Reinhard Eggers

Histologic analysis of thermal effects of laser thermokeratoplasty and corneal ablation using Sirius-red polarization microscopy

Purpose: To evaluate how well several histologic techniques differentiate degrees of thermally induced changes in corneal tissue after laser thermokeratoplasty (LTK) or corneal ablation. Setting: Medical Laser Center Lübeck, Germany. Methods: Corneas of freshly enucleated porcine eyes were treated with a continuous wave laser diode (1.86 &mgr;m) and a pulsed chromium‐thulium‐holmium:YAG laser (2.1 &mgr;m) to produce LTK lesions or ablated with a Q‐switched and a free‐running chromium‐erbium:YSGG laser (2.70 &mgr;m), a free‐running erbium:YAG laser (2.94 &mgr;m), and an argon‐fluoride excimer laser (193 nm). The lesions were evaluated by light microscopy (LM) (hematoxylin and eosin, Azan, van Gieson’s, and Masson‐Goldner’s trichrome stains), transmission electron microscopy (TEM), and polarization microscopy after Sirius‐red staining. Sirius‐red, a strongly elongated, birefringent molecule binding parallel to collagen molecules, was used to enhance corneal birefringence. Results: With routine LM, it was difficult to discriminate the degrees of thermal alterations in LTK lesions. Combined Sirius‐red staining and polarization microscopy distinguished between a strongly coagulated central zone and the transition zone to normal tissue. Sirius‐red uptake was increased in both zones, reflecting the availability of new binding sites. The central zone appeared darker under polarization than normal collagen because of a loss of birefringence. Intrinsic birefringence was greatly reduced; however, form birefringence partly remained as long as some collagen fibrils were intact. In the center of very strong lesions, where the collagen was hyalinized, birefringence was completely lost because of the complete disintegration of the fibrillar structure, which was visible under TEM. The transition zone toward normal cornea showed increased birefringence because the natural birefringence was largely preserved and enhanced by the increased Sirius‐red uptake. Mechanical stretching between neighboring LTK lesions was manifested by increased birefringence. Conclusion: Sirius red offered an improved and simple histologic method for analyzing thermal collagen changes. It may contribute to a better understanding of the working mechanisms of LTK and improve analysis of thermal effects in corneal ablation.

Systematic investigations of the contrast results of histochemical stainings of neurons and glial cells in the human brain by means of image analysis

The investigation of neurohistological specimens by image analysis has become an important tool in morphological neuroscience. The problems which arise during the processing of these images are non-trivial, especially if a pattern recognition of cells in the imaged tissue is intended. One of the major problems faced concerns the segmentation of structures of interest, whether cells or other histologic structures. The segmentation problem is often the result of an inappropriate staining procedure. For serious image analysis to be performed, the material under investigation must be optimally prepared. Spatially complex patterns, e.g. fuzzy-like neighbouring neurons, are easy to recognize for humans. But the integrative and associative performance of current artificial neuronal network schemes is too low to achieve the same recognition quality as humans do. Therefore, a general analysis of staining characteristics was performed, especially with respect to those stains which are relevant to object segmentation. Although most image analytical investigations of tissues are based on stained samples, a study of this type has not been previously conducted. Of the stains and procedures evaluated, the gallocyanin chrome alum combination staining provided the best stain contrast. Furthermore, this staining method shows sufficient constancy within different parts of the human brain. Even the fine nuclear textures are differentiable and can be used for further pattern recognition procedures.

Flat‐bed scanning as a tool for quantitative neuroimaging

The aim of this study was to compare three different imaging techniques which are used to provide data on the laminar structure of the human cerebral cortex. Region V1 of Brodmanns area 17 stained with cresyl violet was investigated, and a conventional semi‐automatic morphometric evaluation, the videomicroscropic procedure and a new transparent flat‐bed scanning technique were compared. The results of each digitizing method were converted into normalized profiles which allow the laminae in the striate cortex to be displayed. It was found that major laminar patterns can be detected by the scanning technique, but that subsidiary laminations are more clearly displayed by morphometry and videomicroscopy. For magnifications up to × 400 a high resolution transparent flat‐bed scanner may be used in place of the videomicroscopy technique.

Pioglitazone Prevents Capillary Rarefaction in Streptozotocin-Diabetic Rats Independently of Glucose Control and Vascular Endothelial Growth Factor Expression

Background/Aims: Reduction of capillary network density occurs early in the development of metabolic syndrome and may be relevant for the precipitation of diabetes. Agonists of the peroxisome proliferator-activated receptor (PPAR)-γ transcription factor are vasculoprotective, but their capacity for structural preservation of the microcirculation is unclear. Methods: Male Wistar rats were rendered diabetic by streptozotocin and treated with pioglitazone in chow for up to 12 weeks. Capillary density was determined in heart and skeletal muscle after platelet endothelial cell adhesion molecule-1 (PECAM-1) immunostaining. Hallmarks of apoptosis and angiogenesis were determined. Results: Capillary density deteriorated progressively in the presence of hyperglycemia (from 971/mm2 to 475/mm2 in quadriceps muscle during 13 weeks). Pioglitazone did not influence plasma glucose, left ventricular weight, or body weight but nearly doubled absolute and relative capillary densities compared to untreated controls (1.2 vs. 0.6 capillaries/myocyte in heart and 1.5 vs. 0.9 capillaries/myocyte in quadriceps muscle) after 13 weeks of diabetes. No antiapoptotic or angiogenic influence of pioglitazone was detected while a reduced expression of hypoxia-inducible factor-3α and PPAR coactivator-1α (PGC-1α) mRNA as well as vascular endothelial growth factor (VEGF) protein possibly occurred as a consequence of improved vascularization. Conclusion: Pioglitazone preserves microvascular structure in diabetes independently of improvements in glycemic control and by a mechanism unrelated to VEGF-mediated angiogenesis.

The ossification centre of the talus

The ossification of the talus was studied in plastinated and histological preparations of normal feet of eight newborn children. Quantitative data on the newborn talus were obtained with the IBAS image analysis system and by point counting methods. In the newborn talus up to 24 percent of the talar anlage already consists of bony tissue. The ossification centre is situated in the neck, which includes the non-articulating surfaces of the talus. Periosteal bone joins the endochondral centre below and, in well-differentiated specimens also above. The basal periosteal collar forms the surfaces of the sinus and canalis tarsi, whereas the cranial bony collar is included in the tibiotalar joint. The histological architecture of these periosteal collars differs. Four arteries contribute to the blood supply of the talar ossification centre.

Quantitative investigations into the histostructural nature of the human putamen. I. Staining, cell classification and morphometry.

Combined staining with aldehyde and cresyl violet allows a reliable morphological distinction to be made between seven different types of neurons in the human putamen. We examined the age distribution of nearly 42,000 neurons in 27 normal putamina, using a semiautomatic morphometric procedure on defined tissue blocks. For morphometric evaluation and stereological calculations a section thickness of 20 microns is recommended. We modified routine aldehyde fuchsin cresyl violet combination staining for nervous tissue, since Braaks original method (Braak 1978, 1980) was developed for thick sections. The results show that neuronal density varies with age for the different types of neurons.

Prophylactic gamma radiation of unaffected vein grafts failed to prevent vein graft disease in a chronic hypercholesterolemic porcine model

OBJECTIVE The value of prophylactic brachytherapy on vein graft disease is unknown. METHODS AND RESULTS Vein bypass grafts in 23 hypercholesterolemic pigs after ex vivo gamma irradiation of the vein grafts (10, 20, and 40Gy) and 16 control veins were analyzed regarding: (1) expression of platelet-derived growth factor (PDGF-AA and -BB, ELISA); (2) smooth muscle cell (SMC) proliferation/cell death (double-immunohistochemistry Mib-1/TUNEL/SMC alpha-actin); and (3) vessel wall dimensions. Planimetric data on vessel wall dimensions revealed no positive effect of gamma radiation on neointima formation and inner lumen diameter. On the contrary, vein grafts subjected to 40Gy were significantly more likely to be occluded and to have reduced inner lumen and increased neointima formation. Radiation therapy had no effect on PDGF expression and SMC proliferation/cell death. The mean inner lumen diameter decreased as PDGF-AA expression increased. CONCLUSIONS Prophylactic gamma radiation of unaffected vein grafts failed to prevent vein graft disease in a hypercholesterolemic porcine model. High-dose radiation (40Gy) resulted in more frequent graft occlusion and vein sclerosis.

Stereologic evaluation of the vasculature in a MX1 xenotransplanted tumour model after combinations of treatment with ifosfamide, hyperthermia and irradiation.

The vascularization of tumours is a critical parameter of their growing and metastatic behaviour. However, little is known about the morphologic reactions of the microvasculature, especially the capillary bed of tumours and the adjacent tissue. In this study, the vessels in MX1 xenotransplants in athymic nu/nu nude mice were quantified and the angioarchitecture was visualized with the aim of presenting stereologic parameters of vessels based on a morphometric analysis of post mortem tissue blocks which were processed by standard histological procedures. In order to study changes of the microvasculature of MX1 tumours, the xenotransplanted nude mice were treated by different therapeutic regimens. Standardized hyperthermia, ifosfamide and irradiation therapies were applied. Special interest was focused on early changes of capillaries and of the pre- as well as post-terminal vascular bed. The stereologic evaluation of capillaries and larger vessels immediately after the therapy with ifosfamide and hyperthermia shows an increase of the mean capillary sizes. Furthermore, tumour samples after the 5th day of irradiation (5 x 2 Gy) and combinations of irradiation and chemotherapy treatment have been investigated. After 5 days of irradiation, a significant decrease of the vascular density was found. The results presented here clearly show that the timing and the mode of therapy influence the capillary morphology and periterminal vasculature of xenotransplanted MX1 tumours.

Darstellung von LTK-Läsionen durch optische Kurzkohärenztomographie (OCT) und Polarisationsmikroskopie nach Sirius-Rot-Färbung*

Hintergrund: Für die Kontrolle der Laserdosis und der postoperativen Entwicklung bei Laserthermokeratoplastik (LTK) sind Informationen über die Ausdehnung und das Ausmaß der thermischen Läsionen von großer Bedeutung. Wir verglichen den Informationsgehalt der Darstellung frischer LTK-Läsionen durch Optische Kurzkohärenztomographie [optical low coherence tomography (OCT)] mit der histologischen Darstellung durch Polarisationsmikroskopie.Methode: Die Kornea von Schweineaugen wurde in vitro durch eine 400-µm-Quarzfaser mit Licht aus einer Laserdiode bestrahlt, die bis zu 300 mW bei λ=1,86 µm emittiert. Es wurden OCT-Querschnittsbilder verschieden starker Läsionen mit etwa 25 µm lateraler und 20 µm axialer Auflösung aufgenommen. Die mit OCT analysierten Bereiche wurden histologisch durch Polarisationsmikroskopie nach Sirius-Rot-Färbung untersucht und mit der Darstellung durch TEM verglichen.Ergebnisse: Es konnten 3 Schadenszonen unterschieden werden: Übergangszone, mäßige und starke Koagulation. In der Übergangszone korrelierte die unter Polarisation sichtbare verstärkte Doppelbrechung mit einer Zunahme des OCT-Signals. Bei mäßiger Koagulation ging eine Abnahme der Doppelbrechung mit einer weiteren Zunahme des OCT Signals einher. Bei starker Koagulation im Zentrum der Läsion ging die Kollagenfaserstruktur weitgehend verloren, wodurch Doppelbrechung und OCT-Signal fast völlig verschwinden.Schlußfolgerungen: OCT liefert zwar eine weniger detaillierte Information über thermische Gewebsveränderungen als eine histologische Untersuchung, gibt aber Aufschlußüber die Ausdehnung und das Ausmaß der Veränderungen und ermöglicht eine nichtinvasive Darstellung und Verlaufskontrolle von LTK-Läsionen ohne Präparationsartefakte. Eine quantitative Analyse der Veränderung der Hornhautdicke ist leichter möglich als mit der Spaltlampe. Zeitaufgelöste Messungen der Lichtstreuung können zur Online-Kontrolle der Laserdosis bei der LTK verwendet werden.Background: Information on the extent and degree of the thermal effect produced is of great importance for control of the laser dosage in laser thermokeratoplasty (LTK) and for postoperative follow-up. We investigated on acute LTK effects which information images obtained by optical low coherence tomography (OCT) offer compared to those obtained by polarization microscopy.Methods: Porcine eyes were irradiated through a 400 µm quartz fiber using light from a laser diode emitting up to 300 mW at a wavelength of 1.86 µm. Thermal lesions of varying strength were scanned using an experimental OCT device with about 25 µm lateral and 20 µm axial resolution. Histologic evaluation of the scanned areas was done by polarization microscopy after Sirius-Red staining, and similar lesions were also analyzed by TEM.Results: Both methods differentiated three damage zones: a transition zone, a zone of mode-rate coagulation, and a central zone of strong coagulation. In the transition zone, increased birefringence was seen in polarization microscopy, which correlated with increased light scattering seen in the OCT images. In the moderately coagulated zone, a decrease in birefringence was associated with an even stronger increase of the OCT signal. In the central zone, a loss of the fibrillar tissue structure was observed, which led to a complete loss of birefringence and a strong reduction of the OCT signal.Conclusions: Although OCT does not provide the detailed information on thermal changes of tissue seen by the histologic method, it offers information on the extent and degree of tissue changes without preparation artifacts and provides a non-invasive method of immediate and follow-up control of LTK lesions. A quantitative analysis of changes in corneal thickness and curvature is much simpler than by a slit lamp. Time-resolved measurements of corneal light scattering may be used for on-line control of the laser-light dosage during LTK.

Mechanism of photodynamic occlusion using liposomal Zn(II)-phtalocyanine

Purpose: To evaluate the potential of liposomal Zinc(II)-phthalocyanine (ZnPc) to selectively target subretinal vasculature. Methods: Photodynamic therapy (PDT) with liposomal Zinc(II)-phtalocyanine was used to induce choroidal occlusion in eyes of pigmented rabbits. Drug doses of 0.16, 0.24, 0.32, and 0.4 mg/kg body weight were administered. Photosensitization was performed at a wavelength of 671 nm and an irradiance of 100 mW/cm2 applying fluences of 5, 10, 20, and 50 J/cm2. Results: Using liposomal ZnPc, occlusion of choroidal vessels was achieved without damage to the overlying neurosensory retina. A tight dose correlation was found with a drug dose of 0.32 mg/kg and a light dose of 10 J/cm2 inducing a selective thrombosis of the subretinal capillary layer. Histology revealed a selective intravascular alteration of the endothelial cells. Conclusions: PDT using liposomal ZnPc allows occlusion of subretinal vasculature with maintenance of neuroretina and RPE. The destructive effect on choroidal vascular endothelium is intensive.