فإن الكتاب أود أن أشكر ميشيل ديوتشاند لاتقان هذه التقنية الاجتثاث العين، تايلور بيركولز للمساعدة في فحص وظيفي، مايكل ليفين عن الأجسام المضادة لمكافحة arrestin، وجونجي Morokuma للحصول على معلومات على لوحات بلتيير. وأيد هذا العمل من منحة SFSA من جامعة ميشيغان الغربية إلى WSB.

<ol>
	<li>Gentile, L., Cebria, F., Bartscherer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+planarian+flatworm:+an+in+vivo+model+for+stem+cell+biology+and+nervous+system+regeneration.">The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration.</a> Dis Model Mech. 4 (1), 12-19 (2011).</li><li>Elliott, S. A., Sanchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+and+enduring+contributions+of+planarians+to+the+study+of+animal+regeneration.">The history and enduring contributions of planarians to the study of animal regeneration.</a> Wiley Interdiscip Rev Dev Biol. 2 (3), 301-326 (2013).</li><li>Emili Sal&#243;, R. B., Tsonis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3.">Chapter 3.</a> Animal Models in Eye Research. , 15-26 (2008).</li><li>Lapan, S. W., Reddien, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dlx+and+sp6-9+Control+optic+cup+regeneration+in+a+prototypic+eye.">dlx and sp6-9 Control optic cup regeneration in a prototypic eye.</a> PLoS Genet. 7 (8), e1002226 (2011).</li><li>Lapan, S. W., Reddien, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+of+the+planarian+eye+identifies+ovo+as+a+specific+regulator+of+eye+regeneration.">Transcriptome analysis of the planarian eye identifies ovo as a specific regulator of eye regeneration.</a> Cell Rep. 2 (2), 294-307 (2012).</li><li>Inoue, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+functional+recovery+of+the+planarian+photosensing+system+during+head+regeneration.">Morphological and functional recovery of the planarian photosensing system during head regeneration.</a> Zoolog Sci. 21 (3), 275-283 (2004).</li><li>Pineda, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+network+of+prototypic+planarian+eye+regeneration+is+Pax6+independent.">The genetic network of prototypic planarian eye regeneration is Pax6 independent.</a> Development. 129 (6), 1423-1434 (2002).</li><li>Paskin, T. R., Jellies, J., Bacher, J., Beane, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarian+Phototactic+Assay+Reveals+Differential+Behavioral+Responses+Based+on+Wavelength.">Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength.</a> PLoS One. 9 (12), e114708 (2014).</li><li>Raffa, R. B., Martley, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+increase+in+planarian+locomotor+activity+and+block+by+UV+light.">Amphetamine-induced increase in planarian locomotor activity and block by UV light.</a> Brain Res. 1031 (1), 138-140 (2005).</li><li>Sandmann, T., Vogg, M. C., Owlarn, S., Boutros, M., Bartscherer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+head-regeneration+transcriptome+of+the+planarian+Schmidtea+mediterranea.">The head-regeneration transcriptome of the planarian Schmidtea mediterranea.</a> Genome Biol. 12 (8), R76 (2011).</li><li>Vasquez-Doorman, C., Petersen, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NuRD+complex+component+p66+suppresses+photoreceptor+neuron+regeneration+in+planarians.">The NuRD complex component p66 suppresses photoreceptor neuron regeneration in planarians.</a> Regeneration (Oxf). 3 (3), 168-178 (2016).</li><li>Deochand, M. E., Birkholz, T. R., Beane, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+regulation+of+planarian+eye+regeneration.">Temporal regulation of planarian eye regeneration.</a> Regeneration. 3 (4), 209-221 (2016).</li><li>Sakai, F., Agata, K., Orii, H., Watanabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+regeneration+ability+of+spontaneous+supernumerary+eyes+in+planarians+-eye+regeneration+field+and+pathway+selection+by+optic+nerves.">Organization and regeneration ability of spontaneous supernumerary eyes in planarians -eye regeneration field and pathway selection by optic nerves.</a> Zoolog Sci. 17 (3), 375-381 (2000).</li><li>Asano, Y., Nakamura, S., Ishida, S., Azuma, K., Shinozawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin-like+proteins+in+planarian+eye+and+auricle:+detection+and+functional+analysis.">Rhodopsin-like proteins in planarian eye and auricle: detection and functional analysis.</a> J Exp Biol. 201 (Pt 9), 1263-1271 (1998).</li><li>Cross, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Maintenance+and+Regeneration+of+Planarian+Eyes+by+ovo.">Control of Maintenance and Regeneration of Planarian Eyes by ovo.</a> Invest Ophthalmol Vis Sci. 56 (12), 7604-7610 (2015).</li><li>Robb, S. M., Gotting, K., Ross, E., Sanchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SmedGD+2.0:+The+Schmidtea+mediterranea+genome+database.">SmedGD 2.0: The Schmidtea mediterranea genome database.</a> Genesis. 53 (8), 535-546 (2015).</li><li>Robb, S. M., Ross, E., Sanchez Alvarado, ., A, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SmedGD:+the+Schmidtea+mediterranea+genome+database.">SmedGD: the Schmidtea mediterranea genome database.</a> Nucleic Acids Res. 36, D599-D606 (2008).</li><li>Beane, W. S., Tseng, A. S., Morokuma, J., Lemire, J. M., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+planar+cell+polarity+extends+neural+growth+during+regeneration,+homeostasis,+and+development.">Inhibition of planar cell polarity extends neural growth during regeneration, homeostasis, and development.</a> Stem Cells Dev. 21 (12), 2085-2094 (2012).</li><li>Forsthoefel, D. J., Waters, F. A., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cell+type-specific+monoclonal+antibodies+for+the+planarian+and+optimization+of+sample+processing+for+immunolabeling.">Generation of cell type-specific monoclonal antibodies for the planarian and optimization of sample processing for immunolabeling.</a> BMC Dev Biol. 14, 45 (2014).</li><li>Ross, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+monoclonal+antibodies+to+study+tissue+regeneration+in+planarians.">Novel monoclonal antibodies to study tissue regeneration in planarians.</a> BMC Dev Biol. 15, 2 (2015).</li><li>Cardona, A., Fernandez, J., Solana, J., Romero, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+situ+hybridization+protocol+for+planarian+embryos:+monitoring+myosin+heavy+chain+gene+expression.">An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression.</a> Dev Genes Evol. 215 (9), 482-488 (2005).</li><li>King, R. S., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+protocol+for+enhanced+detection+of+gene+expression+in+the+planarian+Schmidtea+mediterranea.">In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea.</a> BMC Dev Biol. 13, 8 (2013).</li><li>Pearson, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde-based+whole-mount+in+situ+hybridization+method+for+planarians.">Formaldehyde-based whole-mount in situ hybridization method for planarians.</a> Dev Dyn. 238 (2), 443-450 (2009).</li></ol>

الكتاب ليس لديهم ما يكشف.

تحسن هذه التقنية الاجتثاث العين على الطرق الحالية (مثل ثقب) باستبعاد أنسجة المخ وتقييد الختان أساسا إلى أنسجة العين. مع الممارسة، وهذه التقنية يمكن أن يقوم بها معظم الأفراد، من الفنيين ذوي الخبرة في microsurgeries للطلاب الجامعيين عديم الخبرة ولكن الضميري. فمن المستحسن أن ?...

مستورقات هي قوية نموذج حي لدراسة الجذعية البالغة تجديد بوساطة الخلايا. هذه الديدان المفلطحة المياه العذبة غير الطفيلية تمتلك القدرة على التجدد أي وجميع الأنسجة المفقودة، بما في ذلك نظامهم العصبي المركزي والدماغ 1. درس بقدر ما يعود إلى 1700s في 2، والتقدم التكنولوجي في مجال مستورقة خلال 10-15 سنة الماضية (مثل تسلسل الجينوم، في الموقع التهجين، المناعية، والتدخل RNA (رني)، وtranscriptomics) قمنا بتحديث هذا نموذج حي تاريخي . على وجه التحديد، اكتسبت مستورقات شعبية في الآونة الأخيرة لتكون نموذجا الناشئة للبحوث العين 3. مستورقات لها عيون prototypic مع أنواع الأنسجة اثنين فقط، والخلايا العصبية مستقبلة للضوء والخلايا الصبغية. وقد مكن هذا التوصيف من سكان العين الخلايا الجذعية، وأظهرت أن العديد من نفس الجينات التي تنظم الفقاريات العين دييتم حفظها velopment في مستورقات 4 و 5. تقع الكؤوس البصرية ظهريا وتتكون من البيض، التشعبات unpigmented من الخلايا العصبية مستقبلة للضوء وشبه القمر الخلايا الصبغية الأسود، وعيون عصب الدماغ عن طريق chiasm البصرية. بالإضافة إلى كونه نموذجا لتوضيح عمليات التجدد 6، والعين مستورقة هي مناسبة تماما لدراسة تطور آليات البصرية 7، والاستجابات السلوكية للضوء (مستورقات عرض انجذاب ضوئي سلبي) 8، والأساس العصبي للسلوك 9. تجديد العين في مستورقات وقد درست إلى حد كبير في سياقين رئيسيين: كجزء من تجديد رئيس يلي قطع الرأس 4، 10، وفيما يلي استئصال فقط أنسجة العين 11 و 12 </سوب&gt;. وقد استخدمت معظم الدراسات مستورقة على تجديد العين طريقة قطع الرأس، كما هو بسيط ومباشر. وكان مستورقة طريقة الختان العين الأكثر شيوعا حتى الآن عبر ثقب مع كوب غرامة الشعرية أنبوب 13 و 14، على الرغم من أن بعض الدراسات قد أجريت أيضا عمليات البتر خلف العينين (قطع الرأس الجزئي) 15. ومع ذلك، كل من هذه الأساليب تنطوي على فقدان العديد من الأنسجة الأخرى من مجرد العين (مثل الدماغ والأمعاء وnephridia)، ويحتمل أن تعقيد تفسير النتائج. بروتوكول الاجتثاث العين المعروضة هنا يقيد الختان إلى أنسجة العين (باستثناء المخ على وجه التحديد)، مما أدى إلى البيانات التي هي أكثر تحديدا للعين. بالإضافة إلى ذلك، على عكس الديدان مقطوعة الرأس التي تأخذ 7-14 أيام للبدء في التغذية، والعين ذاب الديدان تتغذى خلال 24 ساعة من الاجتثاث 12، مما يسمح للتجارب رني (حيث يتم تسليم رني عبر الطعام) ليتم performeد متزامن. وعلى الرغم من الاجتثاث العين هو أصعب من الناحية الفنية لأداء بنجاح من قطع الرأس، لم شملت الدراسات الحالية التي تنطوي على استئصال العين تعليمات مفصلة حول إجراءاتها. والهدف من هذه المقالة الفيديو هو لتمكين الباحثين من إزالة باستمرار الكأس البصرية مستورقة دون إزعاج أنسجة المخ الأساسية وإزالة بعض الأنسجة الأخرى كما ممكن. وهذه الطريقة يمكن أن تستخدم في كل من مفردة ومزدوجة الاجتثاث العين وينطبق على مجموعة واسعة من التحقيقات. مثل معظم فحوصات تجديد، والاجتثاث العين مناسبة تماما للمزيج مع كل من (رني) شاشات الدوائية والوراثية، وكذلك الدراسات السلوكية. نحن هنا وصف الطرق لمعالجة الديدان، والحفاظ على مستعمرة مستورقة، وتقنية الاجتثاث العين نفسها.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Instant Ocean sea salts</td>
			<td>Spectrum Brands&#160;</td>
			<td>SS15-10</td>
			<td>&#34;10 Gallon&#34; box&#160; (net weight 3 lbs)</td>
		</tr>
		<tr>
			<td>Kimwipes EX-L lint-free tissue wipe</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td>4.5 x 8.5 in&#160;</td>
		</tr>
		<tr>
			<td>Whatman #2 filter paper</td>
			<td>Sigma&#160;</td>
			<td>WHA1002125</td>
			<td>Circles, 125 mm diameter, white</td>
		</tr>
		<tr>
			<td>Easy Touch Insulin syringe (with needle)</td>
			<td>Pet Health Market</td>
			<td>17175-04</td>
			<td>&#160;U-100 1 cc syringe, 31-gauge 5/16 in needle</td>
		</tr>
		<tr>
			<td>100 mm Petri dish</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td>100 x 15 mm</td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>VWR</td>
			<td>25384-092</td>
			<td>60 x 15 mm</td>
		</tr>
		<tr>
			<td>Dumont #5&#160; forceps&#160;</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>Inox, straight tip , 11 cm</td>
		</tr>
		<tr>
			<td>Transfer pipettes&#160;</td>
			<td>Samco Scientific&#160;</td>
			<td>225</td>
			<td>Graduated, large bulb, 7.5 mL, non sterile</td>
		</tr>
		<tr>
			<td>Parafilm M paraffin film</td>
			<td>Brand&#160;</td>
			<td>701606</td>
			<td>&#160;4 in x 125 ft roll</td>
		</tr>
		<tr>
			<td>12-well untreated tissue culture plate</td>
			<td>VWR</td>
			<td>15705-059</td>
			<td>Untreated, flat bottom, sterile, Falcon brand</td>
		</tr>
		<tr>
			<td>Plastic food containers (for colony)&#160;</td>
			<td>Ziploc</td>
			<td>Large rectangle</td>
			<td>2.25 qt (2.12 L), 10&#34; x 6 -3/4 &#34; x 3 -3/16&#34;&#160;</td>
		</tr>
		<tr>
			<td>Planaria (Girardia tigrina)</td>
			<td>Carolina Biological</td>
			<td>132954</td>
			<td>Sold as &#34;Brown&#34; Planaria; most often they are G. tigrina (aka Dugesia tigrina), but sometimes are G. dorotocephala (aka Dugesia dorotocephala); either will work.</td>
		</tr>
		<tr>
			<td>Planaria (Schmidtea mediterranea)</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>S. mediterranea are not commercially available. At this time animals are only obtainable from laboratories that use them and have extra animals.</td>
		</tr>
		<tr>
			<td>Brown paper towels&#160;</td>
			<td>Grainger</td>
			<td>2U229</td>
			<td>9-3/16 x 9-3/8&#34; 1-Ply Multifold Paper Towel, UNBLEACHED</td>
		</tr>
		<tr>
			<td>Wash bottle (for worm water), optional</td>
			<td>VWR</td>
			<td>16650-275</td>
			<td>Wash Bottles, Low-Density Polyethylene, Wide Mouth, 500 mL</td>
		</tr>
		<tr>
			<td>Anti-synapsin antibody, optional</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>3C11</td>
			<td>Supernatant</td>
		</tr>
		<tr>
			<td>Anti-arrestin antibody, optional</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Not commercially available. Kind gift from Michael Levin, Tufts University</td>
		</tr>
		<tr>
			<td>Nalgene Lowboy carboy with spigot (for storing worm water), optional</td>
			<td>Nalge Nunc International Corporation</td>
			<td>2324-0015</td>
			<td>15 L,&#160; polypropylene, low profile makes it easier to fill plastic colony containers&#160;</td>
		</tr>
		<tr>
			<td>Custom Peltier plate, optional</td>
			<td>Williams Machine, Foxboro, MA&#160;</td>
			<td>n/a</td>
			<td>Design specifics courtesy of Junji Morokuma, Tufts University:&#160; Peltier plate is constructed of a standard thermoelectric heat pump (for example, All Electronics Corp Catalog # PJT-1, 30 mm2).&#160; The square heat pump is covered with a thin mirrored surface, then placed inside a 30 mm2 square hole in a circular plexiglass form (~50 mm in diameter). This form is of similar thickness to the heat pump, and fits flush into a well tooled in the center of a round heat sink (~115 mm in diameter). The form/heat pump is&#160; &#34;anchored&#34; to the sink with silicone base heat sink compound. The leads are threaded through holes drilled through both the form and the the heat sink. The bottom half of the heat sink is tooled into a &#34;foot&#34; that fits into the opening of your microscope&#39;s base plate.&#160;</td>
		</tr>
		<tr>
			<td>DC power source (for Peltier plate), optional</td>
			<td>B &#38; K Precision</td>
			<td>1665</td>
			<td>Regulated Low Voltage DC Power Supply, 1-18 volts (DC), 1-10 amps.</td>
		</tr>
		<tr>
			<td>Other common supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scope with gooseneck lighting</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chopstick rests, optional</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical ablation assay for studying eye regeneration in planarians

في دراسة الخلايا الجذعية للبالغين وآليات التجدد، الديدان المفلطحة مستورقة هي من دعائم النظام في نموذج الجسم الحي. ومن المقرر في جزء كبير منه إلى وفرة المحفزة السكان الخلايا الجذعية وقدرتها على تجديد جميع أنواع الخلايا والأنسجة بعد الإصابات التي سيكون كارثيا بالنسبة لمعظم الحيوانات هذا. في الآونة الأخيرة، اكتسبت شعبية مستورقات كنموذج للتجديد العين. قدرتها على التجدد العين بأكملها (تتألف من أنواع الأنسجة هما: الخلايا الصبغية ومبصرات) تسمح للتشريح الآليات التي تنظم تجديد النظام البصري. العين الاجتثاث لديها العديد من المزايا أكثر من غيرها من التقنيات (مثل قطع الرأس أو ثقب) لدراسة سبل وآليات العين محددة، وأهمها هو أن يقتصر التجديد إلى حد كبير إلى أنسجة العين وحدها. والغرض من هذه المقالة الفيديو هو إظهار كيفية إزالة موثوق الكأس البصرية مستورقة دون إزعاج الدماغ أو الأنسجة المحيطة بها.يوصف أيضا التعامل مع الديدان وصيانة مستعمرة أنشئت. ويستخدم هذا الأسلوب 31 G، 5/16 بوصة الانسولين إبرة لحلج القطن جراحيا من الكأس البصري من مستورقات يجمد على طبق من ذهب الباردة. ويشمل هذا الأسلوب على حد سواء مفردة ومزدوجة الاجتثاث العين، مع عيون تجديد في غضون 1-2 أسابيع، والسماح لمجموعة واسعة من التطبيقات. على وجه الخصوص، هذه التقنية الاجتثاث يمكن الجمع بسهولة مع (التدخل RNA) شاشات الدوائية والجينية لفهم أفضل للآليات التجدد وتطورها، والخلايا الجذعية العين وصيانتها، والاستجابات السلوكية phototaxic وأساسها العصبية.

لأول 1-2 ساعة بعد الجراحة، قد يحمل الحيوانات انخفضت حركة بالمقارنة مع الديدان سليمة (ومع ذلك فإنها لا تزال تتحرك). إذا رغبت في ذلك، والديدان تأكل خلال 24 ساعة من الجراحة (على سبيل المثال، لتغذية رني). عند اتباع تجديد العين في نفس الأفراد مع مرور الوقت، ت?...

Watch this Scientific Journal Video about Surgical Ablation Assay for Studying Eye Regeneration in Planarians at JoVE.com

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

يظهر هذا البروتوكول كيفية استئصال باستمرار عيون مستورقة (أكواب البصرية) من دون إزعاج الأنسجة المحيطة بها. باستخدام إبرة الأنسولين والمحاقن، إما احدة أو كلتا العينين يمكن أن يكون ذاب لتسهيل التحقيقات في آليات تنظيم التجديد العين، وتطور تجديد البصري، والأساس العصبي للسلوك الناجم عن الضوء.

الجراحية تذرية الفحص لدراسة التجديد العين في مستورقات

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

surgical-ablation-assay-for-studying-eye-regeneration-in-planarians

Research

JoVE Journal

Developmental Biology

10.9K Views.  Western Michigan University.  يظهر هذا البروتوكول كيفية استئصال باستمرار عيون مستورقة (أكواب البصرية) من دون إزعاج الأنسجة المحيطة بها. باستخدام إبرة الأنسولين والمحاقن، إما احدة أو كلتا العينين يمكن أن يكون ذاب لتسهيل التحقيقات في آليات تنظيم التجديد العين، وتطور تج?...

Video: Surgical Ablation Assay for Studying Eye Regeneration in Planarians

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

تذرية الكبدية محددة في الزرد لدراسة يحركها صفراوية تجديد الكبد

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

عزل ماوس بطانة الرحم طلائي واللحمية خلايا ل<em&gt; في المختبر</em&gt; Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

دقة الخلوية طريقة الاجتثاث لنمذجة إصابة الكلى الحادة في تطوير الزرد

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

ظهرت مفصص الجزئي: نموذج جراحية لتجديد الكبد مورفولوجك

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

أساليب لدراسة التجديد في ستنتور

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

أسلوب مبسط وفعال لعزل الخلايا الكيراتينيه البشرية الأولية من أنسجة الجلد الكبار

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

فحص قائم علي بروتين الفلورية الخضراء المعززة لدراسة نمو العصب في الخلايا العصبية الاوليه

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

الاستئصال الليزري القائم على المجهر والتجدد في خلايا الحمار الوحشي Interneuromast

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue's (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland's stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells' phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary gland is the key regulator of the body's endocrine system. This article describes the development of organoids from the mouse pituitary as a novel 3D in vitro model to study the gland's stem cell population of which the biology and function remain poorly understood.

تطوير المواد العضوية من الغدة النخامية الفأر كما في المختبر نموذج لاستكشاف بيولوجيا الخلايا الجذعية النخامية

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

إزالة العين في يرقات الزرد الحية لفحص النمو والتطور المعتمد على التعصيب للنظام البصري

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...