TRP channels in neurogastroenterology: opportunities for therapeutic intervention

The members of the superfamily of transient receptor potential (TRP) cation channels are involved in a plethora of cellular functions. During the last decade, a vast amount of evidence is accumulating that attributes an important role to these cation channels in different regulatory aspects of the alimentary tract. In this review we discuss the expression patterns and roles of TRP channels in the regulation of gastrointestinal motility, enteric nervous system signalling and visceral sensation, and provide our perspectives on pharmacological targeting of TRPs as a strategy to treat various gastrointestinal disorders. We found that the current knowledge about the role of some members of the TRP superfamily in neurogastroenterology is rather limited, whereas the function of other TRP channels, especially of those implicated in smooth muscle cell contractility (TRPC4, TRPC6), visceral sensitivity and hypersensitivity (TRPV1, TRPV4, TRPA1), tends to be well established. Compared with expression data, mechanistic information about TRP channels in intestinal pacemaking (TRPC4, TRPC6, TRPM7), enteric nervous system signalling (TRPCs) and enteroendocrine cells (TRPM5) is lacking. It is clear that several different TRP channels play important roles in the cellular apparatus that controls gastrointestinal function. They are involved in the regulation of gastrointestinal motility and absorption, visceral sensation and visceral hypersensitivity. TRP channels can be considered as interesting targets to tackle digestive diseases, motility disorders and visceral pain. At present, TRPV1 antagonists are under development for the treatment of heartburn and visceral hypersensitivity, but interference with other TRP channels is also tempting. However, their role in gastrointestinal pathophysiology first needs to be further elucidated.     

Revascularization of the superficial femoral artery with paclitaxel-coated balloon for claudication

Background: Percutaneous angioplasty of the superficial femoral artery (SFA) with paclitaxel-coated balloon, intended to reduce restenosis, has been proven safe and effective in recent randomized controlled trials.

Objective: To assess outcome results of angioplasty of the SFA with paclitaxel-coated balloon in claudicants in real-world practice of a single center.

Material &; Methods: A continuous prospective cohort study of 53 claudicants (62 lower limbs) from January 2015 to December 2016. Study end points include primary patency, freedom from clinically driven target-lesion revascularization and symptom relief.

Results: It concerns 17 women (32%) and 36 men (68%) with a mean age of 67.8 years, suffering life-style-limiting claudication. Only short to intermediate-length stenoses or occlusions (30.6%), with a mean length of 59.6?mm were selected for percutaneous angioplasty with a paclitaxel-coated balloon. Technical success was 100%. At 16 months, primary patency attained 92.0% (3 early occlusions, 2 restenoses). There were two re-interventions at 6 and 9 months, resulting in a clinically driven target lesion revascularization rate of 3.2%. At the end of the follow-up of 16 months, all but two patients (96.2%) remained symptom-free. Two patients died during follow-up (no procedure-related deaths).

Conclusion: Paclitaxel-coated balloon angioplasty of the SFA gives in routine clinical practice excellent midterm results, with a restenosis rate of 6.5% at 1 year. This procedure has authors’ preference as first-choice technique for correction of short- and intermediate-length symptomatic stenoses of the SFA.     

Use of StarClose for brachial artery closure after percutaneous endovascular interventions

The objective of this study was to evaluate a percutaneous extravascular closure device (StarClose, Abbott Vascular, Redwood City, CA) after brachial endovascular approach. From 2004 to 2006, 29 patients received StarClose for brachial closure. Primary endpoints were successful deployment and absence of procedure-related morbidity, secondary endpoints were brachial artery patency on duplex and absence of late (> 30 days) complications. The device was successfully deployed in all patients. In two patients (6.8%) local complications occurred: one patient developed a large hematoma successfully treated with prolonged compression and a second patient presented with brachial artery occlusion requiring operative intervention. After a mean follow-up of 7.5+/-7.2 months, all patients had a palpable brachial/radial pulse; none had signs of infection, distal embolization or neurological deficits. On ultrasound b-mode imaging, the clip was visible as a 4 mm echolucent area at the outer anterior wall of the artery. Based on the peak systolic velocity ratios between the site of StarClose and proximal brachial artery (mean 1.08+/-0.2), none of the studied patients had a significant stenosis at the site of closure. StarClose is safe and effective in providing hemostasis following interventional procedures through the brachial artery; further advantages include patients comfort and early discharge.     

Penetrating atherosclerosis aortic ulcer: a re-appraisal

Background: Penetrating aortic ulcer is a rare pathology, often clinically silent, but potentially fatal when manifesting as an acute aortic syndrome. It is more often detected in recent years, due to ageing of the population and more widespread use of computed tomography. A literature review aims to define the distinct disease entity of penetrating aortic ulcer.

Methods: Five recent cases of penetrating aortic ulcer, treated in authors’ department are reported. A review of English-language medical literature from 1980 to 2015 was undertaken using PubMed and EMBASE databases, to identify studies reporting surgical (open and endovascular) treatment of penetrating aortic ulcer.

Results: From September 2013 to September 2015, five cases of acute aortic syndrome caused by a penetrating atherosclerotic ulcer of the descending thoracic aorta were observed in authors’ department. This represents 9% of all acute aortic syndromes admitted to our hospital in the same period. All five patients benefitted from thoracic endovascular stent grafting with a 100% success rate. Natural history and optimal management of penetrating aortic ulcer are outlined according to the most recent insights.

Conclusion: Penetrating aortic ulcer represents 2–7% of all acute aortic syndromes. Symptomatic penetrating aortic ulcer requires coverage by thoracic endovascular stent grafting according to the recent guidelines.     

Structurally defined signaling in neuro-glia units in the enteric nervous system

Coordination of gastrointestinal function relies on joint efforts of enteric neurons and glia, whose crosstalk is vital for the integration of their activity. To investigate the signaling mechanisms and to delineate the spatial aspects of enteric neuron-to-glia communication within enteric ganglia we developed a method to stimulate single enteric neurons while monitoring the activity of neighboring enteric glial cells. We combined cytosolic calcium uncaging of individual enteric neurons with calcium imaging of enteric glial cells expressing a genetically encoded calcium indicator and demonstrate that enteric neurons signal to enteric glial cells through pannexins using paracrine purinergic pathways. Sparse labeling of enteric neurons and high-resolution analysis of the structural relation between neuronal cell bodies, varicose release sites and enteric glia uncovered that this form of neuron-to-glia communication is contained between the cell body of an enteric neuron and its surrounding enteric glial cells. Our results reveal the spatial and functional foundation of neuro-glia units as an operational cellular assembly in the enteric nervous system.     

An optimization and refinement of the whole-gut transit assay in mice

Background

Gastrointestinal motility measurements in mice are currently performed under suboptimal conditions, as these nocturnal animals are measured during light conditions. In addition, other stressors, like individual housing, placement in a new cage during observation, and lack of bedding and cage enrichment cause animal discomfort and might contribute to higher variability. Here we aimed to develop a refined method of the widely-used whole-gut transit assay.

Methods

Wildtype mice (N = 24) were subjected to the standard or refined whole-gut transit assay, either with or without a standardized slowing in gastrointestinal motility induced by loperamide. The standard assay consisted of a gavage with carmine red, observation during the light period and individual housing in a new cage without cage enrichment. For the refined whole-gut transit assay, mice were gavaged with UV-fluorescent DETEX®, observed during the dark period, while pairwise housed in their home cage with cage enrichment. Time until excretion of the first colored fecal pellet was assessed, and pellets were collected to assess number, weight, and water content.

Key Results

The DETEX®-containing pellets were UV-detectable, allowing to measure the mice in their active period in the dark. The refined method caused less variation (20.8% and 16.0%) compared to the standard method (29.0% and 21.7%). Fecal pellet number, weight, and water content was significantly different between the standard and refined method.

Conclusions & Inferences

This refined whole-gut transit assay provides a reliable approach to measure whole-gut transit time in mice in a more physiological context, with reduced variability compared to the standard method.     

Neurotransmitters involved in fast excitatory neurotransmission directly activate enteric glial cells

Background The intimate association between glial cells and neurons within the enteric nervous system has confounded careful examination of the direct responsiveness of enteric glia to different neuroligands. Therefore, we aimed to investigate whether neurotransmitters known to elicit fast excitatory potentials in enteric nerves also activate enteric glia directly. Methods We studied the effect of acetylcholine (ACh), serotonin (5‐HT), and adenosine triphosphate (ATP) on intracellular Ca²⁺ signaling using aequorin‐expressing and Fluo‐4 AM‐loaded CRL‐2690 rat and human enteric glial cell cultures devoid of neurons. The influence of these neurotransmitters on the proliferation of glia was measured and their effect on the expression of c‐Fos as well as glial fibrillary acidic protein (GFAP), Sox10, and S100 was examined by immunohistochemistry and quantitative RT‐PCR. Key Results Apart from ATP, also ACh and 5‐HT induced a dose‐dependent increase in intracellular Ca²⁺ concentration in CRL‐2690 cells. Similarly, these neurotransmitters also evoked Ca²⁺ transients in human primary enteric glial cells obtained from mucosal biopsies. In contrast with ATP, stimulation with ACh and 5‐HT induced early gene expression in CRL‐2690 cells. The proliferation of enteric glia and their expression of GFAP, Sox10, and S100 were not affected following stimulation with these neurotransmitters. Conclusions & Inferences We provide evidence that enteric glial cells respond to fast excitatory neurotransmitters by changes in intracellular Ca²⁺. On the basis of our experimental in vitro setting, we show that enteric glia are not only directly responsive to purinergic but also to serotonergic and cholinergic signaling mechanisms.     

Calcium imaging at kHz frame rates resolves millisecond timing in neuronal circuits and varicosities

We have configured a widefield fast imaging system that allows imaging at 1000 frames per second (512x512 pixels). The system was extended with custom processing tools including a time correlation method to facilitate the analysis of static subcellular compartments (e.g. neuronal varicosities) with enhanced contrast, as well as a dynamic intensity processing (DIP) algorithm that aids in data size reduction and fast visualization and interpretation of timing and directionality in neuronal circuits. This system, together with our custom developed processing tools enables efficient detection of fast physiological events, such as action potential dependent calcium steps. We show, using a specific blocker of nerve communication, that with this setup it is possible to discriminate between a pre and post synaptic event in an all optical way.OCIS codes: (100.0118) Imaging ultrafast phenomena, (330.6790) Temporal discrimination, (100.2000) Digital image processing, (170.6920) Time-resolved imaging