James P. Bouley

Normobaric hyperoxia delays perfusion/diffusion mismatch evolution, reduces infarct volume, and differentially affects neuronal cell death pathways after suture middle cerebral artery occlusion in rats

Normobaric hyperoxia (NBO) has been shown to extend the reperfusion window after focal cerebral ischemia. Employing diffusion (DWI)- and perfusion (PWI)-weighted magnetic resonance imaging (MRI), the effect of NBO (100% started at 30 mins after middle cerebral artery occlusion (MCAO)) on the spatiotemporal evolution of ischemia during and after permanent (pMCAO) and transient suture middle cerebral artery occlusion (tMCAO) was investigated (experiment 3). In two additional experiments, time window (experiment 1) and cell death pathways (experiment 2) were investigated in the pMCAO model. In experiment 1, NBO treatment reduced infarct volume at 24 h after pMCAO by 10% when administered for 3 h (P > 0.05) and by 44% when administered for 6 h (P < 0.05). In experiment 2, NBO acutely (390 mins, P < 0.05) reduced in situ end labeling (ISEL) positivity in the ipsilesional penumbra but increased contralesional necrotic as well as caspase-3-mediated apoptotic cell death. In experiment 3, CBF characteristics and CBF-derived lesion volumes did not differ between treated and untreated animals, whereas the apparent diffusion coefficient (ADC)-derived lesion volume essentially stopped progressing during NBO treatment, resulting in a persistent PWI/DWI mismatch that could be salvaged by delayed (3 h) reperfusion. In conclusion, NBO (1) acutely preserved the perfusion/diffusion mismatch without altering CBF, (2) significantly extended the time window for reperfusion, (3) induced lasting neuroprotection in permanent ischemia, and (4) although capable of reducing cell death in hypoperfused tissue it also induced cell death in otherwise unaffected areas. Our data suggest that NBO may represent a promising strategy for acute stroke treatment.

Differences in Ischemic Lesion Evolution in Different Rat Strains Using Diffusion and Perfusion Imaging

Background and Purpose— Interstrain differences in the temporal evolution of ischemia after middle cerebral artery occlusion (MCAO) in rats may considerably influence the results of experimental stroke research. We investigated, in 2 commonly used rat strains (Sprague-Dawley [SD] and Wistar-Kyoto [WK]), the spatiotemporal evolution of ischemia after permanent suture MCAO using diffusion and perfusion imaging. Methods— Serial measurements of quantitative cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) were performed up to 210 min after MCAO. Lesion volumes were calculated by using previously established viability thresholds and correlated with infarct volume defined by 2,3,5-triphenyltetrazolium chloride staining 24 hours after MCAO. Results— While the ADC-derived lesion volume increased rapidly during the first 120 min after MCAO and essentially stopped growing after 3 hours in SD rats, ADC lesion in WK rats increased progressively during the entire 210-min period and was significantly smaller at all time points (P<0.05). The abnormal perfusion volume correlated highly with the TTC-defined infarct size in both groups. In WK rats, the abnormal perfusion volume was significantly larger than the abnormal diffusion volume up to 90 min after MCAO (P<0.001), whereas the diffusion/perfusion mismatch was significant (P<0.001) only at 45 min in SD rats. ADC–CBF scatterplots analysis revealed a slower and less robust ADC decline over time in WK rats in pixels with severe (<20% of normal) and moderate (21 to 40% of normal) CBF reduction. Conclusions— This study demonstrated substantial differences in acute ischemic lesion evolution between SD and WK rats. These interstrain variations must be taken into account when assessing new therapeutic approaches on ischemic lesion evolution in the rat MCAO model.

Normobaric Hyperoxia and Delayed tPA Treatment in a Rat Embolic Stroke Model

In a rat embolic stroke (eMCAO) model, the effects of 100% normobaric hyperoxia (NBO) with delayed recombinant tissue plasminogen activator (tPA) administration on ischemic lesion size and safety were assessed by diffusion- and perfusion (PWI)-weighted magnetic resonance imaging. NBO or room air (Air) by a face mask was started at 30 mins posteMCAO and continued for 3.5 h. Tissue plasminogen activator or saline was started at 3 h posteMCAO. Types and location of hemorrhagic transformation were assessed at 24 h and a spectrophotometry hemoglobin assay quantified hemorrhage volume at 10 h. In NBO-treated animals the apparent diffusion coefficient/PWI mismatch persisted during NBO treatment. Relative to Air groups, NBO treatment significantly reduced 24 h infarct volumes by ~30% and ~ 15% with or without delayed tPA, respectively (P < 0.05). There were significantly more hemorrhagic infarction type 2 hemorrhages in Air/tPA versus Air/saline animals (P < 0.05). Compared with Air/tPA, the combination of NBO with tPA did not increase hemorrhage volume at 10 h (4.0±2.4 versus 6.6±2.6 μL, P = 0.065) or occurrence of confluent petechial hemorrhages at 24 h (P > 0.05), respectively. Our results suggest that early NBO treatment in combination with tPA at a later time point may represent a safe and effective strategy for acute stroke treatment.

Dynamic tracking of acute ischemic tissue fates using improved unsupervised ISODATA analysis of high-resolution quantitative perfusion and diffusion data

High-resolution (200 × 200 × 1,500 μm3) imaging was performed to derive quantitative cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) maps in stroke rats (permanent occlusion) every 30 minutes up to 3 hours after occlusion onset, followed by histology at 24 hours. An improved automated iterative-self-organizing-data-analysis-algorithm (ISODATA) was developed to dynamically track ischemic tissue fate on a pixel-by-pixel basis during the acute phase. ISODATA-resolved clusters were overlaid on the CBF-ADC scatterplots and image spaces. Tissue volume ADC, and CBF of each ISODATA cluster were derived. In contrast to the single-cluster normal left hemisphere (ADC = 0.74±0.02 × 10−3 mm2/s, CBF = 1.36±0.22 mL g−1 min−1, mean±SD, n = 8), the right ischemic hemisphere exhibited three ISODATA clusters, namely: “normal” (normal ADC and CBF), “ischemic core” (low CBF and ADC), and at-risk “perfusion-diffusion mismatch” (low CBF but normal ADC). At 180 minutes, the mismatch disappeared in five rats (Group I, 180-minute “core” lesion volume = 255±62 mm3 and 24-hour infarct volume = 253±55 mm3, P > 0.05), while a substantial mismatch persisted in three rats (Group II, 180-minute CBF-abnormal volume = 198±7 mm3 and 24-hour infarct volume 148±18 mm3, P < 0.05). The CBF (0.3±0.09 mL g−1 min−1) of the “persistent mismatch” (Group II, 0.3±0.09 mL g−1 min−1) was above the CBF viability threshold (0.2 to 0.3 mL g−1 min−1) throughout and its ADC (0.70±0.03 × 10−3 mm2/s) did not decrease as ischemia progressed. In contrast, the CBF (0.08±0.03 mL g−1 min−1) of the analogous brain region in Group I was below the CBF viability threshold, and its ADC gradually decreased from 0.63±0.05 to 0.43±0.03 × 10−3 mm2/s (ADC viability threshold = 0.53±0.02 × 10−3 mm2/s). The modified ISODATA analysis of the ADC and CBF tissue characteristics during the acute phase could provide a useful and unbiased means to characterize and predict tissue fates in ischemic brain injury and to monitor therapeutic intervention.

Neuroprotection by Freezing Ischemic Penumbra Evolution Without Cerebral Blood Flow Augmentation With a Postsynaptic Density-95 Protein Inhibitor

Background and Purpose— The purpose of this study was to determine whether neuroprotection is feasible without cerebral blood flow augmentation in experimental permanent middle cerebral artery occlusion. Methods— Rats were subjected to permanent middle cerebral artery occlusion by the suture occlusion method and were treated 1 hour thereafter with a single 5-minute intravenous infusion of the postsynaptic density-95 protein inhibitor Tat-NR2B9c (7.5 mg/kg) or saline (n=8/group). Arterial spin-labeled perfusion-weighted MRI and diffusion weighted MRI were obtained with a 4.7-T Bruker system at 30, 45, 70, 90, 120, 150, and 180 minutes postmiddle cerebral artery occlusion to determine cerebral blood flow and apparent diffusion coefficient maps, respectively. At 24 hours, animals were neurologically scored (0 to 5), euthanized, and the brains stained with 2–3-5-triphenyl tetrazolium chloride to ascertain infarct volumes corrected for edema. Additionally, the effects of Tat-NR2B9c on adenosine 5′-triphosphate levels were measured in vitro in neurons subjected to oxygen–glucose deprivation. Results— Final infarct volume was decreased by 30.3% in the Tat-NR2B9c-treated animals compared with controls (P=0.028). There was a significant improvement in 24 hours neurological scores in the Tat-NR2B9c group compared with controls, 1.8±0.5 and 2.8±1.0, respectively (P=0.021). Relative to controls, Tat-NR2B9c significantly attenuated diffusion-weighted imaging lesion growth and preserved the diffusion-weighted imaging/perfusion-weighted imaging mismatch (ischemic penumbra) without affecting cerebral blood flow in the ischemic core or penumbra. Tat-NR2B9c treatment of primary neuronal cultures resulted in 26% increase in cell viability and 34% greater adenosine 5′-triphosphate levels after oxygen–glucose deprivation. Conclusions— Preservation of adenosine 5′-triphosphate levels in vitro and neuroprotection in permanent middle cerebral artery occlusion in rats is achievable without cerebral blood flow augmentation using a postsynaptic density-95 protein inhibitor.

The proteasome inhibitor VELCADE® reduces infarction in rat models of focal cerebral ischemia

The potential neuroprotective effects of VELCADE were investigated in two different models of focal cerebral ischemia. For time-window assessment, male Wistar-Kyoto rats were treated with 0.2 mg/kg VELCADE at 1, 2, or 3 h after the induction of permanent middle cerebral artery occlusion (MCAO) using the suture occlusion method (experiment 1). To evaluate effects in a different model, male Sprague-Dawley rats received 0.2 mg/kg VELCADE after embolic MCAO (experiment 2). Infarct volume was calculated based on TTC-staining 24 h postischemia and whole blood proteasome activity was fluorometrically determined in both experiments at baseline, 1 and 24 h post-MCAO. In experiment 1, a dose of 0.2 mg/kg inhibited proteasome activity by 77% and infarct volume was reduced to 175.7+/-59.9 mm3 and 205.9+/-83.9 mm3 (1 and 2 h group, respectively; p<0.05) compared to 306.5+/-48.5 mm3 (control). Treatment at 3 h was not neuroprotective (293.0+/-40.1 mm3). After embolic MCAO, infarct volume was 167.5+/-90.7 mm3 (treatment group) and 398.9+/-141.3 mm3 (control; p=0.002). In conclusion, VELCADE treatment inhibited whole blood proteasome activity and achieved significant neuroprotection in two rat models of focal cerebral ischemia at various time points poststroke.

Effects of intravenous dimethyl sulfoxide on ischemia evolution in a rat permanent occlusion model

Dimethyl sulfoxide (DMSO) has a variety of biological actions that suggest efficacy as a neuroprotectant. We (1) tested the neuroprotective potential of DMSO at different time windows on infarct size using 2,3,5-triphenyltetrazolium staining and (2) investigated the effects of DMSO on ischemia evolution using quantitative diffusion and perfusion imaging in a permanent middle cerebral artery occlusion (MCAO) model in rats. In experiment 1, DMSO treatment (1.5 g/kg intravenously over 3 h) reduced infarct volume 24 h after MCAO by 65% (P<0.00001) when initiated 20 h before MCAO, by 44% (P = 0.0006) when initiated 1 h after MCAO, and by 17% (P = 0.11) when started 2 h after MCAO. Significant infarct reduction was also observed after a 3-day survival in animals treated 1 h after MCAO (P = 0.005). In experiment 2, treatment was initiated 1 h after MCAO and maps for cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) were acquired before treatment and then every 30 mins up to 4 h. Cerebral blood flow characteristics and CBF-derived lesion volumes did not differ between treated and untreated animals, whereas the ADC-derived lesion volume essentially stopped progressing during DMSO treatment, resulting in a persistent diffusion/perfusion mismatch. This effect was mainly observed in the cortex. Our data suggest that DMSO represents an interesting candidate for acute stroke treatment.

Attenuated traumatic axonal injury and improved functional outcome after traumatic brain injury in mice lacking Sarm1

Axonal degeneration is a critical, early event in many acute and chronic neurological disorders. It has been consistently observed after traumatic brain injury, but whether axon degeneration is a driver of traumatic brain injury remains unclear. Molecular pathways underlying the pathology of traumatic brain injury have not been defined, and there is no efficacious treatment for traumatic brain injury. Here we show that mice lacking the mouse Toll receptor adaptor Sarm1 (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) gene, a key mediator of Wallerian degeneration, demonstrate multiple improved traumatic brain injury-associated phenotypes after injury in a closed-head mild traumatic brain injury model. Sarm1(-/-) mice developed fewer β-amyloid precursor protein aggregates in axons of the corpus callosum after traumatic brain injury as compared to Sarm1(+/+) mice. Furthermore, mice lacking Sarm1 had reduced plasma concentrations of the phophorylated axonal neurofilament subunit H, indicating that axonal integrity is maintained after traumatic brain injury. Strikingly, whereas wild-type mice exibited a number of behavioural deficits after traumatic brain injury, we observed a strong, early preservation of neurological function in Sarm1(-/-) animals. Finally, using in vivo proton magnetic resonance spectroscopy we found tissue signatures consistent with substantially preserved neuronal energy metabolism in Sarm1(-/-) mice compared to controls immediately following traumatic brain injury. Our results indicate that the SARM1-mediated prodegenerative pathway promotes pathogenesis in traumatic brain injury and suggest that anti-SARM1 therapeutics are a viable approach for preserving neurological function after traumatic brain injury.

Comparison between coated vs. uncoated suture middle cerebral artery occlusion in the rat as assessed by perfusion/diffusion weighted imaging

Differences among models in the temporal evolution of ischemia after middle cerebral artery occlusion (MCAO) in rats may considerably influence the results of experimental treatment studies. Using diffusion and perfusion imaging, we compared the spatiotemporal evolution of ischemia in Sprague-Dawley rats after permanent MCAO (pMCAO) with different types of sutures. Male Sprague-Dawley rats were randomly assigned to pMCAO produced with either 4-0 silicone coated (n=8), or 3-0 uncoated monofilaments (n=8). Serial determination of quantitative cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) maps were performed up to 3 h after pMCAO. Lesion volumes were calculated by using previously validated thresholds and correlated with infarct volume corrected for edema defined by 2,3,5-triphenyltetrazolium chloride (TTC) staining at 24 h after MCAO. The ADC/CBF-defined mismatch volume in the 4-0 coated suture model was present significantly longer (up to 120 min) compared to the uncoated 3-0 suture model (30 min). The TTC-derived infarct volume was significantly larger in the coated model (290.3+/-32.8 mm(3)) relative to the uncoated model (252.3+/-34.6 mm(3)). This study demonstrates that the type of suture may significantly influence the spatiotemporal evolution of the ADC/CBF-mismatch as well as the final infarct volume. These inter-model variations must be taken into account when assessing new therapeutic approaches on ischemic lesion evolution in the rat MCAO model.

Granulocyte-Colony Stimulating Factor Delays PWI/DWI Mismatch Evolution and Reduces Final Infarct Volume in Permanent-Suture and Embolic Focal Cerebral Ischemia Models in the Rat

Background and Purpose— Granulocyte-colony stimulating factor (G-CSF) is used clinically to attenuate neutropenia after chemotherapy. G-CSF acts as a growth factor in the central nervous system, counteracts apoptosis, and is neuroprotective in rodent transient ischemia models. Methods— We assessed the effect of G-CSF on ischemic lesion evolution in a rat permanent-suture occlusion model with diffusion- and perfusion-weighted magnetic resonance imaging and the neuroprotective effect of G-CSF in a rat embolic stroke model. Results— With a constant perfusion deficit, vehicle-treated animals showed an expanding apparent diffusion coefficient lesion volume that matched the perfusion deficit volume at ≈3 hours, with the 24-hour infarct volume equivalent to the perfusion deficit. In G-CSF–treated rats, the apparent diffusion coefficient lesion volume did not increase after treatment initiation, and the infarct volume at 24 hours reflected the initial apparent diffusion coefficient lesion volume. In the embolic model, we observed a significant decrease in infarct volume in G-CSF–treated animals compared with the vehicle-treated group. Conclusions— These results confirm the potent neuroprotective activity of G-CSF in different focal ischemia models. The magnetic resonance imaging data demonstrate that G-CSF preserved the perfusion/diffusion mismatch.