Urszula Pasławska

Effects of intracoronary delivery of allogenic bone marrow-derived stem cells expressing heme oxygenase-1 on myocardial reperfusion injury

Heme oxygenase-1 (HO-1) decreases apoptosis, inflammation and oxidative stress. The aim of the study was to investigate the effects of intracoronary infusion of allogenic bone marrow cells (BMC) overexpressing HO-1 in the porcine model of myocardial infarction (MI). MI was produced by balloon occlusion of a coronary artery. BMC were transduced with adenoviruses encoding for HO-1 (HO-1 BMC) or GFP (GFP-BMC) genes. Prior to reperfusion animals received HO-1 BMC, control BMC (unmodified or GFP-BMC) or placebo. Left ventricular (LV) ejection fraction (EF), shortening fraction (SF), end-systolic and end-diastolic diameters (EDD, ESD) were assessed by echocardiography before, 30 minutes (min) and 14 days after reperfusion. BMC significantly improved LVEF and SF early (30 min) after reperfusion as well as after 14 days. Early after reperfusion HO-1 BMC were significantly more effective than control BMC, but after 14 days, there were no differences. There were no effect of cells on LV remodelling and diastolic function. Both HO-1 BMC and control BMC significantly reduced the infarct size vs. placebo (17.2 ± 2.7 and 18.8 ± 2.5, respectively, vs. 27.5 ± 5.1, p= 0.02) in histomorphometry. HO-1-positive donor BMC were detected in the infarct border area in pigs receiving HO-1-cells. No significant differences in expression of inflammatory genes (SDF-1, TNF-α, IL-6, miR21, miR29a and miR133a) in the myocardium were found. In conclusion, intracoronary delivery of allogeneic BMC immediately prior to reperfusion improved the LVEF and reduced the infarct size. HO-1 BMC were not superior to control cells after 14 days, however, produced faster recovery of LVEF. Transplanted cells survived in the peri-infarct zone.

Development of porcine model of chronic tachycardia-induced cardiomyopathy

BACKGROUND There are few experimental models of heart failure (HF) in large animals, despite structural and functional similarities to human myocardium. We have developed a porcine model of chronic tachycardia-induced cardiomyopathy. METHODS Homogenous siblings of White Large breed swine (n=6) underwent continuous right ventricular (RV) pacing at 170 bpm; 2 subjects served as controls. In the course of RV pacing, animals developed a clinical picture of HF and were presented for euthanasia at subsequent stages: mild, moderate and end-stage HF. Left ventricle (LV) sections were analyzed histologically and relative ANP, BNP, phospholamban and sarcoplasmic reticulum calcium ATPase 2a transcript levels in LV were quantified by real time RT-PCR. RESULTS In the course of RV pacing, animals demonstrated reduced exercise capacity (time of running until being dyspnoeic: 6.6 ± 0.5 vs. 2.4 ± 1.4 min), LV dilatation (LVEDD: 4.9 ± 0.4 vs. 6.7 ± 0.4 cm), impaired LV systolic function (LVEF: 69 ± 8 vs. 32 ± 7 %), (all baseline vs. before euthanasia, all p<0.001). LV tissues from animals with moderate and end-stage HF demonstrated local foci of interstitial fibrosis, congestion, cardiomyocyte hypertrophy and atrophy, which was not detected in controls and mild HF animals. The up-regulation of ANP and BNP and a reduction in a ratio of sarcoplasmic reticulum calcium ATPase 2a and phospholamban in failing myocardium were observed as compared to controls. CONCLUSIONS In pigs, chronic RV pacing at relatively low rate can be used as an experimental model of HF, as it results in a gradual deterioration of exercise tolerance accompanied by myocardial remodeling confirmed at subcellular level.

Expression and Complex Formation of MMP9, MMP2, NGAL, and TIMP1 in Porcine Myocardium but Not in Skeletal Muscles in Male Pigs with Tachycardia-Induced Systolic Heart Failure

Matrix metalloproteinases (MMPs) are involved in the remodeling of extracellular matrix in various tissues. Their functioning could be related to the formation of complexes, containing MMP9, MMP2, tissue inhibitor of metalloproteinases type 1 (TIMP1), and neutrophil gelatinase-associated lipocalin (NGAL). Such complexes have not been investigated in either myocardial or skeletal muscles. We examined 20 male pigs with heart failure (HF), and 5 sham-operated animals. There were no differences in the mRNA expression of MMP9, MMP2, TIMP1, and NGAL between diseased and healthy animals, in either left ventricle (LV) myocardium or skeletal muscles. In LV from both diseased and healthy animals, in nonreducing and nondenaturing conditions, we demonstrated the presence of high molecular weight (HMW) complexes (130, 170, and 220 kDa) containing MMP9, TIMP1, and NGAL (also MMP2 in 220 kDa complex) without proteolytic activity, and a proteolytically active 115 kDa MMP9 form together with 72 and 68 kDa bands (proMMP2 and MMP2). Proteolytically active bands were also spontaneously released from HMW complexes. In skeletal muscles from both diseased and healthy animals, in nonreducing and nondenaturing conditions, we found no HMW complexes, and proteolytic activity was associated with the presence of 72 and 68 kDa bands (proMMP2 and MMP2).

Cases with manifestation of chemodectoma diagnosed in dogs in Department of Internal Diseases with Horses, Dogs and Cats Clinic, Veterinary Medicine Faculty, University of Environmental and Life Sciences, Wroclaw, Poland

In the period of 3 years, 9 tumours of chemodectoma were supravitally diagnosed and histopathologically verified in dogs. In this period 15 351 dogs were admitted to the Clinic of Dogs and Cats and 2 145 dogs were examined in the cardiological outpatient clinic for dogs. This tumour is located in a typical place - at the base of the heart. Most frequently the tumour manifested in older boxers. Only in one case such a tumour was diagnosed in another breed of dogs. The tumours ranged in size between 3 and 16 cm in diameter. The principal sign accompanying tumours of cardiac base involved dyspnoea but in 3 cases the tumours yielded no clinical signs. All the diagnoses were additionally verified using immunohistochemical examination. We used antibodies to chromogranin A (clone DAK-A3 1:100), synaptophysin (clone SY38 1:20) and neuron-specific enolase (clone BBS/NC/VI-H14 1:150). An immunohistochemical examination is vital for the diagnosis since it allows to differentiate histologically distinct types of neoplasia which may locate in the same site and may manifest a similar histological pattern.

Comparison of P-wave dispersion in healthy dogs, dogs with chronic valvular disease and dogs with disturbances of supraventricular conduction

BackgroundP-wave dispersion (Pd) is a new ECG index used in human cardiology and veterinary medicine. It is defined as the difference between the maximum and the minimum P-wave duration recorded from multiple different ECG leads. So far no studies were performed assessing the importance of P-wave dispersion in dogs.MethodsThe current study was aimed at determining proper value of Pd in healthy dogs (group I), dogs with chronic valvular disease (group II) and dogs with disturbances of supraventricular conduction (group III). The tests were carried out in 53 healthy dogs, 23 dogs with chronic valvular disease and 12 dogs with disturbances of supraventricular conduction of various breeds, sexes and body weight from 1,5 to 80 kg, aged between 0,5 and 17 years, submitted to the ECG examination. ECG was acquired in dogs in a standing position with BTL SD-8 electrocardiographic device and analyzed once the recording was enlarged. P-wave duration was calculated in 9 ECG leads (I, II, III, aVR, aVL, aVF, V1, V2, V4) from 5 cardiac cycles.ResultsThe proper P-wave dispersion in healthy dogs was determined at up to 24 ms. P-wave dispersion was statistically significant increased (p < 0.01) in dogs with chronic valvular disease and dogs with disturbances of supraventricular conduction. In dogs with the atrial enlargement the P-wave dispersion is also higher than in healthy dogs, although no significant correlation between the size of left atria and Pd was noticed (p = 0.1, r = 0,17).ConclusionsThe P-wave dispersion is a constant index in healthy dogs, that is why it can be used for evaluating P wave change in dogs with chronic valvular disease and in dogs with disturbances of supraventricular conduction.

Myocarditis in dogs: etiology, clinical and histopathological features (11 cases: 2007–2013)

BackgroundMyocarditis is a disease caused by numerous etiological factors and characterized by a non-specific course. The only method allowing for precise characterization of inflammatory changes is the histopathological examination of heart muscle specimens. The study was conducted on heart muscle preparations from 11 dogs with ante-mortem diagnosis of cardiac disease. Animals presented with a poor response to an applied treatment or had suspected sudden cardiac death. The heart specimens were taken post-mortem, preserved and stained with haematoxylin and eosin. Subsequently, the presence and intensity of changes, i.e. inflammatory infiltration, the amount of connective tissue and features of cardiomyocyte degeneration were estimated. The specimens from dogs suspected of having a myocarditis of bacteriological etiology underwent additional bacteriological and immunohistochemical examination.ResultsThe examination revealed an inflammatory infiltration of variable intensity combined with the degenerative changes in all dogs. There were vegetative and abnormal cystic forms of Borrelia burgdorferi sensu lato in 6 dogs. A Staphylococcus aureus infection was confirmed in one dog and an acute coronary syndrome with neutrophil infiltration was revealed in another one.ConclusionsAlthough the clinical pattern in patients with myocarditis is diverse, the definitive morphological diagnosis is made based on the histopathological examination. This examination can lead to a better understanding of the pathogenesis of the disease. To the best of our knowledge, this is the first description of myocarditis combined with the presence of spore forms of Borrelia burgdorferi sensu lato in the heart specimens of dogs.

Increased expression of interleukin-1β and its novel splice variant in canine hearts with volume overload

Volume overload frequently caused in dogs by chronic degenerative valvular disease (CDVD), eventually leads to cardiac failure. Experimental and clinical evidences demonstrate that increased interleukin-1beta serum level in patients with heart insufficiency correlates with the severity of failure irrespective of its etiology. Very little is known about the IL-1beta expression in failing vs. non-failing myocardium. IL-1beta transcript level was determined in the CDVD dogs (n=17) and control animals (n=9) without cardiac insufficiency by real-time PCR. IL-1beta transcript level in failing hearts was higher than in the control. In both groups the highest IL-1beta level was detected in the left ventricles. Although IL-1beta is a major pro-inflammatory cytokine most of the CDVD dogs displayed no inflammatory infiltrates into the myocardium. Massive fibrosis was observed in the control group, unlike the failing hearts, in which cardiomyocyte hypertrophy and atrophy dominated. The alternative IL-1beta transcript identified here (IL-1betasv1) was significantly elevated in the failing myocardium compared with the control group. Increased IL-1beta expression seems to be associated with mechanical heart overload. Its endogenous origin, and certain histopathological findings attributed to IL-1beta indicate its importance in cardiac hypertrophy and failure. The lack of some typical IL-1beta actions, i.e. inflammatory, pyrogenic and fibrotic, may suggest a different role of this cytokine in myocardium. It appears that the canine IL-1beta gene can be transcribed in two ways in heart tissue, with the IL-1betasv1 form present mainly in failing hearts.

The effect of physical training on the N-methyl-N-nitrosourea-induced mammary carcinogenesis of Sprague–Dawley rats

The impact of physical activity on carcinogenesis has been demonstrated in many studies. Taking into account the discrepant results of physical exercise on the cell proliferation and apoptosis of breast cancer, we aimed to examine the impact of physical training on N-methyl-N-nitrosourea-(MNU)-induced mammary carcinogenesis. Fifty female rats were divided into four groups according to the intensity of physical activity they undertook. The number of developed tumors, tumor volume, and histopathological diagnoses were noted. Apoptosis and cell proliferation were studied by the number of TUNEL-positive and Ki-67-expressing cells. We demonstrated a statistically significant decrease in the tumor number between all trained groups and the control group. The results were most pronounced in the group with a moderate intensity of training. Moreover, we showed a decrease in tumor volume as training intensity increased, though the differences were not statistically significant. The mean number of TUNEL-positive cancer cells was significantly higher in the training groups than in the control group. These data suggest that physical training, especially of moderate intensity, may alleviate MNU-induced mammary carcinogenesis. The results could suggest that physical exercise-induced apoptosis may be a protective mechanism.

The influence of rapid growth in broilers on florfenicol pharmacokinetics – allometric modelling of the pharmacokinetic and haemodynamic parameters

ABSTRACT 1. The aim of this study was to determine if the pharmacokinetics (PK) of florfenicol (FF) undergo age-dependent changes in broilers. Since drug elimination depends on cardiovascular functions, a haemodynamic study was performed in parallel. 2. Broilers of 0.68, 1.27, 2.45 and 5.13 kg were administered FF in a single intravenous dose of 30 mg/kg body weight. Plasma drug concentrations were determined using high-performance liquid chromatography and PK parameters were calculated using a non-compartmental model. Echocardiography was used to measure haemodynamic functions. 3. During growth, the area under the drug concentration-time curve (AUCinf) increased from 25.7 ± 2.9 to 39.0 ± 8.0 mg h/l. Total body clearance (ClB) gradually decreased from 1.19 ± 0.14 to 0.80 ± 0.15 l/h/kg. Elimination half-life increased from 0.73 ± 0.08 to 1.07 ± 0.07 h, whereas volume of distribution (Vss) remained unchanged. Haemodynamic measurements revealed an increase in cardiac output, from 495 ± 65 to 1303 ± 306 ml/min, in the respective body weight groups. 4. Allometric models for PK and haemodynamic parameters were developed and validated. All models proved to be statistically significant; however, only models for ClB and Vss met stringent validation criteria. Model for ClB was used to calculate an optimal dose for a given age group that provides uniform AUCinf. 5. Age-dependent change in FF kinetics may cause variability in therapeutic response under clinical conditions. A novel approach to the dosing protocol was proposed as a means of optimising therapeutic efficacy.

Normal electrocardiographic and echocardiographic (M-mode and two-dimensional) values in Polish Landrace pigs

BackgroundSwine are recognized animal models of human cardiovascular diseases. Normal values of cardiac morphology and function have been published for swine but for smaller number of pigs and not for swine whose weights ranged up 100 kg. In order to improve the value of results of an investigation on cardiac morphology and function in swine when such data are extrapolated to humans, the aim of this study was to document electrocardiographic and echocardiographic measures of cardiac morphology and function in swine. The study comprised 170 single and repeated measurements that were made in 132 healthy domestic swine (Sus domesticus) whose weights ranged between 20-160 kg and were used as controls in three different experiments. All electrocardiographic and echocardiographic measurements in all swine were done under general anaesthesia.ResultsStatistically significant correlations were found between body weight and heart rate (HR), the duration of the P-wave, the duration of the QRS interval, the duration of the QT interval, and the corrected QT ratio (QTc). Since body weight was positively correlated with age, statistically significant correlations were also found between age and HR, the duration of the P-wave, the duration of the QRS interval, the duration of the QT interval, and the QTc. We found that the thickness of the left ventricular wall and the internal diameter of the left ventricle increased with age and body weight. We also found positive trends between body weight and ejection fraction and body weight and fractional shortening. We also found a positive relationship between age, body weight, and the ratio of the left ventricular internal diameter to its wall thickness, as well as the relative left atrial size.ConclusionMany electro- and echocardiographic measures of cardiac morphology and function of healthy swine are related to their body weight. When the electro- and echocardiographic measures of domestic swine and humans are compared, the most comparable electrocardiographic values are those that were determined in swine whose body weights are not greater than 70 kg. In contrast, the most comparable echocardiographic measures are those that were determined in swine with a body weight of 40–110 kg.