Enterotomy is a surgical procedure commonly performed in veterinary medicine. Dehiscence and enterotomy leakage are potential complications after enterotomy that can have a substantial impact on patient morbidity and mortality rates, with the rate of full-thickness dehiscence in dogs reported to be between 12% and 16%.1–6 Cadaveric tissues have commonly been used to evaluate enterotomy techniques for llamas7 and dogs.8,9 Investigators have used cadaveric intestinal tissue immediately after collection from dogs9,10; within 4 hours after collection from llamas,7 dogs,11 and horses12; within 24 hours after collection from dogs13; and within 72 hours after collection from dogs.14,15
Leak pressure can be tested on occluded intestinal segments by use of fluid or air pressure measured with a transducer. Intraluminal pressure at which leakage of fluid from the incision line is detected is regarded as the leak pressure.7–9,16 It is assumed that postmortem autolysis substantially alters intestinal tissue integrity; thus, it is believed that fresh cadaveric tissues are needed to accurately mimic in vivo conditions. The accepted method for studies of intestinal leak pressure is constrained to the use of fresh tissues, but the reliability of results for intestinal tissues collected > 5 hours after an animal has died has not been evaluated.
The objective of the study reported here was to evaluate whether enterotomy leak pressure could reliably be obtained from fresh, cooled, and fresh-frozen porcine jejunal segments. Our hypothesis was that there would be no difference in the leak pressure among fresh, cooled, or frozen-thawed porcine jejunum.
Materials and Methods
Sample
Samples of jejunum were harvested from cadavers of 3 juvenile female pigs immediately after the pigs were euthanized. Pigs were between 62 and 72 days of age at the time of euthanasia and weighed between 13.5 and 24.8 kg (mean body weight, 18.5 kg). The pigs had been healthy and were euthanized for reasons unrelated to this study.
Procedures
Three sections (approx length, 40 cm) of the mid jejunum were collected from each pig and arbitrarily assigned to 1 of 3 treatment groups (fresh, cooled, and frozen-thawed). For each group, four 8-cm sections were created from each of the 3 longer sections of jejunum; thus, there was a total of 12 segments/group. The mesentery was carefully excised to avoid distortion of the circumference measurement and to prevent bunching of the intestine.
Jejunum in the fresh treatment group was placed in a plastic baga that contained LRS and was transported to our laboratory. Enterotomy and leak pressure testing were completed within 4 hours after euthanasia of the pig and collection of tissue. Jejunum in the cooled treatment group was placed in another plastic baga that contained LRS and stored in a refrigerator at 5°C overnight (approx 16 hours). Enterotomy and leak pressure testing were completed within 4 hours after removal of tissue from the refrigerator the following morning. Jejunum in the frozen-thawed treatment group was placed in a plastic baga that did not contain LRS and stored at −12°C for 8 days. The segments were stored without LRS to minimize thawing time. Segments were removed from the freezer, placed in LRS, and thawed to room temperature (23°C) within 1 hour. Enterotomy and leak pressure testing were completed within 4 hours after the segments were thoroughly thawed.
Jejunal segments were flushed with LRS immediately prior to enterotomy and leak pressure testing and placed in LRS until use. Each 8-cm segment was suspended and occluded with Rochester Pean forceps attached to ring stands (distance between ring stands, 6 cm). A No. 11 scalpel blade was used to make a stab incision on the antimesenteric border of each segment. The incision was then elongated with Metzenbaum scissors to create a longitudinal enterotomy 2 cm in length. The ruler on a scalpel handle was used to confirm length of the enterotomy. All incisions were sutured in a simple continuous pattern by use of 4–0 poliglecaprone with a taper needle swaged to 1 end of each suture.b
Leak pressure was determined after closure of each enterotomy. The jejunal segment remained occluded with Rochester Pean forceps, and two 18-gauge, 1.25-in IV cathetersc were inserted through the intestinal wall into the jejunal lumen at each end of the segment; catheters were inserted adjacent to the occluding forceps (Figure 1). The 18-gauge catheter on the left side of the segment was connected via standard IV tubing to a pressure transducer, transducer amplifier,d and pressure monitoring system.e The transducer and IV tubing were filled with LRS and purged of air, and the transducer was calibrated by use of a manometer.f The manometer then was used to record 2 amounts of pressure, and results were compared with 2 known pressure amounts selected on the pressure monitoring system. The 18-gauge catheter on the right side of the segment was connected to a syringe pumpg via another set of IV tubing. Then, LRS was infused into the segment at a rate of 900 mL/h by use of the syringe pump. The pressure monitoring system had a pressure curve display that was monitored by investigators during infusion of LRS into the jejunal segment. Failure of the incision line was defined as leakage of fluid from the incision line and observation of a concurrent decrease in pressure on the pressure curve display (Figure 2). Pressure at which the incision failed was recorded and defined as the leak pressure.
Photograph of a fresh segment of porcine cadaveric jejunum after enterotomy closure (2-cm-long incision on the antimesenteric border) with a simple continuous pattern (arrow). The segment is suspended between Rochester Pean forceps attached to ring stands. An 18-gauge IV catheter has been inserted into the jejunal lumen at the left (a) and right (b) ends of the segment. Catheter a was connected to a pressure transducer, and catheter b was connected to a syringe pump.
Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.576
Photograph of a fresh segment of porcine cadaveric jejunum after enterotomy closure (2-cm-long incision on the antimesenteric border) with a simple continuous pattern (arrow). The segment is suspended between Rochester Pean forceps attached to ring stands. An 18-gauge IV catheter has been inserted into the jejunal lumen at the left (a) and right (b) ends of the segment. Catheter a was connected to a pressure transducer, and catheter b was connected to a syringe pump.
Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.576
Photograph of a fresh segment of porcine cadaveric jejunum after enterotomy closure (2-cm-long incision on the antimesenteric border) with a simple continuous pattern (arrow). The segment is suspended between Rochester Pean forceps attached to ring stands. An 18-gauge IV catheter has been inserted into the jejunal lumen at the left (a) and right (b) ends of the segment. Catheter a was connected to a pressure transducer, and catheter b was connected to a syringe pump.
Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.576
Photograph of a segment of porcine cadaveric jejunum during leak pressure testing. A drop of LRS (arrow) is visible on the mesenteric aspect of the jejunum, which is evidence of leakage from the incision line.
Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.576
Photograph of a segment of porcine cadaveric jejunum during leak pressure testing. A drop of LRS (arrow) is visible on the mesenteric aspect of the jejunum, which is evidence of leakage from the incision line.
Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.576
Photograph of a segment of porcine cadaveric jejunum during leak pressure testing. A drop of LRS (arrow) is visible on the mesenteric aspect of the jejunum, which is evidence of leakage from the incision line.
Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.576
One investigator (RCB) created all incisions, performed all enterotomy closures, and conducted all leak tests. That same investigator also recorded the data. One month before the study was conducted, that investigator conducted preliminary experiments and became proficient with the suturing technique.
Statistical analysis
Mean leak pressure was compared among treatment groups. Data were analyzed by use of a generalized linear model ANOVA, with leak pressure as the dependent variable and pig (included as a random factor), jejunal segment (nested within pig), and group (fresh, cooled, or frozen-thawed) as independent variables. When significant differences were detected, post hoc pairwise comparisons were performed by use of the Bonferroni method. All analyses were performed with commercial software.h Values of P < 0.05 were considered significant for all analyses.
Results
One of the 18-gauge catheters was dislodged during testing of a segment in the cooled treatment group. The catheter was carefully reinserted and leak pressure testing performed. Leak pressure for that segment was 98.61 mm Hg; that value was included in the statistical analysis. Univariate analysis of the mean data revealed no significant differences in mean leak pressure among pigs (P = 0.30) or jejunal segments (P = 0.70).
Mean ± SD leak pressure for the fresh, cooled, and frozen-thawed treatment groups were 68.3 ± 23.7 mm Hg (range, 24.6 to 118.8 mm Hg), 55.3 ± 28.1 mm Hg (range 10. 0 to 111.4 mm Hg), and 14.4 ± 14.8 mm Hg, (range, 2.1 to 62 mm Hg), respectively. The ANOVA revealed a significant (P = 0.01) difference in mean leak pressure among the 3 treatment groups. Post hoc pairwise comparisons performed by use of the Bonferroni method revealed that frozen-thawed segments had a significantly lower mean leak pressure than did fresh or cooled segments; however, there was no significant difference in mean leak pressure between fresh and cooled segments (Figure 3).
Box-and-whisker plots of leak pressures for fresh, cooled, and frozen-thawed jejunal segments (n = 12 segments/treatment group). Each box represents the 25th to 75th percentiles, the horizontal line in each box is the mean, whiskers represent the SD, and circles are outliers. *Mean value differs significantly (P < 0.05) from the mean value of the other segments.
Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.576
Box-and-whisker plots of leak pressures for fresh, cooled, and frozen-thawed jejunal segments (n = 12 segments/treatment group). Each box represents the 25th to 75th percentiles, the horizontal line in each box is the mean, whiskers represent the SD, and circles are outliers. *Mean value differs significantly (P < 0.05) from the mean value of the other segments.
Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.576
Box-and-whisker plots of leak pressures for fresh, cooled, and frozen-thawed jejunal segments (n = 12 segments/treatment group). Each box represents the 25th to 75th percentiles, the horizontal line in each box is the mean, whiskers represent the SD, and circles are outliers. *Mean value differs significantly (P < 0.05) from the mean value of the other segments.
Citation: American Journal of Veterinary Research 79, 5; 10.2460/ajvr.79.5.576
Discussion
Physiologic intraluminal pressures reach 25 mm Hg during peristalsis in dogs.6 The mean leak pressures of 68.3 and 55.3 mm Hg for fresh and cooled segments of the study reported here were much higher than that value. Although mean leak pressures were different between fresh and cooled jejunal segments, the values did not differ significantly. Mean leak pressures for fresh and cooled cadaveric jejunal segments indicated that leak pressure studies may be performed on cooled intestine up to 24 hours after sample collection with minimal effects of autolysis. Frozen-thawed jejunal segments had a mean leak pressure of 14.4 mm Hg, which was below the maximum physiologic pressure. This would suggest that frozen-thawed tissue loses viability and is not reliable for leak pressure studies. Investigators for a study9 of dogs found that there was no difference between leak pressures ex vivo versus in vivo; however, additional studies should be conducted to compare leak pressure at time of collection with cooled samples at various time points after collection.
A possible weakness of the present study was the small sample size, which was unavoidable because a single investigator performed all enterotomies in a limited time period. However, a significant difference was found among treatment groups. Post hoc pairwise comparison revealed no significant difference in mean leak pressure between fresh and cooled segments. A post hoc sample size calculation for the comparison of mean leak pressure between fresh and cooled segments (difference in means, 13 mm Hg; SD, 25 mm Hg; power, 0.80; and α, 0.05) suggested that 60 segments/treatment group would have been needed to adequately assess significant differences in leak pressure between fresh and cooled segments. However, it is clear from the data that freezing and thawing of segments significantly impacted leak pressure because the mean leak pressure of frozen-thawed segments was approximately 40 and 50 mm Hg less than that of cooled or fresh segments, respectively.
Porcine cadaveric jejunum was used because of its availability and the fact it is comparable in size to canine jejunum. Although overall jejunal size in regard to circumference was comparable to that of canine jejunum, the porcine submucosal layer is relatively thin, which has been observed in other intestinal experiments conducted on porcine intestine.17 The thin submucosa could have contributed to overall lower reported leak pressures, compared with leak pressures for canine intestinal tissues. In 1 study15 conducted to evaluate barbed and nonbarbed suture material inserted in a continuous pattern in dogs, maximal intraluminal leak pressures for nonbarbed suture were between 169.8 and 179.3 mm Hg, whereas the maximal leak pressure recorded in the study reported here was 118.8 mm Hg. Lower leak pressures were also reported in another porcine study17 in which investigators found a mean leak pressure of 27 mm Hg for nonbarbed suture material inserted in a continuous pattern.
Variations in the amount of time to test all 12 segments within each enterotomy group could have played a role in the lack of significant differences between fresh and cooled segments. In the frozen-thawed group, storage of frozen tissue without LRS could have led to variation in tissue strength. Results of preliminary experiments revealed that test samples frozen in LRS required an excessive amount of time to thaw. Because of time constraints during data collection in the present study, we elected to freeze jejunal segments without the addition of LRS. If a similar study were to be performed in the future, investigators should consider wrapping the segments in LRS-soaked gauze to avoid differences in methods between cooled and frozen-thawed segments. Additionally, a study could be performed with frozen tissue stored in LRS versus frozen tissue wrapped in LRS-soaked gauze to determine whether there were significant effects on tissue strength.
Dislodgement of 1 of the catheters and subsequent reinsertion could have resulted in errors in data collection. However, data for the segment in the cooled treatment group was not eliminated because the leak pressure of that segment was much higher than physiologic leak pressure, leak pressure for the segment correlated with leak pressures of other segments in the cooled treatment group, and no visible fluid leakage was evident from the catheter site after reinsertion. Therefore, we believed it unlikely that dislodgement and reinsertion of the catheter affected leak pressure for that segment.
The advantage of only 1 investigator performing the experiments was consistency in incisional closures and evaluation of leak pressure across all segments. A disadvantage was the possibility of observer bias because the investigator was aware of the treatment group of each segment during testing. Ideally, an investigator who had no knowledge of the treatment group for each segment (ie, blinded) would perform the leak pressure testing; however, it would still be possible for a blinded investigator to be biased because the cooled and frozen-thawed segments were visibly more autolyzed than the fresh tissue.
The pressure monitoring system and fluid infusion tubing were purged of air before leak pressure testing. However, there was still likely a small amount of air present within the jejunal segments after enterotomy closure. In future studies, this air could be eliminated by unclamping the forceps from the segment after enterotomy closure, digitally depressing the segment to remove any air, and reclamping the segment with the forceps. The continuous suture pattern would then maintain an airtight seal to prevent air leakage, similar to prevention of fluid leakage. The authors were not aware of any method that could have been used to identify air leakage from segments before fluid leakage during pressure testing. In future studies, segments should be submerged in a solution to detect air leakage.
Our hypothesis was that there would be no difference in leak pressure among the 3 treatment groups. We rejected the hypothesis on the basis that there was a significant difference in mean leak pressure of the fresh or cooled jejunal segments and the frozen-thawed jejunal segments.
Harvested jejunal segments cooled overnight may be used for intestinal leak pressure testing, whereas frozen-thawed segments should not be used. Additional studies could be conducted to help determine more precisely when there is a loss in testing viability of cooled intestine. Loss of viability would be defined as a significant difference from the control segment (leak pressure data obtained immediately after collection of tissues) and that was less than the maximal intraluminal peristaltic pressure of 25 mm Hg.
Acknowledgments
No third-party funding or support was received in connection with this study or the writing or publication of the manuscript. The authors declare that there were no conflicts of interest.
The authors thank Pam Thorne for technical assistance and Dr. Harold Laughlin for use of laboratory space and equipment.
ABBREVIATIONS
| LRS | Lactated Ringer solution |
Footnotes
Ziplock, SC Johnson & Son Inc, Racine, Wis.
Y304 4–0 Monocryl Violet 27” RB-1 taper, Ethicon, Somerville, NJ.
SurfLo, Terumo Medical Corp, Somerset, NJ.
ETH 400, CB Sciences Inc, Dover, NH.
MacLab 4SP, AD Instruments, Colorado Springs, Colo.
Traceable manometer, Fisher Scientific Co, Pittsburgh, Pa.
Medfusion model 3500, Smiths Medical, Saint Paul, Minn.
NCSS 97, Number Cruncher Statistical Software, Kaysville, Utah.
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