8:30am - 8:45am
Attenuated Adverse Health Effects of Occupational Exposure to Multi-Walled Carbon Nanotubes in Chronic Obstructive Pulmonary Disease Monitored in vitro and in vivo
1Department for BioMedical Research, University of Bern, Switzerland; 2Department of Pulmonary Medicine, Inselspital, Bern University Hospital, University of Bern, Switzerland; 3BioNanomaterials, Adolphe Merkle Institute, University of Fribourg, Switzerland; 4Empa Materials, Science and Technology, St. Gallen, Switzerland; 5Institute of Anatomy, University of Bern, Switzerland
Rationale: Manufactured nanoparticles are produced in increasing quantities, resulting in concern about health-effects of occupational nanoparticles, such as multi-walled carbon nanotubes (MWCNT) on the vulnerable respiratory tract environment of healthy and in particular of individuals suffering from respiratory disease.
Methods: To study potential health risks of MWCNT in vitro, primary human bronchial epithelial cells from healthy and chronic obstructive pulmonary disease (COPD) patients, cultured at the air-liquid interface and exposed to aerosolized MWCNT were employed. Differentiation, pro-inflammatory status and viability of cells was monitored 24h post-exposure using laser scanning microscopy (LSM), transmission electron microscopy (TEM), ELISA and RT qPCR.
For in vivo monitoring, we utilized a COPD mouse model in which animals were treated with MWCNT by intratracheal instillation. Differential counts, lung function and phenotype of antigen presenting cell (APC) populations in the respiratory tract were monitored.
Results: In vitro differentiation and epithelial integrity was maintained after exposure to MWCNT (0.16µg/cm2 - 0.34µg/cm2) as visualized by LSM. TEM micrographs showed extra- and intracellular localization of MWCNT and neither RNA expression nor release of pro-inflammatory cytokines was enhanced by MWCNT in vitro.
Preliminary data in vivo showed an enhanced number of total cells in differential counts following exposure to MWCNT, but no up regulation of activation markers in APC. Lung function measurements showed decreased tissue damping (G) and tissue elasticity (H) in COPD mice.
Conclusions: Both, the in vitro and the in vivo model have proved to be complementary and allow in-depth investigation of pulmonary effects of inhalable MWCNT on the healthy and diseased lung. Preliminary data indicate attenuated adverse health effects induced by realistic dosages of MWCNT. These findings require further confirmation in ongoing studies.
8:45am - 9:00am
IP3-Induced SR-Ca2+ Release Functions as an Anti-Arrhythmogenic Mechanism in Ventricular Myocytes
Department of Physiology, University of Bern, Switzerland
In cardiac muscle, besides Ca2+-induced Ca2+ release (CICR), a second Ca2+ release mechanism activated by hormone binding to G-protein coupled receptors is present. This prompt intracellular production of the signaling molecule Inositol 1,4,5-trisphosphate (IP3) which subsequently triggers sarcoplasmic reticulum (SR)-Ca2+ release via the IP3 receptor type 2 (IP3R2). IP3-induced Ca2+ release (IP3ICR) may modulate Ryanodine receptor (RyR2s) function via local interactions and fine-tune excitation-contraction coupling in ventricular myocytes. A functional interplay between IP3R2s and RyR2s may be significantly pronounced under cardiac pathologies where IP3R2 is known to be overexpressed. We examined IP3ICR and CICR in ventricular myocytes on a local scale in a cardiac specific IP3R2-overexpressing mouse model (IP3/tTA) with a phenotype of cardiac hypertrophy. Protein analysis confirmed a 12-fold increase in IP3R2 expression together with a 61.7% reduction in RyR2 levels. IP3-pathway stimulation in wild-type cells increased spontaneous Ca2+ events by 24.2%. Surprisingly, IP3ICR activation in IP3/tTA myocytes induced a decrease in Ca2+ spark frequency by 25.2% together with a reduction of the SR-Ca2+ content by 14.4% that cannot be explained by RyR2 Ca2+ spark occurrence alone. We examined this phenomenon in more detail in intact myocytes by applying specific SR-Ca2+leak/load protocols. We found that in IP3/tTA mice IP3ICR functions below Ca2+ spark threshold, via efficient modulation of the SR-Ca2+ leak. Activation of this pathway acts as a protective mechanism against arrhythmogenic Ca2+ wave occurrence. We conclude that overexpression of IP3R2 in ventricular myocytes may represent a new and so far not recognized anti-arrhythmogenic mechanism to prevent SR-Ca2+ overload and reduce the propensity of aberrant SR-Ca2+ release preconditioned for arrhythmogenicity. This mechanism could be a potential target for precision medicine therapies treating ventricular tachycardia.
9:00am - 9:15am
Neuropathic Pain Impairs Spike-Timing Dependent Long-Term Depression in Layer 5 Pyramidal Neurons of the Anterior Cingulate Cortex
Department of Physiology, University of Bern, Switzerland
Chronic pain is caused by irreversible neuronal alterations in the entire pain processing system. The cellular mechanisms leading to a sensitization of the pain network include changes in cellular excitability, network restructuring and malfunction of synaptic plasticity. Accordingly, it has been suggested that enhanced long-term potentiation of synaptic transmission contributes to the chronic pain state, whereas little is known about the involvement of long-term depression.
We studied spike-timing dependent depression (tLTD) in the anterior cingulate cortex (ACC), a brain region involved in the emotional/affective processing of pain. We found that tTLD at synapses onto layer 5 pyramidal neurons in the ACC was abolished in adult mice when subjected to chronic constriction injury (CCI) of the sciatic nerve, an animal model for neuropathic pain.
Investigating the underlying mechanism in naïve mice revealed that tLTD depended on postsynaptic calcium and the synthesis of nitric oxide (NO). NO acted as a retrograde messenger. In CCI animals, bath application of NO resulted in LTD suggesting that the signaling cascade downstream of NO was still intact. On the postsynaptic site we identified an exclusion of GluN2B‑containing NMDARs from the synapse as the cause of the tLTD phenotype in neuropathic pain.
Then, we expressed channelrhodopsin-2 in various brain areas and activated their axons in the ACC optically to look for a projection specificity of the found LTD phenotype. Finally, we investigated the temporal correlation of pain-associated behavior with the tLTD phenotype.
Our results suggest that neuropathic pain causes a switch in the NMDAR subunit composition blocking the induction of tLTD in the ACC. This block remains even if animals recover from certain pain‑associated behaviors (mechanical hypersensitivity). The change in the NMDAR subunit composition and the resulting changes in synaptic plasticity might represent an engram of the forgoing pain experience.
9:15am - 9:30am
Radiation Induced Liver Disease Mouse Model: A Promising Step Towards Understanding of Normal Liver Tissue Toxicity
1Departement for BioMedical Research, University of Bern, Switzerland; 2Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
Radiation of liver tumors has recently shown promising results in clinics. Its applicability is limited by the life-threatening conditions that can result from hepatic toxicity. The radiation induced liver disease (RILD) is characterized by sinusoid obstruction syndrome and veno-occlusive disease observed few weeks post irradiation (PI), and very little is known about its underlying mechanism. Here we describe our RILD mouse model and ensuing new findings on RILD development.
Based on CT images obtained using exitron6000 nanoparticles as liver contrasting agent, we irradiate a 5mm diameter liver target with 50Gy using small animal image guided irradiation system. 4 collection times were realized at 1day, 6day, 6week and 20week PI. Histological evaluation was done based fixed tissue using eosin hematoxylin, Masson's trichrome, Sirius red and Evans blue staining. Transcriptomic analysis was perused using two samples of RNA per liver (from the target area and not irradiated area; paired analysis).
Mice submitted to irradiation did not show histological change at 1day and 6day PI. 6week PI centrizonal sinusoid were obstructed by collagen fiber and an increased number of Evan’s blue positive ceroid containing macrophage in the target area, 20week PI those observation where further increased. Transcriptomic analysis revealed very different profile at the different time points. After the initial injury, a large number of repair mechanism are ongoing at day 1 PI. At day 6 PI a very few process where find different between high and low irradiated samples. 6 and 20week PI showed an increasing number of process ongoing and among them ECM modulator and immune response as expected from the histology. We hypothesize that the key trigger of RILD is the unresolved injury that we could observe at 6day PI leading to the late fibrosis.
Further characterization RILD marker should allow to better describe RILD mechanistic and further evaluation of prophylactic and therapeutic interventions.