Surrogate markers of diabetic neuropathy are being actively sought to facilitate the diagnosis, measure the progression, and assess the benefits of therapeutic intervention in patients with diabetic neuropathy. We have quantified small nerve fiber pathological changes using the technique of intraepidermal nerve fiber (IENF) assessment and the novel in vivo technique of corneal confocal microscopy (CCM). Fiftyfour diabetic patients stratified for neuropathy, using neurological evaluation, neurophysiology, and quantitative sensory testing, and 15 control subjects were studied. They underwent a punch skin biopsy to quantify IENFs and CCM to quantify corneal nerve fibers. IENF density (IENFD), branch density, and branch length showed a progressive reduction with increasing severity of neuropathy, which was significant in patients with mild, moderate, and severe neuropathy. CCM also showed a progressive reduction in corneal nerve fiber density (CNFD) and branch density, but the latter was significantly reduced even in diabetic patients without neuropathy. Both IENFD and CNFD correlated significantly with cold detection and heat as pain thresholds. Intraepidermal and corneal nerve fiber lengths were reduced in patients with painful compared with painless diabetic neuropathy. Both IENF and CCM assessment accurately quantify small nerve fiber damage in diabetic patients. However, CCM quantifies small fiber damage rapidly and noninvasively and detects earlier stages of nerve damage compared with IENF pathology. This may make it an ideal technique to accurately diagnose and assess progression of human diabetic neuropathy. Diabetes

OBJECTIVEThe accurate quantification of human diabetic neuropathy is important to define at-risk patients, anticipate deterioration, and assess new therapies.RESEARCH DESIGN AND METHODSA total of 101 diabetic patients and 17 age-matched control subjects underwent neurological evaluation, neurophysiology tests, quantitative sensory testing, and evaluation of corneal sensation and corneal nerve morphology using corneal confocal microscopy (CCM).RESULTSCorneal sensation decreased significantly (P = 0.0001) with increasing neuropathic severity and correlated with the neuropathy disability score (NDS) (r = 0.441, P < 0.0001). Corneal nerve fiber density (NFD) (P < 0.0001), nerve fiber length (NFL), (P < 0.0001), and nerve branch density (NBD) (P < 0.0001) decreased significantly with increasing neuropathic severity and correlated with NDS (NFD r = −0.475, P < 0.0001; NBD r = −0.511, P < 0.0001; and NFL r = −0.581, P < 0.0001). NBD and NFL demonstrated a significant and progressive reduction with worsening heat pain thresholds (P = 0.01). Receiver operating characteristic curve analysis for the diagnosis of neuropathy (NDS >3) defined an NFD of <27.8/mm2 with a sensitivity of 0.82 (95% CI 0.68–0.92) and specificity of 0.52 (0.40–0.64) and for detecting patients at risk of foot ulceration (NDS >6) defined a NFD cutoff of <20.8/mm2 with a sensitivity of 0.71 (0.42–0.92) and specificity of 0.64 (0.54–0.74).CONCLUSIONSCCM is a noninvasive clinical technique that may be used to detect early nerve damage and stratify diabetic patients with increasing neuropathic severity.

Painful neuropathy is a common and often distressing complication of diabetes. It has considerable impact on the social and psychological well-being of affected individuals. There are two distinct forms of painful neuropathy: an acute and self-limiting form that resolves within a year or a chronic form that can go on for years. There are now a number of drugs available for the treatment of neuropathic pain. However, some may fail to respond to these drugs or may have unacceptable adverse side effects. When this is the case, the patient's quality of life can be severely affected. Health care professionals need to assess the full impact of painful neuropathy. In this article we review a number of instruments that are used to assess the severity of painful neuropathy and its impact on the quality of life.

Aims/hypothesis The commercially available Neuropad test was developed as a simple visual indicator test to evaluate diabetic neuropathy. It uses a colour change to define the integrity of skin sympathetic cholinergic innervation. We compared the results of Neuropad assessment in the foot with established measures of somatic and autonomic neuropathy. Methods Fifty-seven diabetic patients underwent Neuropad assessment, quantitative sensory and autonomic function testing, and evaluation of intra-epidermal nerve fibre density in foot skin biopsies. Results Neuropad responses correlated with the neuropathy disability score (r s =0.450, p<0.001), neuropathic symptom score (r s =0.288, p=0.03), cold detection threshold (r s = 0.394, p=0.003), heat-as-pain perception threshold visual analogue score 0.5 (r s =0.279, p=0.043) and deep-breathing heart rate variability (r s =−0.525, p<0.001). Intra-epidermal nerve fibre density (fibres/mm) compared with age-and sexmatched control subjects (11.06±0.82) was non-significantly reduced (7.37±0.93) in diabetic patients with a normal Neuropad response and significantly reduced in patients with a patchy (5.01±0.93) or absent (5.02±0.77) response (p= 0.02). The sensitivity of an abnormal Neuropad response in detecting clinical neuropathy (neuropathy disability score ≥5) was 85% (negative predictive value 71%) and the specificity was 45% (positive predictive value 69%). Conclusions/interpretation The Neuropad test may be a simple indicator for screening patients with diabetic neuropathy.

OBJECTIVE -The pathogenesis of painful diabetic neuropathy (PDN) is not clear. Following our in vivo observations of increased sural nerve epineurial blood flow in patients with PDN, we investigated the cutaneous microcirculation of the foot by laser Doppler flowmetry to determine if the epineurial findings were just confined to the nerve or more widespread in other vascular beds.RESEARCH DESIGN AND METHODS -We measured foot skin vasodilator responses to acetylcholine (Ach) and sodium nitroprusside (SNP) and vasoconstrictor responses to sympathetic (deepest possible gasp) stimulation in 5 healthy control subjects, 10 nonneuropathic diabetic (NND) patients, 10 diabetic patients with painless neuropathy (PLDN), and 8 diabetic patients with PDN.RESULTS -In PDN, there were significantly reduced responses to Ach (ANOVA P ϭ 0.003) and vasoconstrictor inspiratory gasp (ANOVA P Ͻ 0.001) but not to SNP (NS). Post hoc analysis showed significant differences in Ach-induced vasodilation between PDN and nondiabetic control subjects (P Ͻ 0.05) as well as between PDN and NND (P Ͻ 0.05) but not PDN and PLDN (NS). There were no significant differences for SNP-induced vasodilation. However, there were significant differences in the vasoconstrictor response between PDN and control, NND, and PLDN (P Ͻ 0.01).CONCLUSIONS -We found an impairment of cutaneous endothelium-related vasodilation and C-fiber-mediated vasoconstriction in PDN. Inappropriate local blood flow regulation may have a role in the pathogenesis of pain in diabetic neuropathy. Prospective studies are required to determine the temporal relationship of these changes in relation to the emergence of neuropathic pain. Diabetes Care 30:655-659, 2007D iabetic peripheral neuropathy is one of the most common long-term complications of diabetes (1) and is associated with cardiovascular risk factors and mortality (2,3). Pain is one of the manifestations of diabetic neuropathy that often prompts the patient to seek medical attention and occurs in ϳ30% of patients with diabetic neuropathy (4).Painful diabetic neuropathy (PDN) is difficult to treat and results in depression and a marked reduction in quality of life (5). Despite considerable research, a complete understanding of the mechanisms of neuropathic pain remains elusive. Consequently, many patients respond poorly to drug therapies. Elucidation of the pathophysiological mechanisms underlying PDN may lead to more effective treatments (3).Many years ago, Archer et al. (6) showed that diabetic patients with PDN have altered blood flow patterns in the lower limbs. More recently, we found an increase in sural nerve epineurial blood flow in patients with PDN (7). We have also shown that patients with severe pain due to insulin neuritis have abnormal epineurial nutrient vessel anatomy and increased epineurial shunt flow, which may lead to a reduction in endoneurial nutritive blood flow (8). These observations linking PDN with alterations in blood flow are supported by two recent trials that have demonstrated significant benefits of...

OBJECTIVE -To assess the relevance of vascular endothelial growth factor (VEGF) in the maintenance of peripheral nerve integrity in diabetic neuropathy we have assessed the expression of VEGF and intra-epidermal nerve fiber density (IENFD) in skin biopsy samples from diabetic patients.RESEARCH DESIGN AND METHODS -Fifty-three diabetic patients and 12 nondiabetic control subjects underwent neurological evaluation, electrophysiology, quantitative sensory, and autonomic function testing. Dermal blood flow responses were evaluated with laser Doppler flowmetry. Skin biopsies were performed on the dorsum of the foot, and IENFD was quantified and compared with the expression of vascular endothelial growth factor A (VEGF-A), its receptor vascular endothelial growth factor receptor 2 (VEGFR-2), hypoxia-inducible factor 1␣ (HIF-1␣), and microvessel density.RESULTS -IENFD decreased progressively with increasing severity of diabetic neuropathy (P Ͻ 0.001). The dermal blood flow response to acetylcholine was reduced in diabetic patients with mild and moderate neuropathy (P Ͻ 0.01), and the intensity of staining for epidermal VEGF-A was significantly reduced in diabetic patients compared with control subjects (P Ͻ 0.01). Epidermal HIF-1␣ and VEGFR-2 expression did not differ between groups.CONCLUSIONS -Progressive endothelial dysfunction, a reduction in VEGF expression, and loss of intra-epidermal nerve fibers occurs in the foot skin of diabetic patients with increasing neuropathic severity.

Recent findings have shed new light on the role of peripheral nerves in the skin and have established a modern concept of cutaneous neurobiology. There is bidirectional rather than unidirectional (conveying information from the periphery) signaling between central and peripheral nerves and the endocrine and immune systems. This interaction is mediated principally by cutaneous small nerve fibers and will influence a variety of physiologic and pathophysiologic functions central to wound healing, which include cellular development, growth, differentiation, immunity, vasoregulation, and leukocyte recruitment. Thus, disease of the small fibers in diabetic patients is frequent and may have a considerable impact on the predisposition and subsequent wound-healing response to foot ulceration. The authors review the basic pathophysiology, clinical consequences, and current methods to evaluate somatic and autonomic nerve fiber dysfunction and damage.

OBJECTIVE -Abnormal small nerve fiber function may be an early feature of diabetic neuropathy and may also underlie painful symptoms. Methods for assessing small-fiber damage include quantitative sensory testing (QST) and determining intraepidermal nerve fiber density. We recently described a reproducible physiological technique, the LDIflare, which assesses small-fiber function and thus may reflect early dysfunction before structural damage. The value of this technique in painful neuropathy was assessed by comparing it with QST and dermal nerve fiber density (NFD).RESEARCH DESIGN AND METHODS -Fifteen healthy control subjects, 10 subjects with type 2 diabetes and painful neuropathy (PFN), and 12 subjects with type 2 diabetes and painless neuropathy (PLN) were studied. LDIflare and QST were performed on the dorsum of the foot, and dermal NFD was determined.RESULTS -Results of both large-and small-fiber quantitative sensory tests were abnormal in patients with PLN but not those with PFN compared with control subjects. Dermal NFD was also significantly reduced in the PLN group compared with control subjects (205.8 Ϯ 165.3 vs. 424.9 Ϯ 176.3 [mean Ϯ SD]; P ϭ 0.003) but not in the PFN group (307.6 Ϯ 164.5). In contrast, the LDIflare (square centimeters) was reduced in both PFN (1.59 Ϯ 0.41) and PLN (1.51 Ϯ 0.56) groups compared with control subjects (4.38 Ϯ 1.4) (P Ͻ 0.001 for both). NFD correlated significantly with the LDIflare (r ϭ 0.57, P Ͻ 0.0001).CONCLUSIONS -The LDIflare demonstrated impaired small-fiber function in patients with PFN when other assessments revealed no abnormality. We believe that this method has potential diagnostic value, particularly because it is noninvasive, has excellent reproducibility, and correlates with NFD. Furthermore, it may have an important role in assessing preventative therapies in early neuropathy. Diabetes Care 32:451-455, 2009