EFFECT OF CHRONICHYPOTHALAMICSTIMULATION UPONCHOLESTEROL-INDUCEDATHEROSCLEROSISIN

THE RABBIT *

By C. G. GUNN, MEYERFRIEDMANAND SANFORD0. BYERS

(From the Departments of Medicine and Physiology, University of Oklahoma Medical Center,Oklahoma City, Okla., and the Harold Brunn Institute, Mount Zion Hospital and

Medical Center, San Francisco, Calif.)

(Submitted for publication July 11, 1960; accepted August 18, 1960)

The possible role of the central nervous system(CNS) in the pathogenesis of experimentalatherosclerosis has received scant investigativescrutiny. Indeed, it can even be stated that themajority of investigators in the field of experi-mental atherosclerosis are frankly skeptical of anypossible etiological relationship between dysfunc-tion of any central neural mechanisms and periph-eral atherosclerosis. Certainly in most of the re-cent American reviews concerned with athero-sclerosis the subject of nervous factors in theetiology of atherosclerosis has either been mini-mized or totally neglected.

Despite this seeming exclusion of the CNSfromlaboratory studies of atherosclerosis by many in-vestigators, certain experimental and clinical datamake this neglect appear rather unfortunate.Myasnikov (1, 2) has demonstrated that whendrugs that depress certain CNSmechanisms (e.g.,barbiturates) were chronically given to cholesterol-fed rabbits, these rabbits exhibited a significant de-crease in hypercholesterolemia and aortic athero-sclerosis. On the other hand, phenamine, a drugwhich excites the CNS, produced the opposite ef-fects, increasing the hypercholesterolemia andatherosclerosis (3). Finally, it has been reportedthat rats fed an atherogenic diet and exposed to aparticular form of stress exhibited greater degreesof hypercholesterolemia and coronary atherosclero-sis than their controls fed the same diet (4).

Several clinical studies also strongly suggestthat changes in psychological behavior influencenot only the level of blood cholesterol (5-8) butalso the incidence of clinically manifested coronaryartery disease (9).

* Aided by grants from the Alameda County HeartAssociation, Life Insurance Medical Research Fund, andthe National Heart Institute (H-3111 and H-119),Bethesda, Md.

If these experimental and clinical reports arevalid, they force us, despite their paucity, to in-clude the CNS in the ambit of investigation con-cerning the pathogenesis of atherosclerosis.

This report describes some effects of chronicminimal electrical stimulation discretely deliveredto electrodes placed in the diencephalon of rabbitsfed a cholesterol-rich diet. It was found that rab-bits exposed to such stimulation exhibited a sig-nificantly greater degree of hyperlipemia and amuch more marked atherosclerotic involvement oftheir aortic and coronary vasculature than didtheir controls.

METHODS

The present study was the result of a joinit effort byindividuals of the University of Oklahoma Medical Center(C. G. G.) and of the Harold Brunn Institute, MountZion Hospital and Medical Center, San Francisco(M. F. and S. 0. B.). All physiological and anatomicalprocedures were done at the University of OklahomaMedical Center, while all biochemical and pathologicalstudies were carried out at the Harold Brunn Institute.In order to eliminate bias, all biochemical and patho-logical data were obtained, assessed and recorded blind,i.e., without knowledge of whether a given specimen wasfrom an experimental or control animal.

Thirty-five male New Zealand white rabbits of thesame age and comparable size from the same inbred herdwere chosen at a large breeding farm. After an ob-servation period of 1 month, 19 of these animals wereoperated upon under pentobarbital anesthesia. By meansof a stereotaxic instrument (10), electrodes were in-serted into or as close as possible to the ventral medialnucleus of the hypothalamus. The electrodes were small,bipolar, concentric and insulated from each other exceptat the tip. The bare electrode tips were separated fromeach other by a distance of 1 mm. After insertion, theelectrodes and their subminiature plug attachment weresecured to the calvarium with dental cement. The sub-cutaneous tissue and skin then were closed in layersleaving the plug outside (see Figure 1) and availablefor connection to either recording or stimulating ap-paratus.

1963

C. G. GUNN, MEYERFRIEDMANAND SANFORD0. BYERS

FIG. 1. RABBIT WITH IMPLANTED ELECTRODESCONNECTEDTO STIMULATOR. The oscilloscopemonitors (voltage or milliamperage) the parameters of stimulation. When not in use, ex-ternal wires and plug are removed without touching the animal.

Following the operation, the animals were rested for1 month. In order to determine the behavioral andautonomic thresholds, preliminary studies were doneutilizing biphasic, 60 cycles per second, monitored cur-

rents of 0.2 to 0.8 ma. The current was administeredat a strength of 1 to 6 v in pulses of 0.2 msec duration.

The threshold parameter (i.e., the current necessary

to produce pupillary and behavioral changes) wNas de-termined for each of the 19 animals. Then they were

given chronic stimulations at a current strength slightlyabove the determined threshold. These chronic stimula-tions were done four times daily, 6 days a week for 3months. Each period or "epoch" of stimulation con-

sisted of ten 30-second episodes of actual stimulationinterspersed with ten 30-second intervals of pause. Thevoltage and amperage of each stimulation wvere moni-tored with an oscilloscope to ensure constancy. Allstimulations were done in the cage without direct han-dling of the animal (see Figure 1). The daily behaviorand response of each rabbit to stimulation were recorded.

Three series of animals were studied for 3 months.Series A consisted of 14 of the 19 animals bearing elec-trodes, that were placed on Purina rabbit chow enrichedwith cottonseed oil (1 per cent) and cholesterol (1 per

cent) immediately following operation. Series B con-

sisted of the remaining 5 of these 19 stimulated rabbitsthat were p)laced on Purina rabbit cliow alone. Series C,a control group, consistedl of 16 initact rabbits that were

given the clholesterol- and oil-enriclhed diet.Stimulationi in the meldial areas of the hypothalamus

in cats has been reported to induce satiety (11), thus

care was taken in the present studies to ensure compara-ble intake of the lipid-enriched food by all of the ani-mals. This was accomplished by restricting the foodallotment for each animal to that quantity completelyconsumed by those rabbits eating least. This latteramount was approximately 100 g per day containiingabout 160 calories.

In order to discover possible effects of acute hypo-thalamic stimulation, some of the animals of the aboveseries also were studied both before and very soon aftera single episode of stimulatioin. Thus after the firstmonth of the experiment, seven rabbits of Series A andfive of Series B were bled (20 ml) and then stimulatedfor two continuous "epochs." One hour later, bloodsamples were again obtained. For control purposes, fiverabbits of Series C were bled at the same time intervals.A sinmilar study (except that the second blood samplewas obtained 5 hours after acute stimulation) was re-peated upoIn seven rabbits of Series A at the end of 3months. In this latter study, four rabbits of Series Calso were bled at the same time intervals.

In the chronic experiment, as distinguished from theabove acute experiments, blood samples were obtainedbefore the beginning of the experimental period and thenrepeated each month, as indicated in Table I. In mostcases, blood samples were analyzed for serum cholesterol(12), phospholipid (13), total lipid (14), at1(d 17-hy(roxy-corticosteroid (15).

At the end of the 3-month period, all animals weresacrificed by intravenious injection of air. The brainwas removed, fixed in formalin, cut at 50 A and stained

1()64

.!PiI. I'w

-!. -..A.

HYPOTHALAMICSTIMULATION IN EXPERIMENTALATHEROSCLEROSIS

04o -04sb uxe r u0 -

C40'0- % 01) %011 0

0 00- - C4- 00

- -1i C0 0

I n) 0-4\0 e n

1)000 N0o 000 %mo'0 ' \0 m - \O0 -u

Ino to o-\0\ r- to

0 0 000 o- in0

11u)040- 0-0 .o004o

C 0No0 m01 0 '0) -

01 na,O01,0 a0 4 0 0 00~~~~~-~ ~ ~ -

0001) '01010 0 00 0- 0% 11

0 0No 0 4-r 4(DI, _00t00Q(0400- 00 -W

\C 00000-u) (DI, co in 0X

ell 10 4 0I0t 0 -0 0-0 o1)

-. 00 0. . .0 . . . in t- .

1)to-U0r C? 0o - - _ o -o) to t o C Wo Mr C

o1 04' 11 040o 0 04 0O

o0-0 oo -1O oooUo _

U X t-U)0 0 0 S 100 . \01 00o " w 0 0 0\%1 )0 %00

CZ

0

m en \0 \0\) \0 en C4O 0

0 00 01)'0'0It 00 0U)

.0 U \0% 00'_ O1)1-O 40 0o

00))044 0404 0C4 en 040C4

1- C4 - 0- 0%04 r0) O0- O 11rO0 ) o

\0 061 '00010 ) 010 a 1)m

~ 0\ 0 00C om0\t

I'd, 00)0 \0) 10) t'01

C4cK] Xo u 4 C4Os + t

L* _-0S __r _-M0 N_U_

Ut en " _n '4_ U" oO m LO "0 LO

00

0%0)

001)o

n -H_0 8

'0H

0

00'O

-H

00 --

\0

0-H00 0

00 00 1

c

00

_ Cd

-3

O4H00 0o

'0 .0n

'n °

0 00

00 C0

-H

-H00

-H

0

g,

0 - 00

-H

0

0 5

0o5

'o0t0.- '0

'0011)11)0 0%00i+ 01T0)) 00)

1) el; 00

e01)0-0 -'0

00-00

0 004

010. 00

-0N

: "tC- -

c -

Ln M

0 00m ~o

C1 r0 _- N

0- .1 '0 .- 00.

0% 40i 0% )0 11)0r0-0-

04 0 %0%o-I 1 No

(Ng CqebX+u

O OeN r*

t t e0-u) 0 1i

010001)0-o40 vb_N4 0% 00O -'0 0% 0% 11)04_

00 0- 0- 11 00 . 0-

oo0qoo

0.-~0) '1)00004'000%ooe0) - -

0 1o 0411Ne NV) Ch _)0 -i 0 0% 00

0% o0 o t-01% 11)..00-

- o11 t- Le)) 0n'0 1 ) 04 Lo 0%

(1 0 -' 00 0-110 0-'4 -

)en (-Ie ooN:o -t o oa, ocro tn r "I 1.1O uxio Q 0_ ion

- - 0-4 0-e 14 )e o0-e) 001e 11

0. - 4

0.0 ^ * uo-t01 Q 0 -0o_ 4Nu 4O

._ e_oo 04.410'001)'00i ) 00in oo 4Le 0

% ).%.0 .0 00.0

0040to

In 0 -40%0 -0-0 " 0- - 0 % 0

05 % 11 .U.- U 01 m0 00 m0%011)'0 0 .'0 - - 0- 1)

in110 .-1)-'in 0% 04 N04 01

r-C7,C~, 01) 00) 01 ~040

0- 0% % 04 o In0-0 00- 0- '0

o0 Xe- e- c4 e6oo ieDO

o '00001)-00 11) '011)000-0% 0- 0%'0

00)0.0 * '0- 4 O N0% -d 0 0

in tnlr- to nC "o

1965

0

-)-

bo2

(12o~

..0

4

0

U

31'U

4S

0

-o

0.0

p0

.0

3

(1'U

d

U)

0Q

0-).It

0-)

0.)

00.4

°t

04Q1

00;

E8-1be

0

E

oo

0

(1

E801

0

S

03

n)

E0

(2

bO

3

3

.co

E

be

0)32

3d

ut

C

r.0

0

E

c

.000

0

._00

0

0

0:U)0lC0

-H

-0%ci-H' 0s

_) 01

-H

0--H

11) 00

'0 0010

-H0 04

00-H

'0

00~

X00

*110

-H

_%-

0

-H

001)

00 -

-H

0

o

0 O4

0._1)

01) O-

0s

u)

08

CZ

*0

IC

't

00rE0

L.

0

o

U)

0

.0L.

0

r.

0

S

10

(L)

.0

va

U)

.0

H_

Go

vCS0t0

.:3Ew

3.oo

._

.0Cd

di&Oqj

C. G. GUNN, MEYERFRIEDMANAND SANFORD0. BYERS

witlh thionin to determine the loci of the implanted elec-trodes.

The heart, total aorta and a(drenials of each animalwere obtained. Thie aorta was first inspected grossly andgraded 0 to + 5 (16) for its degree of atherosclerosis.In addition, an aortic segmeint of 10 cm beginninig at thesemilunar valves was routinely obtained and analyzed forits cholesterol content.

Three cross sections of the heart (base, middle andapex) were obtained aind stained with Sudan IV. Theneach cross section was studied as follows: the total num-ber of large arteries (i.e., vessels exhibiting well definedadventitia, media an(d iintima) were counted; the totalnumber exhibiting sudaniophilic iintimal hyperplasia oc-cupying 20 per ceint or more of the lumen were alsocounited. The percentage of the total number of largearteries in each cross sectioni showing sudanophilia ofthe required degree was thein calculated. A final per-centage of sudainophilia for each heart was obtained byaveraging the three iindividual perceintages calculatedfrom each of the three cross sectioins. Similar observa-tions and calculations were ma(le of the medium-sizedand small coroinary arteries enicounltered in the same threecross sections.

The adrenals were weighed. Sectioins also were ob-tainedl from the adrenal glands of four stimulated and

tlhree nonstimulated rabbits ingesting the lipid-enriclheddiet. These were stained with both Sudan IV and he-matoxylin and eosin.

All animals were housed in similar cages in the sameroom with the temperature controlled between 610 to 710F. Electroenceplhalogramiis were taken mointhly of eachstimiiulate(d animal. Blood pressure determinations (bydirect arterial puncture) were obtaiined initially and thenevery mointlh of the experimental period.

Thirty-four of the 35 rabbits survived the experimeintalperiod of 3 months.

RESULTS

A. Loci of hypothalam1lic stimul lation. Histo-logical examination of the brains of the 14 stimu-lated rabbits fed the lipid-enriched diet revealed(see Table I and Figure 2) that the locus of thestimulating electrodes was in or near the ventralmedial nuicleus hypothalamicus (VMIH) in sixrabbits, the anterior hypothalamic area (AHA)in three, the lateral hypothalamic area (LHA) intwo, the stupra-optictus difftisus (SOD) in two, an(dthe nucleus arcuatus periventrictularis (ARC) inthe remaining rabbit.

SEIectrode Loci0

FIG. 2. DIAGRAM ILLUSTRATING THIE SITE OF ELECTRODEEMPLACEMENTIN TIIE14 EXPERIMENTAL RABBITS. Solid rectangles show electrode positions in thecoronal-section diagrams corresponding to the planes on the midsagittal sche-matic (upper left-han(d diagramii with dashed lines showing coronial pllales)through hypothalamic area (10). Coronal planes are 1 mim apart. OCH=

optic chiasmii; MM= mammillary bodies; III V =third ventricle; DMH=

dorsal medial niucleus hypothalamus; VMH= ventral medlial nucleus hypo-thalamicus; LHA= lateral hypothalamic area; AHA= aniterior hypothalamicarea; SOD= supra-opticus diffusus nucleus; ARC= nuc.leus arcuatuis peri-venitricularis.

1966

HYPOTHALAMICSTIMULATION IN EXPERIMENTALATHEROSCLEROSIS

Histological examiniation of the brains of thefive stilmiulated rabbits fed the stock diet revealedthat the locus of the stimiiulating electrodes was inthe VMHin three of the rabbits; the locus in theremaining two rabbits was in the mesencephalicreticular formation (MesRF).

B. Behavioral and physiological observationsduring stimjtulation and thereafter. When not ac-tually receiving stimulation, all rabbits in any ofthe chronically stimulated groups exhibited be-havior that was identical with that of the non-operated controls. The EEGrecords were in con-formity with this judgment of normality of be-havior between periods of stimulation, since nopermanent EEGabnormality was observed in thechronically stimulated animals. The behavioralthreshold to stimulation remained fairly constantfor all rabbits during the total experimental period.

Nevertheless, during, and sometimes immedi-ately after an "epoch" of stimulation markedchanges in behavior could be observed in the stim-ulated rabbits. During stimulation almost all ani-mals exhibited pupillary dilatation. Those rabbitswhose electrodes were found embedded in theVMH, LHA or AHA exhibited rather charac-teristic differences in behavior from the three rab-bits whose electrodes were found implanted in theSODor ARC. The former group showed increasedactivity with random searching and circling in thecage; they also would display licking and chewingmovements and some would ingest food avidlyduring and only during a phase of stimulationwithin an "epoch." These behavioral and gusta-tory changes were most marked in those animalswhose electrodes were in or near the VMH. Thelatter group, three rabbits whose electrodes wereplaced in the SODor ARC, usually displayed adecrease both in the rate and in the amplitude ofrespiration; moreover, they usually (but not in-variably) remained totally immobile for the dura-tion of the applied current. Rarely they also ex-hibited eating, licking or chewing movementseither during or immediately after stimulation.

No significant chronic change in blood pressurewas observed in either the experimental or thecontrol groups given the lipid-enriched food. Theaverage blood pressure of the control group ofrabbits was 116 mmHg (mean pressure) at thebeginning of the experiment and 114 mmHg atthe end. The average blood pressure of the stim-

ulated rabbits for the saimie two periods was 113anid 117 mmni Hg, respectively. However, stimiiu-lation did evoke a transient rise of 5 to 20 mmHgin five of seven rabbits tested shortly followingan "epoch." It is of interest that the two rabbitsthat failed to show this pressor response werefound to have their electrodes in either the an-terior or posterior area of the medial eminence ofthe hypothalamus (SOD and ARC).

C. Changes in blood lipids and corticosteroidsafter chronic and acuite hypothalainic stimulation.Despite the equivalence of age, amount of food in-gested, and weight, the series of rabbits bothchronically stimulated and also fed the lipid-enriched diet displayed serum cholesterol, phos-pholipid and total lipid values that were consid-erably greater throughout the 3-month periodthan corresponding values in the nonstimulatedrabbits fed the same lipid-enriched diet (see TableI). All increases were significant, with the pvalue at the 0.01 level. On the other hand, rabbitsthat were chronically stimulated but given onlythe (relatively) lipid-poor stock diet (see TableI) failed to exhibit any significant change in theirserum cholesterol levels.

It was of interest to us that apparently the lipidincreases observed in the chronically stimulatedrabbits ingesting the lipid-enriched diet appearedto be about equal in all of the rabbits, regardlessof the exact site of electrode insertion. Thus, theaverage lipid values of the six rabbits having elec-trodes in their VMHwere about the same duringthe 3-month period as those of rabbits having theirelectrodes in the AHA, LHA or SOD. The onlyexception was one animal (no. 55) whose stimula-tion was received in the ARCarea and whose lip-ids after 3 months were simiilar to those of thecontrols.

A single acute stimulation, however, whethercoming after either 1 or 3 months of chronic dailystimulation, failed to provoke any acute rise in theserum lipid values, irrespective of the nature ofthe diet (see Table II). Actually a slight fallusually was observed in the blood lipid valueseither at the 1 or 5 hour period after any particularstimulation. Since a similar slight fall also wasobserved in the nonstimulated controls, this mayreflect a slight dilution of the plasma followingthe initial bleeding prior to stimulation.

The serum content of 17-hydroxycorticosteroids

1967

C. G. GUNN, MEYERFRIEDMANAND SANFORD0. BYERS

0-- C1400 \000 1-

-H

I -H

O O000 ) no 00 4- \Je

00

00 4. _-_

.2

00 0- 0-i a-. U0

oo UO)

_ -<]r11 0 -

00 U-. ot - U00 QV -H 2

C)-4

en 4 14 -.:c) -H a:-. -H

-I .11

rq -I Uf) -40- en - C)eC' -H t14-H

U1) 0m. .2

U) -U) 0

0o 0*-_

= *n,m

dc

U)0.E

0 0

0 0

n- E - E0 0

Cl Cl

a:-c-i

U) 00. .V

C _*n

O = I 4-n0 0 En

Wf)

1968

-tf4

0o

-i

C\

00%

rtf) 0-~

t to

n ooIt)+ 11

00U)

v

ECd

U)

0

Io

U1)

.0

0

0*

U)

0

U)

(.)v2zr

0

0

0

C)0

0

CQ

a1)

0C)

0u

.6

.0P4

0)

.0.C

S

0

(1)

Q

.t-

4

4

CU)

a)

0-

a) U

0

Uq)C/)-

- 2

-2

U)-

2D

.) L.

._

7z UU 0

rn 2; U)

¢>

)-a

2

0

U)_ U

U U)

_C._

_) 2

U U

cU o

U)4._Z

2Ut

-e.)

r)0

1-

0

-0C)

..p:o )0 .1

UC 0 r0%-H = i

00 C-a

-H

o c-CN -

-H

00

-)

*n

1

0

U1)

0DU)

.0Ui)2

0

L._

Q

0

-

*U1)

I..

0Cj)-

U)1-0.0.

00r0 0

0. .°U -

0 0t En.*n

6

r..0.0

C)

d

mU)

I...0

C/)

HYPOTHALAMICSTIMULATION IN EXPERIMENTALATHEROSCLEROSIS

failed to be influenced by either chronic (seeTable I) or acute (see Table II) stimulation ineither of the two stimulated series.

D. Pathological changes after chronic hypothala-mic stimulation. No difference in heart size wasobserved in any of the three groups. However,striking differences in the degree of aortic athero-sclerosis were exhibited by the chronically stimu-

lated rabbits fed the lipid-enriched diet as com-pared with the atherosclerosis shown by controlnonstimulated rabbits fed the same quantities ofthe same diet (see Table III and Figure 3).Thus eight of the 14 stimulated rabbits showedmoderate to marked aortic atherosclerosis (grade2 or above), whereas only two of the 15 controlanimals showed a similar degree of involvement

TABLE III

Vascular and adrenal changes following chronic hypothalamic stimulation

Per cent atheroscleroticAorta involvement

coronary vasculatureElectrode Gross Adrenal

Rabbit no. site grading Cholesterol Large Med. and small wt

g/loo gI. Chronically stimulated rabbits fed lipid-enriched diet

VMHVMHVMHVMHVMHVMHAHAAHAAHALHALHASODSODARC

3.55.03.03.05.01.03.01.01.03.01.51.02.01.0

2.440.37

3.307.374.755.52

11.558.171.882.963.562.693.742.024.492.83

4.63±0.70

0432550127529106614715720

0

344±6.8

mg

38 91014 75045 96837 1,20540 1,14542 2,14575 1,00043 1,45248 80054 97535 1,28533 1,04054 1,00010 1,318

4144.1

1,142484.0

II. Control nonstimulated rabbits fed lipid-enriched diet0.51.04.00.51.01.00.52.50.51.00.50.00.00.50.0

0.9±0.26

0.451.663.281.612.902.852.563.722.054.121.091.000.900.721.03

2.0±0.29

2525223033

0

0

0

3367

0

0

400

0

18±5.1

III. Chronically stimulated rabbits fed regular dietMesRF 0.0 0.35 0MesRF 0.0 0.30 0VMH 0.0 0.42 0VMH 0.0 0.22 0VMH 0.0 0.38 0

0.0 0.33 0

33 1,23517 1,15032 1,57042 1,03535 93024 1,62020 1,01015 1,47315 78539 68515 74517 1,26550 86518 99514 910

26±2.9

0

0

0

0

0

0

1,085±91.0

380600475700600

551

* These rabbits received lipid-enriched diet for only 74 days.---

35*37*4850606534*476339*53436255

AverageSE of mean

334041445152545759616466676869

AverageSE of mean

379

1116

Average

1969

C. G. GUNN, MEYERFRIEDMANAND SANFORD0. BYERS

FIG. 3. AORTAS OF STIMULATED AND CONTROL NON-STIMULATED RABBITS FED CHOLESTEROL. The aorta onI theleft of each photograph is that of a stimulated rabbit, andthe one on the right, of a nonstimulated rabbit of thesame experiment. The greater degree of atheroscleroticinvolvement of the aortas of the stimulated rabbits caneasily be seen.

1970

HYPOTHALAMICSTIMULATION IN EXIPERIMENTAL ATHEROSCLEROSIS

(p value significant at the 0.01 level). This dif-ference observed by gross inspection was corrobo-rated by analysis of the aortic segments for cho-lesterol content. Thus the average cholesterolconcentration (4.63 g per 100 g of dry weight) ofthe 14 aortic segments of the stimulated rabbitswas over twice that (2.0 g per 100 g of dry weight)found in the control nonstimulated rabbits in-gesting the samne diet (p value significant at 0.01level). By the same token (see Table III), agreater percentage of the coronary vasculature ofthe stimulated rabbits was atherosclerotic as com-pared with the vasculature of the control group.

A comparison of the degree of aortic athero-sclerosis with the diencephalic site of electrodeemplacement strongly suggests that the animalsreceiving stimulation in or in the vicinity of theVMHhad excess aortic atherosclerosis and cho-lesterol infiltration. Thus the average grade ofaortic atherosclerosis found in these six animalswas 3.3 as compared to the grade of 2.4 for thetotal group of experimental animals, and the aver-age cholesterol content of their aortic segmentswas 6.76 g per 100 g dry weight as compared to4.63 g for all experimental animals and 2.0 g forthe controls. The relatively small number of ani-mals studied, however, precludes the drawing ofan absolute conclusion concerning the significanceof this difference.

On the other hand, as Table III indicates, stim-ulation of the group of five rabbits that were notfed the lipid-enriched food failed to induce eithergrossly observed or chemically detected athero-sclerosis.

Wewere unable to detect any histological dif-ference in the adrenals of either the stimulatedor the unstimulated rabbits fed the lipid-enricheddiet. In both groups, the adrenals (see Table III)were greatly and, on the average, equally enlarged.This increase in size and weight appeared to bedue to lipid and cholesterol deposition.

DISCUSSION

The preceding data appear to indicate thatrabbits fed a diet excessive in cholesterol and cot-tonseed oil and, in addition, receiving chronicstimulation of their diencephalon in the vicinity ofthe ventral medial nucleus, the lateral and the an-terior hypothalamic areas, will exhibit a greater

degree of chronic lipemiia and a greater degree ofaortic and coronaiy atherosclerosis than their non-stimulated controls. Although we were impressedby the fact that apparently the highest degree ofatherosclerosis was exhibited by the six animalsthat were found to have their stimulating elec-trodes fixed in the ventral medial nucleus, thesmall number of rabbits studied makes the con-clusion of an association between atherosclerosisand stimiiulation of the ventral medial nucleus atentative conclusion at best.

The mechanism(s) whereby this chronic stimu-lation may have brought about the observedchanges in the arterial vessel walls remains to beelucidated. However, it seems clear from theabove study that they were not mediated bychanges in food intake, by chronic changes inblood pressure or in physical activity. Our fail-ure to observe any change in the corticosteroidlevel of the serum either during the period ofchronic stimulation or soon (1 to 5 hours) afteracute stimulation suggests but does not prove thatthe adrenal glands of these rabbits did not play avery important role in the pathogenesis of theatherosclerosis found.

Perhaps the chronically sustained greater de-gree of lipemia observed in the stimulated animalswas responsible for the more extensive aortic andcoronary atherosclerosis found. Certainly thislipemia must have a significant role if only be-cause identical hypothalamic stimulation of rab-bits given ordinary rabbit laboratory chow failedto elicit any detectable change either in the bloodlipids or in the arterial walls. The means wherebychronic diencephalic stimulation (under the con-ditions of the present study) might intensify thehyperlipemic response of rabbits fed controlledrations of lipid-enriched food also remains to beunderstood.

SUMMARY

Evidence has been presented consistent withthe hypothesis that chronic electrical stimulationdelivered to certain hypothalamic areas signifi-cantly intensifies both the rate of increase atfd theamount of hyperlipemia in rabbits fed a lipid-en-riched diet. Such animals also showed a muchgreater atherosclerotic involvement of their aorticand coronary vasculature than did control animalsgiven a similar diet. Those rabbits receiving

197 1

C. G. GUNN, MEYERFRIEDMANAND SANFORD0. BYERS

stimuulation in or near the ventral medial nucleusappeared to exhibit no greater hyperlipemia butconsiderably more atherosclerosis than the ani-mals stimulated elsewhere.

It is concluded that central nervous systemmechanisms exist which, under certain conditions,are capable of significantly influencing arterialatherogenesis.

ACKNOWLEDGMENT

The authors wish to express their deep thanks toHorace W. Magoun, who in 1955 arranged for the ini-tial collaboration of the two participating groups whichmade the present study. They also wish to thank Dr.Shirley St. George for the 17-hydroxycorticosteroidanalyses.

REFERENCES

1. Myasnikov, A. L. Experimenital data oni prophylaxisof arteriosclerosis. Klin. Med. (Mosk.) 1954, 32,no. 6, 9.

2. Myasnikov, A. L. Influence of some factors on devel-opment of experimental cholesterol atherosclero-sis. Circulation 1958, 17, 99.

3. Tsibekmakher, T. D. Effect of phenamine and lu-minal on blood cholesterol anid cholesterol ethers.Ter. Arh. 1955, 27, no. 1, 48.

4. Uhley, H. N., and Friedman, M. Blood lipids, clottingand coronary atherosclerosis in rats exposed to aparticular form of stress. Amer. J. Physiol. 1959,197, 396.

5. Friedman, M., Rosenmiian, R. H., and Carroll, V.Changes in the serum cholesterol and blood clot-ting time in men subj ected to cyclic variation ofoccupational stress. Circulation 1958, 17, 852.

6. Wertlake, P. T., Wilcox, A. A., Haley, M. I., andPeterson, J. E. Ralationship of mental and emo-

tional stress to serum cholesterol levels. Proc.Soc. exp. Biol. (N. Y.) 1958, 97, 163.

7. Grundy, S. M., and Griffin, A. C. Effects of periodicmenital stress oni serum cholesterol levels. Circu-lation 1959, 19, 496.

S. Hammarsten, J. F., Cathey, C. WV., Redmond, R. F.,and Wolf, S. Serum cholesterol, diet and stress inpatients with coronary artery disease (abstract).J. clin. Invest. 1957, 36, 897.

9. Friedman, M., and Rosenman, R. H. Association ofspecific overt behavior pattern with blood andcardiovascular findinigs. Blood cholesterol level,blood clotting time, incidence of arcussenilis, andcliniical coronary artery disease. J. Amer. med.Ass. 1959, 169, 1286.

10. Sawyer, C. H., Everett, J. WV., and Green, J. D.The rabbit diencephaloni in stereotaxic coordinates.J. comp. Neurol. 1954, 101, 801.

11. Anand, B. K., and Dua, S. Feeding responses in-duced by electrical stimulation of the hypothalamusin cat. Indian J. med. Res. 1955, 43, 113.

12. Saifer, A., and Kammerer, 0. F. Photometric de-termination of total cholesterol in plasma or se-rum by modified Liebermann-Burchard reaction.J. biol. Chem. 1946, 164, 657.

13. Zilversmit, D. B., and Davis, A. K. Microdetermina-tioni of plasma phospholipids by trichloroacetic acidprecipitation. J. Lab. clin. Med. 1950, 35, 155.

14. Bragdon, J. H. Colorimetric determination of bloodlipides. J. biol. Chem. 1951, 190, 513.

15. Silber, R. H., anid Porter, C. C. The determination of17,21-dihydroxy-20-ketosteroids in urine and plasma.J. biol. Chem. 1954, 210, 923.

16. Friedman, M., Byers, S. O., and Rosenman, R. H.Resolution of aortic atherosclerotic infiltration inthe rabbit by phosphatide infusion. Proc. Soc.exp. Biol. (N. Y.) 1957, 95, 586.

1972