Mild traumatic brain injury (mTBI) represents a significant public healthcare concern, accounting for the majority of all head injuries. While symptoms are generally transient, some patients go on to experience long-term cognitive impairments and additional mild impacts can result in exacerbated and persisting negative outcomes. To date, studies using a range of experimental models have reported chronic behavioral deficits in the presence of axonal injury and inflammation following repeated mTBI; assessments of oxidative stress and myelin pathology have thus far been limited. However, some models employed induced acute focal damage more suggestive of moderate–severe brain injury and are therefore not relevant to repeated mTBI. Given that the nature of mechanical loading in TBI is implicated in downstream pathophysiological changes, the mechanisms of damage and chronic consequences of single and repeated closed-head mTBI remain to be fully elucidated. This review covers literature on potential mechanisms of damage following repeated mTBI, integrating known mechanisms of pathology underlying moderate–severe TBIs, with recent studies on adult rodent models relevant to direct impact injuries rather than blast-induced damage. Pathology associated with excitotoxicity and cerebral blood flow-metabolism uncoupling, oxidative stress, cell death, blood-brain barrier dysfunction, astrocyte reactivity, microglial activation, diffuse axonal injury, and dysmyelination is discussed, followed by a summary of functional deficits and preclinical assessments of therapeutic strategies. Comprehensive characterization of the pathology underlying delayed and persisting deficits following repeated mTBI is likely to facilitate further development of therapeutic strategies to limit long-term sequelae.

Protein disulfide isomerase (PDI) facilitates proper folding and disulfide bonding of nascent proteins in the endoplasmic reticulum and is secreted by cells and associates with the cell surface. We examined the consequence of over- or underexpression of PDI in HT1080 fibrosarcoma cells for the redox state of cell-surface protein thiols/disulfides. Overexpression of PDI resulted in 3.6–4.2-fold enhanced secretion of PDI and 1.5–1.7-fold increase in surface-bound PDI. Antisense-mediated underexpression of PDI caused 38–53% decreased secretion and 10–33% decrease in surface-bound PDI. Using 5,5′-dithio-bis(2-nitrobenzoic acid) to measure surface protein thiols, a 41–50% increase in surface thiols was observed in PDI-overexpressing cells, whereas a 29–33% decrease was observed in underexpressing cells. Surface thiol content was strongly correlated with cellular (r = 0.998) and secreted (r = 0.969) PDI levels. The pattern of exofacial protein thiols was examined by labeling with the membrane-impermeable thiol reactive compound, 3-(N-maleimidylpropionyl)biocytin. Fourteen identifiable proteins on HT1080 cells were labeled with 3-(N-maleimidylpropionyl)biocytin. The intensity of labeling of 11 proteins was increased with overexpression of PDI, whereas the intensity of labeling of 3 of the 11 proteins was clearly decreased with underexpression of PDI. These findings indicated that secreted PDI was controlling the redox state of existing exofacial protein thiols or reactive disulfide bonds.

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.

Extracellular manipulation of protein disulfide bonds has been implied in diverse biological processes, including penetration of viruses and endotoxin into cells and activation of certain cytokine receptors. We now demonstrate reduction of one or more disulfide bonds in the serine proteinase, plasmin, by a reductase secreted by Chinese hamster ovary or HT1080 cells. Reduction of plasmin disulfide bond(s) triggered proteolysis of the enzyme, generating fragments with the domain structure of the angiogenesis inhibitor, angiostatin. Two of the known reductases secreted by cultured cells are protein disulfide isomerase and thioredoxin, and incubation of plasmin with these purified reductases resulted in angiostatin fragments comparable with those generated from plasmin in cell culture. Thioredoxin-derived angiostatin inhibited proliferation of human dermal microvascular endothelial cells with half-maximal effect at approximately 0.2 μg/ml. Angiostatin made by cells and by purified reductases contained free sulfhydryl group(s), andS-carbamidomethylation of these thiol group(s) ablated biological activity. Neither protein disulfide isomerase nor thioredoxin were the reductases used by cultured cells, because immunodepletion of conditioned medium of these proteins did not affect angiostatin generating activity. The plasmin reductase secreted by HT1080 cells required a small cofactor for activity, and physiologically relevant concentrations of reduced glutathione fulfilled this role. These results have consequences for plasmin activity and angiogenesis, particularly in the context of tumor growth and metastasis. Moreover, this is the first demonstration of extracellular reduction of a protein disulfide bond, which has general implications for cell biology.

Loss of function following injury to the central nervous system is worsened by secondary degeneration of neurons and glia surrounding the injury and 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 semi-quantified 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 days after injury. Injury led to increases in DNA, protein and lipid damage in OPCs and mature oligodendrocytes at 3 days, regardless of proliferative state, associated with a decline in the numbers of OPCs at 7 days. O4+ pre-oligodendrocytes 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 8OHdG immunoreactivity were more likely to be caspase3+. By day 28, newly derived mature oligodendrocytes had significantly reduced MYRF 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 hybridisation have enabled the first demonstration of subpopulation specific oligodendroglial vulnerability to oxidative damage, due to secondary degeneration in vivo. SIGNIFICANCE STATEMENT Injury to the central nervous system is characterised 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 hybridisation 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 MYRF mRNA, yet pre-existing oligodendrocytes are more likely to die.

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.