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.     

鞍侧腔的胚胎发育及其临床意义

目的：研究鞍侧腔形态结构及其内容物的胚胎发育。方法：对45例不同胚(胎)龄时期的鞍旁结构行组织学连续切片，观察鞍侧腔壁结构及其内容物的形态学变化过程。结果：鞍侧腔的发育可分为3个时期：(1)胚龄6～10周时，形成此区的最初边界；(2)胎龄11～16周时，显示鞍侧腔的构建过程，其腔壁约在胚胎14～16周形成。此期鞍侧腔外侧壁外层由脑膜层组成，内层由基质纤维包绕Ⅲ、Ⅳ、Ⅵ和V1神经复合体组成；而其内侧壁主要由垂体囊和基质纤维构成；(3)胎龄17周至出生后鞍侧腔诸壁及其内容物充分发育。结论：(1)鞍侧腔位于蝶鞍外侧区骨膜-硬膜间隙内，由脑神经、颈内动脉、丛状静脉管(海绵窦)及胶原纤维组成。(2)这些静脉管仅有内皮、纤维细胞和结缔组织构成。     

Thinning of fetal pulmonary arterial wall and postnatal remodelling: Ultrastructural studies on the respiratory unit arteries of the pig

Summary Adaptation to extra-uterine life and postnatal remodelling of intra-acinar arteries was followed in 34 Large White pigs, from birth to adult life, applying morphometry to light and electronmicroscopic studies. After birth, percentage wall thickness decreased rapidly due to a reduction in overlap of adjacent smooth muscle cells and an increase in smooth muscle cell surface area/volume ratio, (p<0.01 at 12 h), without a reduction in the volume density of smooth muscle cells. Smooth muscle cells appeared immature at birth and synthetic rather than contractile organelles predominated. Between 3 weeks and 6 months myofilament volume density doubled (p< 0.0001). At all ages, pericytes, intermediate and smooth muscle cells showed similar volume densities of contractile and synthetic organelles. Thus, the high fetal pulmonary vascular resistance appeared to be due to the shape and arrangement of smooth muscle and other contractile cells within the vessel wall, rather than an excessive contractility of these cells. After birth rapid remodelling of arterial wall structure achieved a reduction in wall thickness by 30 min, continuing during the first week of life. After 3 weeks, remodelling involved an increase in wall thickness, connective tissue deposition with more collagen than elastin (p<0.0001), and smooth muscle cell differentiation.     

Serial immunologic studies in patients with mucocutaneous lymph node syndrome (Kawasaki disease)

Twenty cases of mucocutaneous lymph node syndrome (MCLS), from Fall 1984 to April 1986, are included in this study. The serial immunoglobulin levels including IgG, IgA, IgM showed polyclonal increased immunoglobulin levels during the first month. Only one-fourth of patients had high serum IgE levels. There was no correlation with house dust mite antigen skin test and anti-mite IgE levels. By using the polyethylene glycol and ELISA methods, IgE-circulating immune complexes were detectable in 60% of high serum IgE patients. IgG-circulating immune complexes were also detected in 70% by the polyethylene glycol method and 60% by Raji cell method. CH50 and serum properdin factor B level increased during acute febrile phase. Serial T cell subset studies showed OKT4 cells decreased progressively and OKT8 levels were within normal range during the first and second week. By the third week, OKT4 cells increased and OKT8 cells decreased. This progressed to the fifth week and returned to normal range 2 months later. During the acute febrile phase of the first week, Leu2+15+ cells increased and Leu2+15- cells decreased. After the second week, the Leu2+15+ cells decreased and Leu2+15- cells increased. This increase continued to the fifth week when the Leu2+15+ cells were at the lowest level and the Leu2+15- cells at the higher level corresponding to the OKT8 change. After the fifth week, the Leu2+15+ cells and Leu2+15- cells returned to normal. Patients had increased antibody-dependent cell-mediated cytotoxic reaction at the first week of the acute febrile stage. It decreased dramatically in the second week and returned to normal 2 months later.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations of myoepithelial cells in the rat mammary gland during pregnancy, lactation and involution, and after estradiol treatment

Hormone-induced alterations of myoepithelial cells in the mammary gland have not been fully investigated. The aim of the present study was to examine whether myoepithelial cells are altered in response to hormonal conditions. The immunohistochemical findings of smooth muscle actin for myoepithelial cells were studied during pregnancy, lactation and involution, and after estradiol dipropionate (ED) treatment (50, 500, 1000 microg/kg per week for 1-4 weeks) using a total of 71 Wistar female rats. Myoepithelial cells showed a stratified appearance around ducts during pregnancy, extended cytoplasmic processes with wider distance during lactation, and vacuolated cytoplasm after weaning. ED treatment (50-1000 microg/kg per week) for 1 week increased myoepithelial cells to a variable degree, achieving a level similar to that in pregnancy, but ED treatment for 4 weeks reduced them as the dose elevated. The present study showed that the myoepithelial cells became hyperplastic or hypertrophic by low-dose ED treatment within the physiological range, while weaning pups, and excess high-dose ED treatment beyond the physiological range or prolonged ED treatment induced reduction of the myoepithelial cells. Results indicate that myoepithelial cells themselves are also altered by hormonal conditions coordinating the mammary gland development.     

Number of germ cells and somatic cells in human fetal testes during the first weeks after sex differentiation

BACKGROUND: This study presents the number of germ cells and somatic cells in human fetal testes during week 6 to week 9 post conception, i.e. the first weeks following sex differentiation of the testes. METHODS: One testis with attached mesonephros from each of 10 individual legal abortions was used. After recovery of the fetus, the testes were immediately isolated, fixed and processed for histology. The optical fractionator technique, a stereological method, was utilized to estimate the total number of germ cells in ten testes and somatic cells in six of them. RESULTS: The number of germ cells per testis increased from approximately 3000 in week 6 to approximately 30000 in week 9. The ratio of germ cells to Sertoli cells was approximately 1:11 and the ratio of germ cells to somatic cells was approximately 1:44 throughout this period. CONCLUSIONS: For the first time, germ cell and somatic cell number have been determined during early human fetal testis development. Knowledge of the number of germ cells in this period may be very important, because several environmental pollutants are suspected to result in decreased semen quality in men born of mothers exposed to these pollutants during pregnancy.     

Structure of rat ultimobranchial bodies after birth

The evolution of ultimobranchial bodies in Holtzman rats during the first 64 weeks after birth was studied by reconstructing three-dimensional models from serial sections stained by the periodic acid-Schiff technique. Radioautography with ¹²⁵I was made to see if ultimobranchial cells and/or follicular cells lining the lumen of mixed follicles were able to iodinate proteins. The term ultimobranchial body designates herein an embryonic vesicular structure (derived from the third pharyngeal pouch) whose wall is made of a stratified squamous epithelium. During the first week after birth, the vesicular ultimobranchial body elongates rapidly and becomes a canal or a duct. During the second week, cell desquamation brings about local dilatations in the lumen of these ducts; with further enlargement ultimobranchial follicles will appear. In one-day-old rats, mixed follicles are present. Only the follicular component of mixed follicles iodinates proteins as is shown by radioautography. The reconstructed models enlarge rapidly up to the 56th day after birth at which time their weight has increased nineteenfold. These same models show that the three morphological components of ultimobranchial parenchyma, namely ducts, follicles and mixed follicles, are in continuity within the thyroid parenchyma. The formation of new thyroid follicles after birth and the possibility that the ultimobranchial parenchyma may function as an endocrine gland of holocrine type are discussed.     

Division of small intensely fluorescent cells in neonatal rat superior cervical ganglion is inhibited by glucocorticoids

Postnatal genesis of small, intensely fluorescent cells was studied in the rat superior cervical ganglion by combining immunocytochemistry of tyrosine hydroxylase with tritiated thymidine auto-radiography. After injection of tritiated thymidine during the first postnatal week, silver grains were observed over the nuclei of many small cells with intense staining for tyrosine hydroxylase, suggesting that SIF cells are dividing postnatally. Cell counts in ganglia of rats sacrificed 2 h after tritiated thymidine showed that the rate of SIF cell proliferation was highest during the first postnatal week with approximately 20% of SIF cells dividing and that the rate declined thereafter. Counts of labeled SIF cells at 30 days in rats injected with tritiated thymidine on days 0, 2, 4, 6, 8, 10 or 14 revealed a peak of SIF cell birthdays on day 8. In these long-survival experiments, many labeled SIF cells were present in adult superior cervical ganglions. In contrast, only one labeled principal neuron was observed in a total of 450 sections. Glucocorticoid treatment of the rats during the first postnatal week paradoxically increased the number of SIF cells, but inhibited the rate of SIF cell proliferation. Dividing SIF cells immunoreactive for both tyrosine hydroxylase and phenylethanolamineN-methyltransferase were observed in glucocorticoid-treated rats.

These observations suggest that many SIF cells are dividing during the first postnatal week. After cessation of division, these cells either remain SIF cells or die, but do not differentiate into principal neurons. Since glucocorticoids do not stimulate SIF cell proliferation, they must increase the number of SIF cells by biasing the differentiation of precursor cells in the superior cervical ganglion and/or enhancing SIF cell survival.     

Pulmonary epithelial T cells induced by viral infection express T cell receptors alpha/beta.

Cells were recovered from the lungs of mice by bronchoalveolar lavage before and after infection with respiratory syncytial (RS) virus. Uninfected mice yielded mostly macrophages, but after RS virus infection lymphocytes also appeared. Recovered cells were stained for CD4, CD8' and either CD3, T cell receptor (TcR) alpha/beta or TcR gamma/delta and analyzed by 3-color flow cytometry. Both the CD4+CD8- and CD4-CD8+ subpopulations stained uniformly for CD3 and TcR alpha/beta, while none stained for TcR gamma/delta. Of the CD4-CD8- cells, about 5% stained for CD3 and TcR alpha/beta during the first week after infection, although this figure increased during the second week. A small and variable fraction of this subset stained for TcR gamma/delta. These results oppose the view that lymphocytes expressing TcR gamma/delta predominate in initial epithelial immune responses to viral infections.     

Development of intimal lesions after leukocyte migration into the vascular wall.

A model was developed to study the role of leukocytes in the development of vascular lesions. Implantation of an endotoxin-soaked cotton thread in the adventitia on the ventral side of the rat femoral artery resulted in leukocyte migration into the vessel wall exclusively in the ventral half of the vessel. Leukocyte migration occurred from both the luminal and adventitial side and consisted of neutrophils and mononuclear cells. Smooth muscle cell rich intimal lesions localized to the ventral half of the vessel were first observed 1 week after implantation. Lesions remained localized to the ventral half of the vessel wall through the 6th week. When leukocyte migration into the vessel wall was inhibited by treatment with dexamethasone, lesion development did not occur. These results suggest that leukocytes can stimulate smooth muscle cell migration into the intima and result in intimal lesion formation.     

Absence of muscle regeneration after implantation of a collagen matrix seeded with myoblasts

Collagens are widely used as biomaterials for e.g. soft tissue reconstruction. The present study was aimed at reconstruction of abdominal wall muscle using processed dermal sheep collagen (DSC) and myoblast seeding. Myoblasts were harvested from foetal quadriceps muscle of an inbred rat strain, cultured, seeded as non-differentiated cells into DSC-discs and incubated in vitro for 2 h. The discs were implanted in the abdominal wall defects in adult rats. Non-seeded discs functioned as control. Implantation periods till week 6 were chosen. At day 1 and 2 after implantation infiltration of granulocytes and macrophages was clearly more intense in the seeded discs than in the controls. Relatively large numbers of mast cells infiltrated from the side of the adhering omentum. In central areas of the discs, seeded cells were easily recognized till day 5, since non-seeded control discs did not contain such cells. Ingrowth of host cells and tissue at the margins proceeded faster with the seeded discs. Lymphocyte accumulations were observed in the 3 week seeded specimen. At week 3 and week 6, in the seeded discs muscle tissue was not present, in contrast to very large giant-like cells. It is concluded that the chosen method of myoblast seeding did not result in the regeneration of muscle during this observation period. Unfavorable circumstances such as humoral factors, direct cellular interactions (phagocytosis), indirect cellular interactions (cytokines), or initial absence of vascularization, may play a role. Further studies are required.     

The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. III. Cholecystokinin

Summary Immunocytochemistry was used to examine the morphology and distribution of cholecystokinin-like immunoreactive neurons in the visual cortex of the developing and mature albino rat. In the adult, labelled neurons were observed in all cortical layers but were concentrated in layers II and III. The majority of these neurons exhibited morphological features characteristic of bitufted and multipolar non-pyramidal cells described in Golgi preparations.Cholecystokinin immunoreactivity was first observed on postnatal day 3 and was confined to a few immature non-pyramidal cells located in the subplate region. The number of stained cells increased markedly during the latter part of the first postnatal week and by day 8 they were present in all cortical layers. Their morphological maturation occurred gradually during the first three weeks of postnatal life with the exception of a period of pronounced growth during the latter part of the second postnatal week.     

Histochemical Distribution of Carbonic Anhydrase After Ligation of the Pancreatic Duct

The distribution of carbonic anhydrase in the exocrine pancreas of the rat has been examined during the first 3 weeks after duct ligation. The normal pattern of positive reaction in ducts, centroacinar cells and capillaries changed, by the end of the first week, to one in which all the cells in the dilated ducts showed the enzyme. This situation persisted through the third week. By electron microscopy, the enzyme was found on the apical membranes of ductular, centroacinar and acinar cells, as well as on the surface membranes of the modified cells seen at 1 week. The significance of these findings with respect to function and cell origin is discussed.     

Differential role of granule cells in the specification of synapses between climbing fibers and cerebellar Purkinje cells in the rat

The effect of X-irradiation of the cerebellum during postnatal development on the establishment of olivo-cerebellar connections was investigated in the rat. Treatment with 950 R during the first week led to a persistance in the adult of multiple innervation of Purkinje cells (PC) by climbing fibers (CF). On the contrary, the same dose given during the second week was uneffective to prevent the establishment of the one-to-one relationship between CF and PC. These findings suggest that the regression of the multiple innervation mainly depends on early formed granule cells.     

Postnatal proliferation and maturation of olfactory bulb neurons in the rat

Mitral cells are formed prenatally whereas most granule cells originate postnatally. Material was taken from 2-day-old, 14-day-old, 28-day-old, and adult rat olfactory bulbs and processed for rapid Golgi or Cresyl Violet staining. We show that the number of granule cell bodies/mitral cell body increases from 7.0 to 46.3 during the first two weeks of life; most mitral cells appear morphologically functional during the first postnatal week; few granule cells appear to be functional until the second postnatal week; and the number of short axon interneurons increases dramatically during the second postnatal week.We conclude that newborn rats have an intact afferent pathway from olfactory receptors to primary cortex that lacks the extensive interneuronal circuitry characteristic of adults.     

The evolution of crescentic nephritis and alveolar haemorrhage following induction of autoimmunity to glomerular basement membrane in an experimental model of Goodpasture's disease

Goodpasture's, or anti-glomerular basement membrane (GBM), disease presents with rapidly progressive glomerulonephritis and lung haemorrhage, and is caused by autoimmunity to the NC1 domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). This study examines the development of crescentic nephritis and alveolar haemorrhage in a model of Goodpasture's disease, experimental autoimmune glomerulonephritis (EAG), induced in WKY rats by immunization with rat GBM in adjuvant. An increase in circulating anti-GBM antibodies and albuminuria was observed by week 2, which increased further by weeks 3 and 4, while a decrease in creatinine clearance was observed by week 2, which decreased further by weeks 3 and 4. The kidneys of animals with EAG showed linear deposits of IgG on the GBM and a transient glomerular infiltration by CD4+ T cells at week 2. By week 3 there were large deposits of fibrin in Bowman's space, and glomerular infiltration by CD8+ T cells and macrophages, accompanied by focal necrotizing glomerulonephritis with crescent formation. Ultrastructural studies showed glomerular endothelial cell swelling and epithelial cell foot process effacement at week 2. As the lesion progressed, capillary loops became occluded and the mesangium became expanded by mononuclear cells. By week 3 there was detachment of the endothelium from the GBM, and accumulation of fibrin beneath the disrupted endothelial cells and in Bowman's space. Occasional breaks were observed in the continuity of the basement membrane, and cytoplasmic projections from infiltrating mononuclear cells could be seen crossing the capillary wall between the lumen and the crescent. The lungs of animals with EAG showed patchy binding of IgG to the alveolar basement membrane (ABM) at week 2, and infiltration of the interstitium by CD8+ T cells and macrophages by weeks 3 and 4, accompanied by both interstitial and alveolar haemorrhage. Ultrastructural studies showed focal mononuclear cell infiltrates in alveolar walls at week 2. Occasional breaks were observed in the basement membrane and adjacent endothelium by weeks 3 and 4, together with accumulation of surfactant and erythrocytes within the alveolar spaces. This study defines for the first time the relationship between the immunological and pathological events during the evolution of EAG, and provides the basis for further work on the pathogenesis of Goodpasture's disease.     

Differentiation and proliferation of adrenocortical cells during the early stages of regeneration

Changes in the adrenal cortex of the rat were studied by stereologic and autoradiographic techniques during the early phases of regeneration. The adrenal cortex was enucleated on the left side, the right kidney and adrenal gland were removed, and rats were given 1% sodium chloride as drinking solution according to the procedure for inducing adrenal regeneration hypertension. The growth of adrenocortical cells during early stages of regeneration can be divided into two phases. During the first stage (0 to day 3), the cells remaining attached to the capsule recovered from trauma and began to differentiate from zona glomerulosa-like to those characteristic of the zona fasciculata. Mitochondria also divided, and there was increased surface area per cell of total mitochondrial membranes. The number of cells decreased in this initial phase, although the average cell volume increased, in preparation for mitotic division. This conclusion is supported by an increase in the percentage of cells labeled with 3H-thymidine at day 3. The second period (end of day 3 to day 7) was a proliferative phase reflected in a large number of labeled cells and a maximal number of grains per volume (reflecting activity of 3H-thymidine incorporation) followed by a blunting of the wave of proliferation at day 7. As cells divided, the total mitochondrial membranes and cristae per cell decreased. The first week after enucleation is characterized first by preparation for cell division, and in the second phase of the first week a wave of proliferation occurs. At the end of the first week, fewer labeled cells were seen, and there was a decrease in cristae membranes.     

IMMUNO-CYTOCHEMICAL STUDY ON THE DEVELOPMENT AND DIFFERENTIATION OF THE IMMUNE SYSTEM OF THE HUMAN FETUS

The lymphatic tissues of human fetus and infant were studied systematically from an immuno-cytochemical view-point. Among immunoglobulins, γA and γ M first appeared in the primitive mesenchymal cells around the mesenteric and hepatic hilar vessels at the 11th fetal week. These cells were considered to be the primordium of the gut wall lymphoid tissues; the thymus and the bursa type tissue. γM in the reticular cells and γA in the medium-sized lymphoid cells were identified in the thymus after the middle of fetal life. γG, maternal in origin, was observed in the eosinophilic cells, mostly in the thymus. After passing middle fetal life, these three types of immunoglobulins were found in the HassaU's corpuscles, increasing quantitatively toward the end of fetal life. The role of thymus in the development of the immune system during the human fetal life was discussed in relation to other lymphatic tissues.     

Immunochemical studies on the localization and on the concentration of cellular retinol-binding protein in rat liver during perinatal development

Studies were conducted to characterize the localization and the concentration of cellular retinol-binding protein (CRBP) in rat liver during perinatal development. The studies employed both a specific immunohistochemical staining procedure and a sensitive and specific radioimmunoassay for CRBP. Dramatic changes were seen in both the levels and localization of CRBP. Marked increases in hepatic CRBP levels were seen during the last week of gestation and the first postnatal week, resulting in a quadrupling of CRBP levels in this 2-week interval. During the second postnatal week CRBP levels remained very high (approximately 110 to 120 micrograms/gm of wet weight, with peak values at about day 11 of age). During the third postnatal week, CRBP levels declined, followed by a further decline to adult levels (40 to 50 micrograms/ml) postweaning. A changing pattern of immunohistochemical localization of CRBP was seen that correlated with the changes in CRBP levels. In fetal livers at days 11 to 13 of gestation, CRBP was selectively localized in perisinusoidal cells that resembled stellate (fat-storing) cells in their location and shape but that lacked the lipid droplets usually seen in these cells in adult liver. During the final week of fetal development and the first postnatal week, a progressive increase in CRBP in parenchymal cells was seen. By the second postnatal week, very strong staining for CRBP was seen in all parenchymal cells. The staining of parenchymal cells for CRBP then declined, and by weaning (day 21) the pattern of localization of CRBP in liver resembled that seen in the adult, with low to medium staining seen in parenchymal cells and strongly intense staining in perisinusoidal stellate cells. Thus, the rise and then decline in intensity of CRBP staining in parenchymal cells paralleled the pattern of changes seen in CRBP levels during the perinatal period. In the placenta, CRBP was selectively localized in the trophoblast layer of the chorioallantoic placenta and in the entodermal layer of the yolk sac placenta. These findings suggest that either retinol is metabolized or plays a functional role at these placental sites.     

Number of germ cells and somatic cells in human fetal ovaries during the first weeks after sex differentiation

BACKGROUND: This study presents the number of germ cells and somatic cells in human fetal ovaries during week 6 to week 9 post conception, i.e. the first weeks following sex differentiation of the gonads. METHODS: One ovary with attached mesonephros from each of 11 individual legal abortions was used for estimation of cell numbers. After recovery of the fetus, the ovary-mesonephric complexes were immediately isolated, fixed and processed for histology. A stereological method was utilized to estimate the total number of oogonia in all ovaries and somatic cells in seven of them. RESULTS: The number of oogonia per ovary increased from approximately 26,000 in week 6 to approximately 250,000 in week 9 and somatic cells from approximately 240,000 to approximately 1.4 x 10(6). The ratio of oogonia to somatic cells tended to increase throughout the period. The concentration of oogonia was similar in the cranial (mesonephric connected) part and the caudal part of the ovaries. CONCLUSIONS: This is the first stereological estimation of the number of oogonia and somatic cells in human fetal ovaries, and the first estimation of germ cells and somatic cells in ovaries aged <9 weeks. The number of oogonia in week 9 is comparable to the numbers previously published based on non-stereological estimations. We found early stages of meiosis in fetal ovaries from week 9.