Fabrikation og Implantation af Miniature Dual-element strain gauges til måling<em> In Vivo</em> Mave Sammentrækninger i gnavere.
Summary
The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in rodents.
Abstract
Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an area of productive research with over 1,400 published animal studies in just the last 5 years. Numerous techniques have been developed for quantifying smooth muscle activity of the stomach, small intestine, and colon. In vitro and ex vivo techniques offer powerful tools for mechanistic studies of GI function, but outside the context of the integrated systems inherent to an intact organism. Typically, measuring in vivo smooth muscle contractions of the stomach has involved an anesthetized preparation coupled with the introduction of a surgically placed pressure sensor, a static pressure load such as a mildly inflated balloon or by distending the stomach with fluid under barostatically-controlled feedback. Yet many of these approaches present unique disadvantages regarding both the interpretation of results as well as applicability for in vivo use in conscious experimental animal models. The use of dual element strain gages that have been affixed to the serosal surface of the GI tract has offered numerous experimental advantages, which may continue to outweigh the disadvantages. Since these gages are not commercially available, this video presentation provides a detailed, step-by-step guide to the fabrication of the current design of these gages. The strain gage described in this protocol is a design for recording gastric motility in rats. This design has been modified for recording smooth muscle activity along the entire GI tract and requires only subtle variation in the overall fabrication. Representative data from the entire GI tract are included as well as discussion of analysis methods, data interpretation and presentation.
Introduction
Eksperimentelle studier, der optager in vivo gastrointestinal (GI) motilitet på tværs af en række eksperimentelle betingelser stadig et magtfuldt redskab til at forstå de underliggende normale og patofysiologiske processer, der er nødvendige for næringsstof homeostase. Traditionelt talrige eksperimentelle metoder, nogle med ligheder til dem, der findes i klinisk praksis 1, er blevet anvendt til direkte at kvantificere ændringer i GI sammentrækningshastighed 2-5 intraluminale tryk 6, 7 eller GI transit af ikke-absorberbare markører 8, 9 eller stabile isotoper 10-12. Hver af disse teknikker har unikke fordele og ulemper, som er blevet behandlet tidligere i litteraturen. For eksempel har nytten af ballonen manometri at kvantificere trykændringer sat spørgsmålstegn skyldes den iboende overholdelse af ballonen materiale, mens mave genvinding af absorberbare markører kræver euthanizing den eksperimentelle animal for et enkelt datapunkt. For nylig har anvendelsen og validering af en miniature arterietryk kateter blevet rapporteret, der tilbyder en ikke-kirurgisk metode til overvågning gastrisk kontraktilitet i rotter og mus 3. Mens en orogastrisk placeret tryktransducer eliminerer effektivt forstyrrende variable på gastrointestinal funktion ved at undgå invasive kirurgiske procedurer, en sådan fremgangsmåde er kun egnet til bedøvede præparater. Endvidere har manglen på visuelle vejledning ikke tillade ensartet placering af transduceren i specifikke områder af maven. Som sådan er denne ansøgning begrænset til maven eller tyktarmen siden visualisering, kombineret med den relativt stive transduceren ledning inden i duodenum eller ileum er ikke en mulighed.
Tilsvarende har biomagnetic vekselstrøm biosusceptometry (ACB) teknik er valideret for GI sammentrækning analyse 4. Mens ACB teknik giver en noninvasiv approach til måling af gastrointestinale sammentrækninger ACB lider af en lignende begrænsning, at anvendelsen af indtaget magnetisk mediedetektering tillader ikke nøjagtig registrering af specifikke områder af mavetarmkanalen. Denne begrænsning kan overvindes ved kirurgisk implantation af magnetiske markører. Ikke desto mindre ACB teknik kræver, at dyret bedøves til dataindsamling.
Ultrasonomicrometry har været ansat i nogle GI undersøgelser 13, 14 med henblik på at drage fordel af den lille størrelse, rumlige og tidsmæssige fordele ved piezoelektriske krystal sender / modtagere. Bølger af gastrisk glatmuskelsammentrækning er ikke en høj-frekvens begivenhed og forekommer med en hastighed på cirka 3 – 5 cyklusser / min. Derfor kan de tidsmæssige fordele sonomicrometry være unødvendigt at retfærdiggøre udgifterne. Desuden mens lineær bevægelse præcist måles med sonomicrometry, begrænsninger er blevet fremlagt vedrørende præcis mave datafortolkningsproblemer, der måtte opstå som følge af at implantere et utilstrækkeligt antal krystaller 14.
Baseret på den oprindelige design af Bass og kolleger 2, 15 denne visualiseret protokol mere fuldstændigt dokumenterer trin-for-trin fabrikation og eksperimentel anvendelse af miniature dobbelt element, strain gauges, der besidder høj følsomhed og fleksibilitet til optagelse glatte muskelsammentrækninger langs hele GI tarmkanalen. Dimensionerne af strain gage elementer er velegnede til alle gnaver ansøgning, da følsomhed og størrelsen af det færdige stamme gage er mest afhængige af silikone ark indkapsler elementerne. Disse strain gauges er let tilpasses for akut og kronisk anvendelse i bedøvede og frit opfører forsøgsdyrsmodellerne hvilket giver en enkelt teknik til kvantificering glatte muskelsammentrækninger.
Protocol
Representative Results
Discussion
De procedurer, der præsenteres her tillade enkelte laboratorier at fremstille følsomme miniature strain gauges til biologiske applikationer, herunder, men ikke begrænset til, gastrointestinal motilitet i små forsøgsdyr. Da den kommercielle fremstilling af disse strain gauges er ophørt, er laboratorier undersøger gastrointestinal funktion er begrænset til andre teknikker, som kan ikke tillade det fulde spektrum af eksperimentelle programmer, der er til rådighed. Denne rapport giver en opdateret og mere detaljere…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forskningsmidler modtaget gennem National Institute of Neurologiske og Stroke (NS049177 og NS087834). Forfatterne ønsker at anerkende de intellektuelle bidrag fra den afdøde Dr. Paul Bass og hans kolleger i det oprindelige design af strain gauges; og Carol Tollefsrud til fremstilling og markedsføring af strain gauges, indtil ophør af produktion i 2010, samt for hendes indsigtsfulde korrespondance.
Materials
| Strain gage element | Micro-Measurements (Vishay Product Group) | EA-06-031-350 | Linear pattern, foil, stress analysis strain gage (2 required) www.vishaypg.com/micro-measurements/ or http://www.vishaypg.com/docs/11070/031ce.pdf |
| epoxy-phenolic adhesive | M-bond 610 | General purpose adhesive for bonding strain gage elements | http://www.vishaypg.com/docs/11024/wirecable.pdf |
| 3 conductor insulated wire | 336-FTE | Fine gage, flexible general purpose wire | http://www.vishaypg.com/docs/11024/wirecable.pdf |
| Flux and rosin solvent kit | FAR-2 M-Flux AR kit | Liquid solder flux | http://www.vishaypg.com/docs/11023/soldacce.pdf |
| Solder | 361A-20R-25 | Optimized and recommended for strain gage applications | http://www.vishaypg.com/docs/11023/soldacce.pdf |
| Gold socket connector | PlasticsOne | E363/0 | Socket contact for electrode pedestal http://www.plastics1.com/PCR/Catalog/Item.php?item=407 |
| Electrode pedestal | MS363 | Secure platform for wire contacts | http://www.plastics1.com/PCR/Catalog/Item.php?item=499 |
| 6-wire cable | 363 PLUG W/VINYL SL/6 | Pre-fabricated vinyl-coated cable (in customized lengths) with plug adaptor to match electrode pedestal and tinned solder lugs on terminal end | |
| Silicone rubber casting compound | EIS electrical products | Elan Tron E211 | Potting medium for gage/wire solder joints http://www.eis-inc.com |
| HOTweezers | Meisei Corporation | Model 4B | Wire insulation strippers http://www.impexron.us |
| Soldering station | Weller (Apex Tool Group) | WES 51 | High quality soldering equipment http://www.apexhandtools.com/weller/index.cfm |
| Available through http://www.eis-inc.com or http://www.amazon.com | |||
| Silicone sheet | Trelleborg Sealing Solutions Northborough-Life Sciences | Pharmelast 20-20 | Encapsulating strain gauge elements 10 B Forbes Road Northborough, MA 01532 (800) 634-2000 |
| Amplifier | Experimetria Ltd | AMP-01-SG | http://experimetria.com/Biological_amplifiers.php |
References
- Szarka, L. A., Camilleri, M. Methods for measurement of gastric motility. Am. J. Physiol. Gastrointest. Liver Physiol. 296 (3), G461-G475 (2009).
- Pascaud XB, F. A. U., Genton, M. J., Bass, P. A miniature transducer for recording intestinal motility in unrestrained chronic rats. Am. J. Physiol. Endocrinol. Metab. Gastrointest. Physiol. 4 (5), 532-538 (1978).
- Gourcerol, G., Adelson, D. W., Million, M., Wang, L., Tache, Y. Modulation of gastric motility by brain-gut peptides using a novel non-invasive miniaturized pressure transducer method in anesthetized rodents. Peptides. 32 (4), 737-746 (2011).
- Américo, M. F., et al. Validation of ACB in vitro and in vivo as a biomagnetic method for measuring stomach contraction. Neurogastroenterol. Motil. 22 (12), 1340-1374 (2010).
- Fujitsuka, N., Asakawa, A., Amitani, H., Fujimiya, M., Inui, A. Chapter Eighteen – Ghrelin and Gastrointestinal Movement. Ghrelin and Gastrointestinal Movement. , 289-301 (2012).
- Monroe, M. J., Hornby, P. J., Partosoedarso, E. R. Central vagal stimulation evokes gastric volume changes in mice: a novel technique using a miniaturized barostat. Neurogastroenterol. Motil. 16 (1), 5-11 (2004).
- Herman, M. A., et al. Characterization of noradrenergic transmission at the dorsal motor nucleus of the vagus involved in reflex control of fundus tone. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294 (3), 720-729 (2008).
- Gondim, F. A., et al. Complete cervical or thoracic spinal cord transections delay gastric emptying and gastrointestinal transit of liquid in awake rats. Spinal Cord. 37 (11), 793-799 (1999).
- Van Bree, S. H. W., et al. Systemic inflammation with enhanced brain activation contributes to more severe delay in postoperative ileus. Neurogastroenterol. Motil. 25 (8), 540-549 (2013).
- Qualls-Creekmore, E., Tong, M., Holmes, G. M. Gastric emptying of enterally administered liquid meal in conscious rats and during sustained anaesthesia. Neurogastroenterol. Motil. 22 (2), 181-185 (2010).
- Qualls-Creekmore, E., Tong, M., Holmes, G. M. Time-course of recovery of gastric emptying and motility in rats with experimental spinal cord injury. Neurogastroenterol. Motil. 22 (1), 62 (2010).
- Choi, K. M., et al. Determination of gastric emptying in nonobese diabetic mice. Am. J. Physiol. Gastrointest. Liver Physiol. 293 (5), G1039-G1045 (2007).
- Adelson, D. W., Million, M., Kanamoto, K., Palanca, T., Tache, Y. Coordinated gastric and sphincter motility evoked by intravenous CCK-8 as monitored by ultrasonomicrometry in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 286 (2), G321-G332 (2004).
- Xue, L., et al. Effect of modulation of serotonergic, cholinergic, and nitrergic pathways on murine fundic size and compliance measured by ultrasonomicrometry. Am. J. Physiol. Gastrointest. Liver Physiol. 290 (1), G74-G82 (2005).
- Bass, P., Wiley, J. N. Contractile force transducer for recording muscle activity in unanesthetized animals. J. Appl. Physiol. 32 (4), 567-570 (1972).
- Holmes, G. M., Browning, K. N., Tong, M., Qualls-Creekmore, E., Travagli, R. A. Vagally mediated effects of glucagon-like peptide 1: in vitro and in vivo gastric actions. J. Physiol. 587 (19), 4749-4759 (2009).
- Tong, M., Qualls-Creekmore, E., Browning, K. N., Travagli, R. A., Holmes, G. M. Experimental spinal cord injury in rats diminishes vagally-mediated gastric responses to cholecystokinin-8s. Neurogastroenterol. Motil. 23 (2), e69-e79 (2011).
- Miyano, Y., et al. The role of the vagus nerve in the migrating motor complex and ghrelin- and motilin-induced gastric contraction in suncus. PLoS ONE. 8 (5), e64777 (2013).
- Holmes, G. M., Rogers, R. C., Bresnahan, J. C., Beattie, M. S. Thyrotropin-releasing hormone (TRH) and CNS regulation of anorectal motility in the rat. J Auton. Nerv. Syst. 56, 8-14 (1995).
- Ormsbee, H. S., Bass, P. Gastroduodenal motor gradients in the dog after pyloroplasty. Am. J. Physiol. 230, 389-397 (1976).
- Fukuda, H., et al. Impaired gastric motor activity after abdominal surgery in rats. Neurogastroenterol. Motil. 17 (2), 245-250 (2005).
- Browning, K. N., Babic, T., Holmes, G. M., Swartz, E., Travagli, R. A. A critical re-evaluation of the specificity of action of perivagal capsaicin. J. Physiol. 591 (6), 1563-1580 (2013).
