Effects of Shenfu injection on macrocirculation and microcirculation during cardiopulmonary resuscitation

Effects of Shenfu injection on macrocirculation and microcirculation during cardiopulmonary resuscitation

Author’s Accepted Manuscript Effects of Shenfu injection on macrocirculation and microcirculation during cardiopulmonary resuscitation Junyuan Wu, Chu...

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Author’s Accepted Manuscript Effects of Shenfu injection on macrocirculation and microcirculation during cardiopulmonary resuscitation Junyuan Wu, Chunsheng Li, Wei Yuan www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(16)30028-9 http://dx.doi.org/10.1016/j.jep.2016.01.027 JEP9938

To appear in: Journal of Ethnopharmacology Received date: 26 August 2015 Revised date: 21 December 2015 Accepted date: 18 January 2016 Cite this article as: Junyuan Wu, Chunsheng Li and Wei Yuan, Effects of Shenfu injection on macrocirculation and microcirculation during cardiopulmonary r e s u s c i t a t i o n , Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.01.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effects of Shenfu Injection on Macrocirculation and Microcirculation during Cardiopulmonary Resuscitation Junyuan Wu MD, Chunsheng Li* MD, Wei Yuan MD Beijing Key Laboratory of Cardiopulmonary Cerebral Resuscitation (NO:BZ0370), Department of Emergency Medicine, Beijing Chaoyang Hospital, Capital Medical University, Beijing, 100020 China *Corresponding author: Chunsheng Li All authors have made substantial contributions to all of the following: Chunsheng Li: the conception and design of the study Tel: +86-10-85231051; Fax: +86-10-85231051; E-mail: [email protected] Junyuan Wu: acquisition of data, analysis and interpretation of data and drafting the article E-mail:[email protected] Wei Yuan: acquisition of data E-mail:[email protected] of Shenfu Injection on Macrocirculation and Microcirculation during Cardiopulmonary Resuscitation Abstract Aim of the study: To examine the effects of Shenfu injection (SFI) on macrocirculation and microcirculation during ventricular fibrillation (VF) and cardiopulmonary resuscitation (CPR). Materials and Methods: Sixteen female Landrace pigs were used in this study. After anesthesia, coronary perfusion pressure (CPP) was measured, and then the abdominal cavity was opened to observe the mesenteric microcirculation with the aid of sidestream dark field imaging. Following the guidelines, we determined microvascular flow index, perfused vessel density and proportion of perfused vessels both for large˄diameter >20­m˅and small˄diameter <20­m˅microvessels. SFI (1ml/kg) or saline was given by vein injection at 1 h before inducing VF. CPR was initiated after 4 min VF. Results: The shocks and duration of CPR were less in the SFI group compared with saline group. As the occurrence of VF, the CPP suddenly dropped to near zero, and cannot be measured in the both groups. However, there was greater CPP during CPR and at 1 h after return of spontaneous circulation in the SFI group than saline group. Compared with saline, SFI significantly improved 1

the microcirculation parameters of large and small microvessels during VF and CPR. Conclusions: SFI can improve the microvascular blood flow and CPP during VF and CPR, and reduce the shocks and duration of CPR. Key

words:

Microcirculation,

Coronary

Perfusion

Pressure,

Ventricular

Fibrillation,

Cardiopulmonary Resuscitation, Shenfu Injection Chemical compounds studied in this article˖ Ketamine ˄PubChem CID: 3821˅; Propofol˄PubChem CID: 4943˅; Pentobarbital˄PubChem CID: 4734˅; Ginsenoside (PubChem CID: 9918693); Aconitine (PubChem CID: 245005); Ginseng (PubChem CID: 9898279); Radix aconiti lateralis preparata (PubChem CID: 21115231); Aconitum carmichaeli Debx (PubChem CID: 157615); Ranunculaceae (PubChem CID: 353743)

Introduction Shenfu injection (SFI) is a traditional Chinese herbal medicine, and approved by Chinese State Food and Drug Administation (medicine manufacturing approval number: Z51020664). SFI contains ginsenoside and aconitine, which are made from raw material of ginseng (Panax, family: Araliaceae) and fuzi (Radix aconiti lateralis preparata, Aconitum carmichaeli Debx, family: Ranunculaceae) by using multistage countercurrent extraction and macroporous resin adsorption technology. In traditional Chinese medicine, ginseng is used to adjust blood pressure, restore heart function, treat neurasthenia and physical weakness and other symptoms. Aconitine has strong cardiac toxicity, mainly for all kinds of arrhythmia. But in the range of safe dose, there are also pharmacological effects, such as increasing myocardial contraction and antiarrhythmia. The

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compatibility of fuzi and ginseng can reduce the toxicity of aconite. In China, SFI has been widely used in clinic to treat some diseases associated with syndrome of sudden yang collapse, such as septic shock and cardiac shock. As we know, cardiac arrest represent the most sever state of shock. In our previous studies, we found that SFI can improve the post-resuscitation hemodynamics and myocardial dysfunction (Gu et al., 2012; Ji et al., 2013; Ji et al., 2011). Zhang et al (Zhang et al., 2006) found that SFI has a protective effect on gastrointestinal microcirculation after myocardial ischemic-reperfusion injury in rabbits. But there is no direct observation of microcirculation blood flow in their experiment. Sidestream dark field (SDF) imaging which has been described in detail previously can enable microvascular imaging in real time (Goedhart et al., 2007). To determine if SFI can improve microcirculation during Ventricular fibrillation (VF) and cardiopulmonary resuscitation (CPR), we established a porcine model of VF to examine the effects of SFI on the mesenteric microcirculation with the aid of SDF imaging.

Methods This study was approved by the Animal Care and Use Committee of Capital Medical University, China. The humanistic concern for animal complies with the principles of laboratory animal use and care formulated by the Administration Office of Laboratory Animals.

Animal and surgery Sixteen female Landrace pigs (2–3 months of age, 30f5 kg) were used in this study. Animals were fasted overnight except for free access to water. Anesthesia was initiated by intramuscular injection of ketamine (20 mg/kg) and followed by ear vein injection of propofol (1 mg/kg). Anesthesia was maintained by intravenous infusion of pentobarbital (8 mg/kg/h). A cuffed 6.5-mm endotracheal tube was advanced into the trachea, and animals were mechanically

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ventilated with a volume controlled ventilator (Evita 4, Drager Medical, Lubeck, Germany) using a tidal volume of 15 mL/kg, a respiratory frequency of 12 breaths/min, and FiO2 at 0.21. End-tidal PCO2 was measured by in-line infrared capnography and maintained between 35 and 40 mm Hg through adjusting respiratory frequency before induction of VF. Room temperature was controlled at 26oC. Cardiac output (CO) was monitored with the thermodilution technique. An arterial catheter (5F, Pulsiocath PV 2015L20, Pulsion Medical Systems, Germany) was inserted into the descending artery through the left femoral artery for measuring arterial pressure. A 7-Fr central venous catheter was advanced from the right external jugular vein into the right atrium for measuring right atrial pressure and collection of vein blood, and used for injection of iced saline. The arterial and central venous catherters were connected to an integrated bedside monitor (PICCO, Pulsion Medical Systems, Germany) for continuous hemodynamic monitoring. A 7F sheathing canal (Edwards Life Sciences, USA) was inserted into the left femoral vein to place an electrode catheter for induction of VF by a programmed electrical stimulation instrument (GY-600A, Kai Feng Huanan Instrument Limited Company, China). The abdominal cavity was opened to expose the mesentery of small intestine. Mesenteric microcirculation were observed and recorded by Microscan Analysis Software (Automated Vascular Analysis 3.0, University of Amsterdam, Netherlands).

Experimental procedures After surgery, the animals were allowed to equilibrate for 30 minutes to achieve stable resting level, and then were given SFI (1ml/kg) or saline (1ml/kg) by vein injection. The investigators were blinded to the drug treatment. One hour after administration, VF was induced by programed electrical stimulation, mode S1S2 (300/200 milliseconds), 40v, 8:1 proportion, and −10

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milliseconds step length. (Wu et al., 2009) VF was verified by ECG and blood pressure. After successful inducing of VF, mechanical ventilation was discontinued and electrode catheter was extracted. CPR was initiated after 4 min VF. Compression-to-ventilation ratio was 30:2, and ventilation was performed by bag respirator with room air. Defibrillation (Heartstart MRx Monitor/Defibrillator, Philips Medical Systems, Holland) was attempted after 2 min CPR. Defibrillation shocks were administered at 120 J (Smart Biphasic) for the first attempt. All subsequent attempts used the 150 J dose. If the first defibrillation was unsuccessful as indicated by arterial pressure and ECG monitoring for 5 s, another 2 min CPR continued. Mechanical ventilation with 100% oxygen was started at the beginning of the first defibrillation attempt and continued until return of spontaneous circulation (ROSC), after which room air was used. ROSC was defined as maintenance of systolic blood pressure at least 50 mm Hg for a continuous period of at least 10 min. Animals were announced dead if 4 times of defibrillation were attempted but still no ROSC. After ROSC, animals underwent a 4-h intensive care period with administration of Ringer’s solution (20 mL/kg). And then all vascular catheters were removed. The abdominal cavity was closed after obtaining the microcirculatory image of 1 h after ROSC. Animals were allowed to recover from anesthesia, placed in observation cages, and monitored every two hours until 24 h after resuscitation. Water was given during the observation period. Tramadol hydrochloride (50mg) was given by intramuscular injection every 12 hours to ease the pain. The experimental procedure is shown in figure 1. Shenfu injection SFI which contains ginsenoside (0.8 mg/ml) and aconitine (0.1 mg/ml) is an extract of traditional Chinese herbs. Chemical structures of ginsenoside and aconitine are shown in figure 2.

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SFI is produced by Ya’an Sanjiu Pharmaceutical Co, Ltd (No. 110804, Ya’an, China). In order to guarantee the SFI quality from the sourceˈginseng from Jingyu GAP planting base in Jilin province and fuzi from Jiangyou GAP planting base in Sichuan province are used. A four combination approach including complete component fingerprint analysis, main active ingredient detection, toxic ingredients detection and harmful substances limit test is adopted to establish the quality standard system for SFI. SFI was prepared as working procedure (Yang, 2012): the ginseng radix was crushed, extracted with ethanol 4 times under reflux (2 h/time), after soak in ethanol for 2 hours, then combined the ethanol solution concentrated in a rotator evaporator in vacuo to give ginseng radix extract. 22 compounds including 12 kinds of major ginsenosides and 10 kinds of rare ginsenosides were separated from the extracts of ginseng radix as shown in the table 1. Aconite was extracted with water twice (2 h/time), after soakin 1% HCl water solution for 2 hours. The combined solution was concentrated in vacuo, after it cool down, add appropriate amount of ethanol, then concentrated in a rotator evaporator to generate aconite extract. 6 kinds of aconitum alkaloids were separated from the extracts of aconitum as shown in the table 1.

Microcirculatory imaging Mesenteric microcirculation was visualized with the aid of a SDF imaging video microscope (MicroScan, MicroVision Medical, Amsterdam, Netherlands) with a  h optical probe. A hand-held video microscope emits stroboscopic green light, which is absorbed by hemoglobin. The negative image of moving red blood cells is transmitted back to a camera. SDF image show a region of interest of approximately 1000μmh750μm. Individual video of 10 sec was analyzed off-line using a score previously described by Spronk et al (Spronk et al., 2002), in which 0

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represents no flow, 1 represents markedly reduced flow, 2 represents reduced flow, and 3 represents normal flow.

Measurements Hemodynamic data (CO, arterial pressure, right atrial pressure) and ECG were continuously measured and recorded. Coronary perfusion pressure (CPP) was measured from the differences in time-coincident diastolic aortic and right atrial pressures during diastole. During CPR, CPP was measured during the relaxation time of chest compression. Microcirculatory images were obtained at baseline; 1 and 3 min after onset of VF; 1 min after start of CPR; and 1 min and 1 h after ROSC. Mixed venous blood samples for lactate levels analyses were drawn at the same time. Following the guidelines (De Backer et al., 2007),

we determined microvascular flow index (MFI),

perfused vessel density (PVD) and proportion of perfused vessels (PPV) both for large microvessel˄diameter >20μm˅and small microvessel ˄diameter <20μm˅. Two technical personnel performed the analysis independently and their results were averaged.

Statistical analysis Data with normal distribution are reported as mean f SD and those with nonnormal distribution as median (25th and 75th percentiles). Discrete variables, such as ROSC and 24-hour survival were compared with Fisher exact test testing. Continuous variables, including hemodynamic data, microcirculation parameters (PVD, PPV and MFI), lactate levels and duration of CPR were compared by the repeated measures and multivariate analysis of variance. Shocks before ROSC, which is a skewed distribution, were compared by Mann-Whitney Test. A value of p <0 .05 was regarded as being statistically significant. All analyses were conducted using the SPSS 17.0 software (SPSS Inc, Chicago III) and GraphPad PRISM version 5 (GraphPad Software

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Inc.,San Diego, CA).

Results All animals of SFI group were resuscitated. However, there were no significant differences in ROSC and 24-hour survival between SFI group and saline group. The shocks of survivor in the SFI group were less than those in the saline group (p<0.05). And the duration of CPR before ROSC was also less in the SFI group compared with saline group (p<0.05). There were no animals died during the post-operative period. (Table 2) From figure 3, as the occurrence of VF, the CPP and CO suddenly dropped to near zero, and cannot be measured. CPR could effectively improve the CPP and CO level. Compared with baseline, the CPP, CO and MAP was significantly decreased during VF, CPR and at 1 h after ROSC, but the CO and MAP increased at 1 min after ROSC either in the SFI group or in the saline group. The CPP was significantly greater at 1 min of CPR in the SFI group compared with saline group (p<0.05), but there were no significant differences after ROSC. The mean CO of SFI group was greater at 1 h after ROSC than that of saline group (p<0.05), but the differences were not significant at 1 min after ROSC and during CPR. There were no significant differences in mean arterial blood pressures (MAP) between two groups at each time point (p all >0.05). Stills of the acquired video clips of the mesenteric microcirculation were shown in figure 4. In both SFI and saline groups, mesenteric microvascular blood flows were dramatically reduced within the 1 min period as the occurrence of VF, followed by a continuous decrease. Microcirculatory blood flow was improved with the start of CPR, and gradually recovered to near baseline level after ROSC. Compared with saline group, SFI significant improved microcirculatory blood flow in both small microvessel and large microvessel during VF and

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CPR. As shown in figure 5, the microcirculation parameters were significantly decreased during VF and CPR and at 1 min after ROSC than baseline either in the saline group or in the SFI group (p all < 0.05). Compared with baseline, the microcirculation parameters also decreased significantly at 1 h after ROSC in the saline group (p all < 0.05), but there were no significant differences in the SFI group (p all > 0.05). SFI significantly improved the microcirculation during VF and CPR compared with saline. And there were also better microcirculation parameters of large microvessel at 1 min after ROSC in the SFI group than those in the saline group (p all <0.05). Although the PVD and PPV of small microvessel were not significantly different at 1min after ROSC between two groups, the mean MFI of SFI group was greater compared with saline group (2.3 vs 1.7, p<0.05). The differences of microcirculation parameters between two groups were not significant at 1h after ROSC. Lactate level was significantly increased during VF, CPR and after ROSC than baseline. The mean lactate levels of SFI group were lower than those in saline group during CPR and after ROSC (p all <0.05). (Table 3)

Discussion VF is the most common cause of cardiac arrest. The goal of CPR during VF is promoting forward flow of oxygenated blood to maintain the main organs viability until ROSC. CPP values during CPR have been identified as the leading predictor of the success of resuscitation (Paradis et al., 1990; Sanders et al., 1985) . However, the measurement of CPP is limited to clinical patients who need catheters in place in the aorta and the right atrium. In clinic, the sublingual microcirculation monitoring with the aid of SDF imaging which is a noninvasive measurement has

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been widely used in septic and cardiac shock patients, but rarely to assess the human microcirculation during CPR (Elbers et al., 2010). Some studies found that the macrocirculatory and microcirculatory flows were dissociated during cardiogenic and septic shock (De Backer et al., 2004; Sakr et al., 2004). However, Fries et al (Fries et al., 2006a) found that microvascular blood flow was highly correlated with CPP during CPR, and was also predictive of outcome. In the result of this study and our previous experimental results (Wang et al., 2010; Wu et al., 2009), as the occurrence of VF, the mean CPP and CO was very low to near zero so that it could not be measured to evaluate perfusion. Although microvascular blood flow also significantly reduced during VF, flow continued for more than 3 min after onset of VF (Ristagno et al., 2008). Therefore, microvascular blood flow monitoring with the aid of SDF imaging was used in this study to evaluate the effects of SFI on improving the perfusion during VF and CPR. In order to reduce the impact of tracheal intubation and movement during chest compression, we chose to observe the mesenteric microcirculation rather than the sublingual microcirculation. SFI is originated from the ancient prescriptions Shenfu Formula (Panax ginseng C. A. Mey. and Radix Aconitum Carmichaeli), which has been used for 800 years in china. The pharmacological effects of SFI depend on aconitine properties, supplemented by ginsenoside, which can increase heart rate and myocardial contractility. In clinic, SFI is used for treatment of many kinds of diseases, and is especially known for its traditional cardiovascular protective effect on coronary artery dilation, blood pressure stabilization, reduction of ischemia reperfusion injury, and improvement of the heart function (Hu et al., 2009; Li, 2006; Zheng et al., 2004). In our previous studies, animals treated with SFI during CPR had improved left ventricular function, lower troponin I levels, and increased tissue perfusion and oxygen metabolism compared with

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saline (Gu et al., 2012). Treatment with SFI after ROSC produced better maximum rate of left ventricular pressure increase (dp/dt (max)) and maximum rate of left ventricular pressure decline (-dp/dt (max)), cardiac output, and ejection fraction (Ji et al., 2013; Ji et al., 2011). In this study, SFI was given at one hour before inducing VF to observe its effect on hemodynamics and microcirculation during untreated VF. Compared with baseline, the CPP, CO and MAP were significantly decreased at each time point except for 1 min after ROSC either in the SFI group or in the saline group. This is due to the release of a large amount of catecholamine in response to VF, which is then suddenly circulated after ROSC (Wu et al., 2013). From the result, SFI improved the CO at 1 h after ROSC compared with saline. This supports our earlier findings (Gu et al., 2012; Ji et al., 2013; Ji et al., 2011). Fries et al (Fries et al., 2006a; Fries et al., 2006b) found that flow in <20μm microvessel and >20μm microvessel was highly correlated. As the results shown, SFI improved the mesenteric microcirculation both large and small microvessel during VF compared with saline, although the CPP cannot be measured. This suggested the microcirculation parameters were more sensitive to evaluate the perfusion during VF than CPP. Similar to the results of CPP, there were also greater microcirculation parameters during CPR in the SFI group than saline group. Zhang et al (Zhang et al., 2006) also found SFI can improve microcirculation after myocardial ischemic-reperfusion injury, although they evaluated microcirculation only through gastric intramucosal PCO2 and PH which are not direct evidences. The microcirculation parameters were greater at 1 min after ROSC in the SFI group compared with saline group, but there were no significant differences at 1 h after ROSC. Ristagno et al (Ristagno et al., 2008) reported that there were progressive increases in flow velocities after

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ROSC, slightly more rapidly in vessels >20μm, and restoration of normal microvascular blood flow to pre-arrest values occurred within 3 min. These are similar to our results. Cocchi et al (Cocchi et al., 2011) find that initial blood lactate levels are associated with mortality after cardiac arrest. Effective lactate clearance has been found to be associated with reduced mortality in post-cardiac arrest patients (Donnino et al., 2007; Wiklund et al., 2005). In addition, blood lactate level and microcirculation parameter PVD are significantly correlated in critically ill patients (Yeh et al., 2013). From our data, serum lactate levels were significantly lower in the SFI group than saline group during CPR and after ROSC. The reason may be that SFI improve tissue perfusion and alleviate microcirculatory tissue dysfunction during VF. There are several limitations in the interpretation of our results. VF is the critical arrhythmia that most often occurs in patients with severe cardiovascular diseases, such as myocardial infarction. In this study, the experiment was performed on apparently healthy pigs, therefore the results do not reflect the exact changes and complete outcomes that would occur in the human body under similar physiologic conditions. In order to evaluate the effects of SFI on improving the perfusion during VF, SFI was given at one hour before inducing VF. This pretreatment is impossible to achieve in clinic. The mesenteric microcirculation may not reflect other microvascular beds especially brain and heart. And use of the semi quantitative score described by Spronk et al (Spronk et al., 2002) is not an objective measurement. Conclusion We conclude that under these experimental settings, SFI can improve the microvascular blood flow and CPP during VF and CPR, and reduce the shocks and duration of CPR. Acknowledgements

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This work was supported by the National Natural Science Foundation of China (No. 81372025), 2015 annual special cultivation and development project for Technology Innovation Base

of

Beijing

Key

Laboratory

of

Cardiopulmonary

Cerebral

Resuscitation

(NO.Z151100001615056) (to L CS), and Youth Science Fund of Beijing Chaoyang Hospital (to W JY). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References Cocchi, M.N., Miller, J., Hunziker, S., Carney, E., Salciccioli, J., Farris, S., Joyce, N., Zimetbaum, P., Howell, M.D., Donnino, M.W., 2011. The association of lactate and vasopressor need for mortality prediction in survivors of cardiac arrest. Minerva anestesiologica 77, 1063-1071. De Backer, D., Creteur, J., Dubois, M.J., Sakr, Y., Vincent, J.L., 2004. Microvascular alterations in patients with acute severe heart failure and cardiogenic shock. American heart journal 147, 91-99. De Backer, D., Hollenberg, S., Boerma, C., Goedhart, P., Buchele, G., Ospina-Tascon, G., Dobbe, I., Ince, C., 2007. How to evaluate the microcirculation: report of a round table conference. Critical care 11, R101. Donnino, M.W., Miller, J., Goyal, N., Loomba, M., Sankey, S.S., Dolcourt, B., Sherwin, R., Otero, R., Wira, C., 2007. Effective lactate clearance is associated with improved outcome in post-cardiac arrest patients. Resuscitation 75, 229-234. Elbers, P.W., Craenen, A.J., Driessen, A., Stehouwer, M.C., Munsterman, L., Prins, M., van Iterson, M., Bruins, P., Ince, C., 2010. Imaging the human microcirculation during cardiopulmonary resuscitation in a hypothermic victim of submersion trauma. Resuscitation 81, 123-125. Fries, M., Tang, W., Chang, Y.T., Wang, J., Castillo, C., Weil, M.H., 2006a. Microvascular blood flow during cardiopulmonary resuscitation is predictive of outcome. Resuscitation 71, 248-253. Fries, M., Weil, M.H., Chang, Y.T., Castillo, C., Tang, W., 2006b. Microcirculation during cardiac arrest and resuscitation. Critical care medicine 34, S454-457. Goedhart, P.T., Khalilzada, M., Bezemer, R., Merza, J., Ince, C., 2007. Sidestream Dark Field (SDF) imaging: a novel stroboscopic LED ring-based imaging modality for clinical assessment of the microcirculation. Optics express 15, 15101-15114. Gu, W., Li, C., Yin, W., Guo, Z., Hou, X., Zhang, D., 2012. Shen-fu injection reduces postresuscitation myocardial dysfunction in a porcine model of cardiac arrest by modulating apoptosis. Shock 38, 301-306. Hu, Y.H., Wu, H.Q., Qi, X., 2009. [Influence of shenfu injection on heart function and bone marrow stem cell mobilization in patients with chronic heart failure of coronary heart disease]. Zhongguo Zhong xi yi jie he za zhi Zhongguo Zhongxiyi jiehe zazhi = Chinese journal of integrated traditional and Western medicine / Zhongguo Zhong xi yi jie he xue hui, Zhongguo Zhong yi yan jiu yuan zhu ban 29, 309-312. Ji, X.F., Ji, H.B., Sang, D.Y., Wang, S., Yang, L., Li, C.S., 2013. Shen-Fu injection reduces impaired

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myocardial beta-adrenergic receptor signaling after cardiopulmonary resuscitation. Chinese medical journal 126, 697-702. Ji, X.F., Yang, L., Zhang, M.Y., Li, C.S., Wang, S., Cong, L.H., 2011. Shen-fu injection attenuates postresuscitation myocardial dysfunction in a porcine model of cardiac arrest. Shock 35, 530-536. Li, Z.E., 2006. [Clinical research on effects of shenfu injection in different dosage in preventing heart failure occurred in patients of acute myocardial infarction with elevated ST segment]. Zhongguo Zhong xi yi jie he za zhi Zhongguo Zhongxiyi jiehe zazhi = Chinese journal of integrated traditional and Western medicine / Zhongguo Zhong xi yi jie he xue hui, Zhongguo Zhong yi yan jiu yuan zhu ban 26, 555-557. Paradis, N.A., Martin, G.B., Rivers, E.P., Goetting, M.G., Appleton, T.J., Feingold, M., Nowak, R.M., 1990. Coronary perfusion pressure and the return of spontaneous circulation in human cardiopulmonary resuscitation. Jama 263, 1106-1113. Ristagno, G., Tang, W., Sun, S., Weil, M.H., 2008. Cerebral cortical microvascular flow during and following cardiopulmonary resuscitation after short duration of cardiac arrest. Resuscitation 77, 229-234. Sakr, Y., Dubois, M.J., De Backer, D., Creteur, J., Vincent, J.L., 2004. Persistent microcirculatory alterations are associated with organ failure and death in patients with septic shock. Critical care medicine 32, 1825-1831. Sanders, A.B., Kern, K.B., Atlas, M., Bragg, S., Ewy, G.A., 1985. Importance of the duration of inadequate coronary perfusion pressure on resuscitation from cardiac arrest. Journal of the American College of Cardiology 6, 113-118. Spronk, P.E., Ince, C., Gardien, M.J., Mathura, K.R., Oudemans-van Straaten, H.M., Zandstra, D.F., 2002. Nitroglycerin in septic shock after intravascular volume resuscitation. Lancet 360, 1395-1396. Wang, S., Li, C., Ji, X., Yang, L., Su, Z., Wu, J., 2010. Effect of continuous compressions and 30:2 cardiopulmonary resuscitation on global ventilation/perfusion values during resuscitation in a porcine model. Critical care medicine 38, 2024-2030. Wiklund, L., Sharma, H.S., Basu, S., 2005. Circulatory arrest as a model for studies of global ischemic injury and neuroprotection. Annals of the New York Academy of Sciences 1053, 205-219. Wu, J.Y., Li, C.S., Liu, Z.X., Wu, C.J., Zhang, G.C., 2009. A comparison of 2 types of chest compressions in a porcine model of cardiac arrest. The American journal of emergency medicine 27, 823-829. Wu, J.Y., Wang, S., Li, C.S., 2013. Hemodynamic and catecholamine changes after recurrent ventricular fibrillation. The Journal of emergency medicine 44, 543-549. Yang, R.J., 2012. Study on chemical constituents of Shenfu injection. Jilin University. Yeh, Y.C., Wang, M.J., Chao, A., Ko, W.J., Chan, W.S., Fan, S.Z., Tsai, J.C., Sun, W.Z., 2013. Correlation between early sublingual small vessel density and late blood lactate level in critically ill surgical patients. The Journal of surgical research 180, 317-321. Zhang, X.J., Song, L., Zhou, Z.G., Wang, X.M., 2006. Effect of shenfu injection on gastrointestinal microcirculation in rabbits after myocardial ischemia-reperfusion injury. World Journal of Gastroenterology 12, :4389-4391. Zheng, S.Y., Sun, J., Zhao, X., Xu, J.G., 2004. Protective effect of shen-fu on myocardial ischemia-reperfusion injury in rats. The American journal of Chinese medicine 32, 209-220.

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Figure 1. Experimental procedure.

Figure2. Chemical structures of Ginsenoside and Aconitine.

Figure 3. Hemodynamics during VF, CPR and after ROSC. # p < 0.05 vs Baseline * p < 0.05 vs Saline

Figure 4. Stills of video clips of the mesenteric microcirculation at baseline before induction of VF(A,E), 1 min after onset of VF (B,F), 1 min after start of CPR(C,G) and 1 min after ROSC(D,H). A, B, C, D in SFI group; E, F, G, H in Saline group.

Figure 5. Microcirculation parameters during VF, CPR and after ROSC. VF, ventricular fibrillation; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation; PVD, perfused vessel density; PPV, proportion of perfused vessels; MFI, microvascular flow index; SFI, shenfu injection. # p < 0.05 vs Baseline * p < 0.05 vs Saline

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Table 1

Table 1 Composition of Shenfu injection

12 kinds major ginsenosides (content range, µggmlѸ1)

Shenfu injection

Ginsenosides

10 kinds rare ginsenosides (content range, µggmlѸ1)

Aconite

6 kinds aconitum alkaloids (content range, µggml 1) 䳼

Re (58̢89) Rg1 (85̢119) Rf (28̢31) Rg2 (13̢15) Rb1 (198̢227) Rc (97̢114) Rb2 (50̢64) Rb3 (8̢14) Rd (36̢49) F1 (3̢6) F3 F5 Rk1 (8̢15) Rg5 (12̢20) Rk3 (8̢11) Rh4 (6̢11) Rg6 (3̢6) F4 (4̢8) Rg3 (21̢36) 20(R)-Rg3 (16̢30) Rh1 (27̢42) 20(R)-Rh1 (14̢25) Benzoyl deoxyaconitine (0.04-0.1) Benzoyl hypaconitine (0.1-0.7) Benzoyl aconitine (0.2-0.3) Benzoyl mesaconitine (0.2-1.2) Aconine Hypaconine

Table 2

Table 2 Outcomes Outcomes

Saline (n=8)

SFI (n=8)

p

ROSC Shocks before ROSC Duration of CPR before ROSC (s) 24-h survival

6/8 1.8(1.0,2.5) 240 ±100 6/8

8/8 1.1(1.0,1.5) 151 ±46 8/8

0.46 0.01 0.04 0.46

SFI, shenfu injection; ROSC, return of spontaneous circulation; CPR, cardiopulmonary resuscitation;

Table 3

Table 3 Lactate levels of mixed venous blood Lactate

Baseline

VF 1 min

VF 3 min

CPR 1 min

ROSC 1 min

ROSC 1 h

SFI Saline

1.55f0.49 1.50f0.39

2.46f0.53# 2.87f0.52#

3.41f0.63# 4.01f0.40#

4.54f0.83#* 5.85f0.87#

5.36f0.91#* 6.48f0.95#

4.86f0.91#* 5.99f0.52#

SFI, shenfu injection; VF, ventricular fibrillation; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation; # p < 0.05 vs Baseline * p < 0.05 vs Saline

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

*Graphical Abstract