EXCITOTOXIC NMR ENERGY FAILURE IN ISCHEMIC BRAIN SLICES

谷氨酸(Glu)毒性是一种神经细胞损伤的原因 中风是一个主要的健康问题，是导致死亡的第三大原因 也是导致成人残疾的最常见原因。此应用程序建议 大鼠大脑皮层呼吸和呼吸功能的机制研究 神经元/神经胶质细胞能量衰竭和损伤的海马片 外源性谷氨酸毒性和缺血。在每个实验中，时间进程 在20个成年大鼠脑片上监测核磁共振代谢产物的变化 (每亩总湿重=3.2克)。P.I.最近的核磁共振研究。 发现在谷氨酸之后很快就会发生细胞内能量衰竭 曝光。然而，如果停用外源Glu，它可以得到改善 或给予谷氨酸受体拮抗剂。细胞内能量衰竭和 用交错P/H/F核磁共振波谱对钙离子进行非侵入性评估 用于同时测定：ATP、PC、Pi Phi、Mg2+、Ca~(2+)、N- 乙酰天冬氨酸(NAA)和乳酸。细胞外的谷氨酸水平是 通过使用尖端在切片之间的导管来确定的。 组织损伤是通过湿/干测定水分含量来测量的。 热染色切片的重量和光学显微镜 休克蛋白(HSP72)。神经细胞和神经胶质细胞的组织学检查 也使用甲酚紫[Nissl]染色，以及 GFAP和神经元特异性烯醇化酶的免疫细胞化学染色。不是 用放射免疫法测定细胞活性和cGMP水平。五 特定目标统一聚焦于Glu中的三个分子事件 毒性：1)激活谷氨酸受体；2)激活一氧化氮 合酶(NOS)；3)细胞内能量衰竭。我们的目标是将 谷氨酸受体和一氧化氮合酶的缺血型谷氨酸能能量衰竭 激活。目的1)能源故障在非 缺血型谷氨酸毒性和缺血。假设：(A)不可追回 细胞内能量衰竭与NAA减少、增加相关 乳酸、Ca~(2+)和细胞外Glu，神经元损伤加重。 (B)海马神经元比大脑皮层更容易受到损伤 神经元。目的2)地佐西平和NBQX对谷氨酸诱导的小鼠脑缺血再灌注损伤的影响。 缺血引起的能量衰竭。假设：(A)封锁两个NMDA AMPA受体可以减少细胞内钙离子和细胞外Glu的增加， 减少能量衰竭的核磁共振表现，并与减少 神经元损伤。目的3)两种阻断剂NOLA对一氧化氮合酶的抑制作用 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受体激活后能量衰竭 涉及到钙离子的增加和神经元型一氧化氮合酶的激活 钙/钙调蛋白依赖酶。目标4)寻求以下方面的协同效应 NO-阻断和拮抗NMDA型或AMPA型受体。 假设：协同效应是存在的。目的5)果糖-1，6或AMPA的作用 类型受体。假设：FBP通过以下方式保护神经胶质细胞的能量水平 糖酵解激活，减少神经元损伤，并与 细胞外Glu减少，可能是由于胶质细胞摄取增加所致。

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.