Elemental Mapping in a Preclinical Animal Model Reveals White Matter Copper Elevation in the Acute Phase of Central Nervous System Trauma.

Understanding the chemical events following trauma to the central nervous system could assist in identifying causative mechanisms and potential interventions to protect neural tissue. Here, we apply a partial optic nerve transection model of injury in rats and use synchrotron X-ray fluorescence microscopy (XFM) to perform elemental mapping of metals (K, Ca, Fe, Cu, Zn) and other related elements (P, S, Cl) in white matter tracts. The partial optic nerve injury model and spatial precision of microscopy allow us to obtain previously unattained resolution in mapping elemental changes in response to a primary injury and subsequent secondary effects. We observed significant elevation of Cu levels at multiple time points following the injury, both at the primary injury site and in neural tissue near the injury site vulnerable to secondary damage, as well as significant changes in Cl, K, P, S, and Ca. Our results suggest widespread metal dyshomeostasis in response to central nervous system trauma and that altered Cu homeostasis may be a specific secondary event in response to white matter injury. The findings highlight metal homeostasis as a potential point of intervention in limiting damage following nervous system injury.

Secondary Degeneration of Oligodendrocyte Precursor Cells Occurs as Early as 24 h after Optic Nerve Injury in Rats.

Optic nerve injury causes secondary degeneration, a sequela that spreads damage from the primary injury to adjacent tissue, through mechanisms such as oxidative stress, apoptosis, and blood-brain barrier (BBB) dysfunction. Oligodendrocyte precursor cells (OPCs), a key component of the BBB and oligodendrogenesis, are vulnerable to oxidative deoxyribonucleic acid (DNA) damage by 3 days post-injury. However, it is unclear whether oxidative damage in OPCs occurs earlier at 1 day post-injury, or whether a critical 'window-of-opportunity' exists for therapeutic intervention. Here, a partial optic nerve transection rat model of secondary degeneration was used with immunohistochemistry to assess BBB dysfunction, oxidative stress, and proliferation in OPCs vulnerable to secondary degeneration. At 1 day post-injury, BBB breach and oxidative DNA damage were observed, alongside increased density of DNA-damaged proliferating cells. DNA-damaged cells underwent apoptosis (cleaved caspase3+), and apoptosis was associated with BBB breach. OPCs experienced DNA damage and apoptosis and were the major proliferating cell type with DNA damage. However, the majority of caspase3+ cells were not OPCs. These results provide novel insights into acute secondary degeneration mechanisms in the optic nerve, highlighting the need to consider early oxidative damage to OPCs in therapeutic efforts to limit degeneration following optic nerve injury.

Cuprizone feed formulation influences the extent of demyelinating disease pathology.

Cuprizone is a copper-chelating agent that induces pathology similar to that within some multiple sclerosis (MS) lesions. The reliability and reproducibility of cuprizone for inducing demyelinating disease pathology depends on the animals ingesting consistent doses of cuprizone. Cuprizone-containing pelleted feed is a convenient way of delivering cuprizone, but the efficacy of these pellets at inducing demyelination has been questioned. This study compared the degree of demyelinating disease pathology between mice fed cuprizone delivered in pellets to mice fed a powdered cuprizone formulation at an early 3 week demyelinating timepoint. Within rostral corpus callosum, cuprizone pellets were more effective than cuprizone powder at increasing astrogliosis, microglial activation, DNA damage, and decreasing the density of mature oligodendrocytes. However, cuprizone powder demonstrated greater protein nitration relative to controls. Furthermore, mice fed control powder had significantly fewer mature oligodendrocytes than those fed control pellets. In caudal corpus callosum, cuprizone pellets performed better than cuprizone powder relative to controls at increasing astrogliosis, microglial activation, protein nitration, DNA damage, tissue swelling, and reducing the density of mature oligodendrocytes. Importantly, only cuprizone pellets induced detectable demyelination compared to controls. The two feeds had similar effects on oligodendrocyte precursor cell (OPC) dynamics. Taken together, these data suggest that demyelinating disease pathology is modelled more effectively with cuprizone pellets than powder at 3 weeks. Combined with the added convenience, cuprizone pellets are a suitable choice for inducing early demyelinating disease pathology.

Acute Cellular and Functional Changes With a Combinatorial Treatment of Ion Channel Inhibitors Following Spinal Cord Injury.

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 ß-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.

Intravitreal application of AAV-BDNF or mutant AAV-CRMP2 protects retinal ganglion cells and stabilizes axons and myelin after partial optic nerve injury.

Secondary degeneration following an initial injury to the central nervous system (CNS) results in increased tissue loss and is associated with increasing functional impairment. Unilateral partial dorsal transection of the adult rat optic nerve (ON) has proved to be a useful experimental model in which to study factors that contribute to secondary degenerative events. Using this injury model, we here quantified the protective effects of intravitreally administered bi-cistronic adeno-associated viral (AAV2) vectors encoding either brain derived neurotrophic factor (BDNF) or a mutant, phospho-resistant, version of collapsin response mediator protein 2 (CRMP2T555A) on retinal ganglion cells (RGCs), their axons, and associated myelin. To test for potential synergistic interactions, some animals received combined injections of both vectors. Three months post-injury, all treatments maintained RGC numbers in central retina, but only AAV2-BDNF significantly protected ventrally located RGCs exclusively vulnerable to secondary degeneration. Behaviourally, treatments that involved AAV2-BDNF significantly restored the number of smooth-pursuit phases of optokinetic nystagmus. While all therapeutic regimens preserved axonal density and proportions of typical complexes, including heminodes and single nodes, BDNF treatments were generally more effective in maintaining the length of the node of Ranvier in myelin surrounding ventral ON axons after injury. Both AAV2-BDNF and AAV2-CRMP2T555A prevented injury-induced changes in G-ratio and overall myelin thickness, but only AAV2-BDNF administration protected against large-scale myelin decompaction in ventral ON. In summary, in a model of secondary CNS degeneration, both BDNF and CRMP2T555A vectors were neuroprotective, however different efficacies were observed for these overexpressed proteins in the retina and ON, suggesting disparate cellular and molecular targets driving responses for neural repair. The potential use of these vectors to treat other CNS injuries and pathologies is discussed.

Protein corona formation moderates the release kinetics of ion channel antagonists from transferrin-functionalized polymeric nanoparticles.

Transferrin (Tf)-functionalized p(HEMA-ran-GMA) nanoparticles were designed to incorporate and release a water-soluble combination of three ion channel antagonists, namely zonampanel monohydrate (YM872), oxidized adenosine triphosphate (oxATP) and lomerizine hydrochloride (LOM) identified as a promising therapy for secondary degeneration that follows neurotrauma. Coupled with a mean hydrodynamic size of 285 nm and near-neutral surface charge of -5.98 mV, the hydrophilic nature of the functionalized polymeric nanoparticles was pivotal in effectively encapsulating the highly water soluble YM872 and oxATP, as well as lipophilic LOM dissolved in water-based medium, by a back-filling method. Maximum loading efficiencies of 11.8 ± 1.05% (w/w), 13.9 ± 1.50% (w/w) and 22.7 ± 4.00% (w/w) LOM, YM872 and oxATP respectively were reported. To obtain an estimate of drug exposure in vivo, drug release kinetics assessment by HPLC was conducted in representative physiological milieu containing 55% (v/v) human serum at 37 °C. In comparison to serum-free conditions, it was demonstrated that the inevitable adsorption of serum proteins on the Tf-functionalized nanoparticle surface as a protein corona impeded the rate of release of LOM and YM872 at both pH 5 and 7.4 over a period of 1 hour. While the release of oxATP from the nanoparticles was detectable for up to 30 minutes under serum-free conditions at pH 7.4, the presence of serum proteins and a slightly acidic environment impaired the detection of the drug, possibly due to its molecular instability. Nevertheless, under representative physiological conditions, all three drugs were released in combination from Tf-functionalized p(HEMA-ran-GMA) nanoparticles and detected for up to 20 minutes. Taken together, the study provided enhanced insight into potential physiological outcomes in the presence of serum proteins, and suggests that p(HEMA-ran-GMA)-based therapeutic nanoparticles may be promising drug delivery vehicles for CNS therapy.

Comparing modes of delivery of a combination of ion channel inhibitors for limiting secondary degeneration following partial optic nerve transection.

Injury to the central nervous system is exacerbated by secondary degeneration. Previous research has shown that a combination of orally and locally administered ion channel inhibitors following partial optic nerve injury protects the myelin sheath and preserves function in the ventral optic nerve, vulnerable to secondary degeneration. However, local administration is often not clinically appropriate. This study aimed to compare the efficacy of systemic and local delivery of the ion channel inhibitor combination of lomerizine, brilliant blue G (BBG) and YM872, which inhibits voltage-gated calcium channels, P2X7 receptors and Ca2+ permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors respectively. Following a partial optic nerve transection, adult female PVG rats were treated with BBG and YM872 delivered via osmotic mini pump directly to the injury site, or via intraperitoneal injection, both alongside oral administration of lomerizine. Myelin structure was preserved with both delivery modes of the ion channel inhibitor combination. However, there was no effect of treatment on inflammation, either peripherally or at the injury site, or on the density of oligodendroglial cells. Taken together, the data indicate that even at lower concentrations, the combinatorial treatment may be preserving myelin structure, and that systemic and local delivery are comparable at improving outcomes following neurotrauma.

The effects of a combination of ion channel inhibitors on pathology in a model of demyelinating disease.

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.

Elucidating the Inability of Functionalized Nanoparticles to Cross the Blood-Brain Barrier and Target Specific Cells in Vivo.

The adsorption of serum proteins on the surface of nanoparticles (NPs) delivered into a biological environment has been known to alter NP surface properties and consequently their targeting efficiency. In this paper, we use random copolymer (p(HEMA- ran-GMA))-based NPs synthesized using 2-hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA). We show that serum proteins bind to the NP and that functionalization with antibodies and peptides designed to facilitate NP passage across the blood-brain barrier (BBB) to bind specific cell types is ineffective. In particular, we use systematic in vitro and in vivo analyses to demonstrate that p(HEMA- ran-GMA) NPs functionalized with HIV-1 trans-activating transcriptor peptide (known to cross the BBB) and α neural/glial antigen 2 (NG2) (known for targeting oligodendrocyte precursor cells (OPCs)), individually and in combination, do not specifically target OPCs and are unable to cross the BBB, likely due to the serum protein binding to the NPs.

Differential responses to increasing numbers of mild traumatic brain injury in a rodent closed-head injury model.

Following mild traumatic brain injury (mTBI), further mild impacts can exacerbate negative outcomes. To compare chronic damage and deficits following increasing numbers of repeated mTBIs, a closed-head weight-drop model of repeated mTBI was used to deliver 1, 2 or 3 mTBIs to adult female rats at 24 h intervals. Outcomes were assessed at 3 months following the first mTBI. No gross motor, sensory or reflex deficits were identified (p > 0.05), consistent with current literature. Cognitive function assessed using a Morris water maze revealed chronic memory deficits following 1 and 2, but not 3 mTBI compared to shams (p ≤ 0.05). Oxidative damage to DNA was assessed immunohistochemically in the dentate hilus of the hippocampus and splenium of the corpus callosum; no changes were observed. IBA1-positive microglia were increased in size in the cortex following 1 mTBI and in the corpus callosum following 2 mTBI compared to shams (p ≤ 0.05); no changes were observed in the dentate hilus. Glial fibrillary acidic protein (GFAP)-positive astrocyte immunoreactivity was assessed in all three brain regions and no chronic changes were observed. Integrity of myelin ultrastructure in the corpus callosum was assessed using transmission electron microscopy. G ratio was decreased following 2 mTBIs compared to shams (p ≤ 0.05) at post hoc level only. The changing patterns of damage and deficits following increasing numbers of mTBI may reflect dynamic responses to small numbers of mTBIs or a conditioning effect such that increasing numbers of mTBIs do not necessarily result in worsening pathology. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14508.

Comparison of ion channel inhibitor combinations for limiting secondary degeneration following partial optic nerve transection.

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.

The Effects of a Combination of Ion Channel Inhibitors in Female Rats Following Repeated Mild Traumatic Brain Injury.

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 α-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 < 0.05), and a significant decrease in learning-associated improvement in rmTBI in Morris water maze (MWM) trials relative to the sham (p < 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 > 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 < 0.01). The authors' 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.

Inflammation and blood-brain barrier breach remote from the primary injury following neurotrauma.

BACKGROUND: Following 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. METHODS: Adult 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. RESULTS: Immediately 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. CONCLUSIONS: Partial 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.

Oligodendroglia Are Particularly Vulnerable to Oxidative Damage after Neurotrauma In Vivo.

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 vivoSIGNIFICANCE 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.

Three dimensional electron microscopy reveals changing axonal and myelin morphology along normal and partially injured optic nerves.

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.

Retinal genes are differentially expressed in areas of primary versus secondary degeneration following partial optic nerve injury.

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.

Partial Transection of Adult Rat Optic Nerve as a Model of Secondary Degeneration in the Central Nervous System.

Injury to the central nervous system is characterized by damage that spreads from the initial point of impact into the surrounding adjacent tissue, in a phenomenon referred to as secondary degeneration. The optic nerve can be used to effectively model injury and secondary degeneration to white matter tracts. Partial transection of the dorsal aspect of the nerve leaves the ventral aspect initially undamaged but vulnerable to secondary degeneration, allowing study of tissue exclusively vulnerable to secondary degeneration. Thus the partial optic nerve transection model of secondary degeneration is a valuable tool to study the pathology of spreading damage following neurotrauma and can be used to assess potential efficacy of therapeutic strategies.

Specific ion channels contribute to key elements of pathology during secondary degeneration following neurotrauma.

BACKGROUND: Following 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. RESULTS: Following 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. CONCLUSIONS: Data 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.

Repeated mild traumatic brain injury in female rats increases lipid peroxidation in neurons.

Negative outcomes of mild traumatic brain injury (mTBI) can be exacerbated by repeated insult. Animal models of repeated closed-head mTBI provide the opportunity to define acute pathological mechanisms as the number of mTBI increases. Furthermore, little is known about the effects of mTBI impact site, and how this may affect brain function. We use a closed head, weight drop model of mTBI that allows head movement following impact, in adult female rats to determine the role of the number and location of mTBI on brain pathology and behaviour. Biomechanical assessment of two anatomically well-defined mTBI impact sites were used, anterior (bregma) and posterior (lambda). Location of the impact had no significant effect on impact forces (450 N), and the weight impact locations were on average 5.4 mm from the desired impact site. No between location vertical linear head kinematic differences were observed immediately following impact, however, in the 300 ms post-impact, significantly higher mean vertical head displacement and velocity were observed in the mTBI lambda trials. Breaches of the blood brain barrier were observed with three mTBI over bregma, associated with immunohistochemical indicators of damage. However, an increased incidence of hairline fractures of the skull and macroscopic haemorrhaging made bregma an unsuitable impact location to model repeated mTBI. Repeated mTBI over lambda did not cause skull fractures and were examined more comprehensively, with outcomes following one, two or three mTBI or sham, delivered at 1 day intervals, assessed on days 1-4. We observe a mild behavioural phenotype, with subtle deficits in cognitive function, associated with no identifiable neuroanatomical or inflammatory changes. However, an increase in lipid peroxidation in a subset of cortical neurons following two mTBI indicates increasing oxidative damage with repeated injury in female rats, supported by increased amyloid precursor protein immunoreactivity with three mTBI. This study of acute events following closed head mTBI identifies lipid peroxidation in neurons at the same time as cognitive deficits. Our study adds to existing literature, providing biomechanics data and demonstrating mild cognitive disturbances associated with diffuse injury, predominantly to grey matter, acutely following repeated mTBI.

Delayed treatment of secondary degeneration following acute optic nerve transection using a combination of ion channel inhibitors.

Studies have shown that a combined application of several ion channel inhibitors immediately after central nervous system injury can inhibit secondary degeneration. However, for clinical use, it is necessary to determine how long after injury the combined treatment of several ion channel inhibitors can be delayed and efficacy maintained. In this study, we delivered Ca2+ entry-inhibiting P2X7 receptor antagonist oxidized-ATP and AMPA receptor antagonist YM872 to the optic nerve injury site via an iPRECIO@ pump immediately, 6 hours, 24 hours and 7 days after partial optic nerve transection surgery. In addition, all of the ion channel inhibitor treated rats were administered with calcium channel antagonist lomerizine hydrochloride. It is important to note that as a result of implantation of the particular pumps required for programmable delivery of therapeutics directly to the injury site, seromas occurred in a significant proportion of animals, indicating infection around the pumps in these animals. Improvements in visual function were observed only when treatment was delayed by 6 hours; phosphorylated Tau was reduced when treatment was delayed by 24 hours or 7 days. Improvements in structure of node/paranode of Ranvier and reductions in oxidative stress indicators were also only observed when treatment was delayed for 6 hours, 24 hours, or 7 days. Benefits of ion channel inhibitors were only observed with time-delayed treatment, suggesting that delayed therapy of Ca2+ ion channel inhibitors produces better neuroprotective effects on secondary degeneration, at least in the presence of seromas.