이 연구는 파킨슨 병의 연구 2011 신속 대응 혁신 상 프로그램에 대한 Mcihael J. 폭스 재단에 의해 투자되었다. 출자자는 연구 설계, 데이터 수집 및 분석, 게시 할 수있는 의사 결정, 또는 원고의 준비에 역할을했다.

수술에 앞서 수행되는 모든 절차가 IACUC 및 추가 윤리적 또는 법적 당국의 승인을 인간 동물의 치료 방법을 사용하고 있는지 확인합니다. 이것은 불임 수술 조건을 사용하여 IACUC이 방법을 승인하여 마우스를 마취, 수술하는 동안 의사의 연고와 마우스의 눈을 윤활 및 수술 후 관리를 제공하는 단계를 포함한다. 절차의 측면이 승인 여부를 질문이있는 경우에 수술을 진행하지 마십시오. 여기에 수행 된 모든 절차는 보스턴 대학 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 1. 동물의 준비 및 외과 용품 <ol><li> 수술 중에 사용되는 모든 수술 악기를 압력솥. </li><li> 수행 할 수있는 모든 기술이 동물 규제 기관에 의해 승인되어 있는지 확인하십시오. </li></ol> 점막 이식의 2. 수확 <ol><li> 로 비슷한 나이의 유 전적으로 동일한 마우스를 선택했다실험 마우스 (여기 : 자궁 경부 전위 다음에 이소 플루 란 질식) IACUC 승인 된 방법에 안락사. </li><li> 외과 용 가위를 사용하여 두개골을 노출 마우스 머리의 코 지역 주위의 피부를 제거합니다. </li><li> 옆으로 코 지역과 수직으로 두 줄을 연결하는 눈 라인의 세 번째 측면에 두 세 가지의 라인 공압 드릴, 마크. </li><li> 주변 조직에서 비중격을 분리하기 위해 배쪽으로 드릴 다운. 넓은 경로, 그러나 그것은 또한 더 어렵게 멤브레인을 분리 할 것이다 점막의 손상을 방지 할 것이다. 가까운 중간 선에 좁은 컷을 권장합니다. </li><li> 여기에 부착 된 모든 조직에서 무료로 격막을 절단하고 멸균 식염수에 저장하기 위해 가위를 사용합니다. 이 때 수술이 연결된 조직을 제거하는 청소 할 수 있습니다. 이상적인 상황은 피해 점막 멤브레인 연골 격벽의 양​​측에 노출하는 것이다. 한 GR선미는 멤브레인의 표면적이 개두 사이트를 커버하기에 충분한 제공이 생쥐위한 멤브레인을 공급할 수있다. 그것은 이식이 가능한 한 빨리 사용하고 연구자가 즉시 이식이 격리로 3 단계로 진행하는 것이 좋습니다. </li></ol> 점막 이식 3. 외과 이식 <ol><li> 표준, 무균 쥐의 수술 절차를 사용하여, 마취 및 stereological 프레임에 마우스를 탑재합니다. 쥐의 마취 기계를 사용하여 순수한 산소에 약 2 % 이소 플루 란 (isoflurane)을 사용합니다. </li><li> 귀 바, 코 홀더 정위 장치에 마우스를 고정시킨다. 눈에 안과 연고를 적용하고 3 라운드에 대한 베타 딘 75 % 에탄올로 두피를 문질러. 가위 나 머리 트리머를 사용하여, 머리에 모피를 제거합니다. 면도날 두개골을 드러내고 헤드 수평. 투여 할 뇌의 위치 위의 개두술을 수행합니다. 예를 들어, 선조체를 타겟팅은 1.25 mm 컷공기 드릴을 사용 :; (0.88 mm ML 1.00 mm AP를 중심으로) 두개골 직경 원형 구멍. 멸균 식염수로 드릴 된 영역을 적시고 두개골을 제거하기 위해 면도날을 사용합니다. </li><li> 조심스럽게 바늘의 끝 부분을 사용하여 경질을 제거합니다. 또한,이 촉촉한 경막 표면 조직 접착제의 최소량을 적용함으로써 달성 될 수있다. 이 층은 경화되면 면도날 팁의 수평 방향 운동은 멤브레인을 제거하는데 사용될 수있다. </li><li> 멀리 상처에 직면 상피면을 유지하기 위해 극도의주의를 복용 뇌 표면 위의 점막을 놓습니다. 이것은 가장 핀셋 개두술 사이트에 인접한 두개골의 표면에 전체 격막을 전송하여 수행됩니다. 수술 가위의 끝 부분을 사용하여 연골의 할인 및 두개골과 뇌 표면에 막을 당겨. 막 건조 또는 흡수 물질과 접촉하지 마십시오. 이식 아낌없이 개두술의 S의 모든 뼈 가장자리를 오버랩한다ITE. </li><li> 니트릴의 살균 조각으로 이식을 커버. 이것은 치유 중에 그래프트에 피부의 부착을 방지하는 역할을한다. 니트릴은 전체 점막을 커버 할만큼 커야 할 필요가있다. 필요한 경우 과도한 막 트림. 그것은 이식과 접촉되면 니트릴의 움직임을 피하십시오. </li><li> 실행 5-0 멸균 봉합사로 피부를 닫고 마우스가 다음 단계로 진행하기 전에 3~7일에 복구 할 수 있습니다. 피부 폐쇄하는 동안 니트릴 장벽 또는 점막 이식을 교란하지 않도록주의하십시오. </li></ol> 분배 솔루션 4. 관리 <ol><li> 고정대에 마취 된 쥐를 확보 한 후, 가위로 봉합을 잘라 두개골 주위에 여분의 피부를 제거합니다. </li><li> 니트릴 장벽을 제거하고 두개골의 표면을 청소합니다. 이식이 보일 때까지 지역을 청소하는 멸균 식염수 면봉을 사용합니다. D보다 큰 성장하면 면도칼로 이식을 절단해야 할 수도 있습니다표면적 명령 1. </li><li> 실험이 몇 일 이상이 될 경우, 그것은 머리 이식을 강화하기 위해 적어도 두 개의 두개골 나사를 이식하는 것이 현명하다. </li><li> 가장자리가 두개골과 접촉되도록 이식 위의 잘 놓습니다. 잘와 두개골 사이의 교차점에 시아 노 아크릴 레이트 접착제를 적용합니다. 멸균 식염수로 잘 입력하고 확인 누출이 없는지 확인하십시오. 웰스는 컷 주사기 바늘 만들어진다. </li><li> 장소에 잘을 확보하기 위해 두개골에 골 시멘트를 적용합니다. </li><li> 피펫으로 우물에서 염분을 제거합니다. 그 접착제가 원하는 솔루션을 추가 인치 유출되지 확인도 여러 번 세탁; 이 경우 형광 덱스 트란 50 μL가 사용됩니다. 이것은 유사한 극성의 수용성 화합물은 덱스 트란과 동일하게 동작 할 것으로 예상된다. 소수성 화합물 또는 현탁액의 배송이 방법으로 탐구되지 않았습니다. </li><li> 고정 니트릴 원형 조각을 사용하여 웰의 상부 캡시아 노 아크릴 레이트 접착제를 사용하여 맨. 접착제가 아니라 내용에 접촉하지 않도록하십시오. </li></ol> 점막 납품 5. 분석 <ol><li> 원하는 시간이 경과 한 후, 마우스를 마취하고 에반의 청색 염료의 용액으로 웰 내용물을 교환한다. 이 염료는 이식이 손상되었는지 확인하는 데 사용됩니다. </li><li> 30 분 후, 마우스를 심하게 마취 염료 용액을 제거하고, 잘린 통해 안락사. </li><li> 수동으로 임플란트를 제거하고 수술 가위를 사용하여 두뇌를 제거합니다. 장소에 이식 않도록주의하십시오. </li><li> 일단 제거되고, 플래시 동결 이소 부탄의 용액에 뇌는 드라이 아이스 욕에서 냉각시켰다. </li><li> 최적 절단 온도 50 ㎛에서 (10 월) 솔루션과 조각에 뇌를 포함합니다. </li><li> 현미경 슬라이드에 직접 원하는 조각을 놓습니다. </li><li> 이미지 즉시 OCT의 솔루션은 건조되면서 개화 현미경을 사용하여 조각. </li></ol>

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벤자민 S. 같은 Bleier MD는 중추 신경계에 약물 전달의 임시 특허 취재 방법의 리드 발명가입니다.

본원에 기술 된 과정에 가장 어려운 단계는 뇌 표면에 충분히 크기 점막의 성공적인 전송이다. 수확 비중격가 충분하고 잘 청소하는 경우이 단계는 훨씬 쉬워졌다. 격막의 복부 부분이 잘리는 경우, 새로운 이식을 얻을 수 있어야합니다. 드릴링 각도 점막이 드릴에 의해 손상되지 않도록 마우스 헤드에 수직이어야한다. 추천하는보다 넓은 드릴링 경로 촬영할 경우는 격막에 연결되어 여분의 조직...

신경 및 정신 질환의 치료는 심각한 중추 신경계 1-3에 도달하는 모든 잠재적 약제의 95 % 이상을 방지 혈액 - 뇌 장벽 (BBB)의 존재에 의해 방해된다. 예를 들어, 신경교는 신경 친 화성 인자 (GDNF)를 파생은 BBB 4-6을 관통 할 수 없기 때문에 전신적 전달되는 경우에는 비효율적이다, 뇌에 직접 분사하면 파킨슨 병의 치료에 효과적인 것으로 밝혀졌다. 수많은 접근이 문제를 회피하는 것을 시도하기 위해 개발되었다. neurotheraputics의 전신 배달의 개선은 뇌의 모세 혈관 내피 세포에있는 수송 단백질에 대한 선택적인 항체를 포함하는 약물 접합체를 사용하여 증명되었다; 그러나이 방법은 의약품 7,8의 광범위한 적용 할 수 표시되지 않았습니다. 또한, BBB의 삼투압 개방 병원에 사용 된또한 9의 뇌 영역에 대한보다 직접 배달에 반대 동맹, 그러나이 방법은 전신 약물 투여에서 겪고있다. 상당한 노력이 직접 뇌 10-12을 대상으로의 희망에 transnasal 전달을 최적화에 투입되었습니다. 약간의 성공이 달성되었지만, 결정적인 결과는 인슐린의 13, 14 등의 내생 적 수용체를 가지고 의약품에 대한 얻어졌다. 또한 transnasal 전달의 메커니즘은 후각 신경 세포의 흡수를 통해 또는 혈류 (11)를 통해 뇌에 간접적 인 항목을 제안 증거가 논란이되었습니다. 이식 카테터를 사용하여 직접, 두개 배달 그러나이 절차가 매우 침략과 수많은 합병증 (15, 16)와 연결되어, 달성되었습니다. 지금까지 뇌에 고분자 화합물을 제공하는 일반적인 침습적 방법이 없다. 쥐의 수술은 여기에 있습니다 발표즉, 뇌와 반투과성 인터페이스를 만듭니다. 이것은 마우스의 수술 적 개두술의 결함을 통해 점막 이식편 17 engrafting하여 수행됩니다. 이 절차를 사용하여 500 kDa의 최대 가용성 화합물은 시간과 분자량 의존적 (18) 모두에서 중추 신경계 (직접 뇌 실질에뿐만 아니라 뇌척수액에)에 전달 될 ​​수있는 것으로 나타났습니다. BBB를 우회하는이 방법은 transnasal 내시경 수술 19, 20 다음의 두개골에 구멍을 복구하는 혈관 점막 이식을 사용하여 인간의 두개골 바닥의 결함 수리를위한 모델이다.

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heterotopic mucosal engrafting procedure for direct drug delivery to the brain in mice

뇌에 치료제의 전달은 혈액으로부터 뇌 조직으로 극성 및 고 분자량 화합물의 통과를 제한하는 혈액 - 뇌 장벽 (BBB)​​의 존재에 의해 방해된다. 인간의 일부 직접 배달 성공은 두개의 카테터 주입을 통해 달성되었다; 그러나이 방법은 고도의 침략과 수많은 합병증과 관련이 있습니다. 보다 적게 침략적인 대안은 인간의 내시경 transnasal 종양 제거 수술 후 두개골 바닥 결함을 수리하는 데 사용되는 코 점막 등의 이식 수술, 반투막을 통해 뇌를 투약하는 것입니다. 하지만 약물 전달이 막 효과적으로 BBB를 무시하고 뇌와 중추 신경계에 직접으로 확산 될 것이다. 이 방법에 의해 영감을, 마우스의 수술 방법은 extracranial 수술 BBB 결함에 접목 기증자 중격 점막을 사용하는 개발되었다. 이 모델은 효과적으로 표시되었습니다뇌에 고 분자량 화합물의 통과를 허용한다. 많은 약물 후보가 BBB를 횡단하는 능력이 있기 때문에,이 모델은 신경 및 정신 질환에 대한 새로운 치료법의 전임상 시험을 수행 할 가치가있다.

충분히 큰 비중격 이식편을 얻기 후속 단계에 대한 매우 중요합니다. 이것은도 1a에 도시 된 도너 마우스의 두개골에 위치에 드릴에 의해 달성 될 수있다. 그림 1b에서와 같이이 경로를 따라 절단 충분한 크기의 이식편을 생성합니다. 드릴링 깊이가 깊은 충분하지 않은 경우, 그래프트는 잘리고 그 뇌의 표면을 커버하도록 충분히 큰 막을 얻기 어려울 것이다. 이상의 조직 ...

Watch this Scientific Journal Video about Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice at JoVE.com

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

인간의 내시경 두개골 바닥 재건의 마우스 모델은 코 점막 이식을 사용하여 뇌와 코 사이의 반투과성 인터페이스를 만듭니다 개발되었다. 이 방법은 연구자가 체계적으로 관리, 그렇지 않으면 혈액 - 뇌 장벽에 의해 제외되는 고 분자량 치료제의 중추 신경계에 전달을 연구 할 수 있습니다.

쥐의 뇌에 직접 약물 전달 이소성 점막 Engrafting 절차

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

heterotopic-mucosal-engrafting-procedure-for-direct-drug-delivery-to

Research

JoVE Journal

Medicine

10.7K 조회수.  Boston University.  인간의 내시경 두개골 바닥 재건의 마우스 모델은 코 점막 이식을 사용하여 뇌와 코 사이의 반투과성 인터페이스를 만듭니다 개발되었다. 이 방법은 연구자가 체계적으로 관리, 그렇지 않으면 혈액 - 뇌 장벽에 의해 제외되는 고 분자량 치료제의 중추 신경계에 전달을 연구 할 수 있습니다. 

비디오: Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

마이크로 컴퓨터 단층 촬영 영상에 대한 Microfil와 허혈성 뒷발과 혈관의 관류에 삼투의 약물 전달 (Drug Delivery)

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

연구

의학

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 ...

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

직접 마우스 외상 / 이소성 골화의 모델을 굽기

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

마우스의 이소성 자궁 심장 이식

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

마우스 동정맥루 내피에 관내 약물 전달

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Ivacaftor, 그것의 주요 대사 산물, 및 생물 학적 체액의 낭 성 섬유 증 환자에서 Lumacaftor의 임상 샘플의 높은 처리량 분석에 대 한 최적화 된 LC-MS/MS 방법

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

생체 외에서 혈액-뇌 장벽 돼지 뇌 내 피 세포를 기반으로 하는 모델의 설립에 대 한 방법 개선

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

Ototoxicity의 치료에 대 한 트랜스-귀 약물 전달

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

Vivo에서 Photolabeling 신생아 쥐에 있는 조 혈 모 세포의 이동성 잠재력을 평가 하는 콜론에 셀의

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...