Tamoxifen and estradiol improved locomotor function and increased spared tissue in rats after spinal cord injury: their antioxidant effect and role of estrogen receptor alpha

Abstract

17β-Estradiol is a multi-active steroid that imparts neuroprotection via diverse mechanisms of action. However, its role as a neuroprotective agent after spinal cord injury (SCI), or the involvement of the estrogen receptor-alpha (ER-α) in locomotor recovery, is still a subject of much debate. In this study, we evaluated the effects of estradiol and of Tamoxifen (an estrogen receptor mixed agonist/antagonist) on locomotor recovery following SCI. To control estradiol cyclical variability, ovariectomized female rats received empty or estradiol filled implants, prior to a moderate contusion to the spinal cord. Estradiol improved locomotor function at 7, 14, 21, and 28 days post injury (DPI), when compared to control groups (measured with the BBB open field test). This effect was ER-α mediated, because functional recovery was blocked with an ER-α antagonist. We also observed that ER-α was up-regulated after SCI. Long-term treatment (28 DPI) with estradiol and Tamoxifen reduced the extent of the lesion cavity, an effect also mediated by ER-α. The antioxidant effects of estradiol were seen acutely at 2 DPI but not at 28 DPI, and this acute effect was not receptor mediated. Rats treated with Tamoxifen recovered some locomotor activity at 21 and 28 DPI, which could be related to the antioxidant protection seen at these time points. These results show that estradiol improves functional outcome, and these protective effects are mediated by the ER-α dependent and independent-mechanisms. Tamoxifen׳s effects during late stages of SCI support the use of this drug as a long-term alternative treatment for this condition.

Keywords: 17β-estradiol; Estrogen receptor expression; Locomotor recovery; Neuroprotection; Selective estrogen receptor modulators; Trauma.

Fig. 1

Estrogen receptor-α (ER-α) mediates the enhanced locomotor recovery observed with estradiol (E2) after spinal cord injury (SCI). Ovariectomized (OVX) rats pretreated with estradiol (3 mg silastic implants) demonstrated a better locomotor performance in the initial and later stages after SCI. BBB scores for estradiol treated rats were significantly higher at 7 (**p<0.01), 14 (*p<0.05), 21 (*p<0.05), and 28 (***p<0.001) days post injury (DPI) according to Repeated Measures Two-way ANOVA and Bonferroni post-hoc analyses. Data are the mean ± SEM (estradiol, n=17 and control Silastic Empty, n=14). Moreover, rats treated with estradiol + MPP dihydrochloride (an ER-α antagonist, n=7) showed lower locomotor scores than rats with estradiol throughout the 4 weeks after spinal cord injury. Rats treated with MPP dihydrochloride (n=7) scored similar to non-treated control group. Statistical analysis did not show significant difference between the control groups and rats treated with estradiol + MPP dihydrochloride (*p<0.05; **p<0.01; ***p<0.001).

Fig. 2

Estradiol and Tamoxifen treatment promotes white matter sparring during SCI. A: The extent of the lesion cavity in treated and non-treated rats was performed by tissue staining with Luxol (SE-Silastic Empty; E2- Estradiol; MPP- MPP dihydrochloride [estrogen receptor alpha antagonist]; TAM- Tamoxifen). B: Densitometry analysis to determine the lesioned area was performed at 28 DPI using MCID, Imaging Research, Inc. Estradiol (E2) treated rats show a significant increase in white matter tissue at 28 DPI and the effect is mediated through the estrogen receptor alpha because the effect was blocked with MPP dihydrochloride. Rats treated with TAM show a significant white matter sparring at 28 DPI when compared to control, according to One-way ANOVA and Dunnet’s Multiple Comparison post-hoc (n=at least 3, *p<0.05; **p<0.01).

Fig. 3

Estradiol treatment enhances the up-regulation of the ER-α 14 days after SCI. Top panel: The ER-α is up-regulated in injured non-treated animals after 14 DPI when compared to sham group (p<0.005), according to Western blot studies. The up-regulation is more robust in injured animals treated with estradiol (E2) (p<0.005). Uterus (Ut) tissue was used as positive control as seen in lane 1. No changes were observed with GAPDH at each time point and all data was standardized accordingly (n=3). Bottom panel: Densitometric analysis represents the mean ± SEM and the statistical testing was performed at a pre-set alpha of p<0.05 (B). Comparison was made between sham/control and estradiol treated animals using ANOVA followed by Dunnet’s multiple comparisons post-hoc test (*p<0.005).

Fig. 4

Determination of superoxide activity in spinal cord segments 2 days after injury. Superoxides from rostral (A), epicenter (B), and caudal (C) segments (5 mm each) of lesioned cord were determined by lucigenin chemiluminescence in the presence and absence of estradiol (E2). The values shown are the mean ± SEM (n=4/group) and significance evaluated with an unpaired two-tailed student t-test (*p < .05 vs control).

Fig. 5

The effect of estradiol to reduce superoxide production at the lesion epicenter is not receptor mediated. Determination of superoxide activity in spinal cord tissue of estradiol + MPP treated rats 2 days after SCI. Epicenter and caudal segments of lesioned spinal cord 2 days after injury where analyzed for lucigenin chemiluminescence. Estradiol + MPP did not block the effect seen with estradiol (E2) alone. The values shown are the mean ± SEM (n=4/group and *p < .05 when compared to control).

Fig. 6

Levels of Tamoxifen in the plasma of female rats after commercial pellet implantation. The ion chromatogram acquired by UPLC-MS/MS demonstrated the retention time for Tamoxifen (TAM) from serum samples obtained from treated and untreated animals (insert). Serum TAM levels measured by UPLC-MS/MS over time (histogram) demonstrated that TAM was present in serum of all treated groups and absent in control non-treated animals, according to ANOVA analysis (p<0.001). Levels decreased over time in the treated animals. (Data are the mean ± SEM, n=6).

Fig. 7

Tamoxifen enhances functional recovery at late stages of SCI. A: BBB scores from TAM treated animals were not significantly different from control scores at 7 and 14 days post injury (DPI). However, TAM induced a significant change at 21 and 28 DPI (*p<0.05) according to Repeated Measures Two-way ANOVA followed by Bonferroni post hoc test. Results are mean ± SEM (Tamoxifen, n=10 and placebo, n=4). B–D: Determination of superoxide activity in spinal cord segments 28 days after injury in TAM-treated rats. Superoxides from rostral (B), epicenter (C), and caudal (D) segments of lesioned cord were determined by lucigenin chemiluminescence. The values shown are the mean ± SEM (n=4) and significance evaluated with an unpaired two-tailed student t-test (*p < .05 vs control).