PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Muscle Nerve. Author manuscript; available in PMC Nov 1, 2013.
Published in final edited form as:
PMCID: PMC3469745
NIHMSID: NIHMS362271
Corneal confocal microscopy detects small fiber neuropathy in CMT1A patients
Mitra Tavakoli, MSc, PhD, FAAO,1 Andy Marshall, MD, FRCP,2 Siddharth Banka, MBBS, MRCPCH,3 Ioannis N Petropoulos, MSc,1 Hassan Fadavi, MD,1 Helen Kingston, MD, FRCP,3 and Rayaz A Malik, MBChB, PhD, FRCP1
1Division of Cardiovascular Medicine, University of Manchester and Wellcome Trust Clinical Research Facility, Manchester, UK
2Department of Neurophysiology, Manchester Royal Infirmary, UK
3Department of Genetic Medicine, St. Mary’s Hospital, Central Manchester University Hospital NHS Foundation Trust
Corresponding author’s contact information; Professor R.A.Malik, Division of Cardiovascular Medicine, University of Manchester, Manchester, M13 9NT, UK. Tel: 0161 2751196, Fax: 0161 275 1183, rayaz.a.malik/at/man.ac.uk
Although unmyelinated nerve fibers are affected in CMT1A, they have not been studied in detail due to the invasive nature of the techniques needed to study them. We established alterations in C-fiber bundles of the cornea in patients with CMT1A using non-invasive corneal confocal microscopy (CCM).
Twelve patients with CMT1A and twelve healthy control subjects underwent assessment of neuropathic symptoms and deficits, electrophysiology, quantitative sensory testing, corneal sensitivity and corneal confocal microscopy.
Corneal sensitivity, corneal nerve fiber density, corneal nerve branch density, corneal nerve fiber length and corneal nerve fiber tortuosity were significantly reduced in CMT1A patients compared to controls. There was a significant correlation between corneal sensation and CCM parameters with the severity of painful neuropathic symptoms, cold and warm thresholds and median nerve CMAP amplitude.
CCM demonstrates significant damage to C-fiber bundles, which relates to some measures of neuropathy in CMT1A patients.
Keywords: CMT1A, Neuropathy, Electrophysiology, Corneal confocal microscopy, Corneal nerves, C-fibers
Charcot-Marie-Tooth (CMT) disease or hereditary motor and sensory neuropathy (HMSN) is the commonest inherited neuromuscular disorder [1]. The diagnosis of CMT is based on family history, clinical evaluation supported by electrophysiology, and genetic testing. Accurate diagnosis of CMT is difficult in many cases due to its extreme genetic heterogeneity. Family history and electrophysiology are useful components in the diagnosis of CMT and allow patients to be classified into different subtypes. CMT1A is the most frequent sub-type (~70– 80%). It results from duplication of the peripheral myelin protein 22 (PMP22) gene on chromosome 17p11.2 [2]. Patients with CMT1A typically have uniform slowing of nerve conduction velocities (NCVs) consistent with a demyelination, but they also develop reduced amplitudes of compound motor and sensory nerve action potentials (CMAPs and SNAPs) and progressive muscle weakness suggestive of axonal degeneration [35]. Whilst these changes are attributed to primary demyelination with secondary axonal degeneration of the large myelinated fibers [68], it is interesting that there is no correlation between motor or sensory NCVs and neurological disability in patients with CMT1A [3, 6].
While small fiber dysfunction has been demonstrated in CMT1 using quantitative sensory tests (QST) [911] and sympathetic skin response (SSR) [11], preservation of cerebral potentials following laser and electrical stimulation has suggested preservation of small fibers in a patient with CMT [12]. Sural nerve biopsy studies have demonstrated degeneration and regeneration of the unmyelinated fibers [11, 13, 14], but because this procedure is invasive, it is undertaken less frequently, particularly with the development of genetic analyses. However, pathological studies allow definitive assessment of the primary pathology and also provide insights into underlying pathogenetic mechanisms [15]. As an alternative less invasive approach, a detailed immunohistological and electron microscopic study of dermal nerves in skin biopsies from patients with CMT1A has demonstrated shortening of the internodal length with a loss of Meissner corpuscles and accumulation of intra-axonal mitochondria, suggestive of axonal pathology [16]. Comparable pathology has also been demonstrated recently in dermal nerves of foot skin pad and peripheral nerves in a variety of animal models of CMT [17].
There is now increasing literature on the potential for corneal confocal microscopy (CCM) as a means to quantify C-fiber pathology in peripheral neuropathies. A number of detailed morphometric and immunohistological studies have demonstrated that the subbasal nerve fiber bundles studied by CCM are predominantly nociceptive C-fibers [1820]. Indeed it has been applied to evaluate diabetic neuropathy [21, 22], idiopathic small fiber neuropathy [23], Fabry disease [24], and a series of conditions that cause small nerve fiber damage, including hereditary sensory and autonomic neuropathy (HSAN) [25], autoimmune neuropathy [26], Crohn’s disease [27] and neuropathy associated with chemotherapy [27, 28]. We have also shown that corneal nerve damage assessed using CCM relates to the severity of intraepidermal nerve fiber loss [29], is related to a loss of corneal sensitivity [30], and can detect early small nerve fiber regeneration following pancreas transplantation in diabetic patients [31].
CCM may therefore provide a non-invasive means to determine whether there is small fiber pathology in patients with CMT. We have undertaken a detailed evaluation of neuropathy using conventional neurophysiology and quantitative sensory testing in addition to CCM and non-contact corneal aesthesiometry (NCCA), to quantify nerve damage in patients with CMT1A.
Selection of Patients
Twelve patients with CMT1A (6 men, 6 women, average age 43.0 ± 3.5 years) and twelve healthy control subjects (7 men, 5 women, average age: 43.0 ± 3.5 years) were studied. All patients were confirmed to have the PMP22 duplication by dosage analysis.
The study was approved by the Central Manchester Ethics committee, and written informed consent was obtained according to the declaration of Helsinki. All patients were diagnosed and referred from the department of Genetic Medicine, Central Manchester University Hospital NHS Foundation Trust.
Assessment of Neuropathy
All patients and controls underwent a detailed evaluation of their neurological symptoms according to the Neuropathy Symptom Profile (NSP) [32]. The McGill pain analogue score was used to assess the severity of pain. Neurological deficits were assessed using the neuropathy disability score (NDS) and included vibration, pin prick and temperature perception as well as the presence or absence of ankle reflexes to establish the severity of neuropathy (NDS 0–2, no neuropathy; NDS 3–5, mild neuropathy; NDS, 6–8, moderate neuropathy; and NDS, 9–10, severe neuropathy [32]). Vibration Perception Threshold (VPT) was measured using a Neurothesiometer (Horwell, Scientific Laboratory Supplies, Wilford, Nottingham, UK). Quantitative sensory testing included assessment of cold sensation (CS) and cold induced pain (CIP) to assess Að fibers and warm sensation (WS) and heat induced pain (HIP) to assess C fibers on the dorsum of the left foot, using the MEDOC TSA II (Medoc Ltd., Ramat Yishai 30095, Israel). Electrodiagnostic studies were undertaken using a Dantec “Keypoint” system (Dantec Dynamics Ltd, Bristol, UK) equipped with a DISA temperature regulator to keep limb temperature constant between 32 and 35°C. Full electrophysiological assessment in motor (Median, Ulnar, Fibular) and sensory (Radial, Sural) nerves was performed from the right limb. Motor studies were performed using silver-silver chloride surface electrodes at standard sites defined by anatomical landmarks. Compound muscle action potential (CMAP) amplitudes were taken from baseline to negative peak. Motor nerve conduction velocities were calculated after distal and proximal stimulation. Sensory studies were recorded using a bar electrode (cathode-anode distance 3cm) placed at standard sites. Recordings for sural and radial nerves were taken using antidromic stimulation over a distance of 140 and 100mm respectively.
Corneal sensitivity
Corneal sensitivity was quantified using a non-contact corneal aesthesiometer (NCCA) (Glasgow, Caledonian University, UK) which uses a puff of air through a bore 0.5mm in diameter lasting 0.9 seconds and exerting a force expressed in millibars (mbars) [30]. An electronic pressure sensor displays the force exerted in millibars (mbars). The stimulus jet is mounted on a slit lamp. It is positioned 1 cm from the eye, and the air jet is aligned to the centre of the cornea. The subject feels a sensation on the cornea which is most commonly describing as being “cold” or as a ”breeze” and acknowledges this. Each subject is presented with a supramaximal stimulus, and the staircase method is employed by reducing the stimulus strength until the patient does not feel the jet. This is then increased to a threshold level and reduced to the point where the stimulus is not felt. The whole process is repeated three times to derive a threshold. The coefficient of variation for NCCA was 5.6%.
Corneal Confocal Microscopy
Patients underwent examination with the Heidelberg Retina Tomograph (HRT III) (Rostock Cornea Module) in vivo corneal confocal microscopy. The subject’s eyes were anesthetized using a drop of 0.4% benoxinate hydrochloride, and Viscotears were applied on the front of the eye for lubrication. The patient was instructed to fixate on a target with the eye that was not being examined. A drop of viscoelastic gel was placed on the tip of the objective lens, and a sterile disposable Perspex cap was placed over the lens. The gel optically couples the objective lens to the cornea. Several scans of the entire depth of the cornea were recorded by turning the fine focus of the objective lens backwards and forwards for approximately 2 minutes to acquire satisfactory images of all corneal layers providing en face two dimensional images with a lateral resolution of approximately 2 μm/pixel and final image size of 400 pixels × 400 pixels. Images were obtained using the section mode, which enabled manual acquisition and storage of a single image at a time. For the purposes of this study, we obtained high quality images of the sub-basal nerve plexus of the cornea from each patient and control subject. This layer is of particular relevance for defining neuropathic changes, since it is the location of the main nerve plexus that supplies the overlying corneal epithelium. These nerve fiber bundles contain unmyelinated fibers, which run parallel to the Bowman layer before dividing and turning upwards toward the surface to terminate as individual axons underneath the surface epithelium [33,34]. This has been confirmed using electron microscopy, where nerve bundles containing unmyelinated axons were shown to penetrate the Bowman membrane throughout the central and peripheral cornea at approximately 400 sites [35]. Five images per patient from the center of the cornea were selected and examined in a masked and randomized fashion [36].
Four corneal nerve parameters were quantified: (i) Corneal nerve fiber density (CNFD) - the total number of major nerves/mm2 of corneal tissue; Corneal nerve branch density (CNBD) - the number of branches emanating from all major nerve trunks/mm2 of corneal tissue; (iii) Corneal nerve fiber length (CNFL) - the total length of all nerve fibers and branches (mm/mm2) within the area of corneal tissue; and (iv) Corneal nerve fiber tortuosity (CNFT). CNFD and CNFL are considered to reflect overall nerve fiber degeneration, while CNBD reflects nerve fiber regeneration (which is also captured partially by the CNFL).
Statistics
SPSS 16.05.0 for Windows was used to compute the results. Analysis included descriptive and frequency statistics. All data are expressed as mean ± SEM. One-way analysis of variance (ANOVA) with Scheffe post-hoc tests was used to study differences between means. The Pearson test was used to analyze correlations between potentially related variables.
The clinical characteristics and detailed assessment of neuropathy in CMT1A patients and their matched controls are summarized in Table 1.
Table 1
Table 1
Clinical and neurological assessments in control subjects and CMT1A patients. Data are expressed as Mean ± SEM
Symptoms and Neurological Deficits
Neuropathic symptoms assessed with the NSP were significantly increased in CMT1A patients (P<0.0001), as was the severity of pain assessed using the McGill pain analogue (P=0.001). The Neuropathy deficit score (NDS) was significantly increased, consistent with a severe neuropathy in all 12 patients (9.1 ± 0.4, P<0.0001).
Quantitative Sensory Tests
Vibration perception threshold (VPT) (P<0.0001) and CS (P=0.01) were significantly increased in patients compared to control subjects. However, CIP, WS, and WIP did not differ significantly from control subjects.
Electrophysiology
Sural SNAPs and fibular CMAPs were not elicited in all cases. Low voltage, slowed radial and median SNAPs could be obtained in just four cases. As expected, upper limb motor nerve conduction in all patients with CMT1A showed diffuse and uniform slowing of conduction velocity (less than 30m/s) along with prolongation of distal and F-wave latencies. Upper limb CMAP amplitudes were significantly reduced (Table 2).
Table 2
Table 2
Electrophysiology in control subjects and CMT1A patients. Data are expressed as Mean ± SEM
Corneal sensation
Corneal sensitivity was significantly reduced in CMT1A patients compared to control subjects (P=0.01) (Table 3).
Table 3
Table 3
Corneal sensitivity and corneal nerve morphology in control subjects and CMT1A patients. Data are expressed as Mean ± SEM
Corneal Confocal Microscopy
Corneal nerve fiber density (P=0.01) (Fig. 2.a), nerve branch density (P=0.02) (Fig. 2.b), nerve fiber length (P<0.0001) (Fig. 2.c) and nerve fiber tortuosity (P=0.004) (Fig. 2.d) were significantly reduced in CMT1A patients compared to control subjects (Figures 1, ,22 and Table 3). There was no difference in corneal nerve parameters between males and females (NFD- P=0.8; NBD- P=0.6; NFL - P=0.9; NFT - P=0.5; NCCA - P=0.4).
Figure 2
Figure 2
(a) Corneal nerve fiber density (b) corneal nerve branch density (c) corneal nerve fiber length (d) corneal nerve fiber tortuosity (e) corneal sensitivity.
Figure 1
Figure 1
CCM images of the sub-basal layer of cornea: (a) control subject, (b) patient with CMT1A. The red arrows indicate main nerve fibers and yellow arrows indicate branches.
Correlations
Both NDS and NSP showed no significant correlations with NCCA or corneal nerve morphology. However, the severity of pain as judged by the McGill Pain Analogue score correlated significantly with NCCA, NFD and NFL (Table 4). There was no significant correlation with VPT, but NCCA correlated significantly with CS and WS. CCM demonstrated a correlation with CS and WS and reached statistical significance between NFD and CS. Both Median and Ulnar nerve conduction velocity showed no association with NCCA or CCM. However, NCCA correlated significantly with Ulnar nerve amplitude and showed borderline significance with Median nerve amplitude. While CCM did not correlate with Ulnar amplitude, both NBD and NFL showed a significant correlation with Median nerve CMAP amplitude.
Table 4
Table 4
Correlations between corneal sensitivity and corneal nerve morphology with neurological parameters in CMT1A patients.
The diagnosis of CMT has evolved from a purely clinical approach supported by electrophysiology to the currently employed combined clinical/genetic approach. Despite advances in the identification of many of the causative genes for CMT, accurate diagnosis of CMT still requires a detailed knowledge of the clinical and genetic subtypes and their frequencies in different populations. CMT1A is caused by a 1.4-Mb duplication of the PMP22 gene on 17p11.2 [2, 37]. Electrophysiology is an essential component of the diagnosis of CMT and allows patients to be classified into two types: CMT1 (demyelinating) and CMT2 (axonal) subtypes using upper limb NCVs (median or ulnar nerves), where an NCV<38 m/s (commonly around 20 m/s) is strongly suggestive of CMT1A. In this study we have undertaken a systematic evaluation of neuropathy assessing symptoms, neurological deficits, conventional neurophysiology, quantitative sensory testing, NCCA and CCM in patients with CMT1A. The results confirm a significant neuropathy with a markedly elevated NDS and a significant deficit of myelinated nerve fiber function as evidenced by absent lower limb CMAPs and SNAPs and markedly reduced upper limb nerve conduction velocity and amplitudes. Additionally we demonstrate a significant increase in the cold threshold suggestive of Að, thinly myelinated fiber deficits.
With regard to small fiber deficits, patients with CMT1A have clinical evidence of moderate painful neuropathic symptoms, which is probably related to a reduction of the Að afferents [38]. In this study we also show an elevated NSP and McGill pain score, which correlated with damage to corneal nerve fibers. While quantitative sensory testing of small fibers does not demonstrate a significant increase in the thresholds for warmth and heat pain, our studies do demonstrate a significant abnormality in corneal sensation and corneal nerve fiber morphology.
The cornea contains myelinated Að fibers, which respond primarily to mechanical stimuli, and unmyelinated C-fibers, which respond to thermal and chemical stimuli [19]. In this study, we scanned the sub-basal layer of the central cornea to image corneal C-fibers. Stromal nerves representing Að fibers could not be consistently imaged in all patients. Quantitative analysis of stromal nerves imaged using CCM is recognized to be difficult [39], especially to objectively distinguish normal from abnormal nerves [3941]. In a previous sural nerve biopsy study the number of unmyelinated axons/Schwann cell was reduced in patients with CMT1A [14]. We have now demonstrated significant pathology of the corneal C-fiber bundles located in the sub-basal layer using CCM together with reduced corneal sensation in patients with CMT1A. Importantly, these findings closely correlate, particularly with the severity of painful neuropathic symptoms and small fiber deficits as well as median and ulnar nerve CMAP amplitude, the latter reflecting axonal integrity. This is the first time that a correlation between C-fiber pathology and clinical severity of neuropathy has been established in CMT1A. Thus unlike QST, sural nerve biopsy and dermal skin biopsy, NCCA and CCM can be used as rapid non-invasive objective tools for clinical assessment and quantification of small fiber abnormalities in CMT1A. The explanation of these findings may lie in roles of PMP22 in processes other than myelination [42]. PMP22 over expression in myelinating Schwann cells produces abnormal growth and differentiation resulting in defective myelin stability and turnover [43]. We hypothesize that similar PMP22 over expression in non-myelinating Schwann cells could also be the basis of the observed defects in C-fiber bundles in CMT1A. This would be consistent with previous observations in sural nerve biopsies [14], where a reduction in the number of axons/Schwann cell profile has been demonstrated, thus providing insights into the biological role(s) of PMP22 and pathological mechanisms involving its protein.
Furthermore, these observations add to our published studies which demonstrate the clinical utility of CCM in the assessment of patients with a range of peripheral neuropathies, including diabetic neuropathy [22, 44], Fabry Disease [24] and idiopathic small fiber neuropathy [45]. These findings provide the basis for further studies to define whether there are differences in corneal nerve morphology of patients with CMT that primarily affect axons. Ideally, detailed ultra structural morphological studies from the cornea of patients with CMT1 should also be undertaken to be absolutely certain that all the fibers assessed using CCM are indeed unmyelinated. We acknowledge that this is a preliminary study, and further studies are required in a larger group of patients to compare corneal nerve morphology in patients with CMT1 and CMT2 with differing severity of neuropathy. Therefore CCM may aid in earlier diagnosis and to undertake longitudinal or interventional studies in patients with CMT.
Acknowledgments
This work was supported by National Institute of Health Grant R105991. Support from the NIHR Manchester Biomedical Research Centre and Wellcome Trust Clinical Research Facility is acknowledged.
List of Abbreviations
ANOVAAnalysis of variance
CCMCorneal Confocal Microscopy
CNFDCorneal Nerve Fibre Density
CNBDCorneal Nerve Branch Density
CNFLCorneal Nerve Fibre Length
CNFTCorneal Nerve Fibre Tortuosity
CMAPCompound motor action potential
CMTCharcot-Marie-Tooth
CSCold Sensation
CIPCold Induced Pain
HIPHeat Induced Pain
HSANHereditary sensory and autonomic neuropathy
NCCANon Contact Corneal Aesthesiometer
NCVNerve Conduction Velocity
NDSNeuropathy Disability Score
NSPNeuropathy Symptom Profile
PMP22Peripheral myelin protein 22
QSTQuantitative Sensory Testing
SEMStandard error of the mean
SNAPSensory Nerve Action Potential
SSRSympathetic Skin Response
VPTVibration Perception Threshold
WSWarm Sensation

1. Skre H. Genetic and clinical aspects of Charcot-Marie-Tooth's disease. Clin Genet. 1974;6(2):98–118. [PubMed]
2. Lupski JR, Garcia CA. Molecular genetics and neuropathology of Charcot-Marie-Tooth disease type 1A. Brain Pathol. 1992;2(4):337–49. [PubMed]
3. Krajewski KM, et al. Neurological dysfunction and axonal degeneration in Charcot-Marie-Tooth disease type 1A. Brain. 2000;123( Pt 7):1516–27. [PubMed]
4. Reilly MM. Sorting out the inherited neuropathies. Pract Neurol. 2007;7(2):93–105. [PubMed]
5. Shy ME. Charcot-Marie-Tooth disease: an update. Curr Opin Neurol. 2004;17(5):579–85. [PubMed]
6. Dyck P. Hereditary motor and sensory neuropathies. In: Dyck P, Thomas P, editors. Peripheral Neuropathy. Saunders; 1993. pp. 1094–1136.
7. Senderek J, et al. X-linked dominant Charcot-Marie-Tooth neuropathy: clinical, electrophysiological, and morphological phenotype in four families with different connexin32 mutations(1) J Neurol Sci. 1999;167(2):90–101. [PubMed]
8. Hattori N, et al. Demyelinating and axonal features of Charcot-Marie-Tooth disease with mutations of myelin-related proteins (PMP22, MPZ and Cx32): a clinicopathological study of 205 Japanese patients. Brain. 2003;126(Pt 1):134–51. [PubMed]
9. Ericson U, Borg K. Analysis of sensory function in Charcot-Marie-Tooth disease. Acta Neurol Scand. 1999;99(5):291–6. [PubMed]
10. Hanson P, Deltombe T. Preliminary study of large and small peripheral nerve fibers in Charcot-Marie-Tooth disease, type I. Am J Phys Med Rehabil. 1998;77(1):45–8. [PubMed]
11. Zambelis T. Small fiber neuropathy in Charcot-Marie-Tooth disease. Acta Neurol Belg. 2009;109(4):294–7. [PubMed]
12. Lankers J, et al. Ultralate cerebral potentials in a patient with hereditary motor and sensory neuropathy type I indicate preserved C-fiber function. J Neurol Neurosurg Psychiatry. 1991;54(7):650–2. [PMC free article] [PubMed]
13. Ingall TJ, McLeod JG. Autonomic function in hereditary motor and sensory neuropathy (Charcot-Marie-Tooth disease) MuscleNerve. 1991;14(11):1080–3. [PubMed]
14. Koike H, et al. Nonmyelinating Schwann cell involvement with well-preserved unmyelinated axons in Charcot-Marie-Tooth disease type 1A. J Neuropathol Exp Neurol. 2007;66(11):1027–36. [PubMed]
15. Benedetti S, et al. Analyzing histopathological features of rare charcot-marie-tooth neuropathies to unravel their pathogenesis. Arch Neurol. 2010;67(12):1498–505. [PubMed]
16. Saporta MA, et al. Shortened internodal length of dermal myelinated nerve fibers in Charcot-Marie-Tooth disease type 1A. Brain. 2009;132(Pt 12):3263–73. [PMC free article] [PubMed]
17. Dacci P, et al. Foot pad skin biopsy in mouse models of hereditary neuropathy. Glia. 2010;58(16):2005–16. [PMC free article] [PubMed]
18. Muller LJ, et al. Corneal nerves: structure, contents and function. Exp Eye Res. 2003;76(5):521–42. [PubMed]
19. Muller LJ, et al. Architecture of human corneal nerves. Invest Ophthalmol Vis Sci. 1997;38(5):985–94. [PubMed]
20. Ueda S, et al. Peptidergic and catecholaminergic fibers in the human corneal epithelium. An immunohistochemical and electron microscopic study. Acta Ophthalmol Suppl. 1989;192:80–90. [PubMed]
21. Rosenberg ME, et al. Corneal structure and sensitivity in type 1 diabetes mellitus. Invest Ophthalmol Vis Sci. 2000;41(10):2915–21. [PubMed]
22. Tavakoli M, et al. Corneal confocal microscopy: a novel noninvasive test to diagnose and stratify the severity of human diabetic neuropathy. Diabetes Care. 2010;33(8):1792–7. [PMC free article] [PubMed]
23. Tavakoli M, et al. Corneal confocal microscopy: A novel means to detect nerve fiber damage in idiopathic small fiberneuropathy. Exp Neurol. 2009
24. Tavakoli M, et al. Corneal confocal microscopy: a novel noninvasive means to diagnose neuropathy in patients with Fabry disease. Muscle Nerve. 2009;40(6):976–84. [PubMed]
25. Mimura T, et al. In vivo confocal microscopy of hereditary sensory and autonomic neuropathy. Curr Eye Res. 2008;33(11):940–5. [PubMed]
26. Lalive PH, et al. Peripheral autoimmune neuropathy assessed using corneal in vivo confocal microscopy. Arch Neurol. 2009;66(3):403–5. [PubMed]
27. Gemignani F, et al. Non-length-dependent small fiber neuropathy. Confocal microscopy study of the corneal innervation. J Neurol Neurosurg Psychiatry. 2010;81(7):731–3. [PubMed]
28. Ferrari GGF, Macaluso C. Chemotherapy-associated peripheral sensory neuropathy assessed using in vivo corneal confocal microscopy. Arch Neurol. 2010;67(3):364–5. [PubMed]
29. Quattrini C, et al. Surrogate markers of small fiber damage in human diabetic neuropathy. Diabetes. 2007;56(8):2148–54. [PubMed]
30. Tavakoli M, et al. Corneal sensitivity is reduced and relates to the severity of neuropathy in patients with diabetes. Diabetes Care. 2007;30(7):1895–7. [PubMed]
31. Mehra S, et al. Corneal confocal microscopy detects early nerve regeneration after pancreas transplantation in patients with type 1 diabetes. Diabetes Care. 2007;30(10):2608–12. [PubMed]
32. Young MJ, et al. A multicentre study of the prevalence of diabetic peripheral neuropathy in the United Kingdom hospital clinic population. Diabetologia. 1993;36(2):150–4. [PubMed]
33. Belmonte C, Acosta MC, Gallar J. Neural basis of sensation in intact and injured corneas. Exp Eye Res. 2004;78(3):513–25. [PubMed]
34. Guthoff RF, et al. Epithelial innervation of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy. Cornea. 2005;24(5):608–13. [PubMed]
35. Ueda S, et al. Corneal and conjunctival changes in congenital erythropoietic porphyria. Cornea. 1989;8(4):286–94. [PubMed]
36. Tavakoli M, Malik RA. Corneal confocal microscopy: a novel non-invasive technique to quantify small fiber pathology in peripheral neuropathies. J Vis Exp. 2011;(47) [PubMed]
37. Raeymaekers P, et al. Duplication in chromosome 17p11.2 in Charcot-Marie-Tooth neuropathy type 1a (CMT 1a). The HMSN Collaborative Research Group. Neuromuscul Disord. 1991;1(2):93–7. [PubMed]
38. Pazzaglia C, et al. Mechanisms of neuropathic pain in patients with Charcot-Marie-Tooth 1 A: a laser-evoked potential study. Pain. 2010;149(2):379–85. [PubMed]
39. Patel DV, McGhee CN. In vivo confocal microscopy of corneal stromal nerves in patients with peripheral neuropathy. Arch Neurol. 2009;66(9):1179–80. author reply 1180. [PubMed]
40. Mocan MC, et al. Morphologic alterations of both the stromal and subbasal nerves in the corneas ofpatients with diabetes. Cornea. 2006;25(7):769–73. [PubMed]
41. Visser N, McGhee CN, Patel DV. Laser-scanning in vivo confocal microscopy reveals two morphologically distinct populations of stromal nerves in normal human corneas. Br J Ophthalmol. 2009;93(4):506–9. [PubMed]
42. Baechner D, et al. Widespread expression of the peripheral myelin protein-22 gene (PMP22) in neural and non-neural tissues during murine development. J Neurosci Res. 1995;42(6):733–41. [PubMed]
43. Hanemann CO, Muller HW. Pathogenesis of Charcot-Marie-Tooth 1A (CMT1A) neuropathy. Trends Neurosci. 1998;21(7):282–6. [PubMed]
44. Hossain P, Sachdev A, Malik RA. Early detection of diabetic peripheral neuropathy with corneal confocal microscopy. Lancet. 2005;366(9494):1340–3. [PubMed]
45. Tavakoli M, et al. Corneal confocal microscopy: a novel means to detect nerve fiber damage in idiopathic small fiberneuropathy. Exp Neurol. 2010;223(1):245–50. [PMC free article] [PubMed]