This research was supported by the Australian Research Council through the ARC Centre of Excellence in Vision Science (CE0561903), the National Health and Medical Research Council (1049990), and Progetto regionale speciale multiasse 'Reti per l'Alta Formazione' - PO FSE Abruzzo 2007-2013 - Azione 4.II.iii.

수행 모든 실험은 안과 및 비전 연구에서 동물의 사용을위한 ARVO 정책에 따라이었고, L&#39; Aquila의 동물 윤리위원회의 대학과 호주 국립 대학교 동물 실험 윤리위원회의 승인을. 성인 쥐 (P100-200)가이 연구에서 사용 하였다. 1 공중 합체 수지 펠렛을 제조 <ol><li> 흄 후드에서 작은 유리 비커에 20 Elvax 40W 펠렛을 놓습니다. </li><li> 100 % 에탄올 모든 알약을 충당하기 위해 보장과 비커를 입력합니다. </li><li> 파라 필름으로 비커를 커버하고 7-10일 실온에서 100 % 에탄올에 알약을 흡수. 참고 : 이후이 단계에서 처리하거나 수지를 저장하는 금속 또는 플라스틱을 사용하지 마십시오. </li></ol> 2로드의 약물 용액을 제조 <ol><li> 0.1 % 디메틸 술폭 시드 (DMSO)에 원하는 약물을 녹인다. 볼륨 40 μL에 약물 솔루션을 확인하십시오.단일 용량으로 주어질 것이다 약 3 배의 농도로 수지를로드. </li></ol> 3 준비하고 약물 수지 복합을로드 <ol><li> 작은 또 다른 유리 비커에 20 세척 알약을 전송합니다. 약 45 분 동안 4 ml의 디클로로 메탄에 알약을 녹여. 펠릿 용해하는 동안 파라 필름으로 비커를 커버. </li><li> 0.001 ㎎ / ㎖의 농도로 0.1 % DMSO에 용해시켜 패스트 그린 FCF의 용액을 제조 하였다. </li><li> 한 피펫을 가지고, 이전에 제조 된 약물 용액을 40 μl를 작성한다. 빠른 그린 솔루션의 40 μL와 다른 펫을로드합니다. </li><li> 비커에 동시에 두 가지 솔루션을 추가합니다. 믹스가 빠르게 녹색 염료가 균일 혼합물에 분산 될 때까지 교반 봉 유리를 사용. </li><li> 즉시 수지를 동결 금식을 10 분 동안 드라이 아이스에 비커를 전송합니다. </li><li> 용매를 증발 &quot;증발 실&quot;을 설정합니다. 입력황산 칼슘 자갈 3 분의 1에 더 큰 용기입니다. 자갈에 잘 만들기 조심스럽게 실에서 비커를 안정 잘 자갈, 비커의 절반까지 도달 할 정도로에 비커를 놓습니다. </li><li> 파라 필름으로 더 큰 외부 컨테이너를 커버. </li><li> 냉동실에 증발 챔버를 전송합니다. 2~3주 -20 ° C에서 보관하십시오. </li></ol> 4 외과 이식에 대한 약물 - 수지 복합 준비 <ol><li> 냉동실에서 증발 챔버를 제거합니다. 유리 접시에 고체 약물 수지 블록을 이동시켜 얼음에 보관. </li><li> 현미경이나 확대경의 도움으로, 조각보기 직경 약 0.05 mm 및 트레이나 펀칭 공구를 이용하여 블록의 길이가 0.​​1 mm를 잘라. </li><li> 동결 방지 물질 (예를 들면, 알루미늄 호일)에 블록의 나머지 부분을 감싸고 필요한 때까지 -20 ° C에서 보관하십시오. </li></ol> (5) 수술로 임플란트 쥐 눈에 약물 수지 복합 </p&gt; <ol><li> 수술을 위해 동물을 준비합니다. 케타민 (100 ㎎ / ㎏ 체중)과 자일 라진 (xylazine) (12 밀리그램 / kg 체중)의 혼합물을 복강 내 주사하여 마취. 전체 마취가 달성 될 때까지 동물을 관찰한다. NOTE : 동물 (호흡 제외)의 움직임의 손실을 가져야하며 각막과 발가락 핀치 반사의 손실. </li><li> 수술 테이블에 완전히 마취 동물을 전송합니다. 사용 mydriaticum (예를 들어, 아트로핀 황산염) 및 국소 마취제 (예를 들면, 테트라 카인 염산염) 수술을받은 눈 (들)에 삭제합니다. 수술하는 동안 촉촉한 각막을 유지하기 위해 인공 눈물을 적용합니다. 수술을 통해 일회용 바늘 멸균 악기를 사용합니다. </li><li> 동공이 완전히 팽창 된 후에 무딘 집게 곡선의 쌍을 사용하여 눈을 안정화. 25 G 바늘을 이용하여 유리체 윤부로부터 약 2mm 도달하는 전체 깊이 관통상 있도록 운영 현미경을 사용한다. </li><li> t의 준비 조각을 삽입천자 사이트를 관통 할 수있는 미세하고 뾰족한 집게 (예, 핀셋)의 쌍을 사용 관통상으로 그 약물 - 수지 복합체. </li><li> 세균 감염에 대한 예방 적으로, 염증을 방지뿐만 아니라 동물이 회복되는 동안 각막 건조를 방지하기 위해 눈에 항생제 연고를 적용합니다. </li><li> 전체 복구 할 때까지 동물을 관찰한다. 이 의식을 회복 할 때까지 무인 동물을 두지 마십시오. 완전히 회복 될 때까지 다른 동물의 회사에 동물을 반환하지 않습니다. 동물이 스트레스 또는 불편의 흔적이있는 경우, 진통제는 승인 된 윤리 프로토콜에 따라 사용되어야한다. </li><li> 수집 및 조직의 분석을위한 일반적인 실험 절차를 따르십시오. </li><li> 실험의 완료시, 펜 토바 비탈 (60 ㎎ / ㎏ 체중) 2 % 리도카인 염산염 용액을 복강 내 주사로 동물을 안락사. </li></ol> 참고 : Lignocaine 염산염 분지역의 불편을 imizes.

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C., Valter, K., Stone, J., Cervetto, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroretinogram+changes+associated+with+retinal+upregulation+of+trophic+factors:+observations+following+optic+nerve+section.">Electroretinogram changes associated with retinal upregulation of trophic factors: observations following optic nerve section.</a> Neuroscience. 126 (3), 775-783 (2004).</li><li>Maccarone, R., Di Marco, S., Bisti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saffron+supplement+maintains+morphology+and+function+after+exposure+to+damaging+light+in+mammalian+retina.">Saffron supplement maintains morphology and function after exposure to damaging light in mammalian retina.</a> Investigative Ophthalmology &amp; Visual Science. 49 (3), 1254-1261 (2008).</li><li>Tsai, T., Bui, B., Vingrys, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethyl+sulphoxide+dose-response+on+rat+retinal+function.">Dimethyl sulphoxide dose-response on rat retinal function.</a> Documenta Ophthalmologica. 119 (3), 199-207 (2009).</li></ol>

저자는 공개와는 아무 상관이 없습니다.

이 논문은 망막에 약물의 서방 형 전달을위한 약물 - 수지 복합체의 사용을 보여 주었다. 우리는 비교적 저렴​​하고 소형 동물 모델에 적용하기 쉬운 방법을 제시하는 것을 목표로. APB의 함수 글루타메이트 유사체로서 작용하는 것을 감안할 때, 그것은 눈 망막 반응을 차단한다. 결과는 APB는 일주의 postimplantation에 의해 망막 응답의 막힘의 원인이 있음을 보여준다. 이 APB ?...

이들 질환은 일반적으로 장기간 치료를 필요로 당뇨병 및 고혈압과 같은 만성 질환의 치료는 주로 수명, 많은 과제를 제시한다. 이는 빈번한 투여를위한 필요성을 감소 서방 약물 전달 시스템의 개발을 요구하고있다. 이러한 느린 방출 방식의 효과는 당뇨병을 치료하는 데 필요한 인슐린 주사 횟수를 줄이기 위해, 인슐린 펌프의 개발을 통해 증명되었다. 눈의 만성 질환, 그 내부 층에 영향을 미치는 특히이 침략 절차를 통해 약물의 잦은 투여를 필요로합니다. 인간의 눈에 영향을 미치는 하나의 만성 질환은 연령 관련 황반 변성 (AMD)입니다. 이 비전을 개시하기위한 책임을 눈 뒤쪽에 위치한 신경 조직의 층인 중심 망막에 영향을 미친다. AMD는 서양 세계 1 실명의 주요 원인이다. 재 처리와 특정 과제tinal 질병은 종종 약물 전달의 침습적 방법을 필요로 눈이 깊은 층에 도달하기 위해 요구된다는 것이다. 약물은 일반적으로 유리체 강내 주사를 사용하여 유리체 챔버와 망막에 투여한다. 그러나, 각각의 주사 안내 염, 망막 박리, 백내장 및 유리체 출혈이 포함 주 사후 합병증의 위험이있다. 이 위험은 약물의 모든 재 주입을 곱합니다. 다중 주사의 필요성을 감소시키는 것은 AMD의 치료에 큰 이점이 될 것이다. 새로운 혈관의 성장이 특징입니다 AMD의 젖은 양식을 치료하기 위해, 기존의 치료 전략은 VEGF 억제제 3를 사용하여 내피 성장 인자 (VEGF)를 대상으로하는 것입니다. 현재, 이러한 반복 유리체 강내 주사를 통해 전달됩니다. 마찬가지로, 황반 부종, 당뇨 망막 병증의 일반적인 합병증의 치료에 스테로이드 반복 주사를 통해 전달됩니다 <s&gt; 사입니다. 서방의 방법을 통해 이러한 약물의 전달은 참으로 주 사후 합병증의 위험을 줄일 수 있습니다. 느린 방출 약물 전달 시스템의 개념은 먼저 동물 조직 (5)에 작은 분자를 전달하는 실리콘 고무의 차량을 이용하여 설명 하였다. 그 이후로, 다른 서방 방법은 눈을 테스트 한 몇 가지있는 큰 분자를 제공하기 위해 개발되었다. 이러한 생분해 성 마이크로 스피어, 폴리 락 티드 - 코 - 글리콜 라이드 (PLGA) 나노 입자 및 인지질 소포 (리포좀)와 같은 입자 캐리어, 배달 차량 6,7로 유용 할 수 있습니다. PLGA 나노 입자와 리포좀은 7 시간 동안 공막에 걸쳐 항암제를 제공하는 능력에 대한 시험 관내 환경에서 비교되었다. 두 차량은 서서히 약물을 방출 효과가 있었다. 그러나, 연구는 생체 외 환경에서 수행 하였다. Bochot 외. (2002) 8 테스트리포좀의 효과는 생체 내 망막에 분자를 제공합니다. 그들은 리포좀 성공적으로 토끼 망막에 작은 올리고 뉴클레오티드를 제공하는 것을 증명하고있다. 저자는 리포좀은 망막 질환 (8)의 치료에 유용 할 수 있다고 제안했다. 그러나 유리체에 떠있는이 소포의 성격은 가능성이 흐릿하거나 비전 구에 손상을 의미합니다. 오카베는 외. (2003)도 10은 토끼 베타 methasone을 적용하는 33 % 에틸렌 - 비닐 아세테이트로 이루어진 비 생분해 성 중합체 디스크를 사용했다. 그들은 공막 주머니에 디스크를 이식하고 최대 1 개월 10에 대한 유리체와 망막에 약물의 효과적인 방출을 보여 주었다. 그러나,이 특정 프로토콜에, 임플란트가 비교적 크고 강성이고, 큰 공막 절개 봉합 포함한 더 복잡한 외과 수술을 필요. 이전 연구는 tissu 조사전자 토끼 눈의 각막에 그들을 주입함으로써 다양한 중합체 차량에 응답하고, 알코올 세척 에틸렌 - 비닐 아세테이트 공중 합체는 염증 또는 자극을 유발하지 않았다는 것을 발견 하였다. 이러한 착물은 장시간 동물 조직에 큰 화합물의 전달을 유지하기 위해 도시 된, 일부 약물에 따라 11 백일까지 초과. 공중 합체 수지의 그러한 유형 Elvax 40W의 형태로 공업 적으로 개발되어있다 ( &#39;W&#39;아미드 첨가제 중량 에틸렌 - 비닐 아세테이트 코 모노머 함량은 40 % 펠렛 취급을 개선하기 위해). 이 공중 합체 수지는 룸과 체온에서 모두 안정한 비활성 물질이다. 이 생체 조직에 독성 또는 알레르기를 일으키는 것으로 도시되지 않았다. 이 수지는 효과적으로 유방 유관 시스템 (12), 일차 청각 피질 13,14 &lt;같은 다양한 시스템의 기능을 조사 다른 실험 모델에서 약물의 큰 다양성을 전달한/ SUP&gt;, 그리고 개구리의 시각 시스템 15. 이 수지는 거북이 16, 17, 닭 배아 (18, 19)의 개발에 약물을 전달하기 위해 눈을 사용하고 있으며, 성인 흰 족제비는 20 망막. 랫트 중추 신경계에서는, 수지는 뇌 21-23에서 사용되었지만, 쥐 눈에서의 사용은 기록되지 않았다. 다른 방법에 비해 망막 서서히 약물을 전달하기 위해이 공중 합체 수지를 사용하는 장점은, 그것이 눈에 염증이나 자극을 일으키지 않는 안정된 화합물로되어있다. 그것은 일반적 대신 유리체에 떠있는 배달의 사이트에 남아있는 입자 항공사와는 달리, 약물 - 수지 복합체는, 주입 후 시력에 악영향을하지 않을 것이다. 그것은 단지 눈의 각막 윤부 주변 유리체 공동 내로 간단 주입 공정을 필요로하고, 주입 한 후 봉합을 필요로하지 않을 것이다. 최근 같은 여러 신규 한 전달 시스템의 출현이 있었다캡슐화 된 세포 기술 (ECT) 24,25, 26, 및 마이크로 필름 (27) 하이드로 겔. 그러나, 제조 및 약물 - 수지 복합체를 제공하기위한 현재의 연구에서 사용되는 방법은 이에 기초 연구 환경에서 사용하기에 더 유리한 것으로, 모두 수행하기 쉽고 저렴하다. 장기간 약물 치료를 제공하기 위해 이러한 복합체를 사용하는 과제는 적은 강내 주사를 갖는 치료 학적 이점을 극대화 할 약물의 최적 농도를 결정하는 것이다. 이 논문은 성숙한 래트 망막의 장기 치료 용 약물 - 수지 복합체의 사용을 설명하기위한 목적으로한다. 전달 모드의 효능은 약물로서 글루타메이트 유사체 2 - 아미노-4-phosphonobutyrate (APB)를 사용하여 시험한다. APB 블록 망막 28 글루타민산, 내인성 신경 전달 물질을 모방하여 ON 바이폴라 전지의 광 반응. APB는 수용체를 차단 Light 미사용에 대한 글루타메이트와 경쟁 할 때t 응답. APB 망막 기능을 제어 및 전위도 (ERG)과 같은 방법을 사용하여 전기 생리 학적 효과를 측정하는 생체 연구에 사용되었다. 이전 연구에서, APB 단기 (29) 및 현상 망막의 장기 치료에 모두 사용되었다; 후자는 삼십일 30 매일 안구 주사를 통해 단일 용량을 제공하고있었습니다. 이전 작업 28,29,31에 제안 APB의 같은 양 (50 μM의 농도로 멸균 식염수에 0.092 mg을) 모든 주사에 사용되었다. 우리는 눈에 약물을 전달하는 서방 형 차량과 같은 약물 - 수지 복합체를 테스트 할 APB를 선택했다. 본 연구에 제시된 방법은 16, 32 복잡한 약물 수지의 제조와 관련된 전술 한 바와 방법과 유사하다; 그러나 성인 쥐의 눈에 특히 우리는 또한 세부의 사용을. 눈에 APB-로드 수지의 수술 주입 한 후, 망막 전위 APB는 R을 폐지 여부를 설정 하였다etinal 빛 응답은, 따라서 APB 성공적으로 유리체와 망막에 출시되어 있는지 여부.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Elvax 40W Pellets</td>
			<td>Du Pont, DE, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-amino-4-phosphonobutyric acid (APB)</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>A1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>34856</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite, calcium sulfate</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>238910</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine, Ilium Ketamil</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine, Ilium Xylazil-20</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine Sulphate, Minims eye drops</td>
			<td>Bausch &#38; Lomb Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracaine Hydrochloride, Minims eye drops</td>
			<td>Bausch &#38; Lomb Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol, Chlorsig ointment</td>
			<td>Aspen Pharma Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital, Lethabarb</td>
			<td>Virbac Australia Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lignocaine Hydrochloride, Ilium Lignocaine-20</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uni-Core Punch Tool</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Microscope, Zeiss OPMI 99</td>
			<td>Zeiss, West Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25G Insulin Needle</td>
			<td>Terumo Corp., Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

slow-release drug delivery through elvax 40w to the rat retina: implications for the treatment of chronic conditions

망막 질환은 망막 깊은 눈에서 거짓말로 처리하기 어렵다. 약물 전달의 침습성 방법은 종종 이러한 질병을 치료하기 위해 필요하다. 망막 부종이나 신생 혈관 등의 만성 망막 질환은 일반적으로 효과적으로 상태를 치료하기 위해 여러 안구 주사를 필요로한다. 그러나, 이러한 주입과 관련된 위험은 약물 전달 반복적으로 증가한다. 따라서, 다른 전송 방법은 재 주입의 위험을 최소화하기 위하여 확립 될 필요가있다. 다른 몇몇 조사는 눈에 화학 천천히 방출 가능한 재료를 통해 연장 된 시간 동안 약물을 전달하는 방법을 개발했다. 이 조사에서는 성인 쥐의 망막에 약물 전달을위한 비히클로서 작용하는 Elvax 40W, 공중합 수지의 사용을 설명합니다. 수지는 만든 약물로로드됩니다. 약물 - 수지 복합체는 다음 천천히 TI를 통해 약물을 발표 할 예정이다 유리체로 이식나. 이 방법은 2 - 아미노-4-phosphonobutyrate (APB), 블록 망막의 광 반응을 글루탐산 유사체를 사용하여 테스트 하였다. 그것은 APB 천천히 수지에서 출시 된 것을 증명하고, 주입 후 칠일에 의해 망막 반응을 차단할 수이었다. 이것은 서방 형 약물 전달이 공중 합체 수지를 사용하여 망막의 치료에 효과적이며, 또한 테스트를 치료 사용될 수 있다는 것을 나타낸다.

풀 필드 전위도 (ERG)은 망막에 APB의 효과를 검출하기 위해 사용 하였다. ERG이 수행 된 방법에 대한 자세한 내용은 다음과 같은 연구 33, 34를 참조하십시오. 요약하면, 동물을 마취 한 후, 금 전극은 눈의 각막에 배치하고, 상기 기준 전극은 망막의 전기적 활동을 기록하도록, 눈 사이 전방 두피에 넣었다. 자극은 전자 플래시 장치를 사용하여 생성하고, 강도를 막대 당 활성화 감광성 분자의 ...

Watch this Scientific Journal Video about Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions at JoVE.com

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

이 논문은 Elvax 40W가 성인 쥐의 망막에 약물 전달에 대한 서방의 방법으로 사용 할 수있는 방법을 자세히 설명합니다. 준비, 로딩, 및 눈에 약물 - 수지 복합체를 제공하기위한 프로토콜을 설명한다.

쥐의 망막에 Elvax 40W를 통해 서방 약물 전달 : 만성 질환의 치료를위한 시사점

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

slow-release-drug-delivery-through-elvax-40w-to-rat-retina

Research

JoVE Journal

Medicine

11.4K 조회수.  University of L'Aquila.  이 논문은 Elvax 40W가 성인 쥐의 망막에 약물 전달에 대한 서방의 방법으로 사용 할 수있는 방법을 자세히 설명합니다. 준비, 로딩, 및 눈에 약물 - 수지 복합체를 제공하기위한 프로토콜을 설명한다. 

비디오: Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

약시의 억제의 측정 및 치료

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

연구

의학

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

쥐의 만성 영양 주입의 모델

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

마이크로 컴퓨터 단층 촬영 영상에 대한 Microfil와 허혈성 뒷발과 혈관의 관류에 삼투의 약물 전달 (Drug Delivery)

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

쥐의 뇌에 직접 약물 전달 이소성 점막 Engrafting 절차

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

마우스 동정맥루 내피에 관내 약물 전달

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

쥐의 신장에 생물의 혈관 배달

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

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 ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Ivacaftor, 그것의 주요 대사 산물, 및 생물 학적 체액의 낭 성 섬유 증 환자에서 Lumacaftor의 임상 샘플의 높은 처리량 분석에 대 한 최적화 된 LC-MS/MS 방법

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

생체 외에서 혈액-뇌 장벽 돼지 뇌 내 피 세포를 기반으로 하는 모델의 설립에 대 한 방법 개선

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

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 ...