早产儿脑病建模使用产前缺氧缺血大鼠羊膜腔内毒素
Summary
Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.
Abstract
早产儿的脑病（EOP）是包括中枢神经系统（CNS）的异常与早产有关的术语。为了最好预先平移目标，并发现新的治疗策略用于与早产有关的脑损伤，期末的临床前模型必须包括类似的机制产前全球损伤的观察人类和涉及的产妇胎盘，胎儿系统的多个组件。理想的情况下，模型应该产生的功能缺损的类似光谱在成熟动物和概括的病理生理的多个方面。为了模仿人体全身胎盘灌注缺损，胎盘underperfusion和/或绒毛膜羊膜炎与早期早产病原体引起的炎症有关，我们开发了产前瞬时全身缺血缺氧（TSHI）联合羊膜腔内脂多糖（LPS）的模型。在怀孕的SD大鼠，TSHI通过子宫动脉栓塞Øñ胚胎18天（E18）诱导随着胎儿中枢神经系统损伤有关分级胎盘underperfusion缺陷。当羊膜腔内注射LPS结合，胎盘炎症增加，中枢神经系统损伤中配合相关的白质，步态和影像学异常。产前TSHI和TSHI + LPS产前侮辱满足几个的期末模型，包括扼要宫内侮辱的标准，造成神经元，少突胶质细胞，板式损失和功能障碍在模仿那些在极其出生的孩子发现成年动物的损失早产。此外，这种模式使得炎症引起的不同损伤类型的清扫。
Introduction
随着37周之前估计胎龄1出生在美国的婴儿超过12％，围产期脑损伤（PBI）的早产儿是永久性残疾的显著原因。 PBI从早产，早产儿也称为脑病（EOP），影响整个中枢神经系统（CNS）。 CNS损伤往往开始在子宫内，并且由产前过程，包括绒毛膜和产后并发症如低氧和败血症加剧。 PBI从全身辱骂改变神经发育，导致脑性麻痹，癫痫，认知延迟和众多的神经精神障碍影响情绪调节，记忆和执行功能1,2-。虽然已经取得了很大的进步，在有限的理解仍然是来自早产中枢神经系统损伤的细胞和分子的后果如何转化到谁出生的早产儿在众人的神经系统后遗症。这种缺乏知识后的中枢神经系统损伤严重程度ERS实时诊断和新兴干预措施的通知剂量。此外，对于这些弱势患者群体年龄适当的治疗策略仍然难以实现。
宫内炎症是极度早产非常普遍，涉及复杂的胎儿产妇胎盘炎症级联3。宫内感染通常是亚临床型。具体胎盘发现与急性炎症，或组织学绒毛膜羊膜炎一致的，都是胎儿炎症反应的主要决定因素和重合与早产3-5有关的脑损伤。事实上，胎儿的炎症反应有从早产长期预后不同的临床意义。婴儿谁是小于胎龄儿（SGA）或谁遇到感染是非常脆弱的神经功能障碍3,4。绒毛膜羊膜炎是一种典型的病理诊断以下早产<sup> 6,7，和组织学检查发现炎症在胎盘中70％的出生非常早产4例迹象。此外，绒毛膜羊膜炎与认知功能障碍为两年8有关。在出生非常早产儿的产妇胎盘血管underperfusion的证据也与脑瘫儿童9关联。的绒毛膜羊膜炎及胎盘灌注缺损的协同影响在两年10,11岁是很好地说明通过非正常神经成果在该患者人群中显着高风险。
以模仿与病原体诱导的炎症相关的人类全身胎盘灌注缺损和绒毛膜，我们开发产前瞬态全身缺氧缺血（TSHI）结合羊膜腔脂多糖（LPS）的大鼠模型。我们的目标是要调整我们的TSHI模型独自在大鼠12-16包括宫内炎症，以方便与早产有关的中枢神经系统损伤的临床前模型。 TSHI单独揭示少突胶质细胞谱系细胞，皮层神经元的持续损失，增加的细胞死亡，和升高的促炎细胞因子水平，以渐进的缺血间隔导致伤害与产前脑损伤16一致的分级图案。修改该模型的缺血性成分也显示在记忆编码，短期和长期记忆和轻度肌肉骨骼改变障碍研究，因为它们17-19的年龄。实际上，我们以前表明，TSHI + LPS的组合概括期末的病理生理特点，包括少突胶质细胞和神经元损失，轴突损伤，细胞炎症和功能异常20。
Protocol
Representative Results
Discussion
早产儿脑病是困难的，因为病因，神经发育时间的推移，人类的大脑网络形成的复杂性，重叠损伤机制的复杂的相互作用的动物模型，以及中枢神经系统的不同表型侮辱体现在人类的早产儿。期末与特定细胞类型的漏洞相关联（即未成熟的少突胶质细胞）21，以及不同的发育调节的途径（即底板，膜转运和受体亚单位）12,13,22。然而，当动物模型复制人类生存条?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢丹Firl，克里斯·科贝特和杰西·丹森博士。资金是SR提供美国国立卫生研究院NINDS R01 NS060765，在P30铜业试点方案，LJ和儿童健康签名计划，以LJ在新墨西哥大学。
Materials
| Saline Solution, 0.9% | Sigma | S8776 | |
| LPS 011B4 | Sigma | L2630 | |
| Evan's Blue Dye | Sigma | E2129 | |
| Surgical gloves | Biogel | 40870 | |
| OR Towels | Cardinal Health | 287000-008 | Sterile |
| PDI Alcohol Prep Pads | Fisherbrand | 06-669-62 | |
| Mini Arco Rechargeable Clippers | Kent Scientific Corp. | CL8787 | |
| Betadine surgical scrub | Purdue Products L.P. | 67618-151-17 | |
| Eye Lubricant | Refresh Lacri Lube | 00023-0312 | |
| Blunt Forceps | Roboz | RS-8100 | |
| Scissors | Roboz | RS-6808 | |
| Surgical Scissors | Roboz | RS-5880 | |
| Surgical Scissors | F.S.T. | 14002-16 | |
| Syringe | BD | 309628 | 1 ml |
| Needle | BD | 305122 | 25G 5/8 |
| Needle | BD | 305128 | 30G 1 |
| Cotton-tipped Applicators | Fisherbrand | 23-400-114 | Small, 6 inch sterile |
| Cotton Gauze Sponge | Fisherbrand | 22-362-178 | |
| Needle Holders | Kent Scientific Corp. | INS600109 | 12.5 CM STR |
| Vessel Clips | Kent Scientific Corp. | INS600120 | 30G Pressure |
| 3-0 Perma Hand Silk Sutures | Ethicon | 1684G | Black braided, 3-0 (2 metric), 18", non-absorbable, PS-1 24mm needle, 3/8 circle |
| Insulin Syringes | BD | 328438 | 0.3cc 3mm 31G |
| Pentobarbital | |||
| Buprenorphine | |||
| Bupivacaine | |||
| Isoflurane | |||
| Lithium Carbonate | Acros Chemicals | 554-13-2 | |
| Superfrost Plus Microscope Slides | VWR | 48311-703 | |
| Hematoxylin | Leica | 3801521 | Surgipath Gill II Hematoxylin |
| Eosin | Leica | 3801601 | Surgipath Eosin |
| Xylenes | Fisherbrand | X3S-4 | Histological Grade |
| Permount | Fisherbrand | SP15-100 | |
| Coverglass | Fisherbrand | 12-548-5P | Fisher Finest Premium Coverglass |
References
- Blencowe, H., et al. Preterm birth associated neurodevelopmental impairment estimates at regional and global levels for 2010. Pediatr Res. 74, 17-34 (2013).
- Mwaniki, M. K., Atieno, M., Lawn, J. E., Newton, C. R. Long term neurodevelopmental outcomes after intrauterine and neonatal insults a systematic review. Lancet. 379, 445-452 (2012).
- Dammann, O., Leviton, A. Intermittent or sustained systemic inflammation and the preterm brain. Pediatr Res. 75, 376-380 (2014).
- Leviton, A., et al. Microbiologic and histologic characteristics of the extremely preterm infant’s placenta predict white matter damage and later cerebral palsy. the ELGAN study Pediatr Res. 67, 95-101 (2010).
- Redline, R. W. Inflammatory responses in the placenta and umbilical cord. Semin Fetal Neonatal Med. 11, 296-301 (2006).
- Lee, J., et al. Chronic chorioamnionitis is the most common placental lesion in late preterm birth. Placenta. 34, 681-689 (2013).
- Lee, S. M., et al. Acute histologic chorioamnionitis is a risk factor for adverse neonatal outcome in late preterm birth after preterm premature rupture of membranes. PloS one. 8, e79941 (2013).
- Pappas, A., et al. Chorioamnionitis and early childhood outcomes among extremely low gestational age neonates. JAMA Peds. 168, 137-147 (2014).
- Gaillard, R., Arends, L. R., Steegers, E. A., Hofman, A., Jaddoe, V. W. Second and third trimester placental hemodynamics and the risks of pregnancy complications the Generation R Study. Am J Epidemiol. 177, 743-754 (2013).
- Trivedi, S., et al. Fetal placental inflammation but not adrenal activation is associated with extreme preterm delivery. Am J Obstet Gynecol. 206, 236 (2012).
- Yanowitz, T. D., et al. Hemodynamic disturbances in premature infants born after chorioamnionitis association with cord blood cytokine concentrations. Pediatr Res. 51, 310-316 (2002).
- Jantzie, L. L., Corbett, C. J., Firl, D. J., Robinson, S. Postnatal Erythropoietin Mitigates Impaired Cerebral Cortical Development Following Subplate Loss from Prenatal Hypoxia Ischemia. Cereb Cortex. , (2014).
- Jantzie, L. L., et al. Erythropoietin attenuates loss of potassium chloride co transporters following prenatal brain injury. Mol Cell Neurosci. 61, 152-162 (2014).
- Jantzie, L. L., Miller, R. H., Robinson, S. Erythropoietin signaling promotes oligodendrocyte development following prenatal systemic hypoxic ischemic brain injury. Pediatric Res. 74, 658-667 (2013).
- Mazur, M., Miller, R. H., Robinson, S. Postnatal erythropoietin treatment mitigates neural cell loss after systemic prenatal hypoxic ischemic injury. J Neurosurg Peds. 6, 206-221 (2010).
- Robinson, S., et al. Developmental changes induced by graded prenatal systemic hypoxic ischemic insults in rats. Neurobiol Dis. 18, 568-581 (2005).
- Delcour, M., et al. Neuroanatomical sensorimotor and cognitive deficits in adult rats with white matter injury following prenatal ischemia. Brain Pathol. 22, 1-16 (2012).
- Delcour, M., et al. Impact of prenatal ischemia on behavior cognitive abilities and neuroanatomy in adult rats with white matter damage. Behav Brain Res. 232, 233-244 (2012).
- Delcour, M., et al. Mild musculoskeletal and locomotor alterations in adult rats with white matter injury following prenatal ischemia. Intl J Devel Neurosci. 29, 593-607 (2011).
- Jantzie, L. L., et al. Complex pattern of interaction between in utero hypoxia ischemia and intra amniotic inflammation disrupts brain development and motor function. J Neuroinflam. 11, 131 (2014).
- Back, S., et al. Selective vulnerability of late oligodendrocyte progenitors to hypoxia ischemia. J Neurosci. 22, 455-463 (2002).
- Jantzie, L. L., et al. Developmental Expression of N Methyl d Aspartate (NMDA) Receptor Subunits in Human White and Gray Matter Potential Mechanism of Increased Vulnerability in the Immature Brain. Cereb Cortex. 25, 482-495 (2015).
- Jantzie, L. L., Robinson, S. Preclinical Models of Encephalopathy of Prematurity. Devel Neurosci. , (2015).
- Workman, A. D., Charvet, C. J., Clancy, B., Darlington, R. B., Finlay, B. L. Modeling transformations of neurodevelopmental sequences across mammalian species. J Neurosci. 33, 7368-7383 (2013).
- Herlenius, E., Lagercrantz, H. Development of neurotransmitter systems during critical periods. Exper Neurol. 190, S8-S21 (2004).
- Kelsom, C., Lu, W. Development and specification of GABAergic cortical interneurons. Cell Biosci. 3, 19 (2013).
- Kinney, H., Back, S. Human oligodendroglial development Relationship to periventricualr leukomalacia. Semin Pediatr Neuro. 5, 180-189 (1998).
- Robinson, S., Li, Q., DeChant, A., Cohen, M. Neonatal loss of gamma amino butyric acid pathway expression after human perinatal brain injury. J Neurosurg Peds. 104, 396-408 (2006).
- Xu, G., et al. Late development of the GABAergic system in the human cerebral cortex and white matter. J Neuropathol Exp Neurol. 70, 841-858 (2011).
- Segovia, K. N., et al. Arrested oligodendrocyte lineage maturation in chronic perinatal white matter injury. Ann Neurol. 63, 520-530 (2008).
- Robinson, S., Li, Q., Dechant, A., Cohen, M. L. Neonatal loss of gamma aminobutyric acid pathway expression after human perinatal brain injury. J Neurosurg. 104, 396-408 (2006).
- Boksa, P. Effects of prenatal infection on brain development and behavior a review of findings from animal models. Brain Behav Immun. 24, 881-897 (2010).
- Burd, I., Brown, A., Gonzalez, J. M., Chai, J., Elovitz, M. A. A mouse model of term chorioamnionitis unraveling causes of adverse neurological outcomes. Repro Sci. 18, 900-907 (2011).
- Bergeron, J. D., et al. White matter injury and autistic like behavior predominantly affecting male rat offspring exposed to group B streptococcal maternal inflammation. Devel Neurosci. 35, 504-515 (2013).
- Uchida, K., et al. Effects of Ureaplasma parvum lipoprotein multiple banded antigen on pregnancy outcome in mice. J Reprod Immunol. 100, 118-127 .
