Distribution of glial fibrillary acidic protein in central nervous system lesions of tuberous sclerosis

The distribution of glial fibrillary acidic protein (GFAP) in the central nervous system (CNS) lesions of tuberous sclerosis (TS) was examined using antiserum against GFAP and the peroxidase antiperoxidase method of Sternberger. In cortical tubers there were islands of gemistocytic astrocytes staining intensely for GFAP and occasional giant cells having some cytoplasmic staining. The majority of the cortical giant cells had no GFAP. The islands were separated by areas devoid of astrocytes with perikaryal staining. A faintly staining fibrous network was found between these islands. The majority of cells in the subependymal nodules stained. The retinal phakoma stained but not as intensely as the subependymal nodules. There was no staining whatsoever in the giant cell subependymal tumors. Absence of GFAP staining in the subependymal giant cell tumors makes their classification as astrocytomas less certain.     

Ionic and haemodynamic changes influence the release of the excitatory amino acid glutamate in the posterior hypothalamus

Argyrophilic and tau-positive abnormal structures occurring in glial cells are called glial fibrillary tangles. In the astrocyte, a conspicuous tau-positive structure is known to appear in progressive supranuclear palsy (PSP). In this report, another type of argyrophilic and tau-positive astrocytes is reported. The morphology of this new type is quite different from that of the previously reported tau-positive astrocyte in PSP and they are designated here as thorn-shaped astrocytes (TSA). TSA have an apparently argyrophilic cytoplasm with a few short processes and often have a small eccentric nucleus, whose appearance resembles that of a reactive astrocyte. Immunohistochemically, TSA are positive to anti-tau antibodies but are negative for ubiquitin. Simultaneous immunostaining revealed the coexistence of tau and glial fibrillary acidic protein epitopes in the same cytoplasm. Electron microscopically, bundles of 15-nm straight tubules were included in the cytoplasm together with abundant glial filaments. In the vicinity of a cluster of TSA, related structures of perivascular or subpial tau-positive linings, which correspond to astrocytic end-feet, are sometimes observed. In almost all cases, a few TSA are generally located in a confined area of subpial and subependymal regions. Although TSA appear to be intimately associated with some diseases, they are also found in a wide range of cytoskeletal disorders including the aged brain with neurofibrillary tangles. TSA are presumed to be a secondary induced product in relation to astrocytic reaction.     

Developmental qualitative and quantitative aspects of the subependymal layer from the caudato-pallial angel of the rat lateral ventricles

We have studied by both qualitative and quantitative methods the subependymal cells in the caudato-pallial angle (CPA) of the lateral ventricle from 1, 2, 6 and 18 month-old rats. We have chosen the CPA, among other zones, because it is an easily delimited glial proliferative zone. The qualitative analysis was carried out using both Klüver-Barrera and Luxol-Fast-Blue stains. The quantitative study was carried out by a semiautomatized image analysis system. Three types of subependymal nuclei were classified by Klüver-Barrera stain, according to the following parameters: staining intensity, morphology and size. Each of the three type groups was found at different frequency depending of rat age. Luxol-Fast-Blue stained mitotic images in metaphase or anaphase: n = 38 in the 1 month, n = 49 in 2 months, n = 12 in the 6 months, and n = 5 in the 18 month-old rats. The quantitative analysis (length, areas and volume of CPA) were in agreement with the above data, showing a more subependymal activity in the 2 month-old animals than in the 1, 6 and 18 month-old rats.     

Diabetic pigmented spots on the extrapretibial region: immunohistochemical study of type IV collagen in dermal vessel wall

A case of diabetes mellitus associated with multiple pigment spots is reported. The patient had multiple pigmented atrophic patches on the abdominal area and thighs. There were no pigmented spots on the pretibial area. Histologically, dermal vessels showed intimal thickening and deposition of periodic acid-Schiff (PAS)-positive fibrillar material in vessel walls. Clinical and histological features indicated that these pigment spots were diabetic pigmented pretibial patches occurring in the extrapretibial area. Immunohistochemical studies of lesional and non-lesional skin using the antibodies for alpha1-alpha6 chains of type IV collagen revealed that PAS-positive material of vessel walls in both lesional and non-lesional skin was stained with alpha1 and alpha2 chain antibodies but not with alpha3-alpha6 chain antibodies, indicating that alpha1 and alpha2 chains of type IV collagen, which are normal components of the dermal vessel basement membrane, accumulate in the vessel walls in diabetes mellitus.     

Diabetic cardiopathy. Quantitative histological studies of the heart from young juvenile diabetics

A quantitative morphological study, at light microscopical level, of hearts from young diabetics and nondiabetics was performed. The groups were comparable with respect to sex-ratio, age, heart weight and blood pressure. Findings in the hearts were as follows: In arterioles in which the luminal diameter ranged between 15-50 mu, a strongly PAS-positive stained vessel wall occurred at a higher frequency among diabetics than among non-diabetics (75 per cent respectively 33 per cent). The PAS-positive structures, however, occupied the same area of the vessel wall in the two groups. In arterioles from diabetics, the number of cells in tunica media was increased as compared with that in non-diabetics (2 p less than 0.01). The amount of perivascular connective tissue was also increased in the diabetics (2 p less than 0.01). There was no indication of an endothelial cell proliferation in the PAS-positive stained vessels from diabetics and non-diabetics. The wall of the capillaries was not thickened and it was not more PAS-positive in the hearts from diabetics than in those from non-diabetics. Moreover, the number of capillaries per square millimeter of heart muscle was the same in the two groups of hearts. In the present light microscopical study, the diabetic micro-angiography of the heart was demonstrable in the arterioles. The capillaropathy known to occur in other organs was not present in the heart muscle from patients with diabetes of long standing.     

Ontogeny of vesicular monoamine transporter mRNAs VMAT1 and VMAT2. I. The developing rat central nervous system

We used in situ hybridization histochemistry to study the expression of the mRNA of the two vesicular monoamine transporters (VMAT1 and VMAT2) during embryonic and postnatal development of the central nervous system (CNS) in the rat. In the adult rat, VMAT2 mRNA is present exclusively in monoaminergic cell groups of the CNS and VMAT1 mRNA was reported to be present in the adrenal medulla and certain intestinal epithelial cells. In contrast to the above, the expression of VMAT1 mRNA has previously never been detected in the central nervous system. This study shows the first evidence that both transporter molecules are expressed in CNS during ontogenesis. We here demonstrate four main expression patterns detected during development: 1. VMAT2 mRNA expression in monoaminergic neurons of the brainstem beginning as early as embryonic day E13. 2. Expression of VMAT2 mRNA in all major sensory relay nuclei of central nervous system. 3. Co-expression of VMAT1 and VMAT2 mRNA in most limbic structures, basal ganglia, as well as in some hypothalamic nuclei. 4. Exclusive expression of VMAT1 mRNA in the neocortical subventricular zone, in the amygdala at early (E15-18) and late (P1-P28) timepoints, the granular cell layer of cerebellum, and in several brainstem motor nuclei. Based on their distribution during development we suggest that monoamines, released in a controlled fashion, might affect wiring of sensory and also motor circuits. VMAT1 mRNA expression may reflect a specific effect of monoamines in glial differentiation and cerebellar granule cell migration and/or differentiation.     

Neuronal migration and differentiation in the development of the mouse dorsal cochlear nucleus

The dorsal cochlear nucleus (DCN) of mammals displays a cortical structure containing a number of cell types organized into distinct layers. In the present study, the migratory mode of large multipolar cells and granule cells as well as the morphological differentiation of the projection neurons were investigated in the development of the mouse DCN. The classification of the DCN neurons followed that of Ryugo and Willard. The mode of neuronal migration was examined by immunohistochemical bromodeoxyuridine labeling. Large multipolar neurons originated from the primary rhombic lip and small granule cells from the secondary rhombic lip. Large multipolar neurons migrated radially from the ventricular zone into the forming DCN. Granule cells were generated later than the large multipolar neurons and migrated via the subependymal and subpial routes. Large multipolar neurons and small granule cells were thus segregated early in the DCN development and intermixed later during perinatal maturation. Projection neurons retrogradely labeled by DiI application to the contralateral inferior colliculus showed neurite extension between the pial surface and the ventricular zone during migration in the DCN primordium. The retrogradely labeled projection neurons showed a well-differentiated morphology of the large multipolar neurons as early as the late embryonic stage. The arrangement of the radial glial processes coincided with that of the migratory projection neurons. The migratory immature neurons showed close apposition with the radial glial processes, suggesting that glial scaffolds are involved in the migration and settlement of the large multipolar neurons. Thus, it is suggested that the mode of migration and settlement of large multipolar neurons and granule cells in the developing DCN is highly similar to that of Purkinje and granule cell migration in the cerebellar development, based on the findings of this study and the structural similarity between the cerebellum and DCN.     

Neurons with perineuronal sulfated proteoglycans in the mouse brain and spinal cord: their distribution and reactions to lectin Vicia villosa agglutinin and Golgi's silver nitrate

This study demonstrates that many neurons in the somatosensory cortex, cingulate cortex, retrosplenial cortex and hippocampal subiculum of the mouse brain are covered by sulfated proteoglycans which are intensely negative-charged and stained with cationic iron colloid, while being digested with hyaluronidase. Neurons with similar perineuronal proteoglycans are also recognized in the extrapyramidal system (superior colliculus, red nucleus, reticular formation, vestibular nuclei and cerebellar nuclei), in the secondary auditory system (cochlear nuclei, nucleus of trapezoid body, inferior colliculus and nucleus of lateral lemniscus), in the vestibulo-ocular reflex system (vestibular nuclei and extraocular motor nuclei), and in the pupillary reflex system. The neurons with perineuronal sulfated proteoglycans in the cerebral cortices and hippocampal subiculum are usually labeled with the lectin Vicia villosa agglutinin, though those in the cerebellar, vestibular and cochlear nuclei may not be reactive to this lectin. Double staining of the retrosplenial cortex, hippocampal subiculum and cerebellar nuclei with Golgi's silver nitrate and cationic iron colloid indicates that the perineuronal sulfated proteoglycans are identical with the Golgi's reticular coating or glial nets.     

Region-specific DNA synthesis in brains of F344 rats following a six-day bromodeoxyuridine infusion

Prolonged exposure to certain alkylating chemicals induces glial and meningeal tumours in rats, probably resulting from DNA damage to dividing neural cells. The present work evaluated DNA synthesis in the brains of untreated, young adult male F344 rats in order to define a BrdUrd infusion protocol to more adequately assess proliferation in slowly dividing neural cell populations. BrdUrd (2.5 to 160 mg/ml) was administered for 6 days via subcutaneous osmotic pumps. Clinical toxicity was not observed at any dose. The labelling index (LI; % of cells per brain area that incorporated BrdUrd) and unit length labelling index (ULLI; % of cells per meningeal length that incorporated BrdUrd) were calculated for selected regions by counting labelled neural cells in defined areas of the right hemisphere in coronal brain sections. Intensely stained cells were numerous in the cerebral subependymal layer (LI = 35.8%); scattered in cerebral white matter tracts (e.g. corpus callosum and internal capsule; LI = 6.2%) as well as cerebral (ULLI = 4.2%) and cerebellar (ULLI = 3.6%) meninges; and rare in the hippocampus (LI > 0.1%). Mildy stained cells were dispersed in the pons (LI = 2.1%), deep cerebral (LI = 1.8%) and cerebellar (LI = 1.0%) grey matter, and thalamus (LI = 0.3%). Phenotypically, BrdUrd-positive cells in neuropil were glial cell precursors and their progeny, while those associated with meninges were usually located in the superficial subarachnoid space and appeared to be fibrocytes. Using BrdUrd infusion, LI for glial precursors at these sites ranged from two- to 10-fold higher than those reported previously after a brief parenteral pulse dose. These data indicate that continuous BrdUrd infusion for 6 days by subcutaneous osmotic pump is an efficient means of labelling neural cells throughout the brain.     

Ultralow concentrations of proenkephalin and [met5]-enkephalin differentially affect IgM and IgG production by B cells

Adenovirus-mediated gene transfer is a promising method for studies of vascular biology and potentially for gene therapy. Intravascular approaches for gene transfer to blood vessels in vivo generally require interruption of blood flow and have several limitations. We have used two alternative approaches for gene transfer to blood vessels in vivo using perivascular application of vectors. First, replication-deficient adenovirus expressing nuclear-targeted bacterial beta-galactosidase was injected into cerebrospinal fluid via the cisterna magna of rats. Leptomeningeal cells over the major arteries were efficiently transfected, and adventitial cells of large vessels and smooth muscle cells of small vessels were occasionally stained. When viral suspension was injected with the rat in a lateral position, the reporter gene was expressed extensively on the ipsilateral surface of the brain. Thus, adenovirus injected into cerebrospinal fluid provides gene transfer in vivo to cerebral blood vessels and, with greater efficiency, to perivascular tissue. Furthermore, positioning of the head may 'target' specific regions of the brain. Second, vascular gene delivery was accomplished by perivascular injection of virus in peripheral vessels. Injection of the adenoviral vector within the periarterial sheath of monkeys resulted in gene transfer to the vessel wall that was substantial in magnitude although limited to cells in the adventitia. Approximately 20% of adventitial cells expressed the transgene, with no gene transfer to cells in the intima or media. These approaches may provide alternative approaches for gene transfer to blood vessels, and may be useful for studies of vascular biology and perhaps vascular gene therapy.     

Rigid osteosynthesis with the AO plate and Phemister''s spongioplasty in secondary treatment of diaphyseal gunshot fractures

To clarify the histogenesis and differentiation potential of central neurocytoma, a pathological investigation of seven tumors from three patients was conducted using immunohistochemistry and ultrastructural analysis in addition to systematic in vitro studies. Six tumors were studied immunohistochemically and five were examined ultrastructurally. All cases that were immunostained were positive for synaptophysin in nuclear-free neuropil islands. In five tumors, a few tumor cells, in addition to reactive astrocytes, were positive for glial fibrillary acidic protein (GFAP). Vimentin staining was also positive in a few tumor cells of five specimens. Neurofilament staining was always negative. All cases for which ultrastructure was examined showed various synaptic abnormalities. Cultured cells were subdivided into three distinct tumor cell types: neuronal cells which stained for neurofilament proteins with neurosecretory granules; small flat undifferentiated cells with a high nuclear-cytoplasmic ratio and scant cytoplasmic organelles; and small round or multipolar astrocytic cells with 10-nm intermediate filaments which stained for GFAP. Our tissue culture studies disclosed that cultured neurocytoma cells form a cellular mosaic similar to subependymal plate layers that are composed of mitotically active cells, neurons and glia.     

Glial cells in the enteric nervous system contain glial fibrillary acidic protein

The complex nervous networks found throughout the mammalian gut--the enteric nervous system--are histologically, ultrastructurally, and, to some extent, functionally--similar to the central nervous system. The glial cells of the small enteric ganglia are generally classified as Schwann or satellite cells, since they are found in the peripheral nervous system, possess nuclei which ultrastructurally resemble those of Schwann cells and are derived from the neural crest. However, it has been argued that these cells resemble astrocytes of the central nervous system with respect to gross and fine structure, and their relationship with the enteric neurones and their processes. In immunohistochemical studies of these cells, both in frozen sections of gut wall and in tissue culture preparations of the enteric plexuses, we found evidence that the enteric glial cells are rich in glial fibrillary acidic protein (GFAP), a protein associated with the 100 A glial intermediate filaments, and hitherto believed to be specific to astrocytes of the central nervous system only.     

Properties of soluble and membrane intestinal hydrolases in Macaca rhesus monkeys

Evidence from several central nervous system (CNS) inflammatory disease models suggests that intrathecal complement synthesis may contribute to early inflammatory events in the brain. In this study, we examined the expression of the receptor for C5a (C5aR), a potent inflammatory and chemotactic factor, in the brains of transgenic mice with constitutive astrocyte expression of interleukin-3 (IL-3), a hematopoietic and immunomodulatory cytokine. By in situ hybridization, we demonstrated that cells infiltrating the cerebellar meninges, the cerebellum, and demyelinating lesions in the cerebellum were strongly positive for C5aR mRNA. By immunohistochemistry, the infiltrating cells expressing the C5aR were identified as macrophages based on staining with antibodies to the complement receptor type 3 and F4/80, a mouse macrophage-specific marker. In addition, some of the cells in cerebellar lesions were positive for the astrocyte-specific marker, glial fibrillary acidic protein, suggesting that a subpopulation of astrocytes in these lesions express elevated levels of the C5aR. Increased C5aR expression was also observed in cortical neurons in the occipital cortex and in pyramidal neurons in the cornu ammonis and subiculum of the hippocampus, at both the protein and mRNA levels. These data suggest that IL-3 may play an immunomodulatory role in C5aR expression on several cell types in the brain and that increased C5aR expression correlates with the pathology seen in this model. The transgenic mice used in this study provide a useful tool for characterizing the mechanism of regulation of the C5aR expression and for examining the functions of this chemotactic receptor in CNS inflammation.     

Metastatic ability of MXT mouse mammary subpopulations correlates with clonal expression and/or membrane-association of gelatinase A

A vacuolar degeneration affecting primarily the gray matter in the central nervous system (CNS) of young Australian Cattle Dogs is described. An initial presentation of seizures was followed by a progressive spastic tetraparesis. Grossly evident bilateral and symmetrical foci of malacia were in the nuclei of the cerebellum and brain stem and the gray matter of the spinal cord. Microscopically, vacuolation of glial cells, dilation of the myelin sheaths and reactive astrocytosis characterized mild CNS changes. More advanced lesions displayed progressive dissolution of the neuropil, prominent vacuolation of reactive astrocytes, numerous glial fibrillary acidic protein-positive coiled astrocytic processes, neuronal vacuolation and loss with relative sparing of large neurons. Ultrastructurally marked mitochondrial accumulation and swelling were seen in astrocytes. In the appendicular muscles, changes interpreted as long-term denervation atrophy accompanied by widespread expression of the neonatal isoform of myosin were observed. The character of the neurological sings, the nature and the distribution of the lesions within the neuroaxis have not been reported in domestic animals. An inherited biochemical defect, possibly mitochondrial, is proposed as the cause. Selected conditions with a bilateral and symmetrical distribution affecting the gray matter of domestic animals are summarized.     

A transient CD15 immunoreactive sling in the developing mouse cerebellum

The distribution of the 3-fucosyl-N-acetyl-lactosamine (FAL, CD15) epitope in the developing mouse cerebellum was examined with the aid of immunohistochemistry of paraffin sections. CD15 immunoreactivity first appeared at E15 as a discrete bundle of processes lying beneath, and slightly within, the deeper layers of the external granular layer. By E17, when the cerebellar anlagen had completed their midline fusion, these processes could be traced from the germinal trigone at the lateral limits of the cerebellar anlage around the posterior cerebellar midline to the opposite germinal trigone. By P2, this sling was no longer apparent and CD15 immunoreactivity was confined to astrocytes in the cerebellar white matter, surrounding the deep cerebellar nuclei. The CD15 immunoreactive processes pursue an unusual trajectory through the developing cerebellum, unlike any other previously described axonal or glial process bundle in the cerebellum. From its trajectory and association with the ventricular surface it seems that this structure, which we have named the transverse cerebellar sling, is composed of glial processes, although it was not immunoreactive for S-100 or glial fibrillary acidic protein. The transient appearance of this sling encircling the posterior cerebellum is suggestive of a role in prenatal cerebellar morphogenesis.     

Electron microscopic immunolocalization of caltrin proteins in guinea pig seminal vesicles

We report here on five new cases of solid and cystic papillary neoplasm (SCPN) of the pancreas diagnosed by fine-needle aspiration cytology (FNAC). All cytologic samples were obtained by ultrasonography, and the smears were conventionally fixed and stained. Special histochemical and immunocytochemical stains were also performed in some samples. Cytology revealed in all but one case numerous pseudopapillary structures composed of fibrovascular stalks lined with one or more layers of bland-appearing, uniform tumor cells. The tumor cells had round-to-oval euchromatic nuclei with frequently folded smooth contours and one or two small nucleoli. Their cytoplasm often contained eosinophilic, PAS-positive, and diastase-resistant inclusions. Foamy cells, psammoma bodies, blood, and cellular debris were found in the background. The criteria for the differential diagnosis versus other pancreatic lesions are discussed in some detail, as is the role of immunocytochemistry (ICC). In the literature, only 28 cases of cytologically investigated SCPN have been reported to the best of our knowledge. The most helpful criteria for the conclusive identification of SCPN by FNAC include the pseudopapillary arrangement with bland-appearing tumor cells, and, especially, the finding of acidophilic, PAS-positive, and diastase-resistant cytoplasmic granules.     

Transneuronal pathways to the vestibulocerebellum

The alpha-herpes virus (pseudorabies, PRV) was used to observe central nervous system (CNS) pathways associated with the vestibulocerebellar system. Retrograde transneuronal migration of alpha-herpes virions from specific lobules of the gerbil and rat vestibulo-cerebellar cortex was detected immunohistochemically. Using a time series analysis, progression of infection along polyneuronal cerebellar afferent pathways was examined. Pressure injections of > 20 nanoliters of a 10(8) plaque forming units (pfu) per ml solution of virus were sufficient to initiate an infectious locus which resulted in labeled neurons in the inferior olivary subnuclei, vestibular nuclei, and their afferent cell groups in a progressive temporal fashion and in growing complexity with increasing incubation time. We show that climbing fibers and some other cerebellar afferent fibers transported the virus retrogradely from the cerebellum within 24 hours. One to three days after cerebellar infection discrete cell groups were labeled and appropriate laterality within crossed projections was preserved. Subsequent nuclei labeled with PRV after infection of the flocculus/paraflocculus, or nodulus/uvula, included the following: vestibular (e.g., z) and inferior olivary nuclei (e.g., dorsal cap), accessory oculomotor (e.g., Darkschewitsch n.) and accessory optic related nuclei, (e.g., the nucleus of the optic tract, and the medial terminal nucleus); noradrenergic, raphe, and reticular cell groups (e.g., locus coeruleus, dorsal raphe, raphe pontis, and the lateral reticular tract); other vestibulocerebellum sites, the periaqueductal gray, substantia nigra, hippocampus, thalamus and hypothalamus, amygdala, septal nuclei, and the frontal, cingulate, entorhinal, perirhinal, and insular cortices. However, there were differences in the resulting labeling between infection in either region. Double-labeling experiments revealed that vestibular efferent neurons are located adjacent to, but are not included among, flocculus-projecting supragenual neurons. PRV transport from the vestibular labyrinth and cervical muscles also resulted in CNS infections. Virus propagation in situ provides specific connectivity information based on the functional transport across synapses. The findings support and extend anatomical data regarding vestibulo-olivo-cerebellar pathways.