Introduction: What Was Observed?

• All living cells maintain a membrane potential by controlling the flow of ions (such as sodium, potassium, calcium, and chloride) across their membranes.
• This electrical “battery” helps regulate cell behaviors like migration, proliferation, differentiation, and even tissue repair.
• This study focuses on human neurons derived from induced neural stem cells (hiNSC) to understand bioelectric signals and their role in nerve repair.

Understanding the Methods (Step-by-Step)

• Cell Culture:
  • hiNSC are grown on feeder layers and induced to differentiate into neurons over approximately 10 days.
  • By day 10, most cells express the neuronal marker TUJ1 and begin forming extensive neural networks by day 15.
• Live Sensor Dyes:
  • Cell morphology dyes such as Calcein Green and Calcein Red-Orange label live cells, highlighting cell bodies and neurite extensions.
  • Nuclear dyes like DAPI are avoided because they mainly stain dead cells and can be toxic; Hoechst is used more cautiously.
  • These dyes enable real-time imaging of neurons, allowing researchers to “see” cell shape and network formation without harming the cells.
• Ion and Voltage Measurements:
  • CoroNa AM detects intracellular sodium (Na+) by increasing its fluorescence as Na+ levels rise—imagine it as a sensor that “lights up” when sodium goes up.
  • APG2-AM is used for monitoring intracellular potassium (K+) levels, although it may also be influenced by other ions.
  • Fluo4-AM measures intracellular calcium (Ca2+) dynamics, a key signal in neurons. For instance, applying glutamate causes a measurable increase in Ca2+.
  • DiBAC monitors resting membrane potential; as cells depolarize (become less negative), DiBAC’s fluorescence increases.
• Cell Activity Sensors:
  • SNARF-5F AM detects intracellular pH changes, with fluctuations acting as signals of cellular stress or injury.
  • Peroxy Orange 1 (PO1) measures reactive oxygen species (ROS), byproducts of metabolism that indicate cellular stress or damage.

Nerve Repair and Neurite Outgrowth: The Scratch Assay

• A scratch is made in a confluent layer of mature hiNSC-derived neurons to simulate a nerve injury.
• Over the next several days, neurons extend neurites (branch-like projections) into the scratch area, similar to roots growing into an empty space.
• Live dyes help visualize and quantify neurite density within the injured area, providing a measure of how well the nerve repair is progressing.

Effects of Neurotransmitters on Neurite Outgrowth

• Acetylcholine:
  • At lower concentrations, acetylcholine shows little effect initially.
  • At higher concentrations, it exhibits a biphasic effect—first suppressing neurite outgrowth shortly after injury, then later increasing neurite density along the scratch edge.
  • This two-phase effect suggests acetylcholine can both delay and later promote aspects of nerve repair.
• Serotonin:
  • Serotonin significantly enhances neurite outgrowth in a dose-dependent manner.
  • Neurites tend to grow longer and perpendicular to the injury, a pattern associated with more effective nerve regeneration.
• GABA:
  • GABA treatment does not significantly alter neurite outgrowth compared to controls, indicating it may not be a major factor in this repair process.

Effects of Extracellular pH on Neurite Outgrowth

• Acidic conditions (around pH 6) lead to an early increase in neurite density, though the effect may normalize over time.
• Neutral to slightly alkaline conditions (pH 7–8) tend to decrease neurite outgrowth as time progresses.
• This suggests that a slightly acidic environment may promote better nerve repair, much like a specific pH is required for a recipe to “cook” just right.

Key Conclusions and Implications

• The study establishes a suite of bioelectric sensors that can monitor live neuron characteristics including morphology, ion levels, membrane potential, pH, and metabolic stress (ROS).
• These methods provide a clear, step-by-step “recipe” for understanding how neurons respond to injury and initiate repair.
• By manipulating factors such as neurotransmitter levels and extracellular pH, researchers can influence nerve repair and regeneration.
• The findings have potential implications for developing therapies for injuries, congenital defects, and diseases by targeting the bioelectric properties of cells.

观察：研究所见内容简介

• 所有活细胞通过控制钠、钾、钙和氯等离子穿越细胞膜的流动来维持膜电位。
• 这种细胞“电池”调控着细胞的运动、增殖、分化以及组织修复等多种行为。
• 本研究聚焦于由诱导神经干细胞（hiNSC）衍生的人类神经元，以探索生物电信号及其在神经修复中的作用。

方法解析（逐步说明）

• 细胞培养：
  • hiNSC在饲养层上生长，并在大约10天内被诱导分化为神经元。
  • 到第10天，大多数细胞会表达神经元特异性标记TUJ1，并在第15天形成广泛的神经网络。
• 活细胞传感染料：
  • 使用Calcein Green和Calcein Red-Orange等染料标记活细胞，显示细胞体及其神经突（延伸部分）的形态。
  • 由于DAPI容易染色死亡细胞且具有毒性，因此采用Hoechst染料需特别谨慎。
  • 这些染料使研究者能够实时观察神经元的形态和网络形成，而不会对细胞造成损伤。
• 离子及电压测量：
  • CoroNa AM用于检测细胞内钠离子（Na+）水平，当钠离子增多时，其荧光增强，就像一个“亮起”的传感器。
  • APG2-AM用于监测细胞内钾离子（K+）水平，但可能也会受到其他离子的影响。
  • Fluo4-AM用于测量钙离子（Ca2+）动态；例如，添加谷氨酸后细胞内钙离子水平会显著上升。
  • DiBAC用于监测静息膜电位；当细胞去极化（膜电位变得不那么负）时，DiBAC的荧光会增强。
• 细胞活动传感器：
  • SNARF-5F AM用于检测细胞内pH值变化，这种变化就像细胞在受压或受损时发出的信号。
  • Peroxy Orange 1 (PO1)用于检测反应性氧种（ROS），它们是细胞代谢的副产物，可反映细胞的压力或损伤情况。

神经修复与神经突生长：划痕实验

• 在密集的成熟神经元层上制造一条划痕，模拟神经损伤。
• 随后的几天中，神经元会向划痕区域延伸出神经突，就像树根生长进空缺处一样。
• 利用活细胞染料观察并量化划痕区域内神经突的密度，以评估神经修复的效果。

神经递质对神经突生长的影响

• 乙酰胆碱：
  • 低浓度时对神经突生长的影响不大；但在高浓度下，乙酰胆碱呈现双相效应——早期抑制神经突生长，随后在划痕边缘增加神经突密度。
  • 这种双阶段效应表明，乙酰胆碱既可能延迟又可能促进某些神经修复过程。
• 血清素：
  • 血清素能显著促进神经突的生长，效果呈剂量依赖性。
  • 神经突延伸更长且通常呈垂直于损伤方向生长，这种模式与更有效的神经再生有关。
• GABA：
  • 使用GABA处理后，神经突生长与对照组无明显差异，表明GABA在此修复过程中作用不大。

细胞外pH对神经突生长的影响

• 当外部环境变得更酸性（pH约为6）时，早期神经突密度会增加，但这种效应可能在后期趋于平稳。
• 而在中性至略碱性环境（pH 7至8）下，随着时间推移，神经突生长反而会减少。
• 这表明略酸性的环境可能更有利于神经修复，就像某些食谱需要特定pH值才能达到最佳效果一样。

主要结论与启示

• 本研究构建了一套生物电传感器，能够实时监测神经元的形态、离子浓度、膜电位、pH值以及代谢压力（ROS）。
• 这些方法提供了一份详细的“操作指南”，帮助我们理解神经元如何响应损伤并启动修复。
• 通过调控神经递质浓度和细胞外pH值，可以有效影响神经修复和再生。
• 这些发现为开发针对损伤、先天缺陷及疾病的疗法提供了新的思路，即通过调控细胞的生物电特性来促进修复。