저자 아무 승인 있다.

연구 프로토콜 낮은 비전 아카데미의 윤리 위원회에 의해 승인 되었다 고 모든 과목 헬싱키 선언에 따라 서 면된 동의 서명 했다. 이 연구는 러프와 셰필드 대학에서 윤리적인 승인을 받고 있다.
참고: 드라이 연령 관련 황 반 변성 환자를 받을 suprachoroidal 헌 이식 Limoli 망막 복원 기술 (LRRT)에 의해의 포함 및 제외 기준 표 1에 설명 되어 있습니다.
1. 건조 연령 관련 황 반 변성 환자 들의 진단
<ol><li>공초점 레이저 스캐닝 레이저 ophthalmoscope, SD-10 월, 그리고 내 진단을 확인 합니다.</li>
	<li>각 그룹의 BCVA까지 고 거리 근처에 대 한 평가. 측정 포인트 (Pts) 근처 비전 (근접)에 대 한 VA. BCVA 시간 0 (T0), 90 (t 90), 측정 및 180 일 (T180) 비해 초기 치료 당뇨병 성 망막 증 연구 logMAR에 4 미터 (EDTRS) 차트.</li>
	<li>기록 전기 scotopic, mesopic photopic 세포 활동, 또는 임상 전기 생리학의 비전 (ISCEV)11국제 사회에 의해 2009 년에 설정 된 표준 규격에 따라 플래시에.</li>
</ol>2입니다. anesthetization
참고: LRRT 동안 마 취에서 황금 표준 국 소 마 취, 마 취 및 진정의 하위-장부의 침투에 의해 강화 됩니다. 특정 경우에 전신 마 취가 좋습니다.
<ol><li>각 막을 conjunctival 마 취 국 소 로컬 마 취약을 적용 하 여 얻은 lidocaine 4%와 1 %ropivacaine 수술 전에 dropwise 15-20 분 합니다.</li>
	<li>하위 conjunctival에 직접 침투 하 여 마 취 주사와 subtenon의 공간.</li>
	<li>복 부 지역, 지방 조직, 추출 전에 하위 conjunctival 및 하위-장부의 공간, 12 m m는 limbus에서 지역 침투를 사용 합니다. Carbocaine 또는 marcain 1200 IU 피 네 프 린 혼합 지역 마 취 제를 채택 한다.</li>
	<li>반복 된 작은 boli 통해 마약 진통제로 펜타닐을 사용 하 여 제대로 실행 될 수 있는 마 취를 통해 자가 진정을 제공 합니다. 복용량은 일반적으로 bolus 당 midazolam의 1 mg와 펜타닐의 0.025 mg 이다.</li>
</ol>3. Limoli 망막 복원 기술 준비
참고:이 기술을 나타냅니다는 궤도 헌 지방 subscleral 공간1,6,,712이주 Pelaez의 개입의 변종을. 수술로 이식할된 세포 주변 조직, 맥락 막, 및 망막18,,1920,21,22 科 및 angiotrophic 속성을 가진 많은 GFs를 생산할 수 있는 ,23,,2425. LRRT에서 이식할된 헌 셀 및 맥락 막 사이의 거리 깊은 sclerectomy에 의하여 감소 되 고 줄기와 맥락 막 사이의 접촉 영역 확장 안 흐름9, paracrine 헌 세포 분 비를 촉진 하 10,14.
<ol><li>맥락 막 그리고 sclera, Limoli 망막 복원 기술 (LRRT)15,,1617를 호출 하는 프로시저 간의 세포 이식으로 수술 하기 전에 각각의 눈의 적절 한 소독을 수행 합니다.</li>
	<li>콜맨 외 및 복 부 지방에서 sovrachoroidal 공간15,,1617SVF에서 로렌스의 기술은 (그림 1)에 의해 얻은 줄기 이식</li>
	<li>혈소판과 침투 많은 작은 꽃 자루는 다음 단계를 통해 얻은 PRP 젤에서 파생.</li>
	<li>혈액6,12 원심 및 혈소판이 풍부한 혈장 (PRP)을 수집 합니다. 혈소판 degranulation 자극 많은 작은 꽃 자루6,12에 GF 릴리스를 발생합니다.</li>
</ol>4. 기술 사양 및 전략
참고: 지방 조직 수집 이며 로렌스와 콜맨 기술17(테이블의 자료)에 따르면, 환자의 복 부 피하 층에서 정화.
<ol><li>수동으로 수확 10 mL 3 m m 무딘 정을 사용 하 여 각 환자의 복 부 피하 층에서 살 찐 티슈의 로렌스와 콜맨 기술17 (그림 2A/2B)에 따라 잠금 주사기에 연결.</li>
	<li>20 ° c에서 (그림 2C) 1500 x g에 5 분 동안 원심 분리 하 여 혈액, 지방, 기름, 그리고 액체에서 살 찐 티슈의 순수한 SVF를 구분 합니다. SVF 줄기17의 매우 풍부 하다.</li>
	<li>PRP 준비를 위한 별도 튜브 22 G 바늘와 인간의 말 초 혈액의 8 mL를 수집 합니다.</li>
	<li>20 ° c에서 (그림 2D) 1500 x g에 5 분에 대 한 수집 된 혈액 원심 LRRT, 자가 지방 이식, ADSC 확산, 증가 안 관류, 호의의 더 나은 생존 및 지방7만 분 비는 그 요인의 행동의 더 포괄적인 변조에 뒤이어 변화 결과에 11,17.</li>
	<li>궤도 지방에서 얻은 이식 고이 포켓의 잔류 볼륨 SVF는 로렌스에 따르면 얻은 PRP에서 줄기의 혼합물으로 포화 suprachoroidal 포켓 (자세한 내용은 4 단계에서 특정 4.4 및 4.5)을 구축 하 고 콜맨 기술17.</li>
</ol>5. Suprachoroidal LRRT (Limoli 망막 복원 기술)에 의해 Autograft: 수술 및 기술 세부 사항
<ol><li>6-0 비단 봉합 사, 열 등 한 일시적인 limbus 근처와 sclera 앵커.</li>
	<li>5.5"Westcott Tenetomy 곡선된가 위를 사용 하 여 열 등 한 일시적인 limbus에서 11 m m 부근의 subtenonian 공간을 엽니다.</li>
	<li>Limoli-바 실 conjunctival 견인 scleral 외과 분야를 만들기 위하여이 공간에 삽입 합니다.</li>
	<li>5 mm 초승달 칼 각도 경사를 사용 하 여 미리 잘라 플랩에는 limbus에서 8 m m에서 sclera에 측면. 플랩 경첩은 항상 방사형 및 외과 의사의 왼쪽에.</li>
	<li>Limbus에서 8 m m에서 열 등 한 일시적인 사분면에서 초승달 모양의 칼, 각도 경사를 사용 하 여 방사형 경첩에 의해 측면에 약 5 mm의 깊은 scleral 도어를 엽니다. 맥락 막의 슬레이트 색상을 보려면 적절 한 깊이에서 sclerectomy을 실시 합니다.</li>
	<li>후속 suprachoroidal autograft에 혈액 순환을 촉진 하기 위하여는 플랩의 원심 부분에 약간 operculum를 제거 함으로써 간격을 만듭니다.</li>
	<li>안과 집게와 궤도 지방에서에서 추출 열 등 한 비스듬한 근육 위에 간격. 추출 된 지방 그것의 주입 후 살아남을 수 있도록 충분히 나가도록는 다는 것을 확인 하십시오.</li>
	<li>부드럽게 안 침대와 문 근 위 가장자리에 안 6/0 polyglactin 섬유와 봉합에 헌 지방 플랩을 놓습니다.</li>
	<li>피하기 위해 압축 또는 그것의 영양 혈관에 지방 작은 꽃 자루에 scleral 플랩 봉합</li>
	<li>PRP 젤 (혈액 소재의 원심 분리, 구성 요소, 및 혈소판 degranulation26분리 하 여 얻은)의 1 mL와 함께 지방 작은 꽃 자루의 기질에 침투 30 G 각도 (30 °) 정 맥을 사용 하 여.</li>
	<li>결 막의 양쪽을 봉합에 대 한 준비. 그런 다음, conjunctival 견인 제거.</li>
	<li>6/0 polyglactin 섬유를 사용 하 여 결 막 봉합</li>
	<li>폐관, 플랩, 맥락 막, 및 안 autograft 사이의 subscleral 공간에 자가 지방 이식 작은 유연한 플라스틱 튜브를 삽입 하는 공간을 남겨 주세요.</li>
	<li>자가 지방 이식, 맥락 막, scleral 플랩 사이의 잔여 공간 0.5 포화 cc SVF (줄기의 리치)의 이전 scleral 포켓에 삽입 작은 유연한 플라스틱 튜브에 의해 단계 3.2에서에서 준비.</li>
	<li>잔여 공간을 포화 후 봉합을 닫습니다.</li>
	<li>수술 후 3 일간 500 mg azithromycin 항생제 치료의 관리. 또한, 항생제 및 스테로이드 조합으로, 페니 등 디, 약 15-20 일 동안 눈 드롭 치료를 제공 합니다. 참고:는 autograft SVF, 그리고 PRP에서 줄기 지방 세포로 이루어진 지금 되었습니다 취득 했다26. 깊은 sclerectomy paracrine 안 흐름으로 헌 세포의 분 비를 자극 하 여 이식할된 헌 세포와 맥락 막 사이의 거리를 줄일 수 있습니다. 같은 목적을 위해 줄기와 맥락 막 사이의 접촉의 영역을 확장 합니다.</li>
</ol>

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Z., Nebbioso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surgery+and+growth+factors+in+dry+age-related+macular+degeneration:+visual+prognosis+and+morphological+study.">Cell surgery and growth factors in dry age-related macular degeneration: visual prognosis and morphological study.</a> Oncotarget. 7 (30), 46913-46923 (2016).</li><li>Ueki, Y., Reh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+stimulates+M&#252;ller+glial+proliferation+via+a+BMP-dependent+mechanism.">EGF stimulates M&#252;ller glial proliferation via a BMP-dependent mechanism.</a> Glia. 61, 778-789 (2013).</li><li>Kozlowski, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPE+cell+senescence:+A+key+contributor+to+age-related+macular+degeneration.">RPE cell senescence: A key contributor to age-related macular degeneration.</a> Med. Hypotheses. 78, 505-510 (2012).</li><li>Schneider, 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=The+hematopoietic+factor+G-CSF+is+a+neuronal+ligand+that+counteracts+programmed+cell+death+and+drives+neurogenesis.">The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis.</a> J. Clin. Invest. 115, 2083-2098 (2015).</li><li>Yin, Y., 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=Oncomodulin+is+a+macrophage-derived+signal+for+axon+regeneration+in+retinal+ganglion+cells.">Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells.</a> Nat. Neurosci. 9, 843-852 (2006).</li></ol>

발표 ARVO 2015 년 5 월 3-7-덴버, CO-미국.

이 연구의 주된 목적은 adipocytes, SVF, PRP에 줄기의 suprachoroidal 이식 버지니아 및 건조 AMD 영향을 눈에 망막 감도 시간이 지나면서 개선 수 여부를 평가 했다. 또 다른 주요 목적은 여러 전 임상 연구 GF에 기초를 둔 치료 여러 가지 질병에 환자 치료에 도움이 될 수 있는 제안 이후 최근 문학에 따라 이러한 세포의 가능한 치료 효과 입증 했다.
실제로, 일부 연구 자가 인간 유도 만?...

세포 치료, 여러 만성 질환을 치료 하는 부상된 지역에 있는 줄기/뿌리 세포의 조직 또는 지방 주입의 구성 된 지난 10 년간1에 세심 한 관심을 받고 있다. 1990 년대 이후, 성장 인자 (GFs)가 그들의 잠재적으로 치료 역할 망막 위축2에 대 한 연구 되었습니다. 사실, 많은 인간 세포 GFs, 차단 또는 apoptosis, 즉, 셀3의 프로그램 된 죽음을 늦출 수 있는 특정 단백질을 생산할 수 있다.
그것은 건조 연령 관련 황 반 변성 (AMD)은 어디 점진적이 고 돌이킬 수 없는 세포 죽음 관련 포토 리셉터 레이어 및, 따라서, 중앙 시각 기능4의 손실에 부상 위축 한 망막 질환으로 알려져 있습니다. AMD는 선진국 및 모든 황 반 유년까지 효과적인 치료 부족의 80%에 대 한 계정에 55 세 이상의 사람에서 실명의 주요 원인입니다.
몇 가지 연구는 다양 한 소스에서 헌 GFs 얻어질 수 있다 나타났습니다. 이 구성 하는 다른 세포 유형, 궤도, 혈소판 혈소판이 풍부한 혈장 (PRP)에서 파생 된 지방과 지방 유래 줄기 세포 (줄기)의 지방 조직5 stromal 혈관 분수 (SVF)에 포함 된에서 파생 된 지방 기질 세포를 포함 하 여 ,,67. 현재 GF 세트 망막 neuroenhancement, 및 Filatov, Meduri, Pelaez, 연구 및 Limoli는 자가 지방 이식 (후미) 효과적인8,,910은 설명 했다.
또한, 건조 AMD의 영향을 받는 눈11게시물 suprachoroidal 헌 이식, 기록 이전 연구 electroretinogram (에) 데이터에 상당한 개선을 보여주었다. Suprachoroidal 공간에서 수술로 이식할된 조직을 망막 세포, 그들의 apoptosis6,,712연기의 paracrine 분 비 변조. 외부 핵 층 두께 고려 하면 기니 피그의 망막의 조직학 검사 GFs 망막에 영양 영향을 미칠 수 있습니다 나타났습니다. 따라서, GFs의 직접 또는 간접 사용 잠재적으로 분자 inducers 억제제6,,712사이의 균형된 관계를 통해 치료 혜택을 가져올 수 있다.
이 방법의 목적은 시력 (BCVA) 및 microperimetry (내) 응답 건조 AMD 영향을 눈에 수정 adipocytes, 줄기 SVF와 PRP의 suprachoroidal 이식 가장 향상 시킬 수 있는지를 평가 하는. 이 연구 autograft는 인용된 문학6,7,,1213에 따르면 그것의 GF 생산 기초의 치료 효과 입증 하는 것을 목표로.

<table border="0" cellpadding="0" cellspacing="0" style="width:680px;" width="681">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Blunt cannula, 3 mm.&#160;</td>
			<td style="width:179px;">Mentor, Santa Barbara, CA.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luer-LokTM syringe.&#160;</td>
			<td style="width:179px;">BD Biosciences, Franklin Lakes, NJ.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Regen-BCT tube.&#160;</td>
			<td style="width:179px;">RegenKit; RegenLab, Le Mont-sur-Lausanne, CH.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Centrifuge</td>
			<td style="width:179px;">&#160;RegenPRP Centri. RegenLab, Le Mont-sur-Lausanne, CH.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD Venflon Pro Safety 22G x 1.00 inch (0.9 mm x 25 mm).&#160;</td>
			<td style="width:179px;">BD Biosciences, Franklin Lakes, NJ.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SPSS Statistics Version 19.0</td>
			<td style="width:179px;">IBM Corp., Armonk, NY, USA.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Confocal scanning laser ophthalmoscope&#160;</td>
			<td style="width:179px;">Nidek Inc, Fremont, CA</td>
			<td style="width:119px;">Nidek F10&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cirrus 5000 Spectral Domain-Optical Coherence Tomography</td>
			<td style="width:179px;">Carl Zeiss Meditec AG, Jena, Germany</td>
			<td style="width:119px;">&#160;SD-OCT&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Maia 100809 Microperimetry&#160;</td>
			<td style="width:179px;">CenterVue S.p.A., Padua, Italy</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ocular electrophysiology electromedical system,</td>
			<td style="width:179px;">C.S.O., S.r.l., Scandicci, Italy</td>
			<td style="width:119px;"></td>
			<td>&#160;Retimax for ERG&#160;</td>
		</tr>
	</tbody>
</table>

regenerative therapy by suprachoroidal cell autograft in dry age-related macular degeneration: preliminary in vivo report

이 연구 목적으로 검사 suprachoroidal 이식 autologous 세포의 최고의 교정된 시력 (BCVA)을 향상 시킬 수 있습니다 및 눈에 영향을 microperimetry (내)에 대 한 응답을 통해 시간이 지남에 연령 관련 황 반 변성 (AMD) 건조 여부는 생산 및 주변 조직에 성장 인자 (GFs)의 분 비. 환자는 각 연구 그룹에 무작위로 지정 되었다. 모든 환자는 건식 AMD와 BCVA 해상도 (logMAR)의 최소 각도의 1 대 이상의 진단 했다. 11 환자에서 11 눈을 포함 하는 그룹 A에 suprachoroidal 헌 이식 Limoli 망막 복원 기술 (LRRT)에 의해 실시 됐다. 기술은은 adipocytes, stromal 혈관 분수, 및 suprachoroidal 공간에서 혈소판이 풍부한 혈장에서 혈소판에서 얻은 지방 유래 줄기 세포를 이식 하 여 수행 되었다. 반대로, 그룹 B, 14 환자의 14 눈을 포함 하 여 컨트롤 그룹으로 사용 되었다. 각 환자에 대 한 진단 검사 confocal 레이저 ophthalmoscope 및 스펙트럼 도메인 광학 일관성 단층 촬영 (SD-10 월)에 의해 확인 했습니다. 그룹 A, BCVA 향상 0.581 0.504 0.376 logMAR를 90 일에서 180 일에서 (+32.20%) postoperatively. 또한, 내 테스트 12.59 db 11.44 dB에서 180 일에서 증가. 맥락 막 뒤에 투입 하는 다른 세포 유형 상수 GF 분 비 안 흐름에서을 확인 할 수 있었다. 따라서, 결과 이식할된 그룹에서 시각 예민 (VA) 증가 수 나타냅니다 이상 6 개월 후 제어 그룹에서에서.

여기에 제시 된 절차를 사용 하 여, 환자의 건조 AMD 영향, BCVA 해상도 (logMAR)의 최소 각도의 1 대 이상 두 그룹 연구에 등록 했다. 그룹 A, 그룹 B, 14 환자의 14 눈을 포함 한 반면 11 환자, 받은 suprachoroidal 헌 이식 Limoli 망막 복원 기술 (LRRT)에 의해의 11 눈을 포함 하 여, 컨트롤 그룹으로 사용 되었다.
스튜던트 t-검정 및 치 평방 테스...

Watch this Scientific Journal Video about Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report at JoVE.com

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

Suprachoroidal 세포 Autograft 건조 연령 관련 황 반 변성에 의해 재생 치료: 예비 Vivo에서 보고서

이 연구의 목표는 stromal 혈관 분수에 포함 된 지방 유래 줄기 세포와 혈소판 Limoli 망막 복원 기술에 의해 혈소판이 풍부한 혈장에서 파생 된 suprachoroidal 이식 시력을 향상 시킬 수 있는지를 평가 하 고 건조 연령 관련 황 반 변성에 의해 영향을 눈에 망막 감도 응답.

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

regenerative-therapy-suprachoroidal-cell-autograft-dry-age-related

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JoVE Journal

Medicine

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

10.0K 조회수.  Low Vision Research Centre of Milan. 이 연구의 목표는 stromal 혈관 분수에 포함 된 지방 유래 줄기 세포와 혈소판 Limoli 망막 복원 기술에 의해 혈소판이 풍부한 혈장에서 파생 된 suprachoroidal 이식 시력을 향상 시킬 수 있는지를 평가 하 고 건조 연령 관련 황 반 변성에 의해 영향을 눈에 망막 감도 응답.

비디오: Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

VIVO 개 근육 기능 분석에

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

연구

의학

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

암의 생산 및 반응 산소 종의 감지 (ROS)

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

신경근육학 전기적 자극에 의​​해 근육 트레이닝의 Murine 모델

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

세포 추적을위한 생체 19 F MRI에서

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

시간 코스 인도시 아닌 형광 안저 혈관 조영술로 연령 관련 황반 변성의 마우스 모델에서 맥락막 혈관의 이상을 검출

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

순차<em&gt; 생체 내</em&gt; 골절 치료시 골 형성 줄기 / 전구 세포의 이미징

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

마우스에서 관절염 통증의 Monoiodoacetate 모델

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

인간 종양 이종 이식에서 마우스 세포의 고갈은 크게 대상 세포의 다운 스트림 분석을 향상

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

가속도계를 사용하여 일상 생활에서 상지 성능을 정량화하는 방법

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Mechanical Micronization of Lipoaspirates for Regenerative Therapy

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of cell products using collagenase. Here, we present a protocol to generate an injectable mixture of SVF cells and native extracellular matrix from adipose tissue by a purely mechanical process. Lipoaspirates are put into a centrifuge and spun at 1,200 x g for 3 min. The middle layer is collected and separated into two layers (high-density fat at the bottom and low-density fat on the top). The upper layer is directly emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance under the oil layer is collected and defined as the extracellular matrix (ECM)/SVF-gel. The oil on the top layer is collected. Approximately 5 mL of oil is added to 15 mL of high-density fat and emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance is also ECM/SVF-gel. After the transplantation of the ECM/SVF-gel into nude mice, the graft is harvested and assessed by histologic examination. The result shows that this product has the potential to regenerate into normal adipose tissue. This procedure is a simple, effective mechanical dissociation procedure to condense the SVF cells embedded in their natural supportive ECM for regenerative purposes.

Here, we present a protocol to obtain stromal vascular fraction from adipose tissue through a series of mechanical processes, which include emulsification and multiple centrifugations.

재생 치료를 위한 Lipoaspirates의 기계 Micronization

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of ...