Glenn T. Gobbel

Excitotoxicity is Required for Induction of Oxidative Stress and Apoptosis in Mouse Striatum by the Mitochondrial Toxin, 3-Nitropropionic Acid

Excitotoxicity is implicated in the pathogenesis of several neurologic diseases, such as chronic neurodegenerative diseases and stroke. Recently, it was reported that excitotoxicity has a relationship to apoptotic neuronal death, and that the mitochondrial toxin, 3-nitropropionic acid (3-NP), could induce apoptosis in the striatum. Although striatal lesions produced by 3-NP could develop through an excitotoxic mechanism, the exact relationship between apoptosis induction and excitotoxicity after 3-NP treatment is still not clear. The authors investigated the role of excitotoxicity and oxidative stress on apoptosis induction within the striatum after intraperitoneal injection of 3-NP. The authors demonstrated that removal of the corticostriatal glutamate pathway reduced superoxide production and apoptosis induction in the denervated striatum of decorticated mice after 3-NP treatment. Also, the N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801, prevented apoptosis in the striatum after 3-NP treatment for 5 days, whereas the non-NMDA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, was ineffective. The authors also evaluated the initial type of neuronal death by 3-NP treatment for different durations from 1 to 5 days. In early striatal damage, apoptotic neuronal death initially occurred after 3-NP treatment. Our data show that excitotoxicity related to oxidative stress initially induces apoptotic neuronal death in mouse striatum after treatment with 3-NP.

Long-term impairment of subependymal repopulation following damage by ionizing irradiation.

In the mammalian brain, the subependyma (SE) contains stem cells capable of producing neurons and glia. In normal brain these stem cells are responsible, in part, for maintaining the morphologic and functional integrity of the SE; what role the cells of the SE play in brain injury has not yet been elucidated. The present study was designed to determine the long-term regenerative potential of the rat SE after significant depletion of stem cells. Ionizing irradiation was used to deplete cells of the SE and subsequent cellular responses were quantified using immunohistochemical analyses on formalin-fixed, paraffin-embedded tissues. A histomorphometric approach was used to quantify total cell number, number of proliferating cells, number of immature neurons, astrocytes, and undifferentiated components of the SE. Because there are no markers specific for stem cells, we used a repopulation assay as an indirect measure of stem cell response after injury. Our data showed clear radiation dose-dependencies in our quantitative endpoints, implying that there was progressively more stem cell damage with increasing radiation dose. Repopulation of the SE in terms of total cell number, number of proliferating cells and numbers of immature neurons was impaired in a dose-dependent fashion up to 180 days after treatment. These data suggest that after irradiation, surviving stem cells are unable to regenerate the SE. This inability to regenerate after stem cell damage/depletion could have important implications with respect to the normal function of the SE and the function of the SE after brain injury.

Glycolytic glioma cells with active glycogen synthase are sensitive to PTEN and inhibitors of PI3K and gluconeogenesis

Increased glycolysis is characteristic of malignancy. Previously, with a mitochondrial inhibitor, we demonstrated that glycolytic ATP production was sufficient to support migration of melanoma cells. Recently, we found that glycolytic enzymes were abundant and some were increased in pseudopodia formed by U87 glioma (astrocytoma) cells. In this study, we examined cell migration, adhesion (a step in migration), and Matrigel invasion of U87 and LN229 glioma cells when their mitochondria were inhibited with sodium azide or limited by 1% O2. Cell migration, adhesion, and invasion were comparable, with and without mitochondrial inhibition. Upon discovering that glycolysis alone can support glioma cell migration, unique features of glucose metabolism in astrocytic cells were investigated. The ability of astrocytic cells to remove lactate, the inhibitor of glycolysis, via gluconeogenesis and incorporation into glycogen led to consideration of supportive genetic mutations. Loss of phosphatase and tensin homolog (PTEN) releases glycogenesis from constitutive inhibition by glycogen synthase kinase-3 (GSK3). We hypothesize that glycolysis in gliomas can support invasive migration, especially when aided by loss of PTENs regulation on the phosphatidylinositol-3 kinase (PI3K)/Akt pathway leading to inhibition of GSK3. Migration of PTEN-mutated U87 cells was studied for release of extracellular lactic acid and support by gluconeogenesis, loss of PTEN, and active PI3K. Lactic acid levels plateaued and phosphorylation changes confirmed activation of the PI3K/Akt pathway and glycogen synthase when cells relied only on glycolysis. Glycolytic U87 cell migration and phosphorylation of GSK3 were inhibited by PTEN transfection. Glycolytic migration was also suppressed by inhibiting PI3K and gluconeogenesis with wortmannin and metformin, respectively. These findings confirm that glycolytic glioma cells can migrate invasively and that the loss of PTEN is supportive, with activated glycogenic potential included among the relevant downstream effects.

Measurement of regional cerebral blood flow using ultrafast computed tomography. Theoretical aspects.

Theoretical and practical limitations have prevented the measurement of regional cerebral blood flow using dynamic x-ray computed tomography. Development of the ultrafast computed tomography scanner has made it possible to overcome the practical limitations and measure changes in contrast concentration in the brain with excellent time and spatial resolution. By applying modifications of indicator dilution theory, we have derived a method to use these changes in contrast concentration determined using ultrafast computed tomography to measure the fractional vascular volume, mean transit time of blood, and blood flow within specific regions of the brain in a relatively simple and practical manner. This method could theoretically be used in the evaluation of physiological and pathophysiological alterations in cerebral blood flow.

Measurement of regional cerebral blood flow in the dog using ultrafast computed tomography. Experimental validation.

The applicability, feasibility, reproducibility, and accuracy of the method of measuring regional cerebral blood flow using ultrafast computed tomography were evaluated in 25 dogs under varying physiological and pathophysiological conditions. Regional cerebral blood flow values were 75.6 +/- 29.4 ml/100 g/min (mean +/- standard deviation) for the hemisphere, 68.4 +/- 28.2 ml/100 g/min for the basal ganglia, 41.2 +/- 15.0 ml/100 g/min for the internal capsule, and 80.8 +/- 37.2 ml/100 g/min for the neocortex. Measurements made 10 minutes apart were significantly (p less than 0.05) correlated. Simultaneous measurements of regional cerebral blood flow by the microsphere and ultrafast computed tomography methods showed a significant (p less than 0.05) correlation for the hemisphere (r = 0.95), basal ganglia (r = 0.95), and neocortex (r = 0.94) but not for the internal capsule (r = 0.51). Microsphere and ultrafast computed tomography regional cerebral blood flow values were also in agreement in radiation-damaged brain with appreciable blood-brain barrier breakdown, and the two methods demonstrated similar responsiveness of regional cerebral blood flow to alterations in arterial carbon dioxide tension. The accuracy and sensitivity of the ultrafast computed tomography technique suggests that it affords a useful new tool for studying normal and abnormal regional cerebral blood flow.

Apoptosis is induced in the subependyma of young adult rats by ionizing irradiation

To determine if radiation-induced apoptosis occurred in young adult brain, we exposed 2-3-month old rats to single x-ray doses of 5 or 30 Gy. Apoptosis was quantified using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method and a morphologic assessment of nuclear fragmentation. Apoptosis occurred primarily in the subependyma but also in the corpus callosum, peaking 6 h after irradiation. At 48 h there were no apoptotic nuclei observed. These data are the first to show that apoptosis occurs in the young adult rat brain after ionizing irradiation. Further studies are required to define the particular cell type(s) involved and to address the role of this process in the pathogenesis of late radiation injury.

THE INFLUENCE OF OXYGEN FREE RADICALS ON THE PERMEABILITY OF THE MONOLAYER OF CULTURED BRAIN ENDOTHELIAL CELLS

Free radicals have been implicated in the pathogenesis of vasogenic brain edema caused by ischemic or traumatic injury. It has been reported that in transgenic mice overexpressing the human CuZn-superoxide dismutase, brain edema is decreased in many cerebral disorders. To investigate the effects of free radicals on the permeability of the blood brain barrier, we established an in vitro model system of the blood-brain barrier using brain endothelial cells cultivated from transgenic mice and non-transgenic mice. The blood-brain barrier model is originated by a monolayer of brain endothelial cells cultured on a membrane which has 0.45-micron pores. Electrical resistance across the cell monolayer, which reflects the paracellular flux of ionic molecules, was measured. The blood-brain barrier models were incubated with menadione (vitamin K3, an intracellular O2- producing agent), and segmental changes in the electrical resistance across the monolayer were compared between the transgenic and the non-transgenic mice. Superoxide dismutase activity of the cultured brain endothelial cells was 1.7 times higher in the transgenic than in the non-transgenic mice (n = 3, P < 0.001). The electrical resistance was reduced by menadione in the transgenic but not in the non-transgenic mice (n = 7, P < 0.05) in the early stage. Moreover, desferroxamine mesylate (Fe2+ chelating agent) inhibited the menadione-induced early decrease in electrical resistance in the transgenic mice (n = 7, P < 0.05). These results suggest that the permeability of the blood-brain barrier may be affected by hydroxyl radicals and/or peroxynitrite rather than the O2- itself.

Experimental Radiobiological Investigations into Radiosurgery: Present Understanding and Future Directions

LARS LEKSELL BEGAN radiobiological investigations to study the effect of high-dose focused radiation on the central nervous system more than 5 decades ago. Although the effects of radiosurgery on the brain tumor microenvironment are still under investigation, radiosurgery has become a preferred management modality for many intracranial tumors and vascular malformations. The effects and the pathogenesis of biological effects after radiosurgery may be unique. The need for basic research concerning the radiobiological effects of high-dose, single-fraction, ionizing radiation on nervous system tissue is crucial. Information from those studies would be useful in devising strategies to avoid, prevent, or ameliorate damage to normal tissue without compromising treatment efficacy. The development of future applications of radiosurgery will depend on an increase in our understanding of the radiobiology of radiosurgery, which in turn will affect the efficacy of treatment. This article analyzes the current state of radiosurgery research with regard to the nature of central nervous system effects, the techniques developed to increase therapeutic efficacy, investigations into the use of radiosurgery for functional disorders, radiosurgery as a tool for investigations into basic central nervous system biology, and the additional areas that require further investigation.

Apoptosis in the striatum of rats following intraperitoneal injection of 3-nitropropionic acid

The present study investigated the mechanism of cellular degeneration within the striatum following administration of the mitochondrial toxin, 3-nitropropionic (3-NP) acid. Internucleosomal fragmentation typical of apoptosis was present in the DNA of cells from the striatum of 3-NP-treated rats. DNA fragmentation was also evident in this region by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. The data suggest that striatal cells die by apoptosis following administration of 3-NP.

Ischemic Preconditioning in the Rat Brain Enhances the Repair of Endogenous Oxidative DNA Damage by Activating the Base-Excision Repair Pathway

The development of ischemic tolerance in the brain, whereby a brief period of sublethal ‘preconditioning’ ischemia attenuates injury from subsequent severe ischemia, may involve the activation of multiple intracellular signaling events that promote neuronal survival. In this study, the potential role of inducible DNA base-excision repair (BER), an endogenous adaptive response that prevents the detrimental effect of oxidative DNA damage, has been studied in the rat model of ischemic tolerance produced by three episodes of ischemic preconditioning (IP). This paradigm of IP, when applied 2 and 5 days before 2-h middle cerebral artery occlusion (MCAO), significantly decreased infarct volume in the frontal-parietal cortex 72 h later. Correlated with this protective effect, IP markedly attenuated the nuclear accumulations of several oxidative DNA lesions, including 8-oxodG, AP sites, and DNA strand breaks, after 2-h MCAO. Consequently, harmful DNA damage-responsive events, including NAD depletion and p53 activation, were reduced during postischemic reperfusion in preconditioned brains. The mechanism underlying the decreased DNA damage in preconditioned brain was then investigated by measuring BER activities in nuclear extracts. Betapolymerase-mediated BER activity was markedly increased after IP, and this activation occurred before (24 h) and during the course of ischemic tolerance (48 to 72 h). In similar patterns, the activities for AP site and 8-oxodG incisions were also upregulated after IP. The upregulation of BER activities after IP was likely because of increased expression of repair enzymes beta-polymerase, AP endonuclease, and OGG1. These results suggest that the activation of the BER pathway may contribute to IP-induced neuroprotection by enhancing the repair of endogenous oxidative DNA damage after ischemic injury.