Swine as models in biomedical research and toxicology testing.

Swine are considered to be one of the major animal species used in translational research, surgical models, and procedural training and are increasingly being used as an alternative to the dog or monkey as the choice of nonrodent species in preclinical toxicologic testing of pharmaceuticals. There are unique advantages to the use of swine in this setting given that they share with humans similar anatomic and physiologic characteristics involving the cardiovascular, urinary, integumentary, and digestive systems. However, the investigator needs to be familiar with important anatomic, histopathologic, and clinicopathologic features of the laboratory pig and minipig in order to put background lesions or xenobiotically induced toxicologic changes in their proper perspective and also needs to consider specific anatomic differences when using the pig as a surgical model. Ethical considerations, as well as the existence of significant amounts of background data, from a regulatory perspective, provide further support for the use of this species in experimental or pharmaceutical research studies. It is likely that pigs and minipigs will become an increasingly important animal model for research and pharmaceutical development applications.

Mechanism of decreased forward stroke volume in children and swine with ventricular septal defect and failure to thrive.

Children with ventricular septal defect (VSD) often demonstrate failure to thrive (FTT). Such patients usually have reduced systemic cardiac output which has been postulated as a cause for their growth retardation. This study was conducted to ascertain the mechanism of the reduced cardiac output in children with VSD and FTT and also in a porcine model of VSD. Forward stroke volume was reduced in VSD-FTT children, 31 +/- 8 ml/m2, compared to normal children, 49 +/- 15 ml/m2 (P less than 0.05), but was not reduced in children with VSD and normal growth and development (41 +/- 16 ml/m2). Forward stroke volume was also reduced in swine with VSD compared to controls. Contractility assessed by mean velocity of circumferential shortening (Vcf) corrected for afterload was similar in normals and VSD-FTT children. Contractile performance was also similar in normal and VSD swine. Afterload assessed as systolic stress was similar in FTT-VSD children and normal subjects. Preload assessed as end-diastolic stress was increased in the VSD-FTT group. End-diastolic volume was not larger in the VSD-FTT group. We conclude that the reduced stroke volume seen in VSD-FTT children and VSD-swine was not due to reduced contractility, increased afterload or reduced preload. The reduced stroke volume may have been due to failure of end-diastolic volume to increase adequately.

Depressed contractile function due to canine mitral regurgitation improves after correction of the volume overload.

It is known that long-standing volume overload on the left ventricle due to mitral regurgitation eventually leads to contractile dysfunction. However, it is unknown whether or not correction of the volume overload can lead to recovery of contractility. In this study we tested the hypothesis that depressed contractile function due to volume overload in mitral regurgitation could return toward normal after mitral valve replacement. Using a canine model of mitral regurgitation which is known to produce contractile dysfunction, we examined contractile function longitudinally in seven dogs at baseline, after 3 mo of mitral regurgitation, 1 mo after mitral valve replacement, and 3 mo after mitral valve replacement. After 3 mo of mitral regurgitation (regurgitant fraction 0.62 +/- 0.04), end-diastolic volume had nearly doubled from 68 +/- 6.8 to 123 +/- 12.1 ml (P less than 0.05). All five indices of contractile function which we examined were depressed. For instance, maximum fiber elastance (EmaxF) obtained by assessment of time-varying elastance decreased from 5.95 +/- 0.71 to 2.25 +/- 0.18 (P less than 0.05). The end-systolic stiffness constant (k) was also depressed from 4.2 +/- 0.4 to 2.1 +/- 0.3. 3 mo after mitral valve replacement all indexes of contractile function had returned to or toward normal (e.g., EmaxF 3.65 +/- 0.21 and k 4.2 +/- 0.3). We conclude that previously depressed contractile function due to volume overload can improve after correction of the overload.

Cryopreserved human amniotic membrane and a bioinspired underwater adhesive to seal and promote healing of iatrogenic fetal membrane defect sites.

INTRODUCTION: We investigated the ability of cryopreserved human amniotic membrane (hAM) scaffold sealed with an underwater adhesive, bio-inspired by marine sandcastle worms to promote healing of iatrogenic fetal membrane defects in a pregnant swine model. METHODS: Twelve Yucatan miniature pigs underwent laparotomy under general anesthesia at 70 days gestation (term = 114 days). The gestational sacs were assigned to uninstrumented (n = 24) and instrumented with 12 Fr trocar, which was further randomized into four different arms-no hAM patch, (n = 22), hAM patch secured with suture (n = 16), hAM patch with no suture (n = 14), and hAM patch secured with adhesive (n = 9). The animals were euthanized 20 days after the procedure. Gross and histological examination of the entry site was performed for fetal membrane healing. RESULTS: There were no differences in fetal survival, amniotic fluid levels, or dye-leakage from the amniotic cavity between the groups. The fetal membranes spontaneously healed in instrumented sacs without hAM patches. In sacs with hAM patches secured with sutures, the patch was incorporated into the swine fetal membranes. In sacs with hAM patches without sutures, 100% of the patches were displaced from the defect site, whereas in sacs with hAM patches secured with adhesive 55% of the patches remained in place and showed complete healing (p = 0.04). DISCUSSION: In contrast to humans, swine fetal membranes heal spontaneously after an iatrogenic injury and thus not an adequate model. hAM patches became incorporated into the defect site by cellular ingrowth from the fetal membranes. The bioinspired adhesive adhered the hAM patches within the defect site.

Defining appropriate health status and management programs for specific-pathogen-free swine for xenotransplantation.

Swine are expected to be utilized as xenograft donors for both whole-organ and cellular transplantation. In order to meet the criteria for regulatory guidelines, donor animals are going to have to be free of potential zoonoses and other complicating diseases. Screening of animals will have to include tests for viruses, bacteria, parasites, congenital defects, and other inapparent diseases such as neoplasia or metabolic dysfunctions. The term Specific-Pathogen-Free (SPF) swine is a proprietary designation in the U.S. that does not include screening for all appropriate organisms for xenotransplantation. A program for breeding animals as xenograft donors will have to be conducted in a biomedical research facility rather than a conventional farm setting. The research programs at such a facility should include serology, microbiology, necropsy, histology and parasitology. The use of sentinel animal monitoring in a research facility is one method to ensure compliance. It will be impossible to provide complete individual animal screening in a timely fashion prior to performing a xenograft transplant. Quality control measures need to ensure that there is a reasonable confidence that the donor tissue is appropriate for the procedure to be performed. It is suggested that a term such as xenograft-defined flora be used to designate the appropriate health status of donor animals rather than SPF in order to avoid confusion with existing standards.

Ventricular septal defects in a family of Yucatan miniature pigs.

We have studied the hearts from a colony of Yucatan miniature pigs with spontaneously occurring congenital defects. Ventricular septal defect was encountered in 57 of 81 neonates from 15 consecutive litters. Of 73 hearts preserved for morphological assessment, 52 were found to have defects within the ventricular septum remarkably similar to those observed in humans with deficient ventricular septum. The defects, including 3 which had closed spontaneously, were perimembranous in 34, muscular in 12 and doubly committed and juxtaarterial in 6 hearts. Atrial septal defects were found in 12 of the 52 hearts with deficient ventricular septation; only 1 atrial septal defect was seen among 21 hearts with an intact ventricular septum. Anomalies of the aortic arch were associated with ventricular septal defect in 2 cases; 1 with a solitary arterial trunk and one with hypoplasia of the aorta and patent arterial duct. All these findings are replicated in human hearts. This strain of pig provides an ideal large animal model for morphologic and genetic investigations concerning the details of ventricular septation, including potential mechanisms of late spontaneous closure.

Considerations of specific pathogen-free swine (SPF) in xenotransplantation.

The purpose of this manuscript was to introduce the complexities involved in defining both the appropriate health status and the management programs for SPF swine as they may relate to xenotransplantation. One factor that must always be considered in using swine is that they are considered agricultural animals regardless of their purpose and are regulated as such by municipalities, states, and countries. There are nonflexible regulations related to the interstate transportation of swine and even greater hurdles to international transportation. For example, I tried unsuccessfully for three years to transport a single breeding animal with a congenital heart defect to collaborators in the United Kingdom. Bureaucratic factors on both sides of the Atlantic proved insurmountable because the regulations were formulated for economic and isolation purposes and no exceptions could be granted for research animals. Another factor to be considered is the designation of SPF swine, which in this species has a specific proprietary connotation. If the finalized program for production of swine for xenotransplantation proves to be different from the already established SPF program, then a different designation may have to be used. For example, a term like "xenografic defined flora (XDF)" swine could be used to avoid confusion with the already established registry. If xenotransplantation using swine becomes a reality, then it is important that the parameters used to select such animals be defined in advance. It is unlikely that conventionally reared animals of uncertain health status will be widely accepted because of legal and ethical considerations. Likewise, I do not believe that the use of swine for xenotransplantation will be able to avoid the regulatory measures already in place for laboratory animals. The multidisciplinary approach to the problem initiated by the Institute of Medicine workshop is the most appropriate mechanism for resolving the issues.

Swine as models in experimental surgery.

The use of swine in surgical research has undergone a dramatic increase in recent years, due to decreasing availability of dogs as surgical models and a renewed interest in the porcine model based on anatomic and physiologic characteristics. This article reviews the issues involved in selection of swine as experimental surgical subjects including procurement considerations, anesthetic selection, housing methods, and anatomic and physiologic characteristics.

A technique for conducting noninvasive cardiac electrophysiology studies in conscious swine.

A noninvasive method was developed in swine for conducting cardiac electrophysiology (EP) studies without the potential confounding effects of sedatives or anesthetics. Following a 1-week conditioning regimen in the EP laboratory, 1-month-old Hanford miniature swine underwent transvenous pacemaker (PM) implantation under isoflurane anesthesia. Two bipolar screw-in pacing leads were inserted into the left external jugular vein, with one positioned in the right atrial appendage and one in the myocardium at the right ventricular apex. The leads were attached to a multiprogrammable pulse generator, and the pacing system was placed in a subcutaneous pocket. At weekly intervals following implantation, conscious, unsedated swine were remained in a sling for noninvasive programmed stimulation (NIPS) using a programmable telemetry system. A NIPS protocol to induce arrhythmias was performed separately for the atrium and ventricle. Data for this model are reported. Complications occurred in 6 of 26 animals studied and included one infection of the PM pocket, three cases of dislodgement of the atrial lead, and rotation of the generator within the pocket in two animals, preventing communication with the PM. This technique has been used to perform EP studies successfully in swine and has been utilized in a variety of studies of the cardiac conduction system.

Chronic cannulation and fistulization procedures in swine: a review and recommendations.

Swine are important animal models in biomedical research. Some experimental procedures may require chronic access to blood vessels, organs, and structures. Chronic access procedures may be utilized for collection of blood and body fluids as well as for the infusion of test substances. This manuscript reviews methodologies that have been reported to be effective for these procedures. It also makes recommendations on the maintenance of indwelling catheters, cannulas, and fistulas.

Hanford miniature swine model for extracorporeal membrane oxygenation.

Extracorporeal membrane oxygenation (ECMO) is now an accepted therapy for neonatal respiratory failure due to reversible lung disease. There is a continued need to train ECMO team members using animal models. Hanford miniature swine are an appropriate model for this purpose because of the availability and size as well as anatomic and physiologic similarities to humans. This article describes training ECMO team members in venoarterial and venovenous bypass utilizing Hanford miniature swine.

Bretylium and diltiazem in porcine cardiac procedures.

The use of miniature swine as a model for cardiovascular diseases of humans is becoming more popular for many reasons. One of the problems involved in using swine is their propensity for fatal cardiac arrhythmias during surgical procedures requiring general anesthetics, especially cardiac procedures. In preparation for use as a model of human atherosclerotic coronary artery disease, 30 three-month-old (15 kg) Hanford miniature swine underwent left heart catheterization, coronary angiography, and abrasion of the left anterior descending coronary artery. All pigs were treated with diltiazem HCl 30-60 mg (2-4 mg/kg) three times daily and aspirin 25 mg once daily, both given orally for three days before surgery. General anesthesia was induced with a combination of ketamine HCl 25 mg/kg, atropine sulfate 0.1 mg/kg, and acepromazine 0.22 mg/kg, all given intramuscularly. Halothane 1-2% and nitrous oxide 30% were used to maintain general anesthesia after endotracheal intubation. After successful cannulation of the femoral artery, all animals were given 200 units/kg heparin and 5 mg/kg bretylium tosylate intravenously. The electrocardiogram and mean blood pressure were monitored throughout the procedure. No significant change in blood pressure was noted during the procedure. Intracoronary nitroglycerin 200 micrograms was administered prior to abrasion of the left anterior descending with a 2.0-mm angioplasty balloon. The pigs were allowed to recover after routine closure of the incision. One pig (1/30) died during the abrasion procedure as a result of ventricular fibrillation. This represents a low, acceptable mortality using the present regimen compared with other regimens.

Technical aspects of cardiac catheterization of swine.

Long-term cardiovascular studies requiring repeated cardiac catheterizations often pose technical problems for investigator using swine. The purpose of this paper is to describe two reliable methods of cardiac catheterization that are used in swine to acquire complete hemodynamic data. Due to the depth of the vessels, vascular cut-downs are usually required. Hemodynamic, angiographic, and interventional cardiac catheters are introduced into either the femoral vessels or the external jugular vein and internal carotid artery. An alternative technique that obviates the need for multiple surgeries involves implantation of silastic catheters into both atria. This allows the passage of catheters directly into the heart chambers. A disadvantage of repeated vascular cut-downs is that a reduction of vascular access occurs. Chronic lines require careful attention to prevent infection and maintain patency.

Perioperative care protocols for a canine model of a new gastrocolonic pouch for continent urinary diversion.

Traditionally, in continent urinary diversion (CUD), the ileum, ascending colon, or sigmoid colon is utilized to construct a urinary reservoir with a continence mechanism. When CUD was performed in women who had undergone extensive pelvic irradiation therapy for gynecologic cancer, the complication rate was exceedingly high due to the poor vascularity of the irradiated bowel incorporated in the reservoir. In order to avoid this very high complication rate, an animal study was conducted to test the feasibility of constructing a CUD utilizing nonradiated stomach, transverse colon, and proximal ureters. The development of this model in the canine necessitated prolonged abdominal surgery involving both the gastrointestinal and urinary systems. The immediate postoperative challenge included the need for continuous intravenous fluid infusion, maintenance of patent renal stents and neobladder stomal catheters, prevention of hypothermia and hypokalemia, and reduction of postoperative discomfort. Re-establishing an adequate plane of nutrition, preventing colonic ulceration from gastric secretions in the pouch, monitoring renal function, maintaining a patent stoma during healing, and managing the gradual expansion of the pouch were among the chronic care considerations. This procedure required the institution of unique and intensive perioperative care protocols. Using the methods discussed in this paper, three of four dogs in a pilot study survived the procedure and were included in an ongoing study of the new gastrocolonic continent urinary diversion.

High-dose narcotic anesthesia using sufentanil in swine for cardiac catheterization and electrophysiologic studies.

A protocol for high-dose narcotic anesthesia using sufentanil by intravenous infusion was developed in swine undergoing cardiac catheterization and intracardiac electrophysiologic studies. In experiments involving cardiac conduction system ablation, nine swine received intramuscular ketamine, 33 mg/kg, and intramuscular acepromazine, 1.1 mg/kg, as premedication. An infusion of sufentanil (15 mcg/kg/h) was started followed by a loading dose of sufentanil (7 mcg/kg) given as a bolus intravenously. A continuous infusion of sufentanil (15-30 mcg/kg/h) was used for maintenance anesthesia. Since the development of this protocol, we have been able to achieve stable hemodynamics and cardiac rhythms for intracardiac electrophysiologic studies.

Percutaneous serial catheterization in swine: a practical approach.

In a model designed for experimental cryoablation of the His bundle in swine, a study that required multiple and follow-up cardiac catheterizations, femoral percutaneous cannulations were successfully performed in 14 of 15 swine: 6/6 Yucatan miniature, 5/6 Hanford miniature, and 3/3 Yorkshire farm pigs, weight range 15-50 kg. A follow-up cardiac catheterization was performed in 10 of these animals: 5 Hanford and 5 Yucatan. Femoral percutaneous cannulation was successful in 6. A cutdown approach was successful in the remaining 4 after failed attempts at percutaneous vascular access. The results show that percutaneous femoral cardiac catheterization is a feasible and practical technique for cardiovascular studies in swine, especially those that require multiple and serial catheterizations.

An adult canine model of progressive left ventricular pressure overload.

This article details the development of a model of progressive left ventricular pressure overload (LVPO) in the adult dog. LVPO was induced by banding the proximal ascending aorta in 69 adult conditioned dogs. The base of the aorta was exposed through a right thoracotomy. A tunnel was created by blunt dissection between the aorta and pulmonary arteries. An aortic band was constructed by passing umbilical tapes through the lumen of gortex tubing. This band was placed through the tunnel, then tied around a balloon dilatation catheter. The distal end of the balloon catheter was closed with an injection cap and positioned in a subcutaneous pocket. Aortic stenosis was induced by filling the balloon catheter with saline. A predetermined amount of LVPO was created by adjusting the amount of aortic stenosis. At 2, 4, and 6 weeks after aortic banding the LVPO was increased by transcutaneous injection of saline into the balloon catheter. At 8 weeks the dogs were evaluated for sufficiently decreased cardiac contractility and used acutely in one of several studies. The article also discusses perioperative management, postoperative care, and complications that were encountered during the development of the model. Postoperative pain was managed by the combined use of preemptive and postoperative opioids, local nerve blocks, and nonsteroidal anti-inflammatory drugs. Notable intraoperative complications included atrial and ventricular arrhythmias and pulmonary artery laceration during the banding procedure. The most significant postoperative complications were aortic ruptures and congestive heart failure. The success rate of this model has increased from 20% (year 1) to 65% (year 3). This success has been attributed to improvements in band design, surgical technique, and postoperative management.

Surgically induced urologic models in swine.

The porcine urological system is similar anatomically and physiologically to that of humans. Swine have a true multirenculate, multipapillate kidney with a calyceal system like that of humans. The gross anatomic and histologic characteristics of the porcine and the human kidney are more similar than most other commonly used laboratory animals. The physiologic functions of the urinary system including urodynamic parameters are also similar to humans. Swine have been used extensively as models of urologic conditions in humans, most commonly in obstructive urologic syndromes. A large number of investigative procedures can be performed in one experiment both in acute and chronic models. Recently, a new technique of surgically induced antenatal lower urinary tract obstruction in swine has been developed.

Anesthetic and postoperative protocols for a canine model of reversible left ventricular volume overload.

The purpose of this study was to develop anesthetic and postoperative methods that could be used in a canine model of reversible left ventricular volume overload (LVVO). LVVO was created by inducing mitral regurgitation (MR) in adult conditioned dogs. Using fluoroscopy, MR was induced by passing urologic grasping forceps into the left ventricle through a carotid artery and rupturing one or more chordae tendinea. Cardiac catheterization was performed after significant cardiac dysfunction developed (3 months after creation of MR) in order to collect data to confirm reduced contractile function. The MR was repaired by replacing the damaged valve with a bovine pericardial xenograft. Dogs were followed with sequential cardiac catheterizations to determine if the valvular replacement reversed the cardiac dysfunction. To develop this model, unique anesthetic and post-operative care protocols had to be instituted. In order not to exacerbate the existing cardiac dysfunction, a high dose narcotic anesthetic regimen using a sufentanil infusion at 9-13 microgram/kg h-1 was developed in association with cardiopulmonary bypass and hypothermia for the mitral valve replacement surgery. Intensive postoperative care protocols that involved major personnel and pharmaceutic interventions were necessary. Using our methods 11/17 dogs survived the procedure and were included in the protocol for study of mechanisms involved in reversal of cardiac dysfunction.