Download The effect of brain death on cardiovascular function in rats. Part I. Is

Cardiovascular Research 38 Ž1998. 98–106
The effect of brain death on cardiovascular function in rats. Part I. Is the
heart damaged?
Paul Herijgers a , Marcel Borgers b, Willem Flameng
a
a,)
Centre for Experimental Surgery and Anaesthesiology, K.U. LeuÕen, ProÕisorium I, Minderbroedersstraat 17, B-3000 LeuÕen, Belgium
b
Janssen Research Foundation, Beerse, Belgium
Received 4 August 1997; accepted 30 August 1997
Abstract
Objective: Brain death induces important haemodynamic changes in rats, with a drop in arterial blood pressure, left ventricular
developed pressure and dPrdt max to less than 50% of its control value. Myocardial damage was reported to contribute to this paradigm.
The role of potential underlying pathogenetic mechanisms, such as a circulating cardiodepressant factor, NO, endogenous opioid peptides,
vagal or b-adrenergic activation, or hypophyseal dysfunction, were explored, but none of them could be demonstrated as the culprit. This
study investigated whether functionally important intrinsic myocardial damage was induced by brain death in the rat, and whether
coronary endothelial cell dysfunction, possibly causing multifocal ischaemia, contributed to this. Methods: Brain death was induced in
rats by sudden inflation of an intracranial balloon. Extensive haemodynamic measurements, including heart rate, arterial blood pressure,
central venous pressure, left ventricular pressure, and cardiac output, were performed. Hearts excised 1 and 4 h after brain death were
examined histologically. The contractile reserve of these hearts was tested by administration of increasing doses of adrenaline Ž10y9 to
10y6 molrl. in a Langendorff system. The coronary endothelium was tested with regard to its barrier function for macromolecules by
determining the extravasation of injected Evans blue, and with regard to its vasoactive function by testing the effects of serotonin and
nitroglycerin in a Langendorff system. Results: The haemodynamic measurements suggested that the cardiovascular collapse consisted
mainly in alterations in afterload. Contractile reserve, as tested with increasing adrenaline doses, revealed a normal dose–response curve.
No histological myocardial damage was found after brain death in rats. No abnormal extravasation of Evans blue was seen. Coronary
vasoreactivity towards nitroglycerin and serotonin was normal. Conclusion: Myocardial damage, if present at all, contributes only
minimally to the changes in haemodynamic profile seen after brain death in the rat, and the coronary endothelium appears to preserve its
barrier and vasoactive function. q 1998 Elsevier Science B.V.
Keywords: Rat; Brain death; Myocardium; Contractile reserve; Coronary endothelium
1. Introduction
Besides its fundamental interest, the study concerning
the influence of brain death on the heart gained popularity
in the era of heart transplantation. This is caused by the
fact that a considerable part of the potential organ donors
become haemodynamically unstable, precluding the use of
the heart for transplantation w1x. Furthermore a small percentage of the transplanted hearts exhibit poor function,
despite the fact that no immunological or surgical–technical reason is apparent w2x.
Brain death induces a severe haemodynamic collapse in
rats w3,4x, and myocardial damage was reported, propor-
tional to the amount of catecholamines released at the
moment of brain death w4x. Comparable phenomena were
shown in many species, including baboons w5x, pigs w6x,
and dogs w7x. This, however, does not necessarily mean
that the catecholamine storm at the moment of brain death
causes irreversible myocardial damage and that this myocardial damage, in turn, causes the haemodynamic collapse seen after brain death. Especially difficult to reconcile with this hypothesis is the fact that rat hearts explanted
60 min after brain death, when the haemodynamic collapse
is already present in vivo for a considerable time, showed
normal basal contractile function in a Langendorff system
w3x. A possible explanation for the normal contractile func-
)
Corresponding author. Tel.: q32 Ž16. 337298; Fax: q32 Ž16.
337855.
0008-6363r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.
PII S 0 0 0 8 - 6 3 6 3 Ž 9 7 . 0 0 2 8 5 - X
Time for primary review 32 days.
P. Herijgers et al.r CardioÕascular Research 38 (1998) 98–106
tion after explantation and a depressed myocardial function
in vivo after brain death, could be a circulating or neurogenic cardiodepressive factor in vivo. Such a factor, however, is not present, as was elegantly demonstrated by
Galinanes
and Hearse w8x. Another classical hypothesis to
˜
explain the brain death-induced cardiovascular collapse is
thyroidal dysfunction caused by presumed hypophyseal
inactivity, and its ensuing anaerobic cardiac metabolism.
Changes in thyroid function were described after brain
death. However, when hypothyroidism was experimentally
induced by hypophysectomy in rats, cardiac dysfunction
developed only slowly Ždays. and brain death induced after
hypophysectomy resulted in comparable cardiac dysfunction as seen in rats that did not undergo hypophysectomy
w9x. A number of alternative possible pathogenetic mechanisms Že.g. the influence of the b-adrenergic or vagal
pathway, the effect of a chemical sympathectomy, the role
of nitric oxide, the role of endogenous opioid peptides.
explaining the cardiac dysfunction were explored without
providing clear answers w8x.
In a previous study from our centre w7x, it was shown
that the myocardial histological and ultrastructural damage
after brain death in dogs could not be distinguished from
ischaemic damage. Necrosis is multifocal and takes place
in clusters containing few cells. Shanlin et al. w4x reported
that multifocal myocardial damage also occurred in rats
after brain death. This group, moreover, described contraction bands in coronary smooth muscle cells and severe
coronary spasms in coronary casts made with injected
Microfil. The distribution of the lesions in the myocardium
suggests mainly microvascular involvement, with endothelial derived substances as important regulators of microvascular tone. From these observations arose the hypothesis that brain death induces such a profound and
long-lasting coronary vasoconstriction with a complete
cessation of blood supply in certain topographical areas,
that ischaemic myocardial damage occurs disseminated
over the myocardium, possibly caused by coronary endothelial dysfunction.
The discrepancy between the normal basal cardiac function after brain death when the heart was tested in a
Langendorff system and the cardiovascular collapse seen
in vivo made us decide to carefully restudy the importance
of intrinsic myocardial damage induced by brain death.
The second aim of this study was to test coronary endothelial function after brain death, since deranged function
could explain the topographical pattern of the myocardial
damage described above.
2. Methods
2.1. Surgical preparation
The investigation conforms with the Guide for the Care
and Use of Laboratory Animals published by the US
99
National Institutes of Health ŽNIH Publication No. 85-23,
revised 1985.. Male Wistar rats weighing approximately
350 g were anaesthetized with an intraperitoneal injection
of a freshly prepared mixture of urethane Ž600 mgrkg.
and a-chloralose Ž160 mgrkg; both from Sigma, St. Louis,
MO, USA.. A tracheostomy was performed and the rats
were ventilated with a KTR4 small animal ventilator ŽHugo
Sachs, March-Hugstetten, Germany., with oxygen-enriched air, a stroke rate of 60rmin, and a peak inspiratory
pressure of 12–14 mmHg, with 40% inspiratory phase and
10% plateau, and 2 cmH 2 O of PEEP. The inspiratory
pressure and air–oxygen mixture were adjusted every 15
min to keep the partial pressures of arterial blood gases
within the physiological range. ECG lead II was continuously monitored. PE50 catheters ŽIntramedic, Clay Adams,
NY, USA. were inserted in the left femoral artery and
vein. Arterial blood pressure was continuously recorded. A
22-gauge intravenous catheter was inserted into the right
jugular vein, to continuously monitor central venous pressure, kept constant at 4 mmHg by slowly injecting 0.25 ml
of a 50r50 mixture of 0.9% NaCl and Geloplasmaw
ŽInstitut Merieux
Benelux, Brussels, Belgium. as needed.
´
A balloon catheter was placed subdurally ŽFogarty 3F,
Baxter, Irvine, CA, USA.. Brain death was induced by
sudden 300 ml inflation of the intracranial balloon. The
balloon was kept inflated during the entire experiment.
This method was shown to reliably stop all brain perfusion
w10x and electrical activity in the brain w3x.
2.2. Experimental protocols
2.2.1. Protocol 1: haemodynamic changes after brain death
A detailed study of the haemodynamic changes induced
by brain death was performed. After surgery, as described
above, and placement of the intracranial balloon catheter, a
median sternotomy was performed. After opening the pericardium, a micromanometer tipped catheter ŽMillar Instruments, Houston, TX, USA. was inserted in the left ventricle ŽLV. through an apical stab opening. An ultrasonic
transit-time volume flowprobe ŽTransonic Systems, Ithaca,
NY, USA. was placed around the ascending aorta. After
30 min of stabilization, the intracranial balloon was inflated with 300 ml of saline in half of the rats Ž n s 6.,
whereas the others were sham-operated non-brain-dead
controls Ž n s 6.. ECG lead II, mean arterial blood pressure
ŽMABP., central venous pressure ŽCVP., left ventricular
pressure ŽLVP., first derivative of the left ventricular
pressure ŽdPrdt., and aortic flow ŽCO. were continuously
measured. The animals were followed for 60 min after
brain death or time-matched in the sham-operated group.
Preliminary experiments had shown that a stable haemodynamic situation is reached after 1 h and does not change
for the following 3 h, so we decided to restrict the analysis
to the first 60 min. Cardiac index ŽCI. was calculated as
the aortic flow per 100 g body weight, systemic vascular
resistance index was calculated as ŽMABPy CVP.rCI.
Two additional parameters were calculated off-line and
100
P. Herijgers et al.r CardioÕascular Research 38 (1998) 98–106
included in the analysis: Ž1. dPrdt at the common peak
isovolumetric pressure ŽdPrdt at CPIP., as defined by
Mason w11x, was used as largely afterload-independent
parameter for ventricular contractility; and Ž2. maximal
dPrdt divided by the left ventricular pressure at that time
ŽŽdPrdt max .rP., as less preload-dependent measure.
2.2.2. Protocol 2: ex ÕiÕo myocardial contractility
After placing the intracranial balloon, and 30 min of
stabilization, brain death was induced in half of the rats
Ž n s 2 = 6.. Thirty minutes later, the rats were heparinized, and the hearts were rapidly excised via a bilateral
anterior thoracotomy, and mounted on a modified Langendorff isolated heart perfusion system. The hearts were
perfused at a perfusion pressure of 70 mmHg with a
modified Krebs–Henseleit buffer, with final concentrations
Žin mmolrl.: NaCl, 118.3; KCl, 4.7; MgSO4 , 1.2;
KH 2 PO4 , 1.22; CaCl 2 , 1.3; NaHCO 3 , 25.0; glucose, 15.0.
A fluid-filled latex balloon ŽHugo Sachs, March-Hugstetten, Germany. was inserted through a pulmonary vein into
the left ventricle. The balloon connected with a pressure
transducer was used to continuously measure left ventricular diastolic and systolic pressure, and left ventricular
dPrdt and heart rate were continuously derived. Coronary
flow was measured with a 2N in-line flowprobe ŽTransonic, Ithaca, NY, USA. and also continuously recorded.
After stabilization and recording of baseline values, cumulative doses of adrenaline were administered to the perfusion buffer, giving final concentrations of 10y9 , 2 = 10y9 ,
5 = 10y9 , 10y8 , 2 = 10y8 , 5 = 10y8 , 10y7 , 2 = 10y7 ,
5 = 10y7 and 10y6 molrl. Every 90 s, the next higher
concentration was started. To eliminate the chronotropic
effect of adrenaline and its influence on myocardial contractility, isolated hearts were atrially paced at 420
beatsrmin in another series Ž n s 2 = 6.. The same dose–
response curves were constructed.
2.2.3. Protocol 3: morphological alterations
One hour Ž n s 6. and 4 h Ž n s 4. after induction of
brain death in rats, the hearts were excised and perfused
with 2% glutaraldehyde in Sorensen’s
buffer at pH 7.4 for
¨
5 min, further fixed by immersion for at least 2 h, rinsed in
Sorensen’s
buffer overnight, postfixed in OsO4 2% for 1 h,
¨
and routinely embedded in Epon. Two-micrometer-thick
transmural sections were cut, stained with Toluidine blue
and examined with light microscopy. Ultrathin sections
were stained with uranium acetate and lead citrate prior to
examination in an electron microscope. Absence of structural damage was verified by examining transmural sections through the left ventricle at 5 different levels parallel
to the apex, and confirmed by electron microscopy. Hallmark criteria were the absence of contraction band necrosis, subcellular oedema and swelling of mitochondria.
2.2.4. Protocol 4: endothelial barrier function
To test the integrity of the endothelial lining of the
coronary arteries, Evans blue was injected in sham-oper-
ated rats, 30 s before the induction of brain death or 1 h
after brain death Ž n s 3 = 7.. Ten minutes after the injection of Evans blue, the hearts were excised and from every
series, 3 hearts were examined microscopically to examine
Evans blue extravasation. For every time point, the 4
remaining hearts were flushed with the modified Krebs–
Henseleit buffer as described above to rinse the coronary
arteries from Evans blue. Afterwards, the left ventricle was
divided into two parts, one of which was used to determine
the wet weight to dry weight ratio, while the other was put
in formamide ŽSigma, St. Louis, MO, USA; 4 mlrg wet
wt.. for 24 h at 208C, for extraction of extravasated Evans
blue, according to the methodology of Filep et al. w12x. The
amount of extracted Evans blue was measured by spectrophotometry at 624 nm, and expressed as mg Evans blue
per g dry weight of left ventricular tissue.
2.2.5. Protocol 5: endothelial function
As described in protocol 2, hearts were mounted on a
Langendorff perfusion system Ž n s 5 = 6.. After stabilization, coronary vasomotor activity was tested by a 180-s
infusion of serotonin Ž10y6 and 10y5 molrl. and nitroglycerin Ž10y4 molrl. in hearts from sham-operated and
brain-dead rats. This was done in the presence or absence
of L-NAME Ž10y4 molrl. in the perfusion buffer. As fifth
group, hearts were taken from non-brain-dead rats receiving 100 mgrkg of L-NAME i.v. 30 min before excision of
the heart. Coronary flow was continuously measured with
the 2N in-line flow probe. The percentage change in
coronary flow was integrated during the last 120 s of the
infusion of serotonin or nitroglycerin and taken as measure
for the steady state effect of the vasoactive agents.
2.3. Data acquisition and management
All measured variables were continuously digitized at
1000 Hz ŽAT-MIO-16L9, National Instruments, Austin,
TX, USA. and stored on a 486DX4r100 MHz personal
computer, with a custom-made programme developed in
Labview 3.0 ŽNational Instruments, Austin, TX, USA..
Statistical analysis was performed with the statistical
software package Statistica 4.5 ŽStatSoft, Tulsa, OK, USA..
Data are expressed as means " s.e.m. For all tests used,
the a-level for statistical significance was set at 0.05.
Repeated measurements ANOVA was used to test for
significant differences in time for the haemodynamic variables. Two-way repeated measures ANOVA with group
and dose and an interaction term between these two as
independent factors was used for testing differences in the
dose–response curves for adrenaline between the control
and brain-death groups. ANOVA with post-hoc testing
with the LSD test was used for testing differences in
extravasation of Evans blue, and MANOVA for analysis of
the fifth protocol. Changes in basal coronary flow before
and after adding L-NAME were tested with a paired t-test.
P. Herijgers et al.r CardioÕascular Research 38 (1998) 98–106
3. Results
3.1. Haemodynamic changes after brain death
Baseline values were taken after 30 min of stabilization,
immediately before the intracranial balloon was inflated in
the brain death group. In sham-operated animals, heart rate
ŽHR., mean arterial blood pressure ŽMABP., left ventricular dPrdt max ŽLV dPrdt max ., left ventricular dPrdt min
ŽLV dPrdt min ., left ventricular end-diastolic and peak
systolic pressure, cardiac index ŽCI. and systemic vascular
101
resistance index ŽSVRI. were stable throughout the entire
measurement period. The effect of brain death induction
on these parameters can be seen in Fig. 1. Heart rate was
422 " 12 miny1 in baseline conditions, and after a transient bradycardia and tachycardia immediately after brain
death induction, continuously decreased to 350 " 8 miny1
1 h after brain death. Mean arterial blood pressure was
112 " 5 mmHg at baseline, rose to 184 " 10 mmHg 25 s
after brain death and rapidly declined to 50 " 3 mmHg 210
s after brain death, with a stabilization afterwards. The
same profile can be recognized in SVRI, LV peak systolic
Fig. 1. The effect of brain death Ž ns6. on heart rate, mean arterial blood pressure, left ventricular end-diastolic and peak systolic pressure, cardiac index,
systemic vascular resistance index, and left ventricular dPrdt in rats is shown. Brain death is induced at time 0. ) Time point from which the measured
value started to be significantly different from the baseline value before brain death. Differences during the transient changes early after brain death were
statistically significant from 15 s on; they are not marked since they are not essential for the present study. HR s heart rate; MABPs mean arterial blood
pressure; SVRIssystemic vascular resistance index; CI s cardiac index; dPrdt is the first derivative of left ventricular pressure in time.
102
P. Herijgers et al.r CardioÕascular Research 38 (1998) 98–106
control conditions. This parameter is known to be influenced by afterload, a parameter clearly influenced by brain
death.
3.2. Ex ÕiÕo myocardial contractility
Hearts excised 30 min after brain death or from shamoperated time-matched controls exhibited the same basal
contractility when tested in a crystalloid perfused Langendorff system. No differences in dose–response curve for
adrenaline Ž10y9 to 10y6 molrl. could be found between
the two groups for all parameters studied Žpeak systolic
pressure, left ventricular dPrdt, heart rate, and coronary
flow., and both in paced and unpaced conditions. The
effect of adrenaline on left ventricular dPrdt max and
dPrdt min when the hearts were paced at 420 miny1 is
shown in Fig. 3 as example. It is evident that no significant
differences exist between the control hearts and the hearts
from brain-dead rats.
3.3. Morphological alterations
Fig. 2. Upper panel: the effect of brain death in the rat on dPrdt at CPIP,
known to be a largely afterload independent measure of left ventricular
contractility, is depicted. Lower panel: the effect of brain death in the rat
on ŽdPrdt ma x .rP, a calculated variable largely independent of preload.
Brain death is induced at time 0. Both parameters are expressed as
percentage of the value immediately before induction of brain death
Ž ns6.. Differences during the transient changes early after brain death
were statistically significant from 15 s after induction of brain
death.) Time point from which the measured value started to be significantly different from the baseline value before brain death. CPIPs
common peak isovolumetric pressure.
No histological or ultrastructural damage could be detected in the hearts excised 1 or 4 h after brain death. At
the light microscopic level, the hearts showed a regular
pattern of structural organization that was identical to
normal hearts. No signs of degeneration typical of acute
ischaemia, such as the occurrence of contraction bands and
cellular oedema, were seen ŽFig. 4a.. Electron microscopy
revealed the presence of intact substructures. Mitochondria, which are the most sensitive subcellular organelles to
ischaemia, were intact ŽFig. 4b..
3.4. Endothelial barrier function
In none of the hearts examined histologically, could a
significant amount of extravasated Evans blue be detected.
pressure, and dPrdt max and dPrdt min . A transient rise in
LV end-diastolic pressure can be seen at the moment of
brain death, accompanied also by a rise in CVP Žnot
shown., suggestive of a central redistribution of the blood
volume. The CI was 9.6 " 0.6 mlrminr100 g at baseline,
decreased to 8.2 " 1.4 10 s after brain death, and afterwards increased to 12.0 " 0.9 1 h after brain death.
In Fig. 2, the evolution in dPrdt at CPIP and
ŽdPrdt max .rP is shown. These parameters are expressed
as percentage of the baseline value from the same experiment, since the differences in absolute values between the
individual experiments were relatively large when compared with the evolution of the parameter in time. Most
interesting was the comparison of dPrdt at CPIP before
brain death with its final value, since dPrdt at CPIP did
not correct for changes in preload that occurred early after
brain death as evidenced by the changes in LV end-diastolic pressure and CVP. ŽdPrdt max .rP, on the contrary,
was significantly lower late after brain death than in
Fig. 3. The effect of cumulative doses of adrenaline on left ventricular
dPrdt ma x and dPrdt min in isolated Langendorff-perfused hearts from
control or brain-dead rats Ž ns6.. The hearts are paced in the right atrium
at 420 miny1 . No significant differences between the groups can be
detected.
P. Herijgers et al.r CardioÕascular Research 38 (1998) 98–106
103
Fig. 4. Myocardium of a rat examined 1 h after brain death. a: light microscopy showing a completely normal organization of cardiomyocytes ŽCM. and
the extracellular space ŽES.. =360. b: electron microscopy demonstrating the intact structure of sarcomeres Žsm., mitochondria Žm. of cardiomyocytes and
capillary endothelium ŽCAP. =3200.
Fig. 5. The effects of nitroglycerin Ž10y4 molrl., and serotonin Ž10y6 and 10y5 molrl. on coronary flow is shown in 5 groups of Langendorff-perfused
hearts Žeach of ns6.. The bars express the mean percentage coronary vasodilation Žpositive. or vasoconstriction Žnegative. for the different agents, during
the last 120 s of infusion. COs control group with hearts isolated from sham-operated rats; BDs hearts isolated from brain-dead rats; COqL-NAMEs
hearts isolated from sham-operated rats, with 10y4 molrl L-NAME in the perfusion buffer; BDqL-NAMEs hearts isolated from brain-dead rats, with
10y4 molrl L-NAME in the perfusion buffer; L-NAME in vivos hearts isolated from rats that received 100 mgrkg L-NAME in vivo. Statistically
significant differences between the groups are indicated when P - 0.05.
104
P. Herijgers et al.r CardioÕascular Research 38 (1998) 98–106
In control animals, 11.06 " 1.19 mg extravasated Evans
blue per gram dry weight could be extracted, as compared
with 11.33 " 2.13 mg in the group with Evans blue injection immediately before induction of brain death, and
8.81 " .58 mg in the group with Evans blue injection 1 h
after brain death. No significant differences between the
groups could be detected.
3.5. Endothelial function
The coronary vasoactive effects of the tested drugs are
shown in Fig. 5. The vasoactive agents induced significantly different reactions, according to the group of hearts,
as is shown by the overall significance of the interaction
term drug = group when tested with MANOVA Ž P s
0.003.. The results of the post-hoc testing to see where
exactly the differences lie, are also shown in Fig. 5.
Important to notice is the fact that no significant differences exist between the brain death and its corresponding
control group. Adding L-NAME to the perfusion buffer
resulted in a significant decrease in basal coronary flow
Žfrom 15.3 " 1.0 to 11.9 " 1.1 mlrmin, P - 0.001.. After
L-NAME administration, a more pronounced vasodilation
is caused by nitroglycerin infusion than with control buffer,
but this just fails to reach the significance level required in
most pairwise comparisons. Serotonin 10y6 molrl also
induced vasodilatation when L-NAME was present in the
perfusion buffer, serotonin 10y5 molrl sometimes induced
vasoconstriction and sometimes vasodilatation resulting in
a large variation, both in the brain death and control group.
Remarkable was that only in vivo pretreatment with LNAME was able to inhibit serotonin-induced coronary
vasodilation, with a consistent vasoconstriction induced
both by 10y6 and 10y5 molrl serotonin administration.
4. Discussion
With this comprehensive series of experiments concerning the effect of brain death in the rat and its consequences
on myocardial function, we were able to clarify the apparent inconsistencies in published reports. On the one hand,
a severe haemodynamic collapse has been reported in the
rat after brain death and this collapse was attributed to
myocardial damage; but, on the other hand, normal basal
contractile function of hearts explanted 60 min after induction of brain death has also been reported w3x. No circulating or neurogenic cardiodepressant factor is present that
could eventually reconcile both observations w8x. In the
present study, brain death has a tremendous impact on the
haemodynamic situation in the rat as already largely described in previous studies w3,4,10x. An important difference between the present study and the study of Galinanes
˜
and Hearse w3x, however, was that the cardiac output did
not drop after brain death in the present study but, on the
contrary, slightly increased. This led us to the hypothesis
that the haemodynamic changes were not caused by intrinsic myocardial damage, but that these changes might largely
be the consequence of afterload reduction. This was further
corroborated by the stability after brain death of the afterload independent parameter of ventricular contractility
dPrdt at CPIP w11x. The reason for the difference in the
effect of brain death on cardiac output between our study
and that of Galinanes
and Hearse w3x is not entirely clear,
˜
but differences in anaesthetic regimen undoubtedly play an
important role. We have chosen the use of a-chloralose
and urethane because they produce a long-lasting stable
anaesthesia, without the need for additional doses of the
drugs, and because this anaesthesia preserves vascular
reactivity to phenylephrine and nitroglycerin w13x, thus,
probably, also preserving the vascular response to the
intense sympathetic stimulation at the moment of brain
death w10x. Furthermore, a comparable anaesthesia was
used in the study of Shanlin et al. w4x, a study that showed
myocardial damage after brain death in rats. Interestingly,
all haemodynamic parameters after brain death were similar in the study of Galinanes
and Hearse w3x and ours,
˜
despite the highly different starting conditions of cardiac
index, stroke volume index, and systemic vascular resistance index, and, to a lesser extent, heart rate. These
differences in starting value of the haemodynamic parameters, in which stroke volume plays an important role, can
be explained by the known abdominal vasoconstrictive
effects of urethane and chloralose w14x.
Intrinsic myocardial contractility was, furthermore,
studied in isolated Langendorff-perfused hearts. It was
previously shown, in a paracorporeal blood-perfused system, that the basal, unstimulated contractility of hearts
from brain-dead or control rats is equal w3x. In our set-up,
not only basal contractility was equal, but also the inotropic response towards adrenaline was exactly the same.
Galinanes
and Hearse w3x interpreted their results as the
˜
protective effect of explantation against the deleterious
effects of the brain death status. This, however, seems
unlikely since, in our experiments, the situation after brain
death had already stabilized before the hearts were harvested, and from unpublished experiments, we know that
this situation remains stable for at least 4 h. Since elegant
studies of the same group w8x have ruled out a stable
circulating cardiodepressant factor and found no evidence
of a neurogenic cardiodepressant factor, no intrinsic myocardial contractile damage seems to be present. This
interpretation is also compatible with our in vivo haemodynamic measurements, showing that the paramount difference is a strong decrease in afterload.
Histologically or ultrastructurally, we could not find
myocardial damage. On the one hand, this was not surprising given our results of in vivo haemodynamics and ex
vivo contractility, but on the other hand, this is in contradiction with the study of Shanlin et al. w4x, describing
multifocal microscopical myocardial necrosis, myocardial
contraction bands, and even smooth muscle contraction
P. Herijgers et al.r CardioÕascular Research 38 (1998) 98–106
bands in the coronary arteries. In that study, increased
intracranial pressure was maintained, however, for only 15
s, with an abrupt deflation of the intracranial balloon
afterwards. Furthermore, histological examination was only
performed in hearts, after Microfil injection through the
brachial artery to produce coronary artery casts, and without rinsing the coronary arteries of blood. Fixation was
with immersion and not with perfusion as in our experiments. These differences in methodology and tissue
preparatory procedures may account for the histological
damage observed in the latter study. The fact that the same
kind of lesions, although to a lesser extent, were seen in
control rats in the study of Shanlin et al. w4x makes
methodological difficulties even more likely to be the
cause of the difference. Although the myocardial damage
seen after acute stroke, often termed myocytolysis, and its
accompanying ECG changes w15x can be reproduced in rats
w16,17x, this is not necessarily in contradiction with our
results. In our model of brain death, the entire brain,
including brainstem, stops inevitably functioning since very
rapidly brain perfusion is abolished w10x, whereas in the
model of experimental stroke in rats by unilateral occlusion of the middle cerebral artery, the brainstem with the
major cardiocirculatory regulatory centres remains intact,
and even in the cerebral hemispheres, there is an asymmetry of sympathetic and ensuing cardiac consequences of
experimental stroke w16x.
Endothelial damage was hypothesized to be a possible
underlying pathophysiological mechanism, able to induce
the kind of myocardial damage described after brain death
in dogs w7x and rats w4x, although we were unable to
demonstrate this damage in our experiments in rats. The
first approach was to test the integrity of the coronary
endothelial lining. No important extravasation of macromolecules was evident, a fact suggestive of intact endothelial lining, and also suggestive of normal endothelial NO
production, since it is known that inhibiting NO synthase
with L-NAME induces endothelial leakage of Evans blue
w18x. There was even lower extravasation after brain death,
probably related to the lower arterial blood pressure after
brain death. Testing endothelium dependent coronary vasoreactivity by serotonin w19x did not show significant
differences between the brain death and the control group.
This implies that the overall endothelial NO production
and vascular smooth muscle cell reactivity toward this
compound remains intact. An interesting additional observation made in protocol 5 of this study was that only in
vivo pretreatment with L-NAME, but not addition of this
drug to the perfusion buffer, was able to prevent serotonin-induced coronary vasodilatation. Since basal coronary flow decreased significantly after the addition of
L-NAME to the perfusion buffer, an effective concentration of this drug was used during these experiments. This
suggests that: Ž1. L-NAME causes a specific endothelial
andror smooth muscle cell damage when administered in
vivo Žmaybe induced by the pronounced long-lasting
105
hypertension.; or Ž2. that serotonin-induced coronary vasodilatation is not entirely dependent on NO production; or
Ž3. that the involved enzyme cannot be blocked entirely by
this concentration of L-NAME.
What are the implications of this study for the clinical
cardiac transplantation programmes? It is clear that, contrary to what was found in this study, intrinsic myocardial
damage caused by brain death is observed in human
potential donor hearts w20x. This discrepancy can probably
be explained by species differences in autonomic nervous
system regulation, sensitivity of the coronary circulation or
the myocardium for catecholamines, but also by the heterogeneity in patterns of intracranial pressure rise in clinical circumstances. The results of this study, however,
caution against rashly turning down potential donor hearts
from donors that became haemodynamically unstable. It is
not justified to rely exclusively on standard haemodynamic
parameters in this decision-making, since an apparent
haemodynamic collapse per se is not necessarily indicative
of intrinsic myocardial damage.
To conclude this study, we can state that intrinsic
myocardial damage, if present at all, contributes only
minimally to the haemodynamic changes seen after brain
death in the rat. This, of course, raises the question of the
exact pathophysiological mechanism of this so-called collapse.
Acknowledgements
P.H. is an ‘Aspirant van het Fonds voor Wetenschappelijk Onderzoek, Vlaanderen, Belgium’. This work was
supported in part by a grant from the ‘Fonds voor Wetenschappelijk Onderzoek, Vlaanderen, Belgium’.
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