Name: zika virus infection of cultured human fetal brain neural stem cells for immunocytochemical analysis
Uploaded: 2018-02-05T14:30:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis at JoVE.com

이 작품은 존 S. 던 재단과 인간 감염 및 면역 텍사스 대학 의료 지점 (외무장관)의 연구소에서 기금에 의해 지원 되었다.

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertical+transmission+of+Zika+virus+targeting+the+radial+glial+cells+affects+cortex+development+of+offspring+mice.">Vertical transmission of Zika virus targeting the radial glial cells affects cortex development of offspring mice.</a> Cell Res. 26 (6), 645-654 (2016).</li><li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Mol Neurobiol. 34 (3), 153-161 (2006).</li><li>Wu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Region-specific+generation+of+cholinergic+neurons+from+fetal+human+neural+stem+cells+grafted+in+adult+rat.">Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat.</a> Nat Neurosci. 5 (12), 1271-1278 (2002).</li><li>McGrath, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Responses+of+Human+Fetal+Brain+Neural+Stem+Cells+to+Zika+Virus+Infection.">Differential Responses of Human Fetal Brain Neural Stem Cells to Zika Virus Infection.</a> Stem Cell Reports. 8 (3), 715-727 (2017).</li><li>Svendsen, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+rapid+and+long+term+growth+of+human+neural+precursor+cells.">A new method for the rapid and long term growth of human neural precursor cells.</a> J Neurosci Methods. 85 (2), 141-152 (1998).</li><li>Tarasenko, Y. I., Yu, Y., Jordan, P. M., Bottenstein, J., Wu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+growth+factors+on+proliferation+and+phenotypic+differentiation+of+human+fetal+neural+stem+cells.">Effect of growth factors on proliferation and phenotypic differentiation of human fetal neural stem cells.</a> J Neurosci Res. 78 (5), 625-636 (2004).</li><li>Barrows, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Screen+of+FDA-Approved+Drugs+for+Inhibitors+of+Zika+Virus+Infection.">A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection.</a> Cell Host Microbe. 20 (2), 259-270 (2016).</li><li>Jakel, R. J., Schneider, B. L., Svendsen, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+human+neural+stem+cells+to+model+neurological+disease.">Using human neural stem cells to model neurological disease.</a> Nat Rev Genet. 5 (2), 136-144 (2004).</li><li>Lopez-Garcia, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+stretch-induced+neurotrauma+model+for+medium-throughput+screening+in+vitro:+identification+of+rifampicin+as+a+neuroprotectant.">Development of a stretch-induced neurotrauma model for medium-throughput screening in vitro: identification of rifampicin as a neuroprotectant.</a> Br J Pharmacol. , (2016).</li><li>Mich, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+functionally+distinct+stem+cells+and+neurosphere-initiating+cells+in+adult+mouse+forebrain.">Prospective identification of functionally distinct stem cells and neurosphere-initiating cells in adult mouse forebrain.</a> Elife. 3, e02669 (2014).</li></ol>

저자는 선언 하는 관심의 없습니다 충돌 있다.

다양 한 용도로 높은 처리량 약10,11,,1214, 심사에 인간의 질병을 모델링에서 사용할 수 있는 중요 한 도구를 제공 하는 문화 및 hNSCs를 조작 하는 기능 15,,1617. 많은 질문에 어떻게 인간의 태아 두뇌 NSCs 등 해결 되어야 남아 ...

Zika 바이러스 (ZIKV)는 성적으로, 또는 모기 벡터 Aedes aegypti , Aedes albopictus 모기에 의해 전송 flavivirus. ZIKV 전송의 용이성으로 인해 심각한 공중 보건 위협으로 최근 확인 됐다 고 신경 증상1제휴. 하나는 대부분의 감염 된 임산부2,3태어난 태아에 microcephaly의 개발은 신경 효과 관한. Microcephaly 머리가 일반 크기 보다 작은 태아 개발 중 그리고 출생 시, 평균4아래 2 표준 편차 보다 작은 원주와 neurodevelopmental 장애입니다. 작은 머리 둘레 comorbidities 발달 지연, 발작, 비전 및 청력 손실, 및 어려움 먹이 등의 다양 한 일반적으로 동반 됩니다.
최근 연구 동물 모델 또는 유도 만능 줄기 세포 neurodevelopment5,6,,78ZIKV 감염의 효과 연구 하는. 이러한 연구는 ZIKV, 다른 종류의 사용의 우리의 지식에 공헌 했다 또는 유전자 변형된 세포 시간이 소요 될 수 있습니다 또는에 ZIKV의 효과 혼동 수 있습니다 추가적인 변수를 추가 하는 동안5, 세포 신경 개발 6 , 7 , 그러나 8., hNSC 문화, 특히 비 점착 neurosphere 문화가이 프로토콜에서 설명 된 어려움은 문화 문화9를 수행 하는 데 사용 하는 방법에 아주 과민 하다. 중간 구성 요소, 또는 심지어 문화 선박의 물리적 처리에 어떤 변화 든 지 셀9에서 반응을 유도 충분 하다. 이러한 문제를 해결 하기 위해 우리는 생체 외에서 인간 태아 두뇌 파생 된 신경 줄기 세포 (hNSCs) 문화 mechanistically 태아 신경 줄기 세포에 ZIKV의 효과가 개발. 우리의 방법을 사용 하 여, hNSCs는 명백한 phenotypic 변경10없이 80 구절에 대 한 유지 되었다. 또한, trisomy 같은 염색체 변경 했다 없음 또는 최소한의11. 이 hNSC 문화 비 점착 neurosphere 문화로 자 랍니다. Neurospheres의 장점 중 하나는 구체 2 차원 문화9에 비해 vivo에서 틈새의 더 반사 이다 문화에 독특한 환경 틈새를 만들. 이 프로토콜의 또 다른 장점은 그 여러 셀 형식 파생 될 수 있다 hNSC 문화에서 주어진된 변수 hNSC 생존 및 분화에 미치는 영향을 관찰 하는 탐정을 사용. 이 프로토콜은 중앙 신경 시스템 개발 또는 기능 장애에 관한 기계 질문 하고자 하는 개인에 게 적용 됩니다. 다음 프로토콜 위하여 ZIKV, 감염 후 감염 분화 과정에 미치는 영향을 관찰 하는 hNSCs hNSC 문화를 확장 하는 방법을 설명 합니다. 그것은 또한 장기 사용에 대 한 hNSCs를 저장 하 고 hNSCs ZIKV 유도 적자 뇌 기형11에 기여의 더 허용 하는 뉴런의 다양 한 유형으로 차별화 하는 방법을 포함 한다. 우리는이 프로토콜은 또한 감염이 나 신경 줄기 세포 생존 및 분화에 독 소 같은 어떤 환경 자극의 영향을 이해 하고자 하는 수 사관에 대 한 관심의 믿습니다.

<table><tbody><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>F12</td><td>Gibco</td><td>11765-054</td><td></td></tr><tr><td>Glucose (10%)</td><td>Sigma</td><td>G8644</td><td></td></tr><tr><td>HEPES (1M)</td><td>Corning/cellgro</td><td>25-060-c1</td><td></td></tr><tr><td>Pen Strep (100x)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I4011</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>Transferrin</td><td>Sigma</td><td>T2036</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Putrescine</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma</td><td>S5261</td><td></td></tr><tr><td>Heparin Sigma</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>basic fibroblast growth factor</td><td>R&amp;amp;D system</td><td>233-FB</td><td></td></tr><tr><td>epidermal growth factor</td><td>R&amp;amp;D system</td><td>236-EG</td><td></td></tr><tr><td>leukemia inhibitory factor</td><td>R&amp;amp;D system</td><td>7734-LF</td><td></td></tr><tr><td>Laminin</td><td>Invitrogen</td><td>23017-015</td><td></td></tr><tr><td>2.5% trypsin</td><td>Gibco</td><td>15090-046</td><td></td></tr><tr><td>Trypsin inhibitor</td><td>Sigma</td><td>T6522</td><td></td></tr><tr><td>Poly-D-Lysine hydrobromide(PDL)</td><td>Sigma</td><td>P6407-5MG</td><td></td></tr><tr><td>B-27 supplement</td><td>Gibco/Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2650-100ml</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline</td><td>Corning/cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A4378-25G</td><td></td></tr><tr><td>Normal goat serum</td><td>Jackson ImmunoResearch Lab</td><td>005-000-121</td><td></td></tr><tr><td>Triton X-100</td><td>FisherBiotech</td><td>BP151-500</td><td></td></tr><tr><td>Nestin antibody</td><td>BD Transduction Laboratories</td><td>611659</td><td>1:200 dilution</td></tr><tr><td>Class III beta-tubulin antibody (TuJ1)</td><td>Covnce</td><td>MMS-435p</td><td>1:2000 dulution</td></tr><tr><td>GFAP antibody</td><td>Millipore</td><td>AB5804</td><td>1:1000 dilution</td></tr><tr><td>DAPI</td><td>Molecular Probes</td><td>D1306</td><td>1:2000 dilution</td></tr><tr><td>ZIKV antibody</td><td>World Reference Collection for Emerging Viruses and Arboviruses at the University of Texas Medical Branch</td><td></td><td>1:2000 dilution</td></tr><tr><td>Fluoromount G</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Forma</td><td>Model# 3110</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo fisher Scientific</td><td>75004221</td><td></td></tr><tr><td>Biological safety cabinet</td><td>Forma scientific Claas II A/B3</td><td>Model# 1284</td><td></td></tr><tr><td>Freezer (-80 &amp;deg;C)</td><td>Forma scientific,Inc</td><td>model# 8516</td><td></td></tr><tr><td>Microscope(phase contrast image)</td><td>Hp 2230 workstation</td><td>Product#NOE25us#ABA</td><td></td></tr><tr><td>Microscope (epifluorescent image)</td><td>Nikon Eclipse 80i</td><td></td><td></td></tr><tr><td>Confocal Microscope</td><td>Nikon TE2000-E microscope with C1si confocal system</td><td></td><td></td></tr></tbody></table>

zika virus infection of cultured human fetal brain neural stem cells for immunocytochemical analysis

인간의 태아 뇌 신경 줄기 세포는 인간의 발달 신경 생물학에 다양 한 자극의 영향 연구에 고유 비-유전자 변형된 모델 시스템입니다. 오히려 동물 모델 또는 유전자 변형된 유도 만능 세포를 사용 하 여, 보다 인간의 신경 줄기 세포 화면 약물 치료의 효과 검사 하거나 개별 차이 검사에 효과적인 체 외에 시스템을 제공 합니다. 여기, 우리 인간의 태아 뇌 신경 줄기 세포 세럼 무료 미디어, 다양 한 신경 하위 및 다른 못쓰게 절차를 통해 이다 그들을 차별화 하 고 고정 하 고 복구와 문화에서를 확장 하는 데 사용 하는 방법에 대 한 상세한 프로토콜 제공 이러한 세포입니다. 또한, Zika 바이러스 감염 연구를 인간의 태아 뇌 신경 줄기 세포를 사용 하 여 프로시저를 설명 합니다.

그들의 증식 단계에서 교양된 hNSCs 비 점착 neurospheres (그림 1)으로 성장할 것입니다. HNSC 통행 다음 즉시 많은 개별 셀, 집계 및 양식 분야는 앞으로 몇 일 (그림 1A 및 1B)에 시작 있을 것입니다. 건강 한 분야 약 1-2 mm 직경에서 9-10 일 통행 (그림 1C) 이어야 한다. 2 m m 보다 큰 성장 하는 분야는 어?...

Watch this Scientific Journal Video about Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis at JoVE.com

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

이 문서는 문화, 다양 한 신경 하위와 이다, Zika 바이러스 감염 연구 신경 줄기 세포의 사용에 대 한 강조와 함께 그들을 차별화 하는 방법에 있는 인간적 인 태아 두뇌 신경 줄기 세포를 확장 하는 데 사용 되는 방법을 자세히 설명 합니다.

Immunocytochemical 분석에 대 한 교양된 인간 태아 뇌 신경 줄기 세포의 Zika 바이러스 감염

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

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Developmental Biology

6.4K 조회수.  University of Texas Medical Branch. 이 문서는 문화, 다양 한 신경 하위와 이다, Zika 바이러스 감염 연구 신경 줄기 세포의 사용에 대 한 강조와 함께 그들을 차별화 하는 방법에 있는 인간적 인 태아 두뇌 신경 줄기 세포를 확장 하는 데 사용 되는 방법을 자세히 설명 합니다.

비디오: Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

인간의 골격근에서 기본 근육 조직 세포와 섬유 아 세포의 분리 및 정량 면역 세포 화학 특성

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

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발생학

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika 바이러스 감염 세포 배양 시스템과<em&gt; 체외</em인터페론의&gt; 예방 효과

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

면역학 및 감염병학

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

감염 된 Ifnar1/- 생쥐의 Immunoprivileged 장기에서 Zika 바이러스 관련 T 세포 응답의 평가

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

대뇌 Organoids 파생 Zika 바이러스 감염 개발 인간의 두뇌에는 생체 외에서 줄기 세포를 사용 하 여 모델링

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

신경과학

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

Zika 바이러스 유도 신경 장애 Intracerebral 주입 전략을 사용 하 여 마우스 모델을 수립: 배아, 신생아, 및 성인

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

마우스의 여러 장기에서 Zika 바이러스의 격리 및 정량화

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Developing an In Vitro Model of Zika Virus Infection in Cerebral Organoids

Source: Salick, M. R., et al. <a href="https://app.jove.com/v/56404">Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids.</a> J. Vis. Exp. (2017).
This video demonstrates the establishment of an in vitro model of Zika virus infection in a developing brain. Human cerebral organoids containing rosettes of stem cells, neural progenitor cells (NPCs), and neurons are infected with the Zika virus. The virus infects NPCs and triggers apoptosis, disrupting organoid development.

대뇌 오가노이드에서 지카 바이러스 감염의 체외 모델 개발

1. Creating Cerebral Organoids from iPSC / hESC 2D Culture (Day 0)
Note: This protocol assumes that stem cell (SC) maintenance is conducted in xeno-free ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Infectious Diseases

Zika Virus Infection of Fetal Neural Stem Cells

Source: McGrath, E. L., et al., <a href="https://app.jove.com/v/56917">Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.</a> J. Vis. Exp. (2018)
This video demonstrates a procedure to infect human fetal neural stem cells with Zika virus. The infected cells can be used for further studies.

Source: McGrath, E. L., et al., Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis. J. Vis. Exp. (2018)
...

Establishing a Model of Zika Virus-Induced Neurodegeneration in an Adult Mouse

Source: Herrlinger, S. A. et al. <a href="https://app.jove.com/v/56486">Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult.</a> J. Vis. Exp. (2018)
The video demonstrates intracerebral injection of Zika virus suspension in an adult mouse to induce neurodegeneration. An anesthetized mouse is secured in a stereotaxic frame, and a hole is drilled into the exposed skull for injection. A suspension of the Zika virus is injected into the brain tissue. The viral infection causes inflammation, leading to neurodegeneration.

성체 마우스에서 지카 바이러스에 의한 신경퇴행 모델 확립

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Adult ...

Differentiating Human Neural Stem Cells into Neural and Astrocyte Progenitors

Source: McGrath, E. L., et al. <a href="https://app.jove.com/v/56917">Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.</a> J. Vis. Exp. (2018).
This video demonstrates the method to differentiate human neural stem cells from neurospheres into neural and astrocyte progenitors using enzymatic treatment and culturing in a growth factor-rich medium, which eventually leads to maturation into specialized progenitor cells.

인간 신경 줄기 세포를 신경 및 성상 세포 전구 세포로 분화

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Stock medium preparation and stem ...

Immunocytochemical 분석에 대 한 교양된 인간 태아 뇌 신경 줄기 세포의 Zika 바이러스 감염

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