EXCITOTOXIC NMR ENERGY FAILURE IN ISCHEMIC BRAIN SLICES

谷氨酸（Glu）毒性是一种神经元损伤， 中风，一个主要的健康问题，是第三大死亡原因 也是导致成人残疾的最常见原因 本申请提出 在大鼠呼吸皮质和 海马脑片神经元/胶质细胞能量衰竭和损伤 外源性Glu毒性和局部缺血。 在每个实验中， 在20个成年大鼠脑切片中监测NMR代谢物的变化 (each 350亩总湿重=3.2克）。 P.I.最近的P NMR研究。 发现细胞内能量衰竭在glu exposure. 但如果停止外源性glu，则可改善 或Glu受体拮抗剂。 细胞内能量衰竭， 使用交错P/H/F NMR光谱法非侵入性地评估Ca 2 + 用于同时测定：ATP、PC、Pi pHi、Mg 2+、Ca 2+、N- 乙酰天冬氨酸（NAA）和乳酸。 细胞外葡萄糖水平是 通过使用其尖端位于切片之间的导管来确定。 通过从湿/干组织中测定水含量来测量组织损伤。 染色切片的重量和光学显微镜检查以表达热量 休克蛋白（HSP 72）。 神经元和神经胶质的组织学检查是 也使用甲酚紫[Nissl]染色进行，和 GFAP和神经元特异性烯醇化酶免疫细胞化学染色。 没有 活性和cGMP水平用放射免疫测定法测定。 五 具体的目标集中在统一的方式在三个分子事件的葡萄糖 毒性：1）激活Glu受体; 2）激活一氧化氮 合成酶（NOS）; 3）细胞内能量衰竭。 我们的目标是将 Glu受体和NOS的缺血性和缺血性能量衰竭 activation. 目的1）在非核武器期间和之后能源失效的意义 缺血性葡萄糖毒性和局部缺血。 假设：（a）无法收回 细胞内能量衰竭与NAA减少、NAA增加、 乳酸、Ca 2+和细胞外葡萄糖，并增加神经元损伤。 (b)海马神经元比皮层神经元更容易受到损伤 神经元 目的2）地佐环平和NBQX对谷氨酸诱导的 缺血引起的能量衰竭。 假设：（a）阻断两种NMDA AMPA受体将减少Ca 2+和细胞外葡萄糖的增加， 减少能量衰竭的NMR表现，并与减少的 神经元损伤 目的3）NOLA对NOS抑制作用的影响 和L-NMMA对谷氨酸诱导的和缺血诱导的能量衰竭的影响。 假设：NMDA和AMPA受体激活后的能量衰竭 包括Ca 2+增加和NOS的激活，NOS是一种神经元 Ca 2 +/钙调素依赖酶。 目的4）寻找协同作用， NOS-阻断和NMDA型或AMPA型受体的拮抗作用。 假设：存在协同作用。 目的5）果糖-1，6或AMPA- 型受体。 假设：FBP通过以下方式保护神经胶质细胞中的能量水平： 糖酵解激活，减少神经元损伤，并与 细胞外葡萄糖减少，可能是因为神经胶质细胞摄取增加。

Glutamate (glu) toxicity accounts for one kind of neuronal injury from stroke, a major health problem that is the third leading cause of death and the most common cause of adult disability. This application proposes mechanism-oriented studies in respiring rat cerebrocortical and hippocampal slices of neuronal/glial energy failure and injury during exogenous glu toxicity and ischemia. In each experiment the time course of NMR metabolite changes are monitored in 20 adult rat brain slices (each 350 mu total wet wgt=3.2 gm). Recent P NMR studies by the P.I. found that intracellular energy failure occurs very soon after glu exposure. However, it can be ameliorated if exogenous glu is discontinue or glu receptor antagonists are give. Intracellular energy failure and Ca2+ are assessed noninvasively using interleaved P/H/F NMR spectroscopy for concurrent determinations of: ATP, PC, Pi pHi, Mg2+, Ca2+, N- acetylaspartate (NAA), and lactate. Extracellular levels of glu are determined from the use of a catheter whose tip is among the slices. Tissue injury is measured by water content determinations from wet/dry weights and light microscopy of sections stained for expression of heat shock protein (HSP72). Histological examinations of neurons and glia are also performed using Cresyl violet [Nissl] staining, and immunocytochemical stains for GFAP, and neuron-specific enolase. NO activity and cGMP levels ar measured using radioimmunoassay. Five specific aims focus in a unified way on three molecular events in glu toxicity: 1) activation of glu receptors; 2) activation of nitric oxide synthase (NOS); 3) intracellular energy failure. The goal is to link ischemic and glutamatergic energy failure to glu receptor and NOS activation. Aim 1) significance of energy failure during and after non- ischemic glu toxicity and ischemia. Hypotheses: (a) nonrecoverable intracellular energy failure correlates with decreased NAA, increased lactate, Ca2+, and extracellular glu, and with increased neuronal injury. (b) Hippocampal neurons are more susceptible to injury than cortical neurons. Aim 2) effects of dizocilpine and NBQX on glutamate-induced and ischemia-induced energy failure. Hypotheses: (a) Blockade of both NMDA and AMPA receptors will reduce increases in Ca2+ and extracellular glu, reduce NMR manifestations of energy failure, and correlate with decreased neuronal injury. Aim 3) effects of NOS inhibition by two blockers, NOLA and L-NMMA, on glutamate-induced and ischemia-induced energy failure. Hypothesis: Energy failure following NMDA and AMPA receptor activation involves both Ca2+ increases and activation of NOS, a neuronal Ca2+/calmodulin dependent enzyme. Aim 4) search for synergism between NOS-blockade and antagonism of NMDA-type or AMPA-type receptors. Hypothesis: synergism exists. Aim 5) effects of fructose-1,6 or AMPA- type receptors. Hypotheses: FBP protects energy levels in glia by glycolytic activation, reduces neuronal injury, and is associated with decreased extracellular glu, possibly because of increased glial uptake.