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Clinical Hemorheology, Vol. 15, No. 1, pp. 97-105, 1995

Copyright 1995 Elsevier Science Ltd
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0271-5198(94)00080-8

MYOCARDIAL TISSUE HEMATOCRIT: EXISTENCE OF A TRANSMURAL

GRADIENT AND ALTERATIONS AFTER FIBRINOGEN INFUSIONS

Oguz K. Baskurt, Mustafa Edremitlioglu

Akdeniz University Faculty of Medicine, Department of Physiology, Antalya, Turkey

(Received 26.08.1994; accepted 29.11.1994)

ABSTRACT Tissue hematocrit was determined in left ventricular myocardium slices in in situ
frozen rat hearts and the influence of experimental plasma fibrinogen increments was
investigated. Hematocrit values in 100 m thick myocardial slices were estimated by
measuring the activity of two different radionuclides labelling plasma (125I-Albumin)
and red blood cells (99mTc). In the control group with physiological levels of
fibrinogen, myocardial tissue hematocrit was highest at the layer adjacent to
epicardium. It decreased linearly through the depth of myocardium, approaching a
minimum at the myocardial layer closest to the endocardium. In the group with about
two folds higher plasma fibrinogen concentration, plasma viscosity and red blood
cell aggregation were about 50% higher. In this group, tissue hematocrit difference
between epicardial and endocardial layers existed, however this difference was more
likely due to a step change, rather than a linear gradient. These findings confirm those
physiological mechanisms underlying the hematocrit reduction in small blood vessels
might be affected by hemorheological alterations.

INTRODUCTION
Hematocrit reduction in small-sized blood vessels is a well-documented physiological
phenomenon that results in significantly lower hematocrit values in the microcirculation (1,2). This
is an important physiological mechanism favoring blood flow, despite the increased vascular
resistance due to decreased diameter in microcirculation. Hemodynamic and hemo-rheological
mechanisms underlying hematocrit reduction has been discussed in detail, starting with the classical
work of Robin Fhraeus (3) and confirmed by experimental studies (4,5,6). We have learned from
these studies that these mechanisms (i.e., axial migration, plasma skimming, Fhraeus effect) are
significantly affected by the rheological properties of blood (7,8). On the other hand, the degree of
hematocrit reduction also depends on hemodynamic variables (i.e., pressure gradients,
_______________
Key Words: Tissue hematocrit, myocardium, fibrinogen, erythrocyte aggregation.

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local shear forces). Therefore, hematocrit value at the microvascular level is as dynamic variable
and it might be altered due to the disturbances of hemodynamic or hemorheological factors.
As a consequence of this dynamic nature of microvascular hematocrit, this parameter might
change from organ to organ, or in various parts of a given organ paralleling local hemodynamic
conditions. This is most clearly evident in myocardium in which the pressure gradients and flow
conditions may vary considerably (9). A tissue hematocrit gradient has been demonstrated in the left
ventricular myocardium, with the highest value at the myocardial layers adjacent to epicardium and
lowest at the endocardial layers (10,11).
Our previous studies revealed that hemorheological factors might also affect hematocrit values in
various depths of the left ventricular myocardium (11). The hematocrit gradient present under
physiological conditions disappeared after an experimental alteration in red blood cell (RBC)
deformability. In this study, some other hemorheological factors were altered by fibrinogen infusions
and the effect of these alterations on myocardial hematocrit gradient has been investigated, using the
same model and methods.
METHODS
Animals and groups. Two groups of Swiss Albino rats were used in the experiments. There were 10
animals, weighing 270-350 grams in each group. Myocardial hematocrit gradient was determined
after producing hyperfibrinogenemia in one of the groups (Hyperfibrinogenemia group). In the
Control group, myocardial hematocrit gradient was determined without any experimental alteration
in hematological or hemorheological determinants.
Preparation. Under Urethane (1 gr/kg; ip) anaesthesia, tracheotomies were performed and left
carotid artery, left femoral artery and right femoral vein were cannulated. Animals were ventilated
with ambient air. The carotid catheter was positioned at the level of ascending aorta and connected
to a pressure transducer (Statham; P23XL) to monitor arterial blood pressure throughout the
experiment and during the rapid freezing of the heart.
Inducing hyperfibrinogenemia: Hyperfibrinogenemia was produced by fibrinogen injections. Fifty
mg type IV bovine fibrinogen (Sigma; F4753) was dissolved in 1 ml of rat plasma and RBCs were
suspended in this solution at the hematocrit of recipient animal. One ml of blood was sampled from
the femoral artery catheter and used for fibrinogen and microscopic aggregation index (MAI)
determinations. The suspension containing fibrinogen was then slowly injected through the femoral
venous catheter. During this injection, an amount of blood was withdrawn from the arterial catheter
to match with the infused volume, together with the 1 ml sampled prior to the injection of fibrinogen
enriched suspension. Therefore, an amount of blood (less then 2 ml in all cases) was exchanged
isovolemically. Myocardial hematocrit gradient was determined five minutes after this procedure
had been completed.
Fibrinogen used in this study (Sigma, F4753) contains about 15% sodium citrate and 25%
sodium chloride. In order to test the possible interference of these salts infused together with
fibrinogen, 7.5 mg sodium citrate and 12.5 mg of sodium chloride were dissolved in 1 ml of rat
plasma and infused in to a separate group of rats (n=5). Myocardial hematocrit gradient in this group
was also determined as described below.

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Determination of myocardial hematocrit gradient. Tissue hematocrit in the left ventricular

myocardium was estimated by determining the activity of two different radionuclides, labeling the
plasma and RBC in the left ventricular myocardial slices as described by Vicaut and Levy (10).
RBCs were labeled in vitro with 99mTc, and 125I labeled albumin (Amersham) was used to trace
plasma. 125I-Albumin was purified using an ultrafilter with a cut off at 50.000 Da (Microsep) to
eliminate any unbound tracers. 99mTc labelled RBCs (0.2 ml packed cell) and 0.15 ml 125I-Albumin
solution (20 mg/ml albumin, 1 Ci) were suspended in 0.65 ml of saline and injected through the
femoral vein. After 5 minutes, 1 ml of blood was sampled for the determination of 99mTc and 125I
activities, systemic hematocrit, fibrinogen and MAI. Following this, a midsternal thoracotomy was
performed rapidly, and a plastic cylinder was positioned around the heart immediately. This
chamber was rapidly filled with liquid nitrogen, bathing and freezing the heart. The frozen heart was
then excised and kept at -20 C. A 2x3x5 mm tissue block with the long axis perpendicular to the left
ventricular wall was cut and mounted on a cryostat disk, observing that the disk surface be parallel
to the left ventricular wall. This tissue block was then sectioned through the thickness of the left
ventricular wall, from epicardium to endocardium, to obtain 100 m thick myocardial slices, using a
cryostat (Lipshaw, Model 1900). Sectioning was continued until the left ventricular cavity was
approached. Any myocardial block with an unusual geometry observed during slicing (i.e., half
myocardium, half blood appearance in the same tissue slice) was excluded from the study. A
transverse section of the heart was also used to measure the thickness of the left ventricular wall and
to determine whether the heart was arrested during systole or diastole. Only the hearts arrested
during diastole were used in further evaluations.
Radionuclide (99mTc and 125I) activities in the systemic blood specimens and myocardial tissue
slices were measured by a gamma counter (DPC; Gambyte 20). Tissue hematocrit was estimated in
each myocardial slice according to Vicaut and Levy (10). Constants between the radionuclide
activities and the volume of labelled compartments were calculated, using the measurements in
systemic blood specimens for each tracer (K99mTc= (VBlood x Hct)/99mTc cpm; K125I= [VBlood x (1Hct)]/125I cpm). Corresponding constants were multiplied by 99mTc and 125I activities in each 100 m
thick tissue slice to find the total volume of RBCs or plasma (VRBC= K99mTc x 99mTc cpm;
Vplasma =K125I x 125I cpm). Tissue hematocrit in that slice was then calculated as follows:
Hct=VRBC/(VRBC+Vplasma ). Myocardial hematocrit in each slice was expressed as percentage to the
tissue hematocrit in the layer adjacent to epicardium (%Hct/Hctepi). It was then plotted against
normalised depth of that layer, epicardial layer corresponding to 0, and endocardial layer closest to
endocardium to 1. Tissue slices between these two were represented by numbers between 0 and 1,
depending on the depth and the number of 100 m thick slices.
Plasma fibrinogen determinations: Fibrinogen levels in the plasma samples were estimated by
clotting method (Fibri-Prest; Diagnostica Stago) according to Clauss (12).
Microscopic Aggregation Index (MAI) determinations (13): RBCs from each animal were
suspended in autologous plasma at a hematocrit of 0.01 L/L and used for the determination of MAI.
The numbers of cellular units were counted in a hemocytometer, in a humidified chamber at 37 C.
Suspensions using the same blood samples were also prepared in isotonic saline at the same
concentration and cell counts were obtained in a similar set up. MAI was calculated as the number of
cellular units in saline divided by the number of cellular units in plasma.
Plasma viscosity measurements: Plasma viscosity was measured by a capillary tube (0.5 mm ID,
30 mm length) viscometer, which was connected to a microcomputer through an optoelectronic
sensor system. Plasma viscosity calculations were based on the flow-time determinations of about

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500 L sample. All measurements were done at 37 C. The system was routinely calibrated by
standard calibration fluid (Brookfield Engineering).
Statistics. Results are expressed as mean standard error. Comparisons between two means were
done by "Student 't' test". The relationship between the depth of tissue and %Hct/Hctepi at that tissue
level was tested by linear and polynomial regression analysis.
RESULTS
Heart rate, mean arterial pressure and hematocrit values were not significantly different in
control and hyperfibrinogenemia groups. Heart rates were 325 38 /min and 332 47 /min in
control and hyperfibrinogenemia groups respectively. Mean arterial pressure in the control group
was 105 8 mmHg, while it was 102 11 mmHg in the hyperfibrinogenemia group. Systemic
hematocrit values were 0.38 0.05 L/L and 0.39 0.06 L/L in these groups respectively.
Fibrinogen concentrations, MAI and plasma viscosity in control and hyperfibrinogenemia groups
are shown in TABLE-1. All parameters in control animals and in hyperfibrinogenemia group before
fibrinogen infusions were similar, but significantly increased after infusions.
TABLE-1
Fibrinogen concentration, MAI and plasma viscosity in control and hyperfibrinogenemia groups
before and after fibrinogen infusions (*: Difference from control; p<0.01).

Fibrinogen (gr/L)
MAI
Plasma viscosity (cp)

Control Group
(n=10)
1.44 0.08
1.63 0.16
1.21 0.02

Hyperfibrinogenemia Group (n=10)

Before infusion
After infusion
1.41 0.07
2.88 0.20*
1.59 0.13
2.56 0.21*
1.24 0.02
1.81 0.03*

TABLE-2
Tissue hematocrit values in the myocardial layers closest to epicardium (Hctepi) and endocardium
(Hctendo) in control and hyperfibrinogenemia groups.

Hctepi
Hctendo

Control (n=10)
L/L
% Sys. Hct
0.344 0.087
90.2
0.253 0.055
66.5

Hyperfibrinogenemia (n=10)
L/L
%Sys. Hct
0.341 0.074
87.4
0.248 0.087
65.7

Tissue hematocrit values in the layers adjacent to the epicardium and to the endocardium are
presented in TABLE-2. In both groups, tissue hematocrits were about 90% of the systemic
hematocrit in the epicardial layers. In the endocardial layers it decreased to about 65% of the
systemic hematocrit. Tissue hematocrit values in both epicardial and endocardial layers were not
significantly different in control and hyperfibrinogenemic groups. However, tissue hematocrit values
between these two sites changed significantly due to fibrinogen infusions.

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FIG-1 shows the tissue hematocrits at each 100 m thick tissue slice as percentage of Hctepi,
against normalized depth. In the control group, tissue hematocrit in the left ventricular myocardium
decreased between epicardial and endocardial layers almost linearly. On the other hand, in the group
with hyperfibrinogenemia, tissue hematocrit remained at a level that was close to the Hctepi through
the 75% depth of the left ventricular wall. However, after this depth, myocardial hematocrit declined
with a greater slope and at the layer closest to endocardium it approached to the control group value
at the corresponding layer. This clearly indicates a non-linear relationship between the tissue
hematocrit and tissue depth between epicardium and endocardium in the hyperfibrinogenemia group.
Infusion of plasma containing sodium citrate that is present in fibrinogen preparation did not alter
the myocardial hematocrit gradient (data not shown). Therefore, the observed effect seems to be
related to the increased fibrinogen concentration.
%Hct/Hctepi
Control

1 2 0

Hyperfibrinogenemia

1 1 5
1 1 0
1 0 5
1 0 0
9 5
9 0
8 5
8 0
7 5
7 0
6 5
6 0
0

.1

.2

.3

.4

.5

.6

.7

.8

.9

Normalized depth

FIG-1
Myocardial tissue hematocrits in each 100 m thick tissue slice as %Hctepi, plotted against
normalised depth. (Each set of points represents the cumulative results of 10 experiments. 4 -10
values were averaged to obtain each point. Error bars are standard error).

DISCUSSION
It has been observed that tissue hematocrit values determined in 100 m thick tissue slices in the
left ventricular myocardium of rats changes as a function of tissue depth from epicardium. Tissue
hematocrit gradient in the left ventricular wall of rats was first demonstrated by Vicaut and Levy
(10) using the technique utilized in this study. The main advantage of this technique is its ability to
determine the tissue hematocrit values at various depths of the ventricular wall. Additionally, rapid
in situ freezing of the heart allows determinations under physiological conditions. Vicaut and Levy
used pharmacological blockade to obtain heart preparations arrested in systole or diastole prior to in
situ freezing (10). Such a pharmacological manipulation resulted in relatively large pressure drops
before freezing in our preliminary experiments. Therefore we preferred direct freezing of the beating
heart and classified the hearts according to the phase of arrest. However, our efforts to determine
tissue hematocrit values in the left ventricular wall of the hearts arrested in systole were not
successful. The activities of radionuclides tracing RBC and plasma in 100 m thick myocardial
slices were very low, frequently at the level of background activity. These values could not be used

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for the calculation of a reliable hematocrit value. Lower activity of radionuclides in the tissue slices
from systolic heart preparations can be explained by the smaller volume of blood in intramyocardial
blood vessels during systole. Inflow of blood to left ventricular myocardium nearly stops, while
venous outflow makes its peak during systole (9). Therefore, only the data from the hearts arrested in
diastole were used in the evaluation and the results of this study are limited to diastolic conditions.
However, diastolic data concerning the perfusion of left ventricular wall is certainly more important,
as the perfusion of this part is mostly diastolic (9,14).
Left ventricular wall seems to be unique with a reproducible pattern of tissue hematocrit
distribution. The main reason for this should be related to the hemodynamic variability that can be
defined at a particular orientation with the tissue. In the coronary circulation, intramyocardial
pressure is an important determinant of blood flow, as it acts as a Starling resistor (15).
Intramyocardial pressure is mainly a reflection of the pressure in the neighboring heart chambers.
However its influence through the thickness of the myocardium is not uniform. Intramyocardial
pressure is highest in the subendocardial, and lowest in the subepicardial layers (16). Clearly,
intramyocardial pressure (or the extravascular pressure) might be higher than venous pressure, at
least during certain parts of the cardiac cycle. In this case the perfusion pressure for a particular
myocardial layer should be determined by the intramyocardial pressure, according to the waterfall
hypothesis (17). Therefore, perfusion pressure (shear stresses and shear rates accordingly) changes
as a function of myocardial depth. At the end of the systole subendocardial blood vessels are
narrower and have a higher flow resistance when compared to subepicardial vessels (9). The
mechanisms of hematocrit reduction are known to be sensitive to these hemodynamic and vascular
variables. All these diversities in hemodynamic and vascular parameters may contribute to the
establishment of the observed tissue hematocrit gradient. However, both hemodynamic conditions in
the coronary circulation and mechanisms of hematocrit reduction are complex. Further research is
necessary for a clear description of their interactions.
This study shows that myocardial hematocrit gradient might be disturbed, at least in some parts
of the myocardium, after fibrinogen infusions. Fibrinogen concentration of plasma is an important
determinant of blood rheology. It correlates with plasma viscosity (18), as well as RBC aggregation
(13). Both are confirmed in this study; MAI and plasma viscosity both increased about 50% after
fibrinogen infusions. Such a hemorheological alteration might interfere with several aspects of
microcirculatory hemodynamics. Plasma viscosity becomes progressively important as blood moves
toward the microcirculation. It has a dominating effect on in vivo viscosity in the resistance and
exchange vessels (8). Therefore the alterations in the plasma viscosity may significantly contribute to
the resistance in a vascular bed. Increased RBC aggregation also tends to decrease blood fluidity
and disturb the tissue perfusion by increasing flow resistance. As a compensatory mechanism the
vasomotor activity might be altered.
Local hemorheological and hemodynamic conditions are the most important determinants of
microvascular hematocrit in a given tissue (8). Therefore, the hemorheological alterations discussed
above and their consequences would be expected to interfere with tissue hematocrit values. It has
been previously demonstrated that introducing rigidified RBC in the circulation of rats resulted in
tissue hematocrit disturbances in left ventricular myocardium (11). This study extends this
observation to other hemorheological variables (plasma viscosity and RBC aggrega-tion) using
the rat model. The tissue hematocrit values at the myocardial layers adjacent to epicardium and
endocardium in the hyperfibrinogenemic group were not significantly different from the
corresponding values in the control group. However, the gradient patterns between these two values
were clearly not similar in two groups (FIG-1). The non-linearity of the myocardial hematocrit
gradient in the hyperfibrinogenemic group suggests that the influence of increased plasma fibrinogen

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concentration on the mechanisms playing role in the establishment of tissue hematocrit values through
the depth of left ventricular myocardium may not be uniform. The explanation for this varying
influence should be related to the local hemodynamic conditions at different depths of the left
ventricular wall. As discussed above, flow resistance should be elevated after fibrinogen infusions.
This might be compensated by means of the well-developed autoregulatory mechanisms in the
myocardium. The autoregulatory vasodilatation might interfere with the hemodynamichemorheological mechanisms of hematocrit reduction in smaller blood vessels. In the experiments by
Klitzman and Duling (19) capillary hematocrit in the striated muscle increased significantly,
approaching to systemic hematocrit, after a physiological stimulus resulting in vasodilatation.
Abolishment of linear tissue hematocrit gradient in the outer 75% of the left ventricular wall can be
explained by a compensatory vasodilatation. What happens in the inner 25% of the left ventricular
wall, where a steeper gradient has been observed? The autoregulatory response should include the
whole thickness of the ventricular wall. However, there is another mechanism that may affect the
blood vessel diameters in the myocardium. Myocardial contraction and the consequent increment in
the intramyocardial pressure decrease the blood vessel diameters significantly (20). Intramyocardial
pressure increment is more prominent in the subendocardial layers and subendocardial vessels are
narrower than subepicardial vessels during systole (9). This may offset the influence of
autoregulation in the myocardial tissue close to endocardium.
The difference in tissue hematocrit values in subendocardial and subepicardial layers of the left
ventricular wall has been clearly demonstrated. The tissue hematocrit gradient seems to be sensitive
to hemorheological alterations (RBC rigidification (11), elevated plasma viscosity and RBC
aggregation) and appears to be dynamic. However, underlying hemodynamic- hemorheological
mechanisms, physiological significance and clinical aspects are still unclear and need further
investigation.

ACKNOWLEDGMENTS
The authors thank Erol Nizamoglu, Huseyin Isik and Murat Tunc for their technical support.
This study was supported by Turkish Scientific and Technical Research Council (TUBITAK)
Grant TAG-1008.

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