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.

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.

Male fetal pig lower urinary tract function. Part II: free voiding pattern close to term and in the newborn.

PURPOSE: We assessed the development of natural voiding function late in gestation and in the immediate postnatal period in a porcine model. MATERIALS AND METHODS: The study comprised 7 male fetal minipigs (median age 94 days, 0.88 gestation) and 7 male newborn pigs. In all pigs an ultrasonic probe was placed around the subcutaneous urethra and pressure catheters were placed in the bladder and rectum and in the amnionic cavity in the fetal pigs. The catheters were connected to pressure transducers as the flow probe was connected to a flow meter, and the recordings were sampled at a rate of 10 Hz on a personal computer. RESULTS: The newborns had a median voiding frequency of 3.3 times per hour while the fetuses voided a median of 5.9 times per hour (p = 0.16). Both groups voided with a staccato flow at a frequency of 1 to 2 Hz, indicating voiding dyscoordination between the detrusor and urethra. Maximum flow rate increased from median 4.4 ml per minute (range 2.0 to 8.8) in the fetal group to median 10.9 (5.4 to 18.3) in the newborns (p = 0.07). While the minimum opening pressure remained unchanged (median 7.0 cm H2O, range 5.7 to 13.0) vs median 7.6, (range 7.7 to 14.0, p = 0.57) the detrusor pressure at maximum flow decreased from median 22.4 cm H2O (range 16.6 to 39.0) in the fetal pigs to 12.1 cm H2O (8.3 to 22.3) in the newborns. CONCLUSIONS: Fetal and newborn pigs have dyscordinated voiding with a staccato flow. While the urethral opening pressure appears to be unchanged, the detrusor pressure at maximum flow decreases during the last period of gestation, indicating decreased urethral resistance. These findings are in accordance with observations made in human infants.

Effects of feed withdrawal and transport on cecal environment and Campylobacter concentrations in a swine surgical model.

The objective of the present study was to evaluate how feed withdrawal and transportation influenced the cecal environment and cecal populations of Campylobacter in swine. Four miniature Yucatan gilts (8.8 kg), naturally infected with Campylobacter jejuni, were surgically implanted with cecal cannulas. The gilts were fasted for 48 h. Samples of cecal contents were collected for 7 days prior to and for 7 days after the fast, and mean values were determined for pH, volatile fatty acids (VFA), and CFU enumeration of C. jejuni. This was replicated three times. In another trial, gilts (full-fed) were transported in a livestock trailer for 4 h and cecal samples were collected before and after transport and analyzed for pH, VFA, and CFU. Following a 48-h fast, cecal pH increased (P < 0.05) by 1 unit; acetic and propionic acids decreased (P < 0.05) by 61% and 71%, respectively; and there was a twofold log10 increase (P < 0.05) in CFU/g cecal content of C. jejuni. Values of pH, VFA, and CFU of C. jejuni did not change in cecal samples from gilts following transportation. These data are important for food safety considerations because feed withdrawal, commonly associated with shipping and slaughter, can increase Campylobacter concentrations in the pig intestinal tract.

Male fetal pig lower urinary tract function in mid second and early third trimester of gestation.

PURPOSE: Routine use of ultrasound during pregnancy has emphasized the need for greater knowledge about normal and abnormal voiding function during fetal live. We describe the developmental changes in lower urinary tract function from the middle of the second to the early third trimester of gestation. MATERIALS AND METHODS: The study included 22 male fetal pigs of 13 pregnant Goettingen minipigs (gestational period 111 to 115 days) with 11 fetuses in the middle of the second (median 61.5 days, 0.55 of term) and 11 fetuses in the beginning of the third trimester of gestation (median 80 days, 0.72 of term). With the sow under isoflurane anesthesia catheters were placed in the bladder, rectum and amniotic cavity of the fetuses, and a flow probe was placed around the distal urethra. After the sow was at light sleep urethral flow, amniotic pressure, abdominal pressure and bladder pressure were recorded a median of 241 minutes. RESULTS: The recordings of 14 fetuses were evaluable. In the middle of the second trimester the fetuses showed an almost constant dribbling with a median flow of 0.2 ml. per minute and a median of 4 concomitant bladder contractions per minute of median 3.5 cm. H2O (detrusor pressure). At the beginning of the third trimester the fetuses had periods of staccato flow (median 0.2 ml. per minute, staccato frequency 1 to 2 Hz.) on top of contractions (median 4 contractions per minute, median detrusor pressure 5.5 cm. H2O) with no flow between voidings. CONCLUSIONS: Development of storage and voiding function in male pigs occurs between the mid-second and early third trimester of gestation, together with emergence of striated sphincter bursting activity, which interrupts flow in the urethra during the voiding phase.

Non-invasive measurement of blood pressures in the Yucatan micropig (Sus scrofa domestica), with and without midazolam-induced sedation.

Current literature suggests that the effects of midazolam, a water-soluble benzodiazepine, on blood pressure in swine are minimal. The hypothesis of the study reported here was that a light sedative dose would induce a decrease in blood pressure in this species. Healthy female Yucatan Micropigs (n = 20), 16 to 30 (mean, 22) kg, aged four six months, were individually placed in a humane restraint sling and allowed to acclimate. Systolic (SBP), diastolic (DBP), and mean (MBP) blood pressures (mmHg) and heart rate (HR; beats per min [bpm]) were measured by use of oscillometry. The pressure cuff was placed at the base of the tail, and five sets of values were recorded at five-min intervals, beginning at 10 and ending 30 min after cuff placement. Following a three- to four-day rest period, this procedure was repeated with the addition of a dose of 0.5 mg of midazolam HCl/kg of body weight given intramuscularly at the time of cuff placement. A paired one-way Student's t-test was used to compare the means of the five measures between control and midazolam treatment. Mean (+/- SD) differences for SBP, DBP, MBP, and HR were 18.9 (+/- 3.97), 17.8 (+/- 5.27), and 18.6 (+/- 5.09) mmHg and 20.7 (+/- 3.73) bpm, respectively. All four parameters were significantly reduced in the midazolam-sedated group (P < 0.001). The maximal decrease in SBP, DBP, and MBP occurred at 15 and 20 min after dosing. Mean values based on the means of the five measures were 128 (+/- 12.6), 80 (+/- 9.4), and 99 (+/- 9.2) mmHg and 135 (+/- 17.4) bpm, and 109 (+/- 15.4), 63 (+/- 12.6), and 80 (+/- 13.6) mmHg and 115 (+/- 15.5) bpm for SBP, DBP, MBP, and HR in the control (n = 20) and midazolam (n = 20) groups, respectively. The control values can serve as normal oscillometric values for this age, sex, and breed of Micropig. We conclude that midazolam, given intramuscularly at a sedative dosage, negatively affects cardiovascular parameters measured by use of a blood pressure cuff, in sexually mature female Micropigs, compared with values in untreated pigs, which is similar to reports for humans.

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.

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.

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.

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.

Toward optimizing a preshaped catheter and system parameters to achieve single lead DDD pacing.

P wave electrogram amplitudes and atrial stimulation thresholds were determined in eight Hanford miniature swine using a preshaped catheter with an "S" curve in the SVC, and a major lobe in the atrium to enhance electrode contact with the atrial wall. The catheter was designed for pacing and sensing in the DDD mode. P wave amplitudes were also ascertained with two commercially available VDD leads and compared to the data from the experimental catheters. The preshaped catheter used two 6-mm2 platinum iridium atrial electrodes with a 7-mm separation. Both atrial electrodes are on the same side of the catheter, facing outward on the major atrial lobe formed in the catheter. The P wave amplitudes were tested only in the differential bipolar configuration. For the eight preshaped catheters, the mean was 6.6 +/- 3.8 mV while for the conventional leads it was 2.9 +/- 1.6 mV. The mean atrial stimulation thresholds ranged from 1.1 +/- 0.2 V to 2.3 +/- 1.2 V, with still lower thresholds of 0.9-1.3 V when using the parallel unipolar atrial electrode configuration, in which both parts of the bipolar atrial electrode are configured as a unipolar electrode. The data suggest that bipolar simulation may be effective if sequential reverse polarity pulses are used to achieve cathodal stimulation from each electrode of the bipolar pair, on a beat-to-beat basis.

Premorbid determinants of left ventricular dysfunction in a novel model of gradually induced pressure overload in the adult canine.

BACKGROUND: When a pressure overload is placed on the left ventricle, some patients develop relatively modest hypertrophy whereas others develop extensive hypertrophy. Likewise, the occurrence of contractile dysfunction also is variable. The cause of this heterogeneity is not well understood. METHODS AND RESULTS: We recently developed a model of gradual proximal aortic constriction in the adult canine that mimicked the heterogeneity of the hypertrophic response seen in humans. We hypothesized that differences in outcome were related to differences present before banding. Fifteen animals were studied initially. Ten developed left ventricular dysfunction (dys group). Five dogs maintained normal function (nl group). At baseline, the nl group had a lower mean systolic wall stress (96 +/- 9 kdyne/cm2; dys group, 156 +/- 7 kdyne/cm2; P < .0002) and greater relative left ventricular mass (left ventricular weight [g]/body wt [kg], 5.1 +/- 0.36; dys group, 3.9 +/- 0.26; P < .02). On the basis of differences in mean systolic wall stress at baseline, we predicted outcome in the next 28 dogs by using a cutoff of 115 kdyne/cm2. Eighteen of 20 dogs with baseline mean systolic stress > 115 kdyne/cm2 developed dysfunction whereas 6 of 8 dogs with resting stress < or = 115 kdyne/cm2 maintained normal function. CONCLUSIONS: We conclude that this canine model mimicked the heterogeneous hypertrophic response seen in humans. In the group that eventually developed dysfunction there was less cardiac mass despite 60% higher wall stress at baseline, suggesting a different set point for regulating myocardial growth in the two groups.

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.

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.

Fetal surgical protocols in Yucatan miniature swine.

Thirty-nine Yucatan miniature swine were used in three fetal surgical experimental protocols. They involved antiarrhythmic administration, pacemaker implantation, and in-utero diagnosis of ventricular septal defect by intraoperative echocardiography. Because of problems encountered with surgical protocols in the initial stages, modifications were made to prevent fetal hypothermia and intraoperative mortality. These modifications included environmental temperature support, staple surgical techniques to reduce operative time, and development of fetal catheters designed to facilitate cannulation of small vessels. Postoperative care protocols were intensive and included antibiotics, analgesics, and supportive care designed to reduce discomfort and prevent abortion and sepsis. Thirty-seven of 39 sows survived the surgical procedures; experiments were performed on 117 fetuses. Twenty-two fetuses died either intraoperatively or postoperatively because of complications related to the experimental protocols. Modification of surgical and postsurgical protocols for these projects demonstrates the feasibility of using miniature swine as a model for fetal surgery, when their use was appropriate for anatomic and physiologic reasons.