Cerebrospinal fluid is drained primarily via the spinal canal and olfactory route in young and aged spontaneously hypertensive rats
Fluids and Barriers of the CNS
Cerebrospinal fluid is drained primarily via the spinal canal and olfactory route in young and aged spontaneously hypertensive rats
Lucy A Murtha 1
Qing Yang 3
Mark W Parsons 2
Christopher R Levi 2
Daniel J Beard 1
Neil J Spratt 0 1 2
Damian D McLeod 0 1
0 Equal contributors
1 University of Newcastle and Hunter Medical Research Institute, University of Newcastle: School of Biomedical Sciences & Pharmacy , Medical Sciences Building, Callaghan, NSW 2308 , Australia
2 Hunter New England Local Health District: Department of Neurology, John Hunter Hospital , Locked Bag 1, Hunter Region M.C, NSW 2310 , Australia
3 Apollo Medical Imaging Technology Pty Ltd , Suite 611, 365 Little Collins Street, Melbourne, Vic 3000 , Australia
Background: Many aspects of CSF dynamics are poorly understood due to the difficulties involved in quantification and visualization. In particular, there is debate surrounding the route of CSF drainage. Our aim was to quantify CSF flow, volume, and drainage route dynamics in vivo in young and aged spontaneously hypertensive rats (SHR) using a novel contrast-enhanced computed tomography (CT) method. Methods: ICP was recorded in young (2-5 months) and aged (16 months) SHR. Contrast was administered into the lateral ventricles bilaterally and sequential CT imaging was used to visualize the entire intracranial CSF system and CSF drainage routes. A customized contrast decay software module was used to quantify CSF flow at multiple locations. Results: ICP was significantly higher in aged rats than in young rats (11.52 ± 2.36 mmHg, versus 7.04 ± 2.89 mmHg, p = 0.03). Contrast was observed throughout the entire intracranial CSF system and was seen to enter the spinal canal and cross the cribriform plate into the olfactory mucosa within 9.1 ± 6.1 and 22.2 ± 7.1 minutes, respectively. No contrast was observed adjacent to the sagittal sinus. There were no significant differences between young and aged rats in either contrast distribution times or CSF flow rates. Mean flow rates (combined young and aged) were 3.0 ± 1.5 μL/min at the cerebral aqueduct; 3.5 ± 1.4 μL/min at the 3rd ventricle; and 2.8 ± 0.9 μL/min at the 4th ventricle. Intracranial CSF volumes (and as percentage total brain volume) were 204 ± 97 μL (8.8 ± 4.3%) in the young and 275 ± 35 μL (10.8 ± 1.9%) in the aged animals (NS). Conclusions: We have demonstrated a contrast-enhanced CT technique for measuring and visualising CSF dynamics in vivo. These results indicate substantial drainage of CSF via spinal and olfactory routes, but there was little evidence of drainage via sagittal sinus arachnoid granulations in either young or aged animals. The data suggests that spinal and olfactory routes are the primary routes of CSF drainage and that sagittal sinus arachnoid granulations play a minor role, even in aged rats with higher ICP.
Computed tomography; Cerebrospinal fluid dynamics; Contrast; Spontaneously hypertensive rat; Intracranial pressure (ICP); Age; CSF; SHR
Cerebrospinal fluid (CSF) dynamics are thought to be
altered in many pathological conditions including
], idiopathic intracranial hypertension [
intracerebral haemorrhage [
], subarachnoid haemorrhage
], large hemispheric stroke [
], traumatic brain injury
], and in the aging brain [
]. However, relatively
little is known about the exact mechanisms of changes
in CSF dynamics and drainage in these conditions due
to difficulties in quantification.
The role and location of CSF drainage has been studied
in both animals and humans. Traditional interpretation
has been that most CSF drains into the venous sinuses via
arachnoid granulations [
]. The primary site of CSF
reabsorption, however, has become a contentious issue
over the last decade. The importance of the olfactory
perineural pathways and the cervical lymphatics [
in reabsorbing CSF has been studied in several species,
including humans. Postmortem lymphatic vascular
] and radioactive albumin clearance methods
] have demonstrated the major contribution of
these pathways in CSF drainage. Furthermore,
physiological studies by Johnston’s group, suggested that the
arachnoid granulations may only come into play when
intracranial pressure is elevated and that lymphatic
drainage routes may play a major role [
in neonates where arachnoid granulations are sparse
. Both human and animal studies indicate that the
spinal route, either via spinal arachnoid granulations or
via lymphatics around spinal nerve root dural sheaths
may also be important [
]. These lymphatic routes
may have important immunological significance [
To investigate CSF dynamics and drainage within the
entire rat brain we developed a novel contrast-enhanced
computed tomography (CT) technique to image the rat
CSF system in three-dimensions. One unique feature of
this technique is that the dynamic nature of CSF drainage
can be observed in vivo. We chose to compare young and
aged spontaneously hypertensive rats (SHR) because their
cerebral ventricular volume is thought to increase with
], which, in addition to the hypertension, may
affect CSF dynamics. In addition, this effect may also
influence physiological variables such as intracranial
pressure, for example, elevated ICP is seen in some
hydrocephalus patients [
]. Using our novel
contrastenhanced CT method, we hypothesized that the aged
SHRs would have altered CSF dynamics and drainage
and a higher baseline ICP when compared to the young
All animal experiments were conducted in accordance
with the Australian Code of Practice for the Care and Use
of Animals for Scientific Purposes and were approved by
The University of Newcastle Animal Care and Ethics
Committee. Experiments were performed on two cohorts
of male spontaneously hypertensive rats (SHR) (Animal
Resources Centre, Western Australia). One group weighed
200-360 g, aged 2–5 months (n = 5); the other group
weighed 360-400 g, aged 16 months (n = 5). Due to ethical
constraints, we calculated the sample size required to
detect a difference in baseline ICP between two different
rat strains (Wistar and Long Evans; data previously
]). Using an alpha of 0.05 and Power of 0.80, a
sample size of 5 animals per group was required to detect
a 4.2 mmHg difference between baseline ICP in the two
Surgery and physiological monitoring
Animals were anaesthetised with isoflurane (5% induction,
2% maintenance) in 50:50%, N2:O2 via a facemask. It has
previously been reported in dogs that isoflurane causes
no significant change in the rate of CSF production [
Respiratory rate was regularly monitored and core
temperature was maintained at 37°C via a thermocouple
rectal probe and warming plate for the duration of
surgical anaesthesia. Incision sites were shaved, cleaned and
injected subcutaneously with 2 mg/kg 0.05% Bupivacaine
(Pfizer, Australia). To measure arterial blood pressure under
anaesthetic, a fibreoptic microcatheter (SAMBA Sensors,
Sweden) was briefly inserted into the saphenous branch of
the femoral artery, and a steady state baseline arterial blood
pressure trace was recorded.
Intracranial pressure (ICP) was recorded prior to
scanning using a SAMBA microcatheter as previously
] with minor changes. Briefly, animals were
placed in the ear bars of a custom-built CT-compatible
stereotaxic frame. Hollow poly-ether-ether-ketone (PEEK)
screws (Solid Spot LLC, Santa Clara, CA, USA) of 2 mm
in diameter × 5 mm in length were inserted bilaterally
0.3 mm caudal and 1.5 mm lateral to Bregma. The ICP
probe was inserted into the right screw and an airtight seal
made by surrounding both screws in a biocompatiable
caulking material (Silagum, DMG Dental, Hamburg,
Germany). The probe was removed prior to scanning.
Computed tomography (CT) imaging
Following baseline arterial pressure and ICP
measurement, animals remained on the same base plate and
stereotaxic frame, which was positioned on the CT scanner
table. All imaging was performed using a 64-slice clinical
CT scanner (Siemens, Erlangen, Germany) [
CSF imaging protocol was developed specifically for the
current study. Each CT-CSF imaging sequence used
0.6 mm slice thickness with coronal plane image
acquisition, and a total of 90 slices captured from the rat nose
to the cervical vertebrae (C2). Two 1 ml syringes and two
PE-10 intraventricular catheters were then preloaded with
a 1:4 dilution of Ultravist 300 mg/mL (Bayer HealthCare
Pharmaceuticals Inc.) in 0.9% saline. This dilution was
chosen to reduce the viscosity and make it closer to that
of CSF. Care was taken to ensure that no air bubbles were
present within the catheters. The catheter tips were
inserted 3.5 mm below the skull into each lateral ventricle
via the bilateral screws used for ICP monitoring, and a
head CT scan performed (baseline scan) to ensure that
catheter tips were positioned within each lateral ventricle.
Using an automated syringe driver (Harvard Apparatus,
Pump 11 Elite, MA, USA), contrast was injected into each
ventricle at 2 μL/min for 10 minutes, with a CT scan
performed every minute from the start of infusion. The
infusion rate of 2 μL/min was chosen to reduce the
possibility of the infusion affecting the CSF production
(rodent CSF production rate previously reported as
2.66-2.84 μL/min ). Following the cessation of
infusion, serial CT scans were performed every 5 minutes for
60 minutes (Figure 1, an additional movie file shows this
in more detail [see Additional file 1]).
Processing CT images
For each animal, serial CT images were loaded into
MiStar software (Apollo Medical Imaging Technology
Pty Ltd, Melbourne, Australia). Motion correction was
performed, and the resulting images were subtracted
from the baseline non-contrast image.
3D reconstruction of CT images: rat CSF system
A representative 3D reconstruction of the rat CSF system
was created by generating a Maximum Intensity Projection
(MIP).The MIP was loaded into the MiStar fusion 3D
render module. Thresholds were applied to the 3D render
to highlight the CSF system.
Visual inspection of contrast time-course throughout
The time taken for injected contrast to reach specific
anatomical landmarks within the CSF system was
quantified in each animal. The anatomical landmarks included
the cerebral aqueduct, 3rd ventricle, 4th ventricle, spinal
canal, basal cisterns and cribriform plate. The contrast
window range was set to 0–250 Hounsfield Units (HU)
for each animal sequence to prevent the false detection of
signal noise at each image time-point. The time taken for
the contrast to reach each landmark was calculated by
analysing each sequential image.
CSF flow rate calculations
Contrast-enhanced CT images were processed using the
MiStar software decay module specifically written for rat
imaging. CSF flow rate measured at the cerebral aqueduct
was used to estimate CSF production. Flow rates were also
calculated elsewhere within the CSF system. A small
region-of-interest (ROI) with 6 voxels was positioned
within the centre of the aqueduct on the 10 minute
time-point scan (post-infusion). The total change in
Hounsfield Units for voxels within the ROI was plotted
over time, and an exponential decay curve was fitted to
the first five decay data points. The exponential decay
rate constant is directly proportional to flow within the
ROI. This principle was applied to each voxel to
generate the Decay Rate Map, measured in ml/min/100 L
(which was then converted to μl/min/100 ml). The
ROI was then co-registered with the Decay Rate Map
to obtain the flow value. Flow was converted into μl/min
by multiplication with volume (slice thickness × area of
ROI). The process was repeated (with the same fixed ROI)
at the 3rd ventricle and 4th ventricle of each animal.
Brain and intracranial CSF volume calculation
Twenty five 0.6 mm coronal CT slices were analysed
slice-by-slice to calculate brain and CSF volume. On each
slice, a threshold of 100 HU was set and ROI software
tools were used to define the area of total brain tissue
(using baseline scans) or contrast-enhanced CSF within
the cranium. On each slice, the volume was calculated as
slice thickness × area of ROI. The combined sum of the
ROI volumes was calculated.
Statistical tests were performed using GraphPad Prism
Version 6 for Windows (GraphPad Software, USA).
Twotailed Student’s t-test was used to compare differences in
CSF production rate, and CSF volumes between young
and aged groups. Significance was accepted at the p < 0.05
level. T-tests are also reported for physiological variables
for illustrative purposes, with p-values uncorrected for use
of multiple comparisons. Unless otherwise stated, data is
expressed as mean ± standard deviation (±SD).
Arterial blood pressure (systolic/diastolic) was measured
in 3 young animals and 5 aged animals. Mean arterial
blood pressure was lower in the young versus the aged
animals (94 ± 32 mmHg vs. 160 ± 22 mmHg, p = 0.01).
Intracranial pressure (ICP) was measured in all animals
and traces revealed consistent pulse and respiratory
waveforms. A significantly higher ICP was found in
aged rats, 11.52 ± 2.36 mmHg, versus 7.04 ± 2.89 mmHg
in the younger animals, p = 0.03. Cerebral perfusion
pressure (mean arterial blood pressure - intracranial
pressure) was significantly lower in the young versus
the aged animals (86 ± 32 mmHg vs. 148 ± 20 mmHg,
p = 0.02).
Cerebrospinal fluid drainage pathways and time course
CSF was observed to drain into the spinal canal
(subarachnoid space) and via the olfactory pathway (through
the cribriform plate), the optic nerves, and the cervical
lymph nodes in all animals (Figure 2, an additional movie
file shows this in more detail [see Additional file 2]).
Contrast-enhanced CSF was observed at the cerebral
aqueduct 3.2 ± 0.8 minutes post-injection in both cohorts.
It was first observed to enter the 3rd ventricle 1.8 ± 0.4
minutes and 1.6 ± 0.5 minutes post-injection in the young
and aged respectively, the 4th ventricle at 4.4 ± 2.2 minutes
and 3.8 ± 0.8 minutes post-injection, the spinal canal at
11.5 ± 9.1 minutes and 7.2 ± 0.8 minutes post-injection,
and the basal cisterns at 18.5 ± 6.8 minutes and 14.0 ±
4.2 minutes post-injection in the young and the aged
respectively (Figure 3). Contrast could be observed leaving
the cranial vault at the cribriform plate 23.8 ± 6.3 minutes
post-injection in the young and 21 ± 8.2 minutes in the
aged (Figure 3). No contrast-enhanced CSF was observed
at the sagittal sinus [Additional file 1].
Cerebrospinal fluid production and flow rates and total intracranial CSF volume
CSF flow was able to be quantified at multiple sites within
the ventricular system, but flow quantification in the
extraventricular locations was not possible due to
respiratory motion artifact (spinal canal) and contrast dilution,
resulting in reduced signal-to-noise ratio and partial
volume averaging error caused by the extensive surface area
of each drainage route. CSF production rates, as measured
by CSF flow rate at the cerebral aqueduct, were 3.14 ±
2.1 μL/min and 2.79 ± 0.57 μL/min in the young and the
aged, respectively. CSF flow rates were 3.74 ± 1.93 μL/min
and 3.19 ± 0.70 μL/min at the 3rd ventricle and 2.89 ±
0.21 μL/min and 2.70 ± 1.29 μL/min at the 4th ventricle
(Figure 4A- 4C). The total intracranial CSF volume was
204 ± 97 μL in the young and 275 ± 35 μL in the aged
cohort (Figure 4D). None of these values differed
significantly between the young vs. aged cohorts.
Brain volume was 2157 ± 314 mm3 and 2366 ± 233 mm3
in the young and aged rats, respectively (Figure 5). CSF
volumes (and as percentage of brain volume) were
204 ± 97 μL (8.8 ± 4.3%) in the young and 275 ± 35 μL
(10.8 ± 1.9%) in the aged animals. These values were
not significantly different.
This study used young and aged SHRs to demonstrate a
new technique for the measurement of CSF flow, volume
and drainage in vivo. The results of this study suggest
that sagittal sinus arachnoid granulations are not the
primary route of CSF drainage and that CSF is primarily
absorbed via the spinal and olfactory routes. The time
taken for contrast-enhanced CSF to reach each drainage
route did not differ between the young and aged animals.
Our novel CT method also allowed for measurement of
CSF flow and volume and found that there was little
difference between young and aged animals, despite a higher
ICP in the aged animals. This method provides an
alternative avenue for the investigation of the pathophysiological
perturbations occurring in disorders of CSF regulation
and abnormal intracranial pressure.
There is increasing recognition of the importance of
altered CSF and brain interstitial fluid dynamics with
age, not only in conditions such as normal pressure
hydrocephalus but also in dementias such as Alzheimer’s
]. Despite this, most CSF-related research
occurs in young animals. In the current study we
investigated CSF dynamics in an aged population of SHRs, since
this strain is known to develop hypertension,
ventriculomegaly and cerebral volume loss with age [
found little difference in the absolute CSF flow rates or
total brain volume between young (2–5 months) and aged
(16 months) rats. These data are similar to findings in a
previous study of normotensive rats (without
ventriculomegaly) aged from 3–30 months. Chiu et al. found that
peak CSF production occurs at 10 months before steadily
decreasing over time to almost baseline values [
Furthermore, the values they reported in 12–20 month
rats ranged from 2.66-2.84 μL/min, which were
comparable to our reported values in 16 month aged rats and to
previously reported values in 3–4 months old rats in other
]. It is also interesting to note that the CSF
flow rates, when measured at different anatomical
locations within the CSF ventricular system, did not vary
greatly. The lack of a significant difference in in vivo CSF
volumes between young and aged SHRs is inconsistent
with previous in vitro studies, and most likely due to a
lack of statistical power to detect a difference in this
variable. This was contributed to by small animal numbers
and in particular by the significant between-animal
variability in the younger cohort. The point estimate of a 26%
difference is in keeping with previous published data
]. Additionally, it may be that changes would be
more apparent in ‘elderly’ (≈2 years) animals, than in at
16 months of age. This age was chosen to avoid the
tumours and mortality that increase beyond 18 months in
this strain. Our data also demonstrated a significantly
higher intracranial pressure (ICP) and cerebral perfusion
pressure (CPP) in the aged rats when compared to the
young. This is consistent with findings in humans [
and in other rat strains . Additionally, the greater
variability in CPP in the young animals may have contributed
to the greater variance in CSF data, i.e. there was a greater
percentage of variance (relative to the mean) in mean
arterial pressure, and in ICP, in the young vs. aged. It is
plausible that greater variance in CPP resulted in greater
variance in CSF flow and drainage parameters, as changes
in CPP may affect CSF production and drainage.
Although the ICP was higher in aged animals, we
could not see any contrast adjacent to or filling the
sagittal sinus to indicate drainage of the CSF via arachnoid
granulations, even at the later time points. There was,
however, clear evidence of passage of contrast into the
spinal canal, olfactory cavity, along the ophthalmic nerves
and into cervical lymph nodes. Our findings support
recent studies that the primary route of CSF drainage in
most mammalian species is via perineural sheaths. In
anatomical studies using a coloured tracer and anatomical
dissection, CSF drainage into nasal lymphatics via the
olfactory nerves and cribriform plate has been
demonstrated in sheep, pigs, rabbits, rats, mice and monkeys
]. Additionally presence of this route has
been shown in human cadavers . Many studies
have also demonstrated spinal lymphatic CSF drainage
using tracers in several mammalian species including
]. It was calculated that the rate
of spinal CSF absorption was between 38–76% of CSF
production in healthy individuals (higher during activity)
]. At least in the sheep, the venous sinus arachnoid
granulation pathway of CSF absorption appears to be a
secondary pathway only recruited at high intracranial
pressures, for example after a neurological injury [
The calculation of CSF volume is technically challenging
and many techniques have been tried, with varying
success. Values obtained in this study correspond well
to some published data in rats using quite different
techniques, including volumes of 233–240 μL in young
SHRs using the ventriculo-cisternal dilution method
] and volumes from 275–441 μL in Fischer 344/BN
rats using magnetic resonance imaging . However,
some published studies report much higher volumes.
Lai et al. (1983) reported a mean CSF volume of 580 μL
in rats using the formula ‘CSF volume = CSF formation
rate/CSF turnover rate’ [
]. They assumed that the CSF
turnover rate was constant amongst species [
], and used
the human CSF turnover rate of 0.38% per minute
(obtained using ventriculo-lumbar perfusion method from
12 children with subacute sclerosing panencephalitis
and pontine glioma) [
], to calculate CSF volume. We
are not sure that this assumption is well justified. However,
despite these potential limitations, the rat CSF volume and
turnover rate from that paper appear to have become the
accepted values in reviews of the field, perhaps due to the
paucity of other available data [
Our novel CT method provides several possible
advantages, and some limitations, when compared with other
techniques such as the ventriculo-cisternal dilution and
post-mortem dye-tracer methods. First among these is
that it does not require an intracisternal draining catheter,
with potential resultant effects on ICP and possibly on
CSF production, if homeostatic mechanisms are evoked.
Secondly, the ability to image the entire CSF system
simultaneously and sequentially gives a more complete
understanding of the dynamics of CSF flow and drainage. We
were able to monitor the major physiological parameters
thought to influence CSF production rate, that is, ICP
], blood pressure [
], and temperature [
Additionally, flow rates, whole brain volume, and CSF volume
calculations were obtained from the same study. Some
unavoidable limitations also exist with the CT method,
and particular points of the analysis require great care.
In particular, partial volume averaging effects are a
known potential problem when measuring values from
a very small structure such as the cerebral aqueduct.
Great care must be taken to identify the midpoint of the
region of contrast enhancement for placement of a
small region-of-interest. An additional limitation is that
although we could observe and quantify the time taken
for contrast-enhanced CSF to reach the drainage
pathways in vivo, we have been unable to reliably quantify
the volume of CSF draining via these routes.
We have provided in vivo data using CT imaging of CSF
distribution over time, which indicates that the primary
route of CSF drainage in young and aged rats is via the
spinal and olfactory lymphatics, and that drainage into
the sagittal sinus arachnoid granulations plays at most a
minor role. The CT technique we developed provides an
alternative to ventriculo-cisternal dilution methods for
measurement of CSF flow through the cerebral aqueduct,
the widely accepted surrogate measure for CSF
production. It avoids the need to puncture the cisterna magna
and permits visualisation and timing of CSF distribution
and drainage, quantification of CSF flow elsewhere within
the ventricular system and measurement of total brain
and intracranial CSF volumes. Interestingly, this study of
young and aged SHRs suggest that CSF production rates
and volumes are quite similar, and do not change
dramatically with age. The information gathered using our novel
contrast-enhanced CT method may provide much needed
insight into the CSF dynamics and drainage involved in
many neurological diseases.
Additional file 1: Contrast-enhanced cerebrospinal fluid flow
through the cranium over 60 minutes. Radio-opaque contrast (20 μl)
was simultaneously injected into each lateral ventricle at 2 μl/min for
10 min while plain CT images (0.6 mm slice thickness) were taken over
60 minutes. File format: mov.
Additional file 2: 3D render reconstruction of cerebrospinal fluid
system of the rat. The computed tomography images of one rat were
loaded in MiStar software and subtracted from the baseline non-contrast
image. A Maximum Instensity Projection was generation and loaded into
the MiStar fusion 3D render module. File format: mov.
CSF: Cerebrospinal fluid; CT: Computed tomography; HU: Hounsfield Units;
ICP: Intracranial pressure; MIP: Maximum Intensity Projection; ROI: Region of
interest; SHR: Spontaneously hypertensive rat.
The authors declare that they have no competing interests.
LM and DM carried out the surgical and computed tomography
components of the study, analysed and interpreted the data, performed
statistical analysis and drafted the manuscript. QY designed MiStar software
decay module specifically written for this project. DB and NS participated in
the design of the study and helped draft the manuscript. DM, NS, LM, MP
and CL, conceived the study, and participated in its design and coordination.
All authors read and approved the final manuscript.
LM- B. Biomed Sci. (Hons), PhD Candidate (Human Physiology).
QY- PhD (Physics).
MP- PhD, FRACP (Neurology).
CL- MBBS, B Med Sci (Hons), FRACP (Neurology).
DB- B. Biomed Sci. (Hons), PhD Candidate (Human Physiology).
NS- MBBS, PhD, FRACP (Neurology).
DM- PhD (Human Physiology).
We would like to thank the radiographers David Buxton and Marc Heaton
(Hunter Health Imaging, John Hunter Hospital) for CT imaging assistance and
the Faculty of Health Workshop of the University of Newcastle for manufacture
of surgical and anesthetic equipment. Dr. Spratt was supported by an Australian
National Health & Medical Research Council (NHMRC) Career Development
Fellowship (APP1035465). Aspects of this work were supported by the NHMRC
(Project Grant APP1033461), Hunter Medical Research Institute and National
Stroke Foundation (Australia) research grants.
1. Wagshul ME , McAllister JP , Rashid S , Li J , Egnor MR , Walker ML , Yu M , Smith SD , Zhang G , Chen JJ , Benveniste H : Ventricular dilation and elevated aqueductal pulsations in a new experimental model of communicating hydrocephalus . Exp Neurol 2009 , 218 : 33 - 40 .
2. Bateman GA : The pathophysiology of idiopathic normal pressure hydrocephalus: cerebral ischemia or altered venous hemodynamics ? AJNR Am J Neuroradiol 2008 , 29 : 198 - 203 .
3. Preuss M , Hoffmann KT , Reiss-Zimmermann M , Hirsch W , Merkenschlager A , Meixensberger J , Dengl M : Updated physiology and pathophysiology of CSF circulation-the pulsatile vector theory . Childs Nerv Syst 2013 , 29 : 1811 - 1825 .
4. Killer HE , Jaggi GP , Flammer J , Miller NR , Huber AR , Mironov A : Cerebrospinal fluid dynamics between the intracranial and the subarachnoid space of the optic nerve . Is it always bidirectional? Brain 2007 , 130 : 514 - 520 .
5. Shapira Y , Artru AA , Lam AM : Changes in the rate of formation and resistance to reabsorption of cerebrospinal fluid during deliberate hypotension induced with adenosine or hemorrhage . Anesthesiology 1992 , 76 : 432 - 439 .
6. Paradot G , Baledent O , Gondry-Jouet C , Meyer ME , Le Gars D : [Cerebrospinal fluid flow imaging in the meningeal hemorrhage] . Neurochirurgie 2006 , 52 : 323 - 329 .
7. Brinker T , Seifert V , Stolke D : Acute changes in the dynamics of the cerebrospinal fluid system during experimental subarachnoid hemorrhage . Neurosurgery 1990 , 27 : 369 - 372 .
8. Schwab S , Schellinger P , Aschoff A , Albert F , Spranger M , Hacke W : [ Epidural cerebrospinal fluid pressure measurement and therapy of intracranial hypertension in “malignant” middle cerebral artery infarct] . Nervenarzt 1996 , 67 : 659 - 666 .
9. Minnerup J , Wersching H , Ringelstein EB , Heindel W , Niederstadt T , Schilling M , Schabitz WR , Kemmling A : Prediction of malignant middle cerebral artery infarction using computed tomography-based intracranial volume reserve measurements . Stroke 2011 , 42 : 3403 - 3409 .
10. Johanson C , Stopa E , Baird A , Sharma H : Traumatic brain injury and recovery mechanisms: peptide modulation of periventricular neurogenic regions by the choroid plexus-CSF nexus . J Neural Transm 2011 , 118 : 115 - 133 .
11. Schmid Daners M , Knobloch V , Soellinger M , Boesiger P , Seifert B , Guzzella L , Kurtcuoglu V : Age-specific characteristics and coupling of cerebral arterial inflow and cerebrospinal fluid dynamics . PLoS One 2012 , 7 : e37502 .
12. Stoquart-ElSankari S , Baledent O , Gondry-Jouet C , Makki M , Godefroy O , Meyer ME : Aging effects on cerebral blood and cerebrospinal fluid flows . J Cereb Blood Flow Metab 2007 , 27 : 1563 - 1572 .
13. Tripathi R : Tracing the bulk outflow route of cerebrospinal fluid by transmission and scanning electron microscopy . Brain Res 1974 , 80 : 503 - 506 .
14. Tripathi BJ , Tripathi RC : Vacuolar transcellular channels as a drainage pathway for cerebrospinal fluid . J Physiol 1974 , 239 : 195 - 206 .
15. Welch K , Friedman V : The cerebrospinal fluid valves . Brain 1960 , 83 : 454 - 469 .
16. Cserr HF , Harling-Berg CJ , Knopf PM : Drainage of brain extracellular fluid into blood and deep cervical lymph and its immunological significance . Brain Pathol 1992 , 2 : 269 - 276 .
17. Zakharov A , Papaiconomou C , Koh L , Djenic J , Bozanovic-Sosic R , Johnston M : Integrating the roles of extracranial lymphatics and intracranial veins in cerebrospinal fluid absorption in sheep . Microvasc Res 2004 , 67 : 96 - 104 .
18. Kida S , Pantazis A , Weller RO : CSF drains directly from the subarachnoid space into nasal lymphatics in the rat. Anatomy, histology and immunological significance . Neuropathol Appl Neurobiol 1993 , 19 : 480 - 488 .
19. Carare RO , Hawkes CA , Weller RO : Afferent and efferent immunological pathways of the brain. Anatomy, function and failure . Brain Behav Immun 2014 , 36 : 9 - 14 .
20. Johnston M , Zakharov A , Papaiconomou C , Salmasi G , Armstrong D : Evidence of connections between cerebrospinal fluid and nasal lymphatic vessels in humans, non-human primates and other mammalian species . Cerebrospinal Fluid Res 2004 , 1 : 2 .
21. Nagra G , Koh L , Zakharov A , Armstrong D , Johnston M : Quantification of cerebrospinal fluid transport across the cribriform plate into lymphatics in rats . Am J Physiol Regul Integr Comp Physiol 2006 , 291 : R1383 - R1389 .
22. Johnston M , Zakharov A , Koh L , Armstrong D : Subarachnoid injection of Microfil reveals connections between cerebrospinal fluid and nasal lymphatics in the non-human primate . Neuropathol Appl Neurobiol 2005 , 31 : 632 - 640 .
23. Boulton M , Young A , Hay J , Armstrong D , Flessner M , Schwartz M , Johnston M : Drainage of CSF through lymphatic pathways and arachnoid villi in sheep: measurement of 125I-albumin clearance . Neuropathol Appl Neurobiol 1996 , 22 : 325 - 333 .
24. Boulton M , Flessner M , Armstrong D , Mohamed R , Hay J , Johnston M : Contribution of extracranial lymphatics and arachnoid villi to the clearance of a CSF tracer in the rat . Am J Physiol 1999 , 276 : R818 - R823 .
25. Boulton M , Flessner M , Armstrong D , Hay J , Johnston M : Lymphatic drainage of the CNS: effects of lymphatic diversion/ligation on CSF protein transport to plasma . Am J Physiol 1997 , 272 : R1613 - R1619 .
26. Silver I , Li B , Szalai J , Johnston M : Relationship between intracranial pressure and cervical lymphatic pressure and flow rates in sheep . Am J Physiol 1999 , 277 : R1712 - R1717 .
27. Mollanji R , Bozanovic-Sosic R , Zakharov A , Makarian L , Johnston MG : Blocking cerebrospinal fluid absorption through the cribriform plate increases resting intracranial pressure . Am J Physiol Regul Integr Comp Physiol 2002 , 282 : R1593 - R1599 .
28. Papaiconomou C , Zakharov A , Azizi N , Djenic J , Johnston M : Reassessment of the pathways responsible for cerebrospinal fluid absorption in the neonate . Childs Nerv Syst 2004 , 20 : 29 - 36 .
29. Welch K , Pollay M : The spinal arachnoid villi of the monkeys Cercopithecus aethiops sabaeus and Macaca irus . Anat Rec 1963 , 145 : 43 - 48 .
30. Gomez DG , Chambers AA , Di Benedetto AT , Potts DG : The spinal cerebrospinal fluid absorptive pathways . Neuroradiology 1974 , 8 : 61 - 66 .
31. Kido DK , Gomez DG , Pavese AM Jr, Potts DG : Human spinal arachnoid villi and granulations . Neuroradiology 1976 , 11 : 221 - 228 .
32. Tubbs RS , Hansasuta A , Stetler W , Kelly DR , Blevins D , Humphrey R , Chua GD , Shoja MM , Loukas M , Oakes WJ : Human spinal arachnoid villi revisited: immunohistological study and review of the literature . J Neurosurg Spine 2007 , 7 : 328 - 331 .
33. Marmarou A , Shulman K , LaMorgese J : Compartmental analysis of compliance and outflow resistance of the cerebrospinal fluid system . J Neurosurg 1975 , 43 : 523 - 534 .
34. Bozanovic-Sosic R , Mollanji R , Johnston MG : Spinal and cranial contributions to total cerebrospinal fluid transport . Am J Physiol Regul Integr Comp Physiol 2001 , 281 : R909 - R916 .
35. Edsbagge M , Tisell M , Jacobsson L , Wikkelso C : Spinal CSF absorption in healthy individuals . Am J Physiol Regul Integr Comp Physiol 2004 , 287 : R1450 - R1455 .
36. Ritter S , Dinh TT : Progressive postnatal dilation of brain ventricles in spontaneously hypertensive rats . Brain Res 1986 , 370 : 327 - 332 .
37. Ritter S , Dinh TT , Stone S , Ross N : Cerebroventricular dilation in spontaneously hypertensive rats (SHRs) is not attenuated by reduction of blood pressure . Brain Res 1988 , 450 : 354 - 359 .
38. Murtha LA , McLeod DD , McCann SK , Pepperall D , Chung S , Levi CR , Calford MB , Spratt NJ : Short-duration hypothermia after ischemic stroke prevents delayed intracranial pressure rise . Int J Stroke 2013 . doi: 10 .1111/ijs.12181.
39. Artru AA : Isoflurane does not increase the rate of CSF production in the dog . Anesthesiology 1984 , 60 : 193 - 197 .
40. Murtha L , McLeod D , Spratt N : Epidural intracranial pressure measurement in rats using a fiber-optic pressure transducer . J Vis Exp 2012 , 62 :e3689. doi: 10 , 3791 /3689.
41. McLeod D , Parsons M , Hood R , Hiles B , Allen J , McCann S , Murtha L , Calford M , Levi C , Spratt N : Perfusion computed tomography thresholds defining ischaemic penumbra and infarct core: studies in a rat stroke model . Int J Stroke 2013 . doi: 10 .1111/ijs.12147.
42. McLeod DD , Parsons MW , Levi CR , Beautement S , Buxton D , Roworth B , Spratt NJ : Establishing a rodent stroke perfusion computed tomography model . Int J Stroke 2011 , 6 : 284 - 289 .
43. Chiu C , Miller MC , Caralopoulos IN , Worden MS , Brinker T , Gordon ZN , Johanson CE , Silverberg GD : Temporal course of cerebrospinal fluid dynamics and amyloid accumulation in the aging rat brain from three to thirty months . Fluids Barriers CNS 2012 , 9 : 3 .
44. Xie L , Kang H , Xu Q , Chen MJ , Liao Y , Thiyagarajan M , O'Donnell J , Christensen DJ , Nicholson C , Iliff JJ , Takano T , Deane R , Nedergaard M : Sleep drives metabolite clearance from the adult brain . Science 2013 , 342 : 373 - 377 .
45. Sykova E , Vorisek I , Antonova T , Mazel T , Meyer-Luehmann M , Jucker M , Hajek M , Ort M , Bures J : Changes in extracellular space size and geometry in APP23 transgenic mice: a model of Alzheimer's disease . Proc Natl Acad Sci U S A 2005 , 102 : 479 - 484 .
46. Al-Sarraf H , Philip L : Effect of hypertension on the integrity of blood brain and blood CSF barriers, cerebral blood flow and CSF secretion in the rat . Brain Res 2003 , 975 : 179 - 188 .
47. Al-Sarraf H , Philip L : Increased brain uptake and CSF clearance of 14C-glutamate in spontaneously hypertensive rats . Brain Res 2003 , 994 : 181 - 187 .
48. Dunn LT : Raised intracranial pressure . J Neurol Neurosurg Psychiatry 2002 , 73 ( Suppl 1 ): i23 - i27 .
49. Rangel Castilla L , Gopinath S , Robertson CS : Management of intracranial hypertension . Neurol Clin 2008 , 26 : 521 - 541 . x.
50. Hawkins BE , Cowart JC , Parsley MA , Capra BA , Eidson KA , Hellmich HL , Dewitt DS , Prough DS : Effects of trauma, hemorrhage and resuscitation in aged rats . Brain Res 2013 , 1496 : 28 - 35 .
51. Ghersi-Egea JF , Finnegan W , Chen JL , Fenstermacher JD : Rapid distribution of intraventricularly administered sucrose into cerebrospinal fluid cisterns via subarachnoid velae in rat . Neuroscience 1996 , 75 : 1271 - 1288 .
52. Lai YL , Smith PM , Lamm WJ , Hildebrandt J : Sampling and analysis of cerebrospinal fluid for chronic studies in awake rats . J Appl Physiol 1983 , 54 : 1754 - 1757 .
53. Cserr HF : Physiology of the choroid plexus . Physiol Rev 1971 , 51 : 273 - 311 .
54. Cutler RW , Page L , Galicich J , Watters GV : Formation and absorption of cerebrospinal fluid in man . Brain 1968 , 91 : 707 - 720 .
55. Johanson CE , Duncan JA 3rd, Klinge PM , Brinker T , Stopa EG , Silverberg GD : Multiplicity of cerebrospinal fluid functions: New challenges in health and disease . Cerebrospinal Fluid Res 2008 , 5 : 10 .
56. Preston JE : Ageing choroid plexus-cerebrospinal fluid system . Microsc Res Tech 2001 , 52 : 31 - 37 .
57. Redzic ZB , Preston JE , Duncan JA , Chodobski A , Szmydynger-Chodobska J : The choroid plexus-cerebrospinal fluid system: from development to aging . Curr Top Dev Biol 2005 , 71 : 1 - 52 .
58. Hochwald GM , Sahar A : Effect of spinal fluid pressure on cerebrospinal fluid formation . Exp Neurol 1971 , 32 : 30 - 40 .
59. Carey ME , Vela AR : Effect of systemic arterial hypotension on the rate of cerebrospinal fluid formation in dogs . J Neurosurg 1974 , 41 : 350 - 355 .
60. Snodgrass SR , Lorenzo AV : Temperature and cerebrospinal fluid production rate . Am J Physiol 1972 , 222 : 1524 - 1527 .
61. Paxinos G , Watson C : The rat brain in stereotaxic coordinates . 4th edition . San Diego: Academic Press; 1998 .