Research Design and Methods
The study was conducted in accordance with the Declaration of Helsinki and was approved by the ethics committee of Heinrich Heine University, Düsseldorf, Germany. All participants provided a written informed consent. Included were 86 patients with recently diagnosed T2D and 48 age- and sex-matched control subjects. Patients with diabetes were participants of the German Diabetes Study (GDS), which evaluates the long-term course of diabetes and its sequelae (ClinicalTrials.gov Identifier: NCT01055093).[18] Inclusion criteria for entry into the GDS are type 1 diabetes or T2D, known diabetes duration of ≤1 year, and age of 18–69 years at the baseline assessment. Exclusion criteria for the current study were secondary diabetes, pregnancy, severe diseases (cancer), psychiatric disorders, immunosuppressive therapy, limited cooperation ability, corneal disorders, and neuropathy from causes other than diabetes. Inclusion criteria for the control group were age of ≥18 years and a normal result on an oral glucose tolerance test,[22] whereas exclusion criteria were neuropathy from any cause and those applied to the diabetic group. Among T2D subjects from the GDS who were asked to participate in the current study, ~50% agreed. Control subjects included staff from this institution and those recruited by newspaper advertisement.
CCM Examination
CCM was performed using a Heidelberg Retina Tomograph II (HRT II) with the Rostock Cornea Module (RCM; Heidelberg Engineering, Heidelberg, Germany), as previously described.[15,16] The acquired images have a resolution of 384 × 384 pixels and the field of view is ~0.15 mm2. Experienced ophthalmologists (A.Z. and S.P.) performed the examinations and were blinded to all study data, except for CCM and corneal sensation. The HRT II/RCM is equipped with a water contact objective (636/0.95 W, 670 nm; Zeiss, Jena, Germany). The distance from the cornea to the microscope was kept constant by a single-use contact element in sterile packaging (TomoCap). Coupling between the patient's cornea and the cap was facilitated with a thin lubricant layer of Vidisic gel (refractive index, 1.35; Bausch & Lomb/Dr. Mann Pharma, Berlin, Germany). The right eye was anesthetized by instilling Proparakain POS 0.5% eye drops (Ursapharm, Saarbrücken, Germany).
A modified, oscillating volume scan operating mode of the HRT II, in which the focus plane of the microscope is continually shifted back and forth, was used to acquire a number of image stacks (with an axial image distance of 0.5 μm) for each patient, with each stack representing a partial volume of the patient's cornea; particular care was taken that each image stack comprised the subbasal nerves over the entire height of present ACM ridges. A stack size of 96 images (scan depth, 48 μm) was chosen for ridge heights of less than 48 μm, and 120 images (60 μm) otherwise. At least three scans were performed for each patient, and the total duration of the microscopy was ~15 min.
All acquired image stacks were subsequently processed to correct motion artifacts, reconstruct the imaged volume, and compute a depth map of the SNP. An SNP image was composed for each image stack, and all reconstructed SNP images with common overlapping areas were combined into a mosaic image with an expanded field of view. Nerve structures and similar image features in the mosaic images were subsequently segmented. Wrongly segmented structures (reconstruction artifacts, dendritic cells, fibrotic tissue, etc.) were removed based on their morphological properties (size, elongation). In addition to the resulting image of the segmented nerve fibers, the network of fiber centerlines was finally calculated by thinning all segmented nerve fibers to a width of 1 pixel. A more detailed description is available in the Supplementary Data.
The segmentation image and the thinned fiber network image both formed the basis for the automated quantitative morphological and topological assessment of the SNP.[16,23] The following CCM parameters were determined: corneal nerve fiber (CNF) length (CNFL), defined as the total length of all nerve fibers (mm/mm2); CNF density (CNFD), defined as the number of nerve fibers per mm2; corneal nerve branch density (CNBD), defined as the number of branches per mm2; average weighted CNF thickness (CNFTh), measured as mean thickness perpendicular to the nerve fiber course (μm); corneal nerve connecting points (CNCP), defined as the number of nerve fibers crossing area boundary (connections/mm); and average weighted CNF tortuosity (CNFTo), reflecting variability of nerve fiber directions and defined as total absolute nerve fiber curvature. Each fiber segment terminated by branching points, end points, and/or image borders was considered a distinct nerve fiber for the calculation of the above parameters. Weighting of single fibers was based on their contribution to the length of the total fiber network.[15,16]
Morphometrical image analysis was performed for two different populations of segmented nerve fibers. Firstly, all CCM parameters were calculated for the whole fiber network. Thereafter, the determined lengths of all single nerve fiber segments were analyzed to identify an optimal length threshold at 93 μm that provided the most pronounced difference in CNFD between the diabetic and control groups. Finally, segmented nerve fibers shorter than this length threshold were removed, and a second set of CCM parameters was calculated for the remaining major nerve fibers (MNF) with length ≥93 μm only.
Corneal Sensation
Corneal esthesiometry was done using the Cochet-Bonnet esthesiometer (Luneau Ophthalmologie, Chartres, France). The nylon monofilament had a diameter of 0.12 mm and a fully extended length of 60 mm. The central, superior, inferior, nasal, and temporal cornea was touched once on each eye, beginning at a filament length of 60 mm. If a positive answer was not detected, the filament length was shortened in 5-mm steps each time and the procedure repeated until there was a positive response. Corneal sensation was calculated as the mean obtained from the five corneal areas on each eye.
Peripheral Nerve Function
Peripheral nerve function tests were performed as previously described.[24] Motor nerve conduction velocity (NCV) was measured in the median, ulnar, and peroneal nerves, whereas sensory NCV and sensory nerve action potentials (SNAP) were determined in the median, ulnar, and sural nerves at a skin temperature of 33–34°C using surface electrodes (Nicolet VikingQuest; Natus Medical, San Carlos, CA). Quantitative sensory testing included measurement of the vibration perception threshold (VPT) at the second metacarpal bone and medial malleolus using the method of limits (Vibrameter; Somedic, Stockholm, Sweden) and thermal detection thresholds (TDT), including warm and cold thresholds at the thenar eminence and dorsum of the foot using the method of limits (TSA-II NeuroSensory Analyzer; Medoc, Ramat Yishai, Israel). Neurological examination was performed using the Neuropathy Disability Score (NDS) and Neuropathy Symptoms Score (NSS). Clinical DSPN was defined as NDS ≥6 and NSS ≥0, or NDS ≥3 and NSS ≥5 points.[25] These and all other clinical examinations were performed by operators who were blinded to the corneal findings in all subjects.
IENFD
Three-millimeter skin punch biopsy specimens were taken under local anesthesia from the left lateral calf, ~10 cm proximal to the lateral malleolus. The tissue was fixed with 2% periodate-lysine-paraformaldehyde at 4°C for 24 h, rinsed twice for 10 min with 0.1 mol/L Sorensen buffer, and incubated in 33% sucrose for 3 h. After cryoprotection with 0.02 mol/L Sorensen buffer containing 20% glycerol at 4°C overnight, tissue was stored at −80°C.
Serial sections of skin specimens at 50-μm thickness were cut perpendicular to the skin surface at −20°C and −40°C, respectively. Staining of IENF was performed following the free-floating method as described before,[26] with some modifications. In brief, sections were incubated after blocking with a rabbit anti-PGP9.5 antibody (1:1,200; Millipore, Temecula, CA) and a biotinylated anti-rabbit IgG antibody (1:100; Vector Laboratories, Burlingame, CA) for 1 h, followed by 1-h incubation with the Vector ABC kit and 3 min with the Vector SG substrate kit. All steps were performed at room temperature.
For the quantification of IENFD, a method adopted by the European Federation of Neurological Sciences[27] was used.[28] Individual IENFs from four cross-sections per subject were visually counted along the length of the epidermis using a Leica DMRBE inverted microscope (Leica, Wetzlar, Germany) equipped with an Olympus DP73 digital color camera (Olympus, Hamburg, Germany). Only IENFs crossing the dermal-epidermal border were counted. The length of the epidermis was measured using cellSens 1.7 imaging software (Olympus Europa, Hamburg, Germany). IENFD was expressed as IENF/mm epidermis.
Cardiovascular Autonomic Function Tests
Cardiovascular autonomic nerve function was evaluated by measuring heart rate variability (HRV) during spontaneous breathing over 5 min (coefficient of R-R interval variation, spectral analysis), at deep breathing (expiration-to-inspiration ratio), after standing up (maximum-to-minimum 30:15 ratio), and in response to a Valsalva maneuver (Valsalva ratio) using VariaCardio TF5 (MIE Medical Research Ltd., Leeds, U.K.), as previously described.[29]
Microvascular Complications and Physical Activity
Retinopathy was assessed by fundus photography using the panoramic ophthalmoscope P200C (Optos, Bruchsal, Germany) and diagnosed by an experienced ophthalmologist. Albuminuria was measured in 24-h urine samples using a Cobas c 311 analyzer (Roche Diagnostics, Mannheim, Germany) and defined as normal (<20 μg/min), microalbuminuria (20–199 μg/min), and macroalbuminuria (≥200 μg/min). The level of physical activity was defined as low (<1 h/week over <1 month/year), moderate (1–2 h/week over 1–9 months/year), and high (>2 h/week over 9–12 months/year).
Statistical Analysis
Continuous data are expressed as mean ± SD. Categorical data were analyzed by Fisher exact test and are given as absolute or relative frequencies with 95% CI. For normally distributed data, parametric tests (t test or Pearson product-moment correlation), otherwise nonparametric tests (Mann-Whitney U test or Spearman rank correlation) were applied. To determine associations between two variables, univariate correlations and multiple linear regression analyses were performed. The level of significance was set at α = 0.05.
Diabetes. 2014;63(7):2454-2463. © 2014 American Diabetes Association, Inc.
