Cardiovascular and Somatic Reflexes in
Experimental Allergic Encephalomyelitis
By Thomas Baum, Ph.D., and Marvin E. Rosenthale, Ph.D.
With the technical assistance of Allen T. Shropshire, B.A., and Louis Datko, M.S.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
• Experimental allergic encephalomyelitis
(EAE) has been induced in a number of
species by the injection of heterologous, homologous or isologous nervous tissue incorporated
in complete or incomplete Freund's adjuvant.1"3 In rats clinical symptoms occur 11 to
15 days after inoculation and consist of ataxia
or paresis, i.e., grossly irregular gait and
weakness of one or both hind legs, followed
by flaccid paralysis of the hindquarters, urinary incontinence, fecal impaction and weight
loss.2-3 Generally, remission of neurological
signs and clinical improvement are observed
during the third week after inoculation. The
central lesions are characterized by the infiltration of lymphocytes and other mononuclear
cells into the adventitial sheath (VirchowRobin space) of small veins and subpial and
perivascular parenchyma and by perivenous
breakdown of myelin.4"0
Immunologic and pathologic evidence indicates that EAE represents a delayed (cellular or tuberculin) type of reaction to tissue
autoantigen and can thus be classified as an
experimental autoallergic or autoimmune disease.7-8 EAE is thought, to occur as a result
of mechanisms by which animals sensitized to
a component of nervous tissue tend to reject
their own nervous tissue in a manner similar
to the rejection of a tumor transplant or skin
graft.0 Human demyelinating diseases as a
group are characterized by destruction of the
myelin sheaths of nerve fibers, the relative
sparing of the axis cylinders and the presence of perivenous lesions throughout the
brain and spinal cord. Like EAE, the human
diseases are thought to have an allergic etiolFrom the Pharmacological Evaluation Section, Wyeth Laboratories, Inc., Radnor, Pennsylvania.
Accepted for publication July 13, 1965.
118
ogy. EAE has, therefore, been considered a
useful model of the human condition.4' 10> n
The purpose of the present investigation is
to determine whether the paralysis involves
.somatic reflexes and to ascertain whether the
dysfunction extends to the cardiovascular system.
Methods
Male Lewis strain rats (Microbiological Associates, Bethesda, Md.) were used in these experiments. Isologous spinal cord, free from blood and
meninges, was homogenized in distilled water to
give a 40% weight/volume preparation. Phenol
was added to a concentration of 0.5%. An emulsion was prepared from equal volumes of the
homogenate and complete Freund's adjuvant*
(Difco) in a Virtis homogenizer. Single doses of
0.05 ml of the emulsion were injected into the
right hind foot pad in 150 to 200 g rats. Signs
of the disease appeared within 11 days and severe
paralysis became evident on the thirteenth to
fifteenth day after inoculation. Approximately
90 to 95% of the animals developed symptoms
of EAE but only severely paralyzed animals
(about 65% of the total injected) were used for
subsequent experiments.
Animals were anesthetized either with Dialurethane solutiont (0.65 ml/kg) or with chloralose (52 mg/kg) and urethane (520 mg/kg) administered intraperitoneally. Isometric contractions of the gastrocnemius-soleus-plahtaris group
of muscles were recorded from their severed tendons by a Grass model FT03 force transducer and
an Offner oscillograph. A steel pin was driven
into the head of the femur in order to stabilize
the leg. Muscle contractions were induced by
single supramaximal pulses (4 to 6 v in normal
and 3 to 7 v in paralyzed rats) of 1 msec duration applied to the distal end of the ligated sci•Complete Freund's adjuvant contains Arlacel A
(mannide mono-oleate), Bayol F (paraffin oil) and
killed dried tubercle bacilli (4 mg/ml).
t Ciba Pharmaceutical Products Company. Each
ml contains allobarbital 100 mg, urethane 400 mg,
and monoethylurea 400 mg.
Circulation Research, Vol. XVIII, February 1966
119
REFLEXES IN ALLERGIC ENCEPHALOMYELITIS
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atic nerve with bipolar electrodes at intervals
of 10 seconds. An AEL model 104A stimulator
was used. The muscles were extended from their
"initial length" (length at which resting tension
just became detectable) by 4 mm increments at
two-minute intervals. By the end of the twominute interval contractile amplitude had stabilized. Total tension refers to the sum of resting
and active tension. Ten normal and an equal
number of paralyzed rats were used.
Reflex contractions of the anterior tibial muscle (flexor reflex) were obtained by stimulation
of the central end of the ligated ipsilateral posterior tibial nerve at the level of the ankle. Muscles were extended 2 and 3 mm from their initial
lengths. Tension was recorded from the cut tendon of the muscle. Single pulses of supramaximal
voltage (3 to 6 v) and 1 msec duration were
applied every 10 seconds. The sciatic nerve was
then stimulated using the same electrical parameters and muscle contraction recorded at 3 mm
stretch. Each group consisted of seven animals.
The systemic blood pressure response to stimulation of the central end of the ligated sciatic
nerve was recorded from a carotid artery with a
Statham pressure transducer (P23Gb) in eight
normal and eight paralyzed rats. Pulses of 1 msec
duration were applied at frequencies of 10 to
40/second for periods of 5 seconds at two-minute
intervals. At the beginning of each experiment
the voltage producing maximal blood pressure
effects was determined (3 to 5 v for normal rats
and 3 to 6 v for paralyzed rats). Both maximal
and 50% of maximal voltages were used. The
trachea was intubated with polyethylene tubing.
Drugs were administered into a cannulated jugular vein.
The hindquarters of rats were perfused by a
method described by Brody et al.12 The abdominal aorta was exposed by a midline incision and
cannulated in central and peripheral directions
below the renal arteries. Blood was pumped from
the central cannula to the hindquarters by a
Sigmamotor pump (model T8). Blood obtained
from donor animals was used to fill the tubing.
Systemic and perfusion pressures were recorded
from T-tubes located proximally and distally to
the pump, respectively. The output of the Sigmamotor pump was set at a level at which perfusion
pressure approximated systemic blood pressure.
Flow remained constant for the remainder of the
experiment. Over the range of perfusion pressures encountered, pump output was independent
of perfusion pressure; therefore, changes in per-
Resting a Total Tension
Active Tension
600
Normal
500
Resting
H
Active
EH
Paralyzed
^ 400
§ 300
I
200
100
((igl)
0
4
8
12 16
0 4
STRETCH (mm)
8
•i
12 16
FIGURE 1
Response of the gastrocnemius-soleus-plantaris group of muscles to sciatic nerve stimulation in
normal and paralyzed rats. Ordinate: isometric tension. Abscissa: initial stretch of muscles.
Left: resting and total tension. Lower portion of each bar signifies resting tension and the
upper portion indicates active tension. Right: active tension. Top bars were transposed from
the left portion of the figure to a common base line on the right. Height of each bar indicates
the mean response and the line above it, the standard error.
Circulation Research, Vol. XVIII, February 1966
BAUM, ROSENTHALE
120
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fusion pressure were proportional to changes in
vascular resistance. Heparin (10 mg/kg) was
used as the anticoagulant. The central ends of
the severed cervical vagus nerves were stimulated
bilaterally using bipolar electrodes, pulses of 0.5
msec duration, 1 v intensity at a frequency of
50/sec for periods of 10 seconds. The lumbar
sympathetic chains were stimulated at low voltages (1.5 or 2.5 v, divided equally in both
groups) and high voltage (6 v) with pulses of
1 msec duration at frequencies of 5 to 20/second
for periods of 5 seconds. Drugs were injected
intra-arterially into the tubing between the Sigmamotor pump and the hindquarters in volumes
of 2.5 and 5 /xliters. Base line resistance values
were calculated by dividing perfusion pressure
(mm Hg) by flow (ml/min). Ten normal and
10 paralyzed rats were used.
Data are presented as means ± standard errors. The Student t-test was used to indicate
statistically significant differences. Each rat was
used for only one type of experiment.
Anterior tibial muscles were activated reflexly at elongations of 2 and 3 mm. At these
lengths resting tension was 5 ± 1 and 9 ± 1 g,
respectively, for normal rats and 3 ± 1 and
6 ± 2 g for the paralyzed group. The differences were not statistically significant (P >
0.05). In normal animals reflex contraction of
the muscle was elicited by stimulation of the
ipsilateral posterior tibial nerve (flexor reflex,
fig. 2). The active tension developed could
be increased by stretching the muscle from 2
to 3 mm. On the other hand, reflex contraction of the muscle could not be induced in
Flexor Reflex
90
-
I
Results
NERVE-MUSCLE STUDIES
As the gastrocnemius-soleus-plantaris muscles were extended as a group from their "initial lengths" they exerted progressively greater
resting tension (fig. 1). The force of muscular
contraction resulting from sciatic nerve stimulation (active tension) also increased up to a
maximum as muscles were elongated. Maximal active tension was exerted at an increase
in length of approximately 12 mm. The resting
and active tensions developed by the paralyzed rats were similar to the values obtained
for the normal group and no statistically significant differences between groups could be
detected at any of the muscle lengths examined (P>0.05).
Sciatic Stim.
^,3
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50 30
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•
0
0
2
3
STRETCH (mm)
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10
1
-
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j Paralyzed
FIGURE 2
Response of anterior tibial muscles to reflex activation
and to sciatic nerve stimulation. Ordinate: active isometric tension. Abscissa: initial stretch of muscles.
Left: reflexly induced active tension due to ipsilateral
posterior tibial nerve stimulation. Reflex stimulation
yielded no response in paralyzed rats (indicated by
zeros just above base line). Right: active tension induced by sciatic nerve stimulation.
TABLE 1
Hemodynamic Data from Normal and Paralyzed Rats (means ± SE)
Normal
rats
A. Intact rats, sciatic nerve stim. (8)*
Systemic blood pressure, mm Hg
Heart rate, beats/min
B. Rats with perfused hindquarters (10)
Systemic blood pressure, mm Hg
Perfusion pressure, mm Hg
Flow, ml/min
Resistance, pressure/flow
149 ± 5
403 ± 9
90
100
3.6
35
±3
±6
± 0.6
±6
Paralyzed
rats
119 ± 5t
305 ± 12f
83
95
3.3
31
±
±
±
±
6
5
0.3
4
*Numbers in parentheses indicate number of rats in each group.
fP < 0.05, paired comparison <-test.
Circulation Research, Vol. XVlll,
February 1966
121
REFLEXES IN ALLERGIC ENCEPHALOMYELITIS
any of the paralyzed rats. However, stimulation of the motor nerve, i.e., the sciatic,
caused the anterior tibial muscles of both
groups (compared at 3 mm stretch) to contract with approximately equal vigor (P >
0.05, fig. 2). In normal rats sciatic nerve stimulation produced considerably greater active
tension than could be induced by reflex activation.
BLOOD PRESSURE AND HEART RATE
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Blood pressure and heart rate were recorded
in eight normal and eight paralyzed rats. These
animals were used subsequently for determining the blood pressure response to sciatic
nerve stimulation. Pressure and heart rate
were found to be significantly lower in paralyzed rats than in controls (P < 0.05, table
1, part A).
CARDIOVASCULAR REFLEXES
In preliminary experiments it was observed
that sciatic nerve stimulation at various frequencies, voltages, and pulse durations consistently reduced blood pressure. At submaximal and maximal voltages a progressively
greater depressor response was observed as
the frequency of sciatic nerve stimulation was
increased from 10 to 20 to 40 pulses/sec (figs.
3, 4). At each point the magnitude of the
response was significantly less in paralyzed
rats than in the normal group (P < 0.05). The
intravenous injection of methacholine chloride
(0.25 and 1 /xg/kg) also caused a smaller decrease in blood pressure at each dose in paralyzed than in normal rats (P<0.05). The intravenous injection of norepinephrine (1 and
4 jug/kg) caused an approximately equal increase in blood pressure in both groups.
Bilateral central vagal stimulation increased
perfusion pressure in animals whose hindquarters were perfused at constant flow (figs.
r—,1 MIN
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METHACHOLINE
I
4
NOREPINEPHRINE
FIGURE 3
Systemic blood pressure response to sciatic nerve stimulation, methacholine and norepinephrine
in a normal rat. Ordinate: blood pressure in mm Hg. Top panel: sciatic nerve stimulation for
periods of 5 seconds with pulses of 1 msec duration at 2 and 4 v and frequencies of 10 to
40/sec. Bottom panel: effects caused by the intravenous injection of methacholine and
norepinephrine; doses in ng/kg.
Circulation Research, Vol. XVIII, February 1966
BAUM, ROSENTHALE
122
tion of norepinephrine was similar in both
groups.
Systemic blood pressure was slightly lower
in the perfused paralyzed rats than in controls but not to a statistically significant ex-
•5, 6). This response was significantly reduced
in paralyzed rats (P<0.05). However, vasoconstriction resulting from stimulation of the
lumbar sympathetic chains at various parameters or from the intra-arterial administrao—o Normal
•
• Paralyzed
Sciatic Nerve Stimulation
Low Voltage
Norepinephrine
High Voltage
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PULSES/SEC
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0.25 I
^g/kg
FIGURE 4
Comparison of the magnitude of systemic blood pressure responses to sciatic nerve stimulation,
methachoUne and norepinephrine in normal and paralyzed rats. Sciatic nerve stimulation at
low and high voltage and methachoUne reduced blood pressure while norepinephrine increased it. Ordinate: changes of blood pressure in mm Hg. Abscissa: frequencies or intravenous
doses. Points represent mean values and lines above or below them, the standard error.
,1 MIN
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150
CVS
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5/S 10/S 20/S 5/S 10/S 20/S NOREPINEPHRINE
FIGURE 5
Vasoconstrictor response to central vagal stimulation, lumbar sympathetic chain stimulation
and norepinephrine in the perfused hindquarters of a normal rat. Top trace in each panel:
systemic blood pressure (mm Hg). Bottom trace: perfusion pressure (mm Hg). Left panel:
bilateral central vagal stimulation for 10 seconds. Middle and right panels: lumbar sympathetic
chain stimulation for five seconds at voltages and frequencies indicated. Right panel also
illustrates effects of intra-arterially administered norepinephrine in total doses (tig) indicated.
Circulation Research, Vol. XV111, February 1966
123
REFLEXES IN ALLERGIC ENCEPHALOMYELITIS
S
Normal o—o
v77\ P a r a l y z e d •••••• •
Sympathetic Chain Stimulation
Central Vagal
Stimulation
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High Voltage
Norepinephrine
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20
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FIGURE S
Comparison of vasoconstrictor responses to central vagal and lumbar sympathetic chain
stimulation and norepinephrine in the perfused hindquarters of normal and paralyzed rats.
Ordinate: increase of perfusion pressure in mm Hg. Left panel: response to central vagal
stimulation. Middle and right panels: response to sympathetic chain stimulation and to intraarterial norepinephrine at frequencies or total doses indicated. Mean values and their
standard errors are shown.
tent. Perfusion pressure, as well as blood flow
to the hindquarters, was approximately equal
in both groups. The calculated vascular
resistance (pressure/flow, mm Hg/ml/min)
was, therefore, not significantly different.
BODY WEIGHTS
During the first three days after inoculation
the treated rats did not gain weight as rapidly
as the controls (averages of 2.5 and 9 g, respectively). For the next six to eight days
both groups gained at approximately the same
rate. Thereafter, the weight of the treated
animals decreased. At the time that the experiments were done (13 to 16 days after
treatment) the normal rats used in the above
experiments weighed 277 ± 4 g, whereas the
paralyzed group weighed 210 ± 3 g (P <
0.001).
Discussion
At varying degrees of stretch the resting
as well as active (response to motor nerve
stimulation) tensions exerted by the gastrocnemius-soleus-plantaris group of muscles
were similar in normal and paralyzed rats.
Circulation Research, Vol. XVIII, February 1966
Although not studied as extensively analogous responses were obtained with the anterior tibial muscle. On the other hand, reflex
contraction of the anterior tibial muscle, a
response which involves the participation of
the spinal cord, was completely blocked in
EAE rats. Thus, it appears that the functions
of peripheral motor nerves, the neuromuscular
junction, and skeletal muscle are not deficient
in EAE but that spinal cord reflexes are inhibited.
The reflex effects of sciatic nerve stimulation on blood pressure were examined at
several frequencies and voltages. In normal
rats increasing either the frequency or intensity of stimulation caused a progressively
greater reduction in blood pressure although
the slope of the response curve was not particularly steep. The magnitudes of these effects
were significantly less in paralyzed than in
normal animals. However, the reduction of
blood pressure caused by an intravenously administered depressor agent (methacholine)
was also diminished. The reduced responsiveness to methacholine can be attributed, at
124
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least partly, to the lower base line level of
blood pressure since it has been demonstrated
in other systems that the magnitude of depressor or dilator effects depends upon control
pressure.13'14 Therefore, in order to obtain a
more meaningful contrast, the depressor response to each parameter of sciatic nerve
stimulation in each rat was expressed as a percentage of the magnitude of the response to
the lower dose of methacholine. The lower
dose of methacholine was selected because
the magnitude of its effect was more nearly in
the range of the depressor response to sciaticnerve stimulation. Although the mechanisms
involved in the effects of methacholine and
sciatic nerve stimulation are not necessarily
the same, we calculated the responses to nerve
stimulation as percentages of responses to an
internal standard (methacholine) in order to
adjust the effects of nerve stimulation to the
apparent decreased reactivity of the cardiovascular system in EAE. In normal animals the responses to nerve stimulation at low voltage
and frequencies of 10, 20, and 40 pulses/second averaged 59, 65, and 90% and at high
voltage 72, 87, and 100% of those caused by
methacholine. On the other hand, the percentages obtained in paralyzed rats for the
same stimulation parameters were 18, 28, 41,
and 29, 32, 48, respectively. In each case the
difference between normal and paralyzed
animals was significantly different (P < 0.05).
Thus, the responses to nerve stimulation were
reduced out of proportion to the effects of
methacholine in paralyzed rats.
Reflex activation of the sympathetic nervous
system was induced by bilateral afferent vagal stimulation. The magnitude of the vasoconstriction in the perfused hindquarters was
reduced significantly in paralyzed rats. Only
one set of stimulation parameters was used in
comparing the results in both groups because
qualitatively different responses can be obtained when varying frequencies and voltages
are employed. However, additional stimulation parameters were tested in paralyzed
rats to make sure that the parameters used
for comparison did produce optimal results.
Stimulation of the lumbar sympathetic chains
BAUM, ROSENTHALE
caused comparable vasoconstrictor responses
in the hindquarters in both groups as did the
intra-arterial injection of norepinephrine.
These results indicate that the peripheral sympathetic nerves, the release of transmitter material at the nerve endings and the responsiveness of the vascular smooth muscle in the
hindquarters function normally in EAE but
that reflex responses requiring the participation of the spinal cord and higher structures
are inhibited.
The blood pressure and heart rate of animals tested subsequently for response to sciatic nerve stimulation were reduced significantly in paralyzed compared to normal rats.
In perfusion experiments basal blood pressure
tended toward lower levels in paralyzed rats
but in the number of animals studied the reduction was not statistically significant. The
vascular resistance in the hindquarters of the
two groups was very similar.
It is concluded from these experiments that
peripheral nerve-muscle elements in blood
vessels and skeletal muscle respond normally
in EAE but that spinal cord and possibly
higher reflexes are depressed.
Summary
Experimental allergic encephalomyelitis
(EAE) in rats is characterized by flaccid
hindlimb paralysis, urinary incontinence and
fecal impaction. Somatic and cardiovascular
reflexes were studied in these animals. At varying degrees of stretch of the gastrocnemiussoleus-plantaris group of muscles or of the
anterior tibial muscle, stimulation of the sciatic nerve increased the isometric tension of
these muscles similarly in normal and paralyzed rats. Reflex contraction of the anterior
tibial muscle was produced in normal rats by
posterior tibial nerve stimulation. This response was completely blocked in paralyzed
rats. Stimulation of the central end of the
ligated sciatic nerve at various parameters reduced blood pressure. The magnitude of this
effect was less in the paralyzed group. Although vasoconstriction in the perfused hindquarters resulting from bilateral stimulation of
the cervical vagus was also reduced in paraCirculation Research, Vol. Will,
February 1966
125
REFLEXES IN ALLERGIC ENCEPHALOMYELITIS
lyzed rats, the response to lumbar sympathetic
chain stimulation was the same in both groups.
Thus, in EAE the peripheral nerve-muscle elements in blood vessels and the skeletal muscle
responded normally but effects requiring the
participation of the central nervous system
were depressed.
mental allergic encephalomyelitis in the guinea
pig and rabbit. Am. J. Pathol. 41: 135, 1962.
7. KABAT, E. A., WOLF, A., AND BEZER, A E.: The
rapid production of acute disseminated encephalomyelitis in rhesus monkeys by injection
of heterologous and homologous brain tissue
with adjuvants. J. Exptl. Med. 85: 117, 1947.
8. WAKSMAN,
B.
H.:
Experimental
allergic
en-
cephalomyelitis and the "auto-allergic" diseases. Intern. Arch. Allergy Appl. Immunol.
14 (suppl.): 1, 1959.
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Cardiovascular and Somatic Reflexes in Experimental Allergic Encephalomyelitis
Thomas Baum, Marvin E. Rosenthale, Allen T. Shropshire and Louis Datko
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Circ Res. 1966;18:118-125
doi: 10.1161/01.RES.18.2.118
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