Which clip? A prospective comparative study of retention rates of endoscopic clips on normal mucosa and ulcers in a porcine model.

BACKGROUND/AIM: There are currently no data on the relative retention rates of the Instinct clip, Resolution clip, and QuickClip2Long. Also, it is unknown whether retention rate differs when clips are applied to ulcerated rather than normal mucosa. The aim of this study is to compare the retention rates of three commonly used endoscopic clips. MATERIALS AND METHODS: Six pigs underwent upper endoscopy with placement of one of each of the three types of clips on normal mucosa in the gastric body. Three mucosal resections were also performed to create "ulcers." Each ulcer was closed with placement of one of the three different clips. Repeat endoscopy was performed weekly for up to 4 weeks. RESULTS: Only the Instinct and Resolution clips remained attached for the duration of the study (4 weeks). At each time point, a greater proportion of Instinct clips were retained on normal mucosa, followed by Resolution clips. QuickClip2Long had the lowest retention rate on normal mucosa. Similar retention rates of Instinct clips and Resolution clips were seen on simulated ulcers, although both were superior to QuickClip2Long. However, the difference did not reach statistical significance. All QuickClip2Long clips were dislodged at 4 weeks in both the groups. CONCLUSIONS: The Resolution and Instinct clips have comparable retention rates and both appeared to be better than the QuickClip2Long on normal mucosa-simulated ulcers; however this did not reach statistical significance. Both the Resolution clip and the Instinct clip may be preferred in clinical situations when long-term clip attachment is required, including marking of tumors for radiotherapy and anchoring feeding tubes or stents. Either of the currently available clips may be suitable for closure of iatrogenic mucosal defects without features of chronicity.

Extracorporeal fetal support: a new animal model with preservation of the placenta.

BACKGROUND: Previous models of support for premature sheep fetuses have consisted of cesarean delivery followed by catheterization of umbilical or central vessels and support with extracorporeal membrane oxygenation (ECMO). The limitations of these models have been insufficient blood flow, significant fetal edema, and hemorrhage related to anticoagulation. METHODS: We performed a gravid hysterectomy on 13 ewes between 135 and 145days gestational age. The uterine vessels were cannulated bilaterally and circulatory support was provided via ECMO. Successful transition was defined as maintenance of fetal heart rate for 30minutes after establishing full extracorporeal support. Circuit flow was titrated to maintain mixed venous oxygen saturation (SvO2) of 70-75%. RESULTS: Seven experiments were successfully transitioned to ECMO, with an average survival time of 2hours 9minutes. The longest recorded time from cannulation to death was 6hours 14minutes. By delivering a circuit flow of up to 2120ml/min, all but one of the transitioned uteri were maintained within the desired SvO2 range. CONCLUSION: We report a novel animal model of fetal ECMO support that preserves the placenta, mitigates the effects of heparin, and allows for increased circuit flow compared to prior techniques. This approach may provide insight into a technique for future studies of fetal physiology.

Modeling of the renal kinetics of the AT1 receptor specific PET radioligand [11C]KR31173.

PURPOSE: The radioligand [(11)C]KR31173 has been introduced for PET imaging of the angiotensin II subtype 1 receptor (AT1R). The purpose of the present project was to employ and validate a compartmental model for quantification of the kinetics of this radioligand in a porcine model of renal ischemia followed by reperfusion (IR). PROCEDURES: Ten domestic pigs were included in the study: five controls and five experimental animals with IR of the left kidney. To achieve IR, acute ischemia was created with a balloon inserted into the left renal artery and inflated for 60 minutes. Reperfusion was achieved by deflation and removal of the balloon. Blood chemistries, urine specific gravity and PH values, and circulating hormones of the renin angiotensin system were measured and PET imaging was performed one week after IR. Cortical time-activity curves obtained from a 90 min [(11)C]KR31173 dynamic PET study were processed with a compartmental model that included two tissue compartments connected in parallel. Radioligand binding quantified by radioligand retention (80 min value to maximum value ratio) was compared to the binding parameters derived from the compartmental model. A binding ratio was calculated as DVR = DV(S)/DV(NS), where DV(S) and DV(NS) represented the distribution volumes of specific binding and nonspecific binding. Receptor binding was also determined by autoradiography in vitro. RESULTS: Correlations between rate constants and binding parameters derived by the convolution and deconvolution curve fittings were significant (r > 0.9). Also significant was the correlation between the retention parameter derived from the tissue activity curve (Y(ret)) and the retention parameter derived from the impulse response function (f(ret)). Furthermore, significant correlations were found between these two retention parameters and DVR. Measurements with PET showed no significant changes in the radioligand binding parameters caused by IR, and these in vivo findings were confirmed by autoradiography performed in vitro. CONCLUSIONS: Correlations between various binding parameters support the concept of the parallel connectivity compartmental model. If an arterial input function cannot be obtained, simple radioligand retention may be adequate for estimation of in vivo radioligand binding.

A modified heterotopic swine hind limb transplant model for translational vascularized composite allotransplantation (VCA) research.

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression. This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring. Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

Comparison of peritoneal adhesion formation in bowel retraction by cotton towels versus the silicone lap pak device in a rabbit model.

OBJECTIVE: Manipulation of cotton operating room towels within the abdominal cavity in open abdominal surgery has been associated with the formation of peritoneal adhesions. In a rabbit model, the use of standard cotton operating room towels is compared to the Lap Pak, a silicone bowel-packing device, to determine the potential for reducing the risk of adhesions. METHODS: Thirty rabbits were randomly assigned to 3 groups. The rabbits underwent a sham surgery with incision only (n = 10), placement of operating room towels (n = 10), or placement of a Lap Pak (n = 10). After 14 days, the rabbits were sacrificed and the peritoneal cavity explored for adhesions. The number, tenacity, ease of dissection, and density of adhesions were recorded, and the adhesions quantitatively graded using a Modified Hopkins Adhesion scoring system. RESULTS: The operating room towel group had an average adhesion score of 2.5, and 8 (80%) rabbits developed adhesions. The sham group had an average adhesion score of 0.3 and one rabbit (10%) developed adhesions. The Lap Pak group had an average adhesion score of 0.2 and 1 rabbit (10%) developed adhesions. The frequency and severity of adhesions in the operating room towel group were significantly greater from that of the baseline sham group. There was no significant difference between the Lap Pak and sham groups. CONCLUSIONS: In this rabbit laparotomy model, the use of the Lap Pak to retract the bowels resulted in significantly fewer adhesions compared to cotton operating room towels. Lap Pak may be beneficial for bowel packing in general abdominal surgeries.

Large quantities of Abeta peptide are constitutively released during amyloid precursor protein metabolism in vivo and in vitro.

The metabolism of the amyloid precursor protein (APP) has been extensively investigated because its processing generates the amyloid-ß-peptide (Aß), which is a likely cause of Alzheimer disease. Much prior research has focused on APP processing using transgenic constructs and heterologous cell lines. Work to date in native neuronal cultures suggests that Aß is produced in very large amounts. We sought to investigate APP metabolism and Aß production simultaneously under more physiological conditions in vivo and in vitro using cultured rat cortical neurons and live pigs. We found in cultured neurons that both APP and Aß are secreted rapidly and at extremely high rates into the extracellular space (2-4 molecules/neuron/s for Aß). Little APP is degraded outside of the pathway that leads to extracellular release. Two metabolic pools of APP are identified, one that is metabolized extremely rapidly (t1/2;) = 2.2 h), and another, surface pool, composed of both synaptic and extrasynaptic elements, that turns over very slowly. Aß release and accumulation in the extracellular medium can be accounted for stoichiometrically by the extracellular release of ß-cleaved forms of the APP ectodomain. Two α-cleavages of APP occur for every ß-cleavage. Consistent with the results seen in cultured neurons, an extremely high rate of Aß production and secretion from the brain was seen in juvenile pigs. In summary, our experiments show an enormous and rapid production and extracellular release of Aß and the soluble APP ectodomain. A small, slowly metabolized, surface pool of full-length APP is also identified.

Infection during natural orifice transluminal endoscopic surgery: a randomized, controlled study in a live porcine model.

BACKGROUND: Currently reported natural orifice transluminal endoscopic surgery (NOTES) procedures in animals have been done in heterogeneous milieus ranging from nonsterile to sterile procedures, with mixed results, including no infection in those performed in nonsterile settings. OBJECTIVE: To establish the potential frequency of infection during NOTES, comparing sterile to nonsterile approaches. SETTING: Survival experiments on sixteen 50-kg pigs. DESIGN AND INTERVENTIONS: From pilot data (100% infection frequency after nonsterile procedures), sample size (8 animals in each group) was calculated by using a power of 95% and an alpha risk of 0.05. The animals were randomly assigned to two groups: In the transgastric peritoneoscopy study group, liver and ovarian biopsies were performed with sterile overtubes, endoscopes, and accessories and the use of preoperative intravenous antibiotics and antiseptic gastric lavage. In the nonsterile (control) group, the same procedures were performed with nonsterile endoscopes and accessories without the use of gastric lavage and preoperative antibiotics. Complete transmural closure of the transgastric access site was made in all animals. After a 1-week survival time, all animals were killed for necropsy, which included Gram staining and peritoneal cultures. MAIN OUTCOME MEASUREMENTS: Intraperitoneal infection on necropsy. RESULTS: All necropsies revealed intraperitoneal infection (abscesses, fibrinopurulent exudates, and adhesions) in the control group (frequency of infection 100%). Peritoneal bacterial culture grew various aerobic and anaerobic organisms. No gross or bacteriological evidence of infection was seen in the sterile group (frequency of infection 0%, P value = <.0002). LIMITATIONS: Animal experiments. CONCLUSION: Nonsterile conditions invariably lead to intraperitoneal infection. Aseptic techniques during NOTES can prevent intra-abdominal infection. Future studies will determine which infection prevention steps are mandatory and which can be omitted during NOTES procedures.

Successful diagnostic and therapeutic intrauterine fetal interventions by natural orifice transluminal endoscopic surgery (with videos).

BACKGROUND: Despite advances in the development of fetal surgery, morbidity and mortality are substantial. A natural orifice transluminal endoscopic surgery (NOTES)-guided approach to the gravid uterus may offer a less-invasive technique. OBJECTIVE: To assess the feasibility of NOTES for diagnostic and therapeutic intrauterine fetal interventions. SETTING: Survival and nonsurvival experiments on pregnant sheep. DESIGN AND INTERVENTION: Nonsurvival experiments performed in 2 pregnant sheep (80-110 days' gestation). A third ewe underwent NOTES and survived for 4 weeks. Transgastric (nonsurvival ewes) and transvaginal (1 nonsurvival and the survived ewe) peritoneoscopy was performed after standard needle-knife entry into the peritoneal cavity. Endoscopic access to the gravid uterus was assessed. EUS-guided, transuterine injection of saline solution into the fetal cardiac ventricle and vessels was attempted in all. MAIN OUTCOME MEASUREMENTS: Feasibility of NOTES- and EUS-guided intervention in a pregnant animal model, visibility of fetal parts via EUS compared with transabdominal US. RESULTS: Entry into the peritoneal cavity was achieved in each ewe. Access to and complete visualization of the gravid uterus were successful once within the abdominal cavity. Visualization of the fetal parts and the placental cotyledons by EUS was achieved in all animals. EUS-guided amniocenteses and transuterine intracardiac and intravascular injection of saline were successful. There were no complications or preterm delivery after the procedures. LIMITATIONS: Animal model. CONCLUSIONS: NOTES is technically feasible in the pregnant ewe. Intraperitoneal EUS via a NOTES approach provides excellent access and visualization of the intrauterine cavity and fetal parts.

Refinement of canine pancreatitis model: inducing pancreatitis by using endoscopic retrograde cholangiopancreatography.

The causes and treatments of pancreatitis have been studied in diverse species, but the canine pancreatitis model has been used most often due to its similarities to the condition in humans. Although pancreatitis in dogs can be induced readily by numerous methods, managing these dogs can be difficult because they often develop severe abdominal pain, vomiting, inappetance, and lethargy. In an effort to study pancreatitis, we performed a pilot study to determine whether an endoscopic pancreatic procedure would be possible in a dog and whether, through various manipulations, a new method of inducing pancreatitis could be developed. The model uses endoscopic retrograde cholangiopancreatography (ERCP), a common procedure in human gastroenterology that has been associated with postprocedural pancreatitis. Although all 8 dogs used in developing the ERCP model had both biochemical and histologic changes consistent with pancreatitis, 7 of the 8 dogs remained free of classic clinical signs of the disease. This method is presented as a refinement of a canine model and presents an alternative method of inducing pancreatitis, with decreased risk of developing associated clinical signs.

Fas-mediated apoptosis regulates the composition of peripheral alphabeta T cell repertoire by constitutively purging out double negative T cells.

BACKGROUND: The Fas pathway is a major regulator of T cell homeostasis, however, the T cell population that is controlled by the Fas pathway in vivo is poorly defined. Although CD4 and CD8 single positive (SP) T cells are the two major T cell subsets in the periphery of wild type mice, the repertoire of mice bearing loss-of-function mutation in either Fas (lpr mice) or Fas ligand (gld mice) is predominated by CD4(-)CD8(-) double negative alphabeta T cells that also express B220 and generally referred to as B220+DN T cells. Despite extensive analysis, the basis of B220+DN T cell lymphoproliferation remains poorly understood. In this study we re-examined the issue of why T cell lymphoproliferation caused by gld mutation is predominated by B220+DN T cells. METHODOLOGY AND PRINCIPAL FINDINGS: We combined the following approaches to study this question: Gene transcript profiling, BrdU labeling, and apoptosis assays. Our results show that B220+DN T cells are proliferating and dying at exceptionally high rates than SP T cells in the steady state. The high proliferation rate is restricted to B220+DN T cells found in the gut epithelium whereas the high apoptosis rate occurred both in the gut epithelium and periphery. However, only in the periphery, apoptosis of B220+DN T cell is Fas-dependent. When the Fas pathway is genetically impaired, apoptosis of peripheral B220+DN T cells was reduced to a baseline level similar to that of SP T cells. Under these conditions of normalized apoptosis, B220+DN T cells progressively accumulate in the periphery, eventually resulting in B220+DN T cell lymphoproliferation. CONCLUSIONS/SIGNIFICANCE: The Fas pathway plays a critical role in regulating the tissue distribution of DN T cells through targeting and elimination of DN T cells from the periphery in the steady state. The results provide new insight into pathogenesis of DN T cell lymphoproliferation.

Etanercept, a TNF-alpha binding agent, is ineffective in the prevention of post-ERCP pancreatitis in canines.

CONTEXT: The incidence of post-ERCP pancreatitis is 1-22%. It continues to be a difficult problem for endoscopist and patient. Uncovering an agent that may be used to prevent its occurrence is critical. OBJECTIVE: The aim of our study was to investigate the role of etanercept in the prevention of post-ERCP pancreatitis. DESIGN: Endoscopic retrograde pancreatography (ERP)-induced injury was performed in dogs using a previously established endoscopic model of post-ERCP pancreatitis. ANIMALS: Eight study dogs underwent ERP: 4 were pre-treated with etanercept one day before the procedure and 4 were untreated. In addition, three control dogs not undergoing ERP were also studied. MAIN OUTCOME MEASURES: Serum levels of amylase, lipase, and TNF-alpha, as well as the ratio of urinary trypsinogen activation peptide (TAP) and urinary creatinine, were measured before and after ERP. Necropsy was performed on post-operative day 5. All pancreatic specimens were graded by two blinded pathologists according to a validated scoring system. RESULTS: Eight study dogs developed mild to moderate clinical pancreatitis with hyperamylasemia (11,538+/-4,065 U/L vs. 701+/-157 U/L; post-ERP peak levels vs. baseline values: P<0.001) and hyperlipasemia (3,637+/-2,333 U/L vs. 246+/-125 U/L; P=0.003). Mean total injury score was significantly elevated in study dogs compared to control dogs (6.16+/-1.85 vs. 1.06+/-0.49; P=0.001). There were escalating total injury scores concordant with more elaborate methods of endoscopically-induced injury although the trend did not reach the statistical significance (P=0.223). When comparing untreated to etanercept-treated dogs, there were no significant differences in serum amylase levels (P=0.903), serum lipase levels (P=0.771), TAP/creatinine urinary ratio (P=0.912), and pancreatic injury score (P=0.324). CONCLUSION: Etanercept is ineffective in prevention of mild to moderate post-ERCP pancreatitis in canines. ERP-induced pancreatic injury can be used as a reliable animal model for studies investigating therapy and prevention of post-ERCP pancreatitis.

Cervicovaginal evaluation in macaques used as a model for topical microbicide safety studies.

INTRODUCTION: Macaques are a commonly used non-human primate (NHP) model to evaluate safety and efficacy of topically applied vaginal microbicides. Cervicovaginal evaluation for topical microbicide safety studies requires proper technique, equipment, supplies, and sequence of sample collection. MATERIALS AND METHODS: Eleven rhesus macaques received a comprehensive sequential cervicovaginal examination under sedation before treatment and 24 hours post-instillation of test material. Examination was initiated with colposcopy, followed by diagnostics including vaginal culture, pH determination, cervicovaginal lavage, and cervicovaginal biopsy. RESULTS: Overall, the methods performed yielded samples that were appropriate for diagnostic evaluation and interpretation, and the macaques experienced minimal discomfort and complications. DISCUSSION: This paper provides a descriptive summary of compiled techniques required to conduct a safety evaluation for topically applied vaginal microbicides. This novel method-based approach should be methodically executed when evaluating a vaginally-applied, topical microbicide candidate.

Effects of bedding substrates on microsomal enzymes in rabbit liver.

Previous studies have reported that housing rats and mice on softwood beddings induce microsomal enzymes. To date, no published studies investigate effects of softwood beddings on microsomal induction in rabbits. The purpose of this study was to determine whether microsomal enzymes, primarily cytochromes P450 3A and 2B, were induced in rabbits exposed to commonly used bedding substrates. Rabbits were placed in cages 7.6 cm above 1 sheet of 24 x 36 in. postconsumer recycled paper, approximately 16 cups (130 ounces) of pine shavings, or no substrate. Positive-control rabbits were given either rifampin (50 mg/kg) or phenobarbital (60 mg/kg) intraperitoneally once daily for 5 d. At 2, 7, and 14 d after placement in test cages, rabbits were euthanized and the livers harvested. Microsomal pellets were prepared from the livers and used in an erythromycin Ndemethylase assay (to determine CYP3A activity) and a pentoxyresorufin-O-deethylation assay (to determine CYP2B activity). Although the levels of enzyme induction varied slightly in both assays, statistical significance was not reached compared to the positive-control levels. These results indicate that neither CYP450 3A or 2B enzymes are induced by exposure of rabbits to pine shavings or paper substrate as noncontact bedding for up to 14 d.

Complete blood count, clinical chemistry, and serology profile by using a single tube of whole blood from mice.

Clinical pathology is a valuable means for assessing specific organ pathology and a screening tool for general animal health. Routine clinical pathology evaluation in mice usually includes whole blood for a complete blood count (CBC) and a clinical biochemistry analysis. Acquisition and analysis of these samples can be problematic due to the small volumes of blood that can be obtained from a mouse. Typically, a complete blood count requires blood from a tube containing an anticoagulant, whereas a clinical biochemistry profile needs blood from a serum clot tube. Because of the small volume that can be obtained, splitting the blood from a single mouse into 2 different tubes may result in inadequate samples to perform the desired tests or introduce inaccuracies. We explored the feasibility of using a single lithium heparin tube for generation of a CBC, biochemistry profile, and serology profile. We also evaluated the consistency of CBC data, including the quality of a peripheral blood smear taken from a lithium heparin or EDTA tube after various storage times. We found that CBC, biochemistry, and serology profiles could be obtained more readily when blood samples were placed in a single lithium heparin tube than in 2 separate tubes. In addition, the quality of blood smears and CBC results from the lithium heparin tube were comparable (with few exceptions) to those from an EDTA tube after prolonged storage.

Determination of serum organic acids in puppies with naturally acquired parvoviral enteritis.

The purpose of the present study was to investigate the acid-base status and the serum concentration of organic acids in puppies with naturally occurring canine parvoviral enteritis. Between July 1999 and July 2000, 25 client-owned puppies admitted to the St. Louis Animal Emergency Clinic South for treatment of enteritis caused by parvovirus infection were used in our study. Control blood samples were collected from 22 healthy puppies less than 9 months of age. Serum organic acid concentrations were quantitatively determined by HPLC. Puppies infected with parvovirus had significantly lower plasma concentrations of sodium, potassium, chloride, and bicarbonate than controls. Although serum L-lactate tended to increase in some puppies with canine parvoviral enteritis, our study demonstrated that most affected puppies developed only mild compensated metabolic acidosis. None of the affected puppies had an elevated serum D-lactate concentration at admission.