Name: a ferret model of inflammation-sensitized late preterm hypoxic-ischemic brain injury
Uploaded: 2019-11-19T12:30:00
Description: Watch this Scientific Journal Video about A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury at JoVE.com

该模型的开发由比尔和梅林达·盖茨基金会以及NIH赠款5R21NS093154-02（NICHD）资助。

程序是按照NIH《实验室动物护理和使用指南》和华盛顿大学机构动物护理和使用委员会批准的协议的一部分进行的。
1. 准备和LPS管理
注：有关过程的时间表，请参阅图 1。
<ol><li>在开始手术之前，密封、消毒和高压灭菌所有手术器械和手术窗帘。在无菌小瓶中准备术前药物。计算在 8–10 分钟内用实验气体替换缺氧/高...

<ol>
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由于雪铁龙大脑和人脑之间的物理和发育相似性，雪铁龙正越来越多地被用来模拟成人和发育性脑损伤。8，9，10，11，12。然而，迄今为止的研究表明，雪铁龙大脑既对初始损伤有抵抗力，而且具有高度可塑性，行为缺陷会随着时间而减少，即使在可见的病理损...

目前需要大型动物模型，以反映早产和围产期缺氧-缺血症的病理生理学，在其中可以测试婴儿的治疗干预措施。2017年，在美国出生的382，726名婴儿中，9.93%是早产儿，其中84%的婴儿在妊娠32至36周之间出生。在早产儿中，围产期接触感染或炎症很常见，其中由于病毒或细菌病原体而使产妇免疫活化可以启动早产。产后，早产儿有早或晚患败血症的高风险2。早产儿也经常经历缺氧、低血压和高血糖期，原因是其心肺系统不成熟，大气中的氧气紧张程度高于子宫内的婴儿，以及异体性暴露。此外，在早产儿中，抗氧化防御是不成熟的3，亲凋亡因子自然调节4。氧化应激和细胞死亡导致免疫系统的激活和神经炎症。这些综合因素被认为有助于大脑的发育和生理脆弱性，并导致或加剧与早产儿5、6、7发育不良结果相关的脑病。
由于雪铁龙大脑与人脑在物理和发育上的相似性，雪铁龙是一种有吸引力的物种，可以模拟脑损伤8、9、10、11、12。Ferrets也是模拟早产儿大脑的理想候选者，因为它们出生时是脑电，产后发育有陀螺的大脑，这为发育中的大脑暴露在侮辱中提供了一个窗口，模仿早产儿经历的侮辱。出生时，雪铁龙大脑发育类似于13周的人类胎儿，产后（P）17试剂盒被认为相当于妊娠13周32-36周的婴儿。
我们小组最近发布了一个模型，在P10雪铁龙中，通过结合炎症敏感性与大肠杆菌脂多糖（LPS）与随后接触缺氧和高血糖12的异常早产（<28周妊娠）脑损伤。在下面的协议中，我们现在描述了P17雪铁龙的晚期早产模型，LPS敏化之后是双边脑缺血、缺氧和高血糖。这导致一部分动物的损伤更为严重，并更密切地模拟了一些先期婴儿出现脑损伤的长期炎症、缺血、缺氧和氧化应激的复杂相互作用。

<table border="0" cellpadding="0" cellspacing="0" style="width:775px;" width="775">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">80% Oxygen</td>
			<td style="width:196px;">Praxair</td>
			<td style="width:136px;"></td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">9% Oxygen</td>
			<td>Praxair</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Absorbent benchtop protector</td>
			<td style="width:196px;">Kimtech</td>
			<td align="right" style="width:136px;">7546</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Automated catwalk</td>
			<td style="width:196px;">Noldus</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Betadine surgical scrub</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bupivacaine</td>
			<td>Patterson Veterinary</td>
			<td>07-888-9382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buprenorphine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calipers</td>
			<td style="width:196px;">SRA Measurement Products</td>
			<td style="width:136px;">ME-CAL-FP-200</td>
			<td>200mm range, .01 mm resolution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cotton Gauze Sponge</td>
			<td>Fisher Scientific</td>
			<td>22028556</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Curved fine hemostat</td>
			<td>Roboz</td>
			<td>RS-7101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Curved forceps</td>
			<td>World Precision Instruments</td>
			<td>501215</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Curved suture-tying hemostat</td>
			<td>Roboz</td>
			<td>RS-7111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethovision tracking software</td>
			<td style="width:196px;">Noldus</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eye Lubricant</td>
			<td>Rugby</td>
			<td>NDC 0536-1970-72</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ferrets (Mustela putorius furo)</td>
			<td style="width:196px;">Marshall Biosciences</td>
			<td style="width:136px;"></td>
			<td>Outbred (no specific strain)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formalin</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:136px;">SF100-4</td>
			<td>10% (Phosphate Buffer/Certified)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hair Clippers</td>
			<td>Conair</td>
			<td>GMT175N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin Syringes</td>
			<td>BD</td>
			<td>329461</td>
			<td>0.3 cc 3 mm 31G</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>Piramal</td>
			<td>66794-017-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lidocaine</td>
			<td>Patterson Veterinary</td>
			<td>07-808-8202</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LPS</td>
			<td>List Biological</td>
			<td>LPS Ultrapure #423</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygen sensor</td>
			<td>BW Gas Alert</td>
			<td>GAXT-X-DL-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pentobarbital</td>
			<td style="width:196px;"></td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic chamber</td>
			<td>Tellfresh</td>
			<td align="right">1960</td>
			<td>10L; 373x270x135mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Saline Solution, 0.9%</td>
			<td>Hospira</td>
			<td>RL-4492</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel blade</td>
			<td>Integra Miltex</td>
			<td>297</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel handle</td>
			<td>World Precision Instruments</td>
			<td>500236</td>
			<td>#3, 13cm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile suture</td>
			<td>Fine Science Tools</td>
			<td>18020-50</td>
			<td>Braided Silk, 5/0</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical clip applicator</td>
			<td>Fine Science Tools</td>
			<td>12020-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical clip remover</td>
			<td>Fine Science Tools</td>
			<td>12023-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical drapes</td>
			<td>Medline Unidrape</td>
			<td>VET3000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical gloves</td>
			<td>Ansell Perry Inc</td>
			<td>5785004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surigical clips</td>
			<td>Fine Science Tools</td>
			<td>12022-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thermometer (rectal)</td>
			<td>YSI</td>
			<td>Precision 4000A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thermometer (water)</td>
			<td>Fisher Scientific</td>
			<td>14-648-26</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Umbilical tape</td>
			<td>Grafco</td>
			<td>3031</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:136px;">TSCOL19</td>
			<td>19L</td>
		</tr>
	</tbody>
</table>

a ferret model of inflammation-sensitized late preterm hypoxic-ischemic brain injury

目前需要临床上相关的围产期感染和缺氧-缺血（HI）模型，以测试早产症患者的治疗干预措施。铁杉是模拟早产儿大脑的理想候选者，因为它们出生时是脑电，产后发育有陀螺脑。出生时，雪铁龙大脑发育类似于13周的人类胎儿，产后（P）17个试剂盒被认为相当于妊娠32-36周的婴儿。我们描述了P17雪铁龙中的损伤模型，其中脂多糖组分后为双边脑缺血、缺氧和高血糖。这模拟了许多出现脑损伤的新生儿经历的长期炎症、缺血、缺氧和氧化应激的复杂相互作用。受伤的动物表现出一系列严重的损伤严重性，大脑中的形态变化包括缩小多个皮质陀螺和相关的硫化物。受伤的动物也表现出缓慢的反射发育，在自动走道中运动速度较慢和变速，在开阔的田野中减少探索。该模型提供了一个平台，用于测试与炎症和HI相关的新生儿脑病婴儿的假定疗法，研究影响皮质发育的损伤机制，并研究在不受影响的动物。

在受伤群体中，来自6个垃圾中的34只（n = 18男，n=16雌性）动物中，有8只动物（24%;n = 4雄，n=4雌性）在第二个缺氧期（n = 5），在温度管理期间（n = 2），或在侮辱后过夜（n = 1）。在受伤群体中，26名幸存者中有9人（35%）有明显的严重伤害。5只动物（n = 5只雄性）有中度损伤，4只动物（n = 2雄，n =2雌性）有严重伤害，定义为病理评分分别为2⁄5和6+9（图2A）。因?...

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A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury

该方法描述了P17雪铁龙中发炎意识低氧缺血和高氧脑损伤，以模拟一些晚期早产儿经历的长期炎症和氧化性脑损伤之间的复杂相互作用。

炎症敏感晚期缺氧缺血性脑损伤的Ferret模型

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for ...

该协议是重要的，因为它提供了第一个模型的炎症敏感过早脑损伤的雪铁龙，导致严重和持续的伤害。这种技术涉及长期侮辱，其中纳入许多触发因素和生化机制，被认为有助于在早产儿看到的脑损伤类型。展示大脑测量程序的将是凯莉·科里，一个研究科学家在我们的实验室和奥利维亚·怀特，一个本科生。手术开始前30分钟，施用每公斤LPS IP3毫克，以在损伤组中安装成试剂盒，并施用相当于每克三微升的无菌盐水，以控制动物。在开始手术之前，使用小动物剪子去除长方形图案雪铁龙腹颈区域的所有毛发，注意避免剥皮或产生热疹。对剃光区域进行局部麻醉，用三个交替的拭子对皮肤进行碘和七十分之七十乙醇的消毒。在通过头趾捏确认适当的水平的电合后，用暴露颈部区域的无菌一次性切口窗帘盖住动物。对于双边胡萝卜动脉结扎，使用单用数十手术刀刀片在颈部中心做一个1.5厘米的中线切口，并使用精细的半边形和弯曲的钳子钝解到左胡萝卜动脉。将暴露的动脉从相关的神经血管束中解剖。并使用一对弯曲的细钳子在动脉下通过一个循环十厘米长的无菌 5/0 丝缝合线。将缝合线切成两半，并牢固地将两个缝合线长度绑在一起，以连接动脉，在结之间留下至少两毫米。在缝合线之间穿开左角动脉，注意保持神经完好无损，并在右侧重复解剖。用单无菌 1/8 英寸脐带将右侧胡萝卜动脉逆转。用手术性皮肤夹关闭伤口。然后，让动物在温度控制的水浴中恢复至少30分钟，在缺氧前进行监测。对于连续缺氧、高氧和缺氧治疗，将动物放在水浴内气密室中。持续监测室内的氧气浓度，以及至少一个哨兵动物的直肠温度。用加湿的 9% 氧气和 91% 的氮气冲洗腔室，并保持每分钟 3 到 5 升的流速，具体取决于腔室大小。一旦腔室中的氧气浓度达到百分之九，继续输送三十分钟。在缺氧治疗结束时，将供气切换到 80% 的加湿氧气和 20% 的氮气。经过三十分钟的超氧，打开腔室，让其更快速地到达诺莫夏，与室内空气平衡，然后密封室并再次用9%的加湿氧气冲洗。在第二轮缺氧期间持续监测所有动物，注意任何显示过节性呼吸的动物。一旦腔室中的氧气浓度达到百分之九，继续治疗缺氧三十分钟。在治疗结束时，使用弯曲钳子从右肌动脉识别和解开脐带。用手术性皮肤夹重闭伤口。然后将所有试剂盒返回 60 分钟的 jills 进行护理和恢复。在恢复期结束时，将受伤的动物返回37至40摄氏度的水浴场，持续6小时，根据需要调整水温，保持36至37摄氏度的直肠温度。然后，再次返回套件到他们的 jills。在适当的实验终点处进行大脑采集和固定后，将电子卡钳的尖端放在每个大脑的后腹和腹面上，以测量每个受伤和非受伤大脑的高度。将卡钳的尖端放在时间叶的最横向部分，以测量每个大脑的宽度并称量每个大脑。使用卡钳测量纵向裂缝，所有从相应硫化物最明显部分的开始和结束的硫化物，以及从每个相应陀螺的最宽面的所有陀螺。然后，将卡钳的一个尖端放在纵向裂痕的最后点，将卡钳的另一个尖端放在小脑最后部，以测量暴露小脑的数量。在这项研究中，6只动物中的34只受到侮辱，其中5只动物中度受伤，4只动物严重受伤。随着伤害的增加，观察到时间和/或腹叶的陀螺变变窄，在最严重受伤的动物中，纵向裂变和更大的囊组织损失区域会缩短和扩大。在反射测试期间，受伤的动物在负地理轴任务中显示旋转时间较慢。在"悬崖厌恶"任务中，从边缘旋转的较慢的时间，以及向右旋转的较慢的时间。在走道中，受伤的动物表现出与控制装置相似的平均速度，但在每次跑步过程中表现出显著的速度变化。在受伤的动物中，爪子和后爪之间的重量调整距离明显较大，通过四爪的单位爪子区域施加的压力较小。在开阔的田野里，受伤的动物覆盖的总距离更少，停止频率更低，在场地中心停留的时间明显更多，在角落停留的时间更少。在这里，可以观察到有代表性的控制和受伤动物的热图。注意从伴随的神经中解剖动脉，并留出最大的可能缺氧时间。这些步骤将确保大多数动物存活下来，同时也遭受伤害。按照这个程序，任何生化，病理或行为结果可以评估，包括评估通常仅限于啮齿动物模型的脑损伤。

Research

JoVE Journal

Neuroscience

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

侧向液压冲击：创伤性脑损伤小鼠模型

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

科研

神经科学

Preterm EEG: A Multimodal Neurophysiological Protocol

早产儿的脑电图：多式联运神经生理学议定书

Since its introduction in early 1950s, electroencephalography (EEG) has been widely used in the neonatal intensive care units (NICU) for assessment and ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

方法对敌人造成闭合性颅脑创伤性脑损伤<em&gt;果蝇</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

背根神经节内注射和背根挤压伤为感官轴突再生模型

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

小鼠新生儿缺氧缺血性脑损伤模型的神经行为评估

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

A Novel In Vitro Model of Blast Traumatic Brain Injury

一种新的脑外伤体损伤模型

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

创伤性脑损伤诱导受控皮质冲击的母模型

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

小鼠轻度创伤性脑损伤的电磁控制闭头模型

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...