Targeted surface marker screening on neuronal structures in the human choroid.

While choroidal neuronal control is known to be essential for retinal and ocular health, its mechanisms are not understood. Especially, the local choroidal innervation mediated by intrinsic choroidal neurons (ICN) remains enigmatic. Neuronal functionality depends on the synaptic neurotransmitters and neuroregulatory peptides involved as well as from membrane components presented on the cell surface. Since the neuronal surface molecular expression patterns in the choroid are currently unknown, we sought to determine the presence of various cluster-of-differentiation (CD) antigens in choroidal neuronal structures with a particular focus on ICN. Human choroids were prepared for immunohistochemistry and the pan-neuronal marker PGP9.5 was combined with CD15, CD24, CD29, CD34, CD46, CD49b, CD49e, CD56, CD58, CD59, CD71, CD81, CD90, CD146, CD147, CD151, CD165, CD171, CD184, CD200, CD271 and fluorescence- and confocal laser scanning-microscopy was used for documentation. The following antigens were found to be co-localized in PGP.9.5+ nerve fibers and ICN perikarya: CD29, CD34, CD56, CD81, CD90, CD146, CD147, CD151, CD171, CD200 and CD271, while all other CD markers where not detectable. Whereas CD24- and CD59- immunoreactivity was clearly absent in ICN perikarya, some neural processes of the choroidal stroma displayed CD24 and CD59 immunopositivity. While a multitude of the aforementioned CD-markers were indeed detected in nervous structures of the choroid, the CD24+ and CD59+ nerve fibers most likely have extrinsic origin from cranial ganglia since ICN cell bodies were found to lack both markers. These findings illustrate how the detailed analysis of CD molecules described here opens novel avenues for future functional studies on choroidal innervation and its control.

The choroid-sclera interface: An ultrastructural study.

Emmetropization is an active and visually guided process that involves the retina, choroid and sclera, and results in compensatory changes in eye growth. This guided growth is the result of visual cues and possibly mechanical interactions being translated into growth signals via molecular events from the retina into the choroid and sclera, through the choroidal scleral transition zone. If mechanical interactions were a part of the choroid-sclera signaling transduction cascade, specific morphological arrangements should be detectable in this region at the ultrastructural level. The goal of this study was to investigate the ultrastructural features of the choroidal scleral transition zone by comparing avian, non-human primate and human eyes, with the goal to confirm whether specific mechanical structures are present. Choroidal and scleral tissue from chicken, marmoset, and human eyes were imaged using transmission electron microscopy to document the choroid-sclera transition zone. In chicken eyes, fibroblast lamellae bordered the scleral matrix and formed thin end elongated processes that were undercut by scleral collagen fibrils. These processes back-looped into the scleral matrix, and displayed small club-like membrane protrusions. Differences in these arrangements in mature vs young chickens were not detected. The club-like membrane protrusions identified in chickens were rare in marmoset eyes, which instead exhibited two types of collagen fibrils discriminated by size, and were absent in the human eyes investigated. In marmoset and human eyes, elastic components were detected in the transition zone that were absent in chickens. In summary, cellular/membrane specializations indicating a mechanical interaction at the choroid-sclera transition zone were not detected in chicken, non-human primate or human eyes. If mechanotransduction is necessary for scleral growth, matrix integrity or development, alternative structural arrangements might be required.

Lymphatic markers in the human optic nerve.

Tissues of the central nervous system (CNS), including the optic nerve (ON), are considered a-lymphatic. However, lymphatic structures have been described in the dura mater of human ON sheaths. Since it is known that lymphatic markers are also expressed by single non-lymphatic cells, these results need confirmation according to the consensus statement for the use of lymphatic markers in ophthalmologic research. The aim of this study was to screen for the presence of lymphatic structures in the adult human ON using a combination of four lymphatic markers. Cross and longitudinal cryo-sections of human optic nerve tissue (n = 12, male and female, postmortem time = 15.8 ± 5.5 h, age = 66.5 ± 13.8 years), were obtained from cornea donors of the Salzburg eye bank, and analyzed using immunofluorescence with the following markers: FOXC2, CCL21, LYVE-1 and podoplanin (PDPN; lymphatic markers), Iba1 (microglia), CD68 (macrophages), CD31 (endothelial cell, EC), NF200 (neurofilament), as well as GFAP (astrocytes). Human skin sections served as positive controls and confocal microscopy in single optical section mode was used for documentation. In human skin, lymphatic structures were detected, showing a co-localization of LYVE-1/PDPN/FOXC2 and CCL21/LYVE-1. In the human ON however, single LYVE-1+ cells were detected, but were not co-localized with any other lymphatic marker tested. Instead, LYVE-1+ cells displayed immunopositivity for Iba1 and CD68, being more pronounced in the periphery of the ON than in the central region. However, Iba1+/LYVE-1- cells outnumbered Iba1+/LYVE-1+ cells. PDPN, revealed faint labeling in human ON tissue despite strong immunoreactivity in rat ON controls, showing co-localization with GFAP in the periphery. In addition, pronounced autofluorescent dots were detected in the ON, showing inter-individual differences in numbers. In the adult human ON no lymphatic structures were detected, although distinct lymphatic structures were identified in human skin tissue by co-localization of four lymphatic markers. However, single LYVE-1+ cells, also positive for Iba1 and CD68 were present, indicating LYVE-1+ macrophages. Inter-individual differences in the number of LYVE-1+ as well as Iba1+ cells were obvious within the ONs, most likely resulting from diverse medical histories of the donors.

Lymphatic and vascular markers in an optic nerve crush model in rat.

Only few tissues lack lymphatic supply, such as the CNS or the inner eye. However, if the scleral border is compromised due to trauma or tumor, lymphatics are detected in the eye. Since the situation in the optic nerve (ON), part of the CNS, is not clear, the aim of this study is to screen for the presence of lymphatic markers in the healthy and lesioned ON. Brown Norway rats received an unilateral optic nerve crush (ONC) with defined force, leaving the dura intact. Lesioned ONs and unlesioned contralateral controls were analyzed 7 days (n = 5) and 14 days (n = 5) after ONC, with the following markers: PDGFRb (pericyte), Iba1 (microglia), CD68 (macrophages), RECA (endothelial cell), GFAP (astrocyte) as well as LYVE-1 and podoplanin (PDPN; lymphatic markers). Rat skin sections served as positive controls and confocal microscopy in single optical section mode was used for documentation. In healthy ONs, PDGFRb is detected in vessel-like structures, which are associated to RECA positive structures. Some of these PDGFRb+/RECA+ structures are closely associated with LYVE-1+ cells. Homogenous PDPN-immunoreactivity (IR) was detected in healthy ON without vascular appearance, showing no co-localization with LYVE-1 or PDGFRb but co-localization with GFAP. However, in rat skin controls PDPN-IR was co-localized with LYVE-1 and further with RECA in vessel-like structures. In lesioned ONs, numerous PDGFRb+ cells were detected with network-like appearance in the lesion core. The majority of these PDGFRb+ cells were not associated with RECA-IR, but were immunopositive for Iba1 and CD68. Further, single LYVE-1+ cells were detected here. These LYVE-1+ cells were Iba1-positive but PDPN-negative. PDPN-IR was also clearly absent within the lesion site, while LYVE-1+ and PDPN+ structures were both unaltered outside the lesion. In the lesioned area, PDGFRb+/Iba1+/CD68+ network-like cells without vascular association might represent a subtype of microglia/macrophages, potentially involved in repair and phagocytosis. PDPN was detected in non-lymphatic structures in the healthy ON, co-localizing with GFAP but lacking LYVE-1, therefore most likely representing astrocytes. Both, PDPN and GFAP positive structures are absent in the lesion core. At both time points investigated, no lymphatic structures can be identified in the lesioned ON. However, single markers used to identify lymphatics, detected non-lymphatic structures, highlighting the importance of using a panel of markers to properly identify lymphatic structures.

Time-dependent retinal ganglion cell loss, microglial activation and blood-retina-barrier tightness in an acute model of ocular hypertension.

Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, and is the second leading cause of blindness worldwide. Elevated intraocular pressure is a well known risk factor for the development of glaucomatous optic neuropathy and pharmacological or surgical lowering of intraocular pressure represents a standard procedure in glaucoma treatment. However, the treatment options are limited and although lowering of intraocular pressure impedes disease progression, glaucoma cannot be cured by the currently available therapy concepts. In an acute short-term ocular hypertension model in rat, we characterize RGC loss, but also microglial cell activation and vascular alterations of the retina at certain time points. The combination of these three parameters might facilitate a better evaluation of the disease progression, and could further serve as a new model to test novel treatment strategies at certain time points. Acute ocular hypertension (OHT) was induced by the injection of magnetic microbeads into the rat anterior chamber angle (n = 22) with magnetic position control, leading to constant elevation of IOP. At certain time points post injection (4d, 7d, 10d, 14d and 21d), RGC loss, microglial activation, and microvascular pericyte (PC) coverage was analyzed using immunohistochemistry with corresponding specific markers (Brn3a, Iba1, NG2). Additionally, the tightness of the retinal vasculature was determined via injections of Texas Red labeled dextran (10 kDa) and subsequently analyzed for vascular leakage. For documentation, confocal laser-scanning microscopy was used, followed by cell counts, capillary length measurements and morphological and statistical analysis. The injection of magnetic microbeads led to a progressive loss of RGCs at the five time points investigated (20.07%, 29.52%, 41.80%, 61.40% and 76.57%). Microglial cells increased in number and displayed an activated morphology, as revealed by Iba1-positive cell number (150.23%, 175%, 429.25%,486.72% and 544.78%) and particle size analysis (205.49%, 203.37%, 412.84%, 333.37% and 299.77%) compared to contralateral control eyes. Pericyte coverage (NG2-positive PC/mm) displayed a significant reduction after 7d of OHT in central, and after 7d and 10d in peripheral retina. Despite these alterations, the tightness of the retinal vasculature remained unaltered at 14 and 21 days after OHT induction. While vascular tightness was unchanged in the course of OHT, a progressive loss of RGCs and activation of microglial cells was detected. Since a significant loss in RGCs was observed already at day 4 of experimental glaucoma, and since activated microglia peaked at day 10, we determined a time frame of 7-14 days after MB injection as potential optimum to study glaucoma mechanisms in this model.