Structural and microvascular changes of the peripapillary retinal nerve fiber layer in Von Hippel-Lindau disease: an OCT and OCT angiography study.

Von Hippel-Lindau (VHL) disease is an autosomal dominant genetic disease caused by VHL gene mutation. Retinal hemangioblastomas (RH) are vascularized tumors and represent the main ocular manifestation of the disease. Histopathologically, RH are composed of capillary vessels and stromal cells, the neoplastic population of the lesion. The origin of these stromal cells remains controversial, even if they are hypothesized to be glial cells. The aim of the present study was to investigate neuronal and microvascular changes of the peripapillary retinal nerve fiber layer, in which glial cells, neurons and capillaries (the radial peripapillary capillary plexus) interact. VHL patients with or without peripheral RH were enrolled and compared to healthy controls. Mean peripapillary retinal nerve fiber layer (pRNFL) thickness was measured by means of optical coherence tomography (OCT). The following vascular parameters of the radial peripapillary capillary plexus were quantified using OCT angiography: Vessel Area Density,Vessel Length Fraction, Vessel Diameter Index and Fractal Dimension. One hundred and nine eyes of 61 patients, and 56 eyes of 28 controls were consecutively studied. Mean pRNFL was significantly thinner in VHL eyes without RH versus eyes with RH and controls. Mean pRNFL thickness did not differ between VHL eyes with RH and controls. All OCTA vascular parameters were reduced in VHL eyes with or without RH versus controls, with significative difference for Vessel Diameter Index. The same OCTA parameters did not significantly differ between VHL eyes with or without RH. In VHL eyes without RH, pRNFL thinning may be the consequence of impaired perfusion of the radial peripapillary capillary plexus, while the increase of pRNFL thickness in VHL eyes with RH may depend on possible activation and proliferation of the other RNFL resident cells, the glial cells.

Microglia control vascular architecture via a TGFß1 dependent paracrine mechanism linked to tissue mechanics.

Tissue microarchitecture and mechanics are important in development and pathologies of the Central Nervous System (CNS); however, their coordinating mechanisms are unclear. Here, we report that during colonization of the retina, microglia contacts the deep layer of high stiffness, which coincides with microglial bipolarization, reduction in TGFß1 signaling and termination of vascular growth. Likewise, stiff substrates induce microglial bipolarization and diminish TGFß1 expression in hydrogels. Both microglial bipolarization in vivo and the responses to stiff substrates in vitro require intracellular adaptor Kindlin3 but not microglial integrins. Lack of Kindlin3 causes high microglial contractility, dysregulation of ERK signaling, excessive TGFß1 expression and abnormally-patterned vasculature with severe malformations in the area of photoreceptors. Both excessive TGFß1 signaling and vascular defects caused by Kindlin3-deficient microglia are rescued by either microglial depletion or microglial knockout of TGFß1 in vivo. This mechanism underlies an interplay between microglia, vascular patterning and tissue mechanics within the CNS.

Late neuroprogenitors contribute to normal retinal vascular development in a Hif2a-dependent manner.

In the adult central nervous system, endothelial and neuronal cells engage in tight cross-talk as key components of the so-called neurovascular unit. Impairment of this important relationship adversely affects tissue homeostasis, as observed in neurodegenerative conditions including Alzheimer's and Parkinson's disease. In development, the influence of neuroprogenitor cells on angiogenesis is poorly understood. Here, we show in mouse that these cells interact intimately with the growing retinal vascular network, and we identify a novel regulatory mechanism of vasculature development mediated by hypoxia-inducible factor 2a (Hif2a). By Cre-lox gene excision, we show that Hif2a in retinal neuroprogenitor cells upregulates the expression of the pro-angiogenic mediators vascular endothelial growth factor and erythropoietin, whereas it locally downregulates the angiogenesis inhibitor endostatin. Importantly, absence of Hif2a in retinal neuroprogenitor cells causes a marked reduction of proliferating endothelial cells at the angiogenic front. This results in delayed retinal vascular development, fewer major retinal vessels and reduced density of the peripheral deep retinal vascular plexus. Our findings demonstrate that retinal neuroprogenitor cells are a crucial component of the developing neurovascular unit.

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.

Intersublaminar vascular plexus: the correlation of retinal blood vessels with functional sublaminae of the inner plexiform layer.

PURPOSE: Interactions between vasculature and neurons provide important insight into the function of the nervous system, as well as into neurological diseases wherein these interactions are disrupted. This study characterizes a previously unreported retinal vascular plexus and examines potential sites of neurovascular interaction. METHODS: Vascular, neuronal, and glial elements were visualized using immunohistochemical markers. The distribution of vascular layers was measured and compared across eccentricities. Intensity profiles were calculated from confocal image reconstructions to reveal the proximity of vasculature to neuronal and glial processes. RESULTS: Retinal vasculature forms a plexus that coincides with the dendritic processes of OFF cholinergic amacrine cells within the inner plexiform layer. Across eccentricities, this plexus comprises approximately 8% of the total length of horizontally running blood vessels in the retina. Processes of Müller glia and OFF cholinergic amacrine cells colocalize with the blood vessels that form the intersublaminar plexus. CONCLUSIONS: In the retina, vasculature lacks autonomic control, but shows efficient local regulation. Although the source of this regulation is unclear, these results suggest that cholinergic amacrine cells and Müller glia may interact with the intersublaminar plexus to influence vasomotor activity. This may indicate a key role in modulating reciprocal interactions between neuronal activity and blood flow.

Nerve growth factor-mediated vascular endothelial growth factor expression of astrocyte in retinal vascular development.

The angiogenic aspect of neurotrophins and their receptors rather than the neuroscientific aspect has been focused. However, their role in retinal vascular development is underdiscovered. The purpose of this study is to understand the role of neurotrophin receptors in retinal vascular development and the mechanisms of their action. To identify the expression of tropomyosin receptor kinase receptor (Trk) in developing retina, tissues of 4, 8, 12, 16 and 26 day-old mice were prepared for experiments. Immunohistochemistry and immunofluorescence double staining against glial fibrillary acidic protein and type IV collagen were performed. TrkA was expressed mainly along the vessel structure in inner part of retina, especially in retinal astrocyte. In cultured primary astrocyte, recombinant nerve growth factor (NGF) was used to activate TrkA. NGF induced the phosphorylation of TrkA, and it also enhanced the level of activated Akt and vascular endothelial growth factor (VEGF) mRNA. Inhibition of phosphoinositide 3-kinase (PI3K) reversed the NGF-induced activation of these two molecules. This study demonstrated that TrkA activation on NGF leads to VEGF elevation by PI3K-Akt pathway and therefore suggested that TrkA could be a stimulator of retinal vascular development.

Eyeing central neurons in vascular growth and reparative angiogenesis.

The generation of blood vessels is a highly synchronized process requiring the coordinated efforts of several vascular and nonvascular cell populations as well as a stringent orchestration by the tissue being vascularized. Stereotyped angiogenesis is vital for both developmental growth and to restore tissue metabolic supply after ischemic events. Central neurons such as those found in the brain, spinal cord, and retina are vast consumers of oxygen and nutrients and therefore require high rates of perfusion by functional vascular networks to ensure proper sensory transmission. During a metabolic mismatch, such as that occurring during a cerebrovascular infarct or in ischemic retinopathies, there is increasing evidence that central neurons have an inherent ability to influence the vascular response to injury. With a focus on the retina and retinal ischemic disorders, this review explores the ever-growing evidence suggesting that central neurons have the propensity to impact tissue vascularization and reparative angiogenesis. Moreover, it addresses the paradoxical ability of severely ischemic neurons to hinder vascular regrowth and thus segregate the most severely injured zones of nervous tissue. The topics covered here are pertinent for future therapeutic strategies because promoting and steering vascular growth may be beneficial for ischemic disorders.

Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis.

Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5ß1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.

Age-dependent accumulation of lipofuscin in perivascular and subretinal microglia in experimental mice.

Fundus autofluorescence (AF) imaging by confocal scanning laser ophthalmoscopy has been widely used by ophthalmologists in the diagnosis/monitoring of various retinal disorders. It is believed that fundus AF is derived from lipofuscin in retinal pigment epithelial (RPE) cells; however, direct clinicopathological correlation has not been possible in humans. We examined fundus AF by confocal scanning laser ophthalmoscopy and confocal microscopy in normal C57BL/6 mice of different ages. Increasingly strong AF signals were observed with age in the neuroretina and subretinal/RPE layer by confocal scanning laser ophthalmoscopy. Unlike fundus AF detected in normal human subjects, mouse fundus AF appeared as discrete foci distributed throughout the retina. Most of the AF signals in the neuroretina were distributed around retinal vessels. Confocal microscopy of retinal and choroid/RPE flat mounts demonstrated that most of the AF signals were derived from Iba-1+ perivascular and subretinal microglia. An age-dependent accumulation of Iba-1+ microglia at the subretinal space was observed. Lipofuscin granules were detected in large numbers in subretinal microglia by electron microscopy. The number of AF+ microglia and the amount of AF granules/cell increased with age. AF granules/lipofuscin were also observed in RPE cells in mice older than 12 months, but the number of AF+ RPE cells was very low (1.48 mm(-2) and 5.02 mm(-2) for 12 and 24 months, respectively) compared to the number of AF+ microglial cells (20.63 mm(-2) and 76.36 mm(-2) for 6 and 24 months, respectively). The fluorescence emission fingerprints of AF granules in subretinal microglia were the same as those in RPE cells. Our observation suggests that perivascular and subretinal microglia are the main cells producing lipofuscin in normal aged mouse retina and are responsible for in vivo fundus AF. Microglia may play an important role in retinal aging and age-related retinal diseases.

Sympathetic innervation regulates basement membrane thickening and pericyte number in rat retina.

PURPOSE: To determine whether loss of sympathetic innervation alters basement membrane thickness and pericyte loss. METHODS: Sympathetic innervation to the eye was destroyed by surgical removal of the right superior cervical ganglion in rats. Basement membrane changes were assessed by real-time PCR and electron microscopy. The number of pericytes was measured by immunofluorescent staining for NG2 proteoglycan. Steady-state mRNA levels were also evaluated for platelet-derived growth factor-BB (PDGF-BB). RESULTS: Loss of sympathetic innervation caused a significant increase in steady state mRNA levels of fibronectin and a 15% increase in laminin-beta 1 mRNA 3 weeks after surgical sympathectomy. Protein expression also increased at this point. In addition, capillary basement membrane thickness increased significantly. NG2 proteoglycan staining decreased significantly in pericytes in the sympathectomized rat retina. Steady state mRNA for PDGF-BB decreased significantly 6 weeks after surgery. CONCLUSIONS: Sympathetic nerves may be compromised in diabetes, and these findings suggest that they may regulate some complications of diabetic retinopathy. Gene expression levels of fibronectin and laminin-beta 1 changed between 1 and 3 weeks. These data are supported by electron microscopy, which showed the increase in basement membrane thickness in vivo. Loss of sympathetic innervation to the eye also caused a decrease in the number of pericytes. Steady state mRNA expression of PDGF-BB was reduced, suggesting a mechanism for the loss of pericytes in the sympathectomized retina. Overall, these results suggest that sympathetic nerve alterations may function in some complications observed in diabetic retinopathy, and this may be a suitable model to investigate therapies for this disorder.

Cholinergic regulation of pericyte-containing retinal microvessels.

The aim of this study was to test the hypothesis that the neurotransmitter acetylcholine regulates the function of pericyte-containing retinal microvessels. A vasoactive role for acetylcholine is suggested by the presence of muscarinic receptors on pericytes, which are abluminally positioned contractile cells that may regulate capillary perfusion. However, little is known about the response of retinal microvessels to this neurotransmitter. Here we assessed the effects of cholinergic agonists on microvessels freshly isolated from the adult rat retina. Ionic currents were monitored via perforated patch pipettes; intracellular Ca(2+) levels were quantified with the use of fura 2, and microvascular contractions were visualized with the aid of time-lapse photography. We found that activation of muscarinic receptors elevated pericyte calcium levels, increased depolarizing Ca(2+)-activated chloride currents and caused pericytes to contract in a Ca(2+)-dependent manner. Most contracting pericytes were near capillary bifurcations. Contraction of a pericyte caused the adjacent capillary lumen to constrict. Thus acetylcholine may serve as a vasoactive signal by regulating pericyte contractility and thereby capillary perfusion in the retina.

[Effect of infrasound on ultrastructure and permeability of rat's blood-retinal barrier].

OBJECTIVE: To investigate the possible effect of infrasound on the ultra-structure and permeability of rat's blood-retinal barrier (BRB). METHODS: Ultra-structural changes of BRB were observed through the injection of lanthanum nitrate (La), which was used as a tracer to demonstrate the breakdown of the BRB, into blood vessels. Fifteen mature male rats divided into 5 groups were exposed to infrasound at a 8 Hz frequency, 130 dB sound pressure level in a pressure chamber especially designed for the experiment for 0, 1, 7, 14, 21 days, respectively. RESULTS: Under the action of infrasound, along with the prolongation of exposure, the damage of BRB was severer and severer. On the 1st day, there was no significant change in La leakage. On the 7th day, La diffused in the interphotoreceptor space at nuclear level. On the 14th day, La granules could be seen in the space of nervous cells. Finally, on the 21st day, La was found between synapses, synapses and nerve cells, as well as between the nerve cells and supporting cells, then sometimes reached vitreous body. Under the electron microscope, there were no significant morphological changes, but changes related to metabolism, such as edematous mitochondria, dilated rough endoplasmic reticula, precipitation of glycogen grandules, widening of perinuclear space, etc. CONCLUSIONS: The results thus suggest that the exposure to infrasound cause the breakdown of rat's blood-retinal barrier and visual impairment.

Effects of atropine and propranolol on retinal vessel diameters during isometric exercise.

PURPOSE: There is controversy regarding the nervous control of retinal blood vessels in humans. Most in vitro studies indicate that the intraocular part of the central retinal artery lacks autonomic innervation. We investigated the response of retinal vessels to isometric exercise during blockade of beta-receptors (propranolol) or muscarinic receptors (atropine). METHODS: Twelve healthy subjects performed squatting for 6 min during infusion of either propranolol atropine or placebo. Blood pressure and pulse rate were measured non-invasively. Retinal vessel diameters were measured continuously using the Zeiss Retinal Vessel Analyser. RESULTS: Squatting induced a significant increase in blood pressure and pulse rate, which was paralleled by a decrease in retinal vein and artery diameters. Atropine did not change the retinal vessel response to isometric exercise. Propranolol significantly blunted the exercise-induced vasoconstriction in retinal arteries. CONCLUSION: This result likely indicates propranolol-evoked vasoconstriction in the extraocular parts of the central retinal artery during isometric exercise.

Confocal microscopic study of glial-vascular relationships in the retinas of pigmented rats.

Astroglia are interposed between the cerebral vasculature and neurons, where they may mediate the transfer of substances from the circulation to neurons and couple changes in neuronal activity to changes in cerebral blood flow. The retina is a particularly advantageous model system for studying glial-vascular interactions in situ. Confocal microscopy and three-dimensional image reconstruction were used to study the anatomical relationships between glia and the surface vasculature in retinas acutely isolated from adult pigmented rats. Retinas were immunostained using antibodies directed against the basal lamina surrounding the vasculature as well as antibodies directed against glial fibrillary acidic protein. Surface vessels of all calibers were contacted by the processes of astrocytes. The vitreal surfaces of the large retinal vessels were covered by a meshwork of immunoreactive astrocyte processes of a variety of shapes, whereas the scleral surfaces of the vessels were supported by thick bundles of astrocyte processes. In addition, glial cells were filled intracellularly with the gap junction-permeable tracers Lucifer yellow and Neurobiotin. Intracellular fills clearly demonstrated the presence of astrocytes with somata that were closely apposed to the large retinal vessels. Tracer-filled astrocytes displayed a variety and complexity of shapes that was not apparent in immunostained material. Gap junctional coupling was stronger between astrocytes adjacent to the same artery than between periarterial astrocytes and astrocytes located away from arteries. Significantly fewer Müller cells were labeled when Neurobiotin was injected into astrocytes associated with arteries than when Neurobiotin was injected into astrocytes that were distant from arteries.

Horizontal cells invest retinal capillaries in the tree shrew Tupaia belangeri.

Retinal capillaries were studied by transmission electron microscopy, immunohistochemistry and lectin histochemistry in the adult tree shrew Tupaia betangeri. In capillaries from all four vascular layers, adjacent endothelial cells were connected by tight junctions. Up to three layers of pericyte processes were embedded in the subendothelial basal lamina. However, pericytes frequently contacted the endothelial cells. In the innermost vascular layer (capillary layer 4), S-100-immunopositive astrocytes and the vitreal processes of S-100-immunopositive Muller cells entirely ensheathed the capillary basal lamina. However, capillaries revealing an incomplete macroglial investment were observed in the outer vascular layers, predominantly in capillary layers 1 and 2. In sections of capillaries located between the inner nuclear layer and the outer plexiform layer (capillary layer 1), these "gaps" were filled with the perikarya and electron-lucent processes of horizontal cells that ensheathed up to approximately nine tenths of the capillary circumference. Horizontal cells were identified by ultrastructural criteria. They were distinct from microglial cells by not being reactive for Griffonia simplicifolia isolectin-B4. In Tutpaia, vessel-contacting horizontal cells reside in a position reported to be occupied by the processes of Müller cells in other mammals. Current concepts of retinal function and pathology, which are based on the assumption that retinal vessels are strictly isolated from retinal neurons, at least in Tupaia, might deserve reconsideration.

[Structure of the interrelations between retinal neuroglia and ganglion cells]. / Struktura vzaimootnoshenii neiroglii i ganglioznykh kletok setchatki.

Organization of glial environment of bodies and axons of cat retinal ganglion cells (GC) was analysed electron microscopically. Similarities and differences in types of contacts between macroglial cells and GC plasmalemma throughout its length were demonstrated. Regularities of changes of types of glia surrounding different types of GC were established. GC body was surrounded by the processes of Muller gliocytes, while a special glial crown appearing as multiple radial finger-shaped astrocyte processes covers GC initial segment. Possible functional aspects of structural organization of interrelations of GC body and axons with gliocytes are discussed.

Immunohistochemical localization of dopamine-beta-hydroxylase in human and monkey eyes.

PURPOSE: To investigate the pattern of dopamine-beta-hydroxylase (DBH)-containing fibers in human and monkey eyes. METHODS: DBH-containing fibers were detected by immunohistochemistry. The primary antibody used recognized DBH, the key enzyme in the conversion of dopamine to noradrenaline. RESULTS: In the anterior segment, DBH immunoreaction product was found in the peripheral corneal endothelium layer, in both the dilator and sphincter muscles of the iris, as well as in the anterior border layer of the iris. The ciliary muscle and the stroma of the ciliary processes were also zones of concentration. In the posterior segment, staining was seen around blood vessels in the choroid, in the vascular walls of the short posterior ciliary arteries and in the ciliary nerves. The retina was also immunopositive, with specific labeling in cones and rods of photoreceptors, inner and outer plexiform layers and ganglion cell layer. There was no significant difference in the distribution of DBH-related immunoreactivity in human and monkey eyes. CONCLUSIONS: The localization of DBH-related immunoreactivity is generally consistent with the known physiological roles of noradrenaline. However, an apparently high concentration of the enzyme in the anterior border layer of the iris and in retinal photoreceptors raises questions about the possible role of DBH-containing fibers in these structures.

Thyrotropin-like immunoreactivity in human retina: immunoreactive co-localization in ganglion cells and perivascular fibers.

The present immunocytochemical study has demonstrated immunoreactive thyrotropin-like ganglion cell populations as well as perivascular fibers in the human retina by using specific antiserum. Thyrotropin is a pituitary glycopeptide involved in the synthesis and release of thyroid hormones. The existence and functions of peptides in vertebrate retinas are still not well known. Many authors have reported neuropeptide immunoreactivity in the human retina which have had their functions established in the neuroregulatory processes of vision. Moreover, some authors have reported the possibility that the fiber terminal of peptidergic neurons may also be a blood vessel. The appearance of immunoreactive-cells in human retina, e.g. existence of retinal ganglion cells with thyrotropin-like immunoreactivity, indicates the existence of specific mechanisms that would be mediated by these peptides which are located near immunoreactive ganglion cells. We hypothesize that there is an intrinsic mechanism for blood flow control, mediated by retinal ganglion cells which may regulate vessel diameter according to its luminous stimuli. No-one has demonstrated the presence or the functional existence of thyrotropin-like immunoreactive structures in the vertebrate retina, or on the side of the pituitary-thyroid axis. To the best of our knowledge this is the first time that thyrotropin has been immunocytochemically demonstrated in the human retina. Thus, we suggest that thyrotropin acts as a neuromodulator in the human retina, which is implicated in blood flow control.