Ischemic lesion volume determination on diffusion weighted images vs. apparent diffusion coefficient maps

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

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Research Report

Ischemic lesion volume determination on diffusion weighted images vs. apparent diffusion coefficient maps Bernt Tore Bra°tane a,⁎, Birgul Bastan a , Marc Fisher a , James Bouley a , Nils Henninger a,b a

Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01655, USA Department of Internal Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Though diffusion weighted imaging (DWI) is frequently used for identifying the ischemic

Accepted 2 May 2009

lesion in focal cerebral ischemia, the understanding of spatiotemporal evolution patterns

Available online 8 May 2009

observed with different analysis methods remains imprecise. DWI and calculated apparent diffusion coefficient (ADC) maps were serially obtained in rat stroke models (MCAO):

Keywords:

permanent, 90 min, and 180 min temporary MCAO. Lesion volumes were analyzed in a

Stroke

blinded and randomized manner by 2 investigators using (i) a previously validated ADC

MRI

threshold, (ii) visual determination of hypointense regions on ADC maps, and (iii) visual

Suture model

determination of hyperintense regions on DWI. Lesion volumes were correlated with

Focal cerebral ischemia

24 hour 2,3,5-triphenyltetrazoliumchloride (TTC)-derived infarct volumes. TTC-derived

Reperfusion

infarct volumes were not significantly different from the ADC and DWI-derived lesion

ADC

volumes at the last imaging time points except for significantly smaller DWI lesions in the

DWI

pMCAO model (p = 0.02). Volumetric calculation based on TTC-derived infarct also correlated

Interrater agreement

significantly stronger to volumetric calculation based on last imaging time point derived lesions on ADC maps than DWI (p < 0.05). Following reperfusion, lesion volumes on the ADC maps significantly reduced but no change was observed on DWI. Visually determined lesion volumes on ADC maps and DWI by both investigators correlated significantly with threshold-derived lesion volumes on ADC maps with the former method demonstrating a stronger correlation. There was also a better interrater agreement for ADC map analysis than for DWI analysis. Ischemic lesion determination by ADC was more accurate in final infarct prediction, rater independent, and provided exclusive information on ischemic lesion reversibility. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

Magnetic resonance imaging (MRI) is an excellent in vivo technique to determine ischemic lesion evolution in both clinical and experimental stroke studies. It allows for the investigation of treatment efficacy (Schellinger et al., 2007) as

well as studying the natural evolution of cerebral ischemia (Beaulieu et al., 1999) with high spatiotemporal resolution. Diffusion weighted imaging (DWI) is a frequently employed sequence in stroke imaging as it allows for very early detection of cerebral ischemia (Chalela et al., 2007). It is generally accepted that hyperintense regions on DWI

⁎ Corresponding author. Center for Comparative NeuroImaging (CCNI), UMASS Medical School, 303 Belmont Street, Worcester, MA 01604, USA. Fax: +1 508 856 8090. ° tane). E-mail address: [email protected] (B.T. Bra 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.05.002

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183

The purpose of this study was to assess lesion volume determination in a rat middle cerebral artery occlusion (MCAO) model based on DWI using the following approaches: visual determination of hyperintense regions on DWI (DWI), visual determination of hypointense regions on calculated ADC maps (vADC), and a previously validated absolute ADC threshold (tADC) (Meng et al., 2004). In addition, interrater agreement and correlation with final infarct volumes were analyzed.

2.

Results

2.1.

Mortality and data exclusion

One animal in each group (11.1%) died between 16 to 24 h after MCAO. One additional animal in the 180-tMCAO group died before 16 h after MCAO and was excluded from the histological analysis.

Fig. 1 – Spatiotemporal evolution of vADC, tADC, and DWI-derived lesion volumes in (A) pMCAO, (B) 90-tMCAO, and (C) 180-tMCAO groups, respectively. *p < 0.05 between tADC or vADC and DWI-derived lesion volumes, respectively. † p < 0.01 between TTC-derived infarct volumes and tADC, vADC, or DWI-derived lesion volume, respectively. ‡p < 0.05 between the last time point before reperfusion and the post reperfusion time point, valid for both tADC and vADC. Note that data obtained by investigators 1 and 2 were averaged for each modality, respectively. Dashes indicate reperfusion time point.

correspond to tissue experiencing cytotoxic edema and likely subsequent cell death (Moseley et al., 1990). Apparent diffusion coefficient (ADC) maps were introduced to quantify water diffusion, providing researchers and clinicians with the ability to ascertain the acuteness of ischemic changes independent of magnet properties (Ding et al., 2007). Lesion volume measurements are used in experimental stroke studies for interpreting therapeutic efficacy and in clinical stroke research for patient selection and as surrogate markers for clinical outcome (Hacke et al., 2005). Though various methods have been used to analyze DWI lesion volumes in stroke studies (Bardutzky et al., 2005; Girot et al., 2003; Henninger et al., 2007; Luby et al., 2006; Meng et al., 2004), systematic comparisons of different analysis methods are not frequently performed (Na et al., 2004).

Fig. 2 – Bland–Altman plots of interrater agreement for visually determined lesion volumes in (A) ADC maps and (B) DWI. Manual lesion determination based on ADC map analysis produces better interrater agreement than DWI based analysis. Note that data from all experimental groups were pooled. vADC1/DWI1 and vADC2/DWI2 indicate visually determined ADC/DWI lesion volumes from investigators 1 and 2, respectively.

184 2.2.

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Physiological measurements and neurological score

The tADC and vADC lesion volumes at 180 (pMCAO and 90tMCAO) and 270 min (180-tMCAO) were not significantly different (p = 0.99, 0.94, and 0.39, respectively) compared to TTC-derived infarct volumes at 24 h. In the 90-tMCAO group the ADC-derived lesion volume based on the imaging time point preceding reperfusion (90 min) was significantly larger than the 24 hour TTC-derived infarct volume (p < 0.01). DWI-derived lesion volumes at 180 min in the pMCAO group were significantly smaller than final TTC-derived infarct volumes (p < 0.01). There was no significant difference between DWI lesion volume obtained at the last imaging time point in both tMCAO groups and 24 hour TTC-derived infarct volumes (p > 0.05).

Basal physiological parameters, weight, and neurological scores did not differ significantly between experimental groups or time points (data not shown).

2.3. Spatiotemporal lesion evolution of ADC and DWI-derived lesion volumes and correlation with final infarct volumes Fig. 1 shows the spatiotemporal evolution of DWI and ADCderived lesion volumes. In pMCAO, DWI-derived lesion volumes were significantly smaller than ADC-derived lesion volumes from 60 to 180 min (p < 0.05, Fig. 1A). Similarly, in the 90-tMCAO group, DWI-derived lesion volumes were significantly smaller than ADC-derived lesion volumes during occlusion (p < 0.05). Interestingly, following reperfusion ADC lesions partially resolved, matching the unaltered DWIderived lesion volumes (at 120 min, p < 0.05; at 180 min, p > 0.05 Fig. 1B). Unexpectedly, in the 180-tMCAO group there were no statistically significant differences between DWI and ADC-derived lesion volumes during occlusion (p > 0.05). Consistent with 90-tMCAO animals, DWI-derived lesion volumes remained unaltered following reperfusion (p > 0.05, Fig. 1C). Conversely, ADC lesion volumes partially but significantly reversed following reperfusion (p < 0.05, Fig. 1C). The 24 hour edema corrected TTC-derived infarct volumes were 264 ± 58, 176 ± 55, and 214 ± 73 mm3 in pMCAO, 90tMCAO, and 180-tMCAO groups, respectively (Fig. 1). The TTC-derived lesion volumes of the 90-tMCAO group were significantly smaller relative to the pMCAO group (p < 0.01). No significant differences in final infarct volumes were observed between 180-tMCAO vs. pMCAO and 90-tMCAO groups (p > 0.05).

2.4. Interrater agreement of visually derived ADC and DWI lesion volumes Visual determination of ischemic lesion volumes based on ADC maps as well as DWI (pooled data from all time points and groups) showed a significant correlation between the two investigators. However, the former method demonstrated better interrater correlation (p < 0.01, r = 0.93 vs. p < 0.01, r = 0.71). Figs. 2A and B show Bland–Altman plots of vADC and DWI-derived lesion volumes, respectively. Bias and 95% limits of agreement were 9.8 mm3 and − 42.4 to 62.1 mm3 for vADC and 27.2 mm3 and − 71.1 to 125.5 mm3 for DWI, respectively. These results indicate stronger interrater agreement when using ADC maps vs. DWI to analyze ischemic lesion volumes in the employed animal models.

2.5. Intermodality agreement of tADC vs. vADC and DWI-derived lesion volumes Compared to DWI-derived lesion volumes, tADC and vADCderived lesion volumes obtained at 90 (90-tMCAO), 180

Table 1 – Correlation between TTC and final imaging time. A

TTC vs. tADC r

pMCAO 90tMCAO 180tMCAO Pooled

vADC1 p

0.909 0.933 0.884 0.878

0.001 ⁎ 0.000 ⁎ 0.002 ⁎ 0.000 ⁎

r

p

0.889 0.929 0.877 0.879

0.001 ⁎ 0.000 ⁎ 0.002 ⁎ 0.000 ⁎

B

DWI1

r

p

0.816 0.927 0.851 0.862

0.007 ⁎ 0.000 ⁎ 0.004 ⁎ 0.000 ⁎

DWI2

r

p

r

p

0.761 0.909 0.656 0.621

0.017 ⁎ 0.001 ⁎

0.684 0.680 0.377 0.562

0.042 ⁎ 0.044 ⁎ 0.317 0.002 ⁎

0.055 0.001 ⁎

tADC vs. vADC1 Z

pMCAO 90tMCAO 180tMCAO Pooled

vADC2

0.18 0.05 0.05 − 0.02

vADC2 p

0.86 0.96 0.96 0.98

Z 0.60 0.08 0.23 0.23

DWI1 p

0.52 0.94 0.82 0.82

vADC1 vs.

DWI1 vs.

vADC2

DWI2

DWI2

Z

p

Z

p

Z

1.08 0.28 1.05 2.22

0.28 0.78 0.29 0.03 ⁎

1.19 1.48 1.73 2.53

0.23 0.14 0.08 0.01 ⁎

0.47 0.02 0.18 0.24

p 0.64 0.98 0.86 0.81

Z

p

0.28 1.20 0.67 0.31

0.78 0.23 0.50 0.76

A) Correlation analysis for TTC vs. ADC and DWI from last imaging time point. n = 9 in each group except the Pooled group with n = 27. B) Fisher's Z for significance between correlation coefficients. Pooled groups include animals from all of the above groups. vADC1/DWI1 and vADC2/DWI2 indicate visually determined ADC/DWI lesion volumes from investigators 1 and 2, respectively. ⁎ p < 0.05.

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185

Fig. 3 – Bland–Altman plots of intermodality agreement. Threshold-derived ADC lesion volumes agree stronger with vADC-derived lesion volumes than with DWI-derived lesion volumes. Note that data from all experimental groups were pooled. vADC1/DWI1 and vADC2/DWI2 indicate visually determined ADC/DWI lesion volumes from investigators 1 and 2, respectively. tADC indicates threshold determined ADC lesion volumes.

(pMCAO, 90-tMCAO, and 180-tMCAO) and 270 min (180tMCAO) consistently correlated better with final TTC-derived infarct volumes in all three experimental groups (Tables 1A and B). Both tADC and vADC-derived lesion volumes correlated similarly with TTC-derived lesion volumes without consistent between-modality differences in respective p and r values across investigated groups and time points. Since tADC lesion analysis is rater independent, we opted to subsequently test intermodality agreements in Bland–Altman plots against tADC. For both investigators, tADC analysis correlated better with vADC-derived lesion volumes than with DWI-derived lesion volumes (tADC vs. vADC: investigator 1, p < 0.01, r = 0.92; investigator 2, p < 0.01, r = 0.90; tADC vs. DWI: investigator 1, p < 0.01, r = 0.61; investigator 2, p < 0.01, r = 0.48). Bland–Altman plots for intermodality agreement are depicted in Fig. 3. Investigator bias and 95% limits of agreement for tADC vs. vADC analysis were: −9.5 mm3 and −68.3 to 49.3 mm3 (investigator 1, Fig. 3A), and 0.3 mm3 and − 66.5 to 67.2 mm3 (investigator 2, Fig. 3B), respectively. Investigator bias and 95% limits of agreement for tADC vs. DWI were: 13.4 mm3 and − 112.2 to 139.0 mm3 (investigator 1, Fig. 3C), and 40.6 mm3 and − 106.7 to 187.9 mm3 (investigator 2,

Fig. 3D), respectively. These results indicate better agreement of tADC-derived lesion volumes with vADC-derived lesion volumes than with DWI lesion volumes. Overall, investigator 1 (experienced) achieved better intermodality agreement than investigator 2 (inexperienced).

3.

Discussion

In experimental studies of ischemic stroke using MRI, lesion extent is evaluated by visual assessment of DWI (NeumannHaefelin et al., 2000), ADC maps (Bardutzky et al., 2005), or by ADC thresholding (Henninger et al., 2007; Meng et al., 2004; Na et al., 2004). However, in most clinical studies lesions are calculated based on visual determination of DWI abnormalities (Fiebach et al., 2002; Girot et al., 2003; Luby et al., 2006). Few clinical studies used visual determination (Fiehler et al., 2004) or thresholding of ADC maps (Loh et al., 2005; Rana et al., 2003). This study systematically compared these three modalities to determine ischemic lesion volumes in the hyperacute stages of experimental stroke. Although DWI is more widely used to select patients for stroke trials and in daily

186

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practice, our findings suggest that ADC analysis may provide distinct advantages over DWI analysis. Firstly, in this experimental model ADC-derived lesion volumes predicted final infarct volumes more accurately. Secondly, interrater agreement was better with ADC analysis. Thirdly, reperfusion effects were only reliably seen by ADC analysis. Outcome measures used in stroke studies are typically infarct size and functional status. Increasingly MRI has been used to provide clinicians with surrogate markers for these measures to guide clinical decision making (Hacke et al., 2005; Quinn et al., 2008). For example, good correlation between DWIderived lesion volumes and final infarct volume has been previously demonstrated (Barber et al., 1998; Rivers et al., 2006). However, it has been suggested that ADC mapping may provide more detailed information on ischemic tissue fate than DWI and may predict final infarct volume more accurately than DWI (ElKoussy et al., 2002; Oppenheim et al., 2001). Indeed, our results corroborate this notion and extend previous findings by showing high intermodality agreement between vADC and tADC indicating that both methodologies provide analogous information. Nevertheless, ADC thresholding may be the preferred methodology, as it is rater independent and can be performed by automatic segmentation techniques obviating the need for time consuming, manual lesion volume delineation. In experimental studies it has been shown that ischemic tissue fate can be reliably assessed by calculating tissue ADC (Bardutzky et al., 2007; Henninger et al., 2006). Furthermore, it has been suggested that ADC may provide more detailed information than DWI and the latter may underestimate final lesion size (Oppenheim et al., 2001). Indeed, we demonstrate that DWI-derived lesion volumes underestimate final infarct extent. Arguably, since we used a surface coil, reduced signalto-noise in the basal (i.e., the brain tissue most distant from the coil) brain tissues may have caused us to “miss” ischemic tissues on DWI and thus biased the results towards smaller lesion volumes. However, post-hoc comparison with ADC maps did not demonstrate preferential reduction in the DWI lesion volumes in basal parts of the brain. In fact, DWI lesions were “concentrically” reduced indicating that peripheral (penumbral) tissues were the ischemic area most likely to be misclassified on DWI analysis. Therefore we believe that our findings are valid and a fair approximation of the pathophysiologic DWI changes associated with focal cerebral ischemia. By systematically assessing the spatiotemporal evolution in different models of cerebral ischemia we further showed that ischemic tissue damage as assessed by ADC, but not DWI, may partially reverse after up to 180 min of transient ischemia. This finding is important as it extends the previously noted reversibility threshold for ischemic tissue damage of ∼95 min (Bardutzky et al., 2007; Minematsu et al., 1992; NeumannHaefelin et al., 2000) to ∼180 min. This indicates that in addition to the ischemic penumbra (as approximated by reduced perfusion but normal ADC/DWI), at least some parts of the ischemic (abnormal) ADC or DWI lesions may be amenable to late reperfusion therapies indicating that these tissues with abnormal ADC/DWI should not be thought of as entirely irreversibly injured. Further studies are warranted to assess the significance of early vs. late injury reversal, particularly with respect to previously demonstrated secondary declines (Henninger et al., 2006; Li et al., 2000; Sicard et al., 2006a) and yet

unclear consequence for ultimate (tissue) function (Kidwell et al., 2002; Sicard et al., 2006b). Regardless, understanding underlying mechanisms and subsequent imaging correlates may provide additional targets for interventional therapies. Though enlightening, our study is limited in several respects. Firstly, cerebral perfusion was not assessed. As previously mentioned, the perfusion/DWI (or ADC) mismatch, is thought to approximate the ischemic penumbra, which is the main target of reperfusion therapies. Analysis of interaction in the degrees of perfusion restriction with accompanying ADC or DWI changes may therefore provide specific tissue signatures that allow for more accurate prediction of tissue fates. Secondly, our analysis focused on the spatiotemporal lesion evolution rather than regional changes in DWI or ADC maps. It would be interesting to follow the time course of ADC/ DWI in tissue compartments of the brain that are typically involved with focal cerebral ischemia to help define potential tissue fates within tissues with abnormal ADC/DWI values (Henninger et al., 2006). Lastly, we opted to exclusively acquire DWI during the hyperacute phase following MCAO, and only TTC at 24 h. However, this was done intentionally because the effect of vasogenic edema, which typically occurs at later time points (Baird et al., 1997; Barber et al., 1998) may bias results by inducing “pseudonormalization” of ADC and “T2-shine through” on DWI.

3.1.

Summary

In conclusion, our data indicates that ischemic lesion determination by ADC has distinct advantages over DWI in that it is (i) more accurate in final infarct prediction, (ii) less rater dependent, and (iii) provides exclusive information on ischemic damage reversibility.

4.

Experimental procedures

4.1.

Animal preparation

All procedures used in this study were performed in accordance with our institutional guidelines and all experiments were performed in randomized manner. To alleviate pain, animals received 0.05 mg/kg subcutaneous buprenorphine immediately as well as 6 h after the end of anesthesia. Spontaneously breathing male Sprague Dawley rats (n = 27, Taconic Farms, Hudson, NY, USA) weighing 300 g ± 10% were anesthetized with isoflurane (5% for induction, 2% for surgery, 1.2% for maintenance) in room air. PE-50 polyethylene tubing was inserted into the femoral artery for monitoring of mean arterial blood pressure (MABP) and for obtaining blood samples to measure blood gases (pH, PaO2, PaCO2), electrolytes (Na+, K+, Ca2+), and plasma glucose at prior to as well as 90, 180 and 270 min after MCAO. Right MCAO was produced using 4-0 silicon-coated monofilament sutures as previously described (Bouley et al., 2007). In the transient MCAO groups blood flow was reestablished by withdrawing the suture occluder remotely in the MRI scanner 90 or 180 min following MCAO, respectively (Henninger et al., 2007). Neurological evaluation was performed at 5 and 24 h as previously described (Menzies et al., 1992).

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4.2.

Study design

This study consisted of three groups with n=9 each: Permanent MCAO (pMCAO), 90 min of transient MCAO (90-tMCAO), and 180 min of transient MCAO (180-tMCAO). Animals of all groups were imaged at 30, 60, 90, 120, and 180 min after MCAO. Rats in the 180-tMCAO group were additionally imaged at 210, 240, and 270 min. Animals were electively sacrificed at 24 h post-MCAO for 2,3,5-triphenyltetrazolium chloride (TTC) staining (Li et al., 1997).

4.3.

MRI settings

MRI experiments were performed on a 4.7 T/400 mm horizontal magnet equipped with a Biospec Bruker console (Billerica, MA), and a 2.0 G/mm gradient insert (inner diameter = 120 mm, 120 μs rise time). A surface coil (inner diameter= 23 mm) was used for brain imaging (Meng et al., 2004). Three ADC maps were separately acquired with diffusion-sensitive gradients applied along the x, y, or z direction (Meng et al., 2004). Single shot, echoplanar images were acquired over 2.5 min with matrix = 64× 64, spectral width = 200 kHz, TR= 2 s (90° flip-angle), TE= 37.5 ms, b = 8 and 1300 s/mm2, Δ = 24 ms, δ = 4.75 ms, field of view= 25.6× 25.6 mm, seven 1.5 mm slices, and 16 averages (Henninger et al., 2006).

4.4.

Calculation of in vivo lesion size

Images were analyzed using STIMULATE (University of Minnesota) and QuickVol II (http://www.quickvol.com) (Schmidt et al., 2004). Quantitative ADC maps and their corresponding threshold-derived lesion volumes were calculated as described previously (Meng et al., 2004). Two investigators, one experienced and one inexperienced in MRI-data analysis who were blinded to groups and time points, manually outlined ischemic lesion volumes on ADC maps and DWI to assess interrater agreement. For visual lesion volume determination, hyperintense regions on DWI (using the images with high b values and all three directions) and hypointense regions on ADC maps were outlined after optimization of contrast and brightness. The threshold for defining ischemic tissue on ADC maps was 0.53 ± 0.03 × 10− 3 mm2/s as previously validated (Meng et al., 2004). Corresponding DWI and ADC lesion volumes were then calculated by summing the abnormal area on each slice and multiplying by the slice thickness. Lesion volumes determined by the different methodologies were correlated with each other. Lastly, imaging-derived lesion volumes were correlated with 24 hour TTC-derived infarct volumes.

4.5.

Sacrifice and infarct volume analysis

At 24 h post-MCAO, animals were electively euthanized by an overdose of pentobarbital (200 mg/kg, i.p.). Brains were sectioned coronally into seven 1.5 mm-thick slices corresponding to the MR slices and stained with TTC for ex vivo infarct volume calculation with edema correction (Henninger et al., 2007). Animals dying prematurely between 16 and 24 h after stroke were included in the data analysis, as TTC-derived infarct volumes are accurate at these time points (Takano et al., 1997). All histological analyses were performed by investigators blinded to the experimental protocol.

4.6.

187

Statistical analysis

Data are presented as mean ± standard deviation unless otherwise stated. Statistical evaluations (SPSS, v15.0, Chicago, USA) were performed using Spearman correlation, Wilcoxon Test and Fisher's Z. p < 0.05 was considered statistically significant. Bland–Altman plots (Medcalc, v9.3.8.0, Mariakerke, Belgium) were used to assess agreement between raters and modalities (Bland and Altman, 1986). Bland–Altman plots depict the averages of two individual variables (x-axis) versus differences between these variables (y-axis).

Acknowledgments We thank Teri Kleinberg for help with data processing and helpful comments. The authors declare no conflict of interest. This study was funded by institutional grants.

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