American Journal of Pathology, Vol. 129, No. 2, November 1987Copyright X American Association of Pathologists

Spasm of Small Coronary Arteries and IschemicMyocardial Injury Induced by HypothalamicStimulation in the Rat

WILLIAM H. GUTSTEIN, MD,PIERO ANVERSA, MD, andGIANCARLO GUIDERI, PhD

Electrical stimulation of the lateral hypothalamus re-sulted in electrocardiographic evidence of acute myo-cardial ischemia in 35% ofnormal adult rats under an-esthesia. Mean arterial blood pressure was alsoelevated. Study of vascular corrosion casts disclosedthat spasm ofsmaller branches ofthe coronary circula-tion, rather than the major epicardial arteries, was themain cause of the ischemic response. The histologicchanges ofthe same experimental treatment in a sepa-

AN UNEXPLAINED and important problem in car-diology is the presence of various manifestations ofischemic heart disease in the absence of angiographi-cally abnormal coronary arteries. Isolated angina,1myocardial infarction,2 dilated cardiomyopathy,3and sudden cardiac death4 have all been reportedwithout obvious causes, ie, without significant diseaseof the major coronary vessels. Even spasm of theselarge conductance arteries has not always been un-equivocally proven.2 This has led to the suggestionthat in some cases constriction ofthe smaller arterialbranches may be responsible.5-7 With certain cardio-myopathies, both human and experimental, hyperre-activity of the microcirculation has been demon-strated.8 To date, however, there has been no directevidence, either in animals or in humans, that smallintramyocardial arteries, intermediate in size be-tween the major epicardial vessels and those of themicrocirculation, can undergo spasm in a mannercomparable to that of the large conductance arteriesof patients diagnosed as having "coronary arteryspasm."Reduced coronary blood flow, ischemic-like elec-

trocardiographic (ECG) changes, and myocardial tis-sue damage have all been shown to be associated withhypothalamic stimulation (HS) in experimental ani-

From the Departments ofPathology, Pharmacology, andPsychiatry, New York Medical College, Valhalla, New York

rate group of animals revealed multiple focal areas oftissue damage throughout the myocardium, whichwere quantitatively assessed. The results may be rele-vant for the clinical problem ofvarious forms ofische-mic heart disease in which little evidence is found fororganic (atherosclerosis) or dynamic (spasm) stenosisinvolving the major coronary arteries. (Am J Pathol1987, 129:287-294)

mals.9-'5 These alterations have been interpreted to bemediated by coronary vasoconstriction, although thelevel of the coronary circulation involved has notbeen defined. In a recent histologic study reduction ofmean cross-sectional area in the major coronary ar-teries, consistent with spasm, was demonstrated fol-lowing electrical stimulation ofthe lateral hypothala-mus in the rat.16 In the current work, evidence ispresented that intramural branches of the coronaryvasculature may participate in the vasoconstrictor re-sponse to HS and may be associated with ischemicchanges in the ECG and multiple focal areas of myo-cardial necrosis.

Materials and MethodsIn this study the occurrence of acute myocardial

ischemia in the rat was evaluated by the electrocar-

Supported by grants from the Council for Tobacco Re-search-U.S.A., Inc. (Special Project #122R), the NationalInstitutes of Health (Grant Number HL24479), and theWestchester Heart Association.Accepted for publication June 17, 1987.Address reprint requests to William H. Gutstein, MD,

New York Medical College, Department of Pathology,Basic Science Building, Valhalla, NY 10595.

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diographic method developed by Sakai et al.17 Cri-teria for elevation of the S-T segment consisted ofupward displacement ofthe S wave of more than 0.1mV from the isoelectric line in at least one lead. Elec-trocardiograms from limb leads (I and III) and a singleprecordial lead (V) were continuously displayed onthe oscilloscope screen ofan Electronics for MedicineDR- 12 recorder and simultaneously stored on a Hon-eywell Model 5600 tape recording system.

Sixty-eight male Sprague-Dawley rats, weighing285-310 g, were anesthetized (sodium pentobarbital,4 mg/ 100 g body weight, intraperitoneally) and ster-eotaxically implanted bilaterally with fine, stainlesssteel, bipolar electrodes in the hypothalamus. Elec-trode tips were placed within an area bounded bycoordinates: AP=5.0; L=±0.2-±2.1; V=8.2 - 10.3.18 Mean arterial blood pressure was con-tinuously measured with a cannula inserted into theleft femoral artery and connected to a Gould P-23 IDtransducer.The area defined by the coordinates employed was

explored for deviations ofthe S-T segment above theisoelectric line. When S-T segment elevation oc-curred, it was always repeatable at the same locationafter return ofthe segment to baseline. A total of 144locations were stimulated in 68 animals. Final posi-tions of electrode tips were confirmed by histologicexamination'9 and are shown schematically in Fig-ure 1.

Stimulation was delivered from a Grass Model S-88stimulator with a stimulus isolation unit for a periodof 60 seconds at a constant current intensity of 200,A, a frequency of 60 Hz, a pulse width of 0.5 msec,and a monophasic square waveform. Casting was per-

Figure 1-Schematic drawing of histologic section of rat brain."8 Anteropos-terior coordinate = 5.0 mm. Solid triangles (A) illustrate some typical elec-trode tip positions from which ECG responses in the form of S-T segmentelevations were elicited. CC, corpus callosum; V, ventricle; IC, intemal cap-sule; MT, mamillothalamic tract; F, fomix; Lat Hyp, region of the lateral hypo-thalamus.

formed with Batson's resin with modification of thetechniques ofCasellas et al20 and Kardon et a12' forthestudy ofthe morphology ofsmall arterial vessels. Oneout ofthree responding animals was randomly chosenfor casting. Under mechanical ventilation, during theperiod ofS-T segment elevation, the chest was openedrapidly and a 21-gauge needle inserted into the leftventricle at the apex. The right atrium was incised fordrainage and the coronary circulation flushed at a rateof4 ml/min with heparinized saline at 37 C by meansof a Harvard infusion pump. Pressure was main-tained at the same level as measured in vivo. Freshlyprepared Batson's resin (Polysciences, Inc., Warring-ton, Pa) was then delivered through the in situ needleat the same pressure until the left and right coronaryarteries on the epicardial surface filled by direct visu-alization. The animal was left undisturbed for 30 min-utes for initial polymer setting, after which the brainwas removed and fixed for histologic examination.The carcass was placed in the cold room (4 C) for anadditional 16 hours and the heart then carefully re-moved and transferred to KOH solution (340 g/liter)for 24-48 hours at room temperature.A series of eight electrode-implanted but non-

stimulated animals subjected to the same experimen-tal conditions were used as controls, and vascularcasting was also performed in these.The digested casts ofboth groups were gently rinsed

with tap water, air-dried, and inspected under a dis-secting microscope by three independent observerswithout knowledge ofthe animal from which the castwas obtained. The presence of sudden changes in lu-minal diameter along the arterial tree was recordedand the caliber ofthe vessel involved measured. Pho-tography of the three-dimensional casts was per-formed with an Olympus Macro System employing80-mm F4 and 20-mm F2 lenses.

In order to evaluate whether the ischemic-likechanges observed in the ECG were accompanied bytissue injury in the myocardium, we subjected an ad-ditional group of 24 animals, identically treated, toHS. In a subgroup of8 ofthese animals (33%) showingelevation of the S-T segment, the ECG abnormalitywas maintained for a period of 45-60 minutes, be-cause it has been shown that this exceeds the mini-mum required time for irreversible myocardial dam-age of ischemic origin.22'23

After stimulation, electrodes were removed, thescalp sutured closed over the burr holes, antibioticsadministered (chloromycetin sodium succinate, 6.6mg/kg intramuscularly), morphine sulfate (5mg/kgbody weight) given subcutaneously to alleviate post-operative pain, and the animals allowed to recover.Three days later the rats were reanesthetized with

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SPASM OF SMALL CORONARY ARTERIES 289

sodium pentobarbital as before, the heart arrested indiastole by intravenous injection of 1 ml KCl (1 mEq/ml), the chest opened, and the heart removed andfixed in 10% phospate-buffered formalin solution.The 3-day period was employed to allow for the evo-lution of possible histologic changes. For controls ofhistologic myocardial damage, two additional groupsof 1) 8 implanted, stimulated nonresponders and 2) 8implanted, nonstimulated animals were identicallytreated and evaluated.The left ventricle was separated from the right, and

four successive segments, each 2 mm apart, weremade transverse to the longitudinal axis of the heart.The segments were processed and embedded in paraf-fin so that the side facing the base of the heart wasuniformly exposed for sectioning. Histologic sectionswere cut at 5,u in thickness and stained with hematox-ylin and eosin (H&E) for light-microscopic morpho-metry.

Quantitative assessment consisted of counting thenumber of necrotic foci per unit area of tissue. Theventricular wall was equally divided into epicardial,midzonal, and endocardial layers; and 10-15 adja-cent fields in each region were sampled in each ofthefour sections in each animal. This analysis was per-formed at a magnification of 120X for an area of8.8 X 105 sq ,u defined by an ocular reticule (Wild#105844) containing 42 morphometric samplingpoints. The counting rules described by Gunderson24were used.Blood pressure measurements, heart rate values,

and the number of lesions per unit area of myocar-dium are presented as means ± standard deviation.

Differences in heart rate and mean arterial pressurebefore and after stimulation in each experimentalgroup were evaluated for statistical significance bymeans of the unpaired two-tailed Student t test.

Statistical significance for differences in distribu-tion of lesions in the three layers of ventricular myo-cardium were evaluated by analysis ofvariance with amultiple comparison test according to the Bonferronimethod.25 Values ofP < 0.05 were considered signifi-cant.

ResultsElectrocardiographic responses could be elicited

from a total of47 points in 24 ofthe 68 animals (35%)stimulated. Although after histologic examination ofbrain sections the reason the response was limited toone-third of the population was not apparent, whenS-T segment elevation occurred (Figure 2), it alwaysexceeded 0.3 mV in magnitude. Each episode per-sisted 15-20 minutes and was immediate in onset.

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Figure 2-Electrocardiographic recordings of Leads 1, 111, and V (precordial)from top to bottom in the anesthetized rat before (A) and after (B) hypothala-mic stimulation in a susceptible animal. Immediately after stimulation, eleva-tion of the ST segment, indicating the onset of myocardial ischemia, is seen inthe precordial lead.

Other electrocardiographic changes, which in somecases accompanied S-T segment elevation, werepeaking with increased amplitude ofT waves and oc-casionally decreased amplitude of the R wave.

Baseline heart rates (before stimulation) were386 ± 37 bpm for responders and 371 ± 36 bpm fornonresponders. After stimulation these were 390 +45 and 371 ± 44 bpm, respectively, showing that nochanges had occurred.

Baseline mean arterial pressure changed from87 ± 15 to 100 ± 17 mm Hg in responders and re-mained essentially unchanged in nonresponders(from 96 ± 16 to 98 ± 15 mm Hg). The 15% increasein mean arterial pressure in animals with S-T segmentelevation was statistically significant, P < 0.01.

Heart rate and mean arterial pressure in implantednonstimulated controls were 378 ± 25 bpm and93 ± 14 mm Hg and remained constant.Examination of vascular corrosion casts revealed

numerous but scattered constrictions present in thesecond and third degree branches (50-150,u in diame-

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ter) of the arterial bed of the heart in all 8 randomlychosen stimulated animals when viewed indepen-dently by the three observers. These constrictionswere in the form ofabrupt changes in luminal diame-ter ofup to 75% or more giving rise to pinched, beadedor thread-like configurations (Figure 3). Total occlu-sions were often seen with no filling of the terminalportions of the vessels. In some cases the involvedvessels disclosed multiple indentations along theiraxial lengths, suggesting that spasm had occurred in asegmental fashion. Inspection of the three-dimen-sional structures under the dissecting microscopemade it possible to exclude incomplete filling and theviewing of branches on end as possible sources oferror. Smaller branches ofthe size ofprecapillary arte-rioles and capillaries could not be examined with thistechnique.

In 2 of the 8 animals, spasm ofthe major coronaryarteries near their origins was also present.

Examination of vascular corrosion casts of the 8implanted but nonstimulated control rats failed toreveal areas of constricted vessels, with complete fill-ing down to third-degree branches.

Beyond this evaluation of cast morphology it wasnot possible to obtain quantitative estimates of thenumber ofbranches per unit volume oftissue (lengthdensity) involved in the spastic state, because themyocardium was digested away and the individualvessels varied considerably in their orientation andshape. For this reason, histologic evidence ofmyocar-dial injury was assessed qualitatively and quantita-tively in separate groups of animals.

Microscopic examination of the left ventricularmyocardium 3 days after HS revealed multiple scat-tered lesions throughout the wall in animals thatshowed an S-T segment elevation during stimulation(Table 1) with a predilection for the subendocardialregion (Table 2).These lesions were irregular in shape and size and

consisted ofremnants ofmyocytes and foci ofinflam-matory cells, chiefly mononuclear, with moderatecapillary and fibroblast proliferation and some colla-gen deposition (Figures 4-7). An average of nearlyone lesion per square millimeter ofmyocardium wasmeasured (Table 1).As indicated in Table 1, ECG changes and histo-

Figure 3-Vascular corrosion cast of the coronary circulation 15-20 minutes after hypothalamic stimulation in a rat with the electrocardiographic pattem ofFigure 2. Constriction of the left coronary artery near its origin is seen atA with approximately a 50% reduction in luminal diameter. Spasm of smaller branchesgiving beaded or filamentous appearances is seen throughout the cast and is indicated at B, C, and D (arrows). Insets are enlarged views of these smallerbranches. Bar represents 600 p in the whole cast and 480 p in insets.

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SPASM OF SMALL CORONARY ARTERIES 291

Table 1 -Correlation of Histologic Damage and ECG Changes Consistent With Myocardial Ischemiain Hypothalamically Stimulated Animals

Number ECG MAP HR Number oflesions per 10

Group animals changes Before After Before After sq mm of myocardium

HS-R 13 S-T 89± 16 101 ± 23 390± 32 393 ± 35 8.77± 2.63HS-NR 8 None 94 ± 17 96 ± 15 382 ± 40 387 ± 38 None

8 None 94± 18 383±34 None

HS-R, animals subjected to hypothalamic stimulation with ECG responses; HS-NR, animals subject to hypothalamic stimulation without ECG responses;1, animals implanted with electrodes but not receiving hypothalamic stimulation; S-T, elevation of S-T segment; MAP, mean arterial pressure(mm Hg); HR, heartrate (beats per minute).

Table 2-Distribution and Frequency of Myocardial Lesions in theLeft Ventricle of Hypothalamically Stimulated Animals

Epicardium Midmyocardium Endocardium

Area sampled (sqmm) 686 595 457

Number of lesionscounted 191 269 857

Number oflesions/1 0 sqmm 2.79 ±1.46 4.53 ± 2.51 18.75 ± 6.15*

*Indicates a value which is statistically significant atP < 0.0001 from thosein epicardium and midmyocardium.

logic lesions were not observed in the two controlgroups.

Table 2 shows the distribution and frequency ofmyocardial lesions in the inner, middle, and outerlayers ofthe left ventricular wall in HS responder rats3 days after stimulation. The subendocardium c'on-tained almost seven and four times more lesions thanthe epi- and midmyocardial regions, respectively.These differences were found to be statistically signifi-cant (P < 0.0001). The 62% greater concentration oflesions in the midmyocardium with respect to theepicardium was not found to be statistically signifi-cant.

DiscussionThe data indicate that ischemic changes of the

myocardium as judged by electrocardiographic cri-teria were present in 35% of stimulated animals (re-sponders), whereas electrode-implanted but non-stimulated controls did not show such changes. Be-cause responder animals also experienced a moderaterise in blood pressure, which often accompanies hy-pothalamic stimulation as an overall response to con-striction of systemic resistance vessels,26 the possibil-ity arises that the ischemia was due to an increasedoxygen demand secondary to a positive inotropic ef-fect ofthe myocardium, rather than to a primary per-

fusion deficit brought about by a decrease in coronaryblood flow. In this case, S-T segment depression,rather than elevation, should have been observed.27However, because this conclusion is based on findingsin humans only,27 a definitive statement cannot bemade for this animal model.Of the eight randomly chosen responders which

were cast, all had evidence of spasm involvingbranches ofthe second and third degree. These vesselsare in the range of50-150,p in diameter in the rat andare intermediate in size between those traversing theepicardium (250-300 p)16 and those of the microcir-culation (7-25 p), ie, capillaries and precapillary arte-rioles.28 Two ofthese animals also showed narrowingof the major corpnary arteries.- The possibility: thatsimilar large vessel spasm was transiently present inthe other 6 subjects and subsequently disappeared cannot be excluded. However, on the basis of the histo-logic evidence,, the nature, frequency, and distribu-tion of the myocardial "lesions elicited by prolongedHS are consistent with spastic involvementand oc-clusion of the intermediate-sized vessels, as shown inthe vascular corrosion casts. In addition,-the greaterconcentration of myocardial lesions in the subendo-cardial region correlates with the greater number andlength densities of these vessels found in this layer.29Although focal myocardial injury was associated

with ischemic changes in the ECG and segmental nar-rowing in the vascular corrosion casts, a causal rela-tionship with HS cannot be proved at the presenttime. Furthermore, catecholamines have often beenconsidered to be responsible for discrete areas ofmyocardial necrosis independently of their effect onthe coronary vasculature following HS.'2 The lattercannot be excluded currently as a possibility, and thisrelationship still remains a difficult problem.

In previous work, as a result of microvascularspasm, multiple focal areas of myocardial necrosisand replacement scarring have been described in theventricular myocardium ofSyrian hamsters as well asin hypertensive diabetic rats. Moreover, myocardialinjury has been shown to be responsible for impair-

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ment of ventricular function (for review, see Manciniet a130).

Focal ischemic lesions of the type found here, ie,intermediate-sized vessel-induced injury, can bedamaging in a number of ways, because a significantamount of functioning myocardial tissue may be lost.Should similar events occur in humans, a possibleexplanation could be offered for ischemic myocardialdisease of various forms in the absence of significantchanges in the large vessels. Moreover, it may be nec-essary to speak of "coronary artery spasm" as an in-crease of vasomotor tone involving all levels of thecoronary circulation, rather than those of the majorepicardial arteries alone.Although the causes of coronary spasm are un-

known, emotional factors have been implicated insome cases.31 Strong emotional responses have beenelicited in humans by electrical stimulation of thehypothalamus,32'33 which has also often been used tomodel emotional stress in animals, because it pro-duces changes throughout the organism, includingthe brain, which are hardly distinguishable from thoseof "natural stimulation."34 Because hypothalamicstimulation satisfies the criteria for "natural stimula-tion" of neural tissue,34 the present results suggest alink between myocardial ischemia and enhanced ac-tivity ofthe central nervous system, with spasm as themediating mechanism. Whether such a relationshiphas any bearing on the role ofneuropsychologic influ-ences as important risk factors in human ischemicheart disease35 40 remains to be explored.

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Figure 4-Low-power micrograph of the subendocardial region of the left ventricular free wall from a rat 3 days after hypothalamic stimulation. Numerous focalareas of necrosis with cellular infiltrates are present. (H&E, X35) Figure 5-Higher magnification of Figure 4, which shows loss of myocytes withmononuclear cell infiltrates, capillary profiles, and early collagen deposition. (H&E, X90) Figure 6-Necrotic lesions in the subepicardial region of themyocardium. (H&E, X90) Figure 7-Midzone of myocardium showing foci of necrosis and cellular infiltration. (H&E, Xl 80)

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