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Evidence for variation in blood pressure during the day in normotensive and salt-hypertensive rats
Life Sciences, Vol . 21, pp . 199-204, 1977 . Printed In The U .S .A .
Pergamoa Prose
EVIDEZiCE FOR VARIATION IN BIAOD PRSSSURS DURING THE DAY IN NORMOTBriSIVE AND SALT-HYPERTENSIVE RATS C . E . Hall, D . Nasseth and O . Hall Department of Physiology and Biophysics University of Texas Medical Branch Galveston, Texas
77550
(Received in final form June 10, 1977 Summary The systolic blood pressure of unanesthetized normotensive and salt-hypertenaive rats was found to be significantly lower in the afternoon than in the morning . The difference was more marked in hypertensivea than in normotenaivea, perhaps because normal blood pressures are restricted to a narrow range . These findings indicate that blood pressure, like many other physiological functions, varies diurnally . The differences between morning and afternoon pressures, although perhaps less than they would have been at times selected to correspond with maxima and minima of physical activity, were nonetheless large enough to influence the decision as to how long it required for salt excess to cause hypertension and to indicate its rote of progression . It has been amply demonstrated that a considerable number of physiological functions and responses in rata (end other animals) wax 4n8 wane over various periods . Soma of these are endogenous rhythms, whereas others are controlled by external events . Those which cycle within or close to a 24-hour span are termed circadian rhythms . In rata, primarily nocturnal animals, the zenith of physical activity and associated functions occurs in the period between 18 .00 and 06 .00 hours, and the nadir at 06 .00 to 18 .00 hours . This is a circadian rhythm, but can be modified ezperimentally . Occasional animals depart from the norm (1) . Although moat studies of daily rhythmic activity have been conducted with a view to establishing the maxima and minima of functions under study, substantial differences are often noted even if comparisons are made between less than optimal periods . For example, it has been shown that rate and mica detoxify pentobarbital more efficiently, and sleep for shorter periods, when anesthetized at 02 .00 than at 14 .00 hours (2,3) . However, we have found that with a chloralose anesthesia, the difference between sleep time of spontaneously hypertenaive rate of the Okamoto-AOki etrnin is significantly less than that of normotenaive controls when studies are co~uctad between 13 .00 and 17 .00, but not when conducted between 08 .00 and 12 .00 (4) . The foregoing observation suggested that other functions might vary significantly between these two time periods, which delimit the usual working day . The one of greatest interest to us was arterial blood pressure . However useful it might be to make comparisons between time periods yielding the 19.9
20 0
Rat Blood Pressure During 24-Hour Day
Vol . 21, No . 2, 1977
greatest differences in order to establish maxima and minima, the more immediate concern was to ascertain whether discernible differences existed between pressures taken from 0 .800 to 12 .00 and those taken between 13 .00 and 17 .00, Accordingly, the periods circumscribing the routine laboratory work day. following study was undertaken . Materials and Methods Eighteen, four-week old, Sprague-Dnwley rats (Laboratory Associates, All of them had the right kidney removed under St . Louis, MO) were obtained . Half of them were given a 1~ NaCl 5$ sucrose solution to ether anesthesia . drink to facilitate the induction of salt hypertension (5), and . the others were Purina Lab Chow was fed ad lib. Systolic blood given a 5" sucrose solution . pressure was taken once weekly on conscious animals by a tail microphonic method using a programmed electroaphygmomanometer (PS-300) and a DMP-4 Phyaiograph (Narco Biosystems, Houston, Texas) . An average of 3 consecutive pressures agreeing to within 20 mm Hq established the blood pressure of an animal : those above 150 mm Hg were classified hypertensioe . Animals were individually caged in temperature-controlled quarters with light cycled 10 hours on and 14 hours off . Blood pressures were taken between 08 .00 and 11 .30 on one day of each week, between 13 .00 and 16 .30 on the following day and 7 days apart. Thin procedure appeared to be preferable to taking a.m . and p.m . pressures on the same day, which would have subjected the animals to very extended periods in the warming boxes and in the restraining apparatus, and interfered greatly with the normal feeding and drinking patterns . The animals were killed by etherization on the 53rd day. Various organs were placed in neutral l0i formalin for subsequent weighing . When fixed, the organs were removed, blotted, trimmed and weighed on an analytical balance. Statistical comparisons of weights were made using Student's two-tailed "t" test, and of blood pressures using the analysis of variance . Results The blood pressure of control rats fell within a narrow range throughout It was not significantly higher at the and than at the experimental period . the beginning, but in six of the seven weeks the pressures averaged higher in The same difference was true of the high the mornings than in the afternoons . salt intake gros~ in all seven weeks, but in these the blood pressure rose progressively throughout the experiment, by the end of which eight of the nine Employing an analysis of variance procedure, it was rats ware hyperteneive . determined that in both normotenaives (P < 0 .04) and hypertensivea (P < 0.0001) the 08 .00 to 11 .30 group mean blood pressures were significantly above those taken between 13 .00 to 16 .30 over the entire experimental period (Fig . 1) . Hypertensive rata had markedly enlarged hearts and kidneys, but there was no significant difference between weights of the thymus or adrenal glands (Table 1) .
Rat Hlood Pressura Duriag 24-Hour Day
Vol . 21, No . 2, 1977
a
SUCROSE
596
EE
~---~ A M ~---i P M
140
W
cc
201
W 120ac a
u 100O
n
~ 1
00
E E 150,
2 596
3
4 WEEK
SUCROSE+196
5
6
7
NaCI
~---~ A M
W
a 140V
FIG . 1 Systolic blood pressure of normotensive (above) and hypertensive (below) rats .
202
Rat Slood Praseura During 24-Hour Day
Vol . 21, No . 2, 1977
TABLE 1 Organ 9~eights oP Rate on Normal and High Salt Diets Data N Body wt . q . Kidneys Heart Thymus Adrenals
Initial Final
Mg/100 g B.W .
nPn
Sucrose
Sucrose-Saline
Value
9 94 t 2*
9 95 t 2
N .S .
235 t 7
243 t 8
N .S .
499 t 7 263 t 7
683 t 22 330 t 22
122 t 11 20 .8 t 1.1
159 t 15 19 .8 t 1.3
<0 .0001 <0 .02 N .S . N.S .
*Mean t SEM Discussion There appears to be a significant difference between the morning and The afternoon blood prseeura of rate, particularly in salt hypertensives . latter exhibited heart and kidney enlargement . Occasionally a rat had a slightly higher afternoon than morning pressure The reverse, in fact, was the rule . but in no instance did it commonly do so . Even greater differences might have been found had blood pressures been meas ured nt or near the periods of greatest physical activity (around midnight) and contrasted with those taken during periods of least activity (around midday), although these periods themselves might well be altered by human activity in the animal rooms, and by procedures incident to blood pressure measurement. There being no night shift available, nocturnal studies, however useful to establish cycle patterns, are impractical for routine studies . It frequently requires 2-3 hours to obtain satisfactory blood pressures on eighteen animals, which must be warmed and accustomed to necessary restraint before reliable and reproducible pressures are obtained . If such rats are randomly selected on each occasion, as in the present instance, the a.m . and p.m . pressures of a particular animal will acmetimes be measured rather closely together (e .q ., 11 .00 and 13 .00 hours, respectively) and may differ little ; whereas on others intervals will be more temporally separated (e .g ., 08 .00 and 16 .00) and differ more . It is difficult or impossible to compare large numbers of rats at the times most advantageous to demonstrating cyclicity with a single blood pressure apparatus. Had only afternoon blood pressures been obtained, then, with the criterion of 150 mm Hg as the lower limit, it would have required 5 weeks to induce salt hypertension, whereas had only morning pressures been used,3 weeks would have sufficed . If morning pressures one week had alternated with afternoon pressures the next, a saw-toothed blood pressure curve could have been derived, in which longer salt treatment might appear either to be associated with a fall in blood pressure, or with an even greater rise than these data suggest . Fortuitously, the p.m . pressures were measured the day following a.m . pressures. Since the arterial pressure of hypertensivea rose progressively during treatment, higher afternoon pressures, had they occurred, might have been ascribed to the additional day of treatment. Had they not had that slight
Vol . 21, No . 2, 1977
Rat Blood Pressure During 24-Hour Day
203
"advantage," the p,m, pressures of hypertenaivea might have averaged even lower than a .m, pressures than actually was the case . Undoubtedly diurnal variations in blood pressure are linked to other phenomena, such as physical activity, body temperature, metabolic rate, etc which follow circadian rhythms . Being a normal biorhythm it is present in normal animals even though the range of blood pressure fluctuation is small, Hypertension, which greatly increases the upper limit of blood pressure and also expands the range oP variation, is associated with much greater differences between a .m . and p .m . pressures . A number of body functions are known to follow circadian rhythms among them body temperature, blood pressure, pulse rate, and plasma volume (6,7) . It is, therefore, not surprising that normoten~ive and salt hypertenaive rate displayed the variation o~beerved, excepting perhaps that such great differences could occur between periode~separated so slightly in time . In that connection, it may be significant that on a normal diet rata display a circadian rhythm in blood levels of aldosterone, corticosterone, and senior renin activity (S) . The steroids reach maxima at 16,00 hours and minima in the period 24 .00 to 12 .00 hours, whereas serum renin activity follows an inverse pattern . These items would correspond roughly with the periods during which arterial pressures were "low" and "high," but whether a muse and effect relationship exists is unknown. Whether other forms of hypertension behave similarly remains to be seen . In our experience group arterial pressures display greater uniformity when taken during the same time period than they do if taken randomly throughout the day . In following the development of hypertension, or its regression under therapy, conclusions drawn as to the rapidity with vvk~ich the disorder waxes or wanes, might well depend on the time of day that pressures are measured . This study indicates that when blood pressure o£ rata is to be measured periodically, it would be well to make the measurements within the same time frame . Acknowledgem~ent This study was conducted with the aid of grants HL-09911 and HL-15319 from the NIH . References
2. 3. 4. 5. 6. 7.
e.
C . P . RICHTER, Hiologlcal Clocks'in lYedicine an8 Paychtatry, Charley C . Thomas, Springfield, I11 ., p . 3-108 (1965), W . M . RAVIS, Experientia _18 235-237 (1962) . F . M . RADZIALOWSICI and W . F . HOUSQUET, J . Pharm, Exp . Therap . 163 229-238 (1968) . . C . E . HALL, S . AVACHI and O . HALL ; Clin . Exper : Phaimacol . Physiol . Suppl . 3, 83-86 (1976) . C . E . HALL and 0 . HALL, Tex . Rep, Hio1 : Mad . 22 529-549 (1964) . N . XT "R ITMAN and A . RAMSAROOP, Endocrinology 431-20 (1948) . LUCE, G . G . (Ed .), Htologica2 Rhythms in Psychtetry and llfedictne, U . S . Gov t Printing Office, Washington, D .C, (P, 44-45) (1970), C . GOMEZ-SANCHEZ, 0 . B . HOLLAND, J . R . HIGGINS, D . 103M and N . M . 1CAPLAN, Endocrinology 99 567-572 (1976) .
Pergamoa Prose
EVIDEZiCE FOR VARIATION IN BIAOD PRSSSURS DURING THE DAY IN NORMOTBriSIVE AND SALT-HYPERTENSIVE RATS C . E . Hall, D . Nasseth and O . Hall Department of Physiology and Biophysics University of Texas Medical Branch Galveston, Texas
77550
(Received in final form June 10, 1977 Summary The systolic blood pressure of unanesthetized normotensive and salt-hypertenaive rats was found to be significantly lower in the afternoon than in the morning . The difference was more marked in hypertensivea than in normotenaivea, perhaps because normal blood pressures are restricted to a narrow range . These findings indicate that blood pressure, like many other physiological functions, varies diurnally . The differences between morning and afternoon pressures, although perhaps less than they would have been at times selected to correspond with maxima and minima of physical activity, were nonetheless large enough to influence the decision as to how long it required for salt excess to cause hypertension and to indicate its rote of progression . It has been amply demonstrated that a considerable number of physiological functions and responses in rata (end other animals) wax 4n8 wane over various periods . Soma of these are endogenous rhythms, whereas others are controlled by external events . Those which cycle within or close to a 24-hour span are termed circadian rhythms . In rata, primarily nocturnal animals, the zenith of physical activity and associated functions occurs in the period between 18 .00 and 06 .00 hours, and the nadir at 06 .00 to 18 .00 hours . This is a circadian rhythm, but can be modified ezperimentally . Occasional animals depart from the norm (1) . Although moat studies of daily rhythmic activity have been conducted with a view to establishing the maxima and minima of functions under study, substantial differences are often noted even if comparisons are made between less than optimal periods . For example, it has been shown that rate and mica detoxify pentobarbital more efficiently, and sleep for shorter periods, when anesthetized at 02 .00 than at 14 .00 hours (2,3) . However, we have found that with a chloralose anesthesia, the difference between sleep time of spontaneously hypertenaive rate of the Okamoto-AOki etrnin is significantly less than that of normotenaive controls when studies are co~uctad between 13 .00 and 17 .00, but not when conducted between 08 .00 and 12 .00 (4) . The foregoing observation suggested that other functions might vary significantly between these two time periods, which delimit the usual working day . The one of greatest interest to us was arterial blood pressure . However useful it might be to make comparisons between time periods yielding the 19.9
20 0
Rat Blood Pressure During 24-Hour Day
Vol . 21, No . 2, 1977
greatest differences in order to establish maxima and minima, the more immediate concern was to ascertain whether discernible differences existed between pressures taken from 0 .800 to 12 .00 and those taken between 13 .00 and 17 .00, Accordingly, the periods circumscribing the routine laboratory work day. following study was undertaken . Materials and Methods Eighteen, four-week old, Sprague-Dnwley rats (Laboratory Associates, All of them had the right kidney removed under St . Louis, MO) were obtained . Half of them were given a 1~ NaCl 5$ sucrose solution to ether anesthesia . drink to facilitate the induction of salt hypertension (5), and . the others were Purina Lab Chow was fed ad lib. Systolic blood given a 5" sucrose solution . pressure was taken once weekly on conscious animals by a tail microphonic method using a programmed electroaphygmomanometer (PS-300) and a DMP-4 Phyaiograph (Narco Biosystems, Houston, Texas) . An average of 3 consecutive pressures agreeing to within 20 mm Hq established the blood pressure of an animal : those above 150 mm Hg were classified hypertensioe . Animals were individually caged in temperature-controlled quarters with light cycled 10 hours on and 14 hours off . Blood pressures were taken between 08 .00 and 11 .30 on one day of each week, between 13 .00 and 16 .30 on the following day and 7 days apart. Thin procedure appeared to be preferable to taking a.m . and p.m . pressures on the same day, which would have subjected the animals to very extended periods in the warming boxes and in the restraining apparatus, and interfered greatly with the normal feeding and drinking patterns . The animals were killed by etherization on the 53rd day. Various organs were placed in neutral l0i formalin for subsequent weighing . When fixed, the organs were removed, blotted, trimmed and weighed on an analytical balance. Statistical comparisons of weights were made using Student's two-tailed "t" test, and of blood pressures using the analysis of variance . Results The blood pressure of control rats fell within a narrow range throughout It was not significantly higher at the and than at the experimental period . the beginning, but in six of the seven weeks the pressures averaged higher in The same difference was true of the high the mornings than in the afternoons . salt intake gros~ in all seven weeks, but in these the blood pressure rose progressively throughout the experiment, by the end of which eight of the nine Employing an analysis of variance procedure, it was rats ware hyperteneive . determined that in both normotenaives (P < 0 .04) and hypertensivea (P < 0.0001) the 08 .00 to 11 .30 group mean blood pressures were significantly above those taken between 13 .00 to 16 .30 over the entire experimental period (Fig . 1) . Hypertensive rata had markedly enlarged hearts and kidneys, but there was no significant difference between weights of the thymus or adrenal glands (Table 1) .
Rat Hlood Pressura Duriag 24-Hour Day
Vol . 21, No . 2, 1977
a
SUCROSE
596
EE
~---~ A M ~---i P M
140
W
cc
201
W 120ac a
u 100O
n
~ 1
00
E E 150,
2 596
3
4 WEEK
SUCROSE+196
5
6
7
NaCI
~---~ A M
W
a 140V
FIG . 1 Systolic blood pressure of normotensive (above) and hypertensive (below) rats .
202
Rat Slood Praseura During 24-Hour Day
Vol . 21, No . 2, 1977
TABLE 1 Organ 9~eights oP Rate on Normal and High Salt Diets Data N Body wt . q . Kidneys Heart Thymus Adrenals
Initial Final
Mg/100 g B.W .
nPn
Sucrose
Sucrose-Saline
Value
9 94 t 2*
9 95 t 2
N .S .
235 t 7
243 t 8
N .S .
499 t 7 263 t 7
683 t 22 330 t 22
122 t 11 20 .8 t 1.1
159 t 15 19 .8 t 1.3
<0 .0001 <0 .02 N .S . N.S .
*Mean t SEM Discussion There appears to be a significant difference between the morning and The afternoon blood prseeura of rate, particularly in salt hypertensives . latter exhibited heart and kidney enlargement . Occasionally a rat had a slightly higher afternoon than morning pressure The reverse, in fact, was the rule . but in no instance did it commonly do so . Even greater differences might have been found had blood pressures been meas ured nt or near the periods of greatest physical activity (around midnight) and contrasted with those taken during periods of least activity (around midday), although these periods themselves might well be altered by human activity in the animal rooms, and by procedures incident to blood pressure measurement. There being no night shift available, nocturnal studies, however useful to establish cycle patterns, are impractical for routine studies . It frequently requires 2-3 hours to obtain satisfactory blood pressures on eighteen animals, which must be warmed and accustomed to necessary restraint before reliable and reproducible pressures are obtained . If such rats are randomly selected on each occasion, as in the present instance, the a.m . and p.m . pressures of a particular animal will acmetimes be measured rather closely together (e .q ., 11 .00 and 13 .00 hours, respectively) and may differ little ; whereas on others intervals will be more temporally separated (e .g ., 08 .00 and 16 .00) and differ more . It is difficult or impossible to compare large numbers of rats at the times most advantageous to demonstrating cyclicity with a single blood pressure apparatus. Had only afternoon blood pressures been obtained, then, with the criterion of 150 mm Hg as the lower limit, it would have required 5 weeks to induce salt hypertension, whereas had only morning pressures been used,3 weeks would have sufficed . If morning pressures one week had alternated with afternoon pressures the next, a saw-toothed blood pressure curve could have been derived, in which longer salt treatment might appear either to be associated with a fall in blood pressure, or with an even greater rise than these data suggest . Fortuitously, the p.m . pressures were measured the day following a.m . pressures. Since the arterial pressure of hypertensivea rose progressively during treatment, higher afternoon pressures, had they occurred, might have been ascribed to the additional day of treatment. Had they not had that slight
Vol . 21, No . 2, 1977
Rat Blood Pressure During 24-Hour Day
203
"advantage," the p,m, pressures of hypertenaivea might have averaged even lower than a .m, pressures than actually was the case . Undoubtedly diurnal variations in blood pressure are linked to other phenomena, such as physical activity, body temperature, metabolic rate, etc which follow circadian rhythms . Being a normal biorhythm it is present in normal animals even though the range of blood pressure fluctuation is small, Hypertension, which greatly increases the upper limit of blood pressure and also expands the range oP variation, is associated with much greater differences between a .m . and p .m . pressures . A number of body functions are known to follow circadian rhythms among them body temperature, blood pressure, pulse rate, and plasma volume (6,7) . It is, therefore, not surprising that normoten~ive and salt hypertenaive rate displayed the variation o~beerved, excepting perhaps that such great differences could occur between periode~separated so slightly in time . In that connection, it may be significant that on a normal diet rata display a circadian rhythm in blood levels of aldosterone, corticosterone, and senior renin activity (S) . The steroids reach maxima at 16,00 hours and minima in the period 24 .00 to 12 .00 hours, whereas serum renin activity follows an inverse pattern . These items would correspond roughly with the periods during which arterial pressures were "low" and "high," but whether a muse and effect relationship exists is unknown. Whether other forms of hypertension behave similarly remains to be seen . In our experience group arterial pressures display greater uniformity when taken during the same time period than they do if taken randomly throughout the day . In following the development of hypertension, or its regression under therapy, conclusions drawn as to the rapidity with vvk~ich the disorder waxes or wanes, might well depend on the time of day that pressures are measured . This study indicates that when blood pressure o£ rata is to be measured periodically, it would be well to make the measurements within the same time frame . Acknowledgem~ent This study was conducted with the aid of grants HL-09911 and HL-15319 from the NIH . References
2. 3. 4. 5. 6. 7.
e.
C . P . RICHTER, Hiologlcal Clocks'in lYedicine an8 Paychtatry, Charley C . Thomas, Springfield, I11 ., p . 3-108 (1965), W . M . RAVIS, Experientia _18 235-237 (1962) . F . M . RADZIALOWSICI and W . F . HOUSQUET, J . Pharm, Exp . Therap . 163 229-238 (1968) . . C . E . HALL, S . AVACHI and O . HALL ; Clin . Exper : Phaimacol . Physiol . Suppl . 3, 83-86 (1976) . C . E . HALL and 0 . HALL, Tex . Rep, Hio1 : Mad . 22 529-549 (1964) . N . XT "R ITMAN and A . RAMSAROOP, Endocrinology 431-20 (1948) . LUCE, G . G . (Ed .), Htologica2 Rhythms in Psychtetry and llfedictne, U . S . Gov t Printing Office, Washington, D .C, (P, 44-45) (1970), C . GOMEZ-SANCHEZ, 0 . B . HOLLAND, J . R . HIGGINS, D . 103M and N . M . 1CAPLAN, Endocrinology 99 567-572 (1976) .