ORIGINAL PAPER

Improved Techniques for a Murine Model of MyocardialIschemia

Yuan Yuan & Fei Li & Li-Ping Wei & Rong-Qing Zhang &

Hai-Chang Wang

Received: 26 July 2008 /Accepted: 11 January 2009 /Published online: 10 March 2009# Springer Science + Business Media, LLC 2009

AbstractObjective Our study investigated a rapid and reliablemethod of surgical occlusion of coronary vessels in mice.The study improves the chance of success in making amyocardial ischemia model in mice, and provides a novelmethod to a novice and laboratory with limited conditions.Methods and Results Sixty mice were evenly divided intotwo groups, a modified group (oral intubation and self-made rib retractor) and a conventional group (tracheotomyand rib cutting). During the perioperative period, thesuccess rate of model establishment and the survival rateof the mice in the modified group were significantly higherthan those in the conventional group (P<0.01). Also, thestatus of the mice in the modified group after operation wasbetter than that of the conventional group. Moreover,operation times in the modified group were significantlyshorter than those of the conventional group (P<0.01). Theinfarct size, as assessed using triphenyltetrazolium chloridestaining, was similar between the two groups (P>0.05).Conclusion This novel method is simple and efficient andcan be conducted independently, enhancing the success rateof myocardial ischemic model establishment in mice.

Keywords Myocardial infarction .Mouse . Technique .

in vitro model

Introduction

A model for myocardial ischemia is of great value in heartdisease research. In addition, mice are used increasingly forexperimental models of ischemic heart disease as well as othercardiovascular diseases, because they have short reproductivecycles, low consumption, a high degree of homology withhumans in regulatory cardiovascular developmental genes andsignaling pathways, and gene knock out [1–4]. Traditionalthoracotomy on mice needs tracheotomy and rib cutting, andalso needs good instruments, experienced operators and a longperioperative period. Nevertheless, both models of myocardialinfarction and ischemic reperfusion in mice continue to sufferdue to high mortality and low success rates.

Based on previously established methods [5–9], wecreated a novel mouse model of myocardial ischemia witha high success rate, low mortality rate and short operationtime. The major advantage of this method is that it savesoperation time, and causes less trauma and lower mortality.We took acute myocardial infarction (AMI) as an exampleto present the improved techniques.

Materials and methods

Animals Female BALB/c mice (n=60), each weighing 20–25 g, were purchased from the Experimental Animal Centerof the Fourth Military Medical University and housed incages under standard conditions with free access to water.The animals were randomly divided into modified andconventional groups. Experiments were performed inaccordance with the National Institutes of Health Guide-lines on the Use of Laboratory Animals and were approvedby the Fourth Military Medical University Committee onAnimal Care.

J Med Syst (2010) 34:413–417DOI 10.1007/s10916-009-9254-0

Y. Yuan : F. Li (*) : L.-P. Wei : R.-Q. Zhang :H.-C. Wang (*)Department of Cardiology, Xijing Hospital, Fourth MilitaryMedical University,Xi’an 710032, Chinae-mail: lifei01@fmmu.edu.cne-mail: wanghc@fmmu.edu.cn

Experiment protocol Mice in the modified group wereanesthetized with 1% pentobarbital sodium solution(60 mg/kg, intraperitoneal injection) prior to fixing theirlimbs and incisors to a surgical plate with stickers andthread respectively. The shoulders were raised by a 1 mLsyringe. The surgical plate was tilted upright at an angle of60o. While the mouse neck was lighted, the base of thetongue was drawn up with angled tweezers and the glottiswas clearly observed to open and close with breathing(Fig. 1). The mice were gently orally intubated with a 24 GIV catheter and ventilated using a rodent respirator(Medical Instrument Corporation of Zhejiang University,China) at 120 breaths per minute and a tidal volume of1 mL per breath. If the respiratory rhythm coincided withthat of the artificial respirator, intubation was deemedsuccessful. The RM-6000 eight-channel physiologicalrecorder (NIHON KOHDEN, Japan) was employed inmice, and limb leads were used for ECG monitoring. Skinpreparations and sterilizations were performed within theoperative field. A 45o oblique incision next to the left sternumwas made on the skin, and then the pectoralis major andserratus anterior muscles were bluntly separated layer-by-layerusing optical haemostatic forceps. The heart was easilyexposed by opening the rib cage with a self-made rib retractorafter bluntly separating the left fourth intercostal musclewithout the incision of the rib (Fig. 2). The retractor was madeof a Fe-Ni alloy and looked like a micro-eye speculum. Theretractor can be further opened by eye speculum or tissueforceps. Once the pericardium was opened and the left auricleand ventricle were exposed, the left anterior descendingcoronary artery, which descends the anterior wall of the leftventricle from the middle one-third of the left auricle margin,was observed by the stereomicroscope of 10× and ligated with

a 6/0 surgical silk suture. We could see the J-point (TerminalPortion of the QRS) elevation in the ECG immediately afterligation of the coronary artery (Fig. 3). Then we sutured thechest mucle and skin, leaving a tube in the thoracic cavity. Apurse-string suture was sutured around the tube. The lungswere then dilated to remove gas in the thoracic cavity, and thetube was pulled out. Tracheal intubation can be relieved afterpulling out the tube. The operation time was measured fromthe start of anesthesia to the resumption of spontaneousrespiration in mice.

In the conventional group, tracheotomy and rib cuttingwere performed by incisions at the third and forth ribs.All other steps were equivalent to that of the modifiedgroup.

Histology Midventricular slices were processed for histo-chemical analysis. Infarct size was determined on 5%Evans Blue and 1.5% triphenyltetrazolium chloride-stainedsections. The infarct size was calculated as a percentage ofthe total LV area.

Fig. 1 Glottis of a mouse. A 1 mL syringe was placed under theshoulders of the mouse, and the base of the tongue was drawn up withangled tweezers. Note that the glottis of the mouse is translucentlytriangular

Fig. 2 Self-made rib retractor designed according to a blepharostat a.Self-made oral intubation refitted from 24G intravenous catheter b.The heart is well exposed without incisions to the rib, due to the self-made rib retractor c

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Statistical Analysis Data were analyzed using the StatisticalAnalysis System software (SPSS 11.0) and expressed as mean±standard deviation (SD). Operation times were analyzedusing the t-test, while all other comparisons were made usingthe Chi-squared test. Differences were considered statisticallysignificant if P<0.05.

Results

Operation times and success rates The modified groupachieved an oral intubation success rate of 100% (n=30)with no complications such as tracheal perforation or throattissue injury. A significantly increased success rate wasobserved in the modified group, as indicated by a 30%difference between groups (90% vs. 60%, P<0.01) (Fig. 4),in addition to shortened operation times (10±2.2 min vs.55±3.7 min) due to improved techniques. The potential forsurvival, a rise in the J-point, and arched elevation of theST segment in the ECG examined within 72 h post-surgerywere all considered successful.

Perioperative status and survival rate Among the 30 micein the modified group, one died of acute left heart failureafter the intraoperative ligation of the coronary artery, two

died within 12 h postoperatively of heart failure and theremaining mice were able to execute independent activities1 h after recovery from anesthesia without any additionalrespiratory assistance. Of the 30 cases in the conventionalgroup, one died of acute left heart failure after intra-operative ligation of the coronary artery, four died ofdyspnea caused by airway suture, four died within 3 hpostoperatively of respiratory failure, and an additionalthree died within 12 h, one within 24 h, and three within72 h postoperatively. Independent activities and eatingtimes of the conventional group were generally delayed inaddition to displaying varying degrees of decreased appetiteand activity level, poor grooming and restlessness (Fig. 5).

Infarct size The infarct area was similar between the miceof the modified and conventional group three days after MI,which indicated a stability of AMI mice using modifiedtechniques (Fig. 6).

Discussion

Methods of artificial ventilation and thoracotomy are thetwo key links in making MI or IR in mice and in getting ahigh success rate and a low mortality rate.

Fig. 4 Operation times and suc-cess rates of modified and con-ventional groups. Values arepresented as mean ± SD. n=30,*P<0.05, **P<0.01 vs.conventional group

Fig. 3 Changes in ECG before and after coronary artery ligation. ECG shows ambulatory change and J-point elevation immediately after ligationof the coronary artery

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Many laboratories utilize their own artificial ventilationapproaches for establishing myocardial ischemic models.Currently, techniques of endotracheal intubation aregrouped into two categories, invasive and noninvasive.Invasive techniques are subdivided into conventionaltracheotomy[10] and the newly reported forward or reverseintubation for tracheal puncture [11]. Another invasivemethod is exposing the trachea only by metal tubepositioning. These three techniques destroy the normallaryngeal anatomical structure and are rife with complica-tions both during and after intubation, which include lowsurvival rates and comparatively long perioperative periods.Noninvasive techniques need a special laryngoscope tolocalized the glottis and the self-made tube [12]. Since thisapproach requires special instruments, its widespreadapplication is limited.

Thoracotomy on mice often requires the breaking of ribsor the use of extrusion methods. The former is complicatedin manipulation, rather traumatic, and time-consuming,while the latter needs extensive training and is not suitablefor either a novice or an operator in a laboratory withlimited conditions.

Utilizing the methods of Michael and Salto-Tellez et al.and modifying endotracheal intubation and thoracotomy,we have established a safer and more efficient mouse modelof AMI. Our modified method relies on efficient self-madeinstruments that are simple and easily obtained. First, thedirect application of a shorter 24G IV catheter forintubation does not require a self-made sheath tube orguide wire. Second, a lamp or flashlight, rather than alaryngoscope, is sufficient for a light source. Third, bendingtweezers can be used for traction of the tongue base ratherthan an auricular clamp. Finally, the ribs can be distractedwithout being broken using a simple, self-made rib retractordevice. In addition, there are three major characters of this

retractor: i), the materials are easily and cheaply obtained;ii), the retractor is retractable and can be opened by anothereye speculum or tissue forceps to expose the heartadequately; and iii), all sizes of the retractor can be madeby the operator.

Oral intubation creates less trauma and resulted in fasterrecovery, which is a virtuous circle. Oral intubation notonly saves time but also avoids stimulation or secretioninduced by incisions or punctures and maintains trachealanatomic structures. Obviously, the retractor also makes theoperation easier and protects the normal chest tissuestructure to some extent. There was no injury to theesophagus and trachea and no broken ribs were found inthe mice of the modified group. Moreover, ischemia-hypoxia was not serious, so the mice could wake upquickly, recover with movement with ease, and eat withoutpoor grooming and restlessness. However, in the conven-tional group, the mice ribs were broken and ischemia-hypoxia was more serious. Furthermore, the mice woke uplater with poor, decreased appetite and activity level, poorgrooming and restlessness.

In conclusion, these practical techniques can beexecuted without the assistance of special equipment ortraining. We have reliably found that a mouse model ofmyocardial ischemia can be established with a highsuccess rate, using simple surgical tools that are easilymanipulated and independently mastered. The methodintroduced in this article can be practiced easily in a labwith limited conditions or by a novice. Moreover, ourmethod is not only applicable to the establishment ofAMI models, but also provides a reference for theestablishment of chronic infarction and ischemia-reperfusionmodels in mice.

Fig. 6 Heart sections were obtained from the modified andconventional group mice three days after the MI. Bar graphs indicatethe MI area. Results are expressed as mean ± SD (each n=5). P>0.05

Fig. 5 Comparison of survival rates after operation in the two mousegroups. In the modified group, 0, 3, 12, 24, 48, and 72 hour survivalrates were 96.7%, 96.7%, 90%, 90%, 90% and 90%, respectively,(P<0.01), and 83.3%, 70.0%, 60.0%, 56.7%, 56.7% and 50%,respectively, for the conventional group. 0 h indicates “duringoperation”

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Acknowledgments This study was supported by the grants fromNational Natural Science Foundation of China (NO.30600580).

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