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.
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.
Experimental studies that record in vivo gastrointestinal (GI) motility across a number of experimental conditions remain a powerful tool for understanding the underlying normal and pathophysiological processes necessary for nutrient homeostasis. Traditionally, numerous experimental methodologies, some with similarities to those found in clinical practice 1, have been employed to directly quantify changes in GI contraction rate 2-5, intraluminal pressure 6, 7, or the GI transit of non-absorbable markers 8, 9 or stable isotopes 10-12. Each of these techniques has unique advantages and disadvantages, which have been addressed previously in the literature. For example, the utility of balloon manometry to quantify pressure changes has been questioned due to the inherent compliance of the balloon material while gastrointestinal recovery of nonabsorbable markers requires euthanizing the experimental animal for a single data point. Recently, the application and validation of a miniaturized arterial pressure catheter has been reported that offers a non-surgical method for monitoring gastric contractility in rats and mice 3. While an orogastrically placed pressure transducer effectively eliminates confounding variables on gastrointestinal function by avoiding invasive surgical procedures, such an approach is only suitable for anesthetized preparations. Furthermore, the lack of visual guidance does not permit consistent placement of the transducer within specific regions of the stomach. As such, this application is restricted to the stomach or colon since visualization, coupled with the relatively stiff transducer wire, within the duodenum or ileum is not an option.
Similarly, the biomagnetic alternate current biosusceptometry (ACB) technique has been validated for GI contraction analysis 4. While the ACB technique provides a noninvasive approach for measuring gastrointestinal contractions, ACB suffers from a similar limitation in that the use of ingested magnetic detection media does not permit precise recording of specific regions of the GI tract. This limitation can be overcome through the surgical implantation of magnetic markers. Nonetheless, the ACB technique necessitates that the animal be anesthetized for data collection.
Ultrasonomicrometry has been employed in some GI studies 13, 14 in order to take advantage of the small size, spatial, and temporal advantages of piezoelectric crystal transmitter/receivers. Waves of gastric smooth muscle contraction are not a high-frequency event and occur at a rate of approximately 3 – 5 cycles/min. Therefore, the temporal advantages of sonomicrometry may be unnecessary to justify the cost. Furthermore, while linear motion is accurately measured with sonomicrometry, limitations have been presented regarding accurate gastrointestinal data interpretation that may result from implanting an insufficient number of crystals 14.
Based upon the original designs of Bass and colleagues 2, 15 this visualized protocol more fully documents the step-by-step fabrication and experimental application of miniature, dual element strain gages that possess high sensitivity and flexibility for recording smooth muscle contractions along the entire GI tract. The dimensions of the strain gage elements are suitable for any rodent application since sensitivity and size of the finished strain gage are most dependent upon the silicone sheets encapsulating the elements. These strain gages are readily adapted for acute and chronic application in anesthetized and freely behaving laboratory animal models thereby providing a single technique for quantifying smooth muscle contractions.
All procedures followed National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee at the Penn State Hershey College of Medicine. Rats were housed using common vivarium practices. Note: This protocol uses male Wistar rats ≥8 weeks of age and initially weighing 175 – 200 g.
1. Procedures for Fabrication of Strain Gage
2. Surgical Procedures for Acute Implantation of Strain Gage
3. Representative Measurement of Gastric Contractions Following Brainstem Stimulation
Representative data from a Thiobutabarbital-anesthetized rat are shown in Figure 2. The top trace represents the gastric corpus contractions from the rat during the brainstem administration of thyrotropin releasing hormone (TRH, 100 pmol), a known motility-enhancing peptide 3, 19. It shows baseline contractions prior to the increase in phasic gastric smooth muscle activity. Note: Analysis of these peaks in gastric contractions follow the original formula devised by Ormsby and Bass 20
Motility Index= (N1x1) + (N2x2) + (N3x4) + (N4x8)
Based upon this formula, N equals the total number of peaks in a particular milligram range. Therefore, presuming that a 0 mg signal is indicative of no gastric motility, the grouping of peak-to-peak sinusoidal signals may be calculated as 25 – 50 mg, 60 – 100 mg, 110 – 200 mg and signals greater than 210 mg for N1 through N4, respectively. This formula is less sensitive to baseline tone fluctuations that naturally occur across several seconds or minutes. Such fluctuations would have to be subtracted in order to generate valid area using under the curve measurements 3.
The second trace demonstrates a reduction in baseline gastric smooth muscle tone from the same animal in response to the nitric oxide donor, sodium nitroprusside (150 µmol/kg iv). Data representing an inhibition of gastric smooth muscle activity are readily analyzed by the reduction in signal voltage between baseline and maximal response. This voltage signal can then be used to derive the equivalent static load, in grams, if the strain gage was calibrated prior to the experiment. These representative data demonstrate the bidirectional capabilities of a dual element strain gage that has been properly attached to the gastric serosa.
The third trace represents basal smooth muscle contractions recorded by a subminiature strain gage sutured to the serosal surface of the duodenum of a fasted rat. The orientation of the strain gage elements were also in parallel with the circular muscle of the duodenum.
Figure 1. Principal stages of strain gage fabrication. (A) Dual bonded elements that have been trimmed on three of four sides to final dimensions. (B) Representative ends of wires configured for attachment to gage elements (left) and terminal connectors (right). Note that dual red leads are joined only at the terminal end (arrowhead). (C) Representative placement of a strand of copper wire in proximity to fine (1.5 mm) soldering tip. Maintaining fresh solder along this junction (arrowhead) ensures sufficient heat transfer through the micro tip to melt 63% Tin: 36.65% Lead: 0.35% Antimony solder. (D) Representative extent of solder joints between wire leads and solder pads on the gage element. (E) Properly potted solder joints. (F) Representative notch in the internal silicone laminate sheet to accommodate strain gage element without deforming completed element. (G) Bonded layers of silicone sheets (three in total) forming a completed strain gage prior to final sizing. (H) Wire connections to gold plated sockets are reinforced with layers of succeedingly larger diameter shrink tubing before insertion into electrode pedestal. (I) Final shrink wrap affixing of terminal connectors and electrode pedestal. Calibration bars: (A – D), 5 mm; (E), 2 mm; & (F - I), 5 mm. Please click here to view a larger version of this figure.
Figure 2. Representative motility traces generated with fabricated dual element strain gages. Recordings made from the anterior gastric corpus during an increase in gastric contractions (top trace) and during an inhibition of gastric contractions (middle trace) and duodenum (bottom trace) of fasted rats (200 – 250 g).
The procedures presented here allow individual laboratories to fabricate sensitive miniature strain gages for biological applications including, but not limited to, gastrointestinal motility in small laboratory animals. Since the commercial manufacture of these strain gages has ceased, laboratories investigating gastrointestinal function are limited to other techniques which may not permit the full range of experimental applications that are available. This report provides an updated and more detailed description of previously described techniques 15. The text and accompanying video specifically address solutions to common pitfalls that we recognized during development and mastery of the fabrication process.
Each step, as described, presents techniques to successful fabrication. Careful attention to cleanly and securely soldering all connections as well as avoiding damage to the element with excessive heat from the soldering process are the most frequent challenges to success. The fine gage wire is prone to breaking if it is not properly reinforced with shrink tubing or silicone epoxy and will result in an absence of signal when the gage is gently flexed. A strain gage with a broken or disconnected wire in the vicinity of the gold connectors within the plastic terminal pedestal is the most common failure of a previously functional gage. Individual gages can be carefully disassembled by removing the shrink tubing in order to expose the broken wire. After resoldering the wire to the gold connector, the entire gage is reassembled with new shrink tubing.
With a bit of practice and careful attention to fabricating strain gages of uniform dimensions, affixing strain gages relative to clear landmarks (e.g., greater gastric curvature, fundus/corpus boundary), and avoiding damage to the vasculature, novice users will rapidly develop the ability to achieve consistent results.
Encapsulating the dual element in three layers of silicone creates a durable and flexible, yet highly sensitive strain gage that will last over repeated use with proper care. The high sensitivity of an unencapsulated strain gage is minimally affected by any resistance that is imparted by the silicon laminate. Thinner silicone sheets (P/N 20-05) are recommended in order to modify the gage for intestinal applications or for fabricating smaller gages for mice and discrete gut regions such as sphincters and esophagus. Extra caution is required since thinner gages have diminished resistance to tearing of the silicone sheet during implantation.
Surgical difficulties with the use of these gages often result from excessive manipulation of the visceral organs or misalignment of the gage during implantation. The former likely initiates neural and inflammatory processes that directly lead to impaired GI motility, 9, 21 though both pitfalls are easily remedied by refinement of surgical technique. This may include altering the length and starting point of the midline incision into the abdomen as well as minimizing the manipulation of the viscera during exteriorization and replacement of the stomach.
The validity and fidelity of these strain gages have been discussed previously 2, 15. We, and others, routinely measure gastric smooth muscle activity in acute, anesthetized preparations 16, 22. With adequate instrumentation, a single investigator can instrument and acquire data from up to four animals in a single day. Additionally, implantation of multiple gages within the same animal allows one to measure the relationship between adjacent, or distant, regions of the gastrointestinal tract.
In summary, the fabrication of these subminiature strain gages allows for a wider range of studies utilizing a common array of implantation techniques, instrumentation and data analysis. Among applications across the entire gastrointestinal tract, these gages allow for cross comparison of data collected from A) acute and/or chronic experimental designs; B) multiple (simultaneous) recording sites from within a single animal; and C) a wider range of experimental interventions.
The authors have nothing to disclose.
Research funding was received through the National Institute of Neurological Disorders and Stroke (NS049177 and NS087834). The authors wish to acknowledge the intellectual contributions of the late Dr. Paul Bass and his colleagues to the original design of the strain gages; and Carol Tollefsrud for the fabrication and marketing of the strain gages until the cessation of production in 2010 as well as for her insightful correspondence.
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 |