Substance P-like immunoreactive nerve fibers in the pars distalis of the anterior pituitary in the dog.

The pars distalis of the anterior pituitary is known to be regulated by hypothalamic hormones. Recently, we have discovered the presence of substance P-like immunoreactive nerve fibers in the pars distalis of the monkeys. Substance P-like immunoreactivity in the pars distalis of the dog was investigated in this study. A substantial amount of substance P-like immunoreactive nerve fibers with a large amount of varicosities were found. They were widely distributed in the gland, more abundant along its periphery. Most of them were closely related to the glandular tissue, some were located on vascular walls. Substance P-like immunoreactive nerve fibers were also found in the meningeal sheath of the anterior pituitary. They could be followed into the parenchyma of the gland.

Iron and aluminum deposition in the meninges of the lamprey: identification of an aluminum-ferritin inclusion body.

The meningeal tissue of the brain and spinal cord of larval and juvenile adults of lampreys (Petromyzon marinus) was examined by routine electron microscopy, electron microscopic histochemistry, and electron-probe x-ray microanalysis to locate sites of iron deposition. A magnetometer was used for identification of ferromagnetic iron. Ferritin particles, representing ferric iron, are present in abundance within the cytoplasmic matrices and in dense bodies of meningeal cells of both the brain and spinal cord of larvae and juveniles. These round cells of the meninges also contain abundant glycogen and lipid. Small quantities of ferrous iron are associated to the latter inclusion. Aluminum deposits are present within an electron-dense material of many ferritin-containing inclusions of meningeal cells of the larval brain. Ferromagnetic material was not detected in larval and upstream-migrant lampreys. The deposition of iron and aluminum in the meninges of lampreys may be related to physiological and environmental factors, respectively, and/or to an important interaction between the two metals.

Preservation of thrombomodulin antigen on vascular and extravascular surfaces.

The protein C anticoagulant system is mediated by thrombin and is highly accelerated by thrombomodulin. We studied the distribution of thrombomodulin antigen (TM Ag) in the rabbit using an affinity-purified antibody raised in a goat against rabbit thrombomodulin. The preservation of TM Ag was highly dependent on immediate fixation of the surface on which it is located. TM Ag was found on the endothelium of the entire vasculature, whereas it was absent from all connective tissue, smooth and striated muscle, secretory epithelia, cartilage, bone, neural tissue, and all parenchyma examined. A new finding was the presence of TM Ag on nonvascular surfaces of body cavities (the mesothelia of pleura, pericardium, and peritoneum, the synovial membrane, and the arachnoid enveloping the central nervous system). By use of a functional assay, TM activity was recovered in buffered saline/detergent solution which was either injected into the intraperitoneal cavity of rabbits in vivo or incubated with the surface of the arachnoid in vitro. These findings extend the importance of anticoagulant mechanisms to the systems of slowly circulating fluids, in which they might be required for maintenance of the flow, and to mesothelial cavities, in which they could be necessary for preventing adherence between the surfaces, in conditions associated with pathological exudation.

Optical properties of human brain tissue, meninges, and brain tumors in the spectral range of 200 to 900 nm.

Considering the increasing use of lasers in neurosurgery and the increasing number of wavelengths of laser light becoming available, we evaluated optical properties between 200 and 900 nm of meninges, normal human brain tissue, and brain tumors. We used a two-beam spectral photometer with an integrating sphere as the measuring instrument. The material consisted of 13 brains and 1 specimen each of dura mater, falx, and arachnoid obtained at autopsy and 30 samples of brain tumors removed during operation. In tissue samples more than 5 mm thick, the relative levels of absorption and scattering were estimated from the relative level of reflection measured according to the Kubelka-Munk theory. In thin tissue slices, penetration depth was calculated according to Beer's law from measurements of reflection and transmission. Generally, in all tissues there was an increase of reflection, scattering, and penetration depth and a decrease of absorption from the ultraviolet up to the near infrared spectral range interrupted by the absorption bands of hemoglobin. Within the ultraviolet spectral range, no major differences of optical properties were observed. Within the visible and near infrared spectral range, white matter reflected most of the incident power and showed the lowest level of absorption and the shortest penetration depth. Low grade gliomas revealed optical properties similar to those of gray matter. In comparison with normal brain tissue, meningiomas and glioblastomas showed significantly higher levels of absorption calculated according to the Kubelka-Munk theory from reflection measurements in thick tissue samples, but also deeper penetration obtained from measurements of reflection and transmission in thin slices, especially within the near infrared spectral range.

The intragranular location of carboxyl groups in neuromelanin and lipofuscin in human brain and in meningeal melanosomes in mouse brain.

The intragranular location of carboxyl groups was tinctorially determined in human substantia nigra neuromelanin granules, human inferior olive lipofuscin granules, and mouse meningeal melanosomes. Soluble and insoluble lipid was stained with beta naphthol Sudans in unoxidized and oxidized frozen and paraffin sections containing neuromelanin or lipofuscin. Nile blue demonstrated carboxyls in unoxidized neuromelanin, lipofuscin, and melanin, and in oxidized neuromelanin and lipofuscin. Carbodiimide demonstrated carboxyls in unoxidized and oxidized lipofuscin and oxidized neuromelanin. In all instances, staining for carboxyls was inhibited by prior mild methylation, and proof of their presence was obtained by a pre-staining, stepwise, alternating, and repetitive mild demethylation, mild methylation sequence. Structurally, carboxyls were demonstrated in the neuromelanin granule's soluble lipid-free lipofuscin component, in the meningeal melanosome's melanin component, and virtually throughout the lipofuscin granule. The following structural and chemical basis was proposed for the different resistance of Nile blue staining of melanosomes and of neuromelanin and lipofuscin to acetone extraction. Nile blue forms an insoluble complex with melanosomal dopa-melanin's quinonoid, diphenolic, and undissociated carboxyl units. Such complex formation does not occur in neuromelanin's carboxyl-free dopamine-melanin component, however. Instead, Nile blue ionogenicly bonds with dissociated carboxyls belonging to the neuromelanin granule's lipofuscin component.

Neuronal accumulation of silver in brains of progeny from argyric rats.

Using a photochemical method silver was demonstrated in the brains of 1-, 14-, and 45-day-old rats which had been exposed to silver on gestational days 18 and 19. In the brain tissue of new-born rats, silver was found in the lysosomes of neurons and astroglia. Minor changes in the anatomic distribution of silver were observed between days 1 and 45. The pattern seen at day 45 was identical to that observed in silver-exposed adults. At day 1, the choroid plexus and meninges were heavily loaded with silver both intra- and extracellularly. In particular, macrophage-like cells of the meningeal spaces contained large amounts of silver. In these structures, the silver had virtually disappeared by day 45. Silver was always seen in basement membranes of the cerebral vessels.

An immunofluorescence microscopical study of the neurofilament triplet proteins, vimentin and glial fibrillary acidic protein within the adult rat brain.

A collection of antibodies specific to different intermediate filament proteins were applied to frozen sections of adult rat brains. The relative distribution of these proteins was then studied using double label immunofluorescence microscopy. Antibodies specific to each of the neurofilament "triplet" proteins (of approximate molecular weight 68 K, 145 K and 200 K) stained exclusively neuronal structures. The distribution of these three antigens was in general identical, except that certain neurofilament populations such as those in the dendrites and cell bodies of pyramidal cells of the hippocampus and cerebral cortex, contained relatively little if any 200 K protein. Some neurone populations, such as the granule cells of the cerebellar cortex, could not be visualized by neurofilament antibodies, indicating that neurofilaments may not be essential for function of all neurones in vitro. Antibodies to GFA and vimentin stained an entirely different population of processes, none of which stained with any of the neurofilament antibodies. Vimentin antibody stained sheath material around the brain, a monolayer of ependymal cell bodies lining the ventricles, fibrous material associated within the choroid plexus, the walls of blood vessels and capillaries, and the processes of cells in certain regions. GFA antibody stained a second layer of sheath material under the vimentin layer, and numerous processes visible throughout the brain. Some specific populations of GFA-positive processes proved to stain also with vimentin. These included the processes of Golgi "epithelial" cells (Bergmann glial fibres), those of certain astrocytes in bundles of myelinated fibers. In addition, some processes apparently derived from ependymal cells proved to stain for both vimentin and GFA, whilst other could only be reliably visualized by vimentin alone. These results are discussed in terms of the previously described morphological characteristics of the various cell types of the brain.

Brain extracts that promote the proliferation of neuroblasts from chick embryo in culture.

The effects of brain and meningeal extracts from chick embryos on the proliferation of cultured neuroblasts from chick embryo cerebral hemispheres were studied. Morphological observations, counts of the cultured neurons, determination of DNA and protein contents as well as [3H]thymidine incorporation measurements were made to evaluate the proliferative activity of the neuroblasts. We demonstrated a stimulatory effect of brain extract from 8-day-old embryos on the proliferative activity of neuroblasts from chick embryos younger than 7 days. Brain extracts from older chick embryos and meningeal extract possess less stimulatory effect.

Distribution of the LETS protein (fibronectin) in rat cerebellum. An in vitro and in vivo developmental study.

The distribution of the large, external, transformation-sensitive (LETS; fibronectin) protein was investigated in rat cerebellum, both in vitro and in vivo, by biochemical and immunocytochemical methods. Biochemical analyses indicated that LETS protein is not demonstrable on the surfaces of cerebellar neurons from postnatal rats maintained in cell culture for varying periods of time, but is present on the surfaces of at least some fraction of the total nonneuronal cell population in vitro. Indirect immunofluorescence microscopy with an anti-LETS antiserum substantiated these observations and further indicated that LETS-bearing cells of cerebellum maintained in vitro are probably of endothelial and fibroblastic origin. The LETS protein is arranged in a reticular network of filaments spanning the surfaces of the cells, and the filaments are often extensively interdigitated with each other. At all stages of development investigated (two days postnatal to adult) LETS antigen was observed in vivo to be primarily localized in the meninges covering the surface of the cerebellum and between folia, and in the walls of blood vessels within the tissue. Neuroblasts and neurons of the external and internal granule layers of the cerebellum, respectively, were negative for the presence of LETS antigen.

Circulation of cerebrospinal fluid in the bullfrog, Rana catesbiana.

In order to demonstrate features of the cerebrospinal fluid (CSF) path in the amphibian, 2--6 microliters of either Evans Blue-albumin (EBA) or ferrocyanide were injected into the ventricular system of anesthetized bullfrogs. The animals were sacrificed 1 to 135 minutes after injection by either quick freezing (EBA injections) or fixative perfusion (ferrocyanide injections). The contents of the cranial and vertebral cavities were then examined grossly and histologically for distribution of the tracers. In all animals, the tracers were seen throughout the ventricular cavity and in the subarachnoid space surrounding the caudal hindbrain. The site of communication between these two fluid spaces was the posterior tela of the hindbrain. Within this tela, "pores" were found between groups of pavement-like ependymal cells. In many animals, tracer was also observed in the vertebral subarachnoid and epidural spaces, adjacent to spinal nerve roots. In three EBA-injected animals, this tracer was also seen in the subcardinal lymph spaces. These findings suggest that the subarachnoid space in the bullfrog communicates functionally with the ventricular system by way of specialized "pores" in the posterior tela of the hindbrain. There is also indication of movement of fluid within the subarachnoid space which is predominately caudal in direction, with a primary absorptive path for CSF that consists of a perineural route to the lymphatic system.

The third circulation revisited.

The author reviews modern information concerning the formation, flow and functions of the cerebrospinal fluid. Particular attention is given to the lymphatic-like features of the third circulation and to its importance as an internal milieu for nervous tissue.