AMERICAN JOUENAL 01 PEIYSZOLOOY Vol. 230, No. 5, May 1976. Printed in U.S A.
Genetic predisposition to stroke in spontaneously hypertensive
rats
AKINOBU NAGAOKA, HISASHI IWATSUKA, ZIRO SUZUOKI, AND KOZO OKAMOTO Biological Research Laboratories, Central Research Division, Takeda Chemical Industries, Osaka 532, and Faculty of Medicine, Kyoto University, Kyoto, Japan
NAGAOKA, AND Kozo
AKINOBU, OKAMOTO.
HISASHI
IWATSUKA,
ZIRO
SUZUOKI,
Genetic predisposition to stroke in spontaneously hypertensive rats. Am. J. Physiol. 230(5): 1356
1359. 1976. - Hypertension and stroke in spontaneously hypertensive rats (SHR) were investigated genetically using stroke-prone SHR (As), stroke-resistant SHR (C) and their hybrids, hybrid of A3 and C (F,), offspring of F, x F, (F2), and those of backcrossing of F, to the respective parental strains, BC(F, x A,) and BC(F, x C). The average blood pressure measured without anesthesia increased in the following order during the experimental period: C < BC (F, x C),< F, = Fr < BC(F, x A,) c A,. The F2 represented a wider spread of variation than the F,, with some of the pressure extending into the range of both parental strains. When the drinking water was replaced with a 1% salt solution, the blood pressure increased and the onset of stroke markedly accelerated in all groups of SHR. Under the hypertensive conditions, the incidence of stroke was associated with A,-gene concentration rather than with the level of blood pressure. Similar but less dramatic effects of salt were observed in another series of hybrid groups derived from A, and normal Wistar-Kyoto rats. These findings suggest that the genetic factors are of great importance in the development of stroke as well as hypertension in the SHR. cerebral hemorrhage; stroke; salt sensitivity
cerebral
infarction;
genetic
factors
in
IN 1963, SPONTANEOUSLY HYPERTENSIVE RATS (SHR) regarded as a useful animal model for essential hypertension were isolated from the Wistar-Kyoto rat colony by Okamoto and Aoki (15). Later, the SHR colony was further separated by Okamoto and his colleagues into several sublines (16), some of which had a tendency to develop cerebrovascular lesions (cerebral hemorrhage and/or infarction), otherwise known as a stroke. Recently, “stroke-prone SHR” was established by Okamoto et al. (18) from the sublines of A3 and A,-sb. The strokeprone SHR showed a spontaneously high incidence of the cerebrovascular lesion (CVL). In contrast, a subline C could hardly develop the CVL in spite of the presence of hypertension and was called “stroke-resistant SHR.” It is well known that high blood pressure is one of the causative factors of stroke in the human (2, 8-10, 19, 21) and that abnormally high salt intake results in an elevation of the blood pressure in both men and experimental animals (3,4,6,23). As expected, it was recently demonstrated that increased salt intake accelerated the onset of CVL in the stroke-prone SHR (7). In the present
studies, effects of increased salt intake on the CVL were examined in the SHR with different genetic constitutions in order to vallidate a predisposition to the CVL in the stroke-prone SHR, A3. METHODS
The offspring (Fz7-Fz8) of SHR sublines were introduced to our laboratories from Kyoto University in 1972 and had since been bred by selective sibmatings. The two kinds of SHR used in the present study were of the F,, generation in the stroke-prone SHR (A,) and F,, generation in the stroke-resistant SHR (0. These SHR and Wistar-Kyoto rats (WK), used as normotensive control rats, originated from the same ancestors. The degree of inbreeding in these strains was the same as the previous report (18). Animals of F,, FP, and backcrossing (BC) hybrids were produced by reciprocal matings of A, x C and A3 x WK. The males were used in the experiments, because it had been reported that the incidence of stroke was much higher in the males than in the females of the stroke-prone SHR (18). The procedure in the mating of A3 x C and theoretical gene concentration are shown in Fig. 1. The same procedure was used in the mating of A, x WK. However, backcrossing of the F, to WK was not done because the F, rats as well as WK showed no incidence of stroke as described in the item of results. Rats were kept five or six in a cage until 7 wk of age under the controlled conditions of temperature (23. 24”C), humidity (50-60%), and light (7:30 A.M.-~:OO P.M .) and were given a laboratory chow, CA-l, Japan CLEA, containing 0.4% salt and drinking water ad libiturn. Animals in each hybrid group were divided into two groups at 8 wk of age, which were given tap water and 1% salt solution as the drinking fluid for 12 wk (Table 1). In the process for allocating rats into the two groups, it was considered that rats from the same litter were divided into the two groups in about the same ratio, and especially in the series of A3 x WK the rats were assigned more to the salt-loaded group than to the control group, as it was anticipated that the CVL would hardly develop in the latter group for this experimental period. Some of the rats died spontaneously during the experimental period. The organs from these rats and survivors, sacrificed at the beginning of the 13th wk, were fixed with 10% Formalin. The brains were cut in four or five frontal sections for the macroscopic observation to
1354
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GENETIC
PREDISPOSITION
TO
1355
STROKE
following
order: C c BC(F,
x
C) < F, = F, < BC(F,
x
A3) c AB. Blood pressure above 200 mmHg developed in AS, BC(F, x AS), F1, and F, at 2.5,4.5, 6, and 7 wk after the start of the experiment,
respectively,
although
only
the pressure of A3 elevated to 230 mmHg by 7 wk. On the other hand, the blood pressure of BC(F, x C) and C did not reach this level (200 mmHg).
FIG. 1. Mating procedure between stroke-prone SHR (A,) and -resistant SHR (C). Theoretical average gene constitution in each group also is represented, and ratio (%) of A, and C genes is as follows. A, (A, : 100); C (C : 100); F, (A, : 50, C : 50); F, (A, : 50, C : 50) backcross to A, (A, : 75, C : 25); and backcross to C (A, : 25, C : 75). TABLE 1. Number of rats in parental hybrid groups used
Parental
Strains
SHR
A, BW’, WA, F,(F, BC(F,
SHR
A,) C) F,) C)
C BC(F,’ x A,) FI’(A, x WK) JWY x FJ Normal WK
and their
C, although
the mean appeared to be closer to C than
A,. The F, was also generally intermediate but represented a wider range than the F,. Moreover, the blood pressure in the F2 tended to distribute continuously between the parents, and some of their pressure mea-
No. of rats
and Types of Hybrid
x x x x
strains
The blood pressures in AS, C, and their F, and F, hybrid groups are shown in Table 2 in order to determine whether differences between the means indicate different populations sampled. A, had a larger variation than C, but a highly significant difference was observed between the parental strains. The blood pressure of the F, was intermediate between the parents and was significantly lower than that of A, and higher than that of
Tap water
1% salt
12 5 12 12 5
12 12 12 16 12
12 0 12 0 12
12 22 12 24 12
llO=,
A3
C 1
I
a
xA3) I
1
1
1
I
1
L
al
detect the CVL, and then embedded in paraffin to prepare the histological sections, 5 pm in thickness, which were stained with hematoxylin-eosin staining for microscopic observation. Blood pressure was measured weekly using a tail-pulse-pickup method (13) in unanesthetized rats. The animals were previously warmed for 10 min in a chamber which was maintained at 37-38”C, and then they were placed into a rat holder. The average of three recordings of blood pressure taken at approximately 30-s intervals at their rest condition was recorded as systolic blood pressure of the rat. The variation in the three recordings was less than 5%. Volume intake of the drinking water was also measured weekly.
Rats were observed every day for clinical
* E E
110 250
.-u 'zi Y
150
$llO-,
BC(Fq
BC(FjxC)
2509 ........., .... ........ ........ ... ....... - ... ............. ........ .. .. .......... ... ....... ..... ... ..... .......... ............... ...... .. 200-
symptoms
suggestive of stroke or hypertensive encephalopathy, which could be detected visually. Arithemetical means, standard deviation and standard error of the mean were calculated. The difference between the means from two independent samples was analyzed by the Student unpaired-t-test. RESULTS
Hood pressure.
10
O‘n tap water, the systolic blood pres-
sure differed among the six groups in the series of crosses between A3 and C (Fig. 2). Throughout the experimental period, the pressure level was higher in the
lime
0
5
10
(Weeks)
FIG. 2. Time course of systolic blood pressure (BP) in rats receiving a tap water (O0) or 1% salt solution (0-O). Elevation of blood pressure was accelerated by salt loading in all groups except normal WK group. Values are means t SD.
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1356
NAGAOKA,
TABLE 2. Statistical andlysis of blood pressures in As, C, and their F, and F, hybrids Time After Start of Experiment, 2 207 -t 12 171 2 7 187 -e 6 181 + 11
4 212 180 188 192
u 12 + 7 2 8 _+ 15
6 222 185 196 189
8
2 15 ” 8 -+ 12 -’ 15
231 189 203 199
+ 14 2 6 of: 9 2.15
-t
drinking
12 236 197 210 209
+ + f -+
13 10 7 19
8.93* 5.14* 6.11* 1.65
7.94* 5.73*
7.59* 4.69*
2.62t
2.63t
4.82
1.26
9.27* 5.12* 3.60$ 0.72
8.91* 3.84* 5.28* 1.90
OKAMOTO
tap water except for one rat in the A3 group.
The blood pressure of this exceptional
8.02* 5.94* 3.69$ 0.17
Blood pressure values are means * SD. n, number of animals. The number in A, group *p 0.05). The F,’ showed only a slight elevation in the pressure in response to salt. The salt-stimulated blood pressure in the F1’ and F,(F,’ x F1’) was almost the same, but the variation in the pressure was larger in the F, than the F1’. The F, covered the entire range between the pressure of the parental strains. BC(F,’ x A3) showed a much higher blood pressure than the F,’ and FZ. That is, the blood pressure in the F, ‘, FZ, and BC(F1’ x AJ was 171 t 10 (SD), 171 t 25, and 205 t 33 mmHg at the 5th wk, and 185 t 15, 180 t 30, and 206 t 30 mmHg at the 10th wk, respectively. Water or salt solution intake in the parental strains and F, hybrid group is shown in Table 3. There was no significant difference in the intake of either solution
rat reached 230
mmHg at the 5th wk and it died at the 7th wk. The other A? rats, though hypertensive in similar severity,
did not develop any abnormal
t value A, vs. C A, vs. F, C vs. F, F, vs. F,
AND
or cerebral lesions were observed in any group of rats
107
-t
SUZUOKI,
creased with the experimental period. These differences were not observed in tap water intake. Incidence of cerebrovascular lesions. No mortal cases
wk
234 14 190 -t- 9 213 3- 9 202 16
IWATSUKA,
symptoms or the CVL.
Figure 3 illustrates the cumulative incidence of the CVL in AS, C, and their hybrid groups. Many of the saltloaded SHR developed the CVL as well as the severe hypertension. At the 6th-10th wk, the incidence of CVL was increased in association with the concentration of
SHR As-gene in a manner similar to the spontaneous changes in the blood pressure; C < BC(F, x C) < F, = F, < BC(F, x A3) < A,. Thus, the F, and F, were intermediate between the parental strains, but the incidence in the F, was lower than that in the F, at the lOth-12th wk. It is also remarkable from Fig. 3 that the incidence of CVL is increased with the salt-loading period in all six groups of SHR. At the end of the experiments, the incidences were 92% for AS, BC(F, x A3), and F1, 70% for Fz, 83% for BC(F, x C), and 50% for C, respectively. The effect of the genetic constitution on the onset of CVL was observed in the salt-loaded hybrid groups of WK and A,. None of WK and the F1’ showed the CVL until the termination of experiment. The final inciTABLE
3. Intake of tap water or 1% salt solution Tap water
1% Salt Solution
Group 1 wk
SHR FdA, SHR F1’(A, WK
A, x C) C x WK)
3 wk
5 wk
1 wk
1
5 wk
3 wk
18 + 3 18 + 2 20 + 1
ml/l00 g body wt per day 18 + 2 18 k 3 36 + 9 37 k 6 48 2 8 20 + 2 19 2 4 35 -+ 4 38 f 7 47 + 16 20 k 3 18 r 1 35 -+ 6 39 + 4 45 + 8
17 k 2 17 + 3
17 t 3 16 + 4
16 2 4 17 + 4
30 ,+ 5 26 + 3
31 + 8 28 + 7 20 + 3 19 t 2
Values are means * SD. The intake of salt-solution in each group was significantly higher than that of tap water (P < 0.05) at all times except the 5th wk in WK. 1OOr
1
among the six groups of SHR. The intake of salt solution in each group of animals
was higher than that of
tap water. The SHR including
A, took more salt solu-
tion than the control WK. The F, in A, x WK was roughly intermediate in this respect. The difference between SHR and WK in the salt solution intake in-
C FIG. 3. Cumulative AS, C, and their F,,
BC(Fq XC)
Ff,
F1
(F2)
BC(FpA3)
incidence of cerebrovascular and BC hybrid groups.
A3
lesions
(CVL)
in
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GENETIC
PREDISPOSITION
TO
1357
STROKE
dences were 92% (11/12) for As, 41% (g/22) for BC(F,’ x A,), and 4% (l/24) for the Fz, respectively. The one rat in the 24 F, hybrids showed also the severe hypertension as high as the A, rats, as seen in Fig. 4. Relationship between blood pressure and CVL. The highest blood pressure obtained in the individual rats of each salt-loaded group is plotted in Fig. 4. These values were recorded before the onset of the clinical symptoms, which closely related to the cerebral lesions, in order to exclude the changes of blood pressure after the onset of the symptoms. Most of Af, C, and their hybrid rats exhibited the severe hypertension over 230 mmHg. However, in a series of experiments using As, WK, and their hybrids, the blood pressures of F1’ ranged from 165 to 213 mmHg, and those in the F, and BC hybrids were distributed over a wider range, reflecting the difference in their response to salt. The animals with the CVL, irrespective of type of hybrids, showed the severe hypertension. However, some of the animals with the severe hypertension did not develop the lesions. Such animals were frequently observed in the C group, 50% of which did not show both the CVL and clinical symptoms, although they showed sustained hypertension over 230 mmHg. In As, C, and their hybrid rats, which developed the cerebral lesions, the average elapsed time from the onset of the severe hypertension to that of the CVL (i.e., the average duration of the severe hypertension) was shortest in AS, 3.4 + 1.1 (SD) wk, and longest in C, 5.0 ? 1.0 wk, and the time in the F,, FP, and BC hybrids were in the range of the parental strains, A, and C. Moreover, the half of C rats without the CVL attained a blood pressure of 230 mmHg at 5.8 * 1.8 wk. Figure 5 shows the average blood pressure at the 6th wk and the incidence of CVL at the 7th wk after the salt-loading in relation of the A,-gene concentration. The levels of blood pressure of five groups except Azs, which had the highest average pressure, were not differI
!
-
.
I
260
60
2
220
60
g
j
160
40
: 2
d m g
160
20
Q i?
120
; 2 i
0
C
BChK)
FIG. 5. An illustration tion and blood pressure hybrid groups of SHR.
FI
Fz
EiC(F,xAx)
A,
of relationships between genetic constitu(at 6th wk) or incidence of CVL (at 7th wk) in Values for pressure are means i SD.
ent significantly (P > 0.05). Under these conditions, however, the incidence of CVL was highly associated with the theoretical concentration of A,-gene. A similar relationship between the cumulative incidence of the CVL and the average blood pressure or the theoretical gene concentration in At, C, and their hybrid groups was observed at the 6th-10th wk. Classification of CVL and clinical symptoms. The CVL which developed in 71 rats with the salt loading were detected macroscopically in most cases. These lesions were classified simply into hemorrhage (28%), hemorrhagic infarction (58%), and infarction (14%). Initial clinical symptoms were observed in 94% of these 71 rats, and their incidences were as follows: 1) excitement (piloerection, exophthalmus, increased locomotion), 27%; 2) stereotyped lifting of arms, 22%; 3) hyperirritability (jumping, rapid escaping, etc.), 14%; 4) behavioral and psychological depression (decreased locomotion, hyporesponsiveness), 12%; 5) epileptic symptoms (sudden and abnormal hyperkinesia, convulsion of extremities), 6%; 6) hemiplegia, 3%; and 7) sudden death, 10%. After showing the symptoms, the rats lost their weight gradually and died within about 2 wk. Predilection sites of the lesions were the cortex or subcortex of frontal, medial, and occipital areas of telencephalon. DISCUSSION
0
FIG. 4. Highest blood pressure observed in individual rats of saltloaded groups during experimental period. Solid circle, rat with CVL; open circle, rat without CVL. Blood pressures of rats with CVL were higher than 220-230 mmHg.
A possible involvment of genetic factors in the development of CVL has been suggested from the fact that stroke-prone SHR is established by successive selective inbreeding among the sublines of SHR (18). The present studies were carried out in order to clarify the relationship between hypertension and stroke and also to validate the genetic predisposition to stroke in the strokeprone SHR. The blood pressure of stroke-prone SHR (A,,) was significantly higher than that of stroke-resistant SHR (Cl under the nonsalt-loading conditions, which was suggestive of the importance of high blood pressure in the CVL, as described previously (18). The average blood pressure of the F, and F, in A:, x C was roughly intermediate between both parental SHR’s, and those of the backcross groups were also intermediate between F, and A, or C. In addition, the blood pressure in the F,
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1358
NAGAOKA,
distributed continuously between the ranges of those in A, and C , with some of the pressure extending into the range of the two parental strains. The same kind of genetic data was demonstrated in the hybrid groups between A, and WK. That is, the F1’ was intermediate in blood pressure between the parents, and the F, ranged from a blood pressure as high as A, to a blood pressure approaching that of WK, although the experimental conditions were under the salt loading. The findings from A, x C and A, x WK suggested a characteristic pattern for the results of crosses, involving traits dependent on a few genes. If many genes are involved, the extremes in the pressure represented by the parental strains would occur infrequently in the F2 groups. These results seemed to be compatible with the findings by Louis et al. (11) and Tanase et al. (22) which indicated the mode of inheritance of the hypertension in the SHR was polygenic. However, when the small sample size, the large variation in the parental strains, and the fact that the mean pressure measurements for F, and F,
were closer to C or WK than A, were considered, the possibility
that a single-gene
mode of inheritance
might
be involved in the determination of blood pressure in the SHR could not be excluded by this experiment. It has been reported that the incidence of CVL in the SHR is increased by salt loading (7, 17). In the present experiments, it was found that SHR A3, C, and their hybrids were very sensitive to salt, showing a further increase of blood pressure in addition to the spontaneous increase of their blood pressure. The response to salt was not markedly different among all six groups of SHR, although WK did not respond to salt. The F, between A, and WK showed an intermediate response. Therefore, it may be concluded that the extreme response to salt is one of genetic characteristics of SHR. However, the genes participating in this response seem to be different from those determining the vulnerability to stroke, because the incidence of stroke under the saltloading conditions is obviously different among the six groups of SHR in contrast to the salt sensitivity. The magnitude of the blood pressure response to salt was much less in the SHR as compared with a strain of hypertension-prone rats produced by Dahl et al. (5), but an increased turnover of 22Na was observed in both strains of rats (14, 20). The SHR (1, 12, 14) but not Dahl’s strain (25) showed a high preference to salt. These facts may sugges t a possible -importance of salt metabolism and/or salt sensitivity to genetical .ly mediated hypertension as well as some types of experimental hypertension. Hypertension is an important risk factor to the development of stroke in the human as indicated from epidemiological studies (8-10). Also in the present study, it
was demonstrated
that I) the onsets of both hyperten-
sion and CVL were accelerated by the salt loading, 2) all of the rats that developed the CVL showed a severe hypertension irrespective of type of hybrids, and 3) SHR A, has the highest blood pressure and susceptibility to
CVL among all of the groups with
different
genetic
constitutions.
IWATSUKA,
SUZUOKI,
AND
OKAMOTO
Thus, there seems to be no doubt that
hypertension is also one of the great risk factors to the occurrence of CVL in the stroke-prone SHR. This is supported by the observation that the treatment of hypertension with reserpine or hydralazine is associated with a decrease in the incidence of CVL in the SHR under the same salt-loaded conditions (unpublish .ed data) as well as in man (24). However, the presence of the other factors involved in the development of CVL was suggested from the evidence that 1i although the level of blood pressure which was accelerated with salt was almost the same between BC(F, x AZ), F1, Fz, BC(F, x C), and C in the series of crosses of A3 and C,
the incidences of CVL in these groups increased in association with the theoretical concentration of As-gene; 2) in spite of the same magnitude of the highest blood pressure level in these rats, the final incidence of CVL was markedly lower in C group than in the other groups; and 3) the elapsed time from the onset of severe hypertension to the onset of CVL was the shortest in strokeprone’ SHR and the longest in stroke-resistant SHR. From these findings, it was suggested that the onset of stroke in SHR was dependent not only on the severity of hypertension but also on the genetic predisposition. The incidence of CVL in SHR As, C, and their hybrid groups seemed to correlate positively to the A,-gene concentration. Similar but less prominent correlaton was observed in the hybrid groups between A, and WK. Moreover, the incidence of CVL in the F, in A3 x C was intermediate between the parents during the experimental period except the late period (lo-12 wk). The F, was also intermediate in the incidence, although the
final incidence
was lower than that in the F,. On the
other hand, the F, in A, x WK did not show the CVL, but in the F, the CVL was observed in one rat out of 24 rats. These results might be insufficient in the sample size to analyze an exact mode of inheritance of stroke in
the SHR. However, if it is allowed for us to speculate on the mode of inheritance, it would not be dominant or recessive but polygenic. If it were dependent on a dominant single gene, the incidences of CVL in the F,* (A3 x C) and F1’(AQ x WK) would be closer to that in AS. Alternatively, if it were dependent on a recessive gene, the incidence in the &(A, x C) would be closer to that in C, and the incidence (4%) in the F,(A, x WK) would approach the expectation value (25%). At the late period, however, the relationship between the onset of CVL and A,-gene concentration in the cross of A3 and C became unclear, that is, at the 12th wk the final incidence of the CVL was 84-92% in the hybrid groups except F2 and C. This could be explained by the experimental procedure that a salt was loaded as one of the environmental factors accelerating the development of CVL. The salt loading proved of great benefit to narrowing the distribution in age of the development of CVL in the stroke-
prone SHR. Received
for publication
16 April
1975.
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PREDISPOSITION
TO
STROKE
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REFERENCES 1. CATALANOTTO, F., P. J. SCHECHTER, AND R. I. HENKIN. Preference for NaCl in the spontaneously hypertensive rat. Life Sci. 11: 557-564, 1972. 2. COLE, F. M., AND P. 0. YATES. Comparative incidence of cerebrovascular lesions on normotensive and hypertensive patients. NeuroZogy 18: 255-259, 1968. 3. COLEMAN, T. G., AND A. C. GUYTON. Hypertension caused by salt loading in the dog. III. Onset transients of cardiac output and other variables. Circdation Res. 25: 153-160, 1969. 4. DAHL, L. K. Salt, fat and hypertension: the Japanese experience. Nutr. Rev. 18: 97-99, 1960. 5. DAHL, L. K., M. HEINE, AND L. TASSINARI. Effects of chronic excess salt ingestion: evidence that genetic factors play an important role in susceptibility to experimental hypertension. J. ExptZ. Med. 115: 1173-1190, 1962. 6. DAHC, L. K., M. HEINE, AND L. TASSINARI. Effects of chronic excess salt ingestion: role of genetic factors in both DOCA-salt and renal hypertension. J. Exptl. Med. 118: 605-617, 1963. 7. HAZAMA, F., A. OOSHIMA, T. TANAKA, K. TOMIMOTO, AND K. OKAMOTO. Vascular lesions in the various substrains of spontaneously hypertensive rats and the effects of chronic salt ingestion. Japan. CircuZation J. 39: 7-22, 1975. 8. KANNEL, W. B. Current status of the epidemiology of brain infarction associated with occlusive arterial disease. Stroke 2: 295-318, 1971. 9. KANNEL, W. B., P. A. WOLF, J. VERTER, AND P. M. MCNAMARA. Epidemiologic assessment of the role of blood pressure in stroke: the Framingham study. J. Am. Med. Assoc. 214: 301-310, 1970. 10. KATSUKI, S. Epidemiological and clinicopathological study on cerebrovascular disease in Japan. Prog. Bruin Res. 2lB: 64-89, 1966. 11. LOUIS, W. J., R. TABEI, A. SJOERDSMA, AND S. SPECTOR. Inheritance of high blood-pressure in the spontaneously hypertensive rat. Lancet I: 1035-1036, 1969. 12. MCCONNEL, S. D., and R. I. HENKIN. NaCl preference in spontaneously hypertensive rats: age and blood pressure effects. Am. J. Physiol. 225: 624-627, 1973. 13. NAGAOKA, A., K. KIKUCHI, AND Y. ARAMAKI. Participation of tissue electrolytes and water to the spontaneous hypertension in rats. Japan. CircuZation J. 34: 489-497, 1970.
14. NAGAOKA, A., H. IWATSUKA, 2. SUZUOKI, AND K. OKAMOTO. Electrolyte metabolism in substrains of spontaneously hypertensive rats (Abstract). Japan. Heart J. 14: 157-158, 1973. 15. OKAMOTO, K., AND K. AOKI. Development of a strain of spontaneously hypertensive rats. Japan. CircuZation J. 27: 282-293, 1963. 16. OKAMOTO, K., Y. YAMORI, A. OOSHIMA, AND T. TANAKA. Development of substains in spontaneously hypertensive rats; genealogy, isozymes and effects of hypercholesterolemic diet. Japan. CircuZation J. 36: 461-470, 1972. 17. OKAMOTO, K., F. HAZAMA, H. HAEBARA, S. AMANO, T. TANAKA, AND A. OOSHIMA. Pathology of dietary induced cerebrovascular diseases in spontaneously hypertensive rats. In: Spontaneous Hypertension, edited by K. Okamoto. Tokyo: Igaku Shoin, 1972, p. 129-133. 18. OKAMOTO, K., Y. YAMORI, AND A. NAGAOKA. Establishment of the stroke-prone spontaneously hypertensive rat (SHR). CircuZation Res. Suppl. 34 and 35: 143-153, 1974. 19. PRINEAS, J., and J. MARSHALL. Hypertension and cerebral infarction. Brit. Med. J. 1: 14-17, 1966. 20. SCHACKOW, E., AND L. K. DAHL. Effects of chronic excess salt ingestion; lack of gross salt retention in salt-hypertension. Proc. Sot. Exptl. BioZ. Med. 122: 952-957, 1966. 21. STALLONES, R. A., M. L. DYKEN, H. C. H. FANG, A. HEYMAN, R. SELTSER, AND J. STAMLER. Epidemiology for stroke facilities planning. Stroke 3: 360-371, 1972. 22. TANASE, H., Y. SUZUKI, A. OOSHIMA, Y. YAMORI, AND K. OKAMOTO. Genetic analysis of blood pressure in spontaneously hypertensive rats. Japan. CircuZation J. 34: 1197-1212, 1970. 23. TOBIAN, L., AND J. T. BINION. Tissue cations and water in arterial hypertension. CircuZation 5: 754-758, 1952. 24. Veterans Administration Cooperative Study Group on antihypertensive agents. Effects of treatment of morbidity in hypertension. II. Results in patients with diastolic blood pressure averaging 90 through 114 mmHg. J. Am. Med. Assoc. 213: 1143-1152, 1970. 25. WOLF, G., L. K. DAHL, AND N. E. MILLER. Voluntary sodium chloride intake of two strains of rats with opposite genetic susceptibility to experimental hypertension. Proc. Sot. Exptl. BioZ. Med. 120: 301-305, 1965.
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Genetic predisposition to stroke in spontaneously hypertensive
rats
AKINOBU NAGAOKA, HISASHI IWATSUKA, ZIRO SUZUOKI, AND KOZO OKAMOTO Biological Research Laboratories, Central Research Division, Takeda Chemical Industries, Osaka 532, and Faculty of Medicine, Kyoto University, Kyoto, Japan
NAGAOKA, AND Kozo
AKINOBU, OKAMOTO.
HISASHI
IWATSUKA,
ZIRO
SUZUOKI,
Genetic predisposition to stroke in spontaneously hypertensive rats. Am. J. Physiol. 230(5): 1356
1359. 1976. - Hypertension and stroke in spontaneously hypertensive rats (SHR) were investigated genetically using stroke-prone SHR (As), stroke-resistant SHR (C) and their hybrids, hybrid of A3 and C (F,), offspring of F, x F, (F2), and those of backcrossing of F, to the respective parental strains, BC(F, x A,) and BC(F, x C). The average blood pressure measured without anesthesia increased in the following order during the experimental period: C < BC (F, x C),< F, = Fr < BC(F, x A,) c A,. The F2 represented a wider spread of variation than the F,, with some of the pressure extending into the range of both parental strains. When the drinking water was replaced with a 1% salt solution, the blood pressure increased and the onset of stroke markedly accelerated in all groups of SHR. Under the hypertensive conditions, the incidence of stroke was associated with A,-gene concentration rather than with the level of blood pressure. Similar but less dramatic effects of salt were observed in another series of hybrid groups derived from A, and normal Wistar-Kyoto rats. These findings suggest that the genetic factors are of great importance in the development of stroke as well as hypertension in the SHR. cerebral hemorrhage; stroke; salt sensitivity
cerebral
infarction;
genetic
factors
in
IN 1963, SPONTANEOUSLY HYPERTENSIVE RATS (SHR) regarded as a useful animal model for essential hypertension were isolated from the Wistar-Kyoto rat colony by Okamoto and Aoki (15). Later, the SHR colony was further separated by Okamoto and his colleagues into several sublines (16), some of which had a tendency to develop cerebrovascular lesions (cerebral hemorrhage and/or infarction), otherwise known as a stroke. Recently, “stroke-prone SHR” was established by Okamoto et al. (18) from the sublines of A3 and A,-sb. The strokeprone SHR showed a spontaneously high incidence of the cerebrovascular lesion (CVL). In contrast, a subline C could hardly develop the CVL in spite of the presence of hypertension and was called “stroke-resistant SHR.” It is well known that high blood pressure is one of the causative factors of stroke in the human (2, 8-10, 19, 21) and that abnormally high salt intake results in an elevation of the blood pressure in both men and experimental animals (3,4,6,23). As expected, it was recently demonstrated that increased salt intake accelerated the onset of CVL in the stroke-prone SHR (7). In the present
studies, effects of increased salt intake on the CVL were examined in the SHR with different genetic constitutions in order to vallidate a predisposition to the CVL in the stroke-prone SHR, A3. METHODS
The offspring (Fz7-Fz8) of SHR sublines were introduced to our laboratories from Kyoto University in 1972 and had since been bred by selective sibmatings. The two kinds of SHR used in the present study were of the F,, generation in the stroke-prone SHR (A,) and F,, generation in the stroke-resistant SHR (0. These SHR and Wistar-Kyoto rats (WK), used as normotensive control rats, originated from the same ancestors. The degree of inbreeding in these strains was the same as the previous report (18). Animals of F,, FP, and backcrossing (BC) hybrids were produced by reciprocal matings of A, x C and A3 x WK. The males were used in the experiments, because it had been reported that the incidence of stroke was much higher in the males than in the females of the stroke-prone SHR (18). The procedure in the mating of A3 x C and theoretical gene concentration are shown in Fig. 1. The same procedure was used in the mating of A, x WK. However, backcrossing of the F, to WK was not done because the F, rats as well as WK showed no incidence of stroke as described in the item of results. Rats were kept five or six in a cage until 7 wk of age under the controlled conditions of temperature (23. 24”C), humidity (50-60%), and light (7:30 A.M.-~:OO P.M .) and were given a laboratory chow, CA-l, Japan CLEA, containing 0.4% salt and drinking water ad libiturn. Animals in each hybrid group were divided into two groups at 8 wk of age, which were given tap water and 1% salt solution as the drinking fluid for 12 wk (Table 1). In the process for allocating rats into the two groups, it was considered that rats from the same litter were divided into the two groups in about the same ratio, and especially in the series of A3 x WK the rats were assigned more to the salt-loaded group than to the control group, as it was anticipated that the CVL would hardly develop in the latter group for this experimental period. Some of the rats died spontaneously during the experimental period. The organs from these rats and survivors, sacrificed at the beginning of the 13th wk, were fixed with 10% Formalin. The brains were cut in four or five frontal sections for the macroscopic observation to
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GENETIC
PREDISPOSITION
TO
1355
STROKE
following
order: C c BC(F,
x
C) < F, = F, < BC(F,
x
A3) c AB. Blood pressure above 200 mmHg developed in AS, BC(F, x AS), F1, and F, at 2.5,4.5, 6, and 7 wk after the start of the experiment,
respectively,
although
only
the pressure of A3 elevated to 230 mmHg by 7 wk. On the other hand, the blood pressure of BC(F, x C) and C did not reach this level (200 mmHg).
FIG. 1. Mating procedure between stroke-prone SHR (A,) and -resistant SHR (C). Theoretical average gene constitution in each group also is represented, and ratio (%) of A, and C genes is as follows. A, (A, : 100); C (C : 100); F, (A, : 50, C : 50); F, (A, : 50, C : 50) backcross to A, (A, : 75, C : 25); and backcross to C (A, : 25, C : 75). TABLE 1. Number of rats in parental hybrid groups used
Parental
Strains
SHR
A, BW’, WA, F,(F, BC(F,
SHR
A,) C) F,) C)
C BC(F,’ x A,) FI’(A, x WK) JWY x FJ Normal WK
and their
C, although
the mean appeared to be closer to C than
A,. The F, was also generally intermediate but represented a wider range than the F,. Moreover, the blood pressure in the F2 tended to distribute continuously between the parents, and some of their pressure mea-
No. of rats
and Types of Hybrid
x x x x
strains
The blood pressures in AS, C, and their F, and F, hybrid groups are shown in Table 2 in order to determine whether differences between the means indicate different populations sampled. A, had a larger variation than C, but a highly significant difference was observed between the parental strains. The blood pressure of the F, was intermediate between the parents and was significantly lower than that of A, and higher than that of
Tap water
1% salt
12 5 12 12 5
12 12 12 16 12
12 0 12 0 12
12 22 12 24 12
llO=,
A3
C 1
I
a
xA3) I
1
1
1
I
1
L
al
detect the CVL, and then embedded in paraffin to prepare the histological sections, 5 pm in thickness, which were stained with hematoxylin-eosin staining for microscopic observation. Blood pressure was measured weekly using a tail-pulse-pickup method (13) in unanesthetized rats. The animals were previously warmed for 10 min in a chamber which was maintained at 37-38”C, and then they were placed into a rat holder. The average of three recordings of blood pressure taken at approximately 30-s intervals at their rest condition was recorded as systolic blood pressure of the rat. The variation in the three recordings was less than 5%. Volume intake of the drinking water was also measured weekly.
Rats were observed every day for clinical
* E E
110 250
.-u 'zi Y
150
$llO-,
BC(Fq
BC(FjxC)
2509 ........., .... ........ ........ ... ....... - ... ............. ........ .. .. .......... ... ....... ..... ... ..... .......... ............... ...... .. 200-
symptoms
suggestive of stroke or hypertensive encephalopathy, which could be detected visually. Arithemetical means, standard deviation and standard error of the mean were calculated. The difference between the means from two independent samples was analyzed by the Student unpaired-t-test. RESULTS
Hood pressure.
10
O‘n tap water, the systolic blood pres-
sure differed among the six groups in the series of crosses between A3 and C (Fig. 2). Throughout the experimental period, the pressure level was higher in the
lime
0
5
10
(Weeks)
FIG. 2. Time course of systolic blood pressure (BP) in rats receiving a tap water (O0) or 1% salt solution (0-O). Elevation of blood pressure was accelerated by salt loading in all groups except normal WK group. Values are means t SD.
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1356
NAGAOKA,
TABLE 2. Statistical andlysis of blood pressures in As, C, and their F, and F, hybrids Time After Start of Experiment, 2 207 -t 12 171 2 7 187 -e 6 181 + 11
4 212 180 188 192
u 12 + 7 2 8 _+ 15
6 222 185 196 189
8
2 15 ” 8 -+ 12 -’ 15
231 189 203 199
+ 14 2 6 of: 9 2.15
-t
drinking
12 236 197 210 209
+ + f -+
13 10 7 19
8.93* 5.14* 6.11* 1.65
7.94* 5.73*
7.59* 4.69*
2.62t
2.63t
4.82
1.26
9.27* 5.12* 3.60$ 0.72
8.91* 3.84* 5.28* 1.90
OKAMOTO
tap water except for one rat in the A3 group.
The blood pressure of this exceptional
8.02* 5.94* 3.69$ 0.17
Blood pressure values are means * SD. n, number of animals. The number in A, group *p 0.05). The F,’ showed only a slight elevation in the pressure in response to salt. The salt-stimulated blood pressure in the F1’ and F,(F,’ x F1’) was almost the same, but the variation in the pressure was larger in the F, than the F1’. The F, covered the entire range between the pressure of the parental strains. BC(F,’ x A3) showed a much higher blood pressure than the F,’ and FZ. That is, the blood pressure in the F, ‘, FZ, and BC(F1’ x AJ was 171 t 10 (SD), 171 t 25, and 205 t 33 mmHg at the 5th wk, and 185 t 15, 180 t 30, and 206 t 30 mmHg at the 10th wk, respectively. Water or salt solution intake in the parental strains and F, hybrid group is shown in Table 3. There was no significant difference in the intake of either solution
rat reached 230
mmHg at the 5th wk and it died at the 7th wk. The other A? rats, though hypertensive in similar severity,
did not develop any abnormal
t value A, vs. C A, vs. F, C vs. F, F, vs. F,
AND
or cerebral lesions were observed in any group of rats
107
-t
SUZUOKI,
creased with the experimental period. These differences were not observed in tap water intake. Incidence of cerebrovascular lesions. No mortal cases
wk
234 14 190 -t- 9 213 3- 9 202 16
IWATSUKA,
symptoms or the CVL.
Figure 3 illustrates the cumulative incidence of the CVL in AS, C, and their hybrid groups. Many of the saltloaded SHR developed the CVL as well as the severe hypertension. At the 6th-10th wk, the incidence of CVL was increased in association with the concentration of
SHR As-gene in a manner similar to the spontaneous changes in the blood pressure; C < BC(F, x C) < F, = F, < BC(F, x A3) < A,. Thus, the F, and F, were intermediate between the parental strains, but the incidence in the F, was lower than that in the F, at the lOth-12th wk. It is also remarkable from Fig. 3 that the incidence of CVL is increased with the salt-loading period in all six groups of SHR. At the end of the experiments, the incidences were 92% for AS, BC(F, x A3), and F1, 70% for Fz, 83% for BC(F, x C), and 50% for C, respectively. The effect of the genetic constitution on the onset of CVL was observed in the salt-loaded hybrid groups of WK and A,. None of WK and the F1’ showed the CVL until the termination of experiment. The final inciTABLE
3. Intake of tap water or 1% salt solution Tap water
1% Salt Solution
Group 1 wk
SHR FdA, SHR F1’(A, WK
A, x C) C x WK)
3 wk
5 wk
1 wk
1
5 wk
3 wk
18 + 3 18 + 2 20 + 1
ml/l00 g body wt per day 18 + 2 18 k 3 36 + 9 37 k 6 48 2 8 20 + 2 19 2 4 35 -+ 4 38 f 7 47 + 16 20 k 3 18 r 1 35 -+ 6 39 + 4 45 + 8
17 k 2 17 + 3
17 t 3 16 + 4
16 2 4 17 + 4
30 ,+ 5 26 + 3
31 + 8 28 + 7 20 + 3 19 t 2
Values are means * SD. The intake of salt-solution in each group was significantly higher than that of tap water (P < 0.05) at all times except the 5th wk in WK. 1OOr
1
among the six groups of SHR. The intake of salt solution in each group of animals
was higher than that of
tap water. The SHR including
A, took more salt solu-
tion than the control WK. The F, in A, x WK was roughly intermediate in this respect. The difference between SHR and WK in the salt solution intake in-
C FIG. 3. Cumulative AS, C, and their F,,
BC(Fq XC)
Ff,
F1
(F2)
BC(FpA3)
incidence of cerebrovascular and BC hybrid groups.
A3
lesions
(CVL)
in
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GENETIC
PREDISPOSITION
TO
1357
STROKE
dences were 92% (11/12) for As, 41% (g/22) for BC(F,’ x A,), and 4% (l/24) for the Fz, respectively. The one rat in the 24 F, hybrids showed also the severe hypertension as high as the A, rats, as seen in Fig. 4. Relationship between blood pressure and CVL. The highest blood pressure obtained in the individual rats of each salt-loaded group is plotted in Fig. 4. These values were recorded before the onset of the clinical symptoms, which closely related to the cerebral lesions, in order to exclude the changes of blood pressure after the onset of the symptoms. Most of Af, C, and their hybrid rats exhibited the severe hypertension over 230 mmHg. However, in a series of experiments using As, WK, and their hybrids, the blood pressures of F1’ ranged from 165 to 213 mmHg, and those in the F, and BC hybrids were distributed over a wider range, reflecting the difference in their response to salt. The animals with the CVL, irrespective of type of hybrids, showed the severe hypertension. However, some of the animals with the severe hypertension did not develop the lesions. Such animals were frequently observed in the C group, 50% of which did not show both the CVL and clinical symptoms, although they showed sustained hypertension over 230 mmHg. In As, C, and their hybrid rats, which developed the cerebral lesions, the average elapsed time from the onset of the severe hypertension to that of the CVL (i.e., the average duration of the severe hypertension) was shortest in AS, 3.4 + 1.1 (SD) wk, and longest in C, 5.0 ? 1.0 wk, and the time in the F,, FP, and BC hybrids were in the range of the parental strains, A, and C. Moreover, the half of C rats without the CVL attained a blood pressure of 230 mmHg at 5.8 * 1.8 wk. Figure 5 shows the average blood pressure at the 6th wk and the incidence of CVL at the 7th wk after the salt-loading in relation of the A,-gene concentration. The levels of blood pressure of five groups except Azs, which had the highest average pressure, were not differI
!
-
.
I
260
60
2
220
60
g
j
160
40
: 2
d m g
160
20
Q i?
120
; 2 i
0
C
BChK)
FIG. 5. An illustration tion and blood pressure hybrid groups of SHR.
FI
Fz
EiC(F,xAx)
A,
of relationships between genetic constitu(at 6th wk) or incidence of CVL (at 7th wk) in Values for pressure are means i SD.
ent significantly (P > 0.05). Under these conditions, however, the incidence of CVL was highly associated with the theoretical concentration of A,-gene. A similar relationship between the cumulative incidence of the CVL and the average blood pressure or the theoretical gene concentration in At, C, and their hybrid groups was observed at the 6th-10th wk. Classification of CVL and clinical symptoms. The CVL which developed in 71 rats with the salt loading were detected macroscopically in most cases. These lesions were classified simply into hemorrhage (28%), hemorrhagic infarction (58%), and infarction (14%). Initial clinical symptoms were observed in 94% of these 71 rats, and their incidences were as follows: 1) excitement (piloerection, exophthalmus, increased locomotion), 27%; 2) stereotyped lifting of arms, 22%; 3) hyperirritability (jumping, rapid escaping, etc.), 14%; 4) behavioral and psychological depression (decreased locomotion, hyporesponsiveness), 12%; 5) epileptic symptoms (sudden and abnormal hyperkinesia, convulsion of extremities), 6%; 6) hemiplegia, 3%; and 7) sudden death, 10%. After showing the symptoms, the rats lost their weight gradually and died within about 2 wk. Predilection sites of the lesions were the cortex or subcortex of frontal, medial, and occipital areas of telencephalon. DISCUSSION
0
FIG. 4. Highest blood pressure observed in individual rats of saltloaded groups during experimental period. Solid circle, rat with CVL; open circle, rat without CVL. Blood pressures of rats with CVL were higher than 220-230 mmHg.
A possible involvment of genetic factors in the development of CVL has been suggested from the fact that stroke-prone SHR is established by successive selective inbreeding among the sublines of SHR (18). The present studies were carried out in order to clarify the relationship between hypertension and stroke and also to validate the genetic predisposition to stroke in the strokeprone SHR. The blood pressure of stroke-prone SHR (A,,) was significantly higher than that of stroke-resistant SHR (Cl under the nonsalt-loading conditions, which was suggestive of the importance of high blood pressure in the CVL, as described previously (18). The average blood pressure of the F, and F, in A:, x C was roughly intermediate between both parental SHR’s, and those of the backcross groups were also intermediate between F, and A, or C. In addition, the blood pressure in the F,
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1358
NAGAOKA,
distributed continuously between the ranges of those in A, and C , with some of the pressure extending into the range of the two parental strains. The same kind of genetic data was demonstrated in the hybrid groups between A, and WK. That is, the F1’ was intermediate in blood pressure between the parents, and the F, ranged from a blood pressure as high as A, to a blood pressure approaching that of WK, although the experimental conditions were under the salt loading. The findings from A, x C and A, x WK suggested a characteristic pattern for the results of crosses, involving traits dependent on a few genes. If many genes are involved, the extremes in the pressure represented by the parental strains would occur infrequently in the F2 groups. These results seemed to be compatible with the findings by Louis et al. (11) and Tanase et al. (22) which indicated the mode of inheritance of the hypertension in the SHR was polygenic. However, when the small sample size, the large variation in the parental strains, and the fact that the mean pressure measurements for F, and F,
were closer to C or WK than A, were considered, the possibility
that a single-gene
mode of inheritance
might
be involved in the determination of blood pressure in the SHR could not be excluded by this experiment. It has been reported that the incidence of CVL in the SHR is increased by salt loading (7, 17). In the present experiments, it was found that SHR A3, C, and their hybrids were very sensitive to salt, showing a further increase of blood pressure in addition to the spontaneous increase of their blood pressure. The response to salt was not markedly different among all six groups of SHR, although WK did not respond to salt. The F, between A, and WK showed an intermediate response. Therefore, it may be concluded that the extreme response to salt is one of genetic characteristics of SHR. However, the genes participating in this response seem to be different from those determining the vulnerability to stroke, because the incidence of stroke under the saltloading conditions is obviously different among the six groups of SHR in contrast to the salt sensitivity. The magnitude of the blood pressure response to salt was much less in the SHR as compared with a strain of hypertension-prone rats produced by Dahl et al. (5), but an increased turnover of 22Na was observed in both strains of rats (14, 20). The SHR (1, 12, 14) but not Dahl’s strain (25) showed a high preference to salt. These facts may sugges t a possible -importance of salt metabolism and/or salt sensitivity to genetical .ly mediated hypertension as well as some types of experimental hypertension. Hypertension is an important risk factor to the development of stroke in the human as indicated from epidemiological studies (8-10). Also in the present study, it
was demonstrated
that I) the onsets of both hyperten-
sion and CVL were accelerated by the salt loading, 2) all of the rats that developed the CVL showed a severe hypertension irrespective of type of hybrids, and 3) SHR A, has the highest blood pressure and susceptibility to
CVL among all of the groups with
different
genetic
constitutions.
IWATSUKA,
SUZUOKI,
AND
OKAMOTO
Thus, there seems to be no doubt that
hypertension is also one of the great risk factors to the occurrence of CVL in the stroke-prone SHR. This is supported by the observation that the treatment of hypertension with reserpine or hydralazine is associated with a decrease in the incidence of CVL in the SHR under the same salt-loaded conditions (unpublish .ed data) as well as in man (24). However, the presence of the other factors involved in the development of CVL was suggested from the evidence that 1i although the level of blood pressure which was accelerated with salt was almost the same between BC(F, x AZ), F1, Fz, BC(F, x C), and C in the series of crosses of A3 and C,
the incidences of CVL in these groups increased in association with the theoretical concentration of As-gene; 2) in spite of the same magnitude of the highest blood pressure level in these rats, the final incidence of CVL was markedly lower in C group than in the other groups; and 3) the elapsed time from the onset of severe hypertension to the onset of CVL was the shortest in strokeprone’ SHR and the longest in stroke-resistant SHR. From these findings, it was suggested that the onset of stroke in SHR was dependent not only on the severity of hypertension but also on the genetic predisposition. The incidence of CVL in SHR As, C, and their hybrid groups seemed to correlate positively to the A,-gene concentration. Similar but less prominent correlaton was observed in the hybrid groups between A, and WK. Moreover, the incidence of CVL in the F, in A3 x C was intermediate between the parents during the experimental period except the late period (lo-12 wk). The F, was also intermediate in the incidence, although the
final incidence
was lower than that in the F,. On the
other hand, the F, in A, x WK did not show the CVL, but in the F, the CVL was observed in one rat out of 24 rats. These results might be insufficient in the sample size to analyze an exact mode of inheritance of stroke in
the SHR. However, if it is allowed for us to speculate on the mode of inheritance, it would not be dominant or recessive but polygenic. If it were dependent on a dominant single gene, the incidences of CVL in the F,* (A3 x C) and F1’(AQ x WK) would be closer to that in AS. Alternatively, if it were dependent on a recessive gene, the incidence in the &(A, x C) would be closer to that in C, and the incidence (4%) in the F,(A, x WK) would approach the expectation value (25%). At the late period, however, the relationship between the onset of CVL and A,-gene concentration in the cross of A3 and C became unclear, that is, at the 12th wk the final incidence of the CVL was 84-92% in the hybrid groups except F2 and C. This could be explained by the experimental procedure that a salt was loaded as one of the environmental factors accelerating the development of CVL. The salt loading proved of great benefit to narrowing the distribution in age of the development of CVL in the stroke-
prone SHR. Received
for publication
16 April
1975.
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PREDISPOSITION
TO
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REFERENCES 1. CATALANOTTO, F., P. J. SCHECHTER, AND R. I. HENKIN. Preference for NaCl in the spontaneously hypertensive rat. Life Sci. 11: 557-564, 1972. 2. COLE, F. M., AND P. 0. YATES. Comparative incidence of cerebrovascular lesions on normotensive and hypertensive patients. NeuroZogy 18: 255-259, 1968. 3. COLEMAN, T. G., AND A. C. GUYTON. Hypertension caused by salt loading in the dog. III. Onset transients of cardiac output and other variables. Circdation Res. 25: 153-160, 1969. 4. DAHL, L. K. Salt, fat and hypertension: the Japanese experience. Nutr. Rev. 18: 97-99, 1960. 5. DAHL, L. K., M. HEINE, AND L. TASSINARI. Effects of chronic excess salt ingestion: evidence that genetic factors play an important role in susceptibility to experimental hypertension. J. ExptZ. Med. 115: 1173-1190, 1962. 6. DAHC, L. K., M. HEINE, AND L. TASSINARI. Effects of chronic excess salt ingestion: role of genetic factors in both DOCA-salt and renal hypertension. J. Exptl. Med. 118: 605-617, 1963. 7. HAZAMA, F., A. OOSHIMA, T. TANAKA, K. TOMIMOTO, AND K. OKAMOTO. Vascular lesions in the various substrains of spontaneously hypertensive rats and the effects of chronic salt ingestion. Japan. CircuZation J. 39: 7-22, 1975. 8. KANNEL, W. B. Current status of the epidemiology of brain infarction associated with occlusive arterial disease. Stroke 2: 295-318, 1971. 9. KANNEL, W. B., P. A. WOLF, J. VERTER, AND P. M. MCNAMARA. Epidemiologic assessment of the role of blood pressure in stroke: the Framingham study. J. Am. Med. Assoc. 214: 301-310, 1970. 10. KATSUKI, S. Epidemiological and clinicopathological study on cerebrovascular disease in Japan. Prog. Bruin Res. 2lB: 64-89, 1966. 11. LOUIS, W. J., R. TABEI, A. SJOERDSMA, AND S. SPECTOR. Inheritance of high blood-pressure in the spontaneously hypertensive rat. Lancet I: 1035-1036, 1969. 12. MCCONNEL, S. D., and R. I. HENKIN. NaCl preference in spontaneously hypertensive rats: age and blood pressure effects. Am. J. Physiol. 225: 624-627, 1973. 13. NAGAOKA, A., K. KIKUCHI, AND Y. ARAMAKI. Participation of tissue electrolytes and water to the spontaneous hypertension in rats. Japan. CircuZation J. 34: 489-497, 1970.
14. NAGAOKA, A., H. IWATSUKA, 2. SUZUOKI, AND K. OKAMOTO. Electrolyte metabolism in substrains of spontaneously hypertensive rats (Abstract). Japan. Heart J. 14: 157-158, 1973. 15. OKAMOTO, K., AND K. AOKI. Development of a strain of spontaneously hypertensive rats. Japan. CircuZation J. 27: 282-293, 1963. 16. OKAMOTO, K., Y. YAMORI, A. OOSHIMA, AND T. TANAKA. Development of substains in spontaneously hypertensive rats; genealogy, isozymes and effects of hypercholesterolemic diet. Japan. CircuZation J. 36: 461-470, 1972. 17. OKAMOTO, K., F. HAZAMA, H. HAEBARA, S. AMANO, T. TANAKA, AND A. OOSHIMA. Pathology of dietary induced cerebrovascular diseases in spontaneously hypertensive rats. In: Spontaneous Hypertension, edited by K. Okamoto. Tokyo: Igaku Shoin, 1972, p. 129-133. 18. OKAMOTO, K., Y. YAMORI, AND A. NAGAOKA. Establishment of the stroke-prone spontaneously hypertensive rat (SHR). CircuZation Res. Suppl. 34 and 35: 143-153, 1974. 19. PRINEAS, J., and J. MARSHALL. Hypertension and cerebral infarction. Brit. Med. J. 1: 14-17, 1966. 20. SCHACKOW, E., AND L. K. DAHL. Effects of chronic excess salt ingestion; lack of gross salt retention in salt-hypertension. Proc. Sot. Exptl. BioZ. Med. 122: 952-957, 1966. 21. STALLONES, R. A., M. L. DYKEN, H. C. H. FANG, A. HEYMAN, R. SELTSER, AND J. STAMLER. Epidemiology for stroke facilities planning. Stroke 3: 360-371, 1972. 22. TANASE, H., Y. SUZUKI, A. OOSHIMA, Y. YAMORI, AND K. OKAMOTO. Genetic analysis of blood pressure in spontaneously hypertensive rats. Japan. CircuZation J. 34: 1197-1212, 1970. 23. TOBIAN, L., AND J. T. BINION. Tissue cations and water in arterial hypertension. CircuZation 5: 754-758, 1952. 24. Veterans Administration Cooperative Study Group on antihypertensive agents. Effects of treatment of morbidity in hypertension. II. Results in patients with diastolic blood pressure averaging 90 through 114 mmHg. J. Am. Med. Assoc. 213: 1143-1152, 1970. 25. WOLF, G., L. K. DAHL, AND N. E. MILLER. Voluntary sodium chloride intake of two strains of rats with opposite genetic susceptibility to experimental hypertension. Proc. Sot. Exptl. BioZ. Med. 120: 301-305, 1965.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (152.003.102.254) on January 10, 2019.
