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Keywords:

  • cell transplantation;
  • cervical;
  • estrogen;
  • oxidative injury;
  • Schwann cell;
  • spinal cord injury

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

J. Neurochem. (2010) 115, 864–872.

Abstract

Schwann cell (SC) transplantation is a promising repair strategy after spinal cord injury (SCI); however, a large number of SCs do not survive following transplantation. Previous studies have shown that 17β-estradiol (E2) protects several cell types against cytotoxicity. Thus, this study evaluated the protective potential of E2 on SCs in vitro and investigated the effect of E2 on transplanted SC survival in a rat model of SCI. Primary SC cultures were found to robustly express estrogen receptors (ER) and incubation with E2 protected SCs against hydrogen peroxide-induced cell death. This protection was not inhibited by the ER antagonist ICI 182,780, suggesting that genomic signaling is not necessary for protection. In a subsequent experiment, cervical hemicontusion SCI was induced in male rats followed by sustained administration of E2 or placebo. Eight days after SCI, SCs were transplanted into the injury epicenter. E2 treatment significantly increased the number of surviving labeled transplanted SCs evaluated 7 days after transplantation. These data demonstrate that E2 protects SCs against oxidative stress and improves transplanted SC survival, which suggests that E2 administration may be an intervention of choice for enhancing survival of transplanted SCs after SCI.

Abbreviations used:
CFSE

5-(and-6)-carboxyfluorecein diacetate succinimidyl ester

DMSO

dimethylsulfoxide

E2

17β-estradiol

ER

estrogen receptors

MTT

3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide

ROS

reactive oxygen species

SC

Schwann cell

SCI

spinal cord injury

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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Details on all materials and reagents are found in the Appendix S1.

Primary Schwann cell culture

Schwann cells were harvested from the sciatic nerves and brachial plexuses of postnatal day 2 or 3 Sprague–Dawley rats using a protocol described in previous publications with some modifications (Haastert et al. 2007; Brockes et al. 1979). Details of the procedure are found in the Appendix S1. This procedure yielded primary SC cultures of ∼98% (see Figure S1).

Immunocytochemistry of primary Schwann cell cultures

Immunocytochemistry was performed to examine the expression of estrogen receptors-α (ERα) and -β (ERβ) on SCs and the methodology is detailed in the Appendix S1.

In vitro application of test compounds

Water soluble E2 was diluted in Hanks balanced salt solution (HBSS) and an estrogen receptor antagonist ICI 182,780 was diluted in dimethylsulfoxide (DMSO) as a stock solution at a concentration of 1 mM and further diluted to the desired concentration in D-10 medium. Control cells were incubated at the same final DMSO concentration in D-10 medium. Experiments were initiated by plating SCs at a density of 2 × 105 cells/well in poly-D-lysine coated with 24-well plates. After overnight incubation, SCs were treated with E2 by bath application. To test the effect of E2 alone on SCs, cells were incubated with E2 for 24 or 48 h before performing cell viability test. To test the protective effect of E2 on SCs, SCs were pre-incubated with E2 for 2 h followed by co-treatment of E2 and hydrogen peroxide (H2O2) for 24 h. In the experiment involving the estrogen receptor antagonist ICI 182,780, SCs were pre-treated with ICI 182,780 for 30 min before co-incubation with E2 for 2 h followed by incubation with H2O2 for 24 h. H2O2 was freshly diluted from 30% H2O2 stock solution with D-10 medium to a 400 μM final concentration prior to each experiment.

MTT cell viability assay

The 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of SCs. MTT was freshly prepared with HBSS as a stock solution at a concentration of 5 mg/mL and further diluted with serum free Dulbecco’s modified Eagle’s medium without phenol red at a final concentration of 0.7 mg/mL. After application of test compounds and appropriate incubation period, bath solutions were aspirated from each 24-well plate and 250 μL of MTT wash added to each well. Plates were incubated at 37°C for 4 h in the dark. Media were aspirated and the formazan crystal products were solubilized with DMSO. The solutions were transferred to disposable plastic cuvettes where the absorbance of the solution was measured by spectrophotometer (Beckman DU530; Beckman, Fullerton, CA, USA) at a wavelength of 570 nm. The experiments were performed in triplicate with separate cultures.

CFSE labeling of Schwann cells

The fluorescent dye 5-(and-6)-carboxyfluorecein diacetate succinimidyl ester (CFSE) was used to identify transplanted SCs as previously published (Li et al. 2003) with some modifications. To facilitate cell loading, CFSE was dissolved in Pluronic F-127 20% solution in DMSO (Invitrogen, Carlsbad, CA, USA) as a stock solution at a concentration of 10 mM/mL. On the day of transplantation, SCs were trypsinized with 0.25% trypsin–EDTA and washed twice with HBSS. SCs were then re-suspended in HBSS and then incubated in HBSS + CFSE solution (final concentration 5 μM/mL) for 1 min at 37°C. Subsequently, an equal volume of fetal calf serum was added into the cells followed by centrifugation and washing twice with HBSS. SCs were then suspended at a concentration of 2 × 106 cells/10 μL in L15 medium and kept on ice until transplantation.

Spinal cord injury and Schwann cell transplantation

All animal procedures were conducted according to the Guidelines for the Care and Use of Laboratory Animals and under the approval of the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. Eighteen adult male Sprague–Dawley rats (175–200 g) were randomly separated into three groups: (i) SCI + vehicle control (vehicle), (ii) SCI + SC transplantation (SC), (iii) SCI + E2 + SC transplantation (SC + E2), so that = 6 per group. Animals were anesthetized by intraperitoneal injection with a ketamine/xylazine (50/10 mg/kg) cocktail. A midline dorsal skin incision was made between the spinous processes of C2 and T2 and the underlying muscles were separated using muscle retractor. The paravertebral muscles were removed and a C5 laminectomy was made to expose the dorsal surface of the spinal cord. The spinal column was stabilized by clamping at the vertebral body of C2 and spinous process of T2 using toothed Adson forceps connected with supported arms and experimental platform. A right-side hemicontusion injury was induced using the Infinite Horizon Impactor (Precision System, Kentucky, IL, USA) with a 0.8-mm-diameter impactor tip at a force of 200 kdyn and no dwell time. After injury, musculature and skin were sutured in layers with Ethicon Vicryl absorbable suture (Novartis Animal Health, Greensboro, NC, USA). Estrogen treatment animals received subcutaneous implantation of a E2 pellet (5 mg/pellet, 21-day continuous release) 30 min after injury. On day 8 after injury, animals were anesthetized with a ketamine/xylazine cocktail. The injury site was re-exposed and 2 × 106 CFSE-labeled SCs/10 μL were transplanted over 5 min into the injury site using a Hamilton syringe attached with 36-gauge needle (Hamilton Company, Reno, NV, USA). Since 10 μL is a large volume to inject into the rat spinal cord, this slow rate of injection (2 μL/min) was utilized. Vehicle control animals were injected with L15 medium with the same procedure.

Spinal cord tissue processing

Seven days after transplantation, animals were euthanized by barbiturate overdose and then intracardially perfused with ice-cold 0.1 M phosphate-buffered saline and 4% paraformaldehyde in phosphate-buffered saline, pH 7.40. Spinal cord tissue at the lesion epicenter was extracted, cryo-protected, and cryo-sectioned as detailed in the Appendix S1.

Quantification of Schwann cell survival after transplantation

Unbiased stereology was conducted on an Olympus BX-51 microscope (Olympus America Inc., Center Valley, PA, USA) linked to a MicroFire® true color CCD digital camera (Optronics, Goleta, CA, USA) using StereoInvestigator software (Microbrightfield Inc., Williston, VT, USA) at 200–400× magnification. The optical fractionator probe was used to quantify the total number of CFSE-labeled SCs in the epicenter of the lesion in random serial sections as previously described (Kachadroka et al. 2010). Briefly, the contour of the area occupied by CFSE-SCs in each section was drawn and a 200 × 200-μm grid was then overlaid. The CFSE-SCs were counted using a 50 × 50-μm sampling box in every 10th section using standard stereological inclusion criteria.

Statistical analysis

The effect of E2 alone on SCs was analyzed by two-way anova followed by Bonferroni post hoc analysis. All other data were analyzed by one-way anova followed by Tukey’s multiple comparison tests using GraphPad Prism version 5.00 for Windows (GraphPad software, San Diego, CA, USA). For all tests, alpha value of p < 0.05 was considered statistically significant. All data in this study are presented as a mean ± SEM.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Primary rat Schwann cells express both estrogen receptor (ER)-α and -β

The purity of the primary cultures of rat SCs was determined to be 98 ± 0.78% by immunocytochemistry (Figure S1). The expression of ER was evaluated by fluorescent immunocytochemistry using antibodies against the estrogen receptor alpha (ERα) and beta (ERβ). As seen in Fig. 1, SCs at 3 days-in-culture showed extensive expression of both ERα and ERβ. The representative micrographs show the typical pattern of expression observed in all cultures in that ERα was expressed both in nucleus and cytoplasm of SCs (Panel A), whereas ERβ was predominantly localized in nuclear region of SCs (Panel B) confirmed by 4′, 6-diamidino-2-phenylindole (DAPI) counter-staining (Panel C).

image

Figure 1.  Immunostaining of estrogen receptors (ER). (a–d) Micrographs of Schwann cells (SCs) (3 days-in-culture) in an identical field represent (a) ERα (red), (b) ERβ (green), (c) DAPI (blue), (d) double overlay of ERα, ERβ. ERα is expressed in both the nucleus and cytoplasm, whereas ERβ is expressed mainly in the nucleus of SCs. Scale bar, 50 μm.

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High concentrations of 17β-estradiol have a toxic effect on Schwann cells

The effect of E2 on SCs survival was measured after either a 24 or 48 h incubation time (Fig. 2). In this experiment, primary SC cultures were exposed to varying concentrations of E2 in the culture media for 24 or 48 h and cell viability was calculated using the MTT assay. We found that incubation with E2 in a dose range from 10−9 to 10−4 M for 24 h did not induce significant changes in cell survival as compared with cells not incubated with E2. However, after 48 h of incubation with E2, the higher doses of E2 caused a significant reduction in cell survival as compared with control cells not incubated with E2. Specifically, E2 at concentrations of 10−7, 10−6, 10−5, and 10−4 M significantly decreased the percentage of surviving SCs to 84.2 ± 3.1, 84.8 ± 1.4, 74.1 ± 5.5, and 54.2 ± 8.8%, respectively. These data suggest that in pure primary SCs, long exposures to E2 in the micromolar range (i.e. 0.1 μM–1.0 μM) result in ∼20% cell loss and in the millimolar range (i.e. 0.1 mM) result in ∼60% cell loss. Since reported serum levels of E2 in normal, physiological conditions are in the pico- to nanomolar range (i.e. 10−12 to 10−9), the doses that induce toxicity in SCs are likely well above physiological levels.

image

Figure 2.  Toxic effect of 17β-estradiol (E2) on Schwann cell (SC). To test the toxicity of estrogen, SCs were exposed to various concentrations of E2 for 24 or 48 h and SCs viability was estimated using the 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Percentage of cell survival is shown as mean ± SEM. There were no significant differences in cell survival between each concentration of E2 treatment at the 24 h time point. At the 48 h time point, high concentrations of E2 (10−7 to 10−4) significantly decreased the percentage of surviving SCs (*p < 0.05 as compared with cells administered no E2). = 9, from three separate cultures.

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17β-Estradiol protects primary Schwann cell cultures against H2O2-induced cytotoxicity in dose-dependent manner

To evaluate the potential cytoprotective effect of E2 against oxidative stress-induced SC death, SCs were treated with E2 at various concentrations for 2 h followed by exposure to H2O2 at a final concentration of 400 μM for 24 h. SCs viability was estimated using MTT assay. When treated with H2O2 for 24 h, SC viability was significantly decreased to 46.8 ± 1.0% as compared with control. Incubation with E2 at concentrations between 10−7 and 10−4 M 2 h prior to H2O2 treatment significantly increased the percentage of SC survival as compared with H2O2 treatment alone. Specifically, treatment with E2 prior to H2O2 exposure at concentrations of 10−7, 10−6, 10−5, and 10−4 M increased SC survival to 57.4 ± 2.0, 70.6 ± 1.5, 57.0 ± 1.1, and 64.5 ± 2.3%, respectively. There was no significant increase in SC viability when SCs were treated with 10−9 or 10−8 M E2 as compared with H2O2 treatment alone (Fig. 3). These data suggest that E2 in the low micromolar range is protective against oxidative stress in SCs.

image

Figure 3.  Dose response effects of 17β-estradiol (E2) on H2O2-induced Schwann cell (SC) death. To evaluate the protective potential of E2 against H2O2-indueced cytotoxicity in SCs, primary SCs were treated with E2 at various concentrations for 2 h followed by exposure to H2O2 at a final concentration of 400 μM for 24 h. SC viability was estimated using 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Percentage of cell survival is shown as mean ± SEM. Exposure to H2O2 in untreated SCs caused a significant reduction in cell survival over non-exposed control cells, as expected (indicated by **< 0.05). Administration of E2 at concentrations between 10−7 and 10−4 M significantly increased the percentage of SC survival as compared with cells treated with H2O2 alone. (*< 0.05 as compared with H2O2 treatment alone). = 9, from three separate cultures.

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Estrogen receptor antagonist ICI 182,780 fails to attenuate protective effect of E2 against H2O2-induced cytotoxicity on Schwann cells

The estrogen receptor antagonist ICI 182,780 was used to evaluate whether the protective effect of estrogen against oxidative stress-induced SC death is mediated through the ER (Fig. 4). The lowest concentration of E2 that showed significant protective effects on SCs (10−7 M, Fig. 3) was used. For this experiment, SCs were treated with the selective estrogen receptor antagonist ICI 182,780 at a concentration of 10−6 M for 30 min before co-treatment with E2 at a final concentration of 10−7 M for 2 h, and followed by exposure to 400 μM of H2O2 to induce cytotoxicity for 24 h. The viability was estimated using MTT assay. Similar to our previous results, pre-incubation with E2 significantly increased SC survival as compared with H2O2 treatment alone. However, ICI 182,780 co-treatment with E2 also resulted in a significant increase in SC viability as compared with H2O2 treatment alone. Additionally, there was no significant difference in cell survival between cells treated with E2 and cells co-treated with ICI 182,780 and E2. These data indicate that ICI 182,780 did not antagonize the protective effects of E2.

image

Figure 4.  Effect of estrogen receptor antagonist ICI 182,780 on the protective effect of 17β-estradiol (E2) against H2O2-induced Schwann cell (SC) death. To investigate whether the cytoprotective effect of E2 is mediated via the ER, SCs were treated with the estrogen receptor antagonist ICI 182,780 (ICI) at a concentration of 10−6 M for 30 min before co-treatment with E2 at a final concentration of 10−7 M for 2 h, and followed by exposure to 400 μM of H2O2 to induce cytotoxicity for 24 h. Cell viability was estimated using 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Percentage of cell survival is shown as mean ± SEM. Similar to previous results, H2O2 exposure caused a significant reduction in cell survival (*< 0.05 as compared with H2O2 treatment alone). Treatment with E2 and E2 with ICI 182,780 significantly increased the percentage of cell survival (**< 0.05 as compared with H2O2 treatment alone). No statistical difference was found between E2 and E2 + ICI treated conditions (indicated by ns). = 9, from three separate cultures.

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E2 administration increases the number of surviving transplanted Schwann cell in a cervical hemicontusion spinal cord injury

Since we found that E2 protects SCs from oxidative stress in vitro, we hypothesized that co-administration of E2 with SC transplantation after SCI in vivo would increase SC survival. The experimental timeline used to evaluate this hypothesis is diagramed in Fig. 5(a). Briefly, a C5 hemicontusion SCI was induced in adult male rats followed 30 min later by administration of E2 in a slow-release pellet, which maintains elevated levels of E2 for 21 days. At 8 days post-SCI, CFSE-labeled SCs were injected directly into the lesion epicenter and cell survival was evaluated on post-SCI day 15 (7 days after transplantation of SCs). As shown in Fig. 5(b), we observed no CFSE-labeled cells in the vehicle-treated group. Some CFSE-labeled SCs were observed in the hemicontused spinal cord at 7 days post-transplantation in the SC only group (Fig. 5c). As seen in Fig. 5(d), substantially more CFSE-labeled SCs were found in the hemicontused spinal cord in the SC + E2 group. These surviving labeled SCs mainly occupied the injury site; however, some were seen to migrate away from the injury epicenter/injection site (Fig. 5e). To quantify the number of labeled SCs, unbiased stereology was conducted on serial sections throughout the lesion epicenter for animals in the following groups: SCI + vehicle, SCI + SC transplantation, or SCI + E2 + SC transplantation (Fig. 6). As expected, no labeled cells were counted in the vehicle group. In the SCI + SC transplantation group, the number of surviving labeled SCs was 52 572 ± 21 730. Significantly, more labeled SCs were counted in the SCI + E2 + SC transplantation group (315 811 ± 152 361 cells). These data indicated that post-SCI administration of E2 caused a nearly sixfold increase in survival of labeled transplanted SCs and support the hypothesis that E2 protects SCs.

image

Figure 5.  5-(and-6)-Carboxyfluorecein diacetate succinimidyl ester (CFSE)-labeled Schwann cells (SCs) survive in a cervical hemicontused spinal cord at 7 day after transplantation. (a) Diagram of the experimental timeline detailing that a C5 hemicontusion spinal cord injury (SCI) was induced followed 30 min later by administration of 17β-estradiol (E2) in a slow-release pellet. At 8 days post-SCI, CFSE-labeled SCs were injected directly into the lesion epicenter and cell survival was evaluated on post-SCI day 15 (7 days after transplantation of SCs). (b) Representative micrographs showing lack of CFSE-labeled cell in the vehicle (L15-media) injected group and (c) limited numbers of CFSE-labeled SCs (green) in the SC only group. Scale bars, 400 μm. (d) Representative micrograph showing CFSE-labeled SCs in ipsilateral side of the cervical hemicontused spinal cord in the SC + E2 treatment group. (e) A higher magnification micrograph from the box in (d). Labeled transplanted SCs mainly occupied the lesion site (arrow); however, some cells migrated away from the injection site (arrowhead). Scale bar = 200 μm.

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image

Figure 6.  Quantification of Schwann cell (SC) survival. At 7 days post-transplantation, surviving 5-(and-6)-carboxyfluorecein diacetate succinimidyl ester (CFSE)-labeled SCs in hemicontused spinal cord were quantified using unbiased stereology of spinal cord tissue from the following experimental groups: spinal cord injury (SCI) + vehicle (vehicle), SCI + SC transplantation (SC), or SCI + E2 + SC transplantation (SC + E2). The total numbers of SCs in the epicenter of the lesion were estimated using the optical fractionator probe in serial sections. As expected, no cells were counted in the vehicle group. In the SCI + SC transplantation group, the number of surviving labeled SCs was 52 572 ± 21 730. Significantly, more SCs were counted in the SCI + E2 + SC transplantation group (315 811 ± 152 361); *< 0.05 as compared with SC transplanted alone cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This research was supported by NS052559 (CF) and The Thailand Commission on Higher Education Staff Development Project, and Siriraj Graduate Thesis Scholarship (AS).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Appendix S1. Supplementary Materials and methods.

Figure S1. Evaluation of Schwann cell primary culture purity

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