Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, USA.
Department of Neuroscience, University of Kentucky, Lexington, USA.
Fluids Barriers CNS. 2024 Oct 15;21(1):81. doi: 10.1186/s12987-024-00579-9.
Neurovascular deficits and blood-brain barrier (BBB) dysfunction are major hallmarks of brain trauma and neurodegenerative diseases. Oxidative stress is a prominent contributor to neurovascular unit (NVU) dysfunction and can propagate BBB disruption. Oxidative damage results in an imbalance of mitochondrial homeostasis, which can further drive functional impairment of brain capillaries. To this end, we developed a method to track mitochondrial-related changes after oxidative stress in the context of neurovascular pathophysiology as a critical endophenotype of neurodegenerative diseases.
To study brain capillary-specific mitochondrial function and dynamics in response to oxidative stress, we developed an ex vivo model in which we used isolated brain capillaries from transgenic mice that express dendra2 green specifically in mitochondria (mtD2g). Isolated brain capillaries were incubated with 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) or hydrogen peroxide (HO) to induce oxidative stress through lipid peroxidation. Following the oxidative insult, mitochondrial bioenergetics were measured using the Seahorse XFe96 flux analyzer, and mitochondrial dynamics were measured using confocal microscopy with Imaris software.
We optimized brain capillary isolation with intact endothelial cell tight-junction and pericyte integrity. Further, we demonstrate consistency of the capillary isolation process and cellular enrichment of the isolated capillaries. Mitochondrial bioenergetics and morphology assessments were optimized in isolated brain capillaries. Finally, we found that oxidative stress significantly decreased mitochondrial respiration and altered mitochondrial morphology in brain capillaries, including mitochondrial volume and count.
Following ex vivo isolation of brain capillaries, we confirmed the stability of mitochondrial parameters, demonstrating the feasibility of this newly developed platform. We also demonstrated that oxidative stress has profound effects on mitochondrial homeostasis in isolated brain capillaries. This novel method can be used to evaluate pharmacological interventions to target oxidative stress or mitochondrial dysfunction in cerebral small vessel disease and neurovascular pathophysiology as major players in neurodegenerative disease.
神经血管功能障碍和血脑屏障（BBB）功能障碍是脑创伤和神经退行性疾病的主要标志。氧化应激是神经血管单元（NVU）功能障碍的主要原因，并可导致 BBB 破坏。氧化损伤导致线粒体动态平衡失衡，进一步导致脑毛细血管功能障碍。为此，我们开发了一种方法来跟踪神经血管病理生理学背景下氧化应激后与线粒体相关的变化，作为神经退行性疾病的关键内表型。
为了研究氧化应激下脑毛细血管特异性线粒体功能和动力学，我们开发了一种离体模型，其中我们使用在脑毛细血管中特异性表达绿色 dendra2（mtD2g）的转基因小鼠分离脑毛细血管。将分离的脑毛细血管用 2,2'-偶氮双（2-甲基丙脒）二盐酸盐（AAPH）或过氧化氢（HO）孵育，通过脂质过氧化诱导氧化应激。氧化损伤后，使用 Seahorse XFe96 通量分析仪测量线粒体生物能学，使用共聚焦显微镜和 Imaris 软件测量线粒体动力学。
我们优化了脑毛细血管的分离，使其具有完整的内皮细胞紧密连接和周细胞完整性。此外，我们证明了毛细血管分离过程的一致性和分离的毛细血管的细胞丰富度。优化了分离的脑毛细血管中的线粒体生物能学和形态评估。最后，我们发现氧化应激显著降低了脑毛细血管中的线粒体呼吸并改变了线粒体形态，包括线粒体体积和数量。
在离体分离脑毛细血管后，我们证实了线粒体参数的稳定性，证明了这个新开发的平台的可行性。我们还表明，氧化应激对分离的脑毛细血管中的线粒体动态平衡有深远的影响。这种新方法可用于评估药物干预以靶向脑小血管疾病和神经血管病理生理学中的氧化应激或线粒体功能障碍，作为神经退行性疾病的主要参与者。
