INSTITUTE OF PHYSICS PUBLISHING PHYSICS IN MEDICINE AND BIOLOGY

Phys. Med. Biol. 49 (2004) 665–683 PII: S0031-9155(04)65486-8

Non-invasive assessment of radiation injury withelectrical impedance spectroscopy

K Sunshine Osterman1, P Jack Hoopes2, Christine DeLorenzo1,David J Gladstone3 and Keith D Paulsen1

1 Thayer School of Engineering, Dartmouth College, Hanover, NH, USA2 Department of Surgery, Dartmouth Hitchcock Medical Center, Lebanon, NH, USA3 Department of Radiation Oncology, Dartmouth Hitchcock Medical Center, Lebanon, NH, USA

E-mail: kso2003@columbia.edu

Received 30 June 2003Published 6 February 2004Online at stacks.iop.org/PMB/49/665 (DOI: 10.1088/0031-9155/49/5/002)

AbstractA detailed understanding of non-targeted normal tissue response is necessaryfor the optimization of radiation treatment plans in cancer therapy. In thisstudy, we evaluate the ability of electrical impedance spectroscopy (EIS) tonon-invasively determine and quantify the injury response in soft tissue afterhigh dose rate (HDR) irradiation, which is characterized by large localizeddose distributions possessing steep spatial gradients. The HDR after-loadingtechnique was employed to irradiate small volumes of muscle tissue withsingle doses (26–52 Gy targeted 5 mm away from the source). Impedancemeasurements were performed on 29 rats at 1 , 2 and 3 month post-irradiation,employing 31 frequencies in the 1 kHz to 1 MHz range. Over the first 3 months,conductivity increased by 48% and 26% following target doses of 52 Gy and26 Gy 5 mm from the HDR source, respectively. Injury, assessed independentlythrough a grid-based scoring method showed a quadratic dependence ondistance from source. A significant injury (50% of cells atrophied, necroticor degenerating) in 1.2% of the volume, accompanied by more diffuse injury(25% of cells atrophied, necrotic or degenerating) in 9% of the tissue produceda conductivity increase of 0.02 S m−1 (8% over a baseline of 0.24 S m−1). Thiswas not statistically significant at p = 0.01. Among treatment groups, injurydifferences in 22% of the volume led to statistically significant differencesin conductivity of 0.07 S m−1 (23% difference in conductivity). Despitelimitations, the success of EIS in detecting responses in a fraction of thetissue probed, during these early post-irradiation time-points, is encouraging.Electrical impedance spectroscopy may provide a useful metric of atrophyand the development of fibrosis secondary to radiation that could be furtherdeveloped into a low-cost imaging method for radiotherapy monitoring andassessment.

(Some figures in this article are in colour only in the electronic version)

0031-9155/04/050665+19$30.00 © 2004 IOP Publishing Ltd Printed in the UK 665

666 K S Osterman et al

1. Introduction

Radiation doses are prescribed according to a population-based understanding of tumourradiosensitivity and normal tissue tolerance. However, the statistically best treatment for apopulation may not be best for an individual. Factors influencing radiation response includedose, dose rate, radiation quality, field-size and intrinsic radiation sensitivity. The latencyperiod varies with the type of biological structure involved as well as the physiologic healthof the tissue. In the future, non-invasive physiologically based imaging techniques, suchas electrical impedance spectroscopy (EIS), may be used to measure radiation damage andradiation recovery, on a patient-by-patient basis. An accurate assessment of radiation responsecould facilitate radiation dose-escalation and create an increased cure opportunity in manypatients.

There is evidence that histologic assessment of tumour and adjacent normal tissue,during the treatment of cervical cancer, is able to provide information useful in predictingradiosensitivity of tumours and surrounding tissue (Trott 1980). It is not always possible, ordesirable, to obtain biopsy samples during treatment and recovery. Therefore, a non-invasivemeasurement of tissue, which correlates with pathology, might be used directly as a predictorof radiosensitivity and late tissue response.

On the diagnostic end, an electrical impedance system has been approved by the FDA foradjuvant use in the detection of breast cancer (T-Scan 2000, US FDA document P970033).At least two different probes have been developed for use in the detection of pre-cancer andcancer of the cervix (Brown et al 2000, Quek et al 1998). A preliminary analysis of resultsfrom 124 women (8 measurement sites per subject) indicated a sensitivity and specificity of 0.92when using an impedance probe to distinguish between healthy tissue and grade 2/3 cervicalintraepithelial neoplasia (Brown et al 2000). In the field of radiation therapy, impedancemeasurements have been used to quantify post-treatment damage to breast skin (Tamura et al1994, Nuutinen et al 1998, Lahtinen et al 1999, Pigott et al 2000, Warszawski et al 1997).However, there has been virtually no exploration of impedance change secondary to radiationin soft tissue.

Impedance spectroscopy, as a measurement technique, is adept at detecting normal tissueinjury and the development of fibrosis, a common and often serious complication of radiationtherapy. These injuries limit mobility and normal organ function and preclude the use offollow-up radiation should the tumour recur locally. We have previously shown that EIS iscapable of detecting radiation-induced response in muscle after large single uniform doses(70–150 Gy) (Paulsen et al 1999).

Briefly, electrical impedance works as follows. The electrical properties of tissueare dependent upon the structural organization of cells within the tissue. Electric chargeaccumulates at the lipid membranes, restricting current to the extracellular space at lowfrequencies and allowing current to flow through intracellular space at higher frequencies(Foster and Schwan 1989, McRae and Esrick 1996, Sasser et al 1993). The frequency atwhich this transition occurs, from current flow in extracellular space to current flow inintracellular space, is dependent upon the size of the membrane structures. Larger structurestransition at lower frequencies than smaller structures. Collagen, the dominant componentof fibrosis, is highly conductive and non-membranous, thus replacement of healthy cells andtheir complex membrane structures with fibrotic scar will result in an increased conductivity,particularly at low frequencies, and a decreased ability for charge storage (also resulting fromthe loss of cellular membranes) (Chang 1998, Osterman et al 1999).

In this study, we introduce an additional constraint into our model; the injury must bepresent in only a fraction of the tissue being probed. The use of a non-uniform dose, and

EIS detection of radiation response 667

spatially localized injury, allows us to address the important questions, ‘what size and whatmagnitude of injury can be detected with this technique?’ and ‘how different must two injuriesbe for impedance measurements to be able to differentiate one from the other?’. To this end,significant effort was also placed on spatially resolved histology. We have intentionally soughtto examine EIS spectral changes in the face of exposure gradients that occur with HDR, inpart, because HDR is becoming increasingly popular due to the reduced fractionation. One ofthe first subject populations where the EIS approach could be applied is HDR following breastconservation therapies, where cosmesis would be important and the effect of the last fraction(3rd or 4th) may separate ‘adequate’ from ‘overexposure’ vis-a-vis the appearance of un-(orless) desirable late effects.

Specifically, a small, stationary HDR source is employed to irradiate tissue to two differentdose levels. These two levels are identified as ‘26 Gy’ and ‘52 Gy’, delivered a distance 0.5 cmfrom the source. The isodose surface identified by these dose values encloses a tissue volumeof approximately 0.55 cm3. Although the dose distribution in the vicinity of this source has asteep gradient, for convenience the dose levels will be identified throughout the paper by thesingle values, 26 and 52 Gy. A single dose paradigm (instead of fractionated) was adoptedto accelerate the time course of the appearance of radiation-induced late effects. Delivery ofradiation in this manner will also increase the magnitude of the radiation damage.

Measurements are performed on 29 rats using frequencies in the 1 kHz to 1 MHzrange. Assessments are made at 1, 2 and 3 month post-irradiation. Use of a 4-electrodeimpedance probe allows detection of the soft tissue response in a manner insensitive to thehighly variable skin impedance. This probe design increases measurement robustness andsubstantially reduces inter-animal variability over prior 2-electrode data.

2. Methods and materials

2.1. Anaesthesia and animal use

All procedures and measurements were performed on anaesthetized animals (Isofluorancegas anaesthesia) in strict accordance with protocols approved by the Dartmouth CollegeInstitutional Animal Care and Use Committee (IACUC). Animals were maintained at bodytemperature by using a hot water circulating pad. Heart rate and oxygen levels are monitoredthroughout anaesthesia using a pulse oximeter. The latter two points are important due to thedependence of electrical properties on temperature and blood flow. Animals were housed andcared for in an AALAC and USDA approved facility (Animal Resources Center, DartmouthMedical School, Lebanon, NH).

2.2. Electrical impedance spectroscopy (EIS) measurements

Impedance measurements were performed on the caudal muscles of the rat rear limbs witha 4-electrode probe. The two sides of each probe were positioned parallel to one another,in opposing halves of a leg holder, and lowered into full contact with the skin surfaces suchthat the musculature of the hind leg was mildly compressed between the two electrodes. Thisis shown schematically in figure 1. Impedances were recorded at 31 logarithmically spacedfrequencies between 1 kHz and 1 MHz with a Solartron 1260 gain-phase analyser and 1294impedance unit. The 1294 is designed for true differential four-terminal measurements; thevoltage buffers have input resistances to ground of >1 G� and input capacitances of 10 pF toground (1 pF differential).

668 K S Osterman et al

Figure 1. (a) Schematic of rat leg in bottom half of an anatomically shaped positioning device.Compound electrodes are positioned on either side of the musculature. The slot in the holderguides the placement of the radioactive source. (b) The 4-electrode probe: current, I, is driventhrough the outer, annular electrodes, and voltage is sensed at the inner probes. No current flowsthrough the inner, disc electrodes.

Surface hair was removed (clipped and shaved) from the hind leg of the rat prior tomeasurement. A single tape peel (removal of surface keratin and keratinocytes) was thenperformed to reduce the skin impedance to within two orders of magnitude of the underlyingtissue. An isotonic coupling medium, NormlGel R©, was applied to the skin. The use ofthis gel provided good electrical coupling between the probe and the skin while the low ionicconcentration gradient between gel and tissue minimized impedance drift in our measurements.Positioning of the electrodes relative to the hind leg musculature was made simple andrepeatable through the use of an anatomically designed holder.

The impedance probe operates by sending a small alternating current across the tissuebetween two annular driving electrodes (12 mm outer diameter, 5 mm inner diameter). Thevoltage is detected at small sensing electrodes (2 mm diameter), and impedance is calculatedfrom the ratio of the complex voltage and driving current. The tissue sampling volume isessentially the region between the compound electrodes positioned on either side of the leg.A small fringing field causes current to flow within the central region (beneath the senseelectrodes). Thus, the measurements reported here are bulk properties of the caudal musclesbracketed by the probe.

Raw impedance data consist of a real component, resistance = Z′(�), and an imaginarycomponent, reactance = Z′′(�). These were converted to their volume independent tissueproperty equivalents by exploiting the parallel-plate geometry of our electrode-set-up where,

z = (Z′ + jZ′′) ∗ A

d(1)

A is the area under the entire electrode unit, d is the average separation between the electrodes—an approximation of the muscle thickness—and lower-case z is used to indicate theimpedivity or volume independent impedance. This volume independent property, impedivity,will be referred to, for simplicity, as impedance in the rest of this paper. Muscle is highlyanisotropic and all measurements were made transverse to the muscle fibre’s primary axis(Epstein and Foster 1983, Aaron et al 1997).

2.3. Irradiation

A high dose rate (Varian) afterloader was used to irradiate the gastrocnemius and soleusmuscles of the right hind leg of twenty-nine male, Sprague-Dawley rats. The rats were

EIS detection of radiation response 669

divided into two dose groups (26 and 52 Gy) at a target distance of 5 mm from the centreof an 192Ir source (two 2.5 mm long, 0.59 mm diameter seeds encased in a nitonol wire).Treatments were designed (BrachyVision, Varian Medical Systems, Palo Alto, CA) using aCT image volume (fifty-two 1 mm slices) of the leg positioned in the anatomical holder. A slotin the impedance holder (figure 1(a)) acted as a guide during source placement through a smalldiameter (21-gauge) closed-end metal needle, which delivered the radiation with minimalmechanical damage using standard HDR technique. A needle of the same size was insertedinto the opposite leg as a control in ten animals. Dwell times ranged from 83 s to 330 sdepending on target dose and the age of the source. Damage along the needle track wasnegligible or completely absent in histological analysis.

In the studies completed here, we have used a single HDR exposure, which is large butin the same general range (in terms of total dose and dose rate) as fractionated (typically 3–4fractions) clinical treatments with HDR (dose is often specified at distances of 1–2 cm, insteadof the 0.5 cm used here). This technique was chosen in order to accelerate the onset of thetype of late radiation effects observed in humans. Although not delivered conventionally (i.e.in fractions) morphological findings reveal radiation effects similar to late effects which occurin humans.

2.4. Histology: preparation

Five animals from every dose group were measured and sacrificed at each of three time points(1, 2 and 3 month post-irradiation). This enabled histological assessment of injury for everyEIS measurement session. Following sacrifice, the animals were perfusion-fixed, through thedescending aorta with 50 cc of 4% buffered paraformaldehyde, in the same anatomical positionused during EIS measurement. Positioning consistency was important to ensure accurate co-registration of radiation treatment, EIS and assessment of tissue morphology/damage. Theleg was then removed and emersion fixed overnight in 4% neutral buffered formaldehyde,1% gluteraldehyde for optimal fixation prior to trimming and histologic processing. Fulltissue cross-sections were made perpendicular to the muscle fibres’ main axis (and parallelto the direction of current flow during impedance measurements). Histologic sections wereparaffin embedded, cut at 5 µm and stained with hemotoxalin and eosin (H&E) and Masson’strichrome stains.

2.5. Histology: quantification and scoring of injury

A histologic grid-point scheme was developed to accurately assess tissue composition andchange. This grid-point scoring system allowed us to estimate the type and severity of injury,as well as its spatial distribution.

All histological assessment was performed on a cross-section, which both bisected thecentral region of the probe and passed through the seed placement site. A grid with twentyevenly spaced points was placed in the eyepiece of the microscope, and the tissue at eachcrosshair was placed into one of the five categories: (1) normal muscle, (2) necrotic ordegenerating muscle, (3) muscle atrophy with inflammation, (4) muscle atrophy withoutinflammation or (5) connective tissue or fibrosis. This measurement set-up is shown infigure 2. Grid points landing on fixation artefacts or tissue boundaries were excluded from theanalysis.

Two histological studies were performed. First, the tissue residing beneath the probewas analysed using 18 systematically placed fields at a magnification of 40× (360 points of

670 K S Osterman et al

Figure 2. Histology measurement field. Scoring grid superimposed on irradiated muscle withsigns of atrophy, interstitial thickening and fibroplasia. Actual grid resides in microscope eyepiece.Hemotoxalin and eosin stain.

assessment). This amounted to an approximate coverage area of 1.2 cm (electrode diameter)× 1.5 cm (average leg thickness and electrode separation).

A second, and separate study was initiated to explore the spatial distribution of injury.This was performed at a higher magnification of 100×, independently evaluating the tissuein four dose zones. The region delineations were made at isodose levels of (110, 45 and20 Gy) or (180, 90 and 45 Gy), for target doses of 26 Gy and 52 Gy, respectively (figure 3).The highest dose zone (region 1—source location) was comprehensively assessed by threefields, which covered the zone. Six fields were evaluated in region 2 (doses >110 and>180 Gy, respectively), while ten randomly placed fields were assessed in the much largerareas of regions 3 and 4. The approximate volumes circumscribed, from smallest to largest,were 0.02 cm3, 0.15 cm3, 0.4 cm3 and 0.9 cm3. The volume enclosed by the prescription dosewas approximately 0.55 cm3.

Current only flows into or out of the annular drive electrodes, but fringing of the electricfield forces current to pass through the centrally located zones, regions 1 and 2. There will,however, be a small region, lying directly beneath the sense electrodes, which will not influencethe EIS readings.

3. Results

There are two main and complementary components of our study, electrical impedancespectroscopy and histologic assessment. Impedance results are presented first. Plots in thecomplex plane show, simultaneously, both the real and imaginary components of the measuredimpedance over the entire frequency range (1 kHz to 1 MHz). This provides a global overviewof changes and serves to answer the question of whether the electrical impedance signatureof muscle does or does not change in response to focal radiation damage. Conductivity,one component of impedance, is isolated and examined more closely in order to clarifythe relationship between radiation-induced change and impedance shift. Histology suppliesthe details and specifics of the high gradient radiation injury and adds to the relatively sparceliterature on radiation injury in muscle (Hsu et al 1998). It also allows us to determine the

EIS detection of radiation response 671

Figure 3. Muscle cross-section with histology evaluation regions superimposed on treatment area;arrow shows direction of needle entry into leg musculature for seed placement; Mason trichromestain. The dash-enclosed region was evaluated using 18 microscopic fields and includes all themuscle tissue in the plane bisecting the seed and the two-opposing probe faces. The regiondelineations, two to four, are at isodose levels of (110, 45 and 20 Gy) and (180, 90 and 45 Gy),for the target doses of 26 Gy and 52 Gy, respectively. The approximate location of the compoundprobe is indicated by the grey, hatched regions.

magnitude and volume of injury required for statistically significant changes to appear inimpedance readings.

3.1. Impedance curves

Impedance data for irradiated and non-irradiated tissue can be plotted in the complexplane, allowing changes in the real and imaginary component of impedance to be observedsimultaneously (figure 4). For biological tissue, this plotting technique results in a semi-circular curve with the magnitude of the real component (resistivity) read on the x-axis andthe negative of the imaginary component (reactance) read on the y-axis (Ackmann and Seitz1984). At low frequencies, the electrical resistivity is greatest, and it decreases with increasingfrequency. The imaginary component increases from a low at 1 kHz, reaches its peak valueat around 25–30 kHz where the impedance curve crests and then decreases towards 0.25 � mat 1 MHz.

Impedance curves from control legs, from 26 Gy irradiations, and from 52 Gy irradiationsare shown at monthly post-treatment intervals in figure 4. It is important to recall that the26 and 52 Gy designations for the two radiation groups refer to the target dose 5 mm awayfrom the HDR source, but in actuality these exposures consist of a considerable dose gradientwhich is characteristic of the HDR technique. A reduction in curve size is observed withincreasing dose and with increasing time intervals post-irradiation. The dominant shift occurs

672 K S Osterman et al

N=5

N=4

N=5

N=9

High FrequencyImpedance Shift

Low FrequencyImpedance Shift

Figure 4. Complex impedance curves (1 kHz to 1 MHz) for muscle. Data from 29 animals:control (outer curve), 26 Gy (middle curve) and 52 Gy (inner curve). Each panel contains datafrom a subgroup of nine or ten animals measured at a specific time post-irradiation. Error bars are±1 standard deviation based upon 3 × N measurements; 3 measurements per animal; N = numberof animals.

in the low-frequency region of the curve. However, impedance values at all frequencies showsome response to radiation injury. At the highest frequencies, a slight (though statisticallyinsignificant) decrease in both the real and imaginary component of impedance is observed.The stability of high frequency impedance readings suggests that the dominant effect ofradiation is a reduction of muscle fibre volume without a dramatic change in the ionicproperties of the intra and extracellular material or alterations in sub-cellular structures. Amore dramatic shift in high frequency impedance would be expected with large-scale necrosisor inflammation. Inflammation was only observed in two irradiated animals (out of 29) and itoccurred in a small area occupying 1.6% and 0.5% of the tissue sample. In previous studies,using external beam irradiation techniques, we have observed greater levels of inflammation,and recorded larger shifts in high frequency impedance (Pigott et al 2000, Paulsen et al 1999).

In the upper curve of figure 4, ‘1 month post-irradiation’, there is only minimal separationbetween controls and the 26 Gy group. The 52 Gy group impedance values, however,

EIS detection of radiation response 673

are significantly reduced, at low frequencies, relative to both the controls and the 26 Gyirradiations.

At 2 months, the gap between irradiated and non-irradiated tissue widens, with 26 Gy and52 Gy irradiations still distinct from one another, and 26 Gy impedances lower than controls.At 3 months, the separation from controls increases further, and the 26 Gy and 52 Gy curvesbunch slightly. The greatest shift in high frequency impedance is observed in measurementson the 52 Gy, 3 month muscles.

Different time progressions are evident in the two dose groups: with the larger dose, thegreatest change occurs early and further shift between 1 and 3 months is smaller. In contrast,the greatest change for the 26 Gy group occurs later, between 2 and 3 months.

The spectral curves for the control legs demonstrate a small impedance increase withtime. A dummy seed was inserted into the control leg of the ten animals sacrificed at1 month, therefore some disruption due to the mechanical insertion may be present in the1 month controls. This may also explain the somewhat larger error bars in this group.However, there was no control insertion in either the 2 month or the 3 month group, so tissuerepair after mechanical damage is unable to account for the 2–3 month increase. Statistically,differences in controls were not significant.

3.2. Conductivity

Additional insight and understanding is gained by examining the data in terms of itsconductivity since this parameter reflects, exclusively, the conduction properties of the tissue.The relationship between impedance (z), conductivity (σ ) and relative permittivity (εr) is givenby the expression:

z = z′ + jωz′′ = 1

σ + jωε0εr

(2)

where j is the square root of −1, ω is the frequency and ε0 is the permittivity of free space.Since both the real (z′) and imaginary (z′′) component of impedance are measured, bothconductivity and permittivity can be easily calculated for each frequency along the spectrum.

In figure 5, conductivity increases above baseline at both doses. At 1 and 2 monthpost-irradiation, both low and high frequency conductivities appear to increase by a similaramount, suggesting the presence of an electrical shunt pathway in the irradiated tissue.Patho-physiologically, this implies, from the low frequency decrease, a reduced muscle fibrevolume. The high frequency increase would be explained by a change in tissue biochemistry;for example, an average increase in the conductivity of the inter and/or intracellular fluid.Alternatively, a decrease of small membrane-bound structures (mitochondria, for example)could explain a high frequency rise in conductivity. Because of their small size (lowcapacitance), they might not be completely penetrated by a 1 MHz current. A reductionin their density (or an increase in their size) would consequently increase high frequencyconductivity. Low frequency conductivity would not be affected by a change in their numberor structure. The number, shape and size of mitochondria in rat muscle have previously beenshown to change in response to fractionated external beam irradiation, with the number ofmitochondria initially decreasing and then returning to pre-irradiation levels (Hsu et al 1998).

At 3 months, there is a flattening of the conductivity curve for both 26 and 52 Gy (lowfrequency conductivity remains elevated, but high frequency conductivity drops from 1 and 2month levels), suggesting a return towards pre-irradiation biochemistry/sub-cellular structurebut sustained post-irradiation morphology at the cellular level, including muscle fibre atrophyand interstitial thickening.

674 K S Osterman et al

Figure 5. Conductivity (S m−1) as a function of frequency (Hz). (◦) Control, (•) 1 month, (�)2 month and ( ) 3 month post-irradiation.

A number of parameters, including conductivity, permittivity, low-, intermediate- andhigh-frequency impedance values and peak frequency were initially examined. Of these, lowfrequency conductivity appeared to be a fairly robust indicator of damage, and a more carefulanalysis of measurement variability in both controls and irradiated tissue was performed. Theranges of values are compared between animals and within a series of three measurements onsingle animals, so as to capture and address concerns about repeatability.

The full range of conductivity values at 10 kHz in the control legs of 27 animals is shownin figure 6. Mean conductivity values ranged from 0.21 S m−1 to 0.266 S m−1. The maximumdistance from the mean for a single animal was −8% and the average range was ±2.7%.Variability between animals was higher, with a maximum distance from the overall mean,0.239 S m−1, of −12% and a standard deviation of ±7%.

When 10 kHz conductivity values for the control and the irradiated groups are compared,conductivity is observed to increase with time after irradiation and dose (figure 7). The meanconductivity values for the 26 Gy dose (all times) are lower than those for the 52 Gy dose (alltimes). There is no overlap between controls and any of the 14 legs irradiated to a level of52 Gy. Three months after receiving 52 Gy, muscle showed a conductivity increase of 45%.Means and ranges for all treatment groups are shown in table 1.

An analysis of variance was performed on the 10 kHz conductivity values, and a multiplecomparison test (with p < 0.01) was used to further determine which treatments resulted instatistically different conductivity readings. From the analysis of variance, the probability

EIS detection of radiation response 675

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27Means0.2

0.22

0.24

0.26

0.28

0.3Conductivity at 10 kHz: Control Legs

Con

duct

ivity

(S

/m)

Control Subject (index number)

N = 3

Figure 6. Conductivity at 10 kHz for 27 control legs (◦) and mean conductivity for allmeasurements (•); N = 3 measurements per animal. Error bars for individual animals coverthe range of all three measurements. Error bars on point 28 (mean conductivity at 10 kHz for allcontrols) extend to include the range of means.

Control Control Control 26 Gy 26 Gy 26 Gy 52 Gy 52 Gy 52 Gy0.2

0.25

0.3

0.35

0.4Conductivity at 10 kHz: Control and Irradiated

Con

duct

ivity

(S

/m)

1mo 2mo 3mo 1mo 2mo 3mo 1mo 2mo 3mo

Figure 7. Conductivity at 10 kHz for control (•) and irradiated legs (◦); N = 4–5 animals perirradiation category and N = 9–10 animals in each control category. Error bars cover the range ofmeans in each category. Means for each animal in the irradiated groups are indicated by pointsalong the error bars.

Table 1. 10 kHz conductivity values after high dose rate irradiation of skeletal muscle.

Range (S m−1) Mean

Controls at 1 month (10): 0.21–0.27 0.25Controls at 2 months (10): 0.22–0.26 0.24Controls at 3 months (9): 0.21–0.25 0.2326 Gy–1 month (5): 0.23–0.31 0.2626 Gy–2 months (5): 0.26–0.29 0.2726 Gy–3 months (5): 0.26–0.33 0.2952 Gy–1 month (5): 0.29–0.33 0.3252 Gy–2 months (5): 0.31–0.36 0.3352 Gy–3 months (4): 0.30–0.38 0.34

that the measured increase in conductivity was due to chance was less than 2 × 10−12.Controls were grouped together, but each dose/time group was treated separately. Four ofthe dose groups (26 Gy at 3 months, 52 Gy at all times, 1–3 months) had significantly higher10 kHz-conductivities than controls. Also significant were differences between the 26 Gy 1 and2 month irradiations and all 52 Gy irradiations. Differences within the 26 Gy group were

676 K S Osterman et al

1 2 30.6

0.7

0.8

0.9

1Normal Muscle

1 2 30

0.05

0.1

0.15

0.2Degeneration & Necrosis

1 2 30.03

0.04

0.05

0.06

0.07

0.08Mature Fibrosis & Connective Tissue

1 2 30

0.1

0.2

0.3

0.4Atrophy

Months Post–Irradiation Months Post–Irradiation

Figure 8. Histologic assessment of tissue volume over entire measurement-sensitive region of therat hind leg as a function of time post-irradiation. Data from controls (�), 26 Gy irradiations (•) and52 Gy irradiations (♦) are shown. Values are presented as fractions of total point counts in each offour categories, normal muscle (top left), degenerating and necrotic muscle (top right), connectivetissue and mature fibrosis (bottom left) or atrophied muscle with or without inflammation (bottomright). All are plotted as a function of months post-irradiation.

not significant at p = 0.01, nor were the differences within the 52 Gy group. In both groups,however, a trend towards increased conductivity with time was suggested.

3.3. Histological injury assessment

Histological results are separated by injury classification (figure 8) for a more detailed look atinjury progression. The overall increase in connective tissue and mature fibrotic scar is minimalat these relatively early time points (1–3 months). However, fibroplasia is evident undermicroscopic examination, and there is significant thickening of the extracellular matrix/stroma.Muscle degeneration and necrosis increased as a function of dose, with approximately 3.5%and 11% of the tissue showing signs of cell death at 1 month for the 26 Gy and 52 Gy doses,respectively. By 3 months, there is a decrease in necrotic tissue relative to the 1 month finding,and a concurrent increase in atrophied muscle, as would be expected for a tissue further alongin the injury response pathway. The percentage of tissue within the EIS sensitive zone thatshows signs of atrophy ranges from 8–11% in the 26 Gy animals, and peaks at a high of 25%in the 52 Gy dose group 3 months after irradiation.

A dramatic visualization of this increase in extracellular stroma is shown in figure 9.Low frequency current flows exclusively through the extracellular space. In healthy tissue(figure 9(a)), the low frequency current must follow a tortuous and narrow route across the

EIS detection of radiation response 677

(a)

(b)

Figure 9. Fibrotic change in skeletal muscle post-irradiation. Images show muscle taken from(a) control and (b) irradiated legs of a rat. Mason trichrome stain, original magnification of 400×.

muscle. In contrast, the pathway is much more direct, open and conductive in the irradiatedmuscle (figure 9(b)).

The spatial dependence, relative to source proximity, of injury is shown in figure 10.The percentage of muscle characterized as degenerating or necrotic decreases in 1/r2 fashion,proportionally with dose fall-off, where r is the distance from the source to evaluation zone.At 1 and 2 months after receiving a dose of 52 Gy, the volume of tissue characterized asnecrotic ranged from 48–56% in the central region to less than 4% at the periphery.

An analysis of variance was performed on the cumulative injury index (including muscledegeneration, necrosis and atrophy with and without inflammation). For both histology studies(scoring of full tissue-thickness between the two probes ( p < 1×10−9), and scoring of tissue inthe four nested regions (3 × 10−11 < p < 4 × 10−5)) dose and/or time had a significant impact

678 K S Osterman et al

1 2 3 40

0.1

0.2

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0.6

0.7Degeneration and necrosis as a function of distance from source

Region

Fra

ctio

nal t

issu

e vo

lum

e

1 Month 2 Months 3 Months

Figure 10. Degeneration and necrosis as a function of distance from source and determined byhistological point counts. Controls (�), 26 Gy irradiations (•) and 52 Gy irradiations (♦) areshown. Regions correspond with the measurement zones extending out from the radiation source,as identified in figure 2.

on injury score. A multiple comparison test (with p < 0.01) was used to further determinewhich histological scores were statistically different in each of the two studies. Controls weregrouped together, but each dose/time group was treated separately.

In the first study, four of the dose groups (26 Gy at 3 months, 52 Gy at all times,1–3months) had significantly greater injury scores than controls (see figure 11). Also significantwere differences between the 26 Gy 1 and 2 month irradiations and the 52 Gy 3 monthirradiations. Differences within the 26 Gy group were not significant at p = 0.01, nor werethe differences within the 52 Gy group.

Analysis plots from the sub-region histology study are shown in figure 12. While tissueevaluated at 3 month post-irradiation showed statistically greater levels of injury than controlsin all regions, it was only in regions 1 and 2 that the injuries were significantly greater thancontrols at p = 0.01. The approximate volume circumscribed by the outer boundary of region2 was 0.15 cm3. Of additional interest was a significant increase in the volume of injured tissuefound for the 26 Gy dose at 1 month post-irradiation relative to controls in the region closestto the source. The volume of this region was approximately 0.02 cm3, and did not translateinto a statistically significant increase in conductivity. Also of note were larger injuries at 52Gy (3 months) relative to 26 Gy (2 months) which appeared only in region 2 (110 and 180 Gyisodoses enclosing 0.15 cm3) and in region 3 (45 and 90 Gy isodoses enclosing 0.4 cm3), andwhich were not statistically different (p = 0.01) at the radiation source or within the lowestdose region (4).

4. Discussion

Electrical impedance spectroscopy is able to quantitatively assess small volumes of radiationdamage in normal muscle. Impedance curves derived from grouped data tended to averageout inter-animal variability, and differences in both the time progression and the extent of

EIS detection of radiation response 679

–20 0 20 40 60 80 100 120 140 160

52Gy/3mo.

52Gy/2mo.

52Gy/1mo.

26Gy/3mo.

26Gy/2mo.

26Gy/1mo.

Controls

Histologically–Assessed Injury (Full Tissue Cross–Section)

4 groups have population marginal means significantly different from Controls (p=0.01)

Injury Score (on a scale from 0 to 360)

Figure 11. Multiple comparison of means from histologically-assessed injury in the full thicknessof tissue bracketed by the external impedance probes (p = 0.01). This corresponds to an area of1.8 cm2 and assuming cylindrical symmetry, a volume = 1.7 cm3. Data means from controls (�),26 Gy irradiations (•) and 52 Gy irradiations (♦) are shown along with 99% confidence intervals.

impedance change were evident at the two dose levels. The 26 Gy dose group demonstratesan impedance shift increase at each subsequent month. The time progression of EIS change inthe higher dose group (52 Gy) was dominated by an initial impedance change occurring in theshort time period between irradiation and the earliest time point (1 month post-irradiation),with minor changes occurring thereafter.

A small increase in the size of the impedance curve as a function of time post-irradiationwas observed in the controls. It is possible that we detected a systemic response to theirradiation at 1 month post-irradiation, similar to the bystander effects which have beendetected in other contra-lateral organs such as the breast and lung (Nuutinen et al 1998,Mothersill and Seymour 2001), but it is unclear why the impedance would increase over time(impedance decreases in the irradiated legs) and the numbers are too small to give significanceto this shift.

One of the most resilient indicators of damage was conductivity at 10 kHz, and the fullrange of measured values was presented, as well as a statistical analysis of variance in thedata. The average deviation was ±3% in a single animal with a range of ±7% in multi-animalgroups. For comparison, in our previous work with 2-electrode probes, conductivity oftenvaried by over ±15% in a single animal and ±25% in multi-animal groups. Variability inmeasurements on a single animal will be influenced by the repeatability and stability of (1)electrode placement, (2) skin electrode contact and (3) skin condition. Factors contributing tovariability between animals include differences in radioactive seed placement and positioning,radio-sensitivity and skin preparation as well as health and heart function, both of which canaffect vascular perfusion efficiency. Radio-sensitivity differences are believed to be minimal

680 K S Osterman et al

–10 0 10 20 30 40 50 60 70

52Gy/3mo.

52Gy/2mo.

52Gy/1mo.

26Gy/3mo.

26Gy/2mo.

26Gy/1mo.

Controls

Histologically–Assessed Injury (Region 1 – at Source)

5 groups have population marginal means significantly different from Controls

Injury Score (on a Scale from 0 to 60)–20 0 20 40 60 80 100

52Gy/3mo.

52Gy/2mo.

52Gy/1mo.

26Gy/3mo.

26Gy/2mo.

26Gy/1mo.

Controls

Histologically–Assessed Injury (Region 2)

4 groups have population marginal means significantly different from ControlsInjury Score (on a Scale from 0 to 120)

–40 –20 0 20 40 60 80 100 120 140

52Gy/3mo.

52Gy/2mo.

52Gy/1mo.

26Gy/3mo.

26Gy/2mo.

26Gy/1mo.

Controls

Histologically–Assessed Injury (Region 3)

2 groups have population marginal means significantly different from ControlsInjury Score (on a Scale from 0 to 200)

–40 –20 0 20 40 60 80

52Gy/3mo.

52Gy/2mo.

52Gy/1mo.

26Gy/3mo.

26Gy/2mo.

26Gy/1mo.

Controls

Histologically–Assessed Injury (Region 4)

2 groups have population marginal means significantly different from ControlsInjury Score (on a Scale from 0 to 200)

Figure 12. Multiple comparison of means for injury in nested regions 1–4 (p = 0.01). Threefields using a grid of 20 were assessed in region 1 to yield a total possible injury score of 60, sixfields (×20 points) were analysed in region 2, while ten fields (×20 points) were scored in regions3 and 4. Data means from controls (�), 26 Gy irradiations (•) and 52 Gy irradiations (♦) areshown along with 99% confidence intervals.

in this genetically similar animal population. The decreased variability achieved in this studywas attributed to the use of the 4-electrode probe and an isotonic gel.

Conductivity in five of the six dose/time categories showed no significant overlap withcontrols from the same month. The 26 Gy dose group at 1 month post-irradiation was theexception. Muscle has a long latency period, and delays in expression of injury, even withthese very large doses are not surprising. There was generally less separation between dosegroups than there was between irradiated muscle and controls. However, the conductivity washigher (p = 0.01) in the legs treated to 52 Gy (all post-irradiation times) than in the legstreated to 26 Gy (1 and 2 months).

Histological injury scores in muscle 3 months after 52 Gy were significantly higher(p = 0.01) than injury scores in muscle exposed to the lower prescription of 26 Gy (1 and2 months only). In the sub-section-based histology study, injury differences (between low andhigh dose) were only significant (p = 0.01) in regions 2 and 3. A significant difference ininjury between controls and 26 Gy/1 month post-irradiation muscle was detected in region 1(1.2% of the volume), but this injury did not translate into a significant difference in impedancevalues, even though the magnitude of change in this region was large; injury score of 30 out of60. Lesions (26 Gy/2 months and 52 Gy/3 months) showing statistical differences in regions 2and 3 (22% of the volume), but not in regions 1 and 4, led to statistically significant differences

EIS detection of radiation response 681

in conductivity of 0.07 S m−1 (23% difference in conductivity). In this instance, 34% moreof muscle was characterized as injured, on average, in the higher dose group. Finally, asignificant injury (40–60% of muscle classified as altered from pre-irradiation morphology)in regions 1 and 2 (10% of the volume), accompanied by smaller injury levels in regions 3 and4 (10–20% injured), for the 52 Gy/2 month group also led to a statistically significant changein impedance. This provides insight into the minimum lesion size that is required to effect asignificant change in impedance.

The high doses (180 or 110 Gy) delivered at the source provoked an early response. Thedifference between such high doses would not be expected to be biologically meaningful.Overall, radiation injury in region 1 (at the location of the radioactive seed) was similar, asexpected, between the two dose levels. Likewise, at distances towards the periphery of theleg, there is an absence of visible radiation injury in both dose groups. In contrast, it is inthe intermediate regions (regions 2 and 3) that histological differences do emerge between thetwo dose groups. Tissues covering multiple dose regions generate a single EIS signal, thusthe EIS system is less able to resolve differences in tissue damage than the histological systemwhere each signal is from only a single point. This is a natural consequence of the integratingeffect of the measurement technique as applied here.

The introduction of 2D and 3D EIS tomographic techniques, through deployment ofmulti-electrode arrays applied to the surface of the tissue of interest, should improve spatialcorrelation between impedance change and injury, increasing the sensitivity of EIS. Data fromrecent phantom studies suggest that EIS as an imaging technology can discriminate spectralchanges spatially at the 1–2 cm level of resolution (Kerner et al 2000). However, even the1D spectroscopic measurements presented here appear to be capable of quantifying radiation-induced change. There is potential for EIS to be used directly as an indicator of injury, withouthaving to rely upon histology.

5. Conclusions

The quality of the radiation delivered, the dose level, the volume irradiated and the time post-exposure are all factors which combine to determine the magnitude and temporal responseto a treatment. These factors can be controlled, at least in a gross manner, during radiationtherapy. However, genetic variation, age and vascular health combine to produce an injuryresponse which will vary from one individual to another (Burnet et al 1998). In order toimprove our understanding of these responses, we have examined the ability of electricalimpedance spectroscopy to monitor radiation-induced changes on an individual subject basis.This would enable treatments to be individualized in such a way as to improve their efficacyand minimize side effects. In contrast with prior work, which examined uniform (externalbeam) dose distributions (Paulsen et al 1999), the studies described here investigated injuryprogression induced through HDR exposures which allow the examination of localized andspatially resolved responses.

Although EIS, as applied in this study, is a global measurement technique designed todetect change over the tissue volume placed between the electrodes constituting the probe, itwas sensitive enough to record changes in a small, localized region within the measurementzone despite the large dose gradients. Specifically, the recorded impedance change provideda non-invasive measure of interstitial thickening and muscle atrophy, degeneration andnecrosis.

At 3 months, the injury response was still in a dynamic state and the contrast in impedanceand histology at later time points (6 months to 1 year) is expected to be much greater. Theresults from this study are encouraging for the potential employment of EIS in the radiation

682 K S Osterman et al

setting, particularly in applications such as cervical cancer where direct access to the treatmentsite would be possible and in breast, where 2D and 3D implementation would be fairlystraightforward (Osterman et al 2000). No doubt, further work is needed to define the ultimatesensitivity of the technique, first in controlled pre-clinical studies where dosing levels can bemore readily specified and probe measurements can be more easily localized to the exposedtissue. Single doses have been used here as a convenient way of accelerating the time to injuryprogression commensurate with observed clinical late effects, but fractionated schemes mustalso be ultimately evaluated.

Acknowledgments

This work was supported by a grant from the National Institute of Health and the NationalCancer Institute (RO1-CA64588). There are no conflicts of interest associated with thissubmission.

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