Histology and histopathology Vol.13, nº 1 (1998)

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Open Access
Distribution of neuropeptide-containing nerve fibers in the salamander taste organs
(Murcia : F. Hernández, 1998) Kawakami, T.; Kusakabe, T.; Matsuda, H.; Hiruma, H.; Gono, Y.; Kurihara, K.; Takenaka, T.
Salamander taste organs were recognized as oval cell clusters within the dorsal surface of the tongue. A moderate number of SP, CGRP, VIP, NPY, and GAL immunoreactive nerve fibers terminated in the cell clusters, and some of them penetrated into the basal half of the cell clusters. Around the glands, VIP, NPY, and GAL fihers were numerous, and SP and CGRP fibers were less numerous. Immunoreactivity of SOM and FMRF was not detected either in the nerve fibers associated with the cell clusters or those around the glands. These findings suggest that the chemosensory mechanisms of the salamander gustatory organs are under the control of peptidergic innervation. In addition, the present study indicates that the caudate taste organs are structurally primitive but functionally mature.
Open Access
The cytoskeleton in skeletal, cardiac and smooth muscle cells
(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 1998) Stromer, M. H.
The muscle cell cytoskeleton consists of proteins or structures whose primary function is to link, anchor or tether structural components inside the cell. Two important attributes of the cytoskeleton are strength of the various attachments and flexibility to accommodate the changes in cell geometry that occur during contraction. In striated muscle cells, extramyofibrillar and intramyofibrillar domains of the cytoskeleton have been identifi ed , Evidence of the extramyofibrillar cytoskeleton is seen at the cytoplasmic face of the sa rcolemma in striated muscle where vinculin- and dystrophin-rich costameres adjacent to sarcomeric Z lines anchor intermedi a te filaments that span from peripheral myofibrils to the sarcolemma. Intermediate filaments also link Z lines of adjacent myofibrils and may, in some muscles, link successive Z lines within a myofibril at the surface of the myofibril. The intramyofibrillar cytoskeletal domain includes elastic titin filaments from adjacent sarcomeres that are anchored in the Z line and continue through the M line at the center of the sa rcomere; inelastic nebulin filaments also anchored in the Z line and co-extensible with thin filaments; the Z line, which also anchors thin filaments from adjacent sarcomeres; and the M line, which forms bridges between the centers of adjacent thick filaments, In smooth muscle, the cytoskeleton includes adherens junctions at the cytoplasmic face of the sarcolemma, which a nchor B-actin filaments and intermediate filaments of the cytoskeleton, and dense bodies in the cytoplasm, which also anchor actin filaments and intermediate filaments and which may be the interface between cytoskeletal and contractile elements.
Open Access
hu-PBL-SCID mice: an in vivo model of Epstein-Barr virus-dependent lymphoproliferative disease
(Murcia : F. Hernández, 1998) Fuzzati-Armentero, M.T.; Duchosal, M.A.
The Epstein-Barr virus (EBV) exists in most huni:tns as a lifelong latent infection established in host B cells after a primary viral encounter. In immunosuppressed intlivitluals, such as post-transpl~rnt patients, the presence of EBV-infected B cells may lcad to Iq'niplloproliferative disease. Injection of human peripheral blood lymphocytes from EBV-positive clonors into mice wit h severe combined immunodeficiency induces human lymphoproliferative disease i n the recipient closely resembling that of human posttransplant patients. This xenochimeric human-mouse model is increasingly being used to elucidwrc the mcchnnisms of EBV-specific lymphomagenesis and to assess novel therapeutic approaches.
Open Access
Electron probe microanalysis of permanent human enamel and dentine. A methodological and quantitative study
(Murcia : F. Hernández, 1998) Sánchez-Quevedo, M.C.; Nieto-Albano, O.H.; García, J.M.; Gómez de Ferraris, M.E.; Campos, Antonio
Sample preparation of dental tissues for quantitative electron microprobe analysis has not been critically examined because of the highly mineralized nature of these structures. The present study was designed to establish the most suitable method for the electron probe quantitative determination of calcium in human permanent enamel and dentine while preserving the morphological features. Comparisons of quantitative data obtained with air-drying and freeze-drying methods showed that calcium in enamel was more accurately measured in specimens prepared with cryopreservation and freeze-drying. No significant differences between the methods tested were found in dentine although cryopreservation and freeze-drying yielded less statistical variability. Moreover this approach did not modify morphological features of interest. We recommend this combination of processing techniques f o r human permanent teeth not only because it was found the most accurate and least variable in determining calcium concentration, but also because of its potential usefulness in studies of alterations in tooth mineralization.
Open Access
What is the value of proliferation markers in the normal and neoplastic cervix?
(Murcia : F. Hernández, 1998) Heatley, M.K.
Markers of cellular proliferation have been widely applied in cervical disease and include techniques which are applicable to routinely processed tissue including standard hematoxylin and eosin sections, and sections labelled with antibodies to Ki-67 and PCNA proteins. Flow cytometry and in vivo techniques including labelling with Bromodeoxyuridine (BrdU) and radiolabelled thymidine require more specialized facilities. Increases in the mitotic index and the Ki-67 and PCNA labelling indices, the incidence of aneuploidy together with increases in in vivo labelling with BrdU and radiolabelled thymidine have been demonstrated as the grade of cervical intraepithelial neoplasia (CIN) increases. With respect to invasive tumours increases in these parameters are associated with increased tumour size, stage and improved survival after radiotherapy. At present the major practical application of these markers appears to be in distinguishing between postmenopausal atrophy and CIN lesions on histological sections and, in combination with the Papnet system, in identifying high grade dyskaryosis on blood stained cervical smears. Future development may permit the identification of those patients whose CIN lesion will progress, and who require treatment, to be distinguished from those whose lesions will stay static or regress and who can be followed up cytologically. This promises a more rational use of health care resources. Most of the studies to date have been on small numbers of cases. Meta-analysis of existing studies and large, possibly multicentric, prospective studies are needed to elucidate the value of these markers.
Open Access
Histochemical and ultrastructural study of skeletal muscle in patients with sepsis and multiple organ failure syndrome (MOFS)
(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 1998) Díaz, N. L.; Finol, H. J.; Torres, S. H.; Zambrano, C. I.; Adjounian, H.
Muscle biopsies for histochemical and ultrastuctural analysis were obtained from seven critically ill patients admitted to the Intensive Care Unit of the "Domingo Luciani" Hospital, Caracas, Venezuela. The sample included two patients with sepsis of abdominal origin, and five that presented sepsis/MOFS, with renal, hepatic, and respiratory disturbances and muscular weakness. Sections were examined for myosin adenosine triphosphatase (ATPase) after pre-incubation with both acid buffer (pH 4.37 and 4.6) and alkaline buffer (pH 10.3), for reduced nicotinamide dinucleotide diaphorase (NADHd), and for a-glycerophosphate dehydrogenase (a-GPDH). Sections were stained with hematoxilin and eosin to look for pathological changes and examined with a transmission electron microscope. Skeletal muscle of patients in early stage of sepsis showed a normal aspect with light microscopy, but at the ultrastructural level some of the fibres showed atrophy and some capillaries looked altered . Patients with sepsis/MOFS exhibited an evident muscle disorder with oedema. infiltrate, atrophy and segmental necrosis. All fibre types showed decrease in diameter; specially fibre types IIA and lIB . Intramuscular capillaries were thickened and occluded , indexes of capillarity were slightly reduced, and fibre oxidative activity was decreased. At ultrastructural level fibres showed severe atrophy, contractile system disorganization and segmental necrosis. Capillaries were also altered and the mononuclear cell infiltrate was abundant and represented by macrophages. lymphocytes and mastocytes.
Open Access
Programmed cell death in nodular palmar fibromatosis (Morbus Dupuytren)
(Murcia : F. Hernández, 1998) Wilutzky, B.; Berndt, A.; Katenkamp, D.; Kosmehl, H.
The regular loss of cellularity during involutional phase of nodular palmar fibromatosis (Morhus D~lp~lyt r enin)d icates a regulated process known as programmed cell death (apoptosis). Using the TUNEL method apoptosis-related DNA fragmentation is detected in numerous cells as a characteristic feature of fibromatosis noduli of involutional phase. By means of double labelling technique. a-smooth muscle actin immunohistochenlistry and TUNEL method for apoptosis, i t is demonstrated that the cells which underwent apoptotosis are myofibroblasts. As anticipated, the antidote to apoptosis bcl-2 is not detected in involutional phase, but neither it is evidenced in proliferative phase. Immunohistochemically, FasIAPO-l is shown to be existent in a very small number of fibroblasts in involutional phase. However. in view of the high number of TUNEL-stained cells a significance in regulating apoptosis in nodular palmar fibromatosis seems improbable. Taking into account that the development of the fibromatosis noduli, the expression of myofibroblast phenotype, basement membrane formation and growth factor expression including TGFD culminates in involutional phase the initiation of apoptotic cell death can be discussed in relation to these growth factors and matrix protein action and the programmed cell death may be considered as the final step of myofibroblast phenotype evolution.
Open Access
Paracrine control of steroid hormone secretion by chromaffin cells in the adrenal gland of lower vertebrates
(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 1998) Mazzocchi, G.; Gottardo, G.; Nussdorfer, G. G.
The adrenal glands of lowe r vertebrates display a notable intermingling between steroidogenic and chromaffin tissues, which increases from Pisces to Al'es. As in mammals, adrenal chromaffin cells contain and release, in addition to catechol amines, serotonin and several peptides, which may affect the secretory acti vity of steroidogeni c cells in a paracrine manner. Stimulatory molec ul es in c lud e se roto nin , arg inin e-va so tocin. tachykinin s, vasoac ti ve intestinal peptide , pituita ry adenylate cyclase-acti vating peptide and calcitonin generelated peptide: inhibitory molec ul es are dopamine, somatotropic hormone-release inhibiting hormone and ga lanin . Epin ephrine and no repinephrine appe ar to stimulate steroid sec retion in Aves and to inhibit it in Pisces, while their ac tion in Amphibia is controversial. Likewise . atri a l natriureti c peptide exe rts an antisec re ta gog ue ac ti o n in Amphibia and a ma rk ed secretagogue effe ct in Pisces and Aves. The effects of opioids (enkephalins and endorphins) have scarcely been in vesti ga ted a nd th e findin gs obt a ined a re hi g hly qu esti o nabl e. Compared with the ama zin g ma ss of in vestigations carried out in mammals, studies in lower ve rtebrates a re few, and in large part pe rformed in Amphibia and Al'es. It appears that much further work has to be done by comparati ve endocrinologists to fully clari fy the physiolog ica l relevance of th e functi onal interactions between chromaffin and steroidogeni c cells in the adrenal glands of lower vertebrates.
Open Access
Ovarian development in mice bearing homozygous or heterozygous null mutations in zona pellucida glycoprotein gene mZP3
(Murcia : F. Hernández, 1998) Wassarman, P.M.; Liu, C.; Chen, J.; Qi, H.; Litscher, E.S.
The plasma membrane of all mammalian eggs is surrounded by a thick extracellular coat, the zonu pellirc.id(i (ZP), whose paramount function is to regulate species-specific fertilization. The mouse egg ZP is composed of only three glycoproteins, mZPI -3, that are synthesized and secreted exclusively by oocytes during their 2-3 week growth phase. Disruption of the rnZP3 gene by targeted mutagenesis in embryonic stem (ES) cells yields mice heterozygous ( r n ~ P 3 + / -o)r hornozygous ( r n ~ P 3 - / -f)o r the null mutation. As expected, male mice bearing the null mutation are indistinguishable from wild-type males with respect to viability and fertility. Female m ~ ~ 3 +m/ic-e are as fertile as wild-type animals, but their eggs have a thin ZP (-2.7 pm thick) as compared to the ZP (-6.2 pm thick) of eggs from wild-type animals. On the other hand, female rn~P3-/m- ice are infertile and their eggs lack a ZP. The infertility apparently is due to the lack of a sufficient number of eggs in oviducts of superovulated ~ZPJ-lfemales. Light micrographs reveal that development of ovarian follicles is often retarded in rnz~3- l -m ice as compared to wild-type animals. This is manifested as reduced ovarian weights, reduced numbers of Graafian follicles, and reduced numbers of fully-grown oocytes in I ~ Z P ~ -f/em- ales. I t seems likely that the pleiotropic effects of the homozygous null mutation on ovarian development may be due, at least in part, to disruption of intercellular communication between growing oocytes and their surrounding follicle cells.
Open Access
Lectin histochemistry of mixed gliomas demonstrating an intermediate cell type
(Murcia : F. Hernández, 1998) Schill, J.; Cervós-Navarro, J.
18 cases oí' low-graded rnixed gliornas were studied using the two lectins Concanavalin A (Con A) and Peanut lectin (PNA). Con A stained cytoplasm and processes of turnoral astrocytes, whereas PNA stained cell membranes of tumoral oligodendrocytes. Con A and PNA are reliable markers for astrocyte and oligodendrocyte areas of mixed gliornas, respectively. A part of cells were overlappingly positive for both lectins. They expressed an oligosaccharide pattern of both gliorna types and represented a third, interrnediate cell type of rnixed gliomas. The existence of interrnediate cells close to astrocytic and oligodendroglial cell types in mixed gliomas could result frorn transformation processes of neoplastic glial cells or frorn the malignant transformation of a common glial precursor cell.
Open Access
Histochemical study of lymphocystis disease in skin of gilthead seabream, Sparus aurata L.
(Murcia : F. Hernández, 1998) Sarasquete, C.; Gonzalez de Canales, M.L.; Arellano, J.M.; Pérez-Prieto, S.; García-Rosado, E.; Borrego, J.J.
A battery of horseradish peroxidasecorijugated lectiris (Con A, WGA arid DBA). as well as coriventional histochemical techniques (PAS. saponification, Alcian Blue pH 0.1 , 1. 2.5. chlorhydric hydrolisis. sialidasc, Bromophenol blue, Tioglycollate reduction and Ferric-ferricyanide-FeIlI) were used to study the coritent and distribution of carbohydrates, proteins and glycoconjugatc sugar residues on the skin and on the lymphocystis-infected cells of gilthead seabrcain, Spcrrus uurntcr. Variable ari-iounts of glycoproteins containing sialic acid, N-acetyl-Dglucosainine, N-acetyl-D-galactosarnine; mannose andlor glucose residues were observed in the cuticle and mucous cells of the c o r ~ o r a ls kin. tails and f ins . ~ ~ Germinative and epithelial cells of the epidermis contained glycogen. proteins. carboxylated groups, as well as glycoproteins with mannose andlor glucose and N-acetyl-U-galactosamine residues. Hyaline capsule of the mature lyniphocystis-infected cells was strongly stained with PAS, Alcian Blue (pH 0.5 and 2.5) and weakly positive with Alcian Blue (pH 1 ) . Con A reacted with the granular cytoplasm, specially around hyaline capsule. and with the basophilic intracytoplasinic inclusions developed in mature lymphocystis-infected cells of Spcrrrrs crlirntu skin. These sugar residues (niannose andlor glucose), as well as N-acetyl-Drrlucosamine andlor sialic acid and N-acetvl-D- L galactosamine were not detected in the hyaline capsule of the lyr.iphocysti5 disease.
Open Access
Differential signaling through the T cell receptor: from biochemistry to transplantation tolerance
(Murcia : F. Hernández, 1998) Madrenas, J.; Lazarovits, A.I.
Recent advances in our understanding of the structural nature of T cell activation and signal transduction from the T cell receptor for antigen make possible the development of new tolerogenic strategies. Here. we summarize the evidence supporting a critical role for the CO-receptor molecule (CD4 or CD8) and CD45 in determining the pattern of T cell receptormediated signaling. The consequences of this differential signaling can range from T cell proliferation and cytokine production to the establishment of a state of proliferative unresponsiveness known as T cell anergy. Inducing T cell anergy can be an alternative approach for the establishment of transplantation tolerance.
Open Access
Hyperlipidemia and kidney disease: Concepts derived from histopathology and cell biology of the glomerulus
(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 1998) Kamanna, V. S.; Roh, D. D.; Kirschenbaum, M. A.
The association between hyperlipidemia and renal disease was noted by Virchow as earl y as the 19th ce ntury. Subseq ue ntl y, similar histopatho log ica l lipid depo siti o ns we re confirme d in diverse huma n a nd experimental renal diseases. Altho ugh, no studies have been established in man to suggest a causal re lationship between lipid s and the pathogenesis of rena l disease . compe llin g ev id e nce acc umul a te d in experimental animals suggests a direct role of lipids in the initiation and progression of glome rular disease. These studi es showed that cho lesterol-feed ing to various experimental animals induced the development of glomerul ar injury. Furthermore. the treatment of hype rlipidemic a nima ls with lipid lowering drugs prevented the deve lopment of glomenllosclerosis. In this article, we will rev iew recent advances made in understanding various aspects of lipid-mediated rena l injury inc ludin g bioc he mi ca l mec ha nisms of hype rlipidemia, a possible direct role of hyperlipidemi a in the pa th oge nesis o f ren a l disease, pathobiological acc umulation of lipids and lipoprote ins, biochemi cal and histological similarities between systemic atherosclerosis and glomerulosclerosis, and cellular processes invo lved in the development of glomerul ar disease. Furthermore, we will define cellular and mo lecul ar hypotheses that provide putative mechanisms by which hyperlipidemia a nd a theroge ni c lipo pro tei ns indu ce se ri es o f cy toregulatory peptide- med iated eve nts in vo lved in the development of glomerul ar disease.
Open Access
The effect of corticotropin-releasing hormone (CRH) on the adrenal medulla in hypophysectomized rats
(Histology and histopathology, Vol.13, nº 1 (1998), 1998) Hoheisel, G.; Schauer, J.; Scherbaum, W. A.; Bornstein, S. R.
CRH occurs in the adrenal medulla of rats. We were interested to know whether CRH affects medullary chromaffin cells in the absence of ACTH. We investigated the morphological changes of the adrenal medulla in Sprague Dawley rats with light and electron microscopy in normal rats, hypophysectomized rats, and hypophysectomized rats following injections of CRH (10 Jig = 3 nmol for three days). Chromaffin cells were characterized by immunohistochemistry (anti-tyrosine hydroxylase). At light microscopy level chromaffin cells of hypophysectomized rats were reduced in number. On electron microscopy the number of granules and cell organelles were decreased. Following injections of CRH the medulla regained a more compact texture with cell organelles homogenously distributed, but with chromaffin granules still being reduced in number. Immunohistochemistry allowed the identification of chromaffin cells located within the adrenal cortex. In hypophysectomized rats these cells showed fewer signs of alterations compared to cells located within the medulla itself and had recovered better after treatment with CRH. In conclusion. CRH seems to exert a trophic effect on chromaffin cells in the absence of pituitary ACTH. This observation may provide further evidence for a close interaction of the two neuroendocrine stress systems.
Open Access
Localization and functions of steroid hormone receptors
(Murcia : F. Hernández, 1998) Yamashita, S.
This review focuses on the subcellular localization of steroid hormone receptors (SHRs), taking into account the technical problems of immunohistochemistry and the characteristics of nuclear localization signals (NLSs) of each receptor, on the interaction between SHKs and cellular components, and on the possible roles of sex SHRs in the reproductive organs. It is concluded that SHRs are basically localized in the nucleus, regardless of hormonal status, and that considerable amounts of unliganded SHRs may be present in the cytoplasm of target cells in exceptional cases. Most immunohistochemical results that demonstrate nuclear translocation of liganded SHRs seem to be responsible for insufficient fixation. Immunoelectron microscopy shows that SHRs associate with the chromatin in absence or presence of hormones and that intranuclear translocation of liganded SHRs from the condensed chromatin to euchromatin which observed in some cell types, may be a passive process caused by a consequence of conformational changes in the chromatin binding receptors. Histochemical data suggest that the nuclear matrix (NM) is not a main binding site of liganded SHRs in the nucleus. The artificial formation of intermolecular disulfide bonds during NM preparation presumably causes the entrapment of liganded SHRs into the fraction. It seems that heat shock protein 90 (hsp90) does not form stable complexes with unliganded receptors in vivo, and it interacts with SHRs transiently cooperating with other heat shock proteins as a chaperone that helps folding of newly synthesized and refolding of denatured receptors. Estrogens transiently induce a number of nuclear protooncogenes, such as c-fos and c-jun family proteins, which act as transcription factors through estrogen receptor (ER) system in the endometrial epithelium of mature and immature rodents. Therefore, it is suggested that the changes in concentrations of these gene products trigger the proliferation and differentiation of uterine epithelium. In addition, ER system, not only in stroma cells but in the epithelia1 cells appears to participate in the growth response and abnormalities of epithelium elicited by the exogenous estrogen treatment at the neonatal period.
Open Access
Presence of pigment in the ovine pineal gland during embryonic development
(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 1998) Regodón, S.; Franco, A. J.; Gazquez, A.; Redondo, E.
Light- and electron-mi croscopi c analyses were used 10 id entify and desc ribe th e charac teristi c features of cells containing pigment in the ov ine pineal gland during prenatal deve lopment. 72 ov ine embryos (36 male, 36 female) ranging in age from 54 to 150 days were used fo r this study. Ce lls co nt aining pigment granules were a constant fea ture in this gland . Wh en samples from the di f'ferent groups were inspected using the naked eye, the detection of pigment was of 61 % in groups I (54 to 67 days of prenatal development) and II (7 1 to 92 days of prenatal development), ~3 % in group III (9t; to 113 days of prenatal development) and 25% in gro up IV (II R to 150 days of prenatal deve lopment). The morphologica l featu res and histochemical properties of the pineal pigment enab led it to be identified as me lanin . Several types o f pigme nt granules were ultrastru cturall y distinguished; th ese varied in size, shape and loca tion within pinea l cell populations. The pigment g ranul es were de tec ted in pine alocy te s, interstitial cells and pigmented ce lls. The largest amount and the widest variety of pigmented granules were found in pigment ed ce lls. The prese nce of cells containing pigmented gra nules amongst the cell populations of the developing ov in e pineal gland was a nal ysed a nd compared with that of other mammalian species.
Open Access
The distribution, immune complex trapping ability and morphology of follicular dendritic cells in popliteal lymph nodes of aged rats
(Murcia : F. Hernández, 1998) Sato, H.; Dobashi, Michio
Immune system function declines with age, and lymph nodes involute. The aims of the study were to describe the distribution of follicular dendritic cells (FDCs) in the lymphoid follicles of aged rats, and to determine whether these cells have reduced ability to trap immune complexes (ICs). Popliteal lymph nodes of rats aged 24-28 months were immunostained for S-100 protein as a marker of FDCs. Some rats were pretreated with peroxidase-anti-peroxidase complex (PAP) as an 1C. FDCs were densely distributed in lymphoid follicles, which contained many primary follicles and a few secondary follicles. FDCs in primary follicles stained weakly for S-100 protein, and showed weak trapping, while those in secondary follicles stained strongly for both S-100 protein and trapping. Ultrastructurally, in involuted lymphoid follicles FDCs were atrophic. We conclude that FDCs in aged rats are densely distributed in involuted follicles and show reduced trapping ability and atrophic morphology. This seems to reflect the long life of FDCs and the reduced numbers of lymphoid cells in these follicles. We suggest that FDCs in aged rats may show some of their normal functions if fully developed germinal centers are induced, and may not play an important role in the process of involution of the follicles.
Open Access
Chemiluminescence: a sensitive detection system in in situ hybridization
(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 1998) Musiani, M.; Pasini, P.; Zerbini, M.; Roda, A.; Gentilomi, G.; Gallinella, G.; Venturoli, S.; Manaresi, E.
Chemiluminescence is the light emission produced by a chemical reaction in which chemically excited molecules decay to the ground state. The phenomenon is utilized in various analytical techniques in which small amounts of analytes or enzymes can be detected and quantified by measurement of the light emitted by bio- or chemiluminescent reactions. Recently chemiluminescence has been proposed as a valid alternative to radioactive or colorimetric methods in in situ hybridization assays, in which target nucleic acids are localized by labeled probes inside individual cells with the preservation of cell morphology. Chemi-luminescence in situ hybridization is performed using probes that are detected using enzymes with their appropriate chemiluminescent substrates. The luminescent signal from the hybrid formation is detected, analysed and measured with a high performance low light level imaging apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Generally, the instrumental system to detect positive signals after in situ hybridization operates in three steps: firstly tissue structures and cells are recorded in transmitted light then the luminescent signal is measured with an optimized photon accumulation; and then, after a computer elaboration of the luminescent signal with pseudocolors corresponding to the light intensity, an overlay of the two images on the screen provided by the transmitted light and by the luminescent signal allows the spatial distribution of the labeled probe to be localized and evaluated. The main advantages of chemiluminescence in situ hybridization are mainly the sensitivity, the quantifi-cation of the data, the objectivity of the evaluation and the digital imaging of the results. The chemiluminescence in situ hybridization assay, which can be applied to cell smears, archival frozen and paraffin embedded tissue samples, can be a useful tool for a sensitive and specific diagnosis of viral infections and for the detection and study of specific genic sequences inside the cells. The use of the chemi-luminescent in situ hybridization assay is also promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.
Open Access
Participation of angiogenesis from rat femoral veins in the neovascularization of adjacent occluded arteries
(F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 1998) Díaz Flores, L.; Gutierrez, R.; Valera, H.; Valladares, F.; Rancel, N.; Rodríguez, F.; Madrid Cuevas, Juan Francisco
The neovascularization of the arterial wall in human and experimental pathology has been demonstrated. The occlusion of the of the rat femoral artery is a suitable model for the study of these angiogenesis processes. Newly formed capillaries growing into the arterial wall have been described in this model. The origin of these ingrowing capillaries has been attribute to the preformed surrounding venules and capillaries. The contribution of the adjacent femoral vein with a supplementary population of vascular sprouts could also be possible. To test this hypothesis in half of the occluded arteries, the adventitia was removed from the side facing the femoral vein. Between 1 and 3 days after surgery several alterations were found both in the endothelial cells and the smooth muscle cells of the tunica media. Between 3 and 6 days, solid or canalized endothelial sprouts were observed arising from the femoral vein. By days 4 and 6, newly formed capillaries grew into the adventitia and tunica media of the femoral artery. Some of them, penetrated the internal elastic lamina. This microvascular penetration from the femoral vein was more prominent in the area of the ostium of the collateral and when the adventitia was removed. Some ingrowing capillaries were in continuity with the endothelial cells of the arterial neointima. At days 7 and 8, regressing capillaries were observed in the neomicrovasculature network between artery and vein, with a selective loss of the smaller vessels. From day 9 onwards, fewer and larger vascular channels were present between the femoral vein and the femoral artery. An arterial neolumen contained what appeared to be circulating "fresh" blood. Quantitatively, the venous neocapillary density increased from days 4 to 6 and then declined significantly by day 8. The arterial neocapillary density increased form days 4 to 8 and declined significantly by day 12. Moreover, both densities were significantly greater when the arterial adventitia was removed. The perfusion with barium solution showed the presence qf the contrast material in the newly formed vessels, the lumen of the femoral vein, and the neolumen of the occluded arterial segment. The present findings indicate that putative angiogenic molecules released form the occluded arterial segment may reach the adjacent wall of the vein inducing neovascularization from it. The vein vascular sprouts are connected to the ingrowing capillaries in the occluded arterial wall and to the neocapillaries form the preexisting pericytic microvasculature. When the arterial adventitia were removed up to 2 times greater vein neocapillary's density was observed suggesting an easily access of the putative angiogenic factors to the vein.
Open Access
Characterization of natural occurring Pneumocystis carinii pneumonia in pigs by histopathology, electron microscopy, in situ hybridization and PCR amplification
(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 1998) Ramos Vara, J. A.; Lu, J. J.; da Silva, A. J.; Montone, K. T.; Pieniazek, N. J.; Lee, C. H.; Pérez, L.; Steficek, B. A.; Dunstan, R. W.; Craft, D.; Watson, G. L.
Macroscopic. histolog ic . ultrastruc tural. microbiologic. ill sirll hybridi za ti on (ISH ) and PCR detection results in three 8-week-old pi gs naturall y infected with Pllel.llllonsris c{lrinii (PC) are described . All an imals had a nonsu ppurati ve interstitial pneumoni a and intra-alveolar PlleulIlucysris organisms with foamy eos in ophili c and PAS positi ve appearance . Ultrastructurally. PC trophozoites and cysts we re observed in pigs No.2 and NO .3. with the fo rmer being much more numerous. PC organisms we re located on the alveolar surface or within the alveolar septa . Trophozoites had numerous filopodia and were thin-wall ed. Cysts had no o r few filopodia. we re thick- wa ll ed and co nt a ined intracysti c bodies. Using non-isotopic ISH on formalinfixed. paraffin-embedded lung tissue sections. PC DNA from pigs No.2 and No . 3 hyb ridi zed with a probe specific for PC ribosomal RNA (rRNA). Using primers spec ific for mit oc ho ndrial rRNA ge ne (pA Z I0 2- E/pAZ I 02-H). and for the interna l transcriber space rs of ribosomal gene of PC. PCR methods amplified a product in the lung of pigs No.2 and No.3 using either frozen or formalin -fi xed and paraffin -embedded lung tissue. DNA from Pig No. I samples did not amplify with any primer. This is the first time that molecular biology techniques (ill siru hybridi zation and PCR) have been applied to the study of porc ine pneumocystosis.