Assessing Microglial Phagocytosis of Myelin Debris <em>in vitro</em> Under Repeated Magnetic Stimulation

Chenyuan Zhai; Jili Cai; Mei Du; Yuchen Fei; Qi Wu

doi:10.3791/67642

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Assessing Microglial Phagocytosis of Myelin Debris in vitro Under Repeated Magnetic Stimulation

在重复磁刺激下评估髓鞘碎片在体外的小胶质细胞吞噬作用

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08:34 min

June 17, 2025

DOI: 10.3791/67642-v

Chenyuan Zhai¹, Jili Cai², Mei Du³, Yuchen Fei⁴, Qi Wu⁵

¹Department of Rehabilitation Medicine,The Affiliated Suzhou Hospital of Nanjing Medical University, ²Rehabilitation Medicine Center,The First Affiliated Hospital of Nanjing Medical University, ³Department of Children's Rehabilitation,Linyi People's Hospital, ⁴Department of Endocrinology,People's Hospital of Dongxihu District, ⁵Department of Rehabilitation, Hengyang Medical School, The First Affiliated Hospital,University of South China

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Overview

This study investigates the influence of repetitive magnetic stimulation on microglia's phagocytic ability regarding myelin debris using an in vitro co-culture model. The research addresses the therapeutic potential of magnetic stimulation in neuro-rehabilitation.

Key Study Components

Area of Science

Neuroscience
Neuro-rehabilitation
Microglial function

Background

Understanding microglial functions is crucial for neuro-rehabilitation.
Magnetic stimulation has potential neuroprotective effects.
Microglial phagocytosis of myelin debris plays a role in neural repair.

Purpose of Study

To evaluate the effect of magnetic stimulation on microglial phagocytosis.
To explore therapeutic applications in neuro-rehabilitation.
To establish a robust in vitro model for further research.

Methods Used

In vitro co-culture system of microglia and myelin debris.
BV-2 cell line was utilized for assessing phagocytic activity.
Repetitive magnetic stimulation parameters were set at 20 Hz for 2.5 minutes.
Myelin debris was isolated through ultracentrifugation.
Centrifugation and washing steps were used for purification and preservation of myelin samples.

Main Results

Lipopolysaccharide treatment decreased microglial phagocytosis of myelin debris.
Repetitive magnetic stimulation reversed the reduction in phagocytosis.
Quantitative analysis showed increased uptake of myelin debris in magnetically stimulated cells.
Magnetic stimulation significantly increased the percentage of IBA-1 positive microglia co-localized with myelin debris compared to controls.

Conclusions

The study demonstrates magnetic stimulation's capability to enhance microglial function.
Findings support the application of magnetic stimulation in therapeutic settings for neuronal recovery and repair.
This research provides insights into mechanisms involved in microglial response to stimuli in neuro-rehabilitation contexts.

Frequently Asked Questions

What are the advantages of using the BV-2 cell line?

The BV-2 cell line provides a consistent and reproducible model for studying microglial function and responses to stimuli, such as magnetic stimulation.

How is myelin debris isolated for this study?

Myelin debris is isolated through a series of ultracentrifugation steps to ensure purity and yield before use in co-culture with microglia.

What outcomes are measured after magnetic stimulation?

The study measures changes in phagocytosis rates of myelin debris and the presence of IBA-1 positive microglia in the cultures.

How can magnetic stimulation be applied therapeutically?

The findings suggest that magnetic stimulation could be utilized as a therapeutic strategy to enhance microglial function and improve neural repair processes.

What limitations should be considered in this study?

Limitations include the use of an in vitro model, which may not fully replicate in vivo conditions, and the need for further studies to validate findings in clinical settings.

本方案旨在评估重复磁刺激对小胶质细胞吞噬髓鞘碎片能力的影响。为此，已经建立了 体外小 胶质细胞和髓鞘碎片共培养系统。

[导师]我们的研究范围侧重于神经康复和磁模拟治疗的研究。该协议可以为磁刺激探索它如何影响清晰的功能提供新思路。未来我们将专注于神经康复和磁刺激治疗。

[导师]首先，获得一只雌性老鼠的斩首头。在冰上解剖头部。用剪刀将头骨一分为二，使大脑完全暴露并促进其完全切除。使用剪刀和镊子仔细、无菌地切除膜、小脑和海马体。用 PBS 清洁大脑 3 次，以去除任何残留的血液或组织。将清洁后的大脑转移到10毫升0.32摩尔无菌蔗糖溶液中。用显微外科剪刀将脑组织切成小块，得到脑组织-蔗糖混合物。接下来，将脑组织-蔗糖混合物转移到50毫升无菌均质机中。加入30毫升0.32摩尔无菌蔗糖溶液。使用 50 毫升玻璃均质器研磨组织两分钟。获得光滑的脑组织匀浆。用0.32摩尔无菌蔗糖溶液将脑组织匀浆稀释至90毫升，并充分混合。将 20 毫升 0.83 摩尔无菌蔗糖溶液加入六个无菌薄壁聚丙烯超速离心管中。然后慢慢将 15 毫升脑混合物分配到管子的上部。用0.32摩尔无菌蔗糖溶液调平体积。为了收集粗的髓鞘碎片，首先在4摄氏度下预冷和超速离心转子。将样品以75,000 G离心45分钟，然后使用无菌牧场移液器从两个蔗糖密度之间的界面收集髓鞘碎片。对于第一次等渗分离和纯化，将收集的髓鞘碎片溶液转移到 50 毫升离心管中。用预冷的无菌PBS将体积调节至35毫升，然后转移到新的均质管中。均质三分钟后，将髓鞘碎片匀浆均匀分布在六个38.5毫升无菌、薄壁、聚丙烯超速离心管中。用无菌PBS补满体积，然后离心。对于第二次等渗分离和纯化，将固体白色沉淀重悬于 10 毫升预冷的无菌 PBS 中，以获得髓鞘碎片悬浮液。像以前一样将悬浮液分配到超速离心管中，然后离心。离心后，弃去上清液。将固体白色沉淀重悬于六毫升无菌PBS中。将髓鞘碎片悬浮液分成六个1.5离心管，再次离心。最后，弃去上清液后，将沉淀重悬于 100 微升预冷的无菌 PBS 中。将必要的髓鞘碎片在 4 摄氏度或冰水中解冻，然后像以前一样离心 10 分钟。将髓鞘碎片重悬于 200 微升 50 微摩尔 CFSE 溶液中。将悬浮液在室温下在黑暗中孵育30分钟，然后再次离心。弃去上清液，然后用 500 微升无菌 PBS 洗涤样品 3 次。将髓鞘碎片重悬于100微升预冷的无菌PBS中，以获得荧光标记的髓鞘碎片的悬浮液。将样品储存在-80摄氏度，直到进一步使用。对于体外重复磁刺激，将治疗频率设置为 20 赫兹，治疗强度设置为最大输出强度的 1%，刺激时间设置为 5 秒。包括 20 秒的休息时间，并在 2.5 分钟的单次治疗时间内给予 600 次脉冲。预先确定磁刺激参数后，将线圈方向垂直于地面固定并向上定向。用酒精对磁刺激线圈进行消毒，以防止细胞污染。加入50微摩尔CFSE后，将1,000个BV-2细胞接种在24孔板中过夜，然后用移液器吸出旧培养基，并立即加入新鲜的无血清培养基。将培养基改为无血清培养基，并用每毫升脂多糖1微克处理细胞12小时。脂多糖干预12小时后，将每毫升髓磷脂碎片100微克加入培养基中，在黑暗中共培养。如前所述，对细胞进行重复的磁刺激。在将其放回培养箱之前，将酒精喷洒在板上进行灭菌。与髓鞘片段共培养一段时间后，用PBS轻轻洗涤未吸收的髓鞘碎片。用4%多聚甲醛固定细胞15分钟。接下来，将含有 10% 驴血清白蛋白和 0.3% Triton X-100 的 150 微升 PBS 加入孔中并孵育。用PBS洗涤孔后，将一抗溶液以100比200的比例加入孔中，冷孵育。然后，在室温下以 100 比 300 的比例将细胞在二抗中孵育两小时，然后用共聚焦显微镜成像。脂多糖处理后，BV-2小胶质细胞的髓鞘碎片吞噬作用显着降低。在反复磁刺激干预后逆转。定量分析证实，与对照组相比，脂多糖组 BV-2 细胞内髓鞘碎片面积显着减少，脂多糖处理的磁刺激组显着增加。与对照组相比，LPS 组与髓鞘碎片共定位的 IBA-1 阳性小胶质细胞百分比显着降低，而磁刺激显着增加了这一百分比。观察到小胶质细胞吞噬作用的时间依赖性增加，我们组的脂多糖处理磁刺激组显示出显着更高的髓鞘碎片摄取。

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