Falk Schrödl

Intraocular Lymphatics in Ciliary Body Melanomas With Extraocular Extension: Functional for Lymphatic Spread?

OBJECTIVE To assess the functional significance of intraocular tumor-associated lymphatic vessels in ciliary body melanomas with extraocular extension. METHODS Twelve consecutive patients enucleated for a malignant melanoma of the ciliary body with extraocular extension and immunohistochemical presence of intraocular LYVE-1-positive and podoplanin-positive lymphatic vessels were examined for proliferation status and tumor invasion into tumor-associated lymphatics. Proliferating lymphatic vessels were identified using LYVE-1 and podoplanin as specific lymphatic endothelial markers and Ki-67 as the proliferation marker. Tumor invasion into lymphatic vessels was assessed using Melan-A as the melanoma marker. Kaplan-Meier analyses of survival and metastasis were performed. RESULTS Intraocular proliferating lymphatic vessels were detected in all 12 ciliary body melanomas with extraocular extension. The ratio of proliferating lymphatics was significantly higher in the intraocular vs extraocular tumor compartment (P < .001). Extraocular lymphatic invasion by tumor cells was observed in 5 patients (42%), intraocular lymphatic invasion in 4 (33%), and synchronous intraocular and extraocular lymphatic invasion in 3 (25%). Detection of melanoma cells in intraocular and extraocular lymphatic vessels was significantly associated with higher risks of lymphatic spread (P < .001) and lower metastasis-free survival rates (P = .03). CONCLUSIONS Intraocular tumor-associated lymphatic vessels contain proliferating endothelial cells and can be invaded by cancer cells in ciliary body melanomas with extraocular extension. Lymphatic invasion by tumor cells seems to be associated with an increased risk of lymphatic spread and mortality in these affected patients.

Rat choroidal pericytes as a target of the autonomic nervous system

Pericytes are contractile cells that surround blood vessels. When contracting, they change the diameter of the vessel and therefore influence blood flow homeostasis; however, mechanisms controlling pericyte action are less well understood. Since blood flow regulation per se is controlled by the autonomic nervous system, the latter might also be involved in pericyte action. Hence, rat choroidal pericytes were analyzed for such a connection by using appropriate markers. Rat choroidal wholemounts and sections were prepared for immunohistochemistry of the pericyte marker chondroitin-sulfate-proteoglycan (NG2) and the pan-neuronal marker PGP9.5 or of tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP) and choline acetyl transferase (ChAT). Additionally, PGP9.5 and TH were analyzed in the choroid of DCX-dsRed2 transgenic rats, displaying red-fluorescent perivascular cells and serving as a putative model for studying pericyte function in vivo. Confocal laser-scanning microscopy revealed NG2-immunoreactive cells and processes surrounding the blood vessels. These NG2-positive cells were not co-localized with PGP9.5 but received close appositions of PGP9.5-, TH-, VIP- and ChAT-immunoreactive boutons and fibers. In the DCX-dsRed2 transgenic rat, PGP9.5 and TH were also densely apposed on the dsRed-positive cells adjacent to blood vessels. These cells were likewise immunoreactive for NG2, suggesting their pericyte identity. In addition to the innervation of vascular smooth muscle cells, the close relationship of PGP9.5 and further sympathetic (TH) and parasympathetic (VIP, ChAT) nerve fibers on NG2-positive pericytes indicated an additional target of the autonomic nervous system for choroidal blood flow regulation. Similar findings in the DCX-dsRed transgenic rat indicate the potential use of this animal model for in vivo experiments revealing the role of pericytes in blood flow regulation.

Distribution of the regulatory peptide alarin in the eye of various species

Alarin is a recently discovered regulatory peptide with vasoconstrictive properties in murine skin. Control of vasoconstriction/-relaxation is essential for ocular blood flow and hence the eyes homeostasis, and regulatory peptides are involved in regulation of ocular blood flow. Here we describe the existence and distribution of alarin in the eye of human and potential experimental animals (rat, mouse). Eyes of rat, mouse, and human were prepared for immunohistochemistry against murine and human alarin, respectively. Additionally, double staining experiments for alarin and CD31 were performed in human choroidal flat-mount preparations. For documentation, confocal laser scanning microscopy was used while quantitative real-time-PCR was applied to confirm immunohistochemical data and to detect alarin mRNA expression in human retina and choroid. Alarin-like immunoreactivity (alarin-LI) was detected in corneal epi- and endothelium of human, mouse, and rat, as well as in the conjunctiva of mouse and rat. Alarin-LI was found in the iris of all the species investigated and, in humans, was concentrated around blood vessels. All three species showed distinctive alarin-LI in the non-pigmented epithelium of the ciliary body. In the retina of mouse and rat, maximum signals were detected in the outer nuclear and ganglion cell layer, whereas in humans a strong alarin-LI was found around retinal blood vessels and in intrinsic choroidal neurons (ICN). Quantitative RT-PCR in human confirmed alarin mRNA expression retina and choroid. The existence of alarin in cornea and conjunctiva might indicate a role in immune defense, while its presence in the non-pigmented ciliary epithelium favors an involvement in aqueous humor production. Alarin around blood vessels/in ICN might indicate an involvement in ocular blood flow regulation. Since alarin is found widely distributed in the eyes of species investigated, we were able to establish the basis for further functional experiments.

Immunohistochemical Detection of CTGF in the Human Eye

ABSTRACT Purpose/Aim of the study: Connective tissue growth factor (CTGF) is a key player in the control of extracellular matrix remodeling, fibrosis, and angiogenesis. It is also involved in the modification of the trabecular meshwork, thus potentially modulating outflow facility and intraocular pressure (IOP). As a consequence, CTGF might be relevant for the development of elevated IOP, a major risk factor in glaucoma-pathogenesis. While comprehensive information on the origins of CTGF in the human eye is not available, the goal of this study is to identify ocular sources of CTGF using morphological methods. Materials and Methods: Human donor eyes were prepared for immunohistochemical analysis of CTGF, α-smooth muscle-actin (ASMA), and CD31. Confocal laser scanning microscopy was used for documentation. Results: In the cornea, CTGF-immunoreactivity (CTGF-IR) was detected in the epithelium, mainly in basal layers, stromal keratinocytes, and endothelial cells. Adjacent conjunctiva showed also CTGF-IR in epithelial cells. In the iris, both, the sphincter and dilator muscles displayed CGTF-IR, as did iris and ciliary body vessels, deriving at this location from the vascular endothelium, as detected with CD31, but not from vascular smooth muscle cells, as detected with ASMA. In the ciliary body, CTGF-IR was detected in smooth-muscle cells of the ciliary muscle and further in the non-pigmented epithelium. In the retina, CTGF-IR was detected in the NFL and weakly in the IPL/OPL. In the choroid, the choriocapillaris and blood vessels displayed CTGF-IR. Further, few cells in the optic nerve head and the lamina cribrosa were CTGF-positive. Conclusion: CTGF was detected in various structures of the human eye. Since CTGF has been also described in aqueous humor, the identified structures might be the sources of CTGF in the aqueous humor. By means of aqueous flow, CTGF is transported into the trabecular meshwork, where it could change outflow facility and therefore affecting IOP homeostasis.

Retinal Vessel Diameter Responses to Central Electrical Stimulation in the Rat: Effect of Nitric Oxide Synthase Inhibition.

PURPOSE Recent histological data suggest autonomic innervation of the central retinal artery. In the present study, we investigated the effect of electrical brain stem stimulation at the superior salivatory nucleus (SSN) on the retinal vessel diameter in rats and whether nitric oxide mediates a possible effect. METHODS Sprague-Dawley rats (n = 12) were anesthetized using pentobarbital sodium (50 mg/kg intraperitoneally). The animals were artificially ventilated and the femoral artery and vein were cannulated for blood pressure measurement and drug administration. After a craniotomy was performed, a unipolar stainless steel electrode was inserted into the brainstem at the coordinates of the SSN. Stimulations were performed at 20 Hz, 9 μA, 1 ms pulse duration and 200 pulses. Retinal vessel diameters were measured continuously with the Imedos DVA-R, a noncontact fundus camera for rats with image analysis software. After control measurements, L-NAME, a nonspecific inhibitor of NO synthase, was applied intravenously (10 mg/kg), and the SSN stimulations were repeated. RESULTS Stimulation at the SSN coordinates increased the retinal arterial diameter by 6.41% ± 1.65% and the venous diameter by 3.48% ± 1.93% (both P < 0.05). Application of L-NAME reduced the arterial response significantly to 2.93% ± 0.91%, but did not change the venous response. Mean arterial pressure, carotid blood flow, and heart rate remained unaltered (by the stimulation). CONCLUSIONS The present study demonstrates that the retinal circulation reacts to electric stimulation at the SSN coordinates in rats. Nitric oxide is involved in the response, but it is not the sole neurotransmitter.

VIP changes during daytime in chicken intrinsic choroidal neurons

Purpose Ocular autonomic control is mediated by sympathetic and parasympathetic nerve fibres. Their interactions are complemented by primary afferent nerve fibers of and intrinsic choroidal neurons (ICN). As the vasodilatative neuropeptide, vasoactive intestinal peptide (VIP), is expressed in extrinsic and intrinsic ocular neurons, it is of special interest in ophthalmic research. Since circadian changes of ocular blood flow are known in humans and birds, this study aimed at investigating VIP expression at different daytimes in chicken choroid, the preferred model species in ICN research. Methods 12 eyes of 12 chickens were retrieved, slaughtered at 8.00–9.30 a.m. (n = 6) and 8.00 p.m. (n = 6), respectively, and choroidal wholemounts were prepared for immunofluorescence of VIP. VIP‐positive ICN of both groups were quantified and density of VIP‐positive axons assessed semi‐quantitatively. In 28 additional eyes retrieved in the morning (n = 14) and evening (n = 14), choroidal VIP content was determined by ELISA. Morning and evening data were analyzed statistically. NADPH‐diaphorase (NADPH‐d, ICN cell marker) was done at additional 12 whole mount choroids of 12 chicken, retrieved in the morning (n = 6) and evening (n = 6). Results (1) Numbers of VIP positive neurons differed significantly between morning: (239.17 ± 113.9) and evening: (550.83 ± 245.7; p = 0.018). (2) Numbers of VIP‐positive perikarya were significantly more accumulated in the temporal part of the choroid in the evening than in the morning (p = 0.026). (3) VIP positive axon density was found to be similar throughout the choroid in the morning and evening. (4) Number of NADPH‐d positive neurons was not significantly different between morning (848.8 ± 399.5) and evening (945.8 ± 622.1, p > 0.05). (5) ELISA demonstrated a significant difference of VIP content (p = 0.012) in tissues harvested in the morning (145.41 ± 43.3 pg/ml) compared to evening (221.44 ± 106.3 pg/ml). Conclusions As VIP positive axon density was similar in the morning and the evening throughout the choroid, PPG and ICN seemed to contribute equally to the axon network. Yet, changes in the total choroidal VIP content, the numbers of VIP positive perikarya, reflecting the intracellular VIP content, and their topographical distribution at two different days‐times argue for a different status of activation of both neuronal sources in contrast to the equal amount of NADPHD‐d positive neurons. The higher VIP content in the evening, compared to the morning, correlates with a known circadian rhythm of a lower IOP and a higher choroidal thickness at night. Thus, these changes may argue for a potential role of ICN in the regulation of ocular homeostasis and integrity. HighlightsAxons from pterygopalatine and ICN contribute equally to the choroidal network.VIP positive perikarya and total VIP content differ at two different days‐times.VIP positive perikarya accumulate more in temporal choroidal parts in the evening.Different activation of neurons (pterygopalatine and ICN) is proposed.Higher vespertine VIP content correlates with choroidal thickness and IOP.

Retinal Pericytes: Characterization of Vascular Development-Dependent Induction Time Points in an Inducible NG2 Reporter Mouse Model

ABSTRACT Purpose/aim of the study: In the retina, defects in pericytes (PCs) function/loss are associated with various complications; however, the exact pathological mechanisms are still not fully elucidated. Following the behavior of retina-resident PCs during health and disease will reveal new insights for both the understanding of pathological mechanisms and the development of new regenerative therapies for the treatment of retinopathies. The main goal of this study is to determine whether the NG2-reporter mouse (NG2CreERTM-eGFP) is a suitable model to study the fate of retina-resident PCs. Material and methods: Vascular development-dependent reporter induction in retinal PCs was evaluated at different time points [(a) > P21, (b) < P21, and (c) P1 to > P21)] and additionally four different modes of application were tested. Reporter expression was evaluated by enhanced green fluorescent protein (eGFP) immunofluorescence by confocal microscopy and induction efficiency was calculated by analyzing NG2-expressing PCs in comparison to eGFP-labeled PCs in the three capillary layers. Results: eGFP-positive PCs were detected in the three retinal capillary layers at all time points and administration routes tested. Multiple tamoxifen (TAM) applications in adult (> P21) NG2CreERTM-eGFP mice resulted in 3.59% eGFP-positive PCs. 2.37% eGFP-labeled PCs were detected after single intraperitoneal TAM injections at early postnatal days (P2/P5); however, just 1.61% PCs revealed reporter expression upon activation via the lactating mother (P4–P7). The highest number of eGFP-labeled PCs (7.09%) was detected following triple TAM administrations (P10–P12). The number of reporter-positive PCs doubled using homozygous animals. Conclusion: Despite low recombination efficiency in the used PC-specific fate mapping mouse model, changes in NG2 promoter activity of PCs during vascular development are indicated by single and multiple TAM inductions at different developmental time points. Nevertheless, these findings need further confirmation in up-coming studies by using homozygous NG2CreERTM-eGFP mice and additionally by mating the NG2CreERTM with a different reporter mouse to increase the low recombination efficiency.

Distribution of the neuro-regulatory peptide galanin in the human eye

Galanin (GAL) is a neuro-regulatory peptide involved in many physiological and pathophysiological processes. While data of GAL origin/distribution in the human eye are rather fragmentary and since recently the presence of GAL-receptors in the normal human eye has been reported, we here systematically search for sources of ocular GAL in the human eye. Human eyes (n=14) were prepared for single- and double-immunohistochemistry of GAL and neurofilaments (NF). Cross- and flat-mount sections were achieved; confocal laser-scanning microscopy was used for documentation. In the anterior eye, GAL-immunoreactivity (GAL-IR) was detected in basal layers of corneal epithelium, endothelium, and in nerve fibers and keratinocytes of the corneal stroma. In the conjunctiva, GAL-IR was seen throughout all epithelial cell layers. In the iris, sphincter and dilator muscle and endothelium of iris vessels displayed GAL-IR. It was also detected in stromal cells containing melanin granules, while these were absent in others. In the ciliary body, ciliary muscle and pigmented as well as non-pigmented ciliary epithelium displayed GAL-IR. In the retina, GAL-IR was detected in cells associated with the ganglion cell layer, and in endothelial cells of retinal blood vessels. In the choroid, nerve fibers of the choroidal stroma as well as fibers forming boutons and surrounding choroidal blood vessels displayed GAL-IR. Further, the majority of intrinsic choroidal neurons were GAL-positive, as revealed by co-localization-experiments with NF, while a minority displayed NF- or GAL-IR only. GAL-IR was also detected in choroidal melanocytes, as identified by the presence of intracellular melanin-granules, as well as in cells lacking melanin-granules, most likely representing macrophages. GAL-IR was detected in numerous cells and tissues throughout the anterior and posterior eye and might therefore be an important regulatory peptide for many aspects of ocular control. Upcoming studies in diseased tissue will help to clarify the role of GAL in ocular homeostasis.