Article, Cardiology

A simpler cardiac arrest model in rats

Original Contribution

A simpler cardiac arrest model in rats

Meng-Hua Chen MDa, Tang-Wei Liu MDa, Lu Xie DPharmb,*,

Feng-Qing Song MDa, Tao He MDa, Zhi-yu Zeng MDa, Shu-Rong Mo MDb

aInstitute of Cardiovascular Diseases, the First Affiliated Hospital of Guangxi Medical University,

Nanning 530021, P.R. China

bDepartment of Physiology, School of Pre-Clinical Sciences, Guangxi Medical University, Nanning 530021, P.R. China

Received 23 October 2006; revised 22 November 2006; accepted 27 November 2006

Abstract Two disadvantages of electrical induction of cardiac arrest used currently are that it is a technically complicated procedure and the consequent thermal injury, which prompts us to search for a simpler method with less adverse effect to induce ventricular fibrillation (VF) in rats. Different potential (18, 24, 30, and 36 V) of alternating current (AC) were administered to elicit VF in 15 rats via pacing electrode placed in esophagus. Four minutes after onset of VF, conventional cardiopulmonary resuscitation was initiated. restoration of spontaneous circulation was defined as the return of supraventricular rhythm with a mean aortic pressure of 20 mm Hg or greater for a minimum of 5 minute. Ventricular fibrillation was achieved by short interval of AC stimulation in all of the rats. After the termination of prolonged AC stimulation, electrocardiogram indicated VF occurred in 6 of 15 rats, asystole in 3 of 15 rats and pulseless electrical activity in 6 of 15 rats. Before CPR, however, electrocardiogram indicated that only 2 of 15 and 4 of 15 animals remained in VF and pulseless electrical activity, respectively, whereas 9 of 15 animals presented as asystole. After CPR, 11 of 15 animals were resuscitated. Necropsy showed that there was no gross evidence of thermal injury on the surface layer of the heart. Therefore, development of a rat cardiac arrest model by transesophageal AC stimulation is simpler and less adverse effect, which may have practical significance for facilitating experimental investigation on cardiac arrest and CPR.

D 2007

Introduction

Unsatisfactory outcome of cardiopulmonary resuscitation (CPR) prompts vigorous experimental pursuit of a better understanding of cardiac arrest mechanisms and its ap- proach with CPR. However, development of cardiac arrest

This study was supported by Guangxi Department of Health (Z 2006058) and the Guangxi Natural Science Foundation of China (no. 0640081).

* Corresponding author. Tel.: +86 771 5358282; fax: +86 771

5352775.

E-mail address: [email protected] (L. Xie).

(CA) models in animals is a prerequisite. Preparation of small animals is more economic compared to that of large animal preparation. An increasing numbers of investigators use rats as experimental animals in their laboratory studies [1-11]. Nearly all ventricular fibrillation (VF) models in rats are induced by alternating current (AC) delivered to the right ventricular endocardium [12-16].

Unfortunately, this procedure is somewhat complicated because it is an invasive method and possibly produces some pertinent complications during the operation. Fur- thermore, it may produce thermal injury of the endocar- dium at the site of the electrode in the experimental rats as

0735-6757/$ – see front matter D 2007 doi:10.1016/j.ajem.2006.11.033

well [17]. These disadvantages restrict the application of this technique to some extent, which prompt us to search for a simpler method with less adverse effect to induce VF in rat.

It is well known that the heart is located in the chest cavity just anterior to the esophagus, posterior to the breastbone, between the lungs and superior to the dia- phragm. In addition to the vascular approach, electrode or catheter can approach the heart from esophagus. Based on anatomic location of heart, we felt justified to hypothesize that Ventricular tachycardia would be induced by AC delivered to the esophagus just posterior to the heart. It is self-evidenced that advancing an electrode through the esophagus is easier than advancing an electrode to right ventricular endocardium. Furthermore, induction of VF by AC from esophagus would eliminate the thermal injury of the endocardium. The purpose of this study was, therefore, to verify the feasibility of the hypothesis.

Materials and methods

This study was approved by the animal investigation committee of our university and was performed in accor- dance with National Institutes of Health guidelines for ethical animal research.

Animal preparation

Sprague-Dawley rats of both sexes, weighing 230 to 350 g, were fasted overnight except for free access to water. The animals were anesthetized by intraperitoneal injection of 1 g/kg urethane, placed in a supine position on a surgical board, and the extremities were immobilized. The proximal trachea was surgically exposed, and a 14-gauge cannula was inserted through a tracheostomy 10 mm caudal to the larynx. The cannula was advanced for a distance of 1 cm into the trachea and secured by ligature, which was also anchored to the skin.

Via the right external jugular vein, an 18-gauge polyethylene catheter was advanced into the superior vena cava into the right atrium. right atrial pressure was measured with reference to the midchest with a high sensitivity pressure transducer. Another 18-gauge polyeth- ylene catheter was advanced from the left carotid artery into the thoracic aorta for measurement of aortic pressure with the high sensitivity pressure transducer. The dead space of the catheters was filled with physiologic salt solution containing 5 IU/mL of bovine heparin. The core temperature was measured with a rectal temperature probe. Conventional lead II electrocardiogram (ECG) was recorded with the aid of subcutaneous needles.

The ECG (lead II) and aortic and right atrial pressures were continuously recorded on a desktop computer via 4-channel physiologic recorder (BL-420 E Bio-systems, The Chengdu Technology & Market Co Ltd, Chengdu, China) for subsequent analyses. The coronary perfusion pressure

(CPP) was calculated as the difference between the minimum diastolic aortic pressure and simultaneously recorded right atrial pressure [18].

Experimental protocol

15 Sprague-Dawley rats were summated to transesopha- geal AC stimulation. 5F pacing electrode with 2 1-mm ring electrodes and an interelectrode distance of 5 mm was inserted orally into the esophagus of the rats about 7 cm in depth (located in the inferior segment of esophagus just posterior to the heart). First of all, different potentials of AC (18, 24, 30, and 36 V) were delivered to the esophagus through the pacing electrode to stimulate the heart so as to verify the possibility of induction of VF in rat and confirm the optimal potential of inducing VF. Three series of 5 seconds burst stimulation was performed at each different potentials with 2 minutes of rest interval between each burst series. If VF was presented after the burst stimulations, even if it reverted spontaneously within seconds, the definition of VF elicited by transesophageal AC stimulation was estab- lished. The optimal potential of inducing VF was defined as the lowest potential which could elicit 2 or more arrays of VF during 3 series of burst stimulation. Secondly, contin- uous transesophageal AC stimulation was conducted and maintained for at least 60 seconds to prevent spontaneous defibrillation. A pause in AC stimulation was then initiated for a few seconds (1-3 seconds) to observe the change of ECG. As soon as the VF reverted spontaneously, an additional 30-second burst stimulation was performed immediately until the VF reappeared and persisted, possibly until Pulseless electrical activity or asystole occurred. Four minutes after onset of VF, manual chest compres- sion and mechanical ventilation were begun. Ventilation was

Table 1

stimulation

Number

Frequency of VF induced and time of AC

VF-induced

Time of AC

+, VF presented only once; ++, VF presented twice; +++, VF presented

thrice; –, VF was not induced.

18 V 24 V 30 V

36 V

stimulation

1

+++

120

2

+

+

+

+++

90

3

+

+

+

120

4

+++

180

5

++

+++

120

6

+++

90

7

++

+++

60

8

++

+++

150

9

+++

180

10

+++

120

11

+

+++

120

12

++

60

13

+++

120

14

+++

90

15

+

+++

120

Fig. 1 Alternating current stimulation caused the changes of blood pressure and right atrial pressure in the same time frame. BP, blood pressure; RAP, right atrial pressure.

performed by a volume-controlled small animal ventilator (DH-150, the medical instrument of Zhejiang University, Hangzhou, China), with room air at 70 breaths per minute and tidal volume adjusted to 6 mL/kg. Manual chest compression at a rate of 180 compressions per minute with equal compression-relaxation duration was always per- formed by the same investigator who was excluded from the hemodynamic monitor tracings and guided only by

acoustic audio tones. Compression depth was about 30% the anteroposterior chest diameter at maximal compression.

When asystole or PEA was presented or persisted after 1 minute of precordial compression, a dose of epinephrine (40 lg/kg) was administered, and precordial compression

was resumed for several minutes. But when VF presented or persisted, no intervention (including epinephrine and coun- tershocks) was administered in addition to conventional CPR.

Fig. 2 After a short interval of AC stimulation, VF elicited was reverted spontaneously.

Fig. 3 Sustaining VF was presented with the prolongation of AC stimulation.

Restoration of spontaneous circulation was defined as the return of supraventricular rhythm with a mean aortic pressure of 20 mm Hg or greater for a minimum of

5 minutes [19]. When there was a failure to restore spontaneous circulation after 10 minutes, resuscitation efforts were discontinued.

Resuscitated animals were invasively monitored for an additional 30 minutes and then were killed by intra-arterial injection of 2 mL of saturated potassium chloride solution. Necropsy was routinely performed after death, including resuscitated and unresuscitated animals. Thoracic and abdominal organs were examined for gross evidence of traumatic injures that followed intraperitoneal injection of anesthesia, airway management, vascular cannulation, or precordial compression. The researchers paid particular attention to the heart and esophagus and tried to identify

if the AC stimulation would injure the rat heart and esophagus. The position of catheters was documented.

Statistical analyses

Measurements are reported as means F SD. Compar- isons between resuscitated and unresuscitated animals were analyzed with 1-way analysis of variance measurements. P b .05 was considered significant.

Results

Outcome of induction of VF and CPR

Frequency of VF induced by different potentials of AC and time of AC stimulation, which resulted in sustaining VF, PEA or asystole, is shown in Table 1. The time of

Fig. 4 Pulseless electrical activity was presented in some animals.

Fig. 5 Some of the VF elicited developed into asystole over time.

AC stimulation ranges from 60 to 180 seconds (116.0 +-

35.6 seconds).

Ventricular fibrillation was elicited by a short interval of AC stimulation in all of the 15 rats. Furthermore, AC stimulation caused an immediate drop of blood pressure, loss of pulse, and an increase of right atrial pressure in the same time frame (Fig. 1). However, it was found that VF elicited by short interval AC stimulation could easily revert spontane- ously within seconds (Fig. 2). With the prolongation of AC stimulation, the incidences of spontaneous defibrillation in the rats decreased substantially (Fig. 3), whereas incidences of asystole and PEA increased accordingly (Fig. 4). Some of the VF elicited developed into asystole over time (Fig. 5).

After the termination of prolonged AC stimulation, ECG indicated that VF occurred in 6 of 15, asystole in 3 of 15,

and PEA in 6 of 15 rats. Before CPR, however, ECG indicated that only 2 of 15 and 4 of 15 animals remained in VF and PEA, respectively, whereas 9 of 15 animals presented as asystole. After CPR, 11 of 15 animals were successfully resuscitated. Of the remaining 4 unresuscitated animals, 3 died from sustaining VF and only 1 from asystole during 10 minutes of CPR (Table 2). There were no significant differences in weight between resuscitated (295.5 +- 35.3 g) and unresuscitated (287.5 +- 46.5 g) animals. All the resuscitated animals survived for more than 30 minutes.

Hemodynamic observations

No significant differences in baseline hemodynamic measurements were observed between resuscitated and

Table 2 ECG exhibition at the termination of AC stimulation, before CPR and the outcomes of CPR

Number ECG (stop AC) stimulation ECG (before CPR) Outcome of CPR

VF

Asystole

PEA

VF

Asystole

PEA

Resuscitated

Unresuscitated

1

/

/

/ (Sustaining VF)

2

/

/

/

3

/

/

/

4

/

/

/

5

/

/

/

6

/

/

/ (Sustaining VF)

7

/

/

/

8

/

/

/

9

/

/

/ (sustaining VF)

10

/

/

/

11

/

/

/

12

/

/

/

13

/

/

/

14

/

/

/

15

/

/

/

Total

6

3

6

2

9

4

11

4

Fig. 6 Coronary perfusion pressure progress in the early 5 minutes from the beginning of CPR; CPP in resuscitated group was significantly higher than that of unresuscitated group 2 minutes later. Variables are expressed as mean +- SD. *P b .01, **P b .001.

unresuscitated animals. The changes of CPP over the initial 5 minutes of CPR showed significantly different tendency (Fig. 6). Coronary perfusion pressures in survivors were much higher than those in nonsurvivors 2 minutes later ( P b

.01 or b.001, respectively).

Necropsy

Correct placement of catheters was confirmed in all of the animals. No adverse effects of invasive procedures or other traumatic injures were found. There was no gross evidence of electrical thermal injury on the surface layer of the heart. When the esophagus was opened, a visible (mild or moderate) thermal injury to the esophagus at the site of the electrode was found. No esophagus perforation was found in the presented study.

Discussion

It has been reported that the most common initial arrhythmias encountered in cases of out-of-hospital sudden cardiac deaths were VF or VT [20]. This finding calls the increasing investigators’ attention to development of VF animal model. About 10 years ago, Bottiger et al [21,22] developed a rat model, in which cardiac arrest was induced by electrical stimulation (AC; 12 V, 50 Hz) via the esophageal electrode, and an external electrode covered with electrode gel was placed on the animals’ chest [23]. In our preliminary study, however, VF could not be induced most Sprague-Dawley rats by this method. So, we modified the procedure by advancing 5F pacing electrode with 2 ring electrodes into esophagus to stimulate the heart of the rat with AC for the sake of identifying the possibility of inducing VF. It was found that VF could not be induced by the same potential of AC (12 V, 50 Hz) in rats either. With the increase of AC potential, the incidence of VF induced by AC increase.

In the present study, VF could be induced by trans- esophageal AC stimulation in all of the rats. Furthermore, 11 of 15 animals were successfully resuscitated, and only 4 of 15 animals failed to restore spontaneous circulation after CPR. This outcome of CPR seems to be more favorable, compared with the report of Von Planta et al [17], in which only 8 of 14 rats were successfully resuscitated in their established VF model. Therefore, we conclude that our previous hypothesis has been validated by our data.

Two major disadvantages in the development of CA model in rat by AC delivered to the right ventricular endocardium have been overcome in the present study. These include the complicated procedure and the thermal injury of the endocardium at the site of the electrode.

Although electrical induction of CA is the most common technique used currently, which is an invasive method and usually achieved by the transvenous route, it requires some special facilities, expertise, and time. Moreover, it is not without complications such as bleeding, pneumothorax, and cardiac perforation. In certain situations, these complica- tions may be life-threatening and cause a failure of continuing the experiment.

On the contrary, transesophageal AC stimulation used to induce VF in rat would appear to have much to offer because, although being noninvasive, it requires no extra special skills and is easy and quick to perform without adverse effects related to invasive procedure. Although the transesophageal threshold voltage is considerably higher than intracardiac thresholds because of the structures through which AC has to pass, thermal injury caused electrically occurs only on the esophageal membrane, and no significant damage was found on the surface layer of the heart during the experiment. Therefore, it is self-evident that this new and noninvasive method of development of CA model in rat is simpler, safer, and more practical. It may have profound significance for large-scale investigations of the effects of pharmacologic and physical interventions in CA and CPR research.

It is well known that VT reverts spontaneously within seconds in small mammal hearts with very short refractory periods [17,24,25]. To avoid the spontaneous reversion of VF in the experimental rats, the current flow which was delivered to right ventricular endocardium to elicit VF was continued for 3 minutes [17]. In the present study, short interval of transesophageal AC stimulation could elicit a brief VF in all experimental animals. With the prolongation of AC stimulation, the incidences of spontaneous reversion of VF decreased, whereas the incidences of PEA and asystole increased accordingly. Ideally, an investigator would like to have all animals maintain VF after stopping AC stimulation. However, that happened in only 6 of 15 rats. Instead, most animals went into asystole or PEA. Although cardiac arrest in rat and mouse is more often associated with PEA or asystole [25], the mechanism for the phenomenon remains to be established. Nevertheless, whether in settings

of VF, PEA or asystole, the ultimate effect is a failure of the heart to maintain forward blood flow, including coronary, systemic, and pulmonary blood flows, which fulfilled the purpose of developing a CA model in rat.

A notable finding in the present study was that the presentation and maintenance of VF during CPR seems to be associated with the animals’ intrinsic VF threshold. Of 4 unresuscitated animals, for example, the optimal poten- tial of eliciting VF of 3 was 18 V. In other words, these animals’ intrinsic VF thresholds were relatively lower. Interestingly, in these animals, VF was elicited easily and maintained during CPR. We deliberately did not administer countershock for the purpose of identifying whether VF during CPR in rat could revert spontaneously. Unfortu- nately, spontaneous defibrillation did not occur in these animals. There is no doubt that administration of counter- shock would increase the possibilities of resuscitation in sustaining VF animals.

Some limitations of this study should be noted. There is no control group in the present study. Therefore, it is reluctant to some extent to compare our present data with a previously published model. Also, there were no blood gas results and even no end-tidal carbon dioxide monitoring in the present study. Lack of presentation of key data of respiratory and metabolic abnormalities might result in the difficulty to deal with modification of mechanical ventila- tion. Finally, we only monitored the resuscitated animals for an additional 30 minutes after restoration of spontaneous circulation. Survival duration of the resuscitated animals in the model remains to be investigated. But as an initial effect, we were committed to the development of a valid model of cardiac arrest in rat, which would be a simpler and easier procedure. The focus of our investigation, therefore, was that whether VT could be induced via transesophageal AC stimulation in rat and how many animals can restore spontaneous circulation in the experiment. Fortunately, the resulting data fulfilled that purpose.

In conclusion, VF can be induced by transesophageal AC stimulation in rats, and development of a rat CA model in this manner may be simpler and easier than previous methodology. The new rat CA model may have practical significance for facilitating experimental investigation on CA and CPR.

Acknowledgments

The thank the staff of the department of physiology for excellent technical help and constructive criticism.

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