通过脑室内注射血红蛋白模拟新生儿脑室内出血
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
Summary
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。
Abstract
新生儿脑室内出血 （IVH） 是早产的常见后果，可导致脑损伤、出血后脑积水 （PHH） 和终生神经功能缺损。虽然PHH可以通过暂时性和永久性脑脊液（CSF）分流程序（分别为心室储液器和脑室腹腔分流术）进行治疗，但没有药物策略来预防或治疗IVH诱导的脑损伤和脑积水。需要动物模型来更好地了解IVH的病理生理学并测试药物治疗。虽然有现有的新生儿IVH模型，但那些可靠地导致脑积水的模型通常受到大容量注射的必要性的限制，这可能会使病理建模复杂化或引入观察到的临床表型的变异性。
最近的临床研究表明，血红蛋白和铁蛋白会导致脑室内扩大。在这里，我们开发了一种简单的动物模型，该模型利用血液分解产物血红蛋白的小容量脑室内注射来模拟PHH的临床表型。除了可靠地诱导心室扩大和脑积水外，该模型还会导致脑室周围和白质区域的白质损伤、炎症和免疫细胞浸润。本文描述了这种临床相关的简单方法，用于使用脑室内注射模拟新生大鼠的IVH-PHH，并提出了量化注射后心室大小的方法。
Introduction
新生儿脑室内出血性脑室内出血起源于生发基质，生发基质是与发育中的大脑侧脑室相邻的快速细胞分裂部位。这种高度血管结构容易受到与早产相关的血流动力学不稳定的影响。在生发基质出血（GMH）-IVH中，当生发基质内脆弱的血管破裂时，血液被释放到侧脑室。在IV级IVH的情况下，脑室周围出血性梗死也可能导致脑内血液制品的释放。1 GMH-IVH联合使用可能导致PHH，特别是在高度出血（III级和IV级）后1。PHH 可通过放置脑室腹腔分流术进行治疗，但分流放置不能逆转 IVH 可能引起的脑损伤。尽管现代新生儿重症监护降低了 IVH2、 3 的发病率，但一旦发生 IVH 引起的脑损伤或脑积水，尚无特异性治疗方法。开发IVH诱导的脑损伤和PHH的预防性治疗的一个重大限制是对IVH病理生理学的不完全理解。
最近，关键血液分解产物血红蛋白的早期脑脊液水平已被证明与高级别IVH4新生儿PHH的后期发展有关。此外，铁处理途径蛋白（血红蛋白、铁蛋白和胆红素）的脑脊液水平与新生儿 IVH 的心室大小有关。在一项多中心早产儿队列中也显示了这一点，其中较高的脑室脑脊液铁蛋白水平与较大的心室大小5相关。
在这项研究中，我们开发了一个临床相关的IVH诱导的脑损伤和脑积水模型，利用血红蛋白注射到脑室，可以量化脑损伤和PHH，并测试新的治疗策略（图1）6， 7。该IVH模型利用新生大鼠幼崽，在手术过程中将其置于全身麻醉下。在头皮上做一个中线切口，并使用从颅骨标志（前辰或λ）得出的坐标来瞄准侧脑室进行注射。使用输液泵缓慢注射将血红蛋白输送到心室。该协议易于使用，用途广泛，并且可以模拟导致PHH的IVH的不同成分。
Protocol
注意：所有动物方案均已获得机构的动物护理和使用委员会的批准。有关本协议中使用的所有材料、试剂、设备和软件的详细信息，请参阅 材料表 。 1.血红蛋白和脑脊液的制备 通过将 500 μL aCSF 溶液添加到 1.5 mL 微管中并储存在冰上来制备无菌人工 CSF （aCSF） 溶液。 通过在 1.5 mL 微管中将 75 mg 血红蛋白添加到 500 μL aCSF 中制备无菌 150…
Representative Results
通过放射学和免疫组织化学手段证实了注射的成功。通过MRI评估时，接受血红蛋白注射的动物发生中度急性脑室肿大（图2A），与注射aCSF的动物相比，注射血红蛋白后24小时和72小时的侧脑室显着更大（图2B，C）。虽然注射后38天注射血红蛋白和注射aCSF的动物在侧脑室容积方面没有显着差异（图2D），但重要的是要注意，注射后38天的血红蛋…
Discussion
这种利用血红蛋白注射的IVH模型允许研究由血红蛋白特异性介导的IVH的病理学。对于补充研究，血红蛋白也可以很容易地 在体外 递送，并且不会混淆全血中存在的小胶质细胞/巨噬细胞产生的蛋白质的生化测定。
IVH-PHH的主要理论包括脑脊液循环的机械性阻塞，室管膜壁内壁纤毛的破坏，炎症，纤维化和铁毒性10。现有的IVH动物模型，例如胶原酶诱导?…
Declarações
The authors have nothing to disclose.
Acknowledgements
JMS获得了NIH / NINDS R01 NS110793和K12（神经外科医生研究职业发展计划）的资助。BAM获得了NIH / NINDS K08 NS112580-01A1，肯塔基大学神经科学研究优先领域奖和 脑积水协会 创新奖的资助。
Materials
| 0.3 mL insulin syringe | BD Microfine + Insulin Syringe | 230-4533 | 0.3-0.5 mL synringes will work |
| 1.5 mL microtube | USA Scientific | 1615-5500 | Lot No. K194642H -3 511 |
| 4.7T MRI | Agilent/Varian | 4.7T/33 cm | Agilent/Varian DirectDrive 4.7-T (200-MHz) MRI system |
| 6-0 monofilament suture | ETHICON | 667G | |
| 9.4T MRI | Bruker | BioSpec 94/20 | Used in this protocol without the cryoprobe |
| Analytical balance | CCURIS Instruments | W3200-320 | |
| Artificial CSF (aCSF) | Tocris Bioscience | 3525 | Batch No: 72A |
| Betadine | Purdue Products L.P. | 301005-00 | NDC 67618-150-09 |
| Carprofen (injectable) | Zoetis Inc. | PI 4019448 | Rimadyl |
| Ethanol | Decon Laboratories | 2701 | |
| Heating pad | Sunbeam | E12107-819 | UL 612A, Z-1228-001 |
| Hemoglobin | MP Biomedicals | 100714 | LOT NO. SR02321 |
| Isoflurane | Piramal Critical Care | NDC 66794-017-25 | |
| Isoflurane vaporizer | VETEQUIP | 911103 | |
| Light for stereotactic insturment | Dolan-Jenner industries | Fiber-Lite MI-150 | |
| Microinjection syringe pump | World Precision Instruments | MICRO21 | Serial 184034 T08K |
| MRI software | Bruker BioSpin | Paravision 360 3.2 | |
| Oxygen | Airgas Healthcare | UN1072 | LOT NUMBER S1432080XA02 |
| Sprague Dawley rats | Charles River Laboratories | Strain code: 001 | |
| Stereotactic instrument | KOPF Instuments | Model 900LS Lazy Susan | |
| Sterile cotton tipped applicator | Fischerbrand | 23-400-118 | |
| Surgical blade | covetrus | #10 | |
| Topical triple antibiotic | Triple Antibiotic Ointment | NDC 51672-2120-1 | |
| Ventricle volume quantification software | ITK-SNAP | ITK-SNAP 4.0.0 beta |
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Citar este artigo
Miller, B. A., Pan, S., Yang, P. H., Wang, C., Trout, A. L., DeFreitas, D., Ramagiri, S., Olson, S. D., Strahle, J. M. Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin. J. Vis. Exp. (186), e63345, doi:10.3791/63345 (2022).