Ex Vivo Micro-CT in Ophthalmology: Preparation and Contrasting for Non-invasive 3D-Visualisation. / Ex-vivo-Mikro-CT in der Augenheilkunde: Probenbehandlung und -kontrastierung für die 3D-Darstellung.

X-ray-based micro-computed tomography (micro-CT) is a largely non-destructive imaging method for the visualisation and analysis of internal structures in the ex vivo eye and affords high resolution. In contrast to other high-resolution imaging methods, micro-CT enables spatial recording of larger and more complex tissue structures, such as the anterior chamber of the eye. Special contrasting methods help to enhance the absorption properties of soft tissue, that is otherwise only weakly radiopaque. Critical point drying (CPD), as primarily used in scanning electron microscopy, offers an additional tool for improving differential contrast properties in soft tissue. In the visualisation of intraosseous soft tissue, such as the efferent lacrimal ducts, sample treatment by decalcification with ethylenediaminetetraacetic acid and subsequent CPD provides good results for micro-CT. Micro-CT can be used for a wide range of questions in 1. basic research, 2. application-related studies in ophthalmology (e.g. evaluation of the preclinical application of microstents for glaucoma treatment or analysis of the positioning of intraocular lenses) but also 3. as a supplement to ophthalmological histopathology.

Cataract surgery in Kinshasa-Is there a place for "Monovision"?

OBJECTIVES: Monovision is a method of correcting presbyopia where one eye is focused for far and the other for near vision. It is a simple, cost-effective approach to overcome the loss of accommodation with age and to become spectacles independent. METHODOLOGY: About 50 patients, where bilateral cataract extraction was indicated, were included in this study performed at the St. Joseph's Eye Hospital in Kinshasa (DR Congo). Small incision cataract surgery technique (SICS) was applied with the implantation of 6 mm PMMA lenses in the capsular bag. IOL refractive power choice was made to achieve a post-operative refraction of -0.5 dpt for the eye selected for far vision. The second eye received an implant heading for a post-operative myopia of -1.5 dpt suitable for intermediated and near vision. According to the literature, monovision criteria have been regarded as fulfilled when (a) far vision was 0.5 (logMAR) or better and (b) near vision was P3 (0.40, Decimal 32 cm) or better. Spectacle dependence after bilateral cataract surgery heading for monovision was analysed using a dedicated questionnaire. RESULTS: Out of all 50 patients 22 (44%) fulfilled the above defined criteria of monovision in terms of post-operative refraction and visual acuity. About 19 out of these 22 (86.3%) patients were happy without glasses. Two of them used bifocal spectacles, whereas the remaining patient refused spectacles. About 28 patients did not fulfill monovision criteria. Out of these 28 patients, however, 9 (32.1%) of them are happy without glasses. CONCLUSION: In view of the described local circumstances aiming for monovision after bilateral cataract surgery is a suitable approach to optimise cataract surgical outcomes with no extra costs for surgery but considerable improvement of patient's visual performance in daily life.

Assessment of the Efficacy of Microinvasive Glaucoma Surgery Techniques using an Oculopression Stress Test. / Nicht invasiver Belastungstest zur Funktionsprüfung von mikroinvasiven Glaukomdrainage-Implantaten und Laserchirurgie.

BACKGROUND: Glaucoma is one of the most common causes of blindness worldwide. The only evidence-based treatment to slow down the progression of glaucoma is the reduction of intraocular pressure (IOP) using local medication or through surgery. During the last years, a large number of microinvasive glaucoma surgery techniques (MIGS) has been developed, in order to reduce the IOP in glaucoma patients safely and effectively. Until now, efficacy of MIGS has been assessed mainly according to the postoperative IOP and the number of medications used. Results from long-term studies are rare or not available in the majority of the cases. In order to better evaluate the functionality of MIGS, a new examination method has been developed with the help of a new oculopressor device. In this study the efficacy of different MIGS techniques will be examined using the new oculopressor. MATERIAL/METHODS: At first, glaucoma patients that had previously received a MIGS surgery (iStent inject, XEN Stent, ELT) were examined with the new oculopression test. Their results were compared with those of non-operated patients and healthy individuals. Overall, 38 healthy subjects (group 1), 10 non-operated patients (group 2), 19 patients after iStent inject implantation (group 3), 14 patients after XEN Stent implantation (group 4) and 5 patients after ELT (group 5) were examined. The new examination measures the IOP-reduction that occurs after oculopression and can be seen as an indirect measurement of the outflow facility of the eye. RESULTS: The IOP-reduction after oculopression differed among the study groups. Non-operated patients showed a significantly lower IOP-reduction compared to healthy individuals. Patients after iStent inject and XEN stent implantation showed a larger reduction of IOP after oculopression in relation to non-operated patients and their results approximated those of healthy individuals. These patients needed fewer medications postoperatively in relation to non-operated patients. Patients after ELT showed postoperatively a smaller reduction of IOP after oculopression compared to iStent inject and XEN stent patients. CONCLUSION: MIGS can increase the outflow facility of the eye in patients with glaucoma. Though ELT had the lowest impact on the aqueous outflow among the studied procedures in this study. The new test can help in the evaluation of current and further development of new MIGS in the future.

In vivo Histology of the Cornea - from the "Rostock Cornea Module" to the "Rostock Electronic Slit Lamp" - a Clinical "Proof of Concept" Study. / In-vivo-Histologie der Hornhaut ­ vom "Rostocker Cornea Modul" zur "Rostocker Elektronischen Spaltlampe" ­ eine klinische "Proof-of-Concept"-Studie.

INTRODUCTION: Confocal in vivo microscopy is an established method in ophthalmology research. As it requires contact coupling and calibration of the instruments is suboptimal, this method has been only rarely used in clinical routine work. As a result of close collaboration between physicists, information scientists and ophthalmologists, confocal laser scanning microscopy (CLSM) of the eye has been developed in recent years and a prototype can now be used in patients. The present study evaluates possible clinical uses of this method. MATERIAL AND METHODS: The essential innovations in CLSM are (1) a newly designed coupling element with superficial adaptation to corneal curvature and (2) the use of a dual computerised piezo drive for rapid and precise focusing. In post-processing and after elastic imaging registration of the individual images parallel to the surface, it is also possible to produce sagittal sections resembling a split lamp and with resolution in the micrometer range. The concept was tested on enucleated pig bulbi and tested on normal volunteers and selected patients with diseases of the cornea. RESULTS: Simultaneous imaging in planes parallel to the surface and in sagittal planes provided additional information that can help us to understand the processes of wound healing in all substructures of the cornea and the role of immune competent cells. Possible clinical uses were demonstrated in a volunteer with healthy eyes and several groups of patients (keratoconus after CXL, recurrent keratitis, status after PRK). These show that this new approach can be used in morphological studies at cellular level in any desired and appropriate test plane. CONCLUSIONS: It could be shown that this new concept of CLSM can be used clinically. It can provide valuable and novel information to both preclinical researchers and to ophthalmologists interested in corneal disease, e.g. density of Langerhans cells and epithelial stratification in ocular surface diseases.

[Systems Biology in Ophthalmology - Innovative Drug Identification for the Specific Prevention of Postoperative Fibrosis]. / Systembiologie in der Ophthalmologie ­ innovative Wirkstoffidentifikation zur spezifischen Vermeidung postoperativer Fibrose.

BACKGROUND: The long-term success of fistulating therapies for the treatment of glaucoma is essentially limited by excessive scarring reactions (fibrosis). Cytostatic agents such as mitomycin C can prevent fibrosis, but are often associated with side effects. Specific antifibrotics are not currently in clinical use. Therefore, this study describes a systems biology approach using a dedicated bioinformatics technology platform, with which active substances can be identified and repositioned as antifibrotics. MATERIALS AND METHODS: Differential gene expression data of human Tenon fibroblasts (hTF) were collected from untreated hTF and from hTF stimulated with TGF-ß1 ("fibrotic fibroblasts") by next-generation sequencing (NGS) and were used as the basis for the drug identification process. These data were filtered with the bioinformatic tool "FocusHeuristics". In comparison with the Connectivity Map database, antifibrotic agents were identified. The evaluation of a potentially promising drug as an antifibrotic was performed at hTF by indirect immunofluorescence in vitro. RESULTS: The analysis of the gene expression data led to the identification of several interaction networks of genes or proteins involved in fibrotic processes. One of these networks contains the cytokine bone morphogenic protein 6 (BMP6), interleukin 6 (IL6) and fibroblast growth factor 1 (FGF1). Another relevant network has been identified around the cluster of differentiation 34 (CD34) gene. The comparison of these data with those of the Connectivity Map allowed the identification of an inhibitory drug. Its evaluation in the fibrotic cell culture model in vitro using indirect immunofluorescence led to a significant reduction in the expression of the fibrotic marker proteins fibronectin and alpha-smooth muscle actin (α-SMA), which confirmed the predicted antifibrotic effect. CONCLUSION: Systems biological approaches can be used for the identification of antifibrotic drug candidates for the prevention of postoperative fibrosis and should be transferable by the investigating differential gene expression data of further ocular cells or tissues to other ophthalmological fields of application.

Role of Bone Morphogenetic Protein 7 (BMP7) in the Modulation of Corneal Stromal and Epithelial Cell Functions.

In the cornea, healing of the wounded avascular surface is an intricate process comprising the involvement of epithelial, stromal and neuronal cell interactions. These interactions result to the release of various growth factors that play prominent roles during corneal wound healing response. Bone morphogenetic proteins (BMPs) are unique multi-functional potent growth factors of the transforming growth factor-beta (TGF-β) superfamily. Treatment of corneal epithelial cells with substance P and nerve growth factor resulted to an increase in the expression of BMP7 mRNA. Since BMP7 is known to modulate the process of corneal wound healing, in this present study, we investigated the influence of exogenous rhBMP7 on human corneal epithelial cell and stromal cell (SFs) function. To obtain a high-fidelity expression profiling of activated biomarkers and pathways, transcriptome-wide gene-level expression profiling of epithelial cells in the presence of BMP7 was performed. Gene ontology analysis shows BMP7 stimulation activated TGF-β signaling and cell cycle pathways, whereas biological processes related to cell cycle, microtubule and intermediate filament cytoskeleton organization were significantly impacted in corneal epithelial cells. Scratch wound healing assay showed increased motility and migration of BMP7 treated epithelial cells. BMP7 stimulation studies show activation of MAPK cascade proteins in epithelial cells and SFs. Similarly, a difference in the expression of claudin, Zink finger E-box-binding homeobox 1 was observed along with phosphorylation levels of cofilin in epithelial cells. Stimulation of SFs with BMP7 activated them with increased expression of α-smooth muscle actin. In addition, an elevated phosphorylation of epidermal growth factor receptor following BMP7 stimulation was also observed both in corneal epithelial cells and SFs. Based on our transcriptome analysis data on epithelial cells and the results obtained in SFs, we conclude that BMP7 contributes to epithelial-to-mesenchymal transition-like responses and plays a role equivalent to TGF-β in the course of corneal wound healing.

[Development of an Oculopressor to Evaluate the Efficiency of Glaucoma Drainage Devices]. / Entwicklung eines Okulopressors zur Evaluation der Funktionalität von Glaukomdrainage-Implantaten.

PURPOSE: Evaluation of the long-term efficiency of MIGS implants is still challenging, due to the lack of standardized clinical studies of stand-alone procedures. Moreover, the different mechanisms of the various glaucoma drainage devices are not adequately considered. The current study focusses on the development of a method for oculopression to evaluate the efficiency of glaucoma drainage devices. METHODS: Explanted porcine eyes were subjected to pressure or weight load using three oculopressors with different modi operandi. The time-dependent intraocular pressure was measured using an anterior chamber maintainer. The Honan Balloon exerts variable pressure onto the eye via an air bellows, whereas the Taylor oculopressor applies a defined weight loading on the eye. A novel oculopressor with a weight loading of 60 g was developed and manufactured by means of 3-D-printing. RESULTS: The intraocular pressure changes observed during the experiments were similar for all tested oculopression devices, varying only in the absolute pressure values. The Honan Balloon was not suitable for the intended purpose, due to poor standardisation of the applied pressure. Oculopression using a defined weight appeared more suitable. The Taylor oculopressor, however, created intraocular pressure values of up to 203.3 ± 38.4 mmHg, which precludes its use with glaucoma patients. On the basis of these data, the new oculopression device was used in a preliminary trial with healthy human subjects, thereby preparing its use in a clinical trial. CONCLUSIONS: Oculopression represents a potentially suitable tool to analyse the efficiency of glaucoma drainage devices. Commercially available oculopression devices are not directly applicable for this task. Difficult handling, high intraocular pressure, and lack of standardisation complicate the use for glaucoma patients. These difficulties were overcome with the newly designed oculopressor that facilitates a well defined increase in intraocular pressure. The device is currently being used in a clinical study to evaluate the efficiency of MIGS implants.

[Ultra-high-field MRI in the Chicken Embryo in Ovo - a Model for Experimental Ophthalmology]. / Ultrahochfeld-MRT am Hühnerembryo in ovo ­ ein Modell für die experimentelle Ophthalmologie.

Ultra-high-field MRI (UHF-MRI) is an outstanding technique for non-invasive and non-destructive imaging of soft tissues and can provide versatile contrasts and high resolution in the µm range. In vivo imaging of the embryonal chick eye with its filigree anatomical structures imposes these requirements. However, due to the short embryonal development cycle, chicken are a favourite animal model for embryonal research studies. Ultra-high-field MRI allows repeated and longitudinal in ovo investigations on the same embryo. In the present study, the limitations and opportunities of in ovo MR-imaging at 7 T were evaluated and the process of eye growth was described in detail.

MR microscopy of the human fetal upper extremity - a proof-of-principle study.

BACKGROUND: Current knowledge of the human fetal and embryonic development relies on early descriptive studies of humans and from experimental studies of laboratory animals and embryos. Taking the upper extremity as an example, this study explores the potential of magnetic resonance microscopy (MRM) for the assessment of the development of the fetal upper extremity and discusses its correlation with histological findings. METHODS: Ex vivo MRM at 7.1 T (Clin Scan, Bruker Biospin, Germany) was performed in 10 human specimens at 8 to 12 weeks of gestational age (GA). In-plane resolution was 20 µm with a slice thickness of 70 µm. MRM was followed by histological work-up of the specimens. MRM images were then correlated with conventional histology with a focus on the presence of chondrification and ossification. RESULTS: Ossification of the upper human extremity is detectable at 8 weeks GA in the humerus and the long bones of the forearm. There is excellent correlation for location and size of ossification between MRM and conventional histology. MRM imaging is in accordance with historical studies. CONCLUSION: Ex vivo MRM for the non-invasive assessment of the embryonic and fetal development of the upper human extremity is feasible. It may provide an accurate complementary tool for the evaluation of embryological development.

Corneal epithelial and neuronal interactions: role in wound healing.

Impaired corneal innervation and sensitivity are the main causes of corneal neurotrophic keratopathy which simultaneously also leads to poor epithelial wound healing. Restoration of the diminished communication between the corneal epithelium and trigeminal nerve is indispensable for the proper functioning of the epithelium. The present study aims to investigate corneal epithelial and trigeminal neuron interactions to shed light on corneal wound healing during neurotrophic keratopathy. Mouse trigeminal neurons and corneal epithelial cells were cultured according to standard methods. To study the effect of corneal epithelial cells on trigeminal neurons as well as the effect of trigeminal neurons on corneal epithelial cells during wound healing, conditioned media from the cultures of pure trigeminal neurons (CNM) and corneal epithelial cells (CEM) were collected freshly and applied on the other cell type. Neurite outgrowth assay and RT-PCR analysis using primers specific for substance P (SP), Map1a, Map1b were performed on trigeminal neurons in the presence of CEM. We observed an increase in the neurite outgrowth in the presence of CEM and also in co-culture with corneal epithelial cells. Increase in the expression of SP mRNA and a decrease in the expression of Map1b mRNA was observed in the presence of CEM. We also observed the presence of epithelial-to-mesenchymal transition (EMT)-like phenomenon during wound healing using a scratch assay in primary corneal epithelial cultures. This system was further employed to study the effect of CNM on corneal epithelial cells in the context of wound healing to find the effect of trigeminal neurons on epithelial cells. RT-PCR analysis of Pax6 expression in corneal epithelial cell cultures with scratch served as a positive control. Further, we also show the expression of bone morphogenetic protein 7 (BMP7) mRNA in corneal epithelial cells which is decreased gradually along with Pax6 mRNA when cultured together in the presence of CNM. The expression and down regulation of BMP7 in the presence of CNM was further confirmed at the protein level by western blotting. From this study it seems that the epithelial and neuronal interactions in the cornea may contribute to the corneal innervation as well as recovery of corneal epithelial cells during injury. Appraising the differences in the expression of various signalling molecules during EMT of epithelial cells in the presence of SP and BMP7 gives an insight into the detailed dissection of the involved signalling pathways to develop future therapeutics.

γ-Cyclodextrin hydrogel for the sustained release of josamycin for potential ocular application.

Glaucoma is the leading cause of blindness worldwide. However, its surgical treatment, in particular via trabeculectomy, can be complicated by fibrosis. In current clinical practice, application of the drug, Mitomycin C, prevents or delays fibrosis, but can lead to additional side effects, such as bleb leakage and hypotony. Previous in silico drug screening and in vitro testing has identified the known antibiotic, josamycin, as a possible alternative antifibrotic medication with potentially fewer side effects. However, a suitable ocular delivery mechanism for the hydrophobic drug to the surgical site does not yet exist. Therefore, the focus of this paper is the development of an implantable drug delivery system for sustained delivery of josamycin after glaucoma surgery based on crosslinked γ-cyclodextrin. γ-Cyclodextrin is a commonly used solubilizer which was shown to complex with josamycin, drastically increasing the drug's solubility in aqueous solutions. A simple γ-cyclodextrin crosslinking method produced biocompatible hydrogels well-suited for implantation. The crosslinked γ - cyclodextrin retained the ability to form complexes with josamycin, resulting in a 4-fold higher drug loading efficiency when compared to linear dextran hydrogels, and prolonged drug release over 4 days.

Ocular fibroblast types differ in their mRNA profiles--implications for fibrosis prevention after aqueous shunt implantation.

PURPOSE: For an aqueous shunt draining from the anterior chamber into the choroidal space, fibroblasts from the choroidea and/or the sclera are most likely responsible for a fibrotic response around the outflow region of such a shunt. The prevention of fibrosis should extend the operating life of the shunt. A detailed characterization of fibroblasts derived from choroidea and sclera should provide information about whether a fibrosis reaction can be inhibited by cell type-specific agents. METHODS: We generated mRNA profiles of fibroblasts from the choroidea, sclera, and Tenon's space by gene array hybridization to provide a basis on which to search for potential pharmacological targets for fibrosis prevention. Hybridization data were analyzed by the Rosetta Resolver system and Limma to obtain mRNA profiles of the three fibroblast types. RESULTS: The three fibroblast types investigated shared fibroblast-specific gene expression patterns concerning extracellular matrix proteins as collagens and fibronectin, but also showed distinct mRNA patterns. CONCLUSIONS: Individual mRNA species overexpressed in one of the fibroblast types might serve as markers for the identification of the fibroblast type in histological analyses. Future in-depth analyses of the gene expression patterns might help identify pharmacological targets for fibrosis prevention.

Quantitative systems pharmacology of the eye: Tools and data for ocular QSP.

Good eyesight belongs to the most-valued attributes of health, and diseases of the eye are a significant healthcare burden. Case numbers are expected to further increase in the next decades due to an aging society. The development of drugs in ophthalmology, however, is difficult due to limited accessibility of the eye, in terms of drug administration and in terms of sampling of tissues for drug pharmacokinetics (PKs) and pharmacodynamics (PDs). Ocular quantitative systems pharmacology models provide the opportunity to describe the distribution of drugs in the eye as well as the resulting drug-response in specific segments of the eye. In particular, ocular physiologically-based PK (PBPK) models are necessary to describe drug concentration levels in different regions of the eye. Further, ocular effect models using molecular data from specific cellular systems are needed to develop dose-response correlations. We here describe the current status of PK/PBPK as well as PD models for the eyes and discuss cellular systems, data repositories, as well as animal models in ophthalmology. The application of the various concepts is highlighted for the development of new treatments for postoperative fibrosis after glaucoma surgery.

The Antibiotic Kitasamycin-A Potential Agent for Specific Fibrosis Preventing Therapy after Fistulating Glaucoma Surgery?

One major complication after fistulating glaucoma surgeries are fibroblast-mediated scarring processes and their specific prevention is key in the development of novel pharmaceutical concepts. Within this study a possible antifibrotic potential of kitasamycin (KM) in a transforming growth factor (TGF)-ß1-mediated fibroblast model was evaluated in vitro. Primary ocular fibroblasts were isolated, cultivated and a dose-response test including determination of the half maximal effective concentration (EC50) for KM was conducted. Transformation of fibroblasts into myofibroblasts was induced by TGF-ß1and immunofluorescence (IF), and Western blot (WB) analyses were performed with fibroblasts and myofibroblasts. IF analyses were carried out using antibodies against α-smooth muscle actin (α-SMA) and fibronectin, and protein detection of intracellular and extracellular proteins was performed by WB. Using the dose-response test, the viability, cytotoxicity and EC50 of KM after 24 and 48 h were determined. Fibroblasts exposed to various KM concentrations showed no increase in α-SMA and extracellular matrix expression. In TGF-ß1-stimulated myofibroblasts, KM inhibited the expression of α-SMA and fibronectin in a concentration-dependent manner. These findings demonstrate that KM could impair the transformation of fibroblasts into myofibroblasts and the expression of proteins involved in fibrotic processes, representing a potential agent for specific fibrosis prevention in future therapeutic concepts.

MR microscopy of the developing upper extremity of the chicken in ovo using 7 Tesla MRI.

MR microscopy (MRM) is known as ultra-high-field (UHF) magnetic resonance imaging with an in-plane spatial resolution of <100 µm, yields highly resolved non-invasive anatomical imaging and allows longitudinal assessment of embryonic avian development. The aim of the present study was to evaluate the feasibility of in vivo anatomical MRI assessment of the developing upper extremity of the chicken. Thirty-eight fertilized chicken eggs were examined at 7 Tesla acquiring high-resolution T2-weighted images with an in-plane resolution of 74 × 74 µm. To reduce motion artefacts, the eggs were moderately cooled before and during MRI. Development of the upper extremity was anatomically and quantitatively assessed. Chondrification and ossification on MRI were correlated with histological examination. MRM allowed the identification of the embryo from stage D5 onwards. First chondrification of the upper extremity was visible at stage D7, and the differentiation of the forearm was possible from stage D9 throughout the developmental period with excellent correlation to histology. MRM also allowed the differentiation between cortical and medullary bone as well as the detection of chondrified areas. UHF MRM allows the in vivo and in ovo evaluation of the upper limb during embryonic development and provides non-invasive longitudinal anatomical information. This technique allows longitudinal studies of the same embryo during the developmental period and may therefore provide further insights into the development of the upper extremity. With improved coil technique and increasing availability of UHF MR systems, there is great potential regarding several research topics in experimental musculoskeletal radiology.

Development of a Novel Valve-Controlled Drug-Elutable Microstent for Microinvasive Glaucoma Surgery: In Vitro and Preclinical In Vivo Studies.

Purpose: Microinvasive glaucoma surgery (MIGS) has become an important treatment approach for primary open-angle glaucoma, although the safe and long-term effective lowering of intraocular pressure with currently available implants for MIGS is not yet achieved to a satisfactory extent. The study focusses on the development and in vitro and in vivo testing of a novel microstent for MIGS. Methods: A silicone elastomer-based microstent was developed. Implants were manufactured using dip coating, fs-laser cutting, and spray coating. Within the current study no antifibrotic drug was loaded into the device. Sterilized microstents were analyzed in vitro regarding pressure-flow characteristics and biocompatibility. Six New Zealand white rabbits were implanted with a microstent draining the aqueous humor from the anterior chamber into the subconjunctival space. Drainage efficacy was evaluated using oculopressure tonometry as a transient glaucoma model. Noninvasive imaging was performed. Results: Microstents were manufactured successfully and characterized in vitro. Implantation in vivo was successful for four animals with additional device fixation. Without additional fixation, dislocation of microstents was found in two animals. Safe and effective intraocular pressure reduction was observed for the four eyes with correctly implanted microstent during the 6-month trial period. Conclusions: The described microstent represents an innovative treatment approach for MIGS. The incorporation of a selectively antifibrotic drug into the microstent drug-elutable coating will be addressed in future investigations. Translational Relevance: The current preclinical study successfully provided proof of concept for our microstent for MIGS which is suitable for safe and effective intraocular pressure reduction and offers promising perspectives for the clinical management of glaucoma.

Effects of prenatal Doppler ultrasound on the retina of the chick embryo in ovo.

Currently, the effect of prenatal ultrasound on foetal development is intensively discussed and the guidelines for prenatal diagnostics have been changed. However, data supporting these concerns are scarce. Therefore, we used an established in ovo model of the chicken embryo to investigate cell proliferation and apoptosis within the retina. A total of 21 chicken eggs were fenestrated on Day 5 and allocated to either the control (n = 8) or exposition group (n = 13). The exposition group was treated with pulsed-wave Doppler ultrasound (PWD) for 10 min while controls remained without treatment. After subsequent incubation (6-48 h), the eggs were sacrificed, and chicken embryos were examined morphologically (HE-staining) and immunohistochemically. Counting of apoptotic and proliferating cells per retina was performed using antibodies specific for phospho-histone-H3 and active caspase-3 in combination with a biotin-labelled secondary antibody and peroxidase conjugated avidin-biotin complex for chromogenic detection. Due to a rather low number of specimens at each time point after ultrasound exposition, we neglected the effects of incubation time and focused on treatment effects. This approach revealed that the median number of proliferating cells is reduced after 10 min of exposure to PWD (569 vs. 766), while the number of apoptotic cells is fairly comparable between groups (5 vs. 6). Our data contribute to a better understanding of prenatal US on foetal development by suggesting that PWD could have an impact on the number of proliferating cells in the developing chicken retina and therefore justify further investigations.

Different fibroblast subpopulations of the eye: a therapeutic target to prevent postoperative fibrosis in glaucoma therapy.

The aim of this study is the characterization of fibroblasts mainly responsible for fibrosis processes associated with trabeculectomy or microstent implantation for glaucoma therapy. Therefore we isolated human primary fibroblasts from choroidea, sclera, Tenon capsule, and orbital fat tissues. These fibroblast subpopulations were analysed in vitro for expression of the extracellular matrix components which are responsible for postoperative scarring in glaucoma therapy. For scarring the proteins of the collagen family are predominant and so we focused on the expression of collagen I, collagen III and collagen VI in every fibroblast subpopulation. Also, the extracellular matrix protein fibronectin which crosslinks collagen fibres or other extracellular matrix components and cell surfaces, was analyzed. Collagen I, III and VI were prominent in every fibroblast subpopulation. The highest amounts of collagen III were found in hCF and hOF, whereas the signal in hSF and hTF was negligible. Additionally, there is a link between scarring processes and proliferating potential of fibroblasts, in case of microstent implantation triggered through the infiltration of inflammatory cells. Thus we analyzed fibroblast subpopulations for the presence of TGF-ß1 which is one of the most important cytokines involved in proliferation processes. TGF-ß1 was prominent in all fibroblast subpopulations with lowest expression in hCF cultures. To prevent postoperative fibroblast proliferation we analyzed in vitro the proliferation-inhibitors paclitaxel and mitomycin C which are potential candidates in drug eluting drainage systems on ocular fibroblast subpopulations. These inhibitors arrest fibroblast proliferation and viability, being, however, not very specific and have a cytotoxic potential also on healthy tissues surrounding the microstent outflow area. Significant differences in protein synthesis of fibroblasts subpopulations which could be specific targets for inhibition may help to find out fibroblast specific inhibitors to prevent postoperative scarring and could prevent patients from secondary surgery after microstent implantation.

Micro-CT in ophthalmology: ex vivo preparation and contrasting methods for detailed 3D-visualization of eye anatomy with special emphasis on critical point drying.

Background: Micro-computed tomography (micro-CT) provides detailed 3-dimensional (3D) visualization of anatomical structures and encourages morphological reinvestigation of organs with delicate features. The low radiodensity of soft tissues necessitates preceding sample preparation to conduct X-ray imaging with decent contrast between different tissues. In this study, we demonstrate the preparation with three radiopaque agents in combination with elimination of liquids by critical point drying (CPD) introduced for ocular samples. Methods: Enucleated porcine eyes were prepared with ethanolic iodine (EI), aqueous iodine-potassium iodide, or ethanolic phosphotungstic acid (EPTA). Micro-CT scans of the samples were conducted in a moist environment with an isotropic resolution of 9.2-12.5 µm voxel size. Subsequently, samples were chemically dehydrated and critical point (CP) dried to conduct a second scan in a dry environment with a resolution up to 4.7-5.4 µm in voxel size. The visualization effects were qualitatively and semi-quantitatively evaluated with regard to the generated contrast between different ocular tissues. Results: All three contrast agents accumulated well in most of the investigated ocular tissues and lead to an increased X-ray attenuation which allowed for differentiated visualization of ocular structures. Problematic agent penetration into the lens was obvious for iodine-potassium iodide and EPTA. Artificial damages of the lens and thickness reduction for the cornea and sclera due to CPD were noticed. The effects of the different contrasting treatments are described and compared with regard to the effects of CPD. Exclusively CP dried samples that were not treated with contrast agents could also be visualized excellently with a good distinction of different ocular structures from each other. Conclusions: All ocular structures can be visualized by micro-CT. To contrast moist samples, the best results were achieved with iodine potassium iodide (IPI). CPD improved the scan quality in all cases. Even without pretreatment with contrasting agents, the CP dried samples showed a contrast similar to the IPI treated samples.

Predictive value of retinal function by the Purkinje test in patients scheduled for cataract surgery in Kinshasa, DR Congo.

AIM: To assess the retinal function in patients with dense cataracts in resource poor settings in Kinshasa, DR Congo. METHODS: In a tertiary eye care center, the Purkinje entoptic test was performed as part of the ophthalmological examination in 98 eyes in patients with cataract, using a penlight. Totally 92 cataract patients including 86 patients suffered from unilateral and 6 from bilateral cataracts were included in the study. The investigator asked the patients about their perception of the vascular pattern most commonly described as a leafless or dead tree. Visual acuity≥6/60 was considered an indication of good visual function and visual acuity<6/60 of poor function. Following small incision cataract surgery, best corrected visual acuity (BCVA) was determined and compared with the preoperative findings. RESULTS: Out of 98 eyes, there were 80 (81.6%) where the Purkinje entoptic phenomenon was reported by the patients. After cataract surgery, out of the 80 eyes, there were 75 (93.8%) with a BCVA of better than 6/60, whereas in 5 eyes (6.2%), BCVA was 6/60 or less. Out of the 18 eyes (18.4%) where no Purkinje tree was recognized, there were 14 (77.8%) with BCVA of better than 6/60, whereas in 4 (22.2%) BCVA was 6/60 or less. CONCLUSION: The Purkinje entoptic test is successfully used for preoperative assessment of retinal function in patients with dense cataract. However, further investigation and refinement of the test is necessary to validate the method for use in sub-Saharan conditions.