After a traumatic injury to the spinal cord, tissue damage and cavity formation occurs at the epicenter and spreads rostrally and caudally over time. This evolving lesion is accompanied by prevalent oligodendrocyte (OL) apoptosis and loss of myelin around surviving axons (Crowe et al., 1997; Totoiu and Keirstead, 2005; Li et al., 2007).
Demyelination of otherwise intact axons can lead to a conduction failure and may contribute to the functional deficits observed after spinal cord injury (SCI) (Shi and Blight, 1996; Cao et al., 2005; Keirstead et al., 2005). Thus, preventing progression of the demyelination and promoting the remyelination is considered to be one of the desired therapeutic goals to improve functional recovery after SCI (Horner and Gage, 2000; Dobkin and Havton, 2004;
Franklin and Ffrench-Constant, 2008). Remyelination is the process by which new myelin sheath are restored to demylinated axons, enabling them to regain the ability to carry action potentials by salutatory conduction and to recover lost function (Smith et al., 1979; Jeffery and Blakemore, 1997). Even after severe contusion SCI, demyelinated axons persist in the subpial rim of white matter in both humans (Bunge et al., 1993; Guest et al., 2005) and experimental animals (Blight, 1993; Cao et al., 2005; Totoiu and Keirstead, 2005). In addition, glial progenitor cells (GPCs) which possess a potential to become oligodendrocytes exist in the adult spinal cord and proliferate in response to SCI (Horner et al., 2000; McTigue et al., 2001; Zai and Wrathall, 2005; Horky et al., 2006). Although some remyelination by endogenous OLs and invading peripheral Schwann cells occurs (Dusart et al., 1992;
McTigue et al., 2001; Tripathi and McTigue, 2007). However, spontaneous remyelination is
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limited and incomplete. The failure of spontaneous remyelination may be due to inadequate signaling to generate sufficient number of mature OLs from proliferating GPCs (Talbott et al., 2005; Cheng et al., 2007).
OLs are nondividing cells that develop from proliferating, migratory progenitor cells.
The number of OLs in lesion region of injured spinal cord, in principle, will depend upon:
(1) the quantity of oligodendrocyte progenitor cells (OPCs) that migrate into the lesion region; (2) the replicative potential of the resident OPCs prior to entering into the differentiation pathway; (3) the quantity of cell loss occurring during cellular development.
Many studies over the past two decades have examined the processes affecting OLs survival, migration, proliferation and differentiation. It is now appreciated that these cellular decisions are determined by a combination of both cell-extrinsic and –intrinsic factors mediated through the interaction of an ever increasing number of effectors (Raff et al., 1998). Survival, migration, proliferation, and differentiation of oligodendrocytes are regulated by numerous cell-extrinsic molecules. These include growth factors (PDGF, bFGF, BDNF, CNTF, NGF), cytokines (IGF-1, NT-3), hormones (thyroid hormone, glucocorticoid, progesterone), and neurotransmitters (glutamate) that are secreted by neighboring cells and that act in concert to shape normal brain development (Dreyfus, 1998). A variety of extracellular factors may be added to cultures to influence survival, growth, and development of OPCs in vitro. In addition to the extracellular factors, oligodendrocyte development is affected also by intrinsic cellular factors. The transition of an OPC to a mature oligodendrocyte depends on complex factors involving the coordinated expression of particular genes and transcription factors, many of which are oligodendrocyte / myelin specific (Durand and Raff, 2000; Lee et al., 2009). Therefore, it could be conceived that both extracellular and intrinsic factors which
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are important for OL development may have a role in determining the number of OLs in the lesioned spinal cord.
Multiple cell types have been grafted into the demyelinatited spinal cord and remyelinate the demyelinated axons to varying degrees (Cao et al., 2002; Kocsis et al., 2004;
Reier, 2004). Neural stem cells (NSCs) and GPCs have the potential to differentiate into OLs in vitro and in vivo. However, multipotent NSCs mainly differentiate into astrocytes after
transplantation into the injured spinal cord (Chow et al., 2000; Cao et al., 2001). OPCs may be optimal cell grafts because of their potential for more extensive remyelination and their lack of differentiation into astrocytes after transplantation. OPC transplantation is an effective strategy for OL replacement and remyelination after traumatic SCI (Cao et al., 2005; Keirstead et al., 2005). However, cell transplantation studies have demonstrated that exogenous GPCs, which retain strong oligodendrogenic activities in vitro, differentiate only very poorly when grafted into the injured spinal cord (Han et al., 2004; Hill et al., 2004).
Thus, the environment of the injured spinal cord appears to be highly restrictive for differentiation of GPCs. If this environmental restriction can be relieved by certain manipulations, endogenous GPCs may be able to supply new oligodendrocyte.
Endogenous stem or progenitor cells that can differentiate into neurons and glial cells are also present in adult spinal cord (Weiss et al., 1996). The progenitors in glial lineage are stimulated to proliferate in response to SCI (McTigue et al., 2001; Zai and Wrathall, 2005;
Yang et al., 2006; Tripathi and McTigue, 2007). Proliferating glial progenitors are persistently found until several weeks after injury (McTigue et al., 2001), and they are believed to differentiate into mature glial cells, eventually replacing the lost oligodendrocytes and astrocytes (Yang et al., 2006). These findings suggest a promising
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possibility that mobilization of endogenous glial progenitors can provide a therapeutic opportunity to repair the white matter damaged by traumatic SCI. The adult CNS contains a significant number of OPCs (Wolswijk and Noble, 1989; Chang et al., 2000; Horner et al., 2000). Although the turnover of these cells is relatively low under normal conditions, their proliferation increases significantly after SCI (Ishii et al., 2001; McTigue et al., 2001), but spontaneous remyelination is limited and incomplete after SCI.
The present thesis tested a hypothesis that manipulation of post-injury microenvironment or intrinsic transcriptional machinery could promote the endogenous oligodendrogenesis and ultimately improve functional outcome after SCI. In the first part, vascular endothelial growth factor (VEGF) was chosen as an extracellular factor to promote oligodendrogenesis.
VEGF was originally characterized as a potent stimulator of angiogenesis. Later, multifaceted trophic effects of VEGF have been uncovered in nervous tissue (Rosenstein and Krum, 2004). VEGF provides direct protective effects on neurons (Jin et al., 2000; endogenous neural stem or progenitor cells, and VEGF was shown to increase endogenous neurogenesis after stroke (Jin et al., 2002; Sun et al., 2003). Potential effects of VEGF on the glial progenitor cells in the spinal cord after injury have not been investigated yet. In the first
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part of the thesis research, VEGF gene was incorporated into immortalized human neural stem cells and the VEGF overexpressing human neural stem cells were transplanted into the injured spinal cord to achieve a stable and robust expression. Proliferation and differentiation of GPCs and OLs were examined to test if the ex vivo VEGF delivery promoted endogenous oligodendrogenesis.
In the second part of thesis, I examined whether introduction of Olig genes, critical regulators of OL development, can affect endogenous oligodendrogenesis after contusive SCI in rats.
Transcriptional regulation plays an important role in the differentiation of oligodendrocyte lineage cells from initial GPCs to fully mature myelinating oligodendrocytes (Dugas et al., 2006; Nicolay et al., 2007). A pair of closely related basic helix-loop-helix (bHLH) transcription factors Olig1 and Olig2 were identified and shown to be essential for generating oligodendrocyte lineage cells during development (Lu et al., 2000; Zhou et al., 2000; Zhou and Anderson, 2002). Following studies have further shown that both Olig1 and Olig2 in collaboration with other transcription factors play important roles in diverse stages of OL differentiation and maturation (Fu et al., 2002; Cheng et al., 2007; Li et al., 2007; Liu et al., 2007). Given these crucial roles of Olig transcription factors, regulating Olig gene expression in post-injury GPCs could be justified as a rational therapeutic approach to promote the oligodendrogenesis and remyelination after SCI.
Potential functions of Olig1 and Olig2 transcription factor in injured CNS have not been fully studied. Olig1 was required in the remyelination process after demyelinated lesions in mature CNS (Arnett et al., 2004). More recent studies showed the implication of Olig2 transcription factor in the maintain and differentiation of neural and/or glial
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progenitors after injury (Buffo et al., 2005; Lee et al., 2005; Chen et al., 2008). Although more works need to be done, it seems quite apparent that Olig1 and Olig2 have distinct biological properties that could be separable from those of each other after injury as well as during development (Arnett et al., 2004; Buffo et al., 2005; Jakovcevski and Zecevic, 2005;
Xin et al., 2005). In this part of thesis research, therefore, Olig1 and Olig2 genes were separately introduced into proliferating glial progenitor cells, and the results showed that these two genes exerts differential influence on the behavior of GPCs and OLs. Olig1 and olig2 genes were introduced also in combination to find any cooperative effects of these genes.
During the course of experiments in which Olig2 gene was introduced into proliferating GPCs following SCI, glioma formation was unexpectedly observed in the spinal cord. Any kind of strategies to activate Olig transcriptional activity to enhance endogenous oligodendrogenesis may lead to unwanted tumor formation. Several lines of evidence suggest that activity of Olig2 might provide a mechanistic link between growth of malignant glioma. Exposure to glioma-relevant mitogens such as EGF or PDGF (Jackson et al., 2006) stimulates proliferation of Olig2+ rapidly dividing “type C” transit-amplifying cells and glioma-like growths. The other studies have shown that all malignant gliomas, irrespective of grade, express Olig2 in at least some fraction of the malignant cell population (Marie et al., 2001; Ligon et al., 2004). Olig2-regulated lineage-restricted pathway critical for proliferation of normal and tumorigenic CNS stem cells (Ligon et al., 2007).
In the third part of thesis, therefore I studied the detailed mechanism of tumor regulation by Olig genes.
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