Scleraxis expressing scleral cells respond to inflammatory stimulation

Falk Schroedl,Herbert Tempfer,Andreas Traweger,Ghada Atta,Gabriel Spitzer,Ludwig M Heindl,Alexandra Kaser-Eichberger

doi:10.1007/s00418-021-01985-y

Abstract

The sclera is an ocular tissue rich of collagenous extracellular matrix, which is built up and maintained by relatively few, still poorly characterized fibroblast-like cells. The aims of this study are to add to the characterization of scleral fibroblasts and to examine the reaction of these fibroblasts to inflammatory stimulation in an ex vivo organotypic model. Scleras of scleraxis-GFP (SCX-GFP) mice were analyzed using immunohistochemistry and qRT-PCR for the expression of the tendon cell associated marker genes scleraxis (SCX), mohawk and tenomodulin. In organotypic tissue culture, explanted scleras of adult scleraxis GFP reporter mice were exposed to 10 ng/ml recombinant interleukin 1-ß (IL1-ß) and IL1-ß in combination with dexamethasone. The tissue was then analyzed by immunofluorescence staining of the inflammation- and fibrosis-associated proteins IL6, COX-2, iNOS, connective tissue growth factor, MMP2, MMP3, and MMP13 as well as for collagen fibre degradation using a Collagen Hybridizing Peptide (CHP) binding assay. The mouse sclera displayed a strong expression of scleraxis promoter-driven GFP, indicating a tendon cell-like phenotype, as well as expression of scleraxis, tenomodulin and mohawk mRNA. Upon IL1-ß stimulation, SCX-GFP+ cells significantly upregulated the expression of all proteins analysed. Moreover, IL1-ß stimulation resulted in significant collagen degradation. Adding the corticosteroid dexamethasone significantly reduced the response to IL1-ß stimulation. Collagen degradation was significantly enhanced in the IL1-ß group. Dexamethasone demonstrated a significant rescue effect. This work provides insights into the characteristics of scleral cells and establishes an ex vivo model of scleral inflammation.

Highlights

The sclera forms around 85% of the rigid outer tunic coat of the human eyeball, which consists of three anatomical layers: (1) the superficial vascularized episclera which contains a tight network of blood vessels, (2) the scleral stroma resembling the main part, which is mainly avascular, and (3) the lamina fusca, a thin pigmented layer which is located above the uvea, an strongly pigmented layer below the sclera
Despite sclera being considered a quiescent tissue in healthy state, pathologic conditions such as injuries or tumours trigger tissue responses leading to matrix disruption and cellular activation (Harper and Summers 2015)
Collagen Hybridizing Peptide (CHP)-positive areas were reduced to control levels (0.38% ± 0.14; Fig. 2g-i, j)

Summary

Introduction

The sclera forms around 85% of the rigid outer tunic coat of the human eyeball, which consists of three anatomical layers: (1) the superficial vascularized episclera which contains a tight network of blood vessels, (2) the scleral stroma resembling the main part, which is mainly avascular, and (3) the lamina fusca, a thin pigmented layer which is located above the uvea, an strongly pigmented layer below the sclera. The adult sclera shows only a superficial network of blood vessels but lacks lymphatic vessels, thereby creating a lymphatic-free border to the inner eye (Schlereth et al 2019), contributing to the ocular immune privilege. The scleral vascular privilege is compromised and secondary ingrowth of intraocular lymphatic vessels with a significant number of associated LYVE-1 + macrophages takes place, invading the sclera and the inner eye (Schlereth et al 2014a, b) This mechanism supports wound healing, defense against invading microorganisms and autoimmune reactions against intraocular antigens (Schlereth et al 2014a, b)

Objectives

Findings

Methods