Mechanisms of Docosahexaenoic Acid Neuroprotection after Rat Pup TBI

 描述(申请人提供)：创伤性脑损伤(TBI)是导致儿童死亡和残疾的主要原因，导致终身神经功能障碍，目前还没有专门的治疗方法。饮食中的二十二碳六烯酸(DHA)改善了成年大鼠和我们的儿童脑外伤模型严重脑损伤后的神经学结果。然而，DHA的神经保护机制尚不清楚。脑损伤激活小胶质细胞，即大脑中驻留的巨噬细胞，进入M1和M2激活的范围。M1促进氧化应激，而M2则降低氧化应激。在成年动物中，M2的激活与结果的改善有关。目前尚不清楚脑外伤后M2的激活是否与未成熟大脑预后的改善有关，也不知道这种“极化”是如何形成的。我们的初步数据表明，在我们的儿童脑创伤模型中，DHA减少了小胶质细胞的激活，控制了大鼠的皮质撞击(CCI)。我们的DHA饮食增加了调节极化的小胶质细胞转录因子--过氧化物酶体增殖物激活受体(PPARγ)下游的大脑基因表达。PPARγ激动剂使小胶质细胞极化为M2。我们的结果让我们问，DHA的神经保护是否依赖于通过PPARγ依赖的途径促进小胶质细胞M2极化。令人担忧的是，DHA很容易被氧化。大量氧化的DHA会耗尽未成熟大脑有限的抗氧化剂储备，从而导致氧化损伤。另一方面，DHA可通过直接吸收活性氧和减少小胶质细胞自由基的产生来减轻氧化损伤。氧化损伤可以用总抗氧化能力(TAC)来评估。总之，需要对DHA的安全性和机制进行机械性的临床前研究。我们推测，在实验性脑损伤后，DHA将使激活的小胶质细胞极化为M2表型，并通过PPARγ依赖的机制增加脑组织TAC，与改善神经预后相关。为了测试这一点，我们将在CCI或假手术后将大鼠幼鼠暴露于DHA或常规(REG)饮食中，并每天给它们注射PPARγ拮抗剂或载体。我们将使用成像和基因表达来表征M2的激活，TAC来评估氧化，组织学/认知功能测试来评估结果。我们预计DHA将增加大鼠脑损伤后的M2极化和TAC，并被PPARγ拮抗剂所消除，并与损伤体积减少和认知功能障碍相关。我们的建议将提供有关脑外伤后未成熟脑中M2小胶质细胞极化的时间进程和功能重要性的新知识，以及DHA是否对其进行了修改。虽然DHA的可用性和明显的安全性使其成为儿童重型颅脑损伤后的一种有吸引力的候选治疗方法，但由于对其作用机制和在未成熟脑损伤后的安全性的重要认识不足，DHA的临床应用受到阻碍。我们的建议将导致有重点的临床前研究，这些研究将指导严重脑外伤后儿童的DHA临床试验，并有可能减轻儿童脑损伤后神经功能障碍的负担。

 DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is the leading cause of death and disability in children, resulting in life-long neurologic dysfunction for which there is o specific therapy. Dietary docosahexaenoic acid (DHA) improved neurologic outcome after severe TBI in adult rats and in our pediatric TBI model. However, the mechanism of DHA's neuroprotection is not understood. TBI activates microglia, the brain's resident macrophages, into a spectrum ranging between M1 and M2 activation. M1 promotes oxidative stress, while M2 decreases oxidative stress. In adult animals, M2 activation is associated with improved outcome. It is not known if M2 activation after TBI is associated with improved outcome in the immature brain, nor how such "polarization" may be fostered. Our preliminary data suggests that DHA decreases microglial activation in our pediatric TBI model, controlled cortical impact (CCI) in rat pups. Our DHA diet increases brain gene expression downstream of a microglial transcription factor that regulates polarization, the Peroxisome Proliferator Activated Receptor (PPARγ). PPARγ agonists polarize microglia towards M2. Our results led us to ask if DHA's neuroprotection depends on promoting microglial M2 polarization via a PPARγ-dependent pathway. Of concern, DHA is readily oxidized. Large amounts of oxidized DHA could cause oxidative injury by depleting the immature brain's limited antioxidant reserve. On the other hand, DHA could decrease oxidative injury by directly absorbing reactive oxygen species and decreasing microglial free radical production. Oxidative injury may be assessed using total antioxidant capacity (TAC). In sum, mechanistic preclinical studies of DHA safety and mechanism are needed. We hypothesize that DHA will polarize activated microglia towards the M2 phenotype and increase brain TAC in rat pups after experimental TBI, via a PPARγ-dependent mechanism, associated with improved neurologic outcome. To test this, we will expose rat pups to DHA or regular (REG) diet after CCI or SHAM surgery and inject them daily with either a PPARγ antagonist or vehicle. We will use imaging and gene expression to characterize M2 activation, TAC to assess oxidation, and histology/cognitive function testing to assess outcome. We anticipate that DHA will increase M2 polarization and TAC in rat pup brains after TBI, abrogated by PPARγ antagonism and associated with decreased lesion volume and cognitive impairment. Our proposal will provide new knowledge on the time course, and functional importance, of M2 microglial polarization in the immature brain after TBI, and whether these are modified by DHA. While the availability and apparent safety of DHA make it an appealing candidate therapy for children after severe TBI, its clinical use is hindered by important knowledge deficits regarding its mechanism of action and safety in the immature brain after TBI. Our proposal will lead to focused pre-clinical studies that will guide clinical trials o DHA in children after severe TBI and potentially decrease the burden of neurologic disability after childhood TBI.