examinations.

[Monitoring of AMD patients on anti-vascular endothelial growth factor (VEGF) treatment. Practical notes on functional and anatomical examination parameters from drug approval studies, specialist information and case series].

ZusammenfassungDie altersabhängige Makuladegeneration (AMD) gehört zu den häufigsten Ursachen einer Erblindung in westlichen Industrienationen. Die meisten AMD-Patienten leiden an der trockenen Frühform der AMD, die feuchte AMD mit ihrer choroidalen Neovaskularisation (CNV) stellt die Haupterblindungsursache aller AMD-Patienten dar. Neue Perspektiven in der Therapie der AMD wurden mit pharmakologischen Verfahren entwickelt, die zur Behandlung aller Subtypen der exsudativen AMD zur Verfügung stehen. In der Pathophysiologie der feuchten AMD ist eine Vielzahl von inhibierenden und induzierenden Wachstumsfaktoren wie der vaskuläre endotheliale Wachstumsfaktor (VEGF) besonders wichtig. Das sezernierte VEGF scheint durch seine angiogene und permeabilitätssteigernde Wirkung eine entscheidende Rolle bei der Pathogenese von CNV und Makulaödemen zu haben. Diese Erkenntnisse führten zu dem heute verwendeten therapeutischen Ansatz, eine kompetitive VEGF-Blockade durch intravitreal applizierte Anti-VEGF-Medikamente zu erreichen. Durch die Anti-VEGF-Medikamente kommt es zur raschen Abnahme der retinalen Dicke. Die optische Kohärenztomographie (OCT) ist ein wertvolles Instrument in der Verlaufskontrolle, darf aber nur als Entscheidungshilfe gelten. Die klinische Nachkontrolle der Patienten und die weitere Therapieempfehlung müssen sich immer am klinischen Gesamtbild orientieren. Als entscheidendes Kriterium für eine erneute Behandlung gilt der Visus.AbstractAge-related macular degeneration (AMD) is one of the most common causes of blindness in western industrialised nations. Most AMD patients suffer from the dry early form of AMD; however, wet AMD with choroidal neovascularization (CNV) is the main cause of blindness in all AMD patients. New prospects have been developed in AMD treatment using pharmacological methods available for treating all subtypes of exudative AMD. A number of inhibiting and inducing growth factors, such as vascular endothelial growth factor (VEGF), are particularly important in the pathophysiology of wet AMD. The secreted VEGF appears to play a crucial role in the pathogenesis of CNV and macular edemas as a result of its angiogenetic and permeability-enhancing effect. This recognition led to the treatment approach now used, i.e., competitive VEGF blocking through intravitreal adminsitration of anti-VEGF drugs. The anti-VEGF durgs lead to a rapid decrease in retinal thickness. Optical coherence tomography (OCT) is a valuable monitoring tool, but may only be used to assist in decision-making. Clinical follow-up of patients and further treatment recommendations must always be guided by the overall clinical picture. Visual acuity is regarded as the decisive criterion for repeat treatment.Age-related macular degeneration (AMD) is one of the most common causes of blindness in western industrialised nations. Most AMD patients suffer from the dry early form of AMD; however, wet AMD with choroidal neovascularization (CNV) is the main cause of blindness in all AMD patients. New prospects have been developed in AMD treatment using pharmacological methods available for treating all subtypes of exudative AMD. A number of inhibiting and inducing growth factors, such as vascular endothelial growth factor (VEGF), are particularly important in the pathophysiology of wet AMD. The secreted VEGF appears to play a crucial role in the pathogenesis of CNV and macular edemas as a result of its angiogenetic and permeability-enhancing effect. This recognition led to the treatment approach now used, i.e., competitive VEGF blocking through intravitreal adminsitration of anti-VEGF drugs. The anti-VEGF durgs lead to a rapid decrease in retinal thickness. Optical coherence tomography (OCT) is a valuable monitoring tool, but may only be used to assist in decision-making. Clinical follow-up of patients and further treatment recommendations must always be guided by the overall clinical picture. Visual acuity is regarded as the decisive criterion for repeat treatment.

High-resolution optical coherence tomography of subpigment epithelial structures in patients with pigment epithelium detachment secondary to age-related macular degeneration

Background The pathophysiology of pigment epithelial detachment (PED) secondary to age-related macular degeneration (AMD) is as yet incompletely understood and treatment remains challenging. Spectral domain optical coherence tomography (SD-OCT) allows for improved morphological characterisation of the space underneath the retinal pigment epithelium (RPE). Objective To investigate eyes with PED for structures underneath the detached RPE cell layer. Methods In a retrospective observational case study, SD-OCT scans of AMD-related PEDs were assessed for the presence of distinctive morphological features in the space between the detached RPE and inner Bruchs membrane. Results Structures present in the space between the detached RPE and Bruchs membrane were found in 14 of 90 eyes with AMD-related PED. Each of these eyes shows hyper-reflective material underneath the PED, presenting as highly reflective, multilayered, laminar structures, usually orientated parallel to Bruchs membrane. Conclusions The findings indicate that SD-OCT may be useful for a more refined phenotypic stratification of AMD-associated PED. Further studies are warranted to explore the correlates on other imaging modalities, to investigate the composition of this material and to assess the potential prognostic relevance of this new finding.

Monitoring von AMD-Patienten unter Anti-VEGF-Therapie

ZusammenfassungDie altersabhängige Makuladegeneration (AMD) gehört zu den häufigsten Ursachen einer Erblindung in westlichen Industrienationen. Die meisten AMD-Patienten leiden an der trockenen Frühform der AMD, die feuchte AMD mit ihrer choroidalen Neovaskularisation (CNV) stellt die Haupterblindungsursache aller AMD-Patienten dar. Neue Perspektiven in der Therapie der AMD wurden mit pharmakologischen Verfahren entwickelt, die zur Behandlung aller Subtypen der exsudativen AMD zur Verfügung stehen. In der Pathophysiologie der feuchten AMD ist eine Vielzahl von inhibierenden und induzierenden Wachstumsfaktoren wie der vaskuläre endotheliale Wachstumsfaktor (VEGF) besonders wichtig. Das sezernierte VEGF scheint durch seine angiogene und permeabilitätssteigernde Wirkung eine entscheidende Rolle bei der Pathogenese von CNV und Makulaödemen zu haben. Diese Erkenntnisse führten zu dem heute verwendeten therapeutischen Ansatz, eine kompetitive VEGF-Blockade durch intravitreal applizierte Anti-VEGF-Medikamente zu erreichen. Durch die Anti-VEGF-Medikamente kommt es zur raschen Abnahme der retinalen Dicke. Die optische Kohärenztomographie (OCT) ist ein wertvolles Instrument in der Verlaufskontrolle, darf aber nur als Entscheidungshilfe gelten. Die klinische Nachkontrolle der Patienten und die weitere Therapieempfehlung müssen sich immer am klinischen Gesamtbild orientieren. Als entscheidendes Kriterium für eine erneute Behandlung gilt der Visus.AbstractAge-related macular degeneration (AMD) is one of the most common causes of blindness in western industrialised nations. Most AMD patients suffer from the dry early form of AMD; however, wet AMD with choroidal neovascularization (CNV) is the main cause of blindness in all AMD patients. New prospects have been developed in AMD treatment using pharmacological methods available for treating all subtypes of exudative AMD. A number of inhibiting and inducing growth factors, such as vascular endothelial growth factor (VEGF), are particularly important in the pathophysiology of wet AMD. The secreted VEGF appears to play a crucial role in the pathogenesis of CNV and macular edemas as a result of its angiogenetic and permeability-enhancing effect. This recognition led to the treatment approach now used, i.e., competitive VEGF blocking through intravitreal adminsitration of anti-VEGF drugs. The anti-VEGF durgs lead to a rapid decrease in retinal thickness. Optical coherence tomography (OCT) is a valuable monitoring tool, but may only be used to assist in decision-making. Clinical follow-up of patients and further treatment recommendations must always be guided by the overall clinical picture. Visual acuity is regarded as the decisive criterion for repeat treatment.Age-related macular degeneration (AMD) is one of the most common causes of blindness in western industrialised nations. Most AMD patients suffer from the dry early form of AMD; however, wet AMD with choroidal neovascularization (CNV) is the main cause of blindness in all AMD patients. New prospects have been developed in AMD treatment using pharmacological methods available for treating all subtypes of exudative AMD. A number of inhibiting and inducing growth factors, such as vascular endothelial growth factor (VEGF), are particularly important in the pathophysiology of wet AMD. The secreted VEGF appears to play a crucial role in the pathogenesis of CNV and macular edemas as a result of its angiogenetic and permeability-enhancing effect. This recognition led to the treatment approach now used, i.e., competitive VEGF blocking through intravitreal adminsitration of anti-VEGF drugs. The anti-VEGF durgs lead to a rapid decrease in retinal thickness. Optical coherence tomography (OCT) is a valuable monitoring tool, but may only be used to assist in decision-making. Clinical follow-up of patients and further treatment recommendations must always be guided by the overall clinical picture. Visual acuity is regarded as the decisive criterion for repeat treatment.

Accuracy, precision and repeatability in preparing the intravitreal dose with a 1.0-cc syringe

Editor, T he established dose of 0.5 mg ranibizumab (Lucentis, Novartis, Basel, Switzerland) is routinely injected into the vitreous cavity. The diluted drug has to be aspirated from the original glass vial into a disposable 1.0-cc plastic syringe and adjusted to the proposed dose. The actually expelled volume from the syringe may have a significant variance and may critically affect its biological efficacy [Meyer et al. 2010]. Thus, the presumed volume in the syringe and its preparation has to be accurate and precise, although the human error remains unknown (Figs 1 and 2). Raju & Weinberg (2002) filled 1.0-cc syringes with 0.1 versus 0.05 ml distilled water and determined a better accuracy for larger volume compared to smaller volume. Over 71% of the delivered 0.1 ml and 44% of the delivered 0.05 ml injections were within 10% of the intended volume. While this study included only one participant examining two volumes, we decided to perform a study with 11 participants preparing a set of syringes. We replaced ranibizumab by distilled water and measured the actual mass of fluid that is expelled in intravitreal injections (IVT) from individually prepared syringes. We included four nurses (group A) and seven treating physicians (group B), who were routinely involved in the preparation of syringes for IVT. All 11 health care professionals were asked to fill each 40 individual syringes with 0.05 ml distilled water (aqua ad iniectabilia, Delta Pharm, Pfullingen, Germany) in a 1.0-cc disposable syringe (BD Plastipak; Becton Dickinson & Co., Franklin Lakes, NJ, USA) in the prospective blinded study. The fluid in each syringe was slowly injected onto the top loading tray of an AC-powered high-precision weighing balance (AE 160; Mettler Toledo Lab Balances, Greifensee, Switzerland). This procedure was repeated using a total of 440 syringes from 11 individual volunteers. The balance was properly calibrated before the measurements. The mean mass of distilled water for all 11 participants dispensed onto the plastic tray was 53.75 mg (range 24.6 mg to 65.5 mg) compared to the expected mass of 50 mg or 0.05 ml water (Figs 3 and 4). The mean conformity of dispensed water was 7.39% greater than expected. Over 78% were within the 10% of the intended mass, and the calculated accuracy was 3.75 mg with a precision of ±6.38 mg. The lowest mass contained 37.5% of the highest measured. Forty-four samples (29 in group A and 15 in group B) contained <47 mg, and 52 samples (32 in group A and 20 in group B) more than 60 mg water. The mean reproducibility among all 11 participants was 4.8 mg ± 3.1 water. There was no statistical difference in the reproducibility between the two professional health care groups. In 11.8% of all samples, the equivalent ranibizumab dose was 20% or greater than expected (p = 0.02). Fig. 1. A protocol for training of nurses and physicians to prepare IVT was established. Approximately, 0.15 ml distilled water was manually aspirated into a 1.0-cc syringe using an 18-gauge needle (BD Microlance 3; Becton Dickinson & Co.). The 18-gauge needle was then exchanged by a 30-gauge needle (BD Microlance 3; Becton Dickinson & Co.), and possible air bubbles were gently expelled from the wall by tapping the syringe. The surplus of fluid was expelled from the syringe until the position of the plunger tip was adjusted to the 0.05-ml graduation line on the syringe barrel according to the individual estimation of the preparing person. The left image demonstrates the theoretical proper placement of the plugger in the syringe on the 0.05-ml graduation line of the syringe. The lower right image shows the clinical picture of two adjusted syringes, both appearing equally filled. The upper right image demonstrates, under a macroscopic magnification, a reduced volume in the left syringe. The measured mass of water delivered onto the tray is in the right syringe 45 mg, whereas 51 mg in the right syringe. Fig. 2. The graph indicates the relationship of accuracy and precision of repeated measurements as to reference value. Accuracy is defined as the degree of closeness of repeated measurements (conformity) and refers to the agreement between a measured value and the reference value. It indicates how close a measurement is to this value and relates to the quality of a result. Precision refers to the exactness of repeated independent measurements (uniformity) and indicates how close repeated measurements are under unchanged conditions. Precision gives the degree of refinement in the performance and does not relate to the reference value. It is therefore an indication of the repeatability of a result, which describes the variation arising when all efforts are made to keep conditions constant by using the same instrument and operator. Reproducibility describes the variation using the same measurement process among different operators. Thus, precision, repeatability and reproducibility relate to the quality of the performance to obtain the measurement, while accuracy relates to the quality of the values. Acta Ophthalmologica 2012