Reactive species and oxidative stress in optic nerve vulnerable to secondary degeneration

Secondary degeneration contributes substantially to structural and functional deficits following traumatic injury to the CNS. While it has been proposed that oxidative stress is a feature of secondary degeneration, contributing reactive species and resultant oxidized products have not been clearly identified in vivo. The study is designed to identify contributors to, and consequences of, oxidative stress in a white matter tract vulnerable to secondary degeneration. Partial dorsal transection of the optic nerve (ON) was used to model secondary degeneration in ventral nerve unaffected by the primary injury. Reactive species were assessed using fluorescent labelling and liquid chromatography/tandem mass spectroscopy (LC/MS/MS). Antioxidant enzymes and oxidized products were semi-quantified immunohistochemically. Mitophagy was assessed by electron microscopy. Fluorescent indicators of reactive oxygen and/or nitrogen species increased at 1, 3 and 7 days after injury, in ventral ON. LC/MS/MS confirmed increases in reactive species linked to infiltrating microglia/macrophages in dorsal ON. Similarly, immunoreactivity for glutathione peroxidase and haem oxygenase-1 increased in ventral ON at 3 and 7 days after injury, respectively. Despite increased antioxidant immunoreactivity, DNA oxidation was evident from 1 day, lipid oxidation at 3 days, and protein nitration at 7 days after injury. Nitrosative and oxidative damage was particularly evident in CC1-positive oligodendrocytes, at times after injury at which structural abnormalities of the Node of Ranvier/paranode complex have been reported. The incidence of mitochondrial autophagic profiles was also significantly increased from 3 days. Despite modest increases in antioxidant enzymes, increased reactive species are accompanied by oxidative and nitrosative damage to DNA, lipid and protein, associated with increasing abnormal mitochondria, which together may contribute to the deficits of secondary degeneration

Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties

Several recent studies have demonstrated that TAT and other arginine-rich cell penetrating peptides (CPPs) have intrinsic neuroprotective properties in their own right. Examples, we have demonstrated that in addition to TAT, poly-arginine peptides (R8 to R18; containing 8–18 arginine residues) as well as some other arginine-rich peptides are neuroprotective in vitro (in neurons exposed to glutamic acid excitotoxicity and oxygen glucose deprivation) and in the case of R9 in vivo (after permanent middle cerebral artery occlusion in the rat). Based on several lines of evidence, we propose that this neuroprotection is related to the peptide's endocytosis-inducing properties, with peptide charge and arginine residues being critical factors. Specifically, we propose that during peptide endocytosis neuronal cell surface structures such as ion channels and transporters are internalised, thereby reducing calcium influx associated with excitotoxicity and other receptor-mediated neurodamaging signalling pathways. We also hypothesise that a peptide cargo can act synergistically with TAT and other arginine-rich CPPs due to potentiation of the CPPs endocytic traits rather than by the cargo-peptide acting directly on its supposedly intended intracellular target. In this review, we systematically consider a number of studies that have used CPPs to deliver neuroprotective peptides to the central nervous system (CNS) following stroke and other neurological disorders. Consequently, we critically review evidence that supports our hypothesis that neuroprotection is mediated by carrier peptide endocytosis. In conclusion, we believe that there are strong grounds to regard arginine-rich peptides as a new class of neuroprotective molecules for the treatment of a range of neurological disorders

Specific combinations of ion channel inhibitors reduce excessive Ca2+ influx as a consequence of oxidative stress and increase neuronal and glial cell viability in vitro

Combinations of Ca 2+ channel inhibitors have been proposed as an effective means to prevent excess Ca 2+ flux and death of neurons and glia following neurotrauma in vivo. However, it is not yet known if beneficial outcomes such as improved viability have been due to direct effects on intracellular Ca 2+ concentrations. Here, the effects of combinations of Lomerizine (Lom), 2,3-dioxo-7-(1H-imidazol-1-yl)6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]acetic acid monohydrate (YM872), 3,5-dimethyl-1-adamantanamine (memantine (Mem)) and/or adenosine 5'-triphosphate periodate oxidized sodium salt (oxATP) to block voltage-gated Ca 2+ channels, Ca 2+ permeable a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, NMDA receptors and purinergic P2X 7 receptors (P2X 7 R) respectively, on Ca 2+ concentration and viability of rat primary mixed cortical (MC) cultures exposed to hydrogen peroxide (H 2 O 2 ) insult, were assessed. The contribution of ryanodine-sensitive intracellular stores to intracellular Ca 2+ concentration was also assessed. Live cell calcium imaging revealed that a 30 min H 2 O 2 insult induced a slow increase in intracellular Ca 2+ , in part from intracellular sources, associated with loss of cell viability by 6 h. Most combinations of inhibitors that included oxATP significantly decreased Ca 2+ influx and increased cell viability when administered simultaneously with H 2 O 2 . However, reductions in intracellular Ca 2+ concentration were not always linked to improved cell viability. Examination of the density of specific cell subpopulations demonstrated that most combinations of inhibitors that included oxATP preserved NG2+ non-oligodendroglial cells, but preservation of astrocytes and neurons required additional inhibitors. Olig2 + oligodendroglia and ED-1 + activated microglia/macrophages were not preserved by any of the inhibitor combinations. These data indicate that following H 2 O 2 insult, limiting intracellular Ca 2+ entry via P2X 7 R is generally associated with increased cell viability. Protection of NG2+ non-oligodendroglial cells by Ca 2+ channel inhibitor combinations may contribute to observed beneficial outcomes in vivo

Three Ca2+ channel inhibitors in combination limit chronic secondary degeneration following neurotrauma

Following neurotrauma, cells beyond the initial trauma site undergo secondary degeneration, with excess Ca 2+ a likely trigger for loss of neurons, compact myelin and function. Treatment using inhibitors of specific Ca 2+ channels has shown promise in preclinical studies, but clinical trials have been disappointing and combinatorial approaches are needed. We assessed efficacy of multiple combinations of three Ca 2+ channel inhibitors at reducing secondary degeneration following partial optic nerve transection in rat. We used lomerizine to inhibit voltage gated Ca 2+ channels; oxidised adenosine-triphosphate (oxATP) to inhibit purinergic P2X 7 receptors and/or 2-[7-(1H-imidazol-1-yl)-6-nitro-2,3-dioxo-1,2,3,4- tetrahydro quinoxalin-1-yl]acetic acid (INQ) to inhibit Ca 2+ permeable a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Only the three Ca 2+ channel inhibitors delivered in combination significantly preserved visual function, as assessed using the optokinetic nystagmus visual reflex, at 3 months after injury. Preservation of retinal ganglion cells was partial and is unlikely to have accounted for differential effects on function. A range of the Ca 2+ channel inhibitor combinations prevented swelling of optic nerve vulnerable to secondary degeneration. Each of the treatments involving lomerizine significantly increased the proportion of axons with normal compact myelin. Nevertheless, limiting decompaction of myelin was not sufficient for preservation of function in our model. Multiple combinations of Ca 2+ channel inhibitors reduced formation of atypical node/paranode complexes; outcomes were not associated with preservation of visual function. However, prevention of lengthening of the paranodal gap that was only achieved by treatment with the three Ca 2+ channel inhibitors in combination was an important additional effect that likely contributed to the associated preservation of the optokinetic reflex using this combinatorial treatment strategy

A survival model of thoracic contusion spinal cord injury in the domestic pig

Published Online:6 Oct 2022. OnlinePublSpinal cord injury (SCI) frequently results in motor, sensory and autonomic dysfunction for which there is currently no cure. Recent preclinical and clinical research has led to promising advances in treatment; however, therapeutics indicating promise in rodents have not translated successfully in human trials, likely due, in part, to gross anatomical and physiological differences between the species. Therefore, large animal models of SCI may facilitate the study of secondary injury processes that are influenced by scale, and assist the translation of potential therapeutic interventions. The aim of this study was to characterize two severities of thoracic contusion SCI in female domestic pigs, measuring motor function and spinal cord lesion characteristics, over two weeks post-SCI. A custom instrumented weight drop injury device was used to release a 50 g impactor from 10 cm (n=3) or 20 cm (n=7) onto the exposed dura, to induce a contusion at the T10 thoracic spinal level. Hind limb motor function was assessed at 8 and 13 days post-SCI using a 10-point scale. Volume and extent of lesion-associated signal hyperintensity in T2-weighted magnetic resonance (MR) images was assessed at 3, 7 and 14 days post-injury. Animals were transcardially perfused at 14 days post-SCI and spinal cord tissue was harvested for histological analysis. Bowel function was retained in all animals and transient urinary retention occurred in one animal after catheter removal. All animals displayed hind limb motor deficits. Animals in the 10 cm group demonstrated some stepping and weight bearing and scored a median 2- 3 points higher on the 10-point motor function scale at 8 and 13 days post-SCI, than the 20 cm group. Histological lesion volume was 20 % greater, and 30 % less white matter was spared, in the 20 cm group than in the 10 cm group. The MR signal hyperintensity in the 20 cm injury group had a median cranial-caudal extent approximately 1.5 times greater than the 10 cm injury group at all three time points, and median volumes 1.8, 2.5 and 4.5 times greater at day 3, 7 and 14 post-injury, respectively. Regional differences in axonal injury were observed between groups, with amyloid precursor protein immunoreactivity greatest in the 20 cm group in spinal cord sections adjacent the injury epicenter. This study demonstrated graded injuries in a domestic pig strain, with outcome measures comparable to miniature pig models of contusion SCI. The model provides a vehicle for the study of SCI and potential treatments, particularly where miniature pig strains are not available and/or where small animal models are not appropriate for the research question.Christine D. Gayen, Madeleine A. Bessen, Ryan M. Dorrian, Ryan D Quarrington, Adnan Mulaibrahimovic, Ryan L. O’Hare Doig, Brian JC Freeman, Anna V. Leonard, Claire F. Jone

Biodegradable and biocompatible graphene-based scaffolds for functional neural tissue engineering: a strategy approach using dental pulp stem cells and biomaterials

Accepted: 1 July 2021Neural tissue engineering aims to restore function of nervous system tissues using biocompatible cell-seeded scaffolds. Graphene-based scaffolds combined with stem cells deserve special attention to enhance tissue regeneration in a controlled manner. However, it is believed that minor changes in scaffold biomaterial composition, internal porous structure, and physicochemical properties can impact cellular growth and adhesion. The current work aims to investigate in vitro biological effects of 3D graphene oxide (GO)/sodium alginate (GOSA) and reduced GOSA (RGOSA) scaffolds on dental pulp stem cells (DPSCs) in terms of cell viability and cytotoxicity. Herein, the effects of the 3D scaffolds, coating conditions, and serum supplementation on DPSCs functions are explored extensively. Biodegradation analysis revealed that addition of GO enhanced the degradation rate of composite scaffolds. Compared to the 2D surface, the cell viability of 3D scaffolds was higher (p <0.0001), highlighting the optimal initial cell adhesion to the scaffold surface and cell migration through pores. Moreover, the cytotoxicity study indicated that the incorporation of graphene supported higher DPSCs viability. It is also shown that when the mean pore size of the scaffold increases, DPSCs activity decreases. In terms of coating conditions, poly-l-lysine (PLL) was the most robust coating reagent that improved cell-scaffold adherence and DPSCs metabolism activity. The cytotoxicity of GO-based scaffolds showed that DPSCs can be seeded in serum-free media without cytotoxic effects. This is critical for human translation as cellular transplants are typically serum-free. These findings suggest that proposed 3D GO-based scaffolds have favourable effects on the biological responses of DPSCs. This article is protected by copyright. All rights reserved.Negar Mansour, Said Al-Sarawi, Dusan Losic, Jagan Mazumdar, Jillian Clark, Stan Gronthos, Ryan O'Hare Doi

Prolonged glutamate excitotoxicity increases GluR1 immunoreactivity but decreases mRNA of GluR1 and associated regulatory proteins in dissociated rat retinae in vitro

Abstract not availableMichael Challenor, Ryan O'Hare Doig, Paula Fuller, Marcus Giacci, Carole Bartlett, Celeste H. Wale, Greg S. Cozens, Livia Hool, Sarah Dunlop, K. Swaminathan Iyer, Jennifer Rodger, Melinda Fitzgeral

Node/paranode complexes in ventral ON after partial transection.

<p>(a) Representative images of Caspr^+ve paranodes (green) flanking the β-III tubulin^+ve (red) paranodal gap, and β-III tubulin^+ve areas (red) colocalised with Na_v1.6^+ve nodes (blue) in normal ventral ON and at 1 day post injury; colocalised areas are yellow and purple respectively (examples indicated by arrows), scale  = 20 μm. Mean ± SEM length of the paranodal gap (b), paranode length (c) and the ratio of node to paranode lengths (e) in ventral ON of normal animals and 1, 3, 7 days, 1 and 3 months after injury; representative images (d), scale bar  = 1 μm. Orthogonal projection of a representative z stack illustrating a large atypical node/paranode complex well within the stack of images (f, boxed), scale bar  = 5 μm; Caspr^+ve paranodes are green, β-III tubulin^+ve axons are red (note: only projections in the z plane of the identified node/paranode complex are shown in the panels adjacent to the main image). Mean ± SEM percentages of node/paranode complexes that were atypical (hemi – nodes, single paranodes) (g), * significantly different from normal for each complex type (p≤0.05) (n = 6 animals/time point)</p

Oxidative stress indicators in ON after partial transection.

<p>(a) Mean ± SEM ROS/RNS assessed as DCF fluorescence in homogenates of ON including both the dorsal injury site and the ventral region vulnerable to secondary degeneration, from normal animals and 1, 3, 7 days, 1 and 3 months after injury, or (b) from ventral ON only from normal animals and 1 or 7 days after injury (6 animals pooled per time point, assayed in duplicate). (c) Semi – quantification of mean ± SEM intensity of CML immunoreactivity in olig1^+ve oligodendrocytes in ventral ON, assessed by tracing identified cells in single images in the z axis; representative images at 1 day (d), scale bar  = 10 μm, (n = 4–5 animals/time point). Similarly, semi – quantification of mean ± SEM intensity of DHE staining in olig1^+ve (e) or CC1^+ve (f) oligodendrocytes in ventral ON; * significantly different from normal (p≤0.05).</p