Name: a stably established two-point injection of lysophosphatidylcholine-induced focal demyelination model in mice
Uploaded: 2022-05-11T14:00:00
Description: Watch this Scientific Journal Video about A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice at JoVE.com

本研究由国家自然科学基金（基金资助：82071380、81873743）资助。

所有动物程序均经中国华中科技大学同济医学院动物护理委员会批准。本研究使用成年C57BL / 6雄性和雌性小鼠（野生型，WT;20-25g;8-10周龄）。小鼠是从商业来源获得的（见 材料表）。将小鼠饲养在特定的无病原体（SPF）动物设施中 ，随意提供水和食物。在22 °C温度和55%~60%相对湿度的标准条件下，将它们保持在12 h交替的明暗循环中。
1. 液相色谱溶液制...

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MS是中枢神经系统的慢性脱髓鞘疾病，是年轻人神经系统功能障碍的最常见原因之一20.临床上，大约60%-80%的MS患者在发展为继发性进行性MS21，22之前经历复发和缓解的周期，并且最终导致累积的运动障碍和认知缺陷23。目前，没有一个单一的实验模型涵盖了该疾病24的各种临床、病理或免疫学特征。?...

受体介导的溶血磷脂信号传导涉及几乎所有器官系统的不同生理过程1.在中枢神经系统（CNS）中，这种信号传导在自身免疫性神经系统疾病（如多发性硬化症（MS））的发病机制中起着关键作用。多发性硬化症是一种慢性免疫介导的疾病，其特征是病理性脱髓鞘和炎症反应，导致神经系统功能障碍和认知障碍2，3。在疾病早期持续复发和缓解后，大多数患者最终进展到继发性进展阶段，这可能对大脑造成不可逆转的损害并导致残疾4。据信，继发性进展病程的病理标志是由炎症性病变引起的脱髓鞘斑块5。现有的MS治疗方法可以显着降低复发的风险。然而，对于进行性MS6引起的长期脱髓鞘损伤，仍然没有有效的治疗方法。因此，需要一个稳定建立且易于重现的模型来研究专注于白质变性的临床前治疗。
脱髓鞘和髓鞘再生是发展为多发性硬化症的两个主要病理过程。脱髓鞘是由促炎表型7的小胶质细胞诱导的轴突周围髓鞘的丧失，它导致神经冲动的缓慢传导并导致神经元和神经系统疾病的丧失。髓鞘再生是由少突胶质细胞介导的内源性修复反应，其中疾病可能导致神经变性和认知障碍8。炎症反应对整个过程至关重要，影响髓鞘损伤和修复的程度。
因此，持续的炎症性脱髓鞘的稳定动物模型对于进一步探索MS的治疗策略具有重要意义，由于MS的复杂性，已经建立了各种类型的动物模型来模拟 体内脱髓鞘病变，包括实验性自身免疫性脑脊髓炎（EAE），毒性脱髓鞘模型，铜绿素（CPZ）和溶酶磷脂酰胆碱（LPC）9.LPC是一种与炎症相关的内源性溶血磷脂，可诱发快速损伤，对髓鞘脂质有毒性，导致局灶性脱髓鞘。根据以前的报告和研究10，11，提供了具有一些修改的两点注射的详细方案。通常，经典的单点LPC注射模型仅在注射部位产生局部脱髓鞘，并且通常伴有自发性髓鞘再生12，13。然而，两点注射LPC模型可以证明LPC可以直接诱导小鼠胼胝体脱髓鞘，并导致更持久的脱髓鞘，髓鞘再生很少。

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			<td >L-&#945;-Lysophosphatidylcholine from egg yolk</td>
			<td >Sigma-Aldrich</td>
			<td >L4129-25MG</td>
			<td ></td>
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		<tr >
			<td >32 gauge Needle</td>
			<td >HAMILTON</td>
			<td >7762-05</td>
			<td></td>
		</tr>
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			<td >10 &#956;l syringe</td>
			<td >HAMILTON</td>
			<td >80014</td>
			<td></td>
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			<td >high speed skull drill</td>
			<td >strong,korea</td>
			<td >strong204</td>
			<td></td>
		</tr>
		<tr >
			<td >drill</td>
			<td >Hager &#38; Meisinger, Germany&#160;</td>
			<td >REF 500 104 001 001 005</td>
			<td></td>
		</tr>
		<tr >
			<td >Matrx Animal Aneathesia Ventilator</td>
			<td >MIDMARK</td>
			<td >VMR</td>
			<td></td>
		</tr>
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			<td >Portable Stereotaxic Instrument for Mouse</td>
			<td >Reward</td>
			<td >68507</td>
			<td></td>
		</tr>
		<tr >
			<td >Micro syringe</td>
			<td >Reward</td>
			<td >KDS LEGATO 130</td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane&#160;</td>
			<td >VETEASY</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde</td>
			<td >Servicebio</td>
			<td >G1101</td>
			<td></td>
		</tr>
		<tr >
			<td >Phosphate buffer</td>
			<td >BOSTER</td>
			<td >PYG0021</td>
			<td></td>
		</tr>
		<tr >
			<td >LuxoL fast bLue</td>
			<td >Servicebio</td>
			<td >G1030-100ML</td>
			<td></td>
		</tr>
		<tr >
			<td >Suture</td>
			<td >FUSUNPHARMA</td>
			<td >20152021225</td>
			<td></td>
		</tr>
		<tr >
			<td >Brain mold</td>
			<td >Reward</td>
			<td >68707</td>
			<td></td>
		</tr>
		<tr >
			<td >Electron microscope fixative</td>
			<td >Servicebio</td>
			<td >G1102-100ML</td>
			<td></td>
		</tr>
		<tr >
			<td >Neutral red (C.I. 50040), for microscopy Certistain</td>
			<td >Sigma-Aldrich</td>
			<td >1.01376</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-Myelin Basic Protein Antibody&#160;</td>
			<td >Millipore</td>
			<td >#AB5864</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-GST-P pAb</td>
			<td >MBL</td>
			<td >#311</td>
			<td></td>
		</tr>
		<tr >
			<td >Ki-67 Monoclonal Antibody (SolA15)</td>
			<td >Thermo Fisher&#160;Scientific</td>
			<td >14-5698-95</td>
			<td></td>
		</tr>
		<tr >
			<td >Beta Actin Monoclonal Antibody</td>
			<td >Proteintech</td>
			<td >66009-1-Ig&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Myelin Basic Protein Polyclonal Antibody</td>
			<td >Proteintech</td>
			<td >10458-1-AP</td>
			<td></td>
		</tr>
		<tr >
			<td >OLIG2 Polyclonal Antibody</td>
			<td >Proteintech</td>
			<td >13999-1-AP</td>
			<td></td>
		</tr>
		<tr >
			<td >Alexa Fluor 488&#160;AffiniPure&#160;Donkey anti-Rabbit&#160;IgG (H+L)</td>
			<td >YEASEN</td>
			<td >34206ES60</td>
			<td></td>
		</tr>
		<tr >
			<td >Alexa Fluor 594 AffiniPure Donkey Anti-Rat IgG (H+L)&#160;</td>
			<td >YEASEN</td>
			<td >34412ES60</td>
			<td></td>
		</tr>
		<tr >
			<td >Alexa Fluor 594&#160;AffiniPure Donkey Anti-Rabbit IgG (H+L)&#160;</td>
			<td >YEASEN</td>
			<td >34212ES60</td>
			<td></td>
		</tr>
		<tr >
			<td >HRP Goat Anti-Rabbit IgG (H+L)</td>
			<td >abclonal</td>
			<td >AS014</td>
			<td></td>
		</tr>
		<tr >
			<td >HRP Goat Anti-Mouse IgG (H+L)&#160;</td>
			<td >abclonal</td>
			<td >AS003</td>
			<td></td>
		</tr>
		<tr >
			<td >Adult C57BL/6 male and female mice</td>
			<td >Hunan SJA Laboratory Animal Co. Ltd</td>
			<td ></td>
			<td></td>
		</tr>
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</table>

a stably established two-point injection of lysophosphatidylcholine-induced focal demyelination model in mice

受体介导的溶血磷脂信号传导有助于各种神经系统疾病的病理生理学，尤其是多发性硬化症（MS）。溶血磷脂酰胆碱（LPC）是一种与炎症相关的内源性溶血磷脂，可诱发快速损伤，对髓鞘脂质有毒性，导致局灶性脱髓鞘。本文给出了立体定向两点LPC注射的详细方案，可直接导致严重脱髓鞘，并通过外科手术在小鼠中快速稳定地复制实验性脱髓鞘损伤。因此，该模型与脱髓鞘疾病，特别是MS高度相关，并且可以促进相关的临床相关研究的进展。此外，使用免疫荧光和Luxol快速蓝色染色方法来描述注射LPC的小鼠胼胝体中脱髓鞘的时间过程。此外，该行为方法用于评估建模后小鼠的认知功能。总体而言， 通过 立体定位框架两点注射溶血磷脂酰胆碱是一种稳定且可重复的方法，可在小鼠中生成脱髓鞘模型以进行进一步研究。

LPC的两点注射导致更持久的脱髓鞘 LPC主要导致快速损伤，对髓鞘有毒性，轴突完整性15断裂。注射当天被视为第0天。将小鼠保存10-28天（10 dpi和28 dpi）。使用卢克索快速蓝（LFB）染色10来评估这些时间点小鼠脱髓鞘的区域。在两点注射模型中，与假组相比，10 dpi上存在显着的脱髓鞘，表明局部注射LPC可以成功地脱髓胼胝体。在28 dpi处仍然存在相对...

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A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice

本方案描述了 通过 立体定位框架两点注射溶血磷脂酰胆碱以在小鼠中产生稳定且可重复的脱髓鞘模型。

一种稳定建立的溶血磷脂酰胆碱诱导的小鼠局灶性脱髓鞘模型的两点注射

Receptor-mediated lysophospholipid signaling contributes to the pathophysiology of diverse neurological diseases, especially multiple sclerosis (MS). ...

我们的模型与脱髓鞘疾病高度相关，尤其是多发性硬化症。它有助于相关的临床相关研究。这是一个详细的方案，可以通过外科手术直接快速稳定地导致小鼠的严重脱髓鞘。第一次尝试此技术时，请耐心等待，并确保坐标准确无误。首先将 32 号针头连接到 5 微升注射器。取出5微升LPC溶液用于制备注射液。接下来，在呼吸顺畅时通过缺乏脚趾捏反射来确认鼠标的麻醉深度。将鼠标放在立体定位框架中，背侧朝上，并用鼻锥和齿夹固定头部。将鼠标固定在带有双侧耳条的立体定位装置上。确保耳杆水平，头部水平且稳定。用碘酒擦拭头部皮肤进行消毒。然后用手术刀沿着头皮中线切一个小切口约1.5厘米，露出头骨。用蘸有 1% 过氧化氢的棉签擦拭技能，直到后囟暴露在外。将注射器放在立体定位装置上。确保动物头部的水平定位，并定位胼胝体。确定注射部位后，记录坐标，并在头骨上用标记做标记。用骷髅钻轻轻地钻标记部位。注意避免出血。慢慢地将指针移动到给定的坐标。为了诱导胼胝体脱髓鞘，在每个注射部位以每分钟0.4微升的速度注射2微升LPC溶液。注射后，将针头在每个部位再保留10分钟。最后，用4-0缝合缝合皮肤，等待动物在10分钟内醒来。代表性图像显示了通过Luxol快速蓝色染色检测白质军团的过程。从结果来看，在注射后10天和28天可以看到胼胝体的稳定脱髓鞘。此处显示了不同时间点脱髓鞘区域的定量。通过免疫荧光染色降解的髓鞘碱性蛋白，在注射后10 d观察到LPC注射组的dMBP显着增加，这表示胼胝体中的髓鞘丢失。髓鞘碱性蛋白（MBP）表达的蛋白质印迹分析显示注射LPC后MBP明显丢失。β-肌动蛋白用作加载对照来测量基线表达。电子显微镜图像证实了假手术组和LPC注射组胼胝体中的髓鞘超微结构。GST-pi是少突胶质细胞前体细胞或OPC分化成熟的标志物。注射LPC10 d后，GST-pi 与假手术组相比有所下降。同时，少突胶质细胞的增殖能力可以通过少突胶质细胞增殖与总少突胶质细胞谱系细胞的比例来体现。较高的比率表示注射后10天后少突胶质细胞的增殖更多。代表性图像显示了小鼠在每组中的游泳路径，在平台存在的情况下以及移除平台后。结果表明，在两点LPC注射模型中，脱髓鞘小鼠的空间记忆明显受损。在尝试此过程时，请确保注射速率准确，并将针头在每个部位再保持 10 分钟。

Research

JoVE Journal

Neuroscience

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

焦距脑缺血模型大鼠大脑中动脉的血管内缝合闭塞

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

科研

神经科学

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

自体血液的注入模型小鼠自发性脑出血

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

在共同的文化整合模式中GABA能神经元的刺和HEK293细胞抑制性突触形成稳定表达GABA<sub&gt;一个</sub&gt;受体

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

实验脱髓鞘和小鼠脊髓髓鞘再生由溶血卵磷脂的焦点注射

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

大鼠部分视神经切断术: 建立一种新的手术方法评价视网膜神经节细胞二次变性的模型

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

由亲炎细胞因子的内脑注射诱导的广度脑皮质脱叶的鼠模型

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

重组腺相关病毒在成年小鼠中的直接注射

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

双直接注射血液到西斯特纳麦格作为苏巴拉奇内德出血的模型

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

A Rat Model of EcoHIV Brain Infection

生态HIV脑感染的老鼠模型

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...