모든 랫트는 수도 의과 대학 베이징 우호 병원의 동물 실험위원회에서 승인 한 프로토콜에 따라 사용되었습니다.
1. 수술 준비, 마취 및 수술 기술
참고 : 체중이 200-220g 인 총 40 마리의 암컷 Wistar 쥐가 본 연구를 위해 상업적으로 획득되었습니다. 40마리 중 10마리를 무작위로 대조군으로 선정하고 나머지는 시험군으로 처리하였다. 모든 동물은 수도 의과 대학 베이징 우정 병원의 동물 시설에서 멸균 된 환경에 보관되었습니다.
<ol><li>펜토바르비탈 나트륨을 복강 내 투여하여 전신 마취를 수행합니다(40mg/kg). 또는 3%-4% 이소플루란을 사용하여 마취를 유도하고 1%-3%(흡입)로 유지합니다. 건조를 방지하기 위해 눈에 안과 용 연고를 바르십시오. 그런 다음 쥐를 수술 플랫폼에 놓고 열 지원을 제공하십시오. 참고: 시술 시작시 부 프레 노르 핀, 0.05 mg / kg, SC, 0.1-0.2 mL와 같은 진통제를 투여하십시오.</li>
	<li>발가락 꼬집음에 대한 반응이 부족하여 마취 깊이를 확인하십시오. 면도기로 뒤쪽 전체에서 모피를 면도하십시오.</li>
	<li>클로르헥시딘 또는 포비돈 요오드와 같은 2단계 스크럽과 이소프로필 알코올을 최소 3회 주기로 수술 부위를 소독합니다. 수술 용 테이프로 팔다리를 고정하고 수술 용 가위로 뒤쪽을 약 3cm 정도 절개하십시오.</li>
	<li>수술 용 가위를 사용하여 피하 조직을 통해 절개를 깊게하고 척추에 부착 된 근육을 잘라냅니다.</li>
	<li>13번째 갈비뼈를 육안으로 식별하고 노출합니다(해당 갈비 뼈에 연결된 추간 공간은 간격 T13\u2012L1). 봉합사를 사용하여 13번째 갈비뼈를 표시하십시오.</li>
	<li>식별 후 척추에 부착 된 근육을 조심스럽게 절제하고 척추를 노출시킵니다. 척추의 정확한 식별을 위해 극상 인대와 척추 간 인대를 절제합니다. 수술용 가위와 집게로 L4\u2012L5의 레벨을 노출시킵니다. 참고: 극상 인대는 얇은 피하 조직의 존재 때문에 쉽게 식별할 수 있습니다. 극상 인대 절제술 후, 극 과정 사이의 인대는 척간 인대입니다.</li>
	<li>L4-u2012L5 척추 가시돌기와 횡돌기 부분을 켈리 겸자를 사용하여 척수를 노출시키는 부분을 조심스럽게 해부합니다(그림 1).</li>
	<li>L4\u2012L5 수준에서 원추 수질을 완전히 노출시키고 홍채 절제술 가위로 원추 수질을 완전히 횡단합니다. 척수의 회복을 막기 위해 조직 패킹을 삽입하십시오.</li>
	<li>4-0 비 흡수성 봉합사를 사용하여 외부 피부층의 위에 놓인 근육과 피부를 닫습니다.</li>
	<li>대조군의 경우 1.1-7단계를 수행하고 원추 수질을 그대로 둡니다. 1.9단계에 따라 절개 부위를 닫습니다.</li>
</ol>2. 동물 회복
<ol><li>수술 후 첫 1 시간 동안 온도가 조절되는 인큐베이터 (37 ° C)에 쥐를 보관하고 흉골이 생기거나 활발하게 움직일 때까지 모니터링하십시오. 알림: 전체 복구에는 약 30 분이 걸립니다.</li>
	<li>충분한 음식과 물로 동물을 깨끗한 케이지로 옮깁니다. 쥐를 별도의 새장에 보관하십시오. 참고: 절편의 성공은 시험군의 쥐가 앞다리의 도움으로 만 움직이는 반면 대조군의 쥐는 정상적으로 걸을 수있을 때 나타납니다.</li>
</ol>3. 사후 운영 관리
<ol><li>항생제 인 페니실린 G (동물 당 50,000 U / mL)를 복강 주사하십시오. 수술 후 48시간 동안 6-12시간마다 부프레노르핀, 0.05mg/kg, SC, 0.1-0.2mL와 같은 진통제를 투여하십시오.</li>
	<li>배뇨를 돕기 위해 하복부의 방광을 압박하십시오. 6 주 동안 같은 시간에 매일 두 번 (오전 8시와 오후 8시) 수행하십시오. 참고: 배뇨근의 정상적인 수축 손실은 DU의 상징입니다.</li>
	<li>모든 쥐를 대사 케이지에 수용하고, 각각은 배뇨 및 요실금을 모니터링하기 위해 이전에 무게를 측정한 흡수성 종이 위에 놓인 소변 수집 깔때기를 포함합니다.</li>
	<li>공극량(VV)과 잔뇨량을 별도로 나타내는 흡수지의 무게 변화를 수집하고 기록하십시오.</li>
</ol>4. 요역동학 검사
<ol><li>수술 후 6 주에 다음과 같이 요 동력학 측정 장비를 사용하여 방광 조영술을 수행하십시오.<ol><li>복강에 10 % 클로랄 수화물 (3 mL / kg)을 주입하여 쥐를 마취하십시오.</li>
		<li>방광을 압축하여 배뇨시킨 다음 테이프를 사용하여 쥐를 수술 플랫폼에 고정시킵니다.</li>
		<li>경막 외 카테터 (3F)를 방광에 삽입하고 요 역학 측정 장비, 경막 외 카테터 및 주입 펌프를 3 개의 사지 튜브로 연결합니다.</li>
		<li>요역동학 측정을 위해 생리식염수를 0.2mL/min의 속도로 펌핑합니다( 재료 표 참조). MCC 및 DOP 및 방광의 순응도를 기록합니다 (방광 부피δ 배뇨근의 압력으로 나누어 계산δ).</li>
	</ol></li>
</ol>5. 통계 분석
<ol><li>상용 소프트웨어를 사용하여 통계 분석을 수행합니다.</li>
	<li>콜모고로프-스미르노프 검정을 사용하여 데이터의 정규성을 검정합니다.</li>
	<li>정규 분포 변수를 표준 편차가 있는 평균값으로 표현합니다. 양측 쌍을 이루는 스튜던트 t-검정을 사용하여 두 그룹의 방광경 모수를 비교합니다. 참고: p < 0.05는 차이가 통계적으로 유의하다는 것을 나타냅니다.</li>
</ol>

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저자는 공개 할 것이 없습니다.

DU는 남성과 여성 모두에서 하부 요로 증상의 일반적인 원인입니다. 영향을 받은 사람들의 삶의 질(Qol)을 크게 감소시킬 수 있는 치료 옵션이 거의 없는 복잡한 증상 집합체입니다18. DU는 다 인성이라고 믿어 지지만 병인에 대한 이해는 초보적입니다. 연구에 따르면 DU의 발병 기전은 근인성 및 신경성 요인과 관련이 있을 수 있습니다.
근원성 가설에서 DU를 가...

배뇨근 기능 저하 (DU)는 연구 된 전형적인 하부 요로 기능 장애입니다. DU는 국제 요실금 학회(ICS)1에 의해 정의되었지만 이 질병을 지칭하기 위해 "배뇨근 부전", "수축성 방광", "배뇨근 areflexia"2와 같은 다양한 용어가 사용됩니다. 2002 년 국제 요실금 학회 (ICS)에서 정의한 DU는 강도와 지속 시간이 감소하여 방광 비우기 시간이 장기간 증가하여 정상적인 기간 내에 방광 비우기를 완료하지 못하는 수축입니다.
DU는 하부 요로 증상이 있는 남성 (>70세)의 48%와 여성(70세)의 12%에 영향을 미칠 수 있습니다. 그것은 다 인성 인 것처럼 보이며 효과적인 치료법이 없습니다. DU는 다발성 경화증4, 당뇨병5, 파킨슨 병6 또는 뇌졸증7과 같은 신경성 방광 기능 장애 환자에서 어디에나 있는 것으로 보고되었습니다. DU는 또한 복강경 자궁 적출술, 전립선 절제술 또는 작은 골반의 다른 외과 적 개입과 같은 의원 성 신경 손상에 의해 유발 될 수 있습니다8. DU의 병태생리학적 변화와 이용 가능한 치료법은 연구를 위한 적절한 동물 모델이 없기 때문에 여전히 혼란스럽습니다.
배설 반사는 폰틴 배혈 센터, 천골 부교감 신경 핵 및 더 많은 고위 피질 센터9를 결합하는 척수-구근 척추 경로에 의해 제어됩니다. 배변 반사의 활성화 및 유지는 주로 방광에서 더 높은 피질 센터로의 감각 신호의 규칙적인 수송에 달려 있습니다. 감각 기능 장애가 DU에 기여한다고 가정 할 수 있습니다.
하부 요로 기능 장애와 관련된 대부분의 실험 동물 연구는 과민성 방광(OAB) 모델10에 초점을 맞추었습니다. 이러한 모델은 OAB 병태생리학 및 예후에 대한 합리적인 이해를 제공합니다. 그러나 척수상 손상(국소 병변, 쇠퇴 및 중뇌 동맥 폐색), 척수 절개 또는 타박상 손상, 전신(예: 사이클로포스파미드) 또는 자극제 또는 염증제(예: 산, 아크롤레인 및 지질 다당류)의 방광내 투여와 같은 몇 가지 DU 모델만 보고되었습니다.11,12,13,14 . 이러한 방법 중에서, 척수 절개 또는 타박상 손상 방법만이 DU13의 동물 모델을 확립하는데 사용될 수 있다. 폰틴 배뇨 센터와 상급 피질 센터의 부상과 관련된 시도는 심각한 외상으로 인해 포기되었습니다. 따라서 최소한의 부작용으로 DU를 유도하기 위해 배합 반사 센터에서 정확한 위치를 찾는 데 더 많은 관심을 기울이고 있습니다.
앞서 언급했듯이 DU를 유도하는 메커니즘 중 하나는 척수를 손상시켜 배합 반사의 신호 경로를 손상시키는 것입니다. Allen의 체중 감소 방법은 손상된 척수15를 가진 실험실 동물을 확립하기 위해 개발되었습니다. 그러나이 방법에 대한 추가 실험 데이터는 없습니다. 더욱이, 동물의 일부가 DU없이 뇌졸중 후 척추 기능을 회복했기 때문에, DU 동물 모델16을 생성하기 위한 완벽한 방법으로서 간주될 수 없다.
1987년, Bregman은 DU 동물 모델을 생성하기 위해 척수를 횡단하는 과정을 발굴하고 실험 데이터(17)를 획득하였다. 그럼에도 불구하고, 이 방법은 DU 동물 모델을 확립하는데 적용되지 않았다. 그 당시 연구자들은 여전히 DU의 발병 기전에 대해 혼란 스러웠습니다. OAB 또는 DU의 유도와 관련된 척수의 위치가 서로 인접하기 때문에 DU17을 유도하는 척수 손상의 정확한 부위를 찾을 수 없었습니다. OAB 및 DU는 이 방법에 의해 함께 또는 별도로 도입되었습니다. 따라서 이 방법은 DU를 도입했지만 부정확하여 DU의 발생 및 처리를 이해하는 데 사용할 수 없었습니다.
위에서 언급했듯이 DU의 적합한 동물 모델이 없다는 것은 DU의 연구의 주요 장애물 중 하나입니다. 연구원들은 DU의 병리를 시뮬레이션할 수 있는 정확하고 관리 가능한 모델을 지속적으로 찾고 있습니다. DU에 대한 치료 옵션조차도 지난 20 년 동안 크게 개선되지 않았습니다. 총체적으로, DU의 동물 모델을 확립하기 위한 표준 프로토콜을 기술할 필요가 크다.
따라서이 논문에서는 원추 수질 절개에 의한 DU의 쥐 모델을 성공적으로 확립하는 방법을 설명합니다. 원추 수질을 분리하기 위해 L4\u2012L5 수준에서 절개를 수행했습니다. 최대 방광 측정 용량 (MCC), 배뇨근 개방 압력 (DOP) 및 방광의 순응도를 기록하고 분석하여 프로토콜을 검증했습니다. 아래에 명시된 프로토콜은 타당성과 신뢰성을 표준화된 방식으로 결합하여 DU의 발생 및 처리를 시뮬레이션하는 DU 동물 모델을 설정합니다. 이 프로토콜은 DU의 추가 연구를 위한 기술로 사용될 수 있습니다.

<table>
	<tbody>
		<tr>
			<td>0.9% saline</td>
			<td>Wuhan Prosai Company</td>
			<td>EY-C1178</td>
			<td>pump for urodynamic measurement</td>
		</tr>
		<tr>
			<td>10% chloral hydrate</td>
			<td>Shandong Yulong Co., Ltd</td>
			<td>H37022673</td>
			<td>3mL/kg&#65292;administered intraperitoneally</td>
		</tr>
		<tr>
			<td>Buprenorphine Hydrochloride Injection</td>
			<td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co. LTD</td>
			<td>H12020275</td>
			<td>0.05mg/kg subcutaneously 24h and 48h postoperation</td>
		</tr>
		<tr>
			<td>Epidural Catheter</td>
			<td>Shandong Xinghua Co, Ltd</td>
			<td>VABR3L</td>
			<td>for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>Alta Technology Co., Ltd</td>
			<td>1ST5637</td>
			<td>50,000 unit/ml per animal</td>
		</tr>
		<tr>
			<td>pentobarbital</td>
			<td>Beijing solabo Technology Co., Ltd</td>
			<td>NK-WF0001</td>
			<td>40 mg/kg, administered intraperitoneally</td>
		</tr>
		<tr>
			<td>Suture line(4-0)</td>
			<td>ETHICON</td>
			<td>VCP422H</td>
			<td>suture the injury</td>
		</tr>
		<tr>
			<td>Three-limb tube</td>
			<td>Shandong Xinghua Co, Ltd</td>
			<td>VAB3T</td>
			<td>for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Trace infusion pump</td>
			<td>Zhejiang Smith Medical Instrument Co., Ltd</td>
			<td>20162540335</td>
			<td>Pump the saline at a speed of 0.2ml/min for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Urodynamic measurement equipment</td>
			<td>Medical Measurement SystemsB.V.</td>
			<td>08-0467</td>
			<td>urodynamic measurement equipment can not only help the diagnosis of dysuria, but also provide objective materials for treatment and therapeutic effect. It is the most commonly used examination method in clinical diagnosis and treatment of lower urinary tract functional diseases</td>
		</tr>
		<tr>
			<td>Wistar Rats</td>
			<td>HFK Biotechnology Co.Ltd,Beijing ,China</td>
			<td>SCXK2012-0023</td>
			<td>200-220g</td>
		</tr>
	</tbody>
</table>

detrusor underactivity model in rats by conus medullaris transection

제시된 프로토콜의 목표는 원추 수질 절개를 통해 쥐에서 배뇨근 과소 활동 (DU) 모델을 확립하는 것이 었습니다. 후궁 절제술은 체중이 200-220g 인 총 40 마리의 암컷 Wistar 쥐 (대조군 : 10 마리, 시험군 : 30 마리)에서 수행되었으며, 원추 수질은 시험군의 L4-2012L5 수준에서 형이 절제되었습니다. 모든 래트를 6주 동안 동일한 환경 조건 하에서 사육하고 먹이를 주었다. 시험군에서는 6주 동안 매일 2회 소변 배뇨를 실시하고 평균 잔뇨량을 기록하였다. 두 그룹 모두에서 방광 측정이 수행되었습니다. 최대 방광 측정 용량 (MCC), 배뇨근 개방 압력 (DOP) 및 방광의 순응도를 기록하고 계산했습니다. 시험 그룹은 척추 쇼크 동안과 후에 수술 후 상당한 요정체를 보였다. 그러나 대조군에서는 이상이 관찰되지 않았습니다. 대조군과 비교했을 때, 시험군에서 MCC 및 방광의 순응도는 시험군보다 유의하게 높았으며 (3.24 ± 2.261 mL 대 1.04 ± 0.571 mL; 0.43 ± 0.578 mL / cmH 2 O 대 0.032 ± 0.016mL / cmH 2 O), 시험군의 DOP는 대조군보다 낮았다 (20.28 ± 14.022 cmH 2 O 대 35 ± 13.258 cmH2 O). 원추 수질 절개에 의한 DU의 동물 모델을 확립하는이 방법은 DU의 병태 생리학을보다 잘 이해할 수있는 훌륭한 기회를 제공합니다.

원추 수질 절개의 전체 절차는 숙련 된 외과의가 45 분 이내에 완료 할 수 있습니다. 우리 실험실은 100 건 이상의 원추 수질 절개 수술을 수행했습니다. 성공률은 쥐의 생존과 DU의 성공적인 유도에 의해 정의 된 95 % 이상입니다. 요역동학 검사는 DU의 유도를 확인했습니다.
우리의 경험에 비추어 볼 때, DU의 유도는 잔뇨량에 의해 예비 적으로 평가 될 수 있습니다. 수술 직후 소?...

Watch this Scientific Journal Video about Detrusor Underactivity Model in Rats by Conus Medullaris Transection at JoVE.com

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

우리는 쥐에서 원추 수질 절개에 의한 배뇨근 과소 활동 모델을 확립하는 방법을 제시합니다. 배뇨근 기능 저하는이 동물들에서 성공적으로 자극되었습니다. 이 모델은 요로 기능을 연구하는 데 사용할 수 있습니다.

원추 수질 절개에 의한 쥐의 배뇨근 과소 모델

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

detrusor-underactivity-model-in-rats-by-conus-medullaris-transection

Research

JoVE Journal

Neuroscience

2.0K 조회수.  Capital Medical University. 우리는 쥐에서 원추 수질 절개에 의한 배뇨근 과소 활동 모델을 확립하는 방법을 제시합니다. 배뇨근 기능 저하는이 동물들에서 성공적으로 자극되었습니다. 이 모델은 요로 기능을 연구하는 데 사용할 수 있습니다.

비디오: Detrusor Underactivity Model in Rats by Conus Medullaris Transection

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

시신경의 절개 : 중추 신경계에서 신경 세포의 Apoptosis 성인의 모델

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

연구

신경과학

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

포유류의 CNS에서 시신경의 절개 후 실험에 대한 노는 방법

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

마우스 망막 신경절 세포 Transfection로 생체내에 Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

쥐에서 자당의 갈망의 보육을 평가하기위한 일반적인 방법

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

몸에 붙이기 형질 전환 쥐의 발생에 의해<em&gt; 자궁 내</em&gt; 일렉트로 및<em&gt; 생체 내</em&gt; 생물 발광 상영

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

모터 신경 절개와 라이브 제브라 피쉬의 아교 세포 행동의 시간 경과 영상

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

애벌레 Zebrafish의 척수 절개

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

신경 회로 토대 메모리 형성을 해부에 대한 신생아 흰쥐의 편측 냄새 학습 모델

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

쥐에 있는 부분 시 신경 Transection: 망막 신경 절 세포의 2 차 변성을 평가 하기 위해 새로운 수술 방식으로 설립 모델

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, and neuroinflammation is often observed as a co-occurrence. Here, we describe a sub-acute CMHs rat model induced by lipopolysaccharide (LPS) injection, as well as a method for detecting CMHs. Systemic LPS injection is relatively simple, economical, and cost-effective. One major advantage of LPS injection is its stability to induce inflammation. CMHs caused by LPS injection could be detected by gross observation, hematoxylin and eosin (HE) staining, Perl&#39;s Prussian staining, Evans blue (EB) double-labeling, and magnetic resonance imaging-susceptibility weighted imaging (MRI-SWI) technology. Finally, other methods of developing CMHs animal models, including their advantages and/or disadvantages, are also discussed in this report.

We present a protocol to induce and detect CMHs caused by LPS injection in Sprague-Dawley rats, which may be utilized in future research investigations on the pathogenesis of CMHs.

아 급성 대뇌 Microhemorrhages 쥐에서 Lipopolysaccharide 주입에 의해 유도 된

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, ...