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

Dan Ziegler; Nikolaos Papanas; Andrey Zhivov; Stephan Allgeier; Karsten Winter; Iris Ziegler; Jutta Brüggemann; Alexander Strom; Sabine Peschel; Bernd Köhler; Oliver Stachs; Rudolf F. Guthoff; Michael Roden


Diabetes. 2014;63(7):2454-2463. 

In This Article


In this study we demonstrate an early loss of small nerve fibers detected by both CCM and skin biopsy in recently diagnosed T2D, with CNFD-MNF being most sensitive among six measures of CCM. However, these two techniques detect nerve pathologies largely in different groups of patients, suggesting a patchy manifestation pattern of small fiber neuropathy in various organs, possibly due to distinct underlying pathophysiological processes. Because nerve function assessed by nerve conduction studies (NCS), QST, and AFTs was also impaired, we suggest that a parallel involvement of small and large nerve fibers occurs in the early development of DSPN, but prospective studies are required to define the precise temporal sequence.

To the best of our knowledge, this is the first study reporting changes in the corneal SNP using CCM and IENFD using skin biopsy in recently diagnosed T2D. In the current study, the vast majority of the patients with abnormal CNFD showed concomitantly normal IENFD and vice versa. Consequently, small fiber pathology does not appear to develop simultaneously in different organs. This involvement of some but not other anatomical sites may be described as "patchy." However, it remains unclear why some patients develop a corneal neuropathy first, whereas in others, small fiber loss is first observed in the lower limbs. This finding should be further addressed in prospective studies.

Among the six CCM parameters assessed, CNFD-MNF emerged as the most sensitive in detecting corneal nerve pathology in 21% of the patients below the 2.5th percentile of the control group, followed by CNFL and CNBD-MNF with 17% each. This finding somewhat contrasts with a recent study reporting that among four CCM parameters, CNFL best discriminated DSPN case patients from control subjects.[11] It also contrasts with a previous report that demonstrated a significant reduction in CNFD and CNBD but not CNFL in several groups of diabetic patients with mild to severe DSPN with a mean diabetes duration between 18.4 and 25.2 years and no reduction in CNFL and CNFD in patients without clinical DSPN who had diabetes for 16.7 years on average.[12] Furthermore, we found no correlation between CCM measures and AFTs, in contrast to a recent study showing that CNFL, CNFD, and CNBD correlated with HRV during deep breathing in patients with type 1 diabetes and an average diabetes duration of 22.5 years.[30] The reasons for these discrepancies could be due to the differences in study populations, CCM equipment, the size of the corneal area, and the image-processing software algorithms used.

The CCM examination process used in this study is fundamentally based on highly adapted software, comprising a modified version of the microscope control software (oscillating volume scan operating mode) and the entirety of the custom-made image processing algorithms used thereafter, including motion correction, volume reconstruction, SNP layer extraction, mosaicking, segmentation of the subbasal nerve fibers, and quantitative morphometric assessment. This approach enabled us to robustly generate and analyze images of the SNP layer with an extended field of view and devoid of ACM-induced artifacts.[15–17] The entire process also features a higher degree of automation than would have been achievable by using standard image-processing software.

It currently remains an open question whether our approach actually yields a better diagnostic value than recording and examining a small set of CCM images,[4,7,10,12,13] the technique most commonly used by other CCM research groups, and if it does, whether the differences are significant enough to justify the higher effort in examination time. Such comparative studies are presently being conducted.

We used a two-step CCM image analysis, the first for the entire fiber network and the second for MNF only. This approach provides a more detailed insight into the morphological and topological structure of the segmented nerve fibers. Our data indicate that the use of a threshold based on fiber length could be useful to improve the discriminatory power of CNFD and CNBD. Furthermore, this strategy allows a comparison of data generated for research purposes with those used for diagnostic reasons. Comprehensive image processing–oriented approaches aim at the best possible and preferably complete detection of nerve fibers. This includes total elimination of all image artifacts and segmentation even of the faintest fibers. On the other hand, for clinical purposes, identification of MNF to obtain CNFD and CNBD may be sufficient.

Previous studies have shown correlations of CCM measures with VPT,[31] clinical severity of DSPN,[4,10,12,32,33] NCS,[12,32] HRV,[12,30] cold TDT,[12,30] and IENFD[12] in longer-standing diabetes. In the current study, several CCM measures correlated with IENFD and with median and ulnar motor and sensory NCV, as well as sural SNAP, in the entire study population, but the relationship was modest to moderate. This is in line with the aforementioned studies[12,32,34] supporting the notion that the pathophysiology underlying the manifestation of neuropathy in the cornea may be different from DSPN affecting the lower limbs. Indeed, vascular factors including reduced endoneurial blood flow and microvascular alterations appear to contribute to the pathogenesis of DSPN,[35,36] whereas the normal cornea, albeit being the most densely innervated tissue in the human body (several 100-fold higher than skin),[37] is devoid of blood vessels. However, many corneal abnormalities may disrupt the avascular microenvironment and lead to corneal angiogenesis. Recent experimental evidence suggests that sensory nerves and neovessels inhibit each other in the cornea. When vessel growth is stimulated, nerves disappear, and conversely, denervation induces angiogenesis.[38] Hence, whereas angiogenesis may be a consequence of corneal denervation, neuropathy in the lower limbs may be a consequence of reduced angiogenesis and may be treated by promoting angiogenesis.[39] Thus, on one hand, in view of this distinct pathophysiological background, the lack of close correlations between measures of CCM and peripheral nerve tests may not be surprising. On the other hand, advanced glycation end-product immunoreactivity was observed in epithelial cells, epithelial basement membrane, and stromal keratocytes in corneas from diabetic monkeys,[40] suggesting that one possible mechanism for the development of corneal neuropathy could be nonenzymatic glycation, similar to its putative role in the pathogenesis of DSPN.[36]

The relatively high prevalence of micro/macroalbuminuria (18.6%) compared with retinopathy (2.3%) may be explained by the high percentage of treated hypertension (48%). These findings are compatible with those reported by the Hoorn study in newly diagnosed T2D subjects.[41]

The strengths of this work are the inclusion of a relatively large and homogenous study population with recently diagnosed T2D, the use of novel image-processing algorithms to reconstruct SNP images with an extended field of view from three-dimensional image stacks, and the detailed quantitative assessment of neuropathy, including skin biopsy, to detect small fiber neuropathy.

A limitation is the cross-sectional nature of this study so that the predictive value and further course of the described corneal and intraepidermal nerve damage cannot be determined at present. The precise temporal sequence of alterations to small versus large nerve fibers can only be established in prospective studies. Moreover, our approach to compare the value of counting all CNFs with counting MNF only to detect abnormality may be biased toward a better outcome for MNF and is only valid in the present population. Finally, selection bias cannot be excluded, because the patients and control subjects included in this study may not be representative of the general population.

The clinical implications of the current study may be outlined as follows. In recently diagnosed T2D, CCM may be used to detect early evidence of neuropathy in the cornea. The CCM examination could be useful for timely diagnosis, because IENFD may be normal in some patients with CCM abnormalities. However, before CCM can be recommended for use in clinical practice, further validation determining its value in predicting DSPN and its susceptibility to interventions is required.

In conclusion, we have demonstrated corneal nerve pathology in recently diagnosed T2D. In this setting, CCM detects early nerve fiber loss slightly more frequently than a skin biopsy, but not necessarily in the same patients, suggesting a patchy manifestation pattern of small fiber neuropathy. In some patients, CNF loss may even be the first evidence of subclinical DSPN. These results indicate that CCM could be established as a powerful noninvasive tool for an early detection of neuropathy, but prospective studies are required to confirm this notion.