Journal of Cerebral Blood Flow & Metabolism

White matter of the brain and spinal cord is susceptible to anoxia and ischemia. Irreversible injury to this tissue can have serious consequences for the overall function of the CNS through disruption of signal transmission. Myelinated axons of the CNS are critically dependent on a continuous supply of energy largely generated through oxidative phosphorylation. Anoxia and ischemia cause rapid energy depletion, failure of the Na⁺−K⁺-ATPase, and accumulation of axoplasmic Na⁺ through noninactivating Na⁺ channels, with concentrations approaching 100 mmol/L after 60 minutes of anoxia. Coupled with severe K⁺ depletion that results in large membrane depolarization, high [Na⁺]_i stimulates reverse Na⁺–Ca²⁺ exchange and axonal Ca²⁺ overload. A component of Ca²⁺ entry occurs directly through Na⁺ channels. The excessive accumulation of Ca²⁺ in turn activates various Ca²⁺-dependent enzymes, such as calpain, phospholipases, and protein kinase C, resulting in irreversible injury. The latter enzyme may be involved in “autoprotection,” triggered by release of endogenous γ-aminobutyric acid and adenosine, by modulation of certain elements responsible for deregulation of ion homeostasis. Glycolytic block, in contrast to anoxia alone, appears to preferentially mobilize internal Ca²⁺ stores; as control of internal Ca²⁺ pools is lost, excessive release from this compartment may itself contribute to axonal damage. Reoxygenation paradoxically accelerates injury in many axons, possibly as a result of severe mitochondrial Ca²⁺ overload leading to a secondary failure of respiration. Although glia are relatively resistant to anoxia, oligodendrocytes and the myelin sheath may be damaged by glutamate released by reverse Na⁺–glutamate transport. Use-dependent Na⁺ channel blockers, particularly charged compounds such as QX-314, are highly neuroprotective in vitro, but only agents that exist partially in a neutral form, such as mexiletine and tocainide, are effective after systemic administration, because charged species cannot penetrate the blood–brain barrier easily.

These concepts may also apply to other white matter disorders, such as spinal cord injury or diffuse axonal injury in brain trauma. Moreover, whereas many events are unique to white matter injury, a number of steps are common to both gray and white matter anoxia and ischemia. Optimal protection of the CNS as a whole will therefore require combination therapy aimed at unique steps in gray and white matter regions, or intervention at common points in the injury cascades.

Immunogold electron microscopy was used to analyze and quantify the Glut1 glucose transporter in brain tissue from five patients undergoing surgery for treatment of seizures. Samples were prepared from two different regions of each resection: (1) the most actively spiking epileptogenic site, and (2) the least actively spiking region, as indicated by intraoperative EEG monitoring. Two configurations of endothelial cell Glut1 were observed. About one half of the capillary profiles examined displayed abundant Glut1 immunoreactivity on both luminal and abluminal endothelial membranes. In the remainder of the profiles, reduced Glut1 labeling was seen, but adjacent erythrocyte membranes remained highly Glut1 immunoreactive, suggesting that reduced endothelial Glut1 reactivity was not attributable to method artifacts. Immunogold studies using antisera to human glial fibrillary acidic protein and human serum albumin demonstrated increased quantities of these two epitopes in the extravascular regions in which more EEG spiking activity had been demonstrated. These observations were consistent with the hypotheses that capillary integrity was more compromised, and gliosis was quantitatively increased, in the more actively spiking region of the resection. Altered glucose transporter activity in the blood–brain barrier was characterized by a bimodal Glut1 distribution in which the smaller (type B) endothelial cells displayed low Glut1 immunoreactivity, whereas adjacent (and even contiguous) larger (type A) endothelial cells showed 5- to 10-fold greater expression of membrane Glut1 transporter protein. Because this transporter facilitates glucose entry to the brain, small pericapillary volumes of brain tissue may have quite different concentrations of glucose. We hypothesize that in complex partial seizures and other forms of brain insult, an alteration of blood–brain barrier Glut1 glucose transporter activity is indicated by the appearance of these two subpopulations of endothelial cells. In comparison with previous studies of human brain capillaries in hemangioblastoma and brain injury, endothelial Glut1 density was apparently reduced (interictally) in affected temporal lobes of patients with complex partial seizures.

6-[F-18]Fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) has been used to measure the central dopaminergic function in many species, including humans and monkeys. For transport across the blood brain barrier (BBB), FDOPA competes with plasma large neutral amino acids (LNAA). In this article we evaluate the effects of normal physiological LNAA concentration variation on BBB transport (K1) and the FDOPA uptake measurement, Ki. We also investigate a method for reducing the dependency of FDOPA quantitation on LNAA. Adult vervet monkeys (Cercopithecus aethiops sabaeus, n = 19) were fasted overnight before FDOPA positron emission tomography scans. Blood samples were drawn for LNAA determination, metabolite analysis, and compartmental modeling. The estimated K1 and Ki were both negatively correlated with LNAA concentrations (r² = 0.51 and 0.62, respectively). Using an adjustment to K1 and Ki based on these correlations, the LNAA dependency was reduced (SD of the data for K1 was reduced by 33%, for Ki by 40%). Experiments with amino acid loading on an additional six animals indicate that BBB transport can be described using Michaelis-Menten kinetics. Results show a clear dependence of FDOPA uptake on plasma LNAA concentrations, which can be removed to increase the precision of FDOPA quantitation.

The role of tumor necrosis factor-alpha (TNF-α) in brain injury is controversial. We studied the effect of anti–TNF-α antibody in a rat model of reversible middle cerebral artery occlusion. During focal ischemia and early reperfusion, TNF-α was rapidly and transiently released into circulation. Pretreatment with intravenous anti–TNF-α antibody reduced cortical (71%, P < 0.015) and subcortical (58%, P < 0.007) injury, enhanced the cerebral blood flow during reperfusion, and improved the neurologic outcome. This further supports the contention that TNF-α is a deleterious cytokine in stroke, whereas circulating antibody against TNF-α may protect brain from reperfusion injury.

Rats were subjected to the standard four-vessel occlusion model of transient cerebral ischemia (vertebral and carotid arteries). The effects of normothermic ischemia (37°C) followed or not by 30-minute reperfusion, as well as 30-minute postdecapitative ischemia, on translational rates were examined. Protein synthesis rate, as measured in a cell-free system, was significantly inhibited in ischemic rats, and the extent of inhibition strongly depended on duration and temperature, and less on the model of ischemia used. The ability of reinitiation in vitro (by using aurintricarboxylic acid) decreased after ischemia, suggesting a failure in the synthetic machinery at the initiation level. Eukaryotic initiation factor 2 (eIF-2) presented almost basal activity and levels after 30-minute normothermic ischemia, and the amount of phosphorylated eIF-2α in these samples, as well as in sham-control samples, was undetectable. The decrease in the levels of phosphorylated initiation factor 4E (eIF-4E) after 30-minute ischemia (from 32% to 16%) could explain, at least partially, the impairment of initiation during transient cerebral ischemia. After reperfusion, eIF-4E phosphorylation was almost completely restored to basal levels (29%), whereas the level of phosphorylated eIF-2α was higher (13%) than in controls and ischemic samples (both less than 2%). eIF-2α kinase activity in vitro as measured by phosphorylation of endogenous eIF-2 in the presence of ATP/Mg²⁺, was higher in ischemic samples (8%) than in controls (4%). It seems probable that the failure of the kinase in phosphorylating eIF-2 in vivo during ischemia is due to the depletion of ATP stores. The levels of the double-stranded activated eIF-2α kinase were slightly higher in ischemic animals than in controls. Our results suggest that the modulation of eIF-4E phosphorylation could be implicated in the regulation of translation during ischemia. On the contrary, phosphorylation of eIF-2α, by an eIF-2α kinase already activated during ischemia, represents a plausible mechanism for explaining the inhibition of translation during reperfusion

Study investigates the role of endothelin (ET) receptors in mediating early changes in cerebral blood flow—as measured by laser Doppler flowmetry (CBF_LDF)—during experimental pneumococcal meningitis. Meningitis was induced with heat-killed pneumococci and confirmed by a significant increase in CBF_LDF (baseline 100%; 225.3 ± 21.8% after 6 hours; mean ± SD), intracranial pressure (ICP), brain water content, and white blood cell count in the CSF. Intravenous administration of the selective endothelin B (ET_B) receptor antagonist BQ-788 immediately before pneumococcal challenge (but not 4 hours afterward) significantly attenuated these pathophysiologic alterations (e.g., CBF_LDF 6 hours after pneumococcal challenge: 116.7 ± 17.4%). Pretreatment with BQ-123, a selective endothelin A receptor antagonist, had no significant effect on ICP and brain water content, but augmented the increase in CBF_LDF and CSF white blood cell count. Since ET is known to trigger the release of nitric oxide (NO) by ET_B receptor activation, we examined specific ET–NO interactions in primary rat cerebromicrovascular endothelial cells after stimulation with heat-killed pneumococci. Pneumococci induced a significant increase in both ET and NO concentrations in endothelial cell culture medium. Treatment with phosphoramidon, an inhibitor of the endothelin-converting enzyme, prevented the production of endothelin and markedly reduced NO generation. Our data provide evidence that ET is involved as a mediator in early pneumococcal meningitis in the rat and contributes to the increase in CBF_LDF, ICP, brain water content, and CSF pleocytosis, presumably through ET_B receptor–mediated NO production.

Spreading depression (SD) confers either increased susceptibility to ischemic injury or a delayed protection. Because nitric oxide modulates ischemic injury, we investigated if altered expression of nitric oxide synthase (NOS) by SD could account for the effect of SD on ischemia. Furthermore, the identity of cells expressing NOS after SD is important, since SD results in heterogeneous, cell type–specific changes in intracellular environment, which can control NOS activity. Immunohistochemical, computer-based image analyses and Western blotting show that the number of neuronal NOS (nNOS)–positive cells in the somatosensory cortex was significantly increased at 6 hours and 3 days after SD (P < 0.05 and 0.01, respectively), whereas inducible NOS expression remained unchanged. Double-labeling of nNOS and glial fibrillary acidic protein identified these nNOS-positive cells as astrocytes. The effect of altered NO production on induced nNOS expression was examined by treating rats with sodium nitroprusside or N_A-nitro-L-arginine methyl ester (LNAM) during SD. Increased nNOS expression was prevented by sodium nitroprusside and phenylephrine or phenylephrine alone, but not LNAM. Because SD increased astrocytic nNOS expression at time points correlating with both ischemic hypersensitivity and ischemic tolerance, the ability of SD to modulate ischemic injury must be complex, perhaps involving NOS but other factors as well.

In anesthetized piglets, endothelial and neuronal nitric oxide synthase (eNOS and nNOS, respectively) levels were investigated after global cerebral ischemia. Increased intracranial pressure was used to produce 5 or 10 minutes of global ischemia, which was verified visually by observing pial arteriolar blood flow and by a microsphere technique. At 4 to 6 hours of reperfusion, parietal cortex, hippocampus, and cerebellum were collected for immunohistochemical or immunoblot analysis. Immunohistochemical examination localized eNOS only to blood vessels and nNOS only to nonvascular cells, which were primarily neurons in all regions examined. Analysis of immunoblot data revealed significant increases in eNOS levels from 47 ± 22 pixels/μg protein for time controls to 77 ± 36 pixels/μg protein (75% increase) for ischemia in parietal cortex (n = 9 to 10) and 22 ± 10 for control to 40 ± 16 pixels/μg protein (40% increase) for ischemia in hippocampus (n = 7 to 8). Levels of eNOS in cerebellum also tended to be higher but were variable and not significant (n = 5 to 6). In contrast, changes in nNOS levels were not detected at 4 or 6 hours. The increase in eNOS levels detected on immunoblots also was apparent on tissue sections as an increase in intensity of staining. Cyclooxygenase-dependent mechanisms were investigated with respect to the ischemia-induced increase in eNOS levels. Pretreatment with the cyclooxygenase inhibitor indomethacin (5 mg/kg intravenously) abolished the ischemia-induced eNOS increase in parietal cortex and hippocampus (n = 7). Thus, we conclude that the eNOS response is rapid, specific to vessels, and involves an indomethacin-sensitive mechanism.

The acute and delayed effects of anoxia on synaptic transmission and long-term potentiation (LTP) were examined in the CA1 region of rat hippocampal slices. Oxygen deprivation for 20 minutes completely but reversibly depressed excitatory postsynaptic potentials mediated by both N-methyl-d-aspartate receptors (NMDAR) and non-NMDAR. Although LTP was reliably produced by a single tetanus delivered 30 minutes after reoxygenation, LTP could not be induced when a tetanus was delivered 70 to 100 minutes after reoxygenation. A tetanus delivered 100 minutes after reoxygenation produced lasting synaptic enhancement when 100 μmol/L d,l-amino-phosphonovaleric acid (APV), a competitive NMDAR antagonist, was administered during the period of oxygen deprivation. The delayed effects of oxygen deprivation were not blocked when APV was administered after oxygen deprivation. Similarly, the delayed effects on LTP induction were overcome by inhibitors of nitric oxide synthase when the nitric oxide synthase inhibitors were administered during anoxia, but not when administered after oxygen deprivation. These results suggest that untimely activation of NMDAR and nitric oxide release during anoxia produce delayed inhibition of LTP induction and may be involved in the memory defects that occur subsequent to cerebral hypoxia.

Primary cultures of mouse cerebral cortical neurons (GABAergic) were incubated for 4 hours in media without glucose containing 1.0 mmol/L [U-¹³C]lactate in the absence or presence of 0.5 mmol/L glutamine. Redissolved, lyophilized cell extracts were analyzed by ¹³C nuclear magnetic resonance spectroscopy to investigate neuronal metabolism of lactate and by HPLC for determination of the total amounts of glutamate (Glu), γ-aminobutyric acid (GABA), and aspartate (Asp). The ¹³C nuclear magnetic resonance spectra of cell extracts exhibited multiplets for Glu, GABA, and Asp, indicating pronounced recycling of labeled tricarboxylic acid cycle constituents. There was extensive incorporation of ¹³C label into amino acids in neurons incubated without glutamine, with the percent enrichments being approximately 60% for Glu and Asp, and 27% for GABA. When 0.5 mmol/L glutamine was added to the incubation medium, the enrichments for Asp, Glu, and GABA were 25%, 35%, and 25%, respectively. This strongly suggests that glutamine is readily converted to Glu and Asp but that conversion to GABA may be complex. The observation that enrichment in GABA was identical in the absence and presence of glutamine whereas cycling was decreased in the presence of glutamine indicates that only C-2 units derived from glutamine are used for GABA synthesis, that is, that metabolism through the tricarboxylic acid cycle is a prerequisite for GABA synthesis from glutamine. The current study gives further support to the hypothesis that cellular metabolism is compartmentalized and that lactate is an important fuel for neurons in terms of energy metabolism and extensively labels amino acids synthesized from tricarboxylic acid cycle intermediates (Asp and Glu) as well as the neurotransmitter in these neurons (GABA).

To investigate long-term adaptations after unilateral carotid artery ligation, the effect of forebrain ischemia on cerebral blood flow and ATP levels was determined at various times after ligation. Unilateral carotid artery ligation was performed in male Wistar rats 0, 3, or 7 days before forebrain ischemia. Laser-Doppler blood flow was monitored bilaterally over the parietal cortex and ATP was measured in the subadjacent cortex of both hemispheres at the end of a 10-minute episode of ischemia. In the 0-day group, forebrain ischemia reduced cortical blood flow to 12% ± 8% (mean ± SD) of preischemic values and lowered cortical ATP to 26% ± 35% of control levels in the ipsilateral hemisphere. Delaying the onset of forebrain ischemia for 3 days after carotid artery ligation significantly improved cortical blood flow (29% ± 12%, P < 0.05) and ATP levels (92% ± 11%, P < 0.05) in the ipsilateral hemisphere. Delaying forebrain ischemia for 7 days also significantly improved ipsilateral blood flow (36% ± 11%, P < 0.05) and ATP levels (81% ± 29%, P < 0.05) compared with the 0-day group. In the contralateral hemisphere, the reduction in blood flow and ATP levels was not significantly altered by delaying the onset of forebrain ischemia for 3 or 7 days. These results show that unilateral carotid artery ligation induces long-term vascular adaptations that improve the collateral circulation and preserve ATP levels during a subsequent episode of ischemia.