9A7' /V THE EFFECT OF HYPOTHALAMIC STIMULATION ON THE PHAGOCYTIC ACTIVITY OF THE RETICULOENDOTHELIAL SYSTEM THESIS Presented to the Graduate Council of the North Texas State University in Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE By Paul Louis Lambert, B. A, Denton, Texas December, 1979
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9A7'/V
THE EFFECT OF HYPOTHALAMIC STIMULATION
ON THE PHAGOCYTIC ACTIVITY OF THE
RETICULOENDOTHELIAL SYSTEM
THESIS
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
MASTER OF SCIENCE
By
Paul Louis Lambert, B. A,
Denton, Texas
December, 1979
Lambert, Paul Louis, The Effect of Hypothalamic Stimulation on
the Phagocytic Actvity of the Reticuloendothelial System. Master of
A Kopf stereotaxic apparatus (Kopf Instruments, Tujunga,
California) was used in the surgical implantation of elec-
trodes. The electrodes were MS/303 stainless steel, 32 mm in
length and .15 mm in diameter. Self-tapping stainless steel
mounting screws .16 mm in length along with cranioplastic
liquid and powder were utilized in the permanent fixing of
the implanted electrodes. All electrodes, mounting screws,
and cement were supplied by Plastic Products, Inc., Roanoke,
Virginia.
A Bausch and Lomb Spectronic 20 Spectrophotometer with
a wavelength range of from 340 nm to 950 nm was used to
determine clearance rates of blood samples (Bausch & Lomb,
Rochester, New York).
Procedure
Surgery. Animals were first implanted with polyethel-
ene cannulas constructed according to a modification of the
method outlined by Weeks (1967). Animals were anesthetized
with sodium pentobarbitol at a dose of 50 mg per kilogram,
Following anesthesia, the polyethelene cannula was implanted
in the right jugular vein utilizing the procedure described
by Weeks (1967). After 4 days recovery from the above pro-
cedure, each animal was placed in a stereotaxic apparatus
and the skull was exposed by incision approximately 1 inch
long. Following the incision, the skull was trephined and
electrodes were implanted bilaterally in the ventromedial
area of the hypothalamus. The stereotaxic coordinates for
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the implantation of the electrodes were as follows: 6.7 mm
anterior of the interaural line, .75 mm lateral of the mid-
saggital sinus, and 9.5 mm ventral from the dural surface of
the brain (Sherwood & Timeras, 1970).
Following the return of each animal to his preoperative
weight, subjects were removed from their home cates and a
colloidal suspension of carbon particles (Koh-I-Nor Rapido-
graph, Inc., Bloomsbury, New Jersey) was prepared. The col-
loidal suspension was mixed in a ratio of 1:3 with .9%
physiological saline resulting in a concentration of 25mg/ml.
Each animal was injected with this mixture through the jugu-
lar cannula at a ratio of 5 mg/kg of body weight, Prior to
the administration of the suspension, the tail of each animal
was clipped and a .25 ml blood sample was taken from the tail
vein by a "milking" procedure. This first blood sample
served as a baseline measure. Following this procedure, the
carbon suspension was injected into the jugular cannula in
the ratio outlined above and .025 ml aliquots of tail vein
blood were taken at 1-minute intervals for 15 minutes, All
blood samples were lysed in a 4 ml .1% solution of sodium
carbonate (Na2CO3 ). The sodium carbonate solution was a mix-
ture of 1 gm of Na2CO3 to one liter of deionized water. The
samples were next subjected to spectrophotometric analysis
at a setting of 675 nm. Each blood sample was read against
a blank of 4 ml of .1% Na2 CO3 , Optical densities were cal-
culated by subtracting the absorbance value of the baseline
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blood sample from each of the 15 1-minute blood samples taken
after the injection of the carbon suspension, The logarithms
of these optical densities and half-time clearance rates were
calculated for each animal from the slope of the regression
line by means of the formula T = b (Stowe, 1977), where
.301 is the logarithm of 2 and b is the slope of the regres-
sion line based on a least-squares regression equation util-
izing the logarithms of the optical densities across time.
Brain stimulation. Following the determination of the
carbon clearance rates for each animal after the surgical
procedures, a period of 5 days lapsed before the next phase
of the experiment began. At the end of this period, each
animal was placed in the operant chamber where intracranial
brain stimulation was delivered by a Grass S48 brain stimu-
lator through a bifurcated cable. Each animal received 1
train of rectangular wave form pulses every 5 seconds up to
a maximum of 6 volts, with the duration of each train lasting
1 second. The intensity of brain stimulation was adjusted
for each animal by beginning the first session at 1 volt and
moving up in 1-volt increments every 2 minutes until either
the maximum of 6 volts was reached or until an aversive reac-
tion to brain stimulation was observed. If an aversive
reaction was observed, the intensity was lowered by 1 volt
and this parameter was held constant for each animal through-
out the experiment. An aversive reaction was defined as
vocalization or exaggerated motor behavior at the onset of a
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stimulus pulse. Each train of stimulation delivered 100 stim-
ulus pulses per second with the duration of each stimulus
pulse lasting .2 msec. After 2 hours of brain stimulation,
each animal was returned to his home cage for a period of
96 hours. At the end of this period, each animal was sub-
jected to another carbon clearance test. This measurement
was used to assess any long-term effects of brain stimulation.
After 4 days, each animal received intracranial stimulation
using the parameters outlined above and measured using the
carbon clearance test 3, 6, 12, and 24 hours post-brain
stimulation in a counterbalanced sequence. A 4-day period
was imposed between each measurement to allow for carry-over
effects.
Results
Results presented in Figure 1 indicate that ventromedial
hypothalamic stimulation decreases the rate of carbon clear-
ance from the bloodstream. The points along the abcissa
represent the poststimulation measurement periods. The ordi-
nate represents mean difference scores from baseline. The
difference scores were obtained by subtracting the mean half-
time clearance rates for each poststimulation period from
the average half-time carbon clearance rate at baseline. The
half-time carbon clearance rate indicates the amount of time
in minutes taken to clear half of the injected carbon. A
one-way repeated ANOVA of difference scores shows a signifi-
cant overall effect (F = 5.47, p < .01). The summary of
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3.0-
2 .5-
2.0-
1.5-Q)-P
1.0-
0.5-0
00
4, -0. 5-
a)44
-2.0-
-2.5-
3 6 12 24 96
Poststimulation Periods/Hours
Figure 1. Mean difference scores from baseline as a functionof time since hypothalamic stimulation. Negativescores indicate slower carbon clearance ratesrelative to baseline.
15
this analysis is shown in Table 1. A check on the assumption
of compound symmetry of the variance-covariance matrix yields
the parameter 0 = .66. The resulting degrees of freedom are
(k-l) 6 = 3 for the numerator, and (k-1) (n-1) 0 = 19 for the
denominator. The significance level of the obtained F did
not change following the correction for degrees of freedom.
By Newman-Keuls post hoc analysis of the difference scores
the 3-, 6-, 12-, and 24-hour poststimulation periods do not
differ from each other but show a significant increase from
the 4-day measure.
Table 1
Summary Table of the ANOVA of Difference Scores
Source of Variation SS df MS F
Between 282.14 7
Within 115.81 32
Measurement periods 50.82 4 12.71 5,47*
Residual 64.99 28 2.32
Total 397.95 39
*p < .01.
The form of the relationship between brain stimulation
and half-time clearance rates appears to be curvilinear. A
significant quadratic trend supports this observation (F =
11.42, p < .01). In addition, six of the eight subjects
16
showed U-shaped functions when individual curves were
plotted.
The results of histological examination reveal that the
electrodes were generally in the VMN with no indication of
lesion at the tip. There appeared to be a correlation
between electrode placement and changes in reticuloendothel-
ial activity. The six subjects showing distinctive U-shape
functions had electrodes in the VMN adjacent to the third
ventricle, while the other two subjects had one electrode
on the lateral border of the medial hypothalamus,
Discussion
Results of this investigation suggest that central ner-
vous system activity may have an effect on the reticuloendo-
thelial system by depressing macrophage function. This is
consistent with studies reporting effects on other measures
of immune system activity. In this study, carbon clearance
rates are depressed for at least 24 hours poststimulation.
The effect is present at 3 hours, reaches a peak at 12 hours,
is decreasing at 24 hours, and by 96 hours returns to base-
line.
The present experiment should be considered a prelimi-
nary attempt to determine whether measurable changes in
reticuloendothelial activity can be produced by brain stimu-
lation. Because of the nonphysiological amount of stimula-
tion, the pattern of results observed here is not necessarily
indicative of changes following central nervous system
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activation in the intact animal. In fact, stimulation rather
than depression of reticuloendothelial activity seems possi-
ble because stimulation can increase or decrease other mea-
sures of immune system activity such as gamma globulin