Brain Research, 140 (1978) 137 147
137
~F) Elsevier/North-Holland Biomedical Press
C A R D I O V A S C U L A R A L T E R A T I O N BY N U C L E U S LOCUS C O E R U L E U S IN SPONTANEOUSLY HYPERTENSIVE RAT
HIROSHI KAWAMURA, CHESTERFIELD
G.
GUNN and EDWARD
D.
FROHLICH*
Alton Ochsner Medical Foundation, Division of Research, New Orleans, La. (H.K. and E.D.F.), and (C.G.G.) the Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Okla. (U.S.A.)
(Accepted May 4th, 1977)
SUMMARY Biphasic electrical stimulation of the nucleus locus coeruleus (LC) produced frequency-related pressor responses associated with increased heart rate in normotensive rats (WKY) at both 50 and 100 #A. In contrast, spontaneously hypertensive rats (SHR) demonstrated small depressor responses at 50/~A and 50 Hz, but no consistent pressure or heart rate changes at either 50 or 100 #A stimulation. The LC pressor threshold current (current necessary to increase arterial pressure by _> 10 mm Hg) was higher in SHR than W K Y (136 ± 5 #A vs. 84 ± 7 #A; P < 0.001); and only the SHR LC demonstrated a depressor threshold (current necessary to reduce arterial pressure by _> 10 mm Hg) (53 ~ 3 #A). These threshold current levels did not seem to change with acute alterations of arterial pressure produced by either bilateral carotid artery occlusion or carotid sinus stimulation. Therefore, these studies indicate altered levels of responsiveness of the SHR LC to stimulation but that the pressor function of the LC does not seem to be importantly involved in further elevation of abnormally high arterial pressure in the SHR.
INTRODUCTION Various models of experimental hypertension have been utilized to understand pressor mechanisms in clinical hypertension; but at the present time, the OkamotoAoki strain of spontaneously hypertensive rats (SHR), although far from ideal, is generally accepted as the best counterpart for developed essential hypertension in man ~G,2a. Thus, while no experimental model seems to serve as an exact counterpart of * Correspondence to: Edward D. Frohlich, M.D., Vice-President for Research and Education, Alton Ochsner Medical Foundation, 1516 Jefferson Highway, New Orleans, Louisiana 70121.
138 human hypertension, systematic study of underlying mechanisms in various genetic forms of hypertension offers an opportunity for understanding the possible alterations which could be relevant clinically. Although the central nervous system and altered adrenergic mechanisms have been implicated in clinical (essential) hypertension, in neither has a specific abnormality been demonstrated in the central nervous system% Recently, several studies demonstrated certain abnormalities in the S HR nucleus locus coeruleus t LC) ~t),~,~:', 2o. This midbrain nucleus has the highest norepinephrine concentration i~ the brain and is an important center in influencing hypothalamic, cortical, cerebellar, medullary and spinal function 1-:~,6,14,19,2~,'~z as well as in modulating arterial pressure control. As such, delineation of its role in SHR hypertension is most important. Therefore, this study was undertaken to examine the possible participation of LC in the control of arterial pressure, heart rate, and respiration in both spontaneously hypertensive rat (SHR) and its normotensive control strain, the Wi~tar K y o t o rat (WKY). MATERIALS AN D METHODS These studies were performed on female SHR and their age-matched (21-25 weeks) normotensive W K Y control rats. All had been housed in plastic cages and were given regular rat chow and tap water ad libitum. At the time of study, they were anesthetized with a warm mixture of a-chloralose (50 mg/kg) and urethane (250 mg/kg) intraperitoneally. After induction of anesthesia, the rat was placed on her back with arms and legs gently extended and taped in position on the underside of a stereotaxic apparatus. A femoral arterial catheter (PE 50) was advanced centrally and was attached to a strain gauge transducer which, in turn, was connected to a multi-channel recorder. In this fashion, arterial pressure and heart rate could be displayed on separately recorded channels. Mean arterial pressure was calculated from the sum of diastolic pressure and one-third of the pulse pressure. A standard electrocardiogram (lead ll) was also recorded, from which heart rate was measured by counting the complexes over a 10 sec duration before and during LC electrical stimulation. The electroencephalogram (EEG) was monitored to ascertain depth of anesthesia. After midline neck incision and tracheotomy was performed, the animal was allowed to breathe spontaneously and respiratory rate was recorded continuously with a 30 ~) thermistor connected to a lowlevel DC preamplifier. After the stereotaxic frame was returned to its original upright position, the bregma was fixed in 1 mm higher than the lambda. A burr hole was drilled in the calvarium and a bipolar coaxial electrode (tip to barrel distance 0.5 ram) was placed stereotaxically into the left LC, according to the following coordinates: AP, --1.0 ~ - - I . 5 ; L, 1.2 ~ 1.3; H, 2.3 ,~ 2.5; and the zero point was the interaural midpoint intercepting the sagittal, frontal and horizontal planes. Biphasic electrical pulses were delivered using two constant current units, two stimulus isolation units, and an electrical stimulator. The electrical stimulating wave form was closely checked with an oscilloscope just before initiating the stimulation.
139 Parameters of electrical stimulation were: 20 100 Hz; 10-200 # A ; pulse duration 0.1 msec for each phase; and delivery was made in 10-sec train durations of stimulation. The currents necessary for threshold levels were also determined and defined as those levels (in #A) necessary to produce at least a 10 mm Hg increment or decrement from the prestimulation systolic arterial pressure at a constant 50 Hz stimulation setting. To l aise arterial pressure physiologically, bilateral carotid artery occlusion was also performed in some of the experiments by simultaneously placing occluding clips on both common carotid arteries under close EEG observation. Both arterial clips were released before any EEG change occurred (usually within 25 sec). Both carotid sinuses were also stimulated by stretching in order to lower arterial pressure physiologically1~. At the end of each experiment, a localizing brain lesion was made at the electrode tip by passing a 1.5 mA anodal current for 15 sec. Then all animals were perfused with a 10 ~ formalin-saline solution through the left ventricle of the heart for brain fixation: and the brains were kept in the 10°/,, formalin saline for one week. Thereafter, frozen sections were obtained by slicing the brain tissue in 50 #m mounted on slide-glasses at 200/~m intervals. The electrode tips were taken to be at the largest and deepest extent of the brain lesion. Statistical analyses were conducted by using analysis of variance (F-test) and also by using paired and unpaired t-test (two tail). All values were expressed as the mean ~ 1 standard error of the mean. RESULTS
Localization of electrode tips for frequency response stud)'. The precise areas of stimulation for the 17 experiments reported herein are presented in Fig. 1. In this fcequency-response study, the mean arterial pressure of the SHR (n -- 8) was 168 -- 5 mm Hg and significantly higher than that of the control W K Y strain (n .... 9) (107 ± 2 mm Hg, P < 0.005). SHR heart rate, 319 ± 5 beats/rain, was significantly slower than the WKY heart rate (354 ~ 6 beats/rain, P < 0.005); but there was no difference with respect to body weight between SHR and WKY (253 ± 23 vs. 243 :+ 20).
LOCALIZATION OF LOCI ELICITING LC RESPONSE
OL
WKY
SHR
ELECTRODE TIPS FROM 17 EXPERIMENTS WITHIN + 0.5mm
Fig. 1. Electrode tip localization. Localization of electrode tips and area of midbrain stimulation for the 17 reported experiments. These composite illustrations depict the deepest penetrations of the electrode tips within 0.5 mm of the rostral~zaudal direction of the left LC in WKY and SHR. The right LC of both rat strains was left intact.
140
200
WKY
Lc 37
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Fig. 2. The normotensive control (WKY) rat responded to LC stimulation (50 #A, 50 Hz) with a slight rise in systolic and diastolic pressure, usually associated with a little faster heart rate lasting throughout stimulation. After termination of the stimulation, pressure fell to (or below) prestimulation levels. In contrast, small but sustained depressor responses, generally within 3 sec, were obtained in the SHR at the same level of stimulation. After l0 sec, pressure rapidly recovered. (AP, arterial pressure; ECG, electrocardiogram, lead ]I; time marker reflects LC stimulation at current indicated for duration indicated by the one-second marks.)
% CHANGESOF MEAN ARTERIALPRESSURE TO LC STIMULATION 50%[ °WKY • SHR
3o4°] ~o~ 2O
-10
/
~ il 20
I
50
p
137
~F) Elsevier/North-Holland Biomedical Press
C A R D I O V A S C U L A R A L T E R A T I O N BY N U C L E U S LOCUS C O E R U L E U S IN SPONTANEOUSLY HYPERTENSIVE RAT
HIROSHI KAWAMURA, CHESTERFIELD
G.
GUNN and EDWARD
D.
FROHLICH*
Alton Ochsner Medical Foundation, Division of Research, New Orleans, La. (H.K. and E.D.F.), and (C.G.G.) the Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Okla. (U.S.A.)
(Accepted May 4th, 1977)
SUMMARY Biphasic electrical stimulation of the nucleus locus coeruleus (LC) produced frequency-related pressor responses associated with increased heart rate in normotensive rats (WKY) at both 50 and 100 #A. In contrast, spontaneously hypertensive rats (SHR) demonstrated small depressor responses at 50/~A and 50 Hz, but no consistent pressure or heart rate changes at either 50 or 100 #A stimulation. The LC pressor threshold current (current necessary to increase arterial pressure by _> 10 mm Hg) was higher in SHR than W K Y (136 ± 5 #A vs. 84 ± 7 #A; P < 0.001); and only the SHR LC demonstrated a depressor threshold (current necessary to reduce arterial pressure by _> 10 mm Hg) (53 ~ 3 #A). These threshold current levels did not seem to change with acute alterations of arterial pressure produced by either bilateral carotid artery occlusion or carotid sinus stimulation. Therefore, these studies indicate altered levels of responsiveness of the SHR LC to stimulation but that the pressor function of the LC does not seem to be importantly involved in further elevation of abnormally high arterial pressure in the SHR.
INTRODUCTION Various models of experimental hypertension have been utilized to understand pressor mechanisms in clinical hypertension; but at the present time, the OkamotoAoki strain of spontaneously hypertensive rats (SHR), although far from ideal, is generally accepted as the best counterpart for developed essential hypertension in man ~G,2a. Thus, while no experimental model seems to serve as an exact counterpart of * Correspondence to: Edward D. Frohlich, M.D., Vice-President for Research and Education, Alton Ochsner Medical Foundation, 1516 Jefferson Highway, New Orleans, Louisiana 70121.
138 human hypertension, systematic study of underlying mechanisms in various genetic forms of hypertension offers an opportunity for understanding the possible alterations which could be relevant clinically. Although the central nervous system and altered adrenergic mechanisms have been implicated in clinical (essential) hypertension, in neither has a specific abnormality been demonstrated in the central nervous system% Recently, several studies demonstrated certain abnormalities in the S HR nucleus locus coeruleus t LC) ~t),~,~:', 2o. This midbrain nucleus has the highest norepinephrine concentration i~ the brain and is an important center in influencing hypothalamic, cortical, cerebellar, medullary and spinal function 1-:~,6,14,19,2~,'~z as well as in modulating arterial pressure control. As such, delineation of its role in SHR hypertension is most important. Therefore, this study was undertaken to examine the possible participation of LC in the control of arterial pressure, heart rate, and respiration in both spontaneously hypertensive rat (SHR) and its normotensive control strain, the Wi~tar K y o t o rat (WKY). MATERIALS AN D METHODS These studies were performed on female SHR and their age-matched (21-25 weeks) normotensive W K Y control rats. All had been housed in plastic cages and were given regular rat chow and tap water ad libitum. At the time of study, they were anesthetized with a warm mixture of a-chloralose (50 mg/kg) and urethane (250 mg/kg) intraperitoneally. After induction of anesthesia, the rat was placed on her back with arms and legs gently extended and taped in position on the underside of a stereotaxic apparatus. A femoral arterial catheter (PE 50) was advanced centrally and was attached to a strain gauge transducer which, in turn, was connected to a multi-channel recorder. In this fashion, arterial pressure and heart rate could be displayed on separately recorded channels. Mean arterial pressure was calculated from the sum of diastolic pressure and one-third of the pulse pressure. A standard electrocardiogram (lead ll) was also recorded, from which heart rate was measured by counting the complexes over a 10 sec duration before and during LC electrical stimulation. The electroencephalogram (EEG) was monitored to ascertain depth of anesthesia. After midline neck incision and tracheotomy was performed, the animal was allowed to breathe spontaneously and respiratory rate was recorded continuously with a 30 ~) thermistor connected to a lowlevel DC preamplifier. After the stereotaxic frame was returned to its original upright position, the bregma was fixed in 1 mm higher than the lambda. A burr hole was drilled in the calvarium and a bipolar coaxial electrode (tip to barrel distance 0.5 ram) was placed stereotaxically into the left LC, according to the following coordinates: AP, --1.0 ~ - - I . 5 ; L, 1.2 ~ 1.3; H, 2.3 ,~ 2.5; and the zero point was the interaural midpoint intercepting the sagittal, frontal and horizontal planes. Biphasic electrical pulses were delivered using two constant current units, two stimulus isolation units, and an electrical stimulator. The electrical stimulating wave form was closely checked with an oscilloscope just before initiating the stimulation.
139 Parameters of electrical stimulation were: 20 100 Hz; 10-200 # A ; pulse duration 0.1 msec for each phase; and delivery was made in 10-sec train durations of stimulation. The currents necessary for threshold levels were also determined and defined as those levels (in #A) necessary to produce at least a 10 mm Hg increment or decrement from the prestimulation systolic arterial pressure at a constant 50 Hz stimulation setting. To l aise arterial pressure physiologically, bilateral carotid artery occlusion was also performed in some of the experiments by simultaneously placing occluding clips on both common carotid arteries under close EEG observation. Both arterial clips were released before any EEG change occurred (usually within 25 sec). Both carotid sinuses were also stimulated by stretching in order to lower arterial pressure physiologically1~. At the end of each experiment, a localizing brain lesion was made at the electrode tip by passing a 1.5 mA anodal current for 15 sec. Then all animals were perfused with a 10 ~ formalin-saline solution through the left ventricle of the heart for brain fixation: and the brains were kept in the 10°/,, formalin saline for one week. Thereafter, frozen sections were obtained by slicing the brain tissue in 50 #m mounted on slide-glasses at 200/~m intervals. The electrode tips were taken to be at the largest and deepest extent of the brain lesion. Statistical analyses were conducted by using analysis of variance (F-test) and also by using paired and unpaired t-test (two tail). All values were expressed as the mean ~ 1 standard error of the mean. RESULTS
Localization of electrode tips for frequency response stud)'. The precise areas of stimulation for the 17 experiments reported herein are presented in Fig. 1. In this fcequency-response study, the mean arterial pressure of the SHR (n -- 8) was 168 -- 5 mm Hg and significantly higher than that of the control W K Y strain (n .... 9) (107 ± 2 mm Hg, P < 0.005). SHR heart rate, 319 ± 5 beats/rain, was significantly slower than the WKY heart rate (354 ~ 6 beats/rain, P < 0.005); but there was no difference with respect to body weight between SHR and WKY (253 ± 23 vs. 243 :+ 20).
LOCALIZATION OF LOCI ELICITING LC RESPONSE
OL
WKY
SHR
ELECTRODE TIPS FROM 17 EXPERIMENTS WITHIN + 0.5mm
Fig. 1. Electrode tip localization. Localization of electrode tips and area of midbrain stimulation for the 17 reported experiments. These composite illustrations depict the deepest penetrations of the electrode tips within 0.5 mm of the rostral~zaudal direction of the left LC in WKY and SHR. The right LC of both rat strains was left intact.
140
200
WKY
Lc 37
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Fig. 2. The normotensive control (WKY) rat responded to LC stimulation (50 #A, 50 Hz) with a slight rise in systolic and diastolic pressure, usually associated with a little faster heart rate lasting throughout stimulation. After termination of the stimulation, pressure fell to (or below) prestimulation levels. In contrast, small but sustained depressor responses, generally within 3 sec, were obtained in the SHR at the same level of stimulation. After l0 sec, pressure rapidly recovered. (AP, arterial pressure; ECG, electrocardiogram, lead ]I; time marker reflects LC stimulation at current indicated for duration indicated by the one-second marks.)
% CHANGESOF MEAN ARTERIALPRESSURE TO LC STIMULATION 50%[ °WKY • SHR
3o4°] ~o~ 2O
-10
/
~ il 20
I
50
p
