Formation of the external zone of the median eminence of rats in the perinatal period

The formation of interrelations of the axons of neurosecretory cells and of ependyme cells with the capillaries of primary portal plexus in rats from the 14th day of embryogenesis till the 9th day of postnatal life was studied using the light and electron microscope methods. During the whole period under study, the basal processes of the ependyme cells reach the primary portal plexus of the capillaries. The terminals of the basal processes are usually separated from the endothelium of the capillaries by two basal membranes and enclosed pericapillary space. After the birth, some basal process penetrate in the pericapillary space and terminate on the endothelium. The surface of contact of the ependyme cell processes with the external basal membrane increases with the age, this being accompanied by the increase of pinocytotic activity. The neurosecretory axons are found in the median eminence already on the 14th day of embryogenesis, but by the 20th day only they reach the external basal membrane and penetrate sometimes in the pericapillary space. After the birth, the number of axons reaching the external basal membrane and the surface of contact between them increase gradually with, apparently, a concomitant intensification of the transport of neurohormones in the portal circulatory system of the hypophysial-hypothalamic complex.     

Experimental approaches to the study of permeability of the blood-brain barrier for native autogenous proteins in chicken ontogenesis

The composition and distribution of serum proteins in blood, brain vesicles liquid, and nervous tissue has been studied in chicken embryos at 4-20 day of incubation using electrophoresis, immunoelectrophoresis, and fluorescent antibodies. At the 4-5 day of incubation, a difference in the protein composition of brain vesicles liquid and blood serum is apparent. This indicates that the border membrane of brain vesicles plays a part of the barrier. The vessels, preventing the transport of serum proteins to the nervous tissue, which are characteristic of hematoencephalic barrier of adult animals, develop in the embryo brain at the end of incubation period.     

Silver deposition in the central nervous system and the hematoencephalic barrier studied with the electron microscope

For the purpose of studying the hematoencephalic barrier as it is concerned with silver circulating in the blood stream, silver nitrate was vitally administered to rats in their drinking water over periods of 6 to 8 months. The cerebrum, cerebellum, medulla, area postrema, and choroid plexus were prepared for light and electron microscopy. Silver deposition was found in the perivascular spaces in the choroid plexus, area postrema, in the medulla surrounding the area postrema, and in minute quantities in the cerebrum, cerebellum, and most of the medulla. Two levels of the hematoencephalic barrier were apparently demonstrated in our investigations. The endothelial linings of the vessels in the cerebrum, cerebellum, and medulla constitute the first threshold of the hematoencephalic barrier (specifically here, blood-brain barrier). The cell membranes adjacent to the perivascular spaces form the second threshold, as follows:-the neuroglial cell membranes in the cerebrum, cerebellum, and medulla (blood-brain barrier); the membranes of the neuroglial cells in the area postrema (blood-brain barrier); and the membranes of the epithelial cells of the choroid plexus (blood-cerebrospinal fluid barrier). This study deals with silver deposition and does not infer that the penetration of ionic silver, if present in the blood stream, would necessarily be limited to the regions described. Bleb-like structures were observed to cover the epithelial cell surfaces in the choroid plexus. They may be cellular projections increasing the cell surface area or they may be secretory droplets.     

SILVER DEPOSITION IN THE CENTRAL NERVOUS SYSTEM AND THE HEMATOENCEPHALIC BARRIER STUDIED WITH THE ELECTRON MICROSCOPE

For the purpose of studying the hematoencephalic barrier as it is concerned with silver circulating in the blood stream, silver nitrate was vitally administered to rats in their drinking water over periods of 6 to 8 months. The cerebrum, cerebellum, medulla, area postrema, and choroid plexus were prepared for light and electron microscopy. Silver deposition was found in the perivascular spaces in the choroid plexus, area postrema, in the medulla surrounding the area postrema, and in minute quantities in the cerebrum, cerebellum, and most of the medulla. Two levels of the hematoencephalic barrier were apparently demonstrated in our investigations. The endothelial linings of the vessels in the cerebrum, cerebellum, and medulla constitute the first threshold of the hematoencephalic barrier (specifically here, blood-brain barrier). The cell membranes adjacent to the perivascular spaces form the second threshold, as follows:—the neuroglial cell membranes in the cerebrum, cerebellum, and medulla (blood-brain barrier); the membranes of the neuroglial cells in the area postrema (blood-brain barrier); and the membranes of the epithelial cells of the choroid plexus (blood-cerebrospinal fluid barrier). This study deals with silver deposition and does not infer that the penetration of ionic silver, if present in the blood stream, would necessarily be limited to the regions described. Bleb-like structures were observed to cover the epithelial cell surfaces in the choroid plexus. They may be cellular projections increasing the cell surface area or they may be secretory droplets.     

Changes in the subendothelial compartment during the maturation process of cerebral microvessels

Basement lamina and pericytes of growing blood microvessels were analyzed in the chick embryo optic tectum, from the 8th incubation day to hatching. Formation of the basement lamina and morphological changes of the pericytes take place in a short range of time, but late in the embryonic life, when also the blood brain barrier (bbb) devices are developing. The spatial and temporal coincidence between basement lamina formation, endothelium tight junction differentiation, and perivascular arrangement of the astrocytic glia, indicates that these events are correlated and corroborates the hypothesis that the glia needs an extracellular matrix to induce the junctional system maturation in the neural endothelia. Pericytes are irregular in shape during the early neural angiogenesis and smooth and flattened later, as the basement lamina synthesis is taking place; these cells represent a second line of barrier beyond the endothelium when the bbb is immature, owing to their phagocytic and digestive properties.     

Hematoencephalic barrier. Ultrastructure and histophysiology of the endothelium capillary of the neuronal nuclei of the mesencephalon.

The ultrastructure of the dorsal periaqueductal nucleus capillaries of the mesencephalon in the cat was studied under the electron microscope in relation to the hematoencephalic barrier, and its four structural levels: 1. Endothelium; 2. Basal membrane; 3, Pericytes; and 4. Glial prolongations. An analysis was performed of what occurs in these four components (in a non-experimental histophysiological state, and without manipulation by markers) in the thinnest capillaries of the centre of the mesencephalic neuronal nucleus. Special attention was placed on the first diffusion barrier formed by the endothelium capillary as the intimate guardian of the Central Nervous System (C.N.S.) neurons. The C.N.S. capillaries are formed from the continuous endothelium, with no fenestrations, and hermetic joining complexes, without pinocytosis vesicles on both sides of the plasmatic membrane (adluminal and external), and surrounded by a continuous basal membrane. The non-fenestrated capillaries of the C.N.S. are less permeable than those with similar characteristics located in other areas. In the C.N.S. these capillaries form a selective physiological barrier which determines the size of the molecules that are permitted to cross the capillary wall. It is suggested that the electron-dense globules found in the endothelium cytoplasm may be molecules assimilated from the blood, which might represent the first level or step to the selective diffusion entrusted to the hematoencephalic barrier. It is also suggested that the elongated electron-dense particles found in the endothelium cytoplasm and basal membrane may be macromolecules which are normally retained for an active defensive function. They would represent the first and second level or steps of the retention performed by the hematoencephalic barrier which blocks their passage to the confined space of the perivascular capillary.     

Distinct roles of JNK-1 and ERK-2 isoforms in permeability barrier repair and wound healing

c-Jun N-terminal kinases (JNKs, also called stress activated protein kinases) and the extra-cellular signal responsive kinases (ERKs) exert different functions in mitogenesis, maturation and differentiation of immune and epithelial cells. We investigated specific functions of individual JNK and ERK isoforms in skin permeability barrier repair and in wound healing. JNK1, but not JNK2 or JNK3, deficient mice revealed a delay in the permeability barrier repair after superficial injury to the skin (tape-stripping) as well as a delay in the healing of full skin thickness wounds. Skin barrier injury induced an increase in epidermal JNK1 enzyme activity in mouse skin in vivo, and JNK1 activity correlated with the degree of differentiation in organotypic keratinocyte cultures. Skin injury activated epidermal ERK2 enzyme activity with biphasic maxima after 30 min and 3h, and the activity was independent from the differentiation state in keratinocyte culture. In summary, superficial and deep wound healing depends on the differential activity of MAP kinases such as JNK1 in epidermal differentiation and ERK2 in proliferation.     

Development of the capillary system in the neurohypophysis of the rat

Summary The development of the vascular system in the neurohypophysis of the rat shows three periods of structural differentiation. The first period lasts until the 17th fetal day. During this period the superficial and the border plexuses are formed, and rapid vascularization of the neural lobe anlage takes place. The end of this period is marked by the formation of the perivascular space. The second period lasts from the 18th fetal day until the 10th day after birth. Its initial phase is characterized by the appearance of the first endothelial pores. During this period the formation of the internal capillaries proceeds, together with the maturation of the previously formed vascular elements. The third period lasts from the 10th postnatal day until the end of the first month after birth. At the end of this period the vascular net of the neural lobe has attained its fully developed state. The results of this study, together with data from previous investigations, lead to the conclusion that at the 18th fetal day the neural lobe displays signs of functional activity, and that the maturation of the vascular network in the neural lobe occurs more rapidly than the maturation of its other structural components.Research fellow of the Alexander von Humboldt Foundation.Dedicated to Prof. Dr. V. Becker, Department of Pathology, University of Erlangen, on the occasion of his 60th birthday     

AN ELECTRON MICROSCOPIC STUDY OF THE BLOOD-BRAIN BARRIER IN THE RAT, EMPLOYING SILVER NITRATE AS A VITAL STAIN

The intravital deposition of silver in the chorioid plexuses, area postrema, intercolumnar tubercle, neurohypophysis, and pineal body of rats, given 1.5 gm. of silver nitrate per liter of drinking water for periods of up to one year, has been investigated by electron microscopy. Unlike other parts of the central nervous system, these regions store large amounts of silver. In all of these structures, silver is deposited in the form of dense granules in the basement membrane upon which the capillary endothelium rests, in and upon the connective tissue cells and fibers constituting a loose pericapillary sheath, and in an outer membrane separating this sheath from the parenchymatous cells. Parts of the central nervous system which do not store silver, for example the spinal cord, cerebellar cortex, cerebral cortex, and reticular formation, lack a connective tissue investment of the capillaries. In these locations, the glial processes or end-feet are closely applied to the walls of the capillaries. Only a narrow space, filled by an amorphous, moderately electron-dense substance, separates the plasma membranes of the endothelial cells and glial processes. The significance of these observations is discussed with respect to the questions of the Virchow-Robin perivascular spaces, the interstitial ground-substance of the brain, and the location of the hematoencephalic barrier.     

Evidence for the effectiveness of the blood--CSF barrier in the fetal rat choroid plexus. A freeze-fracture and peroxidase diffusion study

The blood--cerebrospinal fluid (CSF) barrier in the choroid plexus is principally constituted of apical junctional complexes between epithelial cells. The effectiveness of this barrier was studied during the fetal development in the rat. Choroid plexuses from fetuses (14th and 18th embryonic day) and newborn (1 and 6 day old) rats were examined after intravascular administration of a proteic tracer (horseradish peroxidase) and investigated by freeze-fracture. From the 14th day of fetal life, apical junctions were seen to constitute a barrier that prevents the passage of peroxidase from blood to CSF; the tight junctions were morphologically similar to those of the mature animals; the junctional fibrils appeared continuous on complementary replicas. These data suggest that, from the 14th day of fetal development, the blood--CSF barrier is both morphologically and physiologically mature.     

Heterogeneous Maturation of Retina of the Pied Flycatcher Ficedula hypoleuca

A method is developed to determine the moment of establishment of functional maturity of cones by the appearance of their lipid droplet. Using this method, the process of differentiation of photoreceptor layer cells is traced and general regularities are elucidated of growth and maturation of the bifoveal retina of chicks of the pied flycatcher Ficedula hypoleuca. Development of retina occurs heterochronously; the first colored lipid droplets determining mature photoreceptors are photooptically revealed locally in the temporal retina area at the 5th day; at the 7th day, the lipid droplets appear within the limits of the central fovea; then the maturation wave moves from the temporal and central fovea towards periphery of retina. By the 14th day the entire area of retina turns out to be filled with mature photoreceptors. It is suggested that the predominant early maturation of photoreceptor cells of the temporal area is due to a necessity of the forestalling development of the retina parts providing the short-focus binocular vision that is the most important for establishment of chick alimentary behavior and start of hunting on their own after flight from the nest.     

The phenomenon of adaptive immunity in exposure to nonionizing microwave radiation]

In experiments with albino rats exposed to microwaves (500 microW/cm2), a model of adaptive immunity was developed by transferring lymphoid cells of exposed animals. The effect of microwave radiation was shown to cause autoimmune disorders that were displayed against the background of the structural and functional disturbances of the hematoencephalic barrier.     

Hematopoietic differentiation cell surface antigen switching in the bone marrow of different aged chickens

Abstract. Immune cytolysis and immunofluorescence were used to examine chicken fetal antigen CFA) and chicken adult antigen (CAA) expression on the differentiation/maturation series of definitive erythroid cells obtained from the bone marrow of different aged chickens. We found that erythroid cells undergo changes in CFA/CAA antigenic expression dependent on their differentiation/maturation stage as well as the developmental age of the chicken. All differentiation/maturation stages of erythroid cells in the bone marrow of 12 and 18-day-old embryos express CFA only. Erythroblasts obtained from 7-day post-hatched chickens express either CFA or CAA. All three CFA/CAA phenotypes (i.e., CFA, CAA, and CFA + CAA) are observed in subsequent maturation stages, but only the CFA + CAA phenotype is observed in mature erythroid cells in the bone marrow of 7day post-hatched chickens. Erythroblasts from 62 day post-hatched chickens exhibit all three CFA/CAA phenotypes. Cells in the subsequent maturation stages express various CFA, CAA, or CFA + CAA phenotypes resulting in a majority of the mature erythrocytes expressing both CFA and CAA, and a small population of mature erythrocytes expressing CAA only. Erythroblasts from adult chickens express both CFA and CAA; however, CFA is lost during erythroid maturation resulting in mature erythrocytes which express CAA only. These studies indicate that both the erythroid differentiation/maturation stage and the developmental age of the chicken influence CFA and CAA antigenic expression on erythroid cells undergoing cellular differentiation/maturation in the bone marrow.     

Nitric oxide mediates the effects of pulsed electromagnetic field stimulation on the osteoblast proliferation and differentiation.

The purpose of this research was to investigate whether the effects of pulsed electromagnetic field (PEMF) stimulation on the osteoblast proliferation and differentiation are mediated by the increase in the nitric oxide (NO, nitrogen monoxide) synthesis. The osteoblasts (MC3T3-E1 cell line) were cultured in the absence (-NMMA group) or in the presence (+NMMA group) of the NO synthase inhibitor L-NMMA. First, osteoblasts were subjected to PEMF stimulation (15 Hz and 0.6 mT) up to 15 days. The DNA content and the NO concentration in the conditioned medium were determined on the 3rd, 7th, and 15th days of culture. Following, osteoblasts were stimulated in the proliferation (P-NMMA and P+NMMA groups) or in the differentiation (D-NMMA and D+NMMA groups) stages of maturation, and the alkaline phosphatase (AlPase) activity was determined on the 15th day of culture for all groups. PEMF stimulation increased significantly the nitrite concentration in the -NMMA group on the 3rd, 7th, and 15th days of culture. However, this effect was partially blocked in the +NMMA group. The DNA content in the -NMMA group, but not in the +NMMA group, increased significantly on the 3rd and 7th days of culture. The AlPase activity in the P-NMMA and D-NMMA groups, but not in the P+NMMA and D+NMMA groups, also increased significantly. In conclusion, the PEMF stimulatory effects on the osteoblasts proliferation and differentiation were mediated by the increase in the NO synthesis.     

THE DEMONSTRATION OF A BLOOD-OCULAR BARRIER IN THE ALBINO RAT BY MEANS OF THE INTRAVITAM DEPOSITION OF SILVER

When silver nitrate is administered to rats in their drinking water for many months, they develop a generalized argyria. In the central nervous system, the deposition of silver follows the pattern of the so called hematoencephalic barrier (Wislocki and Leduc, (2); Dempsey and Wislocki, (3)). The present observations concern the deposition of silver in the rat's eye, investigated by both light microscopy and the electron microscope. In the eye, silver is not detected in the specific neural elements of the retina. Instead, it is heavily deposited in the basement membrane of the epithelium of the ciliary processes and in Bruch's basal membrane between the choriocapillary layer and the retinal epithelium. Traces of silver are visible in the basement membranes of the retinal capillaries with the electron microscope, but cannot be identified with the light microscope. In all of these respects, the pattern of the silver resembles the mode of its deposition in the brain. The heavy accumulation of metal in Bruch's membrane and the ciliary processes is analogous to that observed in the chorioid plexuses, and the traces encountered in the walls of the retinal capillaries correspond to traces observed in the basement membranes of the cerebral capillaries. Hence, with respect to silver, the eye possesses a blood-ocular barrier similar to the hematoencephalic barrier. Silver appears to be restrained from entering the aqueous humor by a barrier in the basement membrane of the ciliary processes, from reaching the photoreceptor elements of the retina by Bruch's basal membrane, and from penetrating the inner layers of the retina by a barrier in the basement membrane surrounding the retinal capillaries.     

Developmental Changes in the Free Amino Acid Pool of Rice(Oryza sativa L. subsp,japonica) Bmbryos

The level of/various amino acids in rice embryos rose sharply during embryo differentiation (7–9 days after anthesis). Then it increased steadily or tended to become stable at mid-maturation stage (13–18 days after anthesis), thereafter continuing to increase, except that the contents of aspartate and glutamate decreased significantly. The total free amino acid pool expanded rapidly during the differential stage. There after the pool capacity showed only a slightly increase until the end of embryogenesis. Both on embryo cell and dry weight bases, the capacity reached the maximum at the 9th day after anthesis, then decreased at the 13th day, and later remained stable. We deemed that the establishment of the free amino acid pool is one of the events which occur in the process of rice embryo differentiation. By the fulfillment of the differentiation (the 13th dray after anthesis), the pool capacity within the embryo cells remained stable on the whole. The free amino acid pool was dominated by serine, alanine, aspartate and glutamate during the differentiation stage. In the maturation stage, serine, alanine, arginine and lysine were the main components. These predominant; amino acids may play an important role in regulating the availability of the whole amino acid pool.     

Dynamics of the changes in the content of testosterone and estradiol receptors in the hypothalamus of male rats in the course of sexual development

The content of receptors to testosterone and estradiol in hypothalamus of the male rats was studied during their sexual maturation (7, 14, 21, 28, 35 and 42 days). In all the age groups of animals the concentration of receptors to testosterone in the cytoplasmic and nuclear fractions of hypothalamus was at a relatively constant level, except in 7 day old males in which the minimal concentration of cytoplasmic and the maximal concentration of nuclear receptors were noted. The highest values of estradiol-binding sites in cytosol of hypothalamus were observed on the 7th and 14th days and in the nuclear fraction on the 28th, 35th and 42nd days of life. The binding of both the hormones with their receptors is a specific process characterized by a high affinity. A suggestion is put forward that receptors both to androgens and estrogens take part in the brain sexual differentiation.     

Histochemical features of the formation of the protective barrier of the stomach in various periods of ontogenesis of mammals

By means of a complex of histochemical methods it has been demonstrated that cytochemical differentiation of the tegmental epitheliocytes and secretory cells of the fundal glands takes place in different time. In Carnivores the tegmental epitheliocytes complete their cytochemical differentiation during the prenatal period, mucocytes--to the time of birth, and chief exocrinocytes--to the period of mixed feeding. In phytophagous animals formation of the protective barrier in the stomach occurs differently: in the mouse and hamster the cytochemical differentiation of the tegmental and glandular epitheliocytes is completed during the prenatal period, and in the rabbit and guinea pig--only by the 30th day after birth.     

Neuronal maturation in an experimental model of brain tissue heterotopia in the lung

Neural maturation involves diverse interaction and signaling mechanisms that are essential to the development of the nervous system. However, little is known about the development of neurons in heterotopic brain tissue in the lung, a rare abnormality observed in malformed babies and fetuses. The aim of this study was to identify the neurons and to investigate their maturation in experimental brain tissue heterotopia during fetal and neonatal periods. The fetuses from 24 pregnant female Swiss mice were used to induce brain tissue heterotopia on the 15th gestational day. Briefly, the brain of one fetus of each dam was extracted, disaggregated, and injected into the right hemithorax of siblings. Six of these fetuses with pulmonary brain tissue implantation were collected on the 18th gestational day (group E18), and six others were collected on the 8th postnatal day (group P8). The brain of each fetus from dams not submitted to any experimental procedure was collected on the 18th gestational day (group CE18) and on the 8th postnatal day (group CP8) to serve as a control for neuronal quantitation and maturation. Immunohistochemical staining of NeuN was used to assess neuron quantity and maturation. The NeuN labeling index was greater in the postnatal period than in the fetal period for the experimental and control groups (P8 > E18 and CP8 > CE18), although there were fewer neurons in experimental than in control groups (P8 < CP8 and E18 < CE18) (P < 0.005). These results indicate that fetal neuroblasts/neurons not only survive a dramatic event such as mechanical disaggregation, in the same way as it happens in human cases, but also they retain their development in heterotopia, irrespective of local tissue influences.     

Ultrastructural differentiation and enzymatic localization of phosphatases in the developing duodenal epithelium of the mouse

Summary The localization of acid and alkaline phosphatase activities during the morphogenesis of the duodenum has been studied at the ultrastructural level in BALB/c mice. The present work deals with the foetal development from the 14th day till birth. It appears that acid phosphatase activity at the 14th day of foetal life, was situated in dense bodies at the base of the intestinal cells. At that moment, the flat apical membrane of the cells lining the intestinal lumen presented a faint alkaline phosphatase activity. With the maturation of the villus cells, the dense bodies migrated to a supra-nuclear zone, and on the 20th day, differentiated into a complex network of dense bodies and dense tubules showing the activity of the two phosphatases. On the other hand, the microvilli were developing from the 16th day, and active pinocytosis appeared on the 18th day. Alkaline phosphatase activity increased rapidly during this differentiation.This study was supported by a research grant no. N.D.G./27 from the Medical Research Council of Canada.The authors are indebted to Mr. Michel Couture for his assistance with the photographs.