이 작품은 국립 보건원 (National Institutes of Health) [그랜트 없음에 의해 지원되었다. R01NS069726] 원조 부여에서 미국 심장 협회 (American Heart Association) 그랜트 [그랜트 없음. 13GRNT17020004] SD에.

참고 : 마우스 (C57BL / 6J, 남성) 세 10-12주이 연구에 사용되었다. 모든 절차는 실험 동물의 관리 및 사용에 대한 NIH 가이드에 따라 수행하고 미주리 기관 동물 관리 및 사용위원회의 대학 (IACUC)에 의해 승인되었다. 1. 사전 수술 <ol><li> 수술 오토 클레이브 전날 모든 수술기구를 소독. (121 O를 C, 15 PSI, 30/30 사이클) 건조 30 분 뒤에 30 분 15 psi에서 121 ℃로 악기와 오토 클레이브를 감 쌉니다. 더 사용할 때까지 깨끗하고 무균 환경에서 악기를 놓습니다. </li><li> 신선한 장미 벵골 (RB) 솔루션 (멸균 식염수에 20 ㎎ / ㎖) 수술 전 모든 시간을 준비합니다. 상기 SUR 중에 사용까지 완전히 RB, 와류 후 19 W. 랩 알루미늄 호일 튜브 빛으로부터 보호의 출력 전력과 50 Hz에서 5 분 동안 초음파 처리 한 다음 튜브를 용해GERY. </li><li> 멸균 식염수에 케타민 / 자일 라진의 혼합물을 준비합니다. 마취 혼합물 1 ㎖의 최종 부피를 만들기 위해 멸균 생리 식염수 550 μL : 및 ketmaine 325 μL (100 ㎎ / ㎖ 재고 농도) : 자일 라진의 125 μL (20 ㎎ / ㎖ 재고 농도)를 추가합니다. </li><li> homeothermic 가열 패드를 미리 따뜻하게. </li><li> 램프 전력을 안정화하기 위해 30 분 동안 메탈 할라이드 램프 (FN1 에피 형광 현미경 용의 광원)을 미리 가온. </li><li> 10X 대물 렌즈 및 직립 FN1 에피 형광 현미경 필드 조리개를 조절함으로써 레티클을 사용하여 1mm의 직경으로 조명 된 영역의 크기를 조정한다. </li></ol> 2. 수술 <ol><li> 케타민 (130 ㎎ / ㎏ B의 중량.)과 자일 라진의 용량으로 마우스를 마취 (10 ㎎ / ㎏ B의 중량.). 1.3 칵테일 바탕 B. 마우스 (WT)의 g 당 4 μL. 필요하다. 알콜 면봉으로 주사 부위를 소독 인트라 통해 마취제를 투여-peritoneal (IP) 경로. 동물의 유도와 회복을 지연 할이 같은 혈관 또는 근육에 마취제를 주입하지 않도록주의하십시오. </li><li> 건조 방지 및 저체온증 방지 가열 패드 위에 동물을 배치 마우스의 양쪽 눈에 인공 눈물 연고를 적용한다. </li><li> 동물의 준비 <ol><li> 발가락 핀치 응답을 사용하여 적절한 수술 수준의 마취의 동물을 확인합니다. </li><li> 동물이 마취 수술 레벨에 도달하면, 전기 머리 트리머를 이용하여 동물의 중심선 주위의 등쪽 표면에 머리카락 클립. betadine 용액 세 번이어서 70 % 에탄올과 수술 부위를 스크럽. 다음 단계까지 무균 수술 드레이프와 사이트를 커버. </li></ol></li><li> 얇은 뼈 수술은 척수를 노출시키기 <ol><li> 수술 플랫폼 (그림 1A)에 homeothermic 가열 패드에 발생하기 쉬운 위치에 마우스를 놓습니다. 마우스를 적절히 확보주둥이 클램프를 사용하여 자세는 가늘고 긴 목 부분 (그림 1A, B)를 유지한다. </li><li> T12에 대한 흉부 척추 T9에서 연장 등의 중간 선을 따라 수술 가위를 사용하여 (약 1cm 길이) 절개를합니다. 수술 영역을 노출 피부를 옆으로 이동합니다. </li><li> 메스를 사용하여 조심스럽게 T9에서 지느러미 쪽을 노출 근육을 취소 - T12 척추를. 멸균 면봉으로 부드럽게 압력을 적용하여 각 단계에서 출혈을 중지합니다. 별도 T10 - T12 주변 근육에서 척추 안정 임의 이동을 방지하기 위해 척추 클램프를 사용하여 고정 (도 1A, B). </li><li> 조심스럽게 부드럽게 얇은, T10이나 T11 척추의 지느러미 표면을 뼈 연마 드릴 비트와 고속 드릴을 사용하면 SC (그림 1C)의 등쪽 표면에 후방 척추 정맥 및 기타 소형 선박을 시각화합니다. </li><li> 인해 씨닝 절차 동안 발생 된 열을 열 손상을 방지하기 위해,파편을 제거하기 위해 일정한 흡입과 함께 생리 식염수의 부드러운 일정한 스트림을 적용합니다. </li><li> 주요 선박이 명확하게 보일 때까지 메스를 사용하여 조심스럽게 뼈의 표면을 부드럽게. 이 과정에서 척수 손상되지 않도록주의하십시오. </li><li> 혈관이 가시화되면, 인슐린 주사기를 사용하여 레트로 궤도 동 경로를 통해 30 mg / kg (체중)의 투여 량을 투여 RB. </li><li> RB 다음 주입 3 분 후, 레이저 도플러 유량계를 사용하여 혈액의 흐름을 측정하고 필요한 경우 (도 2A, B). 전체 절차를 수행하는 동안 무균을 유지한다. </li></ol></li></ol> PT 3. 유도 <ol><li> 높이를 조절할 수있는 실험실 - 잭을 통해 XY 위치 조정 단계에서 동물을 놓습니다. T11 척수의 노출 영역이 FN1의 에피 형광 현미경 (그림 3A)의 10 배 목표 바로 아래에 있도록 마우스의 위치를 조정합니다. </li><li> 리튬의 힘을 설정12 %에서 GHT 소스와는 얇은 척수의 중간에 0.75 mm의 직경 T11 영역을 조사 - 인 580 nm의 녹색 빛 (파장 540와 (주이 지역은 후방 척추 정맥과 다른 모세 혈관을 포함한다) 2 분 동안 10 배 대물 통해 현미경 필터 큐브)에 의해 달성. 시작과 조사의 끝 (그림 3B, C)에서 이미지를 가지고이 시점에서 실험 시간을 기록한다. </li><li> 2.4.8와 같이 척수 상기와 동일한 위치에 레이저 도플러 프로브를 배치하여 10 분 동안 다시 필요한 경우 혈류를 측정 (도 2A, B). </li><li> 어떤 출혈 및 경우 조사 확인 후 아무도 발견은 동물을 봉합를 진행하지 않습니다. 흡수성 봉합사 또는 크기 4-0 실크 봉합사를 이용하여 척추의 어느 한쪽에 따라 근육 근막 피상적 봉합사. 노출 된 SC 손상되지 않도록주의하십시오. 4-0 실크 봉합사로 피부를 봉합. Betadine 또는 요오드 적용봉합 후에 피부의 가장자리. </li></ol> 4. 수술 후 케어 <ol><li> 봉합 후, 복구를 위해 가열 패드에 동물을 배치합니다. 복구 후 두 뒷다리 - 사지의 움직임을 관찰하여 신경 학적 결손의 징후 동물을 확인합니다. 이 흉골 드러 누움을 유지하기 위해 충분한 의식을 회복 할 때까지 무인 동물을 방치하지 마십시오. </li><li> 홈 케이지에 동물을 전송합니다. 완전히 회복 될 때까지 다른 동물의 회사에 수술을 한 동물을 반환하지 않습니다. </li><li> 정기적으로 동물을 확인합니다. 심각한 신경 학적 결손 경우, 방광의 배출, 진통제의 투여 (- 0.1 ㎎ / ㎏ 부 프레 노르 핀, 0.05)과 같은 적절한 치료를 제공합니다. 탈수를 확인하고 심한 경우에는 생리 식염수를 피하을 관리 할 수​​ 있습니다. 일반적으로, 부 프레 노르 핀은 (0.1 ㎎ / ㎏) 수술 부위의 통증을 완화하기 위해 봉합 한 후 투여한다. </li><li> 동물이 아닌 경우 바로동물이 쉽게 음식에 도달 할 수 있도록 수술 후 안락사, 우리는 케이지 바닥에 높은 수분 함량 다이어트를 넣어 것입니다. </li></ol> 5. Transcardial 관류, Nissl 염색 및 면역 염색 <ol><li> 앞서 설명한 바와 같이 17-20로 transcardially 동물 관류. <ol><li> 프로토콜의 앞부분에서 설명한로 transcardially 얼음처럼 차가운 4 % 파라 포름 알데히드 PBS에서 (PFA) 다음 인산염 완충 식염수 (PBS, pH를 7.4)로 관류로 동물을 마취. </li><li> 관류 후, 척수 (SC)를 제거하고 4 ° CO / N에서 PBS에 4 % PFA에서 그것을 포스트 - 수정. 30 % 자당에 PBS에 고정 SC를 전송하고 2 유지 - 그것은 튜브의 바닥에 침몰 할 때까지 3 일. </li><li> 저온 유지 장치를 사용하여 30 μm의 두께 부분으로 척수를 절단하고 젤라틴 코팅 유리 슬라이드에 0.01 M PBS로 48 웰 플레이트에 직렬로 배치합니다. </li></ol></li><li> Nissl 염색 : Nissl staini을 수행 PT에 의한 손상을 검사하려면척수 부분에 NG는 이전에 17-20을 설명했다. <ol><li> 간단히, 0.25 %의 크레 실 바이올렛과 ​​모든 다섯 번째 척수 유리 슬라이드에 슬라이스와 얼룩을 수집합니다. 스테인드 섹션 (그림 4)의 이미지를 가져 가라. </li></ol></li><li> 면역 염색 : 이전 섹션 플로팅 방식 17,18,20를 사용하여 설명 된 바와 같이. <ol><li> , 토끼 항 NeuN 항체 (1 : 300), 토끼 항 - : 간단히, 토끼 항 신경교 원 섬유 성 산성 단백질 (GFAP) 폴리 클로 날 항체 (300 1)와 함께 4 ℃에서 O / N 항온 처리하여 척수 부분 얼룩 Iba1 항체 (1 : 500) 당나귀 항 토끼 알렉사 568 공역의 IgG (1 : 400)에 의해 이어 RT에서 4 시간 동안 이차 항체. 형광 현미경 (그림 5)와 이미지를 가져 가라. </li></ol></li></ol>

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P., Bralet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced,+graded+cerebral+infarction+in+the+mouse+by+laser+irradiation+evolution+of+brain+edema.">Photochemically induced, graded cerebral infarction in the mouse by laser irradiation evolution of brain edema.</a> J Pharmacol Toxicol Methods. 27 (1), 1-6 (1992).</li><li>Kim, G. W., Lewen, A., Copin, J., Watson, B. D., Chan, P. 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Development of a model in the mouse.</a> Stroke. 31 (1), 208-213 (2000).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simplified+spinal+cord+ischemia+model+in+mice.">Development of a simplified spinal cord ischemia model in mice.</a> J Neurosci Methods. 189 (2), 246-251 (2010).</li><li>Labat-gest, V., Tomasi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothrombotic+ischemia:+a+minimally+invasive+and+reproducible+photochemical+cortical+lesion+model+for+mouse+stroke+studies.">Photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies.</a> J Vis Exp. (76), (2013).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+imaging+of+photothrombotic+stroke+in+conscious+and+freely+moving+rats.">Induction and imaging of photothrombotic stroke in conscious and freely moving rats.</a> J Biomed Opt. 19 (9), 96013 (2014).</li><li>Seto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+ischemic+stroke+in+awake+freely+moving+mice+reveals+that+isoflurane+anesthesia+can+mask+the+benefits+of+a+neuroprotection+therapy.">Induction of ischemic stroke in awake freely moving mice reveals that isoflurane anesthesia can mask the benefits of a neuroprotection therapy.</a> Front Neuroenergetics. 6 (1), (2014).</li><li>Bell, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+4-dependent+microglial+activation+mediates+spinal+cord+ischemia-reperfusion+injury.">Toll-like receptor 4-dependent microglial activation mediates spinal cord ischemia-reperfusion injury.</a> Circulation. 128 (11 Suppl 1), S152-S156 (2013).</li><li>Smith, P. 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The authors have nothing to disclose.

본 연구에서는 SC 허혈의 photothrombotic 모델을 설명했다. 때문에 발전에 유전 공학이 가능 SC의 허혈성 병태 생리에 관여하는 특정 유전자의 영향을 연구하기 위해 만든 시판되는 형질 전환 마우스가 급증되고있다. 연구의 목적은 척수 허혈 재현성 마우스 모델을 개발하는 것이었다. 여기에서 우리는 생쥐에서 SCI를 유도하는 대뇌 피질의 PT 모델을 적용. 수술에게 T11 흉부 척추의 수준에...

외상성 척수 손상 (SCI)는 SC의 감각 및 자율 신경계 기능에 영향을 미치는 치명적인 임상 조건이다. SCI 생존 환자는 종종 크게 일상 생활과 삶 (1)의 품질에 영향을 마비 쇠약으로 남아 있습니다. 실험 SCI 모델은 SCI의 병태 생리 및 관련 신경 복구 프로세스를 이해하는 과학적인 조사에 필수적인 도구왔다. 이러한 모델은 기능 회복 겨냥한 다양한 실험 신경 중재 전임상 효능을 테스트하기 위해 사용되어왔다. 현재, 실제로 SCI 모델의 대부분은 기계적으로 혼란과 SC를 손상하는 물리적 둔기의 사용을 사용합니다. 이러한 방법은 SC 2의 타박상, 압축, 전위와 절개를 포함한다. 이 제안되었음을 차 기계적 모독 부상 SC에서의 허혈 세트의 형태로 보조 손상 후<html> 3,4. 차 허혈의 원인은 조직의 부종 5-7으로 혈관의 폐색에 의해 때때로 광범위한 조직 변성, 실질 출혈 등이 포함됩니다. SC의 무결성이 더욱 영향 이차 손상의 결과로서, 뉴런 및 신경 교세포 심각 기능과 생존에 손상되고, 허혈성 주변부 다음 스트로크 성장 유사한 손상의 만성 단계에서 성장 경색에 이르게 아폽토시스를 거칠 8,9. 흥분 독성, 자유 라디칼을 생산하고, 염증과 같은 몇몇 메커니즘 SCI 10,11 다음 허혈성 세포사에 대한 책임이있는 것으로보고되었다. 또한, SC 허혈은 종종 환자 12, 13에서 하반신 마비로 이어질 흉 복부 대 동맥류 수리 수술의 심각한 합병증이다. 높은 임상 적 영향에도 불구하고 높은 재현성과 척수 허혈의 거의 모델은 현재 사용할 수 있습니다. NT는 &quot;&gt; Photothrombosis (PT)이 기술은 높은 재현성 상당히 비 침습적이다. 뇌 14-20에서 국소 허혈의 유도를위한 일반적으로 사용되는 방법이며, 뇌 (17)의 노출 된 영역에서 정확한 초점 허혈성 병소를 생산 -21.이 장미 벵골 (RB) 16-20,22 또는 에리트로 B 등의 광활성 염료의 전신 투여에 의해 달성된다는 적절한 광원과 혈관의 지역화 조사 하였다 (23). 염료의 Photoactivation이 활성 산소의 생성을 일으키는 매끄러운 혈관 내피 세포의 무결성을 파괴하고, 혈소판이어서 용기 (24)에 의해 공급 부위에서 경색에 혈전 결과로. 혈류 폐색 혈전을 형성하는, 축적시킬. 인해가 온 제어 완화 강도와 조사 기간이 절차는 매우 균일하고 재현성 경색을 산출한다. 또한,이 방법은 infarc을 유도하기 위해 사용될 수있다허혈 효과의 공간 (예를 들면, 대 백질 회백질) 이해할 수 있도록 다양한 해부학 적 위치에서 t. 현재의 연구는 마우스에 SC 허혈 쉽고 높은 재현성 모델을 개발하는 것이다. 우리는 마우스에서 SC 허혈의 모델 PT의 절차를 설명했다. 조직학 및 면역 염색 결과는 PT 효과적으로 사우스 캐롤라이나 경색, 신경 세포의 손실 및 반응성 신경교 증을 유도 할 수 있음을 보여 주었다.

<table border="0" cellpadding="0" cellspacing="0" width="1050">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Rose Bengal</td>
			<td style="width:233px;">Sigma-Aldrich</td>
			<td style="width:180px;">330000</td>
			<td style="width:421px;">20 mg/ml in sterile saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57Bl6/J</td>
			<td>Jackson lab</td>
			<td>664</td>
			<td>22-25g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-996-06</td>
			<td>100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-234-11</td>
			<td>100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine solution</td>
			<td>Purdue</td>
			<td>NDC-67618-150-01</td>
			<td>10% povidone iodine topical solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal saline</td>
			<td>Abott Laboratories</td>
			<td>04930-04-10</td>
			<td>For diluting RB, anaesthesia and for preventing tissue from drying</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Artificial tears ointment&#160;</td>
			<td>Rugby</td>
			<td>NDC-0536-6550-91</td>
			<td>83% white petrolatum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Decon labs.Inc</td>
			<td>2716</td>
			<td>70% ethanol for disinfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Metal halide lamp</td>
			<td>EXFO, Canada</td>
			<td>X-Cite 120 PC&#160;</td>
			<td>Set power at 12%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td>Fine Science Tool</td>
			<td>15000-10</td>
			<td>for minor dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors (angled to side)</td>
			<td>Fine Science Tool</td>
			<td>14063-011</td>
			<td>No. 3 handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard scalpel</td>
			<td>Fine Science Tool</td>
			<td>10003-12</td>
			<td>for removing muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blade</td>
			<td>Feather</td>
			<td>2976</td>
			<td>No. 10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (curved)</td>
			<td>Fine Science Tool</td>
			<td>11150-10</td>
			<td>for holding tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (straight)</td>
			<td>Fine Science Tool</td>
			<td>11151-10</td>
			<td>for holding tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle holder&#160;</td>
			<td>Fine Science Tool</td>
			<td>12002-12</td>
			<td>for suturing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue adhesive glue</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
			<td>to adhere to edges of the cut skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monofilament polypropylene&#160;</td>
			<td>USSC Sutures</td>
			<td>VP-521</td>
			<td>Size = 4-0 (for fascia)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perma-hand silk</td>
			<td>Ethicon</td>
			<td>683G</td>
			<td>Size = 4-0 (for skin)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro drill</td>
			<td>Roboz Surgical Instrument Co. Inc.</td>
			<td>RS-6300</td>
			<td>with bone polishing drill bit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser doppler flowmeter</td>
			<td>Moor Instruments</td>
			<td>moorVMS-LDF1</td>
			<td>for monitoring change in blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating pad</td>
			<td>Fine Science Tool</td>
			<td>21052-00</td>
			<td>to prevent hypothermia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab-Jack</td>
			<td>Fisher scientific&#160;</td>
			<td>14-673-50</td>
			<td>4x4 in plate to adjust the height of the animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-Y gliding stage&#160;</td>
			<td>Amscope</td>
			<td>GT100</td>
			<td>for positioning the animal under microscope&#160;&#160;</td>
		</tr>
	</tbody>
</table>

photothrombosis-induced focal ischemia as a model of spinal cord injury in mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

본 연구의 목적은 PT 모델을 사용하여 생쥐의 척수 허혈을 제조 하였다. 척수 (T10 - T12) 위의 뼈의 원하는 영역 후 얇게하고, 로즈 벵갈 복고풍 궤도 동 경로를 통해 주입하고, 허혈은 PT에 의해 유도 된 그림 1A가, B는에 위치 마우스를 보여 맞춤 수술. 수술 중 플랫폼입니다. 마우스가 주둥이 클램프로 고정 된 두 개의 조정 가능한 척추 동물은 척추를 안정화 클램프 그림 1C는</strong...

Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

쥐의 척수 손상의 모델로 초점 국소 빈혈을 Photothrombosis는 유도

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

photothrombosis-induced-focal-ischemia-as-model-spinal-cord-injury

Research

JoVE Journal

Medicine

13.1K 조회수.  University of Missouri. Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice. 

비디오: Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

쥐의 척수를 다친 후에 급성 및 만성 촉각 감각 테스트

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

연구

의학

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

마우스의 제어 자궁 경부 열상 상해

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

쥐에 심각한 척수 손상의 타박상 모델

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

마우스의 급성 신장 손상 및 사후 상해 섬유증의 허혈 - 재관류 모델

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

척수 허혈 - 재관류의 생쥐 모델에서 흉부 대동맥 폐색에 의해 재생산 할 하반신 마비

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

외상성 뇌 손상에 대한 제어 피질 충격 모델

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

무선 원격 측정 시스템은 척수 절개 및 배아 신경 세포 이식 줄기와 흰쥐의 심장 혈관 기능을 모니터링 할 수

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

척수 손상의 실험 Contusive 모델에서 신경 줄기 세포 이식

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

척수 압축 부상의 보정 집게 모델

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

신생아 마우스 척수 압축 부상 모델

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...