Purpose
To determine the extent of retinal and corneal neurodegeneration and investigate the association with intraepidermal neuronal loss and diabetic peripheral neuropathy (DPN) in type 2 diabetes.
Design
Prospective, cross-sectional study.
Methods
Single-center study of 94 patients with type 2 diabetes patients (157 eyes), divided into groups: the groups without diabetic retinopathy (DR) (n = 68); the nonproliferative DR (NPDR) group (n = 48); and the proliferative DR (PDR) group (n = 41). Patients were imaged with optical coherence tomography and confocal microscopy for macular and peripapillary neuroretinal layer thicknesses and corneal nerve length/density, respectively. Distal leg skin punch biopsies and 2 neurological scores were used to depict intraepidermal nerve fiber density (IENFD) and clinical DPN.
Results
Among neuroretinal layers, solely the peripapillary retinal nerve fiber layer was decreased in PDR (96 μm; 95% confidence interval [CI], 92-100 μm) versus no DR (103 μm; 95% CI, 100-106 μm) eyes and only after exclusion of outliers ( P = .01). Corneal nerve fiber length and density were statistically significantly reduced in the NPDR group (23.0 mm/mm 2 ; 95% CI, 20.0-26.00 mm/mm 2 and 14.3 mm; 95% CI, 14.5-16.63 mm, respectively) and the PDR group (18.6 mm/mm 2 ; 95% CI, 14.9-22.30 mm/mm 2 and 11.7 mm; 95% CI, 10.2-13-3 mm, respectively) versus the no DR group (25.5 mm/mm 2 ; 95% CI, 23.3-27.70 mm/mm 2 and 15.6 mm; 95% CI, 14.5-16.6 mm, respectively), and in the PDR versus the NPDR group. IENFD was statistically significantly reduced in the NPDR (2.0/mm; 95% CI, 1.4-2.7/mm) and PDR stage (1.4/mm; 95% CI, 0.9-2.1/mm) versus in eyes without DR (3.6/mm; 95% CI, 2.9-4.6/mm). A low correlation between intraepidermal and corneal fiber loss was found with both neurological scores ( P < .05).
Conclusions
Retinal neurodegenerative changes may develop independently of the microvascular alterations defining DR. Corneal and intraepidermal neuronal loss is more pronounced in advanced stages of DR, indicating a positive severity correlation between DR and DPN.
The prevalence of type 2 diabetes is globally increasing, with patients being at substantially increased risk of developing diabetic retinopathy (DR), its major microvascular complication. Neurodegeneration is an early event in the development of diabetic retinal disease and may be present even in the absence of visible microvasculopathy. The alterations in retinal neural tissue lead to well-defined neurofunctional changes, including a dysfunction in dark adaption ; changes in contrast sensitivity ; altered microperimetric and perimetric test results , ; deficits in the pattern electroretinogram (pattern ERG) ; and increased implicit times in multifocal electroretinography. However, most of these functional tests prove time consuming and cumbersome in clinical routine and for study settings. The structural component of retinal neurodegeneration can precisely be quantified with spectral-domain optical coherence tomography (OCT). Retinal neurodegeneration consists of a selective loss of the inner retinal layers including the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), and inner plexiform layer (IPL), previously demonstrated in patients with type 2 diabetes at all stages of DR and even without visible signs of retinopathy. ,
Hyperglycemic changes also result in a decreased supply of oxygen and nutrients to small nerve fibers in the cornea. The rich network of the sub-basal nerve plexus (SNP) lies between the basal epithelium and Bowman’s membrane. It can be visualized in vivo with corneal confocal microscopy (CCM). This high-resolution method demonstrates a noninvasive, objective means to assess corneal nerve fiber density (CNFD), CNF length (CNFL), and CN branch density (CNBD) in patients with diabetes with high sensitivity and good repeatability. CNFD, CNFL, and CNBD were shown to be decreased in patients with diabetes, but there is no consensus on whether this change is progressive with increasing retinal disease severity. Furthermore, corneal neurodegeneration offers noninvasive visual access to incipient and progressive damage to small fibers, damage predictive of diabetic peripheral neuropathy (DPN). This further diabetic microvascular complication affects 5%-7% of patients with type 2 diabetes at the time of diagnosis and occurs in 50%-90% of patients at some point in the course of their illness. For decades, the gold standard for the diagnosis of DPN has been clinical testing combined with nerve conduction studies (NCS). However, NCS are not sensitive for detecting small-fiber neuropathy (SFN), most likely preceding large-fiber damage. Small fibers constitute 70%-90% of peripheral nerves, and SFN is directly related to the main outcomes of pain and foot ulceration. SFN testing has long been limited to subjective methods such as quantitative sensory testing of thermal thresholds or invasive skin biopsies to quantify intraepidermal nerve fiber density (IENFD). Both IENFD and CCM are sensitive, objective methods for the diagnosis of SFN, but CCM may detect early nerve fiber loss more frequently and with less variability. ,
Early prevention of DR and DPN, as well as individualized, interdisciplinary patient management, can be provided only by means of a sensitive test or relevant biomarker that correlates well with the development and progression of these complications. Signs of retinal and corneal neurodegeneration are key candidates for such prospected biomarkers. However, whether the extent of neuronal damage in the retina and cornea of patients differ, related to the presence and stage of DR, remains controversial. Moreover, the correlation between corneal and intraepidermal small-fiber damage and the potential benefit of corneal nerve fiber morphology over assessment of IENFD for the diagnosis of DPN needs to be clarified.
The purpose of this study was to define the severity of retinal and corneal neurodegeneration in patients with type 2 diabetes according to the presence and stage of DR. An additional aim was to determine the association between corneal nerve fiber morphology and intraepidermal nerve fiber density and their correlation to clinical signs of DPN.
Subjects and Methods
Study investigations were conducted in accordance with the tenets of the Declaration of Helsinki. The study was approved by the Institutional Review Board of the Medical University of Vienna (1341/2016). All patients gave written informed consent prior to inclusion in this prospective, cross-sectional study.
Adult patients with a history of type 2 diabetes for at least 6 months were included. Exclusion criteria consisted of any neuropathy due to a nondiabetic cause; nondiabetic systemic diseases affecting the cornea, corneal disorders, media opacity precluding good quality imaging; other ocular diseases (glaucoma); active intraocular inflammation; laser therapy or intraocular surgery in the previous 3 months; history of any macular laser therapy in the study eye; and presence of diabetic macular edema. The stage of DR was classified based on the Early Treatment Diabetic Retinopathy Study (ETDRS) classification, and each eye was assigned to 1 of 3 groups: no DR, nonproliferative DR (NPDR), and proliferative DR (PDR). Both eyes of a patient were included if eligible. After ETDRS best-corrected visual acuity testing, a standard anterior segment examination, intraocular pressure measurement, and dilated fundoscopy, patients underwent ophthalmic imaging and a skin biopsy as follows on the same day.
Spectral-domain OCT
Macular volume scans (25 B-scans) and a circular scan centered on the optic disc were acquired by using Spectralis OCT (Eye Explorer version 1.10.2.0; Heidelberg Engineering, Germany). Retinal layers were delineated by the inbuilt automated segmentation algorithm and subsequently corrected manually, if applicable, in each B-scan. The software calculates the mean thickness in a 6-mm ETDRS grid centered on the fovea for each layer separately. The individual mean retinal layer thickness was analyzed for each of the 9 subfields of the ETDRS grid to separately report 1-mm central subfield and full grid results. The RNFL, GCL, IPL, and photoreceptor layer and total retinal thickness were analyzed. Peripapillary RNFL (pRNFL) thickness represents the mean distance between the inner limiting membrane and the posterior boundary of the RNFL along the 12-degree radius circle scan centered on the optic disc.
Corneal Confocal Microscopy
CCM was performed using the Rostock cornea module attached to the Heidelberg HRT III retina tomograph (software version 1.9.10.0; Heidelberg Engineering). The study eye was anesthetized with a drop of 1% oxybuprocain (Pharmacy General Hospital Vienna, Austria). Correct alignment and contact of the microscope to the cornea was ensured by using a camera positioned perpendicular to the eye. The distance between the cornea and microscope was kept stable by using a single-use cap (TomoCap, Heidelberg Engineering) coupled optically to the lens with the aid of a viscous gel (Siccaforte, Agepha, Austria). The confocal scanning laser method allows a detailed en face visualization of each layer of the cornea separately within a 400- × 400-μm field of view.
To maintain quality, depth, focus position, and contrast, 3 images of the central SNP (low, medium, high CNFD) of each eye were chosen to avoid under- or overestimation of CNFD. The images were then analyzed by using a fully automated segmentation technique (ACCMetrics version 2; University of Manchester, Manchester, United Kingdom) ( Figure 1 , A through C). , Measurements were averaged to provide the final results for each eye for CNFD (number of major nerve fibers per square millimeter), CNBD (number of branch points on main fibers per square millimeter), and CNFL (total length of nerves in millimeters per square millimeters).
Skin Punch Biopsy
Three-millimeter skin punch biopsy specimens (disposable biopsy punch, Kai Medical, Solingen, Germany) were acquired with the patient under local anesthesia from the distal leg (10 cm proximal to the lateral malleolus) with subsequent adequate wound treatment processes. All specimens were processed as described. IENFD was quantified at the Clinical Institute of Neurology. Indirect immunofluorescence with rabbit polyclonal antiprotein gene product 9.5 (PGP9.5) antibodies in formol (4.5% formaldehyde in water) fixed sections of 50-μm thickness was used to visually count IENFs at high magnification in at least 3 sections per biopsy with confocal microscopy ( Figure 2 and 3 ). Only IENFs crossing the dermal-epidermal border were counted, excluding secondary branching and fragments from quantification. The length of the section was measured to calculate the exact linear epidermal innervation density (IENF per millimeter).
All patients were additionally examined by a neurologist who specialized in polyneuropathies within the first month after study inclusion. Lower limb inspection and a focused neurological examination were performed using 2 validated scores. The Utah Early Neuropathy Scale (UENS) assesses early signs of sensory predominantly small-fiber neuropathy, yielding a score of 0 to 42. Second, part B (consisting of a specific neurological examination) of the Michigan Neuropathy Screening Instrument (MNSI), specifically validated for the evaluation of distal symmetrical polyneuropathy (i.e., large-fiber neuropathy) was used.
Statistical Analysis
Continuous variables are described using median (quartiles) and categorical variables by counts (percentages). Each continuous outcome was compared between groups by using a mixed model to adjust for confounders age, sex, hypertension, hypercholesterolemia, duration of diabetes, and HbA 1c (for retinal outcome additional adjustment for retinal thickness) and to account for patients included with both eyes using a random patient factor. Least squares mean values are reported with 95% confidence intervals (CI). Outcomes for GCL, IPL, IENFD, and CNBD were log-transformed to stabilize residual distributions; their least square means were back-transformed to the original scale. Pairwise comparisons between groups were corrected for multiple testing using the Tukey method. Five retinal outcomes were investigated in the total ETDRS grid and the central subfield; corresponding P values for the pairwise comparisons and the overall (2 df ) test were further adjusted by using the Bonferroni-Holm method. Because various outcomes showed residual outliers and potentially influential observations, the same models were re-estimated excluding these observations. Relevant deviations from the original analysis are reported.
Spearman correlation coefficients quantify the association of variables. Their P values are calculated using 10,000 bootstrap samples to account for patients with both eyes. SAS version 9.4 software (SAS Institute Inc., Cary, North Carolina) was used. Two-sided (corrected) P values ≤5% indicated statistical significance.
Results
A total of 157 eyes were included (13 left eyes) of 94 patients with both eyes of 63 patients (67%) and 1 eye of 31 patients (33%). Sixty-eight eyes (43%) showed no DR, 48 (31%) showed NPDR (mild = 22; moderate = 20; severe = 6), and 41 showed (26%) proliferative DR. Table 1 summarizes baseline characteristics. Among PDR eyes, 39 (95%) had previously undergone panretinal photocoagulation treatment. The mean duration since laser therapy was 28 months (range = 62 months). Retinal neovascularization in the remaining 2 eyes (5%) with PDR had been treated with a total of 5 monthly intravitreal injections of antivascular endothelial growth factor prior to study inclusion. At the time of study inclusion, all PDR eyes were considered “quiescent.”
| Females | 28 (30) |
| Age, y | 61 (53-69) |
| DM duration, y | 15 (8-23) |
| Hemoglobin A 1c , % | 7.4 (6.7-8.1) |
| OAD + insulin a | 50 (53) |
| OAD a | 44 (47) |
| Hypertension b | 71 (76) |
| Hypercholesterolemia b | 51 (54) |
| ETDRS BCVA (letters), Snellen equivalent | 87 (80-91), 20/20-20/15 |
| Lens status | |
| phakic | 65 (69) |
| pseudophakic OD/OS | 2 (2)/2 (2) |
| pseudophakic OU | 25 (27) |
a Oral anti-diabetic (OAD) medication intensified with subcutaneous insulin therapy.
b Based on records provided by the general practitioner or internist.
Retinal Layer Thicknesses and Corneal Nerve Morphology
Table 2 shows retinal layer thickness results for the 6-mm and 1-mm ETDRS areas as well as pRNFL thickness and corneal SNP results for the 3 groups. Table 3 shows the results of the model that compares retinal and corneal neurodegenerative features between the groups.
| No DR | NPDR | PDR | |
|---|---|---|---|
| RT total, μm | 308 (303-319) | 321 (312-328) | 324 (303-345) |
| RNFL total, μm | 26 (25-29) | 27 (25-30) | 33 (29-37) |
| GCL total, μm | 40 (38-43) | 41 (38-43) | 39 (35-44) |
| IPL total, μm | 34 (32-36) | 34 (33-36) | 34 (31-37) |
| PR total, μm | 81 (80-83) | 81 (79-83) | 78 (76-86) |
| RT c, μm | 271 (260-285) | 288 (271-309) | 288 (261-318) |
| RNFL c, μm | 12 (11-14) | 14 (14-15) | 14 (13-17) |
| GCL c, μm | 14 (11-17) | 16 (13-21) | 16 (13-22) |
| IPL c, μm | 20 (18-23) | 22 (19-24) | 22 (19-25) |
| PR c, μm | 88 (83-90) | 86 (84-89) | 82 (80-85) |
| pRNFL, μm | 103 (98-108) | 98 (89-103) | 92 (83-108) |
| CNFL, mm/mm 2 | 15.5 (12.9-17.3) | 13.5 (10.2-22.6) | 10.7 (8.8-13.2) |
| CNFD, n/mm 2 | 25.0 (19.8-29.7) | 19.8 (15.1-25.0) | 14.6 (12.5-18.6) |
| CNBD, n/mm 2 | 37.5 (24.0-56.3) | 28.1 (12.5-34.9) | 18.8 (9.4-31.3) |
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