a b s t r a c t

Objective: Shenfu injection (SFI), a traditional Chinese formulation, has been confirmed to be protective against brain during ischemia and reperfusion injury. In this exploratory study, we investigated the action of SFI in regulating the inflammatory response and Brain edema after cardiopulmonary resuscitation.

Methods: After 8 minutes of untreated ventricular fibrillation (VF), pigs in the cardiopulmonary resuscitation group (n = 24) received a central venous injection of either SFI (SFI group; 1.0 mL/kg), epinephrine (EP group; 0.02 mg/kg), or saline (SA group). Levels of porcine-specific tumor necrosis factor ? and interleukin in sera were measured using enzyme-linked immunosorbent assay at 0.5, 1, 2, 4, 6, and 24 hours after return of spontaneous circulation (ROSC). Surviving pigs were killed 24 hours after ROSC, and the brains were removed for electron microscopy, Western blotting, and quantitative real-time polymerase chain reaction analysis. Results: Compared with the EP and SA groups, SFI decreased the levels of tumor necrosis factor ? and interleukin-6 in serum and the brain (P b .05) and decreased the expression of nuclear factor ?B and aquaporin-4 Messenger RNA in the brain (P b .05). Shenfu injection also inhibited the expression of nuclear factor ?B, matrix metalloproteinase 9, and aquaporin-4 protein after ROSC (P b .05). Observation of brain tissue ultrastructure showed that injury was alleviated in the SFI group compared with the SA and EP groups. Conclusions: Our exploratory experiments demonstrated that SFI reduced cerebral damage in a porcine model of VF, which may be related to suppression of the inflammatory reaction and decreased brain edema after ROSC.

(C) 2013

1. Introduction

It is estimated that 51% of cardiopulmonary resuscitation (CPR) comatose patients will develop severe brain damage [1]. There is clinical evidence that early drug treatment can improve neurologic function and survival after discharge from CPR [2]. For example, prehospital epinephrine (EP) use during CPR is considered beneficial [3]. However, numerous studies have shown that EP is also associated with increased myocardial dysfunction [4] and disturbed cerebral microcirculation [5] after cardiac arrest .

After CPR, the systemic inflammatory response is considered an important post-CA syndrome [6] because immune dysregulation can enhance neuronal damage after successful CPR [7]. Expression of aquaporin-4 (AQP4) in the cerebral cortex is up-regulated after CA [8], whereas expression of matrix metalloproteinase 9 (MMP9) protein and messenger RNA (mRNA) and water content were reported to be

* Corresponding author. Emergency Department of Beijing Chaoyang Hospital, Chaoyang District, Beijing 100020, China. Tel.: +86 010 85231051; fax: + 86

010 85231051.

E-mail address: [email protected] (C. Li).

increased in brain tissue after CPR in pigs [9]. These data suggest that CA can cause cytotoxic and vasogenic cerebral edema.

Brain injury after CPR involves a complex array of signaling mechanisms. Thus, drugs that target multiple pathways have great potential for neuroprotection after CPR. Shenfu injection (SFI) is a well-known traditional Chinese Herbal medicine containing ginseng (Panax; family: Araliaceae) and fuzi (Radix aconiti lateralis preparata, Aconitum carmichaelii Debx; family: Ranunculaceae), and was approved by the Chinese State Food and Drug Administration. A risk-control system for SFI has been constructed [10], and fingerprint technology has been used to ensure that the quality of SFI is consistent over different batches. Fingerprint technology refers to the use of spectroscopy and chromatography to obtain the characteristics of component groups, map or image, combined with computer technol- ogy to analyze information, thereby identifying the authenticity of drugs and the quality control of drugs [10]. Allergic reaction is the most serious clinical Adverse drug reaction to SFI, whereas other adverse effects are mild [11]. The main active components of SFI are ginsenosides and higenamine, which have been used in treating shock for more than 800 years in China. Shenfu injection is one of the most commonly used Traditional Chinese Medicines for heart failure in China [12] and has been shown to reduce myocardial damage and

0735-6757/$ – see front matter (C) 2013 http://dx.doi.org/10.1016/j.ajem.2013.03.020

alleviate the symptoms of cardiac dysfunction after CPR [13,14]. Furthermore, SFI was reported to be neuroprotective after cerebral ischemia in the rat [15].

The protective mechanisms of SFI against cerebral ischemia- reperfusion injury include reduced excitatory amino acid toxicity, blockade of Ca²⁺ overload, and improved antioxidant capacity [16]. However, the effects of SFI on inflammatory pathways and cerebral edema in the brain after CA remain unknown. As such, we performed an exploratory preliminary study to examine the effects of SFI on resuscitation brain injury in our porcine CA model. Inflammatory changes were assessed by expression of porcine-specific tumor necrosis factor-? (TNF-?), interleukin (IL-6), and nuclear factor ?B (NF-?B) expression in serum and brain. Brain edema biomarkers were also evaluated.

1. Materials and methods

This study was conducted with the approval of the Animal Care and Use Committee at the Beijing Chaoyang Hospital of the Capital Medical University, and all experiments complied with the National Research Council’s 1996 Guide for the Care and Use of Laboratory Animals.

1. Animal preparation

Thirty inbred Wu-Zhi-Shan miniature pigs aged 12 to 14 months with an average weight of 30 +- 2 kg were used in this study. The animals were randomly divided into 4 groups: SFI, EP, and saline (SA) resuscitation groups (n = 8, per group) and the sham-operated (SHAM) group (n = 6). All animals were fasted overnight with free access to water. Anesthesia was induced by Intramuscular injection of midazolam ketamine (0.5 mg/kg), followed by an injection of propofol (1.0 mg/kg) into the ear vein. Pentobarbital (8 mg kg^-1 h^-1) was injected to maintain anesthesia. A cuffed 6.5-mm endotracheal tube was advanced into the trachea. All animals were mechanically ventilated by a volume-controlled ventilator (Servo 900c; Siemens, Berlin, Germany) using a tidal volume of 15 mL/kg and a respiratory frequency of 12 breaths/min with room air. The respiratory frequency was adjusted to maintain end-tidal PCO2 between 35 and 40 mm Hg before inducing ventricular fibrillation (VF). aortic pressure was measured with a fluid-filled catheter that was advanced from the left femoral artery into the thoracic aorta. A Swan-Ganz catheter (7F; Edwards Life Science, Irvine, CA) was advanced from the left femoral vein and flow-directed into the pulmonary artery to measure right atrial pressure and cardiac output (CO). Cardiac output was determined by the thermodilution tech- nique. A 5F pacing catheter was advanced from the Right internal jugular vein into the right ventricle to induce VF. A 6F pressure Oxygen metabolism“>catheter was inserted into the right femoral artery to measure mean arterial pressure. All catheters were calibrated before application, and their tip positions were confirmed by the presence of the character- istic pressure traces. The electrocardiographic lead II was continu- ously recorded with a multichannel physiological recorder (BL-420F Data Acquisition & Analysis System; Chengdu TME Technology Co, Ltd, Sichuan, China).

All hemodynamic parameters were monitored using an HP monitor (M1165; Hewlett-Packard Company, Palo Alto, CA). An angiographic catheter was inserted from the femoral artery into the aortic arch to obtain reference blood samples and measure aortic pressure.

Drug

Shenfu injection (contains 0.9 mg ginsenosides and 0.1 mg aconite alkaloid per milliliter) was produced by Ya’an Sanjiu Pharmaceutical Co, Ltd, China.

Experimental protocol

After the operation, the animals were allowed to equilibrate for 30 minutes to obtain a resting level. Ventricular fibrillation was induced in the S1S2 mode (300/200 ms), 40 V, 8:1 proportion, and 10-ms step length to provide a continuous electrical stimulus until VF was obtained using a programmed electrical stimulation instrument (GY- 600A; KaiFeng Huanan Instrument Company, Kaifeng, Henan, China). Ventricular fibrillation was verified by the presence of a characteristic electrocardiogram waveform and an immediate drop in aortic blood pressure. After successful induction of VF, mechanical ventilation was discontinued. After 8 minutes of VF, Manual CPR was performed at a frequency of 100 compressions/min with mechanical ventilation at an FIO2 of 100% and a compression-to-ventilation ratio of 30:2. The Quality of chest compressions was controlled by a HeartStart MRx Monitor/Defibrillator with a Q-CPR (Philips Medical Systems, Best, Holland). After 2 minutes of CPR, the pigs received a central venous injection of SFI (1.0 mL/kg), EP (0.02 mg/kg), or SA. The investigators were blinded to the drug treatments. If VF persisted, defibrillation was attempted once using a diphase of 150 J. If spontaneous circulation was still not achieved, CPR was continued until return of spontaneous circulation (ROSC) was achieved. The same procedure without CA initiation was performed in the Sham group. If resuscitation time was longer than 30 minutes, the animal was considered dead. Return of spontaneous circulation was defined as a systolic blood pressure of greater than 50 mm Hg sustained for at least 10 minutes. After successful resuscitation, the animals underwent intensive care for 6 hours, and mechanical ventilation was resumed with the same settings as those used before induction of VF. At 6 hours after ROSC, all catheters were removed. The animals were then allowed to recover from anesthesia and were placed in observation cages and monitored for a further 18 hours. Finally, the animals were euthanized with 10 mL of 10 mol/L potassium chloride intravenously after a bolus of propofol 100 mg chloride intravenously.

Measurements

1. Hemodynamic and oxygen metabolism measurements

Hemodynamic data (heart rate, mean aortic pressure, and right atrial pressure) were continuously recorded. Coronary perfusion pressure was defined as aortic pressure during relaxation minus right arterial pressure.

Table 1

Baseline characteristics.

Characteristic	SHAM (n = 6)	SA (n = 8)	EP (n = 8)	SFI (n = 8)	P
Weight (kg)	30.50 +- 2.43	32.38 +- 2.62	31.50 +- 2.60	30.63 +- 2.36	.863
HR (ppm)	102.13 +- 6.13	100.50 +- 10.04	102.68 +- 7.96	101.38 +- 8.30	.927
MAP (mm Hg)	102.63 +- 5.32	104.00 +- 5.81	103.52 +- 6.02	101.86 +- 5.22	.735
RAP (mm Hg)	8.88 +- 1.46	8.59 +- 1.66	8.58 +- 1.78	8.99 +- 1.74	.902
CO (L/min)	2.94 +- 0.20	2.98 +- 0.20	2.92 +- 0.28	2.98 +- 0.19	.846

Data are reported as mean +- SD. HR, heart rate; MAP, mean aortic pressure; RAP, right atrial pressure.

IL-6 and TNF-? in the b”>Table 2

Resuscitation outcome.

Resuscitation outcome SA (n = 8) EP (n = 8) SFI (n = 8) Animals with ROSC 7 8 8

No. of shocks 5.5 +- 2.5 2.75 +- 1.66? 2.61 +- 1.03?

Time to ROSC (min) 10.00 +- 3.79 6.00 +- 2.17?? 5.00 +- 1.69??

6-h survival 6 7 7

24-h survival 6 6 6

* P b .05.

?? P b .01 vs SA (1-way repeated-measures ANOVA).

Biochemical assays

Venous blood samples were taken at baseline, 0.5, 1, 2, 4, 6, and 24 hours after ROSC. Blood was centrifuged at 3000 x g 15 minutes. The isolated serum was immediately frozen at -80?C and stored until the time of assay. The serum was processed in duplicate or triplicate according to the kit instructions. A quantitative sandwich enzyme- linked immunosorbent assay was used to measure concentrations of TNF-? and IL-6 (Rapidbio, Sacramento, CA). Levels of TNF-? and IL-6 were expressed in picograms per milliliter. The mean intra-assay coefficients of variation were less than 10%.

Levels of IL-6 and TNF-? in the brain

Brain homogenates were obtained from the cortex and centri- fuged at 3000 x g for 10 minutes to remove cellular debris. The concentrations of IL-6 and TNF-? were measured using specific enzyme-linked immunosorbent assay kits according to the manu- facturer’s instructions (RapidBio). Levels of TNF-? and IL-6 were expressed in picograms per gram. Real-time (RT) quantitative Polymerase chain reaction and Western blotting real-time quantitative PCR and Western blotting tissue samples from the cerebral cortex were collected when the animals were killed at 24 hours after ROSC. The samples were collected immediately and snap-frozen in liquid nitrogen to minimize time-dependent effects. Real-time quantitative PCR (ABI PRISM 7900 Sequence Detection System; Applied Biosystems, Carlsbad, CA) was performed to determine the expression levels of NF-?B, MMP9, and AQP4. Real- time PCR primers were designed by Beacon 7.0 Primer Express software (Applied Biosystems). Protein expressions of NF-?B, MMP9, and AQP4 in cortical tissues were measured by Western blotting, according to the manufacturer’s instructions. Optical densities of the immunoreactive bands were assessed using imaging software. Protein levels were normalized to ?-actin and presented as a ratio.

Brain ultramicrostructure

Brain tissue was preserved in 10% formaldehyde and 4% parafor- maldehyde. The ultramicrostructure of brain tissue was observed using light microscopy and TEM (H-7650; Hitachi, Ibaraki, Japan).

Statistical analysis

Data are expressed as mean +- SD for continuous variables and as percentages for categorical data. Nonparametric data are expressed as median (interquartile range) and analyzed by the Mann-Whitney U test. One-way analysis of variance (ANOVA) was used to compare differences at selected time intervals among groups. Statistical analysis was performed with SPSS 17.0 software (SPSS Inc, Chicago, IL). A 2-tailed probability value of less than .05 was considered statistically significant.

1. Results
   1. Baseline characteristics

Baseline characteristics of the 4 groups are shown in Table 1. No significant changes in body weight, heart rate, mean aortic pressure, right atrial pressure, or CO data were detected in the 4 groups (P N .05).

Resuscitation outcome and survival rates

Eighteen of 24 animals were successfully resuscitated in the 3 CPR subgroups. By comparison, the number of Electric shocks and time to ROSC were significantly lower in the EP and SFI groups than in the SA group (P b .05; Table 2). There was no difference in ROSC time or the number of shocks in the EP and SFI groups (P N .05). Six animals in the SA group and 7 animals in the EP and SFI groups survived to 6 hours, and 6 animals in the 3 CPR subgroups survived to 24 hours. There were no significant differences in 6- and 24-hour survival rates between CPR groups.

Laboratory results

1. Serum cytokine levels

Ventricular fibrillation/CPR resulted in a significant elevation in serum TNF-? and IL-6 levels after 30 minutes of ROSC relative to the SHAM group (P b .01). Compared with the SA group, TNF-? and IL-6 expression in the SFI group was significantly reduced at 1 hour after ROSC. Compared with the EP group, TNF-? and IL-6 expression in the SFI group was significantly reduced at 2 hours after ROSC (Fig. 1).

Fig. 1. Concentrations of TNF-? (A) and IL-6 (B) in serum after CPR. Significant differences vs the sham group are indicated: *P b .05; **P b .01 (1-way ANOVA). Significant differences vs the SA group are indicated: ⁺P b .05; ⁺⁺P b .01 (1-way ANOVA). Significant differences for the SFI vs the EP group are indicated: ^?P b .05; ^??P b .01 (1-way ANOVA). Baseline TNF-? and IL-6 serum levels were not different between the 4 groups. Cardiopulmonary resuscitation groups showed an increase in TNF-? and IL-6 from 30 minutes after VF, which peaked at 1 and 2 hours, respectively, and then began to decrease. Serum TNF-? and IL-6 expression 24 hours after CPR was increased compared with baseline values. Compared with the SA and EP groups, TNF-? and IL-6 expression in the SFI group was significantly reduced after CPR.

Fig. 2. Concentrations of TNF-? (A) and IL-6 (B) in the brain cortex after CPR. Brain TNF-? and IL-6 expression in 24 hours after CPR was increased to the basis of the value. Compared with the SA and EP groups, TNF-? and IL-6 expression in the SFI group significantly reduced after CPR (n = 6 in the SHAM group and n = 3 in the CPR subgroups).

Levels of IL-6 and TNF-? in the brain

The expression of IL-6 and TNF-? in the brains of pigs in the SFI group was significantly lower than that in the SA and EP groups (P b

.01; Fig. 2).

Real-time quantitative PCR

The expression of NF-?B and MMP9 mRNA in the brains of pigs in the SFI group was significantly lower than that in the SA group (P b .01). The expression of AQP4 mRNA in the brains of pigs in the SFI group was significantly lower than that in the SA group (P b .05). The expression of NF-?B and AQP4 mRNA in the brains of pigs in the SFI group was significantly lower than that in the EP group (P b .05; Fig. 3).

Western blotting

The protein expression of NF-?B, MMP9, and AQP4 in the brains of pigs in the SFI group was significantly lower than that in the SA group (P b .01). The protein expression of NF-?B, MMP9, and AQP4 in the brains of pigs in the SFI group was significantly lower than that in the EP group (P b .05; Fig. 4).

Observation of brain ultramicrostructure

Ultramicrostructural changes in the resuscitation control group were observed by electron microscopy. The resuscitation group showed obvious nerve cell damage, including loss of normal nerve cell form, presence of both nuclear deformation and solid shrinkage, mitochondrial swelling, ridge fracture, and cavity changes. In the SA group, glial cell nucleus pyknosis, mitochondrial swelling, and ridge fracture were noted (Fig. 5A). In the EP group, glial cell nuclei injury, mitochondrial swelling, and ridge fracture were observed (Fig. 5B). Animals treated with SFI exhibited little intracellular damage in the

Fig. 3. Messenger RNA expressions of NF-?B, MMP9, and AQP4 are expressed as mean +- SEM (n = 6 in the SHAM group and n = 3 in the CPR subgroups). Quantitative data for NF-?B, MMP9, and AQP4 were significantly higher in the CPR subgroups than in the SHAM group (^ooP b .01); mRNA expression of NF-?B and MMP9 was significantly lower in the SFI group than in the SA group (⁺P b .05); mRNA expression of AQP4 was significantly lower in the SFI group than in the SA group (⁺⁺P b .01); and mRNA expression of NF-?B and AQP4 was significantly lower in the SFI group than in the EP group (^?P b .05).

mitochondrial architecture at 24 hours after Cardiac resuscitation (Fig. 5C).

1. Discussion

Ginsenosides, the active ingredients of SFI, can suppress local inflammation after cerebral ischemia, and ginsenoside treatment has been shown to reduce brain edema and inhibit apoptosis [17-19]. The other active constituent of SFI is higenamine, which can improve myocardial cell pulsation frequency and amplitude, enhance myocar- dial contraction force, increase CO, and reduce myocardial oxygen consumption [20]. As a composite formulation, Shenfu exhibits protective effects on the heart and brain function after ischemia [15]. Based on previous studies, we choose to administer drugs via a topical route.

Our exploratory preliminary study showed that body and brain tissues suffered ischemia-reperfusion damage after ROSC after 8 minutes of VF. We found evidence of an inflammatory response, as indicated by elevated serum and brain levels of TNF-? and IL-6 and overexpression of NF-?B in the brain, as well as overexpression of AQP4 and MMP9 and brain edema. Our experiments demonstrated that SFI significantly suppressed TNF-? and IL-6 levels in the serum and brain and the expression of NF-?B in the brain. Shenfu injection also reduced brain edema by inhibiting AQP4 and MMP9 expression. In addition, brain ultrastructure observations showed that injury was alleviated in the SFI group compared with the SA and EP groups.

The whole body experiences Ischemia-reperfusion injury after CPR, with the brain exhibiting the greatest vulnerability to this insult. When the inflammatory reaction and anti-inflammatory response system are imbalanced, systemic inflammatory response syndrome can occur, which is a serious cause of multiple-organ dysfunction syndrome and death [21]. The Proinflammatory cytokines TNF-? and IL-6 are closely related to reperfusion injury after CPR, which can rapidly increase the inflammatory response system in the body after CPR [22]. Activation of NF-?B can also alter expression of numerous inflammatory factors; promote cell adhesion molecules, chemotactic factors, and response enzymes; and lead to systemic inflammation and cellular apoptosis after ischemia-reperfusion [23,24]. A similar inflammatory response to injury can occur in brain tissue cells after CPR but is markedly more serious [9]. Shenfu injection was previously reported to inhibit endotoxin-induced pulmonary inflam- mation in vivo by decreasing TNF-? and IL-6 expression and NF-?B activation [25]. In the present study, serum TNF-? and IL-6 levels increased immediately after ROSC in the SA group, although TNF-? increased more rapidly. Tumor necrosis factor ? acts as an initiation factor in inflammation [26] and can stimulate the generation of IL-6, resulting in the inflammatory response. Our study showed that the protective effect of SFI may be partly caused by inhibition of the inflammatory response.

Cardiopulmonary resuscitation can result in cytotoxic and vaso- genic brain edema. Aquaporin-4 is highly expressed in brain

Fig. 4. A, Western blots of the expression of NF-?B, MMP9, and AQP4 proteins in brain tissue in the SHAM, SA, EP, and SFI groups at 24 hours after ROSC. B, Quantification of NF-?B, MMP9, and AQP4 protein levels. Quantitative data for NF-?B, MMP9, and AQP4 protein levels were significantly higher in the CPR subgroups than in the SHAM group (^ooP b .01); NF-?B, MMP9, and AQP4 protein levels were significantly lower in the SFI group than in the SA group (⁺⁺P b .01); and NF-?B, MMP9, and AQP4 protein levels were significantly lower in the SFI group than in the EP group (^?P b .05) (n = 6 in SHAM group and n = 3 in the CPR subgroups).

astrocytes and plays an important role in water transport in the central nervous system. Manley et al [27] found that brain tissue water content and swelling of pericapillary astrocytic foot processes were significantly reduced in AQP4-deficient mice. Furthermore, cerebral edema and the severity of neurologic outcomes were associated with AQP4 expression after experimental CPR [7,8]. vasogenic edema is the main factor in cerebral ischemia-reperfusion brain edema. Metalloproteinase 9 plays an important role in the initiation and progression of vasogenic brain edema. For example, MMP9 activation was associated with the extent of the DWI high signal and Poor neurologic outcomes after CPR [28]. A variety of Inflammatory mediators are involved at the transcriptional level to promote MMP9 up-regulation [29]. For example, neuronal damage was significantly reduced in MMP9 gene knockout mice compared with wild types after cerebral ischemia [30], whereas brain edema peaked at 24 to 48 hours after ischemia with increased MMP9 activity in the brain [31]. In the present study, expression of AQP4 and MMP9 was significantly increased in the pig brain after CPR. Furthermore, SFI decreased cerebral cytotoxic and vasogenic edema, potentially by inhibiting the expression of AQP4 and MMP9.

The use of EP before hospital arrival is a positive predictor of short- term survival [32], although intravenous EP was associated with a decreased 1-month survival. Epinephrine dosage has also been reported to influence the outcome of CPR [33]. For example, experimentally, high-dose EP was beneficial for restoration of spontaneous circulation, although this benefit did not translate into improved long-term survival or neurologic outcome [34]. These

findings imply that EP administration might save the heart but not the brain. Unfortunately, EP can also produce deleterious effects on the myocardium [35]. For example, EP increased perfusion pressure and defibrillation rates, but did not significantly alter myocardial ATP during VF reperfusion in an in vivo heart reperfusion flow model [36]. Nevertheless, in the present study, EP effectively reduced the time to ROSC compared with the SA group, although there was no effect on inflammation, cerebral edema, or brain damage. By contrast, SFI injection suppressed the inflammatory reaction and decreased cerebral edema. Furthermore, the SFI group had better hemodynamic and oxygen metabolism parameters compared with the EP and SA groups. Because of the multiple components of SFI, these effects may be mediated by, at least in part, improved CPR.

1. Conclusions

We found that SFI was neuroprotective against brain injury induced by CPR and that the mechanism of action may be related to suppression of the inflammatory reaction and brain edema after ROSC. Thus, SFI might be superior to EP with regard to minimizing postresuscitation cerebral damage.

Acknowledgments

The authors thank Xian-Fei Ji, Shuo Wang, and Jun-Yuan Wu for their technical assistance and Dr Juan Liu for the advice in data analysis.

Fig. 5. Cytoplasmic ultrastructure of the brain neuron under an electron microscope. A, Ischemic pig brain cells ultrastructures in the SA group at 24 hours after cardiac resuscitation. Dotted arrows display brain glial cell nucleus damage, and the arrow shows mitochondrial swelling. B, Perinuclear condensation of chromatin with damaged mitochondria in the EP group at 24 hours after cardiac resuscitation. C, Mitochondrial architecture exhibited little intracellular damage in the SFI group at 24 hours after cardiac resuscitation.

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