Male Minipigs Research Articles

New cardiac expression of two adenosine-2A receptor isoforms in dysfunctioning minipigs

Purpose: Eight A2AR variants are reported in humans while no A2AR isoforms in pigs. The aim of this study was to evaluate potential isoforms presence in cardiac pig tissue to better define possible involvement of A2AR in the cardiovascular pathophysiology.Materials and methods: In adult male minipigs (n = 4) left ventricular dysfunction (LVD) was induced by pacing at 200 bpm in the right ventricular (RV) apex. In these animals and in sham operated pigs (C-SHAM, n = 4) cardiac tissue was collected from LV-septal wall (LV-SW)-close to pacing site-and from lateral (opposite) site (LV-OSW). A2AR specific primers, derived from Sus scrofa AY772412 sequence, were used for Real-Time PCR. The DNA was sequenced using the Sanger method. Histological analysis was also performed.Results: In LV-SW of LVD minipigs the A2AR melting curves were characterized by a sharp peak between 87 and 91 °C (short isoform, 1–94 bp) on the right of the principal peak corresponding to a long A2AR isoform (GenBank: JQ229674.1) 1–213 bp. As for C-SHAM only one peak was observed in LV-OSW region of LVD animals. The short isoform had an alternative promoter region and a specific translated protein. Histology showed in LVD-LV-SW prominent Purkinje cells compared to LV-OSW and C-SHAM. No difference in A2AR expression was observed between LVD animals and C-SHAM although a slight decrease was observed in LVD-LV-OSW.Conclusions: The presence of two different isoforms in the myocardium close to the insertion of pacing is suggestive of a differential state-specific expression of A2AR in cardiac tissue.

Analysis of Serum Cholesterol Efflux Capacity in a Minipig Model of Nonischemic Heart Failure.

Aim: Circulating levels of high-density lipoprotein cholesterol (HDL-C) are decreased in patients with heart failure (HF). We tested whether HDL-C serum levels are associated with cardiac contractile dysfunction in a minipig HF model.Methods: Blood samples were collected from 13 adult male minipigs: 1) before pacemaker implantation, 2) 10 days after surgery, and 3) 3 weeks after high-rate LV pacing. Serum cholesterol efflux capacity (CEC), an index of HDL functionality, was assessed through four mechanisms: ATP Binding Cassette transporter A1 (ABCA1), ATP Binding Cassette transporter G1 (ABCG1), Scavenger Receptor-Class B Type I (SR-BI) and Passive Diffusion (PD).Results: HDL-C serum levels significantly decrease in minipigs with HF compared with baseline (p < 0.0001). Serum CEC mediated by PD and SR-BI, but not ABCA1 or ABCG1, significantly decrease in animals with HF (p < 0.05 and p < 0.005, respectively).Discussion: HDL-C serum levels and partial serum CEC reduction may play a pathophysiological role in the cardiac function decay sustained by high-rate LV pacing, opening new avenues to understand of the pathogenesis of nonischemic myocardial remodeling.

Diagnostic value of [ $$^{18}{\text{F}}$$ 18 F ]FDG-PET/CT in acute hepatic radiation toxicity: a Tibet minipig model

There are few reports with respect to the details of acute effects (during and following RT) of ionizing radiation in liver tissue in epidemiology, pathology, physiology and imageology. Therefore, this study was undertaken to assess the diagnostic value of 2-[\({}^{18}\hbox {F}\)]-fluoro-2-deoxy-D-glucose positron emission tomography with computed tomography ([\({}^{18}\hbox {F}\)]-FDG-PET/CT) in the detection of acute radiation toxicity in normal liver using Tibet minipigs as a model. Thirty-six male Tibetan minipigs were randomly divided into six groups (\(n = 6\)). The irradiation groups were treated with a single dose of \(2, 5, 8, 11 \hbox { and } 14 \hbox { Gy}\) total body irradiation using an 8-MV X-ray linac, at dose rate of \(255 \hbox { cGy/min}\). The control group was not irradiated. The pigs were evaluated with [\({}^{18}\hbox {F}\)]-FDG-PET/CT, and their alanine aminotransferase (AST) and aspartate aminotransferase (ALT) activities and the ALT/AST ratio, INRatio and bilirubin were measured on Day 7 post-TBI. All pigs were killed on Day 7 post-TBI to collect liver tissues for pathological examination. The results showed that liver standardized uptake value (SUV) increased with the dose from 2 to 11 Gy and decreased suddenly at \(14 \hbox { Gy}\). HAI score showed a radiation dose-dependent increase of 2–11 Gy and was positively correlated with SUV (\(p<0.05\)). However, HAI score showed no correlation with liver function. Therefore, we concluded that [\({}^{18}\hbox {F}\)]-FDG-PET/CT has the potential to assess acute radiation-induced hepatic injury on early stage.

Prophylaxis with human serum butyrylcholinesterase protects Göttingen minipigs exposed to a lethal high-dose of sarin vapor

Serum-derived human butyrylcholinesterase (Hu BChE) is a stoichiometric bioscavenger that is being developed as a potential prophylactic nerve agent countermeasure. Previously, we reported the prophylactic efficacy of Hu BChE in Göttingen minipigs against a whole-body exposure to 4.1mg/m3 of sarin (GB) vapor, which produced lethality over 60min. Since the toxicity of nerve agent is concentration-dependent, in the present study, we investigated the toxic effects of an almost 3-fold higher rate of GB vapor exposure and the ability of Hu BChE to protect minipigs against this exposure. Male minipigs were subjected to: (1) air exposure; (2) GB vapor exposure; or (3) pretreatment with 7.5mg/kg of Hu BChE by i.m. injection, 24h prior to whole-body exposure to 11.4mg/m3 of GB vapor for 10min. Electrocardiogram, electroencephalogram, and pupil size were monitored throughout exposure. Blood drawn before and throughout exposure was analyzed for blood gases, electrolytes, metabolites, acetylcholinesterase and BChE activities, and amount of GB bound to red blood cells and plasma. A novel finding was that saline-treated animals exposed to GB vapor did not develop any seizures, but manifested a variety of cardiac and whole blood toxic signs and rapidly died due to respiratory failure. Strikingly, pre-treatment with 7.5mg/kg of Hu BChE not only prevented lethality, but also avoided all cardiac toxic signs manifested in the non-treated cohort. Thus, Hu BChE alone can serve as an effective prophylactic countermeasure versus a lethal high-dose exposure to GB vapor.

Posterolateral lumbar fusion using Escherichia coli–derived rhBMP-2/hydroxyapatite in the mini pig

Background contextHydroxyapatite (HA) is used as a bone graft extender for posterolateral spinal fusion in human. It is also useful as a recombinant human bone morphogenetic protein (rhBMP)-2 carrier because of its high affinity for rhBMP-2. PurposeTo assess the osteoinductivity of Escherichia coli–derived rhBMP-2 (E-BMP-2) using HA granules as a carrier and to evaluate the bone-forming ability depending on the different dosages of E-BMP-2. Study designA mini-pig lumbar posterolateral fusion model using microcomputed tomography (μCT) scanning. Patient sampleThirty-one adult male mini pigs were randomized into a single control group (n=8) without E-BMP-2 and two experimental groups with two different doses of E-BMP-2 (1 mg per side, n=8 and 3 mg per side, n=15). Outcome measuresOutcome was measured by plain radiography, manual palpation, CT, three-dimensional μCT, and histologic examinations. MethodsBilateral intertransverse process arthrodesis was performed, and E-BMP-2 (0, 1.0, 3.0 mg per side) was implanted into the intertransverse space using HA granules as a carrier. ResultsThree mini pigs were removed because of death. Among 28 experimental subjects, 19 animals achieved solid bony union. The fusion rates were 37.5% for control group, 71.4% for 1 mg group, and 84.6% for 3 mg group. Fusion rates were significantly different among groups (p=.031). However, there was no statistically significant difference in fusion rates between 1 and 3 mg groups (p=.587). Thirty-eight intertransverse fusion masses of 19 subjects underwent μCT scanning. The bone volumes determined by μCT were 12,603±3,240 mm3 for control group, 18,718±3,000 mm3 for 1 mg group, and 26,768±7,256 mm3 for 3 mg group, and the difference between groups was statistically significant (p<.001). ConclusionsThis study shows that E-BMP-2 has osteoinductive activity in dose-dependent fashion, and porous HA granule is suitable for E-BMP-2 carrier in a porcine posterolateral fusion model. These preliminary findings suggest that E-BMP-2–adsorbed porous HA granules could be a novel effective bone graft substitute.

Injury in Minipig Parotid Glands following Fractionated Exposure to 30 Gy of Ionizing Radiation

To explore the effects of 30 Gy of (60)Co γ-rays on apoptosis and reactive oxygen species (ROS) levels in minipig parotid cells as a possible mechanism for radiation-induced parotid injury. Experimental study. Department of Radiotherapy, First Affiliated Hospital, Guangxi Medical University, Nanning, China. Forty male minipigs were divided into control and irradiated groups. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling was used for detecting apoptosis in the parotid cells, immunohistochemistry, and western blots were used to test expression of the B-cell lymphoma 2 (Bcl-2) and BCL2-associated X (Bax) proteins, and reverse transcription polymerase chain reaction was used to analyze the expression of Bcl-2, Bax, p53, and caspase-3 messenger ribonucleic acid. An enzyme-linked immunosorbent assay was used to detect ROS levels in the parotid tissue. At each time point, the apoptotic rates in the irradiated group were higher than those in the control group. Furthermore, the ROS and expression levels of Bax, p53, and caspase-3 messenger ribonucleic acid and proteins gradually increased and were higher than those in the control group. Conversely, the expression of Bcl-2 was decreased in the irradiated group (P < .05). Ionizing radiation induces the production of ROS and promotes changes in the expression of several apoptotic proteins, which increases apoptosis and likely contributes to the mechanism of radiation-induced parotid injury.

Accelerated hematopoietic syndrome after radiation doses bridging hematopoietic (H-ARS) and gastrointestinal (GI-ARS) acute radiation syndrome: Early hematological changes and systemic inflammatory response syndrome in minipig

Purpose: To characterize acute radiation syndrome (ARS) sequelae at doses intermediate between the bone marrow (H-ARS) and full gastrointestinal (GI-ARS) syndrome.Methods: Male minipigs, approximately 5 months old, 9–12 kg in weight, were irradiated with Cobalt-60 (total body, bilateral gamma irradiation, 0.6 Gy/min). Endpoints were 10-day survival, gastrointestinal histology, plasma citrulline, bacterial translocation, vomiting, diarrhea, vital signs, systemic inflammatory response syndrome (SIRS), febrile neutropenia (FN).Results: We exposed animals to doses (2.2–5.0 Gy) above those causing H-ARS (1.6–2.0 Gy), and evaluated development of ARS. Compared to what was observed during H-ARS (historical data: ), doses above 2 Gy produced signs of increasingly severe pulmonary damage, faster deterioration of clinical conditions, and faster increases in levels of C-reactive protein (CRP). In the range of 4.6–5.0 Gy, animals died by day 9–10; signs of the classic GI syndrome, as measured by diarrhea, vomiting and bacterial translocation, did not occur. At doses above 2 Gy we observed transient reduction in circulating citrulline levels, and animals exhibited earlier depletion of blood elements and faster onset of SIRS and FN.Conclusions: An accelerated hematopoietic subsyndrome (AH-ARS) is observed at radiation doses between those producing H-ARS and GI-ARS. It is characterized by early onset of SIRS and FN, and greater lung damage, compared to H-ARS.

Comparison of sarin and cyclosarin toxicity by subcutaneous, intravenous and inhalation exposure in Gottingen minipigs

Sexually mature male and female Gottingen minipigs were exposed to various concentrations of GB and GF vapor via whole-body inhalation exposures or to liquid GB or GF via intravenous or subcutaneous injections. Vapor inhalation exposures were for 10, 60 or 180 min. Maximum likelihood estimation was used to calculate the median effect levels for severe effects (ECT50 and ED50) and lethality (LCT50 and LD50). Ordinal regression was used to model the concentration × time profile of the agent toxicity. Contrary to that predicted by Haber’s rule, LCT50 values increased as the duration of the exposures increased for both nerve agents. The toxic load exponents (n) were calculated to be 1.38 and 1.28 for GB and GF vapor exposures, respectively. LCT50 values for 10-, 60- and 180-min exposures to vapor GB in male minipigs were 73, 106 and 182 mg min/m3, respectively. LCT50 values for 10-, 60 - and 180-min exposures to vapor GB in female minipigs were 87, 127 and 174 mg min/m3, respectively. LCT50 values for 10-, 60- and 180-min exposures to vapor GF in male minipigs were 218, 287 and 403 mg min/m3, respectively. LCT50 values for 10-, 60- and 180-min exposures in female minipigs were 183, 282 and 365 mg min/m3, respectively. For GB vapor exposures, there was a tenuous gender difference which did not exist for vapor GF exposures. Surprisingly, GF was 2–3 times less potent than GB via the inhalation route of exposure regardless of exposure duration. Additionally GF was found to be less potent than GB by intravenous and subcutaneous routes.

The Effects of Recombinant Human Bone Morphogenetic Protein‐2‐Loaded Tricalcium Phosphate Microsphere–Hydrogel Composite on the Osseointegration of Dental Implants in Minipigs

Bone formation in tooth defect areas and the osseointegration of dental implants are very important for successful dental implant surgery. The aim of the present study was to assess the strengthening effect of a β-TCP microsphere-hydrogel composite containing recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone healing and implant osseointegration. The molars and premolars on the left and right sides of the maxilla were extracted from six male minipigs, and dental implants were placed using either the β-TCP microsphere-hydrogel carrier alone or the carrier loaded with rhBMP-2 (500 μg). The animals were kept alive for a further 8 weeks. The molars and premolars from the left and the right sides of the mandibles of another six minipigs were extracted, and the animals were kept alive for 4 weeks. Two 5-mm-diameter bone defects were then made on both sides of the mandible. The defects were filled with saline, β-TCP microsphere-hydrogel carrier, or the carrier loaded with rhBMP-2 (300 μg), and dental implant fixtures were inserted. The animals were kept alive for a further 4 weeks. Bone formation was examined using plane radiographs, micro-CT, and the histology of undecalcified specimens. The group treated with the rhBMP-2-loaded carrier composite showed a significantly higher percentage bone volume and a greater trabecular thickness for the newly formed bone in the tooth defect areas when compared to the group treated with the carrier alone. The rhBMP-2 group had a significantly higher osseointegration, a larger percentage bone volume, greater trabecular thickness in the newly formed bone in tooth defect areas, a larger newly formed bone fraction in the fixture pitch, and a greater number of newly formed trabecular bones when compared to the other groups. We confirmed that the rhBMP-2-loaded carrier composite promotes new bone formation after tooth extraction and strengthens osseointegration of dental fixtures by improving the degree of osseointegration around the dental implant fixture.

Cardiac magnetic resonance of acute atrial ablation injury - impact of catheter-myocardium contact force

Background Catheter-myocardium contact force (CF) is a major determinant of ablation lesion generation in the atrium, with higher CF associated with greater energy transfer. Previous publications have suggested that acute atrial injury can be visualized with T2-weighted (T2W) cardiac magnetic resonance (CMR). This study therefore sought to compare T2W images of acute ablation lesions created at low and high CF. Methods Under general anesthesia, femoral venous access was obtained in eight male Gottingen minipigs. In two animals, pre-ablation T2W CMR imaging was performed. Using fluoroscopy, a decapolar reference catheter was placed in the coronary sinus and a force-sensing ablation catheter (SmartTouch, D curve, Biosense Webster) was advanced into the right atrium. Using an electroanatomical mapping system (Carto3-MEM) the geometry of the right atrium was obtained with a 20-pole circular mapping catheter (Lasso Nav, D curve, Biosense Webster). A linear ablation lesion was created from the SVC to the IVC (30W, 48°C, 8 ml/min irrigation). Different target contact forces (>20 g (high force) or <10 g (low force)) were used alternately at the cranial and caudal halves of the ablation line. After the ablation procedure, the animals were immediately transferred for T2W CMR imaging of the ablation lesion. Maximal wall thickness in the posterior right atrium was measured for the caudal and cranial portions of the ablation lines. Results

Innate immune response after acute myocardial infarction and pharmacomodulatory action of tacrolimus in reducing infarct size and preserving myocardial integrity

BackgroundThis study investigated the association between innate immune reaction and myocardial damage after acute myocardial infarction (AMI) and anti-inflammatory role of tacrolimus in reducing infarct size. Male mini-pigs (n=18) were equally categorized into sham control (SC), untreated AMI (by ligation of left anterior descending coronary artery), and AMI-Tacrolimus (AMI-Tac) (0.5 mg intra-coronary injection 30 minutes post-AMI). Cardiac magnetic resonance imaging (MRI) was performed at post-AMI days 2, 5 and 21 before sacrificing the animals.ResultsBy post-AMI day 21, left ventricular ejection fraction (LVEF) was lowest in untreated AMI animals, significantly higher in SC than in AMI-Tac group (all p<0.003). Infarct areas at basal, middle, and apical levels, numbers of CD14+ and iNOS+ cells in infarct area (IA) and peri-IA, and protein expression of CD14, CD68, and Ly6g from circulating inflammatory cells showed an opposite pattern compared with that of LVEF in all groups (all p<0.005). Protein expressions of MCP-1, MIP-1, TNF-α, NF-κB, iNOS, and IL-12 in IA and peri-IA exhibited an identical pattern compared to that of CD14, CD68, and Ly6g from circulating inflammatory cells (all p<0.01). Expressions of myocardial damage biomarkers in IA and peri-IA [γ-H2AX, β-myosin heavy chain (MHC), Smad3, TGF-β] were highest in AMI and higher in AMI-Tac than in SC, whereas expressions of myocardial integrity biomarkers (connexin43, mitochondrial cytochrome-C, α-MHC, BMP-2, Smad1/5) were opposite to those of damage biomarkers (all p<0.001).ConclusionInnate immune responses were markedly augmented and LVEF was significantly reduced after AMI but were remarkably improved after tacrolimus treatment.

Abstract 277: Acute Cardiac Insulin Resistance During Cardiopulmonary Bypass in Mini-pigs: Role of Hexosamine Biosynthesis and Protein O-Glycosylation

Our previous study as well as others has demonstrated a strong positive correlation between cardiac insulin signaling and myocardial function in ischemic/reperfused hearts. This study was designed to determine whether cardiac insulin signaling is impaired during cardiopulmonary bypass (CPB) and the possible mechanisms involved. Twelve male mini-pigs were anesthetized and subjected to CPB for 30 min. Blood samples and left ventricle biopsies were taken at pre-bypass (control), aortic cross clamp (AXC) 5 min and AXC release 120 min. Compared with sham-operated group, both blood glucose and insulin levels went up at 120 min after AXC release. Glucose uptake in heart dropped significantly as measured by attenuated coronary arterio-venous glucose difference and reduced cardiac 18 F-fluorodeoxyglucose uptake by Positron Emission Tomography imaging. Furthermore, myocardial insulin signaling was blunted as manifested by decreased phosphorylation of IRS-1, Akt and GSK-3beta (P&lt;0.01, n=4). Interestingly, these changes were associated with a substantial increase of GFAT activity (the rate-limiting enzyme for hexosamine biosynthesis) and a significant increase in protein O-GlcNAcylation in cardiac tissues (P&lt;0.05, n=6). Moreover, pretreatment with Alloxan, an inhibitor of O-GlcNAc-transferase, blocked both the impaired GSK-3beta phosphorylation and the increase in O-GlcNAcylation. Enhancement of cardiac insulin signaling with glucose-insulin-potassium before AXC decreased protein O-GlcNAcylation and ameliorated myocardial dysfunction after CPB. These data indicate the existence of acute myocardial insulin resistance during CPB which is attributable to elevated hexosamine biosynthesis and protein O-glycosylation.

Effects of a TREM-Like Transcript 1–Derived Peptide During Hypodynamic Septic Shock in Pigs

The objective of this study was to determine the effects of a TREM (triggering receptor expressed on myeloid cells 1)-like transcript 1-derived peptide (LR12) administration during septic shock in pigs. Two hours after induction of a fecal peritonitis, anesthetized and mechanically ventilated adult male minipigs were randomized to receive LR12 (n = 6) or its vehicle alone (normal saline, n = 5). Two animals were operated and instrumented without the induction of peritonitis and served as controls (sham). Resuscitation was achieved using hydroxyethyl starch (up to 20 mL/kg) and norepinephrine infusion (up to 10 μg/kg per minute). Hemodynamic parameters were continuously recorded. Gas exchange, acid-base status, organ function, and plasma cytokines concentrations were evaluated at regular intervals until 24 h after the onset of peritonitis when animals were killed under anesthesia. Peritonitis induced profound hypotension, myocardial dysfunction, lactic acidosis, coagulation abnormalities, and multiple organ failure. These disorders were largely attenuated by LR12. In particular, cardiovascular failure was dampened as attested by a better mean arterial pressure, cardiac index, cardiac power index, and S(v)O(2), despite lower norepinephrine requirements. LR12, a TREM-like transcript 1-derived peptide, exhibits salutary properties during septic shock in adult minipigs.

Evaluation of the efficacy of Escherichia coli-derived recombinant human bone morphogenetic protein-2 in a mini-pig spinal anterior interbody fusion model

We evaluated the efficacy of Escherichia coli-derived recombinant human bone morphogenetic protein-2 (E-BMP-2) in a mini-pig model of spinal anterior interbody fusion. A total of 14 male mini-pigs underwent three-level anterior lumbar interbody fusion using polyether etherketone (PEEK) cages containing porous hydroxyapatite (HA). Four groups of cages were prepared: 1) control (n = 10 segments); 2) 50 μg E-BMP-2 (n = 9); 3) 200 μg E-BMP-2 (n = 10); and 4) 800 μg E-BMP-2 (n = 9). At eight weeks after surgery the mini-pigs were killed and the specimens were evaluated by gross inspection and manual palpation, radiological evaluation including plain radiographs and micro-CT scans, and histological analysis. Rates of fusion within PEEK cages and overall union rates were calculated, and bone formation outside vertebrae was evaluated. One animal died post-operatively and was excluded, and one section was lost and also excluded, leaving 38 sites for assessment. This rate of fusion within cages was 30.0% (three of ten) in the control group, 44.4% (four of nine) in the 50 μg E-BMP-2 group, 60.0% (six of ten) in the 200 μg E-BMP-2 group, and 77.8% (seven of nine) in the 800 μg E-BMP-2 group. Fusion rate was significantly increased by the addition of E-BMP-2 and with increasing E-BMP-2 dose (p = 0.046). In a mini-pig spinal anterior interbody fusion model using porous HA as a carrier, the implantation of E-BMP-2-loaded PEEK cages improved the fusion rate compared with PEEK cages alone, an effect that was significantly increased with increasing E-BMP-2 dosage.

Porcine study on the efficacy of autogenous tooth bone in the maxillary sinus

ObjectivesThis study sought to elucidate the effect of autogenous tooth bone material by experimenting on minipig's maxillary sinus and performing histological and histomorphometric analyses.Materials and MethodsFive 18-24 month-old male minipigs were selected, and right maxillary sinuses were grafted with bone graft material made of their respective autogenous teeth extracted eight weeks earlier. The left sides were grafted with synthetic hydroxyapatite as control groups. All minipigs were sacrificed at 12 weeks after bone graft, which was known to be 1 sigma (σ) period for pigs. Specimens were evaluated histologically under a light microscope after haematoxylin-eosin staining followed by semi-quantitative study via histomorphometric analysis. The ratio of new bone to total area was evaluated using digital software for calculation of area.ResultsAll specimens were available, except one on the right side (experimental group), which was missing during specimen preparation. This study demonstrated new bone at the periphery of the existing bone in both groups, showing evidence of bone remodeling, however, encroachment of new bone on the central part of the graft at the 1 σ period was observed only in the autogenous tooth bone group (experimental group). Histomorphometric analysis showed more new bone formation in the experimental group compared to the control group. Although the difference was not statistically significant (P>0.05), the mean percentage area for new bone for the experimental and control groups were 57.19%±11.16% and 34.07%±13.09%, respectively.ConclusionThe novel bone graft material using autogenous tooth is a good alternative to autogenous bone, comparable to autogenous bone, and outperforming synthetic hydroxyapatite bone graft materials in terms of bone regeneration capacity. Augmentation with autogenous tooth bone materials will reduce donor site morbidity without hampering the safety of the autogenous bone graft.

Regional evidence of modulation of cardiac adiponectin level in dilated cardiomyopathy: pilot study in a porcine animal model

BackgroundThe role of systemic and myocardial adiponectin (ADN) in dilated cardiomyopathy is still debated. We tested the regulation of both systemic and myocardial ADN and the relationship with AMP-activated protein kinase (AMPK) activity in a swine model of non-ischemic dilated cardiomyopathy.Methods and resultsCardiac tissue was collected from seven instrumented adult male minipigs by pacing the left ventricular (LV) free wall (180 beats/min, 3 weeks), both from pacing (PS) and opposite sites (OS), and from five controls. Circulating ADN levels were inversely related to global and regional cardiac function. Myocardial ADN in PS was down-regulated compared to control (p < 0.05), yet ADN receptor 1 was significantly up-regulated (p < 0.05). No modifications of AMPK were observed in either region of the failing heart. Similarly, myocardial mRNA levels of PPARγ, PPARα, TNFα, iNOS were unchanged compared to controls.ConclusionsParadoxically, circulating ADN did not show any cardioprotective effect, confirming its role as negative prognostic biomarker of heart failure. Myocardial ADN was reduced in PS compared to control in an AMPK-independent fashion, suggesting the occurrence of novel mechanisms by which reduced cardiac ADN levels may regionally mediate the decline of cardiac function.

Pacing-Induced Regional Differences in Adenosine Receptors mRNA Expression in a Swine Model of Dilated Cardiomyopathy

The adenosinergic system is essential in the mediation of intrinsic protection and myocardial resistance to insult; it may be considered a cardioprotective molecule and adenosine receptors (ARs) represent potential therapeutic targets in the setting of heart failure (HF). The aim of the study was to test whether differences exist between mRNA expression of ARs in the anterior left ventricle (LV) wall (pacing site: PS) compared to the infero septal wall (opposite region: OS) in an experimental model of dilated cardiomyopathy. Cardiac tissue was collected from LV PS and OS of adult male minipigs with pacing-induced HF (n = 10) and from a control group (C, n = 4). ARs and TNF–α mRNA expression was measured by Real Time-PCR and the results were normalized with the three most stably expressed genes (GAPDH, HPRT1, TBP). Immunohistochemistry analysis was also performed. After 3 weeks of pacing higher levels of expression for each analyzed AR were observed in PS except for A1R (A1R: C = 0.6±0.2, PS = 0.1±0.04, OS = 0.04±0.01, p<0.0001 C vs. PS and OS respectively; A2AR: C = 1.04±0.59, PS = 2.62±0.79, OS = 2.99±0.79; A2BR: C = 1.2±0.1, PS = 5.59±2.3, OS = 1.59±0.46; A3R: C = 0.76±0.18, PS = 8.40±3.38, OS = 4.40±0.83). Significant contractile impairment and myocardial hypoperfusion were observed at PS after three weeks of pacing as compared to OS. TNF-α mRNA expression resulted similar in PS (6.3±2.4) and in OS (5.9±2.7) although higher than in control group (3.4±1.5). ARs expression was mainly detected in cardiomyocytes. This study provided new information on ARs local changes in the setting of LV dysfunction and on the role of these receptors in relation to pacing-induced abnormalities of myocardial perfusion and contraction. These results suggest a possible therapeutic role of adenosine in patients with HF and dyssynchronous LV contraction.

Effect of Tacrolimus on Myocardial Infarction Is Associated with Inflammation, ROS, MAP Kinase and Akt Pathways in Mini-Pigs

This study tested the hypothesis that tacrolimus therapy limited left ventricular (LV) infarct and remodeling by suppressing the inflammatory response, oxidative stress and regulating the mitogen-activated protein kinase (MAPK) and Akt signaling pathways in an acute myocardial infarction (AMI) mini-pig model by ligating the left anterior descending coronary artery (LAD). Twelve male mini-pigs were equally randomized into AMI treated by saline (3.0 mL) (AMI(S)), and AMI treated by tacrolimus (0.5 mg) (AMI(T)). Thirty minutes after the procedure, intra-LAD injections were performed just beyond the ligation. Inflammatory biomarkers at transcription or protein levels [matrix metalloproteinase (MMP9), plasminogen activator inhitor-1, tumor necrotic factor (TNF-α), nuclear factor (NF)-κB] and the cellular level (CD40+ cells) were markedly higher in AMI(S) than in AMI(T) animals (all p<0.001). Fibrosis biomarkers at the protein level (α-smooth muscle actin, transforming growth factor-β) and Sirius-red staining were notably higher in AMI(S) than in AMI(T) animals (all p<0.03). Antioxidant biomarkers at protein or transcription levels (heme oxygenase-1, quinone oxidoreductase-1, glutathione reductase, glutathione peroxidase) were significantly higher in AMI(S) than in AMI(T) animals (all p<0.01). Protein expressions of ERK1, p38 MAPK and Akt were markedly increased in AMI(S) compared to AMI(T) animals (all p<0.001). Significantly aggravated LV infarction and remodeling were noted in AMI(S) compared to AMI(T) animals, whereas LV ejection fraction was markedly decreased in AMI(S) compared to AMI(T) animals (all p<0.001). Intra-coronary administration of tacrolimus attenuated inflammation and MAPK signaling, limited infarct size, and preserved LV function.

Intra-coronary administration of tacrolimus markedly attenuates infarct size and preserves heart function in porcine myocardial infarction

BackgroundWe test the hypothesis that intra-coronary tacrolimus administration can limit infarct size and preserve left ventricular ejection fraction (LVEF) after acute myocardial infarction (AMI) through ligating left anterior descending coronary artery (LAD) in mini-pigs.MethodsTwelve male mini-pigs were randomized into AMI-saline (MI-only) group and AMI-tacrolimus (MI-Tac) group that received intra-coronary saline (3.0 mL) and tacrolimus (0.5 mg in 2.5 mL saline) injection, respectively, beyond site of ligation 30 minutes after LAD occlusion.ResultsLarger infarct area was noted in MI-only group (p < 0.001). Inflammatory biomarkers at protein [oxidative stress, tumor necrotic factor-α, nuclear factor-κB], gene (matrix metalloproteinase-9, plasminogen activator inhibitor-1), and cellular (CD40+, CD68+ inflammatory cells) levels were remarkably higher in MI-only animals (p < 0.01). Conversely, anti-inflammatory biomarkers at gene level (Interleukin-10), gene and protein level (endothelial nitric oxide synthase), and anti-oxidant biomarkers at both gene and protein levels [heme oxygenase 1, NAD(P)H:quinone oxidoreductase] were lower in MI-only group (p < 0.01). Number of apoptotic nuclei and apoptotic biomarkers expressions at gene and protein levels (Bax, caspase 3) were notably higher, whereas anti-apoptotic biomarkers at gene and protein levels (Bcl-2), LVEF, and fractional shortening were markedly lower in MI-only group (p < 0.001).ConclusionIntra-coronary administration of tacrolimus significantly attenuated infarct size and preserved LV function.

Fatal Rectal Perforation Following Boar-to-Boar Mounting

Although abnormal sexual behavior, including boar-to-boar mounting with anal penetration, is recognized in pubescent pigs, reports of the pathologic consequences are scarce. A 7-month-old male minipig, housed with age-matched males, died within 1 day of the onset of lethargy and reluctance to rise. At necropsy, 2 rectal tears were identified as the cause for fibrinous peritonitis, and spermatozoa were identified in the pelvic and peritoneal cavity by light and transmission electron microscopy. According to DNA typing results, using 11 porcine microsatellites, the intraperitoneal semen was from at least 2 pen mates. The prohibition of castration of fattening pigs, implemented or planned in multiple European countries, could increase the risk of rectal perforation in co-housed pigs.