Immature Vessels Research Articles

Vascularization of fetal cell suspension grafts in the excitotoxically lesioned adult rat thalamus

Several studies have considered the establishment of vascularization in intracerebral solid transplants of neural tissue. The widely supported interpretation of the results is that the vascular network of the solid grafts is already present before implantation into the host brain. The situation is different when dissociated fetal tissue is transplanted as a cell suspension because in these conditions the fetal vascular network is disrupted. The present study has, therefore, been undertaken to follow the angiogenesis in a transplant of dissociated fetal cells implanted into the excitotoxically neuron-depleted thalamus. The vascular network is compared to that observed in the intact and in the lesioned thalamus both in terms of morphology of the capillaries and of the function of the blood-brain barrier (BBB). In the transplant, capillaries, stained by Indian ink, are very few in number and have very fine calibers during the first 20 days after grafting. Some structures can be identified as immature blood vessels at the electron microscopic level. The blood vessels are progressively more numerous in the graft and they demonstrate mature ultrastructural features 2 months after grafting. Last, there is no leakage of the BBB for peroxidase. The vascularization seems to follow a pattern of maturation comparable to that described during development in the literature. In contrast, in the lesioned area, there is a reactive angiogenesis: 10 days after the excitotoxic injection (shortest time studied), there are many wide caliber vessels with expanded perivascular spaces engorged with mesodermal cells. A microvascularization also develops transiently during the first two months. Capillaries are abnormal from the functional point of view, since there is a leakage of the BBB to macromolecules. The use of an experimental model in which transplant had to grow in a lesioned area permits to determine two types of vascularization: an apparently normal developmental timetable, normal morphological and functional characteristics, in the transplant; a reactive angiogenesis, in the lesioned area.

Oxygen-Induced Retinopathy in Newborn Rats: Effects of Prolonged Normobaric and Hyperbaric Oxygen Supplementation

An experimental study was conducted to verify the effects of prolonged normobaric and hyperbaric oxygen supplementation on retinal vessels in newborn animals. Nine litters of newborn Wistar rats were used in the following way. Two litters were used as controls. Two other litters received normobaric oxygen supplementation (FiO2 80%) for five days after birth. These rats were then removed and kept for five more days in room air. Retinopathy with marked peripheral retinal neovascularization was seen in these litters. Three other litters were exposed to the same treatment for ten days after birth, removed from oxygen, and kept for 15 more days in room air. Severe retinopathy with extraretinal proliferation and, in some cases, retinal detachment developed in these animals. Two other litters received hyperbaric (1.80 atm) oxygen supplementation (FiO2 80%) for ten days after birth and were then kept for 15 days in normobaric room air. These animals showed no evidence of peripheral retinopathy. It is possible that moderate hyperbarism exerts, in the newborn rat, a protective action against the toxic effects of oxygen supplementation on immature retinal vessels.

Adult Cutaneous Hemangiomas Are Composed of Nonreplicating Endothelial Cells

Thirty-four human "cherry" dermal hemangiomas were studied by electron microscopy, immunohistochemistry, and cell culture to assess the neoplastic nature of these lesions. Electron microscopy of nine hemangiomas revealed a pronounced thickening of the basement membrane (0.6 to 14 micron) in 93% of the total 158 vascular structures examined within the lesions. This increase was caused mainly by multiple layers of basal lamina, which were irregular in outline and frequently associated with pericytes. Basement membrane changes were present both in the periphery of the hemangiomas, as well as in the center of the lesions. Immature vessels could not be identified and mitoses were absent in all endothelial cells. Using an immunohistochemical marker (Ki67) specific for proliferating cells in G2 and S phases, positive staining was not found in the endothelial cells lining the hemangiomatous vessels, whereas basal epidermal keratinocytes in the same preparations and cultured microvascular endothelial cells expressed the antigen. Endothelial cells of nine hemangiomas did not stain with an activation-related antibody (E12) specific for endothelial cells. When endothelial cells from 14 hemangiomas were isolated and cultured under conditions that support the growth of normal human skin microvascular endothelial cells, the cells of hemangiomatous origin failed to grow. We conclude that the adult hemangiomas may not be true neoplasms, but a tissue overgrowth composed of mature vessels resembling dermal venules, lined by endothelial cells with virtually no turnover.

Accumulation of potassium by differentiating metaxylem elements of maize roots

The previous demonstration that the large late metaxylem vessels of field‐grown maize (Zea mays L. cv. Rosella) roots do not lose their crosswalls until they are 20–30 cm from the tip, and that the presence of a soil sheath outside the root was indicative of immature vessels within, greatly strengthened the hypothesis that ion accumulation into these roots was by uptake into living xylem element vacuoles. Proposals that salt movement into the xylem was by leakage or secretion into dead vessels became much less plausible. Potassium concentration in the vacuoles of late metaxylem elements was measured by X‐ray microanalysis in unetched fracture faces of bulk, frozen‐hydrated pieces of sheathed roots, and found to be in the range 150–400 mM. Potassium concentration in open vessels of bare roots, measured both with the microprobe and by spectrophotometry of aspirated sap, was in the range of 5 to 25 mM. It is concluded that uptake of potassium (and possibly other ions) is into living xylem elements, and that its release to the transpiration stream occurs by the breakdown of their crosswalls and the addition of their vacuoles to the solution in the vessels above.

Golgi-electron microscopic study of sprouting endothelial cells in the neonatal rat cerebellar cortex.

Intraparenchymal postnatal development of the CNS vasculature proceeds by a process of vascular 'budding' or 'sprouting' from existing endothelial cells and elongation of these immature vessels. A modified Golgi technique combined with gold toning and deimpregnation was used to identify and characterize sprouting endothelial cells in the neonatal rat cerebellar cortex with light and electron microscopy. Sprouting endothelial cells were the terminal endothelial cells of immature, developing blood vessels and they had characteristic morphologic features. The most distinctive feature was an array of tentacular processes (0.1-0.2 micron in diameter and ca. 20 microns in length) which radiated from the apex of these cells in the presumed direction of vessel growth. Cytoskeletal microtubules and microfilaments were the characteristic organelles of these tentacles. Sprouting endothelial cells were thin (1-3 microns in diameter). lacked a vessel lumen and basement membrane, had abundant cytoplasmic organelles and a prominent nucleus and were closely associated with the subterminal endothelial cell without interendothelial gaps. The developing blood vessels had a blood-brain barrier which excluded intravenously injected colloidal carbon from the neuropil.

Golgi-electron microscopic study of sprouting endothelial cells in the neonatal rat cerebellar cortex

Intraparenchymal postnatal development of the CNS vasculature proceeds by a process of vascular ‘budding’ or ‘sprouting’ from existing endothelial cells and elongation of these immature vessels. A modified Golgi technique combined with gold toning and deimpregnation was used to identify and characterize sprouting endothelial cells in the neonatal rat cerebellar cortex with light and electron microscopy. Sprouting endothelial cells were the terminal endothelial cells of immature, developing blood vessels and they had characteristic morphologic features. The most distinctive feature was an array of tentacular processes (0.1–0.2μm in diameter and ca. 20 μm in length) which radiated from the apex of these cells in the presumed direction of vessel growth. Cytoskeletal microtubules and microfilaments were the characteristic organelles of these tentacles. Sprouting endothelial cells were thin (1–3 μm in diameter), lacked a vessel lumen and basement membrane, had abundant cytoplasmic organelles and a prominent nucleus and were closely associated with the subterminal endothelial cell without interendothelial gaps. The developing blood vessels had a blood-brain barrier which excluded intravenously injected colloidal carbon from the neuropil.

Retinopathy of Prematurity

The clinical and laboratory data on 140 premature infants, 74 cases with retinopathy of prematurity (ROP) and 66 control cases without ROP, were correlated with ROP grades of increasing severity. By using multiple linear regression (MLR), it is shown that for predicting ROP grades the importance of certain variables varies considerably depending on the gestational age. Below 32 weeks of gestation, acidosis, hyperoxemia, gestational age, pathologic paCO2 levels, and multiple birth are strong regressors. Above 31 weeks, the most important regressors are multiple birth and acidosis, while gestational age and duration of FiO2>0.4 are much less influential. Blood transfusions and artificial ventilation do not seem important in the MLR of either group. When ROP is regressed on the number of paO2 values above 100 torr and gestational age or on the number of paCO2 values above 50 torr and gestational age, the regression coefficients of these variables drop to near zero at a gestational age of about 32 weeks. This loss of weight of the two variables paO2 and paCO2 with increasing gestational age coincides with a comparable drop of the relative incidence of ROP and the relative incidence of immature retinal vessels. It is hypothesized that it is the proportion of infants with an immature retinal vasculature in populations of given gestational ages rather than the gestational age itself which is responsible for the widely varying importance of certain factors during the development of ROP.

Retinopathy of prematurity: the influence of gestational age and retinal maturity on the statistical behavior of risk factors.

The clinical and laboratory data on 140 premature infants, 74 cases with retinopathy of prematurity (ROP) and 66 control cases without ROP, were correlated with ROP grades of increasing severity. By using multiple linear regression (MLR), it is shown that for predicting ROP grades the importance of certain variables varies considerably depending on the gestational age. Below 32 weeks of gestation, acidosis, hyperoxemia, gestational age, pathologic paCO2 levels, and multiple birth are strong regressors. Above 31 weeks, the most important regressors are multiple birth and acidosis, while gestational age and duration of FiO2 greater than 0.4 are much less influential. Blood transfusions and artificial ventilation do not seem important in the MLR of either group. When ROP is regressed on the number of paO2 values above 100 torr and gestational age or on the number of paCO2 values above 50 torr and gestational age, the regression coefficients of these variables drop to near zero at a gestational age of about 32 weeks. This loss of weight of the two variables paO2 and paCO2 with increasing gestational age coincides with a comparable drop of the relative incidence of ROP and the relative incidence of immature retinal vessels. It is hypothesized that it is the proportion of infants with an immature retinal vasculature in populations of given gestational ages rather than the gestational age itself which is responsible for the widely varying importance of certain factors during the development of ROP.

Electrophysiological analysis of the nature of adrenoceptors in the rat basilar artery during development.

The nature of adrenoceptors in basilar arteries of neonatal rats was investigated by means of electrophysiological techniques. In immature (2-6 day postnatal) rats, micro-injection of noradrenaline elicited a depolarization which consisted of two components. The initial 'fast' component (time to peak of 0.3-4s) was slightly reduced by phentolamine and was not antagonized by propranolol. The second 'slow' component (time to peak of about 50s) was not blocked by phentolamine but was antagonized by low concentrations (10(-7) M) of propranolol. In immature rats, micro-injection of isoprenaline was more potent than noradrenaline in evoking the 'slow' depolarization but less effective in eliciting the 'fast' response. The pharmacology with respect to adrenoceptor antagonists of both components of the isoprenaline- and noradrenaline-induced depolarizations was similar. There was some evidence of inhibitory beta-adrenoceptors in immature rat basilar vessels. In adult rats (6 week old) noradrenaline produced a large 'fast' depolarization which was followed by a 'slow' tail response. Both components were not antagonized by phentolamine or propranolol. It appears that in the basilar artery of neonatal rats there are excitatory alpha- and inhibitory beta-adrenoceptors but the major responses to noradrenaline and isoprenaline are mediated by gamma- and excitatory beta-receptors. In adult animals the gamma-adrenoceptor predominates. Experiments were carried out in which agonists were applied by ionophoresis. These results confirm the presence of excitatory beta-receptors in neonatal basilar vessels and show the response has slow kinetics and it is likely that the beta-receptors are distributed uniformly over the smooth muscle surface. In adult animals it was not possible to elicit an excitatory beta-receptor-mediated response. The ionophoretic application of noradrenaline never evoked a perceptible depolarization which could be attributed to gamma-adrenoceptor stimulation. This result is discussed in terms of receptor distribution with respect to synaptic function in a syncytium.

Lead-induced permeability changes in immature vessels of the developing cerebellar microcirculation.

The ultrastructure and microcirculatory permeability changes of lead encephalopathy were studied in an animal model using horseradish peroxidase as an intravascular tracer. The fine structure of capillary sprouts in the developing cerebellar microcirculation of lead-poisoned rats were described. Immature vessels, characterized by the presence of endothelial sprouts, were found to have focal areas of endothelial injury with degenerating endothelial cells. These disruptions of the microcirculatory endothelium had tracer extending from the vessel lumen to the surrounding neuropil. The degenerating endothelial cells were found as early as 24-28 h after the first administration of lead acetate by gastric lavage (2-3-day-old rats). The early injury to endothelial cells of immature vessels in the developing microcirculation is suggested as an important component of the vascular permeability changes which characterize lead encephalopathy. Older animals (5-10 days old) had microaneurysmal vascular dilatations which had a complex internal structure formed by endothelial cells. These microaneurysmally dilated vessels may represent an endothelial response to preceding endothelial injury of immature vessels.

Patent ductus arteriosus: a new light on an old problem.

Summary: It has been suggested previously that delayed closure of the ductus arteriosus in premature infants is related to an ineffective constriction in response to an increase in arterial PO2. The contractile effects of increased PO2 and excess K+ were studied in rings of ductus arteriosus from early (70 ± 4 days, n> = 9) and late (137 ± 3 days, n> = 11) gestation fetal lambs. Studies were performed in a laboratory using overhead fluorescent lighting or in a dark, enclosed box. Room light relaxed the oxygen-induced contraction in immature vessels but had no significant effect on the K+-induced contraction. Room light did not alter either the oxygen or K+-induced contractile responses in mature vessels. When comparing oxygen induced contractions in room light in immature vessels (0.27 ± 0.13 g, n> =9) vs. mature vessels (0.82 ± 0.06 g, n> = 11) there appeared to be an increased response to oxygen with advancing gestational age. However, when the oxygen-induced responses of immature (0.59 ± 0.15 g, n> = 9) and mature (0.82 ± 0.06 g, n> = 11) vessels were performed in an environment excluding room light, no significant gestational difference was observed. The role of oxygen in delayed closure of the ductus arteriosus of premature infants will need further evaluation. Speculation: The difference in sensitivity to photorelaxation between rings of ductus arteriosus from immature and mature lambs may be associated with biochemical differences in vessels between early and late gestation.

The developmental response of the ductus arteriosus to oxygen.

We studied the isometric contractile effects of increased PO2 on isolated rings of lamb ductus arteriosus (from three different gestational age groups). When rings were stretched to initial lengths that result in a maximal contractile response for rings in that age group, the oxygen-induced contraction was 5.91 +/- 0.72 g/mm2 (+/-SEM, n = 18) in animals older than 135 days, 5.55 +/- 1.23 g/mm2 (n = 18) in animals between 111 and 130 days, and 3.85 +/- 0.75 g/mm2 (n = 19) in animals between 87 and 110 days. Only in the most immature vessels could a decrease in the tissue's response to oxygen be observed.

The fine structural study on blood vessels within human brain tumors.

The blood vessels of human brain tumors were studied in comparison with normal architecture under electron microscope. 1) In benign glial tumors, their blood vessels were apperently similar to those of normal tissue. 2) In malignant glial tumors, immature vessels were observed as might be expected in young, immature cells. The nucleus-cytoplasm ratio was high, and cytoplasm was filled with an abundance of organelles, especially free ribosomes and mitochondria. These findings suggest a high metabolic activity of endothelium. Opened intercellular junction, increased pinocytotic and coated vesicles were recognized in the endothelial cells, and proliferated endothelial cells had irregular luminal surface and elongated processes. These facts suggest an increased capacity to trasfer materials between the luman and the parenchyma. 3) In non-glial tumors, a fenestrated endothelium was commonly observed. The perivascular basement membrane was often reduplicated and was composed of several layers. 4) Adhesive devices between adjacent endothelial cells presented commonly macula adherens and desmosome-like pattern. 5) Tubular bodies were observed in the vascular endothelial cytoplasm of glioblastoma multiforme, pinealoma, craniopharyngioma, astrocytoma, teratoma with choriocarcinoma and pituitary adenoma. These tubular bodies showed various spectra from immature type to mature, might reflect a marked vascular reaction in angiogenesis with brain tumor formation. 6) Furthermore, in the case of pinealoma, its vascular basement membrane filled with collagen fibers and some dense materials merged continuously into the intercellular space of tumor tissue as seen in meningioma. These facts suggest that the origin of the pinealoma might be the mesodermal tissue.

Effect of oxygen on the developing retinal vessels of the rabbit: II. In vivo experiments

This paper is the second in a series reporting our studies on the effect of hyperoxia on the developing retinal vessels of the rabbit and gives an account of our in vivo experiments. The effects of hyperoxia, with and without air survival, were studied by exposing young rabbits in incubators to hyperoxia for several days, and the vessels were then examined by injection methods, whole mounts, histology and electron microscopy. The oxygen effect was also studied directly by observing the reaction of the vessels through a window inserted in the anterior segment of the eye. It was found that the immature retinal vessels of the rabbit were as readily destroyed by hyperoxia as those of other mammals examined and that the vessels similarly proliferate abnormally on transfer to air. There are, however, features peculiar to the reactions of the rabbit and these are described and discussed. Electron microscopy revealed that the vessels are destroyed through disintegration of their endothelial cells and that this is preceded by the appearance of prominent autophagic vacuoles in the cytoplasm in the presence of normal mitochondria. The significance of these findings is considered and reasons are given for believing the changes to be due to a selective cytocidal action of oxygen, rather than secondary to vessel closure.

The response of newly formed blood vessels in healing wounds to histamine and other permeability factors

Newly formed blood vessels in the granulation tissue of a healing wound in the rat cremaster muscle are resistant to the permeability-increasing action of histamine and other agents until 2–3 wk. after their formation. At the stage when newly formed vessels acquire the ability to react to permeability factors, electron microscopic examination still reveals clear evidence of structural immaturity of endothelial cells. The failure of newly formed vessels to respond to permeability factors may be due to absence of contractile protein from the cytoplasm of young endothelial cells. The failure of immature vessels to respond to the type of permeability-inducing factors normally present in healing wounds may serve a useful purpose in limiting loss of fluid and protein from the surface of extensive wounds. Newly formed blood vessels in the granulation tissue of a healing wound in the rat cremaster muscle are resistant to the permeability-increasing action of histamine and other agents until 2–3 wk. after their formation. At the stage when newly formed vessels acquire the ability to react to permeability factors, electron microscopic examination still reveals clear evidence of structural immaturity of endothelial cells. The failure of newly formed vessels to respond to permeability factors may be due to absence of contractile protein from the cytoplasm of young endothelial cells. The failure of immature vessels to respond to the type of permeability-inducing factors normally present in healing wounds may serve a useful purpose in limiting loss of fluid and protein from the surface of extensive wounds.

A View of Ultrastructure of Cucurbita xylem

1. Certain features of differentiation of helically thickened vessel members were observed. Young vessel members contain cisternoid endoplasmic reticulum, mitochondria, and dictyosomes. During differentiation vesicles with single-layered membranes replace the cisternoid endoplasmic reticulum; mitochondria appear to degenerate; and the dictyosomes cease to be discernible. The changes in the endoplasmic reticulum and mitochondria of maturing vessel members resemble those previously described for maturing sieve elements. 2. Parenchyma cells containing chloroplasts are associated with the vessel members. Cytologically, they resemble phloem-parenchyma cells. Plasmodesmata occur in the primary walls separating parenchyma cells and the immature vessel members.