Volume of saline (0.9% NaCl) solution required to reach maximum peristaltic pressure in cadaveric intact jejunal specimens from dogs of various sizes

Tricia F. Culbertson From the Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.

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 DVM, MS
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Daniel D. Smeak From the Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.

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Sangeeta Rao
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 BVSC, PhD

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Abstract

OBJECTIVE

To compare the volume of saline (0.9% NaCl) solution required to reach a maximum intraluminal peristaltic pressure of 25 mm Hg in dogs of various sizes.

SAMPLES

25 grossly normal jejunal segments from 6 canine cadavers < 20 kg (small dogs) and 25 segments from 5 cadavers ≥ 20 kg (large dogs).

PROCEDURES

Jejunal specimens were obtained within 1.5 hours after euthanasia. Harvested tissue was transected into 12-cm-long segments, mesentery was trimmed, and each segment was measured from the antimesenteric to mesenteric serosal edges. A 10-cm segment was isolated with Doyen forceps, securing a pressure sleeve within the lumen. Intraluminal saline was infused, and the volume was recorded when a pressure of > 25 mm Hg was achieved. Data were analyzed only from specimens in which the pressure remained between 24 and 26 mm Hg for > 5 seconds.

RESULTS

Mean ± SD intestinal measurement for large dogs (17.82 ± 1.44 mm) was greater than that for small dogs (12.38 ± 1.38 mm) as was the volume of saline solution infused (17.56 ± 7.17 mL vs 3.28 ± 1.41 mL, respectively). The volume infused increased by 1.31 mL (95% CI, 1.08 to 1.18) for every 1-mm increase in intestinal measurement and by 1.06 mL (95% CI, 1.052 to 1.068) for every 1-kg increase in body weight.

CONCLUSIONS AND CLINICAL RELEVANCE

The volume of saline solution used for intestinal leak testing should be determined on the basis of patient intestinal measurement or body weight. In vivo studies are necessary to establish the optimal volume for intestinal leak testing.

Abstract

OBJECTIVE

To compare the volume of saline (0.9% NaCl) solution required to reach a maximum intraluminal peristaltic pressure of 25 mm Hg in dogs of various sizes.

SAMPLES

25 grossly normal jejunal segments from 6 canine cadavers < 20 kg (small dogs) and 25 segments from 5 cadavers ≥ 20 kg (large dogs).

PROCEDURES

Jejunal specimens were obtained within 1.5 hours after euthanasia. Harvested tissue was transected into 12-cm-long segments, mesentery was trimmed, and each segment was measured from the antimesenteric to mesenteric serosal edges. A 10-cm segment was isolated with Doyen forceps, securing a pressure sleeve within the lumen. Intraluminal saline was infused, and the volume was recorded when a pressure of > 25 mm Hg was achieved. Data were analyzed only from specimens in which the pressure remained between 24 and 26 mm Hg for > 5 seconds.

RESULTS

Mean ± SD intestinal measurement for large dogs (17.82 ± 1.44 mm) was greater than that for small dogs (12.38 ± 1.38 mm) as was the volume of saline solution infused (17.56 ± 7.17 mL vs 3.28 ± 1.41 mL, respectively). The volume infused increased by 1.31 mL (95% CI, 1.08 to 1.18) for every 1-mm increase in intestinal measurement and by 1.06 mL (95% CI, 1.052 to 1.068) for every 1-kg increase in body weight.

CONCLUSIONS AND CLINICAL RELEVANCE

The volume of saline solution used for intestinal leak testing should be determined on the basis of patient intestinal measurement or body weight. In vivo studies are necessary to establish the optimal volume for intestinal leak testing.

Introduction

Gastrointestinal surgery in companion animals is performed for various reasons, including foreign body retrieval, excision of an intestinal mass, or correction of a malposition or intussusception.1,2,3,4,5 Regardless of the technique used to repair the intestine, it is of utmost importance for surgeons to assess the integrity of the closure for gaps that may lead to leakage of intestinal contents into the abdominal cavity. Although many methods exist for assessing surgical intestinal repair sites for the presence of gaps and leakage, saline (0.9% NaCl) solution pressure leak testing (leak testing) remains one of the most commonly used methods.4,6,7,8,9,10,11,12,13

An argument against performing leak testing is the possibility of creating iatrogenic leaks owing to incorrect technique or the application of too much pressure during testing.8,10,14,15 Multiple researchers have established and reported objective parameters for use during leak testing to provide a more standardized approach. In 2 separate in vivo studies,16,17 similar volumes of saline solution were required to achieve peristaltic pressures of 15 to 25 mm Hg (20 to 34 cm H2O) following completion of enterotomies in dogs that weighed approximately 15 kg. The most appropriate needle and syringe sizes for minimizing iatrogenic leakage during leak testing were determined in another study.18 The specifics from those studies16,17,18 have subsequently been cited as foundational principles for performing leak testing in educational materials.4,6,7,8

Foreign body obstruction necessitating gastrointestinal surgery occurs more commonly in large-breed dogs than in small-breed dogs.19,20,21 However, in clinical settings, veterinarians are likely to perform gastrointestinal surgery in patients of many sizes. In dogs, the intestinal size and wall thickness vary on the basis of age, breed, weight, and health of the subject as well as the location of the gastrointestinal tract.22,23,24,25 Information regarding the volumes of saline solution required to perform leak testing in intestinal segments of various sizes is currently lacking in the veterinary literature. Therefore, the primary objective of the study reported here was to compare the volume of saline solution required to achieve a maximum intraluminal peristaltic pressure of 25 mm Hg (saline volume) in jejunal specimens among dogs of various sizes. A secondary objective was to develop a formula from that data, which could be used to determine leak-testing parameters intraoperatively in clinical settings. We hypothesized that the saline volume required to achieve peak pressure (PP) would be greater in dogs weighing > 20 kg, compared with dogs weighing < 20 kg, and that the intestinal size or patient weight could be used to create a reliable scale for calculation of the saline volume necessary for leak testing in dogs.

Materials and Methods

Specimen collection and preparation

All study protocols and procedures were reviewed and approved by the Colorado State University Veterinary Teaching Hospital Clinical Review Board and the Research Integrity and Compliance Review Office (VCS No. 2018-190). Client-owned dogs presented to the veterinary teaching hospital for euthanasia between August 30, 2019, and July 30, 2020 were considered for study enrollment, provided their owners authorized tissue collection or body use within Colorado State University's College of Veterinary Medicine and Biomedical Sciences facilities for research or teaching purposes on the euthanasia consent form. Concurrent owner authorization for an educational necropsy was not required for a dog to be considered for study inclusion. Dogs with a previously diagnosed gastrointestinal disorder, history of gastrointestinal signs (eg, vomiting or diarrhea) for ≥ 48 hours prior to euthanasia, abdominal effusion, gross intestinal abnormalities detected during tissue collection, or histologic evidence of enteral disease were not included in the study. Historical information about signs of gastrointestinal disease was obtained from the owner and recorded in the medical record. Two purpose-bred dogs from a university research colony that were euthanized for reasons unrelated to the study (Colorado State University Institutional Animal Care and Use Committee protocol No. 19-9679A) were also enrolled in the study. All dogs were euthanized by IV administration of a pentobarbital overdose. Two client-owned dogs underwent necropsy, and the necropsy reports became available 1 week after experimental testing was performed. The reports were reviewed to confirm that those 2 dogs met the study inclusion criteria. Cadavers were allocated to either a small-dog (< 20 kg) or large-dog (≥ 20 kg) group on the basis of body weight.

All jejunal tissue specimens were collected within 1.5 hours after death was confirmed. All specimens were harvested between the duodenojejunal flexure and termination of the antimesenteric vessel of the ileum. The harvested tissue was cut into 12-cm-long segments, flushed with tepid tap water 3 times, then rinsed once with and stored in physiologic saline solution until experimental testing. Segments obtained from the same cadaver were individually stored and sequentially numbered, with segment 1 originating from the most orad location (ie, closest to the duodenum). When experimental testing could not be immediately performed following tissue harvest, the jejunal specimens were refrigerated at 5 °C and allowed to come to room temperature (approx 21 °C) for 1 hour before testing. The maximum time between tissue collection and completion of experimental testing was 4 hours.

Immediately prior to testing, Metzenbaum scissors were used to trim the mesentery from both ends of the jejunal segment. The trimmed segment was laid flat on a table, and a plastic surgical ruler (Cardinal Health Inc) was used to measure the external dimensions of the intestine from the antimesenteric serosal edge to the mesenteric serosal edge at each end of the segment. That intestinal measurement represented half the circumference of the segment. For each segment, the mean of the 2 intestinal measurements was calculated and recorded for subsequent analyses. Following measurement, each jejunal segment was rinsed once with saline solution and assembled for leak testing.

Pressure testing

For each jejunal segment, an 11-cm-long, 6F pressure sleeve (Arrow Pressure Sleeve; Teleflex Inc) was placed within the lumen and a 10-cm portion of segment was isolated by use of 2 straight Doyen intestinal forceps, with the tips of the forceps placed at the mesenteric border of the segment and the ratchets secured to 2 clicks, leaving the pressure sleeve halfway in the lumen. (Figure 1). The pressure sleeve was partially secured by the placement of right-angle forceps on the jejunal segment between the end from which the pressure sleeve exited and the closest Doyen forceps. The right-angle forceps was oriented perpendicular to the segment with the tip positioned parallel to the sleeve and abutting the Doyens forceps. The pressure sleeve was additionally secured with an encircling suture of 3-0 polyglyconate (Medtronic). The entire construct was suspended from dowel rods that were placed on top of wooden platforms. A 3-way stopcock was used to connect the pressure sleeve to a saline solution–primed fluid line and 2 syringes (12-mL syringes were used for testing segments obtained from dogs < 30 kg and 20-mL syringes were used for testing segments obtained from dogs ≥ 30 kg) filled with saline solution in a fluid pump. A catheter with a pressure-sensing transducer tip (Mikro-Tip catheter transducer; Millar Instruments Inc) was placed within the pressure sleeve, with the tip extending approximately 1.5 cm past the end of the sleeve into the lumen. That catheter was attached to a computer with pressure-recording software (LabChart; ADInstruments Group), which was used to zero the pressure measured by the transducer immediately prior to initiation of testing. A checklist was used to verify that the setup was consistent for each experimental construct prior to initiation of testing.

Figure 1
Figure 1
Figure 1

Photographs of the intestinal instrumentation (A) and experimental testing setup (B) used to conduct pressure testing of canine cadaveric jejunal segments in a study conducted to compare the volume of saline (0.9% NaCl) solution required to reach a maximum intraluminal peristaltic pressure of 25 mm Hg (saline volume) in dogs of various sizes. A—Close-up photograph of a jejunal segment in which a 11-cm-long, 6F pressure sleeve surrounding a catheter with a pressure-sensing transducer tip has been inserted in the lumen and a 10-cm portion of the segment has been isolated by the use of 2 straight Doyen intestinal forceps. The pressure sleeve was secured in place with right-angle forceps and an encircling suture of 3-0 polyglyconate. B—The instrumented jejunal segment was suspended from dowel rods placed on top of wooden platforms. A fluid pump was used to infuse saline solution from 2 syringes through the pressure sleeve into the lumen at a constant rate. The catheter with the pressure-sensing transducer was connected to a computer with pressure-recording software (computer not shown).

Citation: American Journal of Veterinary Research 82, 12; 10.2460/ajvr.21.05.0066

For each segment, pressure recording was initiated followed by infusion of saline solution through the pressure sleeve and into the lumen at a rate of 1.5 mL/min for segments assigned to the small-dog group and 2.0 mL/min for segments assigned to the large-dog group. A fluid pump was used to ensure that the infusion rate was accurate and constant. When the intraluminal pressure reached approximately 30 mm Hg, infusion of the saline solution was stopped but pressure recording continued. As soon as the pressure dropped below 23 mm Hg, pressure recording was stopped and the saline volume infused was recorded. When 12-mL syringes were used, the volume was rounded to the nearest 0.2-mL increment. When 20-mL syringes were used, the volume was rounded to the nearest 0.5-mL increment. Segments were excluded from data analysis if they were the first segment to be tested during a given session, the pressure recording was uneven or erratic, droplets of fluid were observed dripping from the segment during testing, or any other steps in the process were missed.

Because our primary objective was to compare the saline solution volume between small and large dogs, we targeted an intraluminal pressure range of 24 to 26 mm Hg for data collection. Data recorded for each segment during pressure testing included the PP, time to PP, time after PP when the pressure fell below 26 mm Hg, and time after PP when the pressure last exceeded 24 mm Hg (Figure 2). The duration between PP and point at which the pressure entered the targeted pressure range (TPP) and duration that the pressure was within the targeted pressure range (TTPR) were calculated from the recorded data. A segment was excluded from statistical analyses if the TPP was > 10 seconds (indicating that too much saline solution was infused) or the TTPR was < 5 seconds (indicating that not enough saline solution was infused). A segment was considered to have reached an intraluminal pressure of 25 mm Hg if the TTPR was ≥ 5 seconds.

Figure 2
Figure 2

Illustration of a pressure tracing obtained during experimental testing of the jejunal constructs described in Figure 1, which depicts when and how key variables were recorded. The peak pressure (PP; 1), time (in seconds) at PP (2), time when intraluminal pressure fell below 26 mm Hg (3), and last time at which the intraluminal pressure was > 24 mm Hg (4) were recorded directly from the tracing. The duration between PP and the point at which the pressure entered the targeted pressure range (TPP) was calculated as point 3 to point 2, and the duration that the pressure was within the targeted pressure range (TTPR) was calculated as point 4 to point 3. *The volume of saline solution infused at PP was recorded and used for analysis purposes.

Citation: American Journal of Veterinary Research 82, 12; 10.2460/ajvr.21.05.0066

Power analysis

Data from a previously conducted pilot study were used to perform sample size calculations for the current study. The pilot study was performed on frozen-thawed cadaveric intestinal tissue specimens to generate data for a sample size calculation. Briefly, 33 intestinal segments from 5 canine cadavers with body weights that ranged between 5 and 32 kg underwent pressure testing as previously described. For the pilot study, the mean ± SD saline volume was 7.31 ± 1.75 mL for 20 segments from dogs weighing < 20 kg (n = 2) and 16.18 ± 3.84 mL for 13 segments from dogs weighing ≥ 20 kg (n = 3). The sample size calculation26 for the present study indicated that a total of 33 intestinal segments would be necessary to detect a saline solution volume difference between small dogs and large dogs, achieving a statistical power of > 90% power and 95% confidence (α = 0.05).

Statistical analysis

The data distribution for each continuous variable was assessed for normality by use of the Shapiro-Wilk test. Results were summarized with the mean ± SD for normally distributed variables and with the median (range) for nonnormally distributed variables. Data for nonnormally distributed variables underwent a logarithmic transformation to approximate a normal distribution for linear regression analysis. Student t tests were used to compare cadaver body weight, saline volume, the intestinal measurement, PP, TPP, and TTPR between the small- and large-dog groups. Linear mixed models were used to assess the respective relationships between saline volume and intestinal measurement, body weight, PP, TPP, and TTPR; between body weight and intestinal measurement; between PP and TPP and TTPR; and between TPP and TTPR. Each model included a random effect to account for the fact that multiple jejunal segments were tested from each dog (ie, repeated measures). The Akaike Information Criterion (AIC) was used to assess the model fit for the 2 linear mixed models assessing the association between saline volume and intestinal measurement and body weight. The model with the lowest AIC was determined to provide the best fit for the data. Values of P < 0.05 were considered significant for all analyses. All analyses were performed with commercially available statistical software (SAS version 9.4; SAS Institute Inc) by 1 investigator (SR). Box-and-whisker plots were created with another statistical software program (Prism version 9.0.0 for Windows; GraphPad Software) by another investigator (TFC).

Results

Dogs from which jejunal specimens were obtained

Fourteen dogs were initially enrolled in the study, but 3 dogs were subsequently excluded from the study owing to the discovery of abdominal effusion (n = 2) or gross intestinal abnormalities (1) during tissue collection. Two dogs included in the study underwent necropsy. One of those dogs was determined to have megaesophagus and pulmonary edema and the other had hepatic nodular hyperplasia, interstitial nephritis, and cardiac valvular endocardiosis. The specimens tested were obtained from 11 dogs with grossly normal intestines. Six dogs weighed < 20 kg and were assigned to the small-dog group, and 5 dogs weighed ≥ 20 kg and were assigned to the large-dog group. No more than 5 jejunal segments were collected from each dog, and there were 25 jejunal specimens tested and analyzed for each group. For 3 dogs in the small-dog group, only 3 or 4 specimens were included in the analysis even though 5 samples were tested because the TTPR did not meet the 5-second minimum requirement for data analysis.

Dogs were euthanized for 1 or more of the following reasons: hindlimb weakness progressing to nonambulatory status (n = 4), declining quality of life (n = 3), dyspnea induced by an intrathoracic mass (1), syncopal episodes (1), diffuse neuromuscular disease (1), congestive heart failure (1), or as part of another research study (2). The study population included 3 mixed-breed dogs, 2 Yorkshire Terriers, and 1 each of the following breeds: Anatolian Shepherd, Boxer, Cairn Terrier, Chihuahua, Labrador Retriever, and Schnauzer.

The mean ± SD age was 10.2 ± 6.4 years for all 11 study dogs and 12.2 ± 5.0 years for the 9 client-owned dogs. The mean ± SD body weight was 20.48 ± 15.6 kg (median, 15.8 kg; range, 2.8 to 47.7 kg) for all 11 dogs and was significantly (P < 0.001) lower for the small-dog group (7.87 ± 4.45 kg [median, 6.85 kg; range, 2.8 to 15.8 kg]) compared with the large-dog group (35.62 ± 7.67 kg [median, 35.5; range, 26.9 to 47.7 kg]; Figure 3). The mean ± SD intestinal measurement was 14.85 ± 3.14 mm (median, 14 mm; range, 10.83 to 19 mm) for all 11 dogs and was significantly (P < 0.001) lower for the small-dog group (12.38 ± 1.38 mm), compared with the large-dog group (17.82 ± 1.44 mm).

Figure 3
Figure 3

Box-and-whisker plots that depict the difference between the 2 experimental groups in the study described in Figure 1 in terms of body weight (A), saline volume (B), intestinal measurement (C), PP (D), TPP (E), and TTPR (F). Twenty-five jejunal segments obtained from the cadavers of 6 dogs weighing < 20 kg (small dogs) and 25 jejunal segments obtained from 5 dogs weighing ≥ 20 kg (large dogs) underwent pressure testing and were eligible for data analysis; ≤ 5 segments were analyzed from each dog. For each plot, the horizontal line within the box represents the median, the plus sign represents the mean, the lower and upper edges of the box represent the interquartile (25th to 75th percentile) range, and the whiskers delimit the minimum and maximum values. Values of P ≥ 0.05 were considered significant. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 82, 12; 10.2460/ajvr.21.05.0066

Pressure testing

The saline volume for jejunal segments in the small-dog group (mean ± SD, 3.28 ± 1.41 mL) was significantly (P < 0.001) lower than that for jejunal segments in the large-dog group (17.56 ± 7.17 mL). The mean ± SD PP in the small-dog group (31.14 ± 1.6 mm Hg) was significantly (P = 0.018) greater than that for the large-dog group (29.03 ± 1.19 mm Hg). However, the TPP and TTPR did not differ significantly between the 2 groups (Figure 3).

Linear mixed modeling

The linear mixed model results were summarized (Table 1). Saline volume was significantly associated with the intestinal measurement (P < 0.001), body weight (P < 0.001), PP (P = 0.022), and TTPR (P = 0.012). The saline volume increased by 1.13 mL (95% CI, 1.08 to 1.18 mL) for each 1-mL increase in intestinal measurement and increased by 1.06 mL (95% CI, 1.05 to 1.07 mL) for each 1-kg increase in patient body weight. The AIC of the model for saline volume and intestinal measurement (35.7) was lower than the AIC of the model for saline volume and body weight (39.9), which suggested that the model with intestinal measurement provided a better fit for the present data. There was a significant (P < 0.001) positive association between body weight and intestinal measurement. For each 1-kg increase in body weight, the intestinal measurement increased by 0.19 mm (95% CI, 0.14 to 0.23 mm).

Table 1

Summary of the results for multiple linear mixed models that depict the association between various variables assessed in a study conducted to compare the volume of saline (0.9% NaCl) solution required to reach a maximum intraluminal peristaltic pressure of 25 mm Hg (saline volume) in cadaveric jejunal specimens obtained from dogs of various sizes.

Outcome Fixed effect Association (95% CI) P value AIC
Saline volume (mL) Intestinal measurement (mm) 1.13 (1.08–1.18) < 0.001 35.7
Saline volume (mL) Body weight (kg) 1.06 (1.05–1.07) < 0.001 39.9
Intestinal measurement (mm) Body weight (kg) 0.19 (0.14–0.23) < 0.001
Saline volume (mL) PP (mm Hg) –1.11 (–1.22 to–1.02) 0.022
TPP (s) PP (mm Hg) 0.89 (0.64–1.14) < 0.001
TTPR (s) PP (mm Hg) NA 0.23
Saline volume (mL) TPP (s) NA 0.403
TTPR (s) TPP (s) 1.10 (1.06–1.14) < 0.001
Saline volume (mL) TTPR (s) 1.05 (1.01–1.09) 0.012

The association represents the change in the outcome variable for each 1-unit increase in the fixed effect; therefore, the units for the association are the same as the units for the outcome variable.

— = Not calculated. AIC = Akaike Information Criterion. NA = Not applicable. PP = Peak pressure. TPP = Duration between PP and the point at which the pressure entered the targeted pressure range (24 to 26 mm Hg). TTPR = Duration that the pressure was within the targeted pressure range.

Discussion

The primary objective of the study reported here was to compare the volume of saline solution required to achieve a maximum intraluminal peristaltic pressure of 25 mm Hg (saline volume) in jejunal specimens among dogs of various sizes. The purpose for conducting the study was to investigate whether the saline volume used for intestinal leak testing should be determined on the basis of patient size. Results indicated that, for intact jejunal specimens, the saline volume required for dogs weighing ≥ 20 kg was significantly greater than that for dogs weighing < 20 kg. For in vivo studies, the volumes of saline solution required to achieve various intraluminal pressures during leak testing following closure of enteric biopsy sites were assessed in 38 dogs with a mean weight of 15.3 kg16 and 19 dogs with a mean weight of 15.7 kg.17 However, the investigators of those studies16,17 did not analyze their data relative to patient weight. This has important clinical ramifications because if surgeons use a standard volume of saline solution to perform leak testing following intestinal repair regardless of intestinal size, it is possible that an inappropriate volume may be used and not accurately identify leaks. The current veterinary literature lacks prospective studies regarding the association between the identification of intestinal leaks during intraoperative leak testing and postoperative dehiscence rates. Results of a retrospective case series report27 indicate that intraoperative leak testing had no significant association with postoperative dehiscence in 62 of 144 dogs; unfortunately, the volume of saline solution used for leak testing was not consistently reported for all dogs in that study.

It is well established that the size of the small intestine varies on the basis of multiple factors. Results of studies22,23 in which ultrasonography was used to compare the wall thickness at various locations of the intestinal tract indicate that smaller dogs have thinner intestinal walls than larger dogs. Results of another study25 indicate that the intestinal wall becomes progressively thinner at its more distal sections. Sumner et al28 postulated that thickened or edematous intestinal tissue, because of its larger size, may require 4.8-mm-long staples, compared with the 3.5-mm-long staples traditionally used for functional end-to-end intestinal anastomoses in dogs. Similarly, White29 proposed that endoscopic staplers be used for intestinal anastomosis in toy breed dogs and cats because the intestinal lumen in those animals is too small to accommodate most gastrointestinal anastomosis stapling instruments. In an effort to examine those variables in the present study, we recorded both the intestinal measurement (half circumference) as well as the location along the intestinal tract for the specimens tested. However, the location of the intestinal tract (ie, jejunum) from where the specimens were collected was not standardized among the study dogs and therefore could not be statistically evaluated. There was a positive association between the intestinal measurement and saline volume, which reinforced the importance of that measurement as it pertains to the volume of saline solution used for leak testing. The intestinal measurement was obtained by laying the intestinal segment flat on a table and measuring the serosal margins from the antimesenteric to the mesenteric surface. From our experience, this method was clinically easier to measure than the true diameter of the lumen, both of which have been used in other studies.30,31 Matz et al17 showed that transverse closure of an enterotomy resulted in a significantly greater circumference, compared with that of longitudinal closure even though the volume of saline solution infused to reach 34 cm H2O (25 mm Hg) did not differ. This could suggest that the intraluminal diameter is a more appropriate measurement than circumference to use when establishing standardized volumes for leak testing. Further research should closely examine the differences in these measurement methods.

Results of the present study indicated that both patient body weight and intestinal measurement were positively associated with saline volume. However, the AIC values for those 2 models suggested that calculation in milliliters per millimeter provided a better model fit for the data. Nevertheless, it is important to note that both associations were significant and could be used to determine volumes for leak testing in a similar cadaveric setting. Before applying these formulas to surgical cases, our experiment should be replicated in vivo to determine more clinically appropriate values.

Intestines, like many hollow visceral organs, are inherently viscoelastic; the wall deforms (ie, stretches) when an intraluminal pressure is applied (ie, the lumen is filled with saline) but can return to its initial state once that force is removed.32 Similarly, once the wall deformation (stretch) ceases, the PP begins to decline despite no further change in the volume.32 This accommodation is due to a decrease in intestinal wall tension caused by relaxation of the circular muscle.33 This biologic feature of hollow organs allows for progressive storage (urine in the bladder) or movement of intraluminal contents (bolus of ingesta through the intestine).31 Intestinal peristalsis is a complicated biomechanical process that involves not only viscoelastic relaxation of collagen fibers, but also muscular contractions and neural responses that detect distension and reflexively cause further propulsion.34 Although peristalsis is not present in a cadaveric experiment like ours, many of the inherent mechanical properties of the intestine affect the stretch and thus potentially the saline volume. Panda and Buist35 documented in rat intestine that there is a nonlinear viscoelastic behavior within intestinal tissues, suggesting that the rate of strain loading can contribute to the material viscosity. Even as early as 1990, Beard et al36 speculated that in leak testing, the anastomotic wall tension was probably more important than the pressure of saline. Wall tension is difficult to measure clinically, and we sought to study parameters more readily quantified in a surgical setting in the present study.

This viscoelasticity of intestine affects leak testing in both research and clinical settings. In many veterinary leak-testing experiments, fluid is infused with a pump to control the rate of administration, thereby removing differences in viscoelasticity as a confounder.29,37,38,39,40,41,42,43 Fluid pumps were not used in clinical studies; instead experimenters injected saline solution by hand.13,16,17,18 For our experiment, we established a minimum total time within a determined pressure range (ie, the TTPR) as an inclusion criterion because of the observed gradual drop in pressure after saline infusion concluded. We chose 5 seconds because, in our experience, this is likely the minimum length of time a surgeon would examine an intestinal repair during leak testing. Initially, fluid was instilled at a rate of 2.0 mL/min, regardless of experimental group. However, the intestinal segments of the small-dog group were significantly smaller than those of the large-dog group, and this same rate of saline infusion resulted in a much faster drop in pressure once the pump was turned off, with no visible leaks in the construct. We were unable to achieve the 5-second TTPR at this rate. This was likely caused by less pressure accommodation in the smaller intestinal segments.33 Therefore, we opted to inject fluid at a slower rate (1.5 mL/min) in the small-dog group. This may seem like a limitation in that a slower rate would inherently result in more fluid infused per area. Even with this likelihood, we still found a significant difference in the saline volume infused between the small-dog and large-dog groups, proving our hypothesis. Despite the different administration rates between groups, we also found no significant difference in TPP and TTPR between groups, suggesting the intestinal relaxation occurred at the same rate. In our opinion, it is acceptable to use different rates of administration because it is unlikely that a surgeon manually injecting fluid in the operating room would achieve a precise steady rate with every leak test. Therefore, the difference in rates between our groups was considered realistic in a clinical setting. In future leak-testing experiments, infusion rates for leak testing should be more closely examined to determine the most suitable rate.

The PP differed significantly between 2 experimental groups of the present study. We acknowledge it is possible this difference in PP between groups could have contributed to the differences in saline volumes, although we think this was unlikely. The difference in mean PP between groups was 2.11 mm Hg, a small enough disparity that it is unlikely detectable by even the most experienced surgeons. There was also no significant association between the PP and TTPR, further supporting the use of the 5-second minimum TTPR for our samples.

Although it seems important to acknowledge that smaller jejunal segments require less saline solution to achieve the same pressure than larger jejunal segments, the necessity for such accuracy in leak testing is not known. For one, there is currently no veterinary literature that proves leak testing helps prevent postoperative dehiscence. Second, multiple peristaltic pressure ranges from 15 to 25 mm Hg16,17,44,45 and from 20 to 40 mm Hg46,47,48 have been reported in the literature, so the clinical significance of leak testing exactly to 25 mm Hg versus a higher limit is unknown. In human medicine, multiple methods for leak testing are reported and each utilizes a different standard. Gilbert and Trapnell14 found that the volume of fluid required to distend the intestinal lumen to 20 cm H2O (as recorded with a manometer) had a wide range (80 to 1,000 mL), reflecting the variability in the size of the bowel. In 1 report,49 saline solution dyed with methylene blue and infused via a gravity-dependent bag was used; no specific pressure limit was monitored. In a more recent retrospective study, Allaix et al50 examined air leak testing in 777 colorectal anastomoses that used a standard 60-mL volume of air. The fact that there is such a wide variety of techniques reported in the literature speaks to the importance of continued research in standardizing these methods.

The present study had multiple limitations, one of which is our use of cadaveric tissue. Only 2 patients underwent necropsy and had histologic examination of tissues performed, of which 1 had neuromuscular disease in the esophagus but not the jejunum. It is possible there may have been pathological lesions that were overlooked. We relied on sometimes vague or incomplete owner history from records to determine whether the study cadavers had gastrointestinal signs at home. Most of these dogs had approximately 24 hours of vague gastrointestinal signs reported, including 1 bout of vomiting or diarrhea or anorexia. However, because our study's patient population included euthanized client-owned dogs, many of these animals were older with an overall declining quality of life, so acute (but not chronic) gastrointestinal signs were a limitation we were willing to accept in our study population. Additionally, the intestines were left intact and an enterotomy was not performed as in previous studies,16,17 which could have affected the saline volume recorded and limited the clinical application of our formulas. Finally, as previously discussed, the rate of saline administration differed between the 2 experimental groups, which did not affect the time parameters used as inclusion criteria but could have had an impact on the volumes recorded.

To our knowledge, the present study was one of the first experiments to document a significant difference in the amount of saline solution needed to perform leak testing up to an intraluminal pressure of 25 mm Hg in small intestinal segments of small dogs (body weight, < 20 kg), compared with large dogs (body weight, > 20 kg). Although this was a cadaveric study in which no histologic examination of intestinal tissues was performed and no enteric defects were created in the tested specimens, the results indicated that a standard approach to leak testing may not be in our canine patients’ best interests. Further in vivo testing should be conducted to clarify more appropriate saline volumes for clinical use.

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.

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