Early nerve fibre regeneration in individuals with type 1 diabetes after simultaneous pancreas and kidney transplantation.

This longitudinal observational study examined neuropathic symptoms, deficits, quantitative sensory testing, neurophysiology, corneal confocal microscopy and skin biopsy results in 32 healthy (non-diabetic) control participants, 29 individuals with type 1 diabetes and severe diabetic peripheral neuropathy [DPN] and 36 individuals with type 1 diabetes after SPK.

Electronic supplementary material The online version of this article (10.1007/s00125-019-4897-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Methods

Participant selection We assessed 36 individuals with type 1 diabetes and end stage renal failure undergoing SPK, 29 individuals with type 1 diabetes and severe diabetic peripheral neuropathy (DPN) and 32 healthy (non-diabetic) control participants from Central Manchester and Manchester Children’s University Hospital. Exclusion criteria were a history of neuropathy due to a non-diabetic cause and any history of corneal trauma or surgery, or systemic or ocular disease that may affect the cornea. The Central Manchester Research and Ethics Committee approved this study and written informed consent was obtained from all participants. This research adhered to the tenets of the declaration of Helsinki. Clinical and metabolic assessment Evaluations were undertaken at baseline, prior to hospital discharge after SPK, and at 6, 12, 24 and 36 months. The control groups were evaluated at the same time points, except 6 months. Study participants underwent assessment of BMI, BP, HbA 1c , lipid profile (total cholesterol, LDL-cholesterol, HDL-cholesterol and triacylglycerols) and eGFR. Neuropathy assessment Symptoms of DPN were assessed using the Neuropathy Symptom Profile (NSP). Neurological deficits were evaluated using the modified Neuropathy Disability Score (NDS). Vibration perception threshold (VPT) was tested using a Neurothesiometer (Horwell, Scientific Laboratory Supplies, Wilford, Nottingham, UK). Cold (CDT) and warm (WDT) detection thresholds were assessed on the foot using the TSA-II NeuroSensory Analyser (Medoc, Ramat-Yishai, Israel). Sural sensory nerve amplitude, conduction velocity and latency, and peroneal motor nerve amplitude, conduction velocity and latency were determined by a consultant neurophysiologist using a Dantec ‘Keypoint’ system (Dantec Dynamics, Bristol, UK). Heart rate variability was assessed with an ANX 3.0 autonomic nervous system-monitoring device (ANSAR Medical Technologies, Philadelphia, PA, USA). Skin biopsy Three millimetre punch skin biopsies were obtained from the dorsum of the foot, approximately 2 cm proximal to the second metatarsal head, under local anaesthesia (1% lidocaine) at baseline ( n = 12), 12 months ( n = 12) and 36 months ( n = 5) after SPK. Fifty micrometre sections were immunostained using anti-human protein gene product 9.5 (PGP9.5) antibody (Abcam, Cambridge, UK) and nerve fibres were demonstrated using SG chromogen (Vector Laboratories, Peterborough, UK). Growth associated protein-43 (GAP-43), a marker found in newly regenerated nerve fibres [ 10 ], was immunolocalised using anti-human GAP-43 antibody (Novus Biologicals, Abingdon, UK). The biopsies were assigned a coded number and all nerve morphology assessments were performed blinded to the case diagnosis. IENFD was quantified according to established international guidelines and expressed as number/mm [ 11 ]. IENFD reflects the number of nerves crossing the epidermal basal membrane into the epidermis and does not account for morphology of the nerve fibres inside the epidermis. Therefore, additional morphological measures of the intraepidermal nerve fibre were quantified. Mean dendritic length (MDL) assessment was performed on PGP9.5-stained sections according to previously described methods [ 12 – 14 ]. The images were captured on a Zeiss AxioImager 2 microscope and Z-stack constructs of six sections were obtained (Axiovision and ZEN lite programmes, Carl Zeiss Microimaging, Jena, Germany). The MDL is the mean value of all of the main nerve fibres from the point of their penetration through the basement membrane to their terminal portion and is expressed as μm [ 14 ]. Total nerve fibre length (TNFL) assessment was performed on Z-stack constructs of six adjacent GAP-43-stained sections. TNFL is the sum of all nerve fibre profiles in the epidermis and is expressed as μm/mm 2 . A recent study has shown that TNFL can identify nerve fibre regeneration in a clinical trial of cibinetide in individuals with small fibre neuropathy due to sarcoidosis [ 15 ].

Corneal Confocal Microscopy

Participants underwent examination with CCM (Heidelberg Retinal Tomograph III Rostock Cornea Module, Heidelberg Engineering, Heidelberg, Germany) according to our established protocol [ 16 ]. A minimum of six non-overlapping images per individual (three per eye) from the centre of the cornea were selected and quantified in a masked fashion. Data derived from these images were averaged for each eye and the mean of both eyes used in subsequent data analysis. Four corneal nerve variables were quantified: CNFD, the total number of major nerves/mm 2 of corneal tissue; corneal nerve branch density (CNBD), the number of branches emanating from the major nerve trunks/mm 2 ; corneal nerve fibre length (CNFL), the total length of all nerve fibres and branches (mm/mm 2 ) within the area of corneal tissue; and corneal nerve fibre area (CNFA), the total area of the nerve fibre net (μm 2 /mm 2 ). CNFA is a two-dimensional measure which accounts for the length and variable thickness of the corneal nerve fibre bundles. Analysis of corneal nerve morphology was performed using automated software (ACCMetrics for CNFD, CNBD and CNFL, and FIJI for CNFA), as previously described [ 15 ].

Statistical analysis

Statistical analyses were carried out using SPSS for Mac (Version 19.0, IBM Corporation, New York, NY, USA) and JMP (Version 11, SAS, Cary, NC, USA). All data contained within Tables 1 and 2 are expressed as mean ± SD. Plots show least squares mean ± SEM. The data were first assessed for normality using the Shapiro–Wilk normality test. Longitudinal change from baseline value for each study variable was estimated using a mixed model repeated measures analysis, which included participant group, time point and group by time point as fixed effects and participant as a random effect. Least mean squares differences within groups were assessed, as well as the difference between the SPK and DPN groups. Using data published in previous studies, a sample size of 21 participants was calculated to have 90% power to detect within-patient change of 4 nerves per mm 2 in CNFD. This assumes a 5% significance level, and that the SD of the within-subject differences is 5.5. Table 1 Demographic and biochemical data of participant groups Variable Group Baseline 6 months 12 months 24 months 36 months Age (years) SPK 48.6 ± 9.2 – – – – DPN 61.9 ± 12.3 – – – – HC 47.7 ± 1.6 Sex (F/M) SPK 11/25 – – – – DPN 13/16 – – – – HC 13/17 – – – – Smoking (cigarettes/day) SPK 1.9 ± 3.4 – – – – DPN 2.1 ± 5.1 – – – – HC 0.4 ± 1.9 – – – – Alcohol consumption (units/week) SPK 6.6 ± 9.9 – – – – DPN 4.7 ± 7.8 – – – – HC 2.8 ± 5.4 – – – – Duration of diabetes (years) SPK 32.3 ± 10.5 – – – – DPN 46.0 ± 13.9 – – – – HC – – – – – BP (mmHg) Systolic SPK 131.8 ± 22.3 130.7 ± 4.2 130.7 ± 4.1 127.6 ± 5.7 123.2 ± 5.0 DPN 141 ± 23.9 – 141.4 ± 27.6 137.1 ± 24.9 132.5 ± 18.0 HC 129.5 ± 19 – 128.4 ± 15.5 127.1 ± 13.2 129.7 ± 10.2 Diastolic SPK 73.9 ± 10.5 70.9 ± 2.6 72.7 ± 2.3 68.5 ± 2.0 64.7 ± 2.7 DPN 72.8 ± 9.8 – 72.2 ± 19.1 68.8 ± 9.8 63.8 ± 8.9 HC 72.3 ± 10 – 70.3 ± 8.5 72.2 ± 7.2 71.9 ± 10.2 BMI (kg/m 2 ) SPK 23.6 ± 5.3 – 25.3 ± 0.9 25.3 ± 0.8 26.2 ± 1.3 DPN 27.1 ± 3.6 – 26.8 ± 0.8 27.1 ± 0.7 26.5 ± 0.7 HC 27.9 ± 4.4 – 27.4 ± 5.1 27.1 ± 6.1 27.3 ± 4.9 HbA 1c (%) SPK 8.4 ± 1.6 5.7 ± 0.9* 5.6 ± 1.1* 5.6 ± 0.9* 5.4 ± 0.7* DPN 8.3 ± 1.3 – 8.3 ± 1.4 8.9 ± 1.9 8.0 ± 1.3 HC 5.7 ± 0.6 – 5.5 ± 0.4 5.5 ± 0.5 5.5 ± 3.0 HbA 1c (mmol/mol) SPK 67.9 ± 16.9 38.4 ± 10.5* 37.6 ± 11.6* 38.3 ± 10.2* 39.0 ± 7.6* DPN 66.7 ± 13.4 – 67.5 ± 14.8 68.3 ± 17.8 65.1 ± 14.8 HC 38.5 ± 3.3 – 37.1 ± 3.1 36.3 ± 3.2 37.1 ± 2.9 eGFR (ml min −1 [1.73 m] −2 ) SPK 14.6 ± 2.4 57.8 ± 4.9* 58.3 ± 4.8* 60.3 ± 4.2* 63.9 ± 5.1* DPN 77.1 ± 20.6 – 73.1 ± 18.3 72.4 ± 17.0 75.2 ± 15.7 HC 83.5 ± 1.8 – 80.2 ± 9.6 83.1 ± 7.1 80.7 ± 8.6 Cholesterol (mmol/l) SPK 4.2 ± 0.9 4.1 ± 0.2 3.9 ± 1.0 4.1 ± 1.1 4.0 ± 0.9 DPN 4.3 ± 0.8 – 4.2 ± 0.8 4.0 ± 0.8 4.0 ± 0.7 HC 5.0 ± 1.5 – 4.9 ± 0.1 5.0 ± 0.6 5.1 ± 0.7 HDL-cholesterol (mmol/l) SPK 1.5 ± 0.5 1.5 ± 0.1 1.4 ± 0.5 1.5 ± 0.5 1.8 ± 0.8* DPN 1.7 ± 0.6 – 1.8 ± 0.4 1.7 ± 0.4 1.7 ± 0.3 HC 1.5 ± 0.6 – 1.6 ± 0.3 1.5 ± 0.4 1.5 ± 0.4 LDL-cholesterol (mmol/l) SPK 2.1 ± 0.7 2.1 ± 0.8 2.1 ± 0.9 2.1 ± 0.9 1.8 ± 0.6 DPN 2.1 ± 0.6 – 2.0 ± 0.6 1.8 ± 0.5 1.8 ± 0.6 HC 3.0 ± 0.6 – 2.8 ± 0.5 2.8 ± 0.5 2.9 ± 0.6 Triacylglycerols (mmol/l) SPK 1.2 ± 0.4 1.2 ± 0.1 1.1 ± 0.5 1.1 ± 0.3 0.8 ± 0.3* DPN 1.1 ± 0.6 – 0.9 ± 0.4 1.0 ± 0.5 0.9 ± 0.3 HC 1.4 ± 1.4 – 1.4 ± 1.1 1.5 ± 0.8 1.4 ± 0.7 Data are mean ± SD * p < 0.05, ** p < 0.01 compared with baseline F, female; HC, healthy control; M, male Table 2 Neuropathy assessments over time in the study groups Variable Group Baseline 6 months 12 months 24 months 36 months NDS SPK 5.2 ± 3.7 5.3 ± 3.8 5.6 ± 3.6 3.1 ± 3.6 4.7 ± 3.7 DPN 4.1 ± 3.5 – 5.4 ± 3.2 5.4 ± 3.2 4.0 ± 3.3 HC 0.3 ± 0.8 – 0.2 ± 0.5 0.2 ± 0.9 0.2 ± 0.5 NSP SPK 5.3 ± 5.9 4.6 ± 6.6 5.1 ± 6.8 3.5 ± 7.5 2.5 ± 4.2* DPN 6.0 ± 7.0 – 6.2 ± 6.1 7.2 ± 7.8 6.0 ± 7.4 HC 0.4 ± 1.1 – 0.4 ± 1.0 0.1 ± 0.4 0.1 ± 0.1 VPT (V) SPK 22.1 ± 13.5 19.5 ± 10.7 21.1 ± 14.3 19.1 ± 12.5 22.8 ± 17.1 DPN 21.6 ± 13.6 – 23.3 ± 13.9 23.3 ± 13.9 23.7 ± 14.6 HC 4.9 ± 3.4 – 6.3 ± 8.7 5.2 ± 3.1 4.4 ± 1.4 Sural amplitude (μV) SPK 3.6 ± 2.5 3.8 ± 2.6 4.4 ± 2.0 5.6 ± 1.9 6.9 ± 2.4 DPN 6.2 ± 6.4 – 5.7 ± 5.7 5.3 ± 4.9 5.9 ± 5.9 HC 19.8 ± 9.8 – 21.3 ± 8.3 21.1 ± 8.6 20.2 ± 7.5 Sural velocity (m/s) SPK 34.4 ± 7.9 36.3 ± 8.7 37.2 ± 8.6 37.0 ± 8.9 37.8 ± 8.2 DPN 38.7 ± 7.8 – 37.7 ± 7.8 36.4 ± 7.2 37.3 ± 8.5 HC 51.0 ± 4.6 – 50.5 ± 4.4 50.3 ± 5.5 50.3 ± 5.0 Peroneal amplitude (mV) SPK 1.4 ± 1.3 1.5 ± 1.4 1.6 ± 1.3 1.4 ± 1.2 1.8 ± 0.7 DPN 2.4 ± 1.9 – 1.8 ± 176 2.4 ± 1.9 2.8 ± 1.9 HC 5.8 ± 2.0 – 6.0 ± 1.7 5.3 ± 1.7 5.8 ± 1.8 Peroneal velocity (m/s) SPK 36.1 ± 5.1 37.9 ± 4.9 37.8 ± 6.3 38.0 ± 7.5* 40.9 ± 3.5** DPN 37.4 ± 8.7 – 36.9 ± 8.5 37.0 ± 8.9 37.0 ± 8.8 HC 49.2 ± 3.4 – 49.0 ± 3.3 48.3 ± 5.0 48.8 ± 4.8 CDT (°C) SPK 16.0 ± 11.1 18.0 ± 11.0 16.8 ± 12.2 17.1 ± 12.1 17.6 ± 12 DPN 23.5 ± 8.2 – 22.3 ± 8.2 22.3 ± 8.2 23.1 ± 7.6 HC 27.8 ± 5.5 – 28.4 ± 2.3 27.1 ± 4.3 28.3 ± 1.6 WDT (°C) SPK 43.8 ± 4.9 44.3 ± 4.3 43.4 ± 4.9 42.8 ± 5.1 40.9 ± 4 DPN 40.8 ± 4.7 – 41.5 ± 4.4 41.5 ± 4.4 42.2 ± 5.1 HC 35.2 ± 6.8 – 38.3 ± 3.4 38.0 ± 3.5 37.1 ± 2.5 DB-HRV (beats/min) SPK 14.6 ± 14.9 10.1 ± 6.0 10.1 ± 7 10.6 ± 9.5 11.3 ± 7.4 DPN 10.0 ± 8.2 – 6.7 ± 8.9 15.3 ± 10.6 16.9 ± 8.4 HC 33.2 ± 10.1 – 31.7 ± 13.6 29.4 ± 13.0 30.5 ± 14.0 CNFD (number/mm 2 ) SPK 9.2 ± 5.8 11.7 ± 6.2* 12.2 ± 8.4** 12.5 ± 6.6** 14.4 ± 5.0** DPN 11.4 ± 5.6 – 12.6 ± 8.1 11.6 ± 6.9 9.0 ± 5.5 HC 30.8 ± 7.3 – 31.8 ± 3.9 28.9 ± 6.0 28.2 ± 5.7* CNBD (number/mm 2 ) SPK 9.7 ± 8.4 10.3 ± 8.3 13.2 ± 13.2 12.7 ± 8.9 15.0 ± 6.6* DPN 10.6 ± 7.7 – 11.4 ± 8.6 10.6 ± 8.8 7.9 ± 4.8 HC 40.0 ± 17.3 – 43.7 ± 16.3 39.7 ± 13.3 36.3 ± 15.2* CNFL (mm/mm 2 ) SPK 7.1 ± 3.0 7.3 ± 2.7 8.2 ± 4.0 8.9 ± 3.5* 10.3 ± 2.0** DPN 7.8 ± 2.8 – 9.2 ± 4.0 8.2 ± 3.3 7.6 ± 2.5 HC 17.8 ± 3.5 – 18.6 ± 2.5 17.1 ± 3.0 16.8 ± 3.0 CNFA (μm 2 /mm 2 ) SPK 10,867 ± 4410 11,554 ± 4537 12,746 ± 6629 13,743 ± 5419* 15,394 ± 4893** IENFD (number/mm) SPK 2.2 ± 2.5 – 2.9 ± 2.8 – 2.9 ± 1.9 DPN 5.2 ± 4.4 – – 3.4 ± 2.9 – HC 9.6 ± 2.8 – – 9.5 ± 1.8 – MDL (μm) SPK 10.9 ± 2.6 – 15.6 ± 4.8* – 18.0 ± 3.0** TNFL (μm/mm 2 ) SPK 2797 ± 3651 – 3954 ± 4927 – 2881 ± 2545 Data are mean ± SD * p < 0.05, ** p < 0.01 compared with baseline HC, healthy control

Electronic supplementary material ESM Table1 (PDF 66 kb)