Introduction
Diabetic sensorimotor polyneuropathy (DSP) is a common complication of diabetes, which typically presents as a distal symmetric polyneuropathy with sensory loss or pain in the feet and hands [
1,
2]. As currently available treatments for neuropathic pain demonstrate modest pain relief [
3], it is essential that we improve our understanding of the mechanisms that contribute to painful DSP [
2,
4], and understand why some patients with DSP develop pain and others do not. Skin nerve fibre morphometric analysis offers potential insights, as differences are observed between individuals with painless and painful DSP [
5].
Intraepidermal nerve fibre density (IENFD) assessment of skin biopsy samples is a validated and sensitive diagnostic tool for the assessment of small fibre neuropathies including DSP, but IENFD is considered the pathological hallmark of DSP [
6‐
8]. There is some uncertainty as to whether IENFD differentiates between patients with painless and painful DSP, as some studies report an inverse correlation between IENFD and pain [
9], while others report no correlation [
10].
Morphometric analysis of nerve fibres detects a change in axonal structures termed axonal swellings, a degenerative change that contains watery axoplasm, neurofilaments and abnormal mitochondria [
11]. It is postulated that axonal swellings, in particular larger swellings, precede small fibre degeneration [
12,
13]. In patients with DSP the relationship of axonal swellings to polyneuropathy progression and symptoms is unclear. In one study an increase of axonal swellings was found in patients with painful DSP [
5]; however, in another study axonal swellings did not differentiate between patients with painful and painless DSP [
14]. In both studies, axonal swellings were higher in participants with DSP when compared with participants with diabetes but without DSP, and with healthy control (HC) participants.
Therefore, it is unclear if axonal swellings are related to type 2 diabetes, DSP or neuropathic pain.
This study investigates whether the axonal swelling ratio (axonal swellings/intraepidermal nerve fibres) is related to DSP, neuropathic pain and clinical variables in a well-characterised and comprehensively phenotyped cohort of study participants with type 2 diabetes mellitus and HC participants [
10,
15].
Methods
Standard protocol approvals, registrations and patient consents
The data presented in this paper are derived from the Pain in Neuropathy Study (PiNS) from the UK and the International Diabetic Neuropathy Consortium (IDNC) study from Aarhus, Denmark. PiNS is a multicentre study approved by the National Research Ethics Service of the UK (No.: 10/H0706/35). The IDNC study was approved by the regional ethics committee (No.: 1-10-72-130-16). All study participants signed written consent forms, in line with the Declaration of Helsinki, before enrolment.
Study protocol
All participants underwent a medical history review and a structured neurological examination. Study participants underwent nerve conduction studies, skin biopsy for IENFD assessment, quantitative sensory testing and diabetes-related biochemistry testing including a single HbA
1c test at the time of study. The clinical examination is described in detail elsewhere [
10,
15]. The Toronto Clinical Scoring System (TCSS) score was calculated for all participants [
16]. TCSS score is used as a surrogate measure of neuropathy severity, and was not used in the diagnosis of DSP.
Participant selection
A total of 249 study participants were recruited as part of two different studies, 204 participants with type 2 diabetes and 45 HC participants without diabetes. Clinical assessment, polyneuropathy grading, neuropathic pain grading and skin staining were performed uniformly using the same methodology. A subset, 142 of the participants (57.0%), were part of the PiNS study, and all were diagnosed with type 2 diabetes [
10]. The remaining 107 participants, 62 individuals with type 2 diabetes and 45 HC participants, were part of the IDNC study [
15] (Electronic supplementary material [ESM] Fig.
1). A detailed description of the clinical assessment and phenotyping of the study participants can be found elsewhere and will be briefly described here [
10,
15].
Selection of IDNC participants
IDNC participants were part of a clinical study of 389 patients conducted in 2016–2018, initially recruited from a questionnaire study on neuropathy and pain of 5514 recently diagnosed individuals with type 2 diabetes from the Danish Centre for Strategic Research in Type 2 Diabetes (DD2) [
15,
17]. Exclusion criteria were cognitive impairment, language difficulties and pregnancy. For more details see references [
15,
17].
Of the 389 patients included at the two IDNC study sites (Aarhus and Odense), 49 were excluded with other causes of neuropathy and significant non-neuropathic pain. We then randomly selected 62 type 2 diabetes participants and 45 HC participants who were included in Aarhus where IENFD and nerve conduction study data were available (ESM Fig.
1).
We ensured that the included HC participants without diabetes, recruited from within the patients’ social circle and by invitational flyers, were as close as possible to the diabetic participants in terms of age and sex. Exclusion criteria for HC participants were diabetes, severe chronic illness, psychiatric or neurologic illness, chronic pain or intake of any pain medication within 3 days before inclusion [
15]. HbA
1c and blood glucose were measured for all HC participants to exclude diabetes (Table
1).
Table 1
Demographics of study participants
Age, years | 62.2 (55.3–68.4) | 62.6 (51.1–68.2) | 67.9 (60.1–72.9) | 66.4 (57.4–71.6) | 0.008 |
Sex, female (%) | 25 (55.6) | 16 (51.6) | 43 (58.1) | 60 (60.6) | 0.83 |
BMI, kg/m2 | 25.4 (22.8–28.1) | 30.5 (25.3–34.3) | 31.4 (26.9–35.7) | 33.1 (28.1–37.9) | <0.001 |
Duration of type 2 diabetes, years | | 5.9 (3.8–7.9) | 10.6 (6.0–17.5) | 12.0 (6.0–18.9) | 0.001 |
HbA1c |
mmol/mol | 37.0 (35.0–39.0) | 50.0 (46.0–57.0) | 52.0 (45.7–61.5) | 58.0 (50.0–69.0) | <0.001 |
% | 5.5 | 6.7 | 6.9 | 7.5 | |
TCSS total score (0–19) | 1.0 (0.0–2.0) | 1.0 (0.0–3.0) | 8.0 (6.0–10.0) | 11.0 (8.0–14.0) | <0.001 |
Selection of PiNS participants
PiNS is an observational cross-sectional multicentre study in which study participants were recruited from primary care practices in London and Oxford, and from tertiary clinics in Oxford, London and Sheffield. Patients with diabetes mellitus aged above 18 years with diagnosed DSP, or patients with symptoms and signs suggestive of DSP, were included. Exclusion criteria were pregnancy, coincident major psychiatric disorders, poor or no English language skills, severe pain at recruitment from a cause other than DSP, documented central nervous system lesions or insufficient mental capacity to provide informed consent or to complete questionnaires. The PiNS participants included in this study were participants with both IENFD and nerve conduction study data.
Nerve conduction studies
Nerve conduction tests were performed with an ADVANCE system (Neurometrix, Waltham, MA, USA) (PiNS study) or Keypoint.Net EMG equipment (Dantec, Skovlunde, Denmark) (Aarhus) and we used conventional reusable electrodes. We performed conventional nerve conduction studies of sural nerves bilaterally and the median, peroneal and tibial nerves unilaterally [
18]. If the median nerve was found to be abnormal, the ulnar nerve was examined on the same side. The results were compared with laboratory controls using
z scores. Polyneuropathy was defined as ≥2 nerves with ≥1 abnormal measure, including at least one abnormal sural nerve [
18].
The diagnosis of definite DSP and definite painful DSP
DSP was defined according to the Toronto Diabetic Neuropathy Expert Group [
22] and painful DSP according to the Neuropathic Pain Special Interest Group (NeuPSIG) criteria [
23] for neuropathic pain.
The following criteria were used for neuropathy and neuropathic pain grading of participants from both cohorts.
Statistical analysis
We used STATA version 14 (StataCorp, TX, USA) for data analysis. Data are reported as medians with IQR. Data were compared across the three groups with Kruskall–Wallis test or between two groups with Mann–Whitney U test. Categorical data were analysed with χ2 test of association. Spearman’s rank correlation analyses were performed to explore associations between swelling ratio and diabetes-related biochemical variables, e.g. HbA1c. Significance was set at p < 0.05.
All comparisons between groups were performed twice: (1) all study participants; and (2) only participants with IENFD greater than 1 fibre/mm. The IENFD cut-off of 1 was set prior to statistical analyses since it was felt from our experience that axonal swellings with lower IENFD (<1) could not be reliably counted without potential bias of our outcomes.
Discussion
In this study, we addressed the question of whether axonal swellings are related to type 2 diabetes, DSP or neuropathic pain. Our key findings are that where IENFD > 1.0 fibre/mm, the axonal swelling ratio is increased in type 2 diabetes when compared with HC participants; axonal swelling ratio did not differ between study participants with or without painless DSP+, or between painless DSP+ and painful DSP+. There was a weak correlation between axonal swelling ratios and HbA1c but not other clinical variables. These findings indicate that patients with type 2 diabetes with preserved intraepidermal nerve fibres have more axonal swellings compared with HC participants; however, the presence of axonal swellings is not associated with DSP or neuropathic pain. This suggests that axonal swellings are pathological and an early marker of sensory neuron injury in type 2 diabetes.
Axonal swellings have been defined and quantified in different ways. Hence, direct comparison between studies is difficult. Axonal swellings in this study were defined by absolute measurements of 1.5 μm [
14], and not by their size relative to adjoining nerve fibres [
5,
13]. We found, as did Cheung et al. [
14], no axonal swelling ratio difference between those with painless DSP and those with painful DSP. In contrast, the studies that defined axonal swellings relative to adjoining nerve fibres, as 3–5 times the diameter of the afferent nerve fibre, found an association between axonal swellings and symptomatology [
5,
13]. Therefore, studies that used axonal swellings relative to axon fibre saw an association with symptomatology, while studies using an absolute size cut-off did not. The reason for these differences is unclear. Such a finding highlights the importance of reaching a consensus on swelling definition and how this should be quantified and measured. We used an absolute cut-off value of 1.5 μm as it is more reliable than multiple measurements of the afferent fibre. Indeed, electron microscopy studies show that C fibre diameters in 95% of HC participants are less than 0.5 μm, with some as low as 0.2 μm. Therefore, in our study the axonal swellings are at least three times the upper limits of normal of healthy C fibres [
24]. Lastly, the discrepancy between our findings and other studies may be due to differences in clinical variables such as age or diabetes duration [
5,
13].
Our findings may indicate that axonal swellings are related to type 2 diabetes rather than to DSP and neuropathic symptoms. There is a weak correlation to single-point HbA1c and no correlation to type 2 diabetes duration or BMI. It is likely that axonal swellings are a sign of nerve injury. A longitudinal study tracking the development of DSP, symptoms and axonal swellings is needed to determine the natural history of axonal swellings and their relationship to DSP and neuropathic pain. It is also not clear what pathological process causes axonal swellings.
Axonal swellings are present in skin biopsies from HC participants, but are clearly higher in patients with polyneuropathies [
5,
14,
20]. Axonal swellings are described in patients with AIDS/HIV [
11,
12], pure small fibre neuropathy [
21], distal symmetric polyneuropathy of various aetiologies [
20], idiopathic neuropathic pain [
11‐
14,
20], bortezomib-induced neuropathy [
25] and amyotrophic lateral sclerosis (ALS) [
26,
27], and in myelinated nerve fibres of patients with Charcot–Marie–Tooth disease [
28]. Axonal swellings may be associated with dysfunctional axonal transport and future nerve fibre loss, or may even be a potential biomarker of axonal regeneration [
5,
12]. Detailed electron microscopy analysis of axonal swellings could yield insights into their potential mechanism, but needs technical modifications in how skin biopsies are processed.
This study has a number of strengths and limitations. Our study consisted of a large cohort of individuals with type 2 diabetes that were well phenotyped. All participants were examined by a clinician and underwent multiple tests to confirm the presence or absence of polyneuropathy and neuropathic pain. Our approach of clinical assessment followed by confirmatory investigations is the current gold standard and most rigorous approach for the diagnosis of polyneuropathy and neuropathic pain. Limitations include the retrospective study design, participants from two distinct study sites and unequal distribution among subgroups. The axonal swelling ratio was only different when IENFD >1 fibre/mm, thus limiting its role in the diagnosis of DSP, where IENFD is often lower. However, this does not negate the importance of studying axonal swellings to improve our understanding of early morphological abnormalities in diabetic neural dysfunction. Lastly, the findings in this study are applicable to type 2 diabetes only, as no patients with type 1 diabetes were included.
Acknowledgements
We would like to thank all patients and healthy volunteers for their participation.
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