Thomas Stahnke

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-β1 which is one of the most important cytokines involved in proliferation processes. TGF-β1 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.

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.

Suppression of TGF-β pathway by pirfenidone decreases extracellular matrix deposition in ocular fibroblasts in vitro

In glaucoma surgery, fibrotic processes occur, leading to impairment of liquid outflow. Activated fibroblasts are responsible for postoperative scarring. The transforming growth factor-β (TGF-β) pathway plays a key role in fibroblast function, differentiation and proliferation. The aim of this study was the characterization of the fibrotic potential of two subtypes of primary human ocular fibroblasts and the attempt to inhibit fibrotic processes specifically, without impairing cell viability. For fibrosis inhibition we focused on the small molecule pirfenidone, which has been shown to prevent pulmonary fibrosis by the decrease of the expression of TGF-β1, TGF-β2 and TGF-β3 cytokines. For in vitro examinations, isolated human primary fibroblasts from Tenon capsule and human intraconal orbital fat tissues were used. These fibroblast subpopulations were analyzed in terms of the expression of matrix components responsible for postoperative scarring. We concentrated on the expression of collagen I, III, VI and fibronectin. Additionally, we analyzed the expression of α-smooth muscle actin, which serves as a marker for fibrosis and indicates transformation of fibroblasts into myofibroblasts. Gene expression was analyzed by rtPCR and synthesized proteins were examined by immunofluorescence and Western blot methods. Proliferation of fibroblasts under different culture conditions was assessed using BrdU assay. TGF-β1 induced a significant increase of cell proliferation in both cell types. Also the expression of some fibrotic markers was elevated. In contrast, pirfenidone decreased cell proliferation and matrix synthesis in both fibroblast subpopulations. Pirfenidone slightly attenuated TGF-β1 induced expression of fibronectin and α-smooth muscle actin in fibroblast cultures, without impairing cell viability. To summarize, manipulation of the TGF-β signaling pathway by pirfenidone represents a specific antifibrotic approach with no toxic side effects in two human orbital fibroblast subtypes. We presume that pirfenidone is a promising candidate for the treatment of fibrosis following glaucoma surgery.

Comparative quantitative assessment of the human corneal sub-basal nerve plexus by in vivo confocal microscopy and histological staining

PurposeThis study was designed to compare and contrast quantitative data of the human corneal sub-basal nerve plexus (SBP) evaluated by two different methods: in vivo confocal microscopy (IVCM), and immunohistochemical staining of ex vivo donor corneas.MethodsSeven parameters of the SBP in large-scale IVCM mosaicking images from healthy subjects were compared with the identical parameters in ex vivo donor corneas stained by β-III-tubulin immunohistochemistry. Corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), average weighted corneal nerve fiber tortuosity (CNFTo), corneal nerve connection points (CNCP), average corneal nerve single-fiber length (CNSFL), and average weighted corneal nerve fiber thickness (CNFTh) were calculated using a dedicated, published algorithm and compared.ResultsOur experiments showed significantly higher values for CNFL (50.2 vs 21.4 mm/mm2), CNFD (1358.8 vs 277.3 nerve fibers/mm2), CNBD (847.6 vs 163.5 branches/mm2), CNFTo (0.095 vs 0.081 μm−1), and CNCP (49.4 vs 21.6 connections/mm2) in histologically staining specimens compared with IVCM images. In contrast, CNSFL values were higher in IVCM images than in histological specimens (32.1 vs 74.1 μm). No significant difference was observed in CNFTh (2.22 vs 2.20 μm) between the two groups.ConclusionsThe results of this study have shown that IVCM has an inherently lower resolution compared with ex vivo immunohistochemical staining of the corneal SBP and that this limitation leads to a systematic underestimation of several SBP parameters. Despite this shortcoming, IVCM is a vital clinical tool for in vivo characterization, quantitative clinical imaging, and evaluation of the human corneal SBP.

Morphological analysis of quiescent and activated keratocytes: a review of ex vivo and in vivo findings.

Abstract Keratocytes are specialized, neural crest-derived mesenchymal cells occupying approximately 3% of the corneal stromal volume. They reside between the collagen lamellae and are responsible for the secretion of extracellular matrix macromolecules, thus contributing to the corneal transparency and integrity. During the regeneration process after infection, traumata and refractive surgery, the keratocytes undergo transition into divergent phenotypes, which are referred to as “activated keratocytes”. Quite shortly after injury, the keratocytes lose their quiescence, enter into the cell cycle and migrate toward the site of injury. In certain types of injury, which affect the integrity of basement membrane, activated keratocytes also participate in wound closure by production of α-smooth muscle actin (α-SMA). Since the activated keratocytes are the major cell type contributing to tissue repair during corneal wound healing, their morphological and biochemical properties have been studied in details in experimental studies using light and electron microscopy. More recently, emerging of in vivo microscopy techniques has opened new possibilities to investigate cornea in vivo. The non-invasive nature of this imaging modality enables repeated examination of the same tissue over time and is an ideal tool to rapidly and accurately investigate corneal wound healing. However, the in vivo data on activated keratocytes are not as uniform as data from experimental ex vivo studies. There is still inconsistency in the literature findings on activated phenotypes, and often the described morphologies cannot be appreciated in in vivo images. In this article, a literature review was performed in order to interpret the morphology of different activated phenotypes, based on biological processes underlying the morphological alterations.

Anatomic and pathological characterization of choroidal melanoma using multimodal imaging: what is practical, what is needed?

Choroidal melanoma is the most frequently occurring intraocular tumor in adults. The aim of this work is to assess the potential of state-of-the art in-vivo and ex-vivo imaging modalities for the characterization of choroidal melanoma. Multimodal imaging of a choroidal melanoma was performed in a 53-year-old male patient. In-vivo ophthalmoscopy, ultrasound microscopy, duplex ultrasound, and 7.0 T MRI were performed. Ex-vivo examination of the enucleated eye included 7.0 and 9.4 T magnetic resonance microscopy as well as histopathology with hematoxylin and eosin staining. Imaging of choroidal melanoma with ultrahigh field MRI and duplex sonography provides detailed morphologic and functional information of the eye. High-spatial-resolution MRI at 9.4 T shows details of the internal texture of melanoma and other structures of the eye with an in-plane spatial resolution of 32 &mgr;m. Ultrahigh field in-vivo MRI at 7.0 T and ex-vivo MRI at 7.0 and 9.4 T correlate well with histologic evaluation. In-vivo ultrahigh field MRI is an emerging technique for the characterization and staging of ocular tumors. The combination of in-vivo ultrahigh-field MRI and duplex sonography has the potential to complement or even substitute complex and invasive biopsies.

Morphologic and biometric evaluation of chick embryo eyes in ovo using 7 Tesla MRI

The purposes of this study were (1) to characterize embryonic eye development during incubation in ovo and (2) to analyze the putative influence of repetitive ultrahigh-field MRI (UHF-MRI) measurements on this development. A population of 38 fertilized chicken eggs was divided into two sub-groups: two eggs (Group A) were examined repeatedly during the developmental period from embryonic day 1 (E1) to embryonic day 20 (E20) to evaluate the influence of daily MRI scanning. A second larger group of 36 eggs was examined pairwise on one day only, from E3 to E20, and the embryos were sacrificed immediately after MR imaging (Group B). Fast T2-weighted MR sequences provided biometric data on the eye with an in-plane resolution of 74 μm. The data show rapid growth of the eye with a steep increase in intraocular dimensions in all axis directions and in eyeball volume during initial development up to E10, followed by a phase of reduced growth rate in later developmental stages. Comparison of the two groups revealed no differences in ocular development.

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.

Hydrophilic silicone elastomer with excellent cell adhesion capability - A promising material for ophthalmic micro-implants

Abstract For minimising tissue reactions of implants hydrophilic silicone elastomers feature a high potential to be utilised as material for micro-implants in ophthalmologic pathology. To estimate the biocompatibility we quantified cell viability of human primary fibroblasts from scleral (hSF) and choroideal (hCF) origin in direct contact test and evaluated cell adhesion and cell morphology with Phalloidin- TRITC stained hSF and EA.hy926. Mechanical properties were analysed by means of uniaxial tensile testing. Direct contact testing revealed a good biocompatibility from 65 to 70% relative cell viability for hCF and moderate viabilities (46 to 63%) for hSF. EA.hy926 and also hSF exhibited typical cell morphology on the silicones. Improvement of cell adhesion was observed with increasing amount of hydrophilic component. Mechanical testing shows a minor decrease in elongation at break from 236% to 155% with increasing amount of hydrophilic component. These findings indicate that the investigated silicones may ensure a reliable integration of implants into the ocular tissues.

Treatment and Pathology of an Unusual Large Carcinoma of the Conjunctiva

Carcinoma of the conjunctiva is a malignant tumor which is mostly detected and surgically treated at an early stage because of medical or cosmetic problems. Exceptions of this rule may occur in developing countries, where patients do not have access to standard medical care systems. We report the case of a conjunctival carcinoma in an African patient with an unusually late presentation. Because of local medical shortcomings, and considering the severe, transmural inflammation of anterior ocular structures, an exenteration was performed although the orbit was not deeply involved with the tumor. The strong nuclear expression of p53 indicates a major role of UV exposure in this case. A total of 16-month follow-up in this patient and larger published series suggest that the risk of metastasis is rather low under those circumstances, even with invasive tumors.