这项工作由奥地利研究促进机构 (FFG; 项目编号 850681) 资助, 应用科学大学上部奥地利基本供资倡议 (项目 GlucoSTAR) 和医学技术创新中心 (定时中心)那接受基本的资金由上部奥地利的省。

根据 2010/63/欧盟的指令, 在胚胎发育的前2/3 阶段, 未孵化的禽胚试验不需要道德委员会的许可。
1. 鸡蛋的贮藏和繁殖
<ol><li>从当地育种者那里获得受精卵 (材料表)。 注: 在使用实验前, 鸡蛋可以在湿润的大气中储存14摄氏度, 最多10天。</li>
	<li>孵化鸡蛋在38°c, 平均湿度为 40-60% 在10或11天的孵化器, 不断打开鸡蛋。</li>
</ol>2. 蛋的选择
<ol><li>孵化11天后, 检查卵子受精。使用烛光进行此步骤。将烛灯的边缘蘸上印泥, 并在鸡蛋的尖面上点燃蜡烛。</li>
	<li>要识别空气中的膀胱, 检查鸡蛋顶部的亮度差异。 注: 气囊呈圆形、更透明的区域, 而蛋白在卵 candled 时呈深色。</li>
	<li>在检测到气囊后, 用以前使用过的墨水按下灯对着鸡蛋进行标记。</li>
	<li>在这个步骤中排除非受精的卵子, 这并不表明蛋白和气囊之间的亮度有差别。</li>
</ol>3. 注射物质
<ol><li>通过在汉克平衡盐溶液 (HBSS) 缓冲液中稀释所需浓度的物质, 制备感兴趣的物质 (例如, 草药提取物或胰岛素)。例如, 在 HBSS 的一个商业上可以使用的胰岛素模拟的 3.3 U/毫升。</li>
	<li>对于所选化合物的应用, 请在气囊的标记区域小心地啄上一双尖头镊子。不要形成比注射器针直径大的洞。注入缓冲液 (300 µL), 包含感兴趣的物质通过注射器的啄区。 注: 使用无菌, 1 毫升单用注射器, 吸管提示, 和血管。给实验戴上手套和实验室大衣。</li>
	<li>为了确定某种物质的降血糖效果, 分别将卵放回孵化器中60、120和180分钟。为确定基础血糖水平, 包括至少 10-15 未治疗的控制卵子。</li>
</ol>4. 毒性试验
<ol><li>在选择的化合物24小时后, 平衡蛋壳膜与磷酸盐缓冲盐水 (PBS) 缓冲 (足以覆盖整个蛋壳膜) 至少三十年代。</li>
	<li>把多余的 PBS 缓冲液倒掉。</li>
	<li>用尖头的镊子小心地拉出蛋壳膜。</li>
	<li>检查血管的病变和确认活力 (例如, 运动) 的小鸡胚。 注意: 请参阅图 1 , 以比较在使用有毒草药提取物后的重要与非重要胚胎。</li>
</ol>5. 血糖值的测定
<ol><li>在各自的时间点, 小心地取出蛋壳上面的气囊和平衡的蛋壳膜与 PBS 缓冲 (足以覆盖整个蛋壳膜)。</li>
	<li>把多余的 PBS 缓冲液倒掉。</li>
	<li>用尖头的镊子小心地拉出蛋壳膜。</li>
	<li>用微剪刀切割和取出胚膜, 使其能很好地接触到合适的血管。切膜至少 2-3 厘米。不要在膜内切开任何大血管, 以避免胚胎不必要的失血。</li>
	<li>找到源自胚胎腹部的大血管。用闭合的微剪刀将这个容器从蛋白中提起, 并将其放在塑料 pH 条上。将 pH 条移动到容器下, 用微剪刀握住, 然后小心地拉回微剪刀。</li>
	<li>用吸管去除胚膜下的 1-2 毫升液体, 以避免下一步稀释血液。将容器和 pH 带用滤纸擦干, 在容器被切开之前。</li>
	<li>在一侧用微剪刀将血管仔细切开。不要把船完全切开。 注: 该船约1毫米厚。不要削减超过一半的船只, 以避免完全通过船只切割, 因为它可能会滑出的条, 没有血液收集是可能的。</li>
	<li>用吸管收集 pH 条上的漏血。用血糖仪测定葡萄糖水平需要十微升的血液。</li>
</ol>6. 统计评价
<ol><li>计算每个时间点的至少10个单独卵的平均值, 并将这些值规范化为控制卵子 (非或缓冲处理的卵子) 之一。</li>
	<li>随后, 确定减少的百分比。此外, 使用胰岛素作为一个积极的控制。 注: 重复每个实验至少三次。</li>
</ol>

<ol>
	<li>Defronzo, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Banting+Lecture.+From+the+triumvirate+to+the+ominous+octet:+a+new+paradigm+for+the+treatment+of+type+2+diabetes+mellitus.">Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus.</a> Diabetes. 58 (4), 773-795 (2009).</li><li>American_Diabetes_Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+classification+of+diabetes+mellitus.">Diagnosis and classification of diabetes mellitus.</a> Diabetes Care. 37, S81-S90 (2014).</li><li>Han Cho, N., Cavan, 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=.">.</a> IDF Diabetes Atlas. 7, (2015).</li><li>Bailey, C. 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=Individualized+glycaemic+targets+and+pharmacotherapy+in+type+2+diabetes.">Individualized glycaemic targets and pharmacotherapy in type 2 diabetes.</a> Diabetes &amp; vascular disease research. 10 (5), 397-409 (2013).</li><li>Maruthur, N. M., 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=Diabetes+Medications+as+Monotherapy+or+Metformin-Based+Combination+Therapy+for+Type+2+Diabetes:+A+Systematic+Review+and+Meta-analysis.">Diabetes Medications as Monotherapy or Metformin-Based Combination Therapy for Type 2 Diabetes: A Systematic Review and Meta-analysis.</a> Annals of internal medicine. 164 (11), 740-751 (2016).</li><li>Aleman-Gonzalez-Duhart, D., Tamay-Cach, F., Alvarez-Almazan, S., Mendieta-Wejebe, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Advances+in+the+Biochemical+and+Physiological+Aspects+of+the+Treatment+of+Type+2+Diabetes+Mellitus+with+Thiazolidinediones.">Current Advances in the Biochemical and Physiological Aspects of the Treatment of Type 2 Diabetes Mellitus with Thiazolidinediones.</a> PPAR research. , 7614270 (2016).</li><li>Cohen, F. J., Neslusan, C. A., Conklin, J. E., Song, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+antihyperglycemic+prescribing+trends+for+US+privately+insured+patients+with+type+2+diabetes.">Recent antihyperglycemic prescribing trends for US privately insured patients with type 2 diabetes.</a> Diabetes care. 26 (6), 1847-1851 (2003).</li><li>Desai, N. R., 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=Patterns+of+medication+initiation+in+newly+diagnosed+diabetes+mellitus:+quality+and+cost+implications.">Patterns of medication initiation in newly diagnosed diabetes mellitus: quality and cost implications.</a> The American journal of medicine. 125 (3), e301-e307 (2012).</li><li>Martel, 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=Anti-obesogenic+and+antidiabetic+effects+of+plants+and+mushrooms.">Anti-obesogenic and antidiabetic effects of plants and mushrooms.</a> Nature reviews. Endocrinology. , (2016).</li><li>Lanzerstorfer, P., 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=Identification+of+novel+insulin+mimetic+drugs+by+quantitative+total+internal+reflection+fluorescence+(TIRF)+microscopy.">Identification of novel insulin mimetic drugs by quantitative total internal reflection fluorescence (TIRF) microscopy.</a> British journal of pharmacology. 171 (23), 5237-5251 (2014).</li><li>Onur, S., St&#246;ckmann, H., Zenthoefer, M., Piker, L., D&#246;ring, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Plant+Extract+Collection+Kiel+in+Schleswig-Holstein+(PECKISH)+Is+an+Open+Access+Screening+Library.">The Plant Extract Collection Kiel in Schleswig-Holstein (PECKISH) Is an Open Access Screening Library.</a> Journal of Food Research. 2 (4), (2013).</li><li>Haselgrubler, R., 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=Gluc-HET,+a+complementary+chick+embryo+model+for+the+characterization+of+antidiabetic+compounds.">Gluc-HET, a complementary chick embryo model for the characterization of antidiabetic compounds.</a> PloS one. 12 (8), e0182788 (2017).</li><li>Stadlbauer, V., 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=Biomolecular+Characterization+of+Putative+Antidiabetic+Herbal+Extracts.">Biomolecular Characterization of Putative Antidiabetic Herbal Extracts.</a> PloS one. 11 (1), e0148109 (2016).</li><li>Greywe, 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=Applicability+and+robustness+of+the+hen's+egg+test+for+analysis+of+micronucleus+induction+(HET-MN):+results+from+an+inter-laboratory+trial.">Applicability and robustness of the hen's egg test for analysis of micronucleus induction (HET-MN): results from an inter-laboratory trial.</a> Mutation research. 747 (1), 118-134 (2012).</li><li>Manakova, E., Hubickova, L., Kostalova, J., Zemanova, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryotoxicity+of+mirtazapine:+a+study+using+Chick+Embryotoxicity+Screening+Test.">Embryotoxicity of mirtazapine: a study using Chick Embryotoxicity Screening Test.</a> Neuro endocrinology letters. 31, 8-10 (2010).</li><li>Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+chorioallantoic+membrane+as+a+useful+tool+to+study+angiogenesis.">Chick embryo chorioallantoic membrane as a useful tool to study angiogenesis.</a> International review of cell and molecular biology. 270, 181-224 (2008).</li><li>Yoshiyama, Y., Sugiyama, T., Kanke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+diabetes+model+in+chick+embryos+treated+with+streptozotocin.">Experimental diabetes model in chick embryos treated with streptozotocin.</a> Biological &amp; pharmaceutical bulletin. 28 (10), 1986-1988 (2005).</li><li>Wolf, T., Niehaus-Rolf, C., Luepke, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+new+methodological+aspects+of+the+hen's+egg+test+for+micronucleus+induction+(HET-MN).">Some new methodological aspects of the hen's egg test for micronucleus induction (HET-MN).</a> Mutation research. 514 (1-2), 59-76 (2002).</li></ol>

作者没有什么可透露的。

在工业12中, 该方法是一种广泛应用于动物试验的替代测试系统。此处描述的蛋系统表示的是一种修改后的多式凸轮检测方法。在其原始形式下, 对化合物的刺激性特性和配方进行了研究, 并对血管生成进行了分析14,15,16, 我们已经适应了测试方法。具有假定的胰岛素模拟特性的化合物12。根据 2010/63/欧盟的指令, 在胚胎发育的前2/3 阶段, 未经孵化的禽胚实验不需要道德委员会的许可, 因为这些实验不被认为是动物实验。最近的研究已经证明了蛋系统的适用性, 以描述在 GLUT4-translocation-based 主屏幕10中确定的草药提取物的功效, 以降低血糖水平缺少胰岛素12,13。对蛋系统的良好性能的关键点包括: 首先, 一个可靠的育种者提供受精卵。罗曼经典棕鸡是一种很好的选择品种。其次, 实施毒性试验, 排除对所研究化合物的毒性作用需要做的。另外, 制备适合血液收集的血管是很重要的。重要的是要避免切割大血管在胚膜本身, 因为这可能导致非最好的失血。此外, 在将容器切割到 pH 条之前, 必须用过滤纸拍干, 以防止收集到的血液稀释。最后, 有足够数量的实验似乎很重要。我们建议每一个时间点使用至少10个鸡蛋, 和三倍的重复实验。
当然, 此蛋方法有一些限制。因为它需要30分钟, 直到物质通过蛋壳膜吸收, 并与胚膜紧密接触, 不可能量化快速反应的胚胎对应用化合物。此外, 一些化合物可能是有毒的应用浓度, 这往往导致血管病变的胚膜。因此, 基于长期孵化的化合物的细胞毒性试验大约24小时似乎是合理的。最后, 我们发现用于化合物的缓冲系统对该方法的性能有显著影响。12当前使用的是 HBSS 缓冲区, 因为它会对胚胎的血糖水平产生最小的影响。
对于将来的应用程序, 可以测试其他缓冲系统作为 HBSS 的替代方法。此外, 草药提取物对其他血液参数如胆固醇、甘油三酯或脂蛋白的影响可能是一个有吸引力的问题。
我们的新的蛋系统是一个有希望和重要的工具, 以测试具有胰岛素模仿特征的物质在一个活的有机体, 而不需要动物实验。因此, 它填补了体外和体内方法之间的差距。与现有的蛋模型系统相比,17没有必要通过链脲佐菌素 (STZ) 治疗来诱发糖尿病, 因为我们使用胚胎在孵化的10天或11。在这个阶段, 胰岛素的生产尚未开始, 但胚胎已经是胰岛素敏感。此外, 如果使用了 14-17 天的胚胎17, 则可能需要道德委员会的许可。此外, 与备选策略18相比, 此处描述的复合应用程序的危害性和费力程度更低, 增加了实验性的通过投入率。
结合起来, 这种蛋方法是一个有吸引力的系统, 如果一个胰岛素模拟物质的减血作用需要在生物体中测试。

糖尿病是一种代谢性疾病的特点是高血糖, 是由缺陷的胰岛素分泌或胰岛素作用的1。90% 的糖尿病病例均为2型糖尿病 (T2DM), 其中个体表现为胰岛素抵抗, 主要为胰岛素缺乏症2。众所周知, 有几个因素会增加 T2DM 的全球发病率, 包括生活方式的改变, 特别是与营养过剩、衰老和身体不活动有关的变化。根据国际糖尿病联合会 (IDF), 全世界约有4亿人患有糖尿病。此数字预计在未来20年3中达到6亿。
高血糖和胰岛素抵抗引起的糖尿病并发症, 如高血压、血脂异常、葡萄糖不耐受、冠心病和脑血管疾病, 导致预期寿命显著降低, 质量为4。受影响的个体需要降糖药物治疗;但是, 药物的使用, 如二甲双胍5通常与戏剧性的副作用6,7,8相关。因此, 进一步的抗糖尿病的方法, 是安全的, 广泛的可用和廉价的是必要的。
不同的草药化合物、提取物、植物和保健品被归因于通过调节各种细胞功能来特征胰岛素模仿的特性。一个重要的特点是刺激葡萄糖转运体 4 (GLUT4) 到血浆膜从细胞内储存室, 从而增加摄取葡萄糖的肌肉和脂肪组织在没有胰岛素, 被称为胰岛素模仿属性9。以前, 我们实施了一种基于荧光显微镜的方法来量化 GLUT410的易位过程。从提取库中分析大量植物提取物11使用这一化验结果, 确定了有前途的候选者。然而, 需要对生物体进行进一步的试验。本文详细描述的方法是一种改进的鸡胚模型, 已被证明是一种很有前景的识别胰岛素样物质的模型。它代表了一个吸引人的工具, 填补了体外和体内研究之间的巨大差距, 并与现有系统 (如可接受的成本、简单的处理过程和适当的吞吐量率) 相比提供了一些优势。此外, 不需要由道德委员会批准。

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:279px;">Phosphate buffered saline (PBS)</td>
			<td style="width:140px;">Biochrom</td>
			<td style="width:123px;">L1825</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#8217;s balanced salt solution (HBSS)</td>
			<td>Thermo Scientific</td>
			<td>SH30268.02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NovoRapid</td>
			<td>Novo Nordisk</td>
			<td>8-0905-82-304-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hens eggs (Lohmann classic brown chicken)</td>
			<td>Local Breeder</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accu-check performa</td>
			<td>Roche</td>
			<td>6454011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accu-check Inform II Teststreifen</td>
			<td>Roche&#160;</td>
			<td>5942861</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator&#160; HEKA- Turbo 288</td>
			<td>HEKA Brutger&#228;te</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe Omnican F 1 mL</td>
			<td>Braun</td>
			<td>09161502S&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Extracts</td>
			<td>PEKISH extract library</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lamp Tempo Nr. 119</td>
			<td>ORBAN</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an in ovo model for testing insulin-mimetic compounds

2型糖尿病 (T2DM) 高血糖水平是一种复杂的多因素代谢疾病, 是由胰岛素抵抗和β细胞衰竭引起的。采取各种策略, 包括注射胰岛素或使用胰岛素增敏药物, 以治疗 T2DM 或至少减少症状。此外, 中草药化合物的应用也引起了越来越多的关注。因此, 有必要找到有效的测试系统来识别和表征胰岛素模拟化合物。在这里, 我们开发了一个改良的小鸡胚模型, 它可以测试合成化合物和草药提取物的胰岛素模仿的性质。使用基于荧光显微镜的主屏幕, 将葡萄糖转运体 4 (Glut4) 的易位量化为等离子膜, 我们能够识别出化合物, 主要是草药提取物, 从而导致细胞内葡萄糖的增加。脂肪细胞中的浓度。然而, 这些物质的功效需要在生物体中进一步验证。因此, 我们使用了一个蛋方法来确定它们的降血糖性能。由于在胚胎发育的头2/3 中使用鸡胚不被认为是动物实验, 所以不需要伦理委员会的批准。在此, 详细介绍了该模型的应用。

Gluc 方法的描述:
孵化后与感兴趣的物质, 受精卵被打开, 并准备通过删除蛋壳的血液收集 (图 2)。进一步的准备工作包括平衡和去除蛋壳膜, 以获得胚膜。然后仔细切开这膜, 为血液收集提供合适的血管。优选地, 起源于胚胎腹部的大血管放置在塑料 pH 带上。为了避免在收集过程中稀释血液, 容器和 pH 带被用滤纸拍干。然后将血管切开, 用吸管将血液泄漏到 pH 带上进行分析。
精选中草药提取物对血糖水平的影响:
通过最近建立的 GLUT4-translocation 定量的主屏幕, 我们能够识别出具有胰岛素模拟特性10的物质。对数以百计的水溶性草药提取物进行筛选, 结果发现了几起命中。这些提取物在我们的 in 蛋模型中进行了测试. 从Combretum 菊花(仰光爬虫) 和非公开提取物 (称为萃取物 0845) 中提取的萃取物用于这些实验。我们溶解在 HBSS 缓冲区中的提取物在300毫克/升, 一个浓度, 结果是适当的以前的研究12,13。将物质应用于孵化11天的胚胎。如图 3所示, 仰光爬虫提取物没有导致显著的血糖降低蛋。然而, 血糖浓度已成功减少, 提取物 0845, 轻微下降后60分钟, 这是可比的效果观察使用商业上可用的胰岛素模拟。当卵孵化时间延长时, 观察到的效果显著。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57237/57237fig1.jpg"> 图 1: 有毒化合物对胚胎活力的影响.卵孵化11天(A 和 C)或治疗与有毒草药提取物在10天为另 24 h (B 和 D).应用提取物导致严重的血管病变的胚膜(D)和胚胎(B)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57237/57237fig2.jpg"> 图 2:蛋模型的说明.从左向右: 蛋壳的打开和移除(1-2);胚膜对蛋壳膜的平衡和去除(3);在 pH 条上制备的容器(4);使用葡萄糖表(5)收集血液和葡萄糖浓度的测定。自 Haselgruebler et al(201712) 改编而成。<a href="https://cloudflare.jove.com/files/ftp_upload/57237/57237fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57237/57237fig3.jpg"> 图 3: 草药提取物对血糖水平的影响与未经治疗或胰岛素治疗的卵子相比.卵孵化了11天, 并与所表明的物质 (商用胰岛素模拟: 3.3 U/毫升; 提取物: 300 毫克/升) 溶解在 HBSS 缓冲 (300 µL 体积), 高达 3 h. 用血糖仪测定血糖水平。结果显示没有缓冲区的影响。误差线基于平均值的标准误差。* p < 0.05 和 ** p < 0.0001, 在相同的孵化时间 HBSS 处理的卵显著减少。自 Haselgruebler et al(201712) 改编而成。<a href="https://cloudflare.jove.com/files/ftp_upload/57237/57237fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about An In Ovo Model for Testing Insulin-mimetic Compounds at JoVE.com

An In Ovo Model for Testing Insulin-mimetic Compounds

在本研究中, 我们描述了一个蛋系统, 作为一个有希望的工具来测试在生物体中的胰岛素模拟化合物。主要的优势包括适当的吞吐量和可接受的成本, 这使植物化学物质的识别与胰岛素模仿的性质。

用于测试胰岛素模拟化合物的蛋模型

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

an-in-ovo-model-for-testing-insulin-mimetic-compounds

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Neuroscience

Unit 1

An In Ovo Model for Testing Insulin-mimetic Compounds

Insulin and Hypoglycemic Drugs

SCIENTISTS IN ACTION

9.6K 次观看.  University of Applied Sciences Upper Austria. 在本研究中, 我们描述了一个蛋系统, 作为一个有希望的工具来测试在生物体中的胰岛素模拟化合物。主要的优势包括适当的吞吐量和可接受的成本, 这使植物化学物质的识别与胰岛素模仿的性质。

视频: An In Ovo Model for Testing Insulin-mimetic Compounds

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

闪光光解笼状化合物在嗅感觉神经元的纤毛

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

科研

神经科学

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

设计和组装的超轻型电动微型硬盘（Microdrive）慢性神经录音的小动物

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

在微RNA在腹鸡中脑大毛表达

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

一个简单的行为分析在检测视觉功能<em&gt;非洲爪蟾</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

一<em&gt;在体外</em&gt;模型细胞病理生理学的球细胞脑​​白质营养不良的研究

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

一种体内评价大鼠缺血性中风模型中血脑屏障的破坏

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

评估大鼠急性中风后脑恢复的临床前模型

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...