EFFECT OF 17β-ESTRADIOL ON SIGNAL TRANSDUCTION PATHWAYS AND ... : Shock (original) (raw)
INTRODUCTION
Spinal cord injury (SCI) is a highly debilitating pathology (1). Although innovative medical care has improved patient outcome, advances in pharmacotherapy for the purpose of limiting neuronal injury and promoting regeneration have been limited. The complex pathophysiology of SCI may explain the difficulty in finding a suitable therapy. The primary traumatic mechanical injury to the spinal cord causes the death of a number of neurons that cannot be recovered and regenerated. Studies indicate that neurons continue to die for hours after traumatic SCI (2). The events that characterize this successive phase to mechanical injury are called "secondary damage." The secondary damage is determined by a large number of cellular, molecular, and biochemical cascades. A large body of recent data suggests the presence of a local inflammatory response, which amplifies the secondary damage (3). The cardinal features of inflammation, namely, infiltration of inflammatory cells (polymorphonuclear neutrophils, macrophage, and lymphocytes), release of inflammatory mediators, and activation of endothelial cells leading to increased vascular permeability, edema formation, and tissue destruction have been extensively characterized in animal models of SCI (4). Because a complex pathophysiology is involved in SCI, the potential therapy should use either multiple agents or a multiactive agent. A previous study has suggested that treatment with the steroid hormone 17β-estradiol (E2) may attenuate several of the damaging pathways initiated after SCI (5). 17β-Estradiol, the most abundant form of estrogen in the body, has been shown to be neuroprotective and produces therapeutic effects in various models of central nervous system disease where inflammation and immune-mediated processes predominate (6-9). 17β-Estradiol exerts its neuroprotective effects, in part, by acting as an anti-inflammatory agent and also as an antioxidant (10). The precise mechanism by which E2 acts as an anti-inflammatory agent, however, is not completely understood.
The aim of the present study was to investigate the effect of estrogen in the modulation of secondary injury in the spinal cord. To characterize the effect of E2 in a model of SCI, we determined the following end points of the inflammatory response: (1) histological damage; (2) motor recovery; (3) neutrophil infiltration; (4) proinflammatory cytokines; (5) nitrotyrosine, iNOS and COX-2 expression; (6) apoptosis (terminal deoxynucleotidyltransferase [TdT]-mediated UTP end labeling [TUNEL] staining); and (7) Bax and Bcl-2 expression. In addition, we investigated the effects of the systemic administration of estrogen receptor antagonist (ICI 182,780) on the above parameters of inflammation.
MATERIALS AND METHODS
Animals
Adult male CD1 mice (25-30 g; Harlan Nossan, Milan, Italy) were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D. M. 116192), as well as with the European Economic Community regulations (O. J. of E.C. L 358/1 12/18/1986).
Spinal cord injury
Mice were anaesthetized using chloral hydrate (400 mg/kg body weight) (11). A longitudinal incision was made on the midline of the back, exposing the paravertebral muscles. These muscles were dissected away, exposing T5-T8 vertebrae. The spinal cord was exposed via a four-level T6-T7 laminectomy, and SCI was produced by extradural compression using an aneurysm clip with a closing force of 24 g. After surgery, 1 mL of saline was administered subcutaneously to replace the blood volume lost during surgery. During the surgery and recovery from anesthesia, the mice were placed on a warm heating pad and covered with a warm towel. The mice were singly housed in a temperature-controlled room at 27°C, and survival was measured during a period of 10 days. Food and water were provided to the mice ad libitum. During this period, the animals' bladders were manually voided twice a day until the mice were able to regain normal bladder function. In all injured groups, the spinal cord was compressed for 1 min. Sham-injured animals were only subjected to laminectomy.
Experimental groups
Mice were randomly allocated into the following groups: (1) saline + SCI group, in which mice received saline subcutaneously and were subjected to SCI (n = 40); (2) E2 group, the same as the saline + SCI group, but E2 was also administered subcutaneously at a dose of 300 μg/kg 1 h before SCI and 3 and 6 h after SCI (n = 40); (3) ICI group, which was the same as the E2 group, but they received ICI 182,780 at a dose of 500 μg/kg subcutaneously 1 h before the administration of E2 (n = 40); (4) saline + sham group, in which mice were subjected to the surgical procedures as the above group, except that the aneurysm clip was not applied (n = 40); (5) sham + E2 group, identical to sham + saline group, but they received an administration of E2; and (6) sham +ICI group, identical to sham + E2 group except that they received an administration of ICI 182,780 (n = 40). In the experiments regarding the motor score, the animals from all the experimental groups were observed and treated daily for 9 days after SCI. At different points (see Fig. 1), the mice (n = 10 from each group for each of the 3 points were killed to evaluate the various parameters as described below.
Mice were killed at different points to evaluate the various parameters (n = 10 mice from each group for each point). 17β-Estradiol was administered subcutaneously at a dose of 300 μg/kg 1 h before SCI and 3 and 6 h after SCI; ICI 182,780 (500 μg/kg, s.c.) was administered 1 h before the administration of E2.
In a separate set of experiments, to elucidate the potential clinical significance of the protective effects of E2, we also investigated whether the posttreatment with E2, administered subcutaneously at a dose of 300 or 600 μg/kg at 3 and 6 h after SCI, attenuates the motor dysfunction assessed by motor score. The dose and the time of treatment of ICI 182,780 (500 µg/kg) were based on in vivo studies from our and other laboratories (12), and the dose of E2 was chosen in agreement with a previous study (13).
Total protein extraction and Western blot analysis for Bax and Bcl-2
Spinal cord tissue, obtained from animals killed 24 h after injury or sham injured, was disrupted by homogenization with an Ultra-turrax T8 homogenizer on ice in lysis buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1.5 μg/mL trypsin inhibitor, 3 μg/mL pepstatin, 2 μg/mL leupeptin, 40 μM benzidamin, 1% NP-40, and 20% glycerol). After 1 h, tissue lysates were obtained by centrifugation at 100,000_g_ for 15 min at 4°C. Protein concentrations were estimated by the Bio-Rad protein assay (Bio-Rad Laboratories, Segrate, Milan, Italy) using bovine serum albumin as standard.
For Western blot analysis, 70 µg protein of lysates was mixed with gel-loading buffer (50 mM Tris, 10% [wt/vol], sodium dodecyl sulfate, 10% [wt/vol] glycerol, 10% [vol/vol] 2-mercaptoethanol, 2 mg/mL bromophenol), boiled for 5 min, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% polyacrylamide). The blot was performed by transferring proteins from a slab gel to nitrocellulose membrane at 240 mA for 40 min at room temperature. The filter was then blocked with 1× phosphate-buffered saline (PBS), 5% nonfat dried milk for 40 min at room temperature and probed with specific monoclonal antibodies against Bax (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif; 1:1,000), or Bcl-2 (Santa Cruz Biotechnology) in 1× PBS, 5% nonfat dried milk, 0.1% Tween-20 at 4°C overnight. The AffiniPure Goat Anti-Rabbit immunoglobulin G coupled to peroxidase secondary antibody (1:2,000; Jackson Immuno Research Laboratories, Inc., Calif) was incubated for 1 h at room temperature. Subsequently, the blot was extensively washed with PBS, developed using SuperSignal West Pico chemiluminescence Substrate (PIERCE, Milan, Italy) according to the manufacturer's instructions, and exposed to Kodak X-Omat film (Eastman Kodak Co., Rochester, NY). An α-tubulin protein (Sigma, St. Louis, Mo; 1:1,000) Western blot was performed to ensure equal sample loading. The protein bands of Bax (~23 kd), or Bcl-2 (~26 kd) on x-ray film were scanned and densitometrically analyzed with a model GS-700 imaging densitometer (Bio-Rad). To ascertain that blots were loaded with equal amounts of protein lysates, they were also incubated in the presence of the antibody against α-tubulin protein (1:10,000; Sigma-Aldrich Corp.). The densitometric data for Western are normalized for loading control values.
Light microscopy
Spinal cord biopsies were taken at 24 h after trauma. Tissue segments containing the lesion (1 cm on each side of the lesion) were paraffin embedded and cut into 5-µm-thick sections. Tissue longitudinal sections (thickness, 5 μm) were deparaffinized with xylene, stained with hematoxylin/eosin, and Luxol fast blue staining (used to assess demyelination), and studied using light microscopy (Dialux 22; Leitz, Wetzlar, Germany). The segments of each spinal cord were evaluated by an experienced histopathologist (R.O.). Damaged neurons were counted, and the histopathological changes of the gray matter were scored on a six-point scale (14): 0, no lesion observed; 1, gray matter contained 1 to 5 eosinophilic neurons; 2, gray matter contained 5 to 10 eosinophilic neurons; 3, gray matter contained more than 10 eosinophilic neurons; 4, small infarction (less than one third of the gray matter area); 5, moderate infarction (one third to one half of the gray matter area); 6, large infarction (more than half of the gray matter area). The scores from all the sections from each spinal cord were averaged to give a final score for an individual mouse. All the histological studies were performed in a blinded fashion.
Grading of motor disturbance
The motor function of mice subjected to compression trauma was assessed once a day for 10 days after injury. Recovery from motor disturbance was graded using the modified murine Basso, Beattie, and Bresnahan (BBB) (15) hind limb locomotor rating scale (16-17). The following criteria were considered: 0, no hind limb movement; 1, slight (<50% range of motion) movement of one to two joints; 2, extensive (>50% range of motion) movement of one joint and slight movement of one other joint; 3, extensive movement of two joints; 4, slight movement in all three joints; 5, slight movement of two joints and extensive movement of one joint; 6, extensive movement of two joints and slight movement of one joint; 7, extensive movement of all three joints; 8, sweeping without weight support or plantar placement and no weight support; 9, plantar placement with weight support in stance only or dorsal stepping with weight support; 10, occasional (0% - 50% of the time) weight-supported plantar steps and no coordination (front/hind limb coordination); 11, frequent (50% - 94% of the time) to consistent (95% - 100% of the time) weight-supported plantar steps and no coordination; 12, frequent to consistent weight-supported plantar steps and occasional coordination; 13, frequent to consistent weight-supported plantar steps and frequent coordination; 14, consistent weight-supported plantar steps, consistent coordination, and predominant paw position is rotated during locomotion (lift off and contact) or frequent plantar stepping, consistent coordination, and occasional dorsal stepping; 15, consistent plantar stepping and coordination, no/occasional toe clearance, paw position is parallel at initial contact; 16, consistent plantar stepping and coordination (front/hind limb coordination) andfrequent toe clearance, and predominant paw position is parallel at initial contact and rotated at lift off; 17, consistent plantar stepping and coordination and frequent toe clearance and predominant paw position is parallel at initial contact and lift off; 18, consistent plantar stepping and coordination and consistent toe clearance and predominant paw position is parallel at initial contact and rotated at lift off; 19, consistent plantar stepping and coordination and consistent toe clearance and predominant paw position is parallel at initial contact and lift off; 20, consistent plantar stepping, coordinated gait, consistent toe clearance, and predominant paw position is parallel at initial contact and lift off and trunk instability; 21, consistent plantar stepping, coordinated gait, consistent toe clearance, and predominant paw position is parallel at initial contact and lift off and trunk stability.
Immunohistochemical localization of nitrotyrosine, iNOS, myeloperoxidase, COX-2, Bax, and Bcl-2
At 24 h after SCI, the tissues were fixed in 10% (wt/vol) PBS-buffered formaldehyde, and 8-μm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (vol/vol) H2O2 in 60% (vol/vol) methanol for 30 min. The sections were permeabilized with 0.1% (wt/vol) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% (vol/vol) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA), respectively. Sections were incubated overnight with antinitrotyrosine rabbit polyclonal antibody (Upstate; 1:500 in PBS, vol/vol), with anti-iNOS polyclonal antibody rat (1:500 in PBS, vol/vol), anti-COX-2 monoclonal antibody (1:500 in PBS, vol/vol), anti-Bax rabbit polyclonal antibody (Santa Cruz Biotechnology; 1:500 in PBS, vol/vol), or with anti-Bcl-2 polyclonal antibody rat (Santa Cruz Biotechnology; 1:500 in PBS, vol/vol), or with antimyeloperoxidase (MPO) polyclonal antibody (Santa Cruz Biotechnology; 1:500 in PBS, vol/vol). Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat antirabbit immunoglobulin G and avidin-biotin peroxidase complex (DBA) and counterstained with nuclear fast red. To verify the binding specificity for nitrotyrosine, iNOS, COX-2, Bax, and Bcl-2, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections, indicating that the immunoreaction was positive in all the experiments carried out. Immunocytochemistry photographs (n = 5) were assessed by densitometry as previously described (18) by using an imaging densitometer (AxioVision; Zeiss, Milan, Italy) and a computer program.
TUNEL assay
Terminal deoxynucleotidyltransferase-mediated UTP end labeling assay was conducted by using a TUNEL detection kit according to the manufacturer's instructions (ApopTag; HRP kit DBA, Milan, Italy). Briefly, sections were incubated with 15 μg/mL proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% H2O2 for 5 min at room temperature and then washed with PBS. Sections were immersed in TdT buffer containing deoxynucleotidyl transferase and biotinylated dUTP in TdT buffer, incubated in a humid atmosphere at 37°C for 90 min, and then washed with PBS. The sections were incubated at room temperature for 30 min with antihorseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine. The number of TUNEL-positive cells/high-power field was counted in 5 to 10 fields for each coded slide as previously described (19).
MPO activity
Myeloperoxidase activity, an indicator of polymorphonuclear neutrophil accumulation, was determined as previously described (20) 24 h after SCI. At the specified time after SCI, spinal cord tissues were obtained and weighed, and each piece was homogenized in a solution containing 0.5% (wt/vol) hexadecyltrimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000_g_ at 4°C. An aliquot of the supernatant was then allowed to react with a solution of 1.6 mM tetramethylbenzidine and 0.1 mM H2O2. The rate of change in absorbance was measured spectrophotometrically at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 µM of peroxide per minute at 37°C and was expressed in milliunits per gram of wet tissue.
Quantitative real-time polymerase chain reaction
A segment of spinal cord (320 ± 124 mg) encompassing lesion epicenter was homogenized in a solution of Tri-reagent (Sigma, Deisenhofen, Germany) according to the manufacturer's instructions. Total cellular RNA was isolated from the aqueous phase, then the levels of IL-1β, TNF-α, monocyte chemoattractant protein (MCP) 1, and IL-6 gene products were determined by real-time polymerase chain reaction (PCR). The amount of each PCR product of gene target was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The RNA extracted from spinal cord of individual mice was reverse-transcripted into first-strand cDNA with random hexamers and purified avian myeloblastosis virus reverse transcriptase (Takara, Japan) in 20 mL of reaction after a DNAse treatment and stored at −80°C until use. Primer pair for each cytokine was designed using the software Oligo (Molecular Biology Insights, Cascade, Colo) (Table 1). The PCR reactions were performed using the following cycle conditions: a denaturation step for 15 min at 95°C and 40 cycles of 30-s denaturation at 94°C, 30 s annealing (TNF-α, 61°C; IL-6, 54°C; IL-1β, 61°C, MCP-1, 54°C, GAPDH 56°C), 15 s at 72°C, and 7 min of final extension at 72°C. The PCR products were visualized by electrophoresis on agarose gel 2% stained by ethidium bromide. The purified PCR products were cloned into a plasmid vector, transformed into the Escherichia coli TOP 10 (Invitrogen) and purified with Turbo Kit (QBIOgene, Irvine, Calif). The recombinant plasmid was linearized upstream the target sequence using the restriction endonuclease _Pme_I (Fermentas, Burlington, Ontario, Canada). Ten-fold dilutions of recombinant plasmid from 109 copies down to 101 copies were used as standards. The real-time PCR assay was developed and evaluated on the Rotor-Gene 3000 system (Corbett Research, Sydney, Australia). Reaction was carried out in a final volume of 25 μL containing 1× SYBR PREMIX Ex Taq Takara, 200 nM each forward and reverse primers, 1× Rox Reference Dye, and 2 μL of cDNA. Cycling parameters were the following: 10 min at 95°C for polymerase activation followed by 40 cycles of 15 s at 95°C, 15 s annealing (TNF-α, 61°C; IL-10, 54°C; IL-6, 54°C; IL-1β, 61°C; MCP-1, 54°C; GAPDH, 56°C) 20 s at 72°C. The signal was acquired on the FAM channel (multichannel machine; source, 470 nm; detector, 510 nm; gain set to 5), with the fluorescence reading taken at the end of each 72°C step. A melt step was added after a cycling run performed with the same parameters of the SYBR Green assay. During the melt cycle, the temperature was increased by increments of 1°C from 72°C to 95°C, and the signal was acquired on the FAM channel (source, 470 nm; detector, 510 nm; gain set to 5).
Primer pairs for PCR analysis and length of amplicons (bp)
Materials
Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company, Ltd. (Milan, Italy). All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter, Milan, Italy), 10% dimethyl sulfoxide, or 10% ethanol.
Statistical evaluation
All values in the figures and text are expressed as mean ± SEM of the number (n) of observations. For the in vivo studies, n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A P value of less than 0.05 was considered significant. Basso, Beattie, and Bresnahan scale data were analyzed by the Mann-Whitney test and considered significant when P value was less than 0.05.
RESULTS
E2 reduces the severity of SCI
The severity of the trauma at the level of the perilesional area, assessed as the presence of edema and alteration of the white matter (Fig. 2B, see histological score in Fig. 2E), was evaluated at 24 h after injury by hematoxylin/eosin. Significant damage to the spinal cord was observed in the spinal cord tissue of control mice subjected to SCI when compared with sham-operated mice (Fig. 2A, see histological score in Fig. 2E). Notably, a significant protection against the SCI-induced histological alteration was observed in E2-treated mice (Fig. 2C, see histological score in Fig. 2E). Myelin structure was observed by Luxol fast blue staining (Fig. 2F-H, see histological score in Fig. 2E). In sham animals (data not shown), myelin structure was clearly stained by Luxol fast blue in both lateral and dorsal funiculi of the spinal cord. At 24 h after the injury, a significant loss of myelin in lateral and dorsal funiculi was observed in control mice subjected to SCI? (Fig. 2F, see histological score in Fig. 2E). In contrast, in E2-treated mice, myelin degradation was attenuated in the central part of lateral and dorsal funiculi (Fig. 2G, see histological score in Fig. 2E). Coadministration of ICI 182,780 and E2 significantly blocked the salutary effects of E2 on the histological alteration (Fig. 2D, see histological score in Fig. 2E) and on the myelin structure (Fig. 2H, see histological score in Fig. 2E).
Effect of E2 on histological alterations of the spinal cord tissue 24 h after injury. No histological alteration was observed in the spinal cord from sham-operated mice (A). Significant damage to the spinal cord in nontreated SCI-operated mice at the perilesional area was assessed 24 h after trauma by the presence of edema and alteration of the white matter (B). Notably, significant protection from the SCI was observed in the tissue collected from E2 SCI-treated mice (C). Myelin structure was observed by Luxol fast blue staining. At 24 h after the injury in nontreated SCI-operated mice (F), a significant loss of myelin was observed. In contrast, in E2 SCI-treated mice, myelin degradation was attenuated (G). Coadministration of ICI 182,780 and E2 significantly blocked the salutary effects of E2 on histological alteration (D) and on myelin structure (H). The histological score (E) was made by an independent observer. This figure is representative of at least three experiments performed on different experimental days. *P < 0.01 vs. SHAM; † P < 0.01 vs. SCI; ‡ P < 0.01 vs. E2. GM indicates gray matter; WM, white matter.
Effects of E2 on neutrophil infiltration
The above-mentioned histological pattern of SCI seemed to be correlated with the influx of leukocytes into the spinal cord. We therefore investigated the role of E2 on neutrophil infiltration by measuring tissue MPO activity. Myeloperoxidase activity was significantly elevated in the spinal cord at 24 h after injury in control mice subjected to SCI when compared with sham-operated mice (Fig. 3A). In E2-treated mice, the MPO activity in the spinal cord at 24 h after injury was significantly attenuated in comparison to that observed in SCI control mice (Fig. 3A). Coadministration of ICI 182,780 and E2 significantly blocked the salutary effects of E2 on neutrophil infiltration (Fig. 3A). In addition, tissue sections obtained at 24 h from SCI-operated mice demonstrate positive staining for MPO mainly localized in the infiltrated inflammatory cells in injured area (Fig. 3C1, see densitometry analysis in Fig. 4). In mice treated with E2 (Fig. 3D, see densitometry analysis in Fig. 4), the staining for MPO was visibly and significantly reduced in comparison with the SCI-operated mice. There was no staining for MPO in spinal cord tissues obtained from the sham group of mice (Fig. 3B, see densitometry analysis in Fig. 4). Coadministration of ICI 182,780 and E2 significantly blocked the salutary effect of E2 on the MPO (Fig. 3EE1, see densitometry analysis in Fig. 4).
Effects of E2 on MPO activity. The MPO activity in spinal cord of nontreated SCI-operated mice was significantly increased at 24 h after the damage in comparison to sham mice (A). Treatment with E2 significantly reduced the SCI-induced increase in MPO activity (A). Coadministration of ICI 182,780 and E2 significantly blocked the salutary effect of E2 on MPO activity (A). In addition, no positive staining for MPO was observed in spinal cord tissues collected from sham-operated mice (B). A significant positive staining for MPO was observed in the spinal cord tissues collected from SCI-operated mice (C; see particle C1). In SCI-operated mice treated with E2 (D), the staining for MPO was visibly and significantly reduced in comparison with the SCI-operated mice. Coadministration of ICI 182,780 and E2 significantly blocked the effect of E2 on MPO formation in the spinal cord tissues (E; see particle E1). Image is a representative of at least three experiments performed on different experimental days. Data are mean ± SE of the mean of 10 mice in each group. *P < 0.05 vs. vehicle; † P < 0.01 vs. SCI; ‡ P < 0.01 vs. E2. GM indicates gray matter; WM, white matter.
Typical densitometry evaluation. Densitometry analysis of immunocytochemistry photographs (n = 5 photos from each sample collected from all mice in each experimental group) for MPO, iNOS, nitrotyrosine, COX-2, Bax, and Bcl-2 from spinal cord tissues was assessed. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU, G3-266). Data are expressed as percentage of the total tissue area. *P < 0.01 vs. sham; † P < 0.01 vs. SCI; ‡ P < 0.01 vs. E2. ND indicates not detectable.
E2 modulates expression of cytokines and chemokines
To determine if E2 modulates the inflammatory process through the regulation of the secretion of several cytokines, we analyzed the tissue levels of TNF-α, IL-1β, IL-6, and MCP-1. When compared with sham-operated mice, SCI caused a significant increase in the tissue levels of TNF-α, IL-1β, IL-6, and MCP-1 in SCI control mice (Fig. 5). The increases in the tissue levels of TNF-α, IL-1β, IL-6, and MCP-1 seen in E2-treated mice subjected to SCI were significantly reduced in comparison with the vehicle-treated mice (Fig. 5). Coadministration of ICI 182,780 and E2 significantly but partially blocked the salutary effects of E2 on cytokine and chemokine production (Fig. 5).
Effects of E2 on spinal cord levels of TNF-α IL-1β, IL-6 and MCP-1. A significant increase of the TNF-α (A), IL-1β (B), IL-6 (C), and MCP-1 (D) mRNA levels was observed in the spinal cord tissues at 24 h after SCI. In the spinal cord tissues of E2-treated SCI mice, the TNF-α (A), IL-1β (B), IL-6 (C), and MCP-1 (D) mRNA levels were significantly reduced in comparison to those of SCI animals measured in the same conditions. Coadministration of ICI 182,780 and E2 significantly blocked the effect of the E2 on TNF-α IL-1β, IL-6, and MCP-1. The results of the quantitative real-time PCR of the TNF-α IL-1β, IL-6, and MCP-1 mRNA expression are expressed as a ratio between the number of copies of the target cytokine and the number of copies of the housekeeper (GAPDH) to have an absolute ratio. *P < 0.05 vs. vehicle; † P < 0.01 vs. SCI; ‡ P < 0.01 vs. E2.
E2 modulates expression of iNOS and the nitrotyrosine formation after SCI
Immunohistological staining for iNOS in the spinal cord was determined 24 h after injury. Sections of spinal cord from sham-operated mice did not stain for iNOS (Fig. 6A, see densitometry analysis in Fig. 4), whereas spinal cord sections obtained from SCI control mice exhibited positive staining for iNOS (Fig. 6B, see densitometry analysis in Fig. 4) mainly localized in inflammatory cells and in nuclei of Schwann cells in the white and gray matter of the spinal cord tissues. Treatment of mice subjected to SCI with E2 reduced the degree of positive staining for iNOS (Fig. 6C, see densitometry analysis in Fig. 4) in the spinal cord. To determine the localization of "peroxynitrite formation" and/or other nitrogen derivatives produced during SCI, nitrotyrosine, a specific marker of nitrosative stress, was measured by immunohistochemical analysis in the spinal cord sections at 24 h after SCI. Sections of spinal cord from sham-operated mice did not stain for nitrotyrosine (Fig. 6E, see densitometry analysis in Fig. 4), whereas spinal cord sections obtained from SCI control mice exhibited positive staining for nitrotyrosine (Fig. 6F, see densitometry analysis in Fig. 4) mainly localized in inflammatory cells and in nuclei of Schwann cells in the white and gray matter of the spinal cord tissues. Treatment of mice subjected to SCI with E2 reduced the degree of positive staining for nitrotyrosine (Fig. 6G, see densitometry analysis in Fig. 4) in the spinal cord. Coadministration of ICI 182,780 and E2 significantly blocked the salutary effects of E2 on iNOS expression (Fig. 6D, see densitometry analysis in Fig. 4) and nitrotyrosine formation (Fig. 6H, see densitometry analysis in Fig. 4).
Immunohistochemical localization of iNOS and nitrotyrosine. No positive staining for iNOS (A) and nitrotyrosine (E) was observed in the spinal cord tissues collected from sham-operated mice. Administration of E2 in SCI-operated mice produced a marked reduction in the immunostaining for iNOS (C) and nitrotyrosine (G) in spinal cord tissue when compared with positive staining for iNOS (B) and nitrotyrosine (F) obtained from the spinal cord tissue of mice 24 h after the injury. Coadministration of ICI 182,780 and E2 significantly blocked the salutary effects of E2 on iNOS (D) and nitrotyrosine (H). This figure is representative of at least three experiments performed on different experimental days. GM indicates gray matter; WM, white matter.
E2 modulates expression of COX-2 after SCI
Immunohistological staining for COX-2 in the spinal cord was also determined 24 h after injury. Sections of spinal cord from sham-operated mice did not stain for COX-2 (Fig. 7A, see densitometry analysis in Fig. 4), whereas spinal cord sections obtained from SCI control mice exhibited positive staining for COX-2 (Fig. 7B, see densitometry analysis in Fig. 4) mainly localized in inflammatory cells and in nuclei of Schwann cells in the white and gray matter of the spinal cord tissues. Treatment of mice subjected to SCI with E2 reduced the degree of positive staining for COX-2 (Fig. 7C, see densitometry analysis in Fig. 4) in the spinal cord. Coadministration of ICI 182,780 and E2 significantly blocked the salutary effects of E2 on COX-2 expression (Fig. 7D, see densitometry analysis in Fig. 4).
Immunohistochemical localization of COX-2. No positive staining for COX-2 (A) was observed in the spinal cord tissues collected from sham-operated mice. Administration of E2 in SCI-operated mice produced a marked reduction in the immunostaining for COX-2 (C) in spinal cord tissue when compared with positive staining for COX-2 (B) obtained from the spinal cord tissue of mice 24 h after the injury. Coadministration of ICI 182,780 and E2 significantly blocked the effect of the E2 on COX-2 (D). This figure is representative of at least three experiments performed on different experimental days. GM indicates gray matter; WM, white matter.
Effect of E2 on apoptosis in spinal cord after injury
To test whether spinal cord damage was associated with cell death by apoptosis, we measured TUNEL-like staining in the perilesional spinal cord tissue. Almost no apoptotic cells were detected in the spinal cord from sham-operated mice (data not shown). At 24 h after the trauma, tissues obtained from SCI-operated mice demonstrated a marked appearance of dark brown apoptotic cells and intercellular apoptotic fragments (Fig. 8A TUNEL+ cells were 3.01 ± 0.13 per field) associated with a specific apoptotic morphology characterized by the compaction of chromatin into uniformly dense masses in perinuclear membrane, the formation of apoptotic bodies, and the membrane blebbing (see particles A1). In contrast, tissues obtained from mice treated with E2 (Fig. 8B TUNEL+ cells were 0.45 ± 0.12 per field) demonstrated a small number of apoptotic cells or fragments. Coadministration of ICI 182,780 and E2 significantly blocked the effect of the E2 on the presence of apoptotic cell (Fig. 8C TUNEL+ cells were 2.80 ± 0.18 per field). Section D demonstrates the positive staining in the Kit-positive control tissue.
Representative TUNEL coloration in rat spinal cord tissue section. The number of apoptotic cells increased at 24 h after SCI (A) associated with a specific apoptotic morphology characterized by the compaction of chromatin into uniformly dense masses in perinuclear membrane, the formation of apoptotic bodies, and the membrane blebbing (see particle a1). In contrast, tissues obtained from E2-treated mice (B) demonstrated a small number of apoptotic cells or fragments. Coadministration of ICI 182,780 and E2 significantly blocked the effect of E2 on the presence of apoptotic cells (C). Positive staining in the Kit-positive control tissue (D). Figure is representative of at least three experiments performed on different experimental days. GM indicates gray matter; WM, white matter.
Effect of E2 on Bax and Bcl-2 expression
The appearance of Bax in homogenates of spinal cord was investigated by Western blot at 24 h after SCI. A basal level of Bax was detected in the spinal cord from sham-operated animals (Fig. 9A, A1). Bax levels were substantially increased in the spinal cord from control mice subjected to SCI (Fig. 9A, A1); however, E2 treatment prevented the SCI-induced Bax expression (Fig. 9A, A1). Coadministration of ICI 182,780 and E2 significantly blocked the salutary effects of E2 on Bax expression (Fig. 9A, A1c).
Representative Western blots of Bax levels (A) and Bcl-2 (B). Western blot analysis was realized in spinal cord tissue collected at 24 h after injury. A, Sham: basal level of Bax was present in the tissue from sham-operated mice. SCI: Bax band is more evident in the tissue from spinal cord-injured mice. SCI + E2: Bax band disappeared in the tissue from spinal cord-injured mice that received E2. SCI + ICI 182,780 + E2: Bax band is more evident in comparison with the band present in the tissue from E2-treated mice; B, Sham: basal level of Bcl-2 was present in the tissue from sham-operated mice. SCI: Bcl-2 band disappeared in the tissue from spinal cord-injured mice. SCI ?+ E2: Bcl-2 band is more evident in the tissue from spinal cord-injured mice that received E2. SCI+ ICI 182 780 + E2: Bcl-2 band is less evident in comparison with the band present in the tissue from E2-treated mice (A1 and B1). The intensity of retarded bands (measured by phosphoimager) in all the experimental groups. A-B, Immunoblotting is representative of one spinal cord tissue of five to six analyzed. The results are expressed as mean ± SEM from five to six spinal cord tissues (A1, B1). *P < 0.05 vs. vehicle; † P < 0.01 vs. SCI; ‡ P < 0.01 vs. E2.
To detect Bcl-2 expression, whole extracts from spinal cord of each rat were also analyzed by Western blot analysis. A low basal level of Bcl-2 expression was detected in spinal cord from sham-operated mice (Fig. 9B, B1). Twenty-four hours after SCI, the Bcl-2 expression was significantly reduced in whole extracts obtained from spinal cord of SCI control mice (Fig. 9B, B1). Treatment of mice with E2 significantly reduced the SCI-induced inhibition of Bcl-2 expression (Fig. 9B, B1). Coadministration of ICI 182,780 and E2 significantly blocked the salutary effect of E2 on the Bcl2 expression (Fig. 9B, B1).
The samples of spinal cord tissue were also taken at 24 h after SCI to determine the immunohistological staining for Bax and Bcl-2. Sections of spinal cord from sham-operated mice did not stain for Bax (Fig. 10A), whereas spinal cord sections obtained from SCI control mice exhibited a positive staining for Bax (Fig. 10B). 17β-Estradiol treatment reduced the degree of positive staining for Bax in the spinal cord of mice subjected to SCI (Fig. 10C). In addition, sections of spinal cord from sham-operated mice demonstrated positive staining for Bcl-2 (Fig. 10D), whereas in SCI control mice, the staining for Bcl-2 was significantly reduced (Fig. 10E). 17β-Estradiol treatment attenuated the loss of positive staining for Bcl-2 in the spinal cord in mice subjected to SCI (Fig. 10F). Coadministration of ICI 182,780 and E2 significantly blocked the salutary effects of E2 on Bax (Fig. 10G) and Bcl-2 (Fig. 10H) expression.
Immunohistochemical expression of Bax and Bcl-2. No positive staining for Bax was observed in the tissue section from sham-operated mice (A). Spinal cord injury caused an increase in the release of Bax expression at 24 h (B). Treatment with E2 significantly inhibited the SCI-induced increase in Bax expression (C). On the contrary, positive staining for Bcl-2 was observed in the spinal cord tissues of sham-operated mice (E). At 24 h after SCI, significantly less staining for Bcl-2 was observed (F). 17β-Estradiol treatment significantly prevents the loss of Bcl-2 expression induced by SCI (G). Coadministration of ICI 182,780 and E2 significantly blocked the salutary effects of E2 on Bax (D) and Bcl-2 (H). Figure is representative of at least three experiments performed on different experimental days. GM indicates gray matter; WM, white matter.
Effect of E2 on motor function
To elucidate the potential clinical significance of the protective effects of E2, we have also investigated whether the pretreatment or posttreatment with E2 modified the motor dysfunction evaluated by the modified BBB hind limb locomotor rating scale score associated with SCI. Although motor function was only slightly impaired in sham mice, mice subjected to SCI had significant deficits in hind limb movement (Fig. 11). A significant amelioration of hind limb motor disturbances was observed in E2-pretreated and posttreated mice (Fig. 11). Coadministration of ICI 182,780 and E2 significantly blocked the effect of E2 on the motor recovery (Fig. 11). In addition, a more significant amelioration of hind limb motor disturbances was observed in the SCI-operated mice that received E2 (600 μg/kg) as posttreatment (Fig. 11).
Effect of E2 on hind limb motor disturbance after SCI. The degree of motor disturbance was assessed every day for 10 days after SCI by BBB criteria. Pretreatment or posttreatment with E2 reduces the motor disturbance after SCI. Coadministration of ICI 182,780 and E2 significantly blocked the salutary effect of E2 on the motor disturbance after SCI. Values shown are mean ± SE of the mean of 10 mice in each group. *P < 0.01 vs. SCI; † P < 0.01 vs. SCI + E2.
DISCUSSION AND CONCLUSIONS
17β-Estradiol has been shown to be therapeutic in a variety of models of neurological disease, including SCI (9). Our results show that E2 exerts beneficial effects in a mouse model of SCI, demonstrating that E2 reduced (1) the degree of spinal cord damage; (2) infiltration of neutrophils; (3) proinflammatory cytokine and chemokine expression; (4) expression of iNOS, nitrotyrosine, and COX-2; and (5) apoptosis.
Our results using histological examination indicate that the increased tissue edema induced by injury was prevented by E2 pretreatment. Application of vascular clips to the dura via a four-level T5-T8 laminectomy resulted in edema and loss of myelin in lateral and dorsal funiculi, and this histological damage was associated with the loss of motor function. However, E2 pretreatment significantly reduced the SCI-induced spinal cord tissue alteration and improved the motor function.
Several studies have clearly demonstrated that an inflammatory response characterized by neutrophil infiltration and microglia activation develops within hours after SCI (22). In this study, we confirmed the infiltration of neutrophils at 24 h after SCI. Moreover, E2 administration reduces this event when compared with the SCI group. The recruitment of inflammatory cells such as the neutrophils is responsible for the production of several immunomodulatory factors, including the cytokines and lipid mediators. Among the cytokines, TNF-α and IL-1 are proinflammatory mediators responsible for leukocyte activation and recruitment (23). Furthermore, there is good evidence that TNF-α and IL-1β are involved in the pathogenesis of SCI (24). Moreover, it has been demonstrated that the expression of proinflammatory cytokines, including TNF-α and IL-1β, at the site of injury controls the cellular events after SCI. In this study, we have confirmed that there is a significant increase in TNF-α, IL-1β, IL-6, and MCP-1 in spinal cord tissues at 24 h after SCI, whereas E2 treatment significantly reduced the expression of these cytokines.
Studies also indicate that cytokines play an important role in the induction of iNOS, which is known to be involved in the development of pathological processes of SCI (25). Our results indicate that E2 treatment reduces the expression of iNOS in SCI-operated mice, and we propose that this attenuation of iNOS expression is secondary to a reduced formation of endogenous TNF-α and IL-1β. In the pathological processes of acute SCI, the up-regulation of COX-2 has also been postulated to be involved. It is well known that COX-1 and COX-2 mRNA and protein are present in the spinal cord tissue, and that COX-2 protein is expressed in white matter astrocytes during basal conditions. Moreover, COX-2 expression is also mediated by TNF-α and IL-1 (26-28). Our results demonstrate that in spinal cord from E2-treated mice, there is a markedly less positive staining for COX-2 when compared with that from SCI mice. These results are in agreement with other observations (29) that reveal a relationship between TNF-α production and the COX-2 expression. In addition, several studies have implicated the formation of reactive oxygen species and reactive nitrogen species in the secondary neuronal damage of SCI (30). To confirm the pathological contributions of peroxynitrite to secondary damage after SCI, we evaluated the formation of nitrotyrosine in the injured tissue. Our results indicate that the immunostaining for nitrotyrosine was reduced in E2-treated mice. Nitrotyrosine formation was initially proposed as a specific marker for the detection of the endogenous formation "footprint" of peroxynitrite (31). There is, however, recent evidence indicating that other reactions can also induce tyrosine nitration, for example, the reaction of nitrite with hypochlorous acid and the reaction of myeloperoxidase with H2O2 (32). Increased nitrotyrosine staining is therefore considered as an indication of "increased nitrosative stress," rather than a specific marker of the peroxynitrite generation.
Recent studies have demonstrated the induction of apoptosis in different cell line in response to reactive oxygen species, peroxynitrite, and NO (33). Using the TUNEL coloration and Western blot analysis, we have confirmed that E2 plays an important role in the attenuation of apoptosis during SCI. In fact, it is well known that Bax is a key regulator of apoptosis (34) and central nervous system injury. It has also been shown that the administration of Bcl-xL fusion protein into injured spinal cords significantly increased neuronal survival, suggesting that SCI-induced changes in Bcl-xL contribute considerably to neuronal death (35). Based on these findings, we identified proapoptotic transcriptional changes, including up-regulation of proapoptotic Bax and down-regulation of antiapoptotic Bcl-2. Antioxidative activities of E2 might in part explain some of the findings reported in the present study. However, the antioxidant activity of E2 is observed at pharmacological concentrations of the hormone and is not blocked by antagonists of the estrogen receptors. We therefore propose that the lack of spinal cord tissue injury in E2-pretreated mice reported here is not due to estrogen's antioxidant action because mice were treated with a dose of the hormone that was not very high. Of significance was the fact that the protective effects of E2 were abrogated by the coadministration of the antagonist of estrogen receptor ICI 182,780, indicating that the observed effects of E2 are receptor-mediated. Taken together, the results of the present study highlighted our current knowledge on E2 in the pathophysiology of spinal cord cell and tissue injury after trauma. Although our results clearly demonstrate that administration of E2 in SCI had protective effects against the cascade of deleterious effects under those conditions, it remains to be determined if administration of E2 after SCI would still have salutary effects on the host inflammatory responses.
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Keywords:
SCI; inflammation; tissue injury; apoptosis
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