Human enteric neurons: morphological, electrophysiological, and neurochemical identification

BACKGROUND: Access to tissue, difficulties with dissection, and poor visibility of enteric ganglia have hampered electrophysiological recordings of human enteric neurons. Here, we report a method to combine intracellular recording with simultaneous morphological identification of neurons in the intact myenteric plexus of human colon ex vivo. METHODS: Specimens of human colon were dissected into flat-sheet preparations with the myenteric plexus exposed. Myenteric neurons were impaled with conventional microelectrodes containing 5% 5,6-carboxyfluorescein in 20 mM Tris buffer and 1 M KC. KEY RESULTS: Electrophysiological recordings identified myenteric neurons with S and AH type properties (n = 13, N = 7) which were dye filled and classified during the recording as Dogiel type I (n = 10), Dogiel type II (n = 2), or filamentous (n = 1) cells. This classification was confirmed after fixation, in combination with immunohistochemical characterization. CONCLUSIONS & INFERENCES: This method allows electrophysiological characterization with simultaneous identification of morphology. It can be used to identify recorded cells immediately after impalement and greatly facilitates recordings of human myenteric neurons in freshly dissected specimens of tissue. It can also be combined with immunohistochemical labeling of recorded cells

Biomimetic calcium carbonate with hierarchical porosity produced using cork as a sustainable template agent

Calcium carbonate has many applications in different fields; its use in environment remediation is particularly considered, due to its non-toxicity and potentially high efficiency. The structure, morphology and surface features of calcium carbonate can greatly affect its performance. Hierarchical porosity, in particular, can be beneficial for several functional properties. In this study, we report the synthesis of biomorphic calcium carbonate using a sustainable template agent – waste cork powder. Pyrolysed cork powder was infiltrated by an appropriate calcium-containing salt and successively thermally treated. Selected precursors, different impregnation-solution concentrations and thermal conditions were tested. The resulting materials were characterised by XRD, Raman spectroscopy and SEM. Surface area and porosity features were studied by BET analysis, with a detailed study on the effect of synthesis on the mesoporosity of the materials, average sizes varying between 4−15 nm. The most valuable results were achieved with calcium acetate followed by pyrolysis performed for relatively short time period. This maintained the porous 3D honeycomb cork structure made of ∼20 μm hexagonal cells, while consisting of highly mesoporous single-phase CaCO3. Such samples showed the highest surface area ever reported for CaCO3 prepared using a plant-based template; moreover, it also exhibited a dual-scale hierarchical porosity as, in addition to micrometer scale cellular macroporosity, it contained a significant mesoporosity in the cell walls, with a very narrow range of 3.6–3.9 nm. These promising characteristics enable the potential employment of cork-derived CaCO3 for environment remediation.publishe

Ascending excitatory neural pathways modulate slow phasic myogenic contractions in the isolated human colon

This is the accepted version of the following article: [Carbone, S. E., Dinning, P. G., Costa, M., Spencer, N. J., Brookes, S. J. H. and Wattchow, D. A. (2013), Ascending excitatory neural pathways modulate slow phasic myogenic contractions in the isolated human colon. Neurogastroenterology & Motility, 25: 670–e510.], which has been published in final form at [http://dx.doi.org/10.1111/nmo.12129]. In addition, authors may also transmit, print and share copies with colleagues, provided that there is no systematic distribution of the submitted version, e.g. posting on a listserve, network or automated delivery.Background In animal models, enteric reflex pathways have potent effects on motor activity; their roles have been much less extensively studied in human gut. The aim of this study was to determine if ascending excitatory interneuronal pathways can modulate spontaneous phasic contractions in isolated preparations of human colonic circular muscle. Methods Human colonic preparations were cut into T shapes, with vertical bar of the ‘T’ pharmacologically isolated. Electrical stimulation and the nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), were applied to the isolated region and circular muscle contractile activity was measured from the cross-bar of the T, more than 10 mm orally from the region of stimulation. Key Results The predominant form of spontaneous muscle activity consisted of tetrodotoxin-resistant, large amplitude, slow phasic contractions (SPCs), occurring at average intervals of 124 ± 68 s. Addition of a high concentration of hexamethonium (1 mmol L-1) to the superfusing solution significantly increased the interval between SPCs to 278.1 ± 138.3 s (P < 0.005). Focal electrical stimulation more than 10 mm aboral to the muscle recording site advanced the onset of the next SPC, and this effect persisted in hexamethonium. However, the effect of electrical stimulation was blocked by tetrodotoxin (TTX, 1 µmol L-1). Application of the nicotinic agonist DMPP (1 mmol L-1) to the aboral chamber often stimulated a premature SPC (n = 4). Conclusions & Inferences The major form of spontaneous contractility in preparations of human colonic circular muscle is SPCs, which are myogenic in origin. Activation of ascending excitatory neural pathways, which involve nicotinic receptors, can modulate the timing of SPCs and thus influence human colonic motility.Australian National Health & Medical Research Counci

Comparative treatments of a green tattoo ink with Ruby, Nd: YAG nano- and picosecond lasers in normal and array mode

The tattoos removal has become an issue upon spread of the tattooing practice worldwide and hindsight regrets. Lasers are typically used for the purpose, though some colours such as green are considered “recalcitrant” to the treatment. In the current investigation, we aim at determining the efcacy of removal of a green ink water dispersion, using 5 laser treatments: Nd:YAG nano- and picosecond lasers in normal and array mode and Ruby nanosecond laser, keeping the total irradiated energy constant. The UV–Vis spectroscopy of the treated samples indicate that Nd:YAG picosecond laser is most efective, and the Ruby nanosecond laser is the least efcient. Fragment compounds generated from the pigment and siloxanes are common to all treatments, whereas hydrocarbon emerge by a larger amount upon Nd:YAG nanosecond treatment. Fibres are formed upon picosecond treatments and when operating in array mode, and lamellae are achieved by Ruby nanosecond laser treatment. Residual particles suspensions are very heterogeneous upon nanosecond treatments

Role of oxidative stress in oxaliplatin-induced enteric neuropathy and colonic dysmotility in mice

BACKGROUND AND PURPOSE: Oxaliplatin is a platinum‐based chemotherapeutic drug used as a first‐line therapy for colorectal cancer. However, its use is associated with severe gastrointestinal side‐effects resulting in dose limitations and/or cessation of treatment. In this study, we tested whether oxidative stress, caused by chronic oxaliplatin treatment, induces enteric neuronal damage and colonic dysmotility. EXPERIMENTAL APPROACH: Oxaliplatin (3 mg·kg(−1) per day) was administered in vivo to Balb/c mice intraperitoneally three times a week. The distal colon was collected at day 14 of treatment. Immunohistochemistry was performed in wholemount preparations of submucosal and myenteric ganglia. Neuromuscular transmission was studied by intracellular electrophysiology. Circular muscle tone was studied by force transducers. Colon propulsive activity studied in organ bath experiments and faeces were collected to measure water content. KEY RESULTS: Chronic in vivo oxaliplatin treatment resulted in increased formation of reactive oxygen species (O(2)ˉ), nitration of proteins, mitochondrial membrane depolarisation resulting in the release of cytochrome c, loss of neurons, increased inducible NOS expression and apoptosis in both the submucosal and myenteric plexuses of the colon. Oxaliplatin treatment enhanced NO‐mediated inhibitory junction potentials and altered the response of circular muscles to the NO donor, sodium nitroprusside. It also reduced the frequency of colonic migrating motor complexes and decreased circular muscle tone, effects reversed by the NO synthase inhibitor, Nω‐Nitro‐L‐arginine. CONCLUSION AND IMPLICATIONS: Our study is the first to provide evidence that oxidative stress is a key player in enteric neuropathy and colonic dysmotility leading to symptoms of chronic constipation observed in oxaliplatin‐treated mice

The VersaLive platform enables microfluidic mammalian cell culture for versatile applications

Microfluidic-based cell culture allows for precise spatio-temporal regulation of microenvironment, live cell imaging and better recapitulation of physiological conditions, while minimizing reagents' consumption. Despite their usefulness, most microfluidic systems are designed with one specific application in mind and usually require specialized equipment and expertise for their operation. All these requirements prevent microfluidic-based cell culture to be widely adopted. Here, we designed and implemented a versatile and easy-to-use perfusion cell culture microfluidic platform for multiple applications (VersaLive) requiring only standard pipettes. Here, we showcase the multiple uses of VersaLive (e.g., time-lapse live cell imaging, immunostaining, cell recovery, cell lysis, plasmid transfection) in mammalian cell lines and primary cells. VersaLive could replace standard cell culture formats in several applications, thus decreasing costs and increasing reproducibility across laboratories. The layout, documentation and protocols are open-source and available online at https://versalive.tigem.it/

Laser vs. thermal treatments of green pigment PG36: coincidence and toxicity of processes

Comparative laser and thermal treatments were carried out on PG36, a green phthalocyanine-based pigment, permitted in European countries where legislation on tattoo composition was issued. Prior to the treatments, PG36 was characterized by SEM imaging, EDX, IR and UV–Vis spectroscopies, revealing an excess of Si and C and O as compared to the pure halogenated Cu-phthalocyanine. Laser treatments were carried out with a Nd:YAG device applied to H2O and propan-2-ol dispersions. Pyrolysis and calcinations were carried out in air or under N2 flow. The outcome of the different procedures was analyzed by UV–Vis spectroscopy, GC–mass spectrometry, X-ray diffraction of the solid residues, SEM microscopy and dynamic light scattering. The comparative analysis indicated the production of different fragment compounds depending on the treatment, (pyrolysis or laser), and, to some extent, to the solvent of the dispersion, with pyrolysis generating a larger number of hazardous compounds. Hydrocarbons and cyclic siloxanes present as additives in PG36 were stable or degraded depending on the treatment. The morphology of the products is also treatment-dependent with nanoparticles &lt; 20 nm and fibers being produced upon laser treatments only. Based on the experimental findings, the equivalence of laser and thermal treatments is evaluated

Predictive Value of Left Ventricular Myocardial Deformation for Left Ventricular Remodeling in Patients With Classical Low-Flow, Low-Gradient Aortic Stenosis Undergoing Transcatheter Aortic Valve Replacement

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