Morphometric analysis of cardiac conduction fibers in horses and dogs, a comparative histological and immunohistochemical study with findings in human hearts.

The principal function of the ventricular conduction system is rapid electrical activation of the ventricles. The aim of this study is to conduct a morphometric study to pinpoint the morphological parameters that define cardiac conduction cells, allowing us to distinguish them from other cells. Five male horse hearts and five male dog hearts were used in the study. The hearts were fixed in a 5% formaldehyde solution. Histological sections of 5 µm thickness were acquired and stained with hematoxylin-eosin and Masson's trichrome and cardiac conduction cells and their junctions were identified by desmin, connexin 40 and a PAS method. We found statistically significant differences in cardiac conduction fibers density and thickness, which was much higher in horses than in dogs (p = 0.000 for both values). By comparing the measured parameters of the cells in both species, we determined that cardiac conduction cells area and diameters were greater in horses than in dogs (p = 0.000 for all values). In dogs there are more junctions (30.8%) than in horses (26.1%), a statistically significant difference (p = 0.041). Our findings regarding the cardiac conduction fibers distribution in the animal species studied becomes new knowledge that contributes to the morphological study of this component of the cardiac conduction system and also makes it possible to locate exactly the site with the highest density of cardiac conduction fibers as a contribution to the cardiological study of these structures that lead to the prevention of ventricular arrhythmias and the identification of their treatment site.

Morphometry and comparative histology of sinus and atrioventricular nodes in humans and pigs and their relevance in the prevention of nodal arrhythmias.

The cardiac conduction system is a network structure that allows the initiation and fast propagation of electrical impulses that trigger the electrical depolarization of the myocardial tissue. The purpose of this work is to study the histological and morphometric characteristics of the different components of the sinus and atrioventricular nodes in humans and pigs and their relationship with supraventricular arrhythmias. In this study, we describe the morphometry of the sinus and atrioventricular nodes of 10 adult humans and 10 pig hearts. A computerized morphometric study has been carried out, where we determined the number of cells that compose the nodes as well as different parameters related to their shape and size. The sinus node in human and pig is a compact structure, whose shape is oblong. Their cells (nodal and transitional cells) are pale and located in the center and the periphery, respectively. The atrioventricular node has also a shape oblong. P cells are pale in both species, but in humans, they are smaller than cardiomyocytes. The T cells are small and pale in both species, identified by hematoxylin-eosin and desmin stains. We have observed through a morphometric profile that the structure of sinus and atrioventricular nodes of pigs and humans show few differences. Pigs can be used as models for hemodynamic applications and experimental studies that include atrial electrical conduction and, in this way, prevent the presentation of arrhythmias that can generate sudden deaths in humans and pigs.

Histological and morphometric study of the components of the sinus and atrioventricular nodes in horses and dogs.

The cardiac nodes are the source of the electrical impulse that is transmitted to the heart, the aim of this work is study the histological and morphometric characteristics of the different components of the sinus and atrioventricular nodes in horses and dogs that help to know the physiopathology of these nodes. A group of ten horse hearts and five dog hearts were used. The region of the sinus and atrioventricular nodes was sectioned serially, and the block of tissue removed for study. The samples were assessed using a morphometric analysis with the Image-Pro Plus 7.1 software and the acquisition of the images using a Leica DMD108 optic microscope. The shape of the horse's sinus node is oblong and its P cells are large. The shape of the dog's sinus is rounded or oblong. The P cells are large and pale. The area of P cells in horses was 976 (SD 223.7) µm2 and in dogs the area for P cells was 106 (SD 30.4) µm2, which indicates that the value for P cells in horses are significantly higher than in dogs (p = .001). The horse atrioventricular node presented an oblong shape and in dogs, presents a spindle shape. The lower cell density in any of the cardiac nodes, especially in P cells of sinus node, can decrease electrical conduction within the nodes and in the internodal tracts, which would reflect the presence of cardiac arrhythmias derived from poor conduction, even in morphologically normal hearts.

Anatomy, immunohistochemistry, and numerical distribution of human splenic microvessels.

The microvascular architecture of the spleen plays an important role in the immunological function of this organ. The different types of vessels are related to different reticular cells each with their own immunomodulatory functions. The present study describes an immunohistochemical and morphometric analysis of the various types of vessels in 21 human autopsy non-pathological splenic samples. On an area of 785,656.37 µm2 for each sample, we classified and quantified the type and number of vascular structures, each according to their morphology and immunohistochemical profile, and obtained the ratios between them. The distribution of trabecular vessels and the characteristics of the venules are reviewed. In our material the so-called "cavernous perimarginal sinus" (anatomical structure previously described by Schmidt et al., 1988) was observed and interpreted as a curvilinear venule shaped by the follicle in contact with the trabecular vein. Our material comprised 261 trabeculae (containing 269 arterial sections and 508 venous sections), 30,621 CD34+ capillaries, 7739 CD271+ sheathed capillaries, 2588 CD169+ sheathed capillaries, and 31,124 CD8+ sinusoids. The total area (TA) (14,765,714.88 µm2) occupied by the sinusoidal sections of the 21 cases was much higher than the TA of the capillary sections (1,700,269.83 µm2). Similarly, the TA (651,985 µm2) occupied by the sections of the trabecular veins was much higher than the TA of the trabecular arteries (88,594 µm2). The total number of CD34+ capillaries and of sinusoids CD8+ was similar for the sum of the 21 cases, nevertheless there were large differences in each case. Statistically the hypothesis that the number of capillaries and sinusoids are present with the same frequency is discarded. In view of the absence of a numerical correlation between capillaries and sinusoids, we postulate that very possibly the arterial and the venous vascular trees are two anatomically independent structures separated by the splenic cords. We believe that this is the first work where splenic microvascularization is simultaneously approached from a morphometric and immunohistochemical point of view.

A quantitative structural and morphometric analysis of the Purkinje network and the Purkinje-myocardial junctions in pig hearts.

The morpho-functional properties of the distal section of the cardiac Purkinje network (PN) and the Purkinje-myocardial junctions (PMJs) are fundamental to understanding the sequence of electrical activation in the heart. The overall structure of the system has already been described, and several computational models have been developed to gain insight into its involvement in cardiac arrhythmias or its interaction with implantable devices, such as pacemakers. However, anatomical descriptions of the PN in the literature have not enabled enough improvements in the accuracy of anatomical-based electrophysiological simulations of the PN in 3D hearts models. In this work, we study the global distribution and morphological properties of the PN, with special emphasis on the cellular and architectural characterization of its intramural branching structure, mesh-like sub-endocardial network, and the PMJs in adult pig hearts by both histopathological and morphometric evaluation. We have defined three main patterns of PMJ: contact through cell bodies, contact through cell prolongations either thick or piliform, and contact through transitional cells. Moreover, from hundreds of micrographs, we quantified the density of PMJs and provided data for the basal/medial/apical regions, anterior/posterior/septal/lateral regions and myocardial/sub-endocardial distribution. Morphometric variables, such as Purkinje cell density and thickness of the bundles, were also analyzed. After combining the results of these parameters, a different septoanterior distribution in the Purkinje cell density was observed towards the cardiac apex, which is associated with a progressive thinning of the conduction bundles and the posterolateral ascension of intramyocardial terminal scattered fibers. The study of the PMJs revealed a decreasing trend towards the base that may anatomically explain the early apical activation. The anterolateral region contains the greatest number of contacts, followed by the anterior and septal regions. This supports the hypothesis that thin distal Purkinje bundles create a junction-rich network that may be responsible for the quick apical depolarization. The PN then ascends laterally and spreads through the anterior and medial walls up to the base. We have established the first morphometric study of the Purkinje system, and provided quantitative and objective data that facilitate its incorporation into the development of models beyond gross and variable pathological descriptions, and which, after further studies, could be useful in the characterization of pathological processes or therapeutic procedures.

Comparative measurement of collagen bundle orientation by Fourier analysis and semiquantitative evaluation: reliability and agreement in Masson's trichrome, Picrosirius red and confocal microscopy techniques.

Measurement of collagen bundle orientation in histopathological samples is a widely used and useful technique in many research and clinical scenarios. Fourier analysis is the preferred method for performing this measurement, but the most appropriate staining and microscopy technique remains unclear. Some authors advocate the use of Haematoxylin-Eosin (H&E) and confocal microscopy, but there are no studies comparing this technique with other classical collagen stainings. In our study, 46 human skin samples were collected, processed for histological analysis and stained with Masson's trichrome, Picrosirius red and H&E. Five microphotographs of the reticular dermis were taken with a 200× magnification with light microscopy, polarized microscopy and confocal microscopy, respectively. Two independent observers measured collagen bundle orientation with semiautomated Fourier analysis with the Image-Pro Plus 7.0 software and three independent observers performed a semiquantitative evaluation of the same parameter. The average orientation for each case was calculated with the values of the five pictures. We analyzed the interrater reliability, the consistency between Fourier analysis and average semiquantitative evaluation and the consistency between measurements in Masson's trichrome, Picrosirius red and H&E-confocal. Statistical analysis for reliability and agreement was performed with the SPSS 22.0 software and consisted of intraclass correlation coefficient (ICC), Bland-Altman plots and limits of agreement and coefficient of variation. Interrater reliability was almost perfect (ICC > 0.8) with all three histological and microscopy techniques and always superior in Fourier analysis than in average semiquantitative evaluation. Measurements were consistent between Fourier analysis by one observer and average semiquantitative evaluation by three observers, with an almost perfect agreement with Masson's trichrome and Picrosirius red techniques (ICC > 0.8) and a strong agreement with H&E-confocal (0.7 < ICC < 0.8). Comparison of measurements between the three techniques for the same observer showed an almost perfect agreement (ICC > 0.8), better with Fourier analysis than with semiquantitative evaluation (single and average). These results in nonpathological skin samples were also confirmed in a preliminary analysis in eight scleroderma skin samples. Our results show that Masson's trichrome and Picrosirius red are consistent with H&E-confocal for measuring collagen bundle orientation in histological samples and could thus be used indistinctly for this purpose. Fourier analysis is superior to average semiquantitative evaluation and should keep being used as the preferred method.

Age-related dermal collagen changes during development, maturation and ageing - a morphometric and comparative study.

The tissue organisation of dermal collagen is gaining importance as a contributing factor both in development and ageing, as well as in skin maturation processes. In this work we aim to study different representative parameters of this structural organisation in 45 human skin samples of assorted ages, by means of image analysis. The variation of these parameters on the basis of age was assessed using several regression models (linear, quadratic and cubic). The area occupied by collagen was significantly reduced as a function of age in the papillary dermis (R(2) = 0.437, P < 0.0001), as well as the thickness of the collagen bundles (R(2) = 0.461, P < 0.0001), following statistical models of cubic and quadratic regression, respectively. The width of the papillary dermis increased in a significant manner over a linear regression model (R(2) = 0.26, P < 0.0001). In the reticular dermis, the cubic regression indicated a significant decline (R(2) = 0.392, P = 0.002) of the area filled with collagen according to the age. Both collagen thickness and bundle orientation parameters fit a quadratic regression over the age in a significant way (R(2) = 0.433 and R(2) = 0.334, respectively, both P < 0.0001). The width of the reticular dermis followed also a significant quadratic distribution according to age (R(2) = 0.193, P = 0.011). These parameters could partially explain the lifelong functional changes taking place in the skin and propose a baseline providing a useful entry point for future investigation.

Microvessel density is high in clear-cell renal cell carcinomas of Ukrainian patients exposed to chronic persistent low-dose ionizing radiation after the Chernobyl accident.

During the 25-year period subsequent to the Chernobyl accident, the morbidity of malignant renal tumors in Ukraine has increased from 4.7 to 10.7 per 100,000 of the total population. Recent studies of our group have shown that increases in morbidity, aggressiveness, and proliferative activity of renal cell carcinomas (RCCs), especially clear-cell renal cell carcinoma (CCRCC), in Ukrainian patients continuously inhabiting the radio-contaminated areas could be explained by specific molecular changes influenced by the so-called "chronic persistent low-dose ionizing radiation" (CPLDIR) exposure. This study aimed to examine the role of angiogenesis in CCRCC carcinogenesis associated with CPLDIR in patients living more than 20 years in cesium 137 ((137)Cs) contaminated areas after the Chernobyl accident in Ukraine. Paraffin-embedded specimens of 106 CCRCs were studied: Control cases were 18 tumors from Spanish patients (group 1), 25 tumors from Ukrainian patients from so-called clean areas without known radio-contamination (group 2), and 63 tumors from Ukrainian patients from radio-contaminated areas (group 3). For intratumoral microvessel density (MVD) determination, anti-CD31 antibody was used. A computerized image analysis program was used to quantitatively calculate the vascular density. Seventy-three percent of group 3 and 72 % of group 2 CCRCCs displayed the highest MVD. A striking increase in MVD was seen in group 3 CCRCCs, in comparison with groups 1 and 2 (p < 0.001). The majority of the hot spot vessels in group 3 was poorly differentiated. Moreover, MVD values for total vessels as well as for capillaries and tumor grade were strongly correlated. When we compared only tumor-node-metastasis tumor stages I and II, the differences remained statistically significant (p < 0.1). The ratio of the average total vessels and capillaries in the Ukrainian groups combined was 1.65:1 in comparison to the Spanish group. Our results provide evidence that CPLDIR exposure increases MVD (particularly capillary) in CCRCCs and is associated with a higher histological grade.

Roflumilast, a phosphodiesterase 4 inhibitor, alleviates bleomycin-induced lung injury.

BACKGROUND AND PURPOSE: The effects of a phosphodiesterase 4 (PDE4) inhibitor, roflumilast, on bleomycin-induced lung injury were explored in 'preventive' and 'therapeutic' protocols and compared with glucocorticoids. EXPERIMENTAL APPROACH: Roflumilast (1 and 5 mg.kg(-1).d(-1), p.o.) or dexamethasone (2.5 mg.kg(-1).d(-1), p.o.) was given to C57Bl/6J mice from day 1 to 14 (preventive) or day 7 to 21 (therapeutic) after intratracheal bleomycin (3.75 U.kg(-1)). In Wistar rats, roflumilast (1 mg.kg(-1).d(-1), p.o.) was compared with methylprednisolone (10 mg.kg(-1).d(-1), p.o.) from day 1 to 21 (preventive) or from day 10 to 21 (therapeutic), following intratracheal instillation of bleomycin (7.5 U.kg(-1)). Analyses were performed at the end of the treatment periods. KEY RESULTS: Preventive. Roflumilast reduced bleomycin-induced lung hydroxyproline, lung fibrosis and right ventricular hypertrophy; muscularization of intraacinar pulmonary vessels was also attenuated. The PDE4 inhibitor diminished bleomycin-induced transcripts for tumour necrosis factor (TNFalpha), transforming growth factor (TGFbeta), connective tissue growth factor, alphaI(I)collagen, endothelin-1 and the mucin, Muc5ac, in lung, and reduced bronchoalveolar lavage fluid levels of TNFalpha, interleukin-13, TGFbeta, Muc5ac, lipid hydroperoxides and inflammatory cell counts. Therapeutic. In mice, roflumilast but not dexamethasone reduced bleomycin-induced lung alphaI(I)collagen transcripts, fibrosis and right ventricular hypertrophy. Similar results were found in the rat. CONCLUSIONS AND IMPLICATIONS: Roflumilast prevented the development of bleomycin-induced lung injury, and alleviated the lung fibrotic and vascular remodeling response to bleomycin in a therapeutic protocol, the latter being resistant to glucocorticoids.

[Genomic profile in high risk neuroblastoma by comparative genomic hybridization]. / Perfil genómico del neuroblastoma de alto riesgo mediante hibridación genómica comparada.

Different subtypes of neuroblastoma (NB) carry associated genetic aberrations that predict their clinical course. Whole chromosome gains are usually associated with early clinical stages and good prognosis, while 1p deletion, 17q gain and MYCN amplification (MNA) are related to advanced stages and poor prognosis. High-risk neuroblastomas (NB-HR) include NB in children aged more than 1 year old, either stage 4 or any stages showing MNA except stage 1. The prognosis of NB-HR patients remains poor, despite aggressive therapy. Only MNA confers poor prognosis. Between January 2000 and February 2005, tumoral specimens from 60 patients with NB-HR were sent to the Spanish Reference Center for NB biological studies. In all cases, MYCN together with 1p36 status was analyzed by fluorescence in situ hybridization (FISH). Comparative genomic hybridization (CGH) was performed in 24 cases. Using FISH we detected 31 MNA cases including 29 with 1p36 deletion; there were 21 cases without MYCN amplification (MNNA) but 7 of these had 1p36 deletion; 8 cases showed MYCN gain (MNG) but 6 of these had 1p36 deletion. CGH showed other chromosomal alterations. Of 11 MNA cases, none had 11q loss and all of them showed 17q gain or 17 disomy. Of the 7 MNNA cases, there were 4 with 11q loss including 2 with 3p loss and all presented 17q gain or 17 disomy. The 6 MNG cases included 4 cases with 11q loss and 5 cases with 17q gain or 17 disomy. Genomic profiling by CGH in NB-HR confirms the interaction among genetic alterations, the prognostic significance of which should be evaluated to establish new treatment criteria.

Perfil genómico del neuroblastoma de alto riesgo mediante hibridación genómica comparada / Genomic profile in high risk neuroblastoma by comparative genomic hybridization

El neuroblastoma presenta alteraciones genéticas que predicen su evolución clínica. Ganancias cromosómicas completas están asociadas a estadios clínicos no avanzados y evolución favorable, mientras que pérdidas de 1p, ganancia de 17q y amplificación del gen MYCN (MNA) son indicativas de estadios clínicos avanzados y pronóstico desfavorable. Son neuroblastomas de alto riesgo (NB-HR) los presentes en niños mayores de un año: estadio 4 o MNA en cualquier estadio de enfermedad, excluyendo estadio 1. El pronóstico de estos enfermos es malo, incluso con tratamientos agresivos. Sólo MNA confiere valor pronóstico negativo. Se remitieron al Centro de Referencia Nacional del neuroblastoma 60 casos de NB-HR. En todos analizamos MYCN y 1p36 con la técnica de hibridación in situ fluorescente (FISH) y en 24 de ellos el perfil genómico con la técnica de hibridación genómica comparada (CGH). Mediante FISH detectamos 31 casos MNA presentando 29 pérdida de 1p36; 21 casos no amplificados (MNNA) con pérdida de 1p36 en 7 casos; y 8 casos con ganancia del gen MYCN (MNG), seis con pérdida de 1p36. Mediante CGH detectamos otros reordenamientos cromosómicos. De 11 casos MNA ninguno presentó pérdida en 11q, todos presentaban disomía del 17 o ganancia 17q. De 7 casos MNNA, cuatro mostraron pérdida de 11q, 2 con pérdida de 3p. Todos mostraron disomía del 17 o ganancia 17q. De 6 casos MNG, cuatro mostraron pérdida de 11q y cinco disomía del 17 o ganancia 17q. El estudio del perfil genómico en NB-HR revela la interacción de alteraciones genéticas cuyo significado pronóstico debe ser evaluado para establecer nuevos criterios terapéuticos

Different subtypes of neuroblastoma (NB) carry associated genetic aberrations that predict their clinical course. Whole chromosome gains are usually associated with early clinical stages and good prognosis, while 1p deletion, 17q gain and MYCN amplification (MNA) are related to advanced stages and poor prognosis. High-risk neuroblastomas (NB-HR) include NB in children aged more than 1 year old, either stage 4 or any stages showing MNA except stage 1. The prognosis of NB-HR patients remains poor, despite aggressive therapy. Only MNA confers poor prognosis. Between January 2000 and February 2005, tumoral specimens from 60 patients with NB-HR were sent to the Spanish Reference Center for NB biological studies. In all cases, MYCN together with 1p36 status was analyzed by fluorescence in situ hybridization (FISH). Comparative genomic hybridization (CGH) was performed in 24 cases. Using FISH we detected 31 MNA cases including 29 with 1p36 deletion; there were 21 cases without MYCN amplification (MNNA) but 7 of these had 1p36 deletion; 8 cases showed MYCN gain (MNG) but 6 of these had 1p36 deletion. CGH showed other chromosomal alterations. Of 11 MNA cases, none had 11q loss and all of them showed 17q gain or 17 disomy. Of the 7 MNNA cases, there were 4 with 11q loss including 2 with 3p loss and all presented 17q gain or 17 disomy. The 6 MNG cases included 4 cases with 11q loss and 5 cases with 17q gain or 17 disomy. Genomic profiling by CGH in NB-HR confirms the interaction among genetic alterations, the prognostic significance of which should be evaluated to establish new treatment criteria

Radial scar versus tubular carcinoma of the breast. A comparative study with quantitative techniques (morphometry, image- and flow cytometry).

The present study is focused on the differential diagnosis between radial scar (RS) and tubular carcinoma (TC) using morphometrical and cytophotometrical analysis (static and flow cytometry) of a number of histologically well-established RS cases (17 lesions) compared with 6 early infiltrating small TC with sclerotic stroma and pseudo-RS fields. One case displayed both RS and TC foci in contiguity. Mean nuclear area was larger in the group of tubular carcinomas (51.0 mu 2) than in the case of radial sclerosis (38.30 mu 2). We also found a larger number of aneuploid cases in tubular carcinomas measured by image cytometry, but both types of lesions were diploid when measured by flow cytometry; only one case of radial scar resulted aneuploid.