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

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.

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.

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).

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/mm². 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].

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/mm² of corneal tissue; corneal nerve branch density (CNBD), the number of branches emanating from the major nerve trunks/mm²; corneal nerve fibre length (CNFL), the total length of all nerve fibres and branches (mm/mm²) within the area of corneal tissue; and corneal nerve fibre area (CNFA), the total area of the nerve fibre net (μm²/mm²). 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 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 mm² in CNFD. This assumes a 5% significance level, and that the SD of the within-subject differences is 5.5.

At baseline, the control, DPN and SPK cohorts did not differ significantly in age, sex or alcohol consumption (Table 1). The SPK group had a significantly higher HbA_1c (p < 0.001) and lower eGFR (p < 0.001), total cholesterol (p < 0.001) and LDL-cholesterol (p < 0.001) compared with control participants. Following SPK, HbA_1c and eGFR improved significantly (p < 0.05). Additionally, there was a significant reduction in triacylglycerols (p < 0.05) and increase in HDL (p < 0.05) (Table 1).

At baseline, the SPK group exhibited marked small and large fibre abnormalities compared with the control cohort. Neuropathic symptoms (NSP), deficits (NDS), quantitative sensory thresholds (VPT, WDT, CDT), cardiac autonomic function (deep breathing heart rate variability [DB-HRV]) and electrophysiology (sural and peroneal nerve amplitude and velocity) were significantly abnormal compared with healthy control participants (Table 2) (all p < 0.001). CNFD, CNBD, CNFL and CNFA on CCM and IENFD on skin biopsy were significantly lower at baseline in the SPK group compared with control participants (all p < 0.001) (Table 2).

There was no significant change in NSP, NDS, VPT, CDT, WDT, DB-HRV, sural and peroneal nerve amplitude and velocity, CNBD, CNFL, or IENFD in the healthy control and DPN groups over 36 months (Table 2). There was a decrease in CNFD of 3.0 ± 1.3 fibres/mm² (least squares mean; p = 0.024) (Fig. 1a), which was an ~10% decrease from baseline, at 36 months in control participants (Fig. 2a).

In the SPK group, corneal nerve fibre regeneration was evident at 6 months and continued over 36 months (Figs. 1a, b and 3). There was a significant improvement in CNFD (Table 2; p = 0.035) 6 months after SPK, but no corresponding change in any other measure of neuropathy (Table 2). At 12 months, there was a significant improvement from baseline in CNFD (p = 0.007), but no significant change in CNFL, CNFA, NSP, NDS, VPT, CDT, WDT, DB-HRV or neurophysiology (Table 2). Corneal nerve morphology continued to improve over follow-up, such that at 24 months CNFD (p < 0.01), CNFL (p < 0.05) and CNFA (p < 0.05) were significantly increased compared to baseline (Table 2) and at 36 months the least squares mean CNFD of the SPK group had increased by 5 fibres/mm² (95% CI 1.8, 8.2; p = 0.003) and CNFL by 3.2 mm/mm² (95% CI 0.9, 5.5; p = 0.006) over the DPN group. The relative percentage change from baseline over the 36 month time period for CNFD and CNFL for the SPK group was ~49% and ~38%, respectively, whereas the DPN group decreased by ~5% and ~7%, respectively (Fig. 2a, b).

A subgroup of SPK participants underwent skin biopsy at baseline (n = 12), 12 months (n = 12) and 36 months (n = 5) (Fig. 4). MDL increased significantly at 12 (p = 0.020) and 36 (p = 0.019) months, but there was no significant change in IENFD or TNFL (Table 2).

There was a reduction in the number of participants followed from baseline (n = 36) to 6 (n = 25), 12 (n = 24), 24 (n = 24) and 36 (n = 15) months. However, there was no significant difference in the baseline clinical and neuropathy measures between the individuals who were followed compared with the individuals who were lost to follow-up (Electronic Supplementary Material Table 1).

The NSP (2.5; 95% CI 0.7, 4.3; p = 0.008; Fig. 1c) improved significantly from baseline in the SPK group at 36 months. Similarly, peroneal nerve conduction velocity improved at 36 months compared with baseline (4.7 m/s; 95% CI 2.2, 7.4; p = 0.0004; Fig. 1d), with no change in sural nerve amplitude (Fig. 1e). The relative change from baseline of NSP at 36 months was ~ −46% (improvement) for the SPK group compared with ~1% for the DPN group (Fig. 2c). The percentage change from baseline for the peroneal nerve conduction velocity was ~10% for the SPK group compared with ~ −3% for the DPN group (Fig. 2d), compared with sural nerve amplitude changes from baseline of ~27% for the SPK group and ~17% for the DPN group (Fig. 2e).