Stefan Leis

Substance-P-induced protein extravasation is bilaterally increased in complex regional pain syndrome

Pain, mechanical hyperalgesia, edema, increased skin temperature, and skin reddening are characteristic symptoms of acute complex regional pain syndrome (CRPS). We have recently demonstrated facilitated neurogenic inflammation on the affected limb. To further elucidate the underlying mechanisms, exogenous substance P (SP) in ascending concentrations (10(-9), 10(-8), 10(-7), 10(-6) M) was intradermally applied to the affected and the unaffected limbs, respectively, in two groups of 11 CRPS patients each using the microdialysis technique. Fourteen healthy volunteers served as controls for SP application, and 9 volunteers and 10 patients served as controls for saline perfusion. Dialysate protein content was measured photometrically to assess plasma protein extravasation. Significant differences in dialysate protein content were found after 10(-9) M SP (affected side, 98.4 +/- 8.4% of baseline value; unaffected side, 104.4 +/- 5.6%; controls, 70.7 +/- 4.1%; P < 0.005) and after 10(-6) M SP (affected, 169.7 +/- 24.2%; unaffected, 189.4 +/- 19.1%; controls, 122.2 +/- 12.0%; P < 0.05). While 10(-9) M SP induced no protein extravasation in controls, it provoked protein extravasation in 6 of 11 patients on the affected and in 5 of 11 patients on the unaffected side (P < 0.01). We conclude that SP-induced plasma protein extravasation is increased in CRPS patients on both the affected and unaffected limbs. The underlying mechanism might be impaired SP inactivation. Thus, our results further support the hypothesis that neurogenic inflammation plays an important role in the initiation of CRPS.

Inhibition of neutral endopeptidase (NEP) facilitates neurogenic inflammation

Neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) are involved in neuropeptide degradation and may modulate neurogenic inflammation. We therefore explored the effect of specific blockers of NEP and ACE on the intensity of neurogenic inflammation. We investigated eight subjects on three occasions. Two pairs of microdialysis fibers equipped with intraluminal wires were inserted intracutaneously into the volar forearms and electrical stimuli were delivered via the intraluminal electrodes. The microdialysis fibers were perfused either with normal saline, phosphoramidon (NEP inhibitor), or captopril (ACE inhibitor). CGRP release was assessed in the microdialysis eluate via a specific EIA and by evaluating the extent and intensity of the neurogenic flare via a laser Doppler imager. The area of hyperalgesia and allodynia was assessed during electrical stimulation. Inhibition of NEP with phosphoramidon increased flare intensity (P < 0.002) and size (P < 0.01), while blocking ACE had no effect on neurogenic vasodilation. CGRP release could be measured in microdialysis samples after phosphoramidon perfusion only (P < 0.03), not in samples with captopril or saline perfusion. No effect on the areas of hyperalgesia and allodynia could be detected. Our findings suggest that NEP but not ACE is most important for CGRP degradation in human skin. This may be of particular importance for the understanding of pain disorders like migraine or complex regional pain syndrome.

Catecholamine release in human skin: a microdialysis study

Dermal microdialysis might be a promising tool to investigate properties of sympathetic neurons in the skin as investigation of peripheral noradrenergic neurons in humans usually relies on highly variable vasoconstrictor reflexes or on indirect measurements like skin temperature recordings. To evaluate this technique, 21 experiments were performed in 15 healthy subjects with four intracutaneous microdialysis fibers (diameter, 200 microm; cutoff, 5 kDa) at hands or feet. After 60 min, saline perfusion tyramine at concentrations of 0.195 to 200 microg/ml was applied for 15 min followed by a 15-min saline perfusion again. Catecholamine concentrations were detected through high-performance liquid chromatography with electrochemical detection. Control experiments were performed in human skin homogenates with and without tyramine incubation. In vivo, norepinephrine (NE) concentration increased from 36.3 +/- 10.2 pg/ml to 84.4 +/- 18.4 pg/ml (P < 0.001) during stimulation with tyramine, dialysate dopamine (DA) concentration increased from 105.2 +/- 36.5 pg/ml to 7162.4 +/- 3972.4 pg/ml (P < 0.001). Both tyramine-induced NE and DA release were dose-dependent (NE: r = 0.438, P < 0.05; DA: r = 0.894, P < 0.001). In skin homogenates, tyramine incubation led to a significant increase of DA concentrations (387.0 +/- 34.8 pg/ml, controls: 13.2 +/- 2.4 pg/ml; P < 0.05), while NE and epinephrine levels remained unchanged. In conclusion, our experiments show that dermal microdialysis is capable of locally measuring catecholamines in human skin. This offers the opportunity to investigate the function of the peripheral sympathetic nervous system. Additional to non-enzymatic oxidation, DA increase probably reflects metabolic degradation of tyramine by non-neuronal pathways and therefore does not reflect local sympathetic innervation.

Angiotensin converting enzyme has an inhibitory role in CGRP metabolism in human skin

The neutral endopeptidase (NEP) is important for calcitonin gene related peptide (CGRP) degradation, while the role of angiotensin converting enzyme (ACE) remains unclear. By using dermal microdialysis we explored the effect of phosphoramidon (NEP blocker), captopril (ACE blocker) and a mixture of both drugs on the intensity of electrically-induced CGRP-mediated neurogenic flare. The results reveal that phosphoramidon elevated flare intensity, but that this was not further increased by adding captopril. In contrast, neurogenic flare was decreased when the drug mixture was applied in compared to NEP only. Electrically released CGRP levels could be measured directly in perfusates containing phosphoramidon and the mixture. Again, CGRP levels were elevated in phosphoramidon treated sites, and significantly reduced upon adding captopril. These findings suggest that NEP and ACE do not have additive effects regarding neuropeptide degradation. In contrast, inhibition of ACE seems to augment CGRP catabolism.

Thoracoscopic sympathectomy at the T2 or T3 level facilitates bradykinin-induced protein extravasation in human forearm skin.

BACKGROUND The endogenous peptide bradykinin (BK) is an inflammatory mediator that induces nociceptor activation and sensitization as well as protein extravasation and vasodilation. OBJECTIVE To test the hypothesis if sympathectomy affects BK-induced inflammation in humans. METHODS Dermal microdialysis was employed on the volar forearm in 10 patients (21-41 years) with regional hyperhidrosis before and three months after preganglionic endoscopic transthoracic sympathetic clipping (ETSC) at the T2 or T3 level and in 10 healthy volunteers (22-36 years). After 60 minutes perfusion with Ringers solution microdialysis fibers were perfused with BK 10(-7) M and 10(-5) M for 30 minutes followed by 30 minutes Ringers solution again. To assess protein extravasation dialysate protein content was measured photometrically and Laser-Doppler imaging was used to quantify axonreflex vasodilation. RESULTS Baseline flux values after ETSC were higher as compared with controls and preoperative values (anova, Bonferroni post hoc test, P < 0.05), but neither BK 10(-7) M nor 10(-5) M led to significant vasodilation. Baseline dialysate protein did not significantly differ between groups. BK 10(-5) M induced protein extravasation while BK 10(-7) M was ineffective, and BK 10(-5) M induced protein extravasation was significantly enhanced after ETSC (P < 0.001). CONCLUSIONS Forearm skin perfusion is increased after ETSC on the T2 or T3 level indicating decreased sympathetic activity while BK-induced protein extravasation was increased. These results show that preganglionic sympathectomy does not diminish bradykinin-induced protein extravasation as found for postganglionic sympathectomy in rats.

Lack of genetic association of neutral endopeptidase (NEP) with complex regional pain syndrome (CRPS).

Complex regional pain syndrome (CRPS) is a condition that is characterized by severe pain and exaggerated neurogenic inflammation, which may develop after injury or surgery. Neurogenic inflammation is mediated by neuropeptides, such as calcitonin gene-related peptide (CGRP) and substance P (SP) that are released from nociceptors. Genetic factors may play a role in CRPS as was suggested by the occurrence of familial cases and several genetic association studies investigating mainly the human leukocyte antigen (HLA) system. Here we investigated the role of neutral endopeptidase (NEP), a key enzyme in neuropeptide catabolism. NEP dysfunction resulting in reduced inactivation of neuropeptides may be a possible pathomechanism in CRPS. To this end, we tested a GT-repeat polymorphism in the NEP promoter region as well as 18 tag-SNPs in six linkage disequilibrium (LD) blocks in the NEP gene region in 320 CRPS patients and 376 controls. No significant genetic association was observed. Thus, we conclude that the NEP gene does not seem to be a major risk factor for CRPS.

Characterization of Somatosensory Profiles in Patients with Crohn's Disease

Crohns disease (CD) is a painful chronic inflammatory bowel disease. It primarily affects terminal ileum, but the involvement of large and small intestines or extraintestinal manifestations is very common. CD may go along with neurogenic inflammation, mediated by substance P and CGRP, which are also key players in pain transmission. This may in turn contribute to hyperalgesia and altered somatosensory function in CD.

Validation of antibody-based tools for galanin research

Antibodies are an integral biomedical tool, not only for research but also as therapeutic agents. However, progress can only be made with sensitive and specific antibodies. The regulatory (neuro)peptide galanin and its three endogenous receptors (GAL1-3-R) are widely distributed in the central and peripheral nervous systems, and in peripheral non-neuronal tissues. The galanin system has multiple biological functions, including feeding behavior, pain processing, nerve regeneration and inflammation, to name only a few. Galanin could serve as biomarker in these processes, and therefore its receptors are potential drug targets for various diseases. For that reason, it is of paramount interest to precisely measure galanin peptide levels in tissues and to determine the cellular and subcellular localization of galanin receptors. A plethora of antibodies and antibody-based tools, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) kits, are commercially available to detect galanin and its receptors. However, many of them lack rigorous validation which casts doubt on their specificity. A goal of the present study was to raise awareness of the importance of validation of antibodies and antibody-based tools, with a specific focus on the galanin system. To that end, we tested and report here about commercially available antibodies against galanin and galanin receptors that appear specific to us. Furthermore, we investigated the validity of commercially available galanin ELISA kits. As the tested ELISAs failed to meet the validation requirements, we developed and validated a specific sandwich ELISA which can be used to detect full-length galanin in human plasma.