Neurotrophins and their receptors in the primary olfactory neuraxis

The primary olfactory pathway is an elegant and simple system in which to study neurogenesis and neuronal plasticity because of the simple fact that olfactory receptor neurons (ORNs) are continually generated throughout the adult lifetimes of vertebrates. Thus, neuronal birth, differentiation, survival, axon pathfinding, target recognition, synapse formation, and cell death are developmental events that can be examined in the mature olfactory epithelium (OE). Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3, and 4/5) are a family of bioactive peptides that exert their effects by interacting with high- and low-affinity receptors on the surfaces of responsive cells, and have been implicated in several stages of neuronal development throughout the central and peripheral nervous system (CNS and PNS). There has been significant interest within the olfactory community as to how these multifunctional peptides might regulate the cycle of degeneration and regeneration of olfactory receptor neurons. The focus of this review is to highlight what is known about the actions of neurotrophins in the primary olfactory pathway, and to pinpoint future directions that will enable us to further understand their role in olfactory receptor neuron development and turnover.     

Regulatory cell interactions between retinal ganglion cells and radial glia during axonal and dendritic outgrowth

Neuronal differentiation and the formation of cell polarity are crucial events during the development of the nervous system. Cell polarity is a prerequisite for directed information flux within neuronal networks. In this article, we focus on neuro-glial cell interactions that influence the establishment of neural cell polarity and the directed outgrowth of axons versus dendrites. The cellular model discussed in detail is the retinal ganglion cell (RGC) of the chick retina, which is investigated by a comprehensive set of in vitro assays. The experiments demonstrate that retinal microenvironment determines axon vs. dendrite formation of RGCs. The instructive differences in different retinal microenvironments are substantially influenced by radial glia. Different glial domains support or inhibit axon vs. dendrite outgrowth. The data support the notion that neuro-glial interactions are crucial for directed neurite outgrowth.     

Neuronal morphology, axonal integrity, and axonal regeneration in situ are regulated by cytoskeletal phosphorylation in identified lamprey central neurons

The CNS of the sea lamprey (Petromyzon marinus) contains giant, individually identifiable neurons that can be microinjected intracellularly in the living animal. We have used the unique accessibility of this system to investigate the role played by serine/threonine kinases and phosphatases in regulating cytoskeletal stability in identified reticulospinal neurons (ABCs) in situ. Injection of broad spectrum kinase and phosphatase inhibitors induce marked changes in ABC gross morphology and in the extent and morphology of sprouts induced by axotomy. The kinase inhibitor K-252a causes regenerating sprouts to be longer and narrower than those seen in control preparations, and significantly reduces the diameters of axon stumps; this latter effect is similar to the effect of microinjecting anti neurofilament (NF) antibodies. By contrast, the phosphatase inhibitor okadaic acid (OA) causes the selective disruption of axonal integrity, blocking axonal regeneration and causing axon stump retraction in axotomized ABCs. The microtubule (MT) disrupting drug colchicine has an effect similar but less marked than OA on ABC axonal morphology. Both OA and colchicine also induce the formation of large somatodendritic swellings in axotomized (but not intact) ABCs by 1-3 weeks post-injection. Immunocytochemical analyses indicate that both colchicine and OA treatments result in the destabilization of MTs and the phosphorylation of NFs, while OA induces the accumulation of phosphorylated tau protein in some dendritic swellings. Control injections of inactive substances have none of these effects. These results suggest that OA does not have its primary effect on NF assembly at the doses used, but may block axonal regeneration by inducing a prolonged disruption of axonal MTs, possibly via an indirect mechanism involving the hyperphosphorylation of tau and other MAPs. K-252a, on the other hand, may interfere with NF assembly and sidearm phosphorylation, thereby reducing NF transport into both axon stumps and sprouts and in turn reducing sprout diameter. The implications of these results for the respective roles of MTs, MAPs, and NFs in axonal regeneration in the vertebrate CNS are discussed.     

Microglia in neuroregeneration

Microglia has the potential to produce and release a range of factors that directly and/or indirectly promote regeneration in the injured nervous system. The overwhelming evidence indicates, however, that this potential is generally not expressed in vivo. Activated microglia may enhance neuronal degeneration following axotomy, thereby counteracting functional recovery. Microglia does not seem to contribute significantly to axonal outgrowth after peripheral nerve injury, since this process proceeds uneventful even if perineuronal microglia is eliminated. The phagocytic phenotype of microglia is highly suppressed during Wallerian degeneration in the central nervous system. Therefore, microglia is incapable of rapid and efficient removal of myelin debris and its putative growth inhibitory components. In this way, microglia may contribute to regeneration failure in the central nervous system. Structural and temporal correlations are compatible with participation by perineuronal microglia in axotomy-induced shedding of presynaptic terminals, but direct evidence for such participation is lacking. Currently, the most promising case for a promoting effect on neural repair by activated microglia appears to be as a mediator of collateral sprouting, at least in certain brain areas. However, final proof for a critical role of microglia in these instances is still lacking. Results from in vitro studies demonstrate that microglia can develop a regeneration supportive phenotype. Altering the microglial involvement following neural injury from a typically passive or even counterproductive state and into a condition where these cells are actively supporting regeneration and plasticity is, therefore, an exciting challenge and probably a realistic goal.     

Regeneration of periodontal Ruffini endings in adults and neonates

We reviewed the regeneration of periodontal Ruffini endings, primary mechanoreceptors in the periodontal ligament, following injury to the inferior alveolar nerve (IAN) in adult and neonatal rats. Morphologically, mature Ruffini endings are characterized by an extensive arborization of axonal terminals and association with specialized Schwann cells, called lamellar or terminal Schwann cells. Following injury to IAN in the adult, the periodontal Ruffini endings of the rat lower incisor ligament regenerate more rapidly than Ruffini endings in other tissues. During regeneration, terminal Schwann cells migrate into regions where they are never found under normal conditions. The development of periodontal Ruffini endings of the rat incisor is closely associated with the eruption of the teeth; the morphology and distribution of the terminal Schwann cells became almost identical to those in adults during postnatal days 15-18 (PN 15-18d) when the first molars appear in the oral cavity, while the axonal elements showed extensive ramification around PN 28d when the functional occlusion commences. When the IAN was injured in neonates, the regeneration of periodontal Ruffini endings was delayed compared with the adults. The migration of terminal Schwann cells is also observed following IAN injury, after which the distribution of terminal Schwann cells became almost identical to that of the adults, i.e., PN 14d. Since the interaction between axon and Schwann cell is important during regeneration and development, further studies are required to elucidate its molecular mechanism during the regeneration as well as the development of the periodontal Ruffini endings.     

Skeletal stem cells and bone regeneration: translational strategies from bench to clinic

Clinical imperatives for new bone to replace or restore the function of traumatized or bone lost as a consequence of age or disease has led to the need for therapies or procedures to generate bone for skeletal applications. Tissue regeneration promises to deliver specifiable replacement tissues and the prospect of efficacious alternative therapies for orthopaedic applications such as non-union fractures, healing of critical sized segmental defects and regeneration of articular cartilage in degenerative joint diseases. In this paper we review the current understanding of the continuum of cell development from skeletal stem cells, osteoprogenitors through to mature osteoblasts and the role of the matrix microenvironment, vasculature and factors that control their fate and plasticity in skeletal regeneration. Critically, this review addresses in vitro and in vivo models to investigate laboratory and clinical based strategies for the development of new technologies for skeletal repair and the key translational points to clinical success. The application of developmental paradigms of musculoskeletal tissue formation specifically, understanding developmental biology of bone formation particularly in the adult context of injury and disease will, we propose, offer new insights into skeletal cell biology and tissue regeneration allowing for the critical integration of stem cell science, tissue engineering and clinical applications. Such interdisciplinary, iterative approaches will be critical in taking patient aspirations to clinical reality.     

Regeneration as an application of gastropod neural plasticity

Gastropod research is providing many insights into mechanisms of neural regeneration. These observations were made possible by the pioneering work of individuals who described the nervous systems of gastropods, mapped prominent neurons and determined their roles and connections, and developed the techniques for culturing them. This information has allowed questions about injury responses, target selection, and pathway cues to be explored at the level of individually identified neurons. Because of gastropod studies, more is known about axon sealing, growth cone formation and behavior, signals that travel from the site of axotomy to the soma, and the second messengers that are activated there. The responses in neurons and non-neuronal cells during neural development and injury are coordinated by chemical messenger systems that are highly conserved, including neurotransmitters, cytokines, and neurotrophins. The nervous system is modified in learning paradigms by some of the same messenger systems activated by injury, because learning and injury both challenge neurons to change. The conservation of basic mechanisms that coordinate neuronal plasticity allows us to approach basic questions in relatively simple nervous systems with reasonable confidence that the findings will be relevant for other nervous systems, including possible applications to the mammalian nervous system.     

Analysis of neuronal networks: A review of techniques for labeling axonal projections

In order to analyze connections between neurons in the vetebrate central nervous system, methods have been developed to label a given population of axons of known origin so that they can be differentiated from other, non-labeled structures. Three such methods are reviewed here: experimentally induced orthograde (Wallerian) degeneration, axon transport of radioactive proteins demonstrated by autoradiography, and axon transport of macromolecules that can be reacted histochemically to yield a visible reaction product. Each of the methods has particular strengths and weaknesses. Degeneration methods may differentiate between different functional classes of axons which have different fiber diameters. However, degeneration distorts the morphology of axon terminals, making them more difficult to interpret, and degenerating terminals may be removed rapidly by phagocytosis. Autoradiography of radioactive terminals preserves normal fine structure, but the necessary exposure times extend the method by weeks or months, and care must be exercised to distinguish labeled axons from other structures exhibiting background or transneuronal radioactivity. Histochemical methods, such as those used to demonstrate horseradish peroxidase conjugated to wheat germ lectin (WGA-HRP), are sensitive and rapid, but the injection site must be carefully characterized, and the presence of transneuronal label may make interpretation of the results difficult. Experimental methods of axonal labeling have been invaluable in studying neuronal networks. Each of the methods described here may be of particular value, given the nature of the system to be analyzed.     

Prospects for tooth regeneration in the 21st century: a perspective

The prospects for tooth regeneration in the 21st century are compelling. Using the foundations of experimental embryology, developmental and molecular biology, the principles of biomimetics (the mimicking of biological processes), tooth regeneration is becoming a realistic possibility within the next few decades. The cellular, molecular, and developmental "rules" for tooth morphogenesis are rapidly being discovered. The knowledge gained from adult stem cell biology, especially associated with dentin, cartilage, and bone tissue regeneration, provides additional opportunities for eventual tooth organogenesis. The centuries of tooth development using xenotransplantation, allotransplantation, and autotransplantation have resulted in many important insights that can enhance tooth regeneration. In considering the future, several lines of evidence need to be considered: (1) enamel organ epithelia and dental papilla mesenchyme tissues contain stem cells during postnatal stages of life; (2) late cap stage and bell stage tooth organs contain stem cells; (3) odontogenic adult stem cells respond to mechanical as well as chemical "signals"; (4) presumably adult bone marrow as well as dental pulp tissues contain "odontogenic" stem cells; and (5) epithelial-mesenchymal interactions are pre-requisite for tooth regeneration. The authors express "guarded enthusiasm," yet there should be little doubt that adult stem cell-mediated tooth regeneration will be realized in the not too distant future. The prospects for tooth regeneration could be realized in the next few decades and could be rapidly utilized to improve the quality of human life in many nations around the world.     

Use of lipophilic dyes in studies of axonal pathfinding in vivo

Fluorescent lipophilic dyes are an ideal tool to study axonal pathfinding. Because these dyes do not require active axonal transport for their spreading, they can be used in fixed tissue. Here, we describe the method we have used to study the molecular mechanisms of commissural axon pathfinding in the embryonic chicken spinal cord in vivo. Based on in vitro studies, different families of molecules had been suggested to play a role in the guidance of developing axons. In order to test their function in vivo, we used the commissural neurons that are located at the dorsolateral border of the chicken spinal cord as a model system [Stoeckli and Landmesser (1995) Neuron 14:1165-1179]. Axonin-1, NgCAM, and NrCAM, three members of the immunoglobulin (Ig) superfamily of cell adhesion molecules (CAMs), were shown to be important for the correct growth pattern of commissural axons. We studied the effect of perturbations of specific CAM/CAM interactions by injection of function-blocking antibodies into the central canal of the spinal cord in ovo. After 2 days, the embryos were sacrificed and fluorescent tracers, such as Fast-DiI, were used to visualize commissural axons, and thus, to analyze their response to these perturbations in two different types of fixed preparations: transverse vibratome sections and whole-mount preparations of the spinal cord. Both pathfinding errors and defasciculation of axons were observed as a result of the perturbation of CAM/CAM interactions.     

Production of mesenchymal stem cell derived-secretome as cell-free regenerative therapy and immunomodulation: A biomanufacturing perspective

The potential of mesenchymal stem cells (MSCs) in regenerative medicine has been largely known due to their capability to induce tissue regeneration in vivo with minimum inflammation during implantation. This adult stem cell type exhibit unique features of tissue repair mechanism and immune modulation mediated by their secreted factors, called secretome. Recently, the utilization of secretome as a therapeutic agent provided new insight into cell-free therapy. Nevertheless, a sufficient amount of secretome is necessary to realize their applications for translational medicine which required a proper biomanufacturing process. Several factors related to their production need to be considered to produce a clinical-grade secretome as a biological therapeutic agent. This viewpoint highlights the current challenges and considerations during the biomanufacturing process of MSCs secretome.     

Changes in membrane trafficking and actin dynamics during axon formation in cultured hippocampal neurons

Neurons begin to polarize when one of the neurites becomes the axon. Hippocampal neurons in cell culture have a sharp transition between their unpolarized and polarized stage revealed by the rapid growth of the future axon. Recent progress shows that both a cytoplasmic membrane flow and actin dynamics govern axon formation, and thereby initial neuronal polarization. We here review these mechanisms, evaluate their physiological role, and show similarities to the transient polarization of migrating fibroblasts. Finally, we present a model how actin dynamics and vectorial membrane flow may interact to achieve axon formation.     

Herpes simplex virus-mediated expression of the axonal protein tau in human model neurons (NT2-N cells)

The establishment of axonal-somatodendritic polarity is an important event during neuronal development. The analysis of the underlying molecular events requires experimental models that display characteristic steps in the development of polarity and that are accessible for experimental manipulations. Here we show that human model neurons (NT2-N cells) can be efficiently infected with an amplicon-based herpes simplex virus (HSV) system that expresses the axonal microtubule-associated protein tau. We demonstrate that the neurons express a high level of exogenous tau, which persists for several days, thus allowing us to analyze the morphological effects of the expressed protein. The intracellular interactions of tau and the effects on the microtubule structure of infected neurons, which were processed for immunocytochemistry, were determined using laser scanning microscopy (LSM). Exogenous tau expression does not result in an increased axon growth of the neurons but promotes neuronal microtubule assembly as indicated by an increased amount of total microtubule polymer as well as a labile, detyrosinated microtubule subpopulation. In contrast, tau expression does not induce a significant microtubule stabilization as judged from the quantitation of acetylated microtubule staining 24 hours after infection. The data demonstrate that HSV-mediated expression of proteins in human model neurons provides a useful system for analysis of the effect of neuronal proteins on the morphology and cytoskeletal organization of terminally differentiated polar neurons. In addition, it suggests a role for tau as a factor which locally promotes tubulin polymerization while the dynamics of axonal microtubules are preserved.     

Role of nerve growth factor in the olfactory system

Olfactory neurons are unique in the mammalian nervous system because of their capacity to regenerate in adult animals. It has been shown that olfactory receptor cells located in the olfactory epithelium are replaced on a continuous basis and in response to injury throughout the life span of most species. NGF, which is one of the neurotrophic factors, is present in many areas of the central and peripheral nervous system. It has been shown that NGF in the olfactory bulb plays a role in the survival of cholinergic neurons in the horizontal limb of the diagonal band (HDB). Recent studies of NGF in the olfactory bulb suggest that it is involved in the development, maintenance, and regeneration of olfactory receptor cells. In this study, we review reports examining the relationship between NGF in the olfactory bulb and neuronal regeneration and development in the mammalian olfactory systems. Low- and high-affinity NGF receptor immunoreactivity is markedly expressed during regeneration and at different stages of development in the mouse olfactory system. This level of immunoreactivity is no longer present after completion of regeneration and at maturation. Other findings indicate that NGF injected into the olfactory bulb is transported retrogradely to the olfactory epithelium. It has also been shown that continuous anti-NGF antibody injection into the olfactory bulb causes degeneration and olfactory dysfunction. Administration of NGF directory into nasal cavity results in an increase in the expression of olfactory marker protein within the olfactory epithelium in axotomized rats. These findings suggested that the presence of NGF in the olfactory bulb plays an essential role in regeneration, maintenance, and development in the olfactory system of mammals.     

Basic techniques for long distance axon tracing in the spinal cord

The regeneration of axons after a spinal cord injury or disease is attracting a significant amount of interest among researchers. Being able to assess these axons in terms of morphology, length and origin is essential to our understanding of the regeneration process. Recently, two specific axon tracers have gained much recognition; biotinylated dextran amine (BDA) 10 kDa as an anterograde tracer and cholera toxin‐B as a retrograde tracer. However, there are still several complexities when using these tracers, including the volume that should be administered and the best administration site so that a significant amount of axons are labeled in the area of interest. In this article, we describe some simple procedures for injecting the tracers and detecting them. We also quantified the number of axons at different locations of the spinal cord. Our results show axons labeled from motor cortex injections traveled down to the lumbosacral spinal cord in 2 weeks, while BDA injections into the lateral vestibular nucleus and reticular formation took 3 weeks to label axons in the lumbosacral spinal cord. Moreover, this protocol outlines some basic procedures that could be used in any laboratory and gives insight into the number of axons labeled and how procedures could be tailored to meet specific researcher's needs. Microsc. Res. Tech. 76:1240–1249, 2013. © 2013 Wiley Periodicals, Inc.     

Analysis of nerve fibers and their distribution in histologic sections of the human brain

The field of quantitative analysis and subsequent mapping of the cerebral cortex has developed rapidly. New powerful tools have been applied to investigate large regions of complex folded gyrencephalic cortices in order to detect structural transition regions that might partition different cortical fields of disjunct neuronal functions. We have developed a new mapping approach based on axoarchitectonics, a method of cortical visualization that previously has been used only indirectly with regard to myeloarchitectonics. Myeloarchitectonic visualization has the disadvantage of producing strong agglomerative effects of closely neighbored nerve fibers. Therefore, single and neurofunctional-relevant parameters such as axonal branchings, axon areas, and axon numbers have not been determinable with satisfying precision. As a result, different staining techniques had to be explored in order to achieve a suitable histologic staining for axon visualization. The best results were obtained after modifying the Naoumenko-Feigin staining for axons. From these contrast-rich stained histologic sections, videomicroscopic digital image tiles were generated and analyzed using a new fiber analysis framework. Finally, the analysis of histologic images provided topologic ordered parameters of axons that were transferred into parameter maps. The axon parameter maps were analyzed further via a recently developed traverse generating algorithm that calculated test lines oriented perpendicular to the cortical surface and white matter border. The gray value coded parameters of the parameter maps were then transferred into profile arrays. These profile arrays were statistically analyzed by a reliable excess mass approach we recently developed. We found that specific axonal parameters are preferentially distributed throughout granular and agranular types of cortex. Furthermore, our new procedure detected transition regions originally defined by changes of cytoarchitectonic layering. Statistically significant inhomogeneities of the distribution of certain axon quantities were shown to indicate a subparcellation of areas 4 and 6. The quantification techniques established here for the analysis of spatial axon distributions within larger regions of the cerebral cortex are suitable to detect inhomogeneities of laminar axon patterns. Hence, these techniques can be recommended for systematic and observer-supported cortical area mapping and parcellation studies.     

Role of nitric oxide and nitric oxide synthases in experimental models of denervation and reinnervation

Nitric oxide (NO) is a short-living free molecule synthesized by three different isoforms of nitric oxide synthases (NOS)—neuronal NOS, endothelial NOS, and inducible NOS—associated with neuromuscular transmission, muscle contractility, mitochondrial respiration, and carbohydrate metabolism in skeletal muscle. Neuronal NOS is constitutively expressed at the muscle fiber sarcolemma linked to the dystrophin-glycoprotein complex and concentrated at the neuromuscular endplate. There is increasing evidence that altered expression of neuronal NOS plays a role in muscle fiber damage in neuromuscular diseases such as dystrophinopathies and denervating disorders. Although there have been some previous conflicting results on the neuronal NOS expression pattern in denervated muscle fibers, it is now well established that denervation is associated with a down-regulation and disappearance of sarcolemmal neuronal NOS at synaptic/extrasynaptic or both sites. As NO has been shown to induce collapse and growth arrest on neuronal growth cones, down-regulation of sarcolemmal neuronal NOS may contribute to axonal regeneration and attraction to muscle fibers aiming at the formation of new motor endplates providing reinnervation and reconstitution of NOS expression. As NO serves as a retrograde messenger, it may trigger structural downstream events responsible for neuromuscular synaptogenesis and preventing polyneural innervation. Nevertheless, decreased NO production in denervation reduces the cytoprotective scavenger function of NO for superoxide anions promoting oxidative stress that is likely to be involved in muscle fiber damage and death. However, the multifaced role of NOS and NO under physiological and pathological conditions remains poorly understood on the basis of the current knowledge. Microsc. Res. Tech. 55:181–186, 2001. © 2001 Wiley-Liss, Inc.     

Electron microscopy and morphometric analyses of microtubules in two differently sized types of axons in the protocerebral tract of a crustacean

Despite several reports on the morphology and functions associated with the morphometry of the vertebrate axoplasm cytoskeleton, the subject has not been thoroughly explored in invertebrates. In vertebrates, among many other functions, microtubules (MTs) serve as scaffolding for axon assembly, and neurofilaments (NFs) as the elements that determine the axon caliber. Intermediate filaments have never been described by electron microscopy in arthropods, although NF proteins have been revealed in the MT side-arms of the axoplasm of certain species, such as the crab Ucides cordatus. Thus, it is not known which elements of the cytoskeleton of invertebrates are responsible for determination of the axon caliber. We studied, by electron microscopy and morphometric analyses, the MT and axon area variability in differently sized axons of the protocerebral tract of the crab Ucides cordatus. Our results revealed differences in the distance between MTs, in MT density and number, and in the areas of differently sized axons. The number of MTs increases with the axon area, but this relationship is not directly proportional. Therefore, MT density is greater in smaller axons than in medium axons, similar to the morphometry of the vertebrate axon MT. The distance between MTs is, however, directly related to the axonal area. On the basis of the results shown here, and on previous reports by us and others, we suggest that MTs may be involved in the determination of the axon caliber, possibly due to the presence of NF proteins found in the side-arms.     

Periodontal regeneration: a challenge for the tissue engineer?

Periodontitis affects around 15 per cent of human adult populations. While periodontal treatment aimed at removing the bacterial cause of the disease is generally very successful, the ability predictably to regenerate the damaged tissues remains a major unmet objective for new treatment strategies. Existing treatments include the use of space-maintaining barrier membranes (guided tissue regeneration), use of graft materials, and application of bioactive molecules to induce regeneration, but their overall effects are relatively modest and restricted in application. The periodontal ligament is rich in mesenchymal stem cells, and the understanding of the signalling molecules that may regulate their differentation has increased enormously in recent years. Applying these principles for the development of new tissue engineering strategies for periodontal regeneration will require further work to determine the efficacy of current experimental preclinical treatments, including pharmacological application of growth factors such as bone morphogenetic proteins (BMPs) or Wnts, use of autologous stem cell reimplantation strategies, and development of improved biomaterial scaffolds. This article describes the background to this problem, addresses the current status of periodontal regeneration, including the background biology, and discusses the potential for some of these experimental therapies to achieve the goal of clinically predictable periodontal regeneration.     

Agmatine inhibits hypoxia-induced TNF-alpha release from cultured retinal ganglion cells

The effect of hypoxia on the release of tumor necrosis factor-alpha (TNF-alpha) in transformed rat retinal ganglion cells (RGCs) and the effect of agmatine on the hypoxia-induced production of TNF-alpha in RGCs were evaluated. RGCs were cultured under hypoxic conditions with 5% oxygen, with or without 100 microM agmatine. The expression levels of TNF-alpha and its receptor-1 (TNF-R1) were investigated by Western blot analysis. After 6 hours of hypoxia, we noted an increase in TNF-alpha production in RGCs. Agmatine significantly reduced TNF-alpha level after 12 hours of hypoxic treatment. The expression of TNF-R1 was not affected by the hypoxia or agmatine treatment. Our results show that agmatine inhibits the TNF-alpha production of RGCs in hypoxic condition. These results demonstrate a possible neuroprotective mechanism for agmatine against hypoxic damage in RGCs.