Choroidal melanocytes: subpopulations of different origin?

Abstract

Background

The human choroid derives from the mesectoderm, except the melanocytes originating from the neuroectoderm. To date, it is unclear whether all choroidal melanocytes share the same origin or might have different origins. The purpose of this study was to screen immunohistochemically for mesenchymal elements in the adult healthy human choroid, in the malignant melanoma of the choroid, as well as in the developing human fetal choroid.

Methods

Human choroids were obtained from cornea donors and prepared as flat whole mounts for paraffin- and cryoembedding. Globes enucleated for choroidal melanoma and eyes from human fetuses between 11 and 20 weeks of gestation were also embedded in paraffin. Sections were processed for immunohistochemistry of the mesenchymal marker vimentin, the melanocyte marker Melan-A, and the macrophage marker CD68, followed by light-, fluorescence-, and confocal laser scanning-microscopy.

Results

The normal choroid contained 499 ± 139 vimentin, 384 ± 78 Melan-A, and 129 ± 57 CD68 immunoreactive cells/mm². The vimentin immunopositive cell density was significantly higher than the density of Melan-A and CD68 immunopositive cells (p < 0.001, respectively). By confocal microscopy, 24 ± 8% of all choroidal melanocytes displayed vimentin immunoreactivity. In choroidal melanomas, numerous melanoma cells of the epithelioid and spindle cell type revealed immunopositivity for both vimentin and Melan-A. The intratumoral density of vimentin immunoreactive cells was 1758 ± 106 cells/mm², significantly higher than the density of Melan-A and CD68 immunopositive cells (p < 0.001, respectively). Comparing to healthy choroidal tissue, the choroidal melanomas revealed significantly higher densities of vimentin, Melan-A, and CD68 immunoreactive cells (p < 0.001, respectively). In the developing human fetal choroid, numerous vimentin and Melan-A immunopositive cells were detected not before the 16th week of gestation, with some of them showing colocalization of vimentin and Melan-A.

Conclusions

The adult healthy human choroid is endowed with a significant number of vimentin immunopositive mesenchymal structures, including a subpopulation of vimentin immunoreactive choroidal melanocytes. These vimentin immunopositive melanocytic cells are also present in choroidal melanomas as well as in the developing human fetal choroid. Therefore, different embryologic origins can be considered for choroidal melanocytes.

Introduction

The choroid forms the middle vascular tunic between the sclera and the neurosensory retina at the posterior pole of the human eye (Nickla and Wallman, 2010; Naumann, 1980; Joussen and Naumann, 2008). With a thickness of about 200-μm at birth decreasing to approximately 80-μm by the age of 90 (Nickla and Wallman, 2010; Naumann, 1980; Joussen and Naumann, 2008; Ramrattan et al., 1994), the highly vascularized choroid serves to supply the outer retina with oxygen and nutrients (Nickla and Wallman, 2010; Naumann, 1980; Joussen and Naumann, 2008; Ramrattan et al., 1994; Linsenmeier et al., 1981), to absorb light by its pigmentation (Nickla and Wallman, 2010), to maintain retinal temperature by heat dissipation (Nickla and Wallman, 2010; Parver, 1991), to adjust the position of the retina in an accommodative mechanism by modifying choroidal thickness (Nickla and Wallman, 2010; Wallman et al., 1995; Read et al., 2010), to modulate the intraocular pressure by uveoscleral drainage of aqueous humor as well as by vasomotor control of blood flow (Nickla and Wallman, 2010; Alm and Nilsson, 2009), and to orchestrate the homeostatic control of eye growth by secreting growth factors and signal molecules with significant impact on the development of myopia and hyperopia (Nickla and Wallman, 2010; Wallman and Winawer, 2004).

Histologically, the choroid consists of five layers, listed here from the retinal side to the scleral side: (1) Bruch’s membrane as the basement membrane of the retinal pigment epithelium as well as the choriocapillaris, (2) the choriocapillaris with a highly anastomozed, single layer network of fenestrated capillaries arising from the arterioles in Sattler’s layer, (3) the two vascular layers including the inner Sattler’s layer of small and medium arteries, arterioles, and veins as well as the outer Haller’s layer of large blood vessels, and (4) the suprachoroid forming a transitional fibrillar zone between choroid and sclera (Nickla and Wallman, 2010). The extravascular tissue of the choroid is comprised of collagen and elastic fibers, fibroblasts, non-vascular smooth muscle cells, numerous large melanocytes as well as numerous mast cells, macrophages, and lymphocytes (Nickla and Wallman, 2010). It harbors intrinsic choroidal neurons receiving sympathetic, parasympathetic, nitrergic and presumably cholinergic innervation (Nickla and Wallman, 2010; Schrödl et al., 2000; Schrödl et al., 2001a, 2001b; Trivino et al., 2002; Schrödl et al., 2003; Stübinger et al., 2010; Hohberger et al., 2018; Nickla and Schrödl, 2019). The normal adult human choroid does not contain typical lymphatic vessels, but it is endowed with numerous single cells expressing some lymphatic markers, most likely compatible with macrophages (Schroedl et al., 2008, 2014; Schroedl et al., 2015). This absence of lymphatic vessels contributes to the so called “immune privilege of the eye” (Streilein, 2003).

This ocular immune privilege is highly conserved during the embryonic and fetal development (Rezai et al., 1999; Niederkorn and Wang, 2005; Nickla and Wallman, 2010; Lutty and McLeod, 2018). At the end of the first month in the development of the human embryo, the choroid matures from the mesenchyme around the two optic vesicles that arise out of the lateral surfaces of the embryonic forebrain (Nickla and Wallman, 2010; Lutty and McLeod, 2018). Interestingly, at around the same time the choroid is invaded by melanocyte precursors from the neural crest that are differentiating into pigmented melanocytes not before the seventh month of gestation (Nickla and Wallman, 2010; Lutty and McLeod, 2018). The choroid is devoid of blood vessels until the second month, before forming the vascularized choriocapillaris out of the mesodermal mesenchyme in closed contact with the neuroectodermally derived retinal pigment epithelium (Nickla and Wallman, 2010; Lutty and McLeod, 2018). Taken together, that means that the entire choroid derives from the mesectoderm, with the exception of the choroidal melanocytes originating from the neuroectoderm (Nickla and Wallman, 2010; Lutty and McLeod, 2018). To date, it is not clear whether indeed all melanocytes share the same origin or might have different origins. This, however, might have tremendous clinical impact for choroidal diseases, especially the malignant melanoma of the choroid showing bad prognosis with currently still rather limited treatment options (Jager et al., 2020). Therefore, we are here investigating mesenchymal elements in the adult healthy human choroid, in the tumor-diseased tissue, as well as in the developing human fetal choroid by using immunohistochemistry and immunofluorescence methods.