Identification of biomarkers for biochemical, pathophysiological and neurological effects of high ammonia concentration on the central nervous system in a preclinical model of neonatal hyperammonemia

摘要 任何原因引起的新生儿高氨血症（HA），包括几种可筛选的疾病，都会导致脑损伤 导致不可逆转的智力和发育障碍，甚至死亡。目前HA的治疗方法有 针对降低血氨水平;虽然他们可以防止脑水肿死亡，他们是 在减少或防止脑损伤方面效率低下。我们对氨毒性机制的理解， 大脑是基于培养细胞和成年动物模型的实验; HA破坏谷氨酰胺，谷氨酸， 星形胶质细胞的K+代谢导致渗透压和脑水肿。然而，不知道如何 HA影响发育中的大脑，因为动物模型适合研究分子，生化， 目前尚不存在HA对新生儿脑的病理生理学和神经学影响。我们有 创建了诱导型HA的动物模型，即N-乙酰谷氨酸合酶敲除（NAGSko）小鼠。 纯合子敲除小鼠存活至成年期，并在用N-氨甲酰谷氨酸和 瓜氨酸，并在治疗停止时产生HA。我们建议使用NAGSko小鼠作为模型， 可诱导新生儿HA。我们的目标是建立可用于临床前和临床的HA生物标志物 测试神经保护药物。我们的具体目标是：1.为了在新生NAGSko小鼠中诱导HA并确定 在尿素生成正常化后，他们大脑中相关的MRS生化变化是否持续存在。 我们将在出生后第13天（P13）在NAGSko小鼠中诱导HA，并测量脑中代谢物的差异 HA NAGSko小鼠和非HA同窝出生的小鼠在HA发作期间和从HA恢复后2周。大脑 将使用质子磁共振波谱（1H-MRS）测量代谢物。2.以确定 新生儿HA发作是否导致与血液相关的持续性异常星形胶质细胞功能 脑损伤的生物标志物。我们将使用我们的新型NAGSko/ALDH 1 L1/GCamp 5G-tdTm小鼠和双光子 显微镜监测新生儿HA发作期间Ca 2+信号的变化及其长期后果 对清醒动物星形胶质细胞Ca 2+信号传导的影响，Ca 2+信号传导的变化将与血清生物标志物相关。 脑损伤S100 B和NSE（神经元特异性烯醇化酶）。3.确定新生儿HA发作是否导致 持续异常的脑电活动我们将评估新生儿HA发作是否会增加频率 和NAGSko小鼠中尿素生成正常化后癫痫发作的严重程度。经过验证，我们计划使用 EEG模式、1H-MRS和脑损伤的血液生物标志物作为HA的临床前和临床生物标志物 评估保护大脑免受氨中毒的药物和疗法。如果成功，药物 这些试验的结果将补充目前的治疗方法， HA患者。

Abstract Neonatal hyperammonemia (HA) from any cause, including several screenable disorders, results in brain injury leading to irreversible intellectual and developmental disabilities, and even death. Current therapies for HA are targeted at reducing blood ammonia levels; although they can prevent death from brain edema, they are inefficient at reducing or preventing brain damage. Our understanding of the mechanism of ammonia toxicity to the brain is based on experiments in cultured cells and adult animal models; HA disrupts glutamine, glutamate, and K+ metabolism in the astrocytes leading to osmotic stress and brain edema. However, it is not known how HA affects the developing brain because animal models suitable for studying molecular, biochemical, pathophysiological, and neurological effects of HA on the neonatal brain currently do not exist. We have created an animal model of inducible HA, the N-acetylglutamate synthase knockout (NAGSko) mouse. Homozygous knockout mice survive into adulthood and reproduces when treated with N-carbamylglutamate and citrulline, and develop HA when treatment is stopped. We propose to use the NAGSko mice as a model of inducible neonatal HA. Our goal is to establish biomarkers of HA that could be used in both preclinical and clinical testing of neuroprotection drugs. Our specific aims are: 1. To induce HA in neonatal NAGSko mice and determine whether associated MRS biochemical changes in their brains persist after ureagenesis has been normalized. We will induce HA in NAGSko mice at postnatal day 13 (P13) and measure metabolite differences in the brains of HA NAGSko mice and non-HA littermates during HA episode and 2 weeks post recovery from HA. Brain metabolites will be measured using proton magnetic resonance spectroscopy (1H-MRS). 2. To determine whether neonatal HA episode causes persistent abnormal astrocyte function that correlates with blood biomarkers of brain damage. We will use our novel NAGSko/ALDH1L1/GCamp5G-tdTm mice and 2-photon microscopy to monitor changes in Ca2+ signaling during neonatal HA episode and its long-term consequences on astrocytic Ca2+ signaling in awake animals, Changes in Ca2+ signaling will be correlated to serum biomarkers of brain injury S100B and NSE (neuron-specific enolase). 3. To determine whether neonatal HA episode causes persistent abnormal brain electrical activity. We will assess whether a neonatal HA episode increases frequency and severity of seizures after normalization of ureagenesis in the NAGSko mice. After validation, we plan to use EEG patterns, 1H-MRS and blood biomarkers of brain damage as biomarkers of HA in pre-clinical and clinical evaluation of drugs and therapies for the protection of the brain from ammonia toxicity. If successful, drugs that result from these trials will complement current treatment approaches and improve the outcome of patients with HA.