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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.

Emmetropization is affected by the temporal parameters of visual stimulation and the spectral composition of light, as well as by autonomic innervation. The goal of the current experiments is to test the hypothesis that different types of visual stimulation interact with ocular innervation in the process of emmetropization. For that, selective lesions of the autonomic nervous system were performed in chickens: involving transection of parasympathetic input to the eye from either the ciliary ganglion, innervating accommodation and pupil responses (CGX; n = 32), or pterygopalatine ganglion, innervating choroidal blood vessels and cornea (PPGX; n = 26). After 1 week of recovery, chicks were exposed to sinusoidally modulated light (3 days, 2 Hz, 680 lux) that was either achromatic (black to white [RGB], or black to yellow [RG]), or chromatic (blue to yellow [B/Y] or red to green [R/G]). Exposure to light stimulation was followed by ocular biometry (Lenstar and a Hartinger refractometer). Surgical conditions revealed a small reduction in anterior chamber depth with CGX but no other significant changes in ocular biometry/refraction under standard light conditions. With RGB achromatic stimulation, CGX eyes produced an effect on ocular components, with a further reduction in anterior chamber depth and an increase in vitreous chamber depth, while RG stimulation showed no effect. No effect was detected in PPGX under both achromatic protocols. With chromatic stimulation, CGX with R/G modulation increased eye length, while PPGX with B/Y modulation decreased eye length. We conclude that the two different types of parasympathetic innervations have antagonistic effects on eye growth and the anterior eye when challenged with the appropriate stimulus, with possible implications for the role of choroidal blood flow in emmetropization.

BackgroundThe choroid is densely innervated by all parts of the autonomic nervous system and further harbours a network of local nerve cells, the intrinsic choroidal neurons (ICN). Their function in ocular control is currently unknown. While morphological data assume a role in intraocular pressure regulation, we here test if increased pressure on isolated choroids may activate ICN.MethodsDonor tissue was transferred into a pressurisable tissue culture chamber, and nasal and temporal choroid halves incubated for 1 or 4 hours, with pressures set to 15 or 50 mm Hg, followed by qRT-PCR expression analysis of the ICN-specific markers VIP, UCN, NOS1, UCH-L1. POL2-normalised data in the different pressure settings, incubation times and localisations were statistically analysed.ResultsThe presence of the ICN-specific markers VIP, UCN, NOS1, UCH-L1 was confirmed using immunohistochemistry, and mRNA of all markers was detected in all experimental conditions. Marker analysis revealed no significant changes of mRNA expression levels between 15 and 50 mm Hg in the different incubation times. When comparing all samples over all experimental conditions, a significant increase of VIP and NOS1 mRNA was detected in temporal versus nasal choroids.ConclusionIn this functional analysis of human ICN in vitro, higher amounts of VIP and NOS1 mRNA were detected in the temporal choroid, that is, the choroidal site with ICN accumulation. Further, our data indicate that elevated pressure is apparently not able to trigger ICN responses via the investigated markers. Alternative markers and stimuli need to be investigated in upcoming studies in order to unravel ICN function.

Collagen rich connective tissues fulfill a variety of important functions throughout the human body, most of which having to resist mechanical challenges. This review aims to compare structural and functional aspects of tendons and sclera, two tissues with distinct location and function, but with striking similarities regarding their cellular content, their extracellular matrix and their low degree of vascularization. The description of these similarities meant to provide potential novel insight for both the fields of orthopedic research and ophthalmology.

To evaluate the presence and appearance of blood and lymphatic vessels in non-functioning bleb capsules of glaucoma drainage devices (GDD).Non-functioning (n=14) GDD-bleb capsules of 12 patients were analyzed by immunohistochemistry for blood vessels (CD31, vascular endothelium), lymphatic vessels (lymphatic vessel endothelial hyaluronan receptor-1 [LYVE-1] and podoplanin) and macrophages (CD68).CD31+++ blood vessels and CD68+ macrophages were detected in the outer layer of all specimens. LYVE-1 immunoreactivity was registered in single non-endothelial cells in 8 out of 14 (57%) bleb capsule specimens. Podoplanin-immunoreactivity was detected in all cases, located in cells and profiles of the collagen tissue network of the outer and/or the inner capsule layer. However, a colocalization of LYVE-1 and podoplanin as evidence for lymphatic vessels was not detected.We demonstrate the presence of blood-vessels but absence of lymphatic vessels in non-functioning bleb capsules after GDD-implantation. While the absence of lymphatic vessels might indicate a possible reason for drainage device failure, this needs to be confirmed in upcoming studies, including animal experiments.

In chicks, axial length and choroidal thickness undergo circadian oscillations. The choroid is innervated by both branches of the autonomic nervous system, but their contribution(s) to these rhythms is unknown. We used two combination lesions to test this. For parasympathectomy, nerve VII was sectioned presynaptic to the pterygopalatine ganglia, and the ciliary post-ganglionics were cut (double lesion; n = 8). Triple lesions excised the sympathetic superior cervical ganglion as well (n = 8). Sham surgery was done in controls (n = 7). 8–14 days later, axial dimensions were measured with ultrasonography at 4-h intervals over 24 h. Rhythm parameters were assessed using a “best fit” function, and growth rates measured. Both types of lesions resulted in ultradian (> 1 cycle/24 h) rhythms in choroidal thickness and axial length, and increased vitreous chamber growth (Exp-fellow: double: 69 µm; triple: 104 µm; p

Ein iatrogenes Ektropium mit Unterlidtiefstand nach unsachgemas exzessiver Unterlidblepharoplastik ist eine ernsthafte Komplikation in der asthetischen Chirurgie. Traditionell ist die freie Hauttransplantation die Korrekturmethode der Wahl. In dieser Ubersicht stellen wir die Wangen-Mittelgesichts-Anhebung (engl. „cheek midface lift“) als eine hilfreiche und wirkungsvolle chirurgische Option fur diese Indikation dar. Mit dieser in Lokalanasthesie durchfuhrbaren Technik konnen funktionell wie auch asthetisch sehr gute Ergebnisse erzielt werden.

Background/Aims: Effective wound-healing generally requires efficient re-vascularization after injury, ensuring sufficient supply with oxygen, nutrients, and various cell populations. While this applies to most tissues, tendons are mostly avascular in nature and harbor relatively few cells, probably contributing to their poor regenerative capacity. Considering the minimal vascularization of healthy tendons, we hypothesize that controlling angiogenesis in early tendon healing is beneficial for repair tissue quality and function. Methods: To address this hypothesis, Bevacizumab, a monoclonal antibody blocking VEGF-A signaling, was locally injected into the defect area of a complete tenotomy in rat Achilles tendon. At 28 days post-surgery, the defect region was investigated using immunohistochemistry against vascular and lymphatic epitopes. Polarization microscopy and biomechanical testing was used to determine tendon integrity and gait analysis for functional testing in treated vs non-treated animals. Results: Angiogenesis was found to be significantly reduced in the Bevacizumab treated repair tissue, accompanied by significantly reduced cross sectional area, improved matrix organization, increased stiffness and Young’s modulus, maximum load and stress. Further, we observed an improved gait pattern when compared to the vehicle injected control group. Conclusion: Based on the results of this study we propose that reducing angiogenesis after tendon injury can improve tendon repair, potentially representing a novel treatment-option.

BackgroundChoroidal vascular regulation is mediated by the autonomic nervous system in order to gain proper blood flow control. While the mechanisms behind this control are unknown, neuroregulatory peptides are involved in this process. To better understand choroidal function, we investigate the presence of urocortin-1 (UCN), a neuroregulatory peptide with vascular effects, in the human choroid and its possible intrinsic and extrinsic origin.MethodsHuman choroid and eye-related cranial ganglia (superior cervical ganglion- SCG, ciliary ganglion-CIL, pterygopalatine ganglion-PPG, trigeminal ganglion-TRI) were prepared for immunohistochemistry against UCN, protein–gene product 9.5 (PGP9.5), substance P (SP), tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT). For documentation, confocal laser scanning microscopy was used.ResultsIn choroidal stroma, UCN-immunoreactivity was present in nerve fibres, small cells and intrinsic choroidal neurons (ICN). Some UCN+ nerve fibres colocalised for VAChT, while others were VAChT. A similar situation was found with SP: some UCN+ nerve fibres showed colocalisation for SP, while others lacked SP. Colocalisation for UCN and TH was not observed. In eye-related cranial ganglia, only few cells in the SCG, PPG and TRI were UCN+, while many cells of the CIL displayed weak UCN immunoreactivity.ConclusionUCN is part of the choroidal innervation. UCN+/VAChT+ fibres could derive from the few cells of the PPG or cells of the CIL, if these indeed supply the choroid. UCN+/SP+ fibres might originate from ICN, or the few UCN+ cells detected in the TRI. Further studies are necessary to establish UCN function in the choroid and its implication for choroidal autonomic control.

Glaucoma is the second leading cause of blindness worldwide. This multifactorial, neurodegenerative group of diseases is characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, leading to irreversible visual impairment and blindness. There is a huge unmet and urging need for the development of new and translatable strategies and treatment options to prevent this progressive loss of RGC. Accumulating evidence points towards a critical role of neuroinflammation, in particular microglial cells, in the pathogenesis of glaucoma. Leukotrienes are mediators of neuroinflammation and are involved in many neurodegenerative diseases. Therefore, we tested the leukotriene receptors CysLT1R/GPR17-selective antagonist Montelukast (MTK) for its efficacy to modulate the reactive state of microglia in order to ameliorate RGCs loss in experimental glaucoma. Ocular hypertension (OHT) was induced unilaterally by injection of 8 μm magnetic microbead (MB) into the anterior chamber of female Brown Norway rats. The contralateral, untreated eye served as control. Successful induction of OHT was verified by daily IOP measurement using a TonoLab rebound tonometer. Simultaneously to OHT induction, one group received daily MTK treatment and the control group vehicle solution by oral gavage. Animals were sacrificed 13-15 days after MB injection. Retina and optic nerves (ON) of OHT and contralateral eyes were analyzed by immunohistochemistry with specific markers for RGCs (Brn3a), microglial cells/macrophages (Iba1 and CD68), and cysteinyl leukotriene pathway receptors (CysLT1R and GPR17). Protein labeling was documented by confocal microscopy and analyzed with ImageJ plugins. Further, mRNA expression of genes of the inflammatory and leukotriene pathway was analyzed in retinal tissue. MTK treatment resulted in a short-term IOP reduction at day 2, which dissipated by day 5 of OHT induction in MTK treated animals. Furthermore, MTK treatment resulted in a decreased activation of Iba1+ microglial cells in the retina and ON, and in a significantly increased RGC survival in OHT eyes. Within the retina, GPR17 and CysLT1R expression was demonstrated in single RCGs and in microglial cells respectively. Further, increased mRNA expression of pro-inflammatory genes was detected in OHT induced retinas. In the ON, OHT induction increased the number of GPR17+ cells, showing a significant reduction following MTK treatment. This study shows for the first time a significantly increased RGC survival in an acute OHT model following treatment with the leukotriene receptor antagonist MTK. These results strongly suggest a neuroprotective effect of MTK and a potential new therapeutic strategy for glaucoma treatment.

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 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.

Purpose Episcleral venous pressure (EVP) greatly influences steady-state IOP and recent evidence suggests a neuronal influence on EVP. Yet little is known about the innervation of the episcleral circulation and, more specifically, the neurotransmitters involved. We identify possible neurotransmitter candidates in the episcleral circulation of rats. Methods Eight immersion-fixated rat eyes taken from four animals were cut into serial sections, followed by standard immunohistochemistry. Antibodies against choline acetyltransferase, dopamine-β-hydroxylase, synaptophysine, PGP 9.5, VIP, neuronal nitric oxide synthase (nNOS), substance P, CGRP, and galanin were used. Additionally, colocalization experiments with smooth muscle actin and neurofilament (200 kDa) were performed. Results In all specimens, the episcleral vessels showed immunoreactivity for smooth muscle actin and were reached by neurofilament (200 kDa)-positive structures. Furthermore, these structures colocalized with immunoreactivity for PGP 9.5, synaptophysine, choline acetyl transferase (ChAT), dopamine-β-hydroxylase, VIP, CGRP, nNOS, substance P and galanin. Conclusions These findings indicate that there is neuronal input to the episcleral circulation. ChAT and VIP as well as dopamine-β-hydroxylase suggest parasympathetic and sympathetic innervation. Further studies are needed on whether the positively-stained structures are of functional significance for the regulation of the episcleral venous pressure and thereby IOP.

Aquaporins (AQPs) are important for ocular homeostasis and function. While AQP expression has been investigated in ocular tissues of human, mouse, rat and dog, comprehensive data in rabbits are missing. As rabbits are frequently used model organisms in ophthalmic research, the aim of this study was to analyze mRNA expression and to localize AQPs in the rabbit eye. The results were compared with the data published for other species. In cross sections of New Zealand White rabbit eyes AQP0 to AQP5 were labeled by immunohistology and analyzed by confocal microscopy. Immunohistological findings were compared to mRNA expression levels, which were analyzed by quantitative reverse transcription real time polymerase chain reaction (qRT-PCR). The primers used were homologous against conserved regions of AQPs. In the rabbit eye, AQP0 protein expression was restricted to the lens, while AQP1 was present in the cornea, the chamber angle, the iris, the ciliary body, the retina and, to a lower extent, in optic nerve vessels. AQP3 and AQP5 showed immunopositivity in the cornea. AQP3 was also present in the conjunctiva, which could not be confirmed for AQP5. However, at a low level AQP5 was also traceable in the lens. AQP4 protein was detected in the ciliary non-pigmented epithelium (NPE), the retina, optic nerve astrocytes and extraocular muscle fibers. For most tissues the qRT-PCR data confirmed the immunohistology results and vice versa. Although species differences exist, the AQP protein expression pattern in the rabbit eye shows that, especially in the anterior section, the AQP distribution is very similar to human, mouse, rat and dog. Depending on the ocular regions investigated in rabbit, different protein and mRNA expression results were obtained. This might be caused by complex gene regulatory mechanisms, post-translational protein modifications or technical limitations. However, in conclusion the data suggest that the rabbit is a useful in-vivo model to study AQP function and the effects of direct and indirect intervention strategies to investigate e. g. mechanisms for intraocular pressure modulation or cornea transparency regulation.

Choroidal all- trans -retinoic acid (atRA) may play a key role in the control of postnatal eye growth in a variety of vertebrates through modulation of scleral extracellular matrix synthesis and may therefore play a crucial role in the development of myopia. In the chick eye, choroidal atRA synthesis is exclusively regulated by its synthesizing enzyme, retinaldehyde dehydrogenase 2 (RALDH2). In chicks and humans, RALDH2 has been detected in a population of hitherto uncharacterized choroidal cells.Therefore, the aim of this study was to identify the RALDH2+ cell type(s) in the choroid and determine how these cells modulate atRA concentrations during periods of visually guided eye growth. Chicks wore translucent goggles on one eye for 10 days and choroids were analyzed for RALDH activity and RALDH2 protein expression at days 0, 1, 4, 7, 15 following removal of the goggle ("recovery"); choroids from contralateral eyes served as controls. The presence of RALDH2+ cells was assessed in chick choroid wholemounts using multiphoton microscopy. RALDH2 protein expression was measured by western blot and RALDH2 activity was assessed via HPLC quantification of atRA. Cell proliferation was assessed by BrdU-labelling in combination with RALDH2-immunohistochemistry. For characterization of RALDH2+ cells, immunohistochemistry for various tissue specific markers was applied in chicken (Ia antigen, CD5, Col1-propeptide, desmin, IgY, L-Cam, Cadherin1, MHC-II; Tcr-γδ, vimentin) and human donor tissue (α-smooth-muscle-actin, CD's 31/34/68/146, desmin, IBA1, LYVE-1, PGP9.5, vimentin) followed by confocal microscopy. In the chick and human choroid, RALDH2+ cells with variable morphology were present in the stroma and adjacent to choroidal blood vessels. In chick wholemounts, RALDH2+ cells were concentrated toward the choriocapillaris, and their number increased nearly linearly between 1 and 7 days of recovery and plateaued between 7 and 15 days compared to corresponding controls. A significant increase in choroidal RALDH2 protein concentration and atRA synthetic activity was observed by four days of recovery (↑107% and ↑120%) by western blot and HPLC, respectively. A 3-fold increase in RALDH2+/BrDU+ cells was observed following 4 days of recovery compared to controls (12.43 ± 0.73% of all RALDH2+ cells in recovering eyes as compared with 4.46 ± 0.63% in control eyes, p < 0.001). In chick choroids, the vast majority of RALDH2+ cells co-expressed Col1-propetide, but did not co-label with any other antibodies tested. In human choroid, some, but not all RALDH2+ cells colocalized with vimentin, but were negative for all other antibodies tested. RALDH2+ cells represent a novel cell type in the chick and human choroid. Our findings that some human RALDH2+ cells were positive for vimentin and all chick RALDH2+ cells were positive for Col1, suggest that RALDH2+ cells most closely resemble perivascular and stromal fibroblasts. The increased number of RALDH2+/BRDU+ cells following 4 days of recovery suggests that choroidal atRA concentrations are partially controlled by proliferation of RALDH2+ cells. The identification of this choroidal cell type will provide a broader understanding of the cellular events responsible for the regulation of postnatal ocular growth, and may provide new avenues for specifically targeted strategies for the treatment of myopia.

Purpose Most organs of the human body are supplied with a dense network of blood and lymphatic vessels. However, some tissues are either hypovascular or completely devoid of vessels for proper function, such as the ocular tissues sclera and cornea, cartilage and tendons. Since many pathological conditions are affecting the human sclera, this review is focussing on the lymphangiogenic and hemangiogenic privilege in the human sclera. Methods This article gives an overview of the current literature based on a PubMed search as well as observations and experience from clinical practice. Results The healthy human sclera is the outer covering layer of the eye globe consisting mainly of collagenous extracellular matrix and fibroblasts. Physiologically, the sclera shows only a superficial network of blood vessels and a lack of lymphatic vessels. This vascular privilege is actively regulated by balancing anti- and proangiogenic factors expressed by cells within the sclera. In pathological situations, such as open globe injuries or ciliary body melanomas with extraocular extension, lymphatic vessels can secondarily invade the sclera and the inner eye. This mechanism most likely is important for tumor cell metastasis, wound healing, immunologic defense against intruding microorganism, and autoimmune reactions against intraocular antigens. Conclusions The human sclera is characterized by a tightly regulated vascular network that can be compromised in pathological situations, such as injuries or intraocular tumors affecting healing outcomes Therefore, the molecular and cellular mechanisms underlying wound healing following surgical interventions deserve further attention, in order to devise more effective therapeutic strategies.

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.

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.

PURPOSE Hereditary hyperferritinemia cataract syndrome (HHCS) is a rare autosomal dominant hereditary disease, characterized by hyperferritinemia but with absence of body iron excess and early onset of bilateral cataracts. Although 5- to 20-fold increased serum ferritin concentrations have been reported in HHCS patients, data of ferritin levels in aqueous humor have not been obtained. We therefore aimed to investigate the ferritin levels in aqueous humor and serum and further present histological and ultrastructural data of the lens. METHODS During cataract extraction and intraocular lens implantation, aqueous humor and lens aspirate of a 37-year-old HHCS patient were obtained from both eyes. Ferritin levels in serum and aqueous humor were quantitatively analyzed via immunoassays in the HHCS patient and healthy control subjects (n = 6). Lens aspirate in HHCS was analyzed histologically and at the ultrastructural level. Further, genetic mutation screening by polymerase chain reaction and DNA sequencing in blood was performed. RESULTS Serum ferritin levels in the control group were 142.2 ± 38.7 μg/L, whereas in the HHCS patient, this parameter was excessively increased (1086 μg/L). Analysis of ferritin in aqueous humor revealed 6.4 ± 3.8 μg/L in normal control subjects and 146.3 μg/L (OD) and 160.4 μg/L (OS) in the HHCS patient. DNA analysis detected a C>A mutation on position +18, a T>G mutation on position +22, a T>C mutation on position +24, and a T>G polymorphism on position +26 in the iron-responsive element of the light-chain ferritin (L-ferritin) gene. CONCLUSIONS In the HHCS patient, a 23-fold (OD) to 25-fold (OS) increased aqueous humor ferritin level was detected. Therefore, the formation of bilateral cataract in HHCS is most likely a result of elevated aqueous humor ferritin. In addition, a novel mutation in this rare disease in the iron-responsive element of L-ferritin gene is reported.

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.

Ciliary ganglionectomy inhibits the development of myopia in chicks (Schmid et al., 1999), but has no effect on the compensatory responses to spectacle lenses (Schmid and Wildsoet, 1996). This study was done to assess the potential influence of the other parasympathetic input to the choroid, the pterygopalatine ganglia, on the choroidal and axial responses to retinal defocus, and to form deprivation. 4-5 week-old chicks had one of the following surgeries to one eye: (1) Section (X) of the autonomic part of cranial N VII (input to the pterygopalatine ganglia) (PPGX, n = 16), (2) PPGX plus ciliary ganglionectomy (PPG/CGX, n = 23) or (3) PPGX plus superior cervical ganglionectomy (PPG/SCGX, n = 10). Experimental eyes were fitted with positive or negative lenses, or diffusers, several days after surgery. In one group of PPG/CGX, eyes did not wear any devices (n = 8). Intact (no surgery) controls were done for all visual manipulations (lenses or diffusers). Sham surgeries were done for the PPG/CGX condition (n = 4). Ocular dimensions were measured using A-scan ultrasonography prior to the surgery, 5 days later when visual devices were placed on the eyes, at the end of lens- or diffuser-wear, and in the case of diffusers, 4 days after diffuser removal to look at "recovery". Refractive errors were measured using a Hartinger's refractometer. IOP was measured in 7 PPG/CGX birds 7d after surgery. PPGX/CGX resulted in choroidal thickening (125 μm) and a decrease in IOP over one week post-surgery. It also prevented the development of myopia in response to form deprivation (X vs intact: 0.2 D vs -4.1 D; p 0.005), by preventing the increase in axial elongation (250 μm vs 670 μm/5d; p 0.005). In fact, growth rate slowed below normal (X vs fellow eyes: 250 μm vs 489 μm/5d; p = 0.002). By contrast, there were no effects of this lesion on the development of myopia in response to negative lenses (X vs intact: -5.4 D vs -5.3 D). All three lesions inhibited the compensatory choroidal thickening in response to myopic defocus (ANOVA, p = 0.0008), but had no effect on the thinning response to hyperopic defocus. These results argue for different underlying mechanisms for the growth responses to form deprivation vs negative lens wear. They also imply that choroidal thickening and thinning are not opposing elements of a single mechanism.

The results of many studies in a variety of species have significantly advanced our understanding of the role of visual experience and the mechanisms of postnatal eye growth, and the development of myopia. This paper surveys and reviews the major contributions that experimental studies using animal models have made to our thinking about emmetropization and development of myopia. These studies established important concepts informing our knowledge of the visual regulation of eye growth and refractive development and have transformed treatment strategies for myopia. Several major findings have come from studies of experimental animal models. These include the eye's ability to detect the sign of retinal defocus and undergo compensatory growth, the local retinal control of eye growth, regulatory changes in choroidal thickness, and the identification of components in the biochemistry of eye growth leading to the characterization of signal cascades regulating eye growth and refractive state. Several of these findings provided the proofs of concepts that form the scientific basis of new and effective clinical treatments for controlling myopia progression in humans. Experimental animal models continue to provide new insights into the cellular and molecular mechanisms of eye growth control, including the identification of potential new targets for drug development and future treatments needed to stem the increasing prevalence of myopia and the vision-threatening conditions associated with this disease.

A thorough understanding of intraocular pressure homeostasis is the biological foundation for the development of new strategies to treat patients with elevated intraocular pressure or glaucoma. However, investigations on the physiology of intraocular pressure homeostasis are also important to gain more comprehensive insights into the pathogenesis of glaucoma and other diseases with associated alterations of intraocular pressure. The present review intends to give alternative insights into the biological and physical aspects of intraocular pressure regulation. The pressure-volume as well as the hydraulic model of intraocular pressure and also the relationship between ciliary blood flow and aqueous humor production, which has moved into the centre of interest because of its possible clinical relevance for glaucoma patients, will be explained. The authors Have attempted to interrelate the different aspects of intraocular pressure genesis and regulation in a comprehensive but understandable way.

We read with interest the recent article by Koina et al.1 suggesting evidence for the presence of lymphatic vessels in the developing and adult human choroid. However, this study does not meet the recently published consensus criteria on the immunohistochemical detection of ocular lymphatic vessels,2 and therefore, in our opinion, requires critical revision. First, appropriate positive and unequivocal negative controls are not presented in the study of Koina et al. In particular, when describing novel anatomical structures for the first time, and in order to change an existing dogma, a detailed documentation of blood and lymphatic vessel detection in the control tissue is mandatory. The provided supplementary data do not fulfill these criteria. Second, the immunohistochemical marker panel used is critical. Endomucin does not represent an established lymphatic marker,3,4 but is rather expressed by “endothelial cells along the whole vascular tree including lymphatic vessels.”5 Thus, an unequivocal discrimination between blood and lymphatic vessels is impossible with this marker. A further discrepancy is the use of the transcription factor prospero-related homebox gene-1 (Prox-1) as an extranuclear lymphatic endothelial precursor marker. Although reports of the extranuclear presence of PROX-1 in cell types other than lymphatic endothelium exist,6–8 PROX-1 clearly shows a nuclear expression in lymphatic endothelia in human,9 as well as mouse10 and avian,11 embryos, retaining its nuclear localization into adulthood.12–14 On the other hand, it is not clear why lymphatic endothelial surface markers, such as podoplanin, lymphatic vascular endothelial-specific hyaluronic acid receptor-1 (LYVE-1), and the vascular endothelial marker CD34 display nuclear expression in this study. Additionally, the only lymphatic endothelial cell marker used in whole mounts is VEGFR-3, which is also expressed in fenestrated blood vessels, and, as such, also in the choriocapillaris.15,16 Morphologically, the supposed lymphatic VEGFR-3–positive vessels are indistinguishable from the honeycomb-like lobular pattern of the choriocapillaris.17 Furthermore, the study of Koina et al. includes a blatant inconsistency in the use and documentation of immunohistochemical markers between fetal and adult eyes. Although one has to acknowledge that certain lymphatic markers might be expressed during embryogenesis, this pattern easily changes during maturation.18 Therefore, such an approach would require extensive comparison of the same markers in different ages, thus representing an extensive survey in its own right. However, this is not the case in the study of Koina et al. Third, the ultrastructural study would be greatly strengthened by immunoelectron microscopy. Indeed, anchoring filaments with a diameter of 40 to 100 A—becoming readily identifiable only at magnifications of 40,000× to 50,000×—are present in lymphatics,19 but could be easily present in the choroid as well without any association to lymphatic vessels,20–22 particularly in aged eyes with typical alterations of the extracellular matrix. For this purpose, as well as for ruling out Weibel-Palade bodies, serial ultrathin sectioning with appropriate labeling would be necessary. Despite possible postmortem tissue alterations, numerous previous studies successfully applied different detection systems for ultrastructural investigations using ocular human donor tissue.23–29 A limited use of immunomarkers for these investigations, as claimed, seems therefore not justified. In regard to the above-mentioned criticisms, the evidence presented in the study of Koina et al. does not justify the hypothesized paradigm shift that functional lymphatic vessels are present in the human choroid. Rather, the findings of Koina et al. confirm previous reports of net-like structures with a “pseudo-vessel” appearance in the human choroid endowed with lymphatic vascular precursor cells (represented as LYVE-1+ macrophages).25 Those “atypical” lymphatic-like cells (i.e., endothelial cells with divergent or uncommon immunohistochemical phenotypes) may also exist in other parts of the eye. For example, the endothelial cells of Schlemm's canal display many, but not all, features of terminally differentiated lymphatic endothelial cells, including responsiveness to VEGF-C–induced lymphangiogenesis.30 In closing, we acknowledge that the work of Koina et al. is a further contribution to our understanding of the choroid, but although the existence of lymphatics in the human choroid cannot be ruled out per se, because of the aforementioned points and the sheer volume of evidence to date, we maintain that the inner human eye and in particular the choroid should still be considered an immune-privileged site devoid of lymphatic vessels. Further unequivocal evidence of “typical lymphatic vessels” in the human choroid is still missing.

Purpose: The normal human cornea is devoid of both blood and lymphatic vessels and actively maintains this avascularity (corneal angiogenic privilege). Whether and when corneal angiogenic privilege is achieved during development is unknown. Methods: This study analyzed whether the cornea is primarily devoid of both blood and lymphatic vessels during intrauterine development or whether secondary regression of pre-existing vessels occurs before delivery. Indirect double immunohistochemistry was performed on 4-μm serial pupil-optic disc sections of paraffin-embedded human eyes stillborn at gestational ages of 17 to 41 weeks with antibodies against von Willebrand factor (vWF; factor VIII-associated antigen) as a panendothelial marker and with antibodies against lymphatic vessel endothelial hyaluronate receptor 1 (LYVE1) as a marker specific for lymphatic vascular endothelium. Results: Human corneas were devoid of both vWF +++ /LYVE-1 - blood vessels and vWF + /LYVE-1 +++ lymphatic vessels at all time-points analyzed. In contrast, there were numerous blood and lymphatic vessels detectable in the adjacent conjunctiva. Conclusion: The normal human cornea is primarily avascular and devoid of both blood and lymphatic vessels. Corneal angiogenic privilege is already achieved very early during fetal intrauterine development. This suggests early and strong expression of both antiangiogenic and antilymphangiogenic factors in the human cornea during development.

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma. Currently, the primary strategy to slow down glaucoma progression is IOP reduction. Numerous studies investigated the IOP lowering effects of neuropeptides, but the complex physiological mechanisms involved are difficult to interpret. Therefore, we investigated the effect of arginine-vasopressin (AVP) on IOP in a multiparameter approach. In a rabbit model the effect of i.v.-applied AVP on IOP, ciliary blood flow (CilBF) and aqueous flow (AqF) was measured simultaneously by manometry, laser-Doppler flowmetry and fluorophotometry. AVP caused a dose-dependent decrease of IOP, CilBF and AqF. Based on the Goldmann equation the decrease of AqF at 0.08 ng/kg/min AVP (low dose) accounted for 77.1% of the IOP reduction (9.56±2.35%), while AqF at 1.33 ng/kg/min AVP (high dose) accounted for 28.4% of the IOP decline (41.02±3.19%). The results indicate that AqF reduction by low dose AVP is mainly caused by inhibition of ciliary epithelium secretion. At high dose AVP AqF reduction is caused by decreased CilBF or more likely by a combination of CilBF decline and secretory inhibition. This conclusion is only possible with the multiparameter measurement approach presented.

Doublecortin (DCX) is predominantly expressed in neuronal precursor cells and young immature neurons of the developing and adult brain, where it is involved in neuronal differentiation, migration and plasticity. Moreover, its expression pattern reflects neurogenesis, and transgenic DCX promoter-driven reporter models have been previously used to investigate adult neurogenesis. In this study, we characterize dsRed2 reporter protein-expressing cells in the adult retina of the transgenic DCX promoter-dsRed2 rat model, with the aim to identify cells with putative neurogenic activity. Additionally, we confirmed the expression of the dsRed2 protein in DCX-expressing cells in the adult hippocampal dentate gyrus. Adult DCX-dsRed2 rat retinas were analyzed by immunohistochemistry for expression of DCX, NF200, Brn3a, Sox2, NeuN, calbindin, calretinin, PKC-a, Otx2, ChAT, PSA-NCAM and the glial markers GFAP and CRALBP, followed by confocal laser-scanning microscopy. In addition, brain sections of transgenic rats were analyzed for dsRed2 expression and co-localization with DCX, NeuN, GFAP and Sox2 in the cortex and dentate gyrus. Endogenous DCX expression in the adult retina was confined to horizontal cells, and these cells co-expressed the DCX promoter-driven dsRed2 reporter protein. In addition, we encountered dsRed2 expression in various other cell types in the retina: retinal ganglion cells (RGCs), a subpopulation of amacrine cells, a minority of bipolar cells and in perivascular cells. Since also RGCs expressed dsRed2, the DCX-dsRed2 rat model might offer a useful tool to study RGCs in vivo under various conditions. Muller glial cells, which have previously been identified as cells with stem cell features and with neurogenic potential, did express neither endogenous DCX nor the dsRed2 reporter. However, and surprisingly, we identified a perivascular glial cell type expressing the dsRed2 reporter, enmeshed with the glia/stem cell marker GFAP and colocalizing with the neural stem cell marker Sox2. These findings suggest the so far undiscovered existence of perivascular associated cell with neural stem cell-like properties in the adult retina.

Previous experiments have shown that arginine-vasopressin (AVP) reduces intraocular pressure (IOP) dose-dependently. The present study investigated the relationships between IOP, ciliary blood flow (CilBF), and aqueous flow (AqF) responses to AVP in anesthetized rabbits.CilBF was measured by laser Doppler flowmetry and AqF by fluorophotometry. Mean arterial pressure (MAP) and IOP were monitored continuously and simultaneously. Perfusion pressure (PP) was varied mechanically. Four experimental protocols were performed: the dose-response (n = 11) and the pressure-flow relationship (n = 8) for CilBF and the effects on CilBF, and AqF at low (0.08 ng/kg/min; n = 14) and high AVP infusion rates (1.33 ng/kg/min; n = 12).AVP decreased CilBF and IOP dose-dependently. At the low AVP infusion rate, AqF was reduced by 21.48% ± 2.52% without changing CilBF significantly. The high AVP infusion rate caused a 24.49% ± 3.53% decrease of AqF and a significant reduction in CilBF (35.60% ± 3.58%). IOP was reduced by 9.56% ± 2.35% at low and by 41.02% ± 3.19% at high AVP infusion rates. Based on the Goldmann equation, the decrease of AqF at the low AVP infusion rate accounted for 77.1% of the IOP reduction, whereas at the high AVP infusion rate, decreased AqF accounted for 28.4% of the IOP decline.The results indicate that AVP can modulate IOP by different dose-dependent physiological mechanisms. The shifts of the CilBF-AqF relationship suggest that the reduction of AqF by the low AVP infusion rate is mainly provoked by inhibiting secretory processes in the ciliary epithelium. In contrast, at the high AVP infusion rate, the AqF reduction is caused by either reduced CilBF or more likely by a combined effect of reduced CilBF and secretory inhibition.

PURPOSE The origin of pericytes (PCs) has been controversially discussed and at least three different sources of PCs are proposed: a neural crest, mesodermal, or bone marrow origin. In the present study we investigated a potential neural crest origin of ocular PCs in a transgenic Rosa26-YFP-Sox10-Cre neural crest-specific reporter mouse model at different developmental stages. METHODS The Rosa26-YFP-Sox10-Cre mouse model expresses the yellow fluorescent protein (YFP) reporter in cells with an active Sox10 promoter and was here used for cell fate studies of Sox10-positive neural crest derived progeny cells. Detection of the YFP signal in combination with double and triple immunohistochemistry of chondroitin sulfate proteoglycan (NG2), platelet derived growth factor receptor β (PDGFRβ), α smooth muscle actin (αSMA), oligodendrocyte transcription factor 2 (Olig2), and lectin was performed and analyzed by confocal microscopy. RESULTS Sox10-YFP-positive cells and profiles were detected in the inner nuclear layer, the ganglionic cell layer, and the axons of the nerve fiber layer in postnatal retinas. An additional population has been identified in the retina, optic nerve, and choroid that displays strong perivascular localization. These cells were colocalized with the PC-specific markers NG2 and PDGFRβ in embryonic (E14.5) as well as postnatal (P4, P12, 6-week-old) vasculature. Beside PCs, vascular smooth muscle cells (vSMCs) were also labeled by the Sox10-YFP reporter protein in all ocular tissues investigated. CONCLUSIONS Since YFP-positive PCs and vSMCs are colocalized with NG2 and PDGFRβ, we propose that capillary PCs and vSMCs in the retina and the optic nerve, both parts of the central nervous system, as well as in the choroid, a tissue of mesodermal origin, derive from the neural crest.

Impaired ocular blood flow is involved in the pathogenesis of numerous ocular diseases like glaucoma or AMD. The purpose of the present study was to introduce and validate a novel, microscope based, non invasive laser Doppler flowmeter (NILDF) for measurement of blood flow in the choroid. The custom made NI-LDF was compared with a commercial fiber optic based laser Doppler flowmeter (Perimed PF4000). Linearity and stability of the NI-LDF were assessed in a silastic tubing model (i.d. 0.3 mm) at different flow rates (range 0.4 – 3 ml/h). In a rabbit model continuous choroidal blood flow measurements were performed with both instruments simultaneously. During blood flow measurements ocular perfusion pressure was changed by manipulations of intraocular pressure via intravitreal saline infusions. The NILDF measurement correlated linearly to intraluminal flow rates in the perfused tubing model (r = 0.99, p

There is currently considerable controversy about existence and classification of “lymphatic vessels” in the eye. Some of the confusion is certainly caused by inappropriate use (or nonuse) of the correct immunohistochemical markers. Many experts in the field expressed the need for a consensus statement, and, in this perspective, authors offer arguments and solutions to reliably continue with immunohistochemical ocular lymphatic research.

To investigate the effects of arginine-vasopressin (AVP) on intraocular pressure (IOP), orbital venous pressure (OVP), and choroidal blood flow (ChorBF) regulation in anesthetized rabbits.Mean arterial pressure (MAP), IOP, and OVP were measured by direct cannulation of the central ear artery, the vitreous, and the orbital venous sinus, respectively. Laser Doppler flowmetry was used to record ChorBF. To change the perfusion pressure (PP), MAP was manipulated mechanically with occluders around the aorta and vena cava. In the first group of animals (n = 11) the dose-response relationship was measured. In the second group of animals (n = 8) pressure-flow relationships were determined at baseline and in response to intravenous application of a low (0.08 ng/kg/min) and a high (1.33 ng/kg/min) infusion rate of AVP.AVP caused a dose-dependent increase of MAP and choroidal vascular resistance (ChorR), whereas IOP, OVP, ChorBF, and heart rate (HR) were decreased. In contrast to the high infusion rate, the low infusion rate of AVP had no effect on baseline ChorBF. However, the pressure-flow relationship was shifted downward significantly by both infusion rates at PP below baseline.AVP reduces IOP and OVP significantly and is a potent vasoconstrictor in the choroidal vascular bed. In the choroid, the effect of AVP is not only dose-dependent, but also PP-dependent, which is indicated by the reduced perfusion relative to control with low-dosed AVP at low PP.

Purpose Lymphatic vessel endothelial hyaluronic acid receptor (LYVE-1) is a newly discovered lymphatic endothelium-specific marker that is also expressed by a subpopulation of macrophages. To date, there is no report on its expression in the posterior human uvea. The purpose of this study was to investigate the expression of LYVE-1 in normal human choroids. Methods Eyes of body/cornea donors (55-89 years of age; 4-9 hours postmortem) were obtained. Choroids were dissected and prepared for cryosections followed by immunohistochemistry with anti-human LYVE-1 antiserum and immunogold labeling. In addition, anti-human antibodies against macrophage markers (CD68, MHC class II) and lymphatic (podoplanin) and blood vascular endothelium (CD31, vWF) were used. For documentation, light-, fluorescence-, confocal laser scanning-, and electron-microscopy were used. Results The normal human choroidal stroma contained 274 +/- 86 LYVE-1 positive cells/mm(2). The cells displayed irregular shapes with a relatively uniform diameter of 32 mum. Costaining with CD68 and negativity for CD31, podoplanin, and melan-A/HMB45, as well as electron microscopic features, suggest these LYVE-1(+) cells to be macrophages. Besides that, no classic LYVE-1(+)/podoplanin(+) lymphatic vessels were detected within the normal adult human choroid. Conclusions The normal adult human choroid does not contain typical lymph vessels, but is endowed with a significant number of LYVE-1 positive macrophages. These cells may be involved in choroidal hyaluronic acid metabolism or contribute to temporary formation of lymphatic channels under inflammatory conditions.