scopolamine-induced tissue damage assessment in dry eye rat models

쥐의 스코폴라민 유도 눈물샘 기능 장애

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.

저자 스포트라이트: 안구건조증 동물 모델 개발의 과제와 향후 연구 방향

스코폴라민에 의해 유도된 눈물샘 기능 장애가 있는 쥐 안구건조증 모델

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.

안구건조증 쥐 모델에서 스코폴라민 유도 조직 손상 평가

시작하려면 쥐의 몸을 안정적으로 잡고 뒷다리를 쭉 뻗습니다. 주사 부위를 알코올로 닦습니다. 그런 다음 엄지와 손가락 사이의 스킨폴드 기저부에 26게이지 바늘이 장착된 1밀리리터 주사기를 삽입합니다.주사기 플런저를 당겨 바늘을 흡입합니다. 필요한 경우 바늘을 제거하고 위치를 변경합니다. 스코폴라민 하이드로브로마이드의 유무에 관계없이 0.5밀리리터의 0.9%염화나트륨을 한 번의 일정한 동작으로 주입합니다.눈물 분비 테스트를 수행하려면 먼저 중심선을 따라 여과지 스트립의 절반을 자릅니다. 스트립의 머리를 다듬어 매끄럽게 만듭니다. 여과지 스트립을 쥐의 아래 눈꺼풀의 결막낭 바깥쪽 1/3에 놓습니다.5분 후 핀셋을 사용하여 여과지 스트립을 마이크로 원심분리기 튜브에 고정합니다. 튜브 벽에 표시를 하여 눈물량을 기록합니다. 쥐 눈의 각막을 염색하려면 0.5 % 플루오레세인 나트륨 용액 0.5 마이크로 리터를 각 눈의 하부 결막 주머니에 떨어 뜨립니다.3분 후 청색광 아래에서 각막을 관찰합니다. 안락사된 쥐의 눈에서 구근결막을 채취합니다. 각 쥐의 오른쪽에서 각막을 절제하고 즉시 4% 파라포름알데히드에 고정합니다.귀와 눈초리를 연결하는 선을 따라 피하 조직에서 두부 표피를 잘라냅니다. 그런 다음 절개 부위를 양쪽으로 확장하고 노란색을 띤 여분의 안와샘을 분리합니다. 다음으로 쥐의 털을 완전히 제거하고 0.9% 염화나트륨 용액으로 여분의 안와샘을 분리합니다.스코폴라민 그룹의 눈물 분비는 현저히 감소했다. 스코폴라민은 각막 착색이 없는 것으로 입증된 바와 같이 각막 상피 결손을 일으키지 않았습니다. 스코폴라민 투여군의 각막 상피는 대조군보다 상대적으로 얇았다.7.5 그룹은 또한 기저층에서 느슨한 세포 내 연결과 진공 구조를 보여주었습니다. 대조군은 각막 상피가 훨씬 더 두꺼웠다. 스코폴라민은 눈물샘에 염증성 변화를 일으키는 것으로 나타났으며, 이는 기능성 눈물샘 손상을 나타냅니다.감소된 점액성 물질은 그룹 2.5 및 5에서 관찰되었으며, 림프구 침윤이 자유로웠다. 또한, 그룹 7.5는 불규칙한 모양의 상피 세포와 세포의 불규칙한 배열을 보여주었습니다. 스코폴라민은 또한 결막의 형태학적 변화를 일으켰다.

Research

JoVE Journal

Medicine

Scopolamine-Induced Tissue Damage Assessment in Dry Eye Rat Models

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

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 ...

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