Quantitative volumetric analysis of a retinoic acid induced hypoplastic model of chick thymus, using Image-J.

OBJECTIVE: To assess the total volume change in a retinoic acid-induced, hypoplastic model of a chick thymus using Image-J. METHODS: This experimental study was carried out at the anatomy department of College of Physicians and Surgeons, Islamabad, Pakistan, from February 2009 to February 2010, and comprised fertilised chicken eggs. The eggs were divided into experimental group A and control group C. Group A was injected with 0.3µg of retinoic acid via yolk sac to induce a defective model of a thymus with hypoplasia. The chicks were sacrificed on embryonic day 15 and at hatching. The thymus of each animal was processed, serially sectioned and stained. The total area of each section of thymus was calculated using Image-J. This total area was summed and multiplied with the thickness of each section to obtain volume. RESULTS: Of the 120 eggs, there were 60(50%) in each group. Image analysis revealed a highly significant decrease in the volume of the chick thymus in the experimental group A than its matched control at the time of hatching (p=0.001). Moreover, volumetric depletion progressed with time, being substantially pronounced at hatching compared to the embryonic stage. CONCLUSIONS: The volume changes were significant and were effectively quantified using Image-J.

[Ultrasound scan of the fetal thymus]. / Evaluación ecográfica del timo fetal.

There is an increased interest nowadays on ultrasound analysis of the fetal thymus. Abnormal fetal thymic growth have been associated with DiGeorge syndrome, conotruncal cardiac malformations, chromosomal abnormalities and adverse outcome in different perinatal conditions as intrauterine growth restriction, preterm birth and others. Different methodologies that measure the fetal thymus by ultrasound have been published, however there is not a consensus of which one is the most useful. Our aim is to describe these methodologies and discuss their clinical applications.

The prophylactic effects of folic acid and vitamin E against valproic acid during fetal thymus development: an ultrastructural study / Los efectos profilácticos del ácido fólico y la vitamina E contra el ácido valproico durante el desarrollo fetal del timo: un estudio ultraestructural

To evaluate histopathologic differences in the thymus of Wistar Albino rat fetuses prenatally exposed to valproic acid (VPA), folic acid (FA) and vitamin E (Vit-E). VPA (400 mg/kg), FA (400 mcg/kg) and Vit -E (250 mg/kg) were administered to rats on each of gestation days 8, 9 and 10. The fetuses (n:24) were divided into four groups: control, VPA, VPA+Vit-E and VPA+FA groups. On the 20th day of gestation, all pregnant rats were sacrificed and the fetuses were extracted. Thin sections from thymus of live fetuses were stained with uranyl acetate-lead citrate and were examined under transmission electron microscope. The histopathological findings of control group was normal. In VPA group, it showed extensive degenerative changes by VPA were on all tissue compartments when compared to controls. In VPA-FA group, vacuoles, mitochondrial cristalysis and swelling were decreased in cytoplasm. In VPA-Vit-E group, lipid storage and vacuolization were observed. Mitochondrial cristalysis decreased. Our aim in the present study is to analyze histopathological changes which may occur in a high risk experimental model after giving of VPA. In addition, protective roles of the administration of FA and Vit-E are assessed.

Se realizó este estudio para evaluar las diferencias histopatológicas en el timo de fetos de ratas Wistar Albinas expuestas prenatalmente a ácido valproico (VPA), ácido fólico (AF) y vitamina E (Vit-E). VPA (400 mg/kg), FA (400 mcg/kg) y vitamina E (250mg/kg) administradas a ratas en los días 8, 9 y 10 de gestación. Los fetos (n=24) fueron divididos en cuatro grupos: control, APV, APV + vitamina E y VPA + FA. En el día 20 de gestación, todas las ratas preñadas fueron sacrificadas y los fetos fueron extraídos. Se obtuvieron secciones delgadas del timo de los fetos y se tiñeron con citrato de uranilo - acetato de plomo, siendo examinados al microscopio electrónico de transmisión. Los hallazgos histopatológicos del grupo control fueron normales. En el grupo VPA, se observaron cambios degenerativos en todos los compartimentos de tejido en comparación con los controles. En el grupo VPA+FA, las vacuolas, cristalisis mitocondrial e inflamación se redujeron en el citoplasma. En grupo VPA + Vitamina E, se observó el almacenamiento de lípidos y vacuolización. La cristalisis mitocondrial disminuyó. El estudio permitió analizar los cambios histopatológicos que pueden ocurrir en un modelo experimental de alto riesgo después de la administración de VPA, además, las funciones de protección por la administración de AF y vitamina E.

Toxoplasma gondii: effect of maternal infection in the development of lymphoid organs of BALB/c neonates.

Toxoplasmosis is one of the worldwide parasitic zoonoses. Alterations in the lymphopoietic system are still poorly studied. We analyzed lymphoid organs of BALB/c mice neonates from Toxoplasma gondii-intraperitoneally-infected mothers on 19th day of gestation, with 30 tachyzoites of strain RH. Normal non-infected pregnant females were used as controls. At 7 days after birth, animals were classified as neonates from infected (NIM) and neonates from non-infected mothers (NNIM). Weight of the thymus and number of thymic cells in NIM were decreased, percentage of apoptosis was significantly increased. Decrease in lymphocytes and monocytes and an increase of plasma cells were observed in bone marrow of NIM. Peripheral blood of NIM showed an increase of monocytes and neutrophils and a decrease in lymphocytes. Infection of the mother during the last day of gestation provokes in the neonates changes in the lymphoid organs that could explain survival of 75% of them.

Genes that code for T cell signaling proteins establish transcriptional regulatory networks during thymus ontogeny.

Gene expression profiling by cDNA microarrays during murine thymus ontogeny has contributed to dissecting the large-scale molecular genetics of T cell maturation. Gene profiling, although useful for characterizing the thymus developmental phases and identifying the differentially expressed genes, does not permit the determination of possible interactions between genes. In order to reconstruct genetic interactions, on RNA level, within thymocyte differentiation, a pair of microarrays containing a total of 1,576 cDNA sequences derived from the IMAGE MTB library was applied on samples of developing thymuses (14-17 days of gestation). The data were analyzed using the GeneNetwork program. Genes that were previously identified as differentially expressed during thymus ontogeny showed their relationships with several other genes. The present method provided the detection of gene nodes coding for proteins implicated in the calcium signaling pathway, such as Prrg2 and Stxbp3, and in protein transport toward the cell membrane, such as Gosr2. The results demonstrate the feasibility of reconstructing networks based on cDNA microarray gene expression determinations, contributing to a clearer understanding of the complex interactions between genes involved in thymus/thymocyte development.

Onset of promiscuous gene expression in murine fetal thymus organ culture.

T-cell differentiation and induction of tolerance to self-antigens occurs mainly in the thymus. Thymic stromal cells, specifically medullary thymic epithelial cells, express a diverse set of genes encoding parenchymal organ-specific proteins. This phenomenon has been termed promiscuous gene expression (PGE) and has been implicated in preventing organ-specific autoimmunity by inducing T-cell tolerance to self antigens. Early thymopoiesis and the critical factors involved in T-cell differentiation can be reproduced in vitro by murine fetal thymus organ culture (FTOC), which mimics the natural thymic microenvironment. To evaluate the occurrence of PGE in FTOC, gene expression profiling during in vitro thymic development in BALB/c mice was performed using a set of nylon cDNA microarrays containing 9216 sequences. The statistical analysis of the microarray data (sam program) revealed the temporal repression and induction of 57 parenchymal and seven lymphoid organ-specific genes. Most of the genes analysed are repressed during early thymic development (15-17 days post-coitum). The expression of the autoimmune regulator (AIRE) gene at 16 days post-coitum marks the onset of PGE. This precedes the induction of parenchymal organ genes during the late developmental phase at 20 days post-coitum. The mechanism of T-cell tolerance induction begins during fetal development and continues into adulthood. Our findings are significant because they show a fine demarcation of PGE onset, which plays a central role in induction of T-cell tolerance.

Hybridization signatures of gamma-irradiated murine fetal thymus organ culture (FTOC) reveal modulation of genes associated with T-cell receptor V(D)J recombination and DNA repair.

In this study, we observed the occurrence of TRBV8.1-DB2.1 V(D)J recombination in murine fetal thymus organ culture (FTOC), in which the thymic microenvironment is mimicked. Since ionizing radiation affects T-cell development, we irradiated FTOCs with gamma rays to evaluate the modulation of genes implicated in TRBV8.1-BD2.1 rearrangements. The nylon cDNA microarray method was employed to monitor the expression of 9216 genes, which were organized in coexpression clusters. Clustering analysis showed similar expression profiling of genes implicated in the V(D)J recombination and DNA double strand break (DSB) repair processes such as XRCC4, RAG-2, Artemis and DNA-PK-cs, thus suggesting overlap between the two processes. The RUNX3 gene, whose coded protein binds to the enhancers of TR genes, was also modulated and the DNA cross-linking LR1 gene, which plays a role in the opening of hairpin DNA structures and whose expression pattern is similar to Artemis, may play a role in the control of V(D)J recombination. Furthermore, our data demonstrate that the FTOC model system and cDNA microarray method are useful tools to evidentiate genes that may play a role in both processes V(D)J recombination and DNA repair.

Hybridization signatures during thymus ontogeny reveals modulation of genes coding for T-cell signaling proteins.

Non-manipulated inbred mouse strains constitutes an interesting model-system for in vivo studies on thymus ontogeny due to the possibility to observe the molecular events of the thymocyte maturation. In previous studies, using RT-PCR method, we have found that several immune system genes such as interleukins and MHC are differentially expressed during ontogeny of the thymus whose genes act as modulators of T-cell differentiation. To determine which other genes are modulated on a large-scale basis, we measured the levels of mRNA expression in mouse fetal thymus (14-17 days of gestation) by hybridization with cDNA microarrays containing 1,576 cDNA sequences derived from the IMAGE MTB library. T-cell maturation was monitored by detection of the T-cell receptor beta TRBV8.1-BD2.1 rearranged DNA segment. Each developmental phase of thymus, displayed a characteristic expression profile, as evaluated by the Cluster and Tree-View softwares. Genes differentially and significantly expressed were selected on the basis of significance analysis of the microarray data (SAM program). With the reclustering of only significantly expressed genes, it was possible to characterize the phases of thymus ontogeny, based on the differential profile of expression. Our method provided the detection of genes implicated in the cell signaling, such as the hematopoietic cell signal transducer gene, genes implicated in T-cell calcium influx (tyrosine phosphatase) and calcium signaling proteins (vesicle transport binding protein 3, proline rich Gla, casein kinase alpha 1 and Down syndrome homolog protein 1) and a gene important for the protein transport, including T-cell receptors chains, towards the cell membrane (Golgi SNAP receptor complex member 2). The results demonstrate that the cDNA microarray used to explore the gene expression was useful for understanding the modulation of several cell-signaling genes, including the calcium cascade pathway, which is important for individual stages of T-cell maturation and control of anergy during thymus ontogeny.

Large scale gene expression analysis of CBA/J mouse strain fetal thymus using cDNA-array hybridizations.

The CBA/J inbred mouse strain constitutes an interesting in vivo model-system for studies on molecular genetics of thymus ontogeny. Using RT-PCR method we have found previously that several immune system related genes as interleukins and MHC are differentially expressed. During this period the onset of T-cell receptor beta rearrangements also occur. To know which other genes are modulated during the ontogeny of the thymus, the mRNA expression levels of fetal thymus (15 and 16 days gestation) of CBA/J mouse strain were measured by hybridization with a set of four macroarrays containing a panel of 6,144 IMAGE cDNA clones from MTB thymus library. We found 145 differentially expressed sequences; 44 were up- and 101 down-regulated in the thymus at 15-16 days gestation. Among these sequences, only 20 are identified as genes whose functions are known and 125 are still unknown. Our data demonstrated that, despite intense research on maturation of the immune system focusing on the activity of several well-characterized genes, the large scale expression profile during thymus ontogeny is still an open matter. The use of cDNA-array technology is an affordable method to identify new genes that may play a role in this phenomenon.

Extrathymic T cells expand in nude mice following different allogeneic stimuli.

We studied extrathymic lymphocyte populations expanded in nude mice after allogeneic stimuli. These were either cells from different tissues or Immunoglobulin (Ig). Although the cells transferred, obtained from Thy-1.1+ donors, were able to induce similar increase in the nude host Thy-1.2+ population, the expanded populations could be qualitatively distinguished from each other by their different expression of mature T cell molecules and by their functional profile. The extrathymic lymphocytes expanded in animals receiving allogeneic fetal thymocytes (FT) were preferentially CD4+ cells and could confer a functional immunocompetent system to the nude host, able to reject allogeneic skin grafts. In contrast, allogeneic adult red blood cells (RBC) led to the expansion of a CD8+ population and to an auto-reactive profile, resulting in the rejection of syngeneic skin grafts by most of the nude hosts. Neither of these profiles was achieved with the other stimuli. These findings support the view that different activation pathways and/or regulatory interactions may lead to the development of distinct extrathymic populations.

Influence of first-wave derived T lymphocytes in the long term functional reconstitution of allogeneic T cell deficient hosts.

The functional immunological reconstitution and the patterns of cytokine secretion were comparatively studied in BALB/c nu/nu mice grafted with allogeneic B6.Thy-1.1+ E14 or E18 embryonic thymus. In spite of equivalent proliferative responses to both mitogen or MLR stimuli, the two groups presented different cytokine patterns. B6 E18-thymus grafted BALB/c nu/nu mice showed a predominant IL-2/IFN-gamma secretion in response to mitogen or to CBA haplotype, with insignificant secretion of either cytokine to the tolerated BALB/c or donor B6 haplotype. In contrast, E14 grafted mice showed a significant IL-10 secretion, both in response to mitogens or to the tolerated haplotypes, even in the absence of a detectable proliferative response. A significant IFN-gamma secretion appeared only accompanying high responses to CBA. The preferential Th2 profile associated to the E14 chimeras was coincident with a longer lifespan of the nude host kept in a conventional environment, higher CD3+ cells frequency in the blood and functional restoration of allogeneic skin graft rejection, not seen on the E18 chimeras. The meaning of these results is discussed in relation to the previously described longer persistence of the first-wave donor derived lymphocytes in the allogeneic BALB/c periphery, also exclusive of the E14 grafted group.

Onset of T-cell receptor Vbeta8.1 and Dbeta2.1 V(D)J recombination during thymus development of inbred mouse strains.

The assembling of T-cell receptor (TCR alpha/beta and gamma/delta) genes depends on the V(D)J recombination occurring in early thymocytes during thymus ontogeny. The V(D)J recombination reaction is directed by a recombinase complex from the RAG-1 and RAG-2 genes, and is modulated by several other gene products. Due to the essential role of the TCRbeta in thymocyte differentiation, it is important to define with precision the temporal emergence of the TCRbeta recombination in normal non-manipulated mouse strains. We analysed the onset of V(D)J recombination between TCRVbeta8.1 and Jbeta2.1 gene segments during fetal development of the thymus in three non-manipulated inbred strains of mice; BALB-c, C57BL/6 and CBA. We show that the emergence of the V(D)J recombination at the TCRbeta locus differs among strains, suggesting an in vivo role of the different genetic backgrounds in driving gene rearrangements.

Upregulated expression of fibronectin receptors underlines the adhesive capability of thymocytes to thymic epithelial cells during the early stages of differentiation: lessons from sublethally irradiated mice.

A 250-cGy whole-body gamma-radiation dose was used to induce thymus regression in mice, and to study the expression and function of extracellular matrix (ECM) receptors in distinct thymocyte subsets emerging during repopulation of the organ. The onset of regeneration was detected from day 2 to 3 postirradiation (P-Ir), when a remarkable increase in the absolute counts of CD3(-)CD25(hi)CD44(+) and CD3(-)CD25(in/hi)CD44(-) cells occurred. Enhanced expression of L-selectin, alpha4, and alpha5 integrin chains (L-selhi alpha4(hi) alpha5(hi)) was also exhibited by these cells. This pattern of expression was maintained until the CD4(+)CD8(+) (DP) young stage was achieved. Afterward, there was a general downregulation of these ECM receptors in DP as well as in CD4(+) or CD8(+) single positive (SP) thymocytes (L-selin alpha4(in) alpha5(in)). In some recently generated SP cells, alpha4 expression was downregulated before the alpha5 chain, and L-selectin was upregulated in half of more mature cells. The expression of the alpha6 integrin chain was downregulated only in maturing CD4(+) cells. Importantly, the increased expression of L-selectin and alpha4 and alpha5 chains in thymocytes was strongly correlated with their adhesiveness to thymic epithelial cells (TEC) in vitro. Blocking experiments with monoclonal antibody or peptides showed the following: (1) that the LDV rather than the REDV cell attachment motif in the IIIC segment of fibronectin is targeted by the alpha4 integrin during thymocyte/TEC adhesion; (2) that the RGD motif of the 120-kD fragment of fibronectin, a target for alpha5 integrin, has a secondary role in this adhesion; and (3) that the YIGSR cell attachment motif of the beta1 chain of laminin/merosin recognized by a nonintegrin receptor is not used for thymocyte adherence. In conclusion, our results show that an upregulated set of receptors endows CD25(+) precursors and cells up to the young DP stage with a high capability of interacting with thymic ECM components.

Epidermal growth factor modulates fetal thymocyte growth and differentiation.

In the present study, we used the fetal organ culture (FTOC) technique in order to study a putative effect of epidermal growth factor (EGF) on the thymus ontogeny. Functional EGF receptors and more recently the EGF molecule itself, respectively, on the membrane of epithelial components of thymic stroma and on a few thymocytes in adult thymus, had been reported in the literature. We could observe a dose-dependent decrease in cellularity and a progressive retention of thymocytes in the double-negative (CD4-/CD8-) stage of differentiation when exogenous EGF was added. Epidermal growth factor interfered with both fetal stroma growth and thymocyte development at a precise moment, that is, in the passage from double-negative to the double-positive (CD4+/CD8+) stage. After a 7-day FTOC in the presence of EGF, most cells recovered were Thy-1.2+, c-kit+, TSA1-/int, CD3-, and one of CD44high/CD25int, CD44-/CD25int, or CD44/CD25-. Some developed into gammadeltaTCR+ cells with a mature (CD3+) phenotype, but not into alphabetaTCR+ thymocytes. It seems that EGF addition makes the cultures "nonpermissible" for alphabetaTCR+ thymocyte generation. We report here the presence of a high Mr "EGF-like" molecule on the membrane of fetal thymocytes, which role in the observed effects is under investigation. Further biochemical characterization of this molecule is still required, because its presence was only evidenced on the basis of its antigenicity.

Emergence of TCR alpha/beta V(D)J recombination and transcription during ontogeny of inbred mouse strains.

The maturation of T-cells depends on V(D)J recombination at the TCR alpha/beta and gamma/delta loci that occurs in the thymus during fetal development. Due to the essential role of the TCRbeta in thymocyte differentiation, it is important to define with precision the temporal emergence of the TCRbeta rearrangement and its expression in normal non-manipulated mouse strains. We studied the onset of the V(D)J recombination and transcription of the TCR Valpha3 and Vbeta11 genes during ontogeny in Balb-c, C57B1/6 and CBA inbred mouse strains. Our data show differences in the emergence of recombination in both TCR alpha and beta loci among strains. The transcriptions of these loci followed respective recombinations and we detected an early germline transcript before TCR beta locus recombination in the CBA strain.

Epidermal growth factor (EGF) modulates fetal thymocyte growth and differentiation: partial reversal by insulin, mimicking by specific inhibitors of EGF receptor tyrosine kinase activity, and differential expression of CD45 phosphatase isotypes.

We have recently reported that epidermal growth factor (EGF) modulates thymocyte development in fetal thymus organ cultures. Exogenously added EGF arrested thymocyte growth and differentiation, acting at the transition from the CD4-CD8- (double-negative (DN)) to the CD4+CD8+ (double-positive (DP)) phenotype. In this study, we further investigate some molecular aspects of this blockade. This inhibitory effect could be mimicked by tyrphostins, which are selective inhibitors of EGF receptor kinase activity. An attempt to use insulin (INS) as a synergizing effector resulted in partial restoration of lobe cellularity, leading to expansion of the CD44-CD25+ DN subset. However, INS did not overcome the EGF-driven blockade of the thymocyte DN --> DP transition. Analysis of CD45 phosphatase showed that this transition was preceded by a rise in CD45RB isotype expression. At the end of a 7-day culture, the remaining DN cells from both EGF- and EGF+INS-treated fetal thymus organ cultures showed a CD45RB- phenotype and were negative for the EGF-immunoreactive molecule described previously on the fetal thymocyte surface. This finding implies that neither molecule is related to the growth capability of cells at this early developmental stage; it is more likely that the molecules are related to subsequent events in the thymocyte pathway to the DP phenotype. Thus, our data support the concept that EGF receptor-related circuitry may be relevant in thymus ontogeny. Additionally, evidence is provided for the duality between growth and differentiation at this particular early stage of thymocyte development.

In vivo and in vitro expression of tenascin by human thymic microenvironmental cells.

Increasing evidence reveals that extracellular matrix components can be regarded as a group of mediators in intrathymic T-cell migration and/or differentiation. Yet, little is known about the expression and putative function of one particular extracellular matrix protein, namely, tenascin in the thymus. Herein we investigated, by means of immunocytochemistry, tenascin expression in normal infant and fetal human thymuses, as well as in cultures of thymic microenvironmental cells. In situ, tenascin distribution is restricted to the medulla and cortico-medullary regions of normal thymuses. This pattern thus differed from that of fibronectin, laminin and type IV collagen, in which subseptal basement membranes were strongly labeled. Interestingly, tenascin did not co-localize with the cytokeratin-defined thymic epithelial cell network. This was in keeping with the in vitro data showing that tenascin-bearing cells were nonepithelial (and probably nonfibroblastic) microenvironmental elements. Studies with fetal thymuses revealed a developmentally regulated expression of tenascin, with a faint but consistent network labeling, in thymic rudiments as early as 12 weeks of gestational age, that progressed to a strong TN expression at 18 weeks of fetal development, which was similar to the distribution pattern observed thereafter, including postnatally. Our results clearly indicated that tenascin is constitutively expressed in the human thymus, since early stages of thymic ontogeny, and suggest that the cell type responsible for its secretion is a nonepithelial microenvironmental cell.

On the size of Hassall's corpuscles in human fetuses.

The areas of prevailingly solid (SHC) and prevailingly cystic (CHC) Hassall's corpuscles in the thymuses of both male and female fetuses 16-39 weeks old were established. The results show that the mean areas of the Hassall's corpuscles increase with fetal age, with the greatest difference between the 16-19 week and 20-23 week age groups. The data indicate that the thymus represent an organ showing a developmental pattern similar to other organs like the spleen's relative growth in human fetus, whose functions are different during the fetal period, being necessary to study its growth in distinct short periods to determinate differentials growth coefficients.

Modulation of cytokeratin expression in the hamster thymus: evidence for a plasticity of the thymic epithelium.

Cytokeratin (CK) expression was investigated, by means of immunocytochemistry, in the hamster thymic epithelium during ontogeny, as well as in primary cultures and upon glucocorticoid hormone treatment in vivo. As compared to the distribution pattern of distinct monoclonal antibody-defined cytokeratins in the normal adult thymus, CK modulation was evidenced in the three situations studied. During thymus ontogeny, both cytokeratins of simple lining epithelia, as CK8 and CK18, as well as the CK1/CK10 pair (typical marker of terminal stage of keratinization), were expressed since early stages of thymus development. They were located in the central region of thymic lobules preceding the cortical-medullary distinctions. This differed from what had been previously shown for mouse thymus ontogeny, revealing that the interspecific diversity in the distribution pattern of thymic cytokeratins occurred early in fetal life. A modulation of CK expression was also detected when hamster thymic epithelial cells (TEC) were led to grow in culture, with a down-regulation of CK19 contrasting with an enhancement of CK18 expression. This diverged from the maintenance of the in situ pattern when human TEC were cultured. Last, in vivo hydrocortisone treatment, known to increase the numbers of KL1+ cells in the mouse thymus medulla, promoted a cortical expression of the CK1/CK10 pair in the hamster thymus. Taken together, our findings demonstrate a continuous plasticity of the thymic epithelium, at least regarding cytokeratin expression, and enlarge the concept of interspecific diversity of intrathymic CK distribution in conditions as morphogenesis, in vitro system, and responsiveness to glucocorticoid hormone treatment.

Neumomediastino por via retroxifoidea vs. tomografía axial computada en Miastenia Gravis: correlación clinico-quirúrgica / Pneumomediastinum by retroxiphoid way vs. tomography axial computed in Myasthemia Gravis: clinico-surgical correlation

Se analizan los resultados de 40 neumomediastinos en 40 pacientes afectados de miastenia gravis, los que venían provistos de pruebas histoquímicas y electrofisiológicas positivas. La vía empleada para la realización del neumomediastino fue la retroxifoidea, incorporando de 60 a 100 c.c. de aire, lo que permite una buena visualización del mediastino anterior y de su contenido con mínimo disconfort del paciente. En 12 casos se efectuó T.C. previa a la realización del neumomediastino. Mientras que la T.C. arrojó resultados falsos positivos y falsos negativos, la positividad diagnóstica del neumomediastino fue del 100 por ciento. Hubo comprobación quirúrgica y anatomopatológica