Journal of Cerebral Blood Flow & Metabolism

The ischemic tolerance is known to show protective effects on the neurons and the restricted Ca²⁺ influx through Ca²⁺ channels might be involved. In α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, ribonucleic acid (RNA) editing of the GluR2 subunit determines receptor desensitization and Ca²⁺ permeability. The authors investigated the effect of ischemic tolerance on the messenger RNA editing of Q/R and R/G sites of GluR2 subunit in hippocampus. It was found that the rate of RNA editing in Q/R site showed no change (100% edited), whereas that in R/G site decreased significantly (83.3% normal editing level to 60.4%) at day 3 (preconditioning period) and returned to normal level at day 14 (after preconditioning period). Further investigation revealed that the decrease of editing rate in ischemic tolerance resulted mainly from the decrease of editing in CA1 area.

Early identification of the potentially salvageable penumbra is critical for the determination of therapeutic intervention strategies in acute focal cerebral ischemia. This study differentiates the ischemic penumbra from the core on the basis of the dynamics of lactate formation. This was tested in a rat model of focal cerebral ischemia by infusion of [1-¹³C]-glucose, using lactate-edited magnetic resonance spectroscopic imaging techniques. The authors detected essentially no enrichment of lactate with ¹³C-label from the infused ¹³C-glucose in the ischemic core. However, in borderzone areas, ¹³C was incorporated into lactate, which could point toward compromised but potentially viable tissue. The authors' findings suggest that this combination of ¹³C-glucose infusion with the proposed magnetic resonance methods may aid in differentiating the penumbra from the core in cerebral ischemia.

Rapid transport of lactate from activated brain regions to blood, perhaps reflecting enhanced metabolite trafficking, would prevent local trapping of labeled metabolites of [6-¹⁴C]glucose and cause underestimation of calculated CMR_glc. Because the identities of glucose metabolites lost from activated structures and major routes of their removal are not known, arteriovenous differences across brains of conscious normoxic rats for derivatives of [6-¹⁴C]glucose were determined under steady-state conditions in blood during K⁺-induced spreading cortical depression. Lactate was identified as the major labeled product lost from brain. Its entry to blood was detected within 2 minutes after a pulse of [6-¹⁴C]glucose, and it accounted for 96% of the ¹⁴C lost from brain within approximately 8 minutes. Lactate efflux corresponded to 20% of glucose influx, but accounted for only half the magnitude of underestimation of CMR_glc when [¹⁴C]glucose is the tracer, suggesting extensive [¹⁴C]lactate trafficking within brain. [¹⁴C]Lactate spreading within brain is consistent with (1) relatively uniform pattern labeling of K⁺-treated cerebral cortex by [6-¹⁴C]glucose contrasting heterogeneous labeling by [¹⁴C]deoxyglucose, and (2) transport of ¹⁴C-labeled lactate and inulin up to 1.5 and 2.4 mm, respectively, within 10 minutes. Thus, newly synthesized lactate exported from activated cells rapidly flows to blood and probably other brain structures.

Functional activation stimulates CMR_glc more than CMRO₂ and raises lactate levels in brain. This has been interpreted as evidence that brain work is supported mainly by energy derived from anaerobic glycolysis. To determine if lactate production accounts for the “excess” glucose consumption, cerebral arteriovenous differences were measured in conscious rats before, during, and 15 minutes after sensory stimulation; the brains were rapidly frozen in situ immediately after completion of blood sampling and assayed for metabolite levels. The molar O₂/glucose uptake ratio fell from 6.1 ± 1.1 (mean ± SD) before stimulation to 5.0 ± 1.1 during activation (P < 0.01); lactate efflux from brain to blood was detectable at rest but not during stimulation. By 15 minutes after activation, O₂ and lactate arteriovenous differences normalized, whereas that for glucose fell, causing the O₂/glucose ratio to rise above preactivation levels to 7.7 ± 2.6 (P < 0.01). Brain glucose levels remained stable through all stages of activity. Brain lactate levels nearly doubled during stimulation but normalized within 15 minutes of recovery. Brain glycogen content fell during activation and declined further during recovery. These results indicate that brain glucose metabolism is not in a steady state during and shortly after activation. Furthermore, efflux from and increased content of lactate in the brain tissue accounted for less than 54% of the “excess” glucose used during stimulation, indicating that a shift to anaerobic glycolysis does not fully explain the disproportionately greater increases in CMR_glc above that of CMRO₂ in functionally activated brain. These results also suggest that the apparent dissociation between glucose utilization and O₂ consumption during functional activation reflects only a temporal displacement; during activation, glycolysis increases more than oxidative metabolism, leading to accumulation of products in intermediary metabolic pools that are subsequently consumed and oxidized during recovery.

The metabolic activity pattern of the monkey visual cortex was mapped quantitatively with [¹⁴C]-2-deoxyglucose during the performance of a visually guided reaching task. After bandpass filtering of the reconstructed two-dimensional metabolic maps of areas V1 and V2, alternating bands of high and low metabolic activity were apparent in control and experimental hemispheres. The spatial arrangement of active bands was studied with two-dimensional spectral analysis, and bands were found to be more organized in the experimental monkey. In area V1 of the control monkey the spectral amplitude was spread over a wider range of directions and frequencies than in the experimental subject. The finding that layer IV is characterized by more complex spectra than layers I through III suggests the coexistence of more than one active columnar system in the geniculorecipient layer. In area V2, stripes running almost perpendicular to the V1/V2 border were found along with superimposed patches of enhanced metabolic activity. In the experimental hemispheres, the corresponding spectra were extremely sharp yielding a constant periodicity. It is suggested that the well-organized columnar arrangement within areas V1 and V2 of the experimental hemispheres emerges from the diffusely organized background network of activity patterns in the control state.

The authors examined the involvement of platelet-activating factor (PAF) in mediating leukocyte adherence to brain postcapillary pial venules and altering blood-brain barrier (BBB) permeability during basal conditions and during reoxygenation after asphyxia in newborn piglets. Intravital epifluorescence videomicroscopy, closed cranial windows, and labeling of leukocytes with rhodamine 6G allowed us to obtain serial measurements of adherent leukocytes within postcapillary venules. Blood-brain barrier breakdown was determined by optical measures of cortical extravascular fluorescence intensity after intravenous sodium fluorescein. Superfusion of PAF over the cortex induced a dose-dependent increase in leukocyte adherence to cerebral venules and leakage of fluorescein; with 1 μmol/L PAF, the magnitude of adherence and BBB breakdown was similar to that seen during reoxygenation after 9 minutes of asphyxia. Both adherence and loss of BBB integrity resulting from either exogenous PAF or asphyxia-reoxygenation could be significantly attenuated by intravenous administration of WEB 2086, a PAF receptor antagonist. Window superfusion of superoxide dismutase with PAF attenuated PAF-induced increases in adherence and associated fluorescein leakage. These findings indicate that PAF exhibits proinflammatory effects in piglet brain and that PAF contributes to leukocyte adherence and BBB breakdown after cerebral ischemia. These PAF effects are mediated by increases in superoxide radical generation.

Although stroke in humans usually afflicts the elderly, most experimental studies on the nature of cerebral ischemia have used young animals. This is especially important when studying restorative processes that are age dependent. To explore the potential of older animals to initiate regenerative processes after cerebral ischemia, the authors studied the expression of the juvenile-specific cytoskeletal protein, microtubule-associated protein (MAP) 1B, and the adult-specific protein, MAP2, in male Sprague-Dawley rats at 3 months and 20 months of age. The levels of MAP1B and MAP2 transcripts and the corresponding proteins declined with increasing age in the hippocampus. In the cortex, the levels of the transcripts did not change significantly with age, but the morphologic features of immunostained fibers were clearly affected by age; that is, cortical MAP1B fibers became thicker, and MAP2 fibers, more diffuse, in aged rats. Focal cerebral ischemia, produced by reversible occlusion of the right middle cerebral artery, resulted in a large decrease in the expression of both MAP1B and MAP2 in the infarct core at the messenger ribonucleic acid and protein levels. However, at 1 week after the stroke, there was vigorous expression of MAP1B and its messenger ribonucleic acid, as well as MAP2 protein, in the border zone adjacent to the infarct of 3-month-old and 20 month-old male Sprague-Dawley rats. The upregulation of these key cytologic elements generally was diminished in aged rats compared with young animals, although the morphologic features of fibers in the infarct border zone were similar in both age groups. These results suggest that the regenerative potential of the aged rat brain appears to be competent, although attenuated, at least with respect to MAP1B and MAP2 expression up to 20 months of age.

The authors used mRNA differential display to identify genes whose expression levels are altered in the adult rat hippocampus 24 hours after global ischemia. At this time after challenge, the basic helix-loop-helix transcription factor, SEF-2, and the 26S proteasome complex subunit, p112, were identified as genes whose expression levels are decreased and increased, respectively, in the hippocampus. To determine the spatial and temporal patterns of expression change for each gene, the authors antisense in situ hybridization to paired brain sections of sham-operated and global ischemia-challenged rats at 6, 12, and 24 hours after reperfusion SEF-2 expression was not significantly altered from that of sham-operated controls in any hippocampal subfield at or before 12 hours after challenge. At 24 hours after ischemia, however, SEF-2 expression levels were significantly diminished in the vulnerable CA1 subfield, but not in the less vulnerable CA3 or dentate granule cell subfields. The proteasome p112 subunit gene displayed no change in expression levels at 6 hours after insult; however, an elevated expression was observed at 12 hours after challenge in the dentate granule cell subfield. By 24 hours after challenge, p112 expression was significantly elevated in both the CA1 and dentate granule cell subfields. These results demonstrate that a member of the basic helix-loop-helix family of transcription factors, SEF-2, and the major subunit of the 26S proteasome complex, p112, display altered gene expression in the hippocampus after transient cerebral ischemia.

Traumatic brain injury evokes multiple axonal pathologies that contribute to the ultimate disconnection of injured axons. In severe traumatic brain injury, the axolemma is perturbed focally, presumably allowing for the influx of Ca²⁺ and initiation of Ca²⁺-sensitive, proaxotomy processes. Mitochondria in foci of axolemmal failure may act as Ca²⁺ sinks that sequester Ca²⁺ to preserve low cytoplasmic calcium concentrations. This Ca²⁺ load within mitochondria, however, may cause colloid osmotic swelling and loss of function by a Ca²⁺-induced opening of the permeability transition pore. Local failure of mitochondria, in turn, can decrease production of high-energy phosphates necessary to maintain membrane pumps and restore ionic balance in foci of axolemmal permeability change. The authors evaluated the ability of the permeability transition pore inhibitor cyclosporin A (CsA) to prevent mitochondrial swelling in injured axonal segments demonstrating altered axolemmal permeability after impact acceleration injury in rat. At the electron microscopic level, statistically fewer abnormal mitochondria were seen in traumatically injured axons from CsA-pretreated injured animals. Further, this mitochondrial protection translated into axonal protection in a second group of injured rats, whose brains were reacted with antibodies against amyloid precursor protein, a known marker of injured axons. Pretreatment with CsA significantly reduced the number of axons undergoing delayed axotomy, as evidenced by a decrease in the density of amyloid precursor protein-immunoreactive axons. Collectively, these studies demonstrate that CsA protects both mitochondria and the related axonal shaft, suggesting that this agent may be of therapeutic use in traumatic brain injury.

Although transcranial Doppler ultrasound (TCD) has been used to detect oscillations in CBF, interpretation is severely limited, since only blood velocity and not flow is measured. Oscillations in vessel diameter could, therefore, mask or alter the detection of those in flow by TCD velocities. In this report, the authors use a TCD-derived index of flow to detect and quantify oscillations of CBF in humans at rest. A flow index (FI) was calculated from TCD spectra by averaging the intensity weighted mean in a beat-by-beat manner over 10 seconds. Both FI and TCD velocity were measured in 16 studies of eight normal subjects at rest every 10 seconds for 20 minutes. End tidal CO₂ and blood pressure were obtained simultaneously in six of these studies. The TCD probe position was meticulously held constant. An index of vessel area was calculated by dividing FI by velocity. Spectral estimations were obtained using the Welch method. Spectral peaks were defined as peaks greater than 2 dB above background. The frequencies and magnitudes of spectral peaks of FI, velocity, blood pressure, and CO₂ were compared with t tests. The Kolmogorov-Smirnov test was used to further confirm that the data were not white noise. In most cases, three spectral peaks (a, b, c) could be identified, corresponding to periods of 208 ± 93, 59 ± 31, and 28 ± 4 (SD) seconds for FI, and 196 ± 83, 57 ± 20, and 28 ± 6, (SD) seconds for velocity. The magnitudes of the spectral peaks for FI were significantly greater (P < 0.02) than those for velocity. These magnitudes corresponded to variations of at least 15.6%, 9.8%, and 6.8% for FI, and 4.8%, 4.2%, and 2.8% for velocity. The frequencies of the spectral peaks of CO₂ were similar to those of FI with periods of 213 ± 100, 60 ± 46, and 28 ± 3.6 (SD) seconds. However, the CO₂ spectral peak magnitudes were small, with an estimated maximal effect on CBF of (±) 2.5 ± 0.98, 1.5 ± 0.54, and 1.1 ± 0.31 (SD) percent. The frequencies of the blood pressure spectral peaks also were similar, with periods of 173 ± 81, 44 ± 8, and 26 ± 2.5 (SD) seconds. Their magnitudes were small, corresponding to variations in blood pressure of (±) 2.1 ± 0.55, 0.97 ± 0.25, and 0.72 ± 0.19 (SD) percent. Furthermore, coherence analysis showed no correlation between CO₂ and FI, and only weak correlations at isolated frequencies between CO₂ and velocity, blood pressure and velocity, or blood pressure and FI. The Kolmogorov-Smirnov test distinguished our data from white noise in most cases. Oscillations in vessel flow occur with significant magnitude at three distinct frequencies in normal subjects at rest and can be detected with a TCD-derived index. The presence of oscillations in blood velocity at similar frequencies but at lower magnitudes suggests that the vessel diameters oscillate in synchrony with flow. Observed variations in CO₂ and blood pressure do not explain the flow oscillations. Ordinary TCD velocities severely underestimate these oscillations and so are not appropriate when small changes in flow are to be measured.

This study evaluates the validity of the transfer function analysis of spontaneous fluctuations of arterial blood pressure (ABP) and blood flow velocity of the middle cerebral artery (MCAFV) as a simple, convenient method to assess human cerebral autoregulation in patients with carotid stenosis. Eighty-three consecutive patients with various degrees of carotid stenosis and 37 healthy controls were enrolled. The carotid stenosis was graded based on the diagnostic criteria of duplex ultrasound. Instantaneous bilateral MCAFV and ABP of all participants were assessed noninvasively using transcranial Doppler sonography and the servocontrolled infrared finger plethysmography, respectively. Spectral analyses of ABP and MCAFV were performed by fast Fourier transform. The fluctuations in ABP as well as in MCAFV were diffracted into three components at specific frequency ranges designated as high-frequency (HF; 0.15 to 0.4 Hz), low-frequency (LF; 0.04 to 0.15 Hz), and very low-frequency (VLF; 0.016 to 0.04 Hz). Cross-spectral analysis was applied to quantify the coherence, transfer phase, and magnitude in individual HF, LF, and VLF components. Transcranial Doppler CO₂ vasomotor reactivity was measured with 5% CO₂ inhalation. The LF phase angle (r = −0.53, P < 0.001); magnitude of VLF (r = −0.29, P = 0.002), LF (r = −0.35, P < 0.001), and HF (r = −0.47, P < 0.001); and CO₂ vasomotor reactivity (r = −0.66, P < 0.001) were negatively correlated with the severity of stenosis. Patients with unilateral high-grade (greater than 90% stenosis) carotid stenosis demonstrated significant reduction in LF phase angle (P < 0.001) and HF magnitude (P = 0.018) on the ipsilateral side of the affected vessel compared with their contralateral side. The study also revealed a high sensitivity, specificity, and accuracy using LF phase angle and HF magnitude to detect a high-grade carotid stenosis. A strong correlation existed between the LF phase angle and the CO₂ vasomotor reactivity test (r = 0.62, P < 0.001), and the correlation between the HF magnitude and the CO₂ vasomotor reactivity (r = 0.44, P < 0.001) was statistically significant as well. We conclude that transfer function analysis of spontaneous fluctuations of MCAFV and ABP could be used to identify hemodynamically significant high-grade carotid stenosis with impaired cerebral autoregulation or vasomotor reserve.