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?...

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。

By 2021, approximately 12% of people are significantly affected by dry eyes1, making it one of the most common chronic eye diseases. Dry eye can be divided into two types: aqueous-deficient dry eye (ADDE) and evaporative dry eye (EDE)2, depending on the different factors that affect the disease. ADDE is further divided into Sj&#246;gren&#39;s syndrome (SS) and non-SS, but the majority of dry eye patients are non-SS patients in clinical3. Chronic dry eye symptoms seriously affect the visual quality of patients. Currently, the conventional treatment of DED involves the application of artificial tears to lubricate the ocular surface and physical therapy of the eyelids. However, dry eye syndrome may not offer a complete cure in many cases. Therefore, studying the pathogenesis of dry eye disease is crucial for the development of new therapies and drugs. Animal models of dry eye syndrome provide a foundation for further research.
There are many ways to construct animal models of dry eye syndrome4, including changing tear secretion levels by altering hormone levels. For example, removing the testes of rats can reduce androgen secretion, increase tear secretion, and decrease the concentration of free secretory component (SC) and IgA in tears5,6. Another method is to indicate autoimmune reactions in the lacrimal gland by removing the eye surface nerves that control the gland. Additionally, directly reducing tear secretion can be achieved by surgically removing the lacrimal gland7. Changing environmental conditions can also accelerate tear evaporation. For example, culturing animals in low humidity and dry ventilation conditions can establish a model of excessive evaporative dry eye8, which can be combined with other methods to increase the severity of dry eye. The main drugs used to induce dry eye experimental models are atropine and scopolamine9. As parasympathetic inhibitors, both can induce pharmacological blockade of cholinergic (muscarinic) receptors in the lacrimal gland and inhibit tear secretion. Compared with dry eyes caused by atropine muscle injection10, scopolamine has a stronger inhibitory effect on secretion glands, a longer duration of drug action, and weaker effects on cardiac, small intestinal, and bronchial smooth muscles. It is one of the most mature drugs for dry-eye animal models.
Different methods can be used to induce dry eye with scopolamine, such as subcutaneous injection, drug pump, or patch application4,11,12. In order to reduce the frequency of drug administration to experimental animals, many researchers apply transdermal patches to the tails of mice or use drug pumps. However, both of these methods have limitations. For example, the absorption of transdermal patches needs to take into account the individual absorption of mice, which can lead to inconsistent drug dosage. Although drug pumps can accurately control the dosage of each administration, they are not always compatible with the drug being delivered or the concentration being used. They also need to be placed surgically &#8211; which is more invasive to the animal, requiring an anesthetic event, and there is potential for post-surgical complications such as dehiscence. Subcutaneous injection, although more cumbersome, can ensure accurate dosage for each administration and maintain consistency in drug administration among different rats. At the same time, it has a lower cost and is suitable for conducting a large number of animal experiments.
This study applies repeated subcutaneous injection of scopolamine to establish a dry eye rat model. We analyze dry eye indicators such as corneal defects, tear secretion levels, and pathological morphology of the cornea, conjunctiva, and lacrimal gland. By combining drug concentration, pathological manifestations, and dry eye symptoms, we further elaborate on the dry eye rat model in detail, providing more accurate experimental data for the study of dry eye treatment and pathological mechanisms. We also describe the modeling process in detail for future researchers.

作者聚焦：开发干眼动物模型的挑战和未来的研究方向

东莨菪碱诱导泪腺功能障碍的大鼠干眼模型

The process of developing a dry eye animal model is a long one. It requires continuous subcutaneous injection of scopolamine in the hind legs of rice to suppress the secretion of the lacrymal gland in rice. So, experimenters need to have enough patience to administer the medication regularly over a long period of time.Our protocol is a very useful method that can created different degrees of dry eye models based on different drug concentrations. It can make it easiest for the future researchers to study the treatment of dry eye disease. Our team will continue to explore the field of lacrimal gland regeneration, very excited to work on reconstructing 3D and conducting transplantation experiments.Our ultimate goal is to develop more advanced and efficient treatment options for individuals suffering from dry eye syndrome.

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?...

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

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。

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

Research

JoVE Journal

Medicine

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

1.2K 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，?...