Effect of endogenous androgens on 17beta-estradiol-mediated protection after spinal cord injury in male rats

Abstract

Several groups have recently shown that 17beta-estradiol is protective in spinal cord injury (SCI). Testosterone can be aromatized to 17beta-estradiol and may increase estrogen-mediated protection. Alternatively, testosterone has been shown to increase excitotoxicity in models of central nervous system (CNS) injury. These experiments test the hypothesis that endogenous testosterone in male rats alters 17beta-estradiol-mediated protection by evaluating a delayed administration over a clinically relevant dose range and manipulating testicular-derived testosterone. Adult male Sprague Dawley rats were either gonadectomized or left gonad-intact prior to SCI. SCI was produced by a midthoracic crush injury. At 30 min post SCI, animals received a subcutaneous pellet of 0.0, 0.05, 0.5, or 5.0 mg of 17beta-estradiol, released over 21 days. Hindlimb locomotion was analyzed weekly in the open field. Spinal cords were collected and analyzed for cell death, expression of Bcl-family proteins, and white-matter sparing. Post-SCI administration of the 0.5- or 5.0-mg pellet improved hindlimb locomotion, reduced urinary bladder size, increased neuronal survival, reduced apoptosis, improved the Bax/Bcl-xL protein ratio, and increased white-matter sparing. In the absence of endogenous testicular-derived androgens, SCI induced greater apoptosis, yet 17beta-estradiol administration reduced apoptosis to the same extent in gonadectomized and gonad-intact male rats. These data suggest that delayed post-SCI administration of a clinically relevant dose of 17beta-estradiol is protective in male rats, and endogenous androgens do not alter estrogen-mediated protection. These data suggest that 17beta-estradiol is an effective therapeutic intervention for reducing secondary damage after SCI in males, which could be readily translated to clinical trials.

FIG. 1.

Effect of post-SCI administration of 17β-estradiol (E2) on the Basso, Beattie, Bresnahan (BBB) score. Hindlimb locomotor function was assessed by two raters naïve to the treatment of the animal, and scores were transformed to improve the metric properties in evaluation of animals with moderate to severe SCI as previously described (Ferguson et al., 2004). Mean scores ± SEM for post-SCI days 3, 7, 14, 21, and 28 are shown for gonadectomized (GDX) male rats in A–C and gonad-intact (intact) male rats in D–F. Data from the each of the vehicle groups (GDX + 0.0 mg or Intact + 0.0 mg) are repeated for comparison to groups treated with 17β-estradiol. *Significantly different than placebo (0.0 mg) pellet on that day. N = 12 for all groups.

FIG. 2.

Effect of post-SCI administration of 17β-estradiol (E2) on wet-tissue weight of the urinary bladder at 28 days post SCI. After euthanasia, the urinary bladder was extracted and the wet-tissue weight was obtained. Average tissue weight of the urinary bladder is shown for gonadectomized animals (A) and for gonad-intact animals (B). *Significantly lower than the placebo (0.0 mg) pellet group. ⁺Laminectomy only (LAM) had significantly lower bladder weight than all SCI groups. N = 12 for all groups.

FIG. 3.

Representative micrographs of immunohistochemistry for myelin basic protein at 28 days post SCI. Micrographs (100 ×) of coronal tissue sections from gonadectomized male rats (A) show intact myelin in tissue from animals that received a laminectomy only (LAM, top panel). Little or no intact myelin was identified in the SCI + 0.0 mg E2 or SCI + 0.05 mg E2 groups (middle two panels), and a larger area of intact myelin was observed in tissue from SCI + 0.5 mg E2 or SCI + 5.0 mg E2 animals (lower two panels, border indicated by arrows). Similarly, in gonad-intact male animals (B), less intact myelin was detected in the SCI + 0.0 mg E2 or SCI + 0.05 mg E2 groups (middle two panels), and more intact myelin was found in the SCI + 0.5 mg E2 or SCI + 5.0 mg E2 groups (lower two panels, border indicated by arrows). Bar = 100 μm.

FIG. 4.

Effect of post-SCI administration of 17β-estradiol (E2) on white-matter sparing at the lesion epicenter. Unbiased stereology using the Cavalieri probe was used to determine the area of intact white matter. The section with the smallest area was designated as the epicenter and averaged with the area of the serial sections immediately rostral and caudal to obtain the percentage of the spared white-matter area for each animal. Mean percentage of white-matter area ± SEM are shown for gonadectomized (A) and gonad-intact (B) groups. *Significantly different than placebo (0.0 mg) pellet group. N = 10 for all groups.

FIG. 5.

Representative micrographs of cresyl violet histochemistry for Nissl substance at 28 days post SCI. Micrographs (200 ×) of transverse tissue sections from gonadectomized male rats (A) illustrate the presence of identifiable neurons (indicated by arrow) in the ventral horn of tissue from the laminectomy only (LAM, top panel), SCI + 0.5 mg E2, or SCI + 5.0 mg E2 (lower two panels) groups. Very few neurons were identified in tissue from the SCI + 0.0 mg E2 or SCI + 0.05 mg E2 groups (middle two panels). Similarly, micrographs (200 ×) from gonad-intact male rats (B) show the presence of neurons (indicated by arrow) in the laminectomy (LAM, upper panel), SCI + 0.5 mg E2, and SCI + 5.0 mg groups (lower two panels), and very few neurons present in the SCI + 0.0 mg E2 or SCI + 0.05 mg E2 groups (middle two panels). Bar = 50μm.

FIG. 6.

Effect of post-SCI administration of 17β-estradiol (E2) on neuronal survival in the ventral horn at 28 days post SCI. Unbiased stereology using the optical fractionator probe was used to determine the number of surviving neurons in the ventral horns. The mean number of motor neurons ± SEM in the ventral horn is shown for gonadectomized (A) and gonad-intact (B) male rats. *Significantly different than placebo (0.0 mg) pellet group. N = 10 for all groups.

FIG. 7.

Effect of post-SCI administration of 17β-estradiol (E2) on apoptosis in the ventral horn at 4 days post SCI. A representative micrograph (400 ×) from a gonadectomized male rat treated with the placebo (0.0 mg) E2 pellet showing darkly stained TUNEL-positive nuclei with a blebbed appearance (dark arrows) and lighter outline of cell soma visualized with neutral red counterstaining (A). Bar = 20 μm. TUNEL+ cells in the ventral horn were counted with unbiased stereology using the optical fractionator probe. The mean number of TUNEL+ cells ± SEM in the ventral horn is shown for gonadectomized (B) and gonad-intact (C) male rats. *Significantly different than placebo (0.0 mg) pellet group. N = 10 for all groups. The stereological counts of TUNEL+ cells was compared across estrogen treatment (D) using the same data from panels B and C. *Significant difference between gonadectomized (GDX) and gonad-intact group at that E2 dose.

FIG. 8.

Effect of post-SCI administration of 17β-estradiol on Bcl-2 family proteins at the epicenter of the lesion. Immunoblots for expression of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-xL were conducted on spinal-cord tissue from the epicenter of the lesion in animals that received either the placebo pellet (0.0 mg) or the 0.5-mg E2 pellet (SCI + E2, A). Mean  ±  SEM relative optical density measurements were normalized to GAPDH for Bax expression (B) or Bcl-xL expression (C), with laminectomy values indicated by the dotted line. *Significant difference between placebo (0.0 mg) and 0.5-mg pellet groups (panels B and C). The ratio of Bax/Bcl-xL was calculated from the normalized relative optical density values (D). N = 5 animals per group, immunoblots in duplicate.

FIG. 9.

Effect of post-SCI administration of 17β-estradiol (E2) on estrogen receptor alpha (ERα) expression at the epicenter of the lesion. Immunoblots for expression of the ERα were conducted on spinal-cord tissue from the epicenter of the lesion in animals that received either the placebo pellet (0.0 mg) or the 0.5 mg E2 pellet (SCI + E2, A). Mean ± SEM relative optical density measurements were normalized to GAPDH for ERα expression (B) with laminectomy values indicated by the dotted line. N = 5 animals per group, immunoblots in duplicate.