Three dimensional electron microscopy reveals changing axonal and myelin morphology along normal and partially injured optic nerves

Following injury to the central nervous system, axons and myelin distinct from the initial injury site undergo changes associated with compromised function. Quantifying such changes is important to understanding the pathophysiology of neurotrauma; however, most studies to date used 2 dimensional (D) electron microscopy to analyse single sections, thereby failing to capture changes along individual axons. We used serial block face scanning electron microscopy (SBF SEM) to undertake 3D reconstruction of axons and myelin, analysing optic nerves from normal uninjured female rats and following partial optic nerve transection. Measures of axon and myelin dimensions were generated by examining 2D images at 5 µm intervals along the 100 µm segments. In both normal and injured animals, changes in axonal diameter, myelin thickness, fiber diameter, G-ratio and percentage myelin decompaction were apparent along the lengths of axons to varying degrees. The range of values for axon diameter along individual reconstructed axons in 3D was similar to the range from 2D datasets, encompassing reported variation in axonal diameter attributed to retinal ganglion cell diversity. 3D electron microscopy analyses have provided the means to demonstrate substantial variability in ultrastructure along the length of individual axons and to improve understanding of the pathophysiology of neurotrauma

Early cellular signaling responses to axonal injury

<p>Abstract</p> <p>Background</p> <p>We have used optic nerve injury as a model to study early signaling events in neuronal tissue following axonal injury. Optic nerve injury results in the selective death of retinal ganglion cells (RGCs). The time course of cell death takes place over a period of days with the earliest detection of RGC death at about 48 hr post injury. We hypothesized that in the period immediately following axonal injury, there are changes in the soma that signal surrounding glia and neurons and that start programmed cell death. In the current study, we investigated early changes in cellular signaling and gene expression that occur within the first 6 hrs post optic nerve injury.</p> <p>Results</p> <p>We found evidence of cell to cell signaling within 30 min of axonal injury. We detected differences in phosphoproteins and gene expression within the 6 hrs time period. Activation of TNFα and glutamate receptors, two pathways that can initiate cell death, begins in RGCs within 6 hrs following axonal injury. Differential gene expression at 6 hrs post injury included genes involved in cytokine, neurotrophic factor signaling (Socs3) and apoptosis (Bax).</p> <p>Conclusion</p> <p>We interpret our studies to indicate that both neurons and glia in the retina have been signaled within 30 min after optic nerve injury. The signals are probably initiated by the RGC soma. In addition, signals activating cellular death pathways occur within 6 hrs of injury, which likely lead to RGC degeneration.</p

Promises of stem cell therapy for retinal degenerative diseases

With the development of stem cell technology, stem cell-based therapy for retinal degeneration has been proposed to restore the visual function. Many animal studies and some clinical trials have shown encouraging results of stem cell-based therapy in retinal degenerative diseases. While stem cell-based therapy is a promising strategy to replace damaged retinal cells and ultimately cure retinal degeneration, there are several important challenges which need to be overcome before stem cell technology can be applied widely in clinical settings. In this review, different types of donor cell origins used in retinal treatments, potential target cell types for therapy, methods of stem cell delivery to the eye, assessments of potential risks in stem cell therapy, as well as future developments of retinal stem cells therapy, will be discussed

Paracrine-mediated neuroprotection and neuritogenesis of axotomised retinal ganglion cells by human dental pulp stem cells:Comparison with human bone marrow and adipose-derived mesenchymal stem cells

We have investigated and compared the neurotrophic activity of human dental pulp stem cells (hDPSC), human bone marrow-derived mesenchymal stem cells (hBMSC) and human adipose-derived stem cells (hAMSC) on axotomised adult rat retinal ganglion cells (RGC) in vitro in order to evaluate their therapeutic potential for neurodegenerative conditions of RGC. Using the transwell system, RGC survival and length/number of neurites were quantified in coculture with stem cells in the presence or absence of specific Fc-receptor inhibitors to determine the role of NGF, BDNF, NT-3, VEGF, GDNF, PDGF-AA and PDGF-AB/BB in stem cell-mediated RGC neuroprotection and neuritogenesis. Conditioned media, collected from cultured hDPSC/hBMSC/hAMSC, were assayed for the secreted growth factors detailed above using ELISA. PCR array determined the hDPSC, hBMSC and hAMSC expression of genes encoding 84 growth factors and receptors. The results demonstrated that hDPSC promoted significantly more neuroprotection and neuritogenesis of axotomised RGC than either hBMSC or hAMSC, an effect that was neutralized after the addition of specific Fc-receptor inhibitors. hDPSC secreted greater levels of various growth factors including NGF, BDNF and VEGF compared with hBMSC/hAMSC. The PCR array confirmed these findings and identified VGF as a novel potentially therapeutic hDPSC-derived neurotrophic factor (NTF) with significant RGC neuroprotective properties after coculture with axotomised RGC. In conclusion, hDPSC promoted significant multi-factorial paracrine-mediated RGC survival and neurite outgrowth and may be considered a potent and advantageous cell therapy for retinal nerve repair

The optic nerve head is the site of axonal transport disruption, axonal cytoskeleton damage and putative axonal regeneration failure in a rat model of glaucoma

The neurodegenerative disease glaucoma is characterised by the progressive death of retinal ganglion cells (RGCs) and structural damage to the optic nerve (ON). New insights have been gained into the pathogenesis of glaucoma through the use of rodent models; however, a coherent picture of the early pathology remains elusive. Here, we use a validated, experimentally induced rat glaucoma model to address fundamental issues relating to the spatio-temporal pattern of RGC injury. The earliest indication of RGC damage was accumulation of proteins, transported by orthograde fast axonal transport within axons in the optic nerve head (ONH), which occurred as soon as 8 h after induction of glaucoma and was maximal by 24 h. Axonal cytoskeletal abnormalities were first observed in the ONH at 24 h. In contrast to the ONH, no axonal cytoskeletal damage was detected in the entire myelinated ON and tract until 3 days, with progressively greater damage at later time points. Likewise, down-regulation of RGC-specific mRNAs, which are sensitive indicators of RGC viability, occurred subsequent to axonal changes at the ONH and later than in retinas subjected to NMDA-induced somatic excitotoxicity. After 1 week, surviving, but injured, RGCs had initiated a regenerative-like response, as delineated by Gap43 immunolabelling, in a response similar to that seen after ON crush. The data presented here provide robust support for the hypothesis that the ONH is the pivotal site of RGC injury following moderate elevation of IOP, with the resulting anterograde degeneration of axons and retrograde injury and death of somas

Network analysis of human glaucomatous optic nerve head astrocytes

<p>Abstract</p> <p>Background</p> <p>Astrocyte activation is a characteristic response to injury in the central nervous system, and can be either neurotoxic or neuroprotective, while the regulation of both roles remains elusive.</p> <p>Methods</p> <p>To decipher the regulatory elements controlling astrocyte-mediated neurotoxicity in glaucoma, we conducted a systems-level functional analysis of gene expression, proteomic and genetic data associated with reactive optic nerve head astrocytes (ONHAs).</p> <p>Results</p> <p>Our reconstruction of the molecular interactions affected by glaucoma revealed multi-domain biological networks controlling activation of ONHAs at the level of intercellular stimuli, intracellular signaling and core effectors. The analysis revealed that synergistic action of the transcription factors AP-1, vitamin D receptor and Nuclear Factor-kappaB in cross-activation of multiple pathways, including inflammatory cytokines, complement, clusterin, ephrins, and multiple metabolic pathways. We found that the products of over two thirds of genes linked to glaucoma by genetic analysis can be functionally interconnected into one epistatic network via experimentally-validated interactions. Finally, we built and analyzed an integrative disease pathology network from a combined set of genes revealed in genetic studies, genes differentially expressed in glaucoma and closely connected genes/proteins in the interactome.</p> <p>Conclusion</p> <p>Our results suggest several key biological network modules that are involved in regulating neurotoxicity of reactive astrocytes in glaucoma, and comprise potential targets for cell-based therapy.</p

Specific ion channels contribute to key elements of pathology during secondary degeneration following neurotrauma

Background: Following partial injury to the central nervous system, cells beyond the initial injury site undergo secondary degeneration, exacerbating loss of neurons, compact myelin and function. Changes in Ca 2+ flux are associated with metabolic and structural changes, but it is not yet clear how flux through specific ion channels contributes to the various pathologies. Here, partial optic nerve transection in adult female rats was used to model secondary degeneration. Treatment with combinations of three ion channel inhibitors was used as a tool to investigate which elements of oxidative and structural damage related to long term functional outcomes. The inhibitors employed were the voltage gated Ca 2+ channel inhibitor Lomerizine (Lom), the Ca 2+ permeable AMPA receptor inhibitor YM872 and the P2X 7 receptor inhibitor oxATP. Results: Following partial optic nerve transection, hyper-phosphorylation of Tau and acetylated tubulin immunoreactivity were increased, and Nogo-A immunoreactivity was decreased, indicating that axonal changes occurred acutely. All combinations of ion channel inhibitors reduced hyper-phosphorylation of Tau and increased Nogo-A immunoreactivity at day 3 after injury. However, only Lom/oxATP or all three inhibitors in combination significantly reduced acetylated tubulin immunoreactivity. Most combinations of ion channel inhibitors were effective in restoring the lengths of the paranode and the paranodal gap, indicative of the length of the node of Ranvier, following injury. However, only all three inhibitors in combination restored to normal Ankyrin G length at the node of Ranvier. Similarly, HNE immunoreactivity and loss of oligodendrocyte precursor cells were only limited by treatment with all three ion channel inhibitors in combination. Conclusions: Data indicate that inhibiting any of a range of ion channels preserves certain elements of axon and node structure and limits some oxidative damage following injury, whereas ionic flux through all three channels must be inhibited to prevent lipid peroxidation and preserve Ankyrin G distribution and OPCs