我们要感谢美国国立卫生研究院和美国国家神经疾病和中风研究所的资助（R01NS079691）。

实验是根据杜克大学和加州大学洛杉矶分校的IUCAC进行的。本研究使用8至12周龄雄性C57Bl / 6J小鼠。这些动物被安置在受控温度（22±2°C）下，具有12小时的光暗循环期，并随意获得颗粒状食物和水。镇痛和镇静方案被描述为由IUCAC批准，但可能与其他实验室使用的方案不同。
如果动物经历过度不安，发声（表明无法控制的疼痛），自我创伤，行动能力下降或受损，皮肤感染的身体体征，食欲不振和/或体重减轻超过10%-15%，作为生存手术的结果，可能会过早安乐死。注射凝胶没有预期的不良影响，因为它是由大脑中天然存在的聚合物组成的。应每天监测动物，包括周末和节假日，并每隔一天重新称重一次。我们实验室和其他人的研究发现，在这些小的皮质或皮质下中风后，小鼠在梳理，体重减轻或戒断或慢性压力的体征/症状方面没有缺陷。如果动物表现出任何局部头部伤口感染的迹象，则应对它们实施安乐死。还应监测动物梳理不良、伤口状况和动物之间打架的证据（背部伤口）。如果有争斗或伤口裂开的证据，动物在接触兽医后应首先接受治疗。这通常涉及水中的抗生素和/或局部局部抗生素的应用。如果这些措施无法治愈，动物应该被安乐死。
1. 准备材料和仪器
<ol><li>在手术前准备光血栓染料。称出15毫克孟加拉玫瑰到2毫升微量离心管中。 注意:孟加拉玫瑰会造成严重的眼睛损伤/刺激。<ol>
<li>将孟加拉玫瑰溶解在1.5毫升无菌过滤的1x PBS中，使工作浓度达到10毫克/毫升。</li>
		<li>涡旋管子，直到看不到团块，表明染料已完全溶解，然后覆盖在箔片中直至进一步使用。 注意:所需的染料量取决于小鼠的数量。将染料以10μL / g的浓度在腹膜内IP内注射，例如，20g小鼠为200μL。</li>
	</ol>
</li>
	<li>打开加热垫，在麻醉（37°C）期间保持小鼠体温。</li>
	<li>准备高压灭菌剪刀，镊子，棉花，兽医眼药膏，手术胶水或缝合材料，蓝色或黑色手术标志物，无菌酒精和β碘湿巾。准备带有无菌钻头的骨钻头。</li>
	<li>将微球灭菌打开至160°C，以允许在小鼠手术之间对器械进行灭菌。留出一个50 mL锥形管无菌盐水或1x PBS溶液，供以后用于维持水合的操作区域。</li>
	<li>将激光连接到立体定位设备。戴上激光安全护目镜，测量激光的功率（mW）。 注意:在手术过程中，只要打开激光，就使用激光安全护目镜。<ol>
<li>按照激光制造说明调整激光控制台上的电流，直到激光功率输出读数为10 mW，并将电流设置为主屏幕控制台上的预设。然后再次调整电流以建立另一个预设，其中激光输出功率读数为40 mW。现在关闭激光，直到进一步使用。 注:10 mW将在使用前用于定向激光器。</li>
	</ol>
</li>
	<li>手术后为小鼠准备一个干净的笼子。然后，将小鼠放入异氟醚浓度为4%的诱导室中麻醉它，直到自发运动停止，其呼吸达到缓慢，稳定的速率。</li>
	<li>一旦睡着，称量鼠标以确定注射多少玫瑰孟加拉染料。然后，将鼠标转移到立体束装置中，维持异氟醚浓度为1.5%。 注意:增加异氟醚浓度可以扩张血管并防止小鼠之间的足够闭塞或大小一致的中风。</li>
	<li>将兽医眼药膏涂抹在双眼上。 注意:在整个过程中注意呼吸和体温，并偶尔捏住脚趾，以确保鼠标感觉不到任何东西。</li>
</ol>2. 光血栓性卒中模型9
<ol><li>剃掉老鼠头上的皮毛，然后用酒精擦拭布和β-碘擦拭物无菌地准备该区域。重复三次。 注意:如有必要，最后使用额外的酒精擦拭巾来清除所有β碘，因为它会引起皮肤刺激，导致小鼠在手术后头骨瘙痒。</li>
	<li>使用小手术剪刀，在耳朵到眼睛之间做一个横向切口，约1-2厘米。为此，用镊子向上拉皮肤以形成帐篷，向下切割一次，然后将剪刀插入开口并形成连续的中线切口。</li>
	<li>分开皮肤，用镊子轻轻按压顶骨以定位胸骨。使用手术标记用点标记胸膜。 注意:骨碎片运动突出显示顶骨的额边缘。胸膜是额骨和顶骨碎片相遇的中心点（图1A）。</li>
	<li>戴上激光安全护目镜，将激光移到鼠标的头骨上，将立体定位装置固定在90°角。选择10 mW预设并打开激光器。<ol>
<li>调整激光的 x 轴和 y 轴，直到它直接超过边角。重置数字显示控制台上的 x 和 y，然后将激光器移动到边距左侧 1.8 mm 处。现在，将激光尽可能靠近颅骨，不要让它接触颅骨。</li>
		<li>将激光移动到边角左侧 2.2 mm 处，以确保它可以在没有任何障碍物的情况下移动。关闭激光。</li>
	</ol></li>
	<li>使用一次性29 G针头腹腔注射适量的Rose Bengal染料（IP）。立即启动定时器7分钟，让染料全身循环。选择预设的 40 mW，无需打开激光器。 注意:一旦熟悉该过程，可以在小鼠无菌制备之前注射染料，并在7分钟结束之前完成以节省时间。</li>
	<li>当7分钟计时器关闭时，戴上激光安全护目镜，打开激光（40 mW），并设置10分钟计时器。<ol>
<li>10分钟后，将激光移动到边角左侧2.2毫米处，不将其关闭，并设置另一个10分钟计时器。</li>
		<li>第二个10分钟计时器关闭后，将激光器切换回10 mW，并将激光器移动到边角左侧2.0 mm处。用手术标记标记标记此位置。然后关闭激光。</li>
		<li>提起激光并从90°角度解锁，以便将其移离鼠标。此时，如果手术区域干燥，请用棉涂抹无菌盐水或PBS。</li>
	</ol></li>
	<li>在垂直于颅骨表面的2.0毫米标记处进行小爆发钻孔。 注意:小心慢慢钻孔，以避免钻孔太深并击中大脑。一旦钻头穿过头骨，阻力就会略有下降/变化，钻孔可能会导致出血。<ol>
<li>使用棉花吸收多余的血液。钻孔后，通常会从颅骨的切口动脉中看到一些血液 - 过多的血液意味着钻头会击中大脑。如果发生这种情况，请记录鼠标标识符，然后继续。</li>
	</ol></li>
	<li>在鼠标旁边放一个小擦拭布。使用镊子，将皮肤拉近以准备粘合。 注意:缝合可以在这里完成。缝合通常只需要3次缝合。<ol>
<li>使用镊子，抓住皮肤的两侧，将它们拉在一起并向上拉。在涂抹到鼠标之前，在擦拭巾的尖端末端轻拍任何大滴的手术胶水。</li>
		<li>小心翼翼地涂抹手术胶水，并用镊子将皮肤固定在一起10秒。继续，直到没有可见的开口。 注意:胶水很快出来 - 不要挤压瓶子。避免将皮肤粘在颅骨上或将胶水粘在眼睛里。</li>
	</ol></li>
	<li>粘合或缝合后，将小鼠放入新笼子中以从麻醉中恢复。动物需要5-10分钟才能恢复，并且有助于将笼子的一半放在加热垫上。</li>
</ol>3. 假中风手术
<ol><li>执行与上述第2节中描述的操作相同的所有程序，除了注射1x PBS或盐水而不是Rose Bengal染料。</li>
</ol>4. 注射水凝胶或其他生物材料
<ol><li>在用户定义的时间注射生物材料。在该实验中，注射在中风后5至7天之间进行。注射2 - 6μL水凝胶（用户定义）。<ol>
<li>打开加热垫，在麻醉（37°C）期间保持小鼠体温。</li>
		<li>准备高压灭菌剪刀，镊子，棉花，兽医眼膏，手术胶水或缝合材料，以及无菌酒精和β碘湿巾。准备带有无菌钻头的骨钻头和带有金属30 G针头的25μL玻璃汉密尔顿注射器。</li>
		<li>将磁珠灭菌打开至160°C，以允许对小鼠之间的器械进行灭菌。</li>
		<li>将喷射泵连接到立体定位装置。将流速设置为 1 μL/min，将体积设置为 4 μL。</li>
		<li>手术后为小鼠准备一个干净的笼子。</li>
	</ol></li>
	<li>将小鼠放入异氟醚浓度为4%的诱导室中麻醉它，直到自发运动停止，其呼吸达到缓慢，稳定的速率。<ol>
<li>一旦睡着，将小鼠转移到立体定位装置中，维持异氟醚浓度为1.5%。</li>
		<li>将兽医眼药膏涂抹在双眼上。</li>
		<li>剃掉任何可能已经重新长出小鼠头部的皮毛，并使用酒精擦拭布然后使用β-碘擦拭物无菌地准备该区域。重复三次。 注意:如有必要，最后使用额外的酒精湿巾来清除所有的β碘，因为它会引起皮肤刺激并导致小鼠在手术后头骨瘙痒。</li>
	</ol></li>
	<li>使用小剪刀和/或镊子，重新打开大脑上方的皮肤。使用含无菌盐水或PBS的棉花清洁颅骨上的任何碎屑。一个白黄色的圆圈（笔程， 图1B）和钻头上的毛刺孔将可见。如果死组织不可见，则可能没有发生中风。如果毛刺孔未在冲程上方居中，请重新钻孔以使其居中。</li>
	<li>将注射泵定向到鼠标上方，并锁定在90°。在回装材料之前，用无菌盐水或PBS溶液清洁注射器。<ol>
<li>清洁后，拆卸玻璃注射器，使没有针头。使用25μL正置换移液器将注射器与10μL水凝胶（或其他生物材料，有或没有细胞）回加载注射器。</li>
		<li>使用注射器柱塞，将凝胶一直推到注射器的前面。然后，重新组装注射器并继续推动，直到凝胶从针头中明显渗出。</li>
	</ol></li>
	<li>将注射器放到泵上。可能需要调整泵的背面，以便注射器适合。为此，请将流速调整为30μL/min，根据需要移动方向选择 "撤液 "或" 注射"， 然后按 "开始"。当泵的背面位于正确的位置时，点击 停止 。<ol>
<li>拧紧泵的背面，使注射器牢固。如果之前进行了调整，请确保将流速设置回1μL / min并设置为注射。</li>
	</ol></li>
	<li>沿 x 和 y 方向移动泵，使其在孔上定向。沿 z 方向向下移动泵，直到注射器的针头接触到孔的顶部。</li>
	<li>重置数字显示控制台上的 z。然后，将注射器沿z方向向下移动0.750 mm。如果注射器弯曲或有阻力，则颅骨愈合或未完全钻孔，需要重新钻孔。</li>
	<li>按泵控制台上的 "开始" 键，以 1 μL/min 的速率注入 4 - 6 μL 水凝胶。</li>
	<li>当泵停止进样时，设置一个5分钟的计时器，以使凝胶开始交联。</li>
	<li>5分钟后，通过转动z nob慢慢拉起注射器。观察是否有任何材料意外拉出或从孔中泄漏。解锁泵，当针头离头骨足够远时，将其移离鼠标。</li>
	<li>使用镊子，手术胶水或缝合线，关闭头部上方的皮肤。将鼠标放入干净的笼子中，观察其恢复情况。小鼠从麻醉中恢复大约需要5-10分钟。</li>
</ol>5. 灌注和样本采集
<ol><li>使用异氟醚麻醉小鼠。</li>
	<li>将动物固定在仰卧位，并用正中切口打开腹腔。（可选）夹住降腹主动脉以使用较少的固定剂和PBS。</li>
	<li>切开隔膜，向上移动肋骨的两侧以打开胸腔。将灌注插管插入左心室，并在右心房切开一个开口。开始缓慢灌注 10-20 mL PBS 或直到液体半透明。</li>
	<li>清除后，换用 4% PFA，再加 10 – 20 mL。松开双臂的销钉允许它们在PFA注入时收缩。一旦他们停止移动，再等30秒。</li>
	<li>斩首动物并拉回皮肤以暴露头骨。使用钝而细的镊子，小心地取出头骨的碎片以暴露大脑。移除卒中部位上方的颅骨时需要特别小心。这里可以使用小型手术剪刀来帮助切除颅骨。最大的挑战是凝胶粘在头骨上，并在头骨被移除时从大脑中出来。</li>
	<li>一旦大脑脱离，放入10-15毫升4%PFA过夜（不超过24小时）。</li>
	<li>第二天将大脑从PFA移动到30%蔗糖。一旦大脑沉入底部（约2-3天），大脑就准备好进行冷冻切除。此时也可以将其保留用于存储。</li>
</ol>6. 冷冻切除和染色
<ol><li>将大脑从蔗糖中取出，放在载玻片上。用剃须刀片切掉大脑后部的一小部分，形成一个平坦的部分，可以安装在卡盘上。<ol>
<li>将一小块最佳切割温度化合物（OCT）放在卡盘上，然后将大脑平放一面朝下，放在OCT中的卡盘上。将此标本放在干冰上，直到冷冻或冷冻块上的低温恒温器中。 注意:可以添加OCT以完全包围大脑，以帮助防止材料在切片过程中与大脑分离。</li>
	</ol>
</li>
	<li>在-20°C的低温恒温器上，在10个明胶包被的载玻片上以30μm厚的部分连续切割大脑。切片时要小心，确保不会丢失水凝胶。如果这部分很困难，请在凝胶上方的大脑顶部放置一些OCT（图3）。储存在-80°C直至进一步使用。</li>
	<li>将载玻片从-80°C中取出，放在染色盘上以使其干燥。尚未冷冻但刚刚切片的载玻片也可以立即染色。</li>
	<li>使用疏水笔，在幻灯片上的脑切片周围画一个圆圈。干燥后，用1x过滤的PBS重新水润脑切片，并在室温下置于摇摇杯上15分钟。每张载玻片需要约1 mL缓冲液，具有9-12个脑切片。</li>
	<li>在室温下用1x过滤的PBS洗涤载玻片5分钟，重复三次。</li>
	<li>在室温下用10%驴血清在0.01%PBS-Triton X中在肚皮舞者身上阻断载玻片30分钟至一小时。每张载玻片大约需要200μL。</li>
	<li>将一抗与0.01%PBS-Triton X在4°C的摇摇杯上孵育10%驴血清中过夜。首先测试一抗以确定正确的浓度。这里使用了GFAP（1:400），Iba1（1:250），Glut1（1:500）（图4）。</li>
	<li>第二天用1x过滤的PBS洗涤载玻片，在室温下洗涤5分钟，重复三次。</li>
	<li>在室温下，将二抗与10%驴血清在0.01%PBS-Triton X和DAPI中孵育2小时。1:1，000 用于所有辅助数据库和 DAPI。</li>
	<li>用1x过滤的PBS洗涤载玻片5分钟。</li>
	<li>让切片在室温下在黑暗中干燥。这可能需要20分钟到4小时;将载玻片放入干燥器中可以加快该过程。</li>
	<li>脱水载玻片在升压浓度的酒精（每个溶液1分钟）为50%，70%，95%，100%和100%。</li>
	<li>在第一次二甲苯浴中脱脂1分钟，然后在第二次二甲苯浴中脱脂5分钟。</li>
	<li>快速将载玻片逐个取出，并在大脑切片被二甲苯润湿时加入安装介质。在顶部快速添加玻璃盖玻片。使用分别用于成像的载玻片和显微镜所需的正确长度和厚度的盖玻片。</li>
	<li>通过用移液器吸头从载玻片中心向外运动轻轻按压盖板下的任何气泡，摆脱盖玻片下的任何气泡。让DPX在室温下干燥4小时，在黑暗中过夜。</li>
	<li>使用剃须刀片刮掉溅到载玻片上的任何干燥安装介质。用镜头清洁剂擦拭幻灯片。载玻片现在可以用荧光显微镜成像。 注意:最好将载玻片存放在黑暗中或4°C下，直到成像完成。</li>
</ol>

<ol>
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R., Weaver, W. M., Scumpia, P., Di Carlo, D., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+wound+healing+by+injectable+microporous+gel+scaffolds+assembled+from+annealed+building+blocks.">Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks.</a> Nature Materials. 14 (7), 737-744 (2016).</li><li>Sideris, E. Y., Aaron, C. J., Carmichael, S. T., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid+particle+hydrogels+decrease+cerebral+atrophy+and+promote+pro-reparative+astrocyte/axonal+infiltration+in+the+core+after+ischemic+stroke.">Hyaluronic acid particle hydrogels decrease cerebral atrophy and promote pro-reparative astrocyte/axonal infiltration in the core after ischemic stroke.</a> bioRxiv. , (2019).</li><li>Yu, H., 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=Combinated+Transplantation+of+Neural+Stem+Cells+and+Collagen+Type+I+Promote+Functional+Recovery+After+Cerebral+Ischemia+in+Rats.">Combinated Transplantation of Neural Stem Cells and Collagen Type I Promote Functional Recovery After Cerebral Ischemia in Rats.</a> The Anatomical Record. 293 (5), 911-917 (2010).</li><li>Jin, K., 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=Transplantation+of+Human+Neural+Precursor+Cells+in+Matrigel+Scaffolding+Improves+Outcome+from+Focal+Cerebral+Ischemia+after+Delayed+Postischemic+Treatment+in+Rats.">Transplantation of Human Neural Precursor Cells in Matrigel Scaffolding Improves Outcome from Focal Cerebral Ischemia after Delayed Postischemic Treatment in Rats.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 30 (3), 534-544 (2009).</li><li>Gelderblom, 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=Temporal+and+spatial+dynamics+of+cerebral+immune+cell+accumulation+in+stroke.">Temporal and spatial dynamics of cerebral immune cell accumulation in stroke.</a> Stroke. 40 (5), 1849-1857 (2009).</li><li>Wei, L., Erinjeri, J. 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作者宣称TS是Tempo Therapeutics的创始人，该公司旨在将MAP水凝胶商业化。

在这里，我们展示了一个易于重复的，微创的，永久性的中风模型，并描述了如何在中风后五天将生物材料注射到梗死中。使用光血栓染料Rose Bengal和连接到立体束装置的520nm准直激光器使我们能够以更高的精度将行程定位在小鼠的运动皮层。卒中五天后，在照射中心用眼睛可以看到梗死的位置，距胸侧2.0mm。然后使用注射泵将水凝胶准确地注射到行程核心中。该模型中手术期间和中风后的死亡率?...

中风是美国残疾的主要原因和第五大死因1。大约87%的卒中是缺血性的，而其余13%中的大多数是出血性2。缺血性卒中被定义为动脉中流向周围组织的血流阻塞。这种闭塞导致缺氧和随后的坏死，这通常导致幸存患者的永久性残疾。虽然中风3的死亡率有所下降，但预计到2030年其患病率将增加到340万人4。残疾幸存者的增加以及随之而来的经济负担导致了对中风研究的推动，该研究的重点是神经修复机制。中风后有一个炎症期，导致疤痕的形成，阻止坏死区域扩张。坏死核心周围的区域被称为"围死期梗死"，并且有强有力的证据表明，该区域的可塑性（包括增加的血管生成，神经发生和轴突发芽）与动物模型和人类观察到的恢复直接相关5。由于没有体外模型可以正确复制中风后的复杂相互作用，因此动物模型对于中风研究至关重要。
有几种体内模型可用于产生缺血性中风。小鼠最常用的模型之一是通过远端或近端（通过动脉内丝）闭塞的脑中动脉闭塞或MCAo。近端模型，也称为细丝MCAo（fMCAo），通常会导致大的缺血性卒中，包括大脑半球的5%至50%，这取决于因素的数量6。在这些模型中，缝合线或细丝从颈内动脉推进到大脑中动脉（MCA）的底部，并保持在适当的位置一段时间。这种闭塞方法可以是暂时的或永久性的，产生以纹状体为中心的梗死，并且可能涉及也可能不涉及覆盖的皮层6。由此产生的笔画大小变化很大，并且需要成像技术（例如激光多普勒）来确认每个小鼠中该程序的有效性。持续超过30分钟的动脉内或腔内灯丝闭塞在尺寸范围的较大末端产生中风。一些研究人员专注于较短的灯丝闭塞时间，这需要大量的实验重点和实验室验证7。小鼠中的细丝MCAo模型遵循与人类中风病例相似的细胞死亡，缺血进展和围死区形成的阶段;然而，较大的中风更接近于恶性脑梗死的疾病状态，这是不太常见，不太可治疗的人类中风6。同时，远端 MCA 闭塞需要更复杂的手术和颅骨切除术。在这个模型中，沿着大脑表面运行的MCA的远端部分被缝合带或烧灼直接闭塞。在该技术的某些变体中，颈动脉是单侧或短暂双侧闭塞的。远端 MCAo 的一个好处是，它会产生基于皮质的卒中，其大小比灯丝模型变化较小。然而，由于颈外动脉（ECA）的横断，远端模型产生较差的行为输出，这也是fMCAo6的一个问题。
另一种已知侵入性较小的卒中模型是光血栓形成（PT）模型。PT模型导致缺血的明确定义位置，并且与高生存率相关8。该技术依赖于腹腔内注射的光敏染料，只需用光或激光照射所需组织9，即可实现血管内光氧化。在激发时，形成氧自由基，引起内皮损伤，从而激活受照区域中的血小板聚集和凝块形成8，9。对行程大小和位置的严格控制，以及PT模型的高再现性，使其成为研究生物材料的理想选择。虽然使用激光和立体自聚焦坐标可以实现精度，但存在一些缺点，可能会使该模型在少数研究中不那么理想。与 fMCAo 模型不同，PT 行程模型不能重新熔化。因此，用于研究针对再灌注后损伤的神经保护剂或再灌注后机制的材料在这里没有用8。此外，由于PT模型的微血管损伤，可见相对较小的缺血性半影。相反，局部血管源性水肿发生，这是人类卒中的特征，使得该模型不适合专注于围梗死区域的临床前药物研究6，8。
中风中生物材料策略的总体目标是提供生物活性剂或作为脑组织生长的替代细胞外基质。我们将使用我们的方法探索的一种策略是将水凝胶直接输送到中风核心，而不是在梗死周围组织，其中许多当前的细胞疗法都提供10。这种方法的基本原理是，递送到核心中发现的坏死组织将避免破坏周围的健康或恢复组织。我们假设生物材料中包含的任何活性剂的扩散将能够从核心到达围陷圈，特别是因为我们发现水凝胶生物材料的输送减少了神经胶质疤痕的厚度11。这一点很重要，因为围陷区域已被证明在卒中后表现出神经可塑性，使其成为一个有吸引力的靶标。此外，将替代基质递送到卒中核心可以加载血管生成12 或神经源性13 因子，以指导新组织的形成，以及用于递送的细胞14。通过使用基质可以大大增强细胞递送，因为它可以保护细胞免受递送过程中存在的苛刻注射力和局部环境的影响，并鼓励分化和植入15。
这些可注射的治疗性生物材料在中风应用中具有临床相关性，因为目前还没有药物疗法可以刺激中风后的神经元恢复。参与恢复的底层神经回路位于与中风核心16相邻的脑组织中，而中风核心本身缺乏活的神经组织。我们预计，将生物材料递送到坏死性卒中核心具有通过前面提到的许多机制刺激相邻组织走向再生过程的潜力，包括生长因子13的库释放，刺激生长中的组织并促进恢复的脑组织发育11，12，免疫反应的改变17，以及干细胞衍生疗法的递送14， 18.然而，为了有效地研究这些应用的可能性，需要一种一致且可重复的方法来诱导中风和注射生物材料。PT 行程模型使用的技术可精确控制行程的方向和位置。连接到立体定位装置的激光引导方向，连接到立体定位装置的泵控制材料的注入速率，而无需其他形式的成像。因此，我们选择描述在小鼠运动皮层中执行PT中风以及将生物材料注射到中风核心的方法。在这里，我们使用微孔退火颗粒（MAP）水凝胶作为注射生物材料，不添加细胞或生长因子。此外，我们解释了如何使用完整的生物材料成功检索大脑，并讨论了用于分析中风结果的免疫化学测定，无论是否注射生物材料。

<table>
	<tbody>
		<tr>
			<td>10% Normal Goat Serum</td>
			<td>VWR</td>
			<td>100504-028</td>
			<td>For blocking buffer</td>
		</tr>
		<tr>
			<td>2-ply alcohol pre pad, Sterile, Medium</td>
			<td>Medline</td>
			<td>MDS090735</td>
			<td></td>
		</tr>
		<tr>
			<td>25uL Hamilton Syringe 702RN, no needle</td>
			<td>Fishcer Scientific</td>
			<td>14824663</td>
			<td>Syringes used to inject biomaterials</td>
		</tr>
		<tr>
			<td>25uL Positive displacement pipette</td>
			<td>Gilson</td>
			<td>M-25</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Beam Expander, 400-650nm</td>
			<td>Thorlabs</td>
			<td>GBE02-A</td>
			<td>Laser beam expander</td>
		</tr>
		<tr>
			<td>Adjustable Stage Platform</td>
			<td>Kopf Instruments</td>
			<td>901</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Glucose Transporter GLUT1 antibody, rabbit</td>
			<td>Abcam</td>
			<td>ab113435</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Iba1 Antibody, goat</td>
			<td>abcam</td>
			<td>ab5076</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Safety-Lok Blood Collection Sets. 25G, 12&#34;</td>
			<td>Medsupply</td>
			<td>367294</td>
			<td>For perfusions</td>
		</tr>
		<tr>
			<td>BKF12- Matte Black Aluminum Foil</td>
			<td>Thorlabs</td>
			<td>BKF12</td>
			<td>To cover anything that is reflective when using laser.</td>
		</tr>
		<tr>
			<td>Bone Iris Mini Scissors - 3-1/2&#34;</td>
			<td>Sklar surgical instruments</td>
			<td>64-2035</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 Mice</td>
			<td>Jackson Laboratory</td>
			<td>000664</td>
			<td>8-12 weeks of age</td>
		</tr>
		<tr>
			<td>Cage Assembly Rob</td>
			<td>Thorlabs</td>
			<td>ER3-P4</td>
			<td>3&#34; Long, diameter 6mm, 4 pack - for attaching laser to sterotax</td>
		</tr>
		<tr>
			<td>Carbon Steel Burrs -0.5mm Diameter</td>
			<td>Fine Science Tools</td>
			<td>19007-05</td>
			<td>For creating burr hole</td>
		</tr>
		<tr>
			<td>Chromium(III) potassium sulfate dodecahydrate</td>
			<td>VWR</td>
			<td>EM1.01036.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact Controller for pigtailed lasers</td>
			<td>Thorlabs</td>
			<td>CLD1010LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Swabs</td>
			<td>VWR</td>
			<td>89031-288</td>
			<td></td>
		</tr>
		<tr>
			<td>CP 25 pipette tips</td>
			<td>Gilson</td>
			<td>F148012</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-goat IgG H&#38;L (488)</td>
			<td>abcam</td>
			<td>ab150129</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-rabbit IgG H&#38;L (647)</td>
			<td>abcam</td>
			<td>ab150075</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-rat IgG H&#38;L (555)</td>
			<td>abcam</td>
			<td>ab150154</td>
			<td></td>
		</tr>
		<tr>
			<td>EMS DPX Mountant</td>
			<td>Elecron Microscopy Sciences</td>
			<td>13512</td>
			<td>Mounting solution for slides</td>
		</tr>
		<tr>
			<td>EMS Gelatin Powder Type A 300 Bloom</td>
			<td>Electron Microscopy Sciences</td>
			<td>16564</td>
			<td>For gelatin coating slides</td>
		</tr>
		<tr>
			<td>EMS Paraformaldehyde, Granular</td>
			<td>VWR</td>
			<td>100504-162</td>
			<td>For making 45 PFA</td>
		</tr>
		<tr>
			<td>ESD Worstation kit</td>
			<td>Elmstat</td>
			<td>WSKK5324SB</td>
			<td>Need for setting up the laser</td>
		</tr>
		<tr>
			<td>Fiber Bench Wall Plate, unthreaded</td>
			<td>Thorlabs</td>
			<td>HCA3</td>
			<td>Need for connecting laser to Kopf shaft</td>
		</tr>
		<tr>
			<td>FiberPort</td>
			<td>Thorlabs</td>
			<td>PAF-X-5-A</td>
			<td>FC/APC&#38; APC, f=4.6mm, 350-700nm, diameter 0.75mm</td>
		</tr>
		<tr>
			<td>Fine Scissors - straight/sharp-blunt/10cm</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP Antibody, rat</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-0300</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Plate</td>
			<td>Kopf Instruments</td>
			<td>HP-4M</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmEdge Hydrophobic Barrier Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td>For staining slides</td>
		</tr>
		<tr>
			<td>IMPAC 6-Integrated Multi Patient Anesthesia Center</td>
			<td>VetEquip</td>
			<td>901808</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine Prep Pads</td>
			<td>Medx Supple</td>
			<td>MED MDS093917H</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewlers Forceps #5</td>
			<td>GFS chemicals</td>
			<td>46085</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Safety Glasses</td>
			<td>Thorlabs</td>
			<td>LG10B</td>
			<td>Amber Lenses, 35% Visible light (googles versions available too)</td>
		</tr>
		<tr>
			<td>M27-1084 Powerful LED Dual Goose-neck</td>
			<td>United Scope</td>
			<td>LED-11C</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical USP Grade Oxygen</td>
			<td>Airgas</td>
			<td>OX USP250</td>
			<td></td>
		</tr>
		<tr>
			<td>Miltex Adson Dressing Forceps, Disecting-grade</td>
			<td>Intefra Miltex</td>
			<td>V96-118</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Cord/Cordless Small Animal Trimmer</td>
			<td>Harvard aparatus</td>
			<td>72-6110</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-pump variable flow</td>
			<td>Thomas Scientific</td>
			<td>70730-064</td>
			<td>Pump for perfusions</td>
		</tr>
		<tr>
			<td>Mouse Brain Matrices, Coronal Slices, 1mm</td>
			<td>Kent Scientific</td>
			<td>RBMA-200c</td>
			<td>For TTC slices</td>
		</tr>
		<tr>
			<td>Mouse Gas Anethesia Head Holder</td>
			<td>Kopf Instruments</td>
			<td>923-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanojet Control Box</td>
			<td>Chemyx</td>
			<td>10050</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanojet pump header</td>
			<td>Chemxy</td>
			<td>10051</td>
			<td>Attach to stereotaxic device for injecting biomaterial</td>
		</tr>
		<tr>
			<td>Needle RN 30G PT STY 3, 0.5 inch</td>
			<td>Fishcer Scientific</td>
			<td>NC9459562</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-rupture ear bars 60&#186;</td>
			<td>Kopf Instruments</td>
			<td>922</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer pH 7.4</td>
			<td>VWR</td>
			<td>97062 338</td>
			<td></td>
		</tr>
		<tr>
			<td>Pigtaled laser 520 nm, 100mW, 5G Pin</td>
			<td>Thorlabs</td>
			<td>LP520-MF100</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive charge glass slides</td>
			<td>Hareta</td>
			<td>AHS90-WH</td>
			<td></td>
		</tr>
		<tr>
			<td>Power engergy meter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>Used to measure your mW laser output</td>
		</tr>
		<tr>
			<td>Puralube Vet Ointment</td>
			<td>Dr. Foster Smith</td>
			<td>9N-76855</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectal Probe Mouse</td>
			<td>kopf Instruments</td>
			<td>Ret-3-ISO</td>
			<td></td>
		</tr>
		<tr>
			<td>Rose Bengal Dye 95%</td>
			<td>Sigma-Aldrich</td>
			<td>330000-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaft Modified 8-32 threaded hole 1/2&#34; depth</td>
			<td>Kopf Instruments</td>
			<td>1770-02</td>
			<td>For connecting laser to sterotaxic device</td>
		</tr>
		<tr>
			<td>Slim photodiode power sensor</td>
			<td>Thorlabs</td>
			<td>S130VC</td>
			<td>Used with power energy meter</td>
		</tr>
		<tr>
			<td>SM1-Threaed 30 mm Cage Plate 0.35&#34; thick 2 Retaining</td>
			<td>Thorlabs</td>
			<td>CP02</td>
			<td>For connecting laser expander</td>
		</tr>
		<tr>
			<td>Sol-M U-100 Insuline syringe with 1/2 unit markings 0.5 mL</td>
			<td>VWR</td>
			<td>10002-726</td>
			<td>To inject rose bengal</td>
		</tr>
		<tr>
			<td>StainTray Slide Staining System</td>
			<td>Simport Scientific</td>
			<td>M920-2</td>
			<td>For staining slides</td>
		</tr>
		<tr>
			<td>Sterotaxic device</td>
			<td>Kopf Instruments</td>
			<td>940</td>
			<td>Small Animal Stereotaxic Instrument</td>
		</tr>
		<tr>
			<td>Student Adson Forceps -1x2 teeth</td>
			<td>Fine Science Tools</td>
			<td>91127-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Fine Forceps - straight/broad Shanks</td>
			<td>Fine Science Tools</td>
			<td>91113-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>Kopf Instruments</td>
			<td>TCAT-2LV</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek OCT compound</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>VWR</td>
			<td>97063-864</td>
			<td></td>
		</tr>
		<tr>
			<td>Upper Bracket Clamp</td>
			<td>Kopf Instruments</td>
			<td>1770-c</td>
			<td>For connecting laser to sterotaxic device</td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhessive 3mL</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-361931</td>
			<td></td>
		</tr>
		<tr>
			<td>Vogue Professional My Manicurist</td>
			<td>Bargin Source</td>
			<td>6400</td>
			<td>For Burrs</td>
		</tr>
		<tr>
			<td>VWR Bead Sterilizers</td>
			<td>VWR</td>
			<td>75999-328</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek OCT compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>For tissue embeding</td>
		</tr>
	</tbody>
</table>

injection of hydrogel biomaterial scaffolds to the brain after stroke

中风是美国残疾的主要原因和第五大死因。大约87%的中风是缺血性中风，被定义为向大脑供血的血管突然阻塞。在阻塞的几分钟内，细胞开始死亡并导致不可挽回的组织损伤。目前的治疗重点是血栓去除或裂解，以允许再灌注并防止更严重的脑损伤。虽然短暂的脑可塑性可能会随着时间的推移挽救一些受损组织，但很大一部分患者留下了永远无法解决的神经功能障碍。缺乏治疗中风引起的神经功能障碍的治疗选择，强调需要制定新的策略来治疗这一不断增长的患者群体。可注射的生物材料目前正在设计中，通过递送活性剂或干细胞来增强大脑可塑性并改善内源性修复。测试这些方法的一种方法是利用啮齿动物中风模型，将生物材料注射到中风核心中，并评估修复情况。了解卒中核心的精确位置对于卒中后的准确治疗至关重要，因此，最好使用导致可预测的卒中位置的卒中模型，以避免在注射前需要成像。以下方案将涵盖如何诱导光血栓性中风，如何以受控和精确的方式注射水凝胶，以及如何提取和冷冻切除大脑，同时保持生物材料完整。此外，我们将重点介绍如何将这些相同的水凝胶材料用于干细胞的共递送。该协议可以推广到使用其他可注射的生物材料进入中风核心。

这种方法的目的是展示如何在中风后将生物材料注射到大脑中。使用带有玫瑰孟加拉和520nm激光的光血栓形成模型来控制卒中病变的大小和位置的方向。卒中五天后，梗死可以在手术过程中（图1B）和TTC和成像IHC染色载玻片（图2）可视化。使用2倍透镜增加激光直径会导致行程病变的视觉增加，其范围超过2.5 mm，而仅使用激光的单个1 mm部分（<strong class="...

Watch this Scientific Journal Video about Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke at JoVE.com

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

中风是一个全球性问题，治疗选择最少，目前没有用于再生丢失的脑组织的临床治疗方法。在这里，我们描述了在啮齿动物的运动皮层中产生精确光血栓性中风的方法，并随后注射水凝胶生物材料，以研究它们对中风后组织再生的影响。

中风后向大脑注射水凝胶生物材料支架

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes ...

injection-of-hydrogel-biomaterial-scaffolds-to-the-brain-after-stroke

Research

JoVE Journal

Biology

4.9K 次观看.  Duke University. 中风是一个全球性问题，治疗选择最少，目前没有用于再生丢失的脑组织的临床治疗方法。在这里，我们描述了在啮齿动物的运动皮层中产生精确光血栓性中风的方法，并随后注射水凝胶生物材料，以研究它们对中风后组织再生的影响。

视频: Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

Intravitreous Injection for Establishing Ocular Diseases Model

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a hemilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given.
To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45&deg; injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes. 
The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.

Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.

玻璃体内注射眼部疾病模型建立

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as ...

科研

生物学

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

斑马鱼脑室注射液

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

到大脑的门户:剖析灵长类动物的眼睛

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

映射后的Theta突发刺激对人类听觉皮层功能成像

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

导管内注射局部给药的小鼠乳腺

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

从大鼠的侧尾静脉血液取样

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

一种从小鼠中分离大脑围取体的高输出方法

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...