Spinal cord injury (SCI) results in extensive demyelination, leading to deleterious axon degeneration and inability of functional recovery. Remyelination has become a part of the fundamental strategy for SCI repair. Endogenous neural progenitor cells (NPCs) respond to SCI producing progenies and provide a possible source of regenerated oligodedrocytes for remyelination. During development of the central nervous system, glycogen synthase kinase-3 isoform beta (GSK-3β) is involved in multiple pathways that regulate oligodendrocyte differentiation and myelination, and thus may also play an important part in remyelination after SCI. This study aims to investigate (1) the role of GSK-3β in the differentiation of adult spinal cord derived-neural progenitor cells (ASC-NPCs); (2) whether AR-A014418 as a GSK-3β inhibitor, can promote oligodendroglial differentiation of ASC-NPCs; (3) the effect of LiCl, another GSK-3β inhibitor, on functional recovery after SCI; (4) the effects of LiCl on the myelin and axonal preservation after SCI. Neurosphere culture from adult mouse spinal cord was performed to test the effect of GSK-3β inhibitors, LiCl and AR-A014418, on differentiation of ASC-NPCs. Phenotyping of differentiated ASC-NPCs by immunocytochemistry (ICC) was performed to identify oligodendroglia progenitor cells (OPCs) at different stages. It was shown that LiCl (1 mM) and AR-A014418 (5 μM) promoted differentiation of OPCs as labeled by oligodendrocyte lineage-specific markers: PDGFR-α, NG2 and O4, while AR-A014418 was more potent in the OPC differentiation. Moreover, preliminary data from western blot confirmed that ARA014418 (5 μM) treatment increased the expression level of pGSK (inactive form of GSK-3) in differentiated ASC-NPCs. This suggests a possible strategy to modulate endogenous NPC response to SCI: to induce the preferential differentiation of NPCs into oligodendrocyte lineage by inhibiting GSK-3β activity and thus leading to enhanced remyelination by the differentiated oligodendrocytes. Basso Mouse Scale (BMS) open field test was used to evaluate the locomotive function of the spinal cord injured mice. The result showed that LiCl (4 mM, 200 μl) administration delivered locally at the lesion site by osmotic pump for 2 weeks improved functional recovery after SCI. Furthermore, immunohistochemistry (IHC) analyses revealed that LiCl treatment inhibited GSK-3β activity in the 〖Olig2〗^+ OPCs/oligodendrocytes, confirming LiCl as a GSK-3β inhibitor in vivo. Moreover, LiCl treatment better preserved myelin and axons detected by myelin basic protein (MBP) immunostaining and neurofilment-200 (NF-200) immunostaining respectively in the injured spinal cords. All together, the data from our in vitro and in vivo experiments suggested that LiCl treatment after spinal cord injury is beneficial for functional recovery by preventing the loss of myelin and axons after SCI and this effect is mediated via GSK-3β inhibition This study provided evidence for the involvement of GSK-3β in the regulation of OPC differentiation and the subsequent remyelination in the injured adult spinal cord. We propose GSK-3β as an important therapeutic target for SCI repair, LiCl as a potential candidate for SCI clinical treatment and the possibility to manipulate endogenous NPCs after SCI to enhance oligodendrocyte differentiation, remyelination, and ultimately better functional recovery.. / published_or_final_version / Anatomy / Master / Master of Philosophy

Several recent experiments on developing chick spinal cord have established a time window when the developing spinal cord changes from a permissive to a restrictive environment for regeneration. This time window occurs during embryonic days 13-14 (E13-E14) of chick development. Recent experiments in adult rat, have found two proteins that actively inhibit axonal regeneration. This study has sought possible inhibitory proteins, in chicks, correlating to this temporal change. Proteins continuously present after this change (E14-E20) but not before (E11) were identified. Two-dimensional gel electrophoresis was used for separatation of the proteins. Seven protein spots of interest demonstrated this correlative late-expressing neural protein (LNP) profile. Although the functions of these proteins could not be ascertained in this study, further investigation is warranted. / Science, Faculty of / Zoology, Department of / Graduate

This work proposes minimally invasive solutions for spinal cord regeneration after trauma. In particular, injectable biomaterials can be precisely positioned in the lesion site, and eventually repetitively injected until the complete regeneration of the tissue. For this application, a silk fibroin functionalized with collagen type IV and laminin-derived peptides, called bio-inspired multifunctionalized silk fibroin (BMS), possessing piezoelectric properties, has been synthesized. Another approach that avoids damages to the spinal cord is proposed in the thesis as a multilayer hydrogel with piezoelectric properties that acts as a bridge between the healthy parts surrounding the injury. The multilayer hydrogel consists of i) a thin-layer of gelatin and fish collagen functionalized with VEGF for blood vessels formation, which helps the survival of the cells integrating with the pia mater of the spinal cord; ii) a BMS layer, which helps the adhesion, migration of neural stem cells and induces the sprouting of the axons thanks to the presence of Netrin (a chemoattractive protein); and iii) an adhesive layer of polydopamine (PDA) to fix the patch on the injured site. The adhesive patch exhibits a potential larger than an injectable hydrogel that could guarantee a long-term cell survival and help the axons to move towards a direction. The adhesive patch will be located on the surface of the spinal cord and the chemoattractive protein will induce the sprouting of the ascendant or descendant axons in the spinal cord to reach the axons present in the patch, restoring a signal connection. Even if not final, the results indicate that the above strategy could be explored further for the regeneration of the spinal cord.

<p dir="ltr">Though mammalian spinal cord injury (SCI) has long been a topic of study, effective therapies that promote functional recovery are not yet available. The axolotl, <i>Ambystoma mexicanum</i>, is a valuable animal model in the investigation of spinal cord regeneration, as this urodele is able to achieve functional recovery even after complete spinal cord transection. Understanding the similarities and differences between the mammalian SCI response and that of the axolotl provides insight into the process of successful regeneration, and bolsters the fundamental knowledge used in the development of future mammalian SCI treatments. This thesis provides a detailed analysis of the ultrastructure of the axolotl meninges, as this has not yet been presented in existing literature, and reveals that the axolotl meninges consist of 3 distinct layers as does mammalian meninges; the dura mater, arachnoid mater, and pia mater. The role of reactive meningeal and ependymal cells is also investigated in regard to the deposition and remodeling of the fibrotic ECM, which is found to be similar in composition to hydrogel scaffolds being studied in mammalian SCI. It is shown that meningeal fibroblasts are the primary source of the extensive fibrillar collagen deposition that fills the entire spinal canal, peaking at approximately 3 weeks post transection and remaining until approximately 5 weeks post transection, and that there is no deposition of type IV collagen within the lesion site. Mesenchymal ependymal cells are shown to contribute to the ECM deposition through the production of glycosaminoglycans that are used in sidechains of both unsulfated and sulfated proteoglycans, while simultaneously remodeling the ECM through the production of MMPs and phagocytosis of cellular debris. Further, this study shows that mesenchymal ependymal cells and a population of foamy macrophages contribute to the degradation of the fibrin clot that forms in the acute phase of injury, and that this fibrin clot provides a necessary and permissive substrate for early mesenchymal outgrowth.</p>

Chondroitin sulfate proteoglycans (CSPGs) are one major class of axon growth inhibitors that are upregulated and accumulated around the lesion site after spinal cord injury (SCI), and result in regenerative failure. To overcome CSPG-mediated inhibition, digestion of CSPGs with chondroitinase ABC (chABC) has been explored and it has shown promising results. chABC digests glycosaminoglycan chains on CSPGs and can thereby enhance axonal regeneration and promote functional recovery when delivered at the site of injury. However, chABC has a crucial limitation; it is thermally unstable and loses its enzymatic activity rapidly at 37 ºC. Therefore, it necessitates the use of repeated injections or local infusions with a pump for days to weeks to provide fresh chABC to retain its enzymatic activity. Maintaining these infusion systems is invasive and clinically problematic. In this dissertation, three studies are reported that demonstrate our strategy to overcome current limitations of using chABC and develop a delivery system for facilitating chABC treatment after SCI: First, we enhanced the thermostability of chABC by adding trehalose, a protein stabilizer, and developed a system for its sustained local delivery in vivo. Enzymatic activity was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dimethylmethylene blue (DMMB), and conformational change of the enzyme was measured via circular dichroism (CD) with and without trehalose. When stabilized with trehalose, chABC remained enzymatically active at 37 ºC for up to 4 weeks in vitro. We developed a lipid microtube-agarose hydrogel delivery system for a sustained release and showed that chABC released from the delivery system is still functionally active and slowly released over 2 weeks in vitro. Second, the hydrogel-microtube system was used to locally deliver chABC over two weeks at the lesion site following a dorsal over hemisection injury at T10. The scaffold consisting of hydrogel and chABC loaded lipid microtubes was implanted at the top of the lesion site immediately following injury. To determine effectiveness of topical delivery of thermostabilized chABC, animal groups treated with single injection or gel scaffold implantation of chABC and penicillinase (P'ase) were included as controls. Two weeks after surgery, the functionality of released chABC and the cellular responses were examined by immunohistological analysis with 3B3, CS-56, GFAP and Wisteria floribunda agglutinin (WFA). The results demonstrated that thermostabilized chABC was successfully delivered slowly and locally without the need for an indwelling catheter by using the hydrogel-microtube delivery system in vivo. The results demonstrated that released chABC from the gel scaffold effectively digested CSPGs, and therefore, there were significant differences in CSPG digestion at the lesion site between groups treated with chABC loaded microtube-hydrogel scaffolds and controls. Third, a long term in vivo study (45 days) was conducted to examine axonal sprouting/regeneration and functional recovery with both a single treatment each of microtube loaded chABC or Neurotrophin-3 (NT-3), and a combination of them by using the hydrogel-microtube delivery system. Over the long term study period, the treated animals showed significant improvement in locomotor function and more sprouting of cholera toxin B subunit (CTB)-positive ascending dorsal column fibers and 5-HT serotonergic fibers around the lesion site. We demonstrated that this significant improvement of chABC thermostability facilitates the development of a minimally invasive method for sustained, local delivery of chABC that is potentially a useful and effective approach for treating SCI. In addition to that, we demonstrated that combinatorial therapy with chABC and neurotrophic factors could provide a synergistic effect on axonal regrowth and functional recovery after SCI.

Indiana University-Purdue University Indianapolis (IUPUI) / Retinoids play an important role in tissue patterning during development as well as in epithelial formation and health. In the mammalian central nervous system, the meninges are a source of retinoids for brain tissue. Retinoid production has been described in juvenile Axolotl ependymal cells. Retinoid effects may possess a significant role in the regeneration-permissive interaction of the meninges and ependyma of the Axolotl spinal cord after penetrating injury. During spinal cord regeneration in urodele amphibians, the pattern of retinoid production changes as the meninges interact with the injury-reactive ependymal cells reconstructing the injured spinal cord. In order to determine which components of the retinoid metabolism and intracellular signaling pathway act in Urodele spinal cord regeneration, we employed antibody/horseradish peroxidase staining of both intact and regenerating Axolotl spinal cord tissues obtained from adult animals as well as cell culture techniques to determine expression of three retinoid pathway components: Cellular Retinoic Acid Binding Protein II (CRABP 2), Cellular Retinol Binding Protein I (CRBP 1), and Retinaldehyde Dehydrogenase II (RALDH 2). Current results demonstrate the following in the intact cord: 1) CRBP 1 is expressed in the pia and dura mater meningeal layers, in gray matter neurons (including their axonal processes), and the ependymal cell radial processes that produce the glia limitans, 2) CRABP 2 is expressed in the arachnoid and/or dura mater meningeal layers surrounding the spinal cord, and 3) RALDH 2 is expressed in the meninges as well as cytoplasm of grey matter neurons and some ependymal/sub-ependymal cells. In the regenerating cord, CRBP 1 is expressed in ependymal cells that are undergoing epithelial-to-mesenchymal transition (EMT), as is CRABP 2. RALDH 2 staining is very strong in the reactive meninges; in addition, expression is also upregulated in the cytoplasmic and perinuclear regions of reactive grey matter neurons, including motor neurons and in the apical region of ependymal. Preliminary studies culturing reactive meninges and ependymal cells together suggested that the meninges could drive re-epithelialization of the reactive ependymal cells. Experiments to characterize this interaction show an unusual proliferation pattern: Proliferating Cell Nuclear Antigen (PCNA) labeling is present in intact and regenerating cord ependymal cells. However, in culture, the presence of meninges results in no proliferation proximal to the explant, but extensive proliferation in leading cell outgrowth; also, the cultured meninges is positive for RALDH2. In summary, the intact adult cord shows meningeal production of RA, which is upregulated following injury; in addition, during this time, RA production is upregulated in the adult ependymal cells as well. In culture, the reactive meninges appears to modulate the behavior of reactive ependymal cells.

Indiana University-Purdue University Indianapolis (IUPUI) / Human traumatic spinal cord injuries (SCI) are primarily incomplete contusion or compression injuries at the cervical spinal level, causing immediate local tissue damage and a range of potential functional deficits. Secondary damage exacerbates initial mechanical trauma and contributes to function loss through delayed cell death mechanisms such as apoptosis and autophagy. As such, understanding the dynamics of cervical SCI and related intracellular signaling and death mechanisms is essential. Through behavior, Western blot, and histological analyses, alterations in phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-kinase (PI3K) signaling and the neuroprotective, functional, and mechanistic effects of administering the protein tyrosine phosphatase (PTP) inhibitor, potassium bisperoxo (picolinato) vanadium ([bpV[pic]) were analyzed following cervical spinal cord injury in rats. Furthermore, these studies investigated the combination of subacute Schwann cell transplantation with acute bpV(pic) treatment to identify any potential additive or synergistic benefits. Although spinal SC transplantation is well-studied, its use in combination with other therapies is necessary to complement its known protective and growth promoting characteristics. v The results showed 400 μg/kg/day bpV(pic) promoted significant tissue sparing, lesion reduction, and recovery of forelimb function post-SCI. To further clarify the mechanism of action of bpV(pic) on spinal neurons, we treated injured spinal neurons in vitro with 100 nM bpV(pic) and confirmed its neurprotection and action through inhibition of PTEN and promotion of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling. Following bpV(pic) treatment and green fluorescent protein (GFP)-SC transplantation, similar results in neuroprotective benefits were observed. GFP-SCs alone exhibited less robust effects in this regard, but promoted significant ingrowth of axons, as well as vasculature, over 10 weeks post-transplantation. All treatments showed similar effects in forelimb function recovery, although the bpV and combination treatments were the only to show statistical significance over non-treated injury. In the following chapters, the research presented contributes further understanding of cellular responses following cervical hemi-contusion SCI, and the beneficial effects of bpV(pic) and SC transplantation therapies alone and in combination. In conclusion, this work provides a thorough overview of pathology and cell- and signal-specific mechanisms of survival and repair in a clinically relevant rodent SCI model.