Annadora J. Bruce-Keller

Food restriction reduces brain damage and improves behavioral outcome following excitotoxic and metabolic insults

Food restriction (FR) in rodents is known to extend life span, reduce the incidence of age‐related tumors, and suppress oxidative damage to proteins, lipids, and DNA in several organ systems. Excitotoxicity and mitochondrial impairment are believed to play major roles in the neuronal degeneration and death that occurs in the brains of patients suffering from both acute brain insults such as stroke and seizures, and chronic neurodegenerative conditions such as Alzheimers, Parkinsons, and Huntingtons diseases. We now report that FR (alternate‐day feeding regimen for 2–4 months) in adult rats results in resistance of hippocampal neurons to excitotoxin‐induced degeneration, and of striatal neurons to degeneration induced by the mitochondrial toxins 3‐nitropropionic acid and malonate. FR greatly increased the resistance of rats to kainate‐induced deficits in performance in water‐maze learning and memory tasks, and to 3‐nitropropionic acid–induced impairment of motor function. These findings suggest that FR not only extends life span, but increases resistance of the brain to insults that involve metabolic compromise and excitotoxicity. Ann Neurol 1999;45:8–15

Cognitive impairment following high fat diet consumption is associated with brain inflammation

C57Bl/6 mice were administered a high fat, Western diet (WD, 41% fat) or a very high fat lard diet (HFL, 60% fat), and evaluated for cognitive ability using the Stone T-maze and for biochemical markers of brain inflammation. WD consumption resulted in significantly increased body weight and astrocyte reactivity, but not impaired cognition, microglial reactivity, or heightened cytokine levels. HFL increased body weight, and impaired cognition, increased brain inflammation, and decreased BDNF. Collectively, these data suggest that while different diet formulations can increase body weight, the ability of high fat diets to disrupt cognition is linked to brain inflammation.

Impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury

Glutamate-induced excitotoxicity involving the formation of reactive oxygen species (ROS) has been implicated in neuronal dysfunction and cell loss following ischemic and traumatic injury to the central nervous system (CNS). ROS are formed in mitochondria when energy metabolism is compromised, and are inactivated by the ROS scavengers superoxide dismutase (SOD), catalase, and glutathione (GSH). ROS can impair the function of several cellular components including proteins, nucleic acids, and lipids. In the present study, we measured indicators of mitochondrial metabolic activity, ROS formation, lipid peroxidation, and antioxidant enzyme activities in synaptosomes obtained from rat spinal cord at early times following traumatic injury. Mitochondrial metabolic activity was found to significantly decrease as early as 1 h following injury, and continued to be compromised over the remaining postinjury time points. ROS formation was found to be significantly increased at 4 and 24 h following injury, while lipid peroxidation levels were found to be significantly increased in the injured spinal cord at 1 and 24 h, but not 4 h following injury. SOD enzyme activity was unchanged at all postinjury time points, while catalase activity and GSH levels were significantly increased at 24 h following injury. These findings indicate that impaired mitochondrial function, ROS, and lipid peroxidation occur soon after traumatic spinal cord injury, while the compensatory activation of molecules important for neutralizing ROS occurs at later time points. Therapeutic strategies aimed at facilitating the actions of antioxidant enzymes or inhibiting ROS formation and lipid peroxidation in the CNS may prove beneficial in treating traumatic spinal cord injury, provided such treatments are initiated at early stages following injury.

Uric acid protects neurons against excitotoxic and metabolic insults in cell culture, and against focal ischemic brain injury in vivo

Uric acid is a well‐known natural antioxidant present in fluids and tissues throughout the body. Oxyradical production and cellular calcium overload are believed to contribute to the damage and death of neurons that occurs following cerebral ischemia in victims of stroke. We now report that uric acid protects cultured rat hippocampal neurons against cell death induced by insults relevant to the pathogenesis of cerebral ischemia, including exposure to the excitatory amino acid glutamate and the metabolic poison cyanide. Confocal laser scanning microscope analyses showed that uric acid suppresses the accumulation of reactive oxygen species (hydrogen peroxide and peroxynitrite), and lipid peroxidation, associated with each insult. Mitochondrial function was compromised by the excitotoxic and metabolic insults, and was preserved in neurons treated with uric acid. Delayed elevations of intracellular free calcium levels induced by glutamate and cyanide were significantly attenuated in neurons treated with uric acid. These data demonstrate a neuroprotective action of uric acid that involves suppression of oxyradical accumulation, stabilization of calcium homeostasis, and preservation of mitochondrial function. Administration of uric acid to adult rats either 24 hr prior to middle cerebral artery occlusion (62.5 mg uric acid/kg, intraperitoneally) or 1 hr following reperfusion (16 mg uric acid/kg, intravenously) resulted in a highly significant reduction in ischemic damage to cerebral cortex and striatum, and improved behavioral outcome. These findings support a central role for oxyradicals in excitotoxic and ischemic neuronal injury, and suggest a potential therapeutic use for uric acid in ischemic stroke and related neurodegenerative conditions. J. Neurosci. Res. 53:613–625, 1998.

Ischemic and excitotoxic brain injury is enhanced in mice lacking the p55 tumor necrosis factor receptor.

Ischemic and excitotoxic insults to the brain induce rapid production of tumor necrosis factor-α (TNF), but the role of TNF in neuronal responses to brain injury are unclear. Two different TNF receptors (p55 and p75) are expressed in neurons and glia, To understand the role of TNF in brain injury, we generated mice that lack p55, p75, or both receptors, We report that neuronal damage after focal cerebral ischemia—reperfusion is significantly increased in mice lacking p55 receptors (85 ± 7 mm3 infarct volume; mean ± SD) compared with wild-type mice (70 ± 8 mm3) and mice lacking p75 receptors (72 ± 6 mm3). Moreover, mice lacking p55 receptors exhibited increased degeneration of CA3 hippocampal neurons after administration of the excitotoxin kainic acid compared with wild-type mice and mice lacking p75 receptors. When taken together with recent data showing that TNF can prevent apoptosis of cultured neurons exposed to oxidative and metabolic insults, our findings suggest that TNF plays a neuroprotective role after acute brain insults.

Astrocytic Gap Junctional Communication Decreases Neuronal Vulnerability to Oxidative Stress-Induced Disruption of Ca2+ Homeostasis and Cell Death

Abstract: We investigated the effect of uncoupling astrocytic gap junctions on neuronal vulnerability to oxidative injury in embryonic rat hippocampal cell cultures. Mixed cultures (neurons growing on an astrocyte monolayer) treated with 18‐α‐glycyrrhetinic acid (GA), an uncoupler of gap junctions, showed markedly enhanced generation of intracellular peroxides (2,7‐dichlorofluorescein fluorescence), impairment of mitochondrial function [(dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction], and cell death (lactate dehydrogenase release) following exposure to oxidative insults (FeSO4 and 4‐hydroxynonenal). GA alone had little or no effect on basal levels of peroxides, mitochondrial function, or neuronal survival. Intercellular dye transfer analyses revealed extensive astrocyte‐astrocyte coupling but no astrocyte‐neuron or neuron‐neuron coupling in the mixed cultures. Studies of pure astrocyte cultures and microscope analyses of neurons in mixed cultures showed that the increased oxidative stress and cell death in GA‐treated cultures occurred only in neurons and not in astrocytes. Antioxidants (propyl gallate and glutathione) blocked the death of neurons exposed to FeSO4/GA. Elevations of neuronal intracellular calcium levels ([Ca2+]i) induced by FeSO4 were enhanced in neurons in mixed cultures exposed to GA. Removal of extracellular Ca2+ and the L‐type Ca2+ channel blocker nimodipine prevented impairment of mitochondrial function and cell death induced by FeSO4 and GA, whereas glutamate receptor antagonists were ineffective. Finally, GA exacerbated kainate‐ and FeSO4‐induced injury to pyramidal neurons in organotypic hippocampal slice cultures. The data suggest that interastrocytic gap junctional communication decreases neuronal vulnerability to oxidative injury by a mechanism involving stabilization of cellular calcium homeostasis and dissipation of oxidative stress.

Bcl-2 Protects Isolated Plasma and Mitochondrial Membranes Against Lipid Peroxidation Induced by Hydrogen Peroxide and Amyloid β-Peptide

Abstract: The bcl‐2 protooncogene product possesses antiapoptotic properties in neuronal and nonneuronal cells. Recent data suggest that Bcl‐2s potency as a survival factor hinges on its ability to suppress oxidative stress, but neither the subcellular site(s) nor the mechanism of its action is known. In this report electron paramagnetic resonance (EPR) spectroscopy analyses were used to investigate the local effects of Bcl‐2 on membrane lipid peroxidation. Using hydrogen peroxide (H2O2) and amyloid β‐peptide (Aβ) as lipoperoxidation initiators, we determined the loss of EPR‐detectable paramagnetism of nitroxyl stearate (NS) spin labels 5‐NS and 12‐NS. In intact cell preparations and postnuclear membrane fractions, Aβ and H2O2 induced significant loss of 5‐NS and 12‐NS signal amplitude in control PC12 cells, but not PC12 cells expressing Bcl‐2. Cells were subjected to differential subcellular fractionation, yielding preparations of plasma membrane and mitochondria. In preparations derived from Bcl‐2‐expressing cells, both fractions contained Bcl‐2 protein. 5‐NS and 12‐NS signals were significantly decreased following Aβ and H2O2 exposure in control PC12 mitochondrial membranes, and Bcl‐2 largely prevented these effects. Plasma membrane preparations containing Bcl‐2 were also resistant to radical‐induced loss of spin label. Collectively, our data suggest that Bcl‐2 is localized to mitochondrial and plasma membranes where it can act locally to suppress oxidative damage induced by Aβ and H2O2, further highlighting the important role of lipid peroxidation in apoptosis.

2-DEOXY-D-GLUCOSE PROTECTS HIPPOCAMPAL NEURONS AGAINST EXCITOTOXIC AND OXIDATIVE INJURY : EVIDENCE FOR THE INVOLVEMENT OF STRESS PROTEINS

Food restriction can extend life span in rodents and was recently reported to increase the resistance of neurons in the brain to excitotoxic and metabolic insults. In principle, administration to ad libitum fed rodents of an agent that reduces glucose availability to cells should mimick certain aspects of food restriction. We now report that administration of 2‐deoxy‐d‐glucose (2DG), a non‐metabolizable analog of glucose, to adult rats results in a highly significant reduction in seizure‐induced spatial memory deficits and hippocampal neuron loss. Pretreatment of rat hippocampal cell cultures with 2DG decreases the vulnerability of neurons to excitotoxic (glutamate) and oxidative (Fe2+) insults. The protective action of 2DG is associated with decreased levels of cellular oxidative stress and enhanced calcium homeostasis. 2DG treatment increased levels of the stress‐responsive proteins GRP78 and HSP70 in hippocampal neurons, without affecting levels of Bcl‐2 or GRP75, suggesting that mild reductions in glucose availability can increase neuronal resistance to oxidative and metabolic insults by a mechanism involving induction of stress proteins. Our findings establish cell culture and in vivo models of “chemical food restriction” which may prove useful in elucidating mechanisms of neuroprotection and in developing preventive approaches for neurodegenerative disorders that involve oxidative stress and excitotoxicity. J. Neurosci. Res. 57:48–61, 1999.

Microglial–neuronal interactions in synaptic damage and recovery

An understanding of the role of microglial cells in synaptic signaling is still elusive, but the neuron–microglia relationship may have important ramifications for brain plasticity and injury. This review summarizes current knowledge and theories concerning microglial–neuronal signaling, both in terms of neuron‐to‐microglia signals that cause activation and microglia‐to‐neuron signals that affect neuronal response to injury. Microglial activation in the brain involves a stereotypical pattern of changes including proliferation and migration to sites of neuronal activity or injury, increased or de novo expression of immunomodulators including cytokines and growth factors, and the full transformation into brain‐resident phagocytes capable of clearing damaged cells and debris. The factors released from neurons that elicit such phenotypical and functional alterations are not well known but may include cytokines, oxidized lipids, and/or neurotransmitters. Once activated, microglia can promote neuronal injury through the release of low‐molecular‐weight neurotoxins and support neuronal recovery through the release of growth factors and the isolation/removal of damaged neurons and myelin debris. Because microglia respond quickly to neuronal damage and have robust effects on neurons, astrocytes, and oligodendrocytes, microglial cells could play potentially key roles in orchestrating the multicell cascade that follows synaptic plasticity and damage. J. Neurosci. Res. 58:191–201, 1999.

Effects of high fat diet on Morris maze performance, oxidative stress, and inflammation in rats: Contributions of maternal diet

This study was undertaken to investigate the effects of prenatal and postnatal exposure to high fat diet on the brain. Female rats were divided into high fat diet (HFD) and control diet (CD) groups 4 weeks prior to breeding and throughout gestation and lactation. After weaning, male progeny were placed on a chow diet until 8 weeks old, and then segregated into HFD or CD groups. At 20 weeks old, rats were evaluated in the Morris water maze, and markers of oxidative stress and inflammation were documented in the brain. In comparison to rats fed CD, cognitive decline in HFD progeny from HFD dams manifested as a decline in retention, but not acquisition, in the water maze. HFD was also associated with significant increases in 3-nitrotyrosine, inducible nitric oxide synthase, IL-6, and glial markers Iba-1 and GFAP, with the largest increases frequently observed in HFD animals born to HFD dams. Thus, these data collectively suggest that HFD increases oxidative and inflammatory signaling in the brain, and further indicate that maternal HFD consumption might sensitize offspring to the detrimental effects of HFD.