Journal of Cerebral Blood Flow & Metabolism

The biochemical pathways to cell death in chronic and acute forms of neurodegeneration are poorly understood, limiting the ability to develop effective therapeutic approaches. As details of the apoptotic and necrotic pathways have been revealed, an appreciation for the decisive role that mitochondria play in life-death decisions for the cell has grown. As a result, the need has arisen to reevaluate the significance to cell viability of mitochondrial Ca²⁺ sequestration, reactive oxygen species generation, and the membrane permeability transition. This review provides basic information on these mitochondrial functions as they relate to control over cell death.

The origins of reflected light changes associated with neuronal activity (optical signals) were investigated in rat somatosensory cortex with optical imaging, microspectrophotometry, and laser-Doppler flowmetry, and dynamic changes in local hemoglobin concentration and oxygenation were focused on. Functional activation was carried out by 2-second, 5-Hz electrical stimulation of the hind limb under chloralose anesthesia. These measurements were performed at the contralateral parietal cortex through a thinned skull. Regional cortical blood flow (rCBF) started to rise 1.5 seconds after the stimulus onset, peaked at 3.5 seconds (26.7% ± 9.7% increase over baseline), and returned to near baseline by 10 seconds. Optical signal responses at 577, 586, and 805 nm showed a monophasic increase in absorbance coincident with the increase in rCBF; however, the signal responses at 605 and 760 nm were biphasic (an early increase and late decrease in absorbance) and microanatomically heterogeneous. The spectral changes of absorbance indicated that the concentrations of both total hemoglobin and oxyhemoglobin increased together with rCBF; deoxyhemoglobin, increased slightly but distinctly (P = 0.016 at 1.0 seconds, P = 0.00038 at 1.5 seconds) just before rCBF increases, then decreased. The authors conclude that activity-related optical signals are greatly associated with a moment-to-moment adjustment of rCBF and metabolism to neuronal activity.

Vibrotactile stimulation of the hand elicits no increase in oxygen consumption commensurate with the increase in blood flow measured in human sensory cortex. To test the hypothesis that previous failures to detect a proportionate increase in oxygen consumption could be an artefact of the sequential bolus, or three-step, method used to measure this parameter in the human brain in vivo, the authors compared the measurements with the results of a novel single bolus, or one-step, method of measuring oxygen consumption. The time of completion of the three-step method was 40 to 50 minutes, whereas the one-step method lasted only 3 minutes. The baseline whole-brain oxygen consumption averaged 185 ± 32 μmol hg⁻¹ min⁻¹ by the three-step method and 153 ± 15 μmol hg⁻¹ min⁻¹ by the one-step method. Vibrotactile stimulation did not elicit a significant increase in oxygen consumption measured by either method. This finding rejects the hypothesis that failure to detect an increase of oxygen consumption could be an artefact caused by limitations of the method used previously. Conversely, it also rejects the hypothesis that observations of an increase of oxygen consumption by the new method are artefacts caused by limitations of the one-step method.

The coupling of oxidative metabolism to the blood flow of the sensory motor hand area is uncertain. The authors tested the hypothesis that continued vibrotactile stimulation ultimately must lead to increased oxygen consumption consumption. Twenty-two healthy right-handed young volunteers underwent positron emission tomography (PET) with the [¹⁵O]water bolus injection method to measure water clearance (K ^{H ₂ O} ₁ an index of blood flow (CBF), and with the [¹⁵O]oxygen bolus inhalation method to measure CMR _{O 2} . The CMR _{O 2} was measured 30 seconds and 20 minutes after onset of intermittent (1 second on, 1 second off) vibrotactile stimulation (110 Hz) and compared with baseline measurements without stimulation. The K ^{H ₂ O} ₁ and CMR _{O 2} changes (ΔK ^{H ₂ O} ₁ and ΔCMR _{O 2} ) were determined using intersubject averaging, together with magnetic resonance imaging based stereotaxic registration technique. The K ^{H ₂ O} ₁ increase was 21 ± 4% and 12 ± 4% at 30 seconds and 20 minutes after onset of stimulation, respectively. No significant increase of CMR _{O 2} was found until 30 minutes after the onset of stimulation. The authors conclude that blood flow and oxidative metabolism undergo uncoupling during sustained phasic stimulation of the sensory hand area. Therefore, neuronal activity stimulated in this manner does not rely on significantly increased oxidative phosphorylation.

To test the hypothesis that brain oxidative metabolism is significantly increased upon adequate stimulation, we varied the presentation of a visual stimulus to determine the frequency at which the metabolic response would be at maximum. The authors measured regional CMR _{O 2} in 12 healthy normal volunteers with the ECAT EXACT HR+ (CTI/Siemens, Knoxville, TN, U.S.A.) three-dimensional whole-body positron emission tomograph (PET). In seven successive activating conditions, subjects viewed a yellow-blue annular checkerboard reversing its contrast at frequencies of 0, 1, 4, 8, 16, 32, and 50 Hz. Stimulation began 4 minutes before and continued throughout the 3-minute dynamic scan. In the baseline condition, the subjects began fixating a cross hair 30 seconds before the scan and continued to do so for the duration of the 3-minute scan. At the start of each scan, the subjects inhaled 20 mCi of ¹⁵O-O₂ in a single breath. The CMR _{O 2} value was calculated using a two-compartment, weighted integration method. Normalized PET images were averaged across subjects and coregistered with the subjects' magnetic resonance imaging in stereotaxic space. Mean subtracted image volumes (activation minus baseline) of CMR _{O 2} then were obtained and converted to z statistic volumes. The authors found a statistically significant focal change of CMR _{O 2} in the striate cortex (x = 9; y = −89; z = −1) that reached a maximum at 4 Hz and dropped off sharply at higher stimulus frequencies.

The effectiveness of 6-[¹⁸F]fluoro-L-m-tyrosine (6FMT) to evaluate dopamine presynaptic integrity was compared to that of 6-[¹⁸F]fluoro-L-dopa (6FDOPA) in vivo by positron emission tomography (PET). Six normal and six 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) -lesioned monkeys received 6FDOPA and 6FMT PET scans on separate occasions with identical scanning protocols. Four measures, the rate of uptake of tracer into striatum using either the arterial input function (K_i) or the activity in the occipital cortex as the input function (K_c), the rate of loss of striatal radioactivity (k_loss), and an index of “effective turnover” of dopamine (k_loss/K_i), were obtained for both tracers during extended PET studies. 6-[¹⁸F]Fluoro-L-m-tyrosine was as effective as 6FDOPA in separating normals from MPTP-lesioned subjects on the basis of the uptake rate constants K_i and K_c. However, in contrast to 6FDOPA, it was not possible to differentiate the normal from the lesioned animal using k_loss or k_loss/K_i for 6FMT. Thus, FMT appears to be a reasonable, highly specific tracer for studying the activity of aromatic dopa decarboxylase enzyme as an index of presynaptic integrity. However, if one is interested in investigating further the metabolic pathway and obtaining an in vivo estimate of the effective turnover of dopamine (after pharmacologic manipulation, for example), 6FDOPA remains the tracer of choice.

The influence of hyperglycemic ischemia on tissue damage and cerebral blood flow was studied in rats subjected to short-lasting transient middle cerebral artery (MCA) occlusion. Rats were made hyperglycemic by intravenous infusion of glucose to a blood glucose level of about 20 mmol/L, and MCA occlusion was performed with the intraluminar filament technique for 15, 30, or 60 minutes, followed by 7 days of recovery. Normoglycemic animals received saline infusion. Perfusion-fixed brains were examined microscopically, and the volumes of selective neuronal necrosis and infarctions were calculated. Cerebral blood flow was measured autoradiographically at the end of 30 minutes of MCA occlusion and after 1 hour of recirculation in normoglycemic and hyperglycemic animals. In two additional groups with 30 minutes of MCA occlusion, CO₂ was added to the inhaled gases to create a similar tissue acidosis as in hyperglycemic animals. In one group CBF was measured, and the second group was examined for tissue damage after 7 days. Fifteen and 30 minutes of MCA occlusion in combination with hyperglycemia produced larger infarcts and smaller amounts of selective neuronal necrosis than in rats with normal blood glucose levels, a significant difference in the total volume of ischemic damage being found after 30 minutes of MCA occlusion. After 60 minutes of occlusion, when the volume of infarction was larger, only minor differences between normoglycemic and hyperglycemic animals were found. Hypercapnic animals showed volumes of both selective neuronal necrosis and infarction that were almost identical with those observed in normoglycemic, normocapnic animals. When local CBF was measured in the ischemic core after 30 minutes of occlusion, neither the hyperglycemic nor the hypercapnic animals were found to be significantly different from the normoglycemic group. Brief focal cerebral ischemia combined with hyperglycemia leads to larger and more severe tissue damage. Our results do not support the hypothesis that the aggravated injury is caused by any disturbances in CBF.

It was previously reported that prosaposin possesses neurotrophic activity that is ascribed to an 18-mer peptide comprising the hydrophilic sequence of the rat saposin C domain. To evaluate the effect of the 18-mer peptide on ischemic neuronal damage, the peptide was infused in the left lateral ventricle immediately after occlusion of the left middle cerebral artery (MCA) in stroke-prone spontaneously hypertensive (SP-SH) rats. The treatment ameliorated the ischemia-induced space navigation disability and cortical infarction and prevented secondary thalamic degeneration in a dose-dependent manner. In culture experiments, treatment with the 18-mer peptide attenuated free radical-induced neuronal injury at low concentrations (0.002 to 2 pg/mL), and the peptide at higher concentrations (0.2 to 20 ng/mL) protected neurons against hypoxic insult. Furthermore, a saposin C fragment comprising the 18-mer peptide bound to synaptosomal fractions of the cerebral cortex, and this binding decreased at the 1st day after MCA occlusion and recovered to the preischemic level at the 7th day after ischemia. These findings suggest that the 18-mer peptide ameliorates neuronal damage in vivo and in vitro through binding to the functional receptor, although the cDNA encoding prosaposin receptor has not been determined yet.

The functions of the epidermal growth factor (EGF) family members in the adult brain are not known. This study investigated the changes in the expression of members of the EGF family following global ischemia employing in situ hybridization and immunohistochemical techniques to elucidate their roles in pathological conditions. EGF mRNA was not detected in either the control or the postischemic rat brain. Although transforming growth factor-α (TGF-α) mRNA was widely expressed in the normal brain, its expression did not change appreciably following ischemia. By contrast, heparin-binding EGF-like growth factor (HB-EGF) mRNA expression was rapidly increased in the CA3 sector and the dentate gyrus of the hippocampus, cortex, thalamus, and cerebellar granule and Purkinje cell layers. EGF receptor mRNA, which was widely expressed, also showed an increase in the CA3 sector and dentate gyrus. Conversely, HB-EGF mRNA did not show any increase prior to ischemic neuronal injury in the CA1 sector, the region most vulnerable to ischemia. Immunohistochemical detection of HB-EGF in the postischemic brain suggested a slight increase of immunostaining in the dentate gyrus of the hippocampus and the cortex. These findings showed that the gene encoding HB-EGF is stress-inducible, indicating the like-lihood that HB-EGF is a neuroprotective factor in cerebral ischemia.

The purpose of this work was to determine whether cerebral endothelial cells have the capacity to synthesize histamine or to express mRNA of receptors that specifically respond to available free histamine. The histamine concentrations and the expression of L-histidine decarboxylase (HDC) and histamine H₁ and H₂ receptor mRNA, both in adult rat brain and in cultured immortalized RBE4 cerebral endothelial cells, were investigated. In this study endothelial cells were devoid of any kind of detectable histamine production, both in vivo and in the immortalized RBE4 cells in culture. Both the immunostainings for histamine and the in situ hybridizations for HDC were negative, as well as histamine determinations by HPLC, indicating that endothelial cells do not possess the capacity to produce histamine. Also, glucocorticoid (dexamethasone) treatment failed to induce histamine production in the cultured cells. Although the cerebral endothelial cells lack histamine production, a nonsaturable uptake in RBE4 cells is demonstrated. The internalized histamine is detected both in the cytoplasm and in the nucleus, which could indicate a role for histamine as an intracellular messenger. Histamine H₁ and H₂ receptor mRNA was expressed in RBE4 cells, and glucocorticoid treatment down-regulated the mRNA levels of both H₁ and H₂ receptors. This mechanism may be involved in glucocorticoid-mediated effects on cerebrovascular permeability and brain edema.

Neuroprotection against cerebral ischemia can be realized if the brain is preconditioned by previous exposure to a brief period of sublethal ischemia. The present study was undertaken to test the hypothesis that nitric oxide (NO) produced from the neuronal isoform of NO synthase (NOS) serves as a necessary signal for establishing an ischemia-tolerant state in brain. A newborn rat model of hypoxic preconditioning was used, wherein exposure to sublethal hypoxia (8% oxygen) for 3 hours renders postnatal day (PND) 6 animals completely resistant to a cerebral hypoxic-ischemic insult imposed 24 hours later. Postnatal day 6 animals were treated 0.5 hour before preconditioning hypoxia with the nonselective NOS inhibitor L-nitroarginine (2 mg/kg intraperitoneally). This treatment, which resulted in a 67 to 81% inhibition of calcium-dependent constitutive NOS activity 0.5 to 3.5 hours after its administration, completely blocked preconditioning-induced protection. However, administration of the neuronal NOS inhibitor 7-nitroindazole (40 mg/kg intraperitoneally) before preconditioning hypoxia, which decreased constitutive brain NOS activity by 58 to 81%, was without effect on preconditioning-induced cerebroprotection, as was pretreatment with the inducible NOS inhibitor aminoguanidine (400 mg/kg intraperitoneally). The protective effects of preconditioning were also not blocked by treating animals with competitive [3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate; 5 mg/kg intraperitoneally] or noncompetitive (MK-801; 1 mg/kg intraperitoneally) N-methyl-D-aspartate receptor antagonists prior to preconditioning hypoxia. These findings indicate that NO production and activity are critical to the induction of ischemic tolerance in this model. However, the results argue against the involvement of the neuronal NOS isoform, activated secondary to a hypoxia-induced stimulation of N-methyl-D-aspartate receptors, and against the involvement of the inducible NOS isoform, but rather suggest that NO produced by the endothelial NOS isoform is required to mediate this profound protective effect.

The reduction of the apparent diffusion coefficient (ADC) of brain tissue water in acute cerebral ischemia, as measured by diffusion-weighted magnetic resonance imaging, is generally associated with the development of cytotoxic edema. However, the underlying mechanism is still unknown. Our aim was to elucidate diffusion changes in the intracellular environment in cytotoxic edematous tissue. The ADC of intracellular metabolites was measured by use of diffusion-weighted ¹H-magnetic resonance spectroscopy after (1) unilateral N-methyl-D-aspartate (NMDA) injection and (2) cardiac arrest-induced global ischemia in neonatal rat brain. The distinct water ADC drop early after global ischemia was accompanied by a significant reduction of the ADC of all measured metabolites (P < 0.01, n = 8). In the first hours after excitotoxic injury, the ADC of water and the metabolites taurine and N-acetylaspartate dropped significantly (P < 0.05, n = 8). At 24 and 72 hours after NMDA injection brain metabolite levels were diminished and metabolite ADC approached contralateral values. Administration of the NMDA-antagonist MK-801 1.5 hours after NMDA injection completely normalized the water ADC but not the metabolite ADC after 1 to 2 hours (n = 8). No damage was detected 72 hours later and, water and metabolite ADC had normal values (n = 8). The contribution of brain temperature changes (calculated from the chemical shift between the water and N-acetylaspartate signals) and tissue deoxygenation to ischemia-induced intracellular ADC changes was minor. These data lend support to previous suggestions that the ischemia-induced brain water ADC drop may partly be caused by reduced diffusional displacement of intracellular water, possibly involving early alterations in intracellular tortuosity, cytoplasmic streaming, or intracellular molecular interactions.