Paul B. Henrich

Nanoscale Topographic and Biomechanical Studies of the Human Internal Limiting Membrane

PURPOSE The purpose of this article was to create a nanometer scale topographic and biomechanical profile of the human internal limiting membrane (ILM) under native conditions. METHODS ILMs from the posterior pole of postmortem human eyes were prepared as flat mounts and investigated by atomic force microscopy (AFM) under physiological conditions. Structural analysis was complemented by transmission electron microscopy. RESULTS Average thickness of the fully hydrated, native ILMs was 3488 ± 460 nm. Thickness variations from 100 nm to 4326 nm characterized the fovea, which displayed a craterlike morphology. Outside the fovea, thickness distribution was uniform. Although mean ILM thicknesses were similar, standard deviation was higher on the retinal than on the vitreal side, indicating greater roughness. Average ILM stiffness was more than fivefold higher on the retinal than on the vitreal side (227 vs. 44 kPa). CONCLUSIONS A detailed topographical and nanomechanical profile of native human ILM was generated using AFM. Thickness values were significantly higher than in previous studies because of the preservation of native conditions. Both thickness and stiffness showed marked variations around the fovea but were relatively uniform outside the foveal area. Interestingly, the foveal ILM displayed a craterlike morphological appearance with four distinct layers separated by comparatively steep thickness increments. ILM stiffness was considerably higher on the retinal than on the vitreal side. AFM opens new possibilities for investigating native basement membranes under physiological and pathological conditions. Transmission electron microscopy revealed higher extracellular matrix protein density on the retinal than on the vitreal side.

Sequential epiretinal membrane removal with internal limiting membrane peeling in brilliant blue G-assisted macular surgery

Purpose To assess the selectivity of brilliant blue G (BBG) staining by analysing the morphological components of unstained and stained tissue obtained during epiretinal membrane (ERM) removal with internal limiting membrane (ILM) peeling in BBG-assisted macular surgery. Methods Twenty-six surgical specimens were removed from 13 eyes with epiretinal gliosis during vitrectomy using BBG for ERM and ILM peeling. We included eyes with idiopathic macular pucker, idiopathic macular hole and vitreomacular traction syndrome. The dye was injected into the fluid-filled globe. Unstained and stained epiretinal tissue was harvested consecutively and placed into separate containers. All specimens were processed for conventional transmission electron microscopy. Results The first surgical specimen of all eyes showed no intraoperative staining with BBG and corresponded to masses of cells and collagen. The second surgical specimen demonstrated good staining characteristics and corresponded to the ILM in all patients included. In seven eyes, the ILM specimens were seen with minor cell proliferations such as single cells or a monolayer of cells. Myofibroblasts, fibroblasts and astrocytes were present. In five cases, native vitreous collagen fibrils were found at the ILM. In six of the eyes, ILM specimens were blank. Conclusion Our clinicopathological correlation underlines the selective staining properties of BBG. The residual ILM is selectively stained by BBG even when a small amount of cells and collagen adheres to its vitreal side. To reduce the retinal exposure to the dye, the surgeon might choose to remove the ERM without using the dye, followed by a BBG injection to identify residual ILM.

New concepts in basement membrane biology

Basement membranes (BMs) are thin sheets of extracellular matrix that outline epithelia, muscle fibers, blood vessels and peripheral nerves. The current view of BM structure and functions is based mainly on transmission electron microscopy imaging, in vitro protein binding assays, and phenotype analysis of human patients, mutant mice and invertebrata. Recently, MS‐based protein analysis, biomechanical testing and cell adhesion assays with in vivo derived BMs have led to new and unexpected insights. Proteomic analysis combined with ultrastructural studies showed that many BMs undergo compositional and structural changes with advancing age. Atomic force microscopy measurements in combination with phenotype analysis have revealed an altered mechanical stiffness that correlates with specific BM pathologies in mutant mice and human patients. Atomic force microscopy‐based height measurements strongly suggest that BMs are more than two‐fold thicker than previously estimated, providing greater freedom for modelling the large protein polymers within BMs. In addition, data gathered using BMs extracted from mutant mice showed that laminin has a crucial role in BM stability. Finally, recent evidence demonstrate that BMs are bi‐functionally organized, leading to the proposition that BM‐sidedness contributes to the alternating epithelial and stromal tissue arrangements that are found in all metazoan species. We propose that BMs are ancient structures with tissue‐organizing functions and were essential in the evolution of metazoan species.

Anatomical and functional outcome in brilliant blue G assisted chromovitrectomy

Purpose:  To evaluate the potential of brilliant blue G (BBG) for intraoperative staining of the inner limiting membrane (ILM) with respect to staining properties and surgical outcome.

The Bi-Functional Organization of Human Basement Membranes

The current basement membrane (BM) model proposes a single-layered extracellular matrix (ECM) sheet that is predominantly composed of laminins, collagen IVs and proteoglycans. The present data show that BM proteins and their domains are asymmetrically organized providing human BMs with side-specific properties: A) isolated human BMs roll up in a side-specific pattern, with the epithelial side facing outward and the stromal side inward. The rolling is independent of the curvature of the tissue from which the BMs were isolated. B) The epithelial side of BMs is twice as stiff as the stromal side, and C) epithelial cells adhere to the epithelial side of BMs only. Side-selective cell adhesion was also confirmed for BMs from mice and from chick embryos. We propose that the bi-functional organization of BMs is an inherent property of BMs and helps build the basic tissue architecture of metazoans with alternating epithelial and connective tissue layers.

Quantification of Contrast Recognizability during Brilliant Blue G- and Indocyanine Green-Assisted Chromovitrectomy.

PURPOSE To evaluate the potential of brilliant blue G (BBG) and indocyanine green (ICG) for intraoperative staining of the internal limiting membrane (ILM) with respect to perceivable contrast. METHODS In a retrospective clinical case series the authors analyzed 26 consecutive chromovitrectomy interventions in 26 patients with macular holes, epiretinal fibrosis, vitreoretinal traction syndromes, or persistent macular edema. Fourteen subjects underwent ICG and 12 subjects, BBG chromovitrectomy. The main outcome measure was the difference in chromaticity between the stained ILM and the unstained underlying retina, measured by means of a novel objective and quantitative video-based analysis method to describe color contrast strengths as they are perceived by the human eye. RESULTS Objective chromaticity measurements of the intraoperative videos of all 26 interventions showed a significantly inferior contrast for BBG compared with that of ICG (BBG = 6.1, ICG = 14.9; P = 3.885 × 10⁻¹⁵). CONCLUSIONS As an adjunct to chromovitrectomy to stain the ILM, BBG yields a significantly less well discernible contrast to the human eye than that of ICG under the premises of this study.

Vital dyes increase the rigidity of the internal limiting membrane

PurposeTo assess the stiffness of the natural human internal limiting membrane (ILM) and evaluate potential changes of the mechanical properties following staining with brilliant blue (BB) and indocyanine green (ICG).MethodsUnstained ILM specimens were obtained during ophthalmic surgical procedures. After removal, the specimens were dissected into five parts. Two fragments were stained with BB and ICG, respectively, for 1 min, another two specimens were stained similarly followed by additional subsequent illumination using a standard light source (PENTA LUX x 50, Ophthalmologische Systeme GmbH Fritz Ruck). The fifth part served as an untreated control. All specimens were then analyzed using atomic force microscopy (AFM) in contact mode with a scan rate of 0.6 Hz. Two scan regions of 10 × 10 μm were chosen and stiffness was determined by using AFM in a force spectroscopy mode. The force curves were plotted with a data rate of 5000 Hz. In all specimens both the retinal side and vitreal side were analyzed.ResultsStaining resulted in a significant increase in tissue stiffness. An increase was seen both for the vitreal (BB: P<0.001; ICG: P<0.01) and retinal side (BB: P<0.01; ICG: P<0.01), with the retinal side being significantly stiffer in all control and stained samples. Additional illumination after staining did further increase tissue rigidity in most samples but not significantly.ConclusionsStaining significantly increases the stiffness of the human ILM. This might explain the fact that the stained ILM can be removed more easily and in larger fragments during vitreoretinal surgical procedures compared with unstained ILM.

The price for reduced light toxicity: Do endoilluminator spectral filters decrease color contrast during Brilliant Blue G–assisted chromovitrectomy?

BackgroundVitreoretinal surgeons have been slow to adopt the use of spectral filters for endoillumination to reduce retinal light toxicity. This study shows that spectral filters can be used without a loss in color contrast during brilliant blue G chromovitrectomy.MethodsTo evaluate the influence of intra operative spectral light filters on perceivable contrast during Brilliant Blue G chromovitrectomy, a prospective, observational clinical study was carried out on 59 consecutive Brilliant Blue G chromovitrectomy interventions in 59 patients admitted for macular holes, macular pucker or vitreomacular traction syndromes. Subsequent to peeling of the internal limiting membrane, six different illumination modes were enabled consecutively: mercury vapor, mercury vapor/xenon, and xenon followed by xenon combined with an amber, green or yellow spectral filter. Main outcome measure was the chromaticity spread between stained internal limiting membrane and unstained retina as a measure for the color contrast perceived by the human eye.ResultsMean chromaticity scores were similar for all light sources: mercury vapor 7.97, mercury vapor/xenon 7.96 (p = 0.96), and xenon 7.41 (p = 0.55). Compared to xenon, the additional use of endoillumination spectral filters did not change contrast recognizability: Chromaticity scores were 9.38 for the amber filter (p = 0.13), 6.63 for the green and 7.02 for the yellow filter (p = 0.37 and 0.64, respectively). When comparing the different filters head-to-head, the amber filter was superior to the green filter (p = 0.03), while the yellow was intermediate and not significantly different from either the amber (p = 0.08) or the green filter (p = 0.51).ConclusionsColor contrast perceptibility during Brilliant Blue G assisted chromovitrectomy is similar with mercury vapor, mercury vapor/xenon or xenon light sources. Spectral filters do not decrease color contrast recognizability. Head-to-head comparison shows a significant advantage for the amber over the green filter with respect to contrast generation, the yellow filter is intermediate. As spectral filters are known to greatly reduce retinal light toxicity, we suggest donor eye studies to validate whether the amber filter should be generally recommended for Brilliant Blue G chromovitrectomy.

Increase in lens capsule stiffness caused by vital dyes

Purpose To assess potential changes in lens capsule mechanical properties after staining with brilliant blue, indocyanine green (ICG), and trypan blue. Setting Department of Ophthalmology and Applied Physics and Center for NanoScience, Ludwig‐Maximilians‐University, Munich, Germany. Design Experimental study. Methods Fifteen unstained lens capsules were dissected into 7 wedge‐shaped parts. Three fragments were stained with brilliant blue 0.025%, ICG 0.05%, and trypan blue 0.06%, respectively, for 1 minute. Another 3 specimens were additionally illuminated using a standard light source. The seventh part served as an untreated control. All specimens were analyzed using atomic force microscopy (AFM) in contact mode with a scan rate of 0.6 Hz. Two scan regions of 10 &mgr;m × 10 &mgr;m were chosen, and stiffness was determined using AFM in a force spectroscopy mode. The force curves were performed with a data rate of 5000 Hz. Results Staining of the samples resulted in an increase in tissue stiffness (brilliant blue: P<.001; ICG: P<.01; trypan blue: P<.05). Additional illumination after staining further increased tissue stiffness, but not significantly. Mean increase in the relative elasticity values were 1.61 ± 0.15 (SD) for brilliant blue, 2.04 ± 0.21 for brilliant blue with illumination, 1.63 ± 0.22 for ICG, 2.01 ± 0.22 for ICG with illumination, 1.23 ± 0.11 for trypan blue, and 1.39 ± 0.11 for trypan blue with illumination. In relation to unstained tissue, the relative elasticity of the stained tissue increased 1.2‐fold after illumination. Conclusion Staining significantly increased the mechanical properties of the human lens capsule. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

Contrast recognizability during brilliant blue G - and heavier-than-water brilliant blue G-assisted chromovitrectomy: a quantitative analysis.

Purpose:  To evaluate the potential of heavier‐than‐water brilliant blue G (BBG‐D20) to stain the internal limiting membrane (ILM) during chromovitrectomy.