Direct neuronal protection by the protease-activated receptor PAR4 antagonist ML354 after experimental stroke in mice.

BACKGROUND AND PURPOSE: Thrombo-inflammation is a key feature of stroke pathophysiology and provides multiple candidate drug targets. Thrombin exerts coagulation-independent actions via protease-activated receptors (PAR), of which PAR1 has been implicated in stroke-associated neuroinflammation. The role of PAR4 in this context is less clear. This study examined if the selective PAR4 antagonist ML354 provides neuroprotection in experimental stroke and explored the underlying mechanisms. EXPERIMENTAL APPROACH: Mouse primary cortical neurons were exposed to oxygen-glucose deprivation (OGD) and simulated reperfusion ± ML354. For comparison, functional Ca2+-imaging was performed upon acute stimulation with a PAR4 activating peptide or glutamate. Male mice underwent sham operation or transient middle cerebral artery occlusion (tMCAO), with ML354 or vehicle treatment beginning at recanalization. A subset of mice received a platelet-depleting antibody. Stroke size and functional outcomes were assessed. Abundance of target genes, proteins, and cell markers was determined in cultured cells and tissues by qPCR, immunoblotting, and immunofluorescence. KEY RESULTS: Stroke up-regulated PAR4 expression in cortical neurons in vitro and in vivo. OGD augments spontaneous and PAR4-mediated neuronal activity; ML354 suppresses OGD-induced neuronal excitotoxicity and apoptosis. ML354 applied in vivo after tMCAO reduced infarct size, apoptotic markers, macrophage accumulation, and interleukin-1ß expression. Platelet depletion did not affect infarct size in mice with tMCAO ± ML354. CONCLUSIONS AND IMPLICATIONS: Selective PAR4 inhibition during reperfusion improves infarct size and neurological function after experimental stroke by blunting neuronal excitability, apoptosis, and local inflammation. PAR4 antagonists may provide additional neuroprotective benefits in patients with acute stroke beyond their canonical antiplatelet action.

Intranasal Lidocaine Administration via Mucosal Atomization Device: A Simple and Successful Treatment for Postdural Puncture Headache in Obstetric Patients.

(1) Background: Postdural puncture headache (PDPH) remains a serious complication in obstetric patients. While the epidural blood patch represents the current gold standard in therapy, a growing number of alternative measures are thought to be beneficial for clinical management. The purpose of this study was to retrospectively analyze the efficacy of intranasal lidocaine administration to treat PDPH in obstetrics at our university hospital; (2) Methods: A retrospective analysis of the medical records of patients with PDPH has been performed focusing on the techniques of administration, dosing, treatment duration, impact on pain intensity as well as side effects of intranasal lidocaine; (3) Results: During the study period, 5610 obstetric patients received neuraxial anesthesia, of whom 43 (0.77%) developed PDPH. About one third of the patients with PDPH after spinal anesthesia (n = 8), epidural anesthesia (n = 5) or both (n = 2) were treated with intranasal lidocaine. Lidocaine was administered either via gauze compresses (GC, n = 4), a mucosal atomization device (MAD, n = 8) or with a second-line mucosal atomization device due to low gauze compress efficacy (n = 3). All patients treated with lidocaine refused the epidural blood patch. Nebulization of lidocaine resulted in a significant reduction in pain intensity after the first dose (p = 0.008). No relevant side effects developed except sporadic temporal pharyngeal numbness. The utilization of the mucosal atomization device averted the necessity for an epidural blood patch, whether employed as the primary or secondary approach; (4) Conclusions: Our data imply that the mucosal atomization device enhances the efficacy of intranasal lidocaine administration in obstetric patients suffering from PDPH.

[Postdural puncture headache in obstetrics : Pathogenesis, diagnostics and treatment]. / Postpunktionskopfschmerz in der Geburtshilfe : Pathogenese, Diagnostik und Therapie.

Postdural puncture headache (PDPH) is one of the most important complications of peripartum neuraxial analgesia. Loss of cerebrospinal fluid volume and pressure as well as compensatory intracranial vasodilation are assumed to be responsible. Potentially severe long-term sequelae necessitate the correct diagnosis of PDPH, exclusion of relevant differential diagnoses (with atypical symptoms and when indicated via imaging techniques) and rapid initiation of effective treatment. Nonopioid analgesics, caffeine and occasionally theophylline, gabapentin and hydrocortisone are the cornerstones of pharmacological treatment, while the timely placement of an autologous epidural blood patch (EBP) represents the gold standard procedure when symptoms persist despite the use of analgesics. Procedures using neural treatment are promising alternatives, especially when an EBP is not desired by the patient or is contraindicated. Interdisciplinary and interprofessional consensus standard procedures can contribute to optimization of the clinical management of this relevant complication.

Evaluation of Recombinant Botulinum Neurotoxin Type A1 Efficacy in Peripheral Inflammatory Pain in Mice.

Well-established efficacy of botulinum neurotoxin type A (BoNT/A) in aesthetic dermatology and neuromuscular hyperactivity disorders relies on canonical interruption of acetylcholine neurotransmission at the neuromuscular junction at the site of the injection. The mechanisms and the site of activity of BoNT/A in pain, on the other hand, remain elusive. Here, we explored analgesic activity of recombinant BoNT/A1 (rBoNT/A1; IPN10260) in a mouse model of inflammatory pain to investigate the potential role of peripheral sensory afferents in this activity. After confirming analgesic efficacy of rBoNT/A1 on CFA-induced mechanical hypersensitivity in C57Bl6J mice, we used GCaMP6s to perform in vivo calcium imaging in the ipsilateral dorsal root ganglion (DRG) neurons in rBoNT/A1 vs. vehicle-treated mice at baseline and following administration of a range of mechanical and thermal stimuli. Additionally, immunohisochemical studies were performed to detect cleaved SNAP25 in the skin, DRGs and the spinal cord. Injection of CFA resulted in reduced mechanical sensitivity threshold and increased calcium fluctuations in the DRG neurons. While rBoNT/A1 reduced mechanical hypersensitivity, calcium fluctuations in the DRG of rBoNT/A1- and vehicle-treated animals were similar. Cleaved SNAP25 was largely absent in the skin and the DRG but present in the lumbar spinal cord of rBoNT/A1-treated animals. Taken together, rBoNT/A1 ameliorates mechanical hypersensitivity related to inflammation, while the signal transmission from the peripheral sensory afferents to the DRG remained unchanged. This strengthens the possibility that spinal, rather than peripheral, mechanisms play a role in the mediation of analgesic efficacy of BoNT/A in inflammatory pain.

Stabilization of Delphinidin in Complex with Sulfobutylether-ß-Cyclodextrin Allows for Antinociception in Inflammatory Pain.

Aims: Delphinidin (DEL) is a plant-derived antioxidant with clinical potential to treat inflammatory pain but suffers from poor solubility and low bioavailability. The aim of the study was to develop a well-tolerated cyclodextrin (CD)-DEL complex with enhanced bioavailability and to investigate the mechanisms behind its antinociceptive effects in a preclinical model of inflammatory pain. Results: CD-DEL was highly soluble and stable in aqueous solution, and was nontoxic. Systemic administration of CD-DEL reversed mechanical and heat hyperalgesia, while its local application into the complete Freund's adjuvant (CFA)-induced inflamed paw dose-dependently reduced mechanical hyperalgesia, paw volume, formation of the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE), and tissue migration of CD68+ macrophages. CD-DEL also directly prevented 4-HNE-induced mechanical hyperalgesia, cold allodynia, and an increase in the intracellular calcium concentration into transient receptor potential ankyrin 1 expressing cells. Both 4-HNE- and CFA-induced reactive oxygen species (ROS) levels were sensitive to CD-DEL, while its capacity to scavenge superoxide anion radicals (inhibitory concentration 50 [IC50]: 70 ± 5 µM) was higher than that observed for hydroxyl radicals (IC50: 600 ± 50 µM). Finally, CD-DEL upregulated heme oxygenase 1 that was prevented by HMOX-1 siRNA in vitro. Innovation:In vivo application of DEL to treat inflammatory pain is facilitated by complexation with CD. Apart from its antioxidant effects, the CD-DEL has a unique second antioxidative mechanism involving capturing of 4-HNE into the CD cavity followed by displacement and release of the ROS scavenger DEL. Conclusion: CD-DEL has antinociceptive, antioxidative, and anti-inflammatory effects making it a promising formulation for the local treatment of inflammatory pain.

[Intranasal lidocaine atomization as novel and noninvasive treatment option for postdural puncture headache : Two case reports from obstetric anesthesiology]. / Intranasale Lidocainvernebelung als neue und nichtinvasive Therapieoption des Postpunktionskopfschmerzes : Zwei Fallberichte aus der geburtshilflichen Anästhesiologie.

BACKGROUND: Postdural puncture headache (PDPH) occurs in up to 11% of patients after spinal anesthesia and in more than 80% after dural perforation upon epidural anesthesia. It represents a severe anesthesiological complication in obstetric patients. If conservative medication measures do not result in a timely relief of symptoms, the current guidelines recommend the early implementation of an epidural blood patch; however, although performing an epidural blood patch is effective to treat PDPH, potential side effects include neurological complications, spinal hematoma and infections. Assumed to reduce cerebral vasodilatation as a potential pathophysiological driver of PDPH, the transnasal block of the sphenopalatine ganglion with local anesthetics is discussed as an alternative approach. METHODS: In this case study a modification of this technique is reported using a mucosal atomization device (MAD) for off-label nasal administration of lidocaine in two obstetric patients suffering from PDPH. Up to now there is no experience with this modified technique in obstetric anesthesiology. RESULTS: The first patient (25-year-old secundigravida, body mass index [BMI] 54.7 kg/m2) displayed a pronounced PDPH with nausea and vomiting during the first day after a cesarean section under spinal anesthesia (3 attempts). The second patient (32-year-old tertiagravida, BMI 27.3 kg/m2) was readmitted to hospital due to PDPH 4 days after a natural birth under epidural anesthesia. Whereas conservative measures and therapeutic attempts with nonopioid analgesics and caffeine did not result in a sufficient treatment success, intranasal lidocaine administration via a MAD led to an immediate and persisting symptom relief. Both patients could be discharged from hospital after 24 h of surveillance and did not report any relevant side effects of the lidocaine administration. CONCLUSION: The described noninvasive and simple procedure represents a valuable addition to previously known treatment options for PDPH and a potential alternative to an epidural blood patch in obstetric patients with PDPH. Prospective studies are needed to validate the findings.

Pain Control by Targeting Oxidized Phospholipids: Functions, Mechanisms, Perspectives.

Within the lipidome oxidized phospholipids (OxPL) form a class of chemically highly reactive metabolites. OxPL are acutely produced in inflamed tissue and act as endogenous, proalgesic (pain-inducing) metabolites. They excite sensory, nociceptive neurons by activating transient receptor potential ion channels, specifically TRPA1 and TRPV1. Under inflammatory conditions, OxPL-mediated receptor potentials even potentiate the action potential firing rate of nociceptors. Targeting OxPL with D-4F, an apolipoprotein A-I mimetic peptide or antibodies like E06, specifically binding oxidized headgroups of phospholipids, can be used to control acute, inflammatory pain syndromes, at least in rodents. With a focus on proalgesic specificities of OxPL, this article discusses, how targeting defined substances of the epilipidome can contribute to mechanism-based therapies against primary and secondary chronic inflammatory or possibly also neuropathic pain.

Redistribution of pulmonary ventilation after lung surgery detected with electrical impedance tomography.

BACKGROUND: Regional ventilation of the lung can be visualized by pulmonary electrical impedance tomography (EIT). The aim of this study was to examine the post-operative redistribution of regional ventilation after lung surgery dependent on the side of surgery and its association with forced vital capacity. METHODS: In this prospective, observational cohort study 13 patients undergoing right and 13 patients undergoing left-sided open or video-thoracoscopic procedures have been investigated. Pre-operative measurements with EIT and spirometry were compared with data obtained 3 days post-operation. The center of ventilation (COV) within a 32 × 32 pixel matrix was calculated from EIT data. The transverse axis coordinate of COV, COVx (left/right), was modified to COVx' (ipsilateral/contralateral). Thus, COVx' shows a negative change if ventilation shifts contralateral independent of the side of surgery. This enabled testing with two-way ANOVA for repeated measurements (side, time). RESULTS: The perioperative shift of COVx' was dependent on the side of surgery (P = .007). Ventilation shifted away from the side of surgery after the right-sided surgery (COVx'-1.97 pixel matrix points, P < .001), but not after the left-sided surgery (COVx'-0.61, P = .425). The forced vital capacity (%predicted) decreased from 94 (83-109)% (median [quartiles]; [left-sided]) and 89 (80-97)% (right-sided surgery) to 61 (59-66)% and 62 (40-72)% (P < .05), respectively. The perioperative changes in forced vital capacity (%predicted) were weakly associated with the shift of COVx'. CONCLUSION: Only after right-sided lung surgery, EIT showed reduced ventilation on the side of surgery while vital capacity was markedly reduced in both groups.

NaV1.9 Potentiates Oxidized Phospholipid-Induced TRP Responses Only under Inflammatory Conditions.

Oxidized phospholipids (OxPL) like oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) were recently identified as novel proalgesic targets in acute and chronic inflammatory pain. These endogenous chemical irritants are generated in inflamed tissue and mediate their pain-inducing function by activating the transient receptor potential channels TRPA1 and TRPV1 expressed in sensory neurons. Notably, prototypical therapeutics interfering with OxPL were shown to inhibit TRP channel activation and pain behavior. Here, we asked how OxPL excite primary sensory neurons of dorsal root ganglia (DRG neurons from mice of either sex). Acute stimulation of sensory neurons with the prototypical OxPL 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) evoked repetitive calcium spikes in small-diameter neurons. As NaV1.9, a voltage-gated sodium channel involved in nociceptor excitability, was previously shown to be essential for the generation of calcium spikes in motoneurons, we asked if this channel is also important for OxPL mediated calcium spike and action potential generation in nociceptors. In wild-type and NaV1.9-deficient neurons, the action potential firing rate and the calcium spike frequency to an acute PGPC stimulus was similar. When preincubated with inflammatory mediators, both, the action potential firing rate and the calcium spike frequency were markedly increased in response to an acute PGPC stimulus. However, this potentiating effect was completely lost in NaV1.9-deficient small-diameter neurons. After treatment with inflammatory mediators, the resting membrane potential of NaV1.9 KO neurons was slightly more negative than that of wild-type control neurons. This suggests that NaV1.9 channels are active under this condition and therefore increases the ease with which action potentials are elicited after OxPL stimulation. In summary, our data suggest that NaV1.9 has a switch function to potentiate the receptor potentials induced by OxPL under inflammatory conditions. Since human NaV1.9 has been shown to mediate painful and painless channelopathies, this study provides new insights into the mechanism by which NaV1.9 amplifies stimuli of endogenous irritants under inflammatory conditions.

Peripheral Interaction of Resolvin D1 and E1 with Opioid Receptor Antagonists for Antinociception in Inflammatory Pain in Rats.

Antinociceptive pathways are activated in the periphery in inflammatory pain, for instance resolvins and opioid peptides. Resolvins are biosynthesized from omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Resolvin D1 (RvD1) and resolvin E1 (RvE1) initiate the resolution of inflammation and control of hypersensitivity via induction of anti-inflammatory signaling cascades. RvD1 binds to lipoxin A4/annexin-A1 receptor/formyl-peptide receptor 2 (ALX/FPR2), RvE1 to chemerin receptor 23 (ChemR23). Antinociception of RvD1 is mediated by interaction with transient receptor potential channels ankyrin 1 (TRPA1). Endogenous opioid peptides are synthesized and released from leukocytes in the tissue and bind to opioid receptors on nociceptor terminals. Here, we further explored peripheral mechanisms of RvD1 and chemerin (Chem), the ligand of ChemR23, in complete Freund's adjuvant (CFA)-induced hindpaw inflammation in male Wistar rats. RvD1 and Chem ameliorated CFA-induced hypersensitivity in early and late inflammatory phases. This was prevented by peripheral blockade of the µ-opioid peptide receptor (MOR) using low dose local naloxone or by local injection of anti-ß-endorphin and anti-met-enkephalin (anti-ENK) antibodies. Naloxone also hindered antinociception by the TRPA1 inhibitor HC-030031. RvD1 did not stimulate the release of ß-endorphin from macrophages and neutrophils, nor did RvD1 itself activate G-proteins coupled MOR or initiate ß-arrestin recruitment to the membrane. TRPA1 blockade by HC-030031 in inflammation in vivo as well as inhibition of the TRPA1-mediated calcium influx in dorsal root ganglia neurons in vitro was hampered by naloxone. Peripheral application of naloxone alone in vivo already lowered mechanical nociceptive thresholds. Therefore, either a perturbation of the balance of endogenous pro- and antinociceptive mechanisms in early and late inflammation, or an interaction of TRPA1 and opioid receptors weaken the antinociceptive potency of RvD1 and TRPA1 blockers.

Inflammatory pain control by blocking oxidized phospholipid-mediated TRP channel activation.

Phospholipids occurring in cell membranes and lipoproteins are converted into oxidized phospholipids (OxPL) by oxidative stress promoting atherosclerotic plaque formation. Here, OxPL were characterized as novel targets in acute and chronic inflammatory pain. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) and its derivatives were identified in inflamed tissue by mass spectrometry and binding assays. They elicited calcium influx, hyperalgesia and induced pro-nociceptive peptide release. Genetic, pharmacological and mass spectrometric evidence in vivo as well as in vitro confirmed the role of transient receptor potential channels (TRPA1 and TRPV1) as OxPAPC targets. Treatment with the monoclonal antibody E06 or with apolipoprotein A-I mimetic peptide D-4F, capturing OxPAPC in atherosclerosis, prevented inflammatory hyperalgesia, and in vitro TRPA1 activation. Administration of D-4F or E06 to rats profoundly ameliorated mechanical hyperalgesia and inflammation in collagen-induced arthritis. These data reveal a clinically relevant role for OxPAPC in inflammation offering therapy for acute and chronic inflammatory pain treatment by scavenging OxPAPC.

Apomorphine is a bimodal modulator of TRPA1 channels.

Apomorphine is a non-narcotic derivative of morphine, which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from Parkinson's disease. Adverse effects of apomorphine treatment include severe emesis and nausea, and ulceration and pain at the injection site. We wanted to test whether sensory transient receptor potential (TRP) channels are a molecular target for apomorphine. Here, we show that rTRPV1, rTRPV2, rTRPV3, and mTRPV4, as well as hTRPM8, and rTRPM3, which are expressed in dorsal root ganglion neurons, are insensitive toward apomorphine treatment. This also applied to the cellular redox sensor hTRPM2. On the contrary, human TRPA1 could be concentration-dependently modulated by apomorphine. Whereas the addition of apomorphine in the low micromolar range produced an irreversible activation of the channel, application of higher concentrations caused a reversible voltage-dependent inhibition of heterologously expressed TRPA1 channels, resulting from a reduction of single-channel open times. In addition, we provide evidence that apomorphine also acts on endogenous TRPA1 in cultured dorsal root ganglion neurons from rats and in the enterochromaffin model cell line QGP-1, from which serotonin is released upon activation of TRPA1. Our study shows that human TRPA1 is a target for apomorphine, suggesting that an activation of TRPA1 might contribute to adverse side effects such as nausea and painful injections, which can occur during treatment with apomorphine.

TRPA1 is functionally expressed in melanoma cells but is not critical for impaired proliferation caused by allyl isothiocyanate or cinnamaldehyde.

Melanoma is the most dangerous form of skin cancer occurring in Caucasians with rising incidence. They are remarkably resistant to conventional anti-tumour therapies like chemotherapy and radiotherapy. Therefore, new treatment strategies are urgently needed. Anti-tumour effects of phytochemicals such as allyl isothiocyanate or cinnamaldehyde have been demonstrated in various melanoma models in vitro and in vivo. Considering their high potency as transient receptor potential A1 (TRPA1)-activating compounds, we examined the functional expression of TRPA1 channels in different melanoma cell lines as well as in non-malignantly transformed primary melanocytes. The presence of TRPA1 transcripts could be detected in most of the melanoma cell lines. Furthermore, single-cell calcium imaging and patch clamp electrophysiology confirmed the presence of functional TRPA1 channels in those cell lines. Proliferation assays revealed that allyl isothiocyanate and cinnamaldehyde clearly reduce the proliferation of melanoma cells, but this effect is independent of an activation of TRPA1 channels, making it unlikely that ionic currents through TRPA1 are responsible for the anti-tumour effects of mustard oil and cinnamaldehyde.