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Background: Nitrovasodilators, such as glyceroltrinitrate (GTN), which produce nitric oxide (NO) in the organism, are known to cause delayed headaches in migraineurs, accompanied by increased plasma levels of calcitonin gene-related peptide (CGRP) in the cranial venous outflow. Increases in plasma CGRP and NO metabolites have also been found in spontaneous migraine attacks. In a rat model of meningeal nociception, infusion of NO donors induced activity of neurons in the spinal trigeminal nucleus.
Methods: Isoflurane-anaesthetised rats were intravenously infused with GTN (250 µg/kg) or saline for two hours and fixed by perfusion after a further four hours. Cryosections of dissected trigeminal ganglia were immunostained for detection of CGRP and neuronal NO synthase (nNOS). The ganglion neurons showing immunofluorescence for either of these proteins were counted.
Results: The proportions of CGRP- and nNOS- as well as double-immunopositive neurons were increased after GTN infusion compared to saline treatment in all parts of the trigeminal ganglion (CGRP) or restricted to the ophthalmic region (nNOS). The size of immunopositive neurons was not significantly different compared to controls.
Conclusion: High levels of NO may induce the expression or availability of CGRP and nNOS. Similar changes may be involved in nitrovasodilator-induced and spontaneous headache attacks in migraineurs.
A facile one-pot synthesis route for the preparation of water-soluble, biocompatible patchy Fe3O4-Au nanoparticles (Fe3O4-Au pNPs) was developed. Biocompatibility was attained through surface functionalization with 1-methyl-3-(dodecylphosphonic acid) imidazolium bromide. The morphology, composition, crystal structure and magnetic properties of the Fe3O4-Au pNPs were investigated by conducting experiments with transmission electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction and superconducting quantum interference device, respectively. Internalization of the Fe3O4-Au pNPs by MCF-7 cells occurred via endocytosis. The performance of the Fe3O4-Au pNPs as X-ray radiosensitizer in tumor cells was compared with that of gold nanocluster and Fe3O4 NPs. For this reason, MCF-7, A549 and MCF-10A cells were loaded with the respective kind of nanoparticles and treated with X-rays at doses of 1, 2 or 3 Gy. The nanoparticle-induced changes of the concentration of the reactive oxygen species (ROS) were detected using specific assays, and the cell survival under X-ray exposure was assessed employing the clonogenic assay. In comparison with the gold nanocluster and Fe3O4 NPs, the Fe3O4-Au pNPs exhibited the highest catalytic capacity for ROS generation in MCF-7 and A549 cells, whereas in the X-ray-induced ROS formation in healthy MCF-10A cells was hardly enhanced by the Fe3O4 NPs and Fe3O4-Au pNPs. Moreover, the excellent performance of Fe3O4-Au pNPs as X-ray radiosensitizers was verified by the quickly decaying radiation dose survival curve of the nanoparticle-loaded MCF-7 and A549 cells and corroborated by the small values of the associated dose-modifying factors.
Background The process of bone resorption by osteoclasts is regulated by Cathepsin K, the lysosomal collagenase responsible for the degradation of the organic bone matrix during bone remodeling. Recently, Cathepsin K was regarded as a potential target for therapeutic intervention of osteoporosis. However, mechanisms leading to osteopenia, which is much more common in young female population and often appears to be the clinical pre-stage of idiopathic osteoporosis, still remain to be elucidated, and molecular targets need to be identified. Methodology/Principal Findings We found, that in juvenile bone the large conductance, voltage and Ca2+-activated (BK) K+ channel, which links membrane depolarization and local increases in cytosolic calcium to hyperpolarizing K+ outward currents, is exclusively expressed in osteoclasts. In juvenile BK-deficient (BK−/−) female mice, plasma Cathepsin K levels were elevated two-fold when compared to wild-type littermates. This increase was linked to an osteopenic phenotype with reduced bone mineral density in long bones and enhanced porosity of trabecular meshwork in BK−/− vertebrae as demonstrated by high-resolution flat-panel volume computed tomography and micro-CT. However, plasma levels of sRANKL, osteoprotegerin, estrogene, Ca2+ and triiodthyronine as well as osteoclastogenesis were not altered in BK−/− females. Conclusion/Significance Our findings suggest that the BK channel controls resorptive osteoclast activity by regulating Cathepsin K release. Targeted deletion of BK channel in mice resulted in an osteoclast-autonomous osteopenia, becoming apparent in juvenile females. Thus, the BK−/− mouse-line represents a new model for juvenile osteopenia, and revealed the BK channel as putative new target for therapeutic controlling of osteoclast activity.
Previous studies have shown that most human myenteric neurons co-staining for vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS) and neurofilaments (NF) display the morphology of spiny type I neurons displaying a descending projection pattern. Here, we estimated the proportions of spiny neurons in human intestines, the amount of congruence of VIP/nNOS-immunoreactive with spiny neurons and whether galanin (GAL) is co-localized with VIP. Three sets of colchicine-pretreated and fixed whole mounts of 21 patients or body donors (median age 65 years; 10 female, 11 male) were stained for VIP, nNOS and NF, for VIP, nNOS and the human neuronal protein Hu C/D (HU) as well as for VIP, nNOS and GAL. The majority of VIP/nNOS-co-reactive neurons were spiny neurons (79/80% in small/large intestine, respectively) and the majority of spiny neurons costained for VIP and nNOS (82/69%). Neurons co-immunoreactive for VIP/nNOS/HU amounted to 7 and 4%, respectively. GAL/VIP-co-immunoreactivity was demonstrated in 69 and 27% of spiny neurons, respectively. We conclude that the number of neurons displaying co-reactivity for VIP and nNOS is a quantitative indicator of spiny neurons in both small and large intestine and that the proportion of spiny neurons is about 7% in small and 4% in large intestines. Since nerve fibres co-staining for NF/VIP/nNOS were found mainly in the circular muscle layer but not the surrounding perikarya of spiny neurons, we suggest that they may represent inhibitory motor neurons rather than descending interneurons.
Für die Zeit des Nationalsozialismus sind an der Friedrich-Alexander-Universität Erlangen über 160 Aberkennungen der Doktorwürde zu verzeichnen. Die Veröffentlichung faßt die Dokumentationen der Gedenkakte der Medizinischen Fakultät (am 12. Juli 2008) und des Fachbereichs Rechtswissenschaft (am 11. Februar 2010) zusammen, ergänzt um Depromotionsfälle aus der Theologischen und der Naturwissenschaftlichen Fakultät. Die Reproduktionen mit der Signatur NLA. HStAH Nds.110W Acc. 8/90 Nr. 183/8 auf den Seiten 22 und 23 wurden mit Genehmigung des Niedersächsischen Staatsarchivs publiziert. Das Archivgut ist Eigentum des Nidersächsischen Landesarchivs. Ohne vorherige schriftliche Zustimmung des Niedersächsischen Landesarchivs dürfen die Abbildungen nicht gespeichert, reproduziert, archiviert, dupliziert, kopiert, verändert oder auf andere Weise genutzt werden.
Background
Nitric oxide (NO) is thought to play an important role in the pathophysiology of migraine. Infusion of the nitrovasodilator glyceroltrinitrate (nitroglycerin, GTN), which mobilizes NO in the organism, is an approved migraine model in humans. Calcitonin gene-related peptide (CGRP) is regarded as another key mediator in migraine. Increased plasma levels of CGRP have been found during spontaneous as well as nitrovasodilator-induced migraine attacks. The nociceptive processes and interactions underlying the NO and CGRP mediated headache are poorly known but can be examined in animal experiments. In the present study we examined changes in immunofluorescence of CGRP receptor components (CLR and RAMP1) and soluble guanylyl cyclase (sGC), the intracellular receptor for NO, in rat trigeminal ganglia after pretreatment with GTN.
Methods
Isoflurane anaesthetised rats were intravenously infused with GTN (1 mg/kg) or saline for four hours and two hours later the trigeminal ganglia were processed for immunohistochemistry. Different primary antibodies recognizing CLR, RAMP1, CGRP and sGC coupled to fluorescent secondary antibodies were used to examine immunoreactive cells in serial sections of trigeminal ganglia with epifluorescence and confocal laser scanning microscopy. Several staining protocols were examined to yield optimized immunolabeling.
Results
In vehicle-treated animals, 42% of the trigeminal ganglion neurons were immunopositive for RAMP1 and 41% for CLR. After GTN pretreatment CLR-immunopositivity was unchanged, while there was an increase in RAMP1-immunopositive neurons to 46%. RAMP1 and CLR immunoreactivity was also detected in satellite cells. Neurons immunoreactive for sGC were on average smaller than sGC-immunonegative neurons. The percentage of sGC-immunopositive neurons (51% after vehicle) was decreased after GTN infusion (48%).
Conclusions
Prolonged infusion of GTN caused increased fractions of RAMP1- and decreased fractions of sGC-immunopositive neurons in the trigeminal ganglion. The observed alterations are likely immunophenotypic correlates of the pathophysiological processes underlying nitrovasodilator-induced migraine attacks and indicate that signalling via CGRP receptors but not sGC-mediated mechanisms may be enhanced through endogenous NO production.
In the 1970s, by using classic histological methods, close topographical relationships between special areas of enteric ganglia and capillaries were shown in the pig. In this study, by application of double and triple immunohistochemistry, we confirmed this neurovascular interface and demonstrated that these zones are mainly confined to nitrergic neurons in the myenteric and the external submucosal plexus. In the upper small intestine of the pig, the respective neurons display type III morphology, i.e. they have long, slender and branched dendrites and a single axon. In another set of experiments, we prepared specimens for electron-microscopical analysis of these zones. Both ganglia and capillaries display continuous basement membranes, the smallest distances between them being 1,000 nm at the myenteric and 300 nm at the external submucosal level. The capillary endothelium was mostly continuous but, at the external submucosal level, scattered fenestrations were observed. This particular neurovascular relationship suggests that nitrergic neurons may require a greater amount of oxygen and/or nutrients. In guinea pig and mouse, previous ischemia/reperfusion experiments showed that nitrergic neurons are selectively damaged. Thus, a preferential blood supply of enteric nitrergic neurons may indicate that these neurons are more vulnerable in ischemia.
Gastro-esophageal reflux disease (GERD) is one of the most common disorders in gastroenterology. Patients present with or without increased acid exposure indicating a nonuniform etiology. Thus, the common treatment with proton pump inhibitors (PPIs) fails to control symptoms in up to 40% of patients. To further elucidate the pathophysiology of the condition and explore new treatment targets, transcriptomics, proteomics and histological methods were applied to a surgically induced subchronic reflux esophagitis model in Wistar rats after treatment with either omeprazole (PPI) or STW5, a herbal preparation shown to ameliorate esophagitis without affecting refluxate pH. The normal human esophageal squamous cell line HET-1A and human endoscopic biopsies were used to confirm our findings to the G-protein-coupled receptor (GPR) 84 in human tissue. Both treatments reduced reflux-induced macroscopic and microscopic lesions of the esophagi as well as known proinflammatory cytokines. Proteomic and transcriptomic analyses identified CINC1-3, MIP-1/3α, MIG, RANTES and interleukin (IL)-1β as prominent mediators in GERD. Most regulated cyto-/chemokines are linked to the TREM-1 signaling pathway. The fatty acid receptor GPR84 was upregulated in esophagitis but significantly decreased in treated groups, a finding supported by Western blot and immunohistochemistry in both rat tissue and HET-1A cells. GPR84 was also found to be significantly upregulated in patients with grade B reflux esophagitis. The expression of GPR84 in esophageal tissue and its potential involvement in GERD are reported for the first time. IL-8 (CINC1-3) and the TREM-1 signaling pathway are proposed, besides GPR84, to play an important role in the pathogenesis of GERD.
Successful primary closure of classic bladder exstrophy (BE) is crucial for development of bladder capacity and voided continence. It is universally agreed that an intensive pain management including the use of caudal epidural anesthesia is an essential cornerstone for the outcome of this complex surgery. Whether and to what extent pain is caused by structural or functional changes is not yet known. The nerve growth factor (NGF) is regarded as a marker for pain in different bladder disorders. This prospective study investigated the role of histological alterations and NGF in patients with BE including 34 patients with BE and 6 patients with congenital vesicoureterorenal reflux (VUR) who served as controls. Between January 2015 and April 2020 transmural bladder biopsies were taken from the posterior bladder wall during delayed primary bladder closure. The samples were stained for histological evaluation and subjected to immunohistochemistry to analyze NGFR p75. Differences in histological alterations were examined with Fisher's exact test, and Mann-Whitney-U-test was used to compare the NGFR p75 staining intensity between patients with BE and controls. Patients with BE showed significantly more often acute inflammation (p < 0.001), squamous metaplasia (p = 0.002), and cystitis glandularis (p = 0.005) as well as NGFR p75 in the urothelium (p = 0.003) than patients with VUR. A limitation of this study is the small number of participants due to the rare disease entity. Similar to other painful bladder disorders, pain transmission in BE after intitial closure may in part be facilitated by elevated NGF signaling through its receptor.
Clinical publications show consistently that headache is a common symptom in the coronavirus disease of 2019 (COVID-19). Several studies specifically investigated headache symptomatology and associated features in patients with COVID-19. The headache is frequently debilitating with manifold characters including migraine-like characteristics. Studies suggested that COVID-19 patients with headache vs. those without headache are more likely to have anosmia. We present a pathophysiological hypothesis which may explain this phenomenon, discuss current hypotheses about how the coronavirus SARS-CoV-2 enters the central nervous system and suggest that activation of the trigeminal nerve may contribute to both headache and anosmia in COVID-19.
Au-Fe3O4 nanoheterodimers (NHD) were functionalized with the natural and synthetic anticancer drugs caffeic acid (CA), quercetin (Q) and 5-fluorocytidine (5FC). Their X-radiation dose-enhancing potential and chemotherapeutic efficacy for bimodal cancer therapy were investigated by designing multicellular tumor spheroids (MCTS) to in vitro avascular tumor models. MCTS were grown from the breast cancer cell lines MCF-7, MDA-MB-231, and MCF-10A. The MCF-7, MDA-MB-231 and MCF-10A MCTS were incubated with NHD-CA, NHD-Q, or NHD-5FC and then exposed to fractionated X-radiation comprising either a single 10 Gy dose, 2 daily single 5 Gy doses or 5 daily single 2 Gy doses. The NHD-CA, NHD-Q, and NHD-5FC affected the growth of X-ray irradiated and non-irradiated MCTS in a different manner. The impact of the NHDs on the glycolytic metabolism due to oxygen deprivation inside MCTS was assessed by measuring lactate secretion and glucose uptake by the MCTS. The NHD-CA and NHD-Q were found to act as X-radiation dose agents in MCF-7 MCTS and MDA-MB-231 MCTS and served as radioprotector in MCF-10A MCTS. X-ray triggered release of CA and Q inhibited lactate secretion and thereupon disturbed glycolytic reprogramming, whereas 5FC exerted their cytotoxic effects on both, healthy and tumor cells, after their release into the cytosol.
Smart Shell‐by‐Shell Nanoparticles with Tunable Perylene Fluorescence in the Organic Interlayer
(2021)
Abstract
A new series of shell‐by‐shell (SbS)‐functionalized Al2O3 nanoparticles (NPs) containing a perylene core in the organic interlayer as a fluorescence marker is introduced. Initially, the NPs were functionalized with both, a fluorescent perylene phosphonic acid derivative, together with the lipophilic hexadecylphosphonic acid or the fluorophilic (1 H,1 H,2 H,2H‐perfluorodecyl)phosphonic acid. The lipophilic first‐shell functionalized NPs were further implemented with amphiphiles built of aliphatic chains and polar head‐groups. However, the fluorophilic NPs were combined with amphiphiles consisting of fluorocarbon tails and polar head‐groups. Depending on the nature of the combined phosphonic acids and the amphiphiles, tuning of the perylene fluorescence can be accomplished due variations of supramolecular organization with the shell interface. Because the SbS‐functionalized NPs dispose excellent dispersibility in water and in biological media, two sorts of NPs with different surface properties were tested with respect to biological fluorescent imaging applications. Depending on the agglomeration of the NPs, the cellular uptake differs. The uptake of larger agglomerates is facilitated by endocytosis, whereas individualized NPs cross directly the cellular membrane. Also, the larger agglomerates were preferentially incorporated by all tested cells.
Our research objective was to develop novel drug delivery vehicles consisting of TiO2 and Al2O3 nanoparticles encapsulated by a bilayer shell that allows the reversible embedment of hydrophobic drugs. The first shell is formed by covalent binding of hydrophobic phosphonic acid at the metal oxide surface. The second shell composed of amphiphilic sodium dodecylbenzenesulfonate emerges by self-aggregation driven by hydrophobic interactions between the dodecylbenzene moiety and the hydrophobic first shell. The resulting double layer provides hydrophobic pockets suited for the intake of hydrophobic drugs. The nanoparticles were loaded with the anticancer drugs quercetin and 7-amino-4-methylcoumarin. Irradiation with X-rays was observed to release the potential anticancer drugs into the cytoplasm. In Michigan Cancer Foundation (MCF)-10 A cells, quercetin and 7-amino-4-methylcoumarin acted as antioxidants by protecting the non-tumorigenic cells from harmful radiation effects. In contrast, these agents increased the reactive oxygen species (ROS) formation in cancerous MCF-7 cells. Quercetin and 7-amino-4-methylcoumarin were shown to induce apoptosis via the mitochondrial pathway in cancer cells by determining an increase in TUNEL-positive cells and a decrease in mitochondrial membrane potential after irradiation. After X-ray irradiation, the survival fraction of MCF-7 cells with drug-loaded nanoparticles considerably decreased, which demonstrates the excellent performance of the double-layer stabilized nanoparticles as drug delivery vehicles.
To date, it has remained unclear whether gastrointestinal symptoms, which are frequently observed in patients with multiple sclerosis (MS), are accompanied by pathology of the enteric nervous system (ENS). Here, the neurotransmitter signature of ENS neurons and morphological alterations of interstitial cells of Cajal (ICCs) were studied in patients with MS and mice with experimental autoimmune encephalomyelitis (EAE), which is an animal model of MS. Immunohistochemical analysis was performed on colonic whole mounts from mice with EAE and on paraffin-embedded sections of intestinal tissue from patients with MS. Antibodies against neurotransmitters or their enzymes (including vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS), and choline acetyltransferase (ChAT)) were used in conjunction with pan-neuronal markers. In addition, the presence of anoctamin 1 (ANO1)-expressing ICCs was studied. ENS changes were observed in the myenteric plexus, but they were absent in the submucosal plexus of both EAE mice and patients with MS. There was a significant decrease in the percentage of ChAT-positive neurons in EAE mice as opposed to a trend toward an increase in patients with MS. Moreover, while ANO1 expression was decreased in EAE mice, patients with MS displayed a significant increase. Although additional studies are necessary to accomplish an in-depth characterization of ENS alterations in MS, our results imply that such alterations exist and may reveal novel insights into the pathophysiology of MS.