Pharmacological Research Communications, VoL 9, No. 1, 1977
79
EFFECT OF THE PROTEINASE INHIBITOR TRASYLOL ~R'(~ ON THE MORTALITY AND INFARCT SIZE IN DOGS WITH ACUTE MYOCARDIAL INFARCTION.
J. Eisenbach* and H. Heine**, Surgical Center, University of Frankfurt* and the Center of Morphology, Frankfurt, Main, Federal Republic of Germany** Received 15 March 1976
SUMMARY
(R)
The proteinase inhibitor Trasylol
was stud%ed for its
effect on the mortality and infarct size in dogs with experimentallyinduced acute myocardial infarction.
The infarction was induced
in the anesthetized dog by means of deep over-lapping pursestring ligatures into the left ventricle, and the infarct size determined both by ultra-violet technique following injection of fluorescein and by post-mortem coronary artery angiograms.
A
total of 45 dogs was studied with approximately half receiving 500,000 units Trasylol intravenously Just prior to infarct induction and 10,000 units/kg intravenously two hours later and thereafter every six hours until death.
Trasylol was found to
increase significantly both survival time and survival rate in those animals with an infarct size in excess of 10% of the heart mass.
While animals in both control and drug-treated groups
experienced circulatory shock, the severity of the circulatory disturbance was distinctly less in the Trasylol-treated animals. Ventrlcular fibrillation in drug-treated dogs also appeared much later compared to control dogs.
Coronary artery angiographic
Pharmacological Research,Communications,, Vol. 9, No. 1, 1977
80
examination in control dogs showed degeneration of minute peripheral branches of the coronary vessels in the border zone of the infarct, whereas a normal coronary pattern within the infarct border zone was seen in the drug-treated group.
Distinct histologlc
differences in the border zone were noted in the drug-treated dogs compared to the controls.
The damaged areas of the hearts from
the drug-treated group exhibited an intense reactive hyperemia not seen in the controls. control dogs.
The infarct size was not contained in the
Other morphological differences were seen which
could account for the differenc~ in survival rate.
The significance
of these effects as related to cellular and biochemical
(enzyme)
changes are discussed. INTRODUCTION At present, myocardial infarction represents one of the m o s t frequent causes of death among disorders of the heart. the accumulation of an immense body of literature,
Despite
new questions
regarding fundamental aspects of the pathophysiology,
histo-
morphology and therapy of this myocardial injury appear to be the center of medical interest. Tschirkov,
et al. (1972, 1973) recently presented results of
studies on experimentally-induced
myocardial infarction in dogs
which gave rise to this present study. METHODS Certain areas of the left ventricle in 45 healthy mongrel dogs were made ischemic by means of deep overlapping purse-strlng ligatures
(Tschirkov, 1972, 1973).
The myocardial infarction
model does allow investigation and interpretation of fundamental events occuring within the center area and border zone of the infarct.
Pharmacological Research Communications, Vol.. 9, No.: 1, 1977
81
The extent of infarctwas determined by ultra-violet light technique following injection of fluorescin.
Additionally, post-
morton coronary artery angiograms were prepared by the injection of a bolus of trilead tetr0xlde and gelatin into the coronary arteries.
The following parameters were recorded continuously or
determined at various time intervals during the course of the experiment : electrocardiogram, aorta and left ventricular pressure, coronary vein and artery gas analysis, electrolyte s in coronary vein and arterial blood and in the infarcted central and border zones, and hist0Pathology.
All tissue preparations were fixed in
formalin, ~%, glycol methacrylate, and toluidine blue (pH 5.5). Section thickness was one micron.
Only the pressur~ and hlstologic
data will be presented in this report. T h e animals were divided into two groups; Group 0 received no treatment while Group T received the proteinase inhibitor Trasylol
i
, 500,000 units intravenously just prior to infarct
induction and i0,000 units/kg intravenously two hours after infarct and thereafter every six hours until death. RESULTS Table i summarizes the survival rate in relation to infarct size.
Both survival time and survival rate appeared closely re-
lated to infarct size.
Animals with infarct size less than 10%
of the heart mass survived up to the time of sacrifice in both the control and drug-treated group.
As the infarct size increased,
the mean survival time decreased in both groups of animals. in those groups where the infarct size was greater than i0%, TrasyloZ significantly prolonged survival time.
1.
Traaylol (R) product of Bayer AG, Leverkusen~ West Germany
However,
Infarct > 15% of heart mass
Group III : 4
1
i0
7
No. of dogs
40 hours
1".7-+ 0.3
Survived
21 -+3.0
All dogs survived
GROUP 0
Survived 18 hours
1
4.0
6.2 -+ 0.3
Survived
31.2+
~ii dogs survived
GROUP T
3
4
8
7
No. of dogs
ANI~gL SURVIVAL TIME (IN HOURS) RELATIVE TO INFARCT SIZE.
Infarct size between I0 and 15% of heart mass
Group II:
Infarct < i0% of heart mass
group, I :
Infarct size
TABLE i.
"~a
~a
CD b~
Pharmacological Research Communications, VoL 9; No, 1, 1977
Figure i.
83
Coronary artery angiograph 48 hours after infarct. Untreated dog, with infarct size 9.4% of heart mass.
Continuous recording of aortic blood pressure revealed that cardiogenlc shock occured in all animals in which the infarct size exceeded 1 0 % of the heart mass. leading to death.
The shock increased in severity
In these animals, the circulatory disturbance
was distinctly less pronounced in the drug-treated dogs.
In the
control animals, ventrlcular fibrillation generally appeared two hours after Infarct induction.
In the Trasylol-treated dogs,
fibrillation appeared 2 - 18 hours later. Coronary artery angiographic examination carried out 12 hours after infarction revealed degeneration of minute peripheral
Pharmacological Research Communications, Vol. 9, No, 1-, 1977
84
~
~
~
.~,p.,,
N..
''
I
t.!.
.
:
-..
,
'
,.
¢.%
F
2
Figure 2.
,
'"
Angiograph 48 hours after infarct. Trasyloltreated d 0 g w i t h infarct s i z e 8.2% of heart mass. Good preservation of ithe idellcate cor0nary':artery branches (ar'r~s i n d i c a t e ligated~ coronary a r t e ~ branches).
branches of the coronary vessels in the border zone of the damaged area in the control dogs. vascularization, Fig. i.
The histologic picture indicated poor Coronary artery angiograms from Trasylol-
treated dogs revealed a normal coronarypattern within the infarct border zone up to 24 hours after infarction, Fig. 2. Histologic examination of thedamaged area indicated the typical pattern of myocardial infarction.
Necrotic areas developed
within the cardiac mass, the myocardial cells lost their nuclei, and the striations had become indistinct within the homogenous
Pharmacological-Research Communications,~ Voi. 9, No,. 1; 1977 cytoplasm~~ Fig. 3.
Twelve hours after the start of the experiment,
a fully developed_pa~£ern of infarction was established. J
.... :
.J
/
85
The
,.
pattern xn tgefcenterTo~ the!damaged area surrounded byathe !igat~res. did not differ:betweehsthe two groups of animals. However, distinct ;hSSt01ogi6 differences were observed in the border-zon%s of the damaged areas.
The hearts from the Trasylol-
treated dogs exhibited-intense reactive hyperemia Within that zone 12 hours after the insult;~: The infarcted area was seperated from the healthy heart ,muscle by an interstitial zone showing leukocyte reaction. I n
contrast~,-specimens from the control group showed
no hyperemia in that zone and only a light leukocyte-infiltration in the damaged-~rea.
The infarct, however, had spread centrifugally
beyond the ligatures into the heart muscle.
In these specimens,
the_border zone was characterized by a typical myocardial cell pattern~indluding hyaline "transverse striations" (Poche and Ohm, .J
1963; Meesan and Poche, 1963) or contraction bonds arranged in a transverse direction to the longitudinal axis of the cells (Krug, 1972).
In addition, interstitial edema was found which, like the
myocardial cells, contained PAS-positive, diastase resistant, metachromatic granules (Yokoyama, et al., 1955; Shnitka and Nachlas, 1963; Bajusz and Jasmin, 1964; Krug, 1972).
The eosin stainability
was reduced typically due to the increased pH in the edema fluid (Krug, 1972). Microscopic examination of the border zone in hearts from control anlmals revealed an interstitial edema rich in proteins. This edema surrounded the vessels and myocardial cells, Fig. 4. The capillary endothelium was fragmented partlally, and the endothelial cells were swollen with enlarged, edematous nuclel. walls of the larger vessels generally appeared "spliced".
The In
B6
Pharmacologica/ Research Communications, VoL 9, No, L 1977
Figure 3. Infarct center from the left ventricle, 48 hours after infarct. Necr0ticmuscle cells 'showinghomogenous cytoplasm, loss of striationsi,iand ldisintegrated nuclei, pyknosis of interstitial cell nuclei~f coagulation necrosis pattern due to acidosis.
Figure 4. Infarct border zone, from the left ventricle, 48 hours after infarct with beginning lqss of striations, appearance of contraction bands (arrows), extensive edeme(0) and largely destroyed arteriole (A), pyknosis of vascularand interstitial cell nuclei, i000 X.
O,harmacolo rical,,Research.CommunicaNons Vol.gsNo. the capilli~ry net:work,, endothelialswelling,
1977
Inpart,
87
was, so
massive thlst' thee-lumen of/ithese/small~'.vessels was llargely occluded.
D,zscuss oN, .The protelnase inhibitor Trasyl01. prol0nged",,the survival time~ in 4,bgs,,subJected-t 0 acute myoC;ardla~Tinfarctig~ involving more, ths',n10% of,' the cardiac', musci~, mass;:::,,Ang!ographlic ,,evidence showed~i:'that/Trasylol; provided,,'pro tectz0n ? for .'the minu'=e branches , ~arteries'wi'thin jthe,~border' zone of the infarcted of thin-'coronary ~ ~~ area~
Thus ',the ~blood Supply to. t~e, t!ss~ue ,in the immediate
vicinity of, the.'infarct remalned',n0rmal. Examination of 'the,border zone, differences between control and ,drug-treated dogs permit~us', to ~ conclude ,that Trasylol ,does not alter the degeneration, of, the heart',muscle within the hypoxic or anoxic zone.
Rather, the blood supply is ensured in the border
zone of the damaged area due to the preservation of the minute coronary artery branches.
Because of this preservation,
the
infarct size is limited to the actual center. The question arises as to the events taking place in the border zone, and why the infarct does not spread in a diffuse manner beyond its actual center into the surrounding tissue. Evidence in this study permits consideration of the manner by which certain processes, normally leading to the destruction of cells and tissue, takes place in an area of tissue no longer anoxic in character.
Microscopic examination of the border zones showed
protein-rlch interstitial edema with endothelial swelling in the capillary network.
It appears that changes occurlng within the
thebeslan system of veins were important (Wearn, 1928).
The
veins represent an interstitial system in the heart muscle which communicates with the capillarle~ arterioles, and venules (Heine,
Pharmacologica/Research .Communications, Vol. 9, No. 1, 7977
88 et al., 1973).
These veins danube traced, back to the trabecular
fissures, 'which, 19 the' embryonlc heart muscle, 'proceed to a ,level Just below,the ~urface O f the heart.
As'a contlnuatlon of ~ the
endocardium, this particular '!venous~system" rests directly~, on the:myocardial cells, ~eing separated' from'the sarcolemma only by t
a very delicate endothelial laYer~ with an underlying basal membranu. In the case of .injurious processes1,,these '..'weak-~walled"vessels, •
"
•
are affected first.
,
1
Since they, on the one hand, a r e located in J
the immediate neighborhood of the myocardia ! .cells, and, o n the other hand, :communicate with the capillary system of the coronary circulation, there can b e little doubt that these veins are of importance for the nutrition of the myocardial cells. It has been known for a long time that the heart is kable to function without disturbance following gradual occlusion of both coronary ostia (Crainlcianu, 1922; Wea'rn, et al., 1933).
Data
from these experimental infarction studies permit the conclusion that tissue electrolytes dan pass even from the infarcted area into the vascular system of the heart by way of the thebesi~n veins as long as these veins are not destroyed as yet. As mentioned, changes occur in the vicinity of the infarct beyond the area actually damaged by anoxia, changes leading to the destruction of myocardial cells.
The particular injury consists
of a traumatization of both the capillary system and the "transmitter stretch".
The cytological and hlstochemlcal changes occuring in
the myocardial cells following anoxia have been studied in great detail (Meesen and Poch, 1963; Buchner, et al,, 1970).
However,
little consideration has been given, in general, to the fact that
d i s t u r b a n c e of the m e t a b o l i c pathways s u p p l y i n g the m y o c a r d i a l c e l l s must precede the hypoxic i n j u r y to the c e l l
(Hauss, e t a l . ,
Pharmacological Research Communications,,-Vol. :9, No.~ 1,..1977 1968).
89
In t h ~ connection, Hauss and :co-workers (1968)have
drawn attention to the importance of the interstitial space between the capillary system and the myocardial cell.
Thls space6 con-
structed of connective ~tissue cells and high .polymer mucopolysaccharide-proteln complexes produced b y the connective tissue cells, is of;decisive importance for the metabolism proceeding between the capillary wall and myocardial cells~. Any prolonged disturbance in the composition, syntheslsor degradation of the proteoglycans along the "transmitter stretch" (Hauss and JungeHulsing, 1963) leads to injury of the myocardial cell.
Brief
e
disturbances, however, can be balanced out at the local level (Hauss, et al., 1968). In the histological preparations presented, disturbance of the "transmitter stretch" already is revealed before any injury of the myocardial cell can be detected by light microscopy.
Under
normal conditions, the connective tissue cells (fibroblasts and mast cells) mediate between the vascular wall and the myocardial cell (Heine and Forster, 1974) or directly between the myocardial cells respectively.
Under hypoxemic conditions~ however, the
substances normally mediated'are subjected to degradation.
The
metachromatic, diastase-resistant and PAS-positive granules (Krug, 1972), which flood the interstitial space, represent the morphological expression of the disturbed metabolism in the matrix or the rates of synthesis and degradation within the matrix, ~ respectively. These granules disappear again following complete development of the infarct with appearance of edema and loss of myocardial cell striations. Thus, the "transmitter stretch" responds to noxae of different types, as oxygen deficiency, by modifying the production and/or
90
Pharmacological Research Communications, Vol. 9, No..1, ~1977
degradation of the pro teoglycans.
If the noxa i~n question exerts
prolonged action, there first occurs depolymerlzation of~ the !
mucopolysaccharlde-protein complexes present in ~the basal membrane of both the capillary system and the "transmitter stretch".
The
molecular sieve effect of~the basal membrane (Buddecke, 1 9 6 5 ) is abolished by the depolymerizati0n, and large protelns, as 7-globulins, are able to penetrate into the interstitial space, leading ultimately to the destruction of the "transmitter space". T h i s
leads to the
failure of metabolic regulatory mechanisms both tc and from the myocardial cell.
Destructi0n of the "transmitter stretch" finally
may lead to the destruction of the myocardial cell due to hypoxidosis, although true anoxia due to disturbed blood flow no longer exists. In the border zone of the infarcted area, the latter events lead to enlargement of the necrotized area around, the infarct which is not caused by general tissue anoxia since the present experimental observations show that the border zone expanded to a considerable extent beyond the area surrounded by the purse-string ligatures.
In answering the question as to the noxae possibly leading to injury of the "transmitter stretch", consideration should be given to the severe changes in the area of the capillaries associated with edema of the endothelial cells and hypoxidotic changes in the myocardial cells that have been described following administration of excessive doses of cortisone, •aldosterone, and administration of metaproterenol (Poche, 1970).
At present~ there can be
little doubt that there exists a series of other substances capable of ellcltlng injury to Both the capillary system and the "transmitter stretch" in the absence of anoxla.
The changes observed
along the "transmitter stretch" are identical to those seen hlstomorphologically in shock caused not only by hypoxla, but also by
PharrnacologicaI,Research Communications, Vol: 9, No. 1, 1977 toxic agents, notably the denatured ~protein bodies (Clowes, 1973 ; Nemir, et al., 1973).
The tissue proteinases appearing in excess
as well as liberated lysosomal enzymes also deserve particular attention.
Injury to the capillary system during shock can be
prevented largely by the pro teinase inhibitor Trasyl01. Experiments in the post-ischemic dog lung and rabbit mesenterium after ischemia have~ demonstrated that ~Trasylol •protects the integrity of the capillary wall (endothelium and light-optical basal membrane)as well as the composition of the basal substance. Swelling of the endothelial cells and breakdown of the permeability r
barrier of the .capillary wall are avoided largely due to this protection. Thus, it can be assumed that the spread of the myocardial infarct into the border zone is prevented by the protective effect of Trasylol o n the "transmitter stretch".
Most likely diffusion
of denatured proteins takes place from the center of the infarcted area into the infarcted border zone, eliciting both depolymerization of the "transmitter stretch" and injury of the capillary system.
The normal exchange processes between capillaries and
myocardial cells remain intact under the effect of Trasylol, and thus the heart muscle cells are not subjected to hypoxidosis. These considerations provide an explanation as to the possible mechanism of the increased survival rate and time in the Trasyloltreated dogs following induction of acute myocardial infarction. By maintaining the function of both the capillary system and "transmitter stretch" within the border zone of the infarcted area, it is possible to limit the infarct to the actual infarct nucleus area.
It is suggested that prompt institution of this
91
92
Pharmacological Research Communications, t/ol. 9, No. 1, 1977
therapeutic measure may improve survival chances of patients suffering acute myocardial infarction.
REFERENCES Bajusz, E., and G. Jasmin: Acta histochem.
18; 222,
(1964).
Buchner, F. : Die Koronarinsuffizienz in alter und neuer S icht. Forum cardiologicum (special issue), Mannheim, Boehringer 1970. Buddecke, E.: Regensburg, J.B. arztl. Fortbildung 13; 189,
(1965).
Clowes, Jr., G.H.A. : New Aspects of Trasylol Therapy 6; 71, (1973), F.K. Schatt~:.ler Verlag, Stuttgart - New York. Crainicianu, A.: Virchows Arch. path. Anat. 238; I, (1922). Hauss, W.H., and G. Junge-Hulsing:
Med. Welt 3; 125,
(1963).
Hause, W.H., G. Junge-Hulsing and O. Gerlach: Die unspezifishen Mesenchym-Reaktionen. Stuttgart, Thieme 1968. Heine, H., F. Tschirkov and D. Manz: Klin. Wschr. 51; 191, Hein, H., and F. J. Forster: Acta anat. 89; 387, Xrug, A.: Arch. Kreisl.-Forsch.
(1973).
(1974).
67; 326, (1972).
Meesen, H., and R. Poche: in_nDas Herz des Menschen (Bargmann, W., and W. Doerr Eds.) Vol. 2, pp 644-734, Stuttgart, G. Thieme 1963. Nemir, Jr., P., W.M. Hamilton, F. Habboushe and J.l. Brody: New Aspects of Trasylol Therapy 6; 149, (1973) F.K. Schattauer Verlag, Stuttgart - New York. Poche, R., and H. G. Ohm: Arch. Kreisl.-Forsch.
41; 86, (1963).
Poche, R.: Forum cardiologicum 13; 27, (1970) Mannheim, Boehringer GmbH. Shnitka, T.K., and N.M. Nachlas: Am. J. Path. 42; 507,
(1963).
Tschirkov, F., J. Greifenstein, J. Eisenbach, P. Rottger and H. Heine: Part i, Med. Welt 23; 1838, (1972); Part 2, Med. Welt 24; 127, (1973). Wearn, J.T., S.R. Mettier, T.G. Klumpp and L. J. Zsieche: Am. Heart J. 9; 143, (1933). Wearn, J.T. : J. exp. reed. 47; 293, (1928). Yokoyama, H.O., R.B. Jennings, G.F. Clabauch and W.B. Wartmann: Arch. path. 59; 347, (1955).
79
EFFECT OF THE PROTEINASE INHIBITOR TRASYLOL ~R'(~ ON THE MORTALITY AND INFARCT SIZE IN DOGS WITH ACUTE MYOCARDIAL INFARCTION.
J. Eisenbach* and H. Heine**, Surgical Center, University of Frankfurt* and the Center of Morphology, Frankfurt, Main, Federal Republic of Germany** Received 15 March 1976
SUMMARY
(R)
The proteinase inhibitor Trasylol
was stud%ed for its
effect on the mortality and infarct size in dogs with experimentallyinduced acute myocardial infarction.
The infarction was induced
in the anesthetized dog by means of deep over-lapping pursestring ligatures into the left ventricle, and the infarct size determined both by ultra-violet technique following injection of fluorescein and by post-mortem coronary artery angiograms.
A
total of 45 dogs was studied with approximately half receiving 500,000 units Trasylol intravenously Just prior to infarct induction and 10,000 units/kg intravenously two hours later and thereafter every six hours until death.
Trasylol was found to
increase significantly both survival time and survival rate in those animals with an infarct size in excess of 10% of the heart mass.
While animals in both control and drug-treated groups
experienced circulatory shock, the severity of the circulatory disturbance was distinctly less in the Trasylol-treated animals. Ventrlcular fibrillation in drug-treated dogs also appeared much later compared to control dogs.
Coronary artery angiographic
Pharmacological Research,Communications,, Vol. 9, No. 1, 1977
80
examination in control dogs showed degeneration of minute peripheral branches of the coronary vessels in the border zone of the infarct, whereas a normal coronary pattern within the infarct border zone was seen in the drug-treated group.
Distinct histologlc
differences in the border zone were noted in the drug-treated dogs compared to the controls.
The damaged areas of the hearts from
the drug-treated group exhibited an intense reactive hyperemia not seen in the controls. control dogs.
The infarct size was not contained in the
Other morphological differences were seen which
could account for the differenc~ in survival rate.
The significance
of these effects as related to cellular and biochemical
(enzyme)
changes are discussed. INTRODUCTION At present, myocardial infarction represents one of the m o s t frequent causes of death among disorders of the heart. the accumulation of an immense body of literature,
Despite
new questions
regarding fundamental aspects of the pathophysiology,
histo-
morphology and therapy of this myocardial injury appear to be the center of medical interest. Tschirkov,
et al. (1972, 1973) recently presented results of
studies on experimentally-induced
myocardial infarction in dogs
which gave rise to this present study. METHODS Certain areas of the left ventricle in 45 healthy mongrel dogs were made ischemic by means of deep overlapping purse-strlng ligatures
(Tschirkov, 1972, 1973).
The myocardial infarction
model does allow investigation and interpretation of fundamental events occuring within the center area and border zone of the infarct.
Pharmacological Research Communications, Vol.. 9, No.: 1, 1977
81
The extent of infarctwas determined by ultra-violet light technique following injection of fluorescin.
Additionally, post-
morton coronary artery angiograms were prepared by the injection of a bolus of trilead tetr0xlde and gelatin into the coronary arteries.
The following parameters were recorded continuously or
determined at various time intervals during the course of the experiment : electrocardiogram, aorta and left ventricular pressure, coronary vein and artery gas analysis, electrolyte s in coronary vein and arterial blood and in the infarcted central and border zones, and hist0Pathology.
All tissue preparations were fixed in
formalin, ~%, glycol methacrylate, and toluidine blue (pH 5.5). Section thickness was one micron.
Only the pressur~ and hlstologic
data will be presented in this report. T h e animals were divided into two groups; Group 0 received no treatment while Group T received the proteinase inhibitor Trasylol
i
, 500,000 units intravenously just prior to infarct
induction and i0,000 units/kg intravenously two hours after infarct and thereafter every six hours until death. RESULTS Table i summarizes the survival rate in relation to infarct size.
Both survival time and survival rate appeared closely re-
lated to infarct size.
Animals with infarct size less than 10%
of the heart mass survived up to the time of sacrifice in both the control and drug-treated group.
As the infarct size increased,
the mean survival time decreased in both groups of animals. in those groups where the infarct size was greater than i0%, TrasyloZ significantly prolonged survival time.
1.
Traaylol (R) product of Bayer AG, Leverkusen~ West Germany
However,
Infarct > 15% of heart mass
Group III : 4
1
i0
7
No. of dogs
40 hours
1".7-+ 0.3
Survived
21 -+3.0
All dogs survived
GROUP 0
Survived 18 hours
1
4.0
6.2 -+ 0.3
Survived
31.2+
~ii dogs survived
GROUP T
3
4
8
7
No. of dogs
ANI~gL SURVIVAL TIME (IN HOURS) RELATIVE TO INFARCT SIZE.
Infarct size between I0 and 15% of heart mass
Group II:
Infarct < i0% of heart mass
group, I :
Infarct size
TABLE i.
"~a
~a
CD b~
Pharmacological Research Communications, VoL 9; No, 1, 1977
Figure i.
83
Coronary artery angiograph 48 hours after infarct. Untreated dog, with infarct size 9.4% of heart mass.
Continuous recording of aortic blood pressure revealed that cardiogenlc shock occured in all animals in which the infarct size exceeded 1 0 % of the heart mass. leading to death.
The shock increased in severity
In these animals, the circulatory disturbance
was distinctly less pronounced in the drug-treated dogs.
In the
control animals, ventrlcular fibrillation generally appeared two hours after Infarct induction.
In the Trasylol-treated dogs,
fibrillation appeared 2 - 18 hours later. Coronary artery angiographic examination carried out 12 hours after infarction revealed degeneration of minute peripheral
Pharmacological Research Communications, Vol. 9, No, 1-, 1977
84
~
~
~
.~,p.,,
N..
''
I
t.!.
.
:
-..
,
'
,.
¢.%
F
2
Figure 2.
,
'"
Angiograph 48 hours after infarct. Trasyloltreated d 0 g w i t h infarct s i z e 8.2% of heart mass. Good preservation of ithe idellcate cor0nary':artery branches (ar'r~s i n d i c a t e ligated~ coronary a r t e ~ branches).
branches of the coronary vessels in the border zone of the damaged area in the control dogs. vascularization, Fig. i.
The histologic picture indicated poor Coronary artery angiograms from Trasylol-
treated dogs revealed a normal coronarypattern within the infarct border zone up to 24 hours after infarction, Fig. 2. Histologic examination of thedamaged area indicated the typical pattern of myocardial infarction.
Necrotic areas developed
within the cardiac mass, the myocardial cells lost their nuclei, and the striations had become indistinct within the homogenous
Pharmacological-Research Communications,~ Voi. 9, No,. 1; 1977 cytoplasm~~ Fig. 3.
Twelve hours after the start of the experiment,
a fully developed_pa~£ern of infarction was established. J
.... :
.J
/
85
The
,.
pattern xn tgefcenterTo~ the!damaged area surrounded byathe !igat~res. did not differ:betweehsthe two groups of animals. However, distinct ;hSSt01ogi6 differences were observed in the border-zon%s of the damaged areas.
The hearts from the Trasylol-
treated dogs exhibited-intense reactive hyperemia Within that zone 12 hours after the insult;~: The infarcted area was seperated from the healthy heart ,muscle by an interstitial zone showing leukocyte reaction. I n
contrast~,-specimens from the control group showed
no hyperemia in that zone and only a light leukocyte-infiltration in the damaged-~rea.
The infarct, however, had spread centrifugally
beyond the ligatures into the heart muscle.
In these specimens,
the_border zone was characterized by a typical myocardial cell pattern~indluding hyaline "transverse striations" (Poche and Ohm, .J
1963; Meesan and Poche, 1963) or contraction bonds arranged in a transverse direction to the longitudinal axis of the cells (Krug, 1972).
In addition, interstitial edema was found which, like the
myocardial cells, contained PAS-positive, diastase resistant, metachromatic granules (Yokoyama, et al., 1955; Shnitka and Nachlas, 1963; Bajusz and Jasmin, 1964; Krug, 1972).
The eosin stainability
was reduced typically due to the increased pH in the edema fluid (Krug, 1972). Microscopic examination of the border zone in hearts from control anlmals revealed an interstitial edema rich in proteins. This edema surrounded the vessels and myocardial cells, Fig. 4. The capillary endothelium was fragmented partlally, and the endothelial cells were swollen with enlarged, edematous nuclel. walls of the larger vessels generally appeared "spliced".
The In
B6
Pharmacologica/ Research Communications, VoL 9, No, L 1977
Figure 3. Infarct center from the left ventricle, 48 hours after infarct. Necr0ticmuscle cells 'showinghomogenous cytoplasm, loss of striationsi,iand ldisintegrated nuclei, pyknosis of interstitial cell nuclei~f coagulation necrosis pattern due to acidosis.
Figure 4. Infarct border zone, from the left ventricle, 48 hours after infarct with beginning lqss of striations, appearance of contraction bands (arrows), extensive edeme(0) and largely destroyed arteriole (A), pyknosis of vascularand interstitial cell nuclei, i000 X.
O,harmacolo rical,,Research.CommunicaNons Vol.gsNo. the capilli~ry net:work,, endothelialswelling,
1977
Inpart,
87
was, so
massive thlst' thee-lumen of/ithese/small~'.vessels was llargely occluded.
D,zscuss oN, .The protelnase inhibitor Trasyl01. prol0nged",,the survival time~ in 4,bgs,,subJected-t 0 acute myoC;ardla~Tinfarctig~ involving more, ths',n10% of,' the cardiac', musci~, mass;:::,,Ang!ographlic ,,evidence showed~i:'that/Trasylol; provided,,'pro tectz0n ? for .'the minu'=e branches , ~arteries'wi'thin jthe,~border' zone of the infarcted of thin-'coronary ~ ~~ area~
Thus ',the ~blood Supply to. t~e, t!ss~ue ,in the immediate
vicinity of, the.'infarct remalned',n0rmal. Examination of 'the,border zone, differences between control and ,drug-treated dogs permit~us', to ~ conclude ,that Trasylol ,does not alter the degeneration, of, the heart',muscle within the hypoxic or anoxic zone.
Rather, the blood supply is ensured in the border
zone of the damaged area due to the preservation of the minute coronary artery branches.
Because of this preservation,
the
infarct size is limited to the actual center. The question arises as to the events taking place in the border zone, and why the infarct does not spread in a diffuse manner beyond its actual center into the surrounding tissue. Evidence in this study permits consideration of the manner by which certain processes, normally leading to the destruction of cells and tissue, takes place in an area of tissue no longer anoxic in character.
Microscopic examination of the border zones showed
protein-rlch interstitial edema with endothelial swelling in the capillary network.
It appears that changes occurlng within the
thebeslan system of veins were important (Wearn, 1928).
The
veins represent an interstitial system in the heart muscle which communicates with the capillarle~ arterioles, and venules (Heine,
Pharmacologica/Research .Communications, Vol. 9, No. 1, 7977
88 et al., 1973).
These veins danube traced, back to the trabecular
fissures, 'which, 19 the' embryonlc heart muscle, 'proceed to a ,level Just below,the ~urface O f the heart.
As'a contlnuatlon of ~ the
endocardium, this particular '!venous~system" rests directly~, on the:myocardial cells, ~eing separated' from'the sarcolemma only by t
a very delicate endothelial laYer~ with an underlying basal membranu. In the case of .injurious processes1,,these '..'weak-~walled"vessels, •
"
•
are affected first.
,
1
Since they, on the one hand, a r e located in J
the immediate neighborhood of the myocardia ! .cells, and, o n the other hand, :communicate with the capillary system of the coronary circulation, there can b e little doubt that these veins are of importance for the nutrition of the myocardial cells. It has been known for a long time that the heart is kable to function without disturbance following gradual occlusion of both coronary ostia (Crainlcianu, 1922; Wea'rn, et al., 1933).
Data
from these experimental infarction studies permit the conclusion that tissue electrolytes dan pass even from the infarcted area into the vascular system of the heart by way of the thebesi~n veins as long as these veins are not destroyed as yet. As mentioned, changes occur in the vicinity of the infarct beyond the area actually damaged by anoxia, changes leading to the destruction of myocardial cells.
The particular injury consists
of a traumatization of both the capillary system and the "transmitter stretch".
The cytological and hlstochemlcal changes occuring in
the myocardial cells following anoxia have been studied in great detail (Meesen and Poch, 1963; Buchner, et al,, 1970).
However,
little consideration has been given, in general, to the fact that
d i s t u r b a n c e of the m e t a b o l i c pathways s u p p l y i n g the m y o c a r d i a l c e l l s must precede the hypoxic i n j u r y to the c e l l
(Hauss, e t a l . ,
Pharmacological Research Communications,,-Vol. :9, No.~ 1,..1977 1968).
89
In t h ~ connection, Hauss and :co-workers (1968)have
drawn attention to the importance of the interstitial space between the capillary system and the myocardial cell.
Thls space6 con-
structed of connective ~tissue cells and high .polymer mucopolysaccharide-proteln complexes produced b y the connective tissue cells, is of;decisive importance for the metabolism proceeding between the capillary wall and myocardial cells~. Any prolonged disturbance in the composition, syntheslsor degradation of the proteoglycans along the "transmitter stretch" (Hauss and JungeHulsing, 1963) leads to injury of the myocardial cell.
Brief
e
disturbances, however, can be balanced out at the local level (Hauss, et al., 1968). In the histological preparations presented, disturbance of the "transmitter stretch" already is revealed before any injury of the myocardial cell can be detected by light microscopy.
Under
normal conditions, the connective tissue cells (fibroblasts and mast cells) mediate between the vascular wall and the myocardial cell (Heine and Forster, 1974) or directly between the myocardial cells respectively.
Under hypoxemic conditions~ however, the
substances normally mediated'are subjected to degradation.
The
metachromatic, diastase-resistant and PAS-positive granules (Krug, 1972), which flood the interstitial space, represent the morphological expression of the disturbed metabolism in the matrix or the rates of synthesis and degradation within the matrix, ~ respectively. These granules disappear again following complete development of the infarct with appearance of edema and loss of myocardial cell striations. Thus, the "transmitter stretch" responds to noxae of different types, as oxygen deficiency, by modifying the production and/or
90
Pharmacological Research Communications, Vol. 9, No..1, ~1977
degradation of the pro teoglycans.
If the noxa i~n question exerts
prolonged action, there first occurs depolymerlzation of~ the !
mucopolysaccharlde-protein complexes present in ~the basal membrane of both the capillary system and the "transmitter stretch".
The
molecular sieve effect of~the basal membrane (Buddecke, 1 9 6 5 ) is abolished by the depolymerizati0n, and large protelns, as 7-globulins, are able to penetrate into the interstitial space, leading ultimately to the destruction of the "transmitter space". T h i s
leads to the
failure of metabolic regulatory mechanisms both tc and from the myocardial cell.
Destructi0n of the "transmitter stretch" finally
may lead to the destruction of the myocardial cell due to hypoxidosis, although true anoxia due to disturbed blood flow no longer exists. In the border zone of the infarcted area, the latter events lead to enlargement of the necrotized area around, the infarct which is not caused by general tissue anoxia since the present experimental observations show that the border zone expanded to a considerable extent beyond the area surrounded by the purse-string ligatures.
In answering the question as to the noxae possibly leading to injury of the "transmitter stretch", consideration should be given to the severe changes in the area of the capillaries associated with edema of the endothelial cells and hypoxidotic changes in the myocardial cells that have been described following administration of excessive doses of cortisone, •aldosterone, and administration of metaproterenol (Poche, 1970).
At present~ there can be
little doubt that there exists a series of other substances capable of ellcltlng injury to Both the capillary system and the "transmitter stretch" in the absence of anoxla.
The changes observed
along the "transmitter stretch" are identical to those seen hlstomorphologically in shock caused not only by hypoxla, but also by
PharrnacologicaI,Research Communications, Vol: 9, No. 1, 1977 toxic agents, notably the denatured ~protein bodies (Clowes, 1973 ; Nemir, et al., 1973).
The tissue proteinases appearing in excess
as well as liberated lysosomal enzymes also deserve particular attention.
Injury to the capillary system during shock can be
prevented largely by the pro teinase inhibitor Trasyl01. Experiments in the post-ischemic dog lung and rabbit mesenterium after ischemia have~ demonstrated that ~Trasylol •protects the integrity of the capillary wall (endothelium and light-optical basal membrane)as well as the composition of the basal substance. Swelling of the endothelial cells and breakdown of the permeability r
barrier of the .capillary wall are avoided largely due to this protection. Thus, it can be assumed that the spread of the myocardial infarct into the border zone is prevented by the protective effect of Trasylol o n the "transmitter stretch".
Most likely diffusion
of denatured proteins takes place from the center of the infarcted area into the infarcted border zone, eliciting both depolymerization of the "transmitter stretch" and injury of the capillary system.
The normal exchange processes between capillaries and
myocardial cells remain intact under the effect of Trasylol, and thus the heart muscle cells are not subjected to hypoxidosis. These considerations provide an explanation as to the possible mechanism of the increased survival rate and time in the Trasyloltreated dogs following induction of acute myocardial infarction. By maintaining the function of both the capillary system and "transmitter stretch" within the border zone of the infarcted area, it is possible to limit the infarct to the actual infarct nucleus area.
It is suggested that prompt institution of this
91
92
Pharmacological Research Communications, t/ol. 9, No. 1, 1977
therapeutic measure may improve survival chances of patients suffering acute myocardial infarction.
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Clowes, Jr., G.H.A. : New Aspects of Trasylol Therapy 6; 71, (1973), F.K. Schatt~:.ler Verlag, Stuttgart - New York. Crainicianu, A.: Virchows Arch. path. Anat. 238; I, (1922). Hauss, W.H., and G. Junge-Hulsing:
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