Myocardial necrosis and electrocardiographic changes related to microcirculatory abnormalities

Myocardial

Necrosis and Electrocardiographic

Changes Related to Microcirculatory Abnormalities* DAVID M.

LONG, JR., KD.,~

MAURICE W. MYER, D.D.s., PH.D., E. B. BROWN, JR.,

PH.D. and

C. WALTON LILLEHEI, M.D., PH.D., F.A.C.C. Minneapolis,

Minnesota

duced by the injection of high molecular weight dextran.4 When myocardial infarctions were produced experimentally by ligation of a coronary artery, prevention of intravascular thrombosis by heparin therapy resulted in a decrease in the size of the infarcted areas5 To evaluate the role of microcirculatory abnormalities in the production of focal myocardial necrosis and electrocardiographic changes, two experimental systems were used. First, the microcirculation was observed directly during prolonged periods of total cardiopulmonary bypass. Under these circumstances, total body perfusion rates and blood pressures were controlled accurately. Total coronary blood flow has been studied previously during extracorporeal circulation and found to be adequate.6 During total cardiopulmonary bypass, cardiac work was practically eliminated and consisted only of the work required to recirculate the bronchial blood flow and the small quantity of coronary venous blood flow returning to the left side of the heart. Thus, the possibility of myocardial hypoxia, due to inadequate total coronary blood flow and increased myocardial work load during stress, was eliminated. The second technic for studying the effects of microcirculatory abnormalities was induction of severe hypercapneic acidosis in the dog. Hypercapnea was a convenient form of experimental stress induced simply by having animals breathe gas mixtures of 30 per cent

N CONSIDERING the etiology of myocardial infarction, primary alterations of blood flow in the arterioles and venules of the myocardium have been neglected. Emphasis has been placed on the mechanical occlusion of the major branches of the coronary arteries. Data from postmortem examinations do not support this approach in all instances. Occlusion and thrombosis of arterioles and venules may, however, play an important role in the initiation and extension of myocardial infarction under certain conditions. Selye’ and Raab2 produced myocardial necrosis experimentally by the administration of steroids and stress. The mechanism by which stress results in myocardial necrosis is not clear. Raab’ has attributed the myocardial necrosis of stress to the necrotizing effects of catecholamines. Changes in the microcirculation, includinq thrombosis of arterioles and venules, have been reported in experimental animals and in human patients in a wide variety of stressful including coronary insufficiency. conditions Petechial myocardial necrosis has been prowho induced intravascular duced by Gelin, aggregation and microthrombosis by the administration of high molecular weight dextran and by experimental trauma. He also demonstrated that the intravascular aggregation was reversible or preventable by the administration of low molecular weight dextran. Electrocardiographic abnormalities have also been associated with intravascular aggregation in-

I

* From the Departments of Surgery and Physiology, University of Minnesota Medical School, Minneapolis. SupMinnesota Heart Association ; Life ported by research grants from the Graduate School, University of Minnesota; Insurance Medical Research Fund; U. S. Public Health Service (Grant No. 830); and Benefactors of Cardiac Surgical Research Fund. This paper was awarded second prize in the Young Investigators’ Award Contest of the American College of Cardiology, May 1961. t Special Research Fellow, National Heart Institute, National Institutes of Health, U. S. Public Health Service. NOVEMBER

1962

695

696 TARIF

Effect of Extracorporeal

Dogs

Group

1.

(no.

High HOWcontrol

5

1

1 on Histology of Myocardium

AveraRe Flow Rat<.

Results of Histologic Examination for Myocardial Necrosis

(cc./k,q. /min.)

112

2.

Low flow control

8

6:

3.

Low How plus dextran

9

66

4.

Low flow plus dextran

3

67

and norepinephrine

Circulation

2 dogs-severe

I

L short term survival dogs--moderaw \ dog --no fibrosis,’ four wrek survival dog ---sc’vere short twm survival dogs- moderate 1 dog - -none dog focal fibrosis and calcification, four week survival dogs- moderatc dog ~~-minimal short trrm survival I, dogs-none 1 dog -~~no fibrosis, four wrek survival 1 dog ~~~~~s~verr 1 2 dogs ___modcrate i short term survival 2 I 1 5 1 1 2 1 5

and 40 pear cent carbon dioxide; injection of stress-inducing drugs which in themselves might influence the microcirculation was not required. Catecholamine levels were elevated during hypercapnea and decreased rapidly with the Brown”*‘” and his reversal of hypercapnea.7s8 associates and Sealy I1 found that potassium concentrations in the plasma increased during long periods of hypercapnea and rose still higher at a rapid late in the first five minutes During, the after reversal of the hypercapnea. reversal of hypercapnric period of rapid acidosis, Brown and Mowleml” reported a loss of potassium from the heart accompanied Coronaryby severe cardiac irregularities. blood floes was elevated during hypercapnva and returned to normal values with the rcversa1 of h\-percapnea. Cardiac irregularities” in human patients have also been produced i))hypercapnca during apneic oxygenation. METHODS Ertracorporral Circulation: Healthy adult mongrel dogs were subjected to periods of total cardiopulmoThe apnary bypass for periods of two to four hours. paratus used consisted of the bubble oxygenator and in the use of Sigmamotor purnl~‘~ with modifications silicone antifoam for the prevention of silicone embolism. The oxygenator was primed with fresh, unmatched, homologous blood collected in siliconized blood bottlrs containing 20 mg. of heparin in 25 cc. of Sterile equipment was used for 0.9 per cent saline. Anesthesia was provided by each experiment. intermittent in.jections of a solution containing 5 per cent thiopental sodium plus 5 per cent gallamine triethiodide (Flaxedila). After thoracotomy the dogs were given heparin. 3

mg.;‘kg. of body weight? to prevent clotting in the A large catheter was placed extracorporeal circuit. in the right atrium to collect venous blood, and an arterial catheter was placed in a femoral artery for the A return of arterialized blood from the oxygenator. tape was passed through the transverse sinus and around the pulmonary artery and aorta. During the bypass period, this tape was tightened to occlude the pulmonary artery but not the aorta. All systemic venous blood and coronary venous blood, except the ‘l‘hebesian venous and bronchial venous return to the left ventricular cavity, was therefore collected and returned to the oxygenator. Cardiac distention was avoided at all times. Cardiac manipulation was limited to opening of the pericardium and insertion of the single large catheter into the right atrium and right ventricle through the right atria1 appendage. Microcirculation During Injections of Dextran and AVorepinephrine: Before, during and after the period of csu.acorporeal circulation, the microcirculation in the vessels of the ocular coIljunctiva was studied through a dissecting microscope with a magnification of 50. In six dogs the microcirculation of the mesentery was also studied at a magnification of 200X, and microphotographic records were obtained for further By using meticulous care to prevent local study. trauma and dehydration, the microcirculation was studied for periods up to eight hours in control experiments without any significant alterations in thr microcirculation. ‘l’he dogs were divided into four groups as illustrated in ‘I’able I. In the dogs in the dextran series and the dextran plus norepinephrine series, low molecular weight dextran* (average molecular weight approximately 40,000) was given in a dosage of 2 to 4.8 gm./kg. of body weight in a 10 per cent solution of dextran in 0.9 per cent saline. Half of * Supplied as Rheomacrode@ tories, Inc., New York, N. Y. THE

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by Pharmacia

JOURNAL

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Labora-

CARDIOLOGY

Myocardial

Necrosis

and Microcirculatory

Abnormalities

697

this dosage was given to the dog during the period of preparatory surgery, and the other half.was added to the priming volume of donor blood in the oxygenator. In the last series, cardiopulmonary bypass was started and continued for the first 10 to 15 minutes as A continuous drip of norin the dextran series. epinephrine was then added to the oxygenator reservoir to maintain the mean blood pressure of the dog between 140 and 160 mm. Hg. The amount of norrpinephrine required varied from 16 to 32 mg. of levoarterenol bitartrate in a three hour period. Following the conclusion of extracorporeal circulatio+ the dogs were either sacrificed with a lethal intravenous dose of pentobarbital sodium or permitted to survive from two hours to four weeks. No attempt was made to attain uniform long term survival. Autopsies were performed immediately on all dogs sacrificed and shortly after death in those dogs dying during the postoperative period. Materials obtained from autopsies were fixed in 10 per cent formalin and stained with routine hematoxylineosin staining technic. Microcirculation During Hypercapneic Acidosis: In the study on hypercapneic acidosis, a cardiac catheter was inserted under fluoroscopic control through the jugular vein into the coronary sinus while the subject was under thiopentothal sodium anesthesia. A catheter was then placed in the femoral artery to measure blood pressure and to collect blood samples. Samples of arterial blood and coronary sinus venous blood were obtained simultaneously and analyzed for pH and plasma potassium. Blood pH was determined with a glass electrode pH meter maintained at 38Oc. Potassium concentration was determined on a protein-free filtrate of plasma with the Beckman DU flame photometer. ‘I‘he microcirculation of the ocular conjunctiva was observed before, during and after hypercapnea. One group of six dogs received low molecular weight dextran, 1 gm./kg. of body weight, before the induction of hypercapneic acidosis and 0.25 to 0.50 gm./kg. of body weight during the four hours of hypercapnea to replace the quantities excreted by the kidneys. In two dogs, a quantity of blood equal to the volume of 10 per cent dextran in 0.9 per cent saline given was drawn off through the femoral artery. The control dogs received a continuous drip of heparinized saline to prevent clotting in the coronary sinus catheter. The experimental group received only a continuous drip of low molecular weight dextran through the cardiac catheter. The volume of fluid administered in this fashion was approximately equal in the two groups. Hypercapneic acidosis was carried out as previously described by Brown and Mowlem.10 An endo-

mixture of 40 per cent carbon dioxide and 60 per cent oxygen for two hours. Samples of expired air were obtained periodically and analyzed for carbon dioxide content. At the end of the four hour period, the endotracheal tube was connected to a respirator and the dog was hyperventilated on 100 per cent oxygen to reverse the hypercapneic acidosis as rapidly as possible. In a few instances, the dog’s own respiratory efl’orts were strong enough to oppose the action of the respirator. In these cases, the endotracheal tube was disconnected from the respirator and the dogs hyperventilated on room air. The electrocardiogram and the blood pressure were recorded intermittently during the experiments and continuously for at least five minutes during the off-carbon dioxide period. Samples of arterial and coronary sinus blood were obtained simultaneously before hypercapnea (control sample l), after two hours of breathing 30 per cent carbon dioxide (sample 2), after two hours of breathing 40 per cent carbon dioxide (sample 3), and four to five minutes after reversal of hypercapnea (sample 4).

tracheal

average

rometer oxygen. mixture

tube The

dogs

passed

and

a mixture were

of 30 per cent

cent oxygen NOVEMBER

was

containing

connected

of carbon allowed

carbon

to

dioxide

to a spidioxide

Befcre bypass

the

observed nary

bypass,

there

in

curred.

Changes

then

control

the

were

velocity total

of

most foI

blood

flow

flow

the

The

blood

peripheral

tendency the

ratio

blood of

particle

accumulation vessels

1,

layer

flow

was

When

Green

circulation,

in of

intra-

develop.

The

the

in

of plasma to

then

that this

accumulate

related

to

the

vessel to the

this stated

an

arterioles.

stated

of the blood

was seen.

observed

distinct

of

corpuscles

size.

to

higher

accumulate

Green’”

blood

of the diameter 10

to

concentric

for

used,

a

was a tendency

center

in thickness. axis

proached

blood

the

to

finding

corpuscles in

were

30 to 60 minutes

began

early

approx-

and that

to the

of

weight

bypass,

aggregation

stream

increased

rates

than

After

initially,

returnecl

of body

period.

conspicuous

axial

in

microcirculation

15 oc-

transient

appeared

cardiopulmonary

vascular

and

less vasoconstriction

the control

10 to

of corpuscular

distention

110 ct./kg.

there

of

alterations

in the velocity

When

was

cardiopulmo-

a period

aggregation

status.

imately

aggregation

inconsistent

venous

intravascular

cardiopulmonary

starting

was

which

movement,

total

After

minutes

but

of

intravascular

rarely.

rxtracorporeal

to a

DURING EXTRACORPOREAL

onset

in dogs,

a

for two hours and were then changed 1962

MICROCIRCULATION CIRCULATION

70 per

breathe and

and

RESULTS

ratio that

apaxial

In these studies during the diameter

of the mesentery

did

not

of the change

698

Long et al.

FIG. 1. Photographs of the microcirculation of the mrwntrry ol’thc doq during cxtracorporc-al circulation. The perfusion rate used in this instancr was 100 cc. /kg. /min. Only minor fluctuations in blood prrssurc occurrrd throughout circulation. “a” dcsisnatcs an the perfusion. (Magnification X200. ) .i3 c:,ntrol. M,.Jrr the. Ons<‘tof cxtracorporral artrriole with two hranchcs. “v” indicates a vcnulr with s(.vcral trihutarirs, and “c” indicates a capillary. B. 25 minutes after the onset of cstracorporral circulation. NotI, thr aqgwqation of thr corpusclrs in the postcapillary vcnulr. the axial accumulation of the corpuscles in the art<-riolr and the, tllickrning of thr layrr of plasma skimming in thr periphery of thr vc~sscl. C:, th(, samr arca after two hours of rxttacorporeal circnlatlon. ‘l‘hr v&city of blood How decreased, and thr arteriole in thr lowrr lrft cx-ncr containrd only plasma and a frw aqgrqatcs of crvthrocytes (arrow). D, aftrr three hours and trn minutrs of (~utracorpurral cirrulation and ten minutes aftrr the administration 1.5 mg./kg. ‘l’he vrlocitv of hl,wtl How incrr~nsrd. tlr rrllrllar aqql-ryatrs dccwasrd or disappeared, of Khcomacrodrx. and the axial accumulation diminishrd.

appreciably (Fiq. l), but the. a\-craye particle size qraduall!, increased as blood corpuscles began to adhcrc to each other. \VhCll rhe conjunctival ~rcssels \vcrc \Gewrd 1)) tile naked eve, onlv the stream of blood corpuscles was thcrc \vai an apparcnl visible. ‘C 0nscqucntly, decrease in the size cf the lumen of tlie vcssrls. However, mar:_nification of the ficlcl rcvcalcd that there ~cas no significant constrickn of tllr vessels. During this please of intra\.asculat a,yqregation, tllcrr \\‘a~ a tcndcncy for onI> plasma to enter some of the branches of tlir arterioles: this resulted in an apparent hcmoconcentration in the distal continuation of thr arterioles. The tenacil y of the adhesion of blood corpuscles to each other then continued to increase Occlusion and the aggrc:gates became larger. of venules at bifurcations was more frequent

(Fiy. 2). After 90 to 120 minutes of extracorporeal circulation, the corpuscular adhesion appcarcd to become stronger. There was less tcndenc). for tht. clumps of cells to become s+~rated 1,) the shcariny forces in the high prcssurc and hi+ \,clocity areas of the arterioles, and artcriolar occlusion then occurred (Fig. 3). ‘lk blood corpuscles also appeared to adhere to the wails of venules. The velocity of blood flow \vas greatly reduced. Arteriovenous shunts also became more apparent at this stay? because of the persistence of higher velocities of blood How through the shunt<:. -l’hc final stages of intravascular aggregation occurred after 120 to 150 minutes of total cardiopulmonar>r bypass. Occlusions of artc.riolcs and venules became permanent, and intravascular thrombosis (Fiy. 4) was present in some vessels in most areas of examination. THE

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Myocardial Necrosis and Microcirculatory

Enlargement of the bottom left panel of FIG. 2. Figure 1. The arrow points to a temporary occlusion of the bifurcation of a venule by a large “arrow-head” shaped aggregate of corpuscles.

Photomicrograph of another area in which FIG. 3. artr-riolar thrombosis (a) and venular occlusion (v) were present after one and one-half hours of extracorporeal circulation.

In some areas of thrombosis, an accumulation of pinkish-stained fluid, presumably plasma, developed outside the lumen of the small vessels. E$Pct of De&an: Little or no evidence of intravascular aggregation was observed when low molecular weight dextran was administered NOVEMBER

1962

Abnormalities

699

FIG. 4. Photograph illustrating thrombosis of a venule at the bifurcation after three hours of extracorporeal circulation. The arrows point to the walls of a venule through which circulation was sluggish. The corpuscles were adhering to the endothelium at the upper arrow and tumbling along the wall of the vein.

prophylactically. Occasionally areas of temporary occlusion of venules or arterioles were found. Permanent occlusion of blood vessels by aggregates was rare in the areas examined microscopically. When low molecular weight dextran was added after two or three hours of extracorporeal circulation, there was a definite improvement in the microcirculation within 10 to 15 minutes (Fig. 1). The aggregates of corpuscles decreased in size, exaggerated axial accumulation decreased, and the velocity of blood flow increased. However, the use of low molecular weight dextran did not affect those vessels in which permanent occlusion or thrombosis had already developed. Norepinephrine: Shortly after starting the norepinephrine drip in the dogs of the final series, the blood vessels of the ocular conjunctiva became severely constricted. The number of blood vessels containing circulating blood gradually diminished. At the end of two hours, there was a severe blanching of the vessels of the ocular conjunctiva, and only a few small blood vessels could be seen. HISTOLOGY

When the dogs were examined numerous incisions were made

post mortem, through the

Long et al,

FIG. 5. Photograph of a histologic section uf the myocardium in a doq sacrificed immediatc*ly after threr hours of extracorporral circulation at a perfusion rate of 100 This dog received no low molecular cc./kg./min. weight dextran. Thr arrows point to myocardial tibers in which there is a dissolution of the myofibrils. The (Hematoxylin nuclei are also swollen and irregular. and cosin

staining:

tcrhnic.)

myocardium, and multiple specimens were taken for histologic examination. Areas with subendocardial hemorrhage and focal areas that appeared darkened and congested in contrast to the surrounding myocardium were always Sections of saved for microscopic examination. kidney, adrenal, liver, pancreas, gastrointestinal tract, brain, and skeletal muscle were also studied histologically. Impressions made on gross examination of the myocardium at autopsy were considered too inexact to be of any value in the dogs sacrificed or dying shortly after tota! cardiopulmonary bypass. Three dogs, one in each series except the norepinephrine series, were permitted to survive for four weeks and were then sacrificed. In the long term survivor from the low-flow control series, there were focal areas of fibrosis the and calcification scattered throughout myocardium. In the other two long term one from the high-flow control survivors, series and one from the dextran series. there was no evidence of chronic fibrosis. The histologic Grading of Histologic Changes: changes noted in the short term experiments were graded arbitrarily according to the extent of early alterations in the myocardial fibers. The characteristics (Fig. .5 and 6) considered abnormal in the sarcoplasm were myocardial fibers in which there was dissolution of the

view of the. rnyocardium of a dog in E‘rc:. 6. Low-powrr which moderate myocardial necrosis occurrrd after tl1r.c.e hours of extracorporeal circulation at a flow rate Note the lalgr numbu- of myocardial of 68 cc./kg./min. cc~lls with fragmentation and disappearancr of the Notr also the capillary engorqrment (c) myofibrils (m). with rrythrocytrs as drscribrd by Reynolds ct a1.16 in the arrrsted heart. No low molrcular weiqht dcstran was used in this pvrfusion. (Hrmatoxylin and eosin staining trchnic.)

myofibrils, particularly around the nucleus, or in \vhich there was actual loss of continuity of the cell wall in addition to dissolution of the myofibrils. The nucleus was considered abnormal when there were swelling and pale staining of the nuclecplasm or when there was actual fragmentation or pyknosis of the nucleus. When none or few myocardial cells showed theue early necrotic changes, the grade fzonr was applied. The grade scwc was applied hvhen focal lesions greater than 3 mm. were encountered containing early necrotic changes in one third or more of the cells. The grade minim& was applied for those hearts in which there were only a felt- foci less than 1 mm. in diameter with approximately one-tenth of the cells showing early necrosis. The grade moderate lvas used to describe those lesions judged to be between the grades of minimal and severe. 7%e analysis of the microscqbic studies revealed that the lesions indicative of early myocardial necrosis were found in all except one doa in the two control series (Table I). In two of the dogs receiving low molecular weight dextran, there was evidence of a moderate degree of One dog showed a minimyocardial necrosis. mal degree of necrosis. In the remaining five short term survival dogs in the dextran series; there was little or no evidence of myocardial THE

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Myocardial

Necrosis

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view of an area of myocardial neFIG. 7. High-power crosis and inflammatory cell infiltration in the heart of a dog subjected to four hours of hypercapneic acidosis No intravascular catheand sacriked three days later. Microscopic terizations were performed in this dog. areas of myocardial necrosis were distributed in the subendocardial layers as well as within the deeper layers of the myocardium. (Hematoxylin and eosin staining technic.)

necrosis. Necrosis of myocardial fibers was found in all three dogs receiving low molecular weight dextran and exogenous norepinephrine. In the one long term survivor of the low-flow control series, there was a diffuse focal fibrosis and petechial calcification of the myocardium. No abnormalities were seen in the myocardial sections from two dogs sacrificed four weeks postoperatively in the high-flow control series and in the low-flow plus dextran series. MICROCIRCULATION

DURING

HYPERCAPNEA

Durrng ty@ercajmeic acidosis in control dogs, changes in the microcirculation occurred (Fig. 7). Within 30 minutes after the induction of high carbon dioxide breathing, intravascular corpuscular aggregates could be seen, and the Changes in velocity of blood flow diminished. the microcirculation similar to those reported above during extracorporeal circulation progressed to more severe stages during the four hours of hypercapnea. The venules and arterioles became distended and engorged with In the majority of the small large aggregates. vessels in the conjunctiva, no passage of blood was discernible for long periods of time. In other vessels the blood flow had a very low velocity alternating with periods of occlusion. Arteriovenous shunts appeared to become more numerous. The velocity of blood flow was more rapid in the arteriovenous shunts, and arterial pulsations were obvious in the passage of NOVEMBER 1962

Abnormalities

701

clumps of blood corpuscles in the venules associated with these shunts. Immediately following the rezjersal of hypercapnea, the aggregates of cells began to break up and pass slowly from occluded vessels. This phenomenon resembled the flow of large blocks of ice in a stream when thawing of a river first The reversal of intravascular aqgregabegins. During tion did not follow a uniform pattern. this period, some vessels were still occluded at a time when the velocity of blood flow in other areas had returned almost to the control state. Vasodilatation persisted in these control dogs. When the dogs became hypotensive, the velocity of blood flow decreased. When ventricular fibrillation occurred, the blood flow in the small vessels ceased. In the dogs pretreated with low molecular weight dextran, no intravascular aggregation was observed, or occasionally a small aggregate of blood corpuscles was found temporarily occluding a venule. There was also an intense vasoconstriction of the arterioles and venules of the ocular conjunctiva in contrast to the engorgement and distention seen in the control The vasoconstriction was consistent with dogs. the situation one would expect in this stress condition. The velocity of corpuscular flow remained rapid throughout the period of hyDuring the period of percapneic acidosis. reversal of hypercapnea, there was a prompt vasodilatation of the arterioles and venules. The velocity of blood flow also decreased slightly during the period of moderate hypotension associated with the vasodilatation in the immediate post-hypercapneic period. ELECTROCARDIOGRAPHIC

CHANGES

The electrocardiographic changes, seen during the period of hypercapnea and in the immediate off-period, were similar to those previousl) During the hypercapneic period reported.g there was a progressive decrease in the amplitude of the QRS complex and an increase in the amplitude of the T wave with spiking of the Within five to ten minutes after rapid T wave. reversal of hypercapneic acidosis was started, frequent premature extrasystoles were observed In four of in the six control dogs (Table II). the six dogs, the premature extrasystoles progressed to ventricular tachycardia and ventricuIn one lar fibrillation with death of the dog. dog, there was a period ofcomplete atrioventricular dissociation which reverted to a normal sinus rhythm in three minutes.

Long et al.

702

TABLEII Plasma

K+

Concentration

in Arterial and Coronary

Sinus Blood CO?

Before,

During

and Following

s 6

NS 8.1 7.2

8.6 8.5 6.0

6 0

6 2

7.0

8.7

4.5 4.6 4.9 0.18

5.9 5.1 5.9 0.15

5.7 4.9 5.8 0.19

7.1 7.0 7.3 0.21

7 6 9.8 8.2 0.52

6.4

6.8

8 1

8.5

NS

n’s

3.9

5.7

5

5.8

5.7

6.8

8.0

3.6

3.6

6.8

6.7

x.7

8.4

8.5

10.0

4

3.3

3.7

7.4

7.3

7.1

7.1

7.3

10.1

5

3.7

3.6

5.3

5.3

7.0

7.1

7.3

7.3

6

3.9

4.2

6.0

5

6 5

6.6

7 0

8.7

Mean

3.7

3.8

6.3

6.3

‘.2

7.2

7.38

8.8

(l-5) SE

0.10

0.10

0.31

0.33

0.43

0.45

0.30

0.55

1 2 3

4 0 3.4 3.8

3.2 3.6

5.9 4.9 5.3

5.6 4.6 4.9

6.2 6.0 6 0

6.0 5.9

4

3.9

3.9

5. 0

5 0

5 6 Mean SE

4.0 3.8 3.8 0.09

3.9 3.5 3.6 0.11

4.4 4.5 5.0 0.22

1

3.9

4.1

2

3.9

3

A = arterial;

3.8

CS

= coronary

sinlm;

NS

0

9

= no sample

In five of the six control does, the plasma potassium level in the coronary sinus blood was greater Lhan that of the arterial blood in the five minutes cff-sample. The one other dog that did not develop ventricular fibrillation also showed no difference in the arterial-coronary sinus potassium levels. During the pried of lzypercajmeic acidosis the dogs that received low molecular weight dextran showed the same electrocardiographic changes However, as seen in the control dogs (Fig. 8). during the period of reversal of acidosis, the electrocardiographic changes were different. Only a few ventricular extrasystolic complexes were observed, and one dog showed a transient period of atrioventricular dissociation for three cardiac cycles. The period of hypotension also developed during the reversal of acidosis but was not as marked as in the control dogs. The configuration of the electrocardiogram

Four

Hours

of High

Breathing

SE

= standard

error

Ventricular fibrillation Ventricular fibrillation Ventricular extrasystoles, survived Atrioventricular dissociation, survived Ventricular fibrillation Ventricular fibrillation

.4trioventricular survived Few ventricular survived No ventricular survived Few ventricular survived Few ventricular survived No ventricular survived

dissociation, extrasystoles, extrasystoles, extrasystoles, estrasystoles, cxtrasystoles,

of mean.

approached that of the control period within All dogs survived the experiment 30 minutes. and regained consciousness within 30 minutes. Plasma potassium elevation also occurred in the dogs given dextran (Table II) during hyperFor technical reasons coronary sinus capnea. blood levels were not obtained in one of the dogs during the post-hypercapneic period. In four dogs, the coronary sinus plasma potassium level was higher than the arterial plasma potassium level during the post-hypercapneic period, and in one dog there was no difference. DISCUSSION The results of these experiments demonstrate that changes occur in the blood flow through the small blood vessels when significant intravascular aggregation is present. Unfortunately, there are no technics for examining the microcirConsequently, the culation of the beating heart. THE

AMERICAN

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CARDIOLOGY

Myocardial

2 Hours

Contro 1

3 Min.

off

Necrosis and Micrxirculatory

CO2

13 Min.

on CO2

off

CO2

703

Abnormalities 4 Hours

30

on CO2

Min.

off

CO2

FIG. 8. Dog 3 (Table II). Representative examples of the electrocardiographic changes (lead II) seen during hypercapneic acidosis experiments in dogs receiving low molecular weight dextran. Note the decrease in amplitude of the R waves, and the reversal and increase in amplitude of the T waves and spiking of the T waves during the period of hypercapneic acidosis and in the immediate period following reversal of the hypercapneic acidosis. These changes were associated with an increase in plasma K+ concentration. One irregularity of rhythm (arrow) lasting one cardiac cvcle was noted in the ten minute off-CO7 Deriod. Note the return of the electrocardiographic pattern toward normal i,‘, the 30 minute, off-CO2 period.

effects of intravascular aggregation on the myocardium must be inferred from postmortem examinations and electrocardiographic observations made in situations of intravascular aggregation observed elsewhere in the body. Reynolds et al.15 used a technic for arresting the circulation through the heart and studied the microcirculation post mortem. These investigators described capillary engorgement with irregular clumps cf corpuscles in the potassium-arrested-reperfused hearts. Their descriptions are similar to those observed in the vital circulation of the ocular conjunctiva and mesentery during prolonged periods of extracorporeal circulation and hypercapneic acidosis. The studies made during extracorporeal circulation support the observations of Gelin who demonstrated the occurrence of myocardial microinfarction following trauma. Gelin showed that these microinfarcts, which were also found in the kidneys and liver, were caused by intravascular aggregation. Both the intravascular aggregation and the microinfarcts were prevented by the administration of low molecular NOVEMBER

1962

weight dextran. Thorsen and HintrE earlier showed that low molecular weight dextran was effective in reversing the elevation of erythrocyte sedimentation rate seen following trauma. They also demonstrated the formation of an adhesive film on the corpuscles during intravascular aggregation and the dissolution of this film when the cells were treated with dextran preparations of appropriate molecular size. The phenomenon of intravascular aggregation is also important in man. The microcirculation during extracorporeal circulation and open heart surgery has been studied in human patients,” with various types of cardiac abnormalities. Intravascular aggregation was frequently present preoperatively, particularly in patients with chronic congestive heart failure. Conceivably, the phenomenon of intravascular aggregation and microthrombosis may be the cause of the focal myocardial fibrosis seen in these patients without coronary artery disease. Madowl attempted to reverse the intravascular aggregation in patients with coronary insutliciency by the use of hydroxpchloroquine

Long et al. EFFECT

OF INTRAVASCULAR AGGREGATION DECREASED

9. Schematic presentation of a method by which intravascular aggregation and changes in the microcirculation might result in ventricular irregularities and fibrillation. During the rapid reversal of hypercapneic acidosis, the areas of the myocardium with normal circulation would be subjected to the increased blood pH, In areas and an efflus of potassium ions would occur. FIG.

of the myocardium where the microcirculation was decreased, there would be little or no rapid change in pH with no efflux of potassium. Adjacent heterogeneous areas of critical size could develop in the myocardium and result in an electrochemical diffrrencc, thus trig,yer-

inq vc-ntricular fibrillation. (Plaquenil”

his study- was still in a stage, he reported that these patients were subjectively improved. Studies of this nature seem warranted in the treatment of coronal-)- insufficiency. The microcirculation has not been studied under the conditions of experimental stress Experireported by Selye’ and by Raab.? mental stress induced by burns, hypothermia, fractures and soft tissue trauma, however. did result in intravascular aggregation of a signifiIn the present study, myocardial cant degree.” necrosis was found in the dogs receiving esogceven though the)- also nous norepincphrine, rccei\.ed low molecular wei,ght dextran. The se\~ere constriction of the arterioles and venules with these pharmacologic doses of exogenous norepinephrine was thought to be the explanation for the necrotic lesions rather than any direct necrotizing effects of the catecholamines. Tfle cardiac irregularities of rajid reversal of Iypercapneic acidosis were attributed by Brown preliminary

1. Although

and Mowlem”’ to the efflttx of potassium from the heart. \lentricular fibrillation was prevented by preventitng the efflux of potassium by, administration of 20 per cent glucose by Scaly” and by the administration of norepinephrine by Coott .h The present studies elaborate on this concept. Although the ventricular irregularities were prevented by preventing intravascular aggregation, the potassium efflux still occurred. During the off-period there was an irregular disintegration of the intravascular aggregates and a return of blood flow in different areas at different times with potassium efflux occurring concomitantly-. It was theorized that these tlvo events produced a difference in potassium ion concentration or areas of heterogeneity within the myocardium resulting in an electrochemical gradient between adjacent areas (Fig. 9i. This phenomenon may be similar to the lowering of ventricular fibrillation threshold produced by oxygen differentials within the myocardium.t” Mvocardial necrosis was also found in dogs surviving periods of hypercapneic acidosis (Fig. 7). The etiology of the intravcrscularaggregation seen with stress or trauma, including extracorporeal circulation, and hypercapneic acidosis has not been defined. Experiments now in progress provide some insight into this vexing problem. _4bdominal evisceration, including enterectomy-, pancrratectomy, and hepatectomy, has been performed on a series of five dogs and total hepatectomy alone in another series of six dogs. In these dogs, intravascular aggregation and ventricular fibrillation did not occur, although comparable changes in pH and pCOz were acidosis. When present with hypercapneic only the gastrointestinal tract or the pancreas was removed from five dogs and six dogs, respectively, intravascular aggregation still occurred and was associated with a high incidence of ventricular irregularities and fibrillation. Studies are now in progress to elucidate the possible role of the liver in this important pathologic reaction to stress and trauma. Samples of arterial blood were obtained from two dogs during hypercapnea and es. amined under dark field illumination. The process of preparing thin smears of blood on a microscope slide was assumed to result in a loss of carbon dioxide and an elevation in pH due to equilibration of the gases in the blood with room air. Nevertheless, the aggregated blood corpuscles were found to be coated with a thin translucent film similar to that described by THE

AMERICAN

JOURNAL

OF CARDIOLOGY

Myocardial

Necrosis

and

Microcirculatory

Thorsen and Hint.r6 Addition of a drop of a dilute solution of low molecular weight dextran to the blood resulted in a disappearance of the coating film and a normal dispersion of the blood cells. This finding, along with the abcve cbservation in dogs with total hepatectomy, indicates that the aggregaticn of blood corpuscles was not due to changes in pH alone or changes in the size or electrochemical charge of the blood cells due to the increased pCOZ. SUMMARY

Studies of the microcirculation of dogs were performed, utilizing the ocular conjuctiva and the mesentery. Intravascular aggregation was noted during total cardiopulmonary by-pass as well as during hypercapneic acidosis. This was associated with microinfarction of the myocardium. The use of low molecular weight dextran prevented intravascular aggregation in the microcirculation of the areas studied and was accompanied by a diminished incidence of microinfarcts in the myocardium. REFERENCES 1. &LYE, H. Conditioning by cortisol for the production of acute massive myocardial necrosis during neuromuscular exertion. Circulation Res., 6: 168, 1958. 2. RAAR, W., STARK, E. and GIGEE, W. R. Role of catecholamines in the origin of stress induced myocardial necrosis. Circulation, 20: 754, 1959. 3. GELIN, L. E. Studies in anemia of injury. Actn chir. scandinav. (Supp.), 210, 1956. 4. SWANK, R. L. and ESCOBAR, A. Effect of dextran injections on blood viscosity in dogs. J. A@l. Physiol., 10: 45, 1957. 5. WRIGHT, I. S. (Ed.) Myocardial Infarction: Its Clinical Manifestation and Treatment with Anticoagulants, p. 4. New York, 1954. Grune & Stratton. 6. READ, R. C., JOHNSON,J. A. and LILLEHEI, C. W. Coronary flow and resistance in the dog during

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total body perfusion. Surg. Forum, 7: 266, 1757. 7. MILI.AR, R. A., BRINDLE,G. F. and GILBERT, R. G. B. Studies with an organic buffer (THAM) during apneic oxygenation in dogs. ht. .I. dw~~fh., 32 : 248, 1960. 8. GOOTT, B., ROSENBERG,J. C., LIL.LEHEI,R. t:. and MILLER, F. A. The relationship of the sympathoadrenal system to potassium flux and cardiac irritability induced by alterations in blood pH. J. Thoracic & Cnrdiovas. Surg.. 40: 625. 1960. 9. BROWN. E. B., JR. and MILLER, F. A. Ventricular fibrillation following a rapid fall in alveolar carbon dioxide concentration. Am. J. Physiol., 169: 56, 1952. 10. BROWN, E. B., JR. and MOWLEM, ii. Potassium loss from the heart during the immediate posthypercapneic period. Am. J. Physiol., 198: 962, 1960. 11. SEALY, W. C.. YOUNG, G., JR. and HARRIS, J. S Studies on cardiac arrest: the relationship of hypercapnia to ventricular fibrillation. J. Thoracic Sq., 28: 447, 1954. 12. FRUMAN, M. J., EPSTEIN, R. M. and COHEN, G. Apneic oxygenation in man. .Innesthesiology. 20: 789, 1959. 13. DEWALL, R. A. and LILLEHEI, C. W. Design and clinical application of the helix reservoir pump oxygenator system for extracorporeal circulation. Postgrad. Med., 23: 561, 1958. 14. GREEN, H. D. Circulation: physical principles. In: Medical Physics. Vol. II, p. 241. Chicago, 1950. The Year Book Publishers, Inc. 15. REYNOLDS, S. R. M., KIRSCH, M. and BING. R. J. Functional capillary beds in the beating, KClarrested and KCl-arrested-perfused myocardium of the dog. Circulation Res., 6: 600, 1958. 16. THORSEN,G. and HINT, H. Aggregation sedimentation and intravascular sludging of eryi hrocytes. Acta chir. scandinav., (Supp.) 154 1950. 17. LONG, D. M., JR.. SANCHEZ, L., VARCO, K. L. and LILLEHEI, C. W. The use of plasma expanders, low molecular dextran and albumin during extracorporeal circulation. Surqrry, 50: 12, 1961. 18. MADOW, B. P. Use of antimalarial drugs as dr.I..-1.‘2f.A., sludging agents in vascular disease. 172: 1630, 1960. 19. BROPMAN, B. L., LEIGHNINGER,D. S. and BECK, C. S. Electric instability of the heart: the roncept of the current of oxygen differential in coronary artery disease. Circulation, 13: 161, 1956.