우리의 연구는 NIH, 국립 안과 연구소의 교내 연구 프로그램에 의해 지원됩니다.

사용된 모든 장비와 시약이 멸균됩니다. 모든 동물 실험은 동물 케어 승인 및 (동물 연구 프로토콜 네이 - 570) 네이 / NIH에서위원회 (ACUC)를 사용하고, NIH의 지침과 규정에 따라 수행 하였다. 1. 1 일에서 망막 신경절 세포의 퇴행성 기준 라벨링 (RGCs) 이 절차의 목적은 삼일 시신경의 호감 부상하기 전에 생쥐의 우수한 colliculus에 신경 추적 염료를 주입하여 망막 신경절 retrogradely 세포 레이블을하는 것입니다. 염료 retrogradely 망막 신경절 세포에 의해 촬영과 생활 RGCs (그림 1)에 대한 마커를 제공하는 것입니다. 이 방법은 약간의 변화 1-5로 가능한 RGCs의 재현성 라벨을 산출. <ol><li> 깊이 마우스를 마취. 머리 위에 머리를 청소합니다. 작은 stereotaxic 장치에서 마우스를 놓습니다. 보호 oinment는 두 눈을에 적용됩니다. 머리 피부를 소독 세 번 iodophor과 70 % 알코올 스크럽을 사용하여 각. </li><li> 두개골을 노출하기 위해 피부에 절개를하고, 3 % 과산화수소를 사용하여 그것이 건조하고 깨끗하게 유지. </li><li> 식별 및 bregma을 표시합니다. bregma 뒤에 2.9 mm 각 반구 7 정중선을 가로 0.5 mm에서 우수한 colliculus 위에 구멍을 드릴. 드릴링하는 동안 구멍이 보조 열 부상을 방지하기 곤란있는 사이트로 살린 적용됩니다. </li><li> stereotaxic 측정 장치와 해밀턴 주사기를 사용하여 두뇌의 골질 표면으로부터 1.6 mm의 깊이 각 반구의 우수한 colliculus로 매우 느리게 FluoroGold 신경 추적 염료 (3 % 1 μl 호수에서 Fluorochrome)을 주입. </li><li> 절개 사이트를 봉합해. 진통제에 대한 buprenorphine의 피하 주사를 제공합니다. </li><li> 따뜻하고 건조한 지역에 마우스를 이동하고 똑바로 자세를 유지하며, 홈 케이지로 돌아갈 수 때까지 그것을 모니터합니다. 라벨 절차 후 3 일 동안, 체계 진통제 (예, buprenorphine) 및 국소 항생 연고는 회 부여되고 마우스가 밀접하게 모니터합니다. </li></ol> 2. 일 4 시신경의 호감 상해 이 절차에서는, 우리는 망막 신경절 세포의 점진적인 죽음에 이르게됩니다 axons에 대한 기본 손상 (그림 2)을 일으킬 수있는 시신경에 호감 부상을 적용합니다. <ol><li> 추적 염료 신청 후 3 일, 마우스 깊이 anesthetized입니다. 해부 현미경을 사용 한 눈 결막은 봄 가위를 사용하여 4시 입장에 대한에 베인 수 있습니다. </li><li> 부드럽게 궤도 근육 비켜 그들을 제쳐두기. 눈 세계로부터 출구에서 흰색 시신경을 폭로. 모든 혈관을 손상하지 않도록 아주 조심 해요. </li><li> 크로스 액션 포셉 한 켤레의 도움으로 약 3 초 동안 안구에서 약 2 mm에서 시신경에 호감 부상을 적용합니다. 출혈을 일으킬 수있는 안과 동맥을 손상하지 않도록 아주 조심 해요. </li><li> 호감 부상의 완료 후, 절개는 봉합합니다. </li><li> 마우스가 마취에서 회복하기 전에 진통제에 대한 buprenorphine의 피하 주사를 제공합니다. </li><li> 그것이 똑바로 자세를 유지하고 홈 케이지로 돌아갈 수 때까지 따뜻하고 건조한 지역에 마우스를 이동하고 모니터링합니다. 호감 부상 절차 후 3 일 동안, 체계 진통제 (예, buprenorphine) 및 국소 항생 연고는 회 부여되고 마우스가 밀접하게 모니터합니다. </li></ol> 3. Retinae를 수확하고 일 11시 RGC 서바이벌 분석 이 절차의 목적은 retinaeto가 망막 신경절 세포의 생존을 분석 수확하는 것입니다. <ol><li> 11 (ONC 매일 7) 하루, 마우스는 CO 2 주입 및 경추 전위에 의해 euthanized있다. </li><li> 눈은 궤도에 압력을 적용하여 포셉 한 켤레의 도움으로 enucleated 있습니다. </li><li> 눈은 두 시간 동안 4 % paraformaldehyde 용액에 고정하고 PBS로 세 번 씻어 있습니다. 망막을 해부하다. 망막의 해부의 과정은 이미 6. Gustmann, S. 외 의해 2010 년 정돈 이전에 출판되어 있으며, 따라서 여기서 설명되지 않습니다. </li><li> 살아 망막 신경절 세포의 이미지는 망막의 정의 지역에서 형광 현미경을 사용하여 촬영하고, 망막 신경절 세포 밀도가 계산하실 수 있습니다. 시신경의 호감 부상 여부에 상관없이 형광 - 표시 망막 신경절 세포의 대표적인 결과는 그림 3에 표시됩니다. </li></ol> 4. 대표 결과 : 망막 신경절 세포의 생존, 시신경의 호감 부상 후 7 일 분석하려면 retinae는 고정, 수확 평평하고 장착했습니다. 망막 이미지는 형광 현미경을 사용하여 촬영되었다. 손상되지 않은 정상적인 R과 비교etina은 (일반, 왼쪽), 실용적 RGCs의 번호는 (retrogradely 우수한 colliculus로 주입 FluoroGold 신경 추적 염료로 표시된 녹색) 시신경의 호감 부상 (오른쪽, ONC) (그림 3)과 retinae 크게 낮은 . <img src="/files/ftp_upload/2685/2685fig1.jpg" alt="그림 1" /> 그림 1. 망막 신경절 세포의베이스 라인 후퇴 라벨링 시신경의 호감 부상 후 다른 시간 지점에서 가능한 망막 신경절 세포를 계산하기 위해 망막 신경절 세포는 (시신경 전에 뇌 사흘 만에 우수한 colliculus에 신경 추적 염료 (녹색 색상)을 주사하여 retrogradely 레이블 아르 적색) 호감 부상. 망막 신경절 세포의 axons이 우수한 colliculus에 거주하고 있으므로, 추적 염료 retrogradely RGCs 의해 촬영과 생활 RGCs에 대한 마커를 제공하는 것입니다. <img src="/files/ftp_upload/2685/2685fig2.jpg" alt="그림 2" /> 그림 2. 시신경의 호감 부상 망막 신경절 세포의 생존의 상태를 분석하기 위해서는, 호감 부상은 axons에 기본 피해를 입을 수있는 시신경 (적색)에 적용됩니다. 이것은 망막 신경절 세포의 점진적인 죽음을 초래합니다. 추적기 색소 (녹색) 분사 후 3 일, 마우스 anesthetized입니다. 눈 하나의 결막이 베인입니다. 궤도 근육 비켜 그들을 제쳐두기. 눈 세계로부터 출구에서 시신경을 쉽게받을 수 있습니다. 크로스 액션 포셉 한 쌍의를 사용하여 약 3 초 동안 안구에서 약 2 mm에서 시신경에 호감 부상을 적용합니다. <img src="/files/ftp_upload/2685/2685fig3.jpg" alt="그림 3" /> 그림 3. 형광 - 표시 시신경의 호감 부상 여부와 상관없이 망막 신경절 세포 망막 신경절 세포의 생존, 시신경의 호감 부상 후 7 일 분석하려면 retinae는 고정, 수확 평평하고 장착했습니다. 망막 이미지는 형광 현미경을 사용하여 촬영되었다. 손상되지 않은 정상적인 망막 (일반, 왼쪽)와 비교할 때, 가능한 RGCs의 숫자 (녹색, retrogradely 우수한 colliculus로 주입 FluoroGold 신경 추적 염료로 표시)은 시신경의 호감 부상 (오른쪽, ONC와 retinae 크게 낮은 ) (그림 3). 스케일 바 : 50 μm의

<ol>
	<li>Yoles, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM1+reduces+injury-induced+metabolic+deficits+and+degeneration+in+the+rat+optic+nerve.">GM1 reduces injury-induced metabolic deficits and degeneration in the rat optic nerve.</a> Investigative ophthalmology &#38; visual science. 33, 3586-3591 (1992).</li><li>Fisher, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+for+neuroprotection+in+the+mouse+optic+nerve:+implications+for+optic+neuropathies.">Vaccination for neuroprotection in the mouse optic nerve: implications for optic neuropathies.</a> J Neurosci. 21, 136-142 (2001).</li><li>Levkovitch-Verbin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGC+death+in+mice+after+optic+nerve+crush+injury:+oxidative+stress+and+neuroprotection.">RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection.</a> Investigative ophthalmology &#38; visual science. 41, 4169-4174 (2000).</li><li>Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-B+inhibits+apoptosis+via+VEGFR-1-mediated+suppression+of+the+expression+of+BH3-only+protein+genes+in+mice+and+rats.">VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats.</a> The Journal of clinical investigation. 118, 913-923 (2008).</li><li>Tang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+effect+of+PDGF-CC+rescues+neurons+from+apoptosis+in+both+brain+and+retina+by+regulating+GSK3beta+phosphorylation.">Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3beta phosphorylation.</a> The Journal of experimental medicine. 207, 867-880 (2010).</li><li>Gustmann, S., Dunker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo-like+organotypic+murine+retinal+wholemount+culture.">In vivo-like organotypic murine retinal wholemount culture.</a> J Vis Exp. , (2010).</li><li>Franklin, K., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li></ol>

관심 없음 충돌 선언하지 않습니다.

시신경의 호감 부상 murine 모델 RGC 죽음과 생존의 과정을 연구하는 데 유용합니다. 이 모델은 또한 종종 RGC apoptosis과 생존에 다른 시약과 유전자의 효과를 조사하는 데 사용됩니다. 이 모델의 장점은 최소한의 변화와 재현성의 높은 수준을 가지고있다는 사실이다. 그러나, 특별한주의가이 모델의 몇 가지 단계가 필요합니다. 첫째, 시신경의 호감 부상을 만들 때, 그것은 그들이 안과 동맥 따라서 이후 망막 국소 빈혈에 손상을 초래할 수 있기 때문에, 너무 무력을 사용하지 않으며 너무 오랫동안 호감하지 필수적입니다. 또한 시신경을 폭로하려고 할 때, 하나는 마우스 눈 주위의 다른 혈관을 손상하지 않도록 큰주의를 기울여야한다.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stereotaxic apparatus</td>
<td>&nbsp;</td>
<td>ASI Instruments</td>
<td>SAS-4100</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>PZMIV-BS </td>
<td>&nbsp;</td>
<tr>
<td>Scalpel handle</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>10003-12 </td>
<td>&nbsp;</td>
<tr>
<td>Scalpel Blades #15</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>10015-00</td>
<td>&nbsp;</td>
<tr>
<td>Microdrill</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>18000-17</td>
<td>&nbsp;</td>
<tr>
<td>Suture needle</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>12050-03</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>16144-13</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>11252-50</td>
<td>&nbsp;</td>
<tr>
<td>Hamilton syringe</td>
<td>&nbsp;</td>
<td>Hamilton Company </td>
<td>88400</td>
<td>&nbsp;</td>
<tr>
<td>Gauze sponges</td>
<td>&nbsp;</td>
<td>Office Depot</td>
<td>674889</td>
<td>&nbsp;</td>
<tr>
<td>Fluorochrome</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>39286</td>
<td>&nbsp;</td>
<tr>
<td>Alcohol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>459844</td>
<td>70%</td>
</tr>
<tr>
<td>H2O2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3410</td>
<td>3%</td>
</tr>		</tbody>
		</table>
		</div>

an optic nerve crush injury murine model to study retinal ganglion cell survival

시신경 손상은 돌이킬 수없는 시력 상실 결과 망막 신경절 세포 (RGCs)의 점진적 죽음 뒤에, axonal 퇴보를 가져올 수 있습니다. 인간의 이러한 질병의 예는 외상 시신경 신경 장해 및 녹내장에서 시신경의 퇴행을 포함합니다. 이것은 시력 손실과 실명으로 이어지는 시신경의 머리에 전형적인 변화, 진보적인 시신경의 퇴행 및 망막 신경절 세포의 손실, 통제하는 경우,이 특징입니다. 시신경의 호감 (ONC) 부상 마우스 모델은 외상성 시신경 신경 장해에 대한 중요한 실험 질병 모델,이 모델에서는 녹내장 등 시신경 점차 망막 신경절 세포의 apoptosis에 이르게하는 호감의 부상이다. 이 질병 모델의 연결을 죽음과 치료 대책의 발전을 위해 필수적이며, 생존의 일반적인 프로세스와 메커니즘을 연구하는 데 사용할 수 있습니다. 또한, 약리 및 분자 방법은 광학 신경 장해 다양한 유형의 치료에 잠재적인 치료 시약을 파악하고 테스트하는이 모델에 사용할 수 있습니다. 여기, 우리는 일 1 망막 신경절 세포의베이스 라인 역행 라벨 (RGCs), 하루 4 (II) 광학 신경 호감 부상, (III) retinae를 수확하고 하루 RGC 생존 분석 (I)의 단계 시범에 의해 단계를 제공합니다 11과 (IV) 대표 결과.

Watch this Scientific Journal Video about An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival at JoVE.com

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

이 프로토콜은 망막 신경절 retrogradely 세포, 및 이후 망막 신경절 세포 생존과 apoptosis를 분석하기 위해서는 시신경의 호감 부상을하기 위해 레이블을하는 방법을 보여줍니다. 그것은 녹내장을 포함하여 시신경 신경 장해의 종류에 대한 실험 질병 모델입니다.

망막 신경절 세포의 생존을 공부 시신경의 호감 상해 Murine 모델

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

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JoVE Journal

Neuroscience

30.4K 조회수.  NIH. 이 프로토콜은 망막 신경절 retrogradely 세포, 및 이후 망막 신경절 세포 생존과 apoptosis를 분석하기 위해서는 시신경의 호감 부상을하기 위해 레이블을하는 방법을 보여줍니다. 그것은 녹내장을 포함하여 시신경 신경 장해의 종류에 대한 실험 질병 모델입니다.

비디오: An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

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

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

유전적으로 망막 신경절 라벨 셀에서 라이트 응답을 기록하기 위해 절연 망막 준비

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

쥐 시신경에 주입을위한 최소 침습 기술

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

실험 시신경의 탈수 초화의 쥐 모델에서 시각 유발 전위 기록

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

망막 신경절 세포의 정의 된 서브 세트의 단일 세포 RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

등쪽 뿌리 신경절 사출 및 지느러미 루트 감각 축삭의 재생을위한 모델로 부상 크러시

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

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 ...

Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen films. Favorable clinical outcomes have been achieved when using these tubes for the treatment of damaged inferior alveolar nerve (IAN). A critical factor for the successful nerve regeneration using PGA-C tubes is blood supply to the surrounding tissue. Cervical sympathetic ganglion block (CSGB) creates a sympathetic blockade in the head and neck region thus increasing blood flow in the area. To ensure an adequate effect, the blockade must be administered with local anesthetics one to two times a day for several consecutive weeks; this poses a challenge when creating animal models for investigating this technique. To address this limitation, we developed an ethanol-induced CSGB in a canine model of long-term increase in blood flow in the orofacial region. We examined whether IAN regeneration via PGA-C tube implantation can be enhanced by this model. Fourteen Beagles were each implanted with a PGA-C tube across a 10-mm gap in the left IAN. The IAN is located within the mandibular canal surrounded by bone, therefore we chose piezoelectric surgery, consisting of ultrasonic waves, for bone processing, in order to minimize the risk of nerve and vessel injury. A good surgical outcome was obtained with this approach. A week after surgery, seven of these dogs were subjected to left CSGB by injection of ethanol. Ethanol-induced CSGB resulted in improved nerve regeneration, suggesting that the increased blood flow effectively promotes nerve regeneration in IAN defects. This canine model can contribute to further research on the long-term effects of CSGB.

We evaluated the effect of cervical sympathetic ganglion block on nerve repair using artificial nerve conduits. Male beagle dogs were each implanted with an artificial nerve across a 10-mm gap in the left inferior alveolar nerve; left cervical sympathetic ganglion was blocked by injecting 99.5% ethanol via lateral thoracotomy.

에탄올을 이용한 자 궁 경부 교감 신경 절 블록 응용 프로그램 사용 하 여 인공 신경 개 열 등 한 치조 신경 재생 촉진을 위한

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

축축한 억제 및 재생을 연구하는 쥐 모델의 안면 신경 수술

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Sciatic 신경 호감 상해의 쥐 모형에 있는 기능 평가를 위한 3D 운동학 분석

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...