Philipp Steven

Optimizing Descemet Membrane Endothelial Keratoplasty Using Intraoperative Optical Coherence Tomography

IMPORTANCE Descemet membrane endothelial keratoplasty (DMEK) is a challenging procedure for the surgeon, particularly because of deficient visibility of the delicate tissue due to the natural en face view through the operating microscope. A cross-sectional view would greatly enhance intraoperative overview and enable the surgeon to better control the procedure. OBJECTIVE To retrospectively analyze the use of intraoperative optical coherence tomography (iOCT) for improving the safety of DMEK. DESIGN Intraoperative OCT during DMEK was performed in 26 eyes of 26 patients. We retrospectively analyzed imaging and video data. SETTING Department of Ophthalmology, University of Cologne. PARTICIPANTS Seven men and 19 women aged 39 to 93 years with corneal endothelial dysfunction undergoing DMEK. EXPOSURE Descemet membrane endothelial keratoplasty. MAIN OUTCOMES AND MEASURES Visibility of surgical steps, overall duration of DMEK, overall time for complete intraoperative air filling of the anterior chamber, and correlation between donor age and Descemet rolling behavior. RESULTS Intraoperative OCT enables visualization of all steps of the DMEK procedure. Overall mean (SD) duration of the DMEK procedure was 25.7 (6.9) minutes when using iOCT. Overall mean (SD) complete intraoperative anterior chamber air-filling time was 236 (108) seconds in contrast to 60 to 90 minutes for standard air-filling time. Descemet membrane rolling behavior showed significant inverse correlation between donor age (range, 39-93 years) and the extent of rolling (R2 = 0.5 [P = .006]). CONCLUSIONS AND RELEVANCE Intraoperative OCT enhances the visibility of graft orientation and unfolding, thereby improving safety of the DMEK procedure. Overall, iOCT is a helpful device that may support surgeons in all steps of DMEK procedures.

Novel anti(lymph)angiogenic treatment strategies for corneal and ocular surface diseases

The cornea is one of the few tissues which actively maintain an avascular state, i.e. the absence of blood and lymphatic vessels (corneal [lymph]angiogenic privilege). Nonetheless do several diseases interfere with this privilege and cause pathologic corneal hem- and lymphangiogenesis. The ingrowths of pathologic blood and lymphatic vessels into the cornea not only reduce transparency and thereby visual acuity up to blindness, but also significantly increases the rate of graft rejections after subsequent corneal transplantation. Therefore great interest exists in new strategies to target pathologic corneal (lymph)angiogenesis to promote graft survival. This review gives an overview on the vascular anatomy of the normal ocular surface, on the molecular mechanisms contributing to the corneal (lymph)angiogenic privilege and on the cellular and molecular mechanisms occurring during pathological neovascularization of the cornea. In addition we summarize the current preclinical and clinical evidence for three novel treatment strategies against ocular surface diseases based on targeting pathologic (lymph)angiogenesis: (a) modulation of the immune responses after (corneal) transplantation by targeting pathologic (lymph)angiogenesis prior to and after transplantation, (b) novel concepts against metastasis and recurrence of ocular surface tumors such as malignant melanoma of the conjunctiva by anti(lymph)angiogenic therapy and (c) new ideas on how to target ocular surface inflammatory diseases such as dry eye by targeting conjunctival and corneal lymphatic vessels. Based on compelling preclinical evidence and early data from clinical trials the novel therapeutic concepts of promoting graft survival, inhibiting tumor metastasis and dampening ocular surface inflammation and dry eye disease by targeting (lymph)angiogenesis are on their way to translation into the clinic.

Intestinal Trefoil Factor/TFF3 Promotes Re-epithelialization of Corneal Wounds

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study using cornea as a model, we demonstrate the importance of trefoil factor 3 (TFF3, also known as intestinal trefoil factor) in re-epithelialization of wounds. In two different models of corneal wound healing, alkali- and laser-induced corneal wounding, we analyzed the wound healing process in in vivo as well as in combined in vivo/in vitro model in wild type (Tff3+/+) and Tff3-deficient (Tff3-/-) mice. Furthermore, we topically applied different concentrations of recombinant human TFF3 (rTFF3) peptide on the wounded cornea to determine the efficacy of rTFF3 on corneal wound healing. We found that Tff3 peptide is not expressed in intact corneal epithelium, but its expression is extensively up-regulated after epithelial injury. Re-epithelialization of corneal wounds in Tff3-/- mice is significantly prolonged in comparison to Tff3+/+ mice. In addition, exogenous application of rTFF3 to the alkali-induced corneal wounds accelerates significantly in in vivo and in combined in vivo/in vitro model wound healing in Tff3+/+ and Tff3-/- mice. These findings reveal a pivotal role for Tff3 in corneal wound healing mechanism and have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.

Intravital Two-Photon Microscopy of Immune Cell Dynamics in Corneal Lymphatic Vessels

Background The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years. Methodology/Principal Findings We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1–5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces. Conclusions To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions.

Optimising deep anterior lamellar keratoplasty (DALK) using intraoperative online optical coherence tomography (iOCT)

Background/aims To describe the use of intraoperative online optical coherence tomography (iOCT) for improving deep anterior lamellar keratoplasty (DALK) surgery. Methods Retrospective case series of 6 eyes of 6 male patients with keratokonus, corneal dystrophy or herpetic stromal scars undergoing DALK were investigated using intraoperative optical coherence tomography and postsurgical image/video analysis. Main outcome measures were: visibility of surgical steps, especially, assessment of placement depth of injection needle, preparation of bare Descemets membrane and drainage of interface fluid. Results iOCT enables real-time visualisation of all surgical steps of DALK procedure in all patients. Placement of air injection needle above Descemets membrane was reliably monitored as was presence of bare Descemets membrane and potential interface fluid. Conclusions iOCT assists with visualisation of injection needle placement and with assessment of bare Descemets membrane as well as interface fluid during the DALK procedure. Overall iOCT may be a helpful device that supports surgeons in all steps of DALK procedure.

Immunreaktionen nach DMEK, DSAEK und DALK

PURPOSE The aim of this study is to describe incidence, diagnosis and therapy for endothelial immune reactions after modern lamellar corneal transplantat surgery (DMEK, DSAEK, DALK). METHODS A PubMed-based literature review and our own clinical and experimental data are evaluated. RESULTS There is no longer an endothelial immune reaction after DALK for keratoconus. DMEK significantly reduces the risk for endothelial immune reactions after surgery for Fuchs dystrophy. CONCLUSIONS Modern lamellar corneal transplant techniques such as DALK and DMEK have nearly abolished the risk for endothelial immune reactions in the avascular recipient bed.

Secondary paracentral retinal holes following internal limiting membrane removal

Aim: To report on a new postoperative finding after delamination of the internal limiting membrane (ILM) for the treatment of different macular pathologies. Methods: Seven patients are described with paracentral retinal holes, all located temporally to the macula, which developed after pars plana vitrectomy and uncomplicated ILM removal for cystoid macular oedema (n = 3), macular pucker (n = 3), and macular hole (n = 1) with the use of indocyanine green (n = 4), trypan blue (n = 1), triamcinolone acetonide (n = 1), or without dye (n = 1). Conclusion: The use of dyes has been incriminated in causing iatrogenic damage to the retina. In this series, the new observation of retinal holes in the macular area is thought to be related to the removal of the ILM itself. The authors speculate that ILM delamination may cause a retinal weakening by Müller cell damage; this may be followed by a structural breakdown and, consequently, hole formation.

Roles of human β-defensins in innate immune defense at the ocular surface: arming and alarming corneal and conjunctival epithelial cells

Human β-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. In this study, the expression and inducibility of β-defensins at the ocular surface were investigated in vitro and in vivo. Expression of human β-defensins (hBD) was determined by RT-PCR and immunohistochemistry in tissues of the ocular surface and lacrimal apparatus. Cultured corneal and conjunctival epithelial cells were stimulated with proinflammatory cytokines and supernatants of different ocular pathogens. Real-time PCR and ELISA experiments were performed to study the effect on the inducibility of hBD2 and 3. Expression and inducibility of mouse β-defensins-2, -3 and -4 (mBD2–4) were tested in a mouse ocular surface scratch model with and without treatment of supernatants of a clinical Staphylococcus aureus (SA) isolate by means of immunohistochemistry. Here we show that hBD1, -2, -3 and -4 are constitutively expressed in conjunctival epithelial cells and also partly in cornea. Healthy tissues of the ocular surface, lacrimal apparatus and human tears contain measurable amounts of hBD2 and -3, with highest concentrations in cornea and much lower concentrations in all other tissues, especially tears, suggesting intraepithelial storage of β-defensins. Exposure of cultured human corneal and conjunctival epithelial cells to proinflammatory cytokines and supernatants of various bacteria revealed that IL-1β is a very strong inductor of hBD2 and Staphylococcus aureus increases both hBD2 and hBD3 production in corneal and conjunctival epithelial cells. A murine corneal scratch model demonstrated that β-defensins are only induced if microbial products within the tear film come into contact with a defective epithelium. Our finding suggests that the tear film per se contains so much antimicrobial substances that epithelial induction of β-defensins occurs only as a result of ocular surface damage. These findings widen our knowledge of the distribution, amount and inducibility of β-defensins at the ocular surface and lacrimal apparatus and show how β-defensins are regulated specifically.

Fluorescence Lifetime Imaging Unravels C. trachomatis Metabolism and Its Crosstalk with the Host Cell

Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ2-NAD(P)H increase inside the chlamydial inclusion. τ2-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity.

Conjunctiva-associated lymphoid tissue - current knowledge, animal models and experimental prospects.

The conjunctiva contains organized lymphoid tissue that consists of intraepithelial lymphocytes, subepithelial lymphoid follicles and adjacent lymphatics and blood vessels. This conjunctiva-associated lymphoid tissue (CALT) is assumed to play a key role in the protection of the ocular surface by initiating and regulating immune responses. The article reviews the current knowledge of CALT in humans and compares its structure and functions with mucosa-associated lymphoid tissues at other mucosal sites. An update is given on CALT animal models and on functional studies on the transepithelial transport of microorganisms and antigenic substances by specialized epithelial cells. The use of two-photon microscopy is reviewed regarding its importance to the understanding of dynamic processes going on within CALT.