104 Figure 6 Second-order polynomial regression plot to calculate the relationship between cornea nerve fiber area (NFA) and cornea nerve fiber length (CNFL). The formula of Brines et al. was compared with the formula calculated with data from this study. A: CCMetrics and NFA FIJI, described by NFA = -4.66 x 10-4 (CNFL)2 + 4.1 (CNFL) - 1580; B: ACCMetrics and NFA FIJI, described by NFA = 3.94 x 10-4 (CNFL)2 + 5.57 x 10-1 (CNFL) + 2041; C: NeuronJ and NFA FIJI, described by NFA = -1.33 x 10-4 (CNFL)2 + 2.68(CNFL) - 1123 Discussion In the present study we did not demonstrate decreased CNFL and NFA in patients with sarcoidosis with and without established SFN compared to healthy controls, see Figure 4. Multiple analytical methods to determine corneal nerve fiber density show a strong correlation. Automatic analysis showed similar results with manual or semi-automatic methods (see Table 3 and Figure 5). NFA calculated with NFA FIJI was comparable to ACCMetrics, but showed a lower 95%-confidence interval. Moreover, a non-linear relationship between NFA and CNFL could be confirmed, see Figure 6. When interpreting CCM results, the consequences of ocular sarcoidosis affecting the cornea should be taken into account. To rule out bias from sarcoidosis or other causes of decreased CNFL rather than pure SFN, eye condition was examined in all participants. Therefore, participants with glaucoma, corneal dystrophy, corneal infection, previous corneal surgery, and contact lens use were excluded from this study. Unexpectedly, CNFL and CNFA were not reduced in patients with sarcoidosis and established SFN compared with patients with sarcoidosis without SFN or healthy controls. Figure 1 shows the course of small nerve fibers during nerve damage. In severe SFN, regeneration of small nerve fibers occurs as a result of sprouting. Regenerated nerves exhibit higher tortuosity. It could be possible that tortuous nerves show higher CNFL in the same area of 400 µm2 compared to less tortuous nerves, which could compensate the decrease of CNFL. Furthermore, sprouting could lead to hyperreinnervation, resulting in higher CNFL.34 It is unclear which phases of nerve damage are present in persistent SFN. There are no studies available for patients with sarcoidosis with SFN that clearly describe the course of persistent SFN. Therefore, it is currently unclear whether persistent SFN is progressive or whether different phases of nerve damage and sprouting present simultaneously. ACCMetrics demonstrated higher CNFL and CNFA in sarcoidosis patients with symptoms and clinical signs of SFN and TTT NOA<2 compared to patients with established SFN. As shown in Figure 1, increased CNFA can be explained by nerve swelling and increased CNFL can be explained by increased nerve tortuosity. However, these phenomena occur in different phases and it therefore seems unlikely that they are the reason for these results, which are present simultaneously. No clear explanation could be found why patients with probable SFN showed increased CNFL and CNFA compared to patients with established SFN. To our knowledge, few studies have compared CNFL results, but none compared NFA FIJI results with NFA calculated with ACCMetrics or results of NFA FIJI with CNFL values from manual methods. Our study confirmed a good correlation between manual analysis (CCMetrics18), semi-automatic analysis 6 109 6
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