scopolamine-induced tissue damage assessment in dry eye rat models

Disfunção da glândula lacrimal induzida por escopolamina em ratos

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.

Autor em destaque: Desafios no desenvolvimento de modelos animais de olho seco e futuras direções de pesquisa

Modelo de Olho Seco de Rato com Disfunção da Glândula Lacrimal Induzida por Escopolamina

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.

Avaliação de Dano Tecidual Induzido por Escopolamina em Modelos de Rato com Olho Seco

Para começar, segure o corpo de um rato firme e estique sua pata traseira. Esfregue o local da injeção com álcool. Em seguida, insira uma seringa de um mililitro, equipada com uma agulha calibre 26 na base da dobra cutânea entre o polegar e o dedo.Puxe o êmbolo da seringa para aspirar a agulha. Retire e reposicione a agulha, se necessário. Injetar 0,5 mililitros de cloreto de sódio a 0,9% com ou sem brometo de escopolamina em um único movimento constante.Para realizar o teste de secreção lacrimal, primeiro corte metade de uma tira de papel filtro ao longo da linha central. Corte a cabeça da tira para deixá-la lisa. Coloque a tira de papel de filtro no terço externo do saco conjuntival da pálpebra inferior do rato.Após cinco minutos, use uma pinça para prender a tira de papel filtro em um tubo de microcentrífuga. Coloque uma marca na parede do tubo para registrar o volume de lágrima. Para manchar a córnea do olho do rato, solte 0,5 microlitros de solução de fluoresceína sódica a 0,5% no saco conjuntival inferior de cada olho.Após três minutos, observe a córnea sob luz azul. Pegue a conjuntiva bulbar dos olhos de um rato eutanasiado. Excisar a córnea do lado direito de cada rato e fixá-la imediatamente em paraformaldeído a 4%.Recorte a epiderme cefálica do tecido subcutâneo ao longo da linha que liga a orelha e o canto externo do olho. Em seguida, expanda a incisão para ambos os lados e isole a glândula orbital extra amarelada. Em seguida, remova completamente o pelo do rato e separe a glândula orbital extra com solução de cloreto de sódio a 0,9%.A secreção lacrimal do grupo escopolamina foi significativamente reduzida. A escopolamina não causou defeitos epiteliais corneanos, evidenciado pela ausência de coloração corneana. O epitélio corneano dos grupos escopolamina foi relativamente mais fino que o grupo controle.O grupo 7,5 também apresentou frouxa conexão intracelular e estrutura vacular na camada basal. Os grupos controle apresentaram epitélios corneanos significativamente mais espessos. A escopolamina pareceu causar alterações inflamatórias nas glândulas lacrimais, indicativas de danos funcionais às glândulas lacrimais.Substância mucinosa reduzida foi observada nos grupos 2,5 e cinco, com infiltração livre de linfócitos. Além disso, o grupo 7.5 mostrou arranjo irregular das células com células epiteliais de forma irregular. A escopolamina também causou alterações morfológicas na conjuntiva.

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 ...

Medicina