Following in vitro ischemia, the nerve growth factor receptor TrkB is activated in the presence of the TrkB agonist 7,8-DHF only in female and not in male cultured hippocampal neurons, leading to increased neuronal survival. Expression of ERα is increased following in vitro ischemia in female but not male hippocampal neurons. The female hippocampal neuronal specific responses to in vitro ischemia are blocked by pre-treatment with testosterone. The data support a model for a female-specific a neuroprotective pathway in hippocampal neurons. The pathway is activated by a TrkB agonist, dependent on ERα and blocked by testosterone. Neonatal hypoxia ischemia (HI) related brain injury is one of the major causes of learning disabilities and memory deficits in children. In both human and animal studies, female neonate brains are less susceptible to HI than male brains. Phosphorylation of the nerve growth factor receptor TrkB has been shown to provide sex-specific neuroprotection following in vivo HI in female mice in an estrogen receptor alpha (ERα)-dependent manner. However, the molecular and cellular mechanisms conferring sex-specific neonatal neuroprotection remain incompletely understood. Here, we test whether female neonatal hippocampal neurons express autonomous neuroprotective properties and assess the ability of testosterone (T) to alter this phenotype. We cultured sexed hippocampal neurons from ERα+/+ and ERα−/− mice and subjected them to 4 h oxygen glucose deprivation and 24 h reoxygenation (4-OGD/24-REOX). Sexed hippocampal neurons were treated either with vehicle control (VC) or the TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following in vitro ischemia. End points at 24 h REOX were TrkB phosphorylation (p-TrkB) and neuronal survival assessed by immunohistochemistry. In addition, in vitro ischemia-mediated ERα gene expression in hippocampal neurons were investigated following testosterone (T) pre-treatment and TrkB antagonist therapy via q-RTPCR. Multifactorial analysis of variance was conducted to test for significant differences between experimental conditions. Under normoxic conditions, administration of 3 µM 7,8-DHF resulted an ERα-dependent increase in p-TrkB immunoexpression that was higher in female, as compared to male neurons. Following 4-OGD/24-REOX, p-TrkB expression increased 20% in both male and female ERα+/+ neurons. However, with 3 µM 7,8-DHF treatment p-TrkB expression increased further in female neurons by 2.81 ± 0.79-fold and was ERα dependent. 4-OGD/24-REOX resulted in a 56% increase in cell death, but only female cells were rescued with 3 µM 7,8-DHF, again in an ERα dependent manner. Following 4-OGD/3-REOX, ERα mRNA increased ~ 3 fold in female neurons. This increase was blocked with either the TrkB antagonist ANA-12 or pre-treatment with T. Pre-treatment with T also blocked the 7,8-DHF- dependent sex-specific neuronal survival in female neurons following 4-OGD/24-REOX. OGD/REOX results in sex-dependent TrkB phosphorylation in female neurons that increases further with 7,8-DHF treatment. TrkB phosphorylation by 7,8-DHF increased ERα mRNA expression and promoted cell survival preferentially in female hippocampal neurons. The sex-dependent neuroprotective actions of 7,8-DHF were blocked by either ANA-12 or by T pre-treatment. These results are consistent with a model for a female-specific neuroprotective pathway in hippocampal neurons in response to hypoxia. The pathway is activated by 7,8-DHF, mediated by TrkB phosphorylation, dependent on ERα and blocked by pre-exposure to T. In the clinical setting, it is well known that in neonates who suffer brain injury due to lack of blood flow and oxygen to the brain, the resulting damage is less severe in females compared to males. Furthermore, males sustaining brain injuries are more likely to exhibit learning and memory deficits as they mature. However, the underlying cellular mechanisms that lead to these sex differences in brain injury outcomes are poorly understood. In this report, we cultured hippocampal neurons from neonatal female and male mice and subjected them to reduced oxygen and glucose to mimic neonatal hypoxia ischemia. We found that the nerve growth factor receptor TrkB in females, as compared to males, is activated following the insult and more responsive to an exogenous nerve growth factor which leads to cell survival. In addition, expression of the estrogen receptor alpha is increased following the insult in females but not in males. Interestingly, the nerve growth factor receptor response in females is dependent on the presence of the estrogen receptor alpha. Both the nerve growth factor response and the increase in estrogen receptor alpha are abolished in females when treated with the male androgen, testosterone. Thus, our results support a model for a female-specific neuroprotective pathway in hippocampal neurons. The pathway is activated by nerve growth factors, dependent on estrogen receptor alpha, and is likely rendered inoperative in males by exposure to neonatal testosterone.

中文翻译：

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女性海马神经元中 TrkB 介导的神经保护是自主的、雌激素受体 α 依赖性的，并被睾酮消除：新生儿海马神经元损伤中性别差异的拟议模型

体外缺血后，神经生长因子受体 TrkB 在 TrkB 激动剂 7,8-DHF 存在的情况下仅在女性而非男性培养的海马神经元中被激活，从而导致神经元存活率增加。体外缺血后，雌性而非雄性海马神经元中 ERα 的表达增加。雌性海马神经元对体外缺血的特异性反应被睾酮预处理所阻断。这些数据支持了海马神经元中女性特有的神经保护通路的模型。该通路由 TrkB 激动剂激活，依赖于 ERα，并被睾酮阻断。新生儿缺氧缺血（HI）相关脑损伤是儿童学习障碍和记忆缺陷的主要原因之一。在人类和动物研究中，女性新生儿大脑比男性大脑更不容易受到HI的影响。神经生长因子受体 TrkB 的磷酸化已被证明可以以雌激素受体 α (ERα) 依赖性方式在雌性小鼠体内 HI 后提供性别特异性神经保护。然而，赋予性别特异性新生儿神经保护作用的分子和细胞机制仍不完全清楚。在这里，我们测试雌性新生儿海马神经元是否表达自主神经保护特性，并评估睾酮 (T) 改变这种表型的能力。我们培养了 ERα+/+ 和 ERα−/− 小鼠的性别海马神经元，并对它们进行 4 小时缺氧葡萄糖和 24 小时复氧（4-OGD/24-REOX）。体外缺血后，用载体对照 (VC) 或 TrkB 激动剂 7,8-二羟基黄酮 (7,8-DHF) 处理有性海马神经元。 24 小时 REOX 时的终点是 TrkB 磷酸化 (p-TrkB) 和通过免疫组织化学评估的神经元存活率。此外，在睾酮 (T) 预处理和 TrkB 拮抗剂治疗后，通过 q-RTPCR 研究了海马神经元中体外缺血介导的 ERα 基因表达。进行多因素方差分析以测试实验条件之间的显着差异。在含氧量正常的条件下，给予 3 µM 7,8-DHF 会导致 ERα 依赖性的 p-TrkB 免疫表达增加，与男性神经元相比，女性神经元的这种增加更高。 4-OGD/24-REOX 后，雄性和雌性 ERα+/+ 神经元中的 p-TrkB 表达均增加了 20%。然而，用 3 µM 7,8-DHF 处理后，雌性神经元中的 p-TrkB 表达进一步增加了 2.81 ± 0.79 倍，并且是 ERα 依赖性的。 4-OGD/24-REOX 导致细胞死亡增加 56%，但只有雌性细胞被 3 µM 7,8-DHF 拯救，同样以 ERα 依赖性方式。 4-OGD/3-REOX 后，雌性神经元中 ERα mRNA 增加约 3 倍。这种增加可以通过 TrkB 拮抗剂 ANA-12 或 T 预处理来阻断。T 预处理还可以阻断 4-OGD/24-REOX 后雌性神经元中 7,8-DHF 依赖性性别特异性神经元存活。OGD/REOX 导致雌性神经元中性别依赖性 TrkB 磷酸化，并且随着 7,8-DHF 治疗进一步增加。 7,8-DHF 磷酸化 TrkB 会增加 ERα mRNA 表达，并优先促进女性海马神经元中的细胞存活。 7,8-DHF 的性别依赖性神经保护作用可被 ANA-12 或 T 预处理阻断。这些结果与海马神经元响应缺氧的女性特异性神经保护途径模型一致。该通路由 7,8-DHF 激活，由 TrkB 磷酸化介导，依赖于 ERα，并通过预先暴露于 T 来阻断。在临床环境中，众所周知，在因血流不足而遭受脑损伤的新生儿中和大脑供氧，与男性相比，女性所造成的损害较男性轻。此外，遭受脑损伤的男性在成熟时更有可能表现出学习和记忆缺陷。然而，导致脑损伤结果性别差异的潜在细胞机制尚不清楚。在本报告中，我们培养了新生雌性和雄性小鼠的海马神经元，并将它们置于减少的氧气和葡萄糖中以模拟新生儿缺氧缺血。我们发现，与男性相比，女性的神经生长因子受体 TrkB 在损伤后被激活，并且对外源性神经生长因子更敏感，从而导致细胞存活。此外，女性中雌激素受体α的表达在受到损伤后增加，但男性中则没有。有趣的是，女性的神经生长因子受体反应取决于雌激素受体α的存在。当用男性雄激素睾酮治疗时，女性的神经生长因子反应和雌激素受体α的增加都会消失。因此，我们的结果支持海马神经元女性特异性神经保护途径的模型。该通路由神经生长因子激活，依赖于雌激素受体α，并且可能因暴露于新生儿睾酮而在男性中失效。然而，导致脑损伤结果性别差异的潜在细胞机制尚不清楚。在本报告中，我们培养了新生雌性和雄性小鼠的海马神经元，并将它们置于减少的氧气和葡萄糖中以模拟新生儿缺氧缺血。我们发现，与男性相比，女性的神经生长因子受体 TrkB 在损伤后被激活，并且对外源性神经生长因子更敏感，从而导致细胞存活。此外，女性中雌激素受体α的表达在受到损伤后增加，但男性中则没有。有趣的是，女性的神经生长因子受体反应取决于雌激素受体α的存在。当用男性雄激素睾酮治疗时，女性的神经生长因子反应和雌激素受体α的增加都会消失。因此，我们的结果支持海马神经元女性特异性神经保护途径的模型。该通路由神经生长因子激活，依赖于雌激素受体α，并且可能因暴露于新生儿睾酮而在男性中失效。然而，导致脑损伤结果性别差异的潜在细胞机制尚不清楚。在本报告中，我们培养了新生雌性和雄性小鼠的海马神经元，并将它们置于减少的氧气和葡萄糖中以模拟新生儿缺氧缺血。我们发现，与男性相比，女性的神经生长因子受体 TrkB 在损伤后被激活，并且对外源性神经生长因子更敏感，从而导致细胞存活。此外，女性中雌激素受体α的表达在受到损伤后增加，但男性中则没有。有趣的是，女性的神经生长因子受体反应取决于雌激素受体α的存在。当用男性雄激素睾酮治疗时，女性的神经生长因子反应和雌激素受体α的增加都会消失。因此，我们的结果支持海马神经元女性特异性神经保护途径的模型。该通路由神经生长因子激活，依赖于雌激素受体α，并且可能因暴露于新生儿睾酮而在男性中失效。