B RA I N R E SE A R CH R E V IE W S 6 5 (2 0 1 1 ) 9 9– 11 2

available at www.sciencedirect.com

www.elsevier.com/locate/brainresrev

Review

Recent insights into a new hydrodynamics of the

cerebrospinal fluid

Marin Bulat⁎, Marijan Klarica

University of Zagreb School of Medicine, Department of Pharmacology and Croatian Institute for Brain Research, 10 000 Zagreb, Croatia

A R T I C LE I N FO AB S T R A C T

Article history: According to the traditional hypothesis, the cerebrospinal fluid (CSF) is secreted inside the
Accepted 24 August 2010 brain ventricles and flows unidirectionally along subarachnoid spaces to be absorbed into
Available online 29 September 2010 venous sinuses across arachnoid villi and/or via paraneural sheaths of nerves into
lymphatics. However, according to recent investigations, it appears that interstial fluid
Keywords: (ISF) and CSF are formed by water filtration across the walls of arterial capillaries in the
Cerebrospinal fluid central nervous system (CNS), while plasma osmolytes are sieved (retained) so that capillary
(CSF) hydrodynamics osmotic counterpressure is generated, which is instrumental in ISF/CSF water absorption into
Cerebral capillaries venous capillaries and postcapillary venules. This hypothesis is supported by experiments
Transcapillary fluid showing that water, which constitutes 99% of CSF and ISF bulk, does not flow along CSF spaces
filtration-reabsorption since it is rapidly absorbed into adjacent CNS microvessels, while distribution of other
Capillary osmotic counterpressure substances along CSF spaces depends on the rate of their removal into microvessels: faster
CSF-interstial fluid mixing removal means more limited distribution. Furthermore, the acute occlusion of aqueduct of
Elimination–distribution rate Sylvius does not change CSF pressure in isolated ventricles, suggesting that the formation and
the absorption of CSF are in balance. Multidirectional distribution of substances inside CSF, as
well as between CSF and ISF, is caused by to-and-fro pulsations of these fluids and their mixing.
Absorption of CSF into venous sinuses and/or lymphatics under the physiological pressure
should be of minor importance due to their minute surface area in comparison to the huge
absorptive surface area of microvessels.
© 2010 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
2. Fate of 3H-water in CSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
3. Residence time and distribution of organic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4. Mixing of CSF and ISF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

⁎ Corresponding author. University of Zagreb School of Medicine, Department of Pharmacology, Šalata 11, 10 000 Zagreb, Croatia. Fax: + 385
1 4596 932.
E-mail address: mklarica@mef.hr (M. Bulat).
Abbreviations: AQP, aquaporin; BP, benzylpenicillin; CB, cisterna basalis; CCC, cisterna corporis callosi; CM, cisterna magna; CNS, central
nervous system; CSF, cerebrospinal fluid; CSS, cortical subarachnoid space; 5-HIAA, 5-hydroxyindoleacetic acid; HPc, capillary hydrostatic
pressure; HPi, interstial hydrostatic pressure; HRP, horseradish peroxidase; ISF, interstial fluid; LSS, lumbar subarachnoid space; LV, lateral
ventricle; OcPc, capillary osmotic counterpressure; OPc, capillary osmotic pressure; RISA, radioiodinated serum albumin; SEM, standard
error of mean; SSS, superior sagittal sinus

0165-0173/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainresrev.2010.08.002
100 B RA I N R ES E A RC H R E V IE W S 6 5 (2 0 1 1 ) 9 9– 11 2

5. Fate of inulin in CSF and CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

6. Fate of proteins in CNS and CSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
7. Intraventricular balance of CSF formation and absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
8. Hydrodynamics of ISF and CSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
8.1. Capillary osmotic counterpressure as regulator of ISF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
8.2. Functional unity of ISF and CSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
9. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

99% of CSF and ISF volume is water, so labeled water is to be used

1. Introduction for investigations of the fate of CSF bulk (Bulat, 1993). When the
distribution of 3H-water and 3H-inulin in CSF were studied in
The cerebrospinal fluid (CSF) is located inside the brain
cats, they showed different fates under the same conditions: 3H-
ventricles and cranial and spinal subarachnoid space. According
water was rapidly absorbed from CSF into adjacent CNS
to the traditional hypothesis, CSF is formed in brain ventricles,
capillaries so its distribution along CSF spaces was very limited.
mostly by secretion from choroid plexuses, and circulates along
On the other hand, 3H-inulin poorly passes across capillary walls
ventricles and the subarachnoid space to be absorbed across
and so is distributed over time along all CSF compartments by
arachnoid villi into dural venous sinuses and/or via the
pulsations and mixing of CSF (Fig. 1). Furthermore, organic acids
paraneural sheaths of cranial and spinal nerves into lymphatics.
such as 5-hydroxyindoleacetic acid (metabolite of serotonin)
Since there occurs an exchange of substances between extra-
and penicillin show relatively limited distribution along CSF
cellular interstial fluid (ISF) of the central nervous system (CNS)
because they are removed into capillaries by powerful active
and CSF, it is assumed that CSF serves as a sink for the removal
transport (Fig. 1) but, when active transport was blocked, their
of various metabolites out of CNS by its pulsatile flow and
absorption (Bradbury and Westorp, 1984; Brodbelt and Stoodley,
2007; Davson 1984; Johanson et al., 2008; Koh et al., 2005).
However, the main postulates of this traditional hypothesis of
CSF and its corollaries were recently questioned, and the main
role in CSF formation and absorption was ascribed to CNS
microvessels (Bulat et al., 2008; Klarica et al., 2009; Vladić et al.,
2009). Furthermore, a critical analysis of the pathophysiology of
hydrocephalus has recently been published (Greitz, 2004; Greitz,
2007; Klarica et al., 2009; Orešković and Klarica, 2010).
It is the intention of this article to discuss some recent
investigations concerning the distribution of substances along
CSF spaces and the regulation of the CSF bulk (water). Three
processes can cause the distribution of substances throughout
various compartments of the body fluid: bulk flow, mixing and
diffusion (Riggs, 1972). Bulk flow (circulation, convection,
volume flow) of the fluid means that the fluid flows unidirec-
tionally, carrying all dissolved substances with the same speed.
Mixing of the fluid by pressure waves disperses the substances Fig. 1 – Scheme of CSF dynamics. CSF compartments in brain
in all directions. Diffusion of the substances in the fluid is a slow ventricles and subarachnoid space with surrounding CNS
process which is efficient only along very short distances. Riggs parenchyma containing blood capillaries and capillaries of
(1972) calculated that, for the diffusion of nitrogen in water at choroid plexuses are shown. Water (bidirectional black
20 °C along distances 0.001, 0.01, 0.1 and 1.0 cm, the time arrows) exchanges rapidly between CSF and capillaries, has a
required to reach 95% equilibrium is 6.3 s, 630 s (11 min), 63,000 s short half-life in CSF and very limited distribution along CSF
(18 hrs) and 6,300,000 s (73 days), respectively; thus, a tenfold spaces (1). On the other hand, substances with very restricted
increase in the diffusion distance means that the diffusion passage from CSF to capillaries such as 3H-inulin
process will require 100 times longer to reach a given degree of (bidirectional white arrows) have long half-lives in CSF and
completion. Therefore, since distances between CSF compart- unlimited bidirectional distribution along CSF spaces (3). An
ments in humans and large animals (e.g. cats and dogs) are long, intermediate group of substances such as organic acids
the diffusion of substances between them is not a relevant (bidirectional gray arrows) with lower passage rate from CSF
process (Bulat et al., 2008; Vladić et al., 2009). into capillaries than water, and intermediate half-lives in CSF
For the study of bulk flow (circulation) and absorption of CSF, shows limited distribution along CSF space (2). Bidirectional
the labeled macromolecules (e.g. proteins, dextrans, inulin) are distribution of substances along CSF spaces is assumed to be
generally used as markers of CSF bulk. However, it is question- due to CSF to-and-fro pulsations. Modified according to Bulat,
able whether macromolecules can show the fate of CSF since 1993 with permission.
 B RA I N R E SE A R CH R E V IE W S 6 5 (2 0 1 1 ) 9 9– 11 2 101

distribution was similar to that of 3H-inulin (Bulat, 1993). In
addition, it has been suggested that CSF bulk (water) is regulated
by hydrostatic and osmotic pressures operating across the
blood–brain barrier (Bulat, 1993).
2. Fate of 3H-water in CSF
If water is absorbed from brain ventricles, after 3H-water
infusion into a lateral ventricle, it should enter the periven-
tricular capillaries and, via the great vena of Galen, reach the
confluence of sinuses (torcular Herophili). Fig. 2 shows that,
during the slow infusion (1.77 μl/min) of 3H-water in physiolog-
ical saline into a lateral ventricle of cats under normal CSF
pressure, its concentrations in the CSF of the cisterna magna
and arterial plasma were equal, while its concentration in the
venous plasma of the confluence of sinuses was several times
higher. The equal concentrations of 3H-water in cisternal CSF
and arterial plasma are explained by the rapid passage of 3H-
water from arterial blood into CNS and CSF: these concentra-
tions reach equilibrium within 5 min in cats (Bulat et al., 2008).
Furthermore, it was shown, in dogs, that deuterium water (D2O)
after intravenous injection reaches the half-time of equilibrium
between the arterial plasma and different parts of the brain in
about 20 s and cisternal CSF in 3 min (Bering, 1952). The delay in
the equilibrium of D2O between brain parenchyma and CSF is
explained by the larger distance of CSF than ISF from cerebral
capillaries.
When infused into cortical CSF, 3H-water concentration in
confluence plasma was several times higher than in arterial
plasma and cisternal CSF, which had equal concentrations
(Fig. 3). A similar phenomenon is observed during 3H-water
infusion in a lateral ventricle (Fig. 2), indicating that 3H-water
passes from cortical subarachnoid space across pia mater and
is absorbed into cerebral microvessels, which drain into
adjacent superior sagittal sinus to reach the confluence
(Bulat et al., 2008). Five days after 3H-water in physiological
saline (1 μl/h) was infused by mini pump into a lateral
ventricle of free-moving cats, its concentrations in the
cisterna magna, cortical, thoracic and lumbar CSF, arterial
plasma and urine were equal, indicating that 3H-water was
absorbed into periventricular capillaries and distributed from
the systemic blood stream to all the body's water compart-
ments (Bulat et al., 2008).
As argued below, the absorption and filtration of water
across the walls of CNS microvessels are simultaneous
processes that maintain constant volumes of ISF and CSF.
The turnover of ISF/CSF water volume seems to be a rapid
process. It was shown during ventriculocisternal perfusion in
dogs that 3H-water passes across ependyma into caudate
nucleus in only a few mm, being rapidly eliminated into
capillaries with the half-time of 1.5 min (Fenstermacher and

Cisternal CSF Confluence plasma Arterial plasma

1200 A 1200 B
1000 1000

800 800

600 600

400 400

200 200
- water (cpm / 50 µl)

0 0
0 1 2 3 0 1 2 3

5000 5000
C D
4000 4000
3H

3000 3000

2000 2000

1000 1000

0 0
0 1 2 3 0 1 2 3
Time (hours)

Fig. 2 – Concentration of 3H-water in the cisternal CSF, confluence and arterial plasma (cpm/50 μl) during infusion (1.77 μl/min)
of 3H-water in saline into lateral ventricle in cats at lower (0.025 mCi/ml, A and B) and five times higher (0.125 mCi/ml, C and D)
concentration of 3H-water. Reproduced from Bulat et al., 2008 with permission.
102 B RA I N R ES E A RC H R E V IE W S 6 5 (2 0 1 1 ) 9 9– 11 2
3500 Confluence plasma
Arterial plasma
3000
Cisternal CSF
2500
- water (cpm / 50 µl)
2000
1500
1000
500
200
150
3H
100
50
0 probenecid pretreated dogs, on the other hand, 3H-BP distri-
0 1 2 3
Time (hours)
Fig. 3 – Concentration of 3H-water in the cisternal CSF,
confluence and arterial plasma (cpm/50 μl) during infusion
(1.77 μl/min) of 3H-water in saline (0.125 mCi/ml) into cortical
subarachnoid space of cats. Results are shown as mean and
SEM (n = 4). Reproduced from Bulat et al., 2008 with
permission.
Kaye, 1988). The rapid absorption of 3H-water is also supported
by the observation that, during slow infusion in the lateral
ventricles of cats, 3H-water concentrations in cisternal CSF
and arterial plasma were equal even when cisternal CSF was
drained under negative (subatmospheric) pressure (Bulat,
1993). Aquaporins (AQP) could mediate rapid water absorp-
tions from CSF and ISF. AQP1 is expressed in the apical
membrane of the choroid plexus epithelium and ependymal
cells, while AQP4 is expressed in astrocytes and ependymal
cells. Furthermore, AQP9 is expressed in astrocytes and in cells
bordering the subarachnoid space and ventricles, including
ependymal cells (Gunnarson et al., 2004). It is not clear,
however, whether the presence of aquaporins in the brain is a
“luxury or necessity” (van Os et al., 2000).
3. Residence time and distribution of organic
acids
That the rate of substance elimination across CNS capillary walls
determines their distribution along CSF spaces is best demon-
strated by the fate of organic acids such as 5-hydroxyindoleacetic
acid (5-HIAA, the main metabolite of serotonin), benzylpenicillin
(BP) and phenolsulphonphtalein (PSP), which are eliminated by
active transport into capillaries unless this transport is compet-
itively inhibited by probenecid. When the active transport of 5-
HIAA is blocked by probenecid, 5-HIAA rises linearly over time in
both CNS and CSF (Bulat, 1974; Bulat and Živković, 1978a). When
PSP is injected into CSF, it passes into CNS parenchyma and from
there is rapidly eliminated into capillaries under control condi-
tions, but in animals pretreated with probenecid, the residence
time of PSP in CNS parenchyma increases greatly since, under
such conditions, PSP can be eliminated across capillary walls
only by a slow diffusional process (Zmajević et al., 2002). These
results show that organic acids pass freely between CNS and CSF
and vice versa and that active transport across capillary walls
acts as a “sink” in their elimination from CNS and CSF.
A study of the distribution of organic acids along CSF spaces
in cats showed that 5-HIAA does not reach lumbar CSF from
cisternal CSF, probably due to its active removal into CNS
capillaries (Bulat, 1977; Bulat and Živković, 1971; Bulat et al.,
1974; Bulat and Živković, 1978a, b). To test this assumption, 3H-
benzylpenicillin (3H-BP) was injected into cisternal CSF in
control and probenecid pretreated dogs under short anesthe-
sia, and its distribution to various CSF compartments was
followed over time in free-moving animals (Vladić et al., 2000).
In the control animals, 3H-BP was distributed from cisternal
CSF to lumbar, ventricular and cortical CSF only in traces; in the
bution greatly increased and, after 5 hours, its concentrations
in cisternal, cortical and lumbar CSF almost equalized (Vladić
et al., 2000). In the control dogs, the disappearance of 3H-BP
from cisternal CSF was a rapid exponential process which can
be fitted on a semi-log scale with a half-time of 30 min (Fig. 4A);
in the probenecid pretreated animals, on the other hand, the
disappearance of 3H-BP from cisternal CSF was retarded and
can be fitted on a log-log scale (Fig. 4B) indicating a slow
multiexponential process (Vladić et al., 2000). Thus, in control
animals, 3H-BP passes from cisternal CSF into CNS parenchy-
ma, where it is eliminated into capillaries by powerful active
transport with the result that its concentration in cisternal CSF
falls rapidly and it cannot be distributed significantly to remote
CSF compartments. When active transport is blocked by
probenecid, however, 3H-BP is eliminated into capillaries by a
slow diffusional process so that its concentration in cisternal
CSF and adjacent CNS parenchyma is maintained at a high
level and, over time, the 3H-BP is efficiently distributed by CSF
mixing to remote CSF compartments (Strikić et al., 1994; Vladić
et al., 2000). It appears, therefore, that the rate of removal of
substances into CNS capillaries correlates with their distribu-
tion along CSF spaces: a faster removal rate into capillaries
results in a poorer distribution along CSF spaces. As discussed
above, the CSF water shows limited distribution along CSF
spaces because it is rapidly absorbed into CNS capillaries.
4. Mixing of CSF and ISF
CNS capillaries form the blood–brain barrier (BBB) and are
characterized by endothelial cells with intracellular junctions
that completely encircle each endothelial cell. As discussed
above, the substances which pass poorly across capillary walls
have a long resistance time in CSF and CNS and can be
efficiently distributed over time along CSF spaces and into
CNS by fluid pulsation and mixing. The mixing of CSF is due to
respiratory and systolic-diastolic changes of CSF pressure with a
to-and-fro displacement of CSF volume. Furthermore, the
distribution of substances between CSF compartments greatly
increases in free-moving dogs in comparison to anesthetized
animals, indicating that physical activity is an important factor
in the displacement and mixing of CSF (Vladić and Bulat, 1988).
The dislocation and mixing of CSF seems to be brought about by
the distensibility of the spinal dura (Martins et al., 1972) and the
compressibility and emptying of the veins in spinal and cranial
cavities (Foltz, 1984). NM imaging shows that, during systolic
vessels expansion, an almost simultaneous craniocaudal CSF
displacement occurs in the aqueduct of Sylvius, the basal cistern
 B RA I N R E SE A R CH R E V IE W S 6 5 (2 0 1 1 ) 9 9– 11 2 103

A B
in CM (dpm / 50 µl)
3H-BP

Time (min) Time (min)

Fig. 4 – Concentration of 3H-BP in cisterna magna (CM) of control (A) and probenecid pretreated (B) dogs after its application in
CM. A, in control dogs concentrations of 3H-BP (dpm/50 ml) over time (min) are shown on semi-log scale. B, in probenecid
pretreated dogs concentrations of 3H-BP (dpm/50 ml) over time (min) are shown on log-log scale. Lines are drawn by hand.
Reproduced from Vladić et al., 2000 with permission.

and the cervical subarachnoid space, while during diastole, the
caudocranial dislocation of CSF appears due to a decrease in
brain blood volume and the recoil of displaced CSF in the spinal
region (Alperin et al., 2005; Enzmann and Pelc, 1991; Feinberg
and Mark, 1987; Greitz et al., 1993).
Simultaneously with CSF pulsation, a similar process occurs
along CNS blood vessels. It was shown by Rennels et al. (1985)
that, after horseradish peroxidase (HRP; m.w. 40,000) was
infused into a lateral ventricle or cisterna magna of cats and
dogs for 4–10 min under normal CSF pressure, it was distributed
in the perivascular (Virchow-Robin) spaces of the whole brain
parenchyma so that all the blood vessels, including capillaries,
were outlined. This rapid perivascular distribution of HRP in the
brain can be prevented by stopping or diminishing the pulsa-
tions of cerebral arteries by aortic occlusion or by partial ligation
of the brachiocephalic artery (Rennels et al., 1985). Thus, the
pulse pressure in CSF and the parenchyma (Di Rocco, 1984)
should be instrumental in the rapid perivascular distribution of
substances from CSF but not in a slow diffusional process
(Rennels et al., 1985). Furthermore, when HRP and some other
substances are infused into the brain parenchyma, they are
distributed along the perivascular spaces (Cserr, 1984; Hadaczek
et al., 2006) and pass into CSF (Cserr, 1984).
Due to the mixing of CSF, the substances in CSF are
distributed multidirectionally along CSF spaces both from
brain ventricles to the subarachnoid space and in the opposite
direction. For example, it was shown, in dogs, that 3H-BP (Vladić
et al., 2000) and 3H-inulin (Vladić et al., 2009) are distributed from
CM to the lateral brain ventricles. Furthermore, it was observed
in patients that tumor cells (Zülch, 1958) and contrast media
(Hindmarsh, 1977) are distributed from the subarachnoid space
into brain ventricles. After lumbar injection in patients,
methotrexate was distributed to the lateral ventricles and, in
most cases, it reached assumed therapeutic concentration
(Shapiro et al., 1975). Since the dynamics of inulin distribution
among various CSF compartments has been studied in detail,
we will summarize the main findings in order to estimate the
intensity of CSF mixing among its compartments.
5. Fate of inulin in CSF and CNS
Since inulin is a macromolecule (m.w. 5,500) which passes
poorly across the walls of CNS capillaries (Crone, 1963; Renkin
and Crone, 1996), it is expected that its passage in the opposite
direction is also a slow process. After injection into the CSF of
monkeys, 14 C-inulin and methotrexate are distributed
throughout the brain parenchyma, and their concentrations
tend to equilibrate in these two compartments over time
(Kimelberg et al., 1978).
When 3H-inulin was slowly infused into a lateral ventricle of
cats, it reached high concentration in cisternal CSF, while its
concentrations in confluence and arterial plasma were very low
and close to background activity (Fig. 5). These results can be
compared to the fate of 3H-water (Fig. 2) since they were obtained
under the same experimental conditions in cats (Bulat et al., 2008).
While 3H-water is efficiently absorbed in periventricular capillar-
ies and reaches a high concentration in the confluence plasma
where these capillaries are drained (Fig. 2), 3H-inulin is poorly
absorbed in periventricular capillaries so its concentration in
confluence plasma is very low (Fig. 5). Since 3H-inulin is poorly
absorbed in periventricular capillaries, its residence time in CSF is
long (Vladić et al., 2009), so it can be distributed by CSF mixing to
all CSF compartments over time.
104 B RA I N R ES E A RC H R E V IE W S 6 5 (2 0 1 1 ) 9 9– 11 2

Cisternal CSF Confluence plasma Arterial plasma

700 A 7000 B
600 6000

500 5000

400 4000

300 3000

200 2000
- inulin (cpm / 50µl)

100 1000

0 0

0 1 2 3 0 1 2 3

2500 2500
C D
3H

2000 2000

1500 1500

1000 1000

500 500

0 0

0 1 2 3 0 1 2 3
Time(hours)

Fig. 5 – Concentration of 3H-inulin in the cisternal CSF, confluence and arterial plasma (cpm/50 μl) during infusion (1.77 μl/min)
of 3H-inulin in saline into lateral ventricle of cats. Radioactivities infused were 0.0013 mCi/ml (A), 0.018 mCi/ml (B), and
0.004 mCi/ml (C and D), respectively. Reproduced from Bulat et al., 2008 with permission.

To study the dynamics of distribution in the CSF system, 3H- When 3H-inulin was injected into LV of dogs, it was
inulin was injected into the cisterna magna, a lateral ventricle or distributed to the contralateral LV, CM and CB, and thereafter
the cisterna corporis callosi of dogs under short anesthesia, and to all other CSF compartments in a similar way as after its
its distribution to various CSF compartments was determined in
free-moving animals over time (Vladić et al., 2009). Fig. 6 shows
the distribution of 3H-inulin in the CSF system during 24 h after 30000
CM
its injection into the cisterna magna (CM) of dogs. An efficient CB
distribution of 3H-inulin from the CM to the cisterna basalis (CB), 25000 LV
H - inulin (dpm/50µl)

CSS
lumbar (LSS) and cortical (CSS) subarachnoid spaces is observed, LSS
while its distribution to the lateral ventricles (LV) is smaller. 20000
From the 7th to the 24th hour, the concentration of 3H-inulin in
LSS is above all other CSF compartments despite the long 15000
distance (about 70 cm) between CM and LSS (Vladić et al., 2009),
which can be explained by the relatively straight spinal 10000
3

subarachnoid space where systolic-diastolic pulsations and

5000
the mixing of CSF are prominent (Alperin et al., 2005; Enzmann
and Pelc, 1991; Feinberg and Mark, 1987; Martins et al., 1972). On
0
the other hand, the distribution of 3H-inulin to LV is limited by 0 5 10 15 20 25
the small cross-sectional area of communication and the high Time(h)
tortuosity of the CSF spaces, which include the foramen of
Magendie and foramina of Luschka, the fourth ventricle, the Fig. 6 – Concentrations of 3H-inulin in CSF (dpm/50 μl) of
aqueduct of Sylvius, the third ventricle and the interventricular cisterna magna (CM), cisterna basalis (CB), lateral ventricle (LV),
foramina (Monro). This, together with the simultaneous passage cortical subarachnoid space (CSS) and lumbar subarachnoid
of 3H-inulin into adjacent parenchyma and slow removal into space (LSS) at different time intervals (h) after 3H-inulin
capillaries, may postpone and limit the distribution of 3H-inulin injection into CM of dogs. Results are shown as mean and SEM
to LV (Vladić et al., 2009). (n = 4). Reproduced from Vladić et al., 2009 with permission.
 B RA I N R E SE A R CH R E V IE W S 6 5 (2 0 1 1 ) 9 9– 11 2
injection into CM. The half-times of 3H-inulin in LV and CM
were 3 h (Vladić et al., 2009). When 3H-inulin was injected into
the cisterna corporis callosi (CCC), which extends along
superior sagittal sinus, its concentrations over 7 h remained
high in CCC and adjacent CSS but were much lower in CM and
CB, and very low in LV and LSS (Vladić et al., 2009). It appears
that the pulsations of large arteries at the base of the brain
(internal carotid arteries and basilar artery) produce CSF
currents toward the top of the brain and postpone the
distribution of 3H-inulin in the opposite direction, i.e. from
CCC toward CM and CB. This assumption can also explain the
efficient distribution of 3H-inulin from CM toward peripherally
located LSS and CSS (Fig. 6) as well as the observation that,
after the lumbar injection of radionuclide in humans, it
remained mostly in the spinal subarachnoid space where it
was slowly absorbed (Edsbagge et al., 2004).
When 3H-inulin was injected into CM (Fig. 6) or CCC of
dogs, its concentrations in venous plasma of the superior
sagittal sinus (SSS) and arterial plasma were low and not
significantly different, while its concentration in urine was
very high (Vladić et al., 2009). Thus, no preferential absorption
of 3H-inulin across the arachnoid villi into SSS was detected.
It is assumed that the low concentration in plasma is
maintained due to several simultaneous processes: slow
absorption of 3H-inulin across capillary walls (see above), its
distribution to peripheral organs and its rapid elimination
into urine, where it reaches very high concentrations (Vladić
et al., 2009).
6. Fate of proteins in CNS and CSF
CSF contains about 250 times fewer proteins than plasma
(Davson, 1984). Some proteins such as horseradish peroxidase
(HRP) and radioiodinated serum albumin (RISA) are often used
to study the fate of proteins after their infusion into CSF. As
already mentioned, 4–10 min after the infusion of HRP under
normal CSF pressure into the LV or CM of dogs and cats, all
blood vessels, including capillaries in the brain, are delineated
by this tracer located in perivascular (Virchow-Robin) spaces
and, over time, in surrounding parenchyma (Rennels et al.,
1985). HRP is removed by micropinocytosis across endothelial
cells into cerebral microvessels and into glia, where it is
degradated by lysosomes (Wagner et al., 1974). In addition,
HRP is rapidly taken up from brain ventricles into the
epithelium of the choroid plexus, where it is partly degradated
by lysosomal apparatus and partly transported by micropino-
cytosis in choroidal microvessels and distributed to some
peripheral organs such as the kidney (van Deurs, 1976; van
Deurs, 1977; van Deurs et al., 1978). Since HRP is rapidly
distributed from CSF into the whole brain parenchyma, where
it is degradated and transported into microvessels which have
a large surface area, it appears that an efficient mechanism for
the removal of proteins from CSF and CNS is available. It is
estimated that the capillaries have a surface area of about
240 cm2/g of brain tissue (Crone, 1963). Taking into account
that the brain weighs 26 g in cats, 88 g in dogs and 1,500 g in
humans, the calculated surface area of cerebral capillaries
would be 6240 cm2 (Bulat et al., 2008), 21,120 cm2 (Vladić et al.,
2009), and 363,000 cm2, respectively.
105
The traditional hypothesis of CSF claims that proteins and
CSF volume are absorbed from the subarachnoid space across
arachnoid villi into the dural venous sinuses, and via the
paraneural sheaths of cranial and spinal nerves into lympha-
tics (see above). We believe that these are accessory pathways
for CSF absorption which may become significant when
substances are infused into CSF under high experimental
pressure. When infused into CSF under normal pressure, HRP
remains in the subarachnoid space and does not enter the
dura due to the layers of arachnoid cells with tight intercel-
lular junctions in close proximity to the dura (Butler, 1984;
Nebeshima et al, 1975). However, when the infusion of HRP is
performed under a CSF pressure exceeding 20 cm H2O, the
arachnoid barrier is damaged and HRP enters the highly
vascularized dura, from where it is removed into vessels by
micropinocytosis (Butler, 1984). Arachnoid villi are covered
with endothelial cells of venous sinuses which are connected
by tight intercellular junctions (Shabo and Maxwell, 1968).
When HRP is infused at resting CSF pressure, it is found in
varying amounts within micropinocytic vesicles opening into
either the subendothelial space or the lumen of venous
sinuses. However, during the infusion of HRP at CSF pressure
exceeding 20 to 25 cm H2O, enlarged vesicles form transcel-
lular channels that function as a pressure-sensitive pathway
for CSF removal and decrease the resistance to fluid infusion
(Butler, 1984). Thus, it appears that high CSF pressure opens
some CSF absorption pathways that are not available at
physiological pressure.
It has been shown in rabbits ands cats that, when RISA was
infused under high CSF pressure into CSF, its concentration
greatly increased in episcleral tissue, optic nerves, olfactory
bulbs and deep cervical lymph nodes, but when it was infused
at normal CSF pressure, only traces of RISA were detected in
these structures (McComb et al., 1982, 1984). Many experi-
ments have been performed with several animal species to
investigate the passage of RISA and some other substances
along the perineural sheaths of olfactory nerves into cervical
lymphatics across the cribriform plate, suggesting that these
substances are removed over time into cervical lymphatics
(Bradbury and Westorp, 1984; Koh et al., 2005). Since the
surface area of arachnoid villi and the space between the
perineural sheaths of cranial and spinal nerves is minute in
comparison to the huge surface area of CNS capillaries where
the absorption of CSF bulk (water) and dissolved substances
takes place (Bulat et al., 2008; Vladić et al., 2009), arachnoid villi
and perineural sheaths of nerves can be regarded as accessory
pathways for CSF absorption that may become significant at
high CSF pressure.
7. Intraventricular balance of CSF formation
and absorption
Due to the high permeability of cerebral capillaries to water
and their very low permeability to plasma osmolytes, it was
proposed that plasma osmolytes are seived (retained) during
water filtration in arterial capillaries under hydrostatic
pressure, causing the plasma osmotic pressure along the
capillaries to increase, and, when it reaches the level of
hydrostatic pressure, to halt water filtration. When this
106 B RA I N R ES E A RC H R E V IE W S 6 5 (2 0 1 1 ) 9 9– 11 2

hypertonic plasma is delivered to venous capillaries and A

24
postcapillary venules, where hydrostatic pressure is low, the

CSF pressure (cmH2O)

osmotic reabsorption of water from ISF occurs, causing the Infusion in lateral ventricle-7µL/min
20
hypertonic plasma to be diluted and finally normalized (Bulat
and Klarica, 2005). During the infusion in LV, 3H-water is 16
reabsorbed into periventricular capillaries and is not delivered
to the cisterna magna (Fig. 2), which indicates that CSF bulk 12

(water) is absorbed from brain ventricles (Bulat et al., 2008).

8
The question arises whether the formation and absorption of BV
CM
CSF in ventricles are in balance. 4
When the aqueduct of Sylvius was occluded in cats, CSF 0 5 10 15 20 25 30 35
pressure in the lateral ventricles and cisterna magna did not Time(min)
differ during 120 min of the experiment, i.e. no transmantle
pressure was developed (Fig. 7A; Klarica et al., 2009). A similar B Infusion in lateral ventricle-13µL/min
24
phenomenon was observed in control cats without aqueductal

CSF pressure (cmH2O)

*
obstruction (Fig. 7B; Klarica et al., 2009). Furthermore, no 20
*
outflow of CSF was observed from cannulated isolated *
16 *
ventricles at physiological CSF pressure (Orešković et al.,
*
2001). This evidence suggests that CSF formation and absorp-
12
tion are in balance in ventricles.
To simulate CSF formation, artificial CSF was infused into 8
BV
isolated ventricles at the rate of 7 μl/min (Fig. 8A) and 13 μl/min CM
(Fig. 8B) over 20 min. At 7 μl/min infusion, statistically significant 4
0 5 10 15 20 25 30 35
transmantle pressure was not developed, while at 13 μl/min
Time(min)
infusion, a significant transmantle pressure was recorded. After
the infusion of artificial CSF was ended, CSF pressures returned
Fig. 8 – Cerebrospinal fluid (CSF) pressure (cm H2O) in cats in
toward control values and transmantle pressure disappeared,
an isolated brain ventricle (BV; black symbols) and in the
indicating that the absorption of infused fluid took place in
cisterna magna (CM; open symbols) during an infusion of the
isolated ventricles (Klarica et al., 2009). In a separate group of cats,
mock CSF (the arrows show start and the end of an infusion)
ventriculography was performed before and two hours after
into the lateral ventricle and thereafter. (A) The rate of the
aqueductal occlusion, but no dilatation of isolated ventricles was
infusion was 7.0 μL/min (n = 5). (B) The rate of the infusion
detected (Klarica et al. 2009). According to the data obtained by
was 13.0 μL/min (n = 5). The values are mean ± S.E.M. *p < 0.05.
Reproduced from Klarica et al, 2009 with permission.
A
CSF pressure (cmH2O)

14
12
10
the ventriculocisternal perfusion method (see below), net CSF
8
formation in ventricles ranges from 15 to 25 μl/min in cats
6
4
(Pollay, 1974). If this is so, there should develop a gradient of CSF
BV
2 CM
pressure between isolated ventricles and CM, but this was not
0 observed (Fig. 7A), indicating that something is wrong with the
0 20 40 60 80 100 120 ventriculocisternal perfusion method (see below). It is suggested
Time (min)
that prolonged occlusion or stenosis of the aqueduct should lead
B 14
to the dilatation of isolated ventricles (hydrocephalus), but it is
CSF pressure (cmH2O)

12 not clear whether transmantle pressure is necessary for such a

10 process (Klarica et al., 2009). Indeed, in patients with communi-
8 cating and noncommunicating hydrocephalus, a transmantle
6 pressure gradient was absent (Stephensen et al., 2002). Further-
4 more, Holtzer and de Lange (1973) observed that hydrocephalus
BV
2 CM did not progress after shunt obstruction in children with
0 communicating and noncommunicating hydrocephalus, indi-
0 20 40 60 80 100 120
Time (min) cating that this pathophysiological process was compensated.
Thus, it appears that CSF formation and absorption in brain
Fig. 7 – Cerebrospinal fluid (CSF) pressure (cm H2O) in the ventricles are in balance both in animals and in patients.
brain ventricles (BV; black symbols) and the cisterna magna The traditionally accepted hypothesis of net CSF formation
(CM; open symbols) in cats with occluded aqueduct (A, n = 5) in brain ventricles is supported by the development of the
and those without such an occlusion (B, n = 5) during a period widely used ventriculocisternal perfusion method for mea-
of two hours. The values are mean ± S.E.M. Reproduced from surement of net ventricular formation of CSF (Heisey et al.,
Klarica et al., 2009 with permission. 1962). In this method, one of the lateral ventricles and the
 B RA I N R E SE A R CH R E V IE W S 6 5 (2 0 1 1 ) 9 9– 11 2
cisterna magna are cannulated, and the ventricles are
perfused with perfusate containing a macromolecular marker
(inulin, albumin or dextran); it is assumed that the marker is
not lost from the perfusate, and its dilution in the perfusate
flowing from the cisternal cannula is taken as the measure of
net CSF formation in ventricles. However, it is known that
markers pass from perfusate into brain parenchyma (Kimel-
berg et al., 1978; Rennels et al., 1985) and into a nonperfused
contralateral ventricle (Strikić et al., 1994; Vladić et al., 2009),
and that it can also be distributed from the cisternal region to
cervical and cranial CSF. All these losses of the marker from
perfusate should give a false calculation of net CSF formation
in ventricles. Furthermore, at lower rates of ventriculocister-
nal perfusion, higher rates of CSF formation are calculated
than at higher perfusion rates (Orešković et al., 2002),
indicating an inherent defect in the method. It appears that
the ventriculocisternal perfusion method should be used with
caution and that it cannot be accepted as a valid method for
determining net CSF formation (Bulat et al., 2008; Orešković
and Klarica, 2010).
Brain ventricles are covered by loosely connected ependy-
mal cells and choroid epithelium cells with tight intercellular
junctions. It was estimated that the surface area of the
ependyma is four times larger than that of the choroid plexus
in the third and lateral ventricles of dogs (Bering and Sato
1963), so the communication of ventricular CSF with sub-
ependymal microvessels may be easier than with choroid
plexus capillaries which are covered by choroidal epithelium
cells connected by tight intercellular junctions. However,
choroid capillaries are mostly fenestrated, so water and
electrolytes can pass easily between them and the choroid
interstitium, and their transcellular passage across epithelial
cells into CSF is controlled (Brown et al., 2004; Johanson et al.,
2008). Various transporters for electrolytes have been demon-
strated at the basolateral membrane of epithelial cells.
Furthermore, several transporters and channels for electro-
lytes have been detected at the apical membrane of epithelial
cells (Brown et al., 2004; Johanson et al., 2008). These processes
have been interpreted to mediate the net transport of Na+, Cl−
and HCO−3 into CSF, which is followed by the osmotic flow of
water, i.e. secretion of CSF (Brown et al., 2004; Johanson et al.,
2008). It appears, however, that these models of CSF secretion
have not been subjected to rigorous experimental testing
(Brown et al., 2004). In the light of the evidence presented
above, the direction of the transport processes of electrolytes
in choroid epithelium cells has to be reconsidered, since
choroid epithelium is a two-way street. It should be men-
tioned that the surgical removal of choroid plexuses from both
lateral ventricles in monkeys (Milhorat, 1969) and in a patient
(Milhorat et al., 1976) did not influence the concentration of
electrolytes in CSF.
8. Hydrodynamics of ISF and CSF
To explain the formation and reabsorption of extracellular
interstial fluid (ISF) and CSF, the role of microvessels (arterial
and venous capillaries and postcapillary venules) in these
processes should be considered. Two different hypotheses are
proposed to explain the control of fluid filtration and
107
reabsorption across microvascular walls: Starling's oncotic
hypothesis (Landis and Pappenheimer, 1963; Michel, 2000;
Renkin and Crone, 1996) and the osmotic counterpressure
hypothesis (Bulat and Klarica, 2005; Bulat et al., 2008). In the
development of Starling's oncotic hypothesis, it was assumed
that only plasma proteins show restricted passage across
microvascular walls, while all other solutes pass freely and
cannot exert a significant osmotic pressure between plasma
and ISF. However, it is known that cerebral capillaries are
negligibly permeable to inorganic ions (Fenstermacher and
Rapoport, 1984; Katzman and Pappius, 1973) and that periph-
eral continuous capillaries significantly restrict the passage of
these solutes (Wolf and Watson, 1989; Yudilevich and Alvarez,
1967). Furthermore, Starling's hypothesis fails to explain fluid
homeostasis when hydrostatic capillary pressure is high (in
feet during orthostasis) and low (in lungs), or when oncotic
plasma pressure is significantly decreased in experiments, or
in some clinical states such as genetic analbuminemia (Bulat
and Klarica, 2005).
The osmotic counterpressure hypothesis (Bulat and Klar-
ica, 2005) claims that, during water filtration across arterial
capillary walls, the passage of plasma osmolytes is restricted
(sieved), generating an osmotic counterpressure (OcPc) in the
arterial capillaries that rises along the length of the capillary
and, when it reaches the level of the hydrostatic capillary
pressure, halts water filtration. When such hyperosmolar
plasma is delivered to venous capillaries and postcapillary
venules where hydrostatic pressure is low, OcPc is instrumen-
tal in water reabsorption from the interstitium, which leads to
the dissipation of OcPc and the delivery of normal plasma
osmolarity to veins (Bulat and Klarica, 2005).
The concentration of osmotically active substances (parti-
cles) defines osmolarity. It is usually given in miliosmoles per
liter of water (mosm/l). The osmolarities of plasma and
interstial fluid are approximately 300 mosm/l (Guyton and
Hall, 1996). The osmotic concentrations of Na+ and Cl− are 142
and 108 mosm/l (Guyton and Hall, 1996), which constitutes
83% of total plasma osmolarity. Other inorganic ions such as
HCO−3, K+, Ca2+, Mg2+, HPO2− − 2−
4 , H2PO4 and SO4 constitute 11% of
the same amount. Proteins constitute 0.4% of total plasma
osmolarity, and other organic substances like glucose, urea,
aminoacids, lactate and creatine contribute only 4.3%.
A modified van't Hoff's equation is used for calculating the
difference in effective osmotic pressure (ΔOP) between two
compartments separated by a membrane or a microvascular
wall (Baumgarten and Feher, 2001):
ΔOPðmmHgÞ = ΔCmosm σ R T ð1Þ
where ΔCmosm represents the difference in the osmolytes'
concentration (mosm/l), σ is the reflection coefficient of
osmolytes, R is the universal gas constant (0.06236 mm Hg
per mosm/l and degree °K) and T is absolute temperature (°K).
According to this Eq. (1), RT (0.06236 × 310) is 19.3 mm Hg
per mosm/l at normal body temperature (37 °C or 310°K)
(Baumgarten and Feher, 2001). The reflection coefficients
(σ) of osmolytes show the way in which the osmolytes are
“reflected” from microvascular walls during their passage,
along with water (bulk flow), under a hydrostatic or
osmotic pressure gradient. Theoretically, they vary from
108 B RA I N R ES E A RC H R E V IE W S 6 5 (2 0 1 1 ) 9 9– 11 2
1, representing complete impermeability (100% “reflection”),
to 0, for a solute permeability equal to that of water (σ = 0)
(Michel, 2000).
8.1. Capillary osmotic counterpressure as regulator of ISF
In the case of plasma filtration through the arterial capillaries'
wall, plasma osmolyte concentration is expected to increase
since more water (σ = 0) than osmolytes (σ > 0) will pass through
(Bulat and Klarica, 2001). This increase in plasma osmolyte
concentration in the arterial capillary creates an osmotic
counterpressure (OcPc) that counteracts water filtration. Based
on Eq. (1), OcPc is dependant on plasma concentration, σ of the
osmolyte, and the water filtration rate. The estimated water
filtration rates in cerebral capillaries (Fenstermacher and
Rapoport, 1984) is lower than in peripheral capillaries (Landis
and Pappenheimer, 1963; Renkin and Crone, 1996), where it
varies from 1 to 4% of plasma volume flow and depends on
changes of HPc. Using the range of capillary water filtration
rates, and the plasma osmolytes' σ, which is known from data
published in the literature for both cerebral and peripheral
capillaries, the OcPc opposing water filtration can be calculated
(see Table 1).
Cerebral capillaries form the blood–brain barrier (BBB) and
are characterized by endothelial cells with tight intercellular
junctions. The permeability of BBB for water is relatively high
(Bolwing and Lassen, 1975), while the passage of proteins and
electrolytes is very poor (Fenstermacher and Rapoport, 1984), so
the estimated σ of proteins is 0.999 (Renkin and Crone, 1996) and
the estimated σ of Na and Cl about 0.98 (Fenstermacher and
Rapoport, 1984; Fraser and Dallas, 1990; Yudilevich and De Rose,
1971). The normal concentration of Na inside the plasma is 142
mosm/l; however, at a water filtration rate of 0.2%, one can
assume that this number of milliosmols is in 0.998 l of plasma
volume due to the loss of water. If recalculation of Na
concentration is made per liter of plasma (142 mosm/0.998 l),
the result is 142.285 mosm/l, an increase of 0.285 mosm/l in
plasma osmolarity. A similar recalculation for Cl gives an
increase from its normal concentration of 108 mosm/l to
108.216 mosm/l, or an increase of 0.216 mosm/l. Taken together,
this means a total increase in Na and Cl osmolarity (0.285 + 0.216)
of 0.501 mosm/l. When calculating osmotic pressure, this total
increase should be multiplied by the osmotic coefficient for
Table 1 – Water filtration rate and osmotic counterpressure
of NaCl in capillaries (OcPc).
Cerebral capillaries Skeletal muscle capillaries
Filtration OcPc of NaCl Filtration OcPc of NaCl
rate (%) (mm Hg) rate (%) (mm Hg)
0.10 4.40 0.25 5.63
0.20 8.81 0.50 11.28
0.40 17.66 1.00 22.66
0.80 35.46 2.00 45.79
1.00 44.41 4.00 93.49
Calculated osmotic counterpressures (OcPc) of NaCl in cerebral and
skeletal muscle capillaries (mm Hg) at different water filtration
rates expressed in percentages (%) of plasma volume flow
(reproduced from Bulat and Klarica, 2005 with permission).
NaCl, or 0.93 (Baumgarten and Feher, 2001), resulting in 0.466
mosm/l (0.501×0.93). If σ of NaCl is 0.98, according to Eq. (1), the
OcPc inside the cerebral capillaries is: 0.466×0.98×19.3=8.81 mm
Hg (Bulat and Klarica, 2005). This shows that there is generation of
an NaCl OcPc of about 9 mm Hg inside the cerebral capillaries, at a
water filtration rate of 0.2%. Table 1 shows some of the NaCl OcPc
values at different rates of water filtration. These OcPc values
cannot be decreased by NaCl diffusion and/or convection through
the capillary wall since they are continuously maintained by fluid
filtration and the plasma flow (Bulat and Klarica, 2005).
For comparative purposes, Table 1 also shows the calcu-
lated values of OcPc of NaCl in continuous peripheral
capillaries of the skeletal muscle, where σ of NaCl is 0.50
(Wolf and Watson, 1989). It appears that the calculated OcPc of
NaCl is about two times higher in cerebral capillaries than in
peripheral continuous capillaries at the same water filtration
rates (Table 1).
When the OcPc of plasma proteins in cerebral and
peripheral capillaries were calculated according to the empir-
ical equation developed by Landis and Pappenheimer (1963),
minute values were obtained (Bulat and Klarica 2005) due to
the very low concentration of plasma proteins in comparison
to plasma NaCl (see above). Furthermore, when plasma
proteins were decreased by 65% in rabbits by plasmapheresis,
no brain water increase was found, indicating that Starling's
oncotic hypothesis is not operative in cerebral capillaries
(Zornow et al., 1987). In the case of peripheral microvessel
perfusion by protein-free or blood-free perfusate, the micro-
vascular walls’ hydraulic conductivity and permeability to the
plasma solutes increased by several times (Huxley and Curry,
1991; Watson, 1983). In these conditions, a decrease in NaCl σ
and OcPc , as well as in other plasma osmolytes, should be
expected. This could compromise normal fluid filtration and
reabsorption through the microvascular walls, leading to an
interstial edema development (Bulat and Klarica, 2005).
The hydrostatic pressure in the cerebral capillaries (HPc) is
as yet unknown. However, the HPc inside the pial arterioles
(25 μm d.) that penetrate the brain parenchyma has been
measured as 55 mm Hg in cats (Shapiro et al., 1971), and the
HPc inside the pial venules (100–200 μm d.) leaving the
parenchyma has been measured as 4 mm Hg in rats (Wiig
and Reed, 1983). All of this suggests that a relatively high axial
hydrostatic pressure gradient is present along the microvas-
cular bed, where both water filtration and reabsorption take
place. Fig. 9 schematically shows the correlation between HPc
and OcPc of NaCl inside the capillaries. Since the HPc decreases
(Fig. 9) and the NaCl OcPc increases along the capillary due to
water filtration and NaCl retention, these pressures should
equalize at a certain point, signifying that filtration has
stopped (there is no further net water filtration). Hypertonic
plasma will then reach the venous capillaries and postcapil-
lary venules in which HPc is lower than OcPc, and this will
induce an osmotic water reabsorption from interstial fluid into
the vessels (Fig. 9). Because of the reabsorption of water, a
dilution of hypertonic plasma in postcapillary venules occurs,
and finally it becomes normal (not shown in Fig. 9), i.e. OcPc is
dissipated.
According to our osmotic counterpressure hypothesis,
osmolarity in microvessels changes from normal due to the
generation of increased osmolarity (OcPc) inside arterial
 B RA I N R E SE A R CH R E V IE W S 6 5 (2 0 1 1 ) 9 9– 11 2
30
HPC
Pressure (mmHg)
20
OcPC
10
0 (Fig. 9). Furthermore, this OcPc in venous capillaries and
0 500 1000
Lenght of capillary (µm)
Fig. 9 – Schematic presentation of osmotic counterpressure
hypothesis of capillaries. HPc, hydrostatic capillary pressure;
OcPc, capillary osmotic counterpressure. Fluid filtration takes
place when HPc > OcPc, while reabsorption occurs when
HPc < OcPc. Reproduced from Bulat and Klarica, 2005 with
permission.
capillaries caused by water filtration and NaCl sieving. This is
followed by a dissipation of the mentioned OcPc inside the
venous capillaries and postcapillary venules due to reabsorption
of water, and finally the plasma delivered to veins has a normal
osmolarity value. In short, when HPc is higher than OcPc, the
filtration of water occurs, whereas when OcPc is higher than HPc,
the reabsorption of water occurs, resulting in the normalization
of plasma osmolarity (Bulat and Klarica, 2005).
We would like to emphasize that other plasma inorganic and
organic osmolytes (see above) should contribute to the devel-
opment of OcPc since they show limited capillary permeability
(Fenstermacher and Rapoport, 1984; Katzman and Pappius,
1973); however, in an attempt to keep this analysis simple, we
have not included them in the explanation of our hypothesis.
Additionally, numerous processes of transport through BBB
could have an important role in the long-term maintenance of
osmotic homeostasis in the brain. Some of them could be: a
facilitated D-glucose (Renkin and Crone, 1996), an aminoacid
influx (Smith and Stoll, 1998), an active organic acid efflux (Bulat,
1974; Bulat and Živković, 1978a; Bulat and Živković, 1978b ; Vladić
et al., 2000; Zmajević et al., 2002), and the transport of some
inorganic ions (Keep et al., 1998).
It can be assumed that water filtration does not change
significantly the volume and osmolarity of interstial fluid.
Cerebral capillaries form a dense and interconnected network
of vessels with the result that water filtration and reabsorp-
tion occur simultaneously everywhere between numerous
adjacent capillary branches, preventing significant changes in
volume and osmolarity in the interstitium. In addition, no
lymphatic system is present in the brain, which means that
blood microvessels are crucial for the reabsorption water and
solutes, contrary to peripheral tissues where this reabsorption
takes place partly into lymphatics.
The rate of water filtration and reabsorption (Jv, volume/
time) across the microvascular walls should depend not only
109
on HPc and OcPc (Fig. 9) but also on hydrostatic pressure inside
the interstitium (HPi), so these relationships can be expressed
by following equation:
Jv = ðHPc –HPi Þ–ΣOcPc ð2Þ
where ΣOcPc is the sum of the counterpressures of all plasma
osmolytes where σ > 0 (Bulat and Klarica, 2005). As mentioned,
this ΣOcPc is mostly due to NaCl and other inorganic ions,
which constitute 94% of total plasma osmolarity. It appears
that ΣOcPc acts as a negative feedback control which opposes
the water filtration rate: a higher HPc and water filtration rate
create a higher OcPc (Table 1), which halts water filtration
postcapillary venules in CNS is instrumental in water reab-
sorption, which dissipates OcPc.
All this raises a question: How would a sudden increase in
HPc in peripheral capillaries and water filtration rates from 1%
to 4% affect the interstial fluid volume? The blood and plasma
volumes in men are 5 l and 3 l, respectively. The capillaries
account for 4% of a total blood volume (0.20 l of blood and 0.12 l
of plasma) (Holtz, 1996; Staub and Dawson, 1996), and the
interstial fluid volume amounts to 12 l. If 1% of the capillary
plasma volume (0.0012 l) filters into 12 l of interstial fluid, its
volume would increase by 0.01%, whereas at a filtration rate of
4% (0.0048 l), it would increase by 0.04%. Thus, if we keep in
mind that the processes of fluid filtration and reabsorption are
active at the same time, minor increases in interstial fluid
volumes are probably easily compensated for by fluid
absorption. Considering that these changes in the volumes
of interstial fluid are very small, we can conclude that the
changes in osmolarity and pressure inside the interstial fluid
are insignificant compared to those inside the microvessels
(Bulat and Klarica, 2005).
Our hypothesis concerning osmotic counterpressure inside
the capillaries implies that the main mode of regulating water
filtration and reabsorption through microvascular walls could
be the plasma osmolytes’ osmotic counterpressure. It could
also be the most important factor for the control of the
interstial fluid volume in physiological conditions. However,
in the case of increased permeability of microvascular walls
following various pathological processes including significant
hypoproteinaemia, a decrease in the reflection coefficient of
plasma osmolytes (σ) would be expected, as well as an
increase in microvascular walls’ hydraulic conductivity. Both
of these processes should lead to the appearance of an
interstitial edema (Bulat and Klarica, 2005).
8.2. Functional unity of ISF and CSF
As mentioned, several lines of evidence indicate that ISF and
CSF bulk (water) constitute a functional unity and are controlled
by changes of hydrostatic and osmotic pressures in micro-
vessels. The filtration of water across arterial capillary walls
under hydrostatic pressure and the reabsorption of water from
interstitium into venous capillaries and postcapillary venules by
osmotic counterpressure create a continuous turnover of ISF
and CSF bulk (water), since they are in continuity and mixed by
to-and-fro fluid pulsations. In such a way, the functional unity
of ISF and CSF is maintained.
110 B RA I N R ES E A RC H R E V IE W S 6 5 (2 0 1 1 ) 9 9– 11 2
It is interesting to analyze the changes in hydrostatic and
osmotic pressures in ISF and CSF when plasma osmolarity is
increased by mannitol (hyperosmotic stress). The mean
hydrostatic pressures of ISF and CSF are equal and positive
when measured at the same hydrostatic level (Wiig and Reed,
1983). However, when plasma osmolarity is enhanced by an
intravenous injection of mannitol, these pressures decrease
and become negative (subatmospheric), the changes in ISF
preceding those in CSF (Wiig and Reed, 1983). Thus, it appears
that increased plasma osmolarity enhances the absorption of
water from ISF and CSF, which causes a fall in intracranial
pressure. In addition, when plasma osmolarity is increased by
mannitol, the concentration of inorganic ions (Na+, Cl-, K+) and
the total osmolarity in CSF significantly increase (Donato et al.,
1994), suggesting selective water absorption in comparison to
inorganic ions, which poorly pass across microvascular walls.
Thus, the increase in osmolarity and the decrease in
hydrostatic pressure in ISF and CSF, when plasma osmolarity
is experimentally increased, should act as counterpressures
that prevent a drastic dehydration of CNS. Since it is known
that the brain does not behave as a passive osmometer when
exposed to high plasma osmolarity (Cserr, 1988), we assume
that the main reason for this phenomenon is the development
of counterpressures in ISF/CSF.
In the process of filtration and reabsorption of ISF/CSF,
some factors are of special importance, such as the surface
area of microvessels and mixing of ISF/CSF. The huge surface
area of CNS microvessels in comparison with the minute
surface area of accessory absorptive sites such as arachnoid
villi and perineural sheaths of cranial and spinal nerves (see
above) indicates that CNS microvessels have a high water
filtration and absorption capacity, since their permeability to
water is relatively high (Bolwing and Lassen, 1975). Further-
more, the surface area of CNS capillaries is 5000 times larger
than that of choroid plexus capillaries according to an
estimate (Raichle, 1983), so the filtration and absorption of
CSF via choroid plexus capillaries should be of minor
importance. The continuous mixing of CSF/ISF (see above)
enables the functional unity of these two fluids and their
control across the CNS microvessels.
9. Concluding remarks
In conclusion, our analysis indicates that CNS microvessels
are instrumental in fluid filtration and reabsorption inside
brain and spinal cord parenchyma, as well as in CSF system.
The osmotic pressure change in CNS microvessels is crucial in
regulation of ISF and CSF volumes which are continuously
mixed by to-and-fro fluid pulsations.
Distribution of substances along CSF spaces depends on
the rate of their removal into microvessels: slow removal of a
substance means that its residence time is long so that it can
be well distributed along all CSF spaces over time. On the other
hand, distribution of water, which constitutes bulk of ISF and
CSF, is very limited due to its rapid turnover across microvas-
cular walls.
Thus, the distribution of substances with long residence
time caused by to-and fro pulsations will always, after its
application into LV, create an illusion of unidirectional bulk
CSF circulation (from LV to CM and cortical and spinal
subarachnoid space). On the contrary, after application of
these substances in other parts of CSF system, their distribu-
tion in all directions occurs (as well as in LV, which is opposite
to classical hypothesis).
Acknowledgments
This work has been supported by the Ministry of Science,
Education and Sport, Republic of Croatia (Project: Pathophys-
iology of the cerebrospinal fluid and intracranial pressure No.
108-1080231-0023).
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