Original Research Articles – Regeneration Science

MRL mice fail to heal the heart in response toischemia-reperfusion injury

IBRAHIM ABDULLAH, MDa; JOHN J. LEPORE, MDb; JONATHAN A. EPSTEIN, MDb; MICHAEL S. PARMACEK,MDb; PETER J. GRUBER, MD, PhDc

The MRL/MpJ mouse strain has been reported to recover after right ventricular cryoinjury without scar forma-tion or evidence of ventricular dysfunction, suggesting that this mouse strain harbors genetic traits that conferthe capacity for adult myocardium to regenerate. We therefore sought to assess the capacity of adult MRLmyocardium to regenerate in a left ventricular ischemia-reperfusion model of myocardial infarction, whichmore closely recapitulates injury that occurs in human disease. MRL (n¼13) and control C57/Bl6 (n¼ 12) miceunderwent transient occlusion of the left anterior descending coronary artery. After 10 weeks, MRL and C57Bl/6 mice were euthanized and the extent of infarcted myocardium quantified with (2,3,5)-triphenyltetrazoliumchloride and trichrome staining. There was no evidence of resistance to cardiac injury or of reduced scarformation in the MRL mice compared to C57/Bl6 controls. Myocardial infarct size (percentage of total heartweight � SEM) did not significantly differ between MRL and C57/Bl6 controls (18.9�1.8% for MRL vs. 15.7þ 1.3%for C57/Bl6, p¼ 0.20). Thickness of the infarcted anterior LV wall at the mid-papillary level normalized to bodyweight was not significantly different between the two groups (0.017þ 0.003 mm/mg for MRL and0.017þ0.002 mm/mg for C57/BL6, p¼0.91). Trichrome staining showed intense scar formation in both C57/BL6 and MRL hearts. We conclude that there appears to be no effect of the MRL genetic background onresistance to myocardial infarction in mice. (WOUND REP REG 2005;13:205–208)

Cardiac regeneration is an elusive goal with as yet noclear demonstration of robust, reproducible native orinducible restorative capacity in mammalian species.1–3

Despite reports of varying strain susceptibility in theresponse to ischemia-reperfusion injury, only MRL/MpJmice have been reported to completely heal wounds.4

The MRL strain was originally bred as a control tothe MRL/MpJ-Tnfrsf6

lpr strain.5 These mice displaysystemic autoimmunity, massive lymphadenopathy, andimmune complex glomerulonephrosis.6 The observation

that MRL mice spontaneously heal following ear punch7

suggests that other tissues may regenerate followinginjury.8–10 Consistent with this finding, it was recentlyreported that MRL mice have the unique ability to com-pletely heal without scar formation following transdiaph-ragmatic cryoinjury to the right ventricle.11,12 Therefore,we examined the capacity of adult MRL mice to regener-ate muscle in a left ventricular ischemia-reperfusionmodel of myocardial infarction. This model of ischemia-reperfusion injury is a quantifiable, reproducible, clin-ically relevant model of cardiac injury for which wefound no significant difference in infarct size or wallthickness between MRL and C57/Bl6 controls.

MATERIALS AND METHODSThis study conformed to the Guide for the Care and

Use of Laboratory Animals (NIH publication no. 85–23,1985) and was conducted under protocols approved by

ECG Electrocardiogram

LAD Left anterior descending

TTC (2,3,5)-triphenyltetrazolium chloride

From the Department of Surgerya, Hospital of the Universityof Pennsylvania; Department of Medicineb,University of Pennsylvania School of Medicine;and The Cardiac Centerc, Children’s Hospital ofPhiladelphia, Philadelphia, Pennsylvania.

Manuscript received: September 1, 2004Accepted in final form: December 17, 2004Reprint requests: Peter J. Gruber, MD, PhD, The Cardiac

Center, Suite 8527, Children’s Hospital ofPhiladelphia, 34th Street and Civic CenterBoulevard, Philadelphia, PA 19104. Fax: (215)59-2715; Email: pgruber@mail.med.upenn.edu.

Copyright # 2005 by the Wound Healing Society.ISSN: 1067-1927.

205

the University of Pennsylvania Institutional AnimalCare and Use Committee.

Ischemia-reperfusion injury modelMRL/MpJ male mice (stock 00486, The Jackson Labora-tory, Bar Harbor, ME) between 8 and 10 weeks of agewere studied. C57Bl/6 male mice (stock 000664, TheJackson Laboratory) between 8 and 10 weeks of agewere used as controls. Animals were anesthetized byintraperitoneal injection with 2% ketamine and intu-bated with PE-60 tubing via a tracheal cut down. Theanimals were ventilated at 100 breaths per minute with100% O2 with a rodent ventilator (Harvard Apparatus,Inc, Holliston, MA). Lead I and lead II electrocardio-grams (ECG) were recorded continuously (PowerLaboratory, ADInstruments, Mountain View, CA).Core-body temperature was monitored with a rectalprobe (Cole-Parmer Instrument Company, Inc., VernonHills, IL) and maintained at 37 ˚C. Under a Leica MZ8steromicroscope (Leica Microsystems, Inc., Bannock-burn, IL), a left anterior thoracotomy and infant eye-speculum retractor (Miltex, Inc., York, PA) were usedto expose the heart. The left anterior descending (LAD)coronary artery was identified 1 mm inferior to the leftatrial appendage at which point a 7–0 silk suture waspassed underneath the artery and tied over a 2 mmsegment of PE-10 tubing. Both visual blanching andST segment elevation on continuous ECG displayconfirmed myocardial ischemia. After 45 minutes, thePE-10 tubing was removed, permitting reperfusion. Thesternotomy wound was closed in two layers with 4–0Vicryl (Ethicon, Inc., Sommerville, NJ) and the mouseextubated.

Infarct area calculation with (2,3,5)-triphenyltetra-zolium chloride stainingAt 24 hours following reperfusion and at 8–10 weeksafter injury, mice were euthanized by CO2 inhalation,the hearts were removed, and the aorta cannulatedwith a 21-gauge blunt-ended needle. Hearts were per-fused with 3 ml of phosphate buffered saline solution,followed by 3 ml of 1% (2,3,5)-triphenyltetrazolium(TTC) at 37 ˚C. The hearts were frozen at �80 ˚C for20 minutes and cut into 1.5-mm slices. Each slice wasindividually weighed and photographed with incidentlight on both sides with a Leica DC200 CCD camera andMZ16 stereomicroscope (Leica Microsystems, Inc.)Infarct area was calculated using NIH Image J software(http://rsb.info.nih.gov/ij/) by the following calculation:The TTC precipitate excluded areas on each side ofeach individual slice were averaged to get a meaninfarct area per slice. This mean/slice was multipliedby the weight/slice and summed over the total slices toget a total infarct weight. This number was divided bythe total heart weight to get percentage infarct. Wall

thickness was reported as the thinnest wall measure-ment in the infarcted anterior wall in the section fromthe mid-papillary level. Images were neither furtherprocessed nor manipulated but were formatted inAdobe InDesign CS (Adobe Systems Inc., San Jose,CA).

Masson’s trichrome stainingHearts from MRL/MpJ and C57BL/6 mice were excisedand perfusion fixed with 4% paraformaldehyde ante-grade through the aortas at constant pressure. Heartswere divided transversely at the mid-papillary level,dehydrated through an ethanol series, paraffinembedded, serially sectioned, and stained with forMasson’s Trichrome (protocols available at http://www.uphs.upenn.edu/mcrc/histology/Masson.html).

RESULTSThe left ventricle of MRL/MpJ (n¼ 13) and C57Bl/6(n¼ 12) control mice was injured using a clinically rele-vant model of 45-minute LAD coronary artery ischemia-reperfusion. Evidence of ischemia was documented bymyocardial blanching distal to the coronary occlusion(Figure 1A, arrows) and ST segment elevation on theECG (Figure 1B, lower trace). After 24 hours, eighthearts were excised (four MRL and four C57/Bl6)and TTC stained showing viable myocardium (red)and areas of infarction (white). There was no differ-ence (P¼ 0.55) in initial injury between MRL/MpJmice (14.5� 2.1%, n¼ 4) and C57BL/6 (13.0� 0.7%,n¼ 4) mice.

After 8–10 weeks, the remainder of the animalswere euthanized, hearts excised, and TTC staining per-formed to quantify infarct size. Similar to the initialtime point, there was no difference (P¼ 0.20) in infarctarea between MRL (18.9� 1.8%, n¼ 9) and C57/Bl6(15.7� 1.3%, n¼ 8) mice (Table 1, Figures 1C and D).Additionally, there was no significant difference(P¼ 0.91) in wall thickness normalized to body weightbetween the MRL (0.017� 0.003 mm/mg) and C57/BL6(0.017� 0.002 mm/mg) mice (Table 1).

We then stained paraffin-sectioned MRL and C57/Bl6 control hearts with Masson’s trichrome stain toassess the extent of scar formation and fibrosis. Bothstrains displayed intense blue staining indicative ofcollagen deposition characteristic of myocardial infarc-tion (Figures 1E and F). In summary, we found nodifference between MRL/MpJ and C57/BL6 mice ininitial injury size, infarct area at 10 weeks, wall thick-ness, or scar formation.

We also investigated direct cryoinjury to the LVfree wall utilizing two exposures of a 2 mm probe for10 seconds according to the protocol of Leferovichet al.11 This resulted in a superficial, epicardial injury.

WOUND REPAIR AND REGENERATION206 ABDULLAH ET AL. MARCH–APRIL 2005

To produce an injury of more than 30% LV wall thick-ness, we required at a minimum, five direct exposuresof a 3 mm cryoprobe for 10 seconds with application tothe LV free wall under direct vision; an increase of 225%exposure area and 250% time.

DISCUSSIONIn mammals, tissue regeneration has been observed intissues such as liver13 and pancreas,14 but not in hearts.Although other species, such as newt,15 axolotl,16 andzebrafish,3 can regenerate partially injured hearts, there

A B

C D

E F

Pre

Post

FIGURE 1. Histological analysis of MRL andC57/Bl6 control mice following left ventricularischemia-reperfusion injury. (A) LAD ligationwith 7–0 silk suture over P10 tubing. Pale areadelineated by arrows shows ischemic region.(B) Lead I ECG of preligation (upper panel)and immediately post reperfusion (lowerpanel) shows ST segment elevat ioncharacteristic of ischemic injury. (C and D)TTC stain of C57/Bl6 control (C) and MRL (D)shows severe anterior left ventricular wallthinning and residual scarring (white). (Eand F) Masson’s trichrome stain of C57/Bl6control (E) and MRL (F) mice shows intenseblue precipitate characteristic of collagendeposition in post-infarction scar.

Table 1. Cardiac measurements upon late harvest (8–10 weeks) of the hearts

C57/Bl6 MRL/MpJ

Animal Weight* Infarct size#

Wall thickness@

Weight Infarct size Wall thickness

1 19 16.5 0.26 38 13.9 1.172 23.5 16.1 0.25 38 18.1 0.83 25.5 12.4 0.71 39 20.8 0.74 26.8 16.1 0.39 44 19.6 0.215 25.5 11.7 0.23 37.5 25.6 0.76 26.5 23.2 0.34 42 14.3 0.717 24.5 11.7 0.95 41 11.5 0.348 27.1 18.2 0.41 39 28.8 0.59 36 17.6 0.68Mean � SEM 24.8� 0.9 15.7� 1.3 0.44� 0.08 39.8� 0.8 18.9� 1.8 0.65� 0.09

* Weight is presented in grams.

# Infarct size indicates percentage of total myocardial weight.

@Wall thickness was determined to be the smallest measurement, mid-papillary, in mm.

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is no reproducible evidence that regeneration, eithernative or induced, occurs in mammals. Previous studiessuggesting that MRL mice can regenerate injuredmyocardium utilized transdiaphragmatic cryoinjury ofthe right ventricle.11 In contrast, we found that ische-mia-reperfusion injury of the left ventricle producedmyocardial infarctions of identical size in both MRLand C57/Bl6 control mice. A recent report also showedthat MRL mice form scar in response to LAD ligation, inagreement with our results.17 Our data confirm andextend these findings by providing a control group forcomparison, by using an ischemia-reperfusion modelrather than simple LAD ligation, and by quantifyinginitial and subacute injury using TTC and Trichromestaining.

It is possible that previous differences in regenera-tive capacity between MRL and C57/Bl6 mice can beexplained by differences in the type of injury modelutilized. A transdiaphragmatic approach with compro-mised heart visualization of the injury site to the RV mayhave resulted in variability in injury location and extentin the initial report.11 It is also conceivable that there areintrinsic differences in regenerative capacity betweenthe right and left myocardium in the MRL strain.However, our results suggest a different explanation.

Our study conclusively shows that the MRL/MpJstrain forms intense scar and wall thinning characteristicof myocardial infarction in a fashion similar to that ofC57Bl/6 mice and suggests that it is not a promisingmodel for the study of the potential regenerative capacityof left ventricular myocardium following ischemic injury.

ACKNOWLEDGMENTSThis study was supported in part by a grant fromthe National Institutes of Health (RO1-HL-56915), theCommonwealth of Pennsylvania (MSP), and theThomas B. and Jeannette E. Laws McCabe Fund (PJG).

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