From the Departments of Nephrology (E.L.A.B., M.S., H.A.K., J.A.J.) and
Experimental Neurology (P.R.D.B.), and Pathology (G.H.J.), University Hospital
Utrecht, and Department of In Vivo NMR, Bijvoet Center, Utrecht University
(E.L.A.B., K.N.), Utrecht, the Netherlands.
Correspondence and reprint requests to Jaap A. Joles DVM, PhD, Department of Nephrology and Hypertension (F03.226), Utrecht University Hospital, PO Box 85500, 3508 GA Utrecht, Netherlands. E-mail nephrology.gdl{at}pobox.ruu.nl
Methods—SHRSP were subjected to 1% NaCl in drinking water.
Cerebral MRI, proteinuria and systolic blood pressure (SBP)
were measured serially. After detection of cerebral edema (T2-weighted
MRI), 6 rats were killed for histology, to confirm the diagnosis of
cerebral edema. The others were followed up for 7 more days while salt
loading was continued (n=10, group 1) or after sodium intake was
normalized (n=7, group 2).
Results—SHRSP invariably developed cerebral edema in 30 days
(range, 8 to 54 days). At this point neurological signs were absent in
16 of 23 rats. SBP rose until 1 week before detection of cerebral
edema, and then stabilized at approximately 265 mm Hg.
Proteinuria invariably preceded cerebral edema, with a concentration
exceeding 40 mg/d predicting development of cerebral edema in 9 days
(range, 3 to 15 days). There was linear correlation
(R=.62, P<.0001) between proteinuria and
cerebral edema (pixels with an intensity above a defined threshold).
Rats in group 1 showed an increase in cerebral edema (from 5.8±1.1%
to 12.5±2.8%; P<.05), and proteinuria remained high
(from 305±44 to 338±29 mg/d); and 2 died spontaneously. Rats in group
2 showed no significant change in edema (from 4.9±0.5% to 6.9±1.3%)
but a marked fall in proteinuria (from 294±24 to 119±10 mg/d;
P<.05), both significantly different from group 1
(P<.05); all survived. SBP remained unaltered in both
groups.
Conclusions—Our data establish MRI as a sensitive method for
detection of cerebral edema, often prior to neurological signs, in
SHRSP. Proteinuria predicts cerebral edema, and these two
variables, both obtained noninvasively, are quantitatively related.
Moreover, in SHRSP normalizing sodium intake after salt loading
attenuates development of cerebral edema and reduces proteinuria.
T2-weighted MRI (T2W MRI) is a noninvasive technique that is uniquely
suited to the assessment of the development, progression, and
regression of brain edema and intracerebral
hemorrhage in vivo. It can define the number spatial
distribution and quantitate the size of brain lesions better than
any other imaging modality.10 11 12 13 14 This technique
offers a novel possibility to do repeated measurements and thus monitor
the development of cerebral lesions as well as the effect of
therapeutic measures at different stages of developing cerebral injury.
The first aim of our study was to describe the development of cerebral
lesions in relation to neurological symptoms in young SHRSP during high
salt intake with use of repeat T2W MRI. If a certain level of
proteinuria inevitably precedes the development of cerebral edema, this
level of proteinuria could be used as a starting point for
prophylactic interventions. Hence, the second goal was to
study the relation between proteinuria and cerebral edema in
salt-loaded SHRSP to ascertain whether proteinuria precedes cerebral
edema in this model, and if so, to define a level of proteinuria that
predicts the appearance of cerebral edema. Low salt intake reduced the
incidence of stroke in humans6 and effectively
prevented proteinuria in uninephrectomized SHR.15
Thus, the third goal was to study the effects of normalized salt intake
in SHRSP, after the initial appearance of cerebral edema, on the
subsequent evolution of proteinuria and cerebral edema.
Protocol
After the appearance of their first abnormalities on T2W MRI, rats were
randomized to three groups. Six rats were killed for pathology, to
evaluate the nature of the MRI changes (group 0). These rats were not
included in the quantitative data analysis. Group 1 (n=10)
continued the high salt intake, and group 2 (n=7) was switched to a
normal salt intake. Groups 1 and 2 were subjected to T2W MRI on days 3
and 7 and to metabolic studies on day 6. After 7 days these
rats were subjected to pathological examination to support
interpretation of the MRI data.
Renal Function and Blood Pressure
Histology
T2W MRI
T2W MRI Evaluation
The standardized threshold, pBRAINstandardized threshold, of
edema in the brain is unique for an individual animal and is defined
as:
Statistics
T2W MRI
Fig 2
Fig 3
Physiological Parameters
Development of Proteinuria and Its Relation to Cerebral
Edema
Effect of Normalizing Salt Intake after Appearance of Cerebral
Edema
T2W MRI
In group 1 it is apparent that continued excessive salt intake
increased the percentage of edematous pixels per slice both in the
central slice as well in the slices surrounding this centrally affected
slice. In group 2 the distribution of edematous pixels in the central
slice, and the nine surrounding slices did not change (Fig 4
Physiological Parameters
Proteinuria (UpV)
MRI allows the longitudinal, noninvasive examination of the brain, and
is the most powerful tool available to assess the number, the spatial
distribution and the size of brain lesions. It has demonstrated its
usefulness and is rapidly gaining importance as a clinical
diagnostic technique.12 18 Using
consecutive brain images, we were able to quantify edema as an increase
in pixel intensity in relation to a defined baseline threshold. In
SHRSP, primary and secondary lesion site and size are variable, and
often spread from the primarily affected side of the cerebrum to the
contralateral side. Therefore we had to relate pixel intensity in the
cerebrum to pixel intensity in a reference area outside the cerebrum.
In the hypertensive rat the cerebellum is always free of
edema.7 19 This was confirmed in the present
study, validating the use of this area as a reference. Use of this
reference area also allowed correction for inter-imaging variability.
This approach allowed us to quantitatively analyze the
progression and regression of cerebral edema, providing a powerful
technique for future studies.
The SHRSP developed cerebral edema at an age of about three months,
that is at a median of 30 days (range: 8 to 54) after the start of salt
loading. The lesions on T2W MRI were characterized by hyperintense
pixels as a result of T2 prolongation. T2 prolongation, a sensitive MRI
indicator of cerebral injury, is associated with
edema.12 16 20 Occasionally, hyperintense pixels
were combined with hypointense pixels, the latter resulting from T2
reduction, indicating hemorrhage. T2 reduction is
consistent with the paramagnetic effects of
deoxyhemoglobin.21 Extensive cerebral
hemorrhage at day 0 was uncommon (3/23 rats). At later times
hemorrhage sometimes did appear, both in the originally
affected area or in more recently affected areas.
Repeated cerebral MRI measurements in our model revealed two important
points. First, approximately 70% of rats developed changes in T2W MRI
before they showed neurologic symptoms. Either the affected brain areas
were not involved in motor function or they were not large enough to
cause disorders in behavior. Thus, T2W MRI is a powerful tool in the
temporal definition of the onset of cerebral lesions. Second, we
observed that the spreading of edema was from a single focus.
Initially, local spreading of edema occurred in gray matter of the
cortex or in the striatum. Then, via the white matter, edema spread
through the whole hemisphere, sometimes even reaching the contralateral
hemisphere via the corpus callosum. The focal origin of cerebral edema
was in contrast to the multifocal leakage sites found in SHRSP by
Fredriksson et al22 23 24 25 through use of
histological techniques. However, the spreading pattern
we observed concurs with their findings. The SHRSP in their
studies22 23 24 25 were considerably older (5 to 9
months of age) than those in our study (2 to 4 months) and therefore
will probably have had edema for a considerably longer period. This may
explain why they did not observe a unifocal origin of edema. Persistent
high NaCl intake after the initial appearance of cerebral edema
resulted in a more than twofold increase in the amount of edema in 1
week. This was the result of an increase of edema both in the slice
that was primarily affected and in the slices surrounding this central
slice. This could mean either that the primary lesion increases in size
or that vascular leakage occurs at multiple sites in close proximity
(within 1 mm) to the original lesion. This could not be
differentiated due to the chosen slice thickness of the MRI sequence.
Alternatively, the capacity to remove extravasated plasma proteins and
fluid25 may gradually become a limiting factor.
This sequence of early pathological changes in the cerebrum of SHRSP
provides a template to which the timing and effect of therapeutic
measures can be related.
The steep increase of proteinuria in the last 2 weeks before the
appearance of cerebral lesions is probably due to intrarenal changes
rather than to the hypertension per se because blood pressure did not
increase over this period. The precedence of proteinuria to cerebral
edema has been recognized in SHRSP, but in the absence of noninvasive
visualization of the brain the time scale could not be
defined.7 8 28 29 Glomerular
filtration rate was not a marker of incipient cerebral damage because
it showed a significant decrease only after day 0. In humans, the
magnitude of proteinuria is a valuable prognostic marker for
progression of malignant hypertension26 and renal
glomerular disease.27 Both in
nondiabetic and in non–insulin-dependent diabetic subjects proteinuria
also independently predicted stroke.9 However,
because of the insidious progression of cerebral damage leading to
stroke, it has not been possible to determine the interval between the
initial occurrence of proteinuria and the appearance of stroke. In the
present study we were able to define the appearance of the first
lesion accurately and thereby identified a proteinuria level of 40 mg/d
above which the SHRSP develops brain edema within a median of 9 days
(range, 3 to 15 days). It should be emphasized that part of the
variability in the time for the development of cerebral edema following
proteinuria is linked to the protocol, particularly at the lower end of
the range. Because urine was collected at weekly intervals and MRI was
performed at intervals of 3 to 4 days, it is quite feasible that an
interval between proteinuria and cerebral edema was underestimated.
Every rat showed proteinuria exceeding 100 mg/d prior to the first
appearance of cerebral edema on T2W MRI. To the best of our knowledge,
such intervals and thresholds have not been described previously in
this model. Even though in patients with severe hypertension the
interval will be much longer, it may be feasible, by combining these
two noninvasive measurements (proteinuria and cerebral T2W MRI), to
define a threshold of proteinuria and the time-span between the moment
of exceeding this threshold and the appearance of cerebral edema.
It is known that sodium intake plays a crucial role in determining
hypertension and that it is positively correlated with a high incidence
of stroke.3 4 5 Benstein et
al15 and Lax et al30 showed
that in uninephrectomized SHR reduced salt intake blunted the increase
in proteinuria and the severity of
glomerulosclerosis, without resulting in a
reduction in hypertension. The present study shows for the first
time that reduction of salt intake stabilizes the amount of edema.
Cerebral areas which showed a reduction in cerebral edema from day 0 to
day 7 did not show any histological sign of damaged
neurons, pointing to improved viability of the tissue. As others found
in SHR, normalization of salt intake did not decrease blood pressure in
SHRSP. In fact, systolic blood pressure tended to increase, as
has been observed in a number of other rat models after reduction of
dietary salt.31 Although tail-cuff pressure
cannot be used to accurately measure small changes in blood pressure,
the latter finding makes it unlikely that a change in
arterial pressure contributed to the stabilization of the
amount of cerebral edema. We have no direct evidence, but speculate
that following blood-brain barrier disruption salt intake in relation
to excretion by the diseased kidney determines the development of
cerebral edema, as is the case in peripheral tissues.
Another explanation could be that the blood-brain barrier, which has
been shown to be disrupted in the malignant phase of
hypertension,32 was restored.
While the rats were on a high salt diet, cerebral edema
correlated well with proteinuria, pointing to a parallel variation in
the permeability of cerebral vasculature and renal
glomerular capillaries. Of course, this does not imply a
causal relation between proteinuria and cerebral edema. Indeed,
stabilization of cerebral edema, due to normalization of sodium intake,
was accompanied by a marked reduction in proteinuria. Possibly, once
sodium intake is normalized, the SHRSP is able to achieve sodium
balance with restitution of preglomerular vascular
resistance. Maintenance of preglomerular vascular
resistance prevents glomerular
hypertension,33 and glomerular
hypertension is a prerequisite for proteinuria in hypertensive renal
disease.34
In conclusion, identifying cerebral edema by repeatedly performing T2W
MRI in salt-loaded SHRSP revealed that in this model proteinuria
invariably precedes cerebral edema and that cerebral edema often
precedes neurological symptoms. Quantitative assessment of the
percentage of the cerebrum that contained edema enabled us to correlate
cerebral edema with proteinuria. In addition, normalizing sodium intake
prevented progression of cerebral edema and decreased proteinuria.
These effects, which were not due to a decrease of the blood pressure,
may be related to restoration of the extracellular fluid volume so that
in the cerebrum extravasation of fluid through the damaged blood-brain
barrier is balanced by removal and in the kidney glomerular
capillary pressure is normalized by restoration of the upstream
vascular resistance. These mechanistic hypotheses await further
study.
Received August 25, 1997;
revision received October 14, 1997;
accepted October 17, 1997.
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Department
of Physiology and Biophysics,
University of Nebraska Medical Center,
Omaha, Nebraska
Using MRI, the investigators examined the development of cerebral edema
in stroke-prone spontaneously hypertensive rats (SHRSP) fed a 1% salt
diet. Histological evaluation of the brains was
completed to confirm the diagnosis of cerebral edema, and the amount of
protein in the urine was measured at various time intervals. The
authors reported a positive correlation between proteinuria and the
development of cerebral edema in SHRSP fed a high salt diet. For the
most part, this increase in cerebral edema preceded the development of
behavioral dysfunction. In contrast, when salt intake was normalized,
there was no significant progression of cerebral edema, and proteinuria
decreased markedly. Thus, it appears that normalization of salt intake
dramatically affects the development of cerebral edema and
proteinuria.
There are three important findings from this study. First, it appears
that MRI is an appropriate methodology for the detection of cerebral
edema in rats. In fact, MRI often predicts the development of cerebral
edema prior to neurological dysfunction. Second, there is a positive
correlation between proteinuria and cerebral edema. Thus, a relatively
noninvasive method can be used as a predictor for the development of
cerebral edema during chronic hypertension. Third, normalization of
salt intake attenuates the development of cerebral edema and reduces
proteinuria. Thus, changes in salt intake in high-risk individuals may
have important consequences for the pathogenesis of cerebral edema and
perhaps stroke.
Received August 25, 1997;
revision received October 14, 1997;
accepted October 17, 1997.
2.
Elliot P. Observational studies of salt and blood
pressure. Hypertension.. 1991;17:I-3-I-8.
3.
Yamori Y, Nara Y, Tsubouchi T, Sogawa Y, Ikeda K, Horie
R. Dietary prevention of stroke and its mechanisms in
stroke-prone spontaneously hypertensive rats: preventive effect of
dietary fibre and palmitoleic acid. J
Hypertens.. 1986;4:S449–S452.
4.
Okamota K, Yamori Y, Nagaoka K. Establishment of
the stroke-prone spontaneously hypertensive rat (SHR).
Circ Res.. 1974;35:143-153.
5.
Nagaoka A, Iwatsuka H, Suzuoki Z, Okamoto K.
Genetic predisposition to stroke in spontaneously hypertensive
rats. Am J Physiol.. 1976;230:H1354–H1359.
© 1998 American Heart Association, Inc.
Original Contributions
Proteinuria Precedes Cerebral Edema in Stroke-Prone Rats
A Magnetic Resonance Imaging Study
Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Background and Purpose—Stroke-prone
spontaneously hypertensive rats (SHRSP) subjected to high sodium intake
develop severe hypertension, cerebral edema, and proteinuria,
culminating in organ damage and early death. MRI, which can be applied
serially, provides the unique opportunity to study temporal and
quantitative relations between these changes and whether diminution of
sodium intake can attenuate established cerebral edema.
Key Words: brain edema • magnetic resonance imaging • proteinuria • sodium, dietary • rats
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Sodium intake plays a
crucial role in determining hypertension1 2 and
the occurrence of stroke.3 4 5 Dietary salt
restriction reduces the risk and mortality of
stroke.6 The SHRSP is an experimental model of
malignant hypertension and has a high incidence (80%) of
cerebrovascular disease.7 Elevation of sodium
intake from 8 weeks of age onward accelerates the increase of blood
pressure and the appearance of cerebral vasogenic edema in
SHRSP.8 Thus, the young SHRSP is an appropriate
model for studies of sodium intake–related development of malignant
hypertension and cerebrovascular disease.7 8 In
addition to cerebral vasogenic edema the SHRSP develops
proteinuria,7 8 which is recognized as being an
important prognostic marker for the occurrence of
stroke.9
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Animals
Male SHRSP (n=23), age 6 weeks, were obtained from IFFA Credo,
L'Arbresle, France. They were housed in constant conditions
(day/night: 12/12; humidity, 55%; temperature, 22° C), given free
access to a standard rat chow (RMH-TM rat chow: protein 22.2%; fat
4.8%; potassium 0.85%; sodium 0.40%; Hope-Farms), and allowed water
ad libitum. The protocol was approved by the Utrecht
University Board for study in experimental animals.
Baseline measurements were collected in all rats at 7 weeks of
age to determine the standardized threshold of brain edema in each
individual rat (see below). Subsequently, at the age of 8 weeks all
rats were switched to a high salt intake by 1% NaCl (170 mmol/L)
in drinking water in order to accelerate the appearance of cerebral
edema.8 The rats were observed daily for overt
neurological symptoms and underwent weekly blood pressure measurements,
T2W MRI of the brain, and metabolic studies, the latter to
assess sodium intake, renal function, and proteinuria (see below). This
was continued until the detection of cerebral abnormalities with T2W
MRI (defined as day 0). If a rat showed behavioral dysfunction,
intercurrent imaging was performed immediately.
For the weekly metabolic studies, rats were weighed,
then housed individually in metabolic cages for 24 hours.
During these measurements, food and water intakes were measured and
urine was collected. Urine volume and sodium, creatinine,
and protein concentrations were determined. To determine plasma
creatinine, blood from the tail artery was collected in
heparin prior to an MRI session and immediately centrifuged for
10 minutes at 3000g. Urinary protein was determined with the
Bradford method. Creatinine in plasma and urine was
determined calorimetrically. Systolic blood pressure was
measured weekly by tail-cuff plethysmography (IITC) in conscious rats
after prewarming at an ambient temperature of 37° C.
Directly after the last MRI session on day 7, the chests of the
anesthetized animals were opened, and a perfusion cannula was
inserted into the left ventricle of the heart for perfusion. A washout
with isotonic heparinized (270 IU/kg) saline was performed (2 to 3
minutes) and immediately followed by perfusion fixation with 4%
formaldehyde in 0.1 mmol/L phosphate buffer at a pressure two
thirds that of the last systolic blood pressure measured by the
plethysmography tail-cuff method. Ten-µm sections of the brain (10
sections at 500-µm intervals) were stained with hematoxylin/eosin and
luxol fast blue. Brains of rats that had died spontaneously were
collected in formaldehyde for histology.
After inducing anesthesia with 1% halothane in
N2O/O2 (70/30), rats were
intubated and mechanically ventilated during the MRI session with the
same mixture. Expiratory CO2 was monitored, and
body temperature was maintained at 37° C with use of a heated water
pad. To prevent movement, the rats were fastened in a
stereotaxic holder and positioned in a 4.7-T, 200- to
400-NMR spectrometer (SIS Co). A 120-mm Helmholtz coil was used for
both transmission and signal reception. After a sagittal scout image,
coronal multislice spin-echo T2W MRI, covering the whole brain (25
slices of 1 mm; TE/TR, 60/3000; matrix, 128x128; field of view,
40x40 mm; two transitions) was performed.
The brain was segmented from the surrounding image by
autocontouring, an analysis that automatically generates
contours by edge tracking on a binary mask set by an upper and lower
threshold of intensity values. From each animal a baseline SIIS was
collected before salt loading. In this SIIS, 25 coronal T2W images
(40x40x1 mm) rostral from the cerebellum were collected in such
a way that the rostral/caudal, left/right, and dorsal/ventral axes of
the brain were positioned parallel to the z, x, and
y axes of the magnet of the NMR spectrometer, respectively.
The four slices caudal to the cerebellum/cerebrum line were considered
a reference area where edema never occurred (REFerence area). This was
confirmed (see "Results"). The remaining part of the brain rostral
to this line until the last slice with a cortical area was
analyzed for the appearance of brain edema. From this brain
area and from the reference area the mean intensity (mi) of
the pixels was computed (p BRAINSIIS
mi and p REFSIIS
mi, respectively). The standard deviation (
) of the
baseline p BRAINSIIS mi
was also calculated.
In every succeeding MRI experiment, the mean pixel
intensity in the reference area, p REFexp mi, was calculated. Multiplying the
p REF exp mi
with p BRAINstandardized
threshold gave us the threshold of the experiment, pBRAINexp threshold. If an
individual pixel intensity in the brain was higher than pBRAINexp threshold it was
considered to be edematous. Cerebral edema formation is associated with
T2 prolongation.16 For
spatial evaluation of the progression/regression of edema, we
identified on day 0 the slice with the primary lesion site from where
edema would eventually progress or regress. The percentage of edematous
pixels was evaluated in ten slices that always fell within the cortex:
five caudal and four rostral to the central slice.
Data were evaluated by two-way ANOVA for repeated measurements,
followed by a pairwise multiple comparison procedure
(Student-Newman-Keuls method). Data are presented as mean±SEM.
P<.05 was considered statistically significant.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Development of Cerebral Lesions
Neurological Symptoms and Mortality
The majority of the rats (16/23) did not show overt behavioral
dysfunction at the time when first lesions on T2W MRI were observed,
although some reduction of activity and ruffling of the fur was usually
present. However, seven had a unilateral intermittent myoclonic
deviation of the head. All animals developed lesions within 30 days
(median) after starting 1% NaCl in the drinking water (range, 8 to 54
days).
By definition, all 23 rats showed areas of high signal intensity
on T2W MRI on day 0, and some also showed areas with low signal
intensity (3 of 23). With this scanning modality it is known that edema
is hyperintense (resulting from T2 prolongation), whereas hemorrhagic
patches are hypointense (resulting from the T2 shortening effect of
iron). This was confirmed histologically in the rats
sacrificed at day 0 (Fig 1
).
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Figure 1. Coronal T2W MRI (pixel resolution 0.31x0.31
mm, slice thickness 1 mm) of a rat sampled at day 0. a, Bregma
-0.20. Hyperintensity is seen in the right frontal cortex, right
cingulum, and corpus callosum. The right lateral ventricle is enlarged.
b, In the histological section of the hyperintense
right frontal cortex, multiple extracellular vacuoles give the tissue a
"spongy" appearance, pointing to the presence of edema.
(Magnification, x300.) c, In the histological section
of the ipsilateral frontal cortex, where hyperintense pixels are
absent, no signs of damage were observed. (Magnification, x300.) d,
Bregma -0.92. Hyperintensity is seen in the right capsula externa,
left and right cingulum, corpus callosum, and the left and right
fimbria hippocampus. e, In the histological section of
the hyperintense right capsula externa, the fibrous white matter is
distended, indicating volume expansion as a result of edema.
(Magnification, x300.) f, In the histological section
of the ipsilateral (not affected) left capsula externa, the white
matter is not rarefied. (Magnification, x300.) shows typical consecutive T2W MRI
made in one rat, illustrating the temporal development of cerebral
damage in the salt-loaded SHRSP. All primary lesions, and therefore the
definition of the central slice (see below), occurred in the grey
matter, mainly in the frontal or forelimb area of the cortex or in the
caudate putamen within a 4-mm window (from bregma -1.80 to bregma
2.2017 ). From there the edema invariably
progressed to the ipsilateral white matter. In some cases edema spread
via the corpus callosum to the contralateral white matter. Edema was
never observed in the cerebellum. In sites with vasogenic edema
secondary hemorrhagic patches occasionally occurred.
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Figure 2. Typical MR images of the temporal development of
chronic cerebral lesions in the salt-loaded SHRSP (group 1). Cerebral
slices (1 mm thickness) positioned to the stereotaxic
reference point bregma (row) at successive time points (column). Day 0:
first lesion detected on T2W MRI. The first cerebral lesions normally
occurred in the grey matter of the occipital cortex, thalamus, and
striatum. In this case the first lesion was in the frontal cortex of
the right hemisphere (bregma 2.20). The edema then progressed to the
ipsilateral white matter and finally via the corpus callosum to the
ipsilateral white matter. A small hemorrhage was observed in
the frontal cortex on day +7. The lateral ventricles were
enlarged. shows the percentage edematous
pixels in the cerebrum, which spanned 13 to14 slices, and included
those slices rostral to the line cerebellum/cerebrum until the first
slice without cortical area. Edema was determined in each rat in
relation to an individual threshold, defined as the mean pixel
intensity ± twice the standard deviation at baseline.
Consequently, 5% of the pixels lie outside this range, by definition
2.5% above and 2.5% below this range. The pixels above this range are
therefore (by definition) "edematous" at baseline. At day 0,
5.8±1.1% of the pixels in the examined part of the brain of the rats
in group 1 and 4.9±0.5% of the pixels in the rats in group 2 were
edematous. These numbers were not different, but both differed
significantly from the preceding time points (day -3 and baseline;
P<.05 versus day 0). Fig 4
shows the distribution of the percentage of edematous pixels in the
slice that showed the primary lesion site on day 0 and its surrounding
slices.
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Figure 3. Changes of the percentage of cerebral edema in the
rats that stayed on high salt intake ( 
) and the rats switched to
normal salt intake (before switch 
, after switch 
). Values are
mean±SEM. +P<.05 vs day 0; #P<.05 vs
normalized salt intake.
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Figure 4. The percentage edema per slice in the slice that
showed the primary lesion (slice 0) and in surrounding slices. Symbols
as in Fig 3 
. Values are mean. +P<.05 vs day -3;
#P<.05 vs normalized salt intake.
At baseline SHRSP had a systolic blood pressure of
~200 mm Hg, which rose to ~245 mm Hg at day 0
during salt loading (P<.05) (Table). Body weight increased
from baseline to day -7. However, from day -7 to day 0 growth was
stunted, and some rats lost weight. Food intake increased until day -7,
but from day -7 to day 0 food intake decreased. Water intake and urine
production increased from baseline to day 0
(P<.05). The glomerular filtration rate
increased slightly from baseline to day -14 and then stabilized to day
0. Sodium intake, both from water (1% Na/l) and food (0.4% Na/kg
food), increased significantly from baseline to day 0. Sodium excretion
followed sodium intake closely (Table). There were no differences
between groups 1 and 2 in this stage.
View this table:
[in a new window]
Table 1. Body Weight, Systolic Blood Pressure, Food and Water
Intake, Urine Volume, Glomerular Filtration Rate, and
Sodium Intake and Excretion
From baseline to day 0, proteinuria (UpV, Fig 5) showed a steep increase and at day 0
UpV was 300±28 mg/d (n=17), and differed significantly from all
preceding days in both groups 1 and 2. All rats developed cerebral
vasogenic edema within a median of 9 days (range: 3–15 days) after
reaching a UpV-level of 40 mg/d. The lowest level of proteinuria at
which a rat showed cerebral edema was 169 mg/d. A linear relation was
found between UpV and the percentage of edematous pixels in the whole
brain. This evaluation included data points from all the rats before
and on day 0, as well as those maintained on the high salt diet between
day 0 and day +7 (group 1). Linear regression led to the following
relationship: percentage cerebral edema=2.02 + (0.0196 x proteinuria in
mg/d) (R=0.619; P<.0001) implying that for approximately
every 50 mg/d increase in proteinuria there was an increase of 1%
edematous pixels in the brain. The intercept of approximately 2% was
mainly due to the high intensity of the fluid-filled ventricles. Free
fluid has a long T2-relaxation time.
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Figure 5. Proteinuria (mg/d). Symbols as in Fig 3 . Values
are mean±SEM. +P<.05 vs day 0; #P<.05
vs normalized salt intake.
Neurological Symptoms and Mortality
Of the 10 rats in group 1 maintained on the high salt diet, two
died spontaneously within a week. Switching back to a normal salt
intake in group 2 tended to ameliorate the condition of the rats. Three
out of four rats that showed unilateral intermittent myoclonic
deviations of the head at day 0 showed regression of this behavior. No
mortality occurred and all rats in group 2 (n=7) could be sacrificed
for histology as scheduled.
Rats in group 1 showed a progressive increase of the percentage of
edematous pixels (day 0: 5.8±1.1%; day +3: 10.8±1.8%; day +7:
12.5±2.8%, both P<.05 versus day 0; Fig 3). In group 2
brain edema showed a dynamic pattern of reduction (3 out of 7),
reappearance, stabilization or even progression (Fig 6). The net result was a slight, but not
significant, increase in edematous pixels (day 0: 4.9±0.5%; day +3:
6.0±1.9%; day +7: 6.9±1.3%. At days +3 and +7 the mean percentage
edematous pixels in group 2 differed significantly from that observed
in group 1 (P<.05). Only 2 out of 10 rats which stayed on a
high salt intake showed a transient reduction of lesion size compared
to a previous MRI session. Histological verification at
day +7 in group 2 showed that areas with a normalization in
T2W-pixel intensity, compared to the preceding
MRI session, showed no edema. In group 2 cerebral hemorrhage
stabilized, whereas in group 1 it tended to progress.
View larger version (154K):
[in a new window]
Figure 6. Typical MRI images of the temporal development of
cerebral lesions in the salt-loaded SHRSP (group 2), showing the
dynamic behavior of regression and progression of cerebral edema after
normalizing salt intake on day 0. For definition of rows and columns
see Fig 2 . The first cerebral lesions occurred in the left caudate
putamen (bregma 2.20). At day +3 the cerebral lesions had almost
completely disappeared. At day +7 lesions reappeared. Note that the
overall pixel intensity at baseline was increased because of a
different setting of the MR spectrometer. In the quantitative
analysis, this type of difference is eliminated by use of the
standardized threshold (see "Materials and Methods").) at day
+3. At day +7 there is an increase of the percentage of edematous
pixels in the central slice and the directly surrounding slices. This
analysis revealed that in this model increases or decreases in
brain edema are due to increased or decreased leakage or spreading from
within one affected area, and not from widespread multiple foci.
At day +7 blood pressure in group 1 was not different from that
found at day 0. Even though the salt intake was normalized in group 2,
blood pressure did not change significantly (Table). After day 0 body
weight continued to decrease in both groups 1 and 2, reflecting the
poor physical condition of the animals. In group 1 food intake
continued to decrease, but in group 2 food intake increased again to
the level found at day -7 and differed from the rats in group 1
(P<.05). In group 2 water intake and urine
production were reduced at day +7 as compared to day 0, and
differed significantly from the high salt intake group at day +7
(P<.05). In group 1 glomerular filtration rate
decreased from day 0 to day +7 (P<.05), but in group 2
glomerular filtration rate remained stable from day 0 to
day +7 and differed significantly from group 1 (P<.05,
Table). In group 1 sodium intake stabilized at day +7, whereas in group
2 sodium intake and excretion decreased significantly from day 0 to day
+7 as a result of the absence of sodium in the drinking water
(Table).
UpV tended to increase from day 0 to day +7 in group 1 (from
305±44 to 338±29 mg/d; NS). However, in group 2 UpV decreased
markedly at day +7 (from 294±24 to 119±10 mg/d; P<.05)
and differed significantly from group 1 (P<.05, Fig 5).
Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
In this study cerebral edema in SHRSP was quantitated by
semi-automatic segmentation of images collected by T2W MRI. This novel
analysis clearly illustrates that in this model variation in
brain edema is due to increased or decreased leakage or spreading
within one affected area, and not from widely-scattered multiple
primary foci. Cerebral edema was positively correlated with proteinuria
during salt loading, and an excretion exceeding 40 mg/d preceded
cerebral edema by 3 to 15 days. Furthermore, salt restriction
stabilized cerebral edema and reduced proteinuria without an effect on
blood pressure.
Selected Abbreviations and Acronyms
SBP
systolic blood pressure
SHRSP
=
stroke-prone spontaneously hypertensive rats
SIIS
=
standard individual image set
T2W MRI
=
T2-weighted magnetic resonance imaging
Acknowledgments
For T2W MRI evaluation the brain image was automatically
segmented from that of surrounding tissue with use of an autocontouring
program kindly supplied by Max A. Viergever, PhD, and J.B. Twan Maintz,
PhD, Image Sciences Institute, University Hospital Utrecht.
The MRI studies were performed at the Netherlands in vivo NMR facility
(Bijvoet Center, Utrecht University), which is financially
supported by the Netherlands Organization for Scientific Research
(NWO). This study was supported by NWO grant 902–18-264 and by grant
93.174 from the Dutch Heart Foundation.
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
1.
Elliot P. Observational studies of salt and blood
pressure. Hypertension. 1991;17(suppl 1):I-3–I-8.
Editorial Comment
A Magnetic Resonance Imaging Study
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Several studies1 2 3 have suggested that sodium intake
contributes to the pathogenesis of chronic hypertension and stroke.
Chronically hypertensive rats fed a diet high in sodium develop severe
hypertension, cerebral edema, and proteinuria.4 5 However,
the temporal relation between the development of these symptoms and
sodium intake remains unclear. The purpose of the preceding study was
to determine (1) the development of cerebral lesions in relation to
neurological symptoms in rats fed a high-salt diet, (2) the relation
between proteinuria and the development of cerebral edema, and (3)
whether normalization of salt intake affects the progression of
proteinuria and cerebral edema.
Selected Abbreviations and Acronyms
SBP
systolic blood pressure
SHRSP
=
stroke-prone spontaneously hypertensive rats
SIIS
=
standard individual image set
T2W MRI
=
T2-weighted magnetic resonance imaging
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
1.
Sadoshima S, Busija D, Brody M, Heistad D.
Sympathetic nerves protect against stroke in stroke-prone
hypertensive rats. Hypertension.. 1981;3:I-124-I-127.
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