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Background Periventricular extracellular oedema, myelin damage, inflammation, and glial reactions are common neuropathological events that occur in the brain in congenital hydrocephalus. The periventricular white matter is the most affected region. The present study aimed to identify altered molecular and cellular biomarkers in the neocortex that can function as potential therapeutic targets to both treat and evaluate recovery from these neurodegenerative conditions. The hyh mouse model of hereditary hydrocephalus was used for this purpose. Methods The hyh mouse model of hereditary hydrocephalus (hydrocephalus with hop gait) and control littermates without hydrocephalus were used in the present work. In tissue sections, the ionic content was investigated using energy dispersive X-ray spectroscopy scanning electron microscopy (EDS-SEM). For the lipid analysis, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) was performed in frozen sections. The expression of proteins in the cerebral white matter was analysed by mass spectrometry. The oligodendrocyte progenitor cells (OPCs) were studied with immunofluorescence in cerebral sections and whole-mount preparations of the ventricle walls. Results High sodium and chloride concentrations were found indicating oedema conditions in both the periventricular white matter and extending towards the grey matter. Lipid analysis revealed lower levels of two phosphatidylinositol molecular species in the grey matter, indicating that neural functions were altered in the hydrocephalic mice. In addition, the expression of proteins in the cerebral white matter revealed evident deregulation of the processes of oligodendrocyte differentiation and myelination. Because of the changes in oligodendrocyte differentiation in the white matter, OPCs were also studied. In hydrocephalic mice, OPCs were found to be reactive, overexpressing the NG2 antigen but not giving rise to an increase in mature oligodendrocytes. The higher levels of the NG2 antigen, diacylglycerophosphoserine and possibly transthyretin in the cerebrum of hydrocephalic hyh mice could indicate cell reactions that may have been triggered by inflammation, neurocytotoxic conditions, and ischaemia. Conclusion Our results identify possible biomarkers of hydrocephalus in the cerebral grey and white matter. In the white matter, OPCs could be reacting to acquire a neuroprotective role or as a delay in the oligodendrocyte maturation.

In species that regenerate the injured spinal cord, the ependymal region is a source of new cells and a prominent coordinator of regeneration. In mammals, cells at the ependymal region proliferate in normal conditions and react after injury, but in humans, the central canal is lost in the majority of individuals from early childhood. It is replaced by a structure that does not proliferate after damage and is formed by large accumulations of ependymal cells, strong astrogliosis and perivascular pseudo-rosettes. We inform here of two additional mammals that lose the central canal during their lifetime: the Naked Mole-Rat (NMR, Heterocephalus glaber) and the mutant hyh (hydrocephalus with hop gait) mice. The morphological study of their spinal cords shows that the tissue substituting the central canal is not similar to that found in humans. In both NMR and hyh mice, the central canal is replaced by tissue reminiscent of normal lamina X and may include small groups of ependymal cells in the midline, partially resembling specific domains of the former canal. However, no features of the adult human ependymal remnant are found, suggesting that this structure is a specific human trait. In order to shed some more light on the mechanism of human central canal closure, we provide new data suggesting that canal patency is lost by delamination of the ependymal epithelium, in a process that includes apical polarity loss and the expression of signaling mediators involved in epithelial to mesenchymal transitions.

Background In obstructive congenital hydrocephalus, cerebrospinal fluid accumulation is associated with high intracranial pressure and the presence of periventricular edema, ischemia/hypoxia, damage of the white matter, and glial reactions in the neocortex. The viability and short time effects of a therapy based on bone marrow-derived mesenchymal stem cells (BM-MSC) have been evaluated in such pathological conditions in the hyh mouse model. Methods BM-MSC obtained from mice expressing fluorescent mRFP1 protein were injected into the lateral ventricle of hydrocephalic hyh mice at the moment they present a very severe form of the disease. The effect of transplantation in the neocortex was compared with hydrocephalic hyh mice injected with the vehicle and non-hydrocephalic littermates. Neural cell populations and the possibility of transdifferentiation were analyzed. The possibility of a tissue recovering was investigated using 1H High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (1H HR-MAS NMR) spectroscopy, thus allowing the detection of metabolites/osmolytes related with hydrocephalus severity and outcome in the neocortex. An in vitro assay to simulate the periventricular astrocyte reaction conditions was performed using BM-MSC under high TNFα level condition. The secretome in the culture medium was analyzed in this assay. Results Four days after transplantation, BM-MSC were found undifferentiated and scattered into the astrocyte reaction present in the damaged neocortex white matter. Tissue rejection to the integrated BM-MSC was not detected 4 days after transplantation. Hyh mice transplanted with BM-MSC showed a reduction in the apoptosis in the periventricular neocortex walls, suggesting a neuroprotector effect of the BM-MSC in these conditions. A decrease in the levels of metabolites/osmolytes in the neocortex, such as taurine and neuroexcytotoxic glutamate, also indicated a tissue recovering. Under high TNFα level condition in vitro, BM-MSC showed an upregulation of cytokine and protein secretion that may explain homing, immunomodulation, and vascular permeability, and therefore the tissue recovering. Conclusions BM-MSC treatment in severe congenital hydrocephalus is viable and leads to the recovery of the severe neurodegenerative conditions in the neocortex. NMR spectroscopy allows to follow-up the effects of stem cell therapy in hydrocephalus.

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo.

et al., Lysophosphatidic acid (LPA) is an intercellular signaling lipid that regulates multiple cellular functions, acting through specific G-protein coupled receptors (LPA1–6). Our previous studies using viable Malaga variant maLPA1-null mice demonstrated the requirement of the LPA1 receptor for normal proliferation, differentiation, and survival of the neuronal precursors. In the cerebral cortex LPA1 is expressed extensively in differentiating oligodendrocytes, in parallel with myelination. Although exogenous LPA-induced effects have been investigated in myelinating cells, the in vivo contribution of LPA1 to normal myelination remains to be demonstrated. This study identified a relevant in vivo role for LPA1 as a regulator of cortical myelination. Immunochemical analysis in adult maLPA1-null mice demonstrated a reduction in the steady-state levels of the myelin proteins MBP, PLP/DM20, and CNPase in the cerebral cortex. The myelin defects were confirmed using magnetic resonance spectroscopy and electron microscopy. Stereological analysis limited the defects to adult differentiating oligodendrocytes, without variation in the NG2+ precursor cells. Finally, a possible mechanism involving oligodendrocyte survival was demonstrated by the impaired intracellular transport of the PLP/DM20 myelin protein which was accompanied by cellular loss, suggesting stress-induced apoptosis. These findings describe a previously uncharacterized in vivo functional role for LPA1 in the regulation of oligodendrocyte differentiation and myelination in the CNS, underlining the importance of the maLPA1-null mouse as a model for the study of demyelinating diseases., This work was supported by the Carlos III Health Institute, State Department of Research, Development and Innovation, Spanish Ministry of Economy and Competitiveness (Grant Numbers PI10/02514—co-funded by European Research Development Fund—, to G.E-T.; SAF2011 to IV-N); Andalusian Regional Ministries of Health (Nicolás Monardes Programme, and Grants PI0187/2008, PI0232/2007 to G.E-T.) and of Economy, Innovation, Science and Employment (CTS643 and CTS433 research group grants to G.E-T. and F.R-DF., respectively); Ramon Areces Foundation (Ramon Areces Fellowship to B.G-D.); and the National Institutes of Health (USA) (Grant Numbers MH051699 and MH01723 to J.C.).

Most cells of the developing mammalian brain derive from the ventricular (VZ) and the subventricular (SVZ) zones. The VZ is formed by the multipotent radial glia/neural stem cells (NSCs) while the SVZ harbors the rapidly proliferative neural precursor cells (NPCs). Evidence from human and animal models indicates that the common history of hydrocephalus and brain maldevelopment starts early in embryonic life with disruption of the VZ and SVZ. We propose that a "cell junction pathology" involving adherent and gap junctions is a final common outcome of a wide range of gene mutations resulting in proteins abnormally expressed by the VZ cells undergoing disruption. Disruption of the VZ during fetal development implies the loss of NSCs whereas VZ disruption during the perinatal period implies the loss of ependyma. The process of disruption occurs in specific regions of the ventricular system and at specific stages of brain development. This explains why only certain brain structures have an abnormal development, which in turn results in a specific neurological impairment of the newborn. Disruption of the VZ of the Sylvian aqueduct (SA) leads to aqueductal stenosis and hydrocephalus, while disruption of the VZ of telencephalon impairs neurogenesis. We are currently investigating whether grafting of NSCs/neurospheres from normal rats into the CSF of hydrocephalic mutants helps to diminish/repair the outcomes of VZ disruption.

The multiciliated ependyma forms an epithelial-like layer that could act as a selective barrier between the brain parenchyma and cerebrospinal fluid. In the present study, tyrosine hydroxylase-containing fibres have been detected in the basal pole of the ependymal cells of the lateral ventricles of rat, mouse and calf. The use of antibodies against at least two different peptide sequences of each D(2), D(3), D(4) and D(5) dopamine receptor subtype has allowed their detection in: (i) sections of mouse, rat and bovine lateral ventricles, by means of immunocytochemistry; and (ii) membrane protein extracts obtained from the ependymal layer of the bovine lateral ventricles, using immunoblotting. The immunocytochemical study has shown the presence of all these subtypes of dopamine receptors in the ependymal cells. Immunoblotting demonstrated similar immunoreactive bands for all receptor subtypes in both ependymal and corpus striatum membrane extracts.

The cytoarchitecture of the walls of the bovine lateral ventricles was investigated by the use of immunocytochemistry. We defined three types of walls. Type 1 lined regions of white matter and had ciliated cuboidal ependyma, a few subependymal cells and a narrow subjacent glial layer. Type 2 lined the striatum and possessed ependymal cells with conspicuous basal processes that extended through a wide subependyma containing many subependymal cells and a wide subjacent glial network. Type 3 lined the rostral horn and displayed ependymal cells with the longest basal processes and wider subependymal and glial layers. Ependymal cells of type 2 and 3 walls were labelled with antibodies against S-100beta protein, vimentin, GFAP, BLBP and nestin. Anti-betaIII-tubulin stained small cells in the subependyma and inside the GFAP- and vimentin-positive subjacent glial network. Anti-PCNA-positive nuclei were abundant in the subependymal and glial layers of type 2 and 3 walls. DiI in vitro tracing studies revealed small bipolar cells in the glial layer at a distance from the site of the label deposit. These results suggest that neurogenesis takes place in adult bovine subependyma mostly in the walls of the striatum and the rostral horn, and that young neuroblasts may migrate in a rostro-ventral direction through the glial network.

Previous studies have shown the existence of proliferating cells in explants from bovine (Bos Taurus) lateral ventricle walls that were maintained for several days in vitro in the absence of serum and growth factors. In this study we have characterized the nature of new cells and have assessed whether the insulin-like growth factor-1 (IGF-1) receptor regulates their survival and/or proliferation. The explants were composed of the ependymal layer and attached subependymal cells. Ependymal cells in culture were labelled with glial markers (S-100, vimentin, GFAP, BLBP, 3A7 and 3CB2) and did not incorporate bromodeoxiuridine when this molecule was added to the culture media. Most subependymal cells were immunoreactive for beta III-tubulin, a neuronal marker, and did incorporate bromodeoxiuridine. Subependymal neurons displayed immunoreactivity for IGF-1 and its receptor and expressed IGF-1 mRNA, indicating that IGF-1 is produced in the explants and may act on new neurons. Addition to the culture media of an IGF-1 receptor antagonist, the peptide JB1, did not affect the incorporation of bromodeoxiuridine to proliferating subependymal cells. However, JB1 significantly increased the number of TUNEL positive cells in the subependymal zone, suggesting that IGF-1 receptor is involved in the survival of subependymal neurons. In conclusion, these findings indicate that neurogenesis is maintained in explants from the lateral cerebral ventricle of adult bovine brains and that IGF-1 is locally produced in the explants and may regulate the survival of the proliferating neurons.

To determine if ventricular zone (VZ) and subventricular zone (SVZ) alterations are associated with intraventricular hemorrhage (IVH) and posthemorrhagic hydrocephalus, we compared postmortem frontal and subcortical brain samples from 12 infants with IVH and 3 nonneurological disease controls without hemorrhages or ventriculomegaly. Birth and expiration estimated gestational ages were 23.0-39.1 and 23.7-44.1 weeks, respectively; survival ranges were 0-42 days (median, 2.0 days). Routine histology and immunohistochemistry for neural stem cells (NSCs), neural progenitors (NPs), multiciliated ependymal cells (ECs), astrocytes (AS), and cell adhesion molecules were performed. Controls exhibited monociliated NSCs and multiciliated ECs lining the ventricles, abundant NPs in the SVZ, and medial vs. lateral wall differences with a complex mosaic organization in the latter. In IVH cases, normal VZ/SVZ areas were mixed with foci of NSC and EC loss, eruption of cells into the ventricle, cytoplasmic transposition of N-cadherin, subependymal rosettes, and periventricular heterotopia. Mature AS populated areas believed to be sites of VZ disruption. The cytopathology and extension of the VZ disruption correlated with developmental age but not with brain hemorrhage grade or location. These results corroborate similar findings in congenital hydrocephalus in animals and humans and indicate that VZ disruption occurs consistently in premature neonates with IVH.

Under normal physiological conditions the cerebrospinal fluid (CSF) is secreted continuously, although this secretion undergoes circadian variations. Mechanisms operating at the vascular side of the choroidal cells involve a sympathetic and a cholinergic innervation, with the former inhibiting and the latter stimulating CSF secretion. There are also regulatory mechanisms operating at the ventricular side of the choroidal cells, where receptors for monoamines such as dopamine, serotonin, and melatonin, and for neuropeptides such as vasopressin, atrial natriuretic hormone, and angiotensin II, have been identified. These compounds, that are normally present in the CSF, participate in the regulation of CSF secretion. Although the mechanisms responsible for the CSF circulation are not fully understood, several factors are known to play a role. There is evidence that the subcommissural organ (SCO)–Reissner's fiber (RF) complex is one of the factors involved in the CSF circulation. In mammals, the predominant route of escape of CSF into blood is through the arachnoid villi. In lower vertebrates, the dilatation of the distal end of the central canal, known as terminal ventricle or ampulla caudalis, represents the main site of CSF escape into blood. Both the function and the ultrastructural arrangement of the ampulla caudalis suggest that it may be the ancestor structure of the mammalian arachnoid villi. RF-glycoproteins reaching the ampulla caudalis might play a role in the formation and maintenance of the route communicating the CSF and blood compartments. The SCO-RF complex may participate, under physiological conditions, in the circulation and reabsorption of CSF. Under pathological conditions, the SCO appears to be involved in the pathogeneses of congenital hydrocephalus. Changes in the SCO have been described in all species developing congenital hydrocephalus. In these reports, the important question whether the changes occurring in the SCO precede hydrocephalus, or are a consequence of the hydrocephalic state, has not been clarified. Recently, evidence has been obtained indicating that a primary defect of the SCO-RF complex may lead to hydrocephalus. Thus, a primary and selective immunoneutralization of the SCO-RF complex during the fetal and early postnatal life leads to absence of RF, aqueductal stenosis, increased CSF concentration of monoamines, and a moderate but sustained hydrocephalus. Microsc. Res. Tech. 52:591–607, 2001. © 2001 Wiley-Liss, Inc.

Stenosis of the cerebral aqueduct seems to be a key event for the development of congenital hydrocephalus. The causes of such a stenosis are not well known. Overholser et al. in 1954 (Anat Rec 120:917-933) proposed the hypothesis that a dysfunction of the subcommissural organ (SCO) leads to aqueductal stenosis and congenital hydrocephalus. The SCO is a brain gland, located at the entrance of the cerebral aqueduct, that secretes glycoproteins into the cerebrospinal fluid that, upon release, assemble into a fibrous structure known as Reissner's fiber (RF). By the permanent addition of new molecules to its rostral end, RF grows and extends along the aqueduct, fourth ventricle, and central canal of the spinal cord. The immunological blockage of the SCO-RF complex has been used to test Overholser's hypothesis. The following was the sequence of events occurring in pregnant rats that had been immunized with RF glycoproteins: the mother produced anti-RF antibodies and transferred them to the fetus through the placenta and to the pup through the milk, and the antibodies reached the brain of the fetus and pup and blocked the SCO-RF complex. This resulted in a permanent absence of RF that was followed by stenosis of the cerebral aqueduct, and then by the appearance of hydrocephalus. The latter was patent until the end of the 6-month observation period. The chronic hydrocephalic state appeared, in turn, to induce new alterations of the SCO. It is concluded that a selective immunological knock out of the SCO-RF complex leads to hydrocephalus.

The neuroepithelium is a germinal epithelium containing progenitor cells that produce almost all of the central nervous system cells, including the ependyma. The neuroepithelium and ependyma constitute barriers containing polarized cells covering the embryonic or mature brain ventricles, respectively; therefore, they separate the cerebrospinal fluid that fills cavities from the developing or mature brain parenchyma. As barriers, the neuroepithelium and ependyma play key roles in the central nervous system development processes and physiology. These roles depend on mechanisms related to cell polarity, sensory primary cilia, motile cilia, tight junctions, adherens junctions and gap junctions, machinery for endocytosis and molecule secretion, and water channels. Here, the role of both barriers related to the development of diseases, such as neural tube defects, ciliary dyskinesia, and hydrocephalus, is reviewed.

Galanin is a brain-gut peptide present in the central nervous system of vertebrates and invertebrates. The distribution of galanin-like immunoreactive perikarya and fibers in the brain of the river lamprey Lampetra fluviatilis (Agnatha) has been studied immunocytochemically by using antisera against rat and porcine galanin. Galanin-like immunoreactive perikarya were seen in the telencephalon and mediobasal diencephalon. In the telencephalon, they were present in the nucleus olfactorius anterior, nucleus basalis, and especially, in the nucleus commissurae anterioris. The diencephalon contained most of the immunoreactive neurons. They were located in the nucleus commissurae praeinfundibularis, nucleus ventralis hypothalami, nucleus commissurae postinfundibularis, nucleus ventralis thalami, and nucleus dorsalis thalami pars medius. Most of the galanin-like immunoreactive infundibular neurons showed apical processes contacting the cerebrospinal fluid. Immunoreactive fibers and terminals were widely distributed throughout the neuraxis. In the telencephalon, the richest galaninergic innervation was found in the nucleus olfactorius anterior, lobus subhippocampalis, corpus striatum, and around the nucleus septi and the nucleus praeopticus. In the diencephalon, the highest density of galanin-like immunoreactive fibers was seen in the nucleus commissurae postopticae, nucleus commissurae praeinfundibularis, nucleus ventralis hypothalami, nucleus dorsalis hypothalami, and neurohypophysis. In the mesencephalon and rhombencephalon, the distribution of immunoreactive fibers was heterogeneous, being most pronounced in a region between the nucleus nervi oculomotorii and the nucleus interpeduncularis mesencephali, in the nucleus isthmi, and in the raphe region. A subependymal plexus of immunoreactive fibers was found throughout the ventricular system. The distribution of immunoreactive neurons and fibers was similar to that of teleosts but different to those of other vertebrate groups. The possible hypophysiotropic and neuroregulatory roles of galanin are discussed.

Rod- and cone-opsin specific antibodies were used in an attempt to immunolabel remaining photoreceptor cells in the mutant rd (retinal degeneration) mouse retina. We identified a region-specific distribution in the pattern of photoreceptor degeneration, with the dorsal retina showing markedly less photoreceptor degeneration than the ventral retina. All rod and cone immunoreactive cells disappeared in the ventral retina by 100-120 days of age. By contrast, both cone and a small number of rod immunopositive cells were identified in the dorsal retina at this time. By 200 days all rod immunoreactive cells had disappeared. At 360 days numerous cone immunoreactive cells remained within a restricted region of the dorsal retina. As rd mice show unattenuated circadian responses to light, these remaining photoreceptor cells within the dorsal retina become candidates for the regulation of circadian physiology by light.

Galanin is a brain-gut peptide present in the central nervous system of fish, amphibians, birds, and mammals. For comparative studies among vertebrates, the distribution of galanin in the brain of reptiles has been investigated. We studied the localization of galanin-like-immunoreactive perikarya and nerve fibers in the brain of the turtle Mauremys caspica by using an antiserum against porcine galanin. In the telencephalon, few immunoreactive perikarya were seen in the amygdaloid complex. The diencephalon contained the majority of the immunoreactive perikarya present in the lamina terminalis, nucleus periventricularis anterior, lateral preoptic area, nuclei hypothalamicus ventromedialis and posterior, nucleus basalis of the anterior commissure, and nucleus ventralis tuberis. Many immunoreactive cells, especially in the infundibulum, contacted the cerebrospinal fluid by an apical process. In the rhombencephalon, immunopositive perikarya were restricted to a few cells in the nucleus tractus solitari. In the mesencephalon, they were absent. Immunoreactive nerve fibers were present in all regions containing labeled perikarya and in 1) telencephalon: septum, nucleus fasciculi diagonalis Brocae; 2) diencephalon: nucleus paraventricularis, nucleus supraopticus, nucleus suprachiasmaticus, subventricular grey, nucleus of the paraventricular organ, nucleus mamillaris, infundibular decussation, outer layer of the median eminence, posterior commissure and subcommissural organ region, habenula, nuclei dorsomedialis anterior, and dorsolateralis anterior of the thalamus; and 3) mesencephalon and rhombencephalon: stratum griseum periventriculare, stratum fibrosum periventriculare, laminar nucleus of the torus semicircularis, periventricular grey, nucleus interpeduncularis, nucleus ruber, substantia nigra, locus coeruleus, raphe nuclei, nuclei of the reticular formation, nucleus motorius nervi trigemini, cochlear and vestibular area, and nucleus spinalis nerve trigemini. Our results suggest that galanin may have hypophysiotropic and central roles in the turtle Mauremys caspica.

A heterogeneous population of ependymal cells lines the brain ventricles. The evidence about the origin and birth dates of these cell populations is scarce. Furthermore, the possibility that mature ependymal cells are born (ependymogenesis) or self-renewed (ependymal proliferation) postnatally is controversial. The present study was designed to investigate both phenomena in wild-type (wt) and hydrocephalic α-SNAP mutant (hyh) mice at different postnatal stages. In wt mice, proliferating cells in the ventricular zone (VZ) were only found in two distinct regions: the dorsal walls of the third ventricle and Sylvian aqueduct (SA). Most proliferating cells were monociliated and nestin+, likely corresponding to radial glial cells. Postnatal cumulative BrdU-labeling showed that most daughter cells remained in the VZ of both regions and they lost nestin-immunoreactivity. Furthermore, some labeled cells became multiciliated and GLUT-1+, indicating they were ependymal cells born postnatally. Postnatal pulse BrdU-labeling and Ki-67 immunostaining further demonstrated the presence of cycling multiciliated ependymal cells. In hydrocephalic mutants, the dorsal walls of the third ventricle and SA expanded enormously and showed neither ependymal disruption nor ventriculostomies. This phenomenon was sustained by an increased ependymogenesis. Consequently, in addition to the physical and geometrical mechanisms traditionally explaining ventricular enlargement in fetal-onset hydrocephalus, we propose that postnatal ependymogenesis could also play a role. Furthermore, as generation of new ependymal cells during postnatal stages was observed in distinct regions of the ventricular walls, such as the roof of the third ventricle, it may be a key mechanism involved in the development of human type 1 interhemispheric cysts.

Background In human foetal hydrocephalus and spina bifida aperta (SBA), the pathogenesis of abnormal cortical development is poorly understood. Previous reports have shown that neuroepithelial/ependymal denudation is involved in the neuropathogenesis of human foetal hydrocephalus and SBA. Interestingly, loss of the neuroepithelium/ ependyma (denudation) at the Sylvian aqueduct is preceded by defective expression of adherent and gap junction proteins. In human foetal hydrocephalus, we aimed to investigate whether abnormal cortical development is similarly associated with intercellular ependymal defects at the telencephalic (sub)ventricular zones.

Neural stem cells persist after embryonic development in the subventricular zone (SVZ) niche and produce new neural cells during postnatal life; ependymal cells are a key component associated with this neurogenic niche. In the animal model of human hydrocephalus, the hyh mouse, the ependyma of the lateral ventricles is progressively lost during late embryonic and early postnatal life and disappears from most of the ventricular surface throughout its life span. To determine the potential consequences of this loss on the SVZ, we characterized the abnormalities in this neurogenic niche in hyh mice. There was overall disorganization and a marked reduction of proliferative cells in the SVZ of both newborn and adult hyh hydrocephalic mice in vivo; neuroblasts were displaced to the ventricular surface, and their migration through the rostral migratory stream was reduced. The numbers of resident neural progenitor cells in hyh mice were also markedly reduced, but they were capable of proliferating, forming neurospheres, and differentiating into neurons and glia in vitro in a manner indistinguishable from that of wild-type progenitor cells. These findings suggest that the reduction of proliferative activity observed in vivo is not caused by a cell autonomous defect of SVZ progenitors but is a consequence of a reduced number of these cells. Furthermore, the overall tissue disorganization of the SVZ and displacement of neuroblasts imply alterations in the neurogenic niche of postnatal hyh mice.

A human brain cDNA clone coding for a novel PDZ-domain protein of 124 amino acids was previously isolated in our laboratory. The protein was termed glutaminase-interacting protein (GIP), because it interacts with the C-terminal region of the human L-type glutaminase (LGA). The pattern of expression and functions of GIP in brain are completely unknown, so its significance remains undefined. Here we describe the expression of GIP mRNA and protein in mammalian brain. Northern blot analysis revealed that GIP mRNA was ubiquitous in most regions of human brain but was particularly abundant in spinal cord. The presence of the protein in rat and monkey brain was studied at the regional, cellular, and subcellular level by immunocytochemistry. The protein was found to be present in both neurons and astrocytes, with a cytosolic and mitochondrial subcellular localization. Double immunofluorescence labeling with anti-GIP and anti-LGA antibodies using confocal microscopy revealed colocalization of both proteins in astrocyte cell processes and their perivascular end feet. Electron microscopy of rat brain neurons revealed GIP immunoreactivity concentrated also in the nuclear envelope and the plasma membrane. The multiple locations for GIP in mammalian brain are in agreement with known protein interaction partners reported for this PDZ protein. The findings presented here support a role of GIP as an important scaffold in both astrocytes and neurons and point toward astrocytic processes and perivascular end feet as plausible anatomical substrates for interaction with glutaminase.

The hyh mouse carrying a point mutation in the gene encoding for soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein alpha (alpha-SNAP) develops inherited hydrocephalus. The investigation was designed to study: (i) the clinical evolution of hyh mice; (ii) factors other than the alpha-SNAP mutation that may influence the expression of hydrocephalus; (iii) the neuropathological features underlying the different forms of clinical evolution. The study included 3017 mice, 22.4% of which were hydrocephalic. The neuropathological study was performed in 112 mice by use of light and electron microscopy. It was found that maternal- and sex-related factors are involved in the heterogeneous expression of hyh phenotype. The clinical evolution recorded throughout a 4-year period also revealed a heterogeneous expression of the hydrocephalic phenotype. Two subpopulations were distinguished: (i) 70% of mice underwent a rapidly progressive hydrocephalus and died during the first 2 months of life; they presented macrocephaly, extremely large expansion of the ventricles, equilibrium impairment and decreased motor activity. (ii) Mice with slowly progressive hydrocephalus (30%) survived for periods ranging between 2 months and 2 years. They had no or moderate macrocephaly; moderate ventricular dilatation and preserved general motor activity; they all presented spontaneous ventriculostomies communicating the ventricles with the subarachnoid space, indicating that such communications play a key role in the long survival of these mice. The hyh mutant represents an ideal animal model to investigate how do the brain "adapt" to a virtually life-lasting hydrocephalus.

mutant mice suffer a congenital hydrocephalus trig-gered by ependyma denudation [1]. The ventricular sur-face in non hydrocephalic newborn mice is lined by theimmature ependyma, which is characterized for beingvimentin (-) and S100β (-), at variance in the adult ani-mals the mature ependyma expresses vimentin and S100 β[2]. On the other hand, in the hydrocephalic mice theependyma begins to denudate on the 12th day of gesta-tion, and at PN8 only some areas of lateral ventricle arestill endowed with ependyma. In parallel, astroglia startsto cover the denuded surface forming a new cell layer, theglial scar, which lines the damaged ventricular surface. Wehave studied the permeability to horseradish peroxidase(HRP) of these four regions at the ventricular wills:mature ependyma, and denuded areas with or withoutglial scar.

The subcommissural organ (SCO) is a brain gland located in the roof of the third ventricle that releases glycoproteins into the cerebrospinal fluid, where they form a structure known as Reissner's fiber (RF). On the basis of SCO-spondin sequence (the major RF glycoprotein) and experimental findings, the SCO has been implicated in central nervous system development; however, its function(s) after birth remain unclear. There is evidence suggesting that SCO activity in adult animals may be regulated by serotonin (5HT). The use of an anti-5HT serum showed that the bovine SCO is heterogeneously innervated with most part being poorly innervated, whereas the rat SCO is richly innervated throughout. Antibodies against serotonin receptor subtype 2A rendered a strong immunoreaction at the ventricular cell pole of the bovine SCO cells and revealed the expected polypeptides in blots of fresh and organ-cultured bovine SCO. Analyses of organ-cultured bovine SCO treated with 5HT revealed a twofold decrease of both SCO-spondin mRNA level and immunoreactive RF glycoproteins, whereas no effect on release of RF glycoproteins into the culture medium was detected. Rats subjected to pharmacological depletion of 5HT exhibited an SCO-spondin mRNA level twofold higher than untreated rats. These results indicate that 5HT down-regulates SCO-spondin biosynthesis but apparently not its release, and suggest that 5HT may exert the effect on the SCO via the cerebrospinal fluid.

Dopamine receptors have been found in certain populations of non-neuronal cells in the brain, viz., discrete areas of ciliated ependyma and the ependymal cells of the choroid plexus. We have studied the presence of both tyrosine-hydroxylase-immunoreactive nerve fibers and dopamine receptors in the subcommissural organ (SCO), an ependymal brain gland that is located in the roof of the third ventricle and that secretes, into the cerebrospinal fluid, glycoproteins that aggregate to form Reissner’s fiber (RF). Antibodies against D2, D3, D4, and D5 dopamine receptors were used in immunoblots of bovine striatum, fresh SCO, and organ-cultured SCO, and in immunocytochemistry of the bovine, rat, and mouse SCO. Only a few tyrosine-hydroxylase fibers appeared to reach the SCO. However, virtually all the secretory ependymal and hypendymal cells of the SCO immunoreacted with antibodies against D2, D4, and D5 receptors, with the last-mentioned rendering the strongest reaction, especially at the ventricular cell pole of the secretory ependymocytes, suggesting that dopamine might reach the SCO via the cerebrospinal fluid. The antibodies against the four subtypes of receptors revealed corresponding bands in immunoblots of striatum and fresh SCO. Although the cultured SCO displayed dopamine receptors, dopamine had no apparent effect on the expression of the SCO-spondin gene/protein or on the release of RF-glycoproteins (SCO-spondin included) by SCO explants, suggesting that dopamine affects the function(s) of the SCO differently from the secretion of RF-glycoproteins.

Two phases may be recognized in the development of congenital hydrocephalus in the hyh mutant mouse. During embryonic life the detachment of the ventral ependyma is followed by a moderate hydrocephalus. During the first postnatal week the cerebral aqueduct becomes obliterated and a severe hydrocephalus develops. The aim of the present investigation was to elucidate the cellular phenomena occurring at the site of aqueduct obliteration and the probable participation of the subcommissural organ in this process. Electron microscopy, immunocytochemistry, and lectin histochemistry were used to investigate the aqueduct of normal and hydrocephalic hyh mice from embryonic day 14 (E-14) to postnatal day 7 (PN-7). In the normal hyh mouse, the aqueduct is an irregularly shaped cavity with 3 distinct regions (rostral, middle, and caudal) lined by various types of ependyma. In the hydrocephalic mouse, these 3 regions behave differently; the rostral end becomes stenosed, the middle third dilates, and the caudal end obliterates. The findings indicate that the following sequence of events lead to hydrocephalus: 1) denudation of the ventral ependyma (embryonic life); 2) denudation of dorsal ependyma and failure of the subcommissural organ to form Reissner fiber (first postnatal week); 3) obliteration of distal end of aqueduct; and 4) severe hydrocephalus. No evidence was obtained that NCAM is involved in the detachment of ependymal cells. The process of ependymal denudation would involve alterations of the surface sialoglycoproteins of the ependymal cells and the interaction of the latter with macrophages.

Hydrocephalic hyh mice are born with moderate hydrocephalus and a normal cerebral aqueduct. At about the fifth postnatal day the aqueduct becomes obliterated and severe hydrocephalus develops. The aim of the present investigation was to investigate the mechanism of this hydrocephalus, probably starting during fetal life when the cerebral aqueduct is still patent. By use of immunocytochemistry and scanning electron microscopy, mutant (n = 54) and normal (n = 61) hyh mouse embryos were studied at various developmental stages to trace the earliest microscopic changes occurring in the brains of embryos becoming hydrocephalic. The primary defect begins at an early developmental stage (E-12) and involves cells lining the brain cavities, which detach following a well-defined temporo-spatial pattern. This ependymal denudation mostly involves the ependyma of the basal plate derivatives. There is a relationship between ependymal denudation and ependymal differentiation evaluated by the expression of vimentin and glial fibrillary acidic protein. The ependymal cells had a normal appearance before and after detachment, suggesting that their separation from the ventricular wall might be due to abnormalities in cell adhesion molecules. The process of detachment of the ventral ependyma, clearly visualized under scanning electron microscope, is almost completed before the onset of hydrocephalus. Furthermore, this ependymal denudation does not lead to aqueductal stenosis during prenatal life. Thus, the rather massive ependymal denudation appears to be the trigger of hydrocephalus in this mutant mouse, raising the question about the mechanism responsible for this hydrocephalus. It seems likely that an uncontrolled bulk flow of brain fluid through the extended areas devoid of ependyma may be responsible for the hydrocephalus developed by the hyh mutant embryos. The defect in these embryos also includes loss of the hindbrain floor plate and a delayed in the expression of Reissner fiber glycoproteins by the subcommissural organ.

The extraretinal and extrapineal photoreceptors of three species of adult lamprey, sea lamprey (Petromyzon marinus), river lamprey (Lampetra fluviatilis) and silver lamprey (Ichthyomyzon unicuspis) were studied using antibodies raised against photoreceptor rod and cone opsins, alpha-transducin and arrestin. In all three species cells in the pineal organ (P), parapineal organ (PP), nucleus preopticus (T5), nucleus commissurae postopticae (D8), nucleus ventralis hypothalami (D10) and nucleus dorsalis hypothalami (D11) were labelled by one or more of the anti-opsin antibodies. In addition, anti-arrestin antibodies labelled cells within the D8 and anti-alpha-transducin antibodies labelled cells within the pineal complex and hypothalamus (primarily D8 and/or D10). A more variable and species dependent pattern of opsin, arrestin and alpha-transducin labelling was observed within the nucleus commissurae postinfundibularis (D12) in an area comprising the nucleus dorsalis thalami pars subhabenularis (D4sh) and nucleus dorsalis thalami pars caudalis/nucleus commissurae posterioris (D4c/M1), and in the proximity of the second Müller cells in the ventrocaudal diencephalon (2.MZ/M6). The majority of the neurons labelled within the pineal and parapineal organs and hypothalamus were periventricular with clear cerebrospinal fluid contacts (CSF-contacting neurons). Labelled neurons in the epithalamic (D4sh and D4c/M1) and caudal diencephalon (2.MZ/M6) had no obvious ventricular contacts. We speculate that the "primitive" vertebrate brain of lampreys represents an ancestral condition in which different populations of encephalic photoreceptors are associated with different behavioural and physiological responses. Image-forming vision needs an eye, but irradiance detection does not require a specialised organ. Rather the photoreceptors could be closely associated with their effector systems within the brain.

Photoreceptor degeneration in the retina of the rd/rd (retinal degeneration) mice has been studied using immunocytochemistry with antisera against cone- and rod-opsin. The rd/rd mice exhibited different regional specific rates of degeneration for rods and cones. As early as postnatal day 25, cells labelled with the rod-opsin and cone-opsin antisera disappeared preferently from the central retina. Whereas in the inferior half of the retina, degeneration subsequently proceeded towards the periphery, this did not occur in the dorsal hemisphere. By the age of 100 days, many cells immunoreactive for the cone-opsin antiserum and a few cells immunoreactive for the rod-opsin antiserum were located in an area of the dorsal retina. The ventral retina lacked labelled elements at this age. Finally, rd/rd mice at one year or 600 days of age contained a similar number of cone-opsin immunopositive cells (approximately 2000–2800 cells), occupying almost the same area in the retina as that found at 100 days of age. A photoreceptor candidate for the entrainment of non-visual photoreception probably remains in the cone population in aged rd/rd mice.

Sunflower (Helianthus annuus) pollen sensitization has been reported as an occupational allergy. In this report, the sensitization of the general population living in sunflower-growing areas to Helianthus pollen was studied. Both RAST results in 32 adults with summer symptoms previously diagnosed as allergic to Artemisia pollen, and cross-reactivity studies between H. annuus and other Compositae suggested that H. annuus pollen was the main allergen involved in the hypersensitivity reaction of those patients. Good correlation was found between RAST and SPT to Helianthus and between RAST and conjunctival provocation test to Helianthus. Bronchial challenge tests performed on 8 of the 32 patients confirmed the clinical implication of Helianthus pollen in suspected subjects. Five workers, handling sunflower pollen, who suffered from related symptoms were subjected to the same study, showing lesser wheal areas and lesser specific IgE levels than a non-worker group. Thirteen patients with RAST valuesor = class 2 showed 2 IgE-binding fractions at 34.0 and 42.8 kD in 65% of sera and 3 IgE-binding fractions at pI 4.9, 9.6 and 10.2 in 54% of sera. By means of micropreparative high-resolution chromatography, it was possible to purify a 34-kD major allergen. Analysis performed by RAST inhibition with sera from atopic patients and ELISA inhibition with experimental anti-Helianthus rabbit sera demonstrated a cross-reactivity between Helianthus and other Compositae, but low affinity of specific anti-Helianthus antibodies for heterologous antigens. Taking into account the above-mentioned data, and the high prevalence of Helianthus pollen in the atmosphere during harvesting (in spite of its entomophilous character), Helianthus pollen should be considered as an allergenic source to be investigated in the general population living in sunflower-growing regions suffering from seasonal summer allergy.

The subcommissural organ is an ependymal gland located at the entrance of the cerebral aqueduct. It secretes glycoproteins into the cerebrospinal fluid, where they aggregate to form Reissner's fiber. This fiber grows along the aqueduct, fourth ventricle, and central canal. There is evidence that the subcommissural organ is involved in the pathogenesis of congenital hydrocephalus. This organ was investigated in the mutant mouse hyh developing a congenital hydrocephalus. The central nervous system of normal and hydrocephalic hyh mice, 1 to 40 days old, was investigated using antibodies recognizing the subcommissural organ secretory glycoproteins, and by transmission and scanning electron microscopy. At birth, the affected mice displayed open communications between all ventricles, absence of a central canal in the spinal cord, ependymal denudation of the ventricles, stenosis of the rostral end of the aqueduct, and hydrocephalus of the lateral and third ventricles and of the caudal end of the aqueduct. Around the 5th postnatal day, the communication between the caudal aqueduct and fourth ventricle sealed, and hydrocephalus became severe. It is postulated that the hyh mice carry a genetic defect affecting the ependymal cell lineage. The subcommissural organ showed signs of increased secretory activity; it released to the stenosed aqueduct a material that aggregated, but it did not form a Reissner's fiber. A large area of the third ventricular wall differentiated into a secretory ependyma synthesizing a material similar to that secreted by the subcommissural organ. It is concluded that the subcommissural organ changes during hydrocephalus; whether these changes precede hydrocephalus needs to be investigated.

The present study was designed to investigate the controversial subject of the existence of a neural input from the pineal organ via the pineal tract to the subcommissural organ (SCO) in teleosts and anurans. Horseradish peroxidase was injected into the pineal organ and pineal tract of Carassius auratus and Rana perezi. Within the pinealofugal fibers the tracer was visualized at the light-and electron-microscopic levels either by immunocytochemistry using an anti-peroxidase serum, or by revealing the enzymatic activity of peroxidase. In both species, labeled myelinated and unmyelinated fibers of the pineal tract were readily traced by means of electron microscopy. In R. perezi, numerous terminals contacting the SCO cells in a synapse-like (synaptoid, hemisynaptic) manner bore the label, whereas a different population of endings was devoid of the tracer, indicating that in this species the SCO receives a dual neural input, one of pineal origin, the other of unknown source and nature. In the SCO of C. auratus, neither labeled nor unlabeled synapse-like contacts were found. Thus, in this latter species, a direct neural input to the SCO is missing. It is concluded that the secretory activity of the SCO can be controlled by different mechanisms in different species, and that more than one neural input mechanism may operate in the same species.

To investigate the role of sialic acid in the ependyma of the rat brain, we injected neuraminidase from Clostridium perfingens into the lateral ventricle of 86 adult rats that were sacrificed at various time intervals. After administration of 10 micrograms neuraminidase, ciliated cuboidal ependymal cells of the lateral ventricles, third ventricle, cerebral aqueduct, and the rostral half of the fourth ventricle died and detached. The ependymal regions sealed by tight junctions such as the choroid plexus and the subcommissural organ were not affected. Debris was removed by infiltrating neutrophils and macrophagic cells. At the same time, after ependymal disappearance, the aqueduct was obliterated. In this region, mitoses were evident and cystic ependymal cells were frequent. Hydrocephalus of the lateral and third ventricles was evident 4 days after neuraminidase injection. Gliosis was restricted to the dorsal telencephalic wall of the injected lateral ventricle. It is thought that cleavage of sialic acid from ependymal surface glycoproteins or glycolipids, likely involved in cell adhesion, led to the detaching and death of the ependymal cells. Thereafter, ependymal loss, together with edema, led to fusion of the lateral walls of the cerebral aqueduct and this in turn provoked hydrocephalus of the third and lateral ventricles. This model of experimental hydrocephalus is compared with other models, in particular those of hydrocephalus after viral invasion of the cerebral ventricles.

Hyh mutant mice develop long-lasting hydrocephalus and represent a good model for investigating neuropathologic events associated with hydrocephalus. The study of their brains by use of lectin binding, bromodeoxyuridine labeling, immunochemistry, and scanning electron microscopy revealed that certain events related to hydrocephalus followed a well-defined pattern. A program of neuroepithelium/ependyma denudation was initiated at embryonic day 12 and terminated at the end of the second postnatal week. After the third postnatal week the denuded areas remained permanently devoid of ependyma. In contrast, a selective group of ependymal areas resisted denudation throughout the lifespan. Ependymal denudation triggered neighboring astrocytes to proliferate. These astrocytes expressed particular glial markers and formed a superficial cell layer replacing the lost ependyma. The loss of the neuroepithelium/ependyma layer at specific regions of the ventricular walls and at specific stages of brain development would explain the fact that only certain brain structures had abnormal development. Therefore, commissural axons forming the corpus callosum and the hippocampal commissure displayed abnormalities, whereas those forming the anterior and posterior commissures did not; and the brain cortex was not homogenously affected, with the cingular and frontal cortices being the most altered regions. All of these telencephalic alterations developed at stages when hydrocephalus was not yet patent at the lateral ventricles, indicating that abnormal neural development and hydrocephalus are linked at the etiologic level, rather than the former being a consequence of the latter. All evidence collected on hydrocephalic hyh mutant mice indicates that a primary alteration in the neuroepithelium/ependyma cell lineage triggers both hydrocephalus and abnormalities in telencephalic development.

Background The hyh (hydrocephalus with hop gait) mouse carries a point mutation in alpha-SNAP protein and develops inherited hydrocephalus. Mutant mice are born with moderate hydrocephalus and a patent Sylvius aqueduct (SA). During the first postnatal week, SA obliterates and a severe hydrocephalus characterized by an enormous expansion of the dorsal third ventricle and of the collicular recess of the SA, develops. Interestingly, neither of these dilated cavities present spontaneous ventriculostomies. The aim of the present investigation was to elucidate some of the cellular phenomena occurring at the ventricular walls that allow such enormous ventricular dilatations.