IOVS Papers in Press. Published on March 17, 2015 as Manuscript iovs.14-15919

The Inferior Whorl for Diabetic Neuropathy The Inferior Whorl For Detecting Diabetic Peripheral Neuropathy Using Corneal Confocal Microscopy.

N Petropoulos 1, 2, †Maryam Ferdousi 2, Andrew Marshall 3, Uazman Alam 2, Georgios Ponirakis 1, 2, Shazli Azmi 2, Hassan Fadavi 2, Nathan Efron 4 , Mitra Tavakoli 2, Rayaz A Malik 1, 2 †Ioannis

† INP and MF contributed equally to this study as first authors.

1

Division of Medicine, Weill Cornell Medical College-Qatar, Qatar Foundation, Education City, Doha, Qatar, 2 Centre for Endocrinology and Diabetes, University of Manchester and Central Manchester NHS Foundation Trust, and 3 Clinical Neurophysiology, Central Manchester NHS Foundation Trust, Manchester Academic Health Science Centre, UK and 4 Queensland University of Technology, School of Optometry and Vision Science, Brisbane, Australia

Word Count: 2,939 Funding source: This research was funded by awards from the National Institutes of Health (R105991) and Juvenile Diabetes Research Foundation International (272008-362). No conflict of interest related to this work for any of the authors.

Correspondence and reprint requests to: Professor Rayaz A. Malik Professor of Medicine Weill Cornell Medical College in Qatar Education City, Qatar Foundation, Doha, Qatar, PO Box 24144 & University of Manchester School of Medicine Centre for Endocrinology and Diabetes T: 00974 4447 8068 | 0044 161 275 1196 E: [email protected]; [email protected] 1 Copyright 2015 by The Association for Research in Vision and Ophthalmology, Inc.

The Inferior Whorl for Diabetic Neuropathy List of Abbreviations AUC: area under the curve CCM: corneal confocal microscopy CIP: cold induced pain CNFD: corneal nerve fiber density CNFL: corneal nerve fiber length CT: cold threshold DPN: diabetic peripheral neuropathy HIP: heat induced pain IWL: inferior whorl length NSP: Neuropathy symptom profile NDS: Neuropathy disability score PMNA: peroneal motor nerve amplitude PMNCV: peroneal motor nerve conduction velocity ROC: receiver operator characteristic SSNA: sural sensory nerve amplitude SSNCV: sural sensory nerve conduction velocity TC: total cholesterol VPT: vibration perception threshold WT: warm threshold

2

The Inferior Whorl for Diabetic Neuropathy Abstract Purpose: in vivo corneal confocal microscopy (CCM) is increasingly used as a surrogate endpoint in studies of diabetic polyneuropathy (DPN). However, it is not clear whether imaging the central cornea provides optimal diagnostic utility for DPN. We therefore compared nerve morphology in the central cornea and inferior whorl, a more distal and densely innervated area located inferior and nasal to the central cornea. Methods: 53 subjects with type 1 / type 2 diabetes and 15 age-matched control subjects underwent detailed assessment of neuropathic symptoms (NPS), deficits [neuropathy disability score (NDS)], quantitative sensory testing [vibration perception threshold (VPT), cold and warm threshold (CT/WT) and cold and heat induced pain (CIP/HIP)] and electrophysiology [sural and peroneal nerve conduction velocity (SNCV/PNCV) and sural and peroneal nerve amplitude (SNA/PNA) to diagnose patients with (DPN+) and without (DPN-) neuropathy. Corneal nerve fibre density (CNFD) and length (CNFL) in the central cornea, and inferior whorl length (IWL) were quantified. Results: Comparing control subjects to DPN- and DPN+ patients, there was a significant increase in: NDS (0 v 2.6±2.3 v 3.3±2.7, P 0.05) when the same observer assessed and re-assessed images of the IW center selected from the same dataset on two separate occasions. The measurements by the same observer on two subsequent days were also strongly correlated (Spearman’s r = 0.99, P < 0.0001) (figure 4C).

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The Inferior Whorl for Diabetic Neuropathy Discussion Over the past decade in vivo CCM has emerged as a powerful endpoint to quantify and stage the severity of human DPN, due to its rapid non-invasive ability to acquire and quantify corneal nerve damage. Several groups have shown significant decreases in corneal sub-basal innervation in patients with minimal and more advanced neuropathy models of diabetes

5, 20, 22, 24-27

and in experimental

28-30

. The majority of studies in humans have visualized

the central and peri-central cornea to quantify sub-basal nerve density and length. A limitation of CCM is the relatively small field of view, which allows only a proportion of the total subbasal nerve plexus to be scanned at any given time. To overcome this studies have proposed alternative image acquisition methods

6

including fully-automated montaging of CCM images

31

.

However, the clinical utility of these techniques remains to be established given the much longer image acquisition times and a lack of validation in larger cohorts of patients. Edwards and colleagues

6

have described a novel, wide-field assessment

technique which allows real time mapping of the sub-basal nerves and visualization of the entire nerve plexus. Their study revealed patchy nerve loss in a patient with severe neuropathy, mostly affecting the central and inferior areas including the inferior whorl, compared to a patient without neuropathy and minimal corneal nerve alterations. This patchy pattern of corneal nerve loss with focal reduction in the IW region has also been described in experimental DPN, and associated with epithelial thinning and reduced basal cell density

16

. Furthermore, Davidson et al.

32

showed that

13

The Inferior Whorl for Diabetic Neuropathy corneal nerve loss occurred predominantly in the IW region and before loss of nerves in the central cornea, leading the authors to suggest that if this could be translated to patients then it may allow the detection of earlier nerve damage, thereby improving the diagnostic ability of CCM. They also showed that only a fraction of the sub-basal nerves were visible on IVCCM, compared to in vitro imaging, and suggested that further enhancement of visualization with CCM would likely improve our ability to detect early neuropathic changes. Recently, Marfurt et al

17

have undertaken detailed immunohistochemical

staining of the entire sub-basal nerve plexus in 16 donor corneas and showed that it is a gentle, spiral-like structure of long, curvilinear nerve fibers that converge on a vortex, located inferior and slightly nasal to the apex. Given that DPN is a length-dependent, symmetrical neuropathy with initial involvement of the most distal sensory nerves

33

, one may expect nerves in

the inferior whorl, the most distal part of the sub-basal plexus, to be depleted before the more proximal central nerves. Indeed in this study we show that patients with DPN had a significantly reduced CNFD, CNFL and IWL compared to patients without DPN and controls. However, in addition we show that patients without DPN showed a significant reduction in IWL but no difference in CNFD or CNFL when compared to controls. This suggests that a reduction in IWL may well be the earliest defect in corneal nerve pathology as previously observed in experimental diabetes 32. We have also shown that that the diagnostic performance of IWL is not as good as CNFD and CNFL in diagnosing diabetic patients with neuropathy. This is not surprising given that IWL is already abnormal in diabetic patients without neuropathy. However, combining IWL with CNFD and CNFL indeed

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The Inferior Whorl for Diabetic Neuropathy all three metrics increased the sensitivity of CCM for the diagnosis of DPN. Hence, a scanning protocol, which incorporates imaging of the central and inferior whorl regions, may offer diagnostic advantage over central scanning alone. Perhaps a real-time mapping technique such as that described by Edwards et al.

6

may be superior to any point scanning methodology.

However, the scanning time of 15-20 minutes per cornea can be uncomfortable to the patient and suboptimal for a healthcare system. Furthermore, if IVCCM is to be deployed for neuropathy screening and indeed in children 34 then scanning should be rapid and well tolerated. This study is cross-sectional and therefore does not address the question of how the IWL may perform prospectively. Identification of the inferior whorl center may be considered to be challenging, especially in patients with a loss of corneal nerve fibres (Figure 2F). To address this limitation we have undertaken an analysis, which demonstrates excellent inter- and intraobserver agreement in identifying the IW and assessing the IWL. However, a larger study should evaluate whether this finding is influenced by neuropathic severity. In conclusion, this study is the first to compare nerve pathology in both central and inferior regions of the cornea in patients with and without diabetic neuropathy. We show that a reduction in corneal nerve length at the inferior whorl occurs earlier than in the central cornea and combining this abnormality with changes in the central cornea may improve the diagnostic performance of in vivo CCM.

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The Inferior Whorl for Diabetic Neuropathy Table 1. Clinical and peripheral neuropathy status Controls (n = 15)

Variable

DPN (-) (n = 28)

DPN (+) (n = 25)

(No Neuropathy)

Duration of Diabetes

N/A

16.2 ± 9.3

24.8 ± 19.5

¶ NSP

0

2.3 ± 5.1*

2.5 ± 2.2 *

¶ NDS

0

1.7 ± 1.5*

3.9 ± 2.5*

¶ VPT (V)

5.8 ± 3.8

6.6 ± 4.3

16.9 ± 9.3**

27.5 ± 4.2 / 38.5 ± 4.3

26.0 ± 5.7 / 39.5 ± 4.0

23.5 ± 6.9** / 41.3 ± 3.9**

¶ CIP / HIP ( C)

12.6 ± 10.5 / 45.7 ± 3.5

12.5 ± 9.0 / 46.1 ± 2.4

7.4 ± 8.0 / 47.9 ± 2.4*

PMNCV (m/s)

47.7 ± 5.5

45.5 ± 3.8

39.0 ± 5.9**

SSNCV (m/s)

49.6 ± 5.7

46.5 ± 4.8

40.0 ± 10.2**

PMNA (mV)

5.4 ± 1.4

5.8 ± 3.2

1.6 ± 1.1**

¶ SSNA (μV)

18.5 ± 7.1

16.2 ± 7.7

7.7 ± 5.6**

CNFD (no./mm2)

37.2 ± 5.9

31.2 ± 8.2

22.4 ± 9.6**

CNFL (mm/mm2)

27.5 ± 4.6

23.4 ± 7.0

17.0 ± 7.5**

IWL (mm/mm2)

31.5 ± 7.7

25.0 ± 9.0*

18.2 ± 8.0**

o

¶ CT / WT ( C) o

Results are expressed as mean ± SD, statistically significant differences using ANOVA / Kruskal-Wallis: § P

The Inferior Whorl For Detecting Diabetic Peripheral Neuropathy Using Corneal Confocal Microscopy.

In vivo corneal confocal microscopy (CCM) is increasingly used as a surrogate endpoint in studies of diabetic polyneuropathy (DPN). However, it is not...
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