The role of neurotrophins in anesthesia-induced developmental neuroapoptosis

描述（由申请人提供）：这是FIRCA奖申请，用于支持主要在塞尔维亚和黑山进行的研究，该研究将在贝尔格莱德大学神经生物系分子神经生物学实验室进行，与弗吉尼亚大学麻醉系Vesna Jevtovic-Todorovic合作，作为NIH/NICHD父母资助#R01 HD 044517的延伸。早产儿、新生儿和非常年幼的儿童经常接受全身麻醉。然而，最近的一些研究结果表明，麻醉药物通过短暂地抑制哺乳动物大脑发育中的神经元活动，干扰正常的大脑发育（如突触发生），从而促进未成熟神经元的凋亡细胞死亡。麻醉诱导的未成熟大鼠的细胞凋亡性神经退行性变性不仅表现为发育中的大脑中严重的广泛的细胞凋亡性神经退行性（如神经细胞凋亡），而且表现为在生命后期检测到的显著且持续的学习/记忆缺陷。神经营养因子是一类生长因子，支持神经元的存活、分化和多种形式的突触可塑性，因此在哺乳动物大脑的突触发生中起着重要作用。因此，神经元活动的广泛抑制可以损害由神经营养因子调节的促进生存的信号。为了研究神经营养因子介导的麻醉诱导的发育性神经细胞凋亡的潜在相关性，我们对幼龄大鼠（在突触发生的高峰-7天大）进行了一项初步研究，这些大鼠暴露于以临床相关浓度（空气中1.5 vol%）吸入麻醉剂异氟烷中，持续2、4或6小时。我们发现异氟醚在两个最脆弱的大脑区域-大脑皮层和丘脑-调节神经营养因子调节的凋亡级联的关键步骤。也就是说，在皮层和丘脑中，阻断凋亡级联的关键成分活化Akt的蛋白水平均显著降低。然而，脑源性神经营养因子（BDNF）蛋白水平在皮层中被差异调节；在丘脑中，BDNF水平迅速而显著地下降。此外，虽然皮质活化的神经酰胺上调，但丘脑活化的神经酰胺没有变化：这些发现表明，异氟醚不仅激活akt调节的凋亡通路，而且在不同的大脑区域以不同的方式激活，最可能是通过激活Trk依赖性和Trk非依赖性，p75NTR依赖性神经营养存活通路。在本次拨款申请中，申请人计划通过对神经营养因子调节的麻醉诱导的神经细胞凋亡的机制进行详细的研究，来扩展在母体拨款中提出的麻醉诱导的发育性细胞凋亡机制的研究。通过扩大我们对麻醉诱导的细胞凋亡级联中所有关键步骤的理解，我们希望提高我们预防全麻对发育中的哺乳动物大脑潜在有害的神经毒性作用的机会。

DESCRIPTION (provided by applicant): This is the FIRCA Award application for the support of the research that will be done primarily in Serbia and Montenegro, in Laboratory for Molecular Neurobiology, the Department of Neurobiology at the University of Belgrade, Belgrade in collaboration with Vesna Jevtovic-Todorovic, Department of Anesthesiology, University of Virginia, as an extension of NIH/NICHD parent grant #R01 HD 044517. Premature infants, neonates and very young children are frequently exposed to general anesthesia. However, some recent findings indicate that anesthetic drugs, by transiently suppressing neuronal activity in the developing mammalian brain, disturb normal brain development (e.g. synaptogenesis) thus promoting apoptotic cell death of the immature neurons. Anesthesia-induced apoptotic neurodegeneration in the immature rats, a species of interest for this proposal, was manifested not only as a severe widespread apoptotic neurodegeneration (e.g. neuroapoptosis) in the developing brain but also as a significant and persistent learning/memory deficits detected latter on in life. The neurotrophins, a family of growth factors, support neuronal survival, differentiation and several forms of synaptic plasticity and therefore play an important role in synaptogenesis of the mammalian brain. Consequently, extensive depression of neuronal activity can impair survival-promoting signals that are regulated by neurotrophins. To investigate the potential relevance of neurotrophin-mediated anesthesia-induced developmental neuroapoptosis we conducted a pilot study on infant rats (at the peak of their synaptogenesis-7 days old) that were exposed to an inhalational anesthetic, isoflurane, at clinically relevant concentration (1.5-vol% in air), for a period of 2, 4 or 6 hrs. We found that isoflurane modulates the key steps in neurotrophin-modulated apoptotic cascade in two most vulnerable brain regions - the cerebral cortex and the thalamus. Namely, the protein levels of activated Akt, a key component in blocking apoptotic cascade, were significantly decreased in both the cortex and the thalamus. However, the BDNF (brain derived neurotrophic factor) protein levels were differentially modulated in the cortex; they were significantly increased, wherein in the thalamus there was a rapid and significant decrease in BDNF levels. In addition, while there was an up-regulation of the activated ceramide in the cortex there were no changes in ceramide activation in the thalamus: These findings suggest that isoflurane not only activates Akt-regulated apoptotic pathways, but does so in differential fashion in different brain regions most likely by activating both Trk dependent and Trk independent, p75NTR dependent neurotrophic survival pathways. In this grant application, the applicants plan to extend the studies of the mechanism of anesthesia-induced developmental apoptosis proposed in the parent grant by performing detailed studies of the mechanism(s) of neurotrophin-modulated anesthesia-induced neuroapoptosis. By expanding our understanding of all the crucial steps in the anesthesia-induced apoptotic cascade we hope to improve our chances of preventing potentially detrimental neurotoxic effects of general anesthetics to the developing mammalian brain.