INTRODUCTION
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T |
he greatest
proportion of the cells that responds to skin temperature is in the nucleus
raphe magnus (NRM), whereas there are few neurons in the raphe dorsalis and
pontis that are influenced [1]. Blood is
diverted from the skin to other organs in hypothermia [2] and the loss of heat
in rats is regulated by blood flow under sympathetic control, through elaborate
system of arteriovenous anastomoses of tail [3,
4]. Skin vasomotor and sudomotor centers are all important in
thermo-regulation and the raphe magnus controls the skin blood flow [5,
6]. Previous studies have shown that
electrical stimulation of the mid to caudal raphe magnus elicited sweat
secretion and skin blood flow rises in the forepaw pads of decerbrate cats
[7]. Excitation of neurons in the raphe
region can cause vasoconstriction in the cutaneous bed without greatly
affecting arterial pressure and changing blood flow in the mesenteric bed [8,
9]. It has also been demonstrated that chemical stimulation of the rostral
venterolateral medulla can reduce tail temperature in hyperthermic,
anesthetized rats [10].
The importance
of medullar raphe in the control of rat tail blood flow (TBF) has been
confirmed by (i) enhancement to tail sympathetic nerve activity after
chemically activation of raphe neurons through glutamate microinjections, (ii)
detection of sympathetic premotor neurons in medullar raphe nuclei after tracer
injections into artery of tail, and (iii), increase in the expression of Fos
immunoreactivity, mainly in the raphe, during hypothermia [11-13].
The aim of this
study is to investigate the inactivation and excitation effects of lidocaine
and glutamate on thermoregulatory processes in NRM of the rats.
MATERIALS AND METHODS
All chemical
reagents were purchased from Sigma Company (
Animals. Male Wistar rats (n = 24, weighting 250-300 g)
were obtained from the Pasteur Institute of Iran (
1°C with free
access to water and food for one week. The rats were anesthetized with sodium
pentobarbital (40 mg/kg, i.p.) and an endotracheal tube was inserted via a tracheostomy
[14]. They were then placed in Kopf stereotaxic instrument and, to access to
the NRM, a 22-gauge stainless steel guide cannula was implanted stereotaxically
into the NRM with the anterior-posterior, mediolateral, and dorsoventral
coordination at 10.52, 0.0, and 10.1 mm, respectively [15].
Laser Doppler
flowmeter. After one week
of recovery, rats were reanesthetized with urethane (l g/kg; i.p.) and a Laser
Doppler probe (1.33 mm probe) was positioned at the tail cutaneous surface
around the tail artery for skin blood flow measurement [6]. The Doppler probe was connected to a
flowmeter and the analog signal was digitized with a Maclab recording system
(Laser Blood Flow Monitor (MBF3), Moor Instruments England, Class 3A Laser
product, Max power 1.7, BSEN 60825, 1991).
Hypothermia. Body
temperature was maintained at 22°C
by wrapping the body with a cooling pad, leaving the tail exposed to the room
temperature. A thermocouple was placed 6 cm past the anal sphincter to record the
body temperature. Tail blood flow was measured before and after cooling the
body. The implanted cannula in NRM was connected to a
Drug treatment
and TBF evaluation. Since the drugs
are solved in artificial cerebrospinal fluid (aCSF), one type of control
injection was performed: a vehicle control injection into the NRM. So, in
control group (n = 8) aCSF and in other groups, lidocaine (1 µl, 2%) and
glutamate (1 µl, 78 pmol/60 nl) were injected [16, 17]. TBF was measured
immediately before and after each injection and in the case of lidocaine
measurement was continued to 10 min.
Histological
confirmation. After
completion of the experiment, the ink was injected through the cannula to
confirm proper implantation of guide cannula and the rats were per fused with
normal saline solution followed by 9% formaldehyde and 30% sucrose
solutions. A section (40 µm) of the
brainstem was stained with Evan's blue dye and proper placement of
the pipette tip in the NRM was verified with histological sections under the
microscope.
Statistical
analysis. Data is
represented as mean ± SEM of 8 male rats in each group. Paired student's t-test was performed
to evaluate the differences in mean of TBF before and after treatment.
All the animal
experiments were performed in accordance with the guideline of Hamadan
University of Medical Sciences and Health Services, Hamadan, Iran (http://www. Umsha.ac.ir)
for experimental animals.
RESULTS AND DISCUSSION
In the present
study, injection of aCSF into the NRM had no significantly effect on TBF
compared to pre-injection values (P>0.05). Following the injection of
lidocaine, TBF decreased from 54.43 ± 5.7 to 46.81 ± 3.4 (P<0.05) and
the significant effect of lidocaine was observed ten min after injection at the
hypothermic temperature (22°C), as shown in
Figure 1A.
Previous
studies have shown that NRM is a necessary component of the central nervous
system thermoregulatory control circuitry in rats [17]
and mid to caudal raphe magnus/pallidus stimulation increases skin
blood flow in the forepaw pads of)decerbrate cats [8]. Therefore, NRM
involves in the modulation of thermal information [15]. In this study,
lidocaine, the fast-acting local anesthetic agent, was used to inactivate the
NRM reversibly. Lidocaine blocks sodium channels, thus it inhibits neuronal
electrical activity in the affected area. A 1.0 µl injection of 2% lidocaine
solution has a 10-15 min effective period. This causes the lidocaineinfusion,
an excellent technique for interrupting local neuronal activity without
permanently altering the system [17]. In hypothermia, lidocaine injections into
the medullary raphe were effective in suppressing the tail vasoconstriction,
and therefore, lidocaine inhibits thermoregulatory effect of NRM and decreases
TBF. Also, it shows another way to confirm previous results.
The investigation of the effect of glutamate injection
on TBF showed a significant increase in TBF from 44.194 ± 0.6 to 98 ± 10, as
indicated in Figure 1B (P<0.05). When tail blood flow was reduced by
lowering the body temperature, the injection of glutamate restored the flow to
levels generally observed in animals maintained at a higher baseline
temperature. Therefore, according to our results, it can be concluded that
chemical excitation of a subpopulation of neurons located in the raphe magnus
region increases the blood flow of the tail cutaneous bed. Since glutamate, an
excitatory neurotransmitter, can stimulate the NRM neurons, it may also
decrease tail vasoconstriction in response to hypothermia. In addition, since
sympathetic fibers play a key role in evoking vasoconstriction of the
superficial vascular beds, it is suitable to propose that a reduction in the
sympathetic outflow by centrally acting glutamate may be responsible for its
hypothermic action in the NRM.
As hypothermia causes peripheral vaso-constriction,
it is concluded that the vasoconstriction effect of hypothermia can be lowered
by glutamate and augmented by lidocaine in NRM.
Our findings add to evidence that the activation of the raphe magnus is
involved in regulatory cutaneous blood flow in response to hypothermia in rats.
In addition, our results highlight the importance of interaction between
glutamate and thermoregulatory pathway in the nervous system. However, further investigations are required
to evaluate NRM thermoregulatory mechanisms.
Fig.
1. Effect of unilateral injection
of (A) lidocaine (1 µl, 2%) and (B) glutamate (78 pmol/60 nl)
into the nucleus raphe magnus on tail blood flow in anesthetized cooled rats.
Data are represented by mean ± SEM. Vertical
bars denote SEM change from baseline values. Each bar represents the average
data from 8 rats. Lido10 = ten min after
lidocaine injection; H, hypothermia; Glu, glutamate injection; *P<0.05.
ACKNOWLEDGEMENTS
We are thankful to the Physiology Department
of Tarbiat Modares University (
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