We sought to determine whether early nerve damage may be detected by corneal confocal microscopy (CCM), skin biopsy, and neurophysiological tests in 86 recently diagnosed type 2 diabetic patients compared with 48 control subjects. CCM analysis using novel algorithms to reconstruct nerve fiber images was performed for all fibers and major nerve fibers (MNF) only. Intraepidermal nerve fiber density (IENFD) was assessed in skin specimens. Neurophysiological measures included nerve conduction studies (NCS), quantitative sensory testing (QST), and cardiovascular autonomic function tests (AFTs). Compared with control subjects, diabetic patients exhibited significantly reduced corneal nerve fiber length (CNFL-MNF), fiber density (CNFD-MNF), branch density (CNBD-MNF), connecting points (CNCP), IENFD, NCS, QST, and AFTs. CNFD-MNF and IENFD were reduced below the 2.5th percentile in 21% and 14% of the diabetic patients, respectively. However, the vast majority of patients with abnormal CNFD showed concomitantly normal IENFD and vice versa. In conclusion, CCM and skin biopsy both detect nerve fiber loss in recently diagnosed type 2 diabetes, but largely in different patients, suggesting a patchy manifestation pattern of small fiber neuropathy. Concomitant NCS impairment points to an early parallel involvement of small and large fibers, but the precise temporal sequence should be clarified in prospective studies.

https://diabetes.diabetesjournals.org/content/diabetes/63/7/2454.full.pdf

Early Detection of Nerve Fiber Loss by Corneal Confocal Microscopy and Skin Biopsy in Recently Diagnosed Type 2 Diabetes

In vivo confocal microscopy (IVCM) is becoming an indispensable tool for studying corneal physiology and disease. Enabling the dissection of corneal architecture at a cellular level, this technique offers fast and noninvasive in vivo imaging of the cornea with images comparable to those of ex vivo histochemical techniques. Corneal nerves bear substantial relevance to clinicians and scientists alike, given their pivotal roles in regulation of corneal sensation, maintenance of epithelial integrity, as well as proliferation and promotion of wound healing. Thus, IVCM offers a unique method to study corneal nerve alterations in a myriad of conditions, such as ocular and systemic diseases and following corneal surgery, without altering the tissue microenvironment. Of particular interest has been the correlation of corneal subbasal nerves to their function, which has been studied in normal eyes, contact lens wearers, and patients with keratoconus, infectious keratitis, corneal dystrophies, and neurotrophic keratopathy. Longitudinal studies have applied IVCM to investigate the effects of corneal surgery on nerves, demonstrating their regenerative capacity. IVCM is increasingly important in the diagnosis and management of systemic conditions such as peripheral diabetic neuropathy and, more recently, in ocular diseases. In this review, we outline the principles and applications of IVCM in the study of corneal nerves in various ocular and systemic diseases.

/pdf/in-vivo-confocal-microscopy-of-corneal-nerves-in-health-and-hnxunvo6by.pdf

In Vivo Confocal Microscopy of Corneal Nerves in Health and Disease

PURPOSE To evaluate the relationship between the in vivo confocal microscopic (IVCM) morphology of subbasal corneal nerves, corneal sensitivity, and clinical parameters in patients with non-Sjogren dry eye disease (NSDD). METHODS Forty-three patients with NSDD and 14 healthy age- and sex-matched control subjects were included. Each patient underwent an evaluation of ocular surface disease symptoms using the Ocular Surface Disease Index (OSDI); tear film break-up time (TBUT); corneal and conjunctival staining (Oxford scale); the Schirmer test; corneal sensation testing using the Cochet-Bonnet esthesiometer; and corneal subbasal nerve analysis with IVCM. One eye of each subject was included in the study. RESULTS Mean corneal sensitivity was significantly lower in the NSDD group as compared with the control group (P = 0.014). Corneal subbasal nerves showed significant changes in NSDD patients as compared with normal subjects: lower density (P < 0.001); increased tortuosity (P < 0.001); number of beadings (P < 0.001); and width (P = 0.041). In patients with NSDD, corneal subbasal nerve density was correlated to age (r = -0.352, P = 0.021); the Oxford scale (r = -0.486, P = 0.01); and central corneal sensitivity (r = 0.383, P = 0.041). The maximum length of nerve fibers within a frame (MaxL) was correlated to the OSDI (r = -0.265, P = 0.019) and the Oxford scale (r = -0.307, P = 0.039). In multivariate analysis after adjustment for age, corneal subbasal nerve density remained correlated to the Oxford score (P < 0.001). The Oxford score was also correlated to central corneal sensitivity (r = -0.304, P = 0.042). CONCLUSIONS NSDD patients have both structural and functional alterations of subbasal corneal nerves and these changes are related to the severity of dry eye.

Corneal nerve structure and function in patients with non-sjogren dry eye: clinical correlations.

The traditional model of dry eye disease based on tear deficiency has presented us with many unanswered questions. Recent studies support the notion that dry eye-like symptoms represent non-specific corneal pain and provide new insights into the mechanisms that sustain the integrity of the optical tear layer. Thus, this enigmatic disease can be viewed with a new perspective, which involves the dysfunctional corneal pain system as a central pathogenetic feature of a series of disorders collectively known today as dry eye.

The Corneal Pain System. Part I: The Missing Piece of the Dry Eye Puzzle

In vivo confocal microscopy (IVCM) is an emerging technology that provides minimally invasive, high resolution, steady-state assessment of the ocular surface at the cellular level. Several challenges still remain but, at present, IVCM may be considered a promising technique for clinical diagnosis and management. This minireview summarizes some key findings in IVCM of the ocular surface, focusing on recent and promising attempts to move ‘‘from bench to bedside’’. IVCM allows prompt diagnosis, disease course follow-up, and management of potentially blinding atypical forms of infectious processes, such as acanthamoeba and fungal keratitis. This technology has improved our knowledge of corneal alterations and some of the processes that affect the visual outcome after lamellar keratoplasty and excimer keratorefractive surgery. In dry eye disease, IVCM has provided new information on the whole-ocular surface morphofunctional unit. It has also improved understanding of pathophysiologic mechanisms and helped in the assessment of prognosis and treatment. IVCM is particularly useful in the study of corneal nerves, enabling description of the morphology, density, and disease- or surgically induced alterations of nerves, particularly the subbasal nerve plexus. In glaucoma, IVCM constitutes an important aid to evaluate filtering blebs, to better understand the conjunctival wound healing process, and to assess corneal changes induced by topical antiglaucoma medications and their preservatives. IVCM has significantly enhanced our understanding of the ocular response to contact lens wear. It has provided new perspectives at a cellular level on a wide range of contact lens complications, revealing findings that were not previously possible to image in the living human eye. The final section of this mini-review provides a focus on advances in confocal microscopy imaging. These include 2D wide-field mapping, 3D reconstruction of the cornea and automated image analysis.

/pdf/in-vivo-confocal-microscopy-of-the-ocular-surface-from-bench-2xil98jhog.pdf

In vivo confocal microscopy of the ocular surface: from bench to bedside.

PURPOSE To overcome the anterior corneal mosaic (ACM) phenomenon in in vivo confocal laser scanning microscopy (CLSM) and to reconstruct undistorted images of the subbasal nerve plexus (SNP), facilitating morphometric analysis in the presence of ACM ridges METHODS CLSM was performed in five healthy volunteers An original image processing algorithm based on phase correlation was used to analyze and reduce motion distortions in volume scan image sequences Three-dimensional tracing of the SNP was performed to reconstruct images containing only the SNP layer, with nerve fibers clearly visible even in ACM areas RESULTS Real-time mapping of the SNP revealed the presence of ridges with K-structures underneath them in all cases The occurrence of K-structures correlated directly with development of ACM observed by slit lamp and resulted in massive deformation at the level of Bowman’s membrane, seriously interfering with examination of SNP structures The average elevation of ACM ridges was 206 m (range, 87‐340 m) The novel method presented permitted reconstruction of the SNP layer in regions of ACM CONCLUSIONS The described method allows the precise analysis and elimination of motion artifacts in CLSM volume scans, in conjunction with the capability to reconstruct SNP structures even in the presence of severe ACM The robustness and automation of the described algorithms require ongoing development, but this will provide a sound basis for extended studies of corneal nerve regeneration or degeneration and for use in clinical practice (Invest Ophthalmol Vis Sci 2011;52: 5022‐5028) DOI:101167/iovs10-6065

Image reconstruction of the subbasal nerve plexus with in vivo confocal microscopy.

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.

Mosaicking the subbasal nerve plexus by guided eye movements.

Die konfokale der Laser-Scanning-Mikroskopie ermoglicht die Erzeugung hochaufgeloster Aufnahmen von Nerven- und Zellstrukturen der Kornea in vivo. Ein Hindernis bei der Generierung grosflachiger Abbildungen durch etablierte Mosaikbildmethoden sind Verzerrungen in den aufgenommenen Bildern, die durch unvermeidbare Augenbewegungen verursacht werden. Diese Arbeit prasentiert ein Verfahren zur elastischen Registrierung solcher Bildserien, welches durch die Berucksichtigung der spezifischen Eigenschaften des Aufnahmesystems in der Lage ist, die Bewegungsartefakte zu erkennen und zu korrigieren.

http://ceur-ws.org/Vol-715/bvm2011_32.pdf

Elastische Registrierung von in-vivo-CLSM-Aufnahmen der Kornea

Hintergrund: Mithilfe der konfokalen Laser-Scanning-Mikroskopie (CLSM) konnen die Nervenstrukturen der Kornea in vivo analysiert werden. Pathologische Veranderungen des peripheren Nervensystems, die sich im kornealen subbasalen Nervenplexus (SNP) widerspiegeln, lassen sich auf diesem Weg nicht invasiv und moglicherweise fruher als mit anderen Methoden diagnostizieren. Das Bildfeld bei In-vivo-CLSM-Aufnahmen der Kornea (ca. 0,4 × 0,4 mm²) ist jedoch nicht ausreichend fur eine zuverlassige Analyse des SNP. Ferner wird die Beurteilung der Bilddaten erschwert durch Gewebedeformationen im Bereich des SNP sowie durch Bildverzerrungen, die durch unvermeidbare Augenbewegungen wahrend der Aufnahme verursacht werden. Die Arbeit prasentiert ein neues Bildverarbeitungsverfahren zur Erzeugung einer verzerrungsfreien Abbildung des SNP mit erweitertem Bildfeld. Methoden: Zur Erprobung des Verfahrens wurden mit einem Heidelberg Retina Tomograph mit Rostock Cornea Module (HRT) bei funf freiwilligen Probanden jeweils acht Fokusserien aufgenommen und ausgewertet. Ein speziell an das Aufnahmesystem angepasstes Registrierverfahren korrigiert die nicht linearen bewegungsbedingten Verzerrungen und rekonstruiert ein Volumen aus jeder Fokusserie. Die basale Grenzflache des Epithels mit dem SNP stellt sich darin deutlich als stark reflektierende zusammenhangende Schicht dar. Durch die Extraktion dieser Schicht werden eine Tiefenkarte und eine zweidimensionale Abbildung des SNP erzeugt. Eine Fusion dieser SNP-Abbildungen liefert eine lateral vergroserte Darstellung. Ergebnisse: Von den insgesamt 40 Fokusserien wurden 34 zu jeweils einer zweidimensionalen Abbildung des SNP verarbeitet, aus sechs Fokusserien konnte kein Volumen rekonstruiert werden, weil extrem schnelle Augenbewegungen wahrend der Aufnahme die durchgangige Registrierung verhinderten. Die 34 erzeugten SNP-Rekonstruktionen weisen eine dargestellte Flache von 94,7 % ( ± 6,2 %) des Bildfelds einer Einzelaufnahme auf. Durch die Fusion der SNP-Abbildungen wurde eine durchschnittliche Vergroserung der dargestellten Flache um den Faktor 2,6 (Minimum: 2,2; Maximum: 3,1) erreicht. Schlussfolgerung: Die vorgestellten Bildverarbeitungsalgorithmen sind in der Lage, die bewegungsbedingten Verzerrungen in CLSM-Fokusserien zu korrigieren und auch bei ausgepragten Gewebedeformationen grosflachige zweidimensionale Abbildungen des SNP zu generieren, die eine zuverlassigere Beurteilung der kornealen Nervenfasern erlauben.

Bildrekonstruktion des subbasalen Nervenplexus der Kornea mit erweitertem Bildfeld aus Fokusserien eines konfokalen Laser-Scanning-Mikroskops

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.

S. Maier

Papers

Image reconstruction of the subbasal nerve plexus with in vivo confocal microscopy.

Mosaicking the subbasal nerve plexus by guided eye movements.

Elastische Registrierung von in-vivo-CLSM-Aufnahmen der Kornea

Bildrekonstruktion des subbasalen Nervenplexus der Kornea mit erweitertem Bildfeld aus Fokusserien eines konfokalen Laser-Scanning-Mikroskops

[Large-scale imaging of corneal nerve fibres by guided eye movements].