The Functional Neuroanatomy of the Cortical Subarachnoid Space- Brain Surface Interface

Dept. Physiology, Membrane permeability Lab Medical School, University of Thessaly, GREECE

current affiliation: Dept. Neurosurgery, Marmarou Lab, VCU, USA

Living on the edge: The functional neuroanatomy of the cortical subarachnoid space- brain surface interface

Aristotelis S. Filippidis M.D., Ph.D. Postdoctoral Fellow

Neurohydrodynamics Meeting Series, Switzerland, EPFL, www.neurohydrodynamics.com, January 2012 What is this and what is this not ?

THIS IS NOT about pure perivascular space anatomy

THIS IS beyond structures, it is about solute-coupled water transport

THIS IS about “Ion & Water mechanics”

THIS IS about identifying analogies

THIS IS about skimming for evidence

THIS IS about teleology*

*Τελεολογία = teleology: a definition induced in philosophy by Plato and Aristotle meaning that the evolution of structures happens always with a purpose

Plato and Aristotle, “The school of Athens”, Raphael 1509 Cortical Meninges Cortical Subarachnoid Space 120 E. T. ZHANG, C. B. E. INMAN AND R. 0. WELLER

Pia mater

J. Anat. (1990), 170, 111-123 111 With 1O figures Printed in Great Britain I , SWaS~~~~~~~~~~~~~~~Cre/~~~~~~~~. Interrelationships of the pia mater and the perivascular (Virchow-Robin) spaces in the human cerebrum*

E. T. ZHANG,tt C. B. E. INMANt AND R. 0. WELLERt t Neuropathology, Southampton University Medical School, UK, and t Department of Anatomy, Beijing Medical University, Beijing, China 'PF (Accepted 7 November 1989)

INTRODUCTION The original concepts regarding the relationships between the perivascular spaces in the brain and the cerebrospinal fluid in the subarachnoid space were strongly supported by the conclusions drawn by Weed (1923) from his physiological studies. Thus it was proposed that a free communication existed between the perivascular CAPS spaces and the subarachnoid space. Despite some discrepancies in the interpretation of Weed's results (see Krisch, Leonhardt & Oksche, 1984; Hutchings & Weller, 1986) histological and ultrastructural studies at first seemed to support a free communication between the subarachnoid space and the Virchow-Robin perivascular spaces Fig. 10. Diagram demonstrating the relationships of the pia mater and intracerebral blood vessels.(Woollam & Millen, 1955; Nelson, Blinzinger & Hager, 1961; Ramsey, 1965; Jones, Subarachnoid space (SAS) separates the arachnoid (A) from the pia mater overlying the cerebral1970; Rascol & Izard, 1972). cortex. An artery on the left of the picture is coated by a sheath of cells derived from the pia mater;Doubts about the free connection between these two spaces, however, were raised by the sheath has been cut away to show that the periarterial spaces (PAS) of the intracerebral theandresults of tracer experiments in which horseradish peroxidase, Evans blue-labelled extracerebral arteries are in continuity. The layer of pial cells becomes perforated (PF) albuminand or radiolabelled tracers injected into the rat brain were concentrated in the incomplete as smooth muscle cells are lost from the smaller branches of the artery. The pial sheathperivascular spaces of the middle cerebral arteries (Bradbury, Cserr & Westrop, 1981; finally disappears as the perivascular spaces are obliterated around capillaries (CAPS). PerivascularSzentistvanyi, Patlak, Ellis & Cserr, 1984; Cserr, 1988). There was a low recovery of spaces around the vein (right of picture) are confluent with the subpial space and only small numbersisotope tracer from bulk CSF. These results suggested that interstitial fluid from the of pial cells are associated with the vessel wall. brain did not drain directly into the cerebrospinal fluid of the subarachnoid space but followed perivascular pathways along major cerebral vessels in the subarachnoid space. An anatomical basis for the separation of the subarachnoid space from the into the subpial space; the absence of such a sheath was a defect in earlier perivascularmodels (Virchow-Robin) spaces has now been established in several mammalian (Krahn, 1982; Hutchings & Weller, 1986; Alcolado et al. 1988). species, including man. It has been shown that the pia mater on the surface of the brain and spinal cord is reflected on to the surface of blood vessels in the subarachnoid Although a small part of the anatomical pathway for the drainage of interstitialspace, thus separating the perivascular and subpial spaces from the subarachnoid fluid from the brain may have been clarified by the present study, the naturespaceof (Krahn,the 1982; Krisch et al. 1984; Hutchings & Weller, 1986; Alcolado, Weller, complete pathway is still unclear. Following injection into the rat brain, tracersParrishappear& Garrod, 1988; Nicholas & Weller, 1988). Nevertheless, the anatomical models suggested for animal and human brain (Krahn, 1982; Hutchings & Weller, in retropharyngeal lymph nodes (Cserr, 1988). Furthermore, obstruction1986; Alcoladoor et al. 1988) still do not correlate exactly with the results of tracer extirpation of cervical lymphatics interferes with drainage of interstitial fluid fromstudiesthein the rat (Bradbury et al. 1981; Szentistvanyi et al. 1984). There are two major discrepancies: firstly, no direct connection between the perivascular spaces of vessels brain (F6ldi, Csillik & Zoltain, 1968). From the present and previous studies itin seemsthe brain and the perivascular spaces of vessels in the subarachnoid space has been that fluid and tracers could pass along periarterial spaces in the brain and intoestablished.the Thus, with the published models, it is likely that fluid or tracers would be perivascular spaces of major arteries in the subarachnoid space. However, tracersdissipatedgo in the subpial space rather than draining directly into the perivascular no further than the entry of the carotid artery into the skull in the rat (Bradbury* etReprintal. requests to Professor R. 0. Weller, Department of Pathology (Neuropathology), Level E, 1981). Furthermore the perivascular space ends at the dura where the vertebralSoutharteryPathology Block, Southampton General Hospital, Southampton S09 4XY, UK. enters the skull in man (Alcolado et al. 1988). Thus there does not seem to be a direct 2%6)%7

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.!452%.%52/3#)%.#%6/,5-%\.5-"%2\./6%-"%2 PIAL SURFACE ARACHNOID SURFACE ARACHNOID

Arachnoid Trabeculae

Artery

Cortical Subarachnoid Space (CSAS) PIA

Filippidis et al. Transmembrane resistance and histology of isolated sheep leptomeninges Neurological Research (2010) vol. 32 (2) pp. 205 ARACHNOID

Subarachnoid space

ARTERY

PIA Pial vessel

Glia limitans BRAIN SURFACE

Filippidis et al. Transmembrane resistance and histology of isolated sheep leptomeninges Neurological Research (2010) vol. 32 (2) pp. 205

Is CSAS important for neurohydrodynamics and CSF disorders ?

J Neurosurg Pediatrics 2:1–11, 2008

The importance of the cortical subarachnoid space in understanding hydrocephalus

HAROLD L. REKATE, M.D.,1,2 TRIMURTI D. NADKARNI, M.CH.,3 AND DONNA WALLACE, R.N., M.S., C.P.N.P.1 1Pediatric Neurosciences, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix; 2Department of Neurosurgery, University of Arizona School of Medicine, Tucson, Arizona; and 3Department of Neurosurgery, King Edward Memorial Hospital, Seth Gordhandas Sunderdas Medical College, Parel, Mumbai, India

Object. In this paper the authors define the role of the cortical subarachnoid space (CSAS) in poorly understood forms of hydrocephalus to cerebrospinal fluid (CSF) dynamics to improve understanding of the importance of the CSAS and its role in selecting patients for endoscopic third ventriculostomy (ETV). The secondary purpose of this work was to define testable hypotheses to explain enigmatic disorders of CSF dynamics and to suggest how these concepts could be tested. Methods. The magnitude of the contribution of the CSAS is explored using the solid geometry of concentric spheres. With this starting point, clinical conditions in which CSF dynamics are not easily understood are explored regarding the potential role of the CSAS. Overall, problems of CSF dynamics are easily understood. Insights may be gained when the results of a pathological process or its treatment vary from what has been expected. Results. Acute changes in ventricular volume at the time that hydrocephalus develops, the failure of shunts, and the changes in ventricular volume with shunt repair may occur very rapidly. Changes in the volume of water in the brain, especially in the brain substance itself, are unlikely to occur at this rapid rate and may be interpreted as a simple redis- tribution of the CSF between the ventricle and CSAS with no initial change in the actual volume of brain parenchyma. Problems such as pseudotumor cerebri, shunt failure with nonresponsive ventricles, and negative-pressure hydrocephalus can be explained by assessing the ability of ventricular CSF to flow to the CSAS and the ability of this fluid to exit this compartment. Ventricular enlargement at the time of shunt failure implies a failure of flow between the ventricles and CSAS, implying that all patients who show this phenomenon are potential candidates for ETV. Conclusions. The important role of the CSAS in the pathophysiology of various forms of hydrocephalus has been largely ignored. Attention to the dynamics of the CSF in this compartment will improve understanding of enigmatic conditions of hydrocephalus and improve selection criteria for treatment paradigms such as ETV. These concepts lead to clearly defined problems that may be solved by the creation of a central database to address these issues. (DOI: 10.3171/PED/2008/2/7/001)

KEY WORDS •biophysics•cortical subarachnoid space• endoscopic third ventriculostomy • hydrocephalus • mathematical model • pseudotumor cerebri

OTH in the physiology laboratory and in clinical prac- final compartment is the SSAS, which lies outside this vol- tice, we have been studying the pathophysiology of umetric constraint.31,35,36 Our early work on this model es- B hydrocephalus based on a mathematical model of sentially ignored the role of the CSAS in the pathophysiol- CSF dynamics. In this multicompartmental model, the skull ogy of abnormalities of CSF dynamics. Instead, changes in is a fixed volume and the 4 ventricles, CSAS, and brain pa- the volume of the ventricles were explained by changes in renchyma are constrained by the volume of the skull. The the actual brain parenchyma itself.26 Later we found that al- most all patients with ventricles that did not expand at the time of shunt failure could be shown to have ventricular Abbreviations used in this paper: CSAS = cortical subarachnoid space; CSF = cerebrospinal fluid; ETV = endoscopic third ventricu- CSF in communication with the upper SSAS and CSAS. lostomy; EVD = external ventricular drain; ICP = intracranial pres- This finding led to the thought that we had underestimated sure; LP = lumboperitoneal; NPH = normal-pressure hydrocephalus; the role played by the CSAS in the pathophysiology of var- NVH = normal-volume hydrocephalus; SSAS = spinal subarachnoid ious forms of hydrocephalus and other abnormalities of space; SVS = slit ventricle syndrome. CSF dynamics.34

J. Neurosurg.: Pediatrics / Volume 2 / July 2008 1 So… The arachnoid and pia mater, line a preformed space (CSAS), with a biological fluid (CSF) which consists of 99% WATER. We need to address the relationship of CSAS with Water… Let’s move on to the cellular level Let’s talk about solute-coupled transport of water Solute-coupled water transport: the analogy Fluid movement, CFD In Macro scale Pressure gradients & pulse amplitude important Water movement Shaffer et al. Cerebrospinal fluid hydrodynamics in type I Chari malformation In Cellular scale Osmotic gradient & cellular permeability important

H2O

Figure 6 Stream traces colored by velocity magnitude from a computational fluid dynamics model of the inferior cranial and superior spinalSchaffer subarachnoid N., spaces Martin of a healthy B., subject Loth geometry F. by Gupta et al.53 demonstrating the three-dimensional complexity of cerebrospinalCerebrospinal fluid motion fluid inhydrodynamics that region. Tracer particlesin type wereI Chiari injected malformation at Plane A, which intersected the basal “Ion & Water Mechanics” pontine and cerebellomedullary cisterns. Neurological Research (2011), 33:3, 247-260 conducting simulations within the spinal subarach- IChiarimalformation.Roldanet al.54 simulated noid space. cerebrospinal fluid flow in rigid geometrically realistic spinal subarachnoid space models based Models of cerebrospinal fluid hydrodynamics — type I Chiari malformation on magnetic resonance images from the spinal canal of a patient and a healthy volunteer. The study Recently, two numerical simulations have been repor- employed the boundary element method, which ted in geometries representative of a patient with type neglects inertial effects, to solve the Navier–Stokes equations. Peak systole and peak diastole were modeled separately as steady flow. The pressure gradient between the inlet and outlet was found to be steeper in the patient model than in a healthy model of the same length. Peak pressures in the patient model were 1.5 times higher than the healthy model. Flow fields were heterogeneous with fluid jets observed anterolaterally to the spinal cord in both models. Qualitatively these results were consistent with findings from several phase-contrast

Published by Maney Publishing (c) W. S. & Son Limited imaging studies.10,25,37 Significant velocity vector components were observed perpendicular to the long axis of the spinal canal (Fig. 7). Linge et al.55 produced similar results using a geometrically idealized model of the posterior cranial fossa and cervical spine. This study examined the effect of anatomic variation on cerebrospinal fluid hydrodynamics. Though the geometry was idealized, spatial variations in flow patterns resembled those observed in in vivo phase-contrast imaging studies. The simulated pressure pulse (0.75 mmHg peak-to- Figure 7 Cerebrospinal fluid velocity patterns obtained peak amplitude) compared favorably with in vivo from a computational fluid dynamics simulation by Roldan cerebrospinal fluid pulse pressure measurements in 54 et al. of flow at peak systole in six different axial levels in a healthy subjects (1–4 mmHg56–58). Other observa- healthy model (left column) and a type I Chiari malformation- tions consistent with priorstudiesweresynchronous affected model (right column) under steady flow conditions. Colors indicate the magnitude of the axial velocity (caudad bidirectional flow and a longitudinal component of direction); arrows indicate the directions and magnitudes of velocity that was larger than the lateral or ante- secondary velocities (anterior, medial, or posterior direction). roposterior components.

Neurological Research 2011 VOL.33 NO.3 253 Solute-coupled water transport: analogy

Newtonian Physics Macro scale

Quantum physics Subatomic scale Thinking beyond structures è “Ion & Water mechanics”

The Critical Mixture for solute-coupled transport

Ion Channels

Water Sodium (Na+) Channels Concentration (Aquaporins) Thinking beyond structures è “Ion & Water mechanics”

Water FOLLOWS Sodium in polarized epithelia (e.g. choroid plexus, pleura, pericardium, omentum, nephron)

Na+

Na+ Na+ H2O Na+

Na+ Na+ Na+ Na+ Na+ Thinking beyond structures è “Ion & Water mechanics”

Water FOLLOWS Chloride in polarized epithelia (e.g. sweat glands, salivary glands, bronchi)

H2O Thinking beyond structures è “Ion & Water mechanics”

Water FOLLOWS osmotic gradient of osmolytes through AQPs

H2O Family of more than 13 water channel proteins

First described in 1991 as aquaporin -1 (AQP1)

Nobel prize in Chemistry 2003 (Peter Agre)

Aquaporin-4 (AQP4) is the dominant form in the brain

Agre et al. Towards a molecular understanding of water homeostasis in the brain. Neuroscience (2004) vol. 129 (4) pp. 849-50 Glia Limitans

Astrocyte foot processes around capillaries that form the Blood-Brain-Barrier (BBB)

Ependymal cells

Supraoptic and suprachiasmatic nuclei of hypothalamus

Cerebellum

Hippocampal dentate gyrus,

Hippocampal areas CA1-CA2

Neocortex

Nucleus of stria terminalis

Medial habenular nucleus Badaut et al. Aquaporins in brain: distribution, physiology, and pathophysiology. J Cereb Blood Flow Metab (2002) vol. 22 (4) pp. 367-78

Filippidis et al. Hydrocephalus and aquaporins: lessons learned from the bench. Childs Nerv Syst. 2011 Jan;27(1):27-33. Epub 2010 Jul 13.

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.!452%.%52/3#)%.#%6/,5-%\.5-"%2\./6%-"%2 AQP4 and the Blood-Brain-Barrier and Cerebral vessels

Endothelial Cell AQP4 H2O

AQP4 AQP4

AQP4

Tight junction AQP4 H2O AQP4 REVIEWS

may sufficiently alter the driving force to favor ion luminal Kϩ conductances were originally shown in influx. This idea is supported by the ventricular lo- the choroid plexus by Zeuthen and coworkers (16, calization of NKCC1 in the choroid plexus (67) and 89, 91). In mammals, two Kϩ conductances have the observation that an increase in extracellular Kϩ been identified in rat choroid plexus: a time-inde- from 3 to 25 mM causes a 40% increase in choroid pendent, inward-rectifying conductance (Kir), and plexus cell volume (87). The fact that this volume a time-dependent, outward-rectifying conduc- increase is inhibited by bumetanide suggests the in- tance (Kv) using whole cell patch-clamp methods volvement of NKCC1 in ion influx rather than in- (49). The inward-rectifying conductance is carried creased Naϩ-Kϩ-ATPase activity or decreased efflux by Kir7.1 channels (27) in the apical membrane of via Kϩ channels. Thus NKCC1 may contribute vitally the choroid plexus (61). The Kv conductance is to the regulation of [Kϩ]intheCSF,althoughitseems thought to be mainly carried by Kv1.1 and Kv1.3 not to be a main Kϩ import pathway under normal channels, which are also expressed in the apical conditions. Most of the imported Kϩ is actually recy- membrane (79). The luminal Kϩ channels also cled into the CSF by the ventricular KCC and Kϩ make a major contribution to the generation of the channels as described below. intracellular negative membrane potential of the choroid plexus epithelial cells. Luminal Kϩ Extruders and Kϩ Recycling The luminal KCC may also be involved in sus- Most of the imported Kϩ leaves the cell, back into taining CSF secretion by the recycling of Kϩ to the Downloaded from the CSF across the luminal membrane, thus ventricle lumen and thereby simultaneously sup- avoiding excessive accumulation of Kϩ in the cells porting the Naϩ-Kϩ-ATPase and mediating ClϪ se- and large changes in cell volume. In amphibians, it cretion at the same time (91). Luminal Kϩ,ClϪ is suggested that at least 90% of Kϩ efflux occurs cotransport was evidenced as a furosemide-sensi- choroid plexus: analogy withϩ Ϫ CSAS ? via ion channels in the luminal membrane, and tive K -andCl -dependent transport in the choroid physiologyonline.physiology.org on August 29, 2011

FIGURE 4. Schematic representation of the ion and water transporters in the choroid plexus epithelium Aquaporin1 (AQP1) is situated in both luminal and basolateral membranes, and the carbonic anhydrase (CA) II and XII are found Damkier in the cytosolic et al. and basolateral compartments, respectively. The Naϩ-Kϩ-ATPase and the Naϩ-Kϩ-2ClϪ cotransporter (NKCC1) are strictly luminal plasma membrane transport proteins along withEpithelial the KCC4, three Pathways Kϩ channels (Kir7.1, in Kv1.1, Choroid and Kv1.3), and Plexus two types of Electrolyte anion conductances ofTransport. unknown mo- ϩ Ϫ Ϫ lecular identity (VRAC and Clir). The electrogenic Na -HCO3 cotransporter NBCe2 or NBC4 is also found at the luminal membrane. Three HCO3 transporters are localized to the plasma membrane domain along with a Naϩ/Hϩ exchanger:Physiology the base extruder (2010), AE2 and the two25, Na-base p 239-249 loaders, NBCn1 and NCBE. KCC3 is the only known candidate for mediating basolateral K extrusion.

246 PHYSIOLOGY • Volume 25 • August 2010 • www.physiologyonline.org Pleura and mesothelial tissues: analogy with CSAS ?

A biological membrane that lines a preformed cavity-space and regulates the turnover of the pleural fluid.

Pleura, Pericardium, Peritoneum are MESOTHELIAL tissues SO WHAT ABOUT CSAS ??? J Appl Physiol 90: 1565–1569, 2001.

Effects of SNP, ouabain, and amiloride on electrical potential proﬁle of isolated sheep pleura

C. H. HATZOGLOU,1 K. I. GOURGOULIANIS,2 AND P. A. MOLYVDAS2 1General Hospital of Larissa and 2Department of Physiology, Medical School, University of Thessaly, 412 22 Larissa, Greece Received 28 December 1999; accepted in ﬁnal form 13 October 2000

Hatzoglou, C. H., K. I. Gourgoulianis, and P. A. Mo- Recently, indirect evidence was provided to support lyvdas. Effects of SNP, ouabain, and amiloride on electrical an active electrolyte transport by mesothelial cells (1, potential proﬁle of isolated sheep pleura. J Appl Physiol 90: 22, 23). Agostoni and Zocchi (1) showed the occurrence 1565–1569, 2001.—The ﬂuid and solute transport properties of a solute-coupled liquid absorption from the pleural of pleural tissue were studied by using specimens of intact space by using inhibitors for the Na1/Cl2,Na1/H1, and visceral and parietal pleura from adult sheep lungs. The 2 2 1 1 Cl /HCO3 double exchange or for the Na -K pump. samples were transferred to the laboratory in a Krebs-Ringer Other experimental data (8) suggest the presence of solution at 4°C within 1 h from the death of the animal. The

both tracheal-vascular and vascular-pleural electrical Downloaded from pleura was then mounted as a planar sheet in a Ussing-type potentials in rat lung and suggested that the potentials chamber. The results that are presented in this study are the 1 means of six different experiments. The spontaneous poten- were generated by active transport of both Na and Cl2 across these barriers. Evidence for a small, active tial difference and the inhibitory effects of sodium nitroprus- 1 side (SNP), ouabain, and amiloride on transepithelial electri- transport of Na from the serosal to the interstitial cal resistance (RTE) were measured. The spontaneous side of the dog parietal pleura in vitro was found by potential difference across parietal pleura was 0.5 6 0.1 mV, D’Angelo and colleagues (5). Other authors suggested Dept. Physiology, Membrane permeability Lab jap.physiology.org whereas that across visceral pleura was 0.4 6 0.1 mV. RTE of that both the visceral and parietal pleura behave as both pleura was very low: 22.02 6 4.1 VzMedicalcm2 for visceral School,highly University permeable membranes,of Thessaly, allowing GREECE free movement pleura and 22.02 6 3.5 Vzcm2 for parietal pleura. There was of water and large proteins (13, 19). an increase in the RTE when SNP was added to the serosal Results from different studies (13–15, 19) support bathing solution of parietal pleura and to the serosal or the hypothesis that pleural capillaries determine the mucosal bathing solution in visceral pleura. The same was rate of liquid and solute movement across the whole observed when ouabain was added to the mucosal surface of pleural membrane and that no water or solute flux on July 25, 2008 visceral pleura and to either the mucosal or serosal surface of takes place in the absence of oncotic or hydrostatic parietal pleura. Furthermore, there was an increase in R TE driving forces. More evidence for this theory is the when amiloride was added to the serosal bathing solution of parietal pleura. Consequently, the sheep pleura appears to benign postpartum pleural effusion, which is attrib- play a role in the fluid and solute transport between the uted to the increase in hydrostatic pressure and de- pleural capillaries and the pleural space. There results sug- crease in the colloid osmotic pressure (11). For water gest that there is a Na1 and K1 transport across both the and solute to be filtered or reabsorbed by the pleural visceral and parietal pleura. capillaries, it must pass through both the capillary endothelium and the pleural mesothelial tissue. physiology; pleural permeability; Ussing system; transpleu- Whereas it has been assumed that pleural tissue offers ral potential; sodium nitroprusside little resistance to fluid movement across either the parietal or the visceral pleura, the permeability of sheep pleural tissue is unknown. FROM EARLY IN THIS CENTURY, there were conflicting the- The objective of this study was to examine the inhib- ories concerning the entry and exit of pleural liquid, itory effects of sodium nitroprusside, ouabain, and including theories that pleural liquid was secreted by amiloride on transepithelial electrical resistance (RTE) pleural mesothelial cells and that pleural liquid en- of sheep pleura. Sodium nitroprusside is a donor of tered from the parietal (high-pressure) pleura and was nitric oxide (NO), which inhibits amiloride-sensitive absorbed in the visceral (low-pressure) pleura. In the cation channels and Na1-K1-ATPase and decreases last decades, a consensus was developed that pleural vectorial Na1 transport. Ouabain inhibits Na1-K1 liquid arises from the systemic pleural vessels in both pump, whereas amiloride inhibits Na1 channels and pleura, flows across the leaky pleural membranes into Na1/H1 exchanger. Because of the anatomic differ- the pleural space, and exits the pleural space via the ences between the visceral and parietal pleura (2, 3, parietal lymphatics (20).

The costs of publication of this article were defrayed in part by the Address for reprint requests and other correspondence: K. I. Gour- payment of page charges. The article must therefore be hereby goulianis, Medical School, Univ. of Thessaly, 22 Papakyriazi, 412 22 marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 Larissa, Greece (E-mail: [email protected]) solely to indicate this fact. http://www.jap.org 8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society 1565 Could this be also the case for CSAS ? Is Solute-coupled transport of water present ?

Skimming for Evidence… Indirect evidence about brain edema (excess water) clearance at this interface Glia limitans – subarachnoid space

ARACHNOID

Subarachnoid space Tait et al. Water movements in the brain: role of aquaporins. Trends Neurosci (2008) vol. 31 (1) pp. 37-43

Reulen et al. Role of pressure gradients and bulk flow in dynamics of vasogenic brain edema. H2O J Neurosurg (1977) vol. 46 (1) pp. 24-35

H2O Monogr. 8, Washington, D.C., 1965), p. 211; dor" (United Nations Resources and Transport Seismometer site J. R. McKnit, ibid., p. 240; G. W. Grindley, Division, Energy Section, New York, 1970). Bull. N.Z. Geol. Surv. 75, 131 (1965). 14. P. C. Whiteford, United Nations Symposium 8. D. M. Evans, Geotimes 10, 11 (1966); J. H. (Pisa, Italy, 1970), in press; G. R. T. Clacy, Healy, W. W. Rubey, D. T. Griggs, C. B. J. Geophys. Res. 73, 5377 (1968). Raleigh, Science 161, 1301 (1968); C. B. 15. Contribution No. 1674 from Lamont-Doherty Raleigh, J. Bohn, J. H. Healy, Trans. Am. Geological Observatory of Columbia Univer- Geophys. Union 51, 351 (1970). tory of Columbia University. This work was 9. M. K. Hubbard and W. W. Rubey, Bull. supported by United Nations purchase order Geol. Soc. An. 70, 115 (1959). 9-20-11471. Special thanks to the staff of the 10. C. H. Scholz, J. Geophys. Res. 73, 3295 (1968). U.N. Resources and Transport Division in El 11. P. Molnar and L. R. Sykes, Bull. Geol. Soc. Salvador and the Comision Ejecutiva Hidro- Am. 80, 1639 (1969). electrica del Rio Lempe', who assisted in the 12. G. Palmason, Soc. Sc. Islandica, No. 40 fieldwork. Drs. Bryan Isa'cks and Jack Oliver (1971). critically reviewed the manuscript. 13. J. J6nsson, "Report to United Nations DP E Survey of Geothermal Resources in El Salva- 29 March 1971 0 -C ,-._ Cerebrospinal Fluid Production by the Choroid Plexus and Brain

0. Abstract. The production of cerebrospinal fluid and the transport of 24Na from 0) the blood to the cerebrospinal fluid were studied simultaneously in normal and 0 choroid plexectomized rhesus monkeys. Choroid plexectomy reduced the production of cerebrospinal fluid by an average of 33 to 40 percent and the rate of appearance of 24Na in the cerebrospinal fluid and its final concentration were pro- portionately reduced. In both normal and plexectomized animals, 24Na levels were found to be markedly greater in the gray matter surrounding the ventricles and in the gray matter bordering the subarachnoid space. That sodium exchanges in these two general areas of the brain may be linked to the formation of the cerebrospinal fluid is discussed here. on May 29, 2008 1 I 7 Although numerous studies (1-3), sus monkeys. The animals ranged in 0 1 2 including the classical experiments of age between 1ϩ/2 to 2 years and varied Distance (km) Dandy and Blackfan (4, 5), have in weight between 2 andMilhorat3 kg. In etsix al. Fig. 3. Cross section through the geo-CSAS-pointed to the choroid Cerebrospinalplexuses as the fluid animals,productionthe bychoroid the choroidplexuses plexusof andboth brain thermal area. Hypocenters of brainearthquakes surfaceexclusive interface site! of cerebrospinal fluid lateralScienceventricles (1971) vol.had 173been (994) removedpp. 330-332 are shown by rectangles. The vertical line (CSF) secretion, this view has not gone 3 to 9 months before the current ex- just left of seismometer site A represents unquestioned (6-8). Evidence has periments [the technique of choroid the well shown in Fig. 1. been advanced, for example, that at plexectomy and the histological conse- www.sciencemag.org least a limited amount of CSF can be quences of the procedure have been re- in the cerebral (9)]. Eleven animals fault directly under the best producing formed extrachoroidally ported elsewhere ventricles (6), the aqueduct of Syl- served as normal, nonplexectomized wells in the Ahuachapan geothermal vius (7), and the subarachnoid space controls. In all animals, the fourth area. Production wells in the future a recent production was an inflat- might best be drilled to intercept this (8). In study, the ventricle obstructed with fault. Such simple microearthquake of CSF after choroid plexectomy was able balloon at the beginning of each estimated to be far more substantial (Fig. 1). A lateral ventri- a method of experiment studies provide powerful Downloaded from than previously reported (9). On the cle-to-lateral ventricle perfusion was mapping active faults and can, there- basis of studies on 76 choroid plex- then arranged, in which a Harvard fore, be of considerable practical and ectomized rhesus monkeys, these con- pump apparatus with an inflow rate economic importance in the location and clusions were reached: (i) The pro- of 0.191 ml/min was used. The ven- utilization of geothermal heat sources. ven- consisted of artificial PETER L. WARD duction of CSF rostral to the fourth tricular perfusate CSF to the tracers KLAUS H. JACOB tricle (determined by ventriculo-aque- which following with were added: (i) 14C-labeled inulin (20 Lamont-Doherty Geological ductal perfusion of the CSF [14C]- inulin) was reduced by only one-third ,uc per 30 ml of perfusate); (ii) dex- Observatory of Columbia University, after choroid plexectomy; (ii) the com- tran 2000 (15 mg per 30 ml of perfus- Palisades, New York 10964 position of the CSF after choroid plex- ate), and (iii) 3H-labeled sucrose (15 References and Notes ectomy was unchanged; and (iii) the ,uc per 30 ml of perfusate). Simulta- an infusion of 1. See, for example, J. Oliver, A. Ryall, J. N. surgical obstruction of the choroid neously, intravenous Brune, D. B. Slemmons, Bull. Seismol. Soc. plexectomized ventricles resulted in 24Na (0.4 mc per experiment) was ad- Am. 56, 899 (1966); L. Seeber, M. Bairazangi, so as a A. Nowroozi, ibid. 60, 1669 (1970). acute and progressive hydrocephalus ministered to establish steady- 2. J. N. Brune and C. R. Allen, ibid. 57, 277 that was only slightly less severe than state level of the isotope in the blood (1967). 3. P. L. Ward, G. Pailmason, C. Drake, J. Geo- that occurring in nonplexectomized (± 3 to 8 percent variation from mean phys. Res. 74, 664 (1969). ventricles with similar obstructions counts per minute per microliter). 4. P. L. Ward and S. Bjornsson, ibid. 76, 3953 (1971). (9). During each experiment, serial samples 5. For California: A. L. Lange and W. H. In this study, we have compared the of plasma and outflow perfusate were Westphal, ibid. 74, 4377 (1969). 6. For Japan: I. Kasuga, J. Geogr. Tokyo 76, production of CSF and the transport of taken at 15-minute intervals. Inflow 76 (1967). 24Na from the blood to the CSF in samples were obtained at the beginning 7. J. W. Elder, in Terrestrial Heat Flow, W. H. K. Lee, Ed. (American Geophysical Union, normal and choroid plexectomized rhe- and end of each perfusion period, and 330 SCIENCE, VOL. 173 176 J. Neurosci., January 1, 1997, 17(1):171–180 Nielsen et al. • Aquaporin-4 Water Channels in Glial Membranes

PIA mater - Glia Limitans lower CSAS interface Black dots ? AQP4

Nielsen et al. Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain J Neurosci (1997) vol. 17 (1) pp. 171-180

Figure 4. Polarized expression and membrane topology of AQP4 in glial cells. Many immunogold particles are present along glial membranes facing blood vessels (A, B, D, E) and pia (C), but few particles overlie membranes facing the neuropil (double-headed arrows indicate the two membranes). B, After preembedding immunogold labeling, silver-intensified immunogold particles localize AQP4 at the cytoplasmic face of the membrane (compare postembedding labeling in A and D). D, Double labeling with antibodies to AQP4 (30 nm gold particles) and the glutamate transporter GLAST (15 nm) reveals distribution of the two antigens to membranes at the opposite poles of the cell. Unlike AQP4, GLAST is concentrated along glial membranes apposed to the neuropil, including those that contact parallel fiber (Pf) synapses with Purkinje cell spines (S). E, Postembedding immunogold labeling of cryosection confirms selective labeling of the perivascular glial membrane. End, Endothelium; Gr, granule cell; Pf, parallel fiber terminal; S, Purkinje cell spines; large asterisk, pial surface; small asterisks, endothelial basal lamina; arrowheads, glial lamellae apposed to parallel fiber synapses. Scale bars: A–D, 0.5 mm; E,1mm. understood after definition of the cellular and subcellular distri- A key finding in this study was the highly selective concentra- bution. Because definition of the membrane distribution of AQP4 tion of AQP4 at astrocyte membrane domains facing blood vessels is still lacking, we have used high-resolution immunogold labeling and pia. This implies that the perivascular glial processes and glia procedures including cryosubstitution and Lowicryl embedding limitans may be primary sites of water flux. Lower levels of AQP4 that had been modified for high sensitivity and optimum preser- expression occurred in the remainder of the glial cell membrane, vation of ultrastructure (Matsubara et al., 1996). Standard immu- with a slight enrichment near certain types of synapse such as the nocytochemical procedures with resolution restricted to the cel- parallel to Purkinje cell synapse in the cerebellum. lular level were deemed inadequate, because previous studies of A functional polarization of glial cells has also been described other tissues demonstrated that aquaporins may exhibit a polar- in relation to K1 flux (Walz, 1989). Investigations of the avascular ized expression that is directly relevant to their physiological roles retina of amphibia provided evidence that excess extracellular K1 (Nielsen et al., 1995a; Terris et al., 1995). resulting from high neuronal activity in the neuropil layers was Let us think with teleology in mind !

ARACHNOID

Subarachnoid space

ARTERY

PIA

Pial vessel AQP4 AQP4 AQP4 Na+ AQP4 + Na Glia limitans Na+ BRAIN SURFACE What is missing ? Water & Ion channels ! Solute-coupled transport Membrane Electrophysiology “Hans Ussing” chambers

ESTABLISHED METHOD FOR SOLUTE-COUPLED TRANSPORT STUDIES

Ussing HH, Zerahn K. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol Scand. 1951 Aug 25;23(2-3):110-27. Ex vivo CSAS model

We get the:

Transmembrane Resistance

R (Ω*cm2)

HIGH transmembrane resistance = LOW ionic permeability LOW transmebrane resistance = HIGH ionic permeability Εικ. 7. Αντλία τύπου Ussing που χρησιµοποιήθηκε στην πειραµατική διαδικασία.

CSAS tissue profiles (facing hemichamber)

Πραγµατοποιούνταν παράλληλα 6 πειράµατα κάθε φορά σεOrientation in between αντίστοιχους θαλάµους και η καταγραφή γινόταν µέσω λογισµικού (Clamphemichambers version 2.14 software: AC Micro-Clamp, Aachen, Germany) σε

ηλεκτρονικό υπολογιστή (Εικ. 8.). Μετά την τοποθέτηση του ιστού

ακολουθούσε στις συνθήκες που αναφέρθηκαν, µια περίοδος ηρεµίας plateau του ιστού διάρκειας 10-30 λεπτών για την καταγραφή της

διαµεµβρανικής αντίστασης µάρτυρα.33

67 Published by Maney Publishing (c) W. S. Maney & Son Limited O 10.1179/174313209X414489 DOI ϩ Gourgoulianis esrmnswr efre ihteUsn ytm itlgclsmlso etmnneltissue leptomeningeal of of resistance 11 samples staining. transmembrane hematoxylin–eosin Histological and of system. microscopy Results: value Ussing light under mean the prepared with and were performed occurrence were the measurements identify state. to basal the is Methods: at study sheep in this turnover. leptomeninges fluid of cerebrospinal new the aim a to for The contribution background the its support suggest will may property tissue This leptomeningeal the on permeability Objective: { { * Filippidis Aristotelis leptomeninges sheep of isolated histology and resistance Transmembrane COMMUNICATION RAPID hsooy eia col nvriyo hsay OBx1400, Box PO for Accepted Thessaly, [[email protected]] 2009. of of Greece. January Larissa, Department publication University 41110 Hatzoglou, Hill, School, C. Mezourlo to: Medical requests Physiology, reprint and Correspondence of ability varied a fluid with properties and morphophysiological its mater) to tissues contribution pia arachnoid of fluid, trabeculae, arachnoid cerebrospinal system granulations, vessels, and complex villi subarachnoid mater, a (arachnoid is sys- leptome- tem the space to leptomeningeal–subarachnoid belong The mater ninges. pia and arachnoid The Introduction Keywords: rnmmrn eitne h o rnmmrn eitneosre scmaal oa‘leaky’ turnover. a This fluid activity. cerebrospinal to of in type comparable involved mesothelial be is a may reflect leptomeninges observed might of resistance resistance property transmembrane novel transmembrane of observation low The epithelium. The resistance. transmembrane Discussion: microscopy. light under humans uaahodsaeadteeedm,aebeing are ependyma, literature the the in and reported the under space surfaces brain subarachnoid as such produc- absorption, of the and sites is tion The extrachoroidal physiological fluid while spaces. cerebrospinal plexus, the preformed the choroid of in site production quantifying turnover main fluid and of aspects studying for aebe soitdwt udtroe nbiological in resistance turnover membranes fluid with transmembrane associated been by have measured variations ytmi h pleura Ussing the with in system conducted studies Electrophysiological ea membranes fetal eateto eprtr eiie nvriyHsia fLrsa eoro ais,Greece Larissa, Mezourlo, Larissa, of Hospital University Medicine, Respiratory of Department and Histopathology of Department eateto hsooy eia col nvriyo hsay ais,Greece Larissa, Thessaly, of University School, Medical Physiology, of Department .S ae o t 2010 Ltd Son & Maney S. W. . 38 ¡ 1 eersia li,mnne,sep rnmmrn eitne Ussing resistance, transmembrane sheep, meninges, fluid, Cerebrospinal . 28 h entasebaerssac ftelpoeigsi he a on obe to found was sheep in leptomeninges the of resistance transmembrane mean The rna etmnneltsusfo 6autsepwr sltd Electrophysiological isolated. were sheep adult 26 from tissues leptomeningeal Cranial 4–7 rnmmrn eitnei esr fincpreblt.Teocrec fionic of occurrence The permeability. ionic of measure a is resistance Transmembrane etmnneltsu eosrtsincpreblt si a niae ytemeasured the by indicated was it as permeability ionic demonstrates tissue Leptomeningeal V hs neetohsooia study electrophysiological an Thus, . cm 7 { aepoie sflbackground useful a provided have acai-dmMolyvdas Paschalis-Adam , 2 4 h itlgclpeaain eeldtehmlg ihtelpoeigsof leptomeninges the with homology the revealed preparations histological The . peritoneum , 8 * oi emaiiyadits and permeability Ionic . oiisZarogiannis Sotirios , 5 pericardium , 6 and 1–3 . nvitro in * * ai Ioannou Maria , Neurol n his Hatzoglou Chrissi and lcrpyilgclpoet lpoeiga trans- fluid (leptomeningeal property cerebrospinal electrophysiological in role its establishing turnover. probably in would space aid subarachnoid the concerning taigtetasebaerssac fthe of resistance sheep. transmembrane in demon- tissue studies leptomeningeal no the are appara- there system space strating knowledge, Ussing our the subarachnoid To using tus. by the sheep in of system resistance) membrane fe h nmlsdah isesmlswere of samples minutes 30 Tissue within air laboratory death. the the tissue to animal’s to tissue leptomeningeal transferred the the of the after exposure dura surrounds minimal outer the ensured and that skull The sheep’s contained excluded. layer the were that of cavity applied tissue samples closed adherent and Tissue or handling tissue. perforations the minimize to to the strain taken the Special zero). was from to of near care ischemia death collected warm and of the (time male were animals after adult immediately Samples 26 slaughterhouse of sheep. hemispheres female obtained brain were the tissue from leptomeningeal of sheets Intact electrophysiology and Isolation Methods h i fti td st eemn h basic the determine to is study this of aim The oe o h td fsbrcni physiology. subarachnoid of study the for model Res. 2010 Mar;32(2):205-8. erlgclResearch Neurological { Konstantinos , * Epub 2010 2009May 8.

VOL 32 NO 2 205 Published by Maney Publishing (c) W. S. Maney & Son Limited 206 al. et Filippidis A. erlgclResearch Neurological 1 rnmmrn eitne(esrdin (measured pairs leptomeningeal resistance Two the transmembrane monitored temperature. electrodes by Ag/AgCl influenced of is ions of 37 port at conditioned temperature n ube ih9%O 95% with bubbled and modified 4 at Dulbecco’s Germany) Munich, in (Sigma- GmbH, animal Chemie Ham Aldrich F-12 the mixture medium/nutrient of Eagle’s death the of distribution the describing diagram Boxplot 1 Figure h eyrto rcs,te eeebde in embedded were they process, After dehydration solution. formaldehyde the were 4% in They fixed sheep. and adult from isolated of obtained hemispheres brain were parietal samples tissue Leptomeningeal Histology raws1cm mounting 1 tissue chamber’s was the area to exposed tissue the 24 igrbcroae xgntdwt 5 O 95% Krebs– with in bathed oxygenated CO sheet so was sample bicarbonate, solution planar tissue Ringer the bicarbonate of a hemichambers side Krebs–Ringer each as Ussing-type with The acrylic mounted filled solution. two was bicarbonate separating sample Krebs–Ringer tissue of ml 4 5 igrbcroaeslto a aacda H7 pH Krebs– at balanced The was was solution solution. bicarbonate chambers Ringer Ussing bicarbonate in K. used Krebs–Ringer Ing. solution Germany). (Dipl. bathing Aachen, The chambers Instruments, Ussing Scientific Mussler in mounted fully n htapaeuvlewudb banda basal a at obtained be would were ensur- value state plateau minutes, samples a 10–30 that tissue for ing equilibrate mounted to allowed The first minute. measured lepto- 1 was The every resistance Instruments). transmembrane Scientific meningeal Mussler v2 K. controlled Software Ing. Clamp was program, acquisition software a Data via mode. circuit open 117 . . 8mo/ MgSO mmol/l 18 5mo/ lcs.Ec eihme contained hemichamber Each glucose. mmol/l 55 . pcmn ftelpoeiga isewr care- were tissue leptomeningeal the of Specimens 9mo/ NaHCO mmol/l 99 2 . mllNC,1 NaCl, mmol/l 5 9 icltdb a it h rs-etoa raof area cross-sectional The lift. gas by circulated . rnmmrn eitneo leptomeninges of resistance Transmembrane experiments n ules otdln ntebxrepresents box the 11 of in value line value mean mean Dotted the with transmem- outliers. along sheep and leptomeningeal in resistance of brane values measured 2 h sigcabrae was area chamber Ussing The . 2010 4 ,2 3 . ,5 2mo/ CaCl mmol/l 52 2 . VOL . 5mo/ NaH mmol/l 15 5 CO /5% 38 u 32 V ic cietrans- active since C . cm 5mo/ KCl, mmol/l 65 2 NO 2 bandfo 26 from obtained tcontained It . 2 V cm . 4(Dipl. 14 2 2 under ) 2 PO 2 u /5% and C. . 4 4 , aafi a.Tsu pcmn eeoinae and orientated were specimens 3 Tissue wax. paraffin xmndudralgtmicroscope. light subsequently a were under examined slides Stained staining. ylin–eosin 8 h odr fsbrcni pc,wihcontains vessels. which subarachnoid space, and trabeculae are subarachnoid arachnoid mater of pia mater and borders pia Arachnoid the cells. and flattened arachnoid are the cells The cross space. which subarachnoid trabeculae, subarachnoid the arachnoid of the cell barrier and upper barrier space, the arachnoid is which specimen the layer, our components: in layer two mater reveals arachnoid The sheep. space in subarachnoid of organization the demonstrates iue2Hsooia mg bandfo apeof sample a from obtained image Histological 2 Figure uha h pleura membranes the serosal of as study- profile for such physiological literature the the apparatus in system ing used extensively Ussing in been The has ions since solution. transported aqueous membrane by of an carried biological are in measure a currents leptomeninges a electrical of is permeability the resistance ionic through Transmembrane transport occurrence sheep. the ionic demonstrate study of our of results The Discussion 11 was tissue ( leptomeningeal across the measured resistance transmembrane mean The Microsoft CA, Results for Richmond, Inc., v11 Software USA). SigmaPlot (Systat Windows using was diagram Boxplot prepared values. the interval are confidence here 95% presented Data USA). mean CA, Diego, Graphpad San using performed v3 InStat was analysis Statistical analysis Statistical n . 74–14 m 5 h itlg rprto ( preparation histology The etoswr u ymcooefrhematox- for microtome by cut were sections m 26; ¡

. E f2 xeiet n hi consequent their and experiments 26 of SEM iue1 Figure 02

h uaahodsae;P,pamtr SaS sub- magniﬁcation, sV, Hemato mater; ginal space; vessels. pia subarachnoid crosses arachnoid arrow), PM, it structure black space); as (and (this appearance subarachnoid trabecula spiderweb-like the a arachnoid has Atr, layer; AM leptomeninges. sheep . bfrMcO Gaha otaeInc., Software (GraphPad X OS Mac for 0b V cm .Te9%cndneitra was interval conﬁdence 95% The ). 2 . 4 h pericardium the , 6 20 ,arachnoidbarriercell yi–oi ti,ori- stain, xylin–eosin iue2 Figure . 38 ¡ 1 6 . clearly ) serosal , 28 V cm 2 CSAS “It is a “leaky” epithelium which bears properties of mesothelium”

Filippidis A, Zarogiannis S, Ioannou M, Gourgoulianis K, Molyvdas PA, Hatzoglou C. Transmembrane resistance and histology of isolated sheep leptomeninges. Neurol Res. 2010 Mar;32(2):205-8. Epub 2009 May 8. Childs Nerv Syst DOI 10.1007/s00381-012-1688-x

ORIGINAL PAPER

Permeability of the arachnoid and pia mater. The role of ion channels in the leptomeningeal physiology

Aristotelis S. Filippidis & Sotirios G. Zarogiannis & Maria Ioannou & Konstantinos Gourgoulianis & Paschalis-Adam Molyvdas & Chrissi Hatzoglou

Received: 30 August 2011 /Accepted: 5 January 2012 # Springer-Verlag 2012

Abstract profound increment in transmembrane resistance. The addition Purpose The purpose of this paper is to study the ionic of amiloride at the arachnoidal or pial side did not produce any permeability of the leptomeninges related to the effect of statistical significant change in the RTM from controls ouabain (sodium–potassium–ATPase inhibitor) and amilor- (p>0.05). Immunohistochemistry confirmed the presence of ide (epithelial sodium channel (ENaC) inhibitor) on the the ATP1A1 and beta- and delta-ENaC subunits at the tissue, as well as identify the presence of ion channels. leptomeninges. Methods Cranial leptomeningeal samples from 26 adult Conclusions In summary, leptomeningeal tissue possesses sheep were isolated. Electrophysiological measurements sodium–potassium–ATPase and ENaC ion channels. The were performed with Ussing system and transmembrane application of ouabain alters the ionic permeability of the 2 resistance values (RTM in Ω*cm )obtainedovertime. leptomeninges thus reflecting the role of sodium–potassi- Experiments were conducted with the application of oua- um–ATPase. Amiloride application did not alter the ionic bain 10ϩ3 M or amiloride 10ϩ5 M at the arachnoidal and pial permeability of leptomeninges possibly due to localization sides. Immunohistochemical studies of leptomeningeal tis- of ENaC channels towards the subarachnoid space, away sue were prepared with alpha-1 sodium–potassium–ATPase from the experimental application sites. The above proper- (ATP1A1), beta-ENaC, and delta-ENaC subunit antibodies. ties of the tissue could potentially be related to cerebrospinal Results The application of ouabain at the arachnoidal side fluid turnover at this interface. raised the transmembrane resistance statistically significantly and thus decreased its ionic permeability. The addition of Keywords Arachnoid . Cerebrospinal fluid . ENaC . ouabain at the pial side led also to a significant but less Meninges . Sodium–potassium–ATPase . Ussing

A. S. Filippidis (*) : S. G. Zarogiannis : P.-A. Molyvdas : Introduction C. Hatzoglou (*) Department of Physiology, Medical School, The laws that govern cerebrospinal fluid (CSF) turnover are University of Thessaly BIOPOLIS, fundamental in understanding the underlying pathophysiology 41110 Larissa, Greece e-mail: [email protected] of related diseases such as hydrocephalus, pseudotumor cere- e-mail: [email protected] bri, idiopathic intracranial hypertension, or choroid plexus tumors. The main production site of the cerebrospinal fluid is M. Ioannou the choroid plexus while extrachoroidal sites of production and Department of Histopathology, University Hospital of Larissa, BIOPOLIS, absorption, such as brain surfaces under the subarachnoid Larissa, Greece space and the ependyma, have been reported [1–4]. The role of ion (e.g., Na+–K+–ATPase) and water channels (such as K. Gourgoulianis aquaporin-1, AQP1 or aquaporin-4, AQP4) is fundamental in Department of Respiratory Medicine, University Hospital of Larissa, BIOPOLIS, CSF turnover at the level of choroid plexus or extrachoroidal Larissa, Greece sites [1, 5, 6]. Interestingly, the application of ouabain, a Sodium-Potassium-ATPase Main source of extracellular Sodium

We tested inhibition with OUABAIN

a1 subunit Sodium-Potassium-ATPase ENaC Epithelial Sodium Channel

We tested inhibition with AMILORIDE

βsubunit of ENaC δsubunit - ENaC Conclusions

CSAS has a vibrant functional anatomy which becomes intriguing at the cellular level

CSAS bears properties of mesothelial tissues

CSAS it is a “leaky epithelium”

Solute-coupled transport can potentially occur at this interface since key structures exist

It shows polarity of ion channels

We do not know if this property is related solely to CSF production or absorption.

More studies needed to explore this new field. “Ἡ γὰρ γένεσις ἕνεκα τῆς οὐσίας ἐστίν, ἀλλ' οὐχ ἡ οὐσία ἕνεκα τῆς γενέσεως.”

“For the process of evolution is for the sake of the thing evolved, and not this for the sake of the process.”

Dept. Physiology Membrane Permeability team:

Sotirios G. Zarogiannis, Ph.D Maria Ioannou, M.D., Ph D. Chrissi Hatzoglou, M.D., Ph.D. Paschalis-Adam Molyvdas, M.D., Ph.D. Konstantinos Gourgoulianis, M.D., Ph.D. Plato and Aristotle, “The school of Athens”, Raphael 1509