Male Minipigs Research Articles

Characterization of clock gene expression and their putative bioactive profiles on development in miniature pig

The aim of this study was to address the differential expression of circadian clock genes and their bioactivity profiles on growth in developmental stages of miniature pig (mini‐pig). In growing stages of piglets of either sex on day 0 (isolated from maternal by C‐section), day 1, 5 as well as parturient subjects were sacrificed. Most of all, differences exist in specific clock‐related gene expression between 0‐, 1‐ and 5‐day‐old neonatal and parturient mini‐pig of either sex, especially in brain. We confirmed the differential expression of clock‐related genes in heart and kidney excluding brain at 0‐, 1‐, 5‐day‐old of male mini‐pig. It is giving us the striking message that not only differential expression of clock genes in varies tissues but also dependency of these gene expression on the distinction of sex in neonatal subjects. Additionally, expression of clock, bmal1 and per1 were predominant in kidney compared to brain and heart but not shown any changing their expression on developmental stages. Taken together, therefore, these findings revealed a tissue‐specific differential expression of clock genes at developmental stages that modulates of development of mini‐pigs, suggesting differential physiological function at different developmental stage, especially neonatal periods in perspective donor for xeno‐transplantation. [Supported by BioGreen21 Program (Code#20070401034006), Republic of Korea].

The histometric analysis of osseointegration in hydroxyapatite surface dental implants by ion beam-assisted deposition

Purpose: This study compared the effects of coating implants with hydroxyapatite (HA) using an ion beam-assisted deposition (IBAD) method prepared with machined, anodized, sandblasted and large-grit acid etched (SLA) surfaces in minipigs, and verified the excellency of coating method with HA using IBAD. Material and Methods: 4 male Minipigs(Prestige World Genetics, Korea), 18 to 24 months old and weighing approximately 35 to 40 kg, were chosen. All premolars and first molars of the maxilla were carefully extracted on each side. The implants were placed on the right side after an 8 week healing period. The implant stability was assessed by resonance frequency analysis (RFA) at the time of placement. 40 implants were divided into 5 groups; machined, anodized, anodized plus IBAD, SLA, and SLA plus IBAD surface implants. 4 weeks after implantation on the right side, the same surface implants were placed on the left side. After 4 weeks of healing, the minipigs were sacrificed and the implants were analyzed by RFA, histology and histometric. Results: RFA showed a mean implant stability quotient (ISQ) of 75.625±5.021, 76.125± 3.739 ISQ and 77.941±2.947 at placement, after 4 weeks healing and after 8 weeks, respectively. Histological analysis of the implants demonstrated newly formed, compact, mature cortical bone with a nearby marrow spaces. HA coating was not separated from the HA coated implant surfaces using IBAD. In particular, the SLA implants coated with HA using IBAD showed better contact osteogenesis. Statistical and histometric analysis showed no significant differences in the bone to implant contact and bone density among 5 tested surfaces. Conclusion: We can conclude that rough surface implants coated with HA by IBAD are more biocompatible, and clinical, histological, and histometric analysis showed no differences when compared with the other established implant surfaces in normal bone. (J Korean Acad Periodontol 2008;38:363-372)

Cells of Primarily Extravascular Origin in Neointima Formation following Stent Implantation

Objectives: In-stent restenosis due to neointima formation is a major limitation following stent implantation. Recently, several studies reported mobilization of primarily extravascular cells to the arterial sites after balloon angioplasty. Therefore, the goal of the present study was to assess the coordinated neointimal expression of endothelial progenitor, dendritic and neural crest-derived cells after stent implantation. Methods: Male minipigs underwent stent implantation in abdominal aortic segments. Animals were sacrificed at 1, 7, 14, 30, 60 or 90 days. Cross sections of the injured vessels were obtained for immunohistochemistry using specific antibodies for the detection of endothelial progenitor (CD133), dendritic (S100) and neural crest-derived cells (GFAP), as well as monocytes/macrophages (CD14) and T lymphocytes (CD3). Results: As a key finding, frequency of CD133, S100, GFAP, CD14 and CD3 (18.5 ± 3.6, 14.9 ± 1.8, 10.6 ± 1.1, 40.2 ± 8.3 and 5.0 ± 0.6%, respectively) in neointima was maximal at day 7. With ongoing neointima enlargement, expression of these cells decreased. In advanced neointima, labeled cells were predominantly localized at luminal and stented sites. Media showed almost no immunoreactivity of the markers studied, whereas adventitial zones of neovascularization revealed some signals. Conclusions: Endothelial progenitor, dendritic, neural crest-derived and inflammatory cells are consistently recruited into arterial neointima, mostly at early time points after stent implantation.

The distribution of esterases in the skin of the minipig

Skin esterases serve an important pharmacological function as they can be utilised for activation of topically applied ester prodrugs. Understanding the nature of these enzymes, with respect to their role and local activity, is essential to defining the efficacy of ester prodrugs. Minipigs are used as models to study the kinetics of absorption of topically applied drugs. Their skin has structural properties very similar to human skin. However, regional distribution differences in esterase activity from site-to-site could influence cross-species extrapolation. Investigation of the regional site variation of minipig skin esterase activity will facilitate standardization of topically applied drug studies. Furthermore, the characterization of regional skin variation, will aid in translation of minipig results to better predictions of human esterase activity. Here we report the variation in rates of hydrolysis by minipig skin taken from different regional sites, using the esterase-selective substrates: phenyl valerate (carboxylesterase), phenyl acetate (arylesterase) and p-nitrophenyl acetate (general esterase). Skin from ears and back of male minipig showed higher activity than female. Skin from minipig ears and the back showed the highest level of esterase activity and was similar to human breast skin used in vitro absorption studies. These results suggest that skin from the minipig back is an appropriate model for preclinical human skin studies, particularly breast skin. This study supports the use of the minipig, with topical application to the back, as a model for the investigation of pharmacokinetics and metabolism of ester prodrugs.

A standardised mini pig model for in vivo investigations of anticholinergic effects on bladder function and salivation

Purpose The objective was to validate an in vivo model for evaluation of pharmacological effects on bladder function taking the most predominant anticholinergic side effect (hyposalivation) into account. Therefore, two anticholinergic properties (propiverine hydrochloride and tolterodine- l(+)-tartrate) were used to test the in vivo model. Sacral anterior root stimulation (SARS) was performed to induce reproducible and standardized bladder contractions. To evaluate hyposalivation standardised salivavary flow measurements by stimulating the lingual nerve was performed in addition to SARS. Materials and methods 10 male mini pigs were anaesthetised. The carotid artery was cannulated for blood pressure measurement and the jugular vein for administration of propiverine 0.4 mg kg −1 b.w. and tolterodine 0.06 mg kg −1 b.w. For stimulation-induced salivary flow measurements both lingual nerves were exposed and a cuff electrode was placed around the nerves. The bladder was exposed and a cystostomy catheter was inserted to performed cystometrographic measurements during SARS. Results In all experiments, for each animal reproducible intravesical pressure values (pves) and salivary flow rates were elicited during electrostimulation before administration of the drug. Bladder pressure: After administration of propiverine, neurostimulation-induced rise in pves had fallen by 60% from the initial value. After administration of tolterodine pves had fallen by about 50%. After additional administration of atropine pves decreased to about 15% of the initial value for both drugs. Salivation: After propiverine salivary flow had fallen by 61%. Inhibition of salivary flow under tolterodine was about 56%. Additional administration of atropine led in both drugs to a nearly complete blockade of salivation. Heart rate (HR) and blood pressure (BP): Directly following intravenous administration of both drugs, a short-term and reversible period of mild but significant fluctuations in HR was observed. There was also a slight but non-significant rise in blood pressure. Conclusions This model allows comparative investigations of various drugs with bladder inhibitory properties in terms of acute efficacy and side effects.

Tracheal Replacement by Allogenic Aorta in the Pig

To assess whether fresh aortic allografts (AAs) can be used for tracheal replacement. Twenty-one male minipigs underwent tracheal replacement using AAs harvested from female pigs. The length of replaced segments exceeded 50% of the trachea. A stent was implanted into the lumen of the AA to prevent collapse. The animals were killed at 3-month intervals, and AAs were assessed for ingrowth of respiratory epithelium and cartilage formation and tested for type II collagen formation and the presence of the SRY gene. A high stent migration rate was observed. Only 10 pigs and 4 pigs made it to follow-up periods exceeding 3 months and 9 months, respectively. Neither rejection nor ischemia were observed. At 3 months, a metaplastic epithelium lined the graft. At 10 months, a posterior membrane could be seen with immature cartilage and disorganized elastic fibers. SRY gene assay showed that the cells engrafted in the AAs, particularly at the level of the newly formed cartilage, were of male origin and thus originated from the recipient. This study confirms that a fresh AA, replacing more than half of the trachea of the pig, transforms into a conduit containing the major tracheal components. These components are relatively immature and do not as of yet replicate the form and function of the native trachea. Questions remain concerning the exact mechanisms of this process. Further research on the role of tracheal replacement is recommended.

Radiographic techniques for medical–dental research with minipigs

Techniques were developed to obtain standardised intra- and extra-oral radiographs in minipigs for use in medical–dental research. Twelve male minipigs (BR-1 Minipigs) were chosen at random. Two animals each at 3, 5, 7, 9, 12 and 15 months of age were anaesthetised and subjected to radiographic examinations to assess six techniques. Three intra-oral and three extra-oral techniques, standardised for humans, were used with variations of the angle of incidence of the X-ray beams, focus-film distance and exposure time. Two film positioners were developed for the intra-oral techniques. Two examiners then chose the radiographs with the least image distortion, greatest clarity and least superimposition of images. For each technique, the suitable angle of incidence of the X-ray beams, the focus-film distance and exposure time that produced the highest quality radiographs were standardised.

Restricted feeding may induce serous fat atrophy in male Göttingen minipigs

Serous atrophy of the fatty tissue of bone marrow is occasionally seen in minipigs. It is not associated with compound toxicity, as it has been observed in both dosing and healthy control groups in toxicity studies, but the etiology and pathogenesis are unknown. However, a nutritional factor is suspected, since minipigs are generally diet restricted. In order to investigate this, newly weaned minipigs were fed different amounts of feed for nine months, after which the bone marrow was evaluated. Serous atrophy was observed in diet-restricted male minipigs fed on a diet based on the nutrient requirements of production pigs, but not in males fed on a diet specially designed for minipigs, at similarly restricted levels. No serous atrophy was observed in females. It was concluded that nutrient requirements of production pigs are not necessarily suitable for minipigs, as a diet based on these nutrient requirements caused total depletion of perirenal fat depots, as well as serous atrophy of the fatty tissue of bone marrow in male Göttingen minipigs at a restricted feeding level.

Comparison of Low-Level Sarin and Cyclosarin Vapor Exposure on Pupil Size of the Gottingen Minipig: Effects of Exposure Concentration and Duration

The current studies estimated effective (miosis) concentrations of the nerve agents' sarin (GB) and cyclosarin (GF) as a function of exposure duration in the Gottingen minipig and determined dependency of the median effective dosage (ECT50) over time. Male and female Gottingen minipigs were exposed to various concentrations of vapor GB or GF for 10, 60, or 180 min. Infrared images of the pig's pupil before, during, and after nerve agent exposure were captured digitally and pupil area was quantified. An animal was classified “positive” for miosis if there was a 50% reduction in pupil area (as compared to baseline) at any time during or after the GB or GF exposure. Maximum likelihood estimation was used on the resulting quantal data to calculate ECT50 (miosis) values, with approximate 95% confidence intervals, for each of the six gender–exposure duration groups. As a group, male minipigs were significantly more sensitive to the pupil constricting effects of GF than were female minipigs. In male minipigs, GF is approximately equipotent to GB for 60-min exposures and more potent for 10- and 180-min exposures. In the female minipig GF is slightly more potent than GB for 10-min exposures but then progressively becomes less potent over the 60- and 180-min durations of exposure. The values of the toxic load exponents were essentially independent of the model fits used: 1.32 ± 0.18 for GB exposures and 1.60 ± 0.22 for GF exposures. Since neither of these intervals overlaps 1, Haber's rule is not an appropriate time-dependence model for these data sets.

Endothelin‐A‐receptor antagonist LU 302146 inhibits electrostimulation‐induced bladder contractions in vivo

Endothelin (ET) is a strong constrictor of smooth muscle structures. The relevance of Endothelin-A receptors in the bladder was demonstrated in several in vitro studies. The aim of this functional study was to evaluate the acute effect of the selective ET-A-antagonist LU 302146 (LU) on neurostimulation-induced bladder contractions in vivo. Eight male mini pigs were anesthesized. The bladder was exposed and a double lumen catheter was inserted to perform intravesical pressure (pves) measurements. Laminectomy was performed for sacral anterior root stimulation (SARS) of S2. Four animals received the selective ET-A-antagonist LU, three atropine and one animal was treated with vehicle. Pves was recorded before and after drug administration as well as before and during neurostimulation. At the end of each LU trial, a supplementary application of 4 mg atropine was administered followed by a final SARS. In all experiments reproducible pves values were elicited during electrostimulation before administration of the test substance. The selective ET-A-antagonist reduced stimulation-induced bladder contraction by a mean of 57%. Additional administration of atropine inhibited the detrusor contraction almost completely during SARS. The vehicle had no effect on bladder contraction. In the presented animal model, ET-1 inhibition with the selective ET receptor-A-antagonist LU 302146 decreases stimulation-induced bladder contraction in vivo. The results suggest that the selective ET-A antagonist LU acts on the atropine-resistant component of efferent detrusor activation since additional administration of atropine almost completely abolish detrusor contraction. This observation in addition to the involvement of ET-1 in bladder smooth muscle proliferation, raises the possibility that ET-receptor antagonists might be beneficial in patients with neurogenic bladder dysfunction or in patients with functional or anatomical BOO.

Heat shock proteins, end effectors of myocardium ischemic preconditioning?

The purpose of this study was to investigate (1) whether ischemia-reperfusion increased the content of heat shock protein 72 (Hsp72) transcripts and (2) whether myocardial content of Hsp72 is increased by ischemic preconditioning so that they can be considered as end effectors of preconditioning. Twelve male minipigs (8 protocol, 4 sham) were used, with the following ischemic preconditioning protocol: 3 ischemia and reperfusion 5-minute alternative cycles and last reperfusion cycle of 3 hours. Initial and final transmural biopsies (both in healthy and ischemic areas) were taken in all animals. Heat shock protein 72 messenger ribonucleic acid (mRNA) expression was measured by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method using complementary DNA normalized against the housekeeping gene cyclophilin. The identification of heat shock protein 72 was performed by immunoblot. In our "classic" preconditioning model, we found no changes in mRNA hsp72 levels or heat shock protein 72 content in the myocardium after 3 hours of reperfusion. Our experimental model is valid and the experimental techniques are appropriate, but the induction of heat shock proteins 72 as end effectors of cardioprotection in ischemic preconditioning does not occur in the first hours after ischemia, but probably at least 24 hours after it, in the so-called "second protection window."

Dental and skeletal characterization of the BR-1 minipig

This study was designed to characterize the development of oral structures in the BR-1 minipig and to compare these data with literature reports on domestic swine and other minipig lineages. Twelve male minipigs were anesthetized and given physical and radiographic examinations. The presence and chronology of tooth eruption from 3 to 15 months of age were assessed, and the radiographic results were interpreted by two certified researchers, who obtained a high index of agreement ( κ > 0.81). The eruption chronology was similar to that reported for domestic swine and other minipig lineages. The results indicate that it is possible to use the BR-1 minipig in dental research.

Protective effects of ulinastatin against multiple organic damage after severe burn injury:experimental and clinic studies

To study the protective effects of ulinastatin (UTI), a human trypsin inhibitor, on the damage to the functions of multiple organs at the early stage of severe burn. (1) Twelve male mini-pigs, inflicted with III degrees burn with 35% total body surface area (TBSA), were randomly divided into two equal groups: Group A (control group, treated with fluid resuscitation immediately after injury and normal saline by intravenous drip tid) and Group B (UTI-treated group, in addition to fluid resuscitation UTI 5000 U/kg was given one hour after injury, 3 times/day). Blood samples were taken for the determination of serum TNF-alpha, IL-6, malonyldiadehyde (MDA), superoxide dismutase (SOD), alanine transaminase (ALT), aspartate transaminase (AST), creatine kinase (CK), MB isoenzyme of CK (CK-MB), blood urea nitrogen (BUN), creatinine (Cr), diamine oxidase (DAO), and D-lactate before and 6, 24, 48, and 72 hours after injury. (2) Forty hospitalized patients with 50%-70% TBSA partial and full thickness burn, 28 males and 12 females, aged 18-60, were randomly divided into routine treatment group (n = 16, receiving routine fluid resuscitation and wound management and normal saline IV drop bid for 7 days) and UTI treatment group (n = 24, in addition to the routine treatment UTI 400,000 U was given IV drop bid for 7 days). Sixteen healthy volunteers served as controls to undergo blood examination. The average body temperature (T), respiratory rate (R), systole blood pressure (BP) were detected 48 hours after burn. Blood samples were taken to determine the plasma lipopolysaccharide (LPS), and serum TNF-alpha, norepinephrine (NE), myeloperoxidase (MPO), ALT, AST, CK, CK-MB, lactic dehydrogenase (LDH), BUN, and Cr 1, 3, and 7 days after burn. (1) The serum TNF-alpha, IL-6, and MDA levels significantly increased, and the serum SOD level significantly decreased 6 hours after burn in the mini-pings in both groups (all P < 0.05), and the changes in Group A were more significant than in Group B (all P < 0.05). The serum AST, CK, CK-MB, BUN, Cr, DAO, and D-lactate levels increased in the mini-pigs of both groups after severe burn in comparison with those before burn (all P < 0.05), especially in Group A in comparison with Group B (all P < 0.05). However, the serum levels of ALT and LDH of Group B did not increase significantly in comparison with those before burn. (2) The serum LPS, TNF-alpha, NE, ALT, AST, CK, CKMB, LDH, BUN, Cr, and MPO significantly increased after burn and began to decrease since the 7th day after burn, and the decrease levels in the UTI group were more significant than in the routine treatment group (all P < 0.05). In the routine treatment group 2 cases of sepsis were found and one case died of multiple organ failure; no complication was found in the UTI treatment group. UTI can significantly lessen the production of inflammatory mediators and oxygen free radical and protect the functions of multiple organs after severe burn.

084 Effects of Fat Autografting on Porcine Skin Wound Healing after Injury

It has been evidenced that the fat may have a potential to secret some growth factors or be a source of stem cells. So, we explore the effects of fat on healing of porcine skin wounds so as to provide a new method for clinical skin wound repair after injury.Forty‐eight full‐thickness skin wounds were produced on both sides of the back in 6 male minipigs (8 wounds in each animal). Then these wounds were randomly divided into 4 groups, which were saline control group, fat autografting group, basic fibroblast growth factor (bFGF) treatment group and epidermal growth factor (EGF) treatment group. At day 3, 7, 14 and 21 after wounding, the area and the volume of wounds were measured and the histological examination was performed to evaluate the velocity and quality of wounds healing in different groups.At day 3 and 7, the amount of granulation tissues and vessel density in fat treatment group were significantly more than that in other groups. Wound areas and volume in fat treatment wounds were markedly decreased in compared with those in other groups (P &lt; 0.01). Regenerated epidermis in fat treatment group was thicker than that in other groups.These results confirmed that the wound healing velocity and quality in wounds treated with fat autografting were enhanced. It indicated that fat has a potential to accelerate velocity and improve the quality of wound healing after skin injury.

Scalp recordings of mid-latency AEP and auditory gating in the Göttingen minipig: a new animal model in information processing research

Early central information processing, measured in humans by the gating of the middle latency auditory evoked potential (AEP) P50 and the effect of attention on AEP N100, is affected in schizophrenia. Exploring the possibility of using miniature pigs in longitudinal studies of chronic neuropsychiatric disorders, we present a method for recording seven channel surface middle latency AEP in the awake animal. The AEP and the AEP gating measured in a paired stimulus paradigm similar to the P50 gating paradigm used in humans, were recorded in six adult male minipigs in two sessions. The AEP had a stable N40/P60/N120/P200 configuration and in the gating paradigm the difference between stimulus one (S1) and two (S2) P60 and N120 amplitudes were significant. Mean AEP P60 gating ratio (S2/S1) at the posterior central electrode was 0.66 (std 0.29) range 0.21–1.08 and corresponding N120 was 0.60 (std 0.19) range 0.28–0.76. The method presented is feasible for scalp recordings of middle latency evoked potentials in the awake animal, but further studies of interval sensitivity and the effect of arousal manipulation are needed to assess the equivalence of the pig components to those of the human at similar latencies.

The long-term behavior of poly-L-lactide screws in a minipig fracture model: preliminary report.

The long-term degradation tissue response to poly-L-lactide (PLLA) screw was investigated for a minipig tibia fracture. As a bone-fracture model, three PLLA screws per animal were used to fix a tibia that had been subjected to a crank-shaped osteotomy. A total of 12 nine-month-old male minipigs were used. In time period from 1 month to 3 years, clinical, radiographic, and pathological studies were conducted. On macroscopic examination, no clear granuloma or fistula formation was noted for 3 years. On microscopic examination, no osteoclasts or giant cells were detected around the screws during the 3 years postoperatively. A substantial degradation of PLLA screw was observed between 2 and 3 years. At 3 years only slight residual breakdown products were found in macrophages. On electric microscopic examination, numerous degraded polymers were seen in a small number of macrophages around the screws. In this model, fixation of tibia fractures using PLLA screws was not associated with any side effects, and was safe and useful for up to 3 years.

Absorption, metabolism, and disposition of [14C]SDZ ENA 713, an acetylcholinesterase inhibitor, in minipigs following oral, intravenous, and dermal administration.

SDZ ENA 713 (rivastigmine) is an acetylcholinesterase inhibitor intended for therapeutic use in Alzheimer's disease. The present study compared the pharmacokinetics of [14C]SDZ ENA 713 after intravenous, oral, and dermal administration to male minipigs, and also examined the effects of dose level and skin abrasion on transdermal absorption. Four groups of 3 minipigs each received a single intravenous (0.1 mg/kg), single oral (1.0 mg/kg), or topical doses of 18 mg or 54 mg of [14C]SDZ ENA 713. Topical doses were administered as dermal patches on two occasions 10 days apart. On Study Day 1, test patches were applied to a virgin skin site. Placebo patches were applied to a separate skin site and were replaced daily during Days 1-10. On Study Day 11, test patches were applied to the site on which the placebo patches had been previously applied. After each dose, serial blood and quantitative urine and feces were collected at designated intervals for 7 days. Concentrations of radioactivity, parent drug, and metabolite ZNS 114-666 were measured in whole blood. Radioactivity was also determined in excreta, skin application sites (at study termination), and on used dermal patches (at 24 hr after application). Oral doses of [14C]SDZ ENA 713 were rapidly (tmax = 0.83 hr) and efficiently (ca. 93%) absorbed, although the bioavailability of the parent drug was low, ca. 0.5%, apparently due to extensive first-pass metabolism. Radioactivity was excreted mainly in the urine (approximately 90%) with a half-life of 56 hr, slightly longer than that observed after an intravenous dose, 46 hr. After dermal administration of [14C]SDZ ENA 713 to a virgin skin site, absorption was 8% at both dose levels investigated. Following daily application of placebo patches for 10 days, absorption from a [14C]SDZ ENA 713 dermal patch increased by approximately twofold, 17% and 19% of the 18 mg and 54 mg doses, respectively. The increase is possibly due to hydration or abrasion of the skin as a result of repeated application and removal of the adhesive patches. Whereas total absorption from the dermal dose was smaller than that from the oral dose, essentially all of the absorbed drug via the dermal route reached the systemic circulation intact, thus yielding a SDZ ENA 713 bioavailability 20-40 times greater than that of the oral dose. Metabolite ZNS 114-666 was rapidly formed and accounted for <4% of total drug-related material in the systemic circulation. Dermal administration in minipigs provided a markedly greater bioavailability of SDZ ENA 713 than the oral route. The extent of absorption was independent of dose within the range tested, and appeared to be enhanced by hydration or abrasion of the skin application site.

Characterization of the P450 system in Göttingen minipigs.

It is essential to establish the activity and regulation of the cytochrome P450 system of species selected for toxicological and pharmacological studies. The minipig has become a popular substitute for the traditional non-rodent species although little information is available on its P450 system. The total P450 and the enzyme activity of the most important drug-metabolizing isoenzymes: CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 were measured in liver microsomes from 4 minipigs and 8 conventional pigs of both sexes. Immunochemical levels were determined for 4 of teh isoenzymes. The total P450 activity was slightly higher in minipigs compared to conventional pigs but no sex difference was detected. CYP1A2 activity (7-ethoxyresorufin) was 4 times higher in female minipigs than in male minipigs. The activity in male minipigs was almost identical to the activity in conventional pigs. The activity of CYP2E1 (chlorzoxazone) was 4 times higher in female than in male minipigs and 2 times higher in female than in male conventional pigs. No activity of CYP2D6 (debrisoquine) and CYP2C19 (mephenytoin) could be detected. The CYP3A4 activity (testosterone) detected in minipigs was higher than the activity in conventional pigs. A weak sex difference was seen in both strains. Western blotting using anti-human CYP2E1 and CYP3A4 confirmed the results obtained in the enzyme activity assays, while only CYP1A2 correlated with the activity in the conventional strain. The total P450 enzyme activity was close to the levels reported for human beings, as were the activities of CYP2E1 and CYP3A4.

Evidence of P2Y-purinoceptor mediated bladder neck smooth muscle post-contractile relaxation in the male mini-pig

The functional role of non-adrenergic non-cholinergic (NANC) nerves in the autonomic control of the male mini-pig bladder neck was investigated in the present study. Electrical stimulation of muscle strips from male mini-pig bladder neck showed biphasic response with initial phasic contraction followed by post-contractile relaxation. Electrical stimulation in the presence of four autonomic blockers (atropine 10−6 M, propanolol 10−6 M, phentolamine 10−6 M and guanethidine 10−6 M) showed suppression of 68±15% of the contractile response (P<0.05, n=8) but no significant change in the relaxation response. Alpha-chymotrypsin 2 U/ml, l-NG-monomethyl-l-arginine acetate (a nitric oxide synthetase inhibitor) 10−4 M, 8-phenylthlophylline (a P1-purinoceptor antagonist) 10−6 M, and pyridoxal-phosphate-6-azophenyl-2′,4′-disulphoric acid tetrasodium salt (a P2Y-purinoceptor antagonist) 3×10−5 M did not alter the NANC response significantly. On the other hand, reactive blue-2 (a P2Y-purinoceptor antagonist) 3×10−5 significantly reduced the relaxation by 79±9%. The result suggested that the P2Y-purinoceptor is involved in the electrically induced NANC post-contractile relaxation of the mini-pig bladder neck smooth muscle.

Positive influence of nifedipine on early-stage minipig shock lung

Mechanical and biochemical lesions can be observed in shock lung. To investigate the influence of the calcium antagonist nifedipine on these lesions, we produced shock lung in male minipigs. Three groups (control, thrombin, and thrombin + nifedipine; n = 5) were formed. In the second and third group thrombin was administered intravenously (400 IU/kg body weight) over a period of 2 h. After that the animals received aminocaproic acid (100 mg/kg body weight/h) throughout the rest of the study. Nifedipine was administered in the third group at a dosage of 46 micrograms/kg body weight/h. During the investigation period coagulation and hemodynamic status were examined as well as blood gas levels. Then the isolated lungs were examined. Without nifedipine there was a significant decrease in arterial PO2, an increase in airway opening pressure (Pi), and a decrease in weight-specific end-inspiratory lung compliance (Ci). These changes were not observed under the influence of nifedipine, after which the results were similar to the control group. This demonstrates a positive influence of nifedipine on the acid-base status and the investigated lung mechanical parameters.