Post-resuscitation Myocardial Dysfunction:Clinical Evaluation and Antioxidant & Hypercarbic Reperfusion Therapy

• Main Authors: Wei-Tien Chang, 張維典
• Other Authors: Yuan-Teh Lee
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/09700140177713984155

博士 === 國立臺灣大學 === 臨床醫學研究所 === 95 === Ｓudden death has been an issue of significant importance in public health. In the United States, more than 400,000 people die outside the hospitol before they have a chance to reach the hospital and receive medical help. Though generally believed to be low, the incidence of sudden death in Taiwan is comparable to those of the western countries. According to the Chin-Shan Community Cardiovascular Cohort (CCCC) study, a population-based epidemiological study held by Prof. Yuan-Teh Lee in National Taiwan University, the incidence of sudden death in Taiwan is 73/100,000 person-year. The incidence is higher in man (108/100,000 person-year), about 2.5 times that of the woman (43/100,000 person-year). Though lower than that of the United States, it is higher than some of the European countries (e.g. 40/100,000 person-year in England, 46100,000 person-year in Israel, 48/100,000 person-year in Finland, and 56/100,000 person-year in Iceland), and is comparable with that of Japan (68-104/100,000 person-year). Therefore, this issue is worthy of extensive exploration in the oriental societies. Effort should be launched to maximize the primary prevention in the general population and strengthen the emergency cardiac care in the communities. The prognosis of out-of-hospital cardiac arrest (OHCA) is generally poor. For example, the rate of return of spontaneous circulation (ROSC) in Taipei was 15.8% in 1993, and only 1.4% survived to hospital discharge.With the progress in emergency medical service system for the last 10 years, the ROSC rate in Taipei has improved to 28.9%. However, the survival to discharge rate was still low (3.1%). The discripency between initial ROSC and survival to discharge has been largely attributed to the gradual decline of survival in the post-resuscitation phase. Considering the cause of death in this post-CPR period, one-third die of cardiac or circulatory failure, one-third of neurological failure, and the rest die of other complications such as infection. Circulatory failure usually occurs early, and is responsible for early mortality. In contrast, neurological failure is associated with prolonged ICU stay, and accounts for most of the late mortalities. Both these consistute the main features of the post-resuscitation syndrome. Myocardial failure usually develops early in the post- resuscitation phase, and has been considered the main cause of early death after initial success in CPR. For the past 10 years, post-resuscitation myocardial dysfunction has attracted keen attention. Resutling from cardiac arrest and CPR, it has been regarded as a specific model of global myocardial ischemia and reperfusion. A lot of studies have been done at the animal level. Experimental studies have implicated a number of factors that might influence its severity, such as the ischemic duration, electrical defibrillation, the number, type, and waveforms of electrical shocks, and the dose of epinephrine. Efforts have also been made to improve such dysfunction via pharmacological or mechanical means. In the real world, however, there are still few studies focusing on characterizing the post-resuscitation myocardial dysfunction in human. Therefore, we launched a prospective observation study using echocardiographic evaluation of the myocardial function in patients suffering from cardiac arrest and CPR, hopefully to identify the factors associated with its severity, as well as the prognostic implications it may have. Between Jan 2001 and Dec 2002, OHCA patients receiving CPR at the emergency department of National Taiwan University Hospital were prospectively followed up. Those who achieved ROSC were admitted to ICU for post-resuscitation care. Echocardiographic evaluation was performed at the sixth hour post-CPR. The left ventricular (LV) size and systolic and diastolic functions were analyzed in correlation to the patients’ underlying diseases and resuscitation factors. The prognostic value of the LV function was also evaluated. During the study period, 58 patients were included. The basic characteristics of the patients were listed in Table 2. The associations between clinical factors and post-resuscitation LV dimensions and functions were shown in Table 3. Both LV end-diastolic (LVEDDi) and end-systolic diameter index values (LVESDi) were significantly higher in patients with cardiac etiology and past histories of ischemic heart disease (IHD) and myocardial infarction (MI). The LVESDi was also higher in patients receiving defibrillation and epinephrine ≧ 5 mg. For LV systolic function, poorer LV ejection fraction (LVEF) was associated with hypertension, past history of MI, CPR duration ≧ 20 minutes, defibrillation, and the use of epinephrine ≧ 5 mg. For LV diastolic function, isovolumic relaxation time (IVRT) was significantly longer in patients with non-cardiac etiology and initial rhythm of non-VF/VT. For patients with myocardial ischemia/infarction as the etiology of cardiac arrest, no significant differences were noted in LV dimensions or functions compared to other cardiogenic patients without evident myocardial ischemia. In multiple regression analysis, past history of IHD was significantly associated with LVEDDi, while epinephrine dose and history of IHD were independent factors for LVESDi (Table 5). For LV systolic function, epinephrine dose and past history of MI were significantly associated with LVEF. For LV diastolic function, the etiology of cardiac arrest was independently associated with IVRT (Table 5). For prognosis, both LVEF and IVRT were significantly associated with the patients’ survival outcomes (p = 0.03 and 0.0004, respectively). The results were demonstrated by Kaplan-Meier survival curves in Figure 8 and 9. Other factors associated with survival included age, initial cardiac rhythm, epinephrine dose, and total CPR duration. After adjusting for these variables in Cox regression analysis, IVRT ≧ 100 ms still served as an independent predictor for poor survival prognosis (hazard ratio 3.3, 95% CI 1.6 to 6.7, p=0.002). Neurological recovery was significantly better in patients with LVEF ≧ 40% (24% vs. 0%, p = 0.04), but this significance no longer existed in multiple regression analysis. Factors independently predicting neurological recovery were initial rhythm of VF/VT, duration of no CPR < 10 minutes, and total CPR time < 20 minutes. According to this study, post-resuscitation LV dysfunction is correlated with a number of clinical factors, among which past history of MI, epinephrine dose, and the etiology of cardiac arrest play independent roles. These results implicate that post-resuscitation myocardial dysfunction may be a combination of the patients’ underlying diseases and the injury resulting from cardiac arrest and CPR. While some of these factors are non-modifiable, certain resuscitation factors can be avoided or substituted by newly developed durgs. For prognosis, both LVEF < 40% and IVRT ≧ 100 ms at the sixth hour post-CPR were associated with poor survival outcomes. This is of prognostic value based on which treatment can be done trying to improve the myocardial function as well as the survival prognosis. As mentioned above, post-resuscitation myocardial dysfunction is a specific model of global myocardial ischemia/reperfusion (I/R) injury. Given the fact that LV dysfunction in the post-resuscitation phase implicates poor survival outcomes, therapeutic interventions trying to limit or treat such I/R injury become the key to eventually improve myocardial function and prognosis. As I/R is inevitably associated with oxidative stress, and the reactive oxygen species (ROS) produced during I/R can cause damages on lipid, protein and DNA, one reasonable intervention to attenuate I/R injury may be antioxidant therapy. Therefore, we sought to investigate the role of antioxidant therapy in mitigating the myocardial injury resulting from I/R. Flavonoids are naturally occurring polyphenolic compounds exhibiting potent antioxidant activities. Their roles in combating oxidative injuries such as reperfusion of the ischemic tissue have been investigated in a variety of in vitro and in vivo models. Typically they are pre-administered as preventive measures for evaluating their protection against an up-coming oxidative insult. However, it may not always be feasible to administer flavonoids as a pretreatment in the clinical settings. Therefore, the protective role of flavonoids in an acute treatment model of I/R, i.e. administration of flavonoids concurrently with I/R, or just at the point of reperfusion, is of greater interest. In this series of study, we first employed electron spin resonance (ESR) spectrometry to evaluate the oxidant scavenging capacities of five selected flavonoids. The oxidants chosen for ESR experiments are superoxide and hydroxyl radical, both being the major ROS produced during the context of I/R. As shown in Fig. 11, addition of the tested flavonoids to xanthine/xanthine oxidase plus DMPO system (25 µM) reduced the DMPO-.OOH signals to different levels compared to that in control. The superoxide scavenging capacity of the five flavonoids was as follows: catechin (90 ± 3%), baicalein (69 ± 5%), procyanidin B2 (68 ± 5%), baicalin (42 ± 4%), and wogonin (8 ± 3%). The hydroxyl radical scavenging effect of the flavonoids was tested using a Fenton reaction system with DMPO as the trapping agent. The addition of flavonoids (200 µM) resulted in differential quenching effects of the DMPO-OH adduct as demonstrated by reduction in the ESR signal intensity (Fig. 12). In general, the scavenging effect of the flavonoids on hydroxyl radicals was weaker than that on superoxide even with higher concentration (200 µM): baicalein (56 ± 4%), catechin (31 ± 2%), procyanidin B2 (28 ± 3%), wogonin (25 ± 5%), and baicalin (15 ± 3%). Only baicalein showed an ESR reduction rate of > 50%. Then we developed three treatment strategies in an established cardiomyocytes model of I/R to evaluate the antioxidant protection of the flavonoids against I/R injury. The protective effects were compared with the oxidant scavenging capacities as well as among different treatment protocols. As shown in Fig. 13, 14 and 15, the protective effects of flavonoids were generally consistent with the flavonoids’oxidant scavenging activities. Meanwhile, chronic treatment exhibited the best protective effects, while in reperfusion treatment only limited protective effects can be seen. The only exception was catechin, whose antioxidant protection against I/R injury seemed less satisfactory given the outstanding oxidant scavenging activities it demonstrated in ESR esperiments. This can probably be explained by the poor lipid solubility of catechin, which may be an important factor when considering cell membrane access and antioxidant activities within the cell. In the second part of the study, we chose the flavonoids with the best antioxidant protection against I/R injury – baicalein – for further study and mechanistic exploration. Since our interest has been the role of acute treatment, we first tried to raise the dose of baicalein in acute treatment protocols to see if the protective effect can be enhanced. As shown in Fig. 17 and 18, as the dose of baicalein was raised from 25 μM to 100 μM, the cell death rates after I/R declined in a reverse dose-response manner, both in I/R treatment and reperfusion treatment. This suggests that if chronic prevention cannot be available in face of an acute ischemic insult, raising therapeutic dose for optimizing protective effects may be a practical approach for maximizing antioxidant protection against I/R injury. Furthermore, as reperfusion ROS burst occurs within minutes of reperfusion, and is associated with tremendous oxidative reperfusion injury thereafter, we employed a delayed reperfusion treatment protocol with different therapeutic time delay in order to justify the critical time window for antioxidant therapy. As shown in Fig. 19, delay of baicalein treatment in the reperfusion phase resulted in attenuation of protection against I/R injury. The protection was completely lost if baicalein was not given until after one hour of reperfusion. This suggests that the therapeutic window for acute antioxidant therapy is within the first 15-30 of reperfusion. In this cardiomyocyte model of I/R, ROS generation was increased during ischemia, followed by a transient but significant ROS burst in the first minutes of reperfusion (Fig. 23). Given that baicalein exhibits antioxidant activities protecting against oxidant injuries, we sought to compare the ROS scavenging capacities of the different treatment protocols, and their correlations with the protective effects against I/R injury. As shown in Fig. 24, either chronic treatment or I/R treatment (25 μM) significantly abolished the reperfusion ROS burst. Moreover, the DCF fluorescence increase during ischemia was abolished as well. In contrast, in reperfusion treatment the ROS generated during ischemia was not affected at all, and the scavenging of reperfusion ROS burst was less effective. These results were basically consistent with the protective effects seen in the corresponding protocols. Likewise, when the baicalein dose was raised from 25 to 100 μM in reperfusion treatment, the reduction of reperfusion ROS burst was also augmented (Fig. 25), which is consistent with the enhanced protective effect (Fig. 18). Nevertheless, if the same effective dose (100 μM) of baicalein treatment was delayed by 15 min into reperfusion, this effect on the reperfusion ROS burst was completely lost (Fig. 25), and the protective effect was diminished (Fig. 19). This again supports the notion that the ROS burst in the first min of reperfusion is important in mediating I/R injury. Acute antioxidant therapy effectively covering this critical window of reperfusion ROS burst is essential for oxidant scavenging and protection against I/R injury. Nitric oxide (NO) is implicated in a number of cardioprotective mechanisms, and has been demonstrated in this cardiomyocyte model mediating cytoprotection in treatment strategies such as preconditioning and hypothermia. To elavuate whether NO, in addition to oxidant scavenging, is involved in baicalein’s protection against I/R injury, we employed DAF-2DA as an indicator of NO and monitored the NO profile of baicalein treatment during I/R. As seen in Fig. 26, in effective treatment strategies such as chronic and I/R treatments (25 μM), there was a sustained increase of NO after the peaking at around 15 min of reperfusion. In contrast, this cannot be seen in less satisfactory treatment protocol such as the reperfusion treatment (25 μM). However, if the baicalein dose was increased from 25 to 100 μM in reperfusion treatment, the NO increased gradually at a later phase of reperfusion (Fig. 27), which may in part accounts for the enhanced protection. Nevertheless, if baicalein treatment was delayed by 15 min at reperfusion, the increase of NO was further delayed and the magnitude was less. These results altogether suggest that NO increase at a later phase of reperfusion may be associated with baicalein’s protection against I/R injury. As NO synthase has been implicated in a number of protective mechanisms, we further tested if the NO increase seen in baicalein treatment was related to activation of the NO synthase. As shown in Fig. 28, co-administration of NO synthase inhibitor L-NAME (200 μM) with baicalein reperfusion treatment (100 μM) resulted in attenuation of the NO increase. Meanwhile, the protective effects was also partially reversed (Fig. 29). This suggests that baicalein’s protection against I/R injury was, at least in part, attributed to NO synthase-mediated NO increase at the later phase of reperfusion. As apoptosis plays a significant role in I/R related cell death, we finally evaluated whether baicalein protects by attenuating apoptotic cell death along the course of I/R. As shown in Fig. 20 and 21, baicalein significantly reduced apoptotic cell death induced by exogenous oxidant injury caused by H2O2. Similarly, in endogenous oxidant injury such as that induced by I/R, the DNA fragmentation was also significantly diminished with the treatment of baicalein (Fig. 22), suggesting that baicalein exhibits antiapoptotic protection against I/R injury. In addition to pharmacological interventions such as antioxidant therapy, there are other methods that can be potentially employed in the compaign of I/R injury. Among these, chemical factors such as CO2 modification, and hence the pH, at the phase of reperfusion may be one that is effective and practically feasible. This is especially true when recent studies suggest that rapid normalization of pH at reperfusion may be associated with increased injury (the pH paradox). In this series of study, we compared hypercarbic (21% O2, 10% CO2, and 69% N2) and hypocarbic (21% O2, 1% CO2, and 78% N2) reperfusion with normocarbic reperfusion (21% O2, 5% CO2, 74% N2) in I/R control, and see if modification of reperfusion CO2 can have impact on reperfusion injury. As shown in Fig. 30, the cell death seen during the 1 hour ischemia prior to hypocarbic, normocarbic or hypercarbic reperfusion was not significantly different. Following ischemia, percent cell death in hypocarbic reperfusion was significantly higher (80.4 ± 4.5%) compared to normocarbic reperfusion (54.8 ± 4.0 %, P < 0.01). By contrast, hypercarbic reperfusion resulted in significantly lower cell death (26.3 ± 2.8%, P < 0.001 vs. normocarbic reperfusion). When comparing the ROS profile among the three protocols, we found that hypocarbic reperfusion was associated with increased ROS burst in the first 10 min of reperfusion, in contrast to reduction of the ROS burst by hypercarbic reperfusion (Fig. 31). The increased ROS induced by hypocarbia was mitochondria in origin since addition of mitochondrial complex III ihibitor stigmatellin (20 nM) significantly decreased the ROS (Fig. 32) and cell death (Fig. 33). On the other hand, in addition to attenuation of the reperfusion ROS burst, hypercarbic reperfusion also resulted in a sustained increase of NO at the later phase of reperfusion, a phenomenon similar to those seen in baicalein (Fig. 26, 27) and hypothermia (Fig. 40). This increase was also mediated by NO synthase activation sicne NO synthase inhibitor L-ANME (200 μM) significantly attenuated such NO increase (Fig.38) as well as the protection (Fig. 39). Therefore, hypercarbic protects cardiomyocytes from I/R injury not only by attenuation of the reperfusion ROS burst, but also activation of NO synthase leading to sustained increase of NO at the later phase of reperfusion. In conclusion, post-resuscitation myocardial dysfunction is associated with a number of clinical and resuscitation factors, and may impact the patient’s prognosis by influencing hymodynamics and organ perfusion in the early post-resuscitation phase. Identification of these factors may help understanding the pathophysiological mechanism involved in post-resuscitation myocardial dysfunction. It may also help avoiding the potentially harmful resuscitation measures such as unnecessary epinephrine doses. On the other hand, development of therapeutic strategies that can reduce I/R injury and augment myocardial recovery may help improve the post-resuscitation myocardial dysfunction and, possibly, prognosis. Though exciting results have been obtained from the basic research at the cell level, further work is needed while trying to translate these into clinical practice. With the establishment of the animal (rat) model of cardiac arrest and resuscitation, application of hypercarbic reperfusion or baicalein treatment for assessing the protective effects in the in-vivo model may offer valuable data and serve as a solid base when further translating these results to clinical use.