MATERIALS AND METHODS

All procedures used in this study were carried out in accordance with the U.K. Animals (Scientific Procedures) Act (1986). Twelve adult male Sprague-Dawley rats (Charles River, U.K., 320 to 350 g) were housed in groups of 2 and maintained under a natural 12-hour light/dark cycle with water ad libitum. Food was restricted during pretraining and at 7 days post-MCAO to facilitate performance on the staircase test which was an appetitively motivated task. The feeding regimen was controlled so that animals gained weight at a rate of 3 to 5 g/w maintaining animals at 85 to 90% of their free-feeding weight. Animals were provided with food ad libitum from 6 hours to 6 days post-MCAO to improve postoperative weight and recovery. Rats were initially trained on the following behavioral tasks before surgery: bilateral sticky label test, beam walking, and the staircase test.

Bilateral sticky label test

The bilateral sticky label test (Schallert and Upchurch, 1983) was used to quantify contralateral neglect/ipsilateral bias and models tactile extinction observed in human stroke patients (Rose et al., 1994). Bilateral stimulation of the radial aspect of the forearm was achieved by placing thin strips of brown parcel/packing tape (1.5 4 cm) firmly around each animal's wrists, so that they covered the hairless part of the forepaw. In instances when the sticky label came partially or completely off without the animal having attempted to remove the stimulus with its mouth, the trial was repeated again. Animals were given three bilateral stimulation trials daily, each lasting up to a maximum of 5 minutes, and the following parameters were recorded: latency to contact label on left and right forearm (in seconds); latency to remove left and right label on forearm (in seconds); order of contact (total number of times left and right forearms were contacted first); and order of removal (total number of times left and right label on forearms were removed first). Care was taken in each trial to apply each stimulus with equal pressure and to randomize the order of application (left versus right). Latency data were assessed on a daily basis, whereas order of contact and removal data were represented as the mean SEM of 2 consecutive days so that a bias analysis could be carried out, i.e., order of contact and removal can be determined according to the protocol of Marshall and Ridley (1996). Animals were trained on this task until a stable baseline for latency and order of contact was established over a period of a week. Final preoperative measurements were carried out over a period of another week before surgery, and there was no bias in order of contact or removal before surgery, indicating that both groups were matched for ability (handedness). This test was applied daily after surgery, i.e, from 1 to 28 days post-MCAO.

Beam walking

Beam walking (modified from De Ryck et al., 1989; Goldstein and Davis, 1990) was used as a measure of hindlimb coordination by means of distance travelled across an elevated 100 cm beam (2.3 cm in diameter, 48 cm off the floor). Rats were systematically trained to walk along the elevated beam from start to finish with the aim of completing the task in 3 minutes. A safe location, i.e, a flat box, was placed at the end of the beam so that the rat was motivated to complete the task. Each rat was trained twice daily for a maximum of 3 minutes per trial, and an acquisition curve demonstrated that rats randomly allocated to either sham-operated or MCAO groups learned the task by 5 days, i.e, a stable baseline was achieved before surgery (data not shown). Each data point represents the mean of two daily sessions. This task was instigated daily after surgery, i.e., from 1 to 28 days post-MCAO.

Staircase test

The staircase test (originally designed by Montoya et al., 1991 and modified by Marston et al., 1995) was used to measure skilled independent forelimb paw reaching, i.e, pellet recovery was only possible with the left paw from the left stair and with the right paw from the right stair. Pellet recovery was not performed under visual guidance but using tactile and possibly olfactory cues. Apart from the top two steps, of the six-step stairway, from which a few rats used their tongues in the early stages of training, retrieval of a food pellet was only possible with a coordinated grasping action using all digits of the paw. Animals were placed on a mild food-restricted diet during pretraining in order to provide motivation for food rewards. Animals were then introduced into a Perspex enclosure (300 67 95 mm long wide high) attached to a holding box. A central plinth (190 20 48 mm) was positioned between the entry aperture and the front wall. Two removable staircases were positioned, either side of the plinth, by insertion through the front wall. Each stair consisted of six steps; each measured 14 17 6 mm with a hemispheric cup (11.5 mm in diameter). Between the back stair and the entrance aperture a V-shaped barrier was positioned on the floor of the box, so food pellets displaced from the steps could be held without the animal getting hold of them, i.e, pellet recovery was only possible with the left paw from the left stair and with the right paw from the right stair. Animals were given two 5-minute sessions in the staircase testing box, approximately 10 minutes between tests, on a daily basis. Each step of the stairs was baited with one chocolate food pellet. Each animal had to retrieve as many pellets as possible (maximum of 6 pellets a side per session were available, i.e., a total of 12 pellets a side for any one day). At the end of the test period, the stairs were removed and the animals were returned to their home cage. Performance was scored as the number of pellets recovered from each stair and the number of pellets displaced but not recovered. Rats were trained over a period of 3 weeks on this task, with a criterion of at least 6 pellets recovered from each side and no more than 4 pellets displaced per side per day over 3 consecutive days. Each data point is represented in blocks, i.e., the mean of 3 daily sessions, from pre- to postoperative blocks, as used by Marston et al. (1995). Essentially, postoperative blocks 1 to 4 correspond to the following postoperative days, respectively: 7 to 9; 13 to 15; 20 to 22; and 26 to 28. This test was begun from 7 days post-MCAO in order to give the animals enough time for recovery before food restriction was reintroduced.

Surgical procedures

After successful training and an overnight fast rats were randomly assigned to receive either tMCAO (90 minutes, n = 6) by means of the intraluminal thread technique by adaption of the method originally described by Koizumi et al. (1986) or sham surgery (n = 6). Surgical procedures were performed under halothane (2% v/v O₂) anesthesia. After exposure of the right common carotid artery through a midline cervical incision, a 3/0 nylon monofilament thread, its leading 5 mm end coated with silicone rubber (diameter of 0.30 to 0.32 mm), was introduced by means of a common carotid arteriotomy and carefully advanced along the internal carotid artery until its tip was positioned 1 mm beyond the origin of the right MCA (rMCA), as verified by a slight resistance. The rMCA was occluded for 90 minutes during which time the rat was allowed to recover from anesthesia. After 90 minutes each rat was briefly reanesthesized with halothane (2% in O₂ v/v) and the thread withdrawn to the common carotid artery to permit retrograde blood flow to the rMCA through the complete circle of Willis. Sham surgery was achieved by introducing the thread into the internal carotid artery immediately followed by careful withdrawal to the common carotid artery. Throughout all surgical procedures, rectal temperature was monitored and maintained at 37 1°C (mean SD) with a heated electrical blanket. Rectal temperature was recorded before occlusion, before reperfusion, and 15 minutes after reperfusion in MCAO animals. Arterial blood samples were obtained just before inserting the intraluminal thread in both MCAO and sham-operated control animals for assessment of blood gas status (pH, PCO₂, PO₂, HCO₃^-). Successful occlusion was verified by applying the Bederson neurological scoring system (grade 0 to 3) after 60 minutes of occlusion. Neurologic status was also monitored after reperfusion over the extended time course of the study as this qualitative grading system is a commonly used scale that assesses neurologic function in experimental stroke models.

MRI methods

For MRI procedures anesthesia was induced and maintained with halothane (1% v/v O₂). Rectal temperature was maintained at 37 1°C using an electric heating pad with feedback rectal temperature probe. MRI was performed at 4.7 Tesla (200 MHz for ¹H) using a SIS-200 imaging spectrometer (Spectroscopy Imaging Systems, Fremont, CA, U.S.A.) and a home-made 75-mm (outside diameter) 8-legged birdcage radiofrequency coil for signal transmission and reception. Proton-density, T₂, and diffusion-weighted MR images were acquired, interleaved with each phase encode step (two averages, NEX = 2) using echo times (TE, in milliseconds)/repetition times (TR, in milliseconds) and diffusion weightings (b, 10^-3 s/mm²) of 23/3000/0, 70/3000/0, and 70/3000/590, respectively. The diffusion weightings (b) of 0 and 590 10^-3 s/mm² were applied along the inferior to superior axis (y-direction) of the brain. Thirty-two contiguous slices starting at the level of the eyes, running rostral to caudal through the brain, were acquired using a 128- 128-image matrix covering a field of view of 4 4 cm and yielding an in-plane resolution of approximately 0.3 mm 0.3 mm. The slice thickness was 0.6 mm to increase the spatial resolution while the total imaging time was approximately 45 minutes in order to minimize the duration of anesthesia for recovery and to allow for subsequent serial measurements. Scanning was used at 1, 7, 14, and 28 days after insult.

MRI image analysis

To calculate MRI-derived lesion volume, a direct 2D Fourier transform (FT) of the raw time domain data was performed on all slices for all of the three differently weighted sets of images produced across the time course. T₂-weighted (T₂W) images were then used to calculate apparent percentage lesion volume by highlighting unilateral regions of hyperintensity on a slice-by-slice basis (in-house display software) to derive total lesion area and then brain volume by summing the total area of the cerebral hemispheres. The threshold for hyperintensity was set as a mean intensity value of the contralateral side + 2 SD as the minimum intensity value, taken from a single representative slice for each animal at each time-point. Images were also smoothed using a three-dimensional anisotropic diffusion filter (Perona and Malik, 1990), and PD, T₂, and ADC maps were then produced. Fitting of the acquired data to monoexponentials was then performed on a pixel-by-pixel basis using 2-point exponential fitting routines. ROI (3 3 pixels) for MCAO and sham-operated groups both ipsilateral and contralateral to the occlusion site were then interrogated to obtain as much pixel information without extending beyond the perimeter of the defined area (Fig. 1). ROI from T₂ and ADC maps were analyzed by measuring the average pixel intensity value in the following anatomically distinct regions: dorsolateral caudate putamen, forelimb cortex (FLC), lower parietal cortex (LPC) at approximately -0.3 mm from bregma, interaural (IA) 8.7 mm and the hindlimb cortex (HLC) at approximately -1.8 mm from bregma, and IA at 7.2 mm according to the rat stereotaxic atlas (Paxinos and Watson, 1986). ROI analysis for PD maps was achieved by using the signal intensity ratio (average pixel intensity ratio, i.e., SIR) between the ipsilateral and contralateral sides for each of the above ROI in both MCAO and sham-operated control groups. In order to confirm consistent average pixel intensity values for each of the specific ROI, we measured adjacent slices either side of the chosen slice for the MCAO group using T₂ maps to provide a consecutive volumetric assessment (1.8 mm, 3 slices) of pixel signal intensity on a slice-by-slice basis. For the caudate putamen (CPU), FLC, and LPC regions, average values were taken on all three slices both ipsilateral and contralateral to the occlusion site at approximately the following co-ordinates from bregma: (1) 0.30 mm; (2) -0.30 mm; (3) -0.90 mm. For the HLC region we acquired average values at the following coordinates from bregma: (1) -1.2 mm; (2) -1.8 mm; (3) -2.4 mm.

Figure 1.

Representative T2W images, at approximately: (A) -0.3 mm from bregma, depicting ROI used for T2, ADC, and PD map analysis across the time course (in days). 1i, ipsilateral CPU; 1c, contralateral CPU; 2i, ipsilateral FLC; 2c, contralateral FLC; 3i, ipsilateral LPC; 3c, contralateral LPC; and (B) -1.8 mm from bregma, depicting the HLC for T2, ADC, and PD map analysis across the time course (in days). 1i, ipsilateral HLC; 1c, contralateral HLC.

Full figure and legend (557K)

Histologic procedures

At 28 days after insult, both MCAO and sham animals were terminally anesthetized and transcardially perfusion-fixed with 0.9% saline followed by 4% paraformaldehyde. Brains were then removed and stored in fixative for about 7 days. Each brain was then cut into 2-mm-thick coronal blocks for a total of 6 blocks per animal using a rat brain matrix. Blocks were then processed for paraffin embedding. Paraffin sections (10 m-thick) were cut and stained with cresyl violet (Nissl) and Luxol fast blue to delineate regions of grey and white matter loss, respectively.

Statistical analysis

All statistical analysis was conducted using the SPSS for Windows 6.0 software package using parametric statistical tests, as the data were shown to be normally distributed, and the variances between the groups were not different after Levene's test for equality of variances. Assessment of blood gases and body/core temperature were analyzed by simple independent and paired t tests, respectively. Data from the MRI experiments were analyzed as follows: A repeated measures analysis of variance (ANOVA) was applied to calculate lesion volume as a percent of hemispheric volume using the T₂W images across the time course (within-subject factor) between MCAO and Sham group (between-subject factor). Similarly, a repeated measures ANOVA was implemented for ROI analyses from T₂ and ADC maps where the time course acted as the within-subject factor whereas group (MCAO and Sham) and side (ipsilateral and contralateral) acted as the between-subject factors. PD map ROI analysis was also performed by a repeated measures ANOVA across the time course, but group (MCAO and Sham) acted as the only between-subject factor. A volumetric assessment was performed on each of the ROI derived from T₂ maps for the MCAO group using a repeated measures ANOVA, where slice number (1, 2, or 3) and side (ipsilateral and contralateral) acted as the between-subject factors across the time course (within-subject factor). Data derived from the bilateral sticky label test (mean SD; latency to contact and remove and order of contact and removal) and the staircase test (total pellets recovered and displaced) were analyzed by a repeated measures ANOVA across the time course (within-subject factor) where group (MCAO and Sham) and side (ipsilateral and contralateral) acted as between-subject factors. Beam walking (total distance travelled) and the Bederson neurological scoring system were also analyzed by a repeated measures ANOVA across the time course, but group (MCAO and Sham) acted as the only between-subject factor. When all tests proved significant, post-hoc Newman Keul's comparisons were implemented to determine differences between groups and sides where appropriate.

Correlational analyses using Pearson's r were then performed to determine relationships between appropriate MR ROI and specific behavioral profiles (reflecting a more continuous and parametric distribution) at the specific time points examined across the course of the study (1, 7, 14, and 28 days), i.e., ipsilateral CPU, FLC, and LPC with latency to contact and remove labels on the bilateral sticky label test and total number of pellets consumed on the staircase test. The HLC was correlated to total distance travelled during beam walking across the time course of the study. These behavioral measures were used in the correlational analyses because they reflect an objective and quantitative assessment rather than the grading of neurologic score which is essentially qualitative. Correlations between qualitative grading on the Bederson scoring system and quantitative MRI data were not performed, as both sham-operated controls and tMCAO animals demonstrated minimal variance when assessed on the Bederson neurologic score. Therefore, correlating quantitative behavioral changes with quantitative MRI changes is a more objective method of attempting to assess a relationship between functional outcome and neuropathology across an extended time course. A hierarchical multiple regression ("sequential regression" statistical model) was finally conducted to attempt to delineate which of the ROI (independent variables) best predicted the behavioral pattern (dependent variable) observed on the staircase test. The staircase test has been demonstrated to be a robust task for behavioral impairment, and a hypothesis was set for predicting which individual ROI best predicted the behavioral profile over and above the other independent variables from the data set. This hierarchical approach to multiple regression allows determination of whether the final predictor or set of predictors entered into the regression equation can explain a significant proportion of the variance in the dependent variable over and above that of which is attributable to earlier data sets. It thus represents a a conservative test of the effect of the variable(s) of interest in that variables first entered are maximized (McLeroy et al., 1984; Rose et al., 1994). An R² change and an F change were reported to explain how much each individual ROI contributed to the variation in the dependent variable. Standardized regression coefficients (beta weightings) together with a significance level, determined by the t statistic, were also included in the analyses to ascertain the best predictor from the regression equation. In all analyses P < 0.05 was considered significant.

RESULTS

Blood gases, measured before insertion of the intraluminal thread, were not significantly different between MCAO and sham-operated control groups (independent t test), respectively (mean SD, n = 5-6: pH, 7.35 0.03, 7.36 0.01; PCO₂ (kPa), 5.26 0.48, 5.39 0.28; PO₂ (kPa), 69.9 2.69, 72.9 2.11; HCO₃^- (mmol/L) 21.8 1.08, 22.9 1.30). Body/core temperature for MCAO rats was initially demonstrated to significantly increase (paired t test) just before occlusion to the point of reperfusion (mean SD, n = 6, before MCAO = 36.9 0.2°C; before reperfusion = 38.3 0.3°C, t(5) = -6.91, P < 0.001), before returning back to baseline levels 15 minutes postreperfusion (37.1 0.6°C; t(5) = -4.95 P < 0.01 compared to prereperfusion values; and t(5) = -0.76, P > 0.45 compared to preocclusion values). All ischemic animals exhibited focal neurologic deficits assessed 60 minutes after MCAO, with failure to fully extend the left forepaw and no resistance to lateral push as well as contralateral circling (Bederson neurologic score, grade 3). No deficits were observed in sham-operated control animals (grade = 0). One rat from the MCAO group died 14 days post-tMCAO after it had received an MRI scan because of compromised upper-airway respiration. MCAO animals lost weight over the first few days but stabilized their weight from 3 to 7 days, just before starting the staircase test task, and subsequently maintained weight up to 28 days (MCAO group, mean SD, total weight loss from preoperative weight in grams, n = 5-6: 1 day, -18.3 15.3; 2 days, -18.3 32.3; 3 days, -5.3 32.2; 4 days, -5.8 29.4; 7 days, -5.0 18.20; 14 days, -2.7 11.3; 28 days, +16.2 26.6).

MRI assessment

Lesion volume analysis. Apparent percent total lesion volume, as revealed by hyperintensity on T₂W images, was initially shown to be high at 1 day post-MCAO, which subsequently decreased to a constant level (remaining hyperintense in comparison to the contralateral side) from 7 to 28 days for the MCAO group (group time interaction: F(3,27) = 4.35, P < 0.05). Selective post-hoc analyses from day 1 post-MCAO revealed significant reductions at day 7 (P < 0.05) and day 28 post-MCAO (P < 0.05) within the MCAO group (Fig. 2). However, there were no significant differences between lesion volumes obtained at 7, 14, and 28 days post-MCAO (P > 0.05, NS). The longitudinal extent of the lesion did not significantly change across the time course (mean SD of number of slices: 1 day = 16.3 1.0; 7 days = 15.8 0.8; 14 days = 14.2 2.1; 28 days = 15.0 1.7, P > 0.05, NS).

Figure 2.

Apparent percent lesion volume (mean SD) assessed by T₂W imaging in MCAO rats across an extended time course in days. () MCAO. *P < 0.05, significantly different from day 1.

Full figure and legend (76K)

ROI analysis. ROI analysis using T₂ maps demonstrated significant changes across the time course as verified by significant group side interactions: CPU (F[1,14] = 18.96, P < 0.01); FLC (F[1,14] = 11.64, P < 0.01); LPC (F[1,14] = 42.95, P < 0.001); HLC [F[1,14] = 6.35, P < 0.05). Selective post-hoc analyses revealed increased relaxation times for ipsilateral CPU, FLC, and LPC at 1, 14, and 28 days post-MCAO, which significantly differed from the contralateral side and sham-operated group (Fig. 3). Elevated T₂ values were also apparent for the ipsilateral HLC at 1 day post-MCAO with a significant decline to contralateral side and sham-operated control group control levels from 7 to 28 days post-MCAO (Fig. 3). Reduced T₂ values were obtained at 7 days post-MCAO for each of the above ROI, but only the ipsilateral CPU, FLC, and LPC remained significantly different from the contralateral side and sham-operated group (Fig. 3). The only ROI that demonstrated a group side time interaction was the LPC (F[3, 42] = 6.79, P < 0.01) which showed a progressive increase in T₂ values ipsilaterally, compared to the contralateral side and sham-operated control group. ROI analysis using ADC maps demonstrated significant changes across the time course as highlighted by significant group side time interactions: CPU (F[3,42] = 8.82, P < 0.001); FLC (F[3,42] = 13.61, P < 0.001); LPC (F[3,42] = 14.01, P < 0.001). There was a trend for the ipsilateral HLC (F[3,42] = 2.66, P = 0.06). Selective post-hoc analyses revealed significant reductions in ADC at 1 day after MCAO for ipsilateral CPU, FLC, and LPC and a subsequent progressive increase from 7 to 28 days post-MCAO compared to the contralateral side and sham group levels (Fig. 4). ADC values for the ipsilateral HLC were shown to initially decline at 1 day but subsequently increased to a level that did not differ from the contralateral side and sham controls from 7 to 28 days post-MCAO (Fig. 4). ROI analysis using PD maps only demonstrated significant changes in group main effects for the FLC (F[1,7] = 6.00, P < 0.05) and the CPU (F[1,7] = 14.94, P < 0.01). A trend was observed for the LPC (F[1,7] = 4.51, P = 0.07) whereas no effect was shown for the HLC (F[1,7] = 1.55, P > 0.24). No group time interactions were observed for any of the above ROI. Restricted post-hoc analyses revealed significant elevations in SIR only within the CPU for the MCAO group at 14 and 28 days post-MCAO (mean SD: 14 days, MCAO, 1.12 0.09, sham, 0.95 0.04, P < 0.05; 28 days, MCAO, 1.15 0.08, sham, 0.95 0.04, P < 0.05) With reference to the volumetric analysis using T₂ maps it was evident that there was no significant slice effects (P > 0.78) or slice side time interactions (P > 0.75) for each of the ROI, indicating that values taken from the initial slices were consistent with values taken longitudinally through those regions across the time course.

Figure 3.

T₂ values (mean SD) derived from T₂ maps, measured across the time course for ROI: (A) CPU; (B) FLC; (C) LPC; (D) HLC. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from respective contralateral MCAO ROI and ipsilateral sham control ROI.

Full figure and legend (135K)

Figure 4.

ADC values (mean SD) derived from ADC maps, measured across the time course for ROI: (A) CPU; (B) FLC; (C) LPC; (D) HLC. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from respective contralateral MCAO ROI and ipsilateral sham control ROI.

Full figure and legend (136K)

Behavioral assessment

The tMCAO and sham groups did not differ significantly in performance across the behavioral tasks before surgery, indicating that both groups were matched for functional ability (P > 0.05, NS). After surgery, results from the standard Bederson neurologic scoring system demonstrated a significant group time interaction (F[7,63] = 7.53, P < 0.001). Individual post-hoc comparisons revealed significant neurologic deficits throughout the time course for MCAO rats, relative to sham-operated controls which showed no deficits (MCAO, mean SD: 1 day, 2.5 0.8, P < 0.001; 7 days, 1.3 0.6, P < 0.001; 14 days, 0.9 0.2, P < 0.001; 28 days, 0.8 0.4, P < 0.01). The bilateral sticky label test revealed significant long-lasting deficits in latency to contact and remove labels from the contralateral forearm for the MCAO group across the time course. Significant group side time interactions were shown for latency to contact (F[6, 108] = 6.36, P < 0.001) and remove (F[6, 108] = 3.68, P < 0.01) sticky labels. Selective post-hoc analyses demonstrated an initially marked impairment followed by improvement in response times across the time course for MCAO animals but remained significantly higher from the preoperative baseline, ipsilateral forearm, and sham-operated control group animals (Fig. 5). A significant group side time interaction was also observed for order of contact (F[6,108] = 19.96, P < 0.001) and removal (F[6,108] = 9.12, P < 0.001) of sticky labels which further reflected a contralateral neglect and an ipsilateral bias to sensory stimuli for MCAO animals (Fig. 5, for post-hoc comparisons).

Figure 5.

Mean latency (seconds SD) to contact (A) and remove (B) adhesive labels placed around right and left forearms by tMCAO and sham control animals. Total number of times that the right and left adhesive labels were contacted (C) and removed (D) first by tMCAO and sham control animals are also depicted (mean SEM) in 2-day blocks across the time course. RA, right forearm; LA, left forearm. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from respective MCAO ipsilateral forearm (RA). +P < 0.05, ++P < 0.01, +++P < 0.001, significantly different from respective sham control contralateral forearm (LA).

Full figure and legend (164K)

On the beam walking task MCAO animals were shown to be initially impaired by a proprioceptive contralateral hindlimb placing deficit which gradually recovered, reflecting improved coordination. A significant group time interaction was shown for total distance travelled (F[8,72] = 23.56, P < 0.001). Individual post-hoc comparisons revealed a marked deficit in coordination from 1 to 7 days post-MCAO followed by a return to preoperative baseline and sham-operated control group performance from 14 to 28 days post-MCAO (Fig. 6).

Figure 6.

Mean distance (cm SD) travelled across a 100-cm elevated beam by tMCAO and sham control animals across the time course. ***P < 0.001, significantly different from sham control group. () MCAO. () SHAM.

Full figure and legend (42K)

The staircase test demonstrated bilateral impairments in the total number of food pellets retrieved across the postoperative blocks by MCAO animals. A significant group side time interaction was found (F[4,72] = 4.19, P < 0.005). Restricted post-hoc analyses indicated that the forelimb contralateral to the lesion exhibited a marked and prolonged impairment across the time course compared to preoperative baseline and sham group performance (Fig. 7). An ipsilateral impairment was also revealed, although not as marked as the contralateral forelimb. However values for ipsilateral paw-reaching food pellet retrieval significantly differed from preoperative baseline and sham group values (Fig. 7). No significant group time side interactions were found for total food pellet displacement across the postoperative blocks (F[4,72] = 0.39, P > 0.8). Although MCAO animals tended to displace more food pellets from the stairs with their ipsilateral forepaw than the sham control group (Fig. 7). Therefore MCAO animals tended to possess long-lasting deficits in fine motor control and sensorimotor neglect across the time course of the study.

Figure 7.

Total number of pellets recovered by contralateral (A) and ipsilateral (B) forelimbs in preoperative and postoperative blocks (each block represents the mean SEM of six trials over a period of 3 days). Total number of pellets displayed by contralateral (D) and ipsilateral (D) forelimbs is also displayed in the same format. **P < 0.01, **P < 0.001, significantly different from the respective sham-control group. () MCAO. () SHAM.

Full figure and legend (105K)

Correlational analysis. T₂ ROI with behavior. (See Tables 1 and 3.) There were significant correlations (Pearson's r > 0.5, P < 0.05) between T₂ relaxation times for ipsilateral CPU (days 1, 7, 14), FLC (days 1, 7), and LPC (1, 7, 14) with latency to contact and remove labels from the contralateral forearm using the bilateral sticky label test in that MCAO animals had high T₂ relaxation times that were associated with high response latency times. In addition there were strong associations between ipsilateral CPU (days 7, 14, 28), FLC (days 7, 14, 28) and LPC (days 7, 14, 28) with contralateral and ipsilateral paw reaching on the staircase test (r > -0.5, P < 0.05), as reflected by high T₂ relaxation times and minimal pellet retrieval by the MCAO animals. A strong relationship was also found between the ipsilateral HLC (day 1) and beam walking (distance travelled, r = -0.82, P < 0.001), as demonstrated by high T₂ values and the short distance travelled on the beam by the MCAO group. This relationship was not evident at the other time points (r < -0.53, P > 0.05, NS).

TABLE 1. - Correlations between T₂ ROI and behavior across an extended time course.

Full table

Table 3 - Correlations (Pearsons r) between HLC and beam walking (total distance traveled [DT]) using T₂ and ADC values, across an extended time-course.

Full table

ADC ROI with behavior. (Tables 2 and 3.) There were significant correlations between ADC values within the ipsilateral CPU (days 1,7), FLC (days 1,7), and LPC (days 1, 7, 14, 28) with latency to contact and remove labels from the contralateral forearm using the bilateral sticky label test (day 1 r > -0.5, P < 0.05 and from day 7 r > 0.5, P < 0.05) in that MCAO animals had low ADC values at day 1 followed by increased ADC values from 7 to 28 days that were related to high-response latency times. Also there were strong associations between ipsilateral CPU (days 7, 14, 28), FLC (days 14, 28), and LPC (days 7, 14, and 28 days) with contralateral paw reaching on the staircase test (r > -0.5, P < 0.05) as reflected by high ADC values and minimal pellet retrieval by MCAO animals. The ipsilateral CPU (days 7, 14, 28), FLC (day 14), and LPC (days 14, 28) were strongly correlated with ipsilateral paw reaching on the staircase test, again demonstrated by high ADC values with minimal pellet recovery. A strong relationship was also found between the ipsilateral HLC (day 1, r = 0.85, P < 0.001 and day 14, r = -0.80, P < 0.01) indicative of behavioral impairment and recovery, respectively, with ADC values for the MCAO group.

TABLE 2. - Correlations between ADC ROI and Behavior across an extended time course.

Full table

Hierarchical multiple regression analysis. T₂ ROI with the staircase test. When ipsilateral CPU and FLC were held constant and LPC was added to the regression equation for contralateral food pellet retrieval at 14 days post-MCAO an additional 21% of the variance was explained (R²_change = 0.21, F_change[1,8] = 7.76, P < 0.03, = 0.80, t = 2.78, P < 0.03) (Table 4). Similarly, when ipsilateral CPU and FLC were held constant and LPC was added to the regression equation for ipsilateral food pellet retrieval at 14 days post-MCAO, an additional 28% of the variance was explained (R²_change = 0.28, F_change[1,8] = 8.99, P < 0.02, = 0.94, t = 2.99, P < 0.02). No improvement in prediction was achieved at 7 and 28 days post-MCAO. This analysis suggests that the ipsilateral LPC revealed on T₂ maps made a unique contribution to both contralateral and ipsilateral pellet retrieval observed on the staircase test at 14 days post-MCAO.

Table 4 - Hierarchical regression analyses demonstrating the contribution made by the LPC from T₂ and ADC maps in explaining the variance of contralateral pellet retrieval (CPR) and ipsilateral pellet retrieval (IPR) on the staircase test across the time-course.

Full table

ADC map ROI with staircase test. When ipsilateral CPU and FLC were held constant and LPC was added to the regression equation for contralateral food pellet retrieval at 14 and 28 days post-MCAO, an additional 24% and 23% of the variance was explained, respectively (14 days = R²_change = 0.24, F_change[1,8] = 8.66, P < 0.02, = 0.64, t = 2.94, P < 0.02; 28 days = R²_change = 0.23, F_change[1,7] = 7.26, P < 0.02, = 0.79, t = 2.70, P < 0.02) (Table 4). Similarly, when the ipsilateral CPU and FLC were held constant and LPC was added to the regression equation for ipsilateral food pellet retrieval at 14 days and 28 days, an additional 42% and 36% of the variance was explained, respectively (14 days = R²_change = 0.42, F_change[1,8] = 22.11, P < 0.01, = 0.86, t = 4.70, P < 0.01; 28 days = R²_change = 0.36, F_change[1,7] = 9.85, P < 0.02, = 0.98, t = 3.14, P < 0.02). No improvements in prediction were found at 7 days post-MCAO. This analysis suggests that the ipsilateral LPC revealed on ADC maps made a unique contribution to contralateral and ipsilateral pellet retrieval observed on the staircase test at 14 and 28 days post-MCAO.

Histopathologic assessment

At 28 days post-MCAO, all MCAO animals demonstrated a large unilateral region of infarction that extended from the FLC to the lower regions of the parietal cortex (LPC) which encompassed the underlying striatum (CPU) (Fig. 8), as represented by scattered cells with pyknotic nuclei and eosinophilic-like cytosol. A large fluid-filled cyst had been formed, signifying cavitation, presumably occupying previously necrotic tissue, which was surrounded by a loose connective tissue matrix which aligned the superficial aspect of the lesion. This profile of damage was also apparent surrounding the ipsilateral HLC (Fig. 9), although the ipsilateral HLC itself was demonstrated to retain structural integrity at 28 days after insult and was visually indistinguishable from the contralateral HLC under higher magnification ( 20: Fig 10A and Fig 10B). Loss of Luxol-fast blue staining was noticeable within the subcortical white matter, relative to the contralateral side, indicative of disorganized myelinated fiber tracts (Fig. 8). This was also further high-lighted in the corpus callosum where disrupted organization was apparent within the ipsilateral hemisphere (Fig. 11A) unlike the equivalent area in the contralateral hemisphere (Fig. 11B). No sham-operated control animals showed any evidence of lesion development at 28 days, corroborating the MRI findings.

Figure 8.

Photomicrograph of cavitation that extends from the ipsilateral dorsolateral cortex (FLC) to the LPC and underlying CPU, visualized by cresyl violet/Luxol fast blue staining. The level is at approximately -0.3mm from bregma, at 28 days after MCAO, in the representive MCAO rat used for the MR images in Fig. 1A (magnification, 1).

Full figure and legend (272K)

Figure 9.

Photomicrograph at the level of ipsilateral HLC, approximately -1.8 mm from bregma, visualized by cresyl violet/Luxol fast blue staining at 28 days after MCAO in the representative MCAO rat used for MR images in Fig. 1 (magnification, 1). Notice the cavitation surrounding the edge of the ipsilateral HLC, which is depicted as hyperintensity on T₂W images in Fig. 1B.

Full figure and legend (302K)

Figure 10.

(A) Ipsilateral HLC at 28 days after MCAO, under higher magnification ( 20, from Fig. 10), depicting part of the sampling area used for MRI analysis. Notice that the integrity of the tissue is similar to that of the contralateral HLC for the same representative MCAO rat (B). Contralateral HLC at 28 days after MCAO, under high magnification ( 20, from Fig. 10), depicting part of the sampling area used for MRI analysis.

Full figure and legend (492K)

Figure 11.

(A) Degeneration of ipsilateral white matter tracts of the corpus callosum in the same representative rat, as revealed by cresyl violet/Luxol fast blue staining at 28 days after MCAO, at the same level as Fig. 8 (magnification, 40). (B) Contralateral white matter tracts of the corpus callosum in an equivalent region to that of (A) in the same representative rat, as revealed by cresyl violet/Luxol fast blue staining at 28 days after MCAO at the same level as Fig. 8 (magnification, 40).

Full figure and legend (404K)

DISCUSSION

This long-term study has delineated dramatic changes in the temporal profile of absolute T₂ and ADC MRI measures after transient MCAO in the rat. To date there have been few published reports that attempt to study extended survival time after the MCAO thread model in the rat, despite a growing interest in potential therapies based on enhancing recovery/regeneration (Jiang et al., 1996; Kawamata et al., 1997, 1998; Stroemer et al., 1998). This study is the first of its kind to address the issue of correlating specific regional areas delineated by MRI with functional outcome. This serial approach is essential for assessing the pathophysiologic and potential plastic events that ensue after stroke and in combination with functional outcome generates crucial information that can be extrapolated from the preclinical (animal model) to the clinical situation.

MRI changes

Our results demonstrate that the size of the lesion initially reduces from 1 to 7 days but remains constant in size from 7 to 28 days after MCAO using a strict threshold criterion. Sham-operated control rats showed no evidence of infarction on MR images (no hyperintense signal intensity on T₂W images) from 1 to 28 days, and no pathology was revealed by cresyl violet/Luxol fast blue staining at 28 days (data not shown). Brain edema has been demonstrated to evolve from 6 hours to 7 days after ischemic insults (Hsu et al., 1989) and correlates with behavioral impairment (Tominaga and Ohnishi, 1989; Rogers et al., 1997) which may have enhanced the over-all apparent percentage total lesion volume visualized by T₂WI at 1 day. Lesion size did not significantly differ at 7, 14, and 28 days after tMCAO while the longitudinal extent of the lesion did not differ across the extended time course of the study, suggesting that total lesion maturation had developed by 7 days after tMCAO. In contrast to the results of this study, Persson et al. (1989) have demonstrated that the infarct area of rats subjected to pMCAO was larger during the first week and then subsequently decreased in size, as a percentage of the contralateral area, at 42 days after insult, using hematoxylin and eosin staining. Furthermore, Clark et al. (1993) have demonstrated that infarct size was maximal at day 1 post-pMCAO in spontaneously hypertensive rats and subsequently decreased in size at days 5, 15, and 30 postinsult, as a percentage of the contralateral hemisphere, using 2,3,5-Triphenyltetrazolium hydrochloride (TTT)-stained sections. However, our MRI assessment of lesion size reduction does correspond to that of Clark et al. (1993), in that a reduced total lesion size was demonstrated at 7 and 28 days when compared to 1 day after MCAO. The reduction in lesion size has been proposed by Clark et al., (1993) as a four-stage organizational matrix of tissue events from 1 to 30 days after pMCAO in which the lesion was characterized by a reduction of edema to resolution of ischemic tissue. Our study measured total lesion size on MR (T₂-W) images as the total area of hyperintensity divided by the total area of the cerebral hemisphere on individual slices that encompassed the whole of the cerebrum, rather than a percentage of the contralateral hemisphere. In this respect taking into account the different surgical approaches (pMCAO versus tMCAO), models (electrocoagulation versus intraluminal thread), and obvious differences in severity and methods of quantifying lesion size, overall estimates of lesion size only differ among studies on the basis of magnitude and not in terms of the direction of change. Therefore, future studies should include comparisons between orthodox histopathologic staining techniques and MRI at the different time points that relate to the chronic phase of ischemia in order to verify the methods used for analysis. However, both Barone et al. (1991) and our group (Smart et al., 1996) have already published on this issue within the acute phase after pMCAO and tMCAO in the rat, respectively, and have demonstrated that lesion size evaluated by T₂WI at 48 and 22 hours, respectively, significantly correlated with lesion size assessed by TTC and hematoxylin and eosin staining at the respective end points. We are therefore confident that the MRI methods used by our group for assessing lesion size are measuring the same pathology revealed by commonly used histopathologic staining methods within the acute period after MCAO.

Further assessment of our data demonstrated that the total area of both cerebral hemispheres across the time course did not dramatically change between MCAO and sham-control groups (data not shown), suggesting that a decrease in swelling may not have been the cause of a reduced lesion volume defined by T₂W hyperintensity at 7 days. These results are in contrast to those of Persson et al. (1989) and Fujie et al. (1990) who demonstrated that the ipsilateral hemisphere (defined as tissue reduction area + infarct area) and ipsilateral thalamus were reduced relative to the percentage of the respective contralateral area using hematoxylin and eosin staining methods. In our total cerebral hemispheric area calculations we included the regions of ipsilateral hemisphere that were damaged as a result of MCAO, from 1 to 28 days, in order to make consistent comparisons between total hemispheric area of tMCAO and sham-operated control rats. An appropriate alternative that should be adopted in future studies would have been to acquire preoperative scans of these animals, which would provide a useful baseline "unaffected" measurement for total cerebral area that could be implemented for later determinations of lesion size and hemispheric swelling (by means of T₂W hyperintensity) after insult. Several other factors should also be taken into account when trying to interpret the apparent reduction in lesion size and hence reduced T₂-hyperintensity at 7 days after tMCAO. One important factor is the role of reperfusion in this model after 90 minutes of occlusion, as retrograde flow from the complete circle of Willis may have "salvaged" penumbral regions, particularly cortical areas at later time points, e.g., the HLC, as verified by absolute T₂ and ADC map analysis. Indeed assessment of the critical threshold or duration for inducing irreversible ischemic injury after tMCAO, using the thread technique, was first demonstrated by Memezawa et al. (1992a, 1992b). Essentially they showed that if reperfusion was initiated early enough after MCAO (up to 90 minutes of MCAO) the penumbral areas were spared, whereas reperfusion initiated after 2 hours of ischemia failed to salvage ischemic tissue when assessed histopathologically, 7 days after MCAO. Clearly one must also consider interanimal differences in collateral blood supply, because variability in perfusion dynamics, as well as the "no-reflow" phenomenon, may ultimately determine regions that are more tolerant than others after reperfusion. In addition, when using the diagnostic modality of T₂WI one must also take into account that a reduction in T₂ may be explained by the paramagnetic influence of iron accumulation (Lang et al., 1994; Schwartz et al., 1996; Nakane et al., 1997), increased gliosis (Schwartz et al., 1996; Ishii et al., 1998), and a decrease in vasogenic edema (Welch et al., 1995). Indeed, a recent study by Ishii et al. (1998) compared changes in T₂W imaging with microtubule-associated protein-2 (MAP2) and glial fibrillary acidic protein (GFAP) immunohistochemical staining for estimating lesion size at 6 to 8 hours and at 7 days after MCAO (rat tMCAO for 2 hours). It was demonstrated that at 7 days the lesion visualized by MRI reduced in conspicuity, confirming our results with T₂W images, and this signal decay was attributable to an increase in glial infiltration around the periphery of the infarct. Attenuating T₂ signal intensity may thus alter the true estimation of lesion size which can only be substantiated by a time series of histopathology in conjunction with immunocytochemical markers for neurons and glial cells, to determine the contribution of different cell types to the observed MRI signal intensity. The work by Ishii et al. (1998) also highlights the fact that assessing lesion size on the basis of hyperintensity by T₂W imaging alone may partially underestimate the ensuing infarction at 7 days which implies ROI analysis using absolute values should be adopted to help resolve this issue. This is also apparent in the clinical situation, in which reduced T₂ signal intensity from 5 to 12 days is observed in patients with MCAO territory infarction (Periera et al., 1997), suggesting that if MRI scans are performed for diagnostic purposes around 7 days, reliance on T₂W imaging may result in the diagnosis of cerebral infarction being missed or the size of the infarct being underestimated.

Regional assessment of the severely damaged CPU using T₂ maps demonstrated that absolute T₂ relaxation times significantly increased across the time course, which has been traditionally considered the "core" of the infarct. This elevation suggests edema and cell necrosis within the acute period was followed by gliosis and cavitation during the subacute to chronic period. The T₂ of tissue is dependent on tissue water, which is distributed between the intracellular and extracellular compartments, and in each compartment exists in either a bound or a free state (Lynch, 1983; Mathur-De Vr'e, 1994). Free water has a much higher T₂ than bound water suggesting that an increase in T₂ relaxation times is indicative of an increase of free water and a reduction in the exchange rate between free and bound protons, arising from the pathological sequelea of cerebral ischemia (Ordidge et al., 1991). Acute decreases in ADC were also demonstrated in the CPU consistent with a contribution from cytotoxic edema. The biophysical basis of reduced diffusion has been viewed as a decrease in extracellular space (Hasegawa et al., 1996) because of a buildup of intracellular water from membrane failure and cell swelling. Increased tortuosity of extracellular diffusion paths (Nicholson et al., 1993) has also been postulated, although reductions in intracellular diffusion by means of cytoplasmic streaming or alterations in the intracellular tortuosity may also play a part (Duong et al., 1998; Dijkhuizen et al., 1999). ADC reductions have also been interpreted as being attributable to the swelling of astrocytes, mainly involving their perivascular processes, as well as the narrowing of the microvascular lumen and shrinkage of the neuronal perikarya, suggesting a complex interplay between cellular compartments and the diffusion of water (Liu et al., 1999). ADC values were then shown to transiently normalize at 7 days (pseudonormalization) followed by a progressive increase from 14 to 28 days after MCAO. Pseudonormalization of ADC has been considered to reflect the emergence of eosinophilic neurones and the start of neuronal necrosis when conducting end point histopathologic examinations (Garcia et al., 1993; Welch et al., 1995). The subsequent rise in ADC during the chronic phase has been postulated to indicate loss of membrane integrity and cell necrosis as the CPU progresses from edema to gliosis and then to cyst formation and cavitation, as revealed by light (Takahashi et al., 1993) and electron (Pierpaoli et al., 1993) microscopy. The histologic profile of cavitation was clear at 28 days after MCAO as illustrated by Nissl stained sections (Fig. 8).

Changes in T₂ relaxation times and ADC were also apparent for the ipsilateral FLC and LPC across the time course reflecting a similar profile to the CPU. These regions traditionally viewed as penumbral or perifocal areas, adjacent to the core of the infarct, demonstrated progressive increases that were greater in magnitude than the CPU across the time course. These results suggest an accumulation of water within the extracellular space of the ipsilateral FLC and LPC, culminating in cavitation through the formation of a loose connective tissue matrix filled with necrotic debris (Chen et al., 1993; Clark et al., 1994). This was verified by Nissl-stained sections acquired at 28 days after MCAO (Fig. 8). Hence these regions were recruited to the lesion by the infarction process across the extended time course. The differences in magnitude between the CPU and other ROI may be explained by a heterogeneous mixture of grey and white matter within the CPU (particularly the striasomes) thus altering signal characteristics.

The HLC demonstrated high T₂ relaxation times and reduced ADC values on day 1 after MCAO, suggesting an impending threat of edema. Conversely, T₂ and ADC values normalized at 7 days and remained at contralateral and sham control levels up to 28 days after MCAO, implying that this region was potentially "salvaged." A number of factors may account for this apparent "recovery" of challenged tissue, such as reperfusion and the role of collateral supply, particularly the anterior cerebral artery which perfuses this anatomic region in addition to the MCA. The neurons within the ipsilateral HLC are presumably "stunned," perturbing metabolism, but some restorative process may then play its part (e.g., metabolic compensation or a trophic influence) to provide enough support to sustain viability. Indeed, histopathologic examination using Nissl-stained sections of the ipsilateral HLC suggests that it remained viable at 28 days after MCAO (Fig 10 and Fig 11A) and did not visually differ from the contralateral HLC (Fig. 11B). Recently, it has been demonstrated that there is no single threshold of ADC reduction that predicts irreversible ischemic injury after variable occlusion times for tMCAO (30, 60, and 90 minutes) in the rat (Mancuso et al., 1997). This suggests that although the reduced ADC occurs in regions that will eventually exhibit ischemic injury, the extent of the ADC reduction is not necessarily related to the likelihood of cell death. Therefore, ADC maps generated within the immediate acute period of ischemia may not be useful to quantitatively predict postreperfusion outcome based on only the magnitude of the ADC reduction. Furthermore, differences between acute changes in ADC, regional water content, and tissue ultrastructure have recently been defined in a cat MCAO model (Kuroiwa et al., 1998). Essentially, the authors found that a significant reduction in ADC was seldom associated with cell membrane disruption or nuclear clamping, which are direct indicators of irreversible injury, implying that the early determination of an ADC threshold value for irreversible ischemic injury is difficult. Alternatively, it may be postulated that ADC changes are useful in providing definitive information about the presence of cellular edema within the acute period, but if assessed serially and in combination with other MR signatures over time delineation of the dynamic changes of ischemic tissue can be optimally identified. Therefore, these results indicate that long-term regional assessment of changes using serial MRI and conventional histopathologic staining methods are necessary to predict irreversible ischemic injury and potentially salvageable tissue.

An important supplementary observation is that of a reduction in T₂ relaxation times at 7 days after MCAO compared to 1 day after MCAO in each of the ROI evaluated. It is clear from the data that the ipsilateral CPU, FLC, and LPC all subsequently increased in magnitude from 14 to 28 days after MCAO. Again possible reasons for this apparent signal decline could be attributable to factors already explained, i.e., the role of iron deposition, gliosis, and/or reduced vasogenic edema. It is also possible that the infiltration of the infarct by macrophages (Myers et al., 1991) increases the cell density in the region of infarction (Petroff et al., 1992) that results in a reduction of T₂ by changing the ratio of free to bound water or the exchange rate between the two states. Work is currently being conducted to determine whether ferric iron deposition, gliosis, and/or infiltration of macrophages contributes to reduced T₂ relaxation times depicted at 7 days after tMCAO, which may have significant bearing on the T₂ findings from stroke patients (Periera et al., 1997) indicative of a T₂ "fogging" effect.

PD map assessment did not prove to be as valuable a marker of ischemic damage across the time course as T₂ and ADC measures. PD images reveal the spatial distribution of a number of MR-visible proton nuclei in the tissue, and significant elevations in PD have only become apparent at 48 hours after pMCAO, which subsequently declined to normal levels at 7 days, particularly within the CPU and penumbral regions (Knight et al., 1991, 1994). Our study has demonstrated significant elevations within the CPU at 14 and 28 days after tMCAO but not consistently in other ROI, implying that although the different MR signatures have distinct temporal profiles (Knight et al., 1994), at later time points PD may be more representative of T₂ and hence edema in severely damaged regions (such as the "core" or CPU). However, the reason that PD measurements did not prove valuable could be attributable to the fact that we were actually looking at very small-intensity ratio changes between the ipsilateral and contralateral hemispheres, and with a small group size statistical validation was proven to be too insensitive. In addition, a 2-point T₂ curve fit was used to derive PD maps, so further values plotted on an exponential curve would provide a more representative signal intensity when evaluating the tissue maps.

Correlations with behaviour

Functional assessment involving the bilateral sticky label test demonstrated a marked disruption followed by a slight improvement in the latency to contact and remove sticky labels from the contralateral forearm, indicating contralateral neglect. In addition, when interpreting the order of contact and removal of labels it was clear that the ipsilateral stimulus was always contacted first throughout the time course of testing regardless of latency, suggesting a long-lasting impairment in ipsilateral bias and contralateral neglect. This test has been shown to be sensitive in detecting sensorimotor deficits in rats after permanent MCAO (pMCAO), although recovery in latencies have been found in some but not all animals from 20 days after MCAO (Markgraf et al., 1992). Recently, Marshall and Ridley (1996) have demonstrated recovery in latency to contact and remove labels from 3 to 8 weeks after pMCAO in the common marmoset. However, an ipsilateral bias/contralateral neglect was evident when analyzing the order of contact data, suggesting a persistent impairment. Tests that can be implemented easily in this manner enable cross-species comparisons to be made, and this is highlighted by the fact that a subpopulation of clinical stroke patients show tactile extinction from 1 to 6 months after MCAO, with varying degrees of recovery which can predict functional status. Indeed, the contralateral neglect observed on this behavioral task has striking similarities to the contralateral neglect syndrome arising from damage to the parietal cortex in humans (Heilman et al., 1985). Correlations between T₂/ADC ROI and latencies to contact and remove labels suggest that the ipsilateral CPU, FLC, and LPC make significant contributions to the functional impairments observed across the time course in this study. This overlapping association is consistent with data derived from selective lesions to each of these areas on contralateral neglect and ipsilateral bias behavioral profiles (Cooper et al., 1979; Price and Fowler, 1981; Schallert et al., 1982; Jones and Schallert, 1992; McDaniel et al., 1995; Forgie et al., 1996) and further substantiates the concept of topographically organized striopetal connections from the cortex (Pisa, 1988; Pisa and Cyr, 1990).

Impairments demonstrated by MCAO rats on the staircase test were bilateral in nature, as both contralateral an ipsilateral paw reaching performance was disrupted. The contralateral deficit was more marked, although significant bilateral deficits suggest that there is possible dysfunction in interhemispheric communication (Marston et al., 1995). The fact that target location was severely impaired on the contralateral side is further corroborated by minimal pellet displacement suggesting a role for spatial neglect of contralateral stimuli as well as a deficit in olfactory/tactile sensing or tactuomotor control of fine motor movements. Indeed, pellets placed on the highest steps were often left untouched. Pellet displacement was more marked on the ipsilateral side suggesting that animals were motivated and preferred the use of the ipsilateral forearm in retrieving pellets yet were ineffective in grasping ability. Immunohistochemical analysis using GFAP and RCA-1 (riccinus communis agglutin-1) markers for astrocytes and microglia, respectively, have shown increased expression in both the ipsilateral contralateral corpus callosum indicative of gliosis and edema at 28 days after MCAO, which could partially explain the hypothesis of a deficit in interhemispheric communication (D. Virley, in preparation). Furthermore, Luxol fast blue staining demonstrated disrupted white matter cytoarchitecture for the striasomes and corpus callosum within the ipsilateral hemisphere, which can been corroborated as axonal injury that has been demonstrated within the acute period after focal cerebral ischemia (Dewar and Dawson, 1997; Yam et al., 1998). Recently impaired dexterity of the ipsilateral hand after stroke has been revealed within the first month for stroke patients who possessed lesions that encroached on the left or right parietal lobe, which was predicted by cognitive (ideomotor apraxia and visuospatial) deficits, affecting perception and control of action (Sunderland et al., 1999). However, ipsilateral sensorimotor loss as well as deficits at the higher cognitive level may be viewed to affect the execution of skilled-motor control of the ipsilateral hand by means of disruption of the ipsilateral motor pathway (Jones et al., 1989; Desroisiers et al., 1996).

Bilateral impairments on the staircase test have also been reported in an alternative transient MCAO model, the endothelin-1 surgical technique (Marston et al., 1995; Sharkey et al., 1996) as well as unilateral lesions of the nigrostriatal (Whishaw et al., 1986, 1992) and cortical (Price and Fowler, 1981; Montoya et al., 1991; Plumet et al., 1990) projections. A pMCAO model using spontaneously hypertensive rats has also demonstrated a marked impairment in contralateral paw reaching which was worse than the ipsilateral side, although comparisons with results obtained by Marston et al. (1995) have indicated differences in strain of rat as well as lesion size and surgical approach in determining performance on this task. Contralateral deficits have also been observed with pMCAO in the marmoset, both on a standard ("Hill") and modified reverse ("Valley") staircase, demonstrating a spatial and fine-motor impairment that was persistent throughout postoperative testing (Marshall and Ridley, 1996). Within the clinical00 context there is often significant recovery in gross larger limb movement although more fine control of finger movement is often persistently affected and leaves a lasting disability. Correlations between ipsilateral T₂/ADC ROI and contralateral paw reaching, as well as ipsilateral paw reaching, again suggest that the ipsilateral CPU, FLC, and LPC all make significant contributions in predicting behavioral impairment on the staircase test. However, hierarchical regression analyses revealed the ipsilateral LPC to be most predictive of contralateral and ipsilateral paw reaching impairments. This result can be reconciled with the notion that unilateral cortical damage can produce bilateral deficits unlike unilateral striatal lesions which produce primarily unilateral (contralateral) deficits (Marston et al., 1995). The cortical output projections in rats, unlike humans, are not fully decussated (Tracey, 1995), implying that the data presented by this study is highly compatible with unilateral neocortical damage. Furthermore, the parietal cortex has recently been implicated as the control center for reaching movements, in that spatial representations of limb position, target locations, and the potential execution of motor actions are expressed as neural activity within this region (Kalaska et al., 1997).

Beam walking as defined by total distance travelled was severely impaired in MCAO rats from 1 to 7 days after MCAO. This was because of a contralateral hindlimb-placing deficit that prevented the animals from initiating movement across the narrow elevated beam. Indeed, MCAO animals tended to fall off the beam more readily, on average within 2 minutes over the first 2 days, but maintained balance from 3 days onwards (data not shown) although still hindered by the placing impairment. Photochemical damage to the sensorimotor cortex that encompasses the HLC of rats has been shown to produce selective and long-lasting deficits in tactile and proprioceptive limb placing (De Ryck et al., 1989, 1992; De Ryck, 1997) which is consistent with the results obtained from the acute to subacute period of this study. Contralateral hindlimb placement recovery was evident from 14 to 28 days after MCAO as total distance travelled improved to the sham-operated control level. De Ryck et al. (1992) has stated that rats with sensorimotor cortical damage will not demonstrate contralateral hindlimb-placing errors if they are engaged in bursts of rhythmic locomotor running without exploratory head scanning on narrow beams. However, tMCAO rats from 14 to 28 days in this study did not always run straight across the beam but also stopped halfway for exploratory activity and still maintained posture to complete the task. Some animals repeated the task from the safe location, actively exploring the exposed start-end of the beam before returning to finish-end of the beam. This is in direct contrast to De Rycks's observation of rats that show impairments on the beam after sensorimotor cortex lesions when locomotor automatism is interrupted. The recovery in beam walking mirrors the normalized T₂ and ADC values acquired from the ipsilateral HLC at 14 to 28 days after MCAO relative to sham control values, suggesting that we are either witnessing reduced regional cell death or directly observing the salvaging of tissue with a preserved functional integrity. Histopathologic inspection of the ipsilateral HLC using Nissl-stained sections at 28 days after MCAO demonstrates normal cell viability, further implying that this region does recover (Fig. 10A). Indeed, correlations between beam-walking measures and MRI signatures were no longer apparent from day 7 to 28 after MCAO, implying that behavioral recovery was related to normalized MRI values within the ipsilateral HLC, other than at day 14 for ADC changes, suggesting that behavioral recovery was not quite complete in all animals and that ADC values for some animals were slightly higher. In addition, at 14 days T₂ values within the contralateral HLC were significantly different from the ipsilateral side in MCAO animals, unlike the ipsilateral side in sham controls, tentatively suggesting that some physiologic changes were being elicited within this contralateral region at the start of behavioral recovery on this task. Hence, one must also consider the role of "plasticity" and structural reorganization of areas involved in contributing to functional restitution on this task.

The precise anatomical, cellular, and molecular mechanisms of functional recovery after stroke remain unknown. In both rodent and nonhuman primate studies, recovery of sensorimotor function after unilateral cortical injury or distal pMCAO is accompanied by new neuronal sprouting and synapse formation in adjacent or penumbral regions surrounding focal infarcts as well as in regions within the contralateral hemisphere (Jones and Schallert, 1992, 1994; Stroemer et al., 1995, 1998; Nudo et al., 1996). From a clinical perspective, functional imaging techniques (functional MRI and positron emission tomography) have demonstrated increased cerebral blood flow and metabolism in intact regions surrounding focal cerebral infarcts and within the contralateral hemisphere in parallel with functional recovery in stroke patients, most likely reflecting increased neuronal activity within these regions (Weiller et al., 1992, 1993; Cramer et al., 1997). This evidence suggests that changes within the ipsilateral penumbral regions of the focal infarct as well as in the corresponding contralateral regions play an influential role in improving the functional impairments that ensue after stroke. This study has demonstrated that the ipsilateral HLC is a penumbral region adjacent to the focal infarct, which recovers in terms of MRI signal characteristics (T₂ relaxation times and ADC) and which contributes to the behavioral recovery on the beam-walking test. The contralateral hemisphere may have been involved with this recovery of hindlimb function, because in the rat the cortical output projections are not fully decussated (Tracey, 1995) suggesting that the intact contralateral cortex may have influenced recovery on this test. Furthermore, Johansson and Ohlsson (1996) have demonstrated improvement on the beam-walking task using a subjective grading scale, with spontaneously hypertensive rats subjected to proximal pMCAO and subsequently housed in an enriched environment. The actual compensatory mechanisms that may be stimulated by an enriched environment have not been examined in any great detail, although in our study the tMCAO rats tested on a daily basis may have experienced a stimulating environment to enhance mechanisms of plasticity in the brain (either ipsilateral or contralateral to the infarct) to improve hindlimb function. However, tMCAO rats persistently demonstrated a contralateral neglect/ipsilateral bias on the bilateral sticky label test and bilateral deficits in skilled forelimb paw reaching on the staircase test. This suggests that even if the daily testing schedule may have provided an enriched environment, contralateral neglect and skilled paw reaching were still severely impaired unlike beam walking. De Ryck et al. (1992) have demonstrated that after unilateral total destruction of the forelimb and parietal cortex, forelimb-placing deficits in the rat do not recover. These findings are consistent with damage to the FLC and LPC in our study, as assessed serially by MRI and terminally by histopathological staining, which were also predictive of forelimb functional deficits. Therefore, an enriched environment may have influenced recovery/compensatory mechanisms within the ipsilateral HLC as demonstrated by MRI and behavior on the beam-walking test, but it certainly did not alter performance on tests probing forelimb function.

More studies are now using functional assessments in conjunction with pathological assessment in permanent and transient MCAO models although correlating lesion size with the degree of impairment is not always clear-cut. Previous studies have assessed functional outcome after MCAO in the rat by incorporating the Bederson neurologic scoring system. We included this scoring system into our experimental design as a relative grade of neurologic deficit but found that more objective tests successfully detected transient or protracted impairments across the time course which are not readily accessible by this simple postural reflex test. Some studies have shown correlations between lesion volume and more objective behavioral tests (Rogers et al., 1997) although because there has also been a lack of correlation across studies we feel a more regional approach would be more sensitive at picking up structure to function relationships. Correlating total lesion size with functional impairment only makes the assumption that the degree or extent of damage associates with the behavioral deficit. In addition, this assumption does not attempt to identify specific structures that may fulfill the specific functions that are being probed by the behavioral task. The importance of selective lesion studies in animals has established the crucial relationship between specific or regional brain damage and specific impairments in behavioral performance. The behavioral tasks are designed to yield information concerning distinct components of behavior that are potentially disrupted by the experimental lesion. Furthermore, Gavrilescu and Kase (1995) have stated that depending on the location of the infarct, clinical syndromes vary in stroke patients, implying that functional impairment is a feature that is contingent on the degree of compromised flow within a particular portion of the vascular territory. Indeed, Gavrilescu and Kase (1995) also highlighted the role MRI and CT scans play in improving our understanding of correlations between the anatomic substrates recruited by the infarction process and the neurologic status of the individual stroke patient. Taking into account these perspectives, our study aims to follow these recommendations by using a regional approach to make sensitive relationships between brain structure and behavioral function. Therefore, this study highlights the fact that specific anatomical substrates recruited by the infarction process rather than lesion size per se may be the critical determinants of behavioral impairment after focal cerebral ischemia.

Recent work has identified anisotropic diffusion patterns within the CPU, FLC, LPC, and HLC to be indicative of fiber projections; hence quantitative assessment of both grey and white matter changes after MCAO in the rat by means of a combination of relaxation parameters and diffusion orientation may be warranted (Lythgoe et al., 1997). The ROI we studied are considered to primarily constitute grey matter, so we used single-axis measurement (isotropic diffusion) rather than diffusion tensor trace estimation (anisotropic diffusion) which can produce ADC maps in three orthogonal directions and consequently increases total scanning time. Indices of anisotropy are influenced primarily by the spatial distribution of barriers to diffusion and hence are inherently more sensitive to the integrity of the structure of tissue. This latter approach should provide a better understanding of lesion conspicuity and maturation, indicative of white matter damage observed in the clinical situation (Jones et al., 1999; Zelaya et al., 1999) which unquestionably contributes to the underlying functional deficits (Pendlebury et al., 1999; Sunderland et al., 1999) exhibited by stroke patients.

In conclusion our results suggest that lesion volume evaluated by T₂W images initially reduces in size and then remains constant from 7 to 28 days and that the MR tissue signature model is appropriate for predicting longterm regional changes in cellular status up to 28 days after tMCAO in the rat. In addition specific behavioral parameters, particularly skilled forelimb paw reaching demonstrated by the staircase test, was profoundly affected throughout the course of the study and correlated highly with changes in T₂ and ADC within ROI that encompass the MCA territory. The bilateral impairments revealed by this task were best predicted by damage to the ipsilateral LPC as revealed by hierarchical multiple regression analyses. One behavioral task, beam walking, that models proprioceptive hindlimb placement and coordination was shown to recover at later time points after tMCAO which was mirrored by a return of the T₂ and ADC values in the ipsilateral HLC to control levels. Therefore this is the first study of its kind that has attempted to correlate long-term changes in cerebral infarction using MRI and functional outcome in an animal model of stroke. In essence, irreversible pathology as visualized by MRI has been associated with persistent functional impairment whereas reversible MRI changes have been shown to be related to recovery of function. It is the view of others (Corbett and Nurse, 1998) and our own group that animal studies that are extended into the chronic phase of ischemia, using both serial MRI and behavior for predicting drug efficacy, will be more predictive of therapeutic benefit within clinical trials.

References

1. Anderson CS, Anderson AB & Finger S. (1991) Neurological correlates of unilateral and bilateral strokes of middle cerebral artery in the rat. Physiol Behav 50: 263−269. | PubMed |
2. Barone FC, Clark RK, Feuerstein G, Lenkinski RE & Sarkar K. (1991) Quantitative comparison of magnetic resonance imaging (MRI) and histological analyses of focal ischemic damage in the rat. Brain Res Bull 26: 285−291. | Article | PubMed | ChemPort |
3. Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL & Bartkowski H. (1986) Rat middle cerebral artery occlusion: evaluation of the model and development of a neurological examination. Stroke 17: 472−476. | PubMed | ISI | ChemPort |
4. Chen H, Chopp M, Schultz L, Bodzin G & Garcia JH. (1993) Sequential neuronal and astrocytic changes after transient middle cerebral artery occlusion in the rat. J Neurol Sci 118: 109−116. | Article | PubMed | ChemPort |
5. Clark RK, Lee EV, Fish CJ, White RF, Price WJ, Jonak ZL, Feuerstein GZ & Barone FC. (1993) Development of tissue damage, inflammation and resolution following stroke: an immunohistochemical and quantitative planimetric study. Brain Res Bull 31: 565−572. | Article | PubMed | ChemPort |
6. Clark RK, Lee EV, White RF, Jonak ZL, Feuerstein GZ & Barone FC. (1994) Reperfusion following focal stroke hastens inflammation and resolution of ischaemic injured tissue. Brain Res Bull 35: 387−392. | Article | PubMed | ChemPort |
7. Cooper RM, Bland BH, Gillespie LA & Whitaker RM. (1979) Unilateral posterior cortical and unilateral collicular lesions and visually guided behaviour in the rat. J Comp Physiol Psychol 72: 286−295.
8. Corbett D & Nurse S. (1998) The problem of assessing effective neuroprotection in experimental cerebral ischaemia. Prog Neurobiol 54: 531−548. | Article | PubMed | ISI | ChemPort |
9. Cramer SC, Nelles G, Benson RR, Kaplan JD, Parker RA, Kwong KK, Kennedy DN, Finklestein SP & Rosen BR. (1997) A functional MRI study of subjects recovered from hemiparetic stroke. Stroke 28: 2518−2527. | PubMed | ChemPort |
10. De Ryck M. (1997) Protection of neurological function in stroke models and neuroprotective properties of lubeluzole. Cerebrovasc Disord 7: 18−30.
11. De Ryck M, van Reempts J, Borgers M, Wauquier A & Janssen PAJ. (1989) Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke 20: 1383−1390. | PubMed | ChemPort |
12. De Ryck M, Van Reempts J, Duytschaever H, Van Deuren B & Clincke G. (1992) Neocortical localization of tactile/proprioceptive limb placing in the rat. Brain Res 573: 44−60. | Article | PubMed | ChemPort |
13. Desroisers J, Bourbonnais D, Bravo G & Roy P-M. (1996) Performance of the "unaffected" upper extremity of elderly stroke patients. Stroke 27: 1564−1570. | PubMed |
14. Dewar D & Dawson DA and. (1997) Changes of cytoskeletal protein immunostaining in myelinated fibre tracts after focal cerebral ischaemia in the rat. Acta Neuropathol 93: 71−77. | Article | PubMed | ChemPort |
15. Dijkhuizen RM, de Graf RA, Tulleken KAF & Nicolay K. (1999) Changes in the diffusion of water and intracellular metabolites after excitotoxic injury and global ischaemia in neonate rat brain. J Cereb Blood Flow Metab 19: 341−349. | PubMed | ChemPort |
16. Duong TQ, Ackerman JJH, Yablonskiy D & Neill JJ. 1998 Extracellular and intracellular apparent diffusion in normal and focally-ischaemic rat brain by means ofave 1H MRS. (In) SMRM Workshop Book of Abstracts Sydney, Australia: Society of Magnetic Resonance in Medicine (p) 342.
17. Forgie ML, Gibb R & Kolb B. (1996) Unilateral lesions of the forelimb area of rat motor cortex: lack of evidence for use-dependent neural growth in the undamaged hemisphere. Brain Res 710: 249−259. | Article | PubMed | ChemPort |
18. Fujie W, Kirino T, Tomukai N, Iwasawa T & Tamura A. (1990) Progressive shrinkage of the thalamus following middle cerebral artery occlusion in rats. Stroke 21: 1485−1488. | PubMed | ChemPort |
19. Garcia JH, Yoshida Y, Chen H, Li Y, Zhang ZG, Lian J, Chen S & Chopp M and. (1993) Progression from ischaemic injury to infarct following middle cerebral artery occlusion in the rat. Am J Pathol 142: 623−635. | PubMed | ChemPort |
20. Gavrilescu T & Kase CS. (1995) Clinical stroke syndromes: clinical-anatomical correlations. Cerebrovasc Brain Metab Rev 7: 218−239. | PubMed | ChemPort |
21. Goldstein LB & Davis JN. (1990) Beam-walking in rats: studies towards developing an animal model of functional recovery after brain injury. J Neurosci Methods 31: 101−107. | Article | PubMed | ChemPort |
22. Gowland C. 1987 Management of hemiplegic upper limb. (In) Stroke Rehabilitation (Brandstater ME, Basmajian JV, eds) Baltimore: Williams & Wilkins (pp) 217−244.
23. Grabowski M, Brundin P & Johansson BB. (1993) Paw-reaching, sensorimotor and rotational behaviour after brain infarction in rats. Stroke 24: 889−895. | PubMed | ChemPort |
24. Hasegawa Y, Formato JE, Latour LL, Gutierrez JA, Liu K-F, Garcia JH, Sotak CH & Fisher M. (1996) Severe transient hypoglycemia causes reversible changes in the apparent diffusion coefficient of water. Stroke 27: 1648−1656. | PubMed | ChemPort |
25. Heilman KM, Watson RT & Valenstein E. 1985 Neglect and related disorders. (In) (Heilman KM, Valenstein E, eds) Clinical Neuropsychology New York: Oxford University Press (pp) 243−294.
26. Hsu CY, Liu TH, Xu J, Hogan EL, Chao J, Sun G, Tai HH, Beckman JS & Freeman BA. (1989) Arachidonic acid and its metabolites in cerebral ischaemia. Ann NY Acad Sci 559: 282−295. | PubMed | ChemPort |
27. Ishii H, Toshiyuki A, Morikawa S, Inubushi T, Tooyama I, Kimura H & Mori K. (1998) Evaluation of focal cerebral ischaemia in rats by magnetic resonance imaging and immunohistochemical analyses. J Cereb Blood Flow Metab 18: 931−934. | Article | PubMed | ChemPort |
28. Jiang N, Finklestein SP, Do T, Caday CG, Charette M & Chopp M. (1996) Delayed intravenous administration of basic fibroblast growth factor (Bfgf) reduces infarct volume in a model of focal cerebral ischemia/reperfusion in the rat. J Neurol Sci 139: 173−179. | Article | PubMed | ChemPort |
29. Jiang Q, Chopp M, Zhang ZG, Knight RA, Jacobs M, Windham JP, Peck D, Ewing JR & Welch KMA. (1997) The temporal evolution of MRI tissue signatures after transient middle cerebral artery occlusion in rat. J Neurol Sci 145: 15−23. | Article | PubMed | ChemPort |
30. Johansson BB & Ohlsson AL. (1996) Environment, social interaction and physical activity as determinants of functional outcome after cerebral infarction in the rat. Exp Neurol 139: 322−327. | Article | PubMed | ISI | ChemPort |
31. Jones RD, Donaldson IM & Parkin PJ. (1989) Impairment and recovery of ipsilateral sensory-motor function following unilateral cerebral infarction. Brain 112: 113−132. | PubMed |
32. Jones DK, Lythgoe D, Horsfield MA, Simmons A, Williams SCR & Markus DM. (1999) Characterization of white matter damage in ischaemic leukoaraiosis with diffusion tensor MRI. Stroke 30: 393−397. | PubMed | ISI | ChemPort |
33. Jones TA & Schallert T. (1992) Overgrowth and pruning of dendrites in adult rats recovering from neocortical damage. Brain Res 581: 156−160. | Article | PubMed | ChemPort |
34. Jones TA & Schallert T. (1994) Use-dependent growth of pyramidal neurons after neocortical damage. J Neurosci 14: 2140−2152. | PubMed | ChemPort |
35. Kalaska JF, Scott SH, Cisek P & Sergio L. (1997) Cortical control of reaching movements. Curr Opin Neurobiol 7: 849−859. | Article | PubMed | ISI | ChemPort |
36. Kawamata T, Dietrich WD, Schallert T, Gotts JE, Cocke RR, Benowitz LI & Finklestein SP. (1997) Intracisternal basic fibroblast growth factor enhances functional recovery and up-regulates the expression of a molecular marker for neuronal sprouting following focal cerebral infarction. Proc Natl Acad Sci USA 94: 8179−8184. | Article | PubMed | ChemPort |
37. Kawamata T, Ren JM, Chan TCK, Charlette M & Finklestein SP. (1998) Intracisternal osteogenic protein-1 enhances functional recovery following focal stroke. Neuroreport 9: 1441−1445. | PubMed | ChemPort |
38. Knight RA, Dereski MO, Helpern JA, Ordidge RJ & Chopp M. (1994) Magnetic resonance imaging assessment of evolving focal cerebral ischaemia: comparison with histopathology in rats. Stroke 25: 1252−1262. | PubMed | ChemPort |
39. Knight RA, Ordidge RJ, Helpern JA, Chopp M, Rodolosi LC & Peck D. (1991) Temporal evolution of ischemic damage in rat brain measured by proton nuclear magnetic resonance imaging. Stroke 22: 802−808. | PubMed | ISI | ChemPort |
40. Koizumi J, Yoshida Y, Nakazawa T & Ooneda G. (1986) Experimental studies of cerebral brain edema. 1. A new experimental model of cerebral embolism in rats in which recirculation can be introduced into the ischaemic area. Jpn J Stroke 8: 1−8.
41. Kuroiwa T, Nagaoka T, Ueki M, Yamada I, Miyasaka N & Akimoto H. (1998) Different apparent diffusion coefficient: water content correlations of gray and white matter during early ischaemia. Stroke 29: 859−865. | PubMed | ChemPort |
42. Lang AE, Curran T, Provias J & Bergeron C. (1994) Striatonigral degeneration: iron deposition in putamen correlates with the slit-like void signal of magnetic resonance imaging. Can J Neurol Sci 21: 311−318. | PubMed | ChemPort |
43. Liu KF, Li FH, Tatlisumak T, Garcia JH, Fisher M & Fenstermacher JD. (1999) Swelling of astrocytes and endothelial cells induced by MCA occlusion are reflected in diffusion-weighted magnetic resonance imaging. J Cereb Blood Flow Metab 19: S666.
44. Lynch LJ. 1983 Water relaxation in heterogenous and biological systems. (In) Magnetic Resonance in Biology (Cohen JS, ed) New York: John Wiley and Sons (pp) 280−296.
45. Lythgoe MF, Busza AL, Calamante F, Sotak CH, King MD, Bingham AC, Williams SR & Gadian DG. (1997) Effects of diffusion anisotropy on lesion delineation in a rat model of cerebral ischaemia. Magn Res Med 38: 662−668. | ChemPort |
46. Mancuso A, Nimura T & Weinstein PR. (1997) Prediction of delayed ischemic injury with diffusion-weighted MRI following temporary middle cerebral artery occlusion in rats. Brain Res 760: 42−51. | Article | PubMed | ChemPort |
47. Markgraf CG, Green EJ, Hurwitz BE, Morikawa E, Dietrich WD, McCabe PM, Ginsberg MD & Schneiderman N. (1992) Sensorimotor and cognitive consequences of middle cerebral artery occlusion in rats. Brain Res 575: 238−246. | Article | PubMed | ChemPort |
48. Markgraf CG, Johnson MP, Braun DL & Bickers MV. (1997) Behavioural recovery patterns in rats receiving the NMDA receptor antagonist MDL 100, 453 immediately post-stroke. Pharmacol Biochem Behav 56: 391−397. | Article | PubMed | ChemPort |
49. Marshall JWB & Ridley RM. (1996) Assessment of functional impairment following permanent middle cerebral artery occlusion in a non-human primate species. Neurodegeneration 5: 275−286. | Article | PubMed | ISI | ChemPort |
50. Marston HM, Faber ES, Crawford JH, Butcher SP & Sharkey J. (1995) Behavioural assessment of endothelin-1 induced middle cerebral artery occlusion in the rat. Neuroreport 6: 1067−1071. | PubMed | ChemPort |
51. Mathur De Vr'e R. (1984) Biological implications of the relaxation behaviour of water related to NMR imaging. Br J Radiol 57: 955−976. | PubMed | ChemPort |
52. McDaniel WF, Via JD, Smith JS, Wells DL, Fu JJ, Bishop JF, Boyd PA & Ledesma HM. (1995) Unilateral injury to the posterior parietal cortex and spatial learning in hooded rats. Behav Brain Res 70: 165−179. | Article | PubMed | ChemPort |
53. McLeroy KR, De Vellis R, Devellis B, Kaplan B & Tool J. (1984) Social support and physical recovery in a stroke population. J Soc Pers Rel 1: 395−413.
54. Memezawa H, Minamisawa ML, Smith ML & Siesjo BK. (1992b) Ischaemic penumbra in a model of reversible middle cerebral artery occlusion in the rat. Exp Brain Res 89: 67−78. | Article | PubMed | ChemPort |
55. Memezawa H, Smith ML & Siesjo BK. (1992a) Penumbral tissues salvaged by reperfusion following middle cerebral artery occlusion in rats. Stroke 23: 552−559. | PubMed | ISI | ChemPort |
56. Montoya CP, Campbell-Hope LJ, Pemberton KD & Dunnett SB. (1991) The "staircase test": a measure of independent forelimb reaching and grasping abilities in rats. J Neurosci Methods 36: 219−228. | Article | PubMed | ISI | ChemPort |
57. Myers R, Manjil LG, Cullen BM, Price GW, Frackowiak RSJ & Cremer JE. (1991) Macrophage and astrocyte populations in relation to (H3)PK 11195 binding in the rat cerebral cortex following local ischaemic lesion. J Cereb Blood Flow Metab 11: 314−322. | PubMed | ChemPort |
58. Nakane M, Tamura A, Nagaoka T & Hirakawa K. (1997) MR detection of secondary changes remote from ischaemia: preliminary observations after occlusion of the middle cerebral artery in rats. AJNR Am J Neuroradiol 18: 945−950. | PubMed | ChemPort |
59. Nicholson C. 1993 Diffusion of ions and micromolecules in the brain. (In) SMRM Workshop Book of Abstracts Berkeley, CA: Society of Magnetic Resonance in Medicine (pp) 1−7. | ChemPort |
60. Nudo RJ, Wise BM, SiFuentes F & Milliken GW. (1996) Neural substrates for the effects of rehabilitative training on motor recovery after ischemic infarct. Science 272: 1791−1794. | PubMed | ISI | ChemPort |
61. Ordidge RJ, Helpern JA, Knight RA, Qing Z & Welch KMA. (1991) Investigation of cerebral ischaemia using magnetization transfer contrast (MTC) MR imaging. Magn Reson Imaging 9: 895−902. | PubMed | ChemPort |
62. Paxinos G & Watson C. 1986 The Rat Brain in Stereotaxic Coordinates (2nd ed.) Orlando, FL: Academic Press.
63. Pendlebury ST, Blaimire AM, Lee MA, Styles P & Matthews PM. (1999) Axonal injury in the internal capsule correlates with motor impairment after stroke. Stroke 30: 956−962. | PubMed | ChemPort |
64. Pereira AC, Howe FA, Doyle VL, Griffiths JR & Brown MM. 1997 The disappearing cerebral infarct. (In) Proceedings for the International Society of Magnetic Resonance in Medicine Book of Abstracts (ISMRM, Poster session) T572.
65. Perona P & Malik J. (1990) Scale-space and edge detection using anisotrophic diffusion. IEE Trans Pattern Analysis Machine Intelligence 12: 629−639.
66. Persson L, Hardemark HG, Bolander HG, Hillered L & Olsson Y. (1989) Neurologic and neuropathological outcome after middle cerebral artery occlusion in rats. Stroke 20: 641−645. | PubMed | ChemPort |
67. Petroff OAC, Graham GD, Blamire AM, Al-Rayess M, Rothman DL, Fayad PB, Brass LM, Shulman RG & Prichard JW. (1992) Spectroscopic imaging of stroke in humans: histopathology correlates of spectral changes. Neurology 42: 1349−1354. | PubMed | ISI | ChemPort |
68. Pierpaoli C, Righini A, Linfante I, Tao-Cheng JH, Alger JR & Di Chiro G. (1993) Histopathologic correlates of abnormal water diffusion in cerebral ischaemia: diffusion-weighted MR imaging and light and electron microscopic study. Radiology 189: 439−448. | PubMed | ChemPort |
69. Pisa M. (1988) Motor somatotopy in the striatum of rat: manipulation, biting and gait. Behav Brain Res 27: 21−35. | Article | PubMed | ChemPort |
70. Pisa M & Cyr J. (1990) Regionally selective roles of the rat's striatum in modality-specific discrimination learning and forelimb reaching. Behav Brain Res 37: 281−292. | Article | PubMed | ChemPort |
71. Plumet J, Cadisseau J & Roger M. (1990) Cortical transplants reduce motor deficits resulting from neonatal damage to the rats frontal cortex. Neurosci Lett 109: 102−106. | Article | PubMed | ChemPort |
72. Price AW & Fowler SC. (1981) Deficits in contralateral and ipsilateral forepaw motor control following unilateral motor cortical ablations in rats. Brain Res 205: 81−90. | Article | PubMed | ChemPort |
73. Rogers DC, Campbell CA, Stretton JL & Mackay KB. (1997) Correlation between motor impairment and infarct volume after permanent and transient middle cerebral artery occlusion in the rat. Stroke 28: 2060−2066. | PubMed | ChemPort |
74. Rose L, Bakel DA, Fung TS, Farn P & Weaver LE. (1994) Tactile extinction and functional status after stroke: a preliminary investigation. Stroke 25: 1973−1976. | PubMed | ChemPort |
75. Schallert T & Upchurch M. (1983) Posture-independent sensorimotor analysis of inter-hemispheric receptor asymmetries in neostriatum. Pharmacol Biochem Behav 18: 753−759. | Article | PubMed | ISI | ChemPort |
76. Schallert T, Upchurch M, Lobaugh S & Farrar SB. (1982) Tactile extinction: distinguishing between sensorimotor and motor asymmetries in rats with unilateral nigrostriatal damage. Pharmacol Biochem Behav 16: 455−462. | Article | PubMed | ChemPort |
77. Schwarz J, Weis S, Kraft E, Tatsch K, Bandmann O, Mehraein P, Vogl T & Oertel WH. (1996) Signal changes on MRI and increases in relative microgliosis, astrogliosis and iron in the putamen of two patients with multiple system atrophy. J Neurol Neurosurg Psychiatry 60: 98−101. | PubMed | ChemPort |
78. Sharkey J, Crawford JH, Butcher SP & Marston HM. (1996) Tacrolimus (FK506) ameliorates skilled motor deficits produced by middle cerebral artery occlusion in rats. Stroke 27: 2282−2286. | PubMed | ChemPort |
79. Smart SC, Beech JS, Lythgoe DJ, Kinchesh P, Everett JR, Swanson AG, Urenjak J & Williams SCR. 1996 In vivo assessment of MK-801 efficacy in a rat model of transient focal cerebral ischaemia. (In) Pharmacology of Cerebral Ischaemia (Krieglstein J, ed) Stuttgart, Germany, Medpharm, Scientific Publishers (pp) 667−681. | ChemPort |
80. Stroemer RP, Kent TA & Hulsebosch CE. (1995) Neocortical neural sprouting, synaptogenesis and behavioural recovery after neocortical infarction in rats. Stroke 26: 2135−2144. | PubMed | ChemPort |
81. Stroemer RP, Kent TA & Hulsebosch CE. (1998) Enhanced neocortical neural sprouting, synaptogenesis and behavioural recovery with D-amphetamine therapy after neocortical infarction in rats. Stroke 29: 2381−2393. | PubMed | ChemPort |
82. Sunderland A, Bowers MP, Sluman SM, Wilcock DJ & Ardron ME. (1999) Impaired dexterity of the ipsilateral hand after stroke and the relationship to cognitive deficit. Stroke 30: 949−955. | PubMed | ChemPort |
83. Takahashi M, Fritz-Zieroth B, Chikugo T & Ogawa H. (1993) Differentiation of chronic lesions after stroke in stroke-prone spontaneously hypertensive rats using diffusion-weighted MRI. Magn Res Med 30: 485−488. | ChemPort |
84. Tominaga T & Ohnishi ST. (1989) Interrelationship of brain edema, motor deficits, and memory impairment in rats exposed to focal ischaemia. Stroke 20: 513−518. | PubMed | ChemPort |
85. Tracey DJ. 1995 Ascending and descending pathways in the spinal cord. (In) The Rat Nervous System (Paxinos G, ed) Sydney, Australia: Academic Press.
86. Weiller C, Chollert F, Friston KJ, Wise RJ & Frackowiak RSJ. (1992) Functional reorganization of the brain in recovery from striatocapsular infarction in man. Ann Neurol 31: 463−472. | Article | PubMed | ChemPort |
87. Weiller C, Ramsay SC, Wise RJS, Friston KJ & Frackowiak RSJ. (1993) Individual patterns of functional reorganization in the human cerebral cortex after capsular infarction. Ann Neurol 33: 181−189. | PubMed | ChemPort |
88. Welch KMA, Windham J, Knight RA, Nagesh V, Hugg JW, Jacobs M, Peck D, Booker P, Dereski MO & Levine SR. (1995) A model to predict the histopathology of human stroke using diffusion and T2-weighted magnetic resonance imaging. Stroke 26: 1983−1989. | PubMed | ChemPort |
89. Whishaw IQ, O'Connor WT & Dunnett SB. (1986) The contributions of motor cortex, nigrostriatal dopamine and caudate-putamen to skilled forelimb use in the rat. Brain 109: 805−843. | PubMed |
90. Whishaw IQ, Pellis SM & Gorny BP. (1992) Skilled reaching in rats and humans: evidence for parallel development or homology. Behav Brain Res 47: 59−70. | PubMed | ChemPort |
91. Yam PS, Dewar D & McCulloch J. (1998) Axonal injury caused by focal cerebral ischaemia in the rat. J Neurotrauma 15: 441−450. | PubMed | ChemPort |
92. Zelaya F, Flood N, Chalk JB, Wang D, Doddrell DM, Strugnell, Benson M, Ostergaard L, Semple J & Eagle S. (1999) An evaluation of the time dependence of the anisotropy of the water diffusion tensor in acute human ischaemia. Magn Res Imaging 17: 331−348. | ChemPort |

Acknowledgements

The authors extend their gratitude to R. Crundwell and D. Bacon at Queen Maryland Westfield College, C. David and A. Tucker at SmithKline Beecham, Harlow, U.K., and A. Chadwick at Institute of Psychiatry, Denmark Hill, London, for providing graphics support. The authors also thank P. Nelson at SmithKline Beecham and A. Riazi at the Institute of Neurology, Queens Square, London, for statistical advice.