Effects of 670 nm light on RGC numbers and visual function.

<p>Mean ± SEM retrograde labelled RGC numbers (central or ventral retinal regions) (a) and responses in the optokinetic nystagmus test of visual function (smooth pursuits or fast resets) (b), in 670 nm treated or control animals 3 months after injury, * significant differences indicated (p≤0.05) (n = 4–5 animals / group), PT is partial ON transection injury.</p

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

Representative TEM images from normal ventral ON (a) and from day 1 after injury (b–d).

<p>Note the disorganisation, lack of definition (arrow head) and multi – layering (arrows) in the paranodal loops from ON vulnerable to secondary degeneration (b, c) and the complete breakdown in structure of one paranode in a node/paranode complex (d), scale bar  = 0.5 μm.</p

Effects of 670 nm light on node/paranode complexes of ventral ON.

<p>Mean ± SEM paranode length (a) and length of the paranodal gap (b) in ventral ON of 670 nm treated and control animals, 1 day after injury; representative images (c). Mean ± SEM percentage of single paranodes in the same groups (d); Caspr^+ve paranodes are green, β-III tubulin^+ve paranodal gaps are red, scale bar  = 5 μm, * significant differences indicated (p≤0.05), PT is partial ON transection injury. Representative TEM images of PT injured (e) and PT 670 nm light treated (f) node/paranode complexes in ventral ON 1 day after injury. Note the increased definition of the paranodal loops in 670 nm light treated animals, but continued disorganisation (arrows). Representative example of a putative hemi – node in ventral ON from 670 nm light treated animal, with one half of the node/paranode complex clearly defined and the other disorganised (g), scale bars for TEM images  = 0.5 μm.</p

Cytochrome<i>c</i> oxidase activity after injury, +/− 670 nm light.

<p>Semi – quantification of mean ± SEM cytochrome <i>c</i> oxidase activity in ON including both the dorsal injury site and the ventral region vulnerable to secondary degeneration (a) or in ventral ON only (b) from normal animals and 1, 3, 7 days, 1 and 3 months after injury. Similarly, mean ± SEM cytochrome <i>c</i> oxidase activity in handled normal, PT handled or PT 670 nm treated animals, encompassing the dorsal injury site and ventral ON (c), or in ventral ON only, 1 day after injury (d). Mean ± SEM cytochrome <i>c</i> oxidase activity encompassing the dorsal injury site and ventral ON (defined by region enclosed in dotted lines in F) in PT handled or PT 670 nm treated ON compared to handled normal, 3 months after injury (e); representative images of cytochrome <i>c</i> oxidase activity histochemistry at 3 months (f), scale bar  = 100 μm (n = 4–5 animals/group), * significant differences indicated (p≤0.05), PT is partial ON transection injury.</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

Effects of 670 nm light treatment on oxidative stress indicators.

<p>(a) Mean ± SEM ROS/RNS assessed as DCF fluorescence in pooled homogenates from dorsal or ventral ON from PT handled or PT 670 nm treated animals, 1 day after injury (6 animals pooled per time point, assayed in duplicate). Semi – quantification of maximum (b) or mean (c) ± SEM intensity of DHE staining in CC1^+ve oligodendrocytes in ventral ON of PT handled or PT 670 nm treated animals, 1 day after injury, assessed by tracing identified cells in single images in the z axis; with (d) representative images, example of identified cells indicated, scale bar  = 10 μm. Similarly, mean ± SEM intensity of CML immunoreactivity in CC1^+ve (e) or olig1^+ve (f) oligodendrocytes in ventral ON of PT handled or PT 670 nm treated animals, 1 day after injury. PT is partial ON transection injury.</p

Paranode Abnormalities and Oxidative Stress in Optic Nerve Vulnerable to Secondary Degeneration: Modulation by 670 nm Light Treatment

Secondary degeneration of nerve tissue adjacent to a traumatic injury results in further loss of neurons, glia and function, via mechanisms that may involve oxidative stress. However, changes in indicators of oxidative stress have not yet been demonstrated in oligodendrocytes vulnerable to secondary degeneration in vivo. We show increases in the oxidative stress indicator carboxymethyl lysine at days 1 and 3 after injury in oligodendrocytes vulnerable to secondary degeneration. Dihydroethidium staining for superoxide is reduced, indicating endogenous control of this particular reactive species after injury. Concurrently, node of Ranvier/paranode complexes are altered, with significant lengthening of the paranodal gap and paranode as well as paranode disorganisation. Therapeutic administration of 670 nm light is thought to improve oxidative metabolism via mechanisms that may include increased activity of cytochrome c oxidase. Here, we show that light at 670 nm, delivered for 30 minutes per day, results in in vivo increases in cytochrome c oxidase activity co-localised with oligodendrocytes. Short term (1 day) 670 nm light treatment is associated with reductions in reactive species at the injury site. In optic nerve vulnerable to secondary degeneration superoxide in oligodendrocytes is reduced relative to handling controls, and is associated with reduced paranode abnormalities. Long term (3 month) administration of 670 nm light preserves retinal ganglion cells vulnerable to secondary degeneration and maintains visual function, as assessed by the optokinetic nystagmus visual reflex. Light at a wavelength of 670 nm may serve as a therapeutic intervention for treatment of secondary degeneration following neurotrauma. © 2013 Szymanski et al