Immunohistochemical Detection in the Lacrimal Gland, Optic Nerve, Fat

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Immunohistochemical Detection in the Lacrimal Gland, Optic Nerve, Fat A RQUIVOS B RASILEIROS DE ORIGINAL ARTICLE Orbital lymphatic vessels: immunohistochemical detection in the lacrimal gland, optic nerve, fat tissue, and extrinsic oculomotor muscles Vasos linfáticos orbitários: identificação imunohistoquímica na glândula lacrimal, no nervo óptico, no tecido adiposo e nos músculos extrínsecos oculomotores Renato Wendell Ferreira Damasceno1,2,3 , Juliana Arôxa Pereira Barbosa1,2, Lucas Rafael Costa Cortez2, Rubens Belfort Jr.3 1. Universidade Estadual de Ciências da Saúde de Alagoas, Maceió, AL, Brazil. 2. Hospital Universitário Professor Alberto Antunes, Universidade Federal de Alagoas, Maceió, AL, Brazil. 3. Department of Ophthalmology and Visual Sciences, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil. ABSTRACT | Purpose: To identify the lymphatic vessels in RESUMO | Objetivos: Identificar vasos linfáticos em espécimes orbital specimens from human cadavers using light microscopy orbitários de cadáveres humanos através de microscopia and immunohistochemical analysis. Methods: A postmortem óptica e análise imunohistoquímica. Métodos: Um estudo study included 10 orbital specimens from 10 human cadavers. postmortem incluiu dez espécimes orbitários provenientes de The orbital specimens were obtained no later than 12 hours dez cadáveres humanos. Todos os espécimes orbitários foram after death. The orbital specimens were dissected into lacri- obtidos até 12 horas após a morte com uma técnica cirúrgica mal gland, optic nerve, fat tissue, and oculomotor muscles. de exenteração orbitária e dissecados em glândula lacrimal, The histologic criteria to qualify as a lymphatic vessel were nervo óptico, gordura órbitária e músculos extraoculares. Para thin-walled channels of endothelium without a well-developed classificar como um vaso linfático, os critérios histológicos basal membrane and with an erythrocyte-free, irregular lumen. incluíram vasos endoteliais de parede única sem membrana The immunohistochemical criteria were irregularly shaped, basal bem desenvolvida, irregulares e lúmen sem hemácias, e thin-walled vessels with an erythrocyte-free, irregular lumen and os critérios imunohistoquímicos incluíram vasos endoteliais immunopositivity for podoplanin D2-40. Results: The lacrimal de parede única, com formato irregular e lúmen sem hemácias gland, optic nerve, fat tissue, and extraocular muscle sections e reagentes a podoplanina D2-40. Resultados: As lâminas were positively stained with podoplanin D2-40. Conclusions: histológicas de glândula lacrimal, nervo óptico, tecido adiposo This study demonstrated lymphatic vessels in the human orbit, e músculos extraoculares reagiram positivamente a podoplanina more precisely, in the lacrimal gland, dura mater of the optic D2-40. Conclusão: Este estudo demonstrou vasos linfáticos nerve, adipose tissue, and extrinsic oculomotor muscles via light na órbita humana, mais exatamente, na glândula lacrimal, no microscopy and immunohistochemistry. nervo óptico, na gordura orbitária e nos músculos extrínsecos Keywords: Lymphatic vessels; Orbit; Optic nerve; Lacrimal extraoculares via microscopia óptica e imunohistoquímica. apparatus; Oculomotor muscles; Adipose tissue; Microscopy Descritores: Vasos linfáticos; Órbita; Nervo óptico; Aparelho lacrimal; Músculos oculomotores; Tecido adiposo; Microscopia INTRODUCTION The lymphatic system is essential for maintaining the normal balance of body fluids(1,2). This system con- Submitted for publication: December 9,2019 Accepted for publication: April 2, 2020 tinually transports interstitial macromolecules from Funding: This study received no specific financial support. the extracellular space into the venous circulation(2). Disclosure of potential conflicts of interest: None of the authors have any potential conflicts of interest to disclose. Additionally, the lymphatic system is responsible for fat Corresponding author: Renato Wendell Ferreira Damasceno. E-mail: [email protected] absorption and transports immune cells to the lymph Approved by the following research ethics committee: Universidade Estadual nodes, thus playing a prime role in immunity(2). Although de Ciências da Saúde de Alagoas (CAAE: 43039315.3.0000.5011) e Universidade Federal de São Paulo (CAAE: 43039315.3.3001.5505). the organization of the lymphatic system is parallel to This content is licensed under a Creative Commons Attributions 4.0 International License. http://dx.doi.org/10.5935/0004-2749.20210035 Arq Bras Oftalmol. 2021;84(3):209-13 ■ 209 Orbital lymphatic vessels: immunohistochemical detection in the lacrimal gland, optic nerve, fat tissue, and extrinsic oculomotor muscles that of the blood system, the lymphatic vessels are not Randomization uniformly distributed throughout the body in a similar Random software (random.org) was used to define way(1). Whereas the ocular adnexa (conjunctiva, eyelids, a number between 1 and 100. The selected side was and lacrimal drainage system) are rich in blood and determined as left if the number was odd or as right if lymphatic vessels, some well-vascularized tissues, such the number was even. as the central nervous system, have no lymphatics(1). The human orbit, an ocular adnexa richly vascula- Modified surgical technique of rized with blood vessels, is a space bounded by rigid orbital exenteration bones and fascial sheaths(1,2). The orbital blood capilla- First, the incision line was marked around the orbit ries are unfenestrated and carry proteins into the extra- just inside the orbital rim. The dissection was then vascular space continuously; however, these proteins performed beneath the orbicularis oculi muscle to the are not readily reabsorbed owing to the concentration orbital rim, and the periorbita was dissected from the gradient(1,2). Thus, this protein-rich interstitial fluid must orbital walls with a periosteal elevator. Enucleation scissors leave the orbit and be returned to the vascular compart- were passed around the orbital tissue to the apex, and ment to avoid an increase in the intraorbital pressure. any residual bleeding vessels were cauterized. At the end, Several studies have shown that lymphatic vessels the orbital cavity was packed with gauze sponges. may play an important role in various disorders(3-15). A better understanding of the orbital lymphatic system Demographic data may significantly increase our knowledge of immu ne- Information was collected about the age and gender asso ciated orbital diseases, such as orbital inflammatory of all subjects. diseases and Graves’ ophthalmopathy, and the spread of malignant neoplasms, including carcinoma, lymphoma, Histologic analysis and melanoma. All specimens (lacrimal gland, optic nerve, fat tissue, The purpose of this study was to determine whether and extraocular muscles) were fixed in a buffered 10% lymphatic vessels are present in orbital specimens from formaldehyde solution, embedded in paraffin, and pro- human cadavers, with special regard to the lacrimal gland, cessed for light microscopy at the Pathology Laboratory, optic nerve, fat tissue, and extraocular muscles, using light Universidade Federal de Alagoas, Maceió, Brazil. For microscopy and immunohistochemical analysis. light microscopy, 4-μm-thick paraffin sections were stained with hematoxylin-eosin and periodic acid-Schiff. METHODS General information Immunohistochemical analysis Immunohistochemical analysis was performed on This postmortem study was performed on 10 orbital 4-μm-thick paraffin sections using the peroxidase-la- specimens from 10 human cadavers between January 1 beled streptavidin-biotin method and an LSAB-Plus Kit and December 31, 2016. The orbital specimens were according to the manufacturer’s instructions (Dako, obtained no later than 12 hours after death using a mo- Glostrup, Denmark). The paraffin sections were incu- dified surgical technique of orbital exenteration at the bated with primary antibody dilutions, biotinylated link Autopsy Service, Universidade Estadual de Ciências da antibody, and peroxidase-conjugated streptavidin for 30 Saúde de Alagoas, Maceió, Brazil. The orbital specimens min each. The 3-chromogenic substrate 3-amino-9-ethyl were dissected into lacrimal gland (palpebral and orbi- carbazole was applied, and the samples were stained tal lobes), optic nerve, fat tissue (lower inner quadrant, with Mayer’s hemalum. upper inner quadrant, lower outer quadrant, and upper A monoclonal antibody against the human lymphatic outer quadrant), and extraocular muscles (levator palpe- vascular endothelial-specific glycoprotein podoplanin brae superioris, superior rectus muscle, inferior rectus D2-40 (mouse, 1:40; AbD Serotec) was used. Positive muscle, lateral rectus muscle, medial rectus muscle, controls were performed on amygdala specimens, and superior oblique muscle, and inferior oblique muscle). negative controls were performed with control immu- The exclusion criteria were prior orbital surgery, orbital noglobulin G. Two investigators analyzed all paraffin disease, or head and neck radiation therapy. sections. 210 Arq Bras Oftalmol. 2021;84(3):209-13 Damasceno RWF, et al. Detection of lymphatic vessels fat tissue, and extraocular muscles, using light micros- The histologic criteria for a lymphatic vessel were copy and immunohistochemical analysis with D2-40, a thin-walled channels of endothelium without a well- specific antibody for lymphatic vessels(9). developed basal membrane and with an erythro cyte- free, irregular lumen. The immunohistochemical criteria Lacrimal gland were irregularly shaped, thin-walled
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