Journal of Cerebral Blood Flow & Metabolism

The ischemic penumbra is defined as tissue with flow within the thresholds for maintenance of function and of morphologic integrity. Penumbra tissue has the potential for recovery and therefore is the target for interventional therapy in acute ischemic stroke. The identification of the penumbra necessitates measuring flow reduced less than the functional threshold and differentiating between morphologic integrity and damage. This can be achieved by multitracer positron emission tomography (PET) and perfusion-weighted (PW) and diffusion-weighted magnetic resonance imaging (DW-MRI) in experimental models, in which the recovery of critically perfused tissue or its conversion to infarction was documented in repeat studies. Neuroimaging modalities applied in patients with acute ischemic stroke—multitracer PET, PW-and DW-MRI, single photon emission computed tomography (SPECT), perfusion, and Xe-enhanced computed tomography (CT)—often cannot reliably identify penumbra tissue: multitracer studies for the assessment of flow and irreversible metabolic damage usually cannot be performed in the clinical setting; CT and MRI do not reliably detect irreversible damage in the first hours after stroke, and even DW-MRI may be misleading in some cases; determinations of perfusion alone yield a poor estimate of the state of the tissue as long as the time course of changes is not known in individual cases. Therefore, the range of flow values in ischemic tissue found later, either within or outside the infarct, was rather broad. New tracers—for example, receptor ligands or hypoxia markers—might improve the identification of penumbra tissue in the future. Despite these methodologic limitations, the validity of the concept of the penumbra was proven in several therapeutic studies in which thrombolytic treatment reversed critical ischemia and decreased the volume of final infarcts. Such neuroimaging findings might serve as surrogate targets in the selection of other therapeutic strategies for large clinical trials.

The involvement of caspase-3 in cell death after hypoxia–ischemia (HI) was studied during brain maturation. Unilateral HI was produced in rats at postnatal day 7 (P7), 15 (P15), 26 (P26), and 60 (P60) by a combination of left carotid artery ligation and systemic hypoxia (8% O₂). Activation of caspase-3 and cell death was examined in situ by high-resolution confocal microscopy with anti-active caspase-3 antibody and propidium iodide and by biochemical analysis. The active caspase-3 positive neurons were composed of more than 90% HI damaged striatal and neocortical neurons in P7 pups, but that number was reduced to approximately 65% in striatum and 34% in the neocortex of P15 pups, and approximately 26% in striatum and 2% in neocortex of P26 rats. In P60 rats, less than 4% of the damaged neurons in striatum and less than 1% in neocortex were positive for active caspase-3. Western blot analysis demonstrated that the level of inactive caspase-3 in normal forebrain tissue gradually declined from a high level in young pups to very low levels in adult rats. Concomitantly, HI-induced active caspase-3 was reduced from a relatively high level in P7, to moderate levels in P15 and P26, to a barely detectable level in P60 rats. The authors conclude that the involvement of caspase-3 in the pathogenesis of cell death after HI declines during neuronal maturation. The authors hypothesize that caspase-3 may play a major role in cell death in immature neurons but a minor role in cell death in mature neurons after brain injury.

The protein kinase Akt/PKB has been implicated in antiapoptosis and neuronal survival. The authors now show that Akt is phosphorylated in the hippocampus during the early reperfusion period after 3.5 minutes bilateral carotid artery occlusion (BCAO) in the gerbil. Repeated sublethal ischemia induces ischemic tolerance, which is known as ischemic preconditioning. Ischemic preconditioning does not affect the amount of Akt protein, but rather decreases the phosphorylation of Akt at Ser-473 after 10 minutes reperfusion after 3.5 minutes BCAO. These results suggest that although Akt may play a role in neuronal survival after ischemia, it may not play a role in ischemic tolerance by preconditioning.

An unexpected decrease of extracellular space (ECS) tortuosity was recently reported in thick (1000 μm) ischemic slices using radiotracers. The current study shows that the tortuosity in thick slices from rat neocortex can increase or decrease depending on experimental conditions, whereas ECS volume fraction remains diminished to approximately 10%. Using diffusion of tetramethylammonium, it was found that tortuosity rose from a normoxic value of 1.66 to 1.99 in thick slices. However, tortuosity dropped to 1.54 when dextran (70,000 molecular weight) was added to the bathing medium. The current results show that dextran enhances diffusion in thick ischemic slices without increasing the size of the ECS.

The authors transplanted adult bone marrow nonhematopoietic cells into the striatum after embolic middle cerebral artery occlusion (MCAO). Mice (n = 23; C57BL/6J) were divided into four groups: (1) mice (n = 5) were subjected to MCAO and transplanted with bone marrow nonhematopoietic cells (prelabeled by bromodeoxyuridine, BrdU) into the ischemic striatum, (2) MCAO alone (n = 8), (3) MCAO with injection of phosphate buffered saline (n = 5), and (4) bone marrow nonhematopoietic cells injected into the normal striatum (n = 5). Mice were killed at 28 days after stroke. BrdU reactive cells survived and migrated a distance of approximately 2.2 mm from the grafting areas toward the ischemic areas. BrdU reactive cells expressed the neuronal specific protein NeuN in 1% of BrdU stained cells and the astrocytic specific protein glial fibrillary acidic protein (GFAP) in 8% of the BrdU stained cells. Functional recovery from a rotarod test (P < 0.05) and modified neurologic severity score tests (including motor, sensory, and reflex;P < 0.05) were significantly improved in the mice receiving bone marrow nonhematopoietic cells compared with MCAO alone. The current findings suggest that the intrastriatal transplanted bone marrow nonhematopoietic cells survived in the ischemic brain and improved functional recovery of adult mice even though infarct volumes did not change significantly. Bone marrow nonhematopoietic cells may provide a new avenue to promote recovery of injured brain.

The purpose of this study was to examine the activation, topographic distribution, and cellular location of three mitogen-activated protein kinases (MAPKs) after permanent middle cerebral artery occlusion (MCAO) in mice. Phosphorylated MAPKs expression in the ischemic region was quantified using Western blot analysis and localized immunohistochemically using the diaminobenzide staining and double-labeled immunostaining. Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), p38 mitogen-activated protein (p38), and c-Jun NH2-terminal kinase or stress-activated protein kinase (SAPK/JNK) were initially activated at 30 minutes, 10 minutes, and 5 minutes, respectively, after focal cerebral ischemia. Peak expression represented a 2.7-fold, 3.7-fold, and 4.8-fold increase in each of these MAPKs, respectively. The immunohistochemical expressions of ERK1, ERK2, p38, and SAPK/JNK protein paralleled the Western blot analysis results. Double-labeled immunofluorescent staining demonstrated that the neurons and astrocytes expressed ERK1, ERK2, p38, and SAPK/JNK during the early time points after MCAO. The current results demonstrate that brain damage after ischemia rapidly triggers time-dependent ERK1, ERK2, p38, and SAPK/JNK phosphorylation, and reveals that neurons and astrocytes are involved in the activation of the MAPK pathway. This very early expression of MAPKs suggests that MAPKs may be closely involved in signal transduction during cerebral ischemia.

Spin-echo and gradient-echo echoplanar functional magnetic resonance imaging (fMRI) studies at 1.5 Tesla (T) were used to obtain blood oxygenation level-dependent (BOLD) contrast images of the whole brain in seven strongly right-handed women during execution of a complex motor task. Five subjects underwent subsequent H₂¹⁵O positron emission tomography (PET) studies while performing the same task. Group-averaged results for changes in the MRI relaxation rates R₂* and R₂ at 1.5T in response to neuronal activation in nine cortical, subcortical, and cerebellar motor regions are reported. Results for each method are grouped according to tissue type—cerebral cortex (precentral gyrus and supplementary motor area), subcortical regions (thalamus and putamen), and cerebellar cortex (superior lobule). The observed changes in R₂* from activation-induced oxygenation changes were more variable across brain regions with different tissue characteristics than observed changes in R₂. The ratio of ΔR₂* to ΔR₂ was 3.3 ± 0.9 for cerebral cortex and 2.0 ± 0.6 for subcortical tissue. ΔR₂*, ΔR₂, and relative blood flow changes were ΔR₂* = −0.201 ± 0.040 s⁻¹, ΔR₂ = −0.064 ± 0.011 s⁻¹, and Δf/f = 16.7 ± 0.8% in the cerebral cortex; ΔR₂* = −0.100 ± 0.026 s⁻¹, ΔR₂ = −0.049 ± 0.009 s⁻¹, and Δf/f = 9.4 ± 0.7% in the subcortical regions; and ΔR₂* = −0.215 ± 0.093 s⁻¹, ΔR₂ = −0.069 ± 0.012 s⁻¹, and Δf/f = 16.2 ± 1.2% in the cerebellar cortex.

Although perfusion-weighted imaging techniques are increasingly used to study stroke, no particular hemodynamic variable has emerged as a standard marker for accumulated ischemic damage. To better characterize the hemodynamic signature of infarction, the authors have assessed the severity and temporal evolution of ischemic hemodynamics in a middle cerebral artery occlusion model in the rat. Cerebral blood flow (CBF) and total and microvascular cerebral blood volume (CBV) changes were measured with arterial spin labeling and steady-state susceptibility contrast magnetic resonance imaging (MRI), respectively, and analyzed in regions corresponding to infarcted and spared ipsilateral tissue, based on 2,3,5-triphenyltetrazolium chloride histology sections after 24 hours ischemia. Spin echo susceptibility contrast was used to measure microvascular-weighted CBV, which had a maximum sensitivity for vessels with radii between 4 and 30 μm. Serial measurements between 1 and 3 hours after occlusion showed no change in CBF (22 ± 20% of contralateral, mean ± SD) or in total CBV (78 ± 13% of contralateral) in regions destined to infarct. However, microvascular CBV progressively declined from 72 ± 5% to 64 ± 11% (P < 0.01) during this same period. Microvascular CBV changes with time were entirely due to decreases in subcortical infarcted zones (from 73 ± 9% to 57±14%, P < 0.001) without changes in the cortical infarcted territory. The hemodynamic variables showed differences in magnitude and temporal response, and these changes varied based on histologic outcome and brain architecture. Such factors should be considered when designing imaging studies for human stroke.

The authors studied the effects of a standardized mild-moderate hypoglycemic stimulus (glucose clamp) on brain functional magnetic resonance imaging (fMRI) responses to median nerve stimulation in anesthetized rats. In the baseline period (plasma glucose 6.6 ± 0.3 mmol/L), the MR signal changes induced by median nerve activation were determined within a fixed region of the somatosensory cortex from preinfusion activation maps. Subsequently, insulin and a variable glucose infusion were administered to decrease plasma glucose. The goal was to produce a stable hypoglycemic plateau (2.8 ± 0.2 mmol/L) for 30 minutes. Thereafter, plasma glucose was restored to euglycemic levels (6.0 ± 0.3 mmol/L). In the early phase of insulin infusion (15 to 30 minutes), before hypoglycemia was reached (4.7 ± 0.3 mmol/L), the activation signal was unchanged. However, once the hypoglycemic plateau was achieved, the activation signal was significantly decreased to 57 ± 6% of the preinfusion value. Control regions in the brain that were not activated showed no significant changes in MR signal intensity. Upon return to euglycemia, the activation signal change increased to within 10% of the original level. No significant activation changes were noted during euglycemic hyperinsulinemic clamp experiments. The authors concluded that fMRI can detect alterations in cerebral function because of insulin-induced hypoglycemia. The signal changes observed in fMRI activation experiments were sensitive to blood glucose levels and might reflect increases in brain metabolism that are limited by substrate deprivation during hypoglycemia.

Gene therapy is being investigated as a putative treatment option for cardiovascular diseases, including cerebral vasospasm. Because there is presently no information regarding gene transfer to human cerebral arteries, the principal objective of this study was to characterize adenovirus-mediated expression and function of recombinant endothelial nitric oxide synthase (eNOS) gene in human pial arteries. Pial arteries (outer diameter 500 to 1000 μm) were isolated from 30 patients undergoing temporal lobectomy for intractable seizures and were studied using histologic staining, histochemistry, electron microscopy, and isometric force recording. Gene transfer experiments were performed ex vivo using adenoviral vectors encoding genes for bovine eNOS (AdCMVeNOS) and Escherichia coli β-galactosidase (AdCMVLacZ). In transduced arteries, studied 24 hours after exposure to vectors, expression of recombinant β-galactosidase and eNOS was detected by histochemistry, localizing mainly to the adventitia (n = 4). Immunoelectron microscopy localized recombinant eNOS in adventitial fibroblasts. During contractions to U46619, bradykinin-induced relaxations were significantly augmented in AdCMVeNOS-transduced rings compared with control and AdCMVLacZ-transduced rings (P < 0.01; n = 6). The NOS inhibitor L-nitroarginine methylester (L-NAME) caused significantly greater contraction in AdCMVeNOS-transduced rings (P < 0.001; n = 4) and inhibited bradykinin-induced relaxations in control and transduced rings (P < 0.001; n = 6). The current findings suggest that in AdCMVeNOS-transduced human pial arteries, expression of recombinant eNOS occurs mainly in adventitial fibroblasts where it augments relaxations to NO-dependent agonists such as bradykinin. Findings from the current study might be beneficial in future clinical applications of gene therapy for the treatment or prevention of cerebral vasospasm.

Dipyridamole is used for secondary prophylaxis in ischemic stroke and as a vasodilator agent in myocardial scintigraphy. An important side effect to administering dipyridamole is headache. The aim of the current study was to investigate the effects of dipyridamole on cerebral blood flow, large artery diameter, and headache induction. Twelve healthy subjects were included in this single-blind placebo-controlled study in which placebo (0.9% NaCl) and dipyridamole 0.142 mg/kg·min were administered intravenously over 4 minutes 1 hour apart. Blood flow velocity in the middle cerebral artery (V_mca) was recorded by transcranial Doppler and regional cerebral blood flow in the middle cerebral artery (rCBF_mca) was measured using single photon emission computed tomography and ¹³³Xenon-inhalation. Blood pressure, heart rate, and pCO₂ were measured repeatedly. Headache response was scored every 10 minutes on a verbal scale from 0 to 10 (10 = worst). Dipyridamole caused a decrease in pCO₂ (P < 0.001). pCO₂ corrected rCBF_mca was 41.7 ± 6.9 mL/100 g ·min after placebo versus 41.2 ± 6.9 after dipyridamole (P ≥ 0.05). pCO₂ corrected V_mca decreased 8.4% ± 11.7 (P < 0.001) after dipyridamole, indicating a mean 5.6% ± 6.7 (P = 0.005) relative increase of the arterial diameter. After dipyridamole the median peak headache score was 2 (range 0 to 7) compared with 0 (range 0 to 3) after placebo (P = 0.02). Dilatation of the middle cerebral artery outlasted the headache response. In conclusion, dipyridamole causes a modest pCO₂ independent dilatation of the MCA, which is time-linked to the onset, but not to the cessation, of headache.

Neuronal injury may be dependent upon the generation of the free radical nitric oxide (NO) and the subsequent induction of programed cell death (PCD). Although the nature of this injury may be both preventable and reversible, the underlying mechanisms that mediate PCD are not well understood. Using the agent nicotinamide as an investigative tool in primary rat hippocampal neurons, the authors examined the ability to modulate two independent components of PCD, namely the degradation of genomic DNA and the early exposure of membrane phosphatidylserine (PS) residues. Neuronal injury was determined through trypan blue dye exclusion, DNA fragmentation, externalization of membrane PS residues, cysteine protease activation, and the measurement of intracellular pH (pH_i). Exposure to the NO donors SIN-1 and NOC-9 (300 μmol/L) alone rapidly increased genomic DNA fragmentation from 20 ± 4% to 71 ± 5% and membrane PS exposure from 14 ± 3% to 76 ± 9% over a 24-hour period. Administration of a neuroprotective concentration of nicotinamide (12.5 mmol/L) consistently maintained DNA integrity and prevented the progression of membrane PS exposure. Posttreatment paradigms with nicotinamide at 2, 4, and 6 hours after NO exposure further demonstrated the ability of this agent to prevent and reverse neuronal PCD. Although not dependent upon pH_i, neuroprotection by nicotinamide was linked to the modulation of two independent components of neuronal PCD through the regulation of caspase 1 and caspase 3-like activities and the DNA repair enzyme poly(ADP-ribose) polymerase. The current work lays the foundation for the development of therapeutic strategies that may not only prevent the course of PCD, but may also offer the ability for the repair of neurons that have been identified through the loss of membrane asymmetry for subsequent destruction.