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J Thorac Cardiovasc Surg 2004;127:1033-1040
© 2004 The American Association for Thoracic Surgery
Cardiopulmonary support and physiology |
a Vanderbilt University Medical Center, Nashville, Tenn, USA
b St Louis University Health Sciences Center, St Louis, Mo, USA
Read at the Eighty-third Annual Meeting of The American Association for Thoracic Surgery, Boston, Mass, May 4-7, 2003.
Received for publication May 2, 2003; revisions received August 29, 2003; accepted for publication September 29, 2003.
* Address for reprints: Davis C. Drinkwater, Jr, MD, William S. Stoney Professor and Chairman, Department of Cardiac and Thoracic Surgery, Vanderbilt University Medical Center, 1161 21st Ave South 2986 TVC, Nashville, TN, USA, 37232-5734
davis.drinkwater{at}vanderbilt.edu
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Abstract |
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METHODS: Piglets underwent normal flow cardiopulmonary bypass (control, n = 4), deep hypothermic circulatory arrest (n = 6), and deep hypothermic circulatory arrest with U0126 (n = 5) at 20°C for 60 minutes. The deep hypothermic circulatory arrest with U0126 group was given 200 µg/kg of U0126 45 minutes prior to initiation of bypass followed by 100 µg/kg at reperfusion. Following 24 hours of post–cardiopulmonary bypass recovery, brains were harvested. Eleven distinct cortical regions were evaluated for neuronal damage using hematoxylin and eosin staining. A section of ischemic cortex was further evaluated by immunohistochemistry with rabbit polyclonal antibody against phosphorylated extracellular signal-regulated kinase 1/2.
RESULTS: The deep hypothermic circulatory arrest and deep hypothermic circulatory arrest with U0126 groups displayed diffuse ischemic changes. However, the deep hypothermic circulatory arrest with U0126 group possessed significantly lower neuronal damage scores in the right frontal watershed zone of cerebral cortex, basal ganglia, and thalamus (P 
.05) and an overall trend toward neuroprotection versus the deep hypothermic circulatory arrest group. This neuroprotection was accompanied by nearly complete blockade of phosphorylated extracellular signal-regulated kinase in the cerebral vascular endothelium.
CONCLUSIONS: In this experimental model of deep hypothermic circulatory arrest, U0126 blocked extracellular signal-regulated kinase activation and provided a significant neuroprotective effect. These results support targeting of the extracellular signal-regulated kinase pathway for inhibition as a novel therapeutic approach to mitigate neuronal damage following deep hypothermic circulatory arrest.
Mitogen-activated protein kinases (MAPKs) represent a family of highly conserved serine/threonine protein enzyme complexes that are critical components of the enzyme cascade systems that connect a wide variety of cell surface receptors to critical cellular targets.8 MAPKs are activated during ischemia and/or reperfusion and contribute to the structural and functional changes associated with ischemia-reperfusion (I/R) injury.9 Recent studies demonstrate that members of the MAPK signal transduction cascade, most notably extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun amino-terminal kinase (JNK), and p38 protein (p38), are activated following I/R in the brain,10,11 heart,12,13 kidney,14,15 and liver.16 The ERK1/2 subfamily, which is activated by MAPK/ERK kinase 1/2 (MEK1/2), has been heavily implicated in the neuronal and endothelial dysfunction witnessed following cerebral I/R injury.10 Pharmacologic studies with MEK1/2-specific inhibitors PD98059,17 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126),18 and SL32719 have shown that inhibition of the ERK1/2 pathway significantly reduces neuronal damage following cerebral ischemia and cerebral I/R injury.
The recognition that neuronal damage following cerebral I/R injury may involve ERK1/2 activation has opened up a new and exciting opportunity for the pharmacologic modulation of cerebral tolerance to DHCA. Previous work by our group demonstrated that the ERK1/2 pathway is activated following DHCA and linked the expression of p-ERK1/2 within cerebral vascular endothelium to the postischemic neuronal damage seen following DHCA (unpublished data). The present study evaluated the ability of the MEK1/2-specific inhibitor (U0126) to block ERK1/2 activation and mitigate ischemic neuronal damage in a clinically applicable model of DHCA (Figure 1). Additionally, immunohistochemistry was performed to qualitatively correlate the presence of phosphorylated ERK1/2 (p-ERK1/2) expression in the cerebral vascular endothelium with the degree of neuronal damage present 24 hours following DHCA.
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Conduct of CPB
The CPB circuit consisted of a Cobe nonpulsatile roller pump (Cobe Cardiovascular Inc, Arvada, Colo), cardiotomy reservoir, and hollow fiber membrane oxygenator (Minimax plus, Medtronic). The circuit was primed with fresh whole blood collected from an adult donor pig 1 day preoperatively, 2000 IU of heparin, 1 g of calcium chloride, and 25 mEq of sodium bicarbonate. Hematocrit was maintained between 26% and 30%. The total priming volume was 700 to 800 mL. CPB was initiated at a flow rate of 100 to 150 mL/kg per minute and adjusted to maintain a mean arterial pressure of 50 to 60 mm Hg.
Piglets were then subjected to 1 of the following conditions. In the CPB control group, piglets underwent normal flow (100 to 150 mL/kg/min), normothermic (37°C) CPB for 60 minutes without aortic crossclamping. In the DHCA and DHCA + U groups, piglets were cooled to a rectal temperature of 20°C using a heat exchanger and cooling blanket. CPB was then discontinued and the piglets were maintained at 20°C for 60 minutes of DHCA. Myocardial protection was afforded by the infusion of high potassium blood cardioplegic solution (15 mL/kg, potassium concentration 20 mEq) and application of iced saline slush during DHCA. Arterial blood gases were controlled using pH stat strategy. After 60 minutes, CPB was reinitiated and the piglets were warmed to a rectal temperature of 37°C. Care was taken to avoid a temperature difference between the perfusate and core temperature of more than 8°C. Once the piglets were normothermic they were weaned from CPB and decannulated. Protamine (1 mg/1000 IU heparin) was administered following separation from CPB.
Administration
U0126 (Calbiochem, La Jolla, Calif), a potent and specific inhibitor of MEK1/2, was administered via the femoral vein in the DHCA + U group (Figure 1). The piglets were given 200 µg/kg of U0126 45 minutes prior to initiation of CPB and 100 µg/kg at the time of reperfusion. The U0126 was dissolved in 0.25 mL of dimethylsulfoxide (Sigma Chemical Co, St Louis, Mo).
Postoperative management
After weaning from CPB, the animals remained intubated and sedated and were allowed to recover for 24 hours. Physiologic data were recorded every hour. To avoid postoperative adverse effect (bias), arterial blood gases, electrolytes, and hemodynamics were maintained within normal limits throughout the recovery period. Upon completion of the experiment, the cranial vault was opened and the fresh whole brain was transected at the level of C1 during deep anesthesia in a state of stable hemodynamics and continuous antegrade cerebral perfusion.
Brain histology and neuronal damage
The resected brains were fixed in 10% neutral buffered formalin and stored at room temperature for 2 weeks. The brains were then sectioned coronally and examined. Sections were taken from the following sites: (1) right frontal watershed area, (2) left frontal watershed area, (3) right parietal watershed area, (4) left parietal watershed area, (5) right temporal lobe and hippocampus, (6) left temporal lobe and hippocampus, (7) basal ganglia, (8) anterior thalamus, (9) cerebellum, (10) mesencephalon, (11) pons, and (12) medulla oblongata. The tissue was processed overnight and subsequently embedded in paraffin. Five-micron sections were cut on a microtome, deparaffinized, and then stained with hematoxylin and eosin. A single neuropathologist (M.J.) blinded to the experimental group examined all specimens and scored the extent of neuronal damage in each slide. A modified grading system of Pulsinelli and colleagues20 was used to quantify neuronal damage. Scores were assigned based on the following scale: 0 = no neuronal damage; 1 = mild neuronal damage (rare or scattered ischemic neurons or a section with scattered neurons and 1 collection less than one 100x magnification field); 2 = moderate neuronal damage (scattered collections of ischemic neurons that collectively occupy one to three 100x magnification fields); 3 = severe neuronal damage (ischemic collections of neurons that collectively occupy four or more 100x magnification fields). Histopathologic scores for each group represented the average among the slides taken from each region. Ischemia was defined as nuclear pyknosis, eosinophilia, and contraction of cytoplasm seen in neurons in a high-powered field (100x).
Immunohistochemistry for phosphorylated ERK1/2
After 2 weeks of fixation, a section of the right frontal watershed zone (WSZ) was examined by immunohistochemistry using polyclonal rabbit antibodies to p-ERK1/2 and total ERK1/2 (Cell Signaling, Beverly, Mass). Immunohistochemistry was performed on a Ventana ES automated immunostainer (Ventana, Tucson, Ariz). Optimal conditions were determined separately for each antibody. To avoid background and artifactual unmasking of epitopes, no pretreatment was used. The antigen–antibody complexes were visualized using an avidin-biotin complex detection system (Vector Laboratories, Inc, Burlingame, Calif) with 3,3'-diaminobenzidine as the chromogen. Sections were counterstained with hematoxylin. No significant immunoreactivity for each antibody was observed when substituting the primary antibodies with nonimmune rabbit serum or by skipping the primary antibodies. The activation of ERK1/2 was assessed by the same blinded neuropathologist (M.J.) as either nonsignificant (no or rare expression of p-ERK1/2) or significant (considerable positive expression of p-ERK1/2).
Statistical analysis
Values are presented as the mean ± standard error of the mean. The continuous variables were compared using an analysis of variance. A general linear model repeated measures analysis of variance was also employed to assess changes among groups from baseline to 6, 12, and 24 hours postoperatively. Differences in neuronal damage among the experimental groups were evaluated using Kruskal-Wallis test. A univariate general linear model was used to assess neuronal damage among groups after adjusting for covariates. Statistical analyses were conducted with the SPSS 11.5 software package (SPSS Inc, Chicago, Ill).
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Activation of ERK1/2
Immunohistochemistry staining revealed significant expression of p-ERK1/2 in the cerebral vascular endothelium in 3 of the 6 DHCA piglets at 24 hours postoperatively. In contrast, nonsignificant expression of p-ERK1/2 appeared in the cerebral vascular endothelium of animals from the CPB control and DHCA + U groups.
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U0126 is a potent and highly specific inhibitor of MEK1/2, the upstream kinase of the ERK1/2 molecule, which noncompetitively binds the MEK1/2 molecule and prevents its phosphorylation and activation.21,22 The use of U0126 blocks the ability of MEK1/2 to phosphorylate and activate the ERK1/2 molecule and the pathway is rendered inactive at this point (Figure 1). U0126 inhibits ERK1/2 activation in a dose-dependent manner both in vitro21 and in vivo in ischemic cortical neurons.18 In our study, the U0126 dosing regimen was based upon the findings of Namura and colleagues,18 who demonstrated maximal cerebral protection with a 200-µg/kg intravenous pretreatment dose of U0126, which resulted in a 42% reduction in infarction volume in a rat stroke model. Based on these findings we administered a 200-µg/kg dose of U0126 45 minutes prior to CPB in an attempt to achieve maximal neuronal protection in our model. Additionally, a half dose of U0126 was given at the time of reperfusion in an attempt to lessen the acute effects of the cerebral reperfusion injury. Using this regimen we obtained nearly complete blockade of ERK1/2 activation in the cerebral vascular endothelium. In preliminary studies by our group, U0126 pretreatment given 10 minutes before DHCA was unable to adequately block ERK1/2 activation (data not shown). However, when U0126 was administered at this time the piglet was on CPB. As a result, the dose of U0126 was diluted by the volume of the CPB prime (700 mL). We believe that not only did these piglets not receive an adequate dose of U0126, but that 10 minutes was not an adequate amount of time for pretreatment of the brain. In contrast, when given 45 minutes prior to the initiation of CPB, the dose of U0126 was not affected by the volume of the priming solution and there was adequate time for pretreatment of the piglet brain. These results suggest that both the timing of U0126 administration and the concentration administered are essential factors in the production of an adequate blockade of ERK1/2 activation.
In the current study, an extensive assessment of the neuronal damage present 24 hours following normothermic CPB, DHCA, and DHCA + U was performed by a single, blinded neuropathologist. Although the DHCA + U group displayed diffuse ischemic changes, the ischemic damage was not significant when compared with the normothermic CPB control group (Figure 2). The treatment of the DHCA + U group with U0126 resulted in significantly lower neuronal damage scores in the right frontal WSZ, basal ganglia, and thalamus and an overall trend toward neuroprotection versus the DHCA group (Figure 2). These results represent the first report of the ability of an inhibitor of the ERK1/2 pathway to attenuate neuronal damage following DHCA. In addition to our neuropathological analysis, sections of the right frontal WSZ were analyzed with immunohistochemistry. All specimens from the normothermic CPB control and the DHCA + U groups were found to have nonsignificant expression of p-ERK1/2 within the cerebral vascular endothelium. In contrast, 3 of the 6 specimens from the DHCA group possessed significant p-ERK1/2 expression. These findings suggest that the ERK1/2 pathway serves as a key mediator of the cerebral I/R injury associated with DHCA and further demonstrate the ability of U0126 to effectively block the activation of the ERK1/2 pathway in our model of DHCA. Overall, the present study supports the selective targeting of the ERK1/2 pathway for inhibition as a means of increasing cerebral tolerance to DHCA and opens up several novel areas for future research.
Limitations of the study
Prior to discussing the implications of our results, it is important to address 2 important methodological aspects of our study. First, the timing of the removal of the piglet brains following DHCA was a critical element within this study. Previous work has shown that following cerebral I/R injury a minimum of 24 hours is required before an accurate neuropathological assessment of the extent of neuronal damage can be made.20 However, the peak time for ERK1/2 activation following cerebral I/R injury has been shown to occur approximately 2 hours following reperfusion.10 Thus, while the optimal time for analysis of cerebral vascular endothelial ERK1/2 activation would have been prior to 24 hours, we chose to perform our analysis at this time to ensure an accurate neuropathologic assessment of the effectiveness of the U0126 molecule as an MEK1/2 inhibitor within our model of DHCA.
Second, we did not utilize the perfusion-fixation method of cerebral extraction. However, our method of rapid extraction (<5 minutes) under a state of stable hemodynamics with continuous antegrade cerebral perfusion was found to be reliable and safe. Analysis of the brains from the CPB control group revealed no significant neuronal injury or expression of p-ERK1/2. These findings demonstrate that our method of extraction has not introduced experimental error or bias into our findings.
Interpretation of results
Although our pathologic and immunohistochemistry data clearly demonstrated a significant neuroprotective effect of U0126 following DHCA, a considerable degree of neuronal damage remained in the DHCA + U group despite its use. This suggests that the neuroprotective effect of U0126 may result from a partial inhibition of the ERK1/2 pathway or the complete inhibition of the ERK1/2 pathway circumvented in some manner by an unknown mechanism of "cross talk" involving the other MAPK pathways (p-38 and JNK). Additionally, the effects of other inflammatory cascades, such as the nuclear factor kappa beta cascade, were not accounted for by this study and may be responsible for the remaining neuronal damage seen in the DHCA + U group. Therefore, future studies are required to elucidate the exact mechanism of the neuroprotective effect of U0126 and to identify the cross talk that exists between the various pathways of the MAPK cascade and between the MAPK cascade and other proinflammatory cascades.
Although the results of our immunohistochemical analyses are encouraging, they must be cautiously interpreted. These results represent the initial evidence of a link between the presence of cerebral vascular endothelial expression of p-ERK1/2 and a more extensive pattern of neuronal damage 24 hours following DHCA. However, we recognize that the significance of such results must be weighed against 2 important factors. Initially, it must be understood that such immunohistochemical analyses yield qualitative and not quantitative results. Secondly, as previously stated, following cerebral I/R injury the peak activation of ERK1/2 has been shown to occur 2 hours following reperfusion. Therefore, the fact that only 3 of the 6 piglets demonstrated significant p-ERK1/2 expression in the cerebral vascular endothelium 24 hours after DHCA was likely due to the timing of cerebral extraction. To establish a more concrete correlation between the cerebral vascular expression pattern of p-ERK1/2 and the extent of neuronal damage seen following DHCA, further studies are required to examine the early activation pattern of the ERK1/2 pathway following DHCA. Ongoing studies in our laboratory are aimed at qualitatively and quantitatively assessing the cerebral vascular endothelial expression pattern of p-ERK1/2 2 hours following DHCA and the effect of U0126 molecule at this time point. We hope the evidence provided by these studies will allow us to definitively correlate the neuronal protection achieved by the use of U0126 with the inhibition of cerebral vascular endothelial p-ERK1/2 expression.
Finally, the promising results of our initial experience with the inhibition of the ERK1/2 pathway of the MAPK signal transduction cascade by U0126 must be viewed with a global perspective of the role of the MAPK cascade in the process of cerebral I/R injury. Cerebral I/R injury not only activates the ERK1/2 subfamily of MAPK but also the p38 and JNK pathways, and the timing and magnitude of their activation within the brain are distinct.10 These MAPK cascades are not simple linear systems separately turned on or off. A complex system of cross talk exists between all 3 of these important signal transduction pathways.23 In this complex network the systematic inhibition of only 1 of these pathways, as in our study, may have unpredictable results. The problem becomes more complicated when the whole body with its multiple different tissue types (organs) is considered. Activation or inhibition of the same MAPK pathway in 2 different tissues may lead to the expression of different mediators with opposing roles.24 Thus, although these MAPK pathways represent novel targets for the therapeutic manipulation of inflammatory states and I/R injury, caution is warranted because the systemic administration of an inhibitor of a single MAPK subfamily targeting 1 organ may achieve markedly different and unintended results in other organs.23 Therefore, the overall benefit of studies such as this depend upon continued research to outline the complicated interaction between the pathways of the MAPK cascade and their effects at a tissue-specific level.
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Dr Mulloy. No, sir, these animals did not receive any preoperative steroids.
Dr Milano. It would be interesting then to see what simple administration of steroids would do in comparison to your inhibitor.
Dr Mulloy. Thank you for your suggestion. We will take that into consideration when planning future experiments with this model.
Dr Frank W. Sellke (Boston, Mass). MAPK is involved in a lot of different mechanisms and pathways, including vasomotion. Was there any hemodynamic effect of giving the MAPK inhibitor?
Dr Mulloy. No, sir, we did not see any significant hemodynamic effects when we gave the inhibitor. The inhibitor was given via a femoral venous line 45 minutes prior to initiation of cardiopulmonary bypass. At this time we had an arterial line in place and the animals were attached to a pulse oximeter. Neither the blood pressure or heart rate demonstrated any significant hemodynamic change when the inhibitor was administered.
Dr Sellke. I have to commend you on your experimental preparation. I put a lot of pigs on bypass and I find it is hard to keep them alive for more than 3 hours after coming off bypass. What was the perfusion pressure after the 24 hours of recovery?
Dr Mulloy. The protocol was to keep the perfusion pressure around 40 to 50 mm Hg. At times we used small doses of dopamine, approximately 3 to 7 mcg/kg/h, to maintain this pressure. Overall, the average was in the 40 to 50 range.
Dr Sellke. MAPK is also involved with apoptosis. Do you think this could have had some role in your beneficial effects that you observed?
Dr Mulloy. Unfortunately we didn't use terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining as a readout in this experiment; however, we are performing some additional experiments at the moment in which we are going to include TUNEL staining as a readout. I think this will be an interesting marker and hope that we will find some beneficial effects of inhibitor administration in regard to apoptosis.
Dr Antonio F. Corno (Lausanne, Switzerland). You studied very well the control of MAPK negative effects on piglets that I presume were normoxic. We know that the vast majority of neonates requiring deep hypothermia circulatory arrest are hypoxic or cyanotic, and also we have seen MAPK implicated in the negative effects not only in ischemia and reperfusion damage but also hypoxia and reoxygenation damage, as we have demonstrated in our research lab with hypoxic myocardium. Did you study any of these protocols in hypoxic piglets, or are you planning to do it?
The second question is, what are the steps between this experimental study and the potential clinical application?
Dr Mulloy. Thank you for your comments. Unfortunately, we did not examine the role of hypoxia in this experiment. This was our first attempt to develop an appropriate model in which we could explore the effect of the MAPK cascade on cerebral ischemia/reperfusion injury, which I think is a very exciting area to explore in cardiac surgery. So we tried to stick with something fairly basic to begin; we are planning to expand from this point on. In response to the first part of your question, no, we didn't examine hypoxic animals, but your suggestion would be a nice study to do in the future.
As far as the potential steps from the bench to the bedside, I think that this work is interesting, but I think it is only the tip of the iceberg. We are looking at cerebral ischemia-reperfusion injury, and, as we all know, there are not only multiple MAPK pathways but there are multiple inflammatory cascades involved as well. However, we were excited and are encouraged that the administration of a single inhibitor was able to produce a significant decrease in neuronal damage. Nevertheless, we realize that we have a long road ahead of us and hope that future studies examining the effects of increasing the dose of the inhibitor and using inhibitors of different pathways of MAPK cascade and, as well, inhibitors of different inflammatory cascades may result in an increased beneficial effect. I think that we are still quite a distance from using this strategy at the bedside, but I think it is exciting initial work and we are happy to proceed with it.
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