Journal of Cerebral Blood Flow and Metabolism
4:103-106
© 1984 Raven Press, New York
Regional Cerebral Blood Flow in Conscious Stroke-Prone
Spontaneously Hypertensive Rats
Kent Fredriksson, *Martin Ingvar, and Barbro B. Johansson
Department of Neurology and *Laboratory for Experimental Brain Research, University of Lund, Lund, Sweden
Summary: Regional cerebral blood flow (rCBF) was
measured autoradiographically with [14Cliodoantipyrine
as a diffusible tracer in two strains of conscious normo­
tensive rats (Wistar Kyoto and local Wistar) and in two
groups of spontaneously hypertensive stroke-prone rats
(SHRSP) with a mean arterial pressure (MAP) below or
above 200 mm Hg, In spite of the large differences in
arterial pressure, rCBF did not differ significantly be-
tween the hypertensive and the normotensive groups in
any of the 14 specified brain structures measured. How­
ever, rCBF increased asymmetrically within part of the
caudate-putamen in two of nine SHRSP with a MAP
above 200 mm Hg, indicating a regional drop in the ele­
vated cerebrovascular resistance. Key Words: Cerebral
blood flow-Focal hyperemia-Spontaneously hyper­
tensive rats.
Whether a spontaneous increase or decrease in
the cerebral blood flow (CBP) occurs in chronic
hypertension in the absence of ischemic or hemor­
rhagic brain lesions is still debated. A rise in blood
pressure induced by an i.v. injection of angiotensin
increases CBP in normotensive as well as in chron­
ically hypertensive individuals, although higher
blood pressure levels are required in hypertensive
individuals (Strandgaard et aI., 1973). Studies on
spontaneously hypertensive rats (SHR) (Okamoto
and Aoki, 1963) and a stroke-prone substrain
(SHRSP) (Okamoto et aI., 1974) are conflicting
even if most investigations indicate that CBP is un­
changed. A spontaneous increase in CBP during the
established phase of chronic hypertension has not
so far been demonstrated. In contrast, two studies
have reported that regional CBP (rCBP) may de­
crease at high blood pressure levels. Thus, with an
invasive hydrogen clearance technique, Yamori and
Horie (1977) observed a decrease in CBP in con­
scious SHRSP when the systolic blood pressure ex­
ceeded 200 mm Hg. Kozniewska et ai. (1982) made
similar observations in SHR using the intracarotid
133Xe technique for CBP determination.
Because of this controversy we measured rCBP
in conscious, minimally restrained normotensive
rats and SHRSP using a noninvasive technique with
high spatial resolution (Sakurada et aI., 1978)' to de­
termine whether spontaneous flow disturbances
were present in localized areas. SHRSP were di­
vided into two subgroups with a mean arterial pres­
sure (MAP) above and below 200 mm Hg to reveal
possible blood pressure-related rCBP changes
within the same breed of hypertensive rats. None
of the SHRSP used in this study showed signs of
brain lesions (e.g., drowsiness, hemiparesis, sei­
zures). Two different strains were used as normo­
tensive controls. The Wistar Kyoto rat (WKY ) is
regarded as the best control for SHR and SHRSP
(Nishiyama et aI., 1976; Nordborg and Johansson,
1980). The local Wistar rat (LWR) has previously
been used for rCBP measurements in conscious,
minimally restrained rats with the same technique
as that used in the present study (Dahlgren et aI.,
1981).
Address correspondence and reprint requests to Dr. Jo­
hansson, Department of Neurology, University Hospital, S-22l
85 Lund, Sweden.
Abbreviations used: LWR, Local Wistar rats; MAP, mean ar­
terial pressure; SHR, spontaneously hypertensive rats; SHRSP,
stroke-prone spontaneously hypertensive rats; rCBF, regional
cerebral blood flow; WKY, Wistar Kyoto rats.
MATERIAL AND METHODS
Animals
The experiments were performed on SHRSP and WKY
from our own colony, 4-8 months old, and age-matched
LWR supplied by M�llegaards Avlslaboratorium (Copen-
103
K. FREDRIKSSON ET AL.
104
hagen, Denmark) with body weights (mean ± SD) of 272
± 8 g (SHRSP, MAP> 200 mm Hg), 333 ± II g (SHRSP,
MAP < 200 mm Hg), 426 ± 17 g (WKY), and 349 ± 18
g (LWR). The animals had free access to food pellets and
tap water until the operation. The number of rats in each
group is given in Tables 1 and 2.
Morphological method
Brain sections between the sections used for autora­
diography were air-dried at room temperature, fixed in
Formalin, and stained with Luxol fast blue (0. 1%) for light
microscopy.
Operative technique
The animals were anesthetized with 3%, and thereafter
1%, halothane in NPI02 (2: 1) via a face mask. One tail
vein and the tail artery were cannulated after local an­
esthetic (lidocaine hydrochloride) had been infiltrated
subcutaneously around the base of the tail. Wound infil­
tration with the local anesthetic was repeated before
rCBF was measured in the conscious rats. The arterial
catheter was used for continuous MAP recording and for
blood sampling for the determination of pH, blood gases,
glucose, and hematocrit. The arterial catheter was cut to
a length of 35 mm before sampling blood for the deter­
mination of tracer concentration. The venous catheter
was used for the injection of heparin (300 IU kg-I) and
the radioactive tracer. Body temperature was recorded
with a small thermistor in the rectum. The rat was placed
in a Perspex cage after termination of anesthesia (for a
detailed description see Dahlgren et aI. , 198 1) and al­
lowed a 2-h recovery period before rCBF was deter­
mined.
Calculations
Regional CBF was calculated according to the method
of Sakurada et al. ( 1978). Statistical differences were
evaluated with analysis of variance using the Newman­
Keuls test to differentiate between groups. Confidence
limits for side differences were calculated on the 99.9%
probability level using Student's t test. Values are given
as means ± SD.
Determination of rCBF
CBF was measured autoradiographically according to
Sakurada et al. ( 1978) using [14C]iodoantipyrine (New En­
gland Nuclear, Boston, MA, U. S. A.) as a diffusible
tracer. For details and modifications of the procedure,
see Abdul-Rahman et al. ( 1979) and Dahlgren et al.
( 198 1). The infusion time for the radioactive tracer, 45 s,
was chosen to be optimal for a rCBF of �1-2 ml g-I
min-I (Eklof et aI. , 1974). The 14C activity in the blood
was determined by liquid scintillation (Rackbeta 12 15,
LKB Wallac, Turku, Finland), and the efficiency in
counting was estimated after adding an internal standard
[14C]hexadecane (Radiochemical Center, Amersham, En­
gland) to each blood sample, which allowed for quench
correction (Ingvar et aI. , 1980). Optical density measure­
ments on the autoradiographs were made bilaterally on
at least three consecutive coronal brain sections (20 ILm
thick) on a specified level (300 ILm apart) using a trans­
mission densitometer (Macbeth TD 50 1, Newburgh, New
York, U. S. A.) with an aperture of 1 mm. The reading
points in different brain structures were chosen according
to a fixed schedule.
RESULTS
The rCBF measurement was made at a stable
MAP level, with Pao2 above 70 mm Hg and Paco2
close to 40 mm Hg. Blood gases, pH, glucose, he­
matocrit, and body temperature did not differ
among the groups. The MAP differed significantly
(p < 0.01) in all groups except for LWR and WKY
(Table 1). The rCBF of specified areas (Table 2) did
not differ significantly among the groups. The fig­
ures represent mean values of five to seven mea­
surements made on both sides of the brain. Only
minor side-to-side differences were seen, with the
exception of two rats with a MAP of 209 and 220
mm Hg. In these two animals distinct rCBF asym­
metry was observed mainly within the middle part
of the caudate-putamen, with a maximum increase
of 73 and 116% in the hyperemic areas compared
with the adjacent ipsilateral and the contralateral
caudate-putamen (see Fig. 1). These side-to-side
differences were outside confidence limits for asym­
metry (± 17.7%), i.e., they were not regarded as
incidental findings (p < 0.001). In one of these rats
rCBF was bilaterally increased within the most pos­
terior part of the caudate-putamen. No decrease in
rCBF was observed. No histopathological abnor­
mality corresponding to the hyperemic regions was
noticed.
Mean arterial pressure (MAP), Puco]> blood glucose, and hematocrit in
normotensive and spontaneously hypertensive rats before the reBF measurement
TABLE 1.
Experimental
group
LWR
WKY
SHRSP,
MAP < 200 mm Hg
SHRSP,
MAP> 200 mm Hg
No.
of
rats
MAP
(mm Hg)
±
Pac02
(mm Hg)
7
7
105
112
±
5
8
40.9
4\.3
7
166
±
13
9
220
±
7
±
Glucose
(fLmol ml-I)
1.5
1.5
6.8
5.8
41.2
± l.6
39.3
±
±
1.3
±
Hematocrit
(%)
0.8
± l.0
46.7
±
1.4
7.5
± l.4
49.3
±
1.4
7.8
± l.9
51.7
± l.0
LWR, local Wistar rats; WKY, Wistar Kyoto rats; SHRSP, stroke-prone spontaneously hyperten­
sive rats. Values are means ± SD. T he MAP differed significantly among the groups (p < 0.01) except
for LWR and WKY (analysis of variance and the Newman-Keuls test).
J Cereb Blood Flow Metabol, Vol. 4, No. I, 1984
rCBF IN CHRONIC HYPERTENSION
105
Regional cerebral blood flow in conscious normotensive and spontaneously
hypertensive rats measured autoradiographically with t4Cjiodoantipyrine as
a diffusible tracer
TABLE 2.
Region
LWR
n
7
=
Frontal cortex
Sensorimotor cortex
Parietal cortex
Cingulate cortex
Auditory cortex
Visual cortex
1.18
1.61
1.63
1.51
2.49
1.31
Caudate-putamen
Globus pallidus
T halamus
Hypothalamus
Hippocampus
Nucleus ruber
Substantia nigra
1.35
0.68
1.70
1.09
0.87
1.46
1.04
Cerebellar cortex
1.48
±
WKY
n
7
=
±
0.09
0.16
0.18
0.31
0.37
0.14
1.22
1.52
1.61
1.54
2.18
1.33
1.25
0.65
1.57
1.14
0.93
1.40
0.95
±
±
0.08
0.12
0.22
0.10
0.09
0.15
0.13
±
0.16
1.42
±
±
±
±
±
±
±
±
±
±
±
MAP
SHRSP,
< 200 mm Hg
n
7
=
±
0.12
0.09
0.12
0.10
0.22
0.11
1.27
1.65
1.74
1.72
2.17
1.42
1.23
0.62
1.72
1.11
0.97
1.37
1.00
±
±
0.07
0.06
0.14
0.08
0.08
0.16
0.10
±
0.13
1.56
±
±
±
±
±
±
±
±
±
±
SHRSP,
MAP> 200 mm Hg
n
9
=
0.15
0.13
0.22
0.26
0.44
0.12
1.32
1.68
1.79
1.76
2.25
1.38
±
0.21
0.10
0.20
0.15
0.14
0.14
0.18
1.28
0.74
1.72
1.03
0.97
1.31
0.98
±
0.13
1.54
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.17
0.23
0.23
0.20
0.24
0.20
±
0.16
0.16
0.18
0.20
0.13
0.15
0.11
±
0.23
±
±
±
±
±
LWR, local Wistar rats; W KY, Wistar Kyoto rats; SHRSP, stroke-prone spontaneously hyperten­
sive rats; MAP, mean arterial pressure. Values are means ± SD (in ml g I min -I). No differences
were significant at the 99% probability level (analysis of variance).
-
DISCUSSION
The P aco2 and glucose values were comparable
in the four groups and did not indicate that the rats
were unduly stressed. Furthermore, the blood pres­
sures recorded for SHRSP corresponded to values
obtained in our laboratory for freely moving
SHRSP of the same age with indwelling catheters
in the aorta (Nordborg et ai., 1983) and were there­
fore regarded as representative of SHRSP from our
breed and not excessively elevated by the experi-
mental conditions. The rCBF measured did not
differ significantly between the hypertensive and
the normotensive groups, and were in agreement
with previously reported values for conscious, min­
imally restrained LWR (Dahlgren et ai., 1981).
Structural arterial alterations including an en­
hanced media/lumen ratio of extraparenchymal ce­
rebral arteries are present in SHR and SHRSP
(Nordborg and Johansson, 1980). The increased
media/radius ratio is associated with a higher resis­
tance at any degree of smooth muscle shortening
FIG. 1. Spontaneous unilateral in­
crease in regional cerebral blood
flow (rCBF) mainly within part of the
caUdate-putamen (arrow) in a con­
scious, minimally restrained stroke­
prone spontaneously hypertensive
rat with a mean arterial pressure of
220 mm Hg. Maximum rCBF within
the hyperemic area was 2.27 ml g-1
min--1 as compared to 1.30 ml g-1
min-1 in the adjacent ipsilateral and
the contralateral caudatoputamen
where flow was within the normal
range. The dark area at the base of
the brain, which in contrast to the
h yperemic region within the cau­
date-putamen was not present in
consecutive sections, represents an
artefact (slice fold).
J Cereb Blood Flow Metabol, Vol. 4, No. I, 1984
106
K. FREDRIKSSON ET AL.
and an exaggerated maximal contractile strength
(Folkow, 1982). If this altered vessel geometry, to­
gether with other factors increasing vascular resis­
tance in chronic hypertension (Overbeck et aI.,
1980), is insufficient to normalize blood pressure
before it reaches the microcirculation, a break­
through in autoregulation with a regional increase
in blood flow could be expected. Whether a spon­
taneous increase in CBF occurs during the devel­
opment of chronic hypertension has, however, been
questioned.
The asymmetric hyperemic areas in two of nine
SHRSP with a MAP above 200 mm Hg were located
mainly within the caudate-putamen but did not
comprise the entire structure. In one rat, part of the
neighbouring globus pallidus was included in the
high flow area. The hyperemia was constantly found
over 8 to 10 consecutive section levels, and it is
thus unlikely that the findings represent technical
artefacts. It is concluded that the focal increase in
CBF represents a pathological cerebrovascular con­
dition spontaneously occurring in SHRSP at severe
degrees of chronic hypertension. With the reser­
vation that the light microscopic investigation was
not optimal because the brains had to be frozen in
situ for the rCBF measurement, the absence of
major histopathological changes indicates a break­
through phenomenon, i.e., the transmural pressure
exceeding the autoregulatory capacity of the ves­
sels.
In a longitudinal study on conscious SHRSP, Ya­
mori and Horie (1977) reported that rCBF, measured
with a hydrogen clearance technique, began to de­
crease in the frontal cortex at the age of 60 days
when the systolic blood pressure measured by a tail
cuff exceeded 200 mm Hg. At the age of 5 months,
frontal cortical blood flow was 0.64 ml g� 1 min� 1
in SHRSP with a systolic blood pressure of 230 mm
Hg, and 1.01 ml g�l min�l in WKY. The hyperten­
sive rats did not have any clinical signs, but since
no pathological investigations were performed until
the rats died spontaneously at a later age, subclin­
ical lesions cannot be ruled out. ManipUlating the
diet (e.g., as to protein and salt content) can mark­
edly influence the development of cerebrovascular
lesions in SHR and SHRSP (Hazama et aI., 1975;
Yamori et al., 1979), and with the Japanese rat chow
used by Yamori et aI. the incidence of stroke is very
high. Whether dietary factors and/or the presence
of subclinical brain lesions can explain the discrep­
ancy between the decrease in CBF shown by Ya­
mori and Horie (1977) and our results remains to be
determined.
Acknowledgment: We thank Karin Jansner and Karin
von Stedingk for skillful technical assistance. We also
J Cereb Blood Flow Metabol, Vol. 4, No. I, 1984
thank Claus Rerup, Institute of P harmacology, University
of Lund, for statistical advice. The study described here
was supported by grants from the Swedish Medical Re­
search Council (project 14X-4968), from Fredrik and In­
grid Thuring's Foundation, Elsa Schmitz's Fund for Neu­
rological and Neurosurgical Research, and from the Med­
ical Faculty, University of Lund.
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