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Einleitung: Eigenserum-Augentropfen (ESAT) werden zur Behandlung des Trockenen Auges in besonders schweren Fällen eingesetzt. Nachdem mit ihrer Anwendung einmal begonnen wurde, gilt eine Rückkehr zu anderen gleitfördernden Augentropfen oder sonstigen Therapieoptionen auf dem Markt als unmöglich. Material und Methoden: In einer randomisierten Studie wurde 11 Patienten, die seit mindestens 3 Monaten mit ESAT behandelt wurden, angeboten, stattdessen Augentropfen mit langkettiger Hyaluronsäure zu verwenden. Die Kontrollgruppe (n = 5) setzte ihre Behandlung mit ESAT fort. Die Verumgruppe (n = 6) wendete Augentropfen mit sehr langkettiger Hyaluronsäure (Comfort Shield®) anstelle der ESAT an. Ergebnisse: Von 4 der anfänglich 6 Patienten in der Verumgruppe, die die Studie abschlossen, entschieden sich 2 (50%) dafür, auch über den Untersuchungszeitraum hinaus bei den Augentropfen mit langkettiger Hyaluronsäure zu bleiben, die anderen beiden kehrten zu ihrer vorherigen Therapie mit ESAT zurück. Die Kontrollgruppe führte ihre Behandlung fort wie zuvor und schloss die Studie nach 8 Wochen ab. Schlussfolgerung: Erstmals erwiesen sich künstliche Augentropfen in Form von langkettigen Hyaluronsäure-Augentropfen als akzeptable Alternative zu ESAT. Einige Patienten empfanden diese Tropfen als noch besser als die Tropfen aus ihrem Eigenserum. Dies ist die erste Evidenz, dass durch Optimierung der Struktur des Hyaluronsäuremoleküls Augentropfen hergestellt werden könnten, die im Vergleich zu anderen Tränenersatzmitteln und sogar zu Eigenserum als besser wahrgenommen werden. Dies eröffnet neue Perspektiven für die Behandlung von Patienten mit schweren Fällen des Trockenen Auges.

Introduction: Autologous serum eye drops (ASED) are used in the treatment of most severe stages of dry eye. Once introduced, it is currently considered impossible to return to other lubricating eye drops or other commercially available therapeutic regimen. Materials and Methods: In a randomized study, non-preserved high-molecular-weight hyaluronic acid eye drops were offered as an alternative to 11 patients using autologous serum treatment for at least 3 months. The control group (n = 5) continued their treatment with ASED. The verum group (n = 6) used very-high-molecular-weight hyaluronic eye drops (Comfort Shield®) instead of the ASED. Results: From four of initially six patients in the verum group that finished the study, 2 (50%) preferred to stay with the very-high-molecular-weight hyaluronic acid eye drops beyond the trial period, the other two returned to the earlier therapy with ASED. The control group continued their treatment as before and finished the study after 8 weeks. Conclusion: For the first time, artificial eye drops, i.e., high-molecular-weight hyaluronic acid eye drops, offered an acceptable alternative to ASED. Some patients perceived these drops as even better than the patient’s own serum. This is the first evidence that optimization of the molecular structure of hyaluronic acid can be used to create eye drops that are perceived to be better than other tested tear substitutes and even patients’ own serum. This offers a new treatment perspective for patients with very severe dry eye disease.

Purpose/Aim of the study: A recently proposed technique enables the generation of continuously increasing mosaic images of the corneal sub-basal nerve plexus (SNP) using in vivo corneal confocal microscopy (CCM). The aim of the present study was to investigate the progression of the corneal nerve fiber length (CNFL) measured in the growing mosaic images with regard to their increasing area.Five large datasets from three healthy volunteers were examined using the proposed CCM technique. Intermediate mosaic images were created and assessed for CNFL.The measured CNFL progression shows both over- and underestimation of the CNFL for small observed areas. Increasing the mosaic image area stabilizes the CNFL values and reduces the moving variance in all five datasets. The relative deviation of means from values of first and second examination of two of the subjects shows high differences for an observed area of1.5 mmThe present examination provides two measures to quantify different area-dependent aspects of the CNFL measured in an expanding mosaic image. The moving variance measures how stable the CNFL can be considered at a certain mosaic size. The relative deviation of means from two repeated CCM examinations on the other hand gives some indication on the level of reliability that can be expected from the measured CNFL. The progression of CNFL in the examined datasets manifests a potentially very high variability for mosaic sizes of less than about 1.5 mm

The capability of corneal confocal microscopy (CCM) to acquire high-resolution in vivo images of the densely innervated human cornea has gained considerable interest in using this non-invasive technique as an objective diagnostic tool for staging peripheral neuropathies. Morphological alterations of the corneal subbasal nerve plexus (SNP) assessed by CCM have been shown to correlate well with the progression of neuropathic diseases and even predict future-incident neuropathy. Since the field of view of single CCM images is insufficient for reliable characterisation of nerve morphology, several image mosaicking techniques have been developed to facilitate the assessment of the SNP in large-area visualisations. Due to the limited depth of field of confocal microscopy, these approaches are highly sensitive to small deviations of the focus plane from the SNP layer. Our contribution proposes a new automated solution, combining guided eye movements for rapid expansion of the acquired SNP area and axial focus plane oscillations to guarantee complete imaging of the SNP. We present results of a feasibility study using the proposed setup to evaluate different oscillation settings. By comparing different image selection approaches, we show that automatic tissue classification algorithms are essential to create high-quality mosaic images from the acquired 3D datasets.

Optical coherence tomography (OCT) of the retina and corneal confocal laser scanning microscopy (CLSM) of the subbasal nerve plexus (SBP) are noninvasive techniques for quantification of the ocular neurodegenerative changes in individuals with type 1 diabetes mellitus (T1DM). In adult T1DM patients these changes are hardly related to T1DM only. Instead, ageing and/or lifestyle associated comorbidities have to be considered as putative confounding variables. Therefore, we investigated pediatric T1DM patients (n = 28; 14.2 ± 2.51 y; duration of disease: 5.39 ± 4.16 y) without clinical signs of diabetic retina disease, neuropathy, vasculopathy or nephropathy and compared our findings with those obtained in healthy controls (n = 46; 14.8 ± 1.89 y). The SBP was characterized by the averaged length, thickness, and tortuosity of nerve fibers as well as the number of branching and connecting points. OCT was used to determine the total thickness of the retina (ALL) and the thickness of each retinal layer. Both methods revealed signs of early neurodegenerative changes, e.g. thinning of distinct retinal layers at the pericentral ring and shortening of corneal nerve fibers that are already present in pediatric T1DM patients. Standardization of instruments and algorithms are urgently required to enable uniform comparison between different groups and define normative values to introduce in the clinical setting.

Die diabetische Neuropathie (DN) gehort zu den haufigsten Folgeerkrankungen des Diabetes mellitus. Dabei treten Schadigungen des sensiblen Nervensystems auf, die sich auch im subbasalen Nervenplexus der Kornea manifestieren. Mittels der konfokalen Laser-Scanning-Mikroskopie sind diese einer In-vivo-Visualisierung zuganglich. Dieser Beitrag untersucht Marker, die Alterationen dieser kornealen Nervenfasern beschreiben. Mittels statistischer Berechnungen und einer interaktiv-visuellen Analyse der Daten aus 2 klinischen Studien werden charakteristische Merkmale und Auspragungen extrahiert, mit dem Ziel, DN-Patienten und gesunde Probanden zu trennen. Durch diese Klassifikation soll die Diagnose der diabetischen Neuropathie unterstutzt werden. Ausgangspunkt fur die visuelle Analyse sind verschiedene Darstellungen, die sowohl die Ergebnisse der statistischen Berechnungen als auch die erfassten Daten im multidimensionalen Zusammenhang zeigen. Auf alle Darstellungen kann der Nutzer interaktiv zugreifen, um sie gesondert anzuzeigen und auszuwerten. Durch diese visuelle Betrachtung soll die Klassifikation der Probanden genauer analysiert werden. Unklare Zuordnungen werden dabei einer separaten Klasse, der so bezeichneten „fuzzy group“ zugewiesen. Fur diese Probanden ist eine weiterfuhrende Diagnostik notwendig.

Hintergrund: Die hohe Auflosung der konfokalen Laser-Scanning-Mikroskopie (CCM) ermoglicht die Abbildung des kornealen subbasalen Nervenplexus (SNP) in vivo. Aufgrund des kleinen Bildfelds (0,16 mm²) ist ein einzelnes CCM-Bild nicht ausreichend fur eine zuverlassige morphometrische SNP-Charakterisierung. Wir entwickeln deshalb derzeit ein automatisiertes Verfahren, das CCM-Bildsequenzen aufnimmt und daraus grosflachige SNP-Abbildungen erzeugt. Methoden: Zur Aufnahme einer Bildserie eines groseren SNP-Areals wird die Blickrichtung des Patienten durch eine computergesteuerte, bewegte Fixationsmarke auf einem Display vor dem nicht untersuchten Auge geeignet gefuhrt. Die CCM-Bildsequenz wird mit 30 Hz aufgezeichnet und bereits wahrend der Aufnahme wird ein Mosaikbild berechnet, an dem der Aufnahmeprozess beobachtet und die resultierende Bildgrose und -qualitat beurteilt werden kann. Verbleibende Bildfehler werden in einem anschliesenden Berechnungsschritt automatisch korrigiert. Ergebnisse: Bei geeignet gewahlter Fixationsmarkenbahn konnte mit einem ersten Prototypsystem ein mittlerer Bildgrosenzuwachs von 0,18 mm²/s erreicht werden. Schlussfolgerung: Mit dem vorgestellten Verfahren konnen grosflachige SNP-Abbildungen erzeugt werden. Das Verfahren ist gekennzeichnet durch einen hohen Automatisierungsgrad und durch kurze Untersuchungszeiten.

Corneal confocal microscopy (CCM) has revealed reduced corneal nerve fiber (CNF) length and density (CNFL, CNFD) in patients with diabetes, but the spatial pattern of CNF loss has not been studied. We aimed to determine whether spatial analysis of the distribution of corneal nerve branching points (CNBPs) may contribute to improving the detection of early CNF loss. We hypothesized that early CNF decline follows a clustered rather than random distribution pattern of CNBPs. CCM, nerve conduction studies (NCS), and quantitative sensory testing (QST) were performed in a cross-sectional study including 86 patients recently diagnosed with type 2 diabetes and 47 control subjects. In addition to CNFL, CNFD, and branch density (CNBD), CNBPs were analyzed using spatial point pattern analysis (SPPA) including 10 indices and functional statistics. Compared to controls, patients with diabetes showed lower CNBP density and higher nearest neighbor distances, and all SPPA parameters indicated increased clustering of CNBPs (all P97.5th percentile of controls in up to 23.5% of patients. When combining an individual SPPA parameter with CNFL, ≥1 of 2 indices were >99th or

BACKGROUND An analysis of the corneal subbasal nerve plexus (SNP) allows an evaluation of the peripheral neuropathy in cases of degenerative diseases. In order to study the SNP structures quantitatively the automatically calculated morphological and topological parameters are required. METHODS In vivo confocal laser scanning microscopy (Heidelberg Retina Tomograph II/Rostock Cornea Module) was performed in healthy volunteers as well as patients with severe diabetic neuropathy. An adapted image processing algorithm was used to preprocess, segment and evaluate quantitatively the nerve fibers of the SNP. Data sets were analysed statistically. RESULTS The developed algorithm allows an automated detection of SNP structures. Furthermore, it allows the collection of data based on morphological and topological parameters. The main parameters that show significant differences between healthy cornea and cases of diabetic neuropathy are nerve fibre density and length, number of branching, tortuosity and number of terminal and crossing points. All parameters of the measurements can be used isolated, combined or weighted for quantification of the SNP networks. CONCLUSION The presented fully automated preprocessing eliminates a large number of motion-induced artefacts. The quality of the resulting pictures allows an automated quantification using characteristic measurements. This represents an in vivo, non-invasive technology analysing degenerative changes of SNP especially in the course of diabetes mellitus.

Background To study the severity of diabetic neuropathy, diabetic retinopathy and grades of diabetic foot syndrome for correlations with corneal subbasal nerve plexus (SBP) changes in Congolese patients with type 2 diabetes. Methodology/Principal Findings Twenty-eight type 2 diabetes patients with diabetes-related foot ulceration were recruited in a diabetic care unit in Kinshasa, Democratic Republic of Congo. Corneal SBP was investigated by confocal laser-scanning microscopy to analyse nerve fibre density (NFD) [µm/ µm²], number of branches [n] and number of connectivity points [n]. Foot ulceration was graded using the Wagner ulcer classification. Corneal sensitivity (Cochet-Bonnet), Neuropathy Symptom Score (NSS), Neuropathy Disability Score (NDS), ankle-brachial index (ABI) and ophthalmological status were evaluated. Foot ulceration was ranked as mild (Wagner 0-1: 13 patients/46.4%), moderate (Wagner 2-3: 10 patients/35.7%) and severe (Wagner 4-5: 5 patients/17.9%). The correlation between Wagner Score and NFD (p=0.017, r = - 0,454), NDS and NFD (p=0,039, r = - 0.400) as well as Wagner Score and HbA1c (p=0,007, r = - 0.477) was stated. Significant differences in confocal SBP parameters were observed between Wagner 0-1 and Wagner 4 5 (number of branches (p=0.012), number of connectivity points (p=0.001), nerve fibre density (p=0.033)) and ABI (p=0.030), and between Wagner 2-3 and Wagner 4-5 (number of branches (p=0.003), number of connectivity points (p=0.005) and nerve fibre density (p=0.014)). Differences in NDS (p=0.001) and corneal sensation (p=0.032) were significant between Wagner 0-1 and Wagner 2-3. Patients with diabetic retinopathy had significantly longer diabetes duration (p=0.03) and higher NDS (p=0.01), but showed no differences in SBP morphology or corneal sensation. Conclusions/Significance While confirming the diabetic aetiology of foot ulceration due to medial arterial calcification, this study indicates that the grade of diabetic foot syndrome correlates with corneal SBP changes and corneal sensation in patients in sub-Saharan Africa.

PURPOSE A growing number of studies provide evidence that the morphology of the corneal subbasal nerve plexus (SNP), examined by corneal confocal microscopy (CCM), is a sensitive marker for diabetic peripheral neuropathy. However, it has been established that the field of view of a single CCM image (≈0.16 mm(2)) is insufficient for reliable assessment of corneal nerve fiber morphology. The present work proposes a highly automated technique for imaging an extended area of the SNP and creating large-scale montages. METHODS A moving fixation target is presented on a small display in front of the nonexamined eye. By guiding the viewing direction of the subject in an expanding spiral pattern, the scanned corneal area is continuously expanded. Specialized software algorithms subsequently assemble a mosaic image from the acquired CCM image data. The proposed technique was applied in 12 healthy subjects. RESULTS Montage images of the SNP were successfully created from all examinations performed. The mean imaged SNP area was 9.86 mm(2) (range, 1.62-18.31 mm(2)). The mean CCM duration was 65.33 seconds (range, 14.58-142.58 seconds). CONCLUSIONS The key advances embodied in the proposed technique are its high degree of integration and automation (both for image acquisition and image processing) and the resulting short duration of CCM. By providing an easy-to-use tool for obtaining large-scale mosaic images of the SNP, this technique has the potential to facilitate larger clinical trials where SNP morphology is used as a surrogate marker for peripheral neuropathy.

Background To quantify the development of radiation neuropathy in corneal subbasal nerve plexus (SNP) after plaque brachytherapy, and the subsequent regeneration of SNP micromorphology and corneal sensation. Methods Nine eyes of 9 melanoma patients (ciliary body: 3, iris: 2, conjunctiva: 4) underwent brachytherapy (ruthenium-106 plaque, dose to tumour base: 523 ± 231 Gy). SNP micromorphology was assessed by in-vivo confocal microscopy. Using software developed in–house, pre-irradiation findings were compared with those obtained after 3 days, 1, 4 and 7 months, and related to radiation dose and corneal sensation. Results After 3 days nerve fibres were absent from the applicator zone and central cornea, and corneal sensation was abolished. The earliest regenerating fibres were seen at the one-month follow-up. By 4 months SNP structures had increased to one-third of pre-treatment status (based on nerve fibre density and nerve fibre count), and corneal sensation had returned to approximately two-thirds of pre-irradiation values. Regeneration of SNP and corneal sensation was nearly complete 7 months after plaque brachytherapy. Conclusions The evaluation of SNP micromorphology and corneal sensation is a reliable and clinically useful method for assessing neuropathy after plaque brachytherapy. Radiation-induced neuropathy of corneal nerves develops quickly and is partly reversible within 7 months. The clinical impact of radiation-induced SNP damage is moderate.

BACKGROUND: The alterations of subbasal nerve plexus (SBP) innervation and corneal sensation were estimated non-invasively and compared with the values in healthy volunteers. Additionally, this study addressed the relation of SBP changes to the retinal status, glycemic control and diabetes duration. METHODOLOGY/PRINCIPAL FINDINGS: Eighteen eyes of diabetic patients with peripheral diabetic neuropathy aged 68.8±8.8 years and twenty eyes of healthy volunteers aged 66.3±13.3 yrs. were investigated with in vivo confocal laser-scanning microscopy (CLSM). An adapted algorithm for image analysis was used to quantify the morphological and topological properties of SBP. These properties were correlated to incidence of diabetic retinopathy (DR) and corneal sensation (Cochet-Bonnet esthesiometer). The developed algorithm allows a fully automated analysis of pre-segmented SBP structures. Altogether, 10 parameters were analysed, and all of them revealed significant differences between diabetic patients and healthy volunteers. The nerve fibre density, total fibre length and nerve branches were found to be significantly lower in patients with diabetes than those of control subjects (nerve fibre density 0.006±0.002 vs. 0.020±0.007 mm/mm(2); total fibre length 6223±2419 vs. 19961±6553 µm; nerve branches 25.3±28.6 vs. 141.9±85.7 in healthy volunteers). Also the corneal sensation was significantly lower in diabetic group when compared to controls (43±11 vs. 59±18 mm). There was found no difference in SBP morphology or corneal sensation in the subgroups with (DR) or without (NDR) diabetic retinopathy. CONCLUSIONS/SIGNIFICANCE: SBP parameters were significantly reduced in diabetic patients, compared to control group. Interestingly, the SBP impairment could be shown even in the diabetic patients without DR. Although automatic adapted image analysis simplifies the evaluation of in vivo CLSM data, image acquisition and quantitative analysis should be optimised for the everyday clinical practice.

PURPOSE. We examined agreement among experts in the assessment of corneal subbasal nerve tortuosity. METHODS. Images of corneal subbasal nerves were obtained from investigators at seven sites (Auckland, Boston, Linkoping, Manchester, Oslo, Rostock, and Sydney) using laser-scanning in vivo confocal microscopy. A set of 30 images was assembled and ordered by increasing tortuosity by 10 expert graders from the seven sites. In a first experiment, graders assessed tortuosity without a specific definition and performed grading three times, with at least 1 week between sessions. In a second experiment, graders assessed the same image set using four focused tortuosity definitions. Intersession and intergrader repeatability for the experiments were determined using the Spearman rank correlation. RESULTS. Expert graders without a specific tortuosity definition had high intersession (Spearman correlation coefficient 0.80), but poor intergrader (0.62) repeatability. Specific definitions improved intergrader repeatability to 0.79. In particular, tortuosity defined by frequent small-amplitude directional changes (short range tortuosity) or by infrequent large-amplitude directional changes (long range tortuosity), indicated largely independent measures and resulted in improved repeatability across the graders. A further refinement, grading only the most tortuous nerve in a given image, improved the average correlation of a given graders ordering of images with the group average to 0.86 to 0.90. CONCLUSIONS. Definitions of tortuosity specifying short or long-range tortuosity and considering only the most tortuous nerve in an image improved the agreement in tortuosity grading among a group of expert observers. These definitions could improve accuracy and consistency in quantifying subbasal nerve tortuosity in clinical studies. uj