Original Research JOURNAL OF MAGNETIC RESONANCE IMAGING 21: (2005)

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1 JOURNAL OF MAGNETIC RESONANCE IMAGING 21: (2005) Original Research Subcortical Lesions after Transient Thread Occlusion in the Rat: T 2 -Weighted Magnetic Resonance Imaging Findings Without Corresponding Sensorimotor Deficits Susanne Wegener, MD, 1,2 * Ralph Weber, MD, 1 Pedro Ramos-Cabrer, PhD, 1 Ulla Uhlenkueken, 1 Dirk Wiedermann, PhD, 1 Korinna Kandal, 1 Arno Villringer, MD, 2 and Mathias Hoehn, PhD 1 Purpose: To investigate infarct evolution and functional consequences of exclusive subcortical or cortico-subcortical strokes, transient middle cerebral artery occlusion (MCAO) was conducted in Wistar rats. Materials and Methods: MCAO was induced in male Wistar rats ( g) for 60 minutes. Lesion volumes and absolute T 2 times on magnetic resonance imaging (MRI) were assessed 1 and 14 days after MCAO using a 4.7-T MRI animal scanner in conjunction with functional testing (adhesive tape removal, cylinder test, and ledged beam walking). Results: Functional test scores were not distinguishable between sham-operated animals (N 5) and those with exclusive caudoputaminal infarct (N 8; group cp), but showed significant deficits in animals with cortico-subcortical infarction (N 10; group cp ). The cp group had lower absolute T 2 times and a more pronounced reduction in T 2 lesion volume over time than the subcortical component in the cp group. There was no correlation of T 2 lesion size or absolute T 2 times and functional impairment in either group. Conclusion: When judged from functional tests alone, subcortical ischemic lesions may not be diagnosed reliably. Furthermore, T 2 -weighted (T 2 -w) MRI does not well anticipate functional deficits in primarily striatal lesions. Key Words: magnetic resonance imaging; animal models; ischemia; middle cerebral artery occlusion; functional tests; sensorimotor behavior J. Magn. Reson. Imaging 2005;21: Wiley-Liss, Inc. 1 Max-Planck-Institute for Neurological Research, Cologne, Germany. 2 Department of Neurology, University Hospital Charité, Berlin, Germany. Contract grant sponsor: German Ministry of Education and Research (BMBF) (Competence Net Stroke B1, B5, Berlin NeuroImaging Center); Contract grant sponsor: Deutsche Forschungsgemeinschaft (DFG, Klinische Forschergruppe); Contract grant sponsor: BMBF through the bi-national German-Israeli project (DIP Project B5.2). *Address reprint requests to: S.W., Max-Planck-Institute for Neurological Research, Cologne, Gleueler Str. 50, Cologne, Germany. wegener@mpin-koeln.mpg.de Received September 24, 2004; Accepted November 22, DOI /jmri Published online in Wiley InterScience ( TRANSIENT MIDDLE CEREBRAL ARTERY occlusion (MCAO) by an intraluminal thread is a widely employed model to study focal cerebral ischemia in rats (1,2). Extent and severity of the ischemic injury depend on the duration of the occlusion, surgical procedure, animal strain, and time of analysis after ischemia (3). The diversity of data is further complicated by the frequent variability of experimentally induced infarctions (4). This problem could be addressed by introducing standardized inclusion criteria (e.g., certain lesion size or location) using magnetic resonance imaging (MRI). Furthermore, MRI has been successfully employed in animal stroke models to follow the course of ischemic damage and allowed a multiparametric analysis of tissue status (5 10). Ideally, the MR parameter based on which the lesion is characterized reflects final infarct volume, correlates with clinical deficit, and can be determined as early as possible after stroke. T 2 -weighted (T 2 -w) images have been employed for this purpose, since it is known that T 2 -w images provide an estimate of the vasogenic edema and blood-brain barrier disruption indicative of irreversible damage (10 15). T 2 times start to rise as early as 2 hours after the onset of ischemia and peak at around hours after MCAO. Thereafter, values decrease again over the following week, either reaching a stable plateau or rising to a second peak around two weeks after ischemia. This secondary increase in T 2 has been correlated to tissue liquefication and cyst formation (6,15 20). The use of T 2 -w MRI for damage assessment emphasizes the need for correlating this parameter to functional deficits. So far, studies on the relationship between functional deficits and T 2 -w MRI in different experimental animal models are scarce (19 21). Aforementioned studies showed a good correlation between behavioral deficits and T 2 lesion size in models where damage was more severe in the cortex of animals. We wanted to test if a correlation between T 2 -w MRI of the lesion and functional impairment can be established in a transient (60-minute) MCAO model in Wistar rats. Thread occlusion in this animal strain results in pri Wiley-Liss, Inc. 340

2 MRI and Function in Subcortical MCAO Lesions 341 marily striatal infarctions with variable cortical portions (22). Sensorimotor tests were chosen with the goal to allow successful discrimination of cortical and subcortical lesions (23 25). We noted positive striatal T 2 -w MRI findings in the absence of functional deficits and conclude a dissociation between these two diagnostic variables. MATERIALS AND METHODS Animal Model All experiments were performed in accordance with the National Institutes of Health animal protection guidelines and approved by the local governmental authorities. Adult male Wistar rats (N 18) weighing g were anesthetized using face mask inhalation of 1.5% halothane in a 2:1 N 2 O:O 2 atmosphere. Temperature was maintained at 37 C by a feedback-controlled heating pad. Focal ischemia was induced for 60 minutes by intraluminal thread occlusion as described previously (1). In short, a 4-0 silicone-coated filament was introduced into the common carotid artery and advanced about mm from the carotid bifurcation, until ipsilateral laser-doppler flowmetry (LDF) indicated a marked drop in perfusion. For sham surgery (N 5), the thread was advanced only about mm from the bifurcation. Animals were reanesthetized for MRI 24 hours and 14 days after MCAO. MRI MRI measurements were performed on a 4.7-T BioSpec animal scanner with a 30-cm horizontal bore magnet (Bruker BioSpin, Ettlingen, Germany). Radio frequency (RF) transmission was achieved with a Helmholtz coil (diameter 12 cm) and the signal was detected with a 22-mm-diameter surface receiver coil, positioned above the skull of the animal. The system was equipped with actively shielded gradient coils (maximum gradient strength 100 mt/m, gradient rise time 250 sec). Gradient-echo fast-imaging pilot scans were used for accurate positioning of the head in the magnet. The T 2 -w images were acquired with a multislice multiecho Carr-Purcell-Meiboom-Gill (CPMG) sequence: T R /T E 3000/12.5 msec, 16 echoes, FOV 4 4cm 2, slice thickness 1.21 mm, interslice distance 1.5 mm, matrix Image Analysis T 2 -w images were analyzed offline using the software package MRIcro (26). Quantitative T 2 maps were calculated by fitting pixel intensities of the multislice multiecho images (16 echoes, TE 12.5 msec) to a monoexponential decay curve, on a pixel-by-pixel basis. To determine the infarct volume on T 2 -w images, the areas of noninfarcted tissue in each hemisphere were delineated manually and integrated over eight slices. Thereafter, the volume of the ipsilateral noninfarcted tissue was subtracted from the contralateral hemisphere to obtain the swelling-corrected lesion volume. Absolute T 2 times in the subcortical and cortical lesion components were determined by manually outlining these lesion areas on T 2 -w images and transferring them to quantitative T 2 maps. To obtain T 2 times of healthy tissue, regions of interest (ROIs) containing about 100 voxels were drawn on one slice over a contralateral subcortical and cortical region corresponding to the ischemic lesion. Functional Tests Limb Use Asymmetry Test (Cylinder Test) This test evaluates forelimb motor deficits and limb use preference (neglect). Forelimb use during explorative activity was analyzed by videotaping rats in a transparent cylinder for 10 complete rears as described previously (27). Forelimb contact to the cylinder during a full rear was counted and classified as left, right, or double (simultaneous contact with left and right forelimb). Behavior was expressed as percent use of right (impaired) and left (nonimpaired) forelimb relative to the total number of limb use observations and simultaneous use of both left and right forelimbs for contacting the cylinder. Adhesive Tape Removal Test This is a test for forelimb motor and sensory deficits (28). Two strips of tape (18 12 mm) were applied with equal pressure to the saphaneous part of the forepaws, in random order. Animals were observed while removing the tapes in their home cages. Latencies and sides (left/right) of first contact and tape removal were recorded in three trials per session, each lasting no longer than 180 seconds. At least five minutes of rest was allowed between trials. Ledged Beam Walking Test Hindlimb motor deficits are assessed by this test. Foot faults while traversing a narrowing beam were videotaped three times per session and scored as described previously (24). Three segments of the beam with wide, middle, and narrow width were distinguished in the scoring system. At least five minutes of rest was allowed between trials. Statistical Analysis Statistical analyses were conducted using SPSS v11.0 for Windows. LDF values and body weight were compared by independent t-test and one-way analysis of variance (ANOVA), respectively. All data are presented as mean standard error of the mean (SEM). Repeatedmeasures ANOVA was performed on behavioral data over time, except for the comparison of baseline performance between groups (one-way ANOVA). Only when the between-subject factor (groups: sham, cp, cp ) was significant, post hoc testing was carried out using Fisher s least significant difference method (LSD) test. As MR data were shown to be not normally distributed, the Mann-Whitney U test was applied for comparisons. Spearman s r was used for correlation analyses. P values are always given as two sided and exact.

3 342 Wegener et al. Physiological Parameters Groups (cp and cp ) were not distinguishable by the decrease in cortical tissue perfusion as assessed by LDF on the ischemic side during the MCAO period (percent preischemic values: cp, %; cp, %; P 0.4). Weight loss after MCAO, previously shown to correlate with lesion size (19), was most pronounced in cp animals, but there was no significant difference in minimum weight after surgery (sham, %; cp, %; cp, %), compared to baseline weight (P 0.1). T 2 Lesion Volumes and Absolute T 2 Times None of the sham-operated animals showed signal increases on T 2 -w MRI at both time points. T 2 times of healthy tissue were msec in subcortical and msec in cortical ROIs contralateral to the infarction, in agreement with previous reports (11,15). In the cp group, the cortical lesion component comprised mm 3, with T 2 times of msec, and the subcortical component mm 3, with T 2 times of msec (Table 1). From day 1 to day 14, cortical lesion volumes slightly increased in size to mm 3 ( %). The subcortical infarct volume decreased to mm 3 ( %) during the same period. T 2 lesion volumes in cp animals were nonsignificantly smaller than the subcortical lesion compartments in cp animals: mm 3 (vs mm 3 ); P From day 1 to day 14, the lesions in the cp group shrank considerably to mm 3 ( % of the size at day 1). This lesion volume decrease was significantly stronger than in cp animals (P 0.001). T 2 values in the cp group were msec on day 1, which was significantly lower than in cp animals (P 0.001), and declined toward day 14 to msec ( %), which was not significantly different from T2 values in cp animals (P 0.07). Functional Characterization Figure 1. Two lesion types on T 2 -w MRI after 60 minutes of MCAO. T 2 -w images of two representative animals 24 hours after the 60 minutes of MCAO depicting the two different lesion types: cp (restricted to the caudoputamen) and cp (additionally involving the cortex). RESULTS Morphological Lesion Characterization Occlusion of the MCA for 60 minutes produced two characteristic lesion types on T 2 -w MRI at day 1: lesions restricted to the caudoputamen (cp) and lesions involving both caudoputamen and cortex (cp ) (Fig. 1). Baseline sensorimotor test scores where not different between groups in one-way ANOVA. Furthermore, functional test scores were not different between sham and cp groups after ischemia or sham procedure. In contrast, cp animals were impaired in all three functional tests at both time points after stroke induction, but improved toward day 14 (Figs. 2 and 3). In the adhesive tape removal test, both the latency to contact the tape on the right (impaired) forepaw and the latency to remove it were significantly longer in cp animals (P 0.01 on days 1 and 14) than in the sham group (Fig. 2a and b). Even though cp animals contacted the tape on the left (unimpaired) forepaw as quickly as sham animals after ischemia, they were impeded in removing it at day 1. While a prolonged latency to first contact the bothersome tape reflects a sensory neglect, motor deficits of the right forepaw might also have affected removal time for the left-sided tape, be-

4 MRI and Function in Subcortical MCAO Lesions 343 Table 1 Comparison of T2 times and lesion volumes on day 1 and 14 after MCAO for the groups cp and cp Group T 2 volume cortical (mm 3 ) T 2 volume subcortical (mm 3 ) P T 2 times cortical (msec) T 2 times subcortical (msec) Day 1 cp cp P P Day 14 cp cp P P % Day 1 cp cp P P P cause removal strategies were often bilateral. Besides, early after stroke some animals in the cp group were slower in their general motor and explorative behavior. In the ledged beam walking test, right (impaired) hindlimb faults were recorded in cp animals after MCAO on all three segments of the beam, but reached statistical significance, compared to sham animals, only on the wide segment at day 1 (Fig. 3a). Animals of the cp group did not have an increased rate of right hindlimb faults. After MCAO, forelimb asymmetry in the cp group changed toward increased use of the left (unimpaired) forepaw (P 0.01 on days 1 and 14), while limb preference of sham or cp animals was not affected (Fig. 3b). Correlation between T 2 -w MRI and Function To evaluate if T 2 times or T 2 lesion volumes are related to sensorimotor function in the two types of lesion, we conducted a correlation analysis taking the latency to contact the tape on the right forepaw from the adhesive tape removal test as a surrogate parameter (Fig. 4). (Other test parameters, such as latency to remove the tape on the right forepaw and forelimb use asymmetry gave similar results in the analysis.) There was no correlation between MR parameter and functional test score in either group on day 1 (Fig. 4) or 14 (data not shown). This lack of functional correlation was confirmed for both the cortical lesion compartment and the total lesion in cp animals. DISCUSSION T 2 is an imaging parameter commonly chosen to depict irreversible ischemic damage. In our present investigation, it was shown to follow a different course in two types of lesion. In lesions involving both caudoputamen and cortex (cp ), T 2 times remained elevated and T 2 lesion size either increased (cortex) or decreased only slightly (caudoputamen) over two weeks. In lesions restricted to the caudoputamen (cp), T 2 times and lesion size drastically decreased within the two-week observation period. The latter group of animals did not show any sensorimotor deficits on days 1 and 14 after MCAO. Although there are no previous reports about the detailed lesion type, as described in our study, and its characteristics on T 2 -w MRI, the lack of functional deficits was not anticipated for the cp group. The dorsolateral striatum affected by focal ischemia in our model is the main striatal subregion for cortical sensorimotor afferents and contains fiber connections to the substantia nigra (29). Besides, others have reported the induction of locally restricted damage to the striatum Figure 2. Adhesive tape removal test. Latency to contact with (a) or removal of (b) the tape of the left (unimpaired) and right (impaired) forepaw (in seconds) at the three time points of testing: Baseline, 1 day after MCAO (day 1) and 14 days after MCAO (day 14). Bars represent mean and SEM of sham group (empty bars), cp group (striped bars), and cp group (filled bars). # indicates statistical significance P 0.05.

5 344 Wegener et al. Figure 3. Ledged beam walking and cylinder test. a: Scores of the ledged beam walking test on the three time points of testing: baseline (D-0), 1 day after MCAO (D-1), and 14 days after MCAO (D-14) for each of the three beam segments (wide, middle, narrow). Negative scores reflect a higher proportion of faults with the right (impaired) hindlimb, while positive scores indicate faults with the left (unimpaired hindlimb). Bars represent mean and SEM. # indicates statistical significance P b: Scores of cylinder test at the three time points of testing: baseline, 1 day after MCAO (day 1), and 14 days after MCAO (day 14). Positive scores reflect preferred use of the left (unimpaired) forepaw for exploration of the cylinder, while negative scores reflect use of the right (affected) forepaw. Bars represent mean and SEM. # indicates statistical significance P and detected deficits on similar tests of sensorimotor function, thus supporting the notion that our selection of functional tests is suited for the assessment of the injured region (25). However, sustained shrinkage of ischemic lesions to less than half of the initial size on T 2 -w MRI after two weeks has not been described before. Our own observations revealed that exclusively subcortical lesions after 60 minutes of MCAO, as induced in our study, were associated with extensive selective neuronal necrosis and gliosis matching the area of initial signal increase on T 2 -w MRI. Fiber tracts traversing through the damaged area remained largely intact. We believe that this may explain the lack of sensorimotor deficits in cp animals (30). We hypothesize that due to collateral or residual flow at the occlusion site in our rat strain and MCAO model, the ischemic injury was not severe enough to cause functionally relevant tissue damage in the cp group. This is supported by a characteristic pattern of lesion evolution on T 2 -w MRI in this group: 1) lower absolute T 2 times than subcortical portions within infarctions also involving the cortex (cp ) and 2) a strong tendency of T 2 changes to resolve within 14 days. While lesion sizes varied considerably, striatal absolute T 2 values showed a very narrow distribution and no overlap between cp and cp groups: msec vs msec. We suggest that transgression of at 2 threshold of about 90 msec in the caudoputamen is an indicator for infarct extension into the cortex and for the observation of functional impairment in our stroke model. Although sample numbers were rather small for our correlation analysis, it is interesting to note that even when subcortical T 2 values increased above this threshold, there was no correlation of lesion T 2 times or lesion volumes with functional deficits within the cp group. The cp lesions were readily visualized on T 2 -w images after 2 weeks, with similar T 2 times and only slightly decreased lesion volumes, compared to day 1. While the severity of ischemia was apparently sufficient to cause loss of function, the lack in correspondence of T 2 times or lesion volumes with our functional test scores might be due to the primarily striatal infarct location with variable cortical involvement. Potentially, with striatal lesions, test scores do not change homogenously with lesion size. Besides, the myelin-rich structure of the striatum might cause more complex and nonlinear signal changes on T 2 -w MRI that do not allow straightforward conclusions on tissue status (such as signal increase in T 2 -w MRI functionally relevant tissue damage).

6 MRI and Function in Subcortical MCAO Lesions 345 Figure 4. Correlation analysis between T 2 lesions (volumes and T 2 times) with function (latency to contact tape on right forepaw) on day 1 after MCAO. Upper panel: Diagrams depicting the results of correlation analyses (Spearman s r) between absolute T 2 times (msec) and latency to contact the tape on the right (impaired) forepaw in the adhesive tape removal test on day 1 after MCAO for the cp group (a), the subcortical lesion component (b), and the total lesion in the cp group (c). Lower panel: Diagrams depicting the results of correlation analyses (Spearman s r) between latency to contact the tape on the right (impaired) forepaw in the adhesive tape removal test on day 1 after MCAO and volumes (mm 3 ) of the lesion in the cp group (d), the subcortical component of the lesion (e), and the total lesion in the cp group (f). There is conflicting data regarding the relationship between infarct size and functional impairment after experimental ischemia in rats, even with similar functional testing approaches (3). In part, this is due to different experimental models used and varying brain areas injured as a consequence. Other groups have demonstrated that function is diminished more severely with increasing size of the infarction on T 2 -w MRI and higher absolute T 2 times using similar tests of sensorimotor function, as in our study (19,20,23). However, the transient intraluminal suture model applied by Palmer et al (19) and Virley et al (20) in Sprague- Dawley rats resulted in large cortical damage with varying degrees of striatal involvement. Although Lindner et al (23) induced small, primarily subcortical lesions in Sprague-Dawley rats, infarct location was different (medial striatal portions, globus pallidus) and animals were older (16 months of age). The fact that application of a similar ischemic duration and occlusion approach may lead to a number of different lesion types with varying disease characteristics emphasizes the impact of variables such as animal strain and age, housing environment, subtleties of surgical procedures, and test conditions on the stroke model in the individual laboratory. Aside from attempting to establish standardized procedures, each lab should determine the relationship between lesion size and functional outcome within its own experimental setting. To further reveal possible functional impairment caused by a cp type lesion, different functional qualities (such as cognition) or other sensorimotor tests, such as the staircase test (forelimb fine motor control) or the placing test (directed forelimb movements in response to sensory stimulus), could be validated for our stroke model (23). In the primarily striatal stroke model applied here, early T 2 -w MRI signal changes do not necessarily indicate functional impairment. Furthermore, if confined to subcortical areas, lesions on T 2 -w MRI decrease rapidly, and animals are without sensorimotor deficits on functional tests. In conclusion, the clinical outcome predicted from an ischemic lesion on T 2 -w MRI is influenced by many factors, such as location and extent of T 2 value increases and time of analysis. Therefore, when judged from functional tests alone, subcortical ischemic lesions may not be diagnosed reliably. This finding highlights the importance of complementary in vivo MRI monitoring and functional testing for outcome assessment after experimental stroke, in particular for trials studying effective neuroprotection. From a clinical perspective, the present practice of determining stroke size from T 2 -w MRI should be subject to further critical investigation, as it may well be insufficient for diagnosis at early time points and in certain (e.g., subcortical) stroke subtypes. REFERENCES 1. Koizumi J, Yoshida Y, Nakazawa T. Experimental studies of ischemic brain edema, I: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Jpn J Stroke 1986;8:1 8.

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