The cytopathology of reactions to ventricular shunts

A common cause of malfunctioning ventricular shunts is the occlusion of either tip by a variety of normal or reactive tissues and foreign substances. A six-year-old girl with communicating hydrocephalus and a meningomyelocele, a 48-year-old man with an ependymoma and an 11-year-old boy with a pineal germinoma had multinucleated histiocytic giant cells and ependymal cells in cerebrospinal fluid obtained from their ventricular shunts. These cellular changes were interpreted as the cytologic counterpart of the foreign-body inflammatory reactions often seen histologically on occluded shunt tips. Numerous clusters of benign choroid plexus epithelium were found in an ascitic fluid from a six-year-old girl with an optic nerve glioma and a ventriculoperitoneal shunt. Such embolism of normal tissues must be distinguished from metastases from intracranial neoplasms.     

Junctional Adhesion Molecule (JAM)-C Deficient C57BL/6 Mice Develop a Severe Hydrocephalus

The junctional adhesion molecule (JAM)-C is a widely expressed adhesion molecule regulating cell adhesion, cell polarity and inflammation. JAM-C expression and function in the central nervous system (CNS) has been poorly characterized to date. Here we show that JAM-C^−/− mice backcrossed onto the C57BL/6 genetic background developed a severe hydrocephalus. An in depth immunohistochemical study revealed specific immunostaining for JAM-C in vascular endothelial cells in the CNS parenchyma, the meninges and in the choroid plexus of healthy C57BL/6 mice. Additional JAM-C immunostaining was detected on ependymal cells lining the ventricles and on choroid plexus epithelial cells. Despite the presence of hemorrhages in the brains of JAM-C^−/− mice, our study demonstrates that development of the hydrocephalus was not due to a vascular function of JAM-C as endothelial re-expression of JAM-C failed to rescue the hydrocephalus phenotype of JAM-C^−/− C57BL/6 mice. Evaluation of cerebrospinal fluid (CSF) circulation within the ventricular system of JAM-C^−/− mice excluded occlusion of the cerebral aqueduct as the cause of hydrocephalus development but showed the acquisition of a block or reduction of CSF drainage from the lateral to the 3^rd ventricle in JAM-C^−/− C57BL/6 mice. Taken together, our study suggests that JAM-C^−/− C57BL/6 mice model the important role for JAM-C in brain development and CSF homeostasis as recently observed in humans with a loss-of-function mutation in JAM-C.     

Myosin IXa Regulates Epithelial Differentiation and Its Deficiency Results in Hydrocephalus

The ependymal multiciliated epithelium in the brain restricts the cerebrospinal fluid to the cerebral ventricles and regulates its flow. We report here that mice deficient for myosin IXa (Myo9a), an actin-dependent motor molecule with a Rho GTPase–activating (GAP) domain, develop severe hydrocephalus with stenosis and closure of the ventral caudal 3rd ventricle and the aqueduct. Myo9a is expressed in maturing ependymal epithelial cells, and its absence leads to impaired maturation of ependymal cells. The Myo9a deficiency further resulted in a distorted ependyma due to irregular epithelial cell morphology and altered organization of intercellular junctions. Ependymal cells occasionally delaminated, forming multilayered structures that bridged the CSF-filled ventricular space. Hydrocephalus formation could be significantly attenuated by the inhibition of the Rho-effector Rho-kinase (ROCK). Administration of ROCK-inhibitor restored maturation of ependymal cells, but not the morphological distortions of the ependyma. Similarly, down-regulation of Myo9a by siRNA in Caco-2 adenocarcinoma cells increased Rho-signaling and induced alterations in differentiation, cell morphology, junction assembly, junctional signaling, and gene expression. Our results demonstrate that Myo9a is a critical regulator of Rho-dependent and -independent signaling mechanisms that guide epithelial differentiation. Moreover, Rho-kinases may represent a new target for therapeutic intervention in some forms of hydrocephalus.     

The PDZ Protein Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) Regulates Planar Cell Polarity and Motile Cilia Organization

Directional flow of the cerebrospinal fluid requires coordinated movement of the motile cilia of the ependymal epithelium that lines the cerebral ventricles. Here we report that mice lacking the Na⁺/H⁺ Exchanger Regulatory Factor 1 (NHERF1/Slc9a3r1, also known as EBP50) develop profound communicating hydrocephalus associated with fewer and disorganized ependymal cilia. Knockdown of NHERF1/slc9a3r1 in zebrafish embryos also causes severe hydrocephalus of the hindbrain and impaired ciliogenesis in the otic vesicle. Ultrastructural analysis did not reveal defects in the shape or organization of individual cilia. Similar phenotypes have been described in animals with deficiencies in Wnt signaling and the Planar Cell Polarity (PCP) pathway. We show that NHERF1 binds the PCP core genes Frizzled (Fzd) and Vangl. We further show that NHERF1 assembles a ternary complex with Fzd4 and Vangl2 and promotes translocation of Vangl2 to the plasma membrane, in particular to the apical surface of ependymal cells. Taken together, these results strongly support an important role for NHERF1 in the regulation of PCP signaling and the development of functional motile cilia.     

The secretory ependymal cells of the subcommissural organ: Which role in hydrocephalus?

Ependyma in the central nervous system gives rise to several specialized cell types, including the secretory ependymal cells located in the subcommissural organ. These elongated cells show large cisternae in their cytoplasm, which are filled with material secreted into the cerebrospinal fluid and toward the leptomeningeal spaces. A specific secretion of the subcommissural organ was named SCO-spondin, regarding its marked homology with developmental proteins of the thrombospondin superfamily (presence of thrombospondin type 1 repeats). The ependymal cells of the subcommissural organ and SCO-spondin secretion are suspected to play a crucial role in cerebrospinal fluid flow and/or homeostasis. There is a close correlation between absence of the subcommissural organ and hydrocephalus in rat and mouse strains exhibiting congenital hydrocephalus, and in a number of mice transgenic for developmental genes. The ependymal cells of the subcommissural organ are under research as a key factor in several developmental processes of the central nervous system.     

The importance of the intrathecal infusion test in the diagnostics of normal pressure hydrocephalus.

OBJECTIVE: The intrathecal infusion test is a reliable method in diagnosing normal pressure hydrocephalus. METHODS: Between May 1982 and January 1997 we investigated 200 patients suspected for a normal pressure hydrocephalus (NPH) by carrying out an intrathecal infusion test in a constant flow technique. The resistance to cerebrospinal fluid outflow (Rout) in the intrathecal infusion test was the main criterion for grouping patients into these with normal pressure hydrocephalus or those with cerebral atrophy. A further differentiation into early stage and late stage was made by measuring the compliance (Cp)--this being the secondary criterion. RESULTS: In 107 patients (54%) the diagnosis of a NPH could be confirmed. Of these, 102 patients (95%) underwent a shunt operation. Graduation of NPH and cerebral atrophy following the results of the infusion test in an early stage and an advanced stage allows the conclusion of prognostic evaluations about the course of disease to be made. Patients with a NPH in an early stage are reporting in the follow up about an improvement of their symptoms after a shunt operation in 65 percent of cases and those with an advanced stage NPH in 50 percent. CONCLUSION: The computer aided infusion test allows a reliable differentiation between patients with NPH and those with cerebral atrophy.     

Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus

Cilia are complex organelles involved in sensory perception and fluid or cell movement. They are constructed through a highly conserved process called intraflagellar transport (IFT). Mutations in IFT genes, such as Tg737, result in severe developmental defects and disease. In the case of the Tg737orpk mutants, these pathological alterations include cystic kidney disease, biliary and pancreatic duct abnormalities, skeletal patterning defects, and hydrocephalus. Here, we explore the connection between cilia dysfunction and the development of hydrocephalus by using the Tg737orpk mutants. Our analysis indicates that cilia on cells of the brain ventricles of Tg737orpk mutant mice are severely malformed. On the ependymal cells, these defects lead to disorganized beating and impaired cerebrospinal fluid (CSF) movement. However, the loss of the cilia beat and CSF flow is not the initiating factor, as the pathology is present prior to the development of motile cilia on these cells and CSF flow is not impaired at early stages of the disease. Rather, our results suggest that loss of cilia leads to altered function of the choroid plexus epithelium, as evidenced by elevated intracellular cAMP levels and increased chloride concentration in the CSF. These data suggest that cilia function is necessary for regulating ion transport and CSF production, as well as for CSF flow through the ventricles.     

The effect of ventricular volume increase in the amplitude of intracranial pressure

We study the impact of vascular pulse in the cerebrospinal fluid (CSF) pressure measured on the lateral cerebral ventricles, as well as its sensitivity with respect to ventricular volume change. Recent studies have addressed the importance of the compliance capacity in the brain and its relation to arterial pulse abortion in communicating hydrocephalus. Nevertheless, this mechanism is not fully understood. We propose a fluid-structure interaction (FSI) model on a 3?D idealized geometry based on realistic physiological and morphological parameters. The computational model describes the pulsatile deformation of the third ventricle due to arterial pulse and the resulting CSF dynamics inside brain pathways. The results show that when the volume of lateral ventricles increases up to 3.5 times, the amplitudes of both average and maximum pressure values, computed on the lateral ventricles surface, substantially decrease. This indicates that the lateral ventricles expansion leads to a dumping effect on the pressure exerted on the walls of the ventricles. These results strengthen the possibility that communicant hydrocephalus may, in fact, be a natural response to reduce abnormal high intracranial pressure (ICP) amplitude. This conclusion is in accordance with recent hypotheses suggesting that communicant hydrocephalus is related to a disequilibrium in brain compliance capacity.     

Intraneural vascular response to defective development of the chick mesencephalon

The development of intraneural vessels was studied in response to an induced hypermorphosis of neural tissue inthe midbrains of 38 chick embryos ranging in age from three days through 14 days of incubation. The pattern of vascularization was compared with that of normal chick embryos at comparable stages of development. In the experimental embryos, the increase in mitotic figures along the ventricular borders of the mesencephalon is accompanied by the establishment of an endoneural plexus approximately one day earlier than is the case during normal vascularization of the midbrain. This plexus also penetrates more deeply and extensively into the ependymal layer. Surface vessels and intraneural vascular elements are dilated, and the cerebrospinal fluid contains varying amounts of blood released from large intraneural vessels which protrude into the ventricle. The most prominent cerebrovascular effects seem to occur between the fourth and eighth days of incubation. Thereafter, the cerebrovascular pattern becomes more normal except for relatively few isolated hemorrhagic areas.     

Altered pH(i) regulation and Na(+)/HCO3(-) transporter activity in choroid plexus of cilia-defective Tg737(orpk) mutant mouse

Tg737^orpk mice have defects in cilia assembly and develop hydrocephalus in the perinatal period of life. Hydrocephalus is progressive and is thought to be initiated by abnormal ion and water transport across the choroid plexus epithelium. The pathology is further aggravated by the slow and disorganized beating of motile cilia on ependymal cells that contribute to decreased cerebrospinal fluid movement through the ventricles. Previously, we demonstrated that the hydrocephalus phenotype is associated with a marked increase in intracellular cAMP levels in choroid plexus epithelium, which is known to have regulatory effects on ion and fluid movement in many secretory epithelia. To evaluate whether the hydrocephalus in Tg737^orpk mutants is associated with defects in ion transport, we compared the steady-state pH_i and Na⁺-dependent transport activities of isolated choroid plexus epithelium tissue from Tg737^orpk mutant and wild-type mice. The data indicate that Tg737^orpk mutant choroid plexus epithelium have lower pH_i and higher Na⁺-dependent HCO₃^– transport activity compared with wild-type choroid plexus epithelium. In addition, wild-type choroid plexus epithelium could be converted to a mutant phenotype with regard to the activity of Na⁺-dependent HCO₃^– transport by addition of dibutyryl-cAMP and mutant choroid plexus epithelium toward the wild-type phenotype by inhibiting PKA activity with H-89. Together, these data suggest that cilia have an important role in regulating normal physiology of choroid plexus epithelium and that ciliary dysfunction in Tg737^orpk mutants disrupts a signaling pathway leading to elevated intracellular cAMP levels and aberrant regulation of pH_i and ion transport activity. cAMP; ion transport     

Fenestration of the epithelium lining the roof of the fourth cerebral ventricle in amphibia

Summary Scanning electron microscopy of the caudal end of the roof of the fourth cerebral ventricle in four amphibian species shows that numerous pores occur between the ependymal cells. These pores have diameters ranging from 5–100 m; they permit bulk flow of cerebrospinal fluid out of the ventricular system into the subarachnoid space.     

Expression of certain proteins in the subfornical organ and cerebrospinal fluid of spontaneously hypertensive rats

The objective of this study was to analyze the proteins in the cerebrospinal fluid of spontaneously hypertensive rats and to study their possible role in the relationship between hydrocephalus, arterial hypertension and variations in the subfornical organ. Brains and cerebrospinal fluid from control Wistar-Kyoto rats and spontaneously hypertensive rats sacrificed with chloral hydrate were used. Cerebrospinal fluid and extract of subfornical organ were processed by protein electrophoresis. Antisera against protein bands of 141, 117 and 48 kDa and Concanavalin A were used for immunohistochemical and western blot study of the subfornical organ, adjacent circumventricular structures and cerebrospinal fluid. Ventricular dilation in the spontaneously hypertensive rats and the presence of quite a lot of protein bands in the cerebrospinal fluid of the hypertensive rats, which were either not observed or scarcely present in the cerebrospinal fluid of the Wistar-Kyoto rats, were confirmed. The subfornical organ, third ventricle ependyma and choroideus plexus showed immunoreactive material for antibodies against 141kDa, 117 and 48 kDa proteins band (anti-B1, anti-B2 and anti-B3). The larger amount of the immunoreactive material was found in the subfornical organ of the spontaneously hypertensive rat. Our results and the alterations observed by other authors in the subfornical organ in hydrocephalic and hypertensive rats support the possibility that this circumventricular organ, some proteins of the cerebrospinal fluid and ventricular dilation could be connected with the physiopathology of this type of hypertension.     

Combined transport of water and ions through membrane channels

The coupling of ion and water flow through membrane channels is under dispute. Among all human aquaporins only aquaporin-6 exhibits ion channel activity. Whether aquaporin-6 functions also as a water channel cannot yet be determined with confidence. Similarly, a comparison of single-channel water permeabilities from ion channels and aquaporins suggests that ion channels may play a secondary role as water channels. However, the fraction of absorbed fluid that crosses epithelial ion channels still remains to be determined.     

The pathogenesis of normal pressure hydrocephalus: A theoretical analysis

Hydrocephalus is an abnormal accumulation of cerebrospinal fluid (CSF) in the cerebral ventricles, usually caused by impaired absorption of the fluid into the bloodstream. Despite obstructed absorption and continued secretion of CSF into the ventricles at a near normal rate, the ventricular CSF pressure (VCSFP) is often normal. We attempt to understand how hydrocephalus can exist with normal VCSFP by exploring the role of the brain parenchyma in absorbing CSF in hydrocephalus. We test three theories: (1) the ventricular wall is impermeable to CSF; (2) ventricular CSF seeps into the parenchyma, from which it is efficiently absorbed; and (3) ventricular CSF seeps into the parenchyma but is absorbed inefficiently. We model the brain as a thick spherical shell consisting of a porous, elastic, solid matrix, containing interstitial fluid and blood. We modify the equations of poroelasticity, which describe flow of fluid through porous solids, to allow for parenchymal absorption. For each of the three theories we calculate the steady state changes in VCSFP and in parenchymal fluid pressure caused by an incremental defect in CSF absorption. We also calculate the steady state changes in fluid content, tissue volume, tissue displacement, and stresses caused by a small increment of VCSFP. We conclude that only the second theory—seepage of CSF with efficient parenchymal absorption—accounts for the clinical features of normal pressure hydrocephalus. These features include sustained ventricular dilatation despite normal VCSFP, increased periventricular fluid content, and localized periventricular white matter damage.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na(+)/K(+)-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Genetic Deletion of Afadin Causes Hydrocephalus by Destruction of Adherens Junctions in Radial Glial and Ependymal Cells in the Midbrain

Adherens junctions (AJs) play a role in mechanically connecting adjacent cells to maintain tissue structure, particularly in epithelial cells. The major cell–cell adhesion molecules at AJs are cadherins and nectins. Afadin binds to both nectins and α-catenin and recruits the cadherin-β-catenin complex to the nectin-based cell–cell adhesion site to form AJs. To explore the role of afadin in radial glial and ependymal cells in the brain, we generated mice carrying a nestin-Cre-mediated conditional knockout (cKO) of the afadin gene. Newborn afadin-cKO mice developed hydrocephalus and died neonatally. The afadin-cKO brain displayed enlarged lateral ventricles and cerebral aqueduct, resulting from stenosis of the caudal end of the cerebral aqueduct and obliteration of the ventral part of the third ventricle. Afadin deficiency further caused the loss of ependymal cells from the ventricular and aqueductal surfaces. During development, radial glial cells, which terminally differentiate into ependymal cells, scattered from the ventricular zone and were replaced by neurons that eventually covered the ventricular and aqueductal surfaces of the afadin-cKO midbrain. Moreover, the denuded ependymal cells were only occasionally observed in the third ventricle and the cerebral aqueduct of the afadin-cKO midbrain. Afadin was co-localized with nectin-1 and N-cadherin at AJs of radial glial and ependymal cells in the control midbrain, but these proteins were not concentrated at AJs in the afadin-cKO midbrain. Thus, the defects in the afadin-cKO midbrain most likely resulted from the destruction of AJs, because AJs in the midbrain were already established before afadin was genetically deleted. These results indicate that afadin is essential for the maintenance of AJs in radial glial and ependymal cells in the midbrain and is required for normal morphogenesis of the cerebral aqueduct and ventral third ventricle in the midbrain.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na⁺/K⁺-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Histochemistry of proteases in ependyma, choroid plexus and leptomeninges

Aminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV) and gamma-glutamyl transferase (GGT) were demonstrated histochemically in cryostat sections of the rat brain to show the reaction pattern of ependyma, choroid plexus and leptomeninges. GGT was only demonstrable in the cell membranes of ependymal cells and in the leptomeninges; however, APA, APM and DAP IV showed a variable degree of activity in the capillary endothelium of the choroid plexus as well as in the leptomeninges. On the basis of these results, it is postulated that peptides in the cerebrospinal fluid can be cleaved extraventricularly by the enzymes demonstrated in the leptomeninges.     

The subcommissural organ expresses D<Subscript>2</Subscript>, D<Subscript>3</Subscript>, D<Subscript>4</Subscript>, and D<Subscript>5</Subscript> dopamine receptors

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 Reissners 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.Financial support was provided by grants PI 030756 and Red CIEN, Instituto de Salud Carlos III, Spain (to J.M.P.F.), and 1030265 from Fondecyt, Chile (to E.M.R.)