The LDIFLARE and CCM Methods Demonstrate Early Nerve Fiber Abnormalities in Untreated Hypothyroidism: A Prospective Study

The LDIFLARE and CCM Methods Demonstrate Early Nerve Fiber Abnormalities in Untreated... Abstract Context Recent studies using skin biopsy suggest presence of small-fiber neuropathy in subclinical hypothyroidism. This study uses two noninvasive methods—the laser Doppler imager flare technique (LDIFLARE) and corneal confocal microscopy (CCM)—to assess small-fiber function (SFF) and small-fiber structure (SFS), respectively, in newly diagnosed hypothyroidism (HT) before and after adequate treatment. Design and Setting Single-center, prospective, intervention-based cohort study. Patients and Participants Twenty patients with newly diagnosed HT (15 with primary HT and 5 with post-radioiodine HT) along with 20 age-matched healthy controls (HCs). Interventions Patients with HT and HCs were assessed neurologically at diagnosis and baseline, respectively. The HT group was reassessed after optimal replacement (defined as TSH level of 0.27 to 4.20 mIU/L) with levothyroxine (LT4) and HCs were reviewed after 1 year. Main Outcome Measures Neurologic assessment for small fibers was performed by using LDIFLARE for SFF and CCM for SFS; large fibers were studied by sural nerve conduction velocity (SNCV) and sural nerve amplitude (SNAP). Results At baseline, both LDIFLARE (mean ± SD) (6.74 ± 1.20 vs 8.90 ± 1.75 cm2; P = 0.0002) and CCM nerve fiber density (CNFD) (expressed as number of fibers per mm2: 50.77 ± 6.54 vs 58.32 ± 6.54; P = 0.002) were significantly reduced in the HT group compared with HCs whereas neither SNCV nor SNAP was different (P ≥ 0.05). After optimal LT4 treatment, both LDIFLARE (7.72 ± 1.12 vs 6.74 ± 1.20 cm2; P ≤ 0.0001) and CNFD (54.43 ± 5.70 vs 50.77 ± 6.54 no./mm2; P = 0.02) improved significantly but remained significantly reduced compared to HCs (P = 0.008 and P = 0.01, respectively) despite normalization of TSH. Conclusions This study demonstrates that dysfunction of small fibers precedes large neural fiber abnormalities in early HT. This can be reversed by replacement therapy to achieve a biochemically euthyroid state, but small-fiber neural outcomes continued to remain low compared with values in HCs. The presence of neuromuscular dysfunction in hypothyroidism (HT) is well recognized, with a prevalence ranging between 42% and 72% (1, 2). Epidemiological neurologic studies have reported HT as a cause in 2% to 5% of cases of polyneuropathy (3–5). Peripheral neuropathy, such as diffuse sensorimotor neuropathy involving myelinated large nerve fibers like Aα and Aβ, have been commonly described; symptoms are usually mild and predominantly sensory (6). Beghi et al. (1) studied 39 consecutive patients with hypothyroidism and detected sensory symptoms in 64%, of whom 72% had neurophysiological evidence and only 33% had clinical signs. Similarly, in another case series comprising of 24 patients with newly diagnosed HT, Duyff et al. (7) reported clinical signs of predominantly sensory peripheral neuropathy in only 42% of patients. Clinical signs in HT are usually subtle and nonspecific, with reduced knee and ankle reflexes along with mild impairment of distal vibration and joint position sense (6). The pathological changes described include axonal degeneration, segmental demyelination, and deposition of mucopolysaccharides in the endoneurial interstitium (2, 8, 9). Such changes have been described to be more related to the duration of HT than to the severity of thyroid failure (10). In many neuropathies, including diabetes and chemotherapy-induced peripheral neuropathy, small-fiber neural involvement affecting the thinly myelinated Aδ and nonmyelinated C fibers has been shown to precede large-fiber involvement (11, 12). However, in HT, limited evidence suggests early small-fiber neural involvement, especially in asymptomatic individuals. In a series of 38 hypothyroid patients, Ørstavik et al. (13) demonstrated significantly abnormal thermal thresholds as compared with controls. In a smaller study of 18 neurologically asymptomatic patients with newly diagnosed overt or subclinical HT, Magri et al. (14) found significantly reduced intraepidermal nerve fiber density (IENFD) in their distal leg skin biopsy specimens but no evidence of large-fiber dysfunction assessed by conventional neurophysiological tests. Subsequently, the same authors showed that such impairment of IENFD was reversible after levothyroxine (LT4) treatment in nine patients (15). With the exception of these studies, we are unaware of any other studies specifically assessing other modalities of small-fiber involv-ement in patients with HT. In this prospective study, we used two noninvasive methods — the laser Doppler imager flare technique (LDIFLARE) and corneal confocal microscopy (CCM) — to assess small-fiber function (SFF) and structure (SFS), respectively, in patients with newly diagnosed HT compared with a cohort of age- and sex-matched healthy controls (HCs), as well as the change before and after adequate monotherapy with LT4. Neither SFF nor SFS has been studied together in HT, and hence this study can be considered as a proof-of-concept study investigating early neuropathic changes in HT. Patients and Methods Ethical approval This study was conducted between 2014 and 2017 in accordance with the Declaration of Helsinki and was approved by the ethics committee of the National Research Ethics Service Committee East of England, Norfolk, United Kingdom (Research Ethics Committee reference: 13/EE/0162). All participants provided written informed consent. Study design and participant selection Twenty patients with newly diagnosed HT along with 20 age- and sex-matched HCs were recruited, the latter by invitation from the local population. HT was diagnosed by a TSH level of ≥10 IU/mL with an abnormally low free T4 (FT4) level (see below). Of the 20 patients with HT, 15 had primary HT (PyHT) and 5 had radioiodine-induced HT (RiHT) occurring at least 6 weeks after administration of radioiodine treatment. We included both types of HT to determine whether the neurologic profile would differ given the difference in cause and duration of the HT. None of the patients with RiHT had any neurologic symptoms before administration of radioiodine. Patients who had a history of HT and were receiving thyroid supplementation at any stage in their life were excluded because we also aimed to look at the effects of LT4 supplementation on neurologic parameters. Patients with a history of impaired fasting glycemia, prediabetes, or diabetes (type 1, type 2, or other specific types) as per the 2018 diagnostic criteria of the American Diabetes Association (16) were excluded on the basis of a fasting blood glucose and glycosylated hemoglobin A1c. Patients were also excluded if they had any history of peripheral arterial disease, hypertriglyceridemia, alcohol abuse, vitamin B12 and folate deficiency, thyroid or connective tissue disorders, renal or hepatic failure, malignancy, inherited neuropathy, or exposure to any toxins (including chemotherapy). Clinical and biochemical assessment All participants underwent baseline biochemical assessment for thyroid fasting glucose, glycosylated hemoglobin A1c, lipids, renal and hepatic profiles, vitamin B12, and folate. Thyroid biochemistry comprised TSH, FT4, and antithyroid peroxidase antibodies (anti-TPOab). Serum concentrations of TSH (noncompetitive sandwich immunoassay; normal range, 0.27 to 4.20 mIU/L) and FT4 (competitive immunoassay; normal range, 12 to 22 pmol/L), and anti-TPOab (normal range, 0 to 34 U/mL) were measured with electrochemiluminescent detection using an automated analyzer (Cobas e602 immunochemical analyzer) employing commercial kits (Roche Diagnostics Ltd, Bugess Hill, UK). The TSH assay has a functional sensitivity of 0.014 mIU/L and limit of detection of 0.005 mIU/L, with interassay coefficients of variation (CVs) of 2.1% and 2.0% at concentrations of 1.2 mIU/L and 7.9 mIU/L, respectively. The FT4 assay has a functional sensitivity of 3.0 pmol/L and limit of detection of 0.5 pmol/L, with interassay CVs of 2.1% and 2.8% at concentrations of 15.4 pmol/L and 39.8 pmol/L, respectively. The anti-TPOab assay has an interassay CV of 8% at 34 U/mL and 5% at 92 U/mL, the limit of detection is 5 U/mL, and the measuring range is 5 to 600 U/mL. A total of 32 patients with HT were initially screened, of whom 8 were excluded because of a new diagnosis of prediabetes, diabetes, or vitamin B12 deficiency. A further four patients were lost during follow-up because of a diagnosis of diabetes (two patients), cancer (one patient), and stroke (one patient). All participants, including HCs, completed the Thyroid Symptom Questionnaire (TSQ)—a 12-point questionnaire validated for use in hypothyroidism (17)—at the beginning and end of the study. Both groups also underwent detailed neurologic examination with the modified Neuropathy Disability Score (18). SFF was assessed with LDIFLARE and SFS was measured by CCM. Vibration perception threshold was measured on the toes by using the Neurothesiometer (Horwell, Scientific Laboratory Supplies, Wilford, Nottingham, UK) and expressed as millivolts (mV). Sural nerve conduction velocity (SNCV) and sural nerve amplitude (SNAP) were assessed in one leg (NC-stat|DPNCheck system; Neurometrix, Waltham, MA). The neurologic assessments were repeated for both groups at the end of the study. LDIFLARE method SFF was assessed by using the noninvasive LDIflare technique as previously published (19). In a thermally controlled room at 30°C, the dorsal foot skin is heated sequentially to 44°C for 2 minutes, 46°C for 1 minute, and finally to 47°C for 3 minutes using a 1-cm2 heating probe. This nociceptive heat results in activation of the C-fiber reflex and the resultant nerve-axon– related hyperemic response is assessed by using a laser scanner (Moor Instruments, Axminster, UK). The size of the hyperemic response is measured in centimeters squared (cm2) by using the Moor V 5.3 software, referred to as the LDIflare (Fig. 1). Flare size relates to SFF; a small flare equates to reduced SFF. The time needed for this assessment is up to 30 minutes. Previous studies have demonstrated that it is a sensitive method to detect C-fiber dysfunction as an early marker of small-fiber neuropathy (SFN) in both type 2 diabetes (20) and impaired glucose tolerance (21). It has significant correlations with IEFND (22) and recently has been shown to be more sensitive than neurophysiological studies to detect SFF in early chemotherapy-induced peripheral neuropathy (12). Figure 1. View largeDownload slide LDIFLARE images. (Left) From a 40-y-old HC, with LDIFLARE at 11.44 cm2. (Right) From a 41-y-old patient with HT. with LDIFLARE at 4.17 cm2. Figure 1. View largeDownload slide LDIFLARE images. (Left) From a 40-y-old HC, with LDIFLARE at 11.44 cm2. (Right) From a 41-y-old patient with HT. with LDIFLARE at 4.17 cm2. CCM method Small-fiber structure was measured by CCM using the laser scanning Retina Tomograph III confocal microscope with Rostock Corneal Module (Heidelberg Engineering GmbH, Dossenheim, Germany) by following an established technique. The total duration of examination of both eyes was 5 to 10 minutes (23, 24). Before scanning, topical anesthesia [oxybuprocaine hydrochloride 0.4% (Minims®); Bausch & Lomb, Kingston upon Thames, Surrey, UK] was applied to the cornea of both eyes, followed by topical application of a viscous gel [carbomer 980 polyacrylic acid 0.2% (Viscotears®); Alcon Eye Care, Frimley, Surrey, UK] to form an aqueous medium between the applanated corneal surface and disposable TomoCap (Heidelberg Engineering Ltd., Hemel Hempstead, UK) covering the objective lens. Several scans of the entire depth of the cornea of both eyes were taken by fine adjustment of the objective lens, resulting in manual acquisition of two-dimensional images with a final image size of 400 × 400 pixels and lateral resolution of 2 mm/pixel of the subbasal nerve plexus of the cornea. Each subbasal nerve fiber bundle contains unmyelinated neural fibers running parallel to the Bowman layer before terminating dividing and terminating as individual axons just underneath the corneal surface epithelium. The six best images from the center of the cornea were manually selected for automated image analysis by using purpose-written, proprietary software (CCM Image Analysis tool v1.1; Imaging Science and Biomedical Engineering, University of Manchester, Manchester, UK) (25). The specific parameters measured per frame were as follows: CCM nerve fiber damage (CNFD; number of fibers/mm2), confocal nerve branch density (CNBD; number of fibers/mm2), and CNFL (length in mm/mm2) (23). Corneal nerve fibers and their branches are shown in Fig. 2. Figure 2. View largeDownload slide CCM images (Left) From a 45-y-old HC, with CNFD at 61.89 no./mm2. (Right) From a 47-y old with HT, with CNFD at 30.12 no./mm2. Red arrows show corneal nerve fibers; yellow arrows show corneal nerve branches. Original magnification ×700. Figure 2. View largeDownload slide CCM images (Left) From a 45-y-old HC, with CNFD at 61.89 no./mm2. (Right) From a 47-y old with HT, with CNFD at 30.12 no./mm2. Red arrows show corneal nerve fibers; yellow arrows show corneal nerve branches. Original magnification ×700. Management of HT All patients with HT were treated with LT4 monotherapy as per 2014 guidelines suggested by the American Thyroid Association (26). The dosage of LT4 was titrated to achieve the TSH reference range of 0.27 to 4.20 mIU/L at 12-weekly intervals. Once a stable TSH was achieved for at least 6 months, the patients with HT thereafter completed the TSQ questionnaire and all the neurologic assessments previously undertaken. Statistical analysis Statistical analysis was performed by using SPSS software, version 20 for Windows (IBM, Armonk, NY) and StatsDirect software, version 3 (StatsDirect Ltd, Cambridge, UK). Analysis included descriptive and frequency statistics. All data are expressed as mean ± SD. Normal distribution of the data was determined with the Kolmogorov-Smirnov test. An independent-sample t test was used to test whether a sample mean differed between the HT and HC groups. A paired sample t test was used to for the comparison between baseline and follow-up data. Nonparametric data were analyzed by using the χ2 test. Pearson bivariate correlation was used to see the relationship between baseline TSH and neurologic assessments. Power was calculated by using the Wilks lambda model and computed by using α = 0.05. Statistical significance was defined at 5% (i.e.,P ≤ 0.05). The CVs for CCM and LDIflare are 7.8% and 8.7%, respectively. Results Differences in baseline characteristics Table 1 shows the clinical characteristics and biochemical assessments of all 20 newly diagnosed patients with HT who were enrolled and completed follow-up in the study. In all 15 patients with PyHT, that condition was due to autoimmune thyroiditis characterized by raised anti-TPOab levels (>34 IU/mL). The remaining five patients had RiHT that developed after radioiodine treatment of Graves thyrotoxicosis (three of five patients) and solitary toxic nodule (two of five patients). None of the patients were receiving any form of thyroid replacement therapy, including LT4, at study entry. Table 1. Clinical and Laboratory Features of Patients With HT Patient No. Age (y) Sex Cause of HT TSH (mIU/L) (0.39–4.2 mIU/L) FT4 (pmol/L) ( 12 – 22 pmol/L ) Anti-TPOab (0–34) TSQ (/12) 1 50 Male PyHT: autoimmune thyroiditis 108.70 3.3 150 8 2 54 Male RiHT: post-radioiodine 77.77 4.2 9 3 3 74 Male RiHT: post-radioiodine 80.70 3.0 10 4 4 38 Female PyHT: autoimmune thyroiditis 150.00 3.9 77 12 5 51 Female PyHT: autoimmune thyroiditis 67.90 5.5 93 8 6 32 Female PyHT: autoimmune thyroiditis 32.50 7.1 178 8 7 63 Female PyHT: autoimmune thyroiditis 89.00 4.7 56 12 8 79 Female PyHT: autoimmune thyroiditis 113.70 3.1 81 9 9 30 Female PyHT: autoimmune thyroiditis 37.78 6.1 71 6 10 56 Male RiHT: post-radioiodine 74.50 3.0 56 3 11 46 Female RiHT: post-radioiodine 79.90 3.8 34 9 12 45 Female PyHT: autoimmune thyroiditis 98.87 6.7 76 7 13 45 Female PyHT: autoimmune thyroiditis 65.54 5.6 55 10 14 31 Male PyHT: autoimmune thyroiditis 75.12 5.4 85 7 15 48 Male PyHT: autoimmune thyroiditis 150 3.0 122 12 16 49 Female PyHT: autoimmune thyroiditis 78.77 4.4 178 8 17 54 Female PyHT: autoimmune thyroiditis 85.5 4.1 25 6 18 55 Male PyHT: autoimmune thyroiditis 79.9 3.2 77 8 19 59 Female RiHT: post-radioiodine 35.56 5.9 25 2 20 32 Male PyHT: autoimmune thyroiditis 87.78 3.2 56 10 Patient No. Age (y) Sex Cause of HT TSH (mIU/L) (0.39–4.2 mIU/L) FT4 (pmol/L) ( 12 – 22 pmol/L ) Anti-TPOab (0–34) TSQ (/12) 1 50 Male PyHT: autoimmune thyroiditis 108.70 3.3 150 8 2 54 Male RiHT: post-radioiodine 77.77 4.2 9 3 3 74 Male RiHT: post-radioiodine 80.70 3.0 10 4 4 38 Female PyHT: autoimmune thyroiditis 150.00 3.9 77 12 5 51 Female PyHT: autoimmune thyroiditis 67.90 5.5 93 8 6 32 Female PyHT: autoimmune thyroiditis 32.50 7.1 178 8 7 63 Female PyHT: autoimmune thyroiditis 89.00 4.7 56 12 8 79 Female PyHT: autoimmune thyroiditis 113.70 3.1 81 9 9 30 Female PyHT: autoimmune thyroiditis 37.78 6.1 71 6 10 56 Male RiHT: post-radioiodine 74.50 3.0 56 3 11 46 Female RiHT: post-radioiodine 79.90 3.8 34 9 12 45 Female PyHT: autoimmune thyroiditis 98.87 6.7 76 7 13 45 Female PyHT: autoimmune thyroiditis 65.54 5.6 55 10 14 31 Male PyHT: autoimmune thyroiditis 75.12 5.4 85 7 15 48 Male PyHT: autoimmune thyroiditis 150 3.0 122 12 16 49 Female PyHT: autoimmune thyroiditis 78.77 4.4 178 8 17 54 Female PyHT: autoimmune thyroiditis 85.5 4.1 25 6 18 55 Male PyHT: autoimmune thyroiditis 79.9 3.2 77 8 19 59 Female RiHT: post-radioiodine 35.56 5.9 25 2 20 32 Male PyHT: autoimmune thyroiditis 87.78 3.2 56 10 View Large Table 1. Clinical and Laboratory Features of Patients With HT Patient No. Age (y) Sex Cause of HT TSH (mIU/L) (0.39–4.2 mIU/L) FT4 (pmol/L) ( 12 – 22 pmol/L ) Anti-TPOab (0–34) TSQ (/12) 1 50 Male PyHT: autoimmune thyroiditis 108.70 3.3 150 8 2 54 Male RiHT: post-radioiodine 77.77 4.2 9 3 3 74 Male RiHT: post-radioiodine 80.70 3.0 10 4 4 38 Female PyHT: autoimmune thyroiditis 150.00 3.9 77 12 5 51 Female PyHT: autoimmune thyroiditis 67.90 5.5 93 8 6 32 Female PyHT: autoimmune thyroiditis 32.50 7.1 178 8 7 63 Female PyHT: autoimmune thyroiditis 89.00 4.7 56 12 8 79 Female PyHT: autoimmune thyroiditis 113.70 3.1 81 9 9 30 Female PyHT: autoimmune thyroiditis 37.78 6.1 71 6 10 56 Male RiHT: post-radioiodine 74.50 3.0 56 3 11 46 Female RiHT: post-radioiodine 79.90 3.8 34 9 12 45 Female PyHT: autoimmune thyroiditis 98.87 6.7 76 7 13 45 Female PyHT: autoimmune thyroiditis 65.54 5.6 55 10 14 31 Male PyHT: autoimmune thyroiditis 75.12 5.4 85 7 15 48 Male PyHT: autoimmune thyroiditis 150 3.0 122 12 16 49 Female PyHT: autoimmune thyroiditis 78.77 4.4 178 8 17 54 Female PyHT: autoimmune thyroiditis 85.5 4.1 25 6 18 55 Male PyHT: autoimmune thyroiditis 79.9 3.2 77 8 19 59 Female RiHT: post-radioiodine 35.56 5.9 25 2 20 32 Male PyHT: autoimmune thyroiditis 87.78 3.2 56 10 Patient No. Age (y) Sex Cause of HT TSH (mIU/L) (0.39–4.2 mIU/L) FT4 (pmol/L) ( 12 – 22 pmol/L ) Anti-TPOab (0–34) TSQ (/12) 1 50 Male PyHT: autoimmune thyroiditis 108.70 3.3 150 8 2 54 Male RiHT: post-radioiodine 77.77 4.2 9 3 3 74 Male RiHT: post-radioiodine 80.70 3.0 10 4 4 38 Female PyHT: autoimmune thyroiditis 150.00 3.9 77 12 5 51 Female PyHT: autoimmune thyroiditis 67.90 5.5 93 8 6 32 Female PyHT: autoimmune thyroiditis 32.50 7.1 178 8 7 63 Female PyHT: autoimmune thyroiditis 89.00 4.7 56 12 8 79 Female PyHT: autoimmune thyroiditis 113.70 3.1 81 9 9 30 Female PyHT: autoimmune thyroiditis 37.78 6.1 71 6 10 56 Male RiHT: post-radioiodine 74.50 3.0 56 3 11 46 Female RiHT: post-radioiodine 79.90 3.8 34 9 12 45 Female PyHT: autoimmune thyroiditis 98.87 6.7 76 7 13 45 Female PyHT: autoimmune thyroiditis 65.54 5.6 55 10 14 31 Male PyHT: autoimmune thyroiditis 75.12 5.4 85 7 15 48 Male PyHT: autoimmune thyroiditis 150 3.0 122 12 16 49 Female PyHT: autoimmune thyroiditis 78.77 4.4 178 8 17 54 Female PyHT: autoimmune thyroiditis 85.5 4.1 25 6 18 55 Male PyHT: autoimmune thyroiditis 79.9 3.2 77 8 19 59 Female RiHT: post-radioiodine 35.56 5.9 25 2 20 32 Male PyHT: autoimmune thyroiditis 87.78 3.2 56 10 View Large Table 2 shows the clinical characteristics of and comparisons between the HT and HC groups at baseline and at the end of the study period. Both groups were matched for age, sex, and body mass index (P ≥ 0.05). Systolic blood pressure was modestly higher in the HT group (P = 0.04) but lipid profiles did not differ (P ≥ 0.05). As expected, the TSH, FT4, anti-TPOab titers, and TSQ score were significantly abnormal in the HT group (P ≤ 0.0001). Both measures of SFN—LDIFLARE (P ≤ 0.0001) and CCM (P ≤ 0.002 for all three parameters)—were significantly lower in patients with HT than in HCs (Fig. 1). None of the three measures of large-fiber neuropathy—SNCV, SNAP, and vibration perception threshold (VPT)—differed between the groups (P = >0.05). Table 2. Baseline Clinical and Neurologic Outcomes For Patients With HT and HCs Baseline Characteristics Baseline a End of Study b HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups Age, y 44.95 ± 14.29 49.55 ± 13.34 NS 45.95 ± 14.29 50.87 ± 13.01 NS Men/women, n/n 8/12 9/11 NS 8/12 9/11 NS BMI, kg/m2 28.96 ± 2.69 28.09 ± 3.04 NS 29.01 ± 2.09 30.02 ± 3.99 NS Mean BP, mm Hg 114.87 ± 10.45 128.65 ± 9.56 0.04 118.11 ± 9.89 122.45 ± 14.21 NS Triglycerides, mmol/L 1.74 ± 0.54 1.86 ± 0.94 NS 1.89 ± 0.60 2.01 ± 1.10 NS Total cholesterol, mmol/L 4.06 ± 1.21 4.31 ± 0.88 NS 4.19 ± 1.45 4.29 ± 0.97 NS TSH, mIU/L 2.18 ± 0.72 83.47 ± 31.18 <0.0001 2.51 ± 0.98 3.01 ± 0.58 0.03 FT4, pmol/L 16.83 ± 2.31 4.46 ± 1.34 <0.0001 16.17 ± 2.45 18.91 ± 4.01 NS anti-TPOab, IU/L 8.35 ± 4.43 75.70 ± 49.40 <0.0001 8.35 ± 4.43 40.23 ± 12.33 <0.0001 TSQ score 1 ± 0.92 7.6 ± 0.54 <0.0001 0.89 ± 1.11 3.67 ± 3.54 0.001 LDIFLARE, cm2 8.90 ± 1.75 6.74 ± 1.20 <0.0001 8.85 ± 1.83 8.45 ± 0.60 0.01 CNFD, no./mm2 58.32 ± 6.54 50.77 ± 6.54 0.002 58.32 ± 6.54 54.43 ± 5.70 0.02 CNBD, no./mm2 38.30 ± 5.93 29.55 ± 6.90 0.001 38.30 ± 5.93 32.81 ± 7.91 0.02 CNFL, mm/mm2 14.24 ± 2.50 10.11 ± 2.33 0.002 14.24 ± 2.50 13.43 ± 2.25 NS SNCV, m/s 50.45 ± 4.66 47.60 ± 5.88 NS 51.19 ± 4.45 49.29 ± 6.89 NS SNAP, µV 19.45 ± 3.30 15.80 ± 5.81 NS 18.01 ± 3.29 16.89 ± 5.01 NS VPT, mV 5.43 ± 1.56 6.65 ± 2.62 NS 5.55 ± 1.67 5.89 ± 1.91 NS Baseline Characteristics Baseline a End of Study b HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups Age, y 44.95 ± 14.29 49.55 ± 13.34 NS 45.95 ± 14.29 50.87 ± 13.01 NS Men/women, n/n 8/12 9/11 NS 8/12 9/11 NS BMI, kg/m2 28.96 ± 2.69 28.09 ± 3.04 NS 29.01 ± 2.09 30.02 ± 3.99 NS Mean BP, mm Hg 114.87 ± 10.45 128.65 ± 9.56 0.04 118.11 ± 9.89 122.45 ± 14.21 NS Triglycerides, mmol/L 1.74 ± 0.54 1.86 ± 0.94 NS 1.89 ± 0.60 2.01 ± 1.10 NS Total cholesterol, mmol/L 4.06 ± 1.21 4.31 ± 0.88 NS 4.19 ± 1.45 4.29 ± 0.97 NS TSH, mIU/L 2.18 ± 0.72 83.47 ± 31.18 <0.0001 2.51 ± 0.98 3.01 ± 0.58 0.03 FT4, pmol/L 16.83 ± 2.31 4.46 ± 1.34 <0.0001 16.17 ± 2.45 18.91 ± 4.01 NS anti-TPOab, IU/L 8.35 ± 4.43 75.70 ± 49.40 <0.0001 8.35 ± 4.43 40.23 ± 12.33 <0.0001 TSQ score 1 ± 0.92 7.6 ± 0.54 <0.0001 0.89 ± 1.11 3.67 ± 3.54 0.001 LDIFLARE, cm2 8.90 ± 1.75 6.74 ± 1.20 <0.0001 8.85 ± 1.83 8.45 ± 0.60 0.01 CNFD, no./mm2 58.32 ± 6.54 50.77 ± 6.54 0.002 58.32 ± 6.54 54.43 ± 5.70 0.02 CNBD, no./mm2 38.30 ± 5.93 29.55 ± 6.90 0.001 38.30 ± 5.93 32.81 ± 7.91 0.02 CNFL, mm/mm2 14.24 ± 2.50 10.11 ± 2.33 0.002 14.24 ± 2.50 13.43 ± 2.25 NS SNCV, m/s 50.45 ± 4.66 47.60 ± 5.88 NS 51.19 ± 4.45 49.29 ± 6.89 NS SNAP, µV 19.45 ± 3.30 15.80 ± 5.81 NS 18.01 ± 3.29 16.89 ± 5.01 NS VPT, mV 5.43 ± 1.56 6.65 ± 2.62 NS 5.55 ± 1.67 5.89 ± 1.91 NS Unless otherwise noted, values are expressed as mean/median ± SD. Abbreviations: BMI, body mass index; NS, not significant (level of significance at 5%). a In HCs and before LT4 in patients with HT. b At 1 y in HCs and after ≥6 months of stable LT4 replacement in patients with HT. View Large Table 2. Baseline Clinical and Neurologic Outcomes For Patients With HT and HCs Baseline Characteristics Baseline a End of Study b HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups Age, y 44.95 ± 14.29 49.55 ± 13.34 NS 45.95 ± 14.29 50.87 ± 13.01 NS Men/women, n/n 8/12 9/11 NS 8/12 9/11 NS BMI, kg/m2 28.96 ± 2.69 28.09 ± 3.04 NS 29.01 ± 2.09 30.02 ± 3.99 NS Mean BP, mm Hg 114.87 ± 10.45 128.65 ± 9.56 0.04 118.11 ± 9.89 122.45 ± 14.21 NS Triglycerides, mmol/L 1.74 ± 0.54 1.86 ± 0.94 NS 1.89 ± 0.60 2.01 ± 1.10 NS Total cholesterol, mmol/L 4.06 ± 1.21 4.31 ± 0.88 NS 4.19 ± 1.45 4.29 ± 0.97 NS TSH, mIU/L 2.18 ± 0.72 83.47 ± 31.18 <0.0001 2.51 ± 0.98 3.01 ± 0.58 0.03 FT4, pmol/L 16.83 ± 2.31 4.46 ± 1.34 <0.0001 16.17 ± 2.45 18.91 ± 4.01 NS anti-TPOab, IU/L 8.35 ± 4.43 75.70 ± 49.40 <0.0001 8.35 ± 4.43 40.23 ± 12.33 <0.0001 TSQ score 1 ± 0.92 7.6 ± 0.54 <0.0001 0.89 ± 1.11 3.67 ± 3.54 0.001 LDIFLARE, cm2 8.90 ± 1.75 6.74 ± 1.20 <0.0001 8.85 ± 1.83 8.45 ± 0.60 0.01 CNFD, no./mm2 58.32 ± 6.54 50.77 ± 6.54 0.002 58.32 ± 6.54 54.43 ± 5.70 0.02 CNBD, no./mm2 38.30 ± 5.93 29.55 ± 6.90 0.001 38.30 ± 5.93 32.81 ± 7.91 0.02 CNFL, mm/mm2 14.24 ± 2.50 10.11 ± 2.33 0.002 14.24 ± 2.50 13.43 ± 2.25 NS SNCV, m/s 50.45 ± 4.66 47.60 ± 5.88 NS 51.19 ± 4.45 49.29 ± 6.89 NS SNAP, µV 19.45 ± 3.30 15.80 ± 5.81 NS 18.01 ± 3.29 16.89 ± 5.01 NS VPT, mV 5.43 ± 1.56 6.65 ± 2.62 NS 5.55 ± 1.67 5.89 ± 1.91 NS Baseline Characteristics Baseline a End of Study b HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups Age, y 44.95 ± 14.29 49.55 ± 13.34 NS 45.95 ± 14.29 50.87 ± 13.01 NS Men/women, n/n 8/12 9/11 NS 8/12 9/11 NS BMI, kg/m2 28.96 ± 2.69 28.09 ± 3.04 NS 29.01 ± 2.09 30.02 ± 3.99 NS Mean BP, mm Hg 114.87 ± 10.45 128.65 ± 9.56 0.04 118.11 ± 9.89 122.45 ± 14.21 NS Triglycerides, mmol/L 1.74 ± 0.54 1.86 ± 0.94 NS 1.89 ± 0.60 2.01 ± 1.10 NS Total cholesterol, mmol/L 4.06 ± 1.21 4.31 ± 0.88 NS 4.19 ± 1.45 4.29 ± 0.97 NS TSH, mIU/L 2.18 ± 0.72 83.47 ± 31.18 <0.0001 2.51 ± 0.98 3.01 ± 0.58 0.03 FT4, pmol/L 16.83 ± 2.31 4.46 ± 1.34 <0.0001 16.17 ± 2.45 18.91 ± 4.01 NS anti-TPOab, IU/L 8.35 ± 4.43 75.70 ± 49.40 <0.0001 8.35 ± 4.43 40.23 ± 12.33 <0.0001 TSQ score 1 ± 0.92 7.6 ± 0.54 <0.0001 0.89 ± 1.11 3.67 ± 3.54 0.001 LDIFLARE, cm2 8.90 ± 1.75 6.74 ± 1.20 <0.0001 8.85 ± 1.83 8.45 ± 0.60 0.01 CNFD, no./mm2 58.32 ± 6.54 50.77 ± 6.54 0.002 58.32 ± 6.54 54.43 ± 5.70 0.02 CNBD, no./mm2 38.30 ± 5.93 29.55 ± 6.90 0.001 38.30 ± 5.93 32.81 ± 7.91 0.02 CNFL, mm/mm2 14.24 ± 2.50 10.11 ± 2.33 0.002 14.24 ± 2.50 13.43 ± 2.25 NS SNCV, m/s 50.45 ± 4.66 47.60 ± 5.88 NS 51.19 ± 4.45 49.29 ± 6.89 NS SNAP, µV 19.45 ± 3.30 15.80 ± 5.81 NS 18.01 ± 3.29 16.89 ± 5.01 NS VPT, mV 5.43 ± 1.56 6.65 ± 2.62 NS 5.55 ± 1.67 5.89 ± 1.91 NS Unless otherwise noted, values are expressed as mean/median ± SD. Abbreviations: BMI, body mass index; NS, not significant (level of significance at 5%). a In HCs and before LT4 in patients with HT. b At 1 y in HCs and after ≥6 months of stable LT4 replacement in patients with HT. View Large Baseline correlates of TSH with small- and large-fiber indices The baseline TSH of all study participants was compared with the initial small- and large-fiber neural indices. Figure 3 demonstrates a significant inverse correlation between baseline TSH and LDIFLARE (r = −0.719; P ≤ 0.0001), CNFD (r = −0.492; P = 0.008), and CNFL (r = −0.382; P = 0.01) but not with CNBD (r = 0.044; P = 0.41). In comparison, none of the large-fiber parameters—SNCV (P = 0.09), SNAP (P = 0.06), and VPT (P = 0.11)—showed any significant correlation with baseline TSH. In HCs, neither small- nor large-fiber parameters demonstrated any significant correlations (P ≥ 0.05) with baseline TSH. There was also a significant correlation between baseline TSH and TSQ scores (r = 0.70; P = 0.008). Comparison of FT4 levels with both large- and small-fiber neurologic parameters revealed no significant correlation (P ≥ 0.05). Figure 3. View largeDownload slide Correlation between baseline TSH and indices of small nerve fibers (LDIFLARE and CCM). ●, correlation of baseline TSH with LDIFLARE; ■, correlation of baseline TSH with CNFD; ♦, correlation of baseline TSH with CNBD; ▬, correlation of baseline TSH with CNFL. Figure 3. View largeDownload slide Correlation between baseline TSH and indices of small nerve fibers (LDIFLARE and CCM). ●, correlation of baseline TSH with LDIFLARE; ■, correlation of baseline TSH with CNFD; ♦, correlation of baseline TSH with CNBD; ▬, correlation of baseline TSH with CNFL. After the initial assessment, all patients with HT began receiving LT4 monotherapy, with doses titrated per 2014 guidelines suggested by the American Thyroid Association (26). After the target TSH was achieved and remained stable for ≥6 months, the patients with HT were reassessed again for neurophysiological changes. Differences in characteristics at study end Table 2 and Fig. 4 depict the changes in neurophysiological indices before and after treatment with LT4. After successful normalization of TSH levels, all small-fiber modalities significantly improved compared with baseline values: LDIFLARE (8.45 ± 0.60 vs 6.74 ± 1.20 cm2; P ≤ 0.0001), CNFD (54.43 ± 5.70 vs 50.77 ± 6.54 no./mm2; P = 0.008), CNBD (32.81 ± 7.91 vs 29.55 ± 6.90 no./mm2; P = 0.002), and CNFL (13.43 ± 2.25 vs 10.11 ± 2.33 mm/mm2; P = 0.01). In contrast, none of the large-fiber modalities—SNCV (P = 0.21), SNAP (P = 0.26), and VPT (P = 0.53)—showed any significant change. The inverse correlations between TSH and small-fiber indices (LDIFLARE, CNFD, and CNFL) observed in the pretreatment period were not seen at the end of the study period (P ≥ 0.05) Figure 4. View largeDownload slide Box plots (with 95% CIs) indicate change of both small- and large-fiber neural outcomes after treatment with LT4. Level of significance indicated with each pair. Figure 4. View largeDownload slide Box plots (with 95% CIs) indicate change of both small- and large-fiber neural outcomes after treatment with LT4. Level of significance indicated with each pair. As shown in Fig. 5, all HCs were reassessed after 1 year and their results were compared with the final outcomes of the HT group. Despite achievement of stable and normal TSH levels in the latter, when compared with HCs, the small neural indices remained significantly lower. The LDIFLARE in the HC group, 8.81 ± 0.99 cm2, remained significantly higher than the value in the HT group. 8.45 ± 0.60 cm2 (P = 0.02). A similar significant difference was observed with CNFD (56.67 ± 6.12 vs 54.43 ± 5.70 no./mm2; P = 0.04) and CNBD (35.86 ± 7.17 vs 32.81 ± 7.91 no./mm2; P = 0.02) but not with CNFL (13.71 ± 3.563 vs 13.43 ± 2.25 mm/mm2; P = 0.05), respectively. None of the large-fiber parameters significantly differed between the HC and HT groups at the end of the study. Finally, no significant differences in baseline or follow-up outcomes were observed between the PyHT and RiHT groups at any level. Figure 5. View largeDownload slide Box plots (with 95% CIs) compare both small (LDIFLARE and CNFD, CNBD, CNFL) and large (SNCV, SNAP, VPT) neural outcomes between HCs and patients with HT at the end of study assessment. Level of significance indicated with each pair. Figure 5. View largeDownload slide Box plots (with 95% CIs) compare both small (LDIFLARE and CNFD, CNBD, CNFL) and large (SNCV, SNAP, VPT) neural outcomes between HCs and patients with HT at the end of study assessment. Level of significance indicated with each pair. Discussion Neuromuscular dysfunction is common in patients with HT and has been historically associated with sensorimotor polyneuropathy and other peripheral neuropathies (1, 27, 28). Previous studies have inconsistently reported the prevalence of such polyneuropathy in hypothyroid patients to vary from 42% to 72% (1, 7). However, these studies, using predominantly markers of large-fiber function, were in symptomatic patients with long-standing hypothyroidism with a wide distribution of neurologic involvement ranging from entrapment neuropathy to more diffuse peripheral neuropathy (29). Such major neurologic sequelae are now rare because of earlier diagnosis and earlier replacement with LT4. However, in neurologically asymptomatic patients, there is conflicting evidence of polyneuropathy when large-fiber function is assessed by measurement of nerve conduction; some studies suggest its presence (30) but others have not (13). In this context, recent studies have demonstrated abnormalities in SFS in HT. In a group of 18 asymptomatic patients with subclinical or overt hypothyroid patients, Magri et al. (14) demonstrated significantly reduced IENFD in skin biopsy specimens from the upper thigh and distal leg, leading to the suggestion that “a considerable number of untreated hypothyroid patients may have preclinical asymptomatic small fibre neuropathy.” However, although IENFD has traditionally been considered as the gold standard for investigating small-fiber neuropathy, its invasiveness may limit repetitive study; moreover, the technique still lacks a consensus reference standard (31). Our proof-of-concept study compared large and small neural measures in 20 patients with untreated HT vs 20 age- and sex-matched HCs. All 15 patients with PyHT were referred to hospital services for management of newly diagnosed HT; however, on the basis of symptoms alone, it is not possible to know the duration of their hypothyroidism because they did not have any records of previous thyroid function tests. The remaining five patients with RiHT were diagnosed during routine biochemical surveillance after radioiodine treatment. Because of the above reasons, we could not correlate duration of disease with neurologic dysfunction or with improvement of small-fiber function with either subgroup, PyHT or RiHT. For SFN, we used two methods not previously applied in HT: LDIFLARE for SFF and CCM for SFS. Although all 20 patients with HT were newly presenting with their diagnosis, their mean TSH was relatively high (83.47 ± 31.18 mIU/L; normal range, 0.27 to 4.20mIU/L); this suggests a relatively profound biochemical abnormality. Despite this, at baseline none of the large-fiber modalities (SNCV, SNAP, and VPT) had significantly abnormal results. In contrast, both small-fiber measures (LDIFLARE and CCM) were significantly reduced (P ≤ 0.0001 and P = ≤ 0.002, respectively) in comparison with values in HCs. This supports the hypothesis that SFN in untreated early HT precedes large-fiber neural involvement and adds to Magri and colleagues findings (14) that IEFND may be abnormal in hypothyroid patients in the presence of normal large-fiber function. Furthermore, in addition to demonstrating structural small-fiber deficits using a different but noninvasive method (CCM), our study for the first time demonstrates significant functional abnormality (LDIFLARE) in patients with early HT. We found that the baseline TSH had a significant inverse correlation with both LDIFLARE and CCM (P ≤ 0.0001 and P ≤ 0.01, respectively) but not with large-fiber methods. Our findings contrast with the results of Magri et al. (14), who did not find any significant correlation between TSH and IENFD. This disparity could be explained by the inclusion of patients with subclinical HT in the latter study; that would also explain the wide TSH range in comparison with the current study (77.19 ± 92.80 mIU/L in the study by Magri et al. vs 83.47 ± 31.18 mIU/L in the current study). Previous studies that used large-fiber methods hypothesized that severity of peripheral neuropathy in HT was related to duration of HT but not to the degree of thyroid failure (2, 10). On the basis of our findings, we suggest that these more sensitive and recent methods of SFN assessment not only detect early neural involvement but also relate to severity of deficiency as indicated by serum TSH levels. After establishment of a euthyroid state with LT4 replacement therapy, both small-fiber indices significantly improved (LDIFLARE, P ≤ 0.0001; CCM, P ≤ 0.02), whereas large-fiber function did not change. We are aware of only one other prospective study, again by Magri et al. (15), whose small study of nine patients with HT (six with overt and three with subclinical disease) demonstrated significant reversibility of previously reduced IENFD. The neural improvement detected by the methods used in our study underlines the sensitivity of these methods in contrast to measures of large-fiber function. In addition, unlike IENFD, these methods are noninvasive and thus have the advantage of being repeatable. Of potential interest was the finding that despite normalization of TSH in all patients with HT, the small-fiber indices remained significantly reduced compared with those in the HCs: LDIFLARE (P = 0.02) and CCM (P ≤ 0.05). This may suggest that a longer period of replacement therapy is necessary before small-fiber function and structure return to normal. Restoration of normal neural cellular function may also require the TSH to be in the lower half of the normal range; however, in this study we did not find a difference between patients with TSH levels in the lower quartile vs those with a normal reference range vs those in the upper quartile. A further study of similar design with more patients in whom a euthyroid state is maintained for a longer period would be necessary to determine whether this observation of persistent reduction of small-fiber neural indices is permanent. This study is not without its limitations. Although these patients were newly diagnosed, they may have had long-standing hypothyroidism, which would explain the very high TSH in the group. It would be of interest to study patients with less biochemical severity, including subclinical hypothyroidism. It is important that our findings be confirmed by other studies, in particular because we studied only 20 patients; however, the high level of significance of the baseline findings and the improvements after treatment suggest that a type II error is unlikely. Conclusion This study demonstrates that small-fiber neuropathy precedes large-fiber dysfunction in early HT. This can be improved by achieving a biochemically euthyroid state with LT4 replacement therapy, but even after 6 months of maintaining this euthyroid state, small-fiber neural indices continued to be remain low when compared with values in HCs. Abbreviations: Abbreviations: anti-TPOab antithyroid peroxidase antibodies CCM corneal confocal microscopy CNBD confocal nerve branch density CNFD corneal confocal microscopy nerve fiber density CV coefficient of variation FT4 free T4 HC healthy control HT hypothyroidism IENFD intraepidermal nerve fiber density LDIFLARE laser Doppler imager flare technique LT4 levothyroxine PyHT primary hypertension RiHT radioiodine-induced hypothyroidism SFF small-fiber function SFN small-fiber neuropathy SFS small-fiber structure SNAP sural nerve amplitude SNCV sural nerve conduction velocity TSQ Thyroid Symptom Questionnaire VPT vibration perception threshold Acknowledgments The authors thank the staff at Ipswich hospital and their friends and family members in Suffolk for their contribution by volunteering for this study. Author Contributions: S.S. was responsible for conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, writing – original draft and writing – review & editing. V.T. was responsible for data curation, investigation, methodology, validation and writing – review & editing. P.V. was responsible for conceptualization, methodology and writing – review & editing. G.R. was responsible for conceptualization, formal analysis, funding acquisition, investigation, methodology, resources, software, supervision, validation, visualization and writing – review & editing. Disclosure Summary: The authors have nothing to disclose. References 1. Beghi E , Delodovici ML , Bogliun G , Crespi V , Paleari F , Gamba P , Capra M , Zarrelli M . Hypothyroidism and polyneuropathy . J Neurol Neurosurg Psychiatry . 1989 ; 52 ( 12 ): 1420 – 1423 . Google Scholar CrossRef Search ADS PubMed 2. Nemni R , Bottacchi E , Fazio R , Mamoli A , Corbo M , Camerlingo M , Galardi G , Erenbourg L , Canal N . Polyneuropathy in hypothyroidism: clinical, electrophysiological and morphological findings in four cases . J Neurol Neurosurg Psychiatry . 1987 ; 50 ( 11 ): 1454 – 1460 . Google Scholar CrossRef Search ADS PubMed 3. Lin KP , Kwan SY , Chen SY , Chen SS , Yeung KB , Chia LG , Wu ZA . Generalized neuropathy in Taiwan: an etiologic survey . Neuroepidemiology . 1993 ; 12 ( 5 ): 257 – 261 . Google Scholar CrossRef Search ADS PubMed 4. Rudolph T , Farbu E . Hospital-referred polyneuropathies—causes, prevalences, clinical, and neurophysiological findings . Eur J Neurol 2007 ; 14 ( 6 ): 603 – 608 . Google Scholar CrossRef Search ADS PubMed 5. Leese GP , Flynn RV , Jung RT , Macdonald TM , Murphy MJ , Morris AD . Increasing prevalence and incidence of thyroid disease in Tayside, Scotland: the Thyroid Epidemiology Audit and Research Study (TEARS) . Clin Endocrinol (Oxf) . 2008 ; 68 ( 2 ): 311 – 316 . Google Scholar PubMed 6. Wood-Allum CA , Shaw PJ . Thyroid disease and the nervous system . Handb Clin Neurol . 2014 ; 120 : 703 – 735 . Google Scholar CrossRef Search ADS PubMed 7. Duyff RF , Van den Bosch J , Laman DM , van Loon BJ , Linssen WH . Neuromuscular findings in thyroid dysfunction: a prospective clinical and electrodiagnostic study . J Neurol Neurosurg Psychiatry . 2000 ; 68 ( 6 ): 750 – 755 . Google Scholar CrossRef Search ADS PubMed 8. Pollard J. Neuropathy in diseases of the thyroid and pituitary glands. In: Dyck PJ, Thomas PK, Griffin JW, Low PA, Poduslo JF, ed. Peripheral Neuropathy. 3rd ed. Philadelphia, PA: Saunders Company; 1993:1266–1274. 9. Shinoda K , Takamatsu J , Mozai T . Peripheral neuropathy in hypothyroidism. I. Clinical and electrophysiological study . Bull Osaka Med Sch . 1987 ; 33 ( 2 ): 137 – 148 . Google Scholar PubMed 10. Somay G , Oflazoğlu B , Us O , Surardamar A . Neuromuscular status of thyroid diseases: a prospective clinical and electrodiagnostic study . Electromyogr Clin Neurophysiol . 2007 ; 47 ( 2 ): 67 – 78 . Google Scholar PubMed 11. Quattrini C , Tavakoli M , Jeziorska M , Kallinikos P , Tesfaye S , Finnigan J , Marshall A , Boulton AJ , Efron N , Malik RA . Surrogate markers of small fiber damage in human diabetic neuropathy . Diabetes . 2007 ; 56 ( 8 ): 2148 – 2154 . Google Scholar CrossRef Search ADS PubMed 12. Sharma S , Venkitaraman R , Vas PR , Rayman G . Assessment of chemotherapy-induced peripheral neuropathy using the LDIFLARE technique: a novel technique to detect neural small fiber dysfunction . Brain Behav . 2015 ; 5 ( 7 ): e00354 . Google Scholar CrossRef Search ADS PubMed 13. Ørstavik K , Norheim I , Jørum E . Pain and small-fiber neuropathy in patients with hypothyroidism . Neurology . 2006 ; 67 ( 5 ): 786 – 791 . Google Scholar CrossRef Search ADS PubMed 14. Magri F , Buonocore M , Oliviero A , Rotondi M , Gatti A , Accornero S , Camera A , Chiovato L . Intraepidermal nerve fiber density reduction as a marker of preclinical asymptomatic small-fiber sensory neuropathy in hypothyroid patients . Eur J Endocrinol 2010 ; 163 ( 2 ): 279 – 284 . Google Scholar CrossRef Search ADS PubMed 15. Magri F , Buonocore M , Camera A , Capelli V , Oliviero A , Rotondi M , Gatti A , Chiovato L . Improvement of intra-epidermal nerve fibre density in hypothyroidism after L-thyroxine therapy . Clin Endocrinol (Oxf) . 2013 ; 78 ( 1 ): 152 – 153 . Google Scholar CrossRef Search ADS PubMed 16. American Diabetes Association . 2. Classification and diagnosis of diabetes: Standards of Medical Care in Diabetes-2018 . Diabetes Care . 2018 ; 41 ( Suppl 1 ): S13 – S27 . CrossRef Search ADS PubMed 17. Saravanan P , Chau WF , Roberts N , Vedhara K , Greenwood R , Dayan CM . Psychological well-being in patients on ‘adequate’ doses of l-thyroxine: results of a large, controlled community-based questionnaire study . Clin Endocrinol (Oxf) . 2002 ; 57 ( 5 ): 577 – 585 . Google Scholar CrossRef Search ADS PubMed 18. Boulton A . Management of diabetic peripheral neuropathy . Clin Diabetes . 2005 ; 23 ( 1 ): 9 – 15 . Google Scholar CrossRef Search ADS 19. Vas PR , Rayman G . Validation of the modified LDIFlare technique: a simple and quick method to assess C-fiber function . Muscle Nerve . 2013 ; 47 ( 3 ): 351 – 356 . Google Scholar CrossRef Search ADS PubMed 20. Krishnan ST , Rayman G . The LDIflare: a novel test of C-fiber function demonstrates early neuropathy in type 2 diabetes . Diabetes Care . 2004 ; 27 ( 12 ): 2930 – 2935 . Google Scholar CrossRef Search ADS PubMed 21. Green AQ , Krishnan S , Finucane FM , Rayman G . Altered C-fiber function as an indicator of early peripheral neuropathy in individuals with impaired glucose tolerance . Diabetes Care . 2010 ; 33 ( 1 ): 174 – 176 . Google Scholar CrossRef Search ADS PubMed 22. Krishnan ST , Quattrini C , Jeziorska M , Malik RA , Rayman G . Abnormal LDIflare but normal quantitative sensory testing and dermal nerve fiber density in patients with painful diabetic neuropathy . Diabetes Care . 2009 ; 32 ( 3 ): 451 – 455 . Google Scholar CrossRef Search ADS PubMed 23. Petropoulos IN , Manzoor T , Morgan P , Fadavi H , Asghar O , Alam U , Ponirakis G , Dabbah MA , Chen X , Graham J , Tavakoli M , Malik RA . Repeatability of in vivo corneal confocal microscopy to quantify corneal nerve morphology . Cornea . 2013 ; 32 ( 5 ): e83 – e89 . Google Scholar CrossRef Search ADS PubMed 24. Tavakoli M , Malik RA . Corneal confocal microscopy: a novel non-invasive technique to quantify small fibre pathology in peripheral neuropathies . J Vis Exp . 2011 ;( 47 ): 2194 . 25. Dabbah MA , Graham J , Petropoulos IN , Tavakoli M , Malik RA . Automatic analysis of diabetic peripheral neuropathy using multi-scale quantitative morphology of nerve fibres in corneal confocal microscopy imaging . Med Image Anal . 2011 ; 15 ( 5 ): 738 – 747 . Google Scholar CrossRef Search ADS PubMed 26. Jonklaas J , Bianco AC , Bauer AJ , Burman KD , Cappola AR , Celi FS , Cooper DS , Kim BW , Peeters RP , Rosenthal MS , Sawka AM , American Thyroid Association Task Force on Thyroid Hormone R. Guidelines for the treatment of hypothyroidism: prepared by the american thyroid association task force on thyroid hormone replacement . Thyroid: Official Journal of the American Thyroid Association 2014 ; 24 : 1670 – 1751 . Google Scholar CrossRef Search ADS PubMed 27. Crevasse LE , Logue RB . Peripheral neuropathy in myxedema . Ann Intern Med . 1959 ; 50 ( 6 ): 1433 – 1437 . Google Scholar CrossRef Search ADS PubMed 28. Dyck PJ , Lambert EH . Polyneuropathy associated with hypothyroidism . J Neuropathol Exp Neurol . 1970 ; 29 ( 4 ): 631 – 658 . Google Scholar CrossRef Search ADS PubMed 29. Khedr EM , El Toony LF , Tarkhan MN , Abdella G . Peripheral and central nervous system alterations in hypothyroidism: electrophysiological findings . Neuropsychobiology . 2000 ; 41 ( 2 ): 88 – 94 . Google Scholar CrossRef Search ADS PubMed 30. El-Salem K , Ammari F . Neurophysiological changes in neurologically asymptomatic hypothyroid patients: a prospective cohort study . J Clin Neurophysiol 2006 ; 23 ( 6 ): 568 – 572 . Google Scholar CrossRef Search ADS PubMed 31. Hlubocky A , Wellik K , Ross MA , Smith BE , Hoffman-Snyder C , Demaerschalk BM , Wingerchuk DM . Skin biopsy for diagnosis of small fiber neuropathy: a critically appraised topic . Neurologist . 2010 ; 16 ( 1 ): 61 – 63 . Google Scholar CrossRef Search ADS PubMed Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

The LDIFLARE and CCM Methods Demonstrate Early Nerve Fiber Abnormalities in Untreated Hypothyroidism: A Prospective Study

Loading next page...
 
/lp/ou_press/the-ldiflare-and-ccm-methods-demonstrate-early-nerve-fibre-FN8GwfVeGB
Publisher
Endocrine Society
Copyright
Copyright © 2018 Endocrine Society
ISSN
0021-972X
eISSN
1945-7197
D.O.I.
10.1210/jc.2018-00671
Publisher site
See Article on Publisher Site

Abstract

Abstract Context Recent studies using skin biopsy suggest presence of small-fiber neuropathy in subclinical hypothyroidism. This study uses two noninvasive methods—the laser Doppler imager flare technique (LDIFLARE) and corneal confocal microscopy (CCM)—to assess small-fiber function (SFF) and small-fiber structure (SFS), respectively, in newly diagnosed hypothyroidism (HT) before and after adequate treatment. Design and Setting Single-center, prospective, intervention-based cohort study. Patients and Participants Twenty patients with newly diagnosed HT (15 with primary HT and 5 with post-radioiodine HT) along with 20 age-matched healthy controls (HCs). Interventions Patients with HT and HCs were assessed neurologically at diagnosis and baseline, respectively. The HT group was reassessed after optimal replacement (defined as TSH level of 0.27 to 4.20 mIU/L) with levothyroxine (LT4) and HCs were reviewed after 1 year. Main Outcome Measures Neurologic assessment for small fibers was performed by using LDIFLARE for SFF and CCM for SFS; large fibers were studied by sural nerve conduction velocity (SNCV) and sural nerve amplitude (SNAP). Results At baseline, both LDIFLARE (mean ± SD) (6.74 ± 1.20 vs 8.90 ± 1.75 cm2; P = 0.0002) and CCM nerve fiber density (CNFD) (expressed as number of fibers per mm2: 50.77 ± 6.54 vs 58.32 ± 6.54; P = 0.002) were significantly reduced in the HT group compared with HCs whereas neither SNCV nor SNAP was different (P ≥ 0.05). After optimal LT4 treatment, both LDIFLARE (7.72 ± 1.12 vs 6.74 ± 1.20 cm2; P ≤ 0.0001) and CNFD (54.43 ± 5.70 vs 50.77 ± 6.54 no./mm2; P = 0.02) improved significantly but remained significantly reduced compared to HCs (P = 0.008 and P = 0.01, respectively) despite normalization of TSH. Conclusions This study demonstrates that dysfunction of small fibers precedes large neural fiber abnormalities in early HT. This can be reversed by replacement therapy to achieve a biochemically euthyroid state, but small-fiber neural outcomes continued to remain low compared with values in HCs. The presence of neuromuscular dysfunction in hypothyroidism (HT) is well recognized, with a prevalence ranging between 42% and 72% (1, 2). Epidemiological neurologic studies have reported HT as a cause in 2% to 5% of cases of polyneuropathy (3–5). Peripheral neuropathy, such as diffuse sensorimotor neuropathy involving myelinated large nerve fibers like Aα and Aβ, have been commonly described; symptoms are usually mild and predominantly sensory (6). Beghi et al. (1) studied 39 consecutive patients with hypothyroidism and detected sensory symptoms in 64%, of whom 72% had neurophysiological evidence and only 33% had clinical signs. Similarly, in another case series comprising of 24 patients with newly diagnosed HT, Duyff et al. (7) reported clinical signs of predominantly sensory peripheral neuropathy in only 42% of patients. Clinical signs in HT are usually subtle and nonspecific, with reduced knee and ankle reflexes along with mild impairment of distal vibration and joint position sense (6). The pathological changes described include axonal degeneration, segmental demyelination, and deposition of mucopolysaccharides in the endoneurial interstitium (2, 8, 9). Such changes have been described to be more related to the duration of HT than to the severity of thyroid failure (10). In many neuropathies, including diabetes and chemotherapy-induced peripheral neuropathy, small-fiber neural involvement affecting the thinly myelinated Aδ and nonmyelinated C fibers has been shown to precede large-fiber involvement (11, 12). However, in HT, limited evidence suggests early small-fiber neural involvement, especially in asymptomatic individuals. In a series of 38 hypothyroid patients, Ørstavik et al. (13) demonstrated significantly abnormal thermal thresholds as compared with controls. In a smaller study of 18 neurologically asymptomatic patients with newly diagnosed overt or subclinical HT, Magri et al. (14) found significantly reduced intraepidermal nerve fiber density (IENFD) in their distal leg skin biopsy specimens but no evidence of large-fiber dysfunction assessed by conventional neurophysiological tests. Subsequently, the same authors showed that such impairment of IENFD was reversible after levothyroxine (LT4) treatment in nine patients (15). With the exception of these studies, we are unaware of any other studies specifically assessing other modalities of small-fiber involv-ement in patients with HT. In this prospective study, we used two noninvasive methods — the laser Doppler imager flare technique (LDIFLARE) and corneal confocal microscopy (CCM) — to assess small-fiber function (SFF) and structure (SFS), respectively, in patients with newly diagnosed HT compared with a cohort of age- and sex-matched healthy controls (HCs), as well as the change before and after adequate monotherapy with LT4. Neither SFF nor SFS has been studied together in HT, and hence this study can be considered as a proof-of-concept study investigating early neuropathic changes in HT. Patients and Methods Ethical approval This study was conducted between 2014 and 2017 in accordance with the Declaration of Helsinki and was approved by the ethics committee of the National Research Ethics Service Committee East of England, Norfolk, United Kingdom (Research Ethics Committee reference: 13/EE/0162). All participants provided written informed consent. Study design and participant selection Twenty patients with newly diagnosed HT along with 20 age- and sex-matched HCs were recruited, the latter by invitation from the local population. HT was diagnosed by a TSH level of ≥10 IU/mL with an abnormally low free T4 (FT4) level (see below). Of the 20 patients with HT, 15 had primary HT (PyHT) and 5 had radioiodine-induced HT (RiHT) occurring at least 6 weeks after administration of radioiodine treatment. We included both types of HT to determine whether the neurologic profile would differ given the difference in cause and duration of the HT. None of the patients with RiHT had any neurologic symptoms before administration of radioiodine. Patients who had a history of HT and were receiving thyroid supplementation at any stage in their life were excluded because we also aimed to look at the effects of LT4 supplementation on neurologic parameters. Patients with a history of impaired fasting glycemia, prediabetes, or diabetes (type 1, type 2, or other specific types) as per the 2018 diagnostic criteria of the American Diabetes Association (16) were excluded on the basis of a fasting blood glucose and glycosylated hemoglobin A1c. Patients were also excluded if they had any history of peripheral arterial disease, hypertriglyceridemia, alcohol abuse, vitamin B12 and folate deficiency, thyroid or connective tissue disorders, renal or hepatic failure, malignancy, inherited neuropathy, or exposure to any toxins (including chemotherapy). Clinical and biochemical assessment All participants underwent baseline biochemical assessment for thyroid fasting glucose, glycosylated hemoglobin A1c, lipids, renal and hepatic profiles, vitamin B12, and folate. Thyroid biochemistry comprised TSH, FT4, and antithyroid peroxidase antibodies (anti-TPOab). Serum concentrations of TSH (noncompetitive sandwich immunoassay; normal range, 0.27 to 4.20 mIU/L) and FT4 (competitive immunoassay; normal range, 12 to 22 pmol/L), and anti-TPOab (normal range, 0 to 34 U/mL) were measured with electrochemiluminescent detection using an automated analyzer (Cobas e602 immunochemical analyzer) employing commercial kits (Roche Diagnostics Ltd, Bugess Hill, UK). The TSH assay has a functional sensitivity of 0.014 mIU/L and limit of detection of 0.005 mIU/L, with interassay coefficients of variation (CVs) of 2.1% and 2.0% at concentrations of 1.2 mIU/L and 7.9 mIU/L, respectively. The FT4 assay has a functional sensitivity of 3.0 pmol/L and limit of detection of 0.5 pmol/L, with interassay CVs of 2.1% and 2.8% at concentrations of 15.4 pmol/L and 39.8 pmol/L, respectively. The anti-TPOab assay has an interassay CV of 8% at 34 U/mL and 5% at 92 U/mL, the limit of detection is 5 U/mL, and the measuring range is 5 to 600 U/mL. A total of 32 patients with HT were initially screened, of whom 8 were excluded because of a new diagnosis of prediabetes, diabetes, or vitamin B12 deficiency. A further four patients were lost during follow-up because of a diagnosis of diabetes (two patients), cancer (one patient), and stroke (one patient). All participants, including HCs, completed the Thyroid Symptom Questionnaire (TSQ)—a 12-point questionnaire validated for use in hypothyroidism (17)—at the beginning and end of the study. Both groups also underwent detailed neurologic examination with the modified Neuropathy Disability Score (18). SFF was assessed with LDIFLARE and SFS was measured by CCM. Vibration perception threshold was measured on the toes by using the Neurothesiometer (Horwell, Scientific Laboratory Supplies, Wilford, Nottingham, UK) and expressed as millivolts (mV). Sural nerve conduction velocity (SNCV) and sural nerve amplitude (SNAP) were assessed in one leg (NC-stat|DPNCheck system; Neurometrix, Waltham, MA). The neurologic assessments were repeated for both groups at the end of the study. LDIFLARE method SFF was assessed by using the noninvasive LDIflare technique as previously published (19). In a thermally controlled room at 30°C, the dorsal foot skin is heated sequentially to 44°C for 2 minutes, 46°C for 1 minute, and finally to 47°C for 3 minutes using a 1-cm2 heating probe. This nociceptive heat results in activation of the C-fiber reflex and the resultant nerve-axon– related hyperemic response is assessed by using a laser scanner (Moor Instruments, Axminster, UK). The size of the hyperemic response is measured in centimeters squared (cm2) by using the Moor V 5.3 software, referred to as the LDIflare (Fig. 1). Flare size relates to SFF; a small flare equates to reduced SFF. The time needed for this assessment is up to 30 minutes. Previous studies have demonstrated that it is a sensitive method to detect C-fiber dysfunction as an early marker of small-fiber neuropathy (SFN) in both type 2 diabetes (20) and impaired glucose tolerance (21). It has significant correlations with IEFND (22) and recently has been shown to be more sensitive than neurophysiological studies to detect SFF in early chemotherapy-induced peripheral neuropathy (12). Figure 1. View largeDownload slide LDIFLARE images. (Left) From a 40-y-old HC, with LDIFLARE at 11.44 cm2. (Right) From a 41-y-old patient with HT. with LDIFLARE at 4.17 cm2. Figure 1. View largeDownload slide LDIFLARE images. (Left) From a 40-y-old HC, with LDIFLARE at 11.44 cm2. (Right) From a 41-y-old patient with HT. with LDIFLARE at 4.17 cm2. CCM method Small-fiber structure was measured by CCM using the laser scanning Retina Tomograph III confocal microscope with Rostock Corneal Module (Heidelberg Engineering GmbH, Dossenheim, Germany) by following an established technique. The total duration of examination of both eyes was 5 to 10 minutes (23, 24). Before scanning, topical anesthesia [oxybuprocaine hydrochloride 0.4% (Minims®); Bausch & Lomb, Kingston upon Thames, Surrey, UK] was applied to the cornea of both eyes, followed by topical application of a viscous gel [carbomer 980 polyacrylic acid 0.2% (Viscotears®); Alcon Eye Care, Frimley, Surrey, UK] to form an aqueous medium between the applanated corneal surface and disposable TomoCap (Heidelberg Engineering Ltd., Hemel Hempstead, UK) covering the objective lens. Several scans of the entire depth of the cornea of both eyes were taken by fine adjustment of the objective lens, resulting in manual acquisition of two-dimensional images with a final image size of 400 × 400 pixels and lateral resolution of 2 mm/pixel of the subbasal nerve plexus of the cornea. Each subbasal nerve fiber bundle contains unmyelinated neural fibers running parallel to the Bowman layer before terminating dividing and terminating as individual axons just underneath the corneal surface epithelium. The six best images from the center of the cornea were manually selected for automated image analysis by using purpose-written, proprietary software (CCM Image Analysis tool v1.1; Imaging Science and Biomedical Engineering, University of Manchester, Manchester, UK) (25). The specific parameters measured per frame were as follows: CCM nerve fiber damage (CNFD; number of fibers/mm2), confocal nerve branch density (CNBD; number of fibers/mm2), and CNFL (length in mm/mm2) (23). Corneal nerve fibers and their branches are shown in Fig. 2. Figure 2. View largeDownload slide CCM images (Left) From a 45-y-old HC, with CNFD at 61.89 no./mm2. (Right) From a 47-y old with HT, with CNFD at 30.12 no./mm2. Red arrows show corneal nerve fibers; yellow arrows show corneal nerve branches. Original magnification ×700. Figure 2. View largeDownload slide CCM images (Left) From a 45-y-old HC, with CNFD at 61.89 no./mm2. (Right) From a 47-y old with HT, with CNFD at 30.12 no./mm2. Red arrows show corneal nerve fibers; yellow arrows show corneal nerve branches. Original magnification ×700. Management of HT All patients with HT were treated with LT4 monotherapy as per 2014 guidelines suggested by the American Thyroid Association (26). The dosage of LT4 was titrated to achieve the TSH reference range of 0.27 to 4.20 mIU/L at 12-weekly intervals. Once a stable TSH was achieved for at least 6 months, the patients with HT thereafter completed the TSQ questionnaire and all the neurologic assessments previously undertaken. Statistical analysis Statistical analysis was performed by using SPSS software, version 20 for Windows (IBM, Armonk, NY) and StatsDirect software, version 3 (StatsDirect Ltd, Cambridge, UK). Analysis included descriptive and frequency statistics. All data are expressed as mean ± SD. Normal distribution of the data was determined with the Kolmogorov-Smirnov test. An independent-sample t test was used to test whether a sample mean differed between the HT and HC groups. A paired sample t test was used to for the comparison between baseline and follow-up data. Nonparametric data were analyzed by using the χ2 test. Pearson bivariate correlation was used to see the relationship between baseline TSH and neurologic assessments. Power was calculated by using the Wilks lambda model and computed by using α = 0.05. Statistical significance was defined at 5% (i.e.,P ≤ 0.05). The CVs for CCM and LDIflare are 7.8% and 8.7%, respectively. Results Differences in baseline characteristics Table 1 shows the clinical characteristics and biochemical assessments of all 20 newly diagnosed patients with HT who were enrolled and completed follow-up in the study. In all 15 patients with PyHT, that condition was due to autoimmune thyroiditis characterized by raised anti-TPOab levels (>34 IU/mL). The remaining five patients had RiHT that developed after radioiodine treatment of Graves thyrotoxicosis (three of five patients) and solitary toxic nodule (two of five patients). None of the patients were receiving any form of thyroid replacement therapy, including LT4, at study entry. Table 1. Clinical and Laboratory Features of Patients With HT Patient No. Age (y) Sex Cause of HT TSH (mIU/L) (0.39–4.2 mIU/L) FT4 (pmol/L) ( 12 – 22 pmol/L ) Anti-TPOab (0–34) TSQ (/12) 1 50 Male PyHT: autoimmune thyroiditis 108.70 3.3 150 8 2 54 Male RiHT: post-radioiodine 77.77 4.2 9 3 3 74 Male RiHT: post-radioiodine 80.70 3.0 10 4 4 38 Female PyHT: autoimmune thyroiditis 150.00 3.9 77 12 5 51 Female PyHT: autoimmune thyroiditis 67.90 5.5 93 8 6 32 Female PyHT: autoimmune thyroiditis 32.50 7.1 178 8 7 63 Female PyHT: autoimmune thyroiditis 89.00 4.7 56 12 8 79 Female PyHT: autoimmune thyroiditis 113.70 3.1 81 9 9 30 Female PyHT: autoimmune thyroiditis 37.78 6.1 71 6 10 56 Male RiHT: post-radioiodine 74.50 3.0 56 3 11 46 Female RiHT: post-radioiodine 79.90 3.8 34 9 12 45 Female PyHT: autoimmune thyroiditis 98.87 6.7 76 7 13 45 Female PyHT: autoimmune thyroiditis 65.54 5.6 55 10 14 31 Male PyHT: autoimmune thyroiditis 75.12 5.4 85 7 15 48 Male PyHT: autoimmune thyroiditis 150 3.0 122 12 16 49 Female PyHT: autoimmune thyroiditis 78.77 4.4 178 8 17 54 Female PyHT: autoimmune thyroiditis 85.5 4.1 25 6 18 55 Male PyHT: autoimmune thyroiditis 79.9 3.2 77 8 19 59 Female RiHT: post-radioiodine 35.56 5.9 25 2 20 32 Male PyHT: autoimmune thyroiditis 87.78 3.2 56 10 Patient No. Age (y) Sex Cause of HT TSH (mIU/L) (0.39–4.2 mIU/L) FT4 (pmol/L) ( 12 – 22 pmol/L ) Anti-TPOab (0–34) TSQ (/12) 1 50 Male PyHT: autoimmune thyroiditis 108.70 3.3 150 8 2 54 Male RiHT: post-radioiodine 77.77 4.2 9 3 3 74 Male RiHT: post-radioiodine 80.70 3.0 10 4 4 38 Female PyHT: autoimmune thyroiditis 150.00 3.9 77 12 5 51 Female PyHT: autoimmune thyroiditis 67.90 5.5 93 8 6 32 Female PyHT: autoimmune thyroiditis 32.50 7.1 178 8 7 63 Female PyHT: autoimmune thyroiditis 89.00 4.7 56 12 8 79 Female PyHT: autoimmune thyroiditis 113.70 3.1 81 9 9 30 Female PyHT: autoimmune thyroiditis 37.78 6.1 71 6 10 56 Male RiHT: post-radioiodine 74.50 3.0 56 3 11 46 Female RiHT: post-radioiodine 79.90 3.8 34 9 12 45 Female PyHT: autoimmune thyroiditis 98.87 6.7 76 7 13 45 Female PyHT: autoimmune thyroiditis 65.54 5.6 55 10 14 31 Male PyHT: autoimmune thyroiditis 75.12 5.4 85 7 15 48 Male PyHT: autoimmune thyroiditis 150 3.0 122 12 16 49 Female PyHT: autoimmune thyroiditis 78.77 4.4 178 8 17 54 Female PyHT: autoimmune thyroiditis 85.5 4.1 25 6 18 55 Male PyHT: autoimmune thyroiditis 79.9 3.2 77 8 19 59 Female RiHT: post-radioiodine 35.56 5.9 25 2 20 32 Male PyHT: autoimmune thyroiditis 87.78 3.2 56 10 View Large Table 1. Clinical and Laboratory Features of Patients With HT Patient No. Age (y) Sex Cause of HT TSH (mIU/L) (0.39–4.2 mIU/L) FT4 (pmol/L) ( 12 – 22 pmol/L ) Anti-TPOab (0–34) TSQ (/12) 1 50 Male PyHT: autoimmune thyroiditis 108.70 3.3 150 8 2 54 Male RiHT: post-radioiodine 77.77 4.2 9 3 3 74 Male RiHT: post-radioiodine 80.70 3.0 10 4 4 38 Female PyHT: autoimmune thyroiditis 150.00 3.9 77 12 5 51 Female PyHT: autoimmune thyroiditis 67.90 5.5 93 8 6 32 Female PyHT: autoimmune thyroiditis 32.50 7.1 178 8 7 63 Female PyHT: autoimmune thyroiditis 89.00 4.7 56 12 8 79 Female PyHT: autoimmune thyroiditis 113.70 3.1 81 9 9 30 Female PyHT: autoimmune thyroiditis 37.78 6.1 71 6 10 56 Male RiHT: post-radioiodine 74.50 3.0 56 3 11 46 Female RiHT: post-radioiodine 79.90 3.8 34 9 12 45 Female PyHT: autoimmune thyroiditis 98.87 6.7 76 7 13 45 Female PyHT: autoimmune thyroiditis 65.54 5.6 55 10 14 31 Male PyHT: autoimmune thyroiditis 75.12 5.4 85 7 15 48 Male PyHT: autoimmune thyroiditis 150 3.0 122 12 16 49 Female PyHT: autoimmune thyroiditis 78.77 4.4 178 8 17 54 Female PyHT: autoimmune thyroiditis 85.5 4.1 25 6 18 55 Male PyHT: autoimmune thyroiditis 79.9 3.2 77 8 19 59 Female RiHT: post-radioiodine 35.56 5.9 25 2 20 32 Male PyHT: autoimmune thyroiditis 87.78 3.2 56 10 Patient No. Age (y) Sex Cause of HT TSH (mIU/L) (0.39–4.2 mIU/L) FT4 (pmol/L) ( 12 – 22 pmol/L ) Anti-TPOab (0–34) TSQ (/12) 1 50 Male PyHT: autoimmune thyroiditis 108.70 3.3 150 8 2 54 Male RiHT: post-radioiodine 77.77 4.2 9 3 3 74 Male RiHT: post-radioiodine 80.70 3.0 10 4 4 38 Female PyHT: autoimmune thyroiditis 150.00 3.9 77 12 5 51 Female PyHT: autoimmune thyroiditis 67.90 5.5 93 8 6 32 Female PyHT: autoimmune thyroiditis 32.50 7.1 178 8 7 63 Female PyHT: autoimmune thyroiditis 89.00 4.7 56 12 8 79 Female PyHT: autoimmune thyroiditis 113.70 3.1 81 9 9 30 Female PyHT: autoimmune thyroiditis 37.78 6.1 71 6 10 56 Male RiHT: post-radioiodine 74.50 3.0 56 3 11 46 Female RiHT: post-radioiodine 79.90 3.8 34 9 12 45 Female PyHT: autoimmune thyroiditis 98.87 6.7 76 7 13 45 Female PyHT: autoimmune thyroiditis 65.54 5.6 55 10 14 31 Male PyHT: autoimmune thyroiditis 75.12 5.4 85 7 15 48 Male PyHT: autoimmune thyroiditis 150 3.0 122 12 16 49 Female PyHT: autoimmune thyroiditis 78.77 4.4 178 8 17 54 Female PyHT: autoimmune thyroiditis 85.5 4.1 25 6 18 55 Male PyHT: autoimmune thyroiditis 79.9 3.2 77 8 19 59 Female RiHT: post-radioiodine 35.56 5.9 25 2 20 32 Male PyHT: autoimmune thyroiditis 87.78 3.2 56 10 View Large Table 2 shows the clinical characteristics of and comparisons between the HT and HC groups at baseline and at the end of the study period. Both groups were matched for age, sex, and body mass index (P ≥ 0.05). Systolic blood pressure was modestly higher in the HT group (P = 0.04) but lipid profiles did not differ (P ≥ 0.05). As expected, the TSH, FT4, anti-TPOab titers, and TSQ score were significantly abnormal in the HT group (P ≤ 0.0001). Both measures of SFN—LDIFLARE (P ≤ 0.0001) and CCM (P ≤ 0.002 for all three parameters)—were significantly lower in patients with HT than in HCs (Fig. 1). None of the three measures of large-fiber neuropathy—SNCV, SNAP, and vibration perception threshold (VPT)—differed between the groups (P = >0.05). Table 2. Baseline Clinical and Neurologic Outcomes For Patients With HT and HCs Baseline Characteristics Baseline a End of Study b HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups Age, y 44.95 ± 14.29 49.55 ± 13.34 NS 45.95 ± 14.29 50.87 ± 13.01 NS Men/women, n/n 8/12 9/11 NS 8/12 9/11 NS BMI, kg/m2 28.96 ± 2.69 28.09 ± 3.04 NS 29.01 ± 2.09 30.02 ± 3.99 NS Mean BP, mm Hg 114.87 ± 10.45 128.65 ± 9.56 0.04 118.11 ± 9.89 122.45 ± 14.21 NS Triglycerides, mmol/L 1.74 ± 0.54 1.86 ± 0.94 NS 1.89 ± 0.60 2.01 ± 1.10 NS Total cholesterol, mmol/L 4.06 ± 1.21 4.31 ± 0.88 NS 4.19 ± 1.45 4.29 ± 0.97 NS TSH, mIU/L 2.18 ± 0.72 83.47 ± 31.18 <0.0001 2.51 ± 0.98 3.01 ± 0.58 0.03 FT4, pmol/L 16.83 ± 2.31 4.46 ± 1.34 <0.0001 16.17 ± 2.45 18.91 ± 4.01 NS anti-TPOab, IU/L 8.35 ± 4.43 75.70 ± 49.40 <0.0001 8.35 ± 4.43 40.23 ± 12.33 <0.0001 TSQ score 1 ± 0.92 7.6 ± 0.54 <0.0001 0.89 ± 1.11 3.67 ± 3.54 0.001 LDIFLARE, cm2 8.90 ± 1.75 6.74 ± 1.20 <0.0001 8.85 ± 1.83 8.45 ± 0.60 0.01 CNFD, no./mm2 58.32 ± 6.54 50.77 ± 6.54 0.002 58.32 ± 6.54 54.43 ± 5.70 0.02 CNBD, no./mm2 38.30 ± 5.93 29.55 ± 6.90 0.001 38.30 ± 5.93 32.81 ± 7.91 0.02 CNFL, mm/mm2 14.24 ± 2.50 10.11 ± 2.33 0.002 14.24 ± 2.50 13.43 ± 2.25 NS SNCV, m/s 50.45 ± 4.66 47.60 ± 5.88 NS 51.19 ± 4.45 49.29 ± 6.89 NS SNAP, µV 19.45 ± 3.30 15.80 ± 5.81 NS 18.01 ± 3.29 16.89 ± 5.01 NS VPT, mV 5.43 ± 1.56 6.65 ± 2.62 NS 5.55 ± 1.67 5.89 ± 1.91 NS Baseline Characteristics Baseline a End of Study b HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups Age, y 44.95 ± 14.29 49.55 ± 13.34 NS 45.95 ± 14.29 50.87 ± 13.01 NS Men/women, n/n 8/12 9/11 NS 8/12 9/11 NS BMI, kg/m2 28.96 ± 2.69 28.09 ± 3.04 NS 29.01 ± 2.09 30.02 ± 3.99 NS Mean BP, mm Hg 114.87 ± 10.45 128.65 ± 9.56 0.04 118.11 ± 9.89 122.45 ± 14.21 NS Triglycerides, mmol/L 1.74 ± 0.54 1.86 ± 0.94 NS 1.89 ± 0.60 2.01 ± 1.10 NS Total cholesterol, mmol/L 4.06 ± 1.21 4.31 ± 0.88 NS 4.19 ± 1.45 4.29 ± 0.97 NS TSH, mIU/L 2.18 ± 0.72 83.47 ± 31.18 <0.0001 2.51 ± 0.98 3.01 ± 0.58 0.03 FT4, pmol/L 16.83 ± 2.31 4.46 ± 1.34 <0.0001 16.17 ± 2.45 18.91 ± 4.01 NS anti-TPOab, IU/L 8.35 ± 4.43 75.70 ± 49.40 <0.0001 8.35 ± 4.43 40.23 ± 12.33 <0.0001 TSQ score 1 ± 0.92 7.6 ± 0.54 <0.0001 0.89 ± 1.11 3.67 ± 3.54 0.001 LDIFLARE, cm2 8.90 ± 1.75 6.74 ± 1.20 <0.0001 8.85 ± 1.83 8.45 ± 0.60 0.01 CNFD, no./mm2 58.32 ± 6.54 50.77 ± 6.54 0.002 58.32 ± 6.54 54.43 ± 5.70 0.02 CNBD, no./mm2 38.30 ± 5.93 29.55 ± 6.90 0.001 38.30 ± 5.93 32.81 ± 7.91 0.02 CNFL, mm/mm2 14.24 ± 2.50 10.11 ± 2.33 0.002 14.24 ± 2.50 13.43 ± 2.25 NS SNCV, m/s 50.45 ± 4.66 47.60 ± 5.88 NS 51.19 ± 4.45 49.29 ± 6.89 NS SNAP, µV 19.45 ± 3.30 15.80 ± 5.81 NS 18.01 ± 3.29 16.89 ± 5.01 NS VPT, mV 5.43 ± 1.56 6.65 ± 2.62 NS 5.55 ± 1.67 5.89 ± 1.91 NS Unless otherwise noted, values are expressed as mean/median ± SD. Abbreviations: BMI, body mass index; NS, not significant (level of significance at 5%). a In HCs and before LT4 in patients with HT. b At 1 y in HCs and after ≥6 months of stable LT4 replacement in patients with HT. View Large Table 2. Baseline Clinical and Neurologic Outcomes For Patients With HT and HCs Baseline Characteristics Baseline a End of Study b HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups Age, y 44.95 ± 14.29 49.55 ± 13.34 NS 45.95 ± 14.29 50.87 ± 13.01 NS Men/women, n/n 8/12 9/11 NS 8/12 9/11 NS BMI, kg/m2 28.96 ± 2.69 28.09 ± 3.04 NS 29.01 ± 2.09 30.02 ± 3.99 NS Mean BP, mm Hg 114.87 ± 10.45 128.65 ± 9.56 0.04 118.11 ± 9.89 122.45 ± 14.21 NS Triglycerides, mmol/L 1.74 ± 0.54 1.86 ± 0.94 NS 1.89 ± 0.60 2.01 ± 1.10 NS Total cholesterol, mmol/L 4.06 ± 1.21 4.31 ± 0.88 NS 4.19 ± 1.45 4.29 ± 0.97 NS TSH, mIU/L 2.18 ± 0.72 83.47 ± 31.18 <0.0001 2.51 ± 0.98 3.01 ± 0.58 0.03 FT4, pmol/L 16.83 ± 2.31 4.46 ± 1.34 <0.0001 16.17 ± 2.45 18.91 ± 4.01 NS anti-TPOab, IU/L 8.35 ± 4.43 75.70 ± 49.40 <0.0001 8.35 ± 4.43 40.23 ± 12.33 <0.0001 TSQ score 1 ± 0.92 7.6 ± 0.54 <0.0001 0.89 ± 1.11 3.67 ± 3.54 0.001 LDIFLARE, cm2 8.90 ± 1.75 6.74 ± 1.20 <0.0001 8.85 ± 1.83 8.45 ± 0.60 0.01 CNFD, no./mm2 58.32 ± 6.54 50.77 ± 6.54 0.002 58.32 ± 6.54 54.43 ± 5.70 0.02 CNBD, no./mm2 38.30 ± 5.93 29.55 ± 6.90 0.001 38.30 ± 5.93 32.81 ± 7.91 0.02 CNFL, mm/mm2 14.24 ± 2.50 10.11 ± 2.33 0.002 14.24 ± 2.50 13.43 ± 2.25 NS SNCV, m/s 50.45 ± 4.66 47.60 ± 5.88 NS 51.19 ± 4.45 49.29 ± 6.89 NS SNAP, µV 19.45 ± 3.30 15.80 ± 5.81 NS 18.01 ± 3.29 16.89 ± 5.01 NS VPT, mV 5.43 ± 1.56 6.65 ± 2.62 NS 5.55 ± 1.67 5.89 ± 1.91 NS Baseline Characteristics Baseline a End of Study b HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups HCs (n = 20) Patients With HT (n = 20) P Value for HC vs HT Groups Age, y 44.95 ± 14.29 49.55 ± 13.34 NS 45.95 ± 14.29 50.87 ± 13.01 NS Men/women, n/n 8/12 9/11 NS 8/12 9/11 NS BMI, kg/m2 28.96 ± 2.69 28.09 ± 3.04 NS 29.01 ± 2.09 30.02 ± 3.99 NS Mean BP, mm Hg 114.87 ± 10.45 128.65 ± 9.56 0.04 118.11 ± 9.89 122.45 ± 14.21 NS Triglycerides, mmol/L 1.74 ± 0.54 1.86 ± 0.94 NS 1.89 ± 0.60 2.01 ± 1.10 NS Total cholesterol, mmol/L 4.06 ± 1.21 4.31 ± 0.88 NS 4.19 ± 1.45 4.29 ± 0.97 NS TSH, mIU/L 2.18 ± 0.72 83.47 ± 31.18 <0.0001 2.51 ± 0.98 3.01 ± 0.58 0.03 FT4, pmol/L 16.83 ± 2.31 4.46 ± 1.34 <0.0001 16.17 ± 2.45 18.91 ± 4.01 NS anti-TPOab, IU/L 8.35 ± 4.43 75.70 ± 49.40 <0.0001 8.35 ± 4.43 40.23 ± 12.33 <0.0001 TSQ score 1 ± 0.92 7.6 ± 0.54 <0.0001 0.89 ± 1.11 3.67 ± 3.54 0.001 LDIFLARE, cm2 8.90 ± 1.75 6.74 ± 1.20 <0.0001 8.85 ± 1.83 8.45 ± 0.60 0.01 CNFD, no./mm2 58.32 ± 6.54 50.77 ± 6.54 0.002 58.32 ± 6.54 54.43 ± 5.70 0.02 CNBD, no./mm2 38.30 ± 5.93 29.55 ± 6.90 0.001 38.30 ± 5.93 32.81 ± 7.91 0.02 CNFL, mm/mm2 14.24 ± 2.50 10.11 ± 2.33 0.002 14.24 ± 2.50 13.43 ± 2.25 NS SNCV, m/s 50.45 ± 4.66 47.60 ± 5.88 NS 51.19 ± 4.45 49.29 ± 6.89 NS SNAP, µV 19.45 ± 3.30 15.80 ± 5.81 NS 18.01 ± 3.29 16.89 ± 5.01 NS VPT, mV 5.43 ± 1.56 6.65 ± 2.62 NS 5.55 ± 1.67 5.89 ± 1.91 NS Unless otherwise noted, values are expressed as mean/median ± SD. Abbreviations: BMI, body mass index; NS, not significant (level of significance at 5%). a In HCs and before LT4 in patients with HT. b At 1 y in HCs and after ≥6 months of stable LT4 replacement in patients with HT. View Large Baseline correlates of TSH with small- and large-fiber indices The baseline TSH of all study participants was compared with the initial small- and large-fiber neural indices. Figure 3 demonstrates a significant inverse correlation between baseline TSH and LDIFLARE (r = −0.719; P ≤ 0.0001), CNFD (r = −0.492; P = 0.008), and CNFL (r = −0.382; P = 0.01) but not with CNBD (r = 0.044; P = 0.41). In comparison, none of the large-fiber parameters—SNCV (P = 0.09), SNAP (P = 0.06), and VPT (P = 0.11)—showed any significant correlation with baseline TSH. In HCs, neither small- nor large-fiber parameters demonstrated any significant correlations (P ≥ 0.05) with baseline TSH. There was also a significant correlation between baseline TSH and TSQ scores (r = 0.70; P = 0.008). Comparison of FT4 levels with both large- and small-fiber neurologic parameters revealed no significant correlation (P ≥ 0.05). Figure 3. View largeDownload slide Correlation between baseline TSH and indices of small nerve fibers (LDIFLARE and CCM). ●, correlation of baseline TSH with LDIFLARE; ■, correlation of baseline TSH with CNFD; ♦, correlation of baseline TSH with CNBD; ▬, correlation of baseline TSH with CNFL. Figure 3. View largeDownload slide Correlation between baseline TSH and indices of small nerve fibers (LDIFLARE and CCM). ●, correlation of baseline TSH with LDIFLARE; ■, correlation of baseline TSH with CNFD; ♦, correlation of baseline TSH with CNBD; ▬, correlation of baseline TSH with CNFL. After the initial assessment, all patients with HT began receiving LT4 monotherapy, with doses titrated per 2014 guidelines suggested by the American Thyroid Association (26). After the target TSH was achieved and remained stable for ≥6 months, the patients with HT were reassessed again for neurophysiological changes. Differences in characteristics at study end Table 2 and Fig. 4 depict the changes in neurophysiological indices before and after treatment with LT4. After successful normalization of TSH levels, all small-fiber modalities significantly improved compared with baseline values: LDIFLARE (8.45 ± 0.60 vs 6.74 ± 1.20 cm2; P ≤ 0.0001), CNFD (54.43 ± 5.70 vs 50.77 ± 6.54 no./mm2; P = 0.008), CNBD (32.81 ± 7.91 vs 29.55 ± 6.90 no./mm2; P = 0.002), and CNFL (13.43 ± 2.25 vs 10.11 ± 2.33 mm/mm2; P = 0.01). In contrast, none of the large-fiber modalities—SNCV (P = 0.21), SNAP (P = 0.26), and VPT (P = 0.53)—showed any significant change. The inverse correlations between TSH and small-fiber indices (LDIFLARE, CNFD, and CNFL) observed in the pretreatment period were not seen at the end of the study period (P ≥ 0.05) Figure 4. View largeDownload slide Box plots (with 95% CIs) indicate change of both small- and large-fiber neural outcomes after treatment with LT4. Level of significance indicated with each pair. Figure 4. View largeDownload slide Box plots (with 95% CIs) indicate change of both small- and large-fiber neural outcomes after treatment with LT4. Level of significance indicated with each pair. As shown in Fig. 5, all HCs were reassessed after 1 year and their results were compared with the final outcomes of the HT group. Despite achievement of stable and normal TSH levels in the latter, when compared with HCs, the small neural indices remained significantly lower. The LDIFLARE in the HC group, 8.81 ± 0.99 cm2, remained significantly higher than the value in the HT group. 8.45 ± 0.60 cm2 (P = 0.02). A similar significant difference was observed with CNFD (56.67 ± 6.12 vs 54.43 ± 5.70 no./mm2; P = 0.04) and CNBD (35.86 ± 7.17 vs 32.81 ± 7.91 no./mm2; P = 0.02) but not with CNFL (13.71 ± 3.563 vs 13.43 ± 2.25 mm/mm2; P = 0.05), respectively. None of the large-fiber parameters significantly differed between the HC and HT groups at the end of the study. Finally, no significant differences in baseline or follow-up outcomes were observed between the PyHT and RiHT groups at any level. Figure 5. View largeDownload slide Box plots (with 95% CIs) compare both small (LDIFLARE and CNFD, CNBD, CNFL) and large (SNCV, SNAP, VPT) neural outcomes between HCs and patients with HT at the end of study assessment. Level of significance indicated with each pair. Figure 5. View largeDownload slide Box plots (with 95% CIs) compare both small (LDIFLARE and CNFD, CNBD, CNFL) and large (SNCV, SNAP, VPT) neural outcomes between HCs and patients with HT at the end of study assessment. Level of significance indicated with each pair. Discussion Neuromuscular dysfunction is common in patients with HT and has been historically associated with sensorimotor polyneuropathy and other peripheral neuropathies (1, 27, 28). Previous studies have inconsistently reported the prevalence of such polyneuropathy in hypothyroid patients to vary from 42% to 72% (1, 7). However, these studies, using predominantly markers of large-fiber function, were in symptomatic patients with long-standing hypothyroidism with a wide distribution of neurologic involvement ranging from entrapment neuropathy to more diffuse peripheral neuropathy (29). Such major neurologic sequelae are now rare because of earlier diagnosis and earlier replacement with LT4. However, in neurologically asymptomatic patients, there is conflicting evidence of polyneuropathy when large-fiber function is assessed by measurement of nerve conduction; some studies suggest its presence (30) but others have not (13). In this context, recent studies have demonstrated abnormalities in SFS in HT. In a group of 18 asymptomatic patients with subclinical or overt hypothyroid patients, Magri et al. (14) demonstrated significantly reduced IENFD in skin biopsy specimens from the upper thigh and distal leg, leading to the suggestion that “a considerable number of untreated hypothyroid patients may have preclinical asymptomatic small fibre neuropathy.” However, although IENFD has traditionally been considered as the gold standard for investigating small-fiber neuropathy, its invasiveness may limit repetitive study; moreover, the technique still lacks a consensus reference standard (31). Our proof-of-concept study compared large and small neural measures in 20 patients with untreated HT vs 20 age- and sex-matched HCs. All 15 patients with PyHT were referred to hospital services for management of newly diagnosed HT; however, on the basis of symptoms alone, it is not possible to know the duration of their hypothyroidism because they did not have any records of previous thyroid function tests. The remaining five patients with RiHT were diagnosed during routine biochemical surveillance after radioiodine treatment. Because of the above reasons, we could not correlate duration of disease with neurologic dysfunction or with improvement of small-fiber function with either subgroup, PyHT or RiHT. For SFN, we used two methods not previously applied in HT: LDIFLARE for SFF and CCM for SFS. Although all 20 patients with HT were newly presenting with their diagnosis, their mean TSH was relatively high (83.47 ± 31.18 mIU/L; normal range, 0.27 to 4.20mIU/L); this suggests a relatively profound biochemical abnormality. Despite this, at baseline none of the large-fiber modalities (SNCV, SNAP, and VPT) had significantly abnormal results. In contrast, both small-fiber measures (LDIFLARE and CCM) were significantly reduced (P ≤ 0.0001 and P = ≤ 0.002, respectively) in comparison with values in HCs. This supports the hypothesis that SFN in untreated early HT precedes large-fiber neural involvement and adds to Magri and colleagues findings (14) that IEFND may be abnormal in hypothyroid patients in the presence of normal large-fiber function. Furthermore, in addition to demonstrating structural small-fiber deficits using a different but noninvasive method (CCM), our study for the first time demonstrates significant functional abnormality (LDIFLARE) in patients with early HT. We found that the baseline TSH had a significant inverse correlation with both LDIFLARE and CCM (P ≤ 0.0001 and P ≤ 0.01, respectively) but not with large-fiber methods. Our findings contrast with the results of Magri et al. (14), who did not find any significant correlation between TSH and IENFD. This disparity could be explained by the inclusion of patients with subclinical HT in the latter study; that would also explain the wide TSH range in comparison with the current study (77.19 ± 92.80 mIU/L in the study by Magri et al. vs 83.47 ± 31.18 mIU/L in the current study). Previous studies that used large-fiber methods hypothesized that severity of peripheral neuropathy in HT was related to duration of HT but not to the degree of thyroid failure (2, 10). On the basis of our findings, we suggest that these more sensitive and recent methods of SFN assessment not only detect early neural involvement but also relate to severity of deficiency as indicated by serum TSH levels. After establishment of a euthyroid state with LT4 replacement therapy, both small-fiber indices significantly improved (LDIFLARE, P ≤ 0.0001; CCM, P ≤ 0.02), whereas large-fiber function did not change. We are aware of only one other prospective study, again by Magri et al. (15), whose small study of nine patients with HT (six with overt and three with subclinical disease) demonstrated significant reversibility of previously reduced IENFD. The neural improvement detected by the methods used in our study underlines the sensitivity of these methods in contrast to measures of large-fiber function. In addition, unlike IENFD, these methods are noninvasive and thus have the advantage of being repeatable. Of potential interest was the finding that despite normalization of TSH in all patients with HT, the small-fiber indices remained significantly reduced compared with those in the HCs: LDIFLARE (P = 0.02) and CCM (P ≤ 0.05). This may suggest that a longer period of replacement therapy is necessary before small-fiber function and structure return to normal. Restoration of normal neural cellular function may also require the TSH to be in the lower half of the normal range; however, in this study we did not find a difference between patients with TSH levels in the lower quartile vs those with a normal reference range vs those in the upper quartile. A further study of similar design with more patients in whom a euthyroid state is maintained for a longer period would be necessary to determine whether this observation of persistent reduction of small-fiber neural indices is permanent. This study is not without its limitations. Although these patients were newly diagnosed, they may have had long-standing hypothyroidism, which would explain the very high TSH in the group. It would be of interest to study patients with less biochemical severity, including subclinical hypothyroidism. It is important that our findings be confirmed by other studies, in particular because we studied only 20 patients; however, the high level of significance of the baseline findings and the improvements after treatment suggest that a type II error is unlikely. Conclusion This study demonstrates that small-fiber neuropathy precedes large-fiber dysfunction in early HT. This can be improved by achieving a biochemically euthyroid state with LT4 replacement therapy, but even after 6 months of maintaining this euthyroid state, small-fiber neural indices continued to be remain low when compared with values in HCs. Abbreviations: Abbreviations: anti-TPOab antithyroid peroxidase antibodies CCM corneal confocal microscopy CNBD confocal nerve branch density CNFD corneal confocal microscopy nerve fiber density CV coefficient of variation FT4 free T4 HC healthy control HT hypothyroidism IENFD intraepidermal nerve fiber density LDIFLARE laser Doppler imager flare technique LT4 levothyroxine PyHT primary hypertension RiHT radioiodine-induced hypothyroidism SFF small-fiber function SFN small-fiber neuropathy SFS small-fiber structure SNAP sural nerve amplitude SNCV sural nerve conduction velocity TSQ Thyroid Symptom Questionnaire VPT vibration perception threshold Acknowledgments The authors thank the staff at Ipswich hospital and their friends and family members in Suffolk for their contribution by volunteering for this study. Author Contributions: S.S. was responsible for conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, writing – original draft and writing – review & editing. V.T. was responsible for data curation, investigation, methodology, validation and writing – review & editing. P.V. was responsible for conceptualization, methodology and writing – review & editing. G.R. was responsible for conceptualization, formal analysis, funding acquisition, investigation, methodology, resources, software, supervision, validation, visualization and writing – review & editing. Disclosure Summary: The authors have nothing to disclose. References 1. Beghi E , Delodovici ML , Bogliun G , Crespi V , Paleari F , Gamba P , Capra M , Zarrelli M . Hypothyroidism and polyneuropathy . J Neurol Neurosurg Psychiatry . 1989 ; 52 ( 12 ): 1420 – 1423 . Google Scholar CrossRef Search ADS PubMed 2. Nemni R , Bottacchi E , Fazio R , Mamoli A , Corbo M , Camerlingo M , Galardi G , Erenbourg L , Canal N . Polyneuropathy in hypothyroidism: clinical, electrophysiological and morphological findings in four cases . J Neurol Neurosurg Psychiatry . 1987 ; 50 ( 11 ): 1454 – 1460 . Google Scholar CrossRef Search ADS PubMed 3. Lin KP , Kwan SY , Chen SY , Chen SS , Yeung KB , Chia LG , Wu ZA . Generalized neuropathy in Taiwan: an etiologic survey . Neuroepidemiology . 1993 ; 12 ( 5 ): 257 – 261 . Google Scholar CrossRef Search ADS PubMed 4. Rudolph T , Farbu E . Hospital-referred polyneuropathies—causes, prevalences, clinical, and neurophysiological findings . Eur J Neurol 2007 ; 14 ( 6 ): 603 – 608 . Google Scholar CrossRef Search ADS PubMed 5. Leese GP , Flynn RV , Jung RT , Macdonald TM , Murphy MJ , Morris AD . Increasing prevalence and incidence of thyroid disease in Tayside, Scotland: the Thyroid Epidemiology Audit and Research Study (TEARS) . Clin Endocrinol (Oxf) . 2008 ; 68 ( 2 ): 311 – 316 . Google Scholar PubMed 6. Wood-Allum CA , Shaw PJ . Thyroid disease and the nervous system . Handb Clin Neurol . 2014 ; 120 : 703 – 735 . Google Scholar CrossRef Search ADS PubMed 7. Duyff RF , Van den Bosch J , Laman DM , van Loon BJ , Linssen WH . Neuromuscular findings in thyroid dysfunction: a prospective clinical and electrodiagnostic study . J Neurol Neurosurg Psychiatry . 2000 ; 68 ( 6 ): 750 – 755 . Google Scholar CrossRef Search ADS PubMed 8. Pollard J. Neuropathy in diseases of the thyroid and pituitary glands. In: Dyck PJ, Thomas PK, Griffin JW, Low PA, Poduslo JF, ed. Peripheral Neuropathy. 3rd ed. Philadelphia, PA: Saunders Company; 1993:1266–1274. 9. Shinoda K , Takamatsu J , Mozai T . Peripheral neuropathy in hypothyroidism. I. Clinical and electrophysiological study . Bull Osaka Med Sch . 1987 ; 33 ( 2 ): 137 – 148 . Google Scholar PubMed 10. Somay G , Oflazoğlu B , Us O , Surardamar A . Neuromuscular status of thyroid diseases: a prospective clinical and electrodiagnostic study . Electromyogr Clin Neurophysiol . 2007 ; 47 ( 2 ): 67 – 78 . Google Scholar PubMed 11. Quattrini C , Tavakoli M , Jeziorska M , Kallinikos P , Tesfaye S , Finnigan J , Marshall A , Boulton AJ , Efron N , Malik RA . Surrogate markers of small fiber damage in human diabetic neuropathy . Diabetes . 2007 ; 56 ( 8 ): 2148 – 2154 . Google Scholar CrossRef Search ADS PubMed 12. Sharma S , Venkitaraman R , Vas PR , Rayman G . Assessment of chemotherapy-induced peripheral neuropathy using the LDIFLARE technique: a novel technique to detect neural small fiber dysfunction . Brain Behav . 2015 ; 5 ( 7 ): e00354 . Google Scholar CrossRef Search ADS PubMed 13. Ørstavik K , Norheim I , Jørum E . Pain and small-fiber neuropathy in patients with hypothyroidism . Neurology . 2006 ; 67 ( 5 ): 786 – 791 . Google Scholar CrossRef Search ADS PubMed 14. Magri F , Buonocore M , Oliviero A , Rotondi M , Gatti A , Accornero S , Camera A , Chiovato L . Intraepidermal nerve fiber density reduction as a marker of preclinical asymptomatic small-fiber sensory neuropathy in hypothyroid patients . Eur J Endocrinol 2010 ; 163 ( 2 ): 279 – 284 . Google Scholar CrossRef Search ADS PubMed 15. Magri F , Buonocore M , Camera A , Capelli V , Oliviero A , Rotondi M , Gatti A , Chiovato L . Improvement of intra-epidermal nerve fibre density in hypothyroidism after L-thyroxine therapy . Clin Endocrinol (Oxf) . 2013 ; 78 ( 1 ): 152 – 153 . Google Scholar CrossRef Search ADS PubMed 16. American Diabetes Association . 2. Classification and diagnosis of diabetes: Standards of Medical Care in Diabetes-2018 . Diabetes Care . 2018 ; 41 ( Suppl 1 ): S13 – S27 . CrossRef Search ADS PubMed 17. Saravanan P , Chau WF , Roberts N , Vedhara K , Greenwood R , Dayan CM . Psychological well-being in patients on ‘adequate’ doses of l-thyroxine: results of a large, controlled community-based questionnaire study . Clin Endocrinol (Oxf) . 2002 ; 57 ( 5 ): 577 – 585 . Google Scholar CrossRef Search ADS PubMed 18. Boulton A . Management of diabetic peripheral neuropathy . Clin Diabetes . 2005 ; 23 ( 1 ): 9 – 15 . Google Scholar CrossRef Search ADS 19. Vas PR , Rayman G . Validation of the modified LDIFlare technique: a simple and quick method to assess C-fiber function . Muscle Nerve . 2013 ; 47 ( 3 ): 351 – 356 . Google Scholar CrossRef Search ADS PubMed 20. Krishnan ST , Rayman G . The LDIflare: a novel test of C-fiber function demonstrates early neuropathy in type 2 diabetes . Diabetes Care . 2004 ; 27 ( 12 ): 2930 – 2935 . Google Scholar CrossRef Search ADS PubMed 21. Green AQ , Krishnan S , Finucane FM , Rayman G . Altered C-fiber function as an indicator of early peripheral neuropathy in individuals with impaired glucose tolerance . Diabetes Care . 2010 ; 33 ( 1 ): 174 – 176 . Google Scholar CrossRef Search ADS PubMed 22. Krishnan ST , Quattrini C , Jeziorska M , Malik RA , Rayman G . Abnormal LDIflare but normal quantitative sensory testing and dermal nerve fiber density in patients with painful diabetic neuropathy . Diabetes Care . 2009 ; 32 ( 3 ): 451 – 455 . Google Scholar CrossRef Search ADS PubMed 23. Petropoulos IN , Manzoor T , Morgan P , Fadavi H , Asghar O , Alam U , Ponirakis G , Dabbah MA , Chen X , Graham J , Tavakoli M , Malik RA . Repeatability of in vivo corneal confocal microscopy to quantify corneal nerve morphology . Cornea . 2013 ; 32 ( 5 ): e83 – e89 . Google Scholar CrossRef Search ADS PubMed 24. Tavakoli M , Malik RA . Corneal confocal microscopy: a novel non-invasive technique to quantify small fibre pathology in peripheral neuropathies . J Vis Exp . 2011 ;( 47 ): 2194 . 25. Dabbah MA , Graham J , Petropoulos IN , Tavakoli M , Malik RA . Automatic analysis of diabetic peripheral neuropathy using multi-scale quantitative morphology of nerve fibres in corneal confocal microscopy imaging . Med Image Anal . 2011 ; 15 ( 5 ): 738 – 747 . Google Scholar CrossRef Search ADS PubMed 26. Jonklaas J , Bianco AC , Bauer AJ , Burman KD , Cappola AR , Celi FS , Cooper DS , Kim BW , Peeters RP , Rosenthal MS , Sawka AM , American Thyroid Association Task Force on Thyroid Hormone R. Guidelines for the treatment of hypothyroidism: prepared by the american thyroid association task force on thyroid hormone replacement . Thyroid: Official Journal of the American Thyroid Association 2014 ; 24 : 1670 – 1751 . Google Scholar CrossRef Search ADS PubMed 27. Crevasse LE , Logue RB . Peripheral neuropathy in myxedema . Ann Intern Med . 1959 ; 50 ( 6 ): 1433 – 1437 . Google Scholar CrossRef Search ADS PubMed 28. Dyck PJ , Lambert EH . Polyneuropathy associated with hypothyroidism . J Neuropathol Exp Neurol . 1970 ; 29 ( 4 ): 631 – 658 . Google Scholar CrossRef Search ADS PubMed 29. Khedr EM , El Toony LF , Tarkhan MN , Abdella G . Peripheral and central nervous system alterations in hypothyroidism: electrophysiological findings . Neuropsychobiology . 2000 ; 41 ( 2 ): 88 – 94 . Google Scholar CrossRef Search ADS PubMed 30. El-Salem K , Ammari F . Neurophysiological changes in neurologically asymptomatic hypothyroid patients: a prospective cohort study . J Clin Neurophysiol 2006 ; 23 ( 6 ): 568 – 572 . Google Scholar CrossRef Search ADS PubMed 31. Hlubocky A , Wellik K , Ross MA , Smith BE , Hoffman-Snyder C , Demaerschalk BM , Wingerchuk DM . Skin biopsy for diagnosis of small fiber neuropathy: a critically appraised topic . Neurologist . 2010 ; 16 ( 1 ): 61 – 63 . Google Scholar CrossRef Search ADS PubMed Copyright © 2018 Endocrine Society

Journal

Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Aug 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off