Investigation of the Modulators of Cerebrovascular Coupling

脑血管耦合调节剂的研究

• 批准号: 7969615
• 负责人: Afonso Silva
• 金额: $ 111.91万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7969615
• 关键词: 7-nitroindazole; Address; Affect; Afferent Pathways; Agreement; Apical; Arachidonic Acids; Architecture; Arginine; Astrocytes; Attenuated; Blood Vessels; Blood capillaries; Blood flow; Bolus Infusion; Brain; Brain region; Cells; Cerebrovascular Circulation; Cerebrum; Characteristics; Chloralose; Citrulline; Cleaved cell; Complex; Coupling; Dendrites; Dependence; Dinoprostone; Disease; Drops; Dyes; Energy Supply; Enzymes; Erythrocytes; Excision; Exhibits; Functional Magnetic Resonance Imaging; Functional disorder; Goals; Hand; Healthcare; Homeostasis; Human; Hyperemia; Immunohistochemistry; Individual; Interneurons; Investigation; Label; Laser Scanning Microscopy; Left; Measurement; Measures; Mediating; Mediator of activation protein; Metabolism; Modeling; Molecular; N-Methyl-D-Aspartate Receptors; Neocortex; Neuroglia; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Organ; PTGS2 gene; Pathway interactions; Perfusion; Physiological; Plasma; Process; Production; Prostaglandins; Protein Isoforms; Rattus; Reaction; Regulation; Relative (related person); Reporting; Research; Rest; Rodent Model; Role; Sensory; Signal Pathway; Signal Transduction; Signaling Molecule; Somatosensory Evoked Potentials; Specificity; Synapses; Techniques; Testing; Thalamic structure; Time; Translating; Variant; Vascular blood supply; Vasodilation; Vasodilator Agents; Veins; Work; attenuation; base; blood oxygenation level dependent response; capillary; cerebrovascular; discs, large (Drosophila) homolog 2 protein, rat; excitatory neuron; glucose metabolism; hemodynamics; hippocampal pyramidal neuron; human NOS3 protein; in vivo; inhibitor/antagonist; interest; meloxicam; neurochemistry; neurovascular unit; partial recovery; postsynaptic density protein; relating to nervous system; response; somatosensory; treatment response; trend; two-photon; wasting

We are interested in understanding the relative contribution, and the spatial/cellular specificity of the different neurosignaling pathways to the HDR elicited by functional brain stimulation. The working hypothesis is that increases in CBF are mediated by substances released by the parenchyma that act on the cerebral vessels and regulate their tone. The outstanding questions are: (i) which substances are released by what cells and under which circumstances? (ii) What is the spatial specificity of with respect to the cortical architecture? (a) The Role of COX-2: COX-2 is an enzyme involved in the conversion of arachidonic acid into substances generally known as prostanoids. In the brain, COX-2 is constitutively expressed and represents the primary isoform under physiological conditions. Involvement of COX-2 products in the neurovascular coupling has long been investigated and recent reports have shown that selective inhibition of COX-2 produces a 50% drop in the CBF response to somatosensory stimulation without affecting baseline CBF levels. However, these studies did not directly measure the neuronal activity following COX-2 inhibition, nor did they administer COX-2 pathway products, leaving open the questions (i) whether the observed attenuation in the CBF response resulted from a decrease in electrical activity or from a disruption of the cerebrovascular coupling; and (ii) whether this effect was reversible. We then hypothesized that: (i) COX-2 inhibition would produce a decrease in the hemodynamic response to functional brain stimulation without concomitant decreases in the neuronal response, thereby directly demonstrating the involvement of COX-2 in cerebrovascular coupling; and (ii) that this effect could be reversed by a systemic administration of a COX-2 derived vasodilatory product of arachidonic acid metabolism, prostaglandin E2 (PGE2). We tested these hypotheses in the alpha-chloralose anesthetized rat model of somatosensory stimulation. CBF and BOLD fMRI responses were measured before and during COX-2 inhibition with Meloxicam (MEL), a preferential COX-2 inhibitor and following a bolus of the major vasodilatory product of the COX-2 pathway, PGE2. The MEL treatment attenuated COX enzymatic activity by 57%, and imposed a progressive attenuation of the CBF response to somatosensory stimulation to 32% of the original pre-treatment response. We did not observe any effects of COX-2 inhibition on somatosensory evoked potentials (SEP), confirming the specific involvement of COX-2 in cerebrovascular coupling. When PGE2 was administered following COX-2 inhibition, we obtained a partial recovery of the CBF response to 52% of the original response, indicating a permissive role of PGE2 in cerebrovascular coupling. Similar results were obtained for the BOLD contrast. We observed a cortical laminar dependence to the CBF and BOLD responses in all pharmacological conditions. In agreement with earlier work, the CBF response prior to pharmacological perturbations was highest in layer IV. Continued administration of MEL caused a gradual attenuation of the CBF response across all layers. The maximum attenuation occurred in layer IV (P < 0.001), essentially flattening the laminar profile of the CBF response. Administration of PGE2 restored the original laminar profile, indicating a preferential sensitivity of the HDR to COX-2 activity in layer IV. On the other hand, the BOLD laminar profile exhibited the maximum change in layer 1 most likely due to the effect of draining veins. While the BOLD profile was maintained throughout the pharmacological manipulation, there was also a trend of layer IV to be more affected than the other layers. The alteration of the cortical CBF profile by COX-2 manipulation has important implications to understanding cerebrovascular coupling and, in particular, to our research aims and goals. Recent immunohistochemistry studies of COX-2 distribution in rat neocortex have found that COX-2 was predominantly expressed in apical dendritic processes of pyramidal neurons in layers II-III and V, as well as in spiny dendrites and axonal terminals of excitatory neurons in layer IV, the latter receiving direct input from the thalamus via the lemniscal pathway. In further support of recent postulates of afferent driven blood flow control, the COX-2-mediated release of PGE2 may thus underlie an important thalamus-driven mechanism of local vasodilatation, providing a way for the microvasculature to follow the layer-specific flow of electrical activity in the cortex. It will be interesting to compare in further detail the cortical expression of COX-2 with the laminar profiles of the CBF response to increased neural activity. (b) The Role of NO: Another major neurosignalling pathway implicated in cerebrovascular coupling is the one that leads to production of nitric oxide (NO). In fact, due to its major function as a potent vasodilator, NO has been frequently interrogated as the most prominent signaling molecule in cerebrovascular coupling. Nitric oxide is synthesized endogenously by nitric oxide synthase (NOS), an enzyme that catalyzes the conversion of L-arginine to L-citrulline and NO. Among the different NOS isoforms, the neuronal NOS (nNOS) has been found in a range of different brain regions, with nNOS rich axonal terminals frequently impinging on cerebral microvessels. Furthermore, nNOS cleaves NO from L-arginine in a Ca2+-dependent reaction and is physically anchored to the Ca2+-permeable NMDA-receptor (NMDA-R) by two postsynaptic density proteins, PSD-93 and PSD-95, and increased NO production after synaptic activity has been reported. Together, these characteristics make NO a prime candidate for a molecular messenger in the direct or neurogenic regulation of focal cerebral blood flow (CBF) following increases in the local neuronal activity. Previous studies in anesthetized rodent models reported significant, yet varying degrees of attenuation (by 30% to 90%) of the CBF response to somatosensory stimulation after NO inhibition. The wide range of attenuation reported likely resulted from differences in the pharmacological paradigms employed and the relative degrees of neuronal versus endothelial NOS inhibition achieved in addition to varying sensitivities of the different techniques for CBF estimation. At the same time, the effect of NOS inhibition on neuronal activity and thus neurovascular coupling remains unclear: some studies reported no concomitant changes in cerebral glucose metabolism or SEP, while others observed a 40% to 60% decrease in SEP amplitudes after NOS inhibition. To investigate the effect of the inhibition of neuronally derived NO on the spatial and temporal profiles of hemodynamic and neuronal responses to functional brain stimulation, using fMRI and SEP measurements in alpha-chloralose anesthetized rats before and after administration of a bolus of 7-nitroindazole (7-NI), a potent in vivo inhibitor of neuronal nitric oxide synthase (nNOS). We reported that administration of 7-NI caused a significant attenuation of the CBF, BOLD, and CBV responses to stimulation in the absence of any statistically significant effect on the resting perfusion and that at all times, the variation of the baseline perfusion was within the standard error of the CBF estimate, below 10%. Treatment of the rats with 7-NI caused complete suppression of the CBF response to somatosensory stimulation, the average across subject CBF response was 53% before treatment and it was nulled by 7-NI to -0.63%. Meanwhile, the average across subject BOLD response was reduced from 4.90% before 7-NI to 1.30% after 7-NI and the average across subject CBV response decreased from 41% before treatment to 4.60% after nNOS inhibition. SEP amplitudes were also affected, albeit to a lesser extent, by 7-NI administration.

我们有兴趣了解不同神经信号通路对功能性脑刺激引起的 HDR 的相对贡献和空间/细胞特异性。工作假设是，CBF 的增加是由实质释放的物质介导的，这些物质作用于脑血管并调节其张力。悬而未决的问题是：（i）什么细胞在什么情况下释放哪些物质？ (ii) 皮质结构的空间特异性是什么？ (a) COX-2 的作用：COX-2 是一种参与花生四烯酸转化为通常称为前列腺素的物质的酶。在大脑中，COX-2 是组成型表达的，代表生理条件下的主要亚型。 COX-2 产品在神经血管耦合中的作用早已被研究，最近的报告表明，选择性抑制 COX-2 可使 CBF 对体感刺激的反应下降 50%，而不影响基线 CBF 水平。然而，这些研究并没有直接测量 COX-2 抑制后的神经元活动，也没有施用 COX-2 通路产品，因此留下了以下问题：(i) 观察到的 CBF 反应衰减是否是由于电活动减少或脑血管耦合破坏所致； (ii) 这种影响是否可逆。然后我们假设：（i）抑制COX-2会降低对功能性脑刺激的血流动力学反应，但不会同时降低神经元反应，从而直接证明COX-2参与脑血管耦合； (ii) 这种效应可以通过全身施用花生四烯酸代谢的 COX-2 衍生的血管舒张产物前列腺素 E2 (PGE2) 来逆转。我们在α-氯醛糖麻醉的体感刺激大鼠模型中测试了这些假设。在用美洛昔康 (MEL)（一种优先的 COX-2 抑制剂）抑制 COX-2 之前和期间以及在推注 COX-2 途径的主要血管舒张产物 PGE2 后测量 CBF 和 BOLD fMRI 反应。 MEL 治疗使 COX 酶活性减弱 57%，并使 CBF 对体感刺激的反应逐渐减弱至原始治疗前反应的 32%。我们没有观察到 COX-2 抑制对体感诱发电位 (SEP) 的任何影响，证实了 COX-2 在脑血管耦合中的特异性参与。当 COX-2 抑制后施用 PGE2 时，我们获得了 CBF 反应部分恢复至原始反应的 52%，表明 PGE2 在脑血管耦合中的许可作用。 BOLD 对比度也获得了类似的结果。 我们观察到在所有药理学条件下皮质层对 CBF 和 BOLD 反应的依赖性。与早期的工作一致，药理学扰动之前的 CBF 响应在第四层最高。持续施用 MEL 导致所有层的 CBF 反应逐渐减弱。最大衰减发生在 IV 层（P < 0.001），基本上使 CBF 响应的层流轮廓变平。 PGE2 的施用恢复了原始层流分布，表明 HDR 对 IV 层中的 COX-2 活性优先敏感。另一方面，BOLD 层流剖面在第 1 层表现出最大变化，很可能是由于引流静脉的影响。虽然在整个药理操作过程中保持了 BOLD 特征，但 IV 层也有比其他层受到更大影响的趋势。 COX-2 操作对皮质 CBF 分布的改变对于理解脑血管耦合，特别是对我们的研究目的和目标具有重要意义。最近对大鼠新皮质中 COX-2 分布的免疫组织化学研究发现，COX-2 主要表达于 II-III 层和 V 层锥体神经元的顶端树突，以及 IV 层兴奋性神经元的多刺树突和轴突末端，后者通过丘脑通路直接接收来自丘脑的输入。为了进一步支持最近传入驱动血流控制的假设，COX-2 介导的 PGE2 释放可能是重要的丘脑驱动的局部血管舒张机制的基础，为微脉管系统遵循皮层中特定层的电活动流动提供了一种方法。进一步详细比较 COX-2 的皮质表达与 CBF 对神经活动增加的反应的层状分布将会很有趣。 (b) NO 的作用：与脑血管耦合有关的另一种主要神经信号传导途径是导致一氧化氮 (NO) 产生的途径。事实上，由于 NO 的主要功能是有效的血管舒张剂，因此经常被质疑为脑血管耦合中最重要的信号分子。一氧化氮由一氧化氮合酶 (NOS) 内源合成，一氧化氮合酶是一种催化 L-精氨酸转化为 L-瓜氨酸和 NO 的酶。在不同的 NOS 亚型中，神经元 NOS (nNOS) 已在一系列不同的大脑区域中发现，富含 nNOS 的轴突末端经常撞击脑微血管。此外，nNOS 在 Ca2+ 依赖性反应中从 L-精氨酸中裂解 NO，并通过两种突触后密度蛋白 PSD-93 和 PSD-95 物理锚定在 Ca2+ 渗透性 NMDA 受体 (NMDA-R) 上，据报道，突触活动后 NO 产量增加。总之，这些特征使得 NO 成为局部神经元活动增加后局部脑血流 (CBF) 的直接或神经源性调节分子信使的主要候选者。先前对麻醉啮齿动物模型的研究报告称，NO 抑制后，CBF 对体感刺激的反应出现了显着但不同程度的减弱（30% 至 90%）。报告的大范围衰减可能是由于所采用的药理学范式的差异以及神经元与内皮 NOS 抑制的相对程度以及 CBF 估计的不同技术的不同灵敏度所致。与此同时，NOS 抑制对神经元活动以及神经血管耦合的影响仍不清楚：一些研究报告称，脑葡萄糖代谢或 SEP 没有伴随变化，而其他研究则观察到 NOS 抑制后 SEP 振幅下降 40% 至 60%。 为了研究神经源性 NO 的抑制对血流动力学和神经元对功能性脑刺激的反应的空间和时间分布的影响，在 α-氯醛糖麻醉的大鼠中使用 7-硝基吲唑 (7-NI)（一种有效的神经元一氧化氮体内抑制剂）推注前后的 fMRI 和 SEP 测量 合酶（nNOS）。我们报道，在对静息灌注没有任何统计学显着影响的情况下，给予 7-NI 会导致 CBF、BOLD 和 CBV 对刺激的反应显着减弱，并且基线灌注的变化始终在 CBF 估计的标准误差内，低于 10%。用7-NI治疗大鼠导致CBF对体感刺激的反应完全抑制，治疗前受试者CBF反应的平均值为53%，7-NI将其消除至-0.63%。同时，受试者的平均 BOLD 反应从 7-NI 前的 4.90% 降低至 7-NI 后的 1.30%，受试者的平均 CBV 反应从治疗前的 41% 降至 nNOS 抑制后的 4.60%。 SEP 振幅也受到 7-NI 给药的影响，尽管程度较小。