Páll Karlsson

Dermal innervation in healthy subjects and small fiber neuropathy patients: a stereological reappraisal.

The aim of this study was to estimate dermal nerve fiber length (DNFL) using a stereological sampling technique in comparison with a previously reported manual estimation. DNFL was analyzed in skin punch biopsy specimens from 24 healthy volunteers and 18 patients with small fiber neuropathy (SFN) using global spatial sampling that yields unbiased and reliable length estimation. The estimation was carried out in 50‐µm biopsy sections after immunostaining with anti‐protein gene product (PGP) 9.5 antibodies. The length of the PGP9.5‐positive dermal nerves from the dermal–epidermal junction and 200 µm down was measured (DNFL mm−2). Results were compared with our previously reported manual method. Patients showed a significantly (p < 0.0001) lower DNFL (105 mm−2 ± 6.4 SD) than healthy subjects (246 mm−2 ± 8.39 SD). Moderate correlation with age was observed for both healthy subjects (Pearsons r = −0.33) and patients (Pearsons r = −0.59). A significant (p < 0.001) correlation between global spatial sampling and manual estimation was observed in both patients and healthy subjects (Pearsons r = 0.62 and 0.61, respectively). These findings provide further evidence on the reliability of dermal nerve morphometry in human skin and strengthen the hypothesis that dermal nerve fibers undergo significant degeneration in SFN.

Epidermal nerve fiber length density estimation using global spatial sampling in healthy subjects and neuropathy patients.

Assessment of intraepidermal nerve fiber density (IENFD) has become a useful tool for the investigation of patients with suspected small-fiber neuropathy (SFN). Here, we estimate epidermal nerve fiber lengths in 12 patients with SFN and 36 healthy controls using global spatial sampling and compare the lengths with IENFD and axonal swelling ratios. Skin biopsies were analyzed on 50-μm-thick free-floating sections immunostained for the neuronal cytoplasmic marker PGP 9.5. Mean IENFD in SFN patients was 2.22 ± 1.63 mm versus 7.51 ± 2.17 mm in controls; mean length density was 112 ± 82.6 mm in SFN patients versus 565 ± 240 mm in controls (p < 0.001 for both). The correlation between the nerve fiber length and the IENFD was r = 0.16 for healthy subjects and r = 0.39 for patients, suggesting that these variables provide different quantitative information. There were significant differences in axonal swelling ratios between healthy subjects and patients, that is, per IENFD and per nerve fiber length. Together, these results suggest that, although length estimation requires more time and additional equipment, it is as effective as IENFD in differentiating SFN patients from healthy subjects. Estimating nerve fiber length may increase mechanistic understanding beyond IENFD estimation and improve efficiency in diagnosing SFN.

Structural and functional assessment of skin nerve fibres in small-fibre pathology.

Damage to nociceptor nerve fibres may give rise to peripheral neuropathies, some of which are pain free and some are painful. A hallmark of many peripheral neuropathies is the loss of small nerve fibres in the epidermis, a condition called small‐fibre neuropathy (SFN) when it is predominantly the small nerve fibres that are damaged. Historically, SFN has been very difficult to diagnose as clinical examination and nerve conduction studies mainly detect large nerve fibres, and quantitative sensory testing is not sensitive enough to detect small changes in small nerve fibres. However, taking a 3‐mm punch skin biopsy from the distal leg and quantification of the nerve fibre density has proven to be a useful method to diagnose SFN. However, the correlation between the nerve fibre loss and other test results varies greatly. Recent studies have shown that it is possible not only to extract information about the nerve fibre density from the biopsies but also to get an estimation of the nerve fibre length density using stereology, quantify sweat gland innervation and detect morphological changes such as axonal swelling, all of which may be additional parameters indicating diseased small fibres relating to symptoms reported by the patients. In this review, we focus on available tests to assess structure and function of the small nerve fibres, and summarize recent advances that have provided new possibilities to more specifically relate structural findings with symptoms and function in patients with SFN.

Structural and functional characterization of nerve fibres in polyneuropathy and healthy subjects

Abstract Objectives Quantification of intraepidermal nerve fibre density (IENFD) is an important small fibre measure in distal symmetric polyneuropathies (DSP), but quantitative evaluation of additional structural and functional factors may help in elucidating the underlying mechanisms, and in improving the diagnostic accuracy in DSP. The literature reports a weak or moderate relationship between IENFD and spontaneous and evoked pain in neuropathies, but the relationship between functional and structural small fibre parameters in patients with DSP is unclear. The objectives of the current study, therefore, were to determine morphological and functional parameters related to small nerve fibres in subjects with distal symmetric polyneuropathy (DSP) and healthy controls, and to characterize the interplay among these parameters in these two groups. Materials and Methods 17 patients with painful DSP (≤4 on 0-10 numerical rating scale) and with symptoms and signs of small fibre abnormality (with or without large fibre involvement) and 19 healthy control subjects underwent comprehensive functional and structural small fibre assessments that included quantitative sensory testing, response to 30 min topical application of 10% capsaicin and analysis of skin biopsy samples taken from the distal leg (IENFD, epidermal and dermal nerve fibre length densities (eNFLD, dNFLD) using global spatial sampling and axonal swelling ratios (swellings/IENFD and swellings/NFLD)). Results DSP patients had reduced sensitivity to cold (median -11.07°C vs. -2.60, P<0.001) and heat (median 46.7 vs. 37.70, P<0.001), diminished neurovascular (median 184 vs. 278 mean flux on laser Doppler, P=0.0003) and pain response to topical capsaicin (median 10 vs. 35 on 0-100 VAS, P=0.0002), and lower IENFD, eNFLD and dNFLD values combined with increased swelling ratios (all P< 0.001) compared to healthy controls. The correlation between structural and functional parameters was poor in DSP patients, compared with healthy controls. In healthy controls eNFLD and dNFLD, IENFD and eNFLD, IENFD and dNFLD all correlated well with each other (r = 0.81; P < 0.001, r = 0.58; P = 0.009, r = 0.60; P = 0.007, respectively). In DSP, on the other hand, only eNFLD and dNFLD showed significant correlation (r = 0.53, P = 0.03). A diagnostic approach of combined IENFD and eNFLD utilization increased DSP diagnostic sensitivity from 82.0% to 100% and specificity from 84.0% to 89.5%. Conclusions This study presents a rigorous comparison between functional and morphological parameters, including parameters such as eNFDL and dNFLD that have not been previously evaluated in this context. The correlation pattern between functional and structural small fibre parameters is different in patients with DSP when compared to healthy controls. The findings suggest a more direct relationship between structure and function of nerve fibres in healthy controls compared to DSP. Furthermore, the findings suggest that combining IENFD with measurement of NFLD improves the diagnostic sensitivity and specificity of DSP. Implications Combining small fibre parameters may improve the diagnostic accuracy of DSP.

Macrophage-to-sensory neuron crosstalk mediated by Angiotensin II type-2 receptor elicits neuropathic pain

Peripheral nerve damage initiates a complex series of cellular and structural processes that culminate in chronic neuropathic pain. Our study defines local angiotensin signaling via activation of the Angiotensin II (Ang II) type-2 receptor (AT2R) on macrophages as the critical trigger of neuropathic pain. An AT2R-selective antagonist attenuates neuropathic, but not inflammatory pain hypersensitivity in mice, and requires the cell damage-sensing ion channel transient receptor potential family-A member-1 (TRPA1). Mechanical and cold pain hypersensitivity that are characteristic of neuropathic conditions can be attenuated by chemogenetic depletion of peripheral macrophages and AT2R-null hematopoietic cell transplantation. Our findings show no AT2R expression in mouse or human sensory neurons, rather AT2R expression and activation in macrophages triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on sensory neurons. Our study defines the precise neuro-immune crosstalk underlying nociceptor sensitization at the site of nerve injury. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic neuropathic pain, and therefore identifies multiple analgesic targets.

Improved sampling and analysis of images in corneal confocal microscopy

Introduction: Corneal confocal microscopy (CCM) is a noninvasive clinical method to analyse and quantify corneal nerve fibres in vivo. Although the CCM technique is in constant progress, there are methodological limitations in terms of sampling of images and objectivity of the nerve quantification. The aim of this study was to present a randomized sampling method of the CCM images and to develop an adjusted area‐dependent image analysis. Furthermore, a manual nerve fibre analysis method was compared to a fully automated method.

Bilaterally Reduced Intraepidermal Nerve Fiber Density in Unilateral CRPS-I

Objective Findings regarding small nerve fiber damage in complex regional pain syndrome type I (CRPS-I) are not uniform, and studies have not included a matched healthy control group. The aim was to assess intraepidermal nerve fiber density (IENFD) in relation to thermal sensitivity of the same skin areas in CRPS-I patients and a gender- and age-matched healthy control group. Methods IENFD was investigated in skin biopsies from the CRPS-affected and contralateral limbs of eight CRPS-I patients and from an equivalent site in eight gender- and age-matched healthy controls (HCs). Thermal thresholds (cold/warm detection, cold- and heat-pain detection) were assessed on the affected limb, the matching contralateral limb, and on the equivalent limbs of HCs, and participants rated the intensity of cold/heat and pain to static thermal stimuli (5 °C and 40 °C). Results IENFD was significantly lower in both the affected and contralateral limbs of CRPS-I patients than HCs, but IENFD did not differ between the affected and contralateral limbs of patients. The heat pain threshold was lower in the affected CRPS-I limb than in HCs, but all other thermal thresholds were similar in both groups. CRPS-I patients rated the cold stimulus as colder and more painful in the affected limb, and the warm stimulus as hotter, bilaterally, than the HCs. Conclusions CRPS-I may be associated with bilateral small fiber damage, and perhaps small fiber neuropathy and bilateral disturbances in thermo-sensory perception. These disturbances could stem from a systemic response to injury or might increase the risk of developing CRPS-I after physical trauma.

Structural, functional, and symptom relations in painful distal symmetric polyneuropathies: a systematic review

Abstract The diagnosis of distal symmetric polyneuropathies (DSPs) relies on the presenting symptomatology and neurological sensory examination, supported by objectively quantified structural and functional changes in sensory nerves. Although these separate components have important diagnostic utility, the associations between the structural vs the symptomatic and functional findings in painful DSP are still unclear. It is assumed that delineation of the correlations, or lack of such, between structure, clinical presentation, and function will contribute to a better understanding and treatment of DSP. This systematic review assessed small fiber morphology in patients with different types of painful DSP, and compared it with symptoms, signs, and nerve fiber function. Overall, 111 papers met the inclusion criteria for the systematic review. The results indicate that epidermal nerve fiber loss, in isolation, is not a useful indicator of painful symptoms or their severity in DSP. Intraepidermal nerve fiber density correlated reasonably well with neuropathy scores on tools assessing signs and symptoms (such as the Michigan Neuropathy Screening Instrument and the Total Neuropathy Score), but less so with symptom measures only. Among various psychophysical sensory measures, warmth detection and heat pain thresholds correlated best with intraepidermal nerve fiber density, particularly when assessed at the same anatomical site. The observed sources of heterogeneity, and the lack of associations between structural and functional measures in several studies are discussed. A framework is proposed for uniform assessment of nerve fiber parameters for investigating clinically relevant mechanisms of neuropathic pain in DSP.

How central is central poststroke pain? The role of afferent input in poststroke neuropathic pain: a prospective, open-label pilot study

Abstract Central poststroke pain (CPSP) is a neuropathic pain disorder, the underlying mechanisms of which are not well understood. It has been suggested that stroke-associated loss of inhibitory neurons in the spinothalamic tract causes disinhibition of thalamic neurons, which autonomously generate ectopic nociceptive action potentials responsible for the pain experience. We hypothesized that CPSP is a result of misinterpretation of afferent sensory input by the sensitized neurons within the brain, rather than generated spontaneously by the damaged central nervous system (CNS) neurons. To test this hypothesis, we prospectively recruited 8 patients with definite CPSP affecting at least 1 extremity. In an open-label intervention, an ultrasound-guided peripheral nerve block with lidocaine was performed to block afferent sensory input from a painful extremity. Spontaneous and evoked pain, neuropathic pain descriptors, and lidocaine plasma concentrations were measured. The blockade of peripheral sensory input resulted in complete abolition of pain in 7 of the 8 subjects within 30 minutes (the primary outcome measure of the study), and >50% pain relief in the remaining participant. Median (interquartile range) spontaneous pain intensity changed from 6.5 (4.3-7.0) at baseline to 0 (0-0) after the block (P = 0.008). All mechanical/thermal hypersensitivity was abolished by the nerve block. The results suggest that it is unlikely that CPSP is autonomously generated within the CNS. Rather, this pain is dependent on afferent input from the painful region in the periphery, and may be mediated by misinterpretation of peripheral sensory input by sensitized neurons in the CNS.

Corneal confocal microscopy as a tool for detecting diabetic polyneuropathy in a cohort with screen-detected type 2 diabetes: ADDITION-Denmark

AIMS In this cross-sectional study, we explored the utility of corneal confocal microscopy (CCM) measures for detecting diabetic polyneuropathy (DPN) and their association with clinical variables, in a cohort with type 2 diabetes. METHODS CCM, nerve conduction studies, and assessment of symptoms and clinical deficits of DPN were undertaken in 144 participants with type 2 diabetes and 25 controls. DPN was defined according to the Toronto criteria for confirmed DPN. RESULTS Corneal nerve fiber density (CNFD) was lower both in participants with confirmed DPN (n = 27) and in participants without confirmed DPN (n = 117) compared with controls (P = 0.04 and P = 0.01, respectively). No differences were observed for CNFD (P = 0.98) between participants with and without DPN. There were no differences in CNFL and CNBD between groups (P = 0.06 and P = 0.29, respectively). CNFD was associated with age, height, total- and LDL cholesterol. CONCLUSIONS CCM could not distinguish patients with and without neuropathy, but CNFD was lower in patients with type 2 diabetes compared to controls. Age may influence the level of CCM measures.