Laminar restriction of retinal macrophagic response to optic nerve axotomy in the rat

Following optic nerve transection, most of the retinal ganglion cells die. Their debris is promptly cleared by phagocytic cells. It is currently not known to what extent peripherally derived macrophages contribute to this activity. Using antibodies OX42 and ED‐1, phagocytic cells were labeled in the retinas of optic nerve lesioned adult rats. To distinguish whether the cells were reactive microglial or macrophagic in origin, blood‐borne monocytes were labeled with fluorescent microspheres while in the systemic circulation. Macrophages invaded the retina, but only in the nerve fiber layer, sparing the ganglion cell and other layers. These macrophages engulfed only the axonal debris from dying ganglion cells, not their degenerating cell bodies. These results indicate that although peripherally derived monocytic cells are recruited into the retrogradely degenerating retina, their role in clearing debris is limited to the optic fiber layer. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 55–66, 1999     

Optic Nerve Head Blood Flow Autoregulation during Changes in Arterial Blood Pressure in Healthy Young Subjects

Aim

In the present study the response of optic nerve head blood flow to an increase in ocular perfusion pressure during isometric exercise was studied. Based on our previous studies we hypothesized that subjects with an abnormal blood flow response, defined as a decrease in blood flow of more than 10% during or after isometric exercise, could be identified.

Methods

A total of 40 healthy subjects were included in this study. Three periods of isometric exercise were scheduled, each consisting of 2 minutes of handgripping. Optic nerve head blood flow was measured continuously before, during and after handgripping using laser Doppler flowmetry. Blood pressure was measured non-invasively in one-minute intervals. Intraocular pressure was measured at the beginning and the end of the measurements and ocular perfusion pressure was calculated as 2/3*mean arterial pressure –intraocular pressure.

Results

Isometric exercise was associated with an increase in ocular perfusion pressure during all handgripping periods (p < 0.001). By contrast no change in optic nerve head blood flow was seen. However, in a subgroup of three subjects blood flow showed a consistent decrease of more than 10% during isometric exercise although their blood pressure values increased. In addition, three other subjects showed a consistent decline of blood flow of more than 10% during the recovery periods.

Conclusion

Our data confirm previous results indicating that optic nerve head blood flow is autoregulated during an increase in perfusion pressure. In addition, we observed a subgroup of 6 subjects (15%) that showed an abnormal response, which is in keeping with our previous data. The mechanisms underlying this abnormal response remain to be shown.     

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (<10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R²>0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.     

Lipid Composition of the Human Eye: Are Red Blood Cells a Good Mirror of Retinal and Optic Nerve Fatty Acids?

Background

The assessment of blood lipids is very frequent in clinical research as it is assumed to reflect the lipid composition of peripheral tissues. Even well accepted such relationships have never been clearly established. This is particularly true in ophthalmology where the use of blood lipids has become very common following recent data linking lipid intake to ocular health and disease. In the present study, we wanted to determine in humans whether a lipidomic approach based on red blood cells could reveal associations between circulating and tissue lipid profiles. To check if the analytical sensitivity may be of importance in such analyses, we have used a double approach for lipidomics.

Methodology and Principal Findings

Red blood cells, retinas and optic nerves were collected from 9 human donors. The lipidomic analyses on tissues consisted in gas chromatography and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS). Gas chromatography did not reveal any relevant association between circulating and ocular fatty acids except for arachidonic acid whose circulating amounts were positively associated with its levels in the retina and in the optic nerve. In contrast, several significant associations emerged from LC-ESI-MS analyses. Particularly, lipid entities in red blood cells were positively or negatively associated with representative pools of retinal docosahexaenoic acid (DHA), retinal very-long chain polyunsaturated fatty acids (VLC-PUFA) or optic nerve plasmalogens.

Conclusions and Significance

LC-ESI-MS is more appropriate than gas chromatography for lipidomics on red blood cells, and further extrapolation to ocular lipids. The several individual lipid species we have identified are good candidates to represent circulating biomarkers of ocular lipids. However, further investigation is needed before considering them as indexes of disease risk and before using them in clinical studies on optic nerve neuropathies or retinal diseases displaying photoreceptors degeneration.     

Mesenchymal stem cell therapy in retinal and optic nerve diseases: An update of clinical trials

Retinal and optic nerve diseases are degenerative ocular pathologies which lead to irreversible visual loss. Since the advanced therapies availability, cell-based therapies offer a new all-encompassing approach. Advances in the knowledge of neuroprotection, immunomodulation and regenerative properties of mesenchymal stem cells(MSCs) have been obtained by several preclinical studies of various neurodegenerative diseases. It has provided the opportunity to perform the translation of this knowledge to prospective treatment approaches for clinical practice. Since 2008, several first steps projecting new treatment approaches, have been taken regarding the use of cell therapy in patients with neurodegenerative pathologies of optic nerve and retina. Most of the clinical trials using MSCs are in Ⅰ/Ⅱ phase, recruiting patients or ongoing, and they have as main objective the safety assessment of MSCs using various routes of administration. However, it is important to recognize that, there is still a long way to go to reach clinical trials phase Ⅲ-Ⅳ. Hence, it is necessary to continue preclinical and clinical studies to improve this new therapeutic tool. This paper reviews the latest progress of MSCs in human clinical trials for retinal and optic nerve diseases.     

Hydrogen clearance: assessment of technique for measurement of skin-flap blood flow in pigs

The hydrogen clearance technique has been used for many years by investigators to determine brain blood flow and has been partially validated in this setting using other methods of blood flow measurement. The method has been modified to allow blood flow measurements in skin, but the accuracy of H2 clearance for measuring skin blood flow has not been determined. Multiple blood flow measurements were performed using H2 clearance and radioactive microspheres on skin flaps and control skin in pigs. On 12 pigs, a total of 117 flap and 42 control skin measurements were available for analysis. There was no significant difference between the two techniques in measuring mean control skin blood flow. In skin flaps, H2 clearance was significantly correlated to microsphere-measured blood flow, but it consistently gave an overestimate. Sources of error may include injury to the tissues by insertion of electrodes, consumption of H2 by the electrodes, or diffusion of H2 from the relatively ischemic flap to its well-vascularized bed. Further studies are necessary to determine the cause of this error and to measure the technique's accuracy in skeletal muscle and other flaps.     

Astrocytes as gate-keepers in optic nerve regeneration--a mini-review

Animals that develop without extra-embryonic membranes (anamniotes--fish, amphibians) have impressive regenerative capacity, even to the extent of replacing entire limbs. In contrast, animals that develop within extra-embryonic membranes (amniotes--reptiles, birds, mammals) have limited capacity for regeneration as adults, particularly in the central nervous system (CNS). Much is known about the process of nerve development in fish and mammals and about regeneration after lesions in the CNS in fish and mammals. Because the retina of the eye and optic nerve are functionally part of the brain and are accessible in fish, frogs, and mice, optic nerve lesion and regeneration (ONR) has been extensively used as a model system for study of CNS nerve regeneration. When the optic nerve of a mouse is severed, the axons leading into the brain degenerate. Initially, the cut end of the axons on the proximal, eye-side of the injury sprout neurites which begin to grow into the lesion. Simultaneously, astrocytes of the optic nerve become activated to initiate wound repair as a first step in reestablishing the structural integrity of the optic nerve. This activation appears to initiate a cascade of molecular signals resulting in apoptotic cell death of the retinal ganglion cells axons of which make up the neural component of the optic nerve; regeneration fails and the injury is permanent. Evidence specifically implicating astrocytes comes from studies showing selective poisoning of astrocytes at the optic nerve lesion, along with activation of a gene whose product blocks apoptosis in retinal ganglion cells, creates conditions favorable to neurites sprouting from the cut proximal stump, growing through the lesion and into the distal portion of the injured nerve, eventually reaching appropriate targets in the brain. In anamniotes, astrocytes ostensibly present no such obstacle since optic nerve regeneration occurs without intervention; however, no systematic study of glial involvement has been done. In fish, vigorously growing neurites sprout from the cut axons and within a few days begin to re-enervate the brain. This review offers a new perspective on the role of glia, particularly astrocytes, as "gate-keepers;" i.e., as being permissive or inhibitory, by comparison between fish and mammals of glial function during ONR.     

Origin of Oligodendrocytes in the Vertebrate Optic Nerve: A Review

One of the unsolved problems in the research field of oligodendrocyte (OL) development has been the site(s) of origin of optic nerve OLs and its precursor cells (OPCs). It is generally accepted that OLs in the optic nerve are derived from the brain, and thus optic nerve OLs are immigrant cells. We previously demonstrated the brain origin of optic nerve OPCs in chick embryos. However, the site of optic nerve OPC origin has not been examined experimentally in developing rodents for the past two decades. We have recently reported that optic nerve OPCs in mice arise in the preoptic area by E12.5 and gradually migrate caudally and enter the optic nerve. These OPCs give rise to myelinating OLs in the optic nerve in the postnatal or adult stages. Surprisingly, there are species differences with respect to the origin of optic nerve OPCs between chicks and mice. Here, we summarize the site of OPC origin in the optic nerve based on our own previous and recent results, and discuss possible mechanisms underlying these species differences.     

Hemodynamic effects of 15-microm-diameter microspheres on the rat pulmonary circulation.

The microsphere method has been used extensively to measure regional blood flow in large laboratory animals. A fundamental premise of the method is that microspheres do not alter regional flow or vascular tone. Whereas this assumption is accepted in large animals, it may not be valid in the pulmonary circulation of smaller animals. Three studies were performed to determine the hemodynamic effects of microspheres on the rat pulmonary circulation. Increasing numbers of 15-microm-diameter microspheres were injected into a fully dilated, isolated-lung preparation. Vascular resistance increased 0.8% for every 100,000 microspheres injected. Microspheres were also injected into an isolated-lung preparation in which vascular tone was increased with hypoxia. Microspheres did not induce vasodilatation, as reported in other vascular beds. Fluorescent microspheres were injected via tail veins into awake rats, and the spatial locations of the microspheres were determined. Regional distributions remained highly correlated when microspheres of one color were injected after microspheres of another color. This indicates that the initial injection did not alter regional perfusion. We conclude that, when used in appropriate numbers, 15-microm-diameter microspheres do not alter regional flow or vascular tone in the rat pulmonary circulation.     

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.     

Intravital microscopic observations of 15-microm microspheres lodging in the pulmonary microcirculation.

Vascular infusions of 15-microm-diameter microspheres are used to study pulmonary blood flow distribution. The sites of microsphere lodging and their effects on microvascular perfusion are debated but unknown. Using intravital microscopy of the subpleural surface of rat lungs, we directly observed deposition of fluorescent microspheres. In a pump-perfused lung model, approximately 0.5 million microspheres were infused over 30 s into the pulmonary artery of seven rats. Microsphere lodging was analyzed for the location in the microvasculature and the effect on local flow after lodging. On average, we observed 3.2 microspheres per 160 alveolar facets. The microspheres always entered the arterioles as singlets and lodged at the inlets to capillaries, either in alveolar corner vessels or small arterioles. In all cases, blood flow continued either around the microspheres or into the capillaries via adjacent pathways. We conclude that 15-microm-diameter microspheres, in doses in excess of those used in typical studies, have no significant impact on pulmonary capillary blood flow distribution.     

Laminar restriction of retinal macrophagic response to optic nerve axotomy in the rat.

Following optic nerve transection, most of the retinal ganglion cells die. Their debris is promptly cleared by phagocytic cells. It is currently not known to what extent peripherally derived macrophages contribute to this activity. Using antibodies OX42 and ED-1, phagocytic cells were labeled in the retinas of optic nerve lesioned adult rats. To distinguish whether the cells were reactive microglial or macrophagic in origin, blood-borne monocytes were labeled with fluorescent microspheres while in the systemic circulation. Macrophages invaded the retina, but only in the nerve fiber layer, sparing the ganglion cell and other layers. These macrophages engulfed only the axonal debris from dying ganglion cells, not their degenerating cell bodies. These results indicate that although peripherally derived monocytic cells are recruited into the retrogradely degenerating retina, their role in clearing debris is limited to the optic fiber layer.     

Cloning of Multiple Forms of Goldfish Vimentin: Differential Expression in CNS

Abstract: In efforts to determine the primary structure of intermediate filament proteins in the goldfish visual pathway, we isolated clones from a retinal λgt11 cDNA expression library that represent goldfish vimentin. We show that there are at least two forms of goldfish vimentin, designated as vimentin α and vimentin β. RNase protection assays indicate that vimentin α mRNA is expressed in low amounts in retina, optic nerve, and brain and in higher amounts in spinal cord. In contrast, vimentin β mRNA is expressed in low amounts in retina, optic nerve, brain, and spinal cord and in very high amounts in eye lens. Immunohistochemical studies show that in the optic nerve, vimentin α is mainly restricted to blood vessels, meninges, and septa. Light staining is observed with this antibody in an astrocytic glial pattern throughout the optic nerve. Two-dimensional gel analysis shows that all of these goldfish vimentins are low abundant components of optic nerve cytoskeletal preparations.     

Myocilin expression in the astrocytes of the optic nerve head

We investigated the expression of myocilin in the optic nerve head of porcine eyes by Western blotting and immunohistochemical staining. Myocilin was localized in the nucleus, centrosome, glial filament, mitochondria, and some parts of the cell membranes of the astrocytes. Myocilin was also detected at the edge-feet portion of the processes of astrocytes adjacent to the inner limiting membrane and blood vessel wall. The astrocytes are the major cell population in the optic nerve head, contributing to the architecture of the nerve axon and blood vessels. Therefore, myocilin gene mutation and change of myocilin protein are likely to affect the architecture of the optic nerve head and induce various forms of glaucomatous optic nerve damage.     

Transport of multiple nicotinic acetylcholine receptors in the rat optic nerve: high densities of receptors containing alpha6 and beta3 subunits

Neuronal nicotinic acetylcholine receptors (nAChRs) are abundant in the rat retina and at least seven heteromeric subtypes have been detected. Axons of retinal ganglion cells form the optic nerve and innervate areas of the brain important for visual processing, including the lateral geniculate nucleus, the superior colliculus, and the pretectal nucleus. Development of eye-specific layers in these projection areas are dependent upon retinal waves which are initially mediated by nAChRs [ Feller et al. , Science 272 (1996), 1182 ; Penn et al. , Science 279 (1998), 2108 ; Bansal et al. , J. Neurosci. 20 (2000), 7672 ]. Unilateral eye-enucleation studies in the rat indicate that nAChRs are on the terminals of optic nerve axons, where they may mediate influences of acetylcholine on visual pathways. In this study, we use radioligand binding and immunoprecipitation with subunit-selective antibodies to investigate the subunit composition of nAChRs in the rat optic nerve. We found multiple nAChR subtypes in the optic nerve, all of which contain the β2 subunit. Most of these receptors are mixed heteromeric subtypes, composed of at least three different subunits. Included among these subtypes is the highest percentage and density of α6- and β3-containing nAChRs of any area of the rat CNS that has been reported.     

Postinjury Changes in Platelet-Derived Growth Factor-Like Activity in Fish and Rat Optic Nerves

The poor regenerative ability of the CNS of mammals has been attributed, at least in part, to the presence of mature oligodendrocytes, which have been shown to inhibit axonal growth. Proliferation of oligodendrocyte progenitor cells in the rat optic nerve during development, and thereby the timing of oligodendrocyte differentiation, has been shown to depend on a factor derived from type 1 astrocytes, later characterized as platelet-derived growth factor (PDGF). In the present study we examine whether injury to the optic nerve induces changes in the levels of PDGF in spontaneously regenerating systems, compared with nonregenerating systems. Soluble substances, derived from nonneuronal cells surrounding injured fish and rat optic nerves, were prepared and examined for the presence of PDGF immunoreactivity and biological mitogenic activity on PDGF-responsive cells. The results suggest that PDGF-like mitogenic activity and immunoreactivity are present in both fish and rat optic nerves. However, in the rat optic nerve PDGF levels increased after axonal injury, whereas in the fish optic nerve injury was accompanied by an apparent decrease in PDGF-like levels. The results are discussed with respect to the possible role of PDGF in regeneration.     

光流信息加工的神经基础

自体运动时,分布在视野内的景物在视网膜上的像旋转,扩张／收缩,浃动,构成光流刺激,光流信息的检测对于人或动物确定前进的方向,速度至关重要,已成为运动信息加工的一个研究热点。本文概括介绍了近年来心理物理和生理学两方面有关光流信息加工研究的主要进展,并讨论了光流信息分析的神经机制。     

Role of polyamines in regulation of uterine blood flow of ovariectomized rats.

Polyamines have been shown to relax several types of smooth muscle including vasculature. In the estrogen-treated uterus, uterine blood flow and polyamine levels in the uterus are increased. The relaxant effect of polyamines on blood vessels suggest that polyamines may act on uterine vasculature to cause uterine hyperemia. In this study, we examined the roles of polyamines in regulating uterine blood flow. Ovariectomized rats were administered with polyamines or estrogen. The uterine blood flow was then measured by employing radioactive microspheres. The direct injection of polyamines into systemic circulation caused a 3-fold increase in uterine blood flow within 30 seconds. The polyamine-induced uterine hyperemia was counteracted by increasing extracellular calcium concentration. When rats were treated with estradiol, uterine blood flow increased. However, the treatment with alpha-difluromethylornithine, a polyamine synthesis inhibitor, did not attenuate the estrogen-induced uterine blood flow. Our results suggest that polyamines may affect uterine blood flow via antagonizing the entry of extracellular calcium. However, the detailed mechanisms via which polyamines involved in estrogenic stimulation of uterine hyperemia may require further studies.     

Quantitative characteristics of degeneration of the fibers of the optic nerve after section

The optic nerve of the dog has been histologically studied. In the intact nerve 160,000 axons have been revealed. In 3 weeks after the nerve section in the orbit and near the nerve disk 72% retinoencephal fibers, 28% encephaloretinal and 0.23% of centrofugal axons are found.