Eur J Cardiothorac Surg 2007;31:899-905. doi:10.1016/j.ejcts.2007.01.049
Copyright © 2007, European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved
Brain oxygen and metabolism is dependent on the rate of low-flow cardiopulmonary bypass following circulatory arrest in newborn piglets
Peter Pastuszkoa,*,
Huiping Liub,
Alberto Mendoza-Paredesb,
Steven E. Schultzc,
Scott D. Markowitzd,
William J. Greeleyd,
David F. Wilsonb,
Anna Pastuszkob
a Department of Surgery, The University of Oklahoma, Oklahoma City, OK, United States
b Department of Biochemistry & Biophysics, The University of Pennsylvania School of Medicine, Philadelphia, PA, United States
c Department of Pediatrics, The University of Miami, Miami, FL, United States
d Department of Anesthesiology & Critical Care, The Children's Hospital of Philadelphia, Philadelphia, PA, United States
Received 14 September 2006;
received in revised form 10 December 2006;
accepted 15 January 2007.
* Corresponding author. Address: The University of Oklahoma Health Sciences Center, Thoracic and Cardiovascular Surgery, 920 Stanton L. Young Blvd., Suite WP2230, Oklahoma City, OK 73104, United States. Tel.: +1 405 271 5789; fax: +1 405 271 3288. (Email: peter-pastuszko{at}ouhsc.edu).
 |
Abstract
|
|---|
Objective: To determine the optimum rate of low-flow hypothermic cardiopulmonary bypass (LF), following circulatory arrest (DHCA) on brain oxygenation (bO2), extracellular dopamine (DA), phosphorylation of select neuroregulatory proteins responsible for neuronal injury, and survival following ischemic brain injury: CREB, Erk1/2, Akt, Bcl-2, and Bax. Methods: The piglets were placed on cardiopulmonary bypass (CPB) and cooled to 18 °C. They were then subjected to 30 min of DHCA followed by 1 h of LF at 20, 50, or 80 ml/(kg/min), rewarmed, separated from CPB, and maintained for 2 h. The bO2 was measured by quenching of phosphorescence; DA by microdialysis; phosphorylation of CREB, ERK1/2, Akt, Bcl-2, and Bax by Western blots. The results are means ± SD for seven experiments. Results: Pre-bypass bO2 was 47.4 ± 4.2 mmHg and decreased to 1.9 ± 0.8 mmHg during DHCA. At the end of LF at 20, 50, and 80 ml/(kg/min), bO2 was 11.8 ± 1.6, 26 ± 1.8, and 33.9 ± 2.6 mmHg, respectively. The DA increased 510-fold relative to control (p
< 0.001) by 15 min of LF-20 with maximum increase occurring at 45 min. With LF-50, increase in DA was not statistically significant and no increase was observed when LF-80 was used. Bcl-2 immunoreactivity increased after LF-50 and LF-80 (140 ± 14.5%, p
< 0.05 and 202 ± 34%, p
< 0.05, respectively). Neither flow increased Bax immunoreactivity. The ratio of Bcl-2/Bax, pCREB, pAkt, pErk increased significantly with increasing the flow rate of LF. Conclusions: The protective effect of LF following DHCA on brain metabolism is dependent on the flow rate. Flow-dependent increase in pCREB, pErk1/2, pAkt, increase in Bcl-2/Bax, and decrease in DA indicated that to minimize DHCA-dependent neuronal injury, LF flow should be above 50 ml/(kg/min).
Key Words: Newborn • Brain injury • Cardiopulmonary bypass • Circulatory arrest • Oxygen
|
1. Introduction
|
|---|
Cardiopulmonary bypass (CPB) and prolonged deep hypothermic circulatory arrest (DHCA) may result in detrimental effects in young, particularly neonatal patients. The early, enthusiastic use of DHCA, particularly in neonates, has been tempered by the finding of significant neurological morbidity associated with its prolonged exposure observed in long-term follow up studies. Stroke, cognitive, and neuropsychological dysfunction, retinal microvascular pathology, impaired level of consciousness, seizures, and spinal cord injury, all can occur after cardiopulmonary bypass [1–4]. Some of the contributing factors of these injuries can be due to DHCA-dependent impaired recovery of cerebral blood flow and cerebral oxygen metabolism [5–9]. These findings have led surgeons to limit, or even eliminate, DHCA and shift to other perfusion techniques such as low-flow cardiopulmonary bypass (LF), intermittent perfusion, and selective cerebral perfusion [10–16]. Another promising strategy for mitigating adverse effects of prolonged DHCA is to use a shorter duration of DHCA followed by LF. However, as for the other techniques, whether this approach affords significant protection from neuronal injury is unknown. Furthermore, the optimum rate of LF has not been determined.
The objective of this study was to examine the optimum CPB flow rate, when initiated after a 30 min period of DHCA, on brain tissue oxygenation, dopamine release, and the activity of select neuronal regulatory proteins. Dopamine release was chosen because this neurotransmitter is widely believed to play a crucial role in the physiology of brain function, and excess release of dopamine following circulatory arrest can play a major role in mediating neuronal damage. The activities of specific neuronal regulatory proteins that play a crucial role during neurodevelopment and are important determinants of the extent of neuronal cell survival and injury were studied. These regulatory proteins include cAMP response element binding protein (CREB), protein kinase B (Akt), extracellular signal-regulated kinase 1/2 (Erk1/2), Bcl-2, and Bax. We hypothesized that the higher CPB flow rates, after a brief duration of DHCA, would improve or mitigate any harmful effects of circulatory arrest, as determined by brain oxygenation, dopamine release, and regulatory protein activity.
|
2. Methods
|
|---|
2.1 Animal model
Newborn piglets, 3–5 days old (1.4–2.5 kg) were anesthetized with halothane, and a tracheotomy was performed. The piglets were then placed on a ventilator, and anesthesia was maintained with fentanyl, isoflurane 0.5%, and pancuronium. Femoral venous and arterial cannulas were placed for the collection of blood samples and for monitoring blood pressure. With the head of the animal in a stereotaxic holder, the scalp was removed and a cranial window approximately 8 mm in diameter was created over the right parietal hemisphere for measurement of cortical oxygenation. A small hole was drilled over the left parietal hemisphere for implantation of a microdialysis probe into the left striatum. After a 2-h stabilization period, cardiopulmonary bypass was initiated. Following bypass, the animals were recovered for 2 h and then euthanized with 4 M KCl.
All animal procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and have been approved by the local Animal Care Committee.
2.2 CPB technique
The circuit was primed with Plasmalyte-A and then 25% albumin was added to the circuit. Donor whole blood was added to maintain a hematocrit of 25–30%. Heparin (1000 units), fentanyl (50 mcg), pancuronium (1 mg), CaCl2 (500 mg), methylprednisolone (60 mg), cefazolin (100 mg), furosemide (2 mg), and NaHCO3 (25 meq.) were then added to the pump prime. A membrane oxygenator (Lilliput) was used as well as a roller pump system (Cobe) and arterial filter (Terumo). For CPB, a median sternotomy was performed. Prior to cannulation, 500 units of heparin was administered i.v. The ascending aorta as well as the right atrial appendage were cannulated. Full CPB flow rate was set at 150 ml/(kg/min). 
-Stat blood gas management was performed in all experiments.
2.3 Experimental protocol
All animals were cooled to a nasopharyngeal temperature of 18 °C over a 30 min period. A total of 28 newborn piglets, 2–4 days of age (1.4–2.5 kg) were randomly assigned to either control-sham (n
= 7), 30 min of DHCA followed by 1 h of LF at either 20 ml/(kg/min) (LF-20, n
= 7), 50 ml/(kg/min) (LF-50, n
= 7), or 80 ml/(kg/min) (LF-80, n
= 7), respectively. All animals were then rewarmed during a 30 min period, separated from CPB and recovered for 120 min under anesthesia, and then euthanized with 4 M KCl. The control animals did not undergo cardiopulmonary bypass. They were anesthetized and then had a sham operation. After euthanasia, the striata were immediately dissected from the brain and frozen for later analysis.
2.4 Measurements of oxygen pressure and oxygen distribution by the oxygen-dependent quenching of phosphorescence
Cortical oxygen pressure was measured using oxygen-dependent quenching of phosphorescence. The technical basis for determining the distribution of oxygen in the microcirculation of tissue from the distribution of phosphorescence lifetimes in the serum of blood has been described in detail [17,18]. The cranial window was prepared, bone wax used to seal the vessels in the bone tissue, and the dura was left intact. A small amount of physiological saline was placed on the dura and then it was covered with a sheet of clear plastic film. The excitation and collection light guides (3 mm diameter fiber bundles with center-to-center distance of about 7 mm) were brought very close (<1 mm) to the plastic film.
A near-infrared oxygen-sensitive phosphor (Oxyphor G2) was injected i.v. at approximately 1.5 mg/kg. The measurements were made using a multi-frequency phosphorescence lifetime instrument (PMOD 5000). The LED excitation lamp (635 nm), modulated by the sum of 37 sinusoidal waves with frequencies spaced between 100 Hz and 40 kHz, was carried to the tissue through a 3 mm light guide. The phosphorescence (
max
= 790 nm) emitted from the tissue was collected through a second light guide, placed against the tissue at approximately 8 mm (center-to-center) from the excitation light guide. This positioning of the light guides allowed effective sampling of brain tissue oxygenation down to approximately 6 mm under the neocortical surface. The phosphorescence signal was amplified, digitised, and analyzed to give distribution of phosphorescence lifetimes (oxygen histogram) in the volume of tissue sampled by the light. Since there were substantial differences among animals with respect to collected phosphorescence, etc., the oxygen histograms were normalized to have the same total amount of signal (integral) for oxygen pressures less than 140 mmHg. Using this method, oxygen measurements have been made over periods of several hours without observable injury to the tissue or evidence of measurement-induced alterations in the oxygen distributions in the tissue.
2.5 Measurement of striatal extracellular levels of dopamine by microdialysis
The extracellular level of dopamine in striatum was measured by in vivo microdialysis as described earlier [15]. Following a 2 h period of stabilization, the dialysis samples were collected at 15 min intervals during the bypass and post-bypass recovery and immediately analyzed for dopamine level by high-pressure liquid chromatography with electrochemical detector. Identification and quantitation of dopamine were conducted by comparison with chromatograms of standard solution of dopamine. The efficiency of the microdialysis probe was determined in vitro at 18 and 37 °C. The levels of dopamine in the dialysate are presented after correction for relative recovery by the microdialysis probe.
2.6 Western blotting
The Western blots of CREB, ERK1/2, Bcl-2, Bax, and Akt were performed as described earlier [19]. The specific antibodies used for incubation with striatal membranes were Erk1 (cross-reacting with Erk2), Bax (N-20), Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); phospho-Erk1/2, phospho-Akt, Akt (Cell Signaling Technology, Beverly, MA, USA); beta-actin (ABCAM, Cambridge, MA, USA); anti-phosphorylated CREB antibodies (a-p-CREB; Upstate Biotechnology, NY, USA; 1:200). Beta-actin antibody served as a loading control.
2.7 Data analysis
Autoradiographic films were analyzed using Scion Image software (NIH). Each blot contained two sets of samples, one for an experimental group and one for the control group. The data were normalized to the values obtained for the untreated control group (assigned a value of 100). Statistical analysis was performed using one-way analysis of variance followed by Mann–Whitney test. A p
< 0.05 was considered statistically significant.
The cortical oxygen pressure and extracellular dopamine values are expressed as means ± SD for seven experiments. Statistical significance was determined using one-way analysis of variance with repeated measures by Wilcoxon signed-rank test. A p
< 0.05 was considered statistically significant.
|
3. Results
|
|---|
3.1 Dependence of cortical oxygen pressure on flow rate of low-flow bypass following circulatory arrest in newborn piglets
Pre-bypass cortical oxygen pressure was 47.4 ± 4.2 mmHg, which decreased to 1.9 ± 0.8 mmHg during 30 min of DHCA. During LF following DHCA, cortical oxygen increased with time, and this increase was dependent on the rate of flow (Fig. 1
). At the end of a 60 min period of LF at flows of 20, 50, and 80 ml/(kg/min), cortical oxygen pressures were 11.8 ± 1.6, 26 ± 1.8, and 33.9 ± 2.6 mmHg, respectively.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1. Effect of DHCA and LF with different flow rates on cortical oxygen in newborn piglets. The cortical oxygen concentration was measured using the oxygen-dependent quenching of phosphorescence. The results are expressed as the means ± SD for seven experiments. a
p
< 0.05–0.001 for significant difference from control (sham-operated) values; b
p
< 0.05 for significant difference between LF-20 and LF-50; c
p
< 0.05 for significant difference between LF-50 and LF-80 as determined by one-way analysis of variance, followed by the Wilcoxon signed-rank test.
|
|
3.2 Effect of DHCA with LF on extracellular striatal level of dopamine
The level of extracellular dopamine in striatum increased by 510 times higher than the control by 15 min of LF-20 with maximum increase occurring at 45 min (785-fold increase) (Fig. 2
). The time before the increase in extracellular dopamine is similar to that for DHCA, described in our earlier study. When a flow of 50 ml/(kg/min) was used, the increase in extracellular dopamine was much less than for LF-20, rising to 39 times higher than control at 15 min and to a maximum of 234-fold higher than control at 30 min. Further increase in the flow to 80 ml/(kg/min) completely eliminated the increase of extracellular dopamine.
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 2. Changes in extracellular striatal dopamine level during DHCA and LF. The three measurements of dopamine during pre-bypass period were averaged and the value considered as the baseline (100%). The results are means ± SD for seven experiments. a
p
< 0.05; b
p
< 0.001 for significant difference from control (sham-operated) values as determined by one-way analysis of variance, followed by the Wilcoxon signed-rank test.
|
|
3.3 Levels of Bcl-2 and N-Bax in striatum following DHCA and different rates of LF
As shown in Fig. 3
, Bcl-2 immunoreactivity was decreased after DHCA with LF-20 (71.0 ± 7.4% as compared to control, p
< 0.05) but was significantly increased after LF-50 and LF-80 (140 ± 14.5%, p
< 0.05 and 202 ± 34%, p
< 0.05, respectively). Neither, LF-20, LF-50, nor LF-80 induced an increase in N-Bax immunoreactivity (115.2 ± 11.5%, 112.5 ± 12.3%, and 104.5 ± 9.3% as compared to control, respectively) (Fig. 4
). The calculated ratio of Bcl-2/Bax increased with increase in the flow rate of LF following DHCA. At a flow rate of 20 ml/(kg/min), the ratio was 0.6, whereas when the flow rate was increased to 50 and 80 ml/(kg/min), the ratios increased to 1.3 and 2, respectively (Fig. 5
).
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 3. Level of Bcl-2 in striatal tissue measured after 2 h recovery following DHCA and LF at different rates. The results are means ± SD for seven experiments. The data are expressed in percent to the corresponding values (100%) obtained for the control group of piglets. a
p
< 0.05 for significant difference from sham-operated animals; b
p
< 0.05 for significant difference between LF-20 and LF-50 and between LF-50 and LF-80, as determined by one-way analysis of variance, followed by Mann–Whitney test. p
< 0.05 was considered statistically significant.
|
|
View larger version (44K):
[in this window]
[in a new window]
|
Fig. 4. Level of N-Bax in striatal tissue measured after 2 h recovery following DHCA and LF at different rates. The results are means ± SD for seven experiments. The data are expressed in percent to the corresponding values (100%) obtained for the control group of piglets.
|
|
View larger version (39K):
[in this window]
[in a new window]
|
Fig. 5. Bcl-2/N-Bax ratio in striatal tissue measured after 2 h recovery following DHCA and LF at different rates.
|
|
3.4 Levels of phosphorylated CREB in striatal tissue measured after 2 h recovery following DHCA and LF
Western blots of proteins isolated from striatal tissues of piglets from control group and the groups subjected to DHCA + LF-20, LF-50, and LF-80, probed with anti-pCREB antibodies, are presented in Fig. 6
. The phospho-CREB immunoreactivity in striata from these three experimental groups of animals was increased when compared to the sham-operated group of animals (4.561 ± 1.281%, p
< 0.01; 5.670 ± 2.197%, p
< 0.01 and 7.158 ± 2.046%, p
< 0.005, respectively). The degree of increase was dependent on the flow rate during low-flow bypass and was highest in LF-80 group.
View larger version (42K):
[in this window]
[in a new window]
|
Fig. 6. Levels of phosphorylated CREB in striatal tissue measured after 2 h recovery following DHCA and LF at different rates. The results are means ± SD for seven experiments. The data are expressed in percent to the corresponding values (100%) obtained for the control group of piglets. a
p
< 0.05 for significant difference from sham-operated animals; b
p
< 0.05 for significant difference between LF-20 and LF-80, as determined by one-way analysis of variance, followed by Mann–Whitney test. p
< 0.05 was considered statistically significant.
|
|
3.5 Levels of Akt in striatum following DHCA and different rates of LF
As can be seen in Fig. 7
, the phospho-Akt (pAkt) immunoreactivity increased in all experimental groups, and percent of increase depended on the rate of low flow used following DHCA. LF-20, LF-50, and LF-80 induced a significant increase in pAkt immunoreactivity (169 ± 52%, p
< 0.05; 207 ± 45%, p
< 0.05; 234 ± 55%, p
< 0.05) as compared to control. The differences in the increase in pAkt between LF-20 and LF-50 were not statistically significant but were statistically significant in the increase between LF-20 and LF-80.
View larger version (50K):
[in this window]
[in a new window]
|
Fig. 7. Levels of phosphorylated Akt in striatal tissue measured after 2 h recovery following DHCA and LF at different rates. The results are means ± SD for seven experiments. The data are expressed in percent to the corresponding values (100%) obtained for the control group of piglets. a
p
< 0.05 for significant difference from sham-operated animals; b
p
< 0.05 for significant difference between LF-20 and LF-80, as determined by one-way analysis of variance, followed by Mann–Whitney test. p
< 0.05 was considered statistically significant.
|
|
3.6 Levels of phosphorylated Erk1/2* in striatal tissue measured after 2 h recovery following DHCA and different rates of LF
The changes in the phospho-Erk (pErk) immunoreactivity in all experimental groups of animals are shown in Fig. 8
. The pErk1/2 immunoreactivity increased in all experimental groups, but the highest increase was observed in LF-80 group. Following LF-80, the increases in pErk1 and pErk2 immunoreactivities, as compared to control, were 2870 ± 140%, p
< 0.005 and 7970 ± 960%, p
< 0.005, respectively. There was significant increase in pErk1 and pErk2 immunoreactivities between LF-80 group and LF-20 and LF-50 groups.
View larger version (38K):
[in this window]
[in a new window]
|
Fig. 8. Levels of phosphorylated Erk1/2 in striatal tissue measured after 2 h recovery following DHCA and LF at different rates. The results are means ± SD for seven experiments. The data are expressed in percent to the corresponding values (100%) obtained for the control group of piglets. a
p
< 0.005 for significant difference from sham-operated animals; b
p
< 0.05 for significant difference between LF-20, LF-50, and LF-80, as determined by one-way analysis of variance, followed by Mann–Whitney test. p
< 0.05 was considered statistically significant.
|
|
|
4. Discussion
|
|---|
The ideal CPB perfusion strategy has not been elucidated. Combination of DHCA and LF may be the most effective approach to repairing the more complex defects, specifically hypoplastic left heart syndrome and other hypoplastic aortic arch lesions. This method might allow for a technically satisfactory result and, at the same time, minimize the risks of neurological injury.
The present study determined the response of newborn brain metabolism to the rate of LF following 30 min of DHCA using -stat strategy. Our earlier experiments had established that when using -stat management the extracellular level of striatal dopamine significantly increased following 30 min of DHCA. The present study extends those observations, asking if this increase in extracellular dopamine can be decreased or abolished by LF, and if so, what flow rate is required. Even if it is accepted that in newborns and children, in contrast to adults, pH-stat blood gas management during CPB provides better protection from DHCA-dependent injury to the brain than does -stat management, the published data are not conclusive [20–23]. The specific time periods used in this study are selected as representative of those used in clinical practice.
Our data show a significant dependence of brain oxygenation following DHCA on the rate of low-flow CPB. Cortical oxygen was measured by using the oxygen-dependent quenching of the phosphorescence. This method measures the oxygen pressure in the microcirculation of the cortex and some underlying white matter, down to about 6 mm from the surface. The presented values are the approximate mean for the sampled volume of tissue. A perfusion flow of 20 ml/(kg/min) does not protect against the release of extracellular dopamine in the striatum observed after 30 min of DHCA and reperfusion at this low rate. Increasing CPB reperfusion flow to 50 ml/(kg/min) significantly suppressed the release of extracellular dopamine compared to a flow of 20 ml/(kg/min), and the increase was no longer significantly different from control. A large standard deviation was present in the data for this group of animals, indicating a varying response of the animals to a flow of 50 ml/(kg/min) after 30 min of DHCA. From the group of seven piglets, four animals showed substantial increase in the dopamine levels, whereas in three animals, extracellular dopamine was not different from the control level. Further increase in the flow to 80 ml/(kg/min) completely abolished the release of dopamine.
Dopamine is widely believed to play a critical role in the pathophysiology of brain function, and the release of dopamine following circulatory arrest during LF-20 can play a major role in mediating neuronal damage, particularly in the striatum. Possible mechanisms of dopamine toxicity have been described in detail in early publications [24,25]. Dopamine could potentiate neuronal damage through overstimulation of the glutaminergic receptors, an increase the production of free radicals, and formation 6-hydroxydopamine, very potent dopaminergic neurotoxin.
The suppression of extracellular dopamine release in the striatum is a good indicator of neuronal integrity. The CPB flow resumed following 30 min DHCA must be above 50ml/(min/kg) to provide suppression of dopamine release and substantial protection to the brain. Our data obtained on protein phosphorylation and gene expression for selected proteins discussed below support this conclusion.
The activities of intracellular regulatory proteins such as Bcl-2, Bax, CREB, Akt, and Erk are important determinants of the extent of neuronal cell survival and injury. Key regulatory factors in apoptotic events are the proteins of Bcl-2 family, which can either promote cell survival (Bcl-2) or promote cell death (Bax). Our study shows that at flow rates of 50 and 80 ml/(kg/min), Bcl-2 expression was significantly higher than control, whereas at a flow of 20 ml/(kg/min), Bcl-2 expression was below the control values. There were no significant differences in expression of Bax among the experimental groups. The relative amounts and ratios of these pro- and anti-apoptotic proteins influence the susceptibility of cells to apoptosis. The calculated ratio of Bcl-2 to Bax was higher following 80 ml/(kg/min) flow than following a flow of 50 ml/(kg/min), suggesting that this flow can be the best in providing protection to the brain from DHCA-dependent injury.
The CREB (cAMP response element binding protein) is a regulatory protein activated by diverse signal transduction pathways influenced by cAMP and calcium, and is thought to be involved in the neuronal signal processing. Our data show that, in all three experimental groups, CREB phosphorylation significantly increased compared to control. The degree of increase was dependent on the CPB flow rate, with the greatest increase in the LF-80 group. This progressive increase in CREB phosphorylation may indicate, similar to the results of Bcl-2 and Bax, that using higher CPB flow rate will more likely be best for protecting the brain from ischemic damage.
Akt protein plays an essential role in neuronal survival and is best known for its ability to inhibit cell death pathways by directly phosphorylating and inactivating proteins involved in apoptosis. Akt targets several key proteins that help stressed cells to survive, including apoptosis regulators and transcription factors. Similar to our CREB findings, the phosphorylation of Akt increased progressively with increase in the rate of LF.
Erk1/2 is a member of the MAPK family that has been proposed to play an important role in the pathogenesis of cerebral ischemic injury and also in neuroprotection. Several lines of evidence suggest that sustained Erk1/2 activation may indicate serious neuronal injury, possibly leading to cell death. On the contrary, it has been suggested that prolonged Erk activation accompanied by an increase in CREB can contribute to cell survival and plasticity. While CREB phosphorylation can be induced by other kinases, activation of Erk1/2 is necessary for inducing expression of CREB target genes, which include Bcl-2, an anti-apoptotic protein. Thus, it appears that the relationship of Erk and CREB phosphorylation may ultimately determine whether cells die or survive within a given region. Our data indicate there is a major increase in phosphorylation of Erk1/2 in the LF-80 group, and this flow also results in the greatest increase in CREB phosphorylation and expression. We conclude that LF-80, given the combination of the largest increases in Erk1/2 and CREB phosphorylation in the striatum, is likely to provide the best conditions for neuronal survival.
In summary, we have shown that a flow of 80 ml/(kg/min) following 30 min of DHCA, and not flows of less than 50 ml/(kg/min), abolished the large increase in extracellular dopamine observed with longer periods of DHCA. This CPB flow rate also caused largest increases in phosphorylation of CREB, Erk1/2, and Akt, and increased the Bcl-2/Bax ratio, which are favorable conditions for neuronal survival.
A limitation of our study was that measurement of phosphorylation and regulatory proteins were done only at one time during the recovery period. Future experiments are already in progress to define the time course of the changes more completely and the degree to which the observed changes, as measured at 2 h, correlate with cell survival and neuronal injury. However, on the basis of the present results, we conclude that, in a newborn animal model, for continued neuronal protection following DHCA, where CPB perfusion is required for additional time for surgical repair, the flow should be above 50 ml/(kg/min), probably between 60 and 80 ml/(kg/min).
|
Acknowledgments
|
|---|
This study was supported by grants HL-58669, HD041484, and NS 31465 from the National Institutes of Health.
|
Footnotes
|
|---|
\#9734; Presented at the joint 20th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 14th Annual Meeting of the European Society of Thoracic Surgeons, Stockholm, Sweden, September 10–13, 2006.
|
References
|
|---|
- Bellinger DC, Jonas RA, Rappaport LA, Wypij D, Wernovsky G, Kuban KC, Barnes PD, Holmes GL, Hickey PR, Strand RD, Walsh AZ, Helmers SL, Constantinou J, Carrazana EJ, Mayer JE, Hanley FL, Castaneda AR, Ware JH, Newburger JW. Developmental and neurologic status of children after heart surgery with hypothermic circulatory arrest or low-flow cardiopulmonary bypass. N Engl J Med 1995;332(9):549-555.[Abstract/Free Full Text]
- Mezrow CK, Gandsas A, Sadeghi AM, Midulla PS, Shiang HH, Green R, Holzman IR, Griepp RB. Metabolic correlates of neurologic and behavioral injury after prolonged hypothermic circulatory arrest. J Thorac Cardiovasc Surg 1995;109(5):959-975.[Abstract]
- Newburger JW, Jonas RA, Wernovsky G, Wypij D, Hickey PR, Kuban KC, Farrell DM, Holmes GL, Helmers SL, Constantinou J, Carrazana EJ, Barlow JK, Walsh AZ, Lucius KC, Share JC, Wessel DL, Hanley FL, Mayer JE, Castaneda AR, Ware JH. A comparison of the perioperative neurologic effects of hypothermic circulatory arrest versus low-flow cardiopulmonary bypass in infant heart surgery. N Engl J Med 1993;329(15):1057-1064.[Abstract/Free Full Text]
- Schell RM, Kern FH, Greeley WJ, Schulman SR, Frasco PE, Croughwell ND, Newman M, Reves JG. Cerebral blood flow and metabolism during cardiopulmonary bypass. Anesth Analg 1993;76(4):849-865.[Free Full Text]
- Greeley WJ, Kern FH, Meliones JN, Ungerleider RM. Effect of deep hypothermia and circulatory arrest on cerebral blood flow and metabolism. Ann Thorac Surg 1993;56(6):1464-1466.[Abstract]
- Greeley WJ, Ungerleider RM, Smith LR, Reves JG. The effects of deep hypothermic cardiopulmonary bypass and total circulatory arrest on cerebral blood flow in infants and children. J Thorac Cardiovasc Surg 1989;97(5):737-745.[Abstract]
- Mault JR, Whitaker EG, Heinle JS, Lodge AJ, Greeley WJ, Ungerleider RM. Cerebral metabolic effects of sequential periods of hypothermic circulatory arrest. Ann Thorac Surg 1994;57(1):96-100discussion 1.[Abstract]
- Tsui SS, Kirshbom PM, Davies MJ, Jacobs MT, Greeley WJ, Kern FH, Gaynor JW, Ungerleider RM. Nitric oxide production affects cerebral perfusion and metabolism after deep hypothermic circulatory arrest. Ann Thorac Surg 1996;61(6):1699-1707.[Abstract/Free Full Text]
- Undar A, Vaughn WK, Calhoon JH. The effects of cardiopulmonary bypass and deep hypothermic circulatory arrest on blood viscoelasticity and cerebral blood flow in a neonatal piglet model. Perfusion 2000;15(2):121-128.[Abstract/Free Full Text]
- Igari T, Hoshino S, Iwaya F, Ando S. Cerebral blood flow and oxygen metabolism during cardiopulmonary bypass with moderate hypothermic selective cerebral perfusion. Cardiovasc Surg 1999;7(1):106-111.[CrossRef][Medline]
- Langley SM, Chai PJ, Miller SE, Mault JR, Jaggers JJ, Tsui SS, Lodge AJ, Lefurgey A, Ungerleider RM. Intermittent perfusion protects the brain during deep hypothermic circulatory arrest. Ann Thorac Surg 1999;68(1):4-12discussion 3.[Abstract/Free Full Text]
- Matsuwaka R, Sakakibara T, Hori T, Shinohara N. Initial experience with cool head, warm body perfusion in aortic arch surgery. J Card Surg 1998;13(6):463-467.[Medline]
- Pigula FA, Siewers RD, Nemoto EM. Regional perfusion of the brain during neonatal aortic arch reconstruction. J Thorac Cardiovasc Surg 1999;117(5):1023-1024.[Free Full Text]
- Schears G, Zaitseva T, Schultz S, Greeley W, Antoni D, Wilson DF, Pastuszko A. Brain oxygenation and metabolism during selective cerebral perfusion in neonates. Eur J Cardiothorac Surg 2006;29(2):168-174.[Abstract/Free Full Text]
- Schultz S, Antoni D, Shears G, Markowitz S, Pastuszko P, Greeley W, Wilson DF, Pastuszko A. Brain oxygen and metabolism during circulatory arrest with intermittent brief periods of low-flow cardiopulmonary bypass in newborn piglets. J Thorac Cardiovasc Surg 2006;132(4):839-844.[Abstract/Free Full Text]
- Yang T, Sun L, Hei F, Liu Y. A cardiopulmonary bypass perfusion technique for the surgical repair of aortic type I dissections requiring the elephant trunk procedure. J Extra Corpor Technol 2000;32(4):220-223.[Medline]
- Vinogradov SA, Fernandez-Seara MA, Dugan BW, Wilson DF. Frequency domain instrument for measuring phosphorescence lifetime distributions in heterogeneous samples. Rev Sci Instrum 2001;72(8):3396-3406.[CrossRef]
- Vinogradov SA, Wilson DF. Recursive maximum entropy algorithm and its application to the luminescence lifetime distribution recovery. Appl Spectrosc 2000;54:849-855.[CrossRef]
- Zaitseva T, Schultz S, Schears G, Pastuszko P, Markowitz S, Greeley W, Wilson DF, Pastuszko A. Regulation of cell death and survival in the brain following cardiopulmonary bypass. Ann Thorac Surg 2006, in press.
- du Plessis AJ, Jonas RA, Wypij D, Hickey PR, Riviello J, Wessel DL, Roth SJ, Burrows FA, Walter G, Farrell DM, Walsh AZ, Plumb CA, del Nido P, Burke RP, Castaneda AR, Mayer Jr. JE, Newburger JW. Perioperative effects of alpha-stat versus pH-stat strategies for deep hypothermic cardiopulmonary bypass in infants. J Thorac Cardiovasc Surg 1997;114(6):991-1000discussion 1.[Abstract/Free Full Text]
- Hindman BJ, Dexter F, Cutkomp J, Smith T. pH-stat management reduces the cerebral metabolic rate for oxygen during profound hypothermia (17 degrees C). A study during cardiopulmonary bypass in rabbits. Anesthesiology 1995;82(4):983-995discussion 24A.[CrossRef][Medline]
- Kurth CD, O’Rourke MM, O’Hara IB. Comparison of pH-stat and alpha-stat cardiopulmonary bypass on cerebral oxygenation and blood flow in relation to hypothermic circulatory arrest in piglets. Anesthesiology 1998;89(1):110-118.[CrossRef][Medline]
- Skaryak LA, Chai PJ, Kern FH, Greeley WJ, Ungerleider RM. Blood gas management and degree of cooling: effects on cerebral metabolism before and after circulatory arrest. J Thorac Cardiovasc Surg 1995;110(6):1649-1657.[Abstract/Free Full Text]
- Pastuszko A. Metabolic responses of the dopaminergic system during hypoxia in newborn brain. Biochem Med Metab Biol 1994;51(1):1-15.[CrossRef][Medline]
- Schultz S, Creed J, Schears G, Zaitseva T, Greeley W, Wilson DF, Pastuszko A. Comparison of low-flow cardiopulmonary bypass and circulatory arrest on brain oxygen and metabolism. Ann Thorac Surg 2004;77(6):2138-2143.[Abstract/Free Full Text]
Top
Abstract
1. Introduction
