injury in on-line fashion during regional and global myocardial

Transcription

1 Online Detetion of Reversible Myoardial shemi njury by Measurement of Myoardial Eletrial mpedane Martin. Ellenby, M.D., Kent W. Small, M.D., Randall M. Wells, B.S., David J. Hoyt, M.D., and James E. Lowe, M.D. ABSTRACT The metaboli and physiologial alterations assoiated with hanges in myoardial tissue eletrial resistivity during ishemia were haraterized to assess the feasibility of using suh resistivity as an online indiator of the onset of ishemi injury. Twelve anesthetized dogs underwent rapid ardia extirpation; 5 served as untreated ontrols, and 7 were pretreated wth metoprolol tartrate. Beta blokade was used to alter the time ourse of ishemi injury as demonstrated previously in studies using this experimental model. n vitro measurement of myoardial resistivity, the detetion of ishemi ontrature, and serial measurements of tissue adenosine triphosphate (ATP) and latate were obtained from totally ishemi left ventriles at 37 C. Myoardial resistivity began to inrease signifiantly before onset of ishemi ontrature in the untreated ontrol group (resistivity at 42.3 k 3.1 minutes, ontrature at 53.8? 3.7 minutes; p < 0.025) as well as the metoprolol group (resistivity at 50.7? 1.5 minutes, ontrature at 70.0 f 3.5 minutes;^ < 0.005). As expeted, ishemi ontrature was delayed in the betabloked group ompared with ontrols (p < 0.01). Similarly, the onset of myoardial resistivity inrease was delayed in the betabloked group (p < 0.025). ATP and latate levels at the onset of myoardial resistivity inrease were onsistent with severe but reversible injury. Resistivity hanges during ishemia orrelated linearly with simultaneous ATP depletion and latate aumulation (r = 0.88 to 0.98; p < 0.05). Furthermore, during global ishemia studied in 3 anesthetized dogs in vivo, the onset of myoardial resistivity inrease ourred after 20 minutes. Finally, 6 anesthetized dogs underwent 60 minutes of in vivo regional ishemia by oronary artery olusion, followed by 60 minutes of reperfusion. Myoardial resistivity in the ishemi region inreased immediately and steadily after oronary olusion, followed by a rapid derease during subsequent reperfusion. These data show that myoardial resistivity may be useful for identifying severe but still reversible ishemi From the Deparhnent of Surgery, Duke University Medial Center, Durham, NC. Presented in part at the 71st Annual Meeting of the Amerian College of Surgeons, Chiago, Otober, Aepted for publiation May 6, Address reprint requests to Dr. Lowe, Department of Surgery, Box 3954, Duke University Medial Center, Durham, NC injury in online fashion during regional and global myoardial ishemia. The surgial orretion of the majority of aquired and ongenital ardia defets requires that the myoardium undergo a period of global ishemia and that the effets of this ishemia be ameliorated by the infusion of old potassium ardioplegia [l]. At present, myoardial temperature is the only ommonly used index of tissue preservation that an be monitored in online fashion during ardia surgial proedures requiring a period of global ishemia (21. However, monitoring myoardial temperature alone does not identify the degree of ongoing ishemi injury that may be ourring. A diret means of identifying the onset of ishemi injury would allow appropriate orretive interventions before the onset of irreversible injury. Totally ishemi myoardium undergoes a progression of metaboli and ultrastrutural hanges, eventually leading to irreversible ishemi ontrature, or ardia rigor mortis [381. To have linial utility, any methodology employed to monitor ishemi injury in online fashion should be sensitive to ellular hanges that our early in the ishemi proess, well before the onset of irreversible injury. For example, a monitor sensitive to shifts in water and ion distribution, gas tensions, alterations in ph, or the earliest loss of sarolemmal funtion would be ideal based on urrent knowledge of the pathogenesis of ishemia. ndeed, reent attempts at online evaluation of myoardial ishemi injury have entered around intramyoardial gastension monitoring and intramyoardial ph determinations [9141. On the other hand, a monitor sensitive only to more marked sarolemmal deterioration or the onset of ishemi ontrature, both of whih are assoiated with irreversible injury, would offer muh less opportunity for the institution of suessful interventions. The marosopi eletrial resistivity of tissues has interested physiologists and biophysiists sine the late nineteenth entury [ Resistivity hanges ourring in dying or ishemially injured tissues have been desribed, but these hanges remain poorly haraderized [15, 18, 2729]. Van Oosterom and assoiates [28] desribed temporal resistivity inreases in regionally ishemi myoardium, but studied only 3 animals. Garrid0 and olleagues [29] measured the tissue resistivity of globally ishemi anine hearts exposed to various degrees of hypothermi and ioni preservation, and showed less marked resistivity inreases with improved 587 Ann Thora Surg 44:587597, De 1987

2 ~ Ad 588 The Annals of Thorai Surgery Vol 44 No 6 Deember 1987 preservation tehniques. However, the diret effets of hypothermia and potassium ardioplegia on myoardial resistivity were not evaluated. Furthermore, none of these investigati ms haraterized inreases in resistivity during ishemia in the ontext of known physiologial or metaboli markers of ishemi injury. Therefore, the present studies were designed to determine the time ourse of hanges in myoardial resistivity during ishemia and to relate these resistivity hanges to onurrent physiologial and metaboli alterations identifying the degree of ishemi injury. Material and Methods A total of 21 mongrel dogs were studied to determine myoardial resistivity hanges during global or regional ishemia. All animals reeived humane are in ompliane with the Priniples of Laboratory Animal Care formulated by the National Soiety for Medial Researh and the Guide for the Care and Use of Laboratory Animals prepared by the National Aademy of Sienes and published by the National nstitutes of Health (NH Publiation No. 8023, revised 1978). n Vitro Study of Total shemia Twelve dogs were anesthetized with sodium pentobarbital, intubated, and ventilated. The heart of eah animal was extirpated through a left thoraotomy. A left ventriular free wall slab was reated from eah heart by inising along the epiardial ourse of the left anterior desending oronary artery (LAD), and removing the right ventrile, base, and apex. The slab was then subdivided into three portions (Fig 1). One portion was instrumented with a foureletrode array for measurement of marosopi myoardial eletrial resistivity during total ishemia. The seond portion was positioned in a tissue ompressibility gauge used to detet the onset of ishemi ontrature. Tissue samples from the third portion of the slab were obtained for serial determinations of adenosine triphosphate (ATP) and latate onentrations. Data were obtained from the time of instrumentation until 90 minutes of total ishemia had elapsed. One instrumented, all myoardial slabs were maintained at 37 C. The 12 animals were subdivided into two groups. The first group, whih onsisted of 5 dogs, did not reeive any pharmaologial pretreatment. The seond group, of 7 dogs, was pretreated with the betaadrenergi bloker metoprolol tartrate. Eah of these dogs reeived 100 mg of metoprolol orally twie daily for two days prior to study, and 0.24 mg per kilogram of body weight as an intravenous bolus 30 minutes prior to ardia extirpation. Previous studies [30] using this same model have shown that betaadrenergi blokade delays the onset of ishemi ontrature, slows the rate of ATP utilization, and delays subellular morphologial hanges during ishemia [31]. DETERMNATON OF MACROSCOPC MYOCARDAL RESS TVTY. A foureletrode tehnique was used to measure marosopi myoardial resistivity. This methodology Resist iuity Measurements J shemi Cantroture Determination ATPond Latote Conentrations Four Eletrode Compressibility Gauge Serial Tissue Samples Army System \ t J Temperature Controlled Water Bath, 370 (Myoardium Sealed in Plosti Bag Fig 1. Total ishemia in vitro: After rapid ardia extirpation, a myoardial slab was reated from the left ventriular free wall, and immediately subdivided into three portions. A foureletrode array was sutured to the endoardial surfae of one portion of the slab to rneasure myoardial resistivity. The seond portion of the slab was positioned in the ompressibility gauge to determine the onset of ishemi ontrature. The third portion was utilized for determination of adenosine triphosphate (ATP) and latate levels. Eah portion was instrumented with a thermistor needle, plaed in a plasti bag, and submerged in a water bath to maintain temperature at 37 C throughout the 90minute duration of the experiment. V Left z13parms,1000hz d.3 mm Fig 2. Foureletrode array method: Resistivity was measured by passing a known ontrolled urrent () between the outer two eletrodes. A resultant potential differene (V) is measured between the inner two eletrodes. The resistivity (p) is diretly related to V as desrribed in the equation. The A is a onstant dependent on the size of the eletrodes relative to the intereletrode distane, and d is the intereletrode distane U91. has the advantages of being simple to implement and of avoiding the ompliations of eletrode polarization [22, 28,321. Four eletrode pins (goldplated brass, mm [0.125 inh] long and mm [0.025 inh] in diameter) were mounted as a linear array on a thin Silasti sheet (Fig 2). The spaing between onseutive pins was fixed at 3 mm. The Silasti sheet with the mounted eletrode pins was sutured to the endoardial surfae of the left

3 589 Ellenby et ak Myoardial Eletrial mpedane as ndiator of shemi njury Onset 1 l O V V REGULATED tdc POWER SUPPLY HGH AND 4 LOW PASS FLTERS OUT, AC DGTAL AC MCROAMMETER MYOCARDAL SPECMEN 4 ELECT RODE ox t LOSCOPE STRP CHART DGTAL DC VOLTMETER ( VoUT 0 Myoordiol Resistivity) Fig 3. Resistivity monitoring system: A foureletrode array tehnique was employed, as shown in Figure 2. A 1 3d rms, 1,000Hz urrent was generated by a bilateral urrent soure and injeted between the outer two eletrodes. The resultant potential differene deteted between the inner two eletrodes was amplified through a highinput impedane instrumentation amplifier. Additional filtering was performed using an operational amplifier bandpass filter iruit. An Rh4StoDC voltage onvertor failitated measurement of the amplified voltage with a digital DC voltmeter. n addition, loal ambient eletrial ativity of the myoardium ould be monitored by turning off the injeted urrent while using the amplifier and filter omponents to reord bipolar eletrograms between the inner two voltagesensing eletrodes. ventriular slab, and the pins were allowed to penetrate into the subendoardium. The eletrode array was maintained in this onstant position for the duration of the experiment. The position of the array relative to musle fiber orientation was random in eah study. To measure resistivity, a 13pArms urrent with a frequeny of 1,000 Hz was injeted and removed from the speimen between the outer two eletrodes of the array for a duration of 2 seonds (see Fig 2). A resultant potential differene was established and measured between the two inner eletrodes through a highinput impedane instrumentation amplifier (Fig 3). A linear relationship exists between the measured voltage and the tissue resistivity, as desribed in Figure 2 [22, 281. The system was alibrated before beginning tissue resistivity measurements by submerging the eletrode array in NaCl solutions of known onentration at room temperature, and therefore of known resistivity [33]. Regression analysis of the known NaCl solution resistivities and the measured voltages yielded a highly orrelated linear relationship that ould then be used to alulate resistivity from a measured voltage. The resolution of the system was? 3 0m. Cardia extirpation, subdivision of the slab, attahment of the eletrode array, submersion in a temperatureontrolled water bath, and subsequent rewarming to normothermia required approximately 15 minutes for eah speimen. Resistivity measurements were made at 5minute intervals for 20 to 40 minutes after extirpation, at 2.5minute intervals from 40 to 60 minutes after extirpation (to inrease resolution when early ishemi injury was expeted), and then one again at 5minute intervals until 90 minutes of total ishemia had elapsed. A %baseline resistivity was defined as the minimum resistivity measured during the 90 minutes of ishemia. Resistivity is expressed as a perentage of hange from baseline. The time to onset of resistivity inrease was defined as the time orresponding to a nonreversible 5% inrease above baseline. DETERMNATON OF SCHEMC CONTRACTURB. The onset of ishemi ontrature was determined using a speially designed tissue ompressibility gauge developed in our laboratory [34]. The onset of ishemi ontrature was deteted by a sudden derease in ompressibility seondary to an inrease in tissue stiffness. This devie has been shown to identify aurately ishemi ontrature onset when ompared with detetion of ishemi ontrature using an intraavitary balloon tehnique [34]. DETERMNATON OF ATP AND LACTATE CONCENTRA TONS. Subendoardial tissue samples weighing 50 to 75 mg were obtained at ontrol and serially at 15minute intervals thereafter for determination of ATP and latate onentrations. Eah tissue sample was weighed immediately on a Cahn Model DTL mirobalane and plaed in 3.6% perhlori aid at 0.5 C and then homogenized using a TriR stirrer. Sample harvest, weighing, and transfer to perhlori aid required 10 to 15 seonds.

4 590 The Annals of Thorai Surgery Vol 44 No 6 Deember 1987 The homogenized mixture was allowed to extrat for 30 minutes at 0.5"C, and then was entrifuged to remove Klo &. The supernatant was neutralized with K2C03 KOH to ph 6.0 to 6.5 and preserved at 70 C until analysis. ATP onentrations were determined in the serial samples with a Waters highperformane liquid hromatography system using the tehnique desribed by HullRyde and assoiates [35]. Lati aid tissue onentrations were measured by spetrophotometry (PerkinElmer Lamda5) in a hydroxeneglyine buffer system. TEMPERATURE CONTROL AND MONTORNG. The temperature of eah portion of the slab was ontinuously monitored with needle thermistor probes onneted to a Shiley digital temperature monitor. Eah speimen was enased in a plasti bag and submerged in a temperatureontrolled water bath to maintain a onstant myoardial temperature of 37 C. STATSTCAL ANALYSS. Cumulative data are expressed as the mean * the standard error of the mean for all phases of this study. Differenes between groups were evaluated by t test. ATP and latate onentrations assoiated with hanges in resistivity were evaluated using linear regression analyses. n Vivo Global shemia To avoid measurement delay aused by instrumentation and transient ooling as ourred during the in vitro total ishemia experiments, and to more losely examine myoardial resistivity during the first minutes of ishemia, in vivo global ishemia was also studied. Three mongrel dogs were anesthetized with pentobarbital, intubated, and ventilated. The heart was exposed through a median sternotomy. A resistivity eletrode array was sutured to the anterior epiardial surfae of the left ventrile in a random orientation. When applying the attahment sutures, are was taken to avoid ligation of superfiial oronary vessels and to keep the array free from blood or saline solution, thereby minimizing loal ishemia and possible error due to eletrode shortiruiting. Baseline resistivity measurements were made at 5minute intervals for at least 30 minutes. The heart was then made globally ishemi by venting both ventriles and by rosslamping the proximal aorta. Resistivity was measured every 2 minutes for the first 10 minutes following rosslamping and at 5minute intervals thereafter until 90 minutes of global ishemia had elapsed. Myoardial temperature was maintained at 37" * 1 C. n Vivo Study of Regional lshemia To study the effets of regional ishemia in vivo, an additional 6 dogs were anesthetized with pentobarbital, intubated, and ventilated. Eah animal was antioagulated with heparin. The heart was exposed through a median sternotomy. A ligature was plaed loosely around the LAD distal to the seond diagonal branh. Two eletrode arrays were sutured to the epiardial surfae of the right anterior ventriular wall. These served as ontrols. Two additional eletrode arrays were positioned in the projeted distribution of the LAD distal to the ligature. After 30 minutes of baseline resistivity measurements taken every 5 minutes, the LAD was oluded, thereby reating a zone of regional ishemia. Following LAD olusion, resistivity was measured at eah site every 2 minutes during the first 10 minutes of ishemia and at 5 minute intervals thereafter. After 60 minutes of regional ishemia, the ligature was released, thus allowing reperfusion of the ishemi region. One again, resistivity was measured every 2 minutes during the first 10 minutes of reperfusion and then at 5minute intervals until 60 minutes of reflow had elapsed. Finally, eah heart was then made globally ishemi in the manner desribed previously, and resistivity measurements were made every 5 minutes for a total duration of 90 minutes. Eah instrumented heart was exised after death. A small plasti atheter was positioned and seured in the LAD with its tip at the site of the previous olusion. A blue dye (Dupont monastral blue) was infused into the LAD bed at a onstant pressure of 140 mm Hg, whih resulted in a learly demarated zone of perfusion [36]. Measurements obtained from the right ventriular array farthest from the perimeter of the bluedyed region provided nonishemi ontrol resistivity. Measurements from the eletrode array most widely enompassed by bluedyed myoardium were treated as "ishemi region" data. n addition, ishemi injury at the eletrode array positioned in the LAD bed was further onfirmed in 2 animals by demonstrating signifiant attenuation of loal eletrial ativation based on bipolar eletrograms reorded through the resistivity eletrode array. To assess the gross eletrophysiologial effet of the injeted urrent, bipolar epiardial eletrograrns were reorded near the right ventriular arrays. These epiardial eletrograms showed no evidene of propagated ativation or alteration in ardia rhythm aused by the injeted urrent of 13 d. Results Total shemia n Vitro The mean absolute baseline resistivities for ontrol and betabloked groups were f 42.7 nm and ~fr am, respetively; there was not a signifiant differene between the values. n 11 of 12 speimens, one an initial small resistivity inrease was deteted (approximately 2%), it ontinued to inrease steadily. n all speimens, a 5% inrease was irreversible. Figure 4 is an example of the timeourse hange in myoardial resistivity during total ishemia in vitro as observed in one representative untreated ontrol speimen. The resistivity of the untreated ontrol group always began to inrease earlier than in the betabloked group (Fig 5). The resistivities of the two groups differed signifiantly for the 40 to 60minute interval after extirpation (p < 0.05). Both groups attained resistivity levels 230% of baseline by 90 minutes of total ishemia (see Fig 5). A omparison of the mean times to onset of resistivity

5 591 Ellenby et al: Myoardial Eletrial mpedane as ndiator of shemi njury Onset Time After Extirpation ( minutes 1 Fig 4. Temporal resistivity hanges from a representative slab speimen during total ishemia in vitro. After the initial 15minute instrumentation and rewarming period, resistivity measurements were made at 5minute intervals during the 20 to 40minute period after extirpation, at 2.5minute intervals from 40 to 60 minutes, and at 5minute intervalsfrom 60 to 90 minutes. Resistivity is expressed as a perentage of baseline. Ajter an initial baseline period of no hange, resistivity began to inrease rapidly and irreversibly. This urve typifies the resistivity hanges observed for in vitro ontrol speimens. 140 lnstrumrnrol~on Time 100 T Time After Extirpation (minutes) Fig 5. Cumulative temporal hanges in resistivity during total ishemia in vitro. Both the ontrol and metoprolol tartratetreated groups are shown. Eah data point is the mean perentage of baseline 2 the standard error of the mean alulated for all speimens in the group at the time indiated. Baseline resistivities did not differ signifiantly between groups (p = not signifiant). However, the two groups did differ signifiantlyfrom 40 to 60 minutes of ishemia (p < 0.05). Pretrmtmmt with metoprolol delayed the onset of resistivity inrease, thus mimiking previously desribed delays in onset of ishemi ontrature in betabloked speimens using this same model of total ishemia. Both groups reahed approximately 230% of baseline by 90 minutes. inrease and ishemi ontrature is shown in the Table and Figure 6. The time to the onset of resistivity inrease ranged from 36 to 54 minutes for the untreated ontrol group (mean, minutes) and from 46 to 58 minutes for the metoprololpretreated group (mean, minutes). The time to onset of ishemi ontrature ranged from 42 to 64 minutes for the ontrol group (mean, minutes) and from 60 to 78 minutes for the metoprololpretreated group (mean, minutes). For both the untreated ontrol group and the betabloked group, the onset of resistivity inrease ourred signifiantly before the onset of ishemi ontrature (p < 0.025). As expeted, based on previous studies using this model [30], the onset of ishemi ontrature was signifiantly delayed in the betabloked group ompared with the untreated ontrols (p < 0.01). nterestingly, the onset of resistivity inrease followed this same trend. Resistivity inreases were deteted signifiantly earlier in the ontrol group than in the betabloked animals (p < 0.025). Conentrations of ATF' and latate assoiated with the onset of resistivity inrease and the onset of ishemi ontrature were alulated from linear regression analyses of the atual ATP and latate onentrations measured at 30, 45, and 60minute sample times (see Table). For both groups, ATP levels at the time of resistivity inrease were signifiantly greater than ATP levels at the onset of ishemi ontrature (p < 0.025) (see Table). Latate levels were lower at the time of resistivity inrease than at the onset of ishemi ontrature for both groups (p < 0.05). Thus, these data demonstrate that ATP depletion and latate aumulation were signifiantly less advaned at the onset of resistivity inrease than at the onset of ishemi ontrature (Fig 7). Adenosine triphosphate and latate levels as a funtion of the orresponding measured resistivity at 30,45, 60, 75, and 90 minutes of ishemia are summarized in Figure 8. Linear regression analyses of these data show that ATP and latate onentrations orrelated signifiantly with resistivity for both groups ( = 0.88 to 0.98, p < 0.05) despite the differing time ourses of resistivity hange between the untreated ontrols and the betabloked group. Changes in myoardial resistivity paralleled ATP depletion and latate aumulation during total ishemia in vitro. Global shemia n Vivo Figure 9 summarizes the timeourse hanges in resistivity for 30 minutes of baseline followed by 90 minutes of global ishemia. The mean absolute resistivity baseline was am. After the onset of global ishemia, resistivity remained near baseline until approximately 20 minutes had elapsed. Then, starting at 20 minutes, resistivity began to inrease rapidly and irreversibly. However, the onset of resistivity inrease in vivo was approximately onehalf of the onset time noted for the untreated ontrol group in vitro. Also of note is that resistivity did not hange markedly during the first 20 minutes of normothermi global ishemia in vivo.

6 ~ The Annals of Thorai Surgery Vol 44 No 6 Deember 1987 Results of Total shemia n Vitro Time to Onset ATP at Onset Latate at Onset Group Method (min) (PmoYgm wet weight) (FmoYgm wet weight) Untreated ontrols h4r 42.3 f f f 2.47 C 53.8 * k t 5.70 ntragroup omparison p < p < p < 0.05 Metoprololtreated dogs MR 50.7 f k f 1.29 C 70.0 f f ntragroup omparison p < p < p < Betweengroup omparison MR C p < p < p = NS p < p = NS p = NS ATP = adenosine triphosphate; MR = myoardial resistivity; C = ishemi ontrature; NS = not signifiant. UNTREATED CONTROLS YETOPROLOL PRETREATED RESSTVTY CONTRACTURE Fig 6. Onset of resistivity inrease and ishemi ontrature: Onset of resistivity inrease was dejined as the time when resistivity underwent a nonreversible 5% inrease above baseline. Onset of ishemi ontrature orresponds to a sudden derease in tissue ompressibility. For both the untreated ontrol and metoprolo1 tartratepretreated groups, resistivity began to inrease signifiantly before the onset of ishemi ontrature. As expeted, ishemi ontrature was delayed in the group relative to the similarly, the blmk& pup hd a delayed time to onset of resistivity inrease relative to the ontrols. Regional lshemia n Vivo Resistivity hanges during regional ishemia, subsequent reflow, and the final period of global ishemia are summarized in Figure 10. Resistivity in the ishemi region inreased in all dogs within 10 minutes after LAD ligation; there was a onomitant smaller derease and plateau of resistivity in the ontrol region. The resistivity in the ishemi region ontinued to inrease steadily throughout the 60 minutes of LAD ligation and attained a level nearly 140% of the initial baseline value. mmediately after release of the LAD ligature, resistivity in the ishemi region dereased rapidly. n ontrast, on w 1.5 3!z \ z w 1.0 a + = W * E 40 \ o l al 30 E a 20 : 10 A p< MYOCARDAL SCH EMlC RESSTVTY CONTRACTURE Fig 7. Mean adenosine triphosphate (ATP) and latate levels at onset of resistivity inrease and ishemi ontrature during total ishemia in vitro. values were frorn linear reflession Of measured onentrations at 30, 45, and 60 minutes. ATP depletion and latate aumulation were signifiantly more advaned at the on. set of ishemi ontrature than at the onset of resistivity inrease. The ATP and latate levels at the onset of resistivity inrease are onsistent with severe but reversible ishemi injury. (NS = not signifiant.) reperfusion, resistivity in the ontrol region rose slightly and leveled off. Finally, after 60 minutes of reflow, the heart was made globally ishemi. The resultant resistivity hanges were similar to the hanges previously desribed for global ishemia in vivo. One again, resistivity began to inrease at approximately 20 minutes of global ishemia (see Fig 10).

7 593 Ellenby et al: Myoardial Eletrial mpedane as ndiator of shemi njury Onset 0. U *UNTREATED CONTROLS~=.~~,~<OOS AMETOPROLOL TREATED (r=bb.p<.a) a. 350 a v1 0 n 300 #.e 500i Resistivity (% of boseline d 200 ONSET OF GLOBAL SCHEMA,/ T 4 T T *UNTREATED CONTROLSlr'.96.p<.Ol AMETOPROLOL TREATED (r=.95. V.025) Resistivity (%of baseline) Fig 8. Adenosine triphosphate (ATP) and latate onentrations versus resistivity for eah sample time from 30 to 90 minutes of ishemia plotted against the assoiated resistivity. Resistivity is expressed as a perentage of baseline resistivity. Sample times are indiated beside eah data point. Linear regression analyses performed on these data yielded signifiant orrelations for both groups, as shown. These orrelations remained sign$ant despite differing time ourse of resistivity, ATP depletion, and latate aumulation between the untreated and betabloked groups. Comment These data show that myoardial eletrial resistivity hanges in a onsistent and preditable fashion during total, global, and regional ishemia under normothermi onditions. Large inreases in resistivity ourred repeatedly in assoiation with measurable metaboli and physiologial markers of ishemi injury. Furthermore, small inreases in resistivity were easily deteted and onsistently predited the more dramati resistivity hanges that inevitably followed. Mean baseline resistivity measurements made in vitro (549.9 & 42.7 am for the untreated ontrol group) and in vivo ( am for all in vivo groups) are onsistent with previous reports onerned with haraterizing the absolute eletrial resistivity of myoardium. Rush and oworkers [22] reported resistivities of 563 R m perpendiular to myoardial musle fiber orientation and 252 am parallel to the fibers. More reently, van Oosterom and olleagues [28] found transmural resistivity to be approximately 410 am, and Roberts and Sher [26] reported gross myoardial resistivities of 213 Rm parallel to fibers and 705 am perpendiular to fiber 100 5Ol f i, Time (minutes) Fig 9. Global ishemia in vim. Cumulative data (mean + the standard error of mean) are shown for resistivity measured in 3 dogs during global ishemia in vivo. Resistivity did not hange markedly until 20 minutes of ishemia had elapsed. Starting at 20 minutes, resistivity inreased rapidly and ontinuously throughout the remaining 70 minutes of the experiment. orientation. All of these studies were performed on normothermi, nonishemi myoardium in vivo, and all used a foureletrode tehnique similar to the one used in the present study. Baseline absolute resistivity values obtained in vivo are probably more reliable than in vitro values beause the intat in vivo preparation does not undergo transient ooling. n addition, the in vivo experimental design provides ample time for injury urrents indued by instrumentation trauma to subside prior to the onset of ishemia. The magnitude of these injury urrents may be on the order of 1 pa before they attenuate [37], whih approahes 10% of the injeted urrent. The maintenane of normothermia was important, sine tissue resistivity will vary slightly with temperature 123, 291, as onfirmed by preliminary studies in our laboratory. The anisotropy of the myoardium was not analyzed. The relatively large intereletrode spaing of 3 mm should result in urrent penetrating deep into the ventriular wall, thus reahing areas of differing fiber orientation [28,32,38]. Therefore, variability in the measured absolute resistivities should be somewhat dereased by using this large intereletrode spaing despite the random orientation of the eletrode arrays relative to the visible fiber orientation. Resistivity data were further normalized by interpreting all temporal hanges in terms of a perentage deviation from baseline rather than by omparing hanges in absolute resistivity mag

8 594 The Annals of Thorai Surgery Vol 44 No 6 Deember W.E 0) 180 v) x.. > 120. v) 100 shemi Region Control Region N=6 it p<05 * A0r Baseline, LAD Olusion, Rellow Perfurion, Global shemia, (60min 160min 1 (90 min bo 2io Time ( minutes) Fig 10. Regional ishemia in vivo. n 6 dogs, in vim instrumentation was arried out for resistivity measurements by positioning an array in the distribution of the right oronary artery (ontrol region) and the left anterior desending oronary artery (LAD) (ishemi region). Shown here are the umulative timeourse hanges in resistivity for the ontrol and ishemi regions (mean 5 standard error of mean). After 30 minutes of baseline measurements, the LAD was oluded. Resistivity in the ishemi region inreased immediately; there was only a slight derease in the ontrol region. After 60 minutes ofregiml ishemia, the LAD ligature was released. The resistivity in the reperfused LAD bed derwd rapidly; there was a simultaneous sub tle inrease in the ontrol region. After 60 minutes of reflow, the heart was made globally ishemi, and subsequent resistivity hanges in both regions were similar to hanges during global ishemia in vivo shown in Figure 9. nitude. n this way, the anisotropi harateristi of the tissue influened temporal trends minimally. During total ishemia in vitro, after a disrete period of little hange, resistivity repeatedly underwent a rapid inrease. The onset of this inrease ourred signifiantly before the onset of ishemi ontrature in all studies. shemi ontrature was deteted at minutes for the untreated ontrols, whih is onsistent with previous reports using the ompressibility gauge [34] or a left ventriular intraavitary balloon tehnique W1. Betaadrenergi blokade was used to alter the time ourse required to reah ishemi injury. Previous studies [30] in our laboratory employing this same in vitro model have demonstrated that beta blokade signifiantly delays the onset of ishemi ontrature. nterestingly, the same trend was noted in the onset of resistivity inrease; the onset was signifiantly delayed in the betabloked group. This trend suggests that the ellular proesses responsible for the observed resistivity hanges parallel the ellular hanges ourring during ishemia and ultimately leading to irreversible injury as marked by the onset of ishemi ontrature. Further more, sine the onset of resistivity inrease was deteted signifiantly before the onset of ishemi ontrature, the initial early resistivity inrease is identifying a period of reversible ishemi injury [4, 391. The ATP and latate onentrations assoiated with the onset of resistivity inrease also suggest that the tissue was in a state of reversible injury. ATP levels of 0.6 to 1.0 pmoygm wet weight have previously been assoiated with the onset of ishemi ontrature and irreversible injury [8, 33, 39, 401. n ontrast, the ATP levels assoiated with the onset of resistivity rise were approximately 2 pmoygm wet weight. n general, a 90% depletion of ATP is assoiated with irreversible ishemi injury [8]. n this study, ATP was 63% depleted at the onset of resistivity inrease. The time to resistivity rise and the degree of ATP depletion at that time have been assoiated with early defets in ell volume regulation and ioni distribution [5,8,41]. These defets may have been partially responsible for the observed resistivity hanges. Furthermore, the observed ATP level of 2 pmoygm wet weight assoiated with the onset of resistivity rise suggests that a large number of myoytes were still viable and had not yet undergone irreversible injury. Latate onentrations were signifiantly lower at the onset of resistivity inrease ompared with onentrations at the onset of ishemi ontrature. This reflets the shorter period of anaerobi glyolysis endured by the tissue prior to the onset of resistivity rise. The latate levels indiate that the degree of ishemi injury, while severe at the onset of resistivity inrease [40], was still less advaned than that at the onset of ishemi ontrature. Continued latate aumulation is assoiated with a deline in tissue ph, whih may also ontribute to alterations in tissue resistivity. However, Lange and assoiates [ 121 found that extraellular ph dereases within seonds of instituting normothermi global ishemia in vivo in dogs. Sine there was always a disrete delay before resistivity rose during global ishemia, re

9 595 Ellenby et al: Myoardial Eletrial mpedane as ndiator of shemi njury Onset sistivity hanges annot be diretly attributed to alterations in ph alone. Linear regression analysis of ATP and latate onentrations versus resistivity yielded signifiant orrelations. Despite the timeourse shift of resistivity rise between the ontrol and the betabloked groups, the preise linear relationship for the two groups was very similar, that is, a speifi resistivity value taken from either group was assoiated with nearly equivalent ATP or latate onentrations, even though the orresponding duration of ishemia differed. This onsistent orrelation strongly suggests that resistivity hanges paralleled the degradation of metabolially dependent ellular proesses. The major tehnial limitation of the in vitro slab model for studying resistivity was the time required for instrumentation. This preluded obtaining measurements of resistivity during the initial minutes of ishemia, and unavoidably exposed the tissue to a transient episode of ooling. Nevertheless, one the tissue was rewarmed to 37T, resistivity tended to remain onstant for several minutes before the harateristi inrease began. While transient ooling may have skewed the resistivity hanges slightly, the prinipal effet would be protetive, delaying the onset of ishemi injury. Thus, if this transient ooling ould be avoided, signifiant inreases in resistivity aused by ishemia might be deteted even earlier than were observed in this study. The resistivity hanges during global ishemia in vivo were similar to the in vitro hanges. The first several minutes of global ishemia in vivo were of partiular interest, sine the initial period of ishemia ould not be studied using the in vitro model. n all animals that underwent global ishemia in vivo, a stable baseline was obtained prior to the onset of ishemia. After the rosslamping of the aorta, resistivity remained essentially unhanged for the first 20 minutes. The onset of resistivity inrease during global ishemia in vivo ourred at approximately onehalf the time that was observed in vitro (20 minutes versus 42 minutes, respetively). The shorter in vivo time may reflet the aelerated depletion of highenergy phosphate resulting from the persistene of mehanial and eletrial ativity for a short period after the aorta was rosslamped [40]. An analogous effet has been demonstrated by paing globally ishemi hearts at various rates, with higher rates resulting in more rapid ATP depletion [39]. n ontrast, the myoardial slabs prepared for in vitro study beame eletrially and mehanially quiesent within seonds after they were reated. The preise ausative mehanisms for the observed resistivity hanges during ishemia annot be determined from these data. The linear relationship between hanges in ATP and resistivity suggests that resistivity is in part dependent on metaboli ellular proesses. t is postulated that the early initial inrease in resistivity during total ishemia in vitro and global or regional ishemia in vivo may reflet alterations in ioni distribution as ATPdependent membrane ioo pumps beome de pressed [461. ntraellular edema may also ontribute to the inrease in resistivity. However, during reflow of the regionally ishemi myoardium, when the most severe ellular swelling has been shown to our [6, 71,resistivity was noted to atually derease. Thus, intraellular edema alone annot explain the observed resistivity hanges. The loal aumulation of anaerobi metabolites along with alterations in interstitial ioni omposition aused by hanging membrane permeabilities may also ontribute to the first hanges in resistivity due to ishemia. nterestingly, in the regional ishemia experiment, resistivity at the distant ontrol site inreased slightly during reperfusion. This effet may result from the washout of ishemi metabolites and ions from the region of ishemia. n the regional ishemia model system, resistivity rose almost immediately after the oronary artery was ligated. n ontrast, after reating global ishemia in vivo, there was an average delay of approximately 20 minutes before resistivity inreased. The reason for this differene is not lear. However, it is well established that ell death during ishemia ours muh earlier during regional ishemia than during global ishemia [34]. Mehanial and eletrial ativity persists longer in the regionally ishemi myoardium ompared with the globally ishemi heart, whih arrests soon after aorti rosslamping. Thus, one hypothesis is that anaerobi metabolites aumulate even more rapidly in the "ative" regionally ishemi myoardium. An alternate hypothesis is that small amounts of ollateral perfusion of the regionally ishemi tissue bed provide additional ions that are, in turn, maldistributed in the injured region, whereas there is muh less ollateral flow in the globally ishemi heart, thereby limiting the supply of additional ions to the injured tissue. During in vitro total ishemia, ollateral flow in the ishemi slab is eliminated altogether and the time to resistivity rise is even greater than during in vivo regional or global ishemia. Thus, as ollateral perfusion of ishemi myoardium inreases, the time to onset of resistivity rise dereases. This trend may be aused by relative onentrations of maldistributed intraellular or extraellular ions. As the duration of myoardial ishemia ontinues, further deleterious and destrutive tissue hanges our. Rigor omplexes form, whih lead to onstrition of intraellular and extraellular spae. n addition, sarolemma1 defets develop, presumably ausing even more maldistribution of eletrolytes as the normal physiologial ion gradients are destroyed [8, 411. With deterioration of the sarolemma, the previously disrete intraellular and extraellular ompartments are dissolved into one unified onduting domain [42,43]. These proesses may also ontribute to the multipliity of fators ausing ontinued inreases in resistivity seen later in the ishemi period. A further understanding of the pathophysiology of ishemi injury must be aquired before the exat mehanism ausing resistivity hanges an be determined.

10 596 The Annals of Thorai Surgery Vol 44 No 6 Deember 1987 n omparison to myoardial resistivity, intramyoardial gastension measurements and ph determinations have reently been used for online evaluation of ishemi injury. A rise in intramural arbon dioxide tension during regional ishemia has been shown to orrespond to redued loal blood flow and histologial signs of injury [9]. n human studies, dereased aumulation of intramyoardial arbon dioxide during global ishemia with hypothermi ardioplegia signifiantly predited less postoperative left ventriular dysfuntion [lo]. However, determination of intramyoardial arbon dioxide involves use of an expensive mass spetrometer with a long alibration time (90 minutes) and a relatively slow turnaround time (approximately 3 minutes) [9]. On the other hand, the tehnique of determining myoardial resistivity as desribed in this study is inexpensive to implement and simple to alibrate, and provides instantaneous results. Measurement of intramyoardial ph using small glass eletrodes has been shown to reflet the ondition of myoardium during ishemia [ll, 121. During human ardia operations involving systemi hypothermia and old ardioplegia, Khuri and assoiates [14] demonstrated that reperfusion aidosis ould be identified and that lower intramyoardial ph was assoiated with depressed preoperative ardia funtion and inreased operative mortality. However, a subutaneous eletrode must be plaed in the patient s arm to omplete the ph measurement iruit, thereby ompliating the methodology [13]. n addition, ph measurements may be sensitive to eletrode movement, eletrode positioning, and the position of the referene eletrode. The ph values must be orreted for alterations in temperature and are affeted by infusion of ardioplegi solution. These fators make interpretation of ph data diffiult and the definition of ritial aidosis, unlear The urrent methods for determining myoardial gas tensions or ph reflet only loal tissue hanges in the immediate viinity of the measurement probes and may be primarily sensitive to extraellular onditions alone (9,111. n ontrast, myoardial resistivity, as determined by the tehnique used in this study, is thought to reflet umulative tissue hanges from intraellular, extraellular, loal, and distant sites [43]. Sine the intereletrode spaing of the resistivity array is relatively wide, the injeted urrent penetrates a wide area of myoardium [32]. The resultant eletrial potential that is sensed represents a summation of interations between the eletrial field and a large area of myoardium. Therefore, resistivity may be a more enompassing or omprehensive representation of omplex ishemi tissue hanges than either ph or gas tension, thus making resistivity a more aurate indiator of injury. Before online resistivity determination an be applied to linial ardia operations, a number of other variables must be haraterized. The effets of temperature and high potassium ardioplegia on resistivity must be determined. Preliminary studies in this laboratory sug gest that myoardial resistivity hanges approximately 5 to 10% per 10 C hange in myoardial temperature. Therefore, it will be neessary to relate temperature to the interpretation of resistivity hanges in hypothermi hearts. n the future, in addition to ardia monitoring, this methodology may also be applied to determination of tissue viability in organ transplantation [M. t may be possible to relate tissue viability to alterations in tissue resistivity to identify in online fashion inadequate tissue preservation during organ prourement and transplantation. Supported by Grants HL 32086, HL 17678, and HL from the National nstitutes of Health. Referenes 1. Kirklin JW, Conti VR, Blakstone EH: Prevention of myoardial damage during ardia operations. N Engl J Med 301:135, Chiu RCJ, Blundell PE, Sott HJ, Cain S The importane of monitoring intramyoardial temperature during hypothermi myoardial protetion. Ann Thora Surg 28:317, Trump BF, Mergner WJ, Kahng MW, Saladino AJ: Studies on the subellular pathophysiology of ishemia. Cirulation 53:Suppl 1:17, Jennings JB: Symposium on the prehospital phase of aute myoardial infartion: 11. Early phase of myoardial ishemi injury and infartion. Am J Cardiol24:753, Jennings JB: Cell volume regulation in aute myoardial ishemi injury. Ata Med Sand [Suppl] 58783, Whalen DA, Hamilton DG, Ganote CE, Jennings RB: Effets of a transient period of ishemia on myoardial ells:. Effets of ell volume regulation. Am J Pathol 74:381, Jennings RB, Ganote CE Strutural hanges in myoardium during aute ishemia. Cir Res 35Suppl3156, Jennings RB, Hawkins HK, Lowe JE, et al: Relation between high energy phosphate and lethal injury in myoardial ishemia in the dog. Am J Pathol92:187, Khuri SF, Kloner RA, Hillis LD, et al: ntramural PC02: reliable index of the severity of myoardial ishemi injury. Am J Physiol237H253, Magovem GJ, Flaherty JT, Kanter KR, et al: Assessment of myoardial protetion during global ishemia with myoardial gas tension monitoring. Surgery 92373, Walters FJM, Wilson GJ, Steward DJ, et al: ntramyoardial ph as an index of myoardial metabolism during ardia surgery. J Thora Cardiovas Surg 78:319, Lange R, Kloner RA, Zierler M, et al: Time ourse of ishemi alterations during normothenni and hypothermi arrest and its refletion by online monitoring of tissue ph. J Thora Cardiovas Surg 86:418, Khuri SF, Marston W, Josa M, et al: First report of intramyoardial ph in man:. Methodology and initial results. 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11 597 Ellenby et al: Myoardial Eletrial mpedane as ndiator of shemi njury Onset 16. Galeotti G: LJber die eletrishe Leitfahigkeit der tierishen Gewebe. 2 Biol25:289, Crile GW, Hosmer HR, Rowland AF: The eletrial ondutivity of animal tissues under normal and pathologial onditions. Am J Physiol 60:59, Hemingway A, Collins DA High and low frequeny eletrial resistane hanges in dying voluntary musle of rabbits. Am J Physiol99:338, Shwan HP, Kay CF Speifi resistane of body tissue. Cir Res 4664, Shwan HP, Kay CF: The ondutivity of living tissues. Ann NY Aad Si 65:1007, Burger HC, van Dongen R Speifi eletri resistane of body tissues. Phys Med Biol5431, Rush S, Abildskov JA, MFee R: Resistivity of body tissues at low frequenies. Cir Res 1240, Geddes LA, Baker LE: The speifi resistane of biologial material a ompendium of data for the biomedial engineer and physiologist. Med Biol Eng 5:271, Weidmann S: Eletrial onstants of trabeular musle from mammalian heart. J Physiol 210:1041, Cler L: Diretional differenes of impulse spread in trabeular musle from mammalian heart. J Physiol255335, Roberts DE, Sher AM: Effet of tissue anisotropy on extraellular potential fields in anine myoardium in situ. Cir Res 5092, Childers RW, Cope T, Lyon R, Holland R Tissue resistane in ventriular ishemia (abstrat). Cirulation 59Suppl 2:110, van Oosterom A, de Boer RW, van Dam RT ntramural resistivity of ardia tissue. Med Biol Eng Comput 17337, Gamdo H, Sueiro J, Rivas J, et a1 Bioeletrial tissue resistane during various methods of myoardial preservation. Ann Thora Surg 36:143, Veronee CD, Lewis WR, Takla MW, et al: Effets of beta blokade on ishemi myoardial metabolism. Surg Forum 36:283, Honer RA, Fishbein MC, Braunwald E, Maroko PR Effet of propranolol on mitohondria1 morphology during aute myoardial ishemia. Am J Cardiol41:880, Robillard PN, Poussart D: Spatial resolution of foureletrode array. EEE Trans Biomed Eng 26465, Weast RC (ed): Handbook of Chemistry and Physis. Boa Raton, FL, CRC Press, 1984, pp Lowe JE, Cummings RG, Adams DH, HullRyde EA: Evidene that ishemi ell death begins in the subendoardim independent of variations in ollateral flow or wall tension. Cirulation 68:190, HullRyde EA, Lewis WR, Veronee CD, Lowe JE: Simple step gradient elution of the major high energy ompounds and their metabolites in ardia musle using high perfomane liquid hromatography. J Chromatogr , Lowe JE, Reimer KA, Jennings RB: Experimental infart size as a funtion of the amount of myoardium at risk. Am J Pathol90363, Anderson GJ, Reiser J, Gough WB, Nydegger CC: ntramyoardial urrent flow in aute oronary olusion in the anine heart. J Am Coll Cardiol1:436, Roberts DE, Hersh LT, Sher AM: nfluene of ardia fiber orientation on wavefront voltage, ondution veloity, and tissue resistivity in the dog. Cir Res 44:701, Lowe JE, Jennings RB, Reimer KA: Cardia rigor mortis in dogs. J Mol Cell Cardiol 11:1017, Jennings RB, Reimer KA, Hill ML, Mayer SE Total ishemia in dog hearts, in vitro: 1. Comparison of high energy phosphate prodution, utilization, and depletion, and of adenine nuleotide atabolism in total ishemia in vitro vs. severe ishemia in vim. Cir Res 49:892, Reimer KA, Jennings RB, Hill ML: Total ishemia in dog hearts, in vitro: 2. High energy phosphate depletion and assoiated defets in energy metabolism, ell volume regulation, and sarolemmal integrity. Cir Res 49:901, Plonsey R, Barr R The foureletrode resistivity tehnique as applied to ardia musle. EEE Trans Biomed Eng 29541, Sperelakis N, Hoshiko T: Eletrial impedane of ardia musle. Cir Res 9:1280, Wojik S, Lambotte L: Extraellular spae determination by eletrial impedane measurement: appliation in organ preservation. n Digest of the 11th nternational Conferene on Medial and Biologial Engineering. Ottawa, Canada, Aug 26, 1976, pp