Sheep Heart Research Articles

B-011-04 ABLATION SCAR IN A SINGLE PULMONARY VEIN CAUSES PROARRHYTHMIC MECHANICAL DESTABILIZATION IN HEALTHY SHEEP ATRIA

Atrial and pulmonary vein (PV) stretch is proarrhythmic, but the mechanical effect of PV ablation scar on atrial fibrillation (AF) arrhythmogenesis is unknown. We hypothesize that discontinuous PV ablation scars are proarrhythmic because they create heterogeneous stretch. To measure local atrial strain and AF inducibility in healthy sheep hearts with and without PV ablation scar. Functional cardiac MRIs acquired in vivo in sheep (n=12) before and 3 months after discontinuous PV ablation by radiofrequency in the right PV (RPV) were analyzed with a feature-tracking algorithm to obtain local strain in the left atrium (LA). Explanted hearts were perfused with 1:4 blood:Tyrode’s solution in a dual-chamber working-heart set-up. Multi-electrode endocardial catheters were positioned in the RPV and left PV (LPV) for premature stimulation during low (∼12mmHg) and high (∼25mmHg) LA pressure. Control hearts (n=12) underwent similar ex vivo investigation. The maximum longitudinal strain of the myocardial wall between the RPV and LPV increased from 20±6 (mean±SD) to 34±16% (pre vs. post-ablation, respectively; p=0.032), whereas the maximum radial motion fraction of the LA septum close to the RPV decreased from 46±10 to 36±7% (pre vs. post, p=0.035). Sustained AF (>30s) was more often induced during stimulation in ablated than in control hearts (22% vs. 9% of induction attempts (n=73 vs. n=87), respectively, p=0.025). In ablated hearts, an increase in LA pressure augmented AF inducibility (11% vs. 34% of induction attempts, n=38 vs. n=35, low vs. high LA pressure, respectively, p=0.024), whereas this was not the case in control hearts (2% vs. 17% of induction attempts (n=45 vs. n=42), low vs. high, p=0.237). Moreover, the diastolic stimulation threshold was higher in ablated than control hearts (90 (60) (median (IQR)) and 75 (40) mA, p=0.007). Local PV ablation scar caused localized disparate mechanical changes and increased inducibility of sustained AF, especially during increased LA pressure. This was associated with decreased tissue excitability. Therefore, ablation scar in a single PV causes an atrial arrhythmogenic substrate. Ablation lesion sets that homogenize atrial mechanics may improve the AF ablation success.

B-PO01-013 ABLATION SCAR IN A SINGLE PULMONARY VEIN CAUSES PROARRHYTHMIC MECHANICAL DESTABILIZATION IN HEALTHY SHEEP ATRIA

Atrial and pulmonary vein (PV) stretch is proarrhythmic, but the mechanical effect of PV ablation scar on atrial fibrillation (AF) arrhythmogenesis is unknown. We hypothesize that discontinuous PV ablation scars are proarrhythmic because they create heterogeneous stretch. To measure local atrial strain and AF inducibility in healthy sheep hearts with and without PV ablation scar. Functional cardiac MRIs acquired in vivo in sheep (n=12) before and 3 months after discontinuous PV ablation by radiofrequency in the right PV (RPV) were analyzed with a feature-tracking algorithm to obtain local strain in the left atrium (LA). Explanted hearts were perfused with 1:4 blood:Tyrode’s solution in a dual-chamber working-heart set-up. Multi-electrode endocardial catheters were positioned in the RPV and left PV (LPV) for premature stimulation during low (∼12mmHg) and high (∼25mmHg) LA pressure. Control hearts (n=12) underwent similar ex vivo investigation. The maximum longitudinal strain of the myocardial wall between the RPV and LPV increased from 20±6 (mean±SD) to 34±16% (pre vs. post-ablation, respectively; p=0.032), whereas the maximum radial motion fraction of the LA septum close to the RPV decreased from 46±10 to 36±7% (pre vs. post, p=0.035). Sustained AF (>30s) was more often induced during stimulation in ablated than in control hearts (22% vs. 9% of induction attempts (n=73 vs. n=87), respectively, p=0.025). In ablated hearts, an increase in LA pressure augmented AF inducibility (11% vs. 34% of induction attempts, n=38 vs. n=35, low vs. high LA pressure, respectively, p=0.024), whereas this was not the case in control hearts (2% vs. 17% of induction attempts (n=45 vs. n=42), low vs. high, p=0.237). Moreover, the diastolic stimulation threshold was higher in ablated than control hearts (90 (60) (median (IQR)) and 75 (40) mA, p=0.007). Local PV ablation scar caused localized disparate mechanical changes and increased inducibility of sustained AF, especially during increased LA pressure. This was associated with decreased tissue excitability. Therefore, ablation scar in a single PV causes an atrial arrhythmogenic substrate. Ablation lesion sets that homogenize atrial mechanics may improve the AF ablation success.

Joint time‐frequency analysis of visible laser reflections in a sheep heart

Limited methods exist to confirm the position of cardiovascular devices in the superior vena cava or right atrium of the heart. The aim of this study was to design, test and validate the feasibility of whether an optical fiber-based instrument could accurately distinguish when a cardiovascular catheter was located in the superior vena cava vs in the right atrium. An optical fiber was placed in a cardiovascular catheter which was inserted into a living sheep and guided to the vicinity of the heart where diode laser-based reflection intensity data were simultaneously gathered from two visible wavelengths of light reflected from the venous and atrial tissue surfaces near the cavoatrial junction. The time series data were postoperatively analyzed using methods of joint time-frequency analysis and validated against catheter positions determined with fluoroscopy and ECG. The system was successful in distinguishing the location of the superior vena cava from the right atrium.

Strategies to Improve Survival from Surgery for Heart Valve Implantation in Sheep.

Sheep are a commonly used and validated model for cardiovascular research and, more specifically, for heart valve research. Implanting a heart valve on the arrested heart in sheep is complex and is often complicated by difficulties in restarting the heart, causing significant on-table mortality. Therefore, optimal cardioprotective management during heart valve implantation in sheep is essential. However, little is known about successful cardioprotective management techniques in sheep. This article reports our experience in the cardioprotective management of 20 female sheep that underwent surgical aortic valve replacement with a stented tissue-engineered heart valve prosthesis. During this series of experiments, we modified our cardioprotection protocol to improve survival. We emphasize the importance of total body hypothermia and external cooling of the heart. Furthermore, we recommend repeated cardioplegia administration at 20 min intervals during surgery, with the final dosage of cardioplegia given immediately before the de-clamping of the aorta. To reduce the number of defibrillator shocks during a state of ventricular fibrillation (VF), we have learned to restart the heart by reclamping the aorta, administering cardioplegia until cardiac arrest, and de-clamping the aorta thereafter. Despite these encouraging results, more research is needed to finalize a protocol for this procedure.

Low Cost Training Models for Videothoracoscopic Surgical Training

Objective: We designed novel practical simulation models for VATS lung nodule palpation and vessel dissection, and subsequently evaluated the performances of the residents in our thoracic surgery program to account for the appropriate level of difficulty and grade the learning experience. Methods: Artificial lung nodules were formed by injecting sheep heart lung blocks with either cyanoacrylate or construction grade silicone diluted with synthetic thinner. Artificial lung and vessel environment was formed using a sponge, with a tube balloon placed inside a tunnel within the sponge and fixed with a flexible glue. Both models were placed in a standard laparoscopy training box, and both conventional and minimally invasive surgery instruments were used as applicable per the attendee's discretion. Results: In the lung nodule simulation, among 4 residents (postgraduate year (PGY) 1, 3, 4 and 4), the average time to palpating the first nodule was 57 seconds,and the average time of whole lung palpation was 7,7 minutes. In the vascular dissection model, of the five residents (PGY 4, 3, 3, 3, 1), median distance dissected at the first attempt was 3,1 cm (1-4,7), and it was shorter in the second attempt, 2,5 cm (2-3,2). Median dissection duration was shorter in the second attempt (5 vs. 3 minutes). All residents were able to complete the dissection of the balloon from the sponge within 9 attempts. Conclusion: Surgical simulation models can be created with minimal resources, allowing for enough difficulty to maintain engagement and accomplish progressive skill through practice. As clinics shift case volume to minimally invasive procedures, resident exposure to open cases can become more scarce; so simulation training in thoracic surgery can not be perceived as a luxury, it has to be accessible even though the learning environment does not have resources to invest in virtual reality sets or computerized simulators.

Anatomical Similarities Between Mitral and Tricuspid Valves in Sheep, Ovis aries, Hearts

With the recent rapid growth in transcatheter mitral and tricuspid valve interventions, it has become increasingly important to understand detailed anatomy of the valves. In this study, we investigated the similarities, differences, and associations between the dimensions of the atrioventricular valves in a sheep model, as sheep heart valves have a similar morphology to human valves. A systematic dissection of twenty-five sheep hearts was performed, with annular circumference measurement, and sub-valvular anatomy documentation. There was a significant association (r=0.865; p=0.007) between the circumference of the mitral and tricuspid annuli. Authors also identified significantly more chordae tendinea in the subvalvular mitral apparatus compared to the tricuspid valve (15.8±1.2 vs. 13.9±1.5; p<0.001). In conclusion, there is a significant association between the size of mitral and tricuspid valve annuli, and the morphology of leaflets and subvalvular apparatus is different between the two valves. These findings could have important implications in transcatheter device design, sizing, and optimal intervention timing.

Clinicopathological and Microscopic Features of Trypanosoma brucei and Trypanosoma evansi Induced Infections in Sheep II

The present study elucidates further on clinical, gross, and microscopic pathologies induced by single or mixed infections with Trypanosoma evansi and Trypanosoma brucei in sheep. Briefly, the experimental animals were divided into four groups of three animals each. Animals in each group were either infected with T. brucei, T. evansi, mixed (T. brucei and T. evansi), or noninfected. Animals were observed for clinical, gross, and microscopic pathologies for 98 days (14 weeks). The clinical pathologies observed included loss of body condition, pale ocular mucus membrane, rough hair coat, scrotal oedema, scrotal degeneration, emaciation, and death. At necropsy, macroscopic or gross lesions included very pale and anaemic carcass composition, congested and pneumonic lungs with severe haemorrhages, serous atrophy of intestinal and body fats, lymphadenopathy, splenomegaly, and hepatomegaly. Microscopic lesions observed in the testes, spleen, liver, lungs, lymphoid, heart, and brain tissues of infected sheep were varied and included swollen kidney with renal tubular degeneration, the proliferation of lymphocytes at the germinal centers of the spleen, degeneration of the bronchioles, severe testicular degeneration with a reduction in the number of spermatogenic cell layers, degenerated Leydig and Sertoli cells with loss of sperm reserves in the seminiferous lumen, congested liver with sinusoidal spaces and the proliferation of monocytes and lymphocytes. The results indicate that trypanosomosis due to experimental T. brucei, T. evansi, or mixed infections may be an important cause of various grades of tissue and organ pathologies in sheep in trypanosome-endemic areas.
 Keywords: Trypanosomosis; Clinico-pathological and microscopic features; Trypanosoma brucei; Trypanosoma evansi; Mixed infections; Sheep

Age Wise Internal Morphometrical Studies on the Heart of Pre-natal Non-descript Sheep

Background: Heart is the central organ of circulatory system that pumps blood into the blood vessels and performs many vital functions. Its development before birth must be studied to safeguard the animal from the occurrence and consequences of various developmental anomalies. The detailed morphometry of different internal parameters of heart especially in pre-natal sheep has not yet been reported. Methods: The foeti of sheep were divided into two age groups viz. mid prenatal (51-100 days) and late prenatal (101 to 150 days) with fifteen animals in each age group. The various internal parameters were recorded by using digital weighing machine, graduated measuring cylinder, digital Vernier’s calliper, non-stretchable nylon thread and graduated scale. The data recorded was statistically analysed by independent t test with IBM SPSS 25.0 version software. Result: The average thickness of left ventricular wall of heart was measured as 2.85±0.15mm and 4.28±0.07mm in mid prenatal and late prenatal stages respectively with significant (p£0.01) differences between the ages. Similarly, the average diameter of left atrio-ventricular opening of heart was measured as 2.06±0.27mm and 6.08±0.36mm in mid prenatal and late prenatal stages respectively with significant (p£0.01) differences between the ages. The average length of septal papillary muscle in right ventricle was measured as 1.12±0.10mm and 3.29±0.21mm in mid prenatal and late prenatal stages respectively with significant (p£0.01) differences between the ages. Further, the average length of moderator band in heart was measured as 1.45±0.11mm and 4.34±0.23mm in mid prenatal and late prenatal stages respectively with significant (p£0.01) differences between the ages. Similarly, the average length of chordae tendinae in right ventricle of heart was measured as 0.97±0.09mm and 3.50±0.34mm in mid prenatal and late prenatal stages respectively with significant (p£0.01) differences between the ages.

Morphometrical Studies on the Exterior of the Heart of Pre-natal Non-descript Sheep

Background: Heart is the principal organ of circulatory system that pumps blood into the blood vessels and performs many vital functions. Its development before birth must be studied to safeguard the animal from the occurrence and consequences of various developmental anomalies. The detailed morphometry of different external parameters of heart especially in pre-natal sheep has not yet been reported.Methods: The foeti of sheep were divided into three age groups viz. early prenatal (up to 50 days), mid prenatal (51-100 days) and late prenatal (101 to 150 days) with fifteen animals in each age group. The various external parameters were recorded by using digital weighing machine, graduated measuring cylinder, digital Vernier’s calliper, non-stretchable nylon thread and graduated scale. The data recorded was statistically analysed by one way ANOVA with IBM SPSS 25.0 version software. Result: It was revealed that the average width of heart at its base was 4.82±0.22mm, 10.52±0.72mm and 21.57±1.03mm in early prenatal, mid prenatal and late prenatal stages respectively. The average dorso-ventral length of left ventricle of heart was measured as 4.82±0.14mm, 12.07±1.16mm and 25.58±0.88mm in early prenatal, mid prenatal and late prenatal stages respectively. The average distance between the left longitudinal groove and cranial border of heart at base was measured as 2.93±0.11mm, 6.38±0.39mm and 9.78±0.41mm in early prenatal, mid prenatal and late prenatal stages respectively. Similarly, the average distance between the right longitudinal groove and cranial border of heart at middle was measured as 1.96±0.09mm, 5.01±0.34mm and 8.31±0.50mm in early prenatal, mid prenatal and late prenatal stages respectively. All these parameters recorded showed significant (p ≤ 0.01) differences among the ages.

3D MRI of explanted sheep hearts with submillimeter isotropic spatial resolution: comparison between diffusion tensor and structure tensor imaging

ObjectiveThe aim of the study is to compare structure tensor imaging (STI) with diffusion tensor imaging (DTI) of the sheep heart (approximately the same size as the human heart).Materials and methodsMRI acquisition on three sheep ex vivo hearts was performed at 9.4 T/30 cm with a seven-element RF coil. 3D FLASH with an isotropic resolution of 150 µm and 3D spin-echo DTI at 600 µm were performed. Tensor analysis, angles extraction and segments divisions were performed on both volumes.ResultsA 3D FLASH allows for visualization of the detailed structure of the left and right ventricles. The helix angle determined using DTI and STI exhibited a smooth transmural change from the endocardium to the epicardium. Both the helix and transverse angles were similar between techniques. Sheetlet organization exhibited the same pattern in both acquisitions, but local angle differences were seen and identified in 17 segments representation.DiscussionThis study demonstrated the feasibility of high-resolution MRI for studying the myocyte and myolaminar architecture of sheep hearts. We presented the results of STI on three whole sheep ex vivo hearts and demonstrated a good correspondence between DTI and STI.

Genotyping of Toxoplasma gondii in sheep and cattle meat using PCR-RFLP technique

Toxoplasma gondii is an obligate, intracellular parasite, with worldwide distribution. The main source of infection for humans is livestock and meat-producer animals. The relationships between Toxoplasma genotype and biological characteristics of the parasite have already been identified. According to the pathogenicity of the parasite in laboratory animals, Toxoplasma is divided into three genotypes included type I, II and III. Understanding the genotype of the parasite, could help us to predict clinical features and severity of disease. The aim of this study was to identify genotypes of T. gondii in cattle and sheep meat and meat products in Ahvaz city southwest of Iran.One hundred and ninety samples of tongue, heart and muscles of sheep and cattle and meat products, including sausages and burgers, were collected from slaughterhouses and stores. To identify Toxoplasma gondii, DNA were extracted from samples and B1 gene were amplified by specific primers. To determine the genotype of T.gondii, PCR-RFLP was done on positive samples using by amplifying GRA6 gene and endonuclease Msel enzyme. Data analysis showed that the strain of the parasite in all positive samples belonged to genotype I.In this study the predominant Toxoplasma genotype was type I which can cause severe clinical symptoms in immunocompromised patients. Further research is needed to determine the genotype of the parasite in humans and other animals.

Investigation of cadaver preservative properties of solution obtained with a mixture of honey and ethyl alcohol in different proportions: A pilot study on sheep heart

The aim of this study is to evaluate the cadaver embalming effects of the honey-based solutions used as an alternative to formaldehyde which is extremely hazardous for human health. In the study, 42 sheep hearts were used. Tissue and swap samples were taken from different honey solution groups on the 3rd, 75th, 150th, and 225th day to perform histological, microbiological, texture and color analyses. In the macroscopic examination, there was a more significant hardness in the hearts kept in the solution I in A and B groups than the solutions II and III. General fixation was found to be positive in the histological analysis. However, the histological structure became deformed later. The microbiological analysis showed no bacterial or mycotic growth. Moreover, according to the comparative results of the hardness scale applied to the groups, the heart tissues which were kept in AI, BII, and BIIIsolutions on the 3rd day, in AII and BIII solutions on the 150th day, in BIII solutions on the 225th day, in AIII on all days werestatistically found to be similar to the fresh heart tissue. Based on the results of the color analysis, the heart tissues of the experimental groups were not statistically similar to the fresh heart tissue in within-group and between-group comparisons of L value. Consequently, it is thought that the positive effect of honey solutions on the sheep heart tissue should be evaluated in organs with different parenchyma tissue and properties and whole cadaver.

Incidence of Hydatidosis in Slaughtered Animals and their Relation to Public Health at Baghdad Province

Hydatid cysts disease is an important public health problem and economic losses especially at developingcountries such, Iraq. A study of incidence of hydatidosis in sheep and cattle, which are slaughtered foodanimals, was carried out at different areas of Baghdad province outside the government abattoirs, duringa period of 18 months from November 2016 to May 2018. Both slaughtered livestock animals (sheep andcattle) which are local breed of both sexes, they are of difference areas in boundary at Baghdad city, and withages having non descriptive features. Hand palpation and visual inspection were followed in this study. Atotal , 2594 carcasses were examined comprising 1632 sheep and 962 cattle, from these 33, 11 ( sheep andcattle) respectively were found to harbour the cysts, recording percentages of (2.0%, 1.1%) respectively. Thepredominant location cysts at the liver (48.4%, 45.4% ) in sheep and cattle respectively, next by the lungs ,in sheep and cattle (39.3%, 36.3%) respectively. Next to the mixed organs (liver + lung), the incidence was(18.1%) in cattle and (9.0%) in sheep. The percentage of cysts in heart of sheep was (3.0%) but in cattle itwas (0%) Zero. The results revealed that fertile cysts only, were present in sheep with (87.8%) fertility. Cystsin the organs ranged(1-13,2-7) in sheep and cattle respectively. It can be observed that , sheep play a majorrole for distributions the disease. Suggestive control measures and public health hazard were mentioned.

Molecular prevalence of Toxoplasma gondii in sheep from Isfahan, Iran as detected in heart samples by loop-mediated isothermal amplification (LAMP)

Toxoplasmosis is considered an important zoonosis that affects not only humans but also livestock worldwide. Small ruminants are important intermediate hosts of T. gondii. This study aimed to determine the molecular prevalence of T. gondii in the slaughtered sheep by loop-mediated isothermal amplification (LAMP) assay in Isfahan, Iran. A total of 250 sheep heart samples were collected from five different slaughterhouses in Isfahan, Iran, from October 2017 to May 2018. The LAMP assay was optimized targeting the sequence of 529 bp DNA fragments of T. gondii and detected T. gondii DNA in 96.4% (241/250) of samples. A significantly higher prevalence of T. gondii was recorded among female animals as compared to male animals (p ≤ 0.05). No statistically significant difference in T. gondii prevalence was noted between the seasons, type of animals, and age groups (p >0.05). To the best of our knowledge, the current study is the first to use the LAMP assay to determine the prevalence of T. gondii in sheep in Iran. The prevalence obtained in this study by the LAMP assay was higher than the prevalence reported by previous studies that used other methods. This study suggests that sheep meat may play a more prominent role as a source of infection of humans and the LAMP technique can be a recommended tool for accurate diagnosis of toxoplasmosis.

The Condition of the Rat Myocardium and Isolated Sheep Heart after Prolonged 24-Hour Hypothermic Preservation in a Pressurized Carbon Monoxide–Oxygen Gas Mixture

High organoprotective properties of a carbon monoxide (CO)–oxygen (O2) gas mixture were confirmed after prolonged (24-h) preservation of the papillary muscle and an isolated rat heart at 4°C. Hypothermic preservation in the high-pressure gas mixture (6 atm) provided efficient restoration of the contractile activity of the isolated rat heart after 24-h storage at 4°C. The isolated retrograde-perfused Langendorff heart performed physically relevant mechanical work, which was similar in duration to that of an intact control heart. Staining with triphenyltetrazolium chloride did not detect infarcted regions in the myocardium. After preservation, the heart tissue was highly capable of performing its function in a test for electrically stimulated contractile activity of papillary muscles. In the test group, The frequency–intensity relationship, the potentiation effect induced by a pause, and the response to stimulation with isoproterenol of test hearts generally corresponded to the parameters of a normal rat myocardium. A sheep heart, which is comparable in size and weight to a human heart, was for the first time successfully preserved using the gas mixture. Normal heartbeat was spontaneously restored after the start of perfusion in all experiments. Histology did not detect a significant difference between test and control sheep hearts. The normal tissue structure of the myocardium was preserved in the test hearts. The 24-h preservation achieved in the study was four times longer than the maximum allowable preservation time of standard static cold storage. The results obtained with the large laboratory animal heart model showed that the hypothermic preservation protocol is promising for prolonged storage of human hearts.

Identification of Novel miRNAs Involved in Cardiac Repair Following Infarction in Fetal and Adolescent Sheep Hearts.

AimsAnimal models have been used to show that there are critical molecular mechanisms that can be activated to induce myocardial repair at specific times in development. For example, specific miRNAs are critical for regulating the response to myocardial infarction (MI) and improving the response to injury. Manipulating these miRNAs in small animal models provides beneficial effects post-MI; however it is not known if these miRNAs are regulated similarly in large mammals. Studying a large animal where the timing of heart development in relation to birth is similar to humans may provide insights to better understand the capacity to repair a developing mammalian heart and its application to the adult heart.MethodsWe used a sheep model of MI that included permanent ligation of the left anterior descending (LAD) coronary artery. Surgery was performed on fetuses (at 105 days gestation when all cardiomyocytes are mononucleated and proliferative) and adolescent sheep (at 6 months of age when all cardiomyocytes contribute to heart growth by hypertrophy). A microarray was utilized to determine the expression of known miRNAs within the damaged and undamaged tissue regions in fetal and adolescent hearts after MI.Results73 miRNAs were up-regulated and 58 miRNAs were down-regulated significantly within the fetal infarct compared to remote cardiac samples. From adolescent hearts 69 non-redundant miRNAs were up-regulated and 63 miRNAs were down-regulated significantly in the infarct area compared to remote samples. Opposite differential expression profiles of 10 miRNAs within tissue regions (Infarct area, Border zone and Remote area of the left ventricle) occurred between the fetuses and adolescent sheep. These included miR-558 and miR-1538, which when suppressed using LNA anti-miRNAs in cell culture, increased cardiomyoblast proliferation.ConclusionThere were significant differences in miRNA responses in fetal and adolescent sheep hearts following a MI, suggesting that the modulation of novel miRNA expression may have therapeutic potential, by promoting proliferation or repair in a damaged heart.

Improving light microscopic detection of collagen by trichrome stain modification

In present study we aimed to introduce modifications in classical protocol applied to stain collagen fibers by Masson's trichrome stain, in order to decrease time and concentration of different steps and chemical in this traditional protocol. The experiment design contains a series of successive amendment steps based on previously successful steps, in which every step where be modified to get the best result, then the next step of staining protocol will be modified in the classical Masson's trichrome staining protocol in a samples of tissue from liver, heart and intestines of sheep diagnosed with chronic fasciolosis. The result showed that the new modified protocol gives the same staining efficiency to collagen fiber when we used Harris hematoxylin as a nuclear strainer or even excluded this stain from staining protocol. In conclusion this new modified staining protocol give a perfect staining reaction to collagen fibers in pathological samples which is similar to that obtained by Masson's trichrome stain, also this new modified procedure is less time consuming and less toxic effect on human and environment than other trichrome stains, and can be easily conducted either by the technician or the pathologists.

Developmental Changes in Total Cardiac mRNA: RNA Complicates Interpretation of Reference Genes

Finding genes that do not vary with age for use as reference or “housekeeping” can be difficult, as can understanding what experimental gene normalization means in a developmental context. We analyzed expression levels of several potential reference genes in the developing sheep heart, as well as global myocardial mRNA levels compared to total nucleic acid levels. Total nucleic acid, total RNA, messenger RNA (mRNA), and individual gene levels were determined by spectroscopy, dot blot, and PCR. Results were analyzed by ANOVA followed by Tukey’s test, and results reported at P<0.05. Potential reference genes hyaluronidase 2, beta‐2‐microglobulin and ribosomal protein L37a were all found to vary by developmental age. The ribosomal RNA 18s did not vary with age. While total RNA relative to total nucleic acid was constant across development, mRNA was a greater fraction of total RNA in the late‐term fetal heart. These results demonstrate of the difficulty of interpreting normalization of myocardial mRNA expression levels across development.Support or Funding InformationR01HL142483

Novel insights into cardiac regeneration based on differential fetal and adult ovine heart transcriptomic analysis.

The adult mammalian cardiomyocyte has a very limited capacity to reenter the cell cycle and advance into mitosis. Therefore, diseases characterized by lost contractile tissue usually evolve into myocardial remodeling and heart failure. Analyzing the cardiac transcriptome at different developmental stages in a large mammal closer to the human than laboratory rodents may serve to disclose positive and negative cardiomyocyte cell cycle regulators potentially targetable to induce cardiac regeneration in the clinical setting. Thus we aimed at characterizing the transcriptomic profiles of the early fetal, late fetal, and adult sheep heart by employing RNA-seq technique and bioinformatic analysis to detect protein-encoding genes that in some of the stages were turned off, turned on, or differentially expressed. Genes earlier proposed as positive cell cycle regulators such as cyclin A, cdk2, meis2, meis3, and PCNA showed higher expression in fetal hearts and lower in AH, as expected. In contrast, genes previously proposed as cell cycle inhibitors, such as meis1, p16, and sav1, tended to be higher in fetal than in adult hearts, suggesting that these genes are involved in cell processes other than cell cycle regulation. Additionally, we described Gene Ontology (GO) enrichment of different sets of genes. GO analysis revealed that differentially expressed gene sets were mainly associated with metabolic and cellular processes. The cell cycle-related genes fam64a, cdc20, and cdk1, and the metabolism-related genes pitx and adipoq showed strong differential expression between fetal and adult hearts, thus being potent candidates to be targeted in human cardiac regeneration strategies.NEW & NOTEWORTHY We characterized the transcriptomic profiles of the fetal and adult sheep hearts employing RNAseq technique and bioinformatic analyses to provide sets of transcripts whose variation in expression level may link them to a specific role in cell cycle regulation. It is important to remark that this study was performed in a large mammal closer to humans than laboratory rodents. In consequence, the results can be used for further translational studies in cardiac regeneration.

A geometrically adaptable heart valve replacement.

Congenital heart valve disease has life-threatening consequences that warrant early valve replacement; however, the development of a growth-accommodating prosthetic valve has remained elusive. Thousands of children continue to face multiple high-risk open-heart operations to replace valves that they have outgrown. Here, we demonstrate a biomimetic prosthetic valve that is geometrically adaptable to accommodate somatic growth and structural asymmetries within the heart. Inspired by the human venous valve, whose geometry is optimized to preserve functionality across a wide range of constantly varying volume loads and diameters, our balloon-expandable synthetic bileaflet valve analog exhibits similar adaptability to dimensional and shape changes. Benchtop and acute in vivo experiments validated design functionality, and in vivo survival studies in growing sheep demonstrated that mechanical valve expansion accommodated growth. As illustrated in this work, dynamic size adaptability with preservation of unidirectional flow in prosthetic valves thus offers a paradigm shift in the treatment of heart valve disease.