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Painful diabetic neuropathy is associated with increased nerve regeneration in patients with type 2 diabetes undergoing intensive glycemic control

Abstract Aims/Introduction Painful diabetic peripheral neuropathy (pDPN) is associated with small nerve fiber degeneration and regeneration. This study investigated whether the presence of pDPN might influence nerve regeneration in patients with type 2 diabetes undergoing intensive glycemic control. Materials and Methods This exploratory substudy of an open‐label randomized controlled trial undertook the Douleur Neuropathique en 4 questionnaire and assessment of electrochemical skin conductance, vibration perception threshold and corneal nerve morphology using corneal confocal microscopy in participants with and without pDPN treated with exenatide and pioglitazone or basal–bolus insulin at baseline and 1‐year follow up, and 18 controls at baseline only. Results Participants with type 2 diabetes, with (n = 13) and without (n = 28) pDPN had comparable corneal nerve fiber measures, electrochemical skin conductance and vibration perception threshold at baseline, and pDPN was not associated with the severity of DPN. There was a significant glycated hemoglobin reduction (P < 0.0001) and weight gain (P < 0.005), irrespective of therapy. Participants with pDPN showed a significant increase in corneal nerve fiber density (P < 0.05), length (P < 0.0001) and branch density (P < 0.005), and a decrease in the Douleur Neuropathique en 4 score (P < 0.01), but no change in electrochemical skin conductance or vibration perception threshold. Participants without pDPN showed a significant increase in corneal nerve branch density (P < 0.01) and no change in any other neuropathy measures. A change in the severity of painful symptoms was not associated with corneal nerve regeneration and medication for pain. Conclusions This study showed that intensive glycemic control is associated with greater corneal nerve regeneration and an improvement in the severity of pain in patients with painful diabetic neuropathy.


INTRODUCTION
In patients with type 1 diabetes, 6.5 years of intensive glycemic control reduced the incidence of diabetic peripheral neuropathy (DPN) by 60%, prevented peroneal nerve conduction velocity slowing 1 and continued to benefit patients 8 years after completion of the DCCT 2 . However, in patients with type 2 diabetes, the United Kingdom Prospective Diabetes Study (UKPDS) 3 and Veterans Affairs Co-operative Study in Type 2 Diabetes Mellitus (VA-CSDM) trial 4 reported no impact on the incidence of DPN, and the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial showed no effect on vibration perception over a period of 6 years 5 . Furthermore, multiple phase III clinical trials failed to show an improvement in diabetic neuropathy, and there are currently no US Food and Drug Administration approved therapies for DPN 6 . It is unclear whether this failure is a consequence of inadequate translation of experimental therapies, inadequate end-points or the enrolment of patients with a widely varying severity of DPN 7 .
The prevalence of painful diabetic peripheral neuropathy (pDPN) and DPN increases with age and duration of diabetes 8 . Hyperglycemia, hyperlipidemia and hypertension are associated with DPN 9 ; whereas obesity, physical inactivity and smoking cigarettes are associated with pDPN 8,10 . Neuropathic pain might be present at any stage of DPN 11 , and has been linked to a complex interplay between ongoing small nerve fiber degeneration and regeneration 12,13 . Indeed, skin biopsy studies have shown comparable intra-epidermal nerve fiber density in patients with and without painful neuropathy 14 and painful diabetic neuropathy 15,16 . However, more detailed immunohistological studies have shown an increase in regenerating intra-epidermal 17,18 and dermal nerve fibers containing substance P and calcitonin gene-related peptide in patients with painful compared with painless diabetic neuropathy 19 . Recently, B€ onhof et al. 20 showed comparable intra-epidermal nerve fiber density, but increased growthassociated protein-43 staining indicative of regenerating dermal nerves in patients with painful diabetic neuropathy. We also utilized corneal confocal microscopy (CCM) to show significantly greater corneal sub-basal nerve plexus degeneration in patients with painful compared with painless DPN 15,21,22 . These studies suggest that patients with painful diabetic neuropathy might have greater small fiber degeneration, but also an increased capacity for nerve regeneration.
CCM has been used to identify early small fiber regeneration in several clinical trials 23 . Indeed, early corneal nerve regeneration occurred 6 months 24 after pancreas and kidney transplantation, and was followed by an improvement in nerve conduction and neuropathic symptoms after 24 months 24,25 . We also recently reported that exenatide and pioglitazone or basal-bolus insulin effectively reduce glycated hemoglobin (HbA1c) 26 and induce corneal nerve regeneration 27 .
The present substudy of the Qatar study 26 assessed whether the presence of pDPN might influence nerve regeneration in patients with type 2 diabetes undergoing intensive glycemic control with exenatide and pioglitazone or insulin.

MATERIALS AND METHODS
This was an exploratory substudy of an open-label, randomized controlled trial (clinicaltrials.gov ID: NCT02887625) 26 , that examined the efficacy of exenatide and pioglitazone versus basal-bolus insulin in patients with poorly controlled type 2 diabetes. This substudy has not been registered in a public clinical trial database. Participants with type 2 diabetes were enrolled from the National Diabetes Center in Hamad General Hospital, Doha, Qatar, and studied at baseline and 1-year follow up, and control participants without diabetes were enrolled from Rumailah Hospital, Doha, Qatar, and studied at baseline only from October 2016 to November 2018.
The present study received ethical approval from the Hamad Medical Corporation IRB (IRB# 13-00076), and all participants consented to participate in the study. The study followed the tenets of the declaration of Helsinki.

Study cohort
Individuals aged 18-75 years with HbA1c >7.5% (>58 mmol/ mol) on near maximum dose of metformin (>1,500 mg/day) and sulfonylurea (>4 mg glimepiride or >60 mg gliclazide); with normal liver and kidney function, and electrocardiogram; and stable bodyweight (-1 kg) in the past year were recruited. The exclusion criteria are described in detail in our previous report 27 , but included any cause of neuropathy apart from diabetes, corneal dystrophies, corneal surgery or trauma in the past year, antidiabetic treatment other than metformin and sulfonylureas, diabetic proliferative retinopathy, and abnormally high albumin excretion.

Interventions
Participants were randomized to receive exenatide and pioglitazone or glargine and aspart insulin treatment to achieve and maintain an HbA1c <7.0% (<53 mmol/mol) 27 .

Demographic and metabolic measures
Age, sex, diabetes duration, bodyweight, body mass index (BMI), blood pressure, HbA1c, total cholesterol, triglyceride, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were recorded from the electronic health record.
DPN assessment pDPN was defined on a Douleur Neuropathique en 4 (DN4) questionnaire score ≥4, as previously described 28 . The DN4 questionnaire has been validated for distinguishing neuropathic pain from non-neuropathic pain 29 in Arabic 30 and for pDPN 28 . It consists of 10 questions relating to symptoms and signs, and each question is equally weighted. A score ≥4 has 80% sensitivity and 92% specificity for pDPN 28 . The questionnaire was administered by the investigator in English or Arabic. Medications for pDPN were recorded.
Sudomotor function was measured by electrochemical skin conductance (ESC) using Sudoscan (Impeto Medical SAS, Paris, France), as described previously 33 . Sudoscan evaluates sympathetic innervation based on sweat chloride concentrations generated by the sweat gland in response to the voltage applied 33 , and is reported as ESC in microSiemens (µS).
Vibration perception threshold (VPT) was measured using a Neurothesiometer (Horwell Scientific Laboratory Supplies, London, UK) on the pulp of the large toe on both feet, and the average value of three measurements was recorded as a VPT in Volts (V) ranging from 0 to 50 V.

Statistical analysis
Continuous variables between controls, participants with type 2 diabetes, with and without pDPN were compared using oneway ANOVA. Continuous variables were compared between the two study groups (with and without pDPN) using the unpaired t-test, whereas categorical variables were compared using the v 2 -test. Within each group, changes between baseline and 1-year follow up were compared for the continuous variables using the paired t-test. Linear regression was used to analyze the association between the change in CCM measures with type of treatment, change in DN4 score, bodyweight, diastolic blood pressure, HbA1c, cholesterol, triglyceride and LDL. No adjustment for multiple testing was carried out, as the study was exploratory in nature. All analyses were carried out using IBM SPSS (version 23; Armonk, NY, USA). A two-tailed P value of ≤0.05 was considered significant.

RESULTS
Difference between participants with and without pDPN and healthy controls A total of 41 participants with type 2 diabetes, with (n = 13) and without (n = 28) pDPN, and 18 control participants were studied ( Table 1). The proportion of those treated with basal-bolus insulin (P = 0.84) or a combination of exenatide and pioglitazone (P = 0.84) were comparable between the two groups. Three out of 13 participants with pDPN (23%) were taking medication to  Numeric variables and frequency distribution for categorical variables are summarized as the meanstandard deviation or n (%), and were compared between patients with and without painful diabetic peripheral neuropathy using the unpaired t-test and v 2 -test, respectively. Variables between controls and patients with type 2 diabetes with and without painful diabetic peripheral neuropathy were compared using one-way ANOVA, and significant differences between them are denoted as ‡ P ≤ 0.05, † P ≤ 0.01, † † P ≤ 0.001, † † † P ≤ 0.0001. BMI, body mass index; BP, blood pressure; DN4, Douleur Neuropathique en 4; CNBD corneal nerve branch density; CNFD, corneal nerve fiber density; CNFL, corneal nerve fiber length; ESC, electrochemical skin conductance; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein; VPT, vibration perception threshold. Bold values are indicates statistically significant.

Association of change in CCM measures and painful symptoms with clinical characteristics
The change in CCM measures was not associated with the type of treatment (P = 0.47) and decrease in HbA1c (P = 0.61). However, it was negatively associated with bodyweight gain, with every 2-kg increase in bodyweight, CNFD decreased by 1 fiber/mm (95% confidence interval -1.0-0.0), but this association just missed statistical significance (P = 0.0501). The change in DN4 score had no association with medications for neuropathic pain at baseline (P = 0.21), decrease in HbA1c (P = 0.81) or gain in bodyweight (P = 0.67).

DISCUSSION
The present study shows that treatment of patients with type 2 diabetes and poor glycemic control with exenatide and pioglitazone or basal-bolus insulin markedly improves glycemic control, and is associated with an improvement in painful diabetic neuropathy and corneal nerve regeneration.
Painful symptoms in DPN have been associated with active nerve degeneration and regeneration 12 . Indeed, although there are no differences in intra-epidermal nerve fiber density between those with and without pDPN 34,35 , there was a significantly lower CNFL in patients with pDPN compared with those with painless DPN 15,21 ; and in another study, CNFD was significantly lower in patients with pDPN 22 . Quantitative sensory testing has also shown increased thermal thresholds in patients with pDPN compared with those with painless DPN 36,37 . More recently, we showed lower intra-epidermal nerve fiber density and corneal nerve fibers in a large group of patients with painful compared with painless diabetic neuropathy 38 . In this study, CCM measures, sudomotor function and vibration perception threshold were comparable between patients with and without pDPN, although the number of patients with painful diabetic neuropathy was much smaller than in previous studies 15,21,22,38 .
A large improvement in HbA1c (>2-3%) has been reported to be associated with treatment-induced neuropathic pain and autonomic neuropathy 39 . However, the present study showed that despite a mean reduction in HbA1c of 3.4% among those without pDPN, and 2.5% among those with pDPN, there was no increase in the DN4 score, consistent with our previous findings 27 . Furthermore, of the 27 patients without pDPN, only one developed pDPN after 1 year of intensive glycemic control. There is a need for better neuropathy phenotyping to enable trial enrichment of participants who are more likely to respond to therapies, whether to reduce the severity of pain with therapies targeting pain or nerve regeneration in clinical trials of disease-modifying therapies for DPN. Thus, there has been a resurgence of interest in identifying biomarkers of specific pain mechanisms that might allow more effective targeted use of existing therapies 35 . Quantitative sensory testing has been used in a phenotype-stratified randomized, double-blind, placebocontrolled study to show that oxcarbazepine had a significantly greater effect in patients with an irritable nociceptor phenotype 40 . Similarly, the conditioned pain modulation test has been used to identify altered descending spinal pathways to predict greater efficacy of duloxetine 41 . We also showed altered rate-dependent depression of the H-reflex, indicative of abnormal descending inhibitory pathways in patients with pDPN 22 . However, a deep phenotyping approach to identify outcomes of disease-modifying therapies has not been undertaken to date. Indeed, despite multiple trials of disease-modifying therapies, there are currently no US Food and Drug Administration-approved therapies for DPN 6 . Several studies showed that subclinical small nerve fiber injury precedes large fiber damage in DPN 42,43 . Furthermore, early small fiber repair has been shown in several small clinical intervention trials 23,44 , and after pancreas and kidney transplantation, normalization of glycemia was associated with corneal nerve regeneration after 6 months, followed by an improvement in neuropathic symptoms and nerve conduction after 24 months 24,25 . More recently, we showed that both exenatide and pioglitazone or basal-bolus insulin effectively reduce HbA1c and induce corneal nerve regeneration, independent of changes in HbA1c, bodyweight and lipids 27 . Preclinical studies have reported that glucagon-like peptide-1 receptor agonists have a neuroprotective effect 45 and suppresses pain hypersensitivity in diabetes 46 , and although earlier clinical trials showed no benefit 47,48 , we recently showed corneal nerve regeneration with exenatide 27 . Thiazolidinediones have been reported to alleviate neuropathic pain by attenuating proinflammatory cytokine expression 49 , and preclinical studies show a prevention of nerve conduction slowing 50,51 . Indeed, the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial reported that rosiglitazone significantly reduced the 4-year cumulative incidence of clinical DPN compared with insulin 52 . Insulin treatment has also been shown to have a neurotrophic effect and reduce tactile allodynia 53 , and intensive insulin treatment might prevent nerve conduction slowing 54 and loss of ankle reflexes 55 .
We acknowledge this is a small exploratory study with potential confounders in relation to the small cohort size and effect of different treatments. Nevertheless, we showed that patients with pDPN have optimal nerve regeneration in response to improved glycemic control. We also showed that nerve regeneration might be limited due to weight gain, and, of course, recently we showed that weight loss with bariatric surgery is associated with corneal nerve regeneration 56 . Diseasemodifying treatments are also more likely to be of benefit in early or mild neuropathy where there is predominantly small fiber damage 57,58 . These findings highlight the complex pathogenesis and risk factors determining outcomes in clinical trials of diabetic neuropathy and argues strongly for pre-trial enrichment of participants. We conclude that pDPN is associated with greater corneal nerve regeneration and improvement in painful neuropathic symptoms in patients with type 2 diabetes after intensive glycemic control. The underlying mechanism is not clear and merits further study.  Numeric variables are summarized as the meanstandard deviation.