Name: a mouse model of orthopedic surgery to study postoperative cognitive dysfunction and tissue regeneration
Uploaded: 2018-02-27T13:00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration at JoVE.com

我们感谢凯西量具, BS (麻醉学系, 杜克大学医学中心, 达勒姆, NC) 的编辑协助。NT 承认来自杜克麻醉学和 NIH/NIA R01 AG057525-01 的梦想创新补助金的支持。

所有动物实验都是按照国家卫生研究院的实验室动物护理和使用指南进行的, 并经杜克大学机构动物护理 & 使用委员会 (IACUC) 批准。
1. 实验动物
<ol><li>将老鼠留在空调环境中, 用12小时的光/暗循环, 并适当地获得标准的食物和水。房子不超过5窝每笼, 并避免条件, 可以鼓励战斗。</li>
	<li>使用雌性 C57BL6/J 小鼠在3月的年龄 (年轻) 或18月的年龄 (老)。</li>
	<li>对于联体共生, 在手术前至少两周将2只老鼠结合在一起。老鼠要么是野生型 C57BL6/J, 要么是 eGFP +。</li>
	<li>每天检查小鼠的身体状况和整体外观。</li>
</ol>2. 联体共生和骨科手术的术前设置
<ol><li>重2只老鼠。</li>
	<li>在恒定的 O2流率为0.2 升/分的情况下, 通过麻醉系统管理全身麻醉. 使用5% 异氟醚在感应腔内诱导。 注意: 麻醉深度可以用脚趾捏来确认。</li>
	<li>把老鼠放在一个仰卧位置的加热垫上。使用直肠探头在整个手术过程中监测体温。</li>
	<li>为了保持麻醉, 将异氟醚吸入浓度降低到 2.0%, 在0.2 升/分钟通过面罩。监测, 以确保足够的麻醉深度。</li>
	<li>使用脉冲脉搏(可选)监测生理参数 (呼吸速率、氧饱和度和心跳)。</li>
</ol>3. 联体共生手术 (1A 号协议)
<ol><li>术后镇痛 (丁丙诺啡, SR/缓释, 0.1 毫克/千克皮下)。在开口前先在侧面注射布比卡因 (0.25%)。涂抹眼部润滑剂。 注意: 联体共生协议可以独立于胫骨骨折进行。</li>
	<li>对于所有内体缝合, 使用 4-0 polydioxanone 缝合材料。对于所有外部缝合, 使用4-0 聚丙烯缝合材料。</li>
	<li>将2只小鼠从肘部、侧面和膝部的相邻线上剃去, 并将其连接在一起。用碘 + 70% 乙醇皮肤擦洗3交替循环, 准备切口。 使用蒸压仪器, 保持无菌场。 注意: 将野生类型的鼠标放在 eGFP 鼠标的右侧, 并准备 eGFP 鼠标的左侧面和用于外科手术的右侧面 (图 1)。</li>
	<li>在每只老鼠身上, 用剪刀做一个沿侧面的皮肤切口, 范围近到膝盖, 只是接近肘部, 而不会扰乱皮肤下面的肌肉。</li>
	<li>加入2中断缝合的动物的三头肌。</li>
	<li>通过连续的7-9 刀道缝合, 沿侧面连接身体壁。</li>
	<li>加入2打断缝合的动物的四头肌。</li>
	<li>关闭 2 parabionts 的皮肤与中断缝线。</li>
	<li>让老鼠在周围的空气中醒来。</li>
	<li>为确保此协议的成功, 必须维护对的健康: 每天监视对对;每周两次进行身体状况评分;每周两次称对。 注: 每对单独存放。 注: 如果使用常规丁丙诺啡 (即不 SR/缓释), 然后管理0.1 毫克/千克皮下1毫升生理盐水每只老鼠每日两次, 3 天。</li>
	<li>允许4周的恢复时间, 以确保 parabionts 之间的共享循环, 如果执行胫骨骨折手术。</li>
</ol>4. 胫骨骨折手术 (1B 号协议)
<ol><li>术后镇痛 (丁丙诺啡, 0.1 毫克/千克皮下注射)。注射布比卡因 (0.25%) 在手术部位近膝前的开口。涂抹眼部润滑剂。</li>
	<li>剃去鼠标右后肢内侧, 暴露手术部位, 用碘 + 70% 酒精皮肤擦洗3交替循环消毒。在整个过程中保持一个无菌的手术场。限制污染;使用蒸压器具和手套;在解剖显微镜下完成手术 (可选);并使用加热垫来保持体温。 注意: 如果协议是在 parabionts 上执行的, 则只有1只鼠标在两个 (右侧的鼠标右胫骨) 中断开。有关 parabionts 上的断开的示意图, 请参见图 1B 。</li>
	<li>使用剪刀, 使一个皮肤切口沿右后肢的内侧侧面的膝盖下部到中段的胫骨在右鼠。</li>
	<li>暴露胫骨的中段, 并在视觉上找到骨干。弹性膝盖和想象胫骨平台使用髌骨股骨韧带作为一个里程碑。</li>
	<li>想象髌骨肌腱;用拇指和食指手动钻孔, 用25口径的针在髓内管中使用0.5 毫米孔。 注: 钻孔将沿胫骨平行运行, 通过胫骨平台。</li>
	<li>插入一个0.38 毫米不锈钢针通过孔约15毫米进入髓腔, 直到阻力, 并削减与胫骨高原使用线切割机 (请参阅辅助视频 1 3D 重建)。</li>
	<li>使用直波恩剪刀, 骨折胫骨中段 (骨干)。有关断开的示意图, 请参见图 1C 。</li>
	<li>观察骨折部位和相邻组织检查骨折视力是否稳定。</li>
	<li>与真皮主食紧密结合。</li>
	<li>把老鼠放在加热的垫子上, 恢复到干净的家庭笼子里。注射1毫升 prewarmed (37 °c) 正常生理盐水皮下在每只老鼠的液体置换。</li>
	<li>每天检查老鼠是否有跛行、感染或流血的迹象。 注: 如果使用常规丁丙诺啡 (即不 SR/缓释) 然后管理0.1 毫克/千克皮下1毫升生理盐水每日3天。</li>
</ol>

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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=beta2-microglobulin+is+a+systemic+pro-aging+factor+that+impairs+cognitive+function+and+neurogenesis.">beta2-microglobulin is a systemic pro-aging factor that impairs cognitive function and neurogenesis.</a> Nat Med. 21 (8), 932-937 (2015).</li><li>Katsimpardi, L., 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=Vascular+and+neurogenic+rejuvenation+of+the+aging+mouse+brain+by+young+systemic+factors.">Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors.</a> Science. 344 (6184), 630-634 (2014).</li><li>Sinha, 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=Restoring+systemic+GDF11+levels+reverses+age-related+dysfunction+in+mouse+skeletal+muscle.">Restoring systemic GDF11 levels reverses age-related dysfunction in mouse skeletal muscle.</a> Science. 344 (6184), 649-652 (2014).</li><li>Castellano, J. 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=Human+umbilical+cord+plasma+proteins+revitalize+hippocampal+function+in+aged+mice.">Human umbilical cord plasma proteins revitalize hippocampal function in aged mice.</a> Nature. 544 (7651), 488-492 (2017).</li></ol>

作者没有什么可透露的。

骨折是常见的临床问题, 仍然是发病率的主要原因, 特别是在快速增长的老年人群中。在这里, 我们引入了一步一步的协议, 一个小鼠胫骨骨折模型的研究机制负责术后 neuroinflammation 和认知障碍。该模型可与联体共生手术结合, 研究神经免疫相互作用, 组织再生和其他信号过程。了解这些机制将提供战略目标, 以尽量减少术后并发症的风险和优化结果。
为研究啮齿目动物的骨修复, 开发了几种矫形模型25。我们采用和修改了这个胫骨骨折的程序, 最初描述哈利et 等。8, 研究骨科手术对脑功能的影响。我们还使用这个骨折模型结合我们的联体共生模型, 以调查的因素, 负责骨愈合和年龄依赖性的组织再生。当在不稳定的全麻下进行时, 这种胫骨骨折程序只需要大约15分钟的每只动物, 结果为零到最低死亡率 (取决于老鼠的年龄和潜在的遗传敏感性), 和概括共同与长骨骨折和骨科手术创伤有关的侮辱。因此, 该模型是最理想的审讯生物通路和执行纵向评估。然而, 重要的是, 截骨和固定是可重复的, 对软组织的损害是一致的。软组织损伤可以调制, 例如通过剥离骨膜和捏周围的肌肉, 使手术更具创伤性。外伤性骨折不固定钝伤或三点弯曲的模型不能提供这样的一致性或准确性。这些程序往往导致再次伤害, 导致长期炎症反应。相反, 涉及刚性固定的骨折模型有一个更温和的炎症, 不完全重述与骨科手术有关的损害26,27。
其他使用钛合金钉扎的模型也被开发来密切模仿人的人工关节成形术, 可能更适合于在小鼠中审问假体不稳定性、骨溶解和假体相关并发症28,29.钻孔模型, 如这里提出的, 提供足够的稳定, 和小鼠可以测试的行为范式没有严重的赤字, 可能会混淆这样的任务, 如恐惧调理或开放领域运动/焦虑测试6 ,7,11,15,19,20。但是, 如果固定不正确锁定, 则会发生旋转畸形。一些模型使用外固定器, 它提供了优越的稳定性, 但在小鼠胫骨中实现是挑战, 虽然它可以成功地实现在鼠标股骨27。
包括谵妄和术后认知功能障碍在内的认知障碍是骨科骨折修复术后常见的并发症, 特别是在老年和虚弱患者中,30。本临床相关的小鼠胫骨骨折手术模型表明, 术后系统性细胞因子释放6,7,17, 受损血脑屏障功能15,19和改变的胶质形态学16,22, 导致记忆障碍, 并可能代表手术后许多患者术后神经系统并发症的关键特征。值得注意的是, 其他手术方法已被用于模型的术后认知功能障碍的小鼠。这些包括腹部31,32,33和血管34手术, 以及肤浅的创伤35,36。联体共生技术适用于所有这些模型, 它们共享类似的端点, 包括炎症、胶质活化和行为缺陷, 这可能由共同的机制介导。
包括联体共生在内的研究揭示了影响衰老动物认知功能、neuroinflammation 和组织复兴的循环因素的新作用 37, 38, 39, 40 ,41,42。我们已经表明, 联体共生可以成功地结合在这里描述的胫骨骨折模型, 以审问再生通路和研究机制涉及血液传播因素, 影响愈合和骨折修复 12.在这里, 我们证明, 当年老的动物与幼兽吻合时, 衰老动物的骨折修复能力是可以恢复的。这种年龄逆转的根源在于骨折部位造血细胞的植入。有趣的是, 通过将幼骨髓移植到老龄小鼠身上, 也可以实现这种复兴。在这方面, 骨髓移植可以被认为是一种更直接和更简单的替代方法联体共生。然而, 联体共生是一种比较健壮的模型, 用于研究循环细胞和因子的功能。我们预计, 结合联体共生和骨科手术模型将发挥重要作用, 回答关键问题的围手术期护理和老化生物学。
总之, 我们介绍了一步一步的协议, 为一个小鼠胫骨骨折模型的研究机制负责的手术后 neuroinflammation 和认知障碍的矫形外科手术程序。该模型可与联体共生程序结合, 研究神经免疫相互作用, 组织再生和其他途径。定义这些机制将提供战略目标, 以尽量减少术后并发症的风险和优化结果。

手术改变了医疗保健系统, 并不断促进尖端技术, 改善安全, 并保持生活质量。不幸的是, 手术还诱发病理生理反应, 可能导致术后并发症, 包括伤口感染, 神经损伤, 甚至死亡率, 特别是在老年患者1,2。骨科手术是例行的, 特别是在老年人, 以改善生活质量和修复常见的骨骼损伤。然而, 多达50% 的骨科手术患者谁是65岁以上的经历神经损伤, 如术后谵妄。这与预后不佳有关,即, 5 倍增加6月死亡率的风险, 持续功能下降, 每位病人的护理时间增加, 住院期限延长, 以及护理家庭安置率较高3,4,5. 已经确定了一些危险因素, 包括晚期年龄, 但对于手术后神经系统损伤的机制知之甚少。
由于骨折在老年人中很常见, 我们建立了一个小鼠胫骨骨折模型, 以确定周围创伤对术后恢复的影响, 包括 neuroinflammation 和脑健康 (认知功能)6,7. 此模型最初由哈利et . 描述。8, 由髓内钉和胫骨骨折在全身麻醉和镇痛, 从而模仿皮肤损伤, 肌肉创伤, 骨修复相关的常见长骨骨折和修复的人。在这个过程之后, 老鼠演示炎症标记物的变化类似于人类观察到的变化9,10, 以及胶质激活在海马, 这是与赤字的声明性记忆和海马可塑性6,7,11。我们以前把这个断裂模型和联体共生结合在一起。联体共生是一种外科模型, 其中2只小鼠吻合, 从而共享循环系统。该模型为了解循环细胞和体液因子对器官功能的调节作用在年龄和疾病的背景下, 提供了突破12,13,14。使用这种方法, 我们最近发现了与年龄相关的骨折愈合相关的系统性因素12。
在这里, 我们引入了一个协议, 结合胫骨骨折模型与联体共生研究骨对脑年龄依赖性机制, 这是有关的再生医学和神经免疫学。协议1A 描述了联体共生过程, 协议1B 详细说明了胫骨骨折过程 (图1A)。根据审讯的性质, 可以独立或组合执行这些操作。

<table width="1234">
	<tbody>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Isoflurane</td>
			<td style="width:272px;">Piramal Healthcare</td>
			<td style="width:175px;">NDC 66794-017-25</td>
			<td style="width:577px;">Other volatile agents or injectable anesthesia can be also used</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Buprenorphine</td>
			<td>Reckitt-Benckiser Pharmaceuticals</td>
			<td>NDC 12496- 6757-1</td>
			<td>Optional and depending on individual Institutional Animal Care and Use Committee recommendations</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Ethanol</td>
			<td>Fisher Scientific</td>
			<td>04-355-451</td>
			<td>70% solution for antiseptic treatment of skin and cleaning</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">10% povidone Iodine</td>
			<td>Dynarex</td>
			<td style="width:175px;"></td>
			<td>For antiseptic treatment of skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">SomnoSuite</td>
			<td style="width:272px;">Kent Scientific</td>
			<td>SS-01</td>
			<td>Low Flow&#160; Anesthesia system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MouseSTAT</td>
			<td style="width:272px;">Kent Scientific</td>
			<td style="width:175px;">PS1161</td>
			<td>Pulse Oximeter &#38; Heart Rate Monitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Shaver</td>
			<td style="width:272px;">Wahl</td>
			<td style="width:175px;">9854L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Stereomicroscope</td>
			<td style="width:272px;">Leica</td>
			<td style="width:175px;">MZ6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle</td>
			<td style="width:272px;">Fine science tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Blades - #11</td>
			<td style="width:272px;">Fine science tools</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps</td>
			<td style="width:272px;">Fine science tools</td>
			<td>11006-12</td>
			<td>Needed for stripping the periosteum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iris Forceps</td>
			<td style="width:272px;">Fine science tools</td>
			<td style="width:175px;">11066-07</td>
			<td>Useful (1x2 teeth) to causing localized muscle/soft tissue trauma</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Bonn Scissors (Straight)</td>
			<td style="width:272px;">Fine science tools</td>
			<td style="width:175px;">14084-08</td>
			<td>Good for osteotomy, note to change regularly as becomes blunt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Scissors</td>
			<td style="width:272px;">Fine science tools</td>
			<td style="width:175px;">14058-09</td>
			<td>Sharp scissors for cutting sutures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22G x 3.5 In Quincke&#160; Spinal Needle</td>
			<td style="width:272px;">BD</td>
			<td style="width:175px;">405181</td>
			<td>Use inner rod for pinning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle Holders</td>
			<td style="width:272px;">Fine science tools</td>
			<td>12001-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Suture</td>
			<td style="width:272px;">Look</td>
			<td style="width:175px;">1079B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">C57BL6/J</td>
			<td style="width:272px;">Jackson Laboratory</td>
			<td style="width:175px;">&#160;stock no. 000664</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:211px;height:63px;">eGFP+ (expressing enhanced green fluorescent protein ubiquitously)</td>
			<td style="width:272px;">Jackson Laboratory</td>
			<td style="width:175px;">&#160;stock no. 003291</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse model of orthopedic surgery to study postoperative cognitive dysfunction and tissue regeneration

手术通常用于改善和维持生活质量。不幸的是, 在诸如老年人等易受伤害的患者中, 可能会出现并发症, 并显著降低结果。事实上, 经过例行矫形外科手术来修复骨折, 多达50% 的老年患者患有谵妄等神经系统并发症。此外, 手术后愈合和再生组织的能力随着年龄的增加而减少, 并可能影响骨折修复的质量, 甚至是骨整合的植入物。因此, 更好地了解推动这些年龄依赖性变化的机制可以提供战略目标, 以尽量减少这种并发症的风险并优化结果。在这里, 我们介绍了一个临床相关的小鼠胫骨骨折模型。这些小鼠的术后变化模仿了人类常规骨科手术后常见的认知障碍。简单地, 在严格无菌条件下, 右后肢进行切口。肌肉是分离的, 一个0.38 毫米不锈钢针插入到胫骨上端, 髓内管内。然后进行截骨术, 并缝合伤口。我们使用这个模型来研究手术创伤对术后 neuroinflammation 和行为改变的影响。应用该骨折模型结合联体共生, 建立了2只小鼠吻合的手术模型, 研究了系统恢复器官功能和损伤后组织再生的细胞和分泌因子。通过遵循我们的循序渐进的协议, 这些模型可以复制高保真度, 并可以适应审问许多生物通路, 被手术创伤改变。

在无菌条件下, 在固定的针脚和正确缝合的情况下进行横截骨术后, 小鼠在手术后左腿没有跛行、感染或出血的迹象。骨科愈合的评估使用射线分析和形态计量学后番红染色 (图 2)。中段骨折胫骨的影像显示, 年轻小鼠骨折愈伤组织沉积比衰老小鼠骨折老茧多。骨折愈伤组织为脱钙, 嵌入石蜡中制备组织学分析。切片染色番红, 组织沉积的量化使用形态计量学分析。幼鼠骨折愈伤组织比衰老小鼠的骨折老茧含有更多的骨性和较少的纤维化。
胫骨骨折诱发全身和中央炎症6,7,11,15,16。事实上, 在骨科手术后, 在小鼠和人类7,17,18中, 抑的炎症细胞因子和危险相关分子模式的外围水平迅速升高。这有助于大脑中胶质细胞的活化, 通过多种信号机制, 包括体液, 细胞和神经通路7,15,19,20, 21。术后, 内皮功能障碍, 血脑屏障开放, 外周巨噬细胞浸润可导致野生类型的急性海马 neuroinflammation 和 Ccr2RFP Cx3cr1GFP/+成体小鼠15,19, 并与随后的内存不足相关联, 类似于人类谵妄和术后认知功能障碍15,19,22。这种 neuroinflammatory 反应在老年动物中加剧, 胶质形态学发生了显著变化, IBA-1 染色 (图 3) 检测到。
利用这里描述的胫骨骨折模型, 我们还发现了短暂性受损的海马神经发生, 这证明了 doublecortin (DCX) 染色在齿状回20中的减少。长期增益 (LTP) 的电生理学测量 (可塑性) 是记忆功能的替代品, 在术后的11中发现了时间依赖性的中断。手术后的小鼠也显示海马依赖性记忆功能的损伤, 例如使用恐惧调理行为评估 (图 4)。在恐惧调理中, 老鼠被放在一个房间里, 暴露在听觉提示之后, 然后是厌恶刺激 (即, 电击)。在胫骨手术三天后, 小鼠在调理室进行测试, 这一次没有任何听觉或厌恶刺激, 冻结行为被记录为内存索引 (详细协议请参阅23)。
高龄是记忆力下降的重要危险因素。然而, 随着年龄的推移, 组织修复和再生的能力也会减少, 尽管这些机制仍然缺乏理解。因此, 我们使用了这里描述的协议来执行 heterochronic 联体共生 (配对年轻/老动物) 和评估骨折愈合在老龄老鼠 (为详细的联体共生协议参见24)。parabiotic 对之间的血液共享已确认, 并发现等于12。接触年轻的循环加强骨修复与早期愈合, 增加骨沉积, 并减少纤维化 (图 5)12。这种恢复骨再生的发生独立于内源性骨钙素阳性成骨细胞 (图 5, 褐色细胞), 而是依赖于从年轻的 parabiont 迁移的 CD45-positive 细胞 (图 5, 蓝色细胞).这些结果表明, CD45-positive 造血细胞分泌一个年轻和健康的利基, 这是能够信号的老年成骨细胞诱导他们变得更加活跃。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56701/56701fig1.jpg"> 图 1: 联体共生和胫骨骨折手术的示意图表示法。(A) 用于执行联体共生和胫骨骨折 (协议 1A) 或仅胫骨骨折 (协议 1B) 的时间线。(B) 在等时或 heterochronic parabiotic 对中有20月大的老鼠胫骨骨折 (右脚的鼠标骨折, 如 X)。灰色小鼠描述 wildtype 小鼠, 而绿色小鼠描绘 eGFP 小鼠。(C) 带髓内钉和中轴压裂的胫骨骨折模型示意图。另请参阅辅助视频 1以3D 重建胫骨的后肢和钉住。<a href="//cloudflare.jove.com/files/ftp_upload/56701/56701fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56701/56701fig2v2.jpg"> 图 2: 在幼鼠和旧老鼠模型中胫骨骨折愈合.损伤后21天, 对胫骨小鼠的骨裂和骨折性老茧进行了研究。(A) 采用影像学和组织学染色 (番红) 对骨折后21天的骨性老茧进行评估。染色显示在红色的蓝色和软骨 (包含在软骨中) 的胶原组织。虚线表示骨折愈伤组织的近似位置。(B) 形态计量学用于评估骨折愈合后21天内沉积的骨、软骨和纤维化组织的数量。数据表示为平均95% 置信区间, * P < 0.05, 统计学意义 (单向方差分析, Dunnett 的测试), 鳞片杆代表2毫米, 和图像获得使用显微镜, 1.25x 目标。n = 9 为每个样品。<a href="//cloudflare.jove.com/files/ftp_upload/56701/56701fig2v2large.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/56701/56701fig3.jpg"> 图 3: 手术导致海马体的年龄依赖性胶质激活.与4月大的 C57BL6/J 小鼠相比, 胫骨骨折手术可诱发老年小鼠 (20 月大) 海马 neuroinflammation。胶质标记 IBA-1 的脑部染色显示术后24小时内手术组的阳性细胞和形态学改变。图像获得了 epifluorescent 显微镜放大100x。缩放栏代表10µm.<a href="//cloudflare.jove.com/files/ftp_upload/56701/56701fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56701/56701fig4.jpg"> 图 4: 胫骨骨折手术后的神经发生受损、远期增效和记忆功能.(A) DCX 是神经发生的定量标记, 在手术后24小时内海马齿状回明显减少。用共焦激光扫描显微镜的目标获得图像, 放大率为 10x;缩放栏代表10µm. (B) 手术后, 从控制或小鼠24小时的海马切片电生理学。长期增效 (LTP) 诱发高频刺激 (痉挛) 和记录超过1小时的现场兴奋后突触电位 (fEPSPs) 记录从 CA1 层 radiatum 使用细胞外记录吸管填充定期人工脑脊液。术后24小时, 与对照鼠相比, LTP 诱导明显降低。数据表示为平均值 s.e.m. n = 3, * p < 0.05 1 路方差分析。(C) 海马依赖性记忆功能 (定义为冻结使用跟踪恐惧调理的%) 在手术后小鼠与仅限于麻醉的控制和动物相比受到损害。数据表示为平均值 s.e.m. n = 9-10, * p < 0.05 1 路方差分析。<a href="//cloudflare.jove.com/files/ftp_upload/56701/56701fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56701/56701fig5.jpg"> 图 5: 联体共生手术导致骨折愈合、血液共享和捐献细胞植入的恢复。建立等时和 heterochronic 联体共生配对, 并对每对衰老的小鼠进行骨愈合的骨折和评估。(A) 使用射线成像对骨折老茧进行了研究。虚线表示骨折愈伤组织的近似位置。(B) 在骨髓中确认了 eGFP + 细胞的植入。(C) 骨折愈伤组织的免疫组化用于从 parabiont 中识别 eGFP+供体细胞 (蓝色) 和骨钙素+成骨细胞 (褐色)。刻度条代表50µm, 并使用40x 目标获得图像。<a href="//cloudflare.jove.com/files/ftp_upload/56701/56701fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
辅助视频 1: 3D 重建胫骨的后肢和钉住的.<a href="http://cloudflare.jove.com/files/ftp_upload/56701/parte_slow.wmv">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration at JoVE.com

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

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