Name: partial heterotopic hindlimb transplantation model in rats
Uploaded: 2021-06-09T14:45:00
Description: Watch this Scientific Journal Video about Partial Heterotopic Hindlimb Transplantation Model in Rats at JoVE.com

这项工作得到了助理国防部长办公室的支持，通过国会指导的医学研究计划，根据第一奖。W81XWH-17-1-0680。意见、解释、结论和建议是作者的意见，不一定得到国防部的赞同。

所有动物都根据《国家卫生研究院护理和使用实验室动物指南》得到人道护理。机构动物护理和使用委员会（IACUC-协议2017N000184）和动物护理和使用审查办公室（ACURO）批准了所有动物协议。近亲繁殖的雄性刘易斯大鼠（250-400克）用于所有实验。
1. 手术
<ol><li>使用异黄素吸入麻醉刘易斯大鼠。诱导感应室中5%异黄素的麻醉，通过呼吸锥体保持1.5-3%异黄酮吸入麻醉。</li>
	<li>手...

<ol>
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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=Penis+transplantation:+first+US+experience.">Penis transplantation: first US experience.</a> Annals of Surgery. 267 (5), 983-988 (2018).</li><li>Grajek, 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=First+complex+allotransplantation+of+neck+organs:+larynx,+trachea,+pharynx,+esophagus,+thyroid,+parathyroid+glands,+and+anterior+cervical+wall:+a+case+report.">First complex allotransplantation of neck organs: larynx, trachea, pharynx, esophagus, thyroid, parathyroid glands, and anterior cervical wall: a case report.</a> Annals of Surgery. 266 (2), 19-24 (2017).</li><li>Pribaz, J. J., Caterson, E. 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A., 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=Vascularized+limb+transplantation+in+the+rat.+II.+Results+with+allogeneic+grafts.">Vascularized limb transplantation in the rat. II. Results with allogeneic grafts.</a> Transplantation. 35 (4), 300-304 (1983).</li><li>Adamson, L. A., 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=A+modified+model+of+hindlimb+osteomyocutaneous+flap+for+the+study+of+tolerance+to+composite+tissue+allografts.">A modified model of hindlimb osteomyocutaneous flap for the study of tolerance to composite tissue allografts.</a> Microsurgery. 27 (7), 630-636 (2007).</li><li>Arav, A., Friedman, O., Natan, Y., Gur, E., Shani, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hindlimb+cryopreservation+and+transplantation:+a+step+toward+"organ+banking".">Rat hindlimb cryopreservation and transplantation: a step toward "organ banking".</a> American Journal of Transplantation. 17 (11), 2820-2828 (2017).</li><li>Gok, 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=Development+of+an+ex-situ+limb+perfusion+system+for+a+rodent+model.">Development of an ex-situ limb perfusion system for a rodent model.</a> ASAIO Journal. 65 (2), 167-172 (2019).</li><li>Gok, E., Rojas-Pena, A., Bartlett, R. H., Ozer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+skeletal+muscle+metabolomic+changes+associated+with+static+cold+storage.">Rodent skeletal muscle metabolomic changes associated with static cold storage.</a> Transplantation Proceedings. 51 (3), 979-986 (2019).</li><li>Brandacher, G., Grahammer, J., Sucher, R., Lee, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+basic+and+translational+research+in+reconstructive+transplantation.">Animal models for basic and translational research in reconstructive transplantation.</a> Birth Defects Research. Part C, Embryo Today. 96 (1), 39-50 (2012).</li><li>Fleissig, Y., 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=Modified+heterotopic+hindlimb+osteomyocutaneous+flap+model+in+the+rat+for+translational+vascularized+composite+allotransplantation+research.">Modified heterotopic hindlimb osteomyocutaneous flap model in the rat for translational vascularized composite allotransplantation research.</a> Journal of Visualized Experiments: JoVE. (146), e59458 (2019).</li><li>Ulusal, A. E., Ulusal, B. G., Hung, L. M., Wei, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+hindlimb+allotransplantation+in+rats:+an+alternative+model+for+immunological+research+in+composite-tissue+allotransplantation.">Heterotopic hindlimb allotransplantation in rats: an alternative model for immunological research in composite-tissue allotransplantation.</a> Microsurgery. 25 (5), 410-414 (2005).</li><li>Jang, Y., Park, Y. E., Yun, C. W., Kim, D. H., Chung, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vest-collar+as+a+rodent+collar+to+prevent+licking+and+scratching+during+experiments.">The vest-collar as a rodent collar to prevent licking and scratching during experiments.</a> Lab Anim. 50 (4), 296-304 (2016).</li><li>Kern, 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=A+novel+rodent+orthotopic+forelimb+transplantation+model+that+allows+for+reliable+assessment+of+functional+recovery+resulting+from+nerve+regeneration.">A novel rodent orthotopic forelimb transplantation model that allows for reliable assessment of functional recovery resulting from nerve regeneration.</a> American Journal of Transplantation. 17 (3), 622-634 (2017).</li><li>Perez-Abadia, G., 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=Low-dose+immunosuppression+in+a+rat+hind-limb+transplantation+model.">Low-dose immunosuppression in a rat hind-limb transplantation model.</a> Transplant International. 16 (12), 835-842 (2003).</li><li>Sucher, 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=Orthotopic+hind-limb+transplantation+in+rats.">Orthotopic hind-limb transplantation in rats.</a> Journal of Visualized Experiments. (41), e2022 (2010).</li><li>Fleissig, Y. Y., Beare, J. E., LeBlanc, A. J., Kaufman, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+rat+hind+limb+transplant+as+an+experimental+model+of+vascularized+composite+allotransplantation:+Approaches+and+advantages.">Evolution of the rat hind limb transplant as an experimental model of vascularized composite allotransplantation: Approaches and advantages.</a> SAGE Open Medicine. 8, 2050312120968721 (2020).</li><li>Lindboe, C. F., Presthus, J. <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+denervation,+immobilization+and+cachexia+on+fibre+size+in+the+anterior+tibial+muscle+of+the+rat.">Effects of denervation, immobilization and cachexia on fibre size in the anterior tibial muscle of the rat.</a> Acta Neuropathologica. 66 (1), 42-51 (1985).</li><li>Nazzal, J. A., Johnson, T. S., Gordon, C. R., Randolph, M. A., Lee, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+limb+allotransplantation+model+to+study+skin+rejection+in+the+rat.">Heterotopic limb allotransplantation model to study skin rejection in the rat.</a> Microsurgery. 24 (6), 448-453 (2004).</li></ol>

在文献15、16、17中描述了啮齿动物的矫肢移植模型：然而，他们需要神经修复，肌肉重新连接，和完美的骨合成股骨，这可能是一个非常困难的步骤。这些模型也与14号啮齿动物的发病率和死亡率较高有关，特别是在短期随访中，因为移植后肢的正常功能的恢复可能需要几个月的时间。然而?...

在过去的二十年里， VCA 已经发展成为一种革命性的治疗患者谁遭受严重毁容，包括脸部1 ，上肢截肢2 ，3 ，和其他复杂的组织缺陷4 ， 5 。然而，终生免疫抑制的后果仍然阻碍这些复杂的重建手术的广泛应用。基础研究对于改进反排斥策略至关重要。增加VCA保存时间对于改善移植物流和增加捐赠者库也至关重要（因为VCA捐赠者必须比实体器官捐献者满足更多的标准，包括肤色、解剖大小、性别）。在此背景下，大鼠肢体移植被广泛应用于6、7、新耐受诱导方案8、保存研究9、10、11等免疫排斥的研究中。因此，这些 VCA 模型是 VCA 转化研究需要掌握的关键元素。
骨皮瓣在文献中被描述为研究8、12、13、14大鼠VCA的可靠模型。虽然正位全肢移植允许长期评估移植功能，但它是一个耗时的程序，与更高的术后发病率和死亡率14。相比之下，异位肢体移植模型是非功能性的，但能够对VCA进行可重复的研究。在开始大鼠VCA移植研究之前，可以可靠地预测术后的结果。本研究报告了大鼠的部分异位骨质皮瓣移植模型，其中包括在三周的随访期内可能出现的频繁可能的结果和术后并发症。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 GA angiocatheter</td>
			<td>BD Insyte Autoguard</td>
			<td>381412</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 suture Black monofilament non absorbable suture</td>
			<td>Ethicon</td>
			<td>1667</td>
			<td>Used to suture the E-collar to the back of the neck</td>
		</tr>
		<tr>
			<td>4-0 suture Coated Vicryl Plus Antibacterial</td>
			<td>Ethicon</td>
			<td>VCP496</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Tissue Forceps, 11 cm, 1 x 2 Teeth with Tying Platform</td>
			<td>ASSI</td>
			<td>ASSI.ATK26426</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar cords</td>
			<td>ASSI</td>
			<td>228000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Black Polyamide Monofilament USP 10-0, 4 mm 3/8c</td>
			<td>AROSurgical</td>
			<td>T04A10N07-13</td>
			<td>Used to perform the microvascular anastomoses</td>
		</tr>
		<tr>
			<td>Buprenorphine HCl</td>
			<td>Pharmaceutical, Inc</td>
			<td>42023-179-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dilating Forceps</td>
			<td>Fine science tools (FST)</td>
			<td>18131-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors 15 cm, Round Handle 8 mm diameter, Straight Slender Tapered Blade 7 mm, Lipshultz Pattern</td>
			<td>ASSI</td>
			<td>ASSI.SAS15RVL</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Micro Clamps 5.5 x 1.5 mm</td>
			<td>Fine science tools (FST)</td>
			<td>18040-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Elizabethan collar</td>
			<td>Braintree Scientific</td>
			<td>EC-R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps 13.5 cm long, Flat Handle, 9 mm wide Straight Tips 0.1 mm diameter (x2)</td>
			<td>ASSI</td>
			<td>ASSI.JFL31</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey Micro Needle Holder</td>
			<td>Fine science tools (FST)</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Lock Flush Solution, USP, 100 units/mL</td>
			<td>BD PosiFlush</td>
			<td>306424</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>14043-704-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewelers Bipolar Forceps Non Stick 11 cm, straight pointed tip, 0.25 mm tip diameter</td>
			<td>ASSI</td>
			<td>ASSI.BPNS11223</td>
			<td></td>
		</tr>
		<tr>
			<td>Lone Star Elastic Stays</td>
			<td>CooperSurgical</td>
			<td>3314-8G</td>
			<td>Used to retract the inguinal ligament for femoral vessels dissection</td>
		</tr>
		<tr>
			<td>Lone Star Self-Retaining Retractors</td>
			<td>CooperSurgical</td>
			<td>3301G</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Mosquito Hemostats</td>
			<td>Fine science tools (FST)</td>
			<td>13010-12</td>
			<td>Used to retract the inguinal fat pad distally</td>
		</tr>
		<tr>
			<td>Needle Holder, 15 cm Round Handle, 8 mm diameter, Superfine Curved Jaw 0.2 mm tip diameter, without lock</td>
			<td>ASSI</td>
			<td>ASSI.B1582</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Suture Black Monolfilament 8-0, 6.5 mm 3/8c</td>
			<td>Ethilon</td>
			<td>2808G</td>
			<td>Used to ligate collateral branches on the femoral vessels</td>
		</tr>
		<tr>
			<td>Offset Bone Nippers</td>
			<td>Fine science tools (FST)</td>
			<td>16101-10</td>
			<td></td>
		</tr>
		<tr>
			<td>S&#38;T Vascular Clamps 5.5 x 1.5 mm</td>
			<td>Fine science tools (FST)</td>
			<td>00398-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Walton scissors</td>
			<td>Fine science tools (FST)</td>
			<td>14077-09</td>
			<td></td>
		</tr>
	</tbody>
</table>

partial heterotopic hindlimb transplantation model in rats

血管化复合异位移植 （VCA） 是患者在复杂组织缺陷后没有自体手术可能性的最高级重建选项。脸部和手部移植改变了毁容患者的生活，赋予他们新的审美和功能社会器官。尽管结果大有希望，但由于终生免疫抑制合并症和传染性并发症，VCA仍然表现不佳。大鼠是研究免疫途径和移植排斥机制 的活体 研究的理想动物模型。大鼠还广泛应用于新型复合组织移植保存技术，包括灌注和低温保存研究。大鼠VCA的模型必须可重复、可靠和高效，术后发病率和死亡率较低。异位肢体移植程序符合这些标准，比矫肢移植更容易执行。掌握啮齿动物显微外科模型需要在显微外科和动物护理方面有扎实的经验。据报道，这是大鼠部分异位骨皮瓣移植、术后结果和预防潜在并发症的方法的可靠和可重复的模型。

在这项单操作者研究中，进行了30例同步异位部分肢体移植。成功定义为术后第21天没有VCA失败或需要安乐死的并发症。移植物的正常演变表现在图3中。接受者部分肢体采购和移植插入的平均持续时间分别为35分钟和105分钟：平均缺血时间是105分钟。在随访过程中，发生两种类型的并发症（表1）早或晚。有的需要安乐死，有的需要抢救，都与兽医人员讨论过（<st...

Watch this Scientific Journal Video about Partial Heterotopic Hindlimb Transplantation Model in Rats at JoVE.com

Partial Heterotopic Hindlimb Transplantation Model in Rats

本文介绍了大鼠部分异位骨质皮瓣移植方案及其中期随访的潜在结果。

大鼠部分异位欣德利姆移植模型

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities ...

我们提出了一个可靠和可重复的手术模型，异位后肢移植在大鼠。该技术是 VCA 中保存或免疫排斥的短期研究的理想之选。分步描述将使受训者能够快速掌握手术。首先，在腿部三分之一处对脚踝上方的皮肤进行包皮切口。使用双极钳将尖骨动脉和流行动脉的末梢部分骨架化和烧灼。烧灼和切割胃内皮，鞋底，头骨前部和二头肌股骨肌肉，直到头骨暴露。在右侧的内皮折痕处切口 2.5 厘米，解剖内侧脂肪垫。使用鱼钩缩回器抓住静脉韧带。用夹钳将内在脂肪垫抱住，并收回脂肪垫以暴露股骨血管。解剖股骨血管，个性化墨菲分支，用8/0尼龙领带唇部。通过使用 27 . 5 测量针头将肝素注射鼻静脉，使供体大鼠肝素化。完成臀部周围的皮肤切口。使用双极钳烧灼二头肌和谷胱甘肽超纤维肌，然后烧灼和切开中股骨长度的坐骨神经。在后股骨峰层处近似地暴露股骨，并在股骨韧带水平上用尼龙领带将股骨容器绑起。在连体下方的股动脉上进行动脉切除术，然后放出 24 量表血管导管。烧灼和切割脚踏下剩余的肌肉，以暴露股骨前侧。使用骨切割机近似切开头骨，尽可能切开股骨。用两毫升肝素盐水冲洗部分后肢，以获得清晰的静脉流出。将后肢放在冰上无菌纱布中，直到微血管转移。解剖内在脂肪垫，并倾斜的内在脂肪垫远程暴露股骨血管。使用钩子缩回静脉韧带和夹钳，以保持在偏心脂肪垫远程。解剖股骨血管，个性化墨菲分支，并像所示，用尼龙领带绑上唇部。使用尼龙领带将两个容器放在表观容器上方。将近似夹子放在近处，稀疏容器透镜，用肝素盐水冲洗。在臀部上方的左翼切口，并创建一个皮下口袋，用皮下隧道进入皮肤折痕。将部分肢体的近部和阴性脂肪垫通过皮下隧道进行微血管转移。使用尼龙缝合线执行静脉和动脉麻醉剂。取下两个近似夹，观察肢体的转速。对两艘船进行挤奶测试，以评估每个麻醉症的轻率。在移植肢体的内侧进行纵向皮肤切口，并插入移植物。去除移植物的多余的皮肤，用单独的缝合线和可吸收的4/0缝合线关闭伤口。用两种独立的可吸收缝合线将移植肢体的阴性脂肪垫和接受者缝合在一起，并在微血管麻醉后关闭阴性折痕。异位后肢模型的演变一直监测到研究结束。头发再生在术后的第一周被观察到，皮肤缩回在两周后出现。血管化复合异体移植失败可能发生在术后的第一周，由于微血管血栓。在20%的病例中，静脉血栓形成是早期安乐死的原因，所有这些病例都发生在术后第五天之前。皮肤看起来是蓝色的，每天都变黑。自残是无敏感移植的严重问题，通常发生在术后第二天和第七天之间。如果仅限于不到三分之一的移植表面，只涉及皮肤，手术脱脂和缝合使用不可吸收的缝合线可以与工作人员兽医讨论。防止自残需要使用缝在颈部的电子衣领，直到术后第七天，并清洁动物手术伤口上的任何血液或地壳。多层移植物的严重自噬导致动物安乐死。皮肤囊肿出现在术后第14天，有时在膀胱前有一个皮肤坏死中心。确保移植放置不会阻止动物流动，并确保在移植内嵌过程中，足部不会扭结或紧绷，这一点至关重要。我们的团队在移植前对所采购的四肢进行了保存研究。评估了不同静态冷藏期的影响以及不同的肢体灌注方案。

Research

JoVE Journal

Immunology and Infection

Murine Model of CD40-activation of B cells

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

Orthotopic Hind-Limb Transplantation in Rats

原位大鼠后肢移植

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

在体内成像方法区分急性和慢性炎症

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

体外和活体评估移植受体的 T、B 和髓细胞抑制活性和体液反应

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

无输血或强心剂剂的大鼠心肺旁路移植模型

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

大鼠异位心脏和心肌细胞移植的免疫模型

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

Optimization of the Cuff Technique for Murine Heart Transplantation

穆林心脏移植袖口技术的优化

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

小鼠骨髓移植程序研究克隆造血症

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

小鼠不变自然杀伤性T细胞的纯化和扩增用于体外和体内研究

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...