Molars to Medicine: A Focused Review on the Pre-Clinical Investigation and Treatment of Secondary Degeneration following Spinal Cord Injury Using Dental Stem Cells.

Spinal cord injury (SCI) can result in the permanent loss of mobility, sensation, and autonomic function. Secondary degeneration after SCI both initiates and propagates a hostile microenvironment that is resistant to natural repair mechanisms. Consequently, exogenous stem cells have been investigated as a potential therapy for repairing and recovering damaged cells after SCI and other CNS disorders. This focused review highlights the contributions of mesenchymal (MSCs) and dental stem cells (DSCs) in attenuating various secondary injury sequelae through paracrine and cell-to-cell communication mechanisms following SCI and other types of neurotrauma. These mechanistic events include vascular dysfunction, oxidative stress, excitotoxicity, apoptosis and cell loss, neuroinflammation, and structural deficits. The review of studies that directly compare MSC and DSC capabilities also reveals the superior capabilities of DSC in reducing the effects of secondary injury and promoting a favorable microenvironment conducive to repair and regeneration. This review concludes with a discussion of the current limitations and proposes improvements in the future assessment of stem cell therapy through the reporting of the effects of DSC viability and DSC efficacy in attenuating secondary damage after SCI.

The Translation of Nanomedicines in the Contexts of Spinal Cord Injury and Repair.

PURPOSE OF THIS REVIEW: Manipulating or re-engineering the damaged human spinal cord to achieve neuro-recovery is one of the foremost challenges of modern science. Addressing the restricted permission of neural cells and topographically organised neural tissue for self-renewal and spontaneous regeneration, respectively, is not straightforward, as exemplified by rare instances of translational success. This review assembles an understanding of advances in nanomedicine for spinal cord injury (SCI) and related clinical indications of relevance to attempts to design, engineer, and target nanotechnologies to multiple molecular networks. RECENT FINDINGS: Recent research provides a new understanding of the health benefits and regulatory landscape of nanomedicines based on a background of advances in mRNA-based nanocarrier vaccines and quantum dot-based optical imaging. In relation to spinal cord pathology, the extant literature details promising advances in nanoneuropharmacology and regenerative medicine that inform the present understanding of the nanoparticle (NP) biocompatibility-neurotoxicity relationship. In this review, the conceptual bases of nanotechnology and nanomaterial chemistry covering organic and inorganic particles of sizes generally less than 100 nm in diameter will be addressed. Regarding the centrally active nanotechnologies selected for this review, attention is paid to NP physico-chemistry, functionalisation, delivery, biocompatibility, biodistribution, toxicology, and key molecular targets and biological effects intrinsic to and beyond the spinal cord parenchyma. SUMMARY: The advance of nanotechnologies for the treatment of refractory spinal cord pathologies requires an in-depth understanding of neurobiological and topographical principles and a consideration of additional complexities involving the research's translational and regulatory landscapes.

Biodegradable and biocompatible graphene-based scaffolds for functional neural tissue engineering: A strategy approach using dental pulp stem cells and biomaterials.

Neural tissue engineering aims to restore the function of nervous system tissues using biocompatible cell-seeded scaffolds. Graphene-based scaffolds combined with stem cells deserve special attention to enhance tissue regeneration in a controlled manner. However, it is believed that minor changes in scaffold biomaterial composition, internal porous structure, and physicochemical properties can impact cellular growth and adhesion. The current work aims to investigate in vitro biological effects of three-dimensional (3D) graphene oxide (GO)/sodium alginate (GOSA) and reduced GOSA (RGOSA) scaffolds on dental pulp stem cells (DPSCs) in terms of cell viability and cytotoxicity. Herein, the effects of the 3D scaffolds, coating conditions, and serum supplementation on DPSCs functions are explored extensively. Biodegradation analysis revealed that the addition of GO enhanced the degradation rate of composite scaffolds. Compared to the 2D surface, the cell viability of 3D scaffolds was higher (p < 0.0001), highlighting the optimal initial cell adhesion to the scaffold surface and cell migration through pores. Moreover, the cytotoxicity study indicated that the incorporation of graphene supported higher DPSCs viability. It is also shown that when the mean pore size of the scaffold increases, DPSCs activity decreases. In terms of coating conditions, poly- l-lysine was the most robust coating reagent that improved cell-scaffold adherence and DPSCs metabolism activity. The cytotoxicity of GO-based scaffolds showed that DPSCs can be seeded in serum-free media without cytotoxic effects. This is critical for human translation as cellular transplants are typically serum-free. These findings suggest that proposed 3D GO-based scaffolds have favorable effects on the biological responses of DPSCs.

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.

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.

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.

Specific combinations of ion channel inhibitors reduce excessive Ca2+ influx as a consequence of oxidative stress and increase neuronal and glial cell viability in vitro.

Combinations of Ca2+ channel inhibitors have been proposed as an effective means to prevent excess Ca2+ flux and death of neurons and glia following neurotrauma in vivo. However, it is not yet known if beneficial outcomes such as improved viability have been due to direct effects on intracellular Ca2+ concentrations. Here, the effects of combinations of Lomerizine (Lom), 2,3-dioxo-7-(1H-imidazol-1-yl)6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]acetic acid monohydrate (YM872), 3,5-dimethyl-1-adamantanamine (memantine (Mem)) and/or adenosine 5'-triphosphate periodate oxidized sodium salt (oxATP) to block voltage-gated Ca2+ channels, Ca2+ permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, NMDA receptors and purinergic P2X7 receptors (P2X7R) respectively, on Ca2+ concentration and viability of rat primary mixed cortical (MC) cultures exposed to hydrogen peroxide (H2O2) insult, were assessed. The contribution of ryanodine-sensitive intracellular stores to intracellular Ca2+ concentration was also assessed. Live cell calcium imaging revealed that a 30min H2O2 insult induced a slow increase in intracellular Ca2+, in part from intracellular sources, associated with loss of cell viability by 6h. Most combinations of inhibitors that included oxATP significantly decreased Ca2+ influx and increased cell viability when administered simultaneously with H2O2. However, reductions in intracellular Ca2+ concentration were not always linked to improved cell viability. Examination of the density of specific cell subpopulations demonstrated that most combinations of inhibitors that included oxATP preserved NG2+ non-oligodendroglial cells, but preservation of astrocytes and neurons required additional inhibitors. Olig2+ oligodendroglia and ED-1+ activated microglia/macrophages were not preserved by any of the inhibitor combinations. These data indicate that following H2O2 insult, limiting intracellular Ca2+ entry via P2X7R is generally associated with increased cell viability. Protection of NG2+ non-oligodendroglial cells by Ca2+ channel inhibitor combinations may contribute to observed beneficial outcomes in vivo.

Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties.

Several recent studies have demonstrated that TAT and other arginine-rich cell penetrating peptides (CPPs) have intrinsic neuroprotective properties in their own right. Examples, we have demonstrated that in addition to TAT, poly-arginine peptides (R8 to R18; containing 8-18 arginine residues) as well as some other arginine-rich peptides are neuroprotective in vitro (in neurons exposed to glutamic acid excitotoxicity and oxygen glucose deprivation) and in the case of R9 in vivo (after permanent middle cerebral artery occlusion in the rat). Based on several lines of evidence, we propose that this neuroprotection is related to the peptide's endocytosis-inducing properties, with peptide charge and arginine residues being critical factors. Specifically, we propose that during peptide endocytosis neuronal cell surface structures such as ion channels and transporters are internalised, thereby reducing calcium influx associated with excitotoxicity and other receptor-mediated neurodamaging signalling pathways. We also hypothesise that a peptide cargo can act synergistically with TAT and other arginine-rich CPPs due to potentiation of the CPPs endocytic traits rather than by the cargo-peptide acting directly on its supposedly intended intracellular target. In this review, we systematically consider a number of studies that have used CPPs to deliver neuroprotective peptides to the central nervous system (CNS) following stroke and other neurological disorders. Consequently, we critically review evidence that supports our hypothesis that neuroprotection is mediated by carrier peptide endocytosis. In conclusion, we believe that there are strong grounds to regard arginine-rich peptides as a new class of neuroprotective molecules for the treatment of a range of neurological disorders.

Prolonged glutamate excitotoxicity increases GluR1 immunoreactivity but decreases mRNA of GluR1 and associated regulatory proteins in dissociated rat retinae in vitro.

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.

Novel combinations of ion channel inhibitors for treatment of neurotrauma.

Neurotrauma results in the progressive degeneration of neurons and glia surrounding the initial injury. Disruptions to myelin structure are a feature of these injuries and are thought to be triggered by excess calcium (Ca2+) influx into myelinating oligodendrocytes and/or their precursor cells. Calcium ions enter oligodendrocytes through a range of receptors including voltage gated calcium channels, N-methyl-D-aspartate (NMDA) receptors, Ca2+ permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and purinergic P2X7 receptors. Inhibitors have been used to limit excess Ca2+ entry following neurotrauma, but clinical success has been limited. We propose that combinations of calcium channel inhibitors may provide an alternative treatment strategy, whereby entry of excess Ca2+ flux through multiple routes is inhibited simultaneously.

Reactive species and oxidative stress in optic nerve vulnerable to secondary degeneration.

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.

Three Ca2+ channel inhibitors in combination limit chronic secondary degeneration following neurotrauma.

Following neurotrauma, cells beyond the initial trauma site undergo secondary degeneration, with excess Ca2+ a likely trigger for loss of neurons, compact myelin and function. Treatment using inhibitors of specific Ca2+ channels has shown promise in preclinical studies, but clinical trials have been disappointing and combinatorial approaches are needed. We assessed efficacy of multiple combinations of three Ca2+ channel inhibitors at reducing secondary degeneration following partial optic nerve transection in rat. We used lomerizine to inhibit voltage gated Ca2+ channels; oxidised adenosine-triphosphate (oxATP) to inhibit purinergic P2X7 receptors and/or 2-[7-(1H-imidazol-1-yl)-6-nitro-2,3-dioxo-1,2,3,4-tetrahydro quinoxalin-1-yl]acetic acid (INQ) to inhibit Ca2+ permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Only the three Ca2+ channel inhibitors delivered in combination significantly preserved visual function, as assessed using the optokinetic nystagmus visual reflex, at 3 months after injury. Preservation of retinal ganglion cells was partial and is unlikely to have accounted for differential effects on function. A range of the Ca2+ channel inhibitor combinations prevented swelling of optic nerve vulnerable to secondary degeneration. Each of the treatments involving lomerizine significantly increased the proportion of axons with normal compact myelin. Nevertheless, limiting decompaction of myelin was not sufficient for preservation of function in our model. Multiple combinations of Ca2+ channel inhibitors reduced formation of atypical node/paranode complexes; outcomes were not associated with preservation of visual function. However, prevention of lengthening of the paranodal gap that was only achieved by treatment with the three Ca2+ channel inhibitors in combination was an important additional effect that likely contributed to the associated preservation of the optokinetic reflex using this combinatorial treatment strategy.

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

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