The Distributional Nexus of Choroid Plexus to Cerebrospinal Fluid, Ependyma and Brain: Toxicologic/Pathologic Phenomena, Periventricular Destabilization, and Lesion Spread

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2011 39: 186 originally published online 28 December 2010Toxicol PatholConrad Johanson, Edward Stopa, Paul McMillan, Daniel Roth, Juergen Funk and Georg Krinke

Toxicologic/Pathologic Phenomena, Periventricular Destabilization, and Lesion SpreadThe Distributional Nexus of Choroid Plexus to Cerebrospinal Fluid, Ependyma and Brain:

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The Distributional Nexus of Choroid Plexus to Cerebrospinal Fluid,Ependyma and Brain: Toxicologic/Pathologic Phenomena,

Periventricular Destabilization, and Lesion Spread

CONRAD JOHANSON1, EDWARD STOPA

1, PAUL MCMILLAN1, DANIEL ROTH

2, JUERGEN FUNK3, AND GEORG KRINKE

4

1Brown University, Providence, Rhode Island, United States2Novartis Institutes for Biomedical Research, Basel, Switzerland

3F. Hoffmann-La Roche Ltd., Pharma Research Non-Clinical Safety, Basel, Switzerland4AnaPath GmbH, Oberbuchsiten, Switzerland

ABSTRACT

Bordering the ventricular cerebrospinal fluid (CSF) are epithelial cells of choroid plexus (CP), ependyma and circumventricular organs (CVOs)

that contain homeostatic transporters for mediating secretion/reabsorption. The distributional pathway (‘‘nexus’’) of CP-CSF-ependyma-brain

furnishes peptides, hormones, and micronutrients to periventricular regions. In disease/toxicity, this nexus becomes a conduit for infectious and

xenobiotic agents. The sleeping sickness trypanosome (a protozoan) disrupts CP and downstream CSF-brain. Piperamide is anti-trypanosomic but

distorts CP epithelial ultrastructure by engendering hydropic vacuoles; this reflects phospholipidosis and altered lysosomal metabolism. CP swelling

by vacuolation may occlude CSF flow. Toxic drug tools delineate injuries to choroidal compartments: cyclophosphamide (vasculature), methylcel-

lulose (interstitium), and piperazine (epithelium). Structurally perturbed CP allows solutes to penetrate the ventricles. There, CSF-borne pathogens

and xenobiotics may permeate the ependyma to harm neurogenic stem cell niches. Amoscanate, an anti-helmintic, potently injures rodent ependyma.

Ependymal/brain regions near CP are vulnerable to CSF-borne toxicants; this proximity factor links regional barrier breakdown to nearby

periventricular pathology. Diverse diseases (e.g., African sleeping sickness, multiple sclerosis) take early root in choroidal, circumventricular, or

perivascular loci. Toxicokinetics informs on pathogen, anti-parasitic agent, and auto-antibody distribution along the CSF nexus. CVOs are suscep-

tible to plasma-borne toxicants/pathogens. Countering the physico-chemical and pathogenic insults to the homeostasis-mediating ventricle-bordering

cells sustains brain health and fluid balance.

Keywords: pegylated drugs; anti-parasitic agents; phospholipidosis; anti-trypanosome agents; tertiary amines; lysosomal storage disease;

multiple sclerosis; epithelial vacuoles.

INTRODUCTION

Integrated Topics and Objectives

Neurons are exquisitely and adversely sensitive to toxicants

and pathogens. Normally the impermeable blood-brain barrier

(BBB) and blood-cerebrospinal fluid (CSF) barrier (BCSFB)

act together to protect the neuronal networks from potentially

injurious agents in blood. Historically, the BBB has received

more pharmacologic, toxicologic, and pathologic attention

than the BCSFB, or choroid plexus (CP). In a disease or toxi-

city context, this is ironic because BCSFB is more vulnerable

(Levine 1987) than cerebral capillaries to many foreign inva-

ders. Often the CP or an ependymal circumventricular organ

(CVO) is the first site of central penetration by a deleterious

substance or pathogen (Siso, Jeffrey, and Gonzales 2010).

CP is also of unique interest to toxicologists and pathologists

because its main secretion, the cerebrospinal fluid, rapidly and

widely disseminates throughout the central nervous system

(CNS) the various substances that have penetrated a breached

BCSFB. Thereafter, the toxicants, pathogens, and cells that

permeate the CSF are readily accessible via bulk flow circula-

tion to neurons and stem cells over wide expanses of brain.

Accordingly, the two aims of this review are to: (a) characterize

the intricate physiology of the CP, CSF, and ependyma and

This review is dedicated to the recently deceased Dr. Seymour Levine, a

neuropathologist at the New York Medical College who was a pioneer

investigator of toxico-pathological phenomena involving the choroid plexus-

cerebrospinal fluid (CSF) system. The authors express their gratitude to A.

Messier for his critique of the manuscript and to J. Johanson for constructing

diagrams. NIH support to EGS (NIH NS/AG 10682) and CEJ (NIH R01

27601 and R01 AG027910) and research support from the Departments of

Neurosurgery and Pathology at Rhode Island Hospital were instrumental for

obtaining information on blood-CSF barrier disruption in disease states.

Address correspondence and reprint requests to Conrad Johanson, Department

of Neurosurgery, Brown Medical School, Providence, RI 02903; e-mail:

[email protected]

Abbreviations: 3V and 4V, third and fourth ventricle; AIDS, acquired

immunodeficiency syndrome; ALA, aminolevulinic acid; AP, area postrema;

APOE4, apolipoprotein E; BBB, blood-brain barrier; BCSFB, blood-CSF bar-

rier; CAD, cationic amphiphilic drug; CBF, cerebral blood flow; CIMZIA, cer-

tolizumab; CNS, central nervous system; CP, choroid plexus; CSF,

cerebrospinal fluid; CVO, circumventricular organ; CY, cyclophosphamide;

EAE, experimental allergic encephalitis; ICV, intracerebroventricular route

of exposure; IGF, insulin-like growth factor; IP, intraperioneal route of expo-

sure; ISF, interstitial fluid; LRP, low density lipophilic receptor-related

protein; MC, methylcholanthrene; ME, median eminence; MS, multiple sclero-

sis; NLP, neural lobe of hypophysis (pituitary); OATP, organic anion transport-

ing polypeptide; OVLT, organum vasculosum of lamina terminalis; PEG,

polyethylene glycol; PEPT2, proton-coupled oligopeptide transporter; PCR,

polymerase chain reaction; PI, pineal gland; PO, per os (oral) route of expo-

sure; PT, proximal tubule; SC, subcutaneous route of exposure; SCO, subcom-

missural organ; SFO, subfornical organ; SVZ, subventricular zone; TGFb,

transforming growth factor b; UDP, uridine 5’-diphospho; VEGF, vascular

endothelial growth factor.

186

Toxicologic Pathology, 39: 186-212, 2011

Copyright # 2011 by The Author(s)

ISSN: 0192-6233 print / 1533-1601 online

DOI: 10.1177/0192623310394214

(b) assess how harmful xenobiotics and microbes (e.g., viruses,

protozoans) damage the CP and ependyma, thereby destabiliz-

ing the brain interior (Krinke et al. 1983; Levine, Sowinski, and

Nochlin 1982; Roth and Krinke 1994). Regions injured include

the delicate neurogenic niches and body-regulatory centers

near ventricular CSF.

Central Nervous System Toxicology/Pathology:

Differential Effects of Barrier Phenomena

Toxicants and pathogens vary greatly in ability to penetrate

particular regions of the central nervous system. Consequently,

knowledge of permeation patterns across diverse transport

interfaces is essential to understand specific disruptions of

brain and CSF. BBB refers to the widespread impermeable

capillaries in the CNS. However, BBB is sometimes used

inappropriately to include the BCSFB and other central sites

of impermeability. Therefore, more precise classification of

CNS transport interfaces is needed. Care should be taken to

delineate properties/reactions of the membrane boundaries that

separate blood, CSF, and brain. Bidirectional secretory and

reabsorptive phenomena should also be considered. This is true

in a physiologic-pharmacologic sense and in relation to

toxicologic-pathologic effects on barrier disruption.

Transport specialists concur that the BBB resides primarily

in the tight junctions of brain microvessels or capillaries.

BCSFB most commonly (and in this review) is used to refer

to tight junctions in CP epithelium (but should be more inclu-

sively defined if meant also to include CNS-inward flux across

arachnoid membrane into subarachnoid space). The lateral ven-

tricular CSF-brain interface, or ependyma, is permeable to

macromolecules, and therefore does not usually act as a barrier.

Certain regions of the ependymal wall contain specialized

CVOs adjacent to CSF. The CVOs, like CP, have permeable

capillaries and are thus open windows to the systemic circula-

tion. Thus, each CVO does not have a BBB.

Because BBB is endothelial and BCSFB epithelial, there are

consequently barrier differences in tight junction (imperme-

ability) properties and in types of transporters expressed at the

respective barrier cell membranes. These are important distinc-

tions in view of how toxicants and pathogens differentially

interact with BBB and BCSFB. Cerebral capillaries and chor-

oid plexuses extensively mediate homeostatic mechanisms to

protect the neuronal microenvironment. When xenobiotic

agents and viruses disrupt BBB and BCSFB, they not only

breach barriers but also may disrupt homeostatic transporter

actions that benefit neurons.

Mechanisms for Keeping a Pristine Environment for

Neurons

To function optimally, the brain requires a clean environ-

ment for the neural parenchyma. Neurons need a stable extra-

cellular or interstitial fluid (ISF) of specialized composition

to maintain an extracellular environment that promotes effi-

cient transmission of impulses along axons and through

synapses. ISF that typically bathes neurons is relatively low

in concentrations of plasma proteins, cytokines, catabolite

waste products, erythrocytes, leukocytes, immune cells, patho-

gens, drug metabolites, and toxicants. Three mechanisms

mainly accomplish CNS microenvironmental cleansing:

(1) an impermeable BBB and BCSFB that restrict diffusion

of plasma water-soluble molecules into CNS; (2) reabsorptive

organic solute transporters at the CNS-facing side of barrier

cells for actively extruding into blood certain organic acids

(anions) and bases (cations) in ISF or CSF; and (3) the sink

action of CSF to remove from the brain various metabolites and

impurities for excretion, by bulk flow, from the ventricles to

downstream venous/lymphatic exit sites.

Fundamental differences exist in the workings of BBB (cer-

ebral capillaries) versus BCSFB (choroid plexus). The latter

purifies the CSF interior and periventricular regions with

homeostatic systems to keep brain and body fluids toxicant free

and in physiologic balance. Attenuation of CP transport capac-

ity can profoundly impair cerebral metabolism and the fluid

environment of neurons (Spector and Johanson 2010a).

Accordingly, we analyze BCSFB transporters, selective CP

permeability, the CSF and peptide secretions that choroid

epithelial cells generate, as well as fluid flow through the ven-

tricular system. CSF streaming down the neuraxis from lateral

to fourth ventricles reaches many regions. The largest portion

of ventricular CSF is eventually convected to the subarachnoid

space for bulk fluid clearance across arachnoid villi and

lymphatic drainage channels that follow the olfactory nerve

and discharge their contents into the nose. However, a small but

significant portion of CSF-borne ions and organic solutes

diffuses across or is taken up by border cells at the ventricular

margins.

Compartmental Aspects of the Physiologic Nexus

Modeling features of the specific distributional pathway, or

‘‘nexus,’’ connecting the CP, CSF, ependyma, and brain are

shown in Figure 1. In a transport physiology sense, a nexus can

be viewed as a series of connected compartments within an

organ, through which substances passively move or are actively

transferred, to effect an action or function at a downstream

target. A nexus example is the facilitated diffusion of plasma

glucose via carrier proteins across brain endothelium into the

interstitial fluid, and then diffusion to nearby neurons for

carrier-mediated uptake of the sugar into axoplasm. Another

nexus pathway is the diffusion of plasma-borne angiotensin

across permeable capillaries of the subfornical organ (a CVO)

into the interstitial space, through which this peptide diffuses to

neurons. Yet another nexus, and the main theme of this review,

is the movement of a micronutrient (or hormone or xenobiotic)

across CP into ventricular CSF for bulk flow to target cells in

the ependyma and underlying periventricular brain (Figure 1).

Nexuses are convenient transport models to delineate precise

point(s) along distributional pathways interrupted by pathogens

or toxicants.

As the medium for distributing materials within ventricles

and to ependyma and subjacent brain, the CSF plays a primary

Vol. 39, No. 1, 2011 TOXICOLOGY OF VENTRICULAR CSF-BORDERING CELLS 187

role in modulating subventricular and hypothalamic regions.

Nexus is a concept applicable to CSF distributional phenomena

from uterine life to senescence. It operates in several states:

1. Ontogenetic: For fetal brain development, the neuro-

genic niches in the subventricular zone (SVZ)

depend on growth factors and neurotrophins secreted

and transported by CP-CSF for promoting stem cell

conversion to neurons (Owen-Lynch et al. 2003).

A steady supply of choroidally derived peptides

(Chodobski and Szmydynger-Chodobska 2001),

nucleosides (Redzic et al. 2005), and vitamins

(Spector and Johanson 2006, 2007b) furnished to

CSF is critical for feeding the periventricular regions

(the domain harboring neurogenic stem cells) that

drive normal brain development. Containing sparse

capillary networks, the early fetal cortex evidently

needs nutritional supplies delivered via CP-CSF as

FIGURE 1.—Central role of the choroid plexus (CP)-cerebrospinal fluid (CSF) nexus in exchanging materials with brain. Ions, water, and organic

molecules passively filter out of CP capillaries (arrow 1) into interstitial fluid (ISF). This is the first step in the distributional nexus to the brain

(green arrows). Solutes diffuse through ISF up to the basolateral membrane of the CP epithelium. Active mechanisms in membranes transfer

solutes sequentially across the basolateral (arrow 2) and apical membranes (arrow 3) into ventricular CSF. As CSF flows (arrow 4) from ventricles

into the cisterna magna, a small fraction of the CSF-borne substances diffuse across ependyma (arrow 5) into periventricular brain or are taken up

by specialized ependymal cells of circumventricular organs (CVOs) in the ventricular walls. Ependyma-penetrating substances diffuse through

brain ISF (arrow 6) for transport into neurons (N); blood-brain barrier (BBB)/capillaries, not depicted, are interspersed among neurons. Material

inflow to CNS interior thus sequentially involves CP, CSF, ependyma/CVOs, and brain. Viewed as a distributional nexus, this CNS-inward path-

way conveys solutes across the blood-CSF barrier (BCSFB) eventually to targets in the caudate nucleus, hippocampus, dentate gyrus, subventri-

cular zone (SVZ), and hypothalamus. In the opposite direction, there is a reverse nexus (red arrows) for catabolites released by neurons/glia into

the brain ISF. Accordingly, cerebral catabolites such as homovanillic acid diffuse through ISF (arrow 7), and down a transependymal concentra-

tion gradient into the CSF (arrow 8). By bulk flow of CSF (arrow 9), catabolites are convected to the subarachnoid space (not depicted) or to the

CP for active removal from the ventricles across the apical surface (arrow 10) followed by extrusion across the basolateral membrane (arrow 11).

Therefore, some endogenous or drug metabolites end up being cleared passively into the blood via microvessels (arrow 12) and venules draining

the CP (Johanson et al. 2000). Overall CSF is simultaneously a source (arrows 1 to 6) and a sink (arrows 7 to 12) for distributing molecules,

depending on the prevailing concentration gradients between ventricular CSF and brain ISF. As such, the CSF and bordering cells constitute a

nexus for mediating trophic (CSF to brain) and excretory (brain to CSF) fluxes.

188 JOHANSON ET AL. TOXICOLOGIC PATHOLOGY

well as plasma. Substantial glycogen deposition in

prenatal CP epithelium points to a BCSFB source

of substrate for energy needs by developing brain

(Kappers 1958).

2. Endocrine: As a hormonal signal relay station, the

lateral ventricle CP epithelium takes up blood-

borne hormones/peptides (Dietrich et al. 2007) by

basolateral endocytosis or carriers (arrow 2, Figure 1)

and releases them apically into CSF (arrow 3,

Figure 1). By CSF bulk flow, these plasma-derived hor-

mones are carried to the third ventricle region where

they readily permeate the arcuate nucleus (Rodriguez,

Blazquez, and Guerra 2010) and bind receptors in

specific hypothalamic nuclei. In this manner, endocrine

feedback loops modulate the hypothalamic-pituitary

axis for regulated secretion of hormones into blood.

Moreover, the co-localized fluid-balancing peptides

arginine vasopressin and basic fibroblast growth

factor, regulated in CP epithelium and hypothalamic

paraventricular nucleus, mediate osmolality homeo-

stasis in CSF and plasma (Gonzalez et al. 2010).

3. Pharmacologic: Therapeutic agents destined for

CNS targets must circumvent the neurovascular and

epithelial barrier systems that thwart agent penetra-

tion into brain. BBB has been widely manipulated

to enhance drug access to neuronal targets, albeit

with limited success. An alternative strategy is to

pharmacologically exploit the BCSFB by altering

CP permeability and transporter capability. Modifi-

cation of BCSFB can alter CP function per se or

expedite drug delivery to brain regions close to CSF

(Johanson, Duncan, et al. 2005).

4. Immunologic: Normally there is highly regulated

traffic of immune cells and molecules across BCSFB

to monitor and adjust CSF immune status. In the relapse

state of certain autoimmune diseases, leukocyte move-

ment across CP into the ventricles and adjacent

periventricular regions is augmented to the brain’s

detriment (Prendergast and Anderton 2009). Finer con-

trol of immunoglobulin and immune cell access to CSF

may prevent exacerbation of CNS immunopathology.

5. Geriatric/therapeutic: With advancing age, there is

progressive deterioration of CP secretory ability and

CSF turnover rate (Silverberg et al. 2001). This

reduces the excretory, lymph-like functions of CSF

and impairs brain metabolism. As a result, neurogenic

niches in the SVZ and dentate gyrus are disrupted.

Consequently, the altered stem cells and hippocampal

neurons near CSF can hasten the decline of cognition.

Therapeutic strategies to reduce aging damage to CP

and ependyma would likely preclude disruption of

subventricular neurogenic and CSF homeostatic-

phenomena in aging (Johanson et al. 2004).

6. Toxicologic/pathologic: CP, ependyma, and CVOs

are vulnerable, to a variable degree, to attacks by

pathogens and toxicants. Some blood-borne viruses

and parasitic agents have a predilection for binding,

damaging, and penetrating the BCSFB. Differential

abilities by xenobiotics (and pathogens) to disturb

BBB endothelial cells versus CP epithelial cells are

manifest by the specific neuropathology inflicted

downstream of the initial insult to the barrier locus.

Toxicology and Pathology Models of Damage to CSF-

Bordering Cells

The CSF fills four ventricles and the mesencephalic aque-

duct of Sylvius in the brain interior. Far from being static sacs

of fluid, the ventricular CSF exerts many dynamic actions, bio-

physical as well as biochemical, on the brain parenchyma.

Basically two types of epithelial cells form the border that

encompasses the CSF: choroidal and ependymal. Thus the

internal (ventricular) surface of the brain is comprised mainly

of a single layer of choroid plexus epithelium or ependyma

(D. E. Smith, Johanson, and Keep 2004). CP epithelial cells

do not generally contact the brain; rather, they extend into the

ventricular CSF. On the other hand, the ependyma intimately

and extensively adjoin the neural tissue. CP has its own inter-

stitium, but the ependymal cells share an underlying interstitial

space with cerebral parenchyma (Figures 1 and 7).

CP is readily dissectable as a tissue tuft for investigations.

Ependyma is not as easily separated intact as a monolayer from

underlying brain. Such architectural or topographical differ-

ences in CP versus ependyma help to explain fundamental dif-

ferences in experimental models evolving in the field. In vitro,

CP is popular due to straightforward tissue excision from the

ventricles (Crossgrove, Li, and Zheng 2005; Q. R. Smith and

Johanson 1985; Sanderson, Khan, and Thomas 2007). Alterna-

tively, the in vivo systemic modeling emphasized in this review

uses microscopy and regional tissue staining to delineate how

structures in the CP-CSF-ependyma-brain nexus (Figure 1)

respond to systemically administered xenobiotics.

Information abounds on CP and ependyma pathology, espe-

cially for tumors (Krinke et al. 2000; Netsky and Shuangshoti

1975). Spontaneous rodent tumors in the brain are less differ-

entiated than their human counterparts. Morphologic character-

istics of CNS tumors in rats and mice, including CP papillomas

and ependymomas, have been thoroughly treated (Krinke et al.

2000). Microbial and immune cell permeation of CP have also

been widely documented (Engelhardt and Sorokin 2009; Petito

2004). Toxicants of CP fall into two major categories:

inorganic agents and organic agents. For example, Zheng and

colleagues extensively recapitulated the toxic effects of the

elements Pb, Fe, Cd, Cu, Hg, and Mn on CP-CSF functions

(Aschner, Vrana, and Zheng 1999; Zheng 2001).

Toxico-Pathologic Considerations for Choroid Plexus-

Cerebrospinal Fluid

A sparsely studied distributional pathway is the transport

route from CP to CSF and then to periventricular regions

(Figure 1). This paucity of attention is surprising in view of the

critical guardian and multiprovider roles of CP-CSF for interior

Vol. 39, No. 1, 2011 TOXICOLOGY OF VENTRICULAR CSF-BORDERING CELLS 189

brain: the hippocampus (Johanson et al. 2000), neurogenic

niches (Miyan, Nabiyouni, and Zendah 2003), and hypothala-

mic nuclei (Rodriguez, Blasquez, and Guerra 2010). In addition

to CSF-mediated delivery of micronutrients, growth factors, and

neurotrophins to periventricular regions (Johanson et al. 2008),

there are toxico-pathologic issues associated with conveyance

of microbes (Levine 1987) and metals (Zheng 2001) along this

nexus. Better control of the spread of pathogens, toxic drug

metabolites, and cancer cells (Glantz and Johanson 2008)

requires additional insight for therapeutically manipulating CSF

distribution (Johanson, Duncan, et al. 2005) (Figure 1).

Due to the CP’s primary role in sustaining CSF dynamics, it

is important to uncover mechanisms by which pathogens and

toxicants alter BCSFB integrity. Upon breaching of the CP

barrier (the first line of defense for the CNS interior) by

CSF-permeating noxious agents, certain additional injuries are

caused downstream in the nexus at the ependymal boundary

(the second protective interface for deep brain regions abutting

CSF-filled cavities). Simultaneous disruption of CP, epen-

dyma, and CVOs renders the brain (and peripheral organs

under control of circumventricular brain centers) vulnerable

to invading agents. Multiple functions can be destabilized.

Therefore, ependymal/subependymal lesions have wide-ranging

effects. Ependymal wall lesions are diffuse or circumscribed.

Phenomenologically, regionalized lesions in the CNS interior

can be related to progressive damage along specific loci in the

CP-CSF-ependyma-brain nexus (Krinke et al. 1983; Levine,

Sowinski, and Nochlin 1982).

Pathogen-disrupted CP initiates the development of some

brain diseases. Phenotypically, the glycoconjugate expression

of the choroid epithelial and ventricular ependymal cells are

such that particular viruses are bound and incorporated

intracellularly (Ormerod and Raseroka 1988). Similarly,

Trypanosoma brucei, the protozoan agent that causes African

sleeping sickness, begins CNS offensive actions by penetrating

and disorganizing CP (Philip et al. 1994). Trypanosome injec-

tion into rodents or goats induces neuropathology akin to the

human African trypanosomiasis syndrome (Darsaud et al.

2003; Moulton 1986; Poltera et al. 1980). Thus, trypanosome

administration to mammals provokes localized (choroidal)

inflammatory reactions that chronically worsen and disrupt

the BCSFB (Van Marck et al. 1981). Following infection of

CP by Trypanosoma brucei, there ensue multiple sequelae that

progressively injure the CSF-brain nexus and finally the BBB

(Sanderson et al. 2008). If left pharmacologically unchecked,

FIGURE 3.—Localization and characteristics of the seven circumventricular

organs (CVOs) in the human brain. Occurring mainly as tiny ‘‘mini-

organs’’ around the ventricular system margin, the CVOs cluster

mainly around the third ventricle. Anatomically the most dorsal CVO

is the subfornical organ (SFO). Going clockwise around the third

ventricle are the subcommissural organ (SCO), pineal gland (PI),

neural lobe of the pituitary gland (NLP), median eminence (ME), and

organum vasculosum of the lamina terminalis (OVLT). Further down

the neuraxis, more distally in the cerebrospinal fluid (CSF) system,

lies the area postrema (AP) near the fourth ventricle. The CVOs are

among few sites in the central nervous system (CNS) lacking a

blood-brain barrier (BBB). Therefore the parenchymal neurons

and ependyma in CVOs sense concentrations of compounds in blood

and make homeostatic adjustments to restore fluid balance in brain

and periphery. Significantly, the diffusing blood-borne factors or

peptide signals are not restricted by the BBB in CVOs. Due to

unavoidable circumstances the above figures were not originals but

were scanned from reprints.

FIGURE 2.—Ultrastructure of typical choroid plexus (CP) epithelium.

The CP ultrastructure reflects an epithelium busily engaged in meta-

bolizing and synthesizing proteins as well as transporting solutes

between blood and cerebrospinal fluid (CSF). Organelles present at

high density include mitochondria (M), endoplasmic reticulum

(ER), lysosomes (L), and Golgi apparatus (G). Tight junctions (J) seal

apical membranes that abut at the CSF-facing pole (upper surface) of

the epithelium. Lush surface areas for transport are extant at the apical

microvilli (Mv) and basal labyrinths (BL); the latter dovetail, one

epithelial cell base with the other. C, centriole. Bar ¼ 2 mm.

190 JOHANSON ET AL. TOXICOLOGIC PATHOLOGY

this intra-CNS pathology cascade that begins with CP invasion

by trypanosomes can be fatal (Moulton 1986).

Another pathogen with a penchant for CP is human immu-

nodeficiency virus (HIV, the cause of acquired immunodeficiency

syndrome [AIDS]). Dendritic cells and monocytes in CP

interstitium are infected with HIV in AIDS patients.

The ratio of CP to brain infection with HIV is generally greater

than twofold. CP infection precedes HIV-induced encephalitis.

Even in asymptomatic AIDS, the CP involvement with

infection precedes that of brain (Petito et al. 1999). Replication-

FIGURE 4.—Cross-sectional area of a choroid plexus (CP) villus. Reconstructed from micrographs, this schematic portrays three major compart-

ments of the CP: an inner vascular core (20% of tissue volume), the intermediate interstitial zone containing a loose stroma and interstitial fluid

(ISF) zone (15% of tissue volume), and an outer circumferential ring of epithelial cells (65% of tissue volume). Solutes in blood readily diffuse out

of CP capillaries into and through the ISF up to the epithelial basal membrane, which restricts diffusion (Johanson and Woodbury 1978). To cross

the blood-cerebrospinal fluid barrier (BCSFB), a substance must be actively transported across the epithelium (transcellular route) and/or diffuse

passively between cells (paracellular route). Diffusion of water-soluble organic solutes is a small proportion of the total molecular flux across CP.

Active transporters, facilitated diffusion mechanisms, ion channels, and aquaporins (regulated water pores) conduct transcellular trafficking

through the basolateral and apical membranes in the BCSFB. Toxic agents or pathogens can damage one, two, or all three compartments of the

villus.

Vol. 39, No. 1, 2011 TOXICOLOGY OF VENTRICULAR CSF-BORDERING CELLS 191

competent HIV in CP is similar to, but not identical with, that in

brain (Burkala et al. 2005). Petito and colleagues (1999)

postulated that CP is the site for hematogenous spread and a

reservoir for productive HIV infection during clinical latency.

Collectively, these histopathologic observations on CP relative

to brain (regional and temporal) implicate CP-CSF nexus

participation in disseminating HIV into CNS. Moreover,

molecular analyses of viral DNA in CP (Burkala et al. 2005)

corroborate early participation of the BCSFB pathway for later

distribution to brain (putatively by the CSF nexus). Unfortu-

nately, CP may provide an environment promoting evolution

of drug-resistant HIV strains with CNS tropism (Chen, Wood,

and Petito 2000). Involvement of CP in initial stages of HIV

infection suggest pharmacological targeting of virus trapped

in CP interstitium (i.e., not yet having accessed CSF). Reme-

dial agents in blood would then not have to pass through the

restrictive BCSFB. Rather, the drugs would readily traverse

leaky capillaries to reach the HIV-laden immune cells in the

CP interstitial space.

Neuropathologic Principles for Analyzing Choroid

Plexus and Ependyma

Pathologists regard CP in the domain of neuropathology

rather than general pathology (Levine 1987). This is understandable

because CP, an invaginating fold of pia within the ventricles, is

physically inside the CNS. However, due to absent tight junc-

tion barriers between endothelial cells in the plexus capillary

network, the CP functions more like a renal-type secretory

organ rather than a neural center (Spector and Johanson

1989). Therefore, a useful pathophysiologic concept is the

comparison between CP epithelium and the renal proximal

tubules (PT), both of which share many functional and ultra-

structural similarities. However, CP acts like a reverse kidney

in that the primary choroidal function is fluid secretion (Johan-

son et al. 2008) rather than reabsorption. This reflects Naþ

pump localization apically in CP (Parmelee and Johanson

1989) but basolaterally in PT. Still, the similar structural adap-

tations and transporter protein expression, CP versus PT,

prompt comparisons of cross-reactions in the respective base-

ment membrane-epithelial systems (Johanson, Stopa, and

McMillan 2010). Many toxicologic effects on CP mimic dam-

age to PT or other nephron regions (Levine 1987).

Great permeability of the choroidal capillaries promotes

interendothelial penetration of macromolecules (arrow 1,

Figure 1). Consequently, the choroid ISF compartment (Q. R.

Smith, Pershing, and Johanson 1981), unlike brain interstitium,

is readily accessible to plasma proteins, cytokines, and immu-

noglobulins. This makes CP parenchyma more vulnerable to

systemic disease (Levine 1987) than is the BBB-protected

brain. Plasma-borne auto-antibodies and immune complexes

(June et al. 1979) wreak havoc with CP basement membrane

and epithelium (Peress, Roxburgh, and Gelfand 1981).

This weakens the BCSFB and places the brain at risk for

CSF-transmitted infections. Immune cell and leukocyte move-

ment through CP displays transfer dynamics different from

BBB due to capillary and barrier cell (epithelial vs. endothelial)

variations (Engelhardt and Sorokin 2009). Paracellular perme-

ability in BCSFB is greater than at BBB (Thomas and Segal

1998). Thus, distinctive properties of the CP explain the differ-

ential barrier penetration of pathogens, xenobiotics, and

immune cells from blood to CSF compared with their transfer

from blood to brain.

Regional differences in structure and function occur in CP

tissues of lateral, third, and fourth ventricles. Ventricular CP

variations include epithelial cell pH, Kþ/Naþ, organic anion

transport capability, water content, and blood flow (Harbut

and Johanson 1986; Murphy and Johanson 1990; Pappenhei-

mer, Heisey, and Jordan 1961; Szmydynger-Chodobska,

Chodobski, and Johanson 1994). Inflammatory responses also

vary in third ventricle CP versus the structure of the CP in

other regions (Levine, Saltzman, and Ginsberg 2008). Given

the regional differences in CP baseline physiology and struc-

ture, one expects variations in how a particular CP responds to

pathogens and toxic agents. Neuropathologic differences

across CP regions have implications for functional variations.

Similar principles apply to comparison of the heterogeneous

ependyma that display unique profiles of cilia and junctional

apparatus. Particular toxicologic effects in ventricular regions

can often be ascribed to peculiar features of each type of

CSF-bordering cell.

Surface epithelium is a term used interchangeably with

CSF-bordering cells. Normally, and especially in disease

states, there is sloughing of choroidal and ependymal cells into

CSF. Surface epithelium was found in 5–10% of human

FIGURE 5.—Adverse effects of cylcophosphamide (CY) on the fourth

ventricle choroid plexus (CP). Two days after CY administration to

adult rats, there is severe choroid plexus inflammation (‘‘plexitis’’).

The CP (shaped like an upside-down triangle) fills the entire fourth

ventricle and is disrupted by hemorrhage, necrosis, and exudates of

plasma/fibrin in the stroma. Neuronal/glial cells in the cerebellum

(top) and medulla (bottom) appear normal. Experimental conditions

are summarized in Table 4 and described by Levine, Sowinski, and

Nochlin (1982). Stain: hematoxylin and eosin, �170. Due to unavoid-

able circumstances the above figures were not originals but were

scanned from reprints.

192 JOHANSON ET AL. TOXICOLOGIC PATHOLOGY

patients with CNS inflammation, neoplasms, compression, and

seizures (Wessmann et al. 2010). However, there were no sig-

nificant associations between specific disease types and the

incidence of surface epithelial cells in CSF. Further methodo-

logical refinements in analyzing surface epithelial cytology

in CSF may yield data to distinguish specific CNS disorders.

COMPLEMENTARY CIRCULATORY SYSTEMS IN THE BRAIN

Understanding differences in molecular trafficking across

BCSFB versus BBB, with patho-toxicologic implications,

depends on precise knowledge of circulatory inflow and vascu-

lar ultrastructures. CNS uses two circulations to convey

trophic/metabolic materials into and out of brain. Such a dual

arrangement of the vascular and CSF circulatory systems sets

up a complex steady state of nutrient influx and catabolite

efflux for neuronal networks (Johanson 2008). Blood supplies

the brain intrahemispherically from the inside via the cerebral

capillaries. Concurrently, from the outside of the hemispheres,

CSF provides supportive materials to neurons from fluid cav-

ities surrounding both the internal (ventricular) and external

(subarachnoid) surfaces of the brain. Both the vascular and

CSF circulations have secretory (influx) as well as reabsorptive

(efflux) components of solute distribution.

Why does the brain need CSF throughput in addition to the

primary vascular perfusion? Complex circulatory physiology,

linking CSF hydrodynamics to cerebral hemodynamics, relates

to the specialized anatomy and needs of the CNS. In a drainage

capacity, the CSF uniquely acts as a quasi-lymphatic system

(Johanson 2008). Moreover, by being a shock absorber, CSF

dampens vascular pulsations (Zou et al. 2008) that would oth-

erwise physically damage the fragile brain capillaries (Stopa

et al. 2008). Third, CSF is proximate to periventricular neuro-

genic niches. Consequently, CSF constituents support stem

cells and neurons via specialized choroidal secretions in fetal

life (Redzic et al. 2005) when brain capillary density is low.

Later in old age when cortical microvessel functions dwindle

(Miller et al. 2008; Silverberg et al. 2010), CP secretions may

become important to support cerebral angiogenesis as well as

neurogenesis. Working in tandem, the CSF and blood provide

complementary circulatory support to maintain cerebral meta-

bolism and structural integrity.

Parallel arterial input of materials to CNS is mediated by

two systems that are foremost in transferring substances: the

BBB (microvascular endothelium) and BCSFB (choroidal

epithelium). Each major transport interface features a distinc-

tive array of ions/molecules exchanged between blood and

FIGURE 6.—Foam cells in the stroma of the choroid plexus (CP) of rats injected with methylcellulose (0.5% Methocel, 5 ml/kg IP daily for 30 days).

The ‘‘foamy’’ cells (enlarged, light-colored cells in the right panel) may be macrophages that ingested methylcellulose in the peritoneum and then

migrated to the CP, where they penetrated the interstitial space and caused swelling of tissue. Control CP (left panel), devoid of foam cells,

displays a normally appearing epithelial cell lining and vascular elements in each villus core. Experimental data are summarized in Table 4 and

described by Roth and Krinke (1994). Hematoxylin and eosin, �400.

Vol. 39, No. 1, 2011 TOXICOLOGY OF VENTRICULAR CSF-BORDERING CELLS 193

CNS regions (Johanson et al. 2008). A prominent feature of CP

is its great secretory abilities. In this case, the nexus mediates

material flow from the CP origin to putative targets in CNS

(Johanson, Miller, et al. 2010). Compartments of the CSF

nexus, diagrammed in Figure 1, are analyzed in the following

in the sequence of the physiological flow.

DISTRIBUTIONAL NEXUS: CHOROID PLEXUS-CSF-EPENDYMA-BRAIN

Substances secreted by CP tissues into the ventricles tra-

verse a series of boundaries and compartments to reach poten-

tial downstream receptors at neuronal and glial membranes.

For perspective on the CSF-brain interior, some distinguishing

features of the tissue compartments and systemic properties of

the nexus are addressed in the following. Fine histological fea-

tures of epithelial cells and organelles are presented further

along in the review.

Choroid Plexus as a Mini-Organ

CP is a secretory engine that actively synthesizes and trans-

ports a plethora of inorganic and organic solutes destined for

brain (Johanson 2008). Collectively, the plexus tissues are a

small fraction (*0.002) of total brain mass. Industrious activ-

ity of the epithelium, however, necessitates a choroidal blood

flow rate that is 5- to 10-fold greater than mean cerebral blood

flow (CBF) (Kadel, Heistad, and Faraci 1990; Szmydynger-

Chodobska, Chodobski, and Johanson 1994). Brisk vascular

perfusion of CP supports the energy and substrate demands

of a high degree of epithelial metabolism. As the main genera-

tor of CSF, the CP epithelia in the four ventricles transfer most

(up to 75%) of the water molecules diffusing from plasma into

CNS. Prolific water movement through CP epithelial aquaporin

1 channels (Oshio et al. 2003) into the ventricles produces the

CSF medium to convey solutes into and out of brain. Continual

fluid production by CP is the driving force behind steady CSF

streaming along the neuraxis.

Ventricular Cerebrospinal Fluid

Understanding how the CP epithelial and ependymal workings

impact the brain is predicated on knowledge of CSF dynamics.

Cells that border the ventricular system affect, and are affected

by, the CSF volume, flow, pressure, and composition. Dynamic

interplay between CP and ependyma, with CSF as the intermedi-

ary, reveals the multiple homeostatic actions of cells lining the

brain’s internal surface. Upon disruption by pathogens and toxi-

cants, the consequent CSF dyshomeostasis impairs brain metabo-

lism. This is presumably due to compromised transport and barrier

mechanisms. Salient features of CSF as a circulating medium

are discussed in the following relative to toxico-pathology.

Regular turnover of CSF in the ventricular system allows

optimal neuronal functioning. Human CSF is renewed three

to four times daily (Silverberg et al. 2002). Flow of CSF is from

lateral ventricles to the third and then through the narrow

mesencephalic aqueduct into the fourth ventricle. CSF

advances toward drainage sites (Grzybowski et al. 2006) by

bulk flow (arrows 4 and 9, Figure 1) driven by a hydrostatic

pressure gradient between CSF and venous blood (Johnston,

Boulton, and Flessner 2000; Pollay 2010). CSF bulk flow dis-

tributes endogenous materials widely throughout the CNS. The

term 3rd circulation has been coined for CSF flow in regard to

its major role in central fluid dynamics. Several minutes after

intracerebroventricular (ICV) injection, radiolabeled test tra-

cers are swept by CSF convection to regions distant from lat-

eral ventricles (Ghersi-Egea et al. 1996). Substances both

beneficial (vitamins) and harmful (cytokines) rapidly distribute

throughout the CNS after gaining access to ventricles and

downstream (subarachnoid) regions (Figure 1).

Lacking true lymphatic capillaries for drainage, the CNS

needs the CSF to continually flow in order to excrete poten-

tially harmful cerebral catabolites and to remove peptides that

leak into the brain. Concentration gradients, from brain ISF to

ventricular CSF, promote net diffusion of cerebral metabolites

and excess proteins into the ventricles (arrow 8, Figure 1). Such

CSF sink action (Parandoosh and Johanson 1982) rids the brain

of metabolic products and toxic agents/pathogens that access

CNS. Overwhelming the clearance or excretory capacity of

CSF by diseases/toxicants may injure the brain and protective

CP/ependymal cells.

Ependyma/Subependymal Regions

Ependymal and periventricular regions quickly take up toxic

as well as trophic substances transported into CSF by CP

FIGURE 7.—Vacuole formation in the choroid plexus (CP) epithelium

of rats treated with a tertiary amine. The cytoplasm of most epithelial

cells is filled with vacuoles that give the tissue the look of hydropic

pallor. This intense vacuolar response was typical of rats injected with

piperamide (500 or 750 mg/kg SC) and then necropsied one day later.

Vacuoles displace the nuclei to a basal position. Ependyma and the

adjacent periventricular brain appear normal (the slight separation

between the ependyma and the brain tissue is an artifact). Congested

CP fills the lateral ventricle. Experimental data are summarized in

Table 4 and interpreted by Levine (1994). Hematoxylin and eosin,

�450. Due to unavoidable circumstances the above figures were not

originals but were scanned from reprints.

194 JOHANSON ET AL. TOXICOLOGIC PATHOLOGY

(arrows 1–5, Figure 1). The permeable CSF-brain interface in

the lateral ventricles permits extensive, dynamic diffusion of

endogenous/xenobiotic agents into (arrow 5, Figure 1) and out

of (arrow 8, Figure 1) the brain. Ependymal destruction by tox-

icants and diseases further distorts the delicate biochemical

balance between CSF and ISF (Johanson 2008). This imbal-

ance neurochemically destabilizes the periventricular regions.

That a steady supply of micronutrients and trophic factors is

critical for ependymal well-being is exemplified by the finding

that malnutrition leads to deformation and metabolic disruption

of ependyma, at least early in development (S. P. Sharma and

Manocha 1977).

Brain Regions Adjacent to Ventricular CSF

Major neuronal networks (cholinergic, serotonergic, etc.) lie

in subependymal, neurogenic regions proximate to CSF

(Miyan, Nabiyouni, and Zendah 2003). Subventricular neurons

are accessible to endogenous and exogenous materials that dif-

fuse from CSF across the ependyma (Figure 1). Modulated

regions include the hippocampus (engaged in memory), the

SVZ and dentate gyrus (neurogenesis), hypothalamus (endo-

crine and autonomic regulation), periaqueductal gray (pain reg-

ulation), and the pons-medulla (respiratory and cardiovascular

control centers). These homeostatically sensitive areas exhibit

compensatory responses to alterations in CSF osmolality, [Kþ],

[Naþ], [Cl�], [Caþþ], pCO2, pO2, and pH. Therefore,

periventricular regions need to be carefully protected against

debilitation by toxic/pathogenic insults from foreign agents in

ventricular fluid.

CHOROID PLEXUS-EPENDYMA DISRUPTION: IMPLICATIONS FOR BRAIN

Brain health relies upon sound barrier/homeostatic mechan-

isms in CP and cerebral capillaries for protection against

blood-borne toxicants. Impermeable tight junctions (Brightman

and Reese 1969) and reabsorptive solute transporters at

BCSFB interfaces (Spector and Johanson 2010b) normally

protect the CNS against attack from foreign agents (Zheng

2001). Advanced aging, infectious agents, and toxic materials,

especially with chronic exposure, can all undermine the integ-

rity of the transport interfaces (Behl et al. 2009; Shi and Zheng

2007). Compromised barriers render the CNS vulnerable to

injury. The BCSFB in CP, as a regulated gateway for molecular

and cellular traffic into the brain (Johanson 2008), will be high-

lighted in the following.

Weakening of BCSFB augments penetration of harmful

plasma-borne materials into CSF and ultimately into the brain.

Collapse of CP-CSF results from many stresses on CNS:

arterial hypertension (Murphy and Johanson 1985), transient

forebrain ischemia (Palm et al. 1995), hyperthermia (H. S.

Sharma, Duncan, and Johanson 2006), traumatic brain injury

(H. S. Sharma, Zimmermann-Meinzingen, and Johanson

2010; Szmydynger-Chodobska et al. 2009), and advanced

aging (Preston 2001). Upon BCSFB damage in pathophysiologic

states, the increased paracellular permeability allows acces-

sion of plasma proteins and other markers to CSF. Commonly,

the elevated protein concentration in CSF leads to ventriculo-

megaly and periventricular edema (H. S. Sharma and Johanson

2007). As a consequence, cognitive/behavioral abilities in

animals are compromised when CSF-bordering hippocampal

and hypothalamic regions are injured by physical stressors

(H. S. Sharma, Duncan, and Johanson 2006). However, there

are relatively few systematic analyses of agent stress on the

CP-CSF-ependyma-SVZ-brain nexus (Figure 1).

Normally the CNS transport interfaces ward off mild to

moderate threats by invading bacteria, viruses, and xenobiotics.

Vigorous challenges by virulent pathogens and toxic drugs

(Levine 1987), though, reduce the homeostatic reserve of trans-

port interfaces. This leads to damage of CSF-contacting neural

regions. It is not feasible to predict exactly the untoward

responses by CP to potent noxious materials. A particular tox-

icant can interfere with tight junctions or with the nutritional/

trophic transporters at the BCSFB (Spector and Johanson

2010b) that vectorially direct solutes to neuronal targets via

CSF. Extreme interference with CP function causes CSF dys-

homeostasis that harms brain.

VENTRICULAR CSF-BORDERING CELLS: HISTOLOGY AND

PHYSIOLOGY

Before reviewing effects of organic agents on CSF-mediated

distribution, we describe normal ultrastructure and function

of ventricle-contacting cells. Three types of cells demarcate

ventricular CSF: choroid epithelial, ependymal, and circum-

ventricular (Table 1). Within these major groupings are

subpopulations of the parenchymal CP, ependyma, and CVOs.

Cellular heterogeneity and phenotype importantly bear on eva-

luations of specific endocrine, pharmacologic and toxicologic

responses. Cells contacting the CSF are part of a dual classifi-

cation of neuroepithelia: those suspended in CSF (CP) or those

comprising the ventricular wall (ependyma and CVOs).

Choroid Plexus Epithelial Cells

Anatomically, the base of the choroid epithelial fronds is

anchored to the brain at fixed points in the lateral, third, and

fourth ventricles. Ramifying outward from where the stalk

attaches to the ventricular wall, the tufts of CP are suspended

within CSF. Physically this arrangement provides enormous

epithelial surface area for translocation of nutrients and drugs

between plasma and CSF. However, this same large transport

area is potentially available for untoward leakage of plasma

proteins and harmful agents into CSF. The basic parenchymal

element in CP is cuboidal epithelium. An ultrastructural image

of a typical CP epithelial cell is portrayed in Figure 2. Large

surface areas promote extensive transport across the basolateral

(plasma-facing) and apical (CSF-facing) membranes. Because

the BCSFB is equipped for heavy-duty transport, severe patho-

logic damage to CP markedly distorts CSF homeostasis and

ventricular configuration.

As a multipurpose organ, the CP carries out diverse tasks:

secretory, synthetic, and reabsorptive (Table 2). Best known

among the secretory functions is CSF formation. CSF forms

Vol. 39, No. 1, 2011 TOXICOLOGY OF VENTRICULAR CSF-BORDERING CELLS 195

at the uniform rate of *0.4 ml/min/g CP in mammals. To initi-

ate CSF formation, the basolateral membrane takes up Naþ,

Kþ, Cl�, and HCO3� via several ion co-transporters and

exchangers (Johanson et al. 2008; P. D. Brown et al. 2004).

Ions and water taken up from the interstitium by epithelial

basolateral membrane (arrow 2, Figure 1) then move through

cytoplasm to the opposite (apical) side. There the apical mem-

brane extrusion mechanisms (arrow 3, Figure 1) include ion

channels, aquaporin 1, and co-transporters. Apical membrane

Naþ pumping (Q. R. Smith and Johanson 1980), by setting

up the appropriate transmembrane ion gradients, actively pro-

pels CSF production.

Drugs that induce renal natriuresis or diuresis consistently

reduce CSF turnover into the ventricles. CSF formation is

inhibited 10–30% by sympathetic, cholinergic, and serotoner-

gic neurotransmitters (Nilsson, Lindvall-Axelsson, and

Owman 1992). Neuropeptides such as arginine vasopressin,

atrial natriuretic peptide, and angiotensin II also slow down

CSF production by 15–20% (Johanson et al. 2008). Upon

secretion, the nascent CSF mixes with brain extracellular fluid

as it flows down the neuraxis. CSF flow decreases in aging.

In neurodegenerative disease (Silverberg et al. 2001) or drug

poisonings, the CSF turnover rate (i.e., formation rate / volume

can be cut in half. Adverse consequences include reduced

clearance, and hence accumulation, of proteins and cerebral

metabolites. ISF retention of uncleared waste products impairs

brain metabolism and behavior. Normally the CSF with its

debris is efficiently reabsorbed into lymph and/or venous blood

at multiple sites in arachnoid tissue (Grzybowski et al. 2006;

Johnston et al. 2004).

In addition to forming CSF, the CP homeostatically

protects the brain (Table 2). Acting like a kidney (Spector and

Johanson 1989), the CP helps to keep the chemical composi-

tion of CNS extracellular fluid in a stable state. In true kidney-

like fashion, the CP actively sets the ionic composition, pH,

and osmolality of extracellular CSF. Low permeability

imposed by tight junctions at the BCSFB allows ion and

molecular gradients to be established secondary to active

transport. For example, Caþþ and Kþ in CSF are held at

concentrations lower than plasma, while Mgþþ and Cl� are

kept at higher levels. CSF titers of numerous growth factors,

fluid-regulating peptides, and proteins are maintained at

0.01 to 0.001 of their respective concentrations in plasma.

Regulated CSF ions benefit the neurons that require finely

maintained extracellular concentrations.

Second, a plethora of reabsorptive solute transporters in the

apical membrane actively removes potentially toxic organic

acids and peptides from CSF (arrow 10, Figure 1). Organic

anion transporting polypeptides (OATPs) are part of an

increasingly appreciated gene superfamily (Spector and Johan-

son 2010b). OATP was initially immunolocalized to the CP

apical membrane (Angeletti et al. 1997), a strategic location

for clearing metabolites from CSF (Ohtsuki et al. 2003).

In BCSFB, the OATPs mediate reabsorptive (excretory) trans-

port of a wide spectrum of amphipathic organic solutes in CSF:

steroid conjugates, neurotransmitter anion catabolites (homo-

vanillic acid, a major metabolite of catecholamine neurotransmit-

ters), bile salts, anionic oligopeptides, drugs, and xenobiotics

(Hagenbuch and Meier 2003). CSF levels of certain drugs are kept

at subtherapeutic low levels by OATP and the P-glycoprotein

transporter, the potential roles of which in CP need evaluation for

therapeutic agents such as suramin and eflornithine, agents that

are used against human African trypanosomiasis (Sanderson

et al. 2008; Sanderson, Khan, and Thomas 2007).

Another CP reabsorptive transporter of organic solutes, with

a wide spectrum of substrate affinity, is the family of low

density lipophilic receptor-related proteins (LRP). Located

apically, the LRP-1 and LRP-2 clearance transporters keep

peptides and peptide fragments in the CSF from building to

toxic levels (arrow 10, Figure 1). As a promiscuous transporter,

LRP-1 can clear from CSF/brain as many as forty types of

molecules including APOE4, amyloid precursor protein, and

amyloid oligopeptide fragments (Crossgrove, Li, and Zheng

2005). LRP-2 (or megalin) has also been implicated in remov-

ing amyloid from CSF (Alvira-Botero and Carro Forthcoming).

Other apically located organic solute transporters, including

PEPT2 (proton-coupled oligopeptide transporter 2), prevent

CSF accumulation of compounds (e.g., 5-aminolevulinic acid

[ALA], a heme precursor) to toxic levels (Hu et al. 2007;

D. E. Smith, Johanson, and Keep 2004). Reabsorptive transport

from CSF is essential because ALA leaks into CSF from blood

and needs to be quickly cleared from the ventricles (arrow 10,

Figure 1) to avoid toxicity (Terr and Weiner 1983).

Third, hepatic-like metabolism in BCSFB epithelium

minimizes toxic drug accumulation in the CSF and CNS. This

liver-type role affords brain an extra line of defense against

TABLE 1.—Characteristics of epithelial cells bordering the ventricular cerebrospinal fluid (CSF) system.

Type of epithelium

Ventricular

regiona

Shape

of cell

Layers of

epithelium

Inter-

epithelial

junctions Presence of cilia Capillaries Specialized functions

Choroid plexus (CP) LV, 3V, 4V Cuboidal Single Tight Some CP cells Permeable CSF secretion and homeostasis

Ependyma LV, 3V, 4V,

and

spinal cord

Columnar Single (adult)

Multiple (infant)

Gapb Some ependymal cells N/A (underlying

capillaries are

of brain)

Regulatory exchange of water/

solutes with brain

Circumventricular

organs (CVOs)

3V, 4V Columnar Single Tight No Permeable Regulation of plasma volume,

composition, and pressure

a LV, 3V, and 4V refer to the lateral, third, and fourth ventricles, respectively.b LV ependyma have intercellular gap junctions, whereas some 3V ependymal cells have tight junctions.

196 JOHANSON ET AL. TOXICOLOGIC PATHOLOGY

toxic drugs and compounds (Strazielle, Khuth, and Ghersi-

Egea 2004). Xenobiotic molecules induce drug-metabolizing

enzymes in liver and CP. In vitro, the expression of

UDP-glucuronosyltransferase (UGT1A6 isoform) is upregu-

lated in rat CP epithelium when tissue is challenged with

3-methycholanthrene (3-MC) or paraquat (Gradinaru et al.

2009), leading to a two- to threefold increase in the choroidal

1-naphthol glucuronidation activity. UGT1A6 mRNA expres-

sion (polymerase chain reaction [PCR] analysis) was also

augmented more than twofold after incubation with these two

drugs. Drug-metabolizing enzymes in the BCSFB transform

xenobiotics that might harm the brain if left unmetabolized.

Fourth, an immune-type function is subserved by CP

through the activity of antigen-presenting cells in the epithelial

lining (Nathanson and Chun 1989), thereby permitting immune

surveillance of CSF. Normally just a few immune cells migrate

from plasma into ventricles to monitor CSF immunologic

status. Under pathologic or toxicologic stress, however, the

upregulation of CP chemokines, integrins, selectins, and matrix

metalloproteinases renders the BCSFB more penetrable

(Prendergast and Anderton 2009). Consequently, many inflam-

matory cells invade CSF and provoke CNS autoimmune

disease. Since CP epithelium engages in diverse tasks to defend

and cleanse CSF, the BCSFB itself needs protection against

toxicant and foreign molecules (Johanson, Silverberg, et al.

2005). High levels of antioxidant glutathione in CP (Cooper

and Kristal 1997) comprise a biochemical defense against

oxidant molecules transported or metabolized by epithelial

cells. Overall, the CP in healthy young adult humans has

sufficient barrier, metabolic, enzymatic, and reabsorptive

capabilities to keep CSF composition sufficiently pure for

optimal neuronal performance.

Finally, over and above its homeostatic role in stabilizing

neurochemical composition and immune health of CSF-CNS,

the CP is the chief supply route for transferring plasma-borne

materials into the ventricles. As much as 70–80% of water, ion,

vitamin, and peptide transfer into CNS occurs across choroidal

epithelium (arrows 2 and 3, Figure 1) (Johanson et al. 2008).

Vitamins B (folate) and C are actively transported into CNS

preponderantly via the blood-CSF interface (Spector and

Johanson 2006). There is also significant neuroendocrine pep-

tide distribution mediated by hormonal transport mechanisms

at the BCSFB interface (Kozlowski 1986). Prolactin, insulin-

like growth factor (IGF)-1, and leptin use CP as a hormonal sig-

nal relay station in being transported from plasma to ventricles

(Dietrich et al. 2008; Redzic et al. 2005). Then these peptides

are convected by CSF bulk flow to hypothalamic regions such

as the arcuate nuclei (Rodriguez, Blazquez, and Guerra 2010).

In this manner, the CP-CSF participates in hormonal signal

transfer within neuroendocrine feedback loops for modulating

feeding/satiety and reproductive behaviors.

Translocation of plasma ions, micronutrients, and water into

CNS is mainly via CP. Serious injury of BCSFB by microbes or

xenobiotics alters brain homeostasis. When the CSF milieu is

substantially changed, brain regions close to ventricles suffer

damage to neurogenesis and cognition. Many pathogens, toxi-

cants, and neurological disorders damage the plexus. Following

stroke and trauma, not only is CSF delivery of trophic factors to

neurons compromised, but also the CSF removal of brain cat-

abolites is diminished (Johanson et al. 2000). Molecules in

TABLE 2.—Homeostatic, secretory, and metabolic functions of choroid plexus (CP) epithelial cells.

Functional aspects

Mechanisms for transport, catalysis,

or secretion

Physiological effects on, or mediation by,

cerebrospinal fluid (CSF) Significance for brain well-being

Kidney-like role (fluid balance) Naþ-Hþ, Cl�-HCO3�, and NaHCO3

transporters (inorganic ions)

Transfers Naþ, Kþ, Cl�, Hþ, or HCO3� to

maintain ion concentrations in the

ventricles

CSF turnover and flow help to buffer

[Hþ], [Kþ], and osmolality in brain

interstitial fluid (ISF)

Organic solute reabsorption

(clearance)

Organic anion transporting

polypeptide (OATP) and

low-density lipophilic

receptor-related protein

(LRP-1)

Removal of CNS catabolites (anions and

peptides) by CP apical reabsorptive trans-

port and CSF bulk flow helps to purify

brain extracellular fluid content

Cleansing effect (by CSF sink action) is

vital for optimal functioning of the

neuronal networks and glia

Liver-like role (for metabolism

of drugs)

Glutathione S-transferase and

NADPH-cyto-chrome P450

reductase in epithelium

By serving as an ‘‘enzymatic barrier,’’

hepatic-like enzymes in CP can metabolize

drugs and prevent their entry into CSF

Important to prevent neurotoxic drugs

from entering CSF and to metabolize

harmful drugs that do penetrate the

blood-brain barrier (BBB)

Immune-related functions Immune cells, such as epiplexus cells

(i.e., macrophage-like elements,

also known as Kolmer cells)

Antigen-presenting cells at the blood-CSF

epithelial interface help regulate the

immune capacity of CSF

Immunosurveillance of CSF by CP works

in concert with BBB activity to

maintain a healthy immune state for

the central nervous system

Supplier of micronutrients MTHF (methylene-tetrahydrofolate)

and SVCT2 (Naþ-activated

ascorbic acid [vitamin C]

transporter-2)

Active transport of folic acid and vitamin C

to maintain CSF concentrations that are

higher than in plasma

Folate and ascorbate in CSF are needed

by brain, but neural tissue does not

receive them by way of cerebral

capillaries

Neuro-endocrine regulation Receptor-mediated transport of

prolactin, insulin-like growth

factor (IGF)-1, and leptin

Peptides that are transported from plasma to

CSF are carried by bulk CSF flow to vari-

ous hypothalamic nuclei

Receptors in the arcuate nuclei bind

specific peptides to integrate

metabolic and reproductive

phenomena

Vol. 39, No. 1, 2011 TOXICOLOGY OF VENTRICULAR CSF-BORDERING CELLS 197

ventricular CSF, even large proteins, have access by diffusion

(down concentration gradients) to periventricular brain regions

(arrow 5, Figure 1). In the reverse direction, catabolically gen-

erated macromolecules in the hemispheres diffuse from inter-

stitium into the ependyma into the ventricular CSF (arrow 8,

Figure 1). Proper steady-state partitioning of molecules

between CP-CSF and brain requires intact ependyma.

Ependymal Cells with Gap Junctions Versus Tight

Junctions

Structurally and functionally, the ependyma lining the cere-

bral ventricles and central canal of the spinal cord is heteroge-

neous. Ontogenetically, there is progression of ependyma as a

multiple layer in the fetus to a single layer in adults. Various

cell types comprise the fetal ependymal wall (Davson and

Segal 1996). These include the tanycyte and ependymal

astrocyte as well as the true ependymal cells that persist into

adulthood. All three cell types have cilia and microvilli at their

CSF-facing apical surfaces. However, structural differentiation

occurs at the basal (brain-facing) sides. Tanycytes and radial

glia send long dense fibers to the CSF. The tanycyte’s other end

projects basally to termini in certain hypothalamic nuclei,

which receive via axonal flow those neuropeptides (‘‘endocrine

signals’’) taken up from the CSF (Rodriguez et al. 2005). The

ependymal astrocyte is a columnar cell with branching periph-

eral processes. After birth, both tanycytes and ependymal astro-

cytes dwindle in number so that the ventricular lining in adults

is mainly ependyma proper. Even in adulthood the ependyma

depend on growth factors such as vascular endothelial growth

factor (VEGF) and transforming growth factor beta (TGFb),

both secreted by CP, to maintain stable cellular structure and

fluid balance with surrounding ISF (Maharaj et al. 2008).

Diminished growth factor support for endothelial and ependy-

mal cells leads to vascular permeability changes and periventri-

cular edema.

Ependymal cells proper are not all equal. Heterogeneity

among ependyma consists of cells having cilia or not, and in

possessing leaky gap junctions or the tighter zonulae occlu-

dentes (not as prevalent). Most lateral ventricle ependymal

cells have intercellular gap junctions just under the apical sur-

face. Gap junctions are highly permeable. Large proteins, the

size of ferritin (mw *445,000), readily diffuse through the

paracellular space to cross the ependyma and thus easily move

between CSF and brain. Accordingly, the ependymal interface

between ventricular fluid and underlying brain is not regarded

as a CSF-brain barrier. Being highly permeable, the ependyma

is thus unlike the restrictive BCSFB and BBB, which impede

paracellular diffusion.

Although the lateral ventricle ependymal system is homoge-

neous, the ependyma lining the third and fourth ventricles are

heterogeneous. In specific regions of third ventricle (3V), there

are tight junctions between specialized ependymal cells. Given

the proximity of 3V to hypothalamus, the structurally modified

ependyma here reflects specialized (regulated) functional rela-

tionships between 3V CSF and the adjacent paraventricular

nucleus. In regions such as the arcuate nucleus (Rodriguez,

Blazquez, and Guerra 2010), the nearby CSF has ‘‘access’’ to

hypothalamus in that hormones exchange via diffusion across

the ependymal interface. Brain midline CVOs (Gross 1992)

in 3V and 4V are especially heterogeneous.

More than being a structural boundary between ventricular

CSF and brain, the ependyma is a dynamic interface mediating

the movement of cilia, CSF, and migrating neuroblasts. On the

ependymal apical surface is a network of cilia that beat in coor-

dination to facilitate CSF circulation. Each cilium is a subcel-

lular organelle emanating from the cell’s interior. Planar cell

polarity signaling may be controlled by cilia (Fischer and

Pontoglio 2009). Cadherin genes (Celsr2 and Celsr3) regulate

planar cell polarity. Celsr genes that are mutated or absent

(knocked out) compromise the development and planar organi-

zation of ependymal cilia. This leads to defective CSF

dynamics and hydrocephalus. Many ciliopathies render CP and

ependyma dysfunctional, thereby distorting the volume and

composition of CSF secretion (Banizs et al. 2005). Migration

of neuroblasts from the lateral ventricle wall to the olfactory

lobe relies upon ciliary-guided flow to forebrain sites for inser-

tion into circuits as interneurons (Sawamoto et al. 2006).

Proper development and operation of the brain depend on nor-

mally functioning cilia in CP and ependyma.

Currently surging research on neurogenesis in the SVZ is

generating additional insights on ependyma. As a delicate

interstitial microenvironment for stem cells (Spector and

Johanson 2007a), the neurogenic niches of dentate gyrus and

SVZ require compositional stability. This assures finely regu-

lated gliogenesis and neurogenesis, even in adulthood. Repair

mechanisms are essential to the integrity of the ependymal

lining disrupted by diseases (hydrocephalus) and disorders

(trauma). Ependyma are also damaged in advanced aging.

One restorative mechanism is SVZ-mediated repair of the

ependymal wall by astrocyte insertion in regions where

ependyma are detached. When new astrocytes incorporate

in gaps between ependymal cells, they take on antigenic and

morphologic characteristics of neighboring ependymal cells

(Luo et al. 2008). This constitutes evidence for non-neuronal

repair as well as the more established neuronal reconstitution

carried out by SVZ elements.

In addition to dynamic interaction between ependyma and

SVZ for repair mechanisms, there is homeostatic interplay

between CSF, ependyma, and the periventricular brain in fluid

balance. Even though the CSF-brain interface permits macro-

molecule diffusion between ependymal cells, implying unregu-

lated water and ion paracellular movements, there is still great

plasticity of expression of ion and water channels in ependyma.

Responding to elevated CSF pressure or a change in CSF Naþ

concentration, the ependyma upregulates aquaporin 4 water

pores and epithelial Naþ channels (H. W. Wang et al. 2010).

This may reflect regulation of ependymal cells per se, or con-

trolled fluid transfer into brain across ependymal regions with

tight junctions. Whatever the explanation for induced channel

expression, the findings point to sensitivity of the ependymal

interface to CSF changes.

198 JOHANSON ET AL. TOXICOLOGIC PATHOLOGY

Circumventricular Organs in Ependymal Wall

Nested within the third and fourth ventricular walls are specia-

lized regions that house ependymal mini-organs, the CVOs (Vio

et al. 2008) (Figure 3). Overall their primary functions can be

sensory, secretory, or both (see Table 3). CVOs bridge endocrine

and autonomic phenomena (Uschakov et al. 2009), particularly

the homeostatic adjustment of fluid composition, volume, and

osmolality (Henry, Grob, and Mouginot 2009). CVOs are sites

of action or release of fluid-regulating peptidergic neurotransmit-

ters, cytokines, and hormones (Feher et al. 2010; Sotthibundhu,

Phansuwan-Pujito, and Govitrapong 2010). Anatomically, CP

is not a CVO because the preponderance of choroidal tissue does

not reside in the ventricular wall. Instead, the CP dangles within

the ventricular spaces away from the ependymal wall.

Lateral ventricle walls do not contain CVO structures.

However, the midline neuraxis running through the third and

fourth ventricles has many CVOs. Each one lacks a BBB. The

third ventricle harbors the subfornical organ (SFO), subcom-

missural organ (SCO), and organum vasculosum of the lamina

terminalis (OVLT) and connects to narrow recesses leading to

the median eminence (ME), neural lobe of the hypophysis

(pituitary gland [NLP]), and the pineal gland (PI). Protruding

into the fourth ventricle is a vasculo-epithelial pouch called the

area postrema (AP). CVOs receive peptide signals from CSF

as well as plasma. Similar to CP, a prominent and distinguishing

feature of SFO, SCO, OVLT, and AP is the permeable capillary

network allowing peptide signals to diffuse from plasma to sen-

sory receptors in the CVO interior. Peptide signaling is trans-

duced by neurons to electrical impulses for neurotransmission

out of CVOs toward central regions that integrate reflex

responses to altered plasma chemistry. The SFO, for example,

receives enhanced angiotensin II signaling from plasma for

integrating the thirst response to dehydration by stimulating

drinking. Each CVO domain is encompassed by an epithelial

ring (with tight junctions) that physically and functionally

separates the mini-organ’s ‘‘business’’ from surrounding brain.

Consequently, the CVO’s extracellular milieu bears a closer

resemblance to plasma than to brain interstitial fluid. CVOs are

‘‘open physiologic windows’’ into the brain. They allow CNS

reception of plasma peptide signaling molecules that would

otherwise be centrally excluded by the ‘‘closed door’’ of the BBB.

CVOs can be categorized into two groups according to their

location in the ventricles: those at the confluence of the

ventricular cavities (SFO, SCO, and AP) and those lining the

ventricular recesses (NLP, OVLT, and PI). Vascular architec-

ture, topographically, varies from one CVO to another

(Duvernoy and Risold 2007), but the cardinal feature of each

CVO is a leaky BBB allowing intimate barrier-free contact

between blood and neural tissue. Open capillaries in CVOs

means that endothelial transporters are not needed to actively

move substrate (e.g., peptides) into CNS.

CVOs are functionally like CP in having highly permeable

capillaries open to nearby ISF. Contrariwise, due to BBB

restriction on diffusion, the brain ISF is not in open passive

communication with blood. However the SFO, SCO, OVLT,

and AP are unlike the CP in that the former CVOs are engaged

in transducing plasma hormonal signals to electrical messages.

On the other hand, CP basolateral receptors take hormones

from blood (e.g., leptin, IGF-2, prolactin) for transepithelial

conveyance into CSF. By endocrine-like CSF bulk flow, these

CP-secreted signaling peptides are conveyed to CNS targets

(Kozlowski 1986; Scott et al. 1977). Generally speaking, the

CVOs and CP take up plasma-borne peptides and relay the

‘‘hormonal information,’’ by neurons or CSF, to peptide-

receptor bearing cells in hypothalamus and other central

regions. Endocrine systems throughout the body can conse-

quently be modulated by compensatory neural and hormonal

TABLE 3.—Salient features of the circumventricular organs (CVOs).

Organ name Ventricular location Main function(s) Physiological and integrative roles in neuroendocrine/autonomic responses

Subfornical organ (SFO) Third Sensory Involved in the thirst response to increased osmolality (and angiotensin II) in plasma

during severe dehydration; sends signals to the medial preoptic nucleus (Henry,

Grob, and Mouginot 2009)

Pineal gland (PI) Third Secretory Integrates circadian rhythms, involving light stimuli, through synthesis and release of

melatonin into cerebrospinal fluid (CSF); melatonin enhances proliferation of stem

cells in subventricular zone (Sotthibundhu, Phansuwan-Pujito, and Govitrapong

2010)

Neural (caudal or posterior)

lobe of the pituitary

gland (NLP)

Third Secretory In response to hypothalamic signals, the NLP releases antidiuretic hormone (ADH) and

oxytocin into plasma to modulate kidney water transfer and uterine muscle

contraction, respectively

Median eminence (ME) Third Secretory Releases neurohormones to regulate the rostral (anterior) part of the pituitary gland;

inactivation of tyrosine hydroxylase in ME is required to induce a prolactin response

(Feher et al. 2010)

Organum vasculosum of

lamina terminalis

(OVLT)

Third Sensory and secretory Detects peptide concentrations in order to control fluid regulatory (homeostatic)

responses; integrates CVO neural signals with hypothalamic nuclei (Uschakov et al.

2009)

Subcommissural organ

(SCO)

Third Secretory Secretes proteins into CSF, such as spondin (which is critical to the normal

development of Reissner’s fiber and the ventricular system–spinal central canal;

Vio et al. 2008)

Area postrema (AP) Fourth Sensory As the ‘‘vomit reflex’’ center, the AP detects noxious substances in blood and

stimulates emesis to clear toxic chemicals from the body

Vol. 39, No. 1, 2011 TOXICOLOGY OF VENTRICULAR CSF-BORDERING CELLS 199

output from CNS. Such feedback loops, involving intermediate

transport/signaling steps via CP and CVOs, constitute homeo-

static machinery for restoring distorted body fluids to a stable,

specialized composition. Hence, toxic/pathological injuries to

CP, hypothalamus, and CVOs, including periventricular

lesions, affect not only the local brain environment but also the

activity of endocrine systems throughout the body.

PERIVENTRICULAR LESIONS AND THE CHOROID PLEXUS-CSF NEXUS

Periventricular lesions are commonly downstream sequelae

of the uncontrolled passage of substances through a compro-

mised BCSFB. Materials that are inappropriately transferred

or that leak across the CP include infectious agents, blood com-

ponents (cells and proteins), and tumor cells. Representative

lesions for each group exemplify types of damage inflicted

on the ventricular wall and underlying brain. For example, the

protozoan Toxoplasma gondii frequently infects the CNS of

immunocompromised AIDS patients. Acting as a staging plat-

form, CP receives and activates Toxoplasma, which in turn

engenders choroid plexus inflammation (Falangola and Petito

1993) that promotes CSF dissemination to periventricular sites

where infectious agent and leukocytes accumulate (Stahl and

Turek 1988). Ependyma and CP in all four ventricles become

diffusely necrotic (Bourgouin et al. 1992). Another infection

of CP-CSF is by ovine lentivirus, which invades periventricular

sites as part of a more generalized encephalomyelitis (Brodie,

Bickle, and DeMartini 1995); the CSF lentiviral markers reflect

CNS pathology. Various blood cells have greatly restricted

access to CSF except in pathophysiologic conditions associated

with autoimmune disease or hemorrhage into CP-ependymal

zones. Augmented leukocyte (T-cell) penetration across the

CP into CSF occurs in relapsing multiple sclerosis (MS) in

humans or experimental allergic encephalitis (EAE) models

of MS, leading to formation of hallmark periventricular lesions

and ependymal/subventricular destabilization. Consequently,

neurogenesis is inhibited by T cell release of granzyme B

(T. Wang 2010). Recent findings in an EAE model point to

T lymphocyte antigen specificity in defining the topography

of periventricular lesions (Berger et al. 1997). Moreover,

extensive erythrocyte permeation across BCSFB distorts

CSF composition and interferes with ependyma/SVZ

functions. Thus, in neonatal brain stressed by hypoxia/ische-

mia, the choroidal/intraventricular and severe subependymal

hemorrhaging perturb the interstitial fluid environment in

periventricular white matter (Bernert et al. 1988). Tumors

(e.g., high-grade B-cell lymphoma) may form directly in the

CP (Cecchi et al. 2008) and migrate across BCSFB to become

entrenched in the periventricular brain. Metastatic CNS

lymphoma, having entered CSF via the CP, can disseminate

to periventricular sites (Kobayashi et al. 2009). Primary intra-

cerebral malignant lymphomas, albeit rarely, fulminate in CP

and putatively escape the BCSFB to permeate subependymal

sites along the lateral, third, and fourth ventricular walls

(Shibata 1989). Specific pharmacologic targeting of CP/BCSFB

may be used to thwart penetration of cancer cells (as well

as viruses and potentially harmful leukocytes) into the

CSF-ependyma-SVZ nexus. Studies of toxicant-induced

disruption to the nexus, as described in the following, furnish

useful basic data for formulating prophylactic strategies

against infection or measures to counter toxicant exposure.

ORGANIC AGENTS USED IN CHOROIDO-EPENDYMAL TOXICOLOGY

STUDIES

Drugs from different classes have been tested for toxicity to

CSF-bordering cells and underlying the brain. Tertiary amines

were extensively analyzed for deleterious effects on BCSFB

and the CSF-brain interface. A series of compounds analyzed

includes molecules with one, two, or three tertiary amines

(Levine 1977). All contain a cyclic structure, either saturated

(piperazine and piperidine) or unsaturated (naphthyridine and

pyridine). Agents used in ventricular cell-CSF investigations,

including piperidine/piperazine derivatives and amoscanate,

are compiled in Table 4. Most of these drugs are anti-parasitic.

Their CSF toxicity is fairly consistent among mammals.

Organic agents listed in Table 4 were used experimentally to

inform on the extent and location of CP-ependyma injuries.

Adult rats were commonly used for in vivo experimentation.

A typical approach involves microscopy and tissue staining,

and time course analysis (days to weeks), to assess development

and reversibility of drug-induced lesions in CP, ependyma, and

brain.

VULNERABILITY OF THE CSF-BRAIN INTERIOR TO TOXICANTS AND

PATHOGENS

A gateway duty has been ascribed to BCSFB (Johanson

et al. 2008). Thus, a significant CNS port of entry in CP is

offered to plasma molecules, pathogens, and immune cells.

Ventricular CSF nourishes and protects brain by an array of

mechanisms controlled at the CP epithelial zone and ependymal

interface. Due to dissimilar structures and molecular expres-

sions of CP versus ependyma, various toxicants and pathogens

exert differential effects on CSF-bordering epithelium.

Upon penetration of a damaged BCSFB, a given substance

flows to the ependyma and regions adjacent to CSF (arrows

2–5, Figure 1). Injury to periventricular brain sites (arrow 6,

Figure 1), causing dyshomeostasis (Aksamit, Parnell, and

Johnson 1999), is often but not always curtailed by enzymatic

defense systems at CSF interfaces that degrade penetrating

toxicants. In this manner the Pi isoform of glutathione

S-transferase that is expressed in ependymal, arachnoid, and

pial cells enzymatically buffers the brain against toxicant

penetration from CSF (Carder et al. 1990).

Many toxicology studies of CNS have observed drug-induced

formation of vacuoles within various neural cell populations.

Regionality of induced vacuoles in brain has been a topic of inter-

est (Wells and Krinke 2008). Vacuolation has clinical importance

because impaired drug elimination from neurons and epithelial

cells, which promotes vacuole generation, may lead to toxicity

and even death. With cell swelling and organelle disruption, the

protective properties of epithelial cells of BCSFB may become

200 JOHANSON ET AL. TOXICOLOGIC PATHOLOGY

seriously compromised. Therefore, it is important to understand

the mechanisms of vacuole formation in CP and ependyma and

to prevent vacuole formation and proliferation or counter any

untoward effects pharmacologically.

Injuries to Choroid Plexus

Delineating the effects of toxic agents involves consider-

ation of CP compartmental anatomy. Each CP villus has an

inner vascular core consisting of a rich network of blood ves-

sels (Figure 4). Nearly 20% of CP mass is vasculature and

blood cells. These elements impart a red cast to the tissue

(Johanson, Reed, and Woodbury 1974). Lying in the intermedi-

ate zone of the CP is the interstitial space. It has a highly devel-

oped extracellular matrix that modulates the location and

functions of both vascular and epithelial cells. The interstitium

comprises about 15% of CP mass (Q. R. Smith, Pershing, and

Johanson 1981). Notably the interstitium of the CP, like that of

brain, lacks true lymphatic capillaries. On the outside perimeter

of the choroidal villi is a single layer of epithelium.

This epithelial ring interfaces with the interstitium on the basal

side and contacts the CSF via a lush microvillous surface at the

apical pole. Severe pathologic changes to the CP may include

damage to all three compartments: blood vessels, interstitium,

and epithelium. Certain drugs destroy one specific compart-

ment, or a single toxicant can perturb all three CP regions

(Levine 1987). It is also useful to identify drug combinations

that intensify damage to multiple compartments within CP.

Vascular impairment: Cyclophosphamide (CY) at high doses

injures the CP vasculature, especially in the fourth ventricle

TABLE 4.—Organic agents used for in vivo toxicology studies of choroid plexus (CP), ependyma, and periventricular brain.

Agent Dosage/route Species

Experimental

duration Main observations Investigators

Piperamide and other

tertiary aminesa

200-1200 mg/kg (SC)

200-1200 mg/kg (oral)

Rat (Lewis)

Male/female

1 day Hydropic degeneration (severe

vacuolation) of all CP; reversal

of CP pathology after 7 days

Levine (1977)

Piperazine and

piperidine

derivatives

200-1200 mg/kg (SC)

200-1200 mg/kg (oral)

Rat (Lewis) 1 day Swollen choroid epithelium, due to

cytoplasmic vacuoles; hydropic

degeneration also in kidney distal

tubule

Levine and Sowinski (1977)

Disobutamide

(piperidine

derivative)

30-250 mg/kg (oral)

45 mg/kg (oral)

90 mg/kg (oral)

Rat

Dog

Monkey

35 days Vacuolation induced in CP epithelium

correlated with drug uptake by CP,

and the amount of drug penetration

into the cerebrospinal fluid (CSF)

Koizumi et al. (1986)

Cyclophosphamide

(alkylating agent)

125-500 mg/kg (SC or IP) Rat (Lewis) 1 to 7 days Edema, hemorrhage, and leukocyte

infiltration of CP; progression to

necrosis of individual choroidal villi

Levine and Sowinski (1973)

Triamine

(pyridine)b or

cyclophosphamide

800 mg/kg (oral)

400 mg/kg (SC)

or 250 mg/kg (SC)

Rat (Lewis)

Male

1 day or 2

days

Pyridine-induced retention of

membrane-limited vacuoles in CP

epithelium; cyclophosphamide-

induced plasma exudation, but not

epithelial vacuoles

Wenk, Levine, and Hoenig

(1979)

N-Methylpropyl-

amine (triamine)

plus cyclophos-

phamide

100-800 mg/kg (SC)

250-375 mg/kg (SC)

Rat (Lewis)

Male/female

1 day Acute necrosis of ventral folium of the

cerebellum; choroid plexitis with

fibrin exudate in the stroma as the

result of CP hemorrhage

Levine, Sowinski, and

Nochlin (1982)

Cyclophosphamide

plus endotoxinsc

250 mg/kg (SC.)

1 mg/ml (SC.)

Rat (Lewis)

Male/female

1 to 2 days Cyclophosphamide-induced hemorrhage

in CP accelerated by endotoxin-

caused thrombocytopenia

Levine and Sowinski (1973)

Amoscanate

(anti-schistosomal

agent, an

isothiocyanate

compound)

250 and 500 mg/kg (oral) Rat

(Sprague-Dawley)

Male/female

28 days Necrosis, Caþþ-positive microgranules,

pyknosis and edema localized in

ependyma/subependyma in the medial

striatum

Clark Kiel, and Parhad

(1982)

Amoscanate

(anti-schistosomal

agent, an uncoupler

of oxidative

phosphorylation)

25-500 mg/kg (oral) Rat

(Sprague-Dawley)

Male/female

Up to 20 days Mononuclear cells in ependymal wall at

20 days; progressive necrosis of

ependyma over 20 days; CP appeared

to be intact

Krinke et al. (1983)

Methycellulose

(vehicle suspender)

5 ml/kg IP of Methocel

(0.5%) daily for 30

days

Rat (LEW/

Tif RA 42)

Male/female

30 days Stroma of CP (lateral and fourth) packed

with large cells containing finely

vacuolated (‘‘foamy’’) cytoplasm

Roth and Krinke (1994)

a See tertiary amine structures in Figure 1 of Levine (1977).b (2,6-di-omega-dimethyl amino ethoxy)-pyridine.c Escherichia coli and Bordella pertussis.

Vol. 39, No. 1, 2011 TOXICOLOGY OF VENTRICULAR CSF-BORDERING CELLS 201

(Levine and Sowinski 1973). Although the plexus vessels bear

the brunt of attack, other compartments of CP eventually

become injured. CY is an experimental tool for rupturing the

BCSFB and promoting penetration of water-soluble drugs into

the ventricles. In rats, a destabilized CP vasculature leads to

hemorrhage, fibrin plugs in capillaries, focal leukocyte infiltra-

tion, and exudation from impaired vessels (Figure 5). Edema

fluid contains phagocytes and erythrocytes. In severe cases,

CY-induced hemorrhagic infarction spreads over large

expanses of CP in one or more ventricles. Substantial passage

of fluid out of choroidal capillaries compresses the epithelium

(Levine and Sowinski 1973). Destabilization of CP initiated by

the grossly ruptured vascular elements and adjacent intersti-

tium is depicted in Figure 5. Due to additive pathologic effects

and BCSFB breaching, the administration of CY followed by a

tertiary amine (acting by a different mechanism) lessens the

amine dose that damages periventricular brain (Levine,

Sowinski, and Nochlin 1982).

Interstitial impairment: The interstitium of CP is accessible to

cells of the peripheral immune system, including macrophages.

Methylcellulose, a vehicle for suspending drugs given orally,

has extensive uses in toxicology assessments. Methylcellulose

dissolved in water and injected intraperitoneally (IP) into

rats for 30 days (see Table 4) substantially modified the inter-

stitium between blood vessels and epithelium of CP (Roth and

Krinke 1994). Following methylcellulose treatment, numerous

foam-like cells appeared within the interstitium (Figure 6).

The foamy cells stained with RCA-1, reflecting a scavenger

characteristic. These large cells in the stroma contained central

nuclei surrounded by finely vacuolated, ‘‘foamy’’ cytoplasm.

This phenomenon in CP (but not in small intestine or lungs)

may be the result of uptake of methycellulose by peritoneal

macrophages that then migrate to CP and infiltrate the stroma

(Roth and Krinke 1994).

Swelling of CP results from interstitial space distension

(Figure 6). Fluid engorgement prompts consideration of two

unfavorable outcomes: edema-associated restriction to diffu-

sion of solutes between blood and CSF and a possible ‘‘mass

effect’’ of enlarged tissue in the ventricles that occludes CSF

flow. Due to macrophage distortion (enlargement) of CP inter-

stitium, a caveat is offered about using methylcellulose vehicle

to study immune cell trafficking into the CP interstitium

(Chinnery, Ruitenberg, and McMenamin 2010; Petito and

Adkins 2005) and through BCSFB (e.g., MS and EAE models).

Toxicology studies of material exchange between CP and CSF

should address the integrity of the stromal-epithelial

relationship.

Epithelial impairment: Tertiary amines cause primary damage

to CP epithelial cells. A relatively selective lesion of the

epithelium is caused by piperamide, a trypanocidal agent that

produces marked CP vacuolation in rodents (Benitz and Kramer

1968). ‘‘Hydropic degeneration’’ describes the extensive epithe-

lial vacuolation after treatment with triamine or tertiary agents:

piperidine, piperazine, disobutamide, and pyridine (Table 4).

Tilorone, a tertiary-amine potent activator of hypoxia-

inducible factor-1, at repeated high doses causes degenerative

vacuolation in CP and kidney (Levine and Sowinski 1977).

Membrane-bound vacuoles that appear in CP do not always

form in ependyma. Enhanced vacuole formation in CP of lateral,

third, and fourth ventricles relates to lysosomal degradation of

invading xenobiotic (tertiary amine).

Lysosomes are small, spherical organelles containing thin,

parallel, concentric lamellae. They break up cellular debris or

foreign agents for excretion. As a result, vacuoles are gener-

ated. Characteristics of tertiary amine-induced vacuoles

(Levine 1977; Wenk, Levine, and Hoenig 1979) are summar-

ized in Table 5. Extensive tertiary-amine elicited vacuoles

compress epithelium and alter CP tissue topography (Figure 7).

Severe vacuolar swelling of CP epithelium may fill ventricles

with enlarged CPs, thereby creating obstructive hydrocephalus

(Benitz and Kramer 1968).

Elucidating vacuole phenomena enhances understanding of

BCSFB toxicokinetics (i.e., the rate and extent of toxicant

transportation into and out of the CSF) and toxicogenomics

(e.g., phospholipidosis induction in lysosomes by cationic

amphiphilic drugs [CAD]). Intense vacuole formation usually

reflects excessive accumulation of xenobiotic material that was

not properly digested within lysosomes, thereby leading to an

iatrogenic lysosomal storage disease. Phospholipidosis in lyso-

somal disorders can be iatrogenic (drug side effects) or genetic

(mutated enzymes or membrane-bound proteins). Lamellated

inclusions characteristic of ‘‘phospholipidosis’’ are also

related to epithelial vacuolation. Various CADs induce generalized

lipidosis of rat CP epithelium, but to different degrees (Frisch and

Lullmann-Rauch 1979); such CAD agents have a hydrophobic

component and a hydrophilic side chain. For instance, chloroquine

produces large cytoplasmic vacuoles. Vacuole ultrastructure after

chloroquine treatment suggested storage of water-soluble materials

in addition to non-water-soluble polar lipids. Quinacrine,

4,4’-diethylaminoethoxyhexestrol, chlorphentermine, iprindole,

1-chloro-amitriptyline, and clomipramine caused formation of

lamellated or crystalloid inclusions as observed in drug-induced

lipidosis.

Drug-lipid complexes in lysosomes, especially those affect-

ing the CP, deserve more pharmacologic attention. Pegylated

(polyethylene glycol [PEG]-coupled) molecules help to reduce

immunogenicity and prolong bioavailabilty of biopharmaceuti-

cal drugs. However, they are typically removed from the

system by phagocytic cells (e.g., macrophages) or secretory

cells (e.g., CP epithelium in the brain, proximal tubule epithe-

lium in the kidney), a process that leads over time to vacuole

formation. This finding may have major implications for

developing pharmaceutical agents. For instance, certolizumab

(CIMZIA1) was not approved by the European Medicines

Agency for treatment of Crohn’s disease based on its look

of efficacy; however, this agent is used to treat other disor-

ders despite producing vacuolation of CP in safety studies.

Two key questions arise in such situations (Wagner et al.

2008): ‘‘How does CP epithelium metabolize pegylated

compounds?’’ and ‘‘Are there sufficient BCSFB disruptions

202 JOHANSON ET AL. TOXICOLOGIC PATHOLOGY

by PEG-induced phospholipidosis to alter CSF composition

and dynamics?’’

A relationship exists between drug concentrations in CP, the

extent of induced vacuole formation, and drug penetration into

the CSF. Testing of a new candidate anti-diabetic drug in rats

daily for 4 or 16 weeks revealed extensive vacuolation and

swelling of the CP (Figure 8), but not ependyma (which also

differed from CP in the expression and distribution of various

cellular elements) (Table 6). The candidate agent may have

been more accessible to CP (plasma side of tissue) relative to

the CSF, by which the ependyma is exposed. Nevertheless,

cumulative evidence demonstrates that several types of drugs

augment epithelial vacuolation and thereby distort CP structure

and function. Future studies of lysosomal storage diseases, for

example, Niemann-Pick disease, should include CP-CSF anal-

yses (Elleder, Jirasek, and Smid 1975). This enables evaluating

possible phospholipidosis-inducing effects on the BCSFB,

material transport into CSF-brain, and ventricular fluid

dynamics.

To date, many CP vacuole assessments have dealt with

tertiary amine effects. However, other major classes of

compounds/disorders predispose toward vacuole formation at

the BCSFB and in various epithelia. Amphiphilic CAD

agents—which are well-known inducers of vacuoles, phospho-

lipidosis, and lysosomal storage disorders—include antidepres-

sants, antiarrythmics, antimalarials, and cholesterol-lowering

agents. Toxicogenomic studies of CAD-induced vacuole

formation and lysosomal expression (e.g., in HepG2 liver-

derived cells) used Affymetrix Microarray Analysis1 to reveal

several mechanisms that putatively result in phospholipidosis:

inhibition of lysosomal phospholipase, inhibition of lysosomal

enzyme transport, enhanced phospholipid biosynthesis, and aug-

mented cholesterol production (Sawada, Takami, and Asahi

2005).

Important practical goals are to identify specific gene

markers for faulty lipid storage and to detect drugs with

phospholipidosis-inducing potential by in vitro screening

(Kasahara et al. 2006). In addition to assessing physicochem-

ical attributes of compounds (alkalinity and lipophilicity) that

induce phospholipidosis, new models are capable of factoring

the physiologic volume of distribution (Hanumegowda et al.

2010). Collectively these new approaches will likely acceler-

ate toxicologic assessments and facilitate the discovery of new

agents to treat lysosomal storage problems encountered with

brain and CP.

Modeling of vacuole formation, in the context of unwanted

drug side effects, should account for pharmacokinetic para-

meters of the induction agent. This can be exemplified with

data for disobutamide distribution in the CP and CSF of

different species. Disobutamide, a bis tertiary amine that was

originally investigated for its anti-arrhythmic properties,

contains 1-piperidinebutanamide; it is a highly alkaline (basic)

molecule (pKa1¼ 8.6; pKa2¼ 10.2) and thus is strongly parti-

tioned into lysosomes due to their acidic pH (4.5). Within

TABLE 5.—Characteristics of vacuole formation in mammalian choroid plexus (CP) epithelium.

Species Microscopy observations Significance, implication, or interpretation Investigators

Rat

Monkey

Dog

CP epithelial cells contain a few small lysosome-like

bodies and a few tiny vacuoles (nontreatment or

vehicle).

Baseline ultrastructural studies of different mammals

yield comparable findings for organelles

Wenk, Levine, and Hoenig (1979)

Koizumi et al. (1986)

Rat Vacuoles have a uniform dispersal within cytoplasm.

Small vacuoles can coalesce to form larger ones. No

evidence of necrosis.

Large vacuoles, when in large numbers, compress other

cellular organelles (that look normal)

Wenk, Levine, and Hoenig (1979)

Rat

Monkey

After drug administration in vivo, some induced vacuoles

appear to be filled with water and minimal solute.

The edematous appearance of most epithelia impart a

hydropic look to the epithelial cells

Wenk, Levine, and Hoenig (1979)

Koizumi et al. (1986)

Rat Some epithelial cells are spared increased vacuolation

after drug treatment.

CP tissues appear heterogeneous when some epithelial

cells are not rendered hydropic

Wenk, Levine, and Hoenig (1979)

Rat Some vacuoles can form aggregates with granules. Occasional vacuoles with whorls of material suggest that

vacuoles may contain different materials

Wenk, Levine, and Hoenig (1979)

Rat

Monkey

Dose-response relationships observed for the ability of

various xenobiotics to induce epithelial vacuoles.

Higher doses of drugs and/or longer periods of exposure

induce vacuole formation more quickly and to a

greater extent

Wenk, Levine, and Hoenig (1979)

Koizumi et al. (1986)

Rat Single dose of piperamide (500 or 750 mg/kg SC) gave

peak vacuolation at 1 day, slight regression by 2 days,

and full recovery at 7 days.

Tertiary amine induction of CP vacuoles is rapid;

recovery starts early (2 days or less), and the vacuole

proliferation can be fully reversed

Levine (1977)

Dog

Rat

Monkey

Species differences in the in vivo route (oral vs. ICV) with

respect to the ability of disobutamide to induce

vacuoles in CP epithelial cells.

Oral disobutamide (45-250 mg/kg) induced vacuoles in

rat and monkey, but not dog; however, ICV drug (3

hours) caused vacuolation in canine CP

Koizumi et al. (1986)

Dog

Rat

Monkey

CSF/plasma for disobutamide (PO) (equivalent peak

serum concentration at 24 hours) was 1.1, 0.4, and 0.2

mM for rat, monkey, and dog, respectively.

Pharmacokinetic data indicate that disobutamide pene-

trated the BCSFB (i.e., crossed the CP) in vivo most

readily in rats, least so in dogs

Koizumi et al. (1986)

Dog

Rat

Monkey

In vitro CP uptake of disobutamide (0.1 mM) over 6 hours

in artificial CSF was greatest in rat, least in dog.

In vitro data point to lower uptake of disobutamide by

CSF due to limited uptake by dog CP from blood

Koizumi et al. (1986)

BCSFB, blood-CSF barrier; ICV, intracerebroventicular; PO, oral; SC, subcutaneous.

Vol. 39, No. 1, 2011 TOXICOLOGY OF VENTRICULAR CSF-BORDERING CELLS 203

FIGURE 8.—Vacuole formation in the choroid plexus (CP) epithelium after treatment with a novel anti-diabetic drug. 8a shows the CP (labeled by

immunohistochemistry to demonstrate pan-cytokeratin) in a control lateral ventricle from a *150-g female rat given vehicle. 8b portrays a swol-

len, hydropic CP, replete with large intra-epithelial vacuoles, in a rat receiving a high dose (1000 mg/kg/day) of an anti-diabetic compound for 16

weeks. Lesions seen in all 10 high-dose rats were as severe as those seen in the right panel. The vacuoles likely contain complexes of drug and

lipid. Ependymal cells on the edge of ventricle also stained for pan-cytokeratin, while blood vessels did not stain. Magnification is 20� for both

images. Pictures provided by J. Funk (unpublished data).

FIGURE 9.—Disobutamide induction of vacuoles in rat choroid plexus (CP) epithelium. The CP villi 5-week-old female rats given vehicle (a) or

disobutamide (250 mg/kg orally for 13 weeks; b). Epithelial cells in the CP, but not the ependyma, are swollen with large, clear cytoplasmic

vacuoles following disobutamide treatment (see Table 4 and Koizumi et al. 1986). Ependyma and cerebral parenchyma, on left side of each image,

appear normal. Magnification is 50� for both images. Due to unavoidable circumstances the above figures were not originals but were scanned

from reprints.

204 JOHANSON ET AL. TOXICOLOGIC PATHOLOGY

lysosomes, this drug reliably induces clear vacuoles, so it is a great

aid in investigating intracellular drug storage (Ruben et al. 1989).

Interestingly, disobutamide in vivo causes vacuolation in CP of

rats and monkeys, but not in dogs (Table 5). However, this CAD

is a weak penetrator of canine CP, attaining an in vivo concentra-

tion only 0.01% of that attained in rat CP (Koizumi et al. 1986).

Still, this drug accesses the dog CP epithelium sufficiently to

induce vacuoles, either in vitro (isolated CP in artificial CSF) or

when given by ICV injection to living animals (Table 5). Thus,

when comparing vacuole induction among species, it is critical

to set the experimental conditions to assure that test agents

adequately permeate the epithelium. Therefore, a combination

of in vitro and in vivo preparations in three species was

required to demonstrate the relationship among disobutamide

uptake by CP, induction of vacuoles in the choroidal epithelium,

and in vivo penetration into CSF across BCSFB (Koizumi et al.

1986). Experimentation reveals the need to explore species

differences in the etiology, incidence, and effect of vacuole

induction development of CADs or other lysosomal-altering

agents (Halliwell 1997).

Disease-induced phospholipidosis also occurs in the CP.

Typically in Reyes disease, there is vacuolation of CP epithe-

lium and possibly associated brain edema (R. E. Brown and

Madge 1972). Are these two hydropic alterations serially

linked by CSF nexus phenomena? Hyperammonemia plus

aspirin or viral infection causes vacuolation in the CP of ferrets,

a common animal model of Reyes disease (Rarey, Davis, and

Deshmukh 1987). Tertiary amines, which also induce vacuoles,

are derived from ammonia. All in all, the vacuolation phenom-

ena in CP suggest the increased passage of pathogenic

molecules into CSF, and then from ventricles into the brain.

Strengthening of this postulate comes from similar observa-

tions in several other models of BCSFB breakdown: protein

penetration into CSF, ventriculomegaly, ependymal damage,

and periventricular edema (H. S. Sharma, Duncan, and

Johanson 2006; Murphy and Johanson 1985).

Injuries to Ependyma

Even though CP epithelium and the ependyma have a

common embryologic origin, there are important differences

in function, structure, and protein expression between these

two cell types. This affects how each cell type responds to a

given drug. The data for amoscanate, an anti-schistosomal

agent (Table 4), exemplify differences in toxicologic effects

on CSF-bordering cells. In several mammalian species, a single

FIGURE 10.—Ultrastructure of choroid plexus (CP) in rats treated with disobutamide. The CP villi in 5-week-old male rats following treatment with

disobutamide (250 mg/kg orally for 13 weeks). Untreated animals (left panel) have healthy lysosomes and lush apical membrane microvilli.

Disobutamide-induced vacuolation (right panel) in the epithelium is extensive, displacing nuclei and organelles; in addition, apical microvilli are

not as evident after drug treatment. Magnification is 1,000� for both images. Images are from Koizumi et al. (1986). Due to unavoidable circum-

stances the above figures were not originals but were scanned from reprints.

Vol. 39, No. 1, 2011 TOXICOLOGY OF VENTRICULAR CSF-BORDERING CELLS 205

curative oral dose is typically 10 to 60 mg/kg (Bueding,

Batzinger, and Petterson 1976). Adsorbable amoscanate given

to an adult induced a lesion in the ependyma but not in CP by

three days after administration of consecutive high oral doses

(125 or 500 mg/kg) (Clark, Kiel, and Parhad 1982; Krinke

et al. 1983). Amoscanate caused necrosis by 28 days to the

lateral walls of both telencephalic ventricles; interestingly, the

lesion (Figure 11) was confined to the caudate/putamen surface

and generally spared the brain, spinal cord, and peripheral

nerves. Significantly, minute lesions occasionally occurred

close to CSF (i.e., in the fornix or septum), thereby providing

a clue about the pharmacokinetics of amoscanate distribution.

Taken together, the neuropathology pointed to CSF (rather

than plasma in periventricular capillaries) as the direct source

of drug that damages regions proximate to the ventricles.

Amoscanate likely penetrated the BCSFB and then flowed

within the CSF, inflicting damage to sensitive cells at the

ventricular border (arrows 2–5, Figure 1). Increasing the num-

ber of consecutive doses resulted in erosion of the ventricular

lining, glial reaction, and granule deposits (Krinke et al. 1983).

Amoscanate also elicited massive ultrastructural damage in

ependymal cells (Figure 12).

Another study of high-dose amoscanate in rats (250 or

500 mg/kg for 28 days) yielded similar findings of lateral

ventricle wall damage, particularly the neuropil of medial stria-

tum (Clark, Kiel, and Parhad 1982). All intoxicated animals, but

no controls, developed discrete necrotic lesions adjacent to the

lateral wall of lateral ventricle, commonly extending 1 mm into

the striatum. Immediately after initial oral dosing, rats displayed

less exploratory activity. Ependymal cells overlying necrotic

brain lesions often survived but were pyknotic. Extracellular

spaces in ependymal/subependymal regions were edematous

and spongiform (Clark, Kiel, and Parhad 1982). Less damage

was inflicted on the medial wall of the lateral ventricle than on

the lateral counterpart. Chronic treatment with high doses of

amoscanate does not injure other parts of the cerebral hemi-

spheres, cerebellum, brainstem, or CP in the third and fourth

ventricles (Clark, Kiel, and Parhad 1982; Krinke et al. 1983).

Similarly, walls of third and fourth ventricles remained intact.

Highly specific adverse responses of the ependyma (certain

regions) to amoscanate have been reproducible. Interesting

issues arise about the differential toxicity of this drug along

points in the distributional nexus: CP to CSF, ependyma, SVZ,

and ultimately the brain (Figure 1). Amoscanate, even in a

high-dose regimen, evidently does not harm CP (as examined

by microscopy). A CP capable of normal transport function

actively concentrates certain endogenous substances and drugs

in CSF relative to plasma (J. H. Wang and Takemori 1972;

Spector and Johanson 2006). One possibility is that certain

membrane transporters of organic solutes by CP (Table 2)

actively pump amoscanate, and other drugs, into CSF (arrow 3,

Figure 1). This notion needs evidence from CSF concentration

data for various therapeutic agents. Exposure of ependyma to

amoscanate in the CSF likely explains the sequential toxicity

TABLE 6.—Immunostaining of choroid plexus (CP) epithelial

and ependymal cells after treatment of rats with a candidate

anti-diabetic agent.a

Antibody

Choroid plexus

epithelium

Ependymal

cells

Pan-cytokeratin (epithelial cytoskeletal

protein)

þþ þþ

Vimentin (connective tissue filament

protein)

–* þþ

S-100 (neural crest marker) – þþFactor VIII (vascular marker) –*; ** –

Adipophilin (lipid droplet- associated

protein)

þ*** –

Ki67 (cell proliferation marker) þ/–**** –

Degree of expression, relative to control: þ ¼ increased expression; – ¼ reduced

expression.a The immunostaining data in the table correspond to the tissue images in Figure 8

(unpublished data from J. Funk).

*Only blood vessels of choroid plexus were positive.

**No increase of blood vessels labeling in high-dose animals.

***Minimal increase in high-dose animals.

****No increase in cell proliferation in high-dose animals.

FIGURE 11.—Destruction of ependyma and periventricular brain by

amoscanate. Sprague-Dawley rats treated with amoscanate (500 mg/

kg/day suspended in oil, given orally for 10 days). In the medial cau-

date/putamen, there was deposition of microgranules. Choroid plexus

was intact, but the ependyma covering the caudate/putamen was

severely damaged. Magnification is 20�. From Krinke et al. (1983).

Cresyl violet.

206 JOHANSON ET AL. TOXICOLOGIC PATHOLOGY

involving ependyma, subependyma, and (at high doses and

after long exposure times) eventually the underlying brain.

Pharmacokinetically, the ependyma overlying caudate/striatal

regions may have been exposed to higher CSF concentrations

of amoscanate than ependyma in other regions. An alternative

but not mutually exclusive explanation for ependymal toxicity

is the specific responsivity (phenotype) of individual cells to a

particular drug. Ependymal heterogeneity, discussed previ-

ously, means that some cells may be more vulnerable than oth-

ers to xenobiotic agents. Ependyma adjacent to the caudate-

putamen is particularly sensitive to some foreign agents in

CSF. Further research will be necessary to ascertain whether

specific ventricular regions are less able to protect caudate/

putamen and striatum against invasion by cancer cells, immune

cells, and pathogens circulating in the CSF.

Injuries to Circumventricular Organs

Systematic toxicologic/pathologic analyses of CVOs are

rare. A recent review, however, emphasizes pathogen entry

into the CNS through CVO portals (Siso, Jeffrey, and Gonzalez

2010). Certain trypanosomes, for example, invade brain by way

of CVO ‘‘open windows’’ (permeable capillaries). Toxicants

also permeate via the CVOs. A compelling example of specific

CVO disruption is by the excitotoxicant domoic acid.

As follow-up to an episode of mussel poisoning in Canada,

experiments in mice tested the effects of high-dose domoic acid

(7 mg/kg). Domoate-induced neuropathology occurred within

30 minutes and was confined to the CNS, mainly to three

CVOs: SFO, OVLT, and AP (Bruni et al. 1991). Particularly

intriguing about domoic acid overload was hypodipsia or

decreased drinking, likely due to damage of the SFO that

normally mediates the thirst response.

Medicinals also toxically stress the CVOs. A prominent

example is adriamycin (doxorubicin), a cytotoxic antibiotic

used to treat malignant tumors. This agent extensively pene-

trates the CVOs and damages circumventricular neurons (to

cause axon terminal degeneration), glia (disrupted nucleus and

nucleolus), and extracellular space (edema). Doses in mice

comparable to those used in chemotherapy in human patients

injured the NLP, ME, and AP. Adverse effects were evident

after 30 days (Bigotte and Olsson 1983). Morphologic altera-

tions in all three CVOs were pronounced.

Enhanced appreciation of the loci, characteristics, and func-

tions of CVOs prompts consideration of the SFO, SCO, OVLT,

and AP as potential pathologic regions of interest. A primary

toxicologic consideration about permeable CVOs is that toxi-

cants do not have to overcome the BBB to access and damage

the organ. Rather, the leaky capillaries in CVOs enable toxic

compounds to reach the inner epithelial parenchyma and neu-

ronal endings. Ironically, the parenchyma of CVOs (unlike

other brain regions protected by BBB) may be more vulnerable

to certain drugs/metabolites in plasma than in CSF.

Can drugs, toxicants, and cells in plasma access the CSF by

moving through CVO structures? Probably, but likely only to a

limited extent. Even though CVO capillaries lack tight junc-

tions between the endothelial cells, the epithelial cells in these

mini-organs have occluding tight junctions. This constitutes a

diffusion barrier between the CVO and CSF. Accordingly, the

CVO perivascular space is segregated by tight junctions from

the CSF-milieu of adjacent neuropil (Krisch, Leonhardt, and

Buchheim 1978). Still, there is some macromolecular and cel-

lular trafficking into, and likely through, the CVOs into nearby

brain and CSF. In EAE models, the CD45þ leukocytes pene-

trated SFO, ME, OVLT, and AP (Schulz and Engelhardt

2005). Enriched EAE inflammatory cell infiltrates were found

outside but near each CVO, intimating immune cell penetration

into CNS/CSF by transit across these four CVOs.

Although considerable evidence indicates that CVOs are

extensively modulated and affected by plasma-borne solutes

and cells, nonetheless there is minimal insight regarding how

CSF-borne hormones, pathogens, toxicants, and cells interact

FIGURE 12.—Ultrastructural view of ependyma damaged by amoscanate.

Top: Normal ependymal cells line the lateral ventricle of a control

female rat. Bottom: Extensive destruction of the ependyma at the same

lateral ventricle site in a female rat treated with three oral doses of

amoscanate (125 mg/kg). Some ependymal/subependymal cells are

shrunken and electron-dense. Ependymal erosion and necrosis were

also observed. Magnification is 5053�. From Krinke et al. (1983).

Due to unavoidable circumstances the above figures were not originals

but were scanned from reprints.

Vol. 39, No. 1, 2011 TOXICOLOGY OF VENTRICULAR CSF-BORDERING CELLS 207

with CVOs from the apical-side of each mini-organ. Tanycyte

fibers contacting CSF pick up ventricular signaling molecules

and convey these neuroendocrine hormones into hypothalamic

portal capillaries (Rodriguez et al. 2005). Putative CVO involve-

ment with tanycyte and hypothalamic homeostatic actions,

especially as modified by CSF-convected toxicants, deserves

more investigation.

PERSPECTIVES ON CEREBROSPINAL FLUID TOXICOLOGY AND

PATHOLOGY

Breakdown of CP by xenobiotic agents and pathogens

compromises CSF homeostasis with profound implications for

many CNS functions. Regulated secretions by CP represent a

lifeline of micronutritional and trophic support for neurons.

In addition, the reabsorption of brain catabolites and drug

metabolites by choroid epithelial cells pivotally maintains CSF

purity. Moreover, fine regulation of immune and neuroendo-

crine phenomena at the BCSFB bears not only on brain well-

being but also that of peripheral organs. Therefore, maintaining

integrity of the blood-CSF transport interface is vital for health.

Throughout life, the CP is challenged by blood-borne

toxicants, pathogens (viruses and protozoans), and various

xenobiotics. Usually the BCSFB remains intact as it success-

fully wards off threatening agents and protects the CNS.

Unfortunately, some stressors, given at high ‘‘doses’’ or for

long periods, overwhelm CP homeostatic capacities so that

foreign agents can invade CSF. Because the ependyma is

readily permeable in many ventricular locations, a CSF-borne

toxicant has easy access to periventricular brain regions. Such

accession challenges neurogenic niches requiring a stable,

clean environment to generate new neurons for supporting cog-

nitive function. Maintaining CP defense systems is therefore of

prime significance. A sound BCSFB thwarts penetration

of harmful molecules into CSF-brain.

Insight is needed on mechanisms underlying CP vulnerability

to certain viruses (HIV) and parasites (e.g., schistosomes and

trypanosomes). Undoubtedly one vulnerability factor is the

great permeability of the CP capillary system and high blood

flow. This assures extensive delivery of materials to intersti-

tium and the epithelial cell basolateral membrane. Another

potential susceptibility factor, quite neglected in research, is

the wide variety of epithelial cell phenotypes revealed by

differential lectin staining of glycoconjugates (McMillan et

al. 2003). Accordingly, one choroid epithelial cell may express

particular glycosylated proteins on its surface while neighbor-

ing epithelium does not; the implication is that some cells at

the BCSFB may bind a particular virus or protein, whereas neigh-

boring cells may not. Since binding is integral to endocytotic and

transcytotic phenomena, it is pertinent to obtain more information

on CP cell receptor affinities.

Certain cells of the immune system migrate through CP

(especially in autoimmune relapse states), perhaps less so

across brain capillaries. Here the epithelial cells of the BCSFB

differ markedly in structure and function from the endothelial

cells of the BBB. These unique barrier phenotypes, including

the expression of proteins (structural and enzymatic), likely

explain most differences in translocational phenomena.

Diseases such as MS and pathogen infections worsen as pro-

teins and particles migrate from CP-CSF into periventricular

regions. This likely contributes to lesion spread into the brain.

Elucidating CSF-brain immunopathology relies on new approaches

for tracking aberrant immune cell and immunoglobulin

permeation (arrows 1–3, Figure 1) at the BCSFB.

Agents for treating parasitic diseases, such as anti-trypano-

somic drugs, can be harsh on CP structure and physiology.

New antiparasitic agents are being sought that are less likely

to perturb the BCSFB. Vacuole formation in CP, provoked

by various types of drugs, is vexing due to potential

deleterious effects on CSF dynamics as well as CP epithelial

phospholipid metabolism. Preservation of lysosomal/Golgi

functions in CP is central to efficient scavenging/reabsorptive

actions to keep CSF clean. Procuring information about lyso-

somal system integrity at the BCSFB will facilitate

development of new strategies and treatments for pharma-

cotherapy and detoxification of central disorders. This includes

minimizing CSF impurities in advanced aging and early

neurodegeneration (Johanson et al. 2004).

The brain, a highly protected and specialized organ, is

exquisitely sensitive to changes in CSF composition. There is

a continuing need for systematic research to delineate and

boost protective mechanisms in CP. Therapeutic strategies

should be developed to stabilize the blood-CSF and CSF-brain

interfaces upon, or before, exposure to toxic and pathogenic

agents.

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