Typically, small nerve fibers are involved in the earlier phases of the natural history of diabetic neuropathy [5]. Severity of small fiber involvement correlates with hyperglycemia and diabetic disease. Patients characterized by impaired glucose tolerance more often develop small fiber neuropathy whereas diabetic patients typically have polyneuropathy involving both small and large fibers [6]. Small fibers are specifically responsible for transmission of information about pain, thermal sensation, and autonomic functions. Therefore, loss or damage of these nerve fibers determines the onset of length-dependent symptoms, such as pain, burning, tingling, or numbness affecting the limbs in a distal-to-proximal gradient [1].

Among the factors associated with neuropathic pain, those directly and indirectly related to glyco-metabolic control are probably the most relevant due to their influence on the pathogenesis and evolution of neuropathy. Chronically elevated glucose levels are the most important trigger for the development of diabetic neuropathy [7]. Support for this hypothesis derives from research in rats suffering from streptozocine-induced type 1 diabetes or Zucker fatty rats with spontaneous type 2 disease [8]. Hyperglycemia launches a cascade culminating in the accumulation of intermediate products of glucose metabolism including excess reactive oxygen species. While these reactive oxygen species are normally controlled by standard antioxidant mechanisms present and working in the cells [9], the normal system of scavenging free radicals can be weakened by diabetes [10]. Eventually persistent hyperglycemia impairs the usual balance between pro-inflammatory conditions and cellular defenses, and excess free radicals overwhelm the intrinsic antioxidant systems [11]. Patients with peripheral neuropathy associated with or without autonomic dysfunction demonstrate increased levels of three relevant pro-oxidative markers, 8-iso-prostaglandin F_2a, levels of superoxide anion generation, and lag time to peroxidation by peroxynitrite, and also decreased levels of the two most important defenses: vitamins C and E [12]. The two most important pathways responsible for this cascade are the advanced glycosylated end products pathway (AGEs) and the Polyol pathway [13]. Prolonged oxidative stress injury eventually causes demyelination of nerve fibers. With time, the damage of multiple myelinated and/or nonmyelinated Schwann cells leads to a clinically significant deficit that may be associated with reduced nerve conduction velocity. One recent validated study also demonstrates that impaired glucose tolerance, a condition sometimes known as “prediabetes,” is commonly associated with earlier stages of peripheral diabetic neuropathy [14]. However, no association has been demonstrated with impaired fasting glucose and DPN. This suggests that only the prolonged postprandial hyperglycemic episodes, and not necessarily chronic hyperglycemia, should correlate with nervous injury [15]. The nerve damage seems to be earlier with respect to the establishment of the hyperglycemic state, and appears to begin to stabilize while the functionality of beta pancreatic cells decreases.

As a consequence of prolonged hyperglycemia a significant modification of the redox state has been demonstrated in both type 1 and type 2 diabetes. The redox state can be defined as the balance between oxidants and antioxidants factors that is involved in the maintenance of normal cellular functions. It is well established that in diabetic neuropathy a reduction of endoneural blood flow causes local nervous tissue hypoxia and eventual ischemia [16]. This reduction in blood flow is related to excess production of endothelial superoxide radicals that reduce nitric oxide synthase (NOs) activity and increase the level of N-nitro-L-arginine, a powerful NOs inhibitor [17]. Nitric oxide levels are particularly relevant to diabetic neuropathic pain. Decreased levels of nitric oxide play a double role: on one hand reducing endoneural circulation, especially at the level of vasa nervorum, and exaggerating and accelerating the direct effect of hyperglycemic conditions on glial cells and neurons, and on the other hand reducing the stimulation on Na⁺/K⁺-ATPase activity, a membrane channel particularly important in the development of painful peripheral neuropathy [18]. Elevated homocysteine levels have been proposed as a marker of this chronic condition of endothelial dysfunction in patients with diabetic neuropathy [19].

There is an increasingly frequent form of peripheral neuropathy in patients shortly after institution of insulin treatment for diabetes mellitus. Described for the first time in 1933, this acute peripheral neuropathy is known as insulin neuritis. It is due to abnormal ectopic sensory responses caused by acute axon regeneration that increases the level of peripheral neuropathic sensitivity [16]. Proliferation of new vessels at the level of epineurium, similar to that observable in diabetic retinopathy, may be diagnosed through fluorescein angiography at the level of sural nerve. This form of acute diabetic neuropathy is present in both forms of diabetes and is related to acute improvement of glycemic control [9]. One likely theory is that this neuropathy is a result of upregulation of cytokines, especially interleukin 1β and 6 and TNF-α, which lead to the developing of artero-venous shunts, responsible for endoneural hypoxia and to the consequent apoptosis related to acute glucose deprivation [20]. The onset of this kind of treatment-induced diabetic neuropathy is generally characterized by an acute pain associated with autonomic symptoms, especially gastrointestinal and genitourinary symptoms [21], and occasionally associated with localized involvement of cranial nerve roots [22]. In the large majority of cases, symptoms resolve over weeks to months [21].

The relation between glycometabolic control and pain in diabetic neuropathy is complex and is influenced both by the chronicity of the disease and by the acute glycemic fluctuations, which acutely worsen pain perception. The level of glycometabolic control affects different forms of diabetic painful neuropathy differently. Indeed, acute and chronic forms of painful neuropathy are distinguishable patterns in patients affected by diabetic neuropathy [7]. Acute pain is typically characterized by allodynia and hyperesthesia and is generally associated with severe sensory and autonomic symptoms as well as a reduction or absence of normal tendon reflexes. Chronic pain is usually characterized by typical symptoms of burning, stabbing, snappy, and pinprick-type pain and is localized primarily to the feet and legs. It is usually worse at night, often affecting sleep, and can sometimes be relieved by cooling the affected area [23].

The acute form of painful neuropathy worsens relative to poor glycometabolic control, as levels of nerve damage increase with occasions of glycometabolic failure. In this acute form of painful neuropathy, it is possible to observe a sudden debut of tight pain, associated with dysesthesia and paresthesias during times of acute hyperglycemia [7]. Not only chronic hyperglycemia but also wide fluctuations in blood glucose levels have been demonstrated to be strictly associated with neuropathic pain in diabetic patients; wider ranging glycemic level worsens the perception of pain in neuropathic patients, while strict metabolic control reduces the perception of pain [7, 15]. The Diabetes Control and Complication Trial (DCCT) and other studies have demonstrated that tight glycometabolic control with near-normal levels of glycated hemoglobin (HbA1c) can prevent or slow the progression of diabetic painful neuropathy [24].

The effect of glycemic control and the evolution of diabetes on chronic neuropathic pain are biphasic. Earlier in the disease, the natural evolution of the pathology leads to increasing nervous damage and worsening of painful symptoms. However, later in the more advanced stages of the disease pain usually subsides because the nerve fibers are extremely damaged and have suffered intense axonal depletion. During these late phases, hypoesthesia is usually the main feature of the disease [25, 26].

According to the ADA recommendations, all patients with diabetes should be screened for DPN at diagnosis of type 2 diabetes and 5 years after the diagnosis of type 1 diabetes and at least annually thereafter for early detection of neuropathy. Prior to diagnostic procedures, the patient’s risk factors for microvascular complications, such as metabolic control, age, and lifestyle, should be reviewed.

Patients should complete a questionnaire for the evaluation of symptoms and signs related to diabetic neuropathy [27]. The more frequently adopted and validated questionnaires are the Michigan Neuropathy Screening Inventory (MNSI) and Neuropathy Disability Score (DNS). The MNSI is used widely for the evaluation of distal symmetrical peripheral neuropathy in diabetes. It includes two separate assessments, a 15-item self-administered questionnaire that is scored by summing abnormal responses, and a lower extremity examination that includes inspection and physical examination of the patient using several different instrumental methods and assigning points for abnormal findings [28]. The DNS modified according to Dyck criteria consists of eight items, two testing muscle strength, one a tendon reflex, and five sensations. The maximum score is 16. A score of >3 points is considered abnormal [29].

Other tests performed in the analysis of initial signs of diabetic neuropathy should include a monofilament examination, analysis of pain and vibration sensibility, and reflex evocation. A monofilament exam can be performed using a Semmes Weinstein 10 g monofilament, a nylon filament embedded in a plastic handle, which allows quantification of light touch perception. Pain sensitivity is tested using a sterile needle applied on the dorsum of the great toe or on the plantar side of the metatarsal heads. Vibration sensitivity is tested using a biothesiometer which applies vibration at increasing voltage until reaching the lower perceptible by the patient, defined as the vibration perception threshold (VPT). A VPT lower than 25 V is an effective predictor of the risk of foot ulceration [30]. The reflex examination should be conducted at both ankles while the patient is sitting or kneeling. If no reflex occurs, the test can be repeated more forcefully [31].

The second-level quantification of peripheral neuropathic dysfunction includes measuring nerve conduction velocity (NCV). NCV is the minimum criteria for diagnosis of DPN related to large myelinated nerves [1, 32]. NCV allows classification of nerve dysfunction by distinguishing axonal damage from demyelination. We can define axonal damage as a reduction of NCV, while demyelination is characterized by decrease in action potential amplitude. Action potential amplitude is defined as the summation of single neuronal cell potentials and is linearly related to intraepidermal nerve fiber density [33].

In the earlier stages of disease, the diagnosis and quantification of small fiber damage are difficult, because the traditional tests are not suited to evaluating small nonmyelinated fibers.

More precise and dedicated methods have been proposed to investigate this component of DPN. Some examples include skin biopsy, quantitative sudomotor axon reflex testing, and quantitative sensory testing. Skin biopsy is a minimally invasive procedure in which 3 mm diameter punch biopsy specimens are taken from distal leg, distal thigh, or proximal thigh of one lower limb [4]. The morphometric quantification of intraepidermal nerve fibers (IENF), generally expressed as number of fibers per length of section, is identified [2]. Quantitative sudomotor axon reflex testing (QSART) is an autonomic study that measures sweat output after stimulation of C-nociceptive fibers by acetylcholine iontophoresis [34]. Quantitative sensory testing is a functional assessment of the thermo-nociceptive pathway from nerve terminals through the entire nervous system. It analyzes the thermal thresholds delivering a stimulus from a baseline temperature of 32°C for an increase or a decrease of 1.0 °C [35].

The analysis of pain symptoms is particularly relevant in the evaluation of chronic disease in patients who are often affected by multiple comorbidities. Pain scales are the most common modality to assess the level of pain throughout treatment. Different scales have been created to be completed by the patient or by a healthcare provider. The most commonly used pain scales are the visual analogue scale (VAS) designed by Scott and Huskisson, and the Numeric Rating Scale [36]. Analysis of the Quality of Life of patients is also important and may be measured through a dedicated questionnaire, specifically developed as a tool for the assessment of patients’ perception of performance in normal activities [37].