We thank members of the Mariani lab for critical reading of the manuscript. Our funding sources were: the Baxter Medical Scholar Research Fellowship (to M.K.S.), USC undergraduate fellowships and the Provost, Dean Joan M. Schaeffer, and Rose Hills Fellowships, (to M.K.S.). We also acknowledge a CIRM BRIDGES fellowship through Pasadena City College (to T.T.T). and the James H. Zumberge Research and Innovation Fund, the USC Regenerative Medicine Initiative, and the NIAMS NIH under Award Number R21AR064462 (to F.V.M).

注：所有的程序都是按照批准的机构动物护理和使用委员会（IACUC）在南加州大学的动物协议。 1.准备手术<ol><li>奠定了必要的外科手术工具和用品（消毒，项目1 - 18 材料清单），并安排一个解剖显微镜和光纤反式照明在引擎盖上或下沉气流表格。 </li><li>将一个性成熟的小鼠（20 - 30克，1 - 3个月）的感应室4％异氟醚诱导麻醉。 </li><li>确认麻醉用爪子和/或尾掐测试。 </li><li>适用软膏鼠标的眼睛，以避免手术过程中眼睛干涩。 </li><li>将鼠标移动到显微镜阶段。将鼠标进入检修软管的鼻锥和横向定位。提供邻近于动物的暖袋，以帮助维持体温。 不E：鼠标的位置是依赖于个人的喜好和/或外科医生的霸道，无论是左边或右边是好的。 </li><li>调整异氟醚为2 - 3％的维修，看呼吸频率。 </li><li>注入丁丙诺啡（0.05毫克/千克）经皮下（横向腿的侧翼），用于手术后疼痛与地下25针。腿可以抽搐这是一种正常的反应。 </li></ol> 2.切口开幕<ol><li>检测所需肋通过触诊，并清除了剃须刀的头发的区域。 注：肋8-10被推荐，因为肋1-7更接近肺和11-13都相当短。计数肋骨应该开始用最喙肋骨为1号。 </li><li>制备具有交替的聚维酮碘和异丙醇（70％）擦拭（3×常被推荐由动物使用的协议）的区域。 </li><li>通过皮肤切成2厘米的切口，正上方，并平行于与中型显微剪刀所需的肋。 Inc这是E到下面的肌肉和脂肪层。 </li><li>将所有3层（皮肤，肌肉，脂肪）到牵开器，以暴露手术区，同时最小切口的大小。 </li></ol> 3.切除肋骨<ol><li>通过肋间肌覆盖肋的所需部分与5.0毫米手术刀切割。位置周围5毫米近侧此切口的chondrocostal接头，这是其中的肋并不像高度弯曲。从小心地用细尖镊子骨分离肌肉。 </li><li>创建一个切除，同时保留在动物的骨膜，切断通过骨膜，沿肋的带5.0毫米手术刀的长度。仔细分离的骨膜从底层骨横向用细尖镊子。操作时要小心，因为骨膜十分细腻并有着一种凝胶状的一致性。 </li><li>在下使横截面穿过骨在一端与细微型剪刀。如果需要的话，测量切除掩模版在显微镜或直尺型测量仪。然后小心地将骨出骨膜和切断的另一端。 注：请谨慎操作。这是最微妙的一步，因为拉起不低于切除肋骨撕裂胸膜，造成气胸。如果胸膜膜被撕裂，这将是容易地明显的，因为胸腔的内部将变得可见。 </li><li>如果出血发生切割骨的结果，将压力施加于切割端用棉签4 - 5秒来止血。 注：在罕见场合继续出血，这可能是必要的，以停止和安乐死的动物作为显著失血会危及恢复（无补液，最大血液量可以安全地丢失是总血液体积的10％或7.7 - 8微升/克对于25克鼠标，这相当于约180 - 200微升9）。 </li><li>立即将取出的肋骨成4％PFA供日后分析。 注意：如果切除两个骨和骨膜的是要进行，省略了骨膜切口和分离（ 步骤3.2）。由于骨膜​​非常紧密附着于胸膜谨慎行事，挑逗肋远离胸膜用钳子以避免撕裂。 </li></ol> 4.关闭切口<ol><li>缝合肋间肌在剩余骨膜套筒用9-0缝合线（2缝线通常足够了）的顶部。放置缝线的肋的切断端部的正上方，作为指标，对手术位置。 </li><li>取出牵开。缝合覆肌肉和脂肪用9-0缝线（3 - 4缝线通常就足够了）。小鼠肌肉和/或脂 ​​肪的特别厚的层，分别缝合各层（ 例如，1层的缝合线的肌肉层和1层缝线的脂肪层）。 </li><li>关闭皮肤缝合7-0（4 - 5缝线通常就足够了）。 </li><li>确保切口缝合胶水，用大钳子捏边在一起。 </li><li>慢慢地戒掉鼠标关闭异氟醚，首先需要调节至1％的几分钟，然后熄灭。 </li><li>将鼠标下加热灯，并离开，直到意识恢复，5 - 10分钟。后意识恢复，并在整个愈合期，鼠标应该移动和走动正常并显示苦恼的迹象。 </li></ol> 5.恢复和分析<ol><li>提供术后疼痛管理，包括丁丙诺啡在0.5毫克口服明胶的形式管理/ kg，每12小时48小时。口服给药不需要抑制这可能会导致疼痛的胸部区域的动物。 </li><li>保持鼠标与在愈合期间自由获取食物和水的笼子里。后切口是干的，雌性动物可以共同收纳而男性s必须保持隔离，以防止战斗。 </li><li>愈合期后，按照批准的程序进行安乐死。拆下肋骨用于固定和分析。 </li></ol>

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The authors have nothing to disclose

当第一次学习此协议，确定在哪里可以找到最初的切口可以是具有挑战性。然而，在小鼠安乐死的做法可以帮助外科医生学会在哪里放置初始切口，暴露所需的肋骨被切除。工作在尸体也提高到具有或不具有骨膜除去肋部分所需的精细运动技能。此外，新的人到该过程可能会发现操纵细的工具和薄缝线是困难的。而打结，多余的拉动缝线可以撕裂组织。因此，一些实践缝合也对尸体可能是有帮助?...

衰弱的骨骼损伤，慢性骨关节炎，以及与重建手术相关的严重问题影响经济生产力，家庭幸福，和生活质量。而小断裂和损伤可以治愈还算不错，人类是不能修复大的缺陷，因此必须依靠重建手术，恢复的结构和功能。重建可能涉及同种异体或heterogeneic移植，骨morcellized，植入支架，或牵张成骨。不幸的是，不仅有与这些处理，但在修复骨骼的原始强度是很少获得持久性相关的发病率的因素。因此，需要新的临床方法。 单向开发创新的方法来治疗节段性缺损的研究中，大型维修时，自然的情况。著名的两栖动物可以再生骨骼元素，而哺乳动物被认为是有限的日是能力。然而，由于20 世纪初期，再生在人肋几个报告已经发表表明人类可能不被如此限制1-4。目前这种现象，最好由整形外科医生谁使用肋材料颚，脸和耳重建已知的，但它不是更广泛地理解5。为了研究这种修复中更详细地说，我们已经开发出使用鼠标的外科模型。使用该协议，研究人员可以识别所涉及的先天因素，并使用该信息，以便在其它地点的骨骼的愈合。 有许多优势，使用肋骨作为模型来研究骨骼修复。首先，将周围的肋提供自然固定器（相对于切除股骨6,7）。这降低了内部和外部固定器的发病风险并简化了外科手术。胸部西澳其次，薄肌层会提供易于访问和良好的能见度使得检测媲美颅骨切除8的便利。第三，在对比其通过膜内骨化，由软骨内骨化肋形式形成，并通过延伸在位于中央骨干的任一端生长板生长长度的颅骨。因此，修复肋可能更媲美修复长骨的阑尾骨架组成。此外，我们已经发现，相对于股骨，肋的骨膜较厚，可以更容易地操纵。因此，调查谁愿意测定骨修复的研究骨膜或测试细胞疗法，药物制剂，和/或组织支架的目的，可能会发现这种手术模式非常有用。总之，这个肋切除模型提供在其内来研究天然大型骨修复哺乳动物中一般使用没有这样的模型当前存在的上下文。

<table border="0" cellpadding="0" cellspacing="0" width="896">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Name of Reagent/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:304px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">Medium sized micro-dissection scissors (Vannas-T&#252;bingen Spring Scissors 5 mm)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Fine micro-dissection scissors (Vannas Spring Scissors - 2mm Cutting Edge)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">15000-04</td>
			<td>curved tip is beneficial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Micro-scalpel 5.0 mm</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>10315-12</td>
			<td>other fine scalpels can be substituted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Dumont 55 forceps</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">11295-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Retractor&#160;</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>17004-05</td>
			<td>adjustability is convenient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Micro-needle holders</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">12060-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">9.0 nylon sutures (Ethilon), taper point best</td>
			<td style="width:160px;">Ethicon</td>
			<td>2819G or similar</td>
			<td>taper point best but reverse cutting is also good</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">7.0 prolene sutures (Prolene)</td>
			<td style="width:160px;">Ethicon</td>
			<td>8700H or similar</td>
			<td>6-0 can be used too, needle point can vary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Large forceps (Adson Forceps)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">11006-12</td>
			<td>other brands are fine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Lubricant Eye Ointment (Akwa Tears)</td>
			<td style="width:160px;">Akorn</td>
			<td style="width:157px;">17478-062-35</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Suture glue (GLUture Topical Tissue Adhesive)</td>
			<td style="width:160px;">Abbot</td>
			<td style="width:157px;">32046-01</td>
			<td>has excellent working time</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Shaver</td>
			<td style="width:160px;">Wahl</td>
			<td>9918-6171 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Clamp lamp</td>
			<td>Zoo Med</td>
			<td style="width:157px;">LF-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Infrared Bulb, 75W</td>
			<td style="width:160px;">Zoo Med</td>
			<td>RS-75</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:275px;">RC2 Rodent Anesthesia System</td>
			<td style="width:160px;">VetEquip&#160;</td>
			<td style="width:157px;">922100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">IsoFlo (Isoflurane)</td>
			<td style="width:160px;">Abbot</td>
			<td style="width:157px;">05260-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Buprenorphine (Buprenex)</td>
			<td style="width:160px;">Reckitt Benckiser</td>
			<td style="width:157px;">12496-0757-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Betadine</td>
			<td style="width:160px;">Purdue Frederick</td>
			<td>67618015017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Flavored Gelatin, raspberry</td>
			<td style="width:160px;">Jell-O</td>
			<td>B000E1FYL0</td>
			<td>made up firm, to the consistency of &#39;jigglers&#39;</td>
		</tr>
	</tbody>
</table>

a surgical procedure for resecting the mouse rib: a model for large-scale long bone repair

该协议研究人员介绍，为大规模的骨修复利用鼠标肋骨的新模式。该过程详细描述了以下内容：准备动物进行手术的，打开胸体壁，从周围肋间肌曝光所需的肋，切除肋的所需部分，而不诱导气胸，并关闭所述切口。相较于四肢骨骼的骨头，肋骨都非常方便。此外，没有任何的内部或外部固定器是必要的，因为在相邻的肋提供自然的固定。手术采用市售的用品，很简单易学，且耐受性良好的动物。该过程可以进行具有或不具有除去周围骨膜，因此骨膜修复的贡献可以被评估。结果表明，如果被保留的骨膜，鲁棒修复发生在1 - 2个月。我们希望利用这个协议会鼓励研究到肋骨修复，而结果将有利于新的方式来刺激骨修复等身体周围地区的发展。

动物通常此过程中快速恢复，切口愈合良好，动物表现出正常的行为。该过程使用市售的用品和实践后，可在1小时内完成的。使用此过程来产生的实验数据前，有必要分析切除在第0天时间点，以确定是否有任何大的骨碎片可能会保留在切除区域内是重要的。评估适当的程序的一种方法是，以评估动物用X射线或显微成像术后，尽管这通常需要特别的辐射的安全认证。相反，另一种简单的评估可以?...

Watch this Scientific Journal Video about A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair at JoVE.com

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

外科手术切除为鼠标肋骨：模型进行大规模的长骨修复

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

a-surgical-procedure-for-resecting-mouse-rib-model-for-large-scale

Research

JoVE Journal

Medicine

13.0K 次观看.  Keck School of Medicine, University of Southern California. The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

视频: A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

科研

医学

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.

Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application.

外科技术治疗脊髓交付：在哥廷根小型猪演示程序

This is a  compact visual description of a combination of surgical technique and device  for the  delivery of (gene and cell) therapies into the spinal ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对无瘢痕修复鼠标胎儿皮肤模型

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

老鼠圆窗途径耳毒性剂递送：一个快速和可靠的技术诱导耳蜗细胞变性

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

动静脉内瘘失败的新型小鼠模型：在详细介绍手术过程

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

猪胫骨移植术后血管生成的动物模型

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

评估在鼠标模型中执行唤醒膀胱测压的过程

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

一种非人类灵长类常见狨中内耳药物的外科手术方法 (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

手术小鼠模型中骨关节炎的标准化形态学评价

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...