本研究得到了广东省临床重点重点专科（SZGSP014）和深圳市自然科学基金（JCYJ20210324125805012）的支持。

按照本协议进行的所有动物实验均在机构动物护理和使用委员会（IACUC）的批准下进行。
1.动物准备
<ol><li>准备 12 只健康的 6 周龄 SPF Wistar 雌性大鼠，体重为 160 克± 20 克。</li>
	<li>使用裂隙灯和检眼镜检查所有大鼠的眼部状况，确保没有眼前节或视网膜疾病。</li>
	<li>将所有老鼠饲养 1 周，并有足够的食物和水源。</li>
	<li>将所有大鼠随机分为正常组，东莨菪碱药物浓度2.5 mg/mL，东莨菪碱药物浓度5 mg/mL，东莨菪碱药物浓度7.5 mg/mL组，每组3只动物。</li>
</ol>2.溶液制备
<ol><li>将东莨菪碱氢溴酸盐溶解在0.9%氯化钠溶液中，制成浓度为7.5mg/mL、5mg/mL和2.5mg/mL的溶液。</li>
	<li>制备不含东莨菪碱氢溴酸盐的0.9%氯化钠溶液，用作对照组大鼠的注射剂。</li>
</ol>3.设备和材料准备
<ol><li>准备一个小动物显微镜。</li>
	<li>准备实验材料，包括 1 mL 带针头的一次性注射器 （26 G）;荧光素钠滴眼试纸;Schirmer 撕裂试纸;无水乙醇;4%多聚甲醛;二甲苯;中性香脂;苏木精、伊红;和高碘酸-希夫染色试剂盒。</li>
</ol>4.皮下注射
注意：此过程需要第二个人的帮助来帮助保护老鼠。
<ol><li>保持老鼠的身体稳定，抓住并伸展它的左（或右）后腿。 注意：助手可以帮助抱着动物。</li>
	<li>用酒精清洁注射部位。</li>
	<li>将 1 mL 带针头 （26 G） 的一次性注射器插入拇指和手指之间的皮肤褶皱底部。</li>
	<li>通过拉回注射器柱塞来吸出注射器。注射器中的任何血液都表明针头放置不当;取出并重新定位针头。</li>
	<li>以稳定的流体运动施用 0.9% 氯化钠溶液，含或不含东莨菪碱氢溴酸盐。</li>
	<li>根据不同浓度注射所有大鼠，每次注射0.5mL，每天4次（9：00,12：00,15：00和18：00），连续19天，左右肢体交替。 注意：这些组的名称如下： 不含东莨菪碱氢溴酸盐组：0组（对照组） 氢溴酸东莨菪碱组 2.5 mg/mL：2.5 组 氢溴酸东莨菪碱组 5 mg/mL：5 组 氢溴酸东莨菪碱组 7.5 mg/mL：7.5 组</li>
	<li>将动物放回笼子并监测呼吸和行为 5-10 分钟。</li>
</ol>5.泪液分泌试验（Schirmer泪液试验，STT）
<ol><li>为大鼠创建改良的滤纸条11.沿中心线（1 毫米× 15 毫米）切割一半的用于人体的滤纸条，并修剪条带的头部使其光滑。 注意：在进行泪液分泌测试之前，请手动约束大鼠的身体，以防止移动并确保暴露大鼠的眼睛。</li>
	<li>将滤纸条放在大鼠下眼睑结膜囊的外1/3处。</li>
	<li>测试时间为 5 分钟。在整个过程中控制大鼠眼睛的闭合。</li>
	<li>测量后，用镊子将滤纸条夹入微量离心管中，并在管壁上做标记，记录撕裂量。</li>
	<li>测量第 0 天、第 1 天、第 3 天、第 5 天、第 7 天、第 11 天、第 15 天和第 19 天的泪液分泌。</li>
</ol>6. 角膜荧光素染色
<ol><li>将0.5μL0.5%荧光素钠溶液滴入每只大鼠的下结膜囊中。</li>
	<li>荧光素滴注后在蓝光下观察角膜3分钟。</li>
	<li>记录每只大鼠角膜的荧光染色，观察是否存在角膜缺损。</li>
	<li>在第 0 天、第 1 天、第 3 天、第 5 天、第 7 天、第 11 天、第 15 天和第 19 天进行角膜荧光素染色。</li>
</ol>7.结膜组织的组织学观察
<ol><li>完成模型开发后，腹膜内注射0.4 mL/100 g 10%水合氯醛水溶液，对大鼠进行深度麻醉，以缓解动物的紧张。然后，通过颈椎脱位对大鼠实施安乐死。</li>
	<li>从每只大鼠的相同区域取球结膜，大小约为2mm x 2mm。</li>
	<li>立即将组织固定在4%多聚甲醛中24小时，并嵌入石蜡13中。</li>
	<li>切割 5 μm 厚的切片，并用苏木精和伊红 （HE）14 和高碘酸 （PAS） 染色（按照制造商的说明进行操作）。</li>
</ol>8.角膜和泪腺组织的组织学观察
<ol><li>完成模型开发后，按照步骤7.1中的说明对大鼠实施安乐死。</li>
	<li>取每只大鼠右侧的角膜，立即将其固定在4%多聚甲醛溶液中。</li>
	<li>沿连接耳朵和外眼角的线切开头皮和皮下组织，将切口扩大到两侧，进一步隔离淡黄色的眼眶外腺。</li>
	<li>彻底去除大鼠的皮毛，用0.9%氯化钠溶液分离眶外腺体。</li>
	<li>将分离的眶外腺体置于4%多聚甲醛溶液中24小时并包埋在石蜡中。</li>
	<li>切割~5μm厚度的连续切片，并用HE染色，用于角膜和眶外腺组织标本。</li>
</ol>9. 统计分析
<ol><li>使用适当的软件对数据进行统计分析。<ol><li>进行单因素方差分析（ANOVA）分析数据，并进行最小显著性差异（LSD）检验以进行组间比较。将统计显著性水平设置为 α = 0.05，P < 0.05 表示统计显著性。 注：SPSS 20软件用于实验数据的统计分析。</li>
	</ol></li>
</ol>

东莨菪碱诱导的大鼠泪腺功能障碍

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作者与该程序中使用的药物和材料没有潜在的利益冲突。

缺水性干眼症（ADDE）是干眼症的重要类型，约占干眼症总人数的1/317，ADDE的主要原因是泪腺病理性损伤和炎症13。对于这种类型的干眼症，最常见的临床治疗方法是人工泪液以缓解症状或局部应用类固醇或环孢菌素18，而泪腺损伤的治疗选择很少。因此，探讨泪腺功能重建对干眼症的影响，建立泪腺功能障碍动物模型非常重要。我们使用重复应用药物的方法抑制大鼠泪腺的分泌，并创建了慢性泪腺功能障碍干眼动物模型。
我们选择大鼠来构建这种干眼症模型，与其他动物模型相比，它具有更多的优势19。例如，兔子的体型较大，需要更多剂量的药物或更频繁的注射才能达到预期的效果。此外，兔子稍微贵一点，这意味着实验成本更高。小鼠也常用于眼科研究，但它们通常用于构建干燥综合征模型20。这些模型侧重于比较器官炎症和淋巴细胞浸润，以探索免疫病理机制。小鼠体型小，泪腺解剖结构复杂，泪液分泌少，因此难以准确反映泪液的产生。大鼠动物模型是一种更适合的干眼症模型，因为它允许方便的药物注射，具有相对简单的喂养条件，适用于临床和实验研究，并且在成本方面也具有优势。它是ADDE的良好动物模型。
我们应用胆碱能受体阻滞剂东莨菪碱抑制大鼠体内胆碱能受体，减少泪腺分泌，改变泪腺细胞的病理结构，从根本上模拟干眼症患者泪腺的状态。与其他方法相比，这种方法可以更好地模拟自然状态下泪腺的受损状况。其它方法，如使用苯扎氯铵滴眼液21或改变外部条件，如降低湿度和增加眼表蒸发4,8，只破坏眼表环境，不改变泪腺的功能状态。因此，它们不适合长期的慢性干眼症。
在测量大鼠泪液分泌量时，我们改进了Schirmer泪液试纸。首先，我们沿着中心线切割人类使用的撕裂试纸。然后，我们将顶部修剪成圆形弧形，并轻轻地将其折叠回顶部，以便于插入大鼠的下结膜囊中。应该注意的是，大鼠是活跃的，并且难以测量5分钟的眼泪。将Schirmer泪液试纸插入大鼠的下结膜囊后，我们手动闭上大鼠的眼睛，以增加它们的舒适度，避免在长时间测量泪液时挣扎，这可能会影响测量结果。
在提取泪腺时，有必要首先找到它。泪腺定位点是耳前部和内眦之间的中点。然后，切割毛皮下的皮肤组织，最大限度地减少碎皮毛的进入，并减少后期的处理过程。在提取过程中，及时清理碎屑皮毛也很重要。特别是在分离泪腺时，使用磷酸盐缓冲盐水 （PBS） 或生理盐水反复冲洗，避免与其他可能影响切片结果的组织混合。
干眼症动物模型开发的优点是，该方案将不同的药物浓度与不同程度的泪腺损伤相关联。这为泪腺功能障碍的治疗研究提供了实验依据。我们改进了建模过程中的一些操作步骤，为研究人员提供更详细的参考资料。此外，我们还增加了干眼动物模型的分析指标，纳入了角膜、结膜和泪腺的形态学指标，计算结膜细胞，并对泪腺损伤程度进行评分。我们评估了炎症、水肿和萎缩引起的泪腺功能。我们选择了最全面、最具成本效益和最准确的分析方法来反映干眼症和泪腺功能障碍的程度。
然而，我们的方法也有一定的局限性。由于构建动物模型的过程很长，实验者需要反复注射。虽然目前有一些方法可以代替手动注射，例如药物泵或透皮贴剂，但它们的使用仍然存在一些并发症。如何应用更好的方法来减少用药频率，避免并发症的发生，并确保剂量的准确性是我们的下一个目标。综上所述，我们改进了东莨菪碱注射引起的干眼动物模型，为ADDE中泪腺功能障碍的研究提供了实验依据。

到 2021 年，大约 12% 的人受到干眼症的严重影响1，使其成为最常见的慢性眼病之一。干眼症可分为两种类型：缺水性干眼症（ADDE）和蒸发性干眼症（EDE）2，具体取决于影响疾病的不同因素。ADDE 进一步分为干燥综合征 （SS） 和非 SS，但大多数干眼症患者在临床3 中为非 SS 患者。慢性干眼症严重影响患者的视力质量。目前，DED的常规治疗包括使用人工泪液润滑眼表和眼睑的物理治疗。然而，在许多情况下，干眼症可能无法完全治愈。因此，研究干眼症的发病机制对于开发新疗法和新药至关重要。干眼症的动物模型为进一步研究奠定了基础。
有许多方法可以构建干眼症4 的动物模型，包括通过改变激素水平来改变泪液分泌水平。例如，切除大鼠的睾丸可以减少雄激素分泌，增加泪液分泌，并降低泪液中游离分泌成分（SC）和IgA的浓度5,6。另一种方法是通过去除控制泪腺的眼表神经来指示泪腺中的自身免疫反应。此外，通过手术切除泪腺可以直接减少泪液分泌7.不断变化的环境条件也会加速泪液蒸发。例如，在低湿度和干燥通风条件下培养动物可以建立过度蒸发干眼症模型8，该模型可以与其他方法相结合，以增加干眼症的严重程度。用于诱导干眼症实验模型的主要药物是阿托品和东莨菪碱9。作为副交感神经抑制剂，两者都能诱导泪腺中胆碱能（毒蕈碱）受体的药理学阻断，并抑制泪液分泌。与注射阿托品肌肉引起的干眼症10相比，东莨菪碱对分泌腺的抑制作用更强，药物作用持续时间更长，对心脏、小肠和支气管平滑肌的作用较弱。它是干眼症动物模型最成熟的药物之一。
不同的方法可用于用东莨菪碱诱导干眼症，例如皮下注射、药物泵或贴剂应用4,11,12。为了减少对实验动物的给药频率，许多研究人员将透皮贴剂涂抹在小鼠的尾巴上或使用药物泵。但是，这两种方法都有局限性。例如，透皮贴剂的吸收需要考虑小鼠的个体吸收，这可能导致药物剂量不一致。尽管药物泵可以准确地控制每次给药的剂量，但它们并不总是与输送的药物或使用的浓度相容。它们还需要通过手术放置——这对动物的侵入性更大，需要麻醉事件，并且有可能出现手术后并发症，例如裂开。皮下注射虽然比较繁琐，但可以确保每次给药的准确剂量，并保持不同大鼠给药的一致性。同时，它成本较低，适合进行大量的动物实验。
本研究应用反复皮下注射东莨菪碱建立干眼症大鼠模型。我们分析干眼症指标，如角膜缺损、泪液分泌水平以及角膜、结膜和泪腺的病理形态。结合药物浓度、病理表现和干眼症状，进一步详细阐述了干眼症大鼠模型，为干眼症治疗及病理机制研究提供了更准确的实验数据。我们还为未来的研究人员详细描述了建模过程。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% sodium chloride solution</td>
			<td>SJZ No.4 Pharmaceutical</td>
			<td>H13023201</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Wuhan Servicebio Technology Co., Ltd</td>
			<td>G1113</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>10009218</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein sodium ophthalmic strips</td>
			<td>Tianjin Yinuoxinkang Medical Device Tech Co., Ltd</td>
			<td>YN-YG-I</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and eosin</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>D006</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Beijing Solarbio Science &#38; Technology Co., Ltd.&#160;</td>
			<td>G8590</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Beijing Solarbio Science &#38; Technology Co., Ltd.</td>
			<td>YA0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic Acid-Schiff Staining Kit</td>
			<td>Beyotime Biotechnology</td>
			<td>C0142S</td>
			<td></td>
		</tr>
		<tr>
			<td>Schirmer tear test strips</td>
			<td>Tianjin Yinuoxinkang Medical Device Tech Co., Ltd</td>
			<td>YN-LZ-I</td>
			<td></td>
		</tr>
		<tr>
			<td>Scopolamine hydrobromide</td>
			<td>Shanghai Macklin Biochemical Co., Ltd</td>
			<td>S860151</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal microscope</td>
			<td>Head Biotechnology Co,. Ltd</td>
			<td>ZM191</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>10023418</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rat dry eye model with lacrimal gland dysfunction induced by scopolamine

缺水性干眼症（ADDE）是一种干眼症，可导致泪液分泌量和质量减少。长时间的异常泪液分泌会导致眼表环境紊乱，包括角膜损伤和炎症。在严重的情况下，ADDE会导致视力丧失甚至失明。目前，干眼症的治疗仅限于滴眼液或物理疗法，只能缓解眼部不适症状，不能从根本上治愈干眼症。为了恢复干眼症中泪腺的功能，我们创建了东莨菪碱诱导的大鼠泪腺功能障碍的动物模型。通过对泪腺、角膜、结膜等因素的综合评估，我们旨在全面了解ADDE的病理变化。与目前的干眼小鼠模型相比，该ADDE动物模型包括泪腺的功能评估，为研究ADDE中的泪腺功能障碍提供了更好的平台。

Schirmer I 测试，SIT I 在实验开始后第0、3、5、7、11、15和19天测量大鼠的泪液量。实验结果表明，与对照组（0组）相比，东莨菪碱组（2.5组、5组、7.5组）泪液分泌量显著降低，差异有统计学意义（P < 0.01）。2.5组、5组和7.5组之间无统计学意义（P > 0.05）。不同组在天数方面没有显著差异（P > 0.05）（图1， 表1）。
角膜荧光素染色 在实验的第 0、3、5、7、11、15 和 19 天进行角膜荧光素染色。结果显示，各组均未出现角膜荧光素染色，表明在20 d不同浓度东莨菪碱药物的实验中未形成明显的角膜上皮缺损（图2）。
角膜上皮的病理分析 实验后，采集每只大鼠的角膜组织进行HE染色，观察角膜上皮的形态，测量角膜上皮层的厚度。对照组角膜上皮由4-6层排列整齐的上皮细胞组成，其中基底层由整齐紧密排列的单层柱状上皮细胞组成。东莨菪碱2.5、5、7组角膜上皮明显偏薄对照组，细胞形态扁平萎缩，细胞结构紊乱。在7.5组中，基底层存在松散的细胞间连接和液泡结构（ 如图3中的红色箭头所示）。与东莨菪碱组的角膜上皮相比，正常对照组的角膜上皮在角膜上皮层厚度上表现出统计学差异（图4）。
泪腺的病理分析 大鼠分泌泪液的主要腺体是眶外泪腺15。观察泪腺切片时，随着东莨菪碱浓度的增加，观察到泪腺上皮细胞形态的变化，伴有炎症和组织水肿。在对照组中没有观察到这种变化。病理结果表明，泪腺炎症改变、细胞水肿和腺上皮细胞萎缩可作为泪腺功能损伤的指标16 （表2）。这些指标可用于测量干眼症的严重程度与泪液分泌量的关系（图5）。
结膜染色结果分析 对照组结膜结构完整，主要由表层和固有层组成。表层为层状柱状上皮细胞，光滑完整，细胞表面有微绒毛。上皮细胞之间存在散在的杯状细胞，细胞体积大，细胞质中有粘液颗粒。3组东莨菪碱药物结膜上皮表层明显变薄，微绒毛和杯状细胞数量减少，细胞排列结构不完整，伴有水肿，HE染色观察到少量炎性细胞（图6）。
通过用PAS染色结膜，计算每只小鼠的三个独立样品中每40x显微视野的平均杯状细胞数，并将其表示为平均值±SD（图7）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66036/66036fig01.jpg"> 图1：每组Schirmer检验值统计（mm） <a href="https://www.jove.com/files/ftp_upload/66036/66036fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66036/66036fig02.jpg"> 图2：大鼠角膜的荧光素钠染色。 在为期20天的荧光素钠实验中，在所有大鼠的角膜中均未观察到阳性结果。 <a href="https://www.jove.com/files/ftp_upload/66036/66036fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66036/66036fig03.jpg"> 图3：角膜上皮染色和厚度测量。 在7.5组中，基底层有松散的细胞间连接和液泡结构（用红色箭头表示） <a href="https://www.jove.com/files/ftp_upload/66036/66036fig03large.jpg" target="_blank">请点击这里查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66036/66036fig04.jpg"> 图4：各组角膜上皮厚度统计。 与东莨菪碱组的角膜上皮相比，正常对照组的角膜上皮在角膜上皮层厚度上表现出统计学差异。 <a href="https://www.jove.com/files/ftp_upload/66036/66036fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66036/66036fig05.jpg"> 图5：大鼠眶外泪腺的HE染色结果。 （A）第0组：在视野中，泪腺呈小叶状结构，由导管和管状腺体组成，导管形态无明显异常，而管状腺体由锥形腺细胞组成，细胞质中粘液物质丰富， 结缔组织无明显水肿，间质血管无明显异常，无明显坏死和炎性细胞浸润。（B） 第 2.5 组：在视野中，观察到泪腺上皮细胞偶尔萎缩，体积减少，腺腔扩张不规则，腔内粘液物质减少（红色箭头表示）。基质中偶尔有游离淋巴细胞浸润（用蓝色箭头表示），但未观察到导管形态明显异常或水肿迹象。（C）第5组：视野中泪道上皮细胞偶有萎缩、体积缩小、腺腔增大、腔内粘液物质减少（红色箭头），基质偶见游离淋巴细胞浸润（蓝色箭头），泪腺小叶间导管形态或结缔组织水肿均未见明显异常。（D）第7.5组：视野可见水肿;泪腺间距增宽，排列不规则（绿色箭头），泪腺上皮细胞常萎缩，体积变小，形状不规则（黄色箭头），偶见腺腔增大，腔内粘液减少（红色箭头），偶游离淋巴细胞浸润（蓝色箭头）， 导管形态无明显异常。 <a href="https://www.jove.com/files/ftp_upload/66036/66036fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66036/66036fig06.jpg"> 图6：大鼠结膜的HE染色。 与对照组的结膜上皮相比，东莨菪碱药物的三组结膜上皮均表现出不同程度的结构损伤。 <a href="https://www.jove.com/files/ftp_upload/66036/66036fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66036/66036fig07.jpg"> 图7：结膜的PAS染色。 （A）正常对照大鼠。（B）东莨菪碱组大鼠。（C）每组高脚杯细胞密度（20x）。黑条 = 100 μm。 <a href="https://www.jove.com/files/ftp_upload/66036/66036fig07large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td>
			<td colspan="7">Schirmer I 检验，SIT（平均值，单位 [mm]）</td>
		</tr><tr><td>群</td>
			<td>0 天</td>
			<td>3天</td>
			<td>5天</td>
			<td>7天</td>
			<td>11天</td>
			<td>15天</td>
			<td>19天</td>
		</tr><tr><td>0</td>
			<td>6</td>
			<td>4.2</td>
			<td>5</td>
			<td>8</td>
			<td>7</td>
			<td>5.5</td>
			<td>6.3</td>
		</tr><tr><td>2.5</td>
			<td>2</td>
			<td>2.7</td>
			<td>2</td>
			<td>2.7</td>
			<td>3.3</td>
			<td>3.7</td>
			<td>3</td>
		</tr><tr><td>5</td>
			<td>2.3</td>
			<td>2.7</td>
			<td>1.7</td>
			<td>2.3</td>
			<td>3.2</td>
			<td>3.7</td>
			<td>3</td>
		</tr><tr><td>7.5</td>
			<td>2.3</td>
			<td>3.2</td>
			<td>2.5</td>
			<td>2.8</td>
			<td>2.7</td>
			<td>3.2</td>
			<td>2.8</td>
		</tr></tbody></table>表1：四组大鼠在不同时间点（mm）的Schirmer试验。 施用药物后，大鼠眼泪的分泌显着减少。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>数</td>
			<td>坏死</td>
			<td>炎症</td>
			<td>水肿</td>
			<td>上皮萎缩</td>
		</tr><tr><td>0 Grp-1型</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr><tr><td>0 Grp -2</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr><tr><td>0 克 -3</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr><tr><td>2.5 克 -1</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
			<td>0</td>
		</tr><tr><td>2.5 克 -2</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr><tr><td>2.5 克 -3</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
			<td>1</td>
		</tr><tr><td>5 Grp -1</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
			<td>1</td>
		</tr><tr><td>5 Grp -2</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
			<td>1</td>
		</tr><tr><td>5 Grp -3</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>1</td>
		</tr><tr><td>7.5 克 -1</td>
			<td>0</td>
			<td>1</td>
			<td>2</td>
			<td>1</td>
		</tr><tr><td>7.5 克 -2</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
			<td>1</td>
		</tr><tr><td>7.5 克 -3</td>
			<td>0</td>
			<td>1</td>
			<td>2</td>
			<td>2</td>
		</tr></tbody></table>表2：大鼠泪腺病理组织评分。 评分标准：0：在正常情况下，考虑到动物年龄、性别和品系等因素，组织被认为是正常的;
1：观察到的变化刚刚超出正常范围;2：可以观察到病变，但还不严重;3：皮损明显，持续恶化;4：病变极其严重，已累及整个组织16.

Watch this Scientific Journal Video about A Rat Dry Eye Model with Lacrimal Gland Dysfunction Induced by Scopolamine at JoVE.com

作者聚焦：开发干眼动物模型的挑战和未来的研究方向

在这里，我们建立了泪腺功能障碍的大鼠模型，为缺水干眼症的研究提供依据。

东莨菪碱诱导泪腺功能障碍的大鼠干眼模型

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal ...

开发干眼症动物模型的过程是一个漫长的过程。它需要在水稻的后腿中连续皮下注射东莨菪碱，以抑制水稻中泪腺的分泌。因此，实验者需要有足够的耐心在很长一段时间内定期服用药物。我们的方案是一种非常有用的方法，可以根据不同的药物浓度创建不同程度的干眼症模型。它可以使未来的研究人员最容易研究干眼症的治疗。我们的团队将继续探索泪腺再生领域，非常高兴能够从事3D重建和移植实验。我们的最终目标是为干眼症患者开发更先进、更有效的治疗方案。

a-rat-dry-eye-model-with-lacrimal-gland-dysfunction-induced

Nanyang Technological University

Research

JoVE Journal

Medicine

A Rat Dry Eye Model with Lacrimal Gland Dysfunction Induced by Scopolamine

1.7K 次观看.  Jinan University. 开发干眼症动物模型的过程是一个漫长的过程。它需要在水稻的后腿中连续皮下注射东莨菪碱，以抑制水稻中泪腺的分泌。因此，实验者需要有足够的耐心在很长一段时间内定期服用药物。我们的方案是一种非常有用的方法，可以根据不同的药物浓度创建不同程度的干眼症模型。它可以使未来的研究人员最容易研究干眼症的治疗。我们的团队将继续探索泪腺再生领域，?...

视频: 作者聚焦：开发干眼动物模型的挑战和未来的研究方向

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

科研

医学

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

实验诱发青光眼通过磁性微球注射眼

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

光化学诱导后部缺血性视神经病变的大鼠模型

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Retroductal颌下腺灌输和本地化分割照射在涎腺功能减退症大鼠模型

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

穿刺诱导的虹膜新生血管虹膜虹膜小鼠模型的建立

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Intense Pulsed Light for the Treatment of Dry Eye Owing to Meibomian Gland Dysfunction

Dry eye disease (DED) is an increasingly common condition and one of the most common complaints of patients. The vast majority of DED is caused by the so-called &#34;evaporative&#34; subtype, that is mainly caused by meibomian gland dysfunction (MGD). Intense pulsed light (IPL) devices employ high intensity pulses of polychromatic lights with a broad range of wavelength (515-1200 nm). IPL treatment has been utilized for years in the field of dermatology, and then its use was applied to ophthalmology for the treatment of MGD. Recently, a new device employing IPL was specifically designed for the periocular application. This procedure determines the thermal selective coagulation and ablation of superficial blood vessels and telangiectasias of the eyelids skin, reducing the release of inflammatory mediators and tear cytokines levels, and improving meibomian glands outflow. IPL treatment is noninvasive and easy to perform, lasts for only a few minutes and can be conducted in an office setting. In the present study, 19 patients underwent 3 sessions of IPL treatment. After treatment, both mean noninvasive break-up time and lipid layer thickness grade significantly increased, as a result of an improvement of tear film stability and quality, respectively. Conversely, no statistically significant changes were found for meibomian gland loss and tear osmolarity. Furthermore, the vast majority of the treated patients (17/19; 89.5% of the total) perceived an improvement of their ocular discomfort symptoms after IPL treatment. Although IPL treatment provides an improvement of both ocular surface parameters and ocular discomfort symptoms after one cycle of three sessions, regular repeated treatments are usually required to maintain the persistence over the time of its beneficial effects.

Dry eye disease is an increasingly common condition, which strongly impair patients&#39; life quality. Recently, a new device employing intense pulsed light, specifically designed for the periocular area, has been shown to improve tear film stability and ocular discomfort symptoms in dry eye disease owing to meibomian gland dysfunction.

梅博米安腺体功能障碍治疗干眼症的强脉冲光

Dry eye disease (DED) is an increasingly common condition and one of the most common complaints of patients. The vast majority of DED is caused by the ...

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

由康卡纳林注射到乳原区引起的水性干眼病的兔子模型:药物功效研究的应用

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

在兔子中使用完全的白眼切除术建立严重干眼模型

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

A Rat Model of Pressure Overload Induced Moderate Remodeling and Systolic Dysfunction as Opposed to Overt Systolic Heart Failure

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation of signal transduction pathways, to counteract, in the short-term, for the cardiac myocyte loss and or the increase in wall stress. However, prolonged activation of these pathways becomes detrimental leading to the initiation and propagation of cardiac remodeling leading to changes in left ventricular geometry and increases in left ventricular volumes; a phenotype seen in patients with systolic heart failure (HF). Here, we describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction (MOD) by ascending aortic banding (AAB) via a vascular clip with an internal area of 2 mm2. The surgery is performed in 200 g Sprague-Dawley rats. The MOD HF phenotype develops at 8-12 weeks after AAB and is characterized noninvasively by means of echocardiography. Previous work suggests the activation of signal transduction pathways and altered gene expression and post-translational modification of proteins in the MOD HF phenotype that mimic those seen in human systolic HF; therefore, making the MOD HF phenotype a suitable model for translational research to identify and test potential therapeutic anti-remodeling targets in HF. The advantages of the MOD HF phenotype compared to the overt systolic HF phenotype is that it allows for the identification of molecular targets involved in the early remodeling process and the early application of therapeutic interventions. The limitation of the MOD HF phenotype is that it may not mimic the spectrum of diseases leading to systolic HF in human. Moreover, it is a challenging phenotype to create, as the AAB surgery is associated with high mortality and failure rates with only 20% of operated rats developing the desired HF phenotype.

We describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction where signal transduction pathways involved in the initiation of the remodeling process are activated. This animal model will aid in identifying molecular targets for applying early therapeutic anti-remodeling strategies for heart failure.

压力过载诱导中度重塑和收缩功能障碍的大鼠模型，而不是过度收缩性心力衰竭

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...