作者承认来自 NSERC 发现赠款 (#05573) 和加拿大创新基金会 (#222084) 的研究经费用于资助活体微 CT. w 病房是加拿大骨骼和肌肉发育方面的研究椅。

这项研究得到了布洛克大学动物保育委员会的批准, 并按照加拿大动物保育理事会制定的指导原则21进行。
1. 使用异氟醚气体进行麻醉
<ol><li>预填充丙烯酸玻璃孵化室与高品质的 O2在一个连续的流速约1-2 升/分钟从一个麻醉机 (补充图 1)。</li>
	<li>先将大鼠转移到孵化室尾部, 然后关闭孵化室盖, 形成密闭密封。</li>
	<li>开始填补孵化室与兽医级的异氟烷在3-4% 伏/v 溶解在 O2在连续流速为1-2 升/分钟 (补充图 1)。 注意: 废弃的麻醉气体会对处理程序产生不利影响。一个清道夫系统 (即, 木炭过滤器或排气直接进入油烟罩) 必须始终到位。</li>
	<li>当老鼠不再能够站立, 将大鼠转移到面罩或鼻锥接受1-3% 的异氟醚在 O2中溶解, 流速为1-2 升/分. 大鼠主要通过鼻子呼吸, 所以, 只要鼻子被面罩或鼻子覆盖锥, 将有足够的麻醉分娩。</li>
	<li>将眼部润滑应用于眼部细腻的细胞膜上, 以保护它们免受任何逃逸的异氟醚气体。 注意: 确保眼部润滑无抗生素, 因为这可能影响干预的结果。</li>
	<li>测量眼睑 (眼睛闪烁反应, 轻柔刺激眼睑开口) 和踏板 (对捏的反应的后肢撤退);随着麻醉深度的增加, 在踏板反射之前眼睑反射将缺席 (补充图 2)。</li>
	<li>当达到适当的麻醉水平和鼠已经失去了眼睑和踏板反射, 维持在 0.5-2% 的异氟醚溶解在 O2在流量为1-2 升/分钟的大鼠。</li>
	<li>持续监测鼠的呼吸率整个过程中, 保持恒定的视觉上的鼠直接与内部监测系统或通过直播视频饲料 (补充图 3)。</li>
</ol>2. 大鼠后肢的定位和抑制
<ol><li>将老鼠放在碳纤维扫描仪床上的仰卧位上 (补充图 4)。</li>
	<li>将右脚放在可锻的泡沫管中, 脚趾伸出管子的末端。用牙蜡把脚紧紧地抱在泡沫内, 然后把管子紧紧地关上。确保保持脚的管子的直径足够牢固地装入塑料管。</li>
	<li>将塑料管滑入 X 射线扫描仪床 (补充图 5)。</li>
	<li>延长鼠后肢, 直到它绷紧。不要 over-extend 腿对老鼠造成任何伤害 (补充图 5), 因为这可能会导致肢体由于呼吸的剧烈运动而引起非自愿的移动。</li>
	<li>拉左腿 (non-scanned 后肢) 与尾巴出扫描视野和朝向躯干, 远离要扫描的扩展腿. </li>
	<li>用掩蔽胶带固定左腿 (non-scanned 后肢) 和位置的尾部。不要使用任何更多或更少的粘性 (即, 管道胶带或画家的磁带), 因为这些材料会伤害鼠时, 他们被删除 (胶带) 或不提供足够强大的持有 (画家的磁带) (补充图 6)。</li>
	<li>将老鼠的身体固定在臀部、肩部和头部, 并用掩蔽胶带。将面罩或鼻锥固定在鼠身上 (补充图 6)。 注: 涂抹胶带的粘合面, 以消除其粘附在鼠毛上的能力。不要涂抹遮蔽胶带的两端, 这样它就可以牢固地固定在扫描床上。</li>
	<li>将大鼠裹在兽医包中以限制热量损耗 (补充图 6)。 注意: 在全身麻醉下, 大鼠由于其大的表面重量比19,20而迅速失去热量。</li>
	<li>通过对大鼠 (直接或通过视频直播) 保持恒定的视觉效果, 在整个手术过程中持续监测大鼠的呼吸速度。 注意: 这里设置了5分钟, 扫描采集是依赖于采集设置, 恢复时间为60分钟。</li>
	<li>继续获取µCT 图像。 注意: 扫描采集的精确规范是针对每种扫描仪类型、软件系统和具体的研究问题, 但是, 在文献中存在着几种方法学出版物1,2,9。</li>
</ol>3. 麻醉恢复
<ol><li>在体内µCT 扫描完成后, 停止对鼠的异氟醚的流动, 但保持 O2的1-2 升/分钟流量。</li>
	<li>当老鼠恢复马达控制 (1-2 分钟), 把它从呼吸器中取出, 并允许它单独在一个 general-purpose 加热垫上放置在低热的笼子里。已知大鼠在全身麻醉下的体温在1° c 以下时会降低体温19。不要让老鼠无人看管, 直到它恢复了足够的意识来维持胸骨卧床。 注意: 从我们的研究小组的轶事证据报告说, 立即从异氟醚麻醉恢复后, 大鼠开始吃, 所以重要的是他们的食物和水可供他们在恢复期间。虽然我们已经观察到这种行为, 重复全身麻醉不会引起食物摄入量或体重的显著增加1,9。</li>
</ol>

<ol>
	<li>Longo, A. B., Sacco, S. M., Salmon, P. L., Ward, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+use+of+micro-computed+tomography+does+not+alter+microarchitecture+of+the+proximal+tibia+in+sham+or+ovariectomized+sprague-dawley+rats.">Longitudinal use of micro-computed tomography does not alter microarchitecture of the proximal tibia in sham or ovariectomized sprague-dawley rats.</a> Calcif Tissue Int. 98 (6), 631-641 (2016).</li><li>Sacco, S. 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=Repeated+irradiation+from+micro-computed+tomography+scanning+at+2,+4+and+6+months+of+age+does+not+induce+damage+to+tibial+bone+microstructure+in+male+and+female+CD-1+mice.">Repeated irradiation from micro-computed tomography scanning at 2, 4 and 6 months of age does not induce damage to tibial bone microstructure in male and female CD-1 mice.</a> Bonekey Rep. 6, 855 (2017).</li><li>Waarsing, J. H., Day, J. S., Verhaar, J. A., Ederveen, A. G., Weinans, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+loss+dynamics+result+in+trabecular+alignment+in+aging+and+ovariectomized+rats.">Bone loss dynamics result in trabecular alignment in aging and ovariectomized rats.</a> J Orthop Res. 24 (5), 926-935 (2006).</li><li>Klinck, R. J., Campbell, G. M., Boyd, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+effects+on+bone+architecture+in+mice+and+rats+resulting+from+in+vivo+micro-computed+tomography+scanning.">Radiation effects on bone architecture in mice and rats resulting from in vivo micro-computed tomography scanning.</a> Med Eng Phys. 30 (7), 888-895 (2008).</li><li>Laperre, 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=Development+of+micro-CT+protocols+for+in+vivo+follow-up+of+mouse+bone+architecture+without+major+radiation+side+effects.">Development of micro-CT protocols for in vivo follow-up of mouse bone architecture without major radiation side effects.</a> Bone. 49 (4), 613-622 (2011).</li><li>Brouwers, J. E., van Rietbergen, B., Huiskes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+effects+of+in+vivo+micro-CT+radiation+on+structural+parameters+and+bone+marrow+cells+in+proximal+tibia+of+wistar+rats+detected+after+eight+weekly+scans.">No effects of in vivo micro-CT radiation on structural parameters and bone marrow cells in proximal tibia of wistar rats detected after eight weekly scans.</a> J Orthop Res. 25 (10), 1325-1332 (2007).</li><li>Francisco, J. I., Yu, Y., Oliver, R. A., Walsh, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+age,+skeletal+site,+and+time+post-ovariectomy+on+bone+mineral+and+trabecular+microarchitecture+in+rats.">Relationship between age, skeletal site, and time post-ovariectomy on bone mineral and trabecular microarchitecture in rats.</a> J Orthop Res. 29 (2), 189-196 (2011).</li><li>Altman, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+skeletal+growth,+modeling,+and+remodeling+by+in+vivo+micro+computed+tomography.">Quantification of skeletal growth, modeling, and remodeling by in vivo micro computed tomography.</a> Bone. 81, 370-379 (2015).</li><li>Longo, A. B., 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=Lifelong+intake+of+flaxseed+or+menhaden+oil+to+provide+varying+n-6+to+n-3+PUFA+ratios+modulate+bone+microarchitecture+during+growth,+but+not+after+OVX+in+Sprague-Dawley+rats.">Lifelong intake of flaxseed or menhaden oil to provide varying n-6 to n-3 PUFA ratios modulate bone microarchitecture during growth, but not after OVX in Sprague-Dawley rats.</a> Mol Nutr Food Res. 61 (8), (2017).</li><li>Sacco, S. M., Saint, C., LeBlanc, P. J., Ward, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+consumption+of+hesperidin+and+naringin+flavanones+exerts+transient+effects+to+tibia+bone+structure+in+female+CD-1+offspring.">Maternal consumption of hesperidin and naringin flavanones exerts transient effects to tibia bone structure in female CD-1 offspring.</a> Nutrients. 9 (3), 250 (2017).</li><li>Campbell, G. M., Buie, H. R., Boyd, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signs+of+irreversible+architectural+changes+occur+early+in+the+development+of+experimental+osteoporosis+as+assessed+by+in+vivo+micro-CT.">Signs of irreversible architectural changes occur early in the development of experimental osteoporosis as assessed by in vivo micro-CT.</a> Osteoporos Int. 19 (10), 1409-1419 (2008).</li><li>De Schaepdrijver, L., Delille, P., Geys, H., Boehringer-Shahidi, C., Vanhove, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+longitudinal+micro-CT+study+of+bent+long+limb+bones+in+rat+offspring.">In vivo longitudinal micro-CT study of bent long limb bones in rat offspring.</a> Reprod Toxicol. 46, 91-97 (2014).</li><li>Perilli, E., 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=Detecting+early+bone+changes+using+in+vivo+micro-CT+in+ovariectomized,+zoledronic+acid-treated,+and+sham-operated+rats.">Detecting early bone changes using in vivo micro-CT in ovariectomized, zoledronic acid-treated, and sham-operated rats.</a> Osteoporos Int. 21 (8), 1371-1382 (2010).</li><li>Bouxsein, M. 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=Guidelines+for+assessment+of+bone+microstructure+in+rodents+using+micro-computed+tomography.">Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.</a> J Bone Miner Res. 25 (7), 1468-1486 (2010).</li><li>Li, H., Zhang, H., Tang, Z., Hu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-computed+tomography+for+small+animal+imaging:+Technological+details.">Micro-computed tomography for small animal imaging: Technological details.</a> Progress in Natural Science. 18 (5), 513-521 (2008).</li><li>Campbell, G. M., Sophocleous, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+bone+and+soft+tissue+by+micro-computed+tomography:+applications+to+ex+vivo+and+in+vivo+studies.">Quantitative analysis of bone and soft tissue by micro-computed tomography: applications to ex vivo and in vivo studies.</a> Bonekey Rep. 3, 564 (2014).</li><li>Meganck, J. A., Kozloff, K. M., Thornton, M. M., Broski, S. M., Goldstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beam+hardening+artifacts+in+micro-computed+tomography+scanning+can+be+reduced+by+X-ray+beam+filtration+and+the+resulting+images+can+be+used+to+accurately+measure+BMD.">Beam hardening artifacts in micro-computed tomography scanning can be reduced by X-ray beam filtration and the resulting images can be used to accurately measure BMD.</a> Bone. 45 (6), 1104-1116 (2009).</li><li>Vazquez, C. M., Molina, M. T., Ilundain, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+rat+large+intestine+in+reducing+diarrhea+after+50%+or+80%+distal+small+bowel+resection.">Role of rat large intestine in reducing diarrhea after 50% or 80% distal small bowel resection.</a> Dig Dis Sci. 34 (11), 1713-1719 (1989).</li><li>Albrecht, M., Henke, J., Tacke, S., Markert, M., Guth, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+isoflurane,+ketamine-xylazine+and+a+combination+of+medetomidine,+midazolam+and+fentanyl+on+physiological+variables+continuously+measured+by+telemetry+in+Wistar+rats.">Effects of isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl on physiological variables continuously measured by telemetry in Wistar rats.</a> BMC Vet Res. 10, 198 (2014).</li><li>Schmitz, S., Tacke, S., Guth, B., Henke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+physiological+parameters+and+anaesthesia+specific+observations+during+isoflurane,+ketamine-xylazine+or+medetomidine-midazolam-fentanyl+anaesthesia+in+male+guinea+pigs.">Comparison of physiological parameters and anaesthesia specific observations during isoflurane, ketamine-xylazine or medetomidine-midazolam-fentanyl anaesthesia in male guinea pigs.</a> PLoS One. 11 (9), e0161258 (2016).</li><li>Stevens, W. C., 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=Comparative+toxicities+of+halothane,+isoflurane,+and+diethyl+ether+at+subanesthetic+concentrations+in+laboratory+animals.">Comparative toxicities of halothane, isoflurane, and diethyl ether at subanesthetic concentrations in laboratory animals.</a> Anesthesiology. 42 (4), 408-419 (1975).</li></ol>

作者没有什么可透露的。

该协议为观众提供了第一个详细的指导方针, 正确的麻醉, 放置和约束的大鼠在在体内µCT 扫描的后肢肢体。这些指南使在体内µCT 扫描系统的用户能够获得高分辨率和高质量的胫骨图像, 可用于量化3维骨微结构。必要的关键步骤, 以确保适当的定位和克制, 包括适当的麻醉的大鼠, 以及延长后肢远离所有其他关键结构, 直到它是绷紧, 但不是在一个不自然的立场。为了获得最佳的影像学结果, 必须将大鼠麻醉到完全放松的程度, 并失去眼睑和踏板的反射。此外, 扫描腿应延长, 整个脚和踝应克制在泡沫。上述方法, 以实现扫描腿的最佳定位, 将确保: 1) 研究中的大鼠后肢在同一方向上始终如一地定向, 从而允许 x 射线束穿过每条腿的同一区域, 因为它旋转在样品附近;2) 不会发生后肢的自发和非自愿运动, 从而最大限度地减少运动工件干扰所采集图像质量的可能性;3) 防止物体的障碍物 (即、尾部), 从而最大限度地减少部分体积效应的可能性, 从而产生不精确的 BMD 和 TMD 测量。这些指南可能会被修改为每一个单独的大鼠, 因为异氟醚的代谢和定位的速度取决于应变和体型大小22。最常见的在体内扫描机器是专为小动物模型 (即, 小鼠, 大鼠, 兔, 豚鼠) 和将有可互换的动物阶段, 允许扫描不同的动物大小。因此, 他们可以容纳广泛的身体重量。
虽然在体内µCT 扫描允许老鼠重新定位和扫描如果从初始扫描获得的图像质量较差, 重复扫描将使大鼠暴露于额外剂量的辐射和异氟醚麻醉 一段长时间。每月重复照射 600 mGy 的大鼠胫骨四月以上对骨微结构没有造成不良影响, 与对侧后肢的1相比, 但这并不能确定两次扫描重复的安全性立即继承。所述技术的进一步限制包括需要延长后肢绷紧的力量, 使其保持静止, 这可能会导致骨骼结构的某些变化。虽然在扫描过程中后肢的限制的严重性取决于每个研究目标, 以前的研究从我们的实验室每月重复在体内µCT 成像的一个后肢导致了不同的皮质微结构参数, 偏心, 相对于对侧后肢, 没有经过重复的扩展, 稳定和扫描1。偏心是一个测量的椭圆形形状的皮质骨和变化的反应, 改变 load-bearing。因此, 当使用这种定位和限制后肢的方法重复在体内µCT 成像时, 应考虑在评估和解释对 load-bearing 微结构参数的变化。
虽然已为骨组织的成像和分析提供了上述的指导方针, 但在对后肢的软组织进行成像时, 必须对该协议作轻微的调整。具体而言, 必须考虑到后肢从躯干延伸和克制的方式, 因为目前的程序畸形在扫描期间将软组织 (肌肉、脂肪组织) 定位为异常定位。因此, 当外推这个模型用于成像软组织的后肢, 应作出一些调整的限制技术, 以减少或消除改变的组织定位的相互关系。
此外, 这些指导方针是根据我们研究小组的经验专门编写的, 但是, 它们可以被修改以适应其他商业上可用的在体内µCT 扫描仪。其他建议的方法来定位和限制后肢可能是由制造商提供的在体内µCT 扫描系统。最商业化的在体内µCT 单位列出聚丙烯, 扩大聚苯乙烯, 和塑料管与牙科蜡举行一个突出的脚作为可接受的材料和方法, 以抑制扫描腿。然而, 该协议提供了更有控制和一致的定位和约束的扫描腿, 并始终如一地产生高质量的图像。本方法所提出的准则要求对大鼠进行麻醉所需的专用设备, 如汽化器、管子、口罩、感应室和氧气。虽然与注射麻醉剂相比, 该设备的成本略高, 但它使研究人员能够迅速和精确地诱导麻醉在特定深度的意识, 这提供了比替代的优势方法.
使用本方法视频中概述的指南, 研究人员利用高分辨率的在体内µCT 技术来调查他们对兴趣的干预将能够正确和始终如一地定位和抑制大鼠后肢的高高质量的 x 射线成像。这将提供一个连续的领域内的在体内µCT 图像获取, 并作为一个步骤, 以优化的一致性和准确性的研究, 并使比较跨研究的文献。同样, 这些协议和方法可以扩展, 以用于其他啮齿动物物种, 包括小鼠, 虽然一些变化将需要2,10。例如, 在泡沫管脚的克制可以包括脚踝, 以尽量减少腿部移动的可能性, 在扫描。此外, 充分的脚将适合于泡沫持有人。因此, 脚趾不伸出的结束的持有人, 因为他们做时, 保护一只老鼠的脚。此外, 鼠标的身体不需要同样的约束与磁带的老鼠。在扫描过程中, 小的鼻锥可以用来维持小鼠的麻醉。如果没有更小的鼻锥, 你可以在可用的鼻锥上固定一个丁腈手套, 并在手套上做一个小切口, 提供一个空间, 使它能适合老鼠的鼻子提供麻醉, 同时保持鼻子周围的密封。
虽然胫骨近端是研究大鼠骨微结构改变的主要部位, 但对其他骨骼部位 (如股骨和腰椎) 的正确和一致定位的指导原则应进行调查, 并建立文学中的一致性。然而, 在进行未来的研究涉及腰椎成像, 必须考虑到脊柱成像提供辐射接触周围的器官和组织。

使用在体内微计算机断层扫描 (µCT) 是一个强大的工具, 包括非破坏性成像的内部结构在高分辨率使用啮齿类动物模型。非破坏性的性质,在体内µCT 允许重复成像相同的啮齿动物随着时间的推移。这一特性不仅减少了实验设计中所需的啮齿动物总数, 从而减少了学科的变异, 而且也使研究人员能够理解对干预的长期反应。随着使用重复的在体内µCT, 实验鼠和大鼠已阐明发展变化的骨骼微结构和骨密度 (BMD) 在整个生命周期1,2,3 ,4,5,6,7,8以及对干预的骨骼健康响应, 如节食9,10,卵巢切除7,11和药理剂8,12,13。骨密度和骨微结构的具体骨骼部位, 即胫骨近端, 股骨和腰椎, 是指整体骨骼健康和维持骨折的风险, 所以在量化反应的主要措施干预.
在体内µCT 图像采集涉及 two-dimensional x 射线投射在多个角度获取, x 射线源和探测器围绕被调查的动物旋转, 14,15。结果图像的质量取决于许多因素, 包括但不限于: 选定的采集参数 (即、空间分辨率、X 射线电压、安培量、旋转步骤、应用滤镜、曝光时间)、µCT 的限制扫描仪 (即, scanner-based 工件, 如环形工件或灰尘, 造成裸奔或部分体积的影响) 和适当的定位和约束的动物。这些因素中的前两个可以由用户在某种程度上纵 , 这取决于特定的扫描机、研究目标以及优化扫描仪功能所需的校正或获取的图像的处理。后者的这些因素, 正确定位的啮齿目动物在扫描之前, 可以实现, 无论是 scanner-based 的限制或获取参数, 被选中, 以达到一个特定的研究目标。虽然许多出版物涉及活体成像已经发表在文献14,15,16,17, 经典的手稿风格是这样详细的 "如何" 信息不能包括在内。因此, 本文和视频指南的目的是填补这一空白。在这里, 我们的目的是指导用户的在体内µCT 扫描仪如何麻醉鼠, 并定位和抑制后肢, 以产生高质量的图像, 可以分析, 以更准确地量化的结果骨微结构。
为了量化最精确的骨密度和骨骼微结构值, 防止 x 射线束通过后肢以外的物体的障碍物是必不可少的。当 x 射线通过不同厚度和密度的物体和组织时, 一些 x 射线被它们通过的材料吸收 (即衰减)。由于样品的质量密度受其厚度和周围组织的存在和厚度的影响, 因此必须以同样的方式扫描用于确定 BMD 的校准幻影。因此, 如果 x 射线光束是通过对象 (即、尾部) 穿过感兴趣区域之前或之后, 这些对象将吸收一些 x 射线能量, 并会干扰所获得的传输图像。此外, 这些扫描将很难模拟时, 扫描的幽灵, 必须密切类似样本扫描。因此, 这些衰减差异导致骨密度测量的不精确性。因此, 为了方便和准确, 最好限制 x 射线源、感兴趣区域和 x 射线探测器之间的障碍物数量。
骨结构的纵向评估从干预的临床前模型涉及重复麻醉的动物, 以限制他们的运动在扫描协议。为了制服接受µCT 扫描的动物, 有几种全身麻醉方法, 包括注射和吸入麻醉1,2,4,5,6,12. 不同于吸入麻醉剂, 如异氟醚, 重复全麻使用可注射麻醉剂导致体重减轻, 手术耐受性和其他生理参数的显著变化, 特别是大鼠和豚鼠, 建议严重禁忌症重复使用18,19,20。虽然异氟醚是高度挥发性和允许快速诱导和恢复, 注射麻醉剂产生不同程度的麻醉和时间在麻醉下取决于应变, 性别, 身体组成, 禁食状态, 和昼夜循环动物.注射麻醉药也对其使用造成额外的障碍, 因为它们受到国家管理机构的高度管制。吸入麻醉然而, 涉及直接分娩进入呼吸系统;这种方法允许更快的诱导和恢复时间, 更好地控制麻醉的长度和深度19,20。吸入麻醉方法的局限性包括其对专用汽化设备的要求以及感应、维护和恢复过程中心率和血压的一些变化18,19。

<table><tbody><tr><td>Isoflurane</td><td>Fresenius Kabi Animal Health</td><td>108737</td><td></td></tr><tr><td>Vaporizer</td><td>Dispomed</td><td>990-1091-3SINEWA</td><td></td></tr><tr><td>Scavengers/Charcoal Filters</td><td>Dispomed</td><td>985-1005-000</td><td></td></tr><tr><td>Micro-CT Scanner</td><td>Bruker microCT</td><td>SkyScan 1176</td><td></td></tr><tr><td>Dental wax</td><td>Kerr Dental Laboratory</td><td>623</td><td></td></tr><tr><td>Foam (Backer Rod)</td><td>Rona</td><td>CF12086</td><td>1&amp;rdquo;x10&amp;rsquo;</td></tr><tr><td>Plastic tube</td><td>Bruker microCT</td><td>SP-3010</td><td></td></tr><tr><td>Carbon-fiber bed</td><td>Bruker microCT</td><td>SP-3002</td><td></td></tr><tr><td>Vet Wrap/Bandage</td><td>Dura-Tech</td><td>17473</td><td></td></tr><tr><td>Ophthalmic Gel</td><td>OptixCare</td><td>006CLC-4256</td><td>Antibiotic-free</td></tr><tr><td>Heating pad</td><td>Sunbeam</td><td>000731-500-000</td><td></td></tr></tbody></table>

proper positioning and restraint of a rat hind limb for focused high resolution imaging of bone micro-architecture using in vivo micro-computed tomography

使用在体内微计算机断层扫描 (µCT) 是一个强有力的工具, 涉及在高分辨率的活体动物模型的内部结构的无损成像。这允许反复成像相同的啮齿动物随着时间的推移。这一特性不仅减少了实验设计中所需的啮齿类动物总数, 从而减少了学科的变异, 同时也使研究人员可以评估对干预的纵向或 life-long 反应。为了获得高质量的图像, 可以进行处理和分析, 以便更准确地量化骨微结构的结果,在体内µCT 扫描仪的用户必须正确麻醉鼠, 并定位和抑制后肢。要做到这一点, 就必须把老鼠麻醉到完全放松的程度, 而踏板反射就会丢失。这些指南可能会被修改为每一个单独的大鼠, 因为异氟醚的代谢速率可以根据应变和身体大小而变化。适当的技术为在体内µCT 图像采集能够准确和一致的测量骨微结构内和跨研究。

这种麻醉方法的大鼠, 并定位和约束的后肢为在体内µCT 成像有助于获得高质量的图像适合分析胫骨微结构。对大鼠后肢的正确定位包括腿部被完全伸展, 整个脚和踝部被抑制在泡沫中 (图 1A), 从而获得足够质量的图像来分析小梁和皮质微结构 (图 1B)。后肢的位置和限制不足 (图 1C) 可能会导致带有移动工件的图像 (图 1D), 而未完全从视图扫描字段 (图 1E) 中删除的尾部将干扰 X 射线通过扫描样本 (图 1F) 进行衰减, 并改变 BMD 和组织矿物密度 (TMD) 测量值。这些放置错误中的任何一个都将导致质量较差的扫描, 而不应进一步分析。获得质量较差的图像将改变细梁网和后肢皮层结构的量化, 并产生不适当或不确定的数据14。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56346/56346fig1.jpg"> 图1。有代表性的大鼠后肢的位置图像和胫骨近端的相应获得的图像。 (A) 大鼠后肢的适当位置, 踝关节完全克制在泡沫中, 腿延长和尾部拉离胫骨提供足够的图像质量 (B) 胫骨及其小梁和皮质的横断面微建筑。(C) 不正确放置大鼠后肢, 腿未完全伸展, 且未完全抑制在泡沫中的踝关节可能导致 (D) 运动工件, 在横断面上被视为裸奔。(E) 干扰视场的物体, 如未从胫骨抽出的尾巴 (F) 会干扰胫骨的 x 射线衰减, 并可能导致骨密度和 TMD 测量的改变, 尽管视觉上并不明显。在面板的左下角F显示在视野中的部分尾巴, 这会干扰随后通过胫骨的 x 射线束。左侧面板中的红色虚线表示右侧面板上显示的剖面线。<a href="//cloudflare.jove.com/files/ftp_upload/56346/56346fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充图1。异氟醚麻醉装置.异氟醚麻醉装置设置为在 O2中以连续流速1-2 升/分钟的速度在全麻中溶解, 以提供3-4% 的异氟醚。<a href="http://cloudflare.jove.com/files/ftp_upload/56346/Suppl_Fig_1.jpg">请单击此处下载此图.</a>
补充图2。确保麻醉深度。通过用面罩或鼻锥捏住接受连续吸入麻醉剂的老鼠的脚趾来测量踏板的反射。当腿部略微延长时, 疼痛反应更明显。非常强的捏或钳子或夹子的用途可能导致组织损伤, 因此不应该使用。<a href="http://cloudflare.jove.com/files/ftp_upload/56346/Suppl_Fig_2.jpg">请单击此处下载此图.</a>
补充图3。屏幕捕获的实时反馈生理监控摄像头的看法.<a href="http://cloudflare.jove.com/files/ftp_upload/56346/Suppl_Fig_3.jpg">请单击此处下载此图.</a>
补充图4。在碳纤维扫描仪床上放置仰卧位的大鼠.<a href="http://cloudflare.jove.com/files/ftp_upload/56346/Suppl_Fig_4.jpg">请单击此处下载此图.</a>
补充图5。右脚的大鼠约束在一个可塑性泡沫管.右脚的大鼠是克制在一个可塑性泡沫管与脚趾延伸出的管 (这里没有图)。泡沫管被限制在塑料支架 (请参阅特定材料/设备表以获取更详细的信息)。<a href="http://cloudflare.jove.com/files/ftp_upload/56346/Suppl_Fig_5.jpg">请单击此处下载此图.</a>
补充图6。大鼠的位置与右腿伸直直.尾巴和左脚被胶带从右腿 (朝向躯干), 臀部是固定的, 鼠躯干被包裹在兽医包装 (蓝色), 以限制热量损失。<a href="http://cloudflare.jove.com/files/ftp_upload/56346/Suppl_Fig_6.jpg">请单击此处下载此图.</a>

Watch this Scientific Journal Video about Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography at 

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

应用活体显微计算机断层成像技术对骨微结构高分辨成像大鼠后肢的定位与抑制

本文指导用户的在体内微计算机断层扫描 (µCT) 扫描仪如何麻醉, 正确定位和抑制后肢的小鼠在高分辨率成像胫骨的最小运动。结果是高质量的图像, 可以处理, 以准确地量化骨微结构。

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

proper-positioning-restraint-rat-hind-limb-for-focused-high

Research

JoVE Journal

Bioengineering

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

9.9K 次观看.  Brock University. 本文指导用户的在体内微计算机断层扫描 (µCT) 扫描仪如何麻醉, 正确定位和抑制后肢的小鼠在高分辨率成像胫骨的最小运动。结果是高质量的图像, 可以处理, 以准确地量化骨微结构。

视频: Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

科研

生物工程

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

计算机断层扫描和成骨血管生成耦合光学成像评估头盖骨自体移植和移植物的整合

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

半自动纵 Microcomputed Tomography-based 裸鼠骨质疏松症相关椎体骨折模型的定量结构分析

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

异位骨化直接鼠标创伤/烧伤模型

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

医学

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

骨折易感性机械测试的原则和方案

Practical Considerations for the Design, Execution, and Interpretation of Studies Involving Whole-Bone Bending Tests of Rodent Bones

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care costs. The ability to understand the mechanisms underlying bone disease and the response to treatment is not only desired, but critical. Mechanical testing of bone serves as a valuable technique for understanding and quantifying a bone&#39;s susceptibility to fracture. While this method appears simple to perform, inappropriate and inaccurate conclusions may be reached if governing assumptions and key steps are disregarded by the user. This has been observed across disciplines as studies continue to be published with misuse of methods and incorrect interpretation of results. This protocol will serve as a primer for the principles associated with mechanical testing along with the application of these techniques-from considerations of sample size through tissue harvesting and storage, to data analysis and interpretation. With this in hand, valuable information regarding a bone&#39;s susceptibility to fracture may be obtained, furthering understanding for both academic research and clinical solutions.

作者聚焦：提高全骨弯曲测试的准确性和可重复性 

Mechanical testing of rodent bones is a valuable method for extracting information regarding a bone's susceptibility to fracture. Lacking proper practical understanding, the results may be overinterpreted or lack validity. This protocol will serve as a guide to ensure mechanical tests are performed accurately to provide valid and functional data.

设计、执行和解释涉及啮齿动物骨骼全骨弯曲试验的研究的实际考虑因素

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care ...

HE 染色和 micro-CT 评估小鼠骨小梁微结构

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

作者聚焦:一种经济有效的小鼠骨质疏松症模型中骨微结构定量评估的方法 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

骨质疏松症小鼠模型中的骨小梁微观结构评估

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

应用活体显微计算机断层成像技术对骨微结构高分辨成像大鼠后肢的定位与抑制

摘要

探索更多视频

此视频中的章节

高分辨率三维成像 EX - VIVO生物样品显微CT

计算机断层扫描和成骨血管生成耦合光学成像评估头盖骨自体移植和移植物的整合

半自动纵 Microcomputed Tomography-based 裸鼠骨质疏松症相关椎体骨折模型的定量结构分析

异位骨化直接鼠标创伤/烧伤模型

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

设计、执行和解释涉及啮齿动物骨骼全骨弯曲试验的研究的实际考虑因素

设计、执行和解释涉及啮齿动物骨骼全骨弯曲试验的研究的实际考虑因素

设计、执行和解释涉及啮齿动物骨骼全骨弯曲试验的研究的实际考虑因素

骨质疏松症小鼠模型中的骨小梁微观结构评估

骨质疏松症小鼠模型中的骨小梁微观结构评估