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Lymphatic Vessels in Human Eyelids: an Immunohistological Study in Dermatochalasis and Chalazion

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Lymphatic Vessels in Human Eyelids: an Immunohistological Study in Dermatochalasis and Chalazion 29 Lymphology 41 (2008) 29-39 LYMPHATIC VESSELS IN HUMAN EYELIDS: AN IMMUNOHISTOLOGICAL STUDY IN DERMATOCHALASIS AND CHALAZION M. Aglianó, P. Lorenzoni, N. Volpi, L. Massai, P. Carbotti, M. Fruschelli, M. Muscettola, C. Alessandrini, G. Grasso Departments of Biomedical Sciences, Anatomy and Histology Section, (MA,PL,NV,LM,PC, CA,GG), Ophthalmology and Neurosurgery (MF), and Physiology (MM), University of Siena, Italy ABSTRACT are modulated by multiple pathophysiological conditions. Thus, vasoactive factors play a role We investigated lymphatic morphology in the physiology of richly vascularized eyelids, and expression of endothelin (ET-1) axis and therefore, morphofunctional characteri- molecules in human eyelids affected by an zation of lymphatic vessels may be useful in inflammatory state (chalazion) and an age- suggesting treatment options. related degenerative condition (dermatocha- lasis). Lymphatics were immunohistologically Keywords: lymphatics, dermatochalasis, detected by D2-40/LYVE-1 staining. chalazion, endothelin-axis, eyelids Absorbing lymphatic vessels were localized in papillary dermis and around skin appendages The main function of lymphatic vessels with distinctive morphology. In chalazion, is to return lymph to the blood circulation D2-40 reactive flattened lymphatic profiles through lymphatic-venous junctions; were compressed by inflammatory infiltrate; moreover, the lymphatic system contributes in dermatochalasis, large fully opened to the immune response, and it is involved lymphatics were observed, with a significantly in numerous pathological conditions such as wider total area (lymphatic lumina/200x field; lymphedema, inflammatory diseases, and p<0.05). The lymphatic density (number/200x tumor metastasis. field) in the two groups was within the same Whereas the detection of lymphatics range. Lymphatic dilation is possibly previously relied exclusively on ultrastruc- dependent on reduction and fragmentation tural criteria (1), recent identification of of the dermal elastic network as well as of several specific markers provides new oxytalanic fibers in the papillary dermis of insights into the lymphatic distribution and dermatochalasis, as shown by Weigert’s pathophysiology (2). These lymphatic reaction. Multifunctional peptide ET-1, markers include VEGFR-3, a tyrosine kinase involved in vasomotion, inflammation and receptor for vascular endothelial growth connective proliferation, was faintly and factor (VEGF)-C and VEGF-D (3); discontinuously localized on lymphatics, as podoplanin, an O-linked transmembrane was its type A receptor. In contrast, the sialoglycoprotein, also recognized by D2-40 consistent expression of type B receptor monoclonal antibody (4-6); transcription indicates that lymphatic endothelium is a factor Prox-1, a homolog of the Drosophila physiological target for ET-1, whose effects homeobox gene prospero (7); and the Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. 30 lymphatic vascular endothelial hyaluronan the human eye (18-20), little information is receptor-1 (LYVE-1) (8). available on ET-1 localization in periocular In the skin, lymphatics originate as a structures (21). network of initial absorbing vessels in the The present study was carried out in superficial dermis which merge in precollec- order to investigate the distribution of human tors draining into larger deep collectors. eyelid lymphatics and their ET-1, ETAR/ Little is known about lymphatic vessels in ETBR expression in dermatochalasis and human eyelids. An histochemical analysis (9) chalazion, disease models of degenerative on lymphatics of monkey and human eyelids, and inflammatory changes of eyelids. performed by 5’-Nase, demonstrated a superficial or pre-tarsal and a deep or post- MATERIALS AND METHODS tarsal lymphatic plexus. In our study, we examined lymphatics in two different Specimens of human upper eyelids were conditions occurring in eyelids: dermatocha- obtained from surgical treatment on seven- lasis and chalazion. Dermatochalasis is an teen patients (seven cases of dermatochalasis aging- related condition of upper or lower and ten cases of chalazion). All samples were eyelids clinically characterized by loose provided by the Department of Ophthalmology redundant skin. Histological investigation and Neurosurgery (S. Maria alle Scotte, shows disruption of elastic fibers and variable University Hospital, Siena, Italy). All research collagen degeneration (10). Chalazion is a procedures involving human subjects were chronic granulomatous inflammatory lesion, carried out according to the Helsinki declara- originating from meibomian glands (11). tion for the use of human tissue in research. The inflammatory infiltrate consists of Written informed consent was obtained from neutrophils, macrophages, T-cells and plasma all patients. The patients age range was 56-75 cells. Chalazion is often associated with years for dermatochalasis (1 man, 6 women) other meibomian gland dysfunctions, such and 20-68 years for chalazion (4 men, 6 as chronic blepharitis, or systemic pathology women). As controls, we used specimens of sebaceous glands, e.g., acne rosacea and obtained from human distal leg or thigh seborrhoea (12). hairy skin. All the samples were routinely Different sets of vasoactive factors inter- processed for light microscopy, fixed in vene in mechanisms of lymph propulsion. Bouin’s solution and paraffin embedded, or Endothelin-1 (ET-1) is a multifunctional snap frozen in liquid nitrogen chilled peptide, first identified as a potent vasocon- isopenthane. Serial sections, 6-7 µm thick, strictor produced by endothelial cells (13). were mounted on SuperFrost Plus microscope ET-1 activity is mediated by specific receptors, slides (Fisher Scientific, Pittsburgh, PA). type A and type B (ETAR and ETBR) (14) For each sample a total length >250 µm was with different levels of expression in various examined, with 35-45 serial sections. Each cell types. Physiological investigations have stain was performed on two slides and shown ET-1/ETAR mediated lymphatic experiments were performed twice. For vasomotion in guinea-pig mesenteric immunohistochemistry (IHC), paraffin- collectors (15). Among its varied biological embedded sections were deparaffinized and effects, ET-1 is also active in inflammation rehydrated, whereas cryostat sections were as a proinflammatory cytokine (16) and in dried and fixed for 10 min in cold acetone. remodelling of extracellular matrix via All sections were then washed in phosphate interactions with fibrogenic cytokines and buffer saline (PBS) and incubated for 30 min metalloproteases (17). Whereas the role of at room temperature (r.t.) in PBS containing ET-1 has been widely investigated in fibro- 5% bovine serum albumin (BSA) (Sigma vascular, neural and epithelial structures of Aldrich, St. Louis, MO) to block aspecific Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. 31 TABLE 1 Specificities, Dilution, and Sources of Primary Antibodies Used in this Study Antibody/antiserum Host Dilution Source D2-40 Mouse 1:50 DakoCytomation# LYVE-1 Rabbit 1:100 Novus Biologicals* CD-31 Mouse 1:20 DakoCytomation# ET-1 Mouse 1:250 Sigma Aldrich^ ETAR Rabbit 1:400 Sigma Aldrich^ ETBR Rabbit 1:400 Sigma Aldrich^ *Novus Biologicals, Inc, Littleton, CO; #DakoCytomation, Carpinteria, CA; ^Sigma Aldrich, St. Louis, MO. binding sites. They were then incubated tion and evaluated with AxioVision 4.6 overnight at 4°C with specific primary software. Density of vessels was expressed antibodies and antisera, diluted in PBS-1% as number of lymphatics/microscopic field BSA, as indicated in Table 1. For lymphatic (1.436x105 µm2). Area was expressed as total immunostaining, D2-40 and LYVE-1 in serial surface of absorbing lymphatic vessels/ sections were utilized. In order to inactivate microscopic field. Counts and measurements endogenous peroxidases, sections were were carried out by two independent incubated with 6% H2O2 in methanol for 30 operators in a blinded fashion, and repeated min at r.t. The reaction was detected by the procedures ensured constant and reproducible EnVision peroxidase polymer (EnVision, results. The results of morphometric analysis + Labelled polymer, DakoCytomation, are expressed as mean ± standard deviation Carpinteria, CA) and visualized with 3,3’- (M±SD). Statistics were performed by one- Diaminobenzidine (DAB, Sigma Aldrich ). way ANOVA followed by Dunnett’s test for All sections were counterstained with Mayer’s multiple comparisons; p<0.05 was considered hemalum (Sigma Aldrich). Negative controls significant. were carried out by omission of primary or secondary antibodies. Images were acquired RESULTS on a Carl Zeiss Axioplan 2 imaging micro- scope using an AxioCam HR CCD camera Lymphatics were recognized by D2-40 and AxioVision 4.6 software (Carl Zeiss, and LYVE-1 antibodies that specifically and Göttingen, Germany). For identification of entirely immunostained lymphatic endothe- elastic fibers Weigert stain was carried out. lium, as further confirmed by panendothelial We performed a quantitative morpho- marker CD-31 serial stain (Fig. 1). metric study in D2-40 stained sections from In dermatochalasis, eyelid lymphatic dermatochalasis, chalazion and normal skin vessels were numerous and characterized by to evaluate the lymphatic density and area. a wide lumen and a tortuous endothelial In each sample, five random microscopic profile (Fig. 2).
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