Porcine coronary arteries: immunohistochemical profile of TNF-alpha, IL-1beta, TGF-beta1 and ICAM-1.

BACKGROUND: In our study we used immunohistochemical technique to demonstrate the presence of the cytokines tumour necrosis factor alpha (TNF-α), interleukin 1beta (IL-1ß), transforming growth factor beta1 (TGF-ß1) and intercellular adhesion molecule-1 (ICAM-1) in porcine coronaries even in physiological conditions. MATERIALS AND METHODS: Inflammatory cytokines are polypeptide mediators which act as a communication signal between immune system cells and other types of cellsin different organs and tissues, both in human and pig coronary circulation. RESULTS: Our results show that pro-inflammatory cytokines TNF-α, IL-1ß, TGF-ß1 and ICAM-1 are also present in the medium tunica of the coronary arteries under physiological conditions. These results may be compared with those found in coronary atherosclerosis, where the increase in TNF-α has a dramatic effect on the function of the left ventricle, and the high value of IL-1 correlates directly with the extent of myocardial necrosis. In our study we observe the damage and activation of endothelial cells; this induces endothelial dysfunction by accumulation and oxidation of low density lipoproteins (LDL). The formation of oxidized LDL could play a central role in the amplification of the inflammatory response causing an increased expression of pro-inflammatory cytokines which promotes leukocyte recruitment in the intimal layer. These leukocytes, after the adhesion to the endothelium, penetrate the intimate tunic. CONCLUSIONS: Therefore inflammatory processes promote the onset and evolution of atheroma and the development of thrombotic complications.

Detecting microcalcifications in atherosclerotic plaques by a simple trichromic staining method for epoxy embedded carotid endarterectomies.

Atherosclerotic plaques have a high probability of undergoing rapid progression to stenosis, becoming responsible of acute coronary syndrome or stroke. Microcalcifications may act as enhancers of atherosclerotic plaque vulnerability. Considering that calcifications with a diameter smaller than 10 mm in paraffin embedded tissue are rather difficult to detect, our aim was to analyze microcalcifications on semithin sections from epoxy resin embedded samples of carotid endarterectomies using an original trichromic stain (methylene blue--azur B--basic fuchsine--alizarin red). We have compared samples stained either with our method, methylene blue-azur B alone or with Von Kossa staining, and methylene blue-azur B -basic fuchsine alone or with Von Kossa staining. Our method resulted to be simple and fast (ca. 2 min), it gives a sharp general contrast for all structures and allows to easy identify collagen and elastin. In addition, gray-green colour associated to intracellular lipid droplets evidences foam cells, which are particularly abundant in endarterectomies samples. Mast cells and their metachromatic granules are also well recognized. Calcifications over 0,5 mm are clearly recognizable. In conclusion, microcalcifications are clearly distinguished from the extracellular matrix in spite of their reduced dimensions. Methylene blue--azur B--basic fuchsine--alizarin red method is easy to use, reproducible, and is particularly suitable for the identification of microcalcifications in the morphological analysis of atherosclerotic plaques.

A singular pancreatico-colic artery: anatomical report and clinical implications.

After dissection of the abdominal cavity of a 59-year-old male, a long artery, extending from the hepatic artery to the transverse colon, and comprised proximally within the neck of the pancreas and distally within the transverse mesocolon, has been detected. This "pancreato-colic" artery (P-C A) was 13 cm in length and 4 mm in diameter at the origin. Its mesocolic part (2.5 mm in diameter) contributed to the formation of the marginal arcade. No middle colic artery from the superior mesenteric was observed. Thus, the transverse colon was supplied by the distal part of the P-C A. Considering both the P-C A caliber and topography, attention should be paid during pancreatic resections and in the interposition of the transverse colon for esophageal replacement in order to avoid serious bleeding and necrosis. Embryologically, the proximal part of the P-C A might be regarded as an intrapancreatic variant of the tract of the longitudinal anastomosis between the ventral segmental arteries, persisting in the adult as dorsal pancreatic artery. Different from its usual retropancreatic location, this part might be entrapped inside the gland by the developing primitive pancreatic anlages. The distal, mesocolic, part of the P-C A might be regarded as a replacing middle colic artery into the dorsal mesentery during midgut rotation.

Microplicae-like structures of the fallopian tube in postmenopausal women as shown by electron microscopy.

Female reproductive aging is associated with several morphological changes of the genital tract with a subsequent decline in fertility; however, ultrastructural changes occurring after menopause have still not been well illustrated. Our aim was to characterize the three-dimensional microanatomy of the luminal surface of the human fallopian tube in perimenopause and postmenopause. Twenty bioptic samples of fallopian tubes were obtained after surgery under the informed consent of the patients. Samples were processed for transmission electron microscopy (TEM) and field-emission scanning electron microscopy (FE-SEM). As age increases the surface epithelium of the fallopian tube appeared somewhat flattened. Correlated TEM/FE-SEM observations showed gradual shortening of microvilli and deciliation. The most interesting finding was the gradual formation of microplicae-like structures on the surface epithelium, particularly from late perimenopause to postmenopause. Microplicae-like structures, associated with other regressive changes, represent an important adaptation of the epithelium of the fallopian tube; these are likely induced by the physiological process of aging, thus better withstanding hormonal changes associated with the advent of the menopause.

Pored domes in adrenocortical capillaries. An ultrastructural study in mammals.

The adrenocortical gland is one of the most vascularized organs of the mammalian body. It undergoes continuous morphological changes dynamically dependent upon special permeability conditions related to various physiological and physiopathological events. The adrenal cortex of different adult mammals (3 pigs, 2 mice, 3 sheep) was studied by means of scanning (SEM) and transmission (TEM) electron microscopy. As seen by SEM it is formed of polyhedral cells which delimit a characteristic continuous labyrinth system of intercellular lacunae occupied by sinusoid-like capillaries. The capillary wall is fenestrated and is lined by flattened endothelial cells with their nuclear part bulging into the capillary lumen. The fenestrae are round or oval pores measuring 50-100 nm in diameter. They are usually clustered to form sieve plates and characteristically present a thin membranous diaphragm. Irregular microelevations and dome-like projections are often seen. These structures are made of thin cytoplasmic plates interrupted by numerous small pores which show a sieve plate profile. The pored-domes on the nuclear portion seem to be identical in structure to those found in the thinner endothelial part; some of them appear to detach from the endothelial cell. The pored-domes are structurally comparable to those reported both in the renal glomerular endothelium and in liver sinusoids. These endothelial structures may be an expression of the high rate of filtration of these tissues and may be also related to the final step of the replacement of the sieve plates during endothelial regeneration.

Cumulus oophorus extracellular matrix in the human oocyte: a role for adhesive proteins.

A viscous elastic matrix secreted by the cumulus oophorus cells represent the "extracellular matrix" surrounding the human mature oocyte obtained from assisted reproductive technology (ART). The cumulus matrix is involved in several reproductive processes, including the pick-up of the oocyte-cumulus complex by the oviduct, the final maturation of the ovum and sperm-egg interaction. As showed by some Authors, the cumulus matrix is rich in hyaluronan, as well as in other proteins including inter-alpha-trypsin inhibitor, a dermatan sulfate proteoglycan, and a pentraxin-3. Proteins and hyaluronan are linked together to form a meshwork comprised of granules and filaments. We found in human cumulus oocyte complexes the presence of specialized cells still capable of producing fibronectin and tenascin-c in the post-ovulatory period. Moreover, fibronectin and tenascin-c are present within the hyaluronan matrix at fertilization during the tubal sojourn of the ovum and the embryo. Since pentraxin3 is important in matrix stabilization and gamete interactions, a possible role for fibronectin and tenascin-c may be postulated in the final maturation of the ovum, in the tubal pick-up and in the complex dialogue with the tubal epithelium.

Pietro Motta's latest studies on the microanatomy of menopause: revised concepts on the beauty of aging.

Menopause signs a hallmark in women's life. When compared to traditional two-dimensional techniques, scanning electron microscopy (SEM) allows to discover the impressive microcosm of the human body and offers original views of the real three-dimensional ultrastructure of reproduction. On the basis of Prof. Motta's last and original book, herein we show a selection of microscopic images that characterize the 3D changes taking place on the surface epithelia of the aging woman's reproductive tract. Biopsies of ovaries, tubes, uteruses, cervices and vaginas were obtained from women (45-72 years old) during surgical or endoscopical procedures and after the informed consent of the patients. Samples were processed for SEM as reported elsewhere. Reproductive aging is associated to epithelial flatness, higher stromal density, absence of ovarian follicles, thickening of vascular walls and lack of glandular secretion. The uterus and the tube show a morphodynamic sequence of gradual microvilli shortening, patchy to complete deciliation and formation of microplicae like structures. The formation of microplicae like structures is interpreted as an adaptive mechanical response of the genital epithelia to the new microenvironment. We can propose that these structures may represent the initial stage of a physiologic metaplasia. In conclusion, imaging of menopause through the use of SEM allows a better understanding of the morphological and physiopathological bases of female aging and represents a perfect blend of art and aesthetics: a further confirmation of the endless beauty of our inner microscopic anatomy.

The human zona pellucida and scanning electron microscopy. Reality or artifacts?

The surface micro-morphology of the zona pellucida (ZP) was investigated in 158 inseminated but unfertilized mature human oocytes derived from assisted reproduction trials (ART) by means of traditional scanning electron microscopy (SEM) techniques (gold coating and conductive staining methods) and saponin-ruthenium red-osmium tetroxide-thiocarbohydrazide method (Sap-RR-Os-TC). The main aspect of the ZP by traditional SEM (122 oocytes) consisted in a porous, net-like structure (97 oocytes), whereas a nearly smooth or compact structure of ZP was detected in 25 oocytes (79.5% vs 20.5%). Using Sap RR-Os-TC method on 36 oocytes, 31 oocytes showed ZP with alternating tight and large meshed networks, whereas 5 oocytes displayed only tight meshed network (86.1% vs 13.9%). Due to our well standardized procedures, to the stabilizing action of the conductive staining on the zona material and similar results obtained with the use of Sap RR-Os-TC method, we confidentially regard the ZP changes, occurring in oocytes of various groups, as genuine features, likely related to their actual maturation status, rather than as artifacts. In addition, we emphasize the concept that a modern view of the ZP surface implies the best evidence of crossing filaments' network. We think that the ZP "spongy" or "compact" appearance is only the result of microfilaments network collapse, not the true three-dimensional (3-D) representation of ZP structure.

In vitro fertilized human eggs. An electron microscopic study.

This paper describes by scanning and transmission electron microscopy the ultrastructure of the human fertilized egg and its vestments (cumulus oophorus and zona pellucida). Data are reported on the ultrastructure of a. conventional in vitro fertilized eggs (pronuclear eggs and cleaving eggs at two-to-four cell stage); b. eggs at the same developmental stage deriving fro intracytoplasmic sperm injection. The present results showed that: 1. The cumulus-enclosed fertilized egg is a highly dynamic structure in which egg vestments play a crucial role, positively affecting fertilization and healthy embryo development; 2. Intracytoplasmic sperm injection technique does not seem to significantly alter fertilized egg morphology.

Study of apoptotic DNA fragmentation in human spermatozoa.

The aim of our work was to define and better understand apoptosis in the spermatozoa of normal subjects, infertile patients and patients affected by specific tumoral diseases employing the method of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling and confirming the results by electron microscopy. We studied 23 healthy, normozoospermic subjects (group A), 29 oligoasthenoteratozoospermic patients, affected by various andrological pathologies (group B), 28 patients with Hodgkin's disease (C1) and 30 patients with testicular cancer (C2). Our data demonstrate that the percentage of apoptosis in normozoospermic subjects (group A) is significantly lower than in all the other groups (B, C1, C2) (P < 0.001). This confirms that high DNA fragmentation is one of the characteristics of spermatogenetic failure. The induction of apoptosis, which can also be a basic response to neoplastic disease, can even act right up to the mature male gamete. Our results suggest that apoptosis could be the final result of various pathologies and of a deregulation of spermatogenesis control systems.

[Laparoscopic and thoracoscopic reintervention after mini-invasive surgery in emergencies]. / Reinterventi laparo e toracoscopici dopo chirurgia mini-invasiva in urgenza.

One-hundred-eighty patients underwent emergency minimally invasive procedures (laparo and thoracoscopy) in the last two years. In twelve postoperative drawbacks another surgical procedure was performed. Laparoscopic or thoracoscopic exploration was undertaken in 8 cases while open surgery was done in 4. Complications and surgical overtiming rate was higher after endoscopic than open surgery. This approach allows us to correct very few and selected disease when a skilled surgical team is available especially during reoperations.

Gastric re-resection in emergency.

BACKGROUND/AIMS: We reviewed a consecutive series of patients with acute complications following gastric resection, in order to evaluate the role of gastric re-resection as the operation of choice in emergency. METHODOLOGY: Records of 90 patients with acute complications following gastric resection observed from January 1991 to January 1996 were retrospectively analyzed. RESULTS: Hemorrhagic events occurred in the vast majority of cases (87), either as acute complications after a long time since surgery (78 cases) or as early postoperative complications (9). Among late acute complications, three cases were related to bleeding cancer of the gastric stump. Anastomotic obstructions presenting as acute complications occurred in 3 cases. Emergency surgery was indicated in three cases of acute obstruction, in 1 case of bleeding cancer of the gastric stump, in 9 (12%) out of the 75 remaining late acute hemorrhagic complications and in 1 (11%) out of 9 early hemorrhagic complications. Completion gastrectomy was chosen in the one case of bleeding cancer of the gastric stump with indication for emergency surgery. Gastric re-resection was performed in 11 cases: 9 for hemorrhagic complications and 2 for obstructive acute complications. In two cases, one for hemorrhage and one for occlusion, other surgical procedures were carried out. CONCLUSIONS: Gastric re-resection can represent the most suitable operation in acute complications following gastric resection.

Bovine cumulus-corona cells in maturing oocytes. A scanning electron microscopic study.

The ultrastructural features of cumulus-corona cells surrounding maturing oocytes in bovine were studied by means of scanning electron microscope in order to provide a detailed description of their surface changes during oocyte maturation. Cumulus corona cell complexes of immature oocytes showed a compact aspect with narrow intercellular spaces. The spaces around mature oocytes enlarged because they were progressively filled with abundant microfibrillar extracellular matrix. In cumulus corona cells complexes of immature oocytes very numerous long and filiform microvilli were observed, whereas the cumulus corona cell surface surrounding mature oocytes showed occasional large cytoplasmic protrusions along with scanty microvilli and numerous blebs.

Egg-sperm interactions in humans: ultrastructural aspects.

A large cumulus mass usually covers the human ovulated oocyte, and voluminous clusters of cumulus cells are still seen after fertilization around the egg. Cumulus cells surround oocytes and fertilized eggs also during in vitro fertilization (IVF) procedures. This study describes, by transmission and scanning electron microscopy, the morphology and the microtopography of the cells forming the human cumulus mass surrounding IVF samples (insemined but not fertilized oocytes and fertilized eggs). Particularly emphasized is their morphodynamic role in sperm-egg interactions. A comparison with the behavior in vivo of cumulus-enclosed oocyte/fertilized eggs has been also performed. All patients have given their informed consent to participate in this protocol. An inner layer (corona radiata cells) and an outer layer (proper cumulus cells) can be microtopographically recognized in the cumulus mass. Numerous cumulus-corona cells, particularly after fertilization, showed ultrastructural characteristics typical for steroid synthetic cells, thus undergoing a sort of "luteinization" parallel to that occurring in the sister granulosa cells of the postovulatory follicle. This steroid synthetic activity, particularly enhanced in vitro but present also in vivo, may be finalized to the release of small amount of steroids (estrogens and progesterone) in the oocyte/fertilized egg milieu. Various proteins, secreted by other cell subpopulations--as revealed in other studies by our research group--, may even enrich this milieu. Lymphocytes and macrophages were often found in the cumulus mass. They may modulate the steroid secretion of the neighboring cumulus cells by production of cytokines, mimicking what occurs in the ovarian follicle and, later, in the corpus luteum. Spermatozoa, both normal (acrosome-intact or--reacted) and abnormal, were frequently seen in the cumulus mass, free in the intercellular spaces or close to the cumulus cells, that can induce sperm capacitation and acrosome reaction. Leukocytes and cumuluscorona cells appeared both capable of actively phagocytizing supernumerary and/or abnormal sperms. Such spermiophagic response is present in a lesser extent around oocytes and eggs fertilized in vivo. In vitro, instead, cumulus spermiophagy leads to the elimination of a large part of the excess spermatozoa that have reached the oocyte, thus restoring in an extracorporeal medium the spermiophagic activity physiologically exerted by leukocytes and epithelial cells in the female and male genital tracts. In conclusion, the cumulus mass surrounding oocytes and fertilized eggs appears as a highly dynamic system, in which various subpopulations of cells cooperate in order to provide a suitable and healthy microenvironment for fertilization and early embryo development.

Ultrastructural features of bovine cumulus-corona cells surrounding oocytes, zygotes and early embryos.

Integrated transmission and scanning electron microscopic (TEM and SEM) techniques have provided the first detailed description of the ultrastructural features of the bovine cumulus-corona (CC) cells surrounding oocytes at the time of final maturation, zygotes and early cleaving embryos (2/4 to 6/8 blastomeres). TEM revealed the presence of rough endoplasmic reticulum and Golgi complexes in the cytoplasm of CC cells surrounding immature, mature and fertilized eggs, and also revealed an increasing amount of smooth endoplasmic reticulation membranes, lipid droplets and mitochondria with villiform and/or tubular cristae in the cytoplasm of CC cells during maturation and fertilization of the oocyte. In addition, a loss of cell-to-cell junctions between CC cells was evident. TEM also demonstrated that a few residual CC cells were still associated with early embryos and that these cells showed rather degenerative or apoptotic patterns, the latter pattern also observed on cells associated with fertilized eggs. SEM revealed that the complex of CC cells of immature oocytes was compact with narrow intercellular spaces, which progressively enlarged in size around mature oocytes. This phenomenon is mostly due to the production of abundant extracellular matrix. Immature CC cell complexes possessed characteristic long and filiform microvilli whereas the surface of CC cells surrounding mature oocytes showed numerous blebs and occasional large cytoplasmic protrusions as well as microvilli. Zygotes and early embryos were covered with a few polyhedral CC cells possessing scarce and short microvilli and a large amount of pleomorphic blebs. This study demonstrated a precocious luteinization occurring in bovine CC cells at ovulation until zygote segmentation, and this process was associated with a progressive apoptotic mechanism that ended in the complete denudation of the zona pellucida covering the early embryo. The presence of CC cells around the maturing oocyte and fertilized egg could have important functions related to the microenvironmental requirements of ovum maturation as well as facilitating activities related to fertilization.

[Clinical and therapeutic aspects of stab and gunshot wounds]. / Aspetti clinici e terapeutici delle ferite d'arma bianca e da fuoco.

In the last three years (1992-1995) 130 stab (114) and gunshot (16) wounds were observed at and admitted to the Emergency Surgical Department of Fatebenefratelli Hospital of Milan. We observed a high incidence of non-EEC patients (62%). Imaging devices (US and CT scan) and surgical minimally invasive procedures have reduced open surgery rate with a remarkable reduction in drawbacks and mortality.

[Carcinoids in emergency surgery]. / I carcinoidi nell'urgenza chirurgica.

The authors report their experience of three cases of carcinoids of the gastroenteric tract which required emergency surgery. Two patients presented symptoms of acute appendicitis caused by appendicular carcinoid, whereas the third presented an occlusive syndrome due to ileal carcinoid.

A new morphological classification during follow-up in patients with celiac disease: a three-dimensional observation by scanning electron microscopy.

The structure and ultrastructure of the villi of small intestinal mucosa was examined in 237 duodenal or jejunal biopsies taken from children with active celiac disease and during gluten-free diet. All biopsies were processed for light and scanning electron microscopy. Conventional histology showed four different morphological aspects: total and subtotal villous atrophy in patients on unrestricted diet, partial villous atrophy and normal mucosa during gluten-free diet. Scanning electron microscopy demonstrated that in active celiac disease the severity of the intestinal lesions was related to individual vulnerability to gluten. Our results showed that during dietary treatment the process of mucosal healing was constant and strictly time-dependent. Furthermore, the ultrastructural examination has been relevant in evaluating the evolution of the villous regeneration. In this study a classification regarding the healing process of the small intestinal mucosa correlated with the time of start of dietary therapy is proposed.

Heterogeneous distribution of fibronectin, tenascin-C, and laminin immunoreactive material in the cumulus-corona cells surrounding mature human oocytes from IVF-ET protocols--evidence that they are composed of different subpopulations: an immunohistochemical study using scanning confocal laser and fluorescence microscopy.

Monoclonal antibodies and immunofluorescence microscopy, including laser confocal microscopy, were used in this study to point out the production of fibronectin, tenascin-c, and laminin in the cumulus-corona (CC) cells surrounding mature human oocytes from IVF-ET protocols in view of their presumptive importance in the coordination of the processes leading to fertilization and early embryo cleavage, including the final maturation of the ovum, the sperm-egg interaction, and the "complex biochemical dialogue" between the gamete and the oviduct through the tubal luminal environment. One hundred fifty mature oocyte-CC complexes were obtained from IVF-ET protocols and fixed in 4.0% buffered paraformaldehyde. Specimens were incubated with a panel of primary monoclonal antibodies (mabs) recognizing different epitopes of fibronectin, tenascin-c, and laminin and then with fluorescein isothiocyanate-conjugated goat anti-mouse IgG. Observations were made by a scanning confocal microscope (Sarastro 2000) and a photomicroscope (Polyvar, Reichert-Jung) equipped with epifluorescence optics. The immunohistochemical data demonstrated that human CC cells are capable of producing fibronectin and tenascin-c but that their production is not homogeneous in the CC population. In fact, fibronectin immunoreactivity was shown mostly by inner CC cells (mainly corona cells), whereas tenascin was produced by some cells scattered in the entire cumulus mass. Moreover, fibronectin and tenascin-c immunoreactive material was observed in the intracytoplasmic areas, at the plasma membrane level as well as in the extracellular matrix. On the contrary, laminin immunofluorescent material was found around plasma membranes of almost all CC cells, but a clear intracytoplasmic reaction was never observed. This leads us to assume that laminin in the extracellular matrix remains entrapped once produced by granulosa follicular cells and that in the postovulatory period no active secretion occurs in CC cells. Even though the functional role of these extracellular matrix proteins remains still unclear, it is reasonable to suggest that they are necessary in various steps of the reproductive process, i.e., from the pick-up of the oocyte, its transport through the oviduct, and fertilization, up until the early cleavage of the embryo. Finally, functional differences between "corona radiata" and "cumulus" cells during the oocyte denudation may be accounted for particular distribution of these adhesive proteins.