James R. Ewing

A rat model of focal embolic cerebral ischemia.

We developed a new model of embolic cerebral ischemia in the rat which provides a reproducible and predictable infarct volume within the territory supplied by the middle cerebral artery (MCA). The MCA was occluded by an embolus in Wistar rats (n = 71). An additional three non-embolized rats were used as a control. Cerebral blood flow (CBF) was measured by means of laser Doppler flowmetry (LDF) and perfusion weighted imaging (PWI) before and after embolization. The evolution of the lesion was monitored by diffusion weighted imaging (DWI). Cerebral vascular perfusion patterns were examined using laser scanning confocal microscopy. Infarct volumes were measured on hematoxylin and eosin (H&E) stained coronal sections. The lodgment of the clot at the origin of the MCA and the ischemic cell damage were examined using light microscopy. Regional CBF in the ipsilateral parietal cortex decreased to 43 +/- 4.1% (P < 0.05) of preischemic levels (n = 10). Confocal microscopic examination revealed a reduction of cerebral plasma perfusion in the ipsilateral MCA territory (n = 6). MRI measurements showed a reduction in CBF and a hyperintensity DWI encompassing the territory supplied by the MCA (n = 4). An embolus was found in all rats at 24 h after embolization. The infarct volume as a percentage of the contralateral hemisphere was 32.5 +/- 3.31% at 24 h (n = 20), 33.0 +/- 3.6% at 48 h (n = 13), and 34.5 +/- 4.74% at 168 h (n = 12) after embolization. This model of embolic focal cerebral ischemia results in ischemic cell damage and provides a reproducible and predictable infarct volume. This model is relevant to thromboembolic stroke in humans and may be useful in documenting the safety and efficacy of fibrinolytic intervention and in investigating therapies complementary to antithrombotic therapy.

Interregional variation of longitudinal relaxation rates in human brain at 3.0 T: Relation to estimated iron and water contents

In a study of interregional variation of the longitudinal relaxation rate (R1) in human brain at 3 T, R1 maps were acquired from 12 healthy adults using a multi‐slice implementation of the T one by multiple readout pulses (TOMROP) sequence. Mean R1 values were obtained from the prefrontal cortex (0.567 ± 0.020 sec–1), caudate head (0.675 ± 0.019 sec–1), putamen (0.749 ± 0.023 sec–1), substantia nigra (0.873 ± 0.037 sec–1), globus pallidus (0.960 ± 0.034 sec–1), thalamus (0.822 ± 0.027 sec–1), and frontal white matter (1.184 ± 0.057 sec–1). For gray matter regions other than the thalamus, R1 showed a strong correlation (r = 0.984, P < 0.0001) with estimated regional nonheme iron concentrations ([Fe]). These R1 values also showed a strong correlation (r = 0.976, P < 0.0001) with estimates of 1/fw obtained from MRI relative proton density measurements, where fw represents tissue water content. When white matter is included in the consideration, 1/fw is a better predictor of R1 than is [Fe]. An analysis based on the fast‐exchange two‐state model of longitudinal relaxation suggests that interregional differences in fw account for the majority of the variation of R1 across gray matter regions. Magn Reson Med 45:71–79, 2001.

Patlak plots of Gd‐DTPA MRI data yield blood–brain transfer constants concordant with those of 14C‐sucrose in areas of blood–brain opening

The blood‐to‐brain transfer rate constant (Ki) of Gd‐DTPA was determined in MRI studies of a rat model of transient cerebral ischemia. The longitudinal relaxation rate, R1, was estimated using repeated Look‐Locker measurements. A model‐independent analysis of ΔR1, the Patlak plot, produced maps of Ki for Gd‐DTPA and the distribution volume of the mobile protons (Vp) with intravascular‐Gd changed R1s. The Kis of Gd‐DTPA were estimated in regions of interest with blood–brain barrier (BBB) opening (regions of interest, ROIs) and compared to those of 14C‐sucrose determined shortly thereafter by quantitative autoradiography. The Kis for both Gd‐DTPA and sucrose were much higher than normal within the ROIs (n = 7); linear regression of Ki for Gd‐DTPA vs. Ki for sucrose yielded a slope of 0.43 ± 0.11 and r2 = 0.72 (P = 0.01). Thus, Ki for Gd‐DTPA varied in parallel with, but was less than, Ki for sucrose. In the ROIs, mean Vp was 0.071 ml g−1 and much higher than mean vascular volume estimated by dynamic‐contrast‐enhancement (0.013 ml g−1) or mean Vp in contralateral brain (0.015 ml g−1). This elevated Vp may reflect increased capillary permeability to water. In conclusion, Ki can be reliably calculated from Gd‐DTPA‐MRI data by Patlak plots. Magn Reson Med 50:283–292, 2003.

Investigation of neural progenitor cell induced angiogenesis after embolic stroke in rat using MRI

Using MRI, we investigated dynamic changes of brain angiogenesis after neural progenitor cell transplantation in the living adult rat subjected to embolic stroke. Neural progenitor cells isolated from the subventricular zone (SVZ) of the adult rat were labeled by superparamagnetic particles and intracisternally transplanted into the adult rat 48 h after stroke (n = 8). Before and after the transplantation, an array of MRI parameters were measured, including high resolution 3D MRI and quantitative T1, T1sat (T1 in the presence of an off-resonance irradiation of the macromolecules of brain), T2, the inverse of the apparent forward transfer rate for magnetization transfer (kinv), cerebral blood flow (CBF), cerebral blood volume (CBV), and blood-to-brain transfer constant (Ki) of Gd-DTPA. The von Willerbrand factor (vWF) immunoreactive images of coronal sections obtained at 6 weeks after cell transplantation were used to analyze vWF immunoreactive vessels. MRI measurements revealed that grafted neural progenitor cells selectively migrated towards the ischemic boundary regions. In the ischemic boundary regions, angiogenesis confirmed by an increase in vascular density and the appearance of large thin wall mother vessels was coincident with increases of CBF and CBV (CBF, P < 0.01; CBV, P < 0.01) at 6 weeks after treatment, and coincident with transient increases of K(i) with a peak at 2 to 3 weeks after cell therapy. Relative T1, T1sat, T2, and kinv decreased in the ischemic boundary regions with angiogenesis compared to that in the non-angiogenic ischemic region (T1, P < 0.01 at 6 weeks; T1sat, P < 0.05 at 2 to 6 weeks; T2, P < 0.05 at 3 to 6 weeks; kinvP < 0.05 at 6 weeks). Of these methods, Ki appear to be the most useful MR measurements which identify and predict the location and area of angiogenesis. CBF, CBV, T1sat, T1, T2, and kinv provide complementary information to characterize ischemic tissue with and without angiogenesis. Our data suggest that select MRI parameters can identify the cerebral tissue destined to undergo angiogenesis after treatment of embolic stroke with cell therapy.

MRI detects white matter reorganization after neural progenitor cell treatment of stroke

We evaluated the effects of neural progenitor cell treatment of stroke on white matter reorganization using MRI. Male Wistar rats (n = 26) were subjected to 3 h of middle cerebral artery occlusion and were treated with neural progenitor cells (n = 17) or without treatment (n = 9) and were sacrificed at 5-7 weeks thereafter. MRI measurements revealed that grafted neural progenitor cells selectively migrated towards the ischemic boundary regions. White matter reorganization, confirmed histologically, was coincident with increases of fractional anisotropy (FA, P < 0.01) after stroke in the ischemic recovery regions compared to that in the ischemic core region in both treated and control groups. Immunoreactive staining showed axonal projections emanating from neurons and extruding from the corpus callosum into the ipsilateral striatum bounding the lesion areas after stroke. Fiber tracking (FT) maps derived from diffusion tensor imaging revealed similar orientation patterns to the immunohistological results. Complementary measurements in stroke patients indicated that FT maps exhibit an overall orientation parallel to the lesion boundary. Our data demonstrate that FA and FT identify and characterize cerebral tissue undergoing white matter reorganization after stroke and treatment with neural progenitor cells.

Effects of administration route on migration and distribution of neural progenitor cells transplanted into rats with focal cerebral ischemia, an MRI study

We tested the hypotheses that administration routes affect the migration and distribution of grafted neural progenitor cells (NPCs) in the ischemic brain and that the ischemic lesion plays a role in mediating the grafting process. Male Wistar rats (n=41) were subjected to 2-h middle cerebral artery occlusion (MCAo), followed 1 day later by administration of magnetically labeled NPCs. Rats with MCAo were assigned to one of three treatment groups targeted for cell transplantation intra-arterially (IA), intracisternally (IC), or intravenously (IV). MRI measurements consisting of T2-weighted imaging and three-dimensional (3D) gradient echo imaging were performed 24 h after MCAo, 4 h after cell injection, and once a day for 4 days. Prussian blue staining was used to identify the labeled cells, 3D MRI to detect cell migration and distribution, and T2 map to assess lesion volumes. Intra-arterial (IA) administration showed significantly increased migration, a far more diffuse distribution pattern, and a larger number of transplanted NPCs in the target brain than IC or IV administration. However, high mortality with IA delivery (IA: 41%; IC: 17%; IV: 8%) poses a serious concern for using this route of administration. Animals with smaller lesions at the time of transplantation have fewer grafted cells in the parenchyma.

Quantitative estimation of permeability surface-area product in astroglial brain tumors using perfusion CT and correlation with histopathologic grade.

BACKGROUND AND PURPOSE: Glioma angiogenesis and its different hemodynamic features, which can be evaluated by using perfusion CT (PCT) imaging of the brain, have been correlated with the grade and the aggressiveness of gliomas. Our hypothesis was that quantitative estimation of permeability surface area product (PS), cerebral blood volume (CBV), cerebral blood flow (CBF), and mean transit time (MTT) in astroglial brain tumors by using PCT will correlate with glioma grade. High-grade gliomas will show higher PS and CBV as compared with low-grade gliomas. MATERIALS AND METHODS: PCT was performed in 32 patients with previously untreated astroglial tumors (24 high-grade gliomas and 8 low-grade gliomas) by using a total acquisition time of 170 seconds. World Health Organization (WHO) glioma grades were compared with PCT parameter absolute values by using Student or nonparametric Wilcoxon 2-sample tests. Receiver operating characteristic (ROC) analyses were also done for each of the parameters. RESULTS: The differences in PS, CBV, and CBF between the low- and high-grade tumor groups were statistically significant, with the low-grade group showing lower mean values than the high-grade group. ROC analyses showed that both CBV (C-statistic 0.930) and PS (C-statistic 0.927) were very similar to each other in differentiating low- and high-grade gliomas and had higher predictability compared with CBF and MTT. Within the high-grade group, differentiation of WHO grade III and IV gliomas was also possible by using PCT parameters, and PS showed the highest C-statistic value (0.926) for the ROC analyses in this regard. CONCLUSIONS: Both PS and CBV showed strong association with glioma grading, high-grade gliomas showing higher PS and CBV as compared with low-grade gliomas. Perfusion parameters, especially PS, can also be used to differentiate WHO grade III from grade IV in the high-grade tumor group.

Magnetic resonance imaging investigation of axonal remodeling and angiogenesis after embolic stroke in sildenafil-treated rats

Interaction between angiogenesis and axonal remodeling after stroke was dynamically investigated by MRI in rats with or without sildenafil treatments. Male Wistar rats were subjected to embolic stroke and treated daily with saline (n = 10) or with sildenafil (n = 11) initiated at 24 h and subsequently for 7 days after onset of ischemia. T*2-weighted imaging, cerebral blood flow (CBF), and diffusion tensor imaging (DTI) measurements were performed from 24 h to 6 weeks after embolization. T*2 and fractional anisotropy (FA) maps detected angiogenesis and axonal remodeling after stroke, respectively, starting from 1 week in sildenafil-treated rats. Areas demarcated by MRI with enhanced angiogenesis, elevated local CBF, and augmented axonal remodeling were spatially and temporally matched, and FA values were significantly correlated with the corresponding CBF values (R = 0.66, P < 4 × 10−5) at 6 weeks after stroke. Axonal projections were reorganized along the ischemic boundary after stroke. These MRI measurements, confirmed by histology, showed that sildenafil treatment simultaneously enhanced angiogenesis and axonal remodeling, which were MRI detectable starting from 1 week after stroke in rats. The spatial and temporal consistency of MRI metrics and the correlation between FA and local CBF suggest that angiogenesis, by elevating local CBF, promotes axonal remodeling after stroke.

Model Selection in Magnetic Resonance Imaging Measurements of Vascular Permeability: Gadomer in a 9L Model of Rat Cerebral Tumor

Vasculature in and around the cerebral tumor exhibits a wide range of permeabilities, from normal capillaries with essentially no blood-brain barrier (BBB) leakage to a tumor vasculature that freely passes even such large molecules as albumin. In measuring BBB permeability by magnetic resonance imaging (MRI), various contrast agents, sampling intervals, and contrast distribution models can be selected, each with its effect on the measurements outcome. Using Gadomer, a large paramagnetic contrast agent, and MRI measures of T1, over a 25-min period, BBB permeability was estimated in 15 Fischer rats with day-16 9L cerebral gliomas. Three vascular models were developed: (1) impermeable (normal BBB); (2) moderate influx (leakage without efflux); and (3) fast leakage with bidirectional exchange. For data analysis, these form nested models. Model 1 estimates only vascular plasma volume, vD, Model 2 (the Patlak graphical approach) vD and the influx transfer constant K1- Model 3 estimates vD, Ki and the reverse transfer constant, kb, through which the extravascular distribution space, ve, is calculated. For this contrast agent and experimental duration, Model 3 proved the best model, yielding the following central tumor means (±s.d.; n = 15): vD = 0.07±0.03 for Ki = 0.0105±0.005 min −1 and ve = 0.10±0.04. Model 2 Ki estimates were approximately 30% of Model 3, but highly correlated (r 0.80, P<0.0003). Sizable inhomogeneity in vD, Ki and kb appeared within each tumor. We conclude that employing nested models enables accurate assessment of transfer constants among areas where BBB permeability, contrast agent distribution volumes, and signal-to-noise vary.

Diffusion-, T2-, and Perfusion-Weighted Nuclear Magnetic Resonance Imaging of Middle Cerebral Artery Embolic Stroke and Recombinant Tissue Plasminogen Activator Intervention in the Rat

Thrombolysis of embolic stroke in the rat was measured using diffusion (DWI)-, T2 (T2WI)-, and perfusion (PWI)-weighted magnetic resonance imaging (MRI). An embolus was placed at the origin of the middle cerebral artery (MCA) by injection of an autologous single blood clot via an intraluminal catheter placed in the intracranial segment of internal carotid artery. Rats were treated with a recombinant tissue plasminogen activator (rt-PA) 1 hour after embolization (n = 9) or were not treated (n = 15). Diffusion-weighted imaging, T2WI, and PWI were performed before, during, and after embolization from 1 hour to 7 days. After embolization in both rt-PA-treated and control animals, the apparent diffusion coefficient of water (ADCw) and cerebral blood flow (CBF) in the ischemic region significantly declined from the preischemic control values (P < 0.001). However, mean CBF and ADCw in the rt-PA—treated group was elevated early after administration of rt-PA compared with the untreated control group, and significant differences between the two groups were detected in CBF (24 hours after embolization, P < 0.05) and ADCw (3, 4, and 24 hours after embolization, P < 0.05). T2 values maximized at 24 (control group, P < 0.001) or 48 hours (treated group, P < 0.01) after embolization. The increase in T2 in the control group was significantly higher at 24 hours and 168 hours than in the rt-PA—treated group (P < 0.05). Significant correlations (r ≥ 0.80, P < 0.05) were found between lesion volume measured 1 week after embolization and CBF and ADCw obtained 1 hour after injection of rt-PA. Within a coronal section of brain, MRI cluster analysis, which combines ADCw and T2 data maps, indicated a significant reduction (P < 0.05) in the lesion 24 hours after thrombolysis compared with nontreated animals. These data demonstrate that the values for CBF and ADCw obtained 1 hour after injection of rt-PA correlate with histologic outcome in the tissue, and that the beneficial effect of thrombolysis of an intracranial embolus by means of rt-PA is reflected in an increase of CBF and ADCw, a reduction in the increase of T2, and a reduction of the ischemic lesion size measured using MRI cluster analysis.