Full text of DOI:10.1177/021849230000800109

MYOCARDIAL DAMAGE INDUCED BY UNCONTROLLED REOXYGENATION
ORIGINAL CONTRIBUCToIOrnNo
MYOCARDIAL DAMAGE INDUCED BY
UNCONTROLLED REOXYGENATION
Antonio F Corno, MD, FRCS, Giuseppina Milano, PhD,
Michele Samaja, PhD1, Ludwig K von Segesser, MD
Department of Cardiovascular Surgery
University Hospital of Vaud
Lausanne, Switzerland
1S Paolo Hospital, University of Milan
Milan, Italy
ABSTRACT
To evaluate myocardial impairment induced by uncontrolled reoxygenation, the
effects of hypoxia-reoxygenation were compared with ischemia-reperfusion in
isolated rat hearts. After stabilization, 2 groups (n = 8) of Langendorff-perfused
rat hearts were exposed to 40 minutes of ischemia (10% of baseline flow) or
hypoxia (10% of baseline oxygen content) followed by a sudden return to baseline
conditions (reperfusion or reoxygenation). The O2 content was identical for the two
groups during baseline conditions, O2 shortage, and O2 readmission. Metabolic
(lactate production) and functional parameters (heart rate, peak systolic pressure,
left ventricular developed pressure, maximal contraction and relaxation rates,
end-diastolic pressure, coronary perfusion pressure) were recorded at the end of
stabilization, after O2 deficiency, and after 2 minutes of reoxygenation. Systolic
function was significantly depressed after ischemia (p < 0.0001) but completely
recovered to baseline values after 2 minutes of reperfusion. In contrast, systolic
function was less severely depressed after hypoxia but failed to return to baseline
after 2 minutes of reoxygenation. Diastolic function, unchanged during ischemia-
reperfusion, remained significantly impaired during hypoxia-reoxygenation.
(Asian Cardiovasc Thorac Ann 2000;8:34–7)
INTRODUCTION
Surgical repair of cyanotic congenital heart defects is
performed under cardiopulmonary bypass (CPB) at an
early age with increasing frequency. Postoperative
tension (arterial PO2) to 300 to 400 mm Hg by priming
the CPB circuit with hyperoxic fluid. This causes an
unintended “reoxygenation phenomenon” that adds to the
subsequent intraoperative stress when surgical ischemia
myocardial dysfunction is the major cause of morbidity is needed for intracardiac repair.
and mortality despite adequate anatomic correction and
In spite of several experimental observations already
it is more severe than after surgery for acquired lesions
in adults with normoxic conditions. The main difference
in intraoperative management of cyanotic children is the
reversal of hypoxemia occurring at the beginning of CPB,
preceding the surgical ischemia necessary to perform the
intracardiac repair. The conventional method of starting
reported by our group and others, the role and nature of
the reoxygenation phenomenon in clinical practice has
yet to be fully established.1–8 This experimental study
was devised to evaluate the metabolic and functional
effects on isolated rat heart of uncontrolled sudden oxygen
CPB in cyanotic children is to suddenly raise the oxygen readmission after a period of oxygen reduction, by
For reprint information contact:
Antonio F Corno, MD, FRCS Tel: 41 21 314 2280 Fax: 41 21 314 2278 email: antonio.corno@chuv.hospvd.ch
University Hospital of Vaud, Department of Cardiovascular Surgery, Rue du Bugnon 46, Lausanne CH1011, Switzerland.
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MYOCARDIAL DAMAGE INDUCED BY UNCONTROLLED REOXYGENATION
comparing reoxygenation after hypoxia with reperfusion At the end of the stabilization period, the hearts were
after ischemia.
exposed to either 40 minutes of ischemia by reducing the
coronary flow to 1.5 mL·min–1 (n = 8), or 40 minutes of
hypoxia by reducing the PO2 to 67 mm Hg. At the end
of ischemia or hypoxia, the hearts were reperfused or
reoxygenated for 2 minutes under the same conditions as
during stabilization. None of the hearts were paced at any
stage. Metabolic and functional measurements were taken
at the end of stabilization (baseline), after ischemia or
hypoxia, and after the reperfusion or reoxygenation
periods. An aliquot of left ventricle was then taken and
weighed (wet weight). The sample was placed in a 65°C-
oven and the dry weight was determined after 24 and 48
hours. Myocardial water content was calculated as (wet
weight – dry weight)/wet weight × 100.
MATERIALS AND METHODS
Male Sprague-Dawley rats (weighing 220 to 280 g) fed
ad libitum, were anesthetized by intraperitoneal injection
of heparinized thiopental sodium 100 mg·kg–1. The hearts
were rapidly excised, placed in isotonic saline (20°C) to
facilitate mounting, and immediately perfused via the
aorta with Krebs-Henseleit buffer containing 2.5 mM
free Ca2+ and 11 mM glucose, pH 7.4, at 37°C. The
ischemic period never exceeded 45 seconds. The perfusion
fluid was equilibrated with either 94% O2 and 6% CO2
or 10% O2, 6% CO2, and 84% N2, giving a PO2 of 670
or 67 mm Hg at constant PCO2 (43 mm Hg). A peristaltic
pump (Ismatec SA, Labortechnik-Analytik, Glattbrugg-
Zurich, Switzerland) delivered the fluid at fixed flow (15
or 1.5 mL·min–1) to an 8-µm pore-size filter (MSI,
Westboro, MA, USA), the micro-oxygenator (Dideco,
Mirandola, Italy), the pre-heater, and the aortic cannula.
All components of the perfusion system were kept at
37°C by an external water bath (Bioblok Scientific, Niles,
IL, USA). The perfusate was not recirculated. A latex
balloon was introduced into the left ventricle through the
left atrial appendage and connected to a pressure transducer
(model mpc-500; Miller Instruments Inc., Houston, TX,
USA) to monitor heart rate (HR), left ventricular
end-diastolic pressure (LVEDP), left ventricular developed
pressure (LVDP) and maximal rates of heart contraction
(+dP/dtmax) and relaxation (–dP/dtmax). A similar pressure
transducer was connected above the aortic cannula to
monitor coronary perfusion pressure to determine the
adequacy of myocardial perfusion. Arterial inflow and
coronary sinus return (from an inflow cannula in the
pulmonary artery) were collected to measure lactate
concentrations. Data were recorded and analyzed by a
LabVIEW system 4.1 for PC (National Instruments,
Austin, TX, USA).
Data were expressed as mean ± standard error. To detect
significant differences, the two-tailed Student’s t test for
unpaired observations was used as appropriate. The level
of significance was set at p = 0.05.
RESULTS
No significant differences were observed between the
two groups of hearts at the end of 30 minutes of
stabilization, with regard to metabolic and functional
parameters (Table 1). The effects of ischemia and reper-
fusion on systolic function are shown in Figure 1. At the
end of ischemia, HR, LVDP, LVDP × HR, and +dP/dtmax
were significantly depressed below baseline levels (p <
0.0001). However, after 2 minutes of reperfusion, the
systolic performance of the ischemic hearts significantly
improved compared with the end of ischemia (p < 0.0001)
and returned to the baseline values. The effects of hypoxia
and reoxygenation on systolic function are shown in
Figure 2. At the end of hypoxia LVDP, LVDP × HR,
and +dP/dtmax were significantly depressed from baseline
(p < 0.0001) but less (p < 0.05) than in the ischemic
hearts. After 2 minutes of reoxygenation, the systolic
performance of hypoxic hearts had improved (p < 0.05)
but remained significantly depressed compared to the
baseline values (p < 0.0001).
The hearts (n = 16) were stabilized for 30 minutes at a
coronary flow rate of 15 mL·min–1 and PO2 of 670 mm
Hg. The volume of the ventricular balloon, adjusted to
achieve LVEDP of 10 ± 0.1 mm Hg, was kept constant. The diastolic function (LVEDP) remained unchanged
Table 1. Functional and Metabolic Data at the End of the Stabilization Period
Parameter
Ischemic Group (n = 8)
Hypoxic Group (n = 8)
Heart rate (beats·min–1
)
267 ± 6
117 ± 5
264 ± 9
123 ± 5
Peak systolic pressure (mm Hg)
+dP/dt (mm Hg·sec–1
)
3768 ± 279
2130 ± 85
10.2 ± 0.1
65 ± 3
4125 ± 171
2236 ± 82
10.4 ± 0.1
72 ± 3
–dP/dt (mm Hg·sec–1
)
LVEDP (mm Hg)
Coronary perfusion pressure (mm Hg)
LVDP (mm Hg)
107 ± 5
112 ± 5
LVDP × heart rate (mm Hg × 10–3·min–1)
28.5 ± 1.3
< 0.1
29.6 ± 1.5
< 0.1
Lactate (mmol·L–1
)
+dP/dt = rate of contraction, –dP/dt = rate of relaxation, LVDP = left ventricular developed pressure, LVEDP= left ventricular end-diastolic pressure.
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MYOCARDIAL DAMAGE INDUCED BY UNCONTROLLED REOXYGENATION
Corno
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Hypoxia
Ischemia
Reoxygenation
Reperfusion
*
*
*
*
NS
*
LVDP
LVDP × HR
+dP/dtmax
LVDP
LVDP × HR
+dP/dtmax
Figure 1. Left ventricular developed pressure (LVPD), left ventricular
developed pressure × heart rate (LVDP × HR), and maximal rate of
contraction (+dP/dtmax) as percentages of the baseline values, at the end
of 40 minutes of ischemia and after 2 minutes of reperfusion. *p <
0.0001.
Figure 2. Left ventricular developed pressure (LVPD), left ventricular
developed pressure × heart rate (LVDP × HR), and maximal rate of
contraction (+dP/dtmax) as percentages of the baseline values, at the end
of 40 minutes of hypoxia and after 2 minutes of reoxygenation. *p < 0.05.
Hypoxia
Ischemia
compared to the baseline at the end of ischemia and after
reperfusion (Figure 3A). In contrast, LVEDP was
significantly increased at the end of hypoxia with respect
to baseline (p < 0.0001) and remained elevated at the end
of reoxygenation (Figure 3B).
35
30
25
20
15
10
5
35
30
25
20
15
10
5
Reoxygenation
Reperfusion
NS
The lactate concentration in the perfusate was higher at
the end of ischemia than at baseline (1.1 ± 0.1 mmol·L–1
versus < 0.10 mmol·L–1, respectively) and returned to
baseline after 2 minutes of reperfusion. At the end of
hypoxia, the perfusate lactate concentration was higher
than baseline (0.4 ± 0 mmol·L–1 versus < 0.1 mmol·L–1,
respectively) but it was lower than the level found after
ischemia. After reoxygenation it returned to baseline, as
observed after reperfusion. No significant differences were
observed between ischemic and hypoxic hearts with regard
to myocardial water content (80% ± 5% and 78% ± 3%,
respectively).
NS
0
0
Ischemic Hearts
Hypoxia Hearts
A
B
Figure 3. Left ventricular end-diastolic pressure (A) after ischemia-
reperfusion and (B) after hypoxia-reoxygenation. NS = not significant.
DISCUSSION
The negative effects of uncontrolled reoxygenation
(reoxygenation phenomenon) on myocardial metabolism
and function have been demonstrated previously by both
experimental and clinical studies.1–9 Since the reoxy-
genation phenomenon seems to occur in the very early
period after oxygen readmission and the previous studies,
including ours, evaluated myocardial function and
metabolism after a prolonged period of reoxygenation,
we decided to analyze the changes during the first 2
function after both ischemia and hypoxia, although to
different extents, confirming that anaerobic metabolism
did not compensate for reduced oxygen availability,
independent of the rate of flow maintained; diastolic
function, unaffected by ischemia-reperfusion, remained
severely impaired during hypoxia-reoxygenation, possibly
due to reduced left ventricular compliance as a result of
increased myocardial water content induced by the full
minutes of oxygen readmission. This study on isolated flow maintained during hypoxia; and whereas uncontrolled
perfused rat hearts allowed the following observations: reperfusion provided prompt recovery of ischemic hearts,
the same degree of oxygen deficit impaired systolic systolic function after hypoxia remained severely impaired
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3. Samaja M, Casalini S, Allibardi S, Corno A, Chierchia S.
Regulation of bioenergetics in O2-limited isolated rat hearts.
J Appl Physiol 1994;77:2530–6.
on reoxygenation and this might be aggravated by the
persisting diastolic dysfunction. These observations
confirm the importance of the reoxygenation phenomenon.
Experimental studies performed by our group as well as
by other researchers, on the negative myocardial effects
of acute reoxygenation have shown that controlled re-
oxygenation, avoiding a sudden rise of arterial PO2 from
a low hypoxic level to full oxygenation, reduced the re-
oxygenation damage.1,10,11
4. Samaja M, Motterlini R, Allibardi S, Casalini S, Merati G,
Corno A, et al. Myocardial metabolism and function in
acutely ischemic and hypoxemic isolated rat hearts. J Mol
Cell Cardiol 1995;27:1213–8.
5. Buckberg GD. Studies of hypoxemic/reoxygenation injury:
linkage between cardiac function and oxidant damage. J
Thorac Cardiovasc Surg 1995;110:1164–70.
6. Guarnieri C, Flamigni F, Caldarera CM. Role of oxygen
in the cellular damage induced by reoxygenation of hypoxic
heart. J Mol Cell Cardiol 1980;12:797–808.
We understand the limitations of our current study. Firstly,
the effects were evaluated only on isolated hearts and we
are aware of the potential effects of reoxygenation on the
entire body, particularly the CPB-generation of reactive
oxygen intermediates that alter neutrophils, complement,
and arachidonic acid metabolism. Secondly, the effect of
reoxygenation was assessed after acute hypoxia, whereas
children with congenital heart defects suffer from chronic
hypoxia. To overcome this limitation, a further study on
chronic hypoxia is planned. In addition, the hearts in this
study were normal at the outset but children come to
surgery after a period of ventricular pressure and/or volume
overload with hypertrophic and/or dilated hearts. To
overcome this limitation, the same evaluations will be
undertaken in hearts with induced hypertrophy and/or
dilatation. However, the clinical inference from these
experimental observations is that uncontrolled reoxy-
genation at the start of CBP in cyanotic children should
be avoided. In fact, preliminary reports are already
available on our experience and that of another group, of
controlled reoxygenation in cyanotic children undergoing
surgical repair, with encouraging results.12–14
7. Hearse DJ, Humphrey SM, Nayler WG, Slade A, Border
D. Ultrastructural damage associated with reoxygenation
of the anoxic myocardium. J Mol Cell Cardiol 1975;7:
315–24.
8. Ihnken K, Morita K, Buckberg GD, Matheis G, Sherman
MP, Allen BS, et al. Studies of hypoxemic/reoxygenation
injury: without aortic clamping. Evidence for reoxygenation
damage. J Thorac Cardiovasc Surg 1995;110:1171–81.
9. del Nido PJ, Mickle DAG, Wilson GJ, Benson LN, Weisel
RD, Coles JG, et al. Inadequate myocardial protection
with cold cardioplegic arrest during repair of tetralogy of
Fallot. J Thorac Cardiovasc Surg 1988;95:223–9.
10. Corno AF, Samaja M, Casalini S, Allibardi S. The effects
of the rate of reoxygenation on the recovery of hypoxemic
hearts. J Thorac Cardiovasc Surg 1995;109:1250–1.
11. Morita K, Ihnken K, Buckberg GD. Studies of hypoxemic/
reoxygenation injury: with aortic clamping. Delay of
cardiac reoxygenation damage in the presence of cyanosis:
a new concept of controlled cardiac reoxygenation. J Thorac
Cardiovasc Surg 1995;110:1265–73.
12. Corno AF, da Cruz E, Lal A, Milella L, Wilson N.
“Controlled reoxygenation” for cyanotic patients. In: Imai
Y, Momma K, editors. Proceedings of 2nd World Congress
of PCCS. New York: Futura, 1998:1127–9.
ACKNOWLEDGMENT
We thank the manufacturer, Dideco, for providing specially
designed micro-oxygenators for this study.
13. Corno AF, von Segesser LK. Is hypothermia necessary in
pediatric cardiac surgery? Eur J Cardio-thorac Surg
1999;15:110–1.
REFERENCES
1. Corno AF, Motterlini R, Tarantola M, Samaja M.
Reperfusion injury of the hypoxemic heart: role of acute
reoxygenation before ischemia. In: Grossi A, Donatelli F,
Corno AF, Brodman R, editors. Cardiology and cardiac
surgery: current topics. New York: Futura, 1992:379–96.
14. Allen BS, Rahman S, Ilbawi MN, Kronon M, Bolling KS,
Halldorsson AO, et al. Detrimental effects of cardio-
pulmonary bypass in cyanotic infants: preventing the
reoxygenation injury. Ann Thorac Surg 1997;64:1381–7.
2. Corno AF, Samaja M. The reoxygenation phenomenon. J
Thorac Cardiovasc Surg 1993;105:373–4.
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