Small fiber neuropathy in Parkinson’s disease: A clinical, pathological and corneal confocal microscopy study.

Twenty-six PD patients and 26 controls underwent CCM of both eyes. 24/26 PD patients and 10/26 controls underwent skin biopsies from the dorsa of both feet. PD patients underwent assessment of parasympathetic function [deep breathing heart rate variability (DB-HRV)], autonomic symptoms [scale for outcomes in Parkinson's disease – autonomic symptoms (SCOPA-AUT)], motor symptoms [UPDRS-III “ON”] and cumulative Levodopa dose.

2 Methods 2.1 Ethical approval NRES Committee/North West (Ref. No 12/NW/0086) approved the study.

Subjects

Thirty-three patients (22 males, 11 females) fulfilling the UK Brain Bank criteria for the diagnosis of Parkinson's disease were recruited from neurology clinics. Patients with a known history of cancer, chemotherapy, diabetes, alcoholism, vitamin deficiencies, celiac disease and autoimmune conditions were excluded. All patients underwent an oral glucose tolerance test and fasting blood screening to include: vitamin B12, folate, methylmalonate, and homocysteine to exclude other causes of neuropathy. Six patients (5 males, 1 female) were excluded due to impaired glucose tolerance, leaving 27 patients who fulfilled the inclusion criteria. CCM was not undertaken in 1 patient due to previous refractive eye surgery. Twenty-six age-matched healthy volunteers served as controls. All participants gave their written informed consent. Disease duration, severity and levodopa exposure PD duration was calculated from reported symptoms onset until the date of assessment. UPDRS-III was used to assess motor severity. Patients were examined in the “ON” state in an outpatient setting. All patients except one were on Levodopa therapy. Cumulative Levodopa dose was calculated from the date of first prescription until the date of assessment.

Non-motor symptoms

Non-motor symptoms were quantified using the non-motor symptoms scale NMSS [22] . Pain was quantified separately using the recently devised King's PD pain scale [23] as well as the short form McGill Pain Questionnaire (SFMPQ).

Autonomic symptoms and function

Autonomic symptoms were assessed using the scale for outcomes in Parkinson's disease – autonomic symptoms (SCOPA-AUT) [24] . Deep breathing heart rate variability (DB-HRV) provided an estimation of cardiac parasympathetic dysfunction and was assessed with an ANX 3.0 autonomic nervous system devise (ANSAR Medical Technologies Inc., Philadelphia, PA). Cardiovascular sympathetic function was assessed by measuring blood pressure and heart rates in the supine position and after standing up for 5 min.

Evaluation of neuropathy

Nerve conduction studies were performed on all PD patients. Patients with evidence of a large fiber neuropathy underwent assessment of immunoglobulins and protein electrophoresis to rule out the presence of a paraproteinemic neuropathy. The neuropathy disability score (NDS) was obtained in all participants. NDS is a bedside measure to stratify diabetic neuropathy [25] . A clinician assesses vibration perception using a tuning fork, temperature perception using a cold and warm metal rods, pinprick perception using Neurotips™ and ankle reflexes using a tendon hammer producing a score from 0 to 10. Quantitative Sensory Testing was assessed using the MEDOC TSA II (Medoc Ltd., Ramat-Yishai 20095, Israel) device on the dorsum of the left foot. Quantitative sensory testing included heat sensation threshold, heat-induced pain threshold, cold threshold and cold-induced pain threshold. Vibration perception threshold was measured using a Neurothesiometer (Horwell, Scientific Laboratory Supplies, Nottingham, UK) on the hallux of both feet. Skin biopsies Two 3-mm punch skin biopsies were taken from the dorsa of both feet. The biopsies were immediately fixed in 4% paraformaldehyde, cryoprotected in graded solutions of sucrose, frozen and cut on a cryomicrotome (HM450, Microm International, Germany). Six 50 μm sections per biopsy were immuno-stained using anti-human PGP 9.5 antibody and nerve fibers were demonstrated using SG chromogen (Vector Laboratories, Peterborough, U.K.). A pathologist blinded to participants' details performed tissue analysis. Intraepidermal nerve fiber density (IENFD), i.e., the number of nerve fibers crossing basement membrane, was quantified according to established criteria and expressed as number per millimetre of epidermal length [26] . The mean between right and left IENFD was calculated for each patient and used for analysis.

Corneal confocal microscopy

Corneal microscopy was performed on both eyes using a Heidelberg Retina Tomograph III with a Rostock Cornea Module (HRT III RCM; Heidelberg Engineering GmbH, Heidelberg, Germany), as previously described [17] . Four to six high-resolution (1–2 μm) images of the sub-basal plexus of each eye were obtained for all participants. A trained investigator who was blinded to participants' details analysed corneal images separately. Corneal Nerve Fiber Density (CNFD): The number of main nerves per square millimetre, Corneal Nerve Branch Density (CNBD): The number of branches emanating from each main nerve per square millimetre and Corneal Nerve Fiber Length (CNFL): The length of all nerve fibers and branches (mm per square millimetre) were quantified and the mean derived from the right and left eye for each parameter. Quantification was undertaken using semi-automated, purpose-written, proprietary software (CCMetrics; M.A. Dabbah, Imaging Science and Biomedical Engineering, Manchester, UK). To estimate the error in measuring CNFD, CNBD and CNFL, we acquired images and determined each of these parameters in 15 subjects on two occasions separated by at least 48 h. The coefficient of variation of these parameters was 12% for CNFD, 24% for CNBD and 9% for CNFL.

Statistical analysis IBM

SPSS version 22 was used to compute the results. All parameters were normally distributed except for cumulative Levodopa dose, vibration and cold perception thresholds as well as heat and cold pain thresholds. Independent samples t -test or Mann–Whitney U test were used to compare means as appropriate. Cohen d was calculated to measure effect size. Two-tailed Pearson's correlation or Spearman's correlations were used as appropriate to determine relationships between continuous variables. Categorical data were compared with Chi square test. P value of