*— ‘
:M 2‘ "'3." W H ‘VH.._I‘_“.. ',-"" u. ,. , m ; ' ‘ ' ‘.. ...A,. ~
‘ '. ‘ V..‘. .. y - 1; - .m. ‘ .~,.'t‘r,-.':‘,-"' Mr.» . M-. H < ‘ - '

 

“$49.4.

ACTIVE TRANS-PORT OF ORGANlC
ANIONS FROM THE BRAIN VENTRICLES
OF THE DOG

Thesis for the Degree of M. S.
MICHIGAN. STATE UNWERSITY
DOUGLAS W. BIERER ~
1972

‘\.-4

Talifil.

L”

LIBRA RY

Michigan State
University

 

JM , Vi's... ._. '

 

V amoma av "'

am & snus' .‘

 

   

r f.

.11.

hlhA

K

‘J

ABSTRACT

ACTIVE TRANSPORT OF ORGANIC ANIONs
FROM THE BRAIN VENTRICLES
OF THE DOG
By

Douglas W. Bierer

The development of an organic anion transport mechanism
in the choroid plexus was studied in vitro in 1 - to 4-week -old and
adult dogs. Lateral ventricular choroid plexuses (LVCP), fourth
ventricular choroid plexuses (FVCP) and diaphragm muscle were
incubated for 1 hour at 37° C in a buffered salt solution containing
p-aminohippuric acid (PAH) and mannitol. Accumulation of PAH
and mannitol by the plexuses was expressed as tissuezmedium
concentration ratios (T/ M) and comparisons made Of the ratios of
PAH and mannitol. T / M mannitol ratios of the adult LVCP and
FVCP are significantly less (p < 0.05) than for animals 1 week old;
T / M mannitol ratios in diaphragm muscle did not change with age.
The active accumulation of PAH for both plexuses was indicated by
accumulation against a concentration gradient, transport saturation,

inhibition by organic anions and a metabolic dependence. PAH

v' .a.

us

fill!

Douglas W. Bierer

transport is poorly developed in the LVCP and FVCP (T/M = 2. 12;
T/M = 1. 97, respectively) of animals 1 week old, but is highly
developed in the adult (T/M = 4. 04; T/M = 3. 84, respectively).
Maximum accumulation of PAH by the LVCP occurs in 2 week
animals whereas maximum accumulation by the FVCP occurs in
the adult.

Prior administration of procaine penicillin G in 1 - and
2 —week —old dogs induced in vitro development of the organic anion
transport system. LVCP' s from 1 -week -old dogs previously
treated with 300, 000 IU penicillin showed a significant increase
(p < 0. 05) in PAH accumulation compared to saline -treated animals.
Noinduction of PAH accumulation was produced by treatment with
600, 000 IU in 1 -week -old dogs or 120, 000 IU in 2 -week -old dogs.
FVCP' s from 1 -week -old dogs treated with 300, 000 or 600, 000 IU
penicillin showed no increase (p > 0. 05) in PAH uptake but FVCP' s
from 2 -week old animals treated with 120, 000 IU showed significantly
greater (p < 0.05) PAH accumulation than controls. The presence of
substrate (penicillin) during development significantly enhanced the
rate of maturation of the organic anion transport mechanism.

The brain ventricular system of the adult dog was perfused
with an artificial cerebrospinal fluid (CSF) containing inulin,

creatinine and radioactively labeled PAH and mannitol. Measurements

Douglas W. Bierer

were made of steady —state rates at which inulin, mannitol and PAH
were removed from the ventricular system. Clearance of inulin
represents bulk absorption of fluid occurring distal to the fourth
ventricle and varied linearly with intraventricular pressure. The
efflux coefficient represents clearance of a molecule by means
other than bulk absorption and for mannitol, a passively diffusing
molecule, efflux is independent of intraventricular pressure. The
efflux of PAH is pressure dependent; PAH efflux increasing over
the range -15 to +15 cm HOH pressure indicating that PAH efflux
may be a function of the perfused surface area of the FVCP. Active
transport of PAH was indicated by competitive inhibition with other
organic anions (Diodrast and penicillin) and self -saturation (150 -
800 (Lg/ml PAH). Efflux coefficients of creatinine and PAH

(46 i 4 FLA/minis; 34 i 4 ill/min, respectively) are significantly
greater (p < O. 05) than mannitol (16 :t 8 FLI/min), suggesting that
creatinine and PAH leave CSF by an active process in addition to

passive diffusion.

ACTIVE TRANSPORT OF ORGANIC ANIONS
FROM THE BRAIN VENTRICLES
OF THE DOG
By
.533

,.
It")
s"
L‘ P

Douglas w‘.” Bierer

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Physiology

1972

ACKNOWLEDGMENTS

Many people have contributed significantly throughout
the course of this investigation, and the author expresses his
sincere appreciation to all.

The author wishes to thank his advisor, Dr. S. R. Heisey,
for his constructive criticism, counsel and encouragement through-
out this program, and the other members of his guidance committee,
Drs. B. Selleck and J. Schwinghammer, for their helpful assistance
in the preparation of this thesis.

Recognition is given to David K. Michael for his assistance
and valuable advice, Dr. J. R. Hoffert for assistance with the
histological study and photography, and Mr. Merlyn Schwab for aid
in procuring animals for this study.

This work is dedicated to my parents, Helen and Glenn
Bierer, without whose deep understanding and support preparation

of this manuscript would not have been possible.

ii

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

IN TRODUC TION

LITERATURE REV IEW .

METHODS
RESULTS
DISCUSSION .
SUMMARY
APPENDIX
1. PREPARATION OF ARTIFICIAL
CEREBROSPINAL FLUID (CSF)
2. p-AMINOHIPPURIC ACID ASSAY
3. INULIN ASSAY
4. CREATININE ASSAY
5. DIAL AND URETHANE SOLUTION .
6. LIQUID SCINTILLATION COUNTING .
7. PROGRAM RATIO: EXTENDED FORTRAN

VERSION FOR THE CALCULATION OF T:M
PAH AND MANNITOL RATIOS .

BIBLIOGRAPHY

iii

Page

iv

21
32
71

85

88
90
93
96

99

. 100

. 105

. 106

J‘

Table

LIST OF TABLE S

Molecules actively transported by the
choroid plexus . . . . .

T/ M ratios of PAH and mannitol of choroid
plexuses and diaphragm from different aged
dogs

PAH/ MAN of choroid plexuses from different
ageddogs

Effect of medium PAH concentration on PAH
accumulation by the 3 -week -old dog choroid
plexus

Inhibition of PAH accumulation in the dog
choroid plexus . . .

Accumulation of PAH by choroid plexuses
from animals treated with procaine
penicillin G

Effects of intraVentricular pressure on the
rate of removal of substances from CSF
in the dog

Inhibition of PAH transport from CSF in
the adult dog . . . . .

iv

Page

19

44

45

46

47

48

49

50

Figure

LIST OF FIGURES

Accumulation of PAH and mannitol by the
in vitro lateral ventricular choroid plexus
(LVCP) in the developing dog

Accumulation of PAH and mannitol by the
in vitro fourth ventricular choroid plexus
(FVCP) in the developing dog

Histological sections (278X) of FVCP from
1 -day -old and adult dogs . . .

Accumulation of PAH by isolated LVCP and
FVCP in the developing dog

Saturation curve of PAH transport by the
LVCP and FVCP of littermate 3-week -old
dogs

Relationship between intraventricular
pressure and outflow cannula height

Clearance of inulin, mannitol and PAH and
its relationship to intraventricular pressure
measured relative to the external auditory
meatus

Efflux coefficients of mannitol and PAH and
its relationship to intraventricular pressure
measured relative to the external auditory
meatus

Page

52

54

56

58

60

62

64

66

Figure

10.

11.

Efflux coefficients of 3 molecules of similar
molecular weight perfused simultaneously
in 5 animals .

Inhibition of PAH transport from CSF by the
addition of Diodrast to the perfusion fluid .

Barium- 133 external standard quench

correlation curve for-differential counting
of tritium and carbon- 14 samples

vi

Page

68

70

. 105

INTRODUCTION

The development of the mammalian blood -brain and
blood -VCSF barriers is of considerable importance in determining
the composition of the cerebrospinal fluid (CSF) and thereby the
maintenance of central nervous system activity and function. In
adults, lipid -soluble basic and acidic dyes, injected intravenously,
do not cross the capillary walls and enter into brain or CSF (Davson,
1967). However, some molecules such as creatinine, urea, K and
Na when perfused into the brain ventricles leave CSF and enter
into blood and brain (Heisey _e_t_a_l_. , 1962). This restriction on the
movement of some molecules between blood and brain or blood and
CSF has led to the concept of blood -brain and blood —CSF barriers.

In the past decade active transport from CSF to blood of
primary, tertiary and quarternary amines (Tochino and Schanker,
1965a; Takemori and Stenwick, 1966; Hug, 1967), inorganic ions
(Becker, 1961; Welch, 1962; Robinson_e_2_t_il_. , 1968) and amino
acids (Lorenzo and Cutler, 1969) has been demonstrated. Pappen-
heimer 3.3.1.“ (1961) showed an active transport mechanism for

Diodrast, p-aminohippuric acid (PAH) and phenolsulphonphthalein

3".

9‘-

.fi.

'(.1

up

u-

[1'

 

‘7-

J

from CSF. The presence of these transport mechanisms from CSF
or brain into blood suggests that the blood -brain and blood -CSF
barriers may consist of a series of active transport mechanisms.

In the fetus and neonate there is evidence that the blood-
brain barrier is not completely developed. In 1927, Stern and
Peyrot reported that the blood -brain barrier to ferrocyanide was
not fully developed in the neonate but developed a few days after
birth. In human fetuses and newborns with kernicterus, bilirubin
passed freely from blood into brain or CSF (Bakay, 1956). Asghar
and Way (1970) demonstrated that intravenous injection of morphine
resulted in higher brain concentrations of morphine in animals less
than 10 days old than in adults. Thus the development of organic
ion transport systems is of biological importance since the removal
of organic ions from CSF and brain extracellular fluid provides a
mechanism for protecting the central nervous system from poten-
tially toxic compounds and for maintaining homeostasis.

It is the aim of this study to determine whether PAH, an
organic anion, is actively transported from CSF in vivo and actively
accumulated by the isolated choroid plexus and to study the postnatal

development of this organic anion transport mechanism.

LITERATURE REVIEW

The cerebrospinal fluid (CSF) is a colorless fluid derived
from blood which fills the brain ventricular cavities and subdural
Spaces of the central nervous system (CNS). Although there are
no ions found in CSF which are not also found in blood, CSF, unlike
blood, contains no cells and only a very small amount of protein
(Millen and Wollam, 1962; Davson, 1967). Investigators have
disagreed about the formation of CSF: some contend CSF is an
ultrafiltrate or dialysate of blood whereas others believe CSF is
formed by active secretion from the cells of the choroid plexus
(Flexner, 1938; Cserr, 1971). If CSF were an ultrafiltrate of blood,
then the ionic compositionof CSF would be the same as a dialysate
of plasma. Flexner (1938) reported that in the pig, Na and Cl con-
centrations were greater in CSF than in a dialysate of plasma and
urea concentrationwwas lower in CSF than in a plasma dialysate.
Dog CSF is similar to that of the pig but the Na concentration in
CSF is the same as that of a plasma dialysate while Mg concentra -
tion is higher in CSF than in a plasma dialysate (Davson, 1967).

Therefore ultrafiltration is not an adequate explanation of CSF

formation since an energy source other than hydrostatic pressure
of the blood must be supplied to give the observed ionic concentra-
tions. Flexner ( 1938) suggests that this extra energy must be
supplied by the source of the CSF, the cells of the choroid plexus.

Ehrlich first observed that with intravenous injection of
acid analine dyes, practically all tissues of the body were stained
with the exception of the CNS (Davson, 1967; Grazer and Clemente,
1958). Grazer and Clemente (1958) reported that Trypan blue
injected into developing rat embryos, ranging in age from, 10 days
prenatal to gestation, did not stain the CNS and this impermeability
of the developing CNS exists at 10 days prenatal when the blood
vessels begin to invade the brain. In icteric adults with high blood
bilirubin concentrations, no bilirubin was found in the CSF or grey
matter of the brain (Bakay, 1956). This restriction on the movement
of molecules between blood and CSF and blood and brain has led to
the terms blood -CSF and blood -brain barriers. Bakay (1956) points
out that the blood —brain and blood —CSF barriers may not be a simple
screening agent but a complex mechanism which is functionally
adapted to the needs of the CNS.

Some molecules such as creatinine, K and Na, when added
to CSF, leave the CSF and enter into blood and brain (Heisey M. ,

1962). Two techniques have been used in vivo to measure the rate

at which molecules are removed from CSF: 1) intracisternal
injection of test substances; and 2) perfusion of the brain ventricular
system with an artificial CSF containing test substances.

Davson §t_a_l_. (1962) observed that 24Na, p-aminohippuric
acid (PAH), sucrose or inulin injected into the cisterna magna of
anesthetized rabbits were removed from CSF at different rates.
One hour afterinjection a large sample of CSF was withdrawn and
analyzed. The rate at which test substances left the CSF was
estimated from concentration differences between injected and
withdrawn fluid. Loss of test substances from CSF, expressed as
a percentage relative to the loss of Na (100%), was always in the
same order: 24Na (100) > PAH (82 i 2) > sucrose (63 i 3. 5) >
inulin (48 :I: 8). Davson assumed that inulin was removed from
CSF only by bulk absorption from the subarachnoid space; sucrose
was removed at a higher rate because in addition to bulk absorption,
it diffused into the extracellular space of the brain. Although the
transfer rate of PAH from blood to CSF or braintwas less than
sucrose (Davson, 1955; Davson and Spaziani, 1959), PAH removal
from CSF was always greater than sucrose. Davson suggested that
PAH might be removed from CSF by a process requiring metabolic
energy.

By perfusing the brain ventricular system of goats,

Pappenheimer et al. (1961, 1962) estimated rates of bulk absorption

."‘(
Etksr

I
n

.
1 .
11!
(1.)
.__‘

n
-rn
4

.auul

.
- -

nu

‘Lc

Fo-

l“

1

of CSF from the subarachnoid spaces, diffusion of molecules through
the ependymal linings and active transport of molecules out of CSF.
Cannulas were implanted in the lateral ventricle and cisterna magna
and an artificial goat CSF containing test molecules infused into the
lateral ventricle and collected from the cisterna magna. Both the
volume and steady -state concentrations of molecules in the inflow
and outflow CSF were measured and the clearance of test molecules
calculated by a formula analogous to that used to calculate renal
clearance. At intraventricular pressures of -15 cm HOH, all
inulin (M. W. = 5000) entering the lateral ventricles was recovered
in the cisternal outflow, indicating that inulin does not diffuse from
the ventricles at any detectable rate. Inulin is assumed to be
removed mainly by bulk absorption from the subarachnoid spaces
distal to the fourth ventricle and inulin clearance was used as a
measure of bulk absorption. Bulk absorption was found to be
dependent on intraventricular pressure since the clearance of
inulin increased linearly with intraventricular pressures greater
than --15 cm HOH. The diffusional loss of molecules ranging in
weight from. labeled water (3HOH; M. W. = 20) to fructose (M. W. =
180) were estimated. As the ventricular pressurewas increased,
clearance of water, creatinine or fructose increased proportionally

with the increase in inulin clearance. Theamount of clearance due

own
"I. A

hov“

'«1' :

v$to~

I!!!

u...~

Ill

u-
13..

'F'
“fly

.P

m..

nA‘
‘H.

V.

.I‘

to diffusion could be estimated and was found to be independent of
intraventricular pressure.

Diodrast was transported from CSF to blood or brain
3 times more rapidly than creatinine. If only molecular size is
considered, creatinine (M.W. = 131) should leave CSF more
rapidly than Diodrast (M.W. = 405). Transfer rates of Diodrast
from CSF to blood were 15 times greater than from blood to CSF;
elevation of plasma Diodrast concentration to twice that in the
perfusate did not alter the transfer rate of Diodrast from CSF to
blood. At elevated CSF concentrations of Diodrast, Pappenheimer

et al. (1961) demonstrated saturation of the transport mechanism.

 

The transfer rate of Diodrast from CSF plotted as a function of
increasing perfusate concentration showed a 2 -component curve:
an active absorption component ard a passive diffusion component.
The amount of Diodrast diffusing from CSF increased linearly with
concentration; the active absorption component was saturated at
high Diodrast concentrations with a transport maximum (Tm) of
2.5 Mg/min.

The ventriculocisternal perfusion technique has been used
to demonstrate active transport of other molecules from CSF,
namely: sulphate and iodide (Cutler £_t_a_l_. , 1968), pertechnetate

and iodide (Oldendorf et a1. , 1970), methotrexate (Rubin et al. ,

1968) and morphine (Asghar and Way, 1970). Flux coefficients
of sulphate out of CSF were 8 times greater than influx coefficients
and plasma sulphate concentrations greater than those in CSF did
not alter the efflux coefficient. Sulphate transport satisfied other
criteria of active transport (Cutler _e_t_al. , 1968): saturation of the
transport mechanism at elevated CSF sulphate concentrations (4 mM/l)
and reduction of the outflux coefficient by the addition of a competi-
tive inhibitor (4 mM/ 1 thiosulphate) to the perfusate. The addition
of 2, 4 dinitrophenol did not inhibit transport of sulphate, indicating
that the transport process may not be dependent on oxidative energy.
Many functional and morphological changes occur in the
brain with age. In the developing rat cerebral cortex, total brain
water is inversely proportional to increases in protein and lipid
concentrations due to the deposition of myelin and increase of
cellular proteins (Vernadakis and Woodbury, 1962). Until 12 days
postnatal, rat brain extraneuronal space consists mostly of inter-
stitial fluid and a few glial cells. As brain weight increases, total
brain chloride Cl) concentration decreases and glial cell density
doubles. Since Cl concentration inside the glial cell is less than Cl
concentration of the extraneuronal space (Tasaki and Chang, 1958),
replacement of extraneuronal space by glial cells could produce the

observed decrease in total brain Cl concentration.

There is little information concerning the exchange of
solutes between blood and CSF in the newborn animal. In contrast
to adults, newborns show an increased permeability of certain
molecules and dyes between CSF and blood or blood and brain. In
kernicteric infants bilirubin in the blood penetrates the grey nuclear
matter of the brain and enters the CSF (Bakay, 1956). Fries and
Chaikoff (1941a, 1941b) injected 32F subcutaneously in the develop-
ing rat and found that uptake by the liver, kidney, skeletal muscle
and blood remained constant or increased slightly with age, while
the uptake by the brain decreased. At birth 32P uptake by all parts
of the CNS was maximal but rapidly declined until animals weighed
50 g.

Cutler §_t_al. (1968) observed that the sulphate carrier
mechanism was poorly developed in the kitten but highly developed
in the adult. In kittens and adult cats injected intravenously with
0.5 mC [kg sulphate 4358), steady -state CSszlasma sulphate
ratios were greater in the 2 —week -old kitten (0. 28 i 0. 02) than in
the adult (0. 07 :t 0. 005). Perfusion of the ventricular system of
adult cats and kittens with 358 showed that influx coefficients for
both kittens and adult cats were the same (0. 003 :t 0.001 ml/min);
however, efflux rates in adult cats were 3 times greater than efflux

rates of the 2 -week -old kitten. Cutler and his colleagues suggest

ul“'
.

-.‘l

1
IT-

(I;

.
.o.

J-u

.p.1

win

”ml

..
~51

" ’I
1.54
A

1‘.-

.b
t.

L.

:n’ I

10

that higher CSF:plasma sulfate ratios in kittens may not be the
result of an increased blood -CSF permeability but are due to
decreased CSF efflux of sulphate.

The choroid plexus is a modification of the pia mater,
located along the walls of the lateral cerebral ventricles and roof
of the third and fourth ventricles, and is composed of both neuro-
epithelial and vascular elements. The choroidal epithelium is
modified cuboidal or columnar ependymal cells arranged in villi
or folds around a core of highly vascularized connective tissue
(Davson, 1967). Blood supply to the lateral ventricular choroid
plexus (LVCP) is supplied by the anterior choroidal artery which is
a branch of the internal carotid artery. The fourth ventricular
choroid plexus (FVCP) is supplied by the posterior choroidal artery;
a branch of the posterior cerebellar artery. The body or glomus of
the plexus is supplied by a large capillary network (Crosby _e_t_al_. ,
1962). Both the LVCP and FVCP are innervated by sympathetic
nerves which terminate on the vascular smooth muscle and by
sensory nerves which arise from the dorsolateral medulla (Kappers
_e_til. , 1960). Galen is reported to credit the discovery of the
LVCP to Herophilus (c. 335-280 B. C.) (Dohrmann, 1970).

The cells of the choroid plexus are structurally similar to

those of other actively secreting tissues of the body such as the

11

renal proximal tubule, intestinal villus and the ciliary body of the
eye. The brush border of the choroidal epithelium is composed

of microvilli as in both renal proximal tubule cells and cells on the
surface of intestinal villi. Similar to the tubule epithelium of the
kidney, the basilar'membrane of the choroid plexus has many in-
foldings which interdigitate'with those of adjacent cells (Davson,
1967).

Many morphological changes occur in the choroid plexus
with age. In humans the primordial choroid plexus appears during
the second month of gestation. At 6 weeks the primordial myel-
encephalic choroid plexus (FVCP) invaginates at the roof of the
fourth ventricle and the telencephalic plexus (LVCP) projects into
the lateral ventricles (Truex and Carpenter, 1969). Meningeal
mesenchyme forms the blood and vascular system as well as the
stroma of the plexus. At 6 weeks the epithelium is pseudostratified
columnar. At 8 weeks the telencephalic plexus becomes enlarged
andlobular and fills almost 75% of the ventricular cavity. Surface
epithelium changes from pseudostratified to low columnar and the
stroma appears as loosely organized gelatinous connective tissue
containing an amorphous, mucoid ground substance. Few capillaries
are present in the stroma and are parallel to and distant from the

epithelium. By 4 months gestation age the size of the plexus

12

decreases and the stroma is reduced due to formation of fibrils
which replace the gelatinous connective tissue. Increases in the
size of the capillary bed and reduction of the stroma brings
capillaries closer to the epithelial surface. At birth the choroid
plexus is a large leaf -like process containing many capillaries
which produce elevations in the epithelium resembling villi (Kaplan
and Ford, 1966; Kappers, 1958).

Smith (1966) reported that the LVCP from 8- to 20-day-
old chick embryos showed 2 morphological alterations: a change in
the epithelium, at 9- 16 days, from pseudostratified columnar to
columnar; and a concomitant change in the number and position of
mitochondria within the epithelial cells. She suggests that epithelial
cell transformations may be caused by the LVCP descending from
the cerebral hemispheres into the lateral ventricles by the twelfth
day. At 8- 9 days mitochondria appeared spherical and randomly
distributed throughout the cytoplasm. As the plexus matured
mitochondria became rod -shaped with well-developed cristae and
they migrated to the brush border of the epithelial cell. Smith
suggests that the location of mitochondria along the brush border
could facilitate ATP production necessary for active carrier
mechanisms between CSF and blood.

The choroid plexus has been suggested as one site for the

production and regulation of CSF composition (de Rougement et a1. ,

13

1960; Welch Eta—l. , 1963). A comparison of hematocrits from
choroidal arterial blood and venous blood draining the choroid
plexus showed that as blood passed through the choroid plexus,
plasma volume was reduced. From venous -arterial hematocrit
ratios and venous blood flow, calculated from the velocity of a
spherule of 1-octanol in the main choroidal vein and venous cross -
sectional area, the CSF production for one lateral ventricular
plexus was estimated at 2. 6 ,LLl/min (Welch _e_t_al_. , 1963).

De Rougement et a1. (1960) observed that the ionic composition of
fluid collected from the surface of the cat choroid plexus closely
resembled the ionic composition of CSF from the cisterna magna
and the cisterna pericallosa. Cl concentrations were higher and

K concentrations lower in fluid collected from the choroid plexus
and cisterna magna than the concentrations measured in a dialysate
of plasma.

The choroid plexus has been suggested as a site for the
regulation of electrolyte and metabolite movement between blood
and CSF. Increased or decreased plasma K concentration does not
significantly alter the CSF K concentration (3. 14 mM/ kg HOH)
(Bekaert and Demeester, 1954; Ames £131; , 1965). Newly formed
CSF from the LVCP showed a damped response to changes in

plasma K concentrations below 3 mM/kg HOH: a 39% decrease in

14

plasma K concentration caused a 21% decrease in CSF K concentration.
However, doubling plasma K concentration caused only a 9% increase
in the K concentration of the newly formed fluid. Ames 2:11: (1965)
suggest a transport mechanism for the regulation of CSF K concen-
tration.

Coben (1969) reported active transport of iodide from CSF
by the dog LVCP. Sodium iodide was injected intraperitoneally and
after 2 hours, when blood iodide reached steady -state concentra-
tions, the animals were sacrificed and the LVCP' s removed. Iodide
concentration, measured in the CSF and LVCP, was expressed as
milligrams iodide per milliliter of CSF or plexus water. As plasma
iodide concentrations increased, CSF iodide concentrations increased
linearly. However, iodide uptake by the LVCP showed both a dif-
fusion and active transport component and became saturated at
plasma iodide concentrations of 0.5 mg/ml or greater. The diffusion
component was calculated from the permeability coefficient of
iodide and CSF iodide concentrations. The active component was
calculated from the difference between the total uptake by the plexus
and the passive diffusion component. The transport maximum for
the active component was 0. 99 mM/hr.

Table 1 lists molecules which are actively accumulated

by the choroid plexus in vitro. The LVCP' s and FVCP' s were

15

incubated for 30 minutes to 2 hours at 37° C in an artificial CSF
solution containing the test molecule and glucose as an energy source.
Accumulation of the radioactively labeled test molecule is expressed

as a tissuezmedium concentration ratio:

T _ counts (CPM)/g tissue (wet wt.)

M _ counts (CPM)/ml medium

 

The tissuezmedium ratio (T/M) is a measure of the concentration
gradient established between the tissue and medium. If a molecule
were distributed between the tissue and medium by passive diffusion
alone, then the expected T:M ratio would indicate distribution in
tissue water and would be less than 1. 0 due to proteins and salts
present within the tissue. Ratios greater than 1 indicate active
accumulation against a concentration gradient (Cross and Tagart,
1950. T:M ratios of all molecules in Table 1 are greater than 1. 0.
Test molecules listed in Table l fulfill other criteria of active
transport: metabolic dependency and competitive inhibition by
structural analogues.

Choroid plexuses from adult rabbits, dogs and cats have
been used to demonstrate active accumulation by this tissue of a
wide variety of compounds ranging from small monovalent ions to

relatively large compounds (Table 1). Active accumulation of

16

organic anions such as PAH, phenolsulfonphthalein, Diodrast and
penicillin have not been studied in the in vitro choroid plexus,
although active transport of certain organic anions have been
reported in vivo (Pappenheimer fl. , 1961). In a few studies,
T:M ratios of the rabbit choroid plexus are higher than T:M ratios
of dog plexuses for the same molecule (Hug, 1967 ; Tochino and
Schanker, 1965a). Only in the rabbit and with only a few molecules
has active accumulation of the FVCP been studied. With the excep-
tion of 1 molecule (5 -hydroxyindoleacetic acid) T:M ratios in the
FVCP are less than ratios in the LVCP. Within and among groups
of molecules there is a wide range of T:M ratios. For example,
2 tertiary amines, morphine and dextrophan, have T:M ratios which
are 10 fold different (37.2 and 3. 1, respectively; Table 1).
Differences in T:M ratios may be due to more transport sites
available to certain molecules, different transport rates for
different molecules or different affinities of molecules for the same
transport mechanism. The T:M ratio is a function of medium
concentration; and since the T:M ratios are not reported either at
saturation concentration or even at the same medium concentration,
T:M ratios of different molecules are not comparable.

The in vitro choroid plexus shows a development of active

transport mechanisms with age. LVCP' s and FVCP' s (from rabbits

17

at various ages incubated for 1 hour in medium containing 1 mM
radioactively labeled morphine) actively accumulate morphine
(Asghar-and Way, 1970). The FVCP T:M ratio decreased with
increasing age (T/M = 4. 3, newborn; T/M = 2. 6, adult); the
LVCP T:M ratio also decreased with age (T/M = 4. 9, newborn;
T/M = 3. 1, adult), but the 15-day LVCP accumulated more
morphine (T/M = 9) than plexuses at other ages. In contrast,
uptake of sulphate by isolated LVCP of fetal rabbits (T/M = 1.7)
was significantly lower than adult LVCP' s (T/ M = 2. 5) but maximal
at 3- 10 days postnatal (T/M = 3. 2) (Robinson _e_t_al. , 1968).
Decreased transport of sulphate in the immature animal could lead
to higher sulphate concentrations in brain extracellular fluid. This
may be advantageous to the immature animal since myelination

requires sulphate for synthesis of sulphatides. Robinson et a1.

 

(1968) reported iodide transport was well developed in the fetus
(T/M = 60, rabbits; T/M = 45, cats) and significantly lower in the
adult (T/M = 25, rabbits, cats). These adult iodide T:M ratios
correspond to those reported by Becker (1961) (Table 1). Iodide
transport in the fetal choroid plexus appears to be better developed
than sulphate transport.

Many functions of the liver and kidney are not fully developed

at birth. In the liver, activity of glycolytic and drug -metabolizing

18

enzymes is low at birth but reaches adult activity in the neonate.
Increased enzymatic activity after birth could be cause by substrate-
induced stimulation of enzymes (Dawkins, 1966). By challenging the
kidney with penicillin, an organic anion, during the period of
development, Hirsch and Hook (1969a) stimulated maturation of PAH
transport. In littermate rabbits, 60, 000 IU procaine penicillin G
was administered subcutaneously bidaily for 3 days to 2 - to 4-week-
old animals. Kidney cortical slices were incubated in vitro with
PAH in the medium and PAH accumulation by the tissue expressed
as T:M PAH concentration ratios. In 2 -week-old treated animals,
PAH accumulation was increased 4 times over nontreated animals
but in 4-week-old animals there was no difference between control
and treated animals. Apparently transport development was com-
plete by 4 weeks of age. Other organic anions, folic acid and
triiodothyronine stimulated PAH accumulation by cortical slices

in young rats and rabbits (Hirsch and Hook. 1969a, 1969b, 1969c).
Presumably stimulation of organic anion transport was selective
since the uptake of N-methylnicotinamide, an organic base, was not
increased by prior treatment with penicillin (Hirsch and Hook,

1970).

19

 

$2 :93 A 3&3. Spam m A A A. .2 mm 5:933
oz: .93 s 3&3. spasm m s A m .5 mm $883.90
82 .93 s 2&3. spasm m .H A m .3 mm 2:230
2.2 .93 A 3&3. Spam 3 .o A m .N A .o A H .m
$2 .95 :pnmm o .o A A .m 03 maficoz
$2 .msm moo o .m
$2 .95 spasm A .H A m .o mcEacoEocEfiQ
C3988 ”$58ch
0.32 .cmxfifim s 0588. spasm A .m A A .S m .H A H .3 mcEfimcamcoz
£2: .coxfifim s 9:88. “Spam H .N A o .m A .m A A .3 62888
mumECm “mofifixw
$2 6:35 Beam 3 .o A S .m osfiaax
mofinsm
AREAS moi no: c5888
monoummom 3534
23m 2:.

 

 

 

 

 

 

 

$3on 393:0 ofi ma nozommcmfi Eofiaom $30302: .H 3an

20

 

 

 

 

88 . .8 8 88838 80 m. .m
88 . .8 8 8858 0.388 m .m 8.858
REMZQ umcodfiw
88 . .8 8 88588 588 8
$8 .880m :88 m .m A m .8 05008
88:85:02 8:3:qu
$8 .880 A 08084 80 m .0 A o .m 2500 05.85
050553 EA.
550A. 058..
E: .88 08> A. 880 0.38m a .0 A A. .5 w .0 A N .m 52... 588
u 208580.813 .m
mEo< 358th
82: .8888 A 0588. 588 m .0 A N .m 85:8
-Gflooficbfimfin .Z
82: .8888 A 05809 0.588 m .0 A A 9 555050585
888 .8888 A. 05808. won A 0 A m A.
82: .8888 A 0588. 588 m o A A .8 55850588
5580884 mnmnumtgmu

 

 

 

 

ME THODS

Organic Anion Accumulation by
the Choroid Plexus In Vitro

 

 

A. Experimental preparation

1. Animals and tissue removal

All animals were obtained from the Michigan State University
Center for Laboratory Animal Resources (C. L.A.R.). Dogs 1 and
2 weeks old were decapitated; dogs older than 3 weeks were
anesthetized with either chloroform (inhalation) or sodium pento-
barbital (60 mg/kg i. v. ; Abbott Laboratories, North Chicago, Ill.)
and decapitated. The skin and muscles of the head and neck were
retracted, the calvaria removed and the dura mater retracted. Two
incisions in the cerebral hemispheres were made 5 mm lateral and
on each side of the midline exposing the left and right lateral ven-
tricles; the cerebellum was retracted exposing the fourth ventricle.
Both the lateral and fourth ventricular choroid plexuses were
excised and immediately placed into separate beakers containing

3 ml of incubation medium. Diaphragm muscle (approximately

21

22

10 mg) obtained from a midline incision, caudal to the rib cage,
was removed and placed into incubation medium to serve as a

control tissue.

2. Incubation medium composition

The incubation medium consisted of a buffered salt solu-
tion (artificial CSF; Appendix 1) containing 0.5 mg/ ml mannitol,
p-aminohippuric acid (PAH; 2 —20 Mg/ml), 0. 1 ch/ml PAH
(glycyl -2 -3H) and 0. 01 MC/ ml D-mannitol-l -14C (New England

Nuclear Corp. , Boston, Mass).

3. Incubation procedure and preparation for analysis

Incubations were performed by shaking (50 times/min)
in a water bath at 37° C for 60 minutes in an atmosphere of 95%
oxygen and 5% carbon dioxide. At the end of the incubation period
the tissue was removed, dipped into non -radioactive artificial
CSF and excess fluid removed by blotting on absorbent paper. Each
choroid plexus and piece of diaphragm muscle was weighted on a
Mettler balance (sensitivity = i 0.01 mg; model B6; Mettler Instru-
ment Co. , Princeton, New Jersey) and homogenized in 0. 5 ml of
10% trichloroacetic acid using a glass rod in a 3 ml centrifuge tube

and placed on a vortex mixer (model S8220; Scientific Products,

23

Allen Park, Mich. ). The homogenate was centrifuged at 2000g for
10 minutes and duplicate 0. 1 m1 samples of supernatant and incuba -
tion medium were added to 10 ml of scintillation fluid (Aquasol;

New England Nuclear Corp. , Boston, Mass. ). Tritium and carbon-
14 were counted differentially in a trichannel liquid scintillation
spectrometer (model Mark 1; Nuclear Chicago Corp. , Des Plaines,
Ill.) (Appendix 6). Non -radioactive PAH concentrations were

determined colorimetrically (Appendix 2).

4. Tissuezmedium ratio

Accumulation of mannitol or PAH by the choroid plexus
or diaphragm muscle is expressed as a tissuezmedium concentra-
tion ratio (T/M) where:

_T_ dpm/g tissue (wet wt.)
M _ dpm/ml medium

 

Numerical calculations of T:M ratios were performed on the Michigan

State University CDC 6500 computer (Appendix 7).

B. Effect of metabolic inhibitors on PAH accumulation

Sodium cyanide or iodoacetic acid was added to the incuba-
tion medium to determine whether metabolic energy is required for

the accumulation of PAH by the choroid plexus. Medium concentrations

24

of PAH and mannitol were 20. 6 ILM/l and 2. 7 mM/l respectively.

4

Inhibitor concentrations in the medium were 1 X 10"3 and 5 X 10- M/l

sodium cyanide and 1 X 10_2 M/l iodoacetic acid.

C. Effect of competitive inhibitors on PAH accumulation

Competitive inhibition of PAH accumulation by the choroid
plexus was studied by incubating the tissue in incubation medium to
which was added 2, 4 dinitrophenol (1 X 10'4 M/l; DNP), iodopyracet

(7 x 10'4

M/l; Diodrast; Winthrop Laboratories, Inc. , New York,
New York), PAH (30-295 FLM/l) or crystalline penicillin G

(12, 500-50, 000 international units (IU)/l; potassium salt; Parke
Davis Co. , Detroit, Mich. ). Medium concentrations of PAH and
mannitol were 20. 6 [AM/l and 2. 7 mM/l respectively. Since 50, 000
IU penicillin G contains 79 meq/l K, KCl equivalent to the amount

of K present in the penicillin preparation was added to the control

incubation medium.

D. Effect of penicillin treatment on PAH accumulation

PAH accumulation was studied in choroid plexuses taken
from 1 - and 2 -week-old littermate dogs previously injected with
penicillin. Young animals were housed in C. L.A.R. facilities with

their mother. From 12 mongrel pups, 4 animals were injected

25

intramuscularly (i. m.) with 75, 000 IU/kg procaine penicillin G
(Duracillin; Eli Lilly and Co. , Indianapolis, Ind.) bidaily and

4 animals were injected with 150, 000 IU/kg bidaily beginning on the
fourth postnatal and continuing through 7 days of age (1 week); the
other 4 animals served as controls, receiving an equivalent volume
of saline intramuscularly. Six Labrador‘retriever pups were injected
with procaine penicillin G (60, 000 IU/kg i. m. ; bidaily) beginning

on the eighth day postnatal and continuing for 7 days (2 weeks) and
another 6 Labrador pups received an equivalent volume of saline

i. m. (controls). Animals were sacrificed 24 hours after the last
injection and the choroid plexus' ability to accumulate PAH was
compared in the 2 groups of animals (penicillin-treated and controls).

Organic Anion Transport from
the Brain Ventricular System

 

 

A. Experimental preparation

1 . Surgical procedure

Mongrel adult dogs (approximately 5 - 12 kg) of either
sex were obtained from the Michigan State University C. L.A.R.
and anesthetized with sodium pentobarbital (60 mg/kg i.v. ; Abbott
Laboratories, North Chicago, III.) or intraperitoneally with Dial

urethane (0. 6 mg/kg; Appendix 5). Femoral arterial pressure was

26

monitored using a Statham pressure transducer (model P23 DC;
Grass Instrument Co. , Quincy, Mass.) and a Grass model 5D
polygraph (Grass Instrument Co. , Quincy, Mass. ). The arterial
pressure transducer was calibrated using a mercury manometer;
the response to pressure was linear over the range 0-200 mmHg.
The animal' 5 ventilation was controlled throughout the experiment
by means of a respiratory pump (model 607; Harvard Apparatus Co. ,
Dover, Mass.) connected to a plastic ”Y" tube in the trachea; the
third arm was open to the air and fitted with a clamp for adjusting
lung inflation. The respiratory pumpwas set to cycle 10- 12 times/
min with a stroke volume of 200 -250 ml.

The dog' 3 head was secured in a model 1504 stereotaxic
frame (David Kopf Instrument Co. , Tujunga, Calif.) by means of
ear bars (inserted into the external auditory meatus) and a clamp
which secured the dog' 3 snout. The animal' 3 neck was flexed so
that the parietal surface of the head was tilted downward at an angle
of 30° from the horizontal position. A midline skin incision was

\.
made extending from a point 5 cm cauxc‘lé‘l to the orbital sockets to
the second cervical vertebra. Muscles were retracted with cauteri-
zation and the parietal and occipital bones exposed. A 8 -inch
trephine hole in the skull, at a point 5 mm rostral and 5 mm lateral
to the junction of the central saggital and coronal sutures, exposed

the dura mater overlying the left cerebral hemisphere.

27

2. Implantation of Cannulas

The cisterna magna was penetrated with a 20-gauge, 2 -inch,
short bevel pointed tube (Vita Needle Co. , Needham, Mass.) held
in a micromanipulator (model MM— 3; Eric Sobotka, Inc. , Farming-
dale, New York) and directed rostrally and parallel to the top of the
skull at the midline. A piece of PE- 90 tubing led from the needle
to a syringe. The needle was first quickly lowered to a depth
5- 8 mm beneath the atlantooccipital membrane and then slowly with-
drawn until cerebrospinal fluid could be withdrawn. Collodion
(Mallinckrodt Chemical Co. , St. Louis, Missouri) or dental acrylic
(Hygienic Dental Mfg. Co. , Akron, Ohio) was used to seal any
. leaks around the cisternal needle.

A 22 -gauge, 1% - inch, thin wall disposable needle held in a
model 1270 standard electrode holder (David Kopf Instrument Co. ,
Tujunga, Calif.) was used to penetrate the lateral cerebral ventricle.
The needle was directed ventrally and normal to the dura mater.
Anterior-posterior and lateral coordinates for the puncture (refer—
enced to the junction of the saggital and coronal sutures) were 5 mm
caudal and 5 mm lateral. The ventricular needle was connected by
a male "T" adaptor and PE -50 tubing to a constant syringe drive
pump (model 975; Harvard Apparatus Co. , Dover, Mass.) and to a

Statham pressure transducer (model P23 AC; Grass Instrument Co. ,

28

Quincy, Mass. ). The pressure transducer was calibrated using a
water-reservoir; output was linear over the range 0-40 cm HOH;
zero pressure was referenced to the level of the stereotaxic earbars.
While artificial CSF was pumped through the ventricular needle,
pressure (representing the resistance of the inflow needle and tubing)
was recorded with the tip of the needle on the dura mater. As the
needle was lowered slowly through the dura mater and brain tissue,
perfusion pressure rose; with the puncture of the lateral ventricular
cavity, there was an abrupt fall in pressure. Injection and withdrawal
of fluid from the syringe connected to the cisternal cannula resulted
in pressure increases and decreases respectively and indicated con-
nection between the ventricular and cisternal cannulas. The PE
tubing from the outflow cannula was connected to a photoelectric

drop recorder (model PTTI; Grass Instrument Co. , Quincy, Mass.)
for monitoring outflow rate. Intraventricular pressure was calcu-
lated by subtracting the pressure with the needle tip on the dura

from that with the needle tip in the lateral ventricle. Intraventricular

pressure could be varied by adjusting the height of the outflow tubing.

3. Perfusion fluid composition

The perfusion fluid consisted of an artificial dog CSF

(Appendix 1) containing: inulin (1.0 mg/ ml), mannitol (0. 5 mg / ml),

29

carbon- 14 labeled mannitol (D -mannitol-1 -14C; 0. 01 ch/ ml; New
England Nuclear Corp. , Boston, Mass. ), PAH (2 -80 ng/ml),
tritiated PAH (glycyl -2 ~3H; 0. 1 [.Lc/ml; New England Nuclear
Corp. , Boston, Mass.) and in some experiments creatinine (1. 0-

2.0 mg/ml).

4. Perfusion technique

Perfusion fluid was pumped at rates of 170-240 [Ll/min
into the lateral cerebral ventricle. Perfusion rate varied between
experiments but was constant in any one experiment. Inflow rate
was determined gravimetrically by collecting fluid from the per-
fusion syringe over timed periods (IO-20 minutes) in tared vials
at the beginning and end of the experiment. Outflow rate was
determined by collecting effluent from the cisternal cannula over
3 timed periods (10 -20 minutes) in tared vials. Inflow and outflow
concentrations were determined on aliquots from the inflow syringe

and from outflow vials, respectively.

B. Effect of inhibitors on PAH transport

Inhibition of PAH transport from the ventriculocisternal
system was studied by using perfusion fluid containing: iodopyracet
(0. 07 -7. 0 mg/ml; Diodrast); PAH (80. 0-8000 [Lg/m1) or crystal-

line penicillin G (10, 000 IU/ml; potassium salt).

30
C. Transport of molecules from CSF

1 . Clearance

The clearance of large molecules such as inulin from CSF

is calculated by an equation described by Heisey et a1. (1962):

Vici - VOCO
C. = (1)

1n C
O

 

where: V rate of flow, [ALI/min

c = concentration, quantity/m1

1, o = subscripts refer to inflow and outflow
. = clearance of inulin, [.Ll/min
in

For small molecules which leave the ventricular system

by diffusion or active transport, clearance is calculated by:

Vici - V c
C : oo (2)

 

8
D”
(D
’1
f?
O
n

clearance of the molecule, [Ll/min

mean ventricular concentration;

estimated by: C = m

31

2 . Efflux coefficient

Molecules smaller than inulin leave the ventricular system
by active transport or passive diffusion in addition to bulk absorption.
The outflux coefficient represents the clearance of any molecule
from CSF by means other than bulk absorption. The bulk absorption
component is estimated by the clearance of inulin times the outflow

concentration of the test molecule divided by its mean ventricular

C.c
1no

 

concentration _ The efflux coefficient is calculated by an
c
equation described by Heisey et a1. (1962):

V,c.-Vc —C.c
11 00 mo

 

K = (3)
O —
c
where: K0 = outflux coefficient, ILI/min
c = concentration of the small molecule
in = clearance of inulin

Statistical Analysis

 

All data obtained were subjected to statistical analysis
using analysis of variance, Student-Newman-Keul' 8 test, student' 8
”t, " paired or group comparisons. The 0. 05. level of probability

was used as the criteria of significance in all statistical tests.

RESULTS

A. In vitro studies

 

Accumulation of PAH and mannitol was studied in the
lateral ventricular choroid plexus (LVCP) and fourth ventricular
choroid plexus (FVCP) from 1 -week, 2-week, 3- to 4-week
postnatal and adult dogs and in diaphragm muscle from 1 - to
4-week dogs (Table 2). Medium concentrations were 2. 7 mM/l
mannitol and 9. 7 [.LM/l PAH. For all ages diaphragm PAH and
mannitol T:M ratios are less than 1. 0 (Table 2). Within each age
group there is no difference (p < 0. 05) between T:M PAH and T:M
mannitol ratios and there is no change in either ratio with age.

The LVCP T:M mannitol ratios are not significantly
greater (P > 0. 05) than 1.0. Shown graphically in Figure 1, the
LVCP T/M mannitol decreases with increasing age; the 3- to
4-week and adult T/M mannitol are significantly less (p < 0. 05)
than those at 1 week postnatal. For all ages LVCP T:M PAH
ratios are significantly greater (p < 0. 05) than 1.0 and greater than
those of mannitol. At 1 week postnatal the LVCP T/M PAH is

approximately 2. 0, increases to 6.0 at 2 weeks and then decreases

32

33

to 2. 5 in the adult. The adult T/ M PAH is not significantly greater
(p > 0.05) than the ratio at 1 week postnatal.

The FVCP T:M mannitol ratio is not significantly greater
(p > 0. 05) than 1. 0 (Table 2; Figure 2) and shows an apparent
decrease with increasing age, although only the adult T/ M mannitol
is significantly less (p < 0. 05) than the ratio at 1 week. The FVCP
T:M PAH ratio does not change significantly (p > 0. 05) with age and
remains at approximately 1. 8. The LVCP T/M PAH at 2 —4 weeks
is greater than those of the FVCP. At 1 week postnatal and in the
adult LVCP and FVCP T/ M PAH are not significantly different
(p > 0.05) from each other and there is no difference (p > 0.05) in
T/M PAH between the 1 -week -old plexus and the adult.

Histological sections of choroid plexuses from 1 -day-old
and adult dogs showed structural changes with age in both the LVCP
and FVCP. Figure 3 is a photograph (magnification = 287X) of a
histological section of the FVCP from 1-day postnatal (left) and
adult (right) dogs. The epithelium of the 1 -day FVCP (left) is
composed of columnar epithelium, approximately 15 ,LL in height
with a large, centrally located nucleus. The majority of the plexus
is composed of a loosely organized, gelatinous connective tissue
with few fibroblasts and fibrocytes present (16-24/100 1L2). Blood

vessels are few and are distant from the epithelial surface (8- 100 1L).

34

In contrast there are an increased number of nuclei in the adult
FVCP (right), indicating an increased number of cells. The height
of the epithelial cells is reduced (7 -8 ,LL) and the cells are low
columnar or cuboidal epithelium. The epithelial cell nuclei are
located near the base of the cell, leavihg more cytoplasm apically:
a feature similar to the proximal tubule cell of the kidney (Davson,
1967). The stromal volume is greatly reduced and is composed of
densely packed connective tissue. Compared to the 1 -day-old
plexus, the vacuolated appearance of the stroma is absent and there
are large numbers of fibroblasts and fibrocytes (40/100 #2) which
indicates proliferation and production of collagen and other compo-
nents of connective tissue (Greep, 1966). In the adult, blood
capillaries are more numerous and lie closer to the epithelial
surface; the distance between the capillary endothelium and
epithelial basement membrane being only 3 -5 11..

These histological changes with age suggest that as the
plexus matures, the space into which mannitol can diffuse decreases.
A decreasing T/M mannitol in both the LVCP and FVCP (Figures 1
and 2) presumably represents a decreasing extracellular space in
the maturing plexus. In order to compare PAH accumulation
between plexuses of different ages, PAH accumulation will be

expressed as T/M PAH/TIM mannitol (PAH/MAN).

35

Data in Table 3, shown graphically in Figure 4, shows PAH
accumulation (PAH/ MAN) of both the LVCP and FVCP for different
aged dogs. Medium concentration of mannitol and PAH were 2. 7 mM/l
and 9. 7 [.LM/l respectively. Although the FVCP shows an apparent
increase in PAH accumulation with increasing age, only the adult
PAH/MAN is significantly greater (p < 0. 05) than at 1 week post-
natal. The 2 -, 3 - to 4-week and adult PAH/MAN of the LVCP are
significantly greater (p < 0.05) than those at 1 week and accumulation
appears to be well developed at 2 and 3 —4 weeks (PAH/MAN = 6. 0).
There is no difference (p > 0. 05) between the PAH/MAN of the FVCP
and LVCP in either the adult or 1—week plexus. . However, the adult
LVCP and FVCP PAH/MAN are significantly greater (p < 0. 05)
than those at 1 week postnatal.

T:M ratios greater than 1. 0 are suggestive of active trans-
port. Further evidence for transport of PAH by the choroid plexus
was: 1) saturation at high medium concentrations; 2) competitive
inhibition and 3) inhibition by metabolic blocking agents. Data in
Table 4 shows effect of medium concentration on PAH accumulation
(T/M PAH) of the LVCP and FVCP from littermate 3-week-old
dogs. T/ M mannitol values were low, resulting in high PAH:MAN
ratios (Figure 5) but T/ M mannitol remained constant (T/ M. = 0.42)

over the range of PAH concentrations used. As the medium

36

concentration of PAH increased, PAH/MAN of the LVCP decreased
approximately from 20 at 29 ILM PAH/l to 3. 2 at 294 [.LM PAH/l
(Figure 5). PAH uptake by the FVCP, although less than that of the
LVCP, also decreased from approximately 8 to 2. 8 over the same
range of medium PAH concentrations. This decrease in PAH
accumulation is non-linear and at high PAH concentrations
(> 200 [.LM/l) transport appears saturated and the amount of
accumulation by the LVCP and FVCP is approximately equal.
Results of adding metabolic and competitive inhibitors to
the incubation medium are shown in Table 5. The dogs were 3 weeks,
6 weeks postnatal or older. The medium PAH concentrationwas
20. 1 FLM/l and mannitol concentration was 2. 7 mM/l. Competitive
inhibition of PAH transport was demonstrated using two organic
anions (Diodrast and penicillin G) and 2, 4 dinitrophenol (DNP).
Diodrast (7 X 104M) completely inhibited PAH accumulation in both
the LVCP and FVCP, decreasing the PAH:MAN ratio to 1.0.
Penicillin G was also an effective inhibitor'over a wide dosage
range (12, 500 — 50, 000 IU) and appeared to inhibit completely both
the LVCP and FVCP. Large variations in penicillin control
PAH:MAN ratios are possible caused by high medium concentra -
tions of potassium. An uncoupler of oxidative phosphorylation,

2, 4 DNP has been shown to be a competitive inhibitor of PAH

ti

37

transport in the kidney (Huang and Lin, 1965) and at a medium
concentration of 1 X 104M appeared to inhibit the FVCP (96%
inhibition) to a greater extent than the LVCP (74% inhibition). Two
metabolic inhibitors produced different effects on PAH accumula-
tion: sodium cyanide, an inhibitor of the electron transport system,
produced no inhibition of PAH accumulation in either the LVCP or

4M or 1 x 10’3M;

FVCP at medium concentrations of 5 X 10-
iodoacetic acid, an inhibitor of glycolysis, decreased the LVCP
PAH:MAN ratio from 7. 2 to 1. 3 and the FVCP PAH:MAN from 4. 2
to 1. O. Iodoacetic acid is also an organic acid and may be acting

as a competitive inhibitor of PAH transport.

Hirsch and Hook (1969a, 1969b, 1969c, 1970) showed that
organic anion transport in renal cortical slices could be induced in
immature kidneys by prior treatment of the animal with penicillin.

A similar study was performed using the choroid plexus to deter-
mine if PAH accumulation could be induced by prior treatment of
animals with penicillin G (Table 6). Incubation medium concentra-
tion was 9. 7, FLM/l PAH and 2. 7 mM/l mannitol. LVCP' s from
dogs 1 week old, preViously treated with 300, 000 IU penicillin G/kg
for 7 days, Showed a significant increase (p < 0. 05) in PAH accumu-
lation (PAH/ MAN) over saline injected animals. However, at twice

the dosage (600, 000 IU/kg) there was no difference (p > 0. 05) in the

38

PAH/MAN between control and penicillin- treated animals. FVCP' s
from the same animals showed no increase in PAH uptake by treat-
ment with either 300, 000 or 600, 000 IU/kg. Two-week-old dogs,
previously injected bidaily with 60, 000 IU penicillin G/kg for 7 days,
showed no significant increase (p > 0. 05) of PAH accumulation by
the LVCP. However the PAH/MAN of the penicillin-treated FVCP
(2. 76 j: 0. 31) was significantly greater than the control FVCP

PAH/MAN (1.80 d: 0.37) (p < 0.05).

B. In vivo studies

Intraventricular pressure is an important determinant of
the clearance of any molecule from CSF since it determines the rate
at which a molecule is removed from the ventricular system by bulk
absorption. The importance of intraventricular pressure on PAH
clearance will be shown later. Intraventricular pressure was varied
by adjusting the height of the cannula draining the cisterna magna.
Data obtained from 27 animals and shown in Figure 6 show a linear
relationship between calculated intraventricular pressure and out-
flow cannula height. The slope of the line is 0.65 cm HOH pressure/
cm change in outflow cannula height, indicating the magnitude of
error associated with estimating intraventricular pressure. Intra-
ventricular pressures shown in Table 7 were estimated from this

figure .

39

The brain ventricular system of the dog was perfused with
an artificial dog CSF containing inulin, mannitol, creatinine and PAH
in order to compare the relative rates of removal of these substances
from the CSF. Table 7 contains measured and calculated quantities
from ventriculocisternal perfusions in 10 adult dogs. Radioactive
counts of mannitol correspond to 0.25 mg/ml mannitol; the first
4 experiments (0(2 - 84) contained no radioactive material. Clear-
ance of inulin, calculated using Equation 1-(Methods); mannitol,
creatinine and PAH, calculated using Equation 2 (Methods) and outflux
coefficients (KO) of mannitol, creatinine and PAH were calculated
using Equation 3 (Methods) between 30- 90 minutes perfusion time.

Figure 7 illustrates calculated inulin, mannitol and PAH
clearances as a function of intraventricular pressure. Lines through
these points were fitted by the method of linear regression and the
slopes of all 3 lines are significantly greater (p < 0.05) than zero.
The clearance of inulin decreases linearly from 85 ,LLl/min at 10 cm
HOH pressure to almost zero at -15 cm HOH. Although mannitol
clearance is always greater than inulin clearance, the slope of the
regressionlines for mannitol and inulin are not significantly differ-
ent from each other (p > 0. 05). Over the pressure range -15 cm HOH
to +15 cm HOH, PAH clearance is always greater than that of inulin

or mannitol and the slope of PAH clearance is significantly greater

HO:

£3“
q 9
l 1

LL.

40

(p < 0. 05) than that of inulin, decreasing from 150 le/min at 10 cm
HOH to 15 FLl/min at -15 cm HOH.

Mannitol and PAH efflux coefficients (KO; Table 7; repre-
senting movement from the ventricular system by means other than
bulk absorption) are plotted as a function of intraventricular
pressure (Figure 8). Efflux coefficients were calculated between
30- 90 minutes perfusion time. The slope of the mannitol efflux is
not significantly greater (p > 0.05) than zero, indicating that the
removal of mannitol is independent of intraventricular pressure.
However, the slope of PAH efflux is significantly greater (p < 0. 05)
than zero and increases linearly from 10 lLl/min at -15 cm HOH
intraventricular pressure to 87 le/min at 10 cm HOH. These data
indicate that the efflux of mannitol, a passively diffusing molecule,
from CSF is independent of intraventricular pressure, whereas the
outflux of PAH in low concentrations (< 10 fig/ml) is pressure
dependent

In 5 experiments (7-3, 7-4, 7-6, A42, A47; Table 7)
the ventricular system was perfused simultaneously with inulin,
mannitol, creatinine and PAH. Clearance of these molecules was
always in the same order: Creatinine > PAH > mannitol > inulin.
Efflux coefficients of these molecules were: Creatinine (46 i 4 [.L1/

min) > PAH (34 d: 4 le/min) > mannitol (16 i 8 [.Ll/min) (Figure 9).

41

Efflux coefficients of both creatinine and PAH are significantly
greater than that of mannitol (p < 0.05), suggesting some mechan—
isms other‘than passive diffusion for their removal.

Outflux coefficients of PAH were always greater than those
of mannitol, suggesting active transport. To test for an active
process, various competitive inhibitors were added to the perfusion:
PAH, Diodrast and penicillin G. Table 8 contains measured and
calculated quantities from ventriculocisternal perfusions in 4 adult
dogs in which inhibitors were added to perfusion fluid. Radioactive
concentrations of mannitol correspond to 0. 25 mg/ml mannitol.
Clearance of inulin and outflux coefficients of mannitol and PAH
were calculated by Equations 1 and 3 respectively (Methods). The
efflux coefficient of mannitol, a passively diffusing molecule, does
not remain constant between periods. In order to compare PAH
efflux between periods, PAH efflux was expressed as a ratio to that
of mannitol (KOPAH/KOMAN).

The addition of increasing amounts of PAH (5- 8, 000 [.Lg/ ml)
to the perfusion fluid inhibited transport of PAH out of CSF. At PAH
concentrations less than 88 FLg/ml (dog, A12) PAH efflux coeffi-
cients are always greater than those of mannitol and KOPAHzKOMAN
ratios-are approximately 2. 0. At a perfusion concentration of

150 [Lg/ml PAH, the ratio decreased to 1.7. PAH concentrations

42

greater than 80 [Lg/ml (dog, sat. 2) showed a large decrease in the
KOPAH:KOMAN ratio, PAH outflux approaching that of mannitol.
The ratio decreased from 8. 2 at 80 jig/ml PAH to 0.7 at 800 ILg/
ml and 1. 0 at 8, 000 [.Lg/ml. Thus PAH transport is probably
saturated between 150 [.Lg/ml and 800 [.Lg/ml PAH since KOPAH/
KOMAN between these 2 concentrations approaches 1. 0.

With perfusion concentrations of mannitol and PAH remain-
ing constant throughout the experiment (0. 25 mg/ml and 10 Hg/ml,
respectively), a series of 10 fold increases in Diodrast concentra-
tion were added to the perfusion fluid (Table 8; Figure 10). With the
addition of 0.07 mg/ml Diodrast, PAH outflux decreased from
83 [.Ll/min during the control period to 62 FLl/min in period 11.
Diodrast concentrations of 0.7 and 7.0 mg/ml result in PAH out-
fluxes of 67 FLl/min and 42 lLl/min respectively. KOPAH/KOMAN
also decreased from 4. 9 to 1.6 upon the addition of 0.07 mg/ml
Diodrast and with the addition of greater concentrations of Diodrast
this ratio approached 1.0, indicating inhibition of PAH transport.

The addition of 10, 000 IU penicillin G/ml to the perfusion
fluid produced only slight inhibition of PAH transport (Table 8;
dog A16). Perfusion fluid concentrations of mannitol (0. 05 mg/ ml)
and PAH (10 11. g / ml) remainedconstant throughout the experiment.

Both mannitol and PAH outfluxes increased slightly on the addition

43

of penicillin to the perfusion fluid; KOPAH/KOMAN decreased from

1.45 to 1.24.

44

88858pr 5 mcofimcfimmno no 858:: u .2 .m .m A888 28 mm cummouaxm mum $305 2:.

 

a V000 A 2.0

A0 80 .0 A 0m .0

880.0 A 8 .0

8 :56 H mm .o

8 30.0 A 8 .0

88:00 A 2.0

a 0A0 .0 A 80.0

a x: .0 A 00.8

A0 8.0 A 38

8:50 A 85

8 EA .0 A A0 .A
8 v8.0 A 00 .8
A0 8.0 A8;

A0c0m0 A :28

838.0 A 8.0

33.8 .0 A80

Amfivwo .o H No .H

.Aomva .o H cm;

8380 A AAA

3“va .o H om .v

3380 A 0A .0

888.0 Am0.m

83A
ammo?
«A: m

mxmmk
m

Rom?
H

 

#32232 SH \ .H.

5.8 2:.

 

#9382832 2 \ H.

 

58 2:.

#030332 2 \ .H.

 

58 2:.

 

ammundfifl

 

nHU>h

 

nHU>IH

 

om<

 

.mMOp vmmw 88.88.36 89¢ Emansmfln cam 855me 30.830 mo “9:588 and mad mo moflwu 2:. I- .m 8an

45

Table 3. --PAH/ MAN of choroid plexuses from different aged dogs.

 

 

 

 

PAH/MAN*
Age
LVCP FVCP
lweek 2.12i0.37 (20) 1.97:t0.47( 9)
2weeks 6.26i0.71 (12) 1.80:0.37( 6)
3-4 weeks 6.34:1: 1.00 (18) 2.44iO.46( 9)
Adult 4.04 :1: 0. 55 (10) 3. 84:!: 1.00 ( 6)

 

 

 

* PAH/MAN values expressed as mean :1: S. E. M. ; number of
observations in parentheses.

46

Table 4. --Effect of medium PAH concentration on PAH accumula-
tion by the 3 -week -old dog choroid plexus.

 

 

 

 

Medium PAH LVCP FVCP
Concentration
(pM/l) T/M PAH T/M MAN T/M PAH T/M MAN
28.8 11.81 0.48 2.41 0.45
7.21 0.34
11.38 0.59 3.90 0.42
6.87 0.44
57.7 6.53 0.30 1.86 0.41
5.04 0.32
87.5 4.53 0.36 1.32 0.27
3.94 0.31
146.7 3.51 0.44 1.72 0.57
3.75 0.49
293.6 1.65 0.46 1.35 0.49
1.32 0.45

 

 

 

 

 

inn... In

I‘CI.I.II!I‘II

47

 

 

 

 

00 A0 .0 A A0 .8 A08 A 00.8. 0803 0 A 088 N.08 x 8 8A0 A808
00. 00 .0 A88 A0 .8 A 08 s 0803 0 A A8 0.08 x 8 8A0 .805
0808.. 088000008
00 00 .0 A 3.0 80 .8 A 00 .0 888A. 82 0-08 x 8 Av
0 00.0 A000 0A0 A080 0803A0 2A-08 x0 8A0 A8058
0 00.0A0e88 00.8A0A88 880088. 820-08 x8 A0
0 5.8A0888 0A.8A0A.0 0803870. 8>8A-08 x0 83.805
QEGQAU Savom
80 00.0A008 8.8.8 A000 08030 A 88888108 x8 EASE
AA 00.0 A120 0A8 A000 08030 A 0,8808 x8 378028
8088058508
008 0 .0 0 .8 0803 0 D8 000 .08 E
008 8. .0 0 .m 0803 0 D8 000 .00 880
008 0 .0 A .N 0803 0 .28 000.00 3 A80;
008 0 .0 0 .0 0803 0 D8 000 .08 880
008 0 .8 0 .08 0803 0 D8 000 .00 E
008 0 .0 N .08 0803 0 D8 000 .00 8: A8028
0 58880888
008 08 .0 A 00 .0 00 .0 A 08 .0 0803 0 s8 A-08 x 8. 800 A808
00 00 .0 A 00.8 00 .8 A 00 .A 0803 0 08 TB x a 800 A828
80800808
Aflcmgummxm 3.88880
8.00 00888588 0008 80 84 8808880000 85 888888
8.888 .m .0 A808 2088,8588

 

 

 

 

 

 

.03me 30.8050 wow

88 5 888805800 88888 00 00888588-- .0 088.8.

48

Table 6. -—Accumulation of PAH by choroid plexuses from animals
treated with procaine penicillin G.

 

 

 

 

Treatment PAH/ MAN*
Age (IU penicillin/

kg/day) LVCP FVCP

1 week Saline 8. 33 :1: 1. 94 (41 3.73 :1: 1.06 (4)
300, 000 14. 38 :1: 0. 97 (4) 4. 04 :1: 0. 18 (4)
600,000 8.701: 2.83 (4) 5.40:1: 1.64 (4)

2 weeks Saline 6. 23 :1: 0. 84 (6) 1. 80 :1: 0. 37 (6)
120,000 6.70:1: 1.00 (6) 2.761: 0.31(4)

 

 

 

 

* PAH/MAN values are expressed as mean i: S. E. M. ; number of
observations in parentheses.

49

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

008888530 08500800

mafia-:30 0.0.398:

 

 

.00 0.8.8 0.008 .008 0.008 00000 00000 00.08 08.08 8.000 8.0 .0 00.0 088 0: 08 8 .05
.00 0 .00 0.08 0 .00 0 .00 .00 0 .00 00000 08080 00.08 00 .0 00 .0 0000 0000 00 .0 00 .8 000 008 0- 8.8-4
.00 0.00 0.08 0.00 0.00 .08 0.0 00000 08000 00.0 00.0 00.0 8000 0000 8.0 .0 00.8 000 008 0. 08-6
.00 0.00 0 .00 0 .00 0 .00 .00 0.08 08.008 00000 80 .0 00.8 00.8 08.08 8.000 00 .0 8 .0 008 008 0- 0K.
.00 0.00 0.0. 0.00 0.00 0 0.0 00008 00080 00.8. 00.0 00.8 008. 08.08 00.0 00.8 000 008 08- TN.
.00 0.00 0.0 0.00 0.08. .00 0.00 2.000 00000 00.0 80.8 80.0 0:. 000 00.0 00.0 000 008 08- 0-x.
.08 0 .08 0 .0 00 .0 08 .0 S .0 00.8 000 008 08- Tm.
8.0 0.008 0.00 00.8 00.0 00.0 00.0 000 008 0 8%
.00 0.00 0.00 00.0 00.0 8.0.0 00.0 080 000 0- 0-00
.08 0 .2. 0 .08. 00 .0 08 .0 00 .0 08 .8 000 000 0- 0.80
o . o
d m. u M d M u m. o 0 o m o A o 8 o u o A
m 0 m m 0 w m u o o o o o o o o u > >
m m m m . . Eon 80.
. I . 7. OEDO
0 8888803 88.503 0808. 088803 088050 38:10 388.0%» 0 000
58:1. 08 8:88:10 0088080 8805 SE 05508.5 8880500 5805 3088 308.0 - .55

 

 

 

.uov 05 S Emu 30.6 2008080528 00 13080.0 no 3a.— ofl 8 0.330.00— 0538580555 we 38E... .0. £2

50

 

 

 

 

 

 

 

 

 

00.8 00 0A 008 00000 80000 0008 0000 80.0 00.8 008 000 008-088 0 .888 88:88 000.08 88
00 .8 08. 80 00 00000 00000 0080 0000 00.0 00.0 000 008 00-00 880.8000 2.6
08.8 00 8.0 00 08.8.00 008.00 0008 0008 0.0.0 8.0.0 0.08 08.8 000-000 80.88.0888 88:88 0.8. CE
08.8 8.0 .00 00 0008.0 08000 088.8 0000 00.0 00.0 008 08.8 000-080 80800888 88008 0.0 :88.
00 .8 00 00 80 00000 088.80 0008 0000 08. .0 00 .0 008 08.8 8.8.8 -008 80800808 88008 8.0.0 8880
00.0 00 8.8 008 00000 00000 08.08 8.000 00.0 00.0 088 08.8 80-80 8808888000 8.0888
00.8 00 00 8.88 00000 8.0000 0008 0008 00.0 00.8 008 000 000-000 .8208 80:01 0.0000 :5
00.0 00 00 00 0008.0 00000. 0000 0000 00.0 00.0 008 000 000-000 884.8 88001 0.000 88.88.
00.0 00 A 0 08.800 00000 0080 0000 00 .0 00.0 000 000 008-008 88.8 80:01 0.00 8880 0.8880
08.8 00 00 00 00000 00000 0008 0008 00.0 00.8 000 008 8.00-800 88.8 80:01 0.008 :6
00.0 00 00 0 08000 00800 8008 0000 00.0 00.0 080 008 000-008 880.88 8.501 0.00 880
08 .8 00 00 0 0008.0 00000 0008 0800 00.0 00.0 080 008 008-008 8888 80:01 0.00 8880
08 .0 00 08 0 00000 00000 8000 0000 00.0 00.0 000 008 00-80 88488 89:01 0.0 5 08-6
0 88488 80885308 58885 00 8o 00 80 00 80 0> 8>
z 08 . .
A.............. 8.... 0.80 :00... 8.0.0 0.08. 0.880... 3mm... .5... :..... ...

 

00880530 8.380800

 

 

0033883 60.885003

 

 

 

 

.000 888.00 0888 5 .000 60.88 8.800385 084.8 80 00888588-- .0 088.8.

51

Figure 1. —-Accumulation of PAH and mannitol by the in vitro lateral
ventricular choroid plexus (LVCP) in the developing dog.
Accumulation of PAH and mannitol (ordinate) expressed as
tissuezmedium concentration ratios (T/M). Medium con-
centration: mannitol, 2. 7 mM/l; PAH, 9. 7 l..LM/1. Data
expressed as mean :t S. E. M. ; numbers of observations

indicated in parentheses.

#430

m0 .m
0n.

 

 

52

 

242 E
140. U

 

 

 

H 0.0st00

m0<

 

 

 

 

AN:

 

 

 

 

2:. ac);

QN

0.?

0.0

0.0

53

Figure 2. —-Accumulation of PAH and mannitol by the in vitro fourth
ventricular choroid plexus (FVCP) in the developing dog.
Accumulation of PAH and mannitol (ordinate) expressed
as tissuezmedium concentration ratios (T/ M). Medium
concentration: mannitol, 2.7 mM/l; PAH, 9.7 #M/l.

Data expressed as mean i S. E. M. ; numbers of observa-

tions indicated in parentheses.

54

 

 

 

 

 

z<2 a
I<m U

 

 

 

 

m ounmfim

vvvvv‘
.
.0.......
' O O
Q. .0
' O O
’0. 0..
.0

a.

 

 

 

2\b ao>u

QN

0.?

06

0.0

55

Figure 3. --Histological sections (287X) of FVCP from 1 -day-old
and adult dogs. A. One-day-old FVCP illustrating
columnar epithelium and large stromal volume contain-
ing few fibroblasts and fibrocytes. B. Adult FVCP
contrasting the epithelium (cuboidal), decreased stromal
volume and increased number of fibroblasts and fibro-
cytes. Epithelium, Ep; stroma, St; fibroblasts and
fibrocytes, fb; artery, a. Sections: 6 FL thick; Hema-

toxin and eosin stain.

56

 

m 90083.00

 

57

Figure 4. --Accumulation of PAH by isolated LVCP and FVCP in the
developing dog. Medium concentrations: mannitol,
2. 7 mM/l; PAH, 9. 7 ,LLM/l. Uptake of PAH (ordinate)
is expressed as T/M PAHzT/M mannitol ratios. Each
bar represents mean i: S. E. M. ; number of animals

indicated parenthetically.

Figure 4

58

 

 

 

 

 

 

 

         
    

 
   

               
            

        

   

 

 

 

 

O. O. ._
Q 0 _,
> > :3
_I II. A - - Q
0 <12
[:1 E =
.8 :3::::::::::::::::::::°:::;
0’9 b’0’o‘o°o°o’o°o°o’o’o’o‘o°« '
0.0 030303030.o?o?o?o?o?o?o?o.c ;
- _ V
I
.. r0
' In
X
N
0":

 

'o'o'o'o'o'o'o'o'o' -

 

 

A
MAN

 

8.0
6.0
4.0
2.0 -

59

Figure 5.--Saturation curve of PAH transport by the LVCP (0—0)

PAH

and FVCP (0—0) of littermate 3 -week-old dogs. m

is plotted on the ordinate as a function of medium PAH
concentration (mM/l). Each point represents 1 observa-

tion. Lines connecting data points were fitted by eye. T/M

mM PAH/gm of wet tissue
mM PAH/ml of medium

 

PAH ratios were calculated as

and T:M ratios of PAH were compared to T/M mannitol.

60

m opswwm

$84.:

oon OmN CON On. 00. on
_ 0 8 O

 

 

 

Q0>m o 242
ao>._ 0 :5. L00

 

 

 

 

61

Figure 6. "Relationship between intraventricular pressure and outflow
cannula height. Both intraventricular pressure (cm HOH
pressure; ordinate) and outflow cannula height (cm;
abscissa) measured relative to external auditory meatus.
Each point represents 1 observation during 30 - 90 minutes
perfusion time. Equation of the line, determined by the
method of linear regression, is: Y = 0. 65 i 0.08X + 1.31

(n = 27).

62

m 0.0089,”.—

.PIOBI 30403.30

mm ON 0. o. n o 0.. OT 0.: ON- mm.-
0 d 0 0 0 A 0 0 d o 0 J ONI

 

 

o.
umammwma

m<...:o.m...zu><¢._.z. om

 

63

Figure 7.—-C1earance (,LLl/min; ordinate) of inulin (x—x), mannitol
(o—- —o) and PAH (o— ——o) and its relationship to intra-
ventricular pressure (cm HOH; abscissa) measured
relative to the external auditory meatus. Clearance of
inulin was calculated by Equation 1 (Methods); that of
PAH and mannitol, by Equation 2 (Methods) and are shown
in Table 7. Perfusion fluid concentrations: inulin,

0. 8-1. 0 mg/ml; mannitol, 0.25 mg/ml; PAH, < 10 #g/ml.
Each point represents 1 observation between 30 - 90 minutes
perfusion time. Equations for lines through data points

were determined by the method of linear regression:

inulin: Y = 3. 45 d: 2. 18X + 50. 67 (n = 10)
mannitol: Y = 3. 89 3: 1.96X+ 62.29 (n = 6)
PAH: Y = 5.00 :t 1.26X+ 91.71 (n = 10)

64

O.
_

m

N. musmwm

mmammumn— ¢<4=o_mkzw><c.—.z_
0

ml

 

O... m...

uoz<¢<msoL

on

00.

on.

 

CON

65

Figure 8, --Efflux coefficients (K0, le/min; ordinate) of mannitol
(o— - -o) and PAH (o- — —o) and its relationship to
intraventricular pressure (cm HOH; abscissa) measured
relative to the external auditory meatus. Efflux coefficients
were calculated by Equation 3 (Methods) and are shown in
Table 7. Perfusion fluid concentrations: mannitol,
0, 25 mg/ml; PAH, < 10 [lg/m1. Each point represents
1 observation during 30- 90 minutes perfusion time.
Equations for lines were determined by the method of

linear regression:

mannitol: Y 0.53 i 2.52X+ 18.65 (n = 6)

PAH: Y 2.97:1: 1.01X+ 55.75 (n = 10)

66

w mhfimwm

mmammmmn. m4..:o.m._.zm><¢._.z_
O

 

m. o. m ms 0... m...
a a

_ q . n

O J—

O
o o .I..|.|.Vo ..
.IIIOIIIOIIIOIIIOIIIO‘OIIIO‘OQII \
\ ..
a
\
o \o -
\
\ l
x u
\ l
\ o
\
\ l
\
\ I
\
\
O\ n
\ o

 

on

00.

67

Figure 9. --Efflux coefficients of 3 molecules of similar molecular
weight perfused simultaneously in 5 animals (original
data in Table 7). Efflux coefficients (K0, ,LLl/min;
ordinate) were calculated by Equation 3 (Methods). Per-
fusion concentrations: creatinine, 1. 0-2. 0 mg/ml;
PAH, < 10 FLg/ml; mannitol, 0.25 mg/ml. Each bar

represents mean at S. E. M.

68

40.52242
iIIJ.

 

 

m mhswwb

72¢

.17

 

 

wz.2_k<wmo

All

 

 

nu

Om

69

Figure 10. --Inhibition of PAH transport from CSF by the addition of
Diodrast to the perfusion fluid. PAH and mannitol efflux
coefficients (K0, {ll/min) are shown on the ordinate and
perfusion fluid Diodrast concentrations (mg/m1) are shown
under each set of bars. Perfusion fluid concentrations:
PAH, < 10 )LLg/ml; mannitol, 0.25 mg/ml. Each bar
represents 1 perfusion period; data taken from Table 8

(Dog Dio. I).

70

S stE

 
      

 

 

 

 

 

 

 

 

 

 

 

 

 

 

hm<¢oo.o
OK 5.0 00
“man” fill i]. _ 11 o
1 ON
ill 10¢
_III I 00
ilL,
24.2 a
I: D _L. - om
ox Poo.

DISCUSSION

Organic anions are products of metabolism which cannot
be further metabolized by the cell. Elimination of organic anions
from the organism is necessary .to prevent slowing of reversible
chemical reactions and accumulation of these compounds to toxic
levels. In the central nervous system high concentrations of some
organic anions cause seizures and convulsions (Pilchner 31:31. ,
1947; Walker 3131. , 1945). Therefore development of an organic
anion transport mechanism is of biological importance since the
active transport may constitute a barrier which prevents toxic
substances in blood from entering cerebrospinal fluid (CSF) and
brain, and also may be responsible for excreting into blood waste
products generated in the brain.

Organic anions are actively transported from blood into
urine by the proximal tubule cells of the kidney. This function in
the kidney is thought to involve transport of a molecule having the

general formula:

R - C:O - NX - (CHR' )n - COOH

71

72

where R or R' may be an aliphatic chain or aromatic ring; X is a
hydrogen or methyl group and when R' is hydrogen, n varies from

1 to 5. Organic anions which lack correspondence to the general
formula are not actively transported (Despopoulus, 1965).
p-Aminohippuric acid (PAH), Diodrast and penicillin share a common
transport mechanism and are actively transported by the renal tubule
cells (Sperber, 1954). PAH conforms to the general formula (R is
an animated benzene ring, X is hydrogen and n equals 1) and its

formula is:
NH2 —©— C:O - HN - CH2 - COOH

Pappenheimer 3111: (1961) perfused the brain ventricular
system of goats with an artificial CSF containing low concentrations
of Diodrast and phenolsulfonphthalein and observed that these
organic anions were actively transported from CSF in the region of
the fourthventricle; possibly by the fourth ventricular choroid
plexus (FVCP). Becker (1961) reported preliminary evidence that
the in vitro lateral ventricular choroid plexus (LVCP) of the rabbit
actively accumulated organic anions. Organic anion transport has
not been studied in the dog, nor have any studies compared the
in vivo and in vitro organic anion transport system. Data presented

in this thesis suggest that PAH, an organic anion, is actively

73

accumulated by both the LVCP and FVCP in vitro and that PAH in
low concentrations is actively transported from CSF in vivo.
Perfusion of an artificial CSF solution through the brain
ventricular system of the dog permitted the study of the exchange
rates of test molecules between CSF, blood and brain. The clearance
of a molecule from CSF is defined as the volume of CSF from which
asubstance is completely removed per unit time; analogous to
clearance as used in renal physiology. This volume depends upon
the amount entering the brain ventricular system, the amount leav-
ing and the concentration of the substance in the ventricle (Equation 2,
Methods; Pappenheimer _e_t_a_l_. , 1961). In the case of a large
molecule such as inulin (M. W. = 5000), Ball and Oppelt (1962)
demonstrated that after 5 hours of perfusion of the ventriculocisternal
system of the dog, only small amounts of inulin penetrated the
ependymal walls of the lateral ventricles and diffused into the
brain parenchyma. . Heisey fl. (1962) found that the trans-
ependymal flux rates of inulin were not detectable and concluded
that the loss of inulin from CSF could be used to estimate bulk
absorptionof fluid distal to the fourth ventricle; possibly through
large Openings on the arachnoid villi leading from the subarachnoid
Spaces into the venous sinus (Davson, 1967). The clearance of

inulin is a linear function of intraventricular pressure (Figure 7)

74

whichsupports observations in the goat (Heisey§_1_:_a_l_. , 1962) and
dog (Bering and Sato, 1963; Cserr, 1965).

The clearance of mannitol, a passively diffusing molecule,
was always greater than the clearance of inulin (Figure 7), but the
slope of mannitol clearance as a function of intraventricular
pressure was equal to that of inulin, indicating that the pressure
dependence of mannitol clearance can be accounted for by the bulk
absorptionof fluid from CSF. The difference between the clearance
of mannitol and inulin represents the diffusional component of
mannitol clearance (Bering and Sato, 1963). Heisey fl. (1962)
observed that the passive diffusion of tritiated water (TOH) from
the brainventricular system in goats was dependent upon intra-
ventricular pressure but the increase in TOH clearance-with intra-
ventricular pre'ssure could be accounted for by the bulk absorption
of CSF.

The outflux coefficient of a molecule represents the
clearance of a molecule by means other-than bulkabsorption
(Equation 3, Methods). The slope of the line relating mannitol
efflux (KO mannitol) to intraventricular pressure is zero (Figure 8),
indicating that the movement of mannitol, a passively diffusing
molecule, out of CSF is independent of intraventricular pressure.

When the bulk absorption component was subtracted from. the

75

clearance of tritiatedwater (TOH), efflux of TOH was also
independent of intraventricular pressure (Heisey 321. , 1962).

In contrast PAH efflux (K0 PAH). increased with intraven-
tricular pressure over the range -15 to +15 cm HOH pressure. A
pressure -dependent efflux of any molecule has not been previously
reported. The pressure-dependent PAH efflux might be explained

g

by the course of the perfusion fluid through the ventriculocisternal

 

system and the location of the FVCP. Perfusion fluid flows from the
lateral ventricle through the third and fourth ventricles, through _;,
the foramina of Luschka, at the lateral recesses of the fourth

ventricular cavity, and into the cisterna magna where the outflow

needle is placed. At increased intraventricular pressures, fluid is

reabsorbed distal to the fourth'ventricle, involving a greatersurface

area of the subarachnoid space. Much of the FVCP projects through

the foramina of Luschka into the subarachnoid space. It is possible

that at low intraventricular pressures, perfusion .fluid takes a more

direct routethrough the foramina of Luschka to the outflow cannula

in the cisterna magna and bypasses much of the subarachnoid space

and the surface of the FVCP; while at increased intraventricular

pressures perfusion fluid passes over more surface of the FVCP en

route to more rostral portions of the subarachnoid space and con-

sequent reabsorption intothe venous sinuses. Therefore, increased

76

intraventricular pressures could effectively increase the surface
area not only for the bulk absorption of fluid but also increase the
perfused surface area of the FVCP for active transport of PAH.

PAH transport exhibits some criteria of active transport,
i.__e_. , saturation of the transport mechanism at high PAH perfusion
fluid concentrations and inhibition of transport by the addition of
competitive inhibitors to the perfusion fluid which are structurally
similar, Diodrast and penicillin (Despopoulus, 1965; Sperber,

1954). At PAH perfusion concentrations between 150 and 800 fig/ml
PAH, the transport system appeared saturated since the ratio of
PAH to mannitol efflux approached 1. 0 (Table 8). Pappenheimer
5131. (1961) reported that the organic anion transport mechanism

in the goat was saturated at 20 - 40 /..Lg/ml. Comparison of these
saturation concentrations would indicate that the dog has a larger
capacity for organic anion transport than the goat.

PAH transport was inhibited by the addition of 70 - 700 [.Lg / ml
Diodrast to the perfusion fluid (Table 8), supporting the observation
that the organic anion transport mechanism is saturated between
150 and 800 ,LLg/ml PAH. Similarly, Pappenheimer £t_a_l. (1961)
reported Diodrast transport was inhibited by the addition of

500 FLg/ml PAH. The addition of 10, 000 IU penicillin/ml to the

perfusion fluid produced slight inhibition of PAH transport (Table 8).

77

Fishman (1966) reported that intracisternal injection of 8 X 10-5

moles PAH in the dog blocked active transport of penicillin from
CSF. It appears that PAH, Diodrast and penicillin share a common
transport system for the removal of these compounds from the CSF.
Although the molecular weights of mannitol, PAH and
creatinine are similar (182, 194 and 113, respectively), efflux
coefficients of both creatinine (46 :l: 4 PIA/min) and PAH (34 :1: 4 f.Ll/
min) are significantly greater (p < 0. 05) than mannitol (16 i 8 FLI/
min; Figure 9), suggesting that creatinine and PAH leave CSF by
a process other than passive diffusion. Others have assumed
creatinine leaves CSF by passive transport (Bering and Sato, 1963;
Heisey 3t_a_l_. , 1962; Cserr, 1965) but none of these investigators
have compared efflux coefficients of creatinine to mannitol. Crea-
tinine efflux coefficients of 27 [.Ll/min (Cserr, 1965) and 47 iLl/
min (Bering and Sato, 1963) were found for dog ventricles; values
which are greater than the mannitol efflux coefficient (16 :1: 8 FLI/
min; Figure 9). Heisey _e_t_a_tl. (1962) reported that the creatinine
efflux coefficient for goat ventricles was 76 :t 15 [.Ll/min; a value
greater‘than those reported for the dog. These differences may be
due to species differences in the surface areas of the walls of the
ventricles and choroid plexuses having an unknown effect upon the
efflux coefficient. My data suggest that creatinine and PAH may be

actively transported from CSF.

78

The tissue:medium concentration ratio (T/M) is a measure

of the concentration gradient maintained between the tissue and
medium in vitro. If a molecule were distributed between the tissue

and medium by passive diffusion alone, then the expected T:M ratio
would be less than 1.0 due to proteins and salts within the tissue;
while T:M ratios greater than 1 indicate active accumulation against

a concentration gradient since energy must be supplied to maintain

this ratio of concentrations. The T:M ratio is a function of medium

concentration and since T:M ratios are not reported at either satura-
tion concentrations or even at the same medium concentration, T:M
ratios of different molecules (Table 1) are not comparable.

In diaphragm muscle neither PAH nor mannitol is actively
accumulated since T/M PAH and T/M mannitol are not greater than
1. 0 (Table-2). Rubin 3121. (1968) reported that methotrexate,
actively transported by the choroid plexus in vitro, is not actively
accumulated by either diaphragm muscle or cerebral cortical
slices; T/M methotrexate ranged from 0.8 to 1. 0.

In both the LVCP and FVCP T:M mannitol ratios decreased
with increasing age, suggesting that the volume into which mannitol

can diffuse decreases. Histological slides of 1 -day and adult

choroid plexuses (Figure 4) provide anatomical evidence that as the

plexus matures the stromal volume of the LVCP and FVCP decreases.

 

 

 

u rdl

79

Kappers (1958) observed a similar change in the choroid plexus

from 6 — week-old human fetuses compared to the fourth month
fetus. In the 6-week-old fetus the choroidal epithelium consisted

of single -,layered cuboidal cells surrounding a stroma composed

of gelatinous connective tissue. By the fourth month, the gelatinous

connective tissue was replaced by dense fibrous connective tissue l

and accompanied by a significant increase in the ratio of epithelium

to stroma. Hilton (1956) observed that the adult human choroid

 

plexus is extremely lobulated with each lobule composed of finer E j

divisions; a phenomenon not observed in the embryo. An increase
in the density of the stromal connective tissue in the developing

plexus could decrease the space into which mannitol diffuses. The
change in T/ M mannitol presumably represents a change in the

extracellular fluid (ECF) of the choroid plexus and in order to

correct for age differences in the ECF, data is expressed as T/M

PAH:T/M mannitol ratios (PAH/MAN). At 1 week of age PAH

transport is poorly developed since neither the LVCP nor the FVCP
is able to concentrate organic anions as can the adult (Figure 4).
TIM for sulphate in cats and rabbits (Robinson et a1. , 1968) also

suggests lower accumulation in the newborn than in the adult. An

increased surface area on the adult might account forthe greater

PAH transport by the adult.

80

Both the adult LVCP and FVCP actively accumulate PAH
and there is no statistical difference between PAH:MAN ratios of
the LVCP and FVCP in the adult. Similarly, Tochino and Schanker

(1965b) reported that accumulation of serotonin and norepinephrine
by the LVCP was not different from the FVCP. In contrast, accumu-
lation of an organic acid, 5—hydroxyindoleacetic acid, was greater
in the FVCP of the adult rabbit than in the LVCP (Cserr and Van
Dyke, 1971). In vivo results of Pappenheimer _e_t_a_l_. (1961) suggest
that Diodrast is actively transported from CSF in the regionof the
fourth ventricle, possibly by the FVCP, and that minimal transport
occurs in the region of the lateral ventricles.

Both the LVCP and FVCP exhibit some criteria for the
active uptake of PAH: PAH is accumulated against a concentration
gradient (T/M. > 1. 0; Table 2); shows a saturable transport mechan-
ism at high medium concentrations of PAH (> 200 mM/l; Figure 7);

is inhibited by other organic anions (Diodrast and penicillin; Table 5)
and has a metabolic energy dependent transport mechanism (iodo-
acetic acid; Table 5).
The LVCP' s ability to accumulate organic anions during
the 2- and 3- to 4-week age is greater than either the adult or
l-week-old dog (Table 1; Figures 1, 2). Maximum transport at

the second week by the LVCP parallels the observations that

 

 

 

81

sulphate transport is maximal in the 10 -day-old rabbit

(Robinson _et_€_tl. , 1968) and morphine transport peaks in the 15-
day-old rabbit (Asghar and Way, 1970). The FVCP PAH/MAN

is greater in the adult than at 1 or 2 weeks of age (Figure 4). The
later development of the FV CP transport mechanism contrasts

anatomical data which indicate that the myelencephalic choroid

 

plexus (FV CP) develops before the telencephalic plexus (LVCP)

in fetal humans (Kappers, 1958) and fetal pigs (Hilton, 1956).

 

Hirsch and Hook (1969a, 1969b, 1969c, 1970) induced :-
organic anion transport in renal cortical slices from animals
previously treated with penicillin. In 2 -week-old rabbits, PAH
transport by cortical slices was increased 4 times by prior treat-
ment with penicillin, but no increase in transport was noted at 4
weeks of age when the organic anion transport system was well
developed. Inthe LVCP, PAH transport was induced at 1 week
postnatal by prior administration of 300, 000 IU but not 600, 000 IU
penicillin G. At 2 weeks no induction was produced in penicillin-
treated animals (Table 6). At 1 week PAH transport by the LVCP
is not well developed but is highly developed at 2 weeks (Figure 4).
Prior administration of 300, 000 IU penicillin induced PAH trans -
port at 1week when the transport mechanism was developing but

failure to produce any effect on PAH transport might be that this

82

dosage exceeded the optimal dosage response for penicillin

induction. In the 1 -week-old FVCP no induction of PAH transport

was produced with either 300, 000 or 600, 000 IU penicillin, but at
2 weeks PAH transport was induced by treatment with 120, 000 IU

penicillin. PAH transport by the FVCP is not developed at 1 or

2 weeks but begins to develop at 3 -4 weeks (Figure 4). Failure of

penicillin to induce PAH transport in the FVCP at 1 week might
either be due to the immaturity of the transport mechanism at that
age or the low dosage of penicillin administered to the animal.
Induction of PAH transport at 2 weeks supports the view that
transport induction occurs at a time before transport normally
develops. These data support the hypothesis that an increased or
prolonged presence of organic anions in the CSF during the period
of transport stimulates maturation or activity of the organic anion
transport system. Hirsch and Hook (1969a) suggest that the
stimulatory effect of penicillin might be caused by enhanced
development of existing transport processes or synthesis of new
enzyme proteins responsible for organic anion transport.
Asghar and Way (1970) suggest that the increased

resistance to drug toxicity in adults could be dependent upon the

development of the blood -brain and blood- CSF barriers. They

observed that brain concentrations of morphine/ gm body weight in

 

 

 

83

younger animals (< 10 days postnatal) are higher than in adults;
a difference which corresponds well to observed toxic effects of
morphine in young animals and adults. Furthermore they suggest
that high brain morphine. levels in the newborn might result from
a decreased outflux of morphine from the CNS. Penicillin, an
organic anion, is actively transported from CSF into blood and
brain (Fishman, 1966) and is a convulsive agent, causing seizures
when topically applied to the cerebral cortex of monkeys (Walker
_e_t__a_t_l_. , 1945) or injected intracisternally (3, 000 IU) or intrathecally
(7, 500 IU) in dogs (Pilchner 3L5}; , 1947). Similar to morphine,
penicillin transport may be poorly developed in the young since
intrathecal injection of 3, 000 IU penicillin G causes convulsions in
children whereas in adults 10, 000 IU penicillin are required to
produce the same effects. Challenging the choroid plexus with
organic anions during the period of development enhances matura-
tion of the organic anion transport mechanism and provides a
mechanism for protecting the CNS of the young from potentially
toxic compounds.

Further studies in the area of transport development are
suggested since it is not known why young animals transport less
organicanions than the adult. Possible explanations could be an

increased permeability of the choroid plexus in the young, or a

 

84

decreased rate of transport in the young compared to the adult or

a combination of both factors. My data indicate that there is an
optimal dosage for penicillin induction; all dosages of penicillin
were not investigated. Also there may be an optimal time after the
last injection of penicillin is administered and PAH accumulation is;

measured.

 

SUMMARY

In vitro incubations

1. T/ M mannitol in LVCP and FV CP from adult animals were
significantly less (p < 0.05) than in animals 1 week old;
T / M mannitol in diaphragmdid not change with age.

2. PAH accumulation by both the LVCP and FVCP was an
active process: PAH was accumulated against a concentra-
tion gradient, suggesting a metabolic energy dependence,
and showed competitive inhibition.

3. PAH accumulation was poorly developed in the LVCP and
FVCP from 1-week-old animals (T/M = 2.12; T/M = 1.97,
respectively) but well developed in the adult (T/M = 4. 04;
T/M = 3. 84, respectively).

4. Maximum accumulation of PAH occurred in the LVCP from
2 -week-—old animals and in the FVCP from adult animals.

5. LVCP' s from 1 -week-old dogs treated with 300, 000 IU
penicillin showed a significant increase (p < 0.05) in PAH

accumulation compared to controls. No induction (p > 0. 05)

85

 

86

of PAH accumulation was produced by treatment with
600, 000 IU in 1 -week-old dogs or 120, 000 IU in 2 —week-
old animals.

6. FVCP' s from 1 «week-old dogs treated with 300, 000 or
600, 000 IU penicillin showed no increase (p > 0.05) in PAH
uptake but FVCP' s from 2 -week -old dogs treated with
120, 000 IU penicillin produced significantly greater

(p < 0.05) PAH accumulation than control animals.

In vivo perfusions

1. Inulin is removed from the ventricular system by bulk
absorption of fluid at rates which vary linearly with intra -
ventricular pressure.

2. Efflux coefficients of mannitol, a passively diffusing
molecule, is independent of intraventricular pressure
over the range -15 to +15 cm HOH pressure.

3. PAH efflux from CSF varies directly with intraventricular
pressure over the pressure range -15 to +15 cm HOH
pressure, indicating that PAH efflux may be a function of
the perfused surface area of the FVCP.

4. Efflux coefficients of creatinine and PAH are significantly

greater (p < 0.05) than mannitol, indicating that creatinine

87

and PAH may be removed from CSF by a process other
than passive diffusion.

Active transport of PAH from CSF was indicated by: an
efflux coefficient of PAH significantly greater (p < 0.05)
than mannitol, inhibition of transport by other organic

anions and self -saturation.

APPENDICES

APPENDIX 1

PREPARATION OF AR TIFICIAL

CEREBROSPINAL FLUID (CSF)

Artificial dog CSF contains:

Cations Anions
Na 150.0 mEq/l (31" 133.2 mEq/l
K 3.0 mEq/l Hcoa' 25.0 mEq/l
Ca++ 2. 3 mEq/l HPO4-2 0.5 mEq/l
Mg++ 1. 6 mEq/l
Reagents:
1. NazHPO4 - 7HOH
2. KC1
3. NaHCO3
4. NaCl
5. CaCl2
6. Mgc12 - 6HOH
7. Dextrose

88

 

89

Solutions :

A.

Dissolve 0. 00345 g Na2HPO 7HOH, 0.2237 g KC1,

4 .

2. 1007 g NaHCO and 7. 2467 g NaCl in distilled water

3
q. S. 1 liter.

MgCl2

,Dissolve 0.8133 g MgCl2 - 6HOH in distilled water;

q. s. 100 ml.
CaCl2

Dissolve 1. 2763 g CaCl in distilled water; q. s. 100 ml.

2

Procedure:

100 ml of solution A is equilibrated with 3 - 9% C02;

0. 1 ml of each of solutions B and C and 0. 7 - 1.0 mg/ml dextrose

are added.

 

APPENDIX 2

p - AMINOHIPPURIC ACID ASSAY

Modified from H. W. Smith, 1956, pp. 212-213.

Principle:

 

A.method in which the p-amino group of p-aminohippuric

acid (PAH) is diazotized with HNO Excess HNO2 is destroyed by

2.
sulfamate and the diazotized group is coupled with N- (1 ~napthyl)
ethylenediamine to yield a colored complex. The intensity of the

color‘is proportional to the amount of PAH present.

Reagents:
1. . HCl
2. Sodium nitrite
3. Ammonium sulfamate (Sigma Chemical Co. , St. Louis,
Missouri)
4. N- (1 -napthyl) ethylenediamine dihydrochloride (Eastman

Kodak Co. , Rochester, New York)

90

91

Solutions:
A. HCl (1. 2N)
Add 10. 0 m1 conc. HCl q. s. 100 ml with distilled water.
B. NaNO2 (1.0 mg/ml)
Dissolve 100 mg NaNO2 q. s. 100 ml with distilled water.
Prepare fresh every 3 days.
C. Ammonium sulfamate (5. 0 mg/ ml)

Dissolve 500 mg ammonium sulfamate q. s. 100 ml with

 

distilled water. Prepare fresh every 2 weeks.

D. N-(l -napthyl) ethylenediamine dihydrochloride (1. 0 mg/ ml)
Dissolve 100 mg N-(l -napthyl) ethylenediamine dihydro-
chloride q. s. 100 ml with distilled water. Store in dark

glass bottle and refrigerate at 4° C.

PAH standard solutions:

Dissolve 1. 0 mg PAH (sodium salt; Sigma Chemical Co. ,
St. Louis, M0.) in distilled water q. s. 100 ml (10 [.Lg/ml PAH).
Dilute 8.0, 6. 0, 4. 0, 2.0 and 1.0 m1 of 10 pg/ml PAH q. s. 10 ml
to obtain 8.0, 6. 0, 4.0, 2. 0 and 1.0 pg/ml PAH standards respec—

tively. Standards are stored at 4° C.

Procedure:
To duplicate 0. 50 m1 unknown samples, PAH standards

and water blank add 0. 10 ml of solution A and 0. 05 m1 of solution B

92

and mix. Not before 3 and not after 5 minutes later add 0. 05 ml
of solution C and mix. Three to 5 minutes later add 0. 05 ml of
solution D and mix. Fifteen minutes after adding reagent D, read

all tubes against the water blank at 540 my. (peak of absorbency).

Calculations:

Absorbency of the standards plotted as a function of PAH
concentration (0- 10 iLg/ ml PAH) yields a straight line. Concen-
tration of PAH in unknown samples can be calculated by multiplying

the optical density of the unknown by the slope of the standard curve:

C
un OD un
std ave
where: C = concentration
OD = optical density
std = standard

un = unknown

ave average

APPENDIX 3

INULIN ASSAY

Direct Resorcinol Method Without Alkali Treatment

Modified from H. W. Smith, 1956, p. 209.

 

Principle:

A method in which inulin is hydrolyzed to fructose by
heating in acid. Fructose molecules combinewith resorcinol to
yield a colored complex; the intensity of the color is proportional

to the amount of fructose present.

Reagents:
1. Resorcinol (Fisher Sci. Co. , Fairlawn, New Jersey)
2. Ethanol (95%)

3. HCl

Solutions:
A. Resorcinol (1. 0 mg/ml)
Dissolve 100 mg resorcinol q. s. 100 ml with 95% ethanol.

Prepare fresh daily.

93

94

B. HCl (approximately 8N)

Add 224 ml of distilled water to 1000 m1 conc. HCl.

Inulin standard solutions:
Dissolve 200 mg inulin (Pfanstiehl Laboratories, Inc. ,
Waukegan, 111.) in distilled water q. s. 100 m1 (2. 0 mg/ml). Dilute

7.5, 5.0, 4.0, 3.0, 2.0 and 1.0ml of 2.0 mg/ml inulin solution to

 

10 ml with distilled water yielding 1. 5, 1. 0, 0. 8, 0.6, 0.5 and

0.2 mg/ml inulin standards. Standards are stored at 4° C.

 

Procedure:

To duplicate 0. 05 ml unknown. samples, inulin standards
and water blank, 1. 0 ml solution A and 2. 5 ml solution B are added
and mixed under a hood. A glass marble is placed on top of the
tubes and tubes are incubated for 25 minutes at 80° C. Tubes are
cooled to room temperature and optical density determined within
1 hour at 490 my. (peak of absorbency) against the water blank in a
Beckman DB spectrophotometer (Beckman Instruments, Inc. ,

Fullerton, Calif. ).

Calculations :
Optical density at 490 Hill. plotted as a function of inulin
concentration yields a straight line over the range 0-2. 0 mg / ml

inulin. Inulin concentration in unknown samples is calculated by

95

multiplying the optical density of the unknown by the slope of the

standard curve:

 

C
c _ Std x CD
un OD un
std ave
where: C = concentration
OD = optical density
std = standard
un = unknown
ave = average

APPENDIX 4

CREATININE ASSAY

Modified from S. Natelson, 1961, pp. 197 -202.

 

Principle:

 

A method in which picric acid forms a colored complex
with creatinine in alkaline solution. Maximum absorbency of the
complex is 490 my. . Color intensity is proportional to the con-
centration of creatinine; color intensity is also dependent on the
concentrations of alkali and picric acid and on temperature and

time, but these factors are maintained constant in the analysis.

Reagents:
1. Picric acid (J. T. Baker Chemical Co. , Phillipsburg,
New Jersey)
2. NaOH

3. HCI

96

97

Solutions:
A. Picric acid (1.0%)
Dissolve 10.0 g picric acid in distilled water q. s. 1 liter.
B. NaOH (10%)
Dissolve 100.0 g NaOH in distilled water q. s. 1 liter.
C. HCl (0. 1 N)

Dilute 8. 5 ml conc. HCl in distilled water q. s. 1 liter.

Creatinine standard solutions:

Dissolve 200 mg creatinine (Pfanstiehl Laboratories, Inc. ,
Waukegan, III.) in 0. l N HCl q. s. 100 ml (2. 0 mg/ml creatinine).
Dilute 8.75, 7. 50, 6.25, 5.00, 3. 75, 2. 50 and 1.25 ml. of 2.0 mg/ml
creatinine solution to 10 ml with 0. 1 N .HCl yielding 1. 75, 1. 50,

1. 25, 1. 00, 0.75, 0.50 and 0. 25 mg/ml creatinine standards respec-

tively. Standards are layered with toluene and stored at 4° C.

Procedure:

Mix 8 parts of solution A with 2 parts of solution B forming
alkaline picrate. Allow mixture to stand for 10 minutes before use.
To duplicate 0. 025 ml unknown samples, standards and water blank
add 2. 5 ml alkaline picrate and mix. After 10 minutes add 2. 5 ml

distilled water and mix. Read all tubes against the water blank at

98

490 mp, (peak of absorbency) before 15 minutes in a Beckman DB

spectrophotometer (Beckman Instruments, Inc. , Fullerton, Calif.).

Calculations:

Optical density of creatinine standards, read at 490 my“
plotted as a function of creatinine concentration yields a straight
line over the range 0 -2. 0 mg/ ml creatinine. Concentration of
creatinine in unknown samples can be calculated by multiplying
the measured optical density of the unknown sample by the slope of

the standard curve:

 

C
C = Std x OD
un OD un
std ave
where C = concentration
OD 2 optical density
std = standard

un = unknown

ave = average

APPENDIX 5
DIAL AND URETHANE SOLUTION
Reagents: j, .

1. Diallyl barbituric acid (crystalline; K & K Laboratories,

Inc. , Plainview, New York).

 

2. Monoethyl urea (Pfaltz and Bauer, Inc. , Flushing, New York).
3. Urethane (Aldrich Chemical Co. , Milwaukee, Wisconsin).
4. Disodium calcium ethylene diamine tetra acetate trihydrate

(Pfaltz and Bauer, Inc. , Flushing, New York).

Procedure:

Dissolve 10. 0 g urethane, 40.0 g monoethyl urea and
40.0 g diallyl barbituric acid in 25 ml of distilled water. Heat in
a water bath to dissolve chemicals. Cool to room temperature,
add 50.0 mg disodium calcium ethylene diamine tetra acetate tri-
hydrate and dilute q. s. 100 ml with distilled water. Store in

stoppered dark glass bottle at room temperature.

99

APPENDIX 6

LIQUID SCINTILLATION COUNTING

Reference: Instruction Manual, Mark I liquid scintillation counter
Model 6860, Nuclear Chicago Corp. , Des Plaines, Ill.
Principle:

Liquid scintillation counting is a method for detecting the
energy of the primary particle emitted by a radioactive molecule.
This energy is converted into light energy by a solution of fluors and
detected by a photomultiplier tube connected to amplifiers and a
scaler circuit. The radioactive substance is placed in close
proximity to the scintillation fluor, making the technique well
adapted for use with-low energy beta emitters such as tritium and

carbon - 14.

Counting channel selection:

The energy spectra of tritium and carbon- 14 overlap; and
when both beta emitters are present in the same sample, they are
counted simultaneously on 2 separate analyzer channels. Channel A

amplifiers are adjusted to give high efficiency of counting tritium

100

101

with minimal interference of carbon- 14; amplifiers of Channel C
were set so that counting efficiency of tritium was insignificant
while efficiency for carbon- 14 was maximal. Channel B amplifiers
were set to maximize energy pulses from an external standard of

known disintegration rate (133Ba).

Quench correction curve:

A non -fluorescent solute or solvent will absorb or quency
energy emitted from the primary particle and reduce the efficiency
of counting the radioactivity. A set of tritium and carbon- 14
standards (Nuclear Chicago Corp. , Des Plaines, Ill.) containing
known amounts of isotope (419, 000 dpm and 255, 000 dpm, respectively)
and varying degrees of quenching for each isotope over the range
used in these. experiments was used to determine:

1. tritium counting efficiency in tritium channel (A)

2. (carbon- 14 counting efficiency in tritium channel (A)

3. carbon- 14 counting efficiency in carbon- 14 channel (C).
The external standard, 133Ba, is used to determine the amount of
quenching present in each sample. The channels ratio relates the
net barium count rate in the barium channel (B) to the net barium
count rate in the tritium channel (A). Since the sample will quench

energy emitted by the gamma source, the channels ratio

channel B (cpm)
channel A (cpm)

 

B . . -
-A— = increases as the amount of quenching increases.

102

The quench correction curve (Figure 11) is a plot of the
efficiencies of tritium and carbon- 14 standards as a function of the
standards' channels ratio (B/A). Counting efficiencies of each
isotope in an unknown sample can be read directly from the quench
correction curve corresponding to the unknown sample' s channels
ratio (B/ A).

The disintegration rates of unknown samples can be calcu-
lated from the count rates of tritium and carbon- 14 and the efficiencies

of counting each isotope in channels A and C.

 

1 2 2 1
D =
H hlcz
N
Dc = 3?-
2
where: : disintegration rate (dpm) of tritium

D
D = disintegration rate (dpm) of carbon-14
N = count rate in Channel A (cpm)
N = count rate in Channel C (cpm)

h = counting efficiency for tritium in
Channel A (‘70)

c = counting efficiency for carbon-14
in Channel A (‘70)

c = counting efficiency for carbon—14
in Channel C (‘70)

103

Figure 11. --Barium- 133 external standard quench correlation curve
for differential counting of tritium and carbon- 14 samples.
Efficiencies calculated as CPM on scaler A or C/dpm of
tritium or carbon- 14 standards X 100, respectively.
Carbon- 14 quench correction curve for carbon- 14
efficiency in Channel C (squares); tritium quench correc -
tion curve for tritium efficiency in Channel A (circles);
and carbon- 14 quench correction curve for carbon- 14

efficiency in Channel A (triangles) are plotted as a

. . B _ Channel B (CPM)
functlon of Channels ratio [A — Channel A (CPM)]'

 

Discriminator settings:

Channel Window Attenuation
A 0. 0 — 2. 5 B524
B 0. 0 - 9. 9 F670

C 1.5-9.9 E738

104

Figure 1 1

5° XEFFICIENOY

 

2.0 4.0 6-0 8.0
RAT I 0 (BIA)

APPENDD§7

PROGRAM RATIO: EXTENDED FORTRAN VERSION FOR THE

CALCULATION OF T:M PAH AND MANNITOL RATIOS

 

PROGRAM RATIO"TffibUT,OUTEOTyTKBEEOQTNEUf{fAoteiEOUTbOTi
REAL MLHOM,ALAss,AANM1,HANM2,MANCP1.MAN1p2,MANH,HANcp,ncp,wHEO,MSM
REAO(60,9u0)_”m__ .__-“--m_.- _“-_.,,. .. ,_ __ ,_---”

 

900

 

FOQMATt‘ «~g,'
HRITE(61,900)
PEAOtsc,901) N

 

901

100

..—'-..._.i.-...._- _. .~.. . 7 -- .- 7 7 7- — -s- 7177..

FORMAT (12)
REA01609100) BOAHg3HANgMLH0M,MLA?S
FORMAT (uF10.2>

 

. m»-.. m ——-..—-.. ._.. .-...._.-. - -._. . . _ . 77 _. . -4- ___._—.——_.._..

KNTP = o
no 300 t=1.N
pEA0160,1n1) HT,DAHH1,PAHnngeflnglfgflCPa_

 

101
C

 

FOQHnTtiF19.5,hFTO}2)
KNTR = KNT? + 1
cowpure HFIGHY 0F TISSUE IN sgfiggsqgsgprso

 

C

102

500

OH = (HT/MLHOM) * MLASS

cowPUTF T/H QATIO 0F 3H-PAH

PAHH = ((PAHM! + PAHH2)/2) - BPAH
fiifififiFEFTTBEFEFT"?“§EEE§2)121 I fiPAH ’ "”_ ' _
PCP = pAHco/cn

Oman = DAHM/NLASS

Psn = POP/PMEO

TOTACT = pep A wt

cFAO(60,102) HANfll,MAN"2,HANUP1,HANCP?
FOQMAT (hF10.?l

coweutr T/w QATIO 0F inc-MANNITOL

MANN = (THANHi + HANH2)/2) — BHAN
MAVCP = ((HANCPl + HANCP?)/2) - RMAN
HOD = MANCO/GM

HME" = MANH/HLASS

«54 = HCP/HHFD

HQITEt61g500) KNT?

FOOMATt’U‘,15)

 

501

wsITE (61,501) uf“““““"
FOQMAT(¥ wFIOHT 0F CHOROIO PLEXUS = v,1p11,1o,v cuss)
"RITE(61,5U?’ 6‘1

 

502

505

F0?HAT(‘ L‘HFIGHT H? TISSUF'ffi“§§“9LE = '51?ii.1d}3'GHS‘)“m
HRITE(61,505) PSH
FORMATt‘ TIM RATIO PAB-EM:;1510.2)

 

508

HPITE(61,508) Hsn ""
FOQMAV(‘ T/M PATIO MANNITOL = '.1F11.2)
HRITE <61,u99) pcs g_

 

800

699

509

FORMATt‘ spffiTFfE‘AETI ITY OF PAH = ‘,1Fi5.21
H91TE(61,509) TOTACT
FORMAT(‘0 TOTAL ACTIVITY IN CHOROIO PLEXUS = ‘,1F15.2)

"COVTTWU?‘” ”

so To 1
£89

105

a——.—..__ . .m.-- a . 7 - . .. .._.-. ._-7..

BIB LIOG RAPHY

BIBLIOGRAPHY

Ames, A. , K. Higashi and F. B. Nesbett. Relation of potassium
concentration in choroid plexus fluid to that of plasma.
J. Physiol. 181:506-515, 1965.

Asghar, K. , and E. L. Way. Active removal of morphine from the
cerebral ventricles. J: Pharmacol. Exp. Ther. 175:75-
83, 1970.

Bakay, L. The Blood-Brain Barrier. C. C. Thomas Co.,
Springfield, 1956.

Becker, B. Cerebrospinalfluid iodide. Amer. J. Physiol. 201:
1149-1151, 1961.

Bekaert, J. , and G. Demeester, Influence of potassium concentra-
tion of the blood on the potassium level of the cerebrospinal
fluid. Exp. Med. Surg. _1_2:480 -501, 1954.

Bering, E. H., and O. S. Sato. Hydrocephalus: Changes in forma-
tion and absorption of cerebrospinal fluid within the cerebral
ventricles. J. Neurosurg. 29:1050-1063, 1963.

Berlin, R. D. Purines: Active transport by isolated choroid
plexus. Science 163:1194-1195, 1969.

Coben, L. Uptake of iodide by choroid plexus in vivo and location
of the iodide pump. Amer. J. Physiol. 217:89-97, 1969.

Crosby, E. C., T. Humphrey and E. W. Lauer. Correlative
Anatomy of the Nervous System. MacMillan Co. , New
York, 1962.

Cross, R. J. , and J. V. Taggart. Renal tubular transport: Accumu-

lation of p-aminohippurate by rabbit kidney slices. Amer.
J. Physiol. 161:181 —190, 1950.

106

107

Cserr, H. F. Potassium exchange between cerebrospinal fluid,
plasma and brain. Amer. J. Physiol. 209:1219—1226,
1965.

Cserr, H. F. Physiology of the choroid plexus. Physiol. Rev.
_5i:273-311, 1971.

Cserr, H. F., and D. H. Van Dyke. 5-Hydroxyindoleacetic acid
accumulation by isolated choroid plexus. Amer. J.
Physiol. 220:718-723, 1971.

Cutler, R. W. P. , R. J. Robinson and A. V. Lorenzo. Cerebro-
spinal fluid transport of sulphate in the cat. Amer. J.
Physiol. 214:448-454, 1968.

 

Davson, H. A comparative study of the aqueous humour and cerebro- E
spinal fluid in the rabbit. J. Physiol. 129:111-133, 1955.

Davson, H. , and E. Spaziani. The blood -brain barrier and the
extracellular space of brain. J. Physiol. 149:135-143,
1959.

Davson, H., C. R. Kleeman and E. Levin. Quantitative studies of
the passage of different substances out of the cerebro-
spinal fluid. J. Physiol. 161:126-142, 1962.

Davson, H. Physiology of the Cerebrospinal Fluid. J. and A.
Churchill Ltd. , London, 1967.

Dawkins, M. J. R. Biochemical aspects of developing function in
newborn mammalian liver. Brit. Med. Bull. 23:27-33,
1966.

Despopoulus, A. A definition of substrate specificity in renal
transport of organic anions. J. Theoret. Biol. 8:163-
192, 1965.

Dohrman, G. J. The choroid plexus: a historical review. Brain
Res. _1_8_:197 -218, 1970.

Fishman, R. A. Blood -brain and CSF barriers to penicillin and
related organic acids. Arch. Neurol. 2:113-124, 1966.

108

Flexner, L. B. Changes in the chemistry and nature of the
cerebrospinal fluid during fetal life in the pig. Amer.
J. Physiol. 124:131-135, 1938.

Fries, B. A. , and I. L. Chaikoff. Factors influencing recovery
of injected labeled phosphorus in various organs of the rat.
J. Biol. Chem. 141:469-478, 1941a.

Fries, B. A. , and I. L. Chaikoff. The potassium metabolism of
the brain as measured with radioactive phosphorus.
J. Biol. Chem. 141:479-485, 1941b.

Grazer, F. M., and C. D. Clemente. Developing blood brain
barrier to Trypan blue. Proc. Soc. Exper. Biol. and
Med. 245758-760, 1957.

Greep, R. O. Histology. 2nd edition, McGraw -Hill Book Co. ,
New York, 1966.

Heisey, S. R., D. Held and J. R. Pappenheimer. Bulk flow and
diffusion in the cerebrospinal fluid system of the goat.
Amer. J. Physiol. 203:775-781, 1962.

Hilton, W. A. Later embryonic development of the fourth ventricle
and lateral plexuses of man and pig. Am. Microscop. Soc.
13:213-217, 1966.

Hirsch, G. H. , and J. B. Hook. Maturation of renal organic acid
transport: Substrate stimulation by penicillin. Sci. 165:
909-910, 1969a.

Hirsch, G. H. , and J. B. Hook. Stimulation of renal PAH transport
by folic acid. Biochem. Pharmacol. 382274-2278, 1969b.

Hirsch, G. H. , and J. B. Hook. Stimulation of PAH transport by
slices of rat renal cortical following in vivo administration
of triiodothyronine. Proc. Soc. Exper. Biol. Med. 131:
513-517, 1969c. _

Hirsch, G. H. , and J. B. Hook. Stimulation of renal organic acid
transport and protein synthesis by penicillin. J. Pharmacol.
Exper. Ther. 174:152 -158, 1970.

 

109

Huang, K. C., and D. S. T. Lin. Kinetic studies on transport of
PAH and other organic acids in isolated renal tubules.
Amer. J. Physiol. 208:391—396, 1965.

Hug, C. C. Transport of narcotic analgesics by choroid plexus and
kidney tissue in vitro. Pharmacol. 163345 -359, 1967.

Kappers, J. A. Structural and functional changes in the telencephalic
choroid plexus during human ontogenesis. in: G. E. W.
Wolstenholme and C. M. O' Conner. The Cerebrospinal
Fluid. Boston, Little, Brown and Co., 1958.

Kappers, C. V. A., G. C. Huber and E. C. Crosby. The Compara-
tive Anatomy of the Nervous System of Vertebrates, Including
Man. Hafner Publishing Co. , New York, 1960.

Kaplan, H. A. , and D- H. Ford. The Brain Vascular System.
Elsevier Publishing Co. , New York, 1966.

Lorenzo, A. V., and R. W. P. Cutler. Amino acid transport by
choroid plexus invitro. J. Neurochem. _1_6_:577-585, 1969.

Millen, J. W. , and D. H. M. Woollam. The Anatomy of the
Cerebrospinal Fluid. Oxford University Press, New York,
1962.

Natelson, S. Microtechniques of Clinical Chemistry. 2nd edition.
C. C. Thomas, Springfield, 1961.

Oldendorf, W. H.,. W. B. Sisson and Y. Iisaka. Affinity of
ventricular 99mTc Pertectnetate and iodide ions for the
choroid plexus. Arch. Neurol. £:74-79, 1970.

Pappenheimer, J. R., S. R. Heisey and E. F. Jordan. Active
transport of Diodrast and phenolsulfonphthalein from cerebro-
spinal fluid to blood. Amer. J. Physiol. 200:1-10, 1961.

Pappenheimer, J. R., S. R..Heisey, E. F. Jordan and J. deC.
Downer. Perfusion of the cerebral ventricular system in
unanesthetized goats. Amer. J. Physiol. 203:763 -775,
1962.

Pilchner, C., W. F. Meachamand and E. R. Smith. Epileptogenic
effects of penicillin injected into dogs. Arch. Intern. Med.
_7_9:465-472, 1947.

 

110

Ball, D. P., W. W. Oppelt and C. S. Patlak. Extracellular space
of brain as determined by diffusion of inulin from the
ventricular system. Life Sci. 1:43 -48, 1962.

Robinson, R. J., R. W. P. Cutler, A. V. Lorenzo and C. F. Barlow.
Development of transport mechanisms for sulphate and
iodide in immature choroid plexus. J. Neurochem. _1_5_:
455-458, 1968.

de Rougement, J., A. Ames, F. B. Nesbett and H., F. Hoffman.
Fluid formed by choroid plexus. A technique for its
collection and a comparison of its electrolytic composition
with serum and cisternal fluids. J. Neurophysiol. 23:
485-495, 1960. "'

Rubin, R. , E. Owens and D. Rall. Transport of methotrexate by
the choroid plexus. Cancer Res. 33:689-694, 1968.

Smith, D. E. Morphological changes occurring in the developing
chick choroid plexus. Comp. Neuro. 127:381-388, 1966.

Smith, H. W. Principles of Renal Physiology. Oxford University
Press, New York, 1956.

Sperber, 1. Competitive inhibition andspecificity of renal tubular
transport mechanisms. Arch. Int. Pharmacodyn. _9_7_:
221-231, 1954.

Stern, L. , and R. Peyrot. Le fonctionnement de la barrier
hemato -encephalique aux devers stades de developpement
chez les diverses especes animals. C. R. Soc. Biol.
16:1124-1127, 1927.

Takemori, A. E. , and M. W. Stenwick. Studies on the uptake of
morphine by the choroid plexus. J. Pharmacol. Exper.
Ther. 154:586 ~592, 1966.

Tasaki, I. , and J. J. Chang. Electric response of glial cells in
the cat brain. Sci. 128:1209-1210, 1958.

Tochino, Y. , and L. S. Schanker. Active transport of quarternary
ammonium compounds by the choroid plexus in vitro.
Amer. J. Physiol. 208:666-673, 1965a.

111

Tochino, Y. , and L. S. Schanker. Transport of serotonin and
norepinephrine by the rabbit choroid plexus in vitro.
Biochem. Pharmacol. 1_4_:1557 -1566, 1965b.

Truex, R. C., and M. B. Carpenter. Human Neuroanatomy.
6th edition. Williams and Wilkins Co. , Baltimore, 1969.

Vernadakis, A. , and D. M. Woodbury. Electrolyte and amino acid
changes in rat brain during maturation. Amer. J. Physiol.
203:748-752, 1962.

Walker, A. E., A. C. Johnson and J. J. Kollros. Penicillin
convulsions: The convulsive effects of penicillin applied to
the cerebral cortex of monkeys and man. Surg. Gynec.
Obstet. fl:692 -701, 1945.

Welch, K. Active transport of iodide by choroid plexus of the rabbit
in vitro. Amer. J. Physiol. 202:757-760, 1962.

Welch, K. Secretion of cerebrospinal fluid by choroid plexus of
the rabbit. Amer. J. Physiol. 205:617 -624, 1963.

I'll!
“I
“2
“1
“7
”9
II“
“I
All.“

12 sulmltl‘w 5 7

ifiifitfluiiu‘u