Fetal and neonatal rat intestinal capillaries: Permeability to carbon,ferritin, hemoglobin,and myoglobin

The function of the mural components involved in vascular exchange (pinocytotic vesicles, fenestrations, intercellular junctions, and the basal lamina) was assessed by intravascular injection of tracers (carbon, carbon plus histamine, ferritin, hemoglobin, and myoglobin) on separate gestational days in fetal and neonatal rat intestinal capillaries. The continuous capillaries present through day 17 of gestation were impermeable, even at junctions, to all the tracers within the maximum circulation time studied (3 minutes). Most of the luminal pinocytotic vesicles were labeled within 3 minutes with myoglobin (3.3-nm diameter) or hemoglobin (5.5-nm diameter), while only occasional vesicles contained ferritin (11-nm diameter) and none contained carbon (25–50nm diameter). Even though luminal pinocytotic vesicles contained the smaller tracers, no vesicular transport and discharge were ever seen. The fenestrated vessels present from day 18 of gestation allowed a rapid extravasation of ferritin and hemoglobin via diaphragmed fenestrations. At no time period did extravasation of carbon take place, nor did the carbon plus histamine solution induce vascular leakage. Once a tracer gained access to the extravascular space, the developing basal lamina did not seem to retard its movement.     

Age-related changes in rat brain capillaries

We have measured several morphological parameters by electron microscopy of frontal cortex (FC) and hippocampal CA1 (HC) capillaries in male Fischer 344 rat 3-, 9- and 24-months old. The results indicate that with increasing age there is an increase in the cross-sectional area of the basement membrane, increase in the friction of endothelial cell and pericyte cytoplasmic area occupied by mitochondria in the FC, increase in the size of the pericyte mitochondria in both the FC and HC, increased capillary lumen area in the FC and decreased capillary lumen area in the HC. Also, the cytoplasmic area occupied by mitochondria in capillary pericytes is larger than in the endothelial cells of both FC and HC. These results suggest that there is regional variation in the age-associated changes in capillary morphometrics.     

Age-related changes in capillaries of rat oral mucosa. A quantitative electron microscopic study

Ultrastructural age changes in capillaries of the buccal mucosa were examined in montages of cross sections made from electron micrographs at 37,500 x. Six rats aged 6 months and 6 aged 30 months were perfused with glutaraldehyde and conventional thin sections obtained. Two capillaries located within a connective tissue papilla were studied from each rat. Capillaries of the old group differed from those of the young group by statistically significant increases in several parameters. The endothelial cell was increased in thickness, especially in the vicinity of junctions. The frequency of pinocytotic vesicles/unit length of the cell circumference was nearly doubled. Junctions were of nearly double length and took a more oblique course. All parts of the basement membranes were thickened, though perhaps less than seems true in skin. In striking contrast to epidermis, the epithelium of oral mucosa undergoes no appreciable thinning with age. We suggest that the observed age increases in frequency of pinocytotic vesicles and in the length of junctions may facilitate blood/tissue exchange, thus compensating for impaired exchange due to the thickened basement membranes. These compensatory changes in conjunction with the unchanged size of the mucosal capillary bed in the aged rat (demonstrated previously) could explain the unchanged thickness of the oral epithelium.     

Myocardial capillaries in the fetal and the neonatal rat: A morphometric analysis of the maturing myocardial capillary bed

Developing myocardial capillaries from 16-day-gestation fetus to adult undergo several morphological changes including a thinning of the lateral extensions of the capillary endothelial cells, the formation of a basal lamina, and an increase in the number of plasmalemmal vesicles. A decrease in the extracellular space, an increase in the number of capillaries, and a decrease in the capillary diameter were also observed during the developmental period. In view of these ultrastructural changes, a morphometric analysis was made on the developing myocardial wall to demonstrate specific quantitative changes. The volumes which were occupied by capillary endothelial cells, capillary lumina, extracellular space, and myocardial myocytes within a reference volume of myocardium were measured; and we found that 8% of the reference myocardial volume was occupied by capillary endothelial cells, 85% was occupied by myocardial myocytes, 4% was occupied by capillary lumina, and, except for a significant change in extracellular space at 16 days gestation, 3% was occupied by extracellular space. Each volume ratio was found to be nearly constant throughout the studied period. In contrast to this constancy in the volume ratios, other parameters which were measured demonstrated significant changes during the developmental period studied. These overall changes include a 135% increase in capillary density, a 63% increase in luminal surface area of capillary endothelial cells, a 24% decrease in capillary diameter, a 12% decrease in diffusion distance, and a 35% decrease in the diameter of the erythrocyte population. The decrease in capillary diameter occurs concomitantly with and may be correlated to the decrease in erythrocyte diameter. Also, the increasing number of capillaries offset the effect of the decrease in capillary diameter, resulting in constant volume ratios. This constancy in the volumes of the measured myocardial compartments during development, despite the considerable changes in morphology and in the other measured parameters which are occurring in the myocardial wall, can be interpreted to mean that some functional constraints are operating to maintain capillary-to-myocardium volume ratios within very narrow limits.     

Lymphatic capillaries of the pig lung: TEM and SEM observations

Pulmonary lymphatic vessels extend within the connective tissue sheets surrounding airways and blood vessels. Frequently in this location they also border the lobular parenchyma, but no lymphatic vessels have been found within intralobular compartments between blood capillaries and alveoli. The presence and distribution of lymphatic vessels in pulmonary tissue are consistent with an important role for the lymphatic system in the clearance of interstitial fluids in the lung. Pulmonary lymphatic channels have structural characteristics of initial lymphatics; their walls are formed only by an endothelial layer, and no muscular cells are present. A network of anchoring filaments and collagen and elastic fibers surrounds the vessel walls. Because the lung is a mobile organ the tissue undergoes compression and distension during respiratory phase. These modifications could have a role in the mechanisms for lymph formation and flow. © 1994 Wiley-Liss, Inc.     

Maturation of myocardial capillaries in the fetal and neonatal rat: An ultrastructural study with a morphometric analysis of the vesicle populations

The ultrastructural morphology of the cellular and extracellular components of the developing myocardial capillary wall—from the 16-day-gestation fetus of the rat to the 21-day neonate—was examined. A morphometric analysis of plasmalemmal vesicles and of coated vesicles and pits of capillary endothelial cells was performed during the same developmental period. As the lateral extensions of the capillary endothelial cells change from irregular to regular in their thickness during development, there is an increase in the number of plasmalemmal vesicles and a progression from clusters of plasmalemmal vesicles to a uniform distribution in the endothelial cell. The ratio of vesicles which are open to the luminal front, which are “free” in the cytoplasm, or which are open to the abluminal front of the endothelial cell was consistent throughout development. The numerical density of plasmalemmal vesicles demonstrates a gradual and significant increase. In contrast, the numbers of coated vesicles and pits are variable within a very narrow range, and no pattern of increase or decrease is discernible during development. Similarly, there is no change in interendothelial cell junctions, which consist of occluding and primitive adhesive junctional types, during development. The lamina densa of the basal lamina gradually develops from discontinuous, patchy densities along the abluminal surface of the endothelial cells to a continuous and distinct layer by 21 days gestation. The presence of the proteoglycan species in the developing basal lamina was assessed with the cationic dye ruthenium red (RR), and the appearance of RR-marked proteoglycans was found to parallel the appearance of lamina densa material. The RR sites appear discontinuously in patches; and later, the RR sites appear in a continuous and regular planar lattice in the lamina rara interna and externa at 21 days gestation. A complete array of RR-stainable anionic sites outside a continuous lamina densa near birth indicates that the basal laminae of developing capillaries in the heart are morphologically, and in part biochemically, mature by the end of the first neonatal week. Our results show that the endothelial cells and the subtending basal lamina of myocardial capillaries gradually mature morphologically during the final days of gestation and the first neonatal week. The finding of tight junctions and small areas of vesicle concentration in fetal endothelial cells could indicate that sites of permeability are limited early in myocardial capillary development and that these vesicular sites increase as gestation proceeds and as the myocardial capillaries mature.     

Pericyte-endothelial gap junctions in developing rat cerebral capillaries: A fine structural study

Background: Fine structural study revealed the intercellular coupling between the pericyte and the endothelial cells via the gap junctions, in the capillaries of the basal forebrain of rat embryos. Results: Gap junctions were constructed by the adluminal plasmalemma of pericyte and the abluminal plasmalemma of endothelial cells. Conclusions: Gap junctions are membranous channels that directly join the cytoplasms of the pericyte and endothelial cell and imply some substantial role for the pericyte on the endothelial proliferation. It is postulated that the function of the pericyte in the prenatal mammals are assigned to the regulation of the development of cerebral microcirculation. © 1995 Wiley-Liss, Inc.     

A correlative SEM/TEM examination of the endothelium surface in neural capillaries.

The modifications of the endothelial surfaces were analyzed in growing neural microvessels by scanning and transmission electron microscopes in the optic tecta of chick embryos and chickens. The endothelial inner aspect appears regular and smooth in the early stages of the vessel growth (7th incubation day). Later (14th incubation day) both the abluminal and luminal surfaces of the endothelium follow a very sinuous course and the luminal ones appear extremely rich in pleomorphic microprojections. When the endothelium differentiation is concluded (5-day-old chicken), the cells are very thin and again exhibit regular and smooth surfaces. These findings reveal a great mobility of the cell membrane of the endothelial cells when they are growing longer and thinner by a moulding process. Moreover, the presence of a number of pinocytotic pits in the embryo vessels would indicate that the neutral vessels, provided with a typically low pinocytotic activity in the adult life, are engaged in this function during development.     

Functional maturation of the capillary wall in the fetal and neonatal rat heart: Permeability characteristics of developing myocardial capillaries

The development of the functional components of the myocardial capillary wall was characterized by time-course studies of transendothelial transport of intravascularly injected probes of graded size from 16 days of gestation in the fetal rat to seven days postpartum. Despite the morphological changes occurring in the developing endothelial cells, the interaction of the probes was similar throughout the developmental period studied. The carbon particles were retained within the capillary lumina without any association with interendothelial junctions or with plasma-lemmal vesicles. Carbon also was associated with coated vesicles. In contrast to carbon, ferritin was localized sequentially, over 60 sec of circulation, in plasmalemmal vesicles on the lumenal surface, in the cytoplasm, and on the ablumenal surface of the endothelial cells as well as in the interstitial space. Ferritin was located also in coated pits and vesicles and, after 90 sec of circulation, in multivesicular bodies. Within 30 sec of circulation, reaction product of myoglobin was located in plasmalemmal vesicles, coated vesicles, and transendothelial cell channels. Also within 30 sec, myoglobin partially filled the interendothelial space from the capillary lumina to the level of the tight junction. At all developmental ages studied, the interendothelial cell junctions appeared structurally tight and were impermeable to all of the probes. Once ferritin or myoglobin had reached the ablumenal space, the basal lamina did not appear to restrain the passage of the probes. Plasmalemmal vesicles are the capillary structures which transendothelially transport ferritin and myo-globin in developing myocardial capillaries.     

Radiation induced changes in the blood capillaries of rat duodenal villi: a corrosion cast, light and transmission electron microscopical study

Disturbances of gastrointestinal function are an important limiting factor in radiotherapy of the abdominal and pelvic regions. The pathogenesis of radiation induced intestinal dysfunction is not completely understood, although the intestinal mucosa has been shown to respond to irradiation by a progressive reduction in villous size. Since blood vessels in other organs have been implicated in the initiation of post-irradiation changes, the present study examines the response of villous blood vessels to an X-ray dose of 10 Gy after 3 days. Vascular corrosion casts and light and transmission electron microscopy (TEM) were used to study the post-irradiation vascular response. In control and sham-irradiated animals, the villous plexus was fountain-like: an arteriole entered the villous base and divided apically into two terminal branches. Villous capillaries apparently derived from the terminal branches, and united to form venules. In capillary loops the vertical inter-capillary distance was greater than the horizontal inter-capillary distance. After irradiation, the vessels became tortuous and the plexus was compressed apico-basally, shown by a decrease in the vertical inter-capillary distance. The capillary luminal diameter, as measured on resin semi-thin sections, was significantly increased. TEM showed that the endothelium was irregular and there was evidence of plasma leakage. These results suggest that villous damage seen after irradiation can include changes in the villous vasculature.     

A cytological study of intestinal absorption in the suckling rat

The distal small intestine of the albino rat has the capacity to absorb protein and particulate matter during the suckling period. Ultrastructural and cytochemical aspects of this absorptive phenomenon were examined in the ileum. Soon after the initial ingestion of milk, a large, yellow, smooth membrane-limited, protein body appears in the immediate supranuclear region of ileal absorptive cells and, also, many small vacuoles and membrane-limited droplets arise between this body and the microvilli. Exogenous protein enters an elaborate superficial tubular system and is segregated in membrane-limited vacuoles and droplets and, then, appears in the supranuclear body. The body and adjacent membrane-limited droplets are basophilic, periodic acid-Schiff positive, and rich in hydrolytic enzymes (acid phosphatase, ATPase, thiamine pyrophosphatase, alkaline phosphatase, esterase). The results suggest the presence of a highly developed lysosomal system during the period of protein absorption. Additional cytological features of the absorptive cells are presented. Ileal absorptive cells are normally free of lipid droplets. When emulsified lipid is introduced into the neonatal ileum, it enters into the cytoplasmic smooth membrane system, including the supranuclear body, and later appeas in the lacteals. This suggests both that the uptake of material may be non-selective and that similar intracellular pathways may be used in transporting protein and lipid through the epithelium.     

Fetal and neonatal development of human spleen: an immunohistological study.

Localization and immunophenotype of lymphocyte subsets in fetal human spleens were studied by employing a panel of monoclonal antibodies (McAb) in an immunoperoxidase staining procedure on frozen tissue sections. Spleens varied from 15 weeks of gestational age (gestational weeks, gw) to newborn. The white pulp consisted of intermediate-sized lymphocytes; no separate compartments could be discerned. Germinal centre development was not observed. Dendritic cells stained for B2, HB5, aC3bR, anti-DRC and OKIa, but in most cases not for immunoglobulin. Although low cellular immunity is observed in neonates, T cells showed adult phenotypes in proportions comparable to the adult situation; immature OKT6(+) lymphocytes were rarely seen. Very few cells stained with anti-NK cell antibody Leu7. B cells all expressed B1, Leu14, aC3bR, T10 and OKIa, were strongly positive for BA1, and mostly stained very weakly for B2 and HB5. Almost all B cells expressed IgM and IgD simultaneously, and very few expressed IgG. IgA-positive cells were absent. At 15 gw a considerable number of IgM(+) B cells showed Leu1 staining, but this decreased during development. These cells may represent the normal counterpart of Leu1(+) IgM(+) cells observed in B-CLL and immunocytic and centrocytic malignant lymphomas. After 25 gw only very few Leu1(+) IgM(+) cells were seen. Altogether, the morphology and immunophenotype of white pulp B cells were different from the predominating adult B-cell subsets, at least until birth. These 'immature' splenic B cells may be precursors for adult splenic B-cell subsets. Considering the presumed role of the marginal zone in the immunity against TI-2 antigens, the absence of a marginal zone at birth may be a main factor in the defective immunity against these antigens in neonates.     

Tanycytes of the third ventricle of the neonatal rat: A golgi study

The morphology and vascular relations of tanycytes in the walls and floor of the third ventricle (3rd V) of neonatal rats (4–10 days of age) were studied with a modification of the rapid Golgi method (Valverde, '70). Serial frontal sections through the infundibular recess revealed that tanycytes may be assigned to two distinct populations based on location and structure. Tanycytes in the ventral one third of the walls as well as the floor of the third ventricle comprise the first group. The cell body of these tanycytes was subependymal in location and possessed two main processes. A centrally directed short neck-process abutted upon the ventricular lumen and often exhibited one or more protrusions extending into the cerebrospinal fluid (CSF). Issuing from the opposite pole of the cell body, a second smooth, relatively unbranched tail process either projected directly to the ventral surface of the brain or terminated in contact with capillaries of the median eminence. A second group of tanycytes was located more dorsally in the walls of the third ventricle. The somatic apical surface of this group generally abutted directly upon the ventricular lumen and exhibited protrusions of diverse configuration projecting into the ventrical lumen. Tanycytes of the latter group were also characterized by a tortuous tail-process which arched laterally and ventrally to terminate either in the adjacent neuropil or at the surface of the brain. Unlike those of the ventral group, these processes exhibited numerous appendages, varicosities and side branches, many of which ended as expansions in direct contact with blood vessels. In addition, segments of beaded afferent fibers of fine caliber were often seen to contact processes of both groups of tanycytes. The observed relationships between CSF, tanycytes and blood vessels support recent hypotheses of tanycyte involvement in neuroendocrine regulation, CSF composition and neuronal metabolism.     

A scanning electron microscopic study of the morphological changes in rat small intestinal mucosa treated by intracolonic indomethacin

Rectal administration of indomethacin induces longitudinal ulcers in the rat small intestine. The current study investigated a sequence of progressive villus injury in this enteropathy, especially by the use of scanning electron microscopy. The initial change was the distortion of several villi on the mesenteric side in the mid-small intestine identified at 0.5 h, even though there was no obvious change under light microscope or dissecting microscope at this time. During the subsequent 2h, distortion of the villi was accompanied by several epithelial defects, and epithelial detachment occurred on the villi tips. Extension of epithelial defects and exposure of the villus core progressed during subsequent periods. The injured villi were confluent with each other on the mesenteric side throughout the 12h after dosing. These findings suggest that the initial mucosal injury induced by the rectal route of administration was not extensive; rather, several villi were focally damaged on the mesenteric side in the mid-small intestine, eventually resulting in a longitudinal ulcer. Although the overall progression after indomethacin administration by the rectal route was similar to that occurrmg after subcutaneous administration, villus change seems to occur much earlier after rectal dosage.This study was presented in part at the 28th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Osaka, October 17–19, 1996.     

Postnatal development of blood vessels (capillaries) in the rat olfactory bulb: A light and ultrastructural study

The increase in density and development of blood vessels (capillaries) were studied in the rat olfactory bulb. The density of blood vessels increased significantly over the second ten-day period of postnatal development. Electron microscopy revealed that blood vessels developed from solid cords of cells with thick endothelial cells, slit-like lumina and ill-defined lamina. The majority of these vessels became luminized during the second ten-day period and were characterized by patent lumina, well defined basal lamina and attenuated endothelial cells.     

Fetal and neonatal transmission of type-b anti-haemophilus influenzae antibodies protecting the newborn rat

Haemophilus influenzae type b is the most common cause of bacterial meningitis in children. Intranasal or intraperitoneal inoculation of infant rats with Haemophilus influenzae type b results in bacteremia and meningitis and has been proved to be a reproductible model of the human disease. For these reasons, it was of interest to use rats as experimental model for the study of anti-Haemophilus influenzae type b vaccine. Immune serum against Haemophilus influenzae type b cell surface extracts was prepared on rabbit. In a first experiment, pregnant rats were passively immunized with either immune rabbit serum or normal rabbit serum or saline. In a second experiment, suckling rat mothers were passively immunized with either immune rabbit serum or normal rabbit serum or saline. All infant rats were inoculated intraperitoneally at 6 days of age with Haemophilus influenzae type b. Bacteremia was determined in all infected rats 24 h after challenge. Only infant rats from immune serum-treated mothers were protected. This protection may be transferred from mother to young rat either before of after the birth.     

Differentiation of fast and slow muscles in the rat after neonatal denervation: A physiological study

Summary Whether an intact innervation is essential for postnatal muscle differentiation was examined in the rat by recording physiological contraction parameters. Muscles in one leg were denervated neonatally (within 24 h of birth) and, between 3–28 days after the operation, their contractions were compared with those of the contralateral control muscles. Experiments were performed on the extensor digitorum longus (edl, a fast muscle) and the soleus (a slow muscle) muscles and contractions were recordedin vitro, at 35 C and with direct stimulation. When compared with the control muscles, 3–4-day-old neonatally denervated fast and slow muscles had longer twitch contractions, higher twitch/tetanus ratios and certain other specific differences in their contraction parameters. These denervation-induced changes in neonatal muscles were essentially similar to those produced 3–7 days after denervation in the differentiated (4-week-old) fast muscle. Despite differences in their absolute values, the contraction parameters of neonatally denervated and control edl muscles changed similarly during development, indicating that postnatal differentiation of fast muscle fibres is independent of a neuronal influence. In the case of the neonatally denervated soleus muscle, the developmental changes in contraction parameters, i.e. shortening of the twitch duration, increase of rate of rise and rate of relaxation in the tetanus and increase of the maximum shortening velocity, were more pronounced than in the control slow muscle; also, there were similarities with the pattern of fast muscle differentiation. Thus, muscle fibre differentiation in soleus becomes altered towards that of a fast muscle after neonatal denervation.     

Ultrastructural changes in odontoblasts and pulp capillaries following cavity preparation in rat molars.

Responses of odontoblasts and pulp capillaries to cavity preparation were investigated in the upper first molar teeth of rats, using light and transmission electron microscopy. At 100 days of age, the blood vessels of the pulp formed a subodontoblastic network consisting of continuous capillaries at a short distance from the odontoblast layer. Cavity preparation caused the displacement of some odontoblasts into the dentinal tubules, while others were separated from the predentin by rapid inflammatory exudation after drilling. The subodontoblastic capillary network under the injured dentin was shifted inwards together with the separated odontoblasts. The endothelium of the shifted capillaries showed a remarkable increase of pinocytotic vesicles, an event thought to be closely related to the formation of the exudative lesion. By one day after cavity preparation, most of the damaged odontoblasts had degenerated. Many cells with high nucleus/cytoplasm (N/C) ratios and prominent nucleoli accumulated around the subodontoblastic capillaries, some of which had many endothelial fenestrae facing these cells. These cells were suggestive of newly differentiating odontoblasts receiving nutritional supply from the capillaries. Three days after cavity preparation, newly differentiating odontoblasts took the place of the degenerated odontoblasts. They began to produce reparative dentin by five days after cavity preparation. Capillaries were located beneath the newly differentiating odontoblasts, but endothelial fenestrae gradually decreased in number. During the active reparative dentin formation, capillaries remained closely beneath the new odontoblast layer. Although the rate of reparative dentin deposition was not significantly lower than that in the primary dentin formation, one could not recognize an invasion of capillaries into the odontoblast layer nor a remarkable increase of endothelial fenestrae, both of which are common in active primary dentin formation. The results suggest that the function of capillaries differs between primary and reparative dentin formation.     

Metabolite changes in neonatal rat brain during and after cerebral hypoxia-ischemia: a magnetic resonance spectroscopic imaging study

Cerebral metabolite concentrations were measured in infant rats using proton magnetic resonance spectroscopic imaging. Measurements were made prior to, during and after exposure of rats (6- and 7-day-old) to unilateral cerebral hypoxia-ischemia (right carotid artery occlusion +2h 8% oxygen). Data clustered according to age and outcome-6-day-old animals with no infarct and 7-day-old animals with infarct. In 6-day-old animals, cerebral lactate concentration increased during hypoxia-ischemia, particularly ipsilateral to the occlusion, and returned to normal soon after the end of hypoxia. There were no major changes in N-acetyl-aspartate levels (NAA) in this group and no regions of hyperintensity on T2 or DW weighted images at 24 h. In the 7-day-old animals, lactate increased during hypoxia-ischemia and remained elevated in the first hour after reperfusion. Furthermore, lactate remained at 258+/-117% and 233+/-56% of pre-hypoxic levels, 24 and 48 h post-hypoxia, respectively. NAA concentrations ipsilateral to the occlusion decreased to 55+/-14% during hypoxia, recovered early post-hypoxia and again decreased to 61+/-25% and 41+/-28% at 24 and 48 h post-hypoxia-ischemia, respectively. The infarct volumes measured by diffusion weighted and T2 weighted MRI at 48 h post-hypoxia were 152+/-40 mm3 and 172+/-35 mm3, respectively. Thus, irreversible damage correlated well with measured in vivo lactate and NAA changes. Those animals in which NAA was unaltered and lactate recovered soon after hypoxia did not show long-term damage (6-day-old animals), whereas those animals in which NAA decreased and lactate remained elevated went on to infarction (7-day-old animals).