Neuropathic pain arises as a consequence of a lesion or disease affecting the somatosensory system [1,2]. Neuropathic pain states are typically characterized by coexistence of positive and negative sensory signs. The diagnosis of a neuropathy is, however, based on tests demonstrating only the sensory deficits. For the diagnosis of a clinically suspected neuropathy of the thick myelinated Aß-fibers, nerve conduction studies (NCS) and somatosensory evoked potentials have been regularly used for decades. These methods, however, do not assess the small nerve fibers (Aδ-and C-fibers), which have come more into focus in the last years, as the clinical relevance of small fiber neuropathies has been rising. Additional tools being able to close this diagnostic gap are skin biopsies and standardized quantitative sensory testing (QST) [3–6]. The function of the unmyelinated and thinly myelinated afferent nerve fibers can be assessed by QST, whereas morphological alterations and a rarefication of the nerve fibers can be depicted by analysis of the intraepidermal nerve fiber density in skin biopsies and more recently also by confocal microscopy of corneal nerve fibers [corneal confocal microscopy (CCM)] [7▪▪]. Other techniques, including microneurography and recording of nociceptive evoked potentials, are currently being used mainly in research and might also give new insights into pathophysiological aspects of neuropathic pain.
The analysis of the somatosensory profile of a single patient with neuropathic pain, including both signs of gain and loss of function, can only be performed by QST. This allows subgrouping of patients into patients with irritable nociceptors, profound deafferentiation or mixed forms (, see Fig. 1) an approach, which might contribute to individualized treatment of neuropathic pain in the future.
The aim of this review was to give a short overview about new developments in the diagnostic procedures of neuropathic pain, discussing the role of the above-mentioned methods for the clinical routine.
CLASSICAL NEUROPHYSIOLOGIC METHODS
Standard neurophysiological methods are widely used to investigate diseases of the peripheral and central nervous system. They include nerve conduction studies, trigeminal reflexes (including the blink reflex) and measurement of somatosensory evoked potentials. These methods assess large non-nociceptive afferent fibers (Aβ-fibers and the dorsal column system, respectively) and are used to confirm lesions of the somatosensory system, thus being a helpful tool in the diagnostic algorithm of neuropathic pain [9–12].
QUANTITATIVE SENSORY TESTING
Quantitative sensory testing (QST) is a noninvasive psychophysical method assessing human responses to painless or painful stimuli (e.g., pressure, pinprick or thermal stimuli, vibration). It assesses both small and large-fiber sensory function including their corresponding central pathways, though without addressing morphological and topographical aspects .
Stimuli of increasing or decreasing intensity are applied, and the patient reports when a sensation first appears or disappears. A threshold is defined as a mean from a series of trials during the continuous stimulus application (method of limits), or from a series of predefined stimuli (method of levels), which is independent of the reaction time, but takes longer than the method of limits. For all sensory qualities, negative (hypoesthesia, hypoalgesia) and positive (allodynia, hyperalgesia, hyperesthesia) phenomena can be assessed. A standardized assessment using standardized instructions and calibrated stimuli applied by highly trained investigators is essential . The protocol of the German Research Network on Neuropathic pain (DFNS) is a multicenter validated QST protocol, widely used in several international QST-labs [8,13]. Normative data for this protocol are available, allowing the use of correction factors for sex, different age groups, including children and adolescents, and different body areas (face, hand, foot, trunk), which can be done manually or by PC-based software (Equista) [14,15▪,16].
QST can be used to detect sensory impairment, for example, in small-fiber neuropathy (SFN). Pure SFN is characterized by isolated impairment of the thinly myelinated Aδ and unmyelinated C-fibers, which typically presents with dysparathesias and paresthesias and/or burning pain with usually normal results in NCS [17▪▪]. In SFN, QST shows abnormally increased thermal detection thresholds with a sensitivity of 36–85% in comparison to an invasive assessment by skin biopsy . The sensitivity of QST varies considerably as peripheral nerve fiber loss can be compensated centrally, especially observed in children [18,19]. Furthermore, QST is able to depict an affection of the Aβ-fibers in patients with chemotherapy-induced polyneuropathy and normal NCS [20▪]. In Restless Legs Syndrome, hyperalgesia to pinprick-stimuli, tactile hypoesthesia and paradoxical heat sensations as a sign of disinhibition because of affected Aδ-fibers also provide evidence for a neuropathic component shown by QST . Using the DFNS protocol, QST offers no diagnostic advantage for the differentiation between complex regional pain syndrome (CRPS) and peripheral nerve injury . Extensions of the protocol can, however, increase the specificity, as pressure hyperalgesia over the proximal interphalangeal joints was a characteristic for CRPS [23▪].
Detailed analysis of the sensory profile using QST enables speculating about the different underlying pathomechanisms in patients with neuropathic pain . This might also strengthen therapeutic decisions in single patients (see Table 1). In a recent prospective, randomized, double-blind trial [25▪▪] and in most retrospective analyses [26,27▪,28,29], the presence of hyperalgesia was shown to be a predictor for better treatment response (see Table 1).
Like all psychophysical methods, QST requires the active participation of the patient. Therefore, the consistency of QST results should be considered during the data evaluation and interpretation. QST is not validated for discrimination between abnormalities because of neuropathy or other non-neuropathic origin, as increased detection thresholds and/or hyperalgesia in each QST parameter alone are not specific for neuropathic pain . The sensitivity can, however, be increased by interpretation of the whole sensory profile . As the whole somatosensory pathways are assessed, no topical attribution of the location of detected lesions is possible.
INTRAEPIDERMAL NERVE FIBER DENSITY ASSESSED BY SKIN BIOPSY
Intraepidermal nerve fibers (IENFD) are the terminal endings of small neurons of dorsal root ganglia, which lose the Schwann cell ensheathment while crossing the dermal–epidermal barrier . Their morphometric analysis in skin biopsies is possible using the antibody directed against the cytoplasmatic protein gene product 9.5 . The European Federation of Neurological Societies and Peripheral Nerve Society have published guidelines on the use of skin biopsies in the diagnosis of SFN . Distal leg skin biopsy with quantification of the density of IENFD, using generally established counting rules, is regarded as a reliable and efficient technique to prove the diagnosis of SFN. One can use material gathered either by a punch biopsy or by the removal of the epidermis alone by applying the blister technique [33,34]. The retest reliability of the IENFD within 3 weeks was good . Using skin biopsies, evidence of SFN has been detected in a variety of conditions [17▪▪], recently also in a subgroup of patients with fibromyalgia [36–39]; thus, skin biopsies were recommended as an important part of diagnostic testing for SFN among FM patients . A decreased IENFD has recently been reported also in patients with amyotrophic lateral sclerosis with spinal, but not with bulbar onset [41▪].
Interestingly, the decreased IENFD recovered after causal treatment of the neuropathy (hormone replacement in hypothyroidism , steroid-sensitive neuropathy  and metabolic improvement in prediabetic neuropathy , as well as because of exercise programs in diabetic patients without neuropathy symptoms and in metabolic syndrome neuropathy [45,46]). A recent study demonstrated no difference in the IENFD between patients with and without neuropathic pain, or between patients with and without ongoing burning pain, confirming previous results . The IENFD was, however, higher in patients with provoked pain than in those without , suggesting that this type of pain might be mediated by spared and sensitized nociceptive afferents, which might have implications for treatment approaches.
Additional parameters are axonal swelling and sweat gland volume and innervation; however, their relevance for the neuropathy diagnosis in the clinical routine still remains unclear [48▪,49].
CORNEAL NERVE FIBERS ASSESSED BY CONFOCAL CORNEAL MICROSCOPY
The nerve fibers in the cornea originate from the ophthalmic branch of the trigeminal nerve and have come in the focus of neuropathic pain research over the last years. The sub-basal plexus lies between the basal epithelium and Bowman's membrane, runs parallel to the surface of the eye and consists of small nerve fibers with a thickness between 0.2 and 10 μm (Aδ or C-fibers with low-threshold polymodal receptors for nociception and mechanical and cold stimuli) .
For the CCM, the eye is prepared with local anesthetic and hypromellose. The microscope is placed over the central cornea to establish physical contact between the microscope and the cornea. The microscope is connected to a retina tomograph enabling high-resolution photographs of the sub-basal nerve plexus. Analysis of the pictures with the best representation of corneal nerve fibers can be done either manually or using software (examples in Fig. 2). The most important parameters to be analyzed are the corneal nerve fiber length (NFL: absolute length of nerves and branches per mm2), nerve fiber density (NFD: number of major nerves per mm2) and nerve fiber branching (NFB: number of branch points per mm2).
CCM has been mostly used in patients with diabetic polyneuropathy and sarcoidosis; for other diseases, there are only few data available so far. A recent meta-analysis of 13 studies with 1680 participants confirmed its value for the detection of early nerve damage in patients with diabetic neuropathy [7▪▪]. Interestingly, patients with impaired glucose tolerance (IGT) already show evidence of neuropathy detected by CCM [51▪]. Further, patients with IGT, who later developed type II diabetes, had significantly lower NFD, NFL and NBD compared with controls [52▪]. Additionally, CCM detects small fiber damage in the absence of other disease complications such as retinopathy or microalbuminuria in patients with type I diabetes . Thus, CCM can help to identify patients at risk who need a stricter treatment regime. There was a correlation between the corneal nerve fiber loss and the severity of diabetic polyneuropathy [54,55]. Currently, many other diseases are being investigated, for example, chronic migraine , Wilson disease , chronic inflammatory demyelinating polyneuropathy  or amyotrophic lateral sclerosis , demonstrating signs of neuropathy detected by CCM. Compared with other tools, the diagnostic efficacy of CCM is comparable or even higher than the quantification of the IENFD [48▪], but has not been compared with QST in larger cohorts so far. The gathering of normative values from healthy volunteers is a further step for enabling the wide clinical use of this technique [60▪] (TavakoliDiabetesCare2015). Interestingly, CCM and skin biopsy both detect nerve fiber loss in recently diagnosed type 2 diabetes, however not always in the same patients, suggesting a patchy manifestation pattern of SFN . A great advantage of CCM seems to be that the corneal nerve density changes more dynamically after treatment than skin innervation or nerve function measured by QST. In patients with diabetes, HbA1c-improvement correlated significantly with nerve fiber density . Similarly, significant pain relief and partial normalization of the corneal NFL and NFD were reported after treatment of sarcoidosis with ARA 290, an 11-amino-acid peptide derived from the structure of erythropoietin .
Limitations for the routine use of CCM are the high acquisition costs and availability only in specialized centers. The exact influence of non-neurological and ophthalmological diseases, such as Sicca syndrome, on the parameters is still unclear. Also, comparison to established methods such as skin biopsy and QST in different diseases is needed for further validation of this method. CCM cannot differentiate between the affection of different fiber systems morphologically.
TOOLS USED IN CLINICAL RESEARCH
Nociceptive evoked potentials
Nociceptive evoked potentials are objective measures assessing the conduction of small nerve fibers . Both laser-evoked potentials (LEPs) and contact heat-evoked potentials (CHEPs) result from a selective activation of Aδ- and C-fibers, whereas pain-related evoked potentials (PREPs) are mostly based on the stimulation of Aδ-fibers .
The main clinically useful LEP signal is a widespread negative–positive complex (N2–P2) with a latency of 150–380 ms and maximum amplitude at the vertex, generated by the anterior cingulate gyrus, and possibly contributed by the bilateral opercular–insular regions, mostly reflecting the Aδ-activation. Depicting the evoked potentials specific of C-fiber stimulation is technically more challenging . Abnormal LEP can represent conduction abnormalities at any point in the pain–temperature pathway, including peripheral nerves, plexus, roots, spinal cord or brainstem .
CHEPs can be induced by a heat-foil contact heat stimulator with extremely rapid heat rising time (70 °C/s), and very rapid elicitation of pain, whereas late CHEPs are associated with Aδ-fiber activation and ultralate CHEPs with C-fiber activation . Though normative data have been recently published, their clinical use is limited by the fact that they cannot be recorded in all healthy individuals .
PREPs are obtained using a concentric planar electrode that delivers electrical stimuli at the pain threshold, limited to the superficial layer of the dermis . PREPs can also be elicited by intraepidermal electrical stimulation by a pin electrode, reflecting mostly selective Aδ-activation [68,69]. Abnormal PREPs have been shown to correlate with IENFD in HIV-neuropathy  and to be affected early in nonsymptomatic diabetic patients .
Microneurography is an invasive and technically demanding neurophysiological technique, recording the action potentials of single sensory nerve fibers in wake humans. Microelectrodes are inserted into a fascicle of a nerve. The nerve can be identified by either electrical stimulation using a needle electrode or by ultrasound monitoring. As even single C-fiber action potentials from nociceptors can be assessed, this method offers valuable information about the pathophysiology of sensory and axonal abnormalities in pain processes. During the assessment, the nerve fibers are stimulated (e.g., by electrical stimuli or touch). On the basis of the responses to the stimulation, the nociceptors can be classified into mechanosensitive or mechano-insensitive [72▪▪].
In patients with diabetic neuropathy, the composition of nociceptors is altered in comparison to healthy controls, as the ratio of mechano-responsive to mechano-insensitive receptors reverses from 2 : 1 in healthy individuals to 1 : 2 in patients. Additionally, patients exhibit many C-fibers having lost their mechanical and heat responsiveness . Further, nociceptors of patients with neuropathic pain reveal higher spontaneous activity, sensitization and hyperexcitability of mechano-insensitive nociceptors .
Although the first randomized, double-blind, controlled study of a highly selective T-type Cav3.2 calcium channel blocker in patients with painful diabetic neuropathy failed to show any changes in the microneurographic recordings , this method remains of great interest having the potential to serve as a biomarker for spontaneous pain.
Diagnosing neuropathic pain requires the confirmation of a lesion or disease affecting the somatosensory system. In addition to the traditional NCS and somatosensory evoked potentials (including LEP, and in the future potentially also PREP), methods assessing the small fiber function and/or morphology should be performed. In case of conclusive abnormal QST findings and clinical signs, a neuropathy can be strongly suspected. In case of normal QST values and/or inconsistency with the clinical appearance, skin biopsies or, especially in case of suspected diabetic neuropathy, CCM should be performed to confirm an SFN (Fig. 3).
QST and morphometric analysis might be used as clinical biomarkers for the prediction of response to treatment in trials, either as inclusion criteria for enrichment of the patient population or as a stratification tool in analysis, and might be helpful in the future to find the best analgesic for the individual patient.
Financial support and sponsorship
E.K.E.K. and T.M. were, and C.M. is part of the EUROPAIN project which has received support from the Innovative Medicines Initiative Joint Undertaking under Grant Agreement no 115007, resources for which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007–2013) and European Federation of Pharmaceutical Industries and Associations (EFPIA) companies’ in kind contribution. E.K.E.K. was also supported by intramural fundings of the Ruhr University Bochum (FoRUM: Grant Number K046-10, Heinemann Award 2013).
Conflicts of interest
There are no conflicts of interest.
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