Christian Runge

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.

The effect of vasopressin on choroidal blood flow, intraocular pressure, and orbital venous pressure in rabbits.

PURPOSE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Rat choroidal pericytes as a target of the autonomic nervous system

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

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

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

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.

The Effect of Vasopressin on Ciliary Blood Flow and Aqueous Flow

PURPOSE 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. METHODS 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). RESULTS 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. CONCLUSIONS 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.

Characterization of dsRed2-positive cells in the doublecortin-dsRed2 transgenic adult rat retina

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. Müller 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.

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

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