저자는이 프로젝트의 초기 단계에서 기술 지원 전문가 축산학 박사 마르코 A. Passini에 대한 존 말스톤양은 감사하고 싶습니다. 이 작품은 CLL로 국립 보건원 (; R01HD054413 R01NS41584)에서 부여에 의해 투자되었다.

1. Intracerebroventricular 주사 <ol><li> 첫 번째 단계는 사출 재고 솔루션의 준비이며 이러한 솔루션은 바이러스 벡터, 플라스미드 DNA, 마약, 그리고 무균 조건 하에서 주입해야합니다. </li><li> 0.05 % 사출 사이트의 시각화를 위해 PBS에 W / V trypan 파랑과 바이러스성 벡터 (5-7 μL 총)의 원하는 titer를 섞습니다. </li><li> 플라스미드 DNA 용액 (오리터 μL 총) D - (+) - 포도당 20% (W / V) (1 μL), trypan 파랑 (0.05 %) PBS (1 μL), 플라스미드 (~ 5 ~ G / ~ L을 포함 ) (2 ~ L) 및 2.5 kDa 선형 PEI 단일 중합체 (150 ㎜) (1 ~ L). </li><li> 1~2분에 대한 cryo - 마취를 통해 쿨러 2 neonates를 무력화. </li><li> 이 주사에 사용되는 바늘은 긴 튜브를 통하여 3ml 3 ML 해밀턴 주사기에 연결된 마이크로 리터 보정 소독 유리 micropipette입니다. </li><li> 두개골에 2mm의 침투에 대한 조정 바늘 (주걱 포함)의 팁을 휴식. 사출 솔루션을 포함하고있는 microcentrifuge 관에서 대각선 바늘을 놓으십시오. 조심스럽게 주사기의 플런저를 당겨 바늘로 주사 솔루션을로드합니다. </li><li> 솔루션의 이해 후, 튜브에서 주사기를 끊을, 추가로 플런저를 끌어 다음 다시 바늘에 첨부합니다. </li><li> 엄지와 검지 손가락 사이의 오른쪽에있는 바늘을 잡고 오른쪽 손바닥을 감동 플런저와 함께 중간 및 링 손가락 사이에 주사기를 놓습니다. </li><li> 단단히 가이드로 사용할 수있는 관련 해부 구조를 조명 광섬유 조명에 왼쪽과 장소를 사용하여 머리 뒤쪽 피부로 고정 마우스를 파악. </li><li> 위치 화살 치료 측면 약 0.25 mm와 신생아 관상 봉합 (그림 1A)에 주동이의 0.50-0.75 mm에서 두개골 표면에 직각으로, 깊은 바늘 2mm를 넣습니다. 그런 다음 손바닥을 사용하여 플런저를 밀어 오른손 아주 천천히 그리고 신중하게. 파열 혈관이나 얼굴의 부기에 대한 모니터링합니다. </li><li> 역류를 방지하기 위해 플런저 운동 중지 후 바늘 15 초 정도 제거합니다. </li><li> , 복구 움직임과 일반적인 대응이 복구 될때까지 따뜻하게 용기에 5-10분에 대한 생쥐를 유지합니다. </li></ol> 2. 시간적 / 안면 정맥을 통해 정맥 주사 <ol><li> 주입되는 솔루션은 준비 및 필터링 녹색 식용 색소를 보충 1:100 희석 시에 무균 조건 하에서 주입해야합니다. </li><li> 100 μL 유리 해밀턴 주사기 끝에 luer 작은 플라스틱을 첨부합니다. luer하려면 33 게이지 0.25 인치 피하 바늘을 첨부합니다. 모든 부품이 안전하게 연결되어 있는지 확보. </li><li> 공기 그릇에 공기 주입으로 주사기에 없다는 것을 보장, 주입으로 볼륨과 주사기를로드하면 치명적입니다. 그것은 종종 pipet parafilm의 일부 이니라 주입하는 볼륨에 도움이됩니다. 이것은 주사기로 솔루션의 이해의 명확 시각화 수 있도록합니다. </li><li> 위 사이트의 Transilluminator 쉽게 neonate의 표면 시간적 정맥, 또는 얼굴 정맥, (그림 2A)를 시각화하는 데 사용됩니다. 주입하기 전에 수술 테이프를 사용하여 transilluminator로 neonate을 확보. 거즈는 동물의 피부를 손상에서 테이프를 방지하기 위해 neonate과 테이프 사이에 위치해야합니다. 테이프가 호흡을 방지하지 않는다는 보장, 그 측면에 transilluminator로 동물을 고정합니다. neonate의 목을 부드럽게 얼굴 정맥을 쉽게 볼 수 있도록 설정되어 있어야하며 코는 머리를 고정 녹화해야합니다. </li><li> 쉽게 시각화 수 있도록 2.25X 밴드의 확대경을 사용하여 천천히 정맥에 바늘을 삽입합니다. 바늘이 피부 아래에 보이는 남아 있어야하므로 정맥은 매우 피상적​​이다. </li><li> 천천히 정맥에 솔루션의 볼륨을 달이다. 녹색 염료는 사출 쉽게 시각화를위한 수 있습니다. </li><li> 완전히 주사기와 매우 좁은 바늘 구멍으로 인해 남아있는 솔루션의 퇴학의 플런저를 우울 사이에 지연이로 바늘을 제거하기 전에 15 초 정도 기다리십시오. </li><li> 바늘을 제거한 후 출혈이 멈추면 때까지 주입 사이트에 압력을 적용하기 위해 거즈를 사용합니다. </li><li> 고통의 흔적에 대한 neonate를 모니터링합니다. 적절한 주사 후, 더 명백한 고통은 관찰되지 않을 것입니다. neonate 그것은 케이지에 반환되기 전에 복구하는 약 5 분 정도 실시한다. </li><li> 주사 바늘에 혈액이 빠르게 인해 작은 구멍으로 방해할되므로 주사 사이에 PBS로 바늘 / 주사기 장치를 플러시. </li></ol> 3. 대표 결과 : ICV 주사 : 두뇌가 서로 다른 시간 포인트 사후 분사에 수확하고 성공적인 사출 기술을 보여주는 시각하실 수 있습니다. 염료가 주입 솔루션에 추가되지 않은 경우, 그것은에의 정확성을 확인하기 어려울 것입니다jection. 심실 중 하나의 적절한 주입은 두뇌의 주입 측면에 trypan 파란색의 배포를 허용합니다 약 10-15분 포스트 사출 (그림 1B). 좌우 대뇌 반구와 후각 구근 모두 trypan 파랑의 균일한 분포가 약 60 분 게시 주입 (그림 1C) 표시됩니다. 이것은 인접한 뇌실에 청색 색소의 보급을 허용합니다 대뇌 심실의 연결에 의한 것입니다. 정확한 주사는 약 12시간 포스트 사출 (그림 1D)에서 주동이의 중앙 척추 운하에있는 청색 색소의 분산에 의해 시각하실 수 있습니다. 부정 확한 주사는 뇌성 반구에있는 파란색의 부족에 의해 구별 수 있습니다. 이 경우 바늘의 깊이가 충분하지되었고 사출 솔루션은 아마도 피부 아래에 배포했다. 다른 가능성은 바늘이이를 뇌실과 솔루션 대뇌 심실 아래납니다 넘어 통과, 두뇌에 너무 깊이 침투한다는 것입니다. IV 주입 : 정확하고 효율적인 시간적 / 안면 정맥 주사는 주사 바로 다음 시각하실 수 있습니다. 솔루션이 주입되면서 녹색 염료 입력하고 얼굴 정맥 (그림 2A)를 통해 흐르는 솔루션의 시각화를위한 수 있습니다. 또한, 마우스의 조작 스킨 색상은 다음과 같은 주사를 관찰합니다. neonate의 정상적인 핑크색 색상 때문에 사출 솔루션에 녹색 식품 색소 (그림 2B)의 존재로, 녹색으로 변환됩니다. neonate에서 관찰 녹색 색깔 변화의 정도는 사용되는 염료의 농도에 의존적일 수 밖에 없습니다. 분사와 연습 기간 동안 간단한 시각화 들어, 1시 50분 희석 권장합니다. 그러나 극적으로 변경 피부 색조는 종종 강아지의 어머니에 의해 거부되고 따라서, 염료 또는 크게 감소 농도의 제거가 실험 과정 중에 고려되어야 결과. 부정 확한 주사는 주사의 사이트에있는 피부 아래에 녹색 색소의 축적의 결과 것입니다. 바늘의 개통 완전히 정맥 내에 삽입되지 않은 경우 발생하는 불완전 주입은, 일부 솔루션은 정맥에 흐르는 때 볼 수 있으며 일부는 피부 아래 축적되어있다. 이것이 관찰되면, 주사 바늘이 혈관에 더 삽입해야합니다. <img src="/files/ftp_upload/2968/2968fig1.jpg" alt="그림 1"/> 그림 1. PDN2 쥐를 성공적으로 ICV 주사의 데모. 심실로 ICV 주사에 이용 사출 사이트) 도식은. B) 좌심실에 PBS에 trypan 블루와 강아지 주입 ICV의 브레인, 사진 15 분 게시 주사를 촬영. C ICV에서) 브레인 좌심실에 PBS에 trypan 블루와 강아지 주입, 촬영한 사진은 60 분 게시 주입. D) 주동이의 중앙 척추 운하는 ICV에 표시됩니다이 좌심실에 PBS에 trypan 블루와 강아지 주입 12 시간 게시 주사를 찍은 사진. <img src="/files/ftp_upload/2968/2968fig2.jpg" alt="그림 2"/> 그림 2. PDN2 생쥐에서 성공 IV 주사의 데모가. IV 주사에 대한 활용 안면 정맥의) 도식은. B) PBS에 녹색 색소와 강아지 주입 IV가 취소 주입 littermate에 비해, 사진은 즉시 게시 주사를 촬영.

<ol>
	<li>Blanchette, M., Fortin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+disruption+in+the+treatment+of+brain+tumors.">Blood-brain barrier disruption in the treatment of brain tumors.</a> Methods Mol. Biol. 686, 447-463 (2011).</li><li>Foust, K. D., Kaspar, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over+the+barrier+and+through+the+blood:+to+CNS+delivery+we+go.">Over the barrier and through the blood: to CNS delivery we go.</a> Cell Cycle. 24, 4017-4018 (2009).</li><li>Snyder, E. Y., Taylor, R. M., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+progenitor+cell+engraftment+corrects+lysosomal+storage+throughout+the+MPS+VII+mouse+brain.">Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain.</a> Nature. 374, 367-370 (1995).</li><li>Passini, M. A., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+gene+delivery+and+structure-specific+patterns+of+expression+in+the+brain+after+intraventricular+injections+of+neonatal+mice+with+an+adeno-associated+virus+vector.">Widespread gene delivery and structure-specific patterns of expression in the brain after intraventricular injections of neonatal mice with an adeno-associated virus vector.</a> J. Virol. 24, 12382-12392 (2001).</li><li>Kienstra, K. A., Freysdottir, D., Gonzales, N. M., Hirschi, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+neonatal+intravascular+injections:+modeling+newborn+disease.">Murine neonatal intravascular injections: modeling newborn disease.</a> J. Am. Assoc. Lab. Anim. Sci. 46, 50-54 (2007).</li><li>Sands, M. S., Barker, 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=Percutaneous+intravenous+injection+in+neonatal+mice.">Percutaneous intravenous injection in neonatal mice.</a> Lab. Anim. Sci. 49, 328-330 (1999).</li><li>Foust, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+the+spinal+muscular+atrophy+phenotype+in+a+mouse+model+by+early+postnatal+delivery+of+SMN.">Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN.</a> Nat. Biotechnol. 28, 271-274 (2010).</li><li>Foust, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat. Biotechnol. 27, 59-65 (2009).</li><li>Passini, M. A., Watson, D. J., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+to+the+mouse+brain+with+adeno-associated+virus.">Gene delivery to the mouse brain with adeno-associated virus.</a> Methods Mol. Biol. 246, 225-236 (2004).</li><li>Coady, T. H., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-splicing-mediated+improvement+in+a+severe+mouse+model+of+spinal+muscular+atrophy.">Trans-splicing-mediated improvement in a severe mouse model of spinal muscular atrophy.</a> J. Neurosci. 30, 126-130 (2010).</li><li>Baughan, T. D., Dickson, A., Osman, E. Y., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+bifunctional+RNAs+that+target+an+intronic+repressor+and+increase+SMN+levels+in+an+animal+model+of+spinal+muscular+atrophy.">Delivery of bifunctional RNAs that target an intronic repressor and increase SMN levels in an animal model of spinal muscular atrophy.</a> Hum. Mol. Genet. 18, 1600-1611 (2009).</li><li>Coady, T. H., Baughan, T. D., Shababi, M., Passini, M. A., Lorson, C. L. <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+single+vector+system+that+enhances+trans-splicing+of+SMN2+transcripts.">Development of a single vector system that enhances trans-splicing of SMN2 transcripts.</a> PLoS One. 3, e3468-e3468 (2008).</li><li>Dickson, A., Osman, E., Lorson, 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=Negatively-Acting+Bifunctional+RNA+Increases+Survival+Motor+Neuron+in+vitro+and+in+vivo.">Negatively-Acting Bifunctional RNA Increases Survival Motor Neuron in vitro and in vivo.</a> Hum. Gene. Ther. 19, 1307-1315 .</li><li>Mattis, V. B., Ebert, A. D., Fosso, M. Y., Chang, C. W., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+a+read-through+inducing+compound,+TC007,+lessens+the+severity+of+a+spinal+muscular+atrophy+animal+model.">Delivery of a read-through inducing compound, TC007, lessens the severity of a spinal muscular atrophy animal model.</a> Hum. Mol. Genet. 18, 3906-3913 (2009).</li><li>Williams, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligonucleotide-mediated+survival+of+motor+neuron+protein+expression+in+CNS+improves+phenotype+in+a+mouse+model+of+spinal+muscular+atrophy.">Oligonucleotide-mediated survival of motor neuron protein expression in CNS improves phenotype in a mouse model of spinal muscular atrophy.</a> J. Neurosci. 29, 7633-7638 (2009).</li><li>Passini, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS-targeted+gene+therapy+improves+survival+and+motor+function+in+a+mouse+model+of+spinal+muscular+atrophy.">CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy.</a> J. Clin. Invest. 120, 1253-1264 (2010).</li><li>Shababi, M., Glascock, J., Lorson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+SMN+Trans-Splicing+and+a+Neurotrophic+Factor+Increases+the+Life+Span+and+Body+Mass+in+a+Severe+Model+of+Spinal+Muscular+Atrophy.">Combination of SMN Trans-Splicing and a Neurotrophic Factor Increases the Life Span and Body Mass in a Severe Model of Spinal Muscular Atrophy.</a> Hum. Gene. Ther. 22, 1-10 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

질병의 마우스 모델을 사용하는 연구는 종종 neonates에 약물이나 기타 물질의 관리를 필요로합니다. 이 비디오에서는, 우리는 입증 단계별로 CNS를 대상으로하는 데 사용할 수있는 분사 전략의 두 가지 유형의 포함하는 단계 절차 : 직접 분사가 intracerebroventricular (ICV) 주사를 이용한 CNS에 1), 또는 시간적 /를 대상으로 2) IV 주입 얼굴 정맥. 이 주사의 타이밍은 매우 중요합니다. ICV 주사는 프리핸드 때...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 그린 식품 염료 </td><td> 맥코믹 </td><td> N / A </td><td> 필터링해야합니다 </td></tr><tr><td> 해밀턴 유리 주사기 (100 μL) </td><td> 시그마 Aldrick </td><td> 20,702 </td><td></td></tr><tr><td> LuerMxF 스레드 스타일 화이트 나일론 </td><td> 소형 부품, 주식 회사 </td><td> VPLF - LC78 - 1-25 </td><td></td></tr><tr><td> 파인 게이지 피하 니들 </td><td> Popper </td><td> 7111 </td><td> 크기 : 33 (SWG) × ¼ &quot;(6.35 MM) </td></tr><tr><td> 위 시야 Transilluminator </td><td> Respironics </td><td> 1,017,920 </td><td></td></tr><tr><td> 2.25X 머리띠 돋보기 </td><td> MagEyes </td><td> 모델 5 호 </td><td> 개별 환경에 맞게 확대를 선택합니다 </td></tr></tbody></table>

delivery of therapeutic agents through intracerebroventricular (icv) and intravenous (iv) injection in mice

혈액 뇌 장벽은 두뇌를 차폐에서 활약하는 보호 역할에도 불구하고, 그것은 중추 신경계 (CNS)에 대한 액세스를 제한하는 neurodegenerative 장애 1,2을위한 잠재적인 치료제의 실패에서 가장 자주 발생합니다. 이러한 척수 근육질 위축 (SMA)로 Neurodegenerative 질병은 낮은 모터 뉴런이 영향을하는, CNS에 치료 에이전트를 도입에서 크게 혜택을 누릴 수 있습니다. 이 비디오의 목적은 출생 직후 신생아 생쥐에 치료 물질을 전달하기 위해 두 개의 서로 다른 사출 패러다임을 설명하는 것입니다. 이러한 방법 중 하나는 뇌척수 3,4 통해 CNS에 자료의 전송 결과 뇌성 측면 심실 (Intracerebroventricular)에 직접 주입합니다. 두 번째 방법은 CNS 5 포함한 체계적인 납품에 이르는, 순환 시스템에 다른 치료제를 소개할 수있는 시간적 정맥 주입 (정맥 주사)입니다. 적절한 바이러스 벡터 및 바이러스 혈청형가 활용되는 경우 CNS의 광범위한 도입이 달성됩니다. 시각화 및 주입을위한 정맥의 시간적 활용도가 출생 후의 일 6 가능합니다. 제공된 자료가 CNS에 도달하기위한 것일 뿐이므로 이러한 경우에는 혈액 뇌 장벽 때문에 그 미숙 상태로 더 투과하는 동안 그러나, 이러한 주사는 가급적 사전에 출생 후의 일 2, 장소 정도 소요됩니다. 완벽하게 개발된 혈액 뇌 장벽은 크게 정맥 ​​전달의 효과를 제한합니다. 수술 적성이 달성되면 전달 시스템 모두 간단하고 효과적입니다. 그들은 광범위한 수술 장치를 필요로하지 않으며 한 사람에 의해 수행할 수 있습니다. 그러나 이러한 기술은 도전없이는되지 않습니다. 출생 후의 일 2 새끼와 후속 작은 대상 영역의 작은 크기는 주사가 수행하기 어려운 및 복제 처음 도전 할 수 있습니다.

Watch this Scientific Journal Video about Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice at JoVE.com

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

신생아 생쥐의 중추 신경계에 치료 요원들을 소개하는 뇌 (intracerebroventricular)와 (정맥 주사) 2) 체계로 : 1)이 문서 분사의 두 가지 방법을 보여줍니다.

마우스에 Intracerebroventricular을 통해 치료 대리점 납품 (ICV)과 정맥 (IV) 사출

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

delivery-therapeutic-agents-through-intracerebroventricular-icv

Research

JoVE Journal

Medicine

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

59.2K 조회수.  University of Missouri. 신생아 생쥐의 중추 신경계에 치료 요원들을 소개하는 뇌 (intracerebroventricular)와 (정맥 주사) 2) 체계로 : 1)이 문서 분사의 두 가지 방법을 보여줍니다.

비디오: Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

대한 인간 루시페라제 태그가 추가된 악성 주변 신경 쉬스 종양 세포 Orthotopic Xenografting 생체내에</em후보 치료 대리점> 테스트

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

연구

의학

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

마우스의 모델링 Intracerebral 출혈 : Autologous 혈액이나 세균 Collagenase의 주입

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

쥐의 망막에 Elvax 40W를 통해 서방 약물 전달 : 만성 질환의 치료를위한 시사점

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

마우스의 초음파 유도 경 흉부 심근 내 주입

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

신경 보호 전략에 대한 치료 요인의 납품을위한 설계 성인 줄기 세포의 높은 처리량 특성

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, antiarrhythmic drugs administered intravenously can produce drops in systemic pressure in the already compromised cardiac patient. While performing open-heart procedures, surgeons will often either create a small port or form a pericardial cradle to create suitable fields for operation. This access yields opportunities for target pharmacological delivery (antiarrhythmic or ischemic preconditioning agents) directly to the myocardial tissue without undesired side effects.
We have developed a swine model for testing pharmacological agents for target delivery within the pericardial fluid. While fully anesthetized, each animal was instrumented with a Swan-Ganz catheter as well as left and right ventricle pressure catheters, and pacing leads were placed in the right atrial appendage and the right ventricle. A medial sternotomy was then performed and a pericardial access cradle was created; a plunge pacing lead was placed in the left atrial appendage and a bipolar pacing lead was placed in the left ventricle. Utilizing a programmer and a cardiac mapping system, the refractory period of the atrioventricular node (AVN), atria and ventricles was determined. In addition, atrial fibrillation (AF) induction was produced utilizing a Grass stimulator and time in AF was observed. These measurements were performed prior to treatment, as well as 30 min&#160;and 60 min&#160;after pericardial treatment. Additional time points were added for selected studies. The heart was then cardiopleged and reanimated in a four chamber working mode. Pressure measurements and function were recorded for 1 hr after reanimation. This treatment strategy model allowed us to observe the effects of pharmacological agents that may decrease the incidence of cardiac arrhythmias and/or ischemic damage, during and after open-heart surgery.

We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated.

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

TRUE 유전자 음소거 : 잠재적 인 암 치료제에 대한 헵 타머 형 소형 가이드 RNA 라이브러리의 심사

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

작은 동물에 대한 치료제의 폐 통기를위한 일회용 Dosators

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

마우스 모델에서 아교 모세포종에 치료 용 줄기 세포 비강 전달

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

경 피 적인 대비 심장 초음파-가이드 Intramyocardial 주입 및 큰 전 임상 모델에서 셀 배달

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...