Acupuncture, Article

The effects of stimulation at acupoint ST36 points against hemorrhagic shock in dogs

Unlabelled imageAmerican Journal of Emergency Medicine (2011) 29, 1188-1193

Brief Report

The effects of stimulation at acupoint ST36 points against hemorrhagic shock in dogs?

Xiao-Yan Suo MD, Zhao-Hui Du PhD?, Hai-Song Wang MD, Jian-Guo Li PhD, Yan-Lin Wang PhD, Shu-Dong Yao MD, Wei-Min Chen MD

Department of Anesthesiology and Critical Care Medicine, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei Province, China

Received 4 February 2010; revised 10 July 2010; accepted 11 July 2010

Abstract The aim of this study is to investigate the effects of electroacupuncturing (EA) zusanli points on levels of basic hemodynamics, lactate, and cytokines in dogs with hemorrhagic shock. Thirty healthy dogs were randomly divided into 5 groups: sham hemorrhagic shocked group, hemorrhagic shocked group, EA group, nonacupuncturing group, and EA after vagotomy group. Zusanli points were electroacupunctured with constant voltage (10-15 V, 30 Hz) for 30 minutes immediately after the shock models were established. Before the stimulation, a blood pressure transducer was implanted into the right femoral artery for continuous recording of mean arterial pressure (MAP), and a 5F Swan-Ganz pediatric catheter was implanted into the pulmonary artery. The levels of serum tumor necrosis factor ? (TNF-?) in the femoral artery were detected at 0, 120, and 180 minutes after hemorrhage. The levels of serum lactate in the femoral artery were detected before hemorrhage (-45 minutes), at 0 minute, and at 180 minutes. In the hemorrhagic shocked group, the levels of MAP, cardiac output, cardiac index, central venous pressure, and pulmonary arterial wedge pressure decreased significantly; at the same time, the levels of serum TNF-? and serum lactate increased significantly. There were no differences between these groups and the hemorrhagic group, but they were different from the sham hemorrhagic shocked group. In the EA group, the levels of MAP, cardiac output, cardiac index, central venous pressure, and pulmonary arterial wedge pressure gradually increased, but the content of serum TNF-? and lactate obviously decreased. The results suggested that EA zusanli points produce a protective effect on hemorrhagic shock in dogs.

(C) 2011

Introduction

? This work was supported by the National Natural Science Foundation of China (grant 200630672727), the Natural Science Foundation of Hubei Province of China (grant 2004ABA161), and the Tackle Key Problems in Science and Technology of Hubei Province of China (grant 2007AA301B46).

* Corresponding author.

E-mail address: [email protected] (Z.-H. Du).

Hemorrhagic shock is a predictor of high mortality and morbidity after hypoperfusion and tissue hypoxia, which tends to progress into multiple-organ failure and death. Hemorrhagic shock induces systemic expression of proin- flammatory cytokines, such as tumor necrosis factor ? (TNF-?) and interleukin 1? (IL-1?) [1,2]. The cytokines are recognized as primary mediators that initiate the elaboration of a cascade of other mediators. Their overproduction may cause systemic inflammatory response syndrome, organ

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damage, and multiple-organ dysfunction [3]. The mechanism of pathogenesis of systemic inflammatory response syn- drome in hemorrhagic shock is complex, and a variety of mechanisms are implicated.

Regarding the duration of hemorrhagic shock, it has been shown that the longer the shock persists, the more intense is the inflammatory response that follows [4]. Similarly, survival rate seems to be improved with early resuscitation; and mortality is high with delayed resuscitation, which is similar to that of unResuscitated animals [5,6].

Acupuncture is the empirical medicine that has been used in China for more than 3000 years [7,8]. Acupuncture medicine has been used in clinical practice in Japan as well for more than 1500 years. In recent years, acupuncture has been rapidly accepted widely throughout the world, including the United States.

Although the underlying mechanisms remain to be studied, increasing data indicate that acupuncture stimulation of ST36 (zusanli) exhibits significant anti-inflammatory effects. For instance, ST36 acupuncture has been shown to inhibit TNF-? production [9], attenuate trauma-induced immunosuppression [10], and reverse sepsis-induced neu- trophil migration impairment in septic rats [11]. ST36 acupuncture has also been shown to activate the parasym- pathetic efferent pathway [12], a pathway thought to have significant anti-inflammatory effects against sepsis [13]. Collectively, these data suggest the therapeutic potential of acupuncture stimulation of ST36 against sepsis [14].

Although little is known about the mechanism(s) of acupuncture, we have recently found the protective effects of electroacupuncture at ST36 points against hemorrhagic shock in rats [15].

To extend the clinical use of ST36 acupuncture in hemorrhagic shock, we also investigated the protective effects of ST36 acupuncture administered after induction of hemorrhagic shock in dogs.

Materials and methods

Acute hypovolemic hemorrhagic shock protocol

This research protocol complied with the regulations regarding animal care published by the National Institutes of Health and was approved by the Institutional Animal Use and Care Committee of the Wuhan University. Thirty mongrel male dogs with age 3 to 9 years and weighing between 13 and 18 kg were used in this study. They were considered healthy after clinical examination and with a normal erythrocyte count. The dogs were fed standard dog chow and water ad libitum for at least 1 week in the divisional kennel before operation. Food was removed at 12 hours and water at 1 hour before administration of anesthesia. The animals were premedicated with 7 mg/kg Intramuscular ketamine. Venous access was achieved with the scalp acupuncture. Anesthesia

was induced with 30 mg/kg intravenous pentobarbital sodium, and a supplement with 50 to 100 mg medication was executed whenever necessary. A tracheal endotracheal tube was inserted, but animals were allowed to breathe spontaneously throughout the experiment.

A 5F Swan-Ganz pediatric catheter was introduced into the delete right external jugular vein and guided to the pulmonary artery. An 18F polyethylene catheter was introduced into the right femoral artery and guided to the abdominal aorta. The correct placement of the catheters was systematically confirmed by the morphology of the pressure curves. ST36 is located at the proximal one fifth of the craniolateral surface of the leg distal to the head of the tibia in a depression between the muscles of the cranial tibia and long digital extensor. Silver needles were inserted at bilateral ST36 (lower limb). Electrostimulation was used in the dense dispersed mode; the current frequency was 30 Hz, and constant voltage varied up to 10 to 15 V for 30 minutes. The nonacupuncturing point is located at the bilateral capitulum fibulae apart from outside the fibulae, with the outside condyle center point to about 1 cm place.

Each catheter was filled with a heparinized solution (saline solution containing 25 IU of heparin per milliliter). The femoral and the Swan-Ganz catheters were connected to pressure transducers cardiac output (CO), cardiac index (CI), central venous pressure , and pulmonary arterial wedge pressure (PAWP); mean arterial pressure (MAP) was measured using a multiparametric monitor (Philips Agilent V24C, San Francisco, CA, USA). All Pressure measurements were performed while the animals were in the supine position, and the 0 reference point in all cases was the cardiac line of the animal.

Hemorrhagic shock model

Hemorrhagic shock was initiated by blood withdrawal, and the MAP was reduced to approximately 35 to 40 mm Hg (defined as the loss of approximately 42% of estimated blood volume) within 15 minutes. This blood pressure was maintained for 30 minutes by further blood withdrawal if the MAP was greater than 45 mm Hg or by injection of a small volume of blood if the MAP was less than 35 mm Hg. During hypotension, the shed blood was kept at Room temperature in a heparinized (200 U) plastic syringe connected to the arterial cannula.

Experimental groups

Thirty healthy dogs were randomly divided into 5 groups: sham hemorrhagic shocked (SHAM) group (sham-shocked dogs underwent all surgical procedures without bleeding); hemorrhagic shocked (HEM) group (HEM was induced by intermittently withdrawing blood until the MAP stabilized for 30 minutes within the range of 35-40 mm Hg); electroacupuncturing (EA) group (zusanli points were

electroacupunctured with constant voltage [10-15 V, 30 Hz] for 30 minutes immediately after the shock models were operated); nonacupuncturing (NON-EA) group (nonacu- puncturing points were the same treatment with EA group for

30 minutes immediately after the shock models were operated); and EA after vagotomy (VGX+EA) group (dogs were subjected to bilateral cervical vagotomy after hemor- rhage shock and then directly EA at zusanli points [10-15 V, 30 Hz]).

Experimental measurements

Hemodynamic variables recorded were MAP, CO, CI, CVP, and PAWP before bleeding (-45 time), immediately after shock (0 time), and every 30 minutes thereafter (30-, 60-, 90-, 120-, 150-, and 180-minute time points). Cardiac output was obtained by thermodilution using an injection of saline solution at a temperature lower than 5?C. Cardiac index (CI = CO/body surface area) was measured in triplicate by thermodilution with 5 mL of 0.75% saline solution at a temperature lower than 5?C. Central temperature was measured directly using the thermistor of the Swan-Ganz catheter located in the pulmonary artery. Blood samples were collected from the femoral and pulmonary arteries. After discarding the fluid occupying dead space in the catheters, the arterial and mixed venous blood were collected in heparin- ized syringes for measurement of blood gases and lactate. The arterial blood samples were collected in heparinized Eppen- dorf tubes for detecting inflammatory cytokines.

Plasma TNF-? levels

Arterial blood samples were taken immediately after shock (0 time) and at 120- and 180-minute time points thereafter. Plasma was obtained by centrefuging at 3000 rpm for 15 minutes. Plasma TNF-? concentrations were determined by an enzyme-linked immunosorbent assay kit (R&D, Minneapolis, MN, USA).

Lactate content of blood

Arterial blood samples were taken immediately in heparinized syringes for measurement of blood gases and lactate before bleeding (-45 time), immediately after shock (0 time), and at the 180-minute time point thereafter. Lactate was measured using the Blood gas analyzer (i-STAT, Princeton, NJ, USA).

Statistical analysis

Data were reported as mean +- SD. Analyses of variance were used to evaluate whether values at the same point were different for the control and treatment groups, and 1-way analysis of variance for repeated measurements with multiple comparisons (Bonferroni) was used. A significant difference was presumed at a P value less than .05.

Results

Mean arterial pressure

At corresponding time points, the HEM group presented with decreasing MAP that differed statistically from that of the Sham group. The NON-EA and VGX+EA groups displayed lower MAP level when compared to the SHAM and EA groups. At corresponding time points 90, 120, 150, and 180 minutes after hemorrhage, the EA group displayed higher MAP levels compared to the HEM group (Fig. 1).

Cardiac output, CI, CVP, and PAWP

The HEM group presented with decreasing CO, CI, CVP, and PAWP levels that differed statistically from the SHAM group. There was no significant difference in CO, CI, CVP, and PAWP levels between the NON-EA, VGX+EA, and HEM groups. The main differences observed during these procedures are summarized in the following sections.

Cardiac output

At 90, 120, 150, and 180 minutes after hemorrhagic challenge, the EA group presented with increasing CO levels that differed statistically from the HEM group. At 120, 150, and 180 minutes after hemorrhage, the CO levels were significantly increased in the EA group compared with those in the NON-EA and VGX+EA groups (Fig. 2).

Cardiac index

At 90, 120, 150, and 180 minutes after hemorrhage, the EA group presented with increasing CI levels that differed statistically from those of the HEM group. At 120, 150, and 180 minutes after hemorrhage, the CI levels were signifi- cantly increased in the EA group compared with those in the NON-EA and VGX+EA groups (Fig. 3).

Fig. 1 Temporal evolution of MAP. The MAP was recorded in the experiment after bilateral cervical vagotomy, direct electric stimulation at ST36 in animals subjected to fatal hemorrhagic shock. The MAP recorded in each time point represents the mean +- SD of 6 animals. *P b .05 vs the HEM group, #P b .05 vs the NON- EA group, and ?P b .05 vs the VGX+EA group.

Fig. 2 Temporal evolution of CO. The CO was recorded after bilateral cervical vagotomy, direct electric stimulation at ST36 in animals subjected to fatal hemorrhagic shock. The CO recorded in each time point represents the mean +- SD of 6 animals. *P b .05 vs

the HEM group, #P b .05 vs the NON-EA group, and ?P b .05 vs the VGX+EA group.

Central venous pressure

At 120, 150, and 180 minutes after hemorrhage, the EA group presented with increasing CVP that differed statisti- cally from those of the HEM, NON-EA, and VGX+EA groups (Fig. 4).

Pulmonary arterial wedge pressure

At 90, 120, 150, and 180 minutes after hemorrhage, the EA group presented with increasing PAWP that differed statistically from that of the HEM group. At 120, 150, and

180 minutes after hemorrhage, the PAWP levels were significantly increased in the EA group compared with those in the NON-EA and VGX+EA groups (Fig. 5).

Plasma TNF-? level

Immediately after shock (0 minute), there was no significant difference among these groups. At 180 minutes after hemorrhage, the EA group significantly reduced plasma TNF-?, which differed statistically from that of the HEM, NON-EA, and VGX+EA groups (Fig. 6).

Fig. 3 Temporal evolution of CI. The CI was recorded after bilateral cervical vagotomy, direct electric stimulation at ST36 in animals subjected to fatal hemorrhagic shock. The CI recorded in each time point represents the mean +- SD of 6 animals. *P b .05 vs the HEM group, #P b .05 vs the NON-EA group, and ?P b .05 vs

the VGX+EA group.

Fig. 4 Temporal evolution of CVP. The CVP was recorded after bilateral cervical vagotomy, direct electric stimulation at ST36 in animals subjected to fatal hemorrhagic shock. The CVP recorded in each time point represents the mean +- SD of 6 animals. *P b .05 vs

the HEM group, #P b .05 vs the NON-EA group, and ?P b .05 vs the VGX+EA group.

Blood lactic acid content

Before hemorrhagic shock, there was no significant difference in lactic acid content among these groups. However, when the hemorrhagic shock model was prepared to complete (0 minute), the dogs in the HEM, EA, NON-EA, and VGX+EA groups presented with higher lactic acid content compared with those in the SHAM group. At 180 minutes after hemorrhage, the EA groups displayed a significantly lower lactic acid content compared with those of the HEM, NON-EA, and VGX+EA groups (Fig. 7).

Discussion

Acupuncture has been used to treat various diseases in Asia for thousands of years. ST36 is on the foot Yang Ming stomach meridian. This acupoint is known to strengthen the vital energy (“qi” in oriental medical terminology). The “vital energy” here means not only the stomach qi, even though this acupoint belongs to the stomach meridian, but

Fig. 5 Temporal evolution of PAWP. The PAWP was recorded after bilateral cervical vagotomy, direct electric stimulation at ST36 in animals subjected to fatal hemorrhagic shock. The PAWP in each time point represents the mean +- SD of 6 animals. *P b .05 vs the HEM group, #P b .05 vs the NON-EA group, and ?P b .05 vs the

VGX+EA group.

Fig. 6 Temporal evolution of plasma levels of TNF-?. The TNF-? level was recorded after bilateral cervical vagotomy, direct electric stimulation at ST36 in animals subjected to fatal hemorrhagic shock. The TNF-? level at each time point represents the mean +- SD of 6 animals. **P b .01 vs the EA group.

also the general qi in the whole body. For this reason, ST36 is the target to treat various diseases in different parts of the body or general symptoms in the whole body, including deficiency and weakness [16-19].

Data from this study demonstrated that ST36 acupuncture significantly improved hemodynamics and attenuated the expression of cytokine and lactate Blood concentration in hemorrhagic shock. These data suggest the anti-inflammatory potential of the use of ST36 acupuncture against hemorrhagic shock. Guarini et al [20] have reported that vagus nerve stimulation counteracts hypotension and increases the survival time during lethal hemorrhagic shock in rats. The mechanisms underlying these beneficial effects are nicotinic receptor-dependent and include inhibition of the activation of nuclear factor ?B and TNF synthesis in the liver.

Stimulation of the parasympathetic efferent pathway has been shown to decrease TNF-? production in septic rats [21]. Agonists of cholinergic receptor, such as acetylcholine, nicotine, and muscarine, have also been reported to attenuate the production of TNF-?, IL-1?, IL-6, and IL-18 in activated

macrophages [13,21]. This has been termed the cholinergic anti-inflammatory pathway by Pavlov and Tracey [13]. They further identified the ?7 subunit of the nicotinic receptor as essential in mediating the effects of the cholinergic anti- inflammatory pathway [22]. ST36 acupuncture has been shown to significantly activate the parasympathetic efferent pathway in freely moving conscious rats [12]. Judging from these data, we speculate that ST36 acupuncture may also act through activating the cholinergic anti-inflammatory path- way and, perhaps, the ?7 subunit of nicotinic receptor to exert its anti-inflammatory effects. In addition, manipula- tions that activate the cholinergic anti-inflammatory path- way, such as vagal electrostimulation or a specific agonist of the ?7 subunit of nicotinic receptor, may very likely offer alternative and clinically relevant solutions to acupuncture. As part of the innate immune system, the cholinergic anti- inflammatory pathway has also been reported to be activated by endotoxin [13,14].

In the previous studies, EA at ST36 promoted the gastric myoelectric activity, which was regulated by the vagus, and

Fig. 7 Temporal evolution of lactate content of blood. The lactate content of blood after bilateral cervical vagotomy, direct electric stimulation at ST36 in animals subjected to fatal hemorrhagic shock. The lactate content of blood in each time point represents the mean +- SD of 6 animals. *P b .05, **P b .01 vs the EA group, #P b .05 vs the SHAM group.

substance P in the dorsal vagal complex may be involved in the excitatory effects. After bilateral vagotomy, the excit- atory effect was completely abolished, suggesting that it was mediated by the vagus. Furthermore, acupuncture increased the number of c-fos immunopositive cells at the ventrolateral medulla, indicating that acupuncture provoked a reflex whose center was within the medulla [23,24].

In our experiments, severe hemorrhagic shock caused histiocyte ischemia and hypoxia that can lead to increased lactic acid values and further induce acidosis and thus reduced oxygen offloading at tissue level. Electrical stimulation of the ST36 in dogs with hemorrhagic shock significantly blunted serum TNF-? levels, reduced the circulating levels of the cytokine, decreased lactate blood concentration, protected against hypotension, and increased CO, CI, CVP, and PAWP to improve tissue perfusion and oxygen supply, which conduce to keep homeostatic mechanisms. Nonacupuncturing or the vagotomy and EA groups decreased basic hemodynamic variables and in- creased the production of TNF-? and the lactate blood concentration, which suggested that anti-inflammatory pathway may be through the vagal nerves and their integrity was essential. None of the methods currently used provide definitive parameters for the monitoring of an adequate oxygen supply to tissues. Cardiac output is one of the most important variables in the context of tissue perfusion. In addition, it participates in the calculations of other hemodynamic and oxygenation variables such as systemic vascular resistance, Pulmonary vascular resistance, and oxygen supply [25]. This antishock effect seems also to influence the cardiovascular apparatus and the overall survival of the animals.

In summary, data from this study would demonstrate that pretreatment with ST36 acupuncture administration had protection effects against hemorrhagic shock.

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