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     

Effects of hormone-releasing stimuli on the area of the perivascular space in the neural lobe of the rat

Summary Neural lobes from rats subjected to neurohypophysial hormone-releasing stimuli were examined electron microscopically following fixation in 4 % tannic acid in 2.5 % glutaraldehyde. This fixation allowed the delineation of the perivascular space in the neural lobe tissue. Measurement of the area of the perivascular space showed that it was significantly increased in the rats subjected to vagal stimulation and intraarterial calcium ions compared to the control rats. The rats which had been subjected to haemorrhage as a hormonereleasing stimulus did not show any significant change in the area of the perivascular space. The significance of these findings in relation to hormone release is discussed.     

On the fine structure of the perivascular cells in the neural lobe of rats

Summary The purpose of this study is to investigate the ultrastructural features of perivascular cells as found in the neural lobe of the hypophysis. In particular, an inquiry was made into the nature of the relationship of such cells to neurosecretory fibers and endings. The latter, in fact, are often invaginated within the perivascular cells and enveloped by their processes; furthermore, they often reveal a certain number of empty granules as well as characteristics of degenerative nature. In the course of this study the localization of the perivascular cells has been investigated as well as that of their processes within the extensive interlobular network typical of the hypophysial neural lobe of rodents. Based on the data gathered, the hypothesis is put forward that the perivascular cells play an important role in the turnover of neurosecretory endings, both under physiological and experimental conditions, contributing thereby to the release of post-hypophysial hormones.With the technical assistance of Vincenzo Panetta.     

Immunocytochemical localization of substance P in the neurohypophysis and hypothalamus of the mouse compared with the distribution of other neuropeptides

Summary Substance-P immunoreactivity has been located in semithin sections of mouse hypothalami and pituitaries and compared with the distribution of other hypothalamic peptides. In the mouse, nerve fibres and terminals reacting with antibodies against substance P (SP) were detected both in the external zone of the median eminence (ME) and in the neural lobe of the pituitary. Immunoreactive SP (ISP) axons of the ME did not react with antibodies against other peptides, i.e. arginine-vasopressin (AVP), oxytocin (OT), somatostatin and enkephalin, and were also negative with an antibody to serotonin. In the neural lobe, SP immunostaining occurred in AVP but not in OT axons. In the hypothalamus, ISP axons were widespread but conspicuously lacking in areas containing AVP neurones, i. e. in the suprachiasmatic nucleus and the clusters of AVP cells in the SO and PV nuclei. In contrast, multiple ISP endings were seen in contact with OT neurones. Immunoreactive cell bodies, only detected after colchicine treatment, belonged to two distinct classes of neurones: 1) single AVP neurones of the SO and PV nuclei; 2) specific (staining only for SP) neurones, scattered or grouped in different areas of the hypothalamus, not showing relationship with any circumscribed nucleus. These results reinforce the opinion that SP can be released as a neurohormone into the vascular portal system and can directly affect the pars distalis. The presence of immunoreactive SP in the neural lobe, which has not been reported in species other than the mouse, may have a different physiological significance.     

Perivascular regions of the rat neural lobe

Summary Neural lobes and portions of occipital cortex from rats were examined electron microscopically following fixation in 4% tannic acid in 2.5% glutaraldehyde. The procedure allowed a clear demonstration of the perivascular space and intercellular spaces in both tissues. The perivascular spaces in the neurosecretory tissue was far more extensive than in the neural tissue and the role of this region in relation to the process of neurosecretion is discussed.     

Ultrastructural changes in the neurohypophysis of the aged male rat

Summary The neurohypophyses (neural lobes of the pituitary) of young adult (3–6 months) and aged (12–30 months) male rats were studied by means of electron microscopy. Stereomorphometric analyses were performed to determine the size, number and relative volume of the hormone-containing vesicles. The principal observations included: 1) the conspicuous depletion in aged rats of the granular hormone-containing vesicles from the axon terminals and the Herring bodies, with a decrease in the relative volume fraction of the vesicles from 4.8 Vv % in the control animals to 1.1 Vv % in the aged rats; 2) a change in the size-distribution of the hormone vesicles; 3) an increase in the extracellular space around the nerve terminals, axons and capillaries; and 4) lipid accumulations and signs of activation in the pituicytes. The possible physiological significance of the findings is discussed in the light of several regulatory functions known to be altered during the process of ageing.     

Specific immunoelectronmicroscopic localization of vasopressin and oxytocin in the neurohypophysis of the rat

Summary An immunoelectronmicroscopic method for the specific localization of neurohypophyseal hormones was developed in neurohypophyses of Wistar and Brattleboro rats, the latter strain being homozygous for diabetes insipidus. If the proper precautions were omitted, a marked cross reactivity between antivasopressin and antioxytocin preparations was found. Cross reaction of an antivasopressin plasma with oxytocin, at a dilution of less than 11600, resulted in electron density of all granules within neurosecretory fibres of the Brattleboro and Wistar neurohypophyses. However, this cross reactivity could be eliminated either by sufficient dilution of the antiplasma, or by its purification. Purification of the antibodies was performed by absorption to agarose beads coated with the cross reacting component. Upon incubation with antivasopressin (diluted unpurified 11600 or purified 180) and unpurified antioxytocin (1400) plasma, sections of a Wistar neurohypophysis revealed two types of neurosecretory fibres, containing either electron dense or lucent granules. Oxytocin and vasopressin containing neurosecretory fibres were found as clusters in the neurohypophysis. The specificity of both unpurified antivasopressin (11600) and antioxytocin (1400) plasma was confirmed on serial sections of a Wistar neurohypophysis, alternately incubated with the solutions of the two antibodies.These data prove that the one-cell-one-hormone hypothesis holds true for the hypothalamic-neurohypophyseal system.The authors wish to thank Dr. L.A. Sternberger (Edgewood Arsenal, Md., U.S.A.) for the peroxidase-anti-peroxidase complex, Dr. J.G. Streefkerk (Free University, Amsterdam) and the members of our project group on neuroendocrinology for their suggestions and critical remarks, and Mrs. M. Mud, Mr. P. Wolters and Mrs. A. van der Velden for their skilful assistance     

The fine structure of the neural lobe of the mouse after transplantation under the kidney capsule

Summary The neurohypophysis of donor mice was implanted under the renal capsule of the recipients. The pituicytes survived while the neurosecretory axons disappeared. The ultrastructure of the glial cells was observed seven and nine weeks after transplantation. There were no signs of phagocytotic activity although remnants of axons were still present at seven weeks. The numerous processes of the pituicytes form a network with intercellular spaces wide in younger and narrower in older implants. The cells are connected by desmosomes and gap junctions. Pituicytes as well as blood vessels preserve their organotypic appearance. The transplant thus represents an experimental model for investigations on pituicytes in vivo in the absence of neurosecretory axons.Fellow of the Alexander von Humboldt-Stiftung. On leave from Department of Histology and Embryology, Institute of Biostructure, Academy of Medicine, Pozna, Poland     

Compartments and perivascular arrangement of the meninges covering the cerebral cortex of the rat

Summary The intercellular clefts of the brain and the leptomeninges, and the perivascular spaces were studied with reference to the results obtained in a previous study (Krisch et al. 1983). The spatial relationships of these compartments were analyzed at the electron-microscopic level. Horse-radish peroxidase (HRP) was injected into the brain or into the contralateral ventricle.The pattern of distribution of HRP depends on the boundary situation in the individual compartments. The inner and outer pial layers accompany the vessels intruding into the brain. In the Virchow-Robin space the pial funnel obliterates within a short distance. The inner arachnoid layer is continuous with the outer arachnoid layer when it covers the vessels traversing the meningeal space. The perivascular compartment is not in communication with the arachnoid space; moreover, the pial funnel within the Virchow-Robin space is sealed off against the arachnoid space.Thus, blood vessels traversing the meningeal spaces and subsequently penetrating the brain surface are exposed to the common intercellular compartment represented by the intercellular clefts of the brain and the leptomeninges; this compartment does not communicate with the other compartments. The cerebrospinal fluid located in this intercellular compartment is preferentially drained into the upper cervical lymph nodes.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/5) and the Stiftung Volkswagenwerk.     

Isolated Anxa5+/Sca-1+ perivascular cells from mouse meningeal vasculature retain their perivascular phenotype in vitro and in vivo

Pericytes are closely associated with endothelial cells, contribute to vascular stability and represent a potential source of mesenchymal progenitor cells. Using the specifically expressed annexin A5-LacZ fusion gene (Anxa5-LacZ), it became possible to isolate perivascular cells (PVC) from mouse tissues. These cells proliferate and can be cultured without undergoing senescence for multiple passages. PVC display phenotypic characteristics of pericytes, as they express pericyte-specific markers (NG2-proteoglycan, desmin, alphaSMA, PDGFR-beta). They also express stem cell marker Sca-1, whereas endothelial (PECAM), hematopoietic (CD45) or myeloid (F4/80, CD11b) lineage markers are not detectable. These characteristics are in common with the pericyte-like cell line 10T1/2. PVC also display a phagocytoic activity higher than 10T1/2 cells. During coculture with endothelial cells both cell types stimulate angiogenic processes indicated by an increased expression of PECAM in endothelial cells and specific deposition of basement membrane proteins. PVC show a significantly increased induction of endothelial specific PECAM expression compared to 10T1/2 cells. Accordingly, in vivo grafts of PVC aggregates onto chorioallantoic membranes of quail embryos recruit endothelial cells, get highly vascularized and deposit basement membrane components. These data demonstrate that isolated Anxa5-LacZ(+) PVC from mouse meninges retain their capacity for differentiation to pericyte-like cells and contribute to angiogenic processes.     

Histology and ultrastructure of the neurohypophysis of the south american lungfish,Lepidosiren paradoxa

Summary The neurohypophysis of the South American lungfish Lepidosiren paradoxa has been studied with light and electron microscopy, including the Falck-Hillarp technique for catecholamines. The pars nervosa hypophyseos is a well-marked, dorsally located subdivision of the pituitary gland composed of lobes or follicles, each one constituted of a central core of ependymal cells, a subependymal hilar region made up of nerve fibers and a peripheric palisade zone of nerve endings which contact capillary vessels. Four types of neurosecretory axons can be distinguished under the electron microscope. Type I, the most common, contains spherical elementary granules of high electron density, 1500–1800 Å in diameter. The scarce type II axons contain irregularly-shaped elementary granules. Type III contains only small clear vesicles, 400–600 Å in diameter. Type IV, mostly present in regions of the gland contacting the pars intermedia, contain large granulated vesicles, 900–1000 Å in diameter. The Falck-Hillarp technique revealed axons with a positive reaction for catecholamines at sites corresponding approximately to the location of type IV of the electron microscope.Ependymal cells are of large size, linking the cerebrospinal fluid, the nerve endings and the blood vessels. A conspicuous membrane-bound, spherical dense material, 1400–2000 Å in diameter, is observed in both the apical and vascular processes of these cells. The ependymal processes which traverse the hilar and palisade regions contain structures resembling degenerated neurosecretory axons. These results are discussed in relation with the currently available information on the comparative anatomy of the pars nervosa. The possible functional significance of ependymal cells and of each type of axon are also discussed.This study was aided by the following grants: NIH NS 06953 to Prof. De Robertis, Consejo Nacional de Investigaciones Científicas y Técnicas to Prof. Zambrano, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires and Comisión de Investigaciones Cientificas de la Universidad Nacional de la Plata: to Prof. Iturriza.The authors are indebted to Prof. De Robertis for his generosity in granting us his laboratory facilities, and to Dr. F. J. J. Risso and Mr. A. Fernández (Resistencia, Chaco) who provided the specimens used in this study. The able microtechnical assistance of Miss L. Riboldazzi and Mrs. R. Raña and the photographic work of Mr. A. Saenz are much appreciated.Members of the Scientific Career, Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

Elementary granules,small vesicles and exocytosis in the rat neurohypophysis after acute haemorrhage

Summary Neural lobes of rats subjected to severe acute haemorrhage under sodium pentobarbitone anaesthesia were examined electron microscopically and the ultrastructure compared with that in anaesthetised and unanaesthetised controls. Changes in the localisation and numerical distribution of elementary granules and small vesicles in the neurohypophysial nerve endings of bled rats were consistent with the occurrence of exocytosis. The occurrence of exocytotic profiles was observed more frequently in freeze-etched tissue samples as compared with the material fixed for conventional electron microscopy. The ratio of small vesicles: elementary granules was shown to be significantly increased (P<0.005) in the nerve endings of neural lobes from bled rats. Equally, the numbers of exocytotic profiles related to 1000 m² of neurohypophysial tissue area were significantly greater (P<0.005) in bled rats.The study was supported by Medical Research Council of Canada. The authors are grateful to Dr. W. Costerton, Biology Department, The University of Calgary, for use of facilities for freeze-etching, and to Miss Y. Carter for technical assistance.Research Associate, Consejo Nacional de Investigationes Cientificas y Tecnicas, Argentina.Associate, Medical Research Council of Canada.     

Immunocytochemical localization of LHRH in the median eminence,infundibular stalk,and neurohypophysis

Summary The distribution of luteinizing hormone-releasing hormone (LHRH) was studied by light-microscopic immunocytochemistry in the hypothalamo-pituitary complex of humans, monkeys, ferrets, bats, and rats. LHRH-immunoreactive fibers were identified in the median eminence of all these species, but the precise location of these fibers varied. In rats, the vast majority of LHRH fibers in the median eminence was confined to the external zone. In contrast, in bats, most of the LHRH fibers were located in the internal zone. While these two species represent opposite extremes in distribution of LHRH fibers within the median eminence, intermediate conditions were found in humans, monkeys, and ferrets, as considerable numbers of fibers occurred in both internal and external zones. In addition to fibers in the median eminence, large numbers of LHRH-immunoreactive fibers were identified traversing the infundibular stalk and entering the neural lobe of the pituitary in all species examined except the rat. In rats, only occasional fibers were observed in the infundibular stalk, and they did not project into the neural lobe. However, in humans, monkeys, ferrets, and bats, groups of LHRH-immunoreactive fibers extended well into the substance of the posterior pituitary. Most of these fibers appeared to terminate near the adenohypophysis, but others coursed away from the anterior lobe and penetrated deeper portions of the neural lobe. These observations, made in several mammalian species, indicate that multiple routes may exist in the median eminence/stalk/pituitary complex for the delivery of LHRH to the anterior pituitary.     

Alterations in the neurohypophysis of rats treated with chlorphentermine or tricyclic antidepressants

Summary Rats were treated with several amphiphilic, cationic compounds that are known to cause generalized lipidosis (chlorphentermine, iprindole, 1-chloro-amitriptyline, clomipramine). After prolonged drug treatment the neurohypophysis showed severe morphologic alterations particularly in Herring bodies (HB), perivascular cells, and pituicytes. HBs displayed the following abnormalities: (a) great accumulation of autophagic vacuoles that contained neurosecretory granules (NSG); (b) numerous coarse osmiophilic conglomerates; (c) masses of multilamellated material; (d) reduced numbers of intact NSGs. Perivascular cells accumulated large lamellated inclusion bodies. Pituicytes contained membrane-bound crystalloid inclusion bodies. The noxious effect of chlorphentermine and 1-chloro-amitriptyline was more pronounced than that of iprindole and clomipramine.The alterations in perivascular cells and in pituicytes are typical of drug-induced lipidosis. The lesions in HBs are tentatively explained as follows: HBs were previously proposed to be the sites of normally occurring intraaxonal disposal of excess neurosecretory material. The present experimental conditions interfere with this catabolic process. Incomplete digestion of the axoplasmic constituents due for disposal might result in abnormal accumulation of NSG-containing autophagic vacuoles, osmiophilic conglomerates, and multilamellated material. This eventually leads to degeneration of HBs.The functional implications of the neurohypophysial lesions remain to be elucidated by functional experiments.This work was supported by the Deutsche Forschungsgemeinschaft (Lu 172/2) and the Stiftung Volkswagenwerk. The skilful technical assistance of Mrs. R. Worm is thankfully acknowledged     

The effects of variation in the arterial pulse waveform on perivascular flow

Cerebrospinal fluid (CSF) enters nervous tissues through perivascular spaces. Flow through these pathways is important for solute transport and to prevent fluid accumulation. Syringomyelia is commonly associated with subarachnoid space obstructions such as Chiari I malformation. However, the mechanism of development of these fluid-filled cavities is unclear. Studies have suggested that changes in the arterial and CSF pressures could alter normal perivascular flow. This study uses an idealised model of the perivascular space to investigate how variation in the arterial pulse influences fluid flow. The model used simulated subarachnoid pressures from healthy controls (N = 9), Chiari patients with (N = 7) and without (N = 8) syringomyelia. A parametric analysis was conducted to determine how features of the arterial pulse altered flow. The features of interest included: the timing and magnitude of the peak displacement, and the area under the curve in the phases of uptake and decline. A secondary aim was to determine if the previously observed differences between subject groups were sensitive to variation in the arterial pulse wave. The study demonstrated that the Chiari patients without a syrinx maintained a significantly higher level of perivascular inflow over a physiologically likely range of pulse wave shapes. The analysis also suggested that age-related changes in the arterial pulse (i.e. increased late systolic pulse amplitude and faster diastolic decay), could increase resistance to perivascular inflow affecting solute transport.     

Extracellular perivascular connective tissue space in the medial terminal nucleus of the accessory optic system in rats

Summary Two types of capillaries were found in the medial terminal nucleus of the rat accessory optic system. Type one capillaries are surrounded by glial processes and lack a perivascular space, whereas the type two capillaries and arterioles show a distinct extracellular perivascular space often filled with collagen fibrils. An internal as well as an external basal lamina lines these spaces. No fenestration of the endothelium was observed.This study was supported by N. I. H. Grant RR-00165 to Yerkes Regional Primate Research Center, and N. I. H. Grants R01-EY 00565-03 and R01-EY 00638-02 to J. Tigges. The excellent technical assistance of Mrs. G. L. Luttrell is gratefully acknowledged. We thank Dr. W. K. O'Steen for providing the rats and Mr. F. H. Kiernan for photographic help. Special thanks are due to Mrs. B. A. Olberding, in charge of the maintenance of the electron microscope.     

The microvascular pattern and perivascular linings of the area postrema

Summary The microvasculature and perivascular linings of the area postrema (A.P.) were studied electron microscopically with the ultrathin section and freeze-etching techniques. Special attention was given to the intercellular contacts of the different cellular entities. Two types of microvascular segments were identified. The endothelium of these vascular segments reveals fenestrations and a high pinocytotic activity. There are no significant differences in the frequency and distribution of the endothelial openings between both types of capillaries. The endothelium of the blood vessels, however, is joined by different types of tight junctions. Focal tight junctions occur between pericytes and the endothelium, and between leptomeningeal cellular elements in the perivascular space. The cell membrane of the perivascular glia shows intramembrane particles which are either distributed at random or organized in the form of membrane-associated orthogonal particle complexes (MOPC, Dermietzel, 1974). The significance of these findings is discussed with respect to the modified blood-brain barrier mechanism in the A.P.Supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 114 Bionach) to R. DermietzelThe authors are indebted to Mrs. D. Schünke and Mr. R. Eichner for technical assistance and Mr. A. Stapper for preparing the diagram     

Intralobular lymphatic vessels and their relationship to blood vessels in the mouse thymus

Summary The spatial distribution and fine structure of the lymphatic vessels within the thymic lobules of normal and hydrocortisone-injected mice were studied by light- and electron microscopy. The lymphatic vessels of the cortex and medulla of normal thymus are irregularly shaped spaces closely associated with branches of the intralobular artery and vein. The overall distribution of these vessels in the greatly involuted thymus of hydrocortisone-treated mice is essentially the same as in the normal thymus. The wall of the lymphatic vessels consists of only a layer of endothelial cells supported by underlying reticular cells. The luminal surface of the endothelial cell is smooth, but trabecular processes are often seen. There are three morphological types of intercellular contacts between contiguous cells, namely, end-to-end, overlapping and interdigitating. The lymphatic vessel has anchoring filaments and collagen fibrils, but a basal lamina is either absent, or if present, is discontinuous. This is in contrast to the continuous basal lamina of the venule. The perivascular space surrounding the postcapillary venule opens into a terminal lymphatic vessel at the cortico-medullary junction and in the medulla. Lymphocytes are seen penetrating the lymphatic endothelium, particularly in acutely involuted thymuses. These findings suggest that the intralobular lymphatic vessels may originate from the vacuities that surround the postcapillary venules, and the lymphatic system may function as a pathway for the migration of lymphocytes into or out of the lymphatic circulation.     

Corticotrophic cells in the rostral zone of the pars intermedia and in the adjacent neurohypophysis of the rat and mouse

Summary In the mouse, the rostral zone of the pars intermedia is almost exclusively composed of typical corticotrophic cells. They are located around and even within the neural stalk, at the level of transition between stalk and neural lobe. In the rat, the corticotrophic cells of the rostral zone are found in scattered islets among the MSH producing cells, and also in the neural lobe. In both the rat and mouse, these cells are in direct contact with various types of nerve terminals. Synaptoid contacts with aminergic and neurosecretory nerve fibers are observed. Furthermore they are also closely related to the hypophysial portal vessels. Following adrenalectomy, the cells located in the neurohypophysis always react more intensely than tose in the rostral zone. The functional significance of these corticotrophic cells which are subject to both humoral and neural regulation remains as yet hypothetical. Their participation in neurogenic stress response seems probable.     

Specific arrangements of membrane particles at sites of exo-endocytosis in the freeze-etched neurohypophysis

Summary Images have been obtained from freeze-etch replicas of neurohypophyses which are consistent with the view that orderly arranged aggregates of membrane particles occur in regions where fragments of membrane are being added to and taken away from the plasma membrane during secretion. Aggregates of particles included rosette-like and necklace-like patterns similar to those described by other authors at sites of exocytosis and endocytosis.Dedicated to Prof. W. Bargmann at the occasion of his 70th birthday. A short account has been presented at the 8th International Congress of Electron Microscopy (Dreifuss, Akert, Sandri and Moor, 1974).This work was supported by grants from the Swiss National Foundation for Scientific Research Nos. 3.257.74, 3.368.0.74, 3.774.72 and 3.045.73; the Hartmann-Müller Foundation for Medical Research in Zürich and the Dr. Eric Slack-Gyr Stiftung.