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Morphometric evaluation of volume shifts between intra- and extra-cellular space before and during global ischemia.

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THE ANATOMICAL RECORD 241:319-327 (1995)
Morphometric Evaluation of Volume Shifts Between Intra- and
Extra-Cellular Space Before and During Global Ischemia
A. SCHMIEDL, G. HAASIS, PH.A. SCHNABEL, M.M. GEBHARD, AND J. RICHTER
Department of Anatomy, Division of Electron Microscopy (A.S., J.R.), Department of
Physiology, Division of Vegetative Physiology and Pathophysiology (G.H., M.M.G.),
University of Gottingen, Gottingen, and Departments of Pathology (PH.A.S.)and
Experimental Surgery (M.M.G.), University of Heidelberg, Heidelberg, Germany
ABSTRACT
Background: It is well known that all forms of cardiac arrest lead to global ischemia combined with alterations in cellular and interstitial volume. The aim of this study was to investigate the nature of
these alterations with respect to different methods of cardiac arrest and
establish the extent of their mutual influence at the onset as well as during
the course of global ischemia.
Methods: Three tested clinical methods were employed to induce cardiac
arrest by a) aortic cross clamping, b) coronary perfusion with the cardioplegic solution St. Thomas, and c ) coronary perfusion with the cardioplegic solution histidine-tryptophane-ketoglutarate(HTK).The arrested
hearts were subjected to global ischemia at 25°C. The size of the myocytes,
as well as the interstitial space of myocytes, was determined morphometrically. The contraction state of myocytes was evaluated according to a
score.
Results: We found that the degree of contraction, as well as nature of
alterations in the cellular and interstitial volumes, depended both on the
form of cardiac arrest and on the duration of ischemia. The following relationships were established. High contraction at the onset of ischemia
leads to expulsion of fluid from the interstitium between bundles of myocytes into the tissue clefts increasing their size. The decrease in contraction
during ischemia leads to narrower tissue clefts. Cellular swelling at the
onset of and during ischemia is caused by volume shifts between intracellular and interstitial space. An increase in cellular volume during global
ischemia and/or additional contraction reduce the interstitium within bundles of myocytes. Sufficient relaxation and/or interstitial edema enlarge the
interstitium.
Conclusions: Cellular and interstitial alterations seen at the onset and
during the course of ischemia are dependent upon the method of cardiac
arrest. Furthermore, a considerable mutual influence is exerted by the alterations in cellular and interstitial spaces. o 1995 WiIey-Liss, Inc.
Key words: Heart, Dog, Cardioplegia, Cardiac arrest, Ischemia, Morphometry, Interstitial space, Contraction state
Myocardial cells show physiological alterations of
their volume and contraction state during the cardiac
cycle (Caulfield and Borg 1979; Borg and Caulfield
1981). On the other hand, cardiac arrest and the resulting global ischemia lead to defined pathological cellular alterations. Mitochondria show loss of their matrix and cristae, as well a s swelling, cellular edema,
and alterations in the contraction state (Katz, 1973;
Hearse et al., 1977; Pine et al., 1979; Poole-Wilson et
al., 1979; Jennings and Reimer, 1981; Tranum-Jensen
et al., 1981; Vanderwee et al., 1981; Schmiedl et al.,
1989; Schnabel et al., 1990).The occurrence and degree
of alterations depend on the method used for cardiac
arrest (Bretschneider, 1964; Paulussen et al., 1968;
0 1995 WILEY-LISS, INC
Schnabel et al., 1987, 1990; Gebhard et al., 1987;
Schmiedl et al., 1990b).
Different procedures can be performed to introduce
cardiac arrest, resulting in a n interruption of blood
flow, and, therefore, in a global myocardial ischemia.
After inflow occlusion by cross clamping of the venae
cavae and additionally the aorta, the heart begins to
fibrillate. The reduction of its creatine phosphate level
Received January 31, 1994; accepted September 15, 1994.
Address reprint requests to Dr. rer. nat. Andreas Schmiedl, Zentrum Anatomie, Kreuzbergring 36, 3400 Gottingen, Germany.
320
A. SCHMIEDL E T AL.
leads to cardiac arrest in systole, combined with a modest ischemic tolerance and a n early occurrence of cellular alterations (Bretschneider, 1964; Paulussen et
al., 1968). Hypothermia and introduction of cardiac arrest by coronary perfusion with cardioplegic electrolyte
solutions leads to a cardiac arrest in diastole. The specific electrolyte composition of these cardioplegic solutions (see Materials and Methods) leads to a cardiac
arrest within seconds without a phase of fibrillating.
Here, creatine phosphate is not used up. The decay of
energy rich phosphates and the cellular alterations
during ischemia take place in slow motion (Paulussen
et al., 1968). Therefore, myocardial protection with cardioplegia leads to significantly better tolerance to ischemic stress (Bretschneider, 1964; Gebhard et al., 1987).
The volume of the interstitial space after cardiac arrest and during global ischemia may be influenced in a
variety of ways. Not only a n interstitial edema but also
a n increase in contraction, as well as swelling of myocytes, for example, can alter the interstitium (PooleWilson et al., 1979; Borg and Caulfield, 1981; Schmiedl
e t al., 1989).
Therefore, for a precise evaluation of interstitial and
intracellular alterations and possible mutual inf luences, both compartments should be investigated in the
same sample. Morphometric methods allow a reproducible, exact definition of all parameters necessary to
evaluate these alterations. Whereas the use of extracellular tracers leads to a remarkable variability of
values (Polimeni, 1974; Reil et al., 1987), the size of the
interstitial space should be defined morphometrically
as discussed earlier (Schmiedl et al., 1991). For morphometry, i t was useful to separate the myocardial interstitial space into two types of reaction spaces, which
were evaluated at different magnifications using a
light microscope (Schmiedl et al., 1990a; Haasis et al.,
1991). The first type, known as a tissue cleft (Henle,
1876), serves as a disposal sink during relaxation-contraction cycles (Feneis, 1944). The second one, called
the interstitium within bundles of myocytes, contains
connective tissue and capillaries.
The aim of this study was to i n v e s t i g a t e
gos. The vena cava superior and inferior, as well as the
aorta and the arteria pulmonalis, were taped with tourniquet bands. Cardiac arrest was introduced by aortic
cross clamping (ACC), in which the taped tourniquet
bands were drawn together. For surface cooling, Tutofusin solution (comp. in mmol/L: 140 N a + , 5 K + , 2.5
C a + + , 1.5 M g + + , 153 C1-; Pfrimmer, Erlangen, Germany) was used. After a phase of fibrillation the lack of
creatine phosphate led to a halt of contractile activity.
For coronary perfusion with cardioplegic solutions, a
polyethylene catheter was advanced up to the aortic
valve via the arteria subclavia. Immediately after
tightening of the taped vessels, coronary perfusion was
started using a roller pump. With the onset of the perfusion the aortic valve closed and the perfusat ran into
the coronary system. For aspiration, the right atrium
was opened. Volume relief of the left ventricle followed
by opening the left atrium and by a n incision near the
apex. The following cardioplegic solutions were used:
1. The cardioplegic solution St. Thomas (composition in mmol/L: 91.1 NaC1, 14.8 KC1, 15 MgCl,, 1.2
CaCl,, 25 NaHCO,, 1.2 procaine.HC1, 1.2 MgSO,, 1.2
KH,PO,; Dr. F. Kohler, Alsbach, Germany).
2. The cardioplegic HTK solution (com. in mmol/L:
15 NaC1, 9 KC1, 4 MgCl,, 180 histidine, 18 histidine.HC1, 30 mannitol, 2 tryptophane, 1 K- Ketoglutarate [Custodial@]; Dr. F. Kohler, Alsbach, Germany).
The coronary perfusion with St. Thomas solution
lasted 4 min; the HTK solution, 11min. After this time
complete equilibration of the interstitial space was
achieved. The high K + content of the St. Thomas solution led to a continued depolarisation of the myocytes
and therefore to a n arrest in diastole within seconds.
The reduction of Na+ and Ca2+ to cytoplasmic values in the HTK solution led to a halt of electrical and
mechanical activity and, therefore, to a n arrest in diastole within seconds.
After excision, the hearts were incubated at 25°C in
the solutions used for cardiac arrest. Samples from the
subendocardial third of the free ventricular wall were
taken a t the onset of global ischemia and at predefined
1. the interstitial and cellular alterations after dif- times during global ischemia at 25°C. During tissue
ferent forms of cardiac arrest and following global isch- processing, the incubated ventricle was reduced from
emia,
the apex to the base, until the samples reached a tissue
2. in what way cellular alterations influence the in- concentration of 2 pmol/gww ATP-the
theoretical
terstitial space a t the onset and during the course of limit of resuscitation (Bretschneider, 1964). Lower
ischemia, and
ATP levels did not lead to a resumption of heart func3. the degree of volume shifts in the arrested and tion during reperfusion.
ischemic ventricular myocardium of the heart.
Samples were taken from the aortic cross clamped
hearts over 3 hours every 15 min, from the St. Thomas
MATERIALS AND METHODS
arrested hearts over 4 hours, and from the HTK arThe experiments were carried out on the left ventric- rested hearts over 6 hours in each case, every 30 min.
ular myocardium of canine hearts. Composition and
Each method of cardiac arrest was carried out on 6
application of the cardioplegic solutions corresponded canines, respectively.
to the original clinically accepted prescriptions (Hearse
Immersion fixation of the samples followed in a fixet al., 1981; Gebhard et al., 1987). Anesthesia and ex- ation solution containing 1.5% glutaraldehyde and
perimental procedure were performed as described 1.5% paraformaldehyde in 0.1 M sodium cacodylate
elsewhere (Preusse et al., 1981).In brief, after premed- buffer a t pH 7.4 (half strength-Karnovsky) (Karication the anaesthesia was introduced with Trapanal@ novsky, 1967) with a n effective osmolarity of 440 mosand then continued with a combination of Dipidolor@ mol/L (Hayat, 1989). Postfixation, dehydration, and
and N 2 0 / 0 2(3:l).Respiration was maintained with a n embedding were carried out continuously and automatendotracheal tube connected to a respirator. Median ically (Histomat, Bio-Med, Theres, Germany). Non-orithoracotomy was followed by ligation of the vena azy- ented semithin sections were stained with 1%azure
32 1
VOLUME SHIFTS IN CELLULAR SPACE
Pathological Contraction
TABLE 1. Multistage sampling technique
Parameters
Interstitial space
Tissue clefts between bundles of myocytes
(VVTJ
Interstitium within bundles of myocytes
(VVIS)
Cellular space
Bundles of myocytes
contraction state
Myocytes
Cellular compartments
Myofibrills (VvMf)
Sarcoplasm (VVsJ
Mitochondria (VVMf)
Nuclei (VNc)
Magnification
x 100
x 1,000
x 20000
x 50000
and methylene blue. Ultrathin sections were stained
with uranyl acetate and lead citrate.
Quantitative investigations according to the point
counting method (Weibel, 1979) were carried out on
line. Light microscopical evaluation was carried out by
using a n ocular integrated 100-point lattice test system
(Integrationsplatte, Zeiss, Oberkochen, Germany). For
electron microscopical evaluation a n EM 10 (Zeiss,
Oberkochen) equipped with a TV camera was used. A
72-point lattice test system was mounted on the monitor-screen. Three samples per canine heart and time
point were evaluated. Parameters were determined according to a multistage sampling technique (Table 1).
The volume densities of the interstitial space were
determined in 30 test fields per sample. The cellular
volume densities were determined in 50 test fields per
sample. The cellular volume densities were related to
the myocardial cells as reference unit. The cellular volume was determined by the fraction V V M ~+ VvsPl
VVMf, which is regarded as a parameter of cellular
edema, independent of the reference space (DiBona and
Powell, 1980). VvMf can be related to the bundles of
myocytes of the left ventricular myocardium. Therefore, VVMf was related to l-VvTe, which corresponds to
the bundles of myocytes as reference space. If this parameter remains constant during ischemia, the relative decrease of VVMf is caused by cell swelling and
VVMf is a n additional parameter for cellular edema.
The contraction state of sarcomeres was determined
in 30 test fields according to a classification, described
elsewhere (Schnabel et al. 1990, Schmiedl et al. 1993).
Physiological Contraction
Relaxation
I-bands are always visible.
Contraction
I-bands have generally disappeared; Z-band spacing
is slightly wider than the A-band, visible under physiological conditions.
Relaxationkontraction (incomplete relaxation)
Relaxed and contracted sarcomeres are found in one
test field.
Overcontraction
Submaximal contractions resulting in A-band compression and apparent thickening of Z-bands.
Hypercontraction
Submaximal to maximal contractions, which lead to
A-band compression and additionally to clumping of
actin and myosin filaments.
Contraction band
Maximal shortening of sarcomeres which, in addition to actidmyosin clumping, leads to distortion of the
contractile system. This formation is regarded as irreversible damage (Ganote, 1983). All results are given
as mean values 2 SD. Significant differences were
noted for P values of 0.05 or less using the WilcoxonMann-Whitney test for unpaired samples and the
Wilcoxon matched paired signed rank test for paired
samples.
RESULTS
In the following, alterations of the cellular and interstitial morphometric parameters after different
types of cardiac arrest at the onset as well as during
ischemia are described. Additionally, the values obtained by morphometry of interstitium and myocytes
are compared with each other in order to obtain informations on possible mutual influences.
Onset of Ischemia
In hearts without ischemic stress, the cellular volume and the degree of contraction, as well as the size of
the interstitial space, depend on the form of cardiac
arrest (Figs. 1-4).
Cellular parameters
The quotient VVMi+ Vvsp.NVMfused as a parameter
for cellular edema differed in the cardioplegically arrested hearts. Myocytes of HTK arrested hearts were
significantly less swollen than myocytes of St. Thomas
arrested hearts. The values for the cellular edema of St.
Thomas arrested hearts corresponded to those of cross
clamped hearts (Fig. 2a).
The percentage of test fields with relaxed sarcomeres
was significantly higher after perfusion with HTK solution than after ACC. The relaxation in St. Thomas
arrested hearts was negligible (Fig. 2b). ACC and cardiac arrest with St. Thomas solution led to a remarkable degree of pathological contractions, e.g., over- and
hypercontractions (Fig. 3).
Interstitial parameters and the influence of
cellular parameters
The size of tissue clefts (VVTc)after aortic cross
clamping (ACC) and after coronary perfusion with cardioplegic solutions differed significantly (Fig. 4a). ACC
resulted in a significantly higher VVTccompared with
hearts arrested by coronary perfusion. In St. Thomas
arrested hearts the values for VVTc were somewhat
higher compared with HTK arrested hearts.
The size of the interstitium within bundles of myocytes (VvIs) in cross clamped hearts (Fig. 4b) was significantly reduced by swelling and contraction of myo-
322
A. SCHMIEDL ET AL.
Fig. 1. Interstitium within bundles of myocytes (a; x 960) and myocytes surrounding a capillary (b; x 5,000) after aortic cross clamping. Contracted or overcontracted (arrow) and more or less swollen
myocytes due to, in part, very small interstitial spaces. Interstitium
within bundles of myocytes fc; x 960) and myocytes (d; x 5,000) after
coronary perfusion with St. Thomas solution and 60 min of global
ischemia. Contracted and swollen myocytes do not lead to a significant decrease of the interstitium within bundles of myocytes because
of interstitial edema. Interstitium within bundles of myocytes (e;
x 960) and myocytes (f; X 5,000) after coronary perfusion with HTK
solution and 120 min of ischemia. The swollen, predominantly relaxed
myocytes do not significantly influence the size of the interstitium.
323
VOLUME SHIFTS IN CELLULAR SPACE
O'"
CELLULAR EDEMA
T
T
St. THOMAS
0.6
HTK
0.1
0.2
0
10
0
a
120
60
180
GLOBAL ISCHEMIA AT 2 5 'C (min)
t
RELAXATION (%)
le3
4
I
,
THOMAS
80
I T
68
0 HTK
18
20
0
b
10
m
GLOBAL ISCHEMIA AT 25 'C (min)
Fig. 2. a: Cellular swelling determined by ,
V
+ vvsp / V, after different forms of cardiac arrest and
global ischemia a t 25°C. b Contraction state of sarcomeres (% of test fields) after different forms of
cardiac arrest and global ischemia at 25°C. *Significant alterations (P< 0.05) compared to the onset of
ischemia
cytes compared to that of cardioplegically arrested
hearts (Fig. 2b). In HTK arrested hearts the significantly less pronounced cellular edema and the high
degree of relaxation led to the highest VvIs (Figs. 2a,b;
4b). After St. Thomas perfusion, myocytes showed high
contraction and swelling. However, a n interstitial
edema led to values of VvIs comparable to those after
HTK perfusion (Fig. 4b)
During Ischemia
Cellular parameters
After ACC, the parameter for cellular swelling, the
quotient VVMi + Vvsp /VVMf,increased continuously
during ischemia. After 60 min the cell volume increased significantly compared with the onset of ischemia. After St. Thomas perfusion, as well as after HTK
perfusion, cell volume after 120 min of ischemia was
significantly higher compared to the onset of ischemia
(Fig. 2a). After ACC, test fields with relaxed sarcomeres increased significantly after 60 min of ischemia,
remained constant between 60 and 120 min, and decreased after 180 min of ischemia. After St. Thomas
perfusion, relaxation was significantly pronounced after 60 min of ischemia. However, the percentage of test
fields exhibiting relaxation was significantly lower
compared with the other methods of cardiac arrest in-
324
A. SCHMIEDL ET AL.
PATHOLOGIC CONTRACTION
- ACC
... S t .
Thomas
HTK
creased after 10 min of ischemia. During the following
time VvIs did not alter significantly, because the swelling of myocytes was compensated for by the almost
complete relaxation (Figs. 2a,b; 4b).
Thus, the decrease and constancy of VvIs during global ischemia also depends on the method used for cardiac arrest and even more on the cellular alterations.
Degree of volume shifts at the onset and during ischemia
0
80
120
[min]
Global Ischemia a t 2 5 ° C
Fig. 3. Pathological contraction after different forms of cardiac arrest and global ischemia at 25°C. *Significant decrease (P < 0.05)
compared to the onset of ischemia.
vestigated (Fig. 2b). After HTK perfusion, myocytes
showed a higher degree of relaxation compared with
the other methods of cardiac arrest. In comparison to
the onset of ischemia, relaxation increased significantly after 180 min (Fig. 2b).
Interstitial parameters and the influence of
cellular parameters
After ACC, the decrease of VvTc over 60 min was
related to the significant relaxation (Fig. 2b, 4a). During further ischemic stress the decrease of relaxation
combined with a renewed occurrence of contraction led
to a n increase of VVTowhich was significant after 180
min, compared to the onset of ischemia (Fig. 4a). After
St. Thomas perfusion, the significant decrease of VvTc
over 10 min was related to a decrease of pathological
contraction (Figs. 3,4a) and a decrease of fluid concentration occurring during the equilibration process. The
slight increase of VvTc during the following time of
ischemia was related to a continued high contraction of
myocytes (Figs. 2b, 4a). After HTK perfusion, VvTcexhibited a significant decrease after 60 min, which was
caused by the significant increase of relaxation (Fig.
2b, 4a). In the following ischemic period only slight
alterations occurred.
Thus, the time course of alterations of the size of
tissue clefts differed from method to method used for
cardiac arrest and was influenced by the occurrence of
cellular alterations.
The size of the interstitium within bundles of myocytes (VvI,) after ACC decreased significantly over 60
min and was related to the significant increase in cell
volume (Figs. 2a, 4b). The constancy of VvIs up to 120
min was related to a simultaneous increase in cellular
volume and relaxation. After 180 min, VvIs remained
almost constant, although swelling and contraction of
myocytes significantly increased (Figs. 2a,b; 4b). After
St. Thomas perfusion, VvIs did not significantly alter
during 120 min of ischemia. The significant swelling
combined with a n insufficient relaxation of myocytes
(cf. Fig. 2b) led only after 180 min of ischemia to a
significantly lower VvIs compared to the onset of ischemia. After HTK perfusion, VvIs significantly de-
The volume density of myofibrils (VVMf)was comparable after ACC and St. Thomas perfusion, but significantly lower than after HTK perfusion. During ischemia VVMf significantly decreased after 60 min in the
aortic cross clamped hearts and after 120 min in the St.
Thomas arrested hearts and the HTK arrested hearts
(Fig. 5a).
The relation of VVMf to the bundles of myocytes
(VvM,/mb) of the left ventricular myocardium additionally included the interstitial space as reference unit.
This parameter showed no alterations after ACC during 120 min of ischemia, but showed a significant decrease following 180 min. After St. Thomas and HTK
perfusion and following ischemia no significant alterations occurred.
The approximate constancy of VVMfrelated to bundles of myocytes during a longer time of ischemia verifies that the relative decrease of VVMfwas caused by
fluid movements from the interstitial to the intracellular space and not at all by loss of myofibrils.
DISCUSSION
Our results show that the size of the interstitial
space of left ventricular myocardium a t the onset of
ischemia as well as the way i t acts during ischemia
varies among the different types of cardiac arrest. The
multistage sampling method for the morphometrical
determination of interstitial and cellular parameters
has never been described before. With our methods,
information on the reactions of interstitial and intracellular space can be obtained. Further, their mutual
influence at the onset and during ischemia after different types of cardiac arrest can be investigated.
Fixation Technique
Recent investigations have shown that the technique
of fixation (physical fixation, chemical fixation by immersion or perfusion) influences only the size of tissue
clefts but not the interstitium between bundles of myocytes (Schmiedl e t al., 1991). Because of this, the application of immersion fixation for the investigation of
different time points is possible without loss of information. The size of tissue clefts after immersion fixation is overestimated compared with physical fixation
(Schmiedl e t al., 1991), but this applies to all methods
of cardiac arrest investigated. The osmolarity of our
fixation and postfixation solution (Hayat, 1989) prevent artificial shrinkage or swelling of the compact tissue (Tranum-Jensen et al., 1981). The volume changes
during dehydration apply to the whole tissue block
(Bone and Denton, 1971) independent of the type of
cardiac arrest and does not lead to fluid shifts between
interstitial and intracellular space. During embedding,
no volume shifts occur (Gerdes et al., 1992).
.
325
VOLUME SHIFTS IN CELLULAR SPACE
21
TISSUE CLEFTS ( X )
T T
zz
20
i3 St. THOMAS
10
I6
11
1%
10
n
b
I
2
0
0
a
10
60
120
188
GLOBAL ISCHEMIA AT 25 'C (rnin)
INTERSTITIAL SPACE ( X )
H ACC
El St. THOMAS
0 HTK
10
b
60
120
180
GLOBAL ISCHEMIA AT 25 'C (rnin)
Fig. 4. a: Size of tissue clefts (VVTJafter different forms of cardiac arrest and global ischemia a t 25°C.
b Size of the interstitium within bundles of myocytes (Vvls)after different forms of cardiac arrest and
global ischemia a t 25°C. *Significant inrease ( P < 0.05) compared to the onset of ischemia.
Interstitial Space and Contractile Activity
At the onset of ischemia the ATP concentrations are
comparable in the differently arrested hearts (Schnabe1 et al. 1987; Schmiedl et al., 1993). Sufficient ATP
levels in combination with physiological Ca2+ or in
combination with the Ca2+ content in the St. Thomas
solution leads to a high degree of contraction and
pathological contraction in cross clamped or St.
Thomas arrested hearts (cf. Figs. Zb, 3). Therefore, the
size of tissue clefts is influenced by the greater or lesser
remaining activity of the hearts and their ability to
contract during contact with the fixation solution (Billingham, 1983; Schnabel et al., 1990; Schmiedl et al.,
1993). This results in fluid shifts into the tissue clefts
and a higher level of VVTc(cf. Fig. 4a).
Using the cardioplegic solution HTK for coronary
perfusion, the reduced values of calcium result in a
significantly higher degree of relaxation without expulsion of fluid into the tissue clefts leading to the
lowest values of VVTc(cf. Figs. 2c, 3, 4a).
In aortic cross clamped and HTK arrested hearts, the
more or less pronounced relaxation during ischemia
minimizes the volume of fluid in the tissue clefts and
leads to a decrease in VVTc(cf. Figs. Zb, 3, 4a). The
increase of this parameter after ACC and 180 min of
ischemia is the result of the onset of rigor mortis associated with the renewed occurrence of contraction
(Hearse et al., 1977; Vanderwee et al., 1981). After St.
Thomas perfusion, the insufficient relaxation of myocytes and a possible interstitial edema lead to continued high values of tissue clefts (cf. Fig. 4a).
326
A. SCHMIEDL ET AL.
lnterstitium and Cellular Alterations
VOLUME DENSITY O F MYOFIBRILS
'
,
*
Y
~
.A
70
-
-ACT
#St
H
T
Thomas
K
~~
80
50
40
v
2
>
30
20
10
0
0
a
80
Global I s c h e m i a a t 2 5 ° C
VOLUME DENSITY O F YYOFIBRILS
RELATED TO BUNDLES O F YYOCYTES
-
70
8o
-
80
El
40
c
Bp
Y
50
P
30
>
P
20
10
0
h
-
0
80
120
180
Global I s c h e m i a at 2 5 ° C
Fig. 5. a: Volume density of myofibrils (V,,)
after different forms
of cardiac arrest and global ischemia a t 25°C. b: VVMfrelated to bundles of myocytes (V,,,,,)
after different froms of cardiac arrest and
following global ischemia a t 25°C. *Significant alterations ( P < 0.05)
compared to the onset of ischemia.
The size of the interstitium between bundles of myocytes (VvIs) was influenced by cellular edema, and by
the different degree of contraction (cf. Figs. 2-4). Our
results confirm earlier observations, which lead to the
conclusion that a higher cell radius of contracting myocytes during systole results in a decrease in the size of
the interstitial space (Pine et al., 1979; Borg and
Caulfield 1981), and that diastole or mechanisms,
which reduce contractility, such as acidosis expand the
interstitial space (Poole-Wilson et al., 1979). However,
the interstitium within bundles of myocytes can be also
modified by additional alterations occurring during
ischemia. The VvIs increased after ACC and 180 min in
spite of additional increase of cellular volume and renewed occurrence of contraction compared to 120 min
of ischemia (cf. Figs. 2a,b; 4b). The cause may be disturbances within the connective tissue leading to a disintegration of the collagen struts combined with a n
insufficient connection to adjacent myocytes (Caulfield
and Borg, 1979; Robinson et al., 1983). This would result in slippage of cells in either transverse or lateral
directions and therefore to a n increase of the interstitium within bundles of myocytes (Borg and Caulfield,
1981). After St. Thomas perfusion and ischemia a n increase in cellular edema and a n insufficient relaxation
occur. Both alterations do not significantly influence
the size of the interstitium within bundles of myocytes,
because the restriction is partly compensated for by the
interstitial edema. After HTK perfusion and ischemia,
VvIs remains almost constant, because the swelling of
myocytes is compensated for by the almost complete
relaxation. Therefore, the size of the interstitium
within bundles of myocytes does not alter.
Interstitial Space and Volume Shifts
The constancy of the relation of VVMf to bundles of
myocytes during a pronounced ischemic period has
shown that the swelling of cells (decrease in VVMf)and
the restriction of interstitium (decrease in VVIJ are the
result of a shift of fluid from the interstitial to the
intracellular space and not of myofibrillolysis. This disintegration of myofibrils seems to occur after ACC and
Size of Tissue Clefts and Orientation of Myocytes
Statements about the alteration in the size of tissue 180 rnin of ischemia, because the VVMf related to bunclefts after immersion fixation should always take into dles of myoctes decrease significantly compared to the
consideration the different orientation of myocytes at onset of ischemia.
Thus, the multistage sampling technique used
the base, equator, and apex of the heart. The orientation of the myocytes a t the base and apex is almost allows investigation of interstitial and cellular alteroblique to epi- and endocardium and at the equator ations during ischemia. With this method, addialmost vertical. This different orientation leads to a tionally, relations between interstitial and cellular pagreater size of the tissue clefts at the apex and at the rameters are possible and volume shifts between
base than at the equator of the heart (Henle, 1876). interstitial and cellular compartments are recorded.
This does not apply to our samples taken from the apex
DEDICATION
of the ventricle only at the onset of ischemia. During
We should like to dedicate this publication to the
ischemia, however, samples were taken from the incubated hearts by continuous diminishing of the ventricle former director of the Department of Physiology, Prof.
from the apex to the equator and to the base (cf. Ma- Dr. Dr. h.c. H.J. Bretschneider, whose sudden death in
terial and Methods). The oblique orientation of myo- December 1993 shocked and saddened us all.
cytes in samples taken from the base may additionally
ACKNOWLEDGMENTS
enlarge the tissue clefts. This may explain the more or
less pronounced increase of VVTcafter ACC and 180
We are indebted to Dr. H. Fehrenbach for encouragmin of ischemia, after St. Thomas perfusion and 120 ing discussions. We thank Ms. S. Freese, Ms. A.
min of ischemia, as well as after HTK perfusion and Gerken, Ms. H. Hiihn, and Ms. M. Scheumann for skill180 min of ischemia (Fig. 4a).
ful technical assistance; Ms. C. Maelicke, B.Sc., for the
VOLUME SHIFTS IN CELLULAR SPACE
English correction; and Ms. H. Altmann for support in
preparing the manuscript.
Parts of the results were presented at the 55th meeting of the Deutsche Gesellschaft fur Herz- und Kreislaufforschung in Mannheim (1989) and at the 86th
meeting of the Anatomische Gesellschaft in Szeged
(1991).
This work was supported by the DFG, SFB 330.
LITERATURE CITED
Billingham, M.E. 1983 The role of endomyocardial biopsy in the diagnosis and treatment of heart disease. In: Cardiovascular Pathology, vol. 2. M.D. Silver, ed. Churchill, Livingstone, New
York, pp. 1205-1224.
Bretschneider, H.J. 1964 Uberlebenszeit und Wiederbelebungszeit
des Herzens bei Normo- und Hypothermie. Verh. Dtsch. Ges.
Kreis1.-Forsch.. 3Ot11-34.
Bone, Q., and E.J. Denton 1971 The osmotic effect of electron microscopic fixatives. Cell Biol., 49:571-581.
Borg, T.K., and J.B. Caulfield 1981 The collagen matrix of the heart.
Fed. Proc., 402037-2041.
Caulfield, J.B., and T.K. Bora 1979 The collagen
- network of the heart.
Lab. Invest., 40:364-372.
DiBona, D.R., and W.J. Powell 1980 Quantitative correlation between
cell swelling and necrosis in myocardial ischemia in dogs. Circ.
Res., 47553-665.
Feneis, H. 1944 Das Gefiige des Herzmuskels bei Systole und Diastole. Gegenbauers Morphol. Jahrb., 89r371-406.
Ganote, C.E. 1983 Contraction band necrosis and irreversible myocardial injury. J. Mol. Cell. Cardiol., 15t67-73.
Gebhard, M.M., H.J. Bretschneider, E. Gersing, and Ph.A. Schnabel
1987 Bretschneider’s histidine-buffered cardioplegic solution:
Concept, application and efficiency. In: Myocardial Protection in
Cardiac Surgery. A.J. Roberts, ed. Marcel Dekker, New York, pp.
95-119.
Gerdes, A.M., J . Kriseman, and S.P. Bishop 1982 Morphometric study
of cardiac muscle-The problem of tissue shrinkage. Lab. Invest.,
64271-274.
Haasis, G., A. Schmiedl, Ph.A. Schnabel, M.M. Gebhard, G. Mall, J .
Richter, and H.J. Bretschneider 1991 Morphometrie des interstitiellen Raumes von schlagenden und stillgestellten Herzen. Verh.
Anat. Ges., 84:285-286.
Hayat, M.A. 1989 Fixation for Electron Microscopy. Macmillan Press,
London.
Hearse, D.J., M.V. Braimbridge, and P. Jynge 1981 Protection of the
Ischemic Myocardium. Raven Press, New York.
Hearse, D.J., P.B. Garlick, and St.M. Humphry 1977 Ischemic contracture of the myocardium: Mechanisms and prevention. Am. J .
Cardiol., 39r986-993.
Henle, J . 1876 Handbuch der systematischen Anatomie des Menschen, vol. IIU1, 2nd ed. Fr Vieweg u Sohn, Braunschweig.
Jennings R.B. and K.A. Reimer 1981 Lethal myocardial ischemic injury. Am. J . Pathol., 102:241-255.
Karnovsky, M.J. 1967 A formaldehyde-glutaraldehyde fixative of
high osmolality for use in electron microscopy. J . Cell Biol., 27:
137-138.
Katz, A.M. 1973 Effects of ischemia on the contractile process of the
heart muscle. Am. J. Cardiol.. 32r456-460.
Paulussen, F., G. Hiibner, D. Grebe, and H.J. Bretschneider 1968 Die
Feinstruktur des Herzmuskels wahrend einer Ischamie mit Senkung des Energiebedarfs durch spezielle Kardioplegie Klin.
Wochenschr., 46:165-171.
327
Pine, M., J.B. Caulfield, O.H.L. Bing, W.W. Brooks, and W.H. Abelmann 1979 Resistance of contracting myocardium to swelling
with hypoxia and glycolytic blockade. Cardiovasc. Res., 13215224.
Polimeni, Ph.1. 1974 Extracellular space and ionic distribution in rat
ventricle. Am. J . Physiol., 229:1299-1304.
Poole-Wilson, P.A., P.D. Bourdillon, and D.P. Harding 1979 Influence
of contractile state on the size of the extracellular space in isolated ventricular myocardium. Basic Res. Cardiol., 73t604-610.
Puff, A. 1961 Uber das Bindegewebssystem im Herzen. Verh. Anat.
Ges., 57331-83.
Preusse, C.J., M.M. Gebhard, and H.J. Bretschneider 1981 Myocardial equilibration processes and myocardial energy turn over during initiation of artificial cardiac arrest with cardioplegic solution-reasons for a sufficiently long cardioplegic perfusion.
Thorac. Cardiovasc. Surg., 29.71-76.
Reil, G.-H., R. Frombach, R. Kownatzki, W. Quante, and P.R. Lichtlen
1987 Ascorbic acid: A non radioactive ECS marker in canine
heart. Am. J . Physiol., 253:1305-1314.
Robinson, T.F., L. Cohen-Gould, and S.M. Factor 1983 Skeletal framework of mammalian heart muscle-Arrangement of inter- and
pericellular connective tissue structures. Lab. Invest., 49t482498.
Schmiedl, A,, Ph.A. Schnabel, M.M. Gebhard, G. Haasis, G. Mall, J .
Richter, and H.J. Bretschneider 1989 Morphometrie der Volumenverschiebungen im Myokard wahrend globaler Ischamie. Z.
Kardiol., 78(Suppl 1):88 (Abstract).
Schmiedl, A,, F. Bach, J. Richter, Ph.A. Schnabel, and H.J.
Bretschneider 1991 Morphometric evaluation of the myocardial
interstitial space after physical and chemical fixation. Acta
Anat., 142r321-325.
Schmiedl, A,, Ph.A. Schnabel, G. Haasis, G. Mall, M.M. Gebhard, J .
Richter, and H.J. Bretschneider 1990a Influence of pretreatment
on interstitial and intracellular space of canine left ventricular
myocardium. Acta Anat., 138:175-1 81.
Schmiedl, A, Ph.A. Schnabel, G. Mall, M.M. Gebhard, D.H. Hunneman, J . Richter, and H.J. Bretschneider 1990b The surface to
volume ratio of mitochondria, a suitable parameter for evaluating mitochondria1 swelling-Correlations during the course of
myocardial global ischemia. Virchows Arch. [A], 416t305-315.
Schmiedl, A,, Ph.A. Schnabel, J . Richter, G. Mall, and H.J.
Bretschneider 1993 Preservation of cardiac myocytes subjected to
different preconditions: A comparative morphometric study of
beating, fibrillating, and cardioplegically arrested canine hearts.
Anat. Rec., 235:425-435.
Schnabel Ph.A., M.M. Gebhard, Th. Pomykaj, A. Schmiedl, C.J.
PreuRe, J . Richter, and H.J. Bretschneider 1987 Myocardial protection: Left ventricular ultrastructure after different forms of
cardiac arrest. Thorac. Cardiovasc. Surg., 35t148-156.
Schnabel, Ph.A., A. Schmiedl, B. Ramsauer, U. Bartels, M.M. Gebhard, J . Richter, and H.J. Bretschneider 1990 Occurrence and
prevention of contraction bands in Purkinje fibres, transitional
cells and working myocardium during global ischemia. Virchows
Arch. [A], 417:463-471.
Tranum-Jensen, J., M.J. Janse, J.W.T. Fidet, W.J.G. Krieger, C.N.
D’Alnoncourt. and D. Durrer 1981 Tissue osmolalitv. cell swelling, and reperfusion in acute regional myocardial iscKemia in the
isolated porcine heart. Circ. Res., 49r364-381.
Vandenvee, M.A., St.M. Humphrey, J.B. Gavin, and L.C. Armiger
1981 Changes in the contractile state, fine structure and metabolism of cardiac muscle cells during the development of rigor
mortis. Virchows Arch. IB1.35t159-167.
Weibel, E.R. 1979 StereologicalMethods, vol. 1. Practical Methods for
Biological Morphometry. Academic Press, New York.
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