Publications by authors named "Shaaban A Mousa"

78 Publications

Functional and Anatomical Characterization of Corticotropin-Releasing Factor Receptor Subtypes of the Rat Spinal Cord Involved in Somatic Pain Relief.

Mol Neurobiol 2021 Jul 31. Epub 2021 Jul 31.

Department of Anesthesiology and Operative Intensive Care Medicine, Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Charité - Universitätsmedizin Berlin, Charité Campus Benjamin Franklin, Hindenburgdamm 30, 12203, Berlin, Germany.

Corticotropin-releasing factor (CRF) orchestrates our body's response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptor subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. Thus, the aim of the present study was to examine CRF-R1 and CRF-R2 expression within the spinal cord of rats with Freund's complete adjuvant-induced unilateral inflammation of the hind paw using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis. Moreover, the antinociceptive effects of intrathecal (i.t.) CRF were measured by paw pressure algesiometer and their possible antagonism by selective antagonists for CRF-R1 and/or CRF-R2 as well as for opioid receptors. Our results demonstrated a preference for the expression of CRF-R2 over CRF-R1 mRNA, protein, binding sites and immunoreactivity in the dorsal horn of the rat spinal cord. Consistently, CRF as well as CRF-R2 agonists elicited potent dose-dependent antinociceptive effects which were antagonized by the i.t. CRF-R2 selective antagonist K41498, but not by the CRF-R1 selective antagonist NBI35965. In addition, i.t. applied opioid antagonist naloxone dose-dependently abolished the i.t. CRF- as well as CRF-R2 agonist-elicited inhibition of somatic pain. Importantly, double immunofluorescence confocal microscopy of the spinal dorsal horn showed CRF-R2 on enkephalin (ENK)-containing inhibitory interneurons in close opposition of incoming mu-opioid receptor-immunoreactive nociceptive neurons. CRF-R2 was, however, not seen on pre- or on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonists inhibit somatic inflammatory pain predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons which finally results in endogenous opioid-mediated pain inhibition.
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http://dx.doi.org/10.1007/s12035-021-02481-zDOI Listing
July 2021

Chronic Naltrexone Therapy Is Associated with Improved Cardiac Function in Volume Overloaded Rats.

Cardiovasc Drugs Ther 2021 Aug 23;35(4):733-743. Epub 2021 Jan 23.

Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Anesthesiology and Operative Intensive Care Medicine, Charité Campus Benjamin Franklin, Hindenburgdamm 30, 12203, Berlin, Germany.

Purpose: Myocardial opioid receptors were demonstrated in animals and humans and seem to colocalize with membranous and sarcolemmal calcium channels of the excitation-contraction coupling in the left ventricle (LV). Therefore, this study investigated whether blockade of the cardiac opioid system by naltrexone would affect cardiac function and neurohumoral parameters in Wistar rats with volume overload-induced heart failure.

Methods: Volume overload in Wistar rats was induced by an aortocaval fistula (ACF). Left ventricular cardiac opioid receptors were identified by immunohistochemistry and their messenger ribonucleic acid (mRNA) as well as their endogenous ligand mRNA quantified by real-time polymerase chain reaction (RT-PCR). Following continuous delivery of either the opioid receptor antagonist naltrexone or vehicle via minipumps (n = 5 rats each), hemodynamic and humoral parameters were assessed 28 days after ACF induction. Sham-operated animals served as controls.

Results: In ACF rats mu-, delta-, and kappa-opioid receptors colocalized with voltage-gated L-type Ca2+ channels in left ventricular cardiomyocytes. Chronic naltrexone treatment of ACF rats reduced central venous pressure (CVP) and left ventricular end-diastolic pressure (LVEDP), and improved systolic and diastolic left ventricular functions. Concomitantly, rat brain natriuretic peptide (rBNP-45) and angiotensin-2 plasma concentrations which were elevated during ACF were significantly diminished following naltrexone treatment. In parallel, chronic naltrexone significantly reduced mu-, delta-, and kappa-opioid receptor mRNA, while it increased the endogenous opioid peptide mRNA compared to controls.

Conclusion: Opioid receptor blockade by naltrexone leads to improved LV function and decreases in rBNP-45 and angiotensin-2 plasma levels. In parallel, naltrexone resulted in opioid receptor mRNA downregulation and an elevated intrinsic tone of endogenous opioid peptides possibly reflecting a potentially cardiodepressant effect of the cardiac opioid system during volume overload.
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http://dx.doi.org/10.1007/s10557-020-07132-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8266787PMC
August 2021

Prostanoid Receptor Subtypes and Its Endogenous Ligands with Processing Enzymes within Various Types of Inflammatory Joint Diseases.

Mediators Inflamm 2020 12;2020:4301072. Epub 2020 Nov 12.

Department of Anaesthesiology and Intensive Care Medicine, Charité-University Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Campus Benjamin Franklin, Berlin, Germany.

A complex inflammatory process mediated by proinflammatory cytokines and prostaglandins commonly occurs in the synovial tissue of patients with joint trauma (JT), osteoarthritis (OA), and rheumatoid arthritis (RA). This study systematically investigated the distinct expression profile of prostaglandin E2 (PGE2), its processing enzymes (COX-2), and microsomal PGES-1 (mPGES-1) as well as the corresponding prostanoid receptor subtypes (EP1-4) in representative samples of synovial tissue from these patients (JT, OA, and RA). Quantitative TaqMan®-PCR and double immunofluorescence confocal microscopy of synovial tissue determined the abundance and exact immune cell types expressing these target molecules. Our results demonstrated that PGE2 and its processing enzymes COX-2 and mPGES-1 were highest in the synovial tissue of RA, followed by the synovial tissue of OA and JT patients. Corresponding prostanoid receptor, subtypes EP3 were highly expressed in the synovium of RA, followed by the synovial tissue of OA and JT patients. These proinflammatory target molecules were distinctly identified in JT patients mostly in synovial granulocytes, in OA patients predominantly in synovial macrophages and fibroblasts, whereas in RA patients mainly in synovial fibroblasts and plasma cells. Our findings show a distinct expression profile of EP receptor subtypes and PGE2 as well as the corresponding processing enzymes in human synovium that modulate the inflammatory process in JT, OA, and RA patients.
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http://dx.doi.org/10.1155/2020/4301072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676943PMC
November 2020

Neuronal aldosterone elicits a distinct genomic response in pain signaling molecules contributing to inflammatory pain.

J Neuroinflammation 2020 Jun 12;17(1):183. Epub 2020 Jun 12.

Department of Anaesthesiology and Intensive Care Medicine, Charité - University Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Campus Benjamin Franklin, Berlin, Germany.

Background: Recently, mineralocorticoid receptors (MR) were identified in peripheral nociceptive neurons, and their acute antagonism was responsible for immediate and short-lasting (non-genomic) antinociceptive effects. The same neurons were shown to produce the endogenous ligand aldosterone by the enzyme aldosterone synthase.

Methods: Here, we investigate whether endogenous aldosterone contributes to inflammation-induced hyperalgesia via the distinct genomic regulation of specific pain signaling molecules in an animal model of Freund's complete adjuvant (FCA)-induced hindpaw inflammation.

Results: Chronic intrathecal application of MR antagonist canrenoate-K (over 4 days) attenuated nociceptive behavior in rats with FCA hindpaw inflammation suggesting a tonic activation of neuronal MR by endogenous aldosterone. Consistently, double immunofluorescence confocal microscopy showed abundant co-localization of MR with several pain signaling molecules such as TRPV1, CGRP, Nav1.8, and trkA whose enhanced expression of mRNA and proteins during inflammation was downregulated following i.t. canrenoate-K. More importantly, inhibition of endogenous aldosterone production in peripheral sensory neurons by continuous intrathecal delivery of a specific aldosterone synthase inhibitor prevented the inflammation-induced enhanced transcriptional expression of TRPV1, CGRP, Nav1.8, and trkA and subsequently attenuated nociceptive behavior. Evidence for such a genomic effect of endogenous aldosterone was supported by the demonstration of an enhanced nuclear translocation of MR in peripheral sensory dorsal root ganglia (DRG) neurons.

Conclusion: Taken together, chronic inhibition of local production of aldosterone by its processing enzyme aldosterone synthase within peripheral sensory neurons may contribute to long-lasting downregulation of specific pain signaling molecules and may, thus, persistently reduce inflammation-induced hyperalgesia.
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http://dx.doi.org/10.1186/s12974-020-01864-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291517PMC
June 2020

Aldosterone Synthase in Peripheral Sensory Neurons Contributes to Mechanical Hypersensitivity during Local Inflammation in Rats.

Anesthesiology 2020 04;132(4):867-880

From the Department of Anaesthesiology and Intensive Care Medicine, Charite Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, Berlin, Germany (D.M.M., M. Shaqura, X.L., S.T., M. Schäfer, S.A.M.) Department of Zoology, Faculty of Science, Aswan University, Aswan, Egypt (D.M.M.) the Departments of Anatomy (M. Shakibaei) Anaesthesiology (A.B.), Ludwig Maximilian University of Munich, Munich, Germany.

Background: Recent emerging evidence suggests that extra-adrenal synthesis of aldosterone occurs (e.g., within the failing heart and in certain brain areas). In this study, the authors investigated evidence for a local endogenous aldosterone production through its key processing enzyme aldosterone synthase within peripheral nociceptive neurons.

Methods: In male Wistar rats (n = 5 to 8 per group) with Freund's complete adjuvant hind paw inflammation, the authors examined aldosterone, aldosterone synthase, and mineralocorticoid receptor expression in peripheral sensory neurons using quantitative reverse transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and immunoprecipitation. Moreover, the authors explored the nociceptive behavioral changes after selective mineralocorticoid receptor antagonist, canrenoate-K, or specific aldosterone synthase inhibitor application.

Results: In rats with Freund's complete adjuvant-induced hind paw inflammation subcutaneous and intrathecal application of mineralocorticoid receptor antagonist, canrenoate-K, rapidly and dose-dependently attenuated nociceptive behavior (94 and 48% reduction in mean paw pressure thresholds, respectively), suggesting a tonic activation of neuronal mineralocorticoid receptors by an endogenous ligand. Indeed, aldosterone immunoreactivity was abundant in peptidergic nociceptive neurons of dorsal root ganglia and colocalized predominantly with its processing enzyme aldosterone synthase and mineralocorticoid receptors. Moreover, aldosterone and its synthesizing enzyme were significantly upregulated in peripheral sensory neurons under inflammatory conditions. The membrane mineralocorticoid receptor consistently coimmunoprecipitated with endogenous aldosterone, confirming a functional link between mineralocorticoid receptors and its endogenous ligand. Importantly, inhibition of endogenous aldosterone production in peripheral sensory neurons by a specific aldosterone synthase inhibitor attenuated nociceptive behavior after hind paw inflammation (a 32% reduction in paw pressure thresholds; inflammation, 47 ± 2 [mean ± SD] vs. inflammation + aldosterone synthase inhibitor, 62 ± 2).

Conclusions: Local production of aldosterone by its processing enzyme aldosterone synthase within peripheral sensory neurons contributes to ongoing mechanical hypersensitivity during local inflammation via intrinsic activation of neuronal mineralocorticoid receptors.
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http://dx.doi.org/10.1097/ALN.0000000000003127DOI Listing
April 2020

Efficacy-Based Perspective to Overcome Reduced Opioid Analgesia of Advanced Painful Diabetic Neuropathy in Rats.

Front Pharmacol 2019 9;10:347. Epub 2019 Apr 9.

Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Budapest, Hungary.

Reduction of the opioid analgesia in diabetic neuropathic pain (DNP) results from μ-opioid receptor (MOR) reserve reduction. Herein, we examined the antinociceptive and antiallodynic actions of a novel opioid agonist 14--methymorphine-6--sulfate (14--MeM6SU), fentanyl and morphine in rats with streptozocin-evoked DNP of 9-12 weeks following their systemic administration. The antinociceptive dose-response curve of morphine but not of 14--MeM6SU or fentanyl showed a significant right-shift in diabetic compared to non-diabetic rats. Only 14--MeM6SU produced antiallodynic effects in doses matching antinociceptive doses obtained in non-diabetic rats. Co-administered naloxone methiodide (NAL-M), a peripherally acting opioid receptor antagonist failed to alter the antiallodynic effect of test compounds, indicating the contribution of central opioid receptors. Reduction in spinal MOR binding sites and loss in MOR immunoreactivity of nerve terminals in the spinal cord and dorsal root ganglia in diabetic rats were observed. G-protein coupling assay revealed low efficacy character for morphine and high efficacy character for 14--MeM6SU or fentanyl at spinal or supraspinal levels ( values). Furthermore, at the spinal level only 14--MeM6SU showed equal efficacy in G-protein activation in tissues of diabetic- and non-diabetic animals. Altogether, the reduction of spinal opioid receptors concomitant with reduced analgesic effect of morphine may be circumvented by using high efficacy opioids, which provide superior analgesia over morphine. In conclusion, the reduction in the analgesic action of opioids in DNP might be a consequence of MOR reduction, particularly in the spinal cord. Therefore, developing opioids of high efficacy might provide analgesia exceeding that of currently available opioids.
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http://dx.doi.org/10.3389/fphar.2019.00347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465774PMC
April 2019

Identification of mineralocorticoid and glucocorticoid receptors on peripheral nociceptors: Translation of experimental findings from animal to human biology.

Brain Res 2019 06 13;1712:180-187. Epub 2019 Feb 13.

Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Anaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum, Berlin, Germany.

Evidence is accumulating that activation of mineralocorticoid (MR) and glucocorticoid (GR) receptors on peripheral sensory neurons modulates pain sensation. While the expression and exact anatomical localization of MR and GR in the various subpopulations of peripheral sensory neurons has been shown in animals, it is still unknown for the human skin. Therefore, we aimed to identify MR and GR mRNA and protein as well as the exact subpopulations of sensory neurons in human versus rat skin. Tissue samples from rat and human skin were subjected to RT-PCR, Western blot and double immunofluorescence confocal analysis of MR and GR with the neuronal markers calcitonin gene-related peptide (CGRP), neurofilament 200 (NF200) and tyrosine hydroxylase (TH). Using RT-PCR we were able to isolate MR as well as GR specific transcripts from human skin. Consistently, Western blot analysis identified MR- as well as GR- specific protein bands at the expected molecular weights of 110 and 87 kD, respectively. Double immunofluorescence confocal microscopy of human skin revealed that MR predominantly colocalized with calcitonin-gene-related peptide (CGRP)-immunoreactive (IR) nociceptive neurons - similar to rat skin - underscoring a pivotal role for MR in the modulation of pain. The majority of GR-immmunoreactivity was localized in peripheral peptidergic CGRP-IR sensory nerve fibers, but in addition on TH-IR sympathetic postganglionic, and NF200-IR myelinated mechanoreceptive nerve fibers, both within human and rat skin. Moreover, GR but not MR were localized in keratinocytes of the epidermal layer of human and rat skin. Overall, our results indicate considerable overlap in sensory neuron expression of MR and GR in humans and rats endorsing a common systems approach in mammals that may modulate the transmission of sensory information by MR and GR activation.
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http://dx.doi.org/10.1016/j.brainres.2019.02.015DOI Listing
June 2019

The Peripheral Versus Central Antinociception of a Novel Opioid Agonist: Acute Inflammatory Pain in Rats.

Neurochem Res 2018 Jun 3;43(6):1250-1257. Epub 2018 May 3.

Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Nagyvárad tér 4, P.O. Box 370, Budapest, 1445, Hungary.

Opioid analgesics devoid of central side effects are unmet medical need in the treatment of acute pain (e.g. post-operative pain). Recently, we have reported on 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU), a novel opioid agonist of high efficacy producing peripheral antinociception in subchronic inflammatory pain in certain doses. The present study focused on the antinociceptive effect of 14-O-MeM6SU compared to morphine in formalin test of an early/acute (Phase I) and late/tonic (Phase II) pain phases. Subcutaneous 14-O-MeM6SU (253-1012 nmol/kg) and morphine (3884-31075 nmol/kg) dose dependently reduced the pain behaviors of both phases. Co-administered naloxone methiodide (NAL-M), a peripherally acting opioid antagonist, abolished the antinociceptive effect of 506 nmol/kg 14-O-MeM6SU. On the other hand, the effects of 14-O-MeM6SU (1012 nmol/kg) and morphine (15538 nmol/kg) were only partially affected by NAL-M, indicating the contribution of CNS to antinociception. Locally injected test compounds into formalin treated paws caused antinociception in both phases. Locally effective doses of test compounds were also injected into contralateral paws. Morphine showed effects in both phases, 14-O-MeM6SU in certain doses failed to produce antinociception in either phase. A NAL-M reversible systemic dose of 14-O-MeM6SU and the lowest systemic effective dose of morphine were evaluated for their sedative effects following isoflurane-induced sleeping (righting reflex). In contrast to morphine, 14-O-MeM6SU in certain antinociceptive doses showed no impact on sleeping time. These data highlight that high efficacy opioids of limited CNS penetration in certain doses mitigate somatic and inflammatory pain by targeting MOR at the periphery.
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http://dx.doi.org/10.1007/s11064-018-2542-7DOI Listing
June 2018

Pro- versus Antinociceptive Nongenomic Effects of Neuronal Mineralocorticoid versus Glucocorticoid Receptors during Rat Hind Paw Inflammation.

Anesthesiology 2018 04;128(4):796-809

From the Department of Anaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charité Mitte, Berlin, Germany (X.L., M. Shaqura, D.M., S.A.M., M. Schäfer); the Department of Anaesthesiology, Ludwig-Maximilians-University Munich, Munich, Germany (A.B.); and the Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kyoto, Japan (S.Y.).

Background: In naive rats, corticosteroids activate neuronal membrane-bound glucocorticoid and mineralocorticoid receptors in spinal cord and periphery to modulate nociceptive behavior by nongenomic mechanisms. Here we investigated inflammation-induced changes in neuronal versus glial glucocorticoid and mineralocorticoid receptors and their ligand-mediated nongenomic impact on mechanical nociception in rats.

Methods: In Wistar rats (n = 5 to 7/group) with Freund's complete adjuvant hind paw inflammation, we examined glucocorticoid and mineralocorticoid receptor expression in spinal cord and peripheral sensory neurons versus glial using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, immunohistochemistry, and radioligand binding. Moreover, we explored the expression of mineralocorticoid receptors protecting enzyme 11-betahydroxysteroid dehydrogenase type 2 as well as the nociceptive behavioral changes after glucocorticoid and mineralocorticoid receptors agonist or antagonist application.

Results: Hind paw inflammation resulted in significant upregulation of glucocorticoid receptors in nociceptive neurons of spinal cord (60%) and dorsal root ganglia (15%) as well as mineralocorticoid receptors, while corticosteroid plasma concentrations remained unchanged. Mineralocorticoid (83 ± 16 fmol/mg) but not glucocorticoid (104 ± 20 fmol/mg) membrane binding sites increased twofold in dorsal root ganglia concomitant with upregulated 11-betahydroxysteroid dehydrogenase type 2 (43%). Glucocorticoid and mineralocorticoid receptor expression in spinal microglia and astrocytes was small. Importantly, glucocorticoid receptor agonist dexamethasone or mineralocorticoid receptor antagonist canrenoate-K rapidly and dose-dependently attenuated nociceptive behavior. Isobolographic analysis of the combination of both drugs showed subadditive but not synergistic or additive effects.

Conclusions: The enhanced mechanical sensitivity of inflamed hind paws accompanied with corticosteroid receptor upregulation in spinal and peripheral sensory neurons was attenuated immediately after glucocorticoid receptor agonist and mineralocorticoid receptor antagonist administration, suggesting acute nongenomic effects consistent with detected membrane-bound corticosteroid receptors.
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http://dx.doi.org/10.1097/ALN.0000000000002087DOI Listing
April 2018

Accessibility of axonal G protein coupled mu-opioid receptors requires conceptual changes of axonal membrane targeting for pain modulation.

J Control Release 2017 Dec 17;268:352-363. Epub 2017 Oct 17.

Dep. of Anaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charite Mitte, Berlin, Germany. Electronic address:

The mechanisms of axonal trafficking and membrane targeting are well established for sodium channels, which are the principle targets for perineurally applied local anaesthetics. However, they have not been thoroughly investigated for G protein coupled receptors such as mu-opioid receptors (MOR). Focusing on these axonal mechanisms, we found that axonal MOR functionality is quite distinct in two different pain states, i.e. hindpaw inflammation and nerve injury. We observed axonal membrane MOR binding and functional G protein coupling exclusively at sites of CCI nerve injury. Moreover at these axonal membrane sites, MOR exhibited extensive co-localization with the membrane proteins SNAP and Na/K-ATPase as well as NGF-dependent enhanced lipid rafts and L1CAM anchoring proteins. Silencing endogenous L1CAM with intrathecal L1CAM specific siRNA, disrupting lipid rafts with the perineurial cholesterol-sequestering agent MβCD, as well as suppressing NGF receptor activation with the perineurial NGF receptor inhibitor K252a abrogated MOR axonal membrane integration, functional coupling, and agonist-elicited antinociception at sites of nerve injury. These findings suggest that local conceptual changes resulting from nerve injury are required for the establishment of functional axonal membrane MOR. Axonal integration and subsequent accessibility of functionally coupled MOR are of great relevance particularly for patients suffering from severe pain due to nerve injury or tumour infiltration.
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http://dx.doi.org/10.1016/j.jconrel.2017.10.016DOI Listing
December 2017

Pathological alterations in liver injury following congestive heart failure induced by volume overload in rats.

PLoS One 2017 21;12(9):e0184161. Epub 2017 Sep 21.

Department of Anaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charité Mitte, Berlin, Germany.

Heart failure has emerged as a disease with significant public health implications. Following progression of heart failure, heart and liver dysfunction are frequently combined in hospitalized patients leading to increased morbidity and mortality. Here, we investigated the underlying pathological alterations in liver injury following heart failure. Heart failure was induced using a modified infrarenal aortocaval fistula (ACF) in male Wistar rats. Sham operated and ACF rats were compared for their morphometric and hemodynamic data, for histopathological and ultrastructural changes in the liver as well as differences in the expression of apoptotic factors. ACF-induced heart failure is associated with light microscopic signs of apparent congestion of blood vessels, increased apoptosis and breakdown of hepatocytes and inflammatory cell inifltration were observed. The glycogen content depletion associated with the increased hepatic fibrosis, lipid globule formation was observed in ACF rats. Moreover, cytoplasmic organelles are no longer distinguishable in many ACF hepatocytes with degenerated fragmented rough endoplasmic reticulum, shrunken mitochondria and heavy cytoplasm vacuolization. ACF is associated with the upregulation of the hepatic TUNEL-positive cells and proapoptotic factor Bax protein concomitant with the mitochondrial leakage of cytochrome C into the cell cytoplasm and the transfer of activated caspase 3 from the cytoplasm into the nucleus indicating intrinsic apoptotic events. Taken together, the results demonstrate that ACF-induced congestive heart failure causes liver injury which results in hepatocellular apoptotic cell death mediated by the intrinsic pathway of mitochondrial cytochrome C leakage and subsequent transfer of activated caspase 3 into to the nucleus to initiate overt DNA fragmentation and cell death.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184161PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608213PMC
October 2017

Histopathological Changes in the Kidney following Congestive Heart Failure by Volume Overload in Rats.

Oxid Med Cell Longev 2017 31;2017:6894040. Epub 2017 Jul 31.

Department of Anaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charité Mitte, Berlin, Germany.

Background: This study investigated histopathological changes and apoptotic factors that may be involved in the renal damage caused by congestive heart failure in a rat model of infrarenal aortocaval fistula (ACF).

Methods: Heart failure was induced using a modified approach of ACF in male Wistar rats. Sham-operated controls and ACF rats were characterized by their morphometric and hemodynamic parameters and investigated for their histopathological, ultrastructural, and apoptotic factor changes in the kidney.

Results: ACF-induced heart failure is associated with histopathological signs of congestion and glomerular and tubular atrophy, as well as nuclear and cellular degeneration in the kidney. In parallel, overexpression of proapoptotic Bax protein, release of cytochrome C from the outer mitochondrial membrane into cell cytoplasm, and nuclear transfer of activated caspase 3 indicate apoptotic events. This was confirmed by electron microscopic findings of apoptotic signs in the kidney such as swollen mitochondria and degenerated nuclei in renal tubular cells.

Conclusions: This study provides morphological evidence of renal injury during heart failure which may be due to caspase-mediated apoptosis via overexpression of proapoptotic Bax protein, subsequent mitochondrial cytochrome C release, and final nuclear transfer of activated caspase 3, supporting the notion of a cardiorenal syndrome.
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http://dx.doi.org/10.1155/2017/6894040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555028PMC
April 2018

Comparative Expression Analyses of Pro- versus Anti-Inflammatory Mediators within Synovium of Patients with Joint Trauma, Osteoarthritis, and Rheumatoid Arthritis.

Mediators Inflamm 2017 20;2017:9243736. Epub 2017 Feb 20.

Department of Anaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charité Mitte, Berlin, Germany.

Synovial injury and healing are complex processes including catabolic effects by proinflammatory cytokines and anabolic processes by anti-inflammatory mediators. Here we examined the expression of pro- versus anti-inflammatory mediators in synovium of patients with diagnostic arthroscopy (control), joint trauma (JT), osteoarthritis (OA), and rheumatoid arthritis (RA). Synovial samples from these patients were subjected to RT-PCR and double immunofluorescence confocal microscopy of pro- and anti-inflammatory mediators as well as immune cell markers. Interestingly, pro- and anti-inflammatory mediators were expressed predominantly in granulocytes in patients with JT and in macrophages, lymphocytes, and plasma cells in patients with OA and RA. Interestingly, parallel to the severity of inflammation, proinflammatory mediators IL-1, TNF-, and 5-LOX specific mRNA as well as immunoreactive (IR) cells were significantly more abundant in patients with RA and JT than in those with OA. However, anti-inflammatory mediators 15-LOX, FPR2, and IL-10 specific mRNA as well as IR cells were significantly more abundant in patients with OA than in those with JT and RA. These findings show that upregulation of proinflammatory mediators contributes to the predominantly catabolic inflammatory process in JT and RA synovium, whereas upregulation of anabolic anti-inflammatory mediators counteracts inflammation resulting in the inferior inflammatory process in OA synovium.
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http://dx.doi.org/10.1155/2017/9243736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5337844PMC
July 2017

Membrane-bound glucocorticoid receptors on distinct nociceptive neurons as potential targets for pain control through rapid non-genomic effects.

Neuropharmacology 2016 12 21;111:1-13. Epub 2016 Aug 21.

Department of Anaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charité Mitte, Berlin, Germany. Electronic address:

Glucocorticoids were long believed to primarily function through cytosolic glucocorticoid receptor (GR) activation and subsequent classical genomic pathways. Recently, however, evidence has emerged that suggests the presence of rapid non-genomic GR-dependent signaling pathways within the brain, though their existence in spinal and peripheral nociceptive neurons remains elusive. In this paper, we aim to systemically identify GR within the spinal cord and periphery, to verify their putative membrane location and to characterize possible G protein coupling and pain modulating properties. Double immunofluorescence confocal microscopy revealed that GR predominantly localized in peripheral peptidergic and non-peptidergic nociceptive C- and Aδ-neurons and existed only marginally in myelinated mechanoreceptive and proprioreceptive neurons. Within the spinal cord, GR predominantly localized in incoming presynaptic nociceptive neurons, in pre- and postsynaptic structures of the dorsal horn, as well as in microglia. GR saturation binding revealed that these receptors are linked to the cell membrane of sensory neurons and, upon activation, they trigger membrane targeted [S]GTPγS binding, indicating G protein coupling to a putative receptor. Importantly, subcutaneous dexamethasone immediately and dose-dependently attenuated acute nociceptive behavior elicited in an animal model of formalin-induced pain hypersensitivity compared to naive rats. Overall, this study provides firm evidence for a novel neuronal mechanism of GR agonists that is rapid, non-genomic, dependent on membrane binding and G protein coupling, and acutely modulates nociceptive behavior, thus unraveling a yet unconsidered mechanism of pain relief.
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http://dx.doi.org/10.1016/j.neuropharm.2016.08.019DOI Listing
December 2016

New Morphine Analogs Produce Peripheral Antinociception within a Certain Dose Range of Their Systemic Administration.

J Pharmacol Exp Ther 2016 10 19;359(1):171-81. Epub 2016 Jul 19.

Departments of Pharmacology and Pharmacotherapy (E.L., P.R., Z.G., L.C., M.B., K.K., Z.S.Z., P.F., S.F., M.A.) and Pharmaceutical Chemistry (A.V., S.H.), Faculty of Medicine, Semmelweis University, Budapest, Hungary; Department of Anaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charité Mitte, Berlin, Germany (S.A.M., M.S.); Department of Pharmacology and Pharmacotherapy, Medical School, Szentágothai Research Centre (K.C., Z.H.), and MTA-PTE NAP B Chronic Pain Research Group, Faculty of Medicine (Z.H.), University of Pécs, Pécs, Hungary

Growing data support peripheral opioid antinociceptive effects, particularly in inflammatory pain models. Here, we examined the antinociceptive effects of subcutaneously administered, recently synthesized 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU) compared with morphine-6-O-sulfate (M6SU) in a rat model of inflammatory pain induced by an injection of complete Freund's adjuvant and in a mouse model of visceral pain evoked by acetic acid. Subcutaneous doses of 14-O-MeM6SU and M6SU up to 126 and 547 nmol/kg, respectively, produced significant and subcutaneous or intraplantar naloxone methiodide (NAL-M)-reversible antinociception in inflamed paws compared with noninflamed paws. Neither of these doses significantly affected thiobutabarbital-induced sleeping time or rat pulmonary parameters. However, the antinociceptive effects of higher doses were only partially reversed by NAL-M, indicating contribution of the central nervous system. In the mouse writhing test, 14-O-MeM6SU was more potent than M6SU after subcutaneous or intracerebroventricular injections. Both displayed high subcutaneous/intracerebroventricular ED50 ratios. The antinociceptive effects of subcutaneous 14-O-MeM6SU and M6SU up to 136 and 3043 nmol/kg, respectively, were fully antagonized by subcutaneous NAL-M. In addition, the test compounds inhibited mouse gastrointestinal transit in antinociceptive doses. Taken together, these findings suggest that systemic administration of the novel compound 14-O-MeM6SU similar to M6SU in specific dose ranges shows peripheral antinociception in rat and mouse inflammatory pain models without central adverse effects. These findings apply to male animals and must be confirmed in female animals. Therefore, titration of systemic doses of opioid compounds with limited access to the brain might offer peripheral antinociception of clinical importance.
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http://dx.doi.org/10.1124/jpet.116.233551DOI Listing
October 2016

Acute mechanical sensitization of peripheral nociceptors by aldosterone through non-genomic activation of membrane bound mineralocorticoid receptors in naive rats.

Neuropharmacology 2016 08 23;107:251-261. Epub 2016 Mar 23.

Department of Anaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charité Mitte, Berlin, Germany.

Recently, there is increasing interest in the role of peripheral mineralocorticoid receptors (MR) to modulate pain, but their localization in neurons and glia of the periphery and their distinct involvement in pain control remains elusive. In naive Wistar rats our double immunofluorescence confocal microscopy of the spinal cord, dorsal root ganglia, sciatic nerve and innervated skin revealed that MR predominantly colocalized with calcitonin-gene-related peptide (CGRP)- and trkA-immunoreactive (IR) nociceptive neurons and only marginally with myelinated trkB-IR mechanoreceptive and trkC-IR proprioreceptive neurons underscoring a pivotal role for MR in the modulation of pain. MR could not be detected in Schwann cells, satellite cells, and astrocytes and only scarcely in spinal microglia cells excluding a relevant functional role of glia-derived MR at least in naïve rats. Intrathecal (i.t.) and intraplantar (i.pl.) application of increasing doses of the MR selective agonist aldosterone acutely increased nociceptive behavior which was reversible by a MR selective antagonist and most likely due to non-genomic effects. This was further substantiated by the first identification of membrane bound MR specific binding sites in sensory neurons of dorsal root ganglia and spinal cord. Therefore, a crucial role of MR on nociceptive neurons but not on glia cells and their impact on nociceptive behavior most likely due to immediate non-genomic effects has to be considered under normal but more so under pathological conditions in future studies.
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http://dx.doi.org/10.1016/j.neuropharm.2016.03.032DOI Listing
August 2016

Protein kinase C-mediated mu-opioid receptor phosphorylation and desensitization in rats, and its prevention during early diabetes.

Pain 2016 Apr;157(4):910-921

Department of Anaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charité Mitte, Berlin, Germany Department of Anatomy, Ludwig Maximilian University Munich, Munich, Germany Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University, Jena, Germany Department of Internal Medicine, Paulinenkrankenhaus, Berlin, Germany.

Painful diabetic neuropathy is associated with impaired opioid analgesia; however, the precise mechanism in sensory neurons remains unclear. This study aimed to identify putative mechanisms involved in modified opioid responsiveness during early streptozotocin-induced diabetes in rats. In this study, we demonstrate that in diabetic animals, impaired peripheral opioid analgesia is associated with a reduction in functional mu-opioid receptor (MOR) G protein coupling. Mu-opioid receptor immunoreactive neurons colocalized with activated forms of protein kinase C (PKC) and with the receptor for advanced glycation end products (RAGE) during streptozotocin-induced diabetes. Moreover, MOR phosphorylation at Thr370 in sensory neurons of diabetic rats, and thus desensitization, was due to RAGE-dependent PKC activation. Importantly, blocking PKC activation using PKC selective inhibitor, silencing RAGE with intrathecal RAGE siRNA, or inhibiting advanced glycation end product (AGE) formation prevented sensory neuron MOR phosphorylation and, consequently, restored MOR G protein coupling and analgesic efficacy. Thus, our findings give the first in vivo evidence of a RAGE-dependent PKC-mediated heterologous MOR phosphorylation and desensitization in sensory neurons under pathological conditions such as diabetic neuropathy. This may unravel putative mechanisms and suggest possible prevention strategies of impaired opioid responsiveness.
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http://dx.doi.org/10.1097/j.pain.0000000000000459DOI Listing
April 2016

Evidence for MOR on cell membrane, sarcoplasmatic reticulum and mitochondria in left ventricular myocardium in rats.

Heart Vessels 2016 Aug 19;31(8):1380-8. Epub 2015 Dec 19.

Department of Anesthesiology and Intensive Care Medicine, Campus Charité Mitte and Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.

Cardiac function is one important determinant to maintain tissue oxygenation and is thus highly regulated. In this context, it is interesting that centrally mediated opioidergic influence on cardiac function has long been known. Only recently, KOR and DOR have been found to be expressed in healthy left ventricular myocardium in rats and colocalized with parts of the excitation-contraction-coupling system. However, several comments in literature exist doubting the existence of MOR in cardiac tissue. We, therefore, aimed to detect MOR in rat left ventricular cardiomyocytes, and to evaluate whether MOR and POMC are regulated during heart failure. After IRB approval, heart failure was induced using a modified infrarenal aortocaval fistula (ACF) in male Wistar rats. All rats of the control and ACF group were characterized by their morphometrics and hemodynamics and the existence of MOR and POMC was investigated by means of radioligand binding, double immunofluorescence confocal analysis, RT-PCR and Western blot. Membrane MOR selective binding sites were detected in the left ventricular myocardium, however, they were lower in abundance than KOR- and DOR-specific binding sites and B max of MOR could not be determined. In left ventricular cardiomyocytes, MOR colocalized with parts of the excitation-coupling mechanism, e.g., Cav1.2 of the cell membrane and invaginated T-tubules as well as the ryanodine receptor of the sarcoplasmatic reticulum. More importantly, MOR strongly colocalized with mitochondria of left ventricular cardiomyocytes. Volume overload was not associated with an altered expression of MOR and POMC on both mRNA and protein level. These findings provide evidence for the existence of MOR on the cell membrane, sarcoplasmatic reticulum and mitochondria in left ventricular cardiomyocytes in rats. However, heart failure does not result in an altered expression of the cardiac MOR-opioid system. Thus, MOR agonist treatment-commonly used in the clinical setting-might directly affect cardiac function, which needs to be evaluated in greater detail in the near future.
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http://dx.doi.org/10.1007/s00380-015-0784-8DOI Listing
August 2016

Upregulation of the kappa opioidergic system in left ventricular rat myocardium in response to volume overload: Adaptive changes of the cardiac kappa opioid system in heart failure.

Pharmacol Res 2015 Dec 10;102:33-41. Epub 2015 Sep 10.

Department of Anesthesiology and Intensive Care Medicine, Campus Charité Mitte and Campus Virchow-Klinikum, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.

Opioids have long been known for their analgesic effects and are therefore widely used in anesthesia and intensive care medicine. However, in the last decade research has focused on the opioidergic influence on cardiovascular function. This project thus aimed to detect the precise cellular localization of kappa opioid receptors (KOR) in left ventricular cardiomyocytes and to investigate putative changes in KOR and its endogenous ligand precursor peptide prodynorphin (PDYN) in response to heart failure. After IRB approval, heart failure was induced using a modified infrarenal aortocaval fistula (ACF) in male Wistar rats. All rats of the control and ACF group were characterized by their morphometrics and hemodynamics. In addition, the existence and localization as well as adaptive changes of KOR and PDYN were investigated using radioligand binding, double immunofluorescence confocal analysis, RT-PCR and Western blot. Similar to the brain and spinal cord, [(3)H]U-69593 KOR selective binding sites were detected the left ventricle (LV). KOR colocalized with Cav1.2 of the outer plasma membrane and invaginated T-tubules and intracellular with the ryanodine receptor of the sarcoplasmatic reticulum. Interestingly, KOR could also be detected in mitochondria of rat LV cardiomyocytes. As a consequence of heart failure, KOR and PDYN were up-regulated on the mRNA and protein level in the LV. These findings suggest that the cardiac kappa opioidergic system might modulate rat cardiomyocyte function during heart failure.
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http://dx.doi.org/10.1016/j.phrs.2015.09.005DOI Listing
December 2015

Cellular localization and adaptive changes of the cardiac delta opioid receptor system in an experimental model of heart failure in rats.

Heart Vessels 2016 Feb 1;31(2):241-50. Epub 2015 Jan 1.

Department of Anesthesiology and Intensive Care Medicine, Campus Charité Mitte and Campus Virchow Klinikum, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.

The role of the cardiac opioid system in congestive heart failure (CHF) is not fully understood. Therefore, this project investigated the cellular localization of delta opioid receptors (DOR) in left ventricle (LV) myocardium and adaptive changes in DOR and its endogenous ligand, the precursor peptide proenkephalin (PENK), during CHF. Following IRB approval, DOR localization was determined by radioligand binding using [H(3)]Naltrindole and by double immunofluorescence confocal analysis in the LV of male Wistar rats. Additionally, 28 days following an infrarenal aortocaval fistula (ACF) the extent of CHF and adaptions in left ventricular DOR and PENK expression were examined by hemodynamic measurements, RT-PCR, and Western blot. DOR specific membrane binding sites were identified in LV myocardium. DOR were colocalized with L-type Ca(2+)-channels (Cav1.2) as well as with intracellular ryanodine receptors (RyR) of the sarcoplasmatic reticulum. Following ACF severe congestive heart failure developed in all rats and was accompanied by up-regulation of DOR and PENK on mRNA as well as receptor proteins representing consecutive adaptations. These findings might suggest that the cardiac delta opioid system possesses the ability to play a regulatory role in the cardiomyocyte calcium homeostasis, especially in response to heart failure.
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http://dx.doi.org/10.1007/s00380-014-0620-6DOI Listing
February 2016

New insights into mechanisms of opioid inhibitory effects on capsaicin-induced TRPV1 activity during painful diabetic neuropathy.

Neuropharmacology 2014 Oct 24;85:142-50. Epub 2014 May 24.

Department of Anaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charite Mitte, Augustenburgerplatz 1, 13353 Berlin, Germany. Electronic address:

Painful diabetic neuropathy is a disease of the peripheral sensory neuron with impaired opioid responsiveness. Since μ-opioid receptor (MOR) activation can inhibit the transient receptor potential vanilloid 1 (TRPV1) activity in peripherally sensory neurons, this study investigated the mechanisms of impaired opioid inhibitory effects on capsaicin-induced TRPV1 activity in painful diabetic neuropathy. Intravenous injection of streptozotocin (STZ, 45 mg/kg) in Wistar rats led to a degeneration of insulin producing pancreatic β-cells, elevated blood glucose, and mechanical hypersensitivity (allodynia). In these animals, local morphine's inhibitory effects on capsaicin-induced nocifensive behavior as well as on capsaicin-induced TRPV1 current in dorsal root ganglion cells were significantly impaired. These changes were associated with a loss in MOR but not TRPV1 in peripheral sensory neurons. Intrathecal delivery of nerve growth factor in diabetic animals normalized sensory neuron MOR and subsequently rescued morphine's inhibitory effects on capsaicin-induced TRPV1 activity in vivo and in vitro. These findings identify a loss in functional MOR on sensory neurons as a contributing factor for the impaired opioid inhibitory effects on capsaicin-induced TRPV1 activity during advanced STZ-induced diabetes. Moreover, they support growing evidence of a distinct regulation of opioid responsiveness during various painful states of disease (e.g. arthritis, cancer, neuropathy) and may give novel therapeutic incentives.
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http://dx.doi.org/10.1016/j.neuropharm.2014.05.026DOI Listing
October 2014

CXCL10 controls inflammatory pain via opioid peptide-containing macrophages in electroacupuncture.

PLoS One 2014 14;9(4):e94696. Epub 2014 Apr 14.

Department of Anesthesiology, University Hospital of Würzburg, Würzburg, Germany.

Acupuncture is widely used for pain treatment in patients with osteoarthritis or low back pain, but molecular mechanisms remain largely enigmatic. In the early phase of inflammation neutrophilic chemokines direct opioid-containing neutrophils in the inflamed tissue and stimulate opioid peptide release and antinociception. In this study the molecular pathway and neuroimmune connections in complete Freund's adjuvant (CFA)-induced hind paw inflammation and electroacupuncture for peripheral pain control were analyzed. Free moving Wistar rats with hind paw inflammation were treated twice with electroacupuncture at GB30 (Huan Tiao--gall bladder meridian) (day 0 and 1) and analyzed for mechanical and thermal nociceptive thresholds. The cytokine profiles as well as the expression of opioid peptides were quantified in the inflamed paw. Electroacupuncture elicited long-term antinociception blocked by local injection of anti-opioid peptide antibodies (beta-endorphin, met-enkephalin, dynorphin A). The treatment altered the cytokine profile towards an anti-inflammatory pattern but augmented interferon (IFN)-gamma and the chemokine CXCL10 (IP-10: interferon gamma-inducible protein) protein and mRNA expression with concomitant increased numbers of opioid peptide-containing CXCR3+ macrophages. In rats with CFA hind paw inflammation without acupuncture repeated injection of CXCL10 triggered opioid-mediated antinociception and increase opioid-containing macrophages. Conversely, neutralization of CXCL10 time-dependently decreased electroacupuncture-induced antinociception and the number of infiltrating opioid peptide-expressing CXCR3+ macrophages. In summary, we describe a novel function of the chemokine CXCL10--as a regulator for an increase of opioid-containing macrophages and antinociceptive mediator in inflammatory pain and as a key chemokine regulated by electroacupuncture.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0094696PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3986408PMC
January 2015

Thoracic epidural anesthesia decreases endotoxin-induced endothelial injury.

BMC Anesthesiol 2014 5;14:23. Epub 2014 Apr 5.

Department of Anesthesiology, Emergency Medicine and Critical Care Medicine, Universitätsmedizin Göttingen, Robert-Koch-Straße 40, Göttingen 37099, Germany.

Background: The sympathetic nervous system is considered to modulate the endotoxin-induced activation of immune cells. Here we investigate whether thoracic epidural anesthesia with its regional symapathetic blocking effect alters endotoxin-induced leukocyte-endothelium activation and interaction with subsequent endothelial injury.

Methods: Sprague Dawley rats were anesthetized, cannulated and hemodynamically monitored. E. coli lipopolysaccharide (Serotype 0127:B8, 1.5 mg x kg(-1) x h(-1)) or isotonic saline (controls) was infused for 300 minutes. An epidural catheter was inserted for continuous application of lidocaine or normal saline in endotoxemic animals and saline in controls. After 300 minutes we measured catecholamine and cytokine plasma concentrations, adhesion molecule expression, leukocyte adhesion, and intestinal tissue edema.

Results: In endotoxemic animals with epidural saline, LPS significantly increased the interleukin-1β plasma concentration (48%), the expression of endothelial adhesion molecules E-selectin (34%) and ICAM-1 (42%), and the number of adherent leukocytes (40%) with an increase in intestinal myeloperoxidase activity (26%) and tissue edema (75%) when compared to healthy controls. In endotoxemic animals with epidural infusion of lidocaine the values were similar to those in control animals, while epinephrine plasma concentration was 32% lower compared to endotoxemic animals with epidural saline.

Conclusions: Thoracic epidural anesthesia attenuated the endotoxin-induced increase of IL-1β concentration, adhesion molecule expression and leukocyte-adhesion with subsequent endothelial injury. A potential mechanism is the reduction in the plasma concentration of epinephrine.
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http://dx.doi.org/10.1186/1471-2253-14-23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4001105PMC
October 2015

Toll like receptor (TLR)-4 as a regulator of peripheral endogenous opioid-mediated analgesia in inflammation.

Mol Pain 2014 Feb 6;10:10. Epub 2014 Feb 6.

Department of Anesthesiology, University Hospital of Wuerzburg, Oberdürrbacher Strasse 6, D-97080 Würzburg, Germany.

Background: Leukocytes containing opioid peptides locally control inflammatory pain. In the early phase of complete Freund's adjuvant (CFA)-induced hind paw inflammation, formyl peptides (derived e.g. from Mycobacterium butyricum) trigger the release of opioid peptides from neutrophils contributing to tonic basal antinociception. In the later phase we hypothesized that toll-like-receptor-(TLR)-4 activation of monocytes/macrophages triggers opioid peptide release and thereby stimulates peripheral opioid-dependent antinociception.

Results: In Wistar rats with CFA hind paw inflammation in the later inflammatory phase (48-96 h) systemic leukocyte depletion by cyclophosphamide (CTX) or locally injected naloxone (NLX) further decreased mechanical and thermal nociceptive thresholds. In vitro β-endorphin (β-END) content increased during human monocyte differentiation as well as in anti-inflammatory CD14+CD16- or non-classical M2 macrophages. Monocytes expressing TLR4 dose-dependently released β-END after stimulation with lipopolysaccharide (LPS) dependent on intracellular calcium. Despite TLR4 expression proinflammatory M1 and anti-inflammatory M2 macrophages only secreted opioid peptides in response to ionomycin, a calcium ionophore. Intraplantar injection of LPS as a TLR4 agonist into the inflamed paw elicited an immediate opioid- and dose-dependent antinociception, which was blocked by TAK-242, a small-molecule inhibitor of TLR4, or by peripheral applied NLX. In the later phase LPS lowered mechanical and thermal nociceptive thresholds. Furthermore, local peripheral TLR4 blockade worsened thermal and mechanical nociceptive pain thresholds in CFA inflammation.

Conclusion: Endogenous opioids from monocytes/macrophages mediate endogenous antinociception in the late phase of inflammation. Peripheral TLR4 stimulation acts as a transient counter-regulatory mechanism for inflammatory pain in vivo, and increases the release of opioid peptides from monocytes in vitro. TLR4 antagonists as new treatments for sepsis and neuropathic pain might unexpectedly transiently enhance pain by impairing peripheral opioid analgesia.
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http://dx.doi.org/10.1186/1744-8069-10-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3922964PMC
February 2014

A modified approach to induce predictable congestive heart failure by volume overload in rats.

PLoS One 2014 31;9(1):e87531. Epub 2014 Jan 31.

Department of Anesthesiology and Intensive Care Medicine, Campus Charité, Mitte and Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, Berlin, Germany.

The model of infrarenal aortocaval fistula (ACF) has recently gained new interest in its use to investigate cardiac pathophysiology. Since in previous investigations the development of congestive heart failure (CHF) was inconsistent and started to develop earliest 8-10 weeks after fistula induction using a 18G needle, this project aimed to induce a predictable degree of CHF within a definite time period using a modified approach. An aortocaval fistula was induced in male Wistar rats using a 16G needle as a modification of the former 18G needle-technique described by Garcia and Diebold. Results revealed within 28 ± 2 days of ACF significantly increased heart and lung weight indices in the ACF group accompanied by elevated filling pressure. All hemodynamic parameters derived from a pressure-volume conductance-catheter in vivo were significantly altered in the ACF consistent with severe systolic and diastolic left ventricular dysfunction. This was accompanied by systemic neurohumoral activation as demonstrated by elevated rBNP-45 plasma concentrations in every rat of the ACF group. Furthermore, the restriction in overall cardiac function was associated with a β1- and β2-adrenoreceptor mRNA downregulation in the left ventricle. In contrast, β3-adrenoreceptor mRNA was upregulated. Finally, electron microscopy of the left ventricle of rats in the ACF group showed signs of progressive subcellular myocardial fragmentation. In conclusion, the morphometric, hemodynamic and neurohumoral characterization of the modified approach revealed predictable and consistent signs of congestive heart failure within 28 ± 2 days. Therefore, this modified approach might facilitate the examination of various questions specific to CHF and allow for pharmacological interventions to determine pathophysiological pathways.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087531PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3909118PMC
October 2014

The presence of mu-, delta-, and kappa-opioid receptors in human heart tissue.

Heart Vessels 2014 Nov 5;29(6):855-63. Epub 2014 Jan 5.

Palliativzentrum Hildegard, Basel, Switzerland,

Functional evidence suggests that the stimulation of peripheral and central opioid receptors (ORs) is able to modulate heart function. Moreover, selective stimulation of either cardiac or central ORs evokes preconditioning and, therefore, protects the heart against ischemic injury. However, anatomic evidence for OR subtypes in the human heart is scarce. Human heart tissue obtained during autopsy after sudden death was examined immunohistochemically for mu- (MOR), kappa- (KOR), and delta- (DOR) OR subtypes. MOR and DOR immunoreactivity was found mainly in myocardial cells, as well as on sparse individual nerve fibers. KOR immunoreactivity was identified predominantly in myocardial cells and on intrinsic cardiac adrenergic (ICA) cell-like structures. Double immunofluorescence confocal microscopy revealed that DOR colocalized with the neuronal marker PGP9.5, as well as with the sensory neuron marker calcitonin gene-related peptide (CGRP). CGRP-immunoreactive (IR) fibers were detected either in nerve bundles or as sparse individual fibers containing varicose-like structures. Our findings offer the first hint of an anatomic basis for the existence of OR subtypes in the human heart by demonstrating their presence in CGRP-IR sensory nerve fibers, small cells with an eccentric nucleus resembling ICA cells, and myocardial cells. Taken together, this suggests the role of opioids in both the neural transmission and regulation of myocardial cell function.
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http://dx.doi.org/10.1007/s00380-013-0456-5DOI Listing
November 2014

Influence of high-dose intraoperative remifentanil with or without amantadine on postoperative pain intensity and morphine consumption in major abdominal surgery patients: a randomised trial.

Eur J Anaesthesiol 2014 Jan;31(1):41-9

From the Department of Anesthesiology and Intensive Care Medicine, Campus Charité Mitte and Campus Virchow-Klinikum (ST, SAM, MS) and Department of Anesthesiology and Intensive Care Medicine, Campus Benjamin Franklin, Charité Universitätsmedizin Berlin, Berlin, Germany (MK, AK).

Background: Human volunteer studies demonstrate ketamine-reversible opioid-induced hyperalgesia, consistent with reports of increased postoperative pain and analgesic consumption. However, recent clinical trials showed controversial results after intraoperative administration of high-dose remifentanil.

Objective: To investigate in lower abdominal surgery patients whether postoperative pain intensity and analgesic consumption are increased following intraoperative high-dose vs. low-dose remifentanil, and whether this could be prevented by preoperative administration of the NMDA antagonist amantadine.

Design: Randomised, placebo-controlled, clinical study.

Setting: University hospital.

Patients: Sixty patients scheduled for elective major lower abdominal surgery.

Interventions: Patients were randomly assigned to one of three anaesthetic regimens. First, in the group 'low-dose remifentanil and preoperative isotonic saline' (n=15), a remifentanil infusion was maintained at a rate of 0.1 μg kg min throughout anaesthesia, and the end-tidal concentration of sevoflurane started at 0.5 minimum alveolar concentration (MAC) and was increased by 0.2% increments according to clinical demand. Preoperatively, 500 ml NaCl 0.9% were infused as study solution. Second, in the group 'high-dose remifentanil and preoperative saline' (n=17), the end-tidal concentration of sevoflurane was maintained at 0.5 MAC throughout anaesthesia. A remifentanil infusion was started at a rate of 0.2 μg kg min and subsequently increased by 0.05 μg kg min increments to clinical demand. Preoperatively, these patients also received a solution of 500 ml NaCl 0.9% as study solution. Third, the group 'high-dose remifentanil and preoperative amantadine' (n=16) received the same anaesthetic protocol as the second group, but the preoperative study solution was substituted by amantadine (200 mg/500 ml).

Main Outcome Measures: Pain intensity measured by the numerical rating scale and cumulative morphine consumption.

Results: The remifentanil dose in both high-dose groups was significantly higher compared with the low-dose remifentanil group (0.20±0.04 and 0.23±0.02 vs. 0.08±0.04 μg kg min; P<0.001). Pain intensity gradually increased up to 45 min postoperatively in all groups, and then decreased again towards low levels in parallel with a linear increase in morphine consumption. Postoperative pain intensity and morphine consumption did not significantly differ between groups. Moreover, preoperative amantadine revealed no additional benefit.

Conclusion: We were not able to demonstrate any influence on routine clinical outcome parameters of pain after high-dose remifentanil. Although not without limitations, these findings are in line with other clinical trials that could not detect an opioid-induced impact on postoperative pain parameters, which might be less sensitive to detect opioid-induced hyperalgesia compared with quantitative sensory testing.

Trial Registration: DRKS00004626.
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http://dx.doi.org/10.1097/01.EJA.0000434967.03790.0eDOI Listing
January 2014

The molecular link between C-C-chemokine ligand 2-induced leukocyte recruitment and hyperalgesia.

J Pain 2013 Sep 16;14(9):897-910. Epub 2013 May 16.

Department of Anesthesiology, University Hospital of Würzburg, Würzburg, Germany.

Unlabelled: The chemokine C-C-chemokine ligand 2 (CCL2) (formerly known as MCP, macrophage chemotactic protein) is one of the important genes upregulated in different types of pain both in animals and humans. CCL2 governs the recruitment of C-C chemokine receptor 2-expressing monocytes into inflamed tissue. In contrast to neutrophilic chemokines, intraplantar injection of CCL2 in Wistar rats recruited macrophages and neutrophils and simultaneously lowered nociceptive thresholds. CCL2-induced hyperalgesia was abolished by prior systemic leukocyte depletion by cyclophosphamide and was reconstituted by local adoptive transfer of donor macrophages but not of neutrophils. Antagonists against transient receptor potential vannilloid 1 inhibited thermal and against transient receptor potential ankyrin 1 blocked mechanical hyperalgesia. Peripheral but not central activation of cyclooxygenase-2 (Cox-2) were critical for CCL2-induced hyperalgesia. In vitro CCL2 did not directly stimulate Cox-2 expression or prostaglandin E2 formation but slightly enhanced the formation of reactive oxygen species in monocytes and macrophages. In vivo, increased immunoreactivity for 4-hydroxy-2-nonenal (4-HNE), a downstream product of reactive oxygen species and known inducer of Cox-2, was observed and colocalized with Cox-2 in ED1 (CD68) positive infiltrating cells. No hyperalgesia, 4-HNE, or Cox-2 immunoreactivity was seen in leukocyte-depleted rats that were reconstituted with macrophages in the absence of CCL2, supporting the important role of CCL2.

Perspective: CCL2 plays a dual role: 1) promoting monocyte/macrophage recruitment into tissue; and 2) potentially stimulating macrophages in the tissue to produce 4-HNE and subsequently Cox-2, all resulting in the induction of hyperalgesia via transient receptor potential vannilloid 1 and transient receptor potential ankyrin 1. This encourages pharmacological efforts targeting CCL2/C-C chemokine receptor 2 and macrophages for treatment of inflammatory pain.
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http://dx.doi.org/10.1016/j.jpain.2013.02.012DOI Listing
September 2013

Peripheral antinociceptive efficacy and potency of a novel opioid compound 14-O-MeM6SU in comparison to known peptide and non-peptide opioid agonists in a rat model of inflammatory pain.

Eur J Pharmacol 2013 Aug 9;713(1-3):54-7. Epub 2013 May 9.

Department of Anesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charite Mitte, Augustnburgerplatz 113353 Berlin, Germany.

This study compared the peripheral analgesic effects of a novel opioid agonist 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU), to that of non-peptide (morphine, fentanyl) and peptide opioid agonists (Met-enkephalin; met-ENK and β-endorphin; β-END) in a model of localized inflammatory pain evoked by intraplantar (i.pl.) Freund's complete adjuvant (FCA). Nociceptive responses to local opioid agonists were measured by pressure paw-withdrawal procedures. In addition, the antinociceptive efficacy and potency of these test compounds in vivo was compared to that in vitro using the rat vas deferens (RVD) bioassay. Intraplantar 14-O-MeM6SU (0.32-2.53 nmol/rat), morphine (14.95-112.15 nmol/rat), fentanyl (0.19-2.36 nmol/rat), met-ENK (0.10-10 nmol/rat) and β-END (0.77-5.00 nmol/rat) dose dependently increased paw pressure thresholds exclusively in inflamed hindpaws. At higher doses analgesic effects were also seen in noninflamed paws for 14-O-MeM6SU, morphine and fentanyl but not for met-ENK or β-END. The maximal possible local analgesic effect (%) measured in inflamed paws was 50.6 ± 2.7, 18.23 ± 1.78, 37.44 ± 2.17, 36.00 ± 1.43, and 40.69 ± 0.91 for 14-O-MeM6SU, morphine, fentanyl, met-ENK and β-END, respectively. Interestingly, i.pl. administered opioid peptides met-ENK and β-END displayed a peripheral analgesic ceiling effect. This local antinociception was antagonized by co-administered opioid antagonist naloxone-methiodide (NAL-M). Similar to the analgesic testing, the RVD showed the following efficacy order of the test compounds: 14-O-MeM6SU>β-END>fentanyl>met-ENK≫morphine. Taken together, 14-O-MeM6SU was more potent than morphine, fentanyl and met-ENK and β-END and displayed superiority in the maximum antinociceptive effects. The superiority of local antinociceptive effects of 14-O-MeM6SU might be due to both pharmacodynamic and pharmacokinetic factors.
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http://dx.doi.org/10.1016/j.ejphar.2013.04.043DOI Listing
August 2013

Reduced number, G protein coupling, and antinociceptive efficacy of spinal mu-opioid receptors in diabetic rats are reversed by nerve growth factor.

J Pain 2013 Jul 24;14(7):720-30. Epub 2013 Apr 24.

Department of Anaesthesiology and Intensive Care Medicine, Charité University Berlin, Campus Virchow Klinikum and Campus Charite Mitte, Berlin, Germany.

Unlabelled: This study investigated putative mechanisms of impaired spinal opioid antinociception such as a downregulation of mu-opioid receptor (MOR) number, coupling, and efficacy in rats with advanced (12 weeks) streptozotocin (STZ)-induced diabetes. Intravenous injection of STZ (45 mg/kg) in Wistar rats led to selective degeneration of insulin-producing pancreatic ß-cells, elevated blood glucose, and mechanical hyperalgesia. In these animals, dose-dependent and naloxone-reversible intrathecal fentanyl antinociception was significantly impaired and associated with a loss in MOR immunoreactivity of calcitonin gene-related peptide-immunoreactive (CGRP-IR) sensory nerve terminals, membrane-bound MOR binding sites, and MOR-stimulated G protein coupling within the dorsal horn of the spinal cord. Intrathecal delivery of nerve growth factor (NGF) in diabetic animals normalized spinal MOR number and G protein coupling and rescued spinal fentanyl-induced antinociception. These findings identify for the first time a loss in functional MOR on central terminals of sensory neurons as a contributing factor for the impaired spinal opioid responsiveness during advanced STZ-induced diabetes that can be reversed by NGF. Moreover, they support growing evidence of a distinct regulation of opioid responsiveness during various painful states of disease (eg, arthritis, cancer, neuropathy) and may give novel therapeutic incentives.

Perspective: In diabetic neuropathy a loss in sensory neuron mu-opioid receptor number and coupling contributes to impaired spinal opioid antinociception that can be reversed by NGF. These findings support growing evidence of a distinct regulation of opioid responsiveness during various painful diseases and may give novel therapeutic incentives.
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http://dx.doi.org/10.1016/j.jpain.2013.01.776DOI Listing
July 2013
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