P.G. Blanco

Doppler ultrasonographic assessment of maternal and fetal blood flow in abnormal canine pregnancy

The aim of this study was to describe the changes of uterine artery, umbilical artery and fetal abdominal aorta, renal and internal carotid arteries blood flow in abnormal canine pregnancy. Twenty-two, Brucella-negative pregnant bitches were retrospectively classified into abnormal (which had either interrupted their pregnancy between days 52 and 60 or had perinatal death >60% of the litter; n=11) and normal (which had delivered healthy puppies at term; n=11). In all the animals, color and pulsed-wave Doppler examinations of uterine artery were conducted every 10 days from Day 20 to 50 from estimated luteinizing hormone peak. Doppler ultrasonography was also conducted in the fetuses to assess umbilical artery, abdominal aorta, renal and internal carotid arteries from Day 40 to 60 of gestation. Throughout the study, resistance index (RI) of uterine, umbilical and fetal renal arteries decreased up to -15% compared to -36% (P<0.01), -11% compared to -23% (P<0.05) and 2% compared to -13% (P<0.05), respectively in the abnormal and normal bitches. Fetal abdominal aorta and internal carotid did not differ between groups (P>0.05). It is concluded that in dogs, uterine artery, umbilical artery and fetal renal artery RI differ between normal and abnormal gestation being useful for the prediction of adverse obstetric outcome.

Doppler Ultrasound in Canine Pregnancy

Objective. The aim of this article is to review the current knowledge of Doppler ultrasound in canine pregnancy. A brief introduction of Doppler principles and their applications in human obstetrics is also included. Methods. A review of the peer‐reviewed published literature was conducted of the articles pertaining to the use of Doppler ultrasound in canine pregnancy, general Doppler principles, and Doppler applications in human obstetrics. Results. In bitches, Doppler ultrasound has been used to assess ovarian, uterine, umbilical, fetal aorta, common carotid artery, and fetal caudal vena cava blood flow during normal gestation. Most of these vessels increase their blood flow during normal pregnancy. The resistive index and systolic/diastolic ratio of the uterine arteries have been recently described in an experimental pharmacologically induced model of abnormal canine gestation. Both indices progressively increase up to abortion, probably because of progesterone deprivation. Moreover, resistive index abnormalities have been found in canine cases of spontaneous abnormal gestations as described in human medicine. Conclusions. Doppler ultrasound is a useful method for assessing development of the placental and fetal circulation during normal and abnormal canine pregnancy. Further studies are still necessary to widely use this technique in clinical practice.

Ultrasonographic assessment of maternal cardiac function and peripheral circulation during normal gestation in dogs.

The aim of this study was to describe changes in cardiac morphology, systolic function and some peripheral hemodynamic parameters during normal pregnancy in dogs. Twenty healthy bitches, 10 pregnant (PG) and 10 non-pregnant controls (CG), were evaluated every 10 days using echocardiography from day 0 of the estrus cycle to parturition or to day 65 for the PG and CG groups, respectively. Systolic blood pressure (SBP) and uterine artery resistance index (RI) were also assessed. Throughout the study, the shortening fraction and cardiac output increased up to 30% vs. 5% (P<0.01) and 45% vs. 2% (P<0.01) in the PG and CG groups, respectively. In contrast, SBP and RI diminished up to 20% vs. 1% (P<0.01) and 29% vs. 0% (P<0.01) in the PG and CG groups, respectively. In conclusion, a decrease in afterload, an increase in cardiac output and cardiac hypertrophy appear to be the result of the hemodynamic modifications occurring during pregnancy in dogs.

Ryanodine receptor phosphorylation by CaMKII promotes spontaneous Ca2+ release events in a rodent model of early stage diabetes: The arrhythmogenic substrate

BACKGROUND Heart failure and arrhythmias occur more frequently in patients with type 2 diabetes (T2DM) than in the general population. T2DM is preceded by a prediabetic condition marked by elevated reactive oxygen species (ROS) and subclinical cardiovascular defects. Although multifunctional Ca2+ calmodulin-dependent protein kinase II (CaMKII) is ROS-activated and CaMKII hyperactivity promotes cardiac diseases, a link between prediabetes and CaMKII in the heart is unprecedented. OBJECTIVES To prove the hypothesis that increased ROS and CaMKII activity contribute to heart failure and arrhythmogenic mechanisms in early stage diabetes. METHODS-RESULTS Echocardiography, electrocardiography, biochemical and intracellular Ca2+ (Ca2+i) determinations were performed in fructose-rich diet-induced impaired glucose tolerance, a prediabetes model, in rodents. Fructose-rich diet rats showed decreased contractility and hypertrophy associated with increased CaMKII activity, ROS production, oxidized CaMKII and enhanced CaMKII-dependent ryanodine receptor (RyR2) phosphorylation compared to rats fed with control diet. Isolated cardiomyocytes from fructose-rich diet showed increased spontaneous Ca2+i release events associated with spontaneous contractions, which were prevented by KN-93, a CaMKII inhibitor, or addition of Tempol, a ROS scavenger, to the diet. Moreover, fructose-rich diet myocytes showed increased diastolic Ca2+ during the burst of spontaneous Ca2+i release events. Mice treated with Tempol or with sarcoplasmic reticulum-targeted CaMKII-inhibition by transgenic expression of the CaMKII inhibitory peptide AIP, were protected from fructose-rich diet-induced spontaneous Ca2+i release events, spontaneous contractions and arrhythmogenesis in vivo, despite ROS increases. CONCLUSIONS RyR2 phosphorylation by ROS-activated CaMKII, contributes to impaired glucose tolerance-induced arrhythmogenic mechanisms, suggesting that CaMKII inhibition could prevent prediabetic cardiovascular complications and/or evolution.

An Experimental Model to Study Resistance Index and Systolic/Diastolic Ratio of Uterine Arteries in Adverse Canine Pregnancy Outcome

The aim of this study was to describe the changes in the resistance index (RI) and systolic/diastolic ratio (S/D) of the uterine arteries during mid-pregnancy abortion induction in the dog. Sixteen 30-35 day pregnant bitches were randomly assigned to either a pharmacological protocol to interrupt gestation (n = 8) or were used as untreated control group (n = 8). Doppler assessments of uterine arteries blood flow were carried out before the initiation of the protocol and then every other day up to abortion (treated group) or parturition (control group). All treated bitches aborted 6 +/- 1.2 days after initiation of the treatment (while none of the non-treated bitches aborted). Pre-treatment RI and S/D did not differ between groups (p > 0.2) while average post-treatment indexes were (mean +/- SD): 0.62 +/- 0.1 vs 0.53 +/- 0.1 (p < 0.01) and 2.96 +/- 0.9 vs 2.23 +/- 0.3 (p = 0.01), for the treated and non-treated group respectively. Correlations between days to abortion and RI or S/D were 0.75 (p < 0.01) and 0.79 (p < 0.01) and, -0.78 (p < 0.01) and -0.73 (p < 0.01) for the treated and non-treated groups respectively. In the treated group, correlations between serum progesterone (P(4)) concentrations and RI and S/D were -0.76 (p < 0.01) and -0.59 (p < 0.01) respectively. It is concluded that, during induction of abortion, RI and S/D of uterine arteries progressively increased while P(4) decreased.

Comparison of Two Doses of the GnRH Antagonist, Acyline, for Pregnancy Termination in Bitches

Gonadotropin-releasing hormone (GnRH) antagonists are particularly useful when a rapid inhibitory effect on the gonadal axis is required. The aim of this study was to test the efficacy and clinical safety of a low and high dose of the third generation GnRH antagonist, acyline, on pregnancy termination in female dogs. The effect of the antagonist on the progesterone (P(4)) serum concentration was also described. Twenty-one mid-pregnant bitches were randomly assigned to a single subcutaneous (SC) dose of a placebo (PLACE; n = 7), a low (ACY-L; 110 microg/kg; n = 6) or high (ACY-H; 330 microg/kg; n = 8) dose of acyline. The animals were followed up for 15 days. All ACY treated but no placebo-treated animals terminated their pregnancy by abortion (p < 0.01). The ACY-L and ACY-H groups interrupted their pregnancy 7 +/- 1.9 and 6.4 +/- 1.3 days after treatment, respectively (p = 0.7). A significant interaction between treatment and day was found (p < 0.01) for P(4) serum concentrations when PLACE was compared with both ACY groups. No difference was found for this hormone between both ACY groups (p > 0.05) where P(4) diminished throughout the study. The decreasing rate varied among animals and was closely related to the time of abortion when P(4) reached basal concentrations. In PLACE animals, gestation progressed normally and P(4) did not change throughout the study (p > 0.05). None of the bitches presented side effects. It was concluded that acyline safely terminated mid-pregnancy by permanently decreasing P(4) serum concentrations.

Doppler ultrasonographic assessment of maternal and fetal arteries during normal feline gestation

The aim of this study was to describe Doppler parameters of uterine, umbilical, fetal abdominal aorta, fetal renal and fetal internal carotid arteries, as well as fetal heart rate (FHR), during normal feline gestation. Fifteen, 1-4 years of age, weighing 2.5-3.9kg, domestic short-hair pregnant queens, which were born in our institutional cat colony were included in this study. Color and pulsed-wave Doppler evaluations of uterine arteries were performed every 10 days (Day 0, 10, 20, 30, 40, 50, 60) from mating. Fetal Doppler and M-mode ultrasonography were performed to assess umbilical, fetal abdominal aorta, fetal renal, fetal internal carotid arteries and FHR. Both peak systolic velocity (PSV) and end diastolic velocity (EDV) of uterine artery increased up to parturition (P<0.01), while resistance index (RI) decreased from Day 10 onwards (P<0.01). From Day 40 onwards, RI of umbilical artery diminished, while PSV and EDV augmented (P<0.01). Fetal abdominal aorta (P<0.01), renal (P<0.01) and internal carotid (P<0.01) arteries diminished their RI from Days 40, 60 and 40 onwards, respectively. Both PSV and EDV of these three arteries increased progressively. Fetal heart rate was first registered on Day 20 when it began to increase up to Day 40 and then diminished to the end of gestation (P<0.01). It is concluded that blood flow of uterine, umbilical, fetal abdominal aorta, fetal renal and fetal internal carotid arteries progressively increased during normal feline pregnancy, while FHR rose to mid gestation and then decreased up to parturition.

Doppler ultrasonographic assessment of uterine arteries during normal canine puerperium.

The aim of this study was to describe Doppler ultrasonographic changes of uterine arteries during normal canine puerperium. Eight healthy, pure-bred bitches, were ultrasonographically assessed during the postpartum period on Days -3, 3, 10, 17, 24, 38, 52 and 80 (Day 0 defined as the day of parturition). Total horn diameters (TD) and endometrium thickness (E) were evaluated. Color Doppler was used to localize uterine arteries at both sides of the body and pulsed-wave Doppler was performed to obtain the waveforms. Peak systolic velocity (PSV) and end diastolic velocity (EDV) were measured. Resistance index [(PSV-EDV)/PSV] was automatically calculated. Values of TD, E, PSV, EDV and RI were analyzed by repeated measures ANOVA followed by LSD test (SPSS 18.0; SPSS, Chicago, IL, USA). A correlation analysis was also carried out between RI and TD. A progressive decrease of TD (P<0.01) and E (P<0.01) was found in the course of the study. A gradual diminution of PSV (P<0.01) and EDV (P<0.01) and an increase of RI (P<0.01) were also found throughout the study period. The resistance index negatively correlated with TD (r=-0.46; P<0.01) and E (r=-0.44; P<0.01) while the ultrasonographic and vascular changes in this period are concurrent with regenerative changes in the glandular and epithelial structures of the uterus. It is concluded that uterine artery RI progressively increased during normal canine puerperium, associated to the two-dimensional ultrasonographic regression of the organ.

Electrocardiographic Changes in Normal and Abnormal Canine Pregnancy

The aim of this study was to describe the canine electrocardiographic changes in the course of normal and abnormal pregnancy. Twenty-three Brucellosis-negative pregnant bitches were retrospectively classified as normal (n = 12) or abnormal (n = 11). A control group of non-pregnant dioestrous bitches (n = 10) was also included. Normal pregnant females delivered healthy puppies at term while abnormal animals interrupted their pregnancy between days 52-60 (from estimated luteinizing hormone peak) or presented perinatal litter death higher than 60%. All the bitches were electrocardiographically evaluated every 10 days from day 0 to day 65 of the oestrous cycle, to parturition or abortion. Percentage heart rate change increased 31.3% from day 40 to 60 in normal gestation while it decreased -1.8% in dioestrous bitches, although it did not change in the abnormal group (p < 0.01). In the abnormal pregnant group but not in the others, percentage QRSa change fell to -34% on day 60 (p < 0.01). At the same time point, percentage QRSd change was 6.2% vs -4.9% in normal gestations and dioestrous animals, respectively (p < 0.05). Corrected QT interval augmented from day 40 onwards up to 9.9% and 4.3% in the normal pregnant and dioestrous groups, respectively, while it remained unchanged in abnormal gestations (p < 0.05). It is concluded that during normal canine pregnancy, some electrocardiographic parameters begin changing from day 40 onwards, and that pathological gestations differ from normality from day 30. The use of electrocardiography in canine obstetrics might contribute to identify abnormal outcomes before they become clinically evident.

Calcium-calmodulin dependent protein kinase mediates the intracellular signaling pathways of cardiac apoptosis in mice with impaired glucose tolerance

Spontaneous sarcoplasmic reticulum (SR) Ca2+ release events increased in fructose‐rich diet mouse (FRD) myocytes vs. control diet (CD) mice, in the absence of significant changes in SR Ca2+ load. In HEK293 cells, hyperglycaemia significantly enhanced [3H]ryanodine binding and Ca2+/calmodulin‐dependent protein kinase II (CaMKII) phosphorylation of RyR2‐S2814 residue vs. normoglycaemia. These increases were prevented by CaMKII inhibition. FRD significantly augmented cardiac apoptosis in WT vs. CD‐WT mice, which was prevented by co‐treatment with the reactive oxygen species scavenger Tempol. Oxidative stress was also increased in FRD‐SR‐autocamide inhibitory peptide (AIP) mice, expressing the SR‐targeted CaMKII inhibitor AIP, without any significant enhancement of apoptosis vs. CD‐SR‐AIP mice. FRD produced mitochondrial swelling and membrane depolarization in FRD‐WT mice but not in FRD‐S2814A mice, in which the CaMKII site on ryanodine receptor 2 was ablated. FRD decreased mitochondrial area, mean Feret diameter and the mean distance between SR and the outer mitochondrial membrane vs. CD hearts. This remodelling was prevented in AC3I mice, with cardiac‐targeted CaMKII inhibition.