저자 아무 승인 있다.

프로토콜 대학 병원 취리히 인간의 연구 윤리 위원회의 지침을 따릅니다.
1입니다. Neuromata와 환자 선택의 진단
<ol><li>수술 전 신경 블록은 싱 경종을 인접 진단 확인 수행 합니다. 팔 뚝의 중간 세 번째에 있는 블록을 평가 하 고 겹치는 신경 분포 영역의 측면 antebrachial 피부 신경 (LACN), 후부 상호 신경 (핀), 척 골 신경 의 지 지점 등 SBRN의 잠재적인 트리거 제외 10.</li>
	<li>상당한 통증 감소 (적어도 5 점 통증에 대 한 시각적 아날로그 규모 (VAS)에)11를 가진 환자에서 서만 수술을 수행 합니다.</li>
</ol>2. 수술 준비
<ol><li>어떤 음식이 나 액체 섭취를 6 h는 수술 전에 환자를 금지 한다.</li>
	<li>비치의 자 위치에 보편적인 요원 테이블에 수술 스위트 중간에 환자를 배치 합니다.</li>
	<li>장소는 팔 나머지에 영향을 받는 팔/손.</li>
	<li>Endotracheal 마 취를 수행 하 마를 물어 (펜타닐 2 µ g/k g, rocuronium 0.6 mg/kg, propofol 150-250 밀리 그램).</li>
	<li>지방 흡입 (예: 허벅지 안쪽 또는 위/아래 복 부)의 영역에 싱 경종 절단의 사이트 povidone 요오드 피부 소독.</li>
	<li>무 균 수술 커튼으로 환자를 커버, 지방 흡입의 사이트와 외과 사이트 노출 떠나.</li>
</ol>3. tumescent 지방 흡입
<ol><li>지방 흡입의 영역에서 #11-블레이드 메스와 함께 약 3-5 m m 직경의 여러 작은 자상 (찔린) 절 개를 확인 합니다. Incise 멀리로 피하 층에 도달.</li>
	<li>포함 하는 희석 lidocaine 피 네 프 린 (500 mL 벨의 젖 산 솔루션, 1 %lidocaine, 1 mg 피 네 프 린의 20 mL) (정 침투) 14 게이지 infiltrator 통해 피하 지방 층 (깊이 3 m m)으로 tumescent 솔루션에 주입 합니다 수확 하는 지방 지역입니다.</li>
	<li>Tumescent 솔루션 적용을 허용 하는 20 분 이상 기다립니다.</li>
	<li>후크는 3 mm 수집 정 (외부 직경 3 mm, 길이 26 cm) 60 mL 투미 주사기를 찔 러 절 개를 통해 지방 수확 시작. 참고: 배치 및 과정은 정 nondominant 손은 지속적으로 제어합니다.</li>
	<li>60 mL 마크 투미 주사기의 플런저를 철수 하 고 주사기에 부정적인 압력을 유지 하 고 지배적인 손으로 연속 진공 생성을 피하기 위해 철회 플런저 따라 모기 클램프 장소.</li>
	<li>투미 주사기 제공된 투미 주사기 스탠드;에 수직 위치에 배치 하 여 지방이 많은 직물의 220 mL의 최소 수확 지방과 액체 로부터 분리 침전 수 있습니다.</li>
	<li>양질의 지방 이식 없이 상당한 양의 혈액을 얻기 위해 특별 한 주의 분리 했습니다. 참고: 증강된 혈액 콘텐츠 발견 지방 흡입의 영역을 변경 합니다.</li>
	<li>간단한 바늘으로 찔 러 절 개를 봉합 하 고 압력으로 (예: 압축 벨트).</li>
	<li>절연 시스템의 단일 사용 멸 균 일회용 세트에는 lipoaspirate를 전송 합니다.</li>
	<li>나중 lipofilling 위한 지방 분수를 얻으려면 제공 된 스탠드에 lipoaspirate와 함께 한 투미 주사기를 유지.</li>
</ol>4. 지방 조직 처리
<ol><li>멸 균 소모품 세트를 닫습니다. 참고: 격리 시스템 젖은 테스트를 수행 하 여 완전성을 확인 ( 재료의 표참조).</li>
	<li>디스플레이에 지시를 따릅니다. 참고: 프로세스 이며 주로 소프트웨어 제어만 컨트롤 패널에서 "뒤로" 또는 "다음" 버튼을 눌러 아래 나열 된 단계에 대 한 수동 확인을 요구 한다. lipoaspirate 조직 수집 용기로 전송 됩니다. 조직 수집 용기에는 lipoaspirate는 무게와 자동으로 잔여 혈액 세포와 일로 솔루션을 제거 하려면 벨소리의 젖 산 용액으로 세척.</li>
	<li>필요한 경우 더 많은 지방 질을 추가 합니다. 참고: 수확된 지방이 많은 직물의 무게에 따라 절연 시스템 요청 더 많은 지방 질을 추가 하려면 사용자 또는 추가할 추가 효소 시 약의 양을 계산 합니다.</li>
	<li>절연 시스템에서 메시지가 표시 되 면 효소 시 약의 필요한 금액을 추가 합니다. 참고: 효소 소화 과정에서 지질 분리는 SVF 이루어집니다. 따라서, 조직 모션 진동 이다입니다. 효소 소화 완료 후는 SVF 셀 농도 원심 챔버로 자동으로 전송 됩니다. 원심 분리기 약 실에는 SVF는 셀 펠 릿에 집중 (원심 분리기 속도 약 3000 x g). 마지막으로, 셀 펠 릿 벨의 젖 산 솔루션 출력에서 잔여 효소 수치를 줄이기 위해 세척의 시리즈를 겪 습. 프로세스가 완료 되 면 맑은 액체 (SVF)의 5 mL는 얻어진 다.</li>
</ol>5. 수술
<ol><li>싱 경종, 접근 하는 팔 뚝의 원심 세 번째에서 이전 진단된 싱 경종 사이트를 통해 피부 절 개를 확인 합니다.</li>
	<li>수술 쌍 안 loupes 돋보기 안경 (2.5-3.5 X)의 사용에서 계속 피하 조직 부 관련된 신경 근 위는 싱 경종을 식별.<ol>
<li>그 후, 신경 distally 따라 고 싱 경종 (검사 기준)를 식별 합니다.</li>
	</ol>
</li>
	<li>전체는 싱 경종은 분명, 일단 바로 트랜스 신경가 위 컷으로는 싱 경종을 삭제할 시작. 없는 싱 경종 식별 됩니다, 신경의 neurolysis만 수행 됩니다.</li>
	<li>손목에 근 위 3 cm까지 나머지 신경 그 루터 기의 neurolysis와 함께 계속. 참고: 신경 그 루터 기 끝 한가운데 주 절 개 해야 합니다.</li>
	<li>표 피 및 진 커티스 주위 주 접근을 통해 메스와 2-4 작은 펑크 (자상) 절 개 (직경에서 1 m m)를 정렬 합니다.</li>
	<li>2 ~ 4 무딘 뉼 장소 (16 게이지, 길이 9 cm) 그 루터 기를 찔 러 절 perineural 통해. 참고:는 뉼의 팁 신경 몸통 쪽으로 이동 한다.</li>
	<li>접착성 드레싱에 의해 장소에서 뉼을 보호 합니다.</li>
	<li>주위는 뉼 바늘 놓고 노드를 확보 하지 않고 현장에서 봉합 사 물자를 두고.</li>
	<li>밀접 하 게 접목의 유출을 방지 하기 위하여 주요 절 개 봉합. 참고: 뉼 제자리에유지.</li>
</ol>6. SVF 농축 지방 이식의 응용
참고: 후 처리의 약 1.5 h, 처리 된 SVF의 5 mL는 추가 사용에 대 한 준비.
<ol><li>10 mL 통신 주사기에서 SVF의 5 mL 발음</li>
	<li>같은 방식으로, 반대 10 mL 주사기에 sedimented 지질 분수의 2 mL 발음.</li>
	<li>SVF의 5 mL 혼합 sedimented 지질 분수의 2 mL와 함께 한 주사기의 플런저를 후퇴 그리고 때까지 충분 한 혼합은 통신, 반대 주사기의 플런저를 잡아당기는.</li>
	<li>SVF 농축 지방 이식 2 ~ 4 10 mL 주사기에 동등 하 게 분할 한다.</li>
	<li>배포는 준비 된 SVF 농축 지방 이식 한 신경 주위에 동등 하 게 다른 무딘 뉼에 주사기를 연결 하 여 그 루터 기. 참고: 적용 하기 전에 이식, 확인는 뉼 올바른 위치에서 보호 되는 뉼 지방 이식 적용 하는 동안 탈 구 하지 않도록 주의 하십시오.</li>
	<li>뉼을 신중 하 게 제거 합니다.</li>
	<li>다음, 작은 빵 꾸 절 steri-스트립과 테이프.</li>
	<li>부 목에 본질적인 플러스 (60-70 °에 근육이 수축 하는 손바닥 (MCP) 관절, 완전히 골 (IP) 관절, 엄지 주먹 프로젝션에서 25 °의 등 굴곡에 손목) 위치를 적용 하 여 손을 무력화. 신경의 neurolysis를 수행 하는 경우에 없는 부 목 필요 하 고 팔과 손목은 10 일 동안 붕대.</li>
</ol>7. 수술 후 환자 처리
<ol><li>환자는 마 취 후 회복 단위 수술 후, 전송 하 고 병 동에 충분 한 모니터링 허용 합니다.</li>
	<li>수술 후 10 일 동안 손목 고정 계속.</li>
	<li>48 h 후 수술 후 붕대의 첫 번째 변화를 수행 하 고 정기적으로 (즉 두 번째 매일 방전까지) 계속.</li>
	<li>Postoperatively는 봉합 12-14 일 제거 합니다.</li>
	<li>2, 6, 12, 후속 동안 통증과 36 개월의 임상 평가 수행 합니다.</li>
</ol>

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저자는 공개 없다.

현재 연구의 목적은 차근차근 증상 끝-neuromata에 대 한 새로운 치료로 지방 이식 SVF 농축의 기술적인 측면을 설명 하는 것입니다.
Neuromata는 SBRN의 3312의 수술 성공률와 연결 됩니다. Vaienti 외. 초기 후속에서 유리한 결과의 시리즈를 발표 perineural 지방 이식 (PFG) 상 지5의 고통 스러운 neuromata 8 환자에 있는 SVF를 ...

관리는 SBRN의 증상 끝-neuromata의 다양 한 기존 수술 치료와 함께 도전 남아 있습니다. 주로, 고 싱 경종 excised 고 나머지 신경 그 루터 기 근육1,2,3뼈 또는 혈관4같은 이웃 구조에 이전. 이것은 밖으로 돋 아 재생 신경 성숙기의 끝-장기의 분위기를 흉내 낸 하 여 방지 하기 위해 간주 됩니다. 그러나, 아무도 이러한 기술을 입증 우수한, 통증 재발 및 부족 한 통증 감소의 높은 속도 자주 관찰 되었다.
최근, 나머지 신경 그 루터 기 주위 지방 이식 유망한 결과 굴복 하 고 상 지5의 고통 스러운 끝-neuromata에 대 한 새로운 치료 옵션으로 주 창 했다. 지속적인 움직임을 방지 하 고 통증을 가볍게 하는 기계적 장벽 역할 외 지방 이식의 유리한 측면은 오히려 세포 수준에 기인 합니다. 인간 지방 조직 preadipocytes, 돛대 세포, pericytes, 내 피 세포, 혈관 평활 근 세포, 섬유 아 세포를 포함 한 여러 세포 유형, 포함 하 고 지방6조 혈 줄기 세포 함께 줄기 세포 (줄기)를 파생. 이 다른 유형의 세포 인구 라고 Adipose-Derived 재생 셀 (ADRCs) 또는 SVF, 고 절연 시스템은 lipoaspirate에서 얻어질 수 있다 ( 재료의 표참조) 및 적용 된 효소 시 약 (참조 테이블의 재료 )7. 행동의 정확한 메커니즘은 아직 완전히 이해 하 고; 그러나, SVF 개선 신생, immunomodulation, 차별화, 및 세포 외 매트릭스8의 분 비와 연결 되었습니다.
최근 연구 결과 밝혀 추가 SVF 없이 지방 이식 12 개월 후 통증 재발 집중된 세포 인구는 SVF의 재흡수 률을 줄이기 위해 고 지방 이식의 재생 잠재력 증가를 목적으로 지방 이식에 추가 됩니다. 5. 또한, 생물 학적 행동 또한 무질서 axonal 돋 아 그 루터 기9 와 신경 함정에 발생할 수 있습니다 후속 흉터 adherences 줄임으로써 신경 자체에 유익한 효과 제공.
SBRN의 증상 끝-neuromata의 치료에 대 한 새로운 수술 옵션이 되었습니다 SVF 농축 뚱뚱한 접목을 설명 하기 위해 첫 번째 임상 연구 최근10출판. 일반적으로, 기술은 사고 또는 의원 성 신경 손상 후 발생 하는 모든 싱 경종에 적용할 수 있습니다. 현재 후속 연구의 목적은 조직적으로이 절차의 기술적 측면을 설명입니다.

<table border="0" cellpadding="0" cellspacing="0" width="688">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Cytori Celution System</td>
			<td style="width:71px;">Cytori</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">isolation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Celase</td>
			<td style="width:71px;">Cytori</td>
			<td style="width:119px;"></td>
			<td>enyzme reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">liposuction system</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">tumescent solution (500 ml Lactated Ringers, 20 ml of 1% lidocaine and 1 mg epinephrine)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">14-gauge infiltrator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 mm-collecting cannula (15, 26, 32 cm)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mL Tommey syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mosquito clamp</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Toomey syringe stand</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">4 cannulas (fat injecting cannulas 1.5 mm, syringe NO.2, 90 mm)</td>
			<td style="width:71px;">Chalybs</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">10 mL syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">connecting device for syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">#11- and #15-blade scalpel</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">stevens scissors</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">pins pincette</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">bipolar electrocautery</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">suture material</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">adhesive dressing</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">binocular loupes magnifier glasses (2.5-3.5x)</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:331px;">Statistical Package for Social Sciences, version 22</td>
			<td>IBM</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

stromal vascular fraction-enriched fat grafting for the treatment of symptomatic end-neuromata

이 연구의 목적은 조직적으로 설명 하 고 실질 혈관 분수 (SVF)의 중요 한 단계를 강조 했다-특히의 증상 끝-neuromata 말 초 감각 신경, 그리고이 연구에서의 새로운 치료로 지방 이식 농축 (SBRN) 광선 신경의 표면 분기 합니다. 다양 한 기존 치료에도 불구 하 고 영구적인 수술 후 통증과 일반적인 통증 재발 지금도 매우 일반적인, 평가 절차의 독립. 싱 경종 microsurgically 삭제에 따라 표준화 된 프로토콜입니다. 근육 이나 뼈, 같은 해 부 구조, 이웃에서 재생 신경 그 루터 기 이전 대신 지방 이식 적용된 perifocally 이며 기계적 장벽 역할. 지방 재흡수 속도 감소 하 고는 이식의 재생 잠재력을 향상, 농축된 SVF는 접목에 통합 된다. SVF는 특정 상업적인 절연 시스템에 의해 lipoaspirate의 효소 및 정비사 분리에 의해 피하 지방에서 격리 됩니다. SVF 농축 지방 이식 기계적 장벽 및 개선 신생, 염증, 섬유 증 등 세포 수준에서 다양 한 생물 학적 효과 제공 합니다. 기계와 생물 학적 효과를 신경 재생 하는 동안 신경 그 루터 기의 무질서 axonal 파생물을 줄이고 따라서 고통 스러운 끝-neuromata의 재발을 방지 도움이.

데이터는 통계 소프트웨어를 사용 하 여 및 ± 표준 편차를 의미 하는으로 분석 되었다. Kolmogorov 스미 르노 프 테스트 연구 인구의 비패라메트릭 분포 확인에 적용 된 고 부정적인 경험적 분포를 확인 합니다. 연구 인구의 비패라메트릭 성격에 따라, Wilcoxon의 테스트 관련된 샘플에 대 한 수술 전 및 수술 후 결과 비교 하 고용 되었다. 통계적 의미는 p-값에 간주 되었다 0.05 ?...

Watch this Scientific Journal Video about Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata at JoVE.com

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

Stromal 혈관 분수 농축 지방 이식 증상 끝-neuromata의 치료에 대 한

연구의 목적은 stromal 혈관 분수 (SVF)의 기술적 절차를 설명 하기 위해-증상 끝-neuromata의 소설 치료로 지방 이식 농축. 이 기술은 기계적 장벽 및 처리 및 집중 SVF에 의해 세포 수준에서 생물 학적 행동의 이점이 있습니다.

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

stromal-vascular-fraction-enriched-fat-grafting-for-treatment

Research

JoVE Journal

Medicine

7.6K 조회수.  University Hospital Zurich. 연구의 목적은 stromal 혈관 분수 (SVF)의 기술적 절차를 설명 하기 위해-증상 끝-neuromata의 소설 치료로 지방 이식 농축. 이 기술은 기계적 장벽 및 처리 및 집중 SVF에 의해 세포 수준에서 생물 학적 행동의 이점이 있습니다.

비디오: Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

인간 Mammary 섬유아 세포에서 암종 - 관련 섬유아 세포의 실험 세대 (CAFs)

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

연구

의학

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

혈관 질병의 연구에 대한 마우스의 Orthotopic 대동맥 이식

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

마우스 Aortocaval 누관의 기술적 측면

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

종양 기질 상호 작용에 대한 세 가지 차원의 공동 문화 모델

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

마우스 난소 지방 패드 속으로 이식

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

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

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

Ototoxicity의 치료에 대 한 트랜스-귀 약물 전달

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

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

지방 조직 유래 Stromal 혈관 분수 잠재적인 연골 재생을 위한 생산의 임상 프로토콜

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

급진적 인 전립선 절제술에 따른 발기 부전의 치료를위한 지방 유래 재생 세포의 분리

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

줄기 세포 치료를위한 심근 경색 심부전의 돼지 모델 설립

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...