Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Brandon A. Miller; Shelei Pan; Peter H. Yang; Catherine Wang; Amanda L. Trout; Dakota DeFreitas; Sruthi Ramagiri; Scott D. Olson; Jennifer M. Strahle

doi:10.3791/63345

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

通过脑室内注射血红蛋白模拟新生儿脑室内出血

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07:57 min

August 25, 2022

DOI: 10.3791/63345-v

Brandon A. Miller^1,2, Shelei Pan³, Peter H. Yang³, Catherine Wang¹, Amanda L. Trout¹, Dakota DeFreitas³, Sruthi Ramagiri³, Scott D. Olson², Jennifer M. Strahle^3,4,5

¹Department of Neurosurgery,University of Kentucky, ²Department of Pediatric Surgery,University of Texas, ³Department of Neurological Surgery,Washington University in St. Louis School of Medicine, ⁴Department of Orthopedic Surgery,Washington University in St. Louis School of Medicine, ⁵Department of Pediatrics,Washington University in St. Louis School of Medicine

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Overview

This study presents a clinically relevant model for neonatal intraventricular hemorrhage (IVH) using rat pups. The introduction of hemoglobin into the ventricles mimics human pathology and allows for the evaluation of therapeutic strategies aimed at post-hemorrhagic hydrocephalus.

Key Study Components

Area of Science

Neuroscience
Neonatal brain injury
Modeling hydrocephalus

Background

Intraventricular hemorrhage (IVH) is a significant cause of neurological issues in neonates.
Current models lack the specificity to study the effects of hemoglobin and iron breakdown products.
Understanding IVH pathophysiology is critical for developing therapeutic interventions.
This study aims to fill the gap in modeling the pathology of IVH.

Purpose of Study

To develop a rat model of IVH that mimics the human condition.
To evaluate the impact of hemoglobin on ventricle size and its inflammatory effects.
To provide a platform for testing new therapies for IVH-related complications.

Methods Used

The protocol involves the stereotactic injection of hemoglobin into neonate rat ventricles.
Rat pups are anesthetized, and a 0.3 cm incision is made to access the skull for injection.
Post-injection MRI is utilized to assess brain changes over specific timeframes.
Measurements include ventricle volumes, inflammatory responses, and astrocyte activation.

Main Results

Hemoglobin injection resulted in significant ventriculomegaly and inflammatory cytokine elevation.
Astrocyte activation was notably higher in hemoglobin-injected pups compared to controls.
Differences in lateral ventricle volumes between treatment groups were observed over time.

Conclusions

This study presents a valuable model for investigating treatments for IVH and its complications.
The findings enhance understanding of IVH pathophysiology and the impact of hemoglobin.
Insights gained could lead to improved therapeutic strategies for neurological outcomes following IVH.

Frequently Asked Questions

What are the advantages of this model for studying IVH?

The model effectively mimics the human pathology of IVH, specifically the impact of hemoglobin breakdown products, making it relevant for therapeutic studies.

How is the hemoglobin injection administered?

Hemoglobin is injected into the lateral ventricles of anesthetized rat pups using a stereotactic apparatus, allowing precise delivery.

What outcomes are measured following the injection?

MRI scans are used to assess ventricular sizes, inflammatory markers, and astrocyte activation in the brain.

Can this method be adapted for other studies?

Yes, the technique allows for further investigations into CSF flow and its alterations stemming from IVH.

What are key limitations in this study?

The main limitation could be the long-term effects of hemoglobin on brain development and functionality, which needs further investigation.

What types of data can be obtained?

Data on ventricle volume changes, inflammatory responses, and astrocyte activity can be obtained through MRI and biochemical assays.

How long should monitoring after injection be conducted?

Monitoring should be conducted for at least 38 days post-injection to assess the long-term effects on ventricular size and inflammation.

我们提出了一种使用大鼠幼崽的新生儿脑室内出血模型，该模型模仿了人类的病理学。

该协议意义重大，因为我们开发了IVH诱导的脑损伤和脑积水的临床相关模型，利用血红蛋白注射到心室，这允许随后量化应用中的心室体积，以测试新的治疗策略。该技术的主要优点是它允许研究由血红蛋白和铁途径血液分解产物特异性介导的IVH的病理学。这种技术也易于使用且用途广泛。

该技术的意义延伸到开发出血后脑积水的治疗方法，因为它可以进一步了解IVH病理生理学和临床相关治疗策略的价值，以预防IVH后的神经系统后遗症。演示该程序的将是澳大利亚实验室的副博士后Sruthi Ramagiri。首先将麻醉大鼠俯卧在立体定向装置中，其鼻子位于麻醉适配器中。

然后通过收紧外耳道上的未破裂耳条来固定头部。要清洁头部，首先触摸头部中央浸泡在Betadine中的无菌棉头涂抹器，然后向外移动以将Betadine打圈。然后用浸泡在70%乙醇中的涂抹器重复该过程。

接下来，使用手术刀，在头部中心垂直做一个0.3厘米的切口，以露出头骨的后膛。然后使用棉头涂抹器擦干该区域。为了设置立体定向注射器，将先前制备的血红蛋白溶液吸入0.3或0.5毫升注射器中，并将注射器置于立体定向注射器系统中。

接下来，打开立体定向进样器界面，点击配置按钮，进入进样量和倍率设置。单击体积，然后将体积设置为 20， 000 纳升。然后单击输注速率，并将速率设置为每分钟 8， 000 纳升。

单击重置位置按钮退出配置。通过单击输注按钮冲洗针尖，直到针尖出现一小颗血红蛋白珠，然后使用棉尖涂抹器轻轻取出珠子。要开始动物注射，通过调整注射器的中外侧和前后位置，将立体定向注射系统上的bregma设置为零。

然后放下注射器针头，轻轻触摸前膛处的颅骨。确定选择的坐标后，将注射器针头抬高到颅骨上方一厘米处以清除头皮。设置中外侧和前后坐标。

然后放下注射器，让针头轻轻接触颅骨，并将背腹坐标设置在 30 秒内。设置坐标后，通过单击立体定向注射器界面上的运行按钮开始注射。注射完成后，将针头留在原位两分钟，以尽量减少溶液的回流。

接下来，沿着背腹坐标缓慢抽出注射器两分钟，直到针尖在头皮上方两厘米处。然后将立体定向注射器臂旋转远离手术区域，并用6-O单丝缝合线关闭头皮。对于 MRI，通过选择 T2 加权快速自旋回波序列进行 T2 加权成像。

按照文本中所述设置 MRI 参数后，单击继续按钮开始序列。对于图像处理和脑容量分析，请在分割软件中打开原生 T2 加权数据。要手动描绘侧心室，请单击画笔模式并选择方形画笔样式，然后将画笔大小调整为一个。

接下来，单击布局检查器，选择轴向视图，然后单击缩放以适合。然后将光标放在图像上并跟踪并填充侧心室空间。最后，单击工具栏中的分段，然后单击卷和统计信息以查看分段的卷。

接受血红蛋白注射的动物在通过MRI评估时出现中度脑室肿大。与注射ACSF的动物相比，注射血红蛋白后24小时和72小时的侧脑室明显较大。虽然两组大鼠在38天后侧心室容积的差异不显著，但MRI扫描显示，接受血红蛋白的大鼠中有44%仍有未解决的脑室肿大。

此外，与ACSF相比，注射血红蛋白导致38天后白质体积显着减少。血红蛋白还促进了体内的炎症反应，这从注射血红蛋白后促炎细胞因子肿瘤坏死因子α的显着升高水平明显，而不是盐水或全血。此外，神经胶质原纤维酸性蛋白的免疫组织化学染色显示，血红蛋白注射后星形胶质细胞的活化明显高于ACSF注射后。

尝试此程序时要记住的最重要的事情是设置正确的注射坐标，以确保血红蛋白输送到侧脑室。按照此程序，可以将脑脊液示踪剂输送到脑脊液间隙，以评估脑脊液循环的变化以及IVH后的外排模式。这种附加方法解决了IVH PHH中脑脊液血流如何改变的问题。

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