The authors have no acknowledgements.

这些协议遵循我院的人类研究伦理委员会的指导方针。 1.常规角膜胶原蛋白CXL（C-CXL） 1.患者准备<ol><li>在手术前5天，放了一天的治疗眼1％毛果芸香碱滴两次。 </li><li>在手术室，在无菌条件下，趴在他/她的背部病人。 </li><li>辖表面麻醉，如奥布卡0.4％。 </li><li>清洁眼睛和眼睛周围的皮肤用碘防腐剂两次。 </li><li>使用盖窥器，以保持眼睛睁开。 </li></ol> 2.上皮去除<ol><li>马可中心9.0毫米用圆圈标记角膜角膜。 </li><li>取出角膜上皮用生硬的抹刀中央7.0至9.0毫米乘机械清除。 </li></ol> 3.核黄素应用<ol><li>适用于日与20％葡聚糖0.1％核黄素<html> Ë角膜每分钟为20分钟。 </li></ol> 4. UVA照射<ol><li>照射具有370nm的波长的UVA光的角膜以3毫瓦/厘米2（5.4焦耳/厘米2表面剂量）的辐照，并在一个5厘米的工作距离为30分钟。 </li></ol><img alt="图1" src="/files/ftp_upload/53119/53119fig1.jpg" /> 图1:在C-CXL UVA照射的角膜以3毫瓦/厘米2（5.4焦耳/厘米2表面剂量）的辐照，并在一个5厘米的工作距离30分钟照射370nm的波长的UVA光。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53119/53119fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="2"><li>在辐照过程中，应用核黄素角膜每隔5分钟滴。 </li><li>在辐照过程中，添加表面麻醉（奥布卡0.4％），如果有必要的。 </li></ol>手术"&gt; 5。完<ol><li>把抗生素滴眼液（妥布霉素0.3％）和人工泪液（透明质酸下降0.18％），到运营的眼睛。 </li><li>将软绷带隐形眼镜在手术结束，直到再上皮化完成。再上皮化通常需要3天。 </li><li>处方止痛药如对乙酰氨基酚（500毫克）加可待因（30毫克），每日6药片。 </li><li>后角膜再上皮，发起局部治疗与类固醇（地塞米松局部为1mg / ml），并继续进行3-4周。此外，使用人工泪液，每天4次，1个月。 </li></ol> 2.加速角膜胶原CXL（A-CXL） 1.患者准备<ol><li>在手术前5天，放了一天的治疗眼1％毛果芸香碱滴两次。 </li><li>在手术室，在无菌条件下，趴在他/她的背部病人。 </li><li>辖表面麻醉，如奥布卡0.4％。 </li><li>清洁电子你们和眼睛周围的碘消毒剂皮肤的两倍。 </li><li>使用盖窥器，以保持眼睛睁开。 </li></ol> 2.上皮去除<ol><li>标记中央9.0毫米角膜的带圈角膜标记</li><li>取出角膜上皮用生硬的抹刀中央7.0至9.0毫米乘机械清除。 </li></ol> 3.核黄素应用<ol><li>在角膜上施加0.1％的核黄素，而不葡聚糖每2分钟进行10分钟。 </li></ol> 4. UVA照射<ol><li>照射具有370nm的波长的UVA光的角膜以30毫瓦/厘米2（5.4焦耳/厘米2表面剂量），并在一个5厘米的工作距离为3分钟的辐照。 </li><li>在照射，增加局部麻醉（oxybuproca＆＃239，是ne 0.4％），如果有必要的。 </li></ol> 5.结束手术<ol><li>将抗生素滴眼液（妥布霉素0.3％）和人工泪液（透明质酸下降0<html> 0.18％）进入操作眼睛。 </li><li>将软绷带隐形眼镜在手术结束，直到再上皮化完成。再上皮化通常需要3天。 </li><li>处方止痛药如对乙酰氨基酚（500毫克）加可待因（30毫克），每日6药片。 </li><li>后角膜再上皮，发起局部治疗与类固醇（地塞米松局部为1mg / ml），并继续进行3-4周。此外，使用人工泪液，每天4次，1个月。 </li></ol> 3，离子透入（I-CXL） 1.患者准备<ol><li>在手术前5天，放了一天的治疗眼1％毛果芸香碱滴两次。 </li><li>在手术室，在无菌条件下，趴在他/她的背部病人。 </li><li>辖表面麻醉，如奥布卡0.4％。 </li><li>清洁眼睛和眼睛周围的皮肤用碘防腐剂两次。 </li><li>使用盖窥器，以保持眼睛睁开。 </li></ol><html> _step"&gt; 2，放置离子电渗装置。 <ol><li>应用粘被动电极的手术视野下的额头。 </li><li>应用活性电极，一个吸环，到打开眼。释放所述抽吸之前围绕角膜的周边上的吸环。 </li></ol><img alt="图2" src="/files/ftp_upload/53119/53119fig2.jpg" /> 图2.离子导入装置，该无源电极施加在手术视野下的额头和活性电极，一个吸环，适用于开放的眼睛。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53119/53119fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.核黄素应用<ol><li>填写吸入环与低渗0.1％核黄素无葡聚糖。 </li></ol><html> JPG"/&gt; 在I-CXL图3.核黄素的应用 。吸入环充满了低渗0.1％核黄素没有葡聚糖。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53119/53119fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="2"><li>启动电流在0.2 mA和逐步增加至1.0毫安的5分钟（图4），总离子电渗疗法的时间。 </li></ol><img alt="图4" src="/files/ftp_upload/53119/53119fig4.jpg" /> 图4.离子导入设备核黄素渗透的电流最初是0.2 mA和逐渐增加至1.0毫安。总离子导入时间为5分钟。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53119/53119fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> EP"&gt; 4。UVA照射<ol><li>照射具有370nm的波长的UVA光的角膜以10毫瓦/厘米2（5.4焦耳/厘米2表面剂量），并在一个5厘米的工作距离为9分钟的辐照。 </li><li>在照射，增加局部麻醉（oxybuproca＆＃239，是ne 0.4％），如果有必要的。 </li></ol> 5.结束手术<ol><li>将抗生素滴眼液（妥布霉素0.3％）和人工泪液（透明质酸下降0.18％），到运营的眼睛。 </li><li>处方止痛药如对乙酰氨基酚（500毫克）加可待因（30毫克），每日6药片。 </li><li>手术后，发起局部治疗与类固醇（地塞米松局部为1mg / ml）和持续3-4周。此外，使用人工泪液，每天4次，1个月。 </li></ol>

<ol>
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S., 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=Pharmacokinetics+and+safety+of+transcorneal+iontophoresis+of+tobramycin+in+the+rabbit.">Pharmacokinetics and safety of transcorneal iontophoresis of tobramycin in the rabbit.</a> Invest Ophthalmol Vis Sci. 29 (9), 1397-1401 (1998).</li><li>Vinciguerra, P., 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=Transepithelial+iontophoresis+corneal+collagen+cross-linking+for+progressive+keratoconus:+initial+clinical+outcomes.">Transepithelial iontophoresis corneal collagen cross-linking for progressive keratoconus: initial clinical outcomes.</a> J Refract Surg. 30 (11), 746-753 (2014).</li><li>Caporossi, 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=Riboflavin-UVA-induced+corneal+collagen+cross-linking+in+pediatric+patients.">Riboflavin-UVA-induced corneal collagen cross-linking in pediatric patients.</a> Cornea. 31 (3), 227-231 (2012).</li><li>Buzzonetti, L., Petrocelli, G., Valente, P., Larossi, G., Ardia, R., Petroni, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iontophoretic+transepithelial+corneal+cross-linking+to+halt+keratoconus+in+pediatric+cases:+15-month+follow-up.">Iontophoretic transepithelial corneal cross-linking to halt keratoconus in pediatric cases: 15-month follow-up.</a> Cornea. 34 (5), 512-515 (2015).</li><li>Baiocchi, S., Mazzotta, C., Cerretani, D., Caporossi, T., Caporossi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+crosslinking:+riboflavin+concentration+in+corneal+stroma+exposed+with+and+without+epithelium.">Corneal crosslinking: riboflavin concentration in corneal stroma exposed with and without epithelium.</a> J Cataract Refract Surg. 35 (5), 893-899 (2009).</li><li>Wollensak, G., Iomdina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+and+histological+changes+after+corneal+crosslinking+with+and+without+epithelial+debridement.">Biomechanical and histological changes after corneal crosslinking with and without epithelial debridement.</a> J Cataract Refract Surg. 35 (3), 540-546 (2009).</li><li>Soeters, N., Wisse, R. P., Godefrooij, D. A., Imhof, S. M., Tahzib, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+versus+epithelium-off+corneal+cross-linking+for+the+treatment+of+progressive+keratoconus:+a+randomized+controlled+trial.">Transepithelial versus epithelium-off corneal cross-linking for the treatment of progressive keratoconus: a randomized controlled trial.</a> Am J Ophthalmol. 159 (5), 821-828 (2015).</li></ol>

The authors have nothing to disclose.

用UVA照射和核黄素CXL的标准治疗为阻止圆锥角膜的进展。核黄素是光敏剂诱导化学共价键（交联）时的UVA 3照射。在角膜上，这种现象造成的胶原纤维能够提高角膜刚度之间交联。虽然这种现象被很好地描述，到现在为止已有角膜内交联的直接证据。然而，一些研究已经报道了的Kmax的稳定化从而显示出的C-CXL 3-8的有效性的步骤之后。这个效果是否是由于交联或在角膜基质等微观结构的变化的问题​​仍然没有答案。 其中一个CXL疗效的间接临床结果是检测为OCT和IVCM1个月CXL后角膜分界线。近日，Kymionis等。表明hyperreflectance EVAluated采用华侨城相当于无细胞和看到的共聚焦显微镜20的蜂窝区之间的过渡区。因此，看到华侨城角膜分界线必须对应于激活的角膜基质和fibroblastes的区域下脱细胞基质及以上正常的。尽管如此，山药及联营公司21未能证明CXL分界线深度之间的相关性最大K值6个月C-CXL后视力的变化。的较大量的CXL是否可导致视力和更大的减少角膜曲率的更大的增强的问题仍然是较长的术后随访研究的主题。另外，关于上IVCM平均前基质角膜密度计，在6个月内观察到显著减少，具有正常化在12个月的具有C CXL和A-CXL，并在6个月与I-CXL 22-24 。因此，不同的微观角膜变化似乎依赖于所用的胶原交联的协议的类型。这一结果和事实，即角膜分界线出现显著更深层次的C-CXL后比后，A-CXL或I-CXL让我们来讨论这三种协议的指示和有效性。 常规的协议已经证明其有效性和安全性，最大的后续的6年3-8。 C-CXL需要具有至少400微米的角膜厚度测量法，以保护内皮细胞25。其主要缺点是与持续时间（1小时），并有必要去除上皮。实际上，这导致患者的不适和疼痛，并可能导致几个并发症如感染性角膜炎和基质混浊9。然而，就目前而言，这个协议仍然建议治疗进行性圆锥角膜，尤其是在进化是积极的。 鉴于对C-CXL的主要缺点之一是第该过程电子工期，加快协议的最初目的是减少操作时间通过提供更高的辐照度角膜26。然而，减少浸泡时间至10分钟可限制核黄素进入角膜的帧内基质的渗透，从而导致所观察到的角膜浅层分界线。即使尚未见报道，尽管事实光子数相同触摸原纤维，它可能是在A-CXL的高出10倍的辐照延伸内皮损伤27的风险，28，在这方面，它是重要此言，缺乏葡聚糖的用于A-CXL核黄素可以解释，尽管高照度的情况下内皮损伤。的确，葡聚糖已知具有通向角膜变薄的过程中，并因此潜在内皮损伤29的渗透作用。因此，加速CXL似乎是一个安全CXL模式。此外，在A-CXL协议小号EEMS是有效;不愧是最大K值保持稳定在6个月随访。然而，作为C-CXL，它的主要限制是导致疼痛和潜在的并发症，如雾度和角膜感染9 desepithelialization。 Touboul 等人进行了使用与A-23 CXL治疗的患者共聚焦显微镜定性研究。事实上，相对于C-CXL的UVA-核黄素的加强效果似乎是最突出于前150-200微米以更大的角膜细胞凋亡和增加的基质的反射率的角膜。这一发现表明，患者角膜薄（350-400微米最小厚度）可以从加速CXL受益。这时，一个防低渗核黄素，导致角膜薄之前C-CXL的肿胀使用，因为该协议仍需要至少400微米的角膜测厚仪，防止血管内皮损伤25。尽管如此，加速CXL可能会喜欢entially用于在未来作为一个更快，更渗透治疗圆锥角膜稳定更薄的角膜。然而，还需要长期的研究，以决定性地关联与对角膜生物力学效果的分界线的深度。 离子电渗疗法CXL是最近开发的，以避免上皮清创12，30。应用的电流力的防低渗核黄素穿透角膜基质1跨上皮协议。 Vinciguerra和同事检查20眼​​而进行离子导入CXL前瞻性研究。他们表明的Kmax是稳定的1年后的程序。然而，分界线并不清楚衡量与华侨城在后续31。同样的，在我们的研究中，角膜分界线评估具有AS十月在不到一半的患者（46.5％），几乎看不到在214微米的平均深度。另外，激光共聚焦显微镜显示少得多角膜APoptosis并增加基质反射后，我-CXL后比其他两个协议。事实上，使用共聚焦显微镜和改性核黄素（Ricrolin TE）Caporossi等。研究跨上皮交联的其他协议。至于离子导入，他们发现角膜基质细胞凋亡是肤浅的（平均140微米的深度）和不均匀见于前基质11。另外，他们确认这外延ON协议导致圆锥角膜进化随访24个月后，加入了值得注意的问题，以它的儿童患者的应用程序，往往从疾病32的更积极的方式受到影响。的确，对于其他跨上皮协议，离子电渗疗法似乎并不保证在地形指标的儿科患者33的改进。此缺席疗效可以通过有限核黄素和UVA渗透原位 11,34-36上皮进行说明。的确，上皮是一个physi校准屏障为核黄素和UVA渗透，限制细胞凋亡的深度，从而角膜生物力学效应11。此外，核黄素伴随用作UV曝光28中的光敏剂和UV阻断剂。因此可以想到的是，作为其他跨上皮协议，离子电渗期间不足核黄素渗透将不仅限制了程序的功效，同时也增加的内皮细胞受损的风险。然而，没有内皮细胞丢失指出尚未离子导入后。最后，在我们的研究中，类似于Vinciguerra 等 31，6个月的I-CXL后的最高K值出现稳定。但是，它仍然从长期随访看到这个新的程序是否依然可靠。因此，与其他外延上的协议，谨慎使用离子导入时需要。尽管如此，热情跨上皮CXL是可以理解的，考虑到潜在的下降CXL并发症。与外延关CXL，发生并发症的基本上是由临时霾9造成箱子的约1％。不幸的是，这种阴霾偶尔留下角膜瘢痕。因此，我们认为，目前，离子导入CXL应慎用于儿童患者使用，我们主要提出这个协议给患者角膜薄，慢慢进展圆锥角膜。 得出结论，对于渗透，传统的CXL协议仍是治疗圆锥角膜进步的标准选项。加速CXL似乎是一种快速，有效，安全的替代治疗尤其是角膜薄。离子电渗疗法用前角膜的损伤小和较不可见分界线相关联，并且因此应考虑以更大的谨慎。

圆锥角膜是一个双边和渐进的角膜扩张通常在一般人群1导致角膜形状的变形例中报告1 2000，从而视力下降2。圆锥角膜通常存在于早期青春期的进展，直到第三次生命不惑之年发病时通常趋于稳定，但进展可以在整个病人的生命变量。通过阻止圆锥角膜进展，交联的目的是推迟或避免角膜移植术。 到目前为止，唯一的有效和安全的治疗进步圆锥角膜证明在临床研究中是常规的角膜胶原交联（C-CXL）协议，其目的是提高刚度，从而制止圆锥角膜级数3-8。为了减少操作时间和对C-CXL其它可能的危险因素，如感染性角膜炎或基质混浊9，几种改进协议具有已有描述。首先，在加速CXL（A-CXL），UVA的更高的辐照度被传递到角膜通过减少的时间10。其次，为了避免的必要性上皮清创，跨上皮的方法已被采用。遗憾的是，他们已经时比现有协议11有限的成功。最近的跨上皮方法CXL期间角膜核黄素递送是离子电渗疗法（Ⅰ-CXL），但这种治疗方法的严格评价还未执行的12。离子电渗疗法是一种在其中一个小电流被施加穿过组织来提高离子化的药物的渗透非侵入性技术。在CXL通过离子电渗，核黄素被离子化通过上皮渗透角膜。 体内共焦显微镜（IVCM）是成像，可以突出显示异常的角膜中的疾病，如圆锥角膜13的细胞改变角膜的方法。事实上，IVCM已经证明改建在圆锥角膜的角膜所有层与在子基底神经丛的密度特定还原和基质角膜13-15。加，IVCM已被证明是对后的C-16 CXL角膜微观结构分析非常方便。 角膜分界线被描述为一个高反射线见于眼前段光学相干断层扫描（OCT AS）1个月后的C-CXL在300微米17,18的深度。 IVCM下面的C-CXL提供有关角膜的结构改变，包括缺乏角膜基​​质的至300μm的深度信息。此无细胞区域的深度，以及在角膜基质内的分界线的深度显示在AS OCT中，似乎与CXL治疗19和角膜分界线深度的测量的有效深度的AS OCT中1到相关联的一个月后CXL已被提出作为一种有效的临床方法的CXL效力18的评价。 在本研究中，我们使用角膜基质分界线测量由AS OCT和共聚焦显微镜研究了三种不同的角膜胶原交联的协议（常规的，加速，离子电渗疗法）的效率。此外，我们使用IVCM定量分析的三种治疗后，角膜组织变化。

<table>
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td>Riboflavin&#160;&#160;&#160;&#160;&#160;&#160;&#160; Product number</td>
			<td></td>
		</tr>
		<tr>
			<td>C-CXL</td>
			<td>Sooft SPA, Montegiorgio, Italy</td>
			<td>Ricrolin&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 468465-6</td>
			<td></td>
		</tr>
		<tr>
			<td>A-CXL</td>
			<td>Avedro Inc, Waltham, Massachusetts</td>
			<td>VibeX&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 520-01863-006</td>
			<td></td>
		</tr>
		<tr>
			<td>I-CXL</td>
			<td>Sooft SPA, Montegiorgio, Italy</td>
			<td>Ricrolin+&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 975481-6</td>
			<td>Passive electrode: PROTENS ELITE 4848LE/ Active electrode: IONTOFOR CXL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>UVA Machine</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Vega</td>
			<td></td>
			<td></td>
			<td>UVA: 3 mW/cm2 30 min</td>
		</tr>
		<tr>
			<td>KXL System</td>
			<td></td>
			<td></td>
			<td>UVA: 30 mW/cm2 10 min</td>
		</tr>
		<tr>
			<td>X-Vega</td>
			<td></td>
			<td></td>
			<td>UVA: 10 mW/cm2 9 min</td>
		</tr>
	</tbody>
</table>

three different protocols of corneal collagen crosslinking in keratoconus: conventional, accelerated and iontophoresis

圆锥角膜是双边和渐进的角膜扩张。为了减缓其进展，角膜胶原交联（CXL）最近已推出了作为有效的治疗选择。在生物和化学科学，交联是指活性分子之间形成新的化学键。因此，角膜胶原CXL的目的是合成地增加在角膜基质中的交联的胶原原纤维之间的形成。尽管常规CXL（C-CXL）协议的效率已经在若干临床研究表明，它可能会受益于改进的过程和除去角膜上皮的持续时间。因此，为了提供两个新的和优化的CXL协议一致的评估，我们研究圆锥角膜患者谁经历了三个CXL治疗之一:离子电渗疗法（Ⅰ-CXL），加速CXL（A-CXL），和常规CXL（ C-CXL）。 A-CXL是6时间快CXL程序ü唱了十次提高的UVA辐射，但仍然包括上皮去除。离子电渗疗法是在其中一个小电流施加到改善整个角膜核黄素渗透跨上皮非侵入性技术。使用眼前段光学相干断层扫描（AS OCT）和体内共焦显微镜（IVCM），我们的结论是关于治疗穿透深度，常规CXL协议仍然用于治疗进行性圆锥形角膜的标准。加速CXL似乎是一个快速，有效，安全的替代治疗角膜薄。利用离子导入还在调查中，应该考虑更加谨慎。

<html>角膜分界线是华侨城可见的情况下，92％在301.6微米的平均深度（SD，73.6） <img alt="图5" src="/files/ftp_upload/53119/53119fig5.jpg" /> C-CXL后图5.分界线。高分辨率的角膜眼前段光学相干断层扫描（华侨城）可视化角膜基质分界线在358微米（白色箭头）1个月传统的角膜胶原蛋白交叉后，平均深度链接（C-CXL）。比例尺250微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53119/53119fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 1个月后C-CXL，而之后A-CXL它在183.1微米（SD，39.6）平均深度见过的病例的85.5％。 加载/ 53119 / 53119fig6.jpg"/&gt; A-CXL后图6.分界线。高分辨率的角膜眼前段光学相干断层扫描（华侨城）可视化角膜基质分界线在176微米（白色箭头）1个月加速角膜胶原蛋白交叉后，平均深度联（A-CXL）。比例尺:250微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53119/53119fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 最后，我-CXL后，角膜分界线仅在214微米（SD，37.5）平均深度见过的病例的46.5％。下面无论是C-CXL，A-CXL或I-CXL角膜分界线深度差异有统计学显著（P &lt;0.001和p = 0.01）。经过C-CXL和A-CXL比我-CXL（P = 0.005）后的分界线存在显著更加频繁。 <img alt="图7" src="/files/ftp_upload/53119/53119fig7.jpg" /> I-CXL后，图7分界线。高分辨率的角膜眼前段光学相干断层扫描（华侨城）可视化角膜基质分界线的238.5微米（白色箭头）1个月后，离子导入（I-CXL平均深度）。比例尺:250微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53119/53119fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 患者随访检测到6个月应用程序的任何的三个协议，其中包括内皮细胞计数无显著差异后的任何内部或术后并发症。此外，最大K值（的Kmax）后6个月的随访保持稳定为每个协议。 <img alt="表格1" src="/files/ftp_upload/53119/53119tab1.jpg" /></p&gt; 表1.疗效和CXL，进化的最大K值（屈光度，D）和内皮细胞密度以下的常规（C-CXL）， 每个协议的安全加速（A-CXL）和离子导入（I-CXL）交联。 对于每个协议，在1-3个月手术后，前基质水肿与细胞外缺陷和零散的角膜基质细胞的细胞核，观察与IVCM。在6个月，前部基质与角膜细胞核复育被看见和更大之后的I-CXL比后两个其他协议。交联和非交联的角膜基质之间的分界被看作是一个区域，其中角膜变得细长，并通过大超反光基质带包围。 <img alt="图8" src="/files/ftp_upload/53119/53119fig8.jpg" /> 图8:微观CC后角膜变化XL常规胶原交联（C-CXL）后获得1个月角膜基质的体内共焦显微镜扫描（IVCM）:前基质水肿与超反射细胞质（白色箭头）和细胞外腔隙（星号）中观察到。比例尺:50μm左右。

Watch this Scientific Journal Video about Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis at JoVE.com

Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Corneal collagen cross-linking (CXL) is the only conservative treatment currently available to halt keratoconus progression by improving the biomechanical rigidity of the corneal stroma. The aim of this manuscript is to highlight the methods of three different protocols of CXL: conventional CXL (C-CXL), accelerated CXL (A-CXL), and iontophoresis CXL (I-CXL).

角膜胶原交联的圆锥角膜的三种不同的协议:常规，加速和离子导入

Keratoconus is a bilateral and progressive corneal ectasia. In order to slow down its progression, corneal collagen cross-linking (CXL) has recently been ...

three-different-protocols-corneal-collagen-crosslinking-keratoconus

Nanyang Technological University

Research

JoVE Journal

Medicine

19.8K 次观看.  Quinze-Vingts National Ophthalmology Hospital. Corneal collagen cross-linking (CXL) is the only conservative treatment currently available to halt keratoconus progression by improving the biomechanical rigidity of the corneal stroma. The aim of this manuscript is to highlight the methods of three different protocols of CXL: conventional CXL (C-CXL), accelerated CXL (A-CXL), and iontophoresis CXL (I-CXL).

视频: Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

人类胰岛染色协议

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

科研

医学

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

角膜新生血管在小鼠碱烧伤模型

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes &#8211; but is not limited to &#8211; hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.

This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.

室颤由传统的封闭胸技术大鼠模型和复苏

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

超声心动图方法和协议心脏瓣膜疾病的综合表型特征小鼠

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导:"电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

心脏磁共振评估疑似心脏血栓:常规和新兴技术

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

使用分裂的角质按钮的孔膜内皮器官培养模型

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Simultaneous Laryngopharyngeal and Conventional Esophageal pH Monitoring

In addition to typical reflux symptoms, many patients with gastroesophageal reflux disease (GERD) present with extraesophageal symptoms such as cough, hoarseness or asthma, which can be caused by laryngopharyngeal reflux (LPR). Due to their multifactorial origin, those symptoms can be a great diagnostic and therapeutic challenge. Esophageal pH-monitoring is commonly used to determine abnormal esophageal acid exposure and confirm the diagnosis of GERD. However, for better evaluation of acid exposure above the upper esophageal sphincter, a new laryngopharyngeal pH measurement system is now available and may lead to more reliable results in patients with predominantly extraesophageal symptoms. This article aims to present a standardized protocol for simultaneous pH measurement using esophageal and laryngopharyngeal pH probes in order to obtain acid exposure scores from both measurements.

Laryngopharyngeal pH monitoring has been specifically designed to measure acid exposure above the upper esophageal sphincter and complements diagnostic evaluation in patients that present with mainly extraesophageal reflux symptoms. Patients with suspected laryngopharyngeal reflux (LPR) were evaluated using distal esophageal and laryngopharyngeal pH testing simultaneously.

同时监测喉咽和常规食管 pH 值

In addition to typical reflux symptoms, many patients with gastroesophageal reflux disease (GERD) present with extraesophageal symptoms such as cough, ...

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, cirrhosis, and/or hepatocellular carcinoma. Liver transplantation is the clinically recommended procedure to improve and extend the life span of patients in advanced disease stages. However, only 10% of transplants are successful, with organ availability, presurgical and postsurgical procedures, and elevated costs directly correlated with that result. Extracellular matrix (ECM) scaffolds have emerged as an alternative for tissue restoration. Biocompatibility and graft acceptance are the main beneficial characteristics of those biomaterials. Although the capacity to restore the size and correct function of the liver has been evaluated in liver hepatectomy models, the use of scaffolds or some kind of support to replace the volume of the extirpated liver mass has not been assessed.
Partial hepatectomy was performed in a rat liver with the xenoimplantation of a collagen matrix scaffold (CMS) from a bovine condyle. Left liver lobe tissue was removed (approximately 40%), and an equal proportion of CMS was surgically implanted. Liver function tests were evaluated before and after the surgical procedure. After days 3, 14, and 21, the animals were euthanized, and macroscopic and histologic evaluations were performed. On days 3 and 14, adipose tissue was observed surrounding the CMS, with no clinical evidence of rejection or infection, as was vessel neoformation and CMS reabsorption at day 21. There was histologic evidence of an insignificant inflammation process and migration of adjacent cells to the CMS, observed with the hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome staining. The CMS was shown to perform well in liver tissue and could be a useful alternative for studying tissue regeneration and repair in chronic liver diseases.

Liver diseases are induced by many causes that promote fibrosis or cirrhosis. Transplantation is the only option for recovering health. However, given the scarcity of transplantable organs, alternatives must be explored. Our research proposes the implantation of collagen scaffolds in liver tissue from an animal model.

三维胶原基质支架植入作为肝脏再生策略

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, ...