Surgical Implantation of Single-Staged Tissue-Engineered Urothelial Tubes in a Minipig Model.

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has been harvesting gastrointestinal tissue for non-orthotopic reconstruction due to its abundance to reestablish normal function in the patient. The clinical outcomes after rearranging native tissues within the body are often associated with significant morbidity; thus, tissue engineering holds specific potential within this field of surgery. Despite substantial advances, tissue-engineered scaffolds have not yet been established as a valid surgical treatment alternative, mainly due to the costly and complex requirements of materials, production, and implantation. In this protocol, we present a simple and accessible collagen-based tubular scaffold embedded with autologous organ-specific tissue particles, designed as a conduit for urinary diversion. The scaffold is constructed during the primary surgical procedure, comprises commonly available surgical materials, and requires conventional surgical skills. Secondly, the protocol describes an animal model designed to evaluate the short-term in vivo outcomes post-implantation, with the possibility of additional variations to the procedure. This publication aims to demonstrate the procedure step-by-step, with special attention to the use of autologous tissue and a tubular form.

Angiogenesis PET Tracer Uptake (68Ga-NODAGA-E[(cRGDyK)]2) in Induced Myocardial Infarction and Stromal Cell Treatment in Minipigs.

Angiogenesis is considered integral to the reparative process after ischemic injury. The αvß3 integrin is a critical modulator of angiogenesis and highly expressed in activated endothelial cells. 68Ga-NODAGA-E[(cRGDyK)]2 (RGD) is a positron-emission-tomography (PET) ligand targeted towards αvß3 integrin. The aim was to present data for the uptake of RGD and correlate it with histology and to further illustrate the differences in angiogenesis due to porcine adipose-derived mesenchymal stromal cell (pASC) or saline treatment in minipigs after induction of myocardial infarction (MI). Three minipigs were treated with direct intra-myocardial injection of pASCs and two minipigs with saline. MI was confirmed by 82Rubidium (82Rb) dipyridamole stress PET. Mean Standardized Uptake Values (SUVmean) of RGD were higher in the infarct compared to non-infarct area one week and one month after MI in both pASC-treated (SUVmean: 1.23 vs. 0.88 and 1.02 vs. 0.86, p < 0.05 for both) and non-pASC-treated minipigs (SUVmean: 1.44 vs. 1.07 and 1.26 vs. 1.04, p < 0.05 for both). However, there was no difference in RGD uptake, ejection fractions, coronary flow reserves or capillary density in histology between the two groups. In summary, indications of angiogenesis were present in the infarcted myocardium. However, no differences between pASC-treated and non-pASC-treated minipigs could be demonstrated.

Angiogenesis PET Tracer Uptake ((68)Ga-NODAGA-E[(cRGDyK)]2) in Induced Myocardial Infarction in Minipigs.

Angiogenesis is part of the healing process following an ischemic injury and is vital for the post-ischemic repair of the myocardium. Therefore, it is of particular interest to be able to noninvasively monitor angiogenesis. This might, not only permit risk stratification of patients following myocardial infarction, but could also facilitate development and improvement of new therapies directed towards stimulation of the angiogenic response. During angiogenesis endothelial cells must adhere to one another to form new microvessels. αvß3 integrin has been found to be highly expressed in activated endothelial cells and has been identified as a critical modulator of angiogenesis. (68)Ga-NODAGA-E[c(RGDyK)]2 (RGD) has recently been developed by us as an angiogenesis positron-emission-tomography (PET) ligand targeted towards αvß3 integrin. In the present study, we induced myocardial infarction in Göttingen minipigs. Successful infarction was documented by (82)Rubidium-dipyridamole stress PET and computed tomography. RGD uptake was demonstrated in the infarcted myocardium one week and one month after induction of infarction by RGD-PET. In conclusion, we demonstrated angiogenesis by noninvasive imaging using RGD-PET in minipigs hearts, which resemble human hearts. The perspectives are very intriguing and might permit the evaluation of new treatment strategies targeted towards increasing the angiogenetic response, e.g., stem-cell treatment.

Myocardial perfusion of infarcted and normal myocardium in propofol-anesthetized minipigs using (82)Rubidium PET.

Cardiac Rubidium-82 ((82)Rb) positron-emission-tomography (PET) is a good method for quantification of myocardial blood flow in man. Quantification of myocardial blood flow in animals to evaluate new treatment strategies or to understand underlying disease is also of great interest but raises some challenges. Animals, which have been anesthetized during PET acquisition, might react differently to used stress medications, and therefore difficulties might exist while evaluating the resulting PET images using standard software packages from commercial vendors optimized for human hearts. Furthermore propofol, used for anesthesia, can influence myocardial perfusion and coronary flow reserve due to its vasorelaxant effect, and interactions might exist between propofol and used stress agents, potentially affecting the result of the examination. We present cardiac (82)Rb-PET studies performed in propofol-anesthetized minipigs with normal and infarcted myocardium stressed with both adenosine and dipyridamole. Despite the mentioned challenges, we were able to trace the small minipig heart with software designed for human cardiac PET and to achieve blood flow measurements comparable with results in humans with both adenosine and dipyridamole. We found dipyridamole to be a superior stress agent for this experimental setup. Finally, we were able to clearly identify the myocardial perfusion defect after an induced myocardial infarction.

Modelling severe Staphylococcus aureus sepsis in conscious pigs: are implications for animal welfare justified?

BACKGROUND: A porcine model of haematogenous Staphylococcus aureus sepsis has previously been established in our research group. In these studies, pigs developed severe sepsis including liver dysfunction during a 48 h study period. As pigs were awake during the study, animal welfare was challenged by the severity of induced disease, which in some cases necessitated humane euthanasia. A pilot study was therefore performed in order to establish the sufficient inoculum concentration and application protocol needed to produce signs of liver dysfunction within limits of our pre-defined humane endpoints. METHODS: Four pigs received 1 × 10(8) cfu/kg BW of S. aureus, and two controls were sham inoculated with saline. A fixed infusion rate of 3 mL/min was used, while the inoculum concentration, i.e., the dose volume, was changed between the pigs. The following dose volumes were used: 10 mL (n = 1), 20 mL (n = 2), and 30 mL (n = 1), corresponding to infusion durations of 3.33, 6.66, and 10 min at dose rates of 3 × 10(7), 1.5 × 10(7), and 1 × 10(7) cfu/min/kg BW, respectively. Blood samples were drawn for complete blood count, clinical chemistry, and inflammatory markers before and every 6 h after inoculation. Prior to euthanasia, a galactose elimination capacity test was performed to assess liver function. Pigs were euthanised 48 h post inoculation for necropsy and histopathological evaluation. RESULTS: While infusion times of 6.66 min, and higher, did not induce liver dysfunction (n = 3), the infusion time of 3.33 min (n = 1) caused alterations in parameters similar to what had been seen in our previous studies, i.e., increasing bilirubin and aspartate aminotransferase, as well as histopathological occurrence of intravascular fibrin split products in the liver. This pig was however euthanised after 30 h, according to humane endpoints. CONCLUSIONS: A usable balance between scientific purpose and animal welfare could not be achieved, and we therefore find it hard to justify further use of this conscious porcine sepsis model. In order to make a model of translational relevance for human sepsis, we suggest that future model versions should use long-term anaesthesia.

Influence of intense neuromuscular blockade on surgical conditions during laparotomy: a pig model.

PURPOSE: Intense neuromuscular block may improve surgical conditions in ileus laparotomies; however, it is difficult to evaluate. The aim of this study was to investigate if neuromuscular block improved surgical conditions in pigs with artificial ileus laparotomy. METHODS: Six pigs were endotracheally intubated, mechanically ventilated, anesthetized with propofol and fentanyl, and randomized into two groups in a cross-over assessor-blinded design. Neuromuscular block was established with rocuronium. Artificial laparotomy for ileus was performed. We investigated the influence of intense neuromuscular block on surgical conditions with a subjective rating scale, force needed to close the fascia, incidences of abdominal contractions while suctioning the lungs, width of the wound diastase and operating time as outcome parameters. RESULTS: In all six pigs no abdominal contractions occurred while suctioning the lungs at intense neuromuscular block. Without neuromuscular block we detected abdominal contractions seen as hiccups and bucking. In all six pigs during intense neuromuscular block we found no visible electromyographic (EMG) activity in the abdominal muscles while suctioning the lungs. Without neuromuscular block suctioning the lungs elicited brief periods of abdominal EMG activity. No difference was found in the force needed to close the fascia when comparing no neuromuscular block with intense neuromuscular block. Furthermore, no significant differences were found in the width of the diastase, operating time and subjective ratings using a four-point rating scale when comparing no neuromuscular block with intense neuromuscular block. However, these outcomes were related to the order of the suturing round. CONCLUSION: Intense neuromuscular block prevented abdominal muscle contractions but did not influence the force needed to close the fascia.

An improved method for lifting and transporting anesthetized pigs within an animal facility.

Transporting anesthetized pigs in a laboratory setting often requires strenuous manual lifting, posing a hazard to the safety of animal care personnel and to the welfare of the pigs. The authors developed an improved approach to lifting and transporting anesthetized pigs weighing up to 350 kg using mechanical lifts. Different equipment was used to accommodate pigs of different sizes as well as the building designs of three animal facilities. Using the lifts, anesthetized pigs are carried on sheets to maintain their comfort while being transported. The approach refines previous methods for handling and transporting anesthetized pigs and reduces the risk of injury to personnel.

Monitoring of intense neuromuscular blockade in a pig model.

Intense neuromuscular blockade (NMB) measured by post tetanic count (PTC) was monitored, reversed and verified in this pig model. In a cross-over assessor blinded design six pigs were randomized to either no NMB followed by intense NMB, or intense NMB followed by no NMB. Neuromuscular measurements were performed with acceleromyography [train-of-four (TOF) Watch SX]. In all pigs, the response to TOF nerve stimulation was stable and intense NMB (PTC 0-1) was established with rocuronium 3 mg/kg. For reversal, the pigs received sugammadex 20-35 mg/kg and returned to TOF-ratio above 0.90 within 2.15 min after injection. We established a pig model for monitoring intense NMB with surface stimulation electrodes and acceleromyography. We verified total relaxation of the diaphragm and the abdominal muscles at the PTC 0-1 by suction test and with surface electromyography. This pig model is suitable for studies with experimental abdominal surgery with monitoring of intense NMB, and where relaxation of the diaphragm and the abdominal muscles are required.

Laparoscopic Roux-en-Y gastric bypass in super obese Göttingen minipigs.

BACKGROUND: The specific mechanisms behind weight loss and comorbidity improvements in obese patients after Roux-en-Y gastric bypass (RYGBP) are still poorly understood. The aim of this study was to establish and evaluate the feasibility of a long-term survival RYGBP model in super obese Göttingen minipigs in order to improve the translational potential relative to current animal models. METHODS: Eleven Göttingen minipigs with diet-induced obesity underwent laparoscopic RYGBP and were followed up to 9 months after surgery. Intra- and post-operative complications, body weight (BW), food intake and necropsy data were recorded. RESULTS: Five minipigs survived without complications to the end of the study. Four minipigs developed surgical related complications and were euthanized while two minipigs died due to central venous catheter related complications. BW and food intake is reported for the six minipigs surviving longer than 4.5 months post-surgery. Weight loss and reduced food intake was seen in all minipigs. After 2-3 months of weight loss, weight regain was evident in all but two minipigs which seemed to continue losing weight. Necropsy revealed some variation in the length of the alimentary, biliary and common limb between minipigs. CONCLUSION: The use of obese Göttingen minipigs as a translational RYGBP model is feasible and has potential for the study of RYGBP-related changes in gut function, type-2 diabetes and appetite regulation. Still, the surgical procedure is technically highly demanding in obese Göttingen minipigs and the peri-operative animal care and follow up requires close monitoring.