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R N Feinberg

Publications and source records attributed to R N Feinberg.

At least 19 recordsLinked to original sources
PubMed1999

Apoptosis in early ocular morphogenesis in the mouse.

Development of the eye requires complex interactions between tissues, extracellular matrix and growth factors. Most cells of the optic primordia grow and differentiate into discrete ocular structures; however, other cells have death as their developmental fate. The most common mechanisms of cell death are apoptosis and necrosis. We have identified the cell death that occurs during ocular morphogenesis in ZRDCT-N mice as apoptosis. Mouse embryos, ages E8.5-E11.5, were embedded in paraffin, sectioned at 5 microns and stained with hematoxylin or by the terminal deoxytransferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The spatial and temporal distribution of apoptotic cells was mapped at 0.5 day intervals using a computerized image analysis system, and 3-D reconstructions were made at each embryonic age. Our data indicate that apoptosis plays a role in normal ocular morphogenesis and provides the groundwork for studies of abnormal ocular development.

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PubMed1995

Macromolecular permeability of chick wing microvessels: an intravital confocal study.

The development of the vertebrate limb requires the formation of a normal vasculature to nurture the soft and hard tissue phenotypes. The pattern of embryonic limb bud vessels has been extensively studied, but little is known about the permeability characteristics of the developing circulation. In the present study, the microvascular endothelial cell phenotype was examined by in vivo confocal microscopy following the systemic injection of a graded series of fluorescent dextrans (40,000, 70,000, 150,000 molecular weight) into chick embryos at stages 21-23 in order to determine how selective is the endothelial lining of microvessels as a partition between the blood vessels and the interstitium. Videodensitometry, over a gray scale range of 0-255, was used to quantitate the amount of tracer found within the interstitial compartment of the limb. The tracers of larger molecular weight (70,000, 150,000) were confined exclusively to the vascular lumina, whereas that of smaller molecular weight (40,000) was found to cause perivascular brightening due to extravasation into the surrounding interstitium. The reported differences in permeability were not dependent upon the stage of the embryo used in this study, but were due to the size of the tracer. These data indicate that embryonic wing microvessels demonstrate permselectivity to macromolecular efflux across the endothelium. The present results provide a basis for additional studies concerned with the dynamic characteristics of the limb microvasculature and challenge our concepts about the role of diffusible morphogens in vertebrate limb development.

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PubMed1991

Experimental analysis of blood vessel development in the avian wing bud.

Shortly after its appearance, the avian limb bud becomes populated by a rich plexus of vascular channels. Formation of this plexus occurs by angiogenesis, specifically the ingrowth of branches from the dorsal aorta or cardinal veins, and by differentiation of endogenous angioblasts within limb mesoderm. However, mesenchyme located immediately beneath the surface ectoderm of the limb is devoid of patent blood vessels. The objective of this research is to ascertain whether peripheral limb mesoderm lacks angioblasts at all stages or becomes avascular secondarily during limb development. Grafts of core or peripheral wing mesoderm, identified by the presence or absence of patent channels following systemic infusion with ink, were grafted from quail embryos at stages 16-26 into the head region of chick embryos at stages 9-10. Hosts were fixed 3-5 days later and sections treated with antibodies that recognize quail endothelial cells and their precursors. Labeled endothelial cells were found intercalated into normal craniofacial blood vessels both nearby and distant from the site of implantation following grafting of limb core mesoderm from any stage. Identical results were obtained following grafting of limb peripheral mesoderm at stages 16-21. However, peripheral mesoderm from donors older than stage 22 did not contain endothelial precursors. Thus at the onset of appendicular development angioblasts are present throughout the mesoderm of the limb bud. During the fourth day of incubation, these cells are lost from peripheral mesoderm, either through emigration or degeneration.

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PubMed1991

Endothelial heterogeneity in the chick wing bud: a morphometric study.

The microvascular endothelium of the chick wing bud at stages 22, 27, and 32 was evaluated by ultrastructural morphometry. The rationale for this study is based on the hypothesis that endothelial cells exhibit variation in structure and function during cytodifferentiation. The microvessels had a luminal diameter range such that they were classified as capillaries. The thin continuous endothelium was devoid of a basal lamina. The endothelium had a very small number of plasmalemmal vesicles; vacuoles were however present for all stages and in some cases were abundant. The temporal findings were that endothelial cell thickness increases, plasmalemmal vesicle densities decrease, and the densities of cytoplasmic vacuoles increase. The spatial results were that endothelial cells in proximal regions of the limb have a greater thickness, contain fewer vesicles and have more vacuoles than those in distal regions. In general, these results indicate that endothelial ultrastructural heterogeneity occurs within a 3 1/2 day time-span of wing bud development. The discussion considers the results with regard to recent reports on endothelial cell heterogeneity.

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PubMed1990

Iloprost attenuates the increased permeability in skeletal muscle after ischemia and reperfusion.

Increased vascular permeability is an early and sensitive indicator of ischemic muscle injury, occurring before significant histologic or radionuclide changes are evident. We investigated the effect of iloprost, a stable prostacyclin analog, on microvascular permeability in a rat striated muscle model. In six control and six experimental animals the cremaster muscle was dissected, placed in a closed-flow acrylic chamber, and suffused with a bicarbonate buffer solution. Dextran labeled with fluorescein was injected intravenously as a macromolecular tracer, and microvascular permeability was determined on the basis of clearance of the fluorescent tracer. Two hours of ischemia were followed by 2 hours of reperfusion. In the experimental group iloprost (0.5 microgram/kg/min) was given in a continuous intravenous infusion. Microvascular permeability increased significantly during reperfusion in both control and experimental animals (p less than 0.0001). Treatment with iloprost, however, significantly attenuated this response compared to the control group, 4.8 +/- 0.3 versus 7.3 +/- 0.5 microliters/gm/min, respectively (p less than 0.0001). Iloprost decreases the rise in vascular permeability after ischemia and reperfusion. Experimental clinical use of iloprost under controlled conditions in the treatment of patients with acute skeletal muscle ischemia appears justified.

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PubMed1989

Mapping of the microcirculation in the chick chorioallantoic membrane during normal angiogenesis.

The microcirculation within the chorioallantoic membrane (CAM) of the chick is particularly well suited for in vivo observation and has been used extensively as an assay to detect angiogenic activity. Although progressive chronological expansion of the CAM capillary network occurs normally during embryogenesis, descriptions of the branching patterns of CAM pre- and postcapillary microvessels during embryonic development have not been recorded. In the present study chick embryos were incubated, using an established shell-less culture technique, and observed in vivo at Days 6, 10, and 14 of embryonic development. Morphometric analyses of photomicrographs of CAM microvessels were based upon the centripetal ordering method of microvascular mapping of the first three orders of pre- and postcapillary microvessels with the capillaries serving as the initial point of reference. For both pre- and postcapillary vessels, the number of first-order vessels exceeded the number of second-order vessels which, in turn, outnumbered third-order vessels during each observation period. First- and second-order vessels progressively increased in number from Day 6 to Day 14; however, the number of third-order vessels remained essentially constant during this period. Further, the number of precapillary vessels was greater than postcapillary vessels in their respective orders at Days 6 and 10; however, by Day 14 the numbers were comparable. Average diameters and lengths of the third-order vessels were greater than the second-order vessels which, in turn, were greater than the first-order vessels in both the pre- and postcapillary compartments. Further, mean lengths of each of the three vessel orders in both compartments decreased progressively and by Day 14 were significantly less than at Day 6. Average diameters of each vessel order, on the other hand, remained unchanged from Day 6 to Day 14. Finally, intercapillary distances, based on measurements from fluorescent micrographs obtained after microinjections of fluorescein isothiocyanate (FITC)-dextran, were substantially less at Day 10 and 14 than at Day 6. Based on these morphometric data, the endothelial precursor responsible for continuous neoformation of first- and second-order microvessels during embryogenesis remains uncertain. Whether existing first-, second-, or third-order vessel endothelia serve as this precursor or histodifferentiation of existing capillaries enables continuous expansion of the first- and second-order microvessels remains to be tested.

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PubMed1988

Vascular histodifferentiation in the chick chorioallantoic membrane: a morphometric study.

This study presents a quantitative description of ultrastructural changes associated with focal histodifferentiation of the chick chorioallantoic membrane (CAM) microvasculature. Early vessels (4-8 days of a 21-day incubation period) were lined by a thin, but continuous endothelium which was characterized by punctate appositional contacts, an incomplete or absent basal lamina, and few plasmalemmal vesicles. During this stage, there was little ultrastructural distinction between CAM arterioles, capillaries, and venules. During the intermediate stage (9-13 days), the arteriolar endothelium displayed more extensive junctional apposition. In addition, the interstitial (periendothelial) space contained more collagen fibrils and increased cell volume densities, some of which resembled presumptive smooth muscle cells. In contrast to the arterioles, endothelial appositions of the venules and capillaries remained punctate during the intermediate stage. The interstitial space of the venules displayed an increase in cell and fibril densities but to a lesser extent than that observed in the arterioles. The capillaries did not exhibit any significant changes in their periendothelial spaces. By the late stage (14-18 days), a circular tunic containing several layers of presumptive smooth muscle cells surrounded only the arteriolar endothelium. The endothelium of both the arterioles and venules increased in mean thickness. The arterioles also displayed complex interdigitating endothelial appositions. All CAM vessels exhibited a progressive increase in the number of endothelial plasmalemmal vesicles and a more complete basal lamina during the developmental stages. These observations served to distinguish differential patterns of vessel maturation along the length of the microvascular system.

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PubMed1987

Quantitative histochemical evaluation of skeletal muscle ischemia and reperfusion injury.

Acute arterial obstruction to the extremities is associated with significant morbidity and mortality. The evaluation of accompanying skeletal muscle injury has thus far been indirect and imprecise. Triphenyltetrazolium chloride (TTC) is an oxidation-reduction indicator which allows for the histochemical quantitation of skeletal muscle injury. In 21 anesthetized nonheparinized adult mongrel dogs, the isolated in vivo gracilis muscle underwent 4, 6, or 8 hr of ischemia with and without reperfusion. The muscles were excised and cut into 1-cm segments, representative muscle biopsies for electron microscopy were taken, each segment was stained in 1% TTC, and the total area of staining was measured with computerized planimetry. All control muscles stained completely with a dark red color. After 4, 6, or 8 hr of ischemia, quantitative measurements of muscle staining indicative of normal tissue were present in 98 +/- 1%, 59 +/- 5%, and 23 +/- 9% of the total muscle areas, respectively. Six hours of ischemia followed by reperfusion was associated with only 36 +/- 9% of the muscle being stained. Segmental TTC staining demonstrated that reperfusion was associated with greater injury, and less TTC staining, in the proximal portion of the gracilis muscle at the site of entry of the major arterial pedicle. The distal muscle did not demonstrate increased damage with reperfusion. It is hypothesized that protection of the distal muscle from reperfusion injury may be due to an absence of reflow farther away from the artery.

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PubMed1987

Regression of blood vessels precedes cartilage differentiation during chick limb development.

We have previously investigated distinct areas of vascular regression in the developing vascular system of the chick limb bud. Avascular areas appear in a characteristic spatial and temporal pattern, and are correlated with the position of developing cartilage. In the present study, we examined limb-bud sections which had been double labeled for endothelial cells and developing cartilage in order to determine the relationship between the appearance of cartilage and the disappearance of capillaries. Endothelial cells, which specifically take up acetylated low-density lipoprotein (acLDL), were labeled by intravenously injecting fluorescent acLDL (DiIacLDL) into chick embryos at Hamburger and Hamilton stages 26-30. Avascular zones, which correspond to the developing digits, were clearly visible within the fluorescently labeled distal vasculature. The same sections were labeled with monoclonal antibodies specific for cartilage. We found that progressing avascularity in the digital regions was followed by increased staining for cartilage antigens in the same areas. Zones of avascularity always developed earlier than morphologically and immunologically detectable cartilage in all planes of section and were always larger than the areas of cartilage. These results demonstrate that blood vessels disappear in predictable areas prior to the overt differentiation of cartilage.

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PubMed1986

Localized vascular regression during limb morphogenesis in the chicken embryo. I. Spatial and temporal changes in the vascular pattern.

Morphogenesis of the vertebrate limb bud depends upon reciprocal interactions between epithelial and mesenchymal tissues. A characteristic limb vascular pattern is essential for normal limb outgrowth. The vascular pattern in the distal portion of the wing bud was examined by ink injection and compared to the sites of cartilage differentiation, as indicated by [35S]-incorporation. During development, avascular areas arose in three distinct locations within the vascularized mesoderm. These areas corresponded to the distal skeletal elements, referred to as digits 2, 3, and 4. Incorporation of radioactive sulfate was high in the avascular areas and low in the adjacent vascular tissue. Examination of autoradiographs of ink-injected limbs suggested that the appearance of avascular regions preceded the accumulation of sulfated cartilage matrix. These results indicate that remodeling of the limb vasculature is related to the formation of the skeletal pattern.

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PubMed1986

Localized vascular regression during limb morphogenesis in the chicken embryo: II. Morphological changes in the vasculature.

The regression of blood vessels in the distal wing bud of chicken embryos from stages 19 to 31 was examined by light and electron microscopy. The vessels were double-labelled by an injection of Monastral blue B (MB) to label the regressing endothelial cells, followed 6-48 hours later with an injection of India ink which marked the lumens of patent vessels. Prior to stage 26 the vessels contained only India ink since the endothelial cells were not phagocytic at this stage. Vessels at stage 26 or later were often double-labelled, with MB sequestered in the endothelial cell cytoplasm and India ink in the vessel lumens. After stage 27 cells not associated with lumens, but labelled with MB, were observed in areas undergoing vascular regression. Ultrastructural changes in the endothelial cells as the vessels regressed included formation of luminal and abluminal processes, long complex junctions, and vacuoles containing MB. In many involuting vessels the endothelial cells appeared normal even though the lumens were collapsed. Occasionally, isolated pyknotic cells were observed in regions that had been previously vascularized. At stage 31 cells in the developing cartilage had vacuoles containing MB. Our study suggests that blood vessels may disappear from the prechondrogenic zone of the distal wing bud by several mechanisms. These could include a type of cell death that does not elicit a cellular infiltrate, migration of the endothelial cells away from vascularized regions, and/or transdifferentiation into cells that resembled chondrocytes.

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PubMed1986

Transport and steady-state concentration of plasma proteins in the vitreous humor of the chicken embryo: implications for the mechanism of eye growth during early development.

The nature and origin of the proteins of the vitreous humor were examined in chickens during embryonic and early posthatching stages. The major proteins of the vitreous humor were similar in electrophoretic mobility to plasma proteins at all ages examined. Earlier studies from our laboratory and experiments described below showed that plasma proteins continuously entered and left the eye throughout its development. From these data it was concluded that the majority of vitreous-humor proteins were derived from the blood. The protein concentration of the vitreous humor was 13% of that of the plasma from embryonic Days 6 through 15 (E6 through E15). After E15, the relative protein concentration in the vitreous humor declined with respect to the plasma and reached 4% of the plasma protein concentration at hatching. Several possibilities were considered to account for how proteins can rapidly enter and leave the eye, yet maintain a steady-state concentration in the vitreous humor that is less than one-seventh of that in the blood.

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PubMed1983

Hyaluronate in vasculogenesis.

Limb buds of chicken embryos contain within the peripheral mesoderm an avascular zone that is rich in hyaluronic acid. Epithelial tissues that synthesize large amounts of hyaluronic acid relative to other glycosaminoglycans caused avascularity when implanted into normally vascular wing mesoderm. Epithelia that synthesize little hyaluronic acid did not cause avascularity. Elvax implants containing hyaluronic acid caused the formation of avascular zones, whereas similar implants containing other glycosaminoglycans did not give rise to avascular zones. Hyaluronic acid may thus play a role in determining the location of blood vessels in the embryo.

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PubMed1983

Ectodermal control of the avascular zone of the peripheral mesoderm in the chick embryo.

Prospective skin ectoderm is underlaid by a relatively thick (100 +/- 20 micrometer) avascular zone of mesoderm in most regions of the early embryo. To determine whether or not the ectoderm exercises a role in the establishment and maintenance of the avascular zone, trypsin-isolated pieces of backskin ectoderm from chick or quail embryos were implanted as a sheet into a slit cut deep into the capillary bed of the wing bud of host chick embryos of stages 19-23. In sham operations, slits were cut at various anteroposterior levels, and the wing was allowed to heal. At intervals of 3-48 hr after these operations, embryos were injected with India ink, fixed, and cleared. Implants formed flattened vesicles, usually in continuity with host ectoderm, but sometimes completely internalized. Periderm cells from each side of the vesicle faced each other, and the cells of the cuboidal layer faced an avascular mesodermal layer at least 100 micrometer thick at all points. The implantation of prospective skin ectoderm resulted in the formation of an avascular zone in normally vascularized mesoderm of the wing bud. In contrast, the vascular bed of the limb bud abutted directly on implants of Millipore filters or of Silastic silicone (Dow Corning). Likewise, the capillary bed came in direct contact with implants of retinal pigment epithelium, an ectodermal derivative normally in close contact with the vascular choroid coat of the eye. These results, taken in conjunction with earlier experiments that show the necessity of the apical ectodermal ridge for the formation of the marginal veins of the limb bud, suggest that epithelial-mesenchymal interactions are involved in important aspects of vasculogenesis in early embryos.

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PubMedDate not supplied

Canine jugular vein endothelial cell monolayers in vitro: vasomediator-activated diffusive albumin pathway.

Cultured canine jugular vein endothelial cells were seeded on polycarbonate filters to create an in vitro permeability assay. The calculated diffusive permeability coefficient for FITC-BSA across untreated monolayers was 1.1 +/- 0.4 x 10(-6) cm/s. After 15-min incubations with either histamine or bradykinin, the resistance to albumin flux across the monolayers was reduced significantly. Diffusive albumin permeability coefficients were 3.4 +/- 1.8 x 10(-6) and 4.1 +/- 2.0 x 10(-6) cm/s, respectively. Ultrastructural morphometric analyses of the endothelial cell monolayers served to define uniform dimensions of intercellular clefts and similar plasmalemmal vesicle densities in the untreated and the vasomediator-activated monolayers. These results are consistent with the interpretation that the vasomediator-activated pathway across the venous endothelial monolayers is not dependent on sustained intercellular gap formation or sustained expansion of the plasmalemmal vesicle population for the 15-min observation periods. Whether the increased albumin flux is dependent on transient gap formation or on physical changes within the venous endothelial cell glycocalyx remains to be tested.

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