Goettingen Minipigs Research Articles

Coronary arterial lesions induced by endothelin antagonists

Endothelins are potent vasoconstrictors and pressor peptides and are important mediators of cardiac, renal and endocrine functions. Increased ET-1 levels in disease states such as congestive heart failure, pulmonary hypertension, acute myocardial infarction, and renal failure suggest the endothelin system as an attractive target for pharmacotherapy. A non-peptidic, selective, competitive endothelin receptor antagonist with an affinity for the ET A receptor in the subnanomolar range was administered by continuous intravenous infusion to beagle dogs, rats, and Goettingen minipigs. It caused mild arteriopathy characterised by segmental degeneration in the media of mid- to large-size coronary arteries in the heart of dog, but not rat or minipig. The lesions only occurred in the atrium and ventricle. Frequency and severity of the vascular lesions was not sex or dose related. No effects were noted in blood vessels in other organs or tissue. Plasma concentrations at steady state, and overall exposure in terms of AUC (0–24h) were higher in minipig and rat than the dog but did not cause cardiac arteriopathy. These findings concur with those caused by other endothelin anatagonists, vasodilators and positive inotropic/vasodilating drugs such as potassium channel openers, phosphodiesterase inhibitors and peripheral vasodilators, and confirm that dogs appear to be uniquely sensitive to the development of cardiac vascular lesions.

Pharmacological effects of epidermal growth factor (EGF) with focus on the urinary and gastrointestinal tracts

Epidermal growth factor (EGF) belongs to a family of growth factor ligands and receptors. At present, five ligands have been recognized which as EGF exert their effects via binding to the same EGF receptor. The family has three other receptors erbB2, erbB3, and erbB4, which have their own ligands (the heregulins). The system is ubiquitously distributed in mammals, and has important roles in normal development, and in regenerative and neoplastic growth. Mouse and human EGF were discovered in 1962 and 1975 by Stanley Cohen and Harry Gregory, respectively, due to EGFs potent systemic effects. EGF accelerated eyelid opening in newborn mice and inhibited gastric acid secretion in humans. Already in the late thirties, a factor in human urine was recognized which prevented or accelerated healing of experimental damage in the gastrointestinal tract. This factor appeared to be EGF. Around 1980, an effect of commercial interest was described-EGF caused shedding of the fleece in sheep. In line with the original observations, several studies have examined effects of EGF on developmental processes. Amongst other effects, EGF accelerates lung and intestinal maturation before birth and in newborn mammals. Due to the possible use of EGF in the wool industry, it was mandatory to know more about EGF. Amongst other effects in mature sheep and other animals are haemodynamic changes, changes in electrolyte homeostasis, and endocrinological changes. In relation to experimental damage, the therapeutic potential of systemic EGF has been demonstrated in all parts of the gastrointestinal tract, in the kidneys, in the liver and in the trachea. EGF has even been tried in humans in gastric ulcer healing and in necrotising enterocolitis. Studies on prolonged treatment with EGF have first recently appeared. We described effects of 4-5 weeks of treatment in Goettingen minipigs and in rats, and two other groups described effects in monkeys and in rats. In summary, species differences were observed. The species of higher order were most sensitive to treatment with EGF. EGF did not consistently change the total body weight despite EGF consistently reduced circulating levels of insulin-like growth factor I (IGF-I) in Goettingen minipigs as well as in rats. Low circulating levels of IGF-I are usually associated with retarded growth. This review mostly focuses on the organs which appeared to be most sensitive to EGF, the urinary and gastrointestinal tracts including the liver and the pancreas. The histopathological changes consisted mainly of epithelial proliferations in the gastrointestinal, urinary and respiratory tracts. These findings match the knowledge obtained from animals overexpressing the EGF agonist, transforming growth factor alpha (TGF alpha), and the mice with a knock out of the gene encoding for the EGF receptor. EGF receptor hyperstimulation (TGF alpha overexpression) in the context of the whole animal leads to epithelial proliferations whereas hypostimulation (EGF receptor knock out) leads to epithelial immaturities. In the minipigs, the epithelia of the oesophagus, ducts of the pancreas, and the urothelium were hyperplastic, the latter two epithelia with accumulation of glycoconjugates. In the rats, the epithelial hyperplasias in these tissues and in the small and large intestines were without glycoconjugate accumulations. In rats, the mucosal proliferations in the intestines resulted in increased mucosal surface area. Mesenchymal growth effects were also noted. In the ureters of the minipigs, smooth muscle cell hyperplasia and hypertrophia were found. The heart of the minipigs was also enlarged, an interesting finding regarding interactions between the different parts of the EGF system, as knock out mice of the receptors erbB2 and erbB4 die due to maldevelopments in the heart. Measurements in blood and serum also revealed consistent changes. (ABSTRACT TRUNCATED)

What is the best possible design of temporary anterior mandibular osteotomy? A histomorphological animal study.

When squamous cell carcinomas in the oral cavity have advanced to the size of T2 to T3 or when they start to encroach on the middle, occasionally it is necessary to divide the mandible at the parasymphysis and swing the hemimandible away from the midline for intraoral exposure of tumor resection. The osteotomy is usually performed in either a straight vertical cut, in a step-cut fashion, or in a dove-tail geometric configuration. The design of the geometric pattern of the osteotomy determines whether or not the postoperative period is prolonged. The authors analyzed the best possible design of temporary anterior mandibular osteotomy. In an animal study of 12 Goettingen minipigs (GMPs) the authors investigated the best possible design of geometric pattern of mandibular osteotomy and the fragment healing process. Their primary interest was directed at the histomorphological bone-healing process after straight vertical bone cut, step-cut fashion, and dove-tail osteotomy of the mandible, and rigid fixation with an osteosynthesis plate. From this study it can be concluded that the dove-tail osteotomy of the mandible is the best possible design of temporary anterior mandibular osteotomy in preventing pseudoarthrosis due to the mainly primary bone contact and gap healing process.

Glycoproteins in the urothelium and in the urine of the epidermal growth factor induced growing urinary tract in rats.

Systemic treatment with epidermal growth factor (EGF) induces growth of all wall layers of the urinary tract in pigs and rats. We have previously described that the EGF stimulated urothelium in Goettingen minipigs accumulates glycoproteins. The aim of the present study was to examine and partly characterize glycoproteins in the urothelium and in the urine from rats treated with EGF. Seventy-two female Wistar rats were allocated into five groups receiving EGF treatment (150 microg/kg per day) for 0 (controls), 1, 2, 3 and 4 weeks before being killed. Glycoconjugates were characterized by means of lectins on tissue sections, and using Western blotting, in bladder extracts and in urine. The characterization mostly focused on the expression of the mucin-type core structures T and Tn using the lectins peanut agglutinin (PNA) and Vicia villosa (VVA) and specific monoclonal antibodies. The thickened EGF-stimulated urothelium retained the normal differentiation pattern as judged from the appearance on electron microscopy and from the expression of carbohydrate structures. Within the urothelium and in the urine there was increased expression of mucin-type glycoproteins suggesting increased urothelial production and excretion of mucin-type glycoproteins. In conclusion, the EGF stimulated hyperplastic urothelium most probably excretes increased amounts of mucin-type glycoproteins to the urine but it retains the normal pattern of differentiation as assessed by lectin characterization.

Systemic treatment with epidermal growth factor in pigs induces ductal proliferations in the pancreas

BACKGROUND & AIMS: Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and the EGF receptor are often overexpressed in chronic pancreatitis and in malignant pancreatic growth. Transgenic mice overexpressing TGF-alpha develop tissue changes in the pancrease resembling changes found in chronic pancreatitis. The effects of systemic treatment with EGF on the porcine pancrease were investigated in this study.METHODS: Mature Goettingen minipigs were treated with solvent (n = 5), EGF (30 micrograms.kg-1.day-1; n = 6) for 4 weeks, or EGF (30 micrograms.kg-1.day-1; n = 5) for 5 weeks followed by 3 weeks of recovery. Pancreata were studied by routine histological examination and electron microscopy and were immunostained for proliferating cell nuclear antigen (PCNA).RESULTS: In the EGF-treated animals, mainly larger interlobular ducts of the pancreas appeared to be considerably hyperplastic, with an increased number of nuclei that stained for PCNA. The epithelia of these ducts were increased in height, with accumulations of glycoconjugates in the columnar cells and in an increased number of goblet cells.CONCLUSIONS: A new approach to experimentally induced hyperplastic changes of the excretory ducts of the pancreas is presented. Because ductal changes with glycoconjugate accumulations are common features of chronic pancreatitis and pancreatic cancer, the findings may be relevant to the pathogeneses of these conditions.(Gastroenterology 1997 Oct;113(4):1367-74)

Chronic systemic treatment with epidermal growth factor induces smooth muscle cell hyperplasia and hypertrophy in the urinary tract of mature Goettingen minipigs.

To determine the effect of systemic treatment with epidermal growth factor (EGF) on the induction of growth in the urinary tract. Mature Goettingen minipigs were treated daily with vehicle (Tris-HCl; n = 5) or EGF (30 micrograms/kg) (n = 6) for 4 weeks. The total number of smooth muscle cells was counted using an optical disector in a 20 microns thick cross-section of the ureter and the mean smooth muscle cell volume estimated. Cell proliferation was detected by immunostaining for the marker Ki67. The ureters of the animals treated with EGF were longer and thicker than those of the controls and the median cross-sectional area of the ureter was 3.3-fold larger; the growth involved all wall layers. The median (range) number of smooth muscle cells in a 20 microns thick cross-section of the ureter was 11 (9-12) x 10(3) in the pigs treated with placebo and 55 (19-80) x 10(3) in those treated with EGF, and the median (range) volume of the smooth muscle cells was 2.3 (2.2-2.4) x 10(3) and 4.0 (3.0-4.5) x 10(3) mm3, respectively. There were two likely mechanisms contributing to smooth muscle cell hyperplasia, the division of fully differentiated smooth muscle cells and division of fibroblasts in the borderline between the submucosal layer and muscular coat, with ensuing differentiation into smooth muscle cells. Treatment with EGF induces the growth of all wall layers in the urinary tract with remarkable hyperplastic and hypertrophic changes of the smooth muscle cells in the muscular coat.

The effect of epidermal growth factor on circulating levels of IGF and IGF-binding proteins in adult Goettingen minipigs.

It has recently been demonstrated that epidermal growth factor (EGF) administration to neonatal rodents causes growth retardation with concomitant reductions in circulation levels of IGF-I. We describe the effects of systemic EGF administration for 4 weeks on circulating levels of IGF-I and IGF-binding proteins (IGFBPs) and on thyroid hormones (tri-iodothyronine, T3; thyroxine, T4) in sexually mature pigs. Goettingen minipigs of either sex were treated with placebo (n = 5) or EGF (30 micrograms/kg per day, n = 6) s.c. for 4 weeks (in relation to an oesophageal sclerotherapy regimen). Blood samples were taken under anaesthesia before and after 1, 2, 3 and 4 weeks of treatment. Circulating levels of IGF-I, insulin, glucose, T3 and T4 were analysed every week and IGFBPs every second week. IGF-I was not reduced significantly after 1 week but significantly reduced after 2 and 3 weeks of EGF treatment. A similar decline was observed for the major IGFBP, IGFBP-3, which was reduced after 2 and 4 weeks. IGFBP-1, IGFBP-2 and IGFBP-4 increased throughout the treatment period (all significantly at week 4). EGF treatment induced increased circulating T3 after 2, 3 and 4 weeks of EGF treatment. In conclusion, we report that EGF treatment for 4 weeks in Goettingen minipigs reduces circulating IGF-I and IGFBP-3, increases circulating IGFBP-1, IGFBP-2 and IGFBP-4, and induces a slight hyperthyroidism as judged from increased circulating levels of T3.

Impedance planimetric characterization of the distal oesophagus in the goettingen minipig

Regional differences in biomechanical properties of the oesophagus were studied in 15 healthy Goettingen minipigs by means of impedance planimetry. The investigation was performed during anaesthesia by stepwise pressure-induced balloon distensions with concomitant measurement of pressure and luminal cross-sectional area (CSA) in the oesophagus 5 and 10 cm above the gastro-oesophageal junction. The circumferential wall tension, circumferential strain and incremental elastic modulus were computed from the measurements of pressure and CSA at steady-state conditions. Probably due to the anaesthesia, only scant peristalsis was recorded and the CSA always reached steady state during the balloon distensions. The CSAs were highest in the distal oesophagus ( P < 0.001). At the highest induced pressure, the CSAs were 605 ± 32 and 453 ± 29 mm 2 (mean ± SEM) for the locations 5 and 10 cm from the gastro-oesophageal junction. The tension-strain distributions were non-linear and the curve obtained 5 cm above the gastro-oesophageal junction was shifted to the right when compared with the curve obtained from 10 cm above this junction. Fitting of the function tension = exp( a + b strain) to the data gave determination coefficients higher than 0.97 and P values lower than 0.001 for both measuring points. The constant a differed between the two locations in the oesophagus ( P < 0.05). In conclusion, the pressure-CSA and the tension-strain distributions differed between the two measuring points suggesting that the elastic properties are different.

Chronic treatment with epidermal growth factor causes esophageal epithelial hyperplasia in pigs and rats

Epidermal growth factor (EGF) is an important factor for maintaining the esophageal functional integrity. Goettingen minipigs were treated with either placebo or subcutaneous EGF (30 micrograms/kg/day) for four weeks. Wistar rats were treated with either placebo or subcutaneous EGF (150 micrograms/kg/day) for four weeks. At sacrifice, esophageal samples were obtained for histology, immunochemistry, and lectin characterization. In pigs, the thickness of the esophageal epithelium was almost doubled in the EGF-treated animals. Characterization with lectins revealed a normal pattern of differentiation. Subcutaneously administered EGF was visualized on cells located basally in the esophageal epithelium. In rats, EGF-treatment increased the esophageal volume of the epithelium, the lamina propria of the mucosa, and the submucosa. In conclusion, systemic EGF challenge induces growth of the esophageal epithelium with an unaltered pattern of differentiation. This supports previous studies demonstrating a beneficial effects of systemic EGF-treatment on sclerotherapy-induced esophageal damage.

Chronic systemic treatment with epidermal growth factor induces hypergastrinaemia in Goettingen minipigs.

Epidermal growth factor (EGF) is an inhibitor of gastric acid secretion. The impact of chronic systemic treatment with EGF on intragastric pH and serum gastrin concentrations has not been investigated previously. Goettingen minipigs were treated with human recombinant EGF (hEGF) or placebo for 4 weeks. Once a week the acidity and protein concentration of gastric juice were determined, and serum gastrin concentrations measured. After 4 weeks, tissue specimens were obtained from the gastric antrum and immunostained for gastrin- and somatostatin-producing G- and D-cells. Furthermore, the development of antibodies against hEGF was evaluated. Subcutaneously administered hEGF, 30 micrograms/kg/day for 4 weeks, included a fourfold increase in basal serum gastrin concentration, increased the number of antral G-cells, and decreased the density of antral D-cells. The acidity of gastric fluid was reduced, and the protein concentration increased. All animals developed low-titred antibodies towards hEGF. The antibodies did not influence the extent to which the individual animal responded to the EGF treatment.

Regional differences in passive elastic wall properties of the oesophagus: an impedance planimetric study in pigs

Abstract Regional differences in biomechanical properties of the oesophagus were studied in seven healthy Goettingen minipigs by means of impedance planimetry. The investigation was done during anaesthesia by stepwise pressure‐induced balloon distensions with concomitant measurement of pressure and luminal cross‐sectional area (CSA) in the oesophagus 5, 10 and 15 em above the gastro‐oesophageal junction (GEJ). The compliance and wall tension were computed from measurements of the pressure and steady‐state CSA. Probably due to the anaesthesia, only scant peristalsis was recorded and the CSA always reached steady state during balloon distension. The CSAs were highest in the distal oesophagus (P &lt; 0.01). At the highest induced pressure, the figures were 665 ± 57, 529 ± 50 and 452 ± 47 mm2 for the locations 5, 10 and 15 cm from the GEJ, respectively. Hysteresis did not differ between the measuring points. The oesophagus was significantly more compliant at low pressures (1–3 kPa) 5 cm above the GEJ compared to the more proximally located measuring points (P &lt; 0.05).In conclusion, regional variations occurred in passive biomechanical properties of the oesophagus with the highest distensibility occurring 5 cm above the GEJ.

Ameliorative Effect of Folic Acid on Pyrimethamine Teratogenesis in Pigs

The effects of oral dosing of folic acid (FA) on pyrimethamine (PYR) teratogenesis were examined in Goettingen minipigs. PYR (3.6 mg/kg/day) was orally administered in the feed to sows with or without FA (2.5, 10 or 50 mg/kg/day) for 12 days, (days 11 - 22 of gestation). Major malformations, such as cleft palate, cleft lips, micrognathia and clubfoot, were observed in all treated groups. The incidence of malformed fetuses decreased dose-dependently as the FA level was raised. This new finding suggests FA administration may prevent newborns from PYR induced malformations. This result contrasts in the observations in rats, in which PYR teratogenesis was potentiated by the concurrent oral dosing of FA. Neither PYR blood concentrations nor plasma protein binding was significantly affected by FA dosing in nonpregnant sows. These results suggest a mechanism other than pharmacokinetic modification was responsible for the protective effect of FA on PYR teratogenesis in minipigs.

Pulmonary capillary pressure and gas exchange after E. coli bacteremia in pigs

In 9 Goettingen minipigs we studied the effect of E. coli bacteremia on effective pulmonary capillary pressure and the longitudinal distribution of pulmonary vascular resistance. Precapillary pressure gradient (dPa) was calculated as the difference between mean pulmonary artery pressure (MPP) and effective pulmonary capillary pressure (Pc) (dPa = MPP-Pc), postcapillary pressure gradient (dPv) as the difference between Pc and left atrial pressure (dPv = Pc-LAP). The disturbance of pulmonary gas exchange was quantified by the AaDO2 quotient 1-PaO2/PAO2. Live E. coli infusion resulted in hypodynamic circulatory failure. Cardiac index fell from 3.7 +/- 0.81 . min-1.m-2 to 2.2 +/- 0.71 .min-1.m-2 after bacteremia lasting for 3.5 h. Simultaneously venous pulmonary vascular resistance rose from 25% of total pulmonary vascular resistance before to 32% after 3.5 h bacteremia, thus raising Pc from 11 mmHg to 16 mmHg. The degree of respiratory insufficiency was correlated with changes of MPP, dPa and dPv: 1-PaO2/PAO2 = 0.2 + 0.035.dPv (r = 0.829). Our results show, that the longitudinal distribution of pulmonary vascular resistance changes during septicemia, thus raising Pc. This may be an important factor in the genesis of septic pulmonary failure.

Experimental induction of splayleg in piglets by pyrimethamine.

Dietary administration of 3.6 mg/kg (BW)/day of pyrimethamine (PYR) to the 10 pregnant Goettingen minipigs for the 1st quarter (during days 11 to 16) or the 2nd quarter (during days 17 to 22) of the organogenetic period elicited the congenital splayleg (CSL) in 20 out of 27 live newborns. Only a splayleg piglet out of 18 was found in the control group. The incidence of stillbirth was higher in the PYR groups than the control group, but no major external malformation was observed in the piglets of the PYR groups. Histological and biochemical examinations of the muscles from the hindleg, foreleg and truncus were carried out. The severity of myofibrillar hypoplasia depended on neither CSL symptoms nor PYR administration. Although a significant decrease of protein content of the muscles was observed in the piglets of the PYR groups, the mechanism underlying the pathogenesis of induced splayleg was obscure.

Role of deacetylation in the nonlinear pharmacokinetics of sulfamonomethoxine in pigs.

The role of deacetylation in the pharmacokinetics of sulfamonomethoxine (SMM) in pigs was studied using 6 Goettingen minipigs and a pig from a commercial breed. The rapid decrease of plasma concentration of the parent compound followed by the rapid increase of plasma concentration of the deacetylated metabolite, SMM, was observed after an i.v. injection of N4-acetylsulfamonomethoxine (AcSMM; 10 mg/kg). The concentration of the metabolite, SMM, was greater than that of AcSMM and after reaching peak, both compounds decreased in parallel on a semilogarithmic graph. On the other hand, the acetylated compound of SMM (AcSMM) appeared in the plasma and reached peak after an i.v. injection of SMM. After reaching the peak, both SMM and AcSMM decreased in parallel. The slopes of the terminal phases of SMM and AcSMM after both injections showed no significant difference. After the i.v. injection of a high dose of SMM (100 mg/kg), a nonlinear pharmacokinetics profile with capacity limited elimination type of SMM was observed. The concentration of plasma AcSMM increased rapidly and the parallel decrease of SMM and AcSMM was observed in both nonlinear and linear phases, while the renal excretion of AcSMM was saturated in the nonlinear phase. The results suggest that the deacetylation in pigs is so strong that it largely affected the pharmacokinetics of SMM in both high and low doses. In spite of the saturation in renal excretion of the main metabolite, AcSMM, after the high dose, the profile of the parallel decrease remained unchanged, which may be due to the rapid conversion of the excess AcSMM to SMM. The converted SMM was added to the existing plasma SMM. As a result, the nonlinear pharmacokinetics of SMM may have occurred after a high dose of SMM in pigs.

Pharmacokinetic analysis of plasma concentration and urinary excretion of sulphamonomethoxine and its metabolite in Goettingen minipigs.

The plasma concentration and renal excretion after a bolus intravenous injection of a low (10 mg/kg) or high (100 mg/kg) dose of sulphamonomethoxine (SMM) were studied in five Goettingen minipigs. The time data of plasma concentration after a low dose decreased rapidly and appeared to be linear on semilog graph paper. On the other hand, a decrease in the plasma concentration after a high dose was slow at first, gradually accelerated, then became rapid, showing that SMM disposition after a high dose (100 mg/kg) seemed to be non-linear with capacity-limited elimination. A large amount of the acetyl derivative of SMM (AcSMM), which was determined to be the main excretory product of SMM in urine, was detected in the plasma after SMM injection. As the ratios of the area under plasma concentration-time curve of AcSMM to that of SMM were not significantly different at either dose, the acetylation of SMM may be unsaturable by injection of 100 mg/kg of SMM. Immediately after the injection of a low dose, a rapid hyperbolic increase of the fraction of the cumulative amount of the excretory products in urine was observed. On the other hand, the fraction curve at the high dose rose slowly at first, then rapidly and hyperbolically. These results suggested that the non-linear drug disposition after a high dose (100 mg/kg) of SMM in pigs may be the result of a limited capacity for renal excretion of SMM and excretory products, especially the acetyl derivative.

Critical period of pyrimethamine teratosis in swine

The teratogenic activity of pyrimethamine(PY) in Goettingen mini pigs was demonstrated by Misawa et al.(1980), who showed that the dose of 3.6mg/kg/day in feed during gestational days 11-35 proved to be teratogenic to swine. The present study was done to examine the incidence of malformation from dams given 3.6mg/kg daily (p.o.) with mashed feed in each of the following 4 subdivided organogenetic periods of pregnancy; days ll-22(group I, Slitters, 24 newborns), days 23-35(group II, 3 litters, 20 newborns), days ll-16(group III, 6 litters, 27 newborns) and days 17-22(group IV, 3 litters, 16 newborns). At farrowing, the newborn piglets having major malformations such as cleft palate, clubfoot, micrognathus etc. were killed and examined for internal abnormalities. Other newborns were observed for one week. The incidence of major malformation «as higher in group 1(54% of newborns), but that in other groups was low (0 - 14%). Newborns which had died within a few days were frequently observed in group 111(41%) and IV(38%).Splayleg was found in many of them. From these findings,it may be suggested that the pig fetuses were susceptible to PY when they were exposed to more than 6 consecutive days during the first half of the organogenetic period.