Matthew J. Hogan

Identification of Penumbra and Infarct in Acute Ischemic Stroke Using Computed Tomography Perfusion–Derived Blood Flow and Blood Volume Measurements

Background and Purpose— We investigated whether computed tomography (CT) perfusion–derived cerebral blood flow (CBF) and cerebral blood volume (CBV) could be used to differentiate between penumbra and infarcted gray matter in a limited, exploratory sample of acute stroke patients. Methods— Thirty patients underwent a noncontrast CT (NCCT), CT angiography (CTA), and CT perfusion (CTP) scan within 7 hours of stroke onset, NCCT and CTA at 24 hours, and NCCT at 5 to 7 days. Twenty-five patients met the criteria for inclusion and were subsequently divided into 2 groups: those with recanalization at 24 hours (n=16) and those without (n=9). Penumbra was operationally defined as tissue with an admission CBF <25 mL · 100 g−1 · min−1 that was not infarcted on the 5- to 7-day NCCT. Logistic regression was applied to differentiate between infarct and penumbra data points. Results— For recanalized patients, CBF was significantly lower (P<0.05) for infarct (13.3±3.75 mL · 100 g−1 · min−1) than penumbra (25.0±3.82 mL · 100 g−1 · min−1). CBV in the penumbra (2.15±0.43 mL · 100 g−1) was significantly higher than contralateral (1.78±0.30 mL · 100 g−1) and infarcted tissue (1.12±0.37 mL · 100 g−1). Logistic regression using an interaction term (CBF×CBV) resulted in sensitivity, specificity, and accuracy of 97.0%, 97.2%, and 97.1%, respectively. The interaction term resulted in a significantly better (P<0.05) fit than CBF or CBV alone, suggesting that the CBV threshold for infarction varies with CBF. For patients without recanalization, CBF and CBV for infarcted regions were 15.1±5.67 mL · 100 g−1 · min−1 and 1.17±0.41 mL · 100 g−1, respectively. Conclusions— We have shown in a limited sample of patients that CBF and CBV obtained from CTP can be sensitive and specific for infarction and should be investigated further in a prospective trial to assess their utility for differentiating between infarct and penumbra.

Cortical Spreading Depression Protects against Subsequent Focal Cerebral Ischemia in Rats

The possibility that cortical spreading depression (CSD) may have neuroprotective action during subsequent focal cerebral ischemia was examined in rats. Three days before the imposition of focal cerebral ischemia CSDs were elicited by applying potassium chloride (KCl) for 2 h through a microdialysis probe implanted in the occipital cortex. Control animals were handled identically except that saline was infused instead of KCl. Focal ischemia was produced by the intraluminal suture method and cortical and subcortical infarct volumes were measured 7 days later. Neocortical infarct volume was reduced from 124.8 ± 49.5 mm3 in the controls to 62.9 ± 59.5 mm3 in the animals preconditioned with CSD (p = 0.012). There was no difference between the two groups in the subcortical infarct volume or in CBF, measured by the hydrogen clearance method, during or immediately after the ischemic interval. Our data indicate that preconditioning CSD applied 3 days before middle cerebral artery occlusion may increase the brains resistance to focal ischemic damage and may be used as a model to explore the neuroprotective molecular responses of neuronal and glial cells.

Cortical spreading depression activates trophic factor expression in neurons and astrocytes and protects against subsequent focal brain ischemia

We recently reported that cortical spreading depression (CSD), used to precondition rat brain, reduced cortical infarction volume resulting from focal cerebral ischemia by middle cerebral artery occlusion (MCAO) 3 days later. The mechanisms underlying this protective effect by CSD remains to be explored. In this study, we confirm that CSD is neuroprotective when KCl is applied epidurally rather than intracortically. Neocortical infarct volume was 101.3+/-48.5 mm3 and 45.3+/-44.1 mm3 in the sham and CSD group, respectively (p<0.05). Using image analysis, we identified the cortical region spared from infarction by the prior CSD. We then determined the distribution of brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) mRNA and the time course of their expression in groups of animals treated with CSD and their controls. We also examined the response of astrocytes to CSD using glial fibrillary acidic protein (GFAP) as a marker. In situ hybridization (done at 0, 3, 12, 24, 72 or 168 h after CSD) showed significant elevation of BDNF mRNA in the cortex immediately after CSD in a distribution surrounding the spared cortex, while bFGF mRNA rose 12 h after CSD and appeared more within the core of the ischemic region. Immunohistochemistry (done at 1, 3 or 7 days after CSD) demonstrated GFAP in the neocortex, with a peak at 3 days after CSD. Heat shock protein 72 (HSP72) expression was not affected by CSD. We concluded that upregulation of trophic factors and activation of glial cells may contribute to the neuroprotection induced by CSD.

Inhibition of Cyclin-Dependent Kinases Improves CA1 Neuronal Survival and Behavioral Performance after Global Ischemia in the Rat

Increasing evidence suggests that cyclin-dependent kinases participate in neuronal death induced by multiple stresses in vitro. However, their role in cell death paradigms in vivo is not well characterized. Accordingly, the authors examined whether cyclin-dependent kinase inhibition resulted in functionally relevant and sustained neuroprotection in a model of global ischemia. Intracerebroventricular administration of the cyclin-dependent kinase inhibitor flavopiridol, immediately or at 4 hours postreperfusion after a global insult, reduced injury in the CA1 of the hippocampus when examined 7 days after reperfusion. No significant protection was observed when flavopiridol was administered 8 hours after reperfusion. The tumor-suppressor retinoblastoma protein, a substrate of cyclin-dependent kinase, was phosphorylated on a cyclin-dependent kinase consensus site after the global insult; this phosphorylation was inhibited by flavopiridol administration. Importantly, flavopiridol had no effect on core body temperature, suggesting that the mechanism of neuroprotection was through cyclin-dependent kinase inhibition but not through hypothermia. Furthermore, inhibition of cyclin-dependent kinases improved spatial learning behavior as assessed by the Morris water maze 7 to 9 days after reperfusion. However, the histologic protection observed at day 7 was absent 28 days after reperfusion. These results indicate that cyclin-dependent kinase inhibition provides an extended period of morphologic and functional neuroprotection that may allow time for other neuroprotective modalities to be introduced.

Calcium-mediated mechanisms of ischemic injury and protection.

Our understanding of calciums role in cerebral ischemia continues to evolve from the initial recognition that it may be harmful to the ischemic cell. A multitude of experiments have supported the hypothesis that excessive influx of calcium into the cell under ischemic conditions is a major mechanism of cell injury and death. Pharmacological intervention to restore cellular calcium homeostasis is protective in many models of cell anoxia. Principle routes of calcium entry are the voltage‐sensitive (VSCC) and N‐methyl‐D‐aspartate linked receptor operated (ROCC) calcium channels. Regional variations in channel densities have been described and it is now known that these classes of channels are located in different regions of the neurons. Activation of both channel types has been identified in in vivo models of cerebral ischemia. Although the ROCC is predominant in number, the VSCC appears to activate at higher cerebral blood flow values suggesting that it is an earlier conduit for calcium than the glutamate‐driven ROCC. Intracellular calcium is well recognized as a second messenger system and there is increasing appreciation that it induces immediate early genes (IEG). Since lEGs function as transcriptional regulating factors, the differential expression of specific target genes may be of importance for determining death or survival of the ischemic tissue.

White Matter Thresholds for Ischemic Penumbra and Infarct Core in Patients with Acute Stroke: CT Perfusion Study

PURPOSE To prospectively determine the parameters derived at admission computed tomographic (CT) perfusion imaging admission that best differentiate ischemic white matter that recovers from that which infarcts, with the latter retrospectively defined at a CT examination performed without contrast material (unenhanced CT) 5-7 days after the event. MATERIALS AND METHODS Ethics committee approval and informed consent were obtained. Thirty patients with stroke underwent unenhanced CT, CT angiography, and CT perfusion studies at admission. Additionally, CT angiography was performed 24 hours after the stroke, and an unenhanced CT study was performed 5-7 days after the stroke. Five patients were excluded; the remaining patients (10 men, 15 women; mean age, 70 years +/- 13 [standard deviation]) were separated into those with recanalization (n = 16) and those without recanalization (n = 9) at 24 hours. For patients with recanalization, the final infarct was outlined on unenhanced CT images obtained 5-7 days after the event and was superimposed on coregistered maps from the CT perfusion study performed at admission. Ischemic white matter tissue (cerebral blood flow [CBF] < 14 mL/min/100 g) was identified at the admission CT perfusion study, and the penumbra was defined as the difference between the ischemic region and the infarct region. RESULTS Infarct regions showed a matched decrease in CBF and cerebral blood volume (CBV) at admission, whereas penumbra regions showed a significant (P < .05) decrease in CBF but no change in CBV (P > .05) from contralateral values. A threshold CBF . CBV value of 8.14 was the most sensitive (95%, 20 of 21 regions) and specific (94%, 32 of 34 regions) parameter for differentiating between regions of ischemic white matter that recovered and regions of ischemic white matter that infarcted. CONCLUSION The product of CBF and CBV derived from CT perfusion data provided the best differentiation between regions of ischemic white matter that infarcted and regions of ischemic white matter that recovered 5-7 days after a stroke.

Preconditioning with Cortical Spreading Depression Decreases Intraischemic Cerebral Glutamate Levels and Down-Regulates Excitatory Amino Acid Transporters EAAT1 and EAAT2 from Rat Cerebal Cortex Plasma Membranes

We previously reported a 50% reduction in cortical infarct volume following transient focal cerebral ischemia in rats preconditioned 3 days earlier with cortical spreading depression (CSD). The mechanism of the protective effect of prior CSD remains unknown. Recent studies demonstrate reversal of excitatory amino acid transporters (EAATs) to be a principal cause for elevated extracellular glutamate levels during cerebral ischemia. The present study measured the effect of CSD preconditioning on (a) intraischemic glutamate levels and (b) regulation of glutamate transporters within the ischemic cortex of the rat. Three days following either CSD or sham preconditioning, rats were subjected to 200 min of focal cerebral ischemia, and extracellular glutamate concentration was measured by in vivo microdialysis. Cortical glutamate exposure decreased 70% from 1,772.4 ± 1,469.2 μM‐min in sham‐treated (n = 8) to 569.0 ± 707.8 μM‐min in CSD‐treated (n = 13) rats (p <0.05). The effect of CSD preconditioning on glutamate transporter levels in plasma membranes (PMs) prepared from rat cerebral cortex was assessed by western blot analysis. Down‐regulation of the glial glutamate transporter isoforms EAAT2 and EAAT1 from the PM fraction was observed at 1, 3, and 7 days but not at 0 or 21 days after CSD. Semiquantitative lane analysis showed a maximal decrease of 90% for EAAT2 and 50% for EAAT1 at 3 days post‐CSD. The neuronal isoform EAAT3 was unaffected by CSD. This period of down‐regulation coincides with the time frame reported for induced ischemic tolerance. These data are consistent with reversal of glutamate transporter function contributing to glutamate release during ischemia and suggest that down‐regulation of these transporters may contribute to ischemic tolerance induced by CSD.

The Effects of a Competitive NMDA Receptor Antagonist (CGS-19755) on Cerebral Blood Flow and pH in Focal Ischemia

We report the effects of intravenous infusion of CGS-19755, a potent competitive N-methyl-D-aspartate (NMDA) antagonist, on local cerebral pH (LCpH) and local CBF (LCBF) in rats with occluded left middle cerebral and common carotid arteries. LCpH and LCBF were determined simultaneously by a double-label autoradiographic technique 4 h after vascular occlusion in three groups: No treatment, carrier infused, and a group receiving CGS-19755 at 10 mg/kg bolus immediately after occlusion followed by infusion at 5 mg kg−1 h−1 for 4 h. Compared with rats receiving carrier, several cortical structures on the side of occlusions showed significantly higher CBF in rats receiving CGS-19755. This drug also corrected the pH in several left cortical structures to values significantly higher than in the rats receiving carrier. The correction in LCpH was not limited to those regions showing significant elevations in LCBF. In the nonoccluded hemisphere, CGS-19755 significantly increased the hemispheric mean blood flow from 122 ± 17 to 221 ± 64 ml 100 g−1 min−1 (mean ± SD of all structures, p < 0.01) without any changes in LCpH. Cortical but not basal ganglia infarct volume was significantly smaller in rats receiving CGS-19755 than in the carrier-treated group. These results suggest that, at least partially, the neuroprotective effect of CGS-19755 in ischemia may be related to changes in CBF and pH in addition to its antagonist effect on the NMDA receptor.

Evidence from cultured rat cortical neurons of differences in the mechanism of ischemic preconditioning of brain and heart

Ca2+ influx and activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) during nonlethal ischemic preconditioning have been implicated in the protection of the heart against subsequent lethal ischemic injury. Thus, we determined if Ca2+ influx, PKC and MAPK also mediate ischemic preconditioning-induced protection in neurons. Preconditioning by exposure of E18 rat cortical cultures to 90 min of nonlethal oxygen-glucose deprivation (OGD) 24 h prior to 180-240 min of lethal OGD was neuroprotective. Exposure to nominally free Ca2+, or blockade of the alpha-amino-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptor with CNQX did not eliminate protection. MAPK activity did not change and PKC activity decreased by 50% relative to normal baseline levels at 0 and 24 h following preconditioning. The sustained decrease in PKC activity was not due to a loss of enzyme as determined from immunoblots using pan and epsilon-, beta- and zeta-specific PKC antibodies. Neuroprotection was maintained with pharmacological inhibition of PKC activity by staurosporine, chelerythrine and calphostin C and MAPK activity by PD 98059 during preconditioning, indicating that activation of these enzymes during preconditioning was not necessary for protection. Therefore, in contrast to cardiac tissue, ischemic preconditioning of neurons does not require activation of PKC and MAP kinase, and protection is maintained with substantial removal of extracellular Ca2+ or blockade of the AMPA receptor.

Time course of cerebral blood flow and histological outcome after focal cerebral ischemia in rats.

Background and Purpose The relation between time-dependent changes in cerebral blood flow and the appearance of infarction after focal cerebral ischemia is still a matter for debate. The aim of this study was to measure perfusion after simultaneous occlusions of the left middle cerebral artery and ipsilateral common carotid artery in rats and correlate it with the timing and distribution of histological changes. Methods We studied histological and cerebral blood flow changes 5 minutes and 4, 24, and 48 hours after the onset of focal ischemia. Blood flow was determined autoradiographically using [14C]iodoantipyrine. A coronal template subdivided into regions of interest was applied to the autoradiographs and the histological data. Results In some regions of the nonoccluded hemisphere, cerebral blood flow 5 minutes after occlusion fell below 50% of normal. Many ischemic structures showed stable blood flow for 48 hours after occlusion, confirming that in this model reperfusion is minimal. Infarction occurred eventually in all areas in which blood flow at 5 minutes fell below 10% of that in control rats, but infarction appeared earlier in regions in which blood flow at 5 minutes was below 5% of that in control rats. When blood flow at 5 minutes rose above 12% of that in control rats, the occurrence of infarction became unpredictable. Conclusions Despite the general dependence of infarction on perfusion levels, blood flow was not a reliable indicator of those regions committed to infarction.