作者要感谢德克萨斯大学西南分校的 Steven Gray、Matthew Rioux、Nanda Regmi 和 Lacey Stearman 对幼年大鼠鞘内注射所带来的挑战进行了富有成效的讨论。这项工作部分得到了Jaguar Gene Therapy（JLFK）的资金支持。

这项研究得到了埃默里大学机构动物护理和使用委员会 （IACUC） 的批准。本研究使用Sprague-Dawley大鼠（28-30日龄，质量范围约为90-135g，雄性和雌性）。
1. 载体的制备
<ol><li>在程序开始时在冰上解冻 AAV9 载体（参见 材料表）。</li>
	<li>在台式离心机中短暂离心含有载体的微量离心管，以确保所有液体都在管的底部。</li>
	<li>轻轻轻弹微量离心管，确保溶液充分混合。</li>
</ol>2. 回收笼的准备
<ol><li>将一个干净的笼子放在电热毯上（参见 材料表），使只有一半的笼子与毯子接触。</li>
	<li>将毯子的温度设置为 ~37 °C。</li>
</ol>3.手术平台的准备
<ol><li>在微波炉或水浴中将等温垫（参见 材料表）加热至39°C，使内容物变成液体。</li>
	<li>将等温垫放在手术平台上，并用干净的吸收性台垫覆盖。</li>
</ol>4. 动物准备
<ol><li>在透明盒子中用异氟醚麻醉大鼠（遵循机构批准的方案）。使用 5% 异氟醚开始麻醉诱导，每分钟降低 1% 直至达到 2%。将动物保持在 2% 再保持 3 分钟。</li>
	<li>将装有动物的盒子移至通风橱中，然后打开盒子。 注意:这限制了外科医生对麻醉剂的暴露。</li>
	<li>使用电动理发器从动物背部去除毛发。 注意:或者，可以使用脱毛膏或手动剃须刀和剃须膏。</li>
	<li>将动物放在手术平台上，其鼻子位于麻醉鼻锥中。 注意:当毛皮从手术部位取下时，动物可能会开始恢复意识。如果发生这种情况，请按照上述方式再次麻醉。</li>
	<li>在每只眼睛上涂抹润滑眼膏，以防止在手术过程中角膜干燥。</li>
	<li>使用聚维酮碘和异丙醇湿巾的三次交替应用对手术区域进行消毒。</li>
	<li>皮下注射镇痛药。 注:丁丙诺啡通常以0.01-0.05mg / kg的剂量使用，每12小时给药一次。或者，该药物的缓释形式可以以 1 mg/kg 给药一次，以提供 72 小时的充分疼痛控制。请咨询该机构的 IACUC 以获取有关疼痛管理的指南。</li>
	<li>将 100 μL 1% 利多卡因皮下注射至 L2 至 L6 棘突上方，以提供局部麻醉。</li>
	<li>将一卷纸巾或直径为 1.5 厘米的管子放在动物下方，刚好靠近臀部。这有助于弯曲脊柱，从而更容易将针头插入两个薄片之间。</li>
	<li>在动物身上放置一个开窗帘（见 材料表），将开窗放在腰椎上方的中心。</li>
</ol>5.暴露腰椎
<ol><li>通过捏住动物的每一只爪子并寻找是否存在戒断反应来确认麻醉的深度。</li>
	<li>使用 #11 手术刀刀片，在从 L2 到 L6 中线的皮肤上创建一个约 3 厘米长的切口。</li>
	<li>通过在肌肉和皮肤之间插入一把无菌弯曲的手术剪刀，然后打开尖端，使皮肤从肌肉上松开。</li>
	<li>移除覆盖 L2-L5 棘突的筋膜。</li>
</ol>6. 注射器的装载
<ol><li>将 25-35 μL 载体（达到所需剂量）移液到无菌微量离心管的盖子中。</li>
	<li>将整个体积吸入胰岛素注射器中。 注意:在此过程中注意不要吸入空气。</li>
</ol>7. 执行注射
<ol><li>识别 L5 和 L4 棘突。 注意: L6 直接位于两个髂嵴之间，其棘突应该很容易通过用钝器探测来识别。然后，可以将仪器轻轻地从后面滑行，以找到 L5 和 L4 过程的边界。</li>
	<li>将一只手轻轻放在动物的尾巴和一条腿上。用手指稳定注射器。</li>
	<li>将注射器的针头放置在L5棘突的左侧，并与其尾端对齐。放置注射器，使其偏离中线约 30°，距工作台平面向上 30°。 注意:使用手术显微镜更好地识别标志并定位针尖可能会有所帮助。</li>
	<li>将注射器针头向前推进约 8 毫米，越过 L5 椎板的顶部，然后在 L4 椎板下方进入腰椎池，直到骨头被击中。正确的放置将导致腿和/或尾巴抽搐，而放在腿/尾巴上的拇指可以看到或感觉到。如果没有抽搐，请取下针头并从左侧尝试该过程。如果仍然没有抽搐，请根据需要在 L4/L3 和 L3/L2 之间重复该过程。</li>
	<li>缓慢按下柱塞约 5 秒。 注意:注射过程中腿部或尾巴可能会抽搐。</li>
	<li>完全按下柱塞后，将注射器保持在原位约 30 秒，以使压力平衡并最大限度地减少针头拔出时注射液的回流。</li>
	<li>慢慢取下针头。</li>
</ol>8. 切口闭合
<ol><li>近似切口的边缘。</li>
	<li>从伤口的一端开始，使用 4-0 缝合线（参见 材料表）或手术钉闭合切口。</li>
</ol>9. 恢复和监控
<ol><li>将动物放入预热的笼子中。</li>
	<li>至少每 15 分钟检查一次动物，直到它完全可以走动。 注意:这应该需要 15 分钟到 45 分钟。</li>
	<li>在接下来的三天里，至少每天进行一次健康检查。在手术后的前 2 天或根据 IACUC 的要求提供镇痛药。</li>
	<li>手术后一周，取下缝合线或订书钉。</li>
</ol>10. 后续程序
注意:为了确定注射技术的准确性，如上所述注射台盼蓝染料，然后立即对动物实施安乐死（遵循机构批准的方案）并进行椎板切除术以可视化结果。
<ol><li>当动物仍处于麻醉状态时，通过 腹膜内 注射以 150 mg/kg 的剂量给予致死剂量的戊巴比妥对其实施安乐死。</li>
	<li>一旦呼吸和心脏活动停止，打开胸腔以确保死亡。将手术切口从背部延伸到颈部。</li>
	<li>在棘突两侧平行于脊柱的肌肉中做一个 4 厘米长的切口，尽可能靠近脊柱突。</li>
	<li>使用细镊子或剪刀，从棘突之间取出肌肉。</li>
	<li>使用 rongeur 从 L6 向下胸椎移除棘突（参见 材料表）。避免扭转动作，因为这可能会损坏 rongeurs。</li>
	<li>将 rongeur 的下尖端插入 L5 椎板下方，并通过从中"咬"几下去除覆盖在脊髓上的骨头。 注意:向后拉 L6 棘突可以更容易地插入 rongeur 的尖端。必须注意防止对脊髓造成损害。</li>
	<li>继续将椎板切除术向喙部扩张至少四个椎板。检查薄片的内表面是否有染料迹象，这可能表明注射失败。</li>
</ol>

幼年大鼠腰椎穿刺 AAV9 递送用于 CNS 基因治疗

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Donsante 博士是 AAV9 载体脑脊液给药的专利的发明人。

多种疾病会影响中枢神经系统。对于那些本质上是隐性和单基因的，如脊髓性肌萎缩症，通过病毒载体提供相关基因的功能拷贝是一种有吸引力的治疗策略。然而，血脑屏障 （BBB） 排除了大多数静脉注射的基因治疗载体11。那些可以穿过 BBB 的药物，例如 AAV9，必须以高剂量给药，以克服由于外周转导引起的载体丢失12。年龄也是一个障碍。对各种 AAV 血清型的?...

近年来，随着 FDA 批准治疗脊髓性肌萎缩症、视网膜营养不良、因子 IX 血友病、癌症等，病毒介导的基因疗法的前景终于得以实现 1,2,3,4。目前有无数其他疗法正在开发中。基因疗法旨在将治疗基因传递到患者的细胞中。这种新基因的产物可以替代缺陷内源性基因缺失的活性，抑制有毒基因，杀死癌细胞，或提供一些其他有益功能。
对于影响中枢神经系统 （CNS） 的疾病，将基因治疗载体直接递送至靶组织通常是可取的。非全身性方法有两个好处:它们最大限度地减少了可能由外周转导引起的脱靶副作用，并且它们大大减少了在靶组织中达到足够转导水平所需的载体量5。
有多种方法可以将基因治疗载体输送到中枢神经系统。脑实质内注射，即将载体直接注射到脊髓或脑组织中，可用于输送到指定区域。然而，对于许多疾病，需要中枢神经系统的广泛转导。这可以通过向脑脊液 （CSF）5 输送载体来实现，脑脊液是流入和流入大脑和脊髓及其周围的液体。有三种主要方法可以将载体输送到 CSF。最具侵入性的方法是脑室内分娩，它涉及在颅骨上钻一个毛刺孔，然后将针头穿过大脑进入侧脑室。这会产生整个大脑的转导。然而，该手术可能导致颅内出血，并且该方法通常仅产生有限的脊髓转导6。注射到颅底的脑池大池中侵入性较小，但有损伤脑干的风险。虽然在动物研究中经常使用5，但注射到大池中在临床上不再常规使用7。腰椎穿刺是进入脑脊液的侵入性最小的方法。这包括在两个腰椎之间放置一根针并插入腰椎池。
用于载体输送的腰椎穿刺在成年大鼠和小鼠以及新生小鼠中常规进行 8,9。这项研究的作者最近对幼年大鼠（28-30 日龄）进行了腰椎穿刺，以提供腺相关病毒血清型 9 （AAV9） 载体。在成年大鼠中，将新生儿腰椎穿刺针垂直放置在 L3 和 L4 椎骨之间9。正确放置会导致尾部甩动和脑脊液向上流入针头储液器。然而，在幼年大鼠中，这些读数都无法实现。然后，作者尝试使用以 L5 和 L610 之间的角度插入的 27 G 胰岛素注射器来适应成年小鼠程序。在通常小于P28大鼠的成年小鼠中，这不会产生尾巴甩动，但是通过注射剂的回流可以明显看出不正确的针头放置。然而，在幼年大鼠中，这种方法一致地导致注射剂是硬膜外给药的，这可能是由于成年小鼠和幼年大鼠之间脊髓周围组织层的弹性不同所致。接下来评估导管入路。具体来说，通过腰椎池硬脑膜的切口引入导管，直至胸椎中脊髓;然而，这种方法导致注射液在分娩过程中大量回流出切口部位。尝试使用导针经皮将导管插入鞘内腔也未成功。由于椎板间宽度较窄，导管可能会撞击喙部椎板而无法前进。
在这里，描述了一种 通过 在幼年大鼠中通过腰椎穿刺实现成功且可重复的溶液递送的方法。这种方法可用于病毒载体，也可能用于细胞、药物和其他疗法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200 &#181;L filtered pipette tips</td>
			<td>MidSci</td>
			<td>PR-200RK-FL</td>
			<td>Pipetting virus</td>
		</tr>
		<tr>
			<td>AAV9-GFP</td>
			<td>Vector Builder</td>
			<td>P200624-1005ynr</td>
			<td>AAV9 vector expressing GFP</td>
		</tr>
		<tr>
			<td>Absorbable Suture with Needle Coated Vicryl Polyglactin 910 FS-2 3/8 Circle Reverse Cutting Needle Size 4 - 0 Braided</td>
			<td>McKesson</td>
			<td>J422H</td>
			<td>Suture</td>
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			<td>Bench pad</td>
			<td>VWR</td>
			<td>56616-031</td>
			<td>Surgery</td>
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		<tr>
			<td>Braintree Scientific Isothermal Pads, 8&#39;&#39; x 8&#39;&#39;</td>
			<td>Fisher Scientific</td>
			<td>50-195-4664</td>
			<td>Maintains body temperature</td>
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			<td>Buprenorphine</td>
			<td>McKesson</td>
			<td>1013922</td>
			<td>Analgesic</td>
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			<td>Buprenorphine-ER (1 mg/mL)</td>
			<td>Zoopharma</td>
			<td></td>
			<td>Extended-release analgesic</td>
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			<td>Cotton swabs</td>
			<td>Fisher Scientific</td>
			<td>19-365-409</td>
			<td>Blood removal</td>
		</tr>
		<tr>
			<td>Drape, Mouse, Clear Plastic, 12&#34; x 12&#34;, with Adhesive Fenestration</td>
			<td>Steris</td>
			<td>1212CPSTF</td>
			<td>Surgical drape</td>
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		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td>Forceps</td>
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			<td>Electric Blanket</td>
			<td>CVS Health</td>
			<td></td>
			<td>CVS Health Series 500 Extra Long Heating Pad</td>
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		<tr>
			<td>Eppendorf Research plus, 1-channel pipette, variable, 20&#8211;200 &#181;L</td>
			<td>Eppendorf</td>
			<td>3123000055</td>
			<td>Pipetting virus</td>
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			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14059-11</td>
			<td>Curved surgical scissors</td>
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			<td>Friedman-Pearson Rongeurs</td>
			<td>Fine Science Tools</td>
			<td>16121-14</td>
			<td>Laminectomy</td>
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		<tr>
			<td>Halsey Needle Holders</td>
			<td>Fine Science Tools</td>
			<td>12001-13</td>
			<td>Needle driver</td>
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		<tr>
			<td>Insulin Syringes with Ultra-Fine Needle 12.7 mm x 30 G 3/10 mL/cc</td>
			<td>BD</td>
			<td>328431</td>
			<td>Syringe</td>
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			<td>Isoflurane</td>
			<td>McKesson</td>
			<td>803250</td>
			<td>Anesthetic</td>
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		<tr>
			<td>Isopropanol wipes</td>
			<td>Fisher Scientific</td>
			<td>22-031-350</td>
			<td>Skin disinfection</td>
		</tr>
		<tr>
			<td>Lidocaine, 1%</td>
			<td>McKesson</td>
			<td>239935</td>
			<td>Local anesthesia</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-137</td>
			<td>Loading the syringe</td>
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		<tr>
			<td>Povidone-iodine</td>
			<td>Fisher Scientific</td>
			<td>50-118-0481</td>
			<td>Skin disinfection</td>
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		<tr>
			<td>Scalpel Handle - #4</td>
			<td>Fine Science Tools</td>
			<td>10004-13</td>
			<td>Scalpel blade holder</td>
		</tr>
		<tr>
			<td>Sure-Seal Induction Chamber</td>
			<td>Braintree Scientific</td>
			<td>EZ-17</td>
			<td>Anesthesia box</td>
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		<tr>
			<td>Surgical Blade Miltex Carbon Steel No. 11 Sterile Disposable Individually Wrapped</td>
			<td>McKesson</td>
			<td>4-111</td>
			<td>#11 Scalpel blade</td>
		</tr>
		<tr>
			<td>SYSTANE NIGHTTIME Eye Ointment</td>
			<td>Alcon</td>
			<td></td>
			<td>Eye ointment</td>
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			<td>Trypan Blue</td>
			<td>VWR</td>
			<td>97063-702</td>
			<td>Injection</td>
		</tr>
	</tbody>
</table>

intrathecal vector delivery in juvenile rats via lumbar cistern injection

基因治疗是一项强大的技术，可以将新基因传递给患者以治疗疾病，无论是引入功能基因、灭活有毒基因，还是提供其产物可以调节疾病生物学的基因。治疗载体的给药方法可以采取多种形式，从用于全身给药的静脉输注到直接注射到靶组织中。对于神经退行性疾病，通常希望将转导偏向大脑和/或脊髓。针对整个中枢神经系统的侵入性最小的方法涉及注射脑脊液 （CSF），使治疗药物能够到达中枢神经系统的大部分。将载体输送到脑脊液的最安全方法是腰椎鞘内注射，将针头引入脊髓的腰椎池。这种技术，也称为腰椎穿刺，已广泛用于新生儿和成年啮齿动物以及大型动物模型。虽然该技术在物种和发育阶段之间是相似的，但鞘内空间周围组织的大小、结构和弹性的细微差异需要在方法中进行调整。本文介绍了一种在幼年大鼠中进行腰椎穿刺以递送腺相关血清型 9 载体的方法。在这里，将 25-35 μL 载体注射到腰椎池中，并使用绿色荧光蛋白 （GFP） 报告基因评估每次注射产生的转导谱。讨论了这种方法的好处和挑战。

为了确定注射技术的准确性，使用染料台盼蓝作为治疗剂的替代物。这种染料很容易与蛋白质结合，因此它通常保持在注射到的结构内。这意味着染料可能无法准确预测治疗剂的注射后分布;它只是用来揭示注射的准确性。当成功引入腰椎池时，台盼蓝会与硬脑膜结合，染上脊髓周边的蓝色。然而，当针头无法穿透硬脑膜时，染料最终会进入硬膜外腔。硬脑膜和周围组织（骨骼表面以及连接椎板的...

Watch this Scientific Journal Video about Author Spotlight: Assessing Intrathecal Gene Therapy Efficacy in Juvenile Rats at JoVE.com

作者聚焦:评估幼年大鼠的鞘内基因治疗疗效

描述了一种外科手术，用于向幼年大鼠的腰椎池进行注射。这种方法已被用于基因治疗载体的鞘内递送，但预计这种方法可用于多种疗法，包括细胞和药物。

通过腰椎池注射在幼年大鼠中鞘内载体给药

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...

我们的研究重点是开发针对影响中枢神经系统的疾病的基因疗法。截至 2023 年 12 月，FDA 已批准基于 AAV 的基因疗法用于少数基因库，包括脊髓性肌萎缩症、Leber 先天性黑朦、杜氏肌营养不良症以及血友病 A 和 B 。为遗传疾病设计安全有效的干预措施需要利用一系列方法，从对患者进行仔细的自然史研究到组织培养和动物模型的临床前工作，而基因治疗实际上只是用于此目的的几种技术之一。许多基因治疗实验使用新生儿或成年动物。很少有人评估青少年的治疗，尽管这代表了遗传疾病的重要患者群体。因此，对于年龄较大的儿童来说，体型和免疫系统对基因疗法的静脉给药构成了重大障碍。鞘内给药可能会克服这一点，但我们需要适当的模型来检验这一假设。我们开发的这种程序将使我们能够评估这种方法在幼年大鼠中的疗效。

intrathecal-vector-delivery-juvenile-rats-via-lumbar-cistern

Research

JoVE Journal

Neuroscience

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

837 次观看.  Emory University. 我们的研究重点是开发针对影响中枢神经系统的疾病的基因疗法。截至 2023 年 12 月，FDA 已批准基于 AAV 的基因疗法用于少数基因库，包括脊髓性肌萎缩症、Leber 先天性黑朦、杜氏肌营养不良症以及血友病 A 和 B 。为遗传疾病设计安全有效的干预措施需要利用一系列方法，从对患者进行仔细的自然史研究到组织培养和动物模型的临床前工作，而基因治疗实际上只是用于此目的?...

视频: 作者聚焦:评估幼年大鼠的鞘内基因治疗疗效

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

科研

神经科学

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 &#181;L; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice

The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice.

在成年小鼠中的亚型腺相关病毒9（AAV9）载体递送

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Laminotomy 对猪腰背根神经节通路和注射的治疗

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

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

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

脂多糖注射液致大鼠亚急性脑 Microhemorrhages

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

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS.&#160;Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

重组腺相关病毒在成年小鼠中的直接注射

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

直接注射慢病毒载体突出大鼠脊髓中的多种运动途径

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

通过对流增强的传递将抗体输送到毛里脑

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

大鼠中枢神经系统中反义寡核苷酸的细胞内传递

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement.

通过宫内注射诱导利普托门宁皮细胞修饰

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the ...