Radiotracers for imaging electroporation

Radiotracers for Imaging Electroporationa

K. L. MATTHEWS II,b J. N. AARSVOLD,c R. A. MINTZER,d C-T. CHEN,d

M. CAPELLI-SCHELLPFEFFER,e M. COOPER,d AND R. C. LEEe

bDepartment of Medical Physics, Rush–Presbyterian–St. Luke’s Medical Center,Chicago, Illinois 60612

cDepartment of Radiology, Emory University, and Nuclear Medicine Service, VeteransAffairs Medical Center, Atlanta, Georgia 30033

dDepartment of RadiologyeDepartment of Surgery, University of Chicago, Chicago, Illinois 60637

INTRODUCTION

Radiotracer imaging can be used to assess the extent of soft tissue damage in vic-tims of electrical trauma; for over 20 years, this has been done.1–5 Radiotracer imag-ing can also be used with in vivo animal models to develop methods for the investiga-tion of electrical injury and for the assessment of therapeutic agents for electrical in-juries. We have applied radiotracer imaging techniques involving a high-resolutionsmall-field-of-view gamma camera to the in vivo study of electrical injury. Specifi-cally, we have used radiotracer imaging and an animal model of electroporation in-jury in skeletal muscle to examine the effects of novel therapies for the treatment ofthe electroporation component of electrical injury. Detailed here is our investigationof four radiotracers that might be used for assessment of such therapies.

ELECTRICAL INJURY

Since the advent of electrical devices and the concurrent development of powerdistribution systems, the number of electrical injuries per year has been a measurablecomponent of the number of accidentally sustained injuries per year.6 Prior to the latenineteenth century, lightning strikes were the only significant cause of electrical in-jury. After Edison’s nineteenth-century invention of electric light, the demand forelectrical devices increased rapidly, resulting in the twentieth century in the ubiqui-tous presence of electrical power and the common occurrence of electrical accidents.

In an electrical accident, a voltage-carrying conductor is shorted to ground poten-tial. The object causing the short could be almost anything—a tree limb, a metal pole,a human body. The severity of a resultant injury is related to the current involved. Atcurrents of a fraction of a milliampere (mA), electric shock may cause muscle twitchand the perception of mild pain; the level of pain increases rapidly with increasingcurrent. Above a few milliamperes, muscle tetanus and severe pain can prevent a vic-

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aSupport for this work was provided by the Department of Energy through Grant No. DE-FG02-86ER60418 and by the Electrical Power Research Institute.

tim from releasing the conductor or calling for help. Respiratory muscle tetanus oc-curs above 10 mA. At progressively higher currents, cardiac excitation, fibrillation,and defibrillation can and likely will occur.7

Electric shock can damage any tissue or organ system in the body. Nerves andskeletal muscles are particularly susceptible to electrical injury as extreme voltagegradients and low resistance can result in a significant current along the long cells ofthese tissues. Burns and cellular disruption can also be produced. These can result di-rectly from heat generated by an electric arc or from the passage of the electric cur-rent over and through the body; they can also result indirectly from heat generated bydebris such as metal fragments or clothing that are heated or ignited as a result of anelectrical shock.6 Electrical accidents can also produce neuropsychological disorders.Such sequelae may be the result of physical trauma to the nervous system or the re-sult of emotional trauma caused by the accident.8

The context of this paper is the study of and the search for therapies for the cellu-lar disruption injury often produced in electrical trauma.

Mechanisms of Electrical Injury

Massive trauma can result from high voltage electrical shock whether producedby lightning or power-frequency currents.9–12 Such trauma often occurs in skeletalmuscles and nerves of the extremities as the current path during shock almost alwaysincludes such tissues. Historically, damage from high voltage shock has been thoughtto be a direct result of and entirely produced by the joule heating of tissue as an elec-trical current passes through it. In this scenario, heating causes macromolecules todeform and become unable to perform their cellular functions; these deformationscan disrupt metabolism and compromise the structural integrity of the cell, possiblyresulting in the death of the cell.

Nerve cells typically have low ohmic resistance. This decreases the likelihood ofjoule heating during the passage of an electrical current. However, such an observa-tion is inconsistent with the facts that nerve cells are often damaged in electricalshock and that they seem particularly susceptible to electrical injury. It is not uncom-mon, for example, for electrical shock victims to present with extensive nerve anddeep muscle damage, simultaneous with minimal signs of thermal injury at the sitesof contact.13 This suggests the presence of a mechanism of damage other than jouleheating.14 We will return to this notion shortly.

A variety of therapeutic approaches are applied to the management of electricalinjuries.15 On the macroscopic level, fluid resuscitation, metabolic and respiratorysupport, surgical debridement, and skin grafting are all common practices, and newtherapeutic methods, such as immunologic therapy and artificial skin application, arebeing researched and tested in clinical trials. On the microscopic level, basic researchinto the mechanisms of electrical injury is providing new insights into cellular-levelapproaches for limiting and repairing damage.16

The traditionally accepted mechanism of tissue damage in electrical trauma isjoule heating; a second mechanism, electroporation, has recently been identified.17,18

The basics of electroporation are as follows. During electric shock, field gradientsforce polar water molecules into molecular-scale defects in the cellular membrane;

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the result is enlargement of the defects such that they become unregulated pores.19 Inelectroporated tissues, ruptured cellular membranes disrupt normal metabolic func-tion. If the membranes are not repaired, the cells will die. If a cell’s membrane can bepatched, the cell may be able to resume metabolic function, repair itself, and thus sur-vive the injury. Poloxamers, a class of surfactants, show promise as suitable patchesfor pores in ruptured membranes.

Poloxamers are nontoxic nonionic surface-active polymers with chemical struc-tures of the form, H(OCH2CH2)a (OCHCH3CH2)b (OCH2CH2)a OH.20 For a specificpoloxamer, the ratio of hydrophilic ethylene oxide units (a subscript) to hydrophobicpropylene oxide units (b subscript) is an indicator of various physical properties ofthat polymer. The structure of two hydrophilic strands joined by a central hydropho-bic region is similar to the geometry of the phospholipids that make up cellular mem-branes. Recent research results indicate poloxamers may be able to associate withdamaged membranes, coat exposed hydrophobic regions of the membrane, and thusplug water-enlarged pores.21,22 Shielded from degrading influences, an electroporat-ed cell might be able to repair its membrane damage and resume normal function. Aspart of a project to investigate the use of poloxamers to treat electroporation injury, aprotocol has been developed for in vivo radiotracer imaging of electroporated ratskeletal muscle. Specifics of that protocol will be discussed shortly.

Radiotracer Imaging of Electrical Injury

Diagnostic strategies for assessment of soft tissue injury include visual assess-ment, histological and blood chemistry analysis, and radiological imaging. Radio-tracer imaging has been particularly useful for assessment of injured soft tissue.23–29

A number of tracers, particularly 99mTc-labeled phosphonates and 99mTc-labeled py-rophosphate (PYP), accumulate in damaged soft tissue.30–35 The degree of tracer ac-cumulation is often a good index of the extent of tissue damage.36,37

Radiotracer imaging has been used for more than 20 years to assess the extent ofdamaged tissue in electrical injury victims.1–5 Often, radiotracer imaging can delin-eate damaged regions from viable tissue. In many cases, areas of abnormal tracer up-take may not appear visually necrotic until hours or days after the injury. Thus, the re-sults of radiotracer imaging can assist significantly in the planning of surgical andtherapeutic management of an injury.38 A variety of radioisotopes may prove usefulfor imaging of electrical injury. This paper describes our assessment of four radio-tracers that might be considered for use with an in vivo model for the study of elec-troporation and therapies for electroporation.

IN VIVO MODEL FOR STUDYING ELECTROPORATION

An animal model has been developed for studying electroporation in vivo.39–42

Experimental validation of the model has been performed and the model has beenused to demonstrate that electroporation can occur in vivo and that it can occur in theabsence of thermal injury.41 Radiotracer imaging combined with the animal modelhas been used to investigate in vivo the effects of a poloxamer therapy on electropo-

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rated skeletal muscle.43 The methods used to identify appropriate and potential radio-tracers for related investigations are the focus of this paper.

A detailed description of the animal model has been given elsewhere.41,43 We pro-vide only a brief description here. The centerpiece of the model is an anesthetized fe-male Sprague-Dawley rat. A jugular cannula is used to introduce directly into thebloodstream of the animal the radiotracer, saline, and any therapeutic agents beinginvestigated. The animal’s hind limb is shocked from the ankle to the base of the tailwith a pulse sequence designed to produce electroporation damage without thermalinjury; validation of the model has shown that electroporation without thermal injuryis achieved.41 All experiments conducted in this research were conducted in accor-dance with institutional-approved animal-care protocols.

To produce electroporation damage, the hind limb of the anesthetized animal isshocked using electrical pulses from a current-regulated high voltage dc power sup-ply. The power supply produces sufficient voltage to deliver a field strength of 150V/cm at a constant current of 1.85 A. A pulse width of 4 ms, equivalent to a 60-Hzsine wave at its root-mean-square (rms) amplitude, is used; this width was chosen be-cause most power distribution systems operate at 60 Hz. Over a 2-min span, 12 puls-es are applied from the ankle to the base of the tail; the 10-s interval between pulsesallows tissues in the limb to dissipate heat, thus producing minimal heat accumula-tion in the limb.

The experimental protocol for investigating poloxamers and other compoundscalls for the investigative drug to be injected through the jugular cannula at 10 min-utes after shock. In control experiments, saline is injected instead.

RADIOTRACER IMAGING AND THE ELECTROPORATION MODEL

In our protocol, radiotracer (e.g., 99mTc PYP) is injected through the jugular can-nula at 30 minutes postshock and serial posterior-view images of the shocked hindlimb are acquired with a high-resolution small-field-of-view (FOV) gamma camera(approximately 2.5 mm full-width-at-half-maximum resolution over a 75 mm × 75mm FOV).43,44 The acquisition time for each image is 2 minutes, with a new imagestarting every 2.4 minutes. The imaging phase extends for 3.5 hours postshock. Re-gion-of-interest (ROI) analysis, using a 5.25 mm × 6.0 mm region positioned over themuscle of the lower limb, is used to compute a time-activity curve (TAC) for each ex-periment. A TAC is a one-dimensional plot of measured radioactivity in an ROI ver-sus elapsed time. In the analysis of data, TACs from individual experiments are aver-aged to yield a mean curve for a data set; the numbers of experiments contributing toeach mean curve vary.

For the results presented below, the number of TACs used to compute each meancurve is noted on the legend of the corresponding figure; bars representing standarderrors are also included. In experiments in which shocks were applied, a direct cur-rent of 1.85 A was applied unless otherwise noted. Either 0.4 cc of isotonic saline or17 mg of poloxamer-188 in 0.4 cc saline was the investigative agent. Each graph in-cludes TACs for unshocked saline-treated and shocked saline-treated animals. Thesehave been included to facilitate comparisons of the various TACs.

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Candidate Radiotracers

We considered four radiotracers for the imaging of rat hind limbs in our in vivoelectroporation model. The radiotracers that we considered were 99mTc pyrophos-phate (PYP), 99mTc diethylenetriamine pentaacetate (DTPA), 99mTc hexakis 2-methoxyisobutyl isonitrile (MIBI), and 201Tl thallous chloride. Each tracer has bio-logical characteristics that make it potentially useful for imaging electroporation in-jury. For the studies involving the 99mTc tracers, an activity of 1.0 ± 0.2 mCi in 0.4 ccsaline was used; for the studies involving 201Tl thallous chloride, 0.5 ± 0.1 mCi in 0.4cc saline was used.

Technetium-99m PYP is a tracer routinely used for assessment of soft tissue in-jury. It is known to accumulate in damaged soft tissue, clears moderately quicklyfrom undamaged soft tissue, and accumulates over time in bone. The mechanism of99mTc PYP accumulation in damaged soft tissue is not well understood; it is believedthat PYP follows calcium in cellular function.45–47 The exact structure of 99mTc PYPis not known. PYP forms a complex with technetium in the presence of a tin catalyst,but the number of PYP molecules per technetium atom has never been determined.Estimates range from one to five PYP molecules per technetium atom; the numbermay not be fixed at a specific value.

Technetium-99m MIBI, also known as sestamibi, is a cationic lipophilic complex.It was developed as a myocardial imaging tracer. Uptake and retention of 99mTc MIBIare dependent on mitochondrial and plasma membrane potentials.48–50 One wouldexpect that MIBI would not accumulate in electroporated cells that cannot maintain atransmembrane potential. In myocardial imaging using MIBI, for example, nonviabletissues present as photopenic areas (cold spots). As noted in the results and discus-sion below, this expected behavior was not seen.

Thallium-201 thallous chloride is another tracer commonly used for cardiac imag-ing. In a cell, thallium mimics the behavior of potassium. Potassium and sodium areintegral components of the cellular machinery involved in the maintenance of energystores and transmembrane potentials. Thallium is pumped actively along with potas-sium into normally functioning cells; diffusion out of normally functioning cells oc-curs immediately. Thallium eventually redistributes to a steady state in plasma andtissues. The significant photon emissions of 201Tl have energies of 68 keV, 70 keV,and 80 keV. These are well below the photopeak energy (140 keV) of 99mTc. Thissuggests that imaging with a 99mTc-labeled tracer could follow imaging with a 201Tl-labeled tracer; such a serial imaging protocol may be useful for monitoring theprogress of novel therapies used in the clinical management of electrical trauma pa-tients.

Technetium-99m DTPA has been investigated for use in electroporation studiesbecause it may be useful in combination with another 99mTc tracer for serial dual-tracer protocols. Its attractive feature is its relatively quick washout from the bloodpool. Technetium-99m DTPA is a perfusion-imaging agent. It is used primarily forrenal imaging because the kidneys readily filter DTPA. The resultant short biologicalhalf-life of DTPA means that a second tracer can be injected in a relatively shorttime, suggesting the possibility of using one tracer postinjury/pretherapy and a sec-ond tracer postinjury/posttherapy. The main determinant for deciding to use 99mTcDTPA in this fashion is the difference between DTPA clearance times from electro-

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porated tissue and normal tissue. If electroporation results in DTPA clearance timesthat are longer than the short clearance times of normal tissue, rapid dual-tracer se-quential imaging using DTPA would likely be problematic. If the clearance times aresimilar, DTPA may be useful.

Tc-99m MIBI, Tc-99m DTPA, and Tl-201 Results

The data in FIGURE 1 show that the TAC for 99mTc MIBI in saline-treated electro-porated tissue has a peak magnitude three times greater than that of unshockedsaline-treated tissue. The data also show that treatment with poloxamer-188 has littleeffect on MIBI uptake in electroporated muscle. As noted previously, the expectationwas that MIBI would show less accumulation in electroporated tissue than in un-shocked tissue because cells with electroporated membranes are not capable of main-taining the transmembrane potentials that are associated with MIBI accumulation.The fact that MIBI accumulation is greater in electroporated tissue than in unshockedtissue means that failure of transmembrane potentials is not the only MIBI-relatedmechanism altered by electrical shock.

Edema and swelling are common responses of the body to many types of trauma.When a limb receives an electrical shock, swelling of the limb may be seen. Fluid,white blood cells, and other material enter the damaged area as a response to the trau-ma of electrical injury. MIBI uptake in edematous material may be the cause of the

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FIGURE 1. Average 99mTc MIBI TACs for unshocked, saline-treated; shocked, saline-treated;and shocked, poloxamer-188-treated muscle. Applied current in all shock studies was 1.85 A.Therapeutic agent was injected 10 minutes postshock (solid line); tracer was injected 30 min-utes postshock (dashed line). Error bars represent standard error.

greater-than-expected uptake observed in electroporated tissue in these experiments.In the assessment of cardiac function, radiotracer imaging is usually not performedimmediately after the occurrence of an infarct or ischemic episode. By the timeimaging occurs, all initial damage responses of the body have subsided. As this is thecase, the use of 99mTc MIBI allows the visualization of reduced uptake in nonviabletissue. In a similar fashion, MIBI imaging of electroporation may be more successfulwhen MIBI is used for delayed assessment of injury. Investigation of this idea wouldrequire keeping experimental animals fully anesthetized or on pain-numbing drugsuntil swelling from injury has dissipated. This period might be 24 hours or more; aninvestigation of this form has not yet been conducted.

Time-activity curves for 99mTc DTPA imaging with the in vivo electroporationmodel are shown in FIGURE 2. The data show that 99mTc DTPA is taken up more read-ily in electroporated muscle than in unshocked muscle. Additionally, the data showthat the uptake and washout kinetics for the two tissue conditions are different. Forunshocked muscle, peak uptake of 99mTc DTPA occurs during the acquisition of thefirst 2-min image. Average peak uptake in saline-treated electroporated muscle wasdelayed by approximately 13 minutes under the conditions of the experiment. Tracerwashout is rapid in unshocked muscle, with tracer concentration decreasing by ap-proximately one-half every 30 minutes. In electroporated muscle, washout is relative-ly slow—the half-time of 99mTc DTPA washout in this case is 210 minutes.

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FIGURE 2. Average 99mTc DTPA TACs for unshocked, saline-treated; shocked, saline-treated;and shocked, poloxamer-188-treated muscle. Applied current in all shock studies was 1.85 A.Therapeutic agent was injected 10 minutes postshock (solid line); tracer was injected 30 min-utes postshock (dashed line). Error bars represent standard error.

In electroporated muscle treated with 17 mg of poloxamer-188, uptake of DTPAis slower than in unshocked muscle. Peak uptake occurs approximately 8 minutes af-ter injection. This is somewhat faster than the uptake in saline-treated electroporatedmuscle. Because DTPA is a perfusion tracer, this could indicate that poloxamer-188is increasing perfusion in electroporated muscle. The peak magnitude for shockedpoloxamer-188-treated muscle is notably larger than for the shocked saline-treatedmuscle. While this may be the result of a poloxamer-mediated increase in perfusion,the increase in peak magnitude could also be due to swelling resulting from the acuteinjury. Poloxamer-treated electroporated muscle shows a faster washout componentthan electroporated muscle treated with saline. DTPA washes out of poloxamer-treat-ed muscle with a half-time of 130 minutes.

FIGURE 3 illustrates the results of 201Tl imaging with the in vivo electroporationmodel. Examination of the TACs shows that saline-treated unshocked muscle accu-mulates 201Tl slowly. In shocked muscle treatment with saline, the initial uptake ismore rapid than in unshocked muscle and the magnitude of uptake is greater by afactor of six over the initial magnitude of uptake of saline-treated unshocked muscle.Four hours postshock, the magnitude is three times greater in shocked muscle than inunshocked muscle. For electroporated muscle treated with 17 mg of poloxamer-188,201Tl uptake at all times is approximately twice as large as the uptake of unshockedmuscle.

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FIGURE 3. Average 201Tl TACs for unshocked, saline-treated; shocked, saline-treated; andshocked, poloxamer-188-treated muscle. Applied current in all shock studies was 1.85 A. Ther-apeutic agent was injected 10 minutes postshock (solid line); tracer was injected 30 minutespostshock (dashed line). Error bars represent standard error.

Tc-99m PYP Results

Technetium-99m PYP is known to accumulate in damaged soft tissue, and experi-mental validation of the in vivo electroporation model confirmed that 99mTc PYP ac-cumulates in electroporated tissue.41 FIGURE 4 shows that the peak magnitudes ofTACs for shocked saline-treated muscle are related monotonically to the current usedto produce electroporation. This result indicates that 99mTc PYP can serve as an indexto the extent of electroporation injury. Higher uptake indicates more damage, andlower uptake indicates less damage. This provides a possible means for in vivo as-sessment of the effect of poloxamer-188 on electroporated muscle. If membranedamage is being reduced by poloxamer, the uptake of PYP should be less than whenpoloxamer is not administered; however, other confounding factors such as edemacould make such an assessment difficult to perform.

Shown in FIGURE 5 are average TACs for 99mTc PYP in unshocked saline-treated,shocked saline-treated, and shocked poloxamer-treated muscle. It is immediately ap-parent that the magnitude of PYP uptake in electroporated muscle is substantiallyhigher than in unshocked muscle. Additionally, while the temporal behaviors of PYPuptake are similar for saline-treated electroporated and saline-treated unshockedmuscle, the temporal behavior of electroporated muscle that has been treated withpoloxamer-188 appears different from the other two cases. The relatively constantvalue of the TAC of PYP in electroporated poloxamer-treated muscle contrastssharply with the exponential shape of the TAC of PYP in saline-treated muscle. Apertinent question here is as follows: “What causes the kinetics of PYP in muscle

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FIGURE 4. Average 99mTc PYP TACs for shocked, saline-treated muscle. Plotted are TACsfor several values of applied current. Saline was injected 10 minutes postshock (solid line);tracer was injected 30 minutes postshock (dashed line). Error bars represent standard error.

treated with 17 mg of poloxamer-188 to be different from the kinetics of saline-treat-ed muscle?”

A possible explanation for the observed difference in 99mTc PYP uptake inpoloxamer-treated muscle as opposed to saline-treated muscle is that poloxamer al-ters PYP kinetics through direct interaction with the molecular structures of PYP.To rule this out, a set of poloxamer-treated unshocked experiments were conducted.FIGURE 6 includes the average TAC for unshocked muscle treated with 17 mg ofpoloxamer-188. The TAC magnitude is marginally higher at all times than that forunshocked muscle treated with saline. This slight difference, possibly the result ofa poloxamer-mediated increase in perfusion, must be taken into account whendrawing conclusions about the effects of poloxamer in electroporated muscle. How-ever, the increase is small relative to the increase seen in shocked muscle. This in-dicates that the observed differences in PYP uptake are probably not the result ofmolecular interaction of 99mTc PYP and poloxamer.

DISCUSSION

Radiotracer imaging and our in vivo model provide tools for quantifying the ex-tent of electroporation injury and the effects of poloxamer-188 and other potentialtherapeutic agents on electroporated muscle. Of the four radiotracers examined in

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FIGURE 5. Average 99mTc PYP TACs for unshocked, saline-treated; shocked, saline-treated;and shocked, poloxamer-188-treated muscle. Poloxamer-188 treatments were 17 mg or 68 mgof poloxamer-188. Applied current in all shock studies was 1.85 A. Therapeutic agent was in-jected 10 minutes postshock (solid line); tracer was injected 30 minutes postshock (dashedline). Error bars represent standard error.

this work, 99mTc PYP was the most useful. Prior to this investigation, 99mTc MIBIseemed to be a promising tracer for use in electrical injury research, but the greateraccumulation of MIBI in shocked muscle compared to unshocked muscle is contraryto the behavior that was expected based on MIBI’s behavior in cardiac tissue. MIBImay prove to be useful for nonacute assessment of tissue damage, after inflammationof a shocked muscle has subsided. However, studies to investigate this possibilityhave not been done.

Technetium-99m DTPA and 201Tl were investigated because of the possibility ofrapid sequential studies to assess the extent of damage both before and after treat-ment with poloxamer. The lengthened washout of 99mTc DTPA in electroporatedmuscle relative to unshocked muscle, whether treated with poloxamer-188 or saline,limits the utility of using DTPA in protocols involving rapid sequential studies. An-other factor that would appear to limit the utility of DTPA for before- and after-thera-py assessments is the fact that peak uptake of DTPA in shocked muscle is greaterwith poloxamer treatment than with saline treatment. This fact makes it difficult todetermine a method for correlating tracer uptake with extent of damage.

Thallium-201 possibly provides a tool for quick assessment of the extent of dam-age prior to treatment with poloxamer—a tool that can be used in conjunction withimaging with 99mTc-labeled tracers. Because the photopeak energy of 201Tl is muchless than that of 99mTc, the presence of thallium would not interfere with subsequentimaging with technetium agents. Clinical diagnosis with a dual 201Tl/99mTc imaging

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FIGURE 6. Average 99mTc PYP TACs for unshocked, saline-treated; shocked, saline-treated;and unshocked, poloxamer-188-treated muscle. Poloxamer-188 treatment was 17 mg of polox-amer-188. Applied current in all shock studies was 1.85 A. Therapeutic agent was injected 10minutes postshock (solid line); tracer was injected 30 minutes postshock (dashed line). Errorbars represent standard error.

protocol would require the development of a body of information to correlate extentof damage via 201Tl with extent of damage with the 99mTc agent. MIBI, PYP, and sev-eral other 99mTc tracers have been used extensively along with 201Tl in cardiac imag-ing. Data from such cardiac studies provide a starting point for acquiring the correla-tive information. However, extensive research is necessary simply to determine ifMIBI and/or Tl uptake will correlate with the extent of tissue damage. Even more re-search is necessary to develop fully a dual-tracer imaging protocol.

Because of its history as a tracer for soft tissue injury, 99mTc PYP was expected tobe a good choice for use in the electroporation research. The results presented aboveshow that PYP uptake is substantially different in electroporated muscle treated with17 mg of poloxamer-188 than in saline-treated electroporated muscle. In particular,PYP reaches a steady state level for the poloxamer case that is significantly less thanthe plateau level for the saline case. In unshocked muscle, PYP uptake is only mar-ginally higher if the muscle is treated with 17 mg of poloxamer-188 than with saline.

Each of the four tracers investigated here was chosen because of some character-istic that might be exploited in the imaging of electroporated muscle. PYP has an ex-tensive history as a tracer for soft tissue injury. MIBI uptake has been shown to de-pend on membrane potentials, a fact that is particularly relevant for studying the elec-troporation of cellular membranes. DTPA has a short biological lifetime in un-shocked tissue and is an excellent perfusion tracer. The gamma emissions of 201Tl arelow enough so as not to interfere with 99mTc imaging when both tracers are present intissue. These tracers are only a few of many tracers and isotopes that are potentiallyuseful for imaging electroporation injury.

Xenon-133 and 18F-labeled fluorodeoxyglucose (FDG) are two tracers that werebriefly considered for this research. Xenon-133 was rejected because of its long half-life, the potential hazard of releasing 133Xe gas into the air, and difficulties in achiev-ing a specific activity of aqueous 133Xe high enough to calibrate the high-resolutiongamma camera used in these investigations.43,44 FDG is expensive to produce, requir-ing a cyclotron and a dedicated radiosynthesis apparatus. Access to a dedicatedsmall-animal positron emission tomography (PET) imager, coupled with the recentdevelopment of regional sites for FDG synthesis and distribution, might justify pur-suit of laboratory in vivo FDG imaging of electroporated muscle. FDG has been usedclinically for assessing skeletal muscle viability,51 but its use for electrical injury pa-tients would require overcoming the difficulties of performing coincidence imagingin an intensive care unit. Such difficulties will not be easy to address.

Two other tracers that might be useful for both research and clinical investigationsare radiolabeled dextran52 and radiolabeled poloxamer. Dextran is commonly used asa plasma expander in electrical injury patients. Radiolabeled poloxamer is especiallyattractive for investigating the interaction of poloxamer with electroporated mem-branes. Development of a 99mTc-labeled poloxamer would be especially fortuitous,but the feasibility of this is uncertain.

CONCLUSIONS

Technetium-99m PYP, among the four tracers studied, was found to be the mostuseful for imaging of electroporation in an in vivo animal model. The tracer kinetics

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of 99mTc PYP in shocked poloxamer-treated muscle were significantly different fromthose in shocked saline-treated muscle—a property useful in this setting. No appre-ciable difference was seen between the 99mTc MIBI TACs for saline-treated andpoloxamer-treated shocked muscle, and 99mTc MIBI demonstrated greater uptake inshocked saline-treated muscle than in unshocked muscle. These observations werecontrary to expectations and suggest that the usefulness of MIBI is uncertain for thissetting. Dual-tracer sequential imaging of electroporation injury with 201Tl and a99mTc-labeled radiotracer may be feasible, but further investigation is needed to cor-relate 201Tl TACs to the extent of electroporation injury and to correlate 201Tl TACsto relevant TACs of 99mTc-labeled tracers. Electroporation significantly slows thewashout of 99mTc DTPA. This fact probably limits the use of 99mTc DTPA for rapidsequential imaging with a second 99mTc tracer such as 99mTc PYP. However, the factthat DTPA has altered kinetics in the presence of electroporation resulting from elec-trical trauma suggests that further investigation of its properties in this setting may beuseful.

REFERENCES

1. CHANG, L. Y. & J. Y. YANG. 1991. The role of bone scans in electric burns. Burns 17(3):250–253.

2. LEWIS, S. E., J. L. HUNT, C. BAXTER, D. C. HICKEY & R. W. PARKEY. 1979. Identification ofmuscle damage in acute electrical burns with technetium-99m pyrophosphate (Tc-99m-PYP) scintigraphy. J. Nucl. Med. 20(6): 646–647.

3. SAYMAN, H. B., I. URGANCIOGLU, I. USLU & T. KAPICIOGLU. 1992. Prediction of muscle via-bility after electrical burn necrosis. Clin. Nucl. Med. 17: 395–396.

4. SPENCER, R. R., A. G. WILLIAMS, JR., F. A. METTLER, JR., J. H. CHRISTIE, R. D. ROSENBERG

& W. D. WEAVER. 1983. Tc-99m-PYP scanning following low voltage electrical injury.Clin. Nucl. Med. 8(12): 591–593.

5. HUNT, J. L., S. LEWIS, R. PARKEY & C. BAXTER. 1979. The use of technetium-99m stannouspyrophosphate scintigraphy to identify muscle damage in acute electric burns. J. Trauma19(6): 409–413.

6. BERNSTEIN, T. 1994. Electrical injury: electrical engineer’s perspective and an historical re-view. Ann. N.Y. Acad. Sci. 720: 1–10.

7. REILLY, J. P. 1994. Scales of reaction to electric shock. Ann. N.Y. Acad. Sci. 720: 21–37.8. KELLEY, K. M., N. PLISKIN, G. MEYER & R. C. LEE. 1994. Neuropsychiatric aspects of

electrical injury: the nature of psychiatric disturbance. Ann. N.Y. Acad. Sci. 720:213–218.

9. HAMMOND, J. S. & S. G. WARD. 1988. High-voltage electrical injuries: management andoutcome of 60 cases. South. Med. J. 81(11): 1351–1352.

10. HOLLIMAN, C. J., J. R. SAFFLE, M. KRAVITZ & G. D. WARDEN. 1982. Early surgical decom-pression in the management of electrical injuries. Am. J. Surg. 144: 733–739.

11. LEE, R. C., E. G. CRAVALHO & J. F. BURKE. 1992. Electrical Trauma: The Pathophysiology,Manifestations, and Clinical Management. Cambridge University Press. London/NewYork.

12. LEE, R. C., L. J. GOTTLIEB & T. J. KRIZEK. 1992. Pathophysiology and clinical manifesta-tions of tissue injury in electrical trauma. In Advances in Plastic and ReconstructiveSurgery. Volume 8, p. 1–29. Mosby/Year Book. St. Louis/Chicago.

13. LEE, R. C. & M. S. KOLODNEY. 1987. Electrical injury mechanisms: dynamics of the ther-mal response. Plast. Reconstr. Surg. 80(5): 663–671.

MATTHEWS et al.: RADIOTRACERS 297

14. LEE, R. C., D. C. GAYLOR, D. BHATT & D. A. ISRAEL. 1988. Role of cell membrane rupturein the pathogenesis of electrical trauma. J. Surg. Res. 44: 709–719.

15. WAYMACK, J. P. & R. L. RUTAN. 1994. Recent advances in burn care. Ann. N.Y. Acad. Sci.720: 230–238.

16. LEE, R. C., A. MYEROV & C. P. MALONEY. 1994. Promising therapy for cell membranedamage. Ann. N.Y. Acad. Sci. 720: 239–245.

17. BHATT, D. L., D. C. GAYLOR & R. C. LEE. 1990. Rhabdomyolysis due to pulsed electricfields. Plast. Reconstr. Surg. 86: 24–34.

18. LEE, R. C. & M. S. KOLODNEY. 1987. Electrical injury mechanisms: electrical breakdownof cell membranes. Plast. Reconstr. Surg. 80(5): 672–679.

19. ABIDOR, I. G., V. B. ARAKELYAN, L. V. CHERNOMORDIK, Y. A. CHIZMADZHEV, V. F. PAS-TUSHENKO & M. R. TARASEVICK. 1979. Electric breakdown of bilayer lipid membranes: I.The main experimental facts and their qualitative discussion. Bioelectrochem. Bioenerg.6: 37.

20. SCHMOLKA, I. R. 1972. Artificial skin. I. Preparation and properties of pluronic F-127 gelsfor treatment of burns. J. Biomed. Mater. Res. 6: 571–582.

21. SCHMOLKA, I. R. 1994. Physical basis for poloxamer interactions. Ann. N.Y. Acad. Sci.720: 92–97.

22. LEE, R. C., P. RIVER, F-S. PAN, L. JI & R. L. WOLLMANN. 1992. Surfactant-induced sealingof electropermeabilized skeletal muscle membranes in vivo. Proc. Natl. Acad. Sci.U.S.A. 89: 4524–4528.

23. BONTE, F. J., R. W. PARKEY, K. D. GRAHAM, J. MOORE & E. M. STOKELY. 1974. A newmethod for radionuclide imaging of myocardial infarcts. Radiology 110: 473–474.

24. BRILL, D. R. 1981. Radionuclide imaging of nonneoplastic soft tissue disorders. Semin.Nucl. Med. 11(4): 277–288.

25. WHEAT, J. 1985. Diagnostic strategies in osteomyelitis. Am. J. Med. 78(6B): 218–224.26. LABBE, R., T. LINDSAY, R. GATLEY, A. ROMASCHIN, D. MICKLE, G. WILSON, S. HOULE & P.

WALKER. 1988. Quantitation of postischemic skeletal muscle necrosis: histochemical andradioisotope techniques. J. Surg. Res. 44(1): 45–53.

27. MATIN, P. 1988. Basic principles of nuclear medicine techniques for detection and evalua-tion of trauma and sports medicine injuries. Semin. Nucl. Med. 18(2): 90–112.

28. MEHTA, R. C. & M. A. WILSON. 1989. Frostbite injury: prediction of tissue viability withtriple-phase bone scanning. Radiology 170: 511–514.

29. MALKI, A. A., A. EL-GAZZAR, T. ASHQAR, A. OWUNWANNE & H. ABDEL-DAYEM. 1992. Newtechnique for assessing muscle damage after trauma. J. R. Coll. Surg. Edinb. 37:131–133.

30. PARKEY, R. W., F. J. BONTE, L. M. BUJA, E. M. STOKELY & J. T. WILLERSON. 1977. Myocar-dial infarct imaging with technetium-99m phosphates. Semin. Nucl. Med. 7(1): 15–28.

31. PURDUE, G. F., S. LEWIS & J. L. HUNT. 1983. Pyrophosphate scanning in early frostbite in-jury. Am. J. Surg. 149: 619–620.

32. HASEMAN, M. K. & J. P. KRISS. 1985. Selective, symmetric, skeletal muscle uptake of Tc-99m pyrophosphate in rhabdomyolysis. Clin. Nucl. Med. 10(3): 180–183.

33. OWUNWANNE, A., A. MALKI, S. SADEK, A. EL-GAZZAR, T. YACOUB & H. M. ABDEL-DAYEM.1989. Investigative study of radiopharmaceuticals useful for imaging skeletal muscle in-jury in experimental animals. Am. J. Physiol. Imag. 4: 62–65.

34. DELPASSAND, E. S., R. D. DHEKNE, B. J. BARRON & W. H. MOORE. 1991. Evaluation of softtissue injury by Tc-99m bone agent scintigraphy. Clin. Nucl. Med. 16(5): 309–314.

35. CHEN, C-T., J. N. AARSVOLD, T. A. BLOCK, K. L. MATTHEWS, R. A. MINTZER, J. MUKHERJEE,N. J. YASILLO, L. P. RIVER, M. COOPER & R. C. LEE. 1994. Radionuclide probes for tissuedamage. Ann. N.Y. Acad. Sci. 720: 181–191.

ANNALS NEW YORK ACADEMY OF SCIENCES298

36. SIEGEL, B. A., W. K. ENGEL & E. C. DERRER. 1975. 99mTc-diphosphonate uptake in skele-tal muscle: a quantitative index of acute damage. Neurology 25: 1055–1058.

37. TIMMONS, J. H., M. F. HARTSHORNE, V. J. PETERS, M. A. CAWTHON & J. M. BAUMAN. 1988.Muscle necrosis in the extremities: evaluation with Tc-99m pyrophosphate scanning—aretrospective review. Radiology 167(1): 173–178.

38. HUNT, J. L., R. M. SATO & C. BAXTER. 1980. Acute electric burns: current diagnostic andtherapeutic approaches to management. Arch. Surg. 115: 434–438.

39. MATTHEWS, K. L., T. A. BLOCK, J. N. AARSVOLD, R. C. LEE, C-T. CHEN, R. A. MINTZER, M.CAPELLI-SCHELLPFEFFER & S. C. TRIPATHI. 1994. Assessment of tissue damage inducedby electrical shock using a miniature gamma camera. Med. Phys. 21(6): 932.

40. AARSVOLD, J. N., T. A. BLOCK, R. C. LEE, K. L. MATTHEWS, C-T. CHEN, M. CAPELLI-SCHELLPFEFFER, S. C. TRIPATHI, R. N. BECK, M. COOPER, R. A. MINTZER & N. J. YASILLO.1995. Small gamma cameras and in vivo animal models for development and investiga-tion of diagnostic and prognostic imaging protocols. In Unity in Diversity: 1995 AAASAnnual Meeting and Innovation Exposition Final Program, p. 139.

41. BLOCK, T. A., R. C. LEE, J. N. AARSVOLD, K. L. MATTHEWS, R. A. MINTZER, L. P. RIVER, M.CAPELLI-SCHELLPFEFFER, R. L. WOLLMANN, S. TRIPATHI & C-T. CHEN. 1995. The 1995Lindberg award: non-thermally mediated muscle injury and necrosis in electrical trauma.J. Burn Care Rehabil. 16: 581–588.

42. LEE, R. C., T. A. BLOCK, J. N. AARSVOLD, K. L. MATTHEWS, C-T. CHEN, R. A. MINTZER &M. CAPELLI-SCHELLPFEFFER. 1995. In vivo evidence of surfactant reduction of muscle in-jury in electrical shock. Fifty-sixth Annual Meeting of the Society of University Sur-geons.

43. MATTHEWS, K. L., II. 1997. Development and application of a small gamma camera. Ph.D.thesis, University of Chicago.

44. AARSVOLD, J. N., R. A. MINTZER, N. J. YASILLO, S. J. HEIMSATH, T. A. BLOCK, K. L.MATTHEWS, X. PAN, C. WU, R. N. BECK, C-T. CHEN & M. COOPER. 1994. A miniaturegamma camera. Ann. N.Y. Acad. Sci. 720: 192–205.

45. SHEN, A. C. & K. B. JENNINGS. 1972. Kinetics of calcium accumulation in acute myocar-dial ischemic injury. Am. J. Pathol. 67: 441–452.

46. JUNG, A., S. BISAZ & H. FLEISCH. 1973. The binding of pyrophosphate and two diphospho-nates by hydroxyapatite crystals. Calcif. Tissue Res. 11: 269–280.

47. LESSEM, J., P. I. POLIMENI, E. PAGE, L. RESNEKOV, P. V. HARPER & V. STARK. 1980. Accumu-lation of technetium-99m pyrophosphate in experimental infarctions in the rat. Cardio-vasc. Res. 14: 352–359.

48. BELLER, G. A. & A. J. SINUSAS. 1990. Experimental studies of the physiologic properties oftechnetium-99m isonitriles. Am. J. Cardiol. 66(13): 5E–8E.

49. PIWNICA-WORMS, D., J. F. KRONAUGE & M. L. CHIU. 1990. Uptake and retention of hexakis(2-methoxyisobutylisonitrile) technetium (I) in cultured chick myocardial cells. Circula-tion 82(5): 1826–1838.

50. CHIU, M. L., J. F. KRONAUGE & D. PIWNICA-WORMS. 1990. Effect of mitochondrial andplasma membrane potentials on accumulation of hexakis (2-methoxyisobutylisonitrile)technetium (I) in cultured mouse fibroblasts. J. Nucl. Med. 31(10): 1646–1653.

51. SMITH, G. T., T. S. WILSON, K. HUNTER, M. C. BESOZZI, K. F. HUBNER, D. B. REATH, M. H.GOLDMAN & E. BUONOCORE. 1995. Assessment of skeletal muscle viability by PET. J.Nucl. Med. 36(8): 1408–1414.

52. ABERNATHY, V. J., N. A. POU, T. L. WILSON, R. E. PARKER, S. N. MASON, J. A. CLANTON,L. J. BAUDENDISTEL & R. J. ROSELLI. 1995. Noninvasive measures of radiolabeled dextrantransport in in situ rabbit lung. J. Nucl. Med. 36(8): 1436–1441.

MATTHEWS et al.: RADIOTRACERS 299