저자는이 모델을 개발 지원 Verlene 헨리와 린지 홈즈 감사하고 싶습니다. 이 작품은 부분적으로 제임스 S. 맥도넬 재단 (# 220,020,173)에 의해 재정 지원되었다.

1. 셀 라인 <ol><li> U87MG glioma 세포 5 % 태아 소 혈청 (FBS)와 보충 DMEM - F12 대형 조직 문화 flasks에서 양식입니다. </li><li> 세포 따뜻한 인산과 함께 두 번 flasks 닦고하여 수확 아르 식염수 (PBS)는 버퍼와 37에서 그들을 잠복기 ° 0.25 %의 트립신과 0.05 % EDTA (에틸렌 다이아 민 테트라 초산)를 포함하는 PBS의 10 ML 5 분 C. 일단 세포들은 문화 미디어와 centrifuged (4 분 300 X G) 10 ML을 포함한 50 ML 튜브에 배치되며, 해제됩니다. </li><li> 세척 후, 세포 10의 농도에 resuspended 아르 X 10 μl 50,000 세포 / 5의 접종에 대한 허용 문화 매체에 6 / ML. </li><li> 전지는 intracranial 사출까지 얼음에 보관됩니다. </li></ol> 2. 탈당 intracranial 가이드 나사 이 절차는 세포의 주입하기 전에 몇 일 진행하실 수 있습니다. 여기에서 설명한 모든 절차가 엄격한 무균 조건 하에서 실시되었습니다. <ol><li> 생쥐 (BALB / C 뉴 / 뉴 여성, 5-6주, 약 18g), 식별을 위해 번호를 무게와 케타민 (100 MG / kg)와 xylazine (5 MG의 혼합물의 intraperitoneal (IP) 주입과 anesthetised 아르 / kg). </li><li> 피부는 요오드 솔루션 닦여 있으며 작은 절개 (2~3mm)은 중간선과 interaural 라인 앞쪽에의 오른쪽을 따라 이루어집니다. 이것은 두개골의 코로나와 화살 봉합를 제공합니다. bregma는이 두 봉합의 교차점에 위치합니다. </li><li> 안내 나사 진입 점은 다음 2.5 mm 수평 및 bregma에 앞쪽에 1mm 지점에서 표시됩니다. 이 시점은 바로 꼬리가있는 핵 위 1에 위치하고 있습니다. </li><li> 1 X 1mm 깊은 구멍은 경질에 두개골을 통해 트위스트 드릴을 개최 살균 손을 뚫고있다. </li><li> 경질로 두개골 표면 아래 1.6 mm를 연장합니다 소독 안내 나사는 다음 스크류 드라이버 구멍으로 나올 수있는 두개골 (그림 1A)와 플러시까지. </li><li> 무균 탐침 또는 스크류 더미는 다음 오프닝 (그림 1B)를 닫습 가이드 나사의 중앙 구멍에 배치됩니다. </li><li> 상처가 Vetbond 조직 접착제 (N - 부틸 cyanoacrylate)와 생쥐와 함께 닫혀있다는 intraperitoneal reversine의 주입 (작은 동물) (0.1 ML / kg)과 진통제에 대한 carprofen을 (5 mg/kg/100 μl) 제공됩니다. 마우스 그런 다음 20 분 소요될 수 온난 매트 (36 ° C)에서 복구하는 허용됩니다. 그들이 완전히 의식 때까지 마우스가 자주 모니터링하고 복구 시간 동안 관찰됩니다. </li></ol> 3. Intracranial 세포 engraftment <ol><li> 무균 수갑에 채워진 해밀턴 주사기는 가이드 나사 콘센트 (그림 1B) 아래 연장하기 위해 바늘의 단지 2mm을 허용 바늘 끝에 작은 플라스틱 반지를 적용하여 준비가되어 있습니다. 최종 접종 지점 따라서 두개골 표면 아래 3.5 mm입니다. </li><li> 가이드 나사 수술 다음과 같은 4 일, 마우스는 위에서 다시 anesthetized 아르 (2.1)와 작은 절개는 탐침을 제거하는 가이드 나사를 통해 이루어집니다. </li><li> 수갑에 채워진 해밀턴 주사기 그러면 어떤 공기 방울을 만들거나 세우하지주의를 복용 잘 혼합 세포의 5 μl로 가득 차 있습니다. </li><li> 주사기는 재관류 펌프로 확보하고 바늘은 가이드 나사 (그림 1C)에 삽입됩니다. 세포는 다음 시간 30 μl의 속도로 주입됩니다. </li><li> 자동 장치 (그림 1D)는 우리가 일정한 유량 한 번에 10 동물까지 주입 수있었습니다. 세포도 수갑에 채워진 해밀턴 주사기를 사용하여 수동으로 분사가 될 수도 있고 아니면 다른 방법으로 주사기는 바늘 끝이 2mm로 안내 나사 넘어 확장할 수 있도록 3mm로 손질되어 200 μl 피펫 팁에 의해 수갑에 채워진 수 있습니다. 주입은 다음 일정한 속도로 지속적으로 수행되어야합니다. </li><li> 주입이 완료되면, 주사기는 신중하게 제거되고 탐침은 가이드 스크류로 대체됩니다. 상처가 닫히고 접착되고 생쥐 (2.7) 이전과 복구 약품을 제공하고 온난 매트에 복구하는 허용됩니다. </li></ol> 4. 치료에 도전 <ol><li> U87MG 세포 접종 후 4 일, 마우스는 무게와 무작위로 제어 및 치료 그룹으로 나누어집니다. </li><li> 치료 그룹이 AMG102 (PBS 100 μl 100 μg)의 IP 주사를 부여하는 동안 컨트롤 그룹은, PBS (100 μl)의 내부 - 복막 (IP) 주사를 주어집니다. </li><li> 주사는 14 일 동안 매초마다 6 일 주사 (그림 2)의 총 반복하고 있습니다. </li><li> 마지막 주사 후, 생쥐는 매일 모니터링 모든 둘째날 무게입니다. </li><li> 쥐가 중요한 신경 장애 (균형 장애, 마비), 탈수, 또는 이상이 10 % 체중 감소 또는 죽어가는을 표시하기 시작하면, 그들은 편안하게 euthanised되었습니다. 이러한 인도적인 엔드 포인트 일은 다음 카플란 - 마이어 생존 곡선에 기록되었다. </li></ol> 5. 치료 효율 평가 </p&gt; <ol><li> GraphPad 소프트웨어, 샌디에고 캘리포니아 미국, 카플란 - 마이어 생존 곡선은 Windows 용 GraphPad 프리즘 4.03를 사용하여 생성되었습니다 <a href="http://www.graphpad.com">www.graphpad.com</a> 3. 사망 또는 euthanizations는 엔드 포인트 이벤트로 표시 1으로 득점했다. 실험의 끝에 살아 남은 동물이 아닌 이벤트로 기록과 0으로 득점했다. </li></ol> 6. 대표 결과 : 제어 동물 세포의 초기 접종 후 신경학적인 장애와 체중 감소 23일의 흔적을 보여주하기 시작했다. 이것은 크게 35일에 AMG102 대우 그룹에서 지연되었습니다. 실제로 35 일, 77 %는 (N은 = 7 / 9) 제어 그룹의이 euthanized했다. 카플란 - 마이어 생존 곡선은 명확하게 AMG102 치료 (그림 3)에 대한 응답으로 생존에 상당한 증가를 보여줍니다. 일 70에 의해, 컨트롤 마우스의 88 % (N = 8 / 9) 37 %와 비교 euthanized했다 AMG102 취급 그룹 (N = 8분의 3). 두뇌의 Histological 분석 컨트롤 그룹에있는 모든 구 동물 개발 종양은 3 8 AMG102 취급 생쥐 (그림 4)에 비해 것으로 확인되었습니다. <img alt="그림 1" src="/files/ftp_upload/3157/3157fig1.jpg" /> 그림 1 : 이미지 (A)는 직접 꼬리가있는 핵 위에있는 지역에 위치 intracranial 가이드 나사를 보여줍니다. 바늘의 2mm는 가이드 초과하고 세포가 꼬리가있는 핵 내부에 3.5 mm를 주입되도록 수갑에 채워진 해밀턴 주사기 (세포와 함께 미리로드)은 다음 가이드 나사 내부에 배치됩니다. 주사기는 나중에 탐침 (B)에 의해 제거 및 교체됩니다. 자동 주입 장치 (C)는 한 번에 10 동물까지 주입 수 있습니다. 오 동시 주사 (D)는 아르 여기 표시됩니다. <img alt="그림 2" src="/files/ftp_upload/3157/3157fig2.jpg" /> 그림 2 : 연구 프로토콜의 도식. 세포 치료 일 4 일 시작과 0에 주입했다. 치료 그룹 AMG102 (100 μg/100 μl) IP의 주사를받은 반면 제어 동물은 PBS (100 μl) IP의 주사를 받았습니다. 이 주사는 10 일 동안 매초마다 주어진 일되었습니다. 6 주사의 총 주어진되었습니다. 동물은 다음 신경 및 신체 장애의 흔적을 70 일 동안 감시했다. <img alt="그림 3" src="/files/ftp_upload/3157/3157fig3.jpg" /> 그림 3 : 제어 및 AMG102 취급 생쥐를 비교 카플란 - 마이어 생존 곡선. 중요한 생존은 AMG102 (P = 0.005가)와 다음과 같은 치료를 얻은 것입니다. <img alt="그림 4" src="/files/ftp_upload/3157/3157fig4.jpg" /> 그림 4 : 제어 동물에서 종양 개발 (A)와 AMG102 치료 동물 튼실한 종양 개발 (B)를 보여주는 코로나 뇌 섹션 Microphotographs. 스케일 바 1mm입니다.

<ol>
	<li>Lal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+implantable+guide-screw+system+for+brain+tumor+studies+in+small+animals.">An implantable guide-screw system for brain tumor studies in small animals.</a> J Neurosurg. 92, 326-333 (2000).</li><li>Jun, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMG+102,+a+fully+human+anti-hepatocyte+growth+factor/scatter+factor+neutralizing+antibody,+enhances+the+efficacy+of+temozolomide+or+docetaxel+in+U-87+MG+cells+and+xenografts.">AMG 102, a fully human anti-hepatocyte growth factor/scatter factor neutralizing antibody, enhances the efficacy of temozolomide or docetaxel in U-87 MG cells and xenografts.</a> Clin Cancer Res. 13, 6735-6742 (2007).</li><li>Pillay, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasticity+of+oncogene+addiction:+implications+for+targeted+therapies+directed+to+receptor+tyrosine+kinases.">The plasticity of oncogene addiction: implications for targeted therapies directed to receptor tyrosine kinases.</a> Neoplasia. 11, 448-458 (2009).</li><li>Buchanan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosensitization+of+glioma+cells+by+modulation+of+Met+signaling+with+the+hepatocyte+growth+factor+neutralizing+antibody.+AMG102.">Radiosensitization of glioma cells by modulation of Met signaling with the hepatocyte growth factor neutralizing antibody. AMG102.</a> J Cell Mol Med. 15, 1999-2006 (2011).</li><li>Wen, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+II+study+evaluating+the+efficacy+and+safety+of+AMG+102+(rilotumumab)+in+patients+with+recurrent+glioblastoma.">A phase II study evaluating the efficacy and safety of AMG 102 (rilotumumab) in patients with recurrent glioblastoma.</a> Neuro Oncol. 13, 437-446 (2011).</li><li>Caretti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+Tumor+Growth+and+Post-Irradiation+Recurrence+in+a+Diffuse+Intrinsic+Pontine+Glioma+Mouse+Model.">Monitoring of Tumor Growth and Post-Irradiation Recurrence in a Diffuse Intrinsic Pontine Glioma Mouse Model.</a> Brain Pathol. 4, 441-451 (2011).</li><li>Jallo, G. I., Volkov, A., Wong, C., Carson, B. S., Penno, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+brainstem+tumor+model:+functional+and+histopathological+characterization.">A novel brainstem tumor model: functional and histopathological characterization.</a> Childs Nerv. Syst. 22, 1519-1525 (2006).</li><li>Johns, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+epidermal+growth+factor+receptor-specific+antibodies+against+gliomaxenografts+is+influenced+by+receptor+levels,+activation+status,+and+heterodimerization.">The efficacy of epidermal growth factor receptor-specific antibodies against gliomaxenografts is influenced by receptor levels, activation status, and heterodimerization.</a> Clin Cancer Res. 13, 1911-1925 (2007).</li><li>Scott, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+clinical+trial+with+monoclonal+antibody+ch806+targeting+transitional+state+and+mutant+epidermal+growth+factor+receptors.">A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 4071-4076 (2007).</li><li>Niyazi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+options+for+recurrent+malignant+glioma.">Therapeutic options for recurrent malignant glioma.</a> Radiother. Oncol. 98, 1-14 (2011).</li></ol>

이 작품은 Amgen 주식 회사에서 연구 그랜트에 의해 지원되었다

여기에 제시 intracranial 안내 나사 방법은 intracranial xenografts의 신속하고 재현성 확립을 가능하게하고 훈련 동물 기술자의 손에,이 절차는 매우 쉽게 stereotactic 장치의 필요없이 수행됩니다. 우리의 방법은 정확하게 생체내 종양 개발에 가장 적합한 두뇌의 지역, 그리고 한 번에 최대 10 동물의 동시 접종을 허용 자동 주입 펌프를 찾으려면 가이드 나사의 사용을 결합한 제품입니다. 안내 나사 확대 절차 수행하는 5 분 미만 소요됩니다. 30 μl / 시간에 세포의 자동 주입은 5 μl 볼륨에 단 10 분 정도 걸립니다. 이것은 일정한 속도로 정확하게 먹이지을 보장하고 증가 intracranial 압력으로 인한 엑스트라 두개골 영역에 셀 퇴학의 위험을 줄일 수 있습니다. 우리는 또한 10 μl 볼륨이 절차를 수행하고 셀룰러 환류으로 인한 엑스트라 두개골 종양의 성장을 관찰하지 않았습니다. 자동 주입 펌프는 여러 동물 우리는 시간당 약 15 동물로 제한됩니다 stereotactic 절차에 비해 시간 60 동물까지 주사할 수있는 동시에 주사 수 있습니다으로서. 위치는 종양이 대량의 최종 위치를 결정하는대로이 절차에서 가장 중요한 단계는 두개골의 가이드 나사의 위치입니다. 가이드 나사가 너무 중앙에있다면, 세포는 가이드가 멀리 전달 배치하는 경우 반면 두 개의 두뇌 반구 사이의 영역으로 주입되며, 세포는 전혀 두뇌에 주입되지 않습니다. 이것은 가이드 측면 2.5 mm와 bregma 1 앞쪽에 1mm를 찾는데 가능한 한 정확하게 그러므로 필수적이다. 플라스틱 가이드 나사는 또한 MRI 나 PET와 같은 뇌 6 이미징을 필요로 그 연구에 사용할 수 있지만 본 연구에서는 우리는 철강 가이드 나사를 사용합니다. 자동 주입 펌프는 일부 그룹에 대한 금지 비용입니다하더라도, 세포는 여전히 수동으로 더 빨리 stereotactic 접근보다 주입 수 있습니다. 전체적으로이 신속, 정확하고 재현성 방법 glioma, medulloblastoma 및 뇌간의 종양 7 공부하는 데 사용할 수 있습니다. 이 절차는 예상치 못한 마취 반응 (&lt;1 %)로 인해 사망과 우리 두 실험실 사이에 500 생쥐 이상에서 수행되었습니다. 가이드 나사와 함께 뛰쳐과 세포가 프로 시저의 결과로 모든 신경 합병증을 죽게하거나 고통 않았와 이후에 주입했다 쥐. 동물은 어떤 합병증이없이 백일 지난 살아있다. 우리가 SCID 같은 머리 hairless 마우스, 단백질 손상된 마우스를 사용하여 원하는 있지만 적절한는 두개골 머리가 사이트를 지키기 위해 싸운다 및 가이드 스크류 간섭 머리카락을 방지하기 위해 수술을하기 전에 제거를 제공 그대로 것입니다. 현재 glioma 80-10의 치료를 위해 개발되고있는 몇 가지 소설 치료제가 있습니다. 최근 우리는 피하 U87MG glioma의 xenografts가 AMG102 3 처리에 의해 저해 수 보여준, 인간화 항체는 현재 glioma 5 임상 평가를 받고 HGF로 이동. 우리는 누드 생쥐의 두뇌에서 intracranial U87MG 종양을 설정하는 방법 여기서 설명하는 데 사용됩니다. 우리의 연구는 명확하게 방법의 유틸리티를 보여줍 AMG102 크게 orthotopic U87MG의 xenografts의 성장을 억제 것을 보여줍니다. 우리는 다른 치료제가 종양 성장의보다 효과적인 억제를 얻을 AMG102와 함께 사용할 수있는 어떤 결정하기 위해이 모델을 계속 사용할 것입니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 12634010 </td><td></td></tr><tr><td> FCS </td><td> Bovogen </td><td> SFBS </td><td></td></tr><tr><td> 트립신 </td><td> Invitrogen </td><td> 15050065 </td><td></td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 시그마 </td><td> E6758 </td><td></td></tr><tr><td> Balb / C 뉴 / 뉴 마우스 </td><td> ARC 퍼스 </td><td></td><td></td></tr><tr><td> 나사 홀더 </td><td> 플라스틱 하나 </td><td> SD - 1 </td><td></td></tr><tr><td> 드릴 홀더 </td><td> 플라스틱 하나 </td><td> DH - 1 </td><td></td></tr><tr><td> 드릴 비트 </td><td> 플라스틱 하나 </td><td> D # 60 </td><td></td></tr><tr><td> 나사 가이드 (금속) </td><td> 플라스틱 하나 </td><td> C212SG </td><td></td></tr><tr><td> 나사 더미 (스타일러스) </td><td> 플라스틱 하나 </td><td> C212SD </td><td></td></tr><tr><td> 해밀턴 주사기 (10 μl 26g 초경 바늘) </td><td> 그레이스 부문 디스커버리 과학 </td><td> 80,075 </td><td></td></tr><tr><td> PHD 2,000분의 22 주입 펌프 </td><td> 하버드 장치 </td><td> 70-2003 </td><td> 옵션 </td></tr><tr><td> Vetbond </td><td> 3M </td><td> 1469SB </td><td></td></tr><tr><td> 써모 패드 </td><td> 하버드 장치 </td><td> 340390 </td><td></td></tr></tbody></table>

a simple guide screw method for intracranial xenograft studies in mice

immunosuppressed 마우스의 두뇌로 인간의 종양 세포의 이식하는 것은 glioblastoma (glioma)과 medulloblastoma 포함한 뇌 암 연구를 위해 설립 방법입니다. 널리 사용되는 stereotactic 접근 방식은 한 번에 하나의 동물의 주입을 허용, 노동 집중하고 고도의 전문 장비를 필요로합니다. 처음 Lal 외. 1가 개발한 가이드 스크류 방식은 성가신 stereotactic 절차를 제거하기 위해 개발되었습니다. 우리는 지금 매우 신속하고 안전 변경 안내 스크류 방식을 설명하는, 중요한 윤리적 고려 둘 중. 특히, 우리의 절차는 지금 동시에 종양 세포로 주입해야하는 10 동물있게 주입 펌프를 포함합니다. HGF / 현재 임상 평가 2-5 겪고 산란 계수로 이동 완전 인간 항체,이 절차의 유틸리티를 입증하기 위해, 우리는 다음 AMG102 취급했다 생쥐에 intracranial xenografts, 인간 U87MG glioma 세포를 설립하였습니다. AMG102의 체계적 주입 크게 intracranial U87MG의 xenografts 모든 생쥐의 생존을 연장하고 완전한 치료의 여러 결과. 이 연구는 가이드 나사 방법은 intracranial xenografts 수립을위한 저렴한 높은 재현성 방법입니다 보여줍니다. 또한, 그것은 뇌 암의 치료를위한 새로운 치료 전략을 검증을 위해 관련 생리적 모델을 제공합니다.

Watch this Scientific Journal Video about A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice at JoVE.com

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

glioma의 치료에 새로운 치료 패러다임을 평가하기 위해, 생리 관련 모델은 필수적입니다. 우리는 stereotactic 접근 방식보다 더 빠르고 안전 intracranial 이종 이식 모델의 설립을위한 implantable 가이드 스크류 절차를 사용합니다.

마우스에 Intracranial 이종 이식 연구에 대한 간단한 안내 스크류 방법

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

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23.0K 조회수.  Monash Institute of Medical Research. glioma의 치료에 새로운 치료 패러다임을 평가하기 위해, 생리 관련 모델은 필수적입니다. 우리는 stereotactic 접근 방식보다 더 빠르고 안전 intracranial 이종 이식 모델의 설립을위한 implantable 가이드 스크류 절차를 사용합니다.

비디오: A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Monochorionic 트윈 Placentas의 혈관 매핑을위한 가이드 와이어 원호 Catheterization과 컬러 염료 사출

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

연구

의학

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

광섬유 압력 변환기를 사용하여 쥐에서 경막외 Intracranial 압력 측정

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

마우스 폐 삽관 법의 간단한 방법

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

소설 고해상도<em&gt; 생체 내</em종양의 성장과 치료학에 두개 내 구조물의 동적 응답을 공부하기&gt; 이미징 기술

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

<em&gt; OVO에서</em육종을위한 Xenograftmodel으로&gt; CAM-분석

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

마우스의 간단한 임계 크기의 대퇴 결함 모델

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is required. In most cases, the preferred route of administration is via oral delivery. At the present time, oral delivery is mostly accomplished using an oral gavage procedure, in part, because it can achieve consistent and precise dosing in the animal model. Although this method is well established it does have complications that can result in a high rate of animal attrition. To this end, the procedure introduced here describes an alternative to the oral gavage method in which the desired drug is incorporated into a tastant, orally dissolving strip (ODS) that can simply be presented to the test animal where it is then rapidly taken up with minimal manipulation of the test subject. Herein, we demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

약리 및 독성 연구에 대한 구두 또한 용해 스트립을 활용 : 동물 용 구강 투여에 대한 간단하고 동물 애호 대안

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

마우스의기도 신속 삽관 용 개선 된 방법

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

가속도계를 사용하여 일상 생활에서 상지 성능을 정량화하는 방법

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...