病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑
Summary
Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.
Abstract
在病毒性脑炎的发病机制的研究中，感染的方法是至关重要的。第一的两个主要感染途径到大脑的是血源性途径，其中涉及内皮细胞和脑的周细胞的感染。第二个是脑室内（ICV）的路线。一旦中枢神经系统（CNS）内，病毒可能会传播到经由脑脊液蛛网膜下腔，脑膜，和脉络丛。在实验模型中，中枢神经系统的病毒式分发的最初阶段不充分表征，并且目前还不清楚只有某些细胞是否最初感染。这里，我们已经分析了在感染的急性期巨细胞病毒（CMV）颗粒的分布，被称为主血症，ICV或血管内（IV）注射以下到新生小鼠的大脑。在ICV注射模型，5微升小鼠CMV（MCMV）或荧光微珠在midpoi分别注入侧脑室NT使用一个27克针10微升注射器耳朵和眼睛之间。在IV注射模型中，使用1-ml注射器用35克针。一个透射被用于可视化颞浅（面）新生小鼠的静脉中。我们注入50微升MCMV或荧光微球进入颞浅静脉。脑收集在不同时间点后喷射。 原位杂交方法，使用该检测MCMV基因组。通过荧光显微镜观察荧光微球或绿色荧光蛋白表达重组MCMV颗粒。这些技术可以应用到许多其它病原体调查脑炎的发病机制。
Introduction
当研究病毒性脑炎，病毒颗粒的初始分布是非常重要的，以了解疾病的发病机制和大脑识别病毒靶。大多数病毒范围从20至300纳米的尺寸，虽然潘多拉病毒是大小为1超过700nm。在感染的急性期的病毒颗粒的分布可取决于颗粒的尺寸，细胞受体的分布，或所述细胞受体的病毒的亲和性。在动物模型中，脑室内（ICV），腹膜内，直接胎盘和静脉内（IV）的感染已被用于研究病毒性脑炎的发病机制。 ICV接种病毒经常被用来建立在小鼠的中枢神经系统（CNS）的感染。使用这种技术的研究报告广泛感染，尤其是在脑室周围区域的细胞，并在与所述脑脊液（CSF），simila直接接触的大脑区域R以病毒ventriculoencephalitis的效果。腺相关病毒（AAV）的颗粒尺寸小（20 -直径为25纳米）有利于在整个ICV感染2-4大脑及其传播。 5腹腔内，胎盘直接6和静脉注射7代表造血全身用药。通过血-脑屏障（BBB）的病毒颗粒的渗透允许它们到达新生儿脑的实质，表示漫小胶质结节8,9。
巨细胞病毒（CMV）是属于疱疹病毒科的共同病毒。在美国，50％ – 在80％的人有过巨细胞病毒感染的40岁CMV感染是很少有害的，但可能会导致免疫功能低下患者和胎儿疾病。所有交付的，为0.2％ – 2％是天生的巨细胞病毒10，产生严重的症状，如小头畸形，脑室周围钙化，小脑发育不全，MICR眼炎，视神经萎缩11,12。此外，智力低下，耳聋，视觉缺陷，癫痫发作和癫痫发生非致命感染巨细胞病毒的婴儿13,14 10％左右。的CNS功能障碍是CMV先天性异常的最常见的典型症状。更多的孩子被永久每年由先天性巨细胞病毒不是唐氏综合症，胎儿酒精综合征或脊柱裂15禁用。有对抗CMV没有接种疫苗可在目前，要求需要一种安全有效的疫苗。研究在感染的最早阶段与它们的受体的CMV颗粒的相互作用是重要的是了解疫苗接种的效果。
Ventriculoencephalitis和小胶质细胞弥漫性结节是CMV的两个主要病理特征脑炎16。 （ – 300纳米150）在感染的急性期传播通过大脑的CMV粒子如何它一直不明朗第二如何细胞受体的分布以及它们的病毒的亲和性向病毒传播。川崎等人已经评估ICV和从颗粒以及它们在病毒感染的早期阶段受体（β1整合）的分布的透视IV感染。我们已发现，巨细胞病毒颗粒和β1整合素的表达的传播感染的两个侧脑室和IV感染8的最早阶段是公相关。 ICV感染是ventriculoencephalitis模型和IV感染是弥漫性结节小胶质细胞的典范。研究病毒或荧光颗粒的动力学将使颗粒尺寸，与细胞受体的病毒的相互作用，和BBB渗透的大脑中的机构的作用的有用信息。以下方案可用于调查在CNS任何病毒感染和病毒载体。
Protocol
Representative Results
Discussion
在动物模型中，ICV，腹膜内，直接胎盘和IV感染已被用于研究病毒性脑炎的发病机制。我们专注于新生小鼠的ICV和静脉注射模型的程序简单和直接喷射粒子的利益为目标区域。虽然腹腔内感染是一个简单的方法，病毒颗粒通过间接方法5,24全身蔓延。胎盘直接感染是一个很好的方法来研究胚胎全身感染。然而，该方法需要特别的训练，以产生稳定的结果，并具有低的成功率。感染的方?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Mr. Masaaki Kaneta, Ms. Hiromi Suzuki, and Ms. Mitsue Kawashima (Department of Regenerative and Infectious Pathology, Hamamatsu University School of Medicine) for their excellent technical assistance. This work was supported by the Japan Society for the Promotion of Science, KAKENHI Grant Number 23590445.
Materials
| Tris; tris(hydroxymethyl)- aminomethane | Sigma-Aldrich | T-6791 | |
| HCl | Sigma-Aldrich | H-1758 | |
| pEGFP-N1 vector | Clontech | #6085-1 | |
| D-sorbitol | Sigma-Aldrich | S-1876 | |
| SPHERO TM Fluorescent Polystyrene Nile Red 0.04-0.06 | Spherotech, Inc. | FP-00556-2 | |
| SPHERO TM Fluorescent Polystyrene Nile Red 0.1-0.3 | Spherotech, Inc. | FP-0256-2 | |
| SPHERO TM Fluorescent Polystyrene Nile Red 1.7-2.2 | Spherotech, inc. | FP-2056-2 | |
| 10% mouse serum | DAKO | X0910 | |
| C57BL/6 mouse | SLC, Inc. | ||
| ICR mouse | SLC, Inc. | ||
| Modified Microliter Syringes (7000 Series) | Hamilton company | ||
| 35-gauge needle | Saito Medical | ||
| A Wee Sight Transilluminator | Phillips Healthcare | 1017920 | |
| O.C.T.Compound | Sakura Finetek | 4583 | |
| RNase A | Sigma-Aldrich | R4642 | |
| Nonidet(R) P-40 | Nacalai | 25223-04 | |
| citrate buffer (pH6) x10 | Sigma-Aldrich | C9999-100ml | |
| pepsin | Sigma-Aldrich | P6887 | |
| EDTA | dojindo | N001 | |
| Formamide | TCI | F0045 | |
| Dextran sulfate sodium salt | Sigma-Aldrich | 42867-5G | |
| Denhardt's Solution (50X) | ThermoFishcer sceintific | 750018 | |
| Yeast tRNA (10 mg/mL) | ThermoFishcer sceintific | AM7119 | |
| SSC x20 | Sigma-Aldrich | S6639 | |
| DAPI | ThermoFishcer sceintific | D1306 | |
| n-Hexane | Sigma-Aldrich | 296090 | |
| superfrost plus glass | ThermoFishcer sceintific | 12-55-18 | |
| Cytokeep II | Nippon Shoji Co. | ||
| FITC-conjugated Griffonia simplicifolia isolectin B4 | Vector laboratories, Inc. | L1104 | |
| Anti-Mouse CD31 (PECAM-1) PE | ebioscience | 12-0311 | |
| ProLong Gold | ThermoFishcer sceintific | P36934 | |
| BIOREVO | KEYENCE | BZ-9000E | |
References
- Philippe, N., et al. Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes. Science. 341 (6143), 281-286 (2013).
- Passini, M. A., et al. Intraventricular brain injection of adeno-associated virus type 1 (AAV1) in neonatal mice results in complementary patterns of neuronal transduction to AAV2 and total long-term correction of storage lesions in the brains of beta-glucuronidase-deficient mice. J Virol. 77 (12), 7034-7040 (2003).
- Kim, J. Y., et al. Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualising and manipulating neuronal circuits in vivo. Eur J Neurosci. 37 (8), 1203-1220 (2013).
- McLean, J. R., et al. Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven AAV9 neonatal intracerebroventricular injection. Neurosci Lett. 576, 73-78 (2014).
- Hsu, K. M., Pratt, J. R., Akers, W. J., Achilefu, S. I., Yokoyama, W. M. Murine cytomegalovirus displays selective infection of cells within hours after systemic administration. J Gen Virol. 90. 90 (Pt 1), 33-43 (2009).
- Sakao-Suzuki, M., et al. Aberrant fetal macrophage/microglial reactions to cytomegalovirus infection. Annals of Clinical and Translational Neruology. 1 (8), 570-588 (2014).
- Gombash Lampe, S. E., Kaspar, B. K., Foust, K. D. Intravenous injections in neonatal mice. J Vis Exp. (93), e52037 (2014).
- Kawasaki, H., et al. Cytomegalovirus initiates infection selectively from high-level beta1 integrin-expressing cells in the brain. Am J Pathol. 185 (5), 1304-1323 (2015).
- Rahim, A. A., et al. Intravenous administration of AAV2/9 to the fetal and neonatal mouse leads to differential targeting of CNS cell types and extensive transduction of the nervous system. FASEB J. 25 (10), 3505-3518 (2011).
- Cannon, M. J., Davis, K. F. Washing our hands of the congenital cytomegalovirus disease epidemic. Bmc Public Health. 5, (2005).
- Frenkel, L. D., Keys, M. P., Hefferen, S. J., Rola-Pleszczynski, M., Bellanti, J. A. Unusual eye abnormalities associated with congenital cytomegalovirus infection. Pediatrics. 66 (5), 763-766 (1980).
- Becroft, D. M. Prenatal cytomegalovirus infection: epidemiology, pathology and pathogenesis. Perspect Pediatr Pathol. 6, 203-241 (1981).
- Conboy, T. J., et al. Intellectual development in school-aged children with asymptomatic congenital cytomegalovirus infection. Pediatrics. 77 (6), 801-806 (1986).
- Fowler, K. B., et al. The outcome of congenital cytomegalovirus infection in relation to maternal antibody status. N Engl J Med. 326 (10), 663-667 (1992).
- Cannon, M. J. Congenital cytomegalovirus (CMV) epidemiology and awareness. J Clin Virol. 46 Suppl 4, S6-S10 (2009).
- Grassi, M. P., et al. Microglial nodular encephalitis and ventriculoencephalitis due to cytomegalovirus infection in patients with AIDS: two distinct clinical patterns. Clin Infect Dis. 27 (3), 504-508 (1998).
- Kawasaki, H., Mocarski, E. S., Kosugi, I., Tsutsui, Y. Cyclosporine inhibits mouse cytomegalovirus infection via a cyclophilin-dependent pathway specifically in neural stem/progenitor cells. J Virol. 81 (17), 9013-9023 (2007).
- Britt, W. J. Human cytomegalovirus: propagation, quantification, and storage. Curr Protoc Microbiol. Chapter 14, Unit 14E 13 (2010).
- Kawasaki, H., Kosugi, I., Arai, Y., Iwashita, T., Tsutsui, Y. Mouse embryonic stem cells inhibit murine cytomegalovirus infection through a multi-step process. PLoS One. 6 (3), e17492 (2011).
- Gage, G. J., Kipke, D. R., Shain, W. Whole animal perfusion fixation for rodents. J Vis Exp. (65), (2012).
- Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. Cutting sections of paraffin-embedded tissues. CSH Protoc. , (2008).
- Chi, V., Chandy, K. G. Immunohistochemistry: paraffin sections using the Vectastain ABC kit from vector labs. J Vis Exp. (8), e308 (2007).
- Wilsbacher, L. D., Coughlin, S. R. Analysis of cardiomyocyte development using immunofluorescence in embryonic mouse heart. J Vis Exp. (97), (2015).
- Ohshima, M., et al. Intraperitoneal and intravenous deliveries are not comparable in terms of drug efficacy and cell distribution in neonatal mice with hypoxia-ischemia. Brain Dev. 37 (4), 376-386 (2015).
- Kim, J. Y., Grunke, S. D., Levites, Y., Golde, T. E., Jankowsky, J. L. Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction. J Vis Exp. (91), e51863 (2014).
- Glascock, J. J., et al. Delivery of therapeutic agents through intracerebroventricular (ICV) and intravenous (IV) injection in mice. J Vis Exp. (56), (2011).
