Melanopsin in the human and chicken choroid.

The choroid embedded in between retina and sclera is essential for retinal photoreceptor nourishment, but is also a source of growth factors in the process of emmetropization that converts retinal visual signals into scleral growth signals. Still, the exact control mechanisms behind those functions are enigmatic while circadian rhythms are involved. These rhythms are attributed to daylight influences that are melanopsin (OPN4) driven. Recently, OPN4-mRNA has been detected in the choroid, and while its origin is unknown we here seek to identify the underlying structures using morphological methods. Human and chicken choroids were prepared for single- and double-immunohistochemistry of OPN4, vasoactive intestinal peptide (VIP), substance P (SP), CD68, and α-smooth muscle actin (ASMA). For documentation, light-, fluorescence-, and confocal laser scanning microscopy was applied. Retinal controls proved the reliability of the OPN4 antibody in both species. In humans, OPN4 immunoreactivity (OPN4-IR) was detected in nerve fibers of the choroid and adjacent ciliary nerve fibers. OPN4+ choroidal nerve fibers lacked VIP, but were co-localized with SP. OPN4-immunoreactivity was further detected in VIP+/SP + intrinsic choroidal neurons, in a hitherto unclassified CD68-negative choroidal cell population thus not representing macrophages, as well as in a subset of choroidal melanocytes. In chicken, choroidal nerve fibers were OPN4+, and further OPN4-IR was detected in clustered suprachoroidal structures that were not co-localized with ASMA and therefore do not represent non-vascular smooth-muscle cells. In the choroidal stroma, numerous cells displayed OPN4-IR, the majority of which was VIP-, while a few of those co-localized with VIP and were therefore classified as avian intrinsic choroidal neurons. OPN4-immunoreactivity was absent in choroidal blood vessels of both species. In summary, OPN4-IR was detected in both species in nerve fibers and cells, some of which could be identified (ICN, melanocytes in human), while others could not be classified yet. Nevertheless, the OPN4+ structures described here might be involved in developmental, light-, thermally-driven or nociceptive mechanisms, as known from other systems, but with respect to choroidal control this needs to be proven in upcoming studies.

The structure and function of the human choroid.

In this manuscript, the structure of the human choroid is reviewed with emphasis of the macro- and microscopic anatomy including Bruch's membrane, choriocapillaris, Sattler's and Haller's layer, and the suprachoroid. We here discuss the development of the choroid, as well as the question of choroidal lymphatics, and further the neuronal control of this tissue, as well as the pathologic angiogenesis. Wherever possible, functional aspects of the various structures are included and reviewed.

Hypertrophic cardiomyopathy is characterized by alterations of the mitochondrial calcium uniporter complex proteins: insights from patients with aortic valve stenosis versus hypertrophic obstructive cardiomyopathy.

Introduction: Hypertrophies of the cardiac septum are caused either by aortic valve stenosis (AVS) or by congenital hypertrophic obstructive cardiomyopathy (HOCM). As they induce cardiac remodeling, these cardiac pathologies may promote an arrhythmogenic substrate with associated malignant ventricular arrhythmias and may lead to heart failure. While altered calcium (Ca2+) handling seems to be a key player in the pathogenesis, the role of mitochondrial calcium handling was not investigated in these patients to date. Methods: To investigate this issue, cardiac septal samples were collected from patients undergoing myectomy during cardiac surgery for excessive septal hypertrophy and/or aortic valve replacement, caused by AVS and HOCM. Septal specimens were matched with cardiac tissue obtained from post-mortem controls without cardiac diseases (Ctrl). Results and discussion: Patient characteristics and most of the echocardiographic parameters did not differ between AVS and HOCM. Most notably, the interventricular septum thickness, diastolic (IVSd), was the greatest in HOCM patients. Histological and molecular analyses showed a trend towards higher fibrotic burden in both pathologies, when compared to Ctrl. Most notably, the mitochondrial Ca2+ uniporter (MCU) complex associated proteins were altered in both pathologies of left ventricular hypertrophy (LVH). On the one hand, the expression pattern of the MCU complex subunits MCU and MICU1 were shown to be markedly increased, especially in AVS. On the other hand, PRMT-1, UCP-2, and UCP-3 declined with hypertrophy. These conditions were associated with an increase in the expression patterns of the Ca2+ uptaking ion channel SERCA2a in AVS (p = 0.0013), though not in HOCM, compared to healthy tissue. Our data obtained from human specimen from AVS or HOCM indicates major alterations in the expression of the mitochondrial calcium uniporter complex and associated proteins. Thus, in cardiac septal hypertrophies, besides modifications of cytosolic calcium handling, impaired mitochondrial uptake might be a key player in disease progression.

IMI-The Dynamic Choroid: New Insights, Challenges, and Potential Significance for Human Myopia.

The choroid is the richly vascular layer of the eye located between the sclera and Bruch's membrane. Early studies in animals, as well as more recent studies in humans, have demonstrated that the choroid is a dynamic, multifunctional structure, with its thickness directly and indirectly subject to modulation by a variety of physiologic and visual stimuli. In this review, the anatomy and function of the choroid are summarized and links between the choroid, eye growth regulation, and myopia, as demonstrated in animal models, discussed. Methods for quantifying choroidal thickness in the human eye and associated challenges are described, the literature examining choroidal changes in response to various visual stimuli and refractive error-related differences are summarized, and the potential implications of the latter for myopia are considered. This review also allowed for the reexamination of the hypothesis that short-term changes in choroidal thickness induced by pharmacologic, optical, or environmental stimuli are predictive of future long-term changes in axial elongation, and the speculation that short-term choroidal thickening can be used as a biomarker of treatment efficacy for myopia control therapies, with the general conclusion that current evidence is not sufficient.

Parasympathetic and sympathetic control of emmetropization in chick.

Emmetropization can be altered by temporal visual stimulation and the spectral properties of the visual environment. The goal of the current experiment is to test the hypothesis that there is an interaction between these properties and autonomic innervation. For that purpose, selective lesions of the autonomic nervous system were performed in chickens followed by temporal stimulation. Parasympathetic lesioning involved transection of both the ciliary ganglion and the pterygopalatine ganglion (PPG_CGX; n = 38), while sympathetic lesioning involved transection of the superior cervical ganglion (SCGX; n = 49). After one week of recovery, chicks were then exposed to temporally modulated light (3 days, 2 Hz, Mean: 680 lux) that was either achromatic (with blue [RGB], or without blue [RG]), or chromatic (with blue [B/Y] or without blue [R/G]). Control birds with lesions, or unlesioned, were exposed to white [RGB] or yellow [RG] light. Ocular biometry and refraction (Lenstar and a Hartinger refractometer) was measured before and after exposure to light stimulation. Measurements were statistically analyzed for the effects of a lack of autonomic input and the type of temporal stimulation. In PPG_CGX lesioned eyes, there was no effect of the lesions one-week post-surgery. However, after exposure to achromatic modulation, the lens thickened (with blue) and the choroid thickened (without blue) but there was no effect on axial growth. Chromatic modulation thinned the choroid with R/G. In the SGX lesioned eye, there was no effect of the lesion 1-week post-surgery. However, after exposure to achromatic modulation (without blue), the lens thickened and there was a reduction in vitreous chamber depth and axial length. Chromatic modulation caused a small increase in vitreous chamber depth with R/G. Both autonomic lesion and visual stimulation were necessary to affect the growth of ocular components. The bidirectional responses observed in axial growth and in choroidal changes suggest that autonomic innervation combined with spectral cues from longitudinal chromatic aberration may provide a mechanism for homeostatic control of emmetropization.

The novel role of lymphatic vessels in the pathogenesis of ocular diseases.

Historically, the eye has been considered as an organ free of lymphatic vessels. In recent years, however, it became evident, that lymphatic vessels or lymphatic-like vessels contribute to several ocular pathologies at various peri- and intraocular locations. The aim of this review is to outline the pathogenetic role of ocular lymphatics, the respective molecular mechanisms and to discuss current and future therapeutic options based thereon. We will give an overview on the vascular anatomy of the healthy ocular surface and the molecular mechanisms contributing to corneal (lymph)angiogenic privilege. In addition, we present (i) current insights into the cellular and molecular mechanisms occurring during pathological neovascularization of the cornea triggered e.g. by inflammation or trauma, (ii) the role of lymphatic vessels in different ocular surface pathologies such as dry eye disease, corneal graft rejection, ocular graft versus host disease, allergy, and pterygium, (iii) the involvement of lymphatic vessels in ocular tumors and metastasis, and (iv) the novel role of the lymphatic-like structure of Schlemm's canal in glaucoma. Identification of the underlying molecular mechanisms and of novel modulators of lymphangiogenesis will contribute to the development of new therapeutic targets for the treatment of ocular diseases associated with pathological lymphangiogenesis in the future. The preclinical data presented here outline novel therapeutic concepts for promoting transplant survival, inhibiting metastasis of ocular tumors, reducing inflammation of the ocular surface, and treating glaucoma. Initial data from clinical trials suggest first success of novel treatment strategies to promote transplant survival based on pretransplant corneal lymphangioregression.

Urocortin-positive nerve fibres and cells are present in the human choroid.

BACKGROUND: Choroidal 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. METHODS: Human 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. RESULTS: In 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. CONCLUSION: UCN 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.

Human intrinsic choroidal neurons do not alter the expression of intrinsic markers in response to pressure.

BACKGROUND: The 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. METHODS: Donor 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. RESULTS: The 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. CONCLUSION: In 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.

Parasympathetic innervation of emmetropization.

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.

Is the human sclera a tendon-like tissue? A structural and functional comparison.

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.

Inhibition of the cysteinyl leukotriene pathways increases survival of RGCs and reduces microglial activation in ocular hypertension.

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 immunofluorescence 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 trend of 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.

Choroidal melanocytes: subpopulations of different origin?

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.

Scleraxis expressing scleral cells respond to inflammatory stimulation.

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.

Absence of lymphatic vessels in non-functioning bleb capsules of glaucoma drainage devices.

PURPOSE: To evaluate the presence and appearance of blood and lymphatic vessels in non-functioning bleb capsules of glaucoma drainage devices (GDD). MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Retinoic acid synthesis by a population of choroidal stromal cells.

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.

The lymphangiogenic and hemangiogenic privilege of the human sclera.

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.

Immunohistochemical Characterization of Neurotransmitters in the Episcleral Circulation in Rats.

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-ß-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-ß-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-ß-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.

Alarin in different human intestinal epithelial cell types.

Alarin (AL), a new member of the galanin family, has been localized in various CNS regions, mainly in rodents. Among other effects, it modulates food intake. Therefore, we analyzed the immunohistochemical distribution pattern of AL in human intestinal epithelia. Cryosections of 12 human bowel samples were immunohistochemically double-stained for AL and α-defensin 5 (αD; first set). Two further sets of sections were quadruple-stained either (second set) for AL, chromogranin (CG), synaptophysin (SY), and somatostatin (SO) or (third set) for AL, CG, Peptide Y (PY), and 5-hydroxytryptamine (5-HT). Slides were digitized and quantitative analysis of co-localization rates was undertaken. Small bowel: most of AL-positive cells (56%) were αD-positive Paneth cells located within the base of the crypts (first set). In the second set, about 27% of AL-labeled cells were co-reactive for SY and CG, likely representing entero-endocrine cells. In the third set, the largest subpopulation of AL-positive cells was not co-reactive for other markers applied (89%); most of them were likely Paneth cells. Large bowel: co-localization of AL with αD was not detected (first set). In the second set, AL was frequently co-localized with the other three markers applied (68%). In the third set, AL was frequently co-localized with 5-HT and CG (31%) as well as with PY and 5-HT (22%). Due to its presence in various enteroendocrine as well as Paneth cells, AL may be involved in different physiological and pathological processes.

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

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.

Bevacizumab Improves Achilles Tendon Repair in a Rat Model.

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.