Ana Patrícia Fontes-Sousa

Echocardiographic evaluation including tissue Doppler imaging in New Zealand white rabbits sedated with ketamine and midazolam.

Limited data are available on the use of more recent echocardiographic parameters in the rabbit. Echocardiographic examination, including conventional echocardiography and tissue Doppler imaging (TDI), was performed on 26 male New Zealand white rabbits under ketamine-midazolam sedation. Particular emphasis was placed on the more recent systolic and diastolic parameters, such as myocardial performance index (Tei index) and mitral annular motion (from septal and lateral sides of the left ventricle) obtained using pulsed TDI. Parameters that assessed systolic and diastolic function (fractional shortening, Tei index, and maximal mitral E- and A-wave velocities) were comparable to those reported in the literature for rabbits in the awake state. The less cardiodepressive anaesthetic protocol could offer a good alternative in performing echocardiographic evaluation whenever such caution is necessary. TDI is feasible in healthy rabbits and potentially suitable for the investigation of left ventricle systolic and diastolic function.

Seroprevalence of heartworm (Dirofilaria immitis) in feline and canine hosts from central and northern Portugal.

Dirofilaria immitis is endemic in Portugal. Several studies have reported the presence of canine heartworm disease, although no previous studies on feline infections have been published. The aim of this study was to determine the prevalence of D. immitis in cats and dogs from central and northern Portugal. Blood samples from 434 cats were tested for circulating anti-D. immitis and anti-Wolbachia antibodies. Furthermore, 386 dogs were tested for circulating D. immitis antigens. Overall feline seroprevalence was 15%, while canine prevalence was 2.1%. The highest feline seroprevalences of 18.7% and 17.6% were found in Aveiro and Viseu, respectively, while the highest canine prevalences of 8.8% and 6.8% were found in Coimbra and Aveiro, respectively. Cats and dogs showing respiratory signs presented higher prevalences of 24.4% and 17%, respectively, while 50% of cats with gastrointestinal signs were seropositive. The present study confirms the seropositivity of D. immitis in the feline population in central and northern Portugal, and suggests the importance of including heartworm disease in the list of differential diagnoses of cats and dogs showing clinical signs compatible with the disease.

Impaired Response to ETB Receptor Stimulation in Heart Failure: Functional Evidence of Endocardial Endothelial Dysfunction?

Inotropic effects of selective ETB receptor stimulation depend on the functional integrity of the endocardial endothelium (EE), which is negative when it is intact and positive when it is damaged. These results have been attributed to the existence of two subtypes of ETB receptors in the heart: (i) ETB1, located on the EE, decreases inotropy; (ii) ETB2, located on myocardial cells, increases inotropy. In the present study we investigated the functional integrity of the EE in a heart failure (HF) model (doxorubicin-induced cardiomyopathy) by evaluating the contractile response to ETB1 receptor stimulation. New Zealand White rabbits were treated with doxorubicin (DOX-HF, 1 mg/kg, iv, twice weekly for 8 weeks) or with saline. Contractile effects of increasing doses of a selective agonist of endothelial ETB receptors, IRL-1620 (10−9 to 10−6 M), were studied in papillary muscles (Krebs-Ringer: 1.8 mM CaCl2, 35°C) from control (n = 10) and DOX-HF rabbits (n = 7). Isotonic and isometric twitches were recorded and analyzed. Reported parameters included active tension (AT) and maximum velocities of tension rise (dT/dtmax) and decline (dT/dtmin). On echocardiography, DOX-HF rabbits had increased left ventricular (LV) end-diastolic and end-systolic diameters and reduced ejection fraction (52% ± 2% vs. 61% ± 1%). Contrary to control papillary muscles, DOX-HF muscles showed a steady decrease in contractility between 1 and 4 Hz. In the control group, IRL-1620 induced dose-dependent negative inotropic and lusitropic effects that decreased at 10−6 M: 26% ± 3%, AT; 17% ± 3%, dT/dtmax; and 16% ± 5%, dT/dtmin. In the DOX-HF group, these effects were significantly reduced. At the same concentration, IRL-1620 decreased AT (8% ± 3%) and dT/dtmax (8% ± 3%), without significantly affecting dT/dtmin. This study showed an impaired response to endothelial ETB receptor stimulation, providing for the first time strong evidence of the occurrence of EE dysfunction in the failing heart and further highlighting the potential use of ETB receptor stimulation as a marker of EE function.

Effects of adrenomedullin on systolic and diastolic myocardial function

Adrenomedullin (AM) effects were studied in rabbit papillary muscles by adding increasing concentrations (10(-10) to 10(-6)M) either alone or after pre-treatment with l-NNA, indomethacin, AM22-52 (AM receptor antagonist), CGRP(8-37) (CGRP receptors antagonist), KT5720 (PKA inhibitor), as well as after endocardial endothelium (EE) removal. Passive length-tension relations were constructed before and after a single concentration of AM (10(-6)M). AM concentration-dependently induced negative inotropic and lusitropic effects, and increased resting muscle length (RL). At 10(-6)M, AT, dT/dt(max) and dT/dt(min) decreased 20.9+/-4.9%, 18.3+/-7.3% and 16.7+/-7.8%, respectively, and RL increased to 1.010+/-0.004L/L(max). Correcting RL to its initial value resulted in a 26.6+/-6.4% decrease of resting tension, indicating decreased muscle stiffness, also patent in the down and rightward shift of the passive length-tension relation. The negative inotropic effect of AM was dependent on its receptor, CGRP receptor, PKA, the EE and NO, while the effects of AM on myocardial stiffness were abolished by EE damage and NO inhibition. This latter effect represents a novel mechanism of acute neurohumoral modulation of diastolic function, suggesting that AM is an important regulator of cardiac filling.

Agreement between echocardiographic techniques in assessment of the left ventricular myocardial performance index in rabbits

OBJECTIVE-To report reference values and examine the agreement in the myocardial performance (Tei) index of the left ventricle (LVTI) as measured by tissue Doppler imaging (TDI), pulsed-wave Doppler imaging (PWD), and M-mode echocardiography in clinically normal rabbits. ANIMALS-26 clinically normal male New Zealand White rabbits. PROCEDURES-Echocardiographic examinations that included TDI, PWD, and M-mode echocardiography were performed. Rabbits were sedated by SC administration of ketamine and midazolam. Intraclass correlation coefficients (ICCs) were used to measure absolute agreement among the 3 echocardiographic techniques. Intraclass correlation coefficients were computed for values a and b and for the equation (a - b)/b used to determine LVTI; value a equals the sum of isovolumic contraction time, ejection time, and isovolumic relaxation time, and value b equals the left ventricular ejection time. Values of ICC > 0.75 indicated good agreement between 2 echocardiographic techniques. RESULTS-For value a, Pearson correlation coefficients between pairs of techniques were all high (r r 0.7). However, only the septal TDI and the lateral wall TDI had good agreement (ICC, 0.86). For value b, correlations were generally low with the exception of the correlation between the septal and the lateral wall TDI. For value b, TDI was the only technique with good agreement (ICC, 0.77). For LVTI, only TDI techniques had a significantly positive correlation. All the other correlations were close to zero with a paradoxic moderate negative correlation between PWD-determined LVTI and lateral wall TDI-determined LVTI. CONCLUSIONS AND CLINICAL RELEVANCE-For LVTI, the absolute agreement was poor between all pairs of techniques.

Image Analysis or Stereology Which to Choose for Quantifying Fibrosis

Dear Editor, In a recent issue of this journal, Lattouf et al. (2014) combined polarization microscopy, immunohistochemistry and connective tissue reconstruction to study the collagen staining of picrosirius red. Using this approach, the authors definitively clarified a frequent misconception with regard to the use of this stain, showing that picrosirius red does not allow a differentiation of collagen types in polarized microscopy. Nowadays, this stain is widely used for studying fibrosis in many organs and, particularly, in the liver (e.g., Huang et al. 2013), kidney (e.g., Farris et al. 2011) and heart (e.g., Hadi et al. 2011). Picrosirius red is considered ideal for collagen staining because it does not fade, is selective and is highly reproducible, more so than Masson’s trichrome or collagen immunohistochemistry (Farris et al. 2011). Notably, the assessment of collagen area with picrosirius red and polarization has been weakly correlated with the area of collagen III detected by immunohistochemistry (Farris et al. 2011) or with Masson’s trichrome (Street et al. 2014). This is well explained by the findings presented by Lattouf et al. (2014). In the paper by Lattouf et al. (2014), the authors state that picrosirius red associated with morphometric image analysis “remains the most powerful method to study and quantify collagen” and we would like to comment on this sentence. Morphometric image analysis is highly influenced by threshold settings, magnification and image resolution (Huang et al. 2013), as it is essentially a two-dimensional procedure (Mandarim-de-Lacerda 2003; Marcos et al. 2012). In contrast, stereology uses test-systems that are applied to twodimensional images (such as histological sections) in order to obtain the three-dimensional information that is inherent to biological tissues (Marcos et al. 2012). Because of the objective and independent nature of stereological methods, these methods are used as the gold standard or reference method for comparative image analysis (Daunoravicius et al. 2014; Hadi et al. 2011). Image analysis methods are generally viewed as more user-friendly and faster, but they can also be time-consuming, particularly when there is a need for the manual exclusion of regions in images (Hadi et al. 2011). We have used stereological point counting and picrosirius red in the livers from normal male Wistar rats, aged 2 to 18 months (n=5 per age; Fig. 1), and have quantified the distribution and total content of collagen in these specimens 592180 JHCXXX10.1369/0022155415592180Marcos et al.Short Title research-article2015

Ion Fluxes through KCa2 (SK) and Cav1 (L-type) Channels Contribute to Chronoselectivity of Adenosine A1 Receptor-Mediated Actions in Spontaneously Beating Rat Atria

Impulse generation in supraventricular tissue is inhibited by adenosine and acetylcholine via the activation of A1 and M2 receptors coupled to inwardly rectifying GIRK/KIR3.1/3.4 channels, respectively. Unlike M2 receptors, bradycardia produced by A1 receptors activation predominates over negative inotropy. Such difference suggests that other ion currents may contribute to adenosine chronoselectivity. In isolated spontaneously beating rat atria, blockade of KCa2/SK channels with apamin and Cav1 (L-type) channels with nifedipine or verapamil, sensitized atria to the negative inotropic action of the A1 agonist, R-PIA, without affecting the nucleoside negative chronotropy. Patch-clamp experiments in the whole-cell configuration mode demonstrate that adenosine, via A1 receptors, activates the inwardly-rectifying GIRK/KIR3.1/KIR3.4 current resulting in hyperpolarization of atrial cardiomyocytes, which may slow down heart rate. Conversely, the nucleoside inactivates a small conductance Ca2+-activated KCa2/SK outward current, which eventually reduces the repolarizing force and thereby prolong action potentials duration and Ca2+ influx into cardiomyocytes. Immunolocalization studies showed that differences in A1 receptors distribution between the sinoatrial node and surrounding cardiomyocytes do not afford a rationale for adenosine chronoselectivity. Immunolabelling of KIR3.1, KCa2.2, KCa2.3, and Cav1 was also observed throughout the right atrium. Functional data indicate that while both A1 and M2 receptors favor the opening of GIRK/KIR3.1/3.4 channels modulating atrial chronotropy, A1 receptors may additionally restrain KCa2/SK activation thereby compensating atrial inotropic depression by increasing the time available for Ca2+ influx through Cav1 (L-type) channels.