ADC characterization of region-specific response ... - Semantic Scholar

Magnetic Resonance in Medicine 47:562–570 (2002) DOI 10.1002/mrm.10103

ADC Characterization of Region-Specific Response to Cerebral Perfusion Deficit in Rats by MRI at 9.4 T Victor E. Yushmanov,1 Lei Wang,1 Serguei Liachenko,1 Pei Tang,1,2 and Yan Xu1,2* Region-specific cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) of water in the rat brain were quantified in vivo by high-field MRI (9.4 T) for 6 –7 h after middle cerebral artery occlusion (MCAO). Upon occlusion, average CBF fell from about 1.5–2 ml/g/min to below 0.5 ml/g/min in cortical areas and the amygdala, and below 0.2 ml/g/min in the caudate putamen. CBF in some of the homologous contralateral areas also decreased by 20 –30%. Average ADC decreased from about 8 䡠 10– 4 to 5 䡠 10– 4 mm2/s in the caudate putamen and parietal cortex. Corresponding changes in ADC were lower in the frontal cortex and negligible in the piriform cortex, suggesting that the perfusion threshold for ADC decrease may be different for different brain regions in the same animal. The area of decreased ADC correlated well with the infarction area revealed by 2,3,5-triphenyltetrazolium chloride (TTC) staining of brain slices in vitro. A better understanding of the mechanisms linking ADC and CBF changes to ischemic cell disorders may prove useful in characterizing the degree of tissue damage, and in developing and evaluating treatment strategies. Magn Reson Med 47:562–570, 2002. © 2002 Wiley-Liss, Inc. Key words: focal cerebral ischemia; magnetic resonance imaging; arterial spin tagging; apparent diffusion coefficient (ADC); cerebral blood flow (CBF)

The apparent diffusion coefficient (ADC) of water, which is measurable noninvasively by MRI, has been proposed as a reliable parameter for early detection of brain ischemia (1–3). ADC was shown to be sensitive to acute ischemia within minutes of occurrence (4). Currently, the intraluminal thread model of middle cerebral artery occlusion (MCAO) in rats (5–7) is generally accepted as a standard model of focal cerebral ischemia. In this model, cerebral blood flow (CBF) measurements are usually carried out to determine adequate occlusion (in both permanent and transient MCAO models) and reperfusion (in the transient model) (8 –14). When describing quantitative changes in cerebral perfusion and ADC values, data are usually reported as averages over 1) very small local regions (of several pixels in size) inside the involved brain structures (9,11), or 2) the whole lesion region, with reference to the symmetric contralateral region or to the whole contralateral hemisphere (12,15). In perfusion and ADC studies with the MCAO model in rats, two issues deserve further elaboration. First, the detailed regional dynamics of ADC and CBF, averaged over specific macroscopic brain regions, remains for the most

1 Department of Anesthesiology and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. 2 Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Grant sponsor: NINDS, NIH; Grant number: 1R01NS36124. *Correspondence to: Dr. Yan Xu, University of Pittsburgh, W-1358 Biomedical Sciences Tower, Pittsburgh, PA 15261. E-mail: [email protected] Received 14 May 2001; revised 21 September 2001; accepted 12 November 2001.

© 2002 Wiley-Liss, Inc.

part unexplored. Second, variability in experimental approaches and animals used in the studies results in a certain diversity of experimental findings. Even more important is that the definition of occlusion based on perfusion measurements is not always sufficiently rigorous, and may lead to different cellular responses in the occluded area. To characterize ischemic damage more quantitatively, stringent criteria are needed. An ADC-based definition of occlusion appears to be more adequate, since it is associated with cellular response to ischemia. This feature also makes ADC a suitable candidate for clinical use. CBF measurements are usually performed by laser-Doppler flowmetry (8,14,16) or perfusion-weighted magnetic resonance imaging (MRI). Qualitative perfusion-weighted images may be produced using paramagnetic contrast agents and echo-planar imaging (EPI) (bolus-tracking), and relative CBF indexes may be obtained (11–13). An alternative technique has been developed that uses arterial spin tagging and thus does not need any exogenous contrast agents (17). The arterial spin tagging technique was shown to be suitable for noninvasive quantitative measurements of CBF (17–19). MRI at high magnetic field (7 T and above) ensures better sensitivity in perfusion measurements (20). The variability in magnetic fields, however, may contribute to lower consistency in results obtained, especially when the difference in magnetic relaxation times is concerned. Variability in animal responses to ischemia has several manifestations. It has been shown that rats of different strains and sizes may yield different results in the MCAO model (10,21). There is a general consensus, however, that the lateral caudate putamen is heavily involved with ischemic injuries. Histologic analysis (22), 2-[14C]deoxyglucose autoradiography (23), and diffusion-weighted MRI (9,11) have also revealed significant involvement of the frontoparietal cortex with ADC decreases. The ADC changes in the frontoparietal cortex, however, were absent in a slightly different MRI setting (15). In some experiments, no lesion was apparent in T2-weighted imaging before reperfusion (12). In others, both early T2 decrease and later T2 increase were observed after transient MCAO in rats (11,24) and permanent MCAO in rats (15) and mice (25). In the transient MCAO model, full recovery of ADC to preocclusion levels usually occurs after 30 – 60 min of reperfusion if occlusion time is less than 30 min; partial recovery is expected if occlusion time does not exceed 90 min, and no recovery until 2–7 d with longer occlusion times (9,11,12,24). ADC recovery may be followed by secondary decline in ADC values (12). In the extreme case, variability in animal responses has been observed even within the same groups of animals: ADC recovers in some but not in others (12,24), and CBF recovers immediately,

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or delays until 2–24 h in the reperfusion period (9). The cause for such variability deserves further investigation. In the present work, we made a quantitative, regionspecific comparison of CBF and ADC in the Sprague-Dawley rat brain during the first 6 –7 h after MCAO, using high-field MRI at 9.4 T. High magnetic field is beneficial for sensitivity in perfusion measurements, and is critical for prospective applications of spectroscopic MRI. Changes in ADC—rather than CBF, as conventionally done—were used as an index for successful occlusion, based on the fact that ADC changes are closely related to an immediate tissue response caused by ischemic damage to the brain. METHODS The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Seventeen male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing 160 –220 g were used. Surgical Procedures Rats were given free access to food and water until the time of the experiment. Anesthesia was induced with 4% isoflurane in a 1:1 mixture of O2 and N2O, and was maintained with 1.25% isoflurane in O2 (350 ml/min)/N2O (450 ml/min) during surgery and 1.00 –1.25% isoflurane in a 1:1 mixture of O2 and air in the magnet during MRI scans. Animals were prepared as described in our earlier work (26). In brief, rats were intubated intratracheally with a plastic catheter, and mechanically ventilated (tidal volume of 1 ml/100 g, 40 strokes/min) using a Harvard rodent ventilator (model 683; Harvard Apparatus, Holliston, MA). Rats received pancuronium bromide (2 mg/kg/h (27–29)) for muscle relaxation. Both femoral arteries were exposed, separated from fascial tissues, and cannulated with PE-50 polyethylene tubing (Becton-Dickinson, Sparks, MD) for monitoring of arterial blood pressure and blood gases and pH. Published procedures (22,30) for MCAO in rats were followed. The rats were placed in the supine position. Under an operating microscope Leica S6E (Leica Microsystems, Bannockburn, IL), a median incision of the neck skin (about 2–3 cm) was made, and the left carotid artery was exposed with careful conservation of the vagus nerve. The branches of the left external (ECA) and internal carotid (ICA) arteries were ligated. A 5-0 silk suture was tied loosely at the origin of the ECA and ligated at the distal end of the ECA. A curved microvascular clip was placed at the left common carotid artery and ICA. To block the origin of the middle cerebral artery (MCA), in 13 of the 17 rats a small puncture was made at the distal end of the ligation in the ECA, and the occluder thread was introduced into the ECA lumen until it could not be advanced further (the thread-traveling distance was about 19 mm). The occluder was a Monosof 3-0 or 4-0 surgical nylon monofilament (USSC, Norwalk, CT) with tips rounded by heating or plastic coating to the diameter of 0.22– 0.26 mm. The suture around the origin of the ECA was tightened around the intraluminal thread to prevent bleeding, and

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the clip on the left common carotid artery was removed. The incision was then closed. Four sham-operated animals underwent the same procedure, except that no puncture and insertion of the occluder into the ECA was made. The animals were subjected to MRI study for 6.5 h after MCAO. After the experiment, in all but six rats the brains were rapidly excised for 2,3,5-triphenyltetrazolium chloride (TTC) staining to verify the location and size of the brain infarct. In the three other rats, the catheters were removed and surgical cuts were sutured under general anesthesia (isoflurane 1.5%). The latter were mechanically ventilated, extubated upon return of spontaneous breathing, and given free access to food and water until they were killed 2–3 d later for staining with hematoxylin and eosin to assess the microscopic cytopathology (26). Physiological Monitoring The arterial blood pressure was monitored using an arterial catheter (2.6 m long, to accommodate in-magnet experiments) connected to a blood pressure transducer (model 53-DTS-260; Baxter Healthcare, Irvine, CA) and a Grass 7D polygraph recorder (Grass Instruments, Quincy, MA) interfaced through a LabNB acquisition board (National Instruments, Austin, TX) to a custom-developed application in LabVIEW 5.1 (National Instruments, Austin, TX). The arterial blood pH, PCO2, and PO2 were determined after placing the rat into the magnet, and several times during the experiment using a Ciba Corning 278 blood gas system (Ciba Corning Diagnostics, Medfield, MA). Ventilation parameters were adjusted to maintain PCO2 at 35– 45 mm Hg. The rat body temperature, measured by a rectal temperature probe (YSI 402; Yellow Springs Instruments, Yellow Springs, OH), was maintained at 37.0 ⫾ 0.3 °C using a feedback-controlled airheating blanket. MRI MRI experiments were carried out using a Chemagnetics CMXW-400SLI NMR spectrometer (Varian Inc., Fort Collins, CO) equipped with a 9.4 T, 111-mm bore superconducting magnet (Magnex Scientific, Concord, CA), 72-mmbore gradient coils, and an imaging kit, operating at 401.10 MHz for 1H. A dedicated NMR probe (31) with a birdcage RF transmitter and receiver coil was used. Rats were positioned in a specially designed cradle inside the probe. The imaging plane was adjusted using conventional spin-echo imaging pilot scans (echo time (TE) ⫽ 30 ms, slice thickness ⫽ 2 mm, 32 in-plane phase-encoding steps, one acquisition, total acquisition time ⫽ 32 s) (Fig. 1) so that the cross-sections for perfusion and diffusion imaging are approximately at brain level 26-28 (if not stated otherwise), according to the anatomy atlas of the rat brain (32). Noninvasive quantitative measurements of the CBF were carried out using the arterial spin-labeling technique (17). An otherwise standard 2DFT spin-echo imaging pulse sequence (with TE ⫽ 14 ms) was modified by addition of an adiabatic arterial spin-tagging pulse, which was 0.8 s in duration and 14 ␮T in field strength, and applied in the presence of a 12 mT/m gradient of magnetic field.

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Data Analysis The T2-weighted images, raw images with arterial tagging, diffusion-weighted images, tissue perfusion, and ADC maps were generated and visualized, and the average values of CBF and ADC over selected brain regions were calculated by custom-written applications using the LabVIEW 5.1 programming software (National Instruments). Perfusion maps were calculated pixel by pixel using the standard equation (17): f ⫽ ␭共M cont ⫺ Mtag兲/2␣T1 Mcont,

FIG. 1. Spin-echo image of the rat brain. Images were acquired individually as anatomic references. Inset: an anatomic brain mask used for image analysis. Regions a– g represent the frontal cortex, parietal cortex, caudate putamen, thalamus, hypothalamus, piriform cortex, and amygdala, respectively. Homologous regions are also taken in the contralateral hemisphere. See text for imaging details.

The relatively low tagging power, which still satisfies the adiabatic condition, was chosen to minimize the power deposition and local tissue heating. The arterial tagging plane was 14 mm away from the imaging plane at the neck level. A 50-ms delay period was added between the end of tagging and the image acquisition. The nontagged images were acquired with the same adiabatic pulses applied on the opposite side relative to the imaging plane at approximately the same distance by changing the sign of the offset frequency, adjusted so as to compensate the magnetization transfer effects (i.e., complete signal cancellation on dead rats). Sine-shaped crusher gradients with amplitude of 180 mT/m and duration of 5 ms were applied in three orthogonal directions at the end of each acquisition. Given the following parameters: two acquisitions, pulse repetition time (TR) ⫽ 1 s, 64 phase-encoding steps, 128 data points per step (matrix size zero-filled to 256 ⫻ 256), and square field of view (FOV) ⫽ 50 mm, the total acquisition time per image was 2.5 min. The tagged and nontagged images were acquired alternately in a train of six images, and perfusion maps were reconstructed from each adjacent pair, as described below. ADC in the brain was measured using a standard 2DFT spin-echo imaging experiment (TR ⫽ 1.3 s, TE ⫽ 25 ms, two acquisitions, 64 phase-encoding steps, 128 data points per step, zero-filled to 256 ⫻ 256 matrix size, FOV ⫽ 50 mm) modified to include two 5-ms diffusion-weighting gradient pulses, one on each side of the refocusing 180° pulse, applied along the readout (y) axis. The diffusionweighting gradient was incremented to obtain a series of five images with gradient strengths of 0, 36, 91, 146, and 182 mT/m, and corresponding diffusion-weighting factor (b) values of 0, 35, 220, 560, and 880 s/mm2, so that the total scan time for the entire sequence was approximately 14.5 min. The anatomic spin-echo T1/T2-weighted images were retrieved from corresponding ADC experiments (the image without additional diffusion weighting).

[1]

where f is the CBF, ␭ is the brain– blood partition coefficient for water (0.9 ml/g for rat brain (33)), T1 is the spin-lattice relaxation time of tissue water protons, ␣ is the degree of arterial spin inversion, and Mcont and Mtag are the pixel intensities of control and tagged images, respectively. The degree of spin inversion (the ␣ value), determined under current experimental conditions as described by Zhang et al. (34), was 0.46 for rats subjected to brain ischemia, and 0.48 for normal or sham-operated rats (35). These measured ␣ values are in agreement with the expected values (34) for the radiofrequency power level of 14 ␮T used in the experiments. Regional T1 maps for normal and ischemic brains were obtained earlier (36), and the average T1 values for different regions are given in Table 1. For different anatomic structures of normal brain, T1 varied between 1.79 and 1.97 s; it increased to between 1.96 and 2.16 s in the ischemic tissue. These regional T1 values are in good agreement with published data at comparable magnetic fields (15,24), and were used in the CBF calculations. Diffusion-weighted images were analyzed on a pixel-bypixel basis by fitting the image intensity M as a function of the b value using an exponential function (37): M/M 0 ⫽ exp共⫺b 䡠 ADC兲,

[2]

where M and M0 are the pixel intensities of the image in the presence and absence of the diffusion-weighting gradient, respectively. For quantitative region-specific analysis of MRI data, an anatomical mask was generated for each individual rat using digital images from a stereotaxic rat brain atlas (32). The masks were digitally scaled to fit the spin-echo image of each individual brain according to its position and size using Adobe Photoshop software (version 5.0.2; Adobe Systems, San Jose, CA). The area of ischemic damage was

Table 1 T1 Values (s) in Different Anatomical Regions of MCA-Occluded (Ipsilateral) and Intact (Contralateral) Areas of the Rat Brain

Frontal cortex Parietal cortex Caudate putamen Thalamus Hypothalamus Piriform cortex Amygdala

Ipsilateral

Contralateral

2.126 2.126 2.157 1.959 1.996 2.157 2.157

1.945 1.945 1.972 1.794 1.871 1.972 1.972

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FIG. 2. Images of the rat brain in the MCAO experiment: (a– c) middle section of the brain, (d–f) anterior section of the brain of another rat, and (g–i) an example of unsuccessful occlusion defined by lack of ADC change and confirmed by TTC staining. a, d, and g: Perfusion maps taken 60 min after MCAO, showing reduced flow in the occluded side; color scale units are ml/g/min. b, e, and h: ADC maps taken 60 –230 min after MCAO; color scale units are 10–3 mm2/s. Note a low ADC region corresponding to CBF occluded area in images b and e, and the absence of ADC changes in h. c, f, and i: Photographs of corresponding brain slices stained by triphenyltetrazolium chloride 7 h after MCAO. Note in c and f the absence of staining in the infarction area corresponding to the areas with low ADC and in i the absence of lesion. See text for imaging details.

determined by counting pixels with ADC values below 5 䡠 10– 4 mm2/s in the ipsilateral brain hemisphere. This ADC threshold provided the best correspondence between infarcted areas determined from ADC and TTC staining data. Statistical analysis was performed using SPSS software (version 10.0.5; SPSS, Chicago, IL). Repeated-measures ANOVA was used to compare arterial blood pressure and heart rate between groups of animals, and CBF and ADC values between injured, contralateral, and control tissues over time. All data are presented as mean ⫾ SD if not stated otherwise. Histological Examination For TTC staining, brains of anesthetized rats were rapidly removed and sliced into transverse sections at 2-mm intervals using a plastic rat brain matrix (Harvard Apparatus, Holliston, MA). Slices were immediately stained with 2% TTC in phosphate-buffered saline (pH 7.4) at 37°C for 20 min (38). Stained sections were fixed in phosphatebuffered 10% formalin before the morphometric analysis, and photographs of these sections were analyzed for infarct size by the same investigator. Staining with hematoxylin and eosin was performed in three rats according to conventional procedures (26). RESULTS The ADC decrease has been established to be an early and reliable manifestation of brain ischemia (1–3); therefore, we used the presence or absence of any change in the ADC in the MCA-supplied area of the brain as a preliminary

index to discriminate between animals with successful or unsuccessful (incomplete) MCAO. Incomplete MCAO was observed in four of the 13 rats. This classification was confirmed later by histological examination (TTC staining). One of the successfully occluded animals developed a subarachnoid hemorrhage, and another one died of cardiac failure after surgery. Those animals with incomplete occlusion, hemorrhage, or failed surgical outcome were excluded from the study. In total, seven experimental and four sham-operated rats were taken for the final analysis. The mean arterial blood pressure of the rats after MCAO was stable during the entire observation period, and did not differ from the blood pressure of sham-operated animals (99 ⫾ 8 and 99 ⫾ 9 mm Hg, respectively; P ⫽ 0.662 by the repeated-measures ANOVA). The heart rate was also similar among groups (350 ⫾ 40 min–1 after MCAO and 360 ⫾ 40 min–1 in the sham animals; P ⫽ 0.830). A typical MRI scan of the rat brain is presented in Fig. 1. The anatomic spin-echo image was used as a reference for localization of the regions of interest in the MRI study. For quantitative region-specific analysis of MRI data, brain masks were used. The inset in Fig. 1 depicts such a mask, with 14 regions marked on the mask for analysis (seven in the ipsilateral hemisphere, and seven homologous regions in the contralateral hemisphere). The selected regions include the frontal, parietal, and piriform cortex, and four core regions: the caudate putamen, thalamus, hypothalamus, and amygdala. The choice of regions was based on the fact that various combinations of these regions appeared damaged by MCAO on ADC maps and after TTC staining.

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FIG. 3. The correlation between areas of ischemic damage determined by triphenyltetrazolium chloride (TTC) staining 7 h after MCAO, and derived from ADC data in the same rat 1– 4 h after MCAO (N ⫽ 8).

Figure 2 (a, d, and g) shows typical appearances of perfusion maps after MCAO. Extensive areas of low perfusion are readily seen in the left hemisphere of the brains. In rats with successful MCAO, these areas correspond to the low-ADC regions, as shown in Fig. 2b and e. The absence of ADC change immediately after the MCAO procedure is used as a stringent criterion for unsuccessful occlusion, even when marked reduction in CBF can be detected. An example of this situation is shown in Fig. 2h. To confirm the location and size of ischemic lesions (or the absence of such), TTC staining was performed in most of the animals. Staining results match nicely with the ADC maps for all animals, as shown in Fig. 2. To validate this observation quantitatively, the infarction areas were determined from TTC staining and ADC data in a larger group of rats, including rats with different MCAO durations. (A larger group was needed to increase the number of available staining samples in order to reach statistical significance.) Damaged regions obtained by the two methods showed significant overlap in the MCA-supplied area in the ipsilateral hemisphere. A significant correlation was observed between the sizes of the damaged regions (Fig. 3). Staining by hematoxylin and eosin performed in one successfully occluded rat 54 h after MCAO confirmed a high degree of ischemic damage to neurons in the affected area. No histological abnormalities were seen in the rats with incomplete MCAO. The ischemia-induced changes in ADC varied among the rats. In six animals, the caudate putamen was mostly involved, whereas in one animal it was almost not involved. Different cortical areas were involved in all animals to a different degree. The hypothalamus was moderately involved in six of the seven rats. The lowest ADC values across the brain reached about 3 䡠 10– 4 mm2/s, compared to the values between 7 䡠 10– 4 and 10–3 mm2/s for normal brain tissues. Figure 4 presents the time courses

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of ADC for three cortical areas of the brain after MCAO, as well as for the striatum and other core areas (caudate putamen, thalamus, hypothalamus, and amygdala), obtained using the mask shown in Fig. 1, and averaged over all seven animals. Compared to the contralateral regions, a statistically significant decrease in ADC after occlusion was observed in all studied brain areas, except for the piriform cortex (Fig. 4f). Overall, the decrease in ADC is most pronounced in the parietal cortex, caudate putamen, thalamus, and amygdala (Fig. 4b– d, and g, respectively). The normal and depressed ADC values in the contra- and ipsilateral hemispheres, respectively, did not vary significantly over time. Presented in Fig. 5 is the dynamics of brain perfusion obtained in different areas of the brain after occlusion. It can be seen that although all brain areas on the occluded side suffered the circulation impairment to some extent, the most involved areas were the caudate putamen, amygdala, and cortex (Fig. 5 a– c, f, and g), and no CBF recovery was seen over the course of the experiment (6.5 h). Given the established role of ADC as an indicator of brain ischemia, it comes as no surprise that both the CBF impairment and the drop in ADC were most pronounced in the

FIG. 4. ADC time course for different regions of the rat brain after MCAO. Charts a– g correspond to regions a– g in Fig. 1, respectively. Open circles, ipsilateral hemisphere; closed squares, contralateral hemisphere. The time of occlusion is defined as time zero. All data are averaged over seven animals. Error bars are SD. The significance of difference between hemispheres in the time range of 75–270 min is (b– d and g) P ⬍ 0.001, (a and e) P ⬍ 0.05, and (f) P ⬎ 0.5 (no difference) by repeated-measures ANOVA.

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amygdala (Fig. 7a, c, and g, respectively) in the shamoperated group as compared to the corresponding contralateral (nonoccluded) areas of the MCAO group. No statistically significant changes in ADC were observed in either brain hemisphere of the sham-operated rats over time. Note that the ADC values in the parietal and piriform cortex (Fig. 6b and f, respectively) and the hypothalamus (Fig. 6e) of the sham-operated rats are somewhat higher than in the corresponding areas in the contralateral hemisphere of rats with MCAO. The signal intensity in the anatomic spin-echo images depends on both T1 and T2 of the protons in the brain. However, Table 1 shows that very small changes in T1 values occur upon ischemia. Therefore, the signal intensity in the spin-echo images reflects mainly the T2 changes, with the contribution of T1 changes being limited to about 7%. In four of the seven MCAO animals included in the analysis, regions of lower intensity (by about 15– 35%) were clearly observed in the ipsilateral cortex, caudate putamen, and amygdala for 190 –230 min postMCAO, as compared to the contralateral ones, whereas a decrease in T2 in the other three animals was not evident. This effect was reversed in a long-term study: we observed T2-hyperintensity (by about 10 – 45%) in the ipsilateral frontoparietal cortex, caudate putamen, and thalamus of five rats subjected to 85-min transient MCAO after 24, 48,

FIG. 5. CBF time course for different regions of the rat brain after MCAO. All data are averaged over seven animals, approximately five images per animal per time point. Significance of difference between hemispheres in the time range of 55–310 min is (b, c, f, and g) P ⬍ 0.001 and (a, d, and e) P ⬍ 0.05 by repeated-measures ANOVA. See legend of Fig. 4 for further details.

same areas (the caudate putamen, parietal cortex, and amygdala). However, a very significant perfusion decrease in the piriform cortex (Fig. 5f) did not necessarily lead to an ADC decrease in all animals, and no statistically significant changes in the ADC value in that area were observed (Fig. 4f). A correlation analysis of percent change in ADC vs. that in CBF did not reveal a significant correlation between the two parameters (R ⫽ 0.294, P ⬎ 0.5). Figures 6 and 7 show the ADC and CBF data for the same brain regions of four sham-operated rats. A comparison of Figs. 5 and 7 shows that, apparently, there was a general trend of an initial increase (between 100 and 200 min) and a subsequent decrease in CBF during the experiment, especially in the cortical areas, thalamus, and caudate putamen. This may be related to either the response of the animals to the surgical procedures and prolonged anesthesia (⬃7 h) or to the vertical position in the magnet (as discussed below), or both. Nevertheless, the CBF is somewhat higher in the frontal cortex, caudate putamen, and

FIG. 6. ADC time course for different regions of the brain in shamoperated rats. All data are averaged over four animals. Significance of difference between contralateral hemispheres of experimental (Fig. 2) and sham-operated rats in the time range of 75–270 min is (b, e, and f) P ⬍ 0.05, and (a, c, d, and g) P ⬎ 0.2 (no difference) by repeated-measures ANOVA. See legend of Fig. 4 for further details.

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FIG. 7. CBF time course for different regions of the brain in shamoperated rats. All data are averaged over four animals. Significance of difference between contralateral hemispheres of experimental (Fig. 3) and sham-operated rats in the time range of 55–310 min is (g) P ⬍ 0.05, (a and c) P ⬍ 0.2, and (b and d–f) P ⬎ 0.2 (no difference) by repeated-measures ANOVA. See legends of Figs. 4 and 5 for further details.

72, and 100 h of reperfusion, and in one rat after 48 h of MCAO (data not shown). DISCUSSION The most important findings of the present work include the quantitative, region-specific characterization of CBF and ADC in the rat brain during the first 6 –7 h after MCAO. This approach, together with the use of an ADC (rather than CBF) decrease as a more rigorous criterion of occlusion, allowed us to gain a clearer understanding of the difference between brain regions in their ADC changes and the underlying perfusion deficits upon MCAO, and also to show that the same degree of the CBF impairment may not necessarily lead to comparable ischemic damage in different parts of the brain. The brain perfusion and ADC data averaged over all animals subjected to MCAO, as presented in Figs. 4 –7, are consistent and statistically significant. However, at the level of individual animals, a certain diversity of individual responses, as given by the standard deviations (SDs) on the plots, was observed. All seven MCAO animals in-

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cluded in the analysis (Fig. 5) showed significant perfusion impairment in the caudate putamen, piriform cortex, and amygdala, whereas in other brain regions the degree of perfusion impairment differed greatly between animals, and was in general lower than in the former three regions. The areas presenting decreased ADC upon occlusion also varied in different rats in our study. In particular, different cortical areas and the hypothalamus were usually, but not always, affected. Other studies have also found this variability. Different methods revealed significant involvement of the frontoparietal cortex with ischemic injuries (22,23), resulting in the decrease in ADC in this region (9,11). However, in slightly different settings, no ADC changes in the frontoparietal cortex were reported, and CBF there remained generally above the level of 20 –30% (15), which is sometimes considered as a maximum residual CBF in cortex after MCAO (14). Variability in individual changes in ADC and CBF within the same groups of animals has also been reported (9,12,24). Changes in ADC are closely connected with cellular ischemic damage. Therefore, these changes were used as an index of successful occlusion. In agreement with numerous reports, MCAO produced using the intraluminal suture model was not 100% successful. The occurrence of inadequate occlusion or intracerebral hemorrhage is well known (8,10,14). It is important to note that the “unsuccessful occlusion” considered in the present work covers a range broader than that defined, e.g., when using laserDoppler flowmetry. Whereas in laser-Doppler studies MCAO is considered unsuccessful if CBF fails to decrease to 20 –30% of baseline (14), in the present work MCAO was considered unsuccessful when it failed to produce any significant decrease in ADC values and did not result in any visible lesion in TTC staining. This definition was used even though CBF in occluded areas might be at or below 10% of baseline according to the perfusion images (i.e., virtually indistinguishable by perfusion MRI from the rats with successful occlusion, as shown in Fig. 2g). Hence, CBF alone may not be a sufficiently reliable index of adequate occlusion. Studies of changes in CBF, T1, and T2 unaccompanied by changes in ADC in a partial MCAO model (oligemia) have been reported recently (39). A region-specific comparison of CBF and ADC changes in MCAO shows significant perfusion impairment in all seven areas in the ipsilateral brain hemisphere (Fig. 5). A statistically significant decrease in ADC after occlusion was also observed in all studied brain areas except for the piriform cortex (Fig. 4). Excluding the piriform cortex, the lowest changes in CBF and ADC were observed in the frontal cortex (usually qualified as ischemic penumbra in this model) and hypothalamus. The study of the MCAO in mice also indicated the presence of a penumbral region, i.e., that the perfusion deficit occupied a larger area than the ADC lesion (25). Recently, a correlation was found between the ADC and perfusion changes in areas with only mild to moderate perfusion deficits toward the lesion periphery in a rat model of MCAO (13). In the ischemic core, the concept of the threshold perfusion suggests that a tiny reduction in CBF near the threshold (e.g., from 0.185 to 0.175 ml/g/min) may cause reversible ischemia to become infarction-inducing ischemia (40). Lesser changes in ADC in the piriform cortex after occlusion, even when CBF

CBF and ADC in Cerebral Ischemia

decreases were significant, suggest that the perfusion threshold for ADC changes may be different not only between animals, but also for different areas in the same animal. This may account for the absence of correlation between changes in ADC and CBF. In the present work, a 20 –30% CBF decline (as compared to the sham-operated group) in the contralateral hemisphere of rats with MCAO has been observed using MRI (Figs. 5 and 7). Previously, laser-Doppler flowmetry detected a CBF decrease in the contralateral hemisphere after MCAO only in the cases of subarachnoid hemorrhage caused by mechanical damage (14). However, other MRI data indicated the possibility of CBF changes in the contralateral brain hemisphere after MCAO over the same time scale (15). Interpretation of the perfusion data requires some methodological considerations. One of these is the fact that animals are in a vertical position inside the magnet bore. CBF may be influenced by this nonphysiological position during relatively long scanning times (30 min to 6.5 h). However, the bulk of MRI data on small animals at high magnetic fields (7 T and above) is being generated using vertical magnets (9,15). Thus, it allows the direct comparison of our data with most related results. Nevertheless, results obtained using different protocols should be compared with caution. For instance, statistically significant anesthetic-dependent variations in CBF values, as well as in the regional distribution of CBF in the rat brain, have been reported recently (41). Another consideration is that absolute values of CBF calculated by Eq. [1] depend on the T1 values and their changes during the experiment. The slight temporal changes in T1 values after MCAO reported recently (15,24) were estimated to affect only marginally the results of CBF calculations from our perfusion data using Eq. [1]. The expected deviation should not exceed about 7%, which falls within the experimental error of the perfusion measurements, and thus should not change the character of the CBF curves presented in Figs. 5 and 7. Therefore, when calculating CBF, possible T1 variations were not taken into consideration, and measurement of small variations in T1 was not pursued. One more factor compromising CBF measurements may be the uncertainty in transit times from the tagging plane to the imaging plane. Longer transit times (and, thus, underestimated CBF) may be expected in areas with low flow, although this factor is not significant in small animal studies at high magnetic fields. One of the consequences of brain ischemia in the medium term (within a few hours) is the development of vasogenic edema due to blood– brain barrier disturbances. The increase in T2 is commonly attributed to this phenomenon. In the present work, the lower T2 regions in the ipsilateral hemisphere as compared to the contralateral one were observed in some of the animals within 3– 4 h of permanent occlusion. T2-hyperintensity was observed in the ipsilateral caudate putamen, frontoparietal cortex, and thalamus in both permanent and transient experiments after 24 –100 h of MCAO (42). In a recent study (15), an early drop in T2 (within a few minutes post-MCAO) followed by a gradual linear increase in occluded areas during about 5 h was also observed. All effects were reported as being within 10 –20%. In a similar mouse model of focal

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ischemia (25), a 1.7-fold increase in signal intensity on the T2-weighted images was observed 5.5 h after MCAO, and a good spatial correspondence between the diffusionweighted images and T2-weighted images of the ischemic territory was found. In some cases, the ischemic-related lesions become identifiable on T2-weighted images only after reperfusion (12). Evaluation of a long-term tissue outcome 48 h after transient 20-min MCAO using T2weighted imaging demonstrated T2-hyperintensity in the ipsilateral caudate putamen, indicating irreversible damage, whereas the cortex showed no change in T2 images (24). These data again highlight variability in the responses of individual animals, as well as, probably, a limited degree of standardization and reproducibility of the model. In summary, using high-field MRI, we performed a quantitative, region-specific comparison of CBF and ADC in the rat brain during the first 6 –7 h after MCAO. We observed different degrees of ischemic lesion in different brain structures that did not always match the relative degree of perfusion deficit. This suggests that the perfusion threshold for ADC decrease may be different for different brain regions. MCAO also resulted in a certain degree of decrease in CBF in several parts of the contralateral brain hemisphere. The combined use of quantitative perfusion and ADC mapping offers a better understanding of the mechanisms of ischemic cell damage, and is useful for characterizing the degree of tissue damage as well as for developing and evaluating treatment strategies.

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