CEREBRA K&WL

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。体内功能磁共振成像和磁共振波谱的发展为评估功能激活的代谢动力学提供了重要的新途径。本应用旨在评估人类高血糖和低血糖时功能激活的病理生理学，并检验酮类药物的作用。重要的是，尽管这一应用强调了对T1DM患者低血糖中常见的认知功能障碍的可能的“治疗”方法，但我们相信这项工作也可能为大脑如何使用激活的燃料（葡萄糖，酮类）提供洞见，这是一个仍在争论的话题。我们特别考虑到RFA NS 02-008（“本RFA征求研究申请，旨在阐明低血糖对神经胶质细胞和神经元细胞的影响……定义不同血糖水平对脑代谢的影响[和]低血糖损伤的病理后果”），因为我们认为，与glia17,18相比，酮优先被神经元氧化，脑低血糖的问题部分与大脑如何能够（或不能够）吸收有关激活备用燃料。本研究研究了正常血糖和低血糖以及有酮时灌注、BOLD激活和乳酸生成的动态变化。我们将使用功能性核磁共振成像、磁共振光谱和脑生理学模型来测试在低血糖期间提供替代燃料选择时大脑的反应。关于功能性活动的代谢需要仍有相当大的争论。虽然在人脑休息时，氧和葡萄糖的使用被认为是耦合的，但功能激活会引起葡萄糖使用的增加（通过氟脱氧葡萄糖测量），相对于氧使用的变化，葡萄糖使用的增加不成比例地高然而，测量大脑燃料使用的方法可能有所贡献。Collins等20通过对受刺激大鼠脑进行定量放射自显影，比较了[14C]脱氧葡萄糖（DG）和[6-14C]葡萄糖的使用，发现DG法的摄食量增加了~80%，而葡萄糖法的摄食量增加了~30%。这种差异，也被其他工作者看到，21已经被解释为激活诱导的葡萄糖使用（糖酵解，硬化）与葡萄糖氧化的差异。然而，随着神经生理学数据显示广泛的星形细胞网络（例如，将生物细胞素微量注射到星形细胞中显示迅速扩散到50-100多个其他细胞，8,9），提出了另一种解释。虽然激活诱导的[6-14C]葡萄糖的扩散是可能的，但磷酸化的-[14C]DG可能无法重新分布。因此，在一项DG研究中，可能通过这种网络正常分布的功能性激发的葡萄糖使用似乎受到空间限制。从这个角度来看，葡萄糖的使用实际上可能与氧气消耗有关，这与更普遍的假设（即，大脑激活主要是糖酵解作用）不同。这对低血糖的相关性被强调，因为“耦合”功能的观点表明，替代燃料可以很容易地支持功能活动。Fox和Raichle观点的重要支持数据是观察到在功能刺激下组织乳酸增加~50-250%，22-24解释为无氧糖酵解燃料激活的结果。然而，乳酸的增加主要意味着产生和清除之间的短暂不匹配，并且可以在专性糖酵解，增加的过敏或氧化中看到。如果葡萄糖的命运是氧化，那么酮类在低血糖中的作用就变得更有可能。实验上，在低血糖中，酮的可用性可能潜在地强烈影响活性诱导的乳酸和激活区域的动态，这可以使用磁共振波谱和功能磁共振成像（fMRI）进行研究。因此，在目的1中，我们将检验灌注动态、BOLD fMRI和乳酸水平受到低血糖显著影响的假设。在正常受试者中，我们将检查底物有效性改变的三种情况：正常血糖（EG，基线），轻度胰岛素诱导的低血糖（IHG，达到60mg/dl或3.45mM的血浆水平）和空腹诱导的低血糖（FHG）。我们预计，与EG状态相比，IHG状态下的灌注增加程度较小，FHG状态下的灌注增加程度最小，反映了IHG状态下葡萄糖不足。同样，我们预计在IHG状态下，BOLD激活和乳酸生成将会减少。相反，我们预计fMRI检测到的激活区域在基线（非禁食）和FHG状态下相似，而在IHG条件下则较少，反映了总氧化燃料（包括葡萄糖和酮类）的可用性。重要的是，如果FHG条件导致的乳酸含量等于或高于EG和IHG条件，这表明糖酵解是功能激活所必需的。基本激活的范例将是一个视觉上交替的放射状棋盘。在目标2中，我们将更严格地测试酮对对照和患者受试者激活的贡献。我们将首先获得低血糖数据，然后注射活化酮。在对照组和1型糖尿病患者中，我们将验证假设，即在低血糖时，酮输注可导致核磁共振检测（灌注、BOLD和乳酸）测量的变化与神经活动的改善相一致。患者将在eeg中进行研究，获取基线和功能激活下的脑血流量和乳酸的测量。IHG将在有/没有功能活动的情况下进行重复MR测量。我们预计IHG状态下的血流和乳酸动力学与EG状态下不同，与目标1相似。在第二项评估患者和对照受试者的研究中，将首先诱导IHG，并在有/没有功能活动的情况下进行MR测量。然后我们将注入酮（IHK），并获得具有/不具有功能活性的MR数据。我们预计IHK研究将显示BOLD活性和灌注恢复到EG水平，同时显示较低的激活诱导的乳酸变化。我们还预计糖尿病患者可能对酮输注表现出更大的敏感性，这反映了他们对替代燃料的长期依赖。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Developments in in vivo functional MRI and MR spectroscopy provide important new avenues to evaluate the metabolic dynamics of functional activation. This application proposes to assess the pathophysiology of functional activation in euglycemia and hypoglycemia in humans, and to examine the effects of ketones. Importantly, although this application suggests an emphasis towards a possible "therapeutic" approach towards the cognitive dysfunction often seen in hypoglycemia in T1DM patients, we believe this work may also provide insight to how the brain uses fuels (glucose, ketones) with activation, a topic that continues to be in debate. We pursue this issue especially given the RFA NS 02-008 ("This RFA solicits applications for studies designed to elucidate the effects of hypoglycemia on glial and neuronal cells...define the effect of varying glycemic levels on cerebral metabolism [and] pathological consequences of¿hypoglycemic insult") because we believe that ketones are preferentially oxidized by neurons compared to glia17,18 and the problem of cerebral hypoglycemia relates in part to how the brain is able (or not) to draw on alternate fuels in activation. This proposal examines the dynamics of perfusion, BOLD activation and lactate generation during euglycemia and hypoglycemia, and then when ketones are available. We will do this using functional MRI, MR spectroscopy and models of cerebral physiology to test how the brain responds when provided the option of alternate fuels during hypoglycemia. Considerable debate remains as to the metabolic needs of functional activity. While in resting human brain oxygen and glucose use are believed to be coupled, functional activation provokes an increase in glucose use (measured by fluorodeoxyglucose, FDG) that is disproportionately high relative to changes in oxygen use.3,19 However, it is possible that the methods used to measure cerebral fuel use may be contributing. Collins et al20 compared [14C]deoxyglucose (DG) and [6-14C]glucose use by quantitative autoradiography of stimulated rat brain, finding that the DG method showed a ~80% rise in uptake while the glucose method showed a ~30% increase. This difference, also seen by other workers,21 has been explained as activation-induced differences in glucose use (glycolysis, anaplerosis) vs. glucose oxidation. However, with neurophysiologic data showing extensive astrocytic networks (e.g., microinjection of biocytin into an astrocyte demonstrates rapid spread into 50-100+ other cells, 8,9), another explanation is raised. While activation-induced spread of [6-14C]glucose is possible, phosphorylated-[14C]DG may not be able to redistribute. Therefore functionally provoked glucose use, which may normally distribute through such a network, would-in a DG study-appear to be spatially restricted. From this view, glucose use may in fact correlate with oxygen consumption, distinct from the more prevalent hypothesis (i.e., brain activation is mostly glycolytic). The pertinence of this for hypoglycemia is emphasized, since the view of "coupled" function would suggest that alternate fuels could readily support functional activity. Important supporting data for the Fox and Raichle view is the observation of a ~50-250% increase in tissue lactate with functional stimulation,22-24 interpreted to result from anaerobic glycolysis fueling activation. However, such an increase in lactate primarily means a transient mismatch between production and clearance, and may be seen either in obligate glycolysis, increased anaplerosis or oxidation. If the fate of glucose is oxidation, then a role for ketones in hypoglycemia becomes a much stronger possibility. Experimentally, the availability of ketones in hypoglycemia may potentially strongly influence the dynamics of activity-induced lactate and regions of activation-which can be studied using MR spectroscopy and functional MRI (fMRI). Thus in aim 1, we will test the hypothesis that the dynamics of perfusion, BOLD fMRI and lactate are significantly influenced by hypoglycemia. In normal subjects, we will examine three conditions of altered substrate availability: euglycemia (EG, baseline), mild insulin-induced hypoglycemia (IHG, reaching a plasma level of 60mg/dl or 3.45mM) and fasting-induced hypoglycemia (FHG). We anticipate that compared to the EG state, the extent of perfusion increase will be less in the IHG state, and the least in the FHG state, reflecting the insufficiency of glucose in the IHG state. Similarly, we anticipate that BOLD activation and lactate generation will be less in the IHG states. In contrast, we anticipate that the fMRI detected areas of activation will be similar in the baseline (non-fasted) and FHG state while it is less under IHG conditions, reflecting the availability of total oxidative fuel (includes glucose and ketones). Importantly, if the FHG condition results in equal or greater lactate than the EG and IHG condition, this suggests that glycolysis is necessary for functional activation. The paradigm of elementary activation will be a visual alternating radial checkerboard. In aim 2, we will more stringently test the hypothesis of ketone contributions towards activation in control and patient subjects. We will do this by initially acquiring hypoglycemic data, followed by infusions of ketones with activation. In both control and type 1 diabetic patients, we will test the hypothesis that in hypoglycemia, ketone infusions can result in changes in MR-detected (perfusion, BOLD and lactate) measures consistent with improved neural activity. Patient subjects will be studied in the EG, acquiring measurements of cerebral blood flow and lactate under baseline and functional activation. IHG will be induced followed by repeat MR measurements with/without functional activity. We anticipate that the dynamics of flow and lactate will be different in the IHG state than under EG, similar to that in aim 1. In a second study evaluating both patients and control subjects, IHG will be induced initially, and MR measurements acquired with/without functional activity. We will then infuse ketones (IHK), and acquire MR data with/without functional activity. We anticipate that the IHK studies will display a return to EG levels of BOLD activity and perfusion, while displaying a lower activation induced lactate change. We anticipate also that the diabetic patients may demonstrate greater sensitivity to the ketone infusion, reflecting a greater chronic reliance on alternate fuels for brain function.