This work was supported by the American Heart Association Scientist Development Grant and the Foundation for Anesthesia Education and Research (MLJ). We would like to thank Talaignair N. Venkatraman PhD for his assistance with magnetic resonance imaging.

윤리 성명 :이 프로토콜은 듀크 대학 기관 애니멀 케어 및 사용위원회에 의해 승인과 동물의 윤리적 사용에 대한 모든 지침을 다음되었습니다. 장비 1. 준비 <ol><li> 수술 전에 수술 도구를 압력솥. </li><li> 70 % 에탄올로 소독 정위 장치. </li><li> 물 목욕을 켜고 42 ℃에서 물 온도를 유지 </li><li> 0.4 ㎕의 당 0.075 U의 농도로 생리 식염수를 입력 IV-S의 클로스 트리 디움 콜라게나 제를 녹여. </li></ol> 이. 콜라게나 사출 모델 <ol><li> 마우스의 무게를. </li><li> 30 % O 70분의 2 % 해당 2에서 5 % 이소 플루 란과 유도 챔버에 마우스를 마취. 적절한 마취 마우스 호흡 초당 1로 둔화 약 2 분 후 신호를. </li><li> 30mm 20 G 정맥 내 카테터를 가진 기관을 삽관. </li><li> 에 카테터를 연결쥐 인공 호흡기와 기계적으로 30 %에서 1.6 % 이소 플루 란과 폐 환기 O 칠십분의이 825 ㎖를 전달 호흡량 분당 105 호흡의 속도 %의 N 2 .. </li><li> 전자 면도기로 두피를 면도. 마우스를 마취하고 삽관되면 면도 다른 작업 스테이션으로 이동하고 수술 벤치로 돌려 보냈다. </li><li> 정위 프레임에 머리를 고정하고, 기준점으로 관상 및 시상 모두 봉합 머리를 수준. </li><li> 눈에 안과 연고를 적용합니다. </li><li> 직장 온도 프로브를 삽입합니다. 물침대 순환 언더 바디를 사용하여 37.0 ± 0.2 ° C에서 직장 온도를 유지합니다. </li><li> 닦아 베타 딘과 수술 영역은 70 % 에탄올에 이어 3 번 반복합니다. </li><li> 1cm의 중간 선 두피 절개를하고 브레 그마를 노출 멸균 면봉으로 옆으로 골막을 닦으십시오. </li><li> 드릴 직경 1mm 버 구멍 2.2 mm는 위도 남아수냉식 드릴 브레 그마하는 ERAL. </li><li> 콜라 유리 병 5 배 회전, 0.5 ㎕의 콜라게나 솔루션 (정위 프레임에 부착) 25 G 바늘로 5 회 (마지막 세척 후 주사기에 콜라게나 솔루션의 0.5 μl를 남겨주세요) 0.5 μL 주사기를 씻어. </li><li> 버 구멍으로 바늘 끝을 정렬 한 후 주사기에서 0.1 μl를 추방하고 폐기 면도칼과 바늘 베벨을 닦으십시오. </li><li> 미세 조작기를 사용하여 피질에 깊은 바늘 3mm를 진행하고 30 초 동안 움직 둡니다. </li><li> 90 초에 0.4 μl를 주입한다. </li><li> 1 % 이소 플루 란을 줄이고 5 분 동안 바늘의 움직임을 둡니다. </li><li> 천천히 바늘을 철회. </li><li> 0.25 %의 부피 바카 인의 피하에 2 방울과 피부를 봉합 - 1을 적용합니다. </li><li> 이소 플루 란 기화기를 끄고 정위 프레임에서 마우스를 제거합니다. </li><li> 마우스는 이후 기관의 발관과 자연 환기를 복구 할 수 있습니다. </li><li> 깨끗한 케이지에 마우스를 반환하고 무료로 액세스 할 수 있도록음식과 물. </li></ol> 3.자가 혈액 주입 모델 <ol><li> 콜라게나 주입 모델의 2.11 - 2.1 단계를 따릅니다. </li><li> 30 G 50 μL 주사기에 멸균 생리 식염수 50 μl를 그립니다. </li><li> 70cm의 PE10 튜브 마이크로 리터 주사기를 연결합니다. </li><li> PE10 튜브에 완전히 해제 공기 튜브에 마이크로 리터 주사기에서 모든 생리 식염수를 추방. </li><li> 저장 과정 중에 염수 및 혈액의 혼합을 방지하기 위해 PE10 튜브 마이크로 리터 주사기 장치의 선단 개구에서 기포를 만들기 위해 아웃 1mm를 마이크로 리터 주사기의 피스톤을 당기. </li><li> 70 % 에탄올로 마우스의 말초 중앙 꼬리 동맥 영역을 닦아, 꼬리 끝 1 cm에 0.5 면도칼로 동맥을 잘라. </li><li> PE10 튜브 마이크로 리터 주사기 장치로 절단 꼬리에서 혈액의 40 μl를 수집합니다. 참고 : 헤파린 바늘, 튜브, 또는 마우스에 사용되지 않았는지 확인합니다. </li><li> 인제에 마이크로 리터 주사기를 부착ction의 펌프. </li><li> PE10 튜브의 단부에 27 G 바늘 캐 뉼러의 금속 부분을 연결하고, 정위 프레임에 미세 조작기에 바늘을 확보. </li><li> 27 G 바늘 밖으로 혈액의 2 μl를 추방하고 폐기 면도칼과 바늘 베벨을 닦으십시오. </li><li> 버 구멍으로 바늘 끝을 맞추고 피질에 깊은 바늘 3mm를 삽입합니다. </li><li> 분 당 2 μL의 비율로자가 혈액의 35 μl를 주입한다. </li><li> 1 % 이소 플루 란을 낮추고 10 분 동안 바늘의 움직임을 둡니다. </li><li> 30 초 이상 바늘을 철회. </li><li> 0.25 %의 부피 바카 인의 피하에 2 방울과 피부를 봉합 - 1을 적용합니다. </li><li> 이소 플루 란 기화기를 끄고 정위 프레임에서 마우스를 제거합니다. </li><li> 마우스 후속 발관과 자연 환기를 복구 할 수 있습니다. </li><li> 깨끗한 케이지에 마우스를 반환하고 음식과 물에 자유롭게 접근 할 수 있습니다. </li></ol> 4. 샴 운영 <ol><li> 콜라게나 사출 성형 및 직접 성형에 대해 동일한 절차를 따르바늘 삽입 후 주입하지 않고 제외 N 모델. </li></ol> 5. 수술 후 케어 <ol><li> 동물의 목 뒤쪽에서 수술의 저녁에 생리 식염수를 피하 0.5 ㎖를 주입한다. </li><li> 케이지의 바닥에 배치 된 작은 플라스틱 컵에 물과 젤 음식을 부드럽게 음식을 제공합니다. 을 7 일 동안 매일 음식을 교체합니다. </li><li> 을 7 일 동안 매일 체중 감소, 상처 치유 및 불편의 흔적을 확인합니다. </li><li> 보다 칠일 회복 간격이 요구되는 경우, 필요한 경우, 봉합사 제거는, (30 % O 70분의 2 % 년 N이 약 1 % 이소 플루 란) 광 흡입 마취 하에서 수행 될 수있다. </li></ol>

<ol>
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L., 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=Intracerebral+hemorrhage+models+in+rat:+comparing+collagenase+to+blood+infusion.">Intracerebral hemorrhage models in rat: comparing collagenase to blood infusion.</a> J Cereb Blood Flow Metab. 28, 516-525 (2008).</li></ol>

Lei, Sheng, Wang, Lascola, Warner, and Laskowitz have no conflicts of interest to declare. James received grant funding by American Heart Association, National Institutes of Health, and Cephalogics.

치료에게 15-18을 약속을위한 전임상 연구 결과 큰 임상 시험을 부상에도 불구하고,이 무형 문화 유산의 결과를 개선하기 위해 입증 어떤 약물의 개입은없고, 치료는 크게지지가 남아있다. 수 치료의 목록은 transcriptomic 및 단백체 작품으로 높은 처리 기술에 의해 생성 될 수있다. 이 기술은 앞으로 잠재적 인 치료 목표에 대한 우리의 지식을 지속적으로 발전 유망한 표적의 뒤로 번역은 최?...

뇌내 출혈 (ICH)는 고통받는 환자의 약 40~50% 30 일 1 ~ 죽어과 뇌 혈관 질환의 비교적 흔한 형태입니다. 불행하게도, 약간의 개선은 지난 20 년 2 이상 사망률을했다. 국민 건강 3의 연구소와 미국 심장 협회 (American Heart Association) 4에서 지침의 보고서는 병태 생리에 대한 이해를 확장하고 새로운 치료 방법의 대상을 개발하는 무형 문화 유산의 임상 적 모델을 개발의 중요성을 강조했다. 몇몇 모델은 인간의 ICH 5을 모방하기 위해 존재합니다. ICH의 병태 생리의 성숙 해지면 이해로서, 다양한 모델이 질환의 다양한 측면을 검사하는 데 사용될 수 있다는 것을 분명하게 하였다. 이전에 사용 된 모델은 쥐의 아밀로이드 혈관 병증 6, 뇌실질 microballoon 삽입과 인플레이션 7, 직접 동맥혈을 포함침투 8,9. 아밀로이드 혈관 병증에서 엽성 출혈은 형질 전환 마우스의 사용과 모델과 별개의 무형 문화 유산의 하위 유형을 대표하고있다. Microballoon 모델은 혈종 형성에서 급성 질량 효과를 모방하여 혈액의 존재로 두뇌의 세포 반응을 포착하는 데 실패. 마지막으로, 직접 동맥혈 침투는 대퇴 동맥에서 동맥 압력에 뇌 과목. 따라서,이 모델을 모방 동맥 압력과 혈액의 존재하지만, 작은 혈관의 파열에서 미세 혈관 부상으로 뇌를주지 않습니다. 또한,이 모델은 본질적으로 높은 변동성을 가지고 있습니다. 그들은 나이가 흥미롭게, 자발적으로 고혈압 쥐 (10)는 자연 무형 문화 유산을 개발한다. ICH 개발 후이 동물의 연구는 ICH에 인간을 걸리기 주요 동반 질환 중 하나의 존재에 질병을 모방 할 수 있습니다. 클로스 트리 디움 콜라게나 제 (11) 또는의 instrastiatal 주입의 다른 모델이 존재하지만, 줄무늬 체 주입utologous 전혈 (12)는 현재 가장 일반적인 두 가지 모델은 전임상 무형 문화 유산의 연구에 사용됩니다. ICH 모델 선택은 종의 선택 및 혈종 형성을 유도하는 방법을 포함 실험 질문의 목적에 기초하여야한다. 예를 들어, 돼지는 쥐에 비해 상대적으로 큰 백질 뇌의 볼륨이 큰 동물입니다. 따라서, 돼지 모델은 ICH 다음과 백질의 병태 생리를 연구하기에 적합합니다. 반면, 설치류의 뇌는 주로 회색 물질이지만, 형질 전환 시스템은 ICH 후 부상과 회복의 분자 메커니즘을 평가하기 위해 설치류 유용합니다. 각 모델에 앞서 실험에 고려되어야하는, 그 고유의 강점과 약점 (표 1)가 있습니다. 다음 프로토콜은 생쥐에서자가 혈액 및 콜라게나 제 주입 모델을 보여줍니다. 이러한 모델은 각각의 원래 쥐에서 개발 된 모델에서 번역에 의한 것13, 14 및 ICH 후 세포의 죽음과 관련된 분자 메커니즘을 탐구하기 위해 널리 사용되는 유전자 변형 기술의 사용을 허용한다. 두 사람의 ICH에서 분명히 다른 부상 메커니즘을 나타내고, 모두 행동 및 조직 학적 측정의 측면에서 완전히 다른 예상 결과가있다. 따라서, 특정 가설은 다른 통해 하나의 모델로 자신을 빌려 수 있지만, 많은 아이디어는 두 모델의 유효성 검사를 필요로 할 수있다. 콜라게나 제 및자가 혈액 주입 뇌출혈 모델의 특성을 표 1. 비교. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td style="width:197px;"></td><td style="width:80px;"> 콜라게나 제 주입 </td><td style="width:66px;"> 혈액 주입 </td></tr><tr><td style="width:197px;"> 사용의 용이성 </td><td style="width:80px;"> + + + </td><td style="width:66px;"> + + </td></tr><tr&gt; <td style="width:197px;"> 재현성 </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> 출혈의 크기 제어 </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + + </td></tr><tr><td style="width:197px;"> 혈액의 역류 </td><td style="width:80px;"> + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> 인간의 질병을 시뮬레이션 </td><td style="width:80px;"> + </td><td style="width:66px;"> - </td></tr><tr><td style="width:197px;"> 간단 </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr><tr><td style="width:197px;"> 여러 종의 사용 </td><td style="width:80px;"> + + </td><td style="width:66px;"> + + </td></tr></tbody></table>

<table border="0" cellpadding="0" cellspacing="0" width="416">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Name</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Stereotactic frame</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51603</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Probe holder with corner clamp</td>
			<td style="width:88px;">Stoelting Co.</td>
			<td style="width:88px;">51631</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Mini grinder</td>
			<td style="width:88px;">Power Glide</td>
			<td style="width:88px;">Model 60100002</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">0.5 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co.</td>
			<td style="width:88px;">86259</td>
			<td style="width:88px;">25 gauge needle</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">50 &#181;l Hamilton syringe</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7637-01</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">26G Hamilton needle</td>
			<td style="width:88px;">Hamilton Co</td>
			<td style="width:88px;">7804-03</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Syringe pump</td>
			<td style="width:88px;">KD Scientific</td>
			<td style="width:88px;">Model 100</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Heat therapy water pump</td>
			<td style="width:88px;">Gaymar Industries, Inc.</td>
			<td style="width:88px;">Model# TP650</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Circulating waterbed</td>
			<td style="width:88px;">CMS Tool &#38; Die, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Rodent ventilator</td>
			<td style="width:88px;">Harvard Apparatus</td>
			<td style="width:88px;">Model 683</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Isoflurane vaporizer</td>
			<td style="width:88px;">Drager</td>
			<td style="width:88px;">Vapor 19.1</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Air flowmeter</td>
			<td style="width:88px;">Cole Parmer</td>
			<td style="width:88px;">Model PMR1-010295</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Induction chamber</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
			<td style="width:88px;">Self made</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Otoscope</td>
			<td style="width:88px;">Welch Allyn</td>
			<td style="width:88px;">22820</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">intravenous catheter</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">381534</td>
			<td style="width:88px;">20-gauge, 1.16 inch Insyte-W</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:152px;">Isoflurane</td>
			<td style="width:88px;">Baxter Healthcare Corporation</td>
			<td style="width:88px;">NDC10019-360-69</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Collagenase Type IV-S</td>
			<td style="width:88px;">Sigma</td>
			<td style="width:88px;">C1889</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE20</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427406</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">Polyethylene tubing PE10</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">427401</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">30G 1 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305128</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:152px;">27G 1 1/4 inch needle</td>
			<td style="width:88px;">Becton-Dickinson</td>
			<td style="width:88px;">305136</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Surgical scissors</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">21-539</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Forceps</td>
			<td style="width:88px;">Miltex</td>
			<td style="width:88px;">17-307</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Needle holder</td>
			<td style="width:88px;">Boboz</td>
			<td style="width:88px;">RS-7840</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Monofilament suture</td>
			<td style="width:88px;">Ethicon</td>
			<td style="width:88px;">8698</td>
			<td style="width:88px;">Size 5-0</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:152px;">Indicating controller</td>
			<td style="width:88px;">YSI</td>
			<td style="width:88px;">73ATD</td>
			<td style="width:88px;"></td>
		</tr>
	</tbody>
</table>

intrastriatal injection of autologous blood or clostridial collagenase as murine models of intracerebral hemorrhage

뇌내 출혈 (ICH)은 뇌 혈관 질환의 일반적인 형태이며 중요한 병적 상태와 사망률과 관련이 있습니다. 지혈 및 혈전 제거하기위한 대규모 임상 시험의 효과적인 치료 실패의 부족은 ICH의 추가 메커니즘을 기반 조사의 필요성을 보여줍니다. 이 연구는 전임상 모델에서 제공하는 프레임 워크를 통해 수행 될 수있다. 대중적인 사용에서 두 쥐 모델은자가 전체 혈액이나 클로스 트리 디움 콜라게나 하나의 줄무늬 체 (기저핵) 주입이 (가) 있습니다. 이후, 각 모델은 ICH 관련된 명백하게 다른 병태 생리 학적 특징을 나타내는, 특정 모델의 사용은 연구 될 질병의 형태 정보에 기초하여 선택 될 수있다. 예를 들어,자가 혈액 주입은 가장 정확하게 뇌실질 내 혈액의 존재에 대한 뇌의 반응을 나타내며, 가장 밀접하게 엽성 출혈을 복제 할 수 있습니다. 클로스 트리 디움 콜라게나 제 주입은 가장 정확하게의를 나타냅니다쇼핑몰 용기 파열과 깊은 출혈의 혈종의 진화 특성. 따라서, 다른 혈종 형성, 신경 염증성 반응, 뇌부종 개발 및 신경 행동 결과의 각 모델 결과. 사칭 치료 적 개입의 견고성이 가장 두 모델을 사용하여 평가 될 수있다. 이 프로토콜에서 두 모델 모두,​​ 부상의 즉각적인 수술 시연, 조기 수술 후 관리 기술을 사용하여 무형 문화 유산의 유도가 입증된다. 두 모델 모두 재현 부상, 혈종의 볼륨 및 신경 행동 적자의 원인. 때문에 인간의 무형 문화 유산의 이질성으로, 다수의 전임상 모델은 철저하게 병태 생리 학적 메커니즘을 탐구하고 잠재적 인 치료 전략을 테스트하기 위해 필요합니다.

혈종의 확장 (그림 2) 발생으로, 콜라게나 제 주입 후 4 시간 -으로 인해 혈종 형성 (그림 1)의 차이로, 동측 회전이 즉시자가 혈액 주입 된 쥐에 대한 웨이크 업 후 (2) 내에 표시됩니다. 동측 회전의 부재는 큰 부상의 부재에 대한 문제를 제기한다. 첫 번째 게시물 부상 날, 두 모델의 마우스는 중요한 신경 학적 결손 (그림 3) 입증해야한다. 주사 후 24 시간에서 ?...

Watch this Scientific Journal Video about Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage at JoVE.com

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

뇌출혈의 쥐과 모델로자가 혈액이나 클로스 트리 디움 콜라게나 제의 줄무늬 체 주입

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

intrastriatal-injection-autologous-blood-or-clostridial-collagenase

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JoVE Journal

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14.6K 조회수.  Duke University. Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase. 

비디오: Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, 2, 3, 4, 5 These signaling events can also lead to sepsis and/or multiple organ dysfunction (MOD). 6, 7, 8, 9 It is critical then to investigate the causes of suppressed immune function and detrimental signaling cascades in order to develop more effective ways to help patients who suffer from traumatic injuries. 10 This fixed pressure Hemorrhagic Shock (HS) procedure, although technically challenging, is an excellent resource for investigation of these pathophysiologic conditions. 11, 12, 13 Advances in the assessment of biological systems, i.e. Systems Biology have enabled the scientific community to further understand complex physiologic networks and cellular communication patterns. 14 Hemorrhagic Shock has proven to be a vital tool for unveiling these cellular communication patterns as they relate to immune function. 15, 16, 17, 18 This procedure can be mastered! This procedure can also be used as either a fixed volume or fixed pressure approach. We adapted this technique in the murine model to enhance research in innate and adaptive immune function. 19, 20, 21 Due to their small size HS in mice presents unique challenges. However due to the many available mouse strains, this species represents an unparalleled resource for the study of the biologic responses. The HS model is an important model for studying cellular communication patterns and the responses of systems such as hormonal and inflammatory mediator systems, and danger signals, i.e. DAMP and PAMP upregulation as it elicits distinct responses that differ from other forms of shock. 22, 23, 24, 25 The development of transgenic murine strains and the induction of biologic agents to inhibit specific signaling have presented valuable opportunities to further elucidate our understanding of the up and down regulation of signal transduction after severe blood loss, i.e. HS and trauma 26, 27, 28, 29, 30. 
There are numerous resuscitation methods (R) in association with HS and trauma. 31, 32, 33, 34 A fixed volume resuscitation method of solely lactated ringer solution (LR), equal to three times the shed blood volume, is used in this model to study endogenous mechanisms such as remote organ injury and systemic inflammation. 35, 36, 38 This method of resuscitation is proven to be effective in evaluating the effects of HS and trauma 38, 39.

The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.

고정 볼륨 또는 고정 압력 : 출혈성 쇼크의 Murine 모델

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, ...

연구

의학

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

경색의 Murine 모델에서 관상 동맥 내고과 Intramyocardial 사출

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

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

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

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

마우스의 모델링 Intracerebral 출혈 : Autologous 혈액이나 세균 Collagenase의 주입

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

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

지주막 하 출혈의 생쥐 모델

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

일시적 또는 영구적 인 뇌허혈 모델에서 백혈구 부분 집단의 Stereological 및 유동 세포 계측법 특성

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

지주막 하 출혈의 급성과 늦은 후유증의 연구에 대한 토끼 피 션트 모델 : 기술적 측면

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

철 염화물에 의한 쥐 혈전증 모델

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

초음파 유도 Intracardiac 주입 실험 선 동적인 장 질병의 Murine 모델에 사용 하기 위해 내장을 추적 증가 인간 중간 엽 줄기 세포의

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

말 왼쪽된 회귀 후 두 신경 근처 헌 혈 줄기 세포의 신경 자극을 유도 주사

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

쥐에서 혈액 샘플 컬렉션의 대체 방법으로 빵 꾸 하 정 맥

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...