Diet-Dependent Changes of the DNA Methylome Using a Göttingen Minipig Model for Obesity.

Objective: Environmental factors can influence obesity by epigenetic mechanisms. The aim of this study was to investigate obesity-related epigenetic changes and the potential for reversal of these changes in the liver of Göttingen minipigs subjected to diet interventions. Methods: High-throughput liquid hybridization capture-based bisulfite sequencing (LHC-BS) was used to quantify the methylation status of gene promotor regions in liver tissue in three groups of male castrated Göttingen minipigs: a standard chow group (SD, N = 7); a group fed high fat/fructose/cholesterol diet (FFC, N = 10) and a group fed high fat/fructose/cholesterol diet during 7 months and reversed to standard diet for 6 months (FFC/SD, N = 12). Expression profiling by qPCR of selected metabolically relevant genes was performed in liver tissue from all pigs. Results: The pigs in the FFC diet group became morbidly obese. The FFC/SD diet did not result in a complete reversal of the body weight to the same weight as in the SD group, but it resulted in reversal of all lipid related metabolic parameters. Here we identified widespread differences in the patterning of cytosine methylation of promoters between the different feeding groups. By combining detection of differentially methylated genes with a rank-based hypergeometric overlap algorithm, we identified 160 genes showing differential methylation in corresponding promoter regions in the FFC diet group when comparing with both the SD and FFC/SD groups. As expected, this differential methylation under FFC diet intervention induced de-regulation of several metabolically-related genes involved in lipid/cholesterol metabolism, inflammatory response and fibrosis generation. Moreover, five genes, of which one is a fibrosis-related gene (MMP9), were still perturbed after diet reversion. Conclusion: Our findings highlight the potential of exploring diet-epigenome interactions for treatment of obesity.

Intermittent mitral regurgitation in Cavalier King Charles spaniels: Short-term progression and influence of stress tests.

In young Cavalier King Charles spaniels (CKCS), intermittent mitral regurgitation (iMR; defined as moderate to severe mitral regurgitation [MR] in a small proportion of heartbeats), has been associated with an increased risk of cardiac death due to myxomatous mitral valve disease (MMVD). It is associated with increased R-R interval variability. Little is known about response to physiological factors and whether iMR is a precursor for developing significant MR. The aim of this study was to determine the effect of stress testing on the presence of iMR and heart rate, and short-term (1-2 year) progression of MR in CKCS with and without iMR. In total, 52 CKCS were included. Substudy 1 enrolled six dogs with iMR and 11 dogs without iMR. Substudy 2 enrolled 14 dogs with iMR and 28 dogs without iMR. Substudy 1 prospectively assessed the influence of stress testing on the presence of iMR and heart rate. Substudy 2 retrospectively evaluated short-term progression of iMR. During stress testing, iMR disappeared in 50% of CKCS and no iMR was recorded at mean heart rates >150 beats/min. Heart rate response did not differ between CKCS with or without iMR. CKCS with iMR did not have a higher odds (odds ratio = 5.2; 95% confidence interval, 0.7-38.2) of MR progression compared to controls (P = 0.1). In conclusion, physical stress influenced the occurrence of iMR in CKCS, but heart rate response was not different from CKCS without iMR. Intermittent mitral regurgitation did not significantly predict short-term MR progression. In stressed CKCS with early disease, iMR may be overlooked.

Urine composition in pigs is regulated in both the ureter and the bladder.

The former perception of the urothelium as an impermeable barrier has been revised during the last decade, as increasing evidence of changes in urine composition during its passage of the urinary tract has been presented. Since differences in urothelial permeability between upper and lower urinary tract have been found, our aim is to demonstrate whether changes in urine composition occur during passage through the ureter. We studied consecutive urine samples from both renal pelvises in six pigs and compared them to samples from the bladder and distal ureter. We further sampled urine during storage in the bladder at a fixed volume. All samples were analysed by measuring osmolality and pH, along with the concentration of the following parameters: Na(+), K(+), Cl(-), creatinine, urea. Urine alkalinity increased significantly during passage of the ureter. Creatinine concentration, pH and K(+) increased significantly during the passage from pelvis to the bladder. All other parameters increased non-significantly during the passage to the bladder. The increase in concentration was more pronounced at low concentrations in the pelvis. During storage in the bladder, there was a significant increase in urea concentration. Changes in the composition of urine occur during its passage from the renal pelvis to the bladder and during storage in the bladder. Despite the brief transit time, significant changes in alkalinity were found already during passage through the ureter.

Urine 5-hydroxyindoleacetic acid in Cavalier King Charles spaniels with preclinical myxomatous mitral valve disease.

Higher concentrations of circulating serotonin have been reported in Cavalier King Charles spaniels (CKCS) compared to other dog breeds. The CKCS is also a breed highly predisposed to myxomatous mitral valve disease (MMVD). The aim of this study was to determine urine concentrations of 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite and excretion product of serotonin, in a population of CKCS with preclinical MMVD, and to evaluate whether urine 5-HIAA concentrations were associated with MMVD severity, dog characteristics, setting for urine sampling, platelet count, and serotonin concentration in serum and platelet-poor plasma (PPP). The study population consisted of 40 privately-owned CKCS (23 females; 17 males) with and without preclinical MMVD as follows: American College of Veterinary Internal Medicine (ACVIM) group A (n = 11), ACVIM group B1 (n = 21) and ACVIM group B2 (n = 8). Urine 5-HIAA concentrations were not significantly associated with preclinical MMVD disease, platelet count or circulating concentrations of serotonin (in serum and PPP; P > 0.05). Females had higher 5-HIAA concentrations than males in morning urine collected at home (females, 3.1 [2.9-3.7] µmol/mmol creatinine [median and quartiles]; males, 1.7 [1.2-2.2] µmol/mmol creatinine; P = 0.0002) and urine collected at the clinic (females, 3.5 [3.1-3.9] µmol/mmol creatinine; males, 1.6 [1.3-2.1] µmol/mmol creatinine; P < 0.0001). Five-HIAA concentrations in urine collected at home and at the clinic were significantly associated (P = 0.0004; r = 0.73), and higher concentrations were found in urine collected at the clinic (P = 0.013). Urine 5-HIAA concentration was influenced by sex and setting of urine sampling. Urine 5-HIAA concentration was not associated with MMVD severity or circulating concentrations of serotonin in CKCS with preclinical disease.

Parental time to pregnancy, medically assisted reproduction and pubertal development in boys and girls.

STUDY QUESTION: Does parental fertility, measured by time to pregnancy (TTP), or use of medically assisted reproduction (MAR) affect pubertal development in the offspring? SUMMARY ANSWER: Neither TTP nor type of MAR treatment had clinically relevant implications for mean age at achieving individual pubertal milestones or overall timing of puberty in boys and girls. WHAT IS KNOWN ALREADY: Parental TTP and MAR have been associated with impaired semen quality in adult sons. Timing of puberty reflects earlier signals of reproductive health, but it remains unclear whether parental fertility or MAR affects pubertal development, especially in the growing generation of children conceived by IVF or ICSI. STUDY DESIGN, SIZE, DURATION: In this study, 15 819 children born by mothers in the Danish National Birth Cohort from 2000 to 2003 participated in a nationwide puberty cohort (participation rate = 70%). Parental TTP and use of MAR were reported by mothers in early pregnancy and children's pubertal development data was self-recorded in web-based questionnaires from 11 years of age and 6 monthly throughout puberty (2012-2018). PARTICIPANTS/MATERIALS, SETTING, METHODS: Pubertal development in children (of planned pregnancies, n = 13 285) born by untreated subfecund (TTP: 6-12 months) (n =2038), untreated severely subfeund (TTP: >12 months) (n = 1242), treated subfecund (n = 230) and treated severely subfecund (n = 1234) parents were compared to children born to more fertile parents (TTP: ≤5 months). We estimated mean monthly differences in mean age at achieving individual pubertal milestones (i.e. age at menarche, voice break, first ejaculation and Tanner stages 2, 3, 4 and 5 for breast or genital development and pubic hair growth) and a combined indicator of timing of puberty. Further, we compared mean age at achieving the individual pubertal milestones in children born by use of IVF or ICSI (n = 480) with children born by controlled ovarian stimulation or ovulation induction with or without intrauterine insemination (n = 902). MAIN RESULTS AND THE ROLE OF CHANCE: We found tendencies towards slightly later mean age at male pubertal timing and slightly earlier mean age at female pubertal timing among children born by untreated subfecund, treated subfecund, untreated severely subfecund and treated severely subfecund parents. There were no specific patterns with increasing TTP, use of MAR nor type of MAR treatment, and the magnitude of the mean differences for individual milestones and overall timing of puberty were small, i.e. 0.9 months (95% CI: -1.0; 2.8) for first ejaculation and -0.5 months (95% CI: -2.0; 1.0) months for age at menarche in boys and girls, respectively, born by treated severely subfecund parents when compared with children born by more fertile parents. LIMITATIONS, REASONS FOR CAUTION: Non-differential misclassification of the self-reported information on parental TTP and pubertal development in the offspring may serve as an alternative explanation of the findings, possibly biasing the estimates towards the null. The information on pubertal development was collected from around 11 years of age and onwards. WIDER IMPLICATIONS OF THE FINDINGS: This study adds to the growing body of literature suggesting only limited harmful effects of parental subfecundity and MAR on offspring's long-term growth and development. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Danish Council for Independent Research [DFF 4183-00152]; and the Faculty of Health at Aarhus University. The authors have no financial relationships or competing interests to disclose.

The biomechanical properties of the urethra in boys with hypospadias: a preliminary study.

PURPOSE: The ventral aspect of the penis in boys with hypospadias is composed of dysplastic tissue of the skin and the urethra. The aim of this study was to assess the pre-operative size and biomechanical properties of urethrae in boys with and without hypospadias using a more objective catheter-based system. MATERIALS & METHODS: In this non-blinded clinical observation study, the study population consisted of 19 boys with hypospadias-the case group (median age 13.9 months [range: 12.2-21.3])-and seven boys without hypospadias-the control group (median age 8.5 months [range: 3.8-18.1]). Modified measurements of impedance were used to assess the size, compliance and viscoelasticity of the urethrae under stepwise increased pressures (between 0, 40 and 60 cmH2O) using a customised Endolumenal Functional Lumen Imaging probe (EndoFLIP®). RESULTS: The sizes of the urethrae in boys with hypospadias are variable but tend towards being narrower and less compliant than those of the control subjects i.e. median diameter for meatus urethra was 3.2 mm (range: 2.98-3.92) in the hypospadias group compared with 3.64 (range: 3.22-4.44) in the control group at 40 cmH2O, and the median change in diameter at meatus urethra was 0.08 mm (range: -0.02 to 0.52) in the hypospadias group compared with 0.23 mm (range: -0.02 to 0.34) when the pressure was increased from 40 to 60 cmH2O. This biomechanical analysis found that there was no significant viscoelasticity of the urethral meatus in both the groups, whereas the remainder of the urethral structure generally had viscoelastic properties in the control group, seen as a creep on the time/diameter curves (Figure). In the group of boys with hypospadias, evaluations of the urethrae revealed varying viscoelastic abilities, ranging from abilities that were comparable with those of the control subjects to no sign of viscoelasticity at all. CONCLUSIONS: This study is the first to measure the biomechanical properties of the urethra in children, which might help to provide an understanding as to the structural and functional changes associated with hypospadias. The urethrae in the subjects with hypospadias were variable in diameter but tended to be narrower overall, especially in the distal portion of the urethra. Furthermore, the urethrae in boys with hypospadias were frequently less viscoelastic than those of controls. CLINICAL RELEVANCE: The EndoFLIP® system may be a future way of objectively estimating the severity of a urethral obstruction and could potentially be included in the postoperative assessment of patients with signs of hampered voiding.

Cardiac procholecystokinin expression during haemodynamic changes in the mammalian heart.

Cardiac myocytes express the cholecystokinin gene (CCK) at propeptide level. We recently reported that cardiac CCK expression is acutely regulated by isoprenaline in a porcine model. The regulation of CCK expression after myocardial infarction, in exercise, and in severe heart failure is, however, unknown. Cardiac tissue was obtained from healthy new-born and adolescent farm pigs. Myocardial infarction was induced by coronary artery occlusion in adult minipigs. Healthy male subjects performed a 3-hour exercise test, and patients with severe heart failure referred for right heart catheterization were included. Extracts of porcine cardiac tissue and human plasma were analysed with specific proCCK radioimmunoassays. Cardiac proCCK expression shifted from the right atrium in new-born piglets to include the left atrium in adolescent pigs. Regional proCCK expression in the adolescent pig heart was mainly confined to the atria without different expression in sinus node tissue. In adult minipigs with myocardial infarction, no changes in overall left ventricular function or proCCK expression were observed after 8 weeks. In healthy adults, proCCK in circulation increased markedly during exercise in parallel with pro-B-type natriuretic peptide. Finally, patients with severe heart failure displayed markedly increased proCCK - but not CCK - concentrations in plasma. Taken together, our data shows that regional proCCK expression reflects haemodynamic changes in the mammalian heart. The data supports the notion that cardiac CCK expression resembles that of cardiac natriuretic peptides in atria. The ventricular content of proCCK, however, differs from natriuretic peptides and suggests a distinct secretory pathway in ventricular cardiomyocytes.

Abrogating fibrinolysis does not improve bleeding or rFVIIa/rFVIII treatment in a non-mucosal venous injury model in haemophilic rodents.

Essentials The efficacy of systemic antifibrinolytics for hemophilic non-mucosal bleeding is undetermined. The effect of systemically inhibiting fibrinolysis in hemophilic mice and rats was explored. Neither bleeding nor the response to factor treatment was improved after inhibiting fibrinolysis. The non-mucosal bleeding phenotype in hemophilia A appears largely unaffected by fibrinolysis. SUMMARY: Background Fibrinolysis may exacerbate bleeding in patients with hemophilia A (HA). Accordingly, antifibrinolytics have been used to help maintain hemostatic control. Although antifibrinolytic drugs have been proven to be effective in the treatment of mucosal bleeds in the oral cavity, their efficacy in non-mucosal tissues remain an open question of significant clinical interest. Objective To determine whether inhibiting fibrinolysis improves the outcome in non-mucosal hemophilic tail vein transection (TVT) bleeding models, and to determine whether a standard ex vivo clotting/fibrinolysis assay can be used as a predictive surrogate for in vivo efficacy. Methods A highly sensitive TVT model was employed in hemophilic rodents with a suppressed fibrinolytic system to examine the effect of inhibiting fibrinolysis on bleeding in non-mucosal tissue. In mice, induced and congenital hemophilia models were combined with fibrinolytic attenuation achieved either genetically or pharmacologically (tranexamic acid [TXA]). In hemophilic rats, tail bleeding was followed by whole blood rotational thromboelastometry evaluation of the same animals to gauge the predictive value of such assays. Results The beneficial effect of systemic TXA therapy observed ex vivo could not be confirmed in vivo in hemophilic rats. Furthermore, neither intravenously administered TXA nor congenital knockout of the fibrinolytic genes encoding plasminogen or tissue-type plasminogen activator markedly improved the TVT bleeding phenotype or response to factor therapy in hemophilic mice. Conclusions The findings here suggest that inhibition of fibrinolysis is not effective in limiting the TVT bleeding phenotype of HA rodents in non-mucosal tissues.

Aquaporin expression in the fetal porcine urinary tract changes during gestation.

The expression of aquaporins (AQPs) in the fetal porcine urinary tract and its relation to gestational age has not been established. Tissue samples from the renal pelvis, ureter, bladder and urethra were obtained from porcine fetuses. Samples were examined by RT-PCR (AQPs 1-11), QPCR (AQPs positive on RT-PCR), and immunohistochemistry. Bladder samples were additionally examined by Western blotting. RNA was extracted from 76 tissue samples obtained from 19 fetuses. Gestational age was 60 (n=11) or 100 days (n=8). PCR showed that AQP1, 3, 9 and 11 mRNA was expressed in all locations. The expression of AQP3 increased significantly at all four locations with gestational age, whereas AQP11 significantly decreased. AQP1 expression increased in the ureter, bladder and urethra. AQP9 mRNA expression increased in the urethra and bladder, but decreased in the ureter. AQP5 was expressed only in the urethra. Immunohistochemistry showed AQP1 staining in sub-urothelial vessels at all locations. Western blotting analysis confirmed increased AQP1 protein levels in bladder samples during gestation. Expression levels of AQP1, 3, 5, 9 and 11 in the urinary tract change during gestation, and further studies are needed to provide insights into normal and pathophysiological water handling mechanisms in the fetus.

Development of contractile properties in the fetal porcine urinary bladder.

BackgroundIn early fetal life, the bladder is merely a conduit allowing urine to pass through freely into the amniotic cavity. As the striated external urethral sphincter evolves, the bladder acquires its reservoir and voiding functions. We characterized the myogenic and neurogenic contractions of the normal fetal porcine bladder from midterm until close to full-term gestation.MethodsContractile responses were measured in vitro using bladder strips from fetuses at 60 (N=23) and 100 days (N=21) of gestation. Spontaneous activity, and the responses to potassium chloride (KCl) solution, electrical field stimulation (EFS), and receptor activation were recorded. The smooth muscle content was evaluated histologically.ResultsHistological studies revealed that the fractional content of smooth muscle doubled between the two time points, and passive tension was adjusted to take that into account. Spontaneous activity was regular at 60 days, changing toward an irregular pattern at 100 days. Contractile force elicited by KCl and carbachol increased significantly with gestational age, while contractions to the purinergic agonist, α-ß-methylene adenosine 5'-triphosphate did not. The responses to EFS were almost completely blocked by atropine.ConclusionSpontaneous myogenic contractions become irregular and contractile responses to muscarinic receptor stimulation increase during gestation, as the bladder reservoir and voiding functions develop.

Evidence of reduced bladder capacity during nighttime in children with monosymptomatic nocturnal enuresis.

INTRODUCTION: Bladder capacity in children with nocturnal enuresis is assessed by maximal voided volumes (MVV) obtained through daytime frequency volume (FV) charts. Although a degree of association has been demonstrated, daytime MVV does not consistently correspond with the nocturnal bladder capacity (NBC) in monosymptomatic nocturnal enuresis (MNE). It was hypothesized that isolated reduced NBC is a common phenomenon in children with nocturnal enuresis, despite normal daytime bladder function. OBJECTIVE: The aim of this study was to evaluate NBC in children with MNE and normal daytime voided volumes. Specifically, it aimed to determine the prevalence and degree of reduced NBC when using nocturnal urine production (NUP) during wet nights as a surrogate estimate of NBC. Furthermore, it aimed to investigate the relationship between NBC and desmopressin response. MATERIALS AND METHODS: Data from 103 children aged 5-15 years consecutively treated for MNE in a tertiary referral centre and with normal MVV on daytime FV charts were collected for this cohort study. Home recordings were completed for 2 weeks at baseline and during desmopressin dose titration. Estimated nocturnal bladder capacity (eNBC) was assessed separately each night as the total NUP causing a wet night. If NUP during a wet night was less than MVV, it was considered to be reduced eNBC during that particular night. RESULTS: Surprisingly, 82% (n = 84) of the children with MNE and normal daytime MVV experienced at least one wet night, with NUP below the daytime MVV indicative of a reduced eNBC. For 84 patients, mean percentage of wet nights with reduced eNBC (NUP below MVV) was 49% (SD ± 31). A total of 11% of children with frequently reduced eNBC (>40% of wet nights with reduced eNBC) responded to desmopressin (Summary Fig.). Of the children with frequently reduced NBC, 91% experienced wet nights, with NUP <65% of expected bladder capacity (EBC). CONCLUSIONS: A significant proportion of children with MNE and normal MVV during the daytime frequently experienced wet nights, with a NUP well below their MVV and even <65% of EBC. This indicated that bladder reservoir dysfunction during sleep is relatively common in MNE. This abnormality was not reflected on daytime recordings, and thus nighttime data with NUP must be collected. This phenomenon may explain treatment failure to desmopressin, despite adequate antidiuretic response.

Mitral Regurgitation Severity and Left Ventricular Systolic Dimension Predict Survival in Young Cavalier King Charles Spaniels.

BACKGROUND: Development and progression of myxomatous mitral valve disease (MMVD) in dogs are difficult to predict. Identification at a young age of dogs at high risk of adverse outcome in the future is desirable. HYPOTHESIS/OBJECTIVES: To study the predictive value of selected clinical and echocardiographic characteristics associated with MMVD obtained at a young age for prediction of long-term cardiac and all-cause mortality in Cavalier King Charles Spaniels (CKCS). ANIMALS: 1125 privately owned CKCS. METHODS: A retrospective study including CKCS examined at the age of 1-3 years. Long-term outcome was assessed by telephone interview with owners. The value of variables for predicting mortality was investigated by Cox proportional hazard and Kaplan-Meier analyses. RESULTS: Presence of moderate to severe mitral regurgitation (MR) (hazard ratio (HR) = 3.03, 95% confidence interval (95% CI) = 1.48-6.23, P = 0.0025) even intermittent moderate to severe MR (HR = 2.23, 95% CI = 1.48-6.23, P = 0.039) on color flow Doppler echocardiography was significantly associated with increased hazard of cardiac death. An interaction between MR and sex was significant for all-cause mortality (P = 0.035), showing that males with moderate to severe MR had a higher all-cause mortality compared to males with no MR (HR = 2.38, 95% CI = 1.27-4.49, P = 0.0071), whereas no difference was found between female MR groups. The risk of cardiac (HR = 1.37, 95% CI = 1.14-1.63, P < 0.001) and all-cause (HR = 1.13, 95% CI = 1.02-1.24, P = 0.016) mortality increased with increasing left ventricular end-systolic internal dimension normalized for body weight (LVIDSN ). CONCLUSIONS AND CLINICAL IMPORTANCE: Moderate to severe MR, even if intermittent, and increased LVIDSN in dogs <3 years of age were associated with cardiac death later in life in CKCS.

Markers of Oxidative Stress in Dogs with Myxomatous Mitral Valve Disease are Influenced by Sex, Neuter Status, and Serum Cholesterol Concentration.

BACKGROUND: Cardiovascular disease has been associated with oxidative stress, which has been suggested to contribute to myocardial remodeling in human patients. Little is known about the relationship between myxomatous mitral valve disease (MMVD) and oxidative stress in dogs. OBJECTIVE: To determine whether clinical stage of MMVD is associated with changes in the plasma concentrations of certain markers of oxidative stress in clinically healthy dogs and dogs with MMVD. ANIMALS: Seventy five privately owned dogs: 59 cavalier King Charles Spaniels (CKCS) with different severities of MMVD and 16 dogs of different breeds with clinical signs of congestive heart failure (CHF) caused by MMVD. METHODS: Markers of oxidative stress including malondialdehyde (MDA), oxidized low-density lipoprotein (oxLDL), and vitamin E (α-tocopherol and γ-tocopherol) were measured in plasma and their association with clinical stage of MMVD was assessed by regression analyses. RESULTS: Plasma oxLDL concentration was significantly lower in female dogs compared with males (P = .01). Significantly higher plasma γ-tocopherol concentrations were found in neutered (P = .003) dogs. Vitamin E (α-tocopherol [P = .0004] and γ-tocopherol [P = .003]) was associated with body condition score (BCS), but the association disappeared when cholesterol was included in the analyses. All markers of oxidative stress (MDA, oxLDL, and vitamin E) were positively associated with serum cholesterol concentration (P ≤ .04), but none were associated with clinical stage of MMVD. CONCLUSIONS: In conclusion, markers of oxidative stress are associated with sex, BCS, neuter status, and cholesterol. The results cannot confirm a relationship between oxidative stress and clinical stage of the disease in dogs with MMVD.

Left Ventricular Function After Prolonged Exercise in Equine Endurance Athletes.

BACKGROUND: Prolonged exercise in human athletes is associated with transient impairment of left ventricular (LV) function, known as cardiac fatigue. Cardiac effects of prolonged exercise in horses remain unknown. OBJECTIVES: To investigate the effects of prolonged exercise on LV systolic and diastolic function in horses. ANIMALS: Twenty-six horses competing in 120-160 km endurance rides. METHODS: Cross-sectional field study. Echocardiography was performed before and after rides, and the following morning, and included two-dimensional echocardiography, anatomical M-mode, pulsed-wave tissue Doppler imaging, and two-dimensional speckle tracking. Correlation between echocardiographic variables and cardiac troponin I was evaluated. RESULTS: Early diastolic myocardial velocities decreased significantly in longitudinal (baseline: -17.4 ± 2.4cm/s; end of ride: -15.8 ± 3.2cm/s (P = .013); morning after: -15.4 ± 3.0cm/s (P = .0033)) and radial directions (-32.8 ± 3.4cm/s; -28.1 ± 5.8cm/s (P < .001); -26.4 ± 5.5cm/s (P < .001)). Early diastolic strain rates decreased significantly in longitudinal (1.58 ± 0.27s(-1) ; 1.45 ± 0.26s(-1) (P = .036); 1.41 ± 0.25s(-1) (P = .013)) and circumferential directions (2.43 ± 0.29s(-1) ; 1.96 ± 0.46s(-1) (P < .001); 2.11 ± 0.32s(-1) (P < .001)). Systolic variables showed ambiguous results. No correlations with serum cardiac troponin I concentrations were evident. CONCLUSIONS AND CLINICAL IMPORTANCE: Prolonged exercise in horses is associated with impaired LV diastolic function. Reduced ventricular filling persisted for 7-21 hours despite normalization of biochemical indicators of hydration status, indicating that the observed changes were not entirely related to altered preload conditions. The clinical relevance of cardiac fatigue in horses remains uncertain.

What is a representative voiding pattern in children with lower urinary tract symptoms? Lack of consistent findings in ambulatory and conventional urodynamic tests.

BACKGROUND: Conventional urodynamics (CU) is a highly standardized evaluation of lower urinary tract function. However, in pediatric patients there is concern that the reliability of measurements could be influenced by development effects and measurement variability, as well as by the unfamiliar clinical environment. Ambulatory urodynamics (AU) provides an alternative to this - it uses natural filling, is measured over a prolonged period, and is conducted in a child-friendly environment. OBJECTIVE: The aim of this study was to conduct a comparative analysis of AU and CU to evaluate the consistency in voiding patterns obtained with these two methods of urodynamic testing. STUDY DESIGN: Urodynamic parameters obtained by AU and CU methods in 50 pediatric patients aged >5 years were retrospectively analyzed. Voiding patterns were categorized into six types: coordinated contraction, detrusor after-contraction, fluctuated contraction, pre-void contraction, relief voiding, and weak or absent contraction. Voiding patterns were used to determine the repeatability within urodynamic tests and to identify consistency between AU and CU tests. Five urodynamic parameters were quantified and compared between AU and CU: voided volume, flow rate, maximum detrusor pressure, and detrusor pressure at peak flow rate. For inter-observer variation analysis, 100 voiding curves were randomly selected and categorized by two independent observers; inter-observer agreement was evaluated using the kappa statistic. RESULTS: A single pattern of voiding was identified in five patients using AU and 37 using CU. Consistency of a single pattern between AU and CU was identified in three patients, and consistency between a predominant pattern with AU, defined by one type of voiding occurring >50% of one's voids, and a single pattern with CU was identified in 10 patients (summary table). Flow rates were similar between methods; however, higher maximum detrusor pressure and detrusor pressure at peak flow and lower voided volume were recorded with AU. DISCUSSION: AU resulted in more diverse voiding patterns. Along with the differences in measured urodynamic parameters challenges the application of findings from one method to form a clinical diagnosis. Furthermore, CU may not be as sensitive as AU to the variability in lower urinary tract pathophysiology. CONCLUSIONS: More diverse voiding patterns were identified in AU compared with CU, with a lack of consistency in identified voiding pattern in both methods. Therefore, the urodynamic findings in children may have to be analyzed in more detail, taking the variations into account.

Increased serum C-reactive protein concentrations in dogs with congestive heart failure due to myxomatous mitral valve disease.

Cardiovascular disease in humans and dogs is associated with mildly increased circulating concentrations of C-reactive protein (CRP). Few studies have evaluated associations between circulating CRP and canine myxomatous mitral valve disease (MMVD) and the results reported have been divergent. The aim of this study was to investigate whether serum concentrations of CRP, determined using a novel automated canine-specific high-sensitivity CRP assay (Gentian hsCRP), were associated with severity of MMVD and selected clinical variables in dogs. The study included 188 client-owned dogs with different severities of MMVD. Dogs were classified based on ACVIM consensus statement guidelines (group A, n = 58; group B1, n = 56; group B2, n = 38; group C, n = 36). Data were analysed using descriptive statistics and multiple regression analysis. Dogs with congestive heart failure (CHF; group C) had significantly higher CRP concentrations (median, 2.65 mg/L; quartile 1-quartile 3, 1.09-5.09) compared to dogs in groups A (median, 0.97 mg/L; quartile 1-quartile 3, <0.50-1.97; P = 0.001), B1 (median, 0.78 mg/L; quartile 1-quartile 3, <0.50-1.73, P <0.0001) and B2 (median, 0.60 mg/L; quartile 1-quartile 3, <0.50-1.23; P <0.0001). Other variables reflecting disease severity, including left atrial to aortic root ratio (P = 0.0002, adjusted r(2) = 0.07) and left ventricular end-diastolic diameter normalised for bodyweight (P = 0.0005, adjusted r(2) = 0.06), were positively associated with CRP concentration, but the association disappeared if dogs with CHF were excluded from analysis. In conclusion, slightly higher CRP concentrations were found in dogs with CHF whereas severity of asymptomatic MMVD showed no association with CRP concentrations.

Breeding Restrictions Decrease the Prevalence of Myxomatous Mitral Valve Disease in Cavalier King Charles Spaniels over an 8- to 10-Year Period.

BACKGROUND: Cavalier King Charles Spaniels (CKCS) are predisposed to myxomatous mitral valve disease (MMVD). Studies have indicated a strong genetic background. OBJECTIVE: The aim of this study was to evaluate the effect of a breeding scheme involving auscultation and echocardiography. ANIMALS: In the Danish Kennel Club mandatory breeding scheme, 997 purebred CKCS were examined during the period 2002-2011. Each dog was evaluated 1-4 times with a total of 1,380 examinations. METHODS: Auscultation and echocardiography were performed to evaluate mitral regurgitation murmur severity and degree of mitral valve prolapse (MVP). The odds of having mitral regurgitation murmur or MVP > grade 1 in 2010-2011 compared to 2002-2003 were estimated using logistic regression analysis including age and sex as covariates. Odds were estimated for dogs that were products of the breeding scheme (defined as dogs with both parents approved by the breeding scheme before breeding) and non-products of the breeding scheme (defined as dogs with at least 1 parent with unknown cardiac status). RESULTS: In 2010-2011, the odds of having mitral regurgitation murmur were 0.27 if dogs were a product of the breeding scheme compared with dogs in 2002-2003, reflecting a 73% decreased risk (P < .0001). If non-products of the breeding scheme examined in 2010-2011 were compared with dogs in 2002-2003, no difference in odds was found (P = .49). CONCLUSION AND CLINICAL IMPORTANCE: A mandatory breeding scheme based on auscultation and echocardiography findings significantly decreased the prevalence of MMVD over the 8- to 10-year period. Such a breeding scheme therefore is recommended for CKCS.

Alpha-smooth muscle actin and serotonin receptors 2A and 2B in dogs with myxomatous mitral valve disease.

Canine Myxomatous mitral valve disease (MMVD) is an age-related disease. Serotonin (5-HT) is implicated in the pathogenesis as locally-produced or platelet-derived. Involvement of the 5-HT2A receptor (R) and 5-HT2BR in the induction of myxomatous-mediating valvular myofibroblasts (MF) has been suggested. In an age-matched population of dogs with non-clinical and clinical MMVD, the objectives were to investigate (1) gene expression of 5-HT2AR and 5-HT2BR, (2) protein expression and spatial relationship of 5-HT2AR, 5-HT2BR and MF in the mitral valve (MV) and the cardiac anterior papillary muscle (AP) and (3) serum 5-HT concentrations. Gene expression of 5-HT2BR was significantly higher in MV and AP among dogs with clinical MMVD. This was not found for 5-HT2BR protein expression, though association of 5-HT2BR with myxomatous pathology and co-localization of 5-HT2BR and MF in MV and AP support a functional relationship, perhaps perpetuation of clinical MMVD. 5-HT2AR-expression and serum 5-HT showed no differences between groups.

Serotonin markers show altered transcription levels in an experimental pig model of mitral regurgitation.

Serotonin (5-hydroxytryptamine, 5-HT) signalling is implicated in the pathogenesis of myxomatous mitral valve disease (MMVD) through 5-HT1B receptor (R), 5-HT2AR and 5-HT2BR-induced myxomatous pathology. Based on increased tryptophan hydroxylase-1 (TPH-1) and decreased serotonin re-uptake transporter (SERT) in MMVD-affected valves, increased valvular 5-HT synthesis and decreased clearance have been suggested. It remains unknown how haemodynamic changes associated with mitral regurgitation (MR) affect 5-HT markers in the mitral valve, myocardium and circulation. Twenty-eight pigs underwent surgically induced MR or sham-operation, resulting in three MR groups: control (CON, n = 12), mild MR (mMR, n = 10) and severe MR (sMR, n = 6). The gene expression levels of 5-HT1BR, 5-HT2AR, 5-HT2BR, SERT and TPH-1 were analysed using quantitative PCR (qPCR) in the mitral valve (MV), anterior papillary muscle (AP) and left ventricle (LV). MV 5-HT2BR was also analysed with immunohistochemistry (IHC) in relation to histological lesions and valvular myofibroblasts. All 5-HTR mRNAs were up-regulated in MV compared to AP and LV (P <0.01). In contrast, SERT and TPH-1 were up-regulated in AP and LV compared to MV (P <0.05). In MV, mRNA levels were increased for 5-HT2BR (P = 0.02) and decreased for SERT (P = 0.03) in sMR vs. CON. There were no group differences in 5-HT2BR staining (IHC) but co-localisation was found with α-SMA-positive cells in 91% of all valves and with 33% of histological lesions. In LV, 5-HT1BR mRNA levels were increased in sMR vs. CON (P = 0.01). In conclusion, these data suggest that MR may affect mRNA expression of valvular 5-HT2BR and SERT, and left ventricular 5-HT1BR in some pigs.

Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and transforming growth factor-ß (TGF-ß) in advanced canine myxomatous mitral valve disease.

This study investigated mitral valve and myocardial protein and gene expression of matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs) and transforming growth factor-ß (TGF-ß) and plasma MMP and TGF-ß concentrations in age-matched dog groups euthanized due to either advanced myxomatous mitral valve disease (MMVD) or other reasons. Furthermore, echocardiographic data and lumen/area ratio were correlated with tissue and plasma levels of MMPs, TIMPs and TGF-ßs. Mitral valve and myocardial gene expression of MMP2, MMP14, TGF-ß1 and TGF-ß2 were increased and plasma MMP9 was decreased in advanced MMVD dogs. Myocardial gene expression of TIMP2 and TIMP3 were increased in advanced MMVD. All affected markers correlated to echocardiographic parameters. Significantly narrowed lumen/area ratio was associated with increased myocardial expression of MMP2, MMP14, TIMP2 and TIMP3. No differences in tissue protein expression were recorded. MMP2, MMP14, TIMP2, TIMP3, TGF-ß1 and TGF-ß2 appear to play a local role in the development of advanced MMVD.