Evaluation of the Use of Intraoperative OCT in Routine Surgery: A Two-year Comparison. / Evaluation der Nutzung von intraoperativer OCT im operativen Alltag: ein 2-Jahres-Vergleich.

BACKGROUND: In recent years, an increasing number of surgical microscopes fitted with an OCT module (intraoperative OCT, iOCT) have become available, providing high-resolution images of the surgical site in real time. While a 2018 survey at our hospital showed that iOCT delivered an additional intraoperative benefit in only 2.4% of all operations, considering that the manufacturer had since revised the hardware and software, we conducted a second user evaluation of this technology. MATERIAL AND METHODS: Prospective monocentric analysis of the application and user-friendliness of an EnFocus Ultra-Deep OCT (Leica Microsystems) over a period of 25 (2018) and 20 working days (2021). A standardized questionnaire was used to assess the surgeons' use of iOCT and its influence on the surgical course. RESULTS: 118 operations were performed over a 25-day period in 2018 and 92 operations were performed over a 20-day period in 2021. In 2018, iOCT was used in 24.6% and in 2021 in 48.9% of all surgeries, with iOCT proving to be "critical" to the surgical course in 2.4% and 3.3% of cases, respectively, as assessed by the surgeons in both years. These were operations in which the intraocular view was limited, e.g., with decompensated cornea, vitreous hemorrhage, or after previous surgery, e.g., after penetrating keratoplasty. CONCLUSION: Further development of the user interface led to an improvement in usability, and the iOCT was used significantly more often. In both years, the iOCT proved to be critical for the course of the surgery in a comparably small number of operations, especially those involving complex situations.

Lacrimal gland regeneration: The unmet challenges and promise for dry eye therapy.

Dry eye disease (DED) is a common multifactorial disease of the tear film and the ocular surface. The problem of DED has gained attention globally, with millions of people affected by the disorder. Although the treatment strategies for DED have significantly evolved over time, most of the existing modalities fall under the category of standard palliative care when viewed from a long-term perspective. To address these limitations, different approaches have been explored by various groups to uncover alternative treatment strategies that can contribute to a full regeneration of the damaged lacrimal gland, which is responsible for producing the major aqueous component of the tear film. For this, multiple groups have investigated the role of lacrimal gland cells in DED based on their regenerating, homing, and differentiating capabilities. In this review, we discuss in detail the therapeutic mechanisms and regenerative strategies that can potentially be applied for lacrimal gland regeneration as well as their therapeutic applications. This review mainly focuses on aqueous deficiency dry eye disease (ADDE) caused by lacrimal gland dysfunction and possible future treatment strategies. The current key findings from cell and tissue-based regenerative therapy modalities that could be utilised to achieve lacrimal gland tissue regeneration are summarized. In addition, this review summarises the available literature from in vitro to in vivo studies, their limitations in relation to lacrimal gland regeneration and the possible clinical applications. Finally, current issues and unmet needs of cell-based therapies in providing complete lacrimal gland tissue regeneration are discussed.

The effect of Rho Kinase inhibition on corneal nerve regeneration in vitro and in vivo.

PURPOSE: Impairment of corneal nerves can lead to neurotrophic keratopathy accompanied with severe ocular surface damage, which due to limited treatment options, can result in severe visual deterioration. This study evaluates a possible new treatment by enhancing the corneal nerve regeneration using a Rho Kinase inhibitor (Y27632). ROCK is known to play an important role in regulating cell morphology, adhesion and motility but little is known about its role in corneal nerve regeneration. METHODS: Effects of ROCK inhibition on murine peripheral nerves was assessed in single cell- and wound healing assays as well as a 3D in vitro model. Furthermore, Sholl analysis evaluating neuronal branching and life-death assays evaluating toxicity of the inhibitor were performed. An in vivo mouse model was established, with monitoring weekly corneal nerve regrowth using confocal microscopy. Additionally, corneal nerve fiber length was evaluated by immunofluorescence staining. Underlying pathways were examined by qrtPCR. RESULTS: ROCK inhibition leads to a significant enhancement of fiber growth in vitro. Sholl analysis revealed a higher degree of branching of treated fibers. Cytotoxicity assay showed no influence of Y27632 on cellular survival. In vivo measurement revealed significant enhanced regeneration after injury in the treated group. QrtPCR of trigeminal ganglia confirmed ROCK knock-down as well as altered pathways. CONCLUSION: The inhibition of ROCK after corneal nerve injury resulted in an enhanced regrowth of fibers in vitro and in vivo. This might be a step towards a new therapeutic concept for the treatment of impaired corneal nerves in diseases such as neurotrophic keratopathy.

High-Throughput Sequencing to Identify Mutations Associated with Retinal Dystrophies.

Retinal dystrophies (RD) are clinically and genetically heterogenous disorders showing mutations in over 270 disease-associated genes. Several millions of people worldwide are affected with different types of RD. Studying the relevance of disease-associated sequence alterations will assist in understanding disorders and may lead to the development of therapeutic approaches. Here, we established a whole exome sequencing (WES) pipeline to rapidly identify disease-associated mutations in patients. Sanger sequencing was applied to identify deep-intronic variants and to verify the co-segregation of WES results within families. We analyzed 26 unrelated patients with different syndromic and non-syndromic clinical manifestations of RD. All patients underwent ophthalmic examinations. We identified nine novel disease-associated sequence variants among 37 variants identified in total. The sequence variants located to 17 different genes. Interestingly, two cases presenting with Stargardt disease carried deep-intronic variants in ABCA4. We have classified 21 variants as pathogenic variants, 4 as benign/likely benign variants, and 12 as variants of uncertain significance. This study highlights the importance of WES-based mutation analyses in RD patients supporting clinical decisions, broadly based genetic diagnosis and support genetic counselling. It is essential for any genetic therapy to expand the mutation spectrum, understand the genes' function, and correlate phenotypes with genotypes.

Effects of temperature and soil fauna on the reduction and leaching of deoxynivalenol and zearalenone from Fusarium graminearum-infected maize stubbles.

A microcosm study was conducted at two different temperatures under laboratory conditions to investigate the regulatory capacity and the interactive performance of two soil fauna species (Aporrectodea caliginosa, earthworms, and Proisotoma minuta, collembolans) on the reduction of Fusarium toxins in contaminated maize stubbles. Single and mixed species treatments were exposed to artificially infected maize stubbles highly contaminated with the mycotoxins deoxynivalenol (DON) (10,462 µg kg-1) and zearalenone (ZEN) (2,780 µg kg-1) at 17 °C and 25 °C for time periods of 3 and 6 weeks. Immediately after the respective end of incubation, the microcosms were heavily watered to determine the leaching potential of DON and ZEN from contaminated maize stubbles. Maize residues, soil, and eluted water (percolate) samples were analysed for mycotoxin content using liquid chromatography coupled to mass spectrometry. The biomass of introduced earthworms and number of collembolans were monitored to get information about their adaptability to the experimental conditions. While the decline of ZEN was temperature-dependent, but not influenced by faunal activities, a reduction of DON due to faunal impact was observed by trend. In the leaching experiment, 67-82% of the DON content in the residual maize stubbles leached from the plant material by irrigation and was detected in the soil (1.9-3.4 µg kg-1) and in the percolate (12-295 µg L-1). In the case of ZEN, 27-50% of the mycotoxin leached from the residual maize stubbles due to watering but was only occasionally detected in traces in the soil and not found in the percolate. The results clearly reveal a leaching potential of both DON and ZEN, respectively, but a mobilisation with water was only observed for DON. Temperature confirmed to be a key factor, affecting the fate of the mycotoxins in the soil by driving the interaction between different soil fauna members as well as functional and trophic levels within the soil food web.

Legacy effects of temporary grassland in annual crop rotation on soil ecosystem services.

The introduction of temporary grassland into an annual crop rotation is recognized to improve soil ecosystem services, and resulting legacies can be beneficial for the following crops. In this context, the aim of the present study was to evaluate legacy effects of introducing temporary grassland into an annual crop rotation on five ecosystem services (i) soil structure maintenance (aggregate stability), (ii) water regulation (saturated hydraulic conductivity), (iii) biodiversity conservation (microbial biomass and microbial metabolic activity, as well as microorganism, enchytraeid, springtail and earthworm communities), (iv) pathogen regulation (soil suppressiveness to Verticillium dahliae), and (v) forage production and quality. Three crop rotation schemes, maintained for twelve years, were compared in four random blocks, one being an annual crop rotation without grassland (0%), another with a medium percentage of grassland (50%, corresponding to 3 years of continuous grassland in the crop rotation), and a third one with a high percentage of grassland in the crop rotation (75%, corresponding to 6 years of continuous grassland in the crop rotation). The results showed that the grassland introduction into an annual crop rotation improved, whatever the duration of the grassland, soil structure maintenance and biodiversity conservation, while it decreased pathogen regulation and did not modify water regulation. Comparing the two crop rotations that included grassland, indicated a stronger beneficial grassland legacy effect for the higher proportion of grassland concerning soil structure maintenance and biodiversity conservation. By contrast, water regulation, pathogen regulation and forage production were not affected by the legacy of the 75% grassland during the rotation. Overall, our findings demonstrated the extent to which grassland legacies are affecting the current state of soil properties and possible ecosystem services provided. To improve ecosystem services, soil management should take legacy effects into account and consider longer timeframes to apply beneficial practices.

Intraoperative OCT - Real-World User Evaluation in Routine Surgery. / Das intraoperative OCT ­ eine Real-World-basierte Nutzerevaluation im operativen Alltag.

BACKGROUND: In recent years, great progress has been made in intraoperative imaging using optical coherence tomography (iOCT). There are now several commercially available iOCT systems that allow high-resolution imaging of all structures of the eye without interrupting surgery. This real-time visualisation can provide additional information to conventional surgical microscopy, but is relatively expensive. The aim of our study was to find out how often OCT integrated into the surgical microscope is used by trained surgeons, or to what extent they consider that iOCT is relevant for intraoperative procedures. PATIENTS AND METHODS: A prospective monocentric analysis was conducted of the field of application and user-friendliness of the EnFocus Ultra-Deep OCT (Leica Microsystems), a mobile device combination of surgical microscope and OCT. The use and benefit were investigated of iOCT, which was not mandatory. Standardised documentation and evaluation using a questionnaire was performed by the respective surgeon (n = 5) immediately after surgery. RESULTS: Over a period of 25 working days, 118 procedures were performed in the operating theatre equipped with the microscope-OCT combination. The iOCT was used in 24.6% of the 118 procedures performed. iOCT was regarded as crucial to the intraoperative procedure in 3 of the 29 patients. In one patient, it was possible to check graft orientation during a DMEK operation in a very opaque cornea and, in the second patient, to visualise the correct positioning of an iris diaphragm in the capsular bag. In the third patient, the risk of developing a pseudoforamen was assessed, and this led to the decision not to perform a full gliosis peel. CONCLUSION: Experienced surgeons in a university eye hospital with a full surgical spectrum considered that intraoperative OCT was decisive for the course of surgery in only a few selected surgical situations, e.g. in case of limited corneal transparency. The impact of the use of iOCT on post-operative outcome quality still needs to be evaluated by larger prospective studies. On the basis of this survey, the cost-benefit ratio is still unclear.

Cell sheet technology: Influence of culture conditions on in vitro-cultivated corneal stromal tissue for regenerative therapies of the ocular surface.

The in vitro reconstruction of stromal tissue by long-term cultivation of corneal fibroblasts is a smart approach for regenerative therapies of ocular surface diseases. However, systematic investigations evaluating optimized cultivation protocols for the realization of a biomaterial are lacking. This study investigated the influence of supplements to the culture media of human corneal fibroblasts on the formation of a cell sheet consisting of cells and extracellular matrix. Among the supplements studied are vitamin C, fetal bovine serum, L-glutamine, components of collagen such as L-proline, L-4-hydroxyproline and glycine, and TGF-ß1, bFGF, IGF-2, PDGF-BB and insulin. After long-term cultivation, the proliferation, collagen and glycosaminoglycan content and light transmission of the cell sheets were examined. Biomechanical properties were investigated by tensile tests and the ultrastructure was characterized by electron microscopy, small-angle X-ray scattering, antibody staining and ELISA. The synthesis of extracellular matrix was significantly increased by cultivation with insulin or TGF-ß1, each with vitamin C. The sheets exhibited a high transparency and suitable material properties. The production of a transparent, scaffold-free, potentially autologous, in vitro-generated construct by culturing fibroblasts with extracellular matrix synthesis-stimulating supplements represents a promising approach for a biomaterial that can be used for ocular surface reconstruction in slowly progressing diseases.

Decellularized porcine conjunctiva as an alternative substrate for tissue-engineered epithelialized conjunctiva.

PURPOSE: The long-term success of visual rehabilitation in patients with severe conjunctival scarring is reliant on the reconstruction of the conjunctiva with a suitable substitute. The purpose of this study is the development and investigation of a re-epithelialized conjunctival substitute based on porcine decellularized conjunctiva (PDC). METHODS: PDC was re-epithelialized either with pre-expanded human conjunctival epithelial cells (PDC + HCEC) or with a human conjunctival explant placed directly on PDC (PDC + HCEx). Histology and immunohistochemistry were performed to evaluate epithelial thickness, proliferation (Ki67), apoptosis (Caspase 3), goblet cells (MUC5AC), and progenitor cells (CK15, ΔNp63, ABCG2). The superior construct (PDC + HCEx) was transplanted into a conjunctival defect of a rabbit (n = 6). Lissamine green staining verified the epithelialization in vivo. Orbital tissue was exenterated on day 10 and processed for histological and immunohistochemical analysis to examine the engrafted PDC + HCEx. A human-specific antibody was used to detect the transplanted cells. RESULTS: From day-14 in vitro onward, a significantly thicker epithelium and greater number of cells expressing Ki67, CK15, ΔNp63, and ABCG2 were noted for PDC + HCEx versus PDC + HCEC. MUC5AC-positive cells were found only in PDC + HCEx. The PDC + HCEx-grafted rabbit conjunctivas were lissamine-negative during the evaluation period, indicating epithelial integrity. Engrafted PDC + HCEx showed preserved progenitor cell properties and an increased number of goblet cells comparable to those of native conjunctiva. CONCLUSION: Placing and culturing a human conjunctival explant directly on PDC (PDC + HCEx) enables the generation of a stable, stratified, goblet cell-rich construct that could provide a promising alternative conjunctival substitute for patients with extensive conjunctival stem and goblet cell loss.

Decellularized human corneal stromal cell sheet as a novel matrix for ocular surface reconstruction.

The shortage of donor corneas as well as the limitations of tissue substitutes leads to the necessity to develop alternative materials for ocular surface reconstruction. Corneal surface substitutes must fulfill specific requirements such as high transparency, low immunogenicity, and mechanical stability combined with elasticity. This in vitro study evaluates a decellularized matrix secreted from human corneal fibroblasts (HCF) as an alternative material for ocular surface reconstruction. HCF from human donors were cultivated with the supplementation of vitamin C to form a stable and thick matrix. Furthermore, due to enhanced cultivation time, a three-dimensional like multilayered construct which partly mimics the complex structure of the corneal stroma could be generated. The formed human cell-based matrices (so-called cell sheets [CS]) were subsequently decellularized. The complete cell removal, collagen content, ultrastructure, and cell toxicity of the decellularized CS (DCS) as well as biomechanical properties were analyzed. Surgical feasibility was tested on enucleated porcine eyes. After decellularization and sterilization, a transparent, thick, cell free, and sterile tissue substitute resulted, which allowed expansion of limbal epithelial stem cells with no signs of cytotoxicity, and good surgical feasibility. DCS seem to be a promising new corneal tissue substitute derived from human cells without the limitation of donor material; however, future in vivo studies are necessary to further elucidate its potential for ocular surface reconstruction.

[If Artificial Tears Aren't Enough. The Importance of Inflammatory Processes in Dry Eye Disease. Practical Aspects of an Anti-Inflammatory Therapy of Dry Eye Disease]. / Wenn Tränenersatzmittel nicht mehr ausreichen: die Bedeutung von Entzündungsprozessen beim Trockenen Auge. Praktische Aspekte einer antientzündlichen Therapie des Trockenen Auges.

Dry eye disease (DED) is a heterogenous disease of the ocular surface. Multiple pathogenetic factors are responsible for the disease process, but DED is generally linked to an increase in the osmolarity of the tear film and to inflammation of the ocular surface. The significance of inflammatory processes in DED is highlighted in the most recent definition of dry eye in the Dry Eye Workshop (DEWS). It is therefore critically important for the management of dry eye disease to understand the pathomechanisms and therapeutic options for the treatment of inflammatory processes. This review summarizes our current knowledge on Inflammation associated with DED and provides practical recommendations for the diagnosis and monitoring of the disease, as well as the use of currently available therapeutic options to counteract inflammation in DED.

Treatment of diabetic retinopathy through neuropeptide Y-mediated enhancement of neurovascular microenvironment.

Diabetic retinopathy (DR) is one of the most severe clinical manifestations of diabetes mellitus and a major cause of blindness. DR is principally a microvascular disease, although the pathogenesis also involves metabolic reactive intermediates which induce neuronal and glial activation resulting in disruption of the neurovascular unit and regulation of the microvasculature. However, the impact of neural/glial activation in DR remains controversial, notwithstanding our understanding as to when neural/glial activation occurs in the course of disease. The objective of this study was to determine a potential protective role of neuropeptide Y (NPY) using an established model of DR permissive to N-methyl-D-aspartate (NMDA)-induced excitotoxic apoptosis of retinal ganglion cells (RGC) and vascular endothelial growth factor (VEGF)-induced vascular leakage. In vitro evaluation using primary retinal endothelial cells demonstrates that NPY promotes vascular integrity, demonstrated by maintained tight junction protein expression and reduced permeability in response to VEGF treatment. Furthermore, ex vivo assessment of retinal tissue explants shows that NPY can protect RGC from excitotoxic-induced apoptosis. In vivo clinical imaging and ex vivo tissue analysis in the diabetic model permitted assessment of NPY treatment in relation to neural and endothelial changes. The neuroprotective effects of NPY were confirmed by attenuating NMDA-induced retinal neural apoptosis and able to maintain inner retinal vascular integrity. These findings could have important clinical implications and offer novel therapeutic approaches for the treatment in the early stages of DR.

Towards Lacrimal Gland Regeneration: Current Concepts and Experimental Approaches.

Dry eye disease (DED) is a complex and multifactorial disease resulting in a continual cycle of tear hyperosmolarity and inflammation. Patients suffering from DED experience severe pain and visual impairments leading to a reduced quality of life. Aqueous-deficient dry eye (ADDE), mainly caused through a loss of functional lacrimal gland tissue, results in the most severe forms of DED. Despite a high prevalence, the current treatments remain palliative and may be insufficient to alleviate the symptoms. Consequently, investigations on experimental approaches for in situ lacrimal gland regeneration are of great clinical interest. This article reviews the current knowledge about processes involved in lacrimal gland regeneration, about lacrimal gland resident stem cells, and offers deductions about possible concepts for in situ lacrimal gland regeneration. Promising starting points might be the utilization of therapeutic proteins, such as bone morphogenetic protein 7, mesenchymal stem cells (MSC) or MSC-based treatments such as conditioned medium, lyophilized cell extracts or adult acinar cells. This review further summarizes current experimental approaches for the treatment of ADDE in animal models and patients. Approaches investigating side population stem cells, epithelial progenitor cells and MSC showed that the transplantation of these cells had therapeutic effects on ADDE. However, the most promising and best-studied experimental approach is the use of MSC for induction/enhancement of in situ lacrimal gland regeneration. Their immunomodulatory effects, low immunogenicity, promotion of tissue regeneration and involvement during spontaneous lacrimal regeneration are favorable traits for clinical applications. In addition, the efficacy and safety of allogeneic MSC transplantation have already been demonstrated in a small patient cohort.

MSC Transplantation Improves Lacrimal Gland Regeneration after Surgically Induced Dry Eye Disease in Mice.

Dry eye disease (DED) is a multifactorial disease characterized by a disrupted tear film homeostasis and inflammation leading to visual impairments and pain in patients. Aqueous-deficient dry eye (ADDE) causes the most severe progressions and depends mainly on the loss of functional lacrimal gland (LG) tissue. Despite a high prevalence, therapies remain palliative. Therefore, it is of great interest to develop new approaches to curatively treat ADDE. Mesenchymal stem/stromal cells (MSC) have been shown to induce tissue regeneration and cease inflammation. Moreover, an increasing amount of MSC was found in the regenerating LG of mice. Therefore, this study investigated the therapeutic effect of MSC transplantation on damaged LGs using duct ligation induced ADDE in mice. Due to the transplantation of sex-mismatched and eGFP-expressing MSC, MSC could be identified and detected until day 21. MSC transplantation significantly improved LG regeneration, as the amount of vital acinar structures was significantly increased above the intrinsic regeneration capacity of control. Additionally, MSC transplantation modulated the immune reaction as macrophage infiltration was delayed and TNFα expression decreased, accompanied by an increased IL-6 expression. Thus, the application of MSC appears to be a promising therapeutic approach to induce LG regeneration in patients suffering from severe DED/ADDE.

Analysis of lacrimal gland derived mesenchymal stem cell secretome and its impact on epithelial cell survival.

In situ regeneration of lacrimal gland (LG) tissue would be a promising approach to curatively treat dry eye disease (DED). Mesenchymal stem cells (MSC) exhibit therapeutic effects in a variety of pathological conditions and our group recently reported that their number increases in regenerating mouse LG. Since the therapeutic effects are suggested to arise from secreted trophic factors, the application of MSC-secreted proteins seems to be a promising approach to induce/enhance LG regeneration. Therefore, this study aims to optimize the isolation of murine LG-MSC and analyze their secretome to investigate its potential for LG epithelial cell survival in vitro. For optimization, LG-MSC were isolated by an explant technique or cell sorting and their secretome was investigated under normal and inflammatory conditions. Results showed that the secretome of MSC had beneficial effects on the viability of ethanol-damaged LG epithelial cells. Additional, Lipocalin-2, prosaposin, ras GTPase-activating protein-binding protein 1 (Rac1) and signal transducer and activator of transcription 1 (STAT1), proteins that were up-regulated under inflammatory conditions, further improved the cell survival of ethanol-damaged LG epithelial cells. Interestingly, recovery of cell viability was highest, when the cells were incubated with STAT1. Summarizing, this study identified promising proteins for further studies on LG regeneration.

Restoring retinal neurovascular health via substance P.

Regulation of vascular permeability plays a major role in the pathophysiology of visually threatening conditions such as retinal vein occlusion and diabetic retinopathy. Principally, several factors such as vascular endothelial growth factor (VEGF), are up-regulated or induced in response to hypoxia thus adversely affecting the blood-retinal barrier (BRB), resulting in retinal edema and neovascularisation. Furthermore, current evidence supports a dysregulation of the inner retinal neural-vascular integrity as a critical factor driving retinal ganglion cell (RGC) death and visual loss. The principal objective of this study was to interrogate whether Substance P (SP), a constitutive neurotransmitter of amacrine and ganglion cells, may protect against N-methyl-d-aspartate (NMDA)-induced excitotoxic apoptosis of ganglion cells and VEGF-induced vessel leakage in the retina. Tight junctional protein expression and a Vascular Permeability Image Assay were used to determine vascular integrity in vitro. The protective effect of SP on RGC was established in ex vivo retinal explants and in vivo murine models. After NMDA administration, a reduction in TUNEL+ cells and a maintained number of Brn-3a+ cells were found, indicating an inhibition of RGC apoptosis mediated by SP. Additionally, SP maintained endothelial tight junctions and decreased VEGF-induced vascular permeability. In conclusion, administration of SP protects against NMDA apoptosis of RGC and VEGF-induced endothelial barrier breakdown.