Special thanks to the Gidday lab for their work in developing the RHP protocol, as well as the Neuro-Models Facility (UTSW) for their assistance in the tMCAo surgeries. This work was supported by grants from the American Heart Association (AMS), The Haggerty Center for Brain Injury and Repair (UTSW; AMS), and The Spastic Paralysis Research Foundation of the Illinois-Eastern Iowa District of Kiwanis International (JMG).

The authors have nothing to disclose.

전신 저산소증 마우스 (즉, 2 시간 11 % O 2)에 하나의 노출은 &quot;일시적으로&quot;짧은 지속되는 저산소 전처리 도전에 후생 유전 학적 반응을 의미하는 결과 tMCAO, 29 일부터 뇌를 보호하고, 기준 표현형 내에서 복원 일. 저산소 전처리 자극의 반복 프리젠 테이션은 크게 신경 표현형의 기간을 연장. 6 많은 연구는 반복적 인 자극 열차의 빈도, 규모 및 기간이 응답의 중...

성인 장애와 사망의 네 번째 주요 원인의 주요 원인으로, 뇌졸중은 미국의 성인 인구가 직면 한 가장 쇠약 질병 상태 중 하나입니다. 뇌졸중의 1 동물 모델은 허혈 손상을 감소시키는 새로운 방법과 실험 조사 허용 졸중 회복을 개선시킨다. 등의 중개 연구에 대한 하나의 새로운 길을 사전 조정된다. 전처리는 후속하는, 더 심한 부상으로부터 손상을 줄이기 위해 비 손상 자극의 의도적 인 사용이다.이 저산소 전처리는 생체 내 및 시험 관내 연구에서 모두 스트로크에 대한 보호를 제공하는 뇌의다면 발현 변화를 생성하는 것으로 나타났다 . (3) 그러나, 저산소증에 하나의 노출은 성인 쥐에서 허혈에 대한 허용 오차 미만의 72 시간을 유도, 단기 신경을 제공합니다., 린 등 기압 성 저산소증에 14 시간 매일 노출의 4도 네 후 주. FO싶게 그 신경은 일주. 5 반복 저산소 전처리 (RHP) 지속 하였다는 주파수, 기간, 저산소 노출의 강도의 확률 적 변화에 의해 특징입니다. 단일 전처리 도전 달리 RHP 쥐 여덟 주까지 지속 cerebroprotective 표현형을 유도한다. 6 RHP 최종 저산소 노출 후 주 경색 부피, 혈액 - 뇌 장벽 (BBB) ​​장애, 혈관 염증 및 백혈구 diapedesis 감소 . RHP 구체적 허혈성 반구에서 B 세포 개체군을 유지하면서, T 세포, 단핵 세포 및 대식 세포 개체군을 감소시켜 허혈성 뇌의 염증을 감소시켰다.도 7은 실제로 RHP 뇌졸중을 포함한 모든 CNS 부상 전에 마우스의 면역 표현형을 유도. RHP 처리 건강한 쥐로부터 분리 RHP 처리 B 세포는 항원 제시 및 항체 생산의 하향 조절 모두에, 고유 항 염증 표현형을 나타내었다.염증성 적응성 면역 기전에 전체적인 감소뿐만 아니라 특정 CNS 염증성 질환에 대한 내인성 면역 억제를 유도하기 위하여 RHP에게 우수한 방법론하게 할뿐만 아니라 염증성 병태를 포함 전신성 상해 또는 질병 모델. 우완는 과도 중간 대뇌 동맥 폐색 (결과 tMCAO) 다음 경색 볼륨 및 BBB 중단을 모두 줄일 수 있습니다. 이러한 일반적으로 사용되는 결과 tMCAO 같이 뇌졸중 동물 모델은 극적 뇌졸중 병태 생리에 대한 이해뿐만 아니라, 더욱 효과적인 neurotherapeutics의 디자인을 향상시킬 수있다. 우선 고이즈미 총리 등의 알에 의해 개발., 1986 년, 8 결과 tMCAO 절차는 설치류에서 뇌졸중 및 재관류 다음과 같은 염증을 조사하기위한 바람직한 방법 중 하나를 유도하는 널리 사용되는 방법이다. 결과 tMCAO위한 방법들이 발전함에 따라, 실리콘 코팅 된 섬유의 최근 이용은 또 다른 모델 9,10 &lt;비교 지주막 하 출혈의 위험을 줄일/ SUP&gt; 불행하게도 결과 tMCAO 자주 경색 볼륨에 다양한 변화를 생산 불구하고, 신뢰성을 향상시킬 수있다. 11-13 이러한 연구의 대부분은 2,3,5- 트리 페닐 클로라이드 (TTC)로 염색하여 관상 뇌 부분에서 경색 영역을 서술 고려 경색 정량 금 표준은 선명한, 복제 가능한 결과를 생성하기위한 간단하고 저렴한 방법이기 때문이다. TTC는 미토콘드리아에 존재하는 탈수소 효소의 기판 역할을합니다. 뇌 조각은 TTC 용액에 노출되면 포르 마잔의 비 수용성 감소 제품, 가능한 미토콘드리아에 깊은 붉은 색 침전 경우, TTC는 선택적으로 살아있는 세포로 촬영됩니다. 때문에 허혈성 조직에서 미토콘드리아 기능 장애의,이 조직 손상과 건강한 조직의 분화를 허용, 흰색 남아있다. (14) 우완 또한 허혈성 반구에서 BBB 중단을 줄일 수 있습니다. (6) 따라서, BBB 무결성의 이중 정량화 같은 B 내에서TTC 기반 경색 볼륨과 비는 내인성 보호의 전체 효과에 대한 유용한 정보, 치료 및 치료 동물에서 BBB 중단 및 경색 사이의 잠재적 인 인과 관계를 제공 할 것 15 결정. 뇌졸중 이차 중단 BBB,,를 통해 말초 혈액의 유입은 궁극적으로 허혈성 뇌졸중 환자에서 감염과 사망률의 비율을 증가, 허혈성 반구에 백혈구 인구, 염증성 사이토 카인, 산화 스트레스, vasogenic 부종, 출혈성 변환을 증가 . 16,17 동물 모델에서 BBB 중단을 측정하는 일반적인 방법은 뇌에 에반스 청 (EB) 염색 누설 정량화 통해서이다. 15,18-21 EB 선택적 알부민, 구형 단백질 혈청에 결합 (MW = 65 kDa의) 즉 손상되지 않은 동물에 BBB를 통과하지 않습니다. (22) 허혈성 뇌졸중에 이어, EB는 뇌를 침투, 광학 밀도 withi의 측정을 허용, 620 nm에서 형광을N 관류 부상 실질. (22) 광학 밀도는 EB는 (rongeur)로하여 사후 대뇌 피질의 혈관에서 세척 된 BBB의 투과성에 직접적으로 비례한다. EB 관리와 동물 TTC 염색 뇌의 즉각적인 처리와 함께, 경색 볼륨과 BBB 중단을 모두 효과적으로 정량화 할 수있다. 이것은 신경 상해 및 BBB 붕괴가 뇌졸중 후 뇌의 프로세스가 수반되지임을 인식하여야한다, 그래서 23,24 희생의 시간의 선택은 중요한 고려 사항이다. 다음 프로토콜 RHP 방법, 임시 동맥 폐색을 유발 결과 tMCAO 방법 자세히 모델링 인간 환자에서 중간 대뇌 동맥 폐색, 신경 및 혈관 뇌졸중 손상 엔드 포인트를 결정하는 이중 조직 학적 방법. TTC는 전체 경색 부피의 정량화를 허용 세포사 누적 조직 손상을 측정매화, EB는 BBB 손상의 반구 정량을 제공한다.

<table border="0" cellpadding="0" cellspacing="0" width="830">
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			<td height="220" style="height:220px;width:249px;">Flowmeters, regulators</td>
			<td style="width:171px;">VetEquip, Inc</td>
			<td style="width:127px;">Specialty order</td>
			<td style="width:283px;">Four flowmeters are attached to 6.0 mm flexible PVC tubing which connects to the inlet port on each induction chamber with a plastic female connector. These flowmeters are bolted to a 6.5&#34; x 1&#34; x 1&#34; metal bar. This metal bar is bolted to a MI-246-P pressure gauge with a DISS outlet. This pressure gauge and flowmeter equipment can be attached to each new gas cylinder with a wrench.</td>
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			<td height="26" style="height:26px;width:249px;">21% O2 tank</td>
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			<td height="26" style="height:26px;width:249px;">11% O2 tank</td>
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			<td style="width:283px;"></td>
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			<td height="26" style="height:26px;width:249px;">8% O2 tank&#160;</td>
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			<td style="width:283px;"></td>
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			<td height="20" style="height:20px;width:249px;">15L induction chambers</td>
			<td style="width:171px;">VetEquip</td>
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			<td height="40" style="height:40px;width:249px;">Zoom Stereo Microscope&#160;</td>
			<td style="width:171px;">NIkon</td>
			<td style="width:127px;">SMZ800</td>
			<td style="width:283px;">Other surgical microscopes may be used.&#160;</td>
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		<tr height="100">
			<td height="100" style="height:100px;width:249px;">Kent Scientific Right Temperature CODA</td>
			<td style="width:171px;">Kent Scientific Corporation</td>
			<td style="width:127px;">Discontinued</td>
			<td style="width:283px;">Recommended replacement is PhysioSuite with RightTemp Temperature Monitoring and Homeothermic Control (Kent Scientific, #PS-RT).</td>
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		<tr height="60">
			<td height="60" style="height:60px;width:249px;">Hovabator Incubator</td>
			<td style="width:171px;">Stromberg&#39;s</td>
			<td style="width:127px;">2362-E</td>
			<td style="width:283px;">Our model is the 2362N. 2362E is a later model and includes an electronic thermostat.&#160;</td>
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		<tr height="120">
			<td height="120" style="height:120px;width:249px;">V010 Anesthesia system&#160;</td>
			<td style="width:171px;">VetEquip</td>
			<td style="width:127px;">901807</td>
			<td style="width:283px;">Includes: ten foot high-pressure oxygen hose, frame, flowmeter, oxygen flush assembly, vaporizer, breathing circuit, chamber, nosecones, waste gas evacuation tubing and two VapoGuard filters</td>
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			<td height="20" style="height:20px;width:249px;">250 mL isoflurane&#160;</td>
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			<td style="width:283px;"></td>
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			<td height="40" style="height:40px;width:249px;">D-6 Vet Trim Animal Cordless Trimmer&#160;</td>
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			<td style="width:283px;">Replacement blades are available from Andis (#23995)</td>
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			<td style="width:171px;">Fisher Scientific</td>
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			<td height="20" style="height:20px;width:249px;">Gauze Pads</td>
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			<td height="20" style="height:20px;width:249px;">Surgical drape</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">GM300&#160;</td>
			<td style="width:283px;"></td>
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			<td height="40" style="height:40px;width:249px;">Silk Sutures&#160;</td>
			<td style="width:171px;">Look/Div Surgical Specialties</td>
			<td style="width:127px;">SP115</td>
			<td style="width:283px;"></td>
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			<td height="40" style="height:40px;width:249px;">Nylon Sutures</td>
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			<td style="width:283px;"></td>
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			<td height="20" style="height:20px;width:249px;">Durmont #5 forceps (2)&#160;</td>
			<td style="width:171px;">Fine Science Tools&#160;</td>
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			<td height="20" style="height:20px;width:249px;">Surgical Scissors</td>
			<td style="width:171px;">Fine Science Tools&#160;</td>
			<td style="width:127px;">14028-10</td>
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			<td height="40" style="height:40px;width:249px;">3mm Vannas</td>
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			<td style="width:127px;">INS600177</td>
			<td style="width:283px;">Straight blade</td>
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			<td height="20" style="height:20px;width:249px;">Hartman Hemostats&#160;</td>
			<td style="width:171px;">Fine Scientific Tools</td>
			<td style="width:127px;">13002-10</td>
			<td style="width:283px;"></td>
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			<td style="width:171px;">Washington University</td>
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			<td style="width:283px;">Filaments are silicone coated at Washington Univeristy and provided to UTSW facilities for a fee.&#160;</td>
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			<td height="20" style="height:20px;width:249px;">Evans Blue</td>
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			<td height="20" style="height:20px;width:249px;">Filter Paper&#160;</td>
			<td style="width:171px;">Sigma Aldritch</td>
			<td style="width:127px;">WHA1001150</td>
			<td style="width:283px;">150 mm, circles, Grade 1&#160;</td>
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			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">02-202-101</td>
			<td style="width:283px;">2.5&#34; diameter</td>
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			<td style="width:283px;"></td>
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			<td height="40" style="height:40px;width:249px;">0.3cc insulin syringe with 29 g needle</td>
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			<td style="width:127px;">309301</td>
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			<td height="20" style="height:20px;width:249px;">Flat bottom restrainer&#160;</td>
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			<td style="width:283px;">2.0&#34; diameter</td>
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			<td height="20" style="height:20px;width:249px;">TTC</td>
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			<td height="20" style="height:20px;width:249px;">10X PBS, pH 7.4</td>
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			<td style="width:283px;">Scintillation tubes with TTC may be manually held under running warm water as an alternative to the water bath.</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Styrofoam board</td>
			<td style="width:171px;">Multiple sellers&#160;</td>
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			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Large Syringe Kit</td>
			<td style="width:171px;">PumpSystems Inc</td>
			<td style="width:127px;">P-SYRKIT-LG</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Perfusion Pump</td>
			<td style="width:171px;">PumpSystems Inc</td>
			<td style="width:127px;">NE-300&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">60 cc syringe</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">NC9203256</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">27g winged infusion set</td>
			<td style="width:171px;">Kawasumi Laboratories, Inc</td>
			<td style="width:127px;">D3K1-25G 1</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">20 ml scintillation vial</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">50-367-126</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Stainless steel spatula</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">14-373-25A</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Alto acrylic 1.0 mm mouse brain, coronal</td>
			<td style="width:171px;">CellPoint Scientific&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">0.21 mm stainless steel blades, 25 pk</td>
			<td style="width:171px;">CellPoint Scientific&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;">Reusable cryostat blades are an inexpensive alternative.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">4% paraformaldehyde</td>
			<td style="width:171px;">Santa Cruz Biotechnology&#160;</td>
			<td style="width:127px;">SC-281692</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Superfrost microscope slides&#160;</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">12-550-15</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">HP Scanjet G4050</td>
			<td style="width:171px;">Multiple sellers&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;">Other commercial scanners are suitable for this step in the protocol.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">ImageJ&#160;</td>
			<td style="width:171px;">National Institute of Health</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Analytical Balance</td>
			<td style="width:171px;">Mettler Toledo&#160;</td>
			<td style="width:127px;">XSE 205U</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Precision Compact Oven&#160;&#160;</td>
			<td style="width:171px;">Thermo Scientific&#160;</td>
			<td style="width:127px;">PR305225M</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">1.7 mL microcentrifuge tubes (Eppendorfs)</td>
			<td style="width:171px;">Denville Scientific&#160;</td>
			<td style="width:127px;">C2170</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Formamide</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">BP228-100</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">96-well plates</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">07-200-9</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Epoch Microplate Spectrophotometer&#160;</td>
			<td style="width:171px;">BioTek&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
		</tr>
	</tbody>
</table>

quantification of neurovascular protection following repetitive hypoxic preconditioning and transient middle cerebral artery occlusion in mice

뇌졸중의 실험 동물 모델은 스트로크의 병리를 이해하고보다 효과적인 치료 전략을 개발하기위한 매우 중요한 도구입니다. 반복적 저산소 전처리 (RHP) 대 이주 프로토콜 국소 허혈성 뇌졸중의 쥐 모델에서 중추 신경계 (CNS) 상해에 대하여 장기간 보호를 유도한다. 우완 두 기간 (2 ~ 4 시간)와 강도 (8 %와 11 % O 2)에 따라 다릅니다 저산소증 9 확률 적 노출로 구성되어 있습니다. 우완는 내인성 중추 신경계 보호 표현형의 장기 유도를 제안, 경색 볼륨, 혈액 - 뇌 장벽 (BBB)​​ 중단 및 저산소증에 마지막 노출 다음 주 이후 행정 염증 반응을 감소시킨다. 경색 볼륨 및 BBB 중단의 이중 정량 방법은 우완 또는 다른 추정 neuroprotectants와 생쥐의 신경 혈관 보호 기능을 평가하는데 효과적이다. 성인 남성 스위스 웹스터 마우스는 21 % O로 우완 또는 기간 - 상응하는 노출에 의해 미리 조정했다 <suB&gt; 2 (즉, 실내 공기). 60 분 과도 중간 대뇌 동맥 폐색 (결과 tMCAO)은 최종 저산소 노출 다음 이주을 유도 하였다. 폐쇄 및 재관류 모두 두개 레이저 도플러 flowmetry에 의해 확인되었다. 재관류 후 스물 두 시간, 에반스 블루 (EB)를 정맥 꼬리 정맥 주사를 통해 투여 하였다. 2 시간 후, 동물을 이소 플루 란과 과량 뇌 섹션에 의해 희생시켰다은 2,3,5- 트리 페닐 테트라 졸륨 클로라이드 (TTC)로 염색 하였다. 경색 볼륨은 정량 하였다. 다음으로, EB는 결과 tMCAO 후 BBB 중단을 결정하기 위해 48 시간 동안 조직으로부터 추출 하였다. 요약하면, RHP 다른 CNS 기반 및 전신성 염증성 질환 상태에 대한 병진 전위, 마우스에서 스트로크 부상 장기 내인성 신경 혈관 보호를 유도하기 위해, 최소한의 비용으로 복제 할 수있는 단순한 프로토콜이다.

이 연구는 연령 10 주 우완으로 무작위 배정의 시작에 있었다 (N = 10) (25) 남성 스위스 웹스터 마우스 또는 21 % O 2 (N = 15) 그룹을 포함. 2 주 최종 우완 노출 후, 수술은 눈을 멀게 그룹으로 수행하고 일 사이에 카운터. 결과 tMCAO 다음, 1 마우스는 수술 후 회복 중 사망하고 재관류 CBF 기준을 충족하지 않았기 때문에 1 마우스는 연구에서 제외되었다. 모두 제외 마우스는 21 % O 2 군에서 ?...

Watch this Scientific Journal Video about Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice at JoVE.com

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

마우스의 반복 저산소 전처리 및 과도 중간 대뇌 동맥 폐쇄 후 신경 혈관 보호의 정량화

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

quantification-neurovascular-protection-following-repetitive-hypoxic

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13.6K 조회수.  University of Texas Southwestern Medical Center. This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

비디오: Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

마우스의 모델링 스트로크 - 필라멘트 모델 중간 대뇌 동맥 폐색

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

연구

의학

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

중간 대뇌 동맥 폐색의 마우스 모델

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

마우스의 레이저 도플러 Flowmetry 안내와 허혈성 뇌졸중에 대한 중간 대뇌 동맥 Intraluminal 폐색 (MCAO) 모델

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

쥐의 레이저 도플러 Flowmetry 안내와 허혈성 뇌졸중에 대한 엔도 테린-1 유도 중간 뇌 동맥 폐색 모델

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

엔도 테린-1 유도 중간 뇌 동맥 폐색 동안 중동 뇌동맥을 시각화하기 위해 두개골 창 활용

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

Intraluminal 중간 대뇌 동맥 폐색의 쥐 뇌졸중 모델에서 뇌 관류 모니터링에 최적화 된 시스템

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

마우스의 모델링 스트로크 : 말초 중간 대뇌 동맥의 영구 응고

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

흰쥐의 동종 혈액 혈전과 허혈성 뇌졸중에 대한 전성 중간 대뇌 동맥 폐색 (MCAO)

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

마우스의 중간 대뇌 동맥 폐색 및 재관류 유도 스트로크에 대한 수술 적 접근

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

P10 쥐에서 신생아 뇌졸중의 과도 중간 대뇌 동맥 폐색 모델

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...