माप tracheal कसना का प्रयोग चूहे की इन विट्रो में
Summary
ट्रांसजेनिक चूहों जीन शारीरिक समारोह ascribing के में अत्यंत उपयोगी है. सामान्य रूप में इस तरह के अनुसंधान, और airway के कार्यात्मक अध्ययन के रूप में, विशेष रूप में, murine मॉडल की ओर एक उल्लेखनीय बदलाव आया है. यहाँ हम प्रोटोकॉल के लिए प्रदान करते हैं<em> इन विट्रो में</em> ट्रेकिआ कसना अध्ययन murine airway में चिकनी मांसपेशी समारोह का मूल्यांकन करने के लिए.
Abstract
Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called “pharmaco-contraction coupling” of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called “excitation-contraction coupling”)4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.
Protocol
Discussion
यहाँ प्रस्तुत करने के लिए airway मांसपेशियों समारोह का आकलन करने के लिए एक शारीरिक तैयारी प्रोटोकॉल प्रदान करता है. हम आम तौर पर 3-4 अंग स्नान की तैयारी एक साथ काम है, तथापि, prepackaged सिस्टम आपूर्तिकर्ताओं कि 8 तैय…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस काम से रोकथाम में अभिनव के लिए केंद्र और श्वासनली रोग, NINDS अनुदान (NS052574), के उपचार और अस्थमा के अनुसंधान के लिए सैंडलर कार्यक्रम से एक अनुदान द्वारा वित्त पोषित किया गया.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Analogue-Digital Converter | ADInstruments | PowerLab 4/35 | |
| Carbachol (Carbamoylcholine Chloride) | Sigma-Aldrich | C4832 | 10-2 M in water (aliquots can be stored at -20°C) |
| Charting Software | ADInstrtuments | LabChart | |
| Heating Circulator | Haake | Mixer Mill MM400 | |
| Isometric Force Transducer | Kent Scientific | TRN001 | |
| Stimulator | Grass Technologies | S88 Dual Output Square Pulse Stimulator | |
| Tissue Bath | WPI | 47264 |
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