이 작품은 JB 마샬 재단에 의해 지원되었다.

1. 절차에 대한 동물 및 장비 준비 <ol><li> 수술을 시작하기 전에 수술 지역과 동물을 준비합니다. PE50 튜브 12의 한쪽 끝 &quot;에 23 게이지 무딘 바늘을 삽입하여 경동맥 카테터를 확인합니다. 카테 테르의 반대편에 약 45 ° 베벨을 잘라 요. 이전 절차 장비를 소독. 머리 모자, 수술 마스크를 착용 및 멸균 장갑. </li><li> 수술이 수행 될 표면에 가열 패드를 놓습니다. 을 활성화하고 따뜻하게 할 수 있습니다. 흡수 벤치 패드와 지역을 커버. </li><li> 당신이 결정하지 않고 솔루션 25 % 마니 톨이 있는지 확인하십시오. 결정이 존재하는 경우, 80 ° C에서 온수 욕조에 병을 배치하고 정기적으로 교반하여 친구를 분해. </li><li> 동물의 질량을 기록합니다. </li><li> 0.8 MG / 0.1 ML / 분의 속도로 kg / 분에서 배달을 위해 propofol 희석하십시오. </li><li> 3 분의 유도 상자에 쥐를 놓습니다. 설정 : 5 % isoflurane, 2 L / 분환풍기에 대한 유량. </li><li> 일회용 수건의 상단에서 목을 모두 안쪽 허벅지를 면도. </li></ol> 2. 쥐를 Intubate <ol><li> 3 분의 유도 상자에 동물을 다시 놓으십시오. </li><li> IV 카테터와 endotracheal 튜브를 준비합니다. 이 크기와 쥐의 변형의 경우, 우리는 18 GA 카테터를 사용합니다. 튜브 손상을 초래하지 않고 적절한 공기 흐름을 허용 할 수있을만큼 큰, 그러나기도에 들어갈만큼 작해야합니다. 플라스틱 카테터의 바늘 아웃을 잡아 당겨 조심스럽게 바늘 약을 깰. 그 날카로운 끝에서 1cm. bunted 바늘은 스타일러스 될 것입니다. 바늘 다시 카테터에 넣어. </li><li> 각진 표면에 날카로운 이빨로 쥐를 잡아. </li><li> 동물의 목에 대해 직접 빛을 발하고 귀 사이에 정렬했습니다. </li><li> 작은 주걱 약을 삽입합니다. oropharynx에 3cm. 부드럽게 입에서 혀를 뽑아 동안 위쪽으로 들어 올려. </li><li> 기도의 개통을 표시합니다. 당신은 SE됩니다EA 작은 구멍은 각 숨 가까이 열고. </li><li> 부드럽게 구멍에 카테터를 삽입합니다. 당신은 가끔 플라스틱 기관의 연골을 입력 느낄 수 있습니다. 기관이 민감하다. 카테터를 강제하지 마십시오. </li><li> 1 L / 분 60 BPM과 2 % isoflurane에 환풍기를 설정합니다. </li><li> 운영 표면에 환풍기와 테이프 튜브에 카테터를 연결합니다. </li><li> 동물이 발가락 핀치을 수행하여 anesthetized 확인합니다. </li></ol> 3. Propofol IV의 마취를 확립 <ol><li> 운영 표면에 사지와 꼬리를 녹화하여 쥐를 고정합니다. </li><li> 면도 목 chlorhexidine 비누로 사타구니 사이트 세 번 씻어. chlorhexidine 세 번 씻어과 다음 씻어. </li><li> IV 확장 집합에 propofol의 주사기를 연결합니다. 주입 펌프에 주사기를 탑재합니다. </li><li> 면도 허벅지의 피부를 잘라내 메스를 사용합니다. 근육을 절단하지 마십시오. </li><li> 힘으로 절개를 엽니 다PS는 넙 다리 정맥을 시각화합니다. </li><li> 혈관을 노출 할 수있는 동맥을 통해 조직을 멀리 당겨. </li><li> 정맥에 26 GA monoject 동물 IV 카테터를 삽입합니다. 바늘이 정맥의 벽을 구멍하면은 밀어 쉽게 될 수있을 것입니다. 바늘을 철회하고 혈관에 카테터를 밀어. </li><li> 0.1 ML / 분에서 propofol 주입을 시작합니다. </li><li> 3-5 분 propofol 주입을 시작 후, 50 % O 2 50 % N 2 O의 혼합물로 마취 가스를 전환하고 isoflurane을 종료합니다. </li></ol> 4. 온목 동맥의 분기를 노출 <ol><li> 메스 블레이드와 목의 중간 선 절개를합니다. </li><li> 가위 또는 메스 두 개의 큰 침샘 사이에 자른다. </li><li> 경동맥을 노출 할 수있는 중간 선에 측면 조직​​을 멀리 해부. 조직의 레이어를 열 집게를 사용하십시오. 귀하는 포셉으로 노출되는 부분을 제거하고 minimall로 결정 가위를 사용할 수 있습니다Y 혈관. 거즈, 면봉, 그리고 소작 필요한 경우에 피를 흘리고을 관리 할 수​​ 있습니다. </li><li> 무거운 출혈에 적당한있을 경우 3 ML 생리 IP를 삽입. </li><li> 경동맥에서 조직을 멀리 끌어 견인기를 사용합니다. 분기를 찾습니다. </li><li> 분기에서 불과 말초 뒤통수 동맥을 쉽게받을 수 있습니다. 포셉 사이에 부식 및 electrocautery로 구분합니다. </li><li> 완전히 경동맥 분기 가능한 ECA의만큼 노출 집게를 사용하십시오. </li><li> 뒤통수 동맥 말초 ECA을 가지 우수한 갑상선 동맥을 쉽게받을 수 있습니다. 포셉 사이에 부식 및 electrocautery로 구분합니다. </li></ol> 5. 외부 경동맥을 ...에 카테 테르를 꽂다 <ol><li> ECA 아래 4 / 0 실크의 2 길이를 놓으십시오. </li><li> 귀하의 hemostat와 봉합의 1 길이를 잡고 (쥐의 발을 향해) 다시 caudally ECA를 철회. </li><li> 수 w로 다른 봉합를 타고 (쥐의 입쪽으로) cranially까지 묶어i 번째 외과 의사의 매듭. 이 ECA를 희생 할 수있는 영구적 인 끈을 형성한다. </li><li> 첫 번째 hemostat (꼬리 장력을 적용 된 1) 제거하고 다른 봉합 (ECA 주변의 매듭이 하나를) 잡아 hemostat와 cranially 부드러운 긴장을 적용 할 수 있습니다. </li><li> 느슨하게 다른 외과 의사의 매듭을 묶게 (보호하지 않습니다 - 거의 묶어두고) 분기 및 영구 끈에 근위에 말초. </li><li> 1 %가 동맥을 휴식을 lidocaine과 관개. </li><li> heparinized 생리와 카테터를 입력합니다. </li><li> 가까이 가능한 분기에 임시 선박 클립을 놓습니다. catheterization을위한 공간을 극대화 할 수있는 각도로 배치 선박 클립. 클립 대한 느슨한 매듭을 슬라이드. </li><li> microscissors를 사용하여 끈으로 단지 근위 ECA의 작은 arteriotomy (컷)을 확인합니다. 클립과 끈이 안전하더라도 출혈이 없어야합니다. </li><li> ECA 벽을 제어하는​​ 집게를 사용하여 혈관에 카테터의 beveled 끝을 먹이. </lI&gt; <li> 느슨한 봉합 매듭으로 카테터를 고정합니다. 카테터 주위의 동맥을 묶어주세요. </li><li> 클립을 제거하고 면봉으로 영역을 청소하십시오. </li><li> 분기에 말초 약 1 mm로 카테터을 부활 시켰습니다. 선박이 고정되어 있는지 확인 할 수있는 매듭에 인접 포셉으로 동맥을 잡아. </li></ol> 6. 마니 톨를 관리 <ol><li> 모든 기포를 생성하지 않고 8 &quot;IV 확장 세트 마니 톨과 6 ML의 주사기를 채우기 37 보육에서이 작업을 저장 ° C. 참고 : 작업을 신속하게 마니 톨과 함께 - 그게 쉽게 상온에서 솔루션에서 침전됩니다.. </li><li> BBBD 시각화하기 위해, 당신은 에반스 청색 색소를 관리 할 수​​ 있습니다하고 장벽이 중단 된 뇌의 영역을 입력 봐. 이 작업을 수행하려면 propofol 주입에 contralateral 다리에 새로운 IV 사이트를 통해 동물에게 2퍼센트 에반스 블루의 2 ML / kg을 제공합니다. </li><li> 필터링 및 공기 온것을 제거 할 마니 톨로 작성 5 μm로 설정 확장을 첨부bles. 그런 다음 거품을 만들지 않고 꼭지이 첨부하십시오. </li><li> 0.09 25 ML / 초 초에 마니 톨을 관리 할 수​​ 있습니다. ICA에 불과 근위 지점에 온목의 흐름과 평형에 도달 할 마니 톨을 보내드릴 것입니다. 이 쥐의 심장 박동 펄스에 나타납니다. 마니 톨은 단순히 마음으로 아래로 흐르는 경우 쥐의 혈액 압력을 극복했습니다. 이 생명을 위협하는 합병증 및 가난한 BBBD가 발생할 수 있습니다. </li></ol>

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관심 없음 충돌이 선언 없습니다.

BBBD의 효능을 극대화 몇 수단이 있습니다. 이 컷 다운 단계에서 출혈을 최소화하는 것이 중요합니다. 혈압과 심장 박동을 상당한 출혈에 의해 영향을받을 수 있으며, 이러한 요소는 BBBD 13 정도에 영향을 미치는 것으로 알려져 있습니다. 출혈은 같은 뛰어난 갑상선과 구분되어야한다 뒤통수 동맥과 같은 주요 혈관을 ligate에 봉합을 사용하여 줄일 수있다. 또한, electrocautery는 혈관을 분할하?...

<table border="1"><tbody><tr><td> 재료 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 논평 </td></tr><tr><td> 긴 에반스 쥐 </td><td> 할란 연구소 </td><td></td><td> 2백10~2백50g 남성 </td></tr><tr><td> PE 50 배관 </td><td> Beckton - 디킨슨 </td><td></td><td></td></tr><tr><td> 18 게이지 X 2.5 &quot;IV 카테터 </td><td> Terumo </td><td></td><td> 동부 표준시 튜브에 대한 </td></tr><tr><td> 30 &quot;IV 확장 설정 </td><td> 애보트 </td><td></td><td></td></tr><tr><td> 26 게이지 수의학 IV 카테터 </td><td> Monoject </td><td></td><td></td></tr><tr><td> 에반스 청색 색소 </td><td> 시그마 </td><td> E2129 </td><td></td></tr><tr><td> 이극의 </td><td> Codman </td><td></td><td></td></tr><tr><td> 필터, 5 μm </td><td> 브라운 </td><td></td><td></td></tr></tbody></table>

rat model of blood-brain barrier disruption to allow targeted neurovascular therapeutics

지하 막과 astrocyte 최종 피트와 함께 꼭 접속점과 내피 세포는 혈액 - 뇌 장벽 하나를 형성하기 위해 대뇌 혈관을 둘러싸고 있습니다. 장벽은 선택적으로 혈액과 크기와 비용에 따라 뇌 간 이동에서 분자를 제외합니다. 이 기능은 신경 장애 치료학의 전달을 방해 할 수 있습니다. chemotherapeutic 약물의 수는 예를 들어, 효율적으로 종양 세포 2를 도달 할 혈액 - 뇌 장벽을 통과하지 않습니다. 따라서, 혈액 - 뇌 장벽에 걸쳐 약물의 전달을 향상하는 것은 관심의 영역입니다. 뇌에 약물의 전달을 향상을위한 가장 널리 방법은 직접 대뇌 주입하고 혈액 - 뇌 장벽 중단 3아르. 치료법은 뇌에 도달 직접 intracerebral 주입 보장하는, 그러나,이 방법은 약물에게 4 분산 할 수있는 제한된 능력을 갖추고 있습니다. 혈액 - 뇌 장벽 장애 (BBBD) 알순환 시스템에서 직접 흐름에 낮은 약물 뇌에 있으며 따라서보다 효율적으로 분산 종양 세포에 도달합니다. 장벽 장애의 세 가지 방법은 삼투 장벽 파괴, 약리 차단 중단하고, microbubbles과 중심 초음파가 포함되어 있습니다. Neuwelt에 의해 개척 삼투 중단은 그들의 꽉 접합을 축소하고 방해하는 원인이되는 혈액 - 뇌 장벽의 세포를 dehydrates 25 % 마니 톨의 hypertonic 솔루션을 사용합니다. 장벽 장애는 또한 히스타민 5, bradykinin 6과 같은 vasoactive 화합물과 pharmacologically 수행 할 수 있습니다. 이 방법은 그러나, 주로 뇌 종양이 장벽을 7 선택합니다. 또한, RMP-7, bradykinin 펩타이드의 아날로그은 25 % 마니 톨 8 삼투 BBBD으로 정면을 비교했을 때 열악한 것으로 발견되었다. 또 다른 방법 microbubble 초음파 대비 에이전트와 함께 집중 초음파 (FUS)는도 표시되었습니다가역적으로 혈액 - 뇌 장벽 9 엽니 다. FUS에 비해하지만, 25 %의 마니 톨은 병진 연구 10-12를위한 입증 된 도구 만드는 인간의 환자 안전의 긴 역사를 가지고 있습니다. BBBD 달성하기 위해, 마니 톨은 직접 뇌의 동맥 순환에 높은 속도로 전달해야합니다. 인간에 ​​endovascular 카테터가 빠른 직접 흐름이 수행 할 수 있습니다 뇌에 안내되어 있습니다. 이 프로토콜 모델 인간 최대한 BBBD. 컷 다운 온목 동맥의 분기에 따라 카테터는 ECA에 역행 삽입과 속목 동맥 (ICA) 순환에 직접 마니 톨을 제공하는 데 사용됩니다. Propofol과 N 2 O의 마취는 장벽 파괴 13의 효과를 극대화 할 수 있는가에 대한 능력을 사용합니다. 제대로 실행하는 경우이 절차를 안전하게, 효율적으로, 그리고 가역적으로 혈액 - 뇌 장벽과 즉흥을 열 수있는 능력을 가지고전자 일반적으로 뇌 8,13,14에 도달하지 약물의 전달.

<img alt="그림 1" src="/files/ftp_upload/50019/50019fig1.jpg" /> 1 그림. 에반스 청색 색소의 넘쳐 흐름을 통해 혈액 - 뇌 장벽 혼란을 시각화. 에반스 청색 색소은 알부민에 바인딩 및 생리 조건 하에서 뇌에 extravasated되지 않는 안료입니다. 뇌의 한쪽에있는 혈액 - 뇌 장벽의 파괴가 아닌 중단 반구는 변함 동안 에반스는 파란색 중단 반구의 파란색를 입력하고 얼룩 ?...

Watch this Scientific Journal Video about Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics at JoVE.com

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

혈액 - 뇌 장벽 장애는 뇌에 특정 약물의 전달을 에이즈. 마니 톨은 물리적 장벽을 방해하기 위해 간 arterially 혈관을 수축 주변 세포를 전달했습니다.

혈액 - 뇌 장벽 장애의 쥐 모델 Neurovascular 치료학 타겟이 분명한 허용하려면

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

rat-model-blood-brain-barrier-disruption-to-allow-targeted

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JoVE Journal

Medicine

19.3K 조회수.  Vanderbilt University School of Medicine. 혈액 - 뇌 장벽 장애는 뇌에 특정 약물의 전달을 에이즈. 마니 톨은 물리적 장벽을 방해하기 위해 간 arterially 혈관을 수축 주변 세포를 전달했습니다.

비디오: Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

쥐 모델에서 Transcranial 집중 초음파를 사용하여 혈액 - 뇌 장벽의 MRI를 유도 장애

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

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의학

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

깨어 마우스로 CNS의 치료학의 비강 관리

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

로 뇌 미세 혈관 내피 세포 (CEnd를)에 기지개<em&gt; 체외</em혈액 뇌 장벽의&gt; 외상성 뇌 손상 모델

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

설정 - 업<em&gt; 체외</em쥐 뇌 혈관 벽의&gt; 모델 (BBB)​​ : BBB 불 침투성 및 수용체 매개 운송에 초점

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

같은 산소 포도당 박탈과 재산 소화<em&gt; 체외</em&gt; 허혈 - 재관류 손상 모델 공부 뇌 혈관 장벽 부전

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

생쥐 경동맥 풍선 손상 모델은 반대로 혈관 리모델링의 치료를 테스트하는

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

타액 기능 저하의 쥐 모델에서 Retroductal 턱밑 글 랜드 점적 및 현지화 된 분획 조사

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

혈액 뇌 장벽 메쉬와 유사한 혈관 형성 및 분열을 평가하기위한 In vitro 시험 법

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

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) ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Myography 와이어를 사용 하 여 마우스 Mesenteric 동맥의 isometric 수축 측정

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

혈액 - 뇌 장벽 장애의 쥐 모델 Neurovascular 치료학 타겟이 분명한 허용하려면

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