Assessment of the MRI and Behavioral Test Results in a Focal Cerebral Ischemia-Reperfusion Model in the Rat after Separate and Combined Use of Mouse-Derived Neural Progenitor Cells, Human-Derived Neural Progenitor Cells and Atorvastatin.

AIM: To assess the efficacy of Neural progenitor cell (NPC) transplantation in ischemic stroke, and to investigate whether atorvastatin enhances therapeutic potency of NPC after stroke. MATERIAL AND METHODS: The focal cerebral ischemia-reperfusion model was performed by transient occlusion of middle cerebral artery. Rats were assigned randomly to receive intracerebral transplantation of mouse NPC alone (mNPC), human NPC alone (hNPC), mouse NPC plus oral atorvastatin (mNPC+A), human NPC plus oral atorvastatin (hNPC+A), oral atorvastatin alone, or intracerebral Dulbecco"s Modified Eagle"s medium injection (control group). Adhesive removal, rotarod, cylinder tests, and magnetic resonance imaging (MRI) were used for assessment of rats during 4 weeks. After sacrification on 28th day, rats were investigated by immunofluorescent staining. RESULTS: The hNPC and mNPC groups showed significantly improved functional outcome and reduced infarct area ratio compared with the control group. The hNPC group had significantly better performance and lower infarct area ratio than the mNPC group. Addition of atorvastatin to stem cell therapy significantly improved functional outcome, although it did not affect the infarct area ratio on MRI. Anti-inflammatory response in the infarct area was higher in the mNPC group. NPC transplantation significantly reduced the amount of microglia and a significant increase in the amount of astrocytes. CD8a+ T lymphocyte and granzyme B activities were not detected in any of the subjects. CONCLUSION: Both hNPC and mNPC treatments significantly improved functional outcome, and reduced infarct area ratio after stroke. Atorvastatin enhanced the therapeutic potency of NPCs, including neurological improvement.

Quantitative assessment of demyelination in ischemic stroke in vivo using macromolecular proton fraction mapping.

A recent MRI method, fast macromolecular proton fraction (MPF) mapping, was used to quantify demyelination in the transient middle cerebral artery occlusion (MCAO) rat stroke model. MPF and other quantitative MRI parameters (T1, T2, proton density, and apparent diffusion coefficient) were compared with histological and immunohistochemical markers of demyelination (Luxol Fast Blue stain, (LFB)), neuronal loss (NeuN immunofluorescence), axonal loss (Bielschowsky stain), and inflammation (Iba1 immunofluorescence) in three animal groups ( n = 5 per group) on the 1st, 3rd, and 10th day after MCAO. MPF and LFB optical density (OD) were significantly reduced in the ischemic lesion on all days after MCAO relative to the symmetrical regions of the contralateral hemisphere. Percentage changes in MPF and LFB OD in the ischemic lesion relative to the contralateral hemisphere significantly differed on the first day only. Percentage changes in LFB OD and MPF were strongly correlated (R = 0.81, P < 0.001) and did not correlate with other MRI parameters. MPF also did not correlate with other histological variables. Addition of T2 into multivariate regression further improved agreement between MPF and LFB OD (R = 0.89, P < 0.001) due to correction of the edema effect. This study provides histological validation of MPF as an imaging biomarker of demyelination in ischemic stroke.

Infarct morphology assessment in patients with carotid artery/middle cerebral artery occlusion using fast fluid-attenuated inversion recovery (FLAIR) vascular hyperintensity (FVH).

We aim to evaluate the value of fast fluid-attenuated inversion recovery (FLAIR) vascular hyperintensity (FVH) in assessing infarct morphology in patients with symptomatic internal carotid artery (ICA) or middle cerebral artery (MCA) occlusions. Magnetic resonance (MR) diffusion-weighted imaging (DWI) FLAIR sequences, and carotid/cerebral magnetic resonance angiography of 102 patients with symptomatic ICA or MCA occlusions were evaluated. The location and score of FVH were determined using Olindo's method; patients were classified as having Low or High FVHs based on FVH score, and either Distal or Proximal FVH based on FVH location. The differences between infarct morphologies were analyzed. FVH were detectable in 62 patients with High FVH and in 40 patients with Low FVHs based on the Olindo's scale. There were no statistically significant differences in age, gender, hypertension, diabetes, hyperlipidemia, smoking history, and vascular occlusive site between High and Low FVHs patients, except for infarct morphology (P<0.01). Patients with Distal FVH presented with significant (P<0.01) perforating artery and border zone infarcts, whereas those with Proximal FVH had significant (P<0.01) large territorial infarcts. The scores and locations of FVH could be a predictive imaging marker for infarct morphology in patients with symptomatic ICA or MCA occlusion.

Assessment of the Neuroprotective Effects of Arginine-Rich Protamine Peptides, Poly-Arginine Peptides (R12-Cyclic, R22) and Arginine-Tryptophan-Containing Peptides Following In Vitro Excitotoxicity and/or Permanent Middle Cerebral Artery Occlusion in Rats.

We have demonstrated that arginine-rich and poly-arginine peptides possess potent neuroprotective properties with arginine content and peptide positive charge being particularly critical for neuroprotective efficacy. In addition, the presence of other amino acids within arginine-rich peptides, as well as chemical modifications, peptide length and cell-penetrating properties also influence the level of neuroprotection. Against this background, we have examined the neuroprotective efficacy of arginine-rich protamine peptides, a cyclic (R12-c) poly-arginine peptide and a R22 poly-arginine peptide, as well as arginine peptides containing tryptophan or other amino acids (phenylalanine, tyrosine, glycine or leucine) in in vitro glutamic acid excitotoxicity and in vivo rat permanent middle cerebral artery occlusion models of stroke. In vitro studies demonstrated that protamine and poly-arginine peptides (R12-c, R22) were neuroprotective. Arginine-tryptophan-containing peptides were highly neuroprotective, with R12W8a being the most potent arginine-rich peptide identified in our laboratory. Peptides containing phenylalanine or tyrosine substituted in place of tryptophan in R12W8a were also highly neuroprotective, whereas leucine, and in particular glycine substitutions, decreased peptide efficacy. In vivo studies with protamine administered intravenously at 1000 nmol/kg 30 min after MCAO significantly reduced infarct volume and cerebral oedema by 22.5 and 38.6%, respectively. The R12W8a peptide was highly toxic when administered intravenously at 300 or 100 nmol/kg and ineffective at reducing infarct volume when administered at 30 nmol/kg 30 min after MCAO, unlike R18 (30 nmol/kg), which significantly reduced infarct volume by 20.4%. However, both R12W8a and R18 significantly reduced cerebral oedema by 19.8 and 42.2%, respectively. Protamine, R12W8a and R18 also reduced neuronal glutamic acid-induced calcium influx. These findings further highlight the neuroprotective properties of arginine-rich peptides and support the view that they represent a new class of neuroprotective agent.

Systematic and detailed analysis of behavioural tests in the rat middle cerebral artery occlusion model of stroke: Tests for long-term assessment.

In order to test therapeutics, functional assessments are required. In pre-clinical stroke research, there is little consensus regarding the most appropriate behavioural tasks to assess deficits, especially when testing over extended times in milder models with short occlusion times and small lesion volumes. In this study, we comprehensively assessed 16 different behavioural tests, with the aim of identifying those that show robust, reliable and stable deficits for up to two months. These tasks are regularly used in stroke research, as well as being useful for examining striatal dysfunction in models of Huntington's and Parkinson's disease. Two cohorts of male Wistar rats underwent the intraluminal filament model of middle cerebral artery occlusion (30 min) and were imaged 24 h later. This resulted in primarily subcortical infarcts, with a small amount of cortical damage. Animals were tested, along with sham and naïve groups at 24 h, seven days, and one and two months. Following behavioural testing, brains were processed and striatal neuronal counts were performed alongside measurements of total brain and white matter atrophy. The staircase, adjusting steps, rotarod and apomorphine-induced rotations were the most reliable for assessing long-term deficits in the 30 min transient middle cerebral artery occlusion model of stroke.

Assessment of Blood-Brain Barrier Permeability by Dynamic Contrast-Enhanced MRI in Transient Middle Cerebral Artery Occlusion Model after Localized Brain Cooling in Rats.

OBJECTIVE: The purpose of this study was to evaluate the effects of localized brain cooling on blood-brain barrier (BBB) permeability following transient middle cerebral artery occlusion (tMCAO) in rats, by using dynamic contrast-enhanced (DCE)-MRI. MATERIALS AND METHODS: Thirty rats were divided into 3 groups of 10 rats each: control group, localized cold-saline (20℃) infusion group, and localized warm-saline (37℃) infusion group. The left middle cerebral artery (MCA) was occluded for 1 hour in anesthetized rats, followed by 3 hours of reperfusion. In the localized saline infusion group, 6 mL of cold or warm saline was infused through the hollow filament for 10 minutes after MCA occlusion. DCE-MRI investigations were performed after 3 hours and 24 hours of reperfusion. Pharmacokinetic parameters of the extended Tofts-Kety model were calculated for each DCE-MRI. In addition, rotarod testing was performed before tMCAO, and on days 1-9 after tMCAO. Myeloperoxidase (MPO) immunohisto-chemistry was performed to identify infiltrating neutrophils associated with the inflammatory response in the rat brain. RESULTS: Permeability parameters showed no statistical significance between cold and warm saline infusion groups after 3-hour reperfusion 0.09 ± 0.01 min(-1) vs. 0.07 ± 0.02 min(-1), p = 0.661 for K(trans); 0.30 ± 0.05 min(-1) vs. 0.37 ± 0.11 min(-1), p = 0.394 for kep, respectively. Behavioral testing revealed no significant difference among the three groups. However, the percentage of MPO-positive cells in the cold-saline group was significantly lower than those in the control and warm-saline groups (p < 0.05). CONCLUSION: Localized brain cooling (20℃) does not confer a benefit to inhibit the increase in BBB permeability that follows transient cerebral ischemia and reperfusion in an animal model, as compared with localized warm-saline (37℃) infusion group.

Spatio-temporal assessment of the neuroprotective effects of neuregulin-1 on ischemic stroke lesions using MRI.

The neuroprotective effects of neuregulin-1 (NRG-1) on stroke lesions were assessed longitudinally in rats with middle cerebral artery occlusion (MCAo) using MRI. Sprague-Dawley rats (n=16, 250±20g) underwent permanent MCAo surgery with cerebral blood flow (CBF) monitored by laser doppler flowmetry at ipsilateral side of bregma for 20min post-occlusion. A single 50µl bolus dose of NRG-1 or vehicle was administered into the left internal carotid artery immediately prior to MCAo. The expansion of the ischemic lesion into the cortex was attenuated by NRG-1 over a 48-hour (h) time span as measured by diffusion weighted imaging (DWI). The final infarct volumes of NRG-1 treated rats were significantly smaller than those of the vehicle treated rats at 48h (264.8±192.1 vs. 533.4±175.5mm(3), p<0.05). The NRG-1 treated rats were further subdivided into 2 subgroups according to their CBF reduction during stroke surgery: mild ischemia (<70% CBF reduction) or severe ischemia (>70% CBF reduction). In particular, ischemic infarction was not usually observed in the cortex of NRG-1 treated rats with mild ischemia at 3 and 48h post-occlusion. Histological results validated the imaging findings and demonstrated that NRG-1 treated rats had fewer injured neurons in peri-infarct areas 48h post-ischemia. In summary, the neuroprotective effect of NRG-1 in the pMCAo stroke model was demonstrated by prevention of ischemic lesion expansion, reduced infarct volume and protection of neurons from ischemic damage.

A blinded randomized assessment of laser Doppler flowmetry efficacy in standardizing outcome from intraluminal filament MCAO in the rat.

BACKGROUND: Laser Doppler flowmetry (LDF) is widely used for estimating cerebral blood flow changes during intraluminal middle cerebral artery occlusion (MCAO). No investigation has systematically examined LDF efficacy in standardizing outcome. We examined MCAO histologic and behavioral outcome as a function of LDF measurement. MATERIALS AND METHODS: Rats were subjected to 90min MCAO by 4 surgeons having different levels of MCAO surgical experience. LDF was measured in all rats during ischemia. By random assignment, LDF values were (Assisted) or were not (Blinded) made available to each surgeon during MCAO (n=12-17 per group). Neurologic and histologic outcomes were measured 7 days post-MCAO. A second study examined LDF effects on 1-day post-MCAO outcome. RESULTS: Pooled across surgeons, intra-ischemic %LDF change (P=0.12), neurologic scores (Assisted vs. Blinded=14±6 vs. 13±7, P=0.61, mean±standard deviation) and cerebral infarct volume (162±63mm(3)vs. 143±86mm(3), P=0.24) were not different between groups. Only for one surgeon (novice) did LDF use alter infarct volume (145±28mm(3)vs. 98±61mm(3), P=0.03). LDF use decreased infarct volume coefficient of variation (COV) by 35% (P=0.02), but had no effect on neurologic score COV. COMPARISON WITH EXISTING METHODS: We compared intraluminal MCAO outcome as a function of LDF use. CONCLUSIONS: LDF measurement altered neither neurologic nor histologic MCAO outcome. LDF did not decrease neurologic deficit COV, but did decrease infarct volume COV. LDF may allow use of fewer animals if infarct volume is the primary dependent variable, but is unlikely to impact requisite sample sizes if neurologic function is of primary interest.

Longitudinal assessment of infarct progression, brain metabolism and behavior following anterior cerebral artery occlusion in rats.

BACKGROUND: Stroke patients suffering from occlusion of the anterior cerebral artery (ACAo) develop cognitive and executive deficits. Experimental models to investigate such functional impairments and recovery are rare and not satisfyingly validated. NEW METHOD: We stereotactically injected the vasoconstrictor endothelin-1 (ET-1) close to the ACA of rats and assessed magnitude and course of CBF reduction using [(14)C]iodoantipyrine autoradiography and [(15)O]H2O-PET. [(18)F]FDG-PET and T2-weighted MRI determined regional metabolic and structural alterations. To test cognitive and executive functions, we analyzed decision-making in a food-carrying task, spatial working memory in a spontaneous alternation task and anxiety in an elevated plus maze test before and 1 month after ACAo. RESULTS: CBF decreased immediately after ET-1 injection, started to recover 1-2h and returned to control 4h thereafter. Metabolic and structural lesions developed permanently in the ACA territory. Hypometabolism occurring bilaterally in the piriform region may reflect diaschisis. Behavioral testing after ACAo revealed context-dependent changes in decision making, exploratory activity and walking speed, as well as decreased anxiety and spatial working memory. COMPARISON WITH EXISTING METHOD(S): Aside from modeling a known entity of stroke patients, ACAo in rats allows to longitudinally study deterioration of cognitive and executive function without major interference by disturbed primary motor function. It complements therefore stroke research since common models using middle cerebral artery occlusion (MCAo) all affect motor function severely. CONCLUSION: The established ACAo model in rats effectively reflects deficits characteristic for ACA stroke in humans. It is furthermore highly suitable for longitudinal assessment of cognitive and executive functions.

Regional distribution of selective neuronal loss and microglial activation across the MCA territory after transient focal ischemia: quantitative versus semiquantitative systematic immunohistochemical assessment.

Histopathologic assessment in transient middle cerebral artery occlusion (MCAo) rodent models generally lacks comprehensiveness and exposes to interobserver bias. Here we compared a novel quantitative assessment of regional infarction, selective neuronal loss (SNL) and microglial activation (MA) across the MCA territory to a previously published semiquantitative visual protocol. NeuN and OX42 immunohistochemistry was applied after either 15 or 45 minutes distal MCAo to maximize SNL and infarction, respectively. Survival times varied from 28 to 60 days to cover potential biases such as delayed tissue shrinkage. Damage was assessed using a template of 44 cytoarchitectonic regions of interest (ROIs) mapped onto a subset of digitized coronal sections spanning the MCA territory. For each ROI were obtained a semiquantitative visually determined index of histopathologic changes (method 1), and lpsilateral/contralesional ratios of remaining neurons and activated microglia cell counts (method 2). There was excellent agreement between the two methods for 28-day survival for both MCAo durations, whereas method 2 more sensitively detected subtle SNL and MA at 45 days and 60 days after 15-minute MCAo. Thus the visual method is accurate for usual degrees of ischemic damage, but absolute cell quantification is superior to detect subtle changes and should therefore be preferred in brief MCAo models, although requires optimal staining quality.

An integrated stroke model with a consistent penumbra for the assessment of neuroprotective interventions.

BACKGROUND/AIM: A longer period of vessel occlusion reduces the coefficient of variation of the infarct lesion size ['infarct variation coefficient' (IVC)] due to a gradual expansion of the lesion within a limited territory defined by the vascular anatomy, but it increases the mortality rate. A crucial issue in the induction of experimental focal cerebral ischemia has been to achieve a low IVC value and a low mortality rate. We attempted to improve IVC and mortality using the 3-vessel occlusion model. METHODS: We introduced a new, transtemporal fascia approach to expose the zygomatic arch, in which the fascia of the temporal muscle is cut and retracted dorsally together with the facial nerve and the vein en bloc. RESULTS/CONCLUSION: The approach avoided traumatic venous bleeding around the zygomatic arch. We established a bloodless model of focal ischemia that can produce a consistent degree of reduction in the regional cerebral blood flow within the ischemic penumbra. The model, characterized by a 15-min ischemia, an IVC of 15-21%, and low mortality after ischemia, is expected to produce reliable preclinical evidence in the assessment of neuroprotective interventions for ischemic stroke. The entire procedure is presented in the online supplementary video (www. karger.com/doi/10.1159/000356048).

Novel MRI detection of the ischemic penumbra: direct assessment of metabolic integrity.

We describe a novel magnetic resonance imaging technique to directly assess the metabolic integrity of penumbral tissue following stroke. For ischemically stressed tissue to be salvageable, it has to be capable of recovering aerobic metabolism (in place of anaerobic metabolism) on reperfusion. We probed ischemic brain tissue by altering the rate of oxygen delivery using a challenge of 100% oxygen ventilation. Any change from anaerobic to aerobic metabolism should alter the rate of lactate production and hence, levels of tissue lactate. Stroke was induced by permanent middle cerebral artery occlusion in rats. In Series 1 (n = 6), changes in tissue lactate during and following 100% oxygen challenge were monitored using (1)H magnetic resonance spectroscopy (MRS). Diffusion weighted imaging (DWI) and perfusion weighted imaging (PWI) were used to locate MRS voxels within the ischemic core, the homotopic contralateral striatum and within PWI/DWI mismatch (i.e. presumed penumbra). After 20 min of oxygen, lactate signal change was -16.1 ± 8.8% (mean ± SD) in PWI/DWI mismatch, +2.8 ± 5.1% in the ischemic core, and -0.6 ± 7.6% in the contralateral striatum. Return to air ventilation for 20 min resulted in a reversal, with lactate increasing by 46 ± 25.3% in the PWI/DWI mismatch, 6.6 ± 6.2% in the ischemic core, and -5 ± 11.4% in the contralateral striatum. In Series 2 (n = 6), a novel form of spectroscopic imaging was used to acquire lactate change maps to spatially identify regions of lactate change within the ischemic brain. This technique has potential clinical utility by identifying tissue that displays anaerobic metabolism capable of recovering aerobic metabolism when oxygen delivery is increased, which could provide a more precise assessment of penumbra.

Diffusion tensor MRI for the assessment of cerebral ischemia/reperfusion injury in the penumbra of non-human primate stroke model.

OBJECTIVES: The purpose of this study is to use diffusion tensor imaging (DTI) parameters to evaluate cerebral ischemia/reperfusion injury in the infarct core (IC) and ischemic penumbra (IP) in a rhesus transient middle cerebral artery occlusion (MCAO) model. METHODS: Seven rhesus monkeys were used to construct the MCAO model. The temporal evolution of the relative apparent diffusion coefficient (rADC) and the relative fractional anisotropy (rFA) in the IC area, infarct growth area (IG), and reversible penumbra area (RP) were investigated. RESULTS: The rADC increased in the three areas in the early stage of reperfusion (1 hour after the reperfusion). However, the rate of rADC improvement was significantly slower in IG than in IC and RP. Different temporal evolutions of rFA were observed in the three areas in the following stage of reperfusion (3-24 hours after the reperfusion), which continued to decline in IG but slightly elevated in IC and RP. DISCUSSION: These findings suggest that the evolution of DTI parameters can help in the assessment of cerebral ischemia/reperfusion injury in the penumbra and predict the growth of the infarction area after stroke.

Multiparametric assessment of acute and subacute ischemic neuronal damage: a small animal positron emission tomography study with rat photochemically induced thrombosis model.

We evaluated sequential changes in rat brain function up to 14 days after focal ischemic insult with a small animal positron emission tomography (PET). Unilateral focal ischemic cerebral damage was induced by left middle cerebral artery occlusion with a photochemically induced thrombosis (PIT) method. PET scans were conducted with [(11)C](R)-PK11195 ([(11)C](R)-PK) for peripheral benzodiazepine receptor (PBR), [(11)C]flumazenil ([(11)C]FMZ) for central benzodiazepine receptor (CBR), and [(18)F]fluoro-2-deoxy-D-glucose ([(18)F]FDG) for glucose metabolism at before (as "Normal") and after PIT. At 1 and 3 days after PIT, [(18)F]FDG indicated lower uptake in the infarct area. Interestingly, unexpectedly high-[(18)F]FDG uptake was observed in the peri-infarct area surrounding the infarct area at day 7. The high-[(18)F]FDG uptake region completely overlapped with the high-[(11)C](R)-PK uptake region at day 7, which resulted in the underestimation of neuronal damage. Immunohistochemical data also suggested that the high-[(18)F]FDG uptake peak at day 7 was caused by inflammation including microglial cell activation. In contrast, imaging with [(11)C]FMZ indicated cortical neuronal damage on days 7 and 14 without any disturbance by microglial formation. These results indicated that [(18)F]FDG might not be a suitable ligand for ischemic neuronal damage detection from acute to subacute phases.

Assessment of collateral flow with multi-phasic CT: correlation with diffusion weighted MRI in MCA occlusion.

PURPOSE: To correlate collateral flow on multiphasic contrast enhancement computed tomography (CT) and graded ischemic changes on diffusion weighted MR in patients with acute middle cerebral artery (MCA) infarction. MATERIALS AND METHODS: A retrospective evaluation of diffusion weighted images (DWIs) and three phasic contrast enhanced CT (CECT) was performed on 11 patients with MCA occlusions. The area of ischemic change on DWIs was graded according to the Alberta Stroke Program Early CT Score (ASPECTS) criteria. To evaluate collateral flow on three phasic CECT, we counted the number of contrast enhancing MCA branches distal to the occlusion site at the sylvian fissure from predetermined axial images. The collateral ratios of counted numbers to those at the normal side were calculated at each phase (CR1, CR2, CR3). We then compared collateral ratios from the three phasic CECT with ASPECTS data from DWIs. RESULTS: Collateral ratios from the three phasic CECT were determined to be CR1 .48 ± .27, CR2 .73 ± .36 and CR3 .72 ± .30. We discovered a correlation between both the CR2 and ASPECTS (r= .675, P= .023) and the CR3 and ASPECTS (r= .664, P= .026). CONCLUSION: The number of contrast enhancing branches distal to the MCA occlusion, as counted in the sylvian fissure on later phase images of multiphasic CECT, reflects the status of collateral flow, and correlates with ASPECTS on DWIs.

Assessment of thrombus in acute middle cerebral artery occlusion using thin-slice nonenhanced Computed Tomography reconstructions.

BACKGROUND AND PURPOSE: We sought to evaluate how accurately length and volume of thrombotic clots occluding cerebral arteries of patients with acute ischemic stroke can be assessed from nonenhanced CT (NECT) scans reconstructed with different slice widths. METHODS: NECT image data of 58 patients with acute ischemic stroke with vascular occlusion proven by CT angiography were reconstructed with slice widths of 1.25 mm, 2.5 mm, 3.75 mm, and 5 mm. Thrombus lengths and volumes were quantified based on these NECT images by detecting and segmenting intra-arterial hyperdensities. The results were compared with reference values of thrombus length and volume obtained from CT angiography images using Bland-Altman analysis and predefined levels or tolerance to find NECT slice thicknesses that allow for sufficiently accurate thrombus quantification. RESULTS: Thrombus length can be measured with high accuracy using the hyperdense middle cerebral artery sign detected in NECT images with slice thicknesses of 1.25 mm and 2.5 mm. We found mean deviations from the reference values and limits of agreement of -0.1 mm+/-0.6 mm with slice widths of 1.25 mm and 0.1 mm+/-0.7 mm for slice widths of 2.5 mm. Thrombus length measurements in NECT images with higher slice width and all evaluated thrombus volume measurements exhibited severe dependence on the level and did not match the accuracy criteria. CONCLUSIONS: The length of the hyperdense middle cerebral artery sign as detected on thin-slice NECT reconstructions in patients with acute ischemic stroke can be used to quantify thrombotic burden accurately. Thus, it might qualify as a new diagnostic parameter in acute stroke management that indicates and quantifies the extent of vascular obliteration.

Neither in vivo MRI nor behavioural assessment indicate therapeutic efficacy for a novel 5HT(1A) agonist in rat models of ischaemic stroke.

BACKGROUND: 5HT1A agonists have previously been shown to promote recovery in animal models of stroke using ex vivo outcome measures which have raised the hopes for a potential clinical implementation. The purpose of this study was to evaluate the potential neuroprotective properties of a novel 5HT1A agonist DU123015 in 2 different models of transient focal ischaemic stroke of varying severities using both in vivo neuroimaging and behavioural techniques as primary outcome measures. For these studies, the NMDA receptor antagonist MK-801 was also utilized as a positive control to further assess the effectiveness of the stroke models and techniques used. RESULTS: In contrast to MK-801, no significant therapeutic effect of DU123015 on lesion volume in either the distal MCAo or intraluminal thread model of stroke was found. MK-801 significantly reduced lesion volume in both models; the mild distal MCAo condition (60 min ischaemia) and the intraluminal thread model, although it had no significant impact upon the lesion size in the severe distal MCAo condition (120 min ischaemia). These therapeutic effects on lesion size were mirrored on a behavioural test for sensory neglect and neurological deficit score in the intraluminal thread model. CONCLUSION: This study highlights the need for a thorough experimental design to test novel neuroprotective compounds in experimental stroke investigations incorporating: a positive reference compound, different models of focal ischaemia, varying the duration of ischaemia, and objective in vivo assessments within a single study. This procedure will help us to minimise the translation of less efficacious compounds.

Post-ischemic leakiness of the blood-brain barrier: a quantitative and systematic assessment by Patlak plots.

The Patlak plot analysis of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) allows estimation of blood-brain barrier (BBB) leakage following temporary focal cerebral ischemia. Thus far, a systematic and quantitative in vivo evaluation of post-ischemic BBB leakage is lacking. Here, using DCE-MRI and the Patlak plot method, we quantitatively assessed BBB leakage in rats at the following time-points after reperfusion: 25 min, 2, 4, 6, 12, 18, 24, 36, 48, and 72 h, and 1, 2, 3, 4, and 5 weeks. Sham-operated animals served as controls. Data collected for each time-point were: the blood-to-brain transfer rate constant (K(i)) of the contrast agent gadolinium, distribution volume (V(p)), ischemic lesion volume, and apparent diffusion coefficient (ADC) values. Compared to controls, K(i), measured at all time-points, except for 5 weeks, appeared significantly different (p<0.001). At several time-points (25 min, 48 and 72 h, 4 and 5 weeks), V(p) was similar compared to that of controls, but for the remaining groups the difference was significant (p<0.001). Analyzing the relationship of K(i) values to time-points, we observed a trend towards a decrease over time (r=-0.61, p=0.014). Both ADC values (r=-0.58, p=0.02) and ischemic lesion volumes (r=0.75, p=0.0015) correlated with K(i) values. These results suggest that after ischemia-reperfusion in rats, BBB leakage is continuous during a 4-week period. Its magnitude diminishes over time and correlates with severity and extent of ischemic injury.

Assessment of arcuate fasciculus with diffusion-tensor tractography may predict the prognosis of aphasia in patients with left middle cerebral artery infarcts.

INTRODUCTION: It is often clinically difficult to assess the severity of aphasia in the earliest stage of cerebral infarction. A method enabling objective assessment of verbal function is needed for this purpose. We examined whether diffusion tensor (DT) tractography is of clinical value in assessing aphasia. METHODS: Thirteen right-handed patients with left middle cerebral artery infarcts who were scanned within 2 days after stroke onset were enrolled in this study. Magnetic resonance data of ten control subjects were also examined by DT tractography. Based on the severity of aphasia at discharge, patients were divided into two groups: six patients in the aphasic group and seven in the nonaphasic group. Fractional anisotropy (FA) and number of arcuate fasciculus fibers were evaluated. Asymmetry index was calculated for both FA and number of fibers. RESULTS: FA values for the arcuate fasciculus fibers did not differ between hemispheres in either the patient groups or the controls. Number of arcuate fasciculus fibers exhibited a significant leftward asymmetry in the controls and the nonaphasic group but not in the aphasic group. Asymmetry index of number of fibers was significantly lower (rightward) in the aphasic group than in the nonaphasic (P = 0.015) and control (P = 0.005) groups. Loss of leftward asymmetry in number of AF fibers predicted aphasia at discharge with a sensitivity of 0.83 and specificity of 0.86. CONCLUSIONS: Asymmetry of arcuate fasciculus fibers by DT tractography may deserve to be assessed in acute infarction for predicting the fate of vascular aphasia.

A chronic 1 year assessment of MRI contrast agent-labelled neural stem cell transplants in stroke.

Non-invasive identification of transplanted neural stem cells in vivo by pre-labelling with contrast agents may play an important role in the translation of cell therapy to the clinic. Understanding the impact of these labels on the cells' ability to repair is therefore vital. In rats with middle cerebral artery occlusion (MCAo), a model of stroke, the transhemispheric migration of MHP36 cells labelled with the bimodal contrast agent GRID was detected on magnetic resonance images (MRI) up to 4 weeks following transplantation. However, compared to MHP36 cells labelled with the red fluorescent dye PKH26, GRID-labelled transplants did not significantly improve behaviour, and performance was akin to non-treated animals. Likewise, the evolution of anatomical damage as assessed by serial, T(2)-weighted MRI over 1 year indicated that GRID-labelled transplants resulted in a slight increase in lesion size compared to MCAo-only animals, whereas the same, PKH26-labelled cells significantly decreased lesion size by 35%. Although GRID labelling allows the in vivo identification of transplanted cells up to 1 month after transplantation, it is likely that some is gradually degraded inside cells. The translation of cellular imaging therefore does not only require the in vitro assessment of contrast agents on cellular functions, but also requires the chronic, in vivo assessment of the label on the stem cells' ability to repair in preclinical models of neurological disease.