Surgical management of congenital extrahepatic portosystemic anomalies is usually achieved by complete or partial occlusion of the shunting vessel.1–3 The goal of surgical treatment is to redirect portal blood through the liver to palliate the clinical signs of hepatic encephalopathy.4 Complete occlusion of the shunt has been associated with a better long-term prognosis than partial ligation or medical management, and various techniques have been used to gradually and completely occlude the shunt without increasing the risk of acute portal hypertension.5–9 Although multiple surgical techniques exist to partially occlude the shunting vessel, the 2 most common approaches are partial ligation with a cellophane band and use of an ameroid ring constrictor, both of which provide gradual shunt occlusion.1,9,10
Ameroid ring constrictors consist of a slotted stainless steel ring surrounding an inner compressed casein band with a key mechanism, permitting the device to be placed around a vessel. Ameroid is a hygroscopic casein derivative and undergoes rapid volumetric expansion that plateaus in 60 days.11,12 As the casein gradually absorbs fluids and expands, the inner diameter (lumen) closes and constricts the shunting vessel.8 The ameroid also triggers an inflammatory reaction around the shunt that contributes to closure13 and possibly thrombosis14 of the shunt. The device is available in multiple sizes (diameters of 3.5, 5, 6.5, 7, 8, and 9 mm). The 3.5- and 5-mm-diameter rings are used most commonly in the management of single extrahepatic portosystemic shunts in dogs.4
One consideration regarding closure of ameroid ring constrictors is the intraoperative selection of constrictor size and need to resterilize the unused constrictor. When a surgeon elects to place an ameroid ring constrictor on the shunting vessel, a decision pertaining to the size of the device may need to be made intraoperatively.15 Because this decision must be made at the time of surgery, a surgeon must have several constrictor ring sizes available. Thus, unused ameroid ring constrictors must be resterilized and returned to stock until the next surgery. However, it is unknown whether repeated sterilization of these restocked constrictors affects the rate of closure or the likelihood that they will close sufficiently.a When unused constrictors are not resterilized after 365 days, they must be discarded, which results in a monetary loss. Currently, gas sterilization is the only means of sterilization for ameroid ring constrictors, and the temperature required to achieve sterilization (60°C) may denature the casein protein within the constrictor.16 Currently, surgeons who resterilize these constrictors risk not knowing whether the casein protein within the ring will have its closure function attenuated by reprocessing. The manufacturer states that the constrictors can only be gas sterilized twice and have a shelf life of 1 year.a
The purpose of the study reported here was to evaluate the effects of repeated gas sterilization on the rate and degree of lumen closure of ameroid ring constrictors in vitro. We hypothesized that rate and degree of lumen closure would not be influenced by repeated gas sterilization in vitro.
Materials and Methods
Twenty-four 3.5-mm ameroid ring constrictorsb were obtained in packaging direct from the manufacturer and allocated to 1 of 4 treatment groups (6/group) to be gas sterilized 0 (control group), 1, 5, or 10 times and subsequently incubated in canine plasma to assess rate and degree of constriction. For gas sterilization, constrictors were placed in an ethylene oxide gas sterilizerc for 360 minutes. A period of 24 to 96 hours separated sterilization sessions.
After treatment, ameroid ring constrictors were incubated in accordance with a previously reported in vitro culture method.4 Briefly, plasma was aseptically obtained from packaged plasma provided by multiple dog donors for use in the plasma bank of the veterinary teaching hospital at Mississippi State University. All plasma was diluted with saline (0.9% NaCL) solution and maintained at a protein concentration of 3 g/dL, as confirmed with a refractometer.d One milliliter of 0.1% streptomycin solution and 1 mL of 0.05% amphotericin B solution were added to the plasma. Each treatment group of constrictors was placed in a standard 10 × 100-mm sterile polystyrene Petri dish containing treated plasma, and Petri dishes were incubated in a standard cell culture incubator for 27 days at 37°C and 5% CO2. Plasma was changed every 48 hours.
A digital camerae was used to photograph the ameroid ring constrictors when received from shipping and during incubation at 0 (prior to incubation), 1, 6, 11, 16, 21, and 27 days. A standard photography ruler was used to ensure accurate, equidistant measurements. The camera was held perpendicular to each Petri dish at a consistent height of 30 cm. The same zoom lens (35 mm) was used for each photograph. The same image quality settings were used for each photograph (International Standards Organization 220; focal length, 2.8; shutter speed, 1/30). Total (outer) area and lumen (inner) area of each constrictor were measured in triplicate at each assessment point with imaging software.f Each set of 3 measurements was then averaged and used for analysis. Differences in areas were calculated, averaged, and recorded as percentage closure (total area – lumen area)/total area × 100).
Statistical analysis
Data were summarized as mean ± SD. Two-factor ANOVA with 1 between-subjects factor treatment (number of sterilization sessions) and 1 within-subjects factor (time or day of measurement) was used to compare treatment groups of ameroid ring constrictors.g The outcome variable was the difference between total and lumen areas of the constrictor, divided by the total area. Because repeated measurements of each constrictor were made over time, the constrictor was included in the model as a random effect. A treatment by time interaction term was also included. Because the interaction term was significant, differences between treatment means on each day were estimated by calculating least square mean value. The 95% CIs of mean differences were calculated, and the importance of significant differences was assessed on this basis.17 End points of these CIs were used to determine whether the estimated differences were clinically important.17 Residuals from the fitted model were examined and tested for normality by means of the Shapiro-Wilk test. Because many pairwise comparisons of means on different days were made, the false discovery rate approach was used to control for multiple testing.18 For all tests, values of P < 0.05 after adjustment were considered significant.
Results
Differences between mean lumen areas measured prior to and after 27 days of plasma incubation were significant for all treatment groups of ameroid ring constrictors (gas sterilized 0, 1, 5, or 10 times; Table 1). Mean ± SD lumen area was 3.64 ± 0.43 mm2 (95% CI, 2.67 to 4.77 mm2) at day 0 and 1.32 ± 0.25 mm2 (95% CI, 0.76 to 2.04 mm2) at day 27. Mean percentage closure (based on the difference between total and lumen areas of the constrictor, divided by total area) for all treatment groups combined was 85.2 ± 1.6% at day 0 (prior to incubation) and 95.4 ± 0.8% at day 27.
Mean ± SD initial* and final lumen areas of ameroid ring constrictors that were gas sterilized 0, 1, 5, or 10 times (n = 6/group) and incubated in canine plasma for 27 days.
| No. of sterilization sessions | Initial area (mm2) | Final area (mm2) | Percentage closure† | Difference between initial and final area (%) | P value |
|---|---|---|---|---|---|
| 0 | 3.73 ± 0.49 | 1.52 ± 0.26 | 95 | 59 | < 0.005 |
| 1 | 3.28 ± 0.37 | 1.21 ± 0.15 | 96 | 63 | < 0.005 |
| 5 | 3.84 ± 0.42 | 1.34 ± 0.21 | 95 | 65 | < 0.005 |
| 10 | 3.26 ± 0.44 | 1.21 ± 0.24 | 95 | 63 | < 0.005 |
| Pooled data | 3.53 ± 0.43 | 1.32 ± 0.22 | 95 | 63 | < 0.005 |
Initial area was measured prior to incubation in canine plasma.
Percentage closure was calculated as follows: (total constrictor area – lumen area)/total constrictor area × 100.
Values of P < 0.05 after adjustment for multiple tests were considered significant.
On days 0, 1, 6, 11, 16, 21, and 27 of plasma incubation, mean lumen area for all treatment groups combined was 3.64 ± 0.43 mm2 (95% CI, 2.67 to 4.77 mm2), 2.93 ± 0.29 mm2 (95% CI, 2.41 to 3.68 mm2), 2.32 ± 0.23 mm2 (95% CI, 1.87 to 3.27 mm2), 2.36 ± 0.30 mm2 (95% CI, 1.82 to 3.23 mm2), 1.89 ± 0.23 mm2 (95% CI, 1.45 to 2.60 mm2), 1.64 ± 0.25 mm2 (95% CI, 1.04 to 2.39 mm2), and 1.32 ± 0.25 mm2 (95% CI, 0.76 to 2.04 mm2), respectively. Significant (adjusted P < 0.05) differences in mean percentage closure were identified between some treatment groups on days 0, 11, and 21 of plasma incubation (Table 2). However, given that the largest of these differences was only 0.024 mm2 (95% CI, 0.015 to 0.034 mm2), the magnitudes of the differences were deemed not clinically important. No significant (P > 0.869) difference was identified among treatment groups in percentage closure over time.
Mean ± SD percentage closure of ameroid ring constrictors that were gas sterilized 0, 1, 5, or 10 times (n = 6/group) at various points of subsequent plasma incubation.
| No. of sterilization sessions | Day 0* | Day 1 | Day 6 | Day 11 | Day 16 | Day 21 | Day 27 |
|---|---|---|---|---|---|---|---|
| 0 | 84.5 ± 1.3a | 87.6 ± 1.1 | 90.1 ± 0.6 | 92.4 ± 0.6a | 92.9 ± 0.7 | 94.6 ± 0.9 | 95.2 ± 0.8 |
| 1 | 85.6 ± 1.1a,b | 87.6 ± 1.2 | 89.8 ± 1.4 | 90.0 ± 0.8b | 92.2 ± 0.7 | 93.9 ± 0.5 | 95.7 ± 0.6 |
| 5 | 84.6 ± 1.4a,b | 88.4 ± 1.3 | 90.5 ± 0.7 | 91.2 ± 0.9a,b | 92.4 ± 0.6 | 93.2 ± 0.8 | 95.0 ± 0.8 |
| 10 | 86.1 ± 1.8b | 87.7 ± 1.1 | 89.7 ± 1.0 | 91.1 ± 1.1a,b | 92.2 ± 1.3 | 93.3 ± 1.1 | 95.6 ± 1.0 |
Day 0 represented prior to incubation.
Within a column, values with different superscript letters differ significantly (adjusted P < 0.05). However, the magnitudes of these differences (0.016 mm2 on day 0 and 0.024 mm2 on day 11) were deemed clinically unimportant.
Discussion
In the study reported here, none of the ameroid ring constrictors completely closed by 27 days after incubation in canine plasma; mean percentage closure was 95% for all treatment groups, regardless of the number of times constrictors were gas sterilized. Additionally, mean lumen area was similar for all groups at the beginning (day 0) and again at the end of the 27-day period. These findings supported those of a previous study4 in which percentage closure of ameroid ring constrictors increased with increasing duration of plasma incubation. In that study,4 high plasma protein concentrations resulted in rapid closure of the lumens of ameroid ring constrictors, and constrictors incubated in canine plasma at a concentration of 1.5 g/dL closed to a larger overall lumen diameter (ie, smaller percentage closure) than did constrictors incubated in canine plasma at a concentration of 3 and 6 g/dL. Investigators therefore concluded that the final diameter of the ameroid lumen was a function of time and protein concentration. We obtained similar results in that a plasma protein concentration of 3 g/dL resulted in large percentage closure. The final lumen size of 1.32 mm2 achieved after 27 days of plasma incubation in the present study would be reasonable for in vivo application, given the supposition that shunts would likely achieve functional closure because of inflammatory thrombosis.19
An unknown variable in the present study was the period the ameroid ring constrictor would be stored on the shelf, as this was beyond the scope of this study. We were unable to identify any reports that indicate the casein component of ameroid ring constrictors becomes denatured when constrictors are stored on the shelf at room temperature (approx 20° to 25°C). To the authors' knowledge, the shelf life of the casein after gas resterilization has also not been reported. Because the ameroid ring constrictors in the present study were purchased directly from the manufacturer and then used, the devices spent little time on the shelf. Additional studies may be warranted to ascertain the effects on percentage closure of extended shelf storage at room temperature alone or in combination with repeated sterilization.
For the plasma incubation stage of the present study, canine plasma samples from multiple donors were pooled and diluted to a protein concentration of 3 g/dL, whereas in the previous study,4 plasma used for incubation originated from 1 donor dog. It is unclear whether use of pooled plasma would have a different effect on the ameroid ring constrictors than would use of plasma from 1 dog, but this was considered unlikely given that our results were similar to those of the previous study4 with regard to closure.
Even with the modest sample size of 6 constrictors/treatment group, some significant differences were found among the groups. However, the largest of these differences was only 0.024 mm2 (95% CI, 0.015 to 0.034 mm2). Even at the upper limit of the estimate (0.034 mm2), this magnitude of difference would be unlikely to be of clinical importance.
The lack of significant differences among the 4 treatment groups after 27 days of plasma incubation suggested that repeated gas sterilization up to 10 times had no effect on percentage closure or overall closure rate and that such constrictors could be resterilized repeatedly in clinical practice with confidence that their closure properties would not change. A larger sample size might have resulted in more differences attaining significance. However, the numeric values would generally be even smaller than the differences observed here and thus would not be clinically important. The finding that the constrictors did not close completely after 27 days of treatment in vitro has been substantiated in other studies4,10,13 and illustrated the need for proper size matching of constrictors to the shunting vessel to minimize potential incomplete occlusion once the lumen has closed to its fullest extent.
Acknowledgments
Supported in part by the Office of Research and Graduate Studies, College of Veterinary Medicine, Mississippi State University.
The authors thank Dr. Cory Langston for technical assistance and laboratory space.
ABBREVIATION
| CI | Confidence interval |
Footnotes
King S, Research Instruments and Manufacturing, Corvallis, Ore: Personal communication, 2012.
Ameroid ring constrictor, Research Instruments and Manufacturing, Corvallis, Ore.
AMSCO Eagle 3017 EO Sterilizer, Steris, Mentor, Ohio.
VET 360 refractometer, Reichert Technologies, Depew, NY.
Digital D-3000 still camera, Nikon, Melville, NY.
ImageJ, National Institutes of Health, Bethesda, Md. Available at: rsb.info.nih.gov/nih-image. Accessed March 1, 2011.
PROC MIXED, SAS, version 9, SAS Institute Inc, Cary, NC.
References
1. Martin RA. Congenital portosystemic shunts in the dog and cat. Vet Clin North Am Small Anim Pract 1993; 23: 609–623.
2. Swalec-Tobias KM. Portosystemic shunts. In: Bojrab MJ, ed. Disease mechanisms in small animal surgery. 2nd ed. Philadelphia: Lea & Febiger, 1993;298–303.
3. Lawrence D, Bellah JR, Diaz R. Results of surgical management of portosystemic shunts in dogs: 20 cases (1985–1990). J Am Vet Med Assoc 1992; 201: 1750–1753.
4. Monnet E, Rosenberg A. Effect of protein concentration on rate of closure of ameroid constrictors in vitro. Am J Vet Res 2005; 66: 1337–1340.
5. Watson PJ, Herrtage ME. Medical management of congenital portosystemic shunts in 27 dogs—a retrospective study. J Small Anim Pract 1998; 39: 62–68.
6. Hottinger HA, Walshaw R, Hauptman JG. Long-term results of complete and partial ligation of congenital portosystemic shunts in dogs. Vet Surg 1995; 24: 331–336.
7. Murphy ST, Ellison GW, Long M, et al. A comparison of the ameroid ring constrictor versus ligation in the surgical management of single extrahepatic portosystemic shunts. J Am Anim Hosp Assoc 2001; 37: 390–396.
8. Vogt JC, Krahwinkel DJJ, Bright RM, et al. Gradual occlusion of extrahepatic portosystemic shunts in dogs and cats using the ameroid ring constrictor. Vet Surg 1996; 25: 495–502.
9. Youmans KR, Hunt GB. Experimental evaluation of four methods of progressive venous attenuation in dogs. Vet Surg 1999; 28: 38–47.
10. Sereda CW, Adin CA. Methods of gradual vascular occlusion and their applications in treatment of congenital portosystemic shunts in dogs: a review. Vet Surg 2005; 34: 83–91.
11. Berman JK. Reminiscences concerning the use of ameroid cylinders and other modalities in research. Rev Surg 1970; 27: 155–169.
12. Elzinga WE. Ameroid ring constrictor: uniform closure rates and a calibration procedure. J Appl Physiol 1969; 27: 419–421.
13. Adin CA, Gregory CR, Kyles AE, et al. Effect of petrolatum coating on the rate of occlusion of ameroid ring constrictors in the peritoneal cavity. Vet Surg 2004; 33: 11–16.
14. Besancon MF, Kyles AE, Griffey SM, et al. Evaluation of the characteristics of venous occlusion after placement of an ameroid ring constrictor in dogs. Vet Surg 2004; 33: 597–605.
15. Kim SE, Giglio RF, Reese DJ, et al. Comparison of computed tomographic angiography and ultrasonography for the detection and characterization of portosystemic shunts in dogs. Vet Radiol Ultrasound 2013; 54: 569–574.
16. Mitchell SL, Berg J. Sterilization. In: Slatter D, ed. Textbook of small animal surgery. 3rd ed. St Louis: WB Saunders Co, 2003;159–160.
17. Braitman LE. Confidence intervals assess both clinical significance and statistical significance. Ann Intern Med 1991; 114: 515–517.
18. Benjamini Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Methodol 1995; 57: 289–300.
19. Berent AC, Tobias KM. Hepatic vascular anomalies. In: Tobias KM, Johnston SA, eds. Veterinary surgery: small animal. St Louis: WB Saunders Co, 2012;1624–1658.
