M .C. Saleh et al. Brain Research 879 2000 105 –114
107
membrane. The catheter was advanced caudally through Changes in nerve activity and phenylephrine-induced
the subarachnoid space placing the tip of the catheter cardiovascular responses were analysed by two-way
immediately rostral to the lumbar enlargement T To ANOVA for repeated measures followed by a Student–
12 .
inject, the catheter was slowly withdrawn over a distance Newman–Keul’s post hoc analysis Sigma Stat. In all
of 5 cm while making 200-nl injections at 1-cm intervals cases, differences were considered significant if P0.05.
between the lumbar enlargement T and the base of the
12
cervical spinal cord T Control injections of saline 2.6. Histology
1 .
0.9; 1 ml preceded intrathecal estrogen injection. Control injections of saline ICI 182,780 0.9; 1 pM; 1
At the end of each experiment animals were perfused ml preceded intrathecal injection of estrogen ICI 182,780.
transcardially with 0.9 saline followed by 10 formalin. The brains were removed and stored in 10 formalin until
2.4. Baroreflex testing and autonomic tone the location of micro-syringe tracks could be verified
measurements histologically in thionin-stained coronal sections 6100
mm. For verification of intrathecal catheter placement, 1 In all animals, the baroreflex was evoked by the
ml of blue ink was injected using the protocol described intravenous administration of phenylephrine hydrochloride
above and the presence of ink in the region of the base of PE; 0.1 mg kg 5 min prior to, and 5, 30, 60, 90 and 120
the cervical spinal cord T to the rostral tip of the lumbar
1
min following the central injection of estrogen and 5 min enlargement T was confirmed.
12
prior to, and 5, 30, and 60 min following the central injection of estrogen ICI 182, 780. As well, the baroreflex
was tested 5 min prior to and 5 and 30 min following
3. Results
control injections of saline and ICI 182,780. Sympatho- vagal balance was assessed at each time point by moni-
3.1. Effect of estrogen on baseline parameters toring changes in renal and vagal efferent nerve activities,
as well as by calculating the ratio of the bradycardic 3.1.1. Estrogen-replaced animals
response to the phenylephrine-evoked pressor response Prior to estrogen injection, mean arterial pressure
Index of Baroreflex Function5DHR DMAP beats per MAP and heart rate HR were 100612 mmHg and
min mmHg. 298619 beats per min respectively n555. Baseline
values for vagal parasympathetic nerve activity VPNA 2.5. Data analysis
and renal sympathetic nerve activity RSNA were 1964 mV n555 and 1565 mV n555 respectively. Control
All data are presented as mean6standard error of the injections of saline and co-injection of estrogen ICI
mean S.E.M.. Changes in blood pressure, heart rate and 182,780 into all nuclei and the intrathecal space had no
nerve activity data were calculated at pertinent time points effect on baseline blood pressure, heart rate or nerve
using the analysis mode of the
POLYVIEW PRO
32 software activities data not shown. Baseline mean arterial pressure
program. Changes in blood pressure and heart rate were and RSNA were significantly decreased 30 min following
analysed by a one-way analysis of variance ANOVA for injection of estrogen into NTS n54, RVLM n54, PBN
repeated measures followed by a Student–Newman– n54, CNA n54 and the intrathecal space n53; Table
Keul’s post hoc analysis Sigma Stat, Jandel Scientific. 1. Injection of estrogen into the IC n54 produced a
Table 1 Effect of estrogen injection in central autonomic nuclei on baseline parameters
Injection site DMAP mmHg
DHR bpm DVPNA
DRSNA E
Saline E
Saline E
Saline E
Saline
2 2
2 2
a a
a a
a a
a a
NTS 22564
22065 23568
22562 4069
3064 23066
22567
a a
a a
Amb 2462
2563 24569
23062 55611
4069 2562
2665
a a
a a
RVLM 23065
22567 2564
2562 865
562 23564
23065
a a
a a
PBN 22065
2965 22565
2764 3065
963 22563
2966
a a
CNA 22565
2865 2663
2865 564
563 22264
2864
a
IC 2563
2564 2864
2766 562
562 22065
2865
a a
a a
a a
a a
Intrathecal 23066
22565 23065
22565 3065
2065 23567
22566
a
Peak changes mean6S.E. in baseline heart rate HR and vagal parasympathetic nerve activity VPNA occurred 5 min following estrogen injection. Peak changes in mean arterial pressure MAP and renal sympathetic nerve activity RSNA occurred 30 min following the bilateral microinjection of
estrogen in estrogen-replaced E and saline-replaced Saline ovariectomized female rats. NTS, nucleus tractus solitarius; Amb, nucleus ambiguus;
2
RVLM, rostral ventrolateral medulla; PBN, parabrachial nucleus; CNA, central nucleus of the amygdala; IC, insular cortex. Asterisk indicates significantly different from pre-injection value ANOVA; P,0.05.
108 M
significant decrease in RSNA only Table 1. Baseline heart rate was significantly decreased and VPNA was
heart rate was significantly decreased and VPNA was significantly increased at 5 min post-estrogen injection into
significantly increased at 5 min post-estrogen injection into NTS, Amb n54 and the intrathecal space Table 1. All
NTS, Amb n54, PBN and the intrathecal space Table baseline parameters returned to pre-injection values 60 min
1. All baseline parameters returned to pre-injection values post-estrogen injection. Injection of estrogen into PBN
60 min post-estrogen injection. n54, CNA n54 and IC n54 had no significant effect
on MAP, HR, VPNA or RSNA Table 1. 3.1.2. Saline-replaced animals
Prior to estrogen injection mean arterial pressure MAP 3.2. Effect of estrogen on baroreflex function
and heart rate HR were 111615 mmHg and 329622 beats per min respectively n553 which were signifi-
3.2.1. Estrogen-replaced animals cantly elevated compared to estrogen-replaced animals
Testing of the baroreflex with phenylephrine PE prior P,0.05. Baseline values for vagal parasympathetic nerve
to central injection of estrogen evoked an increase in MAP activity VPNA and renal sympathetic nerve activity
1762 mmHg; n555 accompanied by a reflexive de- RSNA were 1663 mV n553 and 1865 mV n553
crease in HR 21162 beats per min; n555; Figs. 1A and respectively. Control injections of saline and co-injection
2A. As well, during baroreflex testing VPNA was in- of estrogen ICI 182,780 into all nuclei and the intrathecal
creased 45610 n555 and RSNA was decreased space had no effect on baseline blood pressure, heart rate
2668 n555 relative to baseline levels Fig. 1A. The or nerve activities data not shown. Baseline mean arterial
index of baroreflex function at this time point was pressure and RSNA were significantly decreased 30 min
0.760.05 beats per min mmHg n555; Fig. 4A. RSNA following injection of estrogen into NTS n54, RVLM
and the pressor response to PE were significantly at- n54 and the intrathecal space n54; Table 1. Baseline
tenuated 30 min following injection of estrogen into NTS
Fig. 1. Cardiovascular and autonomic responses to phenylephrine injection PE; 0.1 mg kg; ↑
before baseline and 5 min following the bilateral 100 nl per side micro-injection of either saline 0.9; control or estrogen 0.5 mM; estrogen into the NTS in estrogen-replaced A and saline-replaced B
ovariectomized female rats. Alterations in blood pressure, heart rate and nerve activities measured during baroreflex testing originate at the arrow ↑
and were measured for 1 min following PE injection time scale bar51 min.
M .C. Saleh et al. Brain Research 879 2000 105 –114
109
Fig. 2. Mean changes from baseline in mean arterial pressure MAP and heart rate HR in response to phenylephrine injection 0.1 mg kg; i.v. before and following central injection of saline 0.9, control or estrogen 0.5 mM; 100 nl per side; estrogen in estrogen-replaced A or saline-replaced B
ovariectomized female rats. Asterisks indicate significance P,0.05; ANOVA from pre-injection value before. NTS, nucleus tractus solitarius; Amb, nucleus ambiguus; RVLM, rostral ventrolateral medulla; PBN, parabrachial nucleus; CNA, central nucleus of the amygdala; IC, insular cortex; i.t.,
intrathecal space of the spinal cord.
n54, RVLM n54, PBN n54, CNA n54 and the 2162 mmHg; n553 accompanied by a reflexive de-
intrathecal space n53; Figs. 2A and 3A. Injection of crease in HR 2962 beats min; n553; Figs. 1B and 2B.
estrogen into the IC n54 produced a significant decrease As well, during baroreflex testing VPNA was increased
in RSNA only Fig. 3A. VPNA and the reflexive bradycar- 35611 n553 and RSNA was decreased 2067 n5
dia to PE injection were significantly increased 30 min 53 relative to baseline levels Fig. 1B. The index of
post-estrogen injection into NTS, Amb n54, PBN and baroreflex function at this time point was 0.4560.05 beats
the intrathecal space Figs. 2A and 3A. Baroreflex func- per min mmHg n553 Fig. 4B. RSNA and the pressor
tion was significantly enhanced following injection of response to PE were significantly attenuated 30 min
estrogen into all autonomic nuclei and the intrathecal space following injection of estrogen into NTS n54, RVLM
Fig. 4A. PE-evoked changes in MAP, HR, VPNA and n54 and the intrathecal space n54; Figs. 2B and 3B.
RSNA returned to pre-injection values 90 min post-es- VPNA and the reflexive bradycardia to PE injection were
trogen injection. significantly increased 30 min post-estrogen injection into
NTS, Amb n54 and the intrathecal space Figs. 2B and 3.2.2. Saline-replaced animals
3B. Baroreflex function was significantly enhanced fol- Testing of the baroreflex with phenylephrine PE prior
lowing injection of estrogen into NTS, Amb, RVLM and to central injection of estrogen evoked an increase in MAP
the intrathecal space Fig. 4B. PE-evoked changes in
110 M
Fig. 3. Mean changes in vagal VPNA and renal RSNA efferent nerve activities given as a per cent change from baseline 0 in response to phenylephrine injection 0.1 mg kg; i.v. following central injection of saline 0.9, control or estrogen 0.5 mM; estrogen in estrogen-replaced A or
saline-replaced B ovariectomized female rats. Asterisks indicate significance P,0.05; ANOVA from pre-injection value before. NTS, nucleus tractus solitarius; Amb, nucleus ambiguus; RVLM, rostral ventrolateral medulla; PBN, parabrachial nucleus; CNA, central nucleus of the amygdala; IC, insular
cortex; i.t., intrathecal space of the spinal cord.
MAP, HR, VPNA and RSNA returned to pre-injection unilateral or outside the intended region produced no
values 90 min post-estrogen injection. Injection of estrogen significant effects on baseline parameters nor on PE-
into PBN n54, CNA n54 and IC n54 had no evoked changes in the same parameters data not shown.
significant effect on the PE-evoked changes in MAP, HR, As well, the sites of injection for saline and ICI 182,780
VPNA or RSNA Figs. 2B and 3B. controls have been omitted from Figs. 5 and 6 for clarity.
3.3. Histological verification of cannulae placement
4. Discussion
