Loss of function following injury to the CNS is worsened by secondary degeneration of neurons and glia surrounding the injury and is initiated by oxidative damage. However, it is not yet known which cellular populations and structures are most vulnerable to oxidative damage Using Nanoscale secondary ion mass spectrometry (NanoSIMS), oxidative damage was semiquantified within cellular subpopulations and structures of optic nerve vulnerable to secondary degeneration, following a partial transection of the optic nerve in adult female PVG rats. Simultaneous assessment of cellular subpopulations and structures revealed oligodendroglia as the most vulnerable to DNA oxidation following injury. 5-Ethynyl-2'-deoxyuridine (EdU) was used to label cells that proliferated in the first 3 d after injury. Injury led to increases in DNA, protein, and lipid damage in oligodendrocyte progenitor cells and mature oligodendrocytes at 3 d, regardless of proliferative state, associated with a decline in the numbers of oligodendrocyte progenitor cells at 7 d. O4 preoligodendrocytes also exhibited increased lipid peroxidation. Interestingly, EdU mature oligodendrocytes derived after injury demonstrated increased early susceptibility to DNA damage and lipid peroxidation. However, EdU mature oligodendrocytes with high 8-hydroxyguanosine immunoreactivity were more likely to be caspase3 By day 28, newly derived mature oligodendrocytes had significantly reduced myelin regulatory factor gene mRNA, indicating that the myelination potential of these cells may be reduced. The proportion of caspase3 oligodendrocytes remained higher in EdU cells. Innovative use of NanoSIMS together with traditional immunohistochemistry and hybridization have enabled the first demonstration of subpopulation specific oligodendroglial vulnerability to oxidative damage, due to secondary degeneration Injury to the CNS is characterized by oxidative damage in areas adjacent to the injury. However, the cellular subpopulations and structures most vulnerable to this damage remain to be elucidated. Here we use powerful NanoSIMS techniques to show increased oxidative damage in oligodendroglia and axons and to demonstrate that cells early in the oligodendroglial lineage are the most vulnerable to DNA oxidation. Further immunohistochemical and hybridization investigation reveals that mature oligodendrocytes derived after injury are more vulnerable to oxidative damage than their counterparts existing at the time of injury and have reduced myelin regulatory factor gene mRNA, yet preexisting oligodendrocytes are more likely to die.

Red/near-infrared irradiation therapy (R/NIR-IT) delivered by laser or light-emitting diode (LED) has improved functional outcomes in a range of CNS injuries. However, translation of R/NIR-IT to the clinic for treatment of neurotrauma has been hampered by lack of comparative information regarding the degree of penetration of the delivered irradiation to the injury site and the optimal treatment parameters for different CNS injuries. We compared the treatment efficacy of R/NIR-IT at 670 nm and 830 nm, provided by narrow-band LED arrays adjusted to produce equal irradiance, in four in vivo rat models of CNS injury: partial optic nerve transection, light-induced retinal degeneration, traumatic brain injury (TBI) and spinal cord injury (SCI). The number of photons of 670 nm or 830 nm light reaching the SCI injury site was 6.6% and 11.3% of emitted light respectively. Treatment of rats with 670 nm R/NIR-IT following partial optic nerve transection significantly increased the number of visual responses at 7 days after injury (P≤0.05); 830 nm R/NIR-IT was partially effective. 670 nm R/NIR-IT also significantly reduced reactive species and both 670 nm and 830 nm R/NIR-IT reduced hydroxynonenal immunoreactivity (P≤0.05) in this model. Pre-treatment of light-induced retinal degeneration with 670 nm R/NIR-IT significantly reduced the number of Tunel+ cells and 8-hydroxyguanosine immunoreactivity (P≤0.05); outcomes in 830 nm R/NIR-IT treated animals were not significantly different to controls. Treatment of fluid-percussion TBI with 670 nm or 830 nm R/NIR-IT did not result in improvements in motor or sensory function or lesion size at 7 days (P>0.05). Similarly, treatment of contusive SCI with 670 nm or 830 nm R/NIR-IT did not result in significant improvements in functional recovery or reduced cyst size at 28 days (P>0.05). Outcomes from this comparative study indicate that it will be necessary to optimise delivery devices, wavelength, intensity and duration of R/NIR-IT individually for different CNS injury types.

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.

In the paper “Oligodendroglia are particularly vulnerable to oxidative damage after neurotrauma in vivo,” we determined the extent of oxidative damage to specific cellular subpopulations and structures within regions vulnerable to secondary degeneration and assessed the effect this had on oligodendroglial function. Comparative assessment of oxidative damage demonstrated selective vulnerability of oligodendroglia, specifically oligodendrocyte progenitor cells (OPCs) to DNA oxidation in vivo. Immunohistochemical fate mapping along the oligodendroglial lineage showed a transient susceptibility of these cells to DNA oxidation, protein nitration, and lipid peroxidation, with mature oligodendrocytes derived immediately after injury more vulnerable to DNA oxidation than their counterparts existing at the time of injury or later derived. In situ hybridization demonstrated a reduction in myelin regulatory factor (MyRF) messenger RNA (mRNA) fluorescence in newly derived mature oligodendrocytes, suggesting a compromise in the production and maintenance of the myelin sheath in these cells. The data imply a deficit in the normal differentiation of OPCs to myelinating oligodendrocytes, associated with a transient increase in oxidative damage, which may contribute to the dysmyelinating phenotype seen at chronic time points after injury. Identifying and understanding the sources of this oxidative damage is integral for the development of therapeutic interventions for neurotrauma.

BackgroundFollowing injury to the central nervous system, increased microglia, secretion of pro- and anti-inflammatory cytokines, and altered blood-brain barrier permeability, a hallmark of degeneration, are observed at and immediately adjacent to the injury site. However, few studies investigate how regions remote from the primary injury could also suffer from inflammation and secondary degeneration.MethodsAdult female Piebald-Viral-Glaxo (PVG) rats underwent partial transection of the right optic nerve, with normal, age-matched, unoperated animals as controls. Perfusion-fixed brains and right optic nerves were harvested for immunohistochemical assessment of inflammatory markers and blood-brain barrier integrity; fresh-frozen brains were used for multiplex cytokine analysis.ResultsImmediately ventral to the optic nerve injury, immunointensity of both the pro-inflammatory biomarker inducible nitric oxide synthase (iNOS) and the anti-inflammatory biomarker arginase-1 (Arg1) increased at 7 days post-injury, with colocalization of iNOS and Arg1 immunoreactivity within individual cells. CD11b+ and CD45+ cells were increased 7 days post-injury, with altered BBB permeability still evident at this time. In the lower and middle optic tract and superior colliculus, IBA1+ resident microglia were first increased at 3 days; ED1+ and CD11b+ cells were first increased in the middle and upper tract and superior colliculus 7 days post-injury. Increased fibrinogen immunoreactivity indicative of altered BBB permeability was first observed in the contralateral upper tract at 3 days and middle tract at 7 days post-injury. Multiplex cytokine analysis of brain homogenates indicated significant increases in the pro-inflammatory cytokines, IL-2 and TNFα, and anti-inflammatory cytokine IL-10 1 day post-injury, decreasing to control levels at 3 days for TNFα and 7 days for IL-2. IL-10 was significantly elevated at 1 and 7 days post-injury with a dip at 3 days post-injury.ConclusionsPartial injury to the optic nerve induces a complex remote inflammatory response, characterized by rapidly increased pro- and anti-inflammatory cytokines in brain homogenates, increased numbers of IBA1+ cells throughout the visual pathways, and increased CD11b+ and ED1+ inflammatory cells, particularly towards the synaptic terminals. BBB permeability can increase prior to inflammatory cell infiltration, dependent on the brain region.

BackgroundFollowing 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 Ca2+ 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 Ca2+ channel inhibitor Lomerizine (Lom), the Ca2+ permeable AMPA receptor inhibitor YM872 and the P2X7 receptor inhibitor oxATP.ResultsFollowing 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.ConclusionsData 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.

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BackgroundRed/near-infrared light therapy (R/NIR-LT) has been developed as a treatment for a range of conditions, including injury to the central nervous system (CNS). However, clinical trials have reported variable or sub-optimal outcomes, possibly because there are few optimized treatment protocols for the different target tissues. Moreover, the low absolute, and wavelength dependent, transmission of light by tissues overlying the target site make accurate dosing problematic.ResultsIn order to optimize light therapy treatment parameters, we adapted a mouse spinal cord organotypic culture model to the rat, and characterized myelination and oxidative stress following a partial transection injury. The ex vivo model allows a more accurate assessment of the relative effect of different illumination wavelengths (adjusted for equal quantal intensity) on the target tissue. Using this model, we assessed oxidative stress following treatment with four different wavelengths of light: 450 nm (blue); 510 nm (green); 660 nm (red) or 860 nm (infrared) at three different intensities: 1.93 × 1016 (low); 3.85 × 1016 (intermediate) and 7.70 × 1016 (high) photons/cm2/s. We demonstrate that the most effective of the tested wavelengths to reduce immunoreactivity of the oxidative stress indicator 3-nitrotyrosine (3NT) was 660 nm. 860 nm also provided beneficial effects at all tested intensities, significantly reducing oxidative stress levels relative to control (p ≤ 0.05).ConclusionsOur results indicate that R/NIR-LT is an effective antioxidant therapy, and indicate that effective wavelengths and ranges of intensities of treatment can be adapted for a variety of CNS injuries and conditions, depending upon the transmission properties of the tissue to be treated.