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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. Materials and methods Brains of wild type (non-hydrocephalic) and mutant (hydrocephalic) hyh mice were studied by light and transmission electron microscopy at various age intervals (PN-1 to PN-120). Proliferative activity, especially at the ventricular walls, was studied by PCNA (proliferative cell nuclear antigen) immunocytochemistry, and 5'-Bromo-2'-deoxyUridine (BrdU) labelling. BrdU protocols included pulse and cumulative labelling of postnatal animals, combined with short and long survival periods after the labelling. Results In wild-type mice no BrdU-labelled or PCNA-positive cells were observed in the ependyma of the ventral walls of SA and third ventricle. However, proliferative cells were found in two discrete ependymal regions of the dorsal walls of the third ventricle (3Vd) and the SA (SAd). Here, proliferative activity continued at least during three weeks after birth. The localization, cytology and immunocytochemical properties indicate that both regions originate ependymal cells. Interestingly, in mutant (hydrocephalic) hyh mice, postnatal ependymogenesis occuring in 3Vd and SAd increased several fold. Conclusion 1. In non-hydrocephalic animals all ependymal cells lining the floor of the aqueduct are born during the fetal life; however, in the dorsal wall of the aqueduct and the roof of the third ventricle ependymogenesis continues during postnatal life. 2. In mutant mice, the hydrocephalic process triggers a dramatic increase of proliferative activity in these two ventricular regions letting them to expand without any disruption and, probably, allowing a longer survival. 3. In the cerebral aqueduct of hydrocephalic mice there are various ependymal lineages: one of them detaches, other proliferates while another neither detaches nor proliferates. Since all these ependymal populations are exposed to the same pressure and composition of the CSF, their differential response to the hydrocephalic status can best be explained by their distinct genetic programme. Supported by Fondecyt 1030265-Chile to EMR, CONICYT and DID-UACH D- 2005-12 to LFB, FIS PI030756 and FIS PI060423 to JMPF. [ABSTRACT FROM AUTHOR]

An abstract of the research "Long-lasting hydrocephalus in hyh mutant mice: gain and loss of a brain surviving hydrocephalus" is presented.

Background The hyh (hydrocephalus with hop gait) mutant mice develop inherited hydrocephalus. A key feature in this mutant is that there is a foetal-onset ependymal denudation which precedes cerebral aqueduct obliteration and hydrocephalus [1]. Recently, a point mutation in alpha-SNAP protein has been identified as responsible of the hyh phenotype [2]. However, preliminary findings from our laboratory have suggested clinical and pathological heterogeneity in the expression of hydrocephalus, indicating that other (nongenetic?) factors may influence the degree of severity of this pathology. This is in accordance with findings in other hydrocephalic mutant strains [3,4]. The present investigation was designed to (a) study the clinical evolution of hydrocephalic mice in order to evaluate wether or not clinical heterogeneity does actually occur, (b) identify nongenetic factors (maternal age, multiparity) that may affect such an evolution, and (c) identify neuropathologic events underlying clinical heterogeneity. Materials and methods Mice of the hyh strain (B6C3Fe-a/a-hyh) were used in this investigation. The expression of hydrocephalic phenotype was studied in 1690 hyh mice (231 litters). The clinical evolution of hydrocephalic mice was achieved following up 79 postnatal (PN) hydrocephalic mice, from PN-1 to PN-180. Brain samples of hydrocephalic and non-hydrocephalic mice were studied at different developmental stages with several methods, including light microscopy, immunocytochemistry and scanning electron microscopy. Results In agreement with a monogenetic mendelian recessive disease, 22.4% of newborns developed the hydrocephalic phenotype. The male:female ratio was 1 in non-hydrocephalic mice and 2 in hydrocephalic mice. Multiparous females, as compared to primiparous, had litters with a significant reduction of both, frequency of hydrocephalus and sex ratio. Maternal age did not affect these parameters. Two mortality profiles were identified: (i) 70% of hydrocephalic mice died during the first 8 postnatal weeks and (ii) 30% died during the following months with more than 10% still surviving up to 7 months. The degree of severity of the pathology, as evaluated by the rates of body weight increase and mortality, was higher in males than in females. These results lead us to identify two major forms of clinical evolution, namely (a) rapidly progressive, and (b) slowly progressive. The neuropathological analysis showed that during the first 2 PN months the severity of hydrocephalus was variable ranging from moderate (communicating) hydrocephalus to a very severe (non-communicating) hydrocephalus. A common feature to all pathological groups was ependymal denudation. However, these groups differ in several aspects such as (i) precocity of the onset of aqueductal obliteration; (ii) nature and degree of alterations of periventricular structures, such as the hypothalamus; (iii) presence or absence of spontaneous communications between ventricles and subarachnoid space; (iv) intraventricular haemorrhages and (v) mesencephalic compression. Aspects i, iv, and v showed a high correlation with early mortality, whereas spontaneous ventriculostomies together with the absence of ventricular haemorrhages were associated with a less severe or arrested pathological process leading to a longterm hydrocephalus. Conclusion It is concluded that (1) there are nongenetic (epigenetic) factors related with maternal multiparity (hormones? lactation?) that influence the expression of the mutation, (2) there are sex-related factors (genetic? hormonal?) that determine a higher frequency and severity of the disease in males, (3) there is a correlation between early or late mortality and the nature of the CNS alterations, and (4) the hyh mutant appears as a unique animal model to investigate long-term hydrocephalus [ABSTRACT FROM AUTHOR]

Background In all species developing congenital hydrocephalus in which the SCO has been investigated, changes in the SCOReissner fibre (RF) complex have been reported. However, the question whether these changes precede hydrocephalus, or are a consequence of it, has not been fully clarified. We have reported that in the rat, the maternal transfer of antibodies against RF-glycoproteins to the foetuses and to the pups prevents RF formation and leads to aqueductal stenosis and hydrocephalus [1]. This finding gave support to the early hypothesis of Overholser et al. [2] who had proposed that a maldevelopment of the SCO may result in hydrocephalus. We have now designed new experimental protocols to further test this hypothesis. Materials and methods Since in the rat the first RF is formed around PN-7, we designed a protocol for the postnatal immunoneutralization of the SCO. Antibodies against RF-glycoproteins were perfused into the CSF at PN-2 and PN-5. At PN-30, 74 % of these rats were devoid of RF, had aqueductal stenosis and had developed hydrocephalus. A different strategy has been the study of the SCO in two mutant species developing congenital hydrocephalus, the hyh mouse and the HTx rat. During the first postnatal week the SCO of normal hyh mice forms the first Reissner fibre whereas that of the hydrocephalic littermates does not; the absence of RF preceded the obliteration of the Sylvius aqueduct and the development of a severe hydrocephalus. Similarly, the HTx rat lacks a RF despite having an active SCO. Since the absence of RF appears as a key event in the development of hydrocephalus, studies were performed to clarify the mechanism responsible for the lack of formation of RF. Results It was found (i) that the SCO of hyh mice has a decrease expression of SCO-spondin, the main constituent protein of RF; (ii) an alteration in the pattern of the secretory proteins present in the SCO, suggesting an abnormal processing or degradation. We have succeeded to detect and identify, for the first time, the SCO secretory proteins present in the CSF. It was found that the SCO of the hyh mice and the HTx rats secretes abnormal CSF-soluble proteins, and in the case of the HTx rats these proteins were more abundant. This is surprising since in the HTx rat only the supracommissural portion of the SCO develops; this implies that only about 20–30 % of the secretory cells present in this mutant account for the increased quantity of secretory proteins present in the CSF. In the HTx there are SCO secretory products that are present in the ventricular CSF and missing from the subarachnoidal CSF. Thus, the CSF of both hydrocephalic species presents abnormalities in the quantity and quality of the SCO secretory proteins. Preliminary evidence indicates that CSF of children with congenital hydrocephalus contains secretory proteins which are missing from the CSF of nonhydrocephalic children. Conclusion 1. A primary alteration in the SCO of the mutants hyh and HTx leads to: (i) a modification in the expression of its secretory proteins; (ii) an alteration in the processing of these proteins which, once released into the CSF, do not aggregate normally, resulting in the absence of RF and in the presence in the CSF of abnormal forms of such proteins. 2. The subarachnoidal CSF of HTx rats does not contain certain SCO secretory products. 3. Detection in the human CSF of compounds that would correspond to SCO secretion may open a new field of research in human congenital hydrocephalus. [ABSTRACT FROM AUTHOR]

Background Recent investigations carried out in natural and experimental mutant mice have provided strong evidence that a primary alteration of the ependymal cell lineage triggers a moderate foetal hydrocephalus [1,2]. In human cases of hydrocephalus, however, ependymal loss has been regarded as resulting from the ventricular dilatation due to the accumulation of cerebrospinal fluid [3]. Materials and methods The present investigation was carried out in 16–40 week old human foetuses with a communicating hydrocephalus and displaying a moderate dilatation of the ventricular cavities (n = 8), and foetuses of similar ages with no neuropathological alterations (n = 15). Paraffin sections throughout the walls of the cerebral aqueduct and lateral ventricles were processed for lectin binding and immunocytochemistry using ependyma, astroglia, neuroblasts and macrophague markers. Results Large areas of ependymal denudation were found in the aqueduct and lateral ventricles of all foetuses developing a communicating hydrocephalus. At variance, no ependymal detachment was observed in non-hydrocephalic foetuses. In the youngest foetuses with hydrocephalus, denuded areas were not covered by astrocytes or other organized cell elements, leaving the neuropile directly exposed to the ventricular lumen. The area devoid of ependyma increased as the foetus developed. In the oldest foetuses studied, the denuded areas of the lateral ventricles were lined by a dense plexus of astrocytes. Under the denuded surface the presence of ependymal rosettes was observed. In the denuded areas of the lateral ventricles of hydrocephalic foetuses it was found (i) a loss of the germinal ependymal zone, (ii) disorganization of the subventricular zone and, (iii) abnormal migration of neuroblasts into the ventricular cavity. Conclusion The early loss of ependyma in human hydrocephalic foetuses would be associated to both, the hydrocephalic process and an abnormal migration of neuroblasts. [ABSTRACT FROM AUTHOR]

Background Hyh mutant mice suffer a congenital hydrocephalus triggered by ependyma denudation [1]. The ventricular surface in non hydrocephalic newborn mice is lined by the immature ependyma, which is characterized for being vimentin (-) and S100ß (-), at variance in the adult animals the mature ependyma expresses vimentin and S100ß [2]. On the other hand, in the hydrocephalic mice the ependyma begins to denudate on the 12th day of gestation, and at PN8 only some areas of lateral ventricle are still endowed with ependyma. In parallel, astroglia starts to cover the denuded surface forming a new cell layer, the glial scar, which lines the damaged ventricular surface. We have studied the permeability to horseradish peroxidase (HRP) of these four regions at the ventricular wills: mature ependyma, and denuded areas with or without glial scar. Materials and methods Control and hydrocephalic hyh mice (Jackson Lab., USA) at 3rd and 30th day of post-natal life were injected into a lateral ventricle with 3% HRP. 15 min after the injection the animals were sacrificed under anesthesia. HRP was detected by immunocitochemistry with specific antibodies. Inmunocitochemistry for PCNA (to label proliferating cells) and GFAP, S100ß and vimentin was used Results In non-hydrocephalic mice the immature ependymal layer was impermeable to HRP, whereas the mature ependyma was permeable. In hydrocephalic animal the areas where the ependyma had detached and the glial scar had not yet form were permeable to HRP, diffusing through the parenchyma. The glial scar was recognized for being GFAP positive and surprisingly, vimentin positive. When this barrier was fully developed at PN-30, it was apparently impermeable. However, the presence in the neuropile of cells labelled with HRP might indicates that some HRP has passaged through the glial scar. In adult hydrocephalic animals, there are zones where the ependyma is not denuded. This ependyma and the neighbouring glial scar appear impermeable to HRP. However, the HRP labelling of subventricular structures in these levels suggest that some tracer has passed through. Conclusion The different permeability properties between mature and immature ependymal layers suggest that differences exist in cell adhesion features and permeability. In hydrocephalic mice, denudated areas devoid of glial scar are very permeable to HRP. Thus, ependymal denudation implies the loss of CSF-parenchyma barrier, which could influence the CNS development. In adult hidrocephalic mice there are ependimal patches that do not detach. This particular ependyma, as the glial layer lining the denuded area, prevents partially or completely the passage of HRP. The HRP labelling of subventricular structures in this two regions could be an indication that some HRP has passed through the non-detached ependyma and through the glial sheath by an as yet unknown mechanism. This suggests that these ependymal areas could correspond to an would be a tight ependyma, and that such an ependyma would have the same barrier properties as those of the glial scar. What actually are these barrier properties are being further investigated in our laboratory. specific ependyma population that in the normal animal would be a tight ependyma, and that such an ependyma would have the same barrier properties as those of the glial scar. What actually are these barrier properties are being further investigated in our laboratory. [ABSTRACT FROM AUTHOR]

Background Hyh mutant mice suffer a congenital hydrocephalus triggered by ependyma denudation [1]. Additional pathological events have been observed: the absence of corpus callosum (ACC) and the reduction in the thickness of the cerebral cortex in this mutant. These two alterations frequently appear associated with human and animal hydrocephalus. Crossing the midline by callosal axons requires the presence of three types of midline glial cells (glial wedge, glial sling and indusium griseum glial cells) and also the correct guidance provided by pioneering axons. This crossing occurs about E-16.5 and pioneering axons appear around E-15.5 [2]. Development of cerebral cortex begins at E-12, continues until postnatal life, and requires proliferation and migration of progenitor cells from subventricular regions. The aim of this work is to clarify the nature of the relationship between hydrocephalus and ACC and abnormal neurogenesis. Materials and methods Control and hydrocephalic hyh mice (Jackson Lab., USA) were used. To study the ACC mice from E-15.5 to PN-1 were used. Antibodies against NCAM (callosal axons), GFAP (midline glial cells) and ß-III tubulin (neuroblast) were used. DiI tracing at E-17.5 were used to show crossing of midline by callosal axons. Alteration of the cerebral cortex was studied from 1 to 120 postnatal days. Digital photographs were taken and Noesis Visiolog software was used to measure the thickness of the cerebral cortex. Data obtained were statistically processed using Microsoft Office Excel and Sigmastat32 software. The ventricular surface was studied at E-15.5 by scanning electron microscopy. Organotypic slice culture and DiI labelling at E15.5 were used to analyze growth of pioneering axons and migration of neuroblast [2,3]. Results At PN-1 hydrocephalic mice, corpus callosum is missing. In these animals, however, other commissural formations are present and the lateral ventricles are collapsed, indicating that the ACC is a phenomenon preceding lateral ventricles dilatation. In addition, DiI tracing at E-17.5 shows that in hydrocephalic animals callosal axons do not cross the midline. This led us to study the midline glial cells and pioneering axons. It was found that in E16.5 hydrocephalic mice, the midline glial populations are altered and alterations appear to be associated with the denudation of discrete areas of the lateral ventricles. In hydrocephalic mice, pioneering axon elongation, labelled with DiI at E15.5, has a wrong direction and do not cross the midline. In hydrocephalic mice, cerebral cortex shows a statically significant reduction of its thickness that is especially significant at PN-3, when dilatation on lateral ventricles is not yet apparent. In addition, neuroblast-like cells are detected on the ventricular surface of hydrocephalic mice by use of immunochemistry, scanning electron microscopy, and DiI labelling of organotypic slice culture. [ABSTRACT FROM AUTHOR]

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.

In mutant rodents, ependymal denudation occurs early in fetal life, preceding the onset of a communicating hydrocephalus, and is a key event in the etiology of this disease. The present investigation was designed to obtain evidence whether or not ependymal denudation occurs in 16- to 40-week-old human fetuses developing a communicating hydrocephalus (n = 8) as compared to fetuses of similar ages with no neuropathologic alterations (n = 15). Sections through the walls of the cerebral aqueduct and lateral ventricles were processed for lectin binding and immunocytochemistry using antibodies against ependyma, astroglia, neuroblasts, and macrophages markers. Anticaveolin was used as a functional marker of the fetal ependyma. The structural and functional molecular markers are differentially expressed throughout the differentiation of the human fetal ependyma. Denudation of the ependyma of the aqueduct and lateral ventricles occurred in all fetuses developing a communicating hydrocephalus, including the youngest ones studied. The denuded surface area increased in parallel with the fetus age. The possibility is advanced that in many or most cases of human fetal hydrocephalus there is a common defect at the ependymal cell lineage leading to ependymal detachment. Evidence was obtained that in hydrocephalic human fetuses a process to repair the denuded areas takes place during the fetal life. In hydrocephalic fetuses, detachment of the ependyma of the lateral ventricles resulted in the (i) loss of the germinal ependymal zone, (ii) disorganization of the subventricular zone and, (iii) abnormal migration of neuroblasts into the ventricular cavity. Thus, detachment of the ependymal layer in hydrocephalic fetuses would not only be associated with the pathogenesis of hydrocephalus but also to abnormal neurogenesis.