Regional Cerebral Circulation And Metabolism

这是一个综合性项目，涵盖了本实验室以前开发的方法的开发、验证、改进和应用的各种研究，以检查静息和功能激活时脑血流(CBF)和能量代谢调节的基本生化和生理机制。去年完成并发表的研究表明，存在于血管平滑肌中并能扩张血管的ATP敏感的K+离子通道不参与CBF的功能激活。今年还发表了一项研究，推翻了多巴胺受体参与脑血流功能激活的假设。今年完成并发表的研究证实，葡萄糖利用的途径在一定程度上在星形胶质细胞和神经元之间划分。星形胶质细胞将葡萄糖代谢成乳酸，并将其输出给神经元，神经元将乳酸氧化成二氧化碳和水。然而，这些研究表明，神经元可以容易地氧化葡萄糖和乳酸，但比起细胞内糖酵解产生的丙酮酸/乳酸，神经元更喜欢氧化来自细胞外间隙的乳酸。星形胶质细胞也可以将葡萄糖和乳酸氧化成二氧化碳，但它们这样做很少，而且它们主要将葡萄糖代谢成乳酸。星形胶质细胞的功能受限是由于丙酮酸脱氢酶(PDH)活性有限。这种酶主要以磷酸化的非活性形式存在于星形胶质细胞中，但它可以被二氯乙酸酯激活，我们发现二氯乙酸酯可以刺激星形胶质细胞氧化乳酸，减少其向神经元的乳酸输出。在大鼠体内给药时，大体行为没有明显变化，并导致大脑葡萄糖利用率增加，这可能是由于葡萄糖利用率增加，以弥补从星形胶质细胞输入乳酸的减少。这些研究的结论是，神经元和星形胶质细胞之间确实存在葡萄糖代谢的区域化，但既不是完全的，也不是强制性的，神经元葡萄糖和乳酸的相对氧化速率随细胞外空间乳酸浓度的不同而不同。对两种突变小鼠的研究仍在进行中，这些突变小鼠的α或β甲状腺受体基因发生了改变，使它们不能与L-三碘甲状腺原氨酸结合。部分研究结果已于今年发表。在β-甲状腺激素受体改变的小鼠中，大脑葡萄糖利用完全正常，但在α-受体功能障碍的小鼠中，大脑葡萄糖利用明显而广泛地受到抑制，就像出生时通过放射甲状腺切除而变成克汀病的动物一样。这些结果表明，β-甲状腺激素受体与正常大脑发育几乎没有关系，而且甲状腺激素对大脑发育的影响是通过α-甲状腺激素受体介导的。今年发表的对这些突变小鼠的其他研究表明，尽管大脑结构中的基线葡萄糖利用率显著下降，但神经功能激活引起的葡萄糖利用率增加的百分比相同，表明基线葡萄糖利用率下降是由于突触密度降低，但其余突触功能正常。最近完成并提交发表的研究还显示，甲状腺α受体功能障碍的小鼠心脏大小和心脏葡萄糖利用显着减少，而突变β受体小鼠的心脏大小略大，心脏葡萄糖利用大大增加。正在进行的研究正在检验这样一种假设，即大脑中腺苷水平的增加是导致自然睡眠状态的原因。有报道称，大脑中的腺苷水平在清醒状态下会逐渐升高，然后在睡眠状态下会下降到正常水平。此外，咖啡因是腺苷通道的一种有效的非特异性阻滞剂，众所周知，它会干扰自然睡眠。为了在实验中提高脑腺苷水平，我们一直在向大鼠体内注入抑制酶的药物，这些酶代谢通常在大脑中产生的腺苷。这两种药物分别是抑制腺苷脱氨酶的脱氧辅酶霉素和抑制腺苷激酶的碘结节菌素。当非肠道给药时，这些药物会显著降低动脉血压，这无疑是由于激活血管平滑肌上的腺苷受体而导致的全身血管扩张。为了避免全身性低血压的并发症，我们一直在脑池内给药。到目前为止，同时注入这两种药物可以产生类似睡眠的行为状态，以及类似于快速眼动和非快速眼动睡眠状态的脑电变化。这些研究将继续进行，以确保这些影响的可重复性，并将在接受这些药物治疗的大鼠中测量局部大脑葡萄糖利用，已知在非快速眼动睡眠中显著和广泛地降低。

This is an omnibus project that covers a variety of studies on development, validation, refinement, and applications of methods previously developed in this laboratory to examine basic biochemical and physiological mechanisms underlying the regulation of cerebral blood flow (CBF) and energy metabolism at rest and in response to functional activation. Studies completed and published last year showed that ATP-sensitive K+ ion channels,present in vascular smooth muscle and able to dilate blood vessels, do not contibute to functional activation of CBF. Also published this year was a study that disproved the hypothesis that dopamine receptors are involved in the functional activation of CBF. Studies completed and published this year confirmed that pathways of glucose utilization are to some extent compartmentalized between astroglia and neurons. The astroglia metabolize glucose to lactate and export it to neurons, and the neurons oxidize the lactate to CO2 and H2O. These studies showed, however, that neurons can readily oxidize both glucose and lactate but have a kinetic preference to oxidize lactate derived from the extracellular space over pyruvate/lactate produced intracellularly by glycolysis. Astroglia also can oxidize both glucose and lactate to CO2, but they do so sparingly, and they metabolize glucose mainly to lactate. The limited ability of the astroglia is due to limited pyruvic dehydrogenase (PDH) activity. This enzyme exists in astroglia predominantly in the phosphorylated inactive form, but it can be activated by dichloroacetate, which we found can stimulate astroglia to oxidize lactate and diminish their export of lactate to neurons. When administered in vivo to rats, it produced no obvious changes in gross behavior and led to increases in cerebral glucose utilization, presumably due to increased utilization of glucose to compensate for the diminished import of lactate from the astroglia. The conclusion from these studies is that the compartmentalization of glucose metabolism between neurons and astroglia does exist, but it is neither complete nor obligatory, and the relative rates of neuronal oxidation of glucose and lactate vary with the lactate concentration in the extracellular space. Studies on two strains of mutant mice with either the alpha or the beta thyroid receptor genetically altered so that they could not bind L-triiodothyronine are still in progress. Part of the results were published this year. Cerebral glucose utilization was found to be completely normal in mice with the altered beta thyroid hormone receptor, but markedly and diffusely depressed in mice with the dysfunctional alpha-receptor as in animals made cretinous by radiothyroidectomy at birth. These results indicate that the beta thyroid hormone receptor has little if anything to do with normal brain development and that the effects of thyroid hormone on brain development are mediated by the alpha thyroid hormone receptor. Other studies on these mutant mice that have been published this year showed that although the baseline glucose utilization in cerebral structures is markedly reduced, the percent increases in their rates of glucose utilization evoked by neuronal functional activation reamin the same, indicating that the decreased baseline glucose utilization is due to diminished synaptic density but the remaining synapses are functionally normal. Studies recently completed and submitted for publication also showed marked reductions in heart size and cardiac glucose utilization in the mice with the dysfunctional thyroid alpha-receptors whereas heart size was slightly greater and cardiac glucose utilization were enormously increased in the mice with the mutant beta-receptor. Studies are in progress to examine the hypothesis that increased adenosine levels in brain are responsible for the induction of natural sleep states. There have been reports that brain adenosine levels progressively rise during wakefulness and then decline back to normal levels during sleep. Also, caffeine, which is a potent non-specific blocker of adenosine channels, is known to interfere with natural sleep. In order to raise brain adenosine levels experimentally, we have been infusing into rats drugs that inhibit enzymes which metabolize the adenosine normally produced in brain. These drugs are deoxycoformycin, which inhibits adenosine deaminase, and iodotubericidin, which inhibits adenosine kinase. When administered parenterally, these drugs produce marked falls in arterial blood pressure, undoubtedly due to systemic vasodilatation resulting from activation of adenosine receptors on the vascular smooth muscle. To avoid the complications of this systemic hypotension, we have been administering the drugs intracisternally. Intracisternal infusions of both drugs simultaneusly have thus far been found to produce a sleep-like behavioral state as well as EEG changes similar to those seen in REM and non-REM sleep states. These studies will be continued to assure the reproducibility of these effects, and also local cerebral glucose utilization, which is known to be markedly and diffusely reduced in non-REM sleep, will be measured in rats treated with these drugs.