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Reducing the extent of secondary degeneration following spinal cord injury (SCI) is necessary to preserve function, but treatment options have thus far been limited. A combination of the ion channel inhibitors Lomerizine (Lom), YM872 and oxATP, to inhibit voltage-gated Ca2+ channels, Ca2+ permeable AMPA receptors, and purinergic P2X7 receptors respectively, effectively limits secondary consequences of injury in in vitro and in vivo models of CNS injury. Here, we investigated the efficacy of these inhibitors in a clinically relevant model of SCI. Fischer (F344) rats were subjected to a moderate (150 kD) contusive SCI at thoracic level T10 and assessed at 2 weeks or 10 weeks post-injury. Lom was delivered orally twice daily and YM872 and oxATP were delivered via osmotic mini-pump implanted at the time of SCI until 2 weeks following injury. Open field locomotion analysis revealed that treatment with the three inhibitors in combination improved the rate of functional recovery of the hind limb (compared to controls) as early as 1-day post-injury, with beneficial effects persisting to 14 days post-injury, while all three inhibitors were present. At 2 weeks following combinatorial treatment, the functional improvement was associated with significantly decreased cyst size, increased immunoreactivity of β-III tubulin+ve axons, myelin basic protein, and reduced lipid peroxidation by-products, and increased CC1+ve oligodendrocytes and NG2+ve/PDGFα+ve oligodendrocyte progenitor cell densities, compared to vehicle-treated SCI animals. The combination of Lom, oxATP, and YM872 shows preclinical promise for control of secondary degeneration following SCI, and further investigation of long-term sustained treatment is warranted.

Following neurotrauma, secondary degeneration of neurons and glia adjacent to the injury leads to further functional loss. A combination of ion channel inhibitors (lomerizine + oxATP + YM872) has been shown to be effective at limiting structural and functional loss due to secondary degeneration. Here we assess efficacy of the combination where oxATP is replaced with Brilliant Blue G (BBG), a more clinically applicable P2X7 receptor inhibitor. Partial optic nerve transection was used to model secondary degeneration in adult female rats. Animals were treated with combinations of lomerizine + YM872 + oxATP or lomerizine + YM872 + BBG, delivered via osmotic mini-pump directly to the injury site. Outcomes assessed were Iba1 + and ED1 + microglia and macrophages, oligodendroglial cell numbers, node/paranode structure and visual function using the optokinetic nystagmus test. The lomerizine + BBG + YM872 combination was at least as effective at the tested concentrations as the lomerizine + oxATP + YM872 combination at preserving node/paranode structure and visual function when delivered locally. However, neither ion channel inhibitor combination significantly improved microglial/macrophage nor oligodendroglial numbers compared to vehicle-treated controls. In conclusion, a locally delivered combination of ion channel inhibitors incorporating lomerizine + BBG + YM872 is at least as effective at limiting secondary degeneration following partial injury to the optic nerve as the combination incorporating oxATP.

Background Multiple sclerosis (MS) has been shown to feature oxidative damage, which can be modelled using the cuprizone model of demyelinating disease. Oxidative damage can occur as a result of excessive influx of calcium ions (Ca2+) and oligodendroglia are particularly vulnerable. However, the effects of limiting excess Ca2+ influx on oxidative damage, oligodendroglia and myelin structure are unknown. Objective This study investigated the effects of limiting excess Ca2+ flux on oxidative damage and associated changes in oligodendroglial densities and Node of Ranvier structure in the cuprizone model. Methods The effects of three weeks of cuprizone administration and of treatment with a combination of three ion channel inhibitors (Lomerizine, Brilliant Blue G (BBG) and YM872), were semi-quantified immunohistochemically. Outcomes assessed were protein nitration (3-nitrotyrosine (3NT)) oxidative damage to DNA (8-hydroxy deoxyguanosine (8OHDG)), advanced glycation end-products (carboxymethyl lysine (CML)), immunoreactivity of microglia (Iba1) and astrocytes (glial acidic fibrillary protein (GFAP)), densities of oligodendrocyte precursor cells (OPCs) (platelet derived growth factor alpha receptor (PDGFαR) with olig2) and oligodendrocytes (olig2 and CC1), and structural elements of the Node of Ranvier (contactin associated protein (Caspr)). Results The administration of cuprizone resulted in increased protein nitration, DNA damage, and astrocyte and microglial immunoreactivity, a decrease in the density of oligodendrocytes and OPCs, together with altered structure of the Node of Ranvier and reduced myelin basic protein immunoreactivity. Treatment with the ion channel inhibitor combination significantly lowered protein nitration, increased the density of OPCs and reduced the number of atypical Node of Ranvier complexes; other outcomes were unaffected. Conclusion Our findings suggest that excess Ca2+ influx contributes to protein nitration, and associated changes to OPC densities and Node of Ranvier structure in demyelinating disease.

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 in vivo. 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 in situ hybridization have enabled the first demonstration of subpopulation specific oligodendroglial vulnerability to oxidative damage, due to secondary degeneration in vivo. SIGNIFICANCE STATEMENT 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 in situ 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.

Following mild traumatic brain injury (mTBI), the ionic homeostasis of the central nervous system (CNS) becomes imbalanced. Excess Ca2+ influx into cells triggers molecular cascades, which result in detrimental effects. The authors assessed the effects of a combination of ion channel inhibitors (ICI) following repeated mTBI (rmTBI). Adult female rats were subjected to two rmTBI weight-drop injuries 24 h apart, sham procedures (sham), or no procedures (normal). Lomerizine, which inhibits voltage-gated calcium channels, was administered orally twice daily, whereas YM872 and Brilliant Blue G, inhibiting &alpha, amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and P2X7 receptors, respectively, were delivered intraperitoneally every 48 h post-injury. Vehicle treatment controls were included for rmTBI, sham, and normal groups. At 11 days following rmTBI, there was a significant increase in the time taken to cross the 3 cm beam, as a sub-analysis of neurological severity score (NSS) assessments, compared with the normal control (p &lt, 0.05), and a significant decrease in learning-associated improvement in rmTBI in Morris water maze (MWM) trials relative to the sham (p &lt, 0.05). ICI-treated rmTBI animals were not different to sham, normal controls, or rmTBI treated with vehicle in all neurological severity score and Morris water maze assessments (p &gt, 0.05). rmTBI resulted in increases in microglial cell density, antioxidant responses (manganese-dependent superoxide dismutase (MnSOD) immunoreactivity), and alterations to node of Ranvier structure. ICI treatment decreased microglial density, MnSOD immunoreactivity, and abnormalities of the node of Ranvier compared with vehicle controls (p &lt, 0.01). The authors&rsquo, findings demonstrate the beneficial effects of the combinatorial ICI treatment on day 11 post-rmTBI, suggesting an attractive therapeutic strategy against the damage induced by excess Ca2+ following rmTBI.

Background Partial transection (PT) of the optic nerve is an established experimental model of secondary degeneration in the central nervous system. After a dorsal transection, retinal ganglion cells (RGCs) with axons in ventral optic nerve are intact but vulnerable to secondary degeneration, whereas RGCs in dorsal retina with dorsal axons are affected by primary and secondary injuries. Using microarray, we quantified gene expression changes in dorsal and ventral retina at 1 and 7 days post PT, to characterize pathogenic pathways linked to primary and secondary degeneration. Results In comparison to uninjured retina Cryba1, Cryba2 and Crygs, were significantly downregulated in injured dorsal retina at days 1 and 7. While Ecel1, Timp1, Mt2A and CD74, which are associated with reducing excitotoxicity, oxidative stress and inflammation, were significantly upregulated. Genes associated with oxygen binding pathways, immune responses, cytokine receptor activity and apoptosis were enriched in dorsal retina at day 1 after PT. Oxygen binding and apoptosis remained enriched at day 7, as were pathways involved in extracellular matrix modification. Fewer changes were observed in ventral retina at day 1 after PT, most associated with the regulation of protein homodimerization activity. By day 7, apoptosis, matrix organization and signal transduction pathways were enriched. Discriminant analysis was also performed for specific functional gene groups to compare expression intensities at each time point. Altered expression of selected genes (ATF3, GFAP, Ecel1, TIMP1, Tp53) and proteins (GFAP, ECEL1 and ATF3) were semi-quantitatively assessed by qRT-PCR and immunohistochemistry respectively. Conclusion There was an acute and complex primary injury response in dorsal retina indicative of a dynamic interaction between neuroprotective and neurodegenerative events; ventral retina vulnerable to secondary degeneration showed a delayed injury response. Both primary and secondary injury resulted in the upregulation of numerous genes linked to RGC death, but differences in the nature of these changes strongly suggest that death occurred via different molecular mechanisms.

Glutamate excitotoxicity contributes to damage following injury to the central nervous system via mechanisms including changes in the expression of receptors, calcium overload, oxidative stress and cell death. Excitotoxicity is triggered by glutamate binding to receptors, including calcium permeable AMPA receptors, which can be upregulated under injury conditions. However, the transcriptional response of AMPA receptor regulatory proteins to excitotoxic conditions is unknown. Here, we use real-time quantitative PCR (RT-qPCR), to determine the effect of prolonged glutamate excitotoxicity on the expression of mRNA encoding for GluR1 and AMPA receptor regulatory proteins in dissociated rat retinal cultures that include neuronal (retinal ganglion cell (RGCs)) and glial (Müller) cell populations. mRNA levels of GluR1 and regulators of the GluR1 subunit of AMPA receptors, including Sap97, Cnih2 and Cnih3, decreased following 6, 24 and 48 h incubation with 5 mM glutamate: related regulators not associated with GluR1 were unaffected. In contrast, GluR1 protein, assessed immunohistochemically, was increased in both RGCs and Müller cells after 24 h glutamate exposure: western blotting analysis was inconclusive. Reductions in mRNA of GluR1 and associated regulators occurred prior to cell death, which was first detected at 24 h, and substantial by 48 h. Exposure to glutamate acutely increased the intracellular calcium concentration in the cultures and by 24 h, reactive oxygen species were increased. Our data suggest a negative feedback mechanism in retinal cells, that down-regulates transcription of genes encoding GluR1 regulatory proteins in response to glutamate exposure. Despite this mechanism, GluR1 protein levels remain increased, and are associated with increased reactive species and cell death. Therapeutic strategies targeting calcium permeable AMPA receptors should take into account that increases in GluR1 protein are not necessarily associated with increases in associated mRNA levels over time.

Secondary degeneration is a serious consequence of traumatic injury to the central nervous system (CNS) and involves the progressive loss of neurons and function. However, while disruption to myelin has been observed in spared axons, the ultrastructural abnormalities that occur in myelin and axons spatially separated from the primary injury and susceptible exclusively to secondary degeneration are unknown. We used a model of secondary degeneration in which the dorsal aspect of rat optic nerve (ON) was transected leaving the central/ventral ON undamaged, but vulnerable to secondary degeneration. Transmission electron microscopy of the central/ventral ON at 1 and 3 months was used to quantify secondary changes in axon diameter, myelin sheath thickness and morphology, compared to normal animals. Three months after partial ON transection, cross-sectional nerve area at the injury site was increased (p ≤ 0.05), and changes in axons and myelin sheaths were detected in the central/ventral ON. Although myelin sheath thickness remained normal at both time points, average axon diameter significantly increased at 3 months. The density of the total axon population was decreased by 1 month, reflecting loss of retinal ganglion cells as previously published, with a decrease in the density of normally-myelinated axons, but an increase in unmyelinated axon density (p ≤ 0.05). Myelin basic protein immunoreactivity and fluoromyelin staining were also significantly reduced. Within four subpopulations of abnormally-myelinated axons, there was: no change in lightly-myelinated axons; an increase in axons with excessive myelination (at 1 month); and an increase in the density of axons with partial and fully-decompacted myelin (at 3 months, p ≤ 0.05). Chronic axon swelling and myelin sheath compaction defects are features of secondary degeneration, and may contribute to the reported loss of ON function following partial transection.

RADA16 self-assembling peptide nanofiber scaffolds (SAPNSs) have been shown to have positive effects on neural regeneration following injury to the central nervous system in vivo, but mechanisms are unclear. Here we show that RADA16 SAPNSs form scaffolds of increasing fiber density with increasing peptide concentration which in turn has a concentration-dependent effect on neurons and astrocytes in mixed retinal cultures. Importantly, we report that the final nanoscale fiber architecture is an important factor to consider in designing scaffolds to promote regeneration in the central nervous system.

Secondary degeneration is a form of ‘bystander’ damage that can affect neural tissue both nearby and remote from an initial injury. Partial optic nerve transection is an excellent model in which to unequivocally differentiate events occurring during secondary degeneration from those resulting from primary CNS injury. We analysed the primary injury site within the optic nerve (ON) and intact areas vulnerable to secondary degeneration. Areas affected by the primary injury showed morphological disruption, loss of β-III tubulin axonal staining, reduced myelinated axon density, greater proteoglycan expression (phosphacan), increased microglia and macrophage numbers and increased oxidative stress. Similar, but less extreme, changes were seen in areas of the optic nerve undergoing secondary degeneration. The CNS-specific L- and T-type calcium channel blocker lomerizine alleviated some of the changes in areas vulnerable to secondary degeneration. Lomerizine reduced morphological disruption, oxidative stress and phosphacan expression, and limited early increases in macrophage numbers. However, lomerizine failed to prevent progressive de-myelination of ON axons. Within the retina, secondary retinal ganglion cell (RGC) death was significant in areas vulnerable to secondary degeneration. Lomerizine protected RGCs from secondary death at 4 weeks but did not fully restore behavioural function (optokinetic nystagmus). We conclude that blockade of calcium channels is neuroprotective and limits secondary degenerative changes following CNS injury. However such an approach may need to be combined with other treatments to ensure long-term maintenance of full visual function.

A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.

During development, gradients of EphA receptors (nasal(low)-temporal(high)) and their ligands ephrin-As (rostral(low)-caudal(high)) are involved in establishing topography between retinal ganglion cells (RGCs) and the superior colliculus (SC). EphA5-expressing RGC axons are repulsed by ephrin-A2-expressing SC neurones. In adult rats RGCs maintain graded EphA5 expression but ephrin-A2 expression is down-regulated in the SC to a weak gradient. At 1 month after optic nerve transection, EphA5 expression is reduced in the few remaining RGCs and is no longer graded; by contrast, SC ephrin-A2 is up-regulated to a rostral(low)-caudal(high) gradient. Here we examined expression in adult rat 1 month after bridging the retina and SC with a peripheral nerve graft, a procedure that enhances RGC survival and permits RGC axon regeneration. Double labelling with cell markers revealed preservation of a nasal(low)-temporal(high) EphA5 gradient in RGCs and establishment of a rostral(low)-caudal(high) ephrin-A2 gradient within neurones of the SC. The results suggest a potential for guidance cues to restore the topography of RGC axons in the SC. However, high ephrin-A2 levels were also found in astrocytes surrounding the peripheral nerve graft insertion site. The repulsive ephrin-A2 environment offers at least a partial explanation for the observation that only a limited number of RGC axons can exit the graft to enter target central nervous system tissue.

A novel allograft paradigm was used to test whether adult mammalian central axons regenerate within a peripheral nerve environment containing intact sensory axons. Retinal ganglion cell axon regeneration was compared following anastomosis of dorsal root ganglia grafts or conventional peripheral nerve grafts to the adult rat optic nerve. Dorsal root ganglia grafts comprised intact sensory and degenerate motor axons, whereas conventional grafts comprised both degenerating sensory and motor axons. Retinal ganglion cell axons were traced after 2 months. Dorsal root ganglia survived with their axons persisting throughout the graft. Comparable numbers of retinal ganglion cells regenerated axons into both dorsal root ganglia (1053 ± 223) and conventional grafts (1323 ± 881; P > 0.05). The results indicate that an intact sensory environment supports central axon regeneration.

Eph tyrosine kinase receptors and their ligands, the ephrins, play a key role in the establishment of retinotectal topography during development. Tectal up-regulation of ephrin-A2 in goldfish, coincident with the reestablishment of a retinotectal map, suggests a similar role during optic nerve regeneration. Here we report a complementary study of EphA3, EphA5 and ephrin-A2 expression in the retina. EphA3 and EphA5 are transiently up-regulated as ascending naso-temporal gradients, whereas ephrin-A2 remains uniform. The expression profiles differ from those in developing chick and mouse, suggesting that different combinations of retinal Eph receptors and ligands can generate topographic guidance information.

The addition of polysialic acid (PSA) to neural cell adhesion molecule (NCAM) facilitates axon growth. Here we use Western blots and immunohistochemistry to examine expression of PSA-NCAM during optic nerve regeneration. In lizard, retinal ganglion cell axons become transiently PSA-NCAM positive. By contrast, goldfish RGC axons are PSA-NCAM negative both in normal animals and throughout regeneration with the exception of a PSA-NCAM-positive fascicle arising from newly generated RGCs. Transient sialylation of NCAM in lizard may assist regeneration in the nonpermissive reptilian visual pathway and facilitate the reestablishment of a crude topographic map; down-regulation in the long term may contribute to the breakdown in topography. The lack of sialylation in goldfish presumably reflects the permissive nature of the substrate allowing axon regeneration and the successful reestablishment of a topographic map.

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 7days 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 7days after injury, respectively. Despite increased antioxidant immunoreactivity, DNA oxidation was evident from 1day, lipid oxidation at 3days, and protein nitration at 7days 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 3days. 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.

Rapid changes in Ca(2+) concentration and location in response to injury play key roles in a range of biological systems. However, quantitative analysis of changes in size and distribution of Ca(2+) microdomains in specific cell types in whole tissue samples has been limited by analytical resolution and reliance on indirect Ca(2+) indicator systems. Here, we combine the unique advantages of nanoscale secondary ion mass spectrometry (NanoSIMS) with immunohistochemistry to directly quantify changes in number, size and intensity of Ca microdomains specific to axonal or glial regions vulnerable to spreading damage following neurotrauma. Furthermore, using NanoSIMS allows separate quantification of Ca microdomains according to their co-localization with areas enriched in P. We rapidly excise and cryopreserve optic nerve segments from adult rat at time points ranging from 5 minutes to 3 months after injury, allowing assessment of Ca microdomains dynamics with minimal disruption due to tissue processing. We demonstrate significantly more non-P co-localized Ca microdomains in glial than axonal regions in normal optic nerve. The density of Ca microdomains not co-localized with areas enriched in P rapidly, selectively and significantly decreases after injury; densities of Ca microdomains co-localized with P enriched areas are unchanged. An efflux of Ca(2+) from microdomains not co-localized with P may contribute to the structural and functional deficits observed in nerve vulnerable to spreading damage following neurotrauma. NanoSIMS analyses of Ca microdomains allow quantitative and novel insights into Ca dynamics, applicable to a range of normal, as well as diseased or injured mammalian systems.

During development, a graded expression of ephrin A2 has been implicated in retinotectal map formation. Here we have examined ephrin A2 expression during optic nerve regeneration in the mature goldfish. In the tecta of normal animals, a gradient of ephrin A2 expression is detected in cell bodies within the stratum fibrosum et griseum superficiale with more immunopositive cells caudally than rostrally. The gradient in the mature animal presumably reflects the plasticity associated with continued retinal and tectal neurogenesis. During optic nerve regeneration, expression throughout the tectum is increased by 1 month as a strong rostrocaudal gradient. The gradient declines to normal by 3 months. The up-regulation of ephrin A2 during optic nerve regeneration is likely to be instrumental in reestablishing the retinotectal map.

Partial injury to the central nervous system (CNS) is exacerbated by additional loss of neurons and glia via toxic events known as secondary degeneration. Using partial transection of the rat optic nerve (ON) as a model, we have previously shown that myelin decompaction persists during secondary degeneration. Failure to repair myelin abnormalities during secondary degeneration may be attributed to insufficient OPC proliferation and/or differentiation to compensate for loss of oligodendrocyte lineage cells (oligodendroglia). Following partial ON transection, we found that sub-populations of oligodendroglia and other olig2+ glia were differentially influenced by injury. A high proportion of NG2+/olig2–, NG2+/olig2+ and CC1−/olig2+ cells proliferated (Ki67+) at 3 days, prior to the onset of death (TUNEL+) at 7 days, suggesting injury-related cues triggered proliferation rather than early loss of oligodendroglia. Despite this, a high proportion (20%) of the NG2+/olig2+ OPCs were TUNEL+ at 3 months, and numbers remained chronically lower, indicating that proliferation of these cells was insufficient to maintain population numbers. There was significant death of NG2+/olig2– and NG2−/olig2+ cells at 7 days, however population densities remained stable, suggesting proliferation was sufficient to sustain cell numbers. Relatively few TUNEL+/CC1+ cells were detected at 7 days, and no change in density indicated that mature CC1+ oligodendrocytes were resistant to secondary degeneration in vivo. Mature CC1+/olig2– oligodendrocyte density increased at 3 days, reflecting early oligogenesis, while the appearance of shortened myelin internodes at 3 months suggested remyelination. Taken together, chronic OPC decreases may contribute to the persistent myelin abnormalities and functional loss seen in ON during secondary degeneration.

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.

Secondary degeneration in the central nervous system involves indirect damage to neurons and glia away from the initial injury. Partial transection of the dorsal optic nerve (ON) results in precise spatial separation of the primary trauma from delayed degenerative events in ventrally placed axons and parent somata. Here we conduct an immunohistochemical survey of secondary cellular changes in and around axons and their parent retinal ganglion cell (RGC) somata during the first 3 days after a restricted, dorsal ON transection. This is before the secondary loss of RGCs and axons projecting through the uninjured, ventral portion of the ON. Within 5 min, manganese superoxide dismutase (MnSOD; a marker of oxidative stress) co-localizes within the astrocytic network across the entire profile of the ON. Secondary astrocyte hypertrophy of immunofluorescent labeling was evident from 3 h, with sustained increases in myelin basic protein immunoreactivity across the nerve by 24 h. Increases in NG-2-positive oligodendrocyte precursor cells, ED-1-positive activated microglia/macrophages, and Iba1-positive reactive resident microglia/macrophage numbers were only seen in ON vulnerable to secondary degeneration by 3 days. Changes within RGC somata exclusively vulnerable to secondary degeneration were detected at 24 h, as evidenced by increases in MnSOD immunoreactivity, followed by increases in c-jun immunoreactivity at 3 days. Treatment with the voltage-gated calcium channel blocker lomerizine did not alter any measured outcome. We conclude that oxidative stress spreading via the astrocytic network and from injured axons to parent RGC somata is an early event during secondary degeneration, and containment is likely to be required in order to prevent further damage to the nerve.

The membrane-bound proteins ephrins and their receptors, Eph receptor tyrosine kinases, are known for their key role during development of the central nervous system (CNS). Ligand/receptor interactions as a result of cell–cell contacts activate intracellular signalling pathways which mediate specific cellular responses. Activation can occur bidirectionally in both the receptor and the ligand-bearing cells. Eph receptor and ephrin families have been implicated in synaptic plasticity in the mature brain: effects include long-term potentiation/depression of excitatory transmission (LTP/LTD) and an action on the structure and number of synaptic contacts. However, due to the redundancy of binding between receptors and ligands, the role of individual proteins has not yet been completely elucidated. Ephrin-B1 has been suggested to play a role in synaptic plasticity in the hippocampus, but its expression and localization at pre- or post-synaptic sites has been poorly documented, most likely due to the apparent low activity of the corresponding gene in mature brain. Here we present immunohistochemical data demonstrating a broad but highly regulated cellular distribution of ephrin-B1 in the mature mouse brain. We show that ephrin-B1 is expressed post-synaptically on dendritic spines in the cortex, supporting a role in synaptic plasticity in this region. However, the prevalent extra-synaptic distribution in regions such as the hippocampus and cerebellum suggests an additional structural role, perhaps at the neuron/glia interface.

Topographically ordered projections are established by molecular guidance cues and refined by neuronal activity. Retinal input to a primary visual center, the superior colliculus (SC), is bilateral with a dense contralateral projection and a sparse ipsilateral one. Both projections are topographically organized, but in opposing anterior–posterior orientations. This arrangement provides functionally coherent input to each colliculus from the binocular visual field, supporting visual function. When guidance cues involved in contralateral topography (ephrin-As) are absent, crossed retinal ganglion cell (RGC) axons form inappropriate terminations within the SC. However, the organization of the ipsilateral projection relative to the abnormal contralateral input remains unknown, as does the functional capacity of both projections. We show here that inephrin-A−/−mice, the SC contains an expanded, diffuse ipsilateral projection. Electrophysiological recording demonstrated that topography of visually evoked responses recorded from the contralateral superior colliculus ofephrin-A−/−mice displayed similar functional disorder in all genotypes, contrasting with their different degrees of anatomical disorder. In contrast, ipsilateral responses were retinotopic inephrin-A2−/−but disorganized inephrin-A2/A5−/−mice. The lack of integration of binocular input resulted in specific visual deficits, which could be reversed by occlusion of one eye. The discrepancy between anatomical and functional topography in both the ipsilateral and contralateral projections implies suppression of inappropriately located terminals. Moreover, the misalignment of ipsilateral and contralateral visual information inephrin-A2/A5−/−mice suggests a role for ephrin-As in integrating convergent visual inputs.

The vertebrate visual system is a valuable model for examining recovery after injury to the central nervous system (CNS). It is a relatively “simple” part of the CNS having one major class of projection neuron, the retinal ganglion cells (RGCs), which make topographic connections within well defined visual nuclei, thus recreating visual space within the brain. Topographic maps can be readily assessed electrophysiologically and anatomically and are a critical template for useful visually guided behaviour which can be examined behaviourally. Furthermore, the optic nerve is accessible, an extra-foramenal crush injury severing all RGC axons but leaving the meningeal sheath intact as a conduit for regeneration and preventing gross axonal mis-routing. The procedure also leaves the blood supply to the eye patent, avoiding ischaemic-induced RGC death.

Ephrin ligands and their receptors Eph receptor tyrosine-kinases have received extensive attention for their multiple key roles during development, particularly in the central nervous system (CNS). For example, at early stages of brain and spinal cord development, membrane-bound ephrins provide signals that direct migrating cells and axons. However, much less is known about the role of ephrins and Eph receptors in the adult CNS. Here, we investigated the distribution of ephrin-B2 protein expression in the adult mouse brain to gain insight into its possible function(s). We show that ephrin-B2 is expressed in areas with high levels of synaptic plasticity, such as the cerebral cortex, hippocampus and cerebellum. However, at the cellular level, ephrin-B2 was localized to neuronal cell bodies rather than to the dendritic synaptic sites where mechanisms of long-term modifications of excitatory transmission are located. Our results suggest a role for ephrin-B2 in the membrane at the cell body, possibly in relation to axonal-somatic inhibitory synapses.

EphA receptors and their ligands the ephrin-As, expressed as retinal and tectal gradients, are required for the development of retino-tectal topography [Neuron 25 (2000) 563] and its restoration during goldfish optic nerve regeneration [Mol. Cell. Neurosci. 25 (2004) 56]. We have reported previously that, during regeneration, a transient EphA3/A5 gradient is formed by differential expression across the entire retinal ganglion cell (RGC) population [Neurosci. Abs. 33 (2003) 358.2; Exp. Neurol. 183 (2003) 593]. In retino-recipient tectal layers, ephrin-A2 is normally expressed by only a sub-population of cells, but during regeneration, there is a graded increase with more expressing cells caudally than rostrally [Exp. Neurol. 166 (2000) 196]. Here, we examine the characteristics of tectal ephrin-A2 expression during regeneration. We report that the level of ephrin-A2 expression is comparable for all ephrin-A2-positive cells in normal animals and during regeneration. Using double-labelling immunohistochemistry for ephrin-A2 and specific cell markers (NeuN for neurons, GA5 for astrocytes, NN-1 for microglia/endothelial cells and 6D2 for oligodendrocytes), we demonstrate that ephrin-A2-expressing cells, as in normal animals, are exclusively neuronal. Moreover, double labelling with BrdU showed that ephrin-A2 is expressed in resident cells and not those generated during optic nerve regeneration [Brain Res. 854 (2000) 178, 153 (1978) 345].

During visual system development, interactions between Eph tyrosine kinase receptors and their ligands, the ephrins, guide retinal ganglion cell (RGC) axons to their topographic targets in the optic tectum. Here we show that Eph/ephrin interactions are also involved in restoring topography during RGC axon regeneration in goldfish. Following optic nerve crush, EphA/ephrin-A interactions were blocked by intracranial injections of recombinant Eph receptor (EphA3-AP) or phospho-inositol phospholipase-C. Topographic errors with multiple inputs to some tectal loci were detected electrophysiologically and increased projections to caudal tectum demonstrated by RT-97 immunohistochemistry. In EphA3-AP-injected fish, ephrin-A2-expressing cells in the retino-recipient tectal layers were reduced in number compared to controls and their distribution was no longer graded. The findings, supported by in vitro studies, implicate EphA/ephrin-A interactions in restoring precise topography and in regulating ephrin-A2 expression during regeneration.

PURPOSE. After partial optic nerve (ON) injury, intact retinal ganglion cells (RGCs) undergo secondary death, but the topographic distribution of this death is unknown, and it is unclear which cell death pathways are involved. Although the calcium channel blocker lomerizine reduces RGC death after partial ON injury, it is unknown whether this drug alleviates necrotic or apoptotic death. METHODS. The dorsal ON was transected in adult Piebald-VirolGlaxo (PVG) rats, and the site of secondary RGC death was determined using anterograde and retrograde DiI tracing. RGC death was assessed at 2 and 3 weeks. Retrograde tracing with fluorogold injected into the superior colliculus 3 days before euthanatization was used to identify RGCs undergoing secondary death. Overall cell loss was quantified using III-tubulin immunohistochemistry. Lomerizine (30 mg/kg, oral) or vehicle was given twice daily, and retinal wholemounts were analyzed for necrotic morphology (nucleic acid stain) or anticleaved caspase-3 expression at 2 and 3 weeks. RESULTS. Ventral retina was identified as the site of secondary RGC death, and central and dorsal retinae were defined as sites of both primary and secondary death. Overall RGC loss occurred by 2 weeks in central and ventral retina (P 0.05) and by 3 weeks in dorsal retina (P 0.05). Secondary RGC death was characterized mainly by necrotic morphology, with caspase-3 expression in some RGCs. Lomerizine reduced secondary necrosis at 2 weeks and secondary caspase-3 expression at 3 weeks. CONCLUSIONS. Lomerizine had differential effects on necrotic and apoptotic death with time, but its inability to completely prevent secondary death suggests that full neuroprotection will require combinatorial treatments. (Invest Ophthalmol Vis Sci. 2009;50:5456‐5462) DOI:10.1167/iovs.09-3717

Background Traumatic injury to the central nervous system results in damage to tissue beyond the primary injury, termed secondary degeneration. Key events thought to be associated with secondary degeneration involve aspects of mitochondrial function which may be modulated by red/near-infrared irradiation therapy (R/NIR-IT), but precisely how mitochondria are affected in vivo has not been investigated. Secondary degeneration was modelled by transecting the dorsal aspect of the optic nerve in adult rats and mitochondrial ultrastructure in intact ventral optic nerve vulnerable to secondary degeneration investigated with transmission electron microscopy. Results Despite reported increases in fission following central nervous system injury, we saw no change in mitochondrial densities in optic nerve vulnerable to secondary degeneration in vivo. However, in axons, frequency distributions of mitochondrial profile areas showed higher cumulative probabilities of smaller mitochondrial profiles at day 1 after injury. Glial mitochondrial profiles did not exhibit changes in area, but a more elliptical mitochondrial shape was observed at both day 1 and 7 following injury. Importantly, mitochondrial autophagic profiles were observed at days 1 and 7 in optic nerve vulnerable to secondary degeneration in vivo. Citrate synthase activity was used as an additional measure of mitochondrial mass in ventral optic nerve and was decreased at day 7, whereas mitochondrial aconitase activity increased at day 1 and day 28 after injury in optic nerve vulnerable to secondary degeneration. R/NIR-IT has been used to treat the injured central nervous system, with reported improvements in oxidative metabolism suggesting mitochondrial involvement, but ultrastructural information is lacking. Here we show that R/NIR-IT of injured animals resulted in distributions of mitochondrial areas and shape not significantly different from control and significantly reduced mitochondrial autophagic profiles. R/NIR-IT also resulted in decreased citrate synthase activity (day 7) and increased aconitase activity (day 1) in optic nerve vulnerable to secondary degeneration. Conclusions These findings suggest that mitochondrial structure and activity of enzymes of the citric acid cycle are dynamically altered during secondary degeneration in vivo and R/NIR-IT may protect mitochondrial structure.