Journal of Cerebral Blood Flow & Metabolism

Kainic acid (KA) neurotoxicity was examined in transgenic (Tg) mice overexpressing human CuZn-superoxide dismutase (SOD-1). The doses of KA required to produce seizures, the severity of the seizures, and the regions damaged were similar in SOD-1 Tg and non-transgenic wild-type mice. Intraperitoneal KA injection induced seizure-related neuronal damage in the CA3 and CA1 regions of the hippocampus and in other regions of the brain in both SOD-1 Tg and wild-type mice. These damaged neurons were labeled with the terminal deoxynudeotidyl transferase-mediated uridine 5′—triphosphate-biotin nick end labeling (TUNEL) technique up to 72 h, although no significant difference in the number of TUNEL-positive neurons was observed between SOD-1 Tg and wild-type mice. In situ hybridization showed that c-fos, c-jun, and hsp70 genes were expressed in the hippocampus, cortex, and other regions of the brain after KA treatment. The expression of these genes was maximal 1 to 4 h following KA treatment but persisted longer in the hippocampus and other regions in SOD-1 Tg compared with wild-type mice; however, cell death in the hippocampus, assessed using cresyl violet staining, was similar in SOD-1 Tg and wild-type mice. The data show that superoxide radicals modulate both immediate early gene and heat shock gene expression after KA-induced seizures. The prolonged expression of c-fos, c-jun, and hsp70 in SOD-1 Tg compared with wild-type mice may indicate that hippocampal neurons survive longer in SOD-1 Tg than in wild-type animals; however, cell death as well as the seizure threshold, seizure severity and the pattern of regional vulnerability were not affected substantially by increased levels of SOD in the brain.

A short ischemic episode preceding sustained ischemia is known to increase tolerance against ischemic cell death. We report early-onset long-lasting neuroprotection against in vitro hypoxia by preceding selective chemical inhibition of oxidative phosphorylation: “chemical preconditioning.” The amplitude of CA₁ population spikes (psap) in hippocampal slices prepared from control animals (control slices) was 31 ± 27% (mean ± SD) upon 45-min recovery from 15-min in vitro hypoxia. In slices prepared from animals treated in vivo with 20 mg/kg 3-nitropropionate (3-np) 1–24 h prior to slice preparation (preconditioned slices), psap improved to 90 ± 15% (p < 0.01). Posthypoxic oxygen free radicals were reduced to 65 ± 10% (mean ± SD) of control in preconditioned slices (p < 0.05). Posthypoxic neuronal density improved from 52 ± 15% (mean ± SD) in control slices to 97 ± 23% in preconditioned slices (p < 0.001). Glibenclamide, an antagonist at K_ATP-channels, partly reversed increased hypoxic tolerance. We conclude that chemical preconditioning induces early-onset long-lasting tolerance against in vitro hypoxia. Ultimately, this strategy may be applicable as a neuroprotective strategy in humans.

The in vivo and in vitro bindings of radiolabeled rotenoids to mitochondrial complex I of rat striatum were examined after unilateral intrastriatal injections of quinolinic acid or 1-methyl-4-phenylpyridinium salt (MPP⁺). Quinolinic acid produced significant, similar losses of in vivo binding of [¹¹C]dihydrorotenol ([¹¹C]DHROL: 40%) and in vitro binding of [³H]dihydrorotenone ([³H]DHR: 53%) in the injected striata at 13 days after the injection of neurotoxin. MPP⁺ reduced in vivo binding of [¹¹C]DHROL (up to −55%) as measured 1.5 to 6 h after its administration. Reductions of in vivo [¹¹C]DHROL binding after either quinolinic acid or MPP⁺ injections did not correlate with changes in striatal blood flow as measured with [¹⁴C]iodoantipyrine. These results are consistent with losses of complex I binding sites for radiolabeled rotenoids, produced using cell death (quinolinic acid) or direct competition for the binding site (MPP⁺). Appropriately radiolabeled rotenoids may be useful for in vivo imaging studies of changes of complex I in neurodegenerative diseases.

Reactive oxygen species (ROS) are normally generated in the brain during metabolism, and their production is enhanced by various insults. Low molecular weight antioxidants (LMWA) are one of the defense mechanisms of the living cell against ROS. The reducing capacity of brain tissue (total LMWA) was measured by cyclic voltammetry (CV), which records biological oxidation potential specific to the type of scavenger(s) present and anodic current intensity (I_a), which depends on scavenger concentration. In the present study, the reducing capacity of rat brain following closed head injury (CHI) was measured. In addition, CV of heat-acclimated traumatized rats was used to correlate endogenous cerebroprotection after CHI with LMWA activity. Sham-injured rat brains displayed two anodic potentials: at 350 ± 50 mV (I_a = 0.75 ± 0.06 μA/mg protein) and at 750 ± 50 mV (I_a = 1.00 ± 0.05 μA/mg protein). Following CHI, the anodic waves appeared at the same potentials as in the sham animals. However, within 5 min of CHI, the total reducing capacity was transiently decreased by 40% (p < 0.01). A second dip was detected at 24 h (60%, p < 0.005). By 48 h and at 7 days, the I_a levels normalized. The acclimated rats displayed anodic potentials identical to those of normothermic rats. However, the I_a of both potentials was lower (60% of control, p < 0.001). The I_a profile after CHI was the direct opposite of the normothermic I_a profile: no immediate decrease of I_a and an increase from 4 h and up to 7 days (40–50%, p < 0.001). We suggest that the lowered levels of LMWA in the post-CHI period reflect their consumption due to overproduction of free radicals. The augmented concentration of LMWA found in the brain of the heat-acclimated rats suggests that these rats are better able to cope with these harmful radicals, resulting in a more favorable outcome following CHI.

Interleukin (IL) 8 was measured in CSF of 14 patients with severe traumatic brain injury. IL-8 levels were significantly higher in CSF (up to 8,000 pg/ml) than serum (up to 2,400 pg/ml) (p < 0.05), suggesting intrathecal production. Maximal IL-8 values in CSF correlated with a severe dysfunction of the blood–brain barrier. Nerve growth factor (NGF) was detected in CSF of 7 of 14 patients (range of maximal NGF: 62–12,130 pg/ml). IL-8 concentrations were significantly higher in these patients than in those without NGF (p < 0.01). CSF containing high IL-8 (3,800–7,900 pg/ml) induced greater NGF production in cultured astrocytes (202–434 pg/ml) than samples with low IL-8 (600–1,000 pg/ml), which showed a smaller NGF increase (0–165 pg/ml). Anti-IL-8 antibodies strongly reduced (52–100%) the release of NGF in the group of high IL-8, whereas in the group with low IL-8, this effect was lower (0–52%). The inability of anti-IL-8 antibodies to inhibit the synthesis of NGF completely may depend on cytokines like tumor necrosis factor α and IL-6 found in these CSF samples, which may act in association with IL-8. Thus, IL-8 may represent a pivotal cytokine in the pathology of brain injury.

We examined by immunohistochemistry the expression of ionotropic glutamate receptor subunits (GluRs) in glial cells of the rat dorsal hippocampus 3 to 28 days after transient forebrain ischemia. In general, the expression of GluRs at all time points studied underwent a drastic reduction that was primarily restricted to the CA1 region. In addition to the disappearance of GluRs as a result of neuronal cell death, we observed their expression in reactive glial cells, The time course of expression and the subunits involved were different for astrocytes and microglia. Reactive astrocytes exhibited kainate, GluR5–7, and N-methyl-D-aspartate (NMDA), NR2A/B, receptor subunits, both of which were maximally expressed approximately 4 weeks after ischemia. In contrast, reactive microglia expressed GluR4 and NRI subunits, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and NMDA receptor subtypes, respectively, with maximal expression observed between 3 and 7 days after ischemia. These results demonstrate that specific types of GluRs are expressed in reactive glial cells after ischemia and that, overall, their expression levels peak around or after the periods of maximal astrogliosis and microgliosis. Thus, modulation of GluR expression may be one of the molecular components accompanying the gliotic process.

Using magnetic resonance (MR) echo–planar imaging (EPI), we recently demonstrated the presence of low-frequency fluctuations (<0.1 Hz) in MR signal intensity from the resting human brain that have a high degree of temporal correlation (p < 10^–3) within and across associated regions of the sensorimotor cortex. These fluctuations in MR signal intensity are believed to arise from fluctuations in capillary blood flow and oxygenation. A substantial overlap between the activation map generated by bilateral finger tapping and temporally-correlated voxels from the sensorimotor cortex obtained during rest was observed. In the work reported here, we investigated whether respiratory hypercapnia, which is known to suspend spontaneous oscillations in regional cerebral blood flow, influences these low-frequency fluctuations. The magnitude of low-frequency fluctuations was reversibly diminished during hypercapnia, resulting in a substantial decrease of the temporal correlation both within and across contralateral hemispheres of the sensorimotor cortex. After the breathing mixture was returned to ambient air, the magnitude and spatial extent of the temporal correlation of low-frequency fluctuations returned to normal. Results of this study support the hypothesis that low-frequency physiological fluctuations observed by MR in the human cortex and spontaneous flow oscillations observed in early studies by laser–Doppler flowmetry (LDF) in the cortex of the rat are identical and are secondary to fluctuations in neuronal activity.

Nitric oxide is a potent vasodilator. Infusion of its precursor, L-arginine, results in increased cerebral blood flow (CBF) in experimental animals. We examined the effects of L-arginine infusion on CBF in humans using positron emission tomography and the quantitative H₂¹⁵O method. Six subjects received 500 ml of 0.9% NaCl solution, and six subjects received an infusion of L-arginine (16.7 mg/kg/min; 500 mg/kg). Before and after the i.v. infusion, paired CBF measurements were performed at baseline and with vibrotactile stimulation of the right hand. In scans performed without vibrotactile stimulation, mean whole-brain CBF increased from 34.9 ± 3.7 ml 100 g^–1 min^–1 to 38.2 ± 4.4 ml 100 g^–1 min^–1 (9.5%; p < 0.005) after L-arginine infusion. The temporal pattern of CBF changes differed from that of plasma growth hormone and insulin levels and of arterial pH. In contrast, in the saline group, mean whole-brain CBF did not change significantly (35.8 ± 5.9 ml 100 g^–1 min^–1 to 35.9 ± 6.4 ml 100 g^–1 min^–1; 0.3%). Vibrotactile stimulation produced significant focal increases in CBF, which were unaffected by L-arginine infusion. L-arginine infusion was associated with an increase in plasma L-citrulline, a byproduct of nitric oxide synthesis.

Dopamine receptor density is believed to decline in normal aging. To test this hypothesis, we measured the density of dopamine D₂-like receptors in vivo in the neostriatum of normal living humans by using the graphical method. This method determines the D₂-like dopamine receptor density in the human brain with an occupying ligand (unlabeled haloperidol) and a radioligand (labeled 3-N-methylspiperone). The method was examined critically, and the assumptions underlying the method were shown to be valid. The validation included comparison of the representation of tracer metabolism by high-pressure liquid chromatography and model assays, calculation of the lumped constant D_w from the value of its components, and comparable tracer partition coefficients in vitro and in vivo. In error analysis, the method consistently performed as well as the direct least-squares regression at statistical noise levels appropriate for the tomograph used in these studies. The method revealed that the density of the D₂-like receptors that bind haloperidol in the caudate nucleus of normal humans declined 1% per year after the age of 18 years.

In a previous study of 10 drug-naive schizophrenic patients, the density of D₂ dopamine receptors was found to be elevated in the caudate nucleus. The study raised questions about the influence of the age of the patients, the relationship of receptor density to psychosis, and the accuracy of the method used to obtain this evidence. Using positron emission tomography and constrained analysis of the brain uptake of the radioligand N-[¹¹C] methylspiperone ([¹¹C]NMSP), we tested four questions: Were the assumptions underlying the quantitation valid? Is there an age decline of the density of D₂-like dopamine receptors in drug-naive schizophrenia and bipolar illness? If so, is it different from that observed in normal aging? Are D₂-like dopamine receptors elevated at any age in either drug-naive schizophrenic or psychotic bipolar illness patients? NMSP and haloperidol partition volumes and plasma protein fractions were not significantly different among patient groups and normal volunteers. The model-derived assay of radioligand metabolites in plasma was confirmed by high-performance liquid chromatography in the patient groups. D₂-like dopamine receptors declined with age, and the slope did not differ significantly between the schizophrenic patients, bipolar affective illness patients, and normal controls. Taking the effect of age into account, increases in D₂ dopamine receptor density were found in seven psychotic patients with bipolar affective illness compared with seven nonpsychotic patients and 24 control subjects as well as in 22 drug-naive schizophrenic patients compared with the 24 control subjects.

The in vivo quantification of the benzodiazepine receptor concentration in human brain using positron emission tomography (PET) and ¹¹C-flumazenil (¹¹C-FMZ), is usually based on a three-compartment model and on PET curves measured in a small number of large regions of interest; however, it should be interesting to estimate the receptor concentration for each pixel and to build quantified images of the receptor concentration. The main advantage is to allow screening of the receptor site localization and visual observation of the possible abnormalities. Up to now, all the methods described include complex experimental protocols, difficult to use in routine examinations. In this paper, we propose the partial-saturation approach to obtain parametric images of benzodiazepine receptor concentration and FMZ affinity. It consists of a single FMZ injection with a low specific activity, followed by Scatchard analysis. Like other parametric imaging methods, this partial-saturation approach can lead to a small percentage (<1%) of unrealistic values in receptor-poor regions; however, it is the only method that allows receptor concentration and affinity images to be obtained from a single-injection 40-min experiment without blood sampling. We also propose a second method in which the receptor concentration map is directly deduced from the PET image acquired 5 to 10 min after a partial-saturation injection. This method assumes a known and constant FMZ affinity value but requires only very simple corrections of this PET image. It is robust (negative values are never found) and quite simple to use in routine examination of patients (no blood sampling, single injection, only 10-min experiment).

Deep prepiriform cortex has an important role in modulating neurotransmission during limbic seizures. We used pharmacologic blockade of non-N-methyl-D-aspartate (NMDA) receptors to study excitatory circuitry from the deep prepiriform cortex to the hippocampus during global ischemia in rat. NBQX, a potent non-NMDA glutamate receptor antagonist, was microinjected stereotactically into the deep prepiriform cortex before global ischemia for 10 min. Neuronal cell death in the hippocampus was evaluated quantitatively 72 h after ischemia. The NBQX-injected rats had a greater number of surviving cells in CA1 sector of hippocampus than did saline-injected controls or rats that received NBQX injections 1 mm from the target. Thus, excitatory amino acid-mediated circuitry emanating from deep prepiriform cortex modulates ischemic neuronal injury in the hippocampus.