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Background: Persistent pain after inguinal herniorrhaphy is a disabling condition with a lack of evidence-based pharmacological treatment options. This randomized placebo-controlled trial investigated the efficacy of a capsaicin 8% cutaneous patch in the treatment of severe persistent inguinal postherniorrhaphy pain. Methods: Forty-six patients with persistent inguinal postherniorrhaphy pain were randomized to receive either a capsaicin 8% patch or a placebo patch. Pain intensity (Numerical Rating Scale [NRS 0-10]) was evaluated under standardized conditions (at rest, during movement, and during pressure) at baseline and at 1, 2 and 3 months after patch application. Skin punch biopsies for intraepidermal nerve fiber density (IENFD) measurements were taken at baseline and 1 month after patch application. Quantitative sensory testing was performed at baseline and at 1, 2, and 3 months after patch application. The primary outcome was comparisons of summed pain intensity differences (SPIDs) between capsaicin and placebo treatments at 1, 2 and 3 months after patch application (significance level P<0.01). Results: The maximum difference in SPID, between capsaicin and placebo treatments, was observed at 1 month after patch application, but the pain reduction was not significant (NRS, mean difference [95% CI]: 5.0 [0.09 to 9.9]; P=0.046). No differences in SPID between treatments were observed at 2 and 3 months after patch application. Changes in IENFD on the pain side, from baseline to 1 month after patch application, did not differ between capsaicin and placebo treatment: 1.9 [-0.1 to 3.9] and 0.6 [-1.2 to 2.5] fibers/mm, respectively (P=0.32). No significant changes in sensory function, sleep quality or psychological factors were associated with capsaicin patch treatment. Conclusions: The study did not demonstrate significant differences in pain relief between capsaicin and placebo treatment, although a trend toward pain improvement in capsaicin treated patients was observed 1 month after patch application.
Evaluation der antihyperalgetischen wie neuroregenerativen Potenz von Topiramat nach peripherer Nervenläsion. Untersuchung im Tiermodell nach CCI / Crush-Nervenläsion. Verhaltenstestungen, morphometrisch histologische Analysen, Immunhistochemische Färbungen, elektrophysiologische Studien sowie RT-PCR. Topiramat zeigte hierbei - modulierende Wirkung auf die Entwicklung einer mechanischen Hyperalgesie wie Kälteallodynie nach CCI, auf Hitzehyperalgesie wie Kälteallodynia nach Crush - keine neuroprotektive oder pro-regenerative Wirkung in den von uns verwendeten Läsionsmodellen - eine ausgeprägte Modulation des zellulären Zytokinmilieus distal der Nervenläsion im Sinne einer Hochregulation proinflammatorischer Zytokine.
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.
To investigate the usefulness of pain-related evoked potentials (PREP) elicited by electrical stimulation for the identification of small fiber involvement in patients with mixed fiber neuropathy (MFN). Eleven MFN patients with clinical signs of large fiber impairment and neuropathic pain and ten healthy controls underwent clinical and electrophysiological evaluation. Small fiber function, electrical conductivity and morphology were examined by quantitative sensory testing (QST), PREP, and skin punch biopsy. MFN was diagnosed following clinical and electrophysiological examination (chronic inflammatory demyelinating neuropathy: n = 6; vasculitic neuropathy: n = 3; chronic axonal neuropathy: n = 2). The majority of patients with MFN characterized their pain by descriptors that mainly represent C-fiber-mediated pain. In QST, patients displayed elevated cold, warm, mechanical, and vibration detection thresholds and cold pain thresholds indicative of MFN. PREP amplitudes in patients correlated with cold (p < 0.05) and warm detection thresholds (p < 0.05). Burning pain and the presence of par-/dysesthesias correlated negatively with PREP amplitudes (p < 0.05). PREP amplitudes correlating with cold and warm detection thresholds, burning pain, and par-/dysesthesias support employing PREP amplitudes as an additional tool in conjunction with QST for detecting small fiber impairment in patients with MFN.
Aim of Investigation: The neurological manifestations of Fabry’s disease, a rare, X-linked, multisystem disorder caused by alpha-galactosidase A deficiency and globotriosylceramide (Gb3) accumulation, include both peripheral and central nervous system symptoms. Here we evaluated a prospectively recruited cohort of patients with Fabry’s disease for pain, small nerve fiber function, and skin innervation. Methods: 66 patients (31 male and 35 female) were enrolled,31 patients were on ERT. All patients underwent quantitative sensory testing (QST), electrophysiological examination, and extra- and transcranial Doppler sonography. For pain and mood assessment standardized questionnaires were used. Skin biopsies were performed at the left distal leg in 38 patients for intraepidermal nerve fiber density (IENFD) assessment. Results: Age at examination did not differ significantly between women (40.2+/-16.2 years) and men (38.9+/-13.8; n.s.). 29/31 male and 19/35 female patients complained of acroparesthesias or neuropathic pain. QST abnormalities indicative of small fiber impairment were found in 26/31 male and 28/35 female patients. Electrophysiological examination of large fibers and autonomic fibers revealed pathological findings in 11/31 male and 3/35 female patients. All patients had normal Doppler sonography results. Indicators for depression were present in 14/31 male and 10/35 female patients. 20/31 male and 18/35 female patients had a skin biopsy, the IENFD was significantly reduced in male (2.0+/-2.8 fibers/mm) compared with female patients (6.7 +/- 4.4 fibers/mm). In 10 patients free from neurological symptoms, QST and IENFD abnormalities were still detected. Follow up examination after one year in 12 patients under ERT (2.1+/-1.7 years) showed improvement in some symptoms and in QST and neurophysiology in six patients with normal renal function. 20/35 female patients older than 40 y had concomitant diseases, while none of the 18 younger female patients did. The corresponding radio in male patients was 5/19 (>=40y) and 2/13 (<40y) respectively. Conclusions: Neuropathic pain and sensory deficits of the distal extremities are common in patients with Fabry’s disease. QST and IENFD analysis are important for early diagnosis of nerve involvement in Fabry’s disease. Small fiber function may improve under ERT in patients without severe renal impairment.
microRNA-Genexpressionsprofile in Blut-, Haut- und Nervenproben von Patienten mit Polyneuropathien
(2020)
Die Polyneuropathie (PNP) ist die häufigste Störung des peripheren Nervensystems bei Erwachsenen. Die Suche nach der Ursache bleibt in vielen Fällen erfolglos, ist aber unverzichtbar, da die Therapiewahl von der Ätiologie der Erkrankung abhängt. Geeignete Biomarker könnten die Differentialdiagnose unter Umständen erleichtern. microRNAs (miRNAs) sind in dieser Hinsicht vielversprechend, da in vielen Studien bei Nervende- und regenerationsprozessen sowie in neuropathischen Schmerzmodellen eine Dysregulation beschrieben wurde.
In dieser Studie wurde die Expression zweier miRNAs, miR-103a und miR-let-7d, sowie eines Zielmoleküls der miR-103a, des Kalziumkanals Cav1,2, in einer großen Kohorte von PNP-Patienten unterschiedlicher Ätiologie in Blut, Haut- und Nervenbiopsien untersucht. Insgesamt wurden 116 Patienten und 22 Kontroll-probanden in die Studie eingeschlossen. Nach der Isolation von RNA aus weißen Blutzellen (WBC), Haut- und Nervenbiopsien folgte die Expressionsbestimmung mittels qRT-PCR.
Während sich jeweils Unterschiede zwischen PNP-Patienten und Kontrollen und zwischen Patienten mit entzündlicher und solchen mit nicht-entzündlicher PNP zeigten, wurden keine Unterschiede in der Expression zwischen den ätiologischen Subgruppen oder zwischen Patienten mit schmerzhafter und schmerzloser PNP festgestellt. In den Nervenbiopsien der Patientenkohorte ergab sich eine inverse Korrelation der miR-103a und ihrem Zielgen Cacna1c, die darauf hinweisen könnte, dass Cacna1c von der miR-103a negativ reguliert wird.
Da in unserer Patientenkohorte keine Unterschiede zwischen den PNP-Subgruppen auftraten, scheint der Einsatz der miR-103a und miR-let-7d als diagnostische Biomarker zur ätiologischen Einordnung einer PNP nicht gerechtfertigt. Dennoch deuten unsere Ergebnisse auf eine mögliche Rolle der untersuchten miRNAs bei Entstehung und Verlauf von PNP hin. Für ein tieferes pathophysiologisches Verständnis der miRNAs vor allem bei entzündlichen Neuropathien, könnte die Untersuchung von weiteren miRNAs und Zielgenen Aufschluss geben.
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.
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.
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.
Die Pathophysiologie der PNP wie auch die Entstehung der oft assoziierten neuropathischen Schmerzen ist unklar. Gleichzeitig gibt es bislang keine geeigneten Biomarker, die die oft komplizierte Differentialdiagnose vereinfachen können. Einige Tiermodelle und klinische Studien lieferten bereits Hinweise auf die entscheidende Rolle pro- und anti-inflammatorischer Zytokine in diesen Prozessen. Ziel unserer Studie war es, die systemische Genexpression pro- und anti-inflammatorischer Zytokine in einer großen Kohorte von Patienten mit PNP verschiedener Ätiologie zu charakterisieren. Insgesamt konnten 111 PNP-Patienten und 38 gesunde Kontrollpersonen prospektiv rekrutiert werden. Nach Isolation von PBMC aus Blutproben von 97 Patienten wurde die Genexpression der pro-inflammatorischen Zytokine TNF, IL1, IL2, IL6, IL8 und der anti-inflammatorischen Zytokine IL4 und IL10 mittels qRT-PCR bestimmt. Bei 47 Patienten und 12 Kontrollen wurde zudem die IL6-, IL-8- und TNF-Zytokinproduktion von PBMC in vitro nach Stimulation durch LPS mittels ELISA untersucht. Hauptbefund war ein pro-inflammatorisches Zytokinprofil der PNP-Patienten mit höherer Genexpression von IL1, IL2, IL8 und TNF im Vergleich zu den gesunden Kontrollen. Im Falle der entzündlichen Neuropathien konnte zudem eine niedrigere Genexpression von IL10 im Vergleich zu Gesunden nachgewiesen werden. Sowohl schmerzhafte als auch schmerzlose Verlaufsformen wiesen ein pro-inflammatorisches Zytokingenexpressionsprofil im Vergleich zu Gesunden auf, das bei schmerzhaften PNP deutlich mehr beteiligte pro-inflammatorische Zytokine umfasste; relevante Unterschiede zwischen den PNP-Patienten mit und ohne Schmerz sowie der diagnostischen Subgruppen fanden sich nicht. Eine niedrigere Stimulationsschwelle der PBMC lag bei PNP-Patienten im Vergleich zu Gesunden nicht vor. Insgesamt erscheint die Rolle einzelner Zytokine als systemische Biomarker für die Differenzierung verschiedener PNP-Formen bzw. bezüglich neuropathischen Schmerzes aufgrund einer niedrigen Spezifität deutlich eingeschränkt. Dennoch sprechen unsere Ergebnisse für eine mögliche Rolle eines pro-inflammatorischen Milieus bei der Entstehung bzw. des Verlaufes verschiedener entzündlicher und nicht-entzündlicher Neuropathien und neuropathischen Schmerzes.
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.
In der vorliegenden Studie wurden 32 Patienten (19 Frauen, 13 Männer, medianes Alter 50 Jahren, Spanne: 26-83 Jahre) mit einem klinisch akralen neuropathischen Schmerzsyndrom unterschiedlicher Genese mittels QST, PREP und Hautbiopsie untersucht. Unser Patientenkollektiv bestand aus drei Subgruppen: sechsen Patienten erfüllten die Kriterien einer SFN, acht Patienten hatten eine Neuropathie der großkalibrigen Nervenfasern mit zusätzlicher Beeinträchtigung der kleinkalibrigen Nervenfasern und weitere acht Patienten hatten ein akrales Schmerzsyndrom mit neuropathischen Charakteristika, ohne vorbekannte Diagnose einer Neuropathie der groß- oder kleinkalibrigen Nervenfasern. Die Patienten wurden mittels klinischer neurologischer Untersuchung, elektrophysiologischer Tests, QST, PREP und Hautbiopsie untersucht. Die Patientendaten wurden jeweils mit Daten großer Kontrollgruppen verglichen, die wir in unserer Klinik unter Angehörigen und Freunden unserer Patienten mit deren Einwilligung rekrutiert hatten.
QST und die Hautbiopsie waren bei Patienten mit SFN und PNP jeweils auffällig, bei akralem Schmerzsyndrom unklarer Ätiologie hingegen unauffällig. Nach elektrischer kutaner Stimulation aller drei Körperregionen zeigte sich eine Amplitudenminderung der PREP-Reizantwort in allen Patientensubgruppen (7,5 µV in der SFN-Gruppe, 3,8 µV in der PNP-Gruppe, und 11,3 µV bei den Patienten mit akralem Schmerzsyndrom). Somit konnten wir zeigen, dass eine Kleinfaserpathologie in der Studienpopulation von Patienten mit neuropathischem Schmerzsyndrom besteht. Nur die Amplitudenminderung der PREP bildet diese Pathologie ab.
Diese Daten erlauben uns die eingangs aufgestellte Hypothese, dass PREP zur Diagnostik bei Frage nach Kleinfaserbeteiligung geeignet ist, positiv zu belegen. PREP ist eine nicht-invasive Methode für die Evaluation der Funktion v.a. der Aδ-Faser mit standardisiertem Ablaufprotokoll zur Erhebung von reproduzierbaren Daten. Sie kann bei Patienten mit der Anamnese eines akralen neuropathischen Schmerzsyndroms einen objektiven Hinweis auf eine Dysfunktion der kleinkalibrigen Nervenfasern, auch wenn bereits etablierte Methoden (QST und Hautbiopsie) unauffällig bleiben, erbringen. Entsprechend können die PREP eine wertvolle Ergänzung der klinischen Untersuchungsbatterie für die Evaluation der Funktion der kleinkalibrigen Nervenfasern sein.
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.
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.
Bislang ist ungeklärt, warum PNPs teils schmerzhaft und teils schmerzlos verlaufen. Die in der vorliegenden Arbeit untersuchte Hypothese lautete, dass ein Ungleichgewicht zwischen pro- und anti-inflammatorischen Zytokinen der unterschiedlichen Schmerzausprägung zugrunde liegt.Es wurden 32 Patienten mit schmerzhafter PNP, 20 Patienten mit schmerzloser PNP und 44 Kontrollpersonen auf die Expression und Produktion ausgewählter pro- und anti-inflammatorischer Zytokine untersucht. Zur Messung der Schmerzhaftigkeit wurden etablierte Schmerzfragebögen verwendet. Zusätzlich wurden nahezu alle Patienten mit der Allgemeinen Depressionsskala befragt. Die Diagnose, Ätiologie, Dauer, klinische Manifestation der PNP sowie die Medikation der Patienten wurde auf standardisierten Erhebungsbögen dokumentiert. Zur Messung der Zytokine wurde morgens Blut in EDTA- und Serummonovetten asserviert und entsprechend der Messmethodik weiterverarbeitet. Die relative Genexpression wurde aus Gesamt-RNA mittels reverser Transkription und quantitativer real-time PCR, die Serumproteine mittels enzyme-linked immunosorbant assay gemessen. Die Patienten mit schmerzhafter PNP hatten in der Mehrzahl Neuropathie-typische Plussymptome und mittelstarke Schmerzen, die eine starke bis sehr starke Behinderung darstellten. Die hier untersuchten Zytokinmuster bei Patienten mit schmerzhafter und schmerzloser PNP zeigten eine Verschiebung zu pro-inflammatorischen Zytokinen bei Patienten mit schmerzhafter PNP. Die Zytokinexpression der Patienten mit schmerzhafter PNP war im Vergleich zu Patienten mit schmerzloser PNP und Kontrollen bezüglich der IL-2 und TNF Expression und Produktion signifikant erhöht. Umgekehrt lagen bei Patienten mit schmerzloser PNP die Produktion und die Expression des IL-4 im Vergleich zu Patienten mit schmerzhafter PNP und Kontrollen höher. Die Expression des IL-10 lag bei Patienten mit schmerzloser PNP ebenfalls höher als bei Patienten mit schmerzloser PNP und Kontrollen, unterschied sich aber auf Proteinebene nicht in den drei Gruppen. Die einleitend gestellte Hypothese, dass der schmerzhafte oder schmerzlose Verlauf einer PNP durch unterschiedliche Zytokinprofile bedingt ist, kann durch die vorliegenden Ergebnisse gestützt werden. In Zusammenschau mit den Daten aus der Grundlagenforschung scheint einem pro-inflammatorischen Zytokinmuster eine entscheidende Rolle an der Entstehung und Aufrechterhaltung neuropathischer Schmerzen zuzukommen. Für TNF sind entsprechende pathophysiologische Wirkungen bekannt. Anti-inflammatorische Zytokine, wie IL-4 und IL-10 zeigten analgetische Wirkungen im Tierversuch. Die Mitwirkung des IL-2 an peripheren Opioid-Rezeptoren lässt eine endogene periphere Analgesie vermuten. Hieraus lassen sich Folgerungen für zukünftige Diagnostik und Therapie neuropathischer Schmerzen ziehen. Durch Erkennung von Zytokin-Imbalancen wären schmerzhafte PNPs früher einer adäquaten Therapie zuzuführen. Durch die Modulation von Zytokinprofilen im Rahmen schmerzhafter PNPs könnten sich zusätzlich therapeutische Möglichkeiten eröffnen.
The calpain inhibitor MDL-28710 blocks the early local pro-inflammatory cytokine gene expression in mice after chronic constriction nerve injury (CCI). Onehundred- thirteen wild type mice of C57Bl/6J background received CCI of the right sciatic nerve. Mechanical paw withdrawal thresholds and thermal withdrawal latencies were investigated at baseline and at 1, 3, and 7 days after CCI. Three application regimens were used for MDL-28170: a) single injection 40 min before CCI; b) serial injections of MDL- 28170 40 min before and up to day three after CCI; c) sustained application via intraperitoneal osmotic pumps. The control animals received the vehicle DMSO/PEG 400. The tolerable dose of MDL-28170 for mice was 30 mg/kg body weight, higher doses were lethal within the first hours after application. Mechanical withdrawal thresholds and thermal withdrawal latencies were reduced after CCI and did not normalize after single or serial injections, nor with application of MDL-28170 via osmotic pumps. Although the calpain inhibitor MDL-28170 inhibits the early local cytokine upregulation in the sciatic nerve after CCI, pain behavior is not altered. This finding implies that local cytokine upregulation after nerve injury alone is only one factor in the induction and maintenance of neuropathic pain.
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.
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.
High-Dose Capsaicin for the Treatment of Neuropathic Pain: What We Know and What We Need to Know
(2014)
Neuropathic pain is a frequent and disabling condition with diverse underlying etiologies and is often difficult to treat. Systemic drug treatment is often limited in efficacy. Furthermore, adverse effects may be a limiting factor when trying to reach the necessary dose. Analgesics that can be applied topically have the potential to largely overcome this problem. They may be of particular advantage in localized neuropathic pain syndromes such as postherpetic neuralgia or small fiber neuropathy. Capsaicin, the pungent component of chili peppers, is a natural ligand of the transient receptor potential vanilloid 1 channel and has long been used as topically applicable cream with concentrations of 0.025 to 0.075%. In 2009, a high-concentration transdermal capsaicin 8% patch (Qutenza ; Acorda Therapeutics, Inc., Ardsley, NY, USA; Astellas Pharma Europe Ltd., Chertsey, Surrey, UK) was introduced for the treatment of peripheral neuropathic pain syndromes other than of diabetic origin in adults. It has since been widely used in diverse neuropathic pain disorders. In this review article, we summarize current knowledge on Qutenza, its advantages and problems, and expose unmet needs.
Background
Postherpetic neuralgia (PHN) is the painful complication of a varicella zoster virus reactivation. We investigated the systemic and local gene expression of pro- and anti-inflammatory cytokine expression in patients with PHN.
Methods
Thirteen patients with PHN at the torso (Th4-S1) were recruited. Skin punch biopsies were obtained from the painful and the contralateral painless body area for intraepidermal nerve fiber density (IENFD) and cytokine profiling. Additionally, blood was withdrawn for systemic cytokine expression and compared to blood values of healthy controls. We analyzed the gene expression of selected pro- and anti-inflammatory cytokines (tumor necrosis factor-alpha [TNF] and interleukins [IL]-1β, IL-2, and IL-8).
Results
IENFD was lower in affected skin compared to unaffected skin (p<0.05), while local gene expression of pro- and anti-inflammatory cytokines did not differ except for two patients who had 7fold higher IL-6 and 10fold higher IL-10 gene expression in the affected skin compared to the contralateral unaffected skin sample. Also, the systemic expression of cytokines in patients with PHN and in healthy controls was similar.
Conclusion
While the systemic and local expression of the investigated pro- and anti-inflammatory cytokines was not different from controls, this may have been influenced by study limitations like the low number of patients and different disease durations. Furthermore, other cytokines or pain mediators need to be considered.