A comparison of the anti-inflammatory activities of conjugated estrogens and 17-beta estradiol.

OBJECTIVE AND DESIGN: Unregulated chronic inflammatory process partly due to an estrogen deficiency may render postmenopausal women vulnerable to degenerative conditions such as arthritis, osteoporosis, atherosclerosis, and Alzheimer's disease. Current confusion regarding therapeutic efficacy of estrogen replacement therapy may be due to different estrogen formulations used, short term therapy, as well as advanced stage of the disease. MATERIALS AND METHODS: We compared anti-inflammatory activities of two major estrogen preparations, conjugated equine estrogen (CEE) and 17-beta estradiol, using an animal model (rat mesentery) of in vivo inflammatory reaction to intravenously infused amyloid-beta, examined by video recording and subsequently analyzed by transmission electron microscopy. Cellular markers of inflammation were monitored: leukocyte migration, platelet activation, mast cell activation/degranulation, and endothelial disruption. RESULTS: Low doses of CEE (0.3 mg/kg for 3 weeks) demonstrated significant anti-inflammatory activity, whereas even at high doses (2.0 mg) 17-beta estradiol had only minimal activity. CONCLUSION: CEE, a mixture of several compounds, may have some component(s) with significant anti-inflammatory activity. The anti-inflammatory activity of CEE may have a role in prevention of several degenerative diseases associated with menopause.

A vascular connection to Alzheimer's disease.

Alzheimer"s disease (AD) is characterized by a progressive and debilitating dementia in elderly people. The causes of this disease are not known, but major risk factors include old age and a family history of dementia, Down"s syndrome, female gender, low level of education, and head injury. There is no known cure for Alzheimer"s disease. The disease is characterized by abnormal accumulation of amyloid-beta peptide and the protein Tau in the nerve cells and extracellular space of certain regions of the brain. Cerebral amyloid angiopathy is another marker for Alzheimer"s disease. In autopsies, small cerebral arterial blood vessels and capillaries show signs of inflammation, amyloid accumulations, and a focal breach of the blood-brain barrier. This review focuses on the results of recent investigations of vascular responses to infusion of amyloid-beta(1-40), the means of preventing vascular damage, using a live animal (rat) model, and the combination of intravital video recordings of the mesenteric microvascular bed with electron microscopic analyses of the same vascular segments. We propose that the cerebral vascular changes in patients with Alzheimer"s disease probably precede the neuronal damage and dementia.

Amyloid-beta peptide induced inflammatory reaction is mediated by the cytokines tumor necrosis factor and interleukin-1.

A chronic inflammatory response possibly mediated by amyloid-beta (A beta) is believed to be a major factor in the pathology of Alzheimer's disease (AD). Recently, we demonstrated that in vivo administration of A beta produces an inflammatory response and vascular disruption as seen in the brains of AD patients. In an inflammatory response, leukocyte activation and extravasation involves cytokine production. Previous studies have indicated that immune interactions exist between the central nervous system and the peripheral immune mechanisms in AD. Increased levels of interleukin-1 beta (IL-1 beta) have been detected in brain tissue, cerebrospinal fluid, and blood/serum from AD patients. In addition, A beta stimulated the production of tumor necrosis factor-alpha (TNF-alpha) in brain astrocytes and murine monocytes. Using an animal model we investigated the role of the cytokines, TNF-alpha and IL-1 beta, in the A beta-induced inflammatory response. Adult male rats were perfused via an intra-aortic cannula with either A beta alone, interleukin-1 receptor antagonist (IL-1 ra) plus A beta, tumor necrosis factor-binding protein (TNF-bp) plus A beta or sterile saline. Serum analysis for TNF-alpha, IL-1 beta, A beta and NO showed a significant increase in TNF-alpha and A beta but not in IL-1 beta or NO after the injection of A beta. Control values for serum A beta averaged 1.6 ng/ml and in rats injected with A beta, 99.6% of this peptide was removed from the blood within 30 min. The mesenteric arterioles and venules were video recorded for 1-2 h and then processed for electron microscopy (EM). In rats given A beta alone there was extensive vascular disruption, including endothelial and smooth muscle damage with leukocyte adhesion and migration. Animals receiving either IL-1 ra or TNF-bp before A beta showed no in vivo leukocyte extravasation or vascular damage under EM. Therefore, the cytokines TNF-alpha and IL-1 beta seem to mediate the vascular disruption and inflammatory response initiated by A beta. Antagonism of these pro-inflammatory cytokines may offer new avenues for AD therapy.

Estrogen protects peripheral and cerebral blood vessels from toxicity of Alzheimer peptide amyloid-beta and inflammatory reaction.

Due to increases in life expectancy, women are living 30 years or more beyond menopause. This has led to an increasing interest in the association between postmenopausal estrogen deficiency and degenerative diseases associated with aging such as cardiovascular disease, osteoporosis and dementia. Women are two times more likely to develop late-onset Alzheimer's disease (AD) than age-matched men. A large number of observational reports and a few randomized clinical trials have indicated that estrogen replacement therapy (ERT) may retard the development and severity of dementia in postmenopausal women. The mechanism underlying the protective action of estrogen in AD is under active investigation. A chronic inflammatory reaction mediated by abnormal deposition of proteins such as amyloid-beta (A beta) is central to the pathology of AD. We investigated the effect of low doses of conjugated estrogen (Premarin) in an animal model of A beta-induced vascular disruption and inflammatory reaction. This rodent model allows live videomicroscopic recording and electron microscopic analysis of peripheral vascular disruption and inflammatory reaction triggered by A beta. Estrogen prevented vascular deposition of A beta, endothelial and vessel wall disruption with plasma leakage, platelet and mast cell activation, and characteristic features of an inflammatory reaction: adhesion and transmigration of leukocytes. The beneficial effect was lost when estrogen treatment was discontinued. Estrogen also protected the cerebral blood vessels from endothelial dysfunction induced by A beta. This novel protective effect of estrogen against A beta cytotoxicity in peripheral and cerebral vasculature may contribute to the therapeutic efficacy of estrogen in AD and coronary vascular disease.

In vivo vascular damage, leukocyte activation and inflammatory response induced by beta-amyloid.

beta-amyloid toxicity is central to the pathology of Alzheimer's disease. Recent evidence implicates vascular dysfunction as a contributing factor to the dementia of Alzheimer type. Using intravital microscopy we demonstrate that in vivo administration of beta-amyloid produces extensive vascular disruption including endothelial and smooth muscle damage, adhesion and migration of leukocytes across arteries and venules. Amyloid angiopathy with vascular damage and inflammatory changes are hallmarks in the brains of Alzheimer disease victims. The vascular actions of beta-amyloid are distinct from the neurotoxic properties of the peptide and were prevented by the free radical scavenging enzyme superoxide dismutase. Oxygen radical mediated vascular dysfunction may induce ischemic and inflammatory responses leading to neurodegeneration as seen in Alzheimer's disease.

Differences in microcirculatory bed arrangement in some skeletal muscles of the rat and golden hamster.

The architectural features of the most peripheral parts of the microcirculatory bed were studied in selected skeletal muscles (cremaster, retractor, spinotrapezius, and ventral stripe of spinotrapezius) in rats and golden hamsters ranging in weight between 130 and 330 and 130 and 180 g, respectively. In the cremaster muscle of both species, a very complicated, irregular and heterogenous arrangement of terminal arterioles, as well as of postcapillary and collecting venules was found. In the retractor, the most regular arrangement was along the margins of the middle part of muscle, but an irregular brush-like branching of the terminal arterioles and collecting venules limits the use of these areas. Similarly, in spinotrapezius muscle the most regular vascular architecture, complicated by a different fashion of terminal arterioles and collecting venules, was along the ventral border of muscle. The most regular and simplest vascular arrangement was in the ventral stripe of spinotrapezius of the rat. Therefore, this muscle is recommended as most appropriate for intravital experimental studies of the skeletal muscle microcirculatory bed in all age and weight groups.

Early postnatal growth of skeletal muscle blood vessels of the rat.

The development of blood vessels during the first three postnatal weeks was studied in the ventral stripe of the spinotrapezius muscle of the rat by use of India ink-gelatine injections, and electron microscopy. The number of terminal arterioles and collecting venules remained unchanged postnatally in the observed area. A remarkable proximodistal gradient of vascular development was apparent: while the basic structure of the hilar vessels remained unchanged in the time studied, the intramuscular arteries and veins matured gradually. More peripherally, gradual maturation of terminal and precapillary arterioles was observed. The capillary endothelium and the pericytes showed immature features, and remained unchanged during the time studied. An intense rebuilding activity was found in the endothelial cells of the growing venules, expressed by various forms of gaps, covered by an intact basal lamina and pericytes. Numerous mast cells and macrophages were found along all vessels. Intramuscular lymphatics were not present prior to the first postnatal week.

Circulatory pattern and structure in the tail and tail fins of Xenopus laevis tadpoles.

This investigation was initiated with the intent of study capillary sprouting in the tadpole tail fin microcirculation of the African clawed frog, Xenopus laevis, using a combination of intravital video recordings and electron microscopy. The tadpoles were observed daily for periods up to one hour during one to two weeks. The capillary sprouts originated mostly from postcapillary venules, and within 24-48 h merged with other capillary sprouts, subsequently establishing a capillary loop with blood flow. As the circulatory patterns developed further, capillary regressions also occurred. As the electron microscope analyses of the capillary sprouts progressed, it became obvious that a thorough electron microscope study of the blood vessels of the tadpoles was required in order to explore structural characteristics of arterial and venous blood vessels. Thus, this article deals primarily with the general organization of the tadpole tail circulation, and the ultrastructural characteristics of the vascular walls. A subsequent article will deal with the role of endothelial cells, fibroblasts and pericytes in the process of capillary sprouting and regression, based on intravital recordings and electron microscope analyses.

Capillary growth in the mesentery of normal young rats. Intravital video and electron microscope analyses.

Capillary sprouting was studied by a combination of intravital video recording and subsequent electron microscopy in the mesentery of young female rats without previous experimental manipulation. Selected segments of the mesenteric microvascular bed with capillary sprouts were carefully surveyed and mapped at a monitor magnification of 255 times and submitted to detailed in vivo analysis concerning flow pattern and cells at 2000 times magnification. The mesentery was preserved for light and electron microscopy by a superfusion of glutaraldehyde while observed and recorded on video, confirming earlier investigations that this type of fixation does preserve exceptionally well vascular topography and diameters of the mesenteric microvascular bed. Capillary sprouts originated as endothelial spurs from arteriolar-venular arcades and continued to grow in size through a bipolar rearrangement of endothelial cells, forming a solid sprout tip which progressively lengthened by alternately rapid and slow growth phases. The extended leading tip of the migrating endothelial cells displayed microspikes and pseudopodia denuded of basal lamina. The cytoplasm of the leading tip contained an array of microtubules, 75 A filaments and many small vesicles. In similarity with the situation in nerve growth cones, all these organelles probably participate in cytoplasmic streaming and cell migration. The sprout lumen arose between endothelial cells of the solid sprout. Mesenteric connective tissue fibroblasts approached and settled down on the sprouts, being converted to pericytes as the fibroblasts became enveloped by a basal lamina. The pericytes reinforced the wall of the delicate and fragile capillary sprouts, and trapped plasma, platelets and red blood cells which had leaked out temporarily, in the process assuming an umbrella shape. The arteriolar feeder of the arcades was surrounded by cells which were classified as intermediate between pericytes and true smooth muscle cells. Sprouts that presumably were on the verge of merging with other capillaries were analyzed for indications of how anastomoses are formed.

Perfusion and superfusion fixation effects on rat mesentery microvascular beds. Intravital and electron microscope analyses.

The effects of glutaraldehyde on dimensions and ultrastructure of microvascular beds in rat mesentery were studied in two kinds of experiment, administering the fixative by intra-arterial perfusion at a pressure of 80 mm Hg and by superfusion of the exteriorized mesenteric membrane. The microvascular segments were observed by means of intravital microscopy and recorded on videotape before, during, and after glutaraldehyde reached the microvascular segment being observed. Vascular outer diameters were measured at exactly the same points before and after fixation; in Epon embedded whole-mounts; and in sections analyzed by light and transmission electron microscopy, confirming positively the various segments of the microvascular bed and yielding information concerning the preservation of cellular components. Both experiments confirmed that neither perfusion nor superfusion of glutaraldehyde changes the outer diameter of any segment of the microvascular bed compared to the dimensions 5-10 sec before the blood vessels are reached by the fixative. They remain unaltered also after embedding in epoxy resin. During superfusion, there is a 20-50 sec delay until the blood flow comes to a complete stop. This delay is assumed to give rise to the recorded small undulations of luminal endothelial cell membranes and slight buckling of the entire endothelial layer, probably due to a gradual fall in intravascular pressure. Occasionally, the ultrastructure of some endothelial cells is less well preserved after superfusion fixation. This study demonstrates that intraarterial perfusion of glutaraldehyde renders an instantaneous fixation of mesenteric microvessels, preserving the prefixation dimensions of the various segments and the ultrastructure of the cells. Superfusion of glutaraldehyde is slower in reaching the microvessels and may change slightly the appearance of the vascular wall, and cause some impairment of microvascular functions, such as increased postcapillary leukocyte margination and extravasation.

An electron microscopic study on the type I pneumocyte in the cat: postnatal morphogenesis.

This investigation describes the morphogenesis of the type I pneumocyte from the neonatal stage to the age of 3 months. Cells lining subpleural air spaces were photographed from electron microscopic serial sections and a three-dimensional representation of each cell was obtained by transferring the contours of the cell membranes from micrographs to transparent plastic sheets which were then spaced to scale and stacked. The portion of the reconstructed cell surface taking part in the formation of the blood-air barrier increased extensively in postnatal stages when compared with reconstructed cells of prenatal stages. Reconstructed cell-surface irregularities decrease during distension. A cytoplasmic plate seen in the last stage studied may represent a forming alveolar pore.

[Intravital and electron microscopic analysis of the arterioles by immersion and perfusion technics of fixation of the rat mesentery]. / Prizhiznennyi i élektronno-mikroskopicheskii analiz arteriol pri immersionnom i perfuzionnom sposobakh fiksatsii bryzheiki krysy.

The technique of the intravital microscopic control, videotaping, cinemoregistration and per single-frame analysis made it possible to demonstrate for the first time that a double fixation in glutaraldehyde and osmium has no effect on the arteriolar lumen. Comparing the data on the intravital and a detailed electron microscopic study, it was possible to reveal certain peculiarities and advantages of the fixative perfusion and immersion techniques applied. The investigation establishes a basis for extended morphological studies of small blood vessels and morpho-physiological comparisons.

Combined intravital microscopy and electron microscopy of the blind beginnings of the mesenteric lymphatic capillaries of the rat mesentery. A preliminary report.

The blind beginnings (so-called terminal lymphatics) of the mesenteric lymphatic capillaries have been studied in the rat mesentery by means of intravital microscopy and electron microscopy. Intraluminal objects were observed to move slowly downstream, probably carried by the movement of the lymphatic fluid. No simultaneous changes in capillary lumen could be observed, and the subsequent electron microscope analysis of the same lymphatic capillary demonstrated the absence of smooth muscle cells in the wall of the lymphatic vessel. Interendothelial gaps were present, allowing the uptake of interstitial fluid into the blind ending of the lymphatic capillary.

An electron microscopic study on the type I pneumocyte in the cat: pre-natal morphogenesis.

This investigation describes the pre-natal morphogenesis of the type I pneumocyte subsequent to its differentiation from pulmonary epithelium. Cells lining subpleural alveolar septa were photographed from serial sections with the electron microscope, and a three-dimensional representation of each cell was obtained by transferring the contours of the cell membranes from montages to transparent plastic sheets which were then spaced to scale and stacked. The results of this study indicate that: The nascent blood-air barrier of a 50-day reconstructed cell was twice as thick as the average definitive barrier; definitive barrier thickness was observed in some areas in a 63-day reconstructed cell; the amorphous component of elastic tissue which appears peripherally in septal connective tissue during pre-natal morphogenesis may be directly juxtaposed to the basal lamina of the alveolar epithelium; the orientation of the cell junction between a pneumocyte and its neighboring cells, as observed in sections of alveolar septa, changes as the contour of the pneumocyte changes from simple abutment to overlapping patterns.

An electron microscopic study on the type I pneumocyte in the cat: differentiation.

This investigation describes the differentiation of the type I pneumocyte from undifferentiated pulmonary epithelium. Cells lining subpleural alveolar septa were photographed from serial sections with the electron microscope and a three dimensional representation of each cell was obtained by transferring the contours of the cell membranes from micrographs to transparent plastic sheets which were then spaced to scale and stacked. The results of this study indicate that: (1) the only reliable criterion for differentiating between type I and type II cells is the commencement of attenuation of the type I cell; (2) differentiation of the type I cell occurs via the formation of one or more cytoplasmic attenuations that eventually fuse peripherally, thereby surrounding the unattenuated cell soma; (3) with respect to individual cells, blood-air barriers tend to form in distal areas of the attenuating cytoplasm before proximal areas; (4) both type I and type II pneumocytes retain certain characteristics that reveal their common origin.