Objective
This study determined the influence of a 2-component polyethylene glycol surgical sealant (Coseal) as an adhesion prevention device on sepsis-related mortality and/or systemic bacterial translocation to the spleen.
Study Design
A bacterial inoculum and telemetry probe were implanted in 50 treated and 49 untreated rats. Telemetry probes monitored core-body temperature to determine time of death. Spleens were collected on day 3 for quantitative bacteriology of Escherichia coli and Bacteroides fragilis .
Results
Median survival time and mortality of treated rats (37.0 hours, 54.0%) were noninferior to untreated rats (47.5 hours, 55.1%). Median E coli titers in treated rats (2.24 log colony forming units/spleen) were significantly less than untreated rats (4.32 log colony forming units/spleen). B fragilis titers were not different.
Conclusion
This study demonstrates intraperitoneal administration of a 2-component polyethylene glycol surgical sealant as an adhesion prevention device does not alter time to death or sepsis-related mortality and/or systemic bacterial translocation to the spleen.
Adhesions result from the removal of mesothelial layers of peritoneal organs and ischemia. During surgery, clot formation leads to fibrin deposits on the denuded surfaces of peritoneal organs. Ischemia then reduces fibrinolysis and the clearance of deposited fibrin. The residual fibrin remaining at the time of cellular implantation acts as a scaffold for the formation of adhesions. Consequences of adhesion formation after peritoneal surgery include infertility, pain, complications from laparoscopic entry, bowel obstruction, and prolonged surgical time. Mechanical barriers are used to prevent adhesions by blocking the contact of 2 fibrin deposits preventing the formation of adhesions.
Adhesion prevention devices are placed during surgical manipulation of diseased or infected uterine, vaginal, bowel, or bladder tissue. Surgical manipulation of these organs increases the risk of rupture that would introduce a large number and variety of microorganisms into the peritoneal cavity, which can lead to increased inflammatory cytokines and fatal sepsis. Therefore, a mechanical barrier used to prevent adhesions without potentiating an intraperitoneal infection, which can lead to sepsis, is highly desirable.
A rat intraperitoneal infection model was previously used to investigate whether implantation of adhesion prevention devices affect the course of peritoneal infections.
This study refines these models by measuring time to mortality in combination with quantitative measures that reflect immune function and correlate with septic shock. Quantitative measures include transport of infectious organisms to the periphery (thought to be necessary for the induction of shock) and core body temperature (a measure to quantify time to death).
The purpose of this study was to determine whether intraperitoneal administration of a 2-component polyethylene glycol surgical sealant (Coseal; Baxter Healthcare Corporation, Deerfield, IL) would alter sepsis-related mortality and/or systemic bacterial translocation to the spleen. The primary endpoint of this study was survival time. The secondary endpoints were mortality within the observational period and bacterial enumeration of Escherichia coli and Bacteroides fragilis in the spleen for the subset of animals that did not experience death within the observational period. The 2-component polyethylene glycol sealant has consistently been demonstrated to be an effective local adhesion prevention barrier. This study was performed to determine whether this device exacerbates an intraperitoneal infection.
Materials and Methods
Experimental design
This study consisted of 50 female rats treated with 0.4 mL Coseal (Baxter Healthcare Corporation, Deerfield, IL) and 49 female rats treated with 0.4 mL saline. Each rat received an intraperitoneal bacterial challenge of 35 ± 1 mg, based on animal weight, of a wide-spectrum inoculum. The bacterial challenge level of 35 ± 1 mg was designed to cause 50% mortality based on a pilot evaluation. The bacterial inoculum and a telemetry probe were surgically implanted in rats on day 0 with euthanasia performed on day 3. The telemetry probe monitored core-body temperature for the duration of the study to determine the time of death. The spleens of surviving rats were collected on day 3 and homogenized for quantitative bacteriology of E coli and B fragilis .
Animals
All animal-use activities were approved by the Institutional Care and Use Committee and used according to the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. Female Sprague-Dawley, CD rats, 174-200 g (approximately 7-9 weeks of age), were purchased from Charles River Laboratories (Raleigh, NC). Only animals in good health were assigned to the study. The rats were monitored once daily for general health before surgery and twice daily after surgery. A terminal body weight was obtained 3 days after surgery and the animals were humanely euthanized with CO 2 . The spleen was collected, weighed, and processed for bacterial enumeration. The spleen was suspended in 5 mL cold phosphate buffered saline (PBS). The tissues were disrupted using a Stomacher 80 (Seward Lab Systems, Northampton, Great Britain).
Bacterial inoculum preparation
The wide-spectrum bacterial inoculum consisted of feces and cecal contents of rats that were fed raw ground beef for 2 weeks. The bacterial inoculum was stored frozen at −20°C and aliquots of 500 μL were thawed as needed. On day 0, aliquots were thawed and used to fill a no. 1 gelatin capsule (Electron Microscopy Sciences, Hatfield, PA) with 35 ± 1 mg of the inoculum. Each no. 1 capsule was then placed into a no. 00 capsule (Electron Microscopy Sciences), and stored refrigerated in a sterile covered petri dish until surgical implantation. Three additional samples were collected in sterile cryovials and stored refrigerated until processed on that same day for bacterial enumeration to confirm viability.
Test and control article application
Coseal (Baxter Healthcare Corporation) was sprayed in the peritoneal cavity at 0.4 mL (∼2 mL/kg) using a Coseal Spray Set (Baxter Healthcare Corporation) with the EasySpray Pressure Regulator (Baxter Healthcare Corporation). This dose is approximately equivalent to 1 mL/20 cm 2 , yielding a 0.5-mm thickness. This thickness is similar to that used clinically, where typically 8 mL is used. The 8 mL applied to an average woman of approximately 60 kg, however, results in approximately 0.13 mL/kg. Therefore, the dosage in this study, approximately 2 mL/kg, is 15 times the typical clinical dosage to mimic the worst-case clinical situation. Sterile saline, 0.4 mL, was used in the untreated control group.
Surgical procedure
Each rat was weighed and received an intraperitoneal injection of ketamine/xylazine (60/6 mg/kg). Buprenorphine was administered subcutaneously (0.02 mg/kg). After anesthesia, the abdomen was shaved and aseptically prepared with betadine and 70% alcohol. With sterile instruments, a 6-7 cm celiotomy was performed using standard technique. The capsule containing the respective dose of bacterial inoculum was inserted into the right side of the peritoneal cavity. Coseal was sprayed in the peritoneal cavity of the treated rats. Saline solution was instilled in the peritoneal cavity of the untreated control rats. A 3.5-cm 3 telemetry probe (PhiysioTel TA-F40; Data Sciences International, St. Paul, MN) was inserted into the left side of the peritoneal cavity of each rat. The abdominal musculature was closed with absorbable 4-0 coated polyglactin 910 suture and the skin was closed with stainless steel wound clips. Atipamezole, a xylazine antagonist, was administered subcutaneously (approximately 1 mg/kg) postoperatively. The rats were monitored until sternally recumbent and then returned to their individual housings. Buprenorphine was administered subcutaneously (approximately 0.02 mg/kg) twice daily through day 2, as needed.
Telemetry
After recovery from surgery, animals were monitored for temperature by telemetry using a receiver station beneath the individual housing of each rat connected to a computer using Dataquest A.R.T. v4.1 software (Data Sciences International). The implanted temperature probes monitored the animal’s core temperature every 6 minutes. To estimate the time to death, the temperature curves were evaluated for the inflection point where the curve dropped down to room temperature ( Figure 1 ). This was found to be between 25°C-28°C in most of the animals. Therefore, to allow unbiased comparison between all animals, the time to death was estimated to occur when the internal temperature dropped to 26°C. Thus, the time point nearest to 26°C was selected as time of death unless the animal was euthanized for humane reasons or the telemetry probe failed.
