Are macrophages involved in early myocardial reperfusion injury?

BACKGROUND: Neutrophils are the predominant phagocytes in the early stages of myocardial ischemia-reperfusion response and are also implicated in the development of tissue damage. This study examined the role of recruited macrophages in the evolution of this tissue injury. METHODS: Farm pigs were subjected to 30 minutes of myocardial ischemia followed by 30 minutes of reperfusion. Biopsy samples were taken from the control, ischemic, and ischemic-reperfused left ventricle wall and processed for both morphologic and biochemical analyses. In situ production of tumor necrosis factor-alpha was evaluated by Western blot and immunofluorescence. A full hemodynamic evaluation was also performed. RESULTS: Myocardial ischemia and early reperfusion caused marked neutrophil and macrophage tissue accumulation and tumor necrosis factor-alpha production by the injured tissue. Immunofluorescence studies allowed us to localize tumor necrosis factor-alpha predominantly in tissue-infiltrating macrophages. No depression in the global myocardial contractile function was observed, either during ischemia or after reperfusion. CONCLUSIONS: These data suggest that the newly recruited macrophages within the ischemic and early post-ischemic myocardium may play a role in promoting neutrophil tissue infiltration and subsequent neutrophil-induced tissue dysfunction by producing tumor necrosis factor-alpha.

The anatomical resources on the Internet.

The main anatomical resources found on the Internet are listed and discussed here, concerning the topics of gross anatomy, neuroanatomy, microscopic anatomy and anatomical museums. The search is updated to September 2000, and all the listed Web sites can be used to build a multimedial anatomical lab.

Prevention of corneal keratocyte apoptosis after argon fluoride excimer laser irradiation with the free radical scavenger ubiquinone Q10.

PURPOSE: To assess in vitro the potential of the free radical scavenger ubiquinone Q10 in preventing keratocyte apoptosis after argon fluoride (ArF) excimer laser irradiation. METHODS: Cultured rabbit keratocytes were irradiated at very low single-pulse laser fluences. The cumulative effects generated by three total fluence doses between 12 and 45 mJ/cm2, representative of single-pulse subablative doses during photorefractive keratectomy (PRK) in humans, were evaluated. We employed the following parameters to compare pretreated (10 microM ubiquinone Q10) and untreated samples: 1) number and morphology of living cells by Trypan blue test and ultramicroscopy, respectively; 2) level of free-radical formation assessed by malonaldehyde quantitation; 3) cellular energy level evaluated by ATP assay. RESULTS: Excimer laser irradiation kills cultured keratocytes by inducing apoptosis. The effect increases with the cumulative fluence dose. In the samples pretreated with ubiquinone Q10 there were significantly fewer cumulative apoptotic events than in the untreated ones. Quantitative analysis of malonaldehyde cellular levels suggested this protective action of ubiquinone Q10 was connected with its ability to scavenge laser-generated free radicals. ATP assay also confirmed that it raised cellular energy levels. CONCLUSIONS: The treatment of corneal keratocytes with relatively low concentrations of ubiquinone Q10 can prevent apoptosis after ArF excimer laser irradiation. If these findings are confirmed on human keratocytes this treatment could be usefully exploited in the PRK surgical procedure. That might lead to a reduction in the occurrence of haze and curvature regression triggered by programmed cell death.

Aponecrosis: morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis.

A rat fibroblastic cell line (rat-1/myc-ERtrade mark) was treated with different concentration of Antimycin A, a metabolic poison that affects mitochondrial respiratory chain complex III. The modes of cell death were analyzed by time-lapse videomicroscopy, in situ end-labeling (ISEL) technique, and ultrastructural analysis. Intracellular ATP levels were also measured in order to detect whether the energetic stores were determinant for the type of cell death. It was found that while apoptosis was the prevalent cell death in the fibroblasts treated with low doses, 100 or 200 microM Antimycin A, a new type of cell demise that shared dynamic, molecular, and morphological features with both apoptosis and necrosis represents the most common cell death when the cells were exposed to high doses, 300 or 400 microM, of the hypoxic stimulus. This new type of cell death has been chimerically termed aponecrosis. The inhibition of caspase 3, an enzyme critical for the apoptotic DNA degradation, caused a clear shift from aponecrosis to necrosis in the cell culture, suggesting that this new type of cell death could account for an incomplete execution of the apoptotic program and the following degeneration in necrosis. After being treated with higher doses, i.e., 1000 microM Antimycin A, almost all of the cells died by true necrosis. The analysis of the cellular energetic stores showed that the levels of ATP were a primary determinant in directing toward active cell death (apoptosis), aponecrosis, or necrosis. We conclude that chemically induced hypoxia produces different types of cell death depending on the intensity of the insult and on the ATP availability of the cell, and that the classic apoptosis and necrosis may represent only two extremes of a continuum of intermediate forms of cell demise.

Apoptosis induced by sulindac sulfide in epithelial and mesenchymal cells from human abdominal neoplasms.

We investigated whether the therapeutic action of sulindac, used for the treatment of familial adenomatous polyposis, desmoid tumors, and against colon cancer, could be mediated by its active metabolite, sulindac sulfide, in cell growth and apoptosis on cell lines derived from abdominal neoplasms. Sulindac sulfide actions on cell growth and apoptosis were evaluated in epithelial human colon tumor 8 (HCT8) cell line and mesenchymal cell lines (bovine bone endothelial (BBE) cell line, desmoid tumor-derived cells, human colorectal cancer-derived fibroblasts). Sulindac sulfide (0.1-60 microg/ml) induced a dose-dependent inhibition of cell proliferation of all cell lines tested. Apoptosis was induced at doses of 20 and 40 microg/ml, respectively, in BBE and HCT8 cells with no effect on desmoid tumor cells and colorectal cancer-derived fibroblasts. Since mesenchymal cells respond to clinically effective concentrations of the compound, its preferential action on the stromal compartment of intestinal polyps, desmoid tumors and colon cancer can be proposed, with consequent regression of the tumor.

Vitamin E protects human skeletal muscle from damage during surgical ischemia-reperfusion.

PURPOSE: The biochemical and morphological alterations induced in lower limb skeletal muscle by ischemia-reperfusion (I-R) during aortic surgery and the effect of vitamin E pretreatment were investigated. METHODS: Two groups of patients undergoing aortic aneurysm resection, one untreated and one treated with vitamin E, were examined. Quadricep muscle biopsies were taken after induction of anesthesia, at the end of ischemia, and after reperfusion. The malondialdehyde (MDA) content and morphology of biopsies were examined to assess peroxidative processes. RESULTS: Ischemia did not induce an increase in MDA content but did increase neutrophil infiltration in muscle fibers of untreated patients. Reperfusion led to a significant increase in MDA content and to intermyofibrillar edema and mitochondrial swelling. The MDA content was not increased during ischemia and neutrophil infiltration was minimal in vitamin E treated patients. At reperfusion, the MDA content, the ultrastructural injuries and neutrophil infiltration were significantly reduced by the treatment. CONCLUSIONS: Vitamin E is effective in reducing the oxidative muscle damage occurring after a period of I-R.

Development of the human fetal lung in the pseudoglandular stage: scanning electron microscopic remarks.

The lung buds of 15 human fetuses from the 9th to the 14th week of i.u. life were examined by scanning electron microscopy (SEM). SEM allows to clearly evidentiate the branching pattern of the respiratory tree, the development of the vascular network, the differentiation of the layers in the bronchial wall and the maturation of the epithelium. Particularly, as concerns the epithelium, its pattern in the air conducting ways and in the respiratory zone appears to be different from the very start; the presence of vascular spaces and then of well-defined vessels can be already detected starting from the 9th week of development.

Functional and structural interactions between osteoblastic and preosteoclastic cells in vitro.

Osteoblasts are involved in the bone resorption process by regulating osteoclast maturation and activity. In order to elucidate the mechanisms underlying osteoblast/preosteoclast cell interactions, we developed an in vitro model of co-cultured human clonal cell lines of osteoclast precursors (FLG 29.1) and osteoblastic cells (Saos-2), and evaluated the migratory, adhesive, cytochemical, morphological, and biochemical properties of the co-cultured cells. In Boyden chemotactic chambers, FLG 29.1 cells exhibited a marked migratory response toward the Saos-2 cells. Moreover, they preferentially adhered to the osteoblastic monolayer. Direct co-culture of the two cell types induced: (1) positive staining for tartrate-resistant acid phosphatase in FLG 29.1 cells; (2) a decrease of the alkaline phosphatase activity expressed by Saos-2 cells; (3) the appearance of typical ultrastructural features of mature osteoclasts in FLG 29.1 cells; (4) the release into the culture medium of granulocyte-macrophage colony stimulating factor. The addition of parathyroid hormone to the co-culture further potentiated the differentiation of the preosteoclasts, the cells tending to fuse into large multinucleated elements. These in vitro interactions between osteoblasts and osteoclast precursors offer a new model for studying the mechanisms that control osteoclastogenesis in bone tissue.

Osteolytic processes in human radicular cysts: morphological and biochemical results.

In an attempt to verify the nature of bone resorption processes on the bone surrounding radicular cysts, fragments of cysts with the adjacent bone tissue were studied by morphological, cytochemical and ultrastructural methods. Cyst fluid was analysed for its content of cytokines with osteolytic activity. The cyst wall exhibited several connective tissue extensions which penetrated the adjacent bone. Numerous multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells were seen at the tip of the intraosseous extensions of the cyst capsule and in direct contact with the bone tissue. Typical resorption lacunae were identified on the bone surface by scanning electron microscopy. Moreover, mononuclear TRAP-positive cells were seen within the cyst capsule. High levels of prostaglandin E2 (PGE2) and interleukin-6 (IL-6) were detected in the cyst fluids. In conclusion, active bone resorption may contribute significantly to the growth of these lesions within the jaws.

Expression of E-selectin in ischemic and reperfused human skeletal muscle.

This work was undertaken to assess the role of endothelial E-selectin in the development of neutrophil accumulation into the ischemic and reperfused human skeletal muscle and eventually in the genesis of ischemia-reperfusion syndrome. Twelve patients affected by abdominal aortic aneurysm who were undergoing reconstructive vascular surgery were studied. Muscle biopsies from the right femoral quadriceps were taken (1) immediately after anesthesia, as control samples, (2) before declamping the aorta, as ischemic samples, and (3) 30 minutes after reperfusion and then processed for immunohistochemical and ultrastructural analysis. Immunohistochemistry revealed a strong positive reaction for E-selectin on the venular endothelium during ischemia and reperfusion. Ultrastructural investigation showed that reactivity for E-selectin matched neutrophil accumulation of the skeletal muscle tissue. This phenomenon was dependent upon a complex series of events that included neutrophil adhesion to the inner surface of the postcapillary venules, passage through endothelial intercellular junctions, and migration distally into the interstitial spaces of the skeletal muscle tissue. Neutrophil tissue infiltration was also associated with ultrastructural signs of tissue damage at reperfusion. This is in agreement with accumulating evidence indicating a role for tissue infiltrating neutrophils in the genesis of toxic O2 free radicals. Our data suggest that E-selectin expression on the vascular endothelium of human skeletal muscle may represent a key regulatory point in the process of neutrophil tissue accumulation and indicate an active role for the venular endothelium in the development of human ischemia-reperfusion syndrome.

In vitro structural and functional relationships between preosteoclastic and bone endothelial cells: a juxtacrine model for migration and adhesion of osteoclast precursors.

The role of vascularization in the process of bone resorption has not been clarified. The interactions between vascular endothelium and osteoclast progenitors were analyzed using clonal cell lines of bone-derived endothelial and preosteoclastic cells. Insulin-like growth factor I is a major chemotactic stimulator of preosteoclastic cell migration mediated by bone endothelial cells. Osteoclast precursors rapidly adhered to bone endothelial monolayers. This phenomenon appeared to be cell-specific and mediated through the binding of vitronectin and fibronectin receptors to fibronectin. In addition, direct contact with bone endothelial cells induced osteoclast progenitors to differentiate into more mature elements, with the tendency to cluster together to form large multinucleated cells. These findings demonstrated specific in vitro interactions between bone endothelial cells and osteoclast progenitors, offering a new model for understanding the molecular mechanisms which direct the processes of osteoclast recruitment and ontogeny.

An anatomical study of the healthy human heart by echocardiography with special reference to physiological valvular regurgitation.

110 healthy subjects (45 men and 65 women ranging in age between 24 and 60 years) were studied by 2-dimensional echocardiography. In each subject the diameters of the cardiac chambers, the thickness of the intraventricular septum and posterior wall of the left ventricle in diastole, as well as the diameters and circumferences of the aortic and mitral rings were measured. Moreover, in each subject color doppler echocardiography was performed. Pulmonary valve regurgitation was observed in 47 subjects (42.7%). This finding was not correlated in a statistically significant manner to either age, sex, body weight, or height. The incidence of pulmonary regurgitation, however, tended to decrease with age. In 37 subjects (33.6%), tricuspid regurgitation was detected without any correlation to the above parameters. In 11 cases (10%), mitral regurgitation was observed; its correlation to age was statistically significant (p < 0.05). Aortic regurgitation was noted in 9 (8.2%) cases; its incidence was directly correlated to age (p < 0.01). The observed regurgitations were defined as "physiological" and precise characteristics were chosen so as to distinguish such "physiological" from "pathological" regurgitations.

Development of the chick tongue. A scanning electron microscopical investigation.

With the aim of clarifying certain morphological aspects of lingual development, chick tongues between the 8th day of incubation and hatching and also during the early postincubation period were investigated by means of the Scanning Electron Microscope. During this time, the tongue anlage underwent some remarkable morphogenetic changes, mostly involving the superficial epithelium but also including the appearance of the lingual glands at the level of the lingual root. With regards to the epithelium, it was possible to observe that in the first days of the incubation period examined, the superficial cells appeared dome-shaped, with microvilli on the apical surface; later they tended to become more flattened, and the microvilli were replaced by a thick net of microplicae. During the final days of incubation, and after hatching desquamative phenomena became evident. At no site of the tongue rudiment were taste buds ever observed, possibly because of the different functional role played by the avian tongue in comparison with that of the mammals.

Morphological study of fetal nasopharyngeal epithelium in man.

In 30 human fetuses between 8 and 13 weeks of intrauterine life the lateral wall of the nasopharynx was examined by light microscopy and transmission and scanning electron microscopy. In the subjects between 8 and 9 weeks in utero the mucosa displays still an immature appearance, being mono- or bistratified and lacking the characteristic structures of the respiratory epithelium. Nevertheless, signs of differentiation are to be noticed, with the presence of two distinct cellular types that, in the later periods, will give rise to ciliated cells and microvillus-provided cells. An almost complete differentiation will be reached at 12-13 weeks in utero, even if goblet cells are still lacking in the examined zone during the considered period. Nonrespiratory types of epithelium, such as transitional or squamous, were never found in the studied subjects.

Ultrastructure of human male urethra.

The morphological features of human male urethra lining epithelium have been investigated by means of SEM at the various portions of the organ. The characteristics of the epithelial cells have been described and the significance of different morphological pattern is discussed.

Scanning electron microscopic observations on the epithelium of the human prostatic urethra.

The human prostatic urethra has been investigated by means of scanning electron microscopy. On the posterior wall of the urethra, the seminal colliculus with the orifices of the ejaculatory ducts is clearly detectable. The upper portion of the prostatic urethra shows a typical transitional epithelium with large superficial cells of a ruffled appearance. In the lower portion of the organ (underneath the openings of the ejaculatory ducts), the apical pattern of the cells varies considerably. Four main aspects are recognizable: apices provided with microvilli, dome-shaped apices with an almost smooth surface, large apices with labyrinthic microplicae and ciliated apices. Also, apices showing intermediate characteristics can be noted. The functional significance of the morphological patterns as well as the possibility of a transition among the various types of surface structures are discussed.

The corrected circumference of human pulmonary trunk and arteries in relation to the size of aorta and principal bronchi.

The corrected circumference of the pulmonary trunk, its branches and ascending aorta as well as the maximum diameter of the principal bronchi, were measured in 120 human fresh cadavers of subjects (60 males and 60 females) aged between 27 and 91 years, died of natural causes. The statistical analysis of the obtained data yielded the following main results: 1) the corrected circumference of the pulmonary trunk, smaller than that of the aorta, is not statistically correlated with the age of the subjects and does not present significant degrees between the 2 sexes; 2) the right and left pulmonary arteries have a corrected circumference quite similar in the 2 sexes and statistically correlated with that of the vessel of origin; 3) both the right pulmonary artery and the right principal bronchus have sizes greater than the left ones, possibly due to the greatest volume and weight of the right lung; 4) the lackness of a correlation between the size of the pulmonary arteries and those of the respective bronchi shows once more the difficulty of identifying general rules concerning the interrelationship between vascular and ductal human structures.

Blood vessel size of circulus arteriosus cerebri (circle of Willis): a statistical research on 100 human subjects.

The brain weight of 100 fresh cadavers of Italian subjects (60 males and 40 females), aged between 17 and 84 years, was obtained and the corrected circumference of the following blood vessels was measured: basilar artery, internal carotid arteries, anterior and posterior cerebral arteries, and anterior and posterior communicating arteries. The cerebral 'potential flow' was expressed in each case by adding the circumference of the basilar artery to that of the internal carotid arteries. Moreover, the sides and the perimeter of the circle of Willis as well as the length of the basilar artery were calculated. The statistical analysis of the obtained data yielded the following main results: (1) the brain weight decreases with aging, is lower in females than in males and and is statistically correlated neither with the circumferences of the considered arteries and the 'potential flow' nor with the perimeter of the arterial polygon (circle of Willis); (2) the arteries of the left side appear to be larger than those of the right one; (3) no significant difference exists in the circumference and length of the arteries between males and females; (4) the increase of the perimeter of the arterial polygon is achieved by means of a harmonious increase of all its sides; (5) the anterior and posterior communicating arteries have an anarchic pattern, because of the relatively frequent anomalies and the lack of a correlation between their circumference and that of the vessel of origin or of outlet.