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Int. J. Radiation Oncology Biol. Phys., Vol. 67, No. 3, pp. 879–887, 2007Copyright © 2007 Elsevier Inc.

Printed in the USA. All rights reserved0360-3016/07/$–see front matter

doi:10.1016/j.ijrobp.2006.10.037

IOLOGY CONTRIBUTION

REDUCTION OF PULMONARY COMPLIANCE FOUND WITHHIGH-RESOLUTION COMPUTED TOMOGRAPHY IN IRRADIATED MICE

THOMAS GUERRERO, M.D., PH.D.,* RICHARD CASTILLO, B.S.,* JOSUE NOYOLA-MARTINEZ, M.S.,‡

MYLIN TORRES, M.D.,* XINHUI ZHOU, M.S.,* RUDY GUERRA, PH.D.,‡ DIANNA CODY, PH.D.,†

RITSUKO KOMAKI, M.D.,* AND ELIZABETH TRAVIS, PH.D.*

*Divisions of Radiation Oncology and †Diagnostic Imaging, The University of Texas M. D. Anderson Cancer Center, Houston, TX;and ‡Department of Statistics, Rice University, Houston, TX

Purpose: To demonstrate that high-resolution computed tomography (CT) can be used to quantify loss ofpulmonary compliance in irradiated mice.Methods and Materials: Computed tomography images of three nonirradiated (controls) and three irradiatedmice were obtained 200 days after a single dose of 16-Gy Co (60) thoracic irradiation. While intubated, eachanimal was imaged at static breath-hold pressures of 2, 10, and 18 cm H2O. A deformable image registrationalgorithm was used to calculate changes in air volume between adjacent-pressure CT image pairs (e.g., 2 and 10cm H2O), and functional images of pulmonary compliance were generated. The mass-specific compliance wascalculated as the change in volume divided by the pressure difference between the 2 image sets and the mass oflung tissue.Results: For the irradiated mice, the lung parenchyma mean CT values ranged from �314 (� 11) Hounsfield units(HU) to �378 (� 11) HU. For the control mice, the mean CT values ranged from �549 (� 11) HU to �633 (� 11)HU. Irradiated mice had a 60% (45, 74%; 95% confidence interval) lower mass-specific compliance than did thecontrols (0.039 [� 0.0038] vs. 0.106 [� 0.0038] mL air per cm H2O per g lung) from the 2-cm to 10-cm H2O CT imagepair. The difference in compliance between groups was less pronounced at the higher distending pressures.Conclusion: High-resolution CT was used to quantify a reduction in mass-specific compliance following wholelung irradiation in mice. This small animal radiation injury model and assay may be useful in the study of lunginjury. © 2007 Elsevier Inc.

Thoracic radiation, Pulmonary injury, Computed tomography, Mouse model.

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INTRODUCTION

early all patients undergoing thoracic radiotherapy exhibitome degree of lung injury, as determined by radiography,fter treatment (1). Early symptomatic injury occurs 1 tomonths after radiotherapy and presents as cough, short-

ess of breath, and low-grade fever. Severe cases mayequire hospitalization because of respiratory distress,nd some patients die from respiratory failure. Late tox-city most commonly appears as lung fibrosis on chestadiographs or computed tomography (CT) scans. Pa-ients may experience progressive chronic dyspnea forears after radiotherapy, with a continued reduction inung function (2, 3).

Reprint requests to: Thomas Guerrero, M.D., Ph.D., Departmentf Radiation Oncology, Unit 97, The University of Texas M. D.nderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.el: (713) 563–2300; Fax: (713) 563–2366; E-mail: tguerrero@danderson.orgConflict of interest: none.

cknowledgments—We thank Gabriel Guerrero for performing theanual segmentation of the mice lungs used to develop the auto-

ated algorithm. The University of Texas M.D. Anderson Cancer A

879

Radiation-induced lung injury exhibits a dose–responseelationship. Specifically, a linear relationship has beenbserved between regional radiation dose and reduction ofegional pulmonary perfusion and ventilation; the dose-ependent reductions in ventilation and perfusion were ob-erved 3 months after radiotherapy, and the functions wereartially restored 18 months posttreatment (4, 5). The use ofadioprotector drugs to protect the lung from injury is anctive area of research (6–8). Rodent models of radiationung injury have revealed the role of early molecular events9–11) in development of subsequent pneumonitis (12) andulmonary fibrosis (13). Existing rodent studies have reliedn respiratory rate, survival, histology, or radiographic ap-earance. These studies have failed to evaluate regional

enter’s Physician Scientist Program also provided invaluableupport for this project. The small animal cancer imaging researchacility where all the CT imaging was performed is partiallyupported by Grant No. CA-16672 from the National Cancernstitute.

This work was partially supported by a National Science Foun-ation VIGRE grant (NSF DSM 0240058).Received March 13, 2006, and in revised form Oct 12, 2006.

ccepted for publication Oct 13, 2006.

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unction, or when they did, they applied a terminal assay tossess function. A nonterminal rodent pulmonary functionssay, which will allow longitudinal functional assessment,s needed to study lung injury and interventions designed torotect the lung.The pulmonary function tests and clinical symptoms now

sed to assess lung injury are measures of aggregate func-ional performance and may correlate poorly with regionalhanges. Functional imaging offers the advantage of a non-nvasive in vivo method that allows the longitudinal evalu-tion of the disease or injury course in individual animalsnd within specific regions. However, present pulmonaryunction imaging is poorly suited for evaluating rodententilation. In a recent study measuring global dynamicompliance in mice, a tracheostomy was required on eachnimal to provide a surgically sealed intubation for accurateeasurement of total pulmonary compliance (11). At the

nd of each measurement, the animals were euthanized,recluding a longitudinal study. Our intubation method,reviously reported (14), does not require surgical manipu-ation of the animal, allowing recovery of the animal. Ourethod for in vivo measurement of pulmonary compliance

s noninvasive and may be repeated in the same animalsongitudinally.

Other noninvasive pulmonary imaging studies have beeneveloped for rodents. In tracer-based ventilation imaging,lanar projection images are acquired during the adminis-ration of a radioactive gas or aerosol (15, 16). Traceradioactive gases require dynamic imaging—the imaging ofsequence of images during an uptake to washout period—hereas an aerosol may require only a single acquisition.owever, the single-acquisition aerosol method only allows

ssessment of relative ventilation, not quantitative physio-ogic values. Another option, single photon emission CTSPECT) imaging, provides a three-dimensional (3D) imagef ventilation (17). Although instruments have been devel-ped for small-animal SPECT imaging at submillimeteresolutions (18, 19), these imaging methods are limited byhe long image acquisition times required and the need forynamic imaging for quantitative studies. To our knowl-dge, there have been no reports on the use of SPECT formall-animal ventilation imaging. A third option, positronmission tomography (PET), is an imaging method that usesitrogen (13N) or neon (19Ne) for ventilation imaging (20)nd provides improved spatial resolution over SPECT; how-ver, PET imaging of ventilation would also require arolonged imaging session. Finally, MRI techniques forentilation have been developed recently and applied to aodent model (21). Hyperpolarized noble gases, such asenon (129Xe) or helium (3He), have been used for para-agnetic contrast (22); however, hyperpolarized gas MR

maging techniques require special tracer gases and special-zed equipment, limiting the availability of these methods.n addition, the physiologic meaning of the resulting imagess unclear.

Recent advances in CT scanner instrumentation have led

o the development of CT scanners with submillimeter d

esolution (31 line-pairs/cm) (23–25) and rapid volumetricmaging capability. These CT scanners perform the acqui-ition of an image volume encompassing the entire rodenthorax in a few seconds. We previously reported a novelulmonary ventilation imaging method using CT withoutontrast for patients (26, 27) and applied the same techniqueo rodents to measure the static pulmonary compliancehroughout the physiologic range of pressures (28). Two CTmages are acquired with separate breath-holds at two con-tant pressures. The resulting compliance image representshe spatial distribution of pulmonary compliance at theean of the two pressures. In a recent study in patientsith acute respiratory distress syndrome (ARDS) Gatti-

oni utilized similar measurements; breath-hold CT im-ges acquired at a series of constant pressures (29). Aemiquantitative analysis was performed using CT imageairs at successively higher pressures. Our analyticalpproach of similar data in a rodent animal model is toalculate the regional compliance on a voxel by voxelasis (28).In this study, we evaluated the change in pulmonary

ompliance that occurs following thoracic irradiation inice.

METHODS AND MATERIALS

nimal preparation and imagingThe protocol for this study was approved by the University of

exas M. D. Anderson Cancer Center Institutional Animal Carend Use Committee. Six 30-g C57BL/6J mice (Jackson Laborato-ies, Ben Harbor, ME) used in this study were maintained in thepecific pathogen-free animal colony of the Department of Exper-mental Radiation Oncology. Three of the mice were 200 daysostirradiation to 16 Gy to the entire thorax using a Phillips 250V X-ray unit (1.1 Gy/min, 250 kVp, 15 mA, 80% output). Leadhields were used to block the regions outside of the thorax. Threeice were used as controls and not irradiated.Computed tomography images of the entire (live) thorax were

cquired of the 6 mice with breath-holds at constant pressures of 2,0, and 18 cm H2O using the following procedure: first, the miceere injected with anesthetic (100 mg/kg ketamine) and under-ent endotracheal intubation using disposable Teflon intravenous

atheters (DuPont, Wilmington, DE) (14). Next, the intubatedice were connected to a small animal ventilator (SAR-830/AP;WE, Ardmore, PA) and mechanically ventilated while anesthesiaas maintained with 2% isoflurane and oxygen. There was no sealetween the endotracheal tube and the trachea. An air pressureegulator (Control Air, Amherst, NH) and a traceable manometermodel #06-664-18, Fisher Scientific International, Hampton, NH)t the junction with the endotracheal tube were used to provideonstant pressure during breath-hold episodes for imaging. Com-uted tomography images of the entire (live) thorax were acquiredith breath-holds at constant pressures of 2, 10, and 18 cm H2O.preclinical flat-panel CT unit provided volumetric image acqui-

ition in a single 8-s rotation (General Electric Global Researchenter, Niskayuna, NY) (24, 25). The CT image acquisition pa-

ameters used are given in Table 1. The sequence of CT imagecquisitions was repeated to obtain each acquisition pressure in

uplicate for error analysis. At completion of the study, the inha-

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ation anesthesia was discontinued, and the animals were extu-ated. Awaken, they were placed in a warmed recovery cage.ollowing a 2- to 4-h recovery period, the mice were returned to

he animal care facility.

eformable image registrationThe goal of deformable image registration, for our purposes,

as to find a point-to-point correspondence between the lungarenchyma tissue elements (voxels) represented in two imagesexhalation and inhalation). The algorithm used in this study wasased on an optical flow method. Optical flow methods compriselarge class of deformable image registration algorithms that are

ased on the work of Horn and Schunck (30). Optical flow meth-ds find point-to-point correspondences by computing a displace-ent field that describes the apparent motion depicted in the 2

mages based on image properties (no user intervention is needed).everal reviews of these methods exist (see reference 31 for anxample). We have reported on the application of our optical flowmplementation for tumor motion (32) and ventilation derivedrom CT image pairs (26). In the current study, deformable imageegistration was applied between CT image pairs composed of thedjacent breath-hold pressures within each mouse: 2 and 10 and 10nd 18 cm H2O.

ung parenchyma segmentation and tidalolume determination

To delineate the lung parenchyma on each CT image volume, aegmentation algorithm was applied based on an eight-point con-ectivity scheme and a set of three seed points (33). Voxelsetween �250 and �990 Hounsfield units (HU) for the controlice and �100 and �990 HU for the irradiated mice were

elected as lung parenchyma if they were positive for connectivityo 1 of the 2 lung seed points and not connected to the tracheal-ronchial tree seed point. The threshold for the irradiated mice wasower because of the dense fibrosis and subsequent distribution ofung parenchyma CT values. The tracheal-bronchial tree was sim-larly delineated using a seed point and a cut-off of less than -880U; the resulting structure, which consisted mostly of large air-ays, was subtracted from the lung segmentation. The measured

idal volume was determined from the difference in volumeetween adjacent CT volumes (2 and 10 and 10 and 18 cm

2O). The resulting measured tidal volume was used to estab-ish calibration for the experimental method described in theext paragraph.Simon (34) found a relationship between CT regional values

HU) obtained from exhalation and inhalation breath-hold CTmages �HUex and HUin� and the regional volume change:

�V �HUin � HUex�

Table 1. Computed tomography acquisition parameters used

ube voltage (kVp) 100ube current (mA) 30can time (s) 8umber of projections 1000econstruction filter Standardixel size 0.1 � 0.1 � 0.1 mmmage size 256 � 256 � 255xial coverage (cm) 4.21

Vex

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here ov � vin � vex is the local volume change due to inspiration.quation 1 was applied on a voxel-by-voxel basis to the CT imageairs (2 and 10 and 10 and 18 cm H2O), with the deformable imageegistration mapping providing the link between tissue elementscross the pair. The resulting image sets were images of theractional increase in volume of each voxel between their twoarent CT images. The compliance was calculated by multiply-ng the values in the images by the voxel volume and dividinghe result by the pressure difference (two constant values). Thecalculated) lung volume change was calculated from this quan-ity as

�V � Vex� �V

Vex

dV � Vex� 1000�HUin � HUex�

HUex�1000 � HUin�dV. (2)

he final images were smoothed to a 0.75-mm resolution andisualized using the AMIDE image visualization software (35).

A linear regression analysis was performed to obtain the mea-ured volume from the calculated volume. In our previous studies,mass correction factor was applied to account for differences in

ulmonary perfusion between the two lung image volumes (26). Inhis case, the dense lung fibrosis and difficulty with segmentationf the irradiated mouse lung created artifactual mass differencesetween the segmented lung pairs. Therefore, rather than use ourrior method of correcting for mass differences to obtain quanti-ative results, the results of the linear regression analysis were useds a calibration, where the true (measured) volume change wasalculated from the calculated volume changes. The volumehange and the difference in distending pressure between the CTmage pairs were then used to determine the compliance.

ulmonary compliancePulmonary compliance was defined as the change in volume per

hange in pressure (36, 37):

C ��V

�P· (3)

owever, the value of this quantity was highly dependent on themount of lung and the volume under evaluation. This propertyade comparisons between unequal regions difficult and has been

ecognized as an obstacle for comparison studies (38). In thistudy, in which lungs with differing densities were compared, aeans to normalize the two was needed. Therefore, we usedass-specific compliance to allow such comparisons. The mass of

ach lung was calculated from the CT values using

m � �v�dV � �v �1 �

HU

1000�dV (4)

here HU represents the CT value in Hounsfield units. The inte-ration was performed over the entire volume of interest. Theompliance for a given lung region was divided by its mass toield the mass-specific compliance, or compliance per unit mass ofung tissue. This relationship is given in the equation below:

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882 I. J. Radiation Oncology ● Biology ● Physics Volume 67, Number 3, 2007

he mass-specific compliance was calculated for the segmentedulmonary parenchyma tissue.

tatistical methodsThe measurement data consists of two groups, controls and

rradiated, with three rodents in each group and duplicate mea-urements. A two-factor analysis of variance (ANOVA) was per-ormed using SPSS version 11.5 software (SPSS, Chicago, IL).he ANOVA model assumes the deviations of the mass specificompliance observation from the overall mean may depend on 1)n effect due to radiation treatment, 2) an effect due to pressure,nd 3) random error. The two-factor ANOVA model provided thetandard error estimate and t values used to calculate the 95%onfidence intervals (CI) for the mean mass specific compliance ofrradiated and control mice at the two pressure values 6 and 14 cm

2O. The mean lung mass for each group was compared using the5% confidence intervals. The reduction in compliance was cal-ulated as a ratio of means statistic with a 95% CI (39). Theesulting mass-specific compliances with standard errors wereraphed. All other measured parameters are presented as mean �tandard error.

RESULTS

omputed tomography imagesRepresentative coronal slices through the mid-thorax of

he CT image volumes from one control mouse (Figs. 1–c) and one irradiated mouse (Figs. 1d–f) at each of theistending pressures are displayed in Fig. 1. The imageshown in Fig. 1a and 1d were acquired at 2 cm H2O, Fig. 1bnd 1e were acquired at 10 cm H2O, and Fig. 1c and 1f werecquired at 18 cm H2O. The images are displayed using a

Fig. 1. Constant pressure CT images were acquired forpressure. Coronal sections at each of the pressures, throunonirradiated (control) mouse (a–c) and an irradiated mo

reference in panel a. The lungs expand becoming less dense (d

indow width of 1,150 HU and window level of �450 HUo that the lung parenchyma density changes due to thencreased air content at higher distending pressures arepparent. For both animals, the increasing distension of theirways with increasing pressure can be appreciated. In theontrol animals (Fig. 1a–c), the lung parenchyma darkeneds the pressure increased. In the irradiated animals (Fig.d–f), the lung parenchyma was filled with fibrotic strand-ng and patchy regions that similarly darkened as the pres-ure increased. The CT characteristics of fibrosis, apparentn the irradiated case (Fig. 1d–f), have been previouslyeported for CT images acquired in freely breathing anes-hetized animals (25, 40); however, the parenchymahanges with increased distending pressure have not beeneported. These changes reflect the residual underlyingunction that remains after injury from radiation.

The lung parenchyma was segmented from each of theT image volumes, and a histogram was generated from the

ung CT values. The histogram values for one control mouseFig. 2a) and 1 irradiated mouse (Fig. 2b) were plotted forach of the distending pressures. As the distending pressurencreased, the lung parenchyma histogram values for bothnimals shifted toward more negative values, indicating thencreased air content. The lung parenchyma values for theontrol mouse became more sharply distributed at the higherressures. In contrast, the lung parenchyma values for therradiated mouse became broader with increasing pressure.or the irradiated mice, the lung parenchyma average CTalues ranged from �314 (�11) HU to �378 (�11) HU forhe CT images acquired at 2 and 18 cm H2O, respectively.

sthetized and intubated mice at 2, 10, and 18 cm H2Omid-thorax of the CT image volumes, are shown for a

f) for successive pressures. A 1-cm bar is shown for size

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imilarly, for the control mice, the values ranged from549 (�11) HU to �633 (�11) HU at 2 and 18 cm H2O,

espectively. Per Eq. 4, these values correspond with a lungensity range of 0.37 to 0.45 g/mL (�1.8%) from exhala-ion to inhalation for the normal mice and a density of 0.63o 0.67 g/mL (� 3.2%) for the irradiated mice, respectively.he lung density of the irradiated mice was nearly twice thatf the normal mice at the lowest distending pressure of 2 cm

2O (exhalation). Note that the average CT value wasalculated using a 0.3 mm3 mean surrounding each voxelithin the segmented lungs for those voxels that mapped

nto lung parenchyma in the corresponding CT volumeuring the ventilation calculation.The lung volume and mass were calculated from the lung

ithin the segmented lungs. We found no significant dif-erence between the masses of the control and irradiatedice lungs (0.18 [95% CI, 0.15–0.21] g vs. 0.20 [95% CI,

.17–0.23] g, respectively). Table 2 shows the mean lungolumes vs. distending pressure for irradiated and controlice. The total lung volumes (lung parenchyma and air

(a)

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Fig. 2. The graphs represent the histographic distribut(a) control mouse and (b) mouse 200 days after having bacquired with breath-holds at 2, 10, and 18 cm H2O prautomated segmentation algorithm, as described in Methdata with identifiers (triangle, square, or circle) to refere

Table 2. Total lung volumes vs. distending pressure(� standard error)

Irradiated mice Control mice

2 cm H2O 0.26 (� .05) mL 0.31 (� .05) mL0 cm H2O 0.31 (� .05) mL 0.45 (� .05) mL

h8 cm H2O 0.35 (� .05) mL 0.54 (� .05) mL

ontent) were higher for the control mice at all pressures,eaching statistical significance at 10 and 18 cm H2O dis-ending pressure. From the individual data, and assuminghat the repetitions are independent observations, a simplewo-sample t test was performed at each pressure. The totalung volumes of the irradiated vs. control mice were signifi-antly different at 10 cm H2O (p � 0.01791) and at 19 cm H2Op � 0.00961). As noted previously, the density of the irradi-ted mice lungs was nearly twice as high as that of the controlice lungs, and hence the air content was similarly lower.

eformable image registrationThe deformable image registration algorithm was applied

or the CT image pairs acquired at 2 and 10 cm H2O and 10nd 18 cm H2O. Using the mapping obtained and Eq. 1,ompliance images were generated (Fig. 3) for each animal.

representative coronal slice through the mid-thorax of theompliance image volumes from one control mouse (Fig. 3–c) and one irradiated mouse (Fig. 3d–f) for each pair areisplayed in Fig. 3. The 2 cm and 10 cm H2O coronalection is shown overlying the corresponding CT imagecquired at 10 cm H2O for the control (Fig. 3a) and irradi-ted mice (Fig. 3d). In the subsequent image (Fig. 3b ande), the compliance image were displayed using the Amideisualization software using their NIH color map (35), inhich the maximum value corresponds with a fractional

ncrease in volume of 0.6 (or 60%). There are regions of

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Materials. Each curve represents 150 bins of measurede curve with the acquisition pressure.

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884 I. J. Radiation Oncology ● Biology ● Physics Volume 67, Number 3, 2007

or the control mouse (Fig. 3b) which appear to have lowompliance in the corresponding anatomic regions for therradiated mouse (Fig. 3e). The irradiated mouse had aower compliance within the lung parenchyma. The onlyegions with high compliance in the irradiated mouse cor-esponded with small airways (Fig. 3d). At the higher pres-ure, the control and irradiated mice compliance images,enerated from the CT images acquired at 10 and 18 cm

2O distending pressure, appeared similar.

idal volume and pulmonary complianceThe tidal volume, which is the change in lung volume

etween two image volumes with different distending pres-ure, was calculated for each mouse for 2 and 10 and 10 and8 cm H2O distending pressure. The tidal volume wasalculated using the segmented lung parenchyma volumesmeasured) and the compliance images generated using Eq.

and Eq. 5 (calculated). A graph of the calculated vs.easured tidal volume was made (Fig. 4) using the full set

f 12 pairs, and a linear regression analysis was performed.he following relationship between the measured and cal-ulated tidal volumes was found:

measured � 0.058 � 1.40 � calculated (6)

Fig. 3. A coronal section through the pulmonary complmouse (d–f) for 6 and 14 cm H2O pressure. Each image8 cm H2O pressure difference; for example, the first imagand is referred to as the compliance image at 6 cm H2O.of each voxel between their two parent CT images. Thmultiplied by the voxel volume and divided by the pressusing the National Institutes of Health color scale, wicompliance images. Images a and d show the complianceimage to provide an anatomic reference. The next two indistending pressure.

ith a linear correlation coefficient of R � 0.92. This f

elationship was subsequently applied to the calculated tidalolumes for the irradiated mice. The mass-specific compli-nce was calculated using these results, the lung massalues, and Eq. 5. A graph of Cm for the two midpointressures of 6 and 14 cm H2O was made (Fig. 5). At eachressure, the mass-specific pressure for the irradiated miceas significantly lower than that for the controls. At 6 cm

2O pressure, the Cm value for the irradiated mice was 60%95% CI, 45%–74%) lower than the control value.

DISCUSSION

In this study, we demonstrated the ability to measure theeduction in pulmonary compliance between irradiated micend unirradiated mice; lung compliance was reduced inrradiated mice. A 60% reduction in the pulmonary compli-nce was found between the pressures of 2 cm H2O and 10m H2O. The 95% CI of that reduction was approximately

25%, which places a limit on the degree of difference thatan be measured with these experimental conditions. Auccessful pulmonary radioprotector that provides a 25%eduction (or less) in pulmonary compliance loss wouldequire a larger sample size to demonstrate that difference.xperiments may be designed from an estimate of thexpected degree of radioprotection and the standard errors

images for one control mouse (a–c) and one irradiateded from two constant pressure CT images separated byrived from the CT images obtained at 2 and 10 cm H2Oage sets are images of the fractional increase in volumepliance was calculated from the values in the images

ference (two constant values). The images are displayedscale given below over the range of 0 to 0.6 for theat 6 cm H2O superimposed over the corresponding CTw are the compliance images alone at 6 and 14 cm H2O

ianceis derive is deThe ime com

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In addition to the changes in pulmonary compliance, weound that irradiated mice had higher mean CT values and

nearly twofold increase in lung density, reflecting lesserated lung parenchyma because of fibrosis and scarring.he CT characteristics of radiation-induced fibrosis haveeen reported previously in freely breathing rodents (25, 40,1), but this study is the first to report on the response of therradiated parenchyma to increasing distending pressures (orompliance), changes that reflect underlying lung function.he lung volume and mass were calculated for the tworoups of mice using a lung segmentation algorithm and CTmage values. There were no significant differences in mass,ut the mean lung volumes were higher for the control micehan for the irradiated mice at all pressures. These dataupport our quantitative and qualitative findings of reducedompliance in irradiated mice.

In the past, investigators have used SPECT and PET tossess lung response to radiation in humans. Animal modelssed to investigate changes in pulmonary function haveistorically relied on death, breathing rate, CT density, andistologic change as the primary endpoints (40, 41). Theseerminal assays prevent the use of longitudinal studies,hich are especially needed in studies of radiation injury,hich develops over many months and occurs in distincthases. Furthermore, until recently, imaging studies to de-ermine regional pulmonary function in rodents were notvailable. The MRI techniques using contrast 3He for ven-ilation have been developed recently and applied to a

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ig. 4. The volume differences between the 2 and 10 and 10 and8 cm H2O CT image pairs calculated using Eq. 2 for all controlice and compared with the volume differences acquired from the

olume obtained using the automated lung parenchyma segmen-ation algorithms for the control mice. A linear regression analysisas performed, resulting in a correlation coefficient of 0.92 for theolume differences in the control mice. The inverse relationshipas used as a standard to compute an adjusted volume difference

or the compliance calculations.

odent model of radiation lung injury (21). The apparent f

iffusion coefficient of the hyperpolarized 3He was lower inhe irradiated lungs of rats, reflecting changes in the micro-natomy. Again, no quantitative information regarding re-ional function could be determined using this technique.urthermore, the limited availability of 3He and MR equip-ent make it difficult to broadly apply findings from this

tudy.In our method, to measure pulmonary function after ra-

iation exposure, we assessed regional pulmonary compli-nce in rodents. The compliance images were calculatedrom high-resolution CT images acquired using a flat-panelT (24, 25) and thus have the potential for similar high

esolution. A smoothing filter set the resolution of the com-liance images generated in this study to 0.75 mm. Theighest resolution achievable with this method remains toe determined. The resulting images were quantitative, andheir components were expressed in terms of physiologicuantities; for example, the mass-specific pulmonary com-liance was measured in terms of mL of air per cm H2O perram of lung tissue.Compliance was measured as an indicator of lung func-

ion using high-resolution CT scans. Compliance imagesere generated using a deformable image registration algo-

ithm for CT image pairs acquired at 2 and 10 cm H2O and0 and 18 cm H2O. Regions of high and low complianceere identified. The calculated mass-specific compliance of

ig. 5. The mass-specific pulmonary compliance, Cm, determinedsing Eq. 1 and Eq. 5, plotted for the mean of the control andrradiated mice over the pressure range of 6 to 14 cm H2O. Eachoint was measured independently in duplicate for each of thehree mice in the control and irradiated groups. The standard erroror each measurement point is given by the vertical bars. A 60%eduction in mass-specific compliance was found in the irradiatedice compared with controls. One gram of normal mice lung

issue was more than twice as compliant as one gram of lung tissue

rom the irradiated mice.

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886 I. J. Radiation Oncology ● Biology ● Physics Volume 67, Number 3, 2007

he irradiated mice was significantly lower at the two mid-oint pressures of 6 and 14 cm H2O.

In a recent study of acute lung injury or ARDS patientsere studied with breath-hold CT images at 5, 15, and 45

m H2O pressure (29). Lung regions were classified asonaerated, poorly aerated, normally aerated, or hyperin-ated based on their CT values. The image pairs werevaluated for recruitment with increasing pressure; recruit-ent was defined as a change in classification from nonaer-

ted to aerated. In their method, the percentage of each classs compared between CT images acquired at two pressures.ompliant lung tissue will fill with air when a higherressure is applied, becoming less dense and exhibiting aower CT value. Our method to calculate pulmonary com-liance images first links each voxel from the exhale to thenhale CT image volumes and then calculates a numericalue for the compliance from the local CT values. Thisethodology can also be applied to the patient data in theRDS study yielding estimates of the mechanical propertiesf the pulmonary parenchyma in those patients. In radio-herapy patients, who are usually not intubated, we haveeveloped a technique to measure ventilation under normalesting supine tidal breathing using a similar computationalethod applied to four-dimensional CT (4D CT) images

27). Ventilation will depend on airway factors, chest-wall a

REFEREN

factor protects endothelial cells against radiation-induced pro-

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ompliance, and regional pulmonary compliance. Readersre referred to a textbook of pulmonology for a moreomplete discussion of pulmonary mechanics (42). In adultatients, radiation reduces pulmonary compliance withoutuch effect on chest-wall compliance (1). We anticipate a

egional reduction of ventilation in irradiated patients, sim-lar to the reduction in regional compliance found in thistudy.

Our study is the first to quantify directly pulmonaryompliance in irradiated mice with high-resolution CTcans. Using this novel method, we found a difference inulmonary function between irradiated and nonirradiatedice.

CONCLUSION

In this study, a new method for determining regionaliomechanical compliance was applied to irradiated mice,nd a 60% reduction (95% CI, 45%–74%) in mass-specificompliance was found in irradiated mice compared withontrols. The results of our study indicate that this smallnimal radiation lung injury model and assay may be usefuln the study of lung injury and interventions designed torotect the lung. We will next use this technique to measurehe time course of pulmonary compliance loss in irradiated

nimals with varying radioprotectors.

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