Journal of Cerebral Blood Flow & Metabolism

Interleukin-1 (IL-1) receptor antagonist (IL-1ra) markedly reduces infarct volume induced by middle cerebral artery occlusion (MCAO) in the rat, when injected either centrally (intracerebroventricularly) or peripherally. The site or sites of action of IL-1 in stroke pathology, however, are not known. The present study investigated the site(s) of action of IL-1/IL-1ra in ischemic brain damage by studying the effects of local injection of IL-1ra into the cortex or striatum following permanent MCAO in the rat. Cortical injection of IL-1ra (5 µg) did not affect infarct volume in the cortex or striatum measured 24 h after MCAO. In contrast, striatal injection of IL-1ra ipsilateral to the infarction caused a significant and highly reproducible reduction of cortical (37%, p < 0.001) and striatal damage (27%, p < 0.001, corrected for edema) compared with vehicle-injected animals. Injection of IL-1ra (5 µg) into the striatum, contralateral to the infarction, resulted in a small (9%) but significant (p < 0.001) reduction of ipsilateral cortical damage, with no effect on ipsilateral striatal damage. Injection of a higher dose of IL-1ra (7.5 µg) in the contralateral striatum caused a further inhibition of ipsilateral cortical damage (24%, p < 0.001) and a significant reduction of ipsilateral striatal damage (16%, p < 0.001). In separate groups of rats, it was established that core temperature (measured continuously in free-moving animals with remote radiotelemetry) was not affected by striatal or cortical injection of IL-1ra. These data show that injection of IL-1ra into the striatum but not the cortex reduces infarct volume in both the striatum and the cortex, independently of effects on core temperature. These results imply that blocking striatal IL-1 contributes to IL-1ra-protective effects. We hypothesize that IL-1 may influence striatal distal cortical damage through either the release of specific substances or activation of polysynaptic pathways.

Postischemic cerebral inflammation has been reported to contribute to ischemic brain damage. During inflammation, constituents of the extracellular matrix such as fibronectin and laminin are recognized by certain integrins or proteoglycans and play an important role in the cell adhesion process. The purpose of this study was to evaluate the efficacy of peptides derived from laminin on leukocyte accumulation, infarct size, and neurological outcome in rats subjected to 1 h of cerebral ischemia and 48 h of reperfusion. Forty-four animals were included in this study: transient ischemia without treatment (Group I), treatment with TG-1 peptide (Group II), GD-1 peptide (Group III), and GD-6 peptide (Group IV). Group II showed a significant reduction of the leukocyte accumulation (p < 0.001) and infarct size (p = 0.015) when compared with Group I. The neurological grade of Group II was also significantly better than in Group I at 48 h after reperfusion (p = 0.012). Based on these data, which are the first to explore the therapeutic potential of this peptide in cerebral ischemia, laminin peptide may offer a novel therapeutic approach to allaying injury in ischemic stroke.

Glial inclusions containing the microtubule-associated protein tau are present in a variety of chronic neurodegenerative conditions. We now report a rapid and time-dependent increase of tau immunoreactivity within oligodendrocytes after focal cerebral ischemia in the rat. The number of tau positive oligodendrocytes in the ipsilateral subcortical white matter increased six- to eightfold by 40 minutes after permanent middle cerebral artery occlusion (MCAO). Tau was detected using antibodies that label both the N- and C-terminal of the protein, suggesting accumulation of full-length protein within these cells. Pretreatment with the spin trap agent α-phenyl-tert-butyl-nitrone (PBN)(100mg/kg) reduced the number of tau-positive oligodendrocytes by 55% in the subcortical white matter of the ischemic hemisphere compared with untreated animals at 40 minutes after MCAO. In contrast, pretreatment with glutamate receptor antagonists MK-801 (0.5 mg/kg) or 2,3-dihydroxy-6-nitro-7-sulpfamoylbenzo(f)quinoxaline (NBQX) (2 × 30 mg/kg), failed to reduce the number of tau-positive oligodendrocytes after 40 minutes of ischemia. The results indicate that oligodendrocytes respond rapidly to an ischemic challenge and that free radical-mediated mechanisms are involved in the cascade leading to increased tau immunoreactivity.

Neuronal thread proteins (NTP) are a family of phosphoproteins expressed during neuritic sprouting. The 15 to 18 kD NTP cluster is associated with development and neuronal differentiation, whereas the 21 kD and 39 to 42 kD species are overexpressed in Alzheimer's disease, correlating with neurodegenerative sprouting and synaptic disconnection. Empirical observations suggested that NTP might also be modulated with central nervous system injury and stroke. In this study of both human and experimental (rat) focal cerebral infarcts, in situ hybridization and immunocytochemical staining revealed NTP gene expression up-regulated in perifocal neurons. These findings were confirmed by quantitative Northern and Western blot analyses. Moreover, Western blot analysis demonstrated selectively increased expression of the 15 to 18 kD NTP species during the acute, subacute, and healing phases of cerebral infarction in both humans and experimental animals, corresponding with the expected period of neuronal repair. These results suggest an additional role for the 15 to 18 kD NTP species in neuritic sprouting required for neuronal regeneration after injury in the mature central nervous system.

The prolonged expression of the leucine zipper fos/jun immediate early genes (IEG) has been correlated with neuronal death after cerebral ischemia. In this study, the expression of six zinc finger IEG was examined using in situ hybridization in adult rats after middle cerebral artery occlusion (MCAO) with the suture model. NGFI-A, NGFI-B, NGFI-C, egr-2, egr-3, and Nurr1 mRNA were all induced throughout the ipsilateral cortex at 1 hour to 12 hours after MCAO. The cortical induction for most of the genes was greatest in the anterior cingulate and the anterior cerebral artery (ACA) and middle cerebral artery (MCA) transition zone. All of the zinc finger IEG were induced at 1 hour in all regions of hippocampus. NGFI-A and NGFI-B were induced in ipsilateral thalamus. Within areas of infarction, the basal IEG mRNA expression, and expression of the housekeeping gene cyclophilin A mRNA, decreased below control levels by 12 hours after the ischemia. Immediate early gene expression outside areas of infarction returned to control levels in most brain regions by 24 hours except for egr-3, which continued to be induced in the MCA/ACA transition zone for 24 hours, and NGFI-A, which continued to be expressed in specific regions of the thalamus for 72 hours. The induction of these IEG in the cortex is likely caused by ischemia-induced cortical spreading depression, with the hippocampal and thalamic IEG induction being caused by activation of efferent cortical pathways to these regions. The prominent induction of NGFI-B, NGFI-C, egr-2, and egr-3 in the anterior cingulate cortex, the ACA/MCA transition zone, and medial striatum could reflect the ischemic regions around MCA infarcts. The prolonged NGFI-A expression observed in thalamus in this study, and in CA1 of hippocampus after global ischemia in the gerbil in a previous study, suggests that the prolonged NGFI-A expression could be the result of or the cause of the delayed cell death. Prolonged NGFI-A expression, like c-fos and c-jun, seems to provide a marker for slowly dying neurons.

Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme to produce bile pigments and carbon monoxide. The HO-1 isozyme is induced by a variety of agents such as heat, heme, and hydrogen peroxide. Evidence suggests that the bile pigments serve as antioxidants in cells with compromised defense mechanisms. Because hypoxia-ischemia (HI) increases the level of oxygen free radicals, the induction of HO-1 expression in the brain during ischemia could modulate the response to oxidative stress. To study the possible involvement of HO-1 in neonatal hypoxia-induced ischemic tolerance, we examined the brains of newborn rat pups exposed to 8% O₂ (for 2.5 to 3 hours), and the brain of chronically hypoxic rat pups with congenital cardiac defects (Wistar Kyoto; WKY/ NCr). Heme oxygenase-1 immunostaining did not change after either acute or chronic hypoxia, suggesting that HO-1 is not a good candidate for explaining hypoxia preconditioning in newborn rat brain. To study the role of HO-1 in neonatal HI, 1-week-old rats were subjected to right carotid coagulation and exposure to 8% O₂/92% N₂ for 2.5 hours. Whereas HO enzymatic activity was unchanged in ipsilateral cortex and subcortical regions compared with the contralateral hemisphere or control brains, immunocytochemistry and Western blot analysis showed increased HO-1 staining in ipsilateral cortex, hippocampus, and striatum at 12 to 24 hours up to 7 days after HI. Double fluorescence immunostaining showed that HO-1 was expressed mostly in ED-1 positive macrophages. Because activated brain macrophages have been associated with the release of several cytotoxic molecules, the presence of HO-1 positive brain macrophages may determine the tissue vulnerability after HI injury.

We describe the tracer kinetic analysis of [C-11]-labeled alpha-methyl-tryptophan (AMT), an analogue of tryptophan, which has been developed as a tracer for serotonin synthesis using positron emission tomography (PET) in human brain. Dynamic PET data were acquired from young healthy volunteers (n = 10) as a series of 22 scans covering a total of 60 minutes and analyzed by means of a three-compartment, four-parameter model. In addition, functional images of the K-complex were created using the Patlak-plot approach. The application of a three-compartment model resulted in low identifiability of individual k-values, especially that of k₃. Model identifiability analysis using a singular value decomposition of the final sensitivity matrix showed parameter identifiability to increase by 50% when the Patlak-plot approach was used. K-complex values derived by the Patlak-plot approach overestimated the compartmental values by 10 to 20%, because of the violation of the dynamic equilibrium assumption. However, this bias was fairly constant in all structures of the brain. The rank order of K-complex values from different brain regions corresponded well to the regional concentrations of serotonin in human brain (P < 0.0001). These results indicate that the Patlak-plot method can be readily applied to [C-11]AMT data in order to create functional images of the K-complex, reflecting serotonin synthesis rate, within an acceptable error margin.

To evaluate the effect of the repetition rate of a simple movement on the magnitude of neuronal recruitment in the primary sensorimotor cortex, we used a blood flow-sensitive, echo planar functional magnetic resonance imaging (fMRI) sequence in six normal volunteers. Three of the volunteers also had [¹⁵O]water positron emission tomography (PET) studies using the same paradigm. Previous PET studies had shown an increase in regional CBF (rCBF) with movement frequencies up to 2 Hz and then a plateau of regional cerebral blood flow (rCBF) at faster frequencies. To evaluate the extent of the activation, the correlation coefficient (cc) of the Fourier-transformed time-signal intensity change with the Fourier-transformed reference function was calculated pixel by pixel. The degree of activation was measured as the signal percent change of each region of interest with a cc > 0.5. The left primary sensorimotor cortex was constantly activated at 1, 1.5, 2, and 4 Hz, while there was only inconsistent activation at 0.25 and 0.5 Hz. Percent change in signal intensity linearly increased from 1 to 4 Hz. Area of activation increased up to 2 Hz and showed a tendency to decrease at higher frequencies. Individual analysis of PET data showed activation in the same location as that revealed by fMRI. The combination of progressively increasing signal intensity with an area that increases to 2 Hz and declines at faster frequencies explains the PET finding of plateau of rCBF at the faster frequencies. Functional magnetic resonance imaging shows similar results to PET, but is better able to dissociate area and magnitude of change.

This study was designed to determine age-related changes in autoregulatory responses of the brain stem circulation in vivo. In anesthetized adult (4 to 6 months, n = 8) and aged (24 to 26 months, n = 7) Sprague-Dawley rats, local CBF to the brain stem was determined with laser-Doppler flowmetry and diameters of the basilar artery and its branches were measured through an open cranial window during stepwise hemorrhagic hypotension. In aged rats, the lower limit of CBF auto-regulation shifted upward to 60 to 75 mm Hg from 30 to 45 mm Hg in adult rats. Dilator responses of the basilar artery (baseline diameter: 254 ± 15 μm), large branch (109 ± 23 μm), and small branch (44 ± 10 μm) to hypotension were much smaller in aged rats than in adult rats. The maximum change in diameter of the basilar artery during profound hypotension was significantly smaller in aged rats (11 ± 8%) than that in adult ones (23 ± 12%, P < 0.05); that of the large branch was 12 ± 8% versus 33 ± 17% (P < 0.005); and that of the small branch was 17 ± 7% versus 40 ± 13% (P < 0.0005), suggesting greater attenuation of the responses in the smaller vessels. Thus, this study provides direct evidence that aging diminishes the compensatory dilatation of brain stem arterioles and arteries during hypotension and modifies the autoregulatory plateau of CBF, which seems to increase the risk of the brain stem ischemia during hypotensive conditions.

We earlier reported that electrical stimulation of the rat nucleus basalis of Meynert (NBM) induces large cerebral blood flow increases, particularly in frontal cortical areas but also in some subcortical regions. The present study was designed to address the issue of blood flow control exerted by NBM projections. To this aim, we have determined whether these flow increases were associated with proportionate changes in metabolic activity as evaluated by cerebral glucose utilization (CGU) strictly under the same experimental conditions in the conscious rat. An electrode was chronically implanted in a reactive site of the NBM as determined by laser-Doppler flowmetry (LDF) of the cortical circulation. One to two weeks later, while the cortical blood flow was monitored by LDF, we measured CGU using the [¹⁴C]2-deoxyglucose autoradiographic technique during unilateral electrical stimulation of the NBM, and analyzed the local flow-metabolism relationship. The large increases in cortical blood flow induced by NBM stimulation, exceeding 300% in various frontal areas, were associated with at most 24% increases in CGU (as compared with the control group) in one frontal area. By contrast, strong increases in CGU exceeding 150% were observed in subcortical regions ipsilateral to the stimulation, especially in extrapyramidal structures, associated with proportionate CBF changes. Thus, none of the blood flow changes observed in the cortex can be ascribed to an increased metabolic activity, whereas CBF and CGU were coupled in many subcortical areas. This result indicates that different mechanisms, which do not necessarily involve any metabolic factor, contribute to the regulation of the cerebral circulation at the cortical and subcortical level. Because the distribution of the uncoupling is coincident with that of cholinergic NBM projections directly reaching cortical microvessels, these data strongly support the hypothesis that NBM neurons are capable of exerting a neurogenic control of the cortical microcirculation.

Near infrared spectroscopy (NIRS) is used to measure CBF (CBF_NIRS) in humans, based on Fick's principle, using oxygen as an intravascular tracer. We compared CBF_NIRS with CBF measured by microspheres (CBFμ) and the venous outflow technique (CBFv) in 15 dogs, altering CBF with ventilation-induced changes in PaCO₂. Five hundred forty-nine CBF_NIRS measurements were attempted using an integration time of 2.5 s on the saturation signal from the tongue. One hundred ninety-eight (36.1%) of the measurements fulfilled predefined criteria. The coefficient of variation (CV) for six measurements under stable conditions was 29.1%. The CBF_NIRS measurements correlated best with microsphere-measured blood flows in the cortical gray matter (median 0.43, range 0.16–0.93); the contributions of the skull and dura were variable. The CBFv varied by a median of 12% (range 0–67%) during the CBF_NIRS measurements. The percentage of acceptable CBF_NIRS measurements, the CV, and the correlation coefficients of the CBF_NIRS were improved by using saturation signal directly from the artery and varying the integration time with an estimate of the minimum transit time. The current method of measuring CBF_NIRS in the reflectance mode is inaccurate when compared with other accepted techniques.

Cerebral glucose utilization was higher during the first positron emission tomography (PET) session than during the second session, as assayed using the PET [¹⁸F]fluorodeoxyglucose method in male human volunteers. This difference was due largely to data from subjects with low trait anxiety, since subjects with high anxiety showed similar metabolism in both PET sessions. High-anxiety subjects showed greater right/left ratios of cerebral metabolism than low-anxiety subjects, particularly during the second PET session. These findings suggest that the level of anxiety may be an important variable to consider in PET studies using multiple sessions.