Daniela Bruckner

Brain and Retinal Pericytes: Origin, Function and Role

Pericytes are specialized mural cells located at the abluminal surface of capillary blood vessels, embedded within the basement membrane. In the vascular network these multifunctional cells fulfil diverse functions, which are indispensable for proper homoeostasis. They serve as microvascular stabilizers, are potential regulators of microvascular blood flow and have a central role in angiogenesis, as they for example regulate endothelial cell proliferation. Furthermore, pericytes, as part of the neurovascular unit, are a major component of the blood-retina/brain barrier. CNS pericytes are a heterogenic cell population derived from mesodermal and neuro-ectodermal germ layers acting as modulators of stromal and niche environmental properties. In addition, they display multipotent differentiation potential making them an intriguing target for regenerative therapies. Pericyte-deficiencies can be cause or consequence of many kinds of diseases. In diabetes, for instance, pericyte-loss is a severe pathological process in diabetic retinopathy (DR) with detrimental consequences for eye sight in millions of patients. In this review, we provide an overview of our current understanding of CNS pericyte origin and function, with a special focus on the retina in the healthy and diseased. Finally, we highlight the role of pericytes in de- and regenerative processes.

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

Abstract 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. HighlightsPDPN expression was detected in GFAP positive astrocytes in the rat ON.LYVE‐1 is expressed in cells with microglial/macrophagic activity.Absence of lymphatic structures within the rat ON under physiological conditions.No formation of lymphatic structures within the rat ON following ONC trauma.

Alarin in cranial autonomic ganglia of human and rat

Extrinsic and intrinsic sources of the autonomic nervous system contribute to choroidal innervation, thus being responsible for the control of choroidal blood flow, aqueous humor production or intraocular pressure. Neuropeptides are involved in this autonomic control, and amongst those, alarin has been recently introduced. While alarin is present in intrinsic choroidal neurons, it is not clear if these are the only source of neuronal alarin in the choroid. Therefore, we here screened for the presence of alarin in human cranial autonomic ganglia, and also in rat, a species lacking intrinsic choroidal innervation. Cranial autonomic ganglia (i.e., ciliary, CIL; pterygopalatine, PPG; superior cervical, SCG; trigeminal ganglion, TRI) of human and rat were prepared for immunohistochemistry against murine and human alarin, respectively. Additionally, double staining experiments for alarin and choline acetyltransferase (ChAT), tyrosine hydroxilase (TH), substance P (SP) were performed in human and rat ganglia for unequivocal identification of ganglia. For documentation, confocal laser scanning microscopy was used, while quantitative RT-PCR was applied to confirm immunohistochemical data and to detect alarin mRNA expression. In humans, alarin-like immunoreactivity (alarin-LI) was detected in intrinsic neurons and nerve fibers of the choroidal stroma, but was lacking in CIL, PPG, SCG and TRI. In rat, alarin-LI was detected in only a minority of cranial autonomic ganglia (CIL: 3.5%; PPG: 0.4%; SCG: 1.9%; TRI: 1%). qRT-PCR confirmed the low expression level of alarin mRNA in rat ganglia. Since alarin-LI was absent in human cranial autonomic ganglia, and only present in few neurons of rat cranial autonomic ganglia, we consider it of low impact in extrinsic ocular innervation in those species. Nevertheless, it seems important for intrinsic choroidal innervation in humans, where it could serve as intrinsic choroidal marker.

Lymphatic markers in the human optic nerve

&NA; 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. HighlightsAbsence of lymphatic markers in human optic nerve (ON).Absence of lymphatic markers in human dura mater.Presence of LYVE‐1 positive macrophages in the ON and surrounding tissue.

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

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

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

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

Aquaporin expression and localization in the rabbit eye

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.