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Introduction/Aims
Schwann cell clusters have been described at the murine dermis-epidermis border. We quantified dermal Schwann cells in the skin of patients with small-fiber neuropathy (SFN) compared with healthy controls to correlate with the clinical phenotype.
Methods
Skin punch biopsies from the lower legs of 28 patients with SFN (11 men, 17 women; median age, 54 [range, 19-73] years) and 9 healthy controls (five men, four women, median age, 34 [range, 25-69] years) were immunoreacted for S100 calcium-binding protein B as a Schwann cell marker, protein-gene product 9.5 as a pan-neuronal marker, and CD207 as a Langerhans cell marker. Intraepidermal nerve fiber density (IENFD) and subepidermal Schwann cell counts were determined.
Results
Skin samples of patients with SFN showed lower IENFD (P < .05), fewer Schwann cells per millimeter (P < .01), and fewer Schwann cell clusters per millimeter (P < .05) than controls. When comparing SFN patients with reduced (n = 13; median age, 53 [range, 19-73] years) and normal distal (n = 15, median age, 54 [range, 43-68] years) IENFD, the number of solitary Schwann cells per millimeter (p < .01) and subepidermal nerve fibers associated with Schwann cell branches (P < .05) were lower in patients with reduced IENFD. All three parameters correlated positively with distal IENFD (P < .05 to P < .01), whereas no correlation was found between Schwann cell counts and clinical pain characteristics.
Discussion
Our data raise questions about the mechanisms underlying the interdependence of dermal Schwann cells and skin innervation in SFN. The temporal course and functional impact of Schwann cell presence and kinetics need further investigation.
Both nerve injury and complex regional pain syndrome (CRPS) can result in chronic pain. In traumatic neuropathy, the blood nerve barrier (BNB) shielding the nerve is impaired—partly due to dysregulated microRNAs (miRNAs). Upregulation of microRNA-21-5p (miR-21) has previously been documented in neuropathic pain, predominantly due to its proinflammatory features. However, little is known about other functions. Here, we characterized miR-21 in neuropathic pain and its impact on the BNB in a human-murine back translational approach. MiR-21 expression was elevated in plasma of patients with CRPS as well as in nerves of mice after transient and persistent nerve injury. Mice presented with BNB leakage, as well as loss of claudin-1 in both injured and spared nerves. Moreover, the putative miR-21 target RECK was decreased and downstream Mmp9 upregulated, as was Tgfb. In vitro experiments in human epithelial cells confirmed a downregulation of CLDN1 by miR-21 mimics via inhibition of the RECK/MMP9 pathway but not TGFB. Perineurial miR-21 mimic application in mice elicited mechanical hypersensitivity, while local inhibition of miR-21 after nerve injury reversed it. In summary, the data support a novel role for miR-21, independent of prior inflammation, in elicitation of pain and impairment of the BNB via RECK/MMP9.
The nervous system is shielded by special barriers. Nerve injury results in blood–nerve barrier breakdown with downregulation of certain tight junction proteins accompanying the painful neuropathic phenotype. The dorsal root ganglion (DRG) consists of a neuron-rich region (NRR, somata of somatosensory and nociceptive neurons) and a fibre-rich region (FRR), and their putative epi-/perineurium (EPN). Here, we analysed blood–DRG barrier (BDB) properties in these physiologically distinct regions in Wistar rats after chronic constriction injury (CCI). Cldn5, Cldn12, and Tjp1 (rats) mRNA were downregulated 1 week after traumatic nerve injury. Claudin-1 immunoreactivity (IR) found in the EPN, claudin-19-IR in the FRR, and ZO-1-IR in FRR-EPN were unaltered after CCI. However, laser-assisted, vessel specific qPCR, and IR studies confirmed a significant loss of claudin-5 in the NRR. The NRR was three-times more permeable compared to the FRR for high and low molecular weight markers. NRR permeability was not further increased 1-week after CCI, but significantly more CD68\(^+\) macrophages had migrated into the NRR. In summary, NRR and FRR are different in naïve rats. Short-term traumatic nerve injury leaves the already highly permeable BDB in the NRR unaltered for small and large molecules. Claudin-5 is downregulated in the NRR. This could facilitate macrophage invasion, and thereby neuronal sensitisation and hyperalgesia. Targeting the stabilisation of claudin-5 in microvessels and the BDB barrier could be a future approach for neuropathic pain therapy.
Objectives
The pathogenesis of fibromyalgia syndrome (FMS) is unclear. Transcranial ultrasonography revealed anechoic alteration of midbrain raphe in depression and anxiety disorders, suggesting affection of the central serotonergic system. Here, we assessed midbrain raphe echogenicity in FMS.
Methods
Sixty-six patients underwent transcranial sonography, of whom 53 were patients with FMS (27 women, 26 men), 13 patients with major depression and physical pain (all women), and 14 healthy controls (11 women, 3 men). Raphe echogenicity was graded visually as normal or hypoechogenic, and quantified by digitized image analysis, each by investigators blinded to the clinical diagnosis.
Results
Quantitative midbrain raphe echogenicity was lower in patients with FMS compared to healthy controls (p<0.05), but not different from that of patients with depression and accompanying physical pain. Pain and FMS symptom burden did not correlate with midbrain raphe echogenicity as well as the presence and severity of depressive symptoms.
Conclusion
We found reduced echogenicity of the midbrain raphe area in patients with FMS and in patients with depression and physical pain, independent of the presence or severity of pain, FMS, and depressive symptoms. Further exploration of this sonographic finding is necessary before this objective technique may enter diagnostic algorithms in FMS and depression.
Peripheral neuropathy is accompanied by changes in the neuronal environment. The blood-nerve barrier (BNB) is crucial in protecting the neural homeostasis: Tight junctions (TJ) seal paracellular spaces and thus prevent external stimuli from entering. In different models of neuropathic pain, the BNB is impaired, thus contributing to local damage, immune cell invasion and, ultimately, the development of neuropathy with its symptoms. In this study, we examined changes in expression and microstructural localization of two key tight junction proteins (TJP), claudin-1 and the cytoplasmic anchoring ZO-1, in the sciatic nerve of mice subjected to chronic constriction injury (CCI). Via qPCR and analysis of fluorescence immunohistochemistry, a marked downregulation of mRNA as well as decreased fluorescence intensity were observed in the nerve for both proteins. Moreover, a distinct zig-zag structure for both proteins located at cell-cell contacts, indicative of the localization of TJs, was observed in the perineurial compartment of sham-operated animals. This microstructural location in cell-cell-contacts was lost in neuropathy as semiquantified via computational analysis, based on a novel algorithm. In summary, we provide evidence that peripheral neuropathy is not only associated with decrease in relevant TJPs but also exhibits alterations in TJP arrangement and loss in barrier tightness, presumably due to internalization. Specifically, semiquantification of TJP in cell-cell-contacts of microcompartments could be used in the future for routine clinical samples of patients with neuropathy.
The blood–nerve barrier and myelin barrier normally shield peripheral nerves from potentially harmful insults. They are broken down during nerve injury, which contributes to neuronal damage. Netrin-1 is a neuronal guidance protein with various established functions in the peripheral and central nervous systems; however, its role in regulating barrier integrity and pain processing after nerve injury is poorly understood. Here, we show that chronic constriction injury (CCI) in Wistar rats reduced netrin-1 protein and the netrin-1 receptor neogenin-1 (Neo1) in the sciatic nerve. Replacement of netrin-1 via systemic or local administration of the recombinant protein rescued injury-induced nociceptive hypersensitivity. This was prevented by siRNA-mediated knockdown of Neo1 in the sciatic nerve. Mechanistically, netrin-1 restored endothelial and myelin, but not perineural, barrier function as measured by fluorescent dye or fibrinogen penetration. Netrin-1 also reversed the decline in the tight junction proteins claudin-5 and claudin-19 in the sciatic nerve caused by CCI. Our findings emphasize the role of the endothelial and myelin barriers in pain processing after nerve damage and reveal that exogenous netrin-1 restores their function to mitigate CCI-induced hypersensitivity via Neo1. The netrin-1-neogenin-1 signaling pathway may thus represent a multi-target barrier protector for the treatment of neuropathic pain.
Diabetic neuropathy (DPN) is one of the most severe and yet most poorly understood complications of diabetes mellitus. In vivo imaging of dorsal root ganglia (DRG), a key structure for the understanding of DPN, has been restricted to animal studies. These have shown a correlation of decreased DRG volume with neuropathic symptom severity. Our objective was to investigate correlations of DRG morphology and signal characteristics at 3 Tesla (3T) magnetic resonance neurography (MRN) with clinical and serological data in diabetic patients with and without DPN. In this cross-sectional study, participants underwent 3T MRN of both L5 DRG using an isotropic 3D T2-weighted, fat-suppressed sequence with subsequent segmentation of DRG volume and analysis of normalized signal properties. Overall, 55 diabetes patients (66 ± 9 years; 32 men; 30 with DPN) took part in this study. DRG volume was smaller in patients with severe DPN when compared to patients with mild or moderate DPN (134.7 ± 21.86 vs 170.1 ± 49.22; p = 0.040). In DPN patients, DRG volume was negatively correlated with the neuropathy disability score (r = −0.43; 95%CI = −0.66 to −0.14; p = 0.02), a measure of neuropathy severity. DRG volume showed negative correlations with triglycerides (r = −0.40; 95%CI = −0.57 to −0.19; p = 0.006), and LDL cholesterol (r = −0.33; 95%CI = −0.51 to −0.11; p = 0.04). There was a strong positive correlation of normalized MR signal intensity (SI) with the neuropathy symptom score in the subgroup of patients with painful DPN (r = 0.80; 95%CI = 0.46 to 0.93; p = 0.005). DRG SI was positively correlated with HbA1c levels (r = 0.30; 95%CI = 0.09 to 0.50; p = 0.03) and the triglyceride/HDL ratio (r = 0.40; 95%CI = 0.19 to 0.57; p = 0.007). In this first in vivo study, we found DRG morphological degeneration and signal increase in correlation with neuropathy severity. This elucidates the potential importance of MR-based DRG assessments in studying structural and functional changes in DPN.
MicroRNAs (miRNAs) are increasingly recognized as regulators of immune and neuronal gene expression and are potential master switches in neuropathic pain pathophysiology. miR-21 is a promising candidate that may link the immune and the pain system. To investigate the pathophysiological role of miR-21 in neuropathic pain, we assessed mice deficient of B7 homolog 1 (B7-H1), a major inhibitor of inflammatory responses. In previous studies, an upregulation of miR-21 had been shown in mouse lymphocytes. Young (8 weeks), middle-aged (6 months), and old (12 months) B7-H1 ko mice and wildtype littermates (WT) received a spared nerve injury (SNI). We assessed thermal withdrawal latencies and mechanical withdrawal thresholds. Further, we performed tests for anxiety-like and cognitive behavior. Quantitative real time PCR was used to determine miR-21 relative expression in peripheral nerves, and dorsal root ganglia (DRG) at distinct time points after SNI. We found mechanical hyposensitivity with increasing age of naïve B7-H1 ko mice. Young and middle-aged B7-H1 ko mice were more sensitive to mechanical stimuli compared to WT mice (young: p < 0.01, middle-aged: p < 0.05). Both genotypes developed mechanical and heat hypersensitivity (p < 0.05) after SNI, without intergroup differences. No relevant differences were found after SNI in three tests for anxiety like behavior in B7-H1 ko and WT mice. Also, SNI had no effect on cognition. B7-H1 ko and WT mice showed a higher miR-21 expression (p < 0.05) and invasion of macrophages and T cells in the injured nerve 7 days after SNI without intergroup differences. Our study reveals that increased miR-21 expression in peripheral nerves after SNI is associated with reduced mechanical and heat withdrawal thresholds. These results point to a role of miR-21 in the pathophysiology of neuropathic pain, while affective behavior and cognition seem to be spared. Contrary to expectations, B7-H1 ko mice did not show higher miR-21 expression than WT mice, thus, a B7-H1 knockout may be of limited relevance for the study of miR-21 related pain.
In dorsal root ganglia (DRG) neurons TRESK channels constitute a major current component of the standing outward current IK\(_{SO}\). A prominent physiological role of TRESK has been attributed to pain sensation. During inflammation mediators of pain e.g. lysophosphatidic acid (LPA) are released and modulate nociception. We demonstrate co-expression of TRESK and LPA receptors in DRG neurons. Heterologous expression of TRESK and LPA receptors in Xenopus oocytes revealed augmentation of basal K\(^{+}\) currents upon LPA application. In DRG neurons nociception can result from TRPV\(_{1}\) activation by capsaicin or LPA. Upon co-expression in Xenopus oocytes LPA simultaneously increased both depolarising TRPV\(_{1}\) and hyperpolarising TRESK currents. Patch-clamp recordings in cultured DRG neurons from TRESK[wt] mice displayed increased IK\(_{SO}\) after application of LPA whereas under these conditions IK\(_{SO}\) in neurons from TRESK[ko] mice remained unaltered. Under current-clamp conditions LPA application differentially modulated excitability in these genotypes upon depolarising pulses. Spike frequency was attenuated in TRESK[wt] neurons and, in contrast, augmented in TRESK[ko] neurons. Accordingly, excitation of nociceptive neurons by LPA is balanced by co-activation of TRESK channels. Hence excitation of sensory neurons is strongly controlled by the activity of TRESK channels, which therefore are good candidates for the treatment of pain disorders.
The role of miR-21 in the pathophysiology of neuropathic pain using the model of B7-H1 knockout mice
(2017)
The impact of microRNA (miRNA) as key players in the regulation of immune and neuronal gene expression and their role as master switches in the pathophysiology of neuropathic pain is increasingly recognized. miR-21 is a promising candidate that could be linked to the immune and the nociceptive system. To further investigate the pathophysiological role of miR-21 in neuropathic pain, we assesed mice deficient of B7 homolog 1 (B7-H1 ko), a protein with suppressive effect on inflammatory responses.
B7-H1 ko mice and wildtype littermates (WT) of three different age-groups, young (8 weeks), middle-aged (6 months), and old (12 months) received a spared nerve injury (SNI). Thermal withdrawal latencies and mechanical withdrawal thresholds were determined. Further, we investigated anxiety-, depression-like and cognitive behavior. Quantitative real time PCR was used to determine miR-21 relative expression in peripheral nerves, dorsal root ganglia and white blood cells (WBC) at distinct time points after SNI.
Naïve B7-H1 ko mice showed mechanical hyposensitivity with increasing age. Young and middle-aged B7-H1 ko mice displayed lower mechanical withdrawal thresholds compared to WT mice. From day three after SNI both genotypes developed mechanical and heat hypersensitivity, without intergroup differences. As supported by the results of three behavioral tests, no relevant differences were found for anxiety-like behavior after SNI in B7-H1 ko and WT mice. Also, there was no indication of depression-like behavior after SNI or any effect of SNI on cognition in both genotypes. The injured nerves of B7-H1 ko and WT mice showed higher miR-21 expression and invasion of macrophages and T cells 7 days after SNI without intergroup differences. Perineurial miR-21 inhibitor injection reversed SNI-induced mechanical and heat hypersensitivity in old B7-H1 ko and WT mice.
This study reveals that reduced mechanical thresholds and heat withdrawal latencies are associated with miR-21 induction in the tibial and common peroneal nerve after SNI, which can be reversed by perineurial injection of a miR-21 inhibitor. Contrary to expectations, miR-21 expression levels were not higher in B7-H1 ko compared to WT mice. Thus, the B7-H1 ko mouse may be of minor importance for the study of miR-21 related pain. However, these results spot the contribution of miR-21 in the pathophysiology of neuropathic pain and emphasize the crucial role of miRNA in the regulation of neuronal and immune circuits that contribute to neuropathic pain.