Probing Functional Properties of Nociceptive Axons Using a Microfluidic Culture System

Tsantoulas, Christoforos; Farmer, Clare; Machado, Patrícia Maria Abreu; Baba, Katsuhiro; McMahon, Stephen B.; Raouf, Ramin

doi:10.1371/journal.pone.0080722

Cited by 51 publications

(62 citation statements)

References 66 publications

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“…These mice permit the monitoring of changes in TRPV1-lineage afferent terminals in real time. Dissociated sensory neurons from DRG of TRPV1-GFP mice were plated into multicompartmental microfluidic chambers (MFC), permitting manipulation of axons independent of their somata (22). After approximately 2 weeks, when the axons extended across the compartment, we performed time-lapse imaging of GFP-and tdTomato-expressing axons before and after application of capsaicin to the axonal compartment (supplemental Movie 1 and supplemental Movie 2).…”

Section: Capsaicin Ablates Trpv1-lineage Afferent Terminals In Vitromentioning

confidence: 99%

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Ca2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1

Wang

Asgar

et al. 2017

Journal of Biological Chemistry

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Capsaicin is an ingredient in spicy peppers that produces burning pain by activating transient receptor potential vanilloid 1 (TRPV1), a Ca-permeable ion channel in nociceptors. Capsaicin has also been used as an analgesic, and its topical administration is approved for the treatment of certain pain conditions. The mechanisms underlying capsaicin-induced analgesia likely involve reversible ablation of nociceptor terminals. However, the mechanisms underlying these effects are not well understood. To visualize TRPV1-lineage axons, a genetically engineered mouse model was used in which a fluorophore is expressed under the TRPV1 promoter. Using a combination of these TRPV1-lineage reporter mice and primary afferent cultures, we monitored capsaicin-induced effects on afferent terminals in real time. We found that Ca influx through TRPV1 is necessary for capsaicin-induced ablation of nociceptive terminals. Although capsaicin-induced mitochondrial Ca uptake was TRPV1-dependent, dissipation of the mitochondrial membrane potential, inhibition of the mitochondrial transition permeability pore, and scavengers of reactive oxygen species did not attenuate capsaicin-induced ablation. In contrast, MDL28170, an inhibitor of the Ca-dependent protease calpain, diminished ablation. Furthermore, overexpression of calpastatin, an endogenous inhibitor of calpain, or knockdown of calpain 2 also decreased ablation. Quantitative assessment of TRPV1-lineage afferents in the epidermis of the hind paws of the reporter mice showed that EGTA and MDL28170 diminished capsaicin-induced ablation. Moreover, MDL28170 prevented capsaicin-induced thermal hypoalgesia. These results suggest that TRPV1/Ca/calpain-dependent signaling plays a dominant role in capsaicin-induced ablation of nociceptive terminals and further our understanding of the molecular mechanisms underlying the effects of capsaicin on nociceptors.

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Section: Capsaicin Ablates Trpv1-lineage Afferent Terminals In Vitromentioning

confidence: 99%

“…1A). It was reported that somata and axons of DRG neurons were successfully segregated and independently manipulated using this system (22). Autoclaved rectangular glass coverslips were coated with poly-D-ornithine and laminin.…”

Section: Culture Of Drg Neurons In Mfcmentioning

confidence: 99%

Ca2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1

Wang

Asgar

et al. 2017

Journal of Biological Chemistry

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“…21 Microuidics approach has alleviated some of these drawbacks. 19,20,22 In this study, we have utilized a multicompartment microuidic model based on our previous device, 23 which enables controlled, simultaneous axotomy of a large number of axons. This method maintains the distal as well as proximal axon remains aer lesion, while preserving its supporting substrate coating.…”

Section: Introductionmentioning

confidence: 99%

A microfluidic neuronal platform for neuron axotomy and controlled regenerative studies

et al. 2015

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Understanding the basic mechanisms of neural regeneration after injury is a pre-requisite for developing appropriate treatments. Traditional approaches to model axonal lesions, such as high intensity power laser ablation or sharp metal scratching, are complex to implement, have low throughputs, and generate cuts that are difficult to modulate. We present here a novel reproducible microfluidic approach to model in vitro mechanical lesion of tens to hundreds of axons simultaneously in a controlled manner. The dimensions of the induced axonal injury and its distance from the neuronal cell body are precisely controlled while preserving both the proximal and distal portions of axons. We have observed that distal axons undergo Wallerian-like anterograde degeneration after axotomy; in contrast, proximal portions of the axons remain un-degenerated, possessing the potential to re-grow. More importantly, surpassing the previous axotomy methods performed in Petridishes in which local microenvironments cannot be tailored, our platform holds the capability to implement fine-tuned treatments to lesioned axon stumps in a local, controlled manner. Specifically, molecules such as chondroitin sulphate proteoglycans and its degrading enzyme chondroitinase ABC, hydrogels, and supporting cells have been shown to be deliverable to the lesioned site of injured axons. In addition, this system also permits double interventions at the level of the lesioned axons and the perikaryon. This proves the potential of our model by demonstrating how axonal regrowth can be evaluated under circumstances that are better mimics of biological problems. We believe that this novel mechanical microfluidic axotomy approach is easy to perform, yields high throughput axon lesions, is physiologically relevant, and offers a simplified platform for screening of potential new neurological drugs.

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“…A variety of OAC platforms have been derived from cell lines, stem cells, and/or primary cells, including small and large intestine ( (380)(381)(382)(383), bone (384,385), reproductive tract (386), and blood/endothelium and blood-brain barrier (387)(388)(389)(390)(391)(392)(393), among others. Once developed, these models typically retain their structural and functional integrity for several weeks (model specific), further lending to their experimental tractability.…”

Section: Limitations and Future Directions Of 3-d Organoidsmentioning

confidence: 99%

Modeling Host-Pathogen Interactions in the Context of the Microenvironment: Three-Dimensional Cell Culture Comes of Age

et al. 2018

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Tissues and organs provide the structural and biochemical landscapes upon which microbial pathogens and commensals function to regulate health and disease. While flat two-dimensional (2-D) monolayers composed of a single cell type have provided important insight into understanding host-pathogen interactions and infectious disease mechanisms, these reductionist models lack many essential features present in the native host microenvironment that are known to regulate infection, including three-dimensional (3-D) architecture, multicellular complexity, commensal microbiota, gas exchange and nutrient gradients, and physiologically relevant biomechanical forces (e.g., fluid shear, stretch, compression). A major challenge in tissue engineering for infectious disease research is recreating this dynamic 3-D microenvironment (biological, chemical, and physical/mechanical) to more accurately model the initiation and progression of host-pathogen interactions in the laboratory. Here we review selected 3-D models of human intestinal mucosa, which represent a major portal of entry for infectious pathogens and an important niche for commensal microbiota. We highlight seminal studies that have used these models to interrogate host-pathogen interactions and infectious disease mechanisms, and we present this literature in the appropriate historical context. Models discussed include 3-D organotypic cultures engineered in the rotating wall vessel (RWV) bioreactor, extracellular matrix (ECM)-embedded/organoid models, and organ-on-a-chip (OAC) models. Collectively, these technologies provide a more physiologically relevant and predictive framework for investigating infectious disease mechanisms and antimicrobial therapies at the intersection of the host, microbe, and their local microenvironments.

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Probing Functional Properties of Nociceptive Axons Using a Microfluidic Culture System

Cited by 51 publications

References 66 publications

Ca2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1

Ca2+ and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1

A microfluidic neuronal platform for neuron axotomy and controlled regenerative studies

Modeling Host-Pathogen Interactions in the Context of the Microenvironment: Three-Dimensional Cell Culture Comes of Age

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