Traumatic spinal cord injury (SCI) can lead to lasting motor and sensory deficits at and below the level of the lesion and often results in permanent disability. Because endogenous repair following SCI in adult mammals is restricted, exogenous intervention strategies are necessary to enhance recovery, and among the more promising repair strategies is cell transplantation therapy (Thuret et al. 2006; Eftekharpour et al. 2008). The goals of cell transplantation therapy can vary widely but typically include replacing damaged neurons, filling the cystic cavity, enhancing axonal regeneration by creating a regenerative environment, and supporting or inducing remyelination. Several cell types have been evaluated as therapeutic strategies for post-SCI cell transplantation including embryonic stem cells (Keirstead et al. 2005; Hendricks et al. 2006), fate restricted neural or glial precursor cells (Setoguchi et al. 2004; Cao et al. 2005; Ziv et al. 2006), genetically modified fibroblasts (Pizzi and Crowe 2006; Tobias et al. 2005), olfactory ensheathing cells (Munoz-Quiles et al. 2009; Kocsis et al. 2009; kay-Sim et al. 2008), and Schwann cells (SC) (Pearse et al. 2007; Golden et al. 2007; Fortun et al. 2009; Lavdas et al. 2010). Among these cell types, SCs have several characteristics that favor their use in transplantation strategies. First, activated SCs synthesize and secrete growth factors including brain-derived neurotrophic factor, ciliary neurotrophic factor, neurotrophin-3, and -4/5 (Meyer et al. 1992; Funakoshi et al. 1993; Taylor and Bampton 2004), which are all likely to be beneficial in promoting repair and regeneration after axonal injury. Second, in peripheral nerve injury, SCs have been shown to proliferate after nerve injury and align to form bands of Bunger which can become supportive conduits for axonal growth (Fortun et al. 2009) as well as to produce extracellular matrix and cell adhesion molecules that can provide a substrate for axon growth (Oudega et al. 2005). Third, as the myelinating glial cells of the peripheral nervous system, SCs can restore the integrity of the myelin sheath around damaged axons which would be of therapeutic benefit in SCI (Oudega et al. 2005). Fourth, the methods to obtain and expand highly purified SC culture from several species, including human, are already well-established (Haastert et al. 2007; Baek and Kim 1998). Indeed, it is potentially feasible to obtain a small piece of peripheral nerve from a SCI patient, culture and/or expand SCs in vitro and then conduct autologous implantation to treat SCI. This therapeutic approach would obviate ethical issues surrounding the use of embryonic tissue as well as minimizing immunological rejection.
Several previous studies have demonstrated that SC transplantation post-SCI promotes axonal regeneration, induces remyelination, and enhances functional recovery (Takami et al. 2002; Akiyama et al. 2004; Bachelin et al. 2005; Golden et al. 2007; Papastefanaki et al. 2007; Ban et al. 2009; Dinh et al. 2007). However, a limitation of SC transplantation strategies in SCI is the poor survival rates of transplanted cells (Hill et al. 2006, 2007; Pearse et al. 2007). For example, Hill et al. (2006) reported extensive cell loss of SCs transplanted acutely (1 or 24 h) after a contusional SCI and that delaying transplantation to 7 days post-SCI improved cell survival. In a subsequent report, this group transplanted various densities of SCs at 7 days post-SCI into the lesion epicenter and quantified survival of transplanted SCs at several post-transplantation times. They found that regardless of cell transplantation density, only 22% or 15% of the transplanted cells survived for 7 or 28 days post-transplantation, respectively, and that significant necrotic and apoptotic cell death occurs in transplanted SCs (Hill et al. 2007). Although the mechanisms of SC death after transplantation are not fully elucidated, SC death may be at least partially because of the well-documented ongoing secondary injury processes following initial SCI trauma (Kwon et al. 2004; Onose et al. 2009), which includes excitotoxicity, inflammation, and oxidative stress. Indeed, SCs are particularly sensitive to oxidative stress as indicated by studies of diabetic neuropathy, in which oxidative stress is a critical component (Cameron and Cotter 1999; Eckersley 2002). For example in models of experimental diabetic neuropathy, oxidative stress has been shown to induce SC apoptosis (Wang et al. 2005), which can be ameliorated by pre-treatment with antioxidants (Askwith et al. 2009). Thus, we reason that improving transplanted SC survival after SCI, potentially by reducing secondary injury and the associated oxidative stress, will improve the efficacy and protective potential of the transplanted cells.
Our research group and others have previously shown the post-SCI administration of 17β-estradiol (E2) reduces secondary damage after SCI in male and female rodents (Chaovipoch et al. 2006; Sribnick et al. 2005, 2006; Cuzzocrea et al. 2008; Kachadroka et al. 2010). Additionally, in vitro studies have shown a protective effect of E2 against oxidative stress-induced cell death in several cell types, including neuronal cell lines (Biewenga et al. 2005; Wang et al. 2006), primary neuronal cells (Numakawa et al. 2007; Yu et al. 2004), and oligodendrocytes (Takao et al. 2004). However, the potential protective effects of E2 on SCs have not been described. Thus, this study was conducted to test the hypothesis that E2 has a cytoprotective effect on SCs against oxidative stress in vitro and that the combination of E2 administration and SC transplantation after SCI in a rat model will increase SC survival in the contused spinal cord.
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We report here that E2 protected SCs against H2O2-induced cell death. Although we have provided the first direct experimental evidence of the protective effects of E2 against H2O2 insult in SCs, our findings are similar to other studies which demonstrate the protective effects of E2 against H2O2 insult in several cell types, including primary cortical neurons (Behl et al. 1997; Crossthwaite et al. 2002), neuronal cell lines (Wang et al. 2006; Behl et al. 1995, 1997), oligodendrocytes (Takao et al. 2004), and glial cell lines (Sur et al. 2003). The finding that E2 protects SCs against H2O2-induced damage has significant implications with regard to SCI and therapeutic approaches. Specifically, production of reactive oxygen species (ROS), such as superoxide anion (O2•), hydrogen peroxide (H2O2), hydroxyl radical (OH•), and peroxynitrite (ONOO−) is thought to be one of the major events contributing to secondary injury, the phase following initial mechanical trauma after SCI (Kwon et al. 2004; Hall and Springer 2004). Moreover, elevations in the level of extracellular H2O2 after spinal cord contusion have been observed (Liu et al. 1999). These ROS may therefore be involved, at least in part, in the death of transplanted cells after SCI.
The proposed cytoprotective mechanisms of estrogen can be roughly divided into two mechanisms, a classical estrogen genomic pathway and non-genomic pathways (Singh et al. 2006; Raz et al. 2008). The classical estrogen genomic pathway involves binding of estrogen with intracellular/nuclear ERα and ERβ, dimerization of receptors, and induction of target gene transcription (Behl 2002). Clearly, expression of ER is a requirement for activation of the genomic pathway, and we show in this study that SCs express both ERα and ERβ, which is consistent with other reports of ER expression in human SCs and schwannomas (Fishbein et al. 2007; Patel et al. 2008). We also directly evaluated the ER-dependence of E2-mediated protection against oxidative stress by co-administration of the highly selective ERα/ERβ antagonist ICI 182,780. We found that H2O2-induced SC death was not attenuated by ICI 182,780, which suggests that the protective effect is not mediated through the classical genomic pathway. This result is in agreement with previous observations in other cell types (Wang et al. 2006), but is in contrast to other previous studies, which showed that application of ICI 182,780 significantly attenuated the protective effects of E2 to H2O2 insults (Numakawa et al. 2007; Takao et al. 2004; Urata et al. 2006). This inconsistency may be owing to the differences in cell type; concentrations of E2, ICI 182,780, or H2O2; or administration time point and duration. Alternatively, work from keratinocytes shows that the membrane-impermeable bovine serum albumin-conjugated E2 suppresses H2O2-induced cell death and up-regulates the anti-apoptotic protein Bcl-2, suggesting that the protective effect of estrogen may be mediated via membrane associated-ER (Kanda and Watanabe 2003). However, the involvement of membrane associated-ER and the associated signaling mechanisms mediating cytoprotection, particularly in neurons and glia, remains unclear.
The cytoprotective action of E2 can also be mediated through the non-genomic pathways which include modification of intracellular signaling pathways, mitochondrial mechanisms, and free radical scavenging (Simpkins and Dykens 2008; Winterle et al. 2001; Prokai and Simpkins 2007). Several in vitro studies have shown that E2 protected H2O2-induced cell death by attenuating intracellular calcium overload (Wang et al. 2006; Numakawa et al. 2007; Sur et al. 2003), restoring intracellular ATP levels, reducing lipid peroxidation, and increasing cellular redox regulation of glutathione/glutaredoxin levels (Wang et al. 2006; Urata et al. 2006). In this study, application of ICI 182,780 failed to attenuate protective effect of E2 on SCs suggesting the possibility of involvement of these non-genomic mechanisms. However, the exact mechanism by which E2 protects against oxidative stress needs to be further elucidated.
Intriguingly, we also demonstrate in this study that co-administration of E2 with SC transplantation significantly increased the number of labeled surviving transplanted SCs observed at 1 week post-transplantation, which suggests that E2 administration enhances survival of transplanted SCs after SCI. Previous work clearly demonstrates that delaying transplantation until 7 days following SCI improves SCs survival (Hill et al. 2006), however, poor survival of transplanted SCs is still observed (Pearse et al. 2007; Hill et al. 2007). Thus, a combinatorial intervention of delayed transplantation and pharmacological protection of SCs is likely required to significantly increase SC survival after transplantation. Our previous studies have shown that E2 reduces secondary injury after SCI (Chaovipoch et al. 2006; Kachadroka et al. 2010) and since ROS is a large component of secondary injury, we hypothesized in this study that post-SCI administration of E2 could not only confer protection directly to SCs but also reduce secondary injury to thereby increase the survival of transplanted SCs. We found that when SCs are transplanted into the injury epicenter 1 week after SCI, post-SCI administration of E2 induces a ∼sixfold increase in surviving labeled transplanted SCs. Although the mechanism of this striking protection of transplanted SCs was not evaluated in vivo, our in vitro evidence indicates that E2 has direct protective effects on SCs, which coupled with the known reduction in secondary injury after SCI likely worked in concert to protect the transplanted cells. Thus, we speculate that the substantial increase in SC survival is attributed to both direct protective effects of E2 on SCs and indirect effects, such as reduction in oxidative stress, inflammation, and immunologic responses at the transplanted site. Further experiments are needed to evaluate the effects of E2 on long-term survival and functional outcome following SC transplantation post-SCI, yet the combination of E2 and SC transplantation may be a highly efficacious therapeutic approach to promote repair and return of function after SCI.
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Appendix S1. Supplementary Materials and methods.
Figure S1. Evaluation of Schwann cell primary culture purity
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