The authors would like to thank Prof. Hartwig Wolburg for performing the electron microscopy.

1. 거꾸로 모델 시스템에서 시드 HIBCPP 세포에 대한 세포 배양 필터 삽입을 준비 <ol><li> 5 μg의 / ㎖ 인슐린 보충 전 따뜻한 DMEM / F12 (HAM), 100 U / ㎖ 페니실린, 100 μg의 / ㎖ 스트렙토 마이신 및 10 % 소 태아 혈청 (FCS). </li><li> 거꾸로 12 웰 플레이트 (도 1E)로 3 ㎛의 공극 크기 0.33 cm² 성장 영역 세포 배양 필터 삽입 배치 무균 핀셋을 사용한다. </li><li> 세포 배양 필터 인서트의 하부 구획실 (약 3 mL) 및 필터 인서트의 상부에 100 ㎕의 배지를 채운다. 플레이트뿐만 아니라 매체와 낮은 구획 홍수. 필터 인서트의 하부 구획 (도 1E)를 충전 상태로 유지하는 방식으로 과도한 매체 기음. 참고 :이 단계에 대한 혈청 학적 피펫을 사용하는 것이 좋습니다. </li><li> , 뚜껑을 12 웰 플레이트를 커버하고 인큐베이터에 준비된 세포 배양 필터 삽입을 전송 37 ° C,세포에 첨가 할 때까지 5 % CO 2. </li></ol> 2. 재배 및 HIBCPP 세포의과 Passaging <ol><li> 5 μg의 / ㎖, 100 U / ㎖ 페니실린, 100 μg의 / ㎖ 스트렙토 마이신 및 10 % FCS로 보충 된 DMEM / F12 (HAM)을 준비한다. </li><li> 37 ° C의 물을 욕조에 미리 따뜻한 매체와 PBS. 플라스크에서 대기음 매체. 플라스크 및 소용돌이에 10 ml의 PBS를 추가합니다. 일단이 단계를 반복합니다. </li><li> 플라스크 및 소용돌이에 3 ㎖ 0.25 % 트립신 EDTA를 추가합니다. , 인큐베이터에 37 ° C, 5 % CO 2를 놓습니다. </li><li> 약 20 분 후 인큐베이터에서 플라스크를 제거합니다. 세포가 플라스크의 바닥에서 분리되어 있는지 확인하고 현미경으로 조사 때 둥근 모양을 표시합니다. 주 : 세포가 서로 완전히 분리되지 않고 자주 덩어리에서 발견된다. 통로 (38)까지 세포를 사용하는 것이 좋습니다. </li><li> 17 ml의 매체를 추가 트립신 중지하십시오. 아래로 pipetting에 의해 세포를 재현 탁하고, 현탁액을 전송50 ML 튜브로. 실온에서 10 분 동안 50 XG 원심 분리기. </li><li> 매체의 적절한 부피의 세포를 재현 탁하고 혈구를 이용하여 세포 수를 계산. 주 : 현탁 HIBCPP 세포의 농도가 1 × 106 세포 / ml이어야한다. HIBCPP 세포의 유지 대에는 T75 플라스크에 10 ml의 배지에 1-6 × 106 세포의 양을 전송하기 위해 제안된다. 모든 둘째 날 매체 변경합니다. </li></ol> HIBCPP 세포 3. 시드 반전 세포 배양 필터 삽입 <ol><li> (필터의 바닥면 예) 각 반전 필터 인서트의 위에 세포 현탁액 (예. 8 × 10 4 세포) (그림 1E)의 80 μl를 추가합니다. 참고 : 세포가 균일하게 파종하기 전에 튜브를 반전 현탁액에 분산되어 있는지 확인합니다. </li><li> 인큐베이터에 12 웰 플레이트 및 전송의 뚜껑, 37 ° C, 5 % C와 시드 - 세포 배양 필터 삽입을 커버O 2. </li><li> 첫날, 24 웰 플레이트의 우물에 1 ml의 매체를 입력합니다. 12 웰 플레이트에서 집게를 사용하여 세포 배양 필터 삽입을 들어 올리고, 내부 매체를 폐기 필터 삽입을 켜고 준비 24 웰 플레이트 (그림 1E)에 표준 방향으로 배치합니다. </li><li> 모든 둘째 날 새로운 배지로 세포를 놓습니다. 신선한 매체와 24 우물을 준비하고 여기에 필터 삽입을 전송합니다. 0.5 ml의 신선한 매체와 삽입을 입력합니다. 4 절에 설명 된대로 매일 봉사자를 확인합니다. </li><li> 세포 배양 인서트에 HIBCPP 시드 셀들 티이 값을 초과 할 경우 70 Ω X cm² (약 사일 파종 후) 다음, 1 % FCS, 5 μg의 / ㎖ 인슐린을 포함하는 배지에서 세포 배양을 계속한다. 물론 각 1 ml의 배지로 24 웰 플레이트를 준비합니다. 상단 구획에 제조 된 우물과 교환 매체로 필터 삽입을 전송합니다. 참고 : 컨 플루 후이 혈청 철수의 형성을 유도높은 막전위. </li><li> 필터 실에서 대기음 매체는 잘 준비에 전송하고 500 ㎕의 매체를 입력합니다. 일단이 단계를 반복합니다. , 인큐베이터에 하룻밤 37 ° C, 5 % CO 2 세포를 넣습니다. </li></ol> Transepithelial 전기 저항 4. 측정 (봉사자) <ol><li> 80 % 에탄올에 15 분 동안 상피 조직 voltohmmeter의 전극의 끝 부분을 담가. 후드 voltohmmeter를 전송하고 잠시 전극 건조를 할 수 있습니다. 평형 또 다른 15 분 동안 세포에 사용되는 각각의 배지에 전극을 배치합니다. </li><li> 필터 삽입 실에 짧은 아암을 배치가 하부 구획의 바닥 매번 접촉되도록 상기 전극의 긴 아암을 배치하여 측정을 수행한다. 참고 : 세포 배양 필터 삽입의 저항 값을 배지에서 세포없이이 빈 값을 사용해야합니다 ((측정 값 (Ω) - 빈 값 (Ω)) × 필터표면 (즉, 0.33 cm²)). </li><li> 15 분 동안 80 % 에탄올로 전극 제자리를 측정 한 후. 건조 튜브에 저장합니다. </li></ol> Paracellular 투수 5. 결​​정 <ol><li> 5 μg의 / ㎖ 인슐린을 1 % FCS로 보충 된 200 ㎖의 배지에서 1g FITC-이눌린을 녹인다. 세포의 감염 전에 상부 필터 구획에 용액 50 μg의 / ㎖을 적용합니다. </li><li> 감염 후 많은 이눌린은 셀 층을 통해 필터 실에서 전달 방법을 결정하기 위해 낮은 우물에서 매체 샘플을 수집합니다. </li><li> FITC-이눌린 표준 용액을 조제하고 1 수행 2 희석액, 10 배 (100 %, 50 %, 25 %, 12.5 %, 6.25 %, 3.13 %, 1.56 %, 0.78 %, 0.39 %, 0.2 % 및 0 %) . 마이크로 플레이트 리더의 모든 시료의 측정에 의해 형광을 결정합니다. </li></ol> 세포 배양 필터 삽입에 HIBCPP 세포의 감염에 대한 박테리아의 6. 준비 <ol><li> 실험에 어느 날 이전에, 스크랩 t그는 냉동 N. 뇌수막염은 Vitox 초콜릿 한천에 글리세롤 주식과 행진을 해제 균주. 5 % CO 2, 37 ° C에서 인큐베이터에서 하룻밤 성장. </li><li> 하룻밤 문화에서 20-30 식민지를 추출하고 0.042 %의 NaHCO3, 0.01 M의 MgCl 2, 1 % Polyvitex 보충 8 ml의 Proteose 펩톤 매체를 포함하는 튜브에 전송합니다. </li><li> 220 rpm으로 37 ℃에서 1.25 시간 동안 세균을 흔들어. 실온에서 10 분 동안 2,684g에서 원심 분리하여 균체 펠릿. 뜨는을 취소하고 8 ML의 무 혈청 배지와 세균 펠렛을 재현 탁. 소용돌이는 더욱 서스펜션을 보장합니다. </li><li> 배양 밀도를 결정하기 위해, 600 nm 인 광학 밀도 (OD)로 1:10 희석도를 측정한다. OD 0.1 600 세균 현탁액을 조정합니다. 참고 :이 서스펜션이 약이 포함되어 있습니다. 1 × 10 8 CFU / ㎖. </li><li> 박테리아 세포 농도를 확인하기 위해, 최대 서스펜션 단계적 (1:10) 희석초콜릿 한천 플레이트 상에 10-5의 희석과 접시. 주의 : 유사한 연속 희석 세균 성장​​ 곡선을 계산하기 위해 수행 될 필요가있다. </li></ol> 이중 면역 형광에 의한 세균 침략의 세포 배양 필터 삽입 및 결정에 HIBCPP 세포의 7 감염 <ol><li> 1 % FCS, 5 μg의 / ㎖ 인슐린을 포함하는 배지에서 세포 배양을 계속 한 후, 상피 조직 voltohmmeter를 사용하여 매일 세포를 측정한다. 세포가 약 500 Ω X cm²의 봉사자에 도달 한 경우, 감염을 수행한다. </li><li> 시간의 지정된 기간 동안 5 % CO 2, 37 ℃에서 10 점 감염 인큐베이터에 감염된 세포 (MOI)의 다수의 세균 현탁액을 제조로 세포를 감염. 주 : MOI가 합류 (1.21 × 106 세포 / ㎠)에서 계정에 세포 배양 필터 인서트 당 세포의 수를 고려하여 계산 될 수있다. </li><li> 감염되고 중지1 % 소 혈청 알부민 (BSA)을 함유하는 500 μL의 혈청 배지로 3 회 (SFM)을 세정 이온 하부 구획 필터 구획에 1 ml에 적용 하였다. </li><li> 500 μL의 SFM 필터 실에 1 % BSA 버퍼를 포함하고 비특이적 결합 부위에 대한 항체의 부착을 방지하기 위해 실온에서 20 분 동안 상기 하부 구획의 1 ml의 차단. </li><li> 100 ㎕를 일차 항체 항 N.으로 품어 필터 구획하고 실온에서 20 분 동안 상기 하부 구획의 500 μL : meningtidis α-OMP (200 1). 주 : 필요한 경우, 항체 농도가 적정 될 필요가있다. </li><li> 필터 실로부터 매체를 흡입하여 세포를 세척, 1 % BSA를 함유하는 제조도 1 ML의 SFM에 필터 인서트를 전송하고, 1 % BSA를 함유하는 500 μL의 SFM과 비운 필터 실을 채운다. 두 번이 단계를 반복합니다. </li><li> 필터 안돼요을 500 ㎕의 4 % 포름 알데히드로 세포를 고정rtment 실온에서 10 분 동안 하부 구획 한 용액. </li><li> 필터 실에서 매체를 흡입하여 세포를 씻으, 한 ML PBS와 준비를 잘하여 필터 삽입을 전송하고 500 ㎕의 PBS로 비워 필터 구획을 입력합니다. 일단이 단계를 반복합니다. 참고 : 샘플 4 ° C에서 PBS에 밤새 저장할 수 있습니다. </li><li> 컷 고정 된 세포 배양은 삽입의 필터링 250 ㎕의 PBS가 1 % BSA를 함유 씻는다. 세포 외 박테리아를 얼룩 : 라벨 250 μl의 형광 (여기 파장 594 nm의) 차 항체 닭 반 토끼 (500 일)와 실온에서 15 분 동안 세포를 품어. 주 : 필요한 경우, 항체 농도가 적정 될 필요가있다. </li><li> PBS 250 ㎕를 실온에서 1 시간 동안 1 % BSA 및 0.5 % 트리톤 X-100가 함유 된 세포 Permeabilize 하시려면. 250 ㎕의 PBS가 1 % BSA를 함유하는 세포를 세 번 씻는다. </li><li> 250 μL 차 항체 항 N.으로 품어 뇌수막염 </EM&gt; α-OMP (1 : 200) 30 분 동안은 역외 및 세포 내 세균을 염색합니다. 250 ㎕의 PBS가 1 % BSA를 함유하는 세포를 세 번 씻는다. </li><li> 형광 표지 (여기 파장 660 ㎚) Phalloidin의 (1 : 250) 및 4&#39;- 나프티 diamidino -2- 페닐 인돌 염산염 (DAPI () : 형광 표지 (여기 파장 488 ㎚) 이차 항체 (500 (1)) 250 μL를 적용 1 : 50,000) 실온에서 1 시간 동안 역외 및 세포 내 박테리아, 액틴 세포 골격과 핵을 염색합니다. 주 : 필요한 경우, 항체 농도가 적정 될 필요가있다. </li><li> 250 ㎕의 PBS가 1 % BSA를 함유하는 세포를 세 번 씻는다. 현미경을 통해 검사까지 4 ° C에서 매체 및 저장을 장착의 셀을 포함. </li><li> 미리 정의 된 필드 당 침입 박테리아의 수를 결정합니다. 필터 멤브레인 당 20 필드를 계산하여이 작업을 수행합니다. 침입 세균의 비율을 계산한다. 참고 : 지역 코픽으로 20 현미경 분야의 평균 세균 수를 곱하면ient. 결과는 0.33 cm² 세포 배양 필터 인서트 본 총 박테리아의 양을 나타낸다. 감염 기간 동안 배지에서 자란 균의 양이이 값을 나눈다. </li></ol>

<ol>
	<li>Abott, N. J., Patabendige, A. A. K., Dolman, D. E. M., Yusof, S. R., Begley, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+blood-brain+barrier.">Structure and function of the blood-brain barrier.</a> Neurobiol Dis. 37, 13-25 (2009).</li><li>Wolburg, H., Paulus, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid+plexus:+biology+and+pathology.">Choroid plexus: biology and pathology.</a> Acta Neuropathol. 119, 75-88 (2010).</li><li>Engelhardt, B., Sorokin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+and+the+blood-cerebrospinal+fluid+barriers:+function+and+dysfunction.">The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction.</a> Semin Imunopathol. 31, 497-511 (2009).</li><li>Ishiwata, I., Ishiwata, C., Ishiwata, E., Sato, Y., Kiguchi, K., Tachibana, T., 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=Establishment+and+characterization+of+a+human+malignant+choroid+plexus+papilloma+cell+line+(HIBCPP).">Establishment and characterization of a human malignant choroid plexus papilloma cell line (HIBCPP).</a> Hum Cell. 18, 67-72 (2005).</li><li>Schwerk, C., Papandreou, T., Schuhmann, D., Nickol, L., Borkowski, J., Steinmann, U., 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=Polar+invasion+and+translocation+of+Neisseria+meningitidis+and+Streptococcus+suis+in+anovel+human+model+of+the+blood-cerebrospinal+fluid+barrier.">Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in anovel human model of the blood-cerebrospinal fluid barrier.</a> PloS One. 7, e30069 (2012).</li><li>Bernd, A., Ott, M., Ishikawa, H., Schroten, H., Schwerk, C., Fricker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+efflux+transport+proteins+of+the+human+choroid+plecus+papilloma+cell+line+HIBCPP,+a+functional+in+vitro+model+of+the+blood-cerebrospinal+fluid+barrier.">Characterization of efflux transport proteins of the human choroid plecus papilloma cell line HIBCPP, a functional in vitro model of the blood-cerebrospinal fluid barrier.</a> Pharm Res. , (2014).</li><li>Gr&#252;ndler, T., Quednau, N., Stump, C., Orian-Rousseau, V., Ishikawa, H., Wolburg, H., 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=The+surface+proteins+InlA+and+InlB+are+interdependently+required+for+polar+basolateral+invasion+by+Listeria+monocytogenes+in+a+human+model+of+the+blood-cerebrospinal+fluid+barrier.">The surface proteins InlA and InlB are interdependently required for polar basolateral invasion by Listeria monocytogenes in a human model of the blood-cerebrospinal fluid barrier.</a> Microbes Infect. 15, 291-301 (2013).</li><li>Schwerk, C., Tenenbaum, T., Kwang, S. K., Schroten, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+choroid+plexus+-+a+multi-role+player+during+infectious+diseases+of+the+CNS.">The choroid plexus - a multi-role player during infectious diseases of the CNS.</a> Front Cell Neurosci. 9, 80 (2015).</li><li>Pron, B., Taha, M. K., Rambaud, C., Fournet, J. C., Pattey, N., Monnet, J. 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=Interaction+of+Neisseria+meningtidis+with+the+components+of+the+blood-brain+barrier+correlates+with+increased+expression+of+PilC.">Interaction of Neisseria meningtidis with the components of the blood-brain barrier correlates with increased expression of PilC.</a> J Infect Dis. 176, 1285-1292 (1997).</li><li>Guarner, J., Greer, P. W., Whitney, A., Shieh, W. J., Fischer, M., White, E. H., Carlone, G. M., 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=Pathogenesis+and+diagnosis+of+human+meningococcal+disease+using+immunohistochemical+and+PCR+assays+assays.">Pathogenesis and diagnosis of human meningococcal disease using immunohistochemical and PCR assays assays.</a> Am J Clin Pathol. 122, 754-764 (2004).</li><li>Pizarro-Cerda, J., Kuhbacher, A., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+Listeria+monocytogenes+in+mammalian+epithelial+cells:+an+updated+view.">Entry of Listeria monocytogenes in mammalian epithelial cells: an updated view.</a> Cold Spring Harb Perspect Med. 2, (2012).</li><li>Beutler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbe+sensing,+positive+feedback+loops+and+the+pathogenesis+of+inflammatory+diseases.">Microbe sensing, positive feedback loops and the pathogenesis of inflammatory diseases.</a> Immunol. Rev. 227, 248-263 (2009).</li><li>Wilson, E. H., Weninger, W., Hunter, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+of+immune+cells+in+the+central+nervous+system.">Trafficking of immune cells in the central nervous system.</a> J Clin Invest. 120, 1368-1379 (2010).</li><li>Meeker, R. B., Williams, K., Killebrew, D. A., Hudson, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+trafficking+through+the+choroid+plexus.">Cell trafficking through the choroid plexus.</a> Cell Adh Migr. 6, 390-396 (2012).</li><li>Borkowski, J., Li, L., Steinmann, U., Quednau, N., Stump-Guthier, C., Weiss, C., 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=Neisseria+meningitidis+elicits+a+pro-inflammatory+response+involving+I+kappa+B+zeta+in+a+human+blood-cerebrospinal+fluid+barrier+model.">Neisseria meningitidis elicits a pro-inflammatory response involving I kappa B zeta in a human blood-cerebrospinal fluid barrier model.</a> J Neuroinflammation. 11, 163 (2014).</li><li>Steinmann, U., Borkowski, J., Wolburg, H., Schroppel, B., Findeisen, P., Weiss, C., 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=Transmigration+of+polymorphnuclear+neutrophils+and+monocytes+through+the+human+blood-cerebrospinal+fluid+barrier+after+bacterial+infection+in+vitro.">Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro.</a> J Neuroinflammation. 10, 30 (2013).</li><li>McGuiness, B. T., Clarke, I. N., Lambden, P. R., Barlow, A. K., Poolman, J. T., Heckels, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point+mutation+in+meningococcal+por+A+gene+associated+with+increased+endemic+disease.">Point mutation in meningococcal por A gene associated with increased endemic disease.</a> Lancet. 337, 514-517 (1991).</li><li>Ram, S., Cox, A. D., Wright, J. C., Vogel, U., Getzlaff, S., Boden, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisserial+lipopolysaccharide+is+a+target+for+complement+component+C4b.+inner+core+phosphoethanolamine+residues+define+C4b+linkage+specificity.">Neisserial lipopolysaccharide is a target for complement component C4b. inner core phosphoethanolamine residues define C4b linkage specificity.</a> J Biol Chem. 278, 50853-50862 (2003).</li><li>Claus, H., Maiden, M. C., Maag, R., Frosch, M., Vogel, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+carried+meningococci+lack+the+genes+required+for+capsule+synthesis+and+transport.">Many carried meningococci lack the genes required for capsule synthesis and transport.</a> Microbiology. 148, 1813-1819 (2002).</li><li>Claus, H., Maiden, M. C., Wilson, D. J., Mccarthy, N. D., Jolley, K. A., Urwin, R., 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=Genetic+analysis+of+meningococci+carried+by+children+and+young+adults.">Genetic analysis of meningococci carried by children and young adults.</a> J Infect Dis. 191, 1263-1271 (2005).</li><li>Tenenbaum, T., Papandreou, T., Gellrich, D., Friedrichs, U., Seibt, A., Adam, R., 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=Polar+bacterial+invasion+and+translocation+of+Streptococcus+suis+across+the+blood-cerebrospinal+fluid+barrier+in+vitro.">Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro.</a> Cell Microbiol. 11, 323-336 (2009).</li><li>Laflamme, N., Echchannaoui, H., Landmann, R., Rivest, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+between+toll-like+receptor+2+and+4+in+the+brain+of+mice+challenged+with+cell+wall+components+derived+from+gram-negative+and+gram-positive+bacteria.">Cooperation between toll-like receptor 2 and 4 in the brain of mice challenged with cell wall components derived from gram-negative and gram-positive bacteria.</a> Eur J Immunol. 33, 1127-1138 (2003).</li><li>Laflamme, S., Rivest, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+4:+the+missing+link+of+the+cerebral+innate+immune+response+triggered+by+circulating+gram-negative+bacterial+cell+wall+components.">Toll-like receptor 4: the missing link of the cerebral innate immune response triggered by circulating gram-negative bacterial cell wall components.</a> FASEB J. 15, 155-163 (2001).</li><li>Zughaier, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+meningitidis+capsular+polysaccharides+indice+inflammatory+responses+via+TLR2+and+TLR4-MD-2.">Neisseria meningitidis capsular polysaccharides indice inflammatory responses via TLR2 and TLR4-MD-2.</a> J Leukoc Biol. 89, 469-480 (2011).</li><li>Yamamoto, M., Yamazaki, S., Uematsu, S., Sato, S., Hemmi, M., Hoshino, 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=Regulation+of+Toll/IL-1-receptor+-mediated+gene+expression+by+the+inducible+nuclear+protein+IkappaBzeta.">Regulation of Toll/IL-1-receptor -mediated gene expression by the inducible nuclear protein IkappaBzeta.</a> Nature. 430, 218-222 (2004).</li><li>Lorenz, J., Zahlten, J., Pollok, I., Lippmann, J., Scharf, S., N'Guessan, P. D., 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=Legionella+pheumophila-induced+IkappaBzeta-dependent+expression+of+interleukin-6+in+lung+epithelium.">Legionella pheumophila-induced IkappaBzeta-dependent expression of interleukin-6 in lung epithelium.</a> Eur Respir J. 37, 648-657 (2011).</li><li>Jaerve, A., Muller, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+CNS+injury+and+repair.">Chemokines in CNS injury and repair.</a> Cell Tissue Res. 349, 229-248 (2012).</li><li>Schneider, H., Weber, C. E., Schoeller, J., Steinmann, U., Borkowski, J., Ishikawa, H., 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=Chemotaxis+of+T-cells+after+infection+of+human+choroid+plexus+papilloma+cells+with+Echovirus+30+in+an+in+vitro+model+of+the+blood-cerebrospinal+fluid+barrier.">Chemotaxis of T-cells after infection of human choroid plexus papilloma cells with Echovirus 30 in an in vitro model of the blood-cerebrospinal fluid barrier.</a> Virus Res. 170, 66-74 (2012).</li><li>Chodobski, A., Szmydynger-Chodobska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid+plexus:+Target+for+polypeptides+and+site+of+their+synthesis.">Choroid plexus: Target for polypeptides and site of their synthesis.</a> Microsc. Res. Tech. 52, 65-82 (2001).</li><li>Dickson, P. W., Schreiber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+levels+of+messenger+RNA+for+transthyretin+(prealbumin)+in+human+choroid+plexus.">High levels of messenger RNA for transthyretin (prealbumin) in human choroid plexus.</a> Neurosci. Lett. 66, 311-315 (1986).</li><li>Stylianopoulou, F., Herbert, J., Soares, M. B., Efstratiadis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+insulin-like+growth+factor+II+gene+in+the+choroid+plexus+and+the+leptomeninges+of+the+adult+rat+central+nervous+system.">Expression of the insulin-like growth factor II gene in the choroid plexus and the leptomeninges of the adult rat central nervous system.</a> Proc Natl Acad Sci USA. 85, 141-145 (1988).</li><li>Lim, L., Zhou, H., Costa, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+winged+helix+transcription+factor+HFH-4+is+expressed+during+choroid+plexus+epithelial+development+in+the+mouse+embryo.">The winged helix transcription factor HFH-4 is expressed during choroid plexus epithelial development in the mouse embryo.</a> Proc. Natl Acad. Sci. USA. 94, 3094-3099 (1997).</li><li>Vandenhaute, E., Stump-Guthier, C., Lasierra Losada, M., Tenenbaum, T., Rudolph, H., Ishikawa, H., 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=The+choroid+plexus+may+be+an+underestimated+site+of+tumor+invasion+to+the+brain:+an+in+vitro+study+using+neuroblastoma+cell+lines.">The choroid plexus may be an underestimated site of tumor invasion to the brain: an in vitro study using neuroblastoma cell lines.</a> Cancer Cell Int. , 15-102 (2015).</li></ol>

The authors have nothing to disclose.

제어 포인트의 상피 세포는 혈액 2,3-에서 CSF를 분리 BCSFB을 형성한다. 우리는 최근 BCSFB의 기능적 인간 모델로 HIBCPP 세포주를 확립. 세포는 높은 막 전위의 개발 고분자에 대해 낮은 투과성뿐만 아니라 TJS 5 연속 스트랜드의 존재를 포함하여 시험 관내 BCSFB 중요한 장벽 기능을 표시. TJ 단백질은 세포의 기저 / 혀끝 극성에 기여한다. 극성 표면 수용체에 관한 지역화 높은 중요...

혈액 - 뇌척수액 장벽 (BCSFB)은 혈액과 뇌 (1) 사이의 세 가지 장벽 사이트 중 하나입니다. 그것의 형태 학적 상관 관계가 맥락막 신경총 (CP) 2,3의 상피 세포이며, 강력하게 혈관과 뇌의 뇌실에 위치하고있는 내피 - 상피 컨볼. 제어 포인트는 뇌척수액 (CSF)를 생산뿐만 아니라 혈액에서 후자를 분리하는 역할을한다. 장벽 기능을 달성하기 위해, CP 상피 세포는 낮은 pinocytotic 활성을 나타내는 특정 전송기를 표현하고 조밀 기밀 접합 (TJS) 2,3-의 연속적인 네트워크에 의해 접속되어있다. 일본인 여성 4 악성 맥락막 신경총 유두종 유래의 인간 맥락막 신경총 유두종 (HIBCPP) 세포의 BCSFB의 시험 관내 모델의 기능을 구성하는 데 사용 하였다. HIBCPP 세포는 TJ의 형성 등의 기능 BCSFB의 특성 몇 가지를 보여스트랜드 transepithelial 전기 저항 (티이)로 판정 할 수있는 높은 transepithelial 막전위의 개발 및 거대 분자를위한 작은 투과성. 또한, HIBCPP 세포는 이온 성 미세 환경을 조절하는 역할을하고, 기저 / 혀끝 극성 5,6,7를 표시 할 수 있습니다 특성 수송을 표현한다. BCSFB는 중추 신경계 (CNS)에 8 병원균 (박테리아, 바이러스, 진균)에 대한 엔트리 사이트로서 기능하는 것으로 나타났다. 나이 세리아 뇌수막염 (N. 뇌수막염), 그람 음성 박테리아를 포함하여 병원균의 침입은 뇌막염과 같은 심각한 질병을 일으킬 수 있습니다. 는 CP의 보호 상피 장벽을 극복 증거가 수막 구균 성 질환은 혈관과 CP 상피 세포 9, 10에서 수막 구균의 증가 금액을 나타내는 환자에서 조직 병리학 적 관찰에 의해 지원됩니다. 숙주 세포 바로 항목을 얻으려면cteria들은 중​​재 또는 숙주 세포에있는 특정 표면 수용체에 의해 트리거되는 endocytotic 메커니즘을 하이재킹. 이들 수용체와 병원체의 상호 작용이 종 수 있기 때문에 특정 (11) 동물 모델은 제한된 범위로 참고 될 수있다. HIBCPP 세포주 침입 프로세스뿐만 아니라 인간의 모델 시스템에서 기본이되는 분자 기전을 연구 할 수있는 기회를 제공한다. 세포 배양 삽입을 사용하는 두 가지 세포 측면에서 숙주 세포로 병원균의 상호 작용을 분석하기 위해 우리가 할 수 있습니다. 많은 박테리아를 포함 N. 뇌수막염은 감염 분석하는 동안 중력의 영향을 강하게 될 수 있습니다. 분석법 중 HIBCPP 세포와 병원체의 최적의 상호 작용의 경우, 초기 박테리아는 세포 배양 필터 삽입 시스템의 상부 구획에 첨가 하였다. 각각 체외 시스템의 두 가지 변화가 있었다 ESTA 꼭대기 또는 기저 세포 측에서 감염을 사용하려면blished : 표준 시스템에서 HIBCPP 세포 미생물이 CSF 측에 위치하며 셀 (도 1a, C)의 꼭대기면 접촉에 들어갈 때의 상황을 모방 필터 인서트의 상부 구획으로 시딩 하였다. 반면, 반전 된 세포 배양 필터 인서트 시스템에서 HIBCPP 세포를 사용하여 세균이 혈류를 입력 한 상황을 반영한다. 미생물은 기저 측 (그림 1B, D)에서 혈액과 만남 CP 상피 세포에 전파. 주목할이 모델 시스템에서 세균이 기저 세포 측면에서 특히 5,7- 극성 방식 HIBCPP 세포에 침입하는 것이 밝혀졌다. 이어서 CP의 감염의 병원체가 침입 패턴 - 인식 수용체를 통해 결찰 선천 면역 (PRRS)에 의해 인식 될 수있다. PRRS의 잘 설명 된 회원은 수신자 같은 수용체 (TLR) 가족에 속한다. TLR들 수 빈라고 병원체 관련 분자 패턴 (PAMPs가) 있습니다 감염성 미생물의 특성 구조에 라. 수용체의 결찰 차례로 BCSFB 13,14 걸쳐 윤회 면역 세포를 자극 사이토 카인 및 케모카인 (12)의 발현을 유발 숙주 세포의 활성화 시그널링 캐스케이드로 이끈다. HIBCPP 세포가 여러 가지의 mRNA 수준에서 TLR들과 N.와 그 감염을 표현하는 것으로 밝혀졌다 뇌수막염은 CXCL1-3, IL6, IL8 및 TNFα (15, 16)를 포함한 여러 사이토 카인과 케모카인의 분비를 초래한다. 여기에서는 BCSFB 모방 반전 세포 배양 삽입 시스템에서 배양하고, 인간 세포주 HIBCPP 감염을 설명한다. 이 모델 시스템은 생체 관련 기저 세포 측면뿐만 아니라, 이후 세포 반응과 병원균의 상호 작용을 연구 할 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="573">
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			<td height="21" style="height:21px;width:216px;">0.25% Trypsin-EDTA</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#180;,6 diamidino-2-phenylindole (DAPI)</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-well plates</td>
			<td>Starlab</td>
			<td>CC7682-7512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well plates</td>
			<td>Starlab</td>
			<td>CC7682-7524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti Neisseria meningitidis &#945;-OMP</td>
			<td></td>
			<td></td>
			<td>This antibody was a gift from Drs. H. Claus and U. Vogel (University of W&#252;rzburg, Germany)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 (chicken anti rabbit)</td>
			<td>Invitrogen</td>
			<td>A21441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 (chicken anti rabbit)</td>
			<td>Invitrogen</td>
			<td>A21442</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 660 Phalloidin</td>
			<td>Invitrogen</td>
			<td>A22285</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumine (BSA)</td>
			<td>Calbiochem</td>
			<td>12659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chocolate agar plates</td>
			<td>Biomerieux</td>
			<td>43109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytochalasin D</td>
			<td>Sigma</td>
			<td>C8273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12 + L-Glut + 15 mM HEPES</td>
			<td>Gibco</td>
			<td>31330-095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12 + L-Glut + 15 mM HEPES w/o Phenolred</td>
			<td>Gibco</td>
			<td>11039-047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum (FCS)</td>
			<td>Life Technologies</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-Inulin</td>
			<td>Sigma</td>
			<td>F3272</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin</td>
			<td>Sigma</td>
			<td>19278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma</td>
			<td>2393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3</td>
			<td>Sigma</td>
			<td>55761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS + Mg +Ca</td>
			<td>Gibco</td>
			<td>14040-174</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>MP Biomedicals</td>
			<td>1670049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyvitex</td>
			<td>Biomerieux</td>
			<td>55651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteose peptone</td>
			<td>BD</td>
			<td>211684</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serum-free medium</td>
			<td>Gibco</td>
			<td>10902-096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thincert cell culture inserts for 24-well plates, pore size 3 &#181;m</td>
			<td>Greiner</td>
			<td>662630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture flask 75 cm&#178; red cap sterile</td>
			<td>Greiner</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Volt-Ohm Meter Millicell-ERS2 with MERSSTX01 electrode</td>
			<td>Millipore</td>
			<td>MERSSTX00</td>
			<td></td>
		</tr>
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a choroid plexus epithelial cell-based model of the human blood-cerebrospinal fluid barrier to study bacterial infection from the basolateral side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

여기에서 우리는 역 세포 배양 삽입 시스템에서 배양 및 HIBCPP 세포의 감염을 설명합니다. 이 모델은 우리가 (도 1)을 전파하고, 혈류를 통하여 상피 세포에 진입 박테리아의 생리 학적 상태를 재생 상기 기저 셀 측으로부터 침입 메커니즘 기본 분자 신호 경로를 연구 할 수있다. HIBCPP 셀 최소 레벨에 대한 분자 교?...

Watch this Scientific Journal Video about A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side at JoVE.com

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

인간의 혈액 - 뇌척수액 장벽의 맥락총 상피 세포 기반 모델은 기저 측면에서 세균 감염을 연구하는

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...

a-choroid-plexus-epithelial-cell-based-model-human-blood

Research

JoVE Journal

Medicine

14.1K 조회수.  Heidelberg University. The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

비디오: A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSD의, 신경 Hematologic, 그리고 행동 상관 관계를 누설하는 행위에 대해서는 동물 모델에서 생체

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

연구

의학

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

인간 신경근육학 시스템을 공부하기 Transcranial 자기 자극을 활용

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

인간 내유 동맥 (IMA) 이식과 Stenting : 인 Restenosis 스텐 트의 개발을 공부하는 인간 모델

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

인간 Neuroendocrine 종양 치료의 연구를위한 3 차원 모델로 인간 Neuroendocrine 종양 세포 라인

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

인간의<em&gt; 전의 VIVO</em&gt; 죽상 경화 모델 병변 생물학을 연구하는

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

인간 혈청에있는 N-메틸-D-아스파 라진 산염 수용체 또는 도파민 2 수용체에 대한 항체를 검출하는 높은 처리량 유동 세포 계측법 세포 기반 분석

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

같은 뇌 Microslices의 흥분 독성 자극<em&gt; 체외</em&gt; 스트로크의 모델

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

<em&gt; 생체 내</em&gt; 모델은 인간의 유방암에서 호르몬 행동을 연구하기 위해

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

깊은 뇌 자극과 Saccadometry를 사용하여 정상 및 병적 뇌 기능을 연구하는

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

인간화 된 마우스 모델에 HIV 감염에 대 한 줄기 세포를 기반으로 설계된 내성

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...