Alexandra Kaser-Eichberger

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.

Neural Crest Origin of Retinal and Choroidal Pericytes

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.

A New Nanosecond UV Laser at 355 nm: Early Results of Corneal Flap Cutting in a Rabbit Model

PURPOSE A new 355 nm UV laser was used for corneal flap cutting in an animal model and tested for clinical and morphologic alterations. METHODS Corneal flaps were created (Chinchilla Bastards; n = 25) with an UV nanosecond laser at 355 nm (150 kHz, pulse duration 850 ps, spot-size 1 μm, spot spacing 6 × 6 μm, side cut Δz 1 μm; cutting depth 130 μm) and pulse energies of 2.2 or 2.5 μJ, respectively. Following slit-lamp examination, animals were killed at 6, 12, and 24 hours after treatment. Corneas were prepared for histology (hematoxylin and eosin [HE], TUNEL-assay) and evaluated statistically, followed by ultrastructural investigations. RESULTS Laser treatment was tolerated well, flap lift was easier at 2.5 μJ compared with 2.2 μJ. Standard HE at 24 hours revealed intact epithelium in the horizontal cut, with similar increase in corneal thickness at both energies. Irrespective of energy levels, TUNEL assay revealed comparable numbers of apoptotic cells in the horizontal and vertical cut at 6, 12, and 24 hours, becoming detectable in the horizontal cut as an acellular stromal band at 24 hours. Ultrastructural analysis revealed regular morphology in the epi- and endothelium, while in the stroma, disorganized collagen lamellae were detectable representing the horizontal cut, again irrespective of energy levels applied. CONCLUSIONS This new UV laser revealed no epi- nor endothelial damage at energies feasible for corneal flap cutting. Observed corneal swelling was lower compared with existing UV laser studies, albeit total energy applied here was much higher. Observed loss of stromal keratinocytes is comparable with available laser systems. Therefore, this new laser is suitable for refractive surgery, awaiting its test in a chronic environment.

Rat choroidal pericytes as a target of the autonomic nervous system

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

Sufficient Evidence for Lymphatics in the Developing and Adult Human Choroid

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 Schlemms 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.

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

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

Expression of Lymphatic Markers in the Adult Rat Spinal Cord

Under physiological conditions, lymphatic vessels are thought to be absent from the central nervous system (CNS), although they are widely distributed within the rest of the body. Recent work in the eye, i.e., another organ regarded as alymphatic, revealed numerous cells expressing lymphatic markers. As the latter can be involved in the response to pathological conditions, we addressed the presence of cells expressing lymphatic markers within the spinal cord by immunohistochemistry. Spinal cord of young adult Fisher rats was scrutinized for the co-expression of the lymphatic markers PROX1 and LYVE-1 with the cell type markers Iba1, CD68, PGP9.5, OLIG2. Rat skin served as positive control for the lymphatic markers. PROX1-immunoreactivity was detected in many nuclei throughout the spinal cord white and gray matter. These nuclei showed no association with LYVE-1. Expression of LYVE-1 could only be detected in cells at the spinal cord surface and in cells closely associated with blood vessels. These cells were found to co-express Iba1, a macrophage and microglia marker. Further, double labeling experiments using CD68, another marker found in microglia and macrophages, also displayed co-localization in the Iba1+ cells located at the spinal cord surface and those apposed to blood vessels. On the other hand, PROX1-expressing cells found in the parenchyma were lacking Iba1 or PGP9.5, but a significant fraction of those cells showed co-expression of the oligodendrocyte lineage marker OLIG2. Intriguingly, following spinal cord injury, LYVE-1-expressing cells assembled and reorganized into putative pre-vessel structures. As expected, the rat skin used as positive controls revealed classical lymphatic vessels, displaying PROX1+ nuclei surrounded by LYVE-1-immunoreactivity. Classical lymphatics were not detected in adult rat spinal cord. Nevertheless, numerous cells expressing either LYVE-1 or PROX1 were identified. Based on their localization and overlapping expression with Iba1, the LYVE-1+ cell population likely represents a macrophage subpopulation, while a significant fraction of PROX1+ cells belong to the oligodendrocytic lineage based on their distribution and the expression of OLIG2. The response of these LYVE-1+ and PROX1+ cell subpopulations to pathological conditions, especially in spinal cord inflammatory conditions, needs to be further elucidated.

Aqueous humor ferritin in hereditary hyperferritinemia cataract syndrome.

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 &mgr;g/L, whereas in the HHCS patient, this parameter was excessively increased (1086 &mgr;g/L). Analysis of ferritin in aqueous humor revealed 6.4 ± 3.8 &mgr;g/L in normal control subjects and 146.3 &mgr;g/L (OD) and 160.4 &mgr;g/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.

Immunohistochemical Detection of CTGF in the Human Eye

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

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.